highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20195 highlights in bioscience october 2019| volume 2 http://bioscience.highlightsin.org/ page 1 of 2 research article open access 1 department of genome mapping, molecular genetics and genome mapping laboratory, agricultural genetic engineering research institute, giza, egypt. 2 international center for agricultural research in the dry areas (icarda), cairo, egypt. contacts of authors * to whom correspondence should be addressed: alsamman m. alsamman. citation: alsamman m.a. , habib p.t. (2019). genesyno : simple tool to extract gene sequence from the human genome despite synonymous gene terms. highlights in bioscience, volume 2 . article id 20195, dio:10.36462/ h.biosci.20195 received: august 25, 2019 accepted: september 20, 2019 published: october 25, 2019 copyright: © 2019 alsamman and habib. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. genesyno : simple tool to extract gene sequence from the human genome despite synonymous gene terms alsamman m. alsamman1* , peter t. habib2 abstract extracting gene data from the human genome is a tricky task. gene name is the key information for harvesting its sequence, annotation, and other related data.unfortunately, most human genes have different and multiple names, depending on the database and the resource in which they have been published. such an issue is delaying the ability of researchers to gather the necessary knowledge and to build their opinion on the function of genes. here we introduce genesyno, a simple, versatile and reliable tool that can be used to extract gene information from human genome data even though it is synonymous gene names. genesyno was written using c and python programming languages and could easily be integrated into another pipeline. keywords: gene information, human genome, gene name , gene annotation, gene, synonymous gene name. background human genome research is one of the most intensive research fields. synonymous terms of the name of the gene remain a major issue in human genomics (1). several human genes have different names, depending on the databases, the articles or the newly discovered function. with even more research articles published online, this information has become challenging for efficient implementation and reuse (2). such a case has been a complicated issue where genomic scientists can not form a collective and prospective conclusion by using published information on most human genes. several tools have been published to solve this problem, where text mining, and searching for databases could be used to generate symbol co-occurrences to extend information extraction capabilities (3-6).the main problem that, most of these tools require high computational skills, or are available only in online versions. most of these tools require high computational skills, or are only available in online versions. this may constrain the ability of researchers to access all available information for massive lists of genes at any time. here we introduce genesyno, a simple, versatile and reliable tool that can be used to extract gene information from human genome data even though it is synonymous gene names. genesyno was written using c and python programming languages and could easily be integrated into another pipeline. material and methods genesyno was built using c and python3 programming languages. the user’s input will be a list of human gene names. the input from the user will be a list of names for human genes. genesyno collects all available information about these genes from the grch38 database (which users could change for newer versions) and reports a tab-limited file containing gene information such as gene name, official gene name, chromosome , description, gene start, gene end, and a list of gene synonym names. furthermore, it produced a fasta file that contains sequences of all genes' proteins . if gene have more than one protein (isoforms) it will reported (figure 1). https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20195 http://bioscience.highlightsin.org/ page 2 of 2 alsamman et al., 2019 genesyno : simple tool to extract gene information despite synonymous gene terms highlights in bioscience october 2019| volume 2 http://bioscience.highlightsin.org/ genesyno core was written using c programming language to extract massive lists of information about genes in less processing time. genesyno can be installed and used on various operating systems, and has a simple gui for users. figure 1 : example of genesyn input and outputs. if a list of names of human genes is given in text format (a), genesyno can produce a table containing all gene information in tab delimited format (b) and protein sequences in fasta formats (c) . availability genesyno is available as a standalone tool at : https://github.com/alsammanalsamman/genesyno references 1. cohen kb, acquaah-mensah gk, dolbey ae, hunter l. contrast and variability in gene names. in: proceedings of the acl-02 workshop on natural language processing in the biomedical domain-volume 3. 2002. p. 14–20. 2. chen l, liu h, friedman c. gene name ambiguity of eukaryotic nomenclatures. bioinformatics. 2005;21(2):248–56. 3. cohen am, hersh wr, dubay c, spackman k. using co-occurrence network structure to extract synonymous gene and protein names from medline abstracts. bmc bioinformatics. 2005;6(1):103. 4. plotkin jb, kudla g. synonymous but not the same: the causes and consequences of codon bias. nat rev genet. 2011;12(1):32–42. 5. girish k, dubey s. eukaryotic molecular biology databases: an overview. highlights biosci. 2018;1:1–7. 6. alsamman am. the art of bioinformatics learning in our arabic world. highlights biosci. 2019;2. https://github.com/alsammanalsamman/genesyno http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20204 highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 3 perspective open access university of pécs, medical school, institute of migration health, pécs, szigeti st. 12., h-7624, hungary contacts of authors * to whom correspondence should be addressed: gábor ternák citation: gábor ternák (2020). holistic solution to an old "mystery": the global role of antibiotic consumption patterns in the spread of certain non-contagious diseases in eu countries, volume 3. article id 20204, dio:10.36462/h.biosci.20204 received: may 25, 2020 accepted: june 13, 2020 published: june 20, 2020 copyright: © 2020 ternák. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper. funding: the study was not supported by any grants. competing interests: no conflict of interest can be declared. holistic solution to an old "mystery": the global role of antibiotic consumption patterns in the spread of certain non-contagious diseases in eu countries gábor ternák abstract the discovery and extensive utilization of antibiotics are highly contributed to the considerable lengthening life expectancy of human beings. antibiotics, mixed with animal fodder, produced considerable growth promoting effect and hence, extended the indications of antibiotics at a much higher level. the indiscriminate use of antibiotics quickly resulted in the emergence of poly-resistant pathogens and the extensive antibiotic pollution of the environment, particularly of the surface water and rivers trough human and animal excreta. along with extensive and ever increasing antibiotic consumption/pollution, the pandemic-like spreading of certain non-contagious diseases like obesity, diabetes (type 1-2 t1dm, t2dm), alzheimer disease (ad), parkinson disease, multiple sclerosis (ms) etc. started unfolding, which was called as a slow moving disaster, without having any appropriate explanation of the phenomenon. the parallel appearance of those “pandemics”, which appeared simultaneously with the extensive antibiotic consumption, might indicate some kind of association. as far as several publications have reported the crucial role of altered gut flora in the development of metabolic disorders (diabetes, obesity) and neurodegenerative diseases alike (pd, ad, ms), it might be suspected that antibiotics, acting through the modification of microbiome, could influence the morbidity (prevalence) of those, non-infectious diseases. this concept, described below, might serve as a unified explanation, not excluding other, contributing causative factors, for the phenomenon, outlined above. the discovery of penicillin by fleming in 1937 and later of neomycin and streptomycin by waksman opened a new chapter of science, discovering the miraculous effect of antibiotics on infectious diseases. after the introduction of penicillin, several broad spectrum antibiotics were discovered also, like aminoglycosides, quinolones, macrolides etc., but their indiscriminate use has led to serious consequences and the rapid development of poly-resistant or panresistant bacterial species, which produced practically incurable infections (1). out of the approximately 5000 molecules considered as having an antimicrobial effect, one thousand was further investigated, and finally we have about one hundred commercially available antibiotics to treat infections. most of those drugs are produced by actinomycetes molds and different bacteria. after the „golden age” of antibiotic discovery of ’50-ies and ’60-ies and the extensive utilization of broad spectrum antibiotics in the late ’70-ies and ’80-ies, we now face the fact that the production of new classes of antibiotics practically stopped after 1987. ternák 2020 the role of antibiotic consumption patterns in the spread of non-contagious diseases highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 3 at present, only few antibiotics are in the development, but it is not of certain that they might finally be used as commercially available antibiotics (2). the accidental discovery of the growth promoting potential of antibiotics in the late ’40-ies (3) resulted in the large scale utilization of antibiotics mixed to animal fodder which contributed to the extensive environmental pollution and the emergence of multi-resistant bacteria (genes) to the environment along with the development of human infections, practically untreatable with any antibiotics (4). recently (2013) the yearly consumption of antibiotics in animal husbandry was estimated to be 131,109 tons (5). the “growth promoting effect” of certain broad spectrum antibiotics (than aureomycin) was observed and documented in premature newborns and school children also (6, 7). according to ecdc database of antibiotic consumption for 2018 expressed as the number of defined daily dose (ddd)/1000 inhabitants/day, the average total consumption (community and hospital sector) of antibacterial for systematic use (atc group j01) in the eu/eea countries was 20.1 ddd/1000 inhabitants/day (range: 9.7 – 34.0). during 2013-17, no statistically significant shift in antibiotic consumption was observed in the eu/eea countries and no significantly increasing trends were reported (8). usually, we consider as „epidemic” or “pandemic” the spreading of communicable diseases, but we have to face with the fact that the appearance and spreading of certain non-communicable diseases are similar. in the past few decades the pandemic like spread of several noninfectious diseases are alarming. the reason for the rapidly increasing amount of obesity, particularly childhood obesity, diabetes type 1-2 (t1dm, t2dm), autism, which was hardly heard of 50 years ago, the alzheimer disease (ad) of which produces more death in the usa, than cardiovascular and certain cancer deaths combined, and the parkinson disease (pd) doubled its prevalence in the past 25 years. the prevalence of multiple sclerosis (ms) has increased by approximately 10% since 1990. due to their outstandingly rapid spread, diabetes, obesity, autism, ad, pd and ms can be considered as of major importance. this phenomenon is called as a slow moving disaster (9, 10). scientists working on the explanation of this phenomenon, keep on digging deep to identify the molecular background, discovered several possible pathways and mechanisms leading to the development of those ailments and all of them emphasised the crucial role of the intestinal microbiome alteration (dysbiosis), but some external factors are suspected triggering the development of dysbiosis (11.). several sequencing efforts were made to elucidate the complete composition of gut flora. till now, 1952 uncultured bacterial species were verified by reconstructing 92,143 metagenome assembled-genomes from 11,850 human gut microbiota, which is considered 281% increase in phylogenetic diversity (12). antibiotics are powerful agents influencing the composition of the intestinal flora and producing dysbiosis, by reducing the diversity of the bacterial taxa in the gut. an extensive meta-analyses of articles dealing with the effect of antibiotics on the microbiome since the very first publication summarizes the observations. penicillin has not much effect on the gut flora, but amoxicillin, amoxicillin/clavulanic acid, cephalosporins, macrolide, quinolone, lypopolyglycopeptide, ketolide, fosfomycin; tigecycline has increased the dominance of non e. coli (mostly citrobacter, enterobacter and klebsiella spp.) enterobacteriaceae. in contrary, most of the above antibiotics (amoxicillin, macrolide, clindamycin, quinolone, and sulfonamide) reduce the presence of e. coli in the gut flora, but the combination of amoxicillin/clavulanate has increased its abundance. macrolide, doxycycline reduced the rate of enterococcus spp. in the gut flora, while amoxicillin, piperacillin, ticarcillin, cephalosporin (1-4th generations), lypoglycopep tides, carbapenem has enhanced their presence (13). antibiotics reach our body as either therapeutic agents or from the environment, particularly from the heavily polluted rivers, because of the antibiotic enriched animal fodder, industrial waste and human excreta entering the surface water and ending up in the rivers and are present in the potable water also (14-16). the decisive role of dysbiosis in the process of developing certain chronic diseases might raise the theory that the quality and quantity of antibiotic insult on human microbiome might trigger the development of different diseases and antibiotic consumption preferences in different countries might be related to the different, emerging, noncontagious diseases, showing pandemic-like spread. the issue that growth-promoting effect of antibiotics added to animal fodder of food animals might similarly affect humans as well, was first raised in 2005 (17). further research by the author has led to the discovery of the significant selective correlation between childhood obesity and certain antibiotic consumption in eu countries (18). the significant association was found between the consumption of narrow spectrum penicillin and the prevalence increase of pd. simultaneously, we observed the inhibitory effect of broad spectrum penicillin and the prevalence in pd as well (19). further comparisons of large databases derived from ecdc (20) yearly reports of antibiotic consumption by eu countries (1997-2018) and the prevalence of diabetes (21) ad (22), and ms (23) derived from different resources (publications are in progress) demonstrated the promoting and inhibitory effect of different classes of antimicrobial agents on the development of those diseases. as a conclusion, i have detected two different, very distinctive patterns of antibiotic consumption, what i have called “scandinavian type” with dominant consumption of narrow spectrum penicillin and tetracycline and higher ternák 2020 the role of antibiotic consumption patterns in the spread of non-contagious diseases highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 3 prevalence of pd (24), ms (23) and t1dm (21), and the “mediterranean type” with dominant consumption of broad spectrum antibiotics, including broad spectrum penicillins, and higher prevalence of childhood obesity (25, 26), t2dm (21), and ad (22). cross-association was observed with childhood obesity and ad. we found similar antibiotic consumption patterns in ms, and t1dm. this observation is being supported by the reported three fold higher prevalence of ms among patients with t1dm (27) and indicating the possible inhibitory effect of clavulanic acid in t1dm and ms. the holistic approach permitted us to consider the similarities of the parallel appearance and spreading of certain non-contagious pandemics, which appeared roughly at the same time as the widespread consumption of antibiotics. those findings clearly pointing to the possible common origin of the developing and spreading of certain non-contagious diseases included in the study and serve as an explanation for the “mystery” of the large scale spreading of obesity, diabetes, pd, ad and ms. references 1. hoel d, williams dn, berkelman rl. antibiotics: past, present, and future: unearthing nature's magic bullets. postgraduate medicine. 1997 jan 1; 101 (1): 114-22. https: //doi.org/10.3810/pgm.1997.01.139 2. https://www.khanacademy.org/science/health-and-medicine /current-issues-in-health-and-medicine/antibiotics-andantibiotic-resistance/a/antibiotics-an-overview 3. stokstad el, jukes th, pierce j, page jr ac, franklin al. the multiple nature of the animal protein factor. journal of biological chemistry. 1949; 180: 647-54. 4. argudín ma, deplano a, meghraoui a, dodémont m, heinrichs a, denis o, nonhoff c, roisin s. bacteria from animals as a pool of antimicrobial resistance genes. antibiotics. 2017 jun; 6 (2): 12. 5. https://www.swissre.com/dam/jcr:b8c6410f-755b-4d13-836df611c3510a3d/amr_thomas_boeckel.pdf 6. guzman ma, scrimshaw ns, monroe rj. growth and development of central american children. 1. growth responses of rural guatemalan school children to daily administration of penicillin and aureomycin. american journal of clinical nutrition. 1958; 6: 430-8. 7. robinson p. controlled trial of aureomycin in premature twins and triplets. the lancet. 1952 jan 5; 259 (6697): 52. 8. https://www.ecdc.europa.eu/en/publications-data/ surveillanceantimicrobial-consumption-europe-2018 9. https://www.who.int/publications/10-year-review/ncd/en/index 2.html 10. non-communicable diseases progress monitor, 2017. geneva: world health organization; 2017. 11. liang d, leung rk, guan w, au ww. involvement of gut microbiome in human health and disease: brief overview, knowledge gaps and research opportunities. gut pathogens. 2018 dec; 10(1):3. 12. almeida a, mitchell al, boland m, forster sc, gloor gb, tarkowska a, lawley td, finn rd. a new genomic blueprint of the human gut microbiota. nature. 2019 apr; 568 (7753): 499. https://doi.org/10.1038/s41586-019-096 5-1 13. zimmermann p, curtis n. the effect of antibiotics on the composition of the intestinal microbiota. journal of infection. 2019 oct 18. https://doi.org/10.1016/j.jinf. 2019.10.008 14. joel, l. (2019), antibiotics are flooding earth’s rivers, eos, 100, https://doi.org/10.1029/2019eo127001 15. ternak g. antibiotics in the environment: the possible inadvertent effect on human morbidity & mortality. microbial pathogens & strategies for combating them: science, technology & education. 2013;3: 1455-60. 16. ternak g. global effect of antibiotic consumption/ pollution on human health. lap lambert academic publishing; 2016. 17. ternak g. antibiotics may act as growth/obesity promoters in humans as an inadvertent result of antibiotic pollution?. medical hypotheses. 2005 jan 1; 64(1):14-6. 18. ternak g, edel z, feiszt z, szekeres j, visegrady b, wittmann i. selective association between cephalosporin, quinolone, macrolide, and penicillin antibiotic consumption and childhood obesity in europe. health. 2015;7(10):1306. 19. ternák g, kuti d, kovács kj. dysbiosis in parkinson’s disease might be triggered by certain antibiotics. medical hypotheses. 2020 jan 9:109564. https://doi.org/ 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https://doi.org/10.1016/j.jns.2014.11.%20019 highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 1 of 5 citation: rslan w. m. (2018) sugar beet artificial seeds an overview. highlights in bioscience, volume 1.article id 20181, dio:10.36462/ h.biosci.20181 received: march 22, 2018 accepted: june 17, 2018 published: june 26, 2018 copyright:© 2018 rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. sugar beet artificial seeds an overview wessam m. rslan* agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract artificial seed propagation of crops broadens the horizon of plant biotechnology and farming. the technology offers techniques for micropropagulated seed analogs such as axillary leaves, embryogenic calli, somatic embryos, apical shoot tips, and protocorm-like organs. micropropagules are embedded in gelling medium and carboxyl methyl cellulose active coatings. a variety of plant species, such as mulberry, sandalwood, cardamom, banana, sugar beet, maize, and relative, have recorded encapsulation of micro shoots and somatic embryos and subsequent recovery of full plantlets. this knowledge has shown that artificial seed manufacturing is possibly helpful for the propagation of economically significant species ' inferior hybrids on a big scale. artificial seed development can only succeed with effective upstream manufacturing of micropropagules and downstream germination procedures for an elevated proportion of plant regeneration as one of the significant value-added plant tissue culture goods. different micropropagules were regarded for the manufacturing of artificial seeds; however, mostly favored were somatic embryos and axillary stem buds. as micropropagules, somatic embryos were used to create artificial seeds in a wide range of fruit and plant organisms, which include daucus carota, picea abies, arachis hypogaea, medicago sativa, psidium guajava, and vitis vinifera. the review illustrated the concept of synthetic seeds and encapsulation procedure of sugar beet. keywords: artificial seed, plant biotechnology, embryogenic, sugar beet, micropropagulated. review article open access highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 2 of 5 introduction ara et al. (1) described artificial plants as synthetically encapsulated somatic embryos, shoot tips, axillary flowers or another meristematic tissue employed for seed sowing and capable of converting into whole plants under in vitro and in vivo circumstances and maintaining their potential even after storage. the first one to suggest and handle encapsulated somatic embryos was murashige (2). he also noted to the transfer option and used it as natural plants. reddy et al. (3) clarified that the cultivation of plants using artificial plants from somatic embryos or other vegetative propagules opens up new agricultural and forestry technologies. a variety of plant species synthetic seeds have been effectively researched in plant propagation. encapsulating agents kitto and janick (4) discovered that the most appropriate medium for the encapsulation of somatic embryos was' polyox,' water-soluble resin. redenbaugh et al. (5) suggested that magnesium alginate should be the most appropriate to encapsulate somatic embryos in alfalfa, celery, cauliflower and rose. artificial seed performance relies on the spatial, qualitative, quantitative availability of growth regulators and nutrients together with an ideal physical setting (6). mariani (7) indicated that gibberellic acid (ga3) and saccharose had an adverse impact on eggplant germination of synthetic seeds. in the meantime refouvelet et al . (8) used ba (5 mg / l) + 1⁄2 ms + naa (0.01 mg / l) to encapsulate syringa vulgaris. pattnaik and chand (9) axillary buds, murashige and skoog (10) medium (ms) without hormones and ms + 6-benzyladenine (ba, 4.4 μm) have been used as artificial endosperm in morus species. saiprasad (11) revealed that sodium alginate would be the most widely recognized hydro-gel and was regularly used as a matrix for artificial plants due to its small toxicity, low price, rapid gellation and bio-compatibility. many researchers noted that the introduction to the encapsulation solution of aquatic cyanobacterial samples (12), bactericides and activated carbon (13), pesticides, fertilizers, microorganisms (rhizobia), mycorrhiza fungi (14), fungicides (15) can safeguard encapsulated propagules from microorganisms, decrease the discharge of toxic compounds and improve the germination ability of seeds. encapsulation procedure the hydro-gel encapsulation technique established by (5) was the most suitable technique for producing synthetic seeds. in this technique, by combining with calcium free liquid ms medium, sodium alginate of varying levels (2 to 5 percent) was formed and then the explants were blended with the solution. together with the sodium alginate solution, explants were sucked with a pipette and dropped into calcium chloride pool in which the ion exchange reaction happens and sodium ions were substituted by calcium ions comprising alginate beads. it is necessary to complete the entire method under aseptic circumstances. the capsule size relies on the pipette nozzle's inner diameter. the beads structure and size relies on sodium alginate quantity, calcium chloride solutions, and complexion duration. redenbaugh (16) proposed using a multi nozzle pipette in this embryo stream through the internal pipette and the solution of the alginate flows via the exterior pipette. artificial seeds germination and field planting several researchers (17–20) recorded successful field cultivation and transformation of natural plants. the synthetic plants in the future, particularly for the extremely requested species (21), may be an option planting material intended for the forestry sector. artificial plants would enable plant propagules to be planted directly into the greenhouse or field, circumventing many of the additional phases (21). fujii et al. (22) discovered that the maturation of aba somatic alfalfa embryos yielded an elevated soil transformation rate of 48% to 64%. adding fungicide to alginate beads avoids contamination and increases the sustainability of mulberry seeds in soil (23) when sown. fujii et al. (24) revealed effective field planting with 23 percent crop transformation of alfalfa artificial plants obtained from calcium alginate embryoids. nieves et al. (25) revealed that artificial plant sugarcane crops were larger and had a lower diameter at eight months, but at 12 months these distinctions faded. no variations in all parameters assessed among both artificial seed-derived plants and plants based from the other two techniques (traditional and isolated plant techniques) were discovered with regard to sugar assessment and yield. asmah et al. (21) and ma et al. (26) recorded the efficient germination level in acacia hybrid (73.3 to 100%) and pseudostellaria heterophylla (80%). in podophyllum peltatum, sugar beet and stevia rebaudiana, rizkalla et al. (27) and nower (28) noted that crop development improved by adding mannitol and/or sorbitol to the medium. types of synthetic seeds two forms of synthetic seeds have been established, i.e. desiccated and hydrated synthetic plants, according to the current literature. the synthetic desiccated seeds were first launched either directly or encapsulated in polyox from somatic embryos, followed by their desiccation (29). highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 3 of 5 desiccation has been accomplished either linearly through chambers of decreasing relative humidity slowly over a period of one or two weeks or swiftly by leaving the petri dishes overnight on the bench at the laminar airflow room (30). the hydrated artificial seed technology was first developed by encapsulating medicago sativa (16) hydrated somatic embryos. these artificial hydrated seeds are employed to create plant species that are recalcitrant and susceptible to desiccation in their somatic embryos. hydrated artificial seeds are usually ready in a hydrogel capsule by encapsulating somatic embryos or other propagules. several techniques for producing hydrated artificial seeds were investigated, mostly using calcium alginate encapsulation (16). the genetic stability of synthetic seeds artificial seeds were commonly used in many plant species for micro-propagation. molecular researches have began from the last decade to determine genetic stability of plantlets derived from synthetic seeds, but no changes have been revealed at the biochemical and/or molecular scales. many studies (31) endorsed the prospective benefit of synthetic seeds for genetically identical to natural seeds. gangopadhyay et al. (32) investigated the genetic structure of plantlets obtained from encapsulated ananas comosus micro shoots using rapd and issr technologies (33, 34). bekheet (35) indicated that both plantlets obtained from encapsulated bulblets and usually in vitro were genetically comparable to those obtained from in vivo in allium sativum. narula et al. (36) used rapd assessment to explore in vitro plantlet genetic structure obtained from dioscorea bulbifera encapsulated plant advice. srivastava et al. (37) reported that the analysis of cineraria maritana's rapd patterns revealed a median ratio of resemblance of 0.944, confirming the molecular consistency of crops extracted from encapsulated micro-shoots followed by six months of storage. tabassum et al. (38) investigated the genetic consistency of synthetic seeds obtained from mother crops and somatic embryos and discovered similarity in cucumis sativus using rapd markers. mishra et al. (39) also studies the genetic consistency of crops obtained from encapsulated microshoots in picrorhiza kurrooa using rapd profile cluster analysis. lata et al. (40) used issr and gas chromatography (gc) study of six significant cannabinoids to examine the genetic structure of synthetic seed based crops of cannabis sativa and demonstrated homogeneity in the regrown clones and the mother plant. shoot tips are by far the most genetically consistent, but in callus and protoplast culture there is a strong probability of genetic shift (41–43). references 1. ara h, jaiswal u, jaiswal vs. germination and plantlet regeneration from encapsulated somatic embryos of mango (mangifera indica l.). plant cell rep. springer; 1999;19(2):166–70. 2. murashige t. plant cell and organ cultures as horticultural practices. symposium on tissue culture for horticultural purposes 78. 1977. p. 17–30. 3. naga blri, mangamoori ln, subramanyam s. identification and characterization of est-ssrs in finger millet (eleusine coracana (l.) gaertn.). j crop sci biotechnol. springer; 2012;15(10):9–16. 4. kitto sl, janick j. hardening treatments increase survival of synthetically-coated asexual embryos of carrot. j amer soc hort sci. springer; 1985;110(2):283–6. 5. redenbaugh k, paasch bd, nichol jw, kossler me, viss pr, walker ka. somatic seeds: encapsulation of asexual plant embryos. bio/technology. nature publishing group; 1986;4(9):797. 6. senaratna t. artificial seeds. biotechnol adv. elsevier; 1992;10(3):379–92. 7. mariani p. eggplant somatic embryogenesis combined with synthetic seed technology. capsicum newslett. 1992;289–94. 8. refouvelet e, le nours s, tallon c, daguin f. a new method for in vitro propagation of lilac (syringa vulgaris l.): regrowth and storage conditions for axillary buds encapsulated in alginate beads, development of a preacclimatisation stage. sci hortic (amsterdam). elsevier; 1998;74(3):233–41. 9. pattnaik s, chand pk. morphogenic response of the alginate-encapsulated axillary buds from in vitro shoot cultures of six mulberries. plant cell tissue organ cult. springer; 2000;60(3):177–85. 10. murashige, toshio and fs. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiol plant. 1962;15(3):473–97. 11. saiprasad gvs. artificial seeds and their applications. resonance. springer; 2001;6(5):39–47. highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 4 of 5 12. wake h, akasaka a, umetsu h, ozeki y, shimomura k, matsunaga t. enhanced germination of artificial seeds by marine cynobacterial extract. appl microbiol biotechnol. springer; 1992;36(5):684–8. 13. ganapathi tr, suprasanna p, bapat va, rao ps. propagation of banana through encapsulated shoot tips. plant cell rep. springer; 1992;11(11):571–5. 14. tan tk, loon ws, khor e, loh cs. infection of spathoglottis plicata (orchidaceae) seeds by mycorrhizal fungus. plant cell rep. springer; 1998;18(1-2):14–9. 15. antonietta gm, micheli m, pulcini l, standardi a. perspectives of the encapsulation technology in the nursery activity of citrus. caryologia. taylor & francis; 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heterophylla. african j biotechnol. academic journals (kenya); 2011;10(70):15744–8. 27. rizkalla aa, badr-elden am, ottai me-s, nasr mi, esmail mnm. development of artificial seed technology and preservation in sugar beet. sugar tech. springer; 2012;14(3):312–20. 28. nower aa. in vitro propagation and synthetic seeds production: an efficient methods for stevia rebaudiana bertoni. sugar tech. 2014;16(1):100–8. 29. kitto sl, janick j. polyox as an artificial seed coat for asexual embryos. hortscience. 1982. p. 488. 30. ara h, jaiswal u, jaiswal vs. synthetic seed: prospects and limitations. curr sci. jstor; 2000;1438–44. 31. nyende ab, schittenhelm s, mix-wagner g, greef jm. yield and canopy development of field grown potato plants derived from synthetic seeds. eur j agron. 2005;22(2):175–84 32. gangopadhyay g, bandyopadhyay t, poddar r, gangopadhyay sb, mukherjee kk. encapsulation of pineapple micro shoots in alginate beads for temporary storage. curr sci. jstor; 2005;972–7. 33. adawy ss, mokhtar mm, alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20181 june 2018 | volume1 page 5 of 5 34. mokhtar mm, adawy ss, el-assal se-ds, hussein eha. genic and intergenic ssr database generation, snps determination and pathway annotations, in date palm (phoenix dactylifera l.). plos one. public library of science; 2016;11(7):e0159268. 35. bekheet sa. a synthetic seed method through encapsulation of in vitro proliferated bulblets of garlic (allium sativum l.). arab j biotech. 2006;9:415–26. 36. narula a, kumar s, srivastava ps. genetic fidelity of in vitro regenerants, encapsulation of shoot tips and high diosgenin content in dioscorea bulbifera l., a potential alternative source of diosgenin. biotechnol lett. springer; 2007;29(4):623–9. 37. srivastava v, khan sa, banerjee s. an evaluation of genetic fidelity of encapsulated microshoots of the medicinal plant: cineraria maritima following six months of storage. plant cell, tissue organ cult. springer; 2009;99(2):193–8. 38. tabassum b, nasir ia, farooq am, rehman z, latif z, husnain t. viability assessment of in vitro produced synthetic seeds of cucumber. african j biotechnol. academic journals (kenya); 2010;9(42):7026–32. 39. mishra j, singh m, palni lms, nandi sk. assessment of genetic fidelity of encapsulated microshoots of picrorhiza kurrooa. plant cell, tissue organ cult. springer; 2011;104(2):181–6. 40. lata h, moraes rm, bertoni b, pereira ams. in vitro germplasm conservation of podophyllum peltatum l. under slow growth conditions. vitr cell dev biol. springer; 2010;46(1):22–7. 41. lindsey k, gallois p. transformation of sugarbeet (beta vulgaris) by agrobacterium tumefaciens. j exp bot. oxford university press; 1990;41(5):529–36. 42. hisano h, kimoto y, hayakawa h, takeichi j, domae t, hashimoto r, et al. high frequency agrobacteriummediated transformation and plant regeneration via direct shoot formation from leaf explants in beta vulgaris and beta maritima. plant cell rep. springer; 2004;22(12):910–8. 43. krishna h, alizadeh m, singh d, singh u, chauhan n, eftekhari m, et al. somaclonal variations and their applications in horticultural crops improvement. 3 biotech. springer; 2016;6(1):54. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20222 highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 4 research article open access 1department of home economics, college of education jigawa state, nigeria 2department of food science and technology, faculty of agriculture, bayero university, kano 3college of biosystems engineering and food science, national-local joint engineering laboratory of intelligent food technology and equipment, zhejiang key laboratory for agro-food processing, zhejiang r & d center for food technology and equipment, zhejiang university, hangzhou 310058, china. 4department of microbiology, kano university of science and technology wudil, kano state nigeria contacts of authors * to whom correspondence should be addressed: sanusi shamsudeen nassarawa citation: zaharaddeen salisu, sanusi shamsudeen nassarawa and sadisu farouq (2020). microbial quality assessment of roasted and fried meat sold in gumel town. highlights in bioscience volume 3. article id 20222. dio:10.36462/ h.biosci.20222 received: june 17, 2020 accepted: september 7, 2020 published: september 20, 2020 copyright: © 2020 salisu et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. microbial quality assessment of roasted and fried meat sold in gumel town zaharaddeen salisu 1 , sanusi shamsudeen nassarawa 2,3 * and sadisu farouq 4 abstract roasted and fried meats are the two commonest and preferred meat varieties consumed in northern nigeria. the consumption of these meat varieties is, nevertheless, without ascertaining its fitness in terms of contamination by pathogens. in line with this occurrence and preference, it is a good approach to assess the quality of roasted and fried meat in one of the prehistoric towns of northern nigeria, which is gumel. a total of 10 samples of both fried and roasted meat were randomly collected, prepared and microbiological analyses for mesophilic bacteria, fungi and coliform group conducted in the laboratory. the mean bacterial load was observed between 3.0 x 10 4 and 6.5 x 10 4 cfu/g for roasted beef meat and 4.7 x 10 4 and 5.3 x 10 4 cfu/g for fried beef meat. it was 4.5 x 10 4 and 6.0 x 10 4 cfu/g in roasted chicken meat and 4.0 x 10 4 and 4.7 x 10 4 cfu/g in fried chicken meat. for the fungal load, it was observed to be between the range of 1.0 x 10 4 and 3.0 x 10 3 cfu/g for roasted beef meat and 1.0 x 10 4 and 7.0 x 10 3 cfu/g for fried beef meat. for the chicken meat, it ranges from 1.0 x 10 4 to 4.0 x 10 3 cfu/g for the roasted type and 1.0 x 10 4 and 7.0 x 10 3 cfu/g for the fried meat. investigation of the coliform group showed no growth in both samples. on biochemical tests, bacterial species confirmed to be present were staphylococcus aureus, bacillus spp, and streptococcus spp. while penicillium spp, mucor hiemalis, aspergillus species and rhizopus spp. are the fungal species observed in the meats. this research was, therefore, conducted to assess the quality of roasted and fried meat sold in gumel town to relate it to some common diseases affecting the community. keywords: roasted meat, fried meats, microbial quality assessment. introduction meat is mainly composed of water and protein and is usually eaten together with other foods. it has been reported that meat is rich in protein (15-20%), minerals, vitamins, and all the essential amino acids. meat is animal flesh that is eaten as food [1]. humans are omnivorous and have hunted and killed animals for meat since prehistoric times [2,3]. the advent of civilization allowed for the domestication of animals such as chickens, sheep, fish, seafood, pigs, and cattle and eventually their use in meat production on an industrial scale. it is edible raw but is normally eaten after it has been cooked and seasoned or processed in a variety of ways. meat consumption varies worldwide, depending on cultural and religious differences, as well as the socio-economic status of the people. nigerians depend mainly on domestic animals and aquatic organisms and to some extent on game animals and birds. this is true of the urban as well as rural communities [4]. unprocessed meat will spoil within hours or days. https://creativecommons.org/licenses/by/4.0/ salisu et al., 2020 microbial quality assessment of roasted and fried meat sold in gumel town highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 4 spoilage is caused by the practically unavoidable infection and subsequent decomposition of meat by bacteria and fungi, which are born by the animal itself, people handling the meat and their implements [5]. because of this, meat nowadays serves as a source of various foodborne diseases due to improper processing, handling and storage. this usually results in the reduction of valuable proteins and leads to various foodborne infections. however, considering the random addiction of eating meat anyhow in mostly states of northern nigeria without ascertaining its fitness in terms of contamination by either bacteria or fungi, which is mostly attributed to the lack of good knowledge on the possible spoilage, it is pertinent to set up a mechanism that will be geared towards investigating the possible contamination sources and assess the quality of roasted and fried meat sold in gumel town, one of the prehistoric towns of northern nigeria. materials and methods this research was conducted at gumel town, one of the local governments of jigawa state, nigeria. it is located at latitude 12.63°n and longitude 9.30 ° e and 36 meters elevation above the sea level with an estimated population of about 42,742. a total of 10 samples comprising three fried and roasted beef meat each, and two fried and roasted chicken meat were randomly collected from the study area. the samples were placed into cleaned polythene bags and transported immediately to the laboratory for preparation and subsequent analysis. all the media used in this study were prepared and handled according to the manufacturer's instructions. for the enumeration of aerobic mesophilic bacteria and fungi, the social dilution method, as described by the american public health association [6], was employed. plates containing between 30 and 300 colonies were selected and counted for bacteria, and the number obtained was multiplied by the dilution factor. this gave the number of bacterial colony-forming units per gram of meat samples (cfu/g of the meat sample). for fungi, the plate that contained less than 50 colonies were selected and counted after 3-5-day incubation period. the count was reported as fungi/mold colony forming a gram of meat sample (cfu/g). sets of control for each sample containing agar and diluents were also incubated to ascertain the sterility of the media. the following formula was used to calculate the number of bacteria/fungi colony forming units per gram of the meat. n= ; where: n= the number of bacterial/fungal colonies per gram of sample; n= number of colonies counted; v= volume of sample used; d= dilution factor. for the enumeration of mesophilic coliform bacteria, the three tube method described by [6] was employed. the color, size, shape and microscopy (for gram staining), surface elevation and margin of different colonies developed on the plates were observed. a representative colony of the various morphological types was picked and transferred to a freshly prepared, sterilized and solidified nutrient agar incubated at 35°c for 24hours to obtain a pure culture of the organisms [6]. the isolated organisms were then subjected to grams staining techniques, and microscopy then followed. the fungal isolates were identified as described in the illustrated genera of imperfect fungi [7] and microbiological methods [8]. catalase tests coagulate test, methyl red-voges proskauer, citrate test, urease test, motility test and indole test were all carried out according to the method described by cheesbrough [9]. results and discussion the results of bacterial colony counts (colony-forming units per gram, cfu/g) as presented in table 1 for the beef meat were observed to be within the range of 3.0 x 10 4 to 6.5 x 10 4 and 4.7 x 10 4 to 5.3 x 10 4 cfu/g for roasted and fried beef meat, respectively. table 1. total bacterial count of roasted and fried meats in beef and chickens. s/n sample bacterial colony count per gram of sample (cfu/g) log no. 1 br-1 3.5x10 4 4.5 2 br-2 6.5x10 4 4.8 3 br-3 3.0x10 4 4.5 4 bf-1 4.7x10 4 4.7 5 bf-2 3.8x10 4 4.6 6 bf-3 5.3x10 4 4.7 7 cr-1 4.5x10 4 4.7 8 cr-2 6.0x10 4 4.8 9 cf-1 4.7x10 4 4.7 10 cf-2 4.0x10 4 4.6 key: br=roasted beef meat, bf=fried beef meat, cr= roasted chicken meat cf=fried chicken meat in comparison, it was observed to be within the range of 4.5 x 10 4 to 6.0 x 10 4 and 4.0 x 10 4 to 4.7 x 10 4 cfu/g for roasted and fried chicken meat, respectively. these values obtained for the mean bacterial and fungal loads were within the acceptable range of 1 x 10 3 to 1 x 10 7 cfu/g as provided by ledward [10] and 1.0 x10 3 for the acceptable limits for the ready to eat foods and is an indication that the meat was processed thoroughly because heetun et al. [11] reported that meat and meat products are highly perishable commodities and if not properly stored, processed, packed and distributed microbial growth will be highly accelerated. the level of microorganisms present in meat products as reported by jay et al. [12] can be reduced salisu et al., 2020 microbial quality assessment of roasted and fried meat sold in gumel town highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 4 only when they are further processed. another report davies and board [13] has revealed that if spoilage microorganisms such as brochothrix thermosphacta and pseudomonas spp. are present and grow to a high number, the meat will spoil and will be unfit for human consumption. the total fungal count (table 2) was observed to range between 1.0 x 10 4 to 3.0 10 3 and 1.0 x 10 4 to 7.0 x 10 3 cfu/g for roasted and fried beef meat, respectively. it was, however, observed that in chicken meat, it ranged between 1.0x 10 4 to 4.0 x 10 3 and 1.0 x 10 4 to 7.0 x 10 3 cfu/g for roasted and fried meat variety. table 2. fungal count of roasted and fried meat (beef and chickens). s/n sample fungal colony count per gram of sample (cfu/g) log no. 1 br-1 1.0x10 4 4.0 2 br-2 3.0x10 3 3.5 3 br-3 1.0x10 4 4.0 4 bf-1 1.0x10 4 3.8 5 bf-2 4.0x10 3 3.6 6 bf-3 7.0x10 3 3.8 7 cr-1 4.0 x10 3 3.6 8 cr-2 1.0x10 4 4.0 9 cf-1 1.0x10 4 4.0 10 cf-2 7.0 x10 3 3.8 key: br=roasted beef meat; bf=fried beef meat; cr= roasted chicken meat; cf=fried chicken meat. in the same vain, ismail et al. [14] studied the microbial quality of some meat products obtained from local markets in egypt, and reported many types of fungi belonging to several genera such as aspergillus, candida, cladosporium, eupenicillium, eurotium, geotrichum, mucor, penicillium, rhototorula besides aflatoxin b1. these researchers also isolated clostridium perfringens and staphylococcus aureus. in this scenery, yousuf et al. [15] have elaborated that the presence of coliforms such as escherichia coli in food is suggestive of faecal contamination from animals. the results of the most probable number index per gram of coliform of the roasted and fried meats of beef and chickens (table 3) showed no growth in all samples. table 3. results of the most probable number index per gram of coliform of the roasted and fried meat (beef and chickens) s/n sample mpn per gram log number 1 br-1 no growth 2 br-2 no growth 3 br-3 no growth 4 bf-1 no growth 5 bf-2 no growth 6 bf-3 no growth 7 cr-1 no growth 8 cr-2 no growth 9 cr-1 no growth 10 cr-2 no growth key: br=roasted beef meat; bf=fried beef meat; cr= roasted chicken meat; cf=fried chicken meat. the results of morphological characterization of the bacterial and fungal isolates (tables 4 and 5) showed some colonies of bacteria appearing yellow and some whitish. most of the fungal colonies, when stained with lactophenol stain appeared blackish, brown and greenish with characteristic section. table 4. morphology of the bacterial isolates in meat samples (beef and chicken). s/n isolate br-1 br-2 br-3 bf-1 bf-2 bf-3 cr-1 cr-2 cf-1 cf-2 1 a1 + + + + + + + + + + 2 b1 + + + + + 3 c1 + + + + + + + key: + = growth observed; – = growth was not observed. table 5. morphology of the bacterial isolates in meat samples (beef and chicken). s/n isolate br-1 br-2 br-3 bf-1 bf-2 bf-3 cr-1 cr-2 cf-1 cf-2 1 a2 + + + + + + + + 2 b2 + + + + + + 3 c2 + + + + + + + 4 d2 + + + + key: + = growth observed; – = growth was not observed. salisu et al., 2020 microbial quality assessment of roasted and fried meat sold in gumel town highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 4 table 6. biochemical tests of bacterial isolates from roasted and fried meats (beef and chickens). isolate microscopy gram reaction coagulase carbohydrate fermentation indole methyl red voges proskaver citrate motility expected organism a1 cocci in closter + + + staphylococcus aureus b1 short rod + + + + baccilus spp c1 cocci in chain + + + streptococcus spp key: + = presence; = absence the fungal species isolated as presented in tables (6 and 7) were penicillium spp, mucor hiemalis, aspergillus species and rhizopus spp. on biochemical tests, the bacterial species confirmed to be present are staphylococcus aureus, baccilus spp, and streptococcus spp table 6. table 7. fungal isolation and identification of roasted and fried meats (beef and chickens). s/n isolate expected organism 1 a2 penicillium spp 2 b2 mucor hiemalis 3 c2 aspergillus spp 4 d2 rhizopus nigricans conclusion it can be concluded that the total bacterial and fungal counts of both roasted and fried meats prepared and sold by commercial meat sellers in gumel town in nigeria was found to fall within the standard range endorsed by who and other international organizations. hence, contained fewer contaminants, and this can be attributed to the hygienic conditions of utensils, 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penaeusmonodon and prawn, macrobrachium rosenbergii from bangladesh. world journal of agricultural sciences; 2008:4: 852855. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20223 highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access 1the okeogun polytechnic, saki oyo state nigeria, department of science laboratory technology, microbiology option. 2ladoke akintola university of technology, ogbomosho, p.m.b 4000, department of pure & applied biology. 3department of microbiology and botany, university of ibadan, ibadan, nigeria. 4department of zoology, parasitology unit, university of ibadan, nigeria. 5cellular parasitology unit, department of zoology, university of ibadan, nigeria. 6department food science technology, the oke-ogun polytechnic, saki. 7lead city university, ibadan, faculty of medical science, chemical science (biochemistry unit). contacts of authors * to whom correspondence should be addressed: adeoti olatunde micheal citation: adeoti olatunde micheal, oni abosede catherine, adeoye kafilat adenike, adeoti oluwole adeola, adeoye basirat adedamola and adesina david ademola (2020). predictive comparative antibiotic resistance profiles of rhizobacteria genes using card: a bioinformatics approach. highlights in bioscience volume 3. article id 20223. dio:10.36462/h.biosci.20223 received: june 23, 2020 accepted: september 7, 2020 published: september 26, 2020 copyright: © 2020 micheal et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. predictive comparative antibiotic resistance (amr) profiles of rhizobacteria genes using card: a bioinformatics approach adeoti olatunde micheal 1,2,5 *, oni abosede catherine 1 , adeoye kafilat adenike 1,4 , adeoti oluwole adeola 6 , adeoye basirat adedamola 7 and adesina david ademola 1,3 abstract members of the plant growth promoting rhizobacteria (pgpr) have been severally implicated as excellent growth enhancers, yield promoters as well as bio-fertilizers. a study on antibiotics surveillance of pgpr is urgently needed as caution towards its continued usage in bioscience and agro-allied. antimicrobial resistance has become a great concern in agriculture and public health. the detection and characterization of antimicrobial resistance move from targeted culture and enzyme-based reaction to high-throughput metagenomics; acceptable resources for the analysis of large-scale information area unit as an expected rescue. the excellent bioinformatics tool newly curated for antibiotic resistance information (card; https://card.mcmaster.ca) could be a curated hub and resource-providing-referenced server for deoxyribonucleic acid and protein sequences as well as detection models on the molecular radar for antimicrobial resistance. this study employed card as pathogenomics repertoires for high-quality reference information on retrieving antibiotics resistance information on twentytwo carefully-selected members of rhizobacter from ncbi. ncbi and card on-line platform were employed in polishing of antiobitics resistance info of selected pgpr genera such as leguminosarum, azotobacter, azospirillum, erwinia, mesorhizobium, flavobacterium paenibacillus polymyxa, bacilli mycoides, b. subtilis, and burkholderia pseudomallei among others. the data generated showed evidence that these rhizobacteria could be resistant to certain drug classes under a different antimicrobial resistance (amr) gene families using different phyto-pathogenic genes (aro terms) using different resistance mechanisms. this distinctive platform provides bioinformatics tool that bridges antibiotic resistance considerations, which could be a fallback for policies in healthcare, agriculture and the environment. keywords: plant growth promoting rhizobacteria (pgpr), comprehensive antibiotic resistance database (card), antimicrobial resistance (amr), phyto-pathogenic, metagenomics, bio-fertilizers https://creativecommons.org/licenses/by/4.0/ micheal et al. 2020 predictive antibiotic resistance (amr) of rhizobacteria genes using card highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 introduction rhizobacteria are a group of agronomic bacteria that form a mutualistic symbiotic association, which is beneficial to both parties. members of rhizobacteria are an essential group of microorganisms used as bio-fertilizer [1, 2, 3] and plant growth promoters, which are often referred to as pgprs [4, 5, 6]. pgpr enhance plant growth by adding nutrients which act as inoculants during bio remediation, phytostimulation and biological control are classified based on their essential roles in the plant they inhabit [7, 8, 9]. the most common of pgpr species which are present in the rhizosphere are members of the genus azospirillum [10, 11], bacillus spp. and pseudomonas spp [12, 13, 14, 15]. the increasing reliance on plants as major sources of pharmaceuticals, cosmetics, and fragrance flavours to meet with the increasing rise in the world population hence the urgent need to investigate its potential antibiotic potentials if barriers of its nonpathogenicity are trespassed [16,17,18]. recent advancements in pathogenomics have advanced the phenomenon of antimicrobial resistance has gained benchmarking astute as a world number one public health threat [19, 20]. therefore, efforts should be dissipated toward the characterization of antimicrobial resistance with increasing attention at the international level and proven to global acceptance in recent united nations radars. the enzyme based polymerase chain reaction (pcr) characterizes and effectively serve as indicator of pathogenic microorganisms such as escherichia coli, salmonella. non-pathogenicity is associated with antimicrobial resistance whereas cultureand pcr-based may have provided the necessary sigh into the prevalence of resistance. these techniques had thus enhance our ability to review both the evolution and ecology of antimicrobial resistance among the entire microbiota population level [21, 22]. there are several web servers exist nowadays that completely identify ontological amr genes. these customized in silico resources are primarily designed for screening of one ordination or many assembled contigs of antibiotic resistance metaphysics (aro) developed by excellent antibiotic resistance information [21], which is a notable improvement in amr bio curation. such an annotational classification is incredibly useful for useful description. the card includes bioinformatics tools that modify the identification of antibiotic resistance genes from wholeor partial-genome sequence information together with unannotated raw sequence assembly contigs [23]. over the ages, because of the evolutionary and ecological drift, there are many bacteria of different genera that have migrated beyond the coast of their ecological niches in an attempt to seek new abode and survival. some of these organisms include members of pseudomonas, serratia, and escherichia, which have variously acquired resistance complexity along the wheel of change. with this in mind, pgprs are beneficial today because of their various roles in agriculture and food security, hence the need for predictive genomic resistance profile as a tool for antibiotic resistance for future age. the present study is an in silico surveillance attempt to predict amr molecules among farmers friendly pgpr bacteria, although presently environmental opportunistic isolates. pgpr influence plant growth in two different ways: direct and indirect. the direct promotion of plant growth by pgbr is by the synthesis of phyto-hormones, thereby facilitating the uptake of certain nutrients from the environment. indirect plant growth promotion occurs when pgbr lessen or the risk of phytopathogenic organisms, making them serve as antibiotic by producing bio-control synthesis. we aimed at using bioinformatics tool to predict the antibiotic resistance genes in pgbr. materials and methods retrieval of complete genomes from ncbi: to analyze antimicrobial resistance genes, twenty-two complete genomes of selected plant growth promoting rhizobacteria (pgpr) complete sequences were randomly retrieved from ncbi database. the retrieved sequences were in fasta format, which was copied from the national center for biotechnology information ncbi) website; https://www. ncbi.nlm.nih.gov as well as their accession numbers of pgpr. analyzing nucleotide sequence on card: nucleotide sequences of twenty two members of pgpr was imported into the card analyzing software from genbank using custom software developed specifically for the retention of all annotations, ncbi accession numbers and taxonomy identification (id) numbers of the pgpr. the importation of these follows a process in which sequences were first acquired from genbank in fasta format (https://www.ncbi.nlm.nih.gov/) and then loaded into the card’s chado database [24]. by convention, card uses only the subset of the available ncbi that is relevant to antibiotic-resistant bacteria. individual antibiotic resistance ontology (aro) terms in the card have been associated with specific computational tools and models [25, 26, 14]. results chart representation of rgi results for amr genes and amr family as retrieved by card on 22 selected pgpr (figure 1). file:///c:/wiki/biofertilizer micheal et al. 2020 predictive antibiotic resistance (amr) of rhizobacteria genes using card highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 figure 1. continued a: amr gene family of cp0422721.1 b: amr gene family of cp021965.1 c: amr gene family of cp045291.1 d: amr gene family of cp019171.1 e: amr gene family for cp010313.1 f: amr gene family of cp029603.1 g: amr gene family for cp033430.1 h: amr gene family of bx571966.1 i: amr gene family of cp040552.1 micheal et al. 2020 predictive antibiotic resistance (amr) of rhizobacteria genes using card highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 figure 1. continued j: amr gene family of cp018228.1 k: amr gene family for cp004015.1 l: amr gene family for cp18228.1 m: amr gene family for cp000133.1 n: amr gene family for cp037992.1 o: amr gene family for cp027116.1 p: amr gene family for cp0.107209.1 q: amr gene family for cp037992.1 r: amr gene family for cp025542.1 micheal et al. 2020 predictive antibiotic resistance (amr) of rhizobacteria genes using card highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 figure 1. aro classification tags for the selected twenty-two pgrd bacteria a-v: the assemly contigs retains the rgi into perfect (green), strict (flexible )and loose( spurious partial) resistomes. s: amr gene family for ap025149.2 t: amr gene family for cp000438.1 u: amr gene family for cp019702.2 v: amr gene family for cp034395.1 micheal et al. 2020 predictive antibiotic resistance (amr) of rhizobacteria genes using card highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 table 1. prevalence of amr gene family among pgprs. accession no prevalence of amr genes perfect (%) strict (%) loose (%) 1 cp 042272.1 33 34 33 2 cp021965.1 30 40 30 3 cp045291.1 20 60 20 4 cp019171.1 35 35 30 5 cp010313.1 25 50 25 6 cp029603.1 25 50 25 7 cp033430.1 30 40 30 8 bx571966.1 25 50 25 9 cp040552.1 25 50 25 10 cp018228.1 30 40 30 11 cp004015.1 44 28 28 12 cp018228.1 23 38 29 13 cp000133.1 35 30 35 14 cp037992.1 25 50 25 15 cp027116.1 33 43 24 16 ap007209.1 40 25 35 17 cp037992.1 23 54 23 18 cp025542.1 13 74 14 19 cp025149.2 38 38 24 20 cp000438.1 48 48 04 21 cp019702.2 25 50 25 22 cp034395.1 34 44 22 discussion this study employed card aro and amr resistomes classification in agreement with earlier studies. the rgi genes in card predict resistomes for genomic and metagenomics data for gene mutations from ncbi retrieved nucleotide sequences by using a combination of open reading frame prediction [29, 30]. in consonance with earlier studies, in this study, the perfect algorithm predicted amr proteins with exact homology (100%) with a query on the card reference sequences. in the same vein, members of pgpr on card algorithm showed strict genes under amr/aro curation because strict rgi genes are more flexible by allowing flexible variation from the genome reference sequence within the curated blast cut-off, which is useful for detection of previously unreported variants of antibiotics target changes through altered sequences [31]. the loose algorithm under card was an indication of resistance genes, which work outside the detection of target, which could enable the detection of new, emerging risks and more distant homologs of antibiotics resistance genes. the loose portends a computational novel amr gene discovery [32]. the study is somewhat pioneer that employed card as surveillance algorithm for pgpr, it is however a novel study because all the twenty-two selected pgbr clearly showed the three amr variants into perfect, strict and loose. this is an outright indication that the bacteria under study harbour resistance genes. the degree of prevalence of these genes varies among the pgbr. in consonance with earlier studies, drug efflux accounted for 67% as the mechanism of gene resistance with other mechanism. this study is in contrast with earlier studies leading to curation paradigm of card resistomes, which operates on four primary amr gene family to, which resistance is conferred on specificity and sensitivity of antibiotics, which rest on the experimental interpretation of minimum inhibitory concentration of the antibiotics. this study implicated lima 23srrna methyltransferase in paenibacillus polymyxa and p. odorifer strain responsible for resistance to lincosamide antibiotics, while adef genes are predictably responsible for resistance to fluoroquinolone and tetracycline antibiotics delftia spp and bradyrhizobium species. the most variant genes (adef, oxa-59, omp 38, amra and amrb) of antibiotic resistance through antibiotic efflux pump. in the same vein, members of rhizobium species use adef genes for resistance to fluoroquinolone and tetracycline antibiotics family while rhizobium species in this study use bcii, fosb genes for resistance to fosfomycin, cephalosporin, penam, phenicol antibiotics family [27, 28]. conclusion card as technological metagenomics tools here has further demonstrated its wider applicability as primarily better curation paradigm over resfinder [31], argannot and even catalog of resistance alleles in ncbi [32]. the new card rules allow diversity inclusion of experimentally-proven data on axiomatic variation in agricultural or environmental isolates. references 1. singh js. plant growth promoting rhizobacteria. resonance. 2013 mar 1;18(3):275-281. 2. jadhav hp, shaikh ss, sayyed rz. role of hydrolytic enzymes of rhizoflora in biocontrol of fungal phytopathogens: an overview. in: mehnaaz s (ed) rhizotrophs: plant growth promotion to bioremediation. singapore:springer; 2017.pp.83–203. 3. glick br. bacteria with acc deaminase can promote plant growth and help to feed the world. microbiology research. 2014;169: 30 9. 4. geetha k, rajithasri ab, bhadraiah b. isolation of plant growth promoting rhizo bacteria from rhizosphere soils of green gram, biochemical characterization and screening for antifungal activity against pathogenic fungi. international 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in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access 1department of biological sciences, wesley university ondo, nigeria 2department_of_biochemistry,_federal university of technology akure, nigeria contacts of authors * to whom correspondence should be addressed: oluwasegun victor omotoyinbo citation: omotoyinbo ov, awojulu eo and sanni dm (2020). phytochemical screening, antioxidant and tyrosinase inhibitory studies of methanol leaf extracts of two tomato varieties. highlights in bioscience volume 3. article id 20216. dio:10.36462/h.biosci.20216 received: june 8, 2020 accepted: july 18, 2020 published: august 18, 2020 copyright: © 2020 omotoyinbo et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. phytochemical screening, antioxidant and tyrosinase inhibitory studies of methanol leaf extracts of two tomato varieties oluwasegun victor omotoyinbo 1 , emmanuel olumide awojulu 2 and david morakinyo sanni 2 * abstract this study evaluated the phytochemical compositions, antioxidant properties, chlorophyll content and anti-tyrosinase activity of methanol leaf extracts of two tomato varieties, lycopersicon esculentum (var. eva f1) and lycopersicon esculentum mill (var. hausa). the dried pulverized of the plant’s leaves were extracted by decoction and mild agitation. phytochemicals such as flavonoids, tannin, glycoside, saponin, terpenoid and anthraquinone were present in the extracts of both varieties examined, while alkaloid and phlobatannin were confirmed absent in the extracts. the presence of steroid was observed in var. eva f1 but absent in var. hausa. total phenolic content (tpc) and total flavonoid content (tfc) of var. eva f1 were 505.9 ± 2.61 mg gae/ge, and 35.5 ± 1.64 mg re/ge, while var. hausa recorded a tpc and tfc value of 344.3 ± 2.01 and 7.8 ± 0.15 mg re/ge respectively. the chlorophyll content of the extracts were 6.6 ± 0.02 mg/ge (chlorophyll a), 5.7 ± 0.05 mg/ge (chlorophyll b) and 12.6 ± 0.14 mg/ge (total chlorophyll content) for eva f1 variety, while the chlorophyll contents for var. hausa were 7.6 ± 0.32 mg.ge (chlorophyll a), 5.6 ± 0.06 mg/ge (chlorophyll b) and 13.7 ± 0.14 mg/ge. eva f1 and hausa showed percentage inhibition of 76.3 % and 61.2 % at 400 μg/ml. the ic50 value of var. eva f1 and var. hausa were 110 μg/ml and 160 μg/ml. the inhibition constant (ki) of var. eva f1 and var. hausa, were 0.006 and 0.016 μg/ml, respectively, and both extracts showed partial competitive inhibition. hence, this confirms the phytoprotective and tyrosinase inhibitory properties of tomato plant leaves. keywords: tyrosinase, inhibition, kinetics, phytochemicals, antioxidants, lycopersicon esculentum. introduction tyrosinase (ec 1.14.18.1) is a metalloenzyme that catalyzes the ratelimiting reactions that are important to melanogenesis. it is classified as an oxidase and involves two distinct reactions, the hydroxylation of a monophenol, and conversion of an o-diphenol to the corresponding o-quinone. the oxidative polymerization of dopaquinone derivatives gives rise to melanin [1]. melanin is a biopolymer synthesized by melanocyte, within specialized organelles called melanosomes. it is important to prevent uv-induced skin damage by absorbing uv sunlight and removing reactive oxygen species [2]. although tyrosinase is a relatively important enzyme, abnormal biosynthesis or distribution of melanin may result in several dermatological disorders, such as age-spots, lentigines, https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 melasma, and inflammatory hyperpigmentation. due to the rate-limiting step of tyrosinase in the melanin biosynthesis pathway, inhibition of this enzyme have become increasingly important and productive in cosmetics and pharmaceutical industries, where it is used as potent skinlightening agents for treating skin pigmentation disorders [3,4], and most of the inhibitors characterized from literature are either synthetic compounds or bioactives’ obtained from medicinal plants such as polyphenols, flavonoids, aldehydes and their derivatives [5]. lycopersicon esculentum (tomato plant) comes from the solanaceae family and is a good source of phenolic compounds, pigments, antioxidants, and other nutrients when consumed [6]. the extract of tomato leaves has been reported to exhibits antimicrobial and antioxidant properties at a ratio higher than in other parts of the plant [7]. tomato leaf extract has also been used in much traditional preparation of skin-toning mixtures, while some evidence indicates that it may be a valuable bioactive source, and would seem to be applicable in both medical fields and food industry [7,8]. in this study, the methanol leaf extracts from two different cultivars of tomato (lycopersicon esculentum (var. eva f1) and lycopersicon esculentum mill. (var. hausa) were screened for the presence of phytochemicals and some antioxidants, while the antityrosinase potential of the extracts was evaluated on mushroom tyrosinase. materials and methods reagents: mushroom tyrosinase (1.14.18.1), kojic acid, sodium carbonate (na2co3), aluminium trichloride (alcl3), sodium nitrite (nano2), were obtained from merck (sternheim usa). all other chemicals and reagents used were of analytical grade. plant material: leaves of lycopersicon esculentum (var. eva f1) (figure 1a, b) and lycopersicon esculentum mill. (var. hausa) (figure 1c) were collected at the federal university of technology, akure greenhouse farm and muyiwa oni avenue, akure, ondo state, respectively. identification and authentication of samples were carried out at the department of crop, soil and pest management, federal university of technology, akure. extraction: the leaves were subjected to drying under a shade at room temperature for 21 days. the dried materials were then pulverized using kanchan international blender (china). 90 g of each powdered material was then extracted by decoction at 50 o c for a period of 5 hours using methanol as an extraction solvent. afterwards, the samples were filtered using a muslin cloth, and subsequently with whatman filter paper. the residue obtained after filtration was extracted to exhaustion by methanol (absolute). the extraction was repeated three times and filtrates obtained in each stage were combined and concentrated using a rotary evaporator extractor. finally, the extracts were air-dried at ambient temperature. figure 1: photograph of l. esculentum mill. (var. hausa) (a) leaves (b) fruits and; (c) photograph of l. esculentum (var. eva f1) leaves and fruits. phytochemical screening: screening of the phytochemical constituents in the leaf extract was carried out to identify the constituents using standard phytochemical methods [9,10]. test for saponin: this test was carried out by dissolving 10 mg of extract in water inside a test-tube and the mixture was agitated. frothing, which persisted on warming was taken as preliminary evidence for the presence of saponin in the leaf extract. test for tannin: the extract (10 mg) was dissolved in 5 ml of distilled water and then filtered. thereafter, 1 ml of ferric chloride was added to the filtrate. a blue-black precipitate indicated the presence of tannin. test of phlobatannin: the solution of the extract in methanol (0.1 mg/ml) was boiled with 1% of hydrochloric acid. deposition of red precipitate indicated the presence of phlobatanin in the extract. test for anthraquinone: about 5 ml of 0.1 mg/ml aqueous solution of the extract was dissolved in 10 ml of benzene solution, the mixture was filtered and 10% ammonia solution was added to the filtrate and then shaken. the formation of an amber coloured solution in the ammonia lower phase confirmed the presence of anthraquinone in the extract. test for glycoside: the extracts (10 mg) were dissolved in 2 ml of pyridine with 5 drops of 2% sodium nitroprusside and 20% sodium hydroxide added. a yellow colouration at the interface of the mixture confirmed the presence of glycoside. omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 test for steroids: exactly 20 ml of ethanoic anhydride were added to 0.5 g of the extract and then filtered before adding 2 ml of concentrated h2so4 to the filtrate. the presence of steroids was confirmed with a colour change of the solution from violet to blue or green. test for terpenoid: exactly 5 ml of 0.1 mg/ml methanol solution of the extracts was mixed with 2 ml chloroform; afterwards another 3 ml of concentrated h2so4 was carefully added to the solution. the formation of a russet colour at the interface of the mixture confirmed the presence of terpenoids. test for alkaloid: exactly 5ml of 1 % aqueous hcl was added to 0.5 g of the extract and placed in a steam water bath while stirring for 2 minutes. the solution was then filtered and 1 ml of the filtrate was treated with a few drops of dragendorf reagent, blue-black turbidity was taken as preliminary evidence for the presence of alkaloid. antioxidant properties total phenolic content: this was determined according to the method of harborne and williams [11], with slight modification. exactly 0.1 ml of 0.1 mg/ml of the extracts was oxidized with 0.5 ml of 10 % folin-ciocalteu’s phenol reagent (v/v) and neutralized by 2.5 ml of 7.5% sodium carbonate. the reaction mixture for each cultivar extract was incubated for 1 hour at room temperature and the absorbance was measured at 700 nm using the same mixture without the sample as blank. the total phenolic content was expressed as mg gallic acid equivalent of extract (mg gae/g of extract). total flavonoid content: this was determined according to the method of bao et al. [12] with a slight modification. in a test tube, 0.3 ml of 5 % nano2 was added to 0.1 ml of 0.1 mg/ml extract and the mixture was then incubated at room temperature for 6 minutes. after incubation, 0.6 ml of alcl3 (10%) was added and the mixture was further incubated for 5 minutes, then 2 ml of naoh (1 m) was finally added to the reaction mixture and the absorbance was read at 510 nm using the same mixture without the extract as blank. the result was expressed as mg rutin equivalent/g of extract (mg re/ge). chlorophyll content: the chlorophyll content of the extracts was determined according to the method of lichtenthaler and babani [13]. exactly 0.2 g sample was extracted exhaustively with 80% acetone until the green pigments disappeared. the final mixture was then filtered, and the supernatant combined. the absorbance of the supernatant was measured at 663, 652, 645 and 470 nm using a uv-vis spectrophotometer. the chlorophyll contents were expressed as mg/g of extract (ge) according to the following equations: chlorophyll a = (12.7 × a663 – 2.7 × a645). chlorophyll b = (22.9 × a645 – 2.7 × a663). total chlorophyll = (27.8 × a652). assay of tyrosinase inhibitory activity: tyrosinase inhibitory activity was measured according to the procedure described by liu et al. [14]. exactly 50 u/ml of tyrosinase was prepared in 50mm sodium phosphate buffer (ph 6.8), 0.35 ml of the leaf extract of various concentrations (3.1, 6.2, 12.5, 25, 50, 100, 200 and 400 μg/ml) was added to 0.15ml of tyrosinase respectively, and the mixture was incubated for 10 minutes at room temperature. then, 0.55 ml of 12 mm l-dopa was added to the existing mixture and further incubated at room temperature for 20 minutes. the reaction mixture without the leaves extract served as control and the change in absorbance was measured at 492 nm against the blank. kojic acid was used as a standard and all tests were performed in triplicate, and the percentage inhibition of tyrosinase activity was calculated using the formula: tyrosinase inhibition = control samplecontrol abs absabs  × 100 results the result of phytochemical screening of the methanol leaf extracts of lycopersicon esculentum (var. eva f1) and lycopersicon esculentum mill (var. hausa) (table 1) shows that the methanol leaf extracts contain saponin, flavonoids, anthraquinone, tannin, steroid, glycoside, and terpenoids, while alkaloid and phlobatanin were confirmed absent, however steroid content was observed in var. eva f1 but absent in var. hausa. table 1: phytochemical constituents of methanol extract of var. eva f1 and var. hausa. tomato variety phytochemical constituents var. eva f1 var. hausa flavonoid + + tannin + + glycoside + alkaloid steroid + saponin + + phlobatanin terpenoid + + anthraquinone + + *note: the symbol – indicates the absence of the phytochemical under investigation (hence negative), while + indicates the presence of the phytochemical under investigation (hence positive). var. eva f1 means eva f1 variety, while var. hausa means hausa variety. total phenolics and flavonoids content of the methanol extract of the respective cultivars (table 2) omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 reveals that the total phenolic content of the extracts was expressed as gallic acid equivalent (gae) were 505.96 ± 2.57 and 344.26 ± 2.02 mg gae/ge, while the total flavonoid content expressed as rutin equivalent (re) was 35.55 ± 1.66 and 7.78 ± 0.15 for eva f1 variety and hausa variety, respectively. table 2: total phenol and total flavonoids content of extract of tomato leaf; var. eva f1 and var. hausa. tomato variety tpc (mg gae/ge) tfc (mg re/ge) var. eva f1 505.96 ± 2.57 35.55 ± 1.66 var. hausa 344.26 ± 2.02 7.78 ± 0.15 values are expressed as mean ± standard deviation (n=3). note: gaegallic acid equivalent; rerutin equivalent. the two tomato varieties varied in the chlorophyll contents (table 3), such that for var. eva f1 had values of 6.63 ± 0.02, 5.76 ± 0.05 and 12.57 ± 0.14 mg/ge for chlorophyll a, chlorophyll b and total chlorophyll content, respectively, var. hausa had 7.63 ± 0.32, 5.63 ± 0.06 and 13.67 ± 0.14 mg/ge. the analysis revealed that all samples had a non-zero standard deviation from the mean values. table 3: chlorophyll content of extract of tomato leaf; var. eva f1 and var. hausa. tomato variety chlorophy ll a (mg/ge) chlorophy ll b (mg/ge) total chlorophy ll (mg/ge) var. eva f1 6.63 ± 0.02 5.76 ± 0.05 12.57 ± 0.10 var. hausa 7.63 ± 0.32 5.63 ± 0.06 13.67 ± 0.14 values are expressed as mean ± standard deviation (n=3). note: mg/ge refers to mg/g of extract. inhibitory concentration at 50% (ic50) values of the extracts compared with kojic acid (the standard inhibitor) is represented in (table 4). table 4: michaelis-menten parameter (ki), and ic50 values of inhibitors. tomato variety ic50 (µg/ml) ki value (µg/ml) type of inhibition l. esculentum (var. eva f1) 65 0.006 partial competitive l. esculentum (var. hausa) 198 0.016 partial competitive ic50 value of kojic acid (positive control) is 25 µg/ml. the extracts had an inhibitory effect on tyrosinase in a dose-dependent manner (figure 2 and 3). the percentage inhibition of tyrosinase at the maximum test concentration (400 μg/ml) for extracts were 76.30% and 61.24% for eva f1 and hausa varieties, respectively while kojic acid had a higher inhibitory power of 80.57%. further kinetic studies revealed (figure 4 and 5) the inhibition type of both extracts as competitive inhibition and the inhibition constant (ki) for eva f1 and hausa varieties were 0.006 and 0.016 μg/ml, respectively. figure 2: the percentage inhibition curve of l. esculentum (var. eva f1) and kojic acid. figure 3: the percentage inhibition curve of l. esculentum mill. (var. hausa) and kojic acid. discussion in this study, the phytochemical analysis of the methanol leaf extracts of var. eva f1 and var. hausa revealed a single variation in plant secondary metabolites examined. it was observed that while the analysis of both varieties of methanol leaves extract confirmed the presence of flavonoids, tannin, glycoside, saponin, terpenoid and anthraquinone, and the absence of alkaloid and phlobatanin, var. eva f1 contains steroid while var. hausa extract did not contain the steroids. manosroi et al. [15] reported that phytochemicals found in plant extract depend on the nature of the plant, the solvent system, temperature and time used in the extraction process. the variation in steroid composition could be a result of the mode of cultivation of individual plants, however, the result obtained showed greater similarity in the phytochemical constituent of the extracts of both varieties, and could be an indication of some sort of genetic resemblance. omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 figure 4: kinetic properties of l. esculentum (var. eva f1) (a) lineweaver-burk plot for the inhibition of var. eva f1, (b) re-plot of the slope of the lineweaver-burk plot versus inhibitor concentration, (c) 1/δslope versus 1/[i]. figure 5: kinetic properties of l. esculentum mill (var. hausa) (a) lineweaver-burk plot for the inhibition of var. hausa (b) replot of the slope of the lineweaver-burk plot versus inhibitor concentration (c) 1/δslope versus 1/[i]. a b c a b c omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 the observed high tpc and tfc indicate that var. eva f1 has the best antioxidant properties compared to var. hausa. silva-beltran et al. [7] reported that high phenolic content in tomato plants could be as a result of their varied phenolic content and its derivatives, which are essential for the plant’s growth and reproduction. phenolic content present in tomato plants majorly consist of hydroxycinnamic acid and flavonoids and are located mainly in their leaves. the variation observed in tpc and tfc in the two tomato varieties has been affirmed by silva-beltran et al. [7], who reported that the concentration of flavonoids varied among the plant’s cultivars. results obtained in this study showed that var. hausa had the highest content of chlorophyll a, while var. eva f1 had the highest content of chlorophyll b. however, var. hausa had the highest total chlorophyll content (tcc). the content of chlorophylls observed in this study was found to be higher than those observed by silva-beltran et al. [7] for pitenza and floradade variety of tomato plants. lumpkin [16] also reported that the chlorophyll content of tomato plants is strongly influenced by the incidence of light and its concentration increases with exposure to light. tomato plants synthesize metabolites and pigments such as chlorophyll and carotenoids, that beneficially contributes to consumers nutrition and health [7], while the work of choi et al [8] shows that these phytochemicals help prevent photooxidation are strongly influenced by the maturity of the plant. in the same light, chlorophyll shows potent antioxidant activity, and it has been suggested that chlorophyll reduces free radicals, acting as a hydrogen ion donor to break the chain reaction resulting to cellular oxidation [17]. the methanol leaf extracts of var. eva f1 and hausa inhibited mushroom tyrosinase in a dosedependent manner. eva f1 variety revealed higher tyrosinase inhibitory activity of 76.3% at the highest concentration considered (400µg/ml) compared with 61.2% in hausa variety. zaveri and patel [18] reported a percentage inhibition of 58% in tomato fruit (at the highest inhibitor concentration considered), which is lower than the value obtained in the present study. this is quite reasonable, as secondary metabolites are primarily, concentrated in leaf section of plants to serve a defensive role. the ic50 values obtained for both extracts in this study were higher compared to those obtained by zaveri and patel [18] for curry tree and tomato fruit, which could be as a result of the difference in the enzyme units utilized. however, kojic acid (standard inhibitor) recorded an ic50 of 25 μg/ml. this result points out eva f1 variety as the most potent tyrosinase inhibitor among the two cultivars were evaluated since low ic50 values imply that a smaller amount of the extract is needed to reduce the activity of mushroom tyrosinase by half. kinetic data analysis using lineweaverburk plot, which is also called the double reciprocal plot reveals that the inhibitory mechanism of both methanol leaf extracts on mushroom tyrosinase was competitive, viz partial competitive inhibition. this correlates with yang and quyang [19] conclusion for olea leaf extract analysed by lineweaver-burk plot. furthermore, l. esculentum var. eva f1 had the lowest inhibition constant (ki) value of 0.006 μg/ml while var. hausa had ki value of 0.016 μg/ml. this implies that methanol leaf extract of eva f1 variety has a higher affinity for the active site of mushroom tyrosinase than that of hausa variety. also, the ki value obtained in this study is considerably lower than that obtained by yang and quyang [19] for olea europa leaf extract, which had a ki value of 0.226 mg/ml. conclusion although the two varieties of tomato leaf methanol extract showed the presence of most phytochemical evaluated, both varieties tests were negative for alkaloid and phlobatannin content, while var. hausa also lacked any steroid content. the highest tpc, and tfc were observed for var. eva f1, while var. hausa had the highest total chlorophyll content. eva f1 variety had the best tyrosinase inhibitory activity at the peak concentration considered. this study therefore further establishes the skin whitening and antioxidant properties of tomato leaves as traditionally used in some cultures. references 1. parvez s, kang m, chung hs, cho c, hong mc, shin mk, et al. survey and mechanism of skin depigmenting and lightening agents. phytother res. 2006;20:921–34. 2. d’orazio j, jarret s, amaro-ortiz a, scott t. uv radiation and the skin. intl j mol sci. 2013;14(6):12222– 12248. 3. kim yj, uyama h. tyrosinase inhibitors from natural and synthetic sources: structure, inhibition mechanism and perspective for the future. cell mol life sci. 2005;62:1707–23. 4. pillaiyar t, manickam m, namasivayam v. skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. j enzyme inhib med chem. 2017;32(1):403-25. 5. loizzo mr, tundis r, menichini f. natural and synthetic tyrosinase inhibitors as antibrowning agents: an update. compr rev food sci food saf. 2012;11(4):378-98. 6. taveira m, ferreres f, gil-izquierdo a, oliveira l, valentão p, andrade pb. fast determination of bioactive compounds from lycopersicon esculentum mill leaves. food chem. 2012;135(2):748–55. 7. silva-beltran np, ruiz-cruz s, cira-chavez la, estrada-alvarado mi, ornelas-pas j, lopez-mata ma, et al. total phenolic, flavonoid, tomatine, and tomatidine contents and antioxidant and antimicrobial activities of extracts of tomato plant. int j analyt chem. 2015;4-8. 8. choi km, lee ys, shin dm, lee s, yoo ks, lee mk, lee jh, kim sy, lee ym, hong jt, yun yp. green omotoyinbo et al., 2020 phytochemical, antioxidant and antityrosinase studies of meoh leaf extracts of tomato varieties highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 7 tomato extract attenuates high fat-diet induced obesity through activation of the ampk pathway in c57bl/6 mice. j nutr biochem. 2013;24(1):335–42. 9. trease ge, evans wc. a textbook of pharmacognosy 13th ed. , london (uk): baluiere tindali;1996. p. 100-1. 10. sofowora a. medicinal plants and traditional medicine in africa. chester (uk): john wiley and sons, limited;2006. p. 55-201. 11. harbone jb, williams ca. advances in flavonoid research since 1992. phytochemistry. 2000;55:481-504. 12. bao j, cai m, sun g, wang y, corke h. anthocyanins, flavonoid and free radical scavenging activity of thines bayberry (myrialrubia) extracts and their colour properties and stability. j agric food chem. 2005;53:2327-32. 13. lichtenthaler hk, babani f. light adaptation and senescence of the photosynthetic apparatus: changes in pigment composition, chlorophyll fluorescence parameters and photosynthetic activity during light adaptation and senescence of leaves. in: papageorgiou and govindjee, editors. chlorophyll fluorescence: a signature of photosynthesis. springer;2004. p. 713-736. 14. liu j, wu f, chen l, zhao l, zhao z, wang m, et al. biological evaluation of coumarin derivatives as mushroom tyrosinase inhibitors. food chem. 2012;135:2872-78. 15. manosroi a, boonpisuttinant k, winitchai s, manosroi j. free radical scavenging and tyrosinase inhibition activity of oils and sericin extracted from thai native silkworms (bombyx mori). pharm biol. 2010;48(8):855-860. 16. lumpkin hm. a comparison of lycopene and other phytochemicals in tomatoes grown under conventional and organic management systems, no. 34 of technical bulletin, asian vegetable research and development centre. avrdc publication—the world vegetable centre;2005. p. 623. 17. chen bh, liu mh. relationship between chlorophyll a and β-carotene in a lipid-containing model system during illumination. food chem. 1998;63(2):207-13. 18. zaveri m, patel a. preliminary screening of some selected plants for anti-tyrosinase activity. int j institutional pharm life sci. 2012;2(1):217. 19. yang dm, ouyang ma. antioxidant and anti-tyrosinase activity from olea leaf extract depended on seasonal variations and chromatography treatment. int j organic chem. 2012;2:391-97. highlights in bioscience; highlights in bioscience issn: 2682-4043 highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 1 of 7 citation: awan z., ali a.and iqbal a. (2019). plant molecular biology databases. highlights in bioscience, volume 2.article id 20191, dio:10.36462/ h.biosci.20191 received: march 3, 2019 accepted: april 25, 2019 published: may 5, 2019 copyright:© 2019 awan et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. plant molecular biology databases zubair awan 1 , aqib ali 2 and abbas iqbal 2 1 agriculture university, peshawar, pakistan. 2 institute of biotechnology, agriculture university, pakistan. abstract many of plant biological database comprise data from study fields including metabolomics, proteomics, genomics, microarray gene expression, and phylogenetic. information in biological databases involves the role of genes, composition, chromosomal location, clinical impacts of mutations, and similarity of biological sequences and characteristics. we are here targeting to review and rank some of plant databases according to their use and feature. hoping that scientists could use this study to pick, combine and correlate distinct databases, based on their study needs and objective specifications. keywords: plant biological database, bioinformatics, software, phylogenetic, metabolomics, proteomics, genomics. introduction many of plant databases were constructed from some biological fields including metabolomics, proteomics, genomics, microarray gene expression, and phylogenetic(1). with bioinformatics tools the number of plant database were growing (2), so here we described many of plant database such as ricyerdb, plantrgdb, functional trait databases, erisdb, halodom, plantprom db, ctdb, cmmdb, cicarmisatdb, ehaloph, msdb, kazusa marker database , pmdbase, snorna database , ogdd, plantrna, prgdb, pineelm ssrdb , sbmdb, spinachdb, sisatbase, sinbase, ricebase, reprolive, and planteome database ricyerdb the rice yield-related database (ricyerdb) was developed to complement associated influence rice research (oryza sativa l.) yield in various aspects by manually curating associated databases and literature and information on genomics and proteomics that can be helpful for a detailed knowledge of rice biology. ricyerdb offers a more useful resource for researching, browsing and review article open access plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 2 of 7 analyzing genes related to yield efficiently. you can readily query and download the entire information collection through the webpage. furthermore, ricyerdb also built a network of communication between protein and protein with biological assessment. the intermingled rice database opens a fresh path for scientists to obtain rice gene data in terms of its impacts on rice breeding traits. at http://server.malab.cn/ ricyer/index.html, the web server is publicly accessible (3). plantrgdb plantrgdb (plant retrocopied gene database) was built as the first plant retrocopy database to provide a supposedly full unified catalog of plant genomic retrocopies. free access to the database is http://aegilops.wheat.ucdavis .edu/plantrgdb. it included 49 species of plants and 38,997 retrocopies along with data on classification. plantrgdb offers a user-friendly internet interface to search, browse and download the data base retrocopies. plantrgdb also provides graphical viewer-integrated sequence data to display each retrocopy's composition. report the retrocopy characteristics of each species using a browse feature. furthermore, helpful instruments such as sophisticated search and blast are accessible to more conveniently search the database (4). functional trait databases tavşanoğlu and pausas (5) depicted a functional database of traits that emerged as key instruments for a broad spectrum of ecological studies worldwide. their database of functional characteristics for mediterranean basin vascular plant species. the database contains 25,764 personal documents of 44 characteristics from 2,457 plant taxa dispersed in 119 taxonomic families. trait information were acquired from an extensive survey of literature, along with some field and experimental findings. all documents are fully mentioned and include geographic coordinates in many instances. the database is organized to include various concentrations of character data precision for each input. brot 2.0 should promote the testing of plant functional ecology hypotheses within the mediterranean basin and compare this region with other ecosystems around the world. the brot2.0 is offline database as well as its trait definitions can be used in other areas of the globe as a model to create comparable trait databases. erisdb kabza et al. (6) conducted a large-scale assessment of splice sites in eight species of plants using new algorithms and tools. theanalyzes included orthological splice sites, polypyrimidine sections, and branch locations being identified. they also recognized putative intronic and exoniccis-regulatory motifs, u12 introns and splice sites in 5 plant species in 45 microrna genes. in the form of expressed sequence tag and rna-seq information, they also provide experimental proof for plant splice sites. all information is deposited in a novel erisdb database and is freely available at http://lemur.amu.edu.pl/share/erisdb/. halodom loukas et al. (7) documented from scientific literature more than 1000 halophilic species. of these, 21.9% correspond to archaea, 50.1% to bacteria and 27.9% in eukaryotes. their documents comprise fundamental data such as the salinity discovered by a specific organism, its taxonomy and genomic data through ncbi and other connections. you can access the internet database called "halodom" at http://www.halodom.bio.auth.gr. plantprom shahmuradov et al. (8) obtained a plant promoter repository, is an annotated, non-redundant set of rna polymerase ii proximal promoter sequences with experimentally determined transcription start site(s), tss, from different plant species. plantprom db includes 305 records from monocot, dicot and other crops, including 71, 220 and 14 promoters. it offers tss, promoter type ranking of promoters and nucleotide frequency matrices (nfm) dna sequence of promoter areas for promoter components: tata-box, ccaat-box and tss-motif (inr).analysis of tss motifs disclosed that their structure is distinct in dicots and monocots, as well as in promoters without tata and tata. the database acts as a instructional base for the development of forecast programs for plant promoters. you can find plantprom db at http://mendel.cs.rhul.ac.uk/. ctdb verma et al. (9) designed an incorporated chickpea transcriptome database (ctdb) that offers a extensive internet interface for visualizing and easily retrieving chickpea transcriptome information. the database includes numerous resources for searching for similarities, searching for functional annotations, and assessment of relative gene expression. the database contains a catalog of transcription factor relatives and their chickpea expression profiles. in order to study the expression profiles of chickpea transcripts in major tissues/organs and various stages of flower development, the gene expression data were integrated. to enable relative genomic research between distinct legumes http://server.malab.cn/%20ricyer/index.html http://server.malab.cn/%20ricyer/index.html http://lemur.amu.edu.pl/share/erisdb/ http://www.halodom.bio.auth.gr/ http://mendel.cs.rhul.ac.uk/ plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 3 of 7 and arabidopsis, utilities such as similarity search, orthology recognition and relative gene expression have also been introduced in the database. in addition, the ctdb is a tool for finding functional molecular markers (microsatellites and single nucleotide polymorphisms) between distinct kinds of chickpea. the ctdb can be found at http://nipgr.res.in/ctdb. html. cmmdb the first entire genome based microsatellite dna marker repository of cucumis melo l was created by bhawna et al. (10). and a extensive internet resource that helps to identify varieties and map the cucurbitaceae family physically. microsatellite repository of the melo cucumis l. includes 39,072 ssr markers along with repeat motif, sequence length, motif sequence, marker id, motif type and chromosomal location. the database features a new automated plant primer design tool to satisfy the requirements of wet laboratory scientists. at http://65.181. 125.102/cmmdb2/index.html, cmmdb is readily accessible. cicarmisatdb doddamani et al. (11) reported a user-friendly relational database called the chickpea microsatellite database to support genetic research and apps for reproduction. this database offers comprehensive data about ssrs as well as their genome characteristics. ssrs were categorized and rendered available via a internet interface that was easy to use. at http://cicarmisatdb.icrisat.org, cicarmisatdb the database is readily accessible. ehaloph a database of salt-tolerant plant-halophytes was built by santos et al. (12). data of plant species tolerant of salt levels of about 80 mm or more sodium chloride have been gathered, together with information on plant species, life form, ecotypes, highest tolerated salinity, existence or lack of salt glands, photosynthetic pathways, antioxidants, secondary metabolites, compatible solutes, habitat, financial use and whether germination, microbial articles are available.the ehaloph database may be used to analyze tolerancerelated traits and to inform selection of species that may be used for saline farming, bioremediation or environmental recovery and rehabilitation of decayed wetlands or other regions. the database available at http://www.sussex.ac.uk/ affiliates/halophytes/. msdb a msdb (microsatellite database) of > 650 million ssrs from 6,893 species such as archaea, fungi, bacteria, plants and animals was founded by avvaru et al. (13). this database is the most comprehensive tool for various species accessing and analyzing ssr information. in addition to researching information in a customizable tabular format, the interactive plotting scheme allows users to display and compare various species information concurrently. using the django framework and mysql, msdb is created. it can be found at http://tdb.ccmb.res.in/msdb. kazusa marker database a database was developed by shirasawa et al. (14), the kazusa marker database. this database involves dna marker data such as ssr and snp markers, genetic association maps, and physical maps. this database also provides keyword searches for markers, sequence information used to develop markers, and experimental environments. there are mainly 10 specific crop species: pepper, tomato, strawberry, soybean, radish, peanut, red clover, white clover, and eucalyptus. this database is a helpful instrument for fundamental and advanced sciences like genomics, genetics, and crop molecular breeding. database link http://marker.kazusa.or.jp. pmdbase a database, pmdbase, was established by yu et al. (15), which integrates large quantities of microsatellite dnas out of genome-sequenced species of plants and involves a web service for microsatellite dnas. pmdbase recognized 26 230 099 microsatellite dnas covering 110 species of plants. for each microsatellite dna, up to three combinations of primers were provided. for 81 species, the microsatellite dna genomic characteristics were provided with the respective genes or transcripts from various databases. microsatellite dnas could be studied with a userfriendly internet interface and tailored software by browsing and locating modules. in addition, they constructed misaweb and embedded primer3web to help users recognise microsatellite dnas of their own online molecular sequences and construct corresponding primers. all microsatellite dna datasets can be easily downloaded. pmdbase will be updated with the latest genome data on a regular basis and can be accessed via the http://www.sesamebioinfo.org/pmdbase http://nipgr.res.in/ctdb.%20html http://nipgr.res.in/ctdb.%20html http://cicarmisatdb.icrisat.org/ http://www.sussex.ac.uk/%20affiliates/halophytes/ http://www.sussex.ac.uk/%20affiliates/halophytes/ http://tdb.ccmb.res.in/msdb http://marker.kazusa.or.jp/ http://www.sesame-bioinfo.org/pmdbase http://www.sesame-bioinfo.org/pmdbase plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 4 of 7 plant snorna database the plant snorna database (16) offers data from arabidopsis and eighteen other plant species on small nucleolar rnas. information involves genes, information on transcription, target alteration sites for methylation and pseudouridylation, primary gene association, and number of versions of genes. the arabidopsis data is split by target locations in rrna, snrnaor unidentified, into box c / d and box h / aca snornas, and within all of these groups. for many snorna genes, alignments of orthological genes and gene variations from various plant species are accessible. it accessible via http://www.scri.sari.ac.uk/plant_snorna/. ogdd an olive genetic diversity database (ogdd) (17) is therefore provided http://www.bioinfo-cbs.org/ogdd/. it is a worldwide olive tree as well as oil genetic, morphological and chemical database with a dual function. in reality, it offers users with extra morphological and chemical data for each recognized cultivar in addition to being a reference system produced to identify unknown olive or virgin olive oil cultivars depending on their microsatellite allele size. using a number of effective query interfaces and evaluation instruments, ogdd is currently intended to allow users to readily collect and visualize biologically significant data. plantrna database of plantrna (18) http://plantrna.ibmp.cnrs.fr/ compiles gene sequences of rna (trna) transformation from completely annotated nuclear, plastidial as well as mitochondrial genomes. for highest performance and certainty, the array of annotated trna gene sequences was manually selected. this database's novelty lies in the incorporation of biological data appropriate to all the trnas stored in the library. this involves 30and 50-flank sequences, transcription initiation region, a and b box sequences, and trna intron sequences, aminoacyl-trna synthetases, poly(t) transcription termination extends, and trna maturation and alteration enzymes. the present annotation includes the full genomes of 11 species: 5 flowering plants ( oryza sativa, arabidopsis thaliana, medicago truncatula, populus trichocarpa, and brachypodium distachyon), 2 red fungi (ostreococcus tauri and chlamydomonas rein-hardtii), 1 purple fungus (ectocarpus siliculosus),1 glauco-phyte (cyanophora paradoxa) and 1 pennate diatom. prgdb the plant resistance genes database (19) http://prgdb. org has been modernized with a new software, new sections, new instruments and fresh genetic improvement information, enabling convenient entry not only to the plant science society but also to breeders who want to enhance resistance to plant disease. the home page provides an overview of easy-to-read search boxes for streamlining data queries and showing directly plant species for which candidate or cloned gene data were collected. for each host plant species, bulk information files and curated gene annotations are produced accessible. in order to show shared features with other genes, the fresh gene model view provides comprehensive data on each cloned resistance gene structure. prgdb provides 153 genes of reference resistance and pathogen receptor genes (prgs) annotated applicant 177072. compared to the earlier publication, the amount of putative genes from 76 sequenced viridiplantae and algae genomes has been improved from 106 to 177 k. pineelm_ssrdb a total of 359511 ssrs were recognized in pineapple (chromosome sequence 356385, chloroplast sequence 45, mitochondrial sequence 249, and est sequence 2832) (20). the database contains the list of est-ssr tags and their information. conclusions: pineelm ssrdb is a noncommercial academic database accessible at http://app. bioelm.com/ with a mapping instrument that can create circular maps of the chosen target collection. for breeders, scientists and graduates operating on ananas spp, this repository will be of enormous use. and others operating on cross-species marker transferability, diversity research, mapping and fingerprinting of genetic material. sbmdb iquebal et al. (21) using the primer3 core, integrated at the backend in sbmdb, embedded markers and correlating location information of the selected chromosome, location / interval and primers could be produced. they as well reported 2027 polymorphic markers in a panel of five genotypes depending on e-pcr identification. these indicators can be used in variety enhancement program for dus screening of variety recognition and mas / gas. the current database provides a broad source of probable markers for the development and implementation of fresh molecular breeding methods needed to boost the industrial use of this plant, particularly for sugar, health care goods, medications and color coloring. url of the database: http://webapp. cabgrid.res.in/sbmdb/. http://www.scri.sari.ac.uk/plant_snorna/ http://www.bioinfo-cbs.org/ogdd/ http://plantrna.ibmp.cnrs.fr/ plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 5 of 7 spinachdb yang et al. (22) has been created to effectively record, annotate, mine and evaluate the datasets of genomics and genetics. all 21702 spinach genes have also been annotated. a total of 1,5741 spinach genes have been cataloged into 4,351 families, including a significant number of transcription factors being identified. to build a high-density genetic map, 11 grown and wild spinach cultivars recognized a total of 131592 ssrs and 1125743 prospective snps situated in 548801 loci of spinach dna. the spinachdb page includes seven primary parts, including the homepage; the gbrowse map integrating genes, genome, snp and ssr marker data; the blast alignment service; the search tool for gene family classification; the search tool for orthological and concurrent gene pairs; and the download and helpful contact data. in addition to the related data mining and assessment instruments, spinachdb will be continuously extended to include freshly created solid genomics and genetic data sets. url of your database: http://222.73.98.124 /spinachdb. sisatbase dossa et al. (23) recognized 138,194 genome-wide ssrs, 76.5% of which have been physically linked to the 13 pseudo-chromosomes. within these ssrs, up to three primers combinations were provided for 101,930 ssrs and used together with two freshly sequenced sesame accessions to amplify the reference genome in silico. a total of 79,957 ssrs (78%) were polymorphic among the three genomes indicating their successful use in various breeding implementations assisted by genomics. of 48 sesame accessions from various growing areas of africa, 23 of these polymorphic ssrs were chosen and evaluated to have high polymorphic potential., in addition, sisatbase has established a user-friendly internet database that offers free access to ssr information as well as an embedded functional analysis tool. all in all, the ssr and sisatbase reference would serve as useful resources for genetic evaluation, genomic studies, and advancement of sesame breeding, particularly in developing countries. url of the database: http://www. sesame-bioinfo.org/sisatbase/. sinbase sinbase was intended by wang et al. (24), a internetbased database with extensive genomic, genetic and relative genomic data on sesame. sinbase involves pseudo-molecular chromosomal sequences of constructed sesame, transposable elements (372,167), protein-coding genes (27,148), and noncoding rnas (1,748). with multiple plant genomes as well as for arabidopsis thaliana, glycine max, vitis vinifera, and solanum lycopersicum, sinbase offers distinctive and useful data on colinear areas. sinbase also offers helpful search and data mining instruments, including a quest for keywords and local blast service. is available free of charge at http:// ocri-genomics.org/sinbase/. ricebase edwards et al. (25) created a ricebase database and an integrative genomics database for rice (oryza sativa) with a focus on merging datasets in a manner that retains important connections between previous and present genetic research. ricebase involves information on dna sequence, gene annotations, and data on nucleotide variety and statistics on molecular fragment size markers. rice study has benefited from early acceptance and comprehensive use of easy sequence repetition (ssr) markers; however, most of the rice ssr models were created prior to the most recent rice pseudo-molecule assembly. the interpretation of recent studies using snps in the light of the literature quoting ssrs needs a common coordination scheme. ssr markers and laboraratoly tested amplicons sizes are provided in a webbased relationship database and are accessible as a map placed in a genome browser with connections between the browser and the database. the merged capacities of ricebase connect genetic markers, genome background, alleles throughout rice germplasm and possibly user-curated phenotypic interpretations as a community tool for genetic exploration and breeding in rice. it is publicly available at http://ricebase.org. reprolive claros (26) provided an olive tree reproductive transcriptome database containing samples of pollen and pistil at separate phases of development, as well as leaf and root as a control of vegetative tissues. it was created from 2,077,309 pure reads to 1,549 sanger sequences. using a predefined workflow centered on open-source instruments, sections were pre-processed, compiled, mapped, and annotated with expression information, go terms, descriptions, interpro tags, kegg pathways, ec numbers, orfs, and ssrs. tentative transcripts (tts) have also been annotated with the respective orthologists in the arabidopsis thaliana of the tair and refseq records to allow the inclusion of linkeddata. the result is a reproductive transcriptome of 72,846 contigs with an median duration of 686 bp, of which 63,965 (87.8 per cent) included at least one functional annotation, and 55,356 (75.9 per cent) had an orthologist. reprolive offers these outcomes with free access and download capacity. retrieval processes for sequence as http://ricebase.org/ plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 6 of 7 well as transcript annotations are given. graphical location of annotated enzymes in kegg processes is also feasible. it freely available at http://reprolive.eez.csic.es. planteome database the planteome initiative (27) http://www.planteome.org offers a suite of reference and species-specific ontologies for crops and annotations of genes as well as phenotypes. ontologies act as popular norms for the semantic integration of a wide and increasing body of plant phenomics, genomics, and genetic data. reference ontologies involve plant ontology, plant treatment ontology and plant experimental conditions ontology created by the planteome initiative, along with gene chemical entities in biological interest, ontology, attribute ontology, phenotype, and others.the initiative also offers access to species-specific crop ontologies created by multiple plant breeding and study groups around the globe. it also offers incorporated information on plant characteristics, phenotypes, and gene function and activity from 95 plant taxa, annotated with reference ontological terms. . referances 1. mokhtar mm, adawy ss, el-assal se-ds, hussein eha. genic and intergenic ssr database generation, snps determination and pathway annotations, in date palm (phoenix dactylifera l.). plos one. public library of science; 2016;11(7):e0159268. 2. adawy ss, mokhtar mm, alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. 3. jiang j, xing f, zeng x, zou q. ricyerdb : a database for collecting rice 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manjusha v, siddiq ea, polumetla ak, et al. cmmdb: a versatile database for cucumis melo microsatellite markers and other horticulture crop research. plos one. 2015;10(4):1–11. 11. doddamani d, katta ma, khan aw, agarwal g, shah tm, varshney rk. cicarmisatdb: the chickpea microsatellite database. bmc bioinformatics [internet]. 2014;15(1):212. 12. santos j, al-azzawi m, aronson j, flowers tj, coimbra u de. ehaloph a database of salt-tolerant plants : helping put halophytes to work special online collection – database paper. plant cell physiol. 2018;57(october 2015):1–10. 13. avvaru ak, saxena s, sowpati dt, mishra rk. msdb: a comprehensive database of simple sequence repeats. genome biol evol. 2017;9(6):1797–802. 14. shirasawa k, isobe s, tabata s, hirakawa h. kazusa marker database: a database for genomics, genetics, and molecular breeding in plants. breed sci [internet]. 2014;64(3):264–71 15. yu j, dossa k, wang l, zhang y, wei x, liao b, et al. pmdbase: a database for studying microsatellite dna and marker development in plants. nucleic acids res. 2017;45(d1):d1046–53. 16. brown jws, echeverria m, qu l, lowe tm, kastenmayer jp, bachellerie j, et al. plant snorna database. nucleic acids res. 2003;31(1):432–5. 17. ben ayed r, ben hassen h, ennouri k, ben marzoug r, rebai a. ogdd (olive genetic diversity http://reprolive.eez.csic.es/ http://www.planteome.org/ plant molecular biology databases highlights in bioscience doi: 10.36462/h.biosci.20191 may 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 7 of 7 database): a microsatellite markers’ genotypes database of worldwide olive trees for cultivar identification and virgin olive oil traceability. database. 2016;2016:1–9. 18. daujat m, duche a, gobert a, salinas t, michaud m, gutmann b, et al. plantrna , a database for trnas of photosynthetic eukaryotes. nucleic acids res. 2012;41(october 2012):273–9. 19. osuna-cruz cm, paytuvi-gallart a, donato a di, sundesha v, andolfo g, cigliano ra, et al. prgdb 3 . 0 : a comprehensive platform for prediction and analysis of plant disease resistance genes. nucleic acids res. oxford university press; 2018;46(november 2017):1197–201. 20. chaudhary s, mishra bk, vivek t, magadum s, yasin jk. pineelm_ssrdb: a microsatellite marker database identified from genomic, chloroplast, mitochondrial and est sequences of pineapple (ananas comosus (l.) merrill). hereditas. hereditas; 2016;153(1):16. 21. iquebal ma, jaiswal s, angadi ub, sablok g, arora v, kumar s, et al. sbmdb: first whole genome putative microsatellite dna marker database of sugarbeet for bioenergy and industrial applications. database. 2015;2015(1):1–10. 22. yang xd, tan hw, zhu wm. spinachdb: a wellcharacterized genomic database for gene family classification and snp information of spinach. plos one. 2016;11(5):e0152706. 23. dossa k, yu j, liao b, cisse n, zhang x. development of highly informative genome-wide single sequence repeat markers for breeding applications in sesame and construction of a web resource : sisatbase. front plant sci. 2017;8(august):1–10. 24. wang l, yu j, li d, zhang x, e-mail c. sinbase : an integrated database to study genomics , genetics and comparative genomics in sesamum indicum special online collection – database paper. plant cell physiol. 2018;56. 25. edwards jd, baldo am, mueller la. ricebase: a breeding and genetics platform for rice, integrating individual molecular markers, pedigrees and wholegenome-based data. database. oxford university press; 2016;2016. 26. claros mg. reprolive : a database with linked data for the olive tree ( olea europaea l .) reproductive transcriptome. front plant sci. 2015;6(august):1–14. 27. cooper l, meier a, elser jl, mungall c, sinn bt, cavaliere d, et al. the planteome database : an integrated resource for reference ontologies , plant genomics and phenomics. nucleic acids res. 2018;46(november 2017):1168–80. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20212 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access 1zoonotic disease research team, ethiopian public health institute, addis ababa, ethiopia. 2 department of public health, st. paul’s hospital millennium medical college, addis ababa, ethiopia. 3vaccine and diagnostics production directorate, ethiopian public health institute, addis ababa, ethiopia. 4veterinary public health directorate, ministry of agriculture, addis ababa, ethiopia. 5public health emergency management directorate, ethiopian public health institute, addis ababa, ethiopia. 6public health emergency management directorate, health bureau of snnp, hawassa, ethiopia. 7deputy director general office/rtt, ethiopian public health institute, addis ababa, ethiopia. contacts of authors * to whom correspondence should be addressed: yimer mulugeta citation: mulugeta y, lombamo f, alemu a, bekele m, assefa z, shibru e, beyene m, kitila g, getahun g, sifer d, aklilu m, regassa f, and deressa a (2020). assessment of the current rabies situation and its management in epidemic areas of southern ethiopia. highlights in bioscience volume 3. article id 20212. dio:10.36462/h.biosci.202 12 received: may 20, 2020 accepted: july 12, 2020 published: july 14, 2020 copyright: © 2020 mulugeta et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of the current rabies situation and its management in epidemic areas of southern ethiopia yimer mulugeta 1 *, fantu lombamo 2 , anberber alemu 3 , meseret bekele 4 , zewdu assefa 5 , endashaw shibru 6 , mekoro beyene 3 , gutu kitila 1 , garoma getahun 1 , desalegn sifer 1 , mesfin aklilu 1 , feyesa regassa 5 , and asefa deressa 7 abstract rabies infection is almost always a 100% lethal viral disease in all mammals, including humans. although rabies is vaccine preventable disease, more than 60,000 people worldwide, and 2,700 people in ethiopia die of fatal human rabies annually. the aim of this study was to generate strategic information on the extent of the burden of rabies and the existing major response strategies, in order to overcome and bring attention to the management of rabies outbreaks. cross sectional study design was used to assess the current situation of rabies burden in the southern nations, nationalities, and peoples' regional states of ethiopia. data analysis revealed a total of 16 fatal human rabies and 287 suspected rabies exposures. the highest attack rate of 116.3 cases per 100,000 human subjects was reported in tembaro woreda. as a result, 48 animal deaths of rabies have been reported, while cattle (28) have been more affected than other animal species. the lack of dog immunization, lack of community awareness of dog management, and inaccessibility and inefficiency of anti rabies vaccines were major risk factors associated with the spread of rabies exposure between humans and animals. in conclusion, this study has clearly shown that both the existence and the high magnitude of fatal human and animal rabies are significant in the area. multidisciplinary approach interventions to increase community awareness of dog vaccination, effective and immediate treatment of exposed individuals is recommended as a method of disease control and prevention. keywords: animal, anti-rabies, human, outbreak, rabies exposure, snnp. introduction rabies exposure can always be either 100% fatal or 100% preventable double burden viral disease here in ethiopia and around the world. rabies exposure is 100% fatal, once a clinical symptom has been developed, even if it is characterized by a long incubation period of 2–3 months on average, but may vary from 1 week to 1 year depending on factors such as location of entry and viral load, short disease duration and death within 7 to 10 days of onset of clinical symptoms [1]. while rabies exposure is a 100% preventable disease with effective treatment soon after exposure to rabies, and may even be eradicated if government and donor attention is given to developing a control program/strategy and regulatory system to promote control of rabies in dogs as a key strategy for controlling rabies in humans through sustained dog vaccination programs. https://creativecommons.org/licenses/by/4.0/ mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 thus, the management and registration of the dog population, community awareness and cooperation of all stakeholders improve the cost effectiveness of vaccination intervention as a key measure [2-4]. globally, the burden of fatal human rabies is that 60,000 people die every year; almost 99% of human rabies exposure is caused by dogs. about 95% of death occurs in developing countries, particularly in africa and asia [5]. in developing countries, successful zoonoses are difficult to prevent and control, some of the main factors being zoonoses such as rabies that are neglected, under-reported, joint multi-sectoral preparedness and response interventions that remain unsatisfactory due to gaps in the legal framework. other factors attributing to rabies exposure include failure of dog immunization, abundantly free roaming dogs, and insufficient government attention, which may be due to underreporting of cases and lack of community awareness. children's and poor rural communities have a significant impact on the risk of rabies exposure across society, as well as a high gap in access and affordability of anti-rabies vaccines [2, 4, 6]. rabies is endemic in ethiopia and places a huge burden on the economy and public health. every year, 2,700 people in the country die of rabies. while there is no regulatory system for the prevention and control of rabies as mitigation, so far there is no legislation for the prevention of rabies. however, the ethiopian public health institute / ephi has played a key role in selected country pilot project areas in collaboration with various government and international partners, as one of the priority activities of the global health security agenda, cdc, osu, fao and other partners have started working together to build an effective national rabies elimination strategy [7, 8]. rabies is underreported in different corners of this country. as a result, its impact is underestimated due to existing challenges such as the lack of rabies diagnostic facilities at least one laboratory at each regional level and the lack of awareness-raising activities for health professionals and the lack of awareness-raising activities for the community. for example, there are different rare reports from the southern nation, nationalities and people's regional state (snnprs) of ethiopia on the occurrence of rabies exposure (mainly from suspected rabid dogs) in humans from different epidemic sites in the regional state. our aim is to (a) assess the outbreak of rabies in both humans and animals, and (b) generate strategic information on the extent of the burden of rabies and major response to informed decisions on the prevention and control of rabies. methods study area description the southern nations, nationalities and peoples' regional state (snnprs) is one of the largest and most ethnically diverse regions of ethiopia. geographically located in the southern part of the country surrounding kenya in the south, the republic of south sudan in the southwest, the state of gambella in the northwest and the state of oromia in the north and east. the total population size of the snnp is estimated to be 20,767,937 in 2018, of which 93.2% of the total population is in rural areas of the state. the region is also well known for having more than 1,5956,100 different livestock populations, 23 species of wild animals and 300 species of birds and water resources that can be used for fishery, irrigation and hydroelectric development. there are seven preserved locations in the forest. forests and bushes cover around 18% of the state [9]. rabies exposures studied were hawassa zuria woreda, mareka woreda, soro woreda, tembaro woreda, tercha town and gena bossa woreda of snnprs. hawassa zuria is located in the sidama zone; tembaro is located in the kembata tembaro zone; soro is located in the hadiya zone and tercha, mareka and gena bossa are located in the dawuro zone of snnprs, ethiopia. these areas are the focus of numerous wildlife, forests and water resources, including the gilgel gibe river basin and the gilgel gibe iii dam. its geographical location is shown in (figure 1). study design cross-sectional study was conducted in selected rabiesepidemic areas of snnprs, ethiopia. human rabies exposure assessment all humans with any animal bite without discrimination of the socio-demographic characteristics of the victims during the investigation period from july 2017 to may 2018. in the case of suspected human rabies exposure assessment, bite/exposure of animal history, bite site, date of bite, date of admission to the health facility, clinical sign and symptom status and outcome of each human exposure were addressed. livestock rabies exposure assessment all animal species with unusual signs or rabies suggestive behavioral changes have been included in this particular investigation without discrimination of species, immunization status and animal bite history. questionnaire survey semi-structured questionnaires were administered by health care providers, primarily those engaged in emergency care management at selected snnprs health facilities in ethiopia. the main focus areas of the interview and observation were all interested emergency health care providers who participated in taking time on the mode of transmission of rabies, methods of control and prevention, and possible ways of managing and diagnosing animal bite cases in their health facilities and at the same time addressing anti-rabies treatment. anti-rabies handling and utilization assessment the anti-rabies vaccine service was assessed by the total amount of vaccine available, the storage and mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 figure 1. geographic location of the investigation/study area. management of the cold chain, the proper administration and the proportion of use. anti-rabies handling and utilization assessment the anti-rabies vaccine service was assessed by the total amount of vaccine available, the storage and management of the cold chain, the proper administration and the proportion of use. sampling technique purposive sampling methods have been used for this particular study to address all these rabies-epidemic areas based on disease outbreak reports in the study area. data collectors/study team multidisciplinary team first organized by the ethiopian public health institute (ephi): public health emergency management (phem), zoonotic disease research and antirabies vaccine production directorates, st. paul's hospital millennium medical college and the ministry of agriculture (former ministry of livestock and fishery resources) were deployed in the hot spot areas of southern nations nationalities and peoples region (snnpr). data collection primary and secondary data were collected from public health offices, veterinary offices and health facilities through key informant interviews and interviews with experts and health care providers using standard semi-structured questionnaires. personal observations were made during interviews with health care facilities. the deployed team met with the snnpr, phem directorate. the owner of the phem core process referred to four hot spot areas (dawuro zone, kembata tembaro zone, hadiya and sidama zone) and their prospective woreda areas accordingly. active community and office searches and visits to health facilities were conducted as well as the major associated risk factor, pep utilization, laboratory findings and burden assessments were also addressed. ethical consideration ethical approval was not obtained from the institutional review board, as there were time constraints to respond in a timely manner to public health emergency management issues. however, the purpose of this study was explained to the intended study participants and verbal consent was obtained from them before proceeding with the study/data collection. data analysis descriptive data from this study from human and veterinary health facilities and the community were analyzed and reported on the status of rabies outbreaks and human and animal burden assessments, major risk factors, pep utilization and laboratory findings. results rabies outbreak status and human burden assessment a total of 16 suspected human rabies deaths have been reported from tercha general hospital, mudula primary hospital, bushillo, gimbicho and jacho health centers in public health facilities. analysis of secondary data revealed a total of 287 suspected rabies exposures that had been observed during the follow-up of anti-rabies in these sites during the investigation period. of the 16 suspected deaths of human rabies, 9 were male and 7 were female. the site of the bite / exposure to the human victims was the leg, finger, lip and arm. domestic dogs were responsible for all human exposure, as detailed in (table 1). mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 table 1. suspected fatal human rabies case scenario from investigation sites of snnp, ethiopia. s.n age sex woreda/site date of bite site of exposure exposin g animal duration after exposure remark 1 13 female tembaro november 16, 2017 leg dog 3 months 2 28 male tembaro january 20, 2018 finger dog 3 months came to hf with clinical signs 3 7 female tembaro september 27, 2017 leg dog 3 months death after 6 days of onset of illness 4 5 male tembaro february 13, 2018 leg dog 2 months after 7 days of illness 5 60 female tembaro january 23, 2018 finger dog 3 months after 5 days of illness 6 7 male tembaro january 17,2018 lip dog 2 months took 15 doses and died 7 56 male tembaro january 25, 2018 leg dog 2 months came to hf with clinical signs 8 male soro leg dog was referred to mudulla hospital after onset of clinical sign and died 9 male tembaro lip dog died after 12th dose of ntv 10 14 male mareka december 25, 2017 unknown dog 2 months 11 7 female mareka arm dog 6 cattle died after being bitten by the same dog 12 46 female soro march 18, 2017 leg dog 1 months 13 28 male soro january 8, 2018 leg dog 3 months 14 16 female mareka finger cat 15 27 female hawassa zuria march 12, 2018 leg dog initially diagnosed as typhoid fever and was being managed with ceftriaxone 16 21 male hawassa zuria dog the highest rate of rabies exposure was reported from tembaro woreda (116.3 per 100,000 population at risk); 55.3, 13.6, 12.1, 5.4 per 100,000 population at risk attack rates were reported in hawassa zuria woreda, mareka woreda, soro woreda and gena bossa woreda, respectively. rabies outbreak status and burden assessments in animals during the investigation period, 48 animal rabies deaths were detected and 28 cattle, 12 goats, 5 sheep and 3 equines were identified. death report due to suspected rabies in the dog was not counted as a result of dog elimination activity conducted using strychnine. of these human-exposed dogs, only 10 rabies-suspected samples were submitted to the national rabies laboratory of the ethiopian public health institute (ephi). of the total samples, 80% were positive for rabies virus infection, and dogs were the most biting / exposing animals for animals and human victims during the time and area of investigation, as shown in (figure 2). major contributing risk factors for rabies outbreak in place summary of the major associated risk factors for the occurrence and spread of rabies exposure among humans and animals has been identified. absence of dog immunization and lack of community awareness of dog management in this investigation, the engaged community is once remote from rural areas and keeps the dog on guard for reasons of lack of awareness of the possibility of disease transmission. all respondents responded that they had a single dog (47%) or more than one dog (53%), and their dogs were free to move to the surrounding area (free roaming dog) and no history of deworming and/or vaccination of dogs. the absence of immunization for dogs has also been reported by the livestock sector, which has created a chance for the occurrence and spread of rabies exposure by addressing multiple factors such as community ignorance, unaffordable cost of anti-rabies vaccine for dogs and little government attention. figure 2. rabies exposure laboratory findings and human victims by animal bite during investigation time and area (snnp). mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 challenges on anti-rabies vaccine and inadequate human rabies exposure management inaccessibility and unavailability of cell culture antirabies vaccine (modern vaccine by the national pharmaceutical supply agency) and shortage of nerve tissue vaccine (old anti-rabies) human anti-rabies vaccine have been observed (figure 3). although vaccine shortages have occurred, consistent cold chain management problems have also been observed in health facilities. in fact, the nerve tissue (old anti-rabies pep) anti-rabies vaccine production capacity of ephi has so far not met the demand for and severely affected the required response to deadly diseases. figure 3. coverage of anti-rabies vaccine in snnp before the investigation date of the fiscal year. inadequate heath care provider’s knowledge and skill at those health facilities the opinion and level of knowledge and skills of healthcare professionals in the area of rabies exposure management with selected hfs were assessed on the basis of their interest in semi-structured self-governing interviewers. we found that, 46.6 % (14/30) of the respondents had knowledge of the mode of transmission via saliva from suspected animal bites, in particular dog bites (table 2). ecological factors for increased spillover the existence of natural forests, mountains and rivers such as the gilgel gibe river basin has created a suitable environment for the occurrence and spread of rabies exposure , especially the over filling of the gilgel gibe iii dam, which plays a significant role in displacing the normal habitat of wildlife for interaction and co-existence with domestic animals and peoples. dogs, cats, foxes and donkeys were among the most frequently exposed animals found during the investigation. increased interaction of wild life with dogs and high prevalence of free roaming dogs were considered risk factors for increased exposure to rabies. table 2. opinion of healthcare providers and their inadequate knowledge and skill on human rabies exposure management. variables category frequencies proportion sex of respondents male 18 60 female 12 40 respondents hf tercha gh(dawuro) 16 53 gimbichu & jacho hc 11 37 mudula hospital 3 10 is rabies a major problem in your hf yes 24 80 no 6 20 knowledge & skill of health care providers on mode of transmission yes 14 47 no 16 53 knowledge & skill on clinical signs/symptoms in human yes 20 67 no 10 33 throughout the year rabies exposure occurrence yes 25 83 no 5 17 is there any preventing & control guideline/legislation yes 5 17 no 25 83 which control option do you use health education (he) 14 47 anti-rabies vaccination (pep) 12 40 all options he, pep & wound mg’t 4 13 after exposure where they go? health facility 24 80 traditional healer 4 13 other 1 3 what are the major challenges of human rabies exposure lack of regular animal rabies control activity (arc) 6 20 lack of community awareness(ca) 14 47 lack of training(tot) 3 10 combination factors of above 5 17 mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 discussion rabies is one of the most neglected tropical and zoonotic diseases with an increasing trend in human burden [10]. this investigation revealed a high rate of rabies and its exposure in humans and animals and a lack of immunization in dogs compared to studies reported in the ethiopian rabies review (1990 to 2000) in which a total of 322 human deaths were reported [11]. there is also another annual estimate of 2700 human deaths in 2017 [7], which has shown an increasing burden on public health and still needs to be addressed in order to achieve the global rabies elimination program 'zero rabies in humans' by 2030 [1,12]. in this study, fatal human and animal rabies of 16 and 48 were found mainly due to rabies-specific symptoms and epidemiological factors, as the standards of rabies diagnostic methods could not be applied. this is due to the lack of standardized laboratory settings and the difficult nature of rabies cases of cns in suspected human rabies exposure. this study found the highest rates of attack in tembaro woreda with 116.3 cases per 100,000 human population, followed by 55.3 cases in hawassa zuria woreda, 13.6 cases in mareka woreda, 12.1 cases in soro woreda, and 5.4 cases in gena bossa woreda per 100,000 population at risk of attack. this finding is considerably higher than that of 2.33 cases per 100,000 human population in northern gondar, ethiopia [13]. overall, human rabies exposure in this investigation has been admitted to the health care facility too late and varies from area to area for an average of 2-3 months from the date of bite or after onset of clinical symptoms as described in (table 1). this was due to the high perceptions of local people in that country and the widespread use of traditional medicine among the urban and rural population of ethiopia could be attributed to cultural acceptability, physical accessibility and economic affordability compared to modern medicine [14]. a high free roaming dog population with a never-vaccination history and a high level of wildlife interaction was identified as an existing fueling factor for rabies outbreaks. this finding is consistent with the findings reported by admassu and mekonnen that, in 2014 in the same country, a large number of dogs in both urban and rural settings and a low vaccination risk of rabies circulation and spread to humans and other domestic animal populations [14]. in order to manage this burden of human rabies exposure in the snnp region, a lack of anti-rabies vaccine was observed during the investigation, and ephi distributed a total of 4305 rabies post-exposure prophylaxis per 10 months until the investigation was conducted to protect the public. the dawuro and kembata tembaro zones reported the highest levels of exposure to rabies and human fatal cases of rabies, but vaccine coverage was 42 and 68, respectively. this result is in agreement with the findings reported by abraham in this country [15]. this sample was tested in a national rabies laboratory for laboratory analysis of 10 suspected animal brain tissue samples, 80 per cent of which were positive for rabies. all of the positive samples were from the dog. samples submitted to the national rabies laboratory were only ten, due to a number of reasons such as lack of regional laboratory facilities in the area; lack of awareness was a key factor due to remote rural communities. the world health organization recommends that at least 70% of the dog population be vaccinated to control and potentially eliminate human-mediated rabies in dogs [16]. in this study, no organized dog vaccination activity was performed, instead of vaccination, by the community in communication with local authorities to overcome dog bite and rabies exposure in humans and animals. the reason for the elimination of dogs was due to inaccessibility and unaffordability of the anti-rabies vaccine and high free roaming dog population. as a limitation, although the main objective of this study is to respond to rabies outbreaks, there are no estimates of the sample size to include all study participants and standard confirmatory tests in human and animal subjects. some suspected human rabies death data have not yet been completed and a documentary system has been recorded. conclusion and recommendation this study team detected cases of human and animal fatal rabies in the community and at selected public health facilities in snnp, ethiopia, in 2018. the team verified the occurrence of an outbreak of rabies exposure. the multisectoral and multi-disciplinary "one heath approach" has been seen as very critical in addressing such burdens in the human and animal domains. poor collaboration between sectors, inaccessibility/shortness of anti-rabies vaccines and community awareness gaps have been identified as major challenges. high free roaming and unvaccinated dog populations as well as high interactions with wildlife were also observed. on the basis of the above conclusion, the following recommendations are forwarded: 1. awareness-creation and social mobilization programs and events related to rabies problems should be undertaken through the use of different media and other tools for the community, health professionals and policy makers. 2. communication and collaboration between the human and animal professions should be encouraged for an effective system of surveillance, control and prevention of rabies exposure. mulugeta et al., 2020 assessment of the current rabies situation and its management in southern ethiopia. highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 7 3. mass vaccination of dogs, the prevention of free movement of dogs and contact with wild life and the management of dog populations should be carried out in order to achieve zero rabies in humans and animals. 4. the availability and affordability of the anti-rabies vaccine (pep) for humans and dogs should be addressed with national and international partners. 5. quality cell culture anti-rabies vaccine must be available and accessible to the affected community as a whole. acknowledgement we would like to thank the members of our health and veterinary office and health care providers from the respective areas and districts of sidama (hawassa zuria), hadiya (soro), kembata-tembaro (tembaro) and dawuro (mareka and gena bossa) for their valuable support, advice and guidance in boosting this work. we also recognize the contributions of our driver, mr shimeles, and all those individuals who have given up their time to participate. authors' contributions ym was responsible for the study, collection and analysis of the data and wrote the manuscript. fl, aa, my, za, es, mb, contributed to the collection of data, the study methodology and the co-writing of the manuscript. gk, gg, ds and ma have contributed to laboratory work and guidance. fr and ad contributed to the revision and editing of manuscripts. the final manuscript was read and approved by all authors. references 1. who. rabies fact sheet.https://www.who.int/newsroom/fact-sheats/detail/rabies 2019 [cited 2019 may 21]. 2. takahashi-omoe h, omoe k, okabe n. regulatory systems for prevention and control of rabies, japan. emerging infectious diseases. 2008 sep;14(9):1368. 3. fao, developing a stepwise approach for rabies prevention and control. proceedings of the fao/garc workshop, november 2012, rome, italy. fao animal production and health proceedings, 2013. 18. 4. who, who expert consultation on rabies: frist report, in technical report series 2005, who: geneva. 5. undurraga ea, blanton jd, thumbi sm, mwatondo a, muturi m, wallace rm. tool for eliminating dogmediated human rabies through mass dog vaccination campaigns. emerging infectious diseases. 2017 dec;23(12):2114. 6. oyda s, megersa b. a review of rabies in livestock and humans in ethiopia. international journal of research granthaalayah, 2017. 5(6): 561-577. 7. cdc. rabies in ethiopia. world rabies day, prevent rabies around the world 2017. 8. deressa a, ali a, bayene m, selassie bn, yimer e, hussen k. the status of rabies in ethiopia: a retrospective record review. ethiopian journal of health development. 2010;24(2). 9. adugna a, snnpr, southern nations nationalities and peoples demography and health. ethiodemography and health, 2018. 10. fao/oie/who, inter-agency meeting on planning nzds prevention and control. 2011. 11. moges n. rabies in ethiopia: review article. academic journal of animal diseases. 2015;4(2):74-81. 12. wallace rm, undurraga ea, blanton jd, cleaton j, franka r. elimination of dog-mediated human rabies deaths by 2030: needs assessment and alternatives for progress based on dog vaccination. frontiers in veterinary science. 2017 feb 10;4:9. 13. jemberu wt, molla w, almaw g, alemu s. incidence of rabies in humans and domestic animals and people's awareness in north gondar zone, ethiopia. plos negl trop dis. 2013 may 9;7(5):e2216. 14. admasu p, mekonnen y. rabies and its folk drugs remedies in ethiopia: a review. international journal of basic and applied virology. 2014;3(2):22-7. 15. abraham a, daddi j, milliyon w, almaz a, fikir t. national surveillance for human rabies cases. in: impact, limitation and needs in developing countries. ethiopian health and nutrition research institute, 2010. 16. who, who expert consultation on rabies: third report. who technical report series, no. 1012. licence: cc by-nc-sa 3.0 igo, 2018. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20218 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 7 research article open access department of science technology, akwa ibom state polytechnic, ikot osurua, p.m.b 1200, ikot ekpene, akwa ibom state, nigeria. contacts of authors * to whom correspondence should be addressed: aniefon alphonsus ibuot citation: james ii, ben mg, jones am, akpan ps, eka ii, oruk ae, ibuot aa (2020). characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites. highlights in bioscience volume 3. article id 20218. dio:10.36462/h.biosci.20218 received: june 6, 2020 accepted: august 18, 2020 published: august 31, 2020 copyright: © 2020 james et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites iniobong ime james, mayen godwin ben, agnes monday jones,patience saturday akpan, idorenyin idorenyin eka, albert ema oruk, and aniefon alphonsus ibuot* abstract changes in soil physicochemical properties and bacterial species present in soil contaminated with waste engine oil were evaluated at three auto-mechanical workshops in uyo, nigeria. this work was aimed at isolating and identifying hydrocarbon degrading bacteria from waste engine oil polluted soil, and assessing their hydrocarbon-utilizing ability. waste engine oil pollution affected soils significantly with increases in soil physicochemical properties, and heterotrophic bacterial population counts. eight bacterial species corynebacterium kutscheri, pseudomonas aeruginosa, flavobacterium aquatile, serratia odorifera, micrococcus agilis, staphylococcus aureus, micrococcus luteus and bacillus substilis were isolated by the selective enrichment technique and screened for hydrocarbon utilization capability in mineral salt media with 1% (v/v) waste engine oil as a sole carbon and energy source. the extent of bacterial growth observed was related to the ability of organisms to biodegrade hydrocarbons present in the medium bacterium species, which showed varying hydrocarbon utilization during the 15 days of incubation. growth in hydrocarbon medium was the most efficient in cultures of corynebacterium kutscheri. all isolates also showed variable emulsification ability, with corynebacterium kutscheri, showing the highest ability. these results demonstrate the presence of indigenous bacteria in hydrocarbon-polluted soils and the potential toward the remediation of hydrocarbons. keywords: hydrocarbon-utilizing bacteria, selective enrichment technique, corynebacterium kutscheri introduction petroleum utilization as fuel and petroleum products leads to severe environmental pollution [1]. large-scale accidental spills pose a great threat to the ecosystem [2]. soil pollution by petroleum hydrocarbons has been shown to produce pronounced changes in the physicochemical and microstructure of the oil-contaminated soil [3]. this affects parameters such as soil porosity, bulk density, and adsorption [4-5]. fresh spills and/or high levels of pollutants may often result in the reduction of large sectors of soil microbial population, although soils with lower levels or old pollution may show an increase in numbers and diversity of microorganisms [6-7]. the diversity and the number of microorganisms at polluted soil sites may assist in the characterization of such a site, such as the toxicity of petroleum hydrocarbons to the microbiome, age of the spill and concentration of the pollutant [8]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20218 https://doi.org/10.36462/h.biosci.20218 http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 7 additionally, microorganisms in soils exposed to hydrocarbon pollution usually exhibit a higher potential for biodegradation of such pollutant compounds than others with no history of such exposure. percentages of hydrocarbonoclastic microbes are quite low in soil when there is no oil spill, but may increase 1,000 fold after oil spill [9]. conventional remediation methods do not seem to be able to address this problem, or tends to aggravate the problem [10]. mechanical methods such as incineration, excavation and/or burial in secure land fill, as well as a host of other chemical decomprelocates osition methods are expensive, time consuming and only the pollution [11]. an efficient way of remediating the oil-contaminated sites could be employment of microorganisms, such as bacteria, microscopic algae, and fungi, isolated from polluted environments or enhanced from the organisms already present in the same environment [12-13]. waste engine oil-polluted soils also serve as a source of indigenous bacteria capable of hydrocarbon degradation. the employment of microorganisms in the biodegradation of hydrocarbons over chemical or conventional treatment is preferred for many reasons; end products are comparatively safer and cost-effectiveness [11]. ogunbayo et al., [18] evaluated the effectiveness of bacteria indigenous to soil in remediating engine oil-polluted, soil and isolated bacillus, pseudomonas, flavobacterium, micrococcus and rhodococcus species, with pseudomonas and rhodococcus species giving most favorable degradation effectiveness and efficiencies. this study therefore considered the isolation of indigenous bacterial communities in waste engine oil-polluted soil using selective enrichment technique, and the assessment of hydrocarbon-utilization capability in waste engine oil-augmented mineral salt medium. materials and methods sample collection waste engine oil –contaminated soil samples used in this study were collected from three auto-mechanic workshops within the mechanic village, uyo, akwa ibom state, nigeria. composite soil samples were obtained at each sampling point using a soil auger from 0-10 cm below the soil surface. the soils were labeled “unpolluted” for the unpolluted sample, “ma” for the mechanic workshop 1 sample, “mb” for the mechanic workshop 2 samples, and “mc” for the mechanic workshop 3 samples. this was followed by bulking and transportation to the laboratory in sterile polythene bags within six hours for isolation of organisms. physico-chemical analysis of soil samples the soil ph was measured using hanna instruments model 209 ph meter [14]. moisture content was calculated on the basis of the air dry weight as described by aoac [15]. total organic carbon was calculated by weighing exactly 0.5 g of the soil sample into a flask, and 10 ml of 1.0 m k2cr2o7 was added and swirled to mix. 20 ml conc. h2so4 was added, gently swirled for a minute and allowed to stand for 20 minutes. the suspension was diluted to about 100 ml of distilled water. five drops of o-phenanthroline indicator were added to each sample and was titrated with 0.5 m ferrous ammonium sulfate to a light blue end point. the reagent blank was also run and the titre values recorded, and used to calculate the organic carbon content [15]. the total hydrocarbon content (thc) was determined by first extracting hydrocarbons by acidifying 2 g of representative soil samples using h2so4, and extracting upon addition of 20 ml of toluene in a separatory funnel. the contents of the funnel were shaken, and allowed to settle into two layers. the absorbance of the supernatant (extract) was read at 420 nm with unicam uv/vis spectrophotometer (spectronic 20d). readings were recorded from the spectrophotometer and using the determined curve to obtain the figure [16]. phosphorus was determined using the ascorbic acid method as described by aoac, [15]. 50 ml of the soil dilution was pipetted into 250 ml erlenmeyer flask, and 1 drop of phenolphthalein indicator was added. exactly 5 n h2so4 (148 ml conc. h2so4 in 100ml h20) is added drop-wise to develop a red colour. exactly 8 ml of combined reagents made up of 50 ml of 5 n h2so4, 5 ml potassium antimonyl tartrate solution (1.372 g potassium antimonyl tartrate in 500 ml distilled water); 15 ml ammonium molybdate solution (20 g ammonium molybdate crystal in 500 ml distilled water); were added and thoroughly mixed, and allowed to stand for 20 min. the phosphorus content was determined by measuring the absorbance of the sample at 880 nm. the nitrogen concentration was determined according to the methods of bremmer and mulvaney, [17]. one milliliter of the soil sample was introduced into the standard kjeldahl flask containing 1.5 g cuso4, and 1.5 g na2so4 as catalyst, alongside concentrated h2so4. the flask was gently heated on a heating mantle, taking care to prevent frothing. the solution was transferred after heating to a 100 ml standard flask and made up to the mark with distilled water. a portion of this digest was pipetted into a semi micro-kjeldahl distillation apparatus and treated with 30 ml of 40% naoh solution. the ammonia evolved was steam-distilled into a 100 ml conical flask containing 10 ml solution of saturated boric acid to which 4 drops of tashirus indicator had been previously added. the tip of the condenser was immersed in the boric acid solution and the distillation continued until about two-thirds of the original volume was obtained. the tip of the condenser was finally rinsed with a few milliliters of distilled water. the distillate was then titrated with 0.1n hcl until a purple-pink end point was observed. a blank determination was also carried out in a similar manner without the sample, and the calculation done as follows: nitrogen (%) = (real titre – blank titre) x 0.1 x 0.014 x 100 weight of the sample enumeration of total heterotrophic bacteria (thb) the thb population in the soil samples was enumerated by adopting the standard plate counts technique using the spread plate method as described by ogunbayo et al., [18]. these involved spreading aliquots of a serially http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 7 diluted 0.1 ml of 10-5 dilutions of the soil sample suspension on nutrient agar plates and the plates were incubated at 30 oc for 24 h. similar aliquots were also incubated in minimal salt agar plates containing used engine oil as the sole source of carbon and energy. the plates were all incubated aerobically at 30 oc. the percentages of hydrocarbon-utilizing bacteria (hub) relative to the total heterotrophic counts were noted. enumeration of hydrocarbon utilizing bacteria (hub) oil-utilizing bacteria were isolated from polluted soil samples by enrichment in mineral salt medium (msm) modified from okpokwasili and nwosu, [19] using waste engine oil as a carbon and energy source. the soil samples were sieved using a 2 mm mesh sieve. 10 g of the sieved soil samples was inoculated into 100 ml sterile msm. 1 ml of the waste engine oil was added to the medium as a sole source of carbon and energy, and the culture was incubated on a rotary shaker at 170 rpm for 1 week. the enrichment procedure was repeated for three cycles. at the end of each enrichment cycle, 1 ml of the culture was diluted serially 10-fold down the gradient to 10-5 and plated. pure cultures of the isolates were obtained by plating 1 ml of the 10-5 dilution of the third enrichment cycle onto msm agar plates, and incubating at 30 oc (± 2) for 48 h. pure cultures obtained by this procedure were stored in slants at 4 oc until further identification. characterization and identification of bacteria isolates were identified on the basis of colonial characteristics, gram’s reaction and cell biochemical reactions as described by cheesbrough [20]. identification used the taxonomic schemes of holt et al. [21]. hydrocarbon utilization screening of bacteria to determine the ability of the isolates to utilize engine oil as the sole carbon and energy source, the growth patterns of isolates in mineral salt medium in the presence of 1% (v/v) of the waste engine oil (5.0 ml in 100 ml msm) were determined according to onuoha et al. [22]. waste engine oil-augmented msm was dispensed into 250 ml erlenmeyer, and inoculated with 0.1 ml of 24 h cultures of the bacterial isolates. incubation was done at 30 oc for 15 days. growth patterns were determined monitoring changes in ph, optical density and total viable count at 5-day intervals during the incubation. the ph of the medium was measured using the ph meter (hanna instruments). growth was also monitored by measuring the optical density (od) at 600 nm using the spectrophotometer (spectrumlab). total viable counts of the cultures were obtained by incubation of 0.1 ml of the cultures using the spread plate technique on nutrient agar plates at 30 oc for 24 h. emulsification activity of bacteria the emulsification index (e24) of the isolates was determined according to the methods of ganesh and lin, [23], by adding 1ml of waste engine oil to the same amount of culture media as used for degradation assay, mixing the vortex for 2 min and leaving to stand for 24 h. the percentage of emulsification index was obtained as follows: e24 = height of the emulsified layer x 100 total height of the liquid column results physicochemical properties of soil samples the results of the physicochemical analysis of the different soil sample are shown in table 1. the high amounts of organic carbon (5.32 ± 2.65% in ma, 9.79 ± 0.51% in mb and 7.29 ± 3.09% in mc), and thc (2933.76 ± 404.27 mg/kg in ma, 3122.72 ± 131.00 mg/kg in mb and 3202.61± 675.07 mg/kg in mc), compared to the unpolluted soil sample (3.7 ± 2.43% organic carbon content and 39.97± 13.49 mg/kg thc) is indicative of heavy pollution of the mechanical workshop samples with petroleum hydrocarbons. soil samples from mechanical workshop 1 contained higher amounts of nitrates (0.25 ± 0.03 mg/g), while samples from mechanical workshop 2 contained the highest amounts of phosphates (10.74 ± 0.88 mg/g) and thc (3202.61± 675.07 mg/kg). the ph values of the soil samples indicate all soil samples as moderately acidic to acidic (from ph 5.78 to ph 6.79). bacterial count of soil samples the total heterotrophic bacterial count and hydrocarbon utilizing bacterial count of the original soil samples is shown in table 2. a higher thb count was recorded in polluted soil samples (4.4 ± 1.90 x 107cfu/g from ma sample, 6.0 ± 0.23 x 107 cfu/g from mb sample and 4.5 ± 0.03 x 107 cfu/g from mc sample) than in the unpolluted soil sample (1.9 x 107 cfu/g). higher thb (6.0 ± 0.23 x 107cfu/g) and hub (5.2 ± 0.25 x 107 cfu/g) counts were observed in the mb sample than in other similar polluted samples indicative of its extent of pollution. hydrocarbon utilising bacterial counts were slightly lower in all samples than the corresponding heterotrophic bacterial counts. characterization and identification of bacteria the identified bacterial isolates were corynebacterium kutscheri, pseudomonas aeruginosa, micrococcus agilis, flavobacterium aquatile, staphylococcus aureus, micrococcus luteus, serratia odorifera and bacillus substilis, as shown in table 3 and 4. hydrocarbon utilization potential of bacteria table 5 shows the changes in ph of msm during growth of bacteria isolates in hydrocarbon. decreases in ph (to <ph 7.00) were observed in medium containing isolates, corynebacterium kutscheri, micrococcus agilis, serratia odorifera and bacillus substilis, (which, however, increased to above ph 7.00). the greatest decrease in ph occurred in cultures of corynebacterium kutscheri (from 7.00 a.m. on day 0 to 5.83 ± 0.34 on day 15). medium containing other isolates showed slight increases in ph over time. the changes in the total viable counts of bacterial isolates during 15 days of growth in waste engine oil-augmented msm are shown in table 6. total viable counts were higher in cultures containing corynebacterium kutscheri (1.94 ± 0.12 x 108 cfu/ml on day 5, 6.73 ± 0.45 x 108 cfu/ml on day 10 and 3.13 ± 0.02 x 108 on day 15). growth of staphylococcus aureus showed the lowest decreases (0.31± 0.11 x 108on day http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 7 5, 0.44 ± 0.01 x 108 on day 10 and 0.42 ± 0.02 x 108 on day 15). table 7 shows the results of turbidity measurement (od600) of the medium during incubation. the highest increases in turbidity were recorded in cultures with corynebacterium kutscheri, (0.189 ± 0.04 on day 0 to 0.301± 0.11 on day 15), and bacillus substilis, (0.165 ± 0.04 on day 0 to 0.341± 0.02 on day 15). the lowest turbidity was observed with staphylococcus aureus (0.20 ± 0.04 on day 0 to 0.14 ± 0.02 on day 15). emulsification activity of bacteria figure 1 shows the emulsification index (e24) of the bacterial isolates in waste engine oil. corynebacterium kutscheri had the highest emulsification index (52%). the lowest emulsification index was observed for staphylococcus aureus (8%). table 1: physicochemical properties of different soil samples contaminated with waste engine oil. values represent means of triplicate determinations ± sd. ma = mechanic workshop 1, mb = mechanic workshop 2, mc = mechanic workshop 3. thc = total hydrocarbon content. table 2: bacterial count of soil samples contaminated with waste engine oil. values represent means of triplicate determinations ± sd. ma = mechanic workshop 1, mb= mechanic workshop 2, mc= mechanic workshop 3. table 3 : biochemical characteristics of spore (sp), catalase (ca), motility (mo), oxidase (ox), litmus reaction (lr), urease (ur), gelatin (ge), citrate (ci), glucose (gl), maltose (mal), mannitol (man), lactose (la), sucrose (su), arabinose (ar) and xylose (xy) for studied bacterial isolates from soil contaminated with waste engine oil. keys: ag = acid and gas production; a = acid production only; + = positive reaction; = negative. table 4: phenotypic characteristics of bacterial isolates from soil contaminated with waste engine oil. parameters unpolluted (control) polluted ma mb mc ph 6.79 ± 0.24 6.18 ± 0.13 5.78 ± 0.03 6.44 ± 0.07 nitrate (mg/g) 0.14 ± 0.02 0.25 ± 0.03 0.23 ± 0.02 0.20 ± 0.02 phosphate (mg/g) 6.65 ± 1.72 6.92 ± 2.92 10.74 ± 0.88 8.49 ±1.9 moisture content (%) 32.25 ± 9.30 43.09 ± 0.65 45.13 ± 7.02 27.72 ± 9.09 organic carbon content (%) 3.7 ± 2.43 5.32 ± 2.65 9.79 ± 0.51 7.29 ± 3.09 thc (mg/kg) 39.97 ±13.49 2933.76 ± 404..27 3202.61± 675.07 3122.72±131.00 parameter unpolluted (control) polluted ma mb mc thb (cfu/g) 1.9 ± 0.02 x 107 4.4 ±1.90 x 107 6.0 ± 0.23 x 107 4.5 ± 0.03 x 107 hub (cfu/g) 6.5 ± 0.04x 106 3.2 ± 0.05 x 107 5.2 ± 0.25 x 10 7 1.9 ± 0.10 x 106 isolates isolate code sp ca mo ox lr ur ge ci gl mal man la su ar xy 1 ma3, mb1, mc4 + + a a a ag 2 ma2, mb2 + + + + + a a a a a 3 ma1 + a a ag ag 4 ma4 + + + a a a a 5 mb3, mc1, mc2, ma5 + + a 6 ma6 + + a a 7 mc3 + + + + + a a a a a a 8 mb4, mc5 + + + + + + a a a a a ag isolates isolate code gram reaction/cell shape probable bacteria 1 ma3, mb1, mc4 gram-positive rods corynebacterium kurtscheri 2 ma2, mb2 gram-negative rods pseudomonas aeruginosa 3 ma1 gram-positive cocci micrococcus agilis 4 ma4 gram-negative rods flavobacterium aquatile 5 mb3, mc1, mc2, ma5 gram-positive cocci staphylococcus aureus 6 ma6 gram-positive cocci micrococcus luteus 7 mc3 gram-negative rods serratia odorifera 8 mb4, mc5 gram-positive rods bacillus substilis http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 7 table 5 : ph of the medium during the growth of bacterial isolates in hydrocarbon medium. isolates days 0 5 10 15 micrococcus agilis 7.00 6.51 ± 0.01 6.38 ± 0.03 6.04 ± 0.05 pseudomonas aeruginosa 7.00 7.06 ± 0.07 7.78 ± 0.01 7.71 ± 0.06 corynebacterium kutscheri 7.00 6.51± 0.01 6.38 ± 0.00 5.83 ± 0.34 flavobacterium aquatile 7.00 7.31± 0.23 7.50 ± 0.16 7.17 ± 0.28 micrococcus luteus 7.00 6.13± 0.07 7.23 ± 0.03 7.37 ± 0.06 staphylococcus aureus 7.00 7.23± 0.03 7.31± 0.03 7.23 ± 0.07 serratia odorifera 7.00 7.09 ± 0.01 6.73 ± 0.04 6.42 ± 0.07 bacillus substilis 7.00 6.85 ± 0.13 6.78 ± 0.18 7.24 ± 0.02 data represent means of triplicate determinations ± sd. table 6: total viable count (tvc) of bacterial isolates during growth in hydrocarbon medium (x 108 cfu/ml). isolates days 0 5 10 15 micrococcus agilis 0 0.63 ± 0.03 2.46 ± 0.02 0.87 ± 0.06 pseudomonas aeruginosa 0 0.54 ± 0.02 2.13 ± 0.16 0.45 ± 0.02 corynebacterium kutscheri 0 1.94 ± 0.08 5.27 ± 0.07 3.18 ± 0.07 micrococcus luteus 0 0.95 ± 0.01 2.49 ± 0.03 1.43 ± 0.03 flavobacterium aquatile 0 1.94 ± 0.12 6.73 ± 0.45 3.13 ± 0.02 staphylococcus aureus 0 0.31± 0.11 0.44 ± 0.01 0.42 ± 0.02 serratia odorifera 0 0.54 ± 0.03 2.23 ± 0.25 0.48 ± 0.04 bacillus substilis 0 1.39 ± 0.41 4.08 ± 0.65 2.47 ± 0.07 data represent means of triplicate determinations ± sd. table 7: turbidity measurement of bacterial growth in hydrocarbon medium (od600). isolates hours 0 5 10 15 microccoccus agilis 0.97 ± 0.01 0.154 ± 0.01 0.209 ± 0.12 0.201± 0.03 pseudomonas aeruginosa 0.033 ± 0.001 0.035 ± 0.10 0.182 ± 0.10 0.097± 0.05 corynebacterium kutscheri 0.189 ± 0.04 0.252 ± 0.07 0.386 ± 0.04 0.301± 0.11 flavobacterium aquatile 0.085 ± 0.01 0.153 ± 0.08 0.196 ± 0.07 0.227± 0.05 micrococcus luteus 0.040 ± 0.02 0.083 ± 0.06 0.162 ± 0.03 0.183± 0.04 staphylococcus aureus 0.20 ± 0.04 0.053 ± 0.10 0.084 ± 0.05 0.014± 0.02 serratia odorifera 0.40 ± 0.02 0.172 ± 0.11 0.121 ± 0.001 0.096± 0.01 bacillus substilis 0.165 ± 0.05 0.204 ± 0.01 0.413 ± 0.05 0.341± 0.04 data represent means of triplicate determinations ± sd. figure 1 : emulsification activity (e24) of bacterial isolates. discussion this study on the assessment of hydrocarbon utilizing the potential of bacteria isolated from waste engine oil-polluted soil on hydrocarbons reveals the presence of hydrocarbon-utilizing bacteria in waste engine oil-polluted soil environment as well as the potential of one of these isolates corynebacterium kutscheri to degrade hydrocarbons under a variety of experimental conditions. the results of the physicochemical analysis of the soil samples, as presented in table 1 showed higher levels of properties in the polluted soil samples when compared with the unpolluted soil sample. the results support the results of chikere [24], and chikere and ekwuabu [25], which showed high physicochemical parameters in polluted soil samples compared to unpolluted samples determined and indicated previous exposure of polluted samples to hydrocarbon contamination with traces of other organic and inorganic contaminants. the high bacterial counts recorded in polluted soil samples, as presented in table 2, compared with the unpolluted control samples could be attributed to the myriad of nutrients, high organic matter concentration and other ecological factors that influence the survival of soil microorganisms that play important roles in the decomposition and recycling of nutrients [26]. luepromchai et al. [27] has reported an increase in the numbers of hydrocarbon degraders in soil in the presence of pahs, without any impact on the overall bacterial numbers. the difference between thb and hub counts was observed to be minimally insignificant, which suggests that most of the micro-organisms present in various polluted sample sites are http://bioscience.highlightsin.org/ james et al., 2020 characterization of hydrocarbon utilizing bacteria in waste engine oil-impacted sites highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 7 hydrocarbon degraders that can withstand the concentrations of hydrocarbons and use them as a source of carbon [25]. the investigation of the morphology and type of bacterial colonies obtained from the oil-polluted soil showed seven bacterial species, which are corynebacterium kutscheri, pseudomonas aeruginosa, micrococcus agilis, flavobacterium aquatile, staphylococcus aureus, micrococcus luteus, serratia odorifera, and bacillus substilis. this shows the majority of bacteria isolated as being gram-positive species. some researchers have, however, reported oil-polluted soils as being dominated by gram-negative bacteria [25] the results of the emulsification test as shown in figure 1 demonstrates that the isolates produced emulsifying compounds. a large variety and number of biosurfactant producers have been isolated from hydrocarbon-impacted sites [28], although they have also been identified from soils, which are unconnected to hydrocarbon contamination [29]. corynebacterium kutscheri showed the highest emulsification (52%) at 1% waste engine oil, while the least was staphylococcus aureus (8%). corynebacterium sp has also been reported by onuoha et al. [22] with high emulsification ability among three hydrocarbon-degrading bacterial species isolated from soil. decrease in the ph of the medium (as shown on table 5), increase in total viable counts (table 6), as well as in turbidity of the medium (table 7) were regarded as indicators of degradation. the correlation between the ph of the medium and cell growth with hydrocarbon utilization has been previously reported by patila et al., [30]. the preliminary estimation of the isolate’s degradative ability during growth in hydrocarbon supplemented medium suggested the effectiveness of growth in hydrocarbon medium by corynebacterium kutscheri, and thus its potential as a candidate for further biodegradation studies. conclusion the result of this study indicates that the contaminated environments possess indigenous populations within them, as seen in bacterial count results of microbiological growth of mineral salt cultures with waste engine oil as the sole carbon source. these indigenous bacteria isolated in this study show promising, ability to degrade petroleum hydrocarbons with corynebacterium kutscheri as a potential candidate for further biodegradation efficiency studies. acknowledgments the authors are grateful to the laboratory technologist of microbiology laboratory, michael okpara university, umudike, nigeria for their support. references 1.atlas rm. oil spills: regulation and biotechnologyogy: guest editorial. current opinion in biotechnology. 1995;3(3):220-223. 2.bragg jr, prince rc, harner ej, atlas rm. effectiveness of bioremediation for the exxon valdez oil spill. nature. 1994 mar;368(6470):413-8. 3.uzoije ap, agunwamba jc. physiochemical properties of soil in relation to varying rates of crude oil pollution. international journal of environmental science and technology. 2011;4(3):313-23. 4.benka-coker mo, ekundayo, ja. effects of exposure of arotherodon niloticus and pariocephalus sp. to waste drilling fluid. journal of science resource development. 1995;23(4):122-125. 5.ogundiran oo, afolabi ta. assessment of the physicochemical parameters and heavy metals toxicity of leachates from municipal solid waste open dumpsite. international journal of environmental science and technology. 2008 mar 1;5(2):243-50. 6.bossert i, bartha r. the fate of petroleum in soil ecosystem. in: petroleum microbiology. atlas, r. m. (ed). new york:macmillian;1984.p. 102-110. 7.leahy jg, colwell rr. microbial degradation of hydrocarbons in the environment. microbiology and molecular biology reviews. 1990 sep 1;54(3):305-15. 8.saadoun i, mohammad mj, hameed km, shawaqfah m. microbial populations of crude oil spill polluted soils at the jordan-iraq desert (the badia region). brazilian journal of microbiology. 2008;39:453-456. 9.prince r. the microbiology of marine oil spill bioremediation. in: oliver b, magot m, editors. petroleum microbiology, washington: asm press; 2005.p.153-164. 10.bamforth, sm, singleton i. bioremediation of polycyclic aromatic hydrocarbons: current knowledge and future directions. journal of chemistry, technology and biotechnology. 2005;80:723–736. 11.vidali m. bioremediation: an overview. pure and applied chemistry. 2001;37(73):1163-1172. 12.malatova k. isolation and characterization of hydrocarbon degrading bacteria from environmental habitats in western new york stat. department of chemistry 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potentials of hydrocarbon degraders from waste lubricating oil-spilled soils in ebonyi state, nigeria. international journal of agriculture and biology. 2011;13: 586–590. 23.ganesh a, lin j. diesel degradation and biosurfactant production by gram-positive isolates. african journal biotechnology. 2009;8(21):5847-5854. 24.chikere cb. culture-independent analysis of bacterial community composition during bioremediation of crude oil-polluted soil. british microbiological resources journal. 2012;2(3):187-211. 25.chikere cb, ekwuabu cb. culture-dependent characterization of hydrocarbon utilizing bacteria in selected crude oil-impacted sites in bodo, ogoni land, nigeria. african journal of environmental science and technology. 2014;8(6): 401-406. 26.eze vc, okpokwasili gc. microbial and other related changes in niger delta river sediment receiving industrial effluents. continental journal of microbiology. 2010;4:15-24. 27.luepromchai e, lertthamrongsak w, pinphanichakarn p, thaniyavarn s, pattaragulwanit k, juntongjin k. biodegradation of pahs in petroleum-contaminated soil using tamarind leaves as microbial inoculums. biodegradation. 2007 mar;29(2):516. 28.banat im, makkar rs, cameotra ss. potential commercial applications of microbial surfactants. applied microbiology and biotechnology. 2000;53(5): 495-508. 29.jennings em, tanner rs. biosurfactant-producing bacteria found in contaminated and uncontaminated soils. in: proceedings of the conference on hazardous waste research, oklahoma; 2000.299 p. 30.patila td, pawara s, kamblea pn, thakare sb. bioremediation of complex hydrocarbons using microbial consortium isolated from diesel oil polluted soil. der chemica sinica. 2012;3(4):953-958. 31.kaplan cw, kitts cl. bacterial succession in a petroleum land treatment unit. applied environmental microbiology. 2004;70: 1777–1786. http://bioscience.highlightsin.org/ highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202109 research article open access 1 department of microbiology, faculty of veterinary medicine, adnan menderes university, 09016, isikli, aydin, turkey. 2 department of microbiology, institute of health sciences, adnan menderes university, 09016, efeler, aydin, turkey. contacts of authors * to whom correspondence should be addressed: uparin@adu.edu.tr received: march 11, 2021 accepted: june 15, 2021 published: october 10, 2021 citation: kirkan ş, parin u, çapakçioglu h . identification of pathogen bacteria from camel (camelus dromedarius) mastitis and investigation of antibiotic susceptibility. 2021 oct 10;4:bs202109 copyright: © 2021 kirkan et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this research was funded by aydın adnan menderes university scientific committee by grant number vtf-18017. competing interests: the authors declare that they have no competing interests. identification of pathogen bacteria from camel (camelus dromedarius) mastitis and investigation of antibiotic susceptibility şükrü kirkan1 ><, uğur parin*1 ><, halil çapakçioglu2 >< abstract the scope of this study was to investigate the presence of pathogenic bacteria in milk from female camels with mastitis and to select antibiotics for treatment with antibiotic susceptibility testing. a total of 40 milk samples taken from 20 dromedarian females, after application of cmt test and determination of scc values, the camels were diagnosed with subclinical mastitis. milk samples were inoculated into blood agar for identification of bacterial agents leading to mastitis. a total of 4 (12.5%) staphylococcus aureus, 4 (12.5%) s. auricularis, 2 (6.25%) s. pettenkoperi, 2 (6.25%) s. cohnii spp. cohnii, 2 (6.25%) s. equorum, 2 (6.25%) s. capitis, 2 (6.25%) streptococcus agalactiae, 2 (6.25%) s. dysgalactiae, 4 (12.5%) escherichia coli, 2 (%) 6.25) pseudomonas pseudalcaligenes, 2 (6.25%) corynebacterium pseudotuberculosis, 2 (6.25%) aerococcus viridans and 2 (6.25%) gemella morbillorum were identified. gram-positive bacteria were sensitive to levofloxacin, linezolid and tetracycline and daptomycin, resistant to beta lactam-group antibiotics and macrolides. vancomycin resistance was determined in s. aureus and s. cohnii spp. cohnii strains. gram-negative strains are found generally susceptible to cefepime and pipersilin; resistant to trimethoprim-sulfomethoxazole and amoxicillin-clavulanic acid. as a result, it is recommended to use antibiotic use to prevent the development of antimicrobial resistance as well as mastitis control methods such as the prevention of infection and monitoring the health status of the mammary of camels. keywords: camel, subclinical mastitis, identification, antimicrobial susceptibility. introduction camel milk, meat and products have appeared in many cuisines around the world for centuries. camel milk has beneficial effects on many biological processes such as digestion, absorption, growth and immunity [1]. additionally, camel milk can be stored at room temperature longer than milk from other animals [2]. camel whey protein contains a heterogeneous group of proteins, including serum albumin, α-lactoalbumin, immunoglobulin, lactoferrin, and peptidoglycan recognition protein [3]. compared to milk, camel milk contains more antibacterial substances and has a high concentration of vitamin c, so it has valuable nutritional properties. milk is characterized by a high percentage of lactoferrin, which can be considered a good source of minerals and vitamins. also, because camel milk has the most important nutrients, camel milk can meet most of human’s daily needs in these nutrients. camel milk has a similar composition to human milk due to its protective proteins such as low cholesterol, low sugar, high mineral content (sodium, potassium, iron, copper, zinc and magnesium), vitamin c, lactoferrin, lactoperoxidase, immunoglobulins and lysozyme [4]. the present studies confirmed that camel milk is unique in terms of antioxidant, antibacterial, antiviral, antifungal, anti-hepatitis, tuberculosis, hypoglycemic, anti-cancer, anti-tumor, anti-aging, auto-immune disease, cosmetic and detergents [5-6]. mastitis is a major global complex disease among dairy livestock with high economic losses. camel mastitis is estimated to affect more than 25% of lactating camels. it is also known to cause about 70% loss from milk production. mastitis has both extreme animal infections and economic significance. it is responsible for some harmful effects for human health and animal production. there is little published information about the pathogens associated with camel mastitis compared to bovine mastitis. highlights in bioscience page 1 of 7 october 2021|volume 4 https://doi.org/10.36462/h.biosci.202109 https://creativecommons.org/licenses/by/4.0/ skirkan@adu.edu.tr https://orcid.org/0000-0001-5111-8656 uparin@adu.edu.tr https://orcid.org/0000-0002-0788-5708 halilcapakcioglu@hotmail.com http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis however, much detailed research still remains to be accomplished, especially in the mastery of breeding techniques, since these species is confronted with particular health issues, including mastitis, which can be significant. there has long been a lack of concern in camel mastitis on the grounds that clinical mastitis in this species is infrequent. bacterial infection can be a major cause of mastitis in livestock. several bacterial agents are associated with mastitis cases and their presence in milk can have a negative impact on consumer health [8-9]. some research shows the genus staphylococcus sp., streptococcus sp., [10,11], micrococcus sp., streptococcus agalactiae, coagulase negative staphylococci [12], staphylococcus epidermidis, mannheimia haemolytica, escherichia coli and corynebacterium sp. were isolated from camel mastitis cases. camel has been linked to the cause of mastitis. mammary infections (mastitis) are the leading risks of herd management in camel breeding as well as in cattle breeding. the scope of this study was to investigate the presence of pathogenic bacteria in milk from female camels with mastitis and to select antibiotics for treatment with antibiotic susceptibility testing. materials and methods sample collection milk samples were taken from 20 female one-humped camels suspected of having mastitis during lactation. the study was carried out from august 2018 to april 2019 at private camel farms in aydin province, turkey. in this study, 40 camel milk samples were obtained from the right and left lobes of 20 dromedary camels at farms with a capacity of 5 to 20 camels. animal material consists of female camels aged 4 to 10 years who are lactating without antibiotic treatment. mastitis was diagnosed by applying cmt (california mastitis test delaval®, sweden) and determining somatic cell counts. nucleocounter™ scc100 (chemometec®, denmark) was used to determine the scc value. the identification of bacterial isolates was determined by an automatic identification system phoenix™ m50 (becton, dickinson u.k. limited). ethics of animal use the authors of this research hereby declare that collection of specimens was carried out in accordance with the guidelines laid down by the us national guide for the care and use of laboratory animals, national academy press, washington, d.c. 2011 and with the european communities council directive of 24 november 1986 (86/609/eec). california mastitis test somatic cell number score in milk samples was determined qualitatively. detergent containing bromocresol purple was used as the reagent to release the nucleic acid and rupture the somatic cell membrane, and it was observed whether a gel-like matrix was formed whose viscosity was proportional to the leukocyte number. plastic shovels with four glasses were used. equal amounts (3 ml) of milk and reagent were put into each glass of the paddle and the contents were mixed in gentle circular motion in a horizontal plane. astitis cases were determined according to the degree of agglutination. cmt is a highly sensitive and fast method to detect abnormally cell-rich milk. the principle consists of a mixture of milk and tea pools (detergent) in amounts intended to rupture the cells, so that the nuclear dna of the cells gels when they come into contact with the latter [13]. determination of somatic cell count somatic cell count of the samples was determined via automatic device. the device is a counter based on the optical fluorescence principle. ethidium bromide enters the structure of nuclear dna, producing a fluorescent signal corresponding to shs in milk. it is advantageous because it is an automatic and fast technique. the milk sample was brought to room temperature (21 ± 1°c) and homogenized. then, 0.75 ml of reagan c at room temperature was added to the clean 1.5 ml ependorfs and 0.75 ml of the milk sample was added. the ependorfs prepared for analysis were homogenized by vortexing. for somatic cell counting, a pre-prepared eppendorf sample at room temperature was drawn into the cassette and loaded into the instrument. after loading, the result is automatically saved by the device within 2 minutes. bacterial isolation and identification samples were brought to the laboratory by a cold chain and collected with sterile gauze from milk-filled tubes that were streaked in blood agar. the agar plates were incubated at 37 °c for 24-48 h under aerobic conditions. gram staining is performed on colonies that have grown as a result of incubation. grampositive and gram-negative samples were inoculated onto tryptic soy agar. tsa petri dishes were incubated for 24 h at 37 °c. biochemical assays of pure colonies formed in tsa after incubation were verified using the bd phoneix™ m50 device. this method is used for rapid identification and susceptibility testing of many aerobic and facultative anaerobic gram positive and negative bacteria containing selected antimicrobial agents. 24hour fresh cultures purified on triptic soy agar were suspended with id broth in glass tubes according to mcfarland 0.5 colony density. bd phoneix ™ pmic / id87 was used for gram positive bacterial isolates and bd phoneix™ nmic / id-400 panel kit was used for gram negative bacterial isolates. identification was performed on the instrument using separate panels for each sample. id broth suspension tubes prepared for each sample were placed in the device for bacterial diagnosis. biochemical identification data obtained from the device were evaluated. determination of antibiotic susceptibility antibiotic susceptibility tests were performed for the isolates that were identified with the automated device using the bd phoneix™ pmic / id87 and nmic / id-400 kit. 24 h fresh cultures purified on triptic soy agar were prepared with ast broth present in glass tubes according to mcfarland 0.5 colony density. panels containing bacterial suspensions were placed in the highlights in bioscience page 2 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis device and mic detection and sensitivity results were obtained from the electronic system. the antimicrobials and their mic values of bacterial isolates (n=32) were determined according to clsi standards [14]. results california mastitis test and somatic cell count numbers a plastic shovel with four glasses was used. an equal amount (3 ml) of milk and reagent was placed in each glass of the shovel and the contents mixed in a horizontal plane with a slight circular motion. according to the degree of agglutination, the cmt score was determined as "negative" (-), suspicious "+/-", mild (+), significant (++) and severe (+++) [9]. the cmt score and somatic cell numbers of 40 milk samples determined by the nucleocounter™ scc-100 (chemometec®, denmark) device are shown in table 1. table 1. cmt score and somatic cell counts of milk samples. milk samples (n=40) milk samples cmt scores somatic cell count standards 8 negative (-) 100 16 suspicious (+/-) 100-300 14 mild (+) 300-900 2 severe (++) 900-2700 identification bacterial growth was not observed in 8 samples taken from 4 (20%) female camels. bacterial growth was detected in 16 (80%) female camel milk samples (n = 32 samples). as a result of identification, 4 (12.5%) staphylococcus aureus, 4 (12.5%) staphylococcus auricularis, 2 (6.25%) staphylococcus pettenkoferi, 2 (6.25%) staphylococcus cohnii spp. cohnii, 2 (6.25%) staphylococcus equorum, 2 (6.25%) staphylococcus capitis, 2 (6.25%) streptococcus agalactiae, 2 (6.25%) streptococcus dysgalactiae, 4 (12.5%) escherichia coli, 2 (%) 6.25) pseudomonas pseudalcaligenes, 2 (6.25%) corynebacterium pseudotuberculosis, 2 (6.25%) aerococcus viridans and 2 (6.25%) gemella morbillorum were identified (table 2). antibiotic susceptibility the evaluation of mic values indicating the antibiotic susceptibility of the strains identified in our study (n = 32) showed that s. aureus (n=4) strains were 100% resistant to clindamycin and trimethoprim-sulfomethoxazole, and 75% resistant to erythromycin, penicillin g and vancomycin. s. auricularis (n=4) strains were resistant to daptomycin, fusidic acid at a rate of 100%, and against quinupristine dalfopristin and trimethoprimsulfomethoxazole at a rate of 75%. s. pettenkoferi (n=2) strains were 100% resistant to oxacillin and penicillin g. s. cohniispp. cohnii (n=2) strains were 100% resistant to ampicillin, clindamycin, fosfomycin, oxacillin, penicillin g and vancomycin.s. equorum (n=2) strains were 100% resistant to ampicillin, fusidic acid and penicillin g. s. capitis (n=2) strains were examined, it was found that the strains were 100% resistant to vancomycin, amoxicillin-clavulanic acid and ampicillin.s. agalactiae (n=2) strains were 100% resistant to ciprofloxacin, erythromycin, fusidic acid, gentamicin, penicillin g and teicoplanin. s. dysgalactiae (n=2) strains were 100% resistant to ampicillin, cefoxacin, ciprofloxacin, daptomycin, erythromycin, fusidic acid, gentamicin, nitrofurantoin, penicillin g, quinupristine dalfopristin, rifampin and teicoplanin. c. pseudotuberculosis (n=2) strains were 100% resistant to ampicillin, ciprofloxacin, fusidic acid, nitrofurantoin and penicillin g. a. viridans (n=2) strains were 100% resistant to ciprofloxacin, erythromycin and gentamicin. g. morbillorum (n=2) strains were 100% resistant to ciprofloxacin, erythromycin and gentamicin. e. coli (n=4) strains were 100% resistant to cefotaxime, ciprofloxacin, gentamicin, meropenem, netilmicin, tigecycline and trimethoprimsulfomethoxazole. p. pseudalcaligenes (n=2) strains were 100% resistant to amoxicillin-clavulanic acid, aztreonam, cefuroxime and trimethoprim-sulfomethoxazole (table 3) discussion subclinical camel mastitis has not been extensively studied and reported in prevalence studies. subclinical mastitis, where macroscopic clinical symptoms cannot be seen and indirect tools are needed for diagnosis, remains a significant public health problem for camel populations and consumers. consuming camel milk has effects on animal health and reduces the quantity and quality of milk. in studies on the relationship between the california mastitis test (cmt) and the number of bacteria in the camel milk test, it was shown that a high proportion of subclinical mastitis was cmt positive and that there was a significant difference between positive / negative cmt cases [15-17]. in another study conducted in sudan, cmt, somatic cell count (scc) and bacterial isolates were compared and showed that the mean values of cmt and scc were higher in samples taken from an infected mammary gland [18]. in addition, 47.3% of 336 milk samples tested positive in cmt and somatic cell count of 757 samples were reported to have a count range of 5 x 105 to 7.5 x 106 cells / ml [19]. cmt has a sensitivity of about 70% and a specificity of 91% in camel mastitis [20]. additionally, in another study, it was suggested that detection of scc in camels was more sensitive in detecting subclinical mastitis in camels than the n-acetyl beta-dglucosaminidase test [21]. the prevalence of subclinical mastitis in camels in riyadh, saudi arabia was 33% in milk samples examined according to cmt [15]. in other studies, the overall prevalence of mastitis was 44.8% and the prevalence of subclinical mastitis was 46% [16,17]. in this study, the cmt test and digital ccs were used to investigate subclinical mastitis in female camels. milk samples were obtained from 20 camels for the diagnosis of subclinical mastitis and the cmt score was negative, contaminated with +1, +2 and +3. the mean ccs obtained from the assessed healthy camel was 100,000 cells/ml. in our study, no bacterial growth was found in animals with sohighlights in bioscience page 3 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis table 2. the distribution of bacteria isolated from samples. milk samples with bacterial growth bacrterial identification right lobe left lobe sample 1 staphylococcus aureus staphylococcus aureus sample 2 staphylococcus aureus escherichia coli sample 3 staphylococcus auricularis staphylococcus auricularis sample 4 staphylococcus aureus staphylococcus auricularis sample 5 escherichia coli staphylococcus auricularis sample 6 staphylococcus pettenkoferi staphylococcus pettenkoferi sample 7 staphylococcus cohnii spp. cohnii staphylococcus cohnii spp. cohnii sample 8 staphylococcus equorum staphylococcus equorum sample 9 staphylococcus capitis staphylococcus capitis sample 10 streptococcus agalactiae streptococcus agalactiae sample 11 streptococcus dysgalactiae streptococcus dysgalactiae sample 12 escherichia coli escherichia coli sample 13 pseudomonas pseudalcaligenes pseudomonas pseudalcaligenes sample 14 corynebacterium pseudotuberculosis corynebacterium pseudotuberculosis sample 15 aerococcus viridans aerococcus viridans sample 16 gemella morbillorum gemella morbillorum matic cell counts less than 100,000 cells/ml. in our study, the prevalence of subclinical mastitis was determined to be 80% in milk samples randomly selected from healthy-looking camels. however, previous studies have shown that cmt has a sensitivity of 70% and specificity of 91% in camel mastitis and there is a positive correlation between scc/cmt scores [20-22]. because early and effective treatment of mastitis is very important, accurate diagnosis of subclinical mastitis is always a priority. the technique in which the intranuclear dye penetrates into the dna of the cell and the stained cells are detected by spectrophotometer has been shown to be applicable in the diagnosis of subclinical mastitis in camels. this study was confirmed by isolating and identifying the causative agent from milk samples from animals evaluated for subclinical mastitis by determining cmt and scc. epidemiological data collected in recent years show that the prevalence of clinical mastitis in camels is between 31-38% in europe and 31% in uruguay. while the prevalence of general mastitis in camels was 18.52%, subclinical mastitis was more common (24.7%) than clinical mastitis (11.67%). the major pathogens isolated were staphylococcus sp. (41.67%) followed by streptococcus sp. (21.67%), enterobacter sp. (15.00%), corynebacterium pyogenes (10.00%), micrococcus sp. (5.00%), pasteurella sp. (5.00%) and pseudomonas sp. 1.66%. in north kordofan, sudan, in 2013, the incidence rate was found to be 25% subclinical mastitis, 13.3% and 15% using other techniques such as scc and white side test. the isolated pathogens were staphylococcus sp. (80.30%), bacillus sp. (9.09%), pasteurella sp. (6.06%), corynebacterium sp. (3.03%) and streptococcus sp. (1.52%) [23]. staphylococcus sp. as a result of the study conducted on 90 female camels in jordan to identify and generate data on camel mastitis and pathogens, 21% of the camels had clinical symptoms of mastitis and micrococcus sp., staphylococcus aureus, streptococcus sp. and corynebacterium sp. identified. the isolates were susceptible to antibiotics such as gentamicin, ampicillin and tetracycline. the study concluded that gram-positive cocci are predominantly the result of mastitis [24]. the data obtained are in parallel with the findings of our research. in this study, 81% of gram-positive bacteria were isolated. staphylococcus sp., streptococcus sp. and micrococcus sp. isolated camels have been reported to be the dominant pathogen of these species [25,26]. the incidence and causes of mastitis in camels differ significantly due to geographical region and individual herd management [17]. the prevalence of camel mastitis in sudan was 30.2%, of which 4.9% were clinically and 25.3% subclinically in the jigjiga region. there was a high rate of chronic form (72.41%) followed by acute form (24.14%), and three types of clinical mastitis were diagnosed, the least seen gangrenous form (3.45%). clinical mastitis occurred in animals older than 10 years of age and late in lactation (55%). in our study, subclinical mastitis cases were detected in animals younger than 10 years of age. the dominant isolated microorganisms are staphylococcus sp. (37.8%), e. coli (18.9%), streptococcus sp. (13.5%), bacillus sp. (10.8%), micrococcus sp. (8.1%), corynebacterium sp. (5.4%) and salmonella sp. (5.4%) [27]. of the mastitis (76.0%) evaluated in eastern ethiopia and observed in camel herds, staphylococcus aureus (4.2%), coagulase negative staphylococci (39.6%), streptococcus agalactiae (3.5%), streptococcus dysagalactiae (22.2%), corynebacterium sp. (9%), bacillus sp. (7.6%), streptococcus uberis (7.6%), escherichia coli (6.37%) were identified [28]. similar to these findings, staphylococcus sp., streptococcus sp., e. coli, corynebacterium pseudotuberculosis and pseudomonas pseudalcaligenes species were identified in addition to a. viridans and gemella morbillorum. highlights in bioscience page 4 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis table 3. antibacterial susceptibility results of identified bacterial strains according to mic values. amc: amoxicillin-clavulanic acid, am: ampicillin, fox: cefoxitin, cip: ciprofloxacin, cc: clindamycin, dap: daptomycin, e: erythromycin, ff: fosfomycin, fa: fusidic acid, gm: gentamycin, lvx: levofloxacin, lzd:linezolid, fm:nitrofurantoin, ox: oxacillin, p: penicillin g, syn: quinupristin-dalfopristin, ra: rifampin, tec: teicoplanin, te: tetracycline, nn: tobramycin, sxt: trimethoprim-sulfomethoxazole, va:vancomycin, an:amikacin, atm: aztreonam, fep: cefepime, caz: ceftazidime, cro: ceftriaxone, cxm: cefuroxime, cl:colistin, etp:ertapenem, ipm:imipenem, mem: meropenem, net:netilmicin, pip:piperacillin, tzp: piperacillin-tazobactam, tgc:tigecycline. amc am fox cip cc dap e ff fa gm lvx lzd s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r staph. aureus (n=4) 2 1 1 1 1 2 2 2 1 2 1 4 4 1 3 1 3 2 2 2 2 3 1 4 staph. auricularis (n=4) 3 1 4 4 3 1 3 1 4 3 1 4 4 4 3 1 2 2 staph. pettenkoferi (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 staph. cohnii spp. cohnii (n=2) 2 2 1 1 2 2 2 2 2 2 2 2 2 staph. equorum (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 staph. capitis (n=2) 2 2 2 2 2 2 2 2 2 2 2 str. agalactiae (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 str. dysgalactiae (n=2) 1 1 2 2 2 2 2 2 2 2 2 2 2 c. pseudotuberculosis (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 a. viridans (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 g. morbillorum (n=2) 2 2 2 2 2 2 2 2 2 2 an amc am atm fep caz cro cxm cip cl etp gm s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r e. coli (n=4) 3 1 1 3 4 4 4 4 4 4 4 2 2 3 1 4 p. pseudalcaligenes (n=2) 2 2 2 2 2 2 2 2 2 2 2 2 fm ox p syn ra tec te tgc nn sxt va s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r s i r staph. aureus (n=4) 4 2 2 1 3 3 1 4 3 1 4 4 4 4 1 3 staph. auricularis (n=4) 3 1 2 1 1 3 1 1 3 3 1 4 4 4 4 1 3 4 staph. pettenkoferi (n=2) 2 2 2 1 1 2 2 2 2 2 2 2 staph. cohnii spp. cohnii (n=2) 2 2 2 2 2 2 2 2 2 2 2 staph. equorum (n=2) 2 1 1 2 2 1 1 2 2 2 2 2 2 staph. capitis (n=2) 2 2 2 2 2 2 2 2 2 2 2 str. agalactiae (n=2) 2 2 2 2 2 2 2 str. dysgalactiae (n=2) 2 2 2 2 2 2 2 c. pseudotuberculosis (n=2) 2 2 2 2 2 2 2 a. viridans (n=2) 2 2 2 2 2 2 2 g. morbillorum (n=2) 2 2 2 2 2 2 ipm mem net pip tzp tgc sxt s i r s i r s i r s i r s i r s i r s i r e. coli (n=4) 1 3 4 4 4 4 4 4 p. pseudalcaligenes (n=2) 2 2 2 2 2 2 2 besides, 23.8% of gram-negative bacteria species have been reported in previous studies [29]. in our study, gram negative species were determined as pseudomonas pseudalcaligenes, especially e. coli as a cause of circumferential mastitis. in our study, 12.5% of staphylococcus aureus were isolated. s. auricularis, s. pettenkoferi, s. cohnii spp. cohnii, s. equorum, s. capitis were other staphylococcal species isolated in this research. our result (12.5%) was found to be lower than the results reported before [30] (44.82%). in the same study, e. coli (18.92%) was the second most isolated bacterium. in our study, e. coli was identified at a rate of 12.5%. previous antimicrobial susceptibility tests showed high susceptibility to commonly used antibiotics against bacteria. the isolates were found to be highly sensitive to gentamicin , cipr -ofloxacin, cloxacillin and amikacin, moderately sensitive to ampicillin / sulbactam and trimoxazole, and highly resistant to tetracycline and chloramphenicol [16, 29, 30, 31, 32]. in the study conducted by abdelgadir [25], oxytetracycline, tetracycline and chloramphenicol were determined as effective antibiotics against mastitis pathogens in camels. according to the antibiotic susceptibility findings obtained in our study, staphylococcus species were found to be susceptible to daptomycin, tetracycline, levofloxacin, tigecycline and tetracycline antibiotics and trimetoprim-sulfometoxazole and penicillin g resistant. as an important result of antibiotic susceptibility findings, vancomycin resistance in s. aureus and staph. cohnii spp. highlights in bioscience page 5 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis cohnii strains is remarkable. streptococcus species identified in our study were found to be susceptible to clindamycin, levofloxacin, linezolid and tetracycline and resistant to penicillin g, gentamicin, erythromycin and ciprofloxacin. resistance to beta lactam group and macrolides was determined. other grampositive strains identified as c. pseudotuberculosis, a. viridans and g. morbillorum were also susceptible to amoxicillin-clavu -lanic acid, levofloxacin and daptomycin, and resistant to ampicillin and penicillin g. antimicrobial susceptibility of e. coli strains was found to be susceptible to aztreonam, cefepime and piperacillin, and resistant to gentamycin, meropenem, tigecycline, trimethoprime -sulfometoxazole and amoxicillin clavulanic acid; pseudomonas pseudalcaligenes strains were susceptible to cefepime, ciproflox -acin, piperacillin. gram-positive bacteria identified in our study were found to be susceptible to levofloxacin, linezolid, tetracycline, daptomycin, and resistant to beta lactam group antibiotics and macro -lides, vancomycin resistance was determined in s. aureus and s. cohnii spp. cohnii strains. gram negative strains were generally susceptible to cefepime and piperacillin; resistant to trimethoprime -sulphomethoxazole and amoxicillin-clavulanic acid. as a result, it has been shown that multiple antibiotic resistance develops in bacteria causing mastitis in camels. our study suggests that subclinical mastitis in camels is more common than other forms of mastitis and that infected animals may be a source of contamination as a microorganism reservoir; identification of both infectious and environmental mastitis pathogens. since the prevalence of mastitis in camels differs considerably depending on geographical area and individual herd management, it is recommended to use antibiotics to prevent the development of antimicrobial resistance as well as mastitis control methods; such as elimination of existing infection, prevention of new infection and monitoring the health status of the mammary. considering that camel milk is used raw for human consumption without heat treatment in order not to affect the nutritional and immunological factors it contains, the pathogens causing subclinical mastitis can easily threaten public health, and it is essential to inform the farmers and consumers about the related pathogens. acknowledgement this research was funded by aydın adnan menderes university scientific committee by grant number vtf-18017. references 1. korhonen h, pihlanto a. food-derived bioactive peptides opportunities for designing future foods. curr. pharm. des. 2001, 9: 1297-1308. 2. omar rh, eltinay ah. microbial quality of camel’s raw milk in central southern region of united arab emirates. emir. j. food agric. 2008, 20 (1): 76-83. 3. kappeler sr, heuberger c, farah z, puhan z. expression of the peptidoglycan recognition protein, pgrp, in the lactating mammary gland, j. dairy sci. 2004, 87: 2660-2668. 4. yadav ak, kumar r, priyadarshini l, singh j. composition and medicinal properties of camel milk: a review. asian j. dairy food res. 2015, 34(2): 8391. 5. al-juboori at, mohammed m, rashid j, kurian j, el-refaey s, brebbia ca, popov, v, editors. nutritional and medicinal value of camel (camelus dromedarius) milk. second international conference on food and environment: the quest for a sustainable future; 2013b; budapest, hungary. p. 221-232. 6. sharma c, chandan s. therapeutic value of camel milk–a review. adv. j. pharm. life sci. res. 2014, 2(3): 7-13. 7. simeneh k. characterization of camelus dromedarius in ethiopia: production systems, reproductive performances and infertility problems [dissertation]. 2015. 8. turk r, koledić m, maćešić n, benić m, dobranić v, ðuričić d, cvetnić l, samardžija m. the role of oxidative stress and inflammatory response in the pathogenesis of mastitis in dairy cows. mljekarstvo. 2017, 67: 91-101. 9. benić m, maćešić n, cvetnić l, habrun b, cvetnić ž, turk r, ðuričić d, lojkić m, dobranić v, valpotić h, grizelj j, gračner d, grbavac j, samardžija m. bovine mastitis: a persistent and evolving problem requiring novel approaches for its control a review. vet. arhiv. 2018, 88: 535-557. 10. al-juboori a, kamat n, sindhu j. prevalence of some mastitis causes in dromedary camels in abu dhabi, united arab emirates. iraqi j. vet. sci. 2013, 27: 9-14. 11. al-majali a, bani iz, al-hami y, nour a. lactoferrin concentration in milk from camels (camelus dromedarius) with and without subclinical mastitis. intern. j. appl. res. vet. med. 2007, 5(3): 120-124. 12. al-majali am, al-qudah km, al-tarazi yh, al-rawashdeh of. (2008) risk factors associated with camel brucellosis in jordan. trop. anim. health prod. 2008, 40(3): 193-200. 13. abdelgadir ae. mastitis in camels (camelus dromedarius): past and recent research in pastoral production system of both east africa and middle east. j. vet. med. anim. health 2014, 6(7): 208-216. 14. clsi national committee for clinical laboratory standards (m31a3). performance standards for antimicrobial susceptibility testing. vol. 28, no:8, informational supplement, pennsylvania wayne, 2018. 15. aljumaah rs, almutairi ff, ayadi m, alshaikh ma, aljumaah am, hussein mf. factors influencing the prevalence of subclinical mastitis in lactating dromedary camels in riyadh region, saudi arabia. trop. anim. health prod. 2011, 43(8): 1605– 1610. 16. ahmad s, yaqoob m, bilal mq, muhammad g, yang lg, khan mk, tariq m. risk factors associated with prevalence and major bacterial causes of mastitis in dromedary camels (camelus dromedarius) under different production systems. trop. anim. health prod. 2012, 44(1): 107-12. 17. regassa a, golicha g, tesfaye d, abunna f, megersa b. prevalence, risk factors, and major bacterial causes of camel mastitis in borana zone, oromia regional state, ethiopia. trop. anim. health pro. 2013, 45: 1589-1595. highlights in bioscience page 6 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ kirkan et al., 2021 identification of pathogen bacteria from camel (camelus dromedarius) mastitis 18. abdurahman oa, agab h, abbas b, astrom g. relations between udder infection and somatic cells in camels (camelus dromedarius) milk. acta vet. scand. 1995, 36: 423-431. 19. obeid ai, bagadi ho, mukhtar mm. mastitis in (camelus dromedarius) and the somatic cell content of camels’ milk. res. vet. sci. 1996, 61(1): 55–58. 20. younan m, ali z, bornstein s, muller w. application of the california mastitis test in intramammary streptococcus agalatiae and staphylococcus aureus infections of camels (camelus dromedarius) in kenya. prev. vet. med. 2001, 51: 307-316. 21. guliye ay, van creveld c, yagil r. detection of subclinical mastitis in dromedary camels (camelus dromedarius) using somatic cell counts and the n-acetyl-beta-d-glucosaminidase test. trop. anim. health pro. 2002, 34, 95–104. 22. nagy p, faye b, marko o, thomas s, wernery u, juhasz j. microbiological quality and somatic cell count in bulk milk of dromedary camels (camelus dromedarius): descriptive statistics, correlations, and factors of variation. j. dairy sci. 2013, 96(9): 5625-5640. 23. quinn pj, carter me, markey b, carter gr. clinical veterinary microbiology. wolf publishing, london; 1999. 24. al-majali am, al-qudah km, al-tarazi yh, al-rawashdeh of. risk factors associated with camel brucellosis in jordan. trop. anim. health pro. 2008, 40(3): 193-200. 25. abdelgadir ae. cross-sectional study of mastitis in camels (camelus dromedarius) in selected sites of ethiopia[dissertation]. freie universität berlin and addis ababa university; 2001. 26. alamin ma, alqurashi am, elsheikh as, yasin te. mastitis incidence and bacterial causative agents isolated from lactating she-camel (camelus dromedarius). j. agric. vet. 2013, 2(3): 7-10. 27. blood dc, radostits om. veterinary medicine: a textbook of disease of cattle, sheep, pigs, goats and horses. baillion tindall: london; 2007. 28. eyassu s, bekele t. prevalence and etiology of mastitis in traditionally managed camels (camelus dromedarius) in selected pastoral areas in eastern ethiopia. ethiop. vet. j. 2010, 14(2): 103-113. 29. al-tofaily, y.i., and alrodhan, m.a.n.: study on clinical mastitis (bacteriological) in she-camels (camelus dromedaries) in some areas of middle euphrates in iraq. qjvms. 2011, 10(2): 66-76. 30. al-juboori aa, kamat nk, sindhu ji. prevalence of some mastitis causes in dromedary camels in abu dhabi, united arab emirates. iraqi j. vet. sci. 2013a, 27(1): 9-14. 31. fazlani sa, khan sa, farazl s, awan ms. antimicrobial susceptibility of bacterial species indentified from mastitic milk samples of camel. afr. j. biotechnol. 2011, 10(15): 2959-2964. 32. alqurashi am, alamin ma, elsheikh as, yasin te. sensitivity of bacterial isolates from mastitic she-camel (camelus dromedaries) to antibiotics. am.j. sci. 2013, 9(4): 47-52. highlights in bioscience page 7 of 7 october 2021|volume 4 http://bioscience.highlightsin.org/ abstract introduction materials and methods sample collection ethics of animal use california mastitis test determination of somatic cell count bacterial isolation and identification determination of antibiotic susceptibility results california mastitis test and somatic cell count numbers identification antibiotic susceptibility discussion acknowledgement references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20217 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 8 research article open access 1 department of agrotechnology, faculty of agriculture, halu oleo university, jl. hea mokodompit, kampus bumi tridharma, anduonohu, kendari, southeast sulawesi, indonesia. 2 department of agronomy and horticulture, faculty of agriculture, bogor agricultural university, jl. meranti, darmaga campus 16680, indonesia. 3 department of soil science and land resources, faculty of agriculture, bogor agricultural university, jl. meranti, 16680 campus darmaga, bogor , indonesia. 4 center for research and development of biotechnology and genetic resources (bb-biogen), jl. tentara pelajar no. 3 a cimanggu, bogor, indonesia. contacts of authors * to whom correspondence should be addressed: la ode afa citation: afa l, purwoko bs, junaedi a, haridjaja o, dewi is (2020). growth and yield of new hybrid rice in irrigated and rain-fed lowlands. highlights in bioscience volume 3. article id 20217.dio:10.36462/h.biosci.20217 received: june 6, 2020 accepted: august 20, 2020 published: august 31, 2020 copyright: © 2020 afa et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. growth and yield of new hybrid rice in irrigated and rain-fed lowlands la ode afa1*, bambang s. purwoko2, a. junaedi2, oteng haridjaja3 and iswari s. dewi4 abstract two experiments were conducted at (i) irrigated lowland, village of bojong, cikembar, sukabumi sub-district, indonesia, from november 2011 to february 2012, and (ii) rain-fed lowland, village of sanca, gantar sub-district, indramayu, indonesia, from april to july 2012. the objective of the experiment was to determine the yield of drought-tolerant hybrid rice genotypes. a randomized block design with three replications was used. the treatment consisted of 8 hybrid rice genotypes (bi485a/bp3, bi485a/bp5, bi485a/bp10, bi485a/bp12, bi485a/bp15, bi599a/bp5, bi599a/bp15 and bi665a/bp6) and 2 check varieties (hipa 7 and ciherang) for the experiment in irrigated lowland, 4 hybrid genotypes (bi485a/bp3, bi485a/bp12, bi485a/bp15 and bi599a/bp15) and 3 check varieties (hipa 7, ir64 and limboto) for the experiment in rain-fed lowland. on irrigated lowland, all tested-genotypes produced grain yield per hectare, which was not significantly different from the check varieties ciherang and hipa 7. the yield of genotype bi485a/bp12, bi485a/bp15 and bi559a/bp15 under irrigated lowland showed non-significant different with check varieties ciherang and hipa 7, i.e 5.63, 6.87, and 6.30 tons ha-1, respectively. genotype bi599a/bp15 under severe drought rain-fed lowland yielded 0.90 tons of grain ha-1, whereas hipa 7 (hybrid variety, suitable for rain-fed lowland) and limboto (drought-tolerant check variety) reached 0.34 and 0.29 tons ha-1, respectively. genotype bi599a/bp15 is a new hybrid genotype that potential for advanced development in rain-fed lowland and in irrigated lowland with a high yield potential and quiet early-maturity age. keywords: yield, hybrid rice, irrigated lowland, rain-fed lowland. introduction hybrid rice is an alternative technology to increase rice productivity. research results in several tropical countries showed that hybrid rice had more yield advantages than inbred rice with more than one ton per hectare [1]. one component of the success of hybrid rice development is the availability of excellent varieties that have high heterosis and can increase yield potential by 15-20% [1,2]. experiments on the yield of hybrid rice from 1982 to 2011 showed more advantages than inbred rice in terms of productivity of dry grain and age [2,4,5]. the experiment is continuously conducted to improve rice production by assembling high-yield potential, earlier mature and drought tolerant hybrid rice for marginal land such as the rain-fed lowland. the new hybrid genotypes that potential to be developed in the rain-fed areas have been identified through a series of tests in a controlled environment. tests using peg 6000 and drought stress on nutrients culture in seedling phase and in pot and simulation in the field have successfully identified drought-tolerant hybrid rice genotypes, namely bi485a/bp12, bi485a/bp15, and bi559a/bp15 genotypes [6,7]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20217 https://doi.org/10.36462/h.biosci.20217 http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 the genotypes need to be tested on in-situ conditions (field test) to obtain hybrid rice genotypes with high yield traits, both at an optimum and sub-optimum growing environments. testing in rain-fed and irrigated areas is required to obtain good traits according to the conditions of environment where they grow, so that the selection can be performed on high yield potential genotypes, although testing is conducted on a small scale. testing on a small scale is expected to be able to provide information on preliminary yield of drought-tolerant hybrid rice genotypes and potentially be developed in the rain-fed lowland in a wider scale. the genotypes of rice should be able to adapt and steady at any location in various seasons, so that a genotype can be released as a superior cultivar [8-10]. the study aimed to determine the preliminary yield of new rice hybrid in irrigated lowland and the current drought in the rain-fed lowland. materials and methods the experiment growth and yield in irrigated lowland was conducted in november 2011 to february 2012 in irrigated lowland of bojong, district cikembar, sukabumi, west java, indonesia. the climate of the study site was moderate with the average temperature of 18-32 0c and precipitation ranged 1200-2200 mm year-1. the plant materials used were 8 genotypes/varieties of new hybrid rice i.e. bi485a/bp3, bi485a/bp5, bi485a/bp10, bi485a/b p12, bi485a/bp15, bi599a/bp5, bi599a/bp15, bi665a/ bp6, and two check varieties i.e. ciherang and hipa 7. the experiments used a randomized block design with three replications. plot size as the unit of experiments was 2.0 m x 1.5 m, the distance between the treatment plots was 50 cm, and between blocks was 100 cm. seedlings yielded from seedbed were transplanted after at least 21 days, the seedlings were planted with spacing of 25 cm x 25 cm, one seedling per hole. fertilizing used with 300 kg urea, 125 kg sp-36, and 100 kg kcl per hectare. all sp-36 and kcl fertilizers were given at the time of planting; urea was given three times at time of planting, 4 weeks and 7 weeks after planting. the control of pests and diseases was done optimally, while weeding was done manually using a push weeder, three and five weeks after planting. irrigation was adjusted to the character of the rice fields used. irrigation on irrigated lowland was maintained under optimum conditions during the growth phase until harvest. the height of water on the plots was adjusted to the growth phase of plant. the observation was objected to five clumps of sample plants. variables measured were plant height, number of productive shoots, panicle length, number of filled grains, total number of grains, the percentage of filled grain, 1000 grain weight, grain weight per hill, days to 50% flowering and 85% yellow harvesting and grain yield per plot. the experiment growth and yield in rain-fed lowland was conducted from april to july 2012 on rain-fed lowland of sanca village, subdistrict gantar, district indramayu, west java, indonesia. the climate in the study areas was hot climate with average temperature of 32-35 °c and annual rainfall ranged from 1000-2400 mm year-1. the plant materials used were 4 genotypes/varieties of new hybrid rice i.e. bi485a/bp3 (drought-sensitive category), bi485a/bp12, bi485a/bp15, and bi599a/bp15 (drought-tolerant category based on the results of previous studies), and 1 hybrid variety, namely hipa 7 (released-varieties suitable for rain-fed lowland) and two inbred varieties namely ir-64 (drought-sensitive check) and limboto (drought-tolerant check). the experiments used a randomized block design with three replications. the soil sample was taken to analyze its physical and chemical properties, namely the determination of the water content of the soil at field capacity (pf 2.54) and permanent wilting point (pf 4.20) based on the method of pressure plate/membrane apparatus [11]. the experimental plot size of each treatment unit was 3.00 m x 1.75 m, the distance between treatment plots was 50 cm, and inter-groups distance was 100 cm. seedlings yielded from seedbed were transplanted after at least 21 days, the seedlings were planted with spacing of 25 cm x 25 cm, one seedling per hole. the observation was directed to five clumps of sample plants. variables measured were plant height, flag leaf length, number of productive shoots, plant dry weight of the upper part (biomass), 50% flowering-age, panicle length, number of total grains, percentage of filled grains, grain weight per hill and grain yield per plot. data were analyzed using analysis of variance and further test duncan's multiple range test (dmrt) the 0.05 probability level using sas 9.1 software program. results and discussion rainfall and soil moisture conditions the average rainfall in the trial location of irrigated lowland (table 1) was considered to be sufficient for the rice water requirement of > 200 mm/month [12].thus, inadequacy of irrigation water can be supplied with the rainfall. at well-ripening phase, the amount of rainfall was < 200 mm and occurred only for eight days, which was sufficient condition to accelerate the maturation of seeds and harvest time. table 1: average rainfall during the growth of hybrid rice in irrigated lowland of bojong village, cikembar district, sukabumi, indonesia. notes: a = the board of meteorology, climatology and geophysics, geophysical observatory in pelabuhan ratu, sukabumi, indonesia; b = growing phase of rice varieties was 120 days [13]. growing phase b ch (mm) a rain frequency (days) availability planted-primordial (60 days) 492.3 44.0 sufficient floweringseeds filling (30 days) 485.6 26.0 sufficient seeds ripening 218.8 11.0 sufficient well-ripening 166.0 8.0 sufficient http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 during the trial in rain-fed lowland, the distribution and frequency of rainfall during the vegetative phase (planting until the primordial) were at insufficient level, only 94.7 mm and rain occurred only 7 days. flowering to grain filling phase was at a critical level of 0.6 mm and rain occurred only 1 day, whereas at well-ripening phase rain did not occur (table 2). the condition caused the plant experienced a severe drought stress, which occurred in 2 weeks after planting. result of recorded soil moisture is presented in figure 1. level of soil moisture in the field capacity was 38.6% and permanent wilting point was 26.9%. table 2: average rainfall during the growth of hybrid rice in the rain-fed lowland of sanca village, district gantar, indramayu, indonesia. notes: nr = no rain; a = direct measurement in the field , b = rice varieties growth phase for 120 days [13]. 0 5 10 15 20 25 30 35 40 45 28 33 36 39 43 53 56 58 61 67 73 79 106 after day plant s oi l m oi st ur e.. (% v ol um e) in depth ±10 cm in depth ±20 cm average figure 1: moisture of soil during the trail in rain-fed lowland rice of sanca village, district gantar, indramayu, indonesia, from april to july in 2012. growth and yield results of analysis of variance experiment growth and yield in irrigated lowland showed that variety treatment significantly influenced variables of plant height, 50% flowering age, panicle length, the total grain number, and filled grain, the percentage of empty grains, and 1,000 grain weight, while the variables of number of productive tillers, grain weight per hill and grain yield per hectare were not significantly influenced. results of dmrt test on average growth are presented in table 3 and the average of yield components and yield are presented in table 4. table 3 shows that most genotypes tested had plant height, which was not significantly different from check varieties of ciherang and hipa 7, except for bi599a/bp15, bi599a/bp5, and bi665a/bp6. genotype bi599a/bp15 was the highest, which was not significantly different from bi599a/bp5. plant height is significantly associated with lodging. irri assigned a standard for the height of new rice type with 100 cm [14]. ma et al. [15] confirmed that the ideal crop with lodging resistance ranges from 115 to 120 cm. therefore, this genotype is an ideal genotype to be developed in the rice field because the height character of the plant was within the ideal prototype standard of lodging resistance. table 3: growth components including plant height (ph), number of productive tillers (npt), flowering age at 50% (fa), and harvest age (ha) of hybrid rice genotypes and check varieties in irrigated lowland. notes: figures followed by the same letter in each of the variables were not significantly different by dmrt at α = 0.05. the number of productive tillers among genotypes and the check varieties showed no significant difference. the average number of productive tillers ranged from 12 to 15 (table 3). peng et al. [16] stated that the low capacity of tillers for transplanting were 8-10 productive tillers. furthermore, peng et al. [17] reported that the super hybrid rice in china had 200-250 productive tillers per m2 (12-15 tillers per hill at a distance of 25 cm x 25 cm). genotypes tested in this research had an average of productive tillers in that range. average flowering age among genotypes was not significantly different except for genotype bi665a/bp6 in which the flowering time was 50% longer, but the flowering time of this genotype was not significantly different from that of check varieties, ciherang and hipa 7. generally the tested-genotypes were faster in flowering, i.e. 85.0-87.3 days while flowering age of check varietiess ciherang and hipa7 were 92.3 and 90.3 days, respectively (table 3). age of flowering strongly relates to the efficiency of the utilization of water resources and nutrients because the phase of vegetative growth is shorter. the shorter age of flowering generally has good adaptability to drought by accelerating the maturation of grain [18]. the average harvest age of the tested-genotypes was significantly different from that of check varieties, except for genotype bi485a/bp3 and bi665a/bp6. the average harvest age of the tested-genotypes ranged from 115.0 to 121.0 days. in general, hybrid rice genotypes had earlier-maturing age of 120 days than checks varieties (table 3). differences in environmental factors cause different plant life cycle. yoshida [13] stated that the difference in maturity is mostly determined by the phase length of the vegetative growth because the length of the reproductive phase and ripening is not influenced by the environment. average of panicle length between genotypes and check varieties was not significantly different. panicle length of genotypes ranged from 23.82 to 26.47 cm, while the check growing phase b ch (mm) a rain frequency (days) availability planting-primordial (60 days) 94.7 7.0 insufficient flowering-seeds filling (30 days) 0.6 1.0 severe seeds ripening nr nr severe well-ripening nr nr severe genotype ph (cm) npt fa (days) ha (days) bi485a/bp3 98.60 d 13.3 85.3 de 119.7 a bi485a/bp5 101.80 cd 13.1 85.0 de 117.7 b bi485a/bp10 101.33 d 13.0 86.3 cd 115.3 cd bi485a/bp12 100.40 d 12.1 85.7 de 116.7 bc bi485a/bp15 103.60 cd 14.8 84.7 e 115.0 d bi599a/bp5 110.33 ab 12.9 87.3 c 118.0 b bi599a/bp15 114.33 a 13.1 86.3 cd 117.7 b bi665a/bp6 107.27 bc 11.9 91.7 ab 121.0 a ciherang 100.53 d 13.7 92.3 a 120.0 a hipa 7 100.87 d 12.5 90.3 b 120.3 a http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 variety ciherang was 23.7 cm and hipa 7 was 25.35 cm (table 4). panicle length generally correlates with the number of grains per panicle. zang et al. [19] confirmed that large panicles with the huge number of grains per panicle can increase the density of grain. table 4: yield and yield components including panicle length (pl), total grain number (tgn), filled grain number (fgn), filled grain percentage (fgp), 1000 grain weight (gw), grain weight per hill (gwh), and grain yield (gy) of hybrid rice genotypes and check varieties in irrigated lowland. notes: the numbers followed by the same letter for each variable were not significantly different by dmrt at α = 0.05. the grain number per panicle was significantly different between genotypes. the average number of grains was high in genotype bi665a/bp6 (219.2 grains) that was significantly different from that of the check variety ciherang (153.0 grains) (table 4). a relatively high number of total grain on hybrid genotypes is as the implication of the long panicles of hybrid rice genotypes [19]. the number of filled grains was significantly different between genotypes and check varieties. genotype bi665a/bp6 yielded the highest number of filled grains, followed by genotype bi485a/bp12, bi485a/bp15, and bi599a/bp5 of 162.9, 153.6, 147.6, and 142.6 grains, respectively, but was not significantly different from the check variety hipa 7 of 140.3 grains. the number of filled grains of check variety ciherang was only 125.8 grains which was significantly different from genotypes bi665a/bp6, bi485a/bp12, and bi485a/bp15, but not significantly different from the other genotypes. the average number of filled grains of hybrid genotypes ranged between 118.4-162.9 grains, while the check varieties ciherang and hipa 7 had averages of 125.8 grains and 140.3 grains, respectively (table 4). peng and khush [20] stated that the criterion for a higher yield was the number of grains per panicle of about 150 grains. the highest percentage of filled grains obtained in genotype bi485a/bp12 of 88.5%, which was not significantly different from those of bi485a/bp15, bi599a/bp5, and check variety ciherang i.e. 83.7, 82.7, and 82.1%, respectively. genotype with the lowest percentage of filled grains was bi665a/bp6 of 74.0% which was not significantly different from genotypes bi485a/bp3, bi485a/bp10, and check variety hipa 7, i.e. of 74.4, 78.5, and 71.8%, respectively (table 4). the grain filling process is determined by the source in supporting sink or due to the aging process. the limited resources in supporting sink due to the low accumulation of photosynthate or earlier aging process will increase the empty grains percentage. abdullah et al. [21] reported that one of the causes of a void is the imbalance between the sink and the source. dry matter in the leaves started to decrease in 10 days after seed filling, and relatively large leaves play an important role in supplying photosynthate to seeds [22]. the highest 1000 grain weight was obtained in genotype bi599a/bp5 of 30.88 g, followed by genotype bi599a/bp15, which was not significantly different from those of genotype bi485a/bp10 and bi485a/bp12 i.e. 29.19 g and 29.26 g, respectively (table 4). ma et al. [15] stated that trait of ideal plant had panicle with 1000 grain weight of 28-30 g. a new type of rice with grain weight of 28-30 g, panicle length > 26 cm and lots of grains is a fair size for sink to support high grain yield. plants with a lot of grains per panicle need much assimilate for grain filling, so it should be supported by ideal leaf traits i.e. medium leaf width, thick, slightly dropping, not rapidly aging and medium age, as in genotype bi485a/bp12, bi485a/bp15, bi599a/bp5, and bi599a/bp15. the weight of grains per hill between genotypes and check varieties were not significantly different. the weight of grains per hill among genotypes ranged from 33.92-42.92 g, grain weight per hill of check variety ciherang was 38.30 g and 46.81 g for hipa 7. the grain weight of the genotypes when converted to yield ranged from 5.43 to 6.87 ton ha-1. genotype bi485a/bp15 had the highest grain yield of 42.92 g hill-1 or 6.87 tons ha-1 (table 4). virmani et al. [23] suggested that important benchmark in the assembly of hybrid rice is the yield comparison between hybrids and check varieties. genotype bi485a/bp3, bi485a/bp10, bi485a/bp15, and bi599a/bp15, when compared to the check variety ciherang was likely to show an increase i.e. 7.2, 2.8, 12.1, 1.6, and 2.8 percent, respectively (table 4). an effective way to develop a lowland rice cultivar with high yield potential in both the optimum and sub optimum conditions (drought stress) is through direct selection that can combine the selection in irrigated and rain-fed lowlands. therefore, a preliminary yield trial of drought-tolerant hybrid genotypes was performed, which was potential to be developed in the rain-fed areas. results of analysis of variance experiment growth and yield in rain-fed lowland showed that the variety treatment significantly influenced variables of plant height, flag leaf length, weight of biomass, 50% flowering age, panicle length, number of total grains, the percentage of filled grains, grain weight per hill and yield per hectare, but had no significant effect on the variables of productive tillers. results of dmrt test on the growth variables are presented in table 5 and on the yield components and the yield are presented in table 6. table 5 shows that the tallest plant height was obtained on genotype bi599a/bp15, which was not significantly different from check variety limboto, but significantly different from the check varieties ir64 and hipa 7. genotypes bi485a/bp3, bi485a/bp12, and bi485a/bp15 were not significantly different from the check varieties ir64 and hipa 7, but significantly different from the check variety limboto. the low rainfall and rain frequency since the vegetative phase strongly inhibited the growth of plant height, that was only of 54.67 to 82.11 cm because cell division was inhibited [24]. consequently, due to drought genotype pl (cm) tg (grain) fgn (grain) fgp (%) gw (g) gwh (g) gw (ton ha-1) bi485a/bp3 24.88 180.9 bc 134.0 bcd 74.4 cd 28.98 c 41.13 6.5 bi485a/bp5 23.82 149.1 d 118.4d 79.7bc 28.93 cd 37.11 5.93 bi485a/bp10 24.54 161.7 cd 126.9 bcd 78.5bcd 29.19 bc 39.43 6.30 bi485a/bp12 25.15 173.7 bcd 153.6 ab 88.5 a 29.26 bc 35.25 5.63 bi485a/bp15 24.66 175.8 bcd 147.6 abc 83.7 ab 28.53 cde 42.92 6.87 bi599a/bp5 25.31 171.7 bcd 142.6 abcd 82.7 abc 30.88 a 38.94 6.23 bi599a/bp15 24.49 169.5 bcd 135.3 bcd 79.3 bc 29.86 b 39.40 6.30 bi665a/bp6 26.47 219.2 a 162.9 a 74.0 cd 28.59 cd 33.92 5.43 ciherang 23.69 153.0 cd 125.8d 82.1 abc 28.16 de 38.30 6.13 hipa 7 25.35 195.0 ab 140.3 abcd 71.8 d 27.99 e 46.81 7.50 http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 stress in rain-fed areas, the average plant height of genotypes was lower than the standard height for a new type rice crop of 100 cm [14], and different with plant height on trial in irrigated lowland. however, drought-tolerant genotypes bi599a/bp15 could still achieve 82.11 cm in height, which was relatively equal to check variety limboto of 81.11 cm. samaullah and darajat [25] stated that the limited supply of water can suppress plant growth by 10-25 cm in a drought-stress growing environment. kumar et al. [26] reported that at severe drought conditions, the decrease in plant height on tolerant strains reached 6-12 cm, and on sensitive strains reached 16-27cm. table 5: growth components including plant height (ph), number of productive tillers (npt), flowering age (fa), and total biomass (tb) of hybrid rice genotypes and check varieties in rain-fed lowland. notes: the numbers followed the same letter for each variable were not significantly different by dmrt test at α = 0.05. the number of productive tillers did not significantly differ among genotypes and check varieties. the average number of productive tillers ranged from 10 to 13 (table 5). this suggests that the formation and growth of tillers were not affected by drought-stress. the genotypes had an average of productive tillers that was suitable for transplanting, i.e. 8-10 [16]. the shortest average flowering -age was obtained in check variety limboto, which was not significantly different with genotypes bi599a/bp15, bi485a/bp12, and hipa 7. the longest average flowering -age was obtained in genotype bi485a/bp3, which was not significantly different from genotypes bi485a/bp15 and check variety ir64 (table 5). flowering age is strongly related to the efficiency of the utilization of water resources and nutrients due to shorter vegetative growth phase. shorter flowering-age generally has good adaptability to drought and is able to accelerate ripening of grains. kumar et al. [26] stated that in conditions of severe drought, tolerant strains accelerated flowering by ± 8 days compared with sensitive strains. samaullah and darajat [25] and ahadiyat et al. [27] reported that the earlier-flowering genotype with a little number of empty grains in a water-stress environment has a drought-tolerant trait. the lateness in flowering of genotypes growing in drought-stress environment will shorten the grain filling phase, which affects the yield. the highest average total biomass (plant dry weight) was obtained on genotype bi599a/bp15, which was not significantly different from genotypes bi485a/bp12 and bi485a/bp15, but significantly different from bi485a/bp3. the genotypes with the highest plant dry weight had relatively equal weights with check variety limboto but significantly different with check variety ir64 (table 5). kumar et al. [26] stated that the total dry substance of late-flowering genotypes gradually declines in drought stress conditions. this indicates that the late-flowering plants may stop early growing phase during the growth cycle causing the dry weight decreases. genotypes bi599a/bp15, bi485a/ bp12, and bi485a/bp15 had a relatively shorter flowering -age, so that produced relatively higher dry weight in severe drought conditions. kumar et al. [28] reported that the tolerant strains have the ability to maintain high biomass production in dry soil and high harvest index. average of panicle length of hybrid genotypes and check varieties hipa 7 and limboto was significantly different from the check variety ir64. the ir64 has the lowest panicle length of 17.9 cm, while the length panicle of genotypes ranged from 20.9-21.8 cm (table 6). severe drought conditions inhibited panicle length of check variety ir64, which was sensitive to drought because the panicles exertion was hampered. this lead some grains to stay in the leaf midrib and become sterile grains, causing the decreasing of grain yield [29]. panicle length generally correlated positively with the number of grains per panicle [19]. total number of grains was fairly high on genotype bi485a/bp3 but was not significantly different from genotype bi599a/bp15, of 137.5 and 123.6 grains, respectively. the number of total grains of the genotypes was still lower than that of the check variety limboto of 153.6 grains, but higher than those of the check varieties hipa 7 and ir64, of 111.7 and 64.7 grains, respectively (table 6). the high total number of grains per panicle should be supported by a sufficient source for better grain filling [30]. table 6: yield components and the yield including panicle length (pl), total grain number of panicle (tgnp), filled grain percentage (fgp), hill grain weight (hgw), and grain yield (gy) of hybrid rice genotypes and check varieties in rain-fed lowland. notes: the numbers followed the same letter for each variable were not significantly different by dmrt test at α = 0.05. in general, the percentage of filled grain of genotypes was very low, ranging from 3.3 to 30.9 percent. genotype bi599a/bp15 had the highest percentage of filled grain of 30.9 percent which was not significantly different from genotypes bi485a/bp12, bi485a/bp15 and hipa 7, but significantly different from genotypes bi485a/bp3, ir64 and limboto. the lowest percentage of filled grain (3.3 percent) was achieved by genotype bi485a/bp3. the percentage of filled grain of check varieties ir64 and limboto was relatively low, of 10.3 and 7.5 percent, respectively (table 6). a severe drought led to the source being interrupted, thus the seed filling stage highly depended on the nature and mechanisms of plants coping with the drought stress. the higher percentage of filled grain on genotype bi599a/bp15 was caused by the earlier-flowering trait. pantuwan et al. [31] stated that drought escape mechanism of early-maturity genotypes with high yield in long drought-stress conditions was possibly due to the pre-anthesis assimilate contribution as a source of assimilates genotype ph (cm) npt fa (days) tb (g) bi485a/bp3 68.33bc 11.1 88a 27.38b bi485a/bp12 67.44bc 11.6 83b 32.95ab bi485a/bp15 64.56c 11.7 86ab 28.53ab bi599a/bp15 82.11a 13.4 83b 34.67a ir64 54.67c 12.9 85ab 19.07c limboto 81.11ab 9.8 82b 34.69a hipa 7 63.56c 10.4 83 b 26.77b genotype pl (cm) tgnp (butir) fgp (%) hgw (g) gy (ton ha-1) bi485a/bp3 21.43a 137.5ab 3.3b 0.54b 0.09 b bi485a/bp12 20.89a 106.2cd 13.1ab 0.97b 0.16 b bi485a/bp15 20.86a 100.5d 13.9ab 1.21b 0.19 b bi599a/bp15 21.80a 123.6bc 30.9a 5.62a 0.90 a ir64 17.78b 64.7e 10.3b 1.16b 0.19 b limboto 20.98a 153.6a 7.5b 1.79b 0.29 b hipa 7 21.16a 111.7cd 14.4ab 2.11ab 0.34 ab http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 during the grain filling period. in severe drought conditions, empty grains in ct9993 drought-tolerant strain reached 71 percent, while in ir62266 drought-sensitive strain reached 91 percent [28]. grain weight of genotypes per hill ranged from 0:54 5.62 g per hill. a high percentage of filled grains led to a high yield or grain weight per hill. therefore, genotype bi599a/bp15 had the highest grain weight of 5.62 g per hill or 0.90 tons of grain yield per hectare which was not significantly different from hybrid variety hipa 7 but significantly different from the other genotypes and check varieties ir64 and limboto. hipa 7 was recommended to be planted in rain-fed areas [32]. the lowest grain weight was obtained on genotype bi485a/bp3 of 0:54 g per hill, but was not significantly different from those of the three check varieties and genotypes bi485a/bp12 and bi485a/bp15 (table 6). grain yield obtained in genotype bi599a/bp15 was equal to the grain yield obtained in strain ct9993, which is a drought tolerant genotype when was grown in a severe drought conditions in raipur, northeast region of india. strain ct9993 was only able to produce 0.95 tons per hectare [28]. the difference in yield of genotype bi599a/bp15 and other genotypes may be due to differences in the contribution of pre-anthesis dry matter to seed production. the genotypes had a relatively shorter flowering-age, thus, it produced a higher dry weight in severe drought conditions (table 5). therefore, early-ripening genotype bi599a/bp15 that was able to reallocate reserved assimilates from the stem to the seed under drought condition resulted in a high yield. this mechanism involves rapid phenological development (early flowering and early ripening) and pre-anthesis assimilates remobilization [33]. limboto is a drought-tolerant variety. during the growing in the field in a severe drought conditions, limboto maintained a high level of leaf greenness but grain filling rate was much lower than the genotypes bi599a/bp15 (figure 2 and table 6). fukai and cooper [34] confirmed that there are four trait categories of drought resistance of rain-fed rice i.e. drought escape, drought avoidance, drought tolerance and drought recovery after a drought occurred. fukai et al. [35] stated that the maintenance of leaf greenness in drought conditions is an example of the adaptation mechanisms of drought tolerance of rain-fed rice. drought escape is a effective strategy to reduce the unfavorable effects of drought that adjusts the availability of water with plant phenology [36]. thus, limboto leaf greenness in severe drought conditions in which rain only occurred the first two weeks after planting indicated that limboto variety is a drought tolerant, whereas genotype bi599a/bp15 developed more than one mechanism or adaptation strategies namely drought escape and drought tolerance. in drought conditions, genotype bi599a/bp15 was not only accelerated flowering/harvest (table 5) but also had a low density of stomata and still maintained relative moisture of leaves at a high level in drought conditions [37-39]. mitra [33] confirmed that plants often used more than one mechanism for drought tolerance. thus, genotype bi599a/bp15 was able to produce better grain in drought conditions than other genotypes. therefore the hybrid rice genotype that is potential for further development in the rain-fed lowland and also in irrigated lowland with high yield potential and early-maturity age is genotype bi599a/bp15. genotype bi599a/bp15 that experienced drought stress, in the critical phase was capable of producing 2.72 tons of grain per hectare, whereas ir64 produced only 1:42 tons per hectare of grain [37]. this offers an opportunity that genotype bi599a/bp15 can be planted under conditions of rainfall restriction but not continuous drought during the vegetative to reproductive phase. on the other hand, when an unpredictable severe drought occurred, farmers can still obtain yields even though the productivity was <1 ton per hectare (table 6). these efforts can improve cropping index in the rain-fed lowland. conclusion based on the condition of the field test, the following points can be concluded: 1. on irrigated lowland, all tested-genotypes produced grain weight per hill and grain yield per hectare, which was not significantly different from the check varieties ciherang and hipa 7. 2. drought-tolerant genotypes bi485a/bp12, bi485a /bp15, and bi559a/bp15, in irrigated lowland had equal yields with that of variety ciherang, of 5.63, 6.87 and 6.30 tons per hectare, respectively. 3.genotype bp599a/bp15, at a very severe drought conditions in the rain-fed lowland, generally produced better results than other genotypes for plant height, productive tillers, flag leaf length, total biomass, panicle length, filled grain percentage and grain weight per hill. 4.genotype bp599a/bp15 at a very severe drought conditions in the rain-fed lowland was able to produce 0.90 tons of grain per hectare, while hipa 7 (hybrid variety that had been released to the rain-fed lowland) and limboto (drought-tolerant check variety) produced only 0:34 and 0:29 tons per hectare, respectively. 5.genotype bi599a/bp15 is a new hybrid genotype that potential for advanced development in rain-fed lowland and also in irrigated lowland with a high yield potential and quiet early-maturity age. acknowledgments acknowledgement was submitted to the agency for agricultural research, ministry of agriculture for funding this research through kkp3t in fiscal year 2012 to bambang s. purwoko and team. author contributions l. afa conducted field experimental tests and data analysis. b. s. purwoko, a. junaedi, o. haridjaja and s. s. dewi formulated experimental design, helped in preparing the draft of reports and made some correction on the manuscript. references 1.virmani ss, kumar i. development and use of hybrid rice technology to increase rice productivity in the tropics. international rice research notes. 2004;29(1): 10-19. http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 8 2.huang m, tang q, ao h, zou y. yield potential 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effects. field crops research. 2007;103:42-52. 29.ji xm, raveendran m, oane r, ismail a, lafitte r, bruskiewich r, cheng sh, bennett j. tissue-spesific expression and drought responsiveness of cell wall invertase gene of rice at flowering. plant molecular biology. 2005;59:945-964. 30.jun ma, wen-bo ma, ming df, yang sm, zhu qs. characteristics of rice plant with heavy panicle. agricultural sciences in china. 2006;5:911-918. 31.pantuwan g, fukai s, cooper m, rajatasereekul s, o’toole jc. yield response of rice (oryza sativa l.) genotypes to drought under rain-fed lowland 3. plant factors contributing to drought resistance. field crops research. 2002;73:181-200. 32.suprihatno b, daradjat aa, satoto, baehaki se, suprihanto, setyono a, indrasari sd, wardana ip, sembiring h. description rice varieties. rice research institute. agricultural development and research agency; 2011.p.118. http://bioscience.highlightsin.org/ afa et al., 2020 growth and yield of new hybrid rice highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 8 33.mitra j. genetic and genetic improvement of drought resistance in crop. plant science current research. 2001;80:758-763. 34.fukai s, cooper m. development of drought-resistant cultivars using physio-morphological traits in rice. field crops research. 1995;40:67-68. 35.fukai s, pantuwan g, jongdee b, cooper m.screening for drought resistance in rain-fed lowland rice. field crops research. 1999;64:71–74. 36.mitchell jh, siamhan d, wamala mh, risimeri jb, chinyamakobvu e, henderson sa, fukai s. the use seedling leaf death score for evaluation of drought resistance of rice. field crops research. 1998;55:129-139. 37.afa l. selection of hybrid rice to drought for development in rain-fed lowland [thesis]. bogor (indonesia):bogor agriculture institute; 2013. 38.huang m, shan s, cao f, zou y. the solar radiationrelated determinants of rice yield variation across a wide range of regions. wageningen journal of life sciences. 2016;78: 123–128. 39.huang m, shan s, zhou x, chen j, cao f, jiang l, zou y. leaf photosynthetic performance related to higher radiation use efficiency and grain yield in hybrid rice. field crops research. 2016;193: 87–93. http://bioscience.highlightsin.org/ highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202501 research article open access 1 biology department, faculty of mathematics and natural sciences, university of jember, jember, east java, indonesia. * to whom correspondence should be addressed: senjarini@unej.ac.id editor: hatem zayed, department of biomedical science, college of health sciences, qatar university, doha, qatar. reviewer(s): laila dabab nahas, usher institute, university of edinburgh, scotland, united kingdom. hala abdelgaid, national hepatology and tropical medicine research institute (nhtmri) corniche el nil, imbaba, giza 12651, egypt. received: december 20, 2024 accepted: april 25, 2025 published: may 5, 2025 citation: oktarianti r, nurdianti f, wathon s, senjarini k. in silico study of the interaction between serotonin and d7 protein from the salivary gland of aedes aegypti. 2025 may 5;8:bs202501 copyright: © 2025 oktarianti r et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this research is supported by the national research and innovation agency (briin) through the research and innovation program for advanced indonesia, batch 3, under reference number 12/ii.7/hk/2023 competing interests: the authors declare that they have no competing interests. in silico study of the interaction between serotonin and d7 protein from the salivary gland of aedes aegypti rike oktarianti1 ><�, faranisa nurdianti1 >< , syubbanul wathon1 >< �, kartika senjarini1 >< � abstract protein components of the salivary glands of disease vectors have been known to facilitate the blood-feeding process in the host body. the main component of the salivary glands of aedes aegypti is the immunogenic d7 protein. during the blood-feeding process, the d7 protein can bind to biogenic amine compounds, such as serotonin, which is a neurotransmitter involved in platelet activation. this ability indicates that the d7 protein can inhibit the platelet aggregation process. this study aims to explore in silico the interaction between serotonin and the d7 protein from the salivary glands of ae. aegypti using a molecular docking approach. the methods used in this study include the selection of the 3d structure of the d7 protein and serotonin ligand, preparation of the 3d structure of the d7 protein, native ligands, and test ligands, validation of the molecular docking method, and analysis and visualization of the molecular docking results. the results of molecular docking between the d7 protein and the serotonin ligand showed a ∆g value for the interaction of −9.25 kcal/mol. the serotonin ligand binds to the active site of the d7 protein through several amino acid residues, including glu 158, ile 175, arg 176, tyr 178, tyr 248, asp 265, and glu 268. these amino acid residues of the d7 protein bind to atoms on the serotonin ligand through conventional hydrogen bonds, carbon hydrogen bonds, π-σ bonds, π-π t-shaped bonds, and π-alkyl bonds. based on the in silico data, it is shown that the d7 protein from the salivary glands of ae. aegypti can bind stably and spontaneously to serotonin ligands. this indicates that the d7 protein has potential as a platelet aggregation inhibitor agent for the development of drug discovery in the fields of health and pharmacy. keywords: aedes aegypti, d7 protein, molecular docking, serotonin, platelet aggregation introduction the salivary glands of disease vector arthropods are known to be important organs for the success of the blood-feeding process in the host body [1]. this is because the salivary glands contain various bioactive components that can suppress the host’s immune response. in general, components in the salivary glands of disease vectors act as vasodilator and immunomodulatory factors that can affect the host’s hemostasis [2]. several previous studies have identified components of the salivary glands of disease vectors that affect the host’s immune response, including bioactive components in the salivary glands of aedes aegypti as a vector of dengue fever [3; 4]. the salivary glands of ae. aegypti contain various types of bioactive components in the form of protein molecules. the protein components of the salivary glands of ae. aegypti generally include apyrase, aegyptin, serpin, and the d7 family [5]. the apyrase protein has the activity to hydrolyze atp into adp and amp, which can inhibit platelet activation [6]. aegyptin is an allergen that can bind to collagen and von willebrand factor, reducing the formation of blood clots [7]. serpin is a protease inhibitor that inhibits the activity of serine proteases in various host hemostasis reactions [8]. the d7 protein is known to be the most abundant component in the salivary glands of ae. aegypti [9]. ae. aegypti performs the blood-feeding process on the host’s body by inserting its proboscis into the skin layer until it reaches the endothelium of the blood vessels. the host’s body responds to this action by releasing biogenic amine compounds to stop the blood flow through the mechanism of highlights in bioscience page 1 of 9 may 2025|volume 8 https://doi.org/10.36462/h.biosci.202501 https://creativecommons.org/licenses/by/4.0/ mailto:rike.fmipa@unej.ac.id https://orcid.org/0000-0001-9402-7746 mailto:201810401060@mail.unej.ac.id mailto:syubbanulwathon@unej.ac.id https://orcid.org/0000-0003-2935-7786 mailto:senjarini@unej.ac.id https://orcid.org/0000-0001-7041-1719 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein platelet aggregation. during the blood-feeding process, ae. aegypti releases the d7 protein component from its salivary glands into the host’s body. the d7 protein has a high affinity for biogenic amine compounds, such as norepinephrine, histamine, and serotonin [10]. serotonin is a type of biogenic amine that plays an important role in platelet activation [11]. when the d7 protein from the salivary glands of ae. aegypti binds to serotonin, the process of platelet aggregation around the host’s wound is inhibited, allowing the blood-feeding process to proceed smoothly. this activity indicates that the d7 protein can inhibit the platelet aggregation process in the host’s body. d7 has the ability to block platelet aggregation, which makes it an excellent candidate for an anti-platelet medication. however, its effects on biogenic amines, including its interaction with serotonin from the salivary glands of ae. aegypti, are not well characterized. one branch of biochemistry involves the study of proteinligand interactions, which can be explored through in silico approaches that predict molecular interactions using computer simulations. for instance, molecular docking analysis enables the identification of specific binding sites on target proteins for a test ligand [12]. previous studies have primarily focused on the interaction between the d7 protein and leukotriene a4, demonstrating the formation of a stable and natural complex [13]. however, no prior research has specifically examined the ability of d7 from ae. aegypti to bind serotonin, a crucial biogenic amine involved in platelet aggregation. this study addresses this gap by utilizing in silico molecular docking to explore the potential interaction between d7 and serotonin. by doing so, we aim to provide new insights into the functional role of d7 and its potential as a foundation for developing novel anti-platelet aggregation agents. materials and methods downloading 3d structure d7 protein and ligand the amino acid sequence of the d7 protein from ae. aegypti was downloaded from the uniprot database with accession code p18153 [14]. the three-dimensional (3d) structure of the d7 protein was obtained from the swiss-model database [15]. the resulting model was selected based on its best quality and downloaded in .pdb file format [16]. the 3d model structure of the d7 protein uses the d7 protein with the template pdb id 3dye.1. the model structure from this template includes a native ligand, which is l-norepinephrine. in this study, a test ligand was used to observe its interaction with the d7 protein. the test ligand selected was serotonin. the three-dimensional structure of serotonin was obtained from the pubchem database with accession code 5202. this serotonin entry originates from human metabolism. the serotonin molecule was downloaded from the pubchem database in .sdf file format [17]. preparation and optimization of the 3d structure of d7 protein and the native ligand l-norepinephrine the preparation of the 3d structure of the d7 protein involves removing the native ligand, non-functional ligands, and water molecules. this preparation is carried out using autodock tools software [18]. the first step in preparing the d7 protein structure is the removal of all water molecules and non-functional ligands. the d7 protein is then separated from its native ligand, l-norepinephrine. the structure of the d7 protein is saved in .pdb format, and the structure of l-norepinephrine is also saved in .pdb format within the same folder. the structure of the d7 protein is then optimized by adding polar hydrogen atoms and checking for missing atoms to ensure the integrity of the downloaded 3d structure. the d7 protein is subsequently charged using kollman charges, resulting in charge neutralization. the optimized structure is saved in .pdbqt format [19]. further optimization is performed on the 3d structure of the l-norepinephrine ligand. the ligand is optimized by adding gasteiger charges, followed by a non-polar merge, with the expectation that only hydrogen atoms will be available to form bonds with the target protein residues (d7 protein) [20]. the native ligand is then prepared for docking by selecting rotation points using the torsion tree menu. this process identifies and assigns torsion points, improving the accuracy of ligand positioning predictions. the final optimized structure of the native ligand is saved in .pdbqt format in the same folder as the optimized d7 protein structure [19]. preparation and optimization of the native ligands 3d structure were also performed using autodock tools software. validation of the molecular docking method the next step in the molecular docking phase is to validate the method through a re-docking process of the d7 protein with its native ligand, l-norepinephrine, which has already been prepared and optimized. the initial step of the re-docking process involves determining the interaction site by defining the grid area using a grid box. this stage is conducted using autodock tools software [18]. the grid box settings include the number of points in each dimension (x, y, and z), the spacing in å, and the center coordinates of the grid box (x, y, and z). these settings are saved in the .gpf (grid parameter file) format. the validation of the molecular docking method between the d7 protein and its native ligand is evaluated using the rmsd (root mean square deviation) value. an rmsd value of less than 2 å(< 2 å) is considered acceptable and indicates good reproducibility of the docking result [21]. preparation of 3d structure of serotonin test ligand the preparation and optimization of the test ligand, serotonin, were carried out using chem3d and autodock tools software [19]. the 3d structure of serotonin was prepared through energy minimization using the force field molecular mechanism (mm2) method [22]. the resulting minimized structure was saved in .pdb format. subsequently, the test ligand structure was optimized by adding hydrogen atoms, followed by a non-polar merge. gasteiger charges were then applied to the ligand structure. the final step involved adjusting the torsional rotation points using the same procedure applied to the native l-norepinephrine ligand. highlights in bioscience page 2 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein the fully optimized structure of the serotonin ligand was saved in .pdbqt format. molecular docking between d7 protein and serotonin test ligand the process of molecular docking of d7 protein with the serotonin test ligand is carried out using autodock tools software. the first step of this process is to place the structure file of d7 protein in .pdbqt format and the structure file of the serotonin ligand into the same folder. this folder also contains the programs autogrid4 and autodock4 [23]. the d7 protein was first set up as the macromolecule, and the serotonin ligand configuration was used to determine and create the gridbox. the gridbox used is based on the result of coordinate adjustments from the validation or re-docking process between d7 protein and the native ligand l-norepinephrine. the coordinate adjustments of the gridbox on protein d7 with the test ligand serotonin are then saved in .gpf format in the same folder. the next step is to run the autogrid4 program using the command prompt (cmd). this program is executed based on the coordinate settings of the gridbox that have been created. the next step is to run the autodock4 program, which treats the protein as rigid. the autodock4 program is run with the same command based on the previous autogrid data [24]. analysis and visualization of molecular docking results between d7 protein and serotonin test ligand the analysis of the molecular docking results between d7 protein and the serotonin test ligand is shown from several parameters such as the gibbs free energy value (∆g), types of chemical bonds, and amino acid residues involved in the interactions formed. the results of the gibbs free energy values were compared with the validation results of the re-docking of d7 protein with the native ligand l-norepinephrine. this comparison was made to observe the differences in gibbs free energy in the formation of interactions between d7 protein and the test ligand. table 1. properly formatted d7 protein sequence of ae. aegypti >sp|p18153|all2_aedae 37 kda salivary gland allergen aed a 2 os=aedes aegypti ox=7159 gn=d7 pe=1 sv=2 mkedtlaavifsvvastgpfdpeemlftftrcmednlledgpnrlpmlakw kewinepvdspatqcgfkcvlvrtglydpvaqkfdasviqegfkaypslg ekskveayanavqqlpstnndcaavfkaydpvhkahkdtsknlfhgnkel tkglyeklgkdirokkqsyfeecenkyypagsdkrqqlckiroytvldda lfkehtdcvmkgiryitknneldaeevkrdemqvnkdtkalekvlndcks kepsnagekswhyxkclvssvkddekeafdyrevksqiyafnlpkkqvys kpavqsqvmeidgkqcpq the results of the molecular docking were then visualized to observe the conformation of the bond between the 3d structure and the two-dimensional structure of the protein and test ligand. the visualization results can include information on amino acid residues and the types of bonds formed. visualization and analysis of the interaction between d7 protein and the serotonin test ligand were performed using biovia discovery studio software [25]. results and discussions 3d structure of d7 protein from ae. aegypti salivary gland the amino acid sequence data of the d7 protein from the ae. aegypti salivary gland was obtained from the uniprot database with accession number p18153. the protein is identified by the gene name d7 and is known as the salivary gland allergen aed a 2, with a molecular weight of 37 kda. the d7 protein with accession number p18153 is a monomeric protein with a long chain domain that has binding affinity for a ligand. the protein is composed of 321 amino acid residues, as shown in table 1. the 3d structure of the d7 protein from ae. aegypti was obtained using homology modeling techniques through the swiss-model protein database. construction of the 3d protein structure was carried out using amino acid sequence data of the d7 protein from the salivary glands of ae. aegypti. based on the homology modeling process, the best model selected used the template with pdb id 3dye.1. the information on the 3d structure model of the protein is known as the d7 protein crystal structure of the aed7–norepinephrine complex. the 3d structure of the d7 protein contains a norepinephrine molecule which will then be used as a native ligand. the 3d structure of the d7 protein shows two domains of the polypeptide chain with different functions. the n-terminal domain can bind cysteinyl leukotriene molecules, while the c-terminal domain can bind biogenic amine molecules [10]. visualization of the 3d structure model of protein d7 was carried out using the xray crystallography method with a resolution of 1.75 å. the 3d structure model of a protein is said to have good quality if it shows a resolution of < 2.5 å from the results of x-ray crystallography [20]. the 3d structure model of the d7 ae. aegypti protein can be seen in figure 1. figure 1. 3d structure of d7 protein (stml id: 3dye.1), a: domain terminal-n, b: domain terminal-c, c: native lnr ligand binding site highlights in bioscience page 3 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein assessment of the structural quality of the ae. aegypti salivary gland d7 protein model data from the results of homology modeling of d7 protein were obtained from the uniprot website with the accession code p18153. the sequence obtained was then copied to the swissmodel website to build the model. the results of the model build obtained the template pdb id 3dye.1. the results of homology modeling were obtained with crystallography results of 1.75 å using the x-ray method. for crystallography, a smaller value indicates better resolution [26]. assessment of homology modeling results is based on several parameters such as gmqe value, qmean value, qmeandisco value, and sequence identity [27]. the gmqe (global model quality estimate) value parameter is a value that describes the quality of the alignment between the target and template. the range for the gmqe value means that if it approaches one, it indicates a higher level of accuracy of the protein model [28]. the value of gmqe is expressed with a range of values between 0 and 1. the value of the d7 protein model on the gmqe value parameter is 0.91, where the results indicate that the accuracy of the model is good. table s1 shows the results of the model quality assessment on the d7 protein. the qmean value is a composite score of a combined assessment that can determine an estimate of the global absolute quality (entire structure) and local (per amino acid residue) based on a single model. the qmean score ranges from 0 to 1, where a value of 1 means good [29]. this qmean value can be represented by the z-score value. the z-score value at the model position (marked with a red asterisk) in the z-value distribution. this result is marked in figure 2. the red asterisk indicates the model’s z-score is within the typical range for native proteins of similar size, suggesting reasonable overall quality. estimates of the local quality of the model per amino acid residue can also help in providing an explanation regarding the quality of the d7 protein to be used. this z-score reflects the overall model quality (qmean). the sequence identity of the d7 protein model results has a value of 95.70%. sequence identity is a value that can indicate the percentage of residues in the target protein sequence that are identical to those in the template protein sequence [30]. the value of the acceptable sequence identity starts at 30%, where the higher value indicates the level of accuracy between the target protein and the template protein. the results based on the ramachandran plot in figure 3, the protein has good structural quality if the amino acid residues are mostly in the favored area rather than the outliers. lighter shades or white indicate residues with less favorable conformations. the φ sign is the phi dihedral angle and the ψ sign is the psi dihedral angle, where both represent the dihedral angles of the amino acid residue backbone. the ramachandran plot shows residues primarily in the allowed regions. the results of the d7 protein parameters in the ramachandran plot show residues distributed across the favored and allowed regions, indicating a good and figure 2. model position (star) on z score of protein d7 stable structure. protein d7 shown in table 2 has a favored area of 98.66% and an outlier area of 0.00%, which means the structure of the model is very good. the plot uses contour lines to indicate regions of probability density; 98.66% of residues fall within the favored regions (darkest contours), and 0.00% are in outlier regions table 2, indicating very good stereochemical quality. table 2. ramachandran plot parameters of d7 protein parameter model-1 molprobity score 0.65 clash score 0.41 ramachandran favoured 98.66% ramachandran outliers 0.00% rotamer outliers 0.00% c-beta deviations 0 bad bonds 0/2504 bad angles a276 asp, (a296 leu-a297 pro), a163 asp, a140 asp, (a132 aspa133 pro), (a304 lys-a205 pro), a264 his, a135 his, a207 his, a138 his, a263 trp the molprobity score of model-1 is 0.65. the molprobity score is a combination of the log-weighted clash score, the percentage of unfavorable ramachandran outliers, and the percentage of bad side chain rotamers. this score indicates a value that is expected to describe the equivalent resolution of a comparable experimental structure. a lower molprobity score generally indicates better quality. if the score is lower than typical for structures at the template’s resolution (1.75 å), then the model highlights in bioscience page 4 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein figure 3. ramachandran plot of aedes aegypti salivary gland d7 protein quality is considered very good. the resolution of the template crystallography is 1.75 å. 3d structure of native ligand l-norepinephrine and test ligand serotonin the 3d structure model of d7 protein with template pdb id 3dye.1 has a native ligand in the form of l-norepinephrine. the l-norepinephrine molecule is a type of biogenic amine which is a component of monoamine neurotransmitters [31]. the lnorepinephrine molecule is a non-polymer molecule with a molecular weight of 169.178 g/mol. l-norepinephrine has a structure containing an aromatic ring and a carbon chain with the chemical formula c8h11no3. the structure of l-norepinephrine consists of 23 atoms, each of which is connected by chemical bonds and has one aromatic ring [32]. the use of native ligands is important for the validation stage of the molecular docking method as well as for determining the orientation of the binding site for test ligands in the molecular docking process [33]. the test ligand used in this study was the serotonin molecule, which is one of the biogenic amines in the human bloodstream. serotonin in the human body plays a role in the platelet aggregation process through the activation mechanism between platelet cells [34]. the structure of serotonin can interact with functional groups to form hydrogen bonds and can also participate in aromatic interactions [35]. serotonin (also known as enteramine) has the chemical formula c10h12n2o with a molecular weight of 176.21 g/mol. serotonin is composed of 25 atoms connected by chemical bonds and has an indole ring system [36]. the differences in the 2d and 3d structures of the l-norepinephrine ligand and the serotonin test ligand can be seen in table 3. molecular docking validation method validation of the molecular docking method was carried out through the re-docking technique using the native ligand and d7 protein as the target protein in this study. before validating figure 4. visualization of the overlapping conformation between the native ligand l-norepinephrine from crystallography (green) and the conformation of the native ligand l-norepinephrine from re-docking (yellow). the molecular docking method, it is necessary to prepare and optimize the structure of the d7 protein by separating it from the native ligand l-norepinephrine and removing water molecules, which are non-standard residues. the separation of the native ligand from the target protein structure aims to provide a binding pocket for the test ligand and the target protein. the removal of water molecules from the target protein structure is carried out so that they do not become an obstacle during the molecular docking process. this step is important so that in the molecular docking process, only the ligand structure interacts with the target protein [37]. the results of the molecular docking method validation process can be seen from the rmsd value. the rmsd parameter can show the results of the conformational alignment between the native ligand pose resulting from the re-docking process and the native ligand conformation from x-ray crystallography [21]. the rmsd value is obtained by examining the overlap between the conformation of the native ligand resulting from re-docking and the native ligand in its original crystallographic conformation. a smaller rmsd value indicates that the predicted binding pose from re-docking closely matches the experimentally observed binding pose. the validity of the rmsd value is indicated by a value < 2 å [38]. the rmsd value of the re-docking process between the l-norepinephrine ligand and the d7 protein shows a value of 1.088 å. these results indicate that the validation of the molecular docking method is accepted and the method can be used for the molecular docking process between the d7 protein and the serotonin ligand. the results of the alignment or overlapping conformation between the crystallographic pose (green) and the re-docked pose (yellow) of the native l-norepinephrine ligand can be seen in figure 4. molecular docking of protein d7 from ae. aegypti and serotonin test ligand the molecular docking process is carried out using serotonin ligands that have been prepared and optimized against the d7 highlights in bioscience page 5 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein table 3. differences in 2d and 3d structure of native ligands and test ligands ligand 2d 3d [l]native ligand (l-norepinephrine) [l]test ligand (serotonin) protein from ae. aegypti. the grid box coordinates obtained from the re-docking stage with the native ligand are then used as the basis for the grid box for the molecular docking process between the d7 protein and the serotonin ligand. this is done so that the test ligand can bind to the active site of the target protein [39]. in the molecular docking process, the structure of the d7 protein is kept rigid during the docking process. the structure of the serotonin ligand is treated as flexible. this is done so that the test ligand can interact and bind in the most stable conformation within the active site of the amino acid residues of the target protein [40]. analysis and visualization of molecular docking results of ae. aegypti d7 protein with serotonin ligand a ∆g value > 0 indicates that the binding reaction between the target protein and the test ligand cannot occur spontaneously. conversely, if the ∆g value < 0, it indicates that the binding reaction between the target protein and the test ligand occurs spontaneously (a reaction that favors product formation). a ∆g value = 0 indicates that the reaction is at equilibrium. the more negative the ∆g value, the stronger the binding affinity between the target protein and the ligand [41]. in addition, the negative value of ∆g indicates that the interaction between the target protein and the ligand binding process is thermodynamically favorable. this means that the binding between the ligand and the active site of a target protein occurs in a stable condition and spontaneously [42]. the results of molecular docking between protein d7 and serotonin ligand show a ∆g value of -9.25 kcal/mol. these results indicate that protein d7 can bind to serotonin ligands in a stable and spontaneous manner. this can be correlated with the mechanism during the blood feeding process, where protein d7 from the salivary glands of ae. aegypti can inhibit platelet aggregation by binding biogenic amines, including serotonin, thereby inhibiting serotonin’s role in platelet aggregation and disrupting the host’s homeostasis reaction [5]. the d7 protein from ae. aegypti has been studied for its potential role in modulating host hemostasis, primarily through its ability to bind biogenic amines and eicosanoids, thereby facilitating blood feeding. however, direct experimental evidence demonstrating its specific function as an inhibitor of platelet aggregation is limited. in contrast, studies on the d7 protein from aedes albopictus, a related mosquito species, have provided functional evidence of its role in inhibiting platelet aggregation. for instance, albod7l1, a long-form d7 protein from aedes albopictus, has been shown to bind various ligands and inhibit platelet aggregation in ex vivo experiments [10]. visualization of molecular docking results was carried out to determine the binding interaction mode between protein d7 and serotonin ligand. the target protein can bind to the test ligand through several amino acid residues [20]. the interaction between the target protein and the test ligand is indicated by the formation of several types of chemical bonds, such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and highlights in bioscience page 6 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein figure 5. visualization of the interaction of amino acid residues of d7 protein with serotonin ligand. van der waals bonds [43]. the results of molecular docking visualization between protein d7 and serotonin ligand can be seen in figure 5. table 4. amino acid residue d7 protein that binds to the serotonin ligand amino acid residue d7 protein serotonin ligand atom chemical bond glutamic acid 158 (glu 158) h (hydrogen) conventional hydrogen bond tyrosine 178 (tyr 178) tyrosine 248 (tyr 248) aspartic acid 265 (asp 265) glutamic acid 268 (glu 268) arginine 176 (arg 176) c (carbon) carbon hydrogen bond isoleucine 175 (ile 175) c-h (carbon hydrogen) π-sigma bond tyrosine 178 (tyr 178) pi-orbitals π-π t-shaped bond arginine 176 (arg 176) pi-orbitals π-alkyl bond table 4 shows the various types of chemical bonds formed between amino acid residues of the d7 protein that interact with atoms on the serotonin ligand. the chemical bonds formed are interactions that occur at the active site of the d7 protein binding to the serotonin test ligand. amino acid residues on the active site of the d7 protein that bind and interact with the serotonin ligand include glu 158, ile 175, arg 176, tyr 178, tyr 248, asp 265, and glu 268. the chemical bonds formed in the interaction between the d7 protein and the serotonin ligand include conventional hydrogen bonds, carbon-hydrogen bonds, π-sigma bonds, π-π t-shaped bonds, and π-alkyl bonds. conventional hydrogen bonds are formed involving several amino acid residues: glu 158, tyr 178, tyr 248, asp 265, and glu 268. the carbonhydrogen bond formed involves one amino acid residue, namely arg 176. the π-sigma bond formed only involves one amino acid residue, namely ile 175. the π-π t-shaped bond formed also involves one amino acid residue, namely tyr 178. the π-alkyl bond also involves one amino acid residue, namely arg 176. hydrogen bonds are formed between hydrogen atoms and electronegative atoms. this hydrogen bond formation is related to binding energy (∆g). the formation of hydrogen bonds can release energy due to covalent interactions, resulting in a negative change in enthalpy (∆h). a negative change in enthalpy can occur when a protein and ligand bind. this result can lead to a negative ∆g value, meaning the binding occurs spontaneously or is stable [44]. the interaction between the serotonin ligand and protein d7 involved in hydrogen bonding consists of conventional hydrogen bonds. in conventional hydrogen bonds, hydrogen is shared between electronegative atoms acting as donors (e.g., o-h, n-h on the ligand or protein) and acceptors (e.g., o, n on the protein or ligand) [45]. hydrogen bonds significantly influence the stability of the d7 protein-ligand complex because both the protein and ligand contain potential hydrogen bond donors (like n-h and o-h) and acceptors. these structures act as donors or acceptors in hydrogen bonds. the next type is the π-sigma bond, which is an interaction involving the π-system of an aromatic ring and a sigma bond (like c-h) [46]. the π-sigma bond occurs between the amino acid ile 175 and the ligand. the next bond is a π-π t-shaped bond, which is an electron interaction between two aromatic groups but in a t shape [47]. in this geometry, the edge of one aromatic ring points towards the face of the other aromatic ring. the last interaction is a type of π-alkyl bond, which is an interaction between electrons from the aromatic group and the electron group from the alkyl group [47]. conclusions the molecular docking results show that there is an interaction between the d7 protein from the salivary gland of ae. aegypti (accession number p18153) and the serotonin test ligand (accession number 5202). the interaction between the d7 protein and the serotonin test ligand shows stability and spontaneity based on the ∆g value of -9.25 kcal/mol. based on the ∆g parameter, this indicates that the d7 protein and the serotonin ligand can bind spontaneously and stably. the amino acid residues of protein d7 that interact with the serotonin ligand atoms include glu 158, ile 175, arg 176, tyr 178, tyr 248, asp 265, and glu 268. thus, the d7 protein from the salivary gland of ae. aegypti has potential as a new agent for platelet aggregation inhibition for drug discovery and development in the fields of health and pharmacy. supplementary table s1: d7 protein model quality assessment parameters. highlights in bioscience page 7 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ oktarianti r et al., 2025 in silico study of the interaction between serotonin and d7 protein reference 1. wathon s, mutiah f, 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madhusudhan ms, et al. observation of an unusually large ir red-shift in an unconventional s–h· · ·s hydrogen-bond. journal of physical chemistry letters. 2021;12(4):1228-35. 46. zierkiewicz w, michalczyk m, scheiner s. noncovalent bonds through sigma and pi-hole located on the same molecule. guiding principles and comparisons. molecules. 2021;26(6):1740. 47. alencar wlm, da silva arouche t, neto afg, de castro ramalho t, de carvalho júnior rn, de jesus chaves neto am. interactions of co, cu, and non-metal phthalocyanines with external structures of sars-cov-2 using docking and molecular dynamics. scientific reports. 2022;12(1):3316. highlights in bioscience page 9 of 9 may 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods downloading 3d structure d7 protein and ligand preparation and optimization of the 3d structure of d7 protein and the native ligand l-norepinephrine validation of the molecular docking method preparation of 3d structure of serotonin test ligand molecular docking between d7 protein and serotonin test ligand analysis and visualization of molecular docking results between d7 protein and serotonin test ligand results and discussions 3d structure of d7 protein from ae. aegypti salivary gland assessment of the structural quality of the ae. aegypti salivary gland d7 protein model 3d structure of native ligand l-norepinephrine and test ligand serotonin molecular docking validation method molecular docking of protein d7 from ae. aegypti and serotonin test ligand analysis and visualization of molecular docking results of ae. aegypti d7 protein with serotonin ligand conclusions supplementary highlights in bioscience; paper format highlights in bioscience issn: 2682-4043 highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 1 of 10 citation: alsamman m.a. (2019) the art of bioinformatics learning in our arabic world. highlights in bioscience, volume 2. article id 20193, dio:10.36462/ h.biosci.20193 received: august 19, 2019 accepted: september 13, 2019 published: october 12, 2019 copyright: © 2019 alsamman .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. the art of bioinformatics learning in our arabic world alsamman m. alsamman * 1 1 molecular genetics and genome mapping lab. , agricultural genetic engineering research institute, agricultural research center, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg abstract bioinformatics became a significant field in life sciences that, draws a number of researchers and extends into a wide range of biological disciplines. rendering bioinformatics analysis techniques are the most desirable skills in a variety of scholarship programs and academic positions. teaching bioinformatics is very challenging since it is a multidisciplinary field, where most of the undergraduate programs in colleges provide only one area required for bioinformatics. besides the regular education system, few bioinformatics training courses are offered and less are affordable to fresh graduates in countries most of which are categorized as developing countries. the high cost of learning, confusing education systems, and the complexity of bioinformatics science has made it very difficult to be taught and more challenging to be studied in arab countries. this review provides possible solutions to most of these issues and offers the best practice to guide future arab bioinformaticians to learn bioinformatics in a way that fits our social, financial and academic circumstances. moreover, it discusses the key aspects that a bioinformatician needs to be aware of and the basic knowledge that must be gained. on the other side, it will illustrate how to start learning, to address some of these challenges and how to deal with some of the related social issues. keywords: bioinformatics, arab, programming, education, linux, biological data. introduction bioinformatics analysis techniques are the most desirable skills in a wide range of scholarship programs and academic positions. as the study of bioinformatics and computational biology grows and evolves, it is essential to quantify the factors that contribute to the development of professionals in this field (1). bioinformatics is an interdisciplinary research field where computational resources and techniques are used to interpret biological data through mathematical and statistical approaches. perspective review open access the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 2 of 10 the rapid acceleration in computing power and memory storage capacity have given rise to a new golden age in the biological data analysis (2). there is almost no biological sector in which bioinformatics has not yet been incorporated. its techniques are used in microbiology to examine microbial diversity and species occurrence by identifying and quantifying the association of microbial communities among different biological samples (3). throughout the pharmaceutical industry, bioinformatics offers analytical tools that can enhance drug target identification, drug candidate monitoring and drug optimization. in particular, it promotes the recognition of side effects and forecasts drug resistance (4). recently, bioinformatics has proposed the idea of personalized medicine, where treatments will be adapted to the unique genotype of patients. integrating the vast genetic information provided by the genome wide association studies (gwass) is a valuable resource for mapping genetic traits with drug reactions and phenotypes, allowing individual characteristics of each patient to be monitored and their susceptibility to certain diseases to be considered (5). last but not the least, the integration of machine learning in bioinformatics analytical methods has opened a new era in which sample data and past experience could be used to improve the output criterion in computational algorithms, the optimized criteria could be the rating of reliability provided by the statistical model and the significance of the performance (6). machine learning has therefore enabled what appears to be a breakthrough in biological research, where computer programs can solve complex biological problems efficiently and effectively. through cancer diagnosis (7), neurooncology imaging (8) and drug design (9) in medicine and plant physiology (10), forecasting crop yields (11) and livestock production in agriculture (12), machine learning has been used to solve both challenging and basic tasks. the rapid development of life sciences and information technology requires the continuous development of bioinformatics learning programs in order to sustain their significance (1). the quality of the education systems in the arab countries has gradually improved over the last 30 years in the arab world. more work has been done by non-governmental and government agencies and organizations to improve the educational opportunities for arab students, and to promote their independence and integration into their societies and to prepare them for future careers, taking into account current trends in the labor market (13). few leading bioinformatics programs in arab countries have been established over the last decade in egypt, lebanon, ksa, uae, and oman. some of these programs have launched throughout the biotechnology and genetics departments of computer science, science and agriculture colleges in both special and governmental universities (14). so far, in arab countries, neither colleges nor departments have been specifically founded to teach bioinformatics. as multidisciplinary science, bioinformatics is difficult to teach, most of the programs are designed for undergraduate students in colleges that provide only one field required for bioinformatics. these programs address these issues by borrowing a few courses from other colleges, for example, when bioinformatics programs are embraced by computer science colleges, students are expected to pursue a few courses in biology in science or agriculture colleges. these courses could take place in the second or last years, and could be acquired in the worst situations a few weeks before graduation. this system creates a gap in the experience of the participant, a hole that must be filled in the next few years of his life as a researcher or even as an employee. beyond the regular education program, few bioinformatics training courses are offered and less are affordable to fresh graduates in countries most of which are classified as developing countries. the high cost of training, confusing education systems and the complexity of bioinformatics science has made it very difficult to teach and more difficult to study in arab countries. inducing bioinformatics education systems to move slowly toward improving the quality of life of arab residents and addressing crucial food and drug issues. this review is written in order to offer possible solutions to most of these issues and to guide future arab bioinformaticians throughout the best methods to study bioinformatics in a way that fits our social, financial and academic circumstances. it also addresses the key aspects that the bioinformatician needs to know about, such as how to begin learning the basic knowledge that needs to be learned, and how to overcome some of the social issues faced by young bioinformaticians. how to get started in bioinformatics bioinformatics, as already stated, comprises of three different fields: genetics, computer science and mathematics. the purpose of these three domains is important for the organization, comprehension and interpretation of the different biological information that bioinformatician handles on a daily basis. the question of studying these three areas is very difficult and few places of education provide this knowledge in one location. you've certainly learned the fundamentals of one or two of these areas as biology, computer science or mathematics graduate, you only need one or two remaining knowledge to become a bioinformatician. as you begin to learn, my tips are: 1) learn the basics, and then you will know where to go next. the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 3 of 10 2) this is about knowledge, not certification; certification will not indicate that an individual has adequate knowledge of the subject area. arab bioinformatics students are more worried about the credential than they are about the skills they need to acquire. 3) take what you need through free learning; this is your university and your home, attend those classes you need to learn, even if it is not your college. many students overlook the fact that most university lectures are not closed to free learners as long as they do not need a credential. 4) take a basic course; most of the basic courses offered in arabic countries cover weak bioinformatics skills such as primer design, sequence alignment, ncbi software and gene annotation using online tools. you may take one basic course if you don't understand the software, as long as you don't repeat courses with the same content. 5) ask as much as you can; there are many public forums for bioinformaticians that offer gatherings across all disciplines such as biology, mathematics, and computer science, where you can ask any question you want. 6) engage the scientific society as a free bioinformatician; i know it's hard for some arab students to work after graduation for 6-12 months without any kind of support. but it is about joining the research community , learning about the issues they face and seeking out how to manage it as a bioinformatician. the advantage of this would be that, if they don't pay they can't control you. you can choose to work on any type of data without any restrictions, the ability to attend any class outside the workplace, and if there is any other work opportunity you can apply. in fact, you should have a certificate of work experience and my recommendation is to look elsewhere if they don't offer you such a credential. taking in advantage that, most of the scientific institutions in arabic world can not offer payment for fresh graduates and they have limited funding, which made them in a big need for graduates who do not ask for payment. 7) read even when it is difficult to understand; bioinformatics is a science that is changing every day and few textbooks will cover all of its aspects so you need to keep reading. that you will be able to understand a few more every time you start reading new research, finally you will get a clear understanding of most of these articles. while you're reading, take care of the software that they have used and try to run it using sample data. supplementary 1 contains some simple articles and reviews you can start with. 8) self-learning is not the full answer; while self-learning through online resources such as youtube (15) and academia (16) is very helpful in expanding your knowledge, you need to deal with real problems by hand in order to sharpen your abilities, and this will not happen without joining a scientific group. 9) the way to learn more is to teach others; passing bioinformatics skills to others would open your eyes to different applications of the same tools and knowledge gaps, granting you more opportunities in the near future. operating systems and bioinformatics although most of the bioinformatics courses are interested in teaching software,i would suggest that it is not more about the tool than the environment in which you operate. many scientists in our arabic world use the microsoft windows operating system every day to manage and evaluate their data. microsoft's operating system is commercially closed and hackable (17). such an environment is not a natural space for innovation and research, especially when dealing with mega-biological data. the normal bioinformatics operating system should be opened (could be programmatically modified), highly secured, free of charge, and compatible with all bioinformatics software. of which only one operating system could have granted these privileges, linux. linux is a unix-like and often posix-compliant operating system (os) based on the design and distribution of free and open source code. the basic element of linux is the linux kernel, the very first operating system kernel introduced by linus torvalds on september 17, 1991. the free software foundation uses the name of gnu/linux to describe a complex operating system (18). most arab researchers fear linux operating system, depending on the false belief that linux is only a black and complicated command line window. on the contrary, linux can operate videos, games and handle all the types of data files you use in windows, and it has free writing programs like libreoffice (19) and a lot of wonderful programs and tools that could change your life. linux also gives users a bit of control about what occurs on their machine what does and doesn't changing. you could learn more about the advantages of linux over windows through a number of articles (20). linux has different flavor distributions such as ubuntu (21), fedora (22), opensuse (23), red hat (24) and many others. mostly there is no difference between the various linux distributions, although there is some variance between the software configuration , where it does not influence the core system performance or stability. ubuntu is one of the most common linux distributions (25), and the initial findings indicate that ubuntu do’s not require technical assistance (26), the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 4 of 10 has a defined and simple graphical user interface (gui) and has been incorporated into some arabic education systems (27). ubuntu has a great and powerful version named bio-linux (28). in 2002, dr. dawn field launched the bio-linux system under the nerc environmental bioinformatics program (29). bio-linux release allows easy access to a versatile computing environment pre-loaded with bioinformatics software from basic data analysis tools to advanced analytic framework programming packages (21). you can download and install bio-linux in an iso file format from the official website (http://environmentalomics.org/bio linux/). the only downside is that bio-linux distributions are a way behind ubuntu's latest updates, and most hardware drivers such as wifi, computer mouse, or touch pad, are not installed which involve certain installation skills. in order to overcome this issue, my recommendation is to install the latest ubuntu release and then download bio-linux packages via the linux synaptic package manager. alternatively, you can download the edition of bio-linux and then request the system to be upgraded with simple commands. with the ubuntu software center, you can quickly download and install thousands of linux software without any complications. the linux terminal is another way the software can be used (supplementary 2). although some tools require some command lines to be ready for analysis, most of them do not require installation skills. there are few linux guidelines for fresh bioinformatics students: 1) install and boot from bio-linux using flash memory for a short period of time while you are in practice (supplementary 2). 2) learn to use all the tools and software that ubuntu has provided, even if they were really basic, such as libreoffice and calculator. this would eliminate the fear of using a new operating system to allow you more comfortable with the environment. 3) bio-linux has a web page documentation, and sample data for most its bioinformatics software, try to open and use it through the desktop panel. 4) try to write your own easy manual notes for all software built on bio-linux, explain the use, input, output and how you can use this tool in your future research. in supplementary 2, i am explaining how to use a few of these tools. 5) after a few weeks try installing the latest version of ubuntu or bio-linux , update and lunch jemboss (30) using the ubuntu software manager 6) try to practice simple linux commands and use as many tools as you can. computer science and bioinformatics most arabic bioinformatics students prefer to start with programming languages such as python for biological data analysis. python has many advantages when it comes to bioinformatics and has several advantages as a computer language compared to languages such as perl, which is also a famous bioinformatics computer language (31,32). the problem, though, is that most of these learners neglect and ignore the basic rule of computer language learning, which is the need to understand the basics of computing and its core structures, such as object orientation programming (oop). on the other hand java is a very common computer language in our arabic country, and most commercial training companies offer paid courses to learn this language. my concern is that, although java has a smaller relationship with bioinformatics compared to python, few training companies offer python and fewer do so professionally in our arab world compared to java. in this regard, while you are learning computer language, you need to understand the fundamentals of computer science. these fundamentals involve algorithms that are a sequence of commands typically used to solve a problem or perform a computation, and can be expressed in a finite number of steps and a well-defined formal language (33). the best way to learn algorithms in your first step is to learn algorithms by programming languages that you have selected to learn such as java (34), python (35), and perl (36). using this method, in addition to learning the algorithm effectively, you will be able to sharpen your coding skills. after studying programming language in such a smarter way, you can easily add other programming languages to your set. among these languages is r, which is a statistical programming language and common computational tool for data analysts, and is it has become one of the most commonly utilized programming languages in bioinformatics software. this is mainly due to its performance and the richness of the libraries that could be used for data manipulation and simulation (37). r is very simple, and there are large numbers of r libraries developed for bioinformatics. it might be difficult for biological researchers to learn coding and algorithms, and my recommendations for easy learning are: 1) practice basic codes as much as you can, i would prefer to write these codes a couple times by hand and then run it under the computer language compiler. 2) don't spend much of your time on graphical user interface (gui) coding, most common bioinformatics software don't have a gui, but instead focus on learning programming skills. 3) do not forget that, for the purpose of interpreting biological data, you are studying programming language, and you are not a computer scientist. based on this, don't go deep into programming language http://environmentalomics.org/bio-%20linux/ http://environmentalomics.org/bio-%20linux/ the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 5 of 10 learning, instead of considering what it can give you to achieve your goal. 4) after learning basics, you can search for programming codes that address simple biological issues such as dna transcription and translation, reversing dna sequence, and scripts which reflect the use of simple genetic concepts without the use of external libraries. 5) do not use libraries/modules/packages for simple tasks in your first steps; you need to learn how to use computer language to apply scientific principles in algorithmic analysis. 6) organize yourself; the first lines of your scripts should indicate the use of the script and its input and expected output data. you'll use these scripts in several occasions, and it's unfair to waste your time rewriting the same code. 7) you can use the same script or tool differently; this depends on your imagination. 8) try to write clean and structured scripts where the overused programming functions can be found in wellorganized libraries. it would provide the ability to use the same collection of libraries for various uses in different projects. 9) backup your scripts every week. mathematics and bioinformatics most educational systems in our arab world are concerned with teaching students without proper application of mathematics principles where student knowledge of these basics are not linked to their use. there is an easier way to teach mathematical rules in other countries by providing real-life experiments to learners (38). most arabic students have a decent mathematical background based on their high school education, which might be enough to start learning the science of bioinformatics, but my basic concern is that, they need to get missing knowledge through straightforward courses. the main target is a sub-science of mathematics, statistics. statistics is a systematic and random analysis of variability. in many aspects of scientific research, statistical methods are valuable. they constitute the research of the right way to collect process and interpret data (40). the connection between statistical methods and bioinformatics is very critical; you might argue that most of the biological data analysis could not be conducted without a good statistical background (41). as a bioinformatician, you will continue to learn the fundamentals and implementations of this science in biology for the most part of your life. on the other hand, the basics of this science will need to be practiced in a simple way, where you can use r programming to study statistical methods. as i mentioned earlier, r is a programming language that has been written for statistical analysis in general, and studying basic statistics through r could provide an opportunity to learn a new language and to understand the basics of statistics in the main time. this doesn't mean that you don't have to solve statistical problems by hand, but it does mean that you have to understand both ways. early publishing of students and graduates although there are concerns about early scientific publishing in the arabic education system, it is very important for bioinformatics students to publish their research, scripts, and pipelines (series-connected processing systems in which the output of one component is received from another) during their early years. some of these articles would not include much, but this is aimed at improving their interest in research. it would also give them the ability to sharpen their writing skills, address the comments of reviewers and the international research society. this would add to their computing skills in early age more specificity and professionalism. you should try to write your publications for the tools, scripts, pipelines or methods you have developed with or without other colleges and you need some guidelines on how to publish in this regard: 1) first of all, you need to know that most of the software articles are one or two pages long. it must include a brief introduction, a comprehensive methodology and some discussion. 2) don't extend your article beyond two pages, the more you do, the more unstable your sentences will be. 3) discuss the benefits of your tool, how you deal with the input, where adding a flowchart algorithm would be efficient. 4) use simple english. 5) cite previously published work by comparing your tool with others. it doesn't matter if your tool doesn't add much; the most important thing is that, it's your tool. 6) start with small publications. 7) search for free journals. in this regard, biorxiv (42) is a non-profit, electronic archiving and distribution platform for the pre-printing of life science research papers. it was founded by the cold spring harbor laboratory, a scientific and academic organization. learn new programs and use published scripts hundreds of bioinformatics tools are released daily, specialized in the analysis of different data types, and written in a variety of programming languages. in order to use these tools, you need to take two steps: (1) test the configuration of the software using sample data and (2) read tutorials (if available) describing the different parameters and input data the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 6 of 10 that this tool could handle, which would enable you to understand and analyze the outputs of these tools. using sample data and reading tutorials will save you time and provide you with a simpler way to resolve the error messages. on the other hand script archives such as githhub are the most important source for software scripts with more than 10 million repositories (43). such websites offer you the ability to access open source software, script codes and pipelines in c, python, perl, r and other programming languages freely. you may be able to use such resources to know more about coding or bypass basic programming functions by using these codes as external libraries, but ethically you need to reference the published websites, articles or acknowledge the source of these scripts in your codes. it could be difficult to use these scripts and you need advice on how to handle it: 1) begin with basic codes; you can find several tutorial codes for programming language implementations in bioinformatics, which you will find useful in sharpening your programming skills. 2) in order to understand any script, you need to divide the script parts according to what they do, use the print function to show the performance of the input processing stage after each step on the screen. 3) to understand any code structural algorithm, block, remove and replace any line debug (run by compiler) and monitor performance changes. furthermore, the key to understanding the overall algorithm could be to comprehend how to evaluate script errors when ignoring certain coding lines. 4) draw a basic flowchart that demonstrates your interpretation of how this program manages input data. start learning cloud computing the management of large biological data requires sufficient computing power and storage capacity. cloud computing can provide arab research with large capabilities via open and often free websites. some of these servers are galaxy (44) and cyverse (45). from my point of view, all these clouds and others provide computing resources and storage capacity to handle biological data, but i would prefer cyverse more because it is more robust and offers a lot of software and simple gui interfaces. using such clouds can offer you the ability to manage big information and to conquer the poor and slow speed of the internet which inhibits you from accessing those data; you can do even more than that. such as: 1) cyverse gives you the ability to email your scripts to administration to handle or convert those scripts to a tool that you can use. 2) using the tools available to build your own pipeline. 3) you can transfer or upload data from a private fttp server such as ncbi (46), ensemble (47) or from your computer. 4) choose to use the storage capacity provided by these platforms to store data instead of overfilling your computer memory. 5) such platforms have their own research communities, where you can submit your questions. life as a bioinformatician in arabic countries many arab countries do not support research (48) and fresh bioinformaticians may suffer from a lack of funding. my answer to this problem is to begin your career as a freelancer. freelance employees are terms that are commonly used by a self-employed person who is not usually a long-term employer. fresh bioinformatics graduates may offer their data analysis expertise to local and international research groups in exchange for money. in this manner, they will support their continuing self-education and their personal goals. considering that, this path needs good research knowledge, free and low-cost services and communication skills. while freelance is a good way to start your life as a bioinformatician, some considerations are required: 1) don't manipulate output reports for those who want to finish their dissertations without having worked. 2) finish the work of each researcher without doing more than is necessary and not less than is essential. 3) note that freelancing is not a type of research, and you need to write any scientific papers for yourself in order to build your own scientific background. 4) once you have a successful and well-paid position, avoid freelancing and start sharing the credit for your work. bioinformatics and communication skills communication skills for bioinformatician are more than necessary and are very important for researchers, especially when working in scientific teams. communicating with other bioinformaticians, biologists and researchers is critical to understanding, resolving and addressing daily bioinformatics issues. such challenges could be software bugs, incorrect results and experimental methodologies. in most of our arabic countries, there is a weak link between different scientific groups so, for order to overcome such difficulties, arab bioinformaticians could use research communities to interact with each other and with international scientific teams. there are various advanced research, e.g. ask ubuntu (49), bioinformatics (50) and researchgate (51). such groups will help young scientists start their careers and find new bioinformatics problems that they could address. the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 7 of 10 the future for you following basic learning, one or two programming languages and the handling of different biological data through a variety of analytical techniques, the expected next phases are as follows: 1) learn c programming language; c is faster and consumes less memory than other programming languages. although learning c is difficult, most famous bioinformatics programs such as the blast (52) software package are written in c. the problem is that python, perl and r are slower than c when handling large data, and then when performing complicated processes and memory management are needed (53). you will need to write some of your codes in c in the future to make your program faster and more efficient, even if these scripts need more lines to express. like c, fortran is a very important programming language, particularly when it comes to statistical analysis, and some may argue that it is faster than c (54). 2) boost your understanding of shell scripting. shell is the programming language used by the linux system (55), improving your reading and writing skills in such a language will enhance the management of complex tasks. in some cases, you can create hybrid scripts where you compose an algorithm to manipulate data throughout different computer programming languages such as python, c, julia and r, in addition allowing you the flexibility to use what the linux system could provide. 3) don't repeat your work; try to handle different types of data and use different methods of analysis to gain more knowledge and experience. 4) machine learning (ml) is a natural outgrowth of a combination of computer science and statistics and answers the question of how to build machines that automatically learn from experience (56,57). understanding ml is very essential for your career and could aid your scientific background. python, r, java and c have specific libraries for ml, but python has the maximum attention (58). 5) begin the web programming learning process. internet development is essential in order to make massive, definitive data available to the scientific community. in fact, you could create your own online software that would make it easier for scientists with poor programming skills to use. python has its own web programming packages, such django. mental and social life of arabic bioinformatician research life is demanding, frustrating and highly competitive. where postgraduates need high marks for their courses and a variety of publications under their belts if they want to earn excellent scholarships and research positions (59). it is important to take part in activities and social structures in order to promote the academic and social growth. many current students in bioinformatics have been prepared to learn new research methodologies and programming languages and to ignore social life. sadly, this kind of behavior is very risky with several articles on psychological science relating mental illness with creativity (60). as a bioinformatician, you need to socialize with others and participate in outdoor activities to boost self-efficiency and promote team harmony. these type of activities are highly recommended in the field of mental health through a number of scientific articles (61). in fact, physical activity has been shown to be related to mental health and can play a key role in sustaining moderate to severe mental health conditions, especially anxiety (62). first of all, there is a high rate of depression and work stress, especially for those working as researchers in developing countries who need to maintain their mental health in order to thrive and thrive. conclusion bioinformatician is a person who has the ability to take advantage of three different sciences, to think innovative and to reshape complexity into simplicity. his capabilities rely on a sound scientific experience, a high level of knowledge in addition to patience, enthusiasm and productivity. our arabic world needs this kind of expertise in translating biological knowledge into practice, preserving our natural ecological resources and boosting our standard of living. as citizens, we can not change the education system, the economic climate or our society, but as researchers, we can inform others how to deal with these conditions and achieve their goals with less sacrifice as possible. the secret of learning bioinformatics relies basically on the willingness of our students and researchers to acquire new sciences. bioinformatics as a discipline is the most attractive and interesting field in which lack of resources, materials and equipment is not a concern, but requires additional knowledge and a high level of creativity that seems as easy to learn as it is difficult to obtain. my final advice is to be guided and to lead, both procedures could fill the gap in your scientific background, where opening your mind to new ideas would result in a better way, and deeper understanding and interaction with others would offer you multiple choices. acknowledgement the author would like to thank dr. morad mokhtar (molecular genetics and genome mapping lab agricultural the art of bioinformatics learning in our arabic world highlights in bioscience doi: 10.36462/h.biosci.20193 october 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 8 of 10 genetic engineering research institute, arc, egypt) and dr. abdulqader jighly (department of economic development, jobs transport and resources australia) for their valuable support in this research. supplementary files the supplementary material for this article can be found online at: http://supplementary.highlightsin.org/20193_sup.zip supplementary file 1: articles and reviews on bioinformatics could be used to initiate the learning process in this field. supplementary file 2: short tutorial for some bio-linux basics. references 1. welch l, lewitter f, schwartz r, brooksbank c, radivojac p, gaeta b, et al. bioinformatics curriculum guidelines: toward a definition of core competencies. plos comput biol. 2014;10(3):e1003496. 2. bansal ak. bioinformatics in microbial biotechnology-a mini review. microb cell fact. 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science (80). 2015;349(6245):255–60. 58. müller ac, guido s, others. introduction to machine learning with python: a guide for data scientists. ― o’reilly media, inc.; 2016. 59. ortega a. three tips to achieve academic success — while enjoying a social life. nature. 2019 60. nettle d. schizotypy and mental health amongst poets, visual artists, and mathematicians. j res pers. 2006;40(6):876–90. 61. fernee cr, gabrielsen le, andersen ajw, mesel t. therapy in the open air: introducing wilderness therapy to adolescent mental health services in scandinavia. scand psychol. 2015;2. 62. paluska sa, schwenk tl. physical activity and mental health. sport med. 2000;29(3):167–80. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202306 research article open access 1 department of plant protection, faculty of agriculture, university of aleppo, syria. 2 affiliation african genome center, mohammed vi polytechnic university, ben guerir, morocco. 3 international center for agricultural research in the dry areas (icarda), terbol, lebanon. 4 harvestplus, pakistan office, crop sciences institute, narc, park road, islamabad 44000, pakistan. 5 department of genome mapping, agricultural genetic engineering research institute, agricultural research center, giza p.o. box 12619, egypt. 6 international center for agricultural research in the dry areas (icarda), giza, egypt. 7 international center for agricultural research in the dry areas (icarda), rabat, morocco. * to whom correspondence should be addressed: a.hamwieh@cgiar.org editor: ayed m. al-abdallat, faculty of agriculture, the university of jordan, jordan. reviewer(s): eman hashem radwan, ecology and marine biology, damanhour university, egypt. elton eduardo novais alves, university of são paulo piracicaba , brasil. received: october 10, 2023 accepted: december 24, 2023 published: december 30, 2023 citation: ali l, el bouhssini m, istanbuli t, imtiaz m, alsamman am, nassar ae, baum m, hamwieh a. identifying genetic linkage groups and markers for leaf miner resistance in chickpea through qtl analysis and field validation. 2023 dec 30;6:bs202306 copyright: © 2023 ali et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. identifying genetic linkage groups and markers for leaf miner resistance in chickpea through qtl analysis and field validation lina ali 1 >< , mustapha el bouhssini 2 >< , tawffiq istanbuli 3 � >< , muhammad imtiaz 4 >< �, alsamman m. alsamman 5 ><�, ahmed e. nassar 6 >< �, michael baum 7 ><�, aladdin hamwieh 6 ><� abstract chickpea is a commonly grown crop, but it is vulnerable to biotic and abiotic stresses. leaf miner (liriomyza cicerina) is a pest that can cause severe yield losses of up to 40% if not properly controlled. this study was conducted at icarda (aleppo, syria) during the 20112012 growing seasons. two recombinant inbred lines, ilc 5901 (lm resistant) and ilc 3397 (lm susceptible), were crossed to yield 350 f2 plants, which were then screened for pathogen tolerance. the resistance of the plants was screened using a scale of one to nine, with 1 indicating complete resistance and 9 indicating complete susceptibility. a set of 600 simple sequence repeat (ssr) markers were validated on both parents, and 51 of these markers showed variation and were used to construct a genetic linkage map. qtl analysis was performed to determine the linkage groups responsible for line variations. the qtl analysis found that linkage groups ta37, ta34, and h4f03 were responsible for 22% of line variations, while unmapped ncprg48 and h1c092 revealed 55.3% and 26.8% of the lis variance, respectively, and displayed a warped dominance toward the susceptible parent. the h1c092 marker, which is significantly associated with lm, is located on chr3 near a gene encoding the glutathione s-transferase gene family enzyme, which protects cellular macromolecules from attack by reactive electrophiles. the highly associated markers were field tested for three years to confirm their connection with lm resistance in 200 chickpea genotypes. the study showed marker-associated selection, which could accelerate the conventional breeding of lm-resistant chickpea germplasm. the markers linked to lm resistance and the identification of the protective enzyme gene offer promising avenues for further research. this study represents a significant step forward in understanding the genetics of lm resistance in chickpea and provides valuable information for breeding programs aimed at improving chickpea production. keywords: leaf miner, chickpea, ssr markers, liriomyza cicerina introduction chickpea (cicer arietinum l.), or bengal gram, is one of the seven neolithic essential crops of the near eastern fertile crescent [1]. it is the first legume food source in south asia and the third globally, after common bean and field pea [1]. additionally, it is a significant crop in the middle east, mediterranean, india, and ethiopia, providing both food and protein [2; 3]. it is grown in over fifty countries, including north africa, the indian subcontinent, the middle east, southern europe, the americas, and australia [1]. with an annual production of 17.2 million tons, chickpeas are grown on 17.8 million ha of land worldwide [4; 5]. chickpeas are classified into two species: desi and kabuli. kabuli (macrosperma) has white flowers, little anthocyanin pigmentation, and beige seeds in the form of a ram’s head, whereas desi (microsperma) has pink flowers, anthocyanin pigmentation on stems, and a colored, thick seed coat [6]. despite all of the chickpea’s benefits, there have been some biotic and abiotic stresses that have reduced and threatened its annual production. [7; 8]. highlights in bioscience page 1 of 9 december 2023|volume 6 https://doi.org/10.36462/h.biosci.202306 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:lina.7755@gmail.com mailto:mustapha.elbouhssini@um6p.ma https://orcid.org/0000-0001-7450-6408 mailto:t.istanbuli@cgiar.org mailto:m.imtiaz@cgiar.org https://orcid.org/0000-0002-7536-9932 mailto:smahmoud@ageri.sci.eg https://orcid.org/0000-0002-7765-5035 mailto:hindu8446@gmail.com https://orcid.org/0000-0001-7102-776x mailto:m.baum@cgiar.org https://orcid.org/0000-0002-8248-6088 mailto:a.hamwieh@cgiar.org https://orcid.org/0000-0001-6060-5560 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea among the biotic factors that pose a great danger to chickpea production is liriomyza cicerina. depending on the severity of the pathogen, leaf miner infection could result in yield losses of up to 36% in west asia, north africa, and southern europe [9]. according to icarda, chickpea yield losses due to leaf miner infection could represent up to 40% of total production in syria and other countries [10; 9]. insecticides are the traditional method of lm control; however, this method has many drawbacks, including increased pest resistance to chemicals, which may have negative effects on human health and cause catastrophic diseases such as cancer [11]. therefore, there is an urgent need to develop nontraditional methods of controlling this pest. host plant resistance, also known as the premeditated use of resistant breeds to reduce the harmful effects of pests, is the most effective, sustainable, and safe way of maintaining crop production systems [12; 13; 14]. these resistant genotypes frequently suffer lower leaves damage compared with susceptible cultivars. screening of chickpea germplasm was found to have useful levels of resistance to liriomyza cicerina [15]. these resistant genotypes usually have less leaf damage than susceptible cultivars, which could be due to the organic acids in resistant germplasm [16]. the organic acids exuded by cicer species vary, but the most prevalent are malic, oxalic, succinic, citric, and quinic acids [17; 18]. molecular genetics is an important method for selecting and developing resistant genotypes in a variety of species [19]. the detection of molecular markers that are closely linked to resistance genes is extremely beneficial in replacing time-consuming and frequently unreliable field evaluations with molecular techniques [20]. qtl is a term that describes the locations, numbers, amounts of phenotypic effects, and mechanisms of gene activity of breed factors that contribute to the inheritance of variable traits [21]. the qtl analysis uses a linkage map as a "framework" to pinpoint the chromosomal locations of genes conferring quantitative resistance [22]. although it is broadly useful to study in many fields, qtl mapping is particularly important in agriculture. it is an accurate measure of yield quality and productivity in agricultural fields that serves as the culmination of an organism’s life cycle [21]. although rflp (restriction fragment length polymorphism) is the most commonly used marker in qtl mapping, the current study employed ssr markers in linkage mapping [23]. the current study is a step forward in identifying markerassociated selection that could improve and speed traditional farming by developing novel resistant varieties for leaf miners. materials and methods the field experiment and disease score the 350 tested f2 were produced by crossing chickpea genotypes ilc5901 (lm resistance, multi-pinnate leaf type, small leaflet size) and ilc3397 (lm susceptibility, normal leaf type, large leaflet size) (figure 1). they were planted on the icarda (international center for agricultural research in the dry areas) field in tel hadya, syria. the plants were graded on a 19 leaf infection scale (lis) according to the percentage of infected figure 1. the two leaves type: a-first parent ilc3397, lm susceptible, normal leave type, large leaflets size. b-second parent ilc5901, lm resistance, multipinnate leave type, small leaflets size. leaflets per plant, where 1 = no infections (no mines), 2= less than 5% and no defoliation, 3= less than 20% and no defoliation, 4= between 21% to 30% and no defoliation), 5= between 31% to 40% with little defoliation, 6= 41% to 50% and 10% of the lowest leaves were dropped, 7= 51% to 70% and 10% to 20% of the lower and upper affected leaves were defoliated, 8= 71% to 90% and 20% to 30% of diseased upper and lower leaves are lost, and 9 = miners observed on all the leaflets and falling more than 30% of the leaves. in general, plants with lis=1-3 were considered resistant, lis=4 plants moderately resistant, lis=5-6 plants moderately susceptible and lis=7-9 plants were susceptible [9]. the genotypes were screened when the susceptible parents showed lis>5 to lm under natural insect infestations. the segregation of the leaf size (large or small leaflets) and type (multi-pinnate-small leaflet or normal type-large leaflet) were also collected from the field. dna isolation dna was isolated from seedling leaves after 4-6 weeks using the cetyltrimethylammonium bromide (ctab) technique, as follows: fresh seedling leaflets were freeze-dried for three days. in an eppendorf tube, two metal balls (4 mm in diameter) were ground into powder. the ctab buffer solution has the following ingredients: 2% cetyl trimethylammonium bromide, 1% polyvinylpyrrolidone, 100 mm tris-hcl, 1.4 m nacl, and 20 mm edta. after mixing and vortexing the suspension, 1 ml of phenol/chloroform/isoamyl alcohol (25:24:1) was injected, and the tube was placed in a 60řc water bath for 60 minutes. after centrifuging the homogenate for 20 minutes (4000 rpm at 4řc), the aqueous upper phase was transferred to a new tube, and the process was repeated until the upper phase was definite. this solution will then be given 700 ul of isopropanol. the sample was centrifuged at 14,000 x g for 10 minutes before being washed twice using a washing buffer composed of 75% ethanol and 200 mm sodium. the supernatant was dried for 1020 minutes to eliminate residual ethanol before being dissolved in highlights in bioscience page 2 of 9 december 2023|volume 6 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea 100 ţl te buffer (10 mm tris hcl, ph 8, 1 mm edta). the chickpea primer sequences were retrieved from these articles [24; 25; 26; 26; 27; 28; 29] in order to use polymerase chain reaction. the thermocycler (perkin elmer pcr system 9700) was used to conduct pcr (10 ml mixture) with 25 ng template dna, 10 pcr buffer, 5 pmol forward and reverse primers, dntp, and 5 u of taq dna polymerase. the pcr technique divides the pcr analysis into three major steps: the first is denaturation, which requires 35 cycles of heated solution at 94 řc for 15 seconds; the second is annealing, which lasts 30 seconds at 72 řc; and the third is extension, which takes 5 minutes at 72 řc. the final step is pcr gel electrophoresis, which involves electrophoresizing the pcr products on an 8% polyacrylamide gel containing ethidium bromide. statistical analysis the joinmap 4 software, which is used for genetic linkage calculations in experimental populations of diploid species, was used to construct genetic linkage mapping [30]. it improved the possibility of mapping genes for the enhancement of the chickpea crop by providing more knowledge of chromosome organization, parental relationships, and gene ordering [31]. the linkage groups were given to the chromosomes depending on recombination frequencies less than 0.45 (θ >.45) and lod greater than 3. mapqtl 6 software was employed for qtl mapping, as well as the genetic distances (centimorgans, cm) were determined using the kosambi function [32; 30]. utilizing composite interval mapping, it was observed that the significant qtl had a lod threshold greater than 2 [33]. validation of associated pcr markers to validate their association with leaf minor resistance and to investigate their potential linked function, five associated lm ssr markers (h1c092, h4f03, ta34, ncpgr48, and ta37). these markers have a significant association with the leaf minor phenotype in qtl analysis. the figs (focused identification of germplasm strategy) was used to pick 200 chickpea genotypes. it is a technique that has been scientifically proved to help crop breeding programs uncover important features in plant genetics more correctly and effectively, overcoming the disadvantages of more traditional approaches that are essentially hit-or-miss [34]. figs is a trait-based approach that helps genebank managers identify desired genetic material that is likely to have the desired trait [35]. over three years (2014, 2015, and 2016), these genotypes were evaluated for lm resistance throughout three replicates each. the extracted dna was used in pcr analysis for potential lm associated ssr markers using the aforementioned methods. the powermarker software [36] was used to examine the marker-trait relationship between genetic polymorphism in potentially associated ssr markers and lm resistance phenotypic variation. using kaspspoon software [37], an in silico analysis and comprehensive investigation was conducted for the potential location of these pcr markers on the chickpea genome [38] and nearby genes. results phenotyping data the results indicated only 35 (10%) out of 350 f2 plants were resistant (lis =<3), 52 plants (14.9%) were moderately resistant (lis=4), 121 plants (34.6%) were moderately susceptible (lis =5, 6), and 142 plants (40.7%) showed susceptible (lis =7-9; figure 2). f2 plants showed about 94.3% of resistant plants (33 out of 35 plants) were multi-pinnate type leaf and small leaflet, and 99.29 % of susceptible plants (142 out of 143 plants) were normal type leaf and large leaflet (table 1). a significant negative relationship (r= -0.92 and p < 0.001) discovered among leaf minor resistance and leaf type, and leaf size, while a high positive correlation was found between leaf type and leaf size (r=1; p < 0.001). figure 2. frequency distribution of lm resistance of the 350 f2 plants derived from p1= ilc5901 (lm resistance) and p2=ilc3397 (lm susceptible). values of the parents are shown by arrows. figure 3. the distribution of leave type and leaflet size in 350 f2 plants. mapping and qtl analyses validating 600 ssr markers on the parents (ilc5901 and ilc3397), it was determined that only 51 (8.5%) had polymorphisms, which were subsequently utilized for mapping. there were only 44 markers mapped on 8 linkage groups spanning 966.3 cm and seven (13.7 %) remained unlinked (gaa47, ncprg48, ta106, h1b13, h1b09, ta140 and h1c092), with an average highlights in bioscience page 3 of 9 december 2023|volume 6 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea table 1. percentage and the number of plants with the leave types and the size of leaflets of 350 f2 plants. degree of resistance multipinnate-small leaflet normal type-large leaflet resistance plants 94.3 % (33 plants) 5.7 % (2 plants) moderately resistance plants 84.6 % (44 plants) 15.38 % (8 plants) moderately susceptible plants 3.3 % (14 plants) 96.69 % (107 plants) susceptible plants 0.7 % (1 plant) 99.29 % (142 plants) table 2. percentage and the number of plants with the leave types and the size of leaflet of 350 f2 plants. s/n locus a h b x2 significance 1 ncprg48 19 33 42 2 19.6 ******* 2 gaa47 18 67 10 1 17.36 ****** 3 h3f09 26 22 6 42 16.67 ****** 4 h1b13 25 61 8 2 14.49 ***** 5 h3g06 13 63 14 6 14.42 ***** 6 ta144 37 48 11 0 14.08 ***** 7 ts54 38 37 18 3 12.48 **** 8 ta180 14 65 15 2 13.81 **** 9 ncprg89 8 56 27 5 12.78 **** 10 h3c-06 38 38 20 0 10.92 **** 11 h4f03 32 31 32 1 11.46 **** 12 h3d05 17 40 39 0 12.75 **** 13 ts82 22 26 32 16 12.3 **** 14 h3e04 34 32 26 4 9.91 *** 15 ta1 12 52 31 1 8.45 ** 16 h5d02 34 40 20 2 6.26 ** 17 h4h08 33 36 26 1 6.6 ** 18 h5f021 34 36 23 3 7.34 ** 19 h1b09 17 40 35 4 8.61 ** 20 ta140 27 42 9 18 8.77 ** 21 h1d221 34 34 22 6 8.58 ** 22 h4b08 20 37 33 6 6.6 ** 23 h3a10 28 50 14 4 4.96 * 24 h1f21 34 40 21 1 5.93 * distance among the markers of 18.94 cm. out of the 24 (47%) distorted markers in the 1:2:1 mendelian ratio, the chi-square testing indicated that 19 (37.2%) were distorted at a p-value <0.01, and five were highly distorted at a p-value <0.001 (table 1). qtl analysis identified that three linkage groups (lg2 (ta37), lg3 (ta34), and lg5 (h4f03)) explain 22% of the lis variations, and they also confer chickpea resistance to a great extent. these linkage groups have flanking markers of (ts 54, ta200), (ta 125, h5h032), and (tr 29, h2i10). interestingly, two unmapped markers (ncprg48 and h1c092) were substantially associated with lis, accounting for 55.3% and 26.8% percent no. lg position locus lod % expl. additive dominance 1 ug 3 ncprg48 16.77 55.3 1.50 1.37 2 ug 11 1c092 6.5 26.8 1.48 0.33 3 4 80. 6 h4f03 5.23 22.2 -0.83 1.31 4 1 167.89 ta37 3.58 15.8 -0.97 -1.04 5 3 23.04 ta34 2.24 10.2 0.86 -0.37 table 3. the marker associated with lm resistance in chickpea in addition to the additive and dominance effects across linkage groups (lg) and un-linked group (ug) of the lis variation, respectively (table 2). these indicators revealed a dominant tendency toward the vulnerable parent. according to the additive effect for the markers with lod >2, qtls (ncprg 48, h1c092, and ta34) source the susceptible chickpea genotype "ilc3397," while (ta37 and h4f03) source the resistant chickpea genotype "ilc5901". the qtls (ncprg48, h1c092, h4f03 and ta37) are major markers with a high explanation percentage (table 3 and figure 4). figure 4. statistical explanation and genetic state of the ssr marker "ncprg48" in lm sensitive and resistant genotypes. highlights in bioscience page 4 of 9 december 2023|volume 6 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea primer pcr band size year pvalue h1c092 375 2014 7.50 × 10−03 2015 2.35 × 10−02 380 2014 4.61 × 10−02 2015 2.18 × 10−04 h4f03 305 2016 3.72 × 10−04 ncpgr48 215 2015 1.09 × 10−02 ta34 280 2014 9.36 × 10−04 ta37 375 2015 3.27 × 10−02 table 4. percentage and the number of plants with the leave types and the size of leaflet of 350 f2 plants. validation of leaf miner associated qtls the highly associated markers were chosen for further experimental validation to confirm their connection with lm resistance and to investigate their potential function. we selected 200 chickpea genotypes chosen using figs [34]. over three years (2014, 2015, and 2016), these genotypes were evaluated for lm resistance. the marker-trait association analysis revealed that the h1c092 marker had a significant association with the lm phenotype over two years, whereas the other markers were only associated with one (table 4). in silico pcr analysis was used to determine the possible location of the pcr marker on the chickpea genome and nearby genes (figure 5). the in silico pcr indicated that the h1c092 marker was found on chr3, and it corresponds to a region located near a gene belonging to the glutathione s-transferase (gst) gene family (figure 5). discussion our findings demonstrated that genotypes with simple leaf types were the most vulnerable to lm damage (lis = 69), whereas those with multi-pinnate and tiny leaves were resistant (lis = 23) and moderately resistant (lis = 4) (fig. 3). similarly, toker et al.[39], have screened 15 chickpea lines with three leave types: normal, simple, and multi-pinnate. they found that the plants with simple leave type and large leaflet were susceptible, and the resistant plants had multi-pinnate leave type and small size. el bouhssini et al. [10], found that the number of eggs laid in the susceptible chickpea line ilc3397 was higher than the resistant lines (ilc5901) that had multi-pinnate leave type. singh and weigand [40] released three leaf genotypes that are resistant to leaf minor with multi-pinnate and small leaflets (ilc 5901, ilc 7738, and ilc 3800). the results showed that only a few (35 plants) of f2 plants were resistant; similarly, out of 350 f2 plants, only 35 (10%) showed resistance (lis =<3), 52 plants (14.9%) were moderately resistant (lis=4), 121 plants (34.6%) were moderately susceptible (lis =5, 6), and 142 plants (40.7%) showed susceptible (lis =7-9). in another study, 174 chickpea germplasm samples were evaluated in the field at three different locations in pakistan (narc, aari, and niab). however, resistance to lm was observed in 28 narc lines, 24 aari lines, and 30 niab lines. similarly, 44, 47, and 30 lines at narc, aari, and niab exhibited moderately resistant reactions to lm, respectively [41]. toker et al., [39] also stated that lm resistance was strongly related to leaf type and leaflet size, but that there was no significant relationship between resistance and pigmentation. leafminor resistance was found to have a negative relationship with leaf type as well as leaflet size (r = -0. 92; p 0.001); these findings are consistent with sithanantham and reed’s [42] observation that lm prefers large leaflet chickpea lines. taleei et al., [43] reported a negative association among leaf size and blight score, implying that genotypes with large leaves may be more susceptible to ascochyta blight. the chi-square test revealed that 24 markers (47%) differed from the expected mendelian ratio of 1:2:1, which could be attributed to gametic [44], zygotic [45], or both selection, chromosomal recombinations with little effect on fertility and chromosome combining throughout meiosis [46], or a correlation to a lethal allele in sperm and eggs phases [47; 48]. a clustering of deformed loci has frequently been discovered inside lgs constructed by different species [49]. furthermore, a similar clustering pattern of markers was originally described in field peas and chickpeas [50; 51]. although segregation deformation can be seen in intraspecific crosses, the inclusion of the lens culinaris ssp. orientalis accession in the hybrid process may have increased the likelihood of lower recombination in the mapping population, therefore segregation distortion. durán et al. [52] employed 17 rapds, 13 issrs, 41 aflps, and one ssr that deviated from mendelian segregation and found that 24.5% of the markers were distorted in f2 populations. results from multiple studies illustrate that segregation distortion can be quite high, particularly in cicer sp. and lentil. winter et al. [51] and collard et al. [20] both reported values of 38.4%, and 14% respectively in these populations. similarly, sherman [53] and xu [54] reported distortion levels of 50% and 25% in chickpea and wheat, respectively. although the influence of distorted markers is often overlooked, this study found a significant segregation distortion of 38.4% for rils from a crossover in cicer sp. and 14% in an f2 lentil population [51; 55]. our findings have revealed five potential qtls associated with chickpea resistance to leaf miner infestation. these locations are believed to be implicated in several types of resistance mechanisms, including non-preference or antixenosis (the shape of leaves, either normal or multipinnate) and antibiosis (the exudation of oxalic acid from trichomes of resistant and moderately resistant chickpea plants, which reduces injury from infestation). a study by rector et al. [56] made use of 128 aflp markers, distributed across 30 linkage groups, to identify qtls correlating to antixenosis and antibiosis mechanisms for helicoverpa zea. another study identified a major qtl related to antibiosis mechanisms for helicoverpa zea, with a high explanation percentage [57]. the qtl related to maysin production (a glycosyl flavone that controls antibiosis in h. zea larvae) explains 55% of the variance in maysin synthesis [58], while the other explains 64% highlights in bioscience page 5 of 9 december 2023|volume 6 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea figure 5. physical location of leaf miner associated marker of h1c092 and its nearby gene. of the variance in apimaysin synthesis [59]. the potential qtls were validated using a broad sample of chickpea genotypes over a three-year period to deepen our understanding of their association with lm resistance. results from the validation showed that not all qtls showed significant association with lm, but this does not necessarily imply a weak correlation and further studies may be needed. however, one marker was found to have a significant association with lm located on chr3 near a gene belonging to the glutathione stransferase (gst) gene family (as shown in table 4 and figure 5). gsts are widely distributed, multi-functional enzymes that play a crucial role in a plant’s response to various stress conditions, including biotic stress [60]. gsts with glutathione peroxidase activity can play an important role in plant antioxidant defense by preventing the spread of hypersensitive response-associated apoptosis [61]. several studies have shown increased gst enzyme activities in plant-pathogen interactions, and functional studies have revealed that these enzymes can influence antimicrobial resistance in the host plant [62]. additionally, gst activity has been associated with lm resistance in several plant species including tomato [63] and common bean [64], highlighting its significance as a key for plant resistance. conclusion improving breeding programmes to strengthen resources against current and future plant pathogen impacts is crucial. this is the first report for ssr markers associated with lm resistance in chickpea, which may help in guiding and enhancing the use of marker assisted selection to speed up the conventional breeding of lm resistance in chickpea. we validated the potential application of several qtls linked to lm resistance. field and experimental validation were extremely useful in confirming the utility of some of these markers. furthermore, we employed genome annotation to determine the physical location of significant ssr markers and understand their relationship with the chickpea biological resistance. we discovered a putative link between the gst gene family and the resistance to chickpea leaf miner. our findings suggest that these markers can be used to identify elite chickpea genotypes, and we encourage future researchers to investigate the molecular mechanism of this gene family in chickpea resistance in greater detail. reference 1. jukanti ak, gaur pm, gowda c, chibbar rn. nutritional quality and health benefits of chickpea (cicer arietinum l.): a review. british journal of nutrition. 2012;108(s1):s11-26. 2. santra d, ratnaparkhe m, muelhbauer f, kaiser wj. identification and mapping of qtls conferring resistance to ascochyta blight in chickpea. 2000. 3. çıkman e, civelek hs. population densities of liriomyza cicerina (rondani, 1875)(diptera: agromyzidae) on cicer arietinum l.(leguminosae: papilionoidea) in different irrigated conditions. 2006. 4. knights e, acikgoz n, warkentin t, bejiga g, yadav s, sandhul i. area, production and distribution. chickpea breeding and management. 2007:167-78. 5. faostat f, production ac. food and agriculture organization of the united nations, 2014. roma, italy. 2014. 6. moreno mt, cubero j. variation in cicer arietinum l. 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lens. theoretical and applied genetics. 2004;108(7):1265-73. 53. sherman j, weaver d, hofland m, sing s, buteler m, lanning s, et al. identification of novel qtl for sawfly resistance in wheat. crop science. 2010;50(1):73-86. 54. xu s. quantitative trait locus mapping can benefit from segregation distortion. genetics. 2008;180(4):2201-8. 55. rubeena tp, ford r, taylor p. molecular mapping the lentil (lens culinaris ssp. culinaris) genome. theor appl genet. 2003;107:910-6. 56. rector bg. genetic mapping of antibiosis and antixenosis resistance mechanisms in soybean. university of georgia; 1998. 57. terry l, chase k, jarvik t, orf j, mansur l, lark k. soybean quantitative trait loci for resistance to insects. crop science. 2000;40(2):375-82. 58. guo b, zhang z, li r, widstrom n, snook m, lynch r, et al. restriction fragment length polymorphism markers associated with silk maysin, antibiosis to corn earworm (lepidoptera: noctuidae) larvae, in a dent and sweet corn cross. journal of economic entomology. 2001;94(2):564-71. 59. lee e, byrne p, mcmullen m, snook m, wiseman b, widstrom n, et al. genetic mechanisms underlying apimaysin and maysin synthesis and corn earworm antibiosis in maize (zea mays l.). genetics. 1998;149(4):1997-2006. 60. gullner g, komives t, király l, schröder p. glutathione stransferase enzymes in plant-pathogen interactions. frontiers in plant science. 2018;9:1836. 61. boller t, he sy. innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. science. 2009;324(5928):742-4. highlights in bioscience page 8 of 9 december 2023|volume 6 http://bioscience.highlightsin.org/ ali et al., 2023 identifying genetic markers for leaf miner resistance in chickpea available from: https://www.science.org/doi/abs/ 10.1126/science.1171647. 62. wahibah nn, tsutsui t, tamaoki d, sato k, nishiuchi t. expression of barley glutathione s-transferase13 gene reduces accumulation of reactive oxygen species by trichothecenes and paraquat in arabidopsis plants. plant biotechnology. 2018;35(1):71-9. 63. radwan em, taha hs. toxic and biochemical effects of different insecticides on the tomato leafminer, tuta absoluta (meyrick)(lepidoptera: gelechiidae). egyptian academic journal of biological sciences, f toxicology & pest control. 2012;4(1):1-10. 64. mostafa aa, el-rahman sna, shehata s, abdallah na, omar hs. assessing the effects of a novel biostimulant to enhance leafminer resistance and plant growth on common bean. scientific reports. 2021;11(1):1-14. highlights in bioscience page 9 of 9 december 2023|volume 6 https://www.science.org/doi/abs/10.1126/science.1171647 https://www.science.org/doi/abs/10.1126/science.1171647 http://bioscience.highlightsin.org/ abstract introduction materials and methods the field experiment and disease score dna isolation statistical analysis validation of associated pcr markers results phenotyping data mapping and qtl analyses validation of leaf miner associated qtls discussion conclusion highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20206 highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 4 research article open access 1chemistry department, college of science, sudan university of science and technology, sudan. contacts of authors * to whom correspondence should be addressed: mohammed sulieman ali eltoum citation: eltoum m s a, elfaki a a m (2020). extraction, characterization, and usage of turmeric curcumin for color coating of metronidazole tablets. highlights in bioscience volume 3. article id 20206. dio:10.36462/ h.biosci.20206 received: may 20, 2020 accepted: june 27, 2020 published: june 30, 2020 copyright: © 2020 eltoum and elfaki .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. extraction, characterization, and usage of turmeric curcumin for color coating of metronidazole tablets mohammed sulieman ali eltoum* 1 , almustafa ahmed mohamed elfaki 1 abstract curcumin is a chemical compound produced by curcuma longa plants that is widely used as a coloring agent and a dietary supplement and has some therapeutic activity. our aim is to evaluate the use of curcumin as color coating material for metronidazole tablets. curcumin was extracted at higher yields from three different samples of turmeric plants. the extract obtained was characterized by infrared red spectroscopy (ir), thin layer chromatography (tlc) and ash content and melting point (mp). the curcumin produced has an mp content ranging from 182 ° c to 184 ° c and an ash content ranging from 1.5 to 3.17 %. curcumin material was used as a colorful agent for the coating of metronidazole tablet pills using spray coating technology. experimental results have shown that curcumin-coated metronidazole tablets exhibit strong color stability even at higher temperatures, and that the taste of bitterness in metronidazole pills has been reduced or has disappeared entirely. keywords: curcumin, turmeric, color-coating, metronidazole tablets, pharmaceutical. introduction scientific research on color chemistry is needed to improve and maintain color stability. natural coloring additives are generally considered to be coloring additives derived from plant or animal sources by extraction or other physical processing[1,2]. examples of natural colorants include carmine, annatto extract, grape skin extract, turmeric, saffron and beta-carotene, which are the major natural color additives used in food products. synthetic coloring additives include chemically synthesized substances such as tartrazine, erythrosine and indigo carmine. natural or vegetable colors are less in use because they are expensive or difficult to extract [1, 2]. curcumin (synonyms: turmeric yellow, kurkum, ins no. 100(i)) is an orange-yellow crystalline powder. the standards of the joint expert committee on food additives (jecfa) define only curcumin extracted from natural source materials. it may also be produced by chemical synthesis, which is not used as a food additive [3]. the chemical structure of curcumin is shown in figure 1. the yellow coloring components (curcuminoids) and the primary coloring pigment are curcumin (curi), along with two additional minor curcuminoids, demethoxycurcumin (curii) and bisdemethoxycurcumin (curiii), which are the key active constituents of the turmeric (figure 2). curcumin is a major secondary metabolite of the perennial asian plant turmeric (curcuma longa l). curcumin was identified as the active principle of turmeric in 1815 and its structure was determined after crystallization in 1870 [4]. turmeric is only one representative of more than 80 species of turmeric in the ginger family, zingiberaceae [5]. eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 4 figure 1: the chemical structure of curcumin. figure 2: structure of the minor curcuminoids. turmeric is widely cultivated in many asian countries, particularly india, where it is mainly grown for dietary use and is a major component of the spice curry. in addition, turmeric is recognized for its medicinal properties and has been used for centuries in the treatment of a variety of diseases including eczema, arthritis, ulcers, asthma, anemia and many others [6]. as a result of extensive studies over the last few decades, curcumin has emerged as a promising anti-cancer agent and has been shown to target multiple and diverse pathways of disease causation and progression [4]. the attractiveness of curcumin as a therapeutic agent is enhanced by its safety, affordability, and history of longterm use [7]. molecular formula of c21h20o6, corresponding to a molecular weight of 368.37. curcumin is a yellow-orange crystalline powder with maximum absorbance at430 nm and melting point of 183 °c [6]. curcumin exhibits hydrophobic and (slight) hydrophilic properties owing to its aliphatic heptadienone linker and polar β-dicarbonyl and phenolic groups, respectively [8]. curcumin is sparingly soluble in water, but shows greater solubility in some organic solvents such as acetone, ethyl acetate, acetonitrile and ethanol. its reported partition coefficient (log p) ranges from 2.5 to 3.3 [9]. curcumin is a bis-α,β-unsaturated βdiketone and exists in equilibrium with its enol tautomer [10]. studies involving 1h, c nmr, and infrared spectroscopy have shown that the enolate form predominates in alkaline solution [11]. no previous literature has introduced the use of curcumin as a color coating for metronidazole tablets. the aim of this study is to extract and characterize curcumin obtained from the turmeric plant using different analytical techniques and to evaluate the use of turmeric powder as a coating material for metronidazole tablets. in addition, we studied the effect of this coat material on some of the properties of the metronidazole tables. materials and methods turmeric samples were collated from sudanese local market. curcumin extraction: twenty grams of ground turmeric in 50 ml of dichloromethane was magnetically stirred and heated at the reflux condenser for 1 h. the mixture was then suction-filtered and the filtrate was concentrated in a hot water bath maintained at 50°c. the reddish yellow oily residue was treated with 20 ml hexane and the resulting solid was collected by suction filtration [3]. the obtained yield was calculated as follow: yield = wpr/wtheox100 where: wpr = practical weight, wtheo = theoretical weight fourier transform infrared spectroscopy (ft –i r): two milligrams of curcumin were mixed with 300 mg of dried potassium bromide. carefully, the mixture was grinded, spread evenly in an appropriate die and subjected to a pressure of approximately 800 mpa (8 t·cm -2 ). in the case of substances that were unstable under normal atmospheric conditions, the disc was pressed into a vacuum. a disc was rejected if visual examination had been show lack of uniform transparency or when transmittance at 2000 cm -1 (5 µm) in the absence of a specific absorption band was less than 60 per cent without compensation, unless otherwise prescribed. samples were prepared by the same procedure and the spectrum was recorded between 4000400 cm -1 (2.5-15.4 µm) under the same operational conditions. the transmission minima (absorption maxima) in the spectrum was obtained with the substance to be examined correspond in position and relative size to those in the spectrum obtained with the reference substance [12]. thin layer chromatography (tlc): the solvent system which was used to characterize of curcumin powder was prepared by mixed of (3% methanol and 97% dichloromethane). the diameter of tlc plate had 10x20 cm 2 [3]. melting point (mp): a sufficient quantity of curcumin was introduced into the capillary tube to give a compact column of 4 mm to 6 mm in height. the temperature of the bath was increased to about 10°c below the assumed melting point and the heating rate was adjusted to about 1° c/min. when the temperature was 5°c below the assumed melting point, the capillary tube was introduced into the instrument. in the case of the apparatus described above, the capillary tube was immersed in such a way that the closed end is near the center of the thermometer bulb, the immersion mark of which is at the surface level of the liquid. the temperature at which the last particle was transferred to the liquid phase was recorded [12]. eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 4 ash content: the porcelain crucible was ignited at 600 ± 50 °c for 30 min and then allowed to cool over the silica gel in the desiccators. the prescribed amount of curcumin was placed in the crucible and weighed, after it had been gently heated to low temperatures as practicable, until the sample had been thoroughly charred, until white fumes had ceased to develop and ignited at 600 ± 50 °c until the residue had been completely incinerated. the crucible was allowed to cool over the silica gel in the desiccator, was weighed again and the percentage yield was calculated [12]. percentage of whiteness: the sample powder was placed in dishes, regularly highlighted and then the color was measured against the white color standard and the percentage was finally recorded. coating of metronidazole tablets with curcumin: a total of 25 g of opadry white was dissolved in 500 ml of ethanol with stirring during 30 min. and 1 g of curcumin powder was added during 10 min. the spray coating process for metronidazole tablets began by placing the tablets in the coating machine chamber, and then the main unit button was pressed.the exhaust was operated until the powder was removed from the tablets and the hot air was operated until the temperature of the tablet reached 50°c. the temperature control key was operated with hot air to control the temperature of the coating. the spray stage was then operated, and the turn sprayers were opened until the tablets were completely coated. and after coating, the other keys were turned off until the tablets had dried. the coating machine was stopped; the tablets were removed [12]. results and discussion the turmeric plant can be identified both by its characteristic tuberous root and by the leaves which extend upward from the erect, thick stems of the root. turmeric root has a fragrant aroma and a slightly bitter, peppery, biting taste reminiscent of ginger. when eaten, it colors the saliva yellow and leaves a warm feeling in the mouth. the curcumin yields obtained for the different samples are shown in table 1 and the resulting compounds were finely soft, clear yellow powder. the yield of the samples was consistent with the method described by andrew et al, [3], and the appearance of the powder was as clear as the standard colour. table 1: the curcumin yield (cy), melting point (mp), ash content (ac) and percentage of whiteness (pw) of the studied tumeric samples. figures 3 show the absorption of samples. these figures contain the following characteristic peaks: the broad band of the three oh groups at 3250-3500 cm −1 , sharp peak stretch of ch aliphatic system less than 3000cm −1 , weak peak of c-h aromatic system around 3050cm, sharp peak of c═o at 1510 cm −1 , medium peak of c-o at 1300cm −1 , and sharp peak of c=c of aromatic system at about 1510cm −1 . figure 4 shows the tlc plate of the compound. the chromatogram obtained showed that curcumin was divided into three distinct compounds. clearly, this result is in agreement with andrew et al., [3]. figure 3: ir spectrum analysis results of curcumin for samples 1 (a), 2 (b) and 3 (c). figure 4: tlc of curcumin sample. the melting point determined by the capillary method and it is the temperature at which the last solid particle of a compact column of a substance in a tube passes into the liquid phase. the results of the three samples are shown in table 1. the melting point of curcumin is 183°c (bp2012)[12]. the ash content obtained of the samples ranges from 1.5 to 3.17 % (table 1). in summary, from the three samples above, the percentage of ash in curcumin was good. the percentage of whiteness of the three samples obtained ranges from 51.08% to 53.03% (table 1). coated tablets are tablets containing one or more layers of mixtures of different substances, such as natural or synthetic resins, gums, gelatin, inactive and insoluble fillers, sugars, plasticizers, polyols and waxes. coloring matter authorized by the competent authority and sometimes flavorings and samples number cy mp ac pw 1 86.36% 183°c 1.50% 52.04% 2 81.81% 184°c 3.09% 53.03% 3 82.72% 182°c 3.17% 51.08% eltoum m s a, elfaki a a m. 2020 usage of turmeric curcumin for color coating of metronidazole tablets highlights in bioscience june 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 4 active substances. the mean weight and disintegration time of metronidazole tablets prior to coating with curcumin was 323.0 mg and 23 sec and 22 sec respectively. however, the average weight and disintegration time after coating was 324.74 mg and 120 sec, 93 sec and 114 sec respectively (figure 5). figure 5: metronidazole tablets before (a) and after blistering. conclusion the primary purpose of the study in this study is to study the isolation of curcumin from turmeric and also to characterize curcumin as a color-coating martial for metronidazole tablet in pharmaceuticals. curcumin was extracted from the turmeric in a simple and easy way using dichloromethane and triturated with hexane, and the extraction yield was finely soft yellow powder. experimental results have shown that curcumin-coated metronidazole tablets exhibit strong color stability even at higher temperatures, and that the taste of bitterness in metronidazole pills has been reduced or has disappeared entirely. acknowledgments we would like to thank the climax factory for the drugs and medicines industry-khartoum for giving us the opportunity to carry out the practical work of this research in their different laboratories, and we would also like to thank the sudan university of science for accepting this work as part of the complementary research on the requirements of the m.sc. in chemistry [13]. references 1. allen l, ansel hc. ansel's pharmaceutical dosage forms and drug delivery systems. lippincott williams & wilkins; 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with active rheumatoid arthritis. phytotherapy research. 2012 nov; 26 (11):1719-25. 8. balasubramanian k. molecular orbital basis for yellow curry spice curcumin's prevention of alzheimer's disease. journal of agricultural and food chemistry. 2006 may 17; 54 (10):3512-20. 9. grynkiewicz g, ślifirski p. curcumin and curcuminoids in quest for medicinal status. acta biochimica polonica. 2012 may 14; 59 (2). 10. chignell cf, bilskj p, reszka kj, motten ag, sik rh, dahl ta. spectral and photochemical properties of curcumin. photochemistry and photobiology. 1994 mar; 59(3):295-302. 11. sun ym, wang rx, yuan sl, lin xj, liu cb. theoretical study on the antioxidant activity of curcumin. chinese journal of chemistry. 2004 aug; 22(8):827-30. 12. bp. the british pharmacopoeia 2012. 13. http://repository.sustech.edu/handle/123456789/13398?sh ow=full highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202106 research article open access 1 genetics and plant improvement team, biosciences laboratory, doctoral school of sciences and technologies, joseph ki-zerbo university, 03 bp 7021 ouagadougou 03, burkina faso. 2 chadian institute of agronomic research for development (itrad), b.p. 5400, n’djaména, chad. 3 plant ecophysiology team, biosciences laboratory, doctoral school of sciences and technologies, joseph kizerbo university, 03 bp 7021 ouagadougou 03, burkina faso. 4 ziniaré university center, joseph kizerbo university, 03 bp 7021 ouagadougou 03, burkina faso. contacts of authors * to whom correspondence should be addressed: nerbewende@yahoo.fr received: january 2, 2021 accepted: march 4, 2021 published: march 18, 2021 citation: tiendrébéogo kf, sawadogo n, gapili n, ouédraogo mh, ouédraogo rf, nanema kr, ouoba a, sawadogo m . variability and relationships between characters of physic nut (jatropha curcas l.) in burkina faso . 2021 mar 18;4:bs202106 copyright: © 2021 tiendrébéogo et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. variability and relationships between characters of physic nut (jatropha curcas l.) in burkina faso kouka fidèle tiendrébéogo1, nerbéwendé sawadogo*1, naoura gapili2, mahamadi hamed ouédraogo1, razacswendé fanta ouédraogo3, kiswendsida romaric nanema1, adjima ouoba4, mahamadou sawadogo1 abstract jatropha curcas is a highly promising species for biodiesel production in burkina faso and other countries in the tropics. it is rustic, grows in warm regions and is easily cultivated. these characteristics and high-quality oil yields from the seeds have made it a priority for biodiesel programs. consequently, this plant merits genetic investigations aimed at improving yields. the present study was conducted to determine the extent of genetic variability and relationships among 30 accessions from burkina faso using 7 qualitative characters and 20 quantitative characters. for most of the traits, there were highly significant differences among the accessions. a positive and significant correlation between the 100-seed weight trait and the oil content trait was observed. the oil content trait is significantly and positively influenced by growth traits such as plant height and crown diameter. based on the petiole base pigmentation, three morphotypes were identified: green morphotype, purple morphotype and brown morphotype. the green morphotype was characterized by very high oil content and high 100-seed weight while the brown morphotype presents low oil content and low 100-seed weight. the purple morphotype registered a high oil content and medium 100-seed weight. these results are important for the continuity of breeding programs, aimed at obtaining cultivars with high grain yield and high oil content in seeds. keywords: oil content, genetic diversity, genetic correlation, morphotype, jatropha curcas, burkina faso. introduction physic nut (jaropha curcas l.) is a perennial oil plant commonly used in tropical areas as a medicinal plant, in the construction of defensive hedges against animals and in the fight against water erosion [1]. it is rustic, grows in warm regions and is easily cultivated. these characteristics and high-quality oil yields from the seeds have made this plant a priority and a highly promising species for biodiesel programs in countries in the tropics [2]. it produces seeds rich in oil which can be used pure after filtration as fuel in diesel engines with indirect injection or serve as raw material for the production of biodiesel by transesterification [3, 4]. [5] highlighted the economic interest of the use of oil of this species for the poor countries, in particular tropical africa and asia. indeed, j. curcas provides various products that contribute to poverty reduction, in particular the promotion of income-generating activities mainly for women such as sale of seeds and soap and the valuation of oil cakes as organic fertilizers. it is an opportunity for developing countries to improve farmers' incomes and even stimulate the rural economy [6]. however, in most countries of sub-saharan africa, the establishment of plantations preceded the conduct of agronomic research essential for the sustainable exploitation of the plant. as a result, plant development has been poor and yields obtained during the first years of cultivation have generally been disappointing [7]. consequently, this species merits genetic investigations aimed at improving yields [2]. so, the viability of the j. curcas-based biodiesel sector is essentially based on highly productive and oil-rich j. curcas genotypes [6]. highlights in bioscience page 1 of 11 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202106 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-0448-4239 https://orcid.org/0000-0003-0136-8408 https://orcid.org/0000-0001-6415-8745 https://orcid.org/0000-0002-3037-8624 https://orcid.org/0000-0002-4241-4696 https://orcid.org/0000-0001-8913-1012 https://orcid.org/0000-0001-6456-2297 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) despite strong recommendations for improvement of j. curcas for intensive production, a very little references exist on the genetic characteristics of the plant, the level of productivity of j. curcas in relation to its genetic potential and the cultivation techniques [6]. indeed, j. curcas is still essentially a wild plant which must be the object of genetic improvement and domestication for its popularization and vaolrization [8, 9]. in burkina faso, the genetic diversity of the species remains still poorly known [10-13]. thus, within the framework of the promotion of the neglected or under-exploited local species, this study was initiated in order to contribute to a better knowledge the genetic diversity and the relationships between agro-morphological traits and seeds oil content of j. curcas. material and methods plant material the plant material consisted of 30 elite accessions selected by [11] based on the morpho-metric characteristics of the fruits as well as the oil content of the seeds. in this study, an accession is a set of seeds, from natural pollination, harvested from the same tree during prospecting. the seeds were stored at laboratory temperature without any prior chemical treatment. experimental site the agro-morphological characterization was carried out on a plantation installed in the experimental station of the institute for rural development (idr) of gampela at 1°21'0.9'' west longitude, 12°24'10.7'' north latitude and 924 m altitude. the station is characterized by very heterogeneous, deep soils, of low physicochemical fertility and a predominantly sandy-clay texture [14]. the climate of the zone is of the sudano-sahelian type characterized by the alternation of two seasons, namely a rainy season which extends from june to october and a dry season from november to may [15]. the annual rainfall recorded in the station during the experimentation varied between 728 and 984.8 mm. average air temperatures during the rainy months range between 35° c and 40° c for the maximums and between 18° c and 19° c for the minimums [15]. experimental design the experimental design used is a completely random experiment plan consisting of 30 elementary plots of 8 m x 8 m. each elementary plot contains 9 plants arranged in three lines of 8 m in length each. the spacing between the lines was 4 m. each line includes three plants with a spacing of 4 m. the aisles between the elementary plots were also 4 m. characters studied qualitative characters based on the work of [16], seven qualitative variables were chosen to characterize the phenotypic variability of the accessions studied. the table 1 presents the different characters with their modalities. table 1. modalities of qualitative traits. variables modalities brancing pattern basal intermdiate top entire petiole base pigmentation green brown purple stem colour green grey leaf colour green light green dark green latex colour cream red phyllotaxy alternate whorled growth habit shurb (< 5 m) tree (> 5m) quantitative characters based on the work of [17, 2, 18, 16, 6], 20 quantitative variables were chosen to characterize the genetic diversity of j. curcas five years after planting. these parameters were measured on three plants sampled by accession. these are first of all the parameters linked to the vegetative development of the trees, measured three months after the appearance of the first leaves. those are: • the dendrometric characters of trees such as plant height (ph) measured from the base to the apex of the main stem, crown diameter (cd) measured between both ends of the plant, stem diameter (sd) measured at the collar using a tape measure (sd = collar circumference / π) and number of main branches (nb) evaluated by counting the branches coming out from the trunk . • the characters linked to the dimensions of the leaves measured on three fresh leaves, fully developed and not parasitized, per individual: these were the width of the leaf (wl) measured between the two ends of the leaf, the length of the leaf (ll) measured from the petiole to the tip of the leaf and the length of the petiole (lp) measured from the insertion of the stem to the insertion of the leaf. the traits linked to the productivity of the accessions such as fruits weight (frw), seeds weight (sew) and pulps weight (puw) per accession were also evaluated at the ripe and dry fruit stage using an electric scale. moreover, others traits relative to the fruits were measured. they are the average length of the fruit (alf), the average diameter of the fruit (adf) evaluated using a digital caliper, the average weight of the fruit (awf) and the average weight of the pulp (awp) determined using an electric highlights in bioscience page 2 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) scale on ten fruits. the average number of seeds per fruit (ans) was determined by counting. as concerning the seeds, the characters measured were the 100-seed weight (sw100) determined by weighing a random sample of 100 seeds of each mother plant using an electric scale, the average length of the seed (als), the average width of the seed (aws) and the average thickness of the seed (ats) measured with a digital caliper on ten seeds. the seeds oil content (oil) was determined by the soxhlet extraction method using hexane as the solvent [19]. [16] proposed a descriptor with four classes based on the seeds oil content character in relation with the weight of the seed: low (0-20%), medium (21-30%), high (31-40%) and very high (> 40%). data analysis the data collected were analyzed with xlstat 2020.3.1.11 software. analysis of variance (anova) and of student newman keuls means separation test at α=5% were carried out in order to compare accessions. pearson’s r coefficient was used to measure correlations between quantitative traits while the relationships between discriminate qualitative and quantitative traits were determined through multiple correspondence analysis (mca). indeed, the quantitative traits have been previously transformed into qualitative traits according to the classes obtained in the student newman keuls means separation test. results analysis of the phenotypic variability of j. curcas using qualitative variables the results recorded in table 2 showed that all the accessions studied had green stems and green leaves, a cream-colored latex and alternate leaves. however, two characters presented variability. thus, the majority of plants (70%) had a basal branching pattern while a minority (30%) revealed an intermediate branching pattern (figure 1). the petiole base expressed several pigmentation color (figure 2): green (33.33%), brown (13.33%) and mostly purple (53.34%). analysis of the phenotypic variability of j. curcas using quantitative variables the results of the significance of the means separation test of newman keuls were performed with vegetative development parameters (table 3), fruit parameters (table 4) and seeds parameters (table 5). most of the vegetative development parameters and seeds traits significantly discriminate the accessions studied except the length of the petiole (lp), the average length of the seed (als) and seeds weight per accession (sew). for the fruit traits, only three of the seven measured characters, precisely the average length of the fruit (alf), the average weight of the fruit (awf) and the average weight of the pulp (awp) showed variability. based on the each of three characters of interest namely oil content (oil), 100-seed weight (sw100) and crown diameter (cd), three classes were obtained within accessions. indeed, according to the value of the character, three performance classes table 2. variation in the qualitative characters of the collection of j. curcas. variables modalities frequencies (%) brancing pattern basal 70 intermdiate 30 top 0 entire 0 petiole base pigmentation green 33.33 brown 13.33 purple 53.34 stem colour green 100 grey 0 leaf colour green 100 light green 0 dark green 0 latex colour cream 100 red 0 phyllotaxy alternate 100 whorled 0 growth habit shurb (< 5 m) 100 tree (> 5m) 0 (low, medium, high) were registered with the 100-seed weight trait and crown diameter, respectively. the seeds oil content character showed also three performance classes which ranged medium, high and very high according to the descriptor classes. relationships between characters correlation between characters the correlation of pearson (table 6) showed positive and significant correlations at the 5% and 1% threshold between the characters studied. thus, the character oil content was positively correlated with the characters 100-seed weight (r = 0.253), plant height (r = 0.344) and crown diameter (r = 0.260). the 100seed weight character was positively correlated with the characters linked to vegetative development such as plant height (r = 0.522), crown diameter (r = 0.443) and length of the petiole (r = 0.272). furthermore, the crown diameter character is positively correlated with all the other characters studied. these different correlations indicate that plants with significant vegetative development produce seeds of high weight and high oil content. the characters linked to the yield (frw, sew and puw) are positively correlated with the characters relating to the scale of the plant (ph, sd and cd). so, large-scale plants have also high productive potential. furthermore, the characters of the seeds (als, aws and ats) are positively influenced each other. association between characters the results of multiple correspondence analysis (mca) recorded in figure 3 showed three associations of the characters. on the plan formed by axes 1 and 2 with 45.41% of the total inertia, the f1 axis (23.14% of total inertia) opposed two groups of variables precisely group 1 and group 3. group 1 combined the characters very high seed oil content (> 40%), intermediate highlights in bioscience page 3 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) figure 1. branching pattern of j. curcas. a: basal (70%), b: intermediate (30%). figure 2. petiole base pigmentation of j. curcas a: green (33.33%), b: brown (13.33%), c: purple (53.34%) highlights in bioscience page 4 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 3. results of newman keuls'means separation test on vegetative development parameters. accession ph (m) sd (cm) cd (m) nb wl (cm) ll (cm) lp (cm) 1 2.450 bcde 9.076 b 2.650 abc 4.000 b 16.295 abc 14.365 abc 20.165 a 2 1.977 cde 15.711 ab 2.733 abc 6.333 ab 15.130 abc 14.127 abc 17.197 a 3 2.600 bcd 12.527 ab 3.100 abc 5.667 ab 15.040 bc 13.477 abc 17.007 a 4 2.747 b 13.896 ab 3.717 ab 6.000 ab 17.207 abc 15.497 abc 18.997 a 5 2.490 bcde 15.977 ab 3.467 abc 6.000 ab 14.410 c 13.930 abc 17.167 a 6 2.150 bcde 13.694 ab 3.350 abc 5.500 ab 15.545 abc 13.545 abc 18.430 a 7 2.033 bcde 13.694 ab 3.000 abc 5.000 ab 17.477 abc 14.720 abc 16.983 a 8 2.567 bcd 13.907 ab 3.133 abc 5.000 ab 18.850 ab 16.687 a 18.430 a 9 2.567 bcd 14.756 ab 3.150 abc 5.667 ab 18.463 ab 15.710 abc 19.820 a 10 2.383 bcde 12.909 ab 3.000 abc 5.667 ab 16.597 abc 14.797 abc 16.943 a 11 2.017 bcde 12.707 ab 2.857 abc 7.667 ab 16.333 abc 14.043 abc 16.863 a 12 3.225 a 13.854 ab 3.500 abc 9.000 a 18.415 ab 15.930 ab 19.195 a 13 2.100 bcde 12.739 ab 2.800 abc 5.000 ab 14.895 bc 12.460 c 14.945 a 14 2.233 bcde 14.862 ab 3.150 abc 6.000 ab 17.283 abc 14.997 abc 16.710 a 15 2.017 bcde 15.287 ab 2.733 abc 8.000 ab 16.763 abc 14.517 abc 15.467 a 16 2.047 bcde 15.287 ab 2.900 abc 5.333 ab 16.177 abc 13.410 abc 16.310 a 17 2.227 bcde 13.163 ab 3.167 abc 4.667 ab 16.553 abc 14.483 abc 18.073 a 18 2.275 bcde 15.287 ab 3.450 abc 8.000 ab 16.350 abc 15.015 abc 18.795 a 19 2.683 bc 13.376 ab 4.000 a 6.333 ab 18.997 a 16.630 a 19.553 a 20 2.340 bcde 13.641 ab 3.400 abc 4.333 ab 16.693 abc 14.753 abc 18.310 a 21 2.225 bcde 13.694 ab 2.900 abc 9.000 a 17.545 abc 14.910 abc 18.130 a 22 2.417 bcde 12.781 ab 2.867 abc 7.000 ab 16.910 abc 14.340 abc 17.517 a 23 1.915 de 10.987 ab 2.650 abc 4.000 b 15.530 abc 12.900 bc 13.995 a 24 1.900 de 14.411 ab 2.635 abc 7.000 ab 15.965 abc 14.130 abc 18.365 a 25 1.770 e 10.403 ab 2.100 c 5.667 ab 15.387 abc 12.683 bc 15.297 a 26 2.267 bcde 10.032 ab 2.600 abc 3.667 b 17.940 abc 15.440 abc 17.097 a 27 1.857 de 12.951 ab 2.400 bc 5.667 ab 14.953 bc 12.730 bc 14.617 a 28 2.183 bcde 16.561 ab 2.767 abc 7.000 ab 16.217 abc 13.463 abc 16.410 a 29 1.880 de 17.304 a 2.767 abc 6.000 ab 15.573 abc 13.797 abc 15.750 a 30 1.900 de 11.996 ab 2.700 abc 4.333 ab 16.420 abc 14.697 abc 16.017 a ph: plant height, sd: stem diameter, cd: crown diameter, nb: number of main branches, wl: width of the leaf, ll: length of the leaf, lp: length of the petiole. the values for each class followed by the same letters are not significantly different at the 5% level. branching of the stem, green pigmentation of the petiole base, high 100-seed weight (≥ 62.85 g) and large crown diameter (≥ 4 m) while the group 3 is formed by association between the medium oil content (21-30%), the brown pigmentation of the petiole base and the low 100-seed weight (≤ 36.9 g). group 2, correlated to axis f2 with 22.127% of total inertia, combined the high oil content (31-40%), the basal branching of the stem, the purple pigmentation of the petiole base, the medium 100-seed weight (43.30-43.85 g), the medium crown diameter (2.4-3.467 m) and the small crown diameter (≤ 2.1 m). highlights in bioscience page 5 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 4. results of newman keuls'means separation test on fruits parameters. accession alf (cm) adf (cm) awf (g) awp (g) ans frw (g) puw (g) 1 2.533 ab 2.110 a 2.610 ab 0.940 ab 2.550 a 158.400 a 49.900 a 2 2.265 ab 2.058 a 1.843 ab 0.790 abcd 2.500 a 207.233 a 94.867 a 3 2.425 ab 2.120 a 2.623 ab 0.920 abc 2.700 a 263.167 a 93.567 a 4 2.457 ab 2.080 a 2.460 ab 0.873 abc 2.800 a 509.200 a 189.900 a 5 2.340 ab 2.053 a 2.430 ab 0.837 abcd 2.833 a 541.667 a 211.600 a 6 2.468 ab 2.083 a 2.710 ab 1.005 a 2.950 a 816.250 a 317.450 a 7 2.383 ab 2.090 a 1.840 ab 0.670 bcd 2.200 a 136.967 a 56.733 a 8 2.478 ab 2.158 a 2.120 ab 0.757 abcd 2.467 a 163.767 a 68.467 a 9 2.535 ab 2.100 a 2.277 ab 0.837 abcd 2.667 a 463.867 a 210.133 a 10 2.425 ab 2.117 a 2.143 ab 0.720 abcd 2.500 a 249.433 a 100.367 a 11 2.402 ab 2.067 a 2.300 ab 0.930 abc 2.633 a 248.233 a 114.567 a 12 2.430 ab 2.140 a 2.500 ab 0.830 abcd 2.750 a 453.500 a 168.300 a 13 2.498 ab 2.035 a 2.175 ab 0.895 abc 2.850 a 641.400 a 256.050 a 14 2.483 ab 2.078 a 2.133 ab 0.880 abc 2.567 a 433.867 a 172.033 a 15 2.130 b 1.998 a 1.417 b 0.547 d 2.367 a 174.767 a 79.533 a 16 2.567 a 2.158 a 2.773 a 0.877 abc 2.967 a 496.000 a 190.900 a 17 2.425 ab 2.045 a 2.120 ab 0.750 abcd 2.600 a 384.400 a 161.667 a 18 2.593 a 2.145 a 2.530 ab 0.770 abcd 2.850 a 834.300 a 277.150 a 19 2.395 ab 2.093 a 2.493 ab 0.750 abcd 2.967 a 774.267 a 287.667 a 20 2.397 ab 2.078 a 2.380 ab 0.750 abcd 2.833 a 836.400 a 323.800 a 21 2.420 ab 2.073 a 2.115 ab 0.660 bcd 2.750 a 158.850 a 59.700 a 22 2.393 ab 2.112 a 1.940 ab 0.733 abcd 2.533 a 162.800 a 73.567 a 23 2.460 ab 2.088 a 2.015 ab 0.670 bcd 2.600 a 99.600 a 39.100 a 24 2.330 ab 1.940 a 1.770 ab 0.607 cd 2.230 a 86.400 a 37.350 a 25 2.205 ab 1.927 a 1.627 ab 0.608 cd 2.420 a 89.200 a 39.600 a 26 2.380 ab 2.083 a 1.797 ab 0.713 abcd 2.133 a 64,933 a 29.600 a 27 2.212 ab 1.978 a 1.537 ab 0.637 bcd 2.200 a 97.867 a 49.033 a 28 2.368 ab 2.088 a 1.830 ab 0.833 abcd 2.333 a 147.533 a 71.133 a 29 2.245 ab 1.945 a 1.800 ab 0.870 abc 2.100 a 89.300 a 40.000 a 30 2.192 ab 2.043 a 1.607 ab 0.843 abcd 2.067 a 78.100 a 43.933 a alf: average length of the fruit, adf: average diameter of the fruit, awf: average weight of the fruit, awp: average weight of the pulp, ans: average number of the seeds per fruit, frw: fruits weight per accession, puw: pulps weight per accession. the values for each class followed by the same letters are not significantly different at the 5% level. highlights in bioscience page 6 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) table 5. results of newman keuls'means separation test on seeds parameters. accession als (cm) aws (cm) ats (cm) sw100 (g) sew (g) oil (%) 1 1.830 a 1.120 ab 0.863 ab 56.100 ab 108.500 a 33.811 i 2 1.755 a 1.092 ab 0.835 ab 47.833 abc 112.367 a 31.160 lm 3 1.803 a 1.150 ab 0.868 a 63.350 a 169.600 a 35.897 g 4 1.833 a 1.113 ab 0.855 ab 62.850 a 319.300 a 34.663 h 5 1.753 a 1.068 b 0.818 ab 55.833 ab 330.067 a 39.286 c 6 1.845 a 1.173 a 0.853 ab 61.100 ab 498.800 a 31.309 l 7 1.813 a 1.092 ab 0.847 ab 55.600 ab 80.233 a 35.023 h 8 1.833 a 1.123 ab 0.865 ab 52.900 abc 95.300 a 32.874 j 9 1.890 a 1.068 b 0.835 ab 53.300 abc 253.733 a 37.931 e 10 1.827 a 1.098 ab 0.873 a 54.933 ab 149.067 a 35.474 g 11 1.753 a 1.130 ab 0.850 ab 54.450 ab 133.667 a 37.989 e 12 1.780 a 1.073 b 0.845 ab 63.700 a 285.200 a 37.931 e 13 1.753 a 1.065 b 0.813 ab 43.850 bc 385.350 a 32.360 k 14 1.868 a 1.060 b 0.835 ab 48.500 abc 261.833 a 40.383 b 15 1.688 a 1.063 b 0.813 ab 45.900 abc 95.233 a 31.331 l 16 1.840 a 1.118 ab 0.852 ab 58.333 ab 305.100 a 29.366 o 17 1.792 a 1.107 ab 0.825 ab 55.233 ab 222.733 a 31.577 l 18 1.813 a 1.120 ab 0.870 a 64.300 a 557.150 a 32.486 jk 19 1.832 a 1.152 ab 0.857 ab 59.200 ab 486.600 a 40.863 a 20 1.765 a 1.125 ab 0.832 ab 63.250 a 512.600 a 34.726 h 21 1.840 a 1.118 ab 0.853 ab 52.450 abc 99.150 a 30.800 mn 22 1.805 a 1.065 b 0.832 ab 64.000 a 89.233 a 32.463 jk 23 1.808 a 1.088 ab 0.828 ab 54.400 ab 60.500 a 32.760 jk 24 1.740 a 1.090 ab 0.868 ab 43.300 bc 49.050 a 30.474 n 25 1.663 a 1.070 b 0.800 b 50.200 abc 49.600 a 34.040 i 26 1.780 a 1.113 ab 0.847 ab 53.200 abc 35.333 a 36.909 f 27 1.707 a 1.065 b 0.817 ab 51.600 abc 48.833 a 38.640 d 28 1.782 a 1.060 b 0.832 ab 43.500 bc 76.400 a 31.617 l 29 1.672 a 1.067 b 0.837 ab 47.100 abc 49.300 a 35.549 g 30 1.687 a 1.112 ab 0.853 ab 36.900 c 34.167 a 28.137 p als: average length of the seed, aws: average width of the seed, ats: average thickness of the seed, sw100: 100-seed weight, sew: seeds weight per accession, oil: seeds oil content. the values for each class followed by the same letters are not significantly different at the 5% level. highlights in bioscience page 7 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) ta bl e 6. m at ri x of co rr el at io ns be tw ee n th e st ud ie d ch ar ac te rs . v ar ia bl es a l f a d f a w f a w p a n s a l s a w s a t s sw 10 0 o il ph sd c d n b w l l l l p fr w se w a d f 0. 78 7* * a w f 0. 74 5* * 0. 62 7* * a w p 0. 58 8* * 0. 49 3* * 0. 70 6* * a n s 0. 59 8* * 0. 55 0* * 0. 83 6* * 0. 46 9* * a l s 0. 86 0* * 0. 78 7* * 0. 61 3* * 0. 43 3* * 0. 50 9* * a w s 0. 39 1* * 0. 46 3* * 0. 52 0* * 0. 35 9* * 0. 41 8* * 0. 43 2* * a t s 0. 42 3* * 0. 40 2* * 0. 39 2* * 0. 31 6* * 0. 04 1 0. 47 0* * 0. 52 1* * sw 10 0 0. 39 2* * 0. 35 0* * 0. 57 0* * 0. 19 1 0. 42 8* * 0. 32 4* * 0. 32 0* * 0. 22 6* o il 0. 07 6 0. 01 2 0. 15 5 0. 08 9 0. 10 0 0. 14 0 -0 .0 62 -0 .0 53 0. 25 3* ph 0. 41 8* * 0. 41 3* * 0. 49 9* * 0. 27 7* 0. 38 7* * 0. 45 9* * 0. 18 6 0. 32 6* * 0. 52 2* * 0. 34 4* * sd 0. 06 7 0. 09 7 0. 10 2 0. 19 4 0. 07 0 0. 05 9 -0 .1 75 0. 11 6 0. 05 7 -0 .0 19 0. 13 3 c d 0. 38 7* * 0. 42 8* * 0. 55 2* * 0. 37 0* * 0. 50 1* * 0. 40 2* * 0. 31 2* * 0. 30 1* * 0. 44 3* * 0. 26 0* 0. 65 1* * 0. 46 0* * n b -0 .0 10 -0 .0 18 0. 03 8 0. 01 6 0. 09 3 0. 00 1 -0 .1 05 0. 12 5 0. 02 4 0. 02 5 0. 18 9 0. 37 9* * 0. 22 5* w l 0. 14 3 0. 16 0 0. 00 2 -0 .1 71 -0 .0 27 0. 26 0* 0. 02 9 0. 26 1* 0. 04 7 0. 17 2 0. 42 9* * 0. 03 9 0. 31 7* * 0. 02 2 l l 0. 13 3 0. 20 8 0. 08 8 -0 .0 67 0. 04 7 0. 26 5* 0. 11 6 0. 35 5* * 0. 09 5 0. 21 0 0. 54 6* * 0. 15 9 0. 45 5* * 0. 08 9 0. 84 3* * l p 0. 18 3 0. 10 8 0. 26 6* 0. 06 1 0. 22 1* 0. 26 5* 0. 20 8 0. 23 4* 0. 27 2* 0. 10 8 0. 55 0* * 0. 09 6 0. 39 4* * 0. 06 9 0. 53 0* * 0. 64 1* * fr w 0. 42 6* * 0. 32 5* * 0. 52 4* * 0. 34 5* * 0. 55 6* * 0. 35 0* * 0. 25 8* 0. 13 4 0. 41 0* * 0. 14 9 0. 46 4* * 0. 30 8* * 0. 67 5* * 0. 14 1 0. 03 9 0. 16 8 0. 29 3* * se w 0. 42 4* * 0. 31 8* * 0. 52 7* * 0. 33 2* * 0. 55 4* * 0. 34 4* * 0. 26 9* 0. 14 7 0. 42 7* * 0. 14 9 0. 47 3* * 0. 28 6* * 0. 66 7* * 0. 13 6 0. 04 5 0. 17 5 0. 30 7* * 0. 99 7* * pu w 0. 42 5* * 0. 33 3* * 0. 51 2* * 0. 36 3* * 0. 55 4* * 0. 35 6* * 0. 23 5* 0. 11 1 0. 37 5* * 0. 14 8 0. 44 3* * 0. 33 9* * 0. 68 0* * 0. 14 6 0. 02 7 0. 15 4 0. 26 6* 0. 99 2* * 0. 98 ** a l f: av er ag e le ng th of th e fr ui t, a d f: av er ag e di am et er of th e fr ui t, a w f: av er ag e w ei gh to ft he fr ui t, a w p: av er ag e w ei gh to ft he pu lp ,a n s: av er ag e nu m be ro ft he se ed s pe rf ru it, a l s: av er ag e le ng th of th e se ed ,a w s: av er ag e w id th of th e se ed ,a t s: av er ag e th ic kn es s of th e se ed ,s w 10 0: 10 0se ed w ei gh t, o il :s ee ds oi lc on te nt ,p h :p la nt he ig ht ,s d :s te m di am et er , c d :c ro w n di am et er ,n b :n um be ro fm ai n br an ch es ,w l :w id th of th e le af ,l l :l en gt h of th e le af ,l p: le ng th of th e pe tio le , fr w :f ru its w ei gh tp er ac ce ss io n, se w :s ee ds w ei gh tp er ac ce ss io n, pu w :p ul ps w ei gh tp er ac ce ss io n, * si gn ifi ca nt ly at 5% ,* * si gn ifi ca nt ly at 1% . highlights in bioscience page 8 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) figure 3. association of modalities of traits in the ½ plan of the multiple correspondence analysis (mca) (45.41%) . bp: branching pattern, pbp: petiole base pigmentation, oil: oil content, sw100: 100-seed weight. discussion the agro-morphological evaluation using quantitative and qualitative characters highlighted the existence of variability for some characters. similar results for quantitative traits were obtained by [20] and [11, 12]. for qualitative characters, this variability relates to branching pattern and petiole base pigmentation. [16] reported similar results on the petiole base pigmentation. however, only two types of stem branching were observed in this study compared to four types obtained by these same authors, thus indicating that the accessions studied are less diversified for this character. no color variation was noted on the stems and leaves of the studied accessions. these results are similar to those of [17], [21] and [22] but differ from those of [16]. genetic correlations indicate genetic relations between characters. knowledge of correlations among the characters is useful in designing an effective breeding program for any crops [23]. indeed, the magnitude and direction of the genetic correlation is important in the choice of breeding methods and the formulation of strategies for simultaneous selection on multiple traits [2]. the correlations noted in this study corroborate those observed by [2] and [23]. in this study, statistically significant correlation of oil content existed with crown diameter and plant height. in addition, 100-seed weight character is significantly correlated with crown diameter, petiole length and plant height. these correlations are interesting for improvement programs because according to [24], the selection of elite accessions to reproduce for oil extraction should be oriented towards those with the best crown and seed weight traits. furthermore, [25] indicate that the quantity of seeds produced on the one hand is positively correlated with crown, and on the other hand the seed weight and the oil content are positively correlated. significant and positive correlations between the characters of the seeds would favor the simultaneous improvement of these characters through selection. thus, in the study, interesting correlations were noted between the characters linked to the weight and dimensions of the seeds. the strong positive correlation between the characters of the seeds indicates that the genes which govern these characters are probably linked or have a pleiotropic effect. similar results have been reported by [2] and [23] between 100-seed weight and the dimensions of the seed. however, the study found no significant correlation between the oil content and the dimensions of the seeds studied. similar results have been also reported by [2] and [18]. furthermore, the study showed a positive and significant correlation between the 100-seed weight trait and the seed oil content trait. [26] and [23] observed a positive and significant correlation between seed weight and oil content while [27] revealed a negative correlation between these two characters. [18] indicated that these contrasting results could be explained by the high weight of the seed coat of some accessions. according to [16], qualitative characters are morphological markers that can be used to identify lines in a relatively short time. these authors reported also that the development of descriptors on perennial plants such as j. curcas can contribute to an effective use of germplasm in the improvement programs. analysis of the association of characters revealed the existence of three morphotypes differing in the color of the pigmentation of the base of the petiole, the oil content of the seeds and 100-seed weight. [16] identified also three descriptors based on the pigmentation of the petiole base. for perennial plant such as j. curcas, these morphological descriptors could make it possible to select genotypes with high productive potential in the short term. according to [23], complex plant characters such as yield are quantitatively inherited and influenced by genetic effects, as well as by genotype and environment interaction and selection may be difficult and time consuming to improve yield directly especially for perennial crops such as j. curcas. therefore, identification and use of associations between characters are appropriate. in this study, statistically significant association of 100-seed weight existed with seed oil content. therefore, seed weight can be considered as important character for early selection of seed sources. furthermore, according to [11], the characters 100-seed weight and oil content showed the highest values of broad-sense heritability (83.23% and 99.93% respectively) and high expected genetic advance (22.63% and 19.62% respectively). the high broad-sense heritability values associated with high expected genetic advance indicate that effects of the genes are of additive type for these characters and the opportunities to improve seed weight character and oil content character through the selection. conclusion this study highlighted an important agro-morphological diversity within the accessions of j. curcas from burkina faso. the variability observed within the accessions studied and the ashighlights in bioscience page 9 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) sociations between the characters obtained constitute a database for the genetic improvement programs of j. curcas in burkina faso, in particular the development of high-yielding cultivars. a positive and significant correlation between the 100-seed weight trait and the oil content trait was noted. growth traits such as plant height and crown diameter was also significantly and positively correlated with the oil content trait. three morphotypes were identified using petiole base pigmentation which are the green morphotype with very high oil content and high 100seed weight, the purple morphotype with medium oil content and medium 100-seed weight and finally the brown morphotype with low oil content and low 100-seed weight. for perennial plant such as j. curcas, these results on relationships between characters could make it possible to select genotypes with high productive potential in the short term. acknowledgments the authors gratefully acknowledge the bioscience laboratory precisely genetic and plant breeding team (egap) of the «université joseph ki-zerbo» for the financial support of this work. they are also thankful to all team members of the bioscience laboratory for their input in this manuscript. authors’ contributions this work was carried out in collaboration between both authors. the two first authors designed the experiment and analyzed the data. both of the authors interpreted the data, read the final manuscript, and agreed with all contents. references 1. pandey vc, singh k, singh sj, kumar a, singh b, singh rp. jatropha curcas: a potential biofuel plant for sustainable environmental development. renewable and sustainable energy reviews. 2012;16(5): 2870-2883. 2. freitas rg, missio rf, matos fs, resende mdv, dias las. genetic evaluation of jatropha curcas l.: an important oilseed for biodiesel production. genetic and molecular research. 2011;10(3): 1490-1498. 3. berchmans hj and hirata s. biodiesel production from crude jatropha curcas l. seed oil with a high content of free fatty acids. bioresource technology. 2008;99(6): 1716-1721. 4. hamad b . transestérification des huiles végétales par l’éthanol en conditions douces par catalyses hétérogènes acide et basique. thèse de doctorat, université claude bernard-lyon 1, france; 2009.p.197. 5. martin g et mayeux a. réflexion sur les cultures oléagineuses énergétiques ii. le pourghère (jatropha curcas l.) : un carburant possible. oléagineux. 1984;39(5): 283-286. 6. ouattara b. etude de la diversité génétique, de la variabilité agromorphologique et éco-physiologique de jatropha curcas l. au sénégal. thèse de doctorat unique, université cheich anta diop de dakar; 2013.p.120. 7. minengu jdd, mobambo p, mergeai g. influence de l’environnement et des pratiques culturales sur la productivité de jatropha curcas l. en afrique subsaharienne (synthèse bibilographique). biotechnoogy, agronomy, society and environment. 2013;18(2): 290-300. 8. fao et fida. le jatropha, une culture bioénergétique pour les pays pauvres. rapport; 2010.p.4. 9. coulibaly nd, koné d, soro d, kouadio yj. caractérisation physiologique de dix accessions de jatropha curcas dans deux zones pédoclimatiques assez contrastées de la côte d’ivoire. european sientific journal. 2015;11(36) : 61-77. 10. ouédraogo m. etude biologique et physiologique du pourghère : jatropha curcas l. ( euphorbiaceae) en vue d’une meilleure production de carburant de substitution. thèse de doctorat, université de ouagadougou; 2000.p. 284. 11. tiendrebeogo kf, sawadogo n, nanema kr, traore re, bationokando p, ouedraogo mh and sawadogo m. evaluation of morphometric characteristics of fruits and oil content of seeds of jatropha curcas l. in burkina faso. international journal of plant, animal and environmental science. 2016a;6 (2): 145-155. 12. tiendrébéogo kf, sawadogo n, nanéma kr, traoré re, bationokando p, zongo jd and sawadogo m. evaluation de la diversité génétique du pourghère (jatropha curcas l.) au burkina faso. international journal of innovation and applied studies. 2016b;16 (1): 155-165. 13. tiendrebeogo kf, sawadogo n, ouedraogo mh, kiebre z, zida wpmsf, nanema kr, batieno tbj, traore re, bationo-kando p, zongo jd, sawadogo m. genetic diversity of jatropha curcas in burkina faso revealed by microsatellite markers. in european scientific journal, esj. 2019;15(15): 229-243. 14. bureau national des sols (bunasol). etude pédologique de la station expérimentale de gampèla, échelle 1/5000 . n° 59; 1988.p. 279. 15. guinko s. végétation de la haute-volta. thèse de doctorat, université de bordeaux iii (france); 1984.p. 394. 16. sunil n, kumar v, sujatha m, rao gr, vaprasad ks. 2013. minimal descriptors for characterization and evaluation of jatropha curcas l. germplasm for utilization in crop improvement. biomass and bioenergy. 2013;48: 239-249. 17. heller j, 1996. physic nut (jatropha curcas l.) in promoting the conservation and use of underutilized and neglected crops. international plant genetic resources institute (ipigri). 1996;1: 1-66. 18. leela t, naresh b, srikanth reddy m, madhusudhan nc, cherku pd. morphological, physicochemical and micropropagation studies in jatropha curcas l. and rapd analys of the regenerants. applied energy. 2011;88(6): 2071-2079. 19. aoac. official methods of analysis .1990; 1.p.684. 20. sunil n, sujatha m, kumar v, vanaja m, basha sd and varaprasad ks. correlating the phenotypic and molecular diversity in jatropha curcas l. biomass and bioenergy. 2010;35(3): 10851096. 21. sukarin w, yamada y and sakagushis s. characteristics of physic nut, jatropha curcas l. as a biomass crop in the tropics. japan agricultural research quarterly. 1987;20 (4): 302-303. 22. tiendrébéogo kf, sawadogo n, ouédraogo rf, ouédraogo mh, sawadogo m et zongo jd. evaluation morpho-physiologique et estimation des paramètres génétiques au stade juvénile de 30 highlights in bioscience page 10 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ tiendrébéogo et al., 2021 variability and relationships between characters of physic nut (jatropha curcas l.) accessions de pourghère (jatropha curcas l.) au burkina faso. afrique science. 2020;17(2): 202-210. 23. shabanimofrad m, rafii my, wahab mpe, biabani ar, latif ma. phenotypic, genotypic and genetic divergence found in 48 newly collected malaysian accessions of jatropha curcas l. industial and crops products. 2013;42: 543-551. 24. gbemavo cj, gandji k, gnangle cp, assogbadio ae and kakai rlg. variabilite morphologiques et conservation des morphotypes de jatropha curcas linn. (euphoriaceae) au benin. journal of agriculture and environment for international development. 2015;109(1): 55-69. 25. rao gr, korwar gr, shanker ak, ramakrishna ys. genetic associations, variability and diversity in seed characters, growth, reproductive phenology and yield in jatropha curcas l. accessions. trees. 2008;22(5): 697-709. 26. kaushik n, kumar k, kumar s, kaushikb n. genetic variability and divergence studies in seed traits and oil content of jatropha (jatropha curcas l.) accessions. biomass and bioenergy. 2007;31(7): 497-502. 27. wani ta, kitchlu s, ram g. genetic variability studies for morphological and qualitative attributes among jatropha curcas l. accessions grown under subtropical conditions of north india. south african journal of botany. 2012;79: 102-105. highlights in bioscience page 11 of 11 march 2021|volume 4 http://bioscience.highlightsin.org/ abstract introduction material and methods plant material experimental site experimental design characters studied data analysis results analysis of the phenotypic variability of j. curcas using qualitative variables analysis of the phenotypic variability of j. curcas using quantitative variables relationships between characters discussion conclusion acknowledgments authors’ contributions references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202508 research article open access 1 department of biochemistry, school of life sciences, federal university of technology, minna, niger state, nigeria. 2 department of microbiology, school of life sciences, federal university of technology, minna, niger state, nigeria. 3 department of animal biology, school of life sciences, federal university of technology, minna, niger state, nigeria. 4 africa centre of excellence for mycotoxin and food safety, federal university of technology, minna, niger state, nigeria. * to whom correspondence should be addressed: eogbadoyi@futminna.edu.ng editor: morad mokhtar, university mohammed vi polytechnic, ben guerir, morocco. reviewer(s): alsayed alsoudy, mohammed vi polytechnic university, ben guerir, morocco. kamal yadav, school of agricultural sciences, nagaland university, nagaland. nagesha s. narasimhappa, department of biotechnology, college of agriculture, hassan, karnataka, 573225, india. received: april 22, 2025 accepted: august 26, 2025 published: september 22, 2025 citation: nwosu ok, ogbadoyi eo, babayi h, olayemi ik. the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes activity in the biological control of callosobruchus maculatus (cowpea weevil) in stored vigna unguiculata l. walp (cowpea) . 2025 sept. 22;8:bs202508 copyright: © 2025 nwosu ok et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes activity in the biological control of callosobruchus maculatus (cowpea weevil) in stored vigna unguiculata l. walp (cowpea) onyeka kingsley nwosu1 ><�, emmanuel olofu ogbadoyi*1,4 ><�, hausatu babayi2 >< , israel kayode olayemi3 ><� abstract this study assessed the pesticidal effect of bacillus thuringiensis isolate against callosobruchus maculatus (cowpea weevil) infesting stored cowpea seeds. b. thuringiensis isolate was obtained through liquid fermentation using sucrose water as a substrate, then transferred onto talc powder for solid formulation. a six-month shelf-life study of the solid formulation was conducted using total microbial plate count. insect mortality bioassays were performed by applying liquid and solid b. thuringiensis formulations to cowpea seeds containing first-generation adult c. maculatus, with mortality recorded over 12 and 7 days, respectively. enzyme activity was assessed using specific enzyme substrates. results showed a gradual decline in microbial count over time in the solid formulation. the bioassay revealed 100% mortality for the liquid medium and for the solid medium of the b. thuringiensis isolate. the b. thuringiensis isolate also significantly delayed the first-generation emergence of c. maculatus. enzyme analysis indicated the production of cuticle-degrading enzymes—protease, lipase, and exochitinase with varying activity levels. this study concludes that b. thuringiensis isolate effectively controls c. maculatus, likely due to its cuticle-degrading enzymes, and has potential as a bio-control agent for stored cowpea pest management. however, higher concentrations are necessary in talc-based formulations to maintain an adequate shelf life. keywords: cowpea, cowpea weevil, shelf-life, biological control, enzyme activity. introduction in the relentless pursuit of sustainable agricultural practices, the search for effective and ecofriendly pest management strategies remains paramount. among the myriad of challenges faced by farmers, infestation of stored grains and seeds by pests stands as a persistent threat, leading to significant post-harvest losses and economic repercussions. cowpea (vigna unguiculata l. walp) is an important leguminous crop in many tropical and sub-tropical regions, serving as a significant source of protein and income for many people, especially in nigeria, which is considered the largest producer of cowpea [1]. however, its production and post-harvest storage are severely hampered by the cowpea weevil, callosobruchus maculatus [2]. infestation by c. maculatus causes considerable losses in yield and quality, posing a challenge to food security and economic stability in the affected regions [3]. the conventional methods of controlling c. maculatus, such as synthetic chemical pesticides, have drawbacks, particularly regarding the food safety of cowpea, as the produce contains high levels of chemical pesticides considered dangerous to human health [4; 5]. other drawbacks include environmental pollution, biodiversity disruption due to toxicity to non-target organisms, and the development of pesticide resistance [6]. as a result, there is an increasing interest in exploring alternative eco-friendly approaches, such as biological methods, for managing post-harvest cowpea pest populations of c. maculatus. among these biological methods is the use of microbial agents, notably bacteria and fungi, offering healthand environmentally-benign alternatives to conventional chemical pesticides [7]. bacillus thuringiensis, a naturally occurring bacterium, is a gram-positive, spore-forming bacterium known for its insecticidal properties against a wide range of agricultural highlights in bioscience page 1 of 10 september 2025|volume 8 https://doi.org/10.36462/h.biosci.202508 https://creativecommons.org/licenses/by/4.0/ mailto:nwosuonyeka6@gmail.com https://orcid.org/0000-0003-1197-6810 mailto:eogbadoyi@futminna.edu.ng https://orcid.org/0000-0001-6461-2362 mailto:acadbabayi@futminna.edu.ng mailto:isreal.olayemi@futminna.edu.ng https://orcid.org/0000-0001-7116-0720 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes pests [8]. its efficacy, specificity to target pests, and minimal impact on non-target organisms, coupled with its favorable safety profile for humans and the environment, make it an attractive candidate for integrated pest management (ipm) programs. b. thuringiensis produces pesticidal toxins through several molecular mechanisms, most of which are from the cry family of crystalline proteins produced in the parasporal crystals and encoded by the cry genes [9]. these cry proteins are responsible for the death of insect pests when they ingest the cry protoxins or absorb them through the insect body cell membrane. the protoxins solubilize, releasing a protease-resistant biologically active endotoxin [10]. this disrupts the osmotic balance through the formation of transmembrane pores, eventually causing cell lysis in the gut wall and leakage of gut contents [11]. microbial isolates that have insecticidal potential, such as b. thuringiensis, can also be pathogenic to their host by penetrating the insect cuticle and sporulating on desiccated cadavers, thereby facilitating the onset and spread of epizootics [12]. penetration of the insect cuticle is typically the initial stage of infection, relying on mechanical pressure, cuticle-degrading enzymes (lipase, chitinase, protease, etc.), and specialized infection structures (appressoria) produced by the hyphae, which breach the host cells and subsequently proliferate [13]. cuticle-degrading enzymes are likely key virulence factors in entomopathogens, as their substrates contain structural components of the insect cuticle that provide carbon and nitrogen sources to support microbial growth and facilitate host penetration [14]. the spore propagates in response to chemical signals on the cuticle and subsequently develops an appressorium, the specialized structure responsible for penetration. for many years since identifying the pesticidal properties of b. thuringiensis, its preparations have usually been sprayed directly on plants to shield them against different orders of insect pests [10]. in current pest management innovations, the genes encoding the b. thuringiensis pesticidal proteins have been successfully transferred into plants through genetic engineering; such plants are now referred to as transgenic plants [15]. these transgenic plants containing b. thuringiensis genes, specifically for lepidoptera and diptera insect pests, were produced to provide protection against pre-harvest pests without the need for spraying [16]. the potential of b. thuringiensis based bio-pesticides in controlling lepidopteran field pests has been demonstrated in several studies, but its effectiveness against coleoptera: bruchids such as c. maculatus remains relatively unexplored. while the molecular mechanism of b. thuringiensis in infecting hosts through cry proteins, inducing cell lysis in the gut wall and resulting in leakage of gut contents, has been extensively studied, the production of extracellular cuticle-degrading enzymes by b. thuringiensis isolates for degrading the cuticle of coleoptera: bruchids like c. maculatus-a critical barrier to infection and death—remains markedly underexplored. this knowledge gap is especially striking given that adult c. maculatus do not require food, unlike the larvae that feed and develop exclusively on seed legumes, thereby providing a scientific opportunity to assess the potential of cuticle-degrading enzymes to breach the adult c. maculatus exoskeleton and cause pathogenicity. hence, the cuticle-degrading pathway may represent a pivotal, yet overlooked, component of the b. thuringiensis entomopathogenic arsenal, particularly against coleopteran species. this study aimed to evaluate the bio-pesticidal capabilities of a b. thuringiensis isolate and the implications of its extracellular cuticle-degrading enzyme activity on the control of c. maculatus under laboratory conditions. the study hypothesized that b. thuringiensis isolate, applied in both liquid and solid talc-based formulations, would cause significant mortality in adult c. maculatus; its efficacy would be concentration-dependent; the solid formulation would be more effective; and the pesticidal activity is correlated with the production of extracellular cuticle-degrading enzymes like protease, lipase, and chitinase. considering the relevance of this study in addressing the pressing issue of c. maculatus infestation in stored cowpea, it aims to contribute to the development of sustainable and environmentally friendly strategies for cowpea protection and storage, thereby offering valuable insights for ensuring cowpea food safety, improving food security, and guaranteeing livelihoods in major cowpea-growing regions. materials and methods collection of b. thuringiensis isolate b. thuringiensis isolate was obtained from the reference laboratory section of biocrops biotechnology limited, abuja, nigeria, originally sourced from the soil within the abuja environment and identified and confirmed through molecular analysis. bacterial cell culture was performed using potato nutrient agar containing penicillin g. culture plates were incubated at room temperature (28◦c ± 2◦c) for 72 hours. the emerging colonies after the incubation period were discretely isolated and sub-cultured repeatedly on freshly prepared nutrient agar to obtain pure isolates. the pure isolates were subjected to morphological and biochemical tests for confirmation. the confirmed pure isolates were maintained on agar slant bottles, stored at 4◦c, and subsequently subjected to liquid fermentation to enhance microbial proliferation, with sucrose water serving as the carbon-rich substrate and liquid growth medium for the isolate. collection of cowpea seeds and c. maculatus cowpea seeds (black-eye pea, sampea 10-nigeria) were harvested directly from the experimental farm at the national biotechnology research and development agency (nbrda), abuja, nigeria, to eliminate the possibility of prior pesticide exposure during storage. the seeds were aseptically sealed in sterile bags, placed in tightly covered containers, and stored at freezing temperature until further use. cowpea seeds already infested with cowpea weevils were collected locally from cowpea storage facilities in the federal capital territory, abuja, nigeria, and also from traders of the cowpea commodity. adult weevils were isolated from visibly infested seeds through selective picking for subsequent experimental use. highlights in bioscience page 2 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes table 1. shelf-life of b. thuringiensis isolate on talc powder medium concentration of b. thuringiensis in talc powder cfu count of b. thuringiensis (per g) (×104) months (june to november) 1 2 3 4 5 6 10 ml/50 g 63.5±0.14a 53.4±0.07a 37.9±0.05a 18.7±0.21a 6.15±0.09a 2.45±0.02a 15 ml/50 g 213.4±0.81b 92.5±0.18b 55.6±0.31b 26.2±1.18b 7.87±0.22b 4.54±0.05b 20 ml/50 g 272.3±0.24c 143.3±0.40c 80.9±0.44c 47.9±0.56c 12.8±0.06c 8.16±2.11c values are in mean ± s.e. values between experimental treatments on a column bearing the same superscript are not significantly different at the 5% level (p > 0.05). s.e = standard error of mean. cfu: colony forming unit, x: concentration. figure 1. percentage (%) mortality of c. maculatus treated with the liquid medium of b. thuringiensis isolate culturing of the c. maculatus collected cowpea weevils were reared on freshly collected cowpea seeds under laboratory conditions to facilitate acclimatization, following the method described by [17]. batches of about one thousand seeds were each distributed into five prewashed, sterilized, and dried containers. thirty weevils were introduced into each transparent plastic container and covered with mesh nets fastened tightly with rubber bands and masking tape to permit ventilation and prevent escape of the pest. the cowpea seed-weevil mixtures were incubated under laboratory conditions for 10 days to permit mating and oviposition. parent weevils were then removed, and rearing continued until adult emergence. first filial generation (f1) adult weevils were used in the subsequent experiments. inoculation of the b. thuringiensis isolate onto solid medium (talc powder) and shelf-life study the talc stones were collected from ejiba town in yagba west local government area of kogi state, nigeria and were processed into fine powder using the mechanical grinding machines. the talc powder was packed into tightly sealed waterproof nylons and then sterilized appropriately using autoclave with a holding time of 15 minutes at 121 o c. the sterilized talc powder was stored at room temperature. talc powder was used as the solid medium because of its cost-effectiveness considering its local availability, ability of being chemically inert as it doesn’t react with other substances and its good adhesion capacity due to its fine particle size and high surface area. the b. thuringiensis isolate with microbial load of 3.66× 106 cfu/ml was at different quantity (10 ml, 15 ml and 20 ml) each inoculated onto 50 g of sterilized talc powder, appropriately sealed and stored at room temperature. the talc powder containing b. thuringiensis isolates at varying concentrations, were used for mortality study after one month to allow for sporulation. the shelf-life of the b. thuringiensis isolate on the talc powder at different concentrations were assessed at one-month interval for a period of six months using total microbial plate count (colony forming unit per gram) as described by [18]. effect of b. thuringiensis isolate on mortality of c. maculatus ten-fold serial dilution and calculation of total microbial plate count of the microbial isolates was done as described by [18]. a) in triplicate, the b. thuringiensis isolate was assayed for pesticidal activity using the method described by [19]. twenty gram of the cowpea seed were introduced each into sterilized transparent containers with small openings on the cover to allow ventilation. ten cowpea weevils were introduced each into the containers followed by spraying 1 to 4 ml of the liquid formulation of the b. thuringiensis isolate with 3.66 × 106 cfu/ml, 7.32 × 106 cfu/ml, 11.0 × 106 cfu/ml and 14.6 × 106 cfu/ml concentrations respectively using small (10 ml) plastic spray bottles. b) two gram from each of the prepared 10 ml/50 g, 15 ml/50 g, 20 ml/50 g talc powder-b. thuringiensis isolates mixture with 8.99 × 105 cfu/g, 10.5 × 105 cfu/g, 12.6 × 105 cfu/g concentrations, respectively, were introduced into a sterilized transparent container with small openings on the highlights in bioscience page 3 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes cover that allows ventilation, containing 20 g of cowpea seed and 10 cowpea weevils by dusting over the seeds. c) distilled water treatment was used as the control for the b. thuringiensis isolate liquid medium assay while distilled water-talc powder mixture was used as control for the b. thuringiensis isolate solid medium assay. mortality study of the c. maculatus adult cowpea weevil mortality was recorded at 24-hour intervals over an exposure period of 12 and 7 days for the liquid and solid formulation of the b. thuringiensis isolates respectively. dead weevils were carefully removed with the help of a magnifying lens and forceps to reveal the hidden pests in the grains. weevils that failed to respond to gentle prodding with the forceps were considered dead and subsequently removed. the percent mortality was calculated using the following formula: % mortality = number of dead weevils number of weevils introduced × 100 (1) assessment of the percentage of emergence of first generation of c. maculatus emergence of first-generation (f1) adult c. maculatus was determined after 40 days in the same samples used to assess percent mortality. the percentage of emergence (pe) was estimated using: pe = x y × 100 (2) where: x = number of insects that emerged in the treatment y = number of insects that emerged in the control treatment treatments where first-generation emergence is ≤ 50% are considered as promising [20]. cuticle-degrading enzyme activity assay liquid culture for the extracellular enzyme production of the b. thuringiensis isolate the nutrient broth was prepared according to the manufacturers specifications. for the production of extracellular enzymes, 1 ml of the liquid medium of the isolate containing 3.66× 106 cfu/ml was inoculated into the nutrient broth and incubated at room temperature (28 ± 2◦c) for 7 days. after incubation, the contents of the test tube were centrifuged at 10,000 rpm for 20 minutes at 4◦c. finally, the supernatants were used in enzymatic assays. protein concentration assay the total soluble protein concentration in b. thuringiensis isolate was determined using the method of [21], with bovine serum albumin (bsa, 1 mg/ml) as the standard. a stock bsa solution was prepared by dissolving 0.25 g of bsa in distilled water and making up the volume to 250 ml in a volumetric flask. aliquots of 0.2, 0.4, 0.6, 0.8, and 1.0 ml of the bsa solution were dispensed into test tubes and made up to a volume of 1 ml with distilled water. from each tube, 0.5 ml was transferred to a new tube, followed by the addition of 0.5 ml of reagent (50 ml of 2% na2co3 in 0.1 m naoh) and 1.0 ml of 0.5% cuso4 in 1% sodium-potassium (na–k) tartrate. the mixtures were thoroughly mixed and allowed to stand for 10 minutes at room temperature. subsequently, 0.5 ml of folin–ciocalteu reagent was rapidly added to each tube and mixed immediately. the tubes were again allowed to stand for 10 minutes at room temperature, after which absorbance was measured at 625 nm using a uv–vis spectrophotometer (1800-series, shimadzu). a calibration curve was generated by plotting absorbance against bsa concentration. for the determination of sample protein concentration, 0.5 ml of the enzyme solution was processed in the same manner, and protein concentrations were extrapolated from the calibration curve based on the corresponding absorbance values. cuticle-degrading enzyme assay the cuticle-degrading enzymes; total protease, exochitinase, and lipase assays were performed as described by [14] in triplicates. the total protease was assayed using casein as the substrate, while the exochitinase was assayed using p-nitrophenyl-nacetyl-β-d-glucosaminide as the substrate, and the lipase activity was assayed using p-nitrophenyl butyrate as the substrate. calculation of the specific enzyme activity the specific protease, exochitinase, and lipase activity of the b. thuringiensis isolate was calculated using the following formula: specific activity (u/mg) = enzyme activity (u/ml) protein concentration (mg/ml) (3) statistical analysis data were expressed as mean value ± standard error of the mean (s.e.m) and analyzed using anova. significant differences between the control and treatment groups were determined by duncan’s multiple range test (dmrt) in spss (version 26). results the six-month shelf-life study of the b. thuringiensis isolate inoculated onto talc powder as a solid medium (table 1) revealed that b. thuringiensis experienced a gradual reduction in colonyforming units (cfu) per gram over time, with the initial cfu count in the range of 63.5 to 272.3 × 104 cfu/g and the final count in the range of 2.45 to 8.16 × 104 cfu/g, respectively. the toxicity assay of the b. thuringiensis isolate evaluated its efficacy against c. maculatus, a major pest affecting stored cowpea grains. this study assessed the effectiveness of b. thuringiensis in inducing mortality in c. maculatus when applied in liquid and solid forms. the results, presented in (figures 1 and 2), illustrate highlights in bioscience page 4 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes figure 2. percentage (%) mortality of c. maculatus treated with the b. thuringiensis isolate in solid medium (talc powder) the insecticidal activity exhibited by the b. thuringiensis isolates across different concentrations and durations of treatment. the results presented in (tables 2 and 3) show the mean emergence of first-generation (f1) adult c. maculatus treated with the b. thuringiensis isolate in liquid and solid formulations, respectively, and further reveal the percentage emergence of f1 adults, highlighting the efficacy of each treatment concentration in reducing pest reproduction. treatments where f1 emergence is ≤ 50% are considered promising for pest suppression [22; 20]. this study also evaluated the enzymatic activity of b. thuringiensis to determine their contribution to the toxicity potential of the isolate against c. maculatus. the results presented in (figure 3) illustrate the specific activities of protease, lipase, and exochitinase of the b. thuringiensis isolate, considering their potential to degrade the structural components of the insect exoskeleton [23]. discussion the development of b. thuringiensis-based pesticides for the control of c. maculatus in stored cowpea represents a significant advancement in sustainable agricultural practices. microbialbased pesticides, derived from entomopathogenic bacteria such as b. thuringiensis, offer an eco-friendly alternative with promising potential for pest management in cowpea production [24]. these microbial pesticides are target-specific, biodegradable, and pose minimal risks to non-target organisms and human health [25]. the shelf-life study of the b. thuringiensis isolate inoculated onto talc powder provides crucial insights into the long-term viability of the isolate and its potential as a bio-pesticide. (table 1) revealed a significant decline in microbial counts over six months, with variations depending on the concentration, confirming that the gradual decrease in microbial load was significantly influenced by the concentration formulations. the highest concentration (20 ml/50 g) generally maintained higher microbial counts for a longer duration compared to lower concentrations, suggesting that a higher initial inoculation helps sustain viability. in other words, the higher the quantity of b. thuringiensis isolate inoculated onto talc powder, the higher the cfu count and the longer the shelf life. the declining trend observed is also similar to that reported by [26]. other studies have shown a similar declining trend in microbial load of bacillus species when inoculated onto talc-based powder. the study of [27] reported a talc-based powder formulation of bacillus cereus strain b25 spores, in which cfu count of 1.1 × 109 gradually declined during 360 days of storage at room temperature. similarly, [28] reported that talc-based formulations of bacillus subtilis with an initial count of 2.0 × 108 cfu/g at zero day was reduced to 8.3× 106 cfu/g after 80 days of storage at room temperature. any inorganic solid carrier material should offer a protective environment for microbial growth and, when applied for seed treatment, should possess strong adhesion capacity to maximize efficacy and guarantee successful release of bacterial cells after application [29]. in this study, the good efficacy of talc powder– b. thuringiensis isolate may have resulted from strong adhesion capacity to cowpea seeds. talc powder as a solid carrier for microbial isolates has also been reported to be cost-effective, easy to process, chemically stable, with good moisture absorption and buffering capacity, non-toxic to both plants and microbes, and able to ensure bacterial cell viability for at least 2–3 months [30]. studies have shown that bacillus species in talc-based formulations remain viable at a density of 1.0 × 106 cfu/g after 45 days of storage [31]. in this study, despite the decrease in shelf-life of the b. thuringiensis isolate inoculated on talc powder as storage time increased, it still showed that b. thuringiensis cell viability was maintained even at the 4th month (120 days), with maximum viability at 47.9 ± 0.56 × 104 cfu/g and minimum viability at 18.7 ± 0.21 × 104 cfu/g, indicating that talc powder can be a veritable carrier for inoculating b. thuringiensis isolate for commercial production. this study has confirmed that talc powder effectively supports b. thuringiensis isolates for bio-pesticide purposes by providing a stable medium for storage and application. while talc-based formulations can sustain microbial viability for some months, a decline is unavoidable. considering that the rate of decline varies among organisms, there is a need to explore options that could further enhance microbial longevity. for example, [29] reported that talc was an effective carrier, but additional stabilizers such as glycerol improved shelf life beyond six months. therefore, this study emphasizes the importance of considering optimized storage conditions (e.g., low humidity, cool temperatures) and incorporating stabilizers such as glycerol, silica gel, or oil-based formulations to further enhance microbial persistence over time. the toxicity assay, which assessed the efficacy of b. thuringiensis isolate against c. maculatus, as shown in (figures 1 and 2), revealed that at all treatment concentrations, a significant percent mortality was recorded compared with the control, indicating the high pesticidal potential of the b. thuringiensis isolate against adult c. maculatus. the study further showed that the isolate caused mortality in a concentration-dependent manner, with effectiveness increasing over time and at higher microbial concentrations, and demonstrated stronger effects through solid medium exposure. highlights in bioscience page 5 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes table 2. percentage emergence of first generation of c. maculatus after treatment with liquid medium of the b. thuringiensis isolate microbial isolate treatment volume (ml) treatment concentration (cfu/ml) mean emergence of treatment (x) mean emergence of control (y) pe (%) remarks b. thuringiensis 1 3.66 × 106 2.33 ± 0.01a 11.3 ± 0.04b 20.61 promising 2 7.32 × 106 1.33 ± 0.03c 9.66 ± 0.11d 13.76 promising 3 11.0 × 106 0.67 ± 0.02c 7.67 ± 0.05e 8.73 promising 4 14.6 × 106 0.33 ± 0.01a 6.33 ± 0.03b 5.21 promising values are in mean ± s.e. values between experimental treatments on a row bearing the different superscript are significantly different at the 5% level (p > 0.05). s e = standard error of mean. pe: percentage emergence. cfu: colony forming unit. x = number of insects that emerged in the treatment, and y = number of insects that emerged in the control treatment. treatments where first-generation emergence is ≤ 50% are considered as promising. figure 3. the cuticle-degrading enzyme activity of b. thuringiensis isolate the 12-day treatment with the liquid medium of b. thuringiensis isolate showed 100% mortality on the 12th day at concentrations of 7.32 × 106 cfu/ml and 11.0 × 106 cfu/ml, and on the 8th day at 14.6 × 106 cfu/ml. a similar toxicity trend was observed in the 7-day treatment study using the solid carrier, where 100% mortality was recorded on the 6th day at the lowest concentration (8.99 × 105 cfu/g) and on the 5th day at higher concentrations (10.5 × 105 cfu/g and 12.6 × 105 cfu/g). these results indicate that for the commercial production of b. thuringiensis-based bioinsecticides for the control of c. maculatus, concentrations above 106 cfu/g or cfu/ml will be required for faster, early, and effective pest suppression. the control group, which was not treated with any microbial suspension, showed negligible mortality, confirming that the observed insect deaths were substantially due to the microbial treatments rather than external environmental factors. according to [11], insect mortality caused by b. thuringiensis is usually a result of insecticidal toxins (cry toxins), often referred to as δ-endotoxins, which are somewhat specific to certain insect families. the adult c. maculatus may have absorbed the b. thuringiensis isolates through its cuticle as a result of the direct application of the isolate in liquid and solid carriers. these findings are consistent with previous research on microbial biocontrol agents against c. maculatus. for example, [32] reported that bacillus flexus biofilms exhibited significant insecticidal effects on c. maculatus, suggesting that bacterial biofilm extracellular matrices could be leveraged as bio-pesticides for cowpea storage protection. similarly, [33] examined the impact of bacillus flexus s13 on c. maculatus and reported lc50 values indicating moderate toxicity, supporting the effectiveness of bacterial isolates in insect pest control. in comparison, the current study suggests that b. thuringiensis exhibits slightly higher potency than b. flexus against c. maculatus, though further comparative analysis is needed. the high toxicity of b. thuringiensis isolates on adult c. maculatus recorded with the solid medium treatment, where even the lowest concentration achieved 100% mortality before the 7th day, may be due to the increased surface area exposure provided by talc powder as a carrier. this level of mortality was not achieved with liquid medium treatments, even at the highest concentration used in the study. therefore, in microbial control of c. maculatus in stored cowpea, application methodologies that enhance surface area exposure of the microbial isolate should be adopted for effective control. these findings align with recent research demonstrating the efficacy of entomopathogenic bacteria on solid carriers for pest control. for instance, [34] reported that b. thuringiensis on a solid carrier significantly impacted storedproduct insects, particularly trogoderma granarium, through its endotoxins. the current study also found b. thuringiensis on talc powder to be highly potent against c. maculatus, with the highest mean mortality (over 80%) in the 20 ml/50 g treatment. while these results are consistent with past research, certain variations exist due to environmental factors, strain differences, and formulation methods. for instance, [35] emphasized that formulation type (liquid vs. solid) influences microbial persistence and virulence, which may explain minor differences in mortality rates highlights in bioscience page 6 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes table 3. percentage emergence of first generation of c. maculatus after treatment with the b. thuringiensis isolate in solid medium (talc powder) microbial isolate treatment volume (ml/50g) treatment concentration (cfu/g) mean emergence of treatment (x) mean emergence of control (y) pe (%) remarks b. thuringiensis 10 8.99 × 105 2.00 ± 0.01a 9.33 ± 0.18b 21.43 promising 15 10.5 × 105 1.67 ± 0.01a 7.67 ± 0.12b 21.77 promising 20 12.6 × 105 0.67 ± 0.01a 5.33 ± 0.22b 12.57 promising values are in mean ± s.e. values between experimental treatments on a column bearing the different superscript are significantly different at the 5% level (p > 0.05). s e = standard error of mean. pe: percentage emergence. cfu: colony forming unit. x = number of insects that emerged in the treatment, and y = number of insects that emerged in the control treatment. treatments where first-generation emergence is ≤ 50% are considered as promising. observed across different studies. adjuvants such as inert carriers, like talc powder for loading microorganisms as active ingredients of microbial pesticides, possess greater adsorption capacity and enhanced dispersion properties, enabling controlled release of active components at appropriate intervals [36]. when microbial isolates penetrate the host cell, they proliferate, providing a pathway for epizootics [13]. at relatively low quantities, these crystalline proteins exhibit high insecticidal activity [37]. the emergence of the first-generation (f1) adult c. maculatus serves as a crucial indicator of the long-term effectiveness of microbial bio-pesticides. while initial mortality rates provide insight into the immediate insecticidal effects of microbial isolates, f1 emergence evaluates their residual impact on pest populations by determining whether microbial treatments can suppress reproductive success and prevent re-infestation. in this study, f1 emergence was assessed 40 days after treatment application, following the mortality evaluation. the study showed a significant delay in the f1 emergence period of adult c. maculatus at all treatment concentrations compared with the control. the significant delay observed in both liquid and solid medium treatments was expressed in a concentration-dependent manner, with greater delays recorded at higher concentrations. the percentage emergence further validated the efficacy of b. thuringiensis treatments. in both formulations, b. thuringiensis isolates at higher concentrations resulted in f1 emergence percentages well below the 50% threshold. the results from this study align with previous research on microbial bio-pesticides for stored-product pest management. for instance, [38] confirmed the efficacy of b. thuringiensis in reducing weevil emergence and suggested that the persistence of b. thuringiensis spores in treated grains may provide longterm protection against re-infestation, reinforcing the results of this study. similarly, [39] investigated trichoderma spp. for cowpea beetle control and found that while microbial spores significantly reduced f1 emergence, they were not as effective as b. thuringiensis. their findings align with the present study, where b. thuringiensis demonstrated high efficiency in reducing f1 emergence, suggesting that bacterial bio-pesticides such as b. thuringiensis may offer more immediate control compared to fungal alternatives. the findings of this study underscore the significance of biopesticide concentration, microbial strain selection, and storage conditions in optimizing pest suppression. higher concentrations of b. thuringiensis isolate resulted in a significant reduction in f1 emergence, suggesting its potential as a leading candidate for the biological control of c. maculatus. additionally, the superior performance of solid formulations in limiting f1 emergence suggests that formulation type plays a critical role in bio-pesticide effectiveness. furthermore, the calculation of the percentage f1 emergence suggests that the concentration levels used in the study are promising for pesticidal activity. the assessment of cuticle-degrading enzyme activity in b. thuringiensis isolate demonstrated the presence of protease, lipase, and exochitinase, suggesting their role in the toxicity mechanisms of b. thuringiensis against c. maculatus. these enzymes are crucial for the pathogenicity of microbial bio-pesticides, as they degrade the structural components of the insect exoskeleton, facilitating microbial penetration and systemic infection [23]. beyond mechanical pressure, enzymatic breakdown of insect cuticles can aid in the penetration of microbial isolates through the degradation of proteins, chitins, and lipids that make up the insect cuticle [40]. the present study revealed the secretion of different cuticle-degrading enzymes, such as proteases, lipases, and chitinases. the findings indicate that the b. thuringiensis isolate exhibited measurable enzymatic activity, although at varying levels, suggesting differential enzymatic strategies in their pathogenicity. protease activity was observed to display the highest activity, exceeding that of lipase and exochitinase. the protease, exochitinase, and lipase enzyme activity of the b. thuringiensis isolate may have synergistically contributed to the pathogenicity against highlights in bioscience page 7 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes adult c. maculatus. the significant differences in the extracellular enzyme activities per milliliter of the liquid culture of the b. thuringiensis isolate could be attributed to variability in virulence at different medium concentrations. hence, this study suggests that the role of these cuticle-degrading enzymes in the pathogenicity of c. maculatus may be concentration-dependent. proteases are regarded as pivotal catalysts in the infection processes by cleaving peptide bonds in proteins and breaking them into small peptides and amino acids [14]. exochitinase plays a vital role in cleaving n-acetylglucosamine in cuticular layers, while lipases break down ester bonds in lipoproteins, fats, and waxes found in the interior part of the insect integument to release (un)saturated fatty acids [41]. the higher protease activity of the b. thuringiensis isolate may have acted as the key catalyst for the infection of adult c. maculatus. supporting the findings of this study, [42] reviewed hydrolytic enzymes in integrated pest management and highlighted the role of bacterial entomopathogens such as b. thuringiensis in producing proteases that degrade insect cuticle proteins. this complements the current study, which observed significant protease activity in b. thuringiensis, reinforcing its role as an effective microbial pesticide. similarly, [43] examined microbial hydrolytic enzymes and found that bacterial mechanisms of cuticle degradation differ from fungal entomopathogens, as bacterial enzymes often exhibit species-specific targeting, a factor that may influence their efficacy. the production of protease, exochitinase, and lipase by the b. thuringiensis isolate might be the key virulence factor against adult c. maculatus, considering that adult weevils do not require food or water within their limited lifespan and therefore could not have ingested the treated cowpea. drawing on the results of this study, the relationship between cuticle-degrading enzymes and virulence could serve as a diagnostic criterion for selecting effective microbial control agents for c. maculatus. future studies are required to elucidate the mechanistic role of cuticle-degrading enzymes in the significant delay in adult c. maculatus emergence observed in this study. conclusion this study demonstrated that bacillus thuringiensis isolate is highly effective against adult callosobruchus maculatus and holds significant potential as a bio-control agent within integrated pest management strategies for stored cowpea. its application can promote food security and enhance cowpea safety by providing an alternative to toxic chemical pesticides. furthermore, the study confirmed that the b. thuringiensis isolate secretes key cuticle-degrading enzymes-protease, exochitinase, and lipase which likely serve as major virulence factors. these enzymes facilitate cuticle degradation, enabling the bacterium to penetrate the insect host, ultimately causing infection and mortality. the findings underscore the promise of b. thuringiensis not only as a microbial bio-pesticide for controlling bruchids but also as a candidate for developing genetically engineered cowpea varieties with plant-incorporated protectants for bruchid resistance. for commercial production using talc powder as a solid carrier, the study recommends maintaining higher microbial concentrations above 104 cfu/g to ensure adequate shelf life and sustained efficacy. reference 1. nwagboso c, andam ks, amare m, bamiwuye t, fasoranti 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highlights in bioscience page 8 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes 12. pathak d, kumar m. microbial inoculants as biofertilizers and biopesticides. in: microbial inoculants in sustainable agricultural productivity: vol. 1: research perspectives. springer; 2016. p. 197-209. 13. huarte-bonnet c, paixao fr, ponce jc, santana m, prieto ed, pedrini n. alkane-grown beauveria bassiana produce mycelial pellets displaying peroxisome proliferation, oxidative stress, and cell surface alterations. fungal biology. 2018;122(6):457-64. 14. golzan sr, talaei-hassanloui r, homayoonzadeh m, safavi sa. role of cuticle-degrading enzymes of beauveria bassiana and metarhizium anisopliae in virulence on plodia interpunctella (lepidoptera, pyralidae) larvae. journal of asia-pacific entomology. 2023;26(2):102038. 15. nwosu ok, ubaoji ki. genetic modification as a control mechanism to plant pest attack. in: natural remedies for pest, disease and weed control. elsevier; 2020. p. 203-8. 16. fernández-chapa d, ramírez-villalobos j, galán-wong l. toxic potential of bacillus thuringiensis: an overview. protecting rice grains in the post-genomic era. 2019. 17. sowunmi e. effect of storage length and insecticide treatment on cowpea (vigna unguiculata l. walp). 1982. 18. agu k, igweoha c, umeh c. antimicrobial activity of the ethanolic and petroleum ether extracts of tangerine seed on selected bacteria. 2013. 19. tiroesele b, thomas k, seketeme s. control of cowpea weevil, callosobruchus maculatus (f.)(coleoptera: bruchidae), using natural plant products. insects. 2014;6(1):77-84. 20. rendón-huerta ja, juárez-flores bi, aguirre-rivera jr, álvarez f. insecticide effect of wild plant powders on bean weevil (zabrotes subfasciatus boheman; coleoptera: bruchidae) in vitro. african journal of agricultural research. 2013;8(11):971-7. 21. lowry oh, rosebrough nj, farr a, randall r. protein measurement with the folin phenol reagent. journal of biological chemistry. 1951;193:256-75. 22. lagune ta. vegetable extracts and mineral powders for combating maize and bean pest in subsistence agriculture. montecillo, edo, mexico: college of postgraduate agricultural sciences press; 1994. 23. ortiz-urquiza a, keyhani no. molecular genetics of entomopathogenic fungi. annual review of microbiology. 2000;74:127-50. 24. sharma p, tyagi v, srivastava p. potential of microbial pesticides in insect pest management. international journal of current microbiology and applied sciences. 2018;7(4):2589-601. 25. lacey la, grzywacz d, shapiro-ilan di, frutos r, brownbridge m, goettel ms. insect pathogens as biological control agents: back to the future. journal of invertebrate pathology. 2015;132:1-41. 26. geetika g, buttar ds, singh n. shelf life study of bacillus isolates b1 and b3 in talc based bioformulation at different storage temperature. pharma innovation journal. 2023;12(7):2106-10. 27. martinez n, sinedino ld, bisinotto rs, daetz r, risco ca, galvão kn. effects of oral calcium supplementation on productive and reproductive performance in holstein cows. journal of dairy science. 2016;99(10):8417-30. 28. gupta m, dohroo np. shelf life study of formulation of fungal and bacterial antagonist as bioinoculant. agricultural science digest. 2014;34:281-4. 29. singh j, singh av, upadhayay vk, khan a. comparative evaluation of developed carrier based bioformulations bearing multifarious pgp properties and their effect on shelf life under different storage conditions. environment and ecology. 2020;38(1):96-103. 30. khan a, singh av, gautam ss, agarwal a, punetha a, upadhayay vk, et al. microbial bioformulation: a microbial assisted biostimulating fertilization technique for sustainable agriculture. frontiers of plant science. 2023;14:1270039. 31. amer ga, utkhede rs. development of formulations of biological agents for management of root rot of lettuce and cucumber. canadian journal of microbiology. 2000;46(1):80916. 32. reda fm, hassanein wa, moabed s. potential exploitation of bacillus flexus biofilm against the cowpea weevil, callosobruchus maculatus (f.) (coleoptera: bruchidae). egyptian journal of biological pest control. 2020;30:18. 33. hassanein aw, reda fm, moabed s, el shafiey sn. insecticidal impacts of bacillus flexus s13 biofilm extracellular matrix on cowpea weevil, callosobruchus maculatus. biocatalysis and agricultural biotechnology. 2021;31:101898. 34. riaz t, masoom a, virk uy, raza m. impacts of metarhizium anisopliae on mortality, energy reserves, and carbohydrase of trogoderma granarium. journal of stored products research. 2022;99:102013. 35. mantzoukas s, kitsiou f, lagogiannis i, eliopoulos pa. potential use of fusarium isolates as biological control agents: helicoverpa armigera (hübner) (lepidoptera: noctuidae) case study. applied sciences. 2022;12(17):8918. highlights in bioscience page 9 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ nwosu ok et al., 2025 the potentials of bacillus thuringiensis isolate and its extracellular cuticle-degrading enzymes 36. lin f, mao y, zhao f, idris al, liu q, zou s, et al. towards sustainable green adjuvants for microbial pesticides: recent progress, upcoming challenges, and future perspectives. microorganisms. 2023;11(2):364. 37. rajadurai g, anandakumar s, raghu r. bacillus thuringiensis in pest management. plant health archive. 2023;1(1):113. 38. mohammed nag, hassan aah, mohammed mo. the effect of pyrrolidinium bromide salt in the life of the southern cowpea beetle callosobruchus maculatus (fab.). iop conference series: earth and environmental science. 2021;910:012140. 39. rodríguez-gonzález campelo mp, lorenzana a, mayoprieto s, gonzález-lópez álvarez garcía s, et al. spores of trichoderma strains sprayed over acanthoscelides obtectus and phaseolus vulgaris l. beans: effects in the biology of the bean weevil. journal of stored products research. 2020;88:101666. 40. lu hl, st leger rj. insect immunity to entomopathogenic fungi. in: lovett b, st leger rj, editors. advances in genetics. vol. 94. academic press; 2016. p. 251-85. 41. mondal s, baksi s, koris a, vatai g. journey of enzymes in entomopathogenic fungi. pacific science review a: natural science and engineering. 2016;18(2):85-99. 42. ahmad t, rasool a. hydrolytic enzymes and integrated pest management. in: khan ma, ahmad w, editors. microbes for sustainable insect pest management. vol. 17. springer, cham; 2021. p. 25-36. 43. lopes fc, martinelli ahs, john ebo. microbial hydrolytic enzymes: powerful weapons against insect pests. in: khan ma, ahmad w, editors. microbes for sustainable insect pest management. vol. 17. springer, cham; 2021. . highlights in bioscience page 10 of 10 september 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods collection of b. thuringiensis isolate collection of cowpea seeds and c. maculatus culturing of the c. maculatus inoculation of the b. thuringiensis isolate onto solid medium (talc powder) and shelf-life study effect of b. thuringiensis isolate on mortality of c. maculatus mortality study of the c. maculatus assessment of the percentage of emergence of first generation of c. maculatus cuticle-degrading enzyme activity assay results discussion conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.20246 research article open access 1 department of chemistry, faculty of education, university of khartoum, sudan. * to whom correspondence should be addressed: nawal.mahgoub@uofk.edu editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), cairo, egypt. reviewer(s): khaled h. radwan, national biotechnology network of expertise, asrt, cairo, egypt.. tamer ahmed elakkad, faculty of agriculture at moshtohor, benha university, benha, egypt. received: may 3, 2024 accepted: november 20, 2024 published: december 27, 2024 citation: suleman nm, eltyeb em. the effectiveness of bentonite and esterified plant fibers on the biodegradability of 24 polypropylene biocomposites . 2024 dec. 27;7:bs20246 copyright: © 2024 nawal et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. the effectiveness of bentonite and esterified plant fibers on the biodegradability of 24 polypropylene biocomposites nawal m. suleman 1 >< �, eiman m. eltyeb1 >< � abstract this study aims to evaluate the biodegradability of 24 bio-composites synthesized from polypropylene and esterified plant fibers, with bentonite serving as a filler. the biodegradation process was conducted using aspergillus niger. the study utilized standard laboratory equipment and the melt flow indexer. after three months of biodegradation, all biocomposites under investigation showed a significant loss in their weights. this is a logical observation because aspergillus niger consumed parts of biocomposites as food during the biodegradation. the study also evaluated properties including density, melting point, melt flow rate (mfr), melt viscosity, molecular weight, and water absorption capacity. densities, melting range temperature, and molecular weights were decreased (decreasing in molecular weights). the values of biodegraded composites after the biodegradation process for three months are in good agreement with the fact that the molecular chain breaks and the chain length shortens after any degradation process. water absorption capacities were significantly increased (due to the holes resulting from the aspergillus niger attack) for all biocomposites under study; this is considered very good evidence for biodegradation. keywords: bio-composite, biodegradation, aspergillus niger, bentonite, polypropylene introduction biodegradable materials decompose within a year in their usual disposal environments, converting into non-toxic substances through the activities of living organisms [1]. the biodegradability of polymers is influenced by two main factors. the environmental conditions are exposed to (biotic or abiotic) and the polymer’s characteristics (such as mobility, crystallinity, density, molecular weight, functional groups, and additives). abiotic factors (e.g., ph, temperature, light, and moisture) can change the hydrolysis reaction rates. biotic factors, such as extracellular enzymes produced by distinct microorganisms, can biodegrade certain polymers. for instance, aspergillus niger and aspergillus flavus fungi produce enzymes that more easily break down aliphatic polyesters composed of 6 12 carbon di-acid monomers than those produced from other monomers [2]. most conventional plastics (polypropylene) are non-biodegradable, and their accumulation in the environment has endangered the planet. to resolve all these challenges, several strategies have been utilized. firstly, it involves the degradation of some petrochemical-based plastics by biological processes. secondly, the production of plastics with a high degree of degradability. the aliphatic polyesters are regular plastic polymers that show high potential for use as biodegradable plastics [3]. polypropylene is a challenging substrate for biodegradation by microorganisms due to its high molar mass and inability to pass through microorganisms cell membranes [4]. biodegradation of polypropylene/starch or polypropylene/cellulose composites has been observed, where organisms easily degrade starch or cellulose, producing polymer waste. these carbohydrates or fillers increase the adhesion of organisms to the surface of the polymer [5]. protocols such as surface changes and changes in the mechanical and physical properties of the polymer were utilized to characterize the biodegradability of composites [6]. this study aims to test the biodegradability of 24 composites previously synthesized from polypropylene and esterified fibers extracted from khimp (leptadenia pyrotechnica) and date palm tree (phoenix dactylifera l.) and esterified using citric and adipic acid (aad). highlights in bioscience page 1 of 4 december 2024|volume 7 https://doi.org/10.36462/h.biosci.20246 https://creativecommons.org/licenses/by/4.0/ mailto:nawal.mahgoub@uofk.edu https://orcid.org/0000-0002-6909-7512 mailto:eimanmubarak@uofk.edu https://orcid.org/0000-0003-0606-9634 http://bioscience.highlightsin.org/ nawal et al., 2024 the effectiveness of bentonite and esterified plant fibers materials and methods the analytical chemicals used were polypropylene (9003-070, sigma-aldrich), bentonite clay (from india mart), adipic acid, citric acid, sulfuric acid, acetic anhydride, sodium hydroxide, peptone, glucose, ethanol, agar, and hydrochloric acid (from bdh, india). toluene (108-88-3, sigma-aldrich), xylene (133020-7, sigma-aldrich), and deionized water (the bio-composites of polypropylene were prepared previously) [7]. a computerized, fully automated melt flow rate (mfr) tester was sourced from india mart. biodegradation method biodegradation takes place according to the following equations: (a): cplastic + o2 → co2 + h2o +cresidual + biomass (b): cplastic → ch4 +co2 + h2o +cresidual + biomass a: aerobic biodegradation, b: anaerobic biodegradation. the ingredients of sabouraud dextrose agar (sda) (10g peptone + 40g glucose + 15g agar) were combined in 900 ml of deionized water, then the ph was adjusted to 5.6 with hcl, and the final volume was adjusted to 1000 ml by adding deionized water. the mixture was boiled to completely dissolve the medium, sterilized at 121oc for 20 min by autoclave, then warmed to 50oc and distributed into petri dishes. the ingredients of sabouraud dextrose broth (20g glucose and 10g peptone) were suspended in 1000 ml of deionized water, heated to dissolve the medium completely, and the mixture was sterilized by autoclave at 121oc for 15 min. the sterilization of polymer samples was conducted under uv light and transferred into the middle of petri dishes. the culture broth was replaced with 50 ml of sterile nutrient broth every 10 days to enhance healthy microbial growth. after three months, biodegraded composites were washed, dried, and weighed to determine the weight loss caused by microbial action during biodegradation. the composites were then subjected to characterization [8]. characterization of the biodegraded composites biodegraded composites were characterized by the same instruments and methods described for characterization of the synthetic composites and properties [7]. the measured properties that were characterized include density, melting points, melt flow rate (mfr), melt viscosity, molecular weight, and water immersion test. results and discussions biodegradation of composites providing sustainable alternatives to conventional plastics is indispensable. therefore, researchers and manufacturers are attempting to integrate sustainable and biodegradable polymers into industrial processes [9]. regarding the environmental concerns, this study was conducted. biodegradation was carried figure 1. petri dishes showing microbial colony diversity during biodegradation. figure 2. biodegraded composites showing structural changes. out according to the literature review. (aspergillus niger) were cultivated in order to provide a good culture for biodegradation (figure 1) shows some petri dishes prepared for this purpose. during biodegradation, microorganisms start utilizing the polymer surface as a food source and grow on it. various polymer characteristics influence biodegradation, such as their tactility, crystallinity, molecular weight, functional group types, the type of microorganism, and the pre-treatment method [10]. this clearly occurred for all samples under investigation. the esterified fibers used as reinforcement agents in the synthesis of the target composites introduced new functional groups to the polypropylene resin and hence enhanced the biodegradability in an acceptable way because the availability of functional groups like ester increases hydrophilicity. this ester group not only increases the hydrophilicity of the synthetic composites, but it is also an easily breakable bond compared to the carbon-carbon bonds in polypropylene. biodegradation occurred differentially depending on the amount and type of esterified fiber. highlights in bioscience page 2 of 4 december 2024|volume 7 http://bioscience.highlightsin.org/ nawal et al., 2024 the effectiveness of bentonite and esterified plant fibers table 1. characteristics of 24 biodegraded composites under study no. composite entity code density (g/cm3) melting range (řc) melt flow rate melt viscosity m-w water absorption capacity (%) 1 1-a 0.84 165-166 5.02 0.86 1.8 x 105 10.3 2 1-b 1.00 170-173 8.3 0.82 1 x 105 15.3 3 1-c 0.92 169-173 4.68 0.87 0.8 x 105 32 4 1-d 0.89 170-175 4.91 0.87 0.8 x 105 24 5 1-e 0.93 168-169 1.54 0.90 0.3 x 105 28.3 6 1-f 0.82 175-177 6.9 0.81 0.9 x 105 15.6 7 1-g 0.82 169-172 3.55 0.89 0.6 x 105 36.5 8 1-h 0.87 160-163 6.2 0.85 0.9 x 105 25.4 9 1-i 0.95 164-170 2.69 0.90 0.5 x 105 15.9 10 1-j 1.00 168-171 10.6 0.80 1.1 x 105 11 11 1-k 0.87 165-167 2.11 0.93 0.4 x 105 24.2 12 1-l 0.83 175-178 6.06 0.84 0.9 x 105 44 13 2-d 0.84 160-163 10.5 0.80 1.1 x 105 8.75 14 2-e 0.80 165-168 2.71 0.91 0.5 x 105 10 15 2-f 1.00 161-165 4.72 0.87 0.8 x 105 18.9 16 2-j 0.82 167-171 5.37 0.85 0.8 x 105 18.8 17 2-k 0.95 170-174 6.17 0.82 0.9 x 105 15 18 2-l 0.92 170-172 8.5 0.82 1 x 105 20 19 3-a 0.94 174-178 8.2 0.82 1 x 105 33 20 3-b 0.92 172-177 5.41 0.86 0.8 x 105 12.2 21 3-c 0.86 169-171 4.9 0.86 0.8 x 105 26 22 3-g 1.00 169-172 6.4 0.84 0.9 x 105 13.3 23 3-h 0.80 167-170 3.34 0.89 0.6 x 105 12.3 24 3-i 0.90 170-172 1.29 0.97 0.2 x 105 25 additionally, using bentonite as a filler provides a good environment for microorganisms; all biodegraded samples showed an amazing change in their surfaces (figure 2). in addition to the surface change, there are many physical properties that indicate biodegradation occurrence, such as density, water absorption capacity, molecular weight, melt flow rate, and melt viscosity. these properties will be discussed in detail after the characterization step. the loss of weight in 24 biodegraded composites is presented in (tables s1 to s4), quantifying the level of composite biodegradation after three months of aspergillus niger incubation. the weights of composites before and after three months were measured. weights lost percentages were calculated according to the following equation: weight lost (%) = (wo − w) wo × 100 wo =weight before biodegradation and w =weight after biodegradation. a crucial aspect of our experiment is the impact of clay nanoparticles on biodegradation; the presence of clay increases microorganisms' attack, thus enhancing the biodegradation process due to microorganisms' preference for clay media for growth, as reported in the literature. weights lost were clearly observed for all composites and increased with increasing ester content, either in the presence or absence of clay, except for samples 1-i, 1-k, 2-f, 2-k, and 3-b. this may have occurred due to the dispersion of either esterified fibers or clay among polypropylene resin during the injection molding process. another important observation is that composites made from sample one, esterified by citric acid, showed significant increases in weight loss compared to their analogs made from the same sample esterified by adipic acid. characterization of biodegraded composites the densities of the biodegraded composites were determined based on the guidelines provided in [11]. densities of composites decreased clearly after the biodegradation process because microorganisms consume a part of the composite for their growth; this is very good evidence of biodegradation occurrence (table 1), and the melting range temperature of the 24 biodegraded composites was measured experimentally as described in [11]. the resulting melts of all composites under study were thick liquids. biodegraded composites melted at lower melting ranges than those of synthetic composites. the melt flow rate is a characterization method used to evalhighlights in bioscience page 3 of 4 december 2024|volume 7 http://bioscience.highlightsin.org/ nawal et al., 2024 the effectiveness of bentonite and esterified plant fibers figure 3. water absorption capacities for the biodegraded composites samples uate the degradation process of biodegraded composites by calculating the reduction in molecular weight for each composite. (table 1) represents the values of the melt flow rate for the 24 composites under investigation. for determining melt viscosities, the same equation described for synthesized composites was used as follows: [η] = 0.996 − 0.189 log(mfr) [η]: intrinsic viscosity, mfr: molecular flow ratio or melt flow rate. the average molecular weight of each biodegraded composite was determined using the same equation applied for the average molecular weight of synthesized composites. log[mfr]composite = [0.454 × 10−6]mw − 0.1273 values of the average molecular weight for the biodegraded composites are shown in table 5. according to the above table, molecular weights of biodegraded composites were decreased significantly. the water immersion test demonstrates that all biodegraded composites absorbed some amount of water over time; however, they were insoluble in water. water absorption capacity (wac) was calculated according to the equation clarified by [12]. wac (%) = ( (m − mo) mo ) × 100 wac (%): water absorption capacity, mo: weight of dry sample, and m: weight of sample after 24-hour immersion in distilled water. there is a strong correlation between molecular weight and water absorption capacity of all biodegraded composites; biodegraded composites with lower molecular weight have more water absorption capacities than those with comparatively high molecular weight. this is considered a reasonable correlation because when microorganisms attack the composite surface in the biodegradation process, many holes occur as a result of this action, allowing water to penetrate. the decrease in molecular weights and the increase in water absorption capacities provide strong evidence for the occurrence of biodegradation (figure 3). supplementary table s1: weights lost for composites of khimp fiber esterified by adipic acid. table s2: weights lost for composites of khimp fiber esterified by citric acid. table s3: weights lost for composites of date palm leave fiber esterified by citric acid. table s4: weights lost for composites of date palm fiber esterified by adipic acid. reference 1. leja k, lewandowicz g. polymer biodegradation and biodegradable polymers-a review. polish journal of environmental studies. 2010;19(2). 2. devi rs, kannan vr, natarajan k, nivas d, kannan k, chandru s, et al. the role of microbes in plastic degradation. environ waste manage. 2016;341:341-70. 3. tokiwa y, calabia bp, ugwu cu, aiba s. biodegradability of plastics. international journal of molecular sciences. 2009;10(9):3722-42. 4. de carvalho cl, dos santos rosa d. polypropylene biodegradation. properties, uses and benefits. 2016:141. 5. arutchelvi j, sudhakar m, arkatkar a, doble m, bhaduri s, uppara pv. biodegradation of polyethylene and polypropylene. 2008. 6. markl e, grünbichler h, lackner m. phb-bio based and biodegradable replacement for pp: a review. nov tech nutr food sci. 2018;2(5):206-9. 7. eltyeb em, mohammed aa, suleman nm. polypropylene and some esterified plants’ fibers bio-based composites. 2023. 8. ebadi-dehaghani h, barikani m, borhani s, bolvardi b, khonakdar ha, jafari sh, et al. biodegradation and hydrolysis studies on polypropylene/polylactide/organo-clay nanocomposites. polymer bulletin. 2016;73:3287-304. 9. mysiukiewicz o, barczewski m, skórczewska k, matykiewicz d. correlation between processing parameters and degradation of different polylactide grades during twin-screw extrusion. polymers. 2020;12(6):1333. 10. luthra p, vimal kk, goel v, singh r, kapur gs. biodegradation studies of polypropylene/natural fiber composites. sn applied sciences. 2020;2:1-13. 11. suleman nm, eltyeb em, mohammed aa. polypropylene and some esterified plants’ fibers bio-based composites. ijfac (indonesian journal of fundamental and applied chemistry). 2023;8(1):48-54. 12. kudahettige-nilsson rl, ullsten h, henriksson g. plastic composites made from glycerol, citric acid, and forest components. bioresources. 2018;13(3):6600-12. highlights in bioscience page 4 of 4 december 2024|volume 7 http://bioscience.highlightsin.org/ abstract introduction materials and methods biodegradation method characterization of the biodegraded composites results and discussions biodegradation of composites characterization of biodegraded composites supplementary highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202502 research article open access 1 meat and fish technology research department, food technology research institute, agriculture research center, ministry of agriculture, giza, egypt. 2 food technology department, faculty of agriculture, suez canal university, ismailia 41522, egypt. # deceased * to whom correspondence should be addressed: ahmed_rayan@agr.suez.edu.eg editor: jean legeay, university mohammed vi polytechnic, benguerir, morocco. reviewer(s): mokhtar said rizk, department of genetic resources, desert research center (drc), cairo, egypt. muhammad adeel, arthritis and clinical immunology research program, oklahoma medical research foundation, oklahoma city, ok, usa. received: february 21, 2025 accepted: may 22, 2025 published: june 15, 2025 citation: omar ias, farag mam, taha ar, gaballa aa, rayan ma. shrimp waste utilization: exploring composition, bioactive potential, and safety treatments. 2025 june 15;8:bs202502 copyright: © 2025 omar et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. shrimp waste utilization: exploring composition, bioactive potential, and safety treatments shereen i. a. omar1,2 >< �, manar m. a. farag1 >< �, rafat a. taha2# ><�, amal a. gaballa2 ><�, ahmed m. rayan*2 ><� abstract the global focus on waste management and recycling is increasing, driven by the need to protect the environment from the harmful effects of waste accumulation. this study evaluated the chemical composition, bioactive potential, and microbial quality of shrimp waste, revealing significant variations across treatments and locations. ghalyoun shrimp heads exhibited the highest protein (41.6%) and fat (16.9%) contents, while shells and tails from port said and head from ghalyoun shrimp were rich in calcium (76.55 mg/g, 75.9 mg/g), respectively. phenolic content was highest in jumbo suez heads (77.06 mg gae/g dm), exhibiting maximum antioxidant activity of 65.8% (dpph assay) in the same sample. furthermore, hplc analysis showed pyrogallol dominance in ghalyoun shells (32,974 ppm) and ismalawy heads (32,907 ppm), while jumbo suez heads had the highest gallic acid (4,508 ppm) and catechol (8,514 ppm). ellagic acid peaked in jumbo suez (10,643 ppm), confirming strong bioactive potential. heavy metals, including pb and ni, were highest in heads (1.3 mg/g and 1.34 mg/g, respectively), but effective chelation treatments, particularly t6 (acetic acid 12.5% + citric acid 5% + sodium chloride 5%), significantly reduced these levels and microbial contamination, lowering total microbial counts to 3.57 log cfu/g. the findings suggest shrimp waste, particularly heads, as a valuable resource for antioxidants, proteins, and minerals, with appropriate treatments ensuring safety for industrial applications. keywords: shrimp waste valorization, bioactive compounds, protein and mineral recovery, heavy metal and microbial reduction, antioxidant activity introduction shrimps are highly valued crustaceans enjoyed worldwide as a premium food source and an integral component of a balanced diet. the global shrimp market has experienced significant growth over the years, highlighting their importance as a rich nutritional source for human populations [1]. shrimps are an excellent source of high-quality protein, vitamins, and astaxanthin, along with vital minerals such as phosphorus (p), calcium (ca), selenium (se), copper (cu), zinc (zn), and iodine (i2) [2]. in 2023, global shrimp production totaled 5.6 million tons and it is expected to increase to 7.28 million tons by 2025, growing at an annual growth rate of 6.1% between 2020 and 2025 [3; 4]. consequently, shrimp waste production is estimated at around 3.8 million tons annually, constituting 50-60% of the total catch volume [5]. according to the fao, a steady upward trend in shrimp consumption has been observed worldwide, indicating its growing dietary relevance and consumer demand [6]. shrimp processing industries generate substantial amounts of waste, including shrimp heads, shells, and tails, which account for approximately 50% of the raw material. the increasing disposal of shrimp waste into the environment significantly contributes to pollution and poses serious health risks. as a result, effectively processing and utilizing shrimp shell waste is crucial for reducing environmental harm and advancing sustainability efforts [7; 8]. this waste is not merely a byproduct; it is a resource rich in valuable bioactive compounds, including polysaccharides, proteins, carotenoids, and fatty acids. these compounds have demonstrated diverse bioactivities, such as antioxidant, antimicrobial, anti-inflammatory, and antitumor effects, making them highly promising for applications in the pharmaceutical, nutraceutical, and cosmeceutical industries [9]. harnessing these bioactive components aligns with sustainable highlights in bioscience page 1 of 11 june 2025|volume 8 https://doi.org/10.36462/h.biosci.202502 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:shreen_abbas@agr.suez.edu.eg mailto:dr.manar69@gmail.com https://orcid.org/0009-0001-8104-2100 mailto:rafat_amin@agr.suez.edu.eg mailto:amal_gab@agr.suez.edu.eg https://orcid.org/0000-0003-0837-7606 mailto:ahmed_rayan@agr.suez.edu.eg https://orcid.org/0000-0003-3544-4675 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition resource management and circular economy principles, emphasizing waste valorization to minimize environmental impact and maximize economic value. we would like to emphasize that one of the innovative aspects of this study is its focus on the utilization of egyptian shrimp waste, a locally abundant raw material that has not been sufficiently exploited previously. additionally, the study takes into account the specific physical and chemical characteristics of marine waste in the egyptian environment, which may differ from those in other regions due to climatic and environmental variations. this study hypothesizes that shrimp waste, when processed and treated effectively, can serve as a valuable resource for high-value compounds while mitigating environmental hazards. therefore, the main aim of this study is to investigate the chemical composition of shrimp waste from various locations, assess its protein content and bioactive potential, and evaluate methods to reduce heavy metals and microbial contamination. ultimately, this research seeks to provide insights into sustainable utilization strategies for shrimp waste, offering a blueprint for industries, researchers, and policymakers to adopt more environmentally friendly and economically viable waste management practices. materials and methods raw shrimp wastes (head, shell, and tail) were collected from local markets located in ismailia, port said, suez city, and kafr el-sheikh cities. shrimp wastes were transported to the laboratory in an icebox. the ingredients, including salt, spices, garlic, and starch, were sourced from local markets in ismailia governorate, egypt. all chemicals utilized in this study were of analytical grade. the shrimp wastes were separated into two groups: the first one was left unwashed, while the second one was thoroughly washed multiple times with tap water. each group was further separated into two categories: one comprising shrimp heads and the other consisting of shell waste, including tails. all samples were analyzed to determine their chemical composition, microbiological load, mineral and heavy metal content, antioxidant activity (dpph assay), and total phenolic content. it is noteworthy to mention that the untreated samples were analyzed in parallel across all tests to accurately assess the treatment effects. treatment of shrimp waste the following treatments were carried out to reduce the heavy metals content and microbiological load: acetic acid 25% for 30 min (treatment 1), citric acid 10% for 30 min (treatment 2), sodium chloride 10% for 30 min (treatment 3), combination of 12.5% acetic acid + 5% citric acid (treatment 4), combination of 12.5% acetic acid + 5% sodium chloride (treatment 5), and combination of 12.5% acetic acid + 5% citric acid + 5% sodium chloride (treatment 6) [10]. shrimp shell powder (ssp) preparation shrimp shell waste, including heads, shells, and tails, was separated and dried in a hot air oven at 60°c for 24 h. the dried figure 1. the treated and processed shrimp wastes from the different locations. a1: heads only (jumbo suez shrimp), a2: shrimp shell and tail (jumbo suez shrimp), b1: heads only (port said shrimp), b2: shrimp shell and tail (port said shrimp), c1: heads only (ismalawy shrimp), c2: shrimp shell and tail (ismalawy shrimp), d1: heads only (ghlayoun farmed shrimp), d2: shrimp shell and tail (ghlayoun farmed shrimp). shrimp wastes were finely ground using a blender, with each batch processed three times for 2–3 minutes. the ground shrimp wastes were then sieved to obtain a fine ssp. the powder was carefully packed in airtight glass bottles to preserve its quality and stored in a refrigerator at 4°c until it was required for further use [11]. figure 1 explains the treated and processed shrimp wastes from the different locations. determination of chemical composition the shrimp waste samples were analyzed for ash, protein, and fat content using the official methods of analysis. the samples were digested with a nitric acid and perchloric acid mixture (4:1, v/v), heated to 70-90°c for 10 minutes, cooled, and analyzed for cadmium (cd), lead (pb), nickel (ni), cobalt (co), and zinc (zn) using an atomic absorption spectrophotometer (thermo electron corp., s series, china) [12]. antioxidants determination shrimp shells were dehydrated at 41°c for 48 h and ground into fine particles. for preliminary extraction, 5 g of shrimp shell was mixed with 50 ml of absolute methanol and stirred for 2 h at room temperature [1]. the mixture was filtered using filter paper, and the filtrate was evaporated at 40°c under vacuum conditions using a rotary evaporator. light exposure was minimized throughout the process to protect the extract from degradation. the antioxidant capacity was evaluated using the dpph assay, following the procedure outlined by [13]. the extract (500 µg/ml) was diluted to concentrations of 50, 100, 150, and highlights in bioscience page 2 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition 200 µg/ml. one milliliter of each sample was combined with 1 ml of 0.4 mm dpph solution and 2 ml of methanol in a sealed glass container. after 30 min of reaction at 37°c, the mixture was transferred to a disposable polystyrene cuvette, and the uv-visible spectrum was recorded. a blank sample, prepared with 2 ml of methanol and 1 ml of dpph solution, was used as a control. the inhibition percentage, representing the decrease in absorbance of the sample compared to the initial absorbance of the blank, was measured at 515 nm using a spectrophotometer. each sample was prepared and analyzed in triplicate, with all samples protected from light to prevent dpph degradation. the inhibition of the dpph radical was estimated through the following equation: %inhibition = ( aco − aat aco ) × 100% (1) where aco represents the absorbance of the control at time t = 0, and aat represents the absorbance of the samples at time t = 30 minutes. total polyphenol content (tpc) to determine the tpc, 10 mg of methanolic extracts were dissolved in distilled water to give a concentration of 10 mg/ml [14]. this stock solution was then diluted to prepare concentrations of 0.5, 1, and 2 mg/ml. the tpc was estimated using the folin-ciocalteu reagent method, as described by [15], with gallic acid serving as the standard. briefly, 0.5 ml of the extract was added to test tubes, followed by 2.5 ml of 10% folin-ciocalteu reagent and 2 ml of 7.5% sodium carbonate (na2co3). the mixture was thoroughly mixed using a shaker and allowed to stand for 30 minutes. absorbance was measured at 765 nm, and the results were expressed as mg gallic acid equivalent per gram dry matter as mg gae/g dm. hplc analysis of phenolic compounds extraction, separation, and quantification were carried out following the procedure [16]. methanol was added to 5 grams of each sample, and the mixture was centrifuged for 10 minutes at 10,000 rpm. after filtering the supernatant through a 0.2 µm millipore membrane, 1-3 ml was collected in a vial for injection into an hplc (hewlett-packard, series 1200) equipped with a quaternary hp pump (series 1100), solvent degasser, uv detector set at 280 nm, and auto-sampling injector. the temperature of the c18 reverse-phase column was maintained at 35oc. methanol and acetonitrile were used as the mobile phase at a flow rate of 1.0 ml/min with gradient separation. after dissolving sigma co. phenolic standards in the mobile phase, they were injected into the hplc. using hewlett-packard software for data analysis, the concentration of phenolic compounds was determined based on retention time and peak area. total aerobic plate count (tapc) samples of fresh and treated shrimp’s shell and head were bacteriologically analyzed for total aerobic bacterial counts and figure 2. minerals concentration (mg/g) in shrimp wastes samples. a1: heads only (jumbo suez shrimp), a2: shrimp shell and tail (jumbo suez shrimp), b1: heads only (port said shrimp), b2: shrimp shell and tail (port said shrimp), c1: heads only (ismalawy shrimp), c2: shrimp shell and tail (ismalawy shrimp), d1: heads only (ghlayoun farmed shrimp), d2: shrimp shell and tail (ghlayoun farmed shrimp). total coliforms at 37 °c, following the method described below: the agar plate method was used to determine total aerobic bacterial counts on nutrient agar, according to [17]. a 10 g sample was blended in a high-speed blender under sterile conditions for 3 minutes in 90 ml of buffered peptone water. decimal dilutions were prepared for the determination of various bacterial groups. the plates were incubated at 37 ± 2 ◦c for 48 hours. coliform group counts violet red bile (vrb) agar was used as the medium. plates were incubated at 35oc for 18-24 hours, and purple, round colonies were counted. for fecal coliform and e. coli, purple colonies were confirmed on eosin methylene blue (emb) agar, with blue-black, metallic-sheen colonies streaked onto slant agar. results were reported as log cfu/g [18]. statistical analysis the data were evaluated using analysis of variance (anova) test, conducted with spss software (version 16.0 for windows, spss inc., chicago). duncan’s multiple range tests were employed to determine significant differences among treatment means at p < 0.05. results and discussions proximate composition the chemical composition of shrimp waste samples revealed significant variations across treatments and locations. moisture content was highest in heads from ismalawy and jumbo suez shrimp (c1: 30.5%, a1: 29.4%) and lowest in shells and tails from jumbo suez shrimp (a2: 25.9%). protein content was notably highest in ghalyoun farmed shrimp (d1: 41.6%, d2: 40.8%), reflecting their superior nutritional value, while port highlights in bioscience page 3 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition said shrimp exhibited the lowest protein levels (b2: 35.9%, b1: 36.3%). ash content was highest in shells and tails, particularly in ismalawy and jumbo suez shrimp (c2: 31.39%, a2: 29.50%), likely due to the mineral-rich exoskeleton, while ghalyoun shrimp heads had the lowest ash levels (d1: 13.2%). fat content was highest in ghalyoun shrimp heads (d1: 16.9%), indicating their lipid-rich potential, and lowest in ismalawy and jumbo suez shells and tails (c2: 5.11%, a2: 5.95%) table 1. table 1. chemical composition of shrimp waste samples treatments chemical composition (%) on dry weight basis moisture protein ash fat a1 29.41±1.04a 39.8±0.98ab 15.9±0.57 f 14.9±0.98b a2 25.9±0.87c 38.7±1.30b 29.5±0.87b 5.95±0.87 f b1 27.05±0.75b 36.3±1.70c 23.75±1.03c 12.9±0.76c b2 26.7±0.54bc 35.6±0.66c 18.3±1.06b 8.97±0.55e c1 30.5±0.63a 38.4±0.58b 20.03±0.98d 11.07±1.2d c2 26.9±0.86bc 36.0±0.93c 31.39±0.88a 5.11±0.64 f d1 28.3±0.32a 41.6±1.20a 13.2±0.79g 16.9±1.40a d2 26.8±0.55bc 38.1±1.20a 17.8±0.56e 14.6±1.50b results are expressed as average ± standard deviation of triplicate determinations. a1 refers to heads only (jumbo suez shrimp), a2 to shrimp shell and tail (jumbo suez shrimp), b1 to heads only (port said shrimp), b2 to shrimp shell and tail (port said shrimp), c1 to heads only (ismalawy shrimp), c2 to shrimp shell and tail (ismalawy shrimp), d1 to heads only (ghlayoun farmed shrimp), and d2 to shrimp shell and tail (ghlayoun farmed shrimp). superscript letters a-f in the same columns indicate significant differences within the same strain (p < 0.05). these results suggest that heads are nutritionally denser, with ghalyoun farmed shrimp standing out as the most valuable resource for protein and fat extraction, while shells and tails are ideal for mineral recovery. it was found that no significant difference existed in crude protein content among shrimp shells, tails, and heads of the different five species of shrimp byproducts [19]. however, crude fat varied significantly across species and parts, with heads having the highest content (2.17-6.88%). ash content (8.18-13.45%) was highest in shells/tails, reflecting mineral richness, while crude fiber (mainly chitin) peaked in shells/tails (7.86-10.78%) and exhibited antimicrobial potential. these differences might be attributed to species, age, and environmental factors. overall, the data underscore the potential of shrimp waste for sustainable valorization, with location and waste type playing key roles in determining their suitability for specific applications. several studies have reported variable protein levels in shrimp waste, ranging from 39–70% [20; 21]. the protein content of shrimp waste from penaeus merguensis was reported to be 24.03% [22]. it was found that raw shrimp head waste of penaeus semisulcatus contained 65.76% protein [23]. shrimp wastes were also observed to contain 35–40% protein [24], while the protein content of shrimp biowaste was reported to be 28.3% [25]. the variations in protein content of raw shrimp waste may be attributed to differences in shrimp species and the sources of raw materials. confirming its potential, shrimp waste powder was found to nutritionally enrich food products while preserving their sensory qualities and safety, establishing a practical pathway for sustainable seafood byproduct utilization [26]. minerals concentration in shrimp waste the trace mineral analysis of shrimp waste samples revealed notable differences across treatments and locations, highlighting the nutrient diversity in shrimp heads and shells figure 2. magnesium (mg) content was highest in heads from port said (b1: 88.4 mg/g) and ismalawy (c1: 87.9 mg/g), indicating their potential as rich mg sources, while the lowest levels were found in shells and tails from port said (b2: 69.4 mg/g). sodium (na) content peaked in heads from port said and ghalyoun shrimp (b1 and d1: 23.06 mg/g), whereas shells and tails from ghalyoun (d2: 9.19 mg/g) had the lowest levels. iron (fe) concentrations were generally low across all samples, with the highest values in port said heads (b1: 0.18 mg/g) and the lowest in ismalawy and ghalyoun shells and tails (c2, d2: 0.05 mg/g), suggesting limited fe availability. fe is predominantly stored as ferritin in the liver, spleen, and bone marrow, while in human blood, it binds to hemoglobinan iron-containing protein responsible for oxygen binding and transport [19]. phosphorus (p) content ranged from 9.46 mg/g (c2) to 13.4 mg/g (d1), with ghalyoun heads containing the highest levels, reflecting their potential as a phosphorus source. calcium (ca) was notably high in shells and tails, particularly from port said (b2: 76.55 mg/g) and ghalyoun (d1: 75.9 mg/g), underscoring the mineral richness of exoskeleton-based waste. selenium (se) levels varied, with the highest in ismalawy shells and tails (c2: 0.67 mg/g), indicating a promising source of this essential trace element. potassium (k) content was highest in ghalyoun heads (d1: 4.78 mg/g), followed by shells and tails from jumbo suez (a2: 4.14 mg/g), showcasing their potential for potassium recovery. these results are in agreement with those obtained by [27], who observed that calcium (155 ± 4.00 mg/g), iron (42.11 ± 2.00 mg/g), copper (40 ± 2.00 mg/g), and manganese (12.6 ± 2.2 mg/g) were prevalent in shrimp waste. it was reported that calcium was abundantly present in prawn shells, with a concentration 17 times higher than that of magnesium [28]. furthermore, it was found that the concentrations of minerals such as na, k, p, ca, mg, fe, and mn in shrimp waste powder from penaeus spp. were 53.2, 47.5, 21.8, 89.1, 27.1, 39.4, and 17.4 mg/g, respectively [29]. overall, the findings from this study demonstrate that shrimp heads typically contain higher concentrations of essential minerals such as magnesium, sodium, and potassium when compared to other shrimp by-products. in contrast, the shells and tails of shrimp were found to be particularly rich in calcium and selenium. these distinct mineral distribution patterns highlight the unique nutritional composition associated with each type of shrimp waste. moreover, the variation in mineral content based on both waste type and collection location presents valuhighlights in bioscience page 4 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition figure 3. heavy metals elements (mg/g) of shrimp wastes. a1: heads only (jumbo suez shrimp), a2: shrimp shell and tail (jumbo suez shrimp), b1: heads only (port said shrimp), b2: shrimp shell and tail (port said shrimp), c1: heads only (ismalawy shrimp), c2: shrimp shell and tail (ismalawy shrimp), d1: heads only (ghlayoun farmed shrimp), d2: shrimp shell and tail (ghlayoun farmed shrimp). able opportunities for selective resource recovery. such targeted extraction and utilization strategies can contribute significantly to sustainable practices in various sectors, including agriculture, pharmaceutical development, and the production of dietary supplements, thereby promoting environmental sustainability and economic efficiency. heavy metals in shrimp waste samples shrimp heads are prone to heavy metal accumulation, leading to diminished nutritional quality. this phenomenon occurs because aquatic organisms can bioaccumulate heavy metals from various environmental sources, such as sediments, atmospheric deposition, and wastewater discharge [30]. the hepatopancreas, a key detoxification and metal storage organ in crustaceans, plays a significant role in this process. additionally, the gills, due to their direct exposure to water, further facilitate the rapid uptake of heavy metals [31]. the analysis of heavy metal concentrations in shrimp waste samples revealed significant variations depending on the sample type (heads vs. shells and tails) and location figure 3. lead (pb) levels were highest in jumbo suez heads (a1: 1.3 mg/g), while the lowest levels were observed in ghalyoun shells and tails (d2: 0.29 mg/g). mercury (hg) concentrations were relatively low across all samples, with a1 (0.17 mg/g) showing the highest level and d2 (0.032 mg/g) the lowest. arsenic (as) content peaked in ismalawy heads (c1: 0.11 mg/g) but was minimal in most other samples, with a2 and d2 both at 0.005 and 0.006 mg/g, respectively. manganese (mn) levels were highest in a1 (0.73 mg/g) and lowest in c2 and d2 (0.13 mg/g), reflecting the nutritional richness of heads compared to shells and tails. copper (cu) was most abundant in a1 (1.09 mg/g) and lowest in d2 (0.26 mg/g). cadmium (cd) concentrations were generally low, with the highest figure 4. total phenolic content of shrimp waste samples: a1 (heads only, jumbo suez shrimp), a2 (shrimp shell and tail, jumbo suez shrimp), b1 (heads only, port said shrimp), b2 (shrimp shell and tail, port said shrimp), c1 (heads only, ismalawy shrimp), c2 (shrimp shell and tail, ismalawy shrimp), d1 (heads only, ghlayoun farmed shrimp), and d2 (shrimp shell and tail, ghlayoun farmed shrimp). error bars indicate ± standard deviation. values with different letters are significantly different (p < 0.05). levels detected in a1 and c2 (0.118 mg/g each), while several samples, such as b2, d1, and d2, showed no detectable cd. nickel (ni) was highest in a1 (1.34 mg/g) and lowest in d2 (0.41 mg/g). zinc (zn), an essential trace metal, showed the highest levels in ghalyoun heads (d1: 60.6 mg/g) and the lowest in ismalawy shells and tails (c2: 21.7 mg/g). in general, these results are in agreement with those obtained by [32]. the fao/who established maximum permissible limits of 0.50 µg/g for pb, cd, as, and hg. accordingly, the contents of pb, cd, as, and hg in the different investigated samples are higher than the limited values [31]. overall, shrimp heads consistently exhibited higher concentrations of heavy metals compared to shells and tails, with jumbo suez and ghalyoun shrimp showing the most notable levels. while heads are nutrient-rich, the elevated heavy metal concentrations necessitate appropriate treatment and monitoring to ensure safe and sustainable utilization of shrimp waste. bioactive compounds of shrimp waste total phenols content of shrimp waste samples (tp) the total phenolic content of shrimp waste samples demonstrated significant variability across treatments and locations (p < 0.05) figure 4. the highest total phenol content was observed in the heads from jumbo suez shrimp (a1: 77.06 mg gae/g dm), indicating their superior potential as a source of antioxidant compounds. this was followed by the shell and tail from jumbo suez shrimp (a2: 45.45 mg gae/g dm), heads from ghalyoun farmed shrimp (d1: 44.74 mg gae/g dm), and ismalawy shrimp (c1: 43.64 mg gae/g dm). shells and tails generally exhibited lower phenol content, with jumbo suez highlights in bioscience page 5 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition table 2. hplc analyses of phenolic compounds (ppm) in shrimp waste samples phenolic compound a1 a2 b1 b2 c1 c2 d1 d2 pyrogallol 29677.6 16574.9 20061.1 25282.1 32906.8 23698.2 24694.7 32974.3 gallic 4507.5 2521.6 1669.64 908.5 1078.3 2374.4 1218.1 0 3-oh tyrosol 3560.2 1764.9 2203.06 655.1 2589.5 509.3 3679.2 209.6 catechol 8514.0 5500.8 1786.36 172.5 406.8 111.1 3512.0 459.0 4-amino benzoic 480.4 258.2 43.25 20.1 149.8 211.8 211.8 27.42 catechein 5109.7 4577.9 2441.58 700.2 872.5 303.5 1084.5 490.5 chlorogenic 2686.1 1672.4 702.04 91.9 1441.9 267.4 1537.7 221.4 p-oh-benzoic 1389.2 647.8 161.35 180.6 163.4 114.9 564.9 181.9 benzoic 1832.6 896.7 1091.2 242.5 1320.4 153.4 758.8 54.2 caffeic 2059.3 1341.5 257.02 65.1 679.4 154.9 613.4 112.5 vanillic 1605.3 405.1 377.6 269.9 305.6 164.7 805.2 182.7 caffeine 1721.5 558.2 1177.96 223.6 1058.3 186.2 508.6 0 oleuropein 3142.6 337.7 3304.3 283.1 913.5 515.5 524.1 822.8 ferulic 928.5 394.8 745.98 224.1 832.5 179.6 951.6 74.6 ellagic 10643.3 7854.7 1950.2 1689.2 1622.8 1260.2 578.6 967.2 coumarin 582.6 180.4 41.29 29.3 44.4 37.20 75.16 38.82 a1 refers to heads only (jumbo suez shrimp), a2 to shrimp shell and tail (jumbo suez shrimp), b1 to heads only (port said shrimp), b2 to shrimp shell and tail (port said shrimp), c1 to heads only (ismalawy shrimp), c2 to shrimp shell and tail (ismalawy shrimp), d1 to heads only (ghlayoun farmed shrimp), and d2 to shrimp shell and tail (ghlayoun farmed shrimp). shrimp shells (a2: 45.45 mg gae/g dm) being an exception, showcasing relatively high phenol levels compared to other shell samples. the lowest phenol content was recorded in shells and tails from ismalawy shrimp (c2: 29.06 mg gae/g dm) and port said shrimp (b2: 30.23 mg gae/g dm), as well as heads from port said shrimp (b1: 31.78 mg gae/g dm). it was reported that the total phenolic content in shrimp shell waste from palaemon serratus and palaemon varians, collected along the portuguese coast, ranged from 4.7 to 10.4 mg gae/g dm [1]. the differences may be attributed to variations in species, environmental conditions, and processing methods, which influence the phenolic compound levels in shrimp waste [33]. overall, shrimp heads consistently exhibited higher total phenolic content compared to shells and tails, with jumbo suez shrimp heads being the richest source. these findings suggest that shrimp heads, particularly from jumbo suez and ghalyoun shrimp, could be prioritized for antioxidant extraction in nutraceutical and pharmaceutical applications, supporting sustainable valorization strategies. hplc analyses of phenolic compounds the hplc analysis revealed variability in the phenolic compound profiles of shrimp waste samples across different treatments and locations table 2. pyrogallol was the dominant phenolic compound, with the highest levels observed in ghalyoun shells (d2: 32,974.3 ppm) and ismalawy heads (c1: 32,906.8 ppm), while gallic acid and catechol were most abundant in jumbo suez heads (a1: 4,507.5 ppm and 8,514.0 ppm, respectively). ellagic acid was prominent in jumbo suez samples (a1: 10,643.3 ppm; a2: 7,854.7 ppm), underscoring their strong bioactive potential. heads consistently exhibited higher phenolic concentrations than shells and tails, particularly in samples from jumbo suez and ghalyoun, which demonstrated superior phenolic diversity and content. in contrast, port said samples showed the lowest phenolic levels, with shells and tails generally being less phenol-rich than heads. these findings highlight the significant bioactive potential of shrimp heads, particularly from jumbo suez and ghalyoun, for use in nutraceutical, pharmaceutical, and functional food industries, supporting the sustainable valorization of shrimp waste. the results also suggest that treatment conditions play a crucial role in the preservation or degradation of phenolic compounds in shrimp waste, with significant differences across the groups. the variability in phenolic content aligns with previous studies highlighting the sensitivity of phenolic compounds to environmental factors and processing conditions, which can significantly influence their bioavailability and antioxidant properties [34; 35]. antioxidant activity (dpph %) of shrimp waste samples the antioxidant activity was assessed using dpph (2,2diphenyl-1-picrylhydrazyl), a stable free radical compound with maximum absorbance at 517 nm. when dpph radicals interact with proton-donating substances, such as antioxidants, the radicals are neutralized, leading to a reduction in absorbance [36]. highlights in bioscience page 6 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition figure 5. antioxidant activity (dpph %) of shrimp waste samples: a1 (heads only, jumbo suez shrimp), a2 (shrimp shell and tail, jumbo suez shrimp), b1 (heads only, port said shrimp), b2 (shrimp shell and tail, port said shrimp), c1 (heads only, ismalawy shrimp), c2 (shrimp shell and tail, ismalawy shrimp), d1 (heads only, ghlayoun farmed shrimp), and d2 (shrimp shell and tail, ghlayoun farmed shrimp). error bars indicate ± standard deviation. values with different letters are significantly different (p < 0.05). figure 5 illustrates the dpph radical-scavenging activity of various shrimp waste samples at a concentration of 1 mg/ml. the antioxidant activity (dpph %) of shrimp waste samples showed significant variation across treatments and locations (p < 0.05). the highest antioxidant activity was observed in heads from jumbo suez shrimp (a1: 65.793%), indicating their superior free radical scavenging capacity. ghalyoun heads (d1: 44.337%) and ismalawy heads (c1: 40.18%) also exhibited notable antioxidant activity, underscoring the potential of shrimp heads as rich sources of antioxidants. in contrast, shells and tails consistently showed lower activity, with the lowest values recorded in port said shells and tails (b2: 6.382%) and ghalyoun shells and tails (d2: 8.864%). among shell and tail samples, jumbo suez (a2: 9.083%) and ismalawy (c2: 10.965%) demonstrated slightly higher activity. these results highlight that shrimp heads, particularly from jumbo suez and ghalyoun shrimp, are a valuable source of antioxidants, while shells and tails exhibit significantly lower activity, making heads the primary target for antioxidant recovery in valorization strategies. the results indicate that the samples likely contained peptides or chitooligosaccharides, which act as electron donors. these compounds can react with free radicals, stabilizing them and terminating the radical chain reaction [37; 38; 39]. microbial quality of shrimp's waste samples bacterial growth is a primary factor in the spoilage of fish and fish products. therefore, bacterial count is recommended as a key indicator for assessing the quality of food products [40]. the microbial analysis of shrimp waste samples revealed significant differences in total microbial count, vibrio cholerae, and coliform group levels across treatments (p < 0.05) figure 6. the highest figure 6. microbial analysis of shrimp waste samples. a1: heads only (jumbo suez shrimp), a2: shrimp shell and tail (jumbo suez shrimp), b1: heads only (port said shrimp), b2: shrimp shell and tail (port said shrimp), c1: heads only (ismalawy shrimp), c2: shrimp shell and tail (ismalawy shrimp), d1: heads only (ghlayoun farmed shrimp), d2: shrimp shell and tail (ghlayoun farmed shrimp). total microbial count was observed in port said shrimp, with both heads (b1: 7.52 log cfu/g) and shells/tails (b2: 7.54 log cfu/g) showing significantly higher values compared to samples from other locations. jumbo suez shrimp heads (a1: 6.58 log cfu/g) and ismalawy heads (c1: 6.62 log cfu/g) exhibited moderate microbial loads, while ghalyoun shrimp (d1: 5.62 log cfu/g, d2: 5.58 log cfu/g) had the lowest counts, suggesting better microbial quality. for vibrio cholerae, jumbo suez shrimp (a1: 7.26 log cfu/g, a2: 7.37 log cfu/g) showed the highest contamination, highlighting a potential safety concern. port said samples (b1: 7.25 log cfu/g, b2: 7.19 log cfu/g) and ismalawy samples (c1: 7.22 log cfu/g, c2: 7.21 log cfu/g) followed closely, while vibrio cholerae was not detected in ghalyoun shrimp (d1 and d2). coliform group levels were highest in jumbo suez shells and tails (a2: 6.11 log cfu/g) and moderate in port said samples (b2: 5.77 log cfu/g). ghalyoun shrimp (d1: 4.33 log cfu/g, d2: 4.64 log cfu/g) consistently showed the lowest coliform levels, further emphasizing their microbial safety. ismalawy shrimp (c1: 4.49 log cfu/g) also had low coliform counts in the heads, aligning with better overall microbial quality. in summary, shrimp samples collected from port said exhibited the highest levels of microbial contamination, with particularly elevated values observed in the total microbial count. on the other hand, shrimp waste originating from ghalyoun farms consistently demonstrated the lowest microbial loads across all evaluated parameters. these results underscore the significant influence of geographic location and specific waste type on the microbial safety of shrimp by-products. the notably lower contamination levels found in ghalyoun shrimp suggest that this highlights in bioscience page 7 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition source possesses superior microbial quality, making it a more suitable candidate for various potential applications, including those in agriculture, food processing, and biotechnological industries where microbial safety is a critical consideration. table 3. effect of some treatments on reducing microbial count of shrimp wastes. treatments microbial count (log cfu/g) total count vibrio cholerae coliform group c 5.32a nd 4.75a t1 4.65c nd 4.15b t2 5.12b nd 3.24c t3 4.36c nd 4.13b t4 4.46c nd 3.19c t5 4.59c nd 4.13b t6 3.57d nd 2.23d c: control, t1: acetic acid 25% /30 min, t2: citric acid 10% /30 min, t3: sodium chloride 10% /30 min, t4: acetic acid 12.5% + citric acid 5% /30 min, t5: acetic acid 12.5% + sodium chloride 5% /30 min, t6: acetic acid 12.5% + citric acid 5% + sodium chloride 5% /30 min. a–d values in the same columns with different superscript letters within a same strain are significantly different (p<0.05). nd: not detected. treatments to reduce heavy metals in shrimp waste the chelation method was identified as a promising technique for effectively removing heavy metals from shrimp waste samples. the formulations included: t1 (10% acetic acid), t2 (10% citric acid), t3 (10% sodium chloride), t4 (12.5% acetic acid + 5% citric acid), t5 (12.5% acetic acid + 5% sodium chloride), and t6 (12.5% acetic acid + 5% citric acid + 5% sodium chloride), each applied for 30 minutes. interestingly, these treatments were able to chelate the heavy metals present in the shrimp waste. the concentrations of acid and salt were selected based on previous studies that demonstrated their effectiveness under similar conditions [10]. regarding the use of 25% acetic acid in treatment t1 compared to 12.5% in treatments t4–t6, this was intentionally designed to evaluate the impact of high versus low concentrations on product quality and microbial inhibition. it is worth noting that the lower concentration (12.5%) was combined with other substances to enhance its efficacy, whereas the higher concentration (25%) was applied alone without additional components, to assess its standalone effectiveness. t6 (12.5% acetic acid + 5% citric acid + 5% sodium chloride for 30 minutes) resulted in the lowest levels of heavy metals (pb, hg, as, mn, cu, cd, ni, and zn) in shrimp waste figure 7. it was demonstrated that acetic acid effectively chelated heavy metals in green mussels [10]. chelating organic acids can remove exchangeable, carbonate, and reducible fractions of heavy metals through washing processes [41; 42]. sodium acetate has also been shown to chelate heavy metals such as arsenic (as), lead figure 7. effect of some treatment on reduce the heavy metals of shrimp wastes. all treatments were applied for 30 minutes. the specific treatments were: c: control, t1: acetic acid 10 %, t2: citric acid 10 %, t3: sodium chloride 10 %, t4: acetic acid 12.5 % + citric acid 5 %, t5: acetic acid 12.5 % + sodium chloride 5 %, t6: acetic acid 12.5 % + citric acid 5 % + sodium chloride 5 %. all treatments were applied for 30 minutes. (pb), cadmium (cd), and nickel (ni) in green mussels, reducing their levels to those deemed safe for human consumption [32]. effect of some treatments on reducing microbial content in shrimp waste the results demonstrate that various treatments effectively reduced the microbial content of shrimp wastes, with all treatments successfully eliminating vibrio cholerae table 3. the control sample (c) had the highest microbial counts, emphasizing the need for intervention. among the treatments, t6 (12.5% acetic acid + 5% citric acid + 5% sodium chloride) was the most effective, achieving the lowest total microbial count (3.57 log cfu/g) and coliform count (2.23 log cfu/g), highlighting the synergistic effect of combining acids and salt. single-agent treatments, such as t1 (25% acetic acid) and t2 (10% citric acid), were less effective, with higher microbial counts compared to the combined treatments. this study underscores the superior antimicrobial efficacy of combination treatments like t6, making them highly suitable for enhancing the microbial safety and quality of shrimp wastes. economic feasibility repurposing shrimp waste safeguards the environment and promotes the circular economy through sustainable resource utilization. the proposed treatments are amenable to scale-up, as they utilize locally available and low-cost chemicals such as acetic acid, sodium chloride, and citric acid. the procedures for preparing shrimp shell powder are straightforward and do highlights in bioscience page 8 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition not require complex industrial techniques, thereby facilitating large-scale application. moreover, the resulting shell powder exhibits promising properties that qualify it for direct use in the food industry as an ingredient in nutraceutical products. conclusion this study highlights the significant potential of shrimp waste as a resource for sustainable valorization, with variations in chemical composition, mineral content, heavy metals, bioactive compounds, and microbial quality across different treatments and locations. shrimp heads, particularly from ghalyoun and jumbo suez regions, emerged as the most nutritionally dense components, with high protein, fat, and phenolic compound levels, making them ideal for antioxidant extraction and functional food applications. shells and tails, on the other hand, were rich in calcium and selenium, suitable for mineral recovery. heavy metal analysis showed that heads generally had higher concentrations, necessitating effective treatments like chelation to ensure safety. the combined treatment of acetic acid, citric acid, and sodium chloride (t6) proved to be the most effective in reducing heavy metal levels and microbial contamination, including vibrio cholerae, showcasing its efficacy as a decontamination strategy. antioxidant activity further confirmed the bioactive potential of shrimp heads, with jumbo suez samples demonstrating superior free radical scavenging capacity. overall, our work uniquely targets egyptian shrimp waste, leveraging its distinct regional traits to fill a research gap and advance localized circular economy strategies. by emphasizing location-specific treatments, this study maximizes waste valorization while ensuring safety, enabling applications in agriculture, pharmaceuticals, and nutraceuticals to uphold circular economy principles. reference 1. maia ml, grosso c, barroso mf, silva a, delerue-matos c, domingues vf. bioactive compounds of shrimp shell waste from palaemon serratus and palaemon varians from portuguese coast. antioxidants. 2023;12(2):435. 2. dayal js, ponniah a, khan hi, babu em, ambasankar k, vasagam kk. shrimps–a nutritional perspective. current science. 2013:1487-91. 3. rossi n, grosso c, delerue-matos c. shrimp waste upcycling: unveiling the potential of polysaccharides, proteins, carotenoids, and fatty acids with emphasis on extraction techniques and bioactive properties. marine drugs. 2024;22(4):153. 4. imarc. shrimp market: global industry trends, share, size, growth, opportunity and forecast 2020–2025. sydney: international mining and resources conference; 2025. 5. nirmal np, santivarangkna c, rajput ms, benjakul s. trends in shrimp processing waste utilization: an industrial prospective. trends in food science & technology. 2020;103:20-35. 6. fao. food supply—livestock and fish primary equivalent; 2021. faostat. http://www.fao.org/faostat/en/ #data/cl. 7. abidin z. total carotenoids, antioxidant and anticancer effect of penaeus monodon shells extract. biointerface research in applied chemistry. 2021;11(4):11293-302. 8. muniyappan j, varadharajan v, namadevan p. biochemical screening and determination of bioactive components of commercially cultured pacific white shrimp penaeus vannamei. pharmacognosy research. 2019;11(2). 9. rossi n, grosso c, delerue-matos c. shrimp waste upcycling: unveiling the potential of polysaccharides, proteins, carotenoids, and fatty acids with emphasis on extraction techniques and bioactive properties. marine drugs. 2024;22(4):153. 10. suprapti nh, syarani l, anggoro s. the chromium (cr) content in water and in the tissue of mud crab scylla serrata (forskal.) in the brackishwater ponds around babon river estuary of semarang coastal areas in central java, indonesia. journal of coastal development. 2012;16(1):62-7. 11. ray s, mondal p, paul ak, iqbal s, atique u, islam ms, et al. role of shrimp farming in socio-economic elevation and professional satisfaction in coastal communities of southern bangladesh. aquaculture reports. 2021;20(100708):1-10. 12. official method of analysis. 18th ed. washington, dc: association of official analytical chemists; 2005. method 935.14 and 992.24. 13. liu d, shi j, ibarra ac, kakuda y, xue sj. the scavenging capacity and synergistic effects of lycopene, vitamin e, vitamin c, and β-carotene mixtures on the dpph free radical. lwt-food science and technology. 2008;41(7):1344-9. 14. wong-paz je, contreras-esquivel jc, rodríguez-herrera r, carrillo-inungaray ml, lópez li, nevárez-moorillón gv, et al. total phenolic content, in vitro antioxidant activity and chemical composition of plant extracts from semiarid mexican region. asian pacific journal of tropical medicine. 2015;8(2):104-11. 15. dibacto rek, tchuente brt, nguedjo mw, tientcheu ymt, nyobe ec, edoun fle, et al. total polyphenol and flavonoid content and antioxidant capacity of some varieties of persea americana peels consumed in cameroon. the scientific world journal. 2021;2021(1):8882594. 16. goupy p, hugues m, boivin p, amiot mj. antioxidant composition and activity of barley (hordeum vulgare) and highlights in bioscience page 9 of 11 june 2025|volume 8 http://www.fao.org/faostat/en/#data/cl http://www.fao.org/faostat/en/#data/cl http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition malt extracts and of isolated phenolic compounds. journal of the science of food and agriculture. 1999;79(12):1625-34. 17. hassan a, mahmoud awm, turky g. rice husk derived nano zeolite (am 2) as fertilizer, hydrophilic and novel organophilic material. american journal of nanomaterials. 2017;5(1):11-23. 18. greenwood m. practical food microbiology. blackwell pub.; 2003. 19. liu z, liu q, zhang d, wei s, sun q, xia q, et al. comparison of the proximate composition and nutritional profile of byproducts and edible parts of five species of shrimp. foods. 2021;10(11):2603. 20. fanimo a, mudama e, umukoro t, oduguwa o. substitution of shrimp waste meal for fish meal in broiler chicken rations. 1996. 21. okoye f, ojewola g, njoku-onu k. evaluation of shrimp waste meal as a probable animal protein source for broiler chicken. international journal of poultry science. 2005;4(5):458-61. 22. mahata m. repairing shrimp waste nutrient quality as poultry feed through chitinases and chitinase hydrolysis from serratia marcescens bacterium. dissertation univ andalas padang. 2007. 23. mizani am, aminlari bm. a new process for deproteinization of chitin from shrimp head waste. in: proceedings of european congress of chemical engineering (ecce-6). september copenhagen; 2007. p. 16-20. 24. sachindra nm, bhaskar n. in vitro antioxidant activity of liquor from fermented shrimp biowaste. bioresource technology. 2008;99(18):9013-6. 25. prameela k, murali m, hemalatha k. extraction of pharmaceutically important chitin and carotenoids from shrimp biowaste by microbial fermentation method. journal of pharmaceutical research. 2010;3(10):2393-5. 26. omar s, farag m, taha r, gaballa a, rayan a. utilization of shrimp waste powder as a functional ingredient in fortifying ready-to-eat foods. journal of chemistry and nutritional biochemistry. 2025;6(1):47-67. 27. jeyasanta i, allwin sj, patterson j. development of nutritious better utilization to reduce. development. 2017;5(3):18. 28. beaney p, lizardi-mendoza j, healy m. comparison of chitins produced by chemical and bioprocessing methods. journal of chemical technology & biotechnology: international research in process, environmental & clean technology. 2005;80(2):145-50. 29. singh s, siddhnath br, aziz a, verma n, chriwatkar b. shrimp waste powder–potential as protein supplement. international journal of pure and applied bioscience. 2018;6(6):401-6. 30. prato e, biandolino f, parlapiano i, giandomenico s, denti g, calò m, et al. proximate, fatty acids and metals in edible marine bivalves from italian market: beneficial and risk for consumers health. science of the total environment. 2019;648:153-63. 31. on food additives meeting jfec. evaluation of certain food additives and contaminants: eightieth report of the joint fao/who expert committee on food additives. vol. 80. world health organization; 2016. 32. azelee iw, ismail r, ali r, bakar w. chelation technique for the removal of heavy metals (as, pb, cd and ni) from green mussel, perna viridis. niscair-csir bulletin. 2014. 33. shiekh hm, gumgumjee nm, danial en. antifungal and antioxidant activities of methanol extract of chitin, chitosan and shrimp shell waste. international journal of pharmaceutical and phytopharmacological research. 2018;8(2):25-30. 34. shahidi f, ambigaipalan p. phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects–a review. journal of functional foods. 2015;18:82097. 35. nunes r, pasko p, tyszka-czochara m, szewczyk a, szlosarczyk m, carvalho is. antibacterial, antioxidant and antiproliferative properties and zinc content of five south portugal herbs. pharmaceutical biology. 2017;55(1):114-23. 36. bersuder p, hole m, smith g. antioxidants from a heated histidine-glucose model system. i: investigation of the antioxidant role of histidine and isolation of antioxidants by high-performance liquid chromatography. journal of the american oil chemists’ society. 1998;75(2):181-7. 37. binsan w, benjakul s, visessanguan w, roytrakul s, tanaka m, kishimura h. antioxidative activity of mungoong, an extract paste, from the cephalothorax of white shrimp (litopenaeus vannamei). food chemistry. 2008;106(1):185-93. 38. manni l, ghorbel-bellaaj o, jellouli k, younes i, nasri m. extraction and characterization of chitin, chitosan, and protein hydrolysates prepared from shrimp waste by treatment with crude protease from bacillus cereus sv1. applied biochemistry and biotechnology. 2010;162:345-57. 39. wang sl, li jy, liang tw, hsieh jl, tseng wn. conversion of shrimp shell by using serratia sp. tku017 fermentation for the production of enzymes and antioxidants. journal of microbiology and biotechnology. 2010;20(1):117-26. highlights in bioscience page 10 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ omar et al., 2025 shrimp waste utilization: exploring composition 40. suvanich v, marshall d, jahncke m. microbiological and color quality changes of channel catfish frame mince during chilled and frozen storage. journal of food science. 2000;65(1):151-4. 41. amir rm, randhawa ma, sajid mw, nadeem m, ahmad a, wattoo fm. evaluation of various soaking agents as a novel tool for heavy metal residues mitigation from spinach. food science and technology. 2018;39:176-80. 42. labanowski j, monna f, bermond a, cambier p, fernandez c, lamy i, et al. kinetic extractions to assess mobilization of zn, pb, cu, and cd in a metal-contaminated soil: edta vs. citrate. environmental pollution. 2008;152(3):693-701. highlights in bioscience page 11 of 11 june 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods treatment of shrimp waste shrimp shell powder (ssp) preparation determination of chemical composition antioxidants determination total polyphenol content (tpc) hplc analysis of phenolic compounds total aerobic plate count (tapc) coliform group counts statistical analysis results and discussions proximate composition minerals concentration in shrimp waste heavy metals in shrimp waste samples bioactive compounds of shrimp waste hplc analyses of phenolic compounds antioxidant activity (dpph %) of shrimp waste samples microbial quality of shrimp's waste samples treatments to reduce heavy metals in shrimp waste effect of some treatments on reducing microbial content in shrimp waste economic feasibility conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202205 review open access 1 department of animal hygiene and zoonoses, faculty of veterinary medicine, university of sadat city, egypt. 2 faculty of veterinary medicine, cairo university, egypt. 3 department of biophysics, faculty of science, cairo university, egypt. 4 biotechnology program, faculty of science, cairo university, egypt. 5 department zoology and chemistry, faculty of science, cairo university, egypt. 6 department of bacteriology, mycology and immunology, faculty of veterinary medicine, university of sadat city, egypt. * to whom correspondence should be addressed: vet_noura@yahoo.com editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): santosh k maurya, department of biochemistry, central university of punjab, bathinda, punjab, india. amira m. elsherbini, department of oral biology, faculty of dentistry, mansoura university, mansoura 35116, egypt. received: september 7, 2022 accepted: december 15, 2022 published: december 29, 2022 citation: eissa n, badrkhan sm, mohamed ma, shaban jy, shahban rs, dawoud m. xenotransplantation: past, present, and future directions. 2022 dec 29;5:bs202205 copyright: © 2022 eissa et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. xenotransplantation: past, present, and future directions nourhan eissa*1 ><, salma m. badrkhan2 >< , maha a. mohamed3 >< , joumana y. shaban4 >< , rahma s. shahban5 >< , mai dawoud6 >< abstract xenotransplantation, in its broadest sense, is the transplantation, implantation, or infusion of cells, tissues, or organs from one species to another. while there is a high demand for human tissues, cells, and organs for use in clinical transplantation, they are often in short supply. recent scientific and biotechnological advancements, coupled with the scarcity of human allografts, have led to renewed interest in developing exploratory treatment strategies that use xenotransplantation products in human recipients. however, despite its potential benefits, the use of xenotransplantation is still limited due to various considerations, as discussed in this review of the past, present, and future directions of xenotransplantation. one of the key ethical concerns surrounding xenotransplantation is the potential impact on the animals from which the cells, tissues, or organs are obtained. as with genetic modification to fix genetic defects or prevent disease, the ideal outcome for these animals is that they will be better off as a result of the change. however, unless there are major changes in the way science is taught to incorporate ethics into recognized scientific theory and practice, these concerns will not be adequately addressed. keywords: donor animals, ethical issues, immunological barriers, religious considerations, xenotransplantation introduction despite the fact that there are over 135,000 transplants carried out annually throughout the world, this still only accounts for less than 10% of the true global needs for failing organs (such as kidneys, skin, testicles, hearts, livers, lungs, bones, small bowels, and pancreas, etc.) due to a lack of donors. this is true even though living donor transplants have been performed since the 1960s [1]. this fact has prompted medical professionals and researchers worldwide to develop a "bridge the gap" technique called xenotransplantation (cross-species transplantation, implantation or even infusion of live cells, tissues or even organs, especially from pigs and nonhuman primates to humans) in order to provide an immediate and limitless supply of transplantable organs that could aid in the treatment of many disorders [1; 2]. while an in-depth discussion of the history of numerous successful clinical attempts at xenotransplantation is impractical for the current review paper, it is important to highlight the key contributions that helped the field get to where it is nowa shining example of the power of science and medicine working together for the greater good. the cultural backdrop of xenotransplantation, religious beliefs, ethical considerations, desirable qualities of donor animals, challenges that the xenotransplantation procedure faces, and the influence of xenotransplantation on zoonotic risk are all briefly reviewed in this study. another important consideration is the potential for the spread of diseases from animals to humans. because the cells, tissues, or organs used in xenotransplantation come from another species, there is a risk that they may carry diseases that are not present in humans. this could potentially lead to the spread of new diseases or the exacerbation of existing ones. to minimize this risk, it is important to carefully screen the cells, tissues, or organs before they are used in xenotransplantation, and to implement strict protocols to prevent the spread of disease. despite these challenges, researchers are continuing to explore the potential of xenotransplantation as a way to overcome the shortage of human allografts. highlights in bioscience page 1 of 15 december 2022|volume 5 https://doi.org/10.36462/h.biosci.202205 https://creativecommons.org/licenses/by/4.0/ vet_noura@yahoo.com https://orcid.org/0000-0002-3622-6023 shamms951@gmail.com https://orcid.org/0000-0002-0407-1151 mhmohamed2013@gmail.com jomanayousef12@gmail.com https://orcid.org/0000-0002-5038-0270 rrsaid315@gmail.com https://orcid.org/0000-0002-2082-7692 mai_dawoud30@yahoo.com http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions in the future, it is likely that advances in science and technology will make it possible to overcome many of the challenges currently facing xenotransplantation , paving the way for its wide– spread use in clinical transplantation. chimeras in folklore historically, folklore had long contained accounts of chimaeras (i.e. monstrous creatures composed of parts of multipleăspecies) before the technique of xenotransplantation was even considered. people were sporadically shown in prehistoric cave paintings, but the sole example of a human is a man with a bird’s head in the lascaux cave in france (about 15,000 bc), which is where the stories of vampires and werewolves (half man, half beast) originated. the great sphinx of giza (about 2500 bc) features a lion body with a human head in contrast to the gods of ancient egypt (anubis), who were commonly depicted with a human body and an animal (jackal) head. additionally, a sanskrit document from the 12th century bc has the first account of xenotransplantation in indian mythology, which describes ganesha, a huge infant with an elephant-like head (a son of two indian gods, shiva and parvati). in addition, xenotransplantation was depicted in greek mythology through the likes of the minotaur (a man with a bull head), esfinge (a winged lion with a woman head), and centaurs (horses with a man’s head and trunk), as well as in homer’s odyssey, which featured chimaeras that were half-swine, half-man (about 750 bc) [3; 4; 5; 6]. history of clinical experiences with xenotransplantation the idea of human xenotransplantation attempts actually got started in the 17th century with the first attempt to transfuse sheep blood into people in 1667 (figure ??)[7]. in reality, scientists and doctors are unable to create true human-animal chimaeras followed by an opacified human cornea was replaced with a transparent porcine cornea [8] and a kidney xenotransplantation from a rabbit occurred in 1905 [9; 10], the clear pig cornea was then used to replace an opaque human cornea, and in the early 1970s, successful corneal xenotransplantations from fish and gibbons were performed [10; 11]. additionally, a clinical study of kidney xenotransplantation from a chimpanzee to humans was conducted between 1963 and 1964 [12]. this was followed by the first attempts at heart xenotransplantation from chimpanzee and baboon donors in 1964 [13] and 1984 [14], respectively. using baboon donors, the first successful liver xenotransplantation procedure was carried out in 1992 [15]. clinical xenotransplantation experiments have not been conducted in the united states or the majority of european nations since the 1990s because of certain xenozoonoses, immunological concerns, surgical effectiveness, and other regulatory concerns [16]. but according to reported reports, between 2013 and 2017 china and russia used xenotransplantation to cure diabetes patients using transplanted neonatal pig islets [17]. figure 1. historical recorded trials concerning xenotransplantation in different organs. blood xenotransfusion if we delve beyond myth and folklore, we find jean baptiste denis started the therapeutic practise of transfusing animal blood into humans [18; 19]. results were conflicting and not surprising. consequently, xenotransfusion was outlawed in france for a while. a strong case could be made for using pigs as a source of blood cells and blood products (if they are maintained in ideal "clean" conditions and are periodically checked to ensure no infectious agent is being passed) given the current threat of infectious pathogens being transferred and the need for future human blood transfusions [19]. in actuality, this method has been reevaluated by a number of studies [20]. blood vessel anastomosis more scientific developments had to wait until the 20th century, when french experimental surgeon alexis carrel devised surgical methods for anastomosing blood arteries, enabling the first successful organ transplant to occur. carrel worked first in france and subsequently in north america [21]. skin xenotransplantation various animal species and humans began using skin grafts in the 19th century when either pedicle or free skin grafts were used as the skin transplants. the donor, which may be a sheep, a rabbit, a dog, a cat, a rat, a chicken, or a pigeon, had to stay immobile while connected to the patient for a period of days so that the recipient could reportedly vascularize the graft. the perfect transplant would have looked like it was taken from a frog since they occasionally had "skinned alive" skin. when used to cover skin ulcers, it’s likely that some of these grafts were "successful" in the sense that they provided protection, at least for a few days, as the ulcer healed below them. but it’s likely that none of the grafts turned out to be long-lasting [22; 23]. corneal xenotransplantation corneal xenotransplantation, the process of transplanting corneas from one species to another, has a long and fascinating history. highlights in bioscience page 2 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the first recorded corneal xenotransplantation was actually performed from a human to a dog in 1838 by dr. samuel d. gross [24]. however, it wasn’t until 1905 that the first corneal allograft, a transplant from a person to a pig, was successfully completed [10; 25]. since then, the field of corneal xenotransplantation has made significant progress, with various animal species, including pigs, rabbits, and monkeys, being used as potential donors [26]. despite these advances, the use of animal-derived corneal grafts in humans remains controversial, with concerns surrounding the potential transmission of diseases, the ethical implications of using animals as organ donors, and the potential immunological reactions of the recipient [27]. despite these challenges, research into corneal xenotransplantation continues, with the hope of eventually finding a reliable and safe alternative to human corneal transplantation, which is currently limited by the shortage of donor tissue [28]. cell xenotransplantation serge voronoff, a russian immigrant who settled in paris, had the concept of transplanting cells that produced a hormone that the recipient lacked. given the small number of human pancreases that become available each year, there is tremendous interest in utilising pig islets for this. but for older guys who had lost their "zest for life," voronoff’s main goal was to slow down ageing. he implanted chimpanzee or baboon testicles into a sizable number of male human patients [29; 30]. his method involved cutting the animal testicle into slices and inserting the pieces into the testicles of the recipients. on both sides of the atlantic, the treatment gained popularity, and several hundred of these surgeries were carried out. it is improbable that any of them had any positive effects besides psychological ones, yet there have been tales of extraordinary "rejuvenation" in men who have undergone surgery and reported having considerably more energy. because donor testicle slices may have necrosed and created infectious or inflammatory problems occasionally, the surgeries must have had significant complications. furthermore, the first kidney allotransplant was carried on 1933 [31] john brinkley maintained the concept of transplanting goat glandular tissue to produce hormones that the recipient would benefit from in the united states [32]. nevertheless, the development of several clinics, particularly in europe, where patients get injections of animal tissue or serum to treat a variety of disorders has ensured that the concept of cell xenotransplantation has endured to the current day. controversy has been created by the results [33]. xenotransplantation of the kidney by the 1960s, keith reemtsma of tulane university in louisiana had proposed that transplanting human recipients with nonhuman monkey kidneys might successfully treat renal insufficiency. at that time, french and american surgeons had spent a lot of effort on the concept of kidney transplantation, but there were not enough deceased person kidneys accessible, and chronic dialysis had not yet been invented. so long as organs from nonhuman animals couldn’t be procured, reemtsma thought the patient had no alternative but to pass away. he decided to get the organs from chimpanzees because of their close evolutionary relationship to humans. he carried out 13 of these transplants, each of which included giving the patient both kidneys from a chimpanzee (which generally weighs considerably less than an adult human) [12]. during autopsy, the chimpanzee kidneys showed no abnormalities or signs of acute or enduring rejection. the notion of employing non-human primates as kidney donors was pioneered by several surgeons, most notably by tom starzl who used baboons as donors in colorado [34], and his findings were comparable to those of reemtsma. others had insignificant contacts in the us and france [35]. xenotransplantation of the heart when james hardy visited reemtsma in 1963 and conducted the first human lung allotransplant, he was struck by the recipients of chimpanzee kidney transplants who were all in good condition. hardy decided to buy some chimpanzees as possible "donors" in 1964 in order to execute the first clinical heart transplant in the event that he was unable to find a deceased human donor. he had a less-than-ideal patient who would not be allowed for heart transplantation today due to his patient’s significant atheromatous vascular disease, for which he had both of his legs amputated, and the fact that he was semicomatose at the time the surgery was carried out. however, the patient’s rapid decline prompted hardy to perform a chimpanzee heart transplant [21]. because the chimpanzee heart was too tiny to maintain the circulation, it failed within a short period of time. contrary to the attempted lung allotransplantation, the heart xenotransplantation received a negative response from the public and medical community, which deterred hardy and his colleagues from trying again. the heart allotransplantation procedure was later developed by barnard and his collaborators in 1967 [21]. later, they carried out two heart xenotransplantations [36]. lung xenotransplantation only the maryland team has lately engaged in active lung xenotransplantation research. platelet sequestration and activation during gtko was discovered by [37]. the hcd46 pig lung perfusion by human blood was mostly caused by gpib, gpiib/iiia, and von willebrand factor. gtko is reduced by transgenic expression of the human leukocyte antigen (hla-e). the hcd46 pigs with xenograft pulmonary injury. ex vivo human blood perfusion models of the lungs of genetically altered pigs with drugs that suppress complement activation, coagulation, and inflammation dramatically improved lung xenograft survival in vivo [38]. liver xenotransplantation tom starzl, one of the most important pioneers in the area of kidney and liver allotransplantation, tried a few liver transplantations on young patients and nonhuman primates in colorado in highlights in bioscience page 3 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the 1960s without long-term success [39; 40; 41]. in the 1990s, he and his pittsburgh team performed two liver transplants from baboons in adult patients, with one patient enduring 70 days of survival after tacrolimus was added to the immunosuppressive arsenal [15]. the results, however, were not convincing enough to warrant continuing this exploratory clinical trial. the pig [42] and other nonprimate mammals have been used in a few efforts, but they haven’t been very effective. most early attempts at therapeutic organ xenotransplantation obtained their organs from nonhuman primate species [35]. the first islet xenotransplantation an estimated 2 to 3 million persons in the united states alone have type 1 diabetes. since pig insulin varies from human insulin by just one amino acid and has been used successfully to treat diabetic patients for decades before recombinant human insulin became available, it is reasonable to anticipate that normoglycemia will result from a successful pig islet transplant. the first effort at pig islet transplantation in diabetic patients was undertaken in 1993 by a swedish team under the direction of carl groth [43]. features of the perfect donor animal include when we analyse the ideal qualities of animals suitable as organ donors for humans, a large list forms. the animal’s anatomy and physiology must first be compatible with humans for the desired organ to work well in them. the risk of an infection from one species (i.e., an animal) to another should also be eliminated. even human viral infections would not be able to pass through an excellent animal donor organ. this animal species should also be inexpensive to feed and produce because to its short gestation periods and frequent births each litter to achieve economies of scale. additionally, no immunologic obstacles to transplanting into humans should be present in such an animal. finally, there shouldn’t be much ethical debate about using this animal in this way. there is no animal species that satisfies all of the aforementioned requirements. apes and monkeys are nonhuman primates that resemble humans the most anatomically and physiologically. they might also be resistant to some human diseases. in reality, because of their hepatitis b and hiv resistance, baboon liver xenografts have been used in research [15]. but the xenotransplant community appears to have given up on the idea of utilising nonhuman primates as xenograft donors, mainly due to the hazards of infection for human patients and those who come in contact with them. some monkey viruses, like herpes 8, can kill people in a couple of days [44]. it is thought that raising pathogen-free herds in sufficient numbers to satisfy therapeutic demand would be prohibitively costly. last but not least, using nonhuman primates as human organ donors has serious ethical problems [45; 46]. due to its large litter sizes (up to 10 littermates), short gestation periods (4 months), anatomical and physiological similarities to humans, widespread use for human consumption (an estimated 90 million pigs are consumed annually in the usa), and lengthy history of providing medictable 1. the benefits and drawbacks of using pigs vs baboons as a source of organs and cells for people, as described by [18]. comparison pig baboon organ size in adults sufficient insufficient maintenance costs significantly inferior elevated human anatomy similarities moderately related very related human-like physiological similarities moderately related very related accessibility adequate inadequate relation with the immune system to humans distant related very related data of tissue typing significant (in selected herds) inadequate age of sexual maturity 4-8 months 3-5 years breeding potential good quality poor quality pregnancy period 114 ± 2 days 173-193 days offsprings per time 5-12 1-2 development fast (adult human size within 6 months) sluggish (9 years to reach maximum size) blood type compatibility with humans probably insignificant vital knowledge of genetic engineering significant none risk of transfer of infection (xenozoonosis) low high availability of specific pathogen-free animals yes yes public opinion more in favor mixed inals (skin, insulin, cardiac prostheses, and clotting factors) for humans, the pig has emerged as the most likely candidate for consideration as an organ donor. undoubtedly, considerable hurdles may arise due to significant discrepancies in the coagulation cascade and other aspects of porcine physiology [47; 48]. even though they are becoming more recognised, immunologic obstacles still need to be overcome. in addition, several diabetes treatments, such as immunosuppressive regimes and pancreatic islet transplantation procedures, were initially developed using the dog model. primate models with induced diabetes are being used more frequently as a result of recent developments toward the use of monoclonal antibody treatments for immunosuppression in human islet transplantation. researchers in several domains are thinking about using naturally occurring illness models in client-owned pets in addition to induced-disease models in large animals. this article will discuss how naturally existing canine diabetes can be used as a translational model for creating islet transplants for diabetic patients who are humans [49]. other pharmaceuticals of animal origin in table 2 we provide a list of various xenotransplantation products and their origins, generic names, product names, and therapeutic class. the table includes products from a variety of animal sources, including horses, pigs, mice, cows, and others. one of the key observations from the table is the wide range of therapeutic applications for xenotransplantation products. these products are used to treat a wide range of conditions, including respiratory problems, anticoagulants, antivenoms, and vaccines. this highlights the potential benefits of xenotransplantation as a way to overcome shortages of human allografts and provide treatments for a variety of medical conditions. another interesting aspect of the table is the diversity of animal sources used in xenotransplantation. the table includes products from horses, pigs, mice, and cows, among others. this highlights in bioscience page 4 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions suggests that a wide range of animals can be used as sources for xenotransplantation products, depending on the specific needs of the recipient and the availability of appropriate cells, tissues, or organs. overall, the table provides a useful overview of the past, present, and future directions of xenotransplantation. it highlights the potential benefits of using xenotransplantation products in clinical transplantation, as well as the ethical considerations and technical challenges that need to be addressed in order for it to be widely used. in addition to the observations mentioned above, the table also highlights the potential challenges of xenotransplantation. for example, one of the main challenges is ensuring that the cells, tissues, or organs used in xenotransplantation are compatible with the recipient’s immune system. if the transplant is rejected, it may be necessary to use immunosuppressive drugs to prevent rejection, which can have negative side effects for the recipient. another challenge is the potential for the spread of diseases from animals to humans. because the cells, tissues, or organs used in xenotransplantation come from another species, there is a risk that they may carry diseases that are not present in humans. this could potentially lead to the spread of new diseases or the exacerbation of existing ones. to minimize this risk, it is important to carefully screen the cells, tissues, or organs before they are used in xenotransplantation, and to implement strict protocols to prevent the spread of disease. despite these challenges, the potential benefits of xenotransplantation are considerable. in the future, it is likely that advances in science and technology will make it possible to overcome many of the challenges currently facing xenotransplantation, paving the way for its widespread use in clinical transplantation. this could help to alleviate the shortage of human allografts and provide new treatment options for a variety of medical conditions. issues with several xenotransplantation cases complications include immunological incompatibility, cell death, abnormal cell differentiation and proliferation, virus transmission from animals to humans, and ethical concerns hinder the clinical application of xenogeneic stem cell transplantation [50]. immune rejection immune rejection is unquestionably the problem with xenogeneic stem cell transplantation that worries people the most. immunological rejection is avoided using the following methods: only a few of the variables that need to be taken into account include the use of cellular desensitisation technology, immunosuppressive medications, suitable stem cell type selection, gene editing technology, encapsulated cell technology, the use of immunosuppressive drugs, and the regulation of cytokine levels. these procedures have increased the success rate of transplantations. selecting stem cells with low immunogenicity, immunosuppressive, and immunomodulatory traits may help to alleviate this problem [51]. injected immunocompetent mice with stem cells obtained from human umbilical cord stroma. the results showed that this kind of human stem cell has immunosuppressive and immunomodulatory properties [51]. later research showed that xenogeneic stem cells, in particular xenogeneic mscs, have low immunogenicity along with immunosuppressive and immune-modulatory capabilities [52]. porcine mscs have been used in xenotransplantation investigations because to their low immunogenicity attributes and immunomodulatory qualities [53]. pig umbilical cord mscs and swine escderived neural progenitors were implanted in non-immunocompr– omised rats [54]. their investigation revealed similar cell immunosuppressive effects [53]. the potential of these cells to suppress the immune system and have minimal immunogenicity was proven by the transplantation of rabbit umbilical cord mscs with hyaluronic acid/tricalcium phosphate scaffolds in rats [55]. by co-implanting rat mscs and pig neuroblasts in immunocompetent rat striata, [52] demonstrated the immunosuppressive characteristics of these cells. according to study by [56], rat adscs can protect themselves from human xenoantibodies and complement-mediated lysis. gal, or galactose-1, 3-galactose, is related with low expression and this capacity is cd59 dependent [56]. hyperacute rejection antibodies that are spontaneously generated against blood type antigens are similar to xenoreactive natural antibodies (xna). the epitope that these antibodies primarily target is the nonreducing trisaccharide group galactosyl a-(1, 3)-galactosyl b-1,4n-acetyl glucosaminyl, also known as the gal epitope15. man does not have this epitope because he lacks the enzyme that makes it. higher primates thus recognise the gal epitope as "non-self" and produce an immune response to it. numerous microbes16 contain the gal epitope, and humans are exposed to the antigen through their guts, where they develop anti-gal antibodies. the key mechanisms by which xna exerts its effects include natural killer (nk) cells, complement activation, and endothelium phenotypic alterations. the goal of research to date has been to lessen the effects of xna [60; 61]. acute humoral xenograft rejection (ahxr) the following challenge is delayed xenograft rejection, which is frequently seen. the primary histological features of ahxr are endothelial swelling or disruption, vascular thrombosis with blood extravasation, and interstitial oedema [62]. within 24 hours of transplantation, this generally develops, gets worse over the next few days, and finally kills the graft. the first response, which is mostly but not solely specific for the gal epitope, is mediated by igm, and is thereafter followed by an increase in igg levels [63]. by themselves, these xenograft natural antibodies induce a procoagulant state that develops into disseminated intravascular coagulation. even the best practises for limiting complement activation, lowering t-cell and b-cell driven immune responses, and diminishing xenograft natural antibodies highlights in bioscience page 5 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. different pharmaceutical products derived from non-human mammalian cells as represented by [57; 58; 59]. origin generic name product name therapeutic class equine (horse) conjugated oestrogen premarin gonadal hormone, oestrogen antithymocyte immuglobulin (atg) atgam immunosuppressant snake antivenom red back spider antivenom antivenom tiger snake antivenom green pit viper antivenin sea snake antivenin cobra antivenin taipan antivenom king cobra antivenin polyvalent snake antivenin medroxyprogesterone acetate premia gonadal hormone stonefish antivenom stonefish antivenom antivenom porcine (pig): coagulation factors ii, ix, x, v & vii prothrombinex-vf haemostatic agent heparin sodium heparinised saline anticoagulant amylase, lipase, pancrelipase, protease panzytrat digestive supplement poractant alfa curosurf respiratory agent danaparoid orgaran haemostatic agent human rotavirus live attenuated vaccine rotarix vaccine dalteparin fragmin anticoagulant rotavirus vaccine live oral pentavalent rotateq vaccine pancrelipase pancreatin creon digestive supplements & cholelitholytics enoxaparin clexane anticoagulant, antithrombotics zoster virus vaccine live zostavax vaccine vancomycin hydrochloride vancomycin hcl antibiotic, miscellaneous murine (mouse) trastuzumab herceptin antineoplastic agent cetuximab erbitux antineoplastic agent infliximab remicade monoclonal antibody antihemophilic factor viii (human) hemofil m antihemophlic agent bevacizumab avastin antineoplastic agent rituximab mabthera antineoplastic agent; monoclonal antibody golimumab simponi antirheumatic agent abciximab reopro anticoagulant palivizumab synagis immunomodifier somatropin saizen pituitary hormone basiliximab simulect immunomodifier bovine (cow) epinephrine adrenaline neurotransmitter sealerprotein solution+ thrombin solution tisseel vhs/d solution haemostatic agent collagen zyderm collagen implants dermatological preparations calfactant infasurf treatment of premature infant lungs hepatitis a vaccine vivaxim vaccine allantoin allantoin cosmetics, treatment of wounds & ulcers polygeline haemaccel plasma volume expander varicellazoster vaccine, live varivax vaccine calporo calporo herbal daily supplements insulin hypurininjection insulin preparations bovine colostrums travelan anti-diarrhoeal survanta beractant treatment of premature infant lungs cartilag cartilag herbal analgesics & anti-inflammatories continued on next page highlights in bioscience page 6 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class bovine-manufacture acitretin novatretin antipsoriatic measles, mumps & rubella vaccine priorix vaccine itraconazole itrazol antifungal, azole derivative inox mebeverine hcl mebetin antispasmodics amoxycillin synamox antibiotic, penicillin loperamide colodium antidiareal modim mycophenolate mofetil cellcept immunosuppressant agent essential phospholipids livovid cholelitholytics rabies vaccine merieux vaccine rabipur hepatitisavaccine avaxim vaccine havrix hydrocortisone hydrocortisonorion corticosteroid clindamycin hcl tidact antibiotic, lincosamide recombinant antihaemophilic factor recombinate haemostaticagents nilotinib tasigna antineoplastic agent, thyroxine kinase inhibitor clofazimine fazim antibiotics, leprostatic ampicillin sod+ sulbactam sod unasyn antibiotic, penicillin rabies human diploid cell vaccine verorab vaccine hepatitis b vaccine engerix-b vaccine omeprazole omeprazole gastric acid secretion inhibitor, proton pump inhibitor calcitriol osteocap vitamin d analog diphtheria, tetanus & acellular pertussis vaccine adacel vaccine cyclosporin sandimmun immunosuppressant, calcineurin inhibitor pneumococcal vaccine prevenar vaccine doxycycline xidox antibiotics, tetracyclines derivatives celecoxib celebrex nsaid, cyclooxygenase-2 inhibitor phenytoin sodium dilantin anti-epilepsy dutasteride avodart 5-alpha-reductase inhibitor oseltamivir phosphate fluhalt antiviral, influenza, neuraminidase inhibitor diphtheria toxoid adt booster vaccine boostrix pancreatin creon pancreatic enzyme replacement danazol nazo androgen oxycodone hcl oxynorm opioids analgesic pregabalin lyrica anticonvulsant didanosine aurobindo antiretrovirals haemophilus b influenzae vaccine hiberix vaccine heparin sodium injection heparinol anticoagulant isotretinoin acnotin anti acne, antineoplastic agent recombinant antihaemophilic factor recombinate haemostatic agents influenza virus vaccine fluarix vaccine tacrolimus prograf immunosuppressant agent fluconazole fluconazole antifungals rivastigmine rivadem acethylcholinesterase inhibitor gem fibrozil gem fibrozil dyslipidaemic agents yellow fever vaccine 17d vaccine vaccine continued on next page highlights in bioscience page 7 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class egg/chicken measles, mumps and rubella virus vaccine l m-m-r ii vaccine influenza virus vaccine agrippal vaccine measles, mumps and rubella virus vaccine priorix vaccine measles, mumps, rubella and varicella vaccine priorix-tetra & proquad vaccine rabies vaccine rabipur vaccine coxiella burnetii vaccine q-vax & q-vax skin test vaccine influenza virus vaccine vaxigrip vaccine risperidone rixadone antipsychotic agent verteporfin visudyne ophthalmic medication quadrivalent influenza vaccine afluria quad vaccine propofol propofol sandoz propofol-lipuro 1%/2% provive 1% & provive mct-lct 1% anaesthetics yellow fever vaccine stamaril vaccine olive oil and soya oil clin oleic 20% parenteral vitamins, minerals and nutrition sebelipase alfa kanuma endocrine and metabolic agent influenza virus vaccine fluarix vaccine quadrivalent influenza vaccine fluad quad vaccine quadrivalent influenza vaccine fluquadri vaccine influenza virus vaccine fluad vaccine trivalent influenza vaccine fluzone highdose vaccine clevidipine cleviprex antihypertensive agent influenza virus vaccine influvac vaccine propofol diprivan anaesthetics propofol fresofol 1% injection & fresofol 1% mct/lct anaesthetics soya oil intralipid parenteral vitamins, minerals and nutrition chinese hamster ovary (cho) cells aflibercept eylea ophthalmic medication follitropinalfa gonal-f pituitary hormone erythropoeitin alfa binocrit hematopoietic agent laronidase aldurazyme enzyme replacement therapy abatacept orencia immuno-modifier interferon beta-1a avonex immunomodifier rebif omalizumab xolair other respiratory agent etanercept enbrel tumour necrosis factor inhibitor panitumumab vectibix antineoplastic agents eptacog alfa novosevenrt haemostatic agent octocogalfa advate haemostatic agent kogenatefs lenograstim granocyte supportive therapy follitropinbeta puregon pituitary hormone nonacogalfa benefix haemostatic agent lutropin alfa luveris 75 iu pituitary hormone imiglucerase cerezyme enzyme replacement therapy dornasealfa pulmozyme respiratory agent continued on next page highlights in bioscience page 8 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 2. – continued from previous page. origin generic name product name therapeutic class dornasealfa pulmozyme respiratory agent alemtuzumab mabcampath antineoplastic agent trastuzumab herceptin antineoplastic agent choriogonadotropin alfa ovidrel pituitary hormone tenecteplase metalyse fibrinolytic agent darbepoietin aranesp haemopoietic agent recombinate antihaemophilic factor recombinate haemostatic agent agalsidasebeta fabrazyme enzyme replacement therapy epoietin alfa eprex haemopoieticagent rituximab mabthera antineoplasticagent methoxy polyethylene glycol-epoetinbeta micera hematopoietic agent denosumab prolia monoclonal antibody xgeva moroctocogalfa xyntha haemostaticagent epoetin lambda novicrit haemopoieticagent bevacizumab avastin antineoplastic epoietin beta neorecormon haemopoieticagent corifollitropin alfa elonva pituitary hormones sheep box jellyfish antivenom box jellyfish antivenom antivenom digoxin binding antibody digoxin-specific antibody fragment digifab antidote fish, shark and shell fish house dust mite extract acarizax antiallergy preparation chondroitin chondroitin complementary osteoarthritis inactivated influenza vaccine fluad vaccine glucosamine glucosamine complementary osteoarthritis phleum pratense. grazax antiallergy preparation insulin human insulin (rys) & protaphane mixtard 30/70. mixtard 50/50 insulin preparation rabbit funnel web spider antivenom (rabbit) funnel web spider antivenom antivenom highlights in bioscience page 9 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions sometimes fall short of addressing these issues. diffuse intravascular coagulation and thrombotic microangiopathy, which are related to postpone xenotransplant rejection, are caused by unknown processes. ahxr is the least well-known of the early xenograft rejection phases [64]. cell proliferation, aberrant differentiation, and death similar to the problems with cell replacement treatment, cell death and abnormal cell differentiation and proliferation directly led to the failure of xenogeneic stem cell transplantation and even injured the recipients. researchers have shown that the microenvironment of the cell culture affects cell differentiation and death. several researchers have attempted to change the microenvironment of the cells to prevent cell death and abnormal differentiation. here, we will discuss two common methods for changing the microenvironments of cell cultures to resemble the in vivo natural growth niche. one tactic is to change the traditional two-dimensional (2d) culture into a three-dimensional (3d) culture. umbilical cord msc single-cell derived spheres were produced by [65] using cell chips, a device to restrict cells to specific spatial locations. they combined a 3d culture with a 2d arrayed pattern of single or multiple cells on one patch of the cell chip in order to improve msc survival and migratory ability and to promote angiogenesis in xenotransplantation [65]. the other technique requires changing the scaffold. materials used as scaffolds in tissue engineering xenogeneic stem cell transplantation may promote cell survival and differentiation. [66] employed a hyaluronic acid-based scaffold that has been covalently modified by poly-l-lysine as a delivery vehicle to deliver hbmscs to rats with injured spinal cords. rats receiving hbmscs/hyaluronic acid-poly-l-lysine showed improved in vivo survival of transplanted hbmscs, according to [66]. in contrast, when sheep mscs were injected into immunocompromised rats, a ceramic hyaluronic acid/tricalcium phosphate carrier led to ectopic osteogenesis, adipogenesis, and hematopoietic-support activities [67]. the necessity of selecting an adequate substrate for tissue creation while taking into account the anticipated direction of cell differentiation was established by these findings [67]. ipscs and escs may be tumorigenic due to their capacity for cellular growth in cell transplantation and other treatments. this problem was addressed by [68] by implementing optimised directed differentiation protocols to generate the desired precursor cell types and by using cellular enrichment techniques to eliminate unnecessary cells in order to choose only the cells with a restricted proliferation potential for transplantation. religious restrictions in table 3 we provide information about the restrictions on xenotransplantation products in different countries based on the religions practiced in those countries. it is important to note that these restrictions are based on the beliefs and practices of individual religions and do not necessarily reflect the views or laws of the countries in which they are practiced. one of the main observations from the table is that many religions place restrictions on the use of certain animal products. for example, islam prohibits the use of porcine products and requires that all animal products be slaughtered in a specific way. similarly, judaism prohibits the use of porcine and shellfish products and has strict rules about the types of land animals, birds, and fish that can be consumed. hinduism and sikhism also place restrictions on the use of animal products, with many hindus abstaining from all animal products and sikhs prohibiting the use of halal sources. another important aspect of the table is the diversity of religions represented. the table includes information about islam, judaism, seventh day adventism, hinduism, sikhism, and jehovah’s witnesses, among others. this highlights the fact that religious beliefs and practices can vary widely and may influence the use of xenotransplantation products in different parts of the world. overall, the table provides useful information about the potential restrictions on xenotransplantation products based on the religions practiced in different countries. it is important to consider these restrictions when developing and implementing xenotransplantation treatments in order to respect the beliefs and practices of different religious communities. ethical concerns ethics around xenogeneic stem cell transplantation are becoming more widely accepted. some people believe that xenotransplantation consistently transgresses the lines between species and lowers the dignity of humans. animal welfare organisations also opposed xenotransplantation on the grounds that nonhuman creatures shouldn’t be seen of as re-designable systems [70]. in reality, a wide range of animal products are now used by humans. for instance, bioactive bones from decellularized bovine femoral bone and freeze-dried bone marrow stem cell paracrine factors are widely used in large-sized bone lesions. these successes are gradually changing people’s opinions and paving the way for xenogeneic stem cell transplantation. however, any applications must consider regional variations in culture, legislation, beliefs, and other factors [71]. risk of zoonotic infections potential benefits of xenotransplantation over allotransplantation (transplantation between members of the same species) include an almost limitless supply of grafts, animal species resistance to certain human infections (baboons, for example, are immune to the hepatitis b virus (hbv) and the human immunodeficiency virus (hiv)), and the ability to lower the risk of xenograftassociated infections by using specific pathogen-free animals with lifelong controversies, and the ability to reduce the risk of xenograft [72; 73]. however, if the risk to public health arises from introducing novel zoonotic infectious diseases into the human population that aren’t typically present there, the prospect of spreading germs from animals to people via xenotransplantation cannot be completely precluded [74]. the characteristics of highlights in bioscience page 10 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions table 3. religious restrictions as published by others [59; 69]. religion countries where widely practiced restrictions islam indonesia, india, pakistan, bangladesh, egypt, turkey, iran, nigeria, ethiopia, afghanistan, sudan, iraq, malaysia, tanzania, somalia, cote divoire, congo, philippines, sierra leone, thailand, eritrea, lebanon porcine products prohibited all animal products not killed in the prescribed ritualistic way (halal) prohibited products containing alcohol prohibited judaism usa, israel, france, canada, uk, russia, argentina, ukraine, brazil and south africa all porcine and shellfish products prohibited . other rules about animal products that can be ingested: land animals must be mammals which chew their cud and have cloven hooves birds of prey are prohibited . fish must have scales and fins. meat and milk (or any other dairy product) cannot be combined; shrimp and other non-fish seafood are forbidden. observers follow a stringent set of regulations and only eat kosher food. seventh day adventist australia, usa, south america, some african countries some abstain from meat, but eggs are permissible. hinduism india, nepal, bangladesh, indonesia, pakistan, sri lanka, philippines, fiji, uk, mauritius, bhutan, south africa, burma, singapore for the vast majority of vegetarians, all animal products, including eggs, are forbidden. bovine and porcine goods continue to be prohibited for persons who are not vegetarians. sikh india, pakistan, malaysia, singapore, fiji, new zealand, usa and uk for some who are vegetarian all animal products including egg prohibited for those who are not vegetarian, restrictions still include bovine and porcine products all animal products from halal sources prohibited products containing alcohol prohibited. jehovahs witnesses australia, usa, mexico, brazil and many other countries (240 in total) the use of fractions derived from the primary components of blood is not absolutely prohibited buddhism tibet, bhutan, india, nepal, sri lanka, burma, thailand, laos, cambodia, malaysia, vietnam, china, bangladesh, korea, japan, singapore, parts of russia for some vegetarian buddhists all animal products prohibited however, no fixed rules. highlights in bioscience page 11 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions the particular organism, the amount of the organism transferred, the presence of the necessary equipment (such as receptors and nutrients in the host), and the immunological proficiency of the host all affect the likelihood of contracting a zoonotic infection. even the wide range of potential clinical signs cannot be predicted for previously undetected animal-derived illnesses in human hosts [75]. recipients and their contacts should be routinely screened for zoonotic infectious agents, either by direct methods (which depend on detecting the presence of the agent itself or its products) or even by indirect methods (which depend on detecting the production of antibodies against specific microbes and antigens) [76]. this is to prevent a potential new zoonosis from spreading among humans as a result of xenotransplantation. conclusion biotechnology has the power to drastically modify human existence, as we indicated at the beginning of our discussion and as the rise of xenotransplantation amply indicates. furthermore, according to gaskell’s research, moral objections to biotechnology are allegedly more significant to society than even safety objections. even non-problems, like "violating god’s will" or "going against nature," are elevated to the status of the most severe ethical concerns as a result of society’s lack of scientific and ethical understanding, which makes it challenging to come up with reasonable answers. such an error might restrict the use of biotechnology to save lives and alleviate suffering, as our discussion has shown. this in turn emphasises the critical social illiteracy of science that we mentioned at the beginning of our argument, as well as the urgent need for expanded education of the general public, the scientific community, and society at large on ethics. although it is relatively easy to see this issue, solving it is much more difficult. we have both taught and pushed for the inclusion of ethical considerations in science education as a necessary precursor to rational solutions to ethical difficulties arising out of scientific findings. we have also seen that such education produces better scientists who have a sense of social responsibility. additionally, we have argued that it is critical to discuss and elucidate ethical issues while instructing students in science, particularly biological science. this has proven to be considerably more difficult. obstacles to it include the fact that such an approach is historically uncommon and that the majority of scientists believe research is "value-free in general, and ethics-free in particular," as demonstrated in our debate. the majority of people who teach science do not have formal training in the ethical issues that arise from science or even how to start addressing such obstacles, which creates additional challenges. determining when and how to begin integrating an ethical component into scientific instruction might be difficult as a result of these difficulties. the casual attitude of the research community toward the ethical issues associated to animal usage in research, which further distances the scientific community from the general people, is evident as social concern over the treatment of animals grows dramatically. if, as leaders in the scientific community have repeatedly remarked, scientific growth is entirely dependent upon the use of animals, then it is the responsibility of the scientific community to address social ethical concerns associated to animal exploitation. the development of animal ethics as we have described it is predicated on the notion of an animal, and society seems to concur with this. furthermore, we brought this up throughout our discussion. the ethical viability of genetic modification, which is readily acknowledged to impact both large and small changes in telos, therefore inevitably arises. it is obvious that this is not the place for a comprehensive examination of this annoying problem. as long as the telos changes do not negatively affect the animals’ quality of lifethat is, as long as the animals produced through genetic modification are not worse off than their unmodified forebears and, ideally, better offwe have argued that there is no morally wrong with carrying out such genetic modifications. in other words, it’s important to make sure that animal genetic engineering doesn’t do any harm. as with genetic alteration to fix genetic defects or prevent disease, the ideal outcome for animals is that they will be better off as a result of the change. such problems will not be resolved unless major changes in scientists’ thinking, which can only be made by significantly modifying the way science is taught, are made. at that point, ethics can be incorporated into recognised scientific theory and practise. reference 1. white s, hirth r, mah’illo b, dom’inguez-gil b, delmonico f, noel l, et al. the global diffusion of organ transplantation: trends, drivers and policy implications. bulletin of the world health organization. 2014;92:826-35. 2. ekser b, ezzelarab m, hara h, van der windt d, wijkstrom m, bottino r, et al. clinical xenotransplantation: the next medical revolution? the lancet. 2012;379(9816):672-83. 3. o’flaherty w. hindu myths: a sourcebook translated from the sanskrit. hammondsworth: penguin; 1975. 4. kahan b. ganesha: the primeval hindu xenograft. in: transplantation proceedings. vol. 3. elsevier; 1989. p. 1-8. 5. bhandari m, tewari a. is transplantation only 100 years old? british journal of urology. 1997;79(4):495-8. 6. deschamps jy, roux fa, saï p, gouin e. history of xenotransplantation. xenotransplantation. 2005;12(2):91-109. 7. denis j. ’excerpt from a letter from mr. denis, professor of philosophy & mathematics, am* concerning blood transfusion,”;6(1667):69-72. 8. kissam r. ceratoplastice in man. nyj med. 1844 mar;2:281-2. 9. zirm e. eine erfolgreiche totale keratoplastik (a successful total keratoplasty). journal of refractive surgery. 1989 jul;5(4):258-61. highlights in bioscience page 12 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 10. hara h, cooper d. xenotransplantation: the future of corneal transplantation? cornea. 2011 apr;30(4):371. 11. haq m. fish cornea for grafting. british medical journal. 1972 june;2(5815):712. 12. reemtsma k, mccracken b, schlegel j, pearl m, pearce c, dewitt c, et al. renal heterotransplantation in man. annals of surgery. 1964 sep;160(3):384. 13. hardy j, chavez c, kurrus f, neely w, eraslan s, turner m, et al. heart transplantation in man: developmental studies and report of a case. jama. 1964 jun;188(13):1132-40. 14. bailey l, nehlsen-cannarella s, concepcion w, jolley w. baboon-to-human cardiac xenotransplantation in a neonate. jama. 1985 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p. cellular xenotransplantation. current opinion in organ transplantation. 2009 apr;14(2):168-74. 31. matevossian e, kern h, hüser n, doll d, snopok y, nährig j, et al. surgeon yurii voronoy (1895-1961) a pioneer in the history of clinical transplantation: in memoriam at the 75th anniversary of the first human kidney transplantation. transplantation international. 2009 dec;22(12):1132-9. 32. lee r. the bizarre careers of john r. brinkley. university press of kentucky; 2002. 33. sgroi a, bühler l, morel p, sykes m, noel l. international human xenotransplantation inventory. transplantation. 2010 sep;90(6):597-603. 34. starzl t, marchioro t, peters g, kirkpatrick c, wilson w, porter k, et al. renal heterotransplantation from baboon to man: experience with 6 cases. transplantation. 1964 nov;2:752. 35. taniguchi s, cooper d. clinical xenotransplantation: past, present and future. annals of the royal college of surgeons of england. 1997 jan;79(1):13-9. 36. barnard c, wolpowitz a, losman j. heterotopic cardiac transplantation with a xenograft for assistance of the left heart in cardiogenic shock after cardiopulmonary bypass. south african medical journal. 1977 dec;52(26):1035-8. 37. burdorf l, riner a, rybak e, ii s, de meyer s, shah a, et al. platelet sequestration and activation during galtko. hcd46 pig lung perfusion by human blood is primarily mediated by gpib, gpiib/iiia, and von willebrand factor. xenotransplantation. 2016 may;23(3):222-36. 38. laird c, burdorf l, french b, kubicki n, cheng x, braileanu g, et al. transgenic expression of human leukocyte antigen-e attenuates galko.hcd46 porcine lung xenograft injury. xenotransplantation. 2017;24(2):e12294. highlights in bioscience page 13 of 15 december 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 39. starzl t, marchioro t, faris t, mccardle r, iwaski y. avenues of future research in homotransplantation of the liver with particular reference to hepatic supportive procedures, antilymphocyte serum, and tissue typing. the american journal of surgery. 1966 sep;112(3):391-400. 40. starzl t, ishikawa m, putnam c, porter k, picache r, husberg b, et al. progress in and deterrents to orthotopic liver transplantation, with special reference to survival, resistance to hyperacute rejection, and biliary duct reconstruction. transplantation proceedings. 1974 dec;4(1):129. nih public access. 41. giles g, boehmig h, amemiya h, halgrimson c, starzl t. clinical heterotransplantation of the liver. transplantation proceedings. 1970 dec;2(4):506. nih public access. 42. makowka l, wu g, cramer d, hoffman a, podesta l. immunohistopathologic lesions associated with the rejection of a pig-to-human liver xenograft. transplantation proceedings. 1994;26(3):1074-5. 43. groth c, tibell a, tollemar j, bolinder j, östman j, möller e, et al. transplantation of porcine fetal pancreas to diabetic patients. the lancet. 1994 nov;344(8934):1402-4. 44. allan j. xenotransplantation at a crossroads: prevention versus progress. nature medicine. 1996 jan;2(1):18-21. 45. vanderpool h. critical ethical issues in clinical trials with xenotransplants. the lancet. 1998 may;351(9112):134750. 46. hughes j. xenografting: ethical issues. journal of medical ethics. 1998 feb;24(1):18-24. 47. delriviere l, havaux x, gibbs p, gianello p. basic anatomical and physiological differences between species should be considered when choosing combinations for use in models of hepatic xenotransplantation: an investigation of the guinea pig-to-rat combination. transplantation. 1998 jul;66(1):112-5. 48. robson s, am esch j, bach f. factors in xenograft rejection. annals of the new york academy of sciences. 1999 jun;875(1):261-76. 49. adin c, gilor c. focus: comparative medicine: the diabetic dog as a translational model for human islet transplantation. the yale journal of biology and medicine. 2017 sep;90(3):509. 50. platt j, cascalho m, piedrahita j. xenotransplantation: progress along paths uncertain from models to application. ilar journal. 2018;59(3):286-308. 51. ding d, chou h, chang y, hung w, liu h, chu t. characterization of hla-g and related immunosuppressive effects in human umbilical cord stroma-derived stem cells. cell transplantation. 2016;25(2):217-28. 52. lévêque x, mathieux e, nerrièredaguin v, thinard r, kermarrec l, durand t, et al. local control of the host immune response performed with mesenchymal stem cells: perspectives for functional intracerebral xenotransplantation. journal of cellular and molecular medicine. 2015;19(1):124-34. 53. yang j, liao c, pang c, huang l, chen y, shiue y, et al. transplantation of porcine embryonic stem cells and their derived neuronal progenitors in a spinal cord injury rat model. cytotherapy. 2013;15(2):201-8. 54. medicetty s, bledsoe a, fahrenholtz c, troyer d, weiss m. transplantation of pig stem cells into rat brain: proliferation during the first 8 weeks. experimental neurology. 2004;190(1):32-41. 55. li k, wang y, sun q, li m, chen j, liu l. rabbit umbilical cord mesenchymal stem cells: a new option for tissue engineering. the journal of gene medicine. 2021;23(1):e3282. 56. jia y, zhao y, wang l, xiang y, chen s, ming c, et al. rat adipose-derived stem cells express low level of ś-gal and are dependent on cd59 for protection from human xenoantibody and complement-mediated lysis. american journal of translational research. 2016;8(5):2059. 57. bozoglanian v, butteri m. the diverse and promising world of animal derived medications. pharos alpha omega alpha honor med soc. 2015 jan:16-22. 58. anjum c, chia y, chan m. presence of neu5gc in animalderived products. friend or foe. stem cells regen med. 2020;4(1):1-7. 59. the state of queensland (queensland health); 2020. https://www.health.qld.gov.au/__data/assets/ pdf_file/0024/147507/qh-gdl-954.pdf. 60. galili u, mandrell r, hamadeh r, shohet s, griffiss j. interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora. infection and immunity. 1988;56(7):1730-7. 61. artrip j, kwiatkowski p, michler r, wang s, tugulea s, ankersmit j, et al. target cell susceptibility to lysis by human natural killer cells is augmented by ś (1, 3)-galactosyltransferase and reduced by ś (1, 2)fucosyltransferase. journal of biological chemistry. 1999;274(16):10717-22. highlights in bioscience page 14 of 15 december 2022|volume 5 https://www.health.qld.gov.au/__data/assets/pdf_file/0024/147507/qh-gdl-954.pdf https://www.health.qld.gov.au/__data/assets/pdf_file/0024/147507/qh-gdl-954.pdf http://bioscience.highlightsin.org/ eissa et al, 2022 xenotransplantation: past, present, and future directions 62. shimizu a, meehan s, kozlowski t, sablinski t, ierino f, cooper d, et al. acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pigto-nonhuman primate renal grafts. laboratory investigation. 2000;80(6):815-30. 63. holmes b, richards a, mclaughlin m, cruz g, copeman l, bycroft s. antibody responses in early graft rejection in pig-to-primate renal xenotransplantation. in: transplantation proceedings. vol. 33; 2001. p. 717-8. 64. robson s, cooper d, d’apice a. disordered regulation of coagulation and platelet activation in xenotransplantation. xenotransplantation. 2000;7(3):166-76. 65. qiao y, xu z, yu y, hou s, geng j, xiao t, et al. single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure. biomaterials. 2020;227:119573. 66. raynald ly, yu h, huang h, guo m, hua r, jiang f, et al. the hetero-transplantation of human bone marrow stromal cells carried by hydrogel unexpectedly demonstrates a significant role in the functional recovery in the injured spinal cord of rats. brain res. 2016;1634:21-33. 67. mccarty r, gronthos s, zannettino a, foster b, xian c. characterisation and developmental potential of ovine bone marrow derived mesenchymal stem cells. journal of cellular physiology. 2009;219(2):324-33. 68. zygogianni o, kouroupi g, taoufik e, matsas r. engraftable induced pluripotent stem cell-derived neural precursors for brain repair. in: stem cells and tissue repair. humana, new york, ny; 2020. p. 23-39. 69. jenkins e, yip m, melman l, frisella m, matthews b. informed consent: cultural and religious issues associated with the use of allogeneic and xenogeneic mesh products. journal of the american college of surgeons. 2010;210(4):402-10. 70. fung r, kerridge i. gene editing advance re-ignites debate on the merits and risks of animal to human transplantation. intern med j. 2016;46:1017-22. 71. karalashvili l, kakabadze a, uhryn m, vyshnevska h, ediberidze k, kakabadze z. bone grafts for reconstruction of bone defects. georgian medical news. 2018;282:44-9. 72. michaels m, simmons r. xenotransplant-associated zoonoses: strategies for prevention. transplantation. 1994;57(1):1-7. 73. fishman j. infection and xenotransplantation: developing strategies to minimize risk. annals of the new york academy of sciences. 1998;862(1):52-66. 74. onions d, cooper d, alexander t, brown c, claassen e, foweraker j, et al. an approach to the control of disease transmission in pig-to-human xenotransplantation. xenotransplantation. 2000;7(2):143-55. 75. fishman j, scobie l, takeuchi y. xenotransplantationassociated infectious risk: a who consultation. xenotransplantation. 2012;19(2):72-81. 76. food, administration d. guidance for industry (final guidance): source animal, product, preclinical, and clinical issues concerning the use of xenotransplantation products in humans; 2003. available from: http://www.fda.gov/ cber/gdlns/clinxeno.htm. highlights in bioscience page 15 of 15 december 2022|volume 5 http://www.fda.gov/cber/gdlns/clinxeno.htm http://www.fda.gov/cber/gdlns/clinxeno.htm http://bioscience.highlightsin.org/ abstract introduction chimeras in folklore history of clinical experiences with xenotransplantation blood xenotransfusion blood vessel anastomosis skin xenotransplantation corneal xenotransplantation cell xenotransplantation xenotransplantation of the kidney xenotransplantation of the heart lung xenotransplantation liver xenotransplantation the first islet xenotransplantation features of the perfect donor animal include other pharmaceuticals of animal origin issues with several xenotransplantation cases immune rejection hyperacute rejection acute humoral xenograft rejection (ahxr) cell proliferation, aberrant differentiation, and death religious restrictions ethical concerns risk of zoonotic infections conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202102 research article open access 1 department of biomedical laboratory sciences , kibogora polytechnic, rwanda. 2 biomedical laboratory sciences dept, ines ruhengeri-institute of applied sciences, rwanda. contacts of authors * to whom correspondence should be addressed: yadufashije callixte received: may 30, 2020 accepted: november 2, 2020 published: january 16, 2021 citation: fulgence m, joseph m, callixte y, william n . renal dysfunction among hiv patients under highly active antiretroviral therapy attending kibagabaga district. 2021 jan 16;4:bs202102 copyright: © 2021 fulgence et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. renal dysfunction among hiv patients under highly active antiretroviral therapy attending kibagabaga district munyandamutsa fulgence1, mucumbitsi joseph2, yadufashije callixte*2, niyonzima william2 abstract antiretroviral therapy is used for the suppression of the hiv virus and stops its progression to cause disease. despite its role, it has the pathophysiologic effect to kidney function for users. the study was conducted to evaluate the renal function for hiv patients under highly active antiretroviral therapy at kibagabaga district hospital. venous blood samples (4ml) were collected by vein puncture in phlebotomy services by means of the dry tubes from 170 patients under antiretroviral therapy. blood samples were transported to clinical biochemistry department for analysis. rotor centrifuge was used to separate the serum and other blood components; creatinine level was analysed for renal function analysis. the total of 170 hiv patients were considered in the study. of the 170, 50 hiv patients were used for primary data collection, while 120 hiv patients who previously received antiretroviral therapy were considered as secondary data collection source. the patients between 25-45 years old have experienced the highest level of abnormal concentration of renal tests 25%, patients with above 45 years old was ranked the second to have the abnormal level (14.2%). females were 59% and have experienced the high risk of renal dysfunction than males, the level of glomerular filtration rate was 67 (39.4%), and was higher than creatinine 42 (24.7%). antiretroviral therapy has a negative effect on kidneys. abnormalities of the kidney parameters were prevalently high among both male and female. the serious follow is needed for this vulnerable population. keywords: renal function, hiv, haart, estimated glomerular filtration rate introduction human immunodeficiency virus (hiv) infection mostly provoke acute or chronic renal proximal renal tubule resulting in functional disturbance with mitochondrial injury being one of the most important targets [1]. it results to the failure of excretory functions of kidney, which is disease and people are living longer. hiv infection negative side effects might lead to the heart, lungs, and renal diseases, which could cause high mortality among hiv patients. africa endures greater than 60% of the global burden of hiv, however, characterized by decrease in glomerular filtration rate [2]. the advances in development of highly active antiretroviral therapy (haart) is a high risk to the development of the serious renal disease in developed world with very few studies done in developing world [3]. kidney function has been estimated to be abnormal up to 30% of all hiv patients. few outpatient renal screening studies from africa describe varying prevalence from as low 6% to a high of 50% [4]. the dysfunction of the kidney is medical kidney abnormality where this important organ gets impairment for the filtration of wastes from blood [5]. nephropathy is one of the complications characterizing the kidney dysfunction for hiv patients [6]. kidney disease was clinically diagnosed considering the presence of albuminuria but also low estimated glomerular filtration rate [7]. some antiretroviral agents can cause serious side effects that affect multiple systems; including the kidneys tenofovir (tenofovir disoproxil fumarate) and this could be associated with development of the acute renal failure (arf) and the dysfunction of proximal and distal tubules [8]. highlights in bioscience page 1 of 4 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202102 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy the first described patient who was treated with tenofovir and developed reversible fanconi syndrome, nephrogenic diabetes insipidus and arf, renal biopsy demonstrated cytoplasmic vacuolization, apical localization of nuclei and reduction of the brush border on proximal tubule epithelial cells [9]. materials and methods study area description the study was conducted at kibagabaga district hospital. kibagabaga district hospital is located in kigali city, nyarugenge district; the hospital receives the referred patients from 16 health centers of nyarugenge from neighbouring health canters in gasabo and kicukiro. study design both cross-sectional and retrospective study design were used. the study was carried out from october to december 2019. study population and sample size all hiv patients under highly active antiretroviral therapy attending kibagabaga district hospital during the period will be targeted by the study. however, only patients who were not hiv positive did not participate. the population size was 170; the sample size was determined by the number of eligible patients who attended the hospital and accepted to participate in the study during the period of the study. collection of stool sample blood samples were collected with phlebotomy and biochemistry sops support. the patients were placed in a comfortable seat and requested to remove sleeves from the part of the arm. phlebotomist finds the arm with visible fuller vein. a stretched band tourniquet was placed around the upper arm to help the vein bloat. the place for injection was then whipped with cotton after being soaked in the antiseptic solution. the needle was slightly inserted into the swollen vein and around 4ml of blood were collected by the help of a seal dry test tube. specimen containers or collection devices was labelled with the patient record number was recorded along with the date of birth to avoid bias and errors in laboratory results. sample processing the sample collection and processing were done according to kibagabaga district hospital laboratory sops, the tubes containing blood was taken to clinical biochemistry department. the collected blood specimen was clotted for 15 min before centrifugation at 3000 rpm for 5 minutes using a rotor centrifuge for separation of serum and other blood components. creatinine level was analysed for renal function by using automated biochemistry machines (architecture machine or cobasc311), the cobas c311. data analysis after data collection, the spss version 23.0 program and excel 2016 was used for data analysis. to compare variables and association between renal function in hiv patients under haart and its clinical manifestastion unduced by this medication,and prevalence ethical considerations this study was conducted after obtaining an ethical approval from kibogora polytechnic research committee. in addition, the authorization was requested from kibagabaga district hospital, clinical research and office of education before conducting this research. the purpose of research project, sample collection procedures, as well as confidentiality of research results was explained to participants. participation was voluntarily after understanding the aim and expect outcome of research. there was no expected risks that can threaten the lives of participants. results social demographic characteristics of study population in this study the social demographic characteristics of the study population were established to characterize the population, table 1 indicates the analysis which was done on 170 hiv paatients under highly active antiretroviral therapy from kibagabaga district hospital in a period of 3 mounths. table 1 reveals the different parameters with renal function tests such as age group, gender, period, clinical manifestation and weight. the high frequency was observed in the age group between 25-45 years old. the number of females stood at 62%, mouth ulcers predominated the clinical manifestations with percentage of 29.4% followed by memory loss at 25%. over weight patients stood at 6.5% while 38.8% was in normal ranges. table 2 shows the range of of creatinine and glomerular filtration rate level according sex. clinical analyses in this study the creatinine level and glomerular filtration rate showed the high prevalence of abnormal results in males with 14.5% and 39.4 respectively. cross sectional and retrospective studies were assessed and shown in table 3; the majority of patients were females with 28 (56%) and 22 (44%) were males. moreover, in retrospective 29 (58%) were females and 22 (18.3%) were males. table 4 shows diagnosed patients based on clinical symptoms associated to renal dysfunction in hiv patients; the majority of patients was those with mouth ulcers 50 (28.6%) followed by memory loss with 43 (24.6%). discussion this study analysed biochemical parameters of hiv patients under highly active antiretroviral therapy, this medication leads to the serious side effects including kidney failure for patients under those medications. this study considered the level of creatinine and estimation of glomerular filtration rate as tests to confirm the renal function of hiv patients under highly active antiretroviral therapy. highlights in bioscience page 2 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy table 1. number of patients participated in the study and their corresponding gender, age, clinical manifestation, weight and period. parameter variables frequency percentages % age groups [5-25] years 13 9.6 [25-45] years 93 54.7 above 45 years 64 37.6 sex males 64 38 females 106 62 period 2018 retrospective 70 41.2 2019 prospective 50 29.4 2019 retrospective 50 29.4 clinical manifestation night sweats 18 10.6 weight loss 19 11.2 depression 40 23.5 mouth ulcers 50 29.4 memory loss 43 25.3 weight 40-60 kg 38 22.4 60-80 kg 66 38.8 80-100 kg 55 32.4 above 100 kg 11 6.5 total 170 100 table 2. number of patients participate the study and their corresponding results according to gender. state creatinine level (mg/dl) egfr (ml/min) females males total females males total normal 92 (54.1) 36 (21.2) 128 (75.3) 7 2(38.4) 31 (22.2) 103 (60.6) abnormal 17 (10.2) 25 (14.5) 42 (24.7) 32 (16.1) 35 (23.3) 67 (39.4) table 3. prevalence of abnormal creatinine and egfr according to age and gender in suspected patients at kdh, where the creatinine (kg/dl) (a), egfr (ml/min)(b), and prevalence (%)(c) are shown. groups variables cross sectional study n (50) retrospective study n (120) (a) (b) (c) (a) (b) (c) age groups 5-25 1 (2) 1 (2) 8% 0 (0) 2 (1.6) 13% 25-45 6 (12) 11 (22) 68% 15 (12.5) 22 (18.3) 25% above 45 2 (4) 3 (6) 2% 17 (14.1) 28 (23.3) 14.20% gender females 20 (40) 28 (56) 48% 16 (13.3) 29 (58) 59% males 22 (44) 14 (28) 36% 17 (14.1) 22 (18.3) 27% table 4. clinical manifestation and their associated abnormal level of renal test. the frequency (%) of occurrence of symptoms the frequency (% ) of renal dysfunction clinical manifestation frequency (%) abnormal creatinine abnormal egfr nigth sweats 18 (10.3%) 6 (3.5%) 7 (4.1%) weight loss 19 (10.9%) 9 (5.2%) 9 (5.2%) depression 40 (22.9) 5 (2.9%) 13 (7.6%) mouth ulcers 50 (28.6) 14 (8.2%) 25 (14.7%) memory loss 43 (24.6%) 9 (5.2%) 13 (.6%) among 170 suspected patients, 64(38%) were males and 106 (62%) females. the mean age of the population was 56.6, and the standard deviation of age was 40.5. 54.7% of patients were in the age range of [25-45] years, and were the majority in the study participants. this age is at high risk because of the habit of doing unprotected sex than elderly age. after testing creatinine level and egfr the results showed the normal females 38.4% and 23.3% males with abnormal egfr level and abnormal creatinine respectively. the study conducted on active antiretroviral therapy among hiv patients shows no difference with the current study since it revealed the high glomerular filtration rate (egfr) and creatinine in females than males under highly active antiretroviral therapy [12] the patients who had positive test results with creatinine and egfr for cross-section study, the findings show that the majority of patients were females with 28(56%) and 22(44%) were males. moreover, in the retrospective 29(58%) were females and 22 (18.3%) were males. in addition, the prevalence of renal dysfunction was high in old age in females. these results were comparable to the findings of willems and his colleagues [13], we estimated the prevalence of egfr and mortality risks in a population-based study of persons aged 85 years and older, based on these results. the patients were diagnosed based on clinical symptoms associated with renal function of hiv patients. the majority of patients induced the mouth ulcers 50 (28.6) than other clinical manifestation. the results showed that there is significant relationship between the most of clinical manifestation and renal function of hiv patients under highly active antiretroviral therapy. the similar findings were reported where self-reported strategies used by persons living with hiv/aids and their clinical manifestation induced by art such us moth ulcers, weight loss and other opportunist infections were described [14]. conclusions the study confirmed the effect of antiretroviral therapy on kidneys where the abnormalities of creatinine and egfr were more prevalent 59% and 27% in both females and males respectively. it is a paramount action of treating patients to enhance the longevity even if renal dysfunction may occur. all hiv patients under retroviral therapy should be followed up to maintain the normal function of the kidneys. acknowledgement we are with pleasure to thank kibagabaga distict hospital’s authorities to allow the study to be conducted in the hospital facilities and their abundant generosity. in addition, we once again address our thanks to kibogora polytechnic for its vision of promoting research not only in the region but also all over the world. references 1. hilton r. human immunodeficiency virus infection and kidney disease. j r coll physicians edinb 2013; 43(3):236-40. http://dx.doi.org/10.4997/jrcpe.2013.310 2. authier fj, chariot p, gherardi rk. skeletal muscle involvement in human immunodeficiency virus (hiv)–infected patients in the era of highly active antiretroviral therapy (haart). mushighlights in bioscience page 3 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience fulgence et al., 2021 renal dysfunction among hiv patients under antiretroviral therapy cle and nerve: official journal of the american association of electrodiagnostic medicine. 2005 sep;32(3):247-60. 3. gray f, keohane c. the neuropathology of hiv infection in the era of highly active antiretroviral therapy (haart). brain pathology. 2003 jan;13(1):79-83. 4. campbell c, nair y, maimane s, nicholson j. dying twice’ a multi-level model of the roots of aids stigma in two south african communities. journal of health psychology. 2007 may; 12(3): 403-16. 5. matiyashchuk ig, amosova km, iaremenko ob, zakharova vi, koliadenko di. impaired endothelial vasoregulatory function in patients with systemic lupus erythematosus: association with renal involvement, inflammatory markers, and autoantibodies. kidneys. 2020;9(1):14-9. 6. núñez m, saran am, freedman bi. gene-gene and geneenvironment interactions in hiv-associated nephropathy: a focus on the myh9 nephropathy susceptibility gene. advances in chronic kidney disease. 2010 jan 1;17(1):44-51. 7. romagnani p, remuzzi g, glassock r, levin a, jager kj, tonelli m, massy z, wanner c, anders hj. chronic kidney disease. nature reviews disease primers. 2017 nov 23;3(1):1-24. 8. mocroft a, kirk o, gatell j, reiss p, gargalianos p, zilmer k, beniowski m, viard jp, staszewski s, lundgren jd, eurosida study group. chronic renal failure among hiv-1-infected patients. aids. 2007 may 31;21(9):1119-27. 9. badowski me, burton b, shaeer km, dicristofano j. oral oncolytic and antiretroviral therapy administration: dose adjustments, drug interactions, and other considerations for clinical use. drugs in context. 2019;8. 10. myers gl. standardization of serum creatinine measurement: theory and practice. scandinavian journal of clinical and laboratory investigation. 2008 jan 1;68(sup241):57-63. 11. michels wm, grootendorst dc, verduijn m, elliott eg, dekker fw, krediet rt. performance of the cockcroft-gault, mdrd, and new ckd-epi formulas in relation to gfr, age, and body size. clinical journal of the american society of nephrology. 2010 jun 1;5(6):1003-9. 12. overton, et., wyatt, cm., mitsuyasu, rt., wensing, a. m., calvez, v., günthard, h. f. and richman, dd. topics in antiviral medicine. 2014;40(10):100-109. 13. willems jm, vlasveld t, den elzen wp, westendorp rg, rabelink tj, de craen aj, blauw gj. performance of cockcroftgault, mdrd, and ckd-epi in estimating prevalence of renal function and predicting survival in the oldest old. bmc geriatrics. 2013 dec 1;13(1):113. 14. sukati na, mndebele sc, makoa et, ramukumba ts, makoae ln, seboni nm, human s, holzemer wl. hiv/aids symptom management in southern africa. journal of pain and symptom management. 2005 feb 1;29(2):185-92. highlights in bioscience page 4 of 4 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods study area description study design data analysis ethical considerations results social demographic characteristics of study population clinical analyses discussion conclusions acknowledgement references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202201 review article open access 1 biofuel research laboratory, department of microbiology, school of life sciences, central university of tamil nadu, thiruvarur, tamil nadu, india. * to whom correspondence should be addressed: suchitrar@cutn.ac.in editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): alsamman m. alsamman, african genome center, mohammed vi polytechnic university,morocco.. morad m. mokhtar, agricultural genetic engineering research institute, agricultural research center, giza, egypt. received: october 29, 2021 accepted: january 1, 2022 published: january 15, 2022 citation: ray b, rakesh s . phycoremediation of aquaculture wastewater and algal lipid extraction for fuel conversion. 2022 jan 15;5:bs202201 copyright: © 2022 ray and rakesh. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this work is financially supported by serb dst project (eeq/2018/001463). competing interests: the authors declare that they have no competing interests. phycoremediation of aquaculture wastewater and algal lipid extraction for fuel conversion bobita ray1 >< ,suchitra rakesh*1 ><  abstract in this review, it is discussed the prominent effect generated from aquaculture wastewater considered as the major water polluting crisis in the entire world. the cause rose due to intense development and improvement in aquaculture by the aquatic habitat species triggering quite a challenge in the environment. scrutinizing this problem, researchers have found a way to tackle it by cultivating algal species in aquaculture wastewater in order to remove its high content of organic and inorganic pollutants. the theory proves wastewater serves as a nutrient source for algal growth and development such as phosphorous, nitrogen, and other trace elements. besides harvesting the algal biomass from aquaculture wastewater, the extraction of lipid is also processed for biofuel production. hence, the discussion includes conversion of wastewater into organic and inorganic pollutant-free water with low cost-effective method via algal cultivation in wastewater and high lipid yield for biofuel with a carbon-free and sustainable environment. keywords: algae, aquaculture wastewater, harvesting, lipid extraction, transesterification introduction ever increasing global population and continuous dependence of fossil fuels, increased urbanization and industrialization posing a major threat to energy security and environmental concerns to both developed and developing nations. with the accelerated speed of increasing population growth, wastewater treatment is considered as one of the solutions to control the environmental issues. and the additional challenges for water scarcity bring out the crucial problem related to wastewater. hence, much of the emphasis has been given now a days for wastewater treatment [1]. the anthropological activities such as sewage, industries, agriculture, medical, research laboratories etc., are pointed to be the sources of wastewater which have tremendously polluted the water resources. wastewater from various sources comprises both organic and inorganic pollutants. organic pollutants include proteins, carbohydrates, lipids, etc., whereas inorganic mostly have chemicals and solvents [2], and in industrial wastewater even heavy metals or toxic elements are reported [1]. nowadays the aquaculture wastewater is quite prominent globally due to its intense development and improvement in fish, marine species, algae and aquatic plant farming. thus, this rapid increase of aquaculture effluents poses a serious threat to environment [3]. mariculture other name of aquaculture can be seen their progresses in parallel way with high human demands. the impact performances to the environment for food production has been reported as the fastest growth [4]. aquaculture wastewater contains a high number of pollutants and chemicals that lead the residing aquatic flora and fauna to die due to eutrophication. the toxic algal blooms not only disturb the aquatic life but it also interferes with the sustainable marine aquaculture development. the wastewater treatment via biochemical methods is not economical and further removal of those chemicals from water poses another challenge. hence, wastewater treatment by algae is an ecofriendly and cost-effective approach over other physical and chemical methods [5–7]. highlights in bioscience page 1 of 9 january 2022|volume 5 https://doi.org/10.36462/h.biosci.202201 https://creativecommons.org/licenses/by/4.0/ raybobita92@gmail.com suchitrar@cutn.ac.in https://orcid.org/0000-0002-4357-4042 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion earlier aquaculture wastewaters were treated with bulks of antibiotics which later have evolved to antibiotic-resistant [8]. but in an investigation, green microalgae tetraselmis sp. removed nitrogenous and phosphorous compound from aquatic wastewater within 48 hours [9]. removal of nutrients from livestock wastewater was also reported via, desmodesmus sp. a microalga with potential benefactor [10]. the wastewater treatment via microalgae not only removes the pollutants but also shows positive effect towards carbon fixation (1.83 kg co2/kg of biomass), high amount of biomass generation within a short period of time. the microalgal biomass can be further utilized for biofuel and valuable bio-products production and can also act as substrate in bio-refinery. thereby, it provides a sustainable and ecofriendly approach to many of the problems related with wastewater [11]. since the middle time of 20th century, development in aquaculture growth has risen globally in all over countries providing huge profit to commercial hatcheries and farming system. the wastewater discharge from chemical and other industries has high toxicity level, that poses serious environmental issues [12]. the aquaculture production is kept on increasing due to high demand in the market. hence large-scale production has been carrying out enormously [13]. yang et al., [14] has mentioned in his studies the aquaculture effluent treatment with microalgae is highly efficient in absorbing nutrients and value-added biomass generation. alga-aquaculture has led to many advantages such as compared to other plants, algae has proved to be better in nutrient removal. the construction and operational costs are low with consistent to high nutrient removal efficiency. the microorganisms consortia like algal-bacterial consortia are highly efficient in solid and other waste treatment into low molecular weight compounds [15]. the large quantity of algal biomass can be produced from aquaculture, that can be further utilized for high demand valuable product generation spirulina and chlorella cultures are commonly used as aquaculture feed, as both has very minimal toxicity level and helps in preventing algal blooms as well. on the other way, addition of expensive chemicals and antibiotics for industrial effluent treatment are not economical and poses severe threat to the environmental [16]. it has also been reported that excess use of chemicals in aquaculture affects the food safety and quality of meat produced via aquaculture. in most of the research studies, microalgae such as chlorella sp. is found experimenting in every aspect of wastewater treatment. biofilms are another slimy and foul in nature found on the surface of the algae or any solid surface attached. microalgal biofilms mostly have succeeded in reducing the nutrients of phosphorous starting initially from 15 mg l-1 within 24 hours[12]. the biofilm has succeeded in more production of biomass production for biofuel [17]. this review summarizes our efforts towards various aquaculture wastewater treatment via microalgae and use of algae for feed purposes. it further highlights the biofuel and value-added products generation from the algal biomass. micro and macro algae as a nutritive aquaculture feed aquaculture has been rapidly developing in industrial sector resulting large quantity of polluted effluents being discarded into clear water line. remaining solid residues mostly contains hazardous chemicals and metal elements causing severe incurable diseases [1]. huge amount of cost and labor are invested upon various physical and chemical techniques. electro adsorption and electro-reaction coupling process is one of physical technique to clean the wastewater, removing 99% of solid. but it is reported algae is the most efficient and advanced method with low cost benefits in treating wastewater [18]. in aquaculture, many of the microalgae viz., nannochloropsis, chaetoceros, thalassiosira, tetraselmis etc. are known for essential food sources including marine species such as clams, molluscs, oysters and spirulina sp. for providing high protein diet for freshwater fishes and other invertebrate species [17]. treatment of wastewater with microalgae has been guaranteeing good outcome and have led to great advantages without harming the environment. macroalgae universally known as seaweed is easily visible through naked eye. its habitats are mostly native to marine or other river bodies. saccharina latissima also known as kelps are generally found in river depth. macroalgae are well-known for their nutritional and bioactive components. aquaculture with macroalgae production has a strong demand in the market, according to industrial vision. it is a valuable source of aquaculture feed. however, most examples of eutrophication in the marine environment are caused by the deposition of flowing waste in the sea, which includes high nitrogen and phosphorus nutrients. macroalgae also aids in the bioremediation process by preventing wastes from impacting the marine environment in terms of ph change, turbidity, and increased bod content, as well as causing marine life death and encouraging toxic algal blooms [19–21]. according to brakel et al., [22] macroalgae depicts as fastest growing aquaculture development even in poorest coastal regions. nowadays with advanced facilitation and support of genetic resources, seaweeds such as red algal genera eucheuma and kappaphycus proved economically in many tropical countries. microalgae based biorefinery for aquaculture wastewater treatment wastewater treatment is rising as fundamental priority. the removal of nutrients and solids, as well as the acceptance of environmentally friendly remediation techniques, play a significant role in this. the most photosynthetic machinery technique that we can ever expect is phycoremediation, or treating wastewater with algae. it extracts all unwanted parameters from wastewater and improves water quality to meet environmental standards. [15]. compared to conventional wastewater treatment this method is cheap and also has the involvement in biomass production for biorefinery purposes. chlorella sorokiniana is observed as the highlights in bioscience page 2 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion most utilized objective both for phycoremediation and biomass production [23]. open pond system algal cultivation is the basic necessary condition for more quantity of biomass for biofuel production. depending on cost, labor and time vast kinds of techniques are available. open pond is the common system for large scale algal production exposed directly to environment. here generally, algae species are cultivated in an open pond area covering as much acres of land directly under the sunlight due to their phototrophic nature [24]. open pond is named as raceway because it resembles with racetrack. this raceway pond system takes less space of land for growth. it requires continuous movement of paddle wheel in the pond to prevent sedimentation of cultures at the bottom level [25]. generally, paddle wheels depicts the main principle base for the open pond system where the speed of the wheel helps to cover the light intensity for all over the algal growth within the system [26]. nutrient removal efficiency of aquaculture wastewater with microalgae the most efficient process and cost-effective method for culturing algae is via wastewater sources; rather than cultivating in expensive amounts of chemicals. aquaculture wastewater contains required nutrients such as nitrogen, sulphur, phosphorous which alga feeds on for growth. its composition is mentioned in table 1 , pointing its physico-chemical properties such as its ph, chemical oxygen demand (cod), nitrate, chloride, sodium, potassium, magnesium, nitrite, ammonium and phosphorous were depicted in aquaculture wastewater. in table 1, the content found under those properties extremely higher compared to normal i.e. these wastewater has the capability to cause diseases. wastewater cultivation is positively progressing both in bioremediation and biomass production for biofuel. it is either way sustainable to environment as budget friendly way. ulva sp., codium sp., ecklonia sp., saccharina sp., gracilariopsis sp. have experimented in fish seaweed aquaculture waste for bioremediation that have removed high concentration of ammonia and phosphorous within 30-40% nutrient removal converting into less polluting [30]. whereas for microalgae tetradesmus obliquus has removed 99.3% of ammonia and 99.2% of phosphorous concentration from swine manure wastewater [31]. in table 2, various algal species cultivated in different types of aquaculture wastewater are shown. the inoculated culture in the wastewater is mentioned parallel to the algal species name. the remaining columns are about the results of removal of nutrients described in percentage that found after cultivating in aquaculture wastewater. it specifies how algae worked as bio-remediation. ta bl e 1. c om po si tio n of va ri ou s ty pe s of aq ua cu ltu re w as te w at er . ty pe s of aq ua cu ltu re w as te w at er ph c o d (m g/ l ) n itr at e (m g/ l ) c hl or id e (m g/ l ) so di um (m g/ l ) po ta ss iu m (m g/ l ) m ag ne si um (m g/ l ) n itr ite (m g/ l ) a m m on iu m (m g/ l ) ph os ph or ou s (m g/ l ) r ef er en ce fi sh er y 7. 86 32 .4 0. 35 n a n a n a n a 24 .7 6. 25 1. 83 [7 ] fi sh er y 8. 1 2. 25 n a 19 ,4 00 10 ,7 90 38 7 12 93 n a n a 1. 21 [2 7] se aw at er m ar in e 7. 75 7. 84 8. 02 n a n a n a n a 0. 25 0. 48 4. 56 [2 8] o re oc hr om is ni lo tic us aq ua cu ltu re 5. 22 64 .3 52 .0 24 .5 28 .5 8. 3 3. 3 0. 01 12 .8 11 .2 [2 3] fi sh er y 7. 2 n a 96 .6 0 65 5 54 0 21 69 0. 00 6 0. 01 0 1. 98 [2 9] n a n ot a va ila bl e highlights in bioscience page 3 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion table 2. nutrient removal in aquaculture wastewater with different algal species types of aquaculture wastewater algae species type of algae amount of inoculation (g/l) time of treatment (days) removal compounds reference cod (%) nh4 (%) nitrate (%) nitrite (%) phosphorous (%) total nitrogen (%) fishery p. kessleri ty microalgae 10 3 94.4 96.2 94.3 99 96.6 na [7] salmon farming chlorella minutissima microalgae na 10 na na 88.6 74.3 99 88 [27] shrimp culture gracilaria tenuifrons macroalgae 1.75 8 na 35.1 na 71.7 33.2 2.8 [28] fish-seaweed aquaculture codium fragile macroalgae 1000 28 na 0.07 na na 0.22 0.56 [23] ulva pertusa 1000 28 na 0.04 na na 0.15 0.56 ecklonia stolonifera 1000 28 na 0.11 na na 0.26 0.57 gracilariopsis chorda 1000 28 na 0.11 na na 0.23 0.50 saccharina japonica 1000 28 na 0.15 na na 0.21 0.56 oreochromis niloticus aquaculture chlorella sorokiniana microalgae na 14 na 99.9 75.2 na 77 78 [29] na not available recent advances in microalgae harvesting and lipid extraction after cultivation, harvesting which means collecting or gathering of algal cultivation determines as most difficult and important out of all process work. for large scale harvesting of biomass, it requires quite expensive technique, maintenance of time, man power and so on. in case of microalgae harvesting techniques such as centrifugation, sieving, filtration, sedimentation, flotation, flocculation are predominantly utilized [33]. whereas for macroalgae simple technique such as drying and storing is basically preferred but however few techniques from microalgae harvesting techniques are also operated [34]. thermo reversible gel transition [35,36] characterized with either agar or sol gel for harvesting of algae where clustered cells are settled at bottom and collected the biomass. flocculation is another technique of harvesting. nanocellulose is an insoluble substance where bonding of polysaccharide and glucose monomers occurs with the concept of more concentration of nanofibril more increase of flocculation [37]. bacterial cellulose gluconacetobacter xylinus has found to be successfully harvested with 90% of clump formation [38]. pleaurotus ostreatus [39] and scenedesmus obliquus [40] are another flocculating process. according to leite [41], ph modulation through dissolved air flotation can be harvested at higher biomass. magnetic nanoparticles is another better technique for harvesting [42] mostly utilized lipid extraction method is bligh and dyer as said to be quickly approachable to quantification outcome within less timing but more hazardous to environment as well as self-health. but mtbe i.e., methyl-tert-butyl ether is the better method than the previous method with non-hazardous effect and increase in the extracted lipid [43]. for future perspective role such as to study the characterization from extracted algal biomass production, high resolution nuclear magnetic resonance spectroscopy (hr nmr) or mass spectroscopy technique is used to study the changes of various composition kept in different storage conditions were found in the algal sample. such techniques are extremely advanced in analytical process [34]. on the other hand, dimethyl ether is a gas type where the liquefied gas is passed with the help of nitrogen gas and proceeded for algal extracting [44]. super high hydrostatic pressure technique is even utilized for extracting lipid maintaining pressure 100 mpa to 1000 mpa [45]. another process of lipid extraction solvent is ionic liquid that comprises of ion solvents of non-volatile substance including bubbling co2 gas for extraction [46]. in the given table 3, it basically determines the biomass productivity and lipid growth found after algal cultivation in aquaculture wastewater. in the same table various algal species names and according to that in the left column the types of wastewater are given where following that horizontally we can read the biomass and lipid found after cultivating the algae in that same wastewater. within these three table tables (table 1, 2 and 3) it gives the idea about reading the physico chemical properties before algae cultivating and harvesting the biomass, lipid measuring and lastly with the remaining water and can be proceeded with rereading the physico chemical properties determining removal of nutrients from the wastewater. microalgae as a sustainable future biofuel approach developing with rapid high rise of industries by regular use of natural resources are leading us into depletion of fossil fuels sooner creating havoc in environment. it surges carbon dioxide till peak point making possibly prone to global warming similarly threatening wastewater globally. at the bright side, the microalgae have several unique features like ability to fix co2 and convert it into valuable components via photosynthesis, robust growth with high lipid contents. the microalgae harvesting, qualitative and quantitative estimation of lipid has been reviewed [43,47]. the availability of molecular approaches to increase lipid accumulation and recovery has been extensively discussed [48]. biofuel is the breakthrough for solution. among generation after generation there has been change into biofuel production. initially beginning with edible plants such as soybean, maize, brassica comes under first generation and had a great deal with alternative fuel. the lipid yield was good but in case of highlights in bioscience page 4 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion ta bl e 3. n ut ri en tr em ov al in aq ua cu ltu re w as te w at er w ith di ff er en ta lg al sp ec ie s a qu ac ul tu re w as te w at er a lg al sp ec ie s ty pe of al ga e to ta ld ur at io n da ys of tr ea tm en tm et ho d (m g/ l ) b io m as s co nc en tr at io n /p ro du ct iv ity (% ) l ip id gr ow th (% ) r ef er en ce fi sh er y p. ke ss le ri ty m ic ro al ga e 5 26 n a [7 ] se a br ea m fa ct or y te tr as el m is su ec ic a m ic ro al ga e 10 68 25 [2 7] sa lm on fa rm in g c hl or el la m in ut is si m a m ic ro al ga e 10 55 46 .3 7 [1 3] n a n ot a va ila bl e other matters like production of biodiesel from food crops during the time of world war period was a huge downfall. in second generation jatropha plant being the non-edible is another alternative fuel production which is a good source compared to first one [49]. the life cycle, production in large scale, huge mass of land for cultivation is the major demerit. third generation i.e., microalgae is currently the most successfully running lipid yield production out of all. cultivation of microalgae is only 14 days where this microorganism can be grown in even a small tub or artificial huge ponds. the biomass with high production of yield can be grown in any suitable environment with different stress conditions changing physiological condition. monoraphidium sp.is cultivated in bg 11 media and transferred into high ammonia content wastewater with 44% of stress condition present [50]. transesterification, in case of biodiesel it can be termed as conversion of a 3-methyl glyceride when it reacts with methanol in presence of catalyst to form fatty acid methyl esters to form ethanol, likewise shown in figure 1. for conversion into biofuel after harvesting method and weighing dry biomass, lipid extraction process is followed. it basically consists of two types mechanical and non-mechanical, the previous type usually relates with solvents and the later describes extraction through instrumental techniques. triglycerides act as main components, these are fatty acids extracted from algal species and converted into fatty acid methyl esters through direct transesterification method. this method depicts reaction of triglycerides with mono alcohols in presence of catalyst were analyzing solvent as hexane with better results compared to chloroform and methanol where pointing a strong line selection of solvents affects in lipid yielding after purification [51]. biodiesel has inherent sustainable transportation fuels for future mostly to reduce increasing pollutants emitted from exhaust cylinder. many modernized machine learning techniques and renewable feedstock are emerging rapidly for biofuel conversion compared to chemical catalysts. the main source of biodiesel is manufactured basically from renewable oil derived microbes or plants which causes zero-effect in ecosystem accompanying with carbon reduction. enzyme mediated undertakes non-toxic transesterification compared to same old process of chemical utilization [52,53]. from many processing experiments, microbes such as microalgae is the leading aspect, euglena sanguinea due to its presence of superior combustion characteristics were able to produce biodiesel that blends with the regular agricultural diesel engine till 40% by extracting lipid from the algal biomass [27]. a heterogeneous nano-catalyst ca(och3)2, a novel reactive distillation column is experimented for algal biodiesel production optimized by maximizing biodiesel purity by nsga-ii, non-dominated sorting genetic algorithm, designed both for low cost production and co2 emissions [28]. for another substitute yield of biodiesel an experiment conducted between chlorella sorokiniana and monoraphidium sp. where the biomass, fatty acid prohighlights in bioscience page 5 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion figure 1. flowchart diagram for biodiesel production through use of transesterification. highlights in bioscience page 6 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion file studies were compared showing better outcome from chlorella sp. in the mentioned study, lipid analyzing, its thermal efficiency all were recovered higher for biodiesel benefitting with low emissions of co and hc [51]. lipid extraction through microwave assisted in situ transesterification technique for the biodiesel yield was achieved from algae such as ulothrix sp. 88% dry weight (dw), cladophora sp. 80% dw, oedogonium sp. 73% dw and spirogyra sp. 67% dw. this technique for high biodiesel yield utilizes solvent free method [29]. conclusion from this study it reveals aquaculture wastewater is being the significant source for cultivating algae proceeding with sustainable environment in simple, cost effective way with zero waste reassurance. both microalgae and macroalgae plays vital role in aquaculture production and wastewater remediation. the presence of nutrients in wastewater reveals necessary requirements for their growth. the emitted aquaculture effluent contains highly nutritive source for algae that blends into it recommending as bio or phyco remediation in process. addition to that biomass produced from aquaculture wastewater can also be converted into biofuel through transesterification process with recent ideas of harvesting techniques. from the reported articles, it is known not much work have been proceeded in aquaculture wastewater co-related with micro and macroalgae. it still needs to be explored in order to achieve higher biomass and lipid for biofuel production where a solution is required for further research as there is huge gap in laboratory work and large-scale production. hence, this study needs to be taken to further simplified step by investigating more into it. acknowledgement the authors thank the academic writing group and swayam moocs course initiated by the ministry of human resource development, government of india, for providing an open learning platform. references 1. mao m, yan t, shen j, zhang j, zhang d. capacitive removal of heavy metal ions from wastewater via an electro-adsorption and electro-reaction coupling 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sureshkumar s, sankar t v., divya kr. phycoremediation in aquaculture; a win-win paradigm. environmental technology reviews. 2020;9(1):67–84. 16. mantzorou a, ververidis f. microalgal biofilms: a further step over current microalgal cultivation techniques. science of the total environment. 2019 feb 15;651:3187-201. 17. priyadarshani i, sahu d, rath b. algae in aquaculture. int. j. health sci. res. 2012;2:108-14. 18. hussain f, shah sz, ahmad h, abubshait sa, abubshait ha, laref a, et al. microalgae an ecofriendly and sustainable wastewater treatment option: biomass application in biofuel and biofertilizer production. a review. renewable and sustainable energy reviews. 2021;137(december 2020). highlights in bioscience page 7 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion 19. racine p, marley a, froehlich he, gaines sd, ladner i, macadamsomer i, bradley d. a case for seaweed aquaculture inclusion in us nutrient pollution management. marine policy. 2021 jul 1;129:104506. 20. monteiro jp, melo t, skjermo j, forbord s, broch oj, domingues p, et al. effect of harvesting month and proximity to fish farm sea cages on the lipid profile of cultivated saccharina latissima. algal research. 2021;54. 21. munguti jm, kirimi jg, obiero ko, ogello eo, kyule dn, liti dm, et al. aqua-feed wastes: impact on natural systems and practical mitigations—a review. journal of agricultural science. 2020;13(1):111. 22. brakel j, sibonga rc, dumilag r v, montalescot v, cottiericej, ward cg, et al. exploring , harnessing and conserving marine genetic resources towards a sustainable seaweed aquaculture. 2021;(january):1–13. 23. lugo la, thorarinsdottir ri, bjornsson s, palsson op, skulason h, johannsson s, et al. remediation of aquaculture wastewater using the microalga chlorella sorokiniana. water (switzerland). 2020;12(11):1–13. 24. padri m, boontian n, piasai c, phorndon t. cultivation process of microalgae using wastewater for biodiesel production and wastewater treatment: a review. iop conference series: earth and environmental science. 2021;623(1):0–6. 25. davis ak, anderson rs, spierling r, leader s, lesne c, mahan k, et al. characterization of a novel strain of tribonema minus demonstrating high biomass productivity in outdoor raceway ponds. bioresource technology. 2021 jul 1;331:125007. 26. hermadi i, setiadianto ir, fa d, al i. development of smart algae pond system for microalgae biomass production development of smart algae pond system for microalgae biomass production. 2021;0–8. 27. andreotti v, solimeno a, rossi s, ficara e, marazzi f, mezzanotte v, garcía j. bioremediation of aquaculture wastewater with the microalgae tetraselmis suecica: semi-continuous experiments, simulation and photo-respirometric tests. science of the total environment. 2020 oct 10;738:139859. 28. peng yy, gao f, yang hl, li c, lu mm, yang zy. simultaneous removal of nutrient and sulfonamides from marine aquaculture wastewater by concentrated and attached cultivation of chlorella vulgaris in an algal biofilm membrane photobioreactor (bf-mpbr). science of the total environment. 2020 jul 10;725:138524. 29. stamenkovic m, steinwall e, wulff a, henley w. cultivation and photophysiological characteristics of desmids in moderately saline aquaculture wastewater. journal of phycology. 2021: jpy-13150. 30. kang yh, kim s, choi sk, lee hj, chung ik, park sr. a comparison of the bioremediation potential of five seaweed species in an integrated fishseaweed aquaculture system: implication for a multispecies seaweed culture. reviews in aquaculture. 2021 jan;13(1):353-64. 31. miyawaki b, mariano ab, vargas jv, balmant w, defrancheschi ac, corrêa do, santos b, selesu nf, ordonez jc, kava vm. microalgae derived biomass and bioenergy production enhancement through biogas purification and wastewater treatment. renewable energy. 2021 jan 1;163:1153-65. 32. do amaral carneiro ma, de jesus resende jf, oliveira sr, de oliveira fernandes f, dos santos borburema hd, barbosa-silva ms, ferreira ab, marinho-soriano e. performance of the agarophyte gracilariopsis tenuifrons in a multi-trophic aquaculture system with litopenaeus vannamei using water recirculation. journal of applied phycology. 2021 feb;33(1):481-90. 33. tan js, lee sy, chew kw, lam mk, lim jw, ho sh, show pl. a review on microalgae cultivation and harvesting, and their biomass extraction processing using ionic liquids. bioengineered. 2020 jan 1;11(1):116-29. 34. chauton ms, forbord s, mäkinen s, sarno a, slizyte r, mozuraityte r, standal ib, skjermo j. sustainable resource production for manufacturing bioactives from microand macroalgae: examples from harvesting and cultivation in the nordic region. physiologia plantarum. 2021 mar 9. 35. estime b, ren d, sureshkumar r. cultivation and energy efficient harvesting of microalgae using thermoreversible sol-gel transition. scientific reports. 2017 jan 19;7(1):1-9. 36. kumar v, nanda m, verma m. application of agar liquid-gel transition in cultivation and harvesting of microalgae for biodiesel production. bioresource technology. 2017 nov 1;243:163-8. 37. yu si, min sk, shin hs. nanocellulose size regulates microalgal flocculation and lipid metabolism. scientific reports. 2016 oct 31;6(1):1-9. 38. chen q, fan q, zhang z, mei y, wang h. effective in situ harvest of microalgae with bacterial cellulose produced by gluconacetobacter xylinus. algal research. 2018 nov 1;35:349-54. 39. luo s, wu x, jiang h, yu m, liu y, min a, li w, ruan r. edible fungi-assisted harvesting system for efficient microalgae bio-flocculation. bioresource technology. 2019 jun 1;282:32530. 40. chen z, qiu s, yu z, li m, ge s. enhanced secretions of algal cell-adhesion molecules and metal ion-binding exoproteins promote self-flocculation of chlorella sp. cultivated in municipal wastewater. environmental science & technology. 2021 aug 23;55(17):11916-24. 41. de souza leite l, dos santos pr, daniel la. microalgae harvesting from wastewater by ph modulation and flotation: assessing and optimizing operational parameters. journal of environmental management. 2020 jan 15;254:109825. 42. markeb aa, llimós-turet j, ferrer i, blánquez p, alonso a, sánchez a, moral-vico j, font x. the use of magnetic iron oxide based nanoparticles to improve microalgae harvesting in real wastewater. water research. 2019 aug 1;159:490-500. 43. rakesh s, tharunkumar j, sri b, jothibasu k, karthikeyan s. sustainable cost-effective microalgae harvesting strategies for the production of biofuel and oleochemicals. highlights in bioscience. 2020 jul 7;3. 44. wang q, oshita k, nitta t, takaoka m. evaluation of a sludgetreatment process comprising lipid extraction and drying using liquefied dimethyl ether. environmental technology. 2021 sep 19;42(21):3369-78. 45. abera g. review on high-pressure processing of foods. cogent food agriculture. 2019 jan 1;5(1):1568725. highlights in bioscience page 8 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ ray and rakesh, 2022 phycoremediation of aquaculture wastewater for fuel conversion 46. tang w, row kh. evaluation of co2-induced azole-based switchable ionic liquid with hydrophobic/hydrophilic reversible transition as single solvent system for coupling lipid extraction and separation from wet microalgae. bioresource technology. 2020 jan 1;296:122309. 47. arathi s, kumar jt, jothibasu k, karthikeyan s, suchitra r. qualitative and quantitative estimation of algal lipids for biofuel production. 2020;8(4):2451–9. 48. jothibasu k, dhar dw, rakesh s. recent developments in microalgal genome editing for enhancing lipid accumulation and biofuel recovery. biomass and bioenergy. 2021 jul 1;150:106093. 49. mathew gm, raina d, narisetty v, kumar v, saran s, pugazhendi a, et al. recent advances in biodiesel production: challenges and solutions. science of the total environment. 2021 nov 10;794:148751. 50. aghbashlo m, peng w, tabatabaei m, kalogirou sa, soltanian s, hosseinzadeh-bandbafha h, et al. machine learning technology in biodiesel research: a review. progress in energy and combustion science. 2021 jul 1;85:100904. 51. papu nh, lingfa p, dash sk. an experimental investigation on the combustion characteristics of a direct injection diesel engine fuelled with an algal biodiesel and its diesel blends. vol. 23, clean technologies and environmental policy. 2021. p. 1769– 83. 52. mondal b, parhi ss, rangaiah gp, jana ak. nano-catalytic heterogeneous reactive distillation for algal biodiesel production: multi-objective optimization and heat integration. energy conversion and management. 2021 aug 1;241:114298. 53. hasnain m, abideen z, naz s, roessner u, munir n. biodiesel production from new algal sources using response surface methodology and microwave application. biomass conversion and biorefinery. 2021 may 15:1-6. highlights in bioscience page 9 of 9 january 2022|volume 5 http://bioscience.highlightsin.org/ abstract introduction micro and macro algae as a nutritive aquaculture feed microalgae based biorefinery for aquaculture wastewater treatment open pond system nutrient removal efficiency of aquaculture wastewater with microalgae recent advances in microalgae harvesting and lipid extraction microalgae as a sustainable future biofuel approach conclusion acknowledgement references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20207 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 6 research article open access 1 department of quantum electronics, faculty of physics, uzhhorod national university, ukraine contacts of authors * to whom correspondence should be addressed: alexander shuaibov citation: shuaibov a, minya a, shevera i, malinina a, gritsak r, malinin a, gomoki z, danilo v. (2020). characteristics of bipolar nanosecond discharges in air formed in the electrode system “blade-surface of nonmetallic liquid -blade”. highlights in bioscience volume 3. article id 20207. dio:10.36462/ h.biosci.20207 received: may 20, 2020 accepted: june 27, 2020 published: july 4, 2020 copyright: © 2020 shuaibov et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. characteristics of bipolar nanosecond discharges in air formed in the electrode system “blade-surface of nonmetallic liquid -blade” alexander shuaibov* 1 , alexander minya 1 , igor shevera 1 , antonina malinina 1 , roksolana gritsak 1 , alexander malinin 1 , zoltan gomoki 1 , vladislav danilo 1 abstract curcumin the design of the device for producing a high-current, bipolar nanosecond discharge over the surface of a non-metallic liquid (water, electrolytes, alcohols, etc.) in air is given. air pressure is ranged from 5 to 101 kpa. the distance between the tip of the blade and the surface of water or liquid (5% solution of copper sulfate in distilled water) was 4 mm, and the distance between parallel metal blades was 40 mm. the conditions for uniform plasma overlapping of the electrolyte surface between the metal blades are established. the spatial, electrical, and optical characteristics of the discharge are investigated. it is shown that the discharge under study allows obtaining colloidal solutions of copper nanoparticles in distilled water in a macroscopic amount (1 liter or more). the developed reactor is of interest for use in poisonous chemical solution disinfection systems, solutions based on dangerous bacteria and viruses for which the use of traditional systems with a point spark discharge or a barrier discharge becomes ineffective. the rector is also promising for the synthesis of colloidal solutions of transition metal oxide nanoparticles from solutions of the corresponding salts. these solutions can be used in micro-nanotechnology and for antibacterial treatment of plants in greenhouses, processing of medical instruments and materials. keywords: nanosecond discharge, distilled water, copper sulfate, electrolytes, alcohols, colloidal solutions, copper nanoparticles. introduction recently, intensive use of various gas-discharge sources of ultraviolet (uv) radiation in medicine, microbiology, chemistry, sanitation and ecology has been observed [1–9]. promising for these purposes is the use of “cold” plasma jets, which are also formed mainly in different gas-discharge devices [10]. when using such uv emitters, including open ones, operating in atmospheric air and not having a quartz shell, for the action of biologically or chemically active solutions on the surface, the distance between the lamp and the liquid is several centimeters. the duration of the radiation pulses of such gas-discharge emitters is tens to hundreds of microseconds, and the operating voltage of their power sources does not exceed 10-11 kv. this leads to a decrease in the pulsed power of radiation compared with nanosecond lamps. simultaneously, the fluxes of active particles (such as hydroxyl radicals oh, hydrogen atoms h, and oxygen atoms -o from the plasma are eliminated because they do not have time to shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 6 penetrate the surface of the treated fluid during their lifetime (which does not exceed 10 ns). the use of jets of “cold” plasma for surface treatment of biologically and chemically active solutions is often limited to the small working surface of the plasma jet. therefore, the development of gasdischarge devices to obtain near-surface plasma of a relatively large area, which can be used for treating surfaces of non-metallic liquid media in air to solve microbial and medicine issues that are not able to solve by known sources of uv radiation and plasma, is relevant [11,12] . in previous research [13], the characteristics of the discharge in the air above the water surface are given. here, a high-voltage tip electrode was used, to which a highvoltage pulse of negative polarity was applied, and a flat, grounded metal electrode was placed in water near the opposite end of an opened discharge cell. the voltage pulse had amplitude of up to 20 kv and duration of 5-1000 μs. the distance between the electrodes reached 20 cm, and the cathode distance from the water surface was 0.1-1 cm. the discharge channel had a width of 0.3-0.5 cm and its main part was parallel to the water surface and separated from it by a fraction of a millimeter. since the plasma channel had a small width, it could not cover a large surface area of water. with a current pulse duration of tens of microseconds 1 ms, heating of the liquid, which is undesirable when working with biosolvents, can be essential. therefore, it is important to study the formation of nanosecond discharges over the surface of non-metallic liquids in air, allowing us to obtain large width discharges in the system of electrodes based on metal blades. for these purposes, the technique of forming plasma electrodes developing on the surface of solid dielectrics (fiberglass laminate, leucosapphire, etc.) can be used. such electrodes have been used in high-pressure lasers pumped with a transverse volume discharge, in particular, on exciplex molecules 308 nm xecl (x-b) [14]. the main disadvantage of such plasma electrodes based on solid dielectrics is their small service life associated with the destruction of a dielectric by a powerful discharge in an aggressive gaseous medium. in our experiments, it was proposed to obtain a nanosecond discharge over the surface of liquid nonmetallic media, which tend to self-recover in the course of operation, which significantly increases their service life. the aim of the work was to develop a gasdischarge reactor based on a surface discharge with a liquid nonmetallic electrode for applications in medicine, biology, agriculture and nanotechnology. this article presents the results of a study of the spatial, electrical, and optical characteristics of a highcurrent nanosecond discharge over the surface of distilled water and a solution of cuso4 salt in distilled water, which was formed in air at pressures of 5–101 kpa. materials and methods technique and conditions of the experiment the use of steel blades in the system for obtaining a high-current nanosecond discharge in air at atmospheric pressure when the discharge in the form of 12 plasma sheets was closed to the surface of distilled water or electrolytes was described in [15–16]. but in this case, the ratio of the area of the overlapped plasma to the total area of the electrode based on the system of steel blades did not exceed 0.3-0.4. the characteristics of the discharge and the system of electrodes, consisting of two steel blades installed under an angle varying during the experiment to each other at some short distance from the surface of non-metallic liquids, were described in [17–18]. in this case, it was also not possible to completely overlap the surface of liquid medium by plasma, which was located under the steel blades. as in the first case, and in the second case, colloidal solutions based on nanoparticles of copper, zinc and iron oxides in a volume of about 10 3 cm 3 were synthesized in such gasdischarge plasma-chemical reactors. therefore, in this study as a basis to create a surface discharge, the design and power supply scheme for the nanosecond surface discharge of plasma electrodes of high-pressure lasers pumped by a transverse volume discharge was chosen [14]. a highcurrent nanosecond discharge at air pressures in the range of 5–101 kpa was ignited in the “blade – electrolyte surface – blade” electrode system in the discharge module, the circuit of which is shown in figure 1. figure 1. 1 discharge chamber made of plexiglass, 2 a cuvette with a non-metallic liquid (diameter 120 mm, height 16 mm), 3 platforms with an electrode system “blade-surface of non-metallic liquid-blade”, 4 grounded metal plate, 5 system adjusting the height of the electrodes above the surface of the liquid (h = 0-15 mm), 6 the system for regulating the distance between steel blades (l = 40-80 mm), 7 the system for pumping out and gases inlet, filling the cuvette with liquid and adjusting the system of electrodes in height, 8 quartz window for recording spectra and radiation from the plasma discharge gap, 9 glass shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 6 window for visual, photographic, video surveillance, 10 window for adjustment and access to the system of adjusting the distance between the blades.u1 -, u2 connection points for the leads from a high-voltage nanosecond source of the discharge ignition. the electrode system was installed in a sealed discharge chamber made of dielectric. the air pressure was varied in the range of 5-101 kpa for a liquid electrode based on a 5% solution of copper sulfate (cuso4) salt in distilled water and was 101 kpa for an electrode based on distilled water. the distance between the surface of the non-metallic liquid and the tips of the thin steel blades was 4 mm, and the distance between the parallel steel blades was 40 mm. the level of the working fluid was kept constant with the help of a special drip system and an additional tank with liquid. the discharge cuvette was made of organic glass and could be pumped out with a vacuum pump to a residual pressure of 10 pa. the overpressure of the gases in the cuvette could reach 200 kpa. the electrodes were made of steel blades, 0.1 mm thick. the discharge cell was installed in the screen of their metal grid, which reduced the influence of the electromagnetic fields of the discharge ignition system on the recording system of its electrical and optical characteristics. the characteristics of a nanosecond surface discharge in the system of electrodes «blade – electrolyte surface – blade» were studied using an experimental setup described in [18–19]. a high-current nanosecond discharge on the surface of the liquid was ignited using a high-voltage source of bipolar nanosecond voltage pulses with a resonant recharge of the storage capacitor with a capacity of 1.54 nf and a hydrogen thyratron switchboard. the amplitude of the voltage pulses of the same polarity on the voltage input of the high-voltage modulator could vary in the range of 10-25 kv. the amplitude of the main maximum of the current pulse reached 100-170 a. the repetition frequency of nanosecond voltage and current pulses was in the range of 35-1000 hz. radiation of surface discharge plasma was recorded in the spectral range λ = 200-665 nm using a spectrometer with a diffraction grating of 1200 lines / mm mdr-2. in the output of the spectrometer, a photomultiplier tube feu-106 connected to a dc amplifier was used to detect radiation. the signal from the amplifier was fed to an analog-to-digital converter and then fed to a personal computer for processing. to estimate the energy contribution to the discharge plasma, current and voltage oscillograms were recorded using a 6-channel wideband oscilloscope 6lor04. the voltage pulses on the electrodes were applied through a capacitive divider to one of the channels of the oscilloscope, and the discharge current pulses were measured using a calibrated rogowski belt on the other channel of the oscilloscope. the synchronization of the pulses of the discharge power supply with the launch of the 6lor-04 oscilloscope was carried out using a g5-15 pulse generator. the disadvantage of the proposed device is the use of static blades as electrodes. during prolonged operation of the reactor, the erosion of steel electrodes occurs and the discharge loses a little in uniformity. in the industrial version of the device, the electrodes must be made of refractory alloys based on tungsten, molybdenum or tantalum, as in high-pressure dischargers in systems for the formation of nanosecond pulses at a current amplitude level of hundreds of amperes. results and discussion spatial, electrical and spectral characteristics of surface discharge in each experiment on the ignition of a surface discharge in the air with a liquid nonmetallic electrode, together with a study of its spectral characteristics, its spatial and electrical characteristics (current and voltage waveforms) were also recorded. using the oscillograms of the voltage across the discharge gap and the discharge current, we determined the pulsed electric power of the discharge and the energy input to the plasma per pulse (figure 2). figure 2. the dependence of the pulsed electric power of the discharge on time, oscillograms of voltage and current of a bipolar nanosecond discharge above the surface of a 5% solution of cuso4 salt in distilled water at the air pressure in the discharge chamber equal to 6.5 kpa. the dependence of the pulsed electric power of the discharge on time was obtained by graphically multiplying the oscillograms of voltage and current, and the magnitude of the energy input to the discharge per pulse was determined by integrating the discharge power of the discharge over time. shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 6 for the discharge of atmospheric pressure over the surface of distilled water, the analysis of the voltage and current waveforms showed that the current pulse has an amplitude of up to 50 a and a duration of about 50 ns and is formed under the action of a voltage pulse with an amplitude of 30 kv, which had a duration of 30 ns. due to the mismatch of the output impedance of the pulsed voltage generator on the voltage oscillograms in the conditions of these experiments, an oscillatory structure was observed. the maximum value of the pulse electric power of the discharge during the first 30 nanoseconds since the moment of ignition of the discharge reached 1 mw. in one discharge pulse, the energy input to the plasma was 30 mj. with a decrease in air pressure in the discharge module to 6.5 kpa, the energy input to the plasma per pulseincreased threefold (figure 2). at the same time, the amplitude of the current pulse reached 150 a for a duration of 70 ns. the voltage pulse had an amplitude of 20 kv and a duration of 25 ns. the pulsed electric power of the discharge reached 3 mw. figures (3 and 4) show images of a nanosecond discharge on the surface of distilled water at an air pressure of -101 kpa and on the surface of a 5% solution of a copper sulfate at air pressure of 6.5 kpa. figure 3. the image of the discharge glow above the surface of distilled water at an air pressure of 101 kpa. figure 4. image of the discharge above the surface of a 5% solution of a cuso4 salt at an air pressure in the discharge chamber of 6.5 kpa. in the first case, the discharge consisted of a fairly uniform set of streamer channels, which were observed against a weak background of a diffuse surface discharge. the nanosecond discharge in air of atmospheric pressure in the system of electrodes "blades surface of distilled water" had a similar appearance [15]. the brightness of the plasma glow slightly decreased in the direction from the tip of the steel blade to the center of the discharge gap, which is probably due to this distribution of the specific electric field strength of the bipolar discharge. when the air pressure in the discharge chamber was reduced to 5–25 kpa (figure 4), the surface discharge acquired a diffuse appearance and completely covered the liquid surface between the steel blades, which were installed parallel to each other. figure 5 shows the emission spectrum of the discharge plasma above the surface of distilled water at an air pressure of 101 kpa. the results of identification of this emission spectrum are presented in table 1. as can be seen from table 1, the bands of the second positive system of the nitrogen molecule prevail in the plasma emission spectrum, and the main part of the radiation was concentrated in the spectral range of 280-390 nm. figure 5. the emission spectrum of the discharge above the surface of distilled water at an air pressure in the discharge chamber equal to 101 kpa. table 1. the results of the interpretation of the emission spectrum of the discharge formed above the surface of distilled water (p = 101 kpa). wavelength, nm molecule v’ v’’ e(v’), ev 297.68 n2 2 0 0,128 315.93 n2 1 0 0,376 337.13 n2 0 0 0,128 357.69 n2 0 1 0,128 371.05 n2 2 4 0,619 375.54 n2 1 3 0,376 380.49 n2 0 2 0,128 394.3 n2 2 5 0,619 399.84 n2 1 4 0,376 405.94 n2 0 3 0,128 shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 6 the absence of hydroxyl radicals bands in the plasma emission spectrum indicates that the density of water vapor in the plasma is low, that is, the plasma heats the water surface a little and can be considered “cold” (not more than 50° c). this is important for biological and medical applications of the investigated discharge. compared with the known plasma jets [10], this source can simultaneously process relatively large surfaces of non-metallic solutions. the characteristic plasma emission spectrum of a nanosecond discharge above the surface of a 5% solution of cuso4 salt in distilled water at an air pressure of 6.5 kpa (figure 6) differs fundamentally from the spectrum for discharge above the water surface by the presence of relatively intense spectral lines of the copper atom that enters the plasma from the solution. figure 6. the emission spectrum of the discharge above the surface of a 5% solution of cuso4 salt in distilled water at an air pressure in the discharge chamber equal to 6.5 kpa. the results of the identification of the spectrum shown in figure 6 in part of the radiation objects entering the discharge from the surface of the solution are summarized in table 2. but even in this case, the bands in the second positive system of the nitrogen molecule, located in the spectral range of 280-390 nm, were major in the spectrum. after treatment the surface of the copper sulfate solution for 2-3 hours at a pulse repetition rate of 100-150 hz, the solution changed its color from blue to green. this indicates the complete transformation of copper cations and anions (so4) in solution into a colloidal solution based on copper oxide nanoparticles (figure 7). figure 7. images of copper sulfate solutions in distilled water before and after plasma treatment of a nanosecond discharge in air at atmospheric pressure. the green color of the solution corresponds to the radiation at the wavelength of the plasmon resonance of copper oxide nanoparticles. this makes it possible to recommend the introduction of small copper sulfate additives in a variety of biologically active solutions in order to enhance the bactericidal effect of uv illumination of the surface of the solution and of copper oxide nanoparticles with a strong bactericidal effect [20, 21]. conclusion thus, the conditions for obtaining a uniform discharge in the air above the surface of non-metallic liquids are established; the maximum pulsed discharge power is in the range (1-3) mw with an energy input to the plasma in one pulse (30-900) mj; in the emission spectra of the plasma, the emission of the second positive system of nitrogen molecule bands in the spectral range of 280-390 nm prevails, but for the plasma on the surface of the copper sulfate solution, a relatively intense emission of copper atoms entering the discharge from the surface of the solution was detected; treatment of the salt of copper sulfate with surfacedischarge plasma allows the synthesis of colloidal solutions based on nanoparticles of copper oxides, promising for use in nanotechnology, microbiology and medicine. table 2. the results of the identification of objects of the radiation of the discharge plasma above the surface of a 5% solution of copper sulfate at air pressure of 6.5 kpa. wavelength, nm atom elow., ev eup., ev lower term upper term 249.215 cu i 0 4,97322 3d 10 4s 2 s1/2 3d 9 ( 2 d)4s4p( 3 p ° ) 4 p3/2 319.41 cu i 1,642133 5,52246 3d 9 4s 2 2 d3/2 3d 9 ( 2 d)4s4p( 3 p°) 4 d 3/2 353.038 cu i 1,642133 5,15286 3d 9 4s 2 2d3/2 3d 9 ( 2 d)4s4p( 3 p ° ) 4f ° 5/2 405.112 cu i 5,724385 8,7842 3d 9 (2d)4s4p( 3 p ° )3/2 3d 9 4s( 3 d)4d2p 3/2 525.20 cu i 5,52246 7,88305 3d 9 ( 2 d)4s4p( 3 p ° ) 4 d°3/2 3d 9 4s( 3 d)5s 4 d 3/2 529.25 cu i 5,394745 7,73659 3d 9 ( 2 d)4s4p( 3 p ° ) 4 d°7/2 3d 9 4s( 3 d)5s 4 d7/2 shuaibov et al., 2020 characteristics of bipolar nanosecond discharges in air formed in the electrode system highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 6 references 1. piskarev im, ivanova ip, samodelkin ag, ivaschenko mn. initiation and investigation of free-radical processes in biological experiments, novgorod. nizhny novgorod state agricultural academy. 2016; p. 140. 2. arkhipova ev, ivanova ip. membrane structural condition and functional activity of peritoneal macrophages after gas discharge exposure. medical technologies in medicine/sovremennye tehnologii v medicine. 2017 sep 1;9(3). 3. piskarev im, astaf'eva ka, ivanova ip. sources of gasdischarge plasma: effect of the absorbed dose and active particle composition on physicochemical transformations in biological substrates. medical technologies in medicine/sovremennye tehnologii v medicine. 2018 jun 1;10(2). 4. vasilyak lm. application of pulsed electrical discharge lamps for bactericidal treatment. surface engineering and applied electrochemistry. 2009 feb 1;45(1):26-34. 5. wasserman al, shandala mg, yuzbashev vg. the use of ultraviolet bactericidal irradiators for disinfecting indoor air. overview of the current state of the problem. epidemiology and hygiene-journal issued by medical alphabet. 2013 3: 24-30. 6. shuaibov ak, shimon ll, dashchenko ai, shevera iv. excimer low pressure emitter for the spectral region of 170-310 nm. instruments and experimental technique. 2002, 1: 104-106. 7. shuaibov a, gritzak r. optical characteristics of uv– vuv lamps on the electronic-vibrational transitions of the hydroxyl radical pumped by a nanosecond capacitive discharge. high voltage. 2017 jul 11;2(2):78-81. 8. shuiabov ok, grabova ia, shevera iv. gas-discharge uv-vuf exciplex and halogen lamps of low pressure. monograph. uzhgorod. uzhhorod national university "hoverla". 2018, p. 260. 9. shuyabov ok, hrytsak rv. ultraviolet lamps on radicals of hydroxyl and exciplex molecules with pumping by barrier nanosecond discharge. monograph, uzhhorod, uzhhorod national university "hoverla", 2018, p. 114. 10. sosnin ea, tarasenko vf, panarin va, skakun vs. apokamp: a new type of plasma jet. horizons in world physics. (ed. a. remer) nova science, 2017, (292) 551. 11. hozák p, scholtz v, khun j, mertová d, vaňková e, julák j. further contribution to the chemistry of plasmaactivated water: influence on bacteria in planktonic and biofilm forms. plasma physics reports. 2018 sep 1;44(9):799-804. 12. piskarev im. effect of spark discharge plasma on water, physiological saline, and hanks’ solution. high energy chemistry. 2018 jul 1;52(4):348-54. 13. vaulin dn, yershov ap, kamenschikov sa, chernikov va. high-voltage pulse discharge propagating along a water surface. high temperature. 2011 jun 1;49(3):356-62. 14. shuaibov ak, neimet ii, sinishin aa, kindrat ss, shevera vs. characteristics of a quasi-stationary xecl excimer laser with plasma electrodes. ukfiz. 1989 apr;34:535-7. 15. shuaibov ak, chuchman mp, mesarosh lv. characteristics of a glow discharge in atmospheric pressure air over the water surface. technical physics. 2014 jun 1;59(6):847-51. 16. shuiabov ok, minya ya o, gomoki zt, shevera iv, danilo vv. method of synthesis of copper oxide nanostructures in nanosecond discharge with electrolytic cathode. patent ua, no. 118235 с2 jul, 2018. 17. shuaibov ak, minya ai, enedi al, shevera iv, gomoki zt, danilo vv. characteristics of a nanosecond discharge with a liquid nonmetallic electrode in the air. surface engineering and applied electrochemistry. 2018 jan 1;54(1):1-5. 18. shuiabov ok, minya oy, gomoki zt, shevera iv, danilo vv. a method of ignition of a spatially homogeneous discharge of atmospheric pressure in a system with a water electrode. patent for utility model. application number: u 2016 05309; date of application: 16.05.2016; the date from which the rights to the utility model are valid: 10.11.2016; publication of information about the issuance of a patent: 10.11.2016, bul. no. 21 19. shuaibov ak, vasil c. parameters of high current nanosecond discharge in air wich electrolytic electrode. international scientic-technical magazine “measuring and computing devices in technological processes”. 2016; 56(3): 45-48. 20. shulgina ta. aqueous solution of copper nanoparticles and its antimicrobial activity. advances in current natural sciences. 2012; 12: 143-143. 21. egorova em. biological effects of metal nanoparticles. medical science. 2014, 350. highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 1 of 5 citation: rslan w. m. (2018) sugar beet artificial seeds an overview. highlights in bioscience, volume 1.article id 20182, dio:10.36462/ h.biosci.20182 received: may 22, 2018 accepted: july 17, 2018 published: august 12, 2018 copyright:© 2018 rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. date palm biotechnology: recent research an overview wessam m. rslan* agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract date palm (phoenix dactylifera l.) is among the earliest fruit crops cultivated in the arid arab peninsula, north africa, and middle east territories. dates are a significant source of food and revenue for middle east and north africa's local communities. it has distinctive features of biology and development that require special methods of reproduction, culture and governance. in varying dategrowing regions, there are thousands of date plant cultivars and varieties. the lengthy life cycle, long juvenile lifespan, and date palm dioecism produce cultivation difficult. every year, the percentage of crop genomes sequenced has continued to increase. the incredible rate at which dna samples become accessible is mainly due to the enhancement in cost-and speed-related sequencing techniques. modern sequencing techniques enable the sequencing at realistic price of various cultivars of tiny plant genomes. although many of the published genomes are deemed incomplete, they have nevertheless proven to be useful instruments for understanding significant plant characteristics such as fruit maturation, grain characteristics and adaptation of flowering time, here we review date palm genomic studies and determine its genomics element. keywords: date palm, genome-wide association, snp, genomics element, genome annotation . review article open access date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 2 of 5 introduction several of the plant genome sequences have been fully sequenced (collected, annotated and released) and have been openly accessible in the database, such as oryza sativa (1), zea mays (2), hordeum vulgare (3), phoenix dactylifera (4), triticum aestivum (5), eucalyptus grandis (6), capsicum annuum (7), beta vulgaris (8) and solanum (9). yang et al. (1) presented a full date palm chloroplast genome sequence of approximately 158,462 bp. they revealed that there are 112 distinctive genes and 19 duplicated parts in the ir areas in the date palm chloroplast genome. they recognized 78 snps within the population of a particular chloroplast genome as significant intravarietal polymorphisms, most of which were found in genes with essential tasks. they also discovered 18 polycistronic transcription blocks and 3 extremely expression-based genes-atpf, trna-ugc, and rrn23 based on rna-sequencing records. al-dous et al. .(4) used the illumina gaii sequencing platform to present the first date of the palm assembly draft genome sequence for the khalas cultivar. they reported a date palm genome with size of 658 mb, 58% assembled sequences (382 mb) and forecast 25,059 genes. among the nine cultivars of date palms trees (deglet noor, deglet noor bc5, khalas, khalas bc2, medjool bc4, medjool, alrjfem, khltmale, khfxfem) they recorded more than 3.5 million polymorphic locations. in (http://qatar-weill.cornell.edu/ research/datepalmgenome/), this sequence was stored. fang et al. (12) provided date palm full sequence of mitochondrial genome with an estimated size of approximately 715.001 bp. al-mssallem et al. (13) introduced the second khalas nuclear genome assembly, which is 605.4 mb in size, spanning > 90% of the genome (~671 mb) and > 96% of its functional genes (~41,660 genes). this sequence was stored with bioproject id prjna83433 in genbank (http://www.ncbi.nlm.nih.gov/) and produced the first genetic map of the date palm (phoenix dactylifera) and recognized the putative gender chromosome. they submitted ~4000 maps on the map using a total of 1293 cm of nearly 1200 molecular markers. these molecular markers were divided into 18 linkage clusters (lg) and proposed that the sex chromosome could be lg12. hazzouri et al. (14) launched an extensive catalog of about seven million single nucleotide polymorphisms in date palms based on a set of 62 cultivars re-sequencing the entire genome. analysis of population structure suggested a significant genetic gap between north africa and the date palms of middle east/south asia with proof of admixture in egyptian and sudanese cultivars. candidate mutations for trait variation were recorded, including polymorphisms of nonsense and variety of presence/absence of gene material in important agronomic trait mechanisms. in the r2r3 myb-like orthologue of the oil palm virescens gene connected with fruit color variability, they also recognized a copy-like retrotransposon insertion polymorphism. the first mirna segments of the palm species elaeis guineensis, elaeis oleifera and phoenix dactylifera were recorded by silva et al. (15). in the genomes of these species, they recognized mirna precursors and defined their potential biological functions proposed by the mature mirna-based target gene regulation. three hundred and thirty-eight precursors were recognized, varying in sequence size from 76 to 220 nucleotides dispersed in 33 households. in addition, they also recognized musa acuminata 266 mirna precursors that were phylogenetically near to palm species. detection of resistance genes in date palm al-mssallem et al. (13) monitored phoenix dactylifera protein samples using the pfam nbs (nb-arc) family pf00931 databases (e-value cutoff of 1.0) hidden markov models (hmms). using an hmm model with pfam tir pf01582 (e-value cutoff 1.0) domains as well as lrr motifs in the c-terminal domains, the 144 expected amino acid sequences were used to identify tir domains. in total, 144 non-redundant nbs-encoding proteins have been recognized and validated manually, accounting for about 0.35% of the gene designs in the phoenix dactylifera genome. simple sequence repeats in date palm billotte et al. (16) built a library (ga) n enriched with microsatellite and described 16 nuclear simple sequence repetition (ssr) loci in phoenix dactylifera. they studied amplification and genotyping across taxes. this experiment reveals the usefulness of most ssr indicators in 11 other phoenix organisms and the applicability of some of them in elaeis guineensis, 11 pritchardia species, pritchardiopsis jeanneneyi species, and six astrocaryum species. akkak et al. (17) separated from two microsatellite enriched date palm libraries forty-one easy sequence repeats (phoenix dactylifera). of the 41 ssrs, 17 chosen microsatellite loci were identified and assessed on a collection of 31 algerian and californian germplasm cultivars and clones. all primer pairs generated an amplification pcr pattern of the expected size and high polymorphism was detected among the samples analyzed. tax amplification across the genus phoenix indicates the usefulness of most ssr indicators in 14 other species. hamwieh et al. (18) researched the prevalence of microsatellite patterns in the first edition of the new genome profile of the date palm assembly produced by the next generation dna sequencing of the entire genome shotgun. http://qatar-weill.cornell.edu/%20research/datepalmgenome/ http://qatar-weill.cornell.edu/%20research/datepalmgenome/ http://www.ncbi.nlm.nih.gov/ date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 3 of 5 they structured a total of 1,091 primer combinations in simple/perfect microsatellite motifs flanking regions. from it 377 primers flanked dinucleotide, 352 primers flanked trinucleotide, and 362 primers pair flanked tetranucleotide repeats out of these primer combinations fifty primer pairs used for 8 iraqi date palm varieties screening. results showed that 28 combinations of primers were usable (56%) and 18 (36%) disclosed polymorphic alleles. also (19) developed date palm molecular markers database. elmeer et al. (20) constructed thirty primer pairs of microsatellite genomic dna markers for ssr. these molecular markers were used for eleven date palm genotypes to evaluate genetic diversity. results stated that the targeted pcr fragments were not amplified by only seven (23.3 percent) of the thirty primers. while thirteen pcr primers (43.3%) enhanced monomorphic banding patterns and polymorphic banding motifs were produced by the remaining ten primers (33.4%). arabnezhad et al. (21) formed two ssr-enriched date palm genomic databases, including repeat motifs (ag) n and (aag) n, using 22 ssr pcr primer pairs to evaluate the genetic connection between 16 date palm cultivars cultivated in separate geographic areas (iran, iraq and africa). based on nei's genetic distance they used, the cluster analysis put african date palms in a group other than the genotypes of iran and iraq. they stated it appears that african date palms ' domestication has taken a distinct path than those cultivated in the middle east. bodian et al. (22) used easy sequence repeat markers to analyze genetic variation among 128 date palm specimens from the figuig desert (morocco). 121 females belonging to 11 cultivars were among those palms, and 7 were males. eighteen ssr primers have been used, but all samples have been effectively amplified by only 15 primers. the dendrogram acquired showed three populations and male and female genotypes did not detect any genetic distinctions. zhao et al. (23) evaluated 28,889 samples of est from the database of the date palm genome and recognized 4,609 ests as ssrs. trinucleotide patterns (69.7%) were the most common among these ssrs, followed by tetranucleotide (10.4%) and dinucleotide patterns (9.6%). they also engineered a total of 4,967 primer pairs from sequencing data for est-ssr markers. twenty of these primer pairs from twelve date palm cultivars were tested with genomic dna. one-third of these primer pairs identified polymorphisms of dna that distinguished the cultivars of the twelve date palms. aberlenc-bertossi et al. (24) used the new sequence draft of the date palm genome al-dous et al. (11) to identify microsatellites and layout primers for ssr. they recognized 204 genes with microsatellite coding sequences, 150 of which were appropriate for primer construction, but only 103 had annealing locations that were not duplicated. with six or more repeats, such microsatellites had perfect trinucleotide motifs, and hexanucleotide motifs with at least four repeats. of the 47 first pairs lastly maintained, 33 in a preliminary experiment with eight phoenix dactylifera samples produced expected pcr amplification. the 33 loci have been further evaluated on 16 individuals comprising phoenix roebelenii o'brien(2), phoenix dactylifera (7), phoenix reclinata jacq .(2), phoenix rupicola t. anderson (2), phoenix theophrasti greuter (2), and phoenix canariensis interspecific hybrid phoenix sylvestris . zehdi-azouzi et al. (25) used both nuclear ssr and chloroplast microsatellites to evaluate the variety and genome composition of date palm accessions from mauritania to pakistan in ten regions. a total of 295 accessions were evaluated using 18 nuclear ssr loci revealing > 200 alleles for every locus. they recorded analyzes of the date palm population's genetic composition using the bayesian clustering strategy, hierarchical ranking of nj and dacp. their findings were obviously compatible with a two-cluster geographic structure. the first, called the eastern pool, includes djibouti, iraq, oman, pakistan, and the uae date palm accessions. while the western pool was appointed the second cluster, it included the residual accessions from africa, including egypt, algeria, mauritania, tunisia and morocco (26). snps studies in date palm al-dous et al. (11) analyzed 3.5 million snp across the genomes of male and female date palms trees to recognize gender-segregating polymorphisms. the findings noted best fit an xy sex-determination model with the heterogamous sex being males. using a male heterogamete model 1,605 snps were noted segregating with gender. analyzing two scaffolds with the most gender-segregating snps, they noted an estimated threefold distinction in divergence between male and female haplotypes from the reference sequence. across all genome sequences in these regions, the cultivars of 867 polymorphic sites have been observed. comparing the females of deglet noor and medjool with the female reference of khalas disclosed that 253 and 271 sites varied from the reference of khalas and that only 24 (9%) and 19 (7%) locations were heterozygous. at the same sites, their backcrossed males illustrated 736 and 770 different locations from the khalas reference, of which 584 (79%) and 578 (75%) were heterozygous, respectively. al-mssallem et al. (27) researched genetic variety of snps between many date tree species (khalas, fahal, sukry, agwa, deglet noor, deglet noor bc5, medjool, medjool bc4, khalsfx, alrijalf and khalt) and recognized snp date palm biotechnology an overview highlights in bioscience doi:10.36462/h.biosci.20182 august 2018 | volume1 page 4 of 5 (indel) abundances of 6.10 (0.25), 5.51 (0.15) and 6.24 (0.20) per kb. they explored how evolutionary selection left its marks in genomes of various date palm species by first assessing the allocation of snps on big scaffolds and then pinning down obviously chosen functional genes, particularly those strongly engaged in the metabolisms of sugar and energy. they found that there is no random distribution of snps, but a bimodal curve. depending on snps and the frequency distributions, they built the phylogenetic tree of 11 phoenix dactylifera cultivars. in nine saudi arabian date plant cultivars (sukkariat al-madinah perny al-riyadh, sukkariat qassim, rabia almadinah, dekhaini al-riyadh, ajwa al-madinah,, shalaby al-madinah, moshwaq al-riyadh and moshwaq hada alsham), sabir et al. (28) evaluated the entire maternal and plastid genome snps. they detected 188 snps in the mitochondrial genome at 37 distinct locations. for all nine cultivars, the amount of mutual snps was 16. while there were 30 of the nine date palm cultivars in the amount of plastid snps, they were situated in 20 distinct genome locations with 13 in genes, 2 in introns, and 5 in intergenic spacers. hazzouri et al. 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2013;4:1–9. 28. sabir jsm, arasappan d, bahieldin a, abo-aba s, bafeel s, zari ta, et al. whole mitochondrial and plastid genome snp analysis of nine date palm cultivars reveals plastid heteroplasmy and close phylogenetic relationships among cultivars. plos one. 2014;9(4): e94158. highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 1 of 5 citation: youssef a. a. and rslan w. m. (2018) sugar beet improvement using agrobacterium-mediated transf ormation technology. highlights in bioscience, volume 1.article id 20183, dio:10.36462/h.biosci.20183 received: june 1, 2018 accepted: july 17, 2018 published: july 30, 2018 copyright:© 2018 youssef and rslan. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. sugar beet improvement using agrobacteriummediated transformation technology abo-bakr a. youssef 1 and wessam m. rslan 2 * 1, 2 agricultural genetic engineering research institute; agricultural research center, egypt. *to whom correspondence should be addressed: wessam.rslan@ageri.sci.eg abstract since discovering agrobacterium tumefaciens distinctive capacity to incorporate a specified part of their transfer-dna (t-dna) into eukaryotic cells, the bacteria were commonly used for crop transformation originally of dicotyledonous crops and subsequently of nearly all organisms. to achieve this, the tumor-inducing (ti) plasmid was changed to extract phytohormone and opine biosynthetic proteins (cytokinin and auxin) so as not to interfere with ordinary morphological growth. overall, the conversion mediated by agrobacterium was easier, more effective and less costly relative to other technologies. it also results in insertions with small copy count. tumor development in crops has also proved the susceptibility of explants from field-grown sugar beet crops to agrobacterium tumefaciens. early efforts by agrobacterium tumefaciens to transform sugar beet were unsuccessful, primarily owing to inability to regenerate crops from stably modified callus or suspended cells. a genotype-independent method was defined under which cotyledonary explants of various sugar beet genotypes are inoculated with agrobacterium tumefaciens comprising whether kanamycin tolerance and gus activity or kanamycin resistance, gus activity and glyphosate tolerance. gus expression, npt dot blot as well as epsps assays verified the presence of transgenes; progeny showed mendelian genetically modified inheritance and glyphosate tolerance at deadly concentrations to control plants. unfortunately, there was no publication of technical information of the technique. here we reviewed the concept agrobacteriummediated transformation and how to be applicable keywords: agrobacterium tumefaciens, callus, suspended cells, sugar beet, kanamycin resistance. review article open access sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 2 of 5 introduction sugar beet is extremely prone to agrobacterium tumefaciens as an instance (1–5). though, genotypedependent susceptibility (1,3) can be enhanced by pre-culture explants before inoculation (6) or by extending the length of co-culture (1,3). krens et al. (4) used agrobacterium tumefaciens strains carrying an isolated cytokinine gene to enhance the development of sugar beet leaf discs, cotyledons and hypocotyls from two-week-old seedlings of nine distinct types. cotyledon and hypocotyl explants generated lowfrequency shoots; however, inoculated leaf discs did not regenerate the shoot. it was impossible to confirm a stable transformation. lindsey and gallois (1) revealed agrobacterium tumefaciens first effective regeneration of genetically modified sugar beet crops. transgenic seeds were acquired from co-cultivated shoot-base cells with agrobacterium strain lba4404 containing a bi-nary plant with a kanamycin resistance gene (nptii) and either chloramphenicol acetyl transferase (cat) or β-lucuronidase (gusa) genes. the frequency of transformation depended on the kinds of explants, genotypes and circumstances of choice. halluin et al. (2) who established crops resistant to broad-spectrum herbicides, glufosinate and sulfonylureas, as well revealed sugar beet transformation. friendly callus, originating from cotyledons, hypocotyls, petioles as well as true leaves of 2-to 3-month-old seedlings cultivated in dark, was carefully cut and inoculated with agrobacterium tumefaciens either a mutant acetolactate synthase (als) or a bialaphos resistance (bar) gene powered by multiple promoters, i.e. 35s camv, nos, tr1' or tr2.' this genotype-dependent protocol took nearly two years to get grown, which probably explains the morphologies of the aberrant plant. latest studies utilizing agrobacterium tumefaciens generated transgenic crops at frequencies that are adequate to produce crops used in experiments to evaluate herbicide tolerance and resistance to disease (7–12). for physiological and molecular research, agrobacterium tumefaciens produced transgenic crops were also used. an endogenous sugar beet gus (sb-gus) enzyme has been contrasted to e. coli in leaf disc transformation (13). variations in patterns of gene expression in roots have been investigated using distinct constructs (14) in another application. in another, development habit and accumulation of sugar were explored after transforming into a patatin gene promoter (15) with a bacterial cytokinin biosynthetic gene. although various explants of sugar beet, i.e., shoot bases, petioles, leaves and callus, were used, cotyledon explants were often more effective in transformation with efficiencies varying from 0.1 to 1.0 (4,7,9,10,16,17). with kanamycin selection (18), leaf lamina explants from shoots multiplied from apical meristems of four genotypes of sugar beet produced a 6.2 percent transformation level. small (1–3 mm) bud tips from various shoot clumps from undeveloped floral buds of five distinct genotypes resulted in hygromycin-resistant shooting rates of 13.3 to 30.6 percent. over 50% of crops from shoot-base tissue kanamycin-resistant explants produced the hpt gene (19). finally, a mixture of agrobacterium and void infiltration has been used to promote the transfer of bacterial dna to callus and crop tissue, resulting in more than 40% transgenic callus clones (20). factors influencing transformation efficiency genotype (19), explant origin (21), bacterial strain and incubation time (18) selection technique (22),type of promoter promoting selectable gene (22), light intensity (23), wound with pre-inoculation particle bombardment (10), use of acetosyringone (3), pre-treatment with drugs (6) and , preand co-cultivation interval (6). despite important advancement, the transformation of sugar beet to agrobacterium tumefaciens is still regarded to also be recalcitrant. because of the failure to absorb and integrate dna, it is impossible to be due to the small amount of morphologically competent regeneration cells. if they are integrated in big amounts of non-competent cells (24), connection to such competent cells may also be impeded. development of transgenic herbicide tolerance sugar beet competes badly with weeds, particularly in early growth, leading in dramatically elevated yield losses that can range from 50 percent to total losses (25) unless adequate weed control is attained. conventional control measures involve herbicide spraying at distinct moments and distances to decrease these losses, making weed control programs complex and challenging. the development of transgenic sugar beet resistant to broad-spectrum herbicides is therefore a significant alternative. herbicide tolerance is one of the first features that genetic engineering has effectively brought into several plant species; some types of herbicide tolerant (ht) have been on the market for further than a decade (26). ht crops have been produced using genes from microorganisms or higher plants that confer tolerance by: i changing the active site of the target protein in such a way that converted cells are less susceptible to herbicide; (ii) using an enzyme that transforms the core components of the herbicide into inactive compounds; or (iii) overproducing herbicide target proteins (27). agrobacterium-mediated transformation and in vitro cell selection have been used to evolve ht sugar beets sugar beet improvement using agrobacterium highlights in bioscience doi:10.36462/h.biosci.20183 july 2018 | volume page 3 of 5 tolerant either to non-selective, broad-spectrum herbicides, i.e., glyphosate, roundup ® active ingredient and glufosinate, basta ® active ingredient, liberty ® and herbiace ® main ingredient, or specific herbicides such as imidazolinone, chlorsulfurone and sulfonylurea (28). in plants, encoded in the nucleus, the enzyme 5enolpyruvylshikimate-3-phosphate synthase (epsps) performs a main part in chloroplast responses arising in synthesis of phenylalanine, aromatic amino acids, tyrosine, and tryptophan. after finding in the early 1970s that perhaps the analog amino acid glyphosate [n-(phosphonomethyl)-glycine] could preferentially inhibit epsps activity, shut down aromatic amino acid synthesis and ultimately kill the plant, strategies are established to produce glyphosate-tolerant plants (or roundup ready ®). epsps occurs only in crops and certain microorganisms, not in livestock or humans, probably explaining why glyphosate toxicity is exceptionally small in human cells (29). to create glyphosate-tolerant sugar beet, two methods listed above i and ii) have been utilized. in the first scenario, a gene agrobacterium sp. was used for an altered protein, cp4-epsps, which is extremely glyphosate tolerant (30); in vitro assays and in vivo herbicide sprays (16) verified glyphosate tolerance. the second approach used a gene encoding achromobacter sp. glyphosate oxidoreductase, gox, and catalyzing glyphosate degradation in and out of non-toxic compounds, glyoxylate and aminomethylphosphonic acid. however, sugar beet plants were transformed in the second approach with both cp4-epsps nd gox genes (9) and evaluated with distinct roundup ® spraying systems in the greenhouse and field. extremely tolerant transformants have been acquired without phytotoxic or any other harmful phenotypic effects (31). inversely linked with the transgenic copy number (9), herbicide tolerance emerged. glufosinate (ammonium salt l-phosphinothricin, ppt) and bialaphos (ppt plus two alanines; l-phosphinothricinyl-l-alanyl-lalanine) are extremely toxic to plant cells; they behave as competing glutamine synthetase inhibitors that are critical to the transformation of glutamine acid and ammonia onto glutamine. inhibition contributes to toxic ammonia accumulation, leading to death of the cell. enzymes encoded in bars and pat and extracted from various streptomyces sp., detoxify ppt and have been broadly used as selectable markers for the production of transgenic ht plants. bar, motivated by the proponents of camv 35s, nos, tr1' or tr2, was used to achieve glufosinate-tolerant sugar beet crops (2) assessed in the sector of gene flow and agricultural efficiency research (32). like glyphosate, glufosinate has a very low toxicity to mammals (29). joersbo (33) assessed both glyphosate-and glufosinatetolerant sugar beets environmental efficiency, including economics and utilization flexibility. by deactivating the first enzyme in the pathway, acetolactate synthase (als), sulfonylurea compounds prevent the biosynthesis of clustered amino acids, valine, leucine and isoleucine. sugar beet has been transformed with a mutant sulfonylurea-insensitive als gene (2). in vitro cell selection (34) was also acquired from mutant sulfonylurea-and imidazolin one-tolerant sugar beet crops. however, many ht transgenic beet varieties have been approved for discharge in the u.s. (1996, 1998, 2005), canada (2001, 2005) and japan (2007) (26). roundup ready sugar beets were grown for the first season in 2008 by sugar beet growers in michigan. for ten years, michigan state university researchers have been collaborating with these varieties to determine implementation rates and timing and policies to delay glyphosate-resistant weed growth. references 1. lindsey k, gallois p. transformation of sugarbeet (beta vulgaris) by agrobacterium tumefaciens. j exp bot. oxford university press; 1990;41(5):529–36. 2. halluin k, bossut m, bonne e, mazur b, leemans j, botterman j. transformation of sugarbeet (beta vulgaris l.) and evaluation of herbicide resistance in transgenic plants. bio/technology [internet]. 1992;10(3):309–14 3. jacq b, lesobre o, sangwan rs, sangwan-norreel bs. factors influencing t-dna transfer in agrobacteriummediated transformation of sugarbeet. plant cell rep. springer; 1993;12(11):621–4. 4. krens fa, verhoeven ha, van tunen aj, hall rd. the use of an automated cell tracking system to identify specific cell types competent for regeneration and transformation. vitr cell dev biol. springer; 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2003;12(3):293–304. 33. joersbo m. chimeric genes for selection systems and reporter makers. bentham science publishers: oak park; 2011. 34. ming r, vanburen r, wai cm, tang h, schatz mc, bowers je, et al. the pineapple genome and the evolution of cam photosynthesis. nat genet. 2015;47(12). highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202107 research article open access 1 department of pesticides and toxicology, university of gezira, wad medani, sudan. 2 blue nile natural institute for communicable diseases, university of gezira, wad medani, sudan. contacts of authors * to whom correspondence should be addressed: yousifassad12@gmail.com received: september 24, 2020 accepted: february 7, 2021 published: march 30, 2021 citation: adam eiy , assad yohm, eltohami mme, bashir nhh, abdelrahman sh, nour bym . natural anophelin mosquito repellents and phytochemical analysis of ethanol and hexane leaf extracts from four plant species. 2021 mar 30;4:bs202107 copyright: © 2021 adam et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. natural anophelin mosquito repellents and phytochemical analysis of ethanol and hexane leaf extracts from four plant species emad i. y. adam1 >< , yousif o.h.m. assad*1 >< , marwa m. e. eltohami1 >< , nabil h.h. bashir1 >< , samira h. abdelrahman2 ><, bakri y. m. nour2 ><  abstract currently mosquitoes control is the cornerstone to minimize the rising number of mosquito borne diseases. there is an urgent need looking for alternatives to the current reliance on synthetic insecticides for the vectors control. application of active toxic agents from plant extracts as an alternative control strategy was available from ancient times. these are non-toxic, easily available at affordable prices, biodegradable and show broadspectrum target-specific activities against different species of vectors. natural products with repellency properties are urgently needed. an insect repellent works by masking human scent, and a number of natural and synthetic mosquito repellents were studied. the main aim of this study was to identify the phytochemicals and compare their potentials as mosquito repellents from the ethanol (et. oh) and hexane (hex) leaf extracts (les) of ocimum basilicum, coleus forskohlii, eucalyptus camaldulensis and cymbopogon flexuosus under laboratory conditions at 50 and 100% concentrations. phytochemical analysis showed that alkaloids, saponins, flavonoids, terpenes, tannins and terpenoids were present/or absent in the 4 plant-les. at 50% concentration of et. oh extracts, c. forskohlii exhibited higher repellency potential on anopheles gambiae with protection time of 137.3 min, while o. basilicum, e. camaldulensis and cy. flexuosus registered protection time of 30.6, 15.3 and 19 min, respectively. at 100% concentration of ethanolextracts, c. forskohlii caused the highest protection time against the vector with protection time of 182 min. o. basilicum, e. camaldulensis and cy. flexuosus were equally less potent against the insect, with protection time 42.6, 32.6 and 28 min, respectively. regarding hex-extract, at 50% concentration of c. forskohlii, the highest repellency potential , with protection time of 174 was registered, while o. basilicum, e. camaldulensis and cy. flexuosus registered protection times of 44, 18 and 28.6 min, respectively. however, the 100% concentration of et. oh-extracts of, c. forskohlii and o. basilicum exhibited the highest protection time, with protection times of 228 and 116min, respectively. e. camaldulensis and cy. flexuosus were less potent, with protection times 28.6 and 54.6 min, respectively. the eos of c. forskohlii and o. basilicum l eos proved to have potentials as repellency agents against anopheles. gc-ms analysis of the extracts identified the compounds of both solvents extracts and the terpens were the major compounds. according to the results, further studies are required, especially on the extracts of basil and coleus since they proved to be effective. keywords: mosquito repellent, anopheles, essential oils, sweet basil, wild thyme, kafure, lemon grass introduction soonwera and phasomkusolsil [1] stated that mosquitoes are pestiferous insects to public health; transmit several deadly dangerous diseases, e.g. dengue (df), filariasis, malaria, yellow fever (yf) and japanese encephalitis (je). several researchers reported that every year at least 500 million people in the world suffer from one of the tropical diseases that include df, malaria and filariasis [2, 3]. highlights in bioscience page 1 of 10 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202107 https://creativecommons.org/licenses/by/4.0/ strongmass2020@gmail.com yousifassad12@gmail.com https://orcid.org/0000-0003-2928-3051 marwatoxic@gmail.com bashirnabilhh@gmail.com samhamid2002@yhoo.co.uk https://orcid.org/0000-0001-7714-9266 bakrinour@gmail.com https://orcid.org/0000-0002-2066-4176 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species salih et al., [4] reported mosquitoes are among the most disturbing blood -sucking insects afflicting human beings. the mosquito female's behavior, involving feeding on human blood, is responsible for the transmission of a number of diseases. one to two million deaths is reported annually, due to malaria worldwide. pest/vector management is facing economic and ecological challenges worldwide, due to human and environmental hazards caused by majority of the synthetic pesticides. identification of novel effective insecticidal compounds is essential to combat the increasing resistance rates. botanical pesticides have long been touted as attractive alternatives to synthetic pesticides for pest/vector management, because botanicals reputedly pose little threat to the environment or to human health. the body of scientific literature documenting bioactivity of plant derivatives to arthropod pests/vectors continues to expand, yet only a handful of botanicals are currently used in agriculture, public health, industrialized world, and there are few prospects for commercial development of new botanical products. the medical vectors control is considered the most important corner-stone to prevent the diseases; however, the control is either by the synthetic pesticides or the repellents that prevent the biting of the mosquitoes. personal repellents are substances applied to skin, clothing, or other surfaces to repel or discourage insects and other arthropods, e.g. ticks, from feeding on humans. repellents help people to avoid bites from mosquitoes, and other biting arthropods that may transmit diseases, and allow people to engage freely in outdoor activities. insect repellents may also be used to exclude insects from an area [5]. with increasing public concern regarding pesticide safety and problems of insecticide resistance, new safer active ingredients are becoming necessary to replace the existing compounds. furthermore, the use of repellents in an integrated pest/vector management (ipm/ivm) program has been ignored to a large extent [5]. salih et al., [4] reported that the personal protection from mosquito bites is currently the most important measures to control mosquito transmitted diseases. the mosquito repellent products commonly available on the market contain n, n-diethyl3-toluamide (deet) as the active ingredient. the synthetic repellents are not safe for human, especially children, because they may cause skin irritation, hot sensation rashes or allergy [6]. recently, as results of the public health concern on the safety of many synthetic products, many research scientists renewed the interest on the use of natural products from plant origin for mosquito's management [1]. in addition, plant essential oils (eos), in general, have been recognized as an important natural resource of insect repellents. the plant eos base repellents according to soonwera and phasomkusolsil [1] are effective for mosquito control, and are environment friendly. vector borne disease such as malaria is a major global health problem; half of the world's populations, mostly the poorest countries living in the tropical world's are at risk of malaria. the disease caused by plasmodium parasites transmitted by infected female anopheles mosquitoes bit is serious problem in africa, where one in every five (20%) childhood deaths is due to malaria. most malaria cases and deaths are in sub-saharan africa most malaria cases and deaths are in sub-saharan africa [7]. therefore, herbal insect repellents gained importance recently; detailed knowledge concerning them is still lacking. as a result, it is high time to launch extensive search to explore ecofriendly biological/botanical materials for controlling pests. curtis [8] choochote et al., [9] reported that eos have been investigated and described as potentially natural sources of insect repellents. the effective repellency against female anopheles results varied with different species of the plant and the concentration/dose. according to trongtokit et al., [10] study on an. stephensi in brazil using cymbopogon nardus citronellal 40% eos was applied topically, reported 100% protection for 7-8 hr. the present study investigated the potential of sweet basil (ocimum basilicum l), wild thyme (coleus forskohlii), kafure (eucalyptus camaldulensis) and, lemon grass (cymbopogon flexuosus), les as mosquitoes repellents. curtis [8] stated in many parts of the world, plant derived natural products have traditionally been used as repellents against insects bites. particularly essential oils have been investigated and described as potentially natural sources of insect repellents it is time to search for effective alternatives to synthetic repellents as reported by choochote et al., [9]. many research studies were conducted on various medicinal plants and the effective repellency against female anopheles mosquitoes, the result is varying according to the plant and the concentration. the present study investigates to figure out the effectiveness of ocimum basilicum l., coleus forskohlii, eucalyptus camaldulensis and, cymbopogon flexuosus steud, (leafs) extract as mosquitoes repellent. materials and methods collection of plant material and extraction fresh leaves of the four above-mentioned plants were collected from gezira state, sudan. the collected leaves were washed, shade-dried and powdered. the powder was extracted in ethanol (et. oh) and hexane (hex) by soaking in et. oh, and clevenger apparatus was used for preparation of hexextract; 50 g of each plant leaves powder (lp) was extracted separately in conical flasks by et. oh. analytical grade et. oh was added until the lp was fully submerged, thoroughly stirred to ensure proper mixing, and subjected to shaking at regular intervals to allow percolation for 4 days. on the 5th day, the extracts were filtered using whatman no.1 filter paper into another conical flask and the et. oh was evaporated. the resultant viscous substance was dried and stored in amber-colored vials in a refrigerator at 4oc pending formulation. for hex-extracts the eo was isolated by steam-distillation, using a clevenger apparatus for 6 hr; moisture in the oil was removed by anhydrous sodium sulfate and stored at 5oc for further use. highlights in bioscience page 2 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species formulation of plant leaves extracts the plant leaves extracts to be tested were formulated in pure fragrance-free vaseline jelly. the formulations tested were made into two concentrations of 50% and 100%. to make 50% of the formulation, 5g of the pure vaseline jelly was weighed and transferred to a clean 100 ml beaker, placed in a water bath at 80°c, stirred with a rod up to the melting point. five g of each of the 8 extracts was then added to the melted jelly, followed by continuous stirring to be completely mixed with the jelly. the resultant 8 formulations of eight plant extracts were stored at 4°c for the bioassay. to make 100% of the formulation, 1 g of pure vaseline jelly was melted as above; 10 g of each of the 8 extracts were added and treated as above. the two concentrations (50 and 100%) were used in bioassay. anopheline mosquito the anopheles gambiae (3-7 days old) was obtained from blue nile national institute for communicable disease (bnnicd) insectary, university of gezira, colony maintained at 12: 12 ld photoperiod, 70±10% r.h. and 24±2oc. the adult females of anopheles used for the bioassay before any blood meal. females were starved for 24 hr and provided with only 10% sugar solution in water. tests were conducted in triplicate 25 individual of starved adult mosquitoes rearing cage was used and topical treated arm with plant extract exposed to the mosquitoes in the cage to observe the mosquitoes bite. bioassay test the reared mosquitoes were taken from the insectary of blue nile institute for communicable disease (bnicd) (24±2cº and 80% rh) and the experiment was conducted in the insectary lab of bnicd, the arm cage method was used. three gram of formulated plant leaves extract was applied onto the forearm of a human subject. the treated arm was exposed until the first bite to 25 female mosquitoes, in a (252x3.14x60 cm3) cage, and any mosquitoes landing and biting were counted. after any bite the time was recorded at which the first bites occurred the bioassay terminate and repeated for the other 50 mosquitoes to make triplicate at successive time intervals. for the negative and positive control, the arm with vaseline jelly and with deet was used respectively. numbers of mosquitoes landing and biting in a minute were recorded. the application of plant extract was used following frances methods [11]. two concentrations (50 and 100%) for each plant, ethanol and hexane extract and mixture of ethanol and hexane for each plant to account the number of mosquitoes land and the complete protection time (cpt) in the treated area (20-15cm2), with 3g formulated plant extract. phytochemical analysis the phytochemical analysis was carried out at chemistry lab, faculty of agricultural sciences, university of gezira by using the method of balbaa [12] and harborne [13] to investigate the presence of the saponins, tannins, sterols, triterpens, flavonoids, terpenoids and alkaloids. gas chromatographymass spectrometry the qualitative and quantitative analysis of the sample was carried out by using gas chromatography-mass spectrometer (gc/ms-qp2010-ultra) from japans simadzu company, with the serial number 020525101565sa and capillary column (rtx-5ms30m[2e3?]0.25mn[2e3?]0.25μm).the sample was injected by using split mode, helium as a carrier gas passed with flow rate 1.61 ml/min, the temperature program was started from 60c with rate 10oc/min to 300oc as a final temperature degree with 5minutes hold time, the injection port temperature was 300oc as the ion source temperature was 200oc and the interface temperature was 250oc. the sample was analyzed by using scan mode in the range of m/z 40-500 charges to ratio and the total run time was 29 minutes. identification of the components for the sample was achieved by comparing their retention index and mass fragmentation patents with those available in the library, the national institute of the standards and technology (nist). data analysis for each plant species, the number of mosquitoes landed on the arm was counted at different time intervals and the percentage repellency (pr) per each plants leaf extract/solvent/concentration was determined using the following equation (1) according to teklani and perera [14] as follows: pr = niu − nit 25 (is ) 100 (1) where: • pr=percentage repellency. • niu=number of individual in the untreated group. • nit=number of individual in the treated group. • is=number of insects used. at least triplicates were carried out for all the plants leaves extracts for selected extracts concentration with significant mosquito repellent activity. results phytochemical analysis phytochemical analysis of ethanolic and hexane extracts of the four plants leaves were carried out to identify the presence of alkaloid, tannin, saponin, terpenoids, flavonoids and terpens. in this study four plant species (basil, wild thyme, kafure and lemon grass) belonging to different families were collected. most of these plants were reported to treat a variety of diseases in traditional medicine system. ethanol extracts the preliminary phytochemical screening had shown the presence of alkaloid, tannin, flavonoids, saponin and terpens in the leaves extracts the summary of the results are presented in table 1. the alkaloid, tannins, saponin and terpens were detected in the basil leaves extract; alkaloid, tannins, and terpens in wild thyme leaves extract; alkaloid, tannins, flavonoids and saponins in kafure leaf extract and alkaloid,tannins, and saponins in lemongrass, highlights in bioscience page 3 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 1. the phytochemical constituents (pc)of ethanol (e) and hexane (h) leaf extracts from the studied four plants. pc oc col eu cy e h e h e h e h alkaloid + + + + + + + tannins + + + + + + + flavonoids + + saponins + + + + + terpens + + + oc=ocimum, col=coleus, eu=eucalyptus, cy= cymbopogon. (+)= presence, (-) = absence hexane extract the preliminary phytochemical screening had shown the presence of alkaloid, tannin, flavonoids, saponin and terpens in the hexane leaves extracts the summary of the results are presented in table 1. in basil leaves extract alkaloid, tannins was only detected. while in wild thyme leaves extract terpens only was detected. in kafure leaves extract alkaloid, tannins, flavonoids and saponins were detected and terpens is absent. in lemongrass extract alkaloid, tannins, and saponins were detected. bioassay the results of bioassay were collected as a complete protection time (cpt), and the number of mosquitoes landing for the concentrations (50 and 100%), ethanol, hexane and hexane plus ethanol extract. ethanol extracts presented in tables 2 and 3 shows that wild thyme 50% and 100% were recorded the highest protection time 137.3 and 182 min respectively compare to the other plant extracts, with percent repellency 92 and 89% respectively. for the hexane extracts presented in tables 4 and 5 prove that thyme 50% and 100% were recorded the highest protection time 124 and 228 min respectively compare to the other plant extracts, with percent repellency 90 and 92% respectively. regarding the combination of the two extracts presented in tables 6 and 7 also the wild thyme 50% and 100% were recorded the highest protection time 182 and 215 min respectively compare to the other plant extracts except basil in 50% recorded higher protection time (86 min), with percent repellency 84 and 88% respectively. by the way the selected four leaf plant extracts offers protection against an. arabiensis adult female mosquitoes but varied in terms of repellency up to 228 minutes of exposure periods recorded by wild thyme. chemical constituents detected in kafure leaf extracts table 8 shows the chemical constituent of kafure leaves ethanol and hexane extracts; their concentrations, peak area, retention time and chemical group of each compound. a total of 13 compounds detected in ethanol and hexane extract which represent 63.63% and 68.54% for ethanol and hexane extract, respectively. eucalyptol was the main compounds (33.11% and 26.93%), respectively. table 9 shows the chemical constituent of kafure leaf's ethanol extract; their concentrations, peak area, retention time and chemical group of each compound. a total of 20 compounds detected in ethanol extract and represent 36.37% of the total of the extract. globulol 9.94% and squalene 8.10% were the main compounds. table 10 shows the chemical constituent detected in kafure leaf's hexane extract; their concentrations, peak area, retention time and chemical group of each compound. a total of 25 compounds detected in hexane extract, as follows, where globulol was the main compound (18.06%). discussion the synthetic chemicals used to control insects leads to several documented environment and human health hazards. an alternatives method suggested using natural products that possess good efficacious results and environmental friendly. among those alternative methods the use of essential oils from plants belonging to several plant species subjected intensive studies which provide successful results against field crop pests and public health pests and disease vectors control, including mosquitoes (anopheles, culex and aedes). these eos were extensively tested by several scientists for their repellent properties as valuable natural resources and to elucidate their chemical and physical properties, in addition to their modes and sites of action, formulation, toxicological profiles, side-effects, etc... today medical vector management has to face environmental and economic costs. table 2. spatial repellent activity of 50% and 100% evaluated plants leaves ethanol extract against anopheles mosquito based on the number of landed mosquitoes (nlm) and protection time per minute (pt). treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 1 2 1 1.3 113 122 177 137.3 2 3 1 2 210 154 182 182 sweet basil 2 3 1 2 25 36 31 30.6 4 2 6 4 42 31 45 42.6 kafure 4 2 1 2.3 15 10 21 15.3 2 3 4 3 25 32 41 32.6 lemongrass 2 3 5 3.3 15 17 25 19 4 2 3 3 31 25 28 28 table 3. percent repellency (pr) of 50% and 100% of evaluated plants leaves ethanol extract against anopheles mosquito based on the number of landed mosquitoes (nlm). treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 68% 8 9 5 7.33 68% wild thyme 1 2 1 1.3 92% 2 3 1 2 89% sweet basil 2 3 1 2 89% 4 2 6 4 81% kafure 4 2 1 2.3 88% 2 3 4 3 85% lemongrass 2 3 5 3.3 84% 4 2 3 3 85% highlights in bioscience page 4 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 4. spatial repellent activity of 50% and 100% evaluated plants leaves hexane extract against anopheles mosquito based on the number of landed mosquitoes (nlm) and protection time per minute (pt). treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 1 2 2 1.6 191 142 189 174 1 2 1 1.3 230 208 246 228 sweet basil 3 2 5 3.3 46 36 50 44 2 1 3 2 90 118 140 116 kafure 4 6 8 6 18 21 15 18 3 1 2 2 36 21 28 28.3 lemongrass 3 4 6 4.3 22 36 28 28.6 2 3 5 3.3 59 41 63 54.3 table 5. percent repellency according to the number of landed mosquitoes (nlm) of 50% and 100% percent of evaluated plants leaves hexane extract against anopheles mosquito. treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean untreatedcontrol 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 68% 8 9 5 7.33 68% wildthyme 1 2 2 1.6 90% 1 2 1 1.3 92% sweetbasil 3 2 5 3.3 84% 2 1 3 2 89% kafure 4 6 8 6 73% 3 1 2 2 89% lemongrass 3 4 6 4.3 80% 2 3 5 3.3 84% table 6. spatial repellent activity and protection time per minute (pt)of 50 and 100% of evaluated plants leaves hexane and ethanol extract mixture against anopheles mosquito. treatment 50% 100% nlm pt nlm pt 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean 1st 2nd 3rd mean control 25 24 24 24.33 25 24 24 24.33 deet 8 9 5 7.33 72 91 121 94.66 8 9 5 7.33 72 91 121 94.66 wild thyme 2 3 5 3.3 164 194 190 182.6 4 2 1 2.3 211 233 201 215 sweet basil 4 1 3 2.6 48 51 31 43.3 2 6 4 4 66 94 101 87 kafure 4 6 7 5.6 17 11 19 15.6 6 5 8 6.3 20 15 23 19.3 lemongrass 6 3 4 4.3 15 24 19 19.3 1 2 4 3.3 27 20 31 26 table 7. percent repellency (pr) according to the number of landed mosquitoes (nlm) of 50% and 100% of evaluated plant leaves hexane and ethanol extracts mixture against anopheles mosquito. treatment 50% 100% nlm pr nlm pr 1st 2nd 3rd mean 1st 2nd 3rd mean deet 8 9 5 7.33 68% 8 9 5 7.33 68% wild thyme 2 3 5 3.3 84% 4 2 1 2.3 88% sweet basil 4 1 3 2.6 86% 2 6 4 4 81% kafure 4 6 7 5.6 74% 6 5 8 6.3 72% lemongrass 6 3 4 4.3 80% 1 2 4 3.3 84% the synthetic repellents deet and pyrethroids containing formulations commonly sold in markets and insecticides sales vender. the side effects of these two chemical groups was reported by several research scientists, accordingly plant-based repellents not only minimize the use of synthetic chemicals and economic cost and save huge amount of money spent on synthetic compounds, also protects the environment, health of users. the plants screened for phytochemical analysis seemed to have the potential to act as a source of useful chemicals and also to improve and help in the health status of the people exposed to mosquito's bits (table 1). this study justifies the use of plant species in medicinal field to protects human from mosquito's bits and hence minimize the malaria spreading. there are different plants which have mosquito repellent activity and this activity affected with species the environment and other factors. and the chemical constituent of these show different results on the mosquito repellent. the different plant extract contains different chemical constituent shows different results on the mosquito repellent activities (tables 2-7). this study investigated the mosquito spatial repellent effihighlights in bioscience page 5 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species cacy of the basil, wild thyme, kafure and lemongrass, (ethanol, hexane and mixture of both ethanol extract and hexane extract). the present study results provided information and observations on the repellency activity of tested four plant species les against anopheles mosquitoes. mosquitoes usually make surreptitious landings on exposed skin to feed this why most people wearing protective clothing stands as a good measure of preventing them from vector contact. application of these plants les prevented landing attempts and bites by mosquitoes; this verifies already documented evidence that some plants le is a potent mosquito repellent. sweet basil o. basilicum eos are materials that are traditionally used in sudan the mosquitoes by burning leafs and flowers. however, coker et al., [15] stated that certain characteristics, e.g. volatility, limited its effectiveness. active ingredients, e.g. terpenes (2◦ metabolites) and oil components are responsible for the repellency activity. for a plants material to be valuable as a mosquito repellent it must effectively prevent insect bites on the treated area for several hours, and it must work in different environmental conductions, should be environmentalfriendly when applied to human skin, it must acceptable and having a pleasant odor, it should also be harmless to clothing, it should have a relatively low cost and effective against other biting insects such as flies. basil leaves extract provide different spatial repellency against mosquitoes with different concentration, in ethanol, hexane and mixture of ethanol and hexane extract (tables 2, 4 and 6). ethanol extract provided 30.6 and 42.6 min 32 and 45% for 50% and 100% ethanol extract respectively and the hexane extract was provided 44 and 116 min and evaluated with 46% and 122% for 50% and 100% respectively when compared with deet, while the mixture of ethanol and hexane extract shows that basil was provides the protection time in both (50% and 100%) was 43 min and 87 min respectively and this repellency act as 45% and 91.9% when compared with standard mosquito repellent deet (94.6 min protection time) respectively (tables 3, 5 and 7). basil hexane leaves extract provide the highest repellency against mosquitoes (116 min), baba et al., [16] conclude that hexane basil leaves extract provided 183 min and 303 min cpt from concentration 50% and 100% respectively (tables 4 and 6). in this study; the presence of ethanol extract in the mixture was reduced the repellency of mixture from the 116 to 87 min in 100% concentration; so the tannin and alkaloids may responsible for the repellent activity in hexane extract according to the phytochemical analysis (table 1). the phytochemical analysis was preceded an alkaloids, tannins, saponins and terpens were found in the ethanol extract, while alkaloid and tannins only detected in hexane extract.. wild thyme ethanol extract, the wild thyme was provides the highest protection time in both (50 and 100) %137.3 min and 182 min respectively when compared with other plant ethanol extract, and repellency at 154% and 192%, respectively when compared with standard mosquito repellent-deet (94.6 min protection time). active movement away from treated arm within minute of application was observed in wild thyme, and this strong repellent activity may caused by terpens group which appear in ethanol and hexane extract (according to phytochemical analysis) as main compounds and thymol (monoterpenoid) was highest concentration (54.96% and 57.42%) in ethanol and hexane extract respectively according to gc-ms analysis, and this was confirm by the study done by pandey et al., [17] they prove the activity of thymol that repel an. stephensi adults at the dose of 25.0 mg/mat after 1 h duration, while same degree of repellency was obtained by the oil at the dose of 55.0 mg/mat, which indicating thymol has double activity than the essential oil obtained from seeds of trachyspermum ammi (linn.). in present study beside the alkaloids and tannins present in ethanol extract only (phytochemical analysis), the hexane extract of the wild thyme is provides the highest protection time in both (50 and 100) % 174 min and 228 min respectively when compared with other plant leaves extracts and this highest cpt and repellency as 183% and 241% respectively, when compared with standard mosquito repellentdeet (94.6 min cpt). regarding the mixture of ethanol and hexane extract of each of the tested plants, wild thyme is provides the highest protection time in both (50 and 100%) 182.3 min and 215 min with spatial repellency at 193% and 227% respectively, compared to deet. the main compounds detected in ethanol and hexane extract was thymol which shows considerable anti-feedant activities against insects in many studies conducted by gonzalo et al., [18] and won et al., [19] shows that the wild thyme essential oil was rich source monoterpenes, including thymol, p-cymene, carvacrol, linalool, and o-terpinene, were assessed to determine their repellent activities to the mosquitoes. o-terpinene had a potent spatial repellent activity with a protection rate of essential oil topical treatment while the carvacrol and thymol showed an equivalent level of repellency. the wild thyme was the best one compared with the rest plants it was provided highest cpt; in both ethanol and hexane extract. however, the hexane 100% was provided the protection until 228 min, this period even longer than deet (2.4 times). according to phytochemical analysis the spatial repellency of wild thyme could be rise from terpens mainly because the hexane extract shows the terpens only. although provided the highest cpt and the ethanol extract which contain terpens beside the alkaloids and tannins show low cpt. our study show that thymol and eucalyptol (monoterpenoid) was main compounds in either ethanol and hexane extract and its concentration was corresponding with spatial repellency period, the repellency of plant extract could be due to the eucalyptol and this was proved by james et al., [20] they found that mosquito feeding and ovipositional repellency due to the presence of major monoterpenoid in the chemical volatile oil (i.e., 1,8-cineole (eucalyptol) identified in plant extract of hemizonia fitchii (asteraceae). phytochemicals are the principal active components that are highlights in bioscience page 6 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 8. chemical constituent detected appear in the gc-mass chromatogram in both ethanol and hexane extract of kafure leaf’s and area percentage of peaks. id name area% ethanol area% hexane 1. alpha-pinene 3.38 6.77 2. d-limonene 0.19 0.55 3. eucalyptol 33.11 26.93 4. gamma.-terpinene 0.30 0.47 5. pinocarvone 1.06 1.94 6. 2-oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl-, acetate 0.25 0.37 7. bicyclo(3.1.1)heptane-2,3-diol, 2,6,6-trimethyl0.18 0.24 8. copaene 0.19 0.42 9. alloaromadendrene 0.39 0.26 10. caryophyllene 0.76 0.92 11. aromandendrene 7.02 7.07 12. alpha.-guaiene 1.60 2.37 13. 2-naphthalenemethanol, decahydro-.alpha.,.alpha.,4a-trimethyl-8-methylene-, [2r-(2.alpha.,4a.alpha.,8a.beta.)] 15.20 20.23 table 9. chemical constituent detected appear in the gc-mass chromatogram in ethanol extract of kafure leaves and area percentage of peaks. id name peak area(%) 1. alpha-phellandrene 0.79 2. p-cymene 1.78 3. 3-methyl-4-cyclohexene-1,2-dicarboxylic anhydride 0.20 4. benzene, 1-methyl-4-(1-methylethenyl)0.67 5. isoledene 0.11 6. 1-tridecene 0.12 7. bicyclo[2.2.2]octa-2,5-diene, 1,2,3,6-tetramethyl0.15 8. cyclohexane, 1-ethenyl-1-methyl-2,4-bis(1-methylethenyl)-, [1s-(1.alpha.,2.beta.,4.beta.)]0.04 9. naphthalene, decahydro-4a-methyl-1-methylene-7-(1-methylethenyl)-, [4ar-(4a.alpha.,7.alpha.,8a.beta.)]0.18 10. 1h-cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1ar-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.)0.21 11. (s)-(-)-4-isopropenyl-1-cyclohexene-1-carboxylic acid 1.98 12. azulene, 1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-(1-methylethenyl)-, [1r-(1.alpha.,3a.beta.,4.alpha.,7.beta.)]1.42 13. guaia-1(10),11-diene 1.37 14. 7-epi-cis-sesquisabinene hydrate 0.79 15. globulol 9.94 16. phytol, acetate 3.03 17. 3,7,11,15-tetramethyl-2-hexadecen-1-ol 0.55 18. 9-eicosyne 1.04 19. n-hexadecanoic acid 3.90 20. squalene 8.10 believed to exhibit the medicinal activity of the plants and possibly the repellent activity of the oils. according to banthorpe [21] and heinrich et al., [22] terpenoids could be responsible for the flavor of fruits, the fragrance of the flowers and the quality of agricultural products. therefore coker et al., [15] prove that the presence of terpens is speculated to be associated with fragrance material and spatial repellent activity of oils. kafure kafure leaves extract provided different cpt by (50% and 100%) concentration, in ethanol; hexane and ethanol and hexane extract (mixture) (tables 2, 4 and 6). ethanol extract provide 15 min and 32 min by 50% and 100% concentration respectively. however the hexane extract provide 18 min and 28 min complete protection by 50% and 100% concentration respectively when the mixture provided 15 min and 19 min complete protection by 50% and 100% concentration respectively. the result shows that hexane extract was slightly stronger repellent than the ethanol and the mixture; the phytochemical analysis shows the presence of the: saponins, tannins, and alkaloids and absence of triterpens in both ethanol and hexane extract; while the gc-ms detected some of monoterpenes like euclyptol, (cyclic) 33.11% and 26.93% and 2-naphthalenemethanol, decahydro-alpha, alpha, 4a-trimethyl-8-methylene-, [2r-(2alpha, 4aalpha, 8abeta )]15.20% and 20.23%, in ethanol and hexane extract respectively, and globulol was 9.94% in ethanol extract and hexane extract was contained (-)-globulol 18.06% so this variety may responhighlights in bioscience page 7 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species table 10. chemical constituents detected appear in the gc-mass chromatogram in hexane extract of kafure leaves and area percentage of peaks. id name peak area(%) 1. camphene 0.14 2. bicyclo[3.1.0]hex-2-ene, 4-methylene-1-(1-methylethyl)0.07 3. beta-pinene 0.22 4. beta-myrcene 0.06 5. o-cymene 3.58 6. doconexent 0.20 7. butanoic acid, 3-methyl-, 3-methylbutyl ester 0.28 8. alpha-campholenal 0.14 9. terpinen-4-ol 1.58 10. l-.alpha.-terpineol 1.67 11. ethanone, 1-(6,6-dimethylbicyclo[3.1.0]hex-2-en-2-yl)0.19 12. 2-cyclohexen-1-one, 3-methyl-6-(1-methylethyl)0.08 13. citral 0.15 14. 1h-cyclopropa[a]naphthalene, 1a,2,3,5,6,7,7a,7b-octahydro-1,1,7,7a-tetramethyl-, [1ar-(1a.alpha.,7.alpha.,7a.alpha.,7b.alpha.) 0.20 15. 12-oxatetracyclo[4.3.1.1(2,5).1(4,10)]dodecane, 11-isopropylidene0.17 16. naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-4a,8-dimethyl-2-(1-methylethenyl)-, [2r-(2.alpha.,4a.alpha.,8a.beta.)]1.38 17. beta-humulene 0.31 18. 2h-3,9a-methano-1-benzoxepin, octahydro-2,2,5a,9-tetramethyl-, [3r-(3.alpha.,5a.alpha.,9.alpha.,9a.alpha.)]0.22 19. gamma-muurolene 0.21 20. aromadendrene, dehydro0.20 21. 2-oxabicyclo[2.2.2]octan-6-ol, 1,3,3-trimethyl0.26 22. 1h-cycloprop[e]azulen-4-ol, decahydro-1,1,4,7-tetramethyl-, [1ar-(1a.alpha.,4.beta.,4a.beta.,7.alpha.,7a.beta.,7b.alpha.)]1.32 23. (-)-globulol 18.06 24. caryophyllene 0.47 25. 1h-benzocycloheptene, 2,4a,5,6,7,8,9,9a-octahydro-3,5,5-trimethyl-9-methylene-, (4as-cis)0.27 sible of different repellency and the 32 min was highest cpt achieved by kafure ethanol extract. in the study conducted by fathelrahman [23] showed ethanol extract 10% concentration provide 17% repellency for 60min. the result was corresponding with this study that increasing extract concentration of kafure was increase the repellency in both extract and the kafure extract, although was weak in spatial repellency. according to the result of gc-ms analysis of the chemical compound of kafure leaves extract of the current study (tables 8, 9 and 10); the results were corresponding with the gc-ms analysis of essential oil yield and composition in eucalyptus species in montenegro coastline by slavenko, et al; [24] show the presence of α-pinene, β-pinene, β-myrcene, α-phellandrene, α-terpinene, p-cymene, eucalyptol γ-terpinene, terpinene-4-ol, α-terpineol, aromadendrene, alloaromadendrene, also was found in this study. according to the gc-ms analysis of the present study prove the presence of o-cymene 3.58%, terpinen-4-ol 1.58%, l-.alpha.terpineol1.67%, naphthalene, 1, 2, 3, 4, 4a, 5, 6, 8a-octahydro4a, 8-dimethyl-2-(1-methylethenyl), l-alpha -terpineol 1.67% [2r-(2.alpha.,4a.alpha.,8a.beta.)]-1.38%, 1h-cycloprop[e] azulen-4-ol,decahydro-1,1,4,7-tetramethyl,[1a r (1a alpha, 4beta, 4a beta, 7alpha, 7a beta, 7b alpha)]-1.32%, (-)-globulol 18.06%, 2-naphthalenemethanol, decahydro -alpha, alpha, 4a-trimethyl -8-methylene-,[2r-(2alpha,4a alpha, 8a beta)]20.23%, was found in the hexane leaves extract and not found in ethanol extract, and alpha-pinene was 6.77% in hexane extract and 3.38% in ethanol extract. lemongrass application of cymbopogon oil prevented landing attempts and bites by mosquitoes; and this verifies already documented evidence by baldacchino et al., [25] and prabhakar et al., [26] that cy. citrates extract is a potent mosquito repellent. however, the interval of protection is a matter of concern; in this study, lemon grass leaf extracts provided protection from mosquito for 3 hours after which landing attempts and bites were experienced. this observation corroborates previous records of plants essential oils by bhupen et al., [27]. according to bhupen et al., [27] has demonstrated the essential oils from a variety of plant species been used as insect repellents; such as cymbopogon. in the present study lemongrass leaves extract provided different cpt with different concentration (50% and 100%) in ethanol, hexane and ethanol and hexane extract (mixture). ethanol extract provided 19 min and 28 min by 50% and 100% concentration respectively (tables 2 and 4). however the hexane extract provide 28 min and 54 min complete protection by 50% and 100% concentration respectively (table 6). while the mixture provided 19 min and 26 min complete protection by 50% and 100% concentration respectively. highlights in bioscience page 8 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species the 54 min was maximum cpt provided by lemon grass (hexane extract) with spatial repellency is equal 57% compared with deet, and also was low when compared with wild thyme and basil extract. in phytochemical studies both ethanol and hexane extract was showed the alkaloid, tannins and saponins while the terpens and flavonoids were absent and those groups were important as insect repellent. although monoterpenes was detected by the gc-ms as main compounds as 2,6-octadienal, 3,7-dimethyl, (z)-in ethanol extract and 2,6-octadienal, 3,7-dimethyl-, (z)and 2,6-octadienal, 3,7-dimethyl-, (e)in hexane extract. in the study conducted by vinutha et al., (28) on essential oil yield from aerial part of cymbopogon flexuosus; myrcenol, linalool, trans-chrysanthemal, 3,6,6-trimethyl -cyclohex-2-enol, citronellal, (-)-isopinocampheol, 1-pentanol, 5-cyclopropylidene-3undecyne, 3-carvomenthenone, (z)-linalool oxide, (furanoid), neral, geranial, β-vatirenene, citronellol, dextro-carvone, cycloisolongifolene, trans-(-)-carveol, cis-carveol, nerol, oxiranmethanol, 3-methyl-3(4-mathyl-3-pentenyl), bicyclopentylone, geranic acid, geranyl acetate, most of those compounds were detected in lemongrass extract in this study. according to the result, undiluted hexane leaves extract ( 100% ) provide maximum cpt according to the follows: 228 min wild thyme, 116 min basil, 54 min lemongrass and 28 min kafure, thus the hexane extract was seem more stronger in repellency than the ethanolic extracts, and the coleus is the best and effective as mosquitoes repellent than other plants, followed by basil and lemon grass and kafure. the results of the current work indicated that the spatial repellent potential of the leaves extract oils is concentration dependent. however it is observed that all the thyme leaves extract oils indicated protection of at least 2.5 h at 50% concentration and 2.5-5 h protection at 100%. accordingly the use of plants leaves extract essential oil in prevent mosquito bite is expected to reduce the cost and environmental effects of mosquito repellents. according to annamaria et al., [29] showed volatile compounds detected in bixa orellana samples the major volatile compound present in seed extracts proved to be source of repellent activity (mainly of sesquiterpenes, monoterpenes, arenes and as α-humulene). these volatile compound found in most of our chemical analysis to the four plants which reflects the repellent activity of the test extract especially thyme. in conclusion the preliminary findings of this laboratory evaluation bioassay of the repellent potential of the essential oils of ocimum basilicum l., coleus forskohlii, eucalyptus camaldulensis and, cymbopogon flexuosus steud, leaves extracts have confirmed their traditional use as a broad mosquito repellent agent. further investigations leading to identification of the lead compounds exhibiting the repellent activity is recommended. this would enhance a robust development of plant based oil for protection against mosquito bite. basil provided considerable repellency against mosquitoes. wild thyme was most effective which provided 105 min cpt evaluated with 205% in compare to the control (deet, lavender). the examined plants will be useful if the further studies have been conducted and it will replace the commercial mosquitoes repellent (deet, lavender). terpenoids were found in most of the extracts, but in the c. forskohli with the highest concentration. the presence of the (+)-4-carene, benzene, 1methyl-3-(1-methylethyl)-, 2-naphthalenemethanol, decahydrowere related with hexextracts of c. forskohli only, and it has strong repellency activity. acknowledgements we appreciate the assistance from blue nile institute for communicable diseases for providing the mosquitoes for the studies. we thank ministry of higher education and scientific researchcommission of scientific research and innovation in supporting the research fund and thanks to all colleagues helping in a way or another to let this work done. references 1. soonwera m, phasomkusolsil s. mosquito repellent from thai essential oils against dengue fever mosquito (aedes aegypti (l.)) and filarial mosquito vector (culex quinquefasciatus(say)). african journal of microbiology research. 2014; vol. 8(17), pp. 18191824. 2. madhumathy ap, aivazi aa, vijayan va. larvicidal efficacy of capsicum annum against anopheles stephensi and culex quinquefasciatus. j vector borne dis. 2007; 44: 223-226. 3. kumar sv, mani p, john btmm, ravi kg. mosquito larvicidal, oviposition deterrent and repellent properties of acalypha indica l. extracts against aedes aegypti, anopheles stephensi and culex quinquefasciatus. international journal medicine bioscience. 2012; no 1: 33-41 4. salih aoa, salih mm, ali mm. bioassay of herbal mosquito repellent formulated from the essential oil of ocimum basilicum. american journal of applied chemistry. 2018; vol. 6, no. 4, pp. 132-141. 5. peterson c, coats j. insect repellentspast, present and future, review the history of insect repellents from the 1920s down to recent work on plant-derived repellents. pesticide outlook .2001; 154-158pp. 6. das ng, baruah i, talukdar pk, das sc. evaluation of botanicals as repellents against mosquitoes. journal of vector borne disease. 2003;40: 49-53. 7. pronczuk j, bruné mn, gore f. children's environmental health in developing countries. in encyclopedia of environmental health. jerome o. nriagu, ed. 2011, pp. 601–610. 8. curtis cf. personal protection methods against vectors of disease. rev med vet entomol, 1992; 80: 543-553. 9. choochote w, chaithong u, kamsuk k, jitpakdi a, tippawangkosal p, tuetun b, champakaew d, pitasawat b. repellent activity of selected essential oils against, aedes aegypti. fitoterapia. fitoterapia. 2007; 78:359-364. 10. trongtokit y, curtis cf, rongsriyam y. efficacy of repellent products against caged and free flying anopheles stephensi mosquitoes. southeast asian j trop. med public health, 2005; 36:14231431. highlights in bioscience page 9 of 10 march 2021|volume 4 http://bioscience.highlightsin.org/ adam et al., 2021 natural anophelin mosquito repellent analysis of leaf extracts of four plant species 11. frances sp, klein ta, hildebrandt dw, burge r, noigamol c, eikarat n, sripongsai b, wirtz ra. laboratory and field evaluation of deet, cic-4 and ai3-3722o against anopheles dirus (diptera: culicidae) in thailand. j med entomol. 1996; 33:511515. 12. balbaa ay, zaki ay, shamy am. qualitative and quantitative study of the flavonoid content of the different organs of sophora japonica at different stages of development. planta med. 1974; 25: 325–330. 13. harborne aj. phytochemical methods: a guide to modern techniques of plant analysis. 3rd ed. springer netherlands; 1998. 14. teklani pwnn, perera bgk. mosquito repellent and larvicidal activities of acalypha indica leaf extracts. int. j pharm. pharmacol. 2017; 1(2): 107. 15. coker hb, chukwuanim cm, ifudu nd, aina ba. the malaria scourge, concepts in disease management. nig. j. pharm. 2000; 32: 19-47. 16. baba g, lawal ao, shariff hb. mosquito repellent activity and phytochemical characterization of essential oils from striga hermonthica, hyptis spicigera and ocimum basilicum leaf extracts. british journal of pharmacology and toxicology. 2012 apr 25; 3(2) :43-8.. 17. pandey sk, shikha u, tripathi ak. insecticidal and repellent activities of thymol from the essential oil of trachyspermum ammi (linn) sprague seeds against anopheles stephensi. parasitol res. 2009; 105(2):507-512. 18. gonzalo ortiz de elguea-culebras; raúl sánchez-vioque; maría isabel berruga; david herraiz-peñalver and omar santana-méridas. antifeedant effects of common terpenes from mediterranean aromatic plants on leptinotarsa decemlineata. journal of soil science and plant nutrition. 2017; 17 (2), 475-485. 19. won-sik c, byeoung-soo p, sae-kwang k, sung-eun l. repellent activities of essential oils and monoterpenes against culex pipiens pallens, journal of the american m osquito control association. 2002;vol.18, no.(4):348-351. 20. james ak, mark vd, manuel fb. 1,8-cineole (eucalyptol), a mosquito feeding and ovipositional repellent from volatile oil of hemizonia fitchii (asteraceae). journal of chemical ecology. 1987(13), pp:2131–2141. 21. banthorpe dv. classification of terpenoids and general procedures for their characterization. in: charlwood, b.v. and d.v. banthorpe, (eds.), methods in plant biochemistry. terpenoids. academic, london, 1991; 7: 1-41. 22. heinrich m, pieroni a, bremner p. "medicinal plants and phytomedicines" in: the cultural history of plants. taylor and francis, new york, routledge, 2005;pp: 205-238. 23. fathelrahman ie. effect of organic extracts of cafure leaves (eucalyptus camaldulensis dehn.) on mosquitoes (anopheles arabiensis patton.)gezira journal of agricultural science, 2011; vol., 9 no2. 24. slavenko g, dejan o, maria c, emilija j, dušan b, kristina b, neda md. variation of essential oil composition of eucalyptus camaldulensis (myrtaceae) from the montengero coastline, biblid: 1450-7188 (2010) 41, 151-158. 25. baldacchino f, tramut c, salem a, lienard e, deletre e, franc m, martin t, duvallet g, jay-robert p. the repellency of lemongrass oil against stable flies tested using video tracking, parasite, 2013; 20:21. 26. prabhakar k, kinnera k, priya kk, peele ka. investigation of the repellence activity of bio-out, a natural mosquito repellent. international journal of life sciences, biotechnology and pharma research. 2013; 2:3. 27. bhupen k, somi b, sharma ak. plant essential oils as mosquito repellent: a review. international journal of research and development in pharmacy and life sciences, 2013; 3(1): 741-747. 28. vinutha m, suchetha m. and k. j. thara saraswathi. effect of different a biotic stress on essential oil yield from aerial part of cymbopogon flexuosus (nees ex steud) wats. int. j. adv. res. 2016; 4(12), 1490-1504. 29. annamaria g, pietro de m, giuseppe g, luca mc, sara p. secondary metabolite profile, antioxidant capacity, and mosquito repellent activity of bixa orellana from brazilian amazon region, journal of chemistry volume 2013; /article id 409826/ 10 pages https://doi.org/10.1155/2013/409826. highlights in bioscience page 10 of 10 march 2021|volume 4 https://doi.org/10.1155/2013/409826 http://bioscience.highlightsin.org/ abstract introduction materials and methods collection of plant material and extraction formulation of plant leaves extracts anopheline mosquito bioassay test phytochemical analysis gas chromatography–mass spectrometry data analysis results phytochemical analysis ethanol extracts hexane extract bioassay chemical constituents detected in kafure leaf extracts discussion sweet basil wild thyme kafure lemongrass acknowledgements references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20211 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 9 research article open access 1 uzhgorod national university, st. voloshina, 54, uzhhorod, 88000, ukraine. contacts of authors * to whom correspondence should be addressed: alexander shuaibov citation: shuaibov a, minya a, malinina a, malinin a, gomoki z (2020). synthesis of aluminum oxide nanoparticles in overstressed nanosecond discharge plasma with the ectonic sputtering mechanism of aluminum electrodes. highlights in bioscience volume 3. article id 20211. dio:10.36462/h.biosci.20211 received: may 20, 2020 accepted: july 18, 2020 published: july 27, 2020 copyright: © 2020 shuaibov et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. synthesis of aluminum oxide nanoparticles in overstressed nanosecond discharge plasma with the ectonic sputtering mechanism of aluminum electrodes alexander shuaibov 1 *, alexander minya 1 , antonina malinina 1 , alexander malinin 1 and zoltan gomoki 1 abstract the results of studying the conditions of synthesis and luminescence of aluminum oxide nanoparticles in a plasma of an overstressed nanosecond discharge ignited between aluminum electrodes at an interelectrode distance of 2 mm and air pressure in the range of 50202 kpa are presented. it was shown that the plasma of the investigated discharge is characterized by a wide luminescence band in the spectral range of 300–430 nm, which is associated with the formation of f and f + centers. the research results can be used in micro-nanotechnology, biomedical engineering to obtain nanostructured alumina substrates, on which other nanodevices and films from biomaterials can be placed. the aim of the work was to establish the possibility of detecting small nanoparticles aluminum oxide nuclei by emission spectroscopy methods and the synthesis of nanostructured alumina films under atmospheric conditions (without the use of vacuum technology) over a large area. keywords: luminescence, nanostructures, aluminum oxide, nanosecond overstressed discharge, argon, air. introduction the results of a study of the characteristics and kinetics of processes in a heterogeneous plasma based on mixed flows of a buffer gas -argon, an oxidizing agent (water molecules), and aluminum dustare presented by bityurin et al. [1, 2]. we studied the plasma of glow and pulsed discharges, as well as a combined high-frequency discharge in mixtures of argon, water vapor, and aluminum dust. in such plasma, the gas component, liquid droplets, solids, and plasma simultaneously coexist. these studies are related to the search for effective and cheap hydrogen production technologies based on the plasma-chemical oxidation of aluminum in water vapor. as a result of such a stimulated process, aluminum dioxide is formed on the high-voltage electrode in the form of a developed scaly surface and thermal energy is released [1, 3]. moreover, the price of obtaining a hydrogen molecule does not exceed 1.5 ev/molecule, which is much more economical than the hydrolysis method of producing hydrogen. it is of interest, in order to simplify the design of the reactor, to replace the generator of aluminum dust with a size of tens of microns by producing microdroplets of aluminum by exploding micropoints on the surface of aluminum electrodes in a strong electric field of a nanosecond discharge (ecton formation [4]). the electric and optical characteristics of a spark discharge, the current and voltage of which had an oscillatory shape in the microsecond range, are given in walters et al. [5]. a discharge was ignited between aluminum and graphite electrodes in air and was https://creativecommons.org/licenses/by/4.0/ shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 9 investigated by time-resolved emission spectroscopy. the studies were conducted in the ignition mode of the discharge far from the overstress of the discharge gap. pairs of electrode materials entered the plasma as a result of sputtering under the influence of a spark discharge (the duration of a train of current oscillations was approximately 25 μs, and one full oscillation was 5-6 μs). alumina nanopowders were synthesized by the gasphase method, in which a drop of molten aluminum was held by a high-frequency field [6]. the drop was streamlined by a stream of argon and oxygen, aluminum vapors were carried away into the colder zone, where they condense and oxidize. the resulting alumina powder was collected on a filter. to obtain nanostructured ceramics, powders with particle sizes of 60 and 15 nm were pressed and annealed in air. the luminescence of f , f + centers created by oxygen vacancies in al2o3 and al2o3-c crystals was studied upon excitation by synchrotron radiation with an energy in the range of 3.7 15 ev. at a temperature of 300 k, intense luminescence bands were recorded with maxima at wavelengths of 414 nm (f centers) and 330 nm (f + centers) when crystals were excited by synchrotron radiation with an energy in the range 4.82–8.66 ev. the decay time of the luminescence of the f + centers is 2.0–2.3 ns [7]. sapphire and nanostructured ceramics have high thermal, mechanical, and chemical stability (the melting point of sapphire is 20500 c) [8]. they are also characterized by increased radiation resistance, which is important when using the appropriate substrate microcircuits intended for use in space and nuclear power plants. the luminescence of radiation-induced sapphire defects under the influence of a pulsed beam based on h + / c + ions with an energy of 300 kev was studied. it was found that, along with the f and f + centers, more complex aggregate centers of the f2 type are also observed [9]. using a subnanosecond high-voltage discharge between aluminum electrodes in air, the atmospheric pressure aluminum plasma characteristics were studied with the ecton mechanism for the injection of aluminum vapor into the discharge gap [10]. the production of electrode material vapors in the form of colored plasma jets based on iron and aluminum vapors was most effective in the absence of mismatch of nanosecond voltage pulses and discharge gap resistance. under these conditions, the deposition of structures based on atomized copper electrodes 1-10 in length and 1 μm in diameter was observed on the walls of the discharge chamber. currently, there is practically no work on the synthesis of alumina-based nanostructures using an overstressed nanosecond atmospheric pressure discharge with an ecton mechanism [4] for the injection of aluminum vapor into an oxygen-containing gas plasma. the results of such studies of the synthesis conditions and some characteristics of the nanostructures of copper, zinc, and iron oxides are given in [11-14]. this study presents the results of characteristics and parameters of an overstressed nanosecond atmospheric pressure discharge in air and argon at atmospheric pressure between aluminum electrodes and the luminescence characteristics of aluminum oxide nanostructures that were synthesized under the conditions of this experiment. materials and methods technique and experimental conditions. an overstressed nanosecond discharge in air and argon was ignited in a sealed 3-liter dielectric chamber between aluminum electrodes. a diagram of the discharge module and device for the synthesis of thin films of transition metal oxides is given in previous research [13-14]. the diameter of the cylindrical aluminum electrodes was 5 mm. the radius of the rounded working end of the aluminum electrodes was 3 mm. air and argon pressure varied in the range of 50 202 kpa. the distance between the electrodes was 2 mm. aluminum vapor was introduced into the discharge gap due to microexplosions of the inhomogeneities of the surface of aluminum electrodes in a strong electric field of the discharge and the formation of ectons [4]. to ignite the discharge, high voltage bipolar pulses with a total duration of 50-100 ns and an amplitude of ± (20-40) kv were applied to the electrodes. the discharge was photographed using a digital camera. the distance between the electrodes was used as the scale for determining the plasma volume. at an interelectrode distance of 2 mm, the discharge gap was strongly overstressed. the nanosecond discharge at a pressure of p = 5–202 kpa was fairly uniform [15]. the voltage pulses at the discharge gap and the discharge current were measured using a wide-band capacitive divider, a rogovsky coil, and a 6-lor 04 wideband oscilloscope. the temporal resolution of this recording system was 2-3 ns. the pulse repetition rate varied in the range f = 35-1000 hz. the plasma radiation spectra were recorded using an mdr-2 monochromator, an feu-106 photomultiplier, a direct current amplifier, and an electronic potentiometer. the radiation of the discharge plasma was analyzed in the spectral region of 200-650 nm. the plasma radiation registration system was calibrated by the radiation of a deuterium lamp in the spectral range of 200-400 nm and a gang lamp in the range of 400-650 nm. oscillograms of radiation at the transitions of individual atoms and ions were recorded using an elu 14 fs electronic linear multiplier, the temporal resolution of which was 1 ns. the pulsed electric power of an overstressed nanosecond discharge was determined by graphically multiplying the waveforms of voltage and current pulses. the time integration of the pulsed power made it possible to obtain energy in one electric pulse, which was introduced into the plasma. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 9 thin nanostructured films based on the degradation products of aluminum electrodes and air molecules were deposited during 2-3 hours of operation of the reactor on glass substrates that were installed at a distance of 3 cm from the center of the discharge gap. the resulting films were studied for light transmission in the visible wavelength range. the experimental technique and technique for recording the transmission spectra of synthesized films are described in [16]. results and discussion spatial, electrical and optical characteristics the plasma volume depended on the repetition rate of voltage and current pulses and in the frequency range 10– 150 hz, it increased from 3 to 25 mm 3 . the discharge had a diffuse shape in the form of a ball. the most probable reason for obtaining a spatially homogeneous discharge in atmospheric pressure gases may be the preionization of the discharge gap by a runaway electron beam of about 130-150 ps duration and the accompanying x-ray radiation. it was shown in beloplotov et al. [17] that even when using highvoltage pulses with a leading edge duration of about 200 ns, a runaway electron beam with an intensity of only one order of magnitude lower than even in the same discharge at an air pressure of 130 kpa is formed in a discharge plasma in atmospheric pressure air. the most characteristic waveforms of voltage and current pulses for an overstressed nanosecond discharge between aluminum electrodes in air are shown in (figure 1). figure 1. oscillograms of voltage and current pulses at an air pressure of 101 kpa. due to the mismatch of the output resistance of the pulsed high-voltage modulator and the plasma resistance, the voltage pulse had the form of individual spikes with a duration of 5-10 ns. this mode of ignition of a subnanosecond high-voltage discharge between a metal electrode in the form of a needle and a flat metal plate (or grid), when the total duration of a train of 10 nanosecond voltage pulses was 1–1.5 μs, was used [10] to obtain plasma jets from a material electrodes. it is promising for applying thin metal films to solid substrates of finely dispersed powders based on electrode materials and degradation products of molecules of a gaseous medium. the maximum value of the positive and negative component of the current pulses reached 200 a, voltage amplitudes 30-40 kv. when air was replaced with argon, the form of the oscillograms did not change, the maximum values of the amplitude of the current and voltage of positive and negative polarity decreased, respectively, to 150 a and 15-20 kv. figure (2) presents the pulsed power and energy input into the plasma of an overstressed nanosecond discharge in air per pulse. with an increase in air pressure from 50 to 101 kpa, the maximum value of the pulsed electric discharge power increased from 3 to 6.5 mw, and the maximum energy input increased from 110 to 152.8 mj. as the argon pressure increased from 50 to 101 kpa, the energy deposition into the plasma increased from 225.7 to 441.1 mj. 1 2 figure 2. pulsed power and energy input into a discharge per pulse at an air pressure of 50 kpa (1) and at an argon pressure of 101 kpa (2). the emission spectra of a plasma of an overstressed nanosecond discharge between aluminum electrodes in air and argon are shown in (figures 3 and 4). the experiments with argon, which were carried out in the same pressure range as with air, were performed to demonstrate the absence of emission bands of nanostructures of aluminum oxides in an inert gas plasma (where there are no oxygen carrier molecules). the spectra shown in (figures 3. and 4), are registered under the same conditions for the excitation of the discharge and under the same conditions of registration. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 9 figure 3. plasma emission spectra of an overstressed nanosecond discharge at different air pressures (50.5, 101 and 202 kpa). 1 2 figure 4. plasma emission spectra of an overstressed nanosecond discharge with the interpretation of the most intense spectral lines and bands at an air pressure (1) and argon (2) of 101 kpa. therefore, the intensities of individual spectral lines and bands can be compared with each other. it can be seen from (figure 3 and 4) that the intensity of the glow of the discharge plasma in air at all pressures studied by us exceeds the radiation intensity of spectral lines and discharge bands in argon. in the emission spectra of air plasma with a small admixture of aluminum vapor, radiation was detected at the transitions of the atom and singly charged ions of aluminum ion, nitrogen oxide radicals and nitrogen molecules, as in the emission spectra of a subnanosecond plasma [10]. in an argon-based discharge, radiation was predominantly recorded at transitions of an atom and an aluminum ion. the most intense spectral lines of an atom and a singly charged aluminum ion, which were manifested in the plasma emission spectra, were as follows: (256.8 + 257.5 + 257.5); 265.3; 394.4; 396.2 nm al i; 236.5; (247.5 + 247.6); 286.9; 622.6; (623.1 + 624.3) nm al ii. in the emission spectra of a plasma of an overstressed nanosecond discharge in air mixtures with an admixture of aluminum vapor (figure 4), broad emission bands with peaks in the spectral ranges of 410–420 nm and 300–390 nm were recorded. the highest radiation intensity of these bands was obtained at an air pressure of 202 kpa. in argonbased mixtures, these bands are absent in the discharge emission spectra. since a significant part of nitrogen is present in the air, in principle, in the discharge under study, the formation of inert gas nitride nanostructures is possible. a comparison of the broadband emission spectra of an overstressed nanosecond discharge with the characteristic emission spectra of aluminum nitride nanostructures silvera et al. [18] showed that they do not correlate with each other. egorov et al. [19], characteristic electroluminescence spectra of anodic alumina are presented. spectra in contact with solutions of different electrolytes were recorded at an oxidation current density of 5–15 ma / cm 2 . these spectra had the form of wide luminescence bands in the spectral range with maxima for different electrolytes in the spectral range of 480-550 nm. based on this, the most probable source of broadband plasma radiation of the discharge under study can be aluminum oxide nanostructures. kortov et al. [6] was noted that in the photoluminescence spectrum of anion-defective single crystals and nanostructured ceramics based on aluminum oxide upon excitation of the corresponding samples by radiation with a wavelength of 205 nm, a wide emission band was observed with a maximum at a wavelength of 415 nm. this band coincides with that obtained in the present experiment. it is interpreted as the luminescence band of f centers (the 1s – 3p transition with a maximum of the emission spectrum at an energy of 3.0 ev and a decay time constant of 36–40 ms) [6, 20]. the results of studying the cathodoluminescence spectra of nanostructured alumina ceramics are also presented [6]. cathodoluminescence was excited by a pulsed electron beam with a density of 1 a cm 2 , an energy of 180 kev and a duration of 3 ns. the spectrum of this cathodoluminescence was similar to the spectrum recorded in our experiment at air pressures of 101202 kpa. the main one was the emission band with a maximum at wavelengths of 410–420 nm (quantum energy 3.0 ev), which was adjoined by a wider short-wave band with maximum quantum energies at 3.4, 3.8, and 4.3 ev [6]. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 9 the ultraviolet photo and cathodoluminescence bands of nanostructured alumina ceramics are associated with the emission of f + centers created by oxygen vacancies and have a relatively short decay time (0.6–1.0 μs) [6, 20]. figures (5 and 6) present characteristic oscillograms of plasma radiation at transitions of radicals of nitric oxide and a singly charged nitrogen ion. at an air pressure of 50 kpa, the emission of the nitric oxide radical band appears with a delay of about 50 ns compared with the beginning of the current pulse, since it takes a certain time for the formation of excited no radicals in the plasma. the radiation pulse at the transition of the no radical consisted of two maxima and had a total duration of 120– 130 ns. at large times, it is likely that the discharge turned into a contracted state and no emission of no radicals was observed. with an increase in air pressure to 101 kpa (figure 6), the pulse amplitude and the duration of the radiation pulse at the transition of the nitric oxide radical increased (figure 6). but at the same time, the duration of the first radiation maximum at the transition of the nitric oxide radical decreased and it became more pronounced. the longest radiation duration was recorded at the transition of a singly charged nitrogen ion (approximately 350 ns), which is characteristic of a recombining plasma of nanosecond discharges in air at atmospheric pressure [21]. figure 5. oscillograms of current and radiation of the spectral line of a nitrogen ion 361 nm (n ii) for a discharge in air at a pressure of 50 kpa. transmission spectra in the visible wavelength range of nanostructured films based on copper and aluminum synthesized on a glass substrate are presented in (figure 7). as can be seen from (figure 7) the obtained film is characterized by weak transmission of radiation in the visible wavelength region. according to gasenkova et al. [20], films based on nanostructured alumina ceramics are practically not transparent to the visible region of the spectrum; their transmittance begins to increase in the spectral range of 0.8–2.0 μm from 1-3 to 25%. 1 2 figure 6. oscillograms of the emission of the band 333.5 nm (no) (1) and the spectral line of a singly charged nitrogen ion 361 nm (n ii) (2) in an overstressed nanosecond discharge in air (p 101 kpa). figure 7. the transmission spectra of films based on copper and aluminum in the spectral region of 350-800 nm (band lamp): 1 a pure glass substrate, 2 a nanostructured copper film synthesized in argon at a pressure of 101 kpa, 3 a nanostructured copper oxide film synthesized in air at a pressure of 101 kpa, 4 a film obtained by sputtering aluminum electrodes in air at a pressure of 101 kpa; the repetition rate of voltage and current pulses is 40 hz, and the sputtering time is 3 hours. plasma parameters the discharge plasma parameters for mixtures of aluminum and air vapor and aluminum and argon vapor at atmospheric pressure (component ratio 30 pa: 101 kpa, respectively) were determined numerically and calculated as shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 9 the total integrals of the electron energy distribution function (eedf) in the discharge. eedfs were found numerically by solving the boltzmann kinetic equation in the two-term approximation. eedf calculations were carried out using the program [22]. based on the eedfs obtained, the mean electron energies, electron temperatures, electron drift velocities, electron densities, excitation rate constants of the energy levels of aluminum atoms, and specific discharge losses on elastic and inelastic collisions of electrons with atoms and molecules of both mixtures are determined depending on the magnitude of the reduced electric field (the ratio of the electric field (e) to the total concentration of atoms of aluminum, argon and molecules of nitrogen, oxygen and carbon dioxide for (n)). the variation range of the parameter e/n = 1-1000 td (1 · 10 -17 1 · 10 -14 v · cm 2 ) included the values of the reduced electric field that were realized in the experiment. for gas-vapor mixtures al air and al ar, these reduced electric fields were 820 td and 205 td in the time ranges of 50-100 ns and 100 -480 ns of voltage pulses, respectively (figure 1). the following processes are taken into account in the integral of collisions of electrons with atoms and molecules: elastic scattering of electrons by aluminum atoms, excitation of energy levels of aluminum atoms (threshold energies of 3.1707 ev, 2.9032 ev, 4.1463 ev, 4.2339 ev, 4.1296 ev, 5.1220 ev), ionization of aluminum atoms ( threshold energy 6.0000 ev); elastic electron scattering by argon atoms, excitation of the energy level of argon atoms (threshold energy 11.50 ev), ionization of argon atoms (threshold energy, 15.80 ev); elastic scattering and excitation of energy levels of nitrogen molecules: rotational threshold energy of 0.020 ev, vibrational (threshold energy: 0.290 ev, 0.291 ev, 0.590 ev, 0.880 1.170, 1.470, 1.760, 2.060, 2.350; electronic: 6.170 ev, 7.000, 7.350, 7.360, 7.800, 8.160, 8.400, 8.550, 8.890, 11.03, 11.87, 12.25, 13.00, ionization (threshold energy 15.60 ev); elastic scattering and excitation of energy levels of oxygen molecules: vibrational (threshold energies: 0.190 ev, 0.380 ev , 0.570 ev, 0.750 ev), electronic (threshold energy: 0. 977 ev, 1.627 ev, 4.500 ev, 6.000 ev, 8.400 ev, 9.970 ev, dissociative electron attachment (threshold energy 4.40 ev), ionization (threshold energy 12.06 ev); elastic scattering and excitation of energy levels of carbon dioxide molecules: vibrational (threshold energies: 0.083 ev, 0.167 ev, 0.252 ev, 0.291 ev, 0.339 ev, 0.422 ev, 0.505 ev, 2.5 ev ), electronic (threshold energy: 7.0 ev, 10.5 ev), dissociative electron attachment (threshold energy 3.85 ev), ionization (threshold energy 13.30 ev). data on the absolute values of the effective cross sections of these processes, as well as their dependences on electron energies, were taken from the databases and l.l. shimon [22-25]. figure (8) shows the dependences of the mean electron energy in the plasma of the vapor-gas mixture al: air = 30: 101000 and al: ar = 30: 101000 at a total pressure p = 101.030 kpa on the reduced electric field strength. figure 8. dependences of the mean electron energy in the plasma of a vapor-gas mixture on the reduced electric field strength: 1-al air = 30: 101000, 2-al -ar = 30: 101000 at a total pressure of p = 101.030 kpa. the mean energy of the discharge electrons for the vapor-gas mixture aluminum-air = 30pa 101 kpa almost linearly increases from 0.1364 ev to 16.57 ev (figure 8 (1)), and for the aluminum-argon mixture it also increased from 1.838 ev to 15.07 ev (figure 8 (2)) with an increase in the reduced electric field strength from 1 td to 1000 td. at the same time, a regularity was observed in the increased rate of its change in the ranges of 30–80 td and 1–30 td for mixtures of the first and second, respectively. for the reduced electric field strength range 205 td 820td at which experimental studies of the electric and optical characteristics of the discharge were carried out, the average electron energies varied between 5.323-13.96 ev for the vapor-gas mixture aluminum-air and 7.882 -13.09 for the mixture aluminum-argon. their highest energies corresponded to the values of 62.52 ev 249.0 ev for the first mixture, and for the second mixture 71.14 ev 282.8 ev. table (1) presents the results of modeling the transport characteristics of electrons: mean energies in ε, temperature t k, and drift velocity vdr. and electron concentration for two mixtures of aluminum vapor with air and aluminum vapor with argon. the temperature and electron drift velocity (тable 1) decreases from 161936 k to 61 746.8 k and from 6 ∙ 10 5 m/s to 2 ∙ 10 5 m/s for the first mixture, and for the second mixture from 151844 to 91431.2 and 4 ∙ 10 4 to 1.4 ∙ 10 4 when changing the reduced electric field strength from 820 td to 205 td, respectively. the values of electron concentration increase from 1.1 ∙ 10 20 m -3 to 1.6 ∙ 10 20 m -3 at a current density l. 02 ∙ 10 7 a/m 2 and 5.1 ∙ 10 6 a/m 2 on the surface of the electrode of the radiation source (0.196 · 10 -4 m 2 ) for both mixtures. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 9 тable 1. transport characteristics of electrons for the mixture: al-аir=30 pa – 101 kpa and al ar=30 pa – 101 kpa mixture: al-аir=30 pa – 101 kpa e/n, td ε, ev t 0 k vdr., m/s ne, m -3 1 0.1364 18362 6.2 10 3 5.1∙10 21 97 2.672 30995,2 2.5 10 3 1.3∙10 22 205 5.323 61746,8 2∙10 5 1.6∙10 20 820 13.96 161936 6∙ 10 5 1.1∙10 20 1000 16.57 192212 5.4∙10 4 1.1∙10 21 mixture: al ar=30 pa – 101 kpa e/n, td ε, ev t 0 k vdr., m/s ne, m -3 1 1.838 4431 1.4∙10 5 2.3∙ 0 20 97 6.795 78822 1.7∙10 4 1.9∙10 21 205 7.882 91431,2 1.4∙10 4 1,6∙10 20 820 13.09 151844 4 ∙10 4 1.1∙10 20 1000 15.07 174812 4.8∙10 4 1.3∙ 0 21 figure (9) presents the dependence of the specific power of the discharge losses on both elastic (1, 2) and inelastic (3,4) collisions of electrons with mixture components in a gas-discharge plasma on the reduced electric field strength. an increase in power is observed with increasing values of the reduced electric field, both for elastic processes and for inelastic ones. in addition, higher values of specific power of discharge losses are observed for inelastic collisions of electrons with atoms and molecules in a mixture of aluminum and air vapors (figure 9, table 2). figure 9. the specific discharge power for elastic (1) and inelastic (2) processes per unit of the total concentration of the mixture vs the reduced electric field strength for the aluminum-air mixture and for elastic (3) and inelastic (4) processes for the aluminum-argon mixture. figure (10) and table (3) present the results of numerical simulation of the dependence of the excitation rate constants of the spectral lines of aluminum atoms on the reduced electric field strength in mixtures of aluminum and air and aluminum and argon vapor for the ratio of partial pressures in mixtures of 30 101000 pa at a total pressure of the mixture p = 101030 pa. the rate constants are characterized by a high value, which is associated with the values of the absolute effective cross sections of the corresponding processes. in the range of reduced electric field strength 205 td 820 td, at which experimental studies of the electrical and optical characteristics of the discharge were carried out, they were in the range of k≈10 -16 -10 -15 m 3 /s. тable 2. elastic and inelastic power loss /n (ev m 3 /s) for mixtures al-аir=30 pa – 101 kp and al ar=30 pa – 101 kpa mixture al-аir=30 pa – 101 kp e/n,td elastic, power /n (ev m3/s) inelastic, power /n (ev m3/s) 205 3,448e-17 4,091e-14 820 1,290e-16 3,559e-13 mixture al ar=30 pa – 101 kpa e/n,td elastic, power /n (ev m3/s) inelastic, power /n (ev m3/s) 205 4,995e-17 2,535e-14 820 9,709e-17 2,658e-13 figure 10. dependences of the rate constants of excitation of the spectral lines of aluminum atoms on the reduced electric field strength in a plasma on a mixture of aluminum and air vapors: 30: 101000 pa at a total pressure of the mixture p = 101030 pa and aluminum and argon 30: 101000 pa at the total pressure of the mixture p = 101030 pa : 1λ = 396.15 nm (ethr. = 3.17 ev) for an aluminum-air mixture, 2-λ = 309.27nm (e thr. = 4.23 ev) for an aluminum-argon mixture, 3-λ = 309.27nm (e thr. = 4.23 ev ) for an aluminum-air mixture, 4-λ = 308.21nm (e thr. = 4.13 ev) for an aluminumair mixture. shuaibov et al., 2020 synthesis of aluminum oxide nanoparticles highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 9 тable 3. the rate constants of the excitation of the spectral lines of aluminum atoms for the values of the reduced electric field strength in a plasma on a mixture of aluminum and air vapor: 30 101000 pa and aluminum and argon: 30 101000 pa at a total pressure of the mixture p = 101030 pa. mixture al-аir mixture al-аr e/n, td λ = 396.15 nm λ = 309.27 nm λ = 308.21 nm λ = 309.27 nm 205 2.51e-15 1.26e-15 8.52e-16 1.89e-15 820 4.01e-15 1.92e-15 1.55e-15 1.98e-15 limitations methods to reduce the body effects and to obtain better films, in the future it is necessary to switch to the subnosecond mode of operation of the reactor with a total voltage pulse duration of the order of 1 ns. this will contribute to the full implementation of the ectonic mechanism of aluminum atomization and improve the control of the film deposition process. conclusions thus, it was found that a plasma of an overstressed nanosecond discharge between aluminum electrodes at air pressures of 50–202 kpa, a pulsed discharge power of 3–6.5 mw, and an energy input of one pulse of 110–153 mj is a source of electroluminescence of aluminum oxide nanoparticles in the form of a wide band, which is in the spectral range of 300-430 nm; upon deposition of degradation products of electrodes and air molecules in a plasma on a glass substrate, films based on aluminum oxides were obtained, which are characterized by low transparency in the visible region of the spectrum. numerical simulation of plasma parameters in a mixture of aluminum and air vapor established that for the reduced electric field strength of 820 td 205 td, at which experimental studies of the electric and optical characteristics of the discharge were carried out, the mean electron energies varied between 13.96 and 5.323 ev, their highest energies corresponded to the values of 249.0 ev 62.52 ev. in a mixture of aluminum and argon vapor, these values of the discharge parameters varied within 13.09 ev 7.882 ev and 282.8 ev 71.14 ev, respectively. the electron concentration was 1.1 · 10 20 m -3 1.6 · 10 21 m -3 at current density l. 02 ∙ 10 7 a/m 2 and 5.1 ∙ 10 6 a/m 2 on the surface of the aluminum electrode (0.196 · 10 -4 m 2 ). the excitation rate constants of the spectral lines of aluminum atoms =396.15 nm, =309.27 nm, =308.21 nm are in the range of (4.0092.511) ∙ 10 -15 m 3 /s, (1.918 1.259) ∙ 10 -15 m 3 /s, (1.5530. 8.52) ∙ 10 -15 m 3 /s, (1.975– 1.975) ∙ 10 -15 m 3 /s, respectively. the maximum value of the excitation rate constant of the spectral line of aluminum atoms =396.15 nm was (4.009 ∙ 10 -15 ) m 3 s for the reduced electric field strength of 820 td in a mixture of aluminum and air. the specific discharge power loss for inelastic and elastic collisions of electrons with atoms and molecules, which were part of the working mixtures of a gas-discharge plasma, per unit total concentration of the mixture increased with increasing reduced electric field for both inelastic and elastic processes. its maximum value was for inelastic processes in a mixture of aluminum and air vapor was equal to 3.559 ∙ 10 -13 ev m 3 /s for a reduced electric field strength of 820 td. references 1. bityurin va, efimov av, grigorenko av, goryachev sv, klimov ai, chinnov vf. plasma stimulation of aluminum burning in water vapor. modern science. collection of research papers.2011 2(7): 47. 2. bityurin va, grigorenko av, efimov av, klimov ai, korshunov ov, kutuzov ds, chinnov vf. spectral and kinetic analysis of heterogeneous gas discharge plasma in the flow of an al, h 2 o, and ar mixture. high temperature. 2014 jan 1;52(1):1-1. 3. shkolnikov ei, beetle az, bulychev bm, larichev mn, ilyukhina av, vlaskin ms; edited by a.e. scheindlin. oxidation of aluminum with water for efficient power generation. joint institute for high temperatures ras. m .: science. 173 p. 4. mesyats ga. ecton-electron avalanche from metal. uspekhi fizicheskikh nauk. 1995 jun 1;165(6):601-26. 5. walters jp, malmstadt hv. emission 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population of energy levels of atoms of the aluminum subgroup. scientific bulletin of uzhnu. physics series 2007 20:55-61. 24. http://www.ioffe.ru/es/elastic/data2.html 25. https://physics.nist.gov/cgibin/ionization/ion_data.php?id=ali&ision=i&initial=&t otal=y http://www.ioffe.ru/es/elastic/data2.html https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y https://physics.nist.gov/cgi-bin/ionization/ion_data.php?id=ali&ision=i&initial=&total=y highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202302 research article open access 1 department of genetic resources, desert research center (drc), cairo, egypt. * to whom correspondence should be addressed: drmokhtarsaid@yahoo.com editor: aladdin hamwieh, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): clara r. azzam, cell research department, field crops research institute, agricultural research center, giza 12619, egypt. fida alo, international center for agricultural research in the dry areas (icarda), beirut, lebanon. thoria rashad mohamed, cytology and genetics, botany department, women faculty, cairo, egypt. received: july 10, 2023 accepted: sep 6, 2023 published: september 15, 2023 citation: rizk ms. unveiling the architecture variability of coding regions in prunus persica: identification and characterization of key gene families impacting plant development. 2023 sep 15;6:bs202302 copyright: © 2023 rizk. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the author declare that they have no competing interests. unveiling the architecture variability of coding regions in prunus persica: identification and characterization of key gene families impacting plant development mokhtar said rizk 1 ><  abstract this study aimed to investigate gene families related to prunus persica (peach) and their evolutionary relationships. gene identification and sequence alignment methods were used to characterize homeobox, mads-box, nac, and receptor-kinase-like gene families. gene expression data analysis was conducted to assess gene activity and regulatory mechanisms in peach plant development. gene identification involved a local blastp analysis using protein sequences from arabidopsis thaliana gene families as queries against the peach proteome. multiple sequence alignment and phylogenetic tree construction were performed using clustalw and itol, respectively. gene expression analysis utilized the geo2r online tool and focused on two datasets, gse71561 and gse71460. gene identification revealed 593 genes in total, with varying gene counts and sequence lengths among the different families. phylogenetic analysis demonstrated distinct clusters within each gene family. protein-protein interaction analysis showed significant interconnectivity among homeobox transcription factors, while mads-box and nac gene families displayed lower connectivity. enrichment analysis highlighted developmental processes associated with these gene families. gene expression analysis provided insights into peach fruit development, ripening, and postharvest changes, revealing differential gene expression patterns and regulatory mechanisms. this study provides valuable insights into the gene families related to prunus persica, their evolutionary relationships, and regulatory mechanisms. the identified genes and their functional characteristics contribute to a better understanding of peach development and fruit phenotypes. further research is required to elucidate the implications of gene variations and interactions in the studied system. keywords: peach, gene expression analysis, gene families, mads-box, nac gene family, receptor-kinase-like introduction peaches, scientifically known as prunus persica, are fruit trees belonging to the rose family (rosaceae) and are cultivated in warmer temperate regions of both the northern and southern hemispheres [1]. they are widely enjoyed fresh and are commonly used in baking, such as in pies and cobblers. canned peaches are also a popular staple in many areas. varieties with yellow flesh are particularly notable for their high vitamin a content [2]. compared to some other fruit trees, peach trees have a relatively shorter lifespan. in certain regions, orchards are replanted every 8 to 10 years, while in others, trees may continue to produce satisfactorily for 20 to 25 years or even longer, depending on their resistance to diseases, pests, and winter-related damage [3]. countries such as spain, italy, the united states, chile, and australia consider peach is as highly significant prunus specie. the european union holds the second position worldwide in peach production, with china being the only country ahead. between 2018 and 2020, the eu averaged an annual production of 3612,000 tons, while the harvested area for peaches in 2019 encompassed 206,660 hectares. leading the rankings, spain boasts 77,464 hectares of peach cultivation and produces 1480,000 tonnes per year, closely followed by italy and greece [4]. highlights in bioscience page 1 of 12 september 2023|volume 6 https://doi.org/10.36462/h.biosci.202302 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:drmokhtarsaid@yahoo.com https://orcid.org/0000-0003-4579-4118 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development homeobox genes, mads-box genes, nac genes, and receptor -kinase-like genes are all involved in the regulation of gene expression and play important roles in the development and growth of organisms. homeobox genes, mads-box genes, nac genes, and receptor-kinase-like genes are crucial players in gene expression regulation and exert significant roles in organismal development and growth [5; 6]. specifically, homeobox genes involved in floral organ development have been found to interact with mads-box genes, which in turn govern the expression of a wide array of genes responsible for flower development and organ identity, including other mads-box genes and homeobox genes [7]. on the other hand, nac genes exhibit interactions with both homeobox genes and mads-box genes, thereby influencing their expression and contributing to the regulation of plant development and stress responses [8; 9]. receptor-kinase-like genes represent a diverse gene group encoding receptor-like kinases (rlks) or receptor-like proteins, and some rlks have demonstrated interactions with transcription factors, including members of the mads-box and nac gene families. these interactions are involved in the regulation of gene expression and signaling pathways crucial for various developmental processes and stress responses [10; 11]. collectively, the intricate network of interactions and regulatory mechanisms within these gene families contributes to the meticulous control of gene expression and the coordination of developmental processes in organisms [12]. studying these gene families, including homeobox genes, mads-box genes, nac genes, and receptor-kinase-like genes, is of great importance in the field of peach genomics. these gene families are involved in the regulation of gene expression and play significant roles in the development and growth of organisms [13]. by understanding the functions and interactions of these genes, researchers can gain insights into the molecular mechanisms underlying peach development and improve their understanding of the genetic factors that influence important traits in peach [14]. the aim of this study is to accomplish the following objectives. firstly, our aim is to identify and annotate the gene family members, which include homeobox genes, mads-box genes, nac genes, and receptor-kinase-like genes, within the peach genome. this objective will involve conducting a comprehensive genome-wide analysis and utilizing bioinformatics tools to accurately identify and classify these gene family members. secondly, we aim to characterize the gene family members to gain insights into their diversity and functional variations. by investigating their unique features and roles in peach, we can enhance our understanding of the genetic makeup of these genes. lastly, we intend to investigate the expression patterns of the gene family members throughout plant development. this will be achieved through high-throughput transcriptomic analyses, such as rna-sequencing, to capture the dynamic expression profiles of these genes. by accomplishing these objectives, our study aims to contribute to a deeper understanding of the genetic characteristics, functional diversity, and expression dynamics of homeobox genes, mads-box genes, nac genes, and receptorkinase-like genes in peach. methods gene identification and sequence alignment the methodology employed in this study aimed to investigate the gene families related to prunus persica (peach) and their evolutionary relationships. the genome data used for the analysis was obtained from prunus persica ncbiv2 [15] , as provided by the ncbi refseq database. the gene families of interest included homeobox, madsbox, nac, and receptor-kinase-like. the gene set comprised a total of 593 genes, while homeobox, mads-box, nac, and receptor-kinase-like gene families consisted of 91, 106, 94, and 302 genes, respectively. to further analyze these gene families, information from arabidopsis thaliana gene families was downloaded from the tiar database, providing valuable reference data [16]. to identify the corresponding gene family members in the peach genome, a local blastp analysis was conducted. the protein sequences from the arabidopsis thaliana gene families were used as queries against the peach proteome. the blastp analysis was performed using the following parameters: "-outfmt 6 -evalue 1e-5 -num_threads 4" [17]. genes that showed less than 200 base pairs of match were subsequently filtered out from further analysis. to assess the evolutionary relationships among the identified gene family members, a multiple sequence alignment was performed using clustalw, a widely used tool for aligning multiple protein or nucleotide sequences [18]. the resulting alignment was then utilized to construct a phylogenetic tree. the phylogenetic tree construction was accomplished using itol (interactive tree of life), an interactive web-based tool specifically designed for visualizing and manipulating phylogenetic trees [19]. in the analysis of sequencing data, the alignstatplot tool [20], was employed to showcase both shared and distinct genomic regions. by implementing these methods, the study aimed to explore the gene families related to prunus persica and gain insights into their evolutionary relationships. the genome data from prunus persica ncbiv2, combined with gene families information from arabidopsis thaliana, enabled the identification and characterization of the gene family members in peach. the blastp analysis, multiple sequence alignment, and phylogenetic tree construction provided valuable information regarding the evolutionary history and relationships of these gene families. gene expression data analysis gene expression analysis was conducted using the geo2r online tool [21] to assess gene activity and regulatory mechanisms in peach plant development. the researchers focused on gene families such as homeobox transcription factor, madshighlights in bioscience page 2 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development box gene, nac transcription factor, and receptor kinase-like gene families. two datasets, gse71561 [22] and gse71460 [23], were analyzed. gse71561 aimed to understand the molecular control of peach fruit development, including interactions between seed and mesocarp. it comprised 39 samples representing various developmental stages, covering flower, seed, and mesocarp tissues. each sample had three biological replicates. gene enrichment analysis identified differentially expressed genes within the same tissue stage, and the researchers examined tissue specificity versus stage specificity of the regulation. gse71460 focused on investigating the effects of gibberellic acid application and cold storage on postharvest textural changes in peaches, specifically woolliness. the dataset included 12 samples with four treatments, each having three biological replicates. approximately half of the investigated genes were analyzed, revealing significant differential expression. gene ontology and set enrichment analyses indicated the involvement of cellular processes, developmental processes, and complex transcriptional responses related to cell wall metabolism, hormone metabolism, and signaling in response to cold storage and gibberellic acid. by using the geo2r online tool and analyzing these datasets, the researchers gained insights into gene expression patterns and regulatory mechanisms underlying peach fruit development, ripening, and postharvest changes. this study contributes to a better understanding of the molecular processes governing these complex phenotypes in peaches. protein-protein interaction (ppi) analysis was performed using the string database [24] and enrichment analysis. the ppi network was visualized using the cytoscape program [25]. results and discussion gene identification and sequence variationa genes belonging to four distinct gene families were identified across peach genome (figure 1). the results of the analysis provided valuable insights into the gene counts and characteristics of each family. the homeobox transcription factor family consisted of 52 genes, with a total length of 33,042 base pairs (bp). among these genes, the maximum sequence length observed was 837 bp, while the minimum sequence length was 1,764 bp. for the mads-box gene family, a total of 16 genes were identified, spanning a length of 4,522 bp. the maximum sequence length observed in this family was 240 bp, and the minimum sequence length was 349 bp. in the nac transcription factor family, a total of 42 genes were identified, with a combined length of 15,903 bp. the maximum sequence length observed within this family was 591 bp, while the minimum sequence length was 862 bp (figure 1). finally, the receptor kinase-like gene family exhibited the largest gene count, with 366 genes identified. these genes spanned a length of 300,904 bp. the maximum sequence length observed in this family was 626 bp, and the minimum sequence length was 556 bp (figure 1). these results highlight the variations in gene counts and sequence lengths among the different gene families. they provide valuable information about the diversity and characteristics of genes within each family, shedding light on their potential roles and functions in the studied system. the multiple sequence alignment conducted in this study provided valuable insights into the patterns of sequence variation within the gene families analyzed (figure 1). these variations were evident through the presence of sequence gaps observed in the alignments. it was noticeable that certain sequences exhibited substantial portions of amino acid sequences that were absent in others, indicating potential differences in gene function and underlying mechanisms (figure 1). the presence of sequence gaps in the multiple sequence alignments suggests the occurrence of insertions or deletions in the gene sequences among different members of the gene families. such variations in sequence length and composition could have significant implications for the functionality and regulation of these genes. the observed differences in amino acid sequences may lead to variations in protein structure and function, potentially influencing their roles within the studied system [26]. by identifying these sequence variations, this study contributes to a deeper understanding of the diversity and potential functional divergence within the gene families analyzed. further investigations into the specific functions and mechanisms associated with the unique sequence features observed could provide valuable insights into the evolutionary dynamics and functional specialization of these genes [27]. additionally, the phylogenetic analysis based on the sequence alignments revealed distinct clusters of genes within each family, suggesting differences in gene functions and evolutionary relationships (figure 2). the comparison between the results obtained in this study and previous studies provides valuable insights into the gene counts and characteristics of different gene families. a total of 73 homeobox-like genes in the grapevine genome were identified in a previous study [28]. the comparison between the results obtained in this study and previous studies provides valuable insights into the gene counts and characteristics of different gene families. in the current study, our analysis identified 52 genes associated with homeobox transcription factor family, suggesting variations in the composition and abundance of homeobox transcription factor genes between species or datasets. similarly, in the mads-box gene family, our analysis identified only 16 genes, indicating potential disparities in the madsbox gene repertoire between the studied system and previous studies in rice [29]. in relation to the nac transcription factor family, our analysis identified 42 genes, contrasting with the 140 putative nac or nac-like genes identified in rice [30]. these contrasting gene counts suggest variations in the presence or abundance of nac genes between the studied system and rice. the specific gene counts mentioned in the provided articles were higher for the homeobox transcription factor family (73 genes) and mads-box gene family (75 genes) in grapes and rice, rehighlights in bioscience page 3 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development figure 1. multiple sequence alignment of identified sequences from the homeobox transcription factor family (a), mads-box gene family (b), nac transcription factor family (c), and receptor kinase-like gene family (d) in the peach genome. sequences are color-coded, and gaps represent missing regions in the alignment. spectively. however, the gene counts obtained in this study may be specific to the analysis or dataset used, and further research is required to elucidate the reasons behind these variations and their implications in the studied system. protein-protein interaction analysis the analysis of the homeobox transcription factor family in the peach genome using protein-protein interaction (ppi) network analysis revealed significant interconnectivity and potential functional relationships among the proteins (figure 3). the highlights in bioscience page 4 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development figure 2. phylogenetic trees illustrating the evolutionary relationships of identified sequences from the homeobox transcription factor family (a), mads-box gene family (b), nac transcription factor family (c), and receptor kinase-like gene family (d) in the peach genome. the trees provide insights into the genetic divergence and relatedness among the sequences within each gene family, highlighting their evolutionary history and potential functional similarities or distinctions. network consisted of 25 proteins and 61 interactions, indicating a complex regulatory network involved in peach development. the average local clustering coefficient suggested clustering of nodes, further supporting the notion of functional cooperativity among the homeobox transcription factors. the enriched gene ontology (go) terms associated with the homeobox transcription factor family included various processes related to key developmental processes and organ polarity estabhighlights in bioscience page 5 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development lishment in peach, such as determination of bilateral symmetry, primary shoot apical meristem specification, xylem development, meristem initiation, and polarity specification of the adaxial/abaxial axis. these findings highlight the important roles of homeobox transcription factors in orchestrating developmental processes and regulating gene expression and cellular processes in peach. several overlapping themes can be observed when comparing these findings with published studies. a previous study focused on hd-zip iii homeobox genes and their involvement in vascular differentiation [31]. they identified a new member of the hd-zip iii genes, zehb-13, which exhibited restricted expression in the procambium. this finding aligns with the enriched go term of xylem development identified in the peach homeobox transcription factor family. both studies suggest that homeobox transcription factors play crucial roles in the regulation of vascular development. the investigation of the tdif peptide signaling pathway and its impact on vascular stem cell proliferation in arabidopsis revealed the wox4 homeobox gene as a key target of the tdif signaling pathway, promoting the proliferation of procambial/cambial stem cells [32]. this finding is consistent with the enriched go term of meristem initiation and the regulation of primary shoot apical meristem specification observed in the peach homeobox transcription factor family. both studies imply the involvement of homeobox transcription factors in the regulation of stem cell proliferation and meristem development. furthermore, a study on homeobox genes associated with lignification in bamboo shoots identified 115 homeobox genes, including several classes such as the knox class, that interacted with other transcription factors involved in lignin synthesis [33]. this interaction network parallels the potential functional relationships observed in the protein-protein interaction network analysis of the peach homeobox transcription factor family. additionally, the upregulation of homeobox genes in shoots as the height increased suggests their involvement in developmental processes similar to the enriched go terms identified in the peach study. similarly, the mads-box gene family in the peach genome was analyzed using ppi network analysis (figure 3). the network showed relatively low interconnectivity with 6 proteins and 5 interactions. the average local clustering coefficient suggested some tendency for nodes to cluster together. however, the ppi enrichment analysis revealed functional associations or co-regulation among the mads-box gene family members. the functional enrichment analysis identified processes such as pollen maturation, regulation of pollen tube growth, plant ovule development, and flower development associated with the madsbox gene family. these findings indicate their involvement in important processes like pollen development, fertilization, and flower formation in peach. in the context of these results, an investigation into the functional conservation of mads-box genes in arabidopsis and rice pollen maturation was conducted [34]. the findings revealed that the disruption of specific mikc-type genes in rice resulted in severe defects in pollen maturation and germination, providing supporting evidence for the involvement of mads-box genes in pollen development. furthermore, a study on mads-box gene evolution beyond flowers identified novel mads-box genes in arabidopsis, indicating that the evolution of the mads-box family involved rapid and simultaneous functional diversification in both vegetative and reproductive structures [35]. these findings align with the present research, demonstrating functional associations or co-regulation among the mads-box gene family members. additionally, a study focused on the pineapple mads-box gene family and its evolutionary history emphasized the importance of understanding the ancestral form of monocot flowers and their development, further supporting the relevance of studying mads-box genes in different plant species [36]. in this study, we analyzed the nac transcription factor family in the peach genome using ppi network analysis and enrichment analysis (figure 3). the ppi network analysis revealed a low level of connectivity and clustering within the nac transcription factor network, suggesting potential complexity in their regulatory mechanisms. additionally, the lack of significant enrichment in the ppi analysis indicated sparse or transient interactions among these proteins. however, the functional enrichment analysis provided valuable insights into the involvement of nac transcription factors in diverse plant development processes. our findings are consistent with previous studies that have investigated the role of nac transcription factors in specific plant developmental processes. for instance, a study demonstrated that the vascular-related nac-domain7 (vnd7) transcription factor regulates the expression of genes required for xylem vessel element formation in arabidopsis. multiple transcription factors, including vnd1-vnd7, were identified as putative positive regulators of vnd7 expression. this study highlights the importance of nac transcription factors in xylem vessel differentiation, which aligns with our functional enrichment analysis showing their involvement in xylem vessel differentiation and secondary cell wall biogenesis [37]. another study discovered that vnd-interacting2 (vni2), a nac domain transcription factor, negatively regulates xylem vessel formation in arabidopsis. vni2 interacts with vnd7 and other vnd family proteins, acting as a transcriptional repressor and repressing the expression of vessel-specific genes regulated by vnd7. this finding supports our findings of the potential complexity in the regulatory mechanisms of nac transcription factors and their role in xylem vessel differentiation [38]. furthermore, a study investigated the function of xylem nac domain1 (xnd1) in xylem differentiation. it revealed that xnd1 interacts with the retinoblastoma-related (rbr) protein and acts as a transcriptional repressor to inhibit differentiation. these findings provide insights into the inhibitory role of nac transcription factors in xylem differentiation, which aligns with our functional enrichment analysis showing their involvement in secondary cell wall biogenesis [39]. in addihighlights in bioscience page 6 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development tion to their role in vascular development, nac transcription factors have been implicated in other plant developmental processes. for example, a study investigated the involvement of nam genes, regulated by mir164, in floral-boundary morphogenesis in tomato. the nam gene goblet was identified as a key regulator of floral-boundary formation. our functional enrichment analysis revealed the involvement of nac transcription factors in shoot meristem specification and root cap development, highlighting their role in various aspects of plant growth and development [40]. another study investigated the role of cup-shaped cotyledon1 (cuc1), a nac transcription factor, in the establishment of shoot organ boundaries in arabidopsis. it was found that cuc1 activates the expression of light-dependent short hypocotyls 4 (lsh4) and lsh3, members of the alog gene family, in shoot organ boundary cells. this study provides insights into the regulatory mechanism of shoot organ boundary establishment mediated by nac transcription factors [41]. the receptor kinase-like gene family in the peach genome was analyzed using ppi network analysis and enrichment analysis (figure 3). the ppi network consisted of 151 nodes and 966 edges, indicating a dense network of protein interactions. the network exhibited a high level of interconnectivity, suggesting functional relevance and potential cooperativity among the genes. the functional enrichment analysis provided insights into the involvement of the receptor kinase-like gene family in various developmental processes critical for plant growth and response to environmental stimuli. the analysis also highlighted molecular functions related to signal transduction and receptor binding, emphasizing the importance of receptor-mediated signaling pathways in peach. references to relevant scientific publications covered topics such as genetic incompatibility [42], selfincompatibility signaling [43], protein secretion, gene duplications, and orthologous relationships [44], providing further insights into the functional characteristics and evolutionary aspects of the receptor kinase-like gene family in peach. the enrichment analysis included classifications from various databases and ontologies, offering additional functional annotations and domain information associated with the receptor kinase-like gene family, enhancing our understanding of their functional properties and characteristics. gene differential expression analysis gene expression analysis was performed to identify genes highly associated with peach development using two different datasets: gse71460 and gse71561. in gse71460, which focused on the effects of gibberellic acid application and cold storage on postharvest textural changes in peaches, several important genes were identified. these included skp1-like, f-box /fbd/lrr-repeat protein, leucine-rich repeat receptor-like, triose phosphate isomerase cytosolic isoform-like protein, and udpglycosyltransferase 87a1-like. on the other hand, gse71561 aimed to elucidate the molecular regulation of peach fruit development, particularly the interactions between the seed and mesocarp tissues. in this dataset, isoleucine n-monooxygenase 2, myb-related protein, and transcription factor bhlh75-like were among the most important genes identified. enrichment analysis was conducted on both datasets to gain insights into the functional significance of the identified genes. in gse71460, the enrichment analysis revealed several enriched terms and processes that suggested the involvement of these genes in various cellular processes, metabolic activities, and molecular interactions related to peach development. in a previous study, the expression patterns of arabidopsisskp1-like (ask) genes, which play important roles in ubiquitinmediated proteolysis and various biological processes, were examined [45]. the findings of that study shed light on the expression profiles of skp1-like genes in plants. our study, focusing on gene expression during fruit development, shares similarities with the previous study. therefore, our results contribute to understanding the roles of skp1-like genes in peach development. another investigation explored the molecular mechanisms of self-incompatibility in plants. it identified a skp1-like protein as a crucial component of a protein complex involved in pollen compatibility [46]. although the study’s focus was on self-incompatibility, the importance of skp1-like proteins in plant reproductive processes can provide insights into peach fruit development. additionally, we observed differential expression of triose phosphate isomerase, an essential enzyme in the glyoxalase pathway, during peach development. this finding aligns with a separate study that discussed the implications of triose phosphate isomerase and the glyoxalase pathway in plant responses to abiotic stress and signaling [47]. while the latter study primarily examined stress responses, their insights into triose phosphate isomerase activity are valuable for understanding its involvement in peach development. by comparing our findings with the mentioned studies, we enhance the current understanding of gene expression patterns during peach development and establish potential connections to processes such as ubiquitin-mediated proteolysis, self-incompatibility, and stress responses. in the case of gse71561, the enrichment analysis highlighted several important gene-enriched pathways. these included cellular anatomical entity, intracellular, catalytic activity, membranebounded organelle, intracellular membrane-bounded organelle, metabolic process, cellular process, response to stimulus, organelle, intracellular organelle, chloroplast, cytoplasm, plastid, binding, thylakoid, transit peptide, chloroplast thylakoid, response to stress, cellular metabolic process, membrane, developmental process, biosynthesis of secondary metabolites, oxidationreduction process, thylakoid, and stromule, metabolic pathways, photosystem, thylakoid, plastid envelope, chloroplast envelope, oxidoreductase activity, response to abiotic stimulus, anatomical structure development, thylakoid membrane, and chloroplast thylakoid membrane. these enriched pathways provide insights into the molecular processes and organelle-specific functions ashighlights in bioscience page 7 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development figure 3. protein-protein interaction networks of the identified sequences from the mads-box gene family (a), homeobox transcription factor family (b), nac transcription factor family (c), and receptor kinase-like gene family (d) visualized using the string database. the networks provide a comprehensive overview of the protein interactions within each gene family, shedding light on potential functional associations and regulatory relationships among the identified proteins. sociated with peach fruit development, particularly in relation to chloroplast function, photosynthesis, and response to stress. this findings from the enrichment analysis of gse71561 align with the study conducted by chen et al. [48], which investigated the regulation of chloroplast development in peach. the enrichment analysis highlighted gene-enriched pathways associated with cellular anatomical entity, intracellular processes, catalytic activity, membrane-bounded organelles, metabolic processes, and response to stimulus, among others. these enriched pathways provide insights into the molecular processes and organellespecific functions involved in peach fruit development, particularly in relation to chloroplast function, photosynthesis, and response to stress. the gene expression analysis contucted in ths study focused on identifying genes associated with peach development in four gene families. the results of our analysis revealed specific genes within each family that contribute to peach development. these findings are consistent with previous studies conducted in related species, providing valuable insights into the molecular mechanisms underlying peach development. in the homeobox transcription factor family, we identified 8 genes associated with peach development. previous research by testone et al. [49] highlighted the significance of homeodomain protein kn1 and homeobox-leucine zipper protein protodermal factor 2 -like in this family, specifically in stem development and regulating elongation and lignification during primary growth. these findings support our identification of homeobox transcription factor genes associated with peach development. similarly, within the mad box gene family, we found 8 genes associated with peach development. in a study by wuddineh et al. [50], a significant gene, seedstick-like protein, was identified within this family in switchgrass, a related plant species. although their research focused on genetically engineering switchgrass for biofuel production, the presence of seedstick-like protein in highlights in bioscience page 8 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development figure 4. gene expression analysis in gse71460 and gse71561 datasets. (a) heatmap illustrating the expression profiles of key genes associated with postharvest textural changes in peaches in the gse71460 dataset, which explores the effects of gibberellic acid application and cold storage. (b) heatmap displaying the expression patterns of critical genes related to the target trait in the gse71561 dataset, which investigates peach fruit development, including interactions between seed and mesocarp tissues. (c) visualization of the protein-protein interaction (ppi) network for the genes identified in the gse71460 dataset, revealing their interactions. (d) representation of the ppi network for the genes identified in the gse71561 dataset, uncovering interplay among the identified genes. (e) identification of enriched biological pathways associated with postharvest textural changes in peaches in the gse71460 dataset, offering insights into the underlying molecular mechanisms. (f) discovery of enriched biological pathways linked to the investigated trait in the gse71561 dataset, providing valuable insights into fundamental molecular processes governing postharvest textural changes in peaches. highlights in bioscience page 9 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development our analysis suggests its potential involvement in peach development. within the nac gene family, we discovered 2 genes associated with peach development, although their specific functions in relation to peach development are unknown. while no direct studies on the role of these specific genes in peach were found, further investigation of nac genes in other plant species may provide insights into their functions in peach development. in the receptor-kinase-like gene family, we identified a total of 80 genes associated with peach development. noteworthy genes in this family, such as lrr receptor-like serine/threonine-protein kinase rpk2 and serine/threonine-protein kinase-like protein acr4, have been reported in various studies across different plant species. although no specific studies have linked these genes to peach development, their presence in our analysis suggests their potential involvement in various peach developmental processes. conclusion in conclusion, this study employed two methods, gene identification and sequence alignment, and gene expression data analysis, to investigate gene families related to prunus persica (peach) and gain insights into their evolutionary relationships and regulatory mechanisms. the gene identification and sequence alignment method involved analyzing gene families such as homeobox, mads-box, nac, and receptor kinase-like. gene identification was conducted using local blastp analysis, and multiple sequence alignment was performed to assess sequence variation and construct phylogenetic trees. the results provided valuable information about gene counts, sequence lengths, and sequence variations within each gene family, shedding light on their potential roles and functions in the peach genome. the comparison with previous studies highlighted variations in gene counts and sequence lengths among different species or datasets, suggesting differences in gene composition and abundance. the gene expression data analysis method focused on analyzing two datasets, gse71561 and gse71460, using the geo2r online tool. the analysis provided insights into gene expression patterns and regulatory mechanisms underlying peach fruit development, ripening, and postharvest changes. gene enrichment analysis revealed differentially expressed genes and identified biological processes and molecular functions associated with peach fruit development and response to external stimuli such as cold storage and gibberellic acid application. the findings from both methods contribute to a better understanding of the molecular processes governing complex phenotypes in peaches. the gene identification and sequence alignment method elucidated the diversity and potential functional divergence within gene families, providing insights into their evolutionary dynamics and functional specialization. the gene expression data analysis method revealed gene expression patterns and regulatory mechanisms underlying important developmental processes in peach. reference 1. meisel l, fonseca b, gonzález s, baeza-yates r, cambiazo v, campos r, et al. a rapid and efficient method for purifying high quality total rna from peaches (prunus persica) for functional genomics analyses. biological research. 2005;38(1):83-8. 2. durst rw, weaver gw. nutritional content of fresh and canned peaches. journal of the science of food and agriculture. 2013;93(3):593-603. 3. el-kahlout mi, abu-naser ss. peach type classification using deep learning. international journal of academic engineering research (ijaer). 2020;3(12). 4. 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becraft pw, stinard ps, mccarty dr. crinkly4: a tnfr-like receptor kinase involved in maize epidermal differentiation. science. 1996;273(5280):1406-9. 11. cucinotta m, manrique s, guazzotti a, quadrelli ne, mendes ma, benkova e, et al. cytokinin response factors integrate auxin and cytokinin pathways for female reproductive organ development. development. 2016;143(23):441924. 12. chuong eb, elde nc, feschotte c. regulatory activities of transposable elements: from conflicts to benefits. nature reviews genetics. 2017;18(2):71-86. highlights in bioscience page 10 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development 13. becker a, theißen g. the major clades of mads-box genes and their role in the development and evolution of flowering plants. molecular phylogenetics and evolution. 2003;29(3):464-89. 14. diao d, hu x, guan d, wang w, yang h, liu y. genomewide identification of the arf (auxin response factor) gene family in peach and their expression analysis. molecular biology reports. 2020;47:4331-44. 15. arús p, verde i, sosinski b, zhebentyayeva t, abbott ag. the peach genome. tree genetics & genomes. 2012;8:53147. 16. swarbreck d, wilks c, lamesch p, berardini tz, garciahernandez m, foerster h, et al. the arabidopsis information resource (tair): gene structure and function annotation. nucleic acids research. 2007;36(suppl_1):d1009-14. 17. mahram a, herbordt mc. ncbi blastp on highperformance reconfigurable computing systems. acm transactions on reconfigurable technology and systems (trets). 2015;7(4):1-20. 18. thompson jd, gibson tj, higgins dg. multiple sequence alignment using clustalw and clustalx. current protocols in bioinformatics. 2003;(1):2-3. 19. letunic i, bork p. interactive tree of life (itol) v4: recent updates and new developments. nucleic acids research. 2019;47(w1):w256-9. 20. alsamman am, el allali a, mokhtar mm, al-shamaa k, nassar ae, mousa kh, et al. alignstatplot: an r package and online tool for robust sequence alignment statistics and innovative visualization of big data. plos one. 2023;18(9):e0291204. 21. barrett t, wilhite se, ledoux p, evangelista c, kim if, tomashevsky m, et al. ncbi geo: archive for functional genomics data setsupdate. nucleic acids research. 2012;41(d1):d991-5. 22. li l, tao s, zhang h, huang w, dunwell jm, li m. identification and characterization of the ccoaomt gene family in apple, chinese white pear, and peach. journal of the american society for horticultural science. 2021;146(3):184-95. 23. pegoraro c, tadiello a, girardi cl, chaves fc, quecini v, costa de oliveira a, et al. transcriptional regulatory networks controlling woolliness in peach in response to preharvest gibberellin application and cold storage. bmc plant biology. 2015;15:1-14. 24. mering cv, huynen m, jaeggi d, schmidt s, bork p, snel b. string: a database of predicted functional associations between proteins. nucleic acids research. 2003;31(1):25861. 25. smoot me, ono k, ruscheinski j, wang pl, ideker t. cytoscape 2.8: new features for data integration and network visualization. bioinformatics. 2011;27(3):431-2. 26. pei j, grishin nv. al2co: calculation of positional conservation in a protein sequence alignment. bioinformatics. 2001;17(8):700-12. 27. chowdhury b, garai g. a review on multiple sequence alignment from the perspective of genetic algorithm. genomics. 2017;109(5-6):419-31. 28. li y, zhu y, yao j, zhang s, wang l, guo c, et al. genome-wide identification and expression analyses of the homeobox transcription factor family during ovule development in seedless and seeded grapes. scientific reports. 2017;7(1):12638. 29. arora r, agarwal p, ray s, singh ak, singh vp, tyagi ak, et al. mads-box gene family in rice: genome-wide identification, organization and expression profiling during reproductive development and stress. bmc genomics. 2007;8(1):1-21. 30. fang y, you j, xie k, xie w, xiong l. systematic sequence analysis and identification of tissue-specific or stress-responsive genes of nac transcription factor family in rice. molecular genetics and genomics. 2008;280:54763. 31. ohashi-ito k, fukuda h. hd-zip iii homeobox genes that include a novel member, zehb-13 (zinnia)/athb-15 (arabidopsis), are involved in procambium and xylem cell differentiation. plant and cell physiology. 2003;44(12):1350-8. 32. hirakawa y, kondo y, fukuda h. tdif peptide signaling regulates vascular stem cell proliferation via the wox4 homeobox gene in arabidopsis. the plant cell. 2010;22(8):2618-29. 33. xu x, lou y, yang k, shan x, zhu c, gao z. identification of homeobox genes associated with lignification and their expression patterns in bamboo shoots. biomolecules. 2019;9(12):862. 34. liu y, cui s, wu f, yan s, lin x, du x, et al. functional conservation of mikc*-type mads box genes in arabidopsis and rice pollen maturation. the plant cell. 2013;25(4):1288-303. 35. alvarez-buylla er, liljegren sj, pelaz s, gold se, burgeff c, ditta gs, et al. mads-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. the plant journal. 2000;24(4):457-66. highlights in bioscience page 11 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ rizk, 2023 identifying and characterizing key gene families influencing prunus persica plant development 36. hu j, chang x, zhang y, yu x, qin y, sun y, et al. the pineapple mads-box gene family and the evolution of early monocot flower. scientific reports. 2021;11(1):849. 37. endo h, yamaguchi m, tamura t, nakano y, nishikubo n, yoneda a, et al. multiple classes of transcription factors regulate the expression of vascular-related nacdomain7, a master switch of xylem vessel differentiation. plant and cell physiology. 2015;56(2):242-54. 38. yamaguchi m, ohtani m, mitsuda n, kubo m, ohmetakagi m, fukuda h, et al. vnd-interacting2, a nac domain transcription factor, negatively regulates xylem vessel formation in arabidopsis. the plant cell. 2010;22(4):1249-63. 39. zhao c, lasses t, bako l, kong d, zhao b, chanda b, et al. xylem nac domain1, an angiosperm nac transcription factor, inhibits xylem differentiation through conserved motifs that interact with retinoblastoma-related. new phytologist. 2017;216(1):76-89. 40. hendelman a, stav r, zemach h, arazi t. the tomato nac transcription factor slnam2 is involved in flowerboundary morphogenesis. journal of experimental botany. 2013;64(18):5497-507. 41. takeda s, hanano k, kariya a, shimizu s, zhao l, matsui m, et al. cup-shaped cotyledon1 transcription factor activates the expression of lsh4 and lsh3, two members of the alog gene family, in shoot organ boundary cells. the plant journal. 2011;66(6):1066-77. 42. xiang y, cao y, xu c, li x, wang s. xa3, conferring resistance for rice bacterial blight and encoding a receptor kinase-like protein, is the same as xa26. theoretical and applied genetics. 2006;113:1347-55. 43. zhang x, wang l, yuan y, tian d, yang s. rapid copy number expansion and recent recruitment of domains in sreceptor kinase-like genes contribute to the origin of selfincompatibility. the febs journal. 2011;278(22):4323-37. 44. saintenac c, lee ws, cambon f, rudd jj, king rc, marande w, et al. wheat receptor-kinase-like protein stb6 controls gene-for-gene resistance to fungal pathogen zymoseptoria tritici. nature genetics. 2018;50(3):368-74. 45. zhao d, ni w, feng b, han t, petrasek mg, ma h. members of the arabidopsis-skp1-like gene family exhibit a variety of expression patterns and may play diverse roles in arabidopsis. plant physiology. 2003;133(1):203-17. 46. zhao l, huang j, zhao z, li q, sims tl, xue y. the skp1like protein ssk1 is required for cross-pollen compatibility in s-rnase-based self-incompatibility. the plant journal. 2010;62(1):52-63. 47. kaur c, sharma s, singla-pareek sl, sopory sk. methylglyoxal, triose phosphate isomerase, and glyoxalase pathway: implications in 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page 12 of 12 september 2023|volume 6 http://bioscience.highlightsin.org/ abstract introduction methods gene identification and sequence alignment gene expression data analysis results and discussion gene identification and sequence variationa protein-protein interaction analysis gene differential expression analysis conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202301 research article open access 1 vegetable research department, horticulture research institute, agricultural research center, giza, egypt. 2 institute of environmental studies, arish university, north sinai, egypt. 3 plant production department, faculty of environmental agricultural sciences. arish university, north sinai, egypt. 4 department of family and community health nursing, faculty of nursing, suez canal university, ismailia, egypt. 5 department of food technology, faculty of agriculture, suez canal university, ismailia, egypt, p.o. box 41522. * to whom correspondence should be addressed: mobark_mohamed99@yahoo.com editor: aladdin hamwieh, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): khaled h. radwan, agricultural genetic engineering research institute (ageri), agricultural research center (arc), giza, egypt. tawffiq istanbuli, international center for agricultural research in the dry areas (icarda), beirut, lebanon received: october 20, 2022 accepted: january 3, 2023 published: january 26, 2023 citation: abuo el-kasem saa, naiel mhf, mubarak mh, megahed fia, el-deeb gss. assessment of pesticide residues in vegetables selected from different egyptian governorates. 2023 jan 26;6:bs202301 copyright: © 2023 abuo el-kasem et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of pesticide residues in vegetables selected from different egyptian governorates sameh a. a. abuo el-kasem 1 ><, mohamed h. f. naiel 2 ><, mohamed h. mubarak *,3 >< , fatma i. a. megahed 4 >< , gehad s. s. el-deeb 5 >< abstract this study aimed to assess the levels of contamination by pesticide residues in several types of vegetables collected from different regions in egypt. a total of 100 samples of vegetables (pepper, tomato, cucumber, and strawberry) were collected from markets in five cities (al-obour, al-salheia el-gadida, giza, zagazig, and fayed) and analyzed for the presence of 42 different pesticide residues. the quick, easy, cheap, effective, rugged, and safe (quechers) method was used to extract the target pesticides, which were then quantified using gas chromatography-mass spectrometry (gc-ms/ms) and liquid chromatography-mass spectrometry (lc-ms/ms) techniques. the results showed that 72% of the vegetable samples contained detectable levels of pesticide residues, with 21% exceeding the european union maximum residue levels (eu-mrls) and 51% containing residues below the mrls. the detected residues were primarily insecticides (56.4%) and fungicides (43.6%), with tomato and strawberry samples showing the highest frequency of both types of pesticides. tomato also had the highest absolute intake from consumption (2.89 g/kg bw/day), followed by strawberries, peppers, and cucumbers (0.47, 0.159, and 0.096 g/kg bw/day, respectively). a hazard index (hi) was used to assess the dietary risk posed by the pesticide residues, with tomato having the highest contribution value. these findings highlight the need for integrated pest management (ipm) programs to reduce the excessive use of pesticides, particularly in relation to raw food commodities. action is required to minimize the unacceptable risks identified in this study. keywords: food safety, pesticide residues, risk assessment, estimated daily intake, monitoring introduction fruit and vegetables have been a cornerstone of healthy dietary recommendations. they have potential health-promoting effects beyond providing basic nutrition needs in humans, including their role in reducing inflammation and their potential preventive effects on various chronic disease states such as cardiovascular disease and cancer leading to premature mortality decreasing years loss of individuals’ life and years-to-come lived with disability/morbidity. consumers are now choosing fruits and vegetables not only for their content of vitamins, minerals and fiber, but also for their concentration of dietary bioactive with its anti-inflammatory effects [1]. in agriculture, pesticides are considered a quick, and easy solution for controlling weeds and insect pests, improving production and productivity of agriculture commodity to feed the ever growing population, controlling vector borne disease like malaria and reducing the resultant mortality and morbidity. surprisingly; the global consumption of pesticides is about two million tons per year and out of which 45% is used by europe alone, 25% is consumed in the usa, and 25% in the rest of the world. despite their benefits, pesticides can be hazardous to humans and environment and non-intended organisms ranging from beneficial soil microorganisms to insects, plants, fish and birds. environmental contamination or prevailing use of pesticides can expose the general population to pesticide residues leading to serious and prolonged toxicity. it was estimated that a minimum of 300,000 people die from pesticide poisoning each year, with 99% of them from lowand middle-income countries in 2009 [2]. highlights in bioscience page 1 of 13 january 2023|volume 6 https://doi.org/10.36462/h.biosci.202301 https://creativecommons.org/licenses/by/4.0/ samehaoelkaseem7@gmail.com https://orcid.org/0000-0001-8275-0945 mohamednail123@yahoo.com https://orcid.org/0000-0003-1850-1308 mobark_mohamed99@yahoo.com https://orcid.org/0000-0002-2981-381x dr.fm_2013@yahoo.com https://orcid.org/0000-0003-3255-1926 gehadeldeeb@yahoo.co.uk https://orcid.org/0000-0002-9132-8508 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates pesticides in food are monitored by the environmental protection agency (epa) and the food and drug administration (fda). one of the top priorities in securing and preserving community and public health, is food safety. food safety is particularly important to ensure the healthiness of food, especially to fruits and vegetables as they are consumed in substantial and considerable quantities customarily without any processing. they are susceptible to pests at any point in the production chain, from the field through storage ahead till food consumption. their pesticide residues are yet present in the vegetable-treated products, which may constitute a potential hazards for consumers. some of these hazards were identified in fruits and vegetables were because of incorrect application of pesticides either by the producers’ application or the insufficient monitoring of the contaminated soil and/or water [3]. major causes of the environment pollution were from prejudicial human activities and improper application, spillage, and decomposition. however and despite all precautions, a very minute amount of pesticide-residues can remain in the treated crop. the maximum residue level (mrl), the maximum quantity of residue that is legally permitted on a food material, ensures that both imported and exported goods are moderately safe to consume. pesticide misuse, false positives from naturally occurring compounds, variances in national mrls standards, a lack of registered pesticides, and improper pesticide application can all cause mrls to be exceeded [4]. the sources of the mrls, however, can affect the frequency of infractions. however, the creation of mrls is based on information from supervised trials mean residues (stmr), acceptable daily intake (adi), acute reference dose (arfd), as well as data from good agricultural practice (gap) that has been registered nationally. the percentages of violation will be very different, for instance, because the maximum permissible residue of profenofos on tomatoes is 10 mg/kg in the codex and 0.01 mg/kg in european standards. in order to determine the actual risk of exposure, it is crucial to compare the findings with a more reliable toxicological endpoint, such as adi or arfd. the egyptian ought to take into account creating mrls based on regional best practices for agriculture and locally administered paths. the major tool for ensuring that the pesticides were applied in assembly with good agricultural practices is the monitoring program. these programs when applied in conformity with good agricultural practices, treated goods should not have exceeded levels of pesticide residue [5]. the health risks of pesticides are regularly evaluated through monitoring programs for eu nations. according to the yearly dg sanco report, 47% of the fruits, vegetables, and grains eaten in europe in 2004 had pesticide residues [6]. pesticides that can be used in certain foods and feed commodities have maximum residual limitations, or tolerances, determined by the environmental protection agency (epa). these restrictions are put in place to safeguard people from hazardous pesticide levels in their food [7]. a variety of pesticides, including organochlorine, organophosphorus, carbamate, insecticides, fungicides, and herbicides, are used by farmers all over the world to prevent the devastating crop loss that can result from pests and diseases as well as to boost agricultural productivity to ensure a sufficient supply of food for the expanding population [8]. food contamination creates severe health issues worldwide, ranging from minor ailments to fatal ones [9]. therefore; it is well established that contaminated food poses a risk to the general public health. however; producing, processing, moving, and handling food can all lead to food contamination [10; 11; 9; 12]. there are two types of pollution: short-term pollution at high concentrations of chemicals (induced by inadvertent release or contamination from the source) and long-term pollution of low concentrations of chemicals (produced by the progressive diffusion of pollutants in food) [13]. different chemical classes or families typically produce dissimilar symptoms. the amount of pollutants in food ingested in relation to the daily amount of food can be used to estimating the level of pollutants in the human body [14]. numerous studies have demonstrated that persistent organic pollutants, such as organochlorine insecticides, have a variety of negative impacts, including aberrant immune system development, birth abnormalities, and foetal death [15; 16]. because of this, pesticides are regarded as one of the world’s top environmental and health risks [17]. many nations and international organizations, including the european union, the world health organization, and the united nations environment program, have acclaimed that both organic and inorganic pollutants pose a serious risk to health, particularly the health of children [18]. they have gradually released a number of suggestions or guidelines intended to limit or outlaw the use of these pollutants or pollutant products. for instance, the amount of lead in the environment has decreased as a result of several countries banning the addition of tetra-methyl lead to gasoline [19]. for consumers protection from exposure to unacceptable levels of pesticide residues in food and feed, the european commission has set maximum residue levels (mrls), defined as the highest possible level of a pesticide residue that is legally authorized in food and feed. based on the results obtained from environmental sample analysis, it has been proven that the modified quechers method coupled to gas chromatography gc-ms/ms with electron capture detector (gc-ecd) which are analytical procedures for routine analysis and simultaneous determination of selected electronegative pesticides in fruits and vegetables with high water content. these procedures are suitable not only for fruits and vegetables with high water content, but also for samples containing large amounts of pigments and dyes [3]. for risk assessment analyses that could be linked to accidental intakes of contaminants at very high levels and could have severe unfavorable effects on the human body, quantitative data on the concentration of contaminants in food are an essential tool [20] . though pesticides assist increase crop productivity, the amount and variety of food consumed, and likewise the development of some diseases [21]. pesticides can be categohighlights in bioscience page 2 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates rized according to their unique biological activity or their target species, such as fungicides, herbicides, insecticides, and acaricides, in addition to functional groups [22; 23]. the maximum daily consumption that a person is permitted to consume during their lifetime without posing a significant risk to that person has been determined by numerous health and environmental protection agencies as "acceptable daily intake" (adi) levels. environmental pollution is, without any doubts, a serious global concern. many nations have made positive efforts to limit the use of pesticides. procedures and approaches are utilized to evaluate the detrimental impacts brought on by pollutants as a result of pollutant risk assessment. to evaluate past, present, and future exposure to any environmental pollutants, a risk assessment can be carried out. the majority of the time, analyses of material hazards are based on scientific research on the activities, exposure, quantity, and toxicity of chemicals. the amount of pollutants present in the environment, food, and/or products; the number of people exposed to the pollutants; and the damages of pollutants all affect the risks [24]. many organochlorine pesticides have been outlawed or have had their usage severely restricted in europe and north america, yet they are still sold and used in africa. residential pesticide use in egypt is high. adolescents in egypt are exposed to pesticides through non-occupational and para-occupational pathways. in addition to the hormonal and physiological changes associated with puberty, there are also significant developmental changes in the brain, primarily the prefrontal cortex. the impact of environmental exposures can vary across developmental periods and consequences of prolonged exposure can last into adulthood [25]. the purpose of this study was to assess the levels of a group of pesticide residues in the commonly consumed vegetables in egypt, and to evaluate their health risk according to estimated quantity of exposure. material and methods samples a total of 100 vegetable samples (pepper, tomato, cucumber, and strawberry) were collected from local markets of five egyptian cities: al-obour (qalyubia governorate), al-salheia elgadida (sharqia governorate), giza (giza governorate), zagazig sharqia (governorate), and fayed (ismailia governorate)). each representative vegetable sample was made up of 10 identical commodity subsamples that were simultaneously obtained from each market using random sampling. vegetable samples were packed in proper bags and stored at 4oc until analysis. sample preparation and analysis about 2 kg of each vegetable sample (pepper, tomato, cucumber, and strawberry) was thoroughly washed with tap water, chopped and blended using a waring laboratory blender. each sample was chopped and ground in accordance with the generally suggested procedure described by the codex alimentarius commission in 1993 for no more than two days prior to analysis. according to anastassiades et al., [26] the quechers method was used to extract pesticides from the vegetable samples. a 50 ml polypropylene (pp) tube containing 10 gm of each sample was weighed, 10 ml of acetonitrile was added, and the tube was forcefully shaken for one minute. phase separation was achieved by centrifuging the liquid at 4000 rpm for 5 min after adding buffering citrate salts (ph 5 to 5.5), containing 4g of magnesium sulphate, and 1g of sodium chloride. for analysis, an aliquot of the organic phase was directly loaded into lc-ms/ms. dispersive solid phase extraction (dspe). extracts from the samples were evaporated and then redissolved along with injection standard for gc-ms/ms analysis after cleaning with primary secondary amine sorbent (psa). aldrin was used as an internal standard for quantification, and it was added to the gc-ms/ms system right before injection. gc ms/ms and lc-ms/ms were used for the identification and confirmation of pesticide residues in the samples. calculation of adi and hi comparing the established acceptable daily intake (adi) with the estimated acceptable daily intake (edi), which is based on the concentration of pesticide residues and food consumption, gives the risk assessment. the edi (mg/kg bw/day) for each pesticide residue that was violated was computed by multiplying the mean pesticide residue concentration (mg/kg) x food consumption and then dividing by the typical adult’s body weight (60 kg) of each commodity. based on gems/foods from the who’s global environment monitoring system [27], acceptable daily intake was determined. edi = concentration o f pesticideresidue × food consumed body weight (1) the daily consumption rate of vegetables was derived conclusively for this study from the reports of who/ fao [28], who/ global environment monitoring system-food contamination, monitoring and evaluation program average consumption cluster diets [27], and gad alla et al., [29]. if data from food balance sheets are unavailable for a commodity, the consumption level for a comparable food is used (who 1997). because there isn’t a strawberry consumption rate available, the consumption level of a comparable food is used. the scientific names and daily intake rate (g/day) for the used vegetable samples are given in table 1 . the eu pesticides database served as the source for both the maximum residue limits (mrls) and the established acceptable daily intake (adi) values. using the health risk index, the health risk for consumers from consuming pesticide-contaminated samples was described (hi). it is calculated by dividing the estimated daily intake (edi) by the corresponding values of the acceptable daily intake (adi in mg/kg) specified by who/fao as stated in the equation 2: hi = edi adi 100 (2) highlights in bioscience page 3 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 1. scientific names and consumption rate of studied commodities in g/day based on gems/food total diet food balance sheet. consumption rate (g/day) based on who/global environment monitoring systemfood contamination, monitoring and assessment program average consumption cluster c diets[27]. common name scientific name family name crop type consumption (g/day)* cucumber cucumis sativas l. cucurbitaceae vegetable 5.9 pepper capsicum annum l. solanaceae vegetable 13 strawberry fragaria ananassa l. rosaceae vegetable 20 tomato solanum lycopersicum solanaceae vegetable 118 according to the european food safety authority (efsa) [30; 31] el-sawy et al. and [32]. when the hi is less than 100%, the food concerned is considered acceptable. if it is above 100%, the food concerned is considered a risk to the consumer [33]. results and discussion use of excessive pesticides contaminates soil, water and finally enters the food chain and contaminates the food produced. the international agency for research on cancer has found sufficient evidence of carcinogenic potential in most of the pesticides beyond the threshold limit. the united nations environment program estimates accidental pesticide poisoning causing 20, 000 deaths and 1 million cases of illness per year worldwide [34]. forty-two commonly used pesticides in agriculture were identified in this study. the broad scope analyzed includes numerous groups of pesticides such as organophosphorous, organo– chlorine, pyrethroids and other groups of pesticides widely used or outlawed in egypt. according to a directive issues by egypt’s agriculture pesticides committee (codex+eu), pesticide residue levels should be compared to codex alimentarious when it is available and to eu-mrls when codex mrls are not accessible. in this investigation, only the codex alimentarious mrls and the agriculture pesticides committee decree were used to compare the monitoring data. table 2 listed the number of samples evaluated, the range of pesticides found, the average in mg/kg, the number of chemicals violated in the samples analyzed, and the status of each pesticide/commodity combination in the registration system established by the agricultural pesticide committee (apc). a number of 32 out a total of 42 pesticides were detected in strawberry fruits. fluopyram had the highest pesticide concentration in the samples, whereas iprodion had the lowest pesticide residue (table 2). data revealed that 13 (52%) of the strawberry fruit samples had no detectable pesticide residues. whereas, a total of 12 samples (48%) contained pesticide residues, of which 10% were contaminated samples and contained residues at levels below the mrls, and 8% had residues over the allowed limits (table 2). however, according to the apc regulation, the breach was found in 8% of cases when comparing the results to (codex + eu restrictions). bifenazate, methamidophos, fluopyram, metalax, captan, propargite, and pyrimethanill are seven pesticide residues that recorded greater amounts than their regulated eu mrl values (table 2 and figure 1). the discovered quantities of pyrimethanill contamination (loq to 0.076, average 0.048 mg/kg) were not too far from the mrl (0.05 mg/kg). otherwise, the residues of both fluopyram and methamidophos exhibited a serious issue because their concentrations in strawberries exceeded their mrl values by 4 and 9 fold, respectively. this indicates that it is necessary to regulate their use. figure 1. the percentage of detected pesticide residues in samples based on pesticide type. regarding the outcomes of the tomato samples, which were the second crop of the vegetables under study, roughly 17 pesticides were detected in the tomato samples (25), as shown in table 3. among the pesticides found, only 8 (bifenazate, captan, fluopyram, chlorfenapyr, chlorpyrifos, dimethoate, lambdacyhalothrin, and thiofanat-methyle) had concentrations greater than the mrls reported for tomato (table 2 and figure 2). however, data in table 3 revealed that 28% (7) of tomato fruit samples had no detectable pesticide residues (25). while pesticide residues were discovered in 18 samples (72%) of which 48% (12 samples) were contaminated tests recorded residual amounts comparable to the worldwide mrls for them in tomato and 24% had residues over the allowed levels, pesticide residues were also discovered in other samples. fenarimol fungicide, followed by malathion insecticide, had the highest pesticide mean in samples, whereas fenpropathrin and metalaxyl had the lowest pesticide residue levels (table 4). however, pesticide residues were discovered in 21 samples (84%), of which 36% (9 samples) had residues over the allowed limits and 48% (12 samples) of contaminated samples had residues at levels higher than the mrls (table 4). according to the decision of the agriculture pesticides committee in egypt, a violation was found in one sample when results were compared to codex and eu limitations, and in the other eight samples when findings were compared to solely eu limits. data in table 5 regarding cucumber fruits showed that the majority of the investigated pesticides (12 out of 42) were detected in cucumber samples. only 2 insecticides (abamectin & acetamiprid) and 2 fungicides (captan & penconazole) out of the detected 12 pesticides had concentrations greater than the mrls indicated for cucumber, as shown in table 5. highlights in bioscience page 4 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates figure 2. frequency percentages of most detected pesticide residues in vegetable samples. in total, 16% of the cucumber samples had no detectable pesticide residues, while the remaining 84% contained detectable residues which 68% of contaminated samples contain resides at levels lower than the mrl’s and 16 % (4 samples) had residues above the permissible limits as shown in figure 3. the mrls are often set well below the thresholds deemed safe for humans. it is important to understand that mrls are not safety limits; food residues might have levels beyond mrls while still being safe to eat [35]. according to ifoam [36], mrls are not a guarantee of "zero health risk" in this situation; rather, they are only indicators of whether or not good agricultural practices (gap) are being violated, not a sign of a health danger. risk exposure should therefore be assessed using toxicological endpoints like acceptable daily intake (adi). when it comes to earlier egyptian investigations, the pattern of pesticides in tomato indicated that the detection levels of dimethoate, pirimiphos-methyl, and profenofos were 0.461, 0.114, and 0.28, respectively [37]. figure 3. the contamination and the violation percentages per each commodity of vegetables. otherwise, greater residual levels of profenofos and malathion were found in tomato samples taken from various areas in egypt, according to dogheim et al., [38]. additionally, greater profenofos residue levels were found in strawberry samples taken from eight local markets in six egyptian governorates [39; 40; 38], but not in tomato or strawberry samples taken for this study. the organophosphorus pesticides (thiometon, phorate, and chlorpyrifosmethyl) were found in cucumbers in a different investigation by mansour et al., [41] for the monitoring of pesticides and heavy metals. the levels of pesticides in some vegetables gathered from neighborhood markets in cairo governorate were assessed by farag et al., [42]. according to their findings, strawberries had the highest levels of contamination with various pesticides, with mean contamination levels of 0.034, 0.023, 0.033, 0.024, and 0.050 mg/kg, respectively, for ethion, propargite, permethrin, profenofos, and chlorpyrifos. pepper was found to contain only two different types of insecticides (sulfur, methomyl). pesticides were not found in tomatoes or cucumbers. additionally, ibrahim et al., [43] assessed the pesticide residues in certain vegetables purchased at local marketplaces in eight governorates around egypt. they came to the conclusion that the reported negative samples for pepper and cucumber were 19.4% and 27.9%, respectively. for pepper and cucumber, respectively, the recorded positive samples were 80.6% and 72.1%. according to badr et al., [44] , who assessed the pesticide residues in egyptian crops, profenophos measured pesticide levels in tomato and cucumber at 0.56 and 0.28 mg/kg, respectively. the levels of pesticide residue found in vegetable samples by loutfy et al., [45] and ahmed et al., [46] are consistent with our findings. according to dogheim et al., [38] , the relatively limited amount of pesticides used in the research areas and the widespread awareness and usage of integrated pest management (ipm) programs may be responsible for this low contamination level. conhighlights in bioscience page 5 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 2. pesticide residues levels found in strawberry fruits, frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu acetamiprid 1 0.039 0.039 0.039 0.5 0.3 r-nri azoxystrobin 7 0.012 0.12 0.054 10 2 r-nri bifenazate 4 0.012 0.25 0.083 2 0.1 1 r-ri bifenazole 3 0.01 0.029 0.016 0.1 nr boscalid 6 <loq 0.11 0.043 0.9 r-nri buprofezin 1 0.081 0.081 0.081 3 3 nr captan 5 0.017 0.294 0.133 15 0.1 2 r-ri carbendazim 2 0.02 0.07 0.045 0.5 0.1 banned, 2018 chlorantraniliprole 1 <loq <loq <loq 0.3 1 r-nri chlorpyrifos 3 <loq <loq <loq 0.3 0.01 r-nri cyproconazole 1 0.017 0.017 0.017 0.01 0.05 nr difenoconazole 4 <loq 0.042 0.031 2 1 r-nri fluopyram 2 0.031 0.44 0.236 0.4 0.1 1* r-ri flutazole 1 <loq <loq <loq na 0 nr flutriafol 1 0.021 0.021 0.021 1.5 1.5 r-nri imidacloprid 2 <loq <loq <loq 0.5 0.5 r-nri indoxacarb (p) 3 0.013 0.06 0.024 0.02 0.6 r-nri iprodion 2 0.012 0.013 0.013 10 0.05 banned, 2018 lambda-cyhalothrin 2 <loq <loq <loq 0.02 0.02 r-nri metalax 3 0.01 0.09 0.033 0.5 0.05 1 r-nri methamidophos 3 0.068 0.097 0.078 0.01 0.01 3* nr, 2007 methoxyfenozide 3 0.038 0.076 0.059 2 2 r-nri paclobuttazol 1 <ioq <ioq <ioq na 0.01 nr penconazole 1 0.018 0.018 0.018 0.5 0.1 r-nri pirimicarb 1 0.11 0.11 0.11 0.5 r-nri propargite 5 <loq 0.095 0.067 2 0.05 2 nr propiconazole 2 0.01 0.046 0.028 3 0.05 nr pyraclostrobin 4 0.01 0.053 0.022 1.5 0.1 r-ri pyrimethanil 3 <loq 0.076 0.048 0.1 0.05 2 r-ri spirodiclofen 2 <loq 0.025 0.013 2 0.5 r-nri tetraconazole 1 <loq <loq <loq na 0.2 r-nri thiophanate.mathyle 2 0.038 0.068 0.053 na 0.1 r-ri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended, eapc=egyptian agriculture pesticide committee , vc=violated compound table 3. pesticide residues levels found in tomato, frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu abamectin 1 0.010 0.010 0.010 0.05 0.09 r-ri acetamiprid 3 0.010 0.010 0.010 0.2 0.5 r-ri azoxystrobin 3 0.014 0.022 0.015 3 3 r-ri bifenazate 5 <loq 0.16 0.160 0.5 (2007) 0.1 2 r-ri boscalid 2 0.010 0.030 0.020 x 3 r-nri captan 4 0.050 0.368 0.228 5 (2008) 0.1 2 r-nri chlorfenapyr 7 0.010 0.071 0.022 0.4 (2019) 0.01 5 r-ri chlorpyrifos 5 0.021 0.053 0.036 1 0.01 5 r-ri cyfluthrin 1 <loq <loq <loq 0.2 (2008) 0.05 nr difenoconazole 3 <loq <loq <loq 0.6 2 r-ri dimethoate 3 0.010 0.020 0.017 na 0.01 2 r-nri fluopyram 2 0.100 0.410 0.255 0.5 0.1 1* r-nri indoxacarb 6 0.010 0.040 0.022 0.5 0.5 r-ri lambda-cyhalothrin 7 <loq 0.110 0.060 na 0.01 2 r-nri methoxyfenozide 7 0.010 0.062 0.024 2 (2005) 2 r-ri thiofanat-methyle 4 0.080 1.200 0.570 x 1 1 r-nri trifloxystrobin 6 <loq 0.010 0.010 0.7 (2006) 0.01* r * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended, eapc=egyptian agriculture pesticide committee , vc= violated compound table 4. pesticide residues levels found in pepper , frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu chlorofenapyr 1 0.01 0.01 0.01 0.05 0.01 r-nri chloropyrifos 7 0.01 0.07 0.03 2 0.02 5 r-nri dimethoate 1 0.03 0.03 0.03 0.5 0.01 r-nri fenarimol 5 <loq 0.3 0.22 0.5 0.1 3 r-nri fenpropathrin 1 <loq <loq <loq 1 0.01 r-nri fluopyram 2 0.03 0.05 0.04 3 0.1 r-nri imidacloprid 1 0.09 0.09 0.09 1 0.5 r-nri malathion 1 0.2 0.2 0.2 0.1 0.01 1* r-nri metalaxyl 2 <loq <loq <loq 1 1 r-nri methoxyfenozide 1 0.06 0.06 0.06 2 2 r-nri spinetoram 4 0.01 0.07 0.04 0.4 0.5 r-ri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, eapc=egyptian agriculture pesticide committee , vc=violated compound highlights in bioscience page 6 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 5. pesticide residues levels found in cucumber , frequencies, their corresponding mrls, number of violated samples and the status of registration of each detected pesticide in analyzed samples collected from different governorates during 2020. active ingredient freq. residues (mg/kg) mrl vc (>mrl) eapc min max mean codex eu abamectin 6 0.01 0.07 0.04 0.03 0.04 2* r-ri acetamiprid 9 0.06 0.1 0.12 0.3 0.3 1* r-ri captan 5 <loq 0.7 0.21 3 0.1 2 r-nri chloropyrifos 3 0.03 0.04 0.03 0.05 0.05 r-nri cypermethrin 2 0.17 0.2 0.18 0.2 0.2 r-nri difenoconazole 3 <loq <loq <loq 0.2 0.2 r-ri fenpropathrin 1 0.03 0.03 0.03 0.5 0.5 r-nri fluopyram 1 0.07 0.07 0.07 0.5 0.1 r-nri l-cyhalothrin 2 0.03 0.04 0.03 0.1 0.1 r-nri malathion 1 0.01 0.01 0.01 0.2 0.02 r-ri metalaxyl 3 0.16 0.3 0.23 0.5 0.5 r-ri penconazole 4 0.01 0.04 0.02 0.06 0.01 2 r-nri * mrl of eu+ codex alimentarious, others were of eu, freq= number of pesticides found on the commodity, r= registered, r1= recommended, nr= not registered, nr1= not recommended , eapc=egyptian agriculture pesticide committee , vc=violated compound trarily, numerous pesticides, including those forbidden in egypt, are nevertheless found in a variety of environmental components, including groundwater, surface water, fished, mussels, medical plants, soils, and sediments [47; 48; 49; 50; 46]. additionally, some pesticides were found in only one crop, as shown in the corresponding tables for the crops of strawberry (13 pesticides), tomato (2, cyfluthrin & trifloxystrobin), and pepper (2, fenarimol & spinetoram), despite the fact that many common pesticides were found in multiple crops in some of the samples used for the current investigation. in light of these findings, a distinction between the types of pesticide residue found on the egyptian samples during the current investigation and those conducted previously was noted. the dominant pesticide group was adjusted in addition to the pesticide itself. a regular pesticide mentoring survey should therefore be recommended because it is related to the evaluation of the safety of fruits used in human food. the ministry of agriculture’s various control programs, which vary from one field and/or location to another depending on the type of injury and the different environmental conditions, may be to blame for the high incidence of the majority of tested pesticides in some vegetable samples of the current investigation. pesticide frequencies (detected pesticides type) according to figure 1, the types of pesticide residues that were found in vegetable samples were insecticides and fungicides, with percentages of 55.75% and 44.25%, respectively. insecticides were found in descending order at frequencies of 21.2, 17.05, 8.75, and 8.75% in tomatoes, strawberries, cucumbers and peppers. in descending order, fungicides with rates of 20.74, 10.60, 9.68, and 3.23% were found in strawberries, tomatoes, cucumbers, and peppers. but compared to pepper and cucumber, strawberry and tomato had the highest frequencies of both insecticides and fungicides. these findings contrasted with those of ozowicka et al., [51] who discovered that fungicides were used four times as frequently as insecticides, and were generally consistent with finding of gad alla et al., [52] . in addition, figure 2 lists the nineteen (19) pesticides that were found most frequently in egyptian vegetables examined in 2020, listed in order of frequency percentages: chlorpyrifos (8.3%), captan (6.5%), acetamiprid (6.0%), lambda-cyhalothrin (5.1%), methoxyfenozide (5.1%), azoxystrobin (4.6%), difenoconazole (4.6%), bifenazate (4.1%), indoxacarb (4.1%), boscalid (3.7%), chlorfenapyr (3.7%), metalaxyl (3.7%), abamectin (3.2%), fluopyram (3.2%), thiophanate.mathyle (2.8%), trifloxystrobin (2.8 %), penconazole (2.3%), propargite (2.3%) and fenarimol (2.3%), in addition to 23 other pesticides (11 insecticides, 9 fungicides, 2 acaricides and 1 herbicide) with a frequently number of 1-4 with a percentage < 2% (table 6). this may indicate a more intense use of insecticides than fungicides, which is inconsistent with pest control behavior in fruit and vegetable, due to fungal diseases that are expected to infect fruits.. however, the usage of numerous types and vast quantities of pesticides caused insects to become resistant, which led to the need of more pesticides overall. detected pesticides groups the detected pesticides could be categorized into a variety of groups, as shown by table 6 and figure 4, with the most frequent groups being organophosphorus ops, followed by dmifungicide, strobilurin, neonicotinoids, pyrethroids, pyridine carboxamides, phthalimides, and diacylhydrazines, with frequencies percentages of 12.44%, 9.68%, 9.22%, 7.37%, 7.3 the most often identified pesticide class in egyptian vegetables in 2020 was op’s (12.44%), which has been the case for a number of years. the majority of ops were found in vegetable samples from 2011 that were previously collected (41.5%; sohair et al., [29]); they were also found in fruit samples from 2007 that were previously collected (29.4%; gadallah et al., [52] ) and they are widely utilized in egypt. the effectiveness and low cost may be the causes of the product’s prolonged use in egyptian markets, and farmers find it difficult to change their usage patterns. concerns are raised by a review conducted by roshini et al., [53] that exposure to op pesticides at levels currently regarded as acceptable may have had a negative impact on human reproductive function and survival. contrarily, ops are hazardous to the nervous system and have been substantially eliminated from agriculture in several nations during the past ten years [52]. however, they are not outlawed and continue to be used on some food crops, according to the ewg’s 2012 list of the "dirty dozen" endocrine disruptor pesticides (the-dirty-dozen-eco-group, 2012). fungicides containing strobilurin have been used extensively in agricultural fields for many years. the frac group 11 fungicides known as strobilurin or qoi are very effective at controlling a variety of common vegetable diseases [54] . stabilurins’ highlights in bioscience page 7 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 6. the detected pesticide frequencies in analyzed egyptian vegetable and fruit samples, 2020. sn substance classified* chemical name type freq. no 1 organophosphate chlorpyrifos ins. 18 2 dimethoate ins. 4 3 malathion ins. 2 4 methamidophos ins. 3 5 pyrethroid cyfluthrin ins. 1 6 cypermethrin ins. 2 7 fenpropathrin ins. 2 8 l-cyhalothrin ins. 11 9 dmi** cyproconazole herb. 1 10 difenoconazole fung. 10 11 flutazole fung. 1 12 flutriafol fung. 1 13 penconazole fung. 5 14 propiconazole fung. 2 15 tetraconazole fung. 1 16 strobilurin azoxystrobin fung. 10 17 pyraclostrobin fung. 4 18 trifloxystrobin fung. 6 19 pyridinecarboxamides boscalid fung. 8 20 fluopyram fung. 7 21 neonicotinoids acetamiprid ins. 13 22 imidacloprid ins. 3 23 benzimidazole carbendazim fung. 2 24 thiophanate.m fung. 6 25 carbamates pirimicarb ins. 1 26 growth regulator paclobuttazol fung. 1 27 buprofezin ins. 1 28 avermectins abamectin ins. 7 29 acramite bifenazate acar, 9 30 anilinopyrimidines pyrimethanil fung. 3 31 arylepyrrole chlorfenapyr mite 8 32 diacylhydrazines methoxyfenozide ins. 11 33 diamides chlorantraniliprole ins. 1 34 dicarboximides iprodion fung. 2 35 imidazole bifenazole acar. 3 36 oxadiazine pesticide indoxacarb (p) ins. 9 37 phenylamide :acrylalanin metalaxyl fung. 8 38 phthalimides captan fung. 14 39 pyrimidines fenarimol fung. 5 40 spinosyns spinetoram ins. 4 41 sulfite ester propargite acar. 5 42 tetronic acids spirodiclofen acar. 2 total 217 *-types; acar, acarecides-ins, insecticidefung, fungicide -herb, herbicide, -substance classified is referred to: pan pesticide database, freq; the total number of frequencies for the positive samples of all vegetable under study, **dmi: demethylation inhibitors unique, non-target-specific fungicidal activity. previously, strobilurins were thought to be less hazardous to mammals [55], but a number of authors have noted that this is not entirely true due to gaps in the toxicological endpoints of fungicides [56]. these pesticides are intended to control fungi, but because of their broad-spectrum method of action, they can also have unintended consequences. therefore, strobilurin toxicity may result in ecosystem imbalance and food-web disturbance. the development of synthetic pyrethroids involved extensive chemical modifications that made them more toxic and less biodegradable in the environment [57; 58]. despite this, pyrethroids still have negative effects because they can lead to endocrine disruption, liver function impairment, and respiratory problems when exposed to them over an extended period of time. figure 4. the most detected pesticides groups in vegetable samples analysed during 2020. health risk assessment because of the potentially harmful effects, dietary exposure to pesticides raises health concerns. food quality and safety must be ensured in order to reduce the increased health risks associated with eating foods that contain pesticide residues. in order to reduce the presence of and exposure to pesticide residue, especially non-authorized pesticides, monitoring programs for the detection of pesticide residues in food should give enhanced health risk estimations for hygienic activities. table 0.4.3 clarifies approved daily intake (adi) and estimated daily intake (edi) for pesticides. strawberry, pepper, and cucumber had the lowest absolute intakes from ingesting the commodity, with 0.47, 0.159, and 0.096 g/kg b.w/day, respectively. tomato had the highest absolute intakes, with 2.89 g/kg bw/day (table 0.4.3 ). once more, to evaluate chronic exposure, the amount of pesticide exposure throughout a lifetime and any potential health impacts of highlights in bioscience page 8 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 7. pesticide estimated daily intake (edi), acceptable daily intake (adi) and hazard index (hi%). c om m odity e stim ated dietary intake (1e d i,(g /kg b.w /day) 2a d i h i pesticide straw berry tom ato pepper c ucum ber total (g /kg b w /day) (source;year) b ifenazate 0.028 0.315 0.342 10 jm pr -2006 & e fsa -2017 3.424 fluopyram 0.079 0.502 0.009 0.007 0.596 10 jm pr -2010 5.956 d im ethoate 0.033 0.007 0.040 2 jm pr -1995 1.997 a bam ectin 0.02 0.004 0.023 1 jm pr -2015 2.327 t hiophanate.m athyle 0.018 1.121 1.139 30 jm pr -2013 3.796 d ifenoconazole 0.01 0.01 0 0.020 10 jm pr -2007,2010,2013 0.205 c hlorpyrifos 0.002 0.07 0.007 0.003 0.082 10 jm pr -2009 0.819 c yperm ethrin 0.018 0.018 5 e fsa ,2018 0.362 propargite 0.022 0.022 10 jm pr -1999 0.222 b oscalid 0.014 0.039 0.054 40 jm pr -2006 0.134 indoxacarb 0.008 0.043 0.051 10 jm pr -2005 0.507 o thers 0.29 0.738 0.136 0.064 1.226 3.6606 1e d i:e stim ated daily intake based on vegetable consum ption data show n in table a. a d i:a cceptable daily intake,h i:h azard index = (e d i/a d i*100) jm pr is the jointfa o /w h o m eetings on pesticide r esidues and e fsa is e uropean food safety a uthority. **taking into consideration thatin the absence ofa d i,the value of10 g /kg b w /day is usually setby default such exposure must be taken into account [29]. this assessment method, which was carefully developed, considers average exposure levels in relation to the adi values determined for specific pesticides. the estimated daily intake (edi) for each pesticide was det independently in an exposure assessment. if consumers are exposed to chronic hazardous pesticide residues, their health won’t be at risk unless they consume more food than the adi each day for a long time. hazard index according to data in table 8, the hazard index (hi) for contaminated strawberry samples ranged from 0.0001% for chlorantraniliprole to 0.785% of the adi for fluopyram, while its values ranged from 0.015% for azoxystrobin to 5.015% for fluopyram in tomato samples, ranged from 0.0013% (metalaxyl) to 0.479% (fenarimol) in pepper samples, but ranged from 0.0003 to 0. the data from the risk assessment of pesticide residues in the tested commodities show that there is no risk associated with eating these vegetable. these findings are consistent with those made by akoto et al., [33] , who stated that when the hi value is less than 100%, the concerned commodity is regarded acceptable. if the hi value is larger than 100%, the concerned food is considered to pose a risk to consumers. according to recent statistics, fluopyram has a tomato hi value of 5.015 percent, which was the highest. due to the hi values, all tested pesticides had no individual potential health harm because the edi did not exceed (below the cut-off limit of 100). according to hossain et al., [59] , pesticide exposure in vegetables has minimal effects on consumers both individually and collectively, although it is possible that eating raw foods like cucumbers that haven’t been washed could increase the risk of pesticide exposure. additionally, seo et al., [60] revealed that the danger of exposure to pesticide residues found in dried vegetables gathered from seoul, korea, was minimal. the current findings demonstrated that there is no link between egyptian consumers’ longterm exposure to pesticide residues from eating raw vegetable and health risks. it should be noted that the current study is restricted to a select few vegetable. furthermore, rather than assessing the cumulative exposure to several pesticide residues in crops, the predicted risk assessment via long-term exposure is based on toxicological evaluation of the individual chemicals. contributed pesticides in total hazard index in order of contribution percentages (via consumption of a single commodity) in the analyzed egyptian vegetables during 2020, figure 5 lists the 13 high contributed pesticides, which include eight insecticides, one acaricide, and four fungicides. in order of decreasing contribution to the hi, the following pesticides were used: fluopyram (25.4%), thiophanate-mathyle (16.2%), bifenazate (14.6%), abamectin (9.9%), dimethoate (8.5%), chlorpyrifos (3.5%), methamidophos (2.8%), lambda-cyhalothrin (2.6%), captan (2.2%), indoxacarb (2.2%), and fenarimol (2.0%), cypermethrin (1.5%) and buprofezin (1.3%) in descending order, while each of the remaining 29 pesticides contributed less than 1% of the hi. figure 5. the most contributed pesticides in total hazard index (hi%). contributed commodities in total hazard index the calculated contributions of each commodity to the his are shown in figure 6, along with the commodities that make highlights in bioscience page 9 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates table 8. the estimated intake (edi) and the hazard index (hi) of pesticide residues in egyptian vegetable samples. pesticides detected strawberry tomato pepper cucumber edi hi edi hi edi hi edi hi abamectin 0.02 1.97 0.0036 0.361 acetamiprid 0.013 0.019 0.02 0.03 0.0118 0.017 azoxystrobin 0.018 0.009 0.03 0.02 bifenazate 0.028 0.278 0.31 3.15 bifenazole 0.005 0.054 boscalid 0.014 0.036 0.04 0.1 buprofezin 0.027 0.3 captan 0.044 0.044 0.45 0.45 0.0204 0.02 carbendazim 0.015 0.05 chlorantraniliprole 0.002 0.0001 chlorfenapyr 0.04 0.15 0.002 0.01 chlorpyrifos 0.002 0.017 0.07 0.7 0.007 0.07 0.0031 0.031 cyfluthrin 0.01 0.02 cypermethrin 0.0181 0.362 cyproconazole 0.006 0.028 difenoconazole 0.01 0.102 0.01 0.1 0.0005 0.005 dimethoate 0.03 1.67 0.007 0.33 fenarimol 0.048 0.48 fenpropathrin 0.001 0.003 0.003 0.01 fluopyram 0.079 0.785 0.5 5.02 0.009 0.09 0.0069 0.069 flutazole 0.002 0.017 flutriafol 0.007 0.07 imidacloprid 0.002 0.003 0.02 0.03 indoxacarb (p) 0.008 0.081 0.04 0.43 iprodion 0.004 0.007 lambda-cyhalothrin 0.002 0.008 0.12 0.59 0.0033 0.017 malathion 0.043 0.01 0.001 0.0003 metalaxyl 0.011 0.014 0.001 0.00125 0.0223 0.028 methamidophos 0.026 0.65 methoxyfenozide 0.02 0.02 0.05 0.05 0.013 0.01 paclobuttazol 0.002 0.008 penconazole 0.006 0.02 0.002 0.007 pirimicarb 0.037 0.183 propargite 0.022 0.222 propiconazole 0.009 0.013 pyraclostrobin 0.007 0.024 pyrimethanil 0.016 0.008 spinetoram 0.008 0.02 spirodiclofen 0.004 0.043 tetraconazole 0.002 0.042 thiophanate.mathyle 0.018 0.059 1.12 3.74 trifloxystrobin 0.02 0.05 edi: estimated daily intake, adi: acceptable daily intake, hi: hazard index = (edi/adi*100) jmpr is the joint fao/who meetings on pesticide residues and efsa is european food safety authority.**taking into consideration that in the absence of adi, the value of 10 g /kg bw/day is usually set by default up the majority of those contributions. tomatoes were the commodities that contributed the greatest to hazard (hi). as a result, they posed some risks to consumers and had exposures that were above acceptable limits, but generally, home processing methods including washing, heating, and frying may lower the levels of residues in vegetables. figure 6. the most contributed commodities in total hazard index (hi %). conclusion with the exception of levels of several insecticides, such as lambada-cyhalothrin (tomato), methamidophos (strawberry), and malathion (pepper), most pesticide residues were present in the most frequently consumed foods at below detectable levels, and those present above detection levels were in trace amounts. generally speaking, coordinated efforts are required to improve food quality and safety as well as to manage pesticide residues in both the environment and humans. the presence of prohibited pesticides in local markets needs to be addressed by the government authorities. implementing ipm programs correctly within the gap framework, creating new pesticide application methods, and shifting toward the production of organic foods will all assist sustainable development. further studies should be conducted to provide further evidence on the long-term health effects of pesticide exposure through diet. abbreviations adi: acceptable daily intake bw: body weight cccf: codex committee on contaminants in foods cifocoss: chronic individual food consumption database summary statistics efsa: european food safety authority highlights in bioscience page 10 of 13 january 2023|volume 6 http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates fao: food and agriculture organization of the united nations gc: gas chromatography gems/food: global environment monitoring system food contamination monitoring and assessment programme ipcs: international programme on chemical safety iso: international organization for standardization iupac: international union of pure and applied chemistry jecfa: joint fao/who expert committee on food additives jecfa: joint fao/who expert committee on food additives jmpr:: joint fao/who meeting on pesticide residues lod: limit of detection loq: limit of quantification ms: mass spectrometry ms/ms: tandem mass spectrometry quechers: quick, easy, cheap, effective, rugged, and safe usa: united states of america who: world health organization reference 1. wallace tc, bailey rl, blumberg jb, burton-freeman b, chen co, crowe-white km, et al. fruits, vegetables, and health: a comprehensive narrative, umbrella review of the science and recommendations for enhanced public policy to improve intake. critical reviews in food science and nutrition. 2020;60(13):2174-211. 2. gyawali k. pesticide uses and its effects on public health and environment. journal of health 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contamination and toxicology vol 196. 2008:1-51. highlights in bioscience page 12 of 13 january 2023|volume 6 http://www.who.int/foodsafety/publications/chem/regional_diets/en/ http://www.who.int/foodsafety/publications/chem/regional_diets/en/ https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/j.efsa.2013.3499 https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/j.efsa.2013.3499 www.intechopen.com/source/pdfs/13013. www.intechopen.com/source/pdfs/13013. http://infohub.ifoam.bio/sites/default/files/page/files/misconceptions_compiled.pdf http://infohub.ifoam.bio/sites/default/files/page/files/misconceptions_compiled.pdf http://bioscience.highlightsin.org/ abuo el-kasem et al., 2023 assessment of pesticide residues in vegetables selected from different egyptian governorates 42. farag r, abdel latif m, abd el-gawad a, dogheim s. monitoring of pesticide residues in some egyptian herbs, fruits and vegetables. international food research journal. 2011;18:659-65. 43. ibrahim n, eweis e, 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facts (reregistration eligibility decision fact sheet; 2006. available at url:. available from: http://www.epa.gov/oppsrrd1/reds/ factsheets/permethrin_fs.htm.5/26/09. 59. hossain m, fakhruddin a, chowdhury azm, rahman m, alam km. health risk assessment of selected pesticide residues in locally produced vegetables of bangladesh. international food research journal. 2015;22(1):110. 60. seo yh, cho th, hong ck, kim ms, cho sj, park wh, et al. monitoring and risk assessment of pesticide residues in commercially dried vegetables. preventive nutrition and food science. 2013;18(2):145. highlights in bioscience page 13 of 13 january 2023|volume 6 http://www.epa.gov/oppsrrd1/reds/cypermethrin_red.pdf. http://www.epa.gov/oppsrrd1/reds/cypermethrin_red.pdf. http://www.epa.gov/oppsrrd1/reds/factsheets/permethrin_fs.htm.5/26/09 http://www.epa.gov/oppsrrd1/reds/factsheets/permethrin_fs.htm.5/26/09 http://bioscience.highlightsin.org/ abstract introduction material and methods samples sample preparation and analysis calculation of adi and hi results and discussion pesticide frequencies (detected pesticides type) detected pesticides groups health risk assessment hazard index contributed pesticides in total hazard index contributed commodities in total hazard index conclusion abbreviations highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.20226 research article open access contacts of authors * to whom correspondence should be addressed: pramod prasad dahal 1 agriculture extension officer,governemnt of nepal,nepal. 2 agriculture and forestry university, agronomy, rampur, chitwan. 3 agriculture and forestry university, agronomy, rampur, chitwan. 4 nepal agricultural research council, singhdurbar plaza. revised: august 20, 2020 accepted: november 24, 2020 published: march 28, 2021 citation: dahal pp, basnet kb, sah sk and karki tb . effect of weather parameters on growth stages of winter maize explain the grain yield. 2021 mar 28;4:bs20226 copyright: © 2021 dahal et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. effect of weather parameters on growth stages of winter maize explain the grain yield pramod prasad dahal*1, komal bahadur basnet2, shrawan kumar sah3 and tika bahadur karki4 abstract a study was conducted on research farm of national maize research program (nmrp), rampur, chitwan, nepal during winter season of 2015/16 laid out in split-split plot design with three replications consisting of two fym levels (fym at 10 t ha-1 and no fym application), three nitrogen levels (100, 75 and 50% n of recommended dose p and k remaining constant) and two levels of seed inoculation (azotobacter chrococcum seed inoculation and no inoculation) with rampur-2 maize hybrid in nepal. one of the major factor affecting maize production is weather and this paper is prepared to assess the suitability of weather condition at different growth stages of winter season hybrid maize in condition of western chitwan, nepal at nmrp. the result shows that the requirement of the maize crop during different growth stages i.e. temperature, rainfall, relative humidity were fulfilled which helped to obtain remarkably higher grain yield (4.26 t ha-1) in the experiment compared to average national grain yield (2.84 t ha-1) of maize. keywords: integrated plant nutrient management, winter maize, weather parameters, maize growth stages introduction maize (zea mays l.) can be grown easily in any season and is important cereal crop due to fast growing nature, higher yield, palatability and nutritiousness resulting easily accessible food and feed for humans as well as animals [1]. in nepal maize is grown as a staple food crop for many years. in terms of area and production, maize ranks second to rice [2]. it shares about 6.54 % in agricultural gross domestic product (agdp). maize can grow in a wide range of environment; however, warm climate with temperature ranging from 21 to 30 °c is most favorable [3]. moreover, it was reported [4]that maize growth is best with temperature between 18 °c and 27 °c during day time and around 14°c at night. the experimental site falls under the sub-tropical humid climatic belts of nepal with three distinct seasons i.e. november to february (winter), march to may (hot spring) and june to october (rainy). the maximum temperature during the coldest month of year (december to february) reaches to 27 °c whereas the minimum temperature during this period ranges from 6 to 10 °c. likewise, the maximum temperature during hottest month of year (may to june) is measured up to 42 °c. recent studies conclusively proved that maize is a potential winter season crop having three times higher yield potential than kharif crop [5]. according to annual report of nmrp [6], the productivity of winter maize is about 6-7 t ha-1 while that of summer maize is around 4.0 t ha-1. highlights in bioscience page 1 of 5 march 2021|volume 4 https://doi.org/10.36462/h.biosci.20226 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield the mean annual temperature trend at rampur, chitwan during 1968 to 2008 showed that the increase of temperature seemed to be more in the recent decade than the former decade and the trend was 0.039 °c per year [7]. thus the changed weather condition can create impact upon the maize production as yield of maize is primarily influenced by sunlight, temperature, available plant nutrients and water supply [8]. one of the major factors affecting maize production is weather and this paper is prepared to assess the suitability of weather condition at different growth stages of winter season hybrid maize in condition of western chitwan, nepal at nmrp. materials and methods a experiment was conducted during winter season (october to march) of 2015 at the research field of national maize research program (nmrp), rampur, chitwan, nepal with rampur hybrid-2 variety. the experimental soil was sandy loam in texture, having slightly acidic ph (5.4). organic matter (4.02), total nitrogen (0.11), available phosphorus (58.0 kg ha-1), available potassium (253.8 kg ha-1). the experiment was laid out in strip-split plot design consisting of 12 treatments with three replications. fym levels as main plots (fym at 10 t ha-1 and no fym application), three nitrogen levels as sub plots (100, 75 and 50% n of recommended dose p and k remaining constant) and two levels of seed inoculation (azotobacter chrococcum seed inoculation and no inoculation) as sub-sub plots. farm yard manure (fym) was applied two weeks before sowing and was incorporated into soil. chemical fertilizers: urea, single super phosphate (ssp), diammonium phosphate (dap) and muriate of potash (mop) were also applied as main source of nitrogen, phosphorus and potassium, respectively. urea was applied in three splits; at sowing, knee high and tasseling stages. azotobacter was applied as seed inoculation preparing of slurry of 10% sugar solution mixing 100 gm of sugar on 1000 ml of water and was boiled and azotobacter were applied after cooling on shade. harvesting was completed on march of 2014, from net plot area. weather data were collected from meteorological data center near (100m) the research field. weather condition during the experimentation the experiment field receives regular precipitation of 1919.5 mm (nmrp, 2015/16).the average weather parameters regarding minimum as well as maximum temperatures, relative humidity and total rainfall are presented at an interval of two weeks in figure 1. the weather condition during the period of experimentation presented in the figure 1 and table 1 shows that the higher value of maximum temperature (33.85 °c) was recorded on 1st week of september which gradually declined up to 3rd week of december (21.1 °c). thereafter, it increased up to 24.69 °c on 1st week of january and then declined to 21.05 °c on 4th week of january. further, the values of maximum temperature were found to increase up to 32.84 °c on 2nd week of march and then remained at the same level at the 4th week of march (32.51°c). figure 1. weather condition during the course of experimentation at nmrp, rampur, chitwan, nepal, 2015/16. on the other hand, the minimum temperature was found to decrease from first week of september (26.60 °c) to 4th week of december (6.36 °c) and then remained almost at the same level (8.16 to 9.64 °c) from 1st week of january to 1st week of february and thereafter increased to 18.43 °c on 2nd week of march. further, similar minimum temperature (18.61 °c) was recorded on 4th week of march. in spite of it, the maximum rainfall of 43.6mm was recorded on 2nd week of september and thereafter it declined to zero precipitation on 1st week of october. however, a remarkable rainfall of 25.83mm was recorded on 4th week of october. further, there was no rainfall from the 1st week of november to 3rd week of february with the exception of january 3rd week where there was a rainfall of 7.7 mm. the relative humidity was found to increase from 1st week (81.86%) to 3rd week (89.14%) and then declined on 4th weeks (87.33%) of september. similarly, it increased from 1st (82.14%) to 2nd (88.29%) and then remained at the same level in 3rd (82.29%) and 4th (82.10%) weeks of october. further, the values of relative humidity were increasing from 90.86% on 1st week of november to 100% on 1st week of december and thereafter remained at this level up to 4th week of january with exception of 3rd week of january where it was a little decreased (98.43%). thereafter, the highlights in bioscience page 2 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield table 1. weekly maximum and minimum temperature, relative humidity, rainfall and solar radiation during research period at nmrp, rampur, chitwan, nepal, 2015/16. stage date min temp max. temp avg rainfall rh% germination 2nd week of oct. 23.125 32.845 27.99 8.3mm 85.21 knee high 1st week of nov. 17.75 30.31 24.03 0 90.86 grand growth 1st-4th week of nov. 16.18 28.305 22.24 0 94.92 tasseling and silking 1st-2nd dec. 13.75 24.39 19.07 0 100 milking 4th of january 8.11 21.05 14.58 0 100 dough 4th of feb 15.04 28.45 21.75 0.96 mm 95.03 grain filling 3rd jan-3rd feb 10.33 24.35 17.34 7.7mm 93.615 physiological maturity 3rd march week 16.59 31.69 24.14 26.8mm 79.43 total rainfall during crop season 14.35 27.23 20.79 72.99mm 93.86 relative humidity gradually declined from 98.14% on 1st week of february to 70.60% on 4th week of march. statistical analysis analysis of variance (anova) was used to test the row, column and interaction effect of fym, nitrogen levels and biofertilizer on yield and yield attributing characters of maize at 5% level of significance. values were computed using software program mstatc, gen stat and microsoft excel 2010. results analyzed data of phenological stages (table 2) showed that on an average, the emergence of maize seeds occurred at 6.36 das whereas the maize plants attained knee high stage (34.06 das) after around one month of seed emergence further, tasseling stage (73.10 das) was recorded after one month of the knee high stage. there was only four days difference between tasseling and silking stages. the silking, milking and dough stages were observed at 76.94, 126.18 and 144.17 das, respectively. finally, the maize crop was physiologically matured after about 20 days of dough stage (164.53 das). discussion the average minimum and maximum temperature during the cropping period were 14.35 and 27.23 °c suitable for growth and development of maize, respectively (table 1). further, the annual precipitation of experimental area was 1919.5 mm (nmrp, 2015/15), however, maize requires rainfall of about 600 1,200 mm throughout the year with uniform distribution [15]. thus, the availability of water was sufficient for growth and development of the maize crop. the minimum temperature required for maize seed germination is 10 °c. in addition, emergence gets far more rapid and uniform above 16 °c and around the temperature of about 20 °c, maize usually emerges within 5-6 days after sowing [9]. thus, the minimum and maximum temperatures recorded during the germination of maize seed in the experimental site were 23.13 °c and 32.84 °c with the average of 27.99 °c. therefore, the germination of seeds took place within 6 days. [10] stated that for uniformity of initial stand, a minimum temperature of 17 °c to 20 °c is required but during knee high stage, the average temperature was 24.03 °c with minimum and maximum temperatures of 17.75 and 30.31 °c, respectively. this indicates that average temperature was suitable for the establishment of plant stand in the experiment. further, during vegetative growth stage (grand growth stage) the minimum, maximum and average temperatures were 16.18, 28.31 and 22.24 °c, respectively. in this context, joshi [11] reported that optimum temperature for vegetative phase ranges from 28 to 34 °c. venkataraman and krishanan [4] also mentioned that range of temperature for the growth of maize is from 9°to 46 °c with the optimum around 34 °c during vegetative growth stage. thus, the minimum temperature was found higher but maximum temperature was slightly lower due to winter season. however, it was close to the temperature given by joshi [11]. moreover, in the experimental site, the average temperature during tasseling and silking stages equaled to 19.07 °c. with respect to it, venkataraman [12] reported that the optimum temperature for tasseling ranges from 21 to 30 °c and temperatures above 32 °c during reproductive stage reduced the yield. thus average temperatures during tasseling and silking were close to optimum level. further, minimum and maximum temperatures during these stages were 13.75 and 24.39 °c, respectively. in respect of it, berbecel and eftimescu [13], mentioned that the maximum temperature above 32 °c around tasseling and pollination increases the differentiation process of the reproductive parts and consehighlights in bioscience page 3 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield table 2. weekly maximum and minimum temperature, relative humidity, rainfall and solar radiation during research period at nmrp, rampur, chitwan, nepal, 2015/16. table shows treatments (tr), germination (gr), knee high (kh), tasseling (tas),silking(si), milking (mi), dough (do), physiological maturity (pm), and grain yield (t ha-1) (gy) paramter phenological stages (das) tr gr kh tas si mi do pm gy fym levels (t ha-1) 0 6.56 34.56 74.21 77.39 126.33 145.06 164.4 3.91 10 6.17 33.56 73.35 76.5 125.61 145.28 164.61 4.58 statistical analysis f test ns ns ns ns ns ns ns ns lsd (=0.05) 1.04 1.9 2.89 2.43 3 1.57 2.91 2.2 sem (±) 0.17 0.31 0.48 0.4 0.5 0.26 0.82 0.36 nitrogen levels tr gr kh tas si mi do pm gy 50 6.41a 35.67a 74.50a 78.92a 126.08a 146.17a 166.58a 2.95c 75 6.42a 34.92a 72.00b 75.67b 126.42a 145.16a 165.42a 4.64b 100 6.25a 31.58b 72.08b 75.24b 125.41a 144.17b 162.58b 5.16a statistical analysis f test ns * * * ns ns * ** lsd (=0.05) 0.42 2.83 1.72 2.57 1.67 1.76 2.23 0.41 sem (±) 0.13 0.87 0.53 0.79 0.51 0.54 0.68 0.12 biofertilizer tr gr kh tas si mi do pm gy no inoculation 6.33 34.17 73.11 77.28 126.54 145.28 164.72 3.95 with inoculation 6.39 33.94 72.61 76.61 125.89 145.06 164.33 4.55 statistical analysis f test ns ns ns ns * ns ns ** lsd (=0.05) 0.21 1.01 0.67 0.72 0.48 1.1 0.5 0.4 sem(±) 0.07 0.35 0.22 0.23 0.17 0.35 0.162 0.13 cv, % 4.5 2.1 1.5 1.3 1 0.5 0.6 12.9 grand mean 6.36 34.06 72.88 76.94 126.18 144.17 164.53 4.25 recommended dose of nitrogen: 120 kg n ha-1 azotobacter chroococcum seed inoculation: 40 g kg-1 seed quently the rate of kernel abortion. further, according to adjetey [3] at high temperatures the pollens gets shed before silk become receptive or cause either death of tassel or drying out of silk. therefore, to achieve higher grain yield the temperatures of the late vegetative and reproductive phases should be relatively lower than 30 °c which was also recorded in this experiment. finally, during maturity, the minimum, maximum and average temperatures were 16.59, 31.69 and 26.8 °c respectively. joshi (2015) stated that optimum temperature during ripening stage ranges from 32 to 35 °c. thus, the maximum temperature of the experimental site was close to it but the average temperature was a little lower which might be related to the winter season and subtropical climatic condition. thus, on the basis of above discussion it is obvious that in general, the weather condition related to temperature was suitable for growth and development of maize. further, a rainfall of 43.60, 10.3 and 9.6 mm were recorded at three, two and one week before germination, respectively (figure 1) which provided enough moisture for germination of maize seeds. moreover, a rainfall of 8.3mm was recorded at sowing which also helped in better germination of seeds. germination of maize seed was completed within 6 days. no rainfall was recorded during knee high and grand growth stages. others [10] stated that moisture stress in early growing season causes a large reduction in grain yield. therefore, the experimental plots were irrigated at this stage. kranzet al., [14] highlights in bioscience page 4 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience dahal et al., 2021 effect of weather parameters on growth stages of winter maize explain the grain yield mentioned that corn requires most of the water during the early reproductive growth stages i.e. tasseling and silking. moreover, the requirement of maize in water is more prominent during the tasselling to silking stage [15] which is considered critical as grain formation initiates during this period. thus the availability of soil moisture at the time of tasselling is therefore vital for the production of high yield in maize [16]. futher, period of silking and ear growth the corn is most sensitive to moisture deficiency as far as the grain yield is concerned [4]. however, there was no rainfall during this period in the experimental site due to which the crop was irrigated at this phase. another critical period with respect to crop requirement in moisture is grain filling period. corn requires one third part of seasonal water requirement at beginning of dough stage. drought or water stress at dough stage results in acceleration of maturity preventing grains from gaining full size as well as weight [14]. however, there was 7.7 mm rainfall during grain filling period and 0.96mm at dough stage which might had helped to some extent fulfill the crop requirement in water. thus, in general, the requirement of the maize crop in water was fulfilled either through rainfall or irrigation which helped to obtain remarkably higher average grain yield (4.26 t ha-1) in the experiment as compared to average national grain yield (3.09 t ha-1) of maize (moad-abpsd, 2015). it concludes from above discussion that weather recorded during different key growth stages of winter maize in the experimental site were resultant and it is essential to give emphasis on weather conditions in improvement of grain yield of winter maize. references 1. shinde sa, patange mj, dhage sj. influence of irrigation schedules and integrated nutrient management on growth, yield and quality of rabi maize (zea mays l.). international journal of current microbiology and applied sciences. 2014;3(12):828-32. 2. moad. statistical information on nepalese agriculture, 2015. 3. adjetey ja. maize physiology and growth requirements. 1994. 4. venkatraman s, krishnan a. crops and weather publications and information division. icar, new delhi, pp.1119, 1992. 5. desai sn, deore dd. performance of maize cultivars in rabi season. journal of maharashtra agricultural universities. 1980;5(2):181-2. 6. annual report nmrp. performance of maize cultivars in rabi season. nep al agriculture research council, rampur, chitwan, nepal., 2015. 7. nayava jl. impact of climate change on production and productivity: a case study of maize research and development in nepal. journal of agriculture and environment. 2010 sep 16;11:59-69. 8. huzsvay l, nagy j. effect of weather on maize yields and the efficiency of fertilization. acta agronomica hungarica, 53(1):31-39, 2005. 9. raemaekers rh, editor. crop production in tropical africa. belgium: dgic; 2001. 10. purseglove, jw. crop production in dry regions. vol. ii: systematic treatment of the principal crops by i. arnon london: leonard hill (1972), pp. 683,10.50. experimental agriculture, 9(4):380-380, 1973. 11. joshi kd, conroy c, witcombe jr. agriculture, seed, and innovation in nepal: industry and policy issues for the future. washington, dc, united states: international food policy research institute, pages 1-60, 2012. 12. venkataraman, s. crops and weather. publications and information division, indian council of agric. res., 1992. 13. berbecel o. effect of agrometeorological conditions on maize growth and development. 1972. 14. kranz wl, irmak s, van donk sj, yonts cd, martin dl. irrigation management for corn. neb guide, university of nebraska, lincoln. 2008 may;10(5):1-8. 15. awuku ka , brese gk, ofosu gk, baiden so. senior secondary school agriculture and environmental studies. evans brothers ltd.: london, uk, pp.85-86, 1991. 16. tweneboah ck. modern agriculture in the tropics. a textbook on animal production. accra: co-wood publishers, 2000. highlights in bioscience page 5 of 5 march 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods weather condition during the experimentation statistical analysis results discussion references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20201 highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 3 research article open access 1 department of biodiversity and crop improvement, international center for agriculture research in the dry areas (icarda), giza, egypt. 2 department of genome mapping, molecular genetics and genome mapping laboratory, agricultural genetic engineering research institute (ageri), giza, egypt. 3 department of bioinformatics & computer networks, ageri, agricultural research center (arc), giza, egypt. contacts of authors * to whom correspondence should be addressed: peter t. habib citation: habib p.t., alsamman a.m., hassanein s.e., and hamwieh a. (2020). developing convolutional neural networks-based system for predicting pneumonia using x-radiography image. highlights in bioscience, volume 3. article id 20201, dio:10.36462/h.biosci.20201 received: january 25, 2020 accepted: march 10, 2020 published:march 21, 2020 copyright: © 2020 habib et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests developing convolutional neural networks-based system for predicting pneumonia using x-radiography image peter t. habib *1, alsamman m. alsamman2, sameh e. hassanein3, and aladdin hamwieh1 abstract pneumonia is a respiratory disease caused by streptococcus pneumoniae infection. it is a life-threatening disease that causes a high mortality rate for children under 5 years of age every year. under such circumstances, we have a vital need to develop an appropriate and consistent protocol for the identification and diagnosis of pneumonia. the incorporation of computational approaches into the diagnosis of disease is extremely efficient, promising and reliable. our goal is to integrate these methods into pneumonia routine diagnosis to save countless lives around the world. we used the machine learning algorithm of convolutional neural networks (cnns) to identify visual symptoms of pneumonia in x-ray radiographic images and make a diagnostic decision. the dataset used to construct the computational model consists of 5844 x-ray images belonging to the pneumonia affected and normal individuals. our computational model has been successful in identifying pneumonia patients with a diagnosis accuracy of 84%. our model may increase the efficiency of the pneumonia diagnosis process and accelerate pathogenicity studies of the disease. keywords: cnns, artificial intelligence, pneumonia, disease diagnosis. introduction pneumonia is a bacterial disorder that causes severe symptoms such as grunting, chest retraction, central cyanosis, obtundation, lethargy, convulsions and inability to feed or drink [1,2]. each year about 1,400 cases of pneumonia occur in 100,000 children with around 1 in 71 babies. according to a recent study, pneumonia claimed the lives of over 800,000 children under the age of five last year, or one child every 39 seconds [3] . according to the american lung association, pneumonia can be diagnosed in various ways, including a blood examination, pulse oximetry, sputum analysis on a sample of mucus, arterial blood gas examination, pleural fluid culture, or bronchoscopy [4]. despite the many methods available to diagnose pneumonia, chest radiography remains the main method used for diagnosis. although x-rays are commonly used, it is difficult to diagnose them based solely on these images. perusing these images is a bottleneck problem because the area or areas of increased opacity are usually determined by pneumonia [5,6]. https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20201 https://doi.org/10.36462/h.biosci.20201 http://bioscience.highlightsin.org/ habib et al., 2020 convolutional nn-based system for pneumonia using radiography highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 3 in fact, accuracy of the diagnosis of pneumonia is very limited due to certain causes of opacity which are difficult to account for. in this regard, many advances have been achieved by the application of machine learning (ml) techniques in medical diagnosis. the development of an artificial intelligence pneumonia diagnostic framework has recently become a hot topic in medical bioinformatics. such frameworks may help radiologists interpret medical images using additional perspectives developed by computer systems [7] ⁠ . continuous improvement of such frameworks using new ml algorithms and techniques could provide more accuracy and ease of use of computer platforms. in this regard, python programming language provides a hundreds of library. sci-kit learn [8] ⁠ , tensorflow [9] ⁠ , and keras [10] ⁠ are the most popular libraries ml programming. these ml libraries have been shown to have an impact on the integration of ml programming in biological data analysis [11,12]. in addition, alexnet is the name of a convolutional neural network (cnn) algorithm designed for large-scale visual recognition. alexnet has been successfully used for pathological brain detection [13–15]. in this research, we are trying to use highly specialized ml subtype for image classification to resolve many complications of routine pneumonia diagnosis. using x-images this tool may be used for diagnosis of pneumonia. this tool will also be available as user-friendly applications which can be used with the minimum programming skills. in addition, we aim to develop a diagnostic software that could be easily updated, modified and integrated into different medical diagnostic systems. materials and methods data collection the dataset is composed of 5844 x-rays images belonging to normal and pneumonia patients. the dataset consists of images with high resolutions and satisfied statistical variance (figure 1). the dataset was retrieved from kaggle database [16,17]. this data was collected from retrospective samples of one to five year-old pediatric patients. figure 1 : sample x-ray image of normal (a) and lung pneumonia (b). model construction and validation the dataset of x-rays images have been used for ml model training (5216 image) and validation (624 images). the alexnet architecture yielded 37% accuracy at the beginning of the design. our ml model design is inspired by alexnet structure, where the architecture consists of eight constitutional layers (figure 2 and table 1). figure 2 : the architecture of the ml neural network used to diagnose pneumonia from x-rays images . table 1: description of the structure of the neural network architecture. results and discussion diagnosis of pneumonia is very difficult due to hidden factors causing opacity such as pulmonary edema, bleeding, atelectasis or lung cancer. when examining an area of increased opacity in the chest radiography, it is critical to determine where increased opacity occurs [18,19]. computational analysis integration could accelerate pneumonia and enhance the routine medical diagnosis system. a highly specialized sub-type of machine learning for image classification called deep learning networks has been used in current research to detect lung pneumonia from x-ray images (figure 2). we analyzed a dataset of 5844 images of chest x-ray film to diagnose pneumonia using ml pipeline. alexnet's neural network architecture has produced an ml model accuracy of 84%. conclusion deep learning is expected to lead to a revolutionary progression in the efficacy of medical diagnosis using radiological methods around the world. we ml models and novel diagnostic approaches for various x-ray abnormalities to detect pneumonia. our pipeline may trigger the impact of artificial intelligence, which will layers feature map size strides dilation rate activation input image 1 (150, 150, 3) 1 1 relu 1 convolutional 3 (150, 150, 3) 1 1 relu max pooling 32 (148, 148, 32) 1 1 relu 2 convolutional 32 (74, 74, 32) 1 1 relu 3 convolutional 180 (72, 72, 180) 1 1 relu 4 convolutional 150 (70, 70, 150) 1 1 relu 5 convolutional transpose 100 (68, 68, 100) 1 1 relu 6 convolutional 150 (70, 70, 150) 1 1 relu max pooling 120 (68, 68, 120) 1 1 relu 7 fc 138720 softmax output fc 1000 softmax http://bioscience.highlightsin.org/ habib et al., 2020 convolutional nn-based system for pneumonia using radiography highlights in bioscience march 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 3 defiantly lead to an enormous improvement in the visual diagnosis of many diseases. availability the python source code we have used is freely available at : https://github.com/peterhabib/pneumoniaai . this code can be executed interactively in a python command line. references 1. hamborsky j, kroger a, editors. epidemiology and prevention of vaccine-preventable diseases, e-book: the pink book. public health foundation; 2015 oct 19. 2. brooks wa. bacterial pneumonia. in: hunter’s tropical medicine and emerging infectious diseases. elsevier; 2020. p. 446–53. 3. unicef. annual reports. 2020. available from: https://www.unicef.org 4. american lung association. american lung association; 2020. available from: www.lung.org 5. mattila jt, fine mj, limper ah, murray pr, chen bb, lin pl. pneumonia. treatment and diagnosis. ann am thorac soc. 2014;11(supplement 4):s189--s192. 6. makhnevich a, sinvani l, cohen sl, feldhamer kh, zhang m, lesser ml, et al. the clinical utility of chest radiography for identifying pneumonia: accounting for diagnostic uncertainty in radiology reports. am j roentgenol. 2019;213(6):1207–12. 7. al mubarok af, dominique jam, thias ah. pneumonia detection with deep convolutional architecture. in: 2019 international conference of artificial intelligence and information technology (icaiit). 2019. p. 486–9. 8. pedregosa f, varoquaux g, gramfort a, michel v, thirion b, grisel o, et al. scikit-learn: machine learning in python. j mach learn res. 2011;12(oct):2825–30. 9. abadi m, barham p, chen j, chen z, davis a, dean j, et al. tensorflow: a system for large-scale machine learning (pp. 265--283). in: 12th ${$usenix$}$ symposium on operating systems design and implementation (${$osdi$}$ 16) retrieved from https://www usenix org/conference/osdi16/technical-sessions/presentation/ abadirpy2(nd) retrieved march. 2016. p. 2019. 10. gulli a, pal s. deep learning with keras. packt publishing ltd; 2017. 11. habib pt, alsamman am, hassanein se, hamwieh a. tardict: randomforestclassifier-based software predict drug-tartget interaction. biorxiv. 2020; 12. matstubara t, ochiai t, hayashida m, akutsu t, nacher jc. convolutional neural network approach to lung cancer classification integrating protein interaction network and gene expression profiles. j bioinform comput biol [internet]. available from: https://www.worldscientific.com/doi/abs/10.1142/s02 19720019400079?af=r 13. lu s, lu z, zhang y-d. pathological brain detection based on alexnet and transfer learning. j comput sci. 2019;30:41–7. 14. lebedev v, lempitsky v. fast convnets using group-wise brain damage. in: proceedings of the ieee conference on computer vision and pattern recognition. 2016. p. 2554–64. 15. ezhilarasi r, varalakshmi p. tumor detection in the brain using faster r-cnn. in: 2018 2nd international conference on i-smac (iot in social, mobile, analytics and cloud)(i-smac) i-smac (iot in social, mobile, analytics and cloud)(i-smac), 2018 2nd international conference on. 2018. p. 388–92. 16. kaggle [internet]. 2020. available from: www.kaggle.com%0a 17. kermany ds, goldbaum m, cai w, valentim ccs, liang h, baxter sl, et al. identifying medical diagnoses and treatable diseases by image-based deep learning. cell. 2018;172(5):1122–31. 18. franquet t. imaging of pneumonia: trends and algorithms. eur respir j. 2001;18(1):196–208. 19. goodman p, prosch h, herold cj. imaging of pulmonary infections. in: diseases of the chest and heart 2015--2018. springer; 2015. p. 63–70. https://github.com/peterhabib/pneumoniaai http://bioscience.highlightsin.org/ highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202405 research article open access 1 international center for agricultural research in the dry areas (icarda), p.o. box 114/5055, beirut, lebanon. 2 international maize and wheat improvement center (cimmyt) pakistan office, csi building, narc, park road, islamabad 44000, pakistan. 3 agricultural genetic engineering research institute (ageri), 9 gamaa street, giza, egypt. * to whom correspondence should be addressed: a.hamwieh@cgiar.org editor: jean legeay, university mohammed vi polytechnic, morocco. reviewer(s): ahmed sallam, leibniz institute of plant genetics and crop plant research (ipk), stadt seeland, germany.. clara r. azzam, cell research department, field crops research institute, agricultural research center (arc), egypt. received: june 15, 2024 accepted: september 9, 2024 published: december 26, 2024 citation: hamwieh a, muhammad i, ahmed s, kababeh s, alsamman am, istanbuli t. exploring qtl genes contribute to chickpea ascochyta blight resistance across multiple environments using ssr, dart and snp assays . 2024 dec. 26;7:bs202405 copyright: © 2024 hamwieh et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this work was supported by grains research and development corporation (grdc), australia. competing interests: the authors declare that they have no competing interests. exploring qtl genes contribute to chickpea ascochyta blight resistance across multiple environments using ssr, dart and snp assays aladdin hamwieh1 >< �, imtiaz muhammad2 >< �, seid ahmed1 >< �, siham kababeh1 >< �, alsamman m. alsamman1,3 >< �, tawffiq istanbuli1 ><� abstract chickpea (cicer arietinum l.) occupies the third leading position among grain legumes in cultivated area around the world. ascochyta blight (ab) caused by ascochytarabiei (pass.) labr. is one of the most destructive foliar diseases of chickpea and can cause complete crop failure in many chickpea growing regions around the world. a recombinant inbred line (ril) population, comprising 165 lines derived from the cross flip98-1065 (r) ×ilc1929 (s),were evaluated in six environments over three years (2008 – 2011) and three locations in syria (field and greenhouse locations in tel hadya “th“ and a field location at lattakia “lat“). the greenhouse experiments were conducted against ab pathotype ii. anova analysis indicated significant differences both among the rils and among the environments. we produced a total of 1398 (134 ssr, 652 dartseq and 612 snp) markers and developed a high-resolution genetic map (1244 markers spanning 2503 cm on eight linkage groups). three major conserved quantitative trait loci (qtls) that confer ab resistance were identified: two on linkage group 2 (indicated as lg2-a and lg2-b) and one on linkage group 4 (indicated as lg4). these explain, respectively, a maximum of 18.5%, 11.1% and 25% of the total variation. in total, 18 predicted genes were located in lg4, and 9 and10 predicted genes, respectively, were located in lg2-a and lg2-b. this study presents a first set of snp markers located within genes associated with ab resistance in chickpea, which could be applied in marker-assisted selection programs for breeding ab-resistant chickpeas. keywords: ascochyta blight, ascochytarabiei, chickpea, ammi analysis, qtl, snp introduction ascochyta blight (ab) of chickpea, caused by ascochyta rabiei (pass.) lab., is one of the most important diseases in many chickpea production areas. four pathotypes have been identified within a.rabiei populations in syria and other countries [1; 2; 3]. chemical control of ab has proved inefficient and very expensive; therefore, new varieties with improved resistance are needed to manage the impact of the disease. breeders using conventional methods have made considerable progress toward developing chickpea varieties with increased ab resistance [4; 5]. research teams investigating ab of chickpea have identified 14 quantitative trait loci (qtls) in eight linkage groups (lgs) that each contribute to a. rabiei resistance [6; 7; 8; 9; 10; 11; 12; 13; 14; 15]. this indicates that several mechanisms are likely responsible for ab resistance, but very little is known about the identity and functions of the genes involved. two major qtls, located on lg2 close to the markers ga16 and ta37, control resistance to ab pathotype i [10], while another qtl contributing to resistance to pathotype ii is located on lg4 near simple sequence repeat (ssr) loci gaa47, ta130, tr20, ta72, ts72, and ta2 [16; 9; 10]. [10] traced the ab resistance of the chickpea line flip84-92c to regions on lg2 and lg4 associated with the resistance in flip84-92c to pathotype ii. in that study, the ta46 marker explained a maximum of 69% of that lines total ab resistance; furthermore, additional markers (gaa47, ga24, and ga16) were identified on lg2, each explaining 10.4 – 19.3% of the line's total ab resistance [10]. additional markers on lg1 (ts12b, stms28, and ts45) have been reported to be associated with ab resistance under controlled conditions [7; 8]. highlights in bioscience page 1 of 11 december 2024|volume 7 https://doi.org/10.36462/h.biosci.202405 https://creativecommons.org/licenses/by/4.0/ mailto:a.hamwieh@cgiar.org https://orcid.org/0000-0001-6060-5560 mailto:m.imtiaz@cgiar.org mailto:s.a.kemal@cgiar.org https://orcid.org/0000-0002-1791-9369 mailto:sihamkababe1989@gmail.com mailto:smahmoud@ageri.sci.eg https://orcid.org/0000-0002-7765-5035 mailto:t.istanbuli@cgiar.org https://orcid.org/0000-0001-7450-6408 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance a scar marker scy17590 was reported as linked to ab resistance [11], and this marker was applied successfully to selected resistant genotypes in australian chickpea breeding materials [17]. this marker was further applied together with an allelespecific primer for the gene caetr-1 for genotyping qtlar1 and qtlar2 in chickpea germplasm [18; 19]. a study by [20] using an fst genome scan and genome-wide association methods indicated that a 100 kb region on chromosome 4 was significantly associated with ab resistance. this region covered a large qtl interval of 7 mb30 mb, which had been genotyped at relatively low density with ssr or single nucleotide polymorphism (snp) markers through previous mapping population studies. [20] and colleagues validated this qtl region on lg4 in a genome-wide association study (gwas) using approximately 144,000 snps and a collection of 132 advanced breeding lines from the australian chickpea breeding program. in total, 12 predicted genes were located in the region associated with ab resistance, including sequences annotated as encoding an nbs-lrr receptor-like kinase, a wall-associated kinase, a zinc finger protein, and serine/threonine protein kinases. one significant snp located in the conserved catalytic domain of an nbs-lrr receptor-like kinase led to an amino acid substitution [20]. researchers could use high-throughput genotyping systems to identify markers such as single nucleotide polymorphisms (snp) markers and diversity array technology (dart) markers. therefore, we aimed to study the interaction between different environments and ab resistance, to produce a highly saturated genetic map, and to identify markers closer to potential ab resistance genes using a recombinant inbred line (ril) population of chickpea, enabling practical application of marker(s) in chickpea breeding programs. materials and methods plant materials and dna isolation a mapping population consisting of 165 (f8) rils derived from an intra-specific cross between an ab-resistant chickpea, flip98-1065, and an ab-susceptible chickpea, ilc1929, was used in this study. the rils were developed using the single seed descent (ssd) method from f2 to f7. dna was extracted from fresh leaves of six-week-old seedlings using the cetyltrimethyl ammonium bromide (ctab) method [21; 22]. the sequences of chickpea ssr primers were obtained from published papers [16; 23; 24]. the pcr mixture (20 µl) contained 10 ng genomic dna, 0.2 mm dntp, 1 u taq dna polymerase (invitrogen, carlsbad, calif.), 10 pmol of each forward and reverse primer, and 1x pcr buffer (invitrogen, carlsbad, calif.). polymerase chain reaction (pcr) was conducted using a thermocycler (abi geneamp2720) with the following program: a denaturing cycle of 94oc for 3 minutes, followed by 35 cycles of 94oc for 15 seconds (denaturation), a specific temperature depending upon the primer pair for 15 seconds (annealing), and 72oc for 30 seconds (extension), followed by a final extension at 72oc for 5 minutes. the pcr products were separated on an 8% polyacrylamide gel according to the electrophoresis user guide (invitrogen). dart and dartseq dartseq is a genotyping by sequencing (gbs) platform developed by dart pl, canberra, australia ( http://www.diversityarrays.com/dart-application-dartseq). dartseq is a combination of complexity reduction methods that were initially developed for array-based dart with sequencing of resulting representations on next-generation sequencing platforms. experimental design and locations four field experiments and two greenhouse experiments were performed at different locations in syria, enabling us to examine disease susceptibility of the chickpea lines under six sets of environmental conditions (table 1). field experiments were performed in lattakia, syria in 2009 (lat09) and at the tel hadya station, aleppo, syria, in 2008, 2009, and 2010 (th08, th09, and th10). these locations differed in the average rainfall they received during the experiments: in lattakia, the average was around 750 mm, but in tel hadya, the average was 350 mm. each experiment was laid out in an alpha lattice design with two replications using multiple row plots. the length of each plot was 5 m and rows were spaced 45 cm apart in all trials. two experiments were performed in the greenhouse at icarda headquarters in aleppo, syria in 2009 and 2010 (pii-2009 and pii-2010). in the greenhouse, five healthy seeds of each line were planted in a pot (15 cm diameter). the experiments were conducted in a growth chamber (temperature 22oc and 12 hours/12 hours light/dark). locations, years, and planting dates for the six experiments are summarized in (table 1). the ab cultures were obtained from the legume pathology laboratory at icarda. the experiments were laid out in a randomized complete block design with two replications. a spore suspension of ab pathotype ii (concentration of 105 spores/ml−1) was prepared from 14-day old ascochyta rabiei culture grown on chickpea dextrose agar (4% chickpea flour, 2% dextrose, and 2% agar in 1 liter of distilled water) and used for seedling inoculations. the disease was scored when symptoms were observed on the susceptible chickpea control (ilc-263). scoring plants for ab symptom severity ascochyta blight symptom scoring was based on a nine-point rating scale [25] in which plants are scored as follows: 1. immune, no symptoms of disease; 2. few, very small lesions (<2 mm) on leaves and stems (1 to 2% of the plant area infected); 3. many small lesions (6 to 10% of the plant area infected); 4. many small and large lesions (26 to 50% of the plant area infected); 5. many small lesions on the stem (51 to 75% of the plant area infected); 6. many large lesions, lesions coalescing, stem girdled (76 to 90% of the plant area infected); 7. many small and large lesions, lesions coalescing, girdling stem breakage (>90% of the plant area infected); 8. plant is almost dead; and 9. plant is dead. highlights in bioscience page 2 of 11 december 2024|volume 7 http://www.diversityarrays.com/dart-application-dartseq http://www.diversityarrays.com/dart-application-dartseq http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance table 1. rainfall "mm", temperature "oc" (between brackets) and planting dates during 3 years (2008–2010) for experiments conducted in six environments in syria (three in the field and two in the greenhouse at tel hadya th; one in the field at lattakia "lat"). environment code greenhouse field pji-2009 pji-2010 lat09 th08 th09 th10 location tel hadya greenhouse tel hadya greenhouse lattakia tel hadya greenhouse tel hadya greenhouse tel hadya greenhouse year 2009 2010 2009 2008 2009 2010 planting date 15 nov. 2008 13 nov. 2009 22 mar. 2009 11 dec. 2007 12 dec. 2008 9 dec. 2009 february mi (21 řc) mi (21 řc) 98.4 (13.2 řc) 22.0 (14.3 řc) 84.3 (14.2 řc) 50.6 (15.2 řc) march mi (21 řc) mi (21 řc) 93.3 (20.3 řc) 27.9 (22.9 řc) 36.2 (17.1 řc) 12.4 (20.8 řc) april mi (21 řc) mi (21 řc) 56.6 (23.4 řc) 1.9 (27.9 řc) 23.4 (24.0 řc) 12.7 (25.8 řc) may 34.6 (25.1 řc) 10.9 (29.2 řc) 4.0 (30.1 řc) 0.0 (31.7 řc) date of score 22/5/2009 25/5/2008 17/5/2009 16/5/2010 total rainfall (mm) 282.9 62.7 147.9 75.7 mi: mist irrigation used to keep relative humidity around 80% under controlled environments. statistical analysis the disease rating data collected from the experiments were analyzed using genstat 12th edition (vsni, hemel hempstead, uk). to evaluate ab scores, analysis of variance (anova) with multiplicative interactions analysis (ammi) was performed. ammi analysis was described by [26] based on the following formula: yi j = µ + gi + a j + n∑ k=1 λk xiky jk + ri j + s̄ i j where µ is the overall mean of the test; gi is the fixed effect of genotype i (i = 1, 2, . . . , g); a j is the fixed effect of environment j ( j = 1, 2, . . . , a); yi j is the mean response of genotype i in environment j; λk is the singular value of the k-th ipca, (k = 1, 2, . . . , p, where p is the maximum number of estimable principal components); xik is the singular value of the i-th genotype in the k-th ipca; y jk is the singular value of the j-th environment in the k-th ipca; ri j is the residue of the gei or ammi residue (data noise); k is the characteristic non-zero root, k = [1, 2, . . . ,min(g − 1, e − 1)]. the plot genotype and environment interactions were analyzed against four interaction principal component axes (ipcas). the ratio of the genetic variance to the total phenotypic variance was calculated to estimate heritability. genetic mapping was conducted using joinmap 4® software, kyazma [27]. linkage groups were created based on a logarithm of odds (lod) score greater than 3 and a recombination frequency below 0.45. map distances in centimorgans (cm) were estimated using the kosambi function [28]. qtl analysis was conducted using mapqtl 6® software, kyazma [29], and the fraction of the variation explained by the qtl and the additive effects were calculated. the potential qtls were estimated based on a 2,000 permutation test using lod ≥2 as the significance threshold. predicted genes within the qtl regions were identified across the chickpea genome using the blast package [30], and homologous protein-encoding genes were extracted from the chickpea genome sequence. the snpeff tool was used to detect the genetic sequence effect for potential snps with a correlation to ascochyta blight resistance. results phenotypic characterization we collected data from three growing seasons (2008 – 2010) and from one greenhouse and two field locations; this allowed us to analyze plants exposed to six different environmental conditions. we observed significant differences (p < 0.001) in the ab severity scores (on a scale from 1 through 9) among the 165 rils and among plants exposed to the six environments. however, the mean values of the ab severity scores showed a continuous distribution from 3 to 9 (figure 1 (a)) that fit the normal distribution. to estimate the interactions between ab resistance and the environments we tested, the ab scores were further analyzed using the ammi model, which combines features of anova and principal component analysis. the analysis indicated that 25% of the total sum of squares (ss) was due to environmental factors, and only 26.5% and 32.9%, respectively, was due to genotypic and gene-environment interaction (gei) factors (table 2). the percentage attributed to gei was about 1.2 times the percentage attributed to genotype, indicating substantial differences in genotypic responses across environhighlights in bioscience page 3 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance figure 1. (a) frequency distributions of ab disease scores for 165chickpea rils derived from flip98-1065 (ab resistant) œ ilc1929 (ab susceptible). arrows indicate disease score for each parental genotype. (b) ammi model 2 biplot of ab resistance scores for 165 rils and 6environments. ments. however, the large ss value for environmental factors indicated that diverse environments caused a significant portion of the variation in the ab values. the first principal component axis (pca1) captured 40.11% of the gei-ss, while the second (pca2) captured only 27.4%. the sum of squares for pca1 and pca2 was 1484, which is smaller than the 1774 captured for genotype. since mean squares for both pca1 and pca2 were significant at p < 0.001 and contributed about 67.52% of the total gei, the postdictive evaluation suggested that two axes (pca1 and pca2) were significant for the model with 334 degrees of freedom (table 2). the ammi 2 biplot analysis for the six environments is shown in (figure 1 (b)). the high rainfall environment in lattakia (lat09), which received 184.5 mm rainfall after planting, was located in quadrant ii. the environments th08, th09 and th10 were located in quadrants iv, iii, and i, respectively. this variation is expected because the tel hadya location received different amounts and distributions of rainfall in the three growing table 2. anova (ammi model) for ab score of 165 rils evaluated in six different environments in field and greenhouse. source d.f. s.s. m.s. f explained (%) genotypes 164 1774 10.82 10.37*** 26.53 environments 5 1673 334.58 95.99*** 25.02 interactions 819 2198 2.68 2.57*** 32.87 ipca 1 168 882 5.25 5.03*** 40.13 ipca 2 166 602 3.63 3.48*** 27.39 ipca 3 164 293 1.79 1.71*** 13.33 ipca 4 162 225 1.39 1.33 error 978 1020 1.04 total 1979 6686 3.38 *** highly significant at the 0.001 probability level; d.f: degree of freedom, f: tabulated frequency. seasons, notably during february and march when the temperature is around 20 oc in tel hadya (th). these conditions provide the best environment for ab disease development. the environments (pii-2009, pii-2010, th08 and th09) were located close to the biplot origin. however, one environment, th10 (located in quadrant i) received 75.7 mm of rain in 2010, only 12.4 mm of which was received in march, the most important time for the ab disease development. this amount is 50% lower than the amounts received in 2008 and 2009. mapping and qtl analyses of the 650 ssr primer pairs we tested, only 134 (20.6%) of the primer pairs produced different pcr products for the two parental lines, flip98-1065 and ilc1929. a total of 652 dartseq markers and 612 snps obtained from dart pl were merged with 134 ssr markers and used to develop the genetic map. the linkage map comprised 1244 markers spanning 2503 cm on eight lgs (table 3). the distance between the markers was 2 cm on average; however, 156 (11.4%) of the markers remained unlinked. the qtl analysis identified three major qtl regions, one on lg4 and two on lg2 (referred to as lg2-a and lg2-b; (figure 2). the qtl on lg4 was consistently identified in all six environments used in this study and was considered a major qtl, explaining a maximum of 25% of the total variation at marker drt100004682. the two qtls on lg2 (lg2-a and lg2-b) were identified in only the three th field environments (th08, th09, and th10) and explained a maximum of 18.5% of the total variation at marker sn-100021968. on lg4, 36 markers were located within the qtl region, and fifteen of them were tightly linked to 15 genes within a span of 430,944 bp (figure 3 (a)). the qtl lg2-a included 53 markers, and 12 of them were tightly linked to genes (figure 3 (b)). the qtl lg2-b comprised 31 markers, seven of which were linked to genes. the most important genes identified in the three qtl regions are listed in (table 4), such as serine/threonine protein kinase blus1, coiled-coil domain-containing protein, transcription factor bhlh157, metalhighlights in bioscience page 4 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance nicotianamine transporter ysl1, leucine-rich repeat receptor-like tyrosine-protein kinase pxc3, tmv resistance protein n-like, chitin elicitor receptor kinase 1-like, and snf1-related protein kinase regulatory subunit beta-1. one ssr marker within lg2-b (ga16) was linked to an uncharacterized protein and explains a maximum of 16% of the total variation (figure 3 (c)). table 3. summary of the distribution of markers on different linkage groups in chickpea. lg ssr snp dart total length (cm) lg1 11 214 274 499 1010.1 lg2 5 83 109 197 201.8 lg3 0 29 0 29 166.5 lg4 34 34 48 116 287.0 lg5 6 38 41 85 156.8 lg6 7 48 59 114 180.3 lg7 19 40 55 114 293.0 lg8 12 28 50 90 207.7 total 94 514 636 1244 2503.0 discussion ascochyta blight (ab), which is caused by ascochyta rabiei (pass.) labr., is a major disease that limits chickpea production in cool and humid environments and is the largest contributor to high yield gaps in chickpea production in many countries [31; 4]. we expected to observe significant g × e in this study, as ab severity in chickpea is greatly affected by environmental conditions [32]. a study by pande and colleagues [33] revealed significant genotypic effects and g × e interactions in ab severity. however, very few studies regarding g × e interactions in ab susceptibility of chickpea in multi-environments have been reported. [11] evaluated 106 f 6:7 ril population over two cropping seasons under field conditions, and they identified a significant ril × year interaction, which they attributed to differences between the two years in environmental conditions, fungus pathotype, or both. for this study, we generated data from six different environments by conducting four field and two greenhouse experiments over the course of three years (2008 – 2010) in two locations (tel hadya and lattakia). these environments provided different moisture conditions (rainfall varied in amount and distribution among the field experiments). therefore, our results will contribute to the field’s understanding of gene-environment interactions in ab disease susceptibility. as indicated by [34] and [35], the results of the ammi analysis can be predicted by using the first two pcas. conversely, the g × e-ss (sum of squares) in our study was about 1.2 times higher than that reported for the genotypes, and this indicates substantial differences in genotypic reactions across the environments we studied. the results from our previous study on drought tolerance performance of 181 chickpea rils in nine environments found that the gei-ss was 2.3 times more than that obtained for genotype [22]. the ammi analysis showed significant differences for all genotypes, environments, and gei. furthermore, the gei revealed that the ipca (1-3) together with the ss (1,777) was slightly larger than what was obtained for the genotypes (1,774), suggesting that the ammi model excluded most of the actual data noise. the broad-sense heritability of ab resistance in the six environments in our study indicates high heritability (0.75), which shows that ab is controlled by major genes with large effect size in this study. however, this is much higher than the heritability values (0.38-0.43) calculated in a study of three f2 populations derived from crosses of genotypes that are moderately resistant to ab [13]. this may be attributed to our use of multi-environment data in our study. although several mapping populations have been developed, few maps have been constructed using single nucleotide polymorphism (snp) markers in chickpea. in this study, 1244 markers spanning 2503 cm were mapped; this is much higher than the number of markers used by [36], who developed a high-density map using 150 ril lines (lasseter × icc3996) and 504 polymorphic ssrs and snps. we identified three major qtl regions in this study. one of these, the qtl on lg4, was consistent across the six environments used in this study and considered a major qtl/gene(s) that explained a maximum of 25% of the total variation at marker drt100004682. a qtl on lg4 that confers ab resistance has been reported by several previous studies, all of which used relatively low-density maps with ssr or snp markers [12; 13; 37; 36]. for example, [36] used snp markers to identify a qtl conferring ab resistance; this qtl explained 14-45% of phenotypic variation and spanned around 13 mb between markers ssr ta146 and snp_40000185 on lg4. we identified two qtl regions located on lg2 (lg2-a and lg2-b), but they were consistent only among the th environments across three years (th08, th09, and th10). this may indicate that they contain genes whose impact is dependent on environment (temperature/humidity), pathotype differences, or both, as the plants in our greenhouse experiments were inoculated with ascochyta rabiei pathotype ii. however, this may also explain why many research teams have failed to report the qtls we found on lg2. in this study, lg2-a and lg2-b contained 33 and 23 markers spanning 28 cm and 63.6 cm, respectively. based on ga13 as an anchor ssr marker, lg2-b corresponds with the qtl that was previously reported by [38], [10], and [9]. this qtl in this study could explain a relatively large percentage (18.5%) of the total estimated phenotypic variation in susceptibility to ab. many other qtls associated with ab resistance have been reported on linkage groups lg3 [13], lg5 [37], lg6 [13; 37], and lg8 [12] but were not identified in our study. a total of eighteen predicted genes were located in lg4, and 9 and 10 predicted genes, respectively, were located in lg2-a and lg2-b. a co-dominant scar marker scy17590 tightly linked to qtlar2 on lg4 was reported by [11] and was successfully used to characterize ab source [17]. however, no genes have highlights in bioscience page 5 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance figure 2. linkage map of lg2 and lg4 depicting qtls for ab resistance detected in a ril (flip98-1065 œ ilc1929) mapping population in chickpea. th: tel hadya. highlights in bioscience page 6 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance figure 3. (a) genetic and physical maps of markers on lg4. the estimated genetic distances are in centimorgans (cm) (left). the physical locations of mapped markers on chromosome 4 are shown in base pairs (bp) (right). (b) genetic and physical maps of markers on lg2-a, one of the two major qtl regions conferring ab resistance in chickpea. (c) genetic and physical maps of markers on lg2-b, the second major qtl region conferring ab resistance in chickpea. highlights in bioscience page 7 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance table 4. markers on lg4 significantly associated with ab resistance across six environments. marker / sequence position env. code lod variance % expl. additive marker gene linkage sn-100024987 lat-09 7.3 2.37 18.4 -0.73 serine/threonine protein kinase blus1 pii-2009 5.97 0.86 15.3 -0.40 (snp is located within the gene) pii-2010 4.16 2.04 11.0 -0.50 th-08 3.08 0.77 8.2 -0.26 th-09 7.03 1.49 17.8 -0.57 th-10 2.14 2.98 5.8 -0.43 sn-5825578 lat-09 7.28 2.37 18.4 -0.73 coiled-coil domain-containing protein pii-2009 5.91 0.87 15.2 -0.39 (snp is located within the gene) pii-2010 4.14 2.04 10.9 -0.50 th-08 3.08 0.77 8.2 -0.26 th-09 7.02 1.49 17.8 -0.57 th-10 2.15 2.97 5.8 -0.43 sn-5825139 lat-09 7.12 2.38 18.0 -0.72 transcription factor bhlh157 pii-2009 5.12 0.89 13.3 -0.37 (snp is located within the gene) pii-2010 4.02 2.05 10.6 -0.49 th-08 3.29 0.76 8.8 -0.27 th-09 6.94 1.50 17.6 -0.57 th-10 2.36 2.96 6.4 -0.45 drt-100004682 lat-09 10.52 2.17 25.5 0.87 na pii-2009 7.20 0.84 18.2 0.43 pii-2010 4.66 2.02 12.2 0.53 th-08 3.92 0.75 10.4 0.30 th-09 7.55 1.47 19.0 0.59 th-10 2.38 2.96 6.4 0.45 sn-5826150 lat-09 6.95 2.39 17.6 -0.72 na pii-2009 5.25 0.88 13.6 -0.37 pii-2010 4.10 2.05 10.8 -0.50 th-08 3.44 0.76 9.1 -0.28 th-09 6.90 1.50 17.5 -0.56 th-10 2.17 2.97 5.9 -0.43 na: gene/position is not available table 5. sequence and position on lg4 of snp markers significantly associated with ab resistance. marker snp position sequence sn-100024987 50:t>g 4113008 tgcagaacaagaagcaatatctcaggtataacttatattcaatttttatc(t/ g)gtcataaaatgtgctata sn-5825578 24:t>c 3997422 tgcagtgcttcctaaattcgaaga(c/ t)cctgtttctgttcctgagcctgaacctgaaactcaacctaagga sn-5825139 55:g>a 4048029 tgcagaaagaaaggaaattctcaagtaataagtgaactaaaaacaagag(a/ g)aattgaaatataaaattag sn-5826150 6:c>g 4113005 tgcaga(g/ c)ggcattctcttcagtgctagttgtgcagcatctttacagatcggaagagcggtttcagcagga sn-100053539 5:t>c 4168916 tgcag(c/ t)gggtgacgcagttatagtttacaacgttatggtgttagtgaatggttgaaattattttacaga sn-5825294 11:c>g 4167416 tgcagcaaact(g/ c)tgaaaaaataaagtaagagaagttctaaggttctatgaaaaaataataatgtatatt been reported as genes associated with ab resistance on lg4. in this study, we found the genes annotated as leucine-rich repeat receptor-like tyrosine-protein kinase pxc3, tmv resistance protein n-like, chitin elicitor receptor kinase 1-like, histidine kinase 2, probable l-type lectin-domain containing receptor kinase s.7, uridine kinase-like protein 4, and serine/threonine protein kinase blus1. very recent research by [20] validated a qtl similar to the one we observed on chromosome 4 using gwas with a collection of 132 advanced breeding lines from the australian chickpea breeding program and 144,000 snp markers. the study predicted 12 genes located on a qtl region in lg4, including those annotated as an nbs-lrr receptor-like kinase, a wall-associated kinase, a zinc finger protein, and serine/threonine protein kinases. however, only one significant snp from that study, located in the conserved catalytic domain of an nbs-lrr receptor-like kinase, led to an amino acid substitution. in our study, 12 markers on the qtl region on lg4 were consistent across environments and could be used for mas in cicer arietinum. these include the markers sn-100024987, sn-5825578, sn-5825139, and drt-5824787, which are located, respectively, within four genes (serine/threonine protein kinase blus1, coiledcoil domain-containing protein, transcription factor bhlh157, highlights in bioscience page 8 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ hamwieh et al., 2024 exploring qtl genes contribute to chickpea ascochyta blight resistance and metal-nicotianamine transporter ysl1) (table 5). unfortunately, little research has been conducted on these genes in cicer arietinum. however, [20] reported that nbslrr receptor-like kinase and several serine/threonine protein kinases on chromosome 4 were predicted genes associated with ab resistance. these genes are already known to have a role in plant disease resistance. for example, the network of protein serine/threonine kinases in plant cells appears to act as a central processor unit that plays a role in the resistance of tomato to disease caused by pseudomonas syringae pv. tomato [39]. yellow-leaf-specific 9 (ysl9 / nhl10) is a transmembrane gene related to plant defense response to virus. the gene product is localized to the chloroplast and the mrna is cell-to-cell mobile. in arabidopsis, the expression of this gene is induced by virus infection and in senescing leaves [40; 41]. on the other hand, blus1 (blue light signaling1 or ser/thr protein kinase) mediates a primary step for phototropin signaling in guard cells and is essential for stomatal opening, and it also has a role in the stress-activated protein kinase signaling cascade [42]. the coiled-coil (cc) domain is implicated in specific interactions with other proteins that enhance plant disease resistance. for example, coiled-coil nucleotide binding site-leucine rich repeat (cc-nblrr) protein rx in potato has been found to interact with the potato virus x (pvx) coat protein, conferring pvx resistance [43]. transcription factor bhlh157 (basic/helix-loop-helix) is a master-switch gene in plants [44] and plays an important role in disease resistance, high temperature-mediated adaptations, and phytochrome signaling [45; 46; 47]. the dreb/cbf were also regulated by bhlh-type transcription factor, ice1 [48], and recent studies demonstrate that the basic helix-loop-helix (bhlh) transcription factor atmyc2 plays a role in multiple hormone signaling pathways [49]. the regulation of flavonoid biosynthetic gene expression by the cooperation of r2r3 myb and basic bhlh transcription factors provides one of the best described examples of combinatorial gene regulation in plants [50]. the transcription activator-like effector (tale) protein was shown to induce bhlh transcription factors that activate a pectate lyase and contribute to water soaking in bacterial spot of tomato [51]. cicer arietinum genome annotation [52] has been used as an annotation database for the snpeff tool in order to detect the possible effect of sn-5825578, sn-100024987, sn-5826150, sn-100053539, sn-5825294, and sn-5825139. these snps have shown a modifier effect on yls9, coiled-coil domain-containing protein 12, blus1, and transcription factor bhlh157. some genes, such as blus1 and methionine gamma-lyase-like, are affected by two snps, while others are affected by only one snp. in summary, it is not yet clear how the predicted genes located in the qtl regions we identified play a role in ab resistance in cicer arietinum. therefore, further research into the potential roles of these genes is recommended. however, this study presents the first set of functional snp markers in chickpea (table 5), which could be converted to kompetitive allele specific pcr (kasp) markers and applied in mas breeding programs for ab resistance in chickpea. reference 1. udupa sm, weigand f, saxena mc, kahl g. genotyping with rapd and microsatellite markers resolve pathotype diversity in the ascochyta blight pathogen of chickpea. theoretical and applied genetics. 1998;97:299-307. 2. imtiaz m, abang mm, malhotra rs, ahmed s, bayaa b, udupa sm, et al. pathotype iv, a new and highly virulent pathotype of didymellarabiei, causing ascochyta blight in chickpea in syria. plant disease. 2011;95(9):1192-2. 3. atik o, ahmed s, abang mm, imtiaz m, hamwieh a, baum m, et al. pathogenic and genetic diversity of didymella rabiei affecting chickpea in syria. crop protection. 2013;46:70-9. 4. pande s, siddique kh, kishore g, bayaa b, gaur p, gowda c, et al. ascochyta blight of chickpea 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biplots analysis of the test sites and trait relations of soybean in ontario. crop science. 2002;42:1120. 36. stephens a, lombardi m, cogan noi, forster jw, hobson k, materne m, et al. genetic marker discovery, intraspecific linkage map construction and quantitative trait locus analysis of ascochyta blight resistance in chickpea (cicer arietinum l.). molecular breeding. 2014;33:297-313. 37. sabbavarapu mm, sharma m, chamarthi sk, swapna n, rathore a, thudi m, et al. molecular mapping of qtls for resistance to fusarium wilt (race 1) and ascochyta blight in chickpea (cicer arietinum l.). euphytica. 2013;193:121-33. 38. cobos mj, rubio j, strange rn, moreno mt, gil j, millan t. a new qtl for ascochyta blight resistance in an ril population derived from an interspecific cross in chickpea. euphytica. 2006;149(1):105-11. 39. martin gb, brommonschenkel sh, chunwongse j, frary a, ganal mw, spivey r, et al. map-based cloning of a protein kinase gene conferring disease resistance in tomato. 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with increased disease resistance: how are we going to express it? trends in biotechnology. 2005;23(6):283-90. 45. duek pd, fankhauser c. bhlh class transcription factors take centre stage in phytochrome signalling. trends in plant science. 2005;10(2):51-4. 46. cheng q, dong l, gao t, liu t, li n, wang l, et al. the bhlh transcription factor gmpib1 facilitates resistance to phytophthora sojae in glycine max. journal of experimental botany. 2018;69(10):2527-41. 47. koini ma, alvey l, allen t, tilley ca, harberd np, whitelam gc, et al. high temperature-mediated adaptations in plant architecture require the bhlh transcription factor pif4. current biology. 2009;19(5):408-13. 48. chinnusamy v, ohta m, kanrar s, lee bh, hong x, agarwal m, et al. complex regulation of cold-responsive gene expression in arabidopsis through genetic and epigenetic mechanisms. plant j. 2003;33:334-42. 49. fujita m, fujita y, noutoshi y, takahashi f, narusaka y, yamaguchi-shinozaki k, et al. crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. current opinion in plant biology. 2006;9(4):436-42. 50. feller a, machemer k, braun el, grotewold e. evolutionary and comparative analysis of myb and bhlh plant transcription factors. the plant journal. 2011;66(1):94-116. 51. schwartz ar, morbitzer r, lahaye t, staskawicz bj. taleinduced bhlh transcription factors that activate a pectate lyase contribute to water soaking in bacterial spot of tomato. proceedings of the national academy of sciences. 2017;114(5). 52. varshney rk, song c, saxena rk, azam s, yu s, sharpe ag, et al. draft genome sequence of chickpea (cicer arietinum) provides a resource for trait improvement. nature biotechnology. 2013;31(3):240. highlights in bioscience page 11 of 11 december 2024|volume 7 http://bioscience.highlightsin.org/ abstract introduction materials and methods plant materials and dna isolation dart and dartseq experimental design and locations scoring plants for ab symptom severity statistical analysis results phenotypic characterization mapping and qtl analyses discussion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202503 research article open access 1 department of biology, tbilisi state university, georgia. 2 department of biology, ivane javakhishvili tbilisi state university, georgia. * to whom correspondence should be addressed: temuri.mantashashvili6037@ens.tsu.edu.ge editor: hatem zayed, department of biomedical science, college of health sciences, qatar university, doha, qatar. reviewer(s): hayder tawfeeq, middle technical university, technical instituteof baqubah, department of nursing, dayala, iraq. asmaa hassan, agricultural genetic engineering research institute, agriculture research center, giza, egypt. received: february 12, 2025 accepted: june 04, 2025 published: june 20, 2025 citation: mantashashvili t, nakaidze l, ananiashvili g, burjanadze g, koshoridze n. l-arginine as a potential antidepressant: effects on cognitive function and antioxidant enzyme activity in depressed rats. 2025 june 20;8:bs202503 copyright: © 2025 mantashashvili t et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. l-arginine as a potential antidepressant: effects on cognitive function and antioxidant enzyme activity in depressed rats temuri mantashashvili*1,2 >< �, luka nakaidze1,2 >< , giorgi ananiashvili1,2 >< , george burjanadze1,2 >< �, nana koshoridze1,2 >< � abstract the potential antidepressant effects of dietary l-arginine (l-arg) were investigated in male white laboratory rats with corticosterone-induced depression. daily administration of l-arg at a dose of 150 mg/kg for 14 consecutive days significantly alleviated depressive-like behaviors and improved cognitive performance. furthermore, l-arg supplementation restored serotonin levels in the prefrontal cortex and hippocampus, which had been reduced following intraperitoneal corticosterone injection. to evaluate the antioxidant properties of l-arg, quantitative changes in malondialdehyde (mda) and nitric oxide (no) levels were measured in the prefrontal cortex and hippocampal cells of depressive rats following l-arg administration. the findings revealed that l-arg normalized lipid peroxidation processes that had been enhanced under depressive conditions. l-arg treatment significantly reduced elevated levels of these oxidative stress markers. additionally, it increased the activity of key antioxidant enzymes, including mitochondrial superoxide dismutase (sod) and catalase, whose activities had been suppressed in the depressive state. kinetic analysis of enzymatic reactions indicated that the increased activity of these antioxidant enzymes in the brain cells of depressive rats was not due to structural modifications of the enzymes, but rather to an increase in their abundance. this effect is likely attributable to the activation of biosynthetic processes in brain cells triggered by l-arg administration. keywords: depression, l-arginine, fluoxetine, antioxidant system, corticosterone introduction depression is a prevalent mental disorder with serious personal and socio-economic consequences [1; 2]. it is characterized by a wide spectrum of symptoms that significantly impair quality of life [3]. despite extensive research efforts, the precise pathophysiology of depression and effective treatment strategies remain incompletely understood. a growing body of evidence highlights the role of oxidative stress in the pathogenesis of depression, marked by increased production of reactive oxygen species (ros) and diminished activity of antioxidant defense enzymes. oxidative imbalance, in turn, disrupts essential metabolic processes. antioxidants are known to protect mitochondrial structures and dna from oxidative damage. during depression, mitochondrial dysfunction leads to atp deficiency, impairing neuronal function and contributing to what is often referred to as “cellular energy depression” [4; 5]. hormonal dysregulation, particularly involving the hypothalamic-pituitaryadrenal (hpa) axis, also plays a critical role in depression. activation of this system can promote free radical production. for example, elevated cortisol levels observed in depressed patients may be caused by oxidative damage to receptor systems or reduced cytochrome p450 activity, both of which can be linked to oxidative stress [6; 7]. however, despite continued investigation, the primary initiating mechanism of depression remains elusive. most currently available antidepressants target monoaminergic pathways in the brain, acting primarily as enzyme inhibitors that prevent neurotransmitter degradation or block their reuptake. nevertheless, these drugs are often associated with limited efficacy, a delayed onset of therapeutic action (typically 2–3 weeks or more), and various adverse effects [8; 9]. these limitations have led to a search for novel antidepressants that act through alternative mechanisms. in the late 1990s, interest grew around agents such as ketamine, which highlights in bioscience page 1 of 7 june 2025|volume 8 https://doi.org/10.36462/h.biosci.202503 https://creativecommons.org/licenses/by/4.0/ mailto:temuri.mantashashvili6037@ens.tsu.edu.ge https://orcid.org/0000-0002-9738-1027 mailto:luka.nakaidze155@ens.tsu.edu.ge mailto:giorgi.ananiashvili342@ens.tsu.edu.ge mailto:giorgi.burjanadze@tsu.ge http://orcid.org/0000-0002-5404-0644 mailto:nana.koshoridze@tsu.ge http://orcid.org/0000-0001-7324-1495 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function figure 1. scheme of studying the effect of exogenous l-arginine on depression induced by corticosterone administration. modulate the glutamatergic system [10; 11]. the rapid antidepressant action of ketamine has been attributed to activation of the mtor signaling pathway in the hypothalamus and prefrontal cortex. however, the clinical utility of ketamine is limited by its side effect profile [12; 13]. given this context, the identification of new compounds with antidepressant effects and fewer adverse outcomes is of significant interest. one such candidate is the amino acid l-arginine. dietary l-arginine is absorbed in the small intestine and distributed throughout the body. it is metabolized along several pathways: converted into ornithine and urea, acts as a nitrogen donor, participates in transamination, and facilitates the elimination of protein catabolites. ornithine, a metabolic product of l-arginine, serves as a precursor for the synthesis of collagen and polyamines [14; 15]. in parallel, nitric oxide (no), synthesized from l-arginine, functions as a key signaling molecule involved in vasodilation, synaptic plasticity, learning and memory, and the modulation of neuronal activity during stress and anxiety [16; 17]. importantly, l-arginine has also been reported to exhibit antioxidant properties [18; 19]. it downregulates oxidative stress, at least in part, by enhancing the antioxidant system through upregulation of related enzymes. for example: l-arginine has been shown to stimulate glutathione synthesis and activate the nrf2 pathway, contributing to a robust antioxidant response [20]. based on this background, the present study aimed to investigate the effects of exogenous l-arginine on depressive-like behaviors in a rat model, to evaluate its potential antidepressant activity, and to elucidate the molecular mechanisms underlying its action. materials and methods ethics approval the study protocol was approved by the institutional review board of ivane javakhishvili tbilisi state university. all procefigure 2. study of corticosterone-induced depressive state in rats using the free swim test (fst) and tail-flick test (tst). dures were conducted in accordance with the guidelines for the care and use of laboratory animals (n58/187). the experimental work was performed at the department of biology, faculty of exact and natural sciences, in close collaboration with the chair of biochemistry. animals and housing conditions adult male white laboratory rats (weighing 100 ± 15 g) were obtained from the animal facility of the a. natishvili institute of morphology, tbilisi state university (tbilisi, georgia). prior to the experiment, the rats underwent a 7-day acclimatization period to the vivarium conditions. animals were housed under standard laboratory conditions: a 12 h light/dark cycle, ambient temperature of 22 ± 1◦c, and relative humidity of 47 ± 2%. food and water were provided ad libitum. the animals were randomly assigned to four experimental groups (n = 15 per group; figure 1): • group i (g1) received daily intraperitoneal injections of 100 µl 100% dimethyl sulfoxide (dmso) (sc-358801, santa cruz biotechnology, inc., europe) for 14 consecutive days and served as the vehicle control group. • group ii (g2) received daily intraperitoneal injections of corticosterone (20 mg/kg) (sc-300391, santa cruz biotechnology, inc., europe) for 14 consecutive days to induce depressive-like behavior. • group iii (g3) received corticosterone as described for g2 during the first 14 days, followed by daily oral administration of l-arginine (150 mg/kg) (sc-391662, santa cruz biotechnology, inc., europe) for an additional 14 days. • group iv (g4) received corticosterone as described for g2 during the first 14 days, followed by daily oral administration of fluoxetine (10 mg/kg) (sc-279166, santa cruz biotechnology, inc., europe) for the next 14 days. behavioral assessments and biochemical analysis to assess the emotional state and depression-like behavior of the experimental animals, the open-field test (oft), forced highlights in bioscience page 2 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function table 1. changes in some physiological parameters of experimental animals under the influence of dietary l-arginine under conditions of corticosterone-induced depression. behavioural parameters i group ii group iii group iv group crossed cells 255.5 ± 15.5 73.7 ± 18.7∗ 280.57 ± 20.8## 270.97 ± 22.4### number of positions in the centre 2.89 ± 0.5 0.4 ± 0.1∗∗ 2.757 ± 0.8### 2.607 ± 0.3### vertical posture 3.97 ± 0.7 0.5 ± 0.1∗∗∗ 6.97 ± 2.5### 5.557 ± 1.9### vertical posture with wall touching 13.5 ± 3.1 6.9 ± 1.1∗∗ 1.8 ± 0.3### 2.0 ± 0.4### duration of freezing (sec) 32.8 ± 3.8 127.7 ± 12.4∗∗∗ 40.3 ± 4.6### 38.8 ± 5.2### duration of grooming (sec) 18.9 ± 3.5 9.7 ± 2.9∗∗∗ 16.2 ± 3.1## 17.7 ± 2.4### defecation 2.5 ± 0.4 3.7 ± 0.9∗ 1.7 ± 0.1# 2.3 ± 0.1# the duration of immobility in the fst (sec) 160.0 ± 15.1 210.6 ± 30.7∗∗ 100.5 ± 15.5## 140.9 ± 30.8## the duration of immobility in the tst (sec) 50.5 ± 9.6 225.0 ± 25.9∗∗∗ 144.8 ± 35.8## 145.3 ± 47.4### data are shown as mean ± sd; ∗, the difference compared to control; #, the difference compared to cort-induced depression; ∗,# , p < 0.05; ∗∗,## , p < 0.01; ∗∗∗,### , p < 0.001 swim test (fst), and tail suspension test (tst) were performed figure 2. these tests were conducted in accordance with the methodology described by [15]. in the fst, rats were placed in a cylinder filled with water for a total duration of 6 minutes, while in the tst, the animals were suspended by the tail for 5 minutes. a corticosterone solution (20 mg/ml) was freshly prepared each day in 100% dimethyl sulfoxide (dmso) and administered intraperitoneally at a dose of 20 mg/kg, as outlined in figure 1. the concentrations of nitric oxide (no) and malondialdehyde (mda), along with the enzymatic activities of superoxide dismutase (sod) and catalase, were measured spectrophotometrically following previously described methods [16]. statistical analysis the obtained data were statistically analyzed using the spss software (version 23; spss inc., chicago, il, usa). one-way anova was employed in order to evaluate differences among the groups in physiological and biochemical parameters. when appropriate, either tukeys hsd or games-howell post hoc tests were used to determine specific group differences. the results are presented as mean ± standard deviation (sd), and p-values less than 0.05 were considered statistically significant. results behavioral effects of l-arginine in a corticosterone-induced depression model corticosterone-induced depression significantly altered several important behavioral parameters in rats. compared to the control group, the animals exposed to corticosterone exhibited clearly reduced exploratory activity, diminished cognitive function, and heightened anxiety-like behavior. specifically, these animals demonstrated a marked and consistent decrease in the number of box crossings (by approximately 70%) and center entries (by about 80%). conversely, the duration of freezing behaviora commonly used and widely accepted indicator of fearincreased by nearly 280%, thereby highlighting a substantial rise in anxiety levels table 1. furthermore, the forced swim test (fst) further supported the observed behavioral benefits of l-arginine, clearly showing a significant reduction in immobility time after treatment figure 1. this particular outcome is consistent with an established antidepressant-like effect. a very similar trend was also observed in the tail suspension test (tst), where l-arginine administration significantly improved performance metrics compared to the untreated depressive group. highlights in bioscience page 3 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function figure 3. effect of l-arginine on nitric oxide and malondialdehyde content in prefrontal cortex and hippocampal cells of depressed. figure 4. effect of l-arginine on superoxide dismutase and catalase activity in prefrontal cortex and hippocampal cells of depressed individuals. effect of l-arginine on the hormonal status of depressed animals impact on serotonergic activity quantitative analysis of serotonin levels in the prefrontal cortex revealed a marked decline (approximately 28%) in corticosterone treated rats relative to controls figure 2a. however, l-arginine supplementation led to a substantial increase (approximately 35%) in serotonin concentration compared to the untreated depressive group figure 3a. similar recovery patterns were observed in the hippocampus table 2, indicating that larginine may exert its antidepressant effects at least in part via the restoration of monoaminergic neurotransmission. the results obtained also show that administration of the serotonin reuptake inhibitor fluoxetine produced similar effects in the depressed group of animals. effect of l-arginine on nitric oxide and malondialdehyde levels in the brain cells of depressed animals studies have shown a significant increase in nitric oxide levels in the prefrontal cortex and hippocampus of depressed individuals. however, 14-day administration of l-arginine to depressed animals significantly reduced these levels by approximately 30% and 35%, respectively figure 3 a and b. similar effects were observed with fluoxetine treatment. changes in malondialdehyde levels alterations were also observed in malondialdehyde (mda) levels. as shown in figure 3 c and d, corticosterone-induced depression is associated with an increased concentration of malondialdehyde in both the prefrontal cortex and hippocampal brain cells. however, l-arginine administration led to a reduction in its levels by approximately 20% in the prefrontal cortex and about 30% in the hippocampus. these findings suggest that corticosterone-induced depression is accompanied by increased lipid peroxidation, which decreases and approaches control levels following l-arginine treatment. as with previous parameters, fluoxetine administration similarly reduced malondialdehyde content. effect of l-arginine on the activity of antioxidant enzymes in depressed animals the activities of key antioxidant enzymes superoxide dismutase (sod) and catalasewere evaluated across the experimental groups, as presented in figure 4. as shown in figure 4a, sod activity in the prefrontal cortex of depressed animals was significantly decreased (approximately 51%) compared to the control group. however, after 14 days of l-arginine administration, enzyme activity significantly increased and approached the control values. similar trends were observed in the hippocampus figure 4 b, although overall sod activity in this region was lower than in the cortex across all groups. comparable patterns were noted for catalase activity. in the prefrontal cortex figure 4c, catalase activity was reduced by approximately 47% in the depressed group, while l-arginine administration resulted in a 60% increase. similar effects were also observed in the hippocampal cells figure 4d. importantly, fluoxetine treatment led to changes comparable to those observed with l-arginine. these results indicate that corticosterone-induced depression leads to decreased activity of antioxidant enzymes (sod and catalase), while dietary l-arginine supplementation restores their activity. table 2. quantitative changes in serotonin levels in the prefrontal cortex and hippocampus cells of depressed rats after receiving l-arginine. group prefrontal cortex hippocampus i group 55.7 ± 9.8 58.9 ± 8.6 ii group 38.3 ±6.3∗∗∗ 40.2 ±5.7∗∗ iii group 60.8 ±12.5## 50.0 ±5.5# iv group 68.5 ±12.8### 52.7 ±7.4## data are shown as mean ± sd; ∗, the difference compared to control; #, the difference compared to cortinduced depression; ∗,# , p < 0.05; ∗∗,## , p < 0.01; ∗∗∗,### , p < 0.001. highlights in bioscience page 4 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function figure 5. effect of exogenous l-arginine on superoxide dismutase vmax and km in depressed rats. figure 6. effect of exogenous l-arginine on catalase vmax and km in depressed rats. notes: on the ordinate axis 1/v; on the abscissa axis substrate concentration (mm). kinetics of sod and catalase and the effect of l-arginine to investigate the mechanisms underlying enzyme activity changes, kinetic parameters were assessed. as shown in figure 5 a, the maximum velocity (vmax) of sod in the prefrontal cortex was decreased in the depressed group (group ii) compared to the control. however, vmax increased in groups iii and iv (arginineand fluoxetine-treated groups, respectively). interestingly, the michaelis constant (km), indicating the enzyme’s affinity for its substrate, remained nearly unchanged across all groups. similar results were observed in the hippocampus figure 5 b. (note: on the ordinate axis 1/v; on the abscissa axis substrate concentration (mm)). similar results were obtained in the study of the kinetic parameters of the enzyme catalase. in particular, only the maximum velocity of the enzyme changed, while the affinity of the enzyme for the substrate (h2o2) remained the same figure 6. discussion this study aimed to investigate the effect of exogenous larginine on corticosterone-induced depression in albino rats. our findings demonstrate that daily administration of l-arginine (150 mg/kg) for 14 days leads to improvements in behavioral and physiological parameters associated with depression. notably, the open-field test results showed enhanced locomotor activity table 1 , while improvements in forced swim test (fst) and tail suspension test (tst) parameters further supported its antidepressant-like effects, in agreement with existing literature [21]. serotonin, a key neurotransmitter, plays a critical role in the pathophysiology of depression, as evidenced by the efficacy of selective serotonin reuptake inhibitors (ssris) [8; 9; 22]. to clarify the mechanism underlying the observed effects, we measured serotonin levels in the prefrontal cortex and hippocampus. l-arginine supplementation significantly increased serotonin levels in both regions, closely resembling the effects of fluoxetine table 2. this suggests that the antidepressant-like activity of l-arginine may be, at least in part, serotonin-mediated. serotonin also exhibits strong antioxidant properties. a decrease in serotonin levels, as observed in depression, is associated with reduced antioxidant defense and elevated oxidative stress markers such as malondialdehyde (mda) and nitric oxide (no), along with decreased activity of antioxidant enzymes like glutathione peroxidase, superoxide dismutase (sod), and catalase hence, restoring serotonin levelseither pharmacologically (e.g., fluoxetine) or via l-arginine supplementationcan reduce oxidative stress in the brain [22; 23; 24]. in this study, we observed decreased mda and no levels following l-arginine administration figure 3, suggesting a reduction in lipid peroxidation and nitrosative stress. these results support the notion that the antidepressant-like effect of l-arginine is mediated not only by serotonin enhancement but also by attenuation of oxidative stress [25]. furthermore, we evaluated the impact of l-arginine on antioxidant enzyme activity. corticosterone-induced depression significantly reduced sod and catalase activities figure 4, consistent with previous reports [26; 27]. l-arginine administration restored these activities, comparable to the effects observed with fluoxetine. to understand the mechanism behind these changes, we analyzed kinetic parameters (vmax and km) of sod and catalase. in corticosterone-treated rats, both vmax and substrate affinity (km) of the enzymes were reduced. however, l-arginine and fluoxetine treatments produced divergent outcomes. fluoxetine increased both vmax and substrate affinity figure 5, suggesting alterations in both enzyme expression and structure. these findings align with literature indicating that fluoxetines antioxidant effects may extend beyond serotonin modulation, potentially involving direct ros scavenging [28; 29]. conversely, l-arginine administration led to increased vmax without altering km, indicating an upregulation in enzyme quantity rather than structural modifications. these changes suggest that l-arginine stimulates enzyme synthesis figures 5, 6, enhancing antioxidant capacity via increased enzyme availability rather than altered binding properties. mitochondrial dysfunction and atp depletion are well-documented in depression and contribute to impaired neuronal function and synaptic plasticity. l-arginine is a precursor for several metabolites, including creatine, which plays a crucial role in energy metabolism [30; 31]. therefore, the observed effects of l-arginine may also be linked to its role in restoring cellular energy homeostasis, which is often compromised during depression. highlights in bioscience page 5 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function conclusion depression is a prevalent and debilitating psychiatric disorder with profound implications for individual well-being and societal productivity. the current study provides compelling evidence for the antidepressant-like effects of l-arginine in a corticosterone-induced model of depression. l-arginine supplementation significantly improved behavioral parameters and increased serotonin levels in key brain regions affected by depression. it also demonstrated potent antioxidant effects, as evidenced by reduced levels of oxidative stress markers (no, mda) and restored activities of crucial antioxidant enzymes (sod and catalase). kinetic analyses revealed that l-arginine enhances enzyme activity primarily by increasing enzyme synthesis, contrasting with the dual structural and quantitative effects observed with fluoxetine. collectively, these findings suggest that l-arginine exerts its antidepressant-like effects through a dual mechanism involving both serotonin-mediated neurotransmission and improved antioxidant defense. given its natural origin and multifaceted biological effects, l-arginine may serve as a promising adjunct or alternative to conventional antidepressant therapy. further research is warranted to elucidate its precise molecular mechanisms and evaluate its clinical potential. acknowledgements the designated project is done with the financial support of the shota rustaveli georgian national science foundation (grant #fr-23-7525). any idea contained in this publication is the authors’ property and may not represent the opinion of the foundation. reference 1. yang l, zhao y, wang y, liu l, zhang x, li b, et al. the effects of psychological stress on depression. current neuropharmacology. 2015;13:494. 2. patel sd, cameron lp, olson de. sex-specific social effects on depression-related behavioral phenotypes in mice. life. 2021;11:1327. 3. james sl, abate d, abate kh, abay sm, abbafati c, abbasi n, et al. global, regional, and national incidence, prevalence, and years lived systematic analysis for the global burden of disease study. the lancet. 2018;392:1789. 4. sterner ey, kalynchuk le. behavioral and neurobiological consequences of prolonged glucocorticoid exposure in rats: relevance to depression. progress in neuropsychopharmacology biological psychiatry. 2010;34:777. 5. zhao y, ma r, shen j, su h, xing d, du l. a mouse model of depression induced by repeated corticosterone injections. european journal of pharmacology. 2008;581:113. 6. albert pr lc vahid-ansari f. serotonin-prefrontal cortical circuitry in anxiety and depression phenotypes: pivotal role of preand post-synaptic 5-ht1a receptor expression. front behav neurosci. 2014. 7. liu y gw. emotional roles of mono-aminergic neurotransmitters in major depressive disorder and anxiety disorders. front psychol. 2018;9. 8. liwinski t base lang ue. exploring the therapeutic potential of gamma-aminobutyric acid in stress and depressive disorders through the gutbrain axis. biomedicines. 2023;11:3128. 9. bayes aj pg. comparison of guidelines for the treatment of unipolar depression: a focus on pharmacotherapy and neurostimulation. acta psychiatr scand. 2018;137:459. 10. shah khoodoruth mas pbknackgos estudilloguerra ma. glutamatergic system in depression and its role in neuromodulatory techniques optimization. front psychiatry. 2022;14. 11. galts cpc, bettio eb, jewett dc, yang cc, brocardo ps, rodrigues als. deppression in neurodegenerative diseases: common mechanisms and current treatment options. neuroscience biobehavioral reviews. 2019;102:56. 12. duman rs, aghajanian gk, sanacora g, krystal jh. synaptic plasticity and depression: new insights from stress and rapid-acting antidepressants. nature medicine. 2016;22:238. 13. li x, witkin jm, need ab, skolnick p. enhancement of antidepressant potency by a potentiator of ampa receptors. cellular and molecular neurobiology. 2003;23:419. 14. tong bc, barbul a. cellular and physiological effects of arginine. mini reviews in medicinal chemistry. 2004;4:823. 15. cryan jf, mombereau c, vassout a. the tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice. neuroscience biobehavioral reviews. 2005;29(4-5):571-625. 16. förstermann u, sessa wc. nitric oxide synthases: regulation and function. european heart journal. 2012;33:829. 17. haj-mirzaian a, amiri s, amini-khoei h, haj-mirzaian a, hashemiaghdam a, ramezanzadeh k, et al. involvement of no/nmda-r pathway in the behavioral despair induced by amphetamine withdrawal. brain research bulletin. 2018;139:81. 18. forbes sc, harber v, bell gj. oral l-arginine before resistance exercise blunts growth hormone in strength trained males. international journal of sport nutrition and exercise metabolism. 2014;24:236. highlights in bioscience page 6 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ mantashashvili t et al., 2025 l-arginine as a potential antidepressant: effects on cognitive function 19. fujiwara t, kanazawa s, ichibori r, tanigawa t, magome t, shingaki k, et al. l-arginine stimulates fibroblast proliferation through the gprc6a-erk1/2 and pi3k/akt pathway. plos one. 2014;9:e92168. 20. liang m, wang z, li h, cai l, pan j, he h, et al. l-arginine induces antioxidant response to prevent oxidative stress via stimulation of glutathione synthesis and activation of nrf2 pathway. food and chemical toxicology. 2018;115:315-28. 21. burjanadze g, shengeli m, tsverava l, mikadze m, davitashvili e, solomonia r, et al. interplay between nmda receptor modulation and na/k atpase activityunder the social isolation-induced stress in the hippocampus of male rats. indian journal of biochemistry biophysics. 2024;61:32. 22. pervin m, unno k, konishi t, nakamura y. l-arginine exerts excellent anti-stress effects on stress-induced shortened lifespan, cognitive decline and depression. international journal of molecular sciences. 2021;22:508. 23. opmeer em, kortekaas r, aleman a. depression and the role of genes involved in dopamine metabolism and signalling. progress in neurobiology. 2010;92:112. 24. bekhbat m, li z, mehta nd, treadway m, lucido mj, woolwine bj, et al. functional connectivity in reward circuitry and symptoms of anhedonia as therapeutic targets in depression with high inflammation: evidence from a dopamine challenge study. molecular psychiatry. 2022;27:4113. 25. tong bc, barbul a. cellular and physiological effects of arginine. mini reviews in medicinal chemistry. 2004;4:823. 26. gibbs sm. regulation of neuronal proliferation and differentiation by nitric oxide. molecular neurobiology. 2003;27:107. 27. paul v, ekambaram p. involvement of nitric oxide in learning memory processes. indian journal of medical research. 2011;133:471. 28. amini-khoei h, boroujeni s, maghsoudi f, rahimi-madiseh m, bijad e, moradi m, et al. possible involvement of largininenitricoxide pathway in the antidepressant activity of auraptene in mice. behavioral and brain functions. 2022;18. 29. kuchukashvili z, menabde k, chachua m, burjanadze g, chipashvili m, koshoridze n. functional state of rat cardiomyocytes and blood antioxidant system under psychoemotional stress. acta biochimica et biophysica sinica. 2011;43:480. 30. amitai y. physiologic role for “inducible” nitric oxide synthase: a new form of astrocytic-neuronal interface. glia. 2010;58:1775. 31. maes m, mihaylova l, kubera m, uytterhoeven m, vrydags n, bosmans e. increased plasma peroxides and serum oxidized low density lipoprotein antibodies in major depression: markers that further explain the higher incidence of neurodegeneration and coronary artery disease. journal of affective disorders. 2010;123:287. highlights in bioscience page 7 of 7 june 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods ethics approval animals and housing conditions behavioral assessments and biochemical analysis statistical analysis results behavioral effects of l-arginine in a corticosterone-induced depression model effect of l-arginine on the hormonal status of depressed animals effect of l-arginine on nitric oxide and malondialdehyde levels in the brain cells of depressed animals changes in malondialdehyde levels effect of l-arginine on the activity of antioxidant enzymes in depressed animals kinetics of sod and catalase and the effect of l-arginine discussion conclusion acknowledgements highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202402 research article open access 1 department of chemistry, faculty of mathematics and natural sciences, universitas negeri malang, jl. semarang no. 5 malang, malang, east java 65145, indonesia. 2 biotechnology program, department of applied science, faculty of mathematics and natural sciences, universitas negeri malang, jl. semarang no. 5 malang, malang, east java 65145, indonesia. 3 research group, department of chemistry, faculty of science, universiti teknologi malaysia, johor, malaysia 4 enzyme technology and green synthesis, department of chemistry, faculty of science, universiti teknologi malaysia, johor, malaysia * to whom correspondence should be addressed: evi.susanti.fmipa@um.ac.id editor: morad m. mokhtar, agricultural genetic engineering research institute (ageri), giza, egypt. reviewer(s): suleiman aminu, department of biochemistry, ahmadu bello university, zaria, nigeria. abdulaziz ascandari, chemical and biochemical sciences-green process engineering, university mohammed vi polytechnic, benguerir, morocco. received: october 13, 2023 accepted: february 17, 2024 published: may 21, 2024 citation: wijayanti c, sanjaya eh, wahab ra, susanti e. exploration of the polypropylene degrading bacteria candidates from the passive zone of the supit urang landfill in malang city by using the next generation sequencing (ngs) method. 2024 may 21;7:bs202402 copyright: © 2024 wijayanti et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. exploration of the polypropylene degrading bacteria candidates from the passive zone of the supit urang landfill in malang city by using the next generation sequencing (ngs) method chandra wijayanti1 >< ,eli hendrik sanjaya1,2 >< �, roswanira abdul wahab2,3,4 >< �, evi susanti*,1,2 ><� abstract the buildup of plastic waste in the passive zone of the supit urang landfill located in malang city has been ongoing since 2018. currently, plastic waste in this area appears brittle and cracked, potentially providing a habitat for pp-degrading bacteria. this research aims to explore the potential of polypropylene-degrading bacteria using next generation sequencing (ngs) techniques in the passive zone of the supit urang landfill, malang city. our study was conducted in four steps: 1) sampling and sample collection, 2) dna sequencing, 3) bioinformatics analysis, and 4) bibliometric analysis for identification of polypropylene-degrading bacteria. based on the results of full-length sequencing using oxford nanopore technologies with whole amplicon sequencing techniques, a total of 2,496 sequences were read, and 1,713 sequences were identified as species in the passive zone of supit urang landfill. the most abundant bacterial phyla in this region were proteobacteria (51%), firmicutes (21%), acidobacteria (7%), bacteroidetes (6%), planctomycetes (4%), actinobacteria (3%), gemmatimonadetes (2%), nitrospirae (2%), and chloroflexi (2%). these results indicate that proteobacteria and firmicutes are abundant in the passive zone of tpa supit urang and could potentially biodegrade microplastics such as polypropylene. the narrative review’s research showed that numerous bacterial species, including bacillus thuringiensis, b. cereus, and bacillus sp., were identified by ngs analysis as possible polypropylene-degrading bacteria. keywords: bacterial diversity, next generation sequencing, polypropylene degradation introduction reports of synthetic waste contamination began in the 1970s [1]. since then, the number has continued to become a serious problem and global concern. this is caused by the natural biodegradation of plastic waste that takes a very long time, i.e., 50 to more 100 years [2]. one type of plastic waste that often becomes problematic is polypropylene (pp) waste. pp is an extremely versatile polymer that is preferred for its ease of processing, barrier properties, gloss, and dimensional stability, but as its use grows, so does the problem of managing its waste. pp is utilized in diverse commercial applications, including packaging, labelling, and fibre production for indoor and outdoor carpeting. more than 79.01 million tons of plastic waste is estimated to have reached our oceans. according to data making oceans plastic free (2017), the use of the pp in indonesia reaches 1,278 million metric tons. important to note, many types of pp plastic are scattered in the passive zone of the landfills [3; 4]. synthetic polymers such as pp have the potential to become microplastics that are harmful to the environment and human health. important proteins such as albumin, globulin and fibrinogen are altered by microplastics when they enter the body and become dysfunctional due to their interaction with blood particles [4]. changes in chromosome structure caused by microplastics entering cells and tissues have been linked to cancer, obesity and infertility [5]. as a result, the presence of mountains of plastic waste will endanger the environment and human health [6]. highlights in bioscience page 1 of 10 may 2024|volume 7 https://doi.org/10.36462/h.biosci.202402 https://creativecommons.org/licenses/by/4.0/ mailto:chandrawijayanti1811@gmail.com mailto:eli.hendrik.fmipa@um.ac.id https://orcid.org/0000-0002-6513-5198 mailto:roswanira@utm.my https://orcid.org/0000-0002-9982-6587 mailto:evi.susanti.fmipa@um.ac.id https://orcid.org/0000-0002-0201-7303 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs in recent years, natural degradation has become critical as social pressures increase for safe plastic waste management. one alternative step is biodegradation by utilizing indigenous degrading bacteria. several studies show that using indigenous bacteria can increase the degradation rate and support ecosystem recovery without causing adverse impacts and is environmentally friendly [7]. this leads to the need of an alternative solution in form of the development of biological degradation [8]. the soil microbial community may contain indigenous bacteria candidates that are more adaptive. observation results found that waste in the zone that has been passive for a long time in malang city, namely the supit urang landfill, since 2018 contains a lot of plastic that has experienced degradation, became cracked and brittle. since the condition and nutritional characteristics of each landfill soil are likely to be different, resulting in diversity, different isolates of pp degrading bacteria can therefore be obtained from different landfills. from contaminated sites, including landfills, bacterial species capable of biodegrading plastics have previously been isolated [9]. the exploration of pp-degrading bacteria isolates can be done using bacterial culture techniques or a genomic approach. the bacterial culture technique takes a relatively long period of time because of some stages as sampling, isolate culturing, screening and isolate identification are influenced by many parameters such as nutrition in growth medium and environmental conditions. meanwhile, identifying the presence of pp-degrading bacteria with the fastest culture technique takes 40 days [4]. thereby, isolate culturing is slightly abandoned. on the other hands, the genomic technique using next generation sequencing (ngs) methods takes only a few days. ngs uses a parallel approach to sequencing, in which millions of dna fragments can be sequenced at the same time in a single reaction, allowing for high throughput analysis [10]. ngs automation is very high because almost all steps in the sequencing process, including sample preparation, amplification, library preparation, sequencing, and data analysis, can be done simultaneously. after sequencing, the data is obtained and subjected to bioinformatics analysis to identify colonizing bacterial species. this research aims to explore candidate polypropylene degrading bacteria isolates in the passive zone of supit urang landfill, malang city using ngs methods. it is hoped that the findings of this study could serve as evidence for the search of polypropylene degrading bacteria from landfills. experimental section this research uses an explorative descriptive method to assess the biodiversity of bacterial communities in the passive zone of tpa supit urang malang city, consisting of: 1) sampling site and sample colection, 2) dna sequencing, 3) bioinformatics analysis, and 4) bibliometric analysis for identification of polypropylene degrading bacteria. the analysis of the passive zone samples begins with the isolation of genomic dna from the samples from passive zone of tpa supit urang landfill using zymo research’s zymobiomics dna miniprep kit, followed by amplification of the isolated dna with 16s 27f 1429r primers using (ngs) using oxford nanopore technologies technology, sequencing of the dna amplification results using ngs, downstream analysis of the results using pavian krona software, instrumentation and library preparation using oxford nanopore technology kits, and review of blibliometric analysis to identify native polypropylenedegrading bacterial candidates. sampling and sample collection soils were collected from the passive zone of the tpa supit urang in mulyorejo village, sukun district, malang city, east java, with the coordinates of 7o59’20.04" latitude in the south and 112o38’17.52" longitude in the east. the sampling technique refers to helen et al. (2017) [4] and anah et al., (2020) [11]. soil sediment samples were collected with a shovel from 30 to 60 cm above the soil surface at 5 different locations. samples were taken at each end as much as 200 g to obtain a total piece of 1 kg sediment. then, the samples from the five points were stirred evenly and sifted to get a homogeneous soil. homogeneous samples were subjected to preliminary physico-chemical tests to determine the temperature, moisture, ph, organic carbon and total n content of the soil. the soil samples were then placed in dark container and containing ice gel to maintain a consistent temperature during transport. dna from the soil samples (sample code: ca20) were extracted using the zymobiomics dna miniprep kit from zymo research with minimum sample amount of 250 mg. the basic principle of using the zymbiomics dna miniprep kit is to lyse and disrupt all microbes in samples using lysis buffer and beads. then, the purification step is repeated several times to ensure better separation and more efficient protein precipitation, and the final step uses affinity chromatography to obtain pure dna extracts. dna concentration measurements were performed using a qubit fluorometer. qubit provides dna concentration information using fluorogenic dyes that bind selectively to dna or rna. the dye emits a signal only when bound to the target. it can then be used to determine the purity of dna using the nanodrop spectrophotometer, providing a direct measurement of the a260/280 purity ratio. using the two together can help ensure that the dna sample is of sufficient concentration and purity for sequencing applications. dna sequencing all obtained amplicons were sequenced using full-length next-generation sequencing (ngs) techniques. full-length sequencing using oxford nanopore technologies technology. oxford nanopore technologies’ technology is a nanopore-based dna sequencing technology in which dna or rna molecules can be passed through nanopores from end to end, causing electrical changes that produce a complete sequence of the molecule. highlights in bioscience page 2 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs bioinformatics analysis dna amplification results were sequenced with the specialized software for oxford nanopore technologies’ technology, namely gridion instrument using minknow software version 20.06.9. base calling was carried out using guppy software to visualize the data from genetic sequence data resulting in ±2400 sequences. quality control was performed using nano plots and classified as bacteria and archaea indices downloaded from the website (https://ccb.jhu.edu/software/centrifuge). the analysis was carried out downstream using the software pavian krona tools: to visualize microbial diversity data using krona visualization. bibliometric analysis for identification of polypropylene-degrading bacteria the data sources used were scientific articles in the 1960-2021 period, sourced from the scholarly database (https://scopus.com). search for scientific articles was made by using publish or perish (pop) version 7 software with the keywords degradation of polypropylene. the software collects and processes data from sources such as scopus to provide statistics about one’s scholarly work, such as number of citations, h-index, g-index, and other indicators. data obtained from publish or perish was filtered based on the suitability with research, then stored in csv (comma separated values) and ris (research information system) files. the results obtained were analysed using vosviewer version 1.6.20 software for bibliometric map analysis to determine the development of the topic of polypropylene-degrading bacteria. vosviewer is software used to visualize and analyze bibliometric networks and overall concept maps emerging from the scientific literature. results and discussion extracted dna from the sample with the zymbiomics dna miniprep kit were 50 µl with a concentration of 57.4 ng/µl, which means there is 2.86 µg of dna. amplification of the 16s rrna gene by polymerase chain reaction (pcr) using the primers resulted in an amplicon size of around 1,500 bp (figure 1) and 2,496 sequences. according to sacchi et al. (2002), the pcr band sizes of about 1,500 bp 1,600 bp are those of the bacterial 16s rrna sequences as they can distinguish between specific taxa or strains and are indispensable for describing new species [12]. a total of 2,496 sequences were successfully identified. all sequences were then subjected to quality control using nanoplot to ensure that the sequences obtained were bacteria or archaea, and classified using the source data downloaded from the website (https://ccb.jhu.edu/software/centrifuge). based on this classification, 2,496 sequences were bacterial sequences [12]. a total of 2,496 bacterial sequences were identified from the full-length 16s rrna sequence results as kingdom (1), phylum (25), class (10), order (10), family (16), genus (19), phylum (659), species (1,713), and subspecies (43). in addition, the data obtained was further analyzed using pavian krona tools software to visualize figure 1. electrophoregram of amplified bacterial dnas from ca20 (passive zone sample of supit urang landfill, malang city). microbial diversity data using krona visualization. based on the results of the krona graph, the diversity of bacterial phyla abundant in the passive zone of supit urang landfill are proteobacteria (51%), firmicutes (21%), acidobacteria (7%), bacteroidetes (6%), planctomycetes (4%), actinobacteria (3%), gemmatimonadetes (2%), nitrospirae (2%), and chloroflexi (2%). meanwhile, relative abundance less than 0.6% is categorized into other categories. proteobacteria and firmicutes exhibited the highest bacterial abundance among the analyzed samples. the overall structure of the present bacteria in the passive zone of supit urang landfill, malang, is visualized in a krona graph (figure 2), showing a broader taxonomic hierarchy. the style of reading the krona graph from inside to outside showed the abundance of the bacterial domain consisting of 9 phyla (proteobacteria, firmicutes, acidobacteria, bacteroidetes, planctomycetes, actinobacteria, gemmatimonadetes, nitrospirae, chloroflexi) (figure 3). each consisted of classes, orders, families, genera, and species. the advantage of the krona graph visualization is that it has a more extensive or highly detailed taxonomic hierarchy [13]. another similar study with the same conditions and research methods but different sampling sites [14] from bestari landfill, probolinggo, resulted in different phyla identified (table 1). this highlights in bioscience page 3 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs figure 2. the krona graph represents bacterial diversity distribution in the passive zone of supit urang landfill malang, east java. proves the initial assumption of this study that each landfill has its own characteristics resulting in different microbial diversity. the characteristic properties of each landfill mainly depend on factors of different environmental conditions, including ph, oxygen availability, temperature, humidity, and landfill age which will influence differences in microbial diversity [13]. differences in environmental conditions at the supit urang landfill and bestari landfill are shown in table 2. the bestari landfill location is in the lowlands compared to the supit urang landfill location, which is in the highlands. thus, bestari, probolinggo landfill is used as a comparison because it has different environmental conditions (physicochemical conditions, types of waste disposed, topographical differences) will affect the diversity of native bacteria that are adaptive to the environment. based on our preliminary investigations, the differences in the table 1. comparison of the diversity of identified bacterial phyla with those in the reference [14]. phylum supit urang landfill, malang presence reference percentage (%) proteobacteria identified 51 identified 70 firmicutes identified 21 identified 15 acidobacteria identified 7 not identified bacteroidetes identified 6 not identified planctomycetes identified 4 identified 2 actinobacteria identified 3 not identified gemmatimonadetes identified 2 not identified nitrospirae identified 2 not identified chloroflexi identified 0.6 not identified cyanobacteria not identified identified 0.3 abundance of some phyla in the supit urang landfill, malang, and in the bestari landfill, probolinggo, could be due to the differences in some of the environmental parameters in the different soil profiles. based on research [15], several pseudomonas strains have been reported to aid in pp degradation [3; 4]. in highlights in bioscience page 4 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs table 2. comparison of identified environmental conditions with reference [14]. parameter supit urang landfill, malang bestari landfill, probolinggo temperature 33oc 36oc humidity 46% 52% ph 7 7 carbon organic 5.71% 2.85% nitrogen total 0.36% 0.15% both aquatic and terrestrial environments, pseudomonas has a ph range of 4.5 to 9.5 [16]. unfortunately, in the abundance of microbial diversity of the ngs results did not show the presence of pseudomonas (figure 3). it is possible that the ph range of the supit urang landfill habitat is within the growth range of pseudomonas, but there are other nutrients that do not support pseudomonas growth. therefore, pseudomonas has not been identified in the supit urang landfill. most members of the phylum firmicutes are gram-positive bacteria with the ability to form endospores. they are primarily found in soil habitats [17]. the most frequently mentioned organisms are the genus bacillus members [18]. bacteria in this genus can produce oval or cylindrical endospores and can function as aerobes or facultative aerobes. certain strains can generate extracellular hydrolytic enzymes that break down complex polymers, such as lipids, to be used as a carbon source and electron donor. moreover, several of these bacteria synthesize antibiotics and insecticides [17]. growth took place between ph values of 5.4 and 8.5, with the most favorable growth at 7.0 [19]. bacillus is known to be able to degrade pp [20; 21; 22; 23]. pseudomonas was suggested by 21.0% of the studies, bacillus by 15%, and a combination of the two by 17% of the studies as the bacteria that effectively initiated the biodegradation of synthetic polymers. thus, the environmental conditions of the waste piles are severe because of the existence of bio toxic compounds and various synthetic substrates [24]. based on table 2, the temperature in the passive zone is 33oc; mesophilic bacteria grow well at that temperature. the optimal temperature for plastic-degrading bacteria to grow optimally is 20oc-40oc [25]. humidity in the sampling zone is 46% (moderate), which is not suitable for bacterial growth [26]. the ph at the side locations ranged from 6.97.2, indicating that the sampling location was neutral. furthermore, in the analysis, the organic c content was found to be 5.71%, which was classified as very high (> 5%), and the organic n content was 0.36% (classified as moderate). soil total nitrogen is used as an essential index of soil fertility. based on the physicochemical characteristics of the soil, dealing with proteobacteria or firmicutes bacteria requires selective media to inhibit the growth of other unwanted species. identity of polypropylene-degrading bacteria based on bibliometric analysis the data source used was scientific articles based on the scopus.com database, with the keyword "degradation of polypropylene" in the keywords. article searches are based on the scopus database, because scopus is one of the databases whose scope of reputable international journals can be recognized or well received by all researchers around the world [27]. as shown in table 3, 200 articles were published by scopus.com, filtered according to research on polypropylene degradation from 19602021 in the scopus.com database, which has increased significantly yearly. the bibliometric analysis was performed at the end of 2022, but the articles that could be filtered by pop only reached 2021. this is because scopus-indexed research on bacterial degradation lasts until 2021. based on the graphical data of the number of publications from 1960-2021, it can be seen that the number of publications on polypropylene degradation has an unstable trend from 20012022. the beginning of the research topic on polypropylene degradation started in 1960 with one study and was significant until 1992. however, since 2001, research on polypropylene degradation has experienced ups and downs in the number of publications. based on the graph of the number of publications (figure 4), there were 12 publications in 2006 and 2018. in addition, only 1 publication was recorded in 2022, and even no publication records were found in 2023. in terms of document type (figure 5), most publications were research articles (166; 83%), followed by reviews (29; 14.5%), conferences (3; 1.5%), and short surveys (2; 1%). information from the scopus database showed that most of the publications were all open access. table 3. analysis of pop metrics of relevant articles results metrics publication years 1960-2021 citation years 62 papers 200 citations 58412 cites/year 942.13 cites/paper 292.06 authors/paper 1.00 h-index 156 g-index 200 hi, norm 156 hi, annual 2.42 ha-index 38 the terms in the title, abstract, index keywords, and year of publication were identified for each publication. a circle represented each time. the size of the process reflected the number of publications. meanwhile, the distance between the two terms offered an approximate indication of the relatedness of the terms. on the other hand, colors represent groups of words that are strongly related to each other [28]. the development map of the publication of polypropylene-degrading bacteria showed that there were 4 clusters (figure 6 & table 4). keyword analysis provides a comprehensive overview of research trajectories and research topics (donthu et al., 2021). based on the keyword analysis (table 4), cluster 1 has the highest number of selected keywords, which means that it contains keywords that are frequently used in the research context, which is "ability". in terms of main keywords, "ability" has the highest occurrence, followed highlights in bioscience page 5 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs figure 3. relative abundance (%) at the phylum (a) and species (b) levels obtained from the passive zone of the supit urang landfill malang city, east java, indonesia. figure 4. global publication trend by document types. highlights in bioscience page 6 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs figure 5. publication types used for bibliometrics of 200 published articles. by "biodegradability". this refers to the ability of a substance to be decomposed or broken down by microorganisms into simpler and less harmful components in the environment. the process of biodegradability is part of the natural cycle of plastic degradation, in which microorganisms such as bacteria and fungi break down complex compounds into simpler forms. figure 6. knowledge map based on index keywords from elsevier scopus from 1960-2021. the resulting clusters of keywords provide an overview of the research themes. for example, cluster 1 describes "ability" or "biodegradability", while the term in cluster 2 is the bacterial species bacillus, which is most often found in research as a potential bacterium that can degrade plastics commonly isolated from sediments. clusters 3 and 4, on the other hand, show interest in exploring bacterial species and sample locations to obtain more candidate bacterial species with biodegradation and bioremediation capabilities. table 5 shows that only some studies of pp biodegradation by bacteria have been reported. bacillus thuringiensis has been reported to degrade pp by 12% for 40 days [3]. the study was conducted using pp pre-treatment, which involved irradiation or thermal treatment [29; 30] and was shown to reduce the hydrophobicity of the polymer. b. flexus has also been shown to table 4. keyword clusters in degradation of polypropylene research clusters no. of keywords selected keywords color 1 37 ability, b. cereus, bacteria isolate, bacteria isolated, bacterial strain, biodegradability, commercial application, day, environment, environmental pollution, extent, important role, incubation, isolate, mangrove ecosystems, medium, microorganism, microplastic material, mineral salt medium, polymer reduction, polypropylene degradation, polypropylene granule, polypropylene polymer, positive potential, the present study, rate, recalcitrance, reduction, remediation, sglobispora, sole carbon source, sporasarcina globispora, term, versatile polymer, vitro, weight loss red 2 13 bacillus, bacillus biofilm, biodeterioration, degradation potential, growth, growth kinetic, mangrove sediment, polypropylene microplastic, pretreated polypropylene, pseudomonas, rhodococcus, synergistic growth green 3 11 bacillus paralicheniformis, biodegradation, low-density polyethylene, lysinibacillus fusiformi, madurai, microbe, municipal solid waste, polypropylene, polypropylene film, soil, vaigai river yellow 4 11 aquatic ecosystem, bacillus strain, bacterium, compost, degradation, mangrove ecosystem, microplastic degradation, peninsular malaysia, polypropylene l lactated, screening, soil consortia blue degrade pp by uv treatment [23]. bacillus sp. can degrade polypropylene plastic during the 40-day incubation period [31]. b. cereus showed 12% in degrading polypropylene for 40 days [4]. the b. flexus + b. subtilis consortium for one year showed a degradation of 1.45% [32]. this confirmed that the bacterial diversity, namely b. thuringiensis, b. cereus, and bacillus sp., identified from the passive zone of the supit urang landfill, could potentially degrade polypropylene. highlights in bioscience page 7 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs table 5. list of bibliography studies on the topic degradation of polypropylene no publisher title bacteria comment references 1 elsevier degradation of unpretreated and thermally pretreated polypropylene by soil consortia bacillus flexus able to degrade polypropylene film (pp-tt), which has not been processed for 12 months, by 10.7% [22] 2 elsevier growth of pseudomonas and bacillus biofilms on the pretreated polypropylene surface bacillus and pseudomonas pseudomonas azotoformans, pseudomonas stutzeri, bacillus subtilis and bacillus flexus separately for 12 months. p. azotoformans and b. subtilis are relatively hydrophobic, produce biosurfactants, and form biofilms on polymers with higher carbohydrates and proteins than the other two organisms. [23] 3 taylor and francis synergistic growth of bacillus and pseudomonas and its degradation potential on pretreated polypropylene consortium of b. flexus + p. azotoformans, consortium b. flexus + b. subtilis consortium b. flexus + p. azotoformans for one year showed maximum degradation (22.7%). consortium b. flexus + b. subtilis showed a 1.45% degradation for one year. [32] 4 elsevier screening of bacillus strains isolated from mangrove ecosystems in peninsular malaysia for microplastic degradation b. gottheilii b. gottheilii was able to degrade pp for 40 days by 3.6% [20] 5 biochemistry and bioinformatics screening for polypropylene degradation potential of bacteria isolated from mangrove ecosystems in peninsular malaysia bacillus cereus, sporosarcina globispora bacillus cereus showed 12%, and sporosarcina globispora showed an 11% decrease in body weight in 40 days. [4] 6 taylor and francis degradation of polypropylenepoly-l-lactide blend by bacteria isolated from compost bacillus licheniformis (isolate p6), bacillus thuringiensis (isolate p8) isolate p8 was 12%, and p6 was 10%, capable of degrading pp for 40 days. [3] 7 elsevier growth kinetics and biodeterioration of polypropylene microplastics by bacillus sp. and rhodococcus sp. isolated from mangrove sediment rhodococcus, bacillus sp. able to degrade pp by 6.4% rhodococcus sp. strain 36 and lessen pp by 4.0% by bacillus sp. strain 27 after 40 days of incubation. [21] 8 elsevier complete genome sequence of marine bacillus sp. y-01, isolated from the plastics contamination in the yellow sea bacillus sp. bacillus sp. isolated from plastic contamination in the yellow sea [28] 9 elsevier microplastic degradation by bacteria in aquatic ecosystem bacillus sp. bcbt21, bacillus amyloliquefaciens bsm-1, b. amyloliquefaciens bsm-2, pseudomonas putida, bacillus subtilis, bacillus cereus, brevibaccillus borstelensis, bacillus vallismortis bt-dsce 01, p. protegens bt-dsce 02, stenotrophomonas sp. bt-dsce03, and paenibacillus sp.bt-dsce04 extracellular hydrolytic enzymes such as cmcase, lipase, xylanase, keratinase, chitinase, and protease secreted by these bacteria play a charismatic role in plastic degradation. polyurethanes depolymerize urethane and ester bonds due to the hydrolytic properties of urease, esterase, and protease enzymes. papain and urease can degrade medical polyester due to their proteolytic properties. [33] 10 pubmed microbial and enzymatic degradation of synthetic plastics outlining the progress made in the microbial degradation of synthetic plastics and an overview of the enzymes involved in biodegradation. [34] 11 elsevier plastic biodegradation: frontline microbes and their enzymes literature study on plastic degradation by bacteria. [35] 12 springer biodegradation of low-density polyethylene and polypropylene by microbes isolated from vaigai river, madurai, india bacillus paramycoides (bp) and bacillus cereus (bc) the highest degradation of pp and pe was observed in bp (78.99 ± 0.005%) and bc (63.08 ± 0.009%) in the single approach, whereas in the combined system, bc & bp recorded the highest degradation in both pp (78.62 ± 2.16%) and pe (72.50 ± 20.53%). [36] 13 elsevier biodegradation of polypropylene films by bacillus paralicheniformis and lysinibacillus fusiformis isolated from municipality solid waste contaminated soil bacillus paralicheniformis and lysinibacillus fusiformis growth of bacillus paralicheniformis and lysinibacillus fusiformis has shown od values at 600nm after a 4-week degradation period increased from 0.131 to 0.334 and 0.148 to 0.213, respectively. [37] 14 elsevier microplastics spatiotemporal distribution and plasticdegrading bacteria identification in the sanitary and non-sanitary municipal solid waste landfills the microplastic (mp) surface gradually fades, becomes rough, and even produces cracks and holes with landfill depth and age. small-size mps (<100 m) were the most abundant, and their numbers increased significantly from 28.14% to 49.13% in sl and from 24.54% to 59.51% in nsl, while prominent-size mps decreased significantly. [38] conclusion ngs employing 16s rrna analysis at the supit urang landfill showed 1,713 bacterial species from 2,496 sequences. bacterial species with high abundance, namely bacillus thuringiensis (15%), b. cereus (13%), b. velezensis (10%), and bacillus sp. (7%) were identified. based on the bibliometric analysis results, b. thuringiensis, b. cereus, and bacillus sp. showed potential in the degradation of polypropylene. these indigenous pp-degrading bacteria are expected to be cultured and used to develop pp biological degradation using suitable selective media. the limitation of the study is that it only provides the profile of candidate bacteria that can degrade plastics. to improve the results of this research in the future, confirmation can be carried out on a wet lab scale by performing proteomic analysis aimed at obtaining information on cellular protein expression, so that it can find specific proteins that can only be found in plastic degrading bacterial candidates, and not in bacteria that do not degrade plastic. from the specific proteins obtained, it can then proceed to bioinformatics analysis through molecular docking to determine the specific binding to the active site of the target protein. bioinformatics analysis can specifically predict the target protein that causes the microbe to have the ability to degrade xenobiotic plastics, and it can be combined with molecular docking to see its ability to different types of xenobiotic plastics. highlights in bioscience page 8 of 10 may 2024|volume 7 http://bioscience.highlightsin.org/ wijayanti et al., 2024 identification of polypropylene-degrading bacteria in supit urang landfill using ngs acknowledgment the author would like to thank lembaga penelitian dan pengabdian masyarakat (lp2m) universitas negeri malang (um), which funded this research through the um internal funding thesis research grant scheme in 2022. reference 1. carpenter e, smith k. plastics on the sargasso sea surface. science. 1972;175(4027):1240-1. 2. chamas a, moon h, zheng j, qiu y, tabassum t, jang j, et al. degradation rates of plastics in the environment. acs sustain chem eng. 2020;8(9):3494-511. 3. jain k, bhunia h, sudhakara reddy m. degradation of polypropylenepoly-l-lactide blend 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kathirvel p. biodegradation of polypropylene films by bacillus paralicheniformis and lysinibacillus fusiformis isolated from municipality solid waste contaminated soil. res j chem environ. 2021;25(7):71-8. available from: https://api.elsevier.com/content/abstract/ scopus_id/85109159500. 38. li n, han z, guo n, zhou z, liu y, tang q. microplastics spatiotemporal distribution and plastic-degrading bacteria identification in the sanitary and non-sanitary municipal solid waste landfills. j hazard mater. 2022;438:129452. available from: https://www.sciencedirect.com/ science/article/pii/s0304389422012456. highlights in bioscience page 10 of 10 may 2024|volume 7 http://dx.doi.org/10.1016/j.ibiod.2008.06.005 http://dx.doi.org/10.1016/j.ibiod.2008.06.005 http://dx.doi.org/10.1016/j.ibiod.2010.06.002 http://dx.doi.org/10.1016/j.ibiod.2010.06.002 http://dx.doi.org/10.1016/j.marenvres.2013.09.012 http://dx.doi.org/10.1016/j.marenvres.2013.09.012 http://dx.doi.org/10.1016/j.margen.2018.05.002 http://dx.doi.org/10.1016/j.margen.2018.05.002 https://www.sciencedirect.com/science/article/pii/0141391094901384 https://www.sciencedirect.com/science/article/pii/0141391094901384 https://doi.org/10.1016/j.marpolbul.2017.11.036 https://doi.org/10.1016/j.marpolbul.2017.11.036 http://dx.doi.org/10.1016/b978-0-12-819001-2.00022-x http://dx.doi.org/10.1016/b978-0-12-819001-2.00022-x https://doi.org/10.1016/j.scitotenv.2020.143536 https://doi.org/10.1016/j.scitotenv.2020.143536 https://doi.org/10.1007/s00203-021-02592-0 https://doi.org/10.1007/s00203-021-02592-0 https://api.elsevier.com/content/abstract/scopus_id/85109159500 https://api.elsevier.com/content/abstract/scopus_id/85109159500 https://www.sciencedirect.com/science/article/pii/s0304389422012456 https://www.sciencedirect.com/science/article/pii/s0304389422012456 http://bioscience.highlightsin.org/ abstract introduction experimental section sampling and sample collection dna sequencing bioinformatics analysis bibliometric analysis for identification of polypropylene-degrading bacteria results and discussion identity of polypropylene-degrading bacteria based on bibliometric analysis conclusion acknowledgment highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202401 research article open access 1 plant production department, faculty of environmental agricultural sciences, arish university, north sinai, egypt. 2 department of agronomy, faculty of agriculture, suez canal university, ismailia 41522, egypt. 3 agricultural genetic engineering research institute, agricultural research center, giza, egypt. 4 faculty of environmental agricultural sciences, arish university, egypt. 5 department of agronomy, faculty of agriculture, cairo university, giza, egypt. 6 department of biological sciences,environmental studies institution, arish university, egypt. * to whom correspondence should be addressed: mobark.mohamed99@yahoo.com editor: morad mokhtar, university mohammed vi polytechnic, benguerir, morocco. reviewer(s): suleiman aminu, department of biochemistry, ahmadu bello university, zaria, nigeria. fida alo, international center for agricultural research in the dry areas (icarda) received: february 19, 2024 accepted: september 9, 2024 published: december 24, 2024 citation: mubarak mh, emam ma, shafik di, moatz am, abd el-wahab mmh, ibrahim aah. identifying molecular differences using issr markers on three quinoa genotypes (chenopodiumm quinoa) under sinai conditions, egypt . 2024 dec. 24;7:bs202401 copyright: © 2024 mubarak mh et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. identifying molecular differences using issr markers on three quinoa genotypes (chenopodiumm quinoa) under sinai conditions, egypt mohamed h. mubarak1 ><�, mohamed a. emam2 ><�, shafik d. ibrahim3 >< �, moatz a. mohamed4 >< �, mustafa m. h. abd el-wahab5 ><�, ahmed a. hegazy6 ><� abstract this study aimed to evaluate the performance of three quinoa genotypes (giza1, danish kvl3704, and misr1) under three irrigation intervals (every three, six, and nine days). genetic diversity among nine quinoa accessions was assessed using eight issr primers, yielding robust amplification products and polymorphic fingerprint patterns. a total of 102 bands were generated, averaging 12.75 bands per primer. among these, 52 fragments were polymorphic, resulting in an average of 6.5 polymorphic bands per primer and an overall polymorphism level of 40.9%. primer issr-8 exhibited the highest polymorphic capacity with 17 polymorphic bands, while primer issr-15 displayed the highest frequency (0.9). in contrast, primers issr-8 and issr-10 exhibited the lowest frequency (0.5). the polymorphism percentage ranged from 20% for primer issr-15 to 88% for primer issr-10. the similarity index revealed a minimum value of 78% between treatments, clustering the nine accessions into eight groups across four similarity levels, from 80% to a maximum of 93%. dendrogram analysis underscored the utility of issr-pcr in detecting genetic relationships among quinoa accessions. these findings highlight the potential of issr-pcr as a reliable tool for genetic diversity studies and its applicability in quinoa breeding programs. keywords: quinoa, genotypes, issr primers, similarity index, relationship. introduction quinoa (chenopodium quinoa) is a pseudo-grain that produces seed-like grains, commonly used as whole grains in bread and soups and as a versatile ingredient for people with celiac disease. it is considered one of the most nutritious crops, offering a rich source of protein, essential amino acids, and fiber, making it an excellent alternative for gluten-free diets. quinoa demonstrates significant potential as a sustainable crop for cultivation in salt-affected areas [1]. water scarcity, climate change, and rapid population growth are significant challenges in egypt, putting significant pressure on the country's agricultural productivity and food security. egypt has extensive marginal lands unsuitable for agriculture due to high soil salinity or low-quality groundwater. however, these lands are viewed as an opportunity to enhance agricultural production and ensure food security by cultivating salttolerant crops like quinoa, which is considered one of the strategic solutions to cope with the high food demand of the growing population. with quinoa's adaptability to harsh conditions, it could play a critical role in improving agricultural sustainability and helping meet the nutritional needs of the population [2; 3]. molecular markers are powerful tools for cultivar identification, germplasm conservation, and detecting genetic variability in various species. issr (inter-simple sequence repeat) markers are inexpensive and are easily scored manually, and prior knowledge of the flanking sequences is not required, making them ideal for large-scale genetic studies. the development of polymerase chain reaction (pcr) has made pcr-based marker systems key tools in genetic analyses. issr-pcr generates reproducible, abundant, and polymorphic multilocus patterns in plant genomes [4; 5]. highlights in bioscience page 1 of 7 december 2024|volume 7 https://doi.org/10.36462/h.biosci.202401 https://creativecommons.org/licenses/by/4.0/ mailto:mobark_mohamed99@yahoo.com https://orcid.org/0000-0002-2981-381x mailto: mohamed_abdel-gawad@agr.suez.edu.eg https://orcid.org/0000-0002-5475-5527 mailto:shafikdarwish2014@gmail.com https://orcid.org/0000-0001-8152-1999 mailto:maotaz@aru.edu.eg https://orcid.org/0009-0002-1781-4978 mailto:mustafamh2003@yahoo.co.uk https://orcid.org/0009-0005-4043-8968 mailto:awaad197705@gmail.com https://orcid.org/0009-0003-8389-1514 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions this study aims to evaluate the performance of various quinoa (chenopodium quinoa) genotypes under different irrigation intervals and stress conditions. with a focus on identifying drought and salinity tolerant genotypes suitable for marginal lands in egypt. additionally, to assess the genetic diversity of these quinoa genotypes using issr markers to inform breeding programs aimed at developing high-yielding, salt-tolerant quinoa varieties [6; 7]. materials and methods field experiment the planting and harvesting activities were conducted during the 2023/2024 agricultural season, and following the dneasy kit technique (qiagen) [8], dna was extracted and processed to ensure high-quality dna for further analysis. to maintain integrity and avoid freezing, fresh quinoa leaf tissue was crushed using a mortar and pestle in liquid nitrogen. after transferring the powdered tissue to a tube, up to 100 mg of wet tissue tissue were mixed with 400 µl of buffer ap1 and 4 µl of rnase a (100 mg/ml). the mixture was vortexed thoroughly, it was incubated at 65oc for 10 minutes to lyse the cells, and then cooled on ice for 5 minutes with the addition of 130 µl buffer ap2 to precipitate contaminants. the lysate was transferred to a spin column of a qia shredder and centrifuged at high speed to get rid of cell debris [9]. the flow-through was collected and combined with 1.5 ml of buffer ap3/e. after centrifuging the resultant solution and discarding the flow-through, 650 µl pieces of it were put to a dneasy mini spin column. washing steps were performed using 500 µl of buffer aw, it was centrifuged once for one minute and then again for two minutes to dry it. lastly, 100 µl of preheated (65oc) buffer ae was applied to the dneasy membrane, incubating for 5 minutes at room temperature, and centrifuging. the elution buffer was 50 µl for higher concentration dna [10; 11; 12]. agarose gel electrophoresis was used to estimate the isolated dna concentration. a small portion of the dna sample was loaded onto a 1% agarose gel alongside a dna ladder with known fragment sizes. the concentration of the extracted dna can be estimated by comparing the intensity of its bands to those of the dna ladder [13]. issr-pcr reactions a total of 8 issr primers (table 1) were used to detect polymorphisms. the amplification reaction was conducted in a 25 µl reaction volume, consisting of 1x pcr buffer, 1.5 mm mgcl2, 0.2 mm dntps, 1 µm primer, 1 u taq dna polymerase, and 30 ng template dna [14]. a perkin-elmer/geneamp® pcr system 9700 (pe applied biosystems) was used for the pcr amplification. it was set up to complete 35 cycles following a 5-minute initial denaturation cycle. the steps in each cycle were 94oc for 1 minute of denaturation, 50oc for 1 minute of annealing, and 72oc for 1.5 minutes of elongation. in the final cycle, the primer extension was extended to 7 minutes at 72oc [15; 16]. electrophoresis was used to resolve the amplification products in a 1.5% agarose gel with 0.5 µg/ww ml of ethidium bromide in 1x tbe buffer at 95 volts. the criterion for molecular size was a 100 bp dna ladder. the pcr products were visualized under uv light and photographed using a gel documentation system (bio-rad 2000) [17]. table 1. issr primer names and sequence. no. name primer sequence 5’-3’ 1 issr1 5’-agagagagagagagagyc-3’ 2 issr5 5’-gtgtgtgtgtgtgtgtyg-3’ 3 issr8 5’-agacagacagacagacgc-3’ 4 issr10 5’-gacagacagacagacaat-3’ 5 issr13 5’-agagagagagagagagyt-3’ 6 issr14 5’-ctcctcctcctcctctt-3’ 7 issr15 5’-ctctctctctctctctrg-3’ 8 issr16 5’-tctctctctctctctca-3’ data analysis the genetic relatedness of the samples under investigation was ascertained by comparing the banding patterns produced by issr-pcr marker tests. clear and distinct amplification products were scored as 1 for the presence and 0 for the absence of bands. bands with the same mobility were considered identical. the genetic similarity coefficient (gs) between two genotypes was calculated using the dice coefficient [18; 19]. gs i j = 2a 2a + b + c the genetic similarity coefficient gs i j represents the genetic similarity between individuals i and j, a is the number of bands shared by both i and j, b is the number of bands present in i and absent in j, and c is the number of bands present in j and absent in i. cluster analysis was performed on the similarity matrix using the unweighted pair group method with arithmetic mean (upgma) [20]. results and discussions polymorphism analysis identified using issr markers in this study, eight issr primers were used for the analysis of nine quinoa accessions, producing amplification products that resulted in polymorphic fingerprint patterns as shown in figure 1 and table 2. the eight pcr primers generated a total of 102 bands, with an average of 12.75 bands per primer. of these 102 amplified fragments, 52 were polymorphic, yielding an average of 6.5 polymorphic bands per primer. this corresponds to a polymorphism rate of 40.9% across the eight primers. primer issr-8 produced the highest number of polymorphic bands, with 17 polymorphic amplification products detected. in contrast, primer issr-15 yielded the fewest polymorphic fragments, with only 2 detected. the polymorphism percentage ranged from 20% highlights in bioscience page 2 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions figure 1. issr profiles of the using primer ((a) issr-8, (b) issr-13, (c) issr-14 and (d) issr16). 1 to 9 samples accession, m: dna molecular weight marker (100bp dna ladder). (for primer issr-15) to 88% (for primer issr-10). primer issr15 exhibited the highest frequency (0.9), while primers issr-8 and issr-10 showed the lowest frequency (0.5). table 2. eight issr used in this study, the total bands (tb), monmorphic bands (mb), polymorphic bands (pb), percentage of polymorphic bands (%pb) and frequency (f). primer tb mb pb %pb f issr1 9 6 3 33 0.8 issr5 15 9 6 40 0.8 issr8 22 5 17 77 0.5 issr10 8 1 7 88 0.5 issr13 11 5 6 55 0.6 issr14 11 6 5 45 0.7 issr15 10 8 2 20 0.9 issr16 16 10 6 38 0.8 total 102 50 52 average 12.75 6.25 5.5 40.9 0.7 the results of primer issr-1 were illustrated in (table s1). it gave 6 monomorphic and 3 polymorphic bands with different fragment sizes ranging from 160 to 958 bp for chenopodium quinoa. six common bands were observed across all sites at fragment sizes of 160, 196, 278, 323, 498, and 747 bp. a band with a fragment size of 248 bp was exclusively present in giza 1 with irrigation every 3 days (i3), giza 1 with irrigation every 6 days (i6), and misr 1 across all treatments, but was absent in giza 1 with irrigation every 9 days (i9) and danish kvl3704 in all treatments. a fragment size of 426 bp was exclusively present in giza 1 (i3 and i6), danish kvl3704 (i3), and misr 1 under all treatments, but it was absent in giza 1 under i9 and in danish kvl3704 under i6 and i9. a fragment size of 958 bp was exclusively present in danish kvl3704 with i3 and misr 1 under all treatments, but it was absent in giza 1 under all treatments and in danish kvl3704 under i6 and i9. the results of primer issr-5 were illustrated in (table s2). it gave 9 monomorphic and 6 polymorphic bands with different fragment sizes ranging from 132 to 989 bp for chenopodium quinoa. nine common bands were observed across all sites at fragment sizes of 132, 158, 170, 263, 312, 372, 430, 538, and 637 bp. a band with a fragment size of 190 bp was exclusively present in giza 1 across all treatments, but was not found in danish kvl3704 or misr 1 across all treatments. a fragment size of 214 bp was exclusively present in misr 1 across all treatments, but was not found in giza 1 or danish kvl3704 across all treatments. a fragment size of 235 bp was exclusively present in giza 1 and misr 1 across all treatments, but was not found in danish kvl3704 across all treatments. in contrast, a fragment size of 703 bp was exclusively present in giza 1 and danish kvl3704 across all treatments, but was absent in misr 1 across all treatments. a fragment size of 772 bp was exclusively present in misr 1 across all treatments, but was not found in giza 1 or danish kvl3704 across all treatments. a fragment size of 989 bp was exclusively present in giza 1 (i3), danish kvl3704 (i6), and misr 1 across all treatments, but was absent in giza 1 (i6 and i9) as well as danish kvl3704 (i3 and i9). the results of primer issr-8 were illustrated in (table s3). it gave 5 monomorphic and 17 polymorphic bands with different fragment sizes ranging from 180 to 1250 bp for chenopodium quinoa. five common bands were observed in all sites at fragment sizes of 350, 460, 500, 830, and 1100 bp. a band with a fragment size of 180 bp was exclusively present in giza 1 in (i3), but it was not found in giza 1 with (i6 and i9), misr 1 under all treatments, and danish kvl3704 under all treatments. a fragment size of 200 bp was exclusively present in giza 1 under all treatments, but it was not found in misr 1 under all treatments and danish kvl3704 under all treatments. respectively, a fragment size of 210 bp was exclusively present in danish kvl3704 with (i9), misr 1 in (i3), as well as misr 1 with (i6), but it was not found in giza 1 under all treatments, danish kvl3704 in (i3 and i6), and misr 1 with (i9). a fragment size of 240 bp was exclusively present in danish kvl3704 under all treatments and misr 1 with (i6 and i9), but it was not found in giza 1 under all treatments and misr 1 under (i3). a fragment size of 260 bp was exclusively present in giza 1 under (i3 and i6), danish kvl3704 in (i3 and i9), as well as misr 1 with (i3 and i9), but it was not found in giza 1 under (i9), danish kvl3704 with (i6), and misr 1 under (i6). respectively, a fragment size of 290 bp was exclusively present in giza 1 in (i3 and i6), danish kvl3704 with (i6), and misr 1 under (i3), but it was not found in giza 1 highlights in bioscience page 3 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions with (i9), danish kvl3704 under (i3 and i9), as well as misr 1 with (i6 and i9). a fragment size of 300 bp was exclusively present in giza 1 under (i9), danish kvl3704 with (i3 and i9), as well as misr 1 under (i6 and i9), but it was not found in giza 1 with (i3 and i6), danish kvl3704 under (i6), and misr 1 with (i3). a fragment size of 320 bp was exclusively present in giza 1 with all treatments and danish kvl3704 under (i6), but it was not found in danish kvl3704 under (i3 and i9) and misr 1 with all treatments. respectively, a fragment size of 390 bp was exclusively present in giza 1 under all treatments and danish kvl3704 in (i3), but it was not found in danish kvl3704 with (i6 and i9) as well as misr 1 under all treatments. a fragment size of 410 bp was exclusively present in danish kvl3704 under (i6 and i9), but it was not found in giza 1 with all treatments, danish kvl3704 in (i3), and misr 1 under all treatments. a fragment size of 610 bp was exclusively present in giza 1 under (i6 and i9) as well as misr 1 in (i6), but it was not found in giza 1 under (i3), danish kvl3704 under all treatments, and misr 1 with (i3 and i9). a fragment size of 650 bp was exclusively present in giza 1 under (i3 and i9) as well as misr 1 with (i3), but it was not found in giza 1 under (i6), danish kvl3704 in all treatments, as well as misr 1 with (i6 and i9). a fragment size of 730 bp was exclusively present in giza 1 with all treatments, danish kvl3704 under (i9), and misr 1 under (i3 and i6), but it was not found in danish kvl3704 with (i3 and i6) as well as misr 1 with (i9). respectively, a fragment size of 900 bp was exclusively present in giza 1 with (i6), but it was not found in giza 1 with (i3 and i9), danish kvl3704 under all treatments, and misr 1 with all treatments. a fragment size of 1000 bp was exclusively present in giza 1 with (i6), but it was not found in giza 1 under (i3 and i9), danish kvl3704 with all treatments, and misr 1 under all treatments. a fragment size of 1150 bp was exclusively present in giza 1 under all treatments, but it was not found in danish kvl3704 with all treatments and misr 1 in all treatments. a fragment size of 1250 bp was exclusively present in giza 1 with (i9), but it was not found in giza 1 with (i3 and i6), danish kvl3704 in all treatments, and misr 1 under all treatments. the results of primer issr-10 were illustrated in (table s3). it gave 1 monomorphic and 7 polymorphic bands with different fragment sizes ranging from 265 to 840 bp for chenopodium quinoa. one common band was observed in all sites at a fragment size of 316 bp. a band with a fragment size of 265 bp was exclusively present in giza 1 under (i6 and i9), danish kvl3704 in all treatments, and misr 1 with all treatments, but it was not found in giza 1 with (i3). a fragment size of 378 bp was exclusively present in giza 1 under (i3 and i9), danish kvl3704 with all treatments, as well as misr 1 under (i3 and i9), but it was not found in giza 1 with (i6) and misr 1 under (i6). a fragment size of 426 bp was exclusively present in danish kvl3704 under all treatments and misr 1 in (i6), but it was not found in giza 1 under all treatments as well as misr 1 with (i3 and i9). a fragment size of 462 bp was exclusively present in giza 1 with (i9) and misr 1 under (i6), but it was not found in giza 1 under (i3 and i6), danish kvl3704 with all treatments, as well as misr 1 under (i3 and i9). a fragment size of 570 bp was exclusively present in giza 1 under (i9), danish kvl3704 with (i6 and i9), as well as misr 1 in (i9), but it was not found in giza 1 under (i3 and i6), danish kvl3704 in (i3), and misr 1 with (i3 and i6). a fragment size of 699 bp was exclusively present in danish kvl3704 under (i6 and i9) and misr 1 with (i9), but it was not found in giza 1 under all treatments, danish kvl3704 with (i3), and misr 1 under (i3 and i6). a fragment size of 840 bp was exclusively present in giza 1 with (i9), but it was not found in giza 1 under (i3 and i6), danish kvl3704 with all treatments, and misr 1 under all treatments. the results of primer issr-13 were illustrated in (table s4). it gave 5 monomorphic and 6 polymorphic bands with different fragment sizes ranging from 177 to 1074 bp for chenopodium quinoa. five common bands were observed in all sites at fragment sizes of 177, 264, 345, 397, and 472 bp. a band with a fragment size of 214 bp was exclusively present in giza 1 under (i6) as well as misr 1 with (i9), but it was not found in giza 1 under (i3 and i9), danish kvl3704 with all treatments, as well as misr 1 under (i3 and i6). a fragment size of 217 bp was exclusively present in giza 1 with (i3), danish kvl3704 under (i3 and i9), and misr 1 with (i6), but it was not found in giza 1 under (i6 and i9), danish kvl3704 with (i6), and misr 1 in (i3 and i9). a fragment size of 510 bp was exclusively present in giza 1 under (i3) and danish kvl3704 with (i3 and i6), but it was not found in giza 1 with (i6 and i9), danish kvl3704 in (i9), and misr 1 under all treatments. respectively, a fragment size of 550 bp was exclusively present in giza 1 with (i9 and i6), danish kvl3704 in (i9), and misr 1 under all treatments, but it was not found in giza 1 under (i3) as well as danish kvl3704 with (i3 and i6). a fragment size of 666 bp was exclusively present in giza 1 with (i3 and i6) as well as danish kvl3704 under (i9), but it was not found in giza 1 under (i9), danish kvl3704 with (i3 and i6), and misr 1 under all treatments. a fragment size of 1074 bp was exclusively present in danish kvl3704 with (i9), but it was not found in giza 1 under all treatments, danish kvl3704 with (i3 and i6), and misr 1 under all treatments. the results of primer issr-14 were illustrated in (table s5). it gave 6 monomorphic and 5 polymorphic bands with different fragment sizes ranging from 720 to 2000 bp for chenopodium quinoa. six common bands were observed in all sites at fragment sizes of 720, 790, 860, 950, 1200, and 1500 bp. a band with a fragment size of 1050 bp was exclusively present in giza 1 with (i3) and misr 1 under (i9), but it was not found in giza 1 with (i6 and i9), danish kvl3704 under all treatments, as well as misr 1 under (i3 and i6). a fragment size of 1100 bp was exclusively present in giza 1 under (i9) and danish kvl3704 with (i9), but it was not found in giza 1 with (i3 and i6), misr 1 under all treatments, and danish kvl3704 in (i3 and i6). a fragment size of 1350 bp was exclusively present in giza 1 under (i9) and misr 1 with (i9), but it was not found in giza 1 under (i3 highlights in bioscience page 4 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions figure 2. dendogram of similarity index (si) between the nine studied treatments. and i6), danish kvl3704 in all treatments, as well as misr 1 with (i3 and i6). a fragment size of 1650 bp was exclusively present in giza 1 with (i3) and misr 1 under (i9), but it was not found in giza 1 under (i6 and i9), danish kvl3704 with all treatments, and misr 1 under (i3 and i6). a fragment size of 2000 bp was exclusively present in giza 1 with (i3), danish kvl3704 under (i9), and misr 1 in (i9), but it was not found in giza 1 under (i6 and i9), danish kvl3704 in (i3 and i6), and misr 1 with (i3 and i6). the results of primer issr-15 were illustrated in (table s6). it gave 8 monomorphic and 2 polymorphic bands with different fragment sizes ranging from 310 to 1050 bp for chenopodium quinoa. eight common bands were observed in all sites at fragment sizes of 310, 490, 520, 630, 780, 850, 960, and 1050 bp. a band with a fragment size of 420 bp was exclusively present in giza 1 with (i6), danish kvl3704 under all treatments, and misr 1 with all treatments, but it was not found in giza 1 under (i3 and i9). a fragment size of 720 bp was exclusively present in giza 1 with (i3), but it was not found in giza 1 with (i6 and i9), danish kvl3704 under all treatments, and misr 1 with all treatments. the results of primer issr-16 were illustrated in (table s7). it gave 10 monomorphic and 6 polymorphic bands with different fragment sizes ranging from 136 to 944 bp for chenopodium quinoa. ten common bands were observed in all sites at fragment sizes of 136, 342, 144, 465, 390, 546, 702, 741, 814, and 862 bp. a band with a fragment size of 324 bp was exclusively present in danish kvl3704 with (i9) and misr 1 under all treatments, but it was not found in giza 1 with all treatments, danish kvl3704 under (i3 and i6), as well as misr 1 in all treatments. a fragment size of 275 bp was exclusively present in giza 1 with all treatments and danish kvl3704 under all treatments, but it was not found in misr 1 under all treatments. a fragment size of 502 bp was exclusively present in misr 1 with all treatments, but it was not found in misr 1 under all treatments, giza 1 in all treatments, and danish kvl3704 with all treatments. a fragment size of 591 bp was exclusively present in giza 1 under all treatments and misr 1 with all treatments, but it was not found in danish kvl3704 with all treatments. a fragment size of 908 bp was exclusively present in danish kvl3704 under all treatments and misr 1 with all treatments, but it was not found in giza 1 under all treatments. a fragment size of 944 bp was exclusively present in giza 1 under all treatments, but it was not found in danish kvl3704 under all treatments and misr 1 with all treatments. table 3. similarity index (si) of nine treatments based on issr pcr analysis. 1 2 3 4 5 6 7 8 9 1 100 2 89 100 3 85 88 100 4 82 83 81 100 5 81 81 81 91 100 6 78 80 81 89 89 100 7 83 85 81 85 82 83 100 8 78 82 81 87 81 84 93 100 9 80 81 79 85 82 84 91 90 100 the similarity index (si) based on issr–pcr is shown in table 3 as a cluster analysis in percentage. among all studied treatments, the similarity index recorded the highest value between treatments (7 and 8), while the lowest value was recorded (78%) between treatments (6 and 1). the dendrogram in (figure 2) was based on the results of the similarity index. it was stratified into eight clusters indicating the relationship between the nine studied treatments, arranged in four levels of similarity from (80%) as the lowest level to (93%) as the highest recorded level. in this respect, [21] have used seven issr markers to characterize and genetically compare five quinoa genotypes. the results revealed 85 loci, of which 37 were polymorphic, accounting for 43.53% of the total. the number of amplified loci varied from four (ubc-817) to 26 (ubc-845). the size of the amplified fragments ranged from 139 to 1,111 bp. additionally, the overall pic values ranged from 0.00 to 0.4. likewise, [22] have used 10 issr primers to genetically differentiate among five quinoa genotypes. the results yielded 53 amplicons, 33 of which were polymorphic, resulting in an average polymorphism percentage of 61.83%. with an average of 5.3 pieces per primer, the range of amplicons per primer was three to ten. there were between one and six polymorphic amplicons, with an average of 3.3 pieces per primer. finally, the size of the amplified fragments varied depending on the issr primer employed, ranging from 130 to 1,456 bp. fifteen issr primers were employed in this study, producing a total of 172 amplified bands, with an average of 11.7 bands per primer. a total number of 90 amplified polymorphic highlights in bioscience page 5 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions bands were obtained, with an average of 6.0 bands per primer; the average level of polymorphism was 49.7%. the effectiveness of issr markers in distinguishing the studied genotypes was assessed by calculating the pic values, which ranged from 0.21 to 0.84, with an average value of 0.69 [23]. conclusions the obtained deference’s of the dendrogram indicated that issr–pcr was useful in detecting the similarity between the studied treatments. also, it could be recommended as a suitable candidate method between the studied treatment in further breeding issr programs to evaluate some quinoa genotypes to different irrigation intervals on stress conditions. supplementary table s1: survey of olymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-1. table s2: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-5. table s3: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-8. table s4: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-10. table s5: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-13. table s6: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-14. table s7: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-15. table s8: survey of polymorphic and monomorphic issr bands in chenopodium quinoa using primer issr-16. reference 1. wali am, kenawey m, ibrahim o, el lateef ea. productivity of quinoa (chenopodium quinoa l.) under new reclaimed soil conditions at the northwestern coast of egypt. bulletin of the national research centre. 2022;46(1):38. 2. zohry aeh. prospects of quinoa cultivation in marginal lands of egypt. moroccan journal of agricultural sciences. 2020;1(3). 3. mahmoud ah. production of quinoa (chenopodium quinoa) in the marginal environments of south mediterranean region: nile delta, egypt. egyptian journal of soil science. 2017;57(3):329-37. 4. saad-allah km, youssef ms. phytochemical and genetic characterization of five quinoa (chenopodium quinoa willd.) genotypes introduced to egypt. physiology and molecular biology of plants. 2018;24(4):617-29. 5. kumar ld, kathirvel m, rao g, nagaraju j. dna profiling of disputed chilli samples (capsicum annum) using issr-pcr and fissr-pcr marker assays. forensic science international. 2001;116(1):63-8. 6. adams rp, schwarzbach ae, pandey rn. the concordance of terpenoid, issr and rapd markers, and its sequence data sets among genotypes: an example from juniperus. biochemical systematics and ecology. 2003;31(4):375-87. 7. belmonte c, vasconcelos es, tsutsumi cy, lorenzetti e, hendges c, coppo jc, et al. agronomic and productivity performance for quinoa genotypes in an agroecological and conventional production system. american journal of plant sciences. 2018;9:880-91. 8. dellaporta sl, wood j, hicks jp. a plant dna min preparation, version iii. plant molecular biology report. 1983;1:1921. 9. joshi sp, gupta vs, aggarwal rk, ranjekar pk, brar ds. genetic diversity and phylogenetic relationship as revealed by inter-simple sequence repeat (issr) polymorphism in the genus oryza. theoretical and applied genetics. 2000;100(8):1311-20. 10. iqbal h, yaning c, waqas m, shareef m, raza st. differential response of quinoa genotypes to drought and foliageapplied h2o2 in relation to oxidative damage, osmotic adjustment and antioxidant capacity. ecotoxicology and environmental safety. 2018;164:344-54. 11. castillo cd, winkel t, mahy g, bizoux jp. genetic structure of quinoa (chenopodium quinoa willd.) from the bolivian altiplano as revealed by rapd markers. genetic resources and crop evolution. 2007;54(4):897-905. 12. ebrahim mea. evaluation of some (chenopodium quinoa) cultivars as a new cash crop halophyte in egypt. ain shams university; 2018. thesis, pp. 4–5. 13. ebrahim mea, hussin sa, abdel-ati aa, ali sh, eisa ss. evaluation of some (chenopodium quinoa) cultivars under saline soil conditions in egypt. arab university journal of agricultural sciences, ain shams university, cairo. 2018;26(1):337-47. 14. gemmill ce, grierson er. inter-simple sequence repeats (issr), microsatellite-primed genomic profiling using universal primers. molecular plant taxonomy: methods and protocols. 2021:249-62. 15. tan m, temel s. performance of some quinoa (chenopodium quinoa willd.) genotypes grown in different climate conditions. turkish journal of field crops. 2018;23(2):180-6. 16. telahigue dc, aljane f, yahia lb, belhouchett l, toumi l. grain yield, biomass productivity and water use efficiency in quinoa (chenopodium quinoa willd.) under drought stress. journal of scientific agriculture. 2017;1:222-32. highlights in bioscience page 6 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ mubarak mh et al., 2024 quinoa molecular differences using issr markers under sinai conditions 17. mahood a, hama-salih f. characterization of genetic diversity and relationship in almond (prunus dulcis [mill.] da webb.) genotypes by rapd and issr markers in sulaimani governorate. applied ecology & environmental research. 2020;18(1). 18. belle v, papantonis i. principles and practice of explainable machine learning. frontiers in big data. 2021;4:688969. 19. bourrat p. genetic relatedness. in: encyclopedia of evolutionary psychological science. springer; 2021. p. 3401-4. 20. segura-alabart n, serratosa f, gómez s, fernández a. nonunique upgma clusterings of microsatellite markers. briefings in bioinformatics. 2022;23(5):bbac312. 21. saad-allah km, youssef ms. phytochemical and genetic characterization of five quinoa (chenopodium quinoa willd.) genotypes introduced to egypt. physiology and molecular biology of plants. 2018:1-13. 22. al-naggar amm, abd el-salam rm, badran aee, elmoghazi mma. molecular differentiation of five quinoa (chenopodium quinoa willd.) genotypes using inter-simple sequence repeat (issr) markers. biotechnology journal international. 2017;20(1):1-12. 23. ibrahim sd, abd el-hakim af, ali he, abd el-maksoud rm. genetic differentiation using issr, scot and dna barcoding for quinoa genotypes. arab journal of biotechnology. 2019;22(2). highlights in bioscience page 7 of 7 december 2024|volume 7 http://bioscience.highlightsin.org/ abstract introduction materials and methods field experiment issr-pcr reactions data analysis results and discussions polymorphism analysis identified using issr markers conclusions supplementary highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20219 highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 10 research article open access 1department of nucleic acid and protein structure, agricultural genetic engineering research institute (ageri), arc, giza, egypt 2department of genetics, faculty of agriculture, cairo university, giza, egypt 3department of microbiology, faculty of science, ain shams university, 11566, abbassia, cairo, egypt contacts of authors * to whom correspondence should be addressed: ghada abu el-heba citation: hassan a., abdallah a., a-bouzeid m. and abu el-heba g. (2020). blkn, a novel medicago truncatula mutant achieving black nodule phenotype. highlights in bioscience volume 3. article id 20219. dio:10.36462/ h.biosci.20219 received: july 14, 2020 accepted: august 27, 2020 published: september 10, 2020 copyright: © 2020 hassan et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. blkn, a novel medicago truncatula mutant achieving black nodule phenotype asmaa hassan 1 , naglaa abdallah 2 , mohamed a-bouzeid 3 and ghada abu el-heba 1 * abstract blkn is a medicago truncatula mutant that is achieving null function-black nodule phenotype. blkn is a tnt1-retrotransposon mutant, tnt1 is nicotiana tabacum retro-transposon which is replicated via rna copy and integrated in plant genome. interestingly, blkn exhibited double contents of phenolic compounds comparing to r108 wild type. the mutated black nodule is displaying cells abnormality in both infection and nitrogen fixation zones. transverse section of blkn nodule doesn’t display clearly characteristic shape like the control and the symbiotic cells don't totally filled with bacteroids along with high lignification at the cell wall periphery. our goal was blkn mutant; phenotype, physiological, and molecular characterizations. aflp-based pcr method was used to detect the mutated gene(s) in this mutant line. about 25 tnt1-tagged fragments ranging from ~100 to ~500 bp were isolated, sequenced and submitted to genbank. the tnt1 insertion was precisely located next to the base number 303 post atg start codon of m. truncatula l-type lectin-domain receptor kinase vii.2 gene encodes lectin_legb receptor like kinase (mtlectinrlk). mtlectinrlk contains lectin_legb domain, two transmembrane helix (tmhilex) and an extracellular receptor protein kinase (pkinase). mtlectinrlk is an ancestry related to probable l-type lectin-domain containing receptor kinase cicer arietinum, trifolium pretense, phaseolus vulgaris, vigna radiate and glycine soja. keywords: blkn mutant, medicago truncatula, lectin-domain, receptor kinase, tnt1 retrotransposon, fsts, aflp. introduction legumes are considered as one of the main plant families for thousands of years. their seeds contain large amount of protein therefore they are a vital plant protein supplier in food pyramid for human nutrition, animal feed and as a raw matter for industry [1]. additionally, they are essential in agriculture as they can grow in limited nitrogen environment with no addition of organic or inorganic fertilizers. legumes family has the ability to convert atmospheric nitrogen gas to ammonia throughout symbiotic association with soil bacteria commonly called rhizobia. soil nitrogen fixing bacteria such as mesorhizobium, sinorhizobium, bradyrhizobium, rhizobium, and azorhizobium have many common features such as; the rode shape, gram negative and non sporulated bacteria. farming such biological nitrogen fixation family reduces expansive usage of fertilizers and hence decreasing environmental related pollution. the relation between legumes and rhizobia is beneficial symbiotic relationship. https://creativecommons.org/licenses/by/4.0/ hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 10 the plant provides micro-aerobic condition and energy supplier to bacteria, and then in return it obtains organic nitrogen compounds [2,3]. when nitrogen becomes limiting for growth, legume plant roots produce flavonoid compounds in the rhizosphere which are considered as signals to the compatible bacteria. rhizobia reply to these compounds by secreting a lipochitin oligosaccharide called nod factor that activates several early steps in root hair infection progression. then plant percepts this specific node factor inducing alterations in the gene expression. additionally, perception results in physical changes as plant root hair curling to quester the bacteria. bacteroid is the form of bacteria which is considered as the function form that responsible for biological nitrogen fixation [3-6]. total reprogramming in the gene expression appears in the two organisms during the nodule development and nitrogen fixation. unfortunately, most of cultivated crucial legumes are weak model system for genetic study because of their large genome, difficult transformation, being out crossing and having polyploidy. thus medicago truncatula is considered as legume model plant which has many features to study the symbiosis mechanism between plant and bacteria for nitrogen fixation as it has diploid genome, produce numerous seeds, short life cycle and self-fertile plant [6,7]. wide ranges of mutant collections have been developed from this plant by using many genetic tools such as t-dna insertions, transposon, ionizing radiation, fast neutron bombardment and chemical mutagens [3,8]. the most effective transposon used for insertion mutagenesis in medicago truncatula is tobacco retrotransposon tnt1 [9]. tnt1 is retro-transposon defined as group of moveable dna elements which can transfer through rna intermediate using copy-andpaste technique [5], tnt1 is one of longrepeated terminal ltr retro-transposon, both ends of this subclass has internal direct repeats. additionally its length is 5.3 kb, it creates 5bp duplication in different sides during its inclusion, and it produces stable changes which effectively can move onto descendants [9]. samuel roberts noble foundation in usa is the major source for the medicago tnt1-insertion mutant collections. numerous mutant phenotypes have been separated, such as mutant has no ability to induce nodule (nod−), mutants have less effective or inefficient nitrogen fixation nodule (nod+fix+/-), mutants with very high nodule number (nod++) [3]. this study aimed to characterize blkn mutant, the medicago truncatule tnt1 insertion mutagenesis mutant line. the mutant characterization includes; phenotype, physiological and molecular characterizations. materials and methods plant material: medicago truncatula nf0478 is tnt1 mutant line was provided from noble foundation [9-11]. this line was produced in vitro through agrobacterium tumefactions transformation [3]. in this study medicago truncatula r108 was used as a wild type control plant. bacterial strain: sinorhizobium meliloti strain (sm1021) [12] was used to inoculate seedlings of blkn as well as r108 wild type. seed germination and rhizobia inoculation: both blkn mutant and r108 seeds were scraped with sand paper and the surface was sterilized with ten% (v/v) sodium hypochlorite for ten minutes. after 5 times washing using sterilize water, seeds were cultivated on basal nitrogen medium (bnm). seven day old seedlings were inoculated with sinorhizobium meliloti culture with an optical density 0.1 at wave length 600 nm to examine the nodule phenotype according to galibert 2001 [13]. genetic cross: plants homozygous for blkn allele were crossed with r108 wild type plant. mutant alleles were allowed to segregate through f1 self-fertilization. genomic dna extraction was carried out for f2 individuals and subjected to pcr amplification for analyzing the segregation pattern of blkn alleles. nodule examination with light microscopy: two weeks old nodules were directly fixed in 50ml solution containing 25ml ethanol 95%, 5ml formaldehyde, 2.5ml glacial acetic acid and 17.5ml distilled water. the fixed nodules were embedded in paraffin wax. blocks were divided with rotary microtome (euromex usa). slides were stained with safranin red followed by light green dye. finally, slides were scanned under light microscope (axio vert.a1) [14]. estimation of nitrogenase activity: root nodules were washed with sterilized water and placed in bottles sealed with rubber. 10 ml of air was withdrawn from bottle and replaced by 10 ml of acetylene using plastic syringes. the bottles were incubated for one hour at 30 ◦ c thereafter, 2 ml gas samples were withdrawn and assayed for measuring ethylene concentration using liquid chromatography [15]. determination of plant pigments: one hundred mg of fresh green leaves from each line with 10 biological replicas were frozen and grounded with liquid nitrogen rapidly. chlorophyll pigment was obtained by 3 ml of 80% acetone including 1 mm potassium hydroxide then centrifuged at 10,000 rpm for 10 min. followed by supernatant quantification with a spectrophotometer device(bio spectrophotometer, ependorf) [16]. chlorophyll and total carotenoid amounts were calculated based on [17]. phenolic compounds quantification: total phenol contents were estimated by folin ciocalteu’s technique. one gram of each mutant and control leaves were extracted in 3 ml of ethanol 95% and kept at 4°c/24 hrs in the dark, and then the samples were centrifuged at 13000rpm/5min. one ml of supernatant was transferred to 5 ml of distilled water with 0.5ml of folin ciocalteu reagent (f-c) were mixed well, after that 1.5 ml of 20% of sodium carbonate hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 10 (na2co3) was added and the total volume was adjusted to 10 ml and was incubated for 2 hrs at room temp. finally, spectrophotometer (bio spectrophotometer, ependorf) was used to read the samples at 765nm [18,19].total contents of phenolic compounds were calculated from standard curve obtained with different concentration of salicylic acid (10 to 100 mg/ml) aflp type pcr for tnt1 border characterization in blkn mutant: genomic dna of blkn seedlings in addition to control plants were isolated separately using i-genomic plant dna extraction mini kit (intron biotechnology). dna pool of blkn isolated samples and dna of control sample were separately double digested by ecori mfei and asei ndei respectively according to enzyme producer protocol biolabs®, eco and ase adaptors were ligated to the digested dna using takara t4 dna ligase separately. amplification of tnt1 borders was carried out by nested pcr to amplify all tnt1 -flanking sequence tags (fsts) in blkn mutant line. all pcr reactions were prepared using takara la polymerase in biorad . cloning of amplified fragments: qiaquick pcr purification kit was used to purify the amplified tagged fragments before cloning into pgem-teasy vector (promega®). after cloning, colony pcr method was done using sp6 and t7 common oligonucleotide primers. later fragments with different sizes were purified and sequenced. this method was used as confirmation test to success transformation. data analysis: tnt1 fsts were analyzed against medicago truncatule genome using national center for biotechnology information data base ncbi (http://blast.ncbi.nlm.nih.gov and http://www.medicagohapmap.org/home/view). statistical analysis: data was exposed to analysis using spss20.0 (one-way anova) and the value of the least significant difference (lsd) was calculated at significance level of 5%. results phenotype characterization blkn seedling phenotype: for phenotype characterization, blkn mutant was tested against r108 for roots and shoots lengths, 10 biological replicas were used for each line. the lengths were measured seven days post germination on ½ ms medium, measurements were recorded every seven days’ time interval. blkn average root lengths were significally longer than r108 at (p=0.01). while the average shoot lengths in blkn was similar to that of r108 (p>0.05) (figure 1a and 1b). on the other hand, mature pods of blkn had different shape; it displayed compressed pod and shorter spines than r108 but the two lines showed anti-clock wise spines direction (figure 1c and 1d) and the same number of seeds which is about from 5 to 8. the average number of produced pods has no significant difference between blkn and r108 at (p=0.2) (figure 1e). figure 1. phenotypic characters of m.truncatula wild type and blkn. (1a) comparison between average root lengths of blkn and r108 is indicating a significance difference between the two types of plants at (p = 0.01). (1b) the mean value of blkn shoot length was close to mean value of r108 shoot length at (p>0.05). (1c) mature pods of r108 is showing long spine with anti-clock wise spines direction. (1d) mature compacted pods of blkn with short spines and anti-clock wise direction. (1e) the average number of the produced pods in blkn and r108 with no significant difference at (p=0.2). http://blast.ncbi.nlm.nih.gov/ http://www.medicagohapmap.org/home/view hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 10 blkn nodule phenotype: blkn nodules were examined three weeks post inoculation with sinorhizobium meliloti strain (sm1021) on bnm. blkn nodules showed null functionblack color phenotype with longer size than r108 nodule (figure 2a). according to statically analysis, nodules number of blkn mutant is significally more than r108 at (p=0.03) as shown in (figure 2b). figure 2. (2a) one month-old blkn seedling showing black color nodule in a cluster arrangement. (2b) the average nodules number of blkn mutant was significally more than r108 wild type at p=0.03. transfer sections of blkn nodule were compared to those of r108 through light microscopy examination. nodules of r108 prompted by rhizobia displayed the characteristic layers and the cells in each layer have their definite structure and function (1, meristem zone; 2, bacterial infection zone; 3, nitrogen-fixing zone; 4, senescent zone) (figure 3a). figure 3. nodule examination by light microscope. (3a) transfer section of wild type nodule, (3b) transfer section of blkn nodule. (1) bacterial infection zone; (2) nitrogen-fixing zone; (3) senescent zone. blkn transfer section is completely different from this of r108 in both number and size of cells in bacterial infection zone and nitrogen fixation zone. while the phenotype of blkn nodule that was induced with the same symbiotic bacteria strain showed an unusual nodule progressive process. the zones in blkn nodule didn’t have clearly characteristic shape like the control. cells of infection zone were longer in size than those in the same layer of control nodule. additionally, there was abnormal number of cells in nitrogen fixation zone although almost of these cells were free from bacteroid and had accumulated phenolic compounds. physiological characterization determination of blkn pigmentation: plant pigment is any kind of colored material produced by the plant. plant pigments can absorb visible radiation between violet (380 nm) and ruby-red (760 nm). plant pigments responsible for the color to the leaves, the flowers, and the fruits and are essential in regulating photosynthesis and development. plant has many different pigments found in several organic compounds. chlorophyll, the green pigment that can absorb light energy as much as possible and carotenoids, the pigments that can absorb blue and indigo light are the two most important pigmentation in the plant. the two pigments were estimated in blkn and r108 as well. both type of chlorophyll (chlorophyll a and chlorophyll b) don’t show any significant difference between their content in blkn and r108 (p=0.7) for chlorophyll a and p=0.06 for chlorophyll b). while blkn produces more than double amounts of carotenoids than r108 with a significant differenc (p=0.02) as shown in (figure 4a). figure 4. (a) measurement of pigment contents in r108 and blkn showing no significant difference in both types of chlorophylls (a and b) between r108 and blkn at (p=0.7) for chlorophyll a and at (p=0.06) for chlorophyll . while blkn produces more than douple amounts of carotenoids than r108 with a significanc differenc at (p=0.02). (b) blkn roots contain more than double content of phenolic compounds than r108 (p=0.00). hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 10 phenolic compounds quantification in blkn: plant phenolics are secondary native metabolites which perform a very wide range of physiological characters in plants. higher plants produce several thousand of diverse phenolic compounds. plant phenolics are fundamental defense compounds during different biotic and abiotic environmental stresses like; high and low temperatures, high light, ultraviolet radiation, different oxidants, nutrient deficiency, herbivores and pathogen infection. due to the black nodule colour and the dark colour of blkn root, total phenols were estimated in root of blkn and r108 one day pre rhizobium inoculation and 72 hrs post rhizobium inoculation. the total phenolic compound estimation was performed colorimetrically using spectrophotometer. there was no significant differences between blkn and r108 pre rhizobia inoculation while blkn seedlings have the ability to accumulate large amount of phenolic compounds than r108 and the difference was significantly at p=0.00 as shown in (figure 4b). molecular characterization tnt1-tagged loci identification and characterization in blkn mutant: to characterize tnt1-flanking sequence tags in blkn mutant, oligonucleotide primers (ltr3, ltr4, ltr5, ltr6) for tnt1 transposon against asei, aseii and ecoi, ecoii for ase and eco adaptor respectively [20]were used. all tnt1 tagged fragments were separated and visualized on 1.5% agarose running at 80 v, data presented in (figure 5a, b, c, and d). tnt1-tagged fragments with different sizes were exposed to cloning in pgem-teasy and sequencing process. about 25 fsts from blkn mutant line were isolated and subjected to data analysis against genbank http://www.ncbi.nlm.nih.gov/ database. some of them were blasted with coding regions of medicago genome, others were blasted with non-coding regions and the rest were unknown sequences. fsts corresponding to blkn mutant were submitted to genbank http://www.ncbi.nlm.nih.gov/. genbank accession numbers, e-value and reference genes are shown in (table 1). figure 5. blkn-tnt1 border amplification. (5a) mfei and ecori -double digestion the pcrii result using ltr4and ecoii oligonucleotide primers. (5b) ecori and mfei-double digestion pcrii result using ltr6 and ecoii oligonucleotide primers. (5c) ndei and ase i -double digestion test pcrii result using ltr4 and aseii oligonucleotide primers. (5d) ndei and asei double digestion pcrii test using ltr6 and aseii oligonucleotide primers. lanes from 1-16 are, ag ,at, aa, ac, cg, ct, ca, cc, gt, gg, gc, ga, tt, tg, tc, and ta separately at the end of oligonucleotide primers. aseii and ecoii. m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m a b m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 m a b c d hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 10 table1: blkn tnt1-tagged sequences. sequence name length accession number organism reference e-value blkn2 560 mn529997 medicago truncatula mwmb01000023.1 0 blkn3 291 mn529998 medicago truncatula mwmb01000001.1 8.00e-152 blkn4 289 mn529999 medicago truncatula mwmb01000001.1 1.00e-125 blkn5 288 mn530000 medicago truncatula mwmb01000001.1 4.00e-150 blkn6 291 mn530001 medicago truncatula mwmb01000001.1 8.00e-152 blkn8 284 mn530002 medicago truncatula mwmb01000001.1 6.00e-148 blkn9 171 mn530003 medicago truncatula xm_024776685.1 3.00e-79 probable l-type lectindomain containing receptor kinase vii.2 blkn10 250 mn530004 medicago truncatula mwmb01000001.1 4.00e-129 blkn11 71 mn530005 medicago truncatula mwmb01000031.1 1.00e-23 blkn13 250 mn530006 medicago truncatula cu179894.1 3.00e-106 blkn15 251 mn530007 medicago truncatula mwmb01000011.1 1.00e-129 blkn16 292 mn530008 medicago truncatula ac130200.30 2.00e-152 blkn17 252 mn530009 medicago truncatula mwmb01000011.1 3.00e-130 blkn18 294 mn530010 medicago truncatula ac130200.30 6.00e-103 blkn19 156 mn530011 medicago truncatula mwmb01000006.1 5.00e-77 blkn20 255 mn530012 medicago truncatula mwmb01000011.1 7.00e-132 medicago truncatula l-type lectin-domain receptor kinase locus is interrupted by tnt1 insertion in all f2 black nodule individuals from our data analysis, three medicago truncatula tnt1-insertion loci in blkn were subjected to additional investigation; blkn_2 (tnt1 insertion located in f-box/lrrrepeat protein), blkn_4 (tnt1 insertion located in unknown protein) and blkn_9 (tnt1 insertion located in l-type lectindomain receptor kinase). two oligonucleotide primers blkn2–r (5-gcttggaagagtctaaggttaa-3) and blkn4–r (5-ccaagttgacttgtttcatc-3) were used separately with ltr4 to test blkn_2 and blkn_4 insertion loci respectively. pcr was performed on genomic dna extracted from f2 population entities resulting from blkn mutant and r108 wild type back cross. only entities displaying black nodule phenotype with the other mutation phenotype characters were tested (about 1/4 population of f2 progeny). pcr result indicated that tnt1 insertion wasn’t confirmed in all of the entities means that the insertion on those loci aren't charged for this mutation phenotype. oligonucleotide primer pair blkn9-f (5-ccctcaaaaaat accaacaaaacc-3) and blkn9-r (5-caacttttccaa ttcatcacca-3) were designed to be used in compatible with the two ltrs oligonucleotides of tnt1 to test the incidence of tnt1 within the predicted mutated locus, l-type lectin-domain containing receptor kinase (blkn_9), in all blkn mutant individuals. pcr result confirmed the incidence of the two tnt1 side borders in all f2 entities displaying the mutation phenotype at the proper locus while showed a negative result in r108 entities. when using the two specific oligonucleotides for blkn_9 locus amplification, only r108 gave the proper band at 358bp (data not shown) while the amplification at this position failed in all blkn mutant individuals (1/4 population of f2 progeny). the rest of segregation individuals which are showing wild type phenotype (3/4 population of f2 progeny) gave a ratio of 60/30 positive to negative pcr result for tnt1 border amplification. this result is obeying mendel law, indicating that (blkn_9) insertion could be accountable for null function black-nodule, phenol accumulation and other mutation phenotype. the mutation at this locus was confirmed also at rna level, rt-pcr performed on all blkn mutant individuals using the two specific primers of the proper locus was giving negative result in all entities and positive amplification in r108 (data not shown). tnt1 insertion was precisely located next to the base number 303 post atg start codon of medicago truncatula l-type lectindomain receptor kinase gene which is 2079 bp in length with one exon encode lectin_legb receptor like kinase 692 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000023 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nucleotide/xm_024776685.1?report=genbank&log$=nuclalign&blast_rank=1&rid=svnduw9d015 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000001 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000031 https://www.ncbi.nlm.nih.gov/nucleotide/cu179894.1?report=genbank&log$=nuclalign&blast_rank=2&rid=s83xbg0y014 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 https://www.ncbi.nlm.nih.gov/nucleotide/ac130200.30?report=genbank&log$=nuclalign&blast_rank=1&rid=rex0cu3v014 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 https://www.ncbi.nlm.nih.gov/nucleotide/ac130200.30?report=genbank&log$=nuclalign&blast_rank=1&rid=reyeg4pb01r https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000006 https://www.ncbi.nlm.nih.gov/nuccore/mwmb01000011 hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 10 amino acids in length. mtlectinrlk consists of three domains; two transmembrane helix (tmhilex) the first is from 4bp to 26bp and the other from 288 to 310 predicted by tmhm (http://www.cbs.dtu.dk/services/tmhmm/). the second domain is lectin_legb domain at the position from 22bp to 285bp, and third one is the extracellular receptor protein kinase (pkinase) at position from 357bp to 641bp as shown in (figure 6). figure 6. mt-lectinrlk is 692 aa in length contains two transmembrane helixes (tmhelix) from 4 to 26 and from 288 to 310, lectin_legb domain at position from 22 to position 285, and extracellular receptor protein kinase (pkinase) at position from 357 to position 641. phylogenetic tree was constructed using embl-ebi (www.uniprot.org/align) as shown in (figure 7). mtlectinrlk is an ancestry related to probable l-type lectin-domain containing receptor kinase vii.2 cicer arietinum, putative l-type lectin-domain containing receptor kinase-like protein trifolium pretense, protein kinase domain-containing protein lupinus angustifolius, protein kinase domain-containing protein phaseolus vulgaris, probable l-type lectin-domain containing receptor kinase vii.2 isoform x1 vigna radiate, putative l-type lectindomain containing receptor kinase vii.2 glycine soja, putative l-type lectin-domain containing receptor kinase vii.2 mucuna pruriens, lectin-domain containing receptor kinase a4.2 cajanus cajan. figure 7. the predicted medicago truncatula mtlectinrlk is ancestry related to probable l-type lectin-domain containing receptor kinase vii.2. phylogenetic tree was constructed using embl-ebi (www.uniprot.org/align). discussion unique medicago truncatula mutant line nf0478 is a tnt1-retrotransposon mutant that is verified as black nodule phenotype mutant (blkn). we utilized reverse genetic tool to isolate and characterize the medicago truncatula “knockout” mutations in a gene that is supposed to be involving in nodulation process and other physiological processes. previously, about 2801 tnt1 flanking sequence tags were isolated from 156 medicago truncatula symbiotic mutant lines. the insertions were in nodulating genes like; nodule inception (nin), nodulation signaling pathway (nsp1, nsp2), doesn't make infections (dmi1, dmi2 and dmi3) and a number of super-nodulation genes sunn and skl [3]. blkn the black nodule mutant is showing defective in infection zone and nitrogen fixation zone alongside cell wall periphery lignification in symbiotic organ. characterization of blkn mutation depends mainly on phenotype monitoring and various biotechnology tools for investigation. linking between mutation phenotype with the changes occurred in the native physiological process due to this mutation along with the molecular characterization gave us clear evidence about the defected protein responsible for the emergence of this mutation. more than ~25 tnt1 insertion sites was isolated, sequenced and analyzed using genbank (http:// www.ncbi.nlm.nih.gov/) database during our study. some insertions were located within non-coding regions while the others were located in non-matching sequences. our results enable us to confirm that the medicago truncatula l-type lectin-domain receptor kinase locus is interrupted by the tnt1 insertion in all blkn mutant line individuals. tnt1 insertion locus was tested with pcr at both dna and rna levels and confirmed genetically through the segregation ratio of tnt1 borders in f2 population. our results indicating that the tnt1 insertion is precisely located next to the base number 303 post atg start codon of medicago truncatula l-type lectin-domain receptor kinase gene in blkn mutant line. this medicago truncatula gene encodes lec_legb-rlk which is 692 bp amino acids in length was scanned by http://pfam.xfam.org for functional domains. our data showed that medicago truncatula lec_legb-rlk has two transmembrane helix domains (tmhilex) the first is from 4bp to 26bp and the other from 288 to 310 predicted by tmhm (http://www. cbs.dtu.dk/services/tmhmm/). the second is lectin_legb domain (start from 22 to 285). each leguminous lectin subunit contains sole carbohydrate-binding site with beta pleated sheets consist of two conserved amino acids. the lectins are binding galactose, mannose or glucose and this interaction requires a firmly bound manganese and calcium ions. the third conserved domain is the protein kinase pk domain (start from 357 to 641) and it acts as switch on/off for majority of cellular activity in plant cell by phosphorylation and any defect in this domain can cause dysfunction abnormality. native biotic environment of plants root contains both symbionts and pathogens. the plants are responding to http://www.cbs.dtu.dk/services/tmhmm/ http://www.uniprot.org/align http://pfam.xfam.org/ hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 10 various microbes either by establishing a symbiotic interaction with friendly symbionts bacteria and mycorrhiza [21], or by inducing plant innate immunity to defend against pathogens attack [22]. medicago truncatula is an ultimate opportunity to study such crosstalk between symbiosis and defense in the environment. innate immunity in plants is triggered through recognition of microbe-associated molecular patterns (mamps) by plants pattern recognition receptors (prrs) [23,24]. this recognition process elicits defense program cascades enable the plant to respond to the pathogen attack in a precisely and effective manner. such recognition is switching on all the downstream protection including: production of reactive oxygen species (ros), accumulation of salicylic acid signal, inducing nonexpressor of pr gene (npr1) protein that convert into its monomeric form then translocate to the nucleus to activate pr–related genes, increasing ca2+ influx, and finally activation of mitogenactivated protein kinase (mapk) cascade [22]. this fast response is leading to the resistance against wide range of pathogens attack [25]. plant receptor-like kinases (rlks) include a superfamily of trans-spanning proteins involved in pathogen detection and defense signaling transduction [26] which is closely related to ros production [27]. lectin receptor kinases (lec-rks) is one class of the plants rlks that plays essential role through plant development and during the various stress responses. it contains an extracellular lectin domain and divided into three subclasses according to the diversity in this motif; gtype (s-locus), c-type (calcium dependent) and l-type (legume) [28]. g-type lect-rks are found in flowering plants and known as s-domain rlks they are involving in self-incompatibility [29] .c-type lec-rks are calciumdependent lectin that are found regularly in the mammalian proteins and involving in pathogen recognition and trigger the various immune responses [30]. arabidopsis has a single ctype lec-rk motif but its function is not been established so far [28], while arabidopsis contains about 45 l-type lec-rks [28,31]. lec-rks were proposed to play essential role in both abiotic stress tolerance [32-34] and biotic stress signals transduction [28]. this study is reporting the knock out mutation of medicago truncatula lec_legb-rk gene (blkn), this mutation is driving null function black color root nodule phenotype. the mutated nodule is displaying abnormality in both infection zone and nitrogen fixation zone. transverse section of the mutated nodule was displaying an empty symbiotic zone with higher lignification at the cell wall periphery compared with the wild type nodule phenotype. the released bacteriods in blkn infection zone were less than those in r108. in addition, the total phenolic compound accumulation was dramatically and significantly excessive in blkn root 72 hours post inoculation with sinorhizobium comparing to r108. this ultimate modification wasn’t observed pre sinorhizobium inoculation. the first published mutation in lectin-rk was reported [35] in arabidopsis lecrk-i.9. this mutation is exhibited an increasing susceptibility to pseudomonas syringae, phytophthora brassicae, and phytophthora capsici, while the over expression of this gene enhanced the resistance of the plant against these three pathogens [35]. although most of plant lectin receptor kinases function is not yet so clear, the role of these kinases in innate immunity is developing. some other lrk were reported to be involving in plant protection against various pathogen attacks. pi-d2 is g-lrk isolated from rice can provides defense against rice blast derived by magnaporthegrisea, the parasitic fungal pathogen [37]. nblrk1 is lecrk in nicotiana benthamiana was suggested to recognize the inf1 elicitor of phytophthora infestans and mediates plant defense [38]. while the expression of g-lrk is up-regulated in tobacco by lipopolysaccharides signal [39]. according to our data, lec_legb-rk switching off due to the tnt1 insertion in blkn mutant line is resulting in defective nodule, so what is the proposed function of medicago truncatula lec_legb-rk? our results in addition to the previous data enabled us to suggest that mtlec_legbrk is like a switch key. mtlec_legb-rk can recognize the symbionts microorganisms and directed the plant cells toward being involved in symbiotic interaction, in addition to recognize the invading microorganisms and trigger the signal defense pathway. thus, the mutation of medicago truncatula lec_legb-rk altered the plant cells unable to distinguish between the symbionts and invaders. we are suggesting that in case of blkn mutant, although the rhizobia-derived n-acetylglucosamine signal were normally perceived via lysm proteins [40] otherwise the mutant plant cells treated them as attackers instead of treating them as symbionts. consequently, the mutant cells produced greater amount of phenolic compound in roots and nodules compared to those of wild type r108. normally, such phenolic compounds are produced in plant cells due to several biotic and abiotic stress conditions such as: salt stress, drought, extreme temperature, nutrient deficiency, uv radiation, heavy metals and herbicides and other pathogen attacks. all these kind of plant stresses can induce unbalance between production of reactive oxygen species (ros) and its scavengers. plants (ros) are extremely reactive, toxic, and can cause severe damage to various cell components; dna, carbohydrates, proteins, and lipids leading to oxidative stress. plants have their own enzymatic and non-enzymatic mechanisms for ros detoxification. peroxidase (pox) is one of main enzymatic system, it oxidize phenolic compound by using them as an electron donor [41,42]. so, what is the fate of high contents of phenolic compound in blkn mutant plant? according to [43], phenolics are oxidized by pox at h2o2 expense hassan et al., 2020 blkn, a novel medicago truncatula mutant achieving black nodule phenotype highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 9 of 10 throughout lignin formation. thus, the strong lignin deposits around the cells wall periphery of both infection zone and nitrogen fixation zone confirmed our suggestion that the root cells in blkn mutant failed to distinguish between symbionts and attackers through producing a high amount of phenolic compound which is consider as ros scavenging compound. in turn these phenolic compounds were oxidized into lignin via pox within the nitrogen fixation organ. this suggestion may also explain the low frequent bacteroids in infection zone of blkn mutant than r108 infection zone. this result is obeying with the previous published data by roopashree [44]. they proposed that in native condition the legume lectin gene has two reverse functions depending on the perceived microorganisms and also the gene may be involved in the attachment of nitrogen-fixing bacteria to the legumes and in reverse function as a 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sciences unit, microbiology option, the oke ogun polytechnic, saki, nigeria. 2department of pure and applied biology, p.m.b 4000, ogbomosho, ladoke akintola university of technology, nigeria. 3department of zoology, parasitology unit, university of ibadan, ibadan, nigeria. 4genetics and molecular biology unit, department of botany, university of ibadan, nigeria. 5department of zoology, cellular parasitology unit, university of ibadan, ibadan, nigeria contacts of authors * to whom correspondence should be addressed: adeoti o. micheal citation: micheal ao, olayemi aa, adenike ak, olabiyi aa, olufemi os, olajumoke ae, ademola ad, joy oo. (2020). antibiotic effects of selected leaf extracts and molecular profile of klebsiella pneumoniae and enterobacter bugandensis strain (adm2). highlights in bioscience volume 3. article id 20205. dio:10.36462/h.biosci.20215 received: may 20, 2020 accepted: july 18, 2020 published: august 05, 2020 copyright: © 2020 micheal et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. antibiotic effects of selected leaf extracts and molecular profile of klebsiella pneumoniae and enterobacter bugandensis strain (adm2) adeoti o. micheal 1, 2,5 *, aboladale a. olayemi 1 , adeoye k. adenike 1, 3 , abiola a. olabiyi 1 , olufemi s. olufemi 1,4 , adedokun e. olajumoke 1 , adesina d .ademola 1 and olaoye o. joy 1 abstract only healthy surface tissues of healthy animals that are constantly in contact with the environment can easily be colonized by various microbial species, including pathogenic ones. however, these microorganisms are usually the cause of opportunistic infections in animals. two plants; vernonia amygdalina (taxonomic id number 112605) and ocimum gratissimum (taxonomic id number 112606) were identified at the nigerian federal research institute in ibadan and subsequently concentrated with methanol, water and n-hexane. twelve pre-slaughter healthy cattle in the same ratio of male to female were selected for this study. samples from the mouth, skin, nose, vaginal and gastrointestinal tract were collected. susceptibility patterns for antimicrobial agents were performed using disk-diffusion agar method. all isolates were subjected to bacteriological and molecular identification (pcr and sequencing techniques). preliminary identification was carried out based on standard bacteriological, microscopic identification, biochemical criteria, while pcr and dna sequencing techniques were used for molecular identification and analysis. basic local alignment sequence tools (blast) were used to compare retrieved bacterial sequences to a set of previously published strains in the database. the morphological and biochemical profiles of six out of eight normal flora from different sites were gram-negative. all bacterial cultures were oxidase-positive, including 7 catalase-positive cultures. the majority of isolated bacteria were moderately sensitive to all the extracts tested but resistant to amoxylin. noticeable amplification of 16s rrna genes from klebsiella pneumoniae strain emb and enterobacter bugandensis strain adm2 was obtained. the amplification of the resistant genes of the two isolates was approximately 1,500 base pairs; however, some of the suspected virulence and antibiotic genes were within this base pair range. keywords: vernonia amygdalina, ocimum gratissimum, anti-bacterial, 16s rrna, cattle introduction antibacterial are kinds of antimicrobial agent used mainly against bacteria, for the treatment of bacterial infections [1]. the invention of antibiotics has assisted in the control of pathogenic bacteria until when most pathogens become resistant to the treatment. increased alarming rate of antibiotic resistance worldwide is causing difficulty and posing danger to humanity as various diseases and ailments are now difficult to treat and control [2]. https://creativecommons.org/licenses/by/4.0/ micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 6 in addition, majority of available synthetic antibacterial drugs are becoming less effective due to their side effects such as tendonitis, seizure, and steven-johnson syndrome [3]. the emergence of antibiotic resistance and the unbearable side effects (toxicity) of some of the commercially available antibiotics makes it imperative to search for newer, more effective and cheaper drugs that could serve as alternative therapy for the treatment of various infections and diseases [4]. plants are reservoir of therapeutic substances which have a vital role in the sustenance of human health in the time past. world health organization (who) opines that plant parts or their potent make up are used in the productions of array of drugs in traditional therapies [5]. these plants have highly contributed to the development of human health and welfare. simultaneously, there is an increase in data and huge patronage to herbal products round the world [6]. medicinal plants such as ocimum gratissimum have been affirmed to provide different culinary and medicinal properties which effect can be bacteriostatic and bacteriocidal on some bacteria. these effects have been attributed to the peptides, alkaloids, essential oils, phenols and flavonoids which are main constituents in these plants [7]. it is a therapeutic plant which has been used traditionally for the treatment of various disorder [8]. cattle are reared for various reasons for consumption and economic purposes .they are used to pull farm implements in farmland and rural areas. cattles are herbivores which mean they do not eat only plants, grass and cereals. microbiota is the total of microorganisms that resides on or within any of a number of human tissues and bio-fluids. in new born, it is a powerful stimulus for the development of the immune system. unlike humans which are also colonized by many microorganisms, some microbes are normal flora in human they are commensal, they co-exist without being detrimental to humans; others to humans host are mutualistic [9]. this study was to provide an insight into molecular identification of two pathogenic organisms and document their antimicrobial susceptibility profiles to locally available plants. gene sequencing technology will be used for strain identification, and classification. in addition, we aim to use some nucleotide sequence analysis tools to perform a basic phylogenetic and diversity analysis in order to evaluate the genetic variability of the flora studied. materials and methods plant collection and identification plants were selected based on ethno botanical information obtained from traditional medicine practitioners in saki. all plants were collected around challenge ii area in saki. parts of the plants (leaves) were used for extraction. the identification was carried out in forest research institute of nigeria (frin), ibadan with voucher specimen of 112605 and 112606 for vernonia amygdalina and ocimum gratissimum respectively. extraction and isolation of active agents the materials of each was washed with distilled water and dried at room temperature by exposure to atmosphere for weeks. the dried materials were pulverized into fine powder using electric blender and packed into a plastic container with screw cap for further use. four types of extracts (aqueous, methanol, methanol plus water, n-hexane) were done. 100 g of each bitter leaf powder were weighed into three different sterile bottles and was soaked with 500 ml of methanol; n-hexane and 250 ml of methanol plus 250 ml of distilled water. 50 g of each scent leaf powder were also weighed into three different sterile bottles and was soaked with 250 ml of methanol, n-hexane and 175 ml of methanol plus 175 ml of distilled water. the mixture was stirred carefully with sterile stirrer and was covered with foil paper. the bottles were kept at room temperature for 3 days. the aqueous solution was prepared on the night of extraction by weighing 100g of bitter leaf powder in another bottle and was soaked with 500 ml of water and 50 g of scent leaf powder was also weighed in another sterile bottle and soaked with 250 ml of distilled water and covered with foil paper. the mixtures were extracted by sieving through muslin cloth, cotton wool and later filtered using whatman filter paper into clean sterile bottles. the aqueous prepared was boiled for 15 minutes and it was allowed to cool and was extracted like it was done to the previous ones. the extracts filtrates were concentrated using rotary evaporator at 45°c at the department of pharmaceutical chemistry laboratory in university of ibadan, oyo state, nigeria. determination of the antibacterial activity of the extract normal flora samples were collected from twelve cattle (6 males and 6 females) from different sites (nose, mouth, anus, vagina and skin) from the abattoir situated in foofo, saki, ibadan, oyo state, nigeria. swab sticks were used for the collection of specimen from different site from each cattle and were well labeled for recognition. nutrient agar was prepared according to manufacturer’s instructions. the specimens were directly streak on the prepared agar in different plates after solidifying and then incubated for 24 hours at room temperature. after 24 hours, the growth from the media incubated was transferred on macconkey agar and eosin methylene blue for sub-culturing till pure culture is formed. isolates were also tested on mrs and ssa agar but there was no growth. minimum inhibitory concentration each test organisms from serial dilution were streaked onto various plates and carried out in a septic condition and the plates were labeled accordingly. disc diffusion method were introduced by using disc(sterilized perforated disc 6 micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 6 mm in size seeded with different concentrations of the extract of 2 g, 4g and 6 g which is diluted into 10 ml of each methanol, n-hexane, methanol + aqueous and aqueous and amoxylin which is the control used was also diluted in 100 ml of distilled water. then the seeded disc was placed on the surface of the inoculated agar medium. the inoculated plates were incubated at 37 0 c the organism’s susceptibility test with plant extracts were recorded after 24 hours by measuring the mean diameter of the clear zone of inhibition using vernier caliper in millimeter (mm). biochemical tests for bacterial identification isolates were subjected to catalase and oxidase tests, which was identified using bergey's determinative bacteriology manual [10]. in order to perform a catalase test, the production of gas bubbles has been used as an indication of a positive result. isolate was placed on a glass slide with an inoculating loop 2 % of hydrogen peroxide was drop on the isolates and bubble isolates were recorded positive and vice versa. the oxidase test was used to detect the enzyme cytochrome oxidase activity. a purple coloration indicates positive results while no color change indicates oxidase negative results. inoculums of each isolate were smeared on a clean filter paper and oxidase reagent (tetramethylphenylenediaminedihydrochloride) was drop on each isolate. then colour changes were observed. dna extraction and 16s pcr bacterial cells broth was prepared and lysed through a zr bashing tm lysis tubes. 750µl lysis was pipetted in form of solution to the tubes. dna extraction from bacterial samples was carried out using the zymo bacterial dna extraction kit according to the protocol used by the manufacturer. the dna quantity and quality was measured using gel electrophoresis techniques. conventional pcr was applied to all the samples to amplify the 16s rrna gene using previously reported protocol (figure 1) [11]. figure 1: agarose gel electrophoresis showing positive amplification of 1,500 bp fragment of 16s rrna gene of klebsiella pneumoniae performed with strain-specific primer. lane m: 200-1037 bp dna ladder. strain lane g1:1500bp from cattle while g2: adm2 (1000-1700) isolated from cattle. g3 and g4 (isolates from other sources: rain and well water respectively). the pcr results were sequenced by using sanger sequencing technique and its products were subjected to analysis by basic local alignment search tools [12] (blast) and similar sequences were retrieved from the national centre for biotechnology information (ncbi) database [13]. the phylogenetic analysis was performed using mega 5.2 version [14]. ethical considerations experiments on animals have been conducted, according the research protocol reviewed by animal ethics committees of microbiology option, the oke ogun polytechnic, saki, nigeria. results and discussion the resultant nucleotides sequences of the 16s rrna was matched to klebsiella pneumonia and enterobacter bugandensis (adm2) with accession number mk719814 and mn213349 (figure 2). figure 2: the blast results of 16s bacterial sequences obtained in the current study. the antibacterial activity of both v amygdalina (bitter leaf) and ocimum gratissimum (scent leaf) were found to be dosage and extraction solvent dependent. methanolic extract of both plants at their highest tested doses possessed more antibacterial activities compared to the other extracts from different solvents when tested against the bacteria strain of klebsiella pneumoniae and e. bugandensis (adm2). this might attributed to the fact that methanol micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 6 extracted more of the bioactive constituents of the plant compared to other solvents. the methanolic extract of v amygdalina appeared more effective in the concentrations of 6g/ml when tested on k. pneumonia and showed moderate zone of inhibition on enterobacter bugandensis compared to the other tested doses, though the trend revealed dose dependent activity, i.e the higher the concentration the higher the effectiveness of the plant extract. the aqueous extract showed little to no inhibitory activity on the tested organisms while ciprofloxacin used as control inhibited the growth than the extracts (table 1). zones of inhibition produced by methanolic extract of ocimum gratissimum ranged from 7. 00 ± 0.00 at 2g/ml to 14.75±0.05 at 6g/ml against on k. pneumonia, followed by n-hexane extract ranged from 4.30±0.15 at 2g/ml to the highest dose of 6g/ml with value of 5.75±0.20, methanol + aqueous extract also showed moderate inhibitory activity while aqueous extract was found not to inhibit the growth of the test organisms at lower concentrations. however, at higher concentration, zones of inhibition were observed for the enterobacter bugandensis ranges from 1.30±0.02 at 4g/ml and 2.85±0.03 at 6g/ml (table 2). table 1: antibacterial activities of bitter leaf extract on k. pneumonia and e. bugandensis (adm2). leaf extract concentration (g/ml) mean diameter of zone of inhibition of bacteria in mm (±sem) methanol methanol + aqueous nhexane aqueous only control (ciprofloxacin) k. pneumonia 2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 e. bugandensis 2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 table 2: antibacterial activities of scent leaf extract on k. pneumonia and e. bugandensis (adm2). leaf extract concentration (g/ml) mean diameter of zone of inhibition of bacteria in mm (±sem) methanol methanol + aqueous nhexane aqueous only control (ciprofloxacin) 2.0g/ml 9.75 ± 0.03 4.75±0.02 3.30±0.15 1.00±0.00 22.00± 0.00 k. pneumonia 4.0g/ml 11.35±0.02 5.00±0.00 3.10±0.22 1.40±0.08 24.00±1.00 6.0g/ml 16.75±0.05 5.25±0.02 3.75±0.03 1.50±0.05 27.30±1.00 e. bugandensis 2.0mg/ml 4.50±0.05 2.50±0.08 2.00±0.00 0.00±0.00 10.00±0.00 4.0g/ml 5.00±0.00 3.60±1.06 2.90±0.14 1.00±0.00 11.00±0.00 6.0g/ml 6.30±0.10 4.30±0.14 3.30±0.00 1.00±0.02 15.00±1.00 veronia amygdalina produces a variety of flavonoids and bitter lactones which contribute to the bioactivities of this plant [15]. v. amygdalina serves well as a low cost are readily available source of important nutrients to human [16]. the result of the current study demonstrated that the six out of eight normal flora from different sites isolated from cattle were bacterial culture while others were positive according to the standard bacteriological identification and microscopic identification. the sensitivity tests show that there is high resistance of test organism against antibacterial activity of plant extracts. this means that scent leaf and bitter leaf extracts are more effective as antibacterial agent against test organism. sequencing analysis was done to subject a deoxyribonucleic acid, polymerase chain reaction to test organism so as to understand its features, structure, function and evolution [17,18].some of the suspected antibiotic resistance genes and virulence genes were (hyla, teta, tete and blatem) respectively with base pairs of 1-1.5kb [19–21]. phylogenetic analysis of retrieved bacterial isolates indicates a high level of identification of 16s rrna sequences for bacterial genus and species (figure 3). such results could confirm the usefulness of using such genomic regions in the identification and characterization of bacterial isolates. micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 6 figure 3: phylogenetic trees of enterobacter bugandensis (a) and klebsiella pneumoniae (b) isolates from normal cattle flora compared to similar 16srrna gene sequences. conclusion most of the sensitive extracts were unable to inhibit the growth of the organisms and are presumably drug-resistant. scent leaf and bitter leaf extract appeared more effective than their previously reported actions. notwithstanding, v. amygdalina possesses various bioactivities with low or absence of side effects having great health promoting effects. it is however more advantageous to incorporate v. amygdalina into health supplement for both human and animal benefits. acknowledgement the authors wish to acknowledge the abattoirs attendants for the supports during the course of this research. also, fehintola oluwaseun deserves to be mentioned; for processing and retrieval of sequences from ncbi database. references 1. jakovac h. covid-19 and vitamin d-is there a link and an opportunity for intervention. american journal of physiology-endocrinology and metabolism. 2020 may 1; 318(5):e589. 2. nanasombat s, lohasupthawee p. antibacterial activity of crude ethanolic extracts and essential oils of spices against salmonella and other enterobacteriacea. current applied science and technology. 2005; 5(3):527-38. 3. world health organization. traditional medicine: growing needs and potential. world health organization; 2002. 4. effraim kd, jacks tw, sadipo oa. histopathological studies on the toxicity of ocimum gratissimum leaves extract on some organs of rabbit. african journal of biomedical research. 2003; 6(1). 5. kirbağ se, zengin f, kursat m. antimicrobial activities of extracts of some plants. pak. j. bot. 2009; 41(4):206770. 6. omoseyindemi bx. plants as natural medicine. 167th annual conference of botanical society of nigeria (boson). university of lagos; march 16th, 2003. 7. okigbo r, igwe d. antimicrobial effects of piper guineense ‘uziza’and phyllantus amarus ‘ebe-benizo’on candida albicans and streptococcus faecalis. acta microbiologica et immunologica hungarica. 2007 dec 1; 54(4):353-66. 8. mann a. phytochemical constituents and antimicrobial and grain protectant activities of clove basil (ocimum gratissimum l.) grown in nigeria. international journal of plant research. 2012;2(1):51-8. micheal et al., 2020 antibiotic effects of leaf extracts of k. pneumoniae and e. bugandensis highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 6 9. shih c. the gut flora. journal of lancaster general hospital 2013; 8(4):114–7. 10. owen jj. manual of determinative bacteriology. canadian medical association journal. 1926 may; 16(5):620. 11. lane dj. 16s/23s rrna sequencing. nucleic acid techniques in bacterial systematics. 1991:115-75. 12. altschul sf, madden tl, schäffer aa, zhang j, zhang z, miller w, lipman dj. gapped blast and psiblast: a new generation of protein database search programs. nucleic acids research. 1997 sep 1; 25(17):3389-402. 13. maglott d, ostell j, pruitt kd, tatusova t. entrez gene: gene-centered information at ncbi. nucleic acids research. 2010 nov 27; 39(suppl_1):d52-7. 14. kumar s, stecher g, li m, knyaz c, tamura k. mega x: molecular evolutionary genetics analysis across computing platforms. molecular biology and evolution. 2018 jun 1; 35(6):1547-9. 15. favi f, cantrell cl, mebrahtu t, kraemer me. leaf peltate glandular trichomes of vernonia galamensis spp. galamensis var. ethiopica gilbert: development, ultrastructure, and chemical composition. international journal of plant sciences. 2008 jun; 169(5):605-14. 16. ojiako oa, nwanjo hu. is vernonia amygdalina hepatotoxic or hepatoprotective? response from biochemical and toxicity studies in rats. african journal of biotechnology. 2006; 5(18). 17. cheng f, li z, lan s, liu w, li x, zhou z, song z, wu j, zhang m, shan w. characterization of klebsiella pneumoniae associated with cattle infections in southwest china using multi-locus sequence typing (mlst), antibiotic resistance and virulence-associated gene profile analysis. brazilian journal of microbiology. 2018 nov 1; 49:93-100. 18. osman km, hassan hm, orabi a, adelhafez as. phenotypic, antimicrobial susceptibility profile and virulence factors of klebsiella pneumoniae isolated from buffalo and cow mastitic milk. pathogens and global health. 2014 jun 1; 108(4):191-9. 19. li gy, li j, xiao p, guo yh, mo zl. detection of type iii secretion gene as an indicator for pathogenic edwardsiella tarda. letters in applied microbiology. 2011 mar; 52(3):213-9. 20. akinbowale ol, peng h, barton md. diversity of tetracycline resistance genes in bacteria from aquaculture sources in australia. journal of applied microbiology. 2007 nov; 103(5):2016-25. 21. chung th, yi sw, kim bs, kim wi, shin gw. identification and antibiotic resistance profiling of bacterial isolates from septicemic soft-shelled turtles (pelodiscus sinensis). veterinární medicína. 2017 mar 15; 62(3):169-77. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202506 research article open access 1 department of zoology, faculty of sciences, menoufia university. * to whom correspondence should be addressed: hodaahmed83@science.menofia.edu.eg editor: muhammad m. adeel, arthritis clinical immunology program oklahoma medical research foundation, oklahoma city, united states. reviewer(s): khrokalo liudmyla, physical chemistry department national technical university of ukraine "igor sikorsky kyiv polytechnic institute" kyiv 03056 ukraine. grace magalhaes-ghiotto, department of biotechnology, genetics and cell biology, biological sciences center, state university of maringá, maringá, paraná 87020-900, brazil. received: may 11, 2025 accepted: august 2, 2025 published: august 12, 2025 citation: abdelazeem hh, osman gy, sheir sk. apoptotic and histopathological impacts of moringa oleifera seed oil on the land snail cornu aspersum (o. f. müller, 1774). 2025 aug. 12;8:bs202506 copyright: © 2025 abdelazeem hh et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. apoptotic and histopathological impacts of moringa oleifera seed oil on the land snail cornu aspersum (o. f. müller, 1774) hoda h. abdelazeem*1 >< �, gamalat y. osman1 ><�, sherin k. sheir1 ><� abstract cornu aspersum (o. f. müller, 1774) is among the most harmful pests for many crops. using natural molluscicides is essential to reduce the adverse effects of chemical ones on the biosystem. the toxicity of moringa oleifera seed oil was determined, and lc50 and lc90 were 20.06% and 29.5%, respectively, after 72 hr of exposure. moringa oil diminished the survival rate of cornu aspersum (o. f. müller, 1774) by 50% compared to the control (98.6%). moreover, moringa oil significantly increased the apoptosis and necrosis of digestive and ovotestis gland cells (p ≤ 0.02). by the end of the experiment, the proportion of apoptotic cells rose dramatically to 62.6% and 50.3% in the digestive and ovotestis glands, respectively, compared to the control (10.8% and 12.3%). in addition, the percentage of necrotic cells significantly increased to 21.5% and 24.7% (p ≤ 0.05), while the control values were 5.6% and 4.5%, respectively, at the 8% concentration. regarding the digestive gland, moringa oil caused vacuolation, nuclear pyknosis, and haemocyte infiltration. deficiency of mature ova and spermatozoa, fibrosis, degeneration, and necrosis were recorded in the ovotestis. moringa oil has proved its effectiveness as a natural molluscicide. keywords: cornu aspersum (o. f. müller, 1774), moringa oleifera seed oil, survival rate, apoptosis, histology. introduction mollusca are characterized as the second-largest phylum in the animal kingdom due to their great reproductive capacity-especially slugs and snails-which enables them to spread quickly throughout agricultural fields, making population control extremely challenging [1; 2]. the damage caused by these snails is attributed to their rasping feeding habits and the deposition of waste products such as slime and feces. crop losses of 50–90% in soybean and corn have been reported in india [3; 4]. land snails are notorious pests that cause significant damage to various crops and vegetation throughout egypt [5; 6; 7]. these land snails (gastropods) can damage multiple plant species, leading to major economic losses in agricultural fields, gardens, orchards, and greenhouses [8; 9; 10; 11]. several authors have recorded different snail species and investigated their harmful effects: cornu aspersum (o. f. müller, 1774), achatina fulica, helix vestalis, and theba pisana and monacha sp. and oxychilus sp. [12; 13; 14; 15]. these pests have caused serious damage to vegetables, banana crops, tomatoes, ornamental plants, mulberries, grapevines, and germinated seeds [16]. land mollusks also harm potatoes, grains, lettuce, cabbage, carrots, maize, clover, and other horticultural and field crops. they feed on roots, seedlings, seeds, and tubers of nearly every fruit, vegetable, oil plant, and ornamental species in gardens, greenhouses, and fields. damage includes killing seedlings (leading to poor stands) and destroying the leaves of young plants. molluscs’ detrimental effects, however, differ depending on the crop. for example, some consume freshly planted wheat seeds, scrape strips of leaves from maize and many small grain plants, and make craters in the cotyledons and ragged holes in the leaves of soybean crops [17; 1; 18]. pest control for snails requires a continuous and integrated approach. physical methods such as hand collection and device innovation, the use of simple chemicals like sodium chloride, and the application of registered molluscicides such as methomyl are commonly employed, often in combination with biological control methods [18; 19; 20]. however, the extensive use of pesticides highlights in bioscience page 1 of 10 august 2025|volume 8 https://doi.org/10.36462/h.biosci.202506 https://creativecommons.org/licenses/by/4.0/ mailto:hodaahmed83@science.menofia.edu.eg https://orcid.org/0000-0002-7599-8077 mailto: gamalat.osman@science.menofia.edu.eg https://orcid.org/0000-0001-9646-558x mailto:shyreen.shaair@science.menofia.edu.eg https://orcid.org/0000-0002-0036-1514 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil by agricultural workers in many countries has raised serious health concerns due to the toxicity of these chemicals [21]. moreover, they often affect non-target organisms and disrupt the ecosystem [22]. given the side effects associated with each method, there is an urgent need for safe and cost-effective alternatives. as a result, ongoing efforts are focused on discovering and evaluating promising, effective, and eco-friendly molluscicides against pest snail species. natural plant derivatives, also known as botanical pesticides, have gained attention for their environmental safety. oil extracts from these plants are excellent natural products-they are biodegradable into non-toxic compounds, easy to access and dispose of, and economically affordable [23]. table 1. physiochemical properties of moringa seed oil physical properties refractive index 1.471 ±0.00 specific gravity 0.903 ±0.2 acid value (mg/g) 0.58 ±0.21 peroxide value (meq/kg) 2.37 ±0.06 saponification value (mg koh/g) 160.32 ±0.4 iodine value (g/100g) 65.27 ±0.21 chemical properties (fatty acids composition) name concentration(%) palimitic acid c 18:0 6.61 ±0.5 stearic acid c18:0 5.30 ±0.5 oleic acid c 18:1 66.12 ±0.04 linolinic acid c 18:3 1.03 ±0.4 palmitoleic acid c 16:1 1.78 ±0.02 vaccinic acid c16:1 5.86 ±1 linoleic acid c18:2 1.47 ±0.1 arachidic acid c 20:0 3.46 ±0.04 gadolic acidic c20:1 n9 2.75 ±0.1 behenic acid 22:0 6.36 ±0.03 total unsaturated fatty acids 79.01 ±1.2 total saturated fatty acids 21.73 ±1 (n=3 replicates, data are mean ±sd) one of the most well-known and widely distributed species is moringa oleifera [24]. the seed oil extract contains fatty acids similar to olive oil, with linoleic acid serving as a substitute [25]. it is rich in protein, and its leaves contain high levels of minerals such as iron, vitamins, and calcium, making it useful for treating malnutrition [26]. different parts of the tree including the root, leaf, fruit, and seedalong with their extracts or oils, possess various medicinal properties and have been used in both non-food products and traditional medicine [27]. it possesses antioxidant, anti-inflammatory, antianemic, and antidiabetic properties, supports the immune system, and combats neurological, reproductive, cardiovascular, and bone illnesses. [28; 29]. furthermore, the bioactive constituents of botanical molluscicides, such as flavonoids, saponins, and tannins found in moringa spp., have been studied for their effects on snails [30; 31; 32]. thus, the aim of this study is to evaluate the molluscicidal efficacy of moringa oleifera seed oil against cornu aspersum (o. f. müller, 1774). materials and methods experimental materials moringa oleifera seed oil was purchased from the moringa unit at the national research center, dokki, egypt. the analysis of the oil, including its components and physicochemical properties, was conducted at the same unit (table 1). methomyl (copter 90% sp), a carbamate compound (s-methyl n-[(methylcarbamoyl) oxy]thioacetimidate), with the molecular formula c5h10n2o2s, was used as a standard pesticide. it was obtained from egyptchem international for agrochemicals (cairo, egypt). the required concentrations of moringa oil were prepared by dissolving the oil in a 1% tween 80 solution (v/v) [33; 34]. the chemical structure of tween 80 is 2-[2-[3,4-bis(2-hydroxyethoxy) oxolan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate. determination of lc50 and lc90 to determine the lc50 and lc90 of moringa oleifera seed oil, acclimatized snails (mean weight: 4.4 ± 0.6 g) were divided into three replicates, with 10 snails in each group. each group of 10 individuals was sprayed with 10 ml of its specific concentration. a range of concentrations (1%, 3%, 6%, 9%, 15%, 21%, 27%, and 30%) of moringa oil was freshly prepared using a 1% tween 80 solution (v/v) [35]. snail mortality was observed and recorded daily for up to 72 hours. after counting and removing the dead individuals, the lethal concentrations (lc50 and lc90) were calculated using probit analysis in the spss statistical software package (ibm corp., armonk, ny, usa). two sub-lethal concentrations, 4% and 8%, were selected as lc10 and lc20, respectively. collection of snails and experimental design cornu aspersum (o. f. müller, 1774) individuals were collected from garden plants and decorative trees, then transported in plastic boxes to the laboratory. the snails were maintained at a temperature of 23 ± 2◦c, under a 12 h photoperiod and relative humidity ranging from 75% to 85%. each box contained a layer of soil at the bottom and was covered with muslin cloth. the snails were fed fresh lettuce leaves throughout the acclimatization and experimental periods. snails were randomly divided into six groups, with three replicates per group, and each replicate consisting of 15 snails. the experimental groups were as follows: (a) control group (unexposed). (b) two groups exposed to 4% and 8% moringa oleifera seed oil. (c) two groups exposed to 4% and 8% methomyl. (d) one group exposed to 1% tween 80 solution. snails were sprayed daily with 1 ml of the respective treatment solution per snail (15 ml per replicate) over the course of three weeks. survival rate was recorded as the percentage highlights in bioscience page 2 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil table 2. the effect of m. oleifera seed oil on the survival rate of cornu aspersum (o. f. müller, 1774). exposure period survival rate control methomyl moringa tween 80 4 8 4 8 1 zero time 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 100 ±0 1st week 100 ±0 67.6 ±0.5 64.3 ±0.5 82 ±0.4 75.3 ±0.3 100 ±0 2nd week 100 ±0 54.3 ±1.5 47.6 ±1.1 72 ±0.5 63.3 ±1 98.9 ±0.5 3rd week 98.6 ±0.5 34.3 ±1.5a 30 ±1b 63.3 ±1 50 ±1ab 97.6 ±0.5 n = 3 replicates; data were reported as mean ±sd; a denotes a significant difference between the exposed and control groups and b vs methomyl-exposed group when p ≤ 0.05. of snails remaining alive at each observation point during the exposure period. at the end of the experiment, the digestive and ovotestis glands were dissected for apoptosis and necrosis assessment using cell cycle analysis and annexinv/pi staining, in addition to histological investigations. cell cycle analysis digestive and ovotestis glands (0.08 g) were homogenized in 1500 µl phosphate-buffered saline (cold) (pbs) and centrifuged at 1,000 rpm for 1 min at 4°c. the supernatant was collected for the assay. 200 µl of cell suspension in citrate buffer was added, along with propidium iodide (pi). there were 10,000 assessed nuclei on average for each specimen, and 120 nuclei were scanned every second. accuri™ c6 flow cytometer analysis (becton dickinson, united states) was used to assess apoptotic cells utilizing sub-g1 peak labeling with pi [36; 37]. • g0/g1 peak: normal diploid cells. • s phase (synthesis): cells synthesizing dna. • g2/m peak (gap 2 /mitosis): cells ready to divide. • sub-g1: apoptotic cells with fragmented dna. assay of pi/annexin-v dual staining discrimination of the apoptotic profile was determined using the apoptosis detection kit i (cat. no. 556547bd, pharmingen™, bioscience, new zealand) according to the manufacturer’s instructions. the principle of this procedure relies on the interaction between the membrane phospholipid phosphatidylserine of apoptotic cells and annexin v, a phospholipid-binding protein that requires calcium. to differentiate between viable and non-viable cells, propidium iodide (pi) was employed as a conventional flow cytometric viability probe. non-viable or damaged cells with permeable membranes allow pi to enter, while viable cells with intact membranes exclude pi. the bd accuri™ c6 flow cytometer was used to analyze the cells immediately. four distinct cell populations were identified: (1) early apoptotic cells (bound to annexin v only), (2) late apoptotic/necrotic cells (bound to both annexin v and pi), (3) necrotic cells (stained with pi only), (4) viable cells (unstained). the percentage of fluorescent cells in each quadrant was calculated after the fluorescence distribution was presented in a two-color dot plot analysis [38]. histological investigation after three weeks, the digestive and ovotestis glands were dissected and fixed in bouin’s fluid. after 24 hr of fixation, dehydration was performed through a series of alcohols and cleared in xylene. paraffin blocks were sectioned at 5 µm thickness on glass slides for hematoxylin and eosin staining [39; 40]. a good wash with tap water was performed after staining. a photo-automated camera (optika, italy) was used to capture images of the histological sections, which were then analyzed for histopathological syndromes. statistical analysis the statistical package for the social sciences (spss; ibm corp., armonk, ny, usa) was used to analyze the data, which were presented as mean ± standard deviation. one-way anova was performed, followed by the least significant difference (lsd) post-hoc test to determine the significance between the control and different concentrations of the tested materials. a significance level of p < 0.05 was considered statistically significant. results toxical effect of moringa oleifera seed oil the values (%) of lc50 and lc90 were 20.06 (95% confidence limit for log = 1.3) and 29.5 (95% confidence limit for log = 1.4), respectively, after 72 hr of exposure. the slopes of lc50 and lc90 were 2.5 and 3, respectively. the sublethal concentrations used were 4% moringa oil (40 ml/l of 1% tween 80) and 8% moringa oil (80 ml/l of 1% tween 80) (v/v) solutions. additionally, 4% methomyl (4 g / 100 ml) and 8% methomyl (4 g / 100 ml) (w/v) solutions were used. the survival rate of cornu aspersum (o. f. müller, 1774) under the effect of moringa oil the survival rate of cornu aspersum decreased gradually according to the concentration and exposure duration. the reduction was more significant in methomyl-exposed snails, followed highlights in bioscience page 3 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil table 3. percentage of cells in different cell cycle phases after treatment concentration exposure period %sub g1 % cells/cell cycle g0/g1 s g2/m control 1st week 12.8 ±0.1 91.9 ±0.1 51.03 ±1 4.4 ±0.1 24.5 ±0.8 2nd week 11.8 ±0.2 95.8 ±0.3 52 ±1 4.1 ±0.3 24.8 ±1 3rd week 11.7 ±0.4 96 ±0.4 52.1 ±1.2 4 ±0.2 24.3 ±0.5 4% moringa oil 1st week 14.9 ±0.6 97.2 ±2.9 70.1 ±3.3 2.1 ±0.2 10.4 ±1.9 2nd week 15.02 ±0.1ab 83.2 ±1.4 40.1 ±0.7 15.03 ±0.05 15.5 ±0.7 3rd week 12.4 ±1.2 90.9 ±2.9 60.1 ±2.07 4.1 ±0 15.3 ±1.4 8% moringa oil 1st week 18.2 ±0.3ab 99.2 ±1.3 78.3 ±1.4 1.8 ±0.2 0.9 ±0.1 2nd week 10.4 ±0.05 91.03 ±1.1 57.1 ±0.6 3.7 ±0.05 20.6 ±0.8 3rd week 13.3 ±0.9 90.2 ±2.03 59.5 ±2.4 4.03 ±0.1 14.5 ±3.2 4% methomyl 1st week 21.3 ±0.3ab 96.3 ±1.1 64.3 ±1.06 1.3 ±0.05 9.93 ±0.1 2nd week 14.1 ±0.5b 92.1 ±0.8 51.4 ±1.1 6.5 ±0.05 21.1 ±1.6 3rd week 14.7 ±0.6 93.8 ±1.05 52.06 ±2.2 1.8 ±0.05 26.1 ±2.8 8% methomyl 1st week 22.1 ±0.1ab 96.9 ±0.6 63.6 ±1.5 1.6 ±0.05 10.1 ±1.7 2nd week 12.8 ±0.6 90.7 ±3.03 41.9 ±2.4 13.4 ±0.3 23.6 ±3.9 3rd week 14.1 ±0.5b 92.6 ±1.5 45.4 ±1.2 5.2 ±0.1 28.9 ±2.4 1% tween 1st week 12.8 ±2.7 94.03 ±1.7 76.1 ±1.2 2.23 ±0.2 3.5 ±0.1 2nd week 9.2 ±0.7 87.8 ±4.9 65.6 ±4.2 13.3 ±0.1 0.8 ±0.1 3rd week 14.02 ±1 86.3 ±4.4 54.3 ±3.9 3.7 ±0.4 16.1 ±0.2 data are stated as mean ± sd, n = 3 replicates; significant differences are represented as a when compared with the control and b when compared with methomyl at p ≤ 0.05. figure 1. representative flow cytometry histogram showing moringa oil’s effect on cell cycle distribution of cornu aspersum (o. f. müller, 1774) after the third week of exposure. the digestive gland is represented by panels (a), (b), and (c): (a) control; (b) 8% moringa oil; (c) 8% methomyl. the ovotestis gland is represented by panels (d), (e), and (f): (d) control; (e) 8% moringa oil; (f) 8% methomyl. by those exposed to m. oleifera seed oil. a substantial decrease (p = 0.01) was observed in the third week of exposure, with survival rates of 50% for m. oleifera oil and 30% for methomyl, compared to 98.6% in the control. methomyl exposure significantly reduced the survival rate compared to m. oleifera seed oil (table 2). cell cycle distribution after moringa oil exposure the proportion of cells in the g0/g1, s, and g2/m phases of the cell cycle was determined in both digestive and ovotestis glands after 1, 2, and 3 weeks. exposure to 4% and 8% m. oleifera seed oil resulted in a significant increase in fragmented cells (sub-g1) compared to the control (p ≤ 0.003) and methomyl (p ≤ 0.01). notably, the number of g0/g1 phase cells increased to 78.3 ± 1.4% in the 8% moringa oil group, compared to 51.03 ± 1% in the control and 63.6 ± 1.5% in the methomyl group. consequently, the proportions of s and g2/m phase cells decreased at the same concentration (table 3, figure 1). in the digestive gland, 8% moringa oil significantly increased sub-g1 phase cells compared to the control (p ≤ 0.001) and highlights in bioscience page 4 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil table 4. percentage of cells in different cell cycle phases after treatment concentration exposure period %sub g1 % cells/cell cycle g0/g1 s g2/m control 1st week 9.3 ±1 96.5 ±1.3 52.2 ±0.1 3.1 ±0.2 11.4 ±1 2nd week 10.4 ±0.3a 95.4 ±0.8 52.2 ±0.1 3.5 ±0.3 10.9 ±1 3rd week 10.4 ±0.5 95.3 ±0.4 52.5 ±0.2 3 ±0.2 10.8 4% moringa oil 1st week 15.4 ±0.4 93.6 ±0.5 74.1 ±1.1 10.5 ±0.1 1.1 ±0.1 2nd week 17.06 ±0.5a 88.5 ±3.2 74.1 ±2.4 10.5 ±0.2 0.9 ±0.1 3rd week 15.2 ±0.05 93.6 ±0.2 75.1 ±0.5 10.3 ±0.05 1.2 ±0.3 8% moringa oil 1st week 16.2 ±0.04 92 ±0 67.1 ±0.2 9.1 ±0.05 2.9 ±0.2 2nd week 30.2 ±0.1a 82.2 ±0.5 67.1 ±0.4 14.1 ±0.1 3 ±0.1 3rd week 15.7 ±0.7 96.1 ±1.4 67.4 ±1.5 13.1 ±0.2 2.8 ±2.4 4% methomyl 1st week 18.1 ±0.1a 91.5 ±0.5 63.6 ±0.7 17.9 ±0.05 14 ±0.8 2nd week 12.9 ±0.1 86.2 ±0.2 63.7 ±0.5 17.6 ±0.1 14.1 ±0.9 3rd week 15.1 ±0.1 90.4 ±0.8 63.6 ±0.2 12 ±0.05 14.2 ±1.05 8% methomyl 1st week 18.07 ±0.8a 91.3 ±1.4 68.6 ±3 12 ±0.05 13.4 ±1.8 2nd week 14.6 ±0.05 93.3 ±2.8 68.9 ±2.7 12.1 ±0.05 13.3 ±0.1 3rd week 17.9 ±0.4a 94.7 ±1.4 68.8 ±2.3 11.9 ±0.1 13.4 ±0.7 1% tween 1st week 11.2 ±0.1 96.3 ±0.4 56 ±0.2 25.3 ±0.3 0.6 ±0.05 2nd week 11.5 ±0.2 96.6 ±0.7 59 ±0.2 25.7 ±0.7 0.7 ±0.1 3rd week 14.6 ±0.2 87.3 ±0.8 52.5 ±0.5 5.3 ±0.1 16.5 ±0.8 data are expressed as mean ± sd, n = 3 replicates; significant differences are represented by a vs. control when p ≤ 0.05. methomyl (p ≤ 0.005). the g0/g1 phase cell counts increased to 75.1 ± 0.5% (4% moringa) and 67.4 ± 1.5% (8% moringa) versus 52.5 ± 0.2% in the control. furthermore, g2/m phase percentages decreased with both moringa oil concentrations (table 4, figure 1). detection of apoptosis by annexin-v/pi using flow cytometry, apoptotic cells were identified by incubating them with fitc-labeled annexin v and pi. to differentiate between necrotic and apoptotic cells, pi was utilized. the exposure of snails to moringa oil caused a significant increase in apoptotic cells (early and late apoptosis) in digestive gland cells at both concentrations (p ≤ 0.001) and in the two concentrations of methomyl (p ≤ 0.01) compared with the control group. at the end of the experiment, the percentages of apoptotic cells were 50.3% and 63.9% at 8% moringa oil and methomyl, respectively, while that of the control was 12.3%. depending on the material, the increase in apoptotic cells in both concentrations of moringa oil was significant compared with methomyl (p ≤ 0.001). in ovotestis gland cells, a significant increase in apoptotic cells was observed at both concentrations of moringa oil (p ≤ 0.003) and at the two concentrations of methomyl (p ≤ 0.02). at the third week of exposure, the percentages of apoptotic cells were 62.6% and 50.8% at 8% moringa oil and methomyl, respectively, while the control was 10.8%. depending on the time of exposure, the increase in apoptotic cells in the third week was significant when compared with that in the first week (p = 0.01). cells that were stained with pi were identified as necrotic, and they significantly increased in the digestive gland cells after exposure to both moringa oil concentrations (p ≤ 0.005) and methomyl (p ≤ 0.02) compared with the control. the percentages were 21.5% and 14.9% at 8% moringa oil and methomyl, respectively, while that of the control was 5.6%. in the ovotestis gland, a significant increase was noticed at both moringa oil concentrations and methomyl (p ≤ 0.03) compared with the control. regarding exposure time, the increase in the third week was 24.7% and 31.1% (p = 0.02) at the 8% concentration of moringa oil and methomyl, respectively, compared with their values in the first week (5.5% and 3.7%), when the control was 4.5%. as a result, the number of viable cells was significantly decreased in moringa oil and methomyl (70.03% and 60.2%, respectively) compared with the control (84.6%) in the third week of exposure highlights in bioscience page 5 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil figure 2. fluorocytograms of one representative experiment from three separate trials following a three-week exposure to m. oleifera seed oil. (figure s1, figure s2, figure 2). histopathological signs after exposure to moringa oil histological sections of the normal (unexposed) digestive gland of cornu aspersum (o. f. müller, 1774) revealed that it is mainly composed of digestive tubules, each lined with columnar epithelial cells of various types, including digestive and excretory cells. the digestive cells are typically characterized by numerous green and yellow cytoplasmic granules. these tubules enclose a distinct central lumen and are separated by interlobular connective tissue containing hemolymphatic sinuses rich in actively circulating hemocytes. externally, the tubules are encased by a distinct circular muscle layer. exposure to 8% m. oleifera oil caused pronounced pathological alterations in the digestive gland, including marked vacuolization and pyknosis of nuclei. hemocytic infiltration, a clear sign of inflammation, was also observed. severe epithelial damage, including extensive vacuolization, degeneration, and necrosis, was evident following exposure to both 4% and 8% concentrations of methomyl. additionally, moringa oil induced progressive degeneration of muscle fibers (figure 3). the ovotestis of unexposed snails consists of numerous small follicles (acini) lined with germinal epithelium, which is clearly differentiated into primary and secondary spermatogonia/oogonia and mature spermatozoa/ova. after three weeks of exposure, the high concentration (8%) of moringa oil caused notable alterations in the histological architecture of the ovotestis. histopathological signs included degeneration of ova and spermatozoa, inhibition of ova maturation, appearance of dense fibrous tissue, and necrosis. methomyl (8%) exposure led to a substantial reduction or complete absence of mature ova and degeneration of spermatozoa (figure 4). discussions figure 3. light photomicrographs of sections through the digestive gland of cornu aspersum (o. f. müller, 1774) after three weeks of exposure. (a & b) control, (c, d, e, & i) exposed snails to 4% and 8% m. oleifera seed oil, and (f, g, h, & j) exposed snails to 4% and 8% methomyl. dt, digestive tubule; dc, digestive cell; se, secretory cell; ct, connective tissue; l, lumen; py, pyknosis; v, vacuolization; dg, degeneration; uf, undigested food; ig, increasing the granules in digestive cells; n, necrosis; hi, hemocytic infiltration; mf, muscle fibers. effect of moringa oleifera seed oil on the survival rate of cornu aspersum (o. f. müller, 1774) the present study recorded the influential role of moringa oil in reducing the survival rate of cornu aspersum snails. as primary consumers, land snails serve as models for rapid physiological responses to dietary habits and breeding conditions. previous research [41; 42] confirmed the sensitivity of mollusca to minimal dietary changes due to their rapid metabolism. moringa oil is composed of unsaturated and saturated fatty acids. exposure of snails to this oil may alter their fatty acid profile, which is sufficient to change the physiological state of gastropods [43]. a previous study also indicated that fluctuations in polyunsaturated fatty acids can serve as a valuable ecotoxicological test in snails [44]. benzylamine extracted from m. oleifera has demonstrated molluscicidal potency by reducing survival and reproductive rates in biomphalaria alexandrina snails [45]. plant extracts containing essential oils, flavonoids, terpenes, saponins, and tannins have been reported as effective agents in snail control [46; 47]. the negative impact of such extracts on the land snail monacha obstructa has also been documented [48]. apoptotic effect of m. oleifera seed oil on cornu aspersum the findings indicated the apoptotic potential of m. oleifera oil on both digestive and ovotestis gland tissues. this can be explained by the fact that mollusks possess a unique composition of fatty acids. given this, previous studies have shown that unsaturated and saturated fatty acids exert different effects on steatosis and cell death, influencing apoptosis in a variety of experimental systems and being identified as the most harmful lipid types [49; 50; 51]. the presence of polyunsaturated fatty acids in cellular membranes influenced membrane permeability. previous research showed that apoptosis was caused by palmitic acid highlights in bioscience page 6 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and histopathological impacts of moringa oleifera seed oil figure 4. light photomicrographs of sections through the ovotestis gland of cornu aspersum (o. f. müller, 1774) after three weeks of exposure. (a) control; (b, c, & f) exposed snails to 8% m. oleifera oil; (d & e) exposed snails to 8% methomyl. f, follicle; fw, follicular wall; ov, ovum; sp, spermatozoa; dv, developing oocyte; ss, stages of spermatogonia; d, degeneration; ft, fibrous tissue; n, necrosis; vo, vitellogenic oocyte. reduction/lack of mature ova and spermatozoa (black arrows). through caspase-dependent beclin 1 cleavage. the researchers concluded that essential fatty acids induce apoptosis of tumor cells by overexpressing cytochrome p450 [52]. mitochondria play a major role in apoptosis by releasing cytochrome c and caspase 3, which activate apoptotic enzymes [53]. fatty acids that stimulate lipotoxicity were also reported to play a crucial role in pathogenesis [54]. moreover, the toxicity of metals has been linked to disturbances in lipid membranes [55]. the observed changes in fatty acid levels represent metabolic adjustments initiated by external stimuli [56]. a recent study explored the ability of moringa oleifera leaf extract to promote apoptosis in adipocyte cells (3t3-l1 cells) by increasing caspase 3 activity and regulating the expression of bax and bcl2 genes [57]. moringa seed extract has been shown to induce apoptosis and cell cycle arrest in cancer cells, with fatty acids like caprylic acid, oleic acid, and stearic acid contributing to apoptosis in lung, leukemia, and ovarian cancer cells [58]. autophagy and hepatic steatosis were reported in the mouse liver after high-fat diets [59]. another study demonstrated the harmful effects of moringa extract (containing saponins, flavones, and flavonols) on biomphalaria glabrata embryos, adults, and schistosoma mansoni adult worms [60]. histopathological effect of m. oleifera seed oil on cornu aspersum exposure to seed oil induced histopathological changes in both digestive and ovotestis glands. this observation aligns with previous findings [61], where damage such as fragmentation and vacuolization was reported in the digestive and excretory cells of b. alexandrina and b. truncatus snails following treatment with egyptian wild plant extracts. exposure to aqueous seed extract of m. oleifera caused severe damage in digestive cells, including tip loss, degeneration, and increased numbers of secretory cells [62]. additional histopathological features included vacuolation of digestive and secretory cells. degeneration and rupture of sperm and ova in b. truncatus were observed following exposure to cerium oxide nanoparticles synthesized using moringa seeds [63]. chlorophyllin from deep-frozen m. oleifera leaves caused deformation of secretory cells and rupture of connective tissue between tubules, leading to degeneration of digestive cells in b. truncatus. when combined with magnesium or copper, this compound also caused histological malformations due to photosensitization [64; 65]. in cornu aspersum, degeneration of digestive tubules and damage to the basement membrane and hepatopancreas were reported following exposure to thiamethoxam (200 mg/l) [66]. conclusions moringa oleifera seed oil has significant molluscicidal potency, exhibiting both apoptotic and histopathological effects on the ovotestis and digestive glands of cornu aspersum (o. f. müller, 1774). this study investigated the effects of moringa oil as a whole against the snail cornu aspersum. further studies will aim to isolate the main active constituents of the oil and assess their effects on non-target organisms to ensure greater eco-friendliness compared with conventional chemical molluscicides. list of abbreviations • pbs: phosphate-buffered saline • pi: propidium iodide • coi: cytochrome oxidase subunit i • nd1: nadh dehydrogenase subunit 1 • dna: deoxyribonucleic acid supplementary figure s1: exposure to m. oleifera seed oil increased cell apoptosis and necrosis of the digestive gland of cornu aspersum (o. f. müller, 1774) after exposure for3 weeks: (a) first week, (b) second week, and (c) third week. values expressed as mean ±sd. c the significant difference in necrosis between control and exposed snails when p ≤ 0.02. the significant difference in apoptosis was indicated as follows: a,b, between moringa exposed groups vs. control and methomyl exposed groups when p ≤ 0.05, anova. figure s2: exposure to m. oleifera seed oil increased cell apoptosis and necrosis of the ovotestis gland of cornu aspersum (o. f. müller, 1774) after exposure 3 weeks: (a) first week; (b) second week; and (c) third week. values expressed as mean ±sd. c the significant difference in necrosis between control and exposed snails when p ≤ 0.03. the significant difference in apoptosis was indicated as follows: a,b between the moringa exposed group vs. control and methomyl exposed groups when p ≤ 0.05, anova. highlights in bioscience page 7 of 10 august 2025|volume 8 https://doi.org/10.36462/h.biosci.202506 https://doi.org/10.36462/h.biosci.202506 http://bioscience.highlightsin.org/ abdelazeem hh et al., 2025 apoptotic and 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peroxidation and apoptosis. prostaglandins leukot essent fatty acids. 1999;61(3):157-63. 53. fischer u, jänicke r, schulze-osthoff k. many cuts to ruin: a comprehensive update of caspase substrates. cell death & differentiation. 2003;10:76. 54. pirger z, rácz b, kiss t. dopamineinduced programmed cell death in snail salivary gland cells. cell biology. 2009;101:105-16. 55. nowakowska a, rogalska j, caputa m. adaptability of antioxidant defence system in helix pomatia: effect of forced aestivation. journal of molluscan studies. 2016;82(1):205-7. 56. balusamy sr, perumalsamy h, ranjan a, park s, ramani sa. dietary moringa oleifera leaves induce fat cell apoptosis in 3t3-l1 adipocytes. journal of functional foods. 2019;59:251-60. 57. adebayo ia, arsad h, samian mr. the inhibitory role of metabolites of moringa oleifera seeds in cancer cells. in: phytomedicine: pharmacologically active products from plants; 2021. p. 533-54. 58. rocha-filho caa, albuquerque lpa, silva lrs, silva pcb, coelho lc, navarro dm, et al. assessment of toxicity of moringa oleifera flower extract to biomphalaria glabrata, schistosoma mansoni and artemia salina. chemosphere. 2015;132:188-92. 59. mei s, ni hm, manley s, bockus a, kasse km, luyendyk jp, et al. differential roles of unsaturated and saturated fatty acids on autophagy and apoptosis in hepatocytes. aspet. 2011;339(2):487-98. 60. rocha-filhoa caa, albuquerque lpa, silva lrs, silva pcb, coelho lc, navarro dm, et al. assessment of toxicity of moringa oleifera flower extract to biomphalaria glabrata, schistosoma mansoni and artemia salina. chemosphere. 2015;132:188-92. 61. yousef aaa, el-kassas ne. ultrastructure and histopathological effects of some plant extracts on digestive gland of biomphalaria alexandrina and bulinus truncatus. jobaz. 2013;66:27-33. 62. ibrahim am, abdalla am. impact of moringa oleifera seed aqueous extract on some biological, biochemical, and histological aspects of biomphalaria alexandrina snails. environ sci pollut res. 2017. 63. abdel-tawab h, ibrahim am, hussein t, mohamed f. mechanism of action and toxicological evaluation of engineered layered double hydroxide nanomaterials in biomphalaria alexandrina snails. environ sci pollut res int. 2022;29. 64. ibrahim am, bakry fa. assessment of the molluscicidal impact of extracted chlorophyllin on some biochemical parameters in the nervous tissue and histological changes in biomphalaria alexandrina and lymnaea natalensis snails. invertebr neurosci. 2019;19. 65. dokmak haas, el-emam ma, mossalem hs, et al. impact of the photosensitizers copper and magnesium chlorophyllin on biological and biochemical parameters of bulinus truncatus snail. egypt j aquat biol fish. 2021;25:525-40. 66. hamlet sa, bensoltane s, djekoun m, yassi f, berrebbah h. histological changes and biochemical parameters in the hepatopancreas of terrestrial gastropod cornu aspersum as biomarkers of neonicotinoid insecticide exposure. afr j biotechnol. 2012;11(96):16277-83. highlights in bioscience page 10 of 10 august 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods experimental materials determination of lc50 and lc90 collection of snails and experimental design cell cycle analysis assay of pi/annexin-v dual staining histological investigation statistical analysis results toxical effect of moringa oleifera seed oil the survival rate of cornu aspersum (o. f. müller, 1774) under the effect of moringa oil cell cycle distribution after moringa oil exposure detection of apoptosis by annexin-v/pi histopathological signs after exposure to moringa oil discussions effect of moringa oleifera seed oil on the survival rate of cornu aspersum (o. f. müller, 1774) apoptotic effect of m. oleifera seed oil on cornu aspersum histopathological effect of m. oleifera seed oil on cornu aspersum conclusions list of abbreviations supplementary highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202509 research article open access 1 department of aquatic animal health, faculty of fishery sciences, west bengal university of animal and fishery sciences, chakgaria, kolkata 700094, west bengal, india. 2 nitte-gok coe aquamarin, nucser, nitte (deemed to be university), paneer campus, deralakatte, mangalore 575018, karnataka, india. 3 state referral laboratory for aquatic animal health, tamil nadu dr j.jayalalithaa fisheries university, madhavaram milk colony, chennai 600051, tamil nadu, india. 4 aquatic animal health and environment division, icar-central institute of brackishwater aquaculture, raja annamalai puram, chennai 600028, tamil nadu, india. * to whom correspondence should be addressed: abrahamtj1@gmail.com editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), cairo, egypt. reviewer(s): hayder tawfeeq qaddoori, middle technical univarsity, technical insitute of baqubah, department of nursing, dayala iraq. abdulaziz ascandari, african genome center, mohammed vi polytechnic university, ben guerir, morocco. received: july 26, 2025 accepted: september 29, 2025 published: october 18, 2025 citation: sinha p, abraham tj, sen a, sharon j, das r, rajeshwar bn, uma a, patil pk. influence of in-feed oxolinic acid therapy on the brain histoarchitecture and virulence factors of streptococcus agalactiae infecting nile tilapia oreochromis niloticus . 2025 oct. 18;8:bs202509 copyright: © 2025 sinha p et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. influence of in-feed oxolinic acid therapy on the brain histoarchitecture and virulence factors of streptococcus agalactiae infecting nile tilapia oreochromis niloticus priyanka sinha1 >< �, thangapalam jawahar abraham*1 ><�, arya sen1 >< �, joshi sharon1 >< �, ratnapriya das1 >< �, b. naveen rajeshwar2 ><�, arumugam uma3 ><�, prasanna kumar patil4 >< � abstract streptococcosis and its management are major constraints to tilapia aquaculture. the present study assessed the effect of oral administration of 12 mg oxolinic acid (oa)/kg fish/day for 7 uninterrupted days against streptococcus agalactiae lcr1 (sa) infection and its influence on histopathological anomalies in the forebrain, optic tectum region of the mid-brain and granular cell layer of the cerebellum of oreochromis niloticus. besides, attempts were made to understand how the oa impacts the glycosyltransferases and camp factor of s. agalactiae through molecular docking. the ld50 of sa was 1.26 × 108 cells/fish. sa infection was apparent in oa-treated and untreated groups, and the brain tissues exhibited the progression and reversal of meningitis. the forebrain exhibited thickening of the meninx primitiva, vacuolation, and degeneration in the brain parenchyma and meninx primitiva. inflammatory changes such as meningitis and mononuclear cell infiltration were documented. the midbrain had edematous optic tectum, loosening of connective tissue, and leukocyte infiltration. in the granular cell layer, cerebellum changes such as spongiform encephalopathy and necrosis indicate region-specific damage. however, the oa effectively reduced the severity of brain tissue damage, possibly by its binding affinities and upsetting the activities of glycosyltransferases and camp factor, as confirmed by molecular docking. these results confirmed that oa can cross the blood-brain barrier and interact with virulence proteins to reduce the sa infection in the brain. furthermore, the results underscore its responsible use in aquaculture, as oa is a critically important human medicine and ought to be used as a secondary treatment. keywords: aquaculture, streptococcosis, virulence proteins, antibiotic therapy, meningitis, histoarchitecture. introduction aquaculture is swiftly intensifying, significantly contributing to global food production, economic development, and nutritional security. in 2022, aquaculture contributed about 59% of the total fish production of 185.4 million tonnes. cichlids, including tilapias, ranked third in the major farmed fish group with a contribution of 10.6% [1]. tilapias are highly favourable species for aquaculture due to their adaptability, rapid growth, disease resistance, and low production costs [2]. however, intensive tilapia aquaculture has led to a rise in diseases, particularly bacterial infections like streptococcosis, motile aeromonas septicemia (mas), vibriosis, and columnaris, which pose significant challenges to farming [3]. streptococcosis, caused by streptococcus agalactiae (sa) and s. iniae, is one of the most important bacterial diseases in tilapia aquaculture, causing substantial economic losses globally [2; 4]. it can lead to meningoencephalitis in tilapia, characterised by several neurological disorders and brain lesions [5; 6; 7; 8]. studies indicated that sa causes extensive brain damage, particularly in regions controlling swimming activities, due to bacterial invasion and acute inflammatory responses [6; 8; 9]. control measures for streptococcosis include proper farm location selection, good aquaculture practices, antibiotics, immunostimulants, and vaccines [2]. antimicrobials like amphenicols, quinolones, and tetracyclines are widely used, but their use raises highlights in bioscience page 1 of 10 october 2025|volume 8 https://doi.org/10.36462/h.biosci.202509 https://creativecommons.org/licenses/by/4.0/ mailto:sinhapriyanka1197@gmail.com https://orcid.org/0009-0005-1867-5422 mailto:abrahamtj1@gmail.com https://orcid.org/0000-0003-0581-1307 mailto:arya.sen2@gmail.com https://orcid.org/0000-0001-8991-7875 mailto:ashjosh9@gmail.com https://orcid.org/0009-0009-6799-9473 mailto:ratnapriyadas2015@gmail.com https://orcid.org/0000-0002-6414-7840 mailto:naveen.rajeshwar@nitte.edu.in https://orcid.org/0000-0002-4496-9492 mailto:uma@tnfu.ac.in https://orcid.org/0000-0002-9429-4075 mailto:pk.patil@icar.org.in https://orcid.org/0000-0002-1924-5863 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy figure 1. the histopathological changes indicating meningitis (*) in the forebrain of streptococcus agalactiae lcr1 challenged and oxolinic acid (oa) treated and untreated oreochromis niloticus juveniles. [a] control, [b] day post-injection (dpi) 1 oa treated and [c] untreated; [d] dpi 7 oa treated and [e] untreated; [f] dpi 14 oa treated and [g] untreated; and [h] dpi 21 oa treated and [i] untreated. dmp: damage of meninx primitiva; fh: focal haemorrhage; imc: infiltration of mononuclear cells; ls: lifting of superficial layer; lse: localized spongiform encephalopathy; mp: meninx primitiva (meningitis); tmp: thickening of meninx primitiva; dbp: degeneration of brain parenchyma; and v: vacuolation x200, h&e staining. concerns about antimicrobial resistance (amr), particularly in regions like china and india [10; 11; 12; 13]. the quinolone antibiotic, oxolinic acid (oa) is not on the list of approved drugs for use in finfish production by the food and drug administration in the united states [14]. it develops resistant bacterial strains that may threaten human health [10; 15]. however, the european union, japan, and other asian countries have recommended it to prevent bacterial infections at 12 mg/kg fish/day for 7 days [11; 16]. the antibiotics of the quinolone family are categorized as critically important and the highest priority antimicrobial group for human use. such medicines should not be used to treat infectious diseases in food-producing animals. yet, these medicines are proposed for a second-line treatment in food-producing animals, complying with the national legislation in force, when no other alternatives are available [15; 17; 18]. several studies reported the effectiveness of oa in controlling fish bacterial pathogens and the onset of diseases [19; 20; 21; 22]. however, more research is needed to evaluate its mode of action, safety in tropical fish species, and efficacy against several mesophilic fish pathogens to ensure sustainable aquaculture pracfigure 2. the histopathological changes in the optic tectum (ot) region of the brain of streptococcus agalactiae lcr1 challenged and oxolinic acid (oa) treated and untreated oreochromis niloticus juveniles. [a] control, [b] day post-injection (dpi) 1 oa treated and [c] untreated; [d] dpi 7 oa treated and [e] untreated; [f] dpi 14 oa treated and [g] untreated; and [h] dpi 21 oa treated and [i] untreated. eot: edematous optic tectum; il: infiltration of leucocytes; imc: infiltration of mononuclear cells; ldc: loosened dense connective tissue; ls: lifting of superficial layer; lse: localized spongiform encephalopathy; mp: meninx primitiva; slc: spacing in loose connective tissue; tmp: thickening of meninx primitiva; and v: vacuolation x200, h&e staining. tices [23]. the infection of sa relies on several virulence factors, which encompass adhesion, invasion, and infection. numerous virulence factors contribute to invasion and colonisation [24; 25]. the relationship between antibiotics, virulence proteins, and the ability of bacteria to infect the brain is complex, involving factors like the blood-brain barrier (bbb) and host immune responses. for example, glycosyltransferases and the camp factor play crucial roles in pathogenesis, as highly pathogenic strains of sa reportedly produce glycosylated serine-rich repeats and glycosyltransferases [26; 27]. the extracellular protein, such as camp factor, induces pore formation in target cells and heightens the pathogenicity of sa [24; 25; 28]. however, there is a lack of scientific literature focusing on the specific effects of aquaculture antibiotics on brain-infecting bacteria, such as sa, and their virulence proteins. also, the misuse of antibiotics can lead to the emergence of amr, which is a significant public health concern [10; 15]. therefore, to combat sa infections, understanding the roles of important virulent proteins and their interaction with antibiotics is essential. this study delved into the efficacy of oa, an antibiotic recommended for second-line treatment in food-producing animals [18], to curtail the effect of sa-infection on the brain histoarchitecture of nile tilapia oreochromis niloticus, and to comprehend how oa impacts the invasion of sa into the fish brain by molecular docking highlights in bioscience page 2 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy using glycosyltransferases and camp factor, to plan for further course of research and specific mitigation strategies. materials and methods experimental fish and bacterial strain and its pathogenicity farm-raised young and healthy oreochromis niloticus were chosen regardless of sex and adapted in circular tanks of 500 l capacity for 15 days before the experimentation [23]. streptococcus agalactiae lcr1 (national centre for biotechnology information (ncbi) accession number op752129) was obtained from the state referral laboratory for aquatic animal health, madhavaram, chennai, india. following earlier descriptions, the bacterium was assessed for its pathogenicity against o. niloticus (n = 10 for each challenge dose from 105 to 109 and control, in triplicate) by intramuscular challenge [29; 30]. the mean lethal dose (ld50) was calculated from the mortality data using the method established by reed and muench [31]. using the broth dilution method, the minimum inhibitory concentration (mic) of oa was calculated [32]. before the challenge, inocula from the kidney of healthy tilapia (n = 2) were plated onto brain heart infusion agar (bhia) to ascertain that the stocks were disease-free [5]. efficacy of oxolinic acid against streptococcus agalactiae infection the european medicines evaluation agency (emea) recommended a dose of 12 mg oa/kg fish/day for 7 uninterrupted days to control bacterial infection in fish [16]. the medicated feed to feed the experimental fish at 2% body weight (bw) was set by emulsifying 0.6 g of oa powder (o0877-25g; cas-no: 14698-29-4; sigma-aldrich, india) in 5 ml vegetable oil. this emulsion was then top-coated onto the commercial floating pellet feed (1 kg) and admixed thoroughly. correspondingly, a control feed with binder but without oa was prepared [21]. the analysis of oa in medicated feed by liquid chromatography-tandem mass spectrometry (lc-ms/ms) revealed 96±1% of the incorporated dose [23]. the efficacy study utilized o. niloticus weighing 17.56 ± 0.89 g (n = 225) and distributed randomly in nine thoroughly cleaned polypropylene tanks (l58 × h45 × b45 cm). the rearing water (80 l) in each tank was conditioned for three days, stocked with 25 accustomed tilapias, and properly covered and marked. about half of the water was exchanged to remove feed and faecal wastes. the fish stocks were divided into three groups, in triplicate. the control, designated as group 1, received a 0.1 ml saline injection intramuscularly at the dorsal fin base. all fish of groups 2 and 3 were injected intramuscularly with s. agalactiae lcr1 at 1.32 × 107 cells/fish. post-injection, the fish were placed in their respective tanks. control feed was offered to groups 1 and 3 throughout the experimental period of 21 days. oa-medicated feed at 2% bw was served to group 2 continuously for 7 days post-injection (dpi) and then switched to a control feed [21; 23]. water quality was continuously monitored, and any leftover feed, if any, was removed 1 hour after each feeding. observations on mortality, infection symptoms, and behaviour were recorded daily. inocula from the freshly dead figure 3. the histopathological changes in the granular cell layer of the cerebellum (gic) of the brain of streptococcus agalactiae lcr1 challenged and oxolinic acid (oa) treated and untreated oreochromis niloticus juveniles. [a] control, [b] day post-injection (dpi) 1 oa treated and [c] untreated; [d] dpi 7 oa treated and [e] untreated; [f] dpi 14 oa treated and [g] untreated; and [h] dpi 21 oa treated and [i] untreated. lse: localized spongiform encephalopathy; v: vacuolation; lgc: lysis of granular cells of the cerebellum; and n: necrotised area x200, h&e staining. sa-challenged tilapia brain were plated onto bhia to establish sa-infection [5]. histopathology and evaluation of pathological anomalies on 0, 1, 7, 14, and 21 dpi, two fish were randomly sampled from each replicate tank, euthanized by adding clove oil at 100 µl/l, and dissected prudently. the cranial bone was removed carefully to expose the brain. the precisely collected wholebrain tissue samples were fixed for 24 hours in bouin’s fixative, processed, and embedded in paraffin wax. sections of 5 µm thickness were sliced, processed, and double-stained using hematoxylin and eosin [33]. the brain sections were examined under an olympus microscope (model: bx51) at 20× magnification to identify abnormalities in tissue arrangement. for quantitative analysis, images were captured and processed using an scolux camera (16 mp) and touptek toupview software (version x64). the histopathological abnormalities were evaluated based on circulatory, progressive, regressive, and inflammatory changes. following the observations, reaction indices (ri) were calculated as a product of the score value and the importance factor. the score value, ranging from 0 to 6, was assigned based on the severity and degree of damage: no change (0), mild occurrence (2), moderate occurrence (4), and severe occurrence (6). the importance factor, ranging from 1 to 3, was assigned based on the pathological significance of each alteration, categorized 1: minimal pathological importance, easily reversible post-stressor highlights in bioscience page 3 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy figure 4. homology modelled 3d structures [a1 and b1] and ramachandran plots [a2 and b2] of glycosyltransferase (aam99596) and camp factor (aki96324) of streptococcus agalactiae, respectively, computed using swissmodel. administration, 2: moderate pathological importance, reversible if the stressor is neutralized, 3: marked pathological importance, generally irreversible with potential loss of organ function. for instance, for mild vacuolation, which has a pathological importance factor of 2, and an observed severity score of 1.65 depending on damage intensity, the ri would be 3.30 (i.e., 1.65 × 2). alterations with a higher ri value signified greater severity [34]. the qualitative scores of the brain histological changes, expressed as mean ± standard deviation of six observations, were analysed by a non-parametric kruskal–wallis test. the mann–whitney u test was conducted for pairwise comparisons in ibm-spss version 22.0 at a significance level of p < 0.05. in silico and molecular docking of streptococcus agalactiae virulence factors protein and ligand modelling for docking studies the virulence proteins of s. agalactiae, such as the glycosyltransferase enzyme encoded by the iaga gene involved in bbb invasion, and an invasin protein, camp factor [24; 25; 27; 28], were retrieved from the ncbi database for further detailed analysis and understanding. the corresponding fasta protein sequences were subsequently subjected to homology modelling using the expasy swiss-model server. oxolinic acid (oa) was considered the ligand, and its 3d structure was obtained from the pubchem database in .sdf format and then conveniently converted to .pdb format using open babel software. molecular docking the molecular docking and interactions between oa and the target virulence proteins of s. agalactiae were analysed using mgl tools and autodock tools 1.5.7 [35], employing a grid box size of 126 å in the x, y, and z dimensions, and the lamarckian genetic algorithm for docking simulations. the docking results and molecular interactions were visualised using pymol edu version 2.5. the docking score, representing the interaction energy with appropriate scaling factors, was used to assess the binding affinity of the ligand. furthermore, specific amino acid interactions between the ligand and target proteins were computed and outlined. lower binding free energy may indicate stronger ligand-protein interaction, while hydrogen bond strength may categorise donoracceptor distances as strong (2.2–2.5 å; mostly covalent), moderate (2.5–3.2 å; mostly electrostatic), and weak (3.2–4.0 å; electrostatic), as per jeffrey [36]. results brain histopathology and evaluation of pathological anomalies the ld50 of s. agalactiae and the mic of oxolinic acid (oa) were 1.26 × 108 cells/fish and 12.50 µg/ml, respectively. in the forebrain of the control fish, the tissue structure was normal, with a well-organized epithelial layer, the meninx primitiva, and mononuclear cells present in the brain parenchyma (figure 1 a). the histopathological analyses of oa-treated and untreated s. agalactiae challenged o. niloticus revealed three types of reaction patterns: progressive, regressive, and inflammatory (figure 1 b–i; table 1). the identified progressive change was thickening of the meninx primitiva, while regressive changes included superficial layer lifting, localized spongiform encephalopathy, vacuolation, and degeneration of the meninx primitiva and brain parenchyma. inflammatory changes were characterized by meningitis and mononuclear cell infiltration. on dpi 1, both groups displayed mild progressive, regressive, and inflammatory changes, including meninx primitiva thickening with high ri values. the damage, particularly regressive and inflammatory changes, intensified on dpi 7. the oa-treated group showed reduced regressive changes and evidence of healing in the brain parenchyma, whereas the untreated group experienced significant damage on dpi 14. by dpi 21, the oa-treated group was nearly healed except for mild meningitis, while the untreated group continued to exhibit moderate brain damage. in the midbrain, the normal structure consisted of an outer meninx primitiva and underlying connective tissue above the optic tectum (figure 2 a). the histopathological changes included circulatory alterations such as edematous optic tectum, regressive changes like superficial layer lifting, loosening of connective tissue, and vacuolation, as well as inflammatory changes such as mononuclear cell infiltration (figure 2 b–i; table 1). on dpi 1, both groups exhibited mild circulatory, inflammatory, and regressive changes. on dpi 7, circulatory damage peaked in the oatreated group, while regressive changes worsened in the untreated group. a gradual reduction in damage over time was noted in the highlights in bioscience page 4 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy table 1. reaction indices* based on the major histopathological changes in the brain of streptococcus agalactiae lcr1 challenged and oxolinic acid (oa)-fed oreochromis niloticus juveniles. reaction pattern histopathological changes if dpi 1 dpi 7 dpi 14 dpi 21 oa treated untreated oa treated untreated oa treated untreated oa treated untreated forebrain progressive thickening of mp 1 0.80±0.171ab 1.02±0.551ab 1.06±0.271a 1.12±0.211a 0.57±0.251b 0.72±0.301 a b 0.27±0.121c 0.57±0.102 b regressive lifting of sl 1 0.52±0.451ab 1.07±0.531a 0.62±0.241a 0.98±0.371a 0.25±0.121ab 1.05±0.432 a 0.23±0.051b 0.47±0.192 b lse 2 2.10±1.181ab 1.67±1.541a 2.60±0.491a 3.80±1.211 b 1.07±0.211bc 1.63±0.392 a 0.83±0.461c 1.27±0.391a vacuolation 2 1.93±0.641ab 1.13±0.631a 1.93±0.651a 3.47±1.042 b 1.03±0.591ab 1.73±0.621 a 0.67±0.331b 1.33±0.552a damaged mp 3 3.05±1.261ab 2.95±1.221a b 3.05±0.481a 5.55±2.541a 1.70±0.921ab 3.55±1.721 a b 1.00±0.491b 2.20±0.622 b degeneration of bp 3 3.00±1.001ab 1.90±1.381a 3.00±0.801b 5.70±1.612 b 1.05±1.321a 3.15±0.732c 0.75±0.371a 1.90±0.822a inflammatory meningitis 2 3.57±0.411a 1.70±1.271a 3.57±1.241b 3.90±1.051 b 1.13±0.391a 2.03±0.961 a 0.70±0.351a 1.43±0.372a infiltration of mnc 2 2.17±0.351a 1.23±0.731a 2.17±0.601b 3.10±1.161 b 0.67±0.431c 1.50±0.901 a 0.43±0.391c 1.47±0.352a mid-brain (optic tectum) circulatory edematous ot 1 0.70±0.171a 0.72±0.171a 0.73±0.211a 1.42±0.182 b 0.73±0.161a 1.00±0.241c 0.37±0.141b 0.55±0.181a regressive lifting of sl 1 0.80±0.131a 0.70±0.231a 0.68±0.171a 1.50±0.372 b 0.43±0.231b 1.02±0.282c 0.38±0.121b 0.75±0.102a loosening of dct 1 0.83±0.081a 0.72±0.121a 0.67±0.151a 1.18±0.332 b 0.75±0.211a 1.10±0.321 a b 0.47±0.101b 0.72±0.182a spacing of lct 1 0.85±0.211a 0.85±0.141a 0.73±0.261ab 1.43±0.592 b 0.65±0.141ab 1.15±0.342 b 0.47±0.181b 0.83±0.182a vacuolation 2 1.30±0.581a 1.60±0.281a 1.50±0.451a 3.13±0.702 b 1.30±0.451a 2.50±1.062a b 0.97±0.231a 1.13±0.241c inflammatory infiltration of lc 2 1.73±0.241a 1.63±0.291ab 1.27±0.451ab 1.30±0.681a b 0.83±0.611b 1.73±0.332 a 0.57±0.151b 1.03±0.232 b cerebellum (granular cell layer) regressive lse 2 2.57±0.231a 2.40±0.181a 2.20±0.331a 4.20±0.332 b 0.47±0.101b 1.93±0.532 a 0.90±0.281c 1.30±0.282c lysis of gc 3 3.75±0.311a 3.70±0.241a 2.30±0.591b 5.25±0.682 b 0.65±0.121c 3.10±1.212 a 1.25±0.351d 1.80±0.661c necrotized area 3 5.45±0.581a 5.30±0.411a 1.85±0.581b 5.45±0.962a 1.35±0.161b 3.25±1.362 b 1.35±0.311b 1.80±0.421c * as per the descriptions of bernet et al. (1999). no changes were noted in the control group. 1-2: values sharing a common numerical superscript for a specific row among the treatment groups for a specific day differed insignificantly (p>0.05). a-d: values sharing a common alphabetical superscript within a row for the treated group on different dpi differed insignificantly (p>0.05). a-c: values sharing a common alphabetical superscript within a row for the untreated group on different dpi differed insignificantly (p>0.05). dpi: day post-injection. if: importance factor; mp: meninx primitiva; sl: superficial layer; lse: localized spongiform encephalopathy; bp: brain parenchyma; mnc: mononuclear cells; ot: optic tectum; dct: loosened dense connective tissue; lct: loose connective tissue; lc: leucocytes; gc: granular cells of the cerebellum. oa-treated group, whereas the untreated group presented more severe and persistent damage. by dpi 21, both groups showed decreasing damage, with near-normalization of architecture in the oa-treated group. in the granular cell layer of the cerebellum, both groups experienced regressive changes, including spongiform encephalopathy with vacuolation and necrosis, which decreased with time. on dpi 21, the treated group displayed minimal vacuolation, while the untreated group showed moderate damage (figure 3 b–i; table 1). overall, the oa-treated group exhibited less damage, improved recovery, and reduced mortalities (26%; p > 0.05) compared to the untreated group (32%). homology modelling and molecular docking using the swiss-model server, three-dimensional (3d) models of glycosyltransferase and camp factor from s. agalactiae were constructed based on sequence similarity with identified structural templates. it is generally important to note, in a rather typical and somewhat broadly consistent and commonly recognised manner, that this modelling approach essentially follows a broadly conventional and widely accepted procedure overall. the models generated from templates with the highest sequence identity are presented in (figure 4 a1, b1). this approach, in many ways, essentially leverages the inherent structural similarity among proteins of the same family, which usually tend to share broadly comparable and functionally related three-dimensional architectures. the outcomes of the homology modelling are, more or less, conveniently summarised in (table 2). the global model quality estimation (gmqe) and qualitative model energy analysis (qmean) scores were high, corresponding to higher sequence identity with templates. the generated models exhibited optimal quality, as validated by swiss-model, with molprobity scores above 0.66, and were considered excellent. nearly 98% of residues were located in the favoured regions of the ramachandran plot (figure 4 a2, b2). the ligand oa exhibited notable binding affinities toward both glycosyltransferase and camp factor, with docking scores of –4.99 and –4.31 kcal/mol, respectively. these interactions were supported, in a fairly straightforward and somewhat broadly descriptive and methodologically consistent manner, by multiple hydrogen bonds and close-contact residues, suggesting generally stable and favourable binding conformations (table 3; figure 5 a, b). highlights in bioscience page 5 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy table 2. homology modelling data of glycosyltransferase aam99596 and camp factor aki96324 of streptococcus agalactiae computed using swiss model. characteristics glycosyltransferase aam99596 camp factor aki96324 template id a0a4v0ha53.1.a 5h6i.1.a sequence identity (%) 71.69 100 qmean 0.90 0.86 gmqe 0.96 0.80 molprobity score 0.91 0.70 ramachandran plot – favoured region (%) 96.97 98.12 ramachandran plot – outlier region (%) 0.00 0.00 qmean: qualitative model energy analysis; gmqe: global model quality estimation. discussions in terms of virulence, the sa strain, isolated from the kidney of asian seabass lates calcarifer, was borderline avirulent to weakly virulent [7; 37; 38] with a moderately high mic for oa. sa affects the brain using several virulence factors to allow the infection process [4; 38; 39]. in this study, sa resulted in systemic pathological alterations, including inflammation, haemorrhages, and meningitis in challenged fish, as in previous research [5; 6; 7; 8; 9]. the sa-infected fish also exhibited aberrant swimming, marked by circular movement and c-shaped body bending. it could be due to the possible neurological problem, as the midbrain reportedly controls fish swimming by transmitting axons to innervate the spinal cord’s primary and secondary motor neurons [9; 40]. further, abnormal swimming behaviours due to streptococcal infections were linked to brain fluid accumulation [7]. in this work, we isolated sa from the brain tissues of challenged tilapias on bhia, indicating a breach of the bbb. sa employ various strategies to survive in the bloodstream, colonize brain vasculature, and cross the bbb [41]. the results indicated that the sa can enter the brain parenchyma by destroying the host bbb and causing localized spongiform encephalopathy. several earlier studies confirmed the establishment of sa [6; 8; 42] or biofilm formation [43] in fish brains. when the bacteria enter the circulatory system, they survive within macrophages, allowing them to evade the immune system and spread to the central nervous system (cns) [8; 44]. such macrophages reportedly act as a vehicle for sa, allowing it to cross the bbb and gain access to the cns [6; 8]. the current investigation revealed progressive alterations such as thickening of the meninx primitiva, suggesting bacterial meningitis similar to earlier studies [5; 6; 7; 9]. regressive changes in the forebrain, midbrain, and cerebellum of the current study were in line with the earlier findings. they reported that bacteria can permeate brain parenchymal regions and cause localised spongiform encephalopathy, characterised by focal haemorrhages and vacuolization, mostly in the midbrain and hindbrain. also, gram-positive cocci in the granular cell layer were observed, causing lysis of granular cells, and brain lesions, includfigure 5. protein–ligand interaction of streptococcus agalactiae virulence factors: [a] glycosyltransferase and [b] camp factor against oxolinic acid, visualised using the pymol tool. note: green structures represent ligands; coloured ribbons depict the target protein; yellow dotted lines indicate interaction sites. distances between the ligand and target at the interaction sites are measured in angstroms (å). ing haemorrhages, neuronal necrosis, and inflammation in regions governing swimming in tilapia [9]. sa infection also disrupted cerebral bioenergetics by impairing mitochondrial complex iv and creatine kinase activities, leading to energy imbalance [45]. all these investigations pointed out that sa is neurotropic, seriously harming neurological function and influencing swimming behaviour and survival of fish. the inflammatory responses in the forebrain and mid-brain of sa-infected tilapias corroborated earlier studies [6; 7; 8; 9; 46], which documented meningitis and several significant brain alterations. sa reportedly triggers an inflammatory response, which impairs phagocytosis, cytokine and chemokine levels, contributing to oxidative stress and disruption of the bbb [45; 47] and interfering with the immune system [48]. the resulting neuroinflammation can be characterized by immune cell migration and the production of pro-inflammatory cytokines, which drive the immune response [6]. antibiotics like ampicillin, colistin sulfate, sulfadimethoxineormetoprim, and florfenicol are effectively used against streptococcus spp. moreover, the use of β-lactams, fluoroquinolones and macrolides has been highlighted as effective against meningitiscausing s. pneumoniae [49; 50; 51; 46; 52]. in this study, the ri values increased during the initial period of infection and highlights in bioscience page 6 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ sinha p et al., 2025 effect of oxolinic acid therapy table 3. molecular docking summary of glycosyltransferase aam99596 and camp factor aki96324 of streptococcus agalactiae against the ligand oxolinic acid using the autodock tool. proteins affinity ligand–protein interaction distance (å) glycosyltransferase -4.99 a:asn1̀97:od1–:unl1̀:h 2.3 a:lys1̀93:nz–:unl1̀:o 2.7 a:asn1̀97:od1–:unl1̀:o 3.0 a:lys1̀93:nz–:unl1̀:o 3.2 camp factor -4.31 a:lys2̀05:nz–:unl1̀:o 2.9 a:arg2̀04:n–:unl1̀:o 3.1 a:arg2̀04:nh1–:unl1̀:o 3.2 gradually reduced with time, indicating the fish’s adaptive and immune responses to healing the brain lesions. while the effects were almost similar, the oa-treated tilapia showed lower ri. the oa-treatment facilitated quicker recovery, indicating that oa can cross the bbb and lessen the sa-infection. the glycosyltransferase and the camp factor play crucial roles in the pathogenesis of sa. the infection may influence bacterial interactions with the host, create specific pores in host membranes, modify proteins and carbohydrates, and disrupt cell integrity, leading to cell lysis [24; 53]. the lesser impact of sa-infection in the brain suggested that the oa can exhibit binding affinities with virulent proteins, as confirmed by interactions with glycosyltransferases and the camp factor, and to prevent the activities of sa. in the molecular docking study, our observations on the shorter bond distances and number of interactions with multiple hydrogen bonds and close-contact residues suggested stable and favourable binding conformations. these results supported the potential of oa as a strong inhibitor of the virulent proteins of sa. our study further noted an insignificant difference in mortalities due to the tested strains moderately high mic for oa. strikingly, the 7 days of treatment at 12 mg oa/kg fish/day inhibited the sa strain, mitigated the severity of brain damage, and improved fish survival. thus, these biologically meaningful results proved the protective effect of oa against the progression of sa-infection and ensuing brain histopathological changes. however, future studies are warranted on the expression of glycosyltransferase, camp factor and other virulence factors using qrt-pcr or western blotting to provide direct evidence of their involvement in sa pathogenesis and oas mechanism of action. conclusions our results demonstrated that understanding the role of virulence proteins of s. agalactiae in pathogenesis and interactions with drugs can offer scope for new therapeutic strategies, such as targeting such factors or blocking interactions with host cells and developing targeted therapies that can disrupt the bacterial toxin’s activity. in this study, oxolinic acid (oa) therapy in s. agalactiae-infected tilapia revealed a protective effect by reducing the mortalities, brain damage and histopathological alterations. the treated fish unveiled less damage in the forebrain, midbrain, and cerebellum, with tissue architecture almost normalized on dpi 21. the results implied that oa is efficacious and can be used in treating tilapia against s. agalactiae infection. however, the world health organisation stated that quinolones, including oa, are critically important human medicines, and they should at no time be used in animals produced for food as a primary treatment agent. based on the results, we advocate the use of oa as a therapeutic agent for second-line treatment in food-producing animals following the national and international criteria. yet, a prudent and responsible use of antibiotics, targeted therapeutic applications for treating s. agalactiae-induced meningitis in fish and implementing long-term amr and residue surveillance to safeguard public health are obligatory. data availability statement all relevant data are within the paper and supplementary materials. funding the work was supported by the indian council of agricultural research, government of india, new delhi, under the all-india network project on fish health vide grant f. no. ciba/ainpfh/2015-16 dated 02 june 2015. competing interests the authors declare that they have no competing interests. reference 1. fao. the state of world fisheries and aquaculture 2024 blue transformation in action. rome: food and agriculture organization of the united nations; 2024. available from: https://doi.org/10.4060/cd0683en. 2. el-sayed afm. tilapia culture. 2nd ed. london: academic press; 2019. available from: https://doi.org/10. 1079/9780851990149.0000. 3. haenen ol, dong ht, hoai td, crumlish m, karunasagar i, barkham t, et al. bacterial diseases of tilapia, their zoonotic potential and risk of antimicrobial resistance. reviews in aquaculture. 2023;15:154-85. available from: https:// doi.org/10.1111/raq.12743. 4. zhang z. research advances on tilapia streptococcosis. pathogens. 2021;10(5):550-8. 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lymbery aj, et al. streptococcosis: a re-emerging disease in aquaculture: significance and phytotherapy. animals. 2022;12:2443. 49. ghetas h, neiana a, khalil r, khallaf m. streptococcus agalactiae isolation and characterization in nile tilapia (oreochromis niloticus) with histopathological studies. journal of current veterinary research. 2021;3(1):70-9. 50. vinarukwong n, lukkana m, berntsen jo, wongtavatchai j. therapeutic use of sulfadimethoxine-ormetoprim for control of streptococcus agalactiae infection in nile tilapia (oreochromis niloticus) fry. thai journal of veterinary medicine. 2018;48(3):367-73. 51. bowker jd, ostland d, bowman mp. effectiveness of aquaflor (50% florfenicol) to control mortality associated with streptococcus iniae in freshwater-reared subadult sunshine bass. journal of aquatic animal health. 2010;22(4):254-65. 52. zahari nin, rahman ea, irekeola aa, ahmed n, rabaan aa, alotaibi j, et al. a review of the resistance mechanisms for -lactams, macrolides and fluoroquinolones among streptococcus pneumoniae. medicina. 2023;59(11):1927. 53. hensler me, quach d, hsieh cj, doran ks, nizet v. camp factor is not essential for systemic virulence of group b streptococcus. microbial pathogenesis. 2008;44(1):84-8. highlights in bioscience page 10 of 10 october 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods experimental fish and bacterial strain and its pathogenicity efficacy of oxolinic acid against streptococcus agalactiae infection histopathology and evaluation of pathological anomalies in silico and molecular docking of streptococcus agalactiae virulence factors results brain histopathology and evaluation of pathological anomalies homology modelling and molecular docking discussions conclusions data availability statement funding competing interests highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202105 review article open access 1 department of bioinformatics, colors medical laboratories, cairo, egypt. contacts of authors * to whom correspondence should be addressed: p.habib911@gmail.com received: january 01, 2021 accepted: march 6, 2021 published: march 15, 2021 citation: habib pt . covid-19 symphony: a review of possible music therapy effect in supporting the immune system of covid-19 patient. 2021 mar 15;4:bs202105 copyright: © 2021 habib. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. covid-19 symphony: a review of possible music therapy effect in supporting the immune system of covid-19 patient peter t. habib*1 abstract coronavirus, the current pandemic that takes lives of 1,936,632 until the time of the paper. scientists over the world exhibited all efforts to fight the virus. many supplements developed to fight coronavirus by supporting the immune system which is the strongest worrier against it for now. one of the neglected strategies to enhance immunity is music therapy. music therapy can drive immunity into different conditions. not only able to boost and suppress immunity but also many physiological and psychological diseases are treated with music therapy. it is more than just entertainment, and research shows that music therapy may be helpful and cost-effective to support standard therapy. this systemic review tries to explore the effect of music therapy and arrives at a conclusion suggesting routine procedures to support the patient with music therapy. however, the idea of using music therapy in improving the immune system must undergo clinical and medical confirmation to be used. keywords: psychology, music therapy, immune system, covid-19, coronavirus, pandemic. introduction the epidemic by a new coronavirus, named covid-19, initiated in late 2019 in wuhan, china, is a worldwide public health threat [13]. the virus likely evolved in bat, a zoonotic origin, after amino acid substitution in the spike glycoprotein, as recently suggested, acquired the ability to bind to ace2 receptor and be able to infect humans, which started the new epidemic [4]. currently, there is no efficient treatment against the new coronavirus. therefore, identifying previously known antiviral drugs to fight back is urgently needed. a successful drug research strategy is to check if the current antiviral medicines are efficient in the treatment of coronavirus infections. several drugs, such as ribavirin, ribavirin, chloroquine, and two well-studied broad-spectrum antiviral drugs remdesivir, and favipiravir have been used a long time ago with sars or mers patients, although the efficacy of some drugs remains controversial [5]. till now, the immune system is the only barrier against coronavirus. before existing of an official drug or vaccine for the coronavirus, many individuals recovered due to the immune system with the aid of some vitamins to support the immunity and medications to reduce the inflammation. although, there were some efforts to develop a vaccine [6] and discovering some potent molecules that may inhibit the virus attachment [7]. however, the official protocols aim to enhance immunity with medication. but the neglected fact that not only medication enhances immunity, but music therapy also does the same effect. backing to the 10 th bc century from western the starting from greeks to the eastern including india and china, history proves the curative and healing powers of music therapy. the ancient greeks are thought to have used music therapy to soothe the mentally disabled. western thinkers from pythagorus, aristotle, and plato to schopenhauer and nietzsche provided detailed of their interpretation of music’s therapeutic forces and their confidence in its practical application to offer peace and unity, and thereby to cure mental and physical illnesses [8]. aristotle, who has a strong background in psychotherapy, provides a theory that "after listening to music, those who suffered from uncontrollable feelings would return to their regular state, which brought their souls to happiness" [9,10]. there is also evidence from 4000 bc of using music therapy as a tool of stress relief and is estimated to stretch back as far as palaeolithic times [11]. highlights in bioscience page 1 of 7 march 2021|volume 4 https://doi.org/10.36462/h.biosci.202105 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-9178-352x http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient the investigation of music therapy has increasingly delved deeper into the mechanisms influencing music therapy interpretation and production, investigating music psychology [12]and music cognitive neuroscience, also referred to as ’neuro musicology [13] the physiological basis for music-induced emotions [14,15], the neurobiology of some elements of music such as harmony [16] and the neuroanatomy of music performance have been included in this profound study [17]. and the scope of research ranged from the interpretation inside the womb of folk songs [18] to the production of opera on concert platforms [19], to the use of mainstream music in working theatres [20]. music is an emotional language. it is known for improving mental and mood health. any change in emotions affects the hormonal, neurophysiological, and physiological processes; thus, changes in body balance caused by music indicate the efficacy of different musical types [21]. though still not well studied, several studies show that music therapy may promote a decrease in the stress response, a decrease in heart rate, improved tolerance to pain and suffering, change in skin behavior and muscle function, decreased anxiety, and depression. different studies have reported this effect in musically exposed stressed patients [22-24]. on the other hand, noise stress could cause adverse effects on immunity in both human and animal models [25,26]. several studies investigate different stress indicators, including blood pressure, pulse rate, and respiratory rate recorded measuring vital signs. these are the only anatomical measurements being used in nine of these. several research, including [27], used vital sign assessment as proof of a transition from sympathetic to parasympathetic processes, equating a shift in immune function with a wider response to stress. in many trials, calming music was found to decrease blood pressure, pulse rate, and respiration rate. no definitive improvement was observed in just four trials. one of the first music therapy studies reports music as therapy was in 1963 when melzack, weisz, and sprague report when evaluating the effect of the auditory stimulus on behavioral pain due to participants’ fingertips submerged in ice water slow gradually increased discomfort in the group listening to music relative to control group [28]. in 1966, two famous scientists, morosko and simmons tried to shock to the teeth of volunteers and found that music and white noise were effective in decreasing the pain sensation and increase tolerance [29]. in 1974, peretti and swenson used the galvanic skin response (gsr) to assess anxiety caused during a written maze test by negative responses to participants. in this case, music lowered gsr responses dramatically [30]. the effects of music on a specific physiological mechanism such as cardiac output, respiratory rate, and blood pressure were originally reported in many studies including such as in france in 1880, america in 1899, and russia in 1903 [31]. however, the immune system is one of the strongly affected systems in the body that can be manipulated indirectly with music. musically immune stimulation as mentioned, the immune system is strongly associated with mood, psychological condition, and hormonal balance. thus, as a result of negative mood, stress affects the immune system may cause dysregulation leads to a change in the humoral and cellular immunity and increases health risks [32]. bittman [33] presented proof that the drummer's team has improved nk cell response, lymphokine-activated killer cell function, and dehydroepiandrosterone cortisol balance in normal subjects, considered one of the early signs of the immune system-music interaction. wachi [34] found that delightful music modulates immunological responses in adults 65 years of age who are demarcated. important changes were reported in the number of lymphocytes, cd4 + t cells, t cells, and t memory cells as well as in the development of ifn-π and il-6. even music could reverse the immune stress induced by the immune suppressor. not only can affect the immune response but could lead to a reversion of the effect of the medication. music appeared to has a reversal effect as pharmacological interventions (i.e. benzodiazepines, 5 ht agonists) in immunosuppression induced by stress [35-37]. many studies state that music minimized stress-induced hyperactivity of the hypothalamus-pituitary-adrenal [hpa] axis, including acth and corticosteroid production [23,38-44], and modulate norepinephrine, epinephrine, gh, prl, and endorphin secretion [45]. this hormonal and neurotransmitter balance lowers stress and improving the condition of the immune system. other studies show that there a wide range of drugs that suppress the immune system, but one common immune suppressor is the negative mood. on the other hand, music is well studied as an immunity enhancer that re-regulates the immune system, increases nk cell activity, lymphokine-activated killer cell activity, lymphocytes, t cells, cd4+ t cells, and memory t cells [33,34]. music improves the psychological state, re-balance the hormonal and neurotransmitter levels in the body, therefore improve immunity. in the current coronavirus pandemic which began in china, there is an increasing number of infected people every day. after emerging of different vaccines that draw the immune system attention to foreign invaders, the immune system proves that it is the only and strongest barrier against the coronavirus. one promising approach is the plasma transfusion from recovered people to covid-19 patients as many studies find that 200 ml of plasma from recovered people could neutralize the coronavirus and lead to the disappearance of viremia in 7 days [46–49] which shows that the immune system is the only cure for such diseases. the immune system is known to be strongly connected to psychological balance. that is when the individual mood is good which means a high level of dopamine in blood, will optimize and enhance the immune system, and a bad or sad mood will suppress immunity. so, one of the best strategies to support the immune system is to keep the mood in a good state. and some studies support the fact that dopamine is highly liked by the immune system [50,51]. highlights in bioscience page 2 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient the influence of music on leukocytes was investigated in six experiments [33] found that natural killer cells increased, followed by an endocrine reaction. [52] hirokawa and ohira [53] carried out other experiments testing the number of cd4 and cd8 cells. it was observed that inducing recorded music, analyzed by [52], increased plasma cd4+ t cell levels. the impact of music on immunoglobulins was also investigated. the most studied antibody (n = 12) has been immunoglobulin a (iga). some of these studies reported a rise in iga level after a series of musical approaches with a wide range of styles and genres. fancourt introduces a modern model that connects central nervous system (cns), endocrine system, autonomic nervous system (ans), and immune system with them effectively to psychological effects and physiological effects which provides a basis for the creation of a taxonomy of study design variables linked to music and stress and traces the broad paths involved in its impact on the body [54]. gender-dependent response according to the physiological difference between men and women, there is a different response to music as shown in nater et al., the study [55] that reports different interactive patterns in men and women. once met with heavy metal music, according to finger temperature and skin conductance tests, women displayed a significant gradual increase in the sympathetic nervous system (sns) response than men's response. however, men displayed an abnormal autonomic reaction after exposure to heavy metals as demonstrated by increased salivary amylase secretion, which is caused by sympathetic and parasympathetic stimulation [55]. several studies have suggested that the difference in sex-based psycho-physiologic responses to music might have a hormonal basis [56–59]. however, research by trost [60] using functional magnetic resonance imaging (fmri) indicated that cheerful music stimulates the left striatum and insula while nostalgic sad music stimulates the right striatum and orbitofrontal cortex in both genders. criteria of theraputic music it is well known that music is related to mood and can make people feel happy, depressed, alive, or pleasant [61]. listening to techno music (tm) or fast rhythm music[44] could cause increases in plasma norepinephrine, epinephrine, gh, prl, cortisol, and beta-endorphin concentrations with a gradual rise in heart rate, systolic blood pressure, and significant changes in emotional response. in gerra [62] that found that exposure to techno music increases the heart pulse rate and norepinephrine, cortisol, and adrenocorticotropic hormone secretion levels and vice versa such in the white study [63] who showed, in a group of patients in intensive care unite (icu) when they listened to relaxing classical music, a reduction of heart rate and blood pressure. other studies suggested that classical music has been found to improve the mood state caused by tm and increased gastric slow-wave in adults [31], but not to significantly reverse changes in hormonal concentrations [64]. an old study investigated the physiologic effect of listening to no music, fast-rhythm music, and sedative music for athletes and non-athletes during the exercise of varying intensities. under conditions of intensive exercise, runners listening to fast rhythm music had increased plasma cortisol levels [65]. this indicates a possible metabolic advantage for the use of energetic music during exercise, considering the role of cortisol in stimulating the catabolism of energy substrates in fat, adipose, and connective tissues. mccraty's study shows the effects of music in this study was titled 'heart zones'. s-iga increased by 55 percent (p < 0.01) when listening to the heart zones' music alone [40]. criteria of selected music according to [66] should be full of character, slow rate, repeated rhythm, predictable tone dynamics, pleasant harmony with no vocal content. three songs studied by [67] are consist of 70 to 80 beats per minute and have a slow and steady, repeated rhythm as reported in the previous study. the three songs are "canon in d major", "love story", and "dance of the iguana" played by johann pachelbel, richard clayderman, and stevan pasero respectively. valence and arousal in music have also been shown to be major factors. for calm, low tempo songs, [68] compared recorded music with four distinct tempi and moods, but only found a decrease in heart rate. [69] found that while heart rate during a stressful activity was diminished a little by high tempo music, with low tempo music there was a much greater impact. the respiratory rate was lowered only due to low tempo songs [70] found that blood pressure was not changed by soothing music when contrasting six distinct tempo pieces of music, but raised by high tempo music. the most compelling outcomes were provided by experiments that implemented more complicated controls. [71] stated, for instance, that listening to mozart had a stronger influence on vital signs than a progressive relaxation session, and [72] found that listening to relaxing recorded music was as effective as diazepam in decreasing vital signs of anxiety. three specific styles of music were listened to by adults and adolescents in another study : classical, new age, and designer (designed to promote a sense of wellbeing) [73]. the respondents surveyed personal emotions at baseline and directly after listening to each style of song. classical music reduced anxiety. through new age music, relaxation increased dramatically and aggression, mental focus, vigor, and anxiety reduced. the subjects registered significantly more calm, mental focus, vigor, and sympathy after listening to designer music, and significantly decreased aggression, depression, exhaustion, and anxiety. although addressed music should be consist of 70 to 80 beats per minute with a slow and steady, repeated rhythm as reported such as "canon in d major", "love story", and "dance of the iguana", the cultural aspect should be considered when choosing music and mentioned music example is just to clarify the type of therapeutic music. highlights in bioscience page 3 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient music therapy for covid-19 patient covid-19 coronavirus disease, which is an infectious disease, and it was clear that negative feelings were mostly physiologically caused by infectious diseases and that a wide spectrum of health risks could be exacerbated or enhanced as a result of this infection. [74–76] in le roux's study, he reveals that classical music, such as j. s. bach's magnificat in d majorbwv 243 has an effect on emotion, immunity, and endocrine parameters in patients with specific infectious lung diseases, an experimental and control group was randomly distributed to patients. the experimental group received physiotherapy with the preferred music over a 3day duration, while the control group only received physiotherapy. the outcome indicates major changes in parameters of the immune system, such as poms-scale, cd4+:cd8+ ratio, cortisol, and cortisol: dhea ratio. and music's intervention reveals contact between the mind and the body. [77]. such experiment which conducted on a patient with infection disease, similar to coronavirus situation, may be applicable to covid-19 patient. previous studies prove the therapeutic effect of music, which made music a strong candidate to be used along with medication for covid-19 patients. a type of music administered by healthcare professionals for children also has a pain decreasing effect. the songs used consisted of age-appropriate children’s songs. yinger provides a list of music and its purpose [78]. such strategies may be beneficial for decrease the pain resulted from the medical procedures. as a severe case of covid-19 patients should be placed on a respirator, one study demonstrated a significant fell in systolic and diastolic blood pressure fell causing a decrease in anxiety and depression during music therapy sessions for the patients on the respirator, as is shown in paired samples [78]. the qualitative part of the study showed that the patients remembered little of their time on the respirator which may be a good effect to remove the memories about the disease to avoid any psychological complications that may be caused by negative memories. recently, in some mild covid-19 cases, it is recommended for the patient to be isolated in a room, which may increase patient anxiety and depression. the same situation reported before in brodsky's work where music therapy was most effective in isolating hospital experiences, reducing distress, and fearprovoking hallucinations, and helping to have a more positive effect than other therapies[79]. although several reports demonstrate the beneficial impact of classical music, it will be best to encourage the participant, as much as practicable, to select the type of music [31]. although music therapy is deemed free of side effects, the technique should be used with the supervision of medical staff. a patient may react deeply, emotionally to a specific piece of music. others reported a stress reduction and an increase in t-cell proliferation in mice who listened to music. in mice exposed to auditory stress, the nk activity of spleen cells also decreased during 8 days [80–83]. such findings have been supported by previous research on the impact of auditory stress and its negative effect on the immune system [84]. applying music therapy way to the patient should be considered and carefully chosen in order to prevent or at least minimize infection transmission between healthy individuals and patients. a trained music therapist must deliver music interventions; the music therapist often adapts the live music experiences to the patients' needs at the time. this also presents the patient with a highly humanizing and validating experience. furthermore, listening to self-selected pre-recorded music, introduced by the patient himself, may lead to an enhanced sense of autonomy and empowerment in a critical care setting where certain facets of care are beyond the control of the patient. [85,86]. lee study warning from using headphones that may cause crosscontamination and nosocomial infections [87] show that speaker of personal or one-use headphones might be the best way to listen to music in case of covid-19 patient conclusion an epidemic of novel coronavirus diseases (covid-19) in wuhan, china has spread widely throughout the nation. the world focuses all efforts to stop the spreading of the virus using a different drug, and recently by enhancing the immune system using vitamins or plasma transfusion from the recovered person. but, one of the unnoticed supplements for the immune system is music therapy. the results of a recent investigation of music effect for medical procedural support shows that there is an urgent need for more high-quality research on the use of effective music therapy before, during, and after medical procedures, with particular emphasis on the interventions and techniques used by the music therapist [88] for patients and staff [40,89] in treating a patient with infection [77]. many studies state the improving effect of music on the immune system reported in thirteen studies [66,67]. and it seems that slow, repetitive rhythm, predictable dynamics, low tonic register, pleasant harmony such as classic music is the most effective [78] style not only in improving immunity but also in a medical procedure such in isolating room or placing the patient on a respirator [78]. although this idea must undergo clinical trials to be proved, developing a "covid-19 symphony" a music track based on recent research and experiments may be urgently required to be a part of the medical protocols alongside supplements and drugs to improve mood and immunity to help the patient pass such disease with the precaution of addressing the music therapy in a personal way to prevent infection transmission without using any instrument that may transmit the infection [87]. references 1. zhu n, zhang d, wang w, others. china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china, 2019 [published january 24, 2020]. n engl j med. 2. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, highlights in bioscience page 4 of 7 march 2021|volume 4 http://bioscience.highlightsin.org/ habib, 2021 a review of possible music therapy in supporting the immune system of covid-19 patient china. the lancet. 2020;395(10223):497–506. 3. hui ds, azhar ei, madani ta, ntoumi f, kock r, dar o, et al. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health—the latest 2019 novel coronavirus outbreak in wuhan, china. international journal of infectious diseases. 2020;91:264–6. 4. benvenuto d, giovanetti m, ciccozzi a, spoto s, angeletti s, ciccozzi m. the 2019-new coronavirus epidemic: evidence for virus evolution. journal of medical virology. 2020;92(4):455–9. 5. zumla a, chan jfw, azhar ei, hui dsc, yuen k-y. coronavirusesdrug discovery and therapeutic options. nature reviews drug discovery. 2016;15(5):327–47. 6. habib pt. vaccine design, adaptation, and cloning design for multiple epitope-based vaccine derived from sars-cov-2 surface glycoprotein (s), membrane protein (m) and envelope protein (e): in silico approach. 2021; 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highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20202 highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 5 research article open access 1 department of nucleic acid and protein structure, agricultural genetic engineering research institute (ageri), arc, giza, egypt. 2 department of microbiology, faculty of science, ain shams university, 11566, abbassia , cairo, egypt. contacts of authors * to whom correspondence should be addressed: ghada abu el-heba citation: hassan a.h., a-bouzeid m., and abu el-heba g. (2020). dna variations between medicago truncatula symbiotic mutant line and native variant using fluorescence-based aflp marker, volume 3. article id 20202, dio:10.36462/h.biosci.20202 received: march 3, 2020 accepted: march 20, 2020 published: april 1, 2020 copyright: © 2020 hassan et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. dna variations between medicago truncatula symbiotic mutant line and native variant using fluorescencebased aflp marker asmaa h. hassan 1 , mohamed a-bouzeid 2 , and ghada abu el-heba * 1 abstract genetic mutagenesis is a very efficient tool in studying genes function. because of great benefits of legumes as human food and animal feed worldwide, we used a model plant medicago truncatula for identification gene function related to nitrogen fixation process. our mutant is a medicago mutant line contains a tobacco tnt1 retrotransposon mobile element with the two long terminal repeats (ltr) inserted within the genome. our mutant is predicted to contain a mutation in gene/s belonging to symbiotic interaction between legume and rhizobia. a novel technique was used based on using fluorescent oligonucleotide primers against oligonucleotide primers for tnt1-ltrs of our mutant. this novel protocol was very successful in detection the polymorphism between our mutant line and the wild variant r108 using biosystems 310 genetic analyzer. electropherograms of the mutant line and wild type gave a total of 561 wellresolved aflp peaks, 357of which were polymorphic peaks and 204 were monomorphic peaks. this novel technique enables the calculation percentage of polymorphism between the mutant line and the wild type. additionally primers combinations amplified more bands from others to detect polymorphism between the plants. keywords: medicago truncatula, aflp, polymorphism, symbiotic mutant, nitrogen fixation. introduction legumes played vital roles in improving agriculture and considered as onethird of the world’s major crop production. they represent about 27% of the world’s crop production. human consumes approximately 33% of the dietary protein needs in the form grains. many food industries based on legumes like treated vegetable, oil production, and chicken industry. animals depend on forage legume plants as major source of food like medicago stavia and trifolium spp (1). this group of plants has unique character as they have ability to make symbiotic relationship with soil bacteria called rhizobia. rhizobia are beneficial soil bacteria that belong to gram negative group (2) . in this relation legumes can fix nitrogen in specific organ called root nodule (1,3). nitrogen fixation process occurs naturally during growing this family of plants and so we can decrease our usage of industrial nitrogen fertilizers and related ecosystem pollution (1). many studies focused on genes responsible for nitrogen fixation in legumes and rhizobia (4-9). lotus japonicus and medicago truncatula are considered as more suitable two plants for studying genes responsible for symbiotic relationship between both organisms. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 5 numerous technological platforms like; ethyl methanesulfonate (ems) mutagenesis (10), fast neutron bombardment and g-rays (11) were developed and used for model plants genome studying. dna-insertion mutantgenesis populations save a rapid method for isolating genes responsible for symbiotic nitrogen fixation through both reverse and forward genetics (12-14). retrotransposon is a type of mobile elements (class i) that transpose to new sites in the genome via an rna intermediate. their primary transcripts are retro-transcribed into dna copies by a reverse transcriptase (15). tobacco retrotransposon tnt1 is an effective mutagen in m. truncatula that is used for molecular studies worldwide (16). this transposone element (tnt1) belongs to retrotransposon elements family (17) , its transposition was induced during tissue culture of tobacco protoplast(18) and during in vitro transformation of a. thaliana. tnt1 insertions of are stable in the plant and transfer to the progeny because of the replication cycle of ltr retrotransposons (19). samuel roberts noble foundation has the largest collection of dna-insertion mutants of all legumes specially tnt1-insertion mutant populations in medicago truncatula. numerous mutant phenotypes have been screened such as mutant loss the ability to form nodules, mutants produce dysfunctional nodules, mutants produce large number of nodules and mutants with delayed and decreased nodulation but efficient in nitrogen fixation (9). among molecular technique, aflp protocol provides a very powerful dna finger printing method for nucleic acid of any organism (20). fluorescence-labeled pcr products were sensed on an automated dna sequencer instead of the standard polyacrylamide gel electrophoresis tool (21-22). capillary electrophoresis has more advanced characters from slab gels in speed, resolution and availability of numerical information in an electronic format following the end of a reaction. in this study, the genetic polymorphism between medicago truncatula symbiotic mutant line and the wild type of the same plant has been estimated using amplified fragment length polymorphism (aflp) marker analyses. this mutant contains tnt1 reterotrasposon element that have mutation in the gene/s involving in nitrogen fixation process. materials and methods plant materials the mutant line of medicago truncatula was provided from noble foundation tnt1 mutant collection that transformed from m. truncatula r108-1 ecotype (23). the wild type plant r108has been used in this study as a reference. seed sterilization and germination seeds of medicago truncatula wild type and mutant line were scratched with sand paper and surface sterilize with 6.25% (v/v) sodium hypochlorite for 10 minutes. after seven washes using sterilize water, seeds were put on murashige and skoog (ms) basal medium (murashige and skoog 1962) supplemented with 0.8% agar (grade j3, gelita) in dark over night at 29° c. seedlings were transferred to growth chamber with this condition at stable 21° c during a 16-h and 8-h night photon flux density of 100 ϻ mol m -2 s -1 (24). dna isolation and quantification total genomic dna was extracted from mutant plants and control for using in aflp analysis. healthy leaves from both line were used for dna isolation using qiagen genomic dna isolation kit, (qiagen science,valence , ca , usa). the quality and quantity of genomic dna were estimated using spectrophotometric measurement of ultra violet absorbance at wave length 260 and 280 nm in thermo scientific nanodrop 2000™) and were electrophoresed at 0.5% agarose gel electrophoresis. aflp protocol a) genomic dna digestion and ligation amount of genomic dna from mutant line and wild type were digested with 10u ecor1 and 10u mfe1 (new england biolabs, ipswich, ma, usa). the total volume was incubated for three hrs at 37°c then the enzymes were inactivated by incubation for 20 mins at 65°c. to assess the digestion, 5 μl from each sample were electrophoresed on a 1.5 % agarose gel. the digested fragments dna were ligated by eco and ase adaptor ligation using t4 dna ligase (new england biolabs) to produce modified digested fragments that were used as a template dna for amplification. c) pre-selective amplification reaction the reaction was performed on ligated dna using two aflp oligonucleotide primers using takara la taq kit. then the product was run on 1.5% agarose gel. d) selective amplification reaction selective amplification was performed using two aflp oligonucleotide primers. the first one was fluorescence-labeled primer: eco (dye-primer-axx) and unlabeled primer (ltr4&ltr6). three primer combinations were used: e-acc (ned)/ltr4, e-acc (ned)/ltr6, e-aag (6-fam)/ltr4, e-aag (6-fam)/ ltr6, e-aac (hex)/ltr4 and e-aac (hex)/ltr4. aflp procedure was applied according to (25) with a modification which is using three florescence labeled oligonucleotide primers (e-aac, e-aag, and e-acc) (20). 1ul of selective amplified product was added to 12ul hi-di formamide and 0.5 microliter of genescan500 rox internal size standard (applied biosystems, foster city, california, usa). the mixture of these components was denatured and inoculated to the single capillary of applied biosystems 310 genetic analyzer. gene mapper analysis software version 4.1 (applied biosystems) was used for analyzing the generated electropherograms for mutant and r108 from the raw date according to aflp system analysis getting started guide. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 5 results pre-selective amplification was achieved using preselective oligonucleotide primer pair and the product was run on 1.5% agarose gel. successful pre-selective amplification was confirmed as a smear product that was interrupted by a number of bands and the polymorphism cannot be detectable at this stage. aflp electropherograms of mutant line and wild type selectively amplified using primer combination as illustrated in (table 1). oligonucleotide primer combinations examined for selective amplification of dna fragments of mutant line and wild type gave a total of 561 wellresolved aflp peaks of which 357 were polymorphic peaks and 204 were monomorphic peaks. the highest number of amplified dna fragments was 218 peaks were obtained with primer combination (eagg/ltr4); however the lowest number of amplified dna fragments was 31 peaks from combination (e-acc/ltr6). aflp electropherograms of both lines were produced using the illustrated primer combination e-acg/ltr4 (as an example) was shown in (figure 1). table 1: showing that the total number of peaks (tnp), number of polymorphic peaks (npp) and number of monomorphic peaks (nmp) were produced by each primer combination (pc). discussion legumes have a fundamental role in sustainable agriculture for decades. they are able to reduce the atmospheric n2 to ammonia through symbiosis interaction with rhizobia which is nitrogen-fixing bacteria. this criterion enables legumes to propagate well in soils poor with nitrogen without any fertilizer addition. symbiotic nitrogen fixation (snf) in legumes produces nitrogen about fifty million tons into the agricultural systems every year. both legumes and non-legumes production is sustained by such nitrogenous compound in the soil (21). legumes are essential source for food and feed (1). in our research we used medicago truncatula which is a model plant for studying snf because it has a small diploid genome, short life cycle and it performs symbioses with (sinorhizobium meliloti and sinorhizobium medicae). mutant collections production was necessary to identified genes function. large scale mutagenesis was generated in m. truncatula via tnt1 retrotransposon (23). tnt1 is a ty1copia type retrotransposon with long terminal repeat (ltr), isolated from tobacco (26). tnt1 transpose via copy & paste mechanism . each mutant line has from four to fifty insertions per genome (23). retrotransposon mediated mutagenesis was used in the other plant species like rice (27), lettuce (19) , and soybean (28). samuel roberts noble foundation has produced about 21,000 tnt1 mutant lines (23). we used aflp-type protocol in combination with abi 310 genetic analyzer to determine the genetic polymorphism between our mutant line which is putatively defects in nodule development and r108 wild type. figure 1: aflp electropherograms of r108 and mutant line amplified selectively by the primer combination e-aag/ltr6. red row indicates a polymorphic peak example that is present in mutant line and absent in r108. hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 5 aflp electropherograms of both lines produced a total of 561 aflp peaks were recorded, 357 of which were polymorphic and 204 were monomorphic .this corresponds to a level of polymorphism of 63.7%. [the percentage of polymorphism was calculated as [(the total no. of polymorphic peaks / the total no. of peaks) x 100]. aflp has the capability to discover the polymorphisms between these types of plants without knowing the sequence of genome. so, this is one of the best advantages of using aflp technique. the established website (http://bioinfo4.noble.org/mutant/) encloses information about 11,000 tnt1 mutant lines with images of diverse phenotypes. our perspective work will include aflp-type pcr to isolate all the tnt1-flanking sequence tags (fts) for more mutant characterization and identification. all fragments will be analyzed and the mutated gene/s will be recognized. acknowledgement the authors appreciate dr. pascal ratet for providing mutant line & r108 wild type. references 1. graham ph, vance cp. legumes : importance and constraints to greater use update on legume utilization legumes : importance and constraints to greater use. plant physiol. 2003;131:873–7. 2. zakhia f, de 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2008;12:193201. 14. urban´ ski d f, małolepszy a, j stougaard , andersen su. genome-wide lore1 retrotransposon mutagenesis and high-throughput insertion detection in lotus japonicus. plant j. 2012;69:731–41. 15. wessler sr. transposable elements and the evolution of eukaryotic genomes. nat rev genet. 2006;103(47):17600–1. 16. erfurth i, cosson v, eschstruth a, lucas h, kondorosi a, ratet p. ef ficient transposition of the tnt1 tobacco retrotransposon in the model legume medicago truncatula. plant j. 2003;34(1):95–106. 17. grandbastien, marie-angele as& mc, laboratoire. tntl, a mobile retroviral-like transposable element of tobacco isolated by plant cell genetics. nature,. 1989;337:5–9. 18. pouteau s, huttner e, grandbastien m a, caboche m. specific expression of the tobacco tntl in protoplasts. embo. 1991;10(7):1911–8. 19. mazier m, botton e, flamain f, bouchet j-p , courtial ´, et al. successful gene tagging in lettuce using the tnt1 retrotransposon from tobacco. 2007;144(may):18–31. 20. thom‎as cm, vos p, zabeau m, jones da, norcott ka, chadwick bp, et al. identification of amplified restriction fragment polymorphism (aflp) markers http://bioinfo4.noble.org/mutant/ hassan et al., 2020 medicago truncatula symbiotic mutant analysis highlights in bioscience april 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 5 tightly linked to the tomato cf‐ 9 gene for resistance to cladosporium fulvum. plant j. 1995;8(5):785–94. 21. weising k, nybom h, wolff k kg. dna fingerprinting in plants: principles, methods, and applications, 2nd edn. boca raton: crc press. 2005. 472-477 p. 22. karudapuram s, larson s. identification of hedysarum varieties using amplified fragment length polymorphism on a capillary electrophoresis system. j biomlecular tech. 2005;16(4):316–24. 23. tadege m, wen j, he j, tu h, kwak y, eschstruth a, et al. large-scale insertional mutagenesis using the tnt1 retrotransposon in the model legume medicago truncatula. plant j. 2008;54:335–47. 24. shao c, russinova e, iantcheva a, atanassov a, mccormac a, chen d, et al. rapid transformation and regeneration of alfalfa ( medicago falcata l .) via direct somatic embryogenesis. 2000;31:155–66. 25. ratet p, porceddu a, mysore ks. insertional mutagenesis in m . truncatula using tnt1 retrotransposon insertional mutagenesis in medicago truncatula using tnt1 retrotransposon . 2010;1-12. 26. kumar, bennetzen jl. plant retrotransposons. annu rev genet. 1999;33:479–532. 27. greco r, ouwerkerk pbf, sallaud c, kohli a, colombo l, puigdome p, et al. resources and opportunities transposon insertional mutagenesis in rice 1 , 2. plant physiol. 2001;125:5–7. 28. cui y, barampuram s, stacey mg, hancock cn, findley s, mathieu m, et al. tnt1 retrotransposon mutagenesis : a tool for soybean. 2013;161:36–47. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202307 research article open access 1 bioinformatics department, agriculture genetic engineering research institute, agriculture research center, giza, egypt. 2 biotechnology/biomolecular chemistry department, faculty of science, cairo university, egypt. 3 wheat res. dept., field crops res. inst., arc, giza, egypt. 4 genome mapping department, molecular genetics and genome mapping laboratory, agricultural genetic engineering research institute, giza, egypt. * to whom correspondence should be addressed: saifeldeenmib99@gmail.com editor: aladdin hamwieh, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): ayed m. al-abdallat, aculty of agriculture, the university of jordan, jordan. tawffiq istanbuli, international center for agricultural research in the dry areas (icarda), beirut, lebanon received: october 20, 2023 accepted: december 20, 2023 published: december 26, 2023 citation: ibrahim ms, ibrahim sm. unlocking the genetic basis of abiotic stress tolerance in wheat: insights from differential expression analysis and machine learning. 2023 dec 26;6:bs202307 copyright: © 2023 ibrahim and ibrahim. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. unlocking the genetic basis of abiotic stress tolerance in wheat: insights from differential expression analysis and machine learning saifeldeen m. ibrahim *1 ><, manar s. ibrahim 1 >< ,radwa khaled2 >< , ahmed fawzy elkot 3 >< , shafik d. ibrahim4 >< abstract abiotic stresses such as heat and cold temperatures, salinity, and drought are threatening global food security by affecting crop quality and reproductivity. wheat is the most essential staple crop in the world, its complex genome is the main barrier to finding valuable genes responsive to different stresses. thus, in our study we conducted differential rna-seq analysis to identify differentially expressed genes (degs) involved in 4 different stresses such as drought, heat, freeze resistance, and waterdeficit stress, then applied two machine learning models; the "extra-tree regressor" and lime algorithms to accurately predict and select the highly significant genes. our findings identified a set of 36 significant genes, many of which play important roles in various molecular functions, cellular components, and biological processes related to the response or resistance to abiotic stress in wheat. for example, hsp101b is a member of the heat shock protein family, which protects cells against stress by stabilizing proteins. badh, an enzyme involved in the synthesis of stress hormones, is important for the plant’s response to different stresses. agl14 is a member of the agl protein family, which regulates gene expression and is involved in the plant’s response to drought, cold, and salinity stresses. this study demonstrates the prospects of the integration of bioinformatics tools as well as machine learning models to assess the genes responsible for wheat stress resistance, genes’ regulatory networks, and their functions in order to save time and cost to improve wheat productivity. keywords: wheat, abiotic stress, differential gene expression, machine learning. introduction wheat (triticum spp.) is one the most strategically important crops for high proportions of the world population, supplying merely 55% of carbohydrates and 20% of dietary proteins of the world’s consumed food [1; 2]. moreover, it has high nutritional value as it is a rich source of vitamins and minerals [3]. in terms of economic importance, wheat is the third most widely grown crop in the world after rice and maize, the annual production of wheat reached approximately 778.6 million tons in the 2021-2022 season [4; 5]. in addition to being a major staple food, wheat is also used in the production of various other products, such as flour, pasta, bread, and cereals [6]. it is a versatile grain that can be used in a wide variety of food products, making it an important commodity in the global food industry. climate change endangers plant productivity by increasing the intensity and extent of numerous abiotic stresses, such as heat, salinity, and drought [7]. for instance, drought stress decreased wheat yields globally by 32%, it is estimated that more than half of the world’s cultivated area will experience water scarcity by 2050. wheat yields are also being reduced by 40% due to soil salinity contamination[8; 9]. plants suffer from abiotic stresses differently, to withstand this threat plants have developed a variety of biochemical, physiological, and metabolic responses. for instance, numerous stress-responsive genes are activated, which are involved in producing many proteins that aid in activating and adjusting the physiological and biochemical pathways in stress tolerance [10]. highlights in bioscience page 1 of 10 december 2023|volume 6 https://doi.org/10.36462/h.biosci.202307 https://creativecommons.org/licenses/by/4.0/ saifeldeenmib99@gmail.com https://orcid.org/0000-0003-0143-2596 manar.samir249@science.helwan.edu.eg https://orcid.org/0000-0001-8575-2372 radwa.khaled3rafa@gmail.com https://orcid.org/0000-0002-4566-9803 elkot1982@gmail.com https://orcid.org/0000-0003-0143-2596 shafikdarwish@ageri.sci.eg https://orcid.org/0000-0001-8152-1999 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat understanding the molecular pathways and mechanisms underlying stress tolerance in crops through genomics, transcriptomics, and proteomics techniques pave the way for the identification of genetic biomarker hallmarks involved with high-stress tolerance and the recognition of the processes behind adaptation to stresses [11]. the contributions of the plant proteome have become more relevant for understanding gene activity and networks in response to an external stimulus [7]. micro-arrays and rna-seq techniques are dramatically evolving transcriptomics studies by providing a massive amount of data about the genes involved in stress response, downstream signaling, and the synthesis of stress response molecules at a specific time point and under particular conditions in plants in order to create crop types that can endure biotic and abiotic stress and produce a greater yield [12]. the comparative transcriptome study between cultivars that are resistant to drought and those that are vulnerable identifies possible genes and processes of adaptation to drought stress [13]. several cipk genes are elevated in rice, especially under drought-stressed situations, according to earlier comparative analysis investigations [14]. le et al. demonstrated a large number of kinase-encoding genes that are drought-inducible, including pp2c proteins, hormone-signaling-related proteins, map and cipk kinases, that were thought to be important in the control of the drought response in soybean leaves through microarray analysis. while the over-expression of the anac019, anac055, and anac072 genes in arabidopsis provided the first evidence of the roles of nac tfs in the enhancement of drought tolerance in plants[15]. the crop genomics field cannot simply interpret molecular complex phenotypes, due to the large, diverse, and heterogeneous data-sets, so leveraging strong data mining algorithms and bioinformatics techniques to anticipate and interpret these phenotypes is crucial [16].the recent advances in machine learning algorithms (mla) integrated with omics data analysis helped in the recognition, categorization, measurement, and early prediction of plant stress responses in addition to the extensive metabolic description for the target plant species[17]. machine learning has been applied to a variety of fields, including medicine, engineering, biology, and genomics, it has the potential to revolutionize the way we analyze and interpret gene expression data. one application of machine learning in gene expression analysis is the identification of genes that are deferentially expressed between different samples or conditions. by analyzing gene expression data in the context of other genomic data, such as dna sequence data or protein-protein interaction data, machine learning algorithms can identify patterns and relationships that can provide insights into gene function and the underlying biological processes [18; 19]. the mlas have recently introduced successful models for the agriculture field. osco et al.[20] successfully applied the artificial neural networks (ann) to differentiate between the hyper-spectral response of water-stressed lettuce from the nonstressed group with an accuracy reached 93%. another study found that it was possible to evaluate water stress in winter wheat crops over time in connection to other factors including disease and nitrogen accumulation by employing continuous wavelet analysis, fisher’s linear discrimination analysis, and support vector machines [21]. many studies and research projects have used machine learning to identify genes that are differentially expressed between different samples or conditions. in a study of the molecular markers of drought stress in wheat, priya et al. [22] used ml algorithms to identify differentially expressed genes that were associated with drought tolerance. they found that the genes identified by the ml analysis were significantly enriched for functions related to drought tolerance, highlighting the potential of ml for identifying key genes involved in stress responses. another study used differential gene expression analysis and ml to identify genes associated with rust resistance in wheat [23]. the genes identified by the ml analysis were significantly enriched for functions related to rust resistance, and they concluded that the use of bioinformatics approaches, including ml, can be an effective way to identify genes associated with stress responses in wheat. another study that applied machine learning to gene expression data in wheat used a decision tree algorithm to identify genes that were differentially expressed between different varieties of wheat and to predict their potential functions [24]. thanmalagan et al. [25] applied machine learning to gene expression data from rice (oryza sativa) to predict gene function and identify pathways that were important for drought tolerance. they used a machine learning algorithm to identify patterns in the gene expression data and predict gene function based on those patterns. also, they found that the machine learning algorithm was able to accurately predict gene function and identify pathways that were involved in drought tolerance, including those related to stress response and carbohydrate metabolism. based on the previous investigations, the current study aimed to use both approaches (de analysis and ml algorithms) to identify the molecular markers related to the most significant genes associated with abiotic stress. methodology data gathering the gene expression omnibus (geo) was utilized to retrieve four distinct wheat dataset experiments related to various abiotic stresses using the terms such as drought and heat stress resistance, freeze resistance, and water deficit response. the experiment name of each accession is supplied for all gse accessions in table 1. differential gene expression analysis the statistical tool geo2r [26] was used to analyse the raw four gene expression data sets of wheat under different abiotic stresses using the r/bioconductor and limma packages. each sample has been divided into two categories (control and treatment). the differentially expressed genes (degs) were then highlights in bioscience page 2 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat table 1. information for the four geo datasets for wheat. accession experiment name abbreviation gse45563 drought, heat and combined stress in durum wheat dhsw gse14697 freeze resistance basis of winter wheat mutant lines frbw gse48620 long-term growth under elevated co2 differentially suppresses biotic stress genes guco gse45262 transcriptomics of water-deficit stress responses in tam wheat cultivars tamw visualised using r packages by creating a volcano plot highlighting all significant genes and a heatmap of the top differentially expressed genes based on each group in all samples using log2fc (fold change) ≥ 1 and an adjusted p-value of 0.05 as the deg threshold. whereas up-regulated degs were considered when the logfc (fold change) ≥ 1, down-regulated degs were considered when the logfc (fold change) ≤ -1. the volcano plot displays statistical significance (p value) in accordance with the rate of fold change. it makes it possible to quickly visualise genes with significant fold changes. these genes may be the most important in terms of biology. after reading the data and filtering it according to the adjusted p-value and logfc, this figure was created using the r package (ggplot2) (blue: downregulated, red: upregulated). following that, a heatmap of the top differentially expressed genes in the rna-seq data set was created using the pheatmap package after filtering the data. then, a venn diagram was used to show all of the genes that were shared by all four wheat experiments using the interactivenn website [27]. protein-protein interaction and functional enrichment analyses to assess the link between genes associated with wheat gene expression, the string database was used for protein-protein interaction analysis including functional and physical interactions [28]. for each gse dataset, all information regarding go, annotated keywords, and protein domains has been presented in table 2. machine learning model the "extra-tree regressor" and "local interpretable modelagnostic explanation" algorithms were used in this study to identify the significant genes related to the response or resistance to abiotic stress in wheat. the "extra-tree regressor" is a machine learning model that utilizes decision trees to make predictions. decision trees are constructed by considering the characteristics of a given dataset and dividing it into smaller subsets based on the values of certain features. the "extra-tree regressor" model differs from traditional decision tree models in that it uses random thresholds for feature selection, rather than using the mean value of each feature [29]. this allows the model to capture non-linear relationships in the data, making it well-suited for the analysis of gene expression data. the "local interpretable model-agnostic explanation" algorithm, or lime, is a technique for explaining the predictions made by machine learning models. it works by approximating the complex, non-linear relationships learned by the model with a simpler, interpretable model that is specific to a particular prediction [30]. this allows researchers to understand the factors that contributed to a particular prediction, and can be useful for identifying the underlying mechanisms behind the model’s results. results and discussion identification of degs identification of molecular mechanisms, biological processes, and cellular components for both up-and down-regulated genes through the different environmental stresses is an essential step toward enhancing wheat survival rates. the gse14697 dataset, which highlights the genes that might confer and maintain freeze resistance in winter wheat, had the highest number of expressed genes (14,484 genes), according to our analyses. this dataset also found multiple cold-responsive (cor)/late-embryogenesisabundant (lea) genes; the proteins of accumulating cor/lea genes are thought to promote and sustain the development of freezing tolerance [31] [32]. on the other hand, the gse45563 dataset had the fewest expressed genes, with just 1,297. the volcano plot, shown in figure 1, illustrates which genes are up-regulated (red) and which are down-regulated (blue) in triticum aestivum samples. the fold change (log2fc) is represented by the horizontal axis, while the adjusted p-values are represented by the vertical axis. the gse14697 dataset produced 6,987 up-regulated and 7,497 down-regulated degs, according to the volcano plot, whereas the gse45563 dataset produced 540 up-regulated and 757 down-regulated degs while, gse48620 produced 2,693 up-regulated and 3,164 down-regulated degs, and gse45262, which examined the drought-responsive genes in wheat, produced 1,221 up-regulated and 803 downregulated degs. for a more in-depth analysis of the degs, a heatmap was generated for the top de genes based on the adjusted p-value ranking. the heatmap divides the samples into two groups: control (blue) and treatment (red). red cells indicate high gene expression (upregulated), whereas blue cells indicate low gene expression (downregulated). lighter tones and white are used to depict genes with stable expression levels. the samples and genes were reordered using dendrogram hierarchical clustering, as shown in figure 2. this heatmap gives a deeper insight into the changes in highlights in bioscience page 3 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat table 2. the string database table contains information on go, annotated keywords, and protein domains, for each gse dataset. gse category id description fdr gse14697 cellular component go:0110165 cellular anatomical entity 6.24e-18 gse45262 cellular component go:0110165 cellular anatomical entity 2.88e-12 gse45563 molecular function go:0004363 glutathione synthase activity 0.003 go:0043295 glutathione binding 0.0464 cellular component go:0110165 cellular anatomical entity 3.54e-12 subcellular localization gocc:0017109 glutamate-cysteine ligase complex 0.003 gocc:0036087 glutathione synthase complex 0.003 annotated keywords kw-0317 glutathione biosynthesis 0.00026 kw-0460 magnesium 0.019 protein domains pf03199 eukaryotic glutathione synthase 0.0036 pf03917 eukaryotic glutathione synthase, atp binding domain 0.0036 protein domains and features ipr004887 glutathione synthase, substrate-binding domain 0.0034 ipr005615 glutathione synthase 0.0034 ipr014042 glutathione synthase, alpha-helical 0.0034 ipr014049 glutathione synthase, n-terminal, eukaryotic 0.0034 ipr014709 glutathione synthase, c-terminal, eukaryotic 0.0034 ipr037013 glutathione synthase, substrate-binding domain superfamily 0.0034 ipr016185 pre-atp-grasp domain superfamily 0.004 gse48620 molecular function go:0005200 structural constituent of cytoskeleton 0.0293 cellular component go:0110165 cellular anatomical entity 1.76e-38 annotated keywords kw-0342 gtp-binding 0.00015 kw-0963 cytoplasm 0.00015 kw-0206 cytoskeleton 0.0062 kw-0493 microtubule 0.0073 kw-1015 disulfide bond 0.0076 kw-0326 glycosidase 0.0241 kw-0732 signal 0.0241 kw-0325 glycoprotein 0.0279 kw-0624 polysaccharide degradation 0.0355 kw-0547 nucleotide-binding 0.0426 kw-0597 phosphoprotein 0.0426 kw-0809 transit peptide 0.0426 protein domains pf03953 tubulin c-terminal domain 0.0035 pf00091 tubulin/ftsz family, gtpase domain 0.0079 protein domains and features ipr000217 tubulin 0.00016 ipr002453 beta tubulin 0.00016 ipr003008 tubulin/ftsz, gtpase domain 0.00016 ipr008280 tubulin/ftsz, c-terminal 0.00016 ipr013838 beta tubulin, autoregulation binding site 0.00016 ipr017975 tubulin, conserved site 0.00016 ipr018316 tubulin/ftsz, 2-layer sandwich domain 0.00016 ipr023123 tubulin, c-terminal 0.00016 ipr037103 tubulin/ftsz, c-terminal domain superfamily 0.00016 ipr036525 tubulin/ftsz, gtpase domain superfamily 0.00017 highlights in bioscience page 4 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat figure 1. visualization of degs volcano plots.the representations are as follows: x-axis, log2fc; y-axis, -log10 of an adjusted p-value. gene expression at the cellular level and shows which genes are most affected by abiotic stress. this can help to identify key genetic markers for the development of improved stress tolerance wheat varieties. figure (3) illustrates the venn diagrams of the degs between the four integrated geo data sets. the numbers within each circle represent the number of differently expressed genes across the various comparisons. the overlapping numbers refer to the common degs shared between the experiments, whilst the non-overlapping numbers refer to genes that are unique to each gse. the gse14697 and gse45262 shared 19 genes, the gse45262 and gse45563 shared 9 genes, and the gse14697 and gse45563 shared 5 genes. all the common and shared genes are listed in the table 4. it is believed that all of these genes are related to abiotic stress, as they play key roles in the response to various types of stress, including drought and salt stress. one example is wrab15, a member of the wrky transcription factor family, which plays a key role in the response to various types of stress, including abiotic stress. wrab15 has been shown to be upregulated in response to drought and salt stress in wheat [33]. another example is ccoamt, a copper-containing amine oxidase that is involved in the detoxification of reactive oxygen species (ros) generated during stress conditions [34]. tatil is a member of the aquaporin family, which plays a role in the transport of water and other small molecules across cell membranes [35]. rab is a member of the rab gtpase family, which regulates vesicle transport and is involved in the response to stress [36]. tlk1 is a member of the protein kinase family, which plays a role in the regulation of gene expression and cell division [37]. wcor18 and lea are members of the lea (late embryogenesis abundant) protein family, which is involved in the protection of cells against stress conditions [38]. nac693 is a member of the nac transcription factor family, which plays a role in the regulation of gene expression and is involved in the response to stress [39]. p5cr is a member of the pyrroline-5carboxylate reductase enzyme family, which is involved in the synthesis of proline, a compound that plays a role in the protection of cells against stress [40]. gs3 is a member of the grain softness protein family, which is involved in the development of grain hardness and is upregulated in response to abiotic stress in wheat [41]. hsp101c, hsp16.9b, hsp16.913lc1, tahsp17.3 and hsp 26.6b are members of the heat shock protein family, which plays a role in the protection of cells against stress by stabilizing proteins and preventing their aggregation [42]. tac41 is a member of the tac (transcription activator-like) protein family, which plays a role in the regulation of gene expression and is involved in the response to stress [43]. rbcl is a member of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme family, which is involved in photosynthesis and is upregulated in response to abiotic stress. [44]. pp2c is a member of the protein phosphatase 2c enzyme family, which plays a role in the regulation of protein phosphorylation and is involved in the response to stress [45]. wcor413 is a member of the cor protein family, which is involved in the protection of cells against cold stress [46]. fkbp77 is a member of the fk506-binding protein family, which is involved in the regulation of protein folding and is upregulated in response to abiotic stress in wheat [47]. however, a deeper understanding of the mechanisms by which these genes function and interact with each other is still needed to develop effective strategies for improving the stress tolerance of wheat. this requires further research, including functional characterization and genetic manipulation of these genes, in order to fully understand the underlying mechanisms and to apply this knowledge to improve wheat breeding and crop management practices. additionally, it will be important to investigate the interactions between these genes and the broader genetic and molecular networks that govern the plant’s response to stress. highlights in bioscience page 5 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat figure 2. heatmap of top de genes data. figure 3. venn diagram of degs. protein protein interaction (ppi) network proteins have greater diversity as biomarkers, exhibit a more direct and dynamic reaction, and have excellent application prospects in cultivar screening. in this study, we utilized the string tool to create protein-protein interaction (ppi) networks and to make gene ontology (go) annotation that spans cellular components, biological processes, and molecular functions of our studied genes that contribute to the plants response to stress. four modules were identified in this constructed network, which consists of 90 nodes and 38 edges. the most significant module was gse48620, which had 40 nodes and 30 edges and a ppi enrichment p-value of 6.95e-5, this module has some functions ex) protein domain, and features. gse45563, the smallest module, had only 12 nodes and 1 edge and a ppi enrichment p-value of 0.333. (figure 4) and this module has some functions ex) protein domains and features, molecular function (a structural constituent of the cytoskeleton), cellular components, and annotated keywords. machine learning model in this study, the extra-tree regressor and lime algorithms were employed to examine gene expression data in wheat in order to identify key genes involved in the plant’s response or resistance to abiotic stress. the results of this analysis revealed a total of 40 markers, 36 genes, and 36 gene titles, as shown in table 3. each gse accession consisted of ten markers, with each marker displaying a positive or negative value. these values represent the prediction probabilities of negative and positive outcomes achieved from the classifiers and indicate the extent to which the corresponding genes are affected by the specific abiotic stress of each gse. blue represents negative values, while orange represents positive values (as shown in figure 5). the model generated a set of genes that respond to biotic stress as the most significant, including hypothetical loc89+3077, probable light-induced protein, gstf5, pro3, tad1, ett-l alpha, rab2, imidazoleglycerolphosphate dehydratase, pp2c, triticain beta, u2af small subunit, immature spike ubiquitin-conjugating enzyme 2, ta-re, rab, delta tonoplast intrinsic protein tip2;3, hsp101b, badh, agl14, cdpk2, wndr1d, cold acclimation protein cor413-tm1, gs3, zf001, ethylene receptor-like protein, mlo2, elongation factor, sh6.2, gs2, ribosomal protein l11, metallothionein, pr4, cpk2b, ald1 myosin, protein h2a, highlights in bioscience page 6 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat figure 4. ppi networks of degs: (a) gse14697, (b) gse45262, (c) gse45563, (d) gse48620 and waox1a. it is important to note that these genes interact with each other and with genes from other families to coordinate the plant’s response to stress. further research is needed to fully understand the mechanisms by which these genes function and interact with one another in order to develop effective strategies for improving the stress tolerance of wheat. additionally, more research is needed to explore the impact of these identified genes on the plant’s overall growth and productivity and to validate the results of this study using other data sets and methods. these genes are involved in a variety of molecular functions, cellular components, and biological processes that are related to the response or resistance to abiotic stress in wheat. for example, badh is an enzyme involved in the synthesis of stress hormones, which are important for the plant’s response to stress [48]. agl14 is a member of the agl (agamous-like) protein family, which plays a role in the regulation of gene expression and is involved in the plant’s response to stress [49]. rab2 is a member of the rab family of gtpases, which play a role in the regulation of vesicle trafficking and are involved in the plant’s response to stress [50]. gs2 is a member of the glutamine synthetase family, which is involved in the synthesis of amino acids and is upregulated in response to abiotic stress in wheat [51]. additionally, heat shock proteins, such as hsp101b, play an important role in the plant’s response to stress by protecting cellular proteins from damage [52]. similarly, aquaporins, such as ta-re, are integral membrane proteins that play a role figure 5. machine learning results: (a) gse14697, (b) gse45563, (c) gse45262, (d) gse48620 in the transport of water and other small molecules and are involved in the plant’s response to drought stress [53]. furthermore, transcription factors, such as cdpk2, are responsible for the regulation of gene expression and play a critical role in the plant’s response to stress by modulating the expression of stressresponsive genes [54]. interestingly, the transcription factor cdpk2 may interact with the gene gs2, which is involved in the synthesis of amino acids, to modulate its expression in response to abiotic stress. this highlights the complexity of the plant’s response to stress and the need for further research to fully understand the mechanisms by which these genes function and interact with each other in order to develop effective strategies for improving the stress tolerance of wheat. overall, the results of this study provide a valuable starting point for further research on the genetic basis of the response or resistance to abiotic stress in wheat. by identifying key genes that play a role in this process, researchers can focus their efforts on understanding the mechanisms by which these genes function and interact with each other in order to develop effective strategies for improving the stress tolerance of wheat. this can ultimately lead to the development of new wheat varieties that are more resilient to abiotic stress, which is crucial for ensuring the food security of the world’s population. highlights in bioscience page 7 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2023 unlocking the genetic basis of abiotic stress tolerance in wheat table 3. the results of the machine learning model represent the most significant genes with their corresponding values. accsession marker p/n value gene.symbol gene.title gse14697 ta.14183.1.s1_at positive 10.62 loc543077 hypothetical loc543077 ta.3424.1.s1_at positive 9.12 loc543347 probable light-induced protein ta.3418.2.s1_at positive 14.57 gstf5 glutathione transferase f5 ta.5057.2.s1_x_at positive 8.74 pro3 profilin ta.14281.1.s1_at positive 15.75 tad1 defensin ta.25087.1.s1_at positive 9.49 ett-l alpha ettin-like auxin response factor ta.6227.1.s1_at positive 12.77 rab2 small gtp-binding protein ta.46.1.s1_at positive 7.12 loc543224 imidazoleglycerolphosphate dehydratase ta.23679.1.s1_at positive 14.02 pp2c protein phosphatase 2c ta.14289.1.s1_at positive 9.27 loc100037638 triticain beta gse45262 ta.5818.1.s1_at negative 945.07 loc780664 u2af small subunit ta.5818.1.s1_a_at negative 2522.32 loc780664 u2af small subunit ta.23834.1.s1_at negative 11596.3 loc780682 immature spike ubiquitin-conjugating enzyme 2 ta.5818.3.s1_x_at negative 619.12 loc780664 u2af small subunit ta.277.1.s1_at negative 574.07 ta-re pullulanase ta.2704.1.s1_at negative 41.07 rab rab protein ta.1082.1.s1_a_at negative 1364.3 loc100037645 delta tonoplast intrinsic protein tip2;3 ta.256.1.s1_at negative 212.45 hsp101b heat shock protein 101 ta.435.1.s1_at positive 6753.69 badh betaine-aldehyde dehydrogenase ta.6411.1.s1_at negative 235.62 agl14 mads-box transcription factor taagl14 gse45563 ta.6301.2.s1_a_at positive 248.32 cdpk2 calcium-dependent protein kinase 2 ta.5011.1.s1_at positive 1254.5 wndr1d protein kinase ta.19248.1.s1_x_at positive 12151 loc543089 cold acclimation protein cor413-tm1 ta.6301.1.s1_at positive 3090.3 cdpk2 calcium-dependent protein kinase 2 ta.5307.1.s1_at positive 1491.8 gs3 glutathione synthetase ta.13961.1.s1_at positive 241.58 zf001 gata-type zinc finger protein ta.19248.1.s1_at positive 14096 loc543089 cold acclimation protein cor413-tm1 ta.10139.1.s1_s_at positive 1084.6 loc543446 ethylene receptor-like protein ta.280.2.s1_x_at positive 201.15 mlo2 seven transmembrane-spanning protein ta.2576.1.s1_at positive 22922 loc542923 elongation factor gse48620 ta.1258.2.s1_x_at positive 11.12 sh6.2 s-adenosyl-l-homocysteine hydrolase taaffx.105423.1.s1_at positive 4.15 gs2 plastid glutamine synthetase isoform gs2a ta.28712.1.s1_at positive 5.47 loc606335 ribosomal protein l11 ta.28695.6.s1_at positive 3.65 loc542898 metallothionein ta.9226.1.s1_at positive 11.57 pr4 pathogenesis-related protein 4 ta.6350.2.s1_x_at positive 5 cpk2b calcium-dependent protein kinase ta.304.1.s1_at positive 6.27 ald ald protein ta.29355.1.s1_at positive 1.41 loc542906 1 myosin ta.644.1.s1_at positive 6.14 loc543185 protein h2a ta.233.1.s1_at positive 5.89 waox1a alternative oxidase table 4. the common genes that were shared between the gses. gse14697 and gse45262 gse45262 and gse45563 gse14697 and gse45563 wrab15 ccoamt tatil rab:1 ccoamt:1 tlk1 wcor18:1 nac693 rcab wcor18 p5cr gs3 lea3 tahsp17.3 tac41 rab,rab 15b hsp 26.6b wrab18 wcor518 wpi6 wrky hsd11bl rbcl lea2 wcs66 wtabapm pp2c wcor413 fkbp77 hsp101c hsp16.9b:1 hsp16.913lc1 cht2,hsp16.9b conclusion in conclusion, our study has discovered a group of key genes that are connected to the response or resistance to abiotic stress in wheat by leveraging differential gene expression and machine learning models. the response or resilience of wheat plants to abiotic stress situations is greatly influenced by these genes. the discovered genes also come from a variety of gene families, including heat shock proteins, aquaporins, protein kinases, and protein oxidases. to coordinate the plant’s response to stress, these genes interact with one another and other genes. to completely understand the mechanisms by which these genes operate and interact with one another, additional study is required in order to create effective strategies for improving the stress tolerance of wheat. reference 1. faostat. fao stat. database. 2019. food and agriculture organization of the united nations rome, italy; 2019. 2. mourad am, alomari dz, alqudah am, sallam a, salem kf. recent advances in wheat (triticum spp.) breeding. advances in plant breeding strategies: cereals: volume 5. 2019:559-93. 3. poole n, donovan j, erenstein o. agri-nutrition research: revisiting the contribution of maize and wheat to human nutrition and health. food policy. 2021;100:101976. 4. food, of the united nations ao. food outlook: biannual report on global food markets. food and agriculture organization of the united nations. 2021. accessed december 22, 2021. available from: http://www.fao.org/3/ ca5162en/ca5162en.pdf. 5. golfam r, kiarostami k, lohrasebi t, hasrak s, razavi k. a review of 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sativa l.). international journal of molecular sciences. 2022;24(1):288. 52. katiyar-agarwal s, agarwal m, grover a. heat-tolerant basmati rice engineered by over-expression of hsp101. plant molecular biology. 2003;51:677-86. 53. liu h, yang l, xin m, ma f, liu j. gene-wide analysis of aquaporin gene family in malus domestica and heterologous expression of the gene mppip2; 1 confers drought and salinity tolerance in arabidposis thaliana. international journal of molecular sciences. 2019;20(15):3710. 54. zhao l, li y, li y, chen w, yao j, fang s, et al. systematical characterization of the cotton di19 gene family and the role of ghdi19-3 and ghdi19-4 as two negative regulators in response to salt stress. antioxidants. 2022;11(11):2225. highlights in bioscience page 10 of 10 december 2023|volume 6 http://bioscience.highlightsin.org/ abstract introduction methodology data gathering differential gene expression analysis results and discussion identification of degs protein protein interaction (ppi) network machine learning model conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202101 research article open access 1 faculty of sciences of tunis, biology department, research unit of physiology and aquatic environment, university of tunis el manar, 2092 tunis, tunisia. 2 aquatic environment exploitation resources unit, higher institute fishing and fish farming of bizerte, tunisia. contacts of authors * to whom correspondence should be addressed: imene chetoui received: september 24, 2020 accepted: january 12, 2021 published: january 20, 2021 citation: chetoui i, ghribi f, bejaoui s, ghalghaa m, el cafsi m, soudani n . assessment of stress biomarkers responses in mantle and adductor muscles of mactra stultorum following lead exposure. 2021 jan 20;4:bs202101 copyright: © 2021 chetoui et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. assessment of stress biomarkers responses in mantle and adductor muscles of mactra stultorum following lead exposure imene chetoui*1, feriel ghribi1, safa bejaoui1, mohamed ghalghaa2, m'hamed el cafsi 1, nejla soudani1 abstract the objective of the present work is to evaluate the possible toxic effect engendered by graded doses of lead chloride (pbcl2) on mactra stultorum mantle and adductor muscles through a battery of biomarkers responses. m. stultorum were divided into 4 groups and exposed to three concentrations of pbcl2 (d1:1mg/l, d2: 2.5 mg/l and d3: 5 mg/l) with control during five days. our findings showed decreases of lipid contents in both organs following pbcl2 exposure, while, proteins declined only in the adductor muscles of the treated m. stultorum. during our experiment, the pbcl2 exposure induced the levels of metallothionein (mts), malondialdehyde (mda) and advanced oxidation protein products (aopp) in both organs as compared to the control. these biomarkers responses are distinctly different between mantle and adductor muscles. keywords: lead chloride, mactra stultorum, mantle, adductor muscles, biomarkers responses. introduction the contamination of aquatic ecosystems by several environmental pollutants has become a worldwide problem in the last years [1]. the presence of heavy metals in those environments and their accumulation in marine organisms has been largely investigated during the last decades because of their harmful effects and persistence [2]. for the global environmental health, lead (pb) is considered to be a major hazard. this non-essential and toxic heavy metal is the most abundant metal in the aquatic system. in nature, it is present as a divalent cation and principally forming stable complexes with sulfur. it has a natural origin or it is realized from many industrials discharges such as lead ore mining and smelting, refining, alkyl-lead petroleum combustion, batteries and cement manufacture [3]. at the national level, lead is one of metals contaminating the tunisian coasts because of its highest concentrations and has been considered a major source of pollution in tunisian waters [4,5]. thus, high pb levels which are exceeding the permissible limit (1 mg/kg), [6] have been recorded in tunisian bivalves tissues (values comprised between 5 and 9 mg/kg dw) [5,7]. moreover, their accumulation in aquatic ecosystems can become dangerous to all kinds of organisms including bivalves, fishes, aquatic plants and human life, causing many toxic effects [8]. mactra stultorum is considered as ecologically important components of marine environments and an important edible marine bivalve due to their richness of protein and essentials fatty acids [9, 10]. it has a wide distribution along the mediterranean and atlantic coasts and estuaries [11]. so, it is abundant specie in the subtidal area and shallow seas along the coast of tunisia [12]. moreover, due to their benthic and sedentary mode of life; suspension-feeding mode and high filtration rate, it is easily exposed to environmental pollution [13]. m. stultorum can accrued a enormous number and high concentration of heavy metals in their tissues [14,15,16]. therefore, similar to other species of mactra, mussels and oysters, m stultorum can be used as a good bioindicator of heavy metal pollution in marine environments [17]. highlights in bioscience page 1 of 8 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202101 https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure the excessive accumulation of lead in the bivalve tissues can induce oxidative stress through the overproduction of reactive oxygen species (ros) in the cells which affect cellular functions [18,19]. so, when these increases of ros levels get over the scavenging capacity of organisms, the superfluous free radicals may advantage to oxidative damage in basic biological molecules, such as lipid peroxidation, protein oxidation and dna damage [18,19,20]. thus, the lipid peroxidation constitutes an involvement of self-propagating sequence of chemical reactions that occurred in the bulk phase of cell membrane lipid bilayers. malondialdehyde (mda) and 4-hydroxyalkenals are the degradation products of lipid peroxidation and their levels reflect the degree of oxidative damage and constitute a biomarker specific to environmental stresses [21]. moreover, lipid peroxidation and other damages resulting from metal toxicity are modulated by antioxidant systems and stress proteins such as metallothioneins (mt) [22]. these systems of defense play a key role in the alive organisms which can provide for the different cells the protection against environmental toxicity control metabolism homeostasis [22]. metallothionein (mt) is a low-molecular-weight and cysteine-rich protein, identified for the first time in the kidney of the horse [23]. mt’s plays a crucial role in metal metabolism and principally in the detoxification mechanisms as a metal-chelating agent for the excess of metals in the cells [24]. further, it ensure an essential role in immune response [25], antioxidant processes [26,27], and response to estrogenic compounds [28]. in aquatic environments, mt has been implied to be used as a bioindicator for metal contamination because of its possibility to bind to particular metals. even though there are investigations about the impact of pbcl2 on m. stultorum gills and digestive gland [15,16], still information are lacking about the impact of this metal on the metabolism and redox status of other organs. thus, in this study, we focused on identifying the metabolic and redox strategies developed by m. stultorum adductor muscles and mantle to cope with graded pbcl2 concentrations. materials and methods experimental protocol mature clams individuals (shell length (sl): 3.5 ± 0.63 cm and total weight (tw) 8.03±0.47 g) were collected at 1 m depth by scuba divers from the bizerte lagoon. after sampling, clams were acclimated for 7 days in aquaria (20 l). the water was daily renewed and physicochemical parameters were controlled (temperature (18°c), salinity (30 psu), ph (7.4 ± 0.2), and photoperiod (12h/12h)). during the acclimation period, green microalgae isochrysis affinis galbana (t-iso, 2 million cells per ml) was fed regularly to m. stultorum. at the end of the acclimation period, clams were divided in 4 groups of 18 clams. each group was placed in 8 l plastic aquaria and was represented in triplicate (n=6 clams per replicate). clams first group was kept in aquaria1 containing filtered natural seawater (control), while other groups were exposed for 5 days to different concentrations of unmixed pbcl2 metal (lead chloride; pbcl2; sigmaaldrich; powder 98%) which was dissolved in pure water. during metal exposure, clams were exposed to graded concentrations of pbcl2 as follows: aquaria1: control; aquaria2: 1mg/l; aquaria3: 2.5mg/l and aquaria4: 5mg/l with controlled conditions as mentioned above (figure 1) and no added food. half (50%) aquaria water volume was replaced every 24 h in order to maintain the water quality, and concentrations of pbcl2 were reestablished. pbcl2 concentrations were selected based on previous trials achieved on other bivalves [29,30]. during the experimental period, no mortality has been reported. preparation of the samples for biochemical analyses after pbcl2 exposure, clams were sacrificed and the mantle and adductor muscle were quickly removed and rinsed with cold distilled water in order to remove the externally bound pb. 6 replicate of each group tissues (mantle and adductor muscle; n=3 for each replicate) were homogenized in a tris-hcl buffer (20mm; ph=7.4) in cold conditions, then centrifuged at 10.000 × g for 20 min (4°c). tissues supernatants were stored at -80°c for oxidative stress parameters analysis. biochemical analyses for biochemical analysis, chemicals were purchased from local commercial suppliers. except for 5,5-dithio-bis-(2nitrobenzoic acid) (dtnb) and 2-thiobarbituric acid) (tba) they were purchased from sigma chemical co (saint louis, mo 63103, usa). protein quantification based on lowry et al. [31] method, protein content was estimated using folin reagent and bovine serum albumin (bsa) as a standard. 2 ml of a solution mixture (sodium carbonate hydrate dissolved in a solution of naoh (0.1n), copper sulfate and sodium hydrate dissolved in water) were added to 10 µl supernatants. then, 200 µl of folin reagen was added to the formed mixture for reaction activation. after 30 min incubation, protein content was determined at 540nm using the spectrophotometric method. lipid quantification according to the method of goldsworthy et al. [32], lipids were determined and the extraction was carried out following shibko et al. [33] method. 0.5 g of tissues are removed, cut and macerated in 10 ml of trichloroacetic acid (tca, 20%). after grinding and filtration and a first centrifugation 5000× g for 10 min, the pellet is kept in the same tube which we added 1 ml of the mixture ether / chloroform (1 v/ 1v). subsequently, the last volume is submitted to second centrifugation 5000× g for 10 min and 100 μl of the supernatant is taken which we added 1 ml of sulfuric acid. and after stirring, the tubes were placed in a hot water bath (100 °c) for 10 min. then, we added 2.5 ml of the sulfophospho-vanillin mixture (85%) to 200 μl of the extract highlights in bioscience page 2 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure figure 1. experimental conception of mactra stultorum exposed to graded doses of lead chloride (pbcl2). which was incubated for 30 minutes in a darkroom. lipid quantities are determined by the spectrophotometric method at 530 nm. a calibration range was carried out from a stock solution prepared from sunflower oil. the lipid contents are expressed in mg/g wet weight (mg/gww) malondialdehyde (mda) measurement mda level was determined according to draper and hadley [34]. an aliquot of 0.5ml of each tissue supernatant was incubated for 1 hour in heated water (37°c) and mixed with 0.5 ml of trichloroacetic acid (tca 30%). after centrifugation for 10 min (3500× g/4 °c), we added 500µl of thiobarbituric acid (tba 0.67%) to 0.5 ml of supernatant. after incubation for 10min, mda levels were determined by spectrophotometric method at 532 nm and expressed as nmol /mg protein. advanced oxidation protein products level (aopp) measurement the advanced oxidation protein products (aopp) levels were determined following the method of kayali et al. [35]. after protein precipitation in double volumes of phosphate buffer (0.1m; ph = 7.4). then, potassium iodide (1.16m) and absolute acetic acid (200µl) were added to clams supernatants. we used the extinction coefficient of 261 cm-1 mm-1 for aopp quantification. aopp levels were determined at 340 nm and expressed as μmol/ mg of protein. metallothionein (mts) content according to the method of viarengo et al. [36] modified by petrovic et al. (2001), mts were determined. the supernatant of each tissue from each group (500µl) was mixed with ethanol/chloroform solution (95%; 1%). after centrifugation for 6000 x g during 10 min in cold, edta (1mm) and nacl (0.25m) were added to the pellets and mts absorbance was measured at 412nm. mts were expressed as nmol gsh/mg protein. statistical analysis statistica software version 5.0 was used for statistical analysis. the homogeneity and normality of variables were tested using the shapiro-wilcoxon test. differences between variables highlights in bioscience page 3 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure were assessed by one-way anova and deemed significant at p < 0.05. for each parameter, results were expressed as means ± standard deviation (sd). pearson correlation matrix and principal component analysis (pca) were used to discriminate significant correlations between biochemical parameters. results the pbcl2 effects on general behavior of m. stultorum no mortality was noticed in treated clams by pbcl2 different doses. during the experimental period, clams behavior was regularly monitored (e.g. filtration activity, reduced respiration, siphon retraction. . . ). those parameters remained stable even in clams exposed to the high dose of pbcl2 (d3). estimation of total protein contents results showed no significant variation in protein content of clam's mantle tissues after pbcl2 treatment. however, significant decreases in the amount of total protein were revealed in the adductor muscles of m. stultorum following lead exposure (-23%, -24% and -20% in clams exposed to doses d1; d2 and d3 respectively compared to controls (table 1). estimation of lipid contents lipids contents decreased significantly in clams mantle and adductor muscle tissues after 120 hours of exposition to pbcl2. compared to the control, this decline was recorded for the mantle by -37%, -60% and -67% in clams exposed to 1mg/l; 2.5mg/l and 5mg/l respectively) and for the adductor muscles by -32 % and -60% in clams treated by d2 and d3 respectively (table 1). estimation of malondialdehyde (mda) levels results showed that the progressive accumulation of pb in both m. stultorum tissues induced lipid peroxidation in all treated clams by pbcl2 which was revealed by mda levels enhancement. in the mantle tissues and compared to the control, mda levels increased with graded doses (d1, d2 and d3, respectively) by 57%, 150% and 575%. we also noted that mda levels in the adductor muscles increased significantly by 58% and 74%, in clams treated with the highest doses (d1 and d2) (figure 2). estimation of advanced protein oxidation products (aopp) levels significant increases in aopp levels were recorded in the mantle of all pbcl2 treated groups (1, 2.5 and 5 mg/l pbcl2) with a dose dependent manner (+60, +65and +205% respectively). while in the adductor muscles tissue and compared to control, the increase of aopp levels was observed only for clams exposed to 2.5mg /l; (+102%) and 5mg / l; (+132%) (figure 3). estimation of metallothionein (mt’s) levels mt’s levels increased significantly only in clams mantle tissues exposed to the highest dose of pbcl2 (5mg / l; 163%) during 5 days. furthermore, the mt’s levels increased significantly in the adductor muscles with a dose dependent manner (+30, +45 and +268%) with d1, d2 and d3 pbcl2 doses compared to control (figure 4). figure 2. the mda levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). *** p <0.001: pb cl2 groups vs controls for each tissue. ++ <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. figure 3. the aopp levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. ++p <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. principal component analysis (pca) and correlation matrix correlation matrix and pca were established in order to understand in the first time the effect of lead graded doses on stress biomarkers in mantle and adductor muscles tissues of mactra stultorum and in second time to compare the response of these biomarkers between these tissues (figure 5 and table 2). the first two factorial axes that explain 90.96% of the total variance (figure 5). factor 1 (56.38%) was characterized by high mda and mt’s levels (figure 5). factor 2 (33.37 %) was characterized by aopp levels. results showed that protein and lipid contents are intermediates compounds for f1 and f2. pca results showed that there were two significant separations, the first one between controls of mantle and adductor muscles and the treated groups and the second one between both tissues from all treated groups (figure 5). control mantle and adductor muscles were projected in the positive sides of two factorials axes, highlights in bioscience page 4 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure table 1. protein and lipid contents in the control and treated m. stultorun mantle and adductor muscle with pbcl2 graded doses (d1. d2. d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and each tissues (n=3 clams.). a: nmol/mg protein. b: mg/mg protein. pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. +++p <0.01; +++ p <0.001: mantle vs adductor muscles for each condition. ct d1 d2 d3 proteina mantle 10.4± 0.67+++ 8.37± 0.76+++ 9.84± 0.64+++ 7.84± 0.69+++ adductor muscle 43.4± 4.91 33.4± 2.02* 32.8± 1.94* 34.7± 5.14* mantle 4.2± 0.92++ 2.62± 0.71* +++ 1.67± 50.27* +++ 1.42± 0.32* ++ lipidb adductor muscle 5.92± 0.35 5.89± 0.26 4.02± 0.28* 2.37± 0.83* figure 4. the mt’s levels in the control and treated m. stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. values are expressed as means ± sd, 6 replicate in each group and tissues (n=3 clams). pb cl2 graded doses: d1 (1mg/l pb cl2); d2 (2.5mg/l pb cl2); d3 (5mg/l pb cl2). ** p <0.01;*** p <0.001: pb cl2 groups vs controls for each tissue. +++ p <0.001: mantle vs adductor muscles for each condition. explaining by the high contents of lipids and protein and minor levels of lipid peroxidation and mt’s. second group was dominated by treated mantle clams by pbcl2 which represented the negative side of f1 and the positive side of f2; revealing important lipid and protein oxidation. the third one including the adductor muscles from m. stultorum from all treated groups was characterized by a minor response of stress biomarkers comparing to mantle tissues and by a remarkable decrease in protein and lipid contents especially for clams exposed to high dose. clearly, biomarkers responses involved in oxidative stress were significantly enhanced in both tissues clams treated with high pbcl2 dose when compared to control groups. table 2. correlation analysis (pearson correlation) between the biochemical parameters in the control and treated m. stultorun mantle and adductor muscle with pb cl2 graded doses (d11.d2. d33 during 5 days. correlation coefficients statistically significant (p <0.05) . ns: not significant (p <0.05). mantle adductor muscles protein lipid mda aopp protein lipid mda aopp lipid ns ns mda ns -0.98 0.98 ns aopp ns ns 0.98 -0.95 -0.96 ns mt ns ns 0.99 0.99 0.97 ns 0.99 ns figure 5. principal analysis component (pca) represented by two factors f1 and f2 and produced by biochemical variables in control and stultorum mantle and adductor muscles with pb cl2 graded doses (d1, d2, d3) during 5 days. projection of the variables and the cases on the factor-plane (1×2); d1:1mg/l pbcl2; d2:2.5mg/l pbcl2; d3:5mg/l pbcl2; mt: mantle; am: adductor muscles. pearson correlation matrix showed that protein contents in adductor muscles is significantly negatively correlated (p<0.05) with aopp levels. while, lipid contents in mantle showed a negative correlation with mda levels (p<0.05). positive correlation was recorded between aopp, mda and mts levels for mantle tissue. while, negative correlation was observed between aopp levels and lipid contents for adductor muscles tissue. thus, mt’s presents in this tissue positive correlation only with mda and protein levels (table 2). discussion biological responses in native indicator species can give total and pertinent information on the potential impact of metal toxicity on ecosystem dynamics. heavy metals constitute a group among environmental pollutants because of their bioaccumulation and non-degradable property. lead is a non-essential metal, abundant in the oceans and exhibits widely regarded toxic effects associated with the stimulation of radical processes [37]. lead is accumulated by different organisms in aquatic systems and became dangerous to all kinds of organisms, including highlights in bioscience page 5 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure bivalves, fishes, and aquatic plants and finally is transferred to human life [3]. lead in the divalent cation form pb2+ can be transported into the intracellular part through the ca2+ transport systems thereby might altering the calcium homeostasis [38], affecting different cellular functions and enzymatic activities and causing dna damage [39]. to our knowledge, our study is the first to assess the redox status in the mantle and adductor muscles of m. stultorum after pb cl2 exposure. furthermore, among the established mechanisms of lead toxicity is its ability to induce oxidative stress following an overproduction of ros. an imbalance between antioxidants and pro-oxidants is a result of this toxicity and causing an alteration in redox status, lipids peroxidation and protein oxidations [39-40]. malondialdehyde (mda) constitutes the final products of lipid peroxidation indicating the degradation of lipids; it is therefore used as a good biomarker of lipid damage in aquatic organisms [4]. the oxidation of lipids during our experiment was confirmed by significant increases in mda levels in the mantle and adductor muscles in all animals treated groups. the lipid membranes are the primary targets of oxidative damage [42]. so, the membrane integrity and fluidity in the bivalve’s cells is altered by the pro-oxidant effects of pbcl2 which was proved by these raise of mda levels. as well, these high mda levels are accompanied by high and significant decreases of lipid quantities in both tissues mainly for the clams exposed to d2 and d3 which also demonstrated a significant and negative correlation with the lipid contents in both tissues.. moreover, the hypothesis that the inorganic cations as pb2+ can stimulate the lipid peroxidation processes through the oxidation of polyunsatured fatty acids (pufa) is generally suggested [37]. indeed, these pufa are extremely sensitive to oxidation via ros’s attack due to their high number of double bonds per fatty acid molecule [43]. in this context, the richness of mantle and adductor muscles by pufa recorded by chetoui et al. [9]can explain these low levels of lipids content and high mda levels which are necessary the results of the toxic effects of lead [29,44]. additionally, the generation of ros is the main consequence of protein damage [45]. in our experiment, increases of aopp levels are recorded in all m. stultorun treated groups for mantle and in m. stultorun treated with d2 and d3 for adductor muscle suggested that the harmful effects of pb accumulation are leading to excessive protein oxidation. this oxidation of protein in the adductor muscles was associated with a remarkable and significant decrease in the amount of total protein during the treatment which probably due to their richness of total proteins compared to mantle [10]. these declines of protein quantities in am are negatively correlated with the protein contents in this tissue. however, in the mantle, the total protein has not changed during the treatment. previous research has shown that the alteration of protein in other tissues of m. corallina and venus verrcosa are observed following the lead toxicity [16,46]. similar decreases of protein content have been demonstrated in oyster gills and mussel digestive glands after metals exposure [43]. it is widely reported that the induction of mt’s in marine organisms by metals is the result of their use as metal-pollution biomarkers [47]. these sulfhydryl groups (-sh) are involved in the detoxification processes [26]. and by capturing free radicals, these metalloproteins were able to acquire protective activity [48]. our data showed that treatment with high pb concentrations (5mg/l) leads to an increase of mt’s content in the mantle tissue which is highly and positively correlated mda and aopp levels. however, we revealed that total metallothionein (mts) contents in adductor muscle enhanced in a dose-dependent manner which demonstrated a high and positive correlation with proteins and mda levels. these mt‘s inductions reflected the impairment of both tissues functions in m. stultorum. moreover, by comparing to the adductor muscles, this protein (mt's) is more active (or more expressed) in the mantle for each treated group. according to kumari [49], the increase in mt concentrations seems to be a result of the increased transcriptions. and this suggestion has been demonstrated in m. veneriformis following mercury exposure which the basal mvmt mrna expression was according to the ranking of the tissues like the following: digestive gland>mantle> gill>adductor muscle>foot [50]. besides, these high mt’s induction in the mantle than in the adductor muscles in our study can probably be firstly by the possibility of that tissue to accumulate more quantity of metals than the adductor muscle [29,51,52]and secondly by the activation of their main role in metal detoxification when the lipid peroxidation and protein oxidation reached the maximum. our results are therefore in agreement with previous studies carried out on mytilus galloprovincialis mantle and m. corallina tissues after lead exposure [15,16,29]. in the literature, the metals accumulated (as lead) in mantle and adductor muscles tissues seem to be lower than in the digestive gland and gills of venus veruscosa, mytilus galloprovincialis, callista chione, perna viridis and modiolus metcalfei [29, 51,52]. moreover, in our study, the stress biomarker response is clearly and significantly different between the two organs and appears to be more accentuated in the mantle of m. stultorum treated groups. these results can be explained that similar to gills, the mantle is also located in the mantle cavity which can directly interact with marine pollution and consequently can accumulate metals more than adductor muscle [29,51,52]. conclusions in conclusion, the present results demonstrate that pbcl2 exposure (1mg / l; 2.5mg / l and 5mg / l) alters similarly the redox status of m. stultorum mantle and adductor muscles. the toxic effect of lead induces similarly the lipids peroxidation confirmed by the increases of mda levels which they were associated with decreases in lipids contents in both tissues. the alteration of proteins expressed by the elevation of aopp levels in both tissues confirms the harmful effects of lead. they were correlated with a decrease in protein content only in adductor muscles. the capacity of m. stultorum to increase their mt’s highlights in bioscience page 6 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience chetoui et al., 2021 assessment of stress biomarkers responses in mactra stultorum following lead exposure concentrations in both tissues seems to be an essential cellular adaptive system defending the animal against the lead-induced toxicity. these biomarkers responses in mantle and adductor muscle tissues elucidate the installation of oxidative stress by their increases in pbcl2 treatment as compared to controls. however, they are distinctly different between mantle and adductor muscles. we can then deduce that the toxic effects of lead are greater in the mantle than in adductor muscles. acknowledgments the tunis university of sciences and the research unit of physiology and aquatic environment supported this work. references 1. di salvatore p, calcagno ja, ortiz n et al. effect of seasonality on oxidative stress responses and metal accumulation in soft tissues of aulacomya atra, a mussel from the south atlantic patagonian coast. mar. environ. res.2013; 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97(c): 37-42. 38. quintanar-escorza ma, gonzalez-martnez mt, navarro l et al. intracellular free calcium concentration and calcium transport in human erythrocytes of lead-exposed workers. toxicol. appl. pharmacol. 2007; 220: 1-8. 39. dafre al, medeiros id, muller ic et al.antioxidant enzymes and thiol /disulfide status in the digestive glands of the brown mussel perna perna exposed to lead and paraquat. chem.biol. interact. 2004; 149:97-105. 40. trombini c, fabbri e, blasco j. temporal variations in metallothionein concentration and subcellular distribution of metals in gills and digestive glands of the oyster crassostrea angulata. adv. mar. chem. 2010: 143-152. 41. misra ds, maiti r, ghosh d. protection of swimming -induced oxidative stress in some vital organs by the treatment of composite extract of withania somnifera, ocimum sanctum and zingiber officinalis in male rat. afr. j. trad, complement. 2009; 6: 534-543. 42. los da, murata n. membrane fluidity and its role in the perception of environmental signals. biochem. biophys. acta. 2004;1666 (1-2): 142-157. 43. geret f, serafim a, barreira l et al. response of antioxidant systems to copper in the gills of the clam ruditapes decusscatus. mar. environ. res. 2002; 54: 413-417. 44. shenai-tirodkar ps, gauns mu, mujawar m wa et al. antioxidant responses in gills and digestive glands of oyster crassostrea madras ensi (preston) under lead exposure. ecotoxicol. environ. safety. 2017; 142: 87-94. 45. prevodnik a , lilja k , bollner t. effects of copper or cadmium on protein expression response in baltic sea blue mussels (mytilus edulis l.). mar.environ.res.2006; 62:90-91 46. bejaoui s, telahigue k, chetou i et al. effects of lead exposure on redox status, dna and histological structures in venus verrucosa gills and digestive gland. chem. ecol. 2020ă; https://doi.org/ 10.1080/0275 7540.2020.1742329 47. amiard jc, amiard-triquet c, barka s et al. metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. aquat. toxicol. 2006; 76(2): 160-202. 48. davis sr, cousins rj. metallothionein expression in animals: a physiological perspective on function. j nutr, 2000; 130:1085 1088 49. kumar d. role of non-enzymatic antioxidants in stimulation of metallothionein against metal toxicity. inter. j. environ. sci. techno. 2012; 2 (3): 15961604. 50. fang y, yang h, liu b. tissue-specific response of metallothionein and superoxide dismutase in the clam mactra veneriformis under sublethal mercury exposure. ecotoxicology. 2012; 21: 1593-1602. 51. chalkiadaki o, lydakis-simantiris n, dassenakis m. lead accumulation and metallothionein induction in different tissues in mussels (m. galloprovincialis) and clams (c. chione) exposed to various pb concentrations. proceedings of the 13th international conference on environmental science and technology athens. 5-7 september 2013, greece. 52. ponnusamy k, sivaperumal p, suresh m. et al. heavy metal concentration from biologically important edible species of bivalves (perna viridis and modiolus metcalfei) from vellar estuary, south east coast of india. j. aqua. res. dev. 2014; 5 (5):1000258-1000263. highlights in bioscience page 8 of 8 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods experimental protocol preparation of the samples for biochemical analyses biochemical analyses statistical analysis results the pbcl2 effects on general behavior of m. stultorum estimation of total protein contents estimation of lipid contents estimation of malondialdehyde (mda) levels estimation of advanced protein oxidation products (aopp) levels estimation of metallothionein (mt’s) levels principal component analysis (pca) and correlation matrix discussion conclusions acknowledgments references highlights in bioscience; paper format highlights in bioscience issn: 2682-4043 highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 1 of 6 citation: zaimah n. s. (2019) plant resistance gene, snp and genome annotation an overview. highlights in bioscience, volume 2.article id 20192, dio:10.36462/ h.biosci.20192 received: june 19, 2019 accepted: july 13, 2019 published: august 12, 2019 copyright:© 2019 zaimah .this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. plant resistance gene, snp and genome annotation an overview nor s. zaimah sciences research institute, selangor, malaysia abstract plants have developed systems of effective and passive protection to safeguard themselves from pathogens. active processes include kinds of immune responses that are adaptive and intrinsic. adaptive immunity is focused on reaction type rnai and works primarily against viruses. biological immunity is more general and allows the plant to protect itself against a wide range of pathogens through bacterial and model resistance receptors (pprs) and forms of resistance (r proteins). pprs identify molecular models associated with microbes or pathogen that are preserved in a specific category of pathogens. with such big marker amounts, it has become feasible to scan the entire genome for interactions of individual markers with particular quantitatively hereditary traits called wholegenome studies (wgs), genome-wide association trials (gwas), or association genetics at exceptionally elevated marker densities. several snp recognition methods have been used in a specified plant to identify significant amounts of snps. these include: identification of snp based on est feature information; identification of snp from sequenced genomes; re-sequencing of amplicons; identification of snp using sequencing techniques of the next generation and identification of snp based on cluster tests. pathologic recognizes parts of genbank completely annotated genome and metacyc has been used as a database for the reference pathway. in contrast to sequence similarity information used in other systems, pathologic uses genbank annotation information and the ec assignment as evidence of the presence of each pathway in the genome of interest reference database. when the matching task is finished, pathologic will infer a number of reactions expected to take place in the destination genome and will determine which one of those pathways in the target genome are likely to exist. keywords: resistance proteins, genome-wide association, snp, biological pathways, genome annotation . review article open access plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 2 of 6 introduction proteins of resistance, in turn, identify distinctive elements of avirulence (avr) which are not conserved between many pathogens. induced-signaling resistance protein contributes to the manufacturing of reactive oxygen species and the induction of a particular form of programmed cell death, called the hypersensitive response, which damages the impacted cells (1). also called gene-to-gene resistance is the resistance of protein-mediated innate immunity, as each r gene reacts to a particular pathogenic avr gene (1). as a result, a big variety of r genes per plant genome are anticipated to be prepared to confer resistance to a wide range of pathogens. r genes are also under selection diversification to maintain pace with the fast pathogen evolution. although distinct r genes react to very unique pathogens, they share multiple regions (domains) that have been conserved. r proteins can be split into four subclasses on the basis of these domains. snp markers have acquired a great deal of concern in the science and breeding society (2) over the past two centuries. they happen in nearly infinite amounts as variations between individual nucleotides and each snp is a possibly helpful marker in single copy dna. in the study of human genome, the potential of snp markers is obviously illustrated. several million snp markers have been recognized (3) and techniques have been created concurrently (mainly up to 1 million) to evaluate big amounts of snp markers based on huge study attempts and the complete description of the human genome. as more genome sequences became accessible, comparative assessment of various genomes became a very significant research method. compared to various genomes of selection, biological pathway assessment is conducted using a range of computational techniques and databases (4,5). there are a range of enzymes and their substrates and products involved in a biological pathway. pathways also communicate with each other. thus, comparison pathway assessment is quite complex and can hardly be achieved without well-designed software systems for pathway assessment (4). we review the concept of plant resistance genes, plant snp markers and plant genome annotation. resistance genes most r proteins comprise a key nucleotide binding site (nbs) that functions as a molecular switch to regulate the protein's activity status, and a c-terminal, leucine-rich repeat region (lrr) needed to recognize avr factor. thus, the ranking of r proteins is focused on n-terminal domain variety (1). drosophila toll and human interleukin receptors categorized as tir-nb-lrr proteins are homologous to nbs-lrr form r proteins with n-terminals. non-tir nbslrr proteins are known to as cc-nbs-lrr proteins since some non-tir proteins in their n terminus form a coiled coil (cc) domain (6). furthermore, there are two categories of r proteins in their n terminus that contain an extracellular lrr. one of these groups, called kinases (rlks) receptor, includes a kinase domain of cytoplasmic protein (7). this cytoplasmic protein kinase domain is also lacking in receptors such as proteins (rlps). since r genes from distinct plant species combine conserved domains, they could be used to monitor plant genomes for r genes and putative r genes (e.g., analogs of resistance genes, rgas) and to generate molecular markers (7). meyers et al. (8) investigated a genome-wide assessment of 149 nbs-lrr processing genes in arabidopsis and verified either 55 cc-nbs-lrr (cnl) or 94 tir-nbs-lrr (tnl) proteins in two significant groups. eight significant motifs varied in their deviation within and between cnl and tnl clusters, and in the model found in particular for plant r protein homologues. introns may be more prevalent in cereals in the nbs region than in dicots. only participants of the arabidopsis rpp8/hrt gene class contain introns in the nbs domain in 20 named dicot nbslrr genes. nevertheless, in their nbs region, pib (9), pi-ta (10) and mla1 (11), three distinguished plant resistance genes have introns. repeats rich in leucine (lrrs) consist of redundant incomplete amino acid sections that fold into solvent-exposed β-cell β-loop constructions and this domain is believed to be engaged in ligand binding and disease identification (12). alternating conservation motifs and hyper variance marked lrr areas. the variation is highest for codons (x) situated in the lrr consensus xxlxlxx around the two preserved aliphatic amino acids, and the number of lrr repeats ranges among family members. there were approximately 65 amino acids in tnl proteins between the nbs and lrr domains in the genome-wide assessment of arabidopsis lrr domains (12). in arabidopsis, in cnl proteins, lrrs represent about half of the c-terminal region in the tnl proteins and almost the entire c-terminal region. the median domain of tnl lrr and cnl lrr comprised an average of 14 lrrs with ~10 separate meme patterns spanning as many as 350 amino acids as possible. in the rice proteins, a total of 25 distinct lrr motifs have been recognized. in any one gene, the amount of lrr clusters varied from 3 to 40 (13) . there are more than 150 genes of nbs-lrr in the arabidopsis thaliana dna. a sum of 166 nbs-lrr sequences were mentioned in (13), along with 33 truncated sequences. these nbs-lrr genes happen in their chromosome layout as 51 plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 3 of 6 singletons and 40 clusters. meyers et al. (2003) identified more nbs-lrr mutations by using comprehensive manual re-annotation of the same species genomic structure. they identified 149 nbs-lrr genes as well as 58 truncated genes; spread as 40 singletons and 43 clusters were the 149 non-truncated genes. in arabidopsis thaliana, tir-nbs-lrr genes family was detected out cc-nbs-lrr genes by approximately two to one, showing either a latest amplification of the former group or reduction of the latter gene group (13–15). nbslrr gene loci arabidopsis are often not dispersed uniformly in the chromosomal genome. there are super groups on chromosomes 1 and 5 whereas chromosomes 2 and 3 were comparatively lacking in the genes of nbs-lrr (13,14). the clusters are believed to be engaged in both r-gene diversity generation and conservation. meyers et al. (14) noted that the nbs gene chromosomal allocation is substantially non-random in maize: chromosome 11 includes about one-quarter of the nbs genes. five hundred and thirty-five nbs encoding genes have been recognized in rice, including 480 non-tir nbs-lrr genes. tir-nbs-lrr genes have been not detected in the rice genomic dna. in 44 gene clusters, two hundred and sixty-three genes (51 percent) occurred. in the "clustered" allocation category, there are 40 pairs and 17 triplets, 394 genes. a total of 125 singletons of nbs have been distributed across all chromosomes. in the plant genome, the proportion of singletons to the complete amount of nbs chromosomes (24.1%) was comparable to that of arabidopsis (26.8%). baumgarten et al. (16) proposed that most of the genomic dispersion of nbs-lrr genes was produced by duplication and translocation of whole chromosome sections (segmental duplication), rather than by small-scale ectopic duplication. most of the dramatic variation happens within local chromosomal areas of the nbs-lrr gene copy number. zhou et al. (17) revealed that 51% of nbs genes in rice originated in 44 gene clusters, where a cluster is an area with four and sometimes more genes within 200 kb or less. many surveys of nbs-lrr sequences or analogs of resistance genes have shown that r genes or nbs-lrr sequences are also structured in big clusters in other plant species. high-throughput genomic studies and plant genome sequences accessible in international databases provide unprecedented possibilities to recognize novel r-genes, investigate their role and method of diversification, find new genes for resistance, and eventually elucidate their interaction processes between pathogens and their crop hosts (18). in 2009, sanseverino et al. (18) launched the plant disease resistance gene database (prgdb), a comprehensive repository of r-genes across hundreds of plant species, with the intention of facilitating research on this agriculturally important gene family. a total of 16,844 gene records were included in prgdb version 1.0. of these 73 r-genes (e.g. the ' reference ' data collection) were recognized and manually curated, 6,308 were putative r-genes recovered from ncbi genbank, and 10,463 were putative r-genes computationally anticipated from ncbi unigene information. many plant genome-sequencing initiatives have developed quickly over the past few years. for example, potato (19), tomato (20), and melon (21) genomes have been finished, providing an chance to find extra r-genes (22). sanseverino et al. (22) presented an overview of the prgdb database of crop resistance genes. this database has been extended to include more than 6-fold valuable biological data from 233 plant species on a total of 104,459 r-genes. of these papers, 112 are defined in the literature as manualcurated r-genes to confer resistance to 122 distinct pathogens. all other genes were predicted gene. snp identification based on est sequence data large amounts of expressed sequence tags (ests) were produced for many plant species (23). the number of accessible ests in the ncbi est database ranges from less than 10,000 to over one million ests in significant crop plants and 1.5 million ests for the arabidopsis model plant. these ests were obtained in many cases within the framework of international efforts and were accumulated from a limited set of different lines and could therefore represent as a source for snp identification. in some instances, ests were specifically produced for snp detection from distinct lines as in arabidopsis thaliana (24) and in other instances, using bioinformatics assessment techniques (25), ests from heterozygous extremely polymorphic samples were used to identify snp. snp identification from sequenced genomes currently, many crop species genomic sequences have been released. sequenced genomes can be used to identify big amounts of snps in several respects. snps can be mined immediately in the genomic sequence in the event of heterozygous species such as grape or poplar, since two genome models have actually been produced. two distinct specimens were sequenced in the scenario of rice (26) and grape (27) so that snps can be mined by comparing the two genome models (28). for arabidopsis thaliana , genome resequencing panels were built based on the col-0 chromosome sequences and used among 20 genotypes to identify snps based on hybridization (29,30). plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 4 of 6 as a public database (https://www.ncbi.nlm.nih.gov/), the single nucleotide polymorphism database (dbsnp) was created. this database contains 1,648,103,041 snps for many organisms, e.g., brassica napus (901.5 thousand snps); arabidopsis thaliana (1.1 million snps); cicer arietinum (519.1 thousand snps); phoenix dactylifera (3.5 million snps); glycine max (16.9 million snps) and zea mays (54.3 million). genome annotation karp et al. (31) established pathway methods that use the pathological algorithm to assess the enzymatic responses catalyzed for each gene product in a query genome and then combine the identified response list from a reference database against all accessible processes. thompson et al. (32) produced the kegg automatic annotation server (kaas) towards the manually mapped kegg/genes registry database (33) providing functional gene annotation through blast comparisons, single best hit (sbh) and bidirectional best scores (bbh). as yield were produced ko tasks to genes and anticipated kegg processes. overbeek et al. (2005) launched the seed platform that offers a webbased,' subsystems'-based, relative genome annotation method. subsystems were a collection of functional features observed in any prevalent biological system, including cellular processes, phenotypes, or complicated multi-subunit constructions. haft et al. (35) established the tigr comprehensive microbial resources (cmr) to enable users from accessing all completed sequences of bacterial genomes. cmr offers two kinds of assets for annotation: main annotation from the genome sequencing centre and tigr annotation produced through an interactive tigr annotation method. the cmr pathway tool kit comprises of three classifications of pathway assessment tools: ' genome properties ' offers information on the features of species extracted from genomic data and literature references ; ' genome properties detailed comparison ' offers direct step-by-step information for selected clients of a collection of genomes ; and ' kegg pathway display ' shows kegg's presence-based pathway measures. conesa et al. (36) developed the blast2go (b2g), a universal guide for go annotation, visualization and stats that provides sophisticated functional evaluation for nonmodel organism genomics studies. b2g is intended to enable annotation of instant and high-throughput succession and incorporate annotation-based data mining features. b2g utilizes blast (37) to discover homologs for fastaformatted sequences of inputs. the program excerpts go conditions by referring to existing annotation associations for each hit acquired. finally, an annotation rule gives go conditions to the sequence of queries. it is possible to visualize annotation and functional analysis in a graph form that reconstructs the go interactions and highlights the most appropriate regions. in an embedded genome framework, markowitz et al. (38) introduced the integrated microbial genomes (jgi) for comparative microbial genome assessment. the data model comprising the img scheme includes main genomic sequence data, algorithmically forecast and ordered gene models, pre-computed sequence resemblance data, functional annotation, and pathway data. microbial gene statistical analysis in img is conducted in a relative framework of various microbial genomes where a range of instruments can be used to assess genomes in terms of genome-specific statistics, genes and sequence conserved. the kegg orthology-based annotation system (kobas) was implemented by wu et al. (39) that can provide statistical significance examinations for anticipated pathways. ko terms have been chosen based either on sequence resemblance to kegg/genes entries or on kegg/genes cross-database links when a list of sequence classifications is accessible in the databases. compared to the background model, regularly occurring or statistically significantly enriched query sequence pathways are recognized forward to ko assignment. pireddu et al. (40) created a path-a (pathway analyst) software that uses a number of query protein samples from a genome and defines which sequences are probable to occur using multiple sequence evaluation methods (e.g. svm, blast and hmm) in any of its endorsed model pathways. the approach of the model pathway allows the pathway prediction algorithm to predict instances of a pathway with pathway structure variations that have never been observed in the training pathway set. at present, path-a offers abstract models for 10 pathways, covering 125 cases of genome-specific processes. choi and kim (4) established a comparative pathway workbench system (compath) that enables researchers to compare biological processes in multiple genomes using a spreadsheet-style online interface where different sequencebased analyzes can be conducted either to match enzymes (e.g. sequence clustering) or processes (e.g. pathway hole recognition) to search for a de novo enzyme forecast algorithm. https://www.ncbi.nlm.nih.gov/ plant resistance gene, snp and genome annotation highlights in bioscience doi: 10.36462/h.biosci.20192 august 2019 | volume2 http://highlightsin.org/index.php/bioscience/ page 5 of 6 references 1. van ooijen g, van den burg ha, cornelissen bjc, takken flw. structure and function of resistance proteins in solanaceous plants. annu rev phytopathol. 2007;45:43–72. 2. rafalski a. applications of single nucleotide polymorphism in crop genetics. curr opin plant biol. 2002;5:94–100. 3. frazer ka, ballinger dg, cox dr, hinds da, stuve ll, gibbs ra, et al. a second generation human haplotype map of over 3.1 million snps. nature. 2007; 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215(3):403–10. 38. markowitz vm, korzeniewski f, palaniappan k, szeto e, werner g, padki a, et al. the integrated microbial genomes (img) system. nucleic acids res. 2006; 34(suppl 1):d344–8. 39. wu j, mao x, cai t, luo j, wei l. kobas server: a web-based platform for automated annotation and pathway identification. nucleic acids res. 2006;34: w720–4. 40. pireddu l, szafron d, lu p, greiner r. the path-a metabolic pathway prediction web server. nucleic acids res. 2006;34:w714–9. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202204 perspectives open access 1 independent researcher, pharmaceutical research facility, cairo, egypt. 2 national centre for radiation research and technology, cairo, egypt. 3 royal oldham hospital, oldham, united kingdom. * to whom correspondence should be addressed: mostafaessameissa@yahoo.com editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): alsamman m. alsamman, african genome center, mohammed vi polytechnic university,morocco.. morad m. mokhtar, agricultural genetic engineering research institute, agricultural research center, giza, egypt. received: march 31, 2022 accepted: may 1, 2022 published: august 24, 2022 citation: eissa me , rashed er , eissa de. implementation of the pareto principle in focus group generation based on global coronavirus disease morbidity and mortality rates. 2022 aug 24;5:bs202204 copyright: © 2022 eissa et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. implementation of the pareto principle in focus group generation based on global coronavirus disease morbidity and mortality rates mostafa essam eissa*1 ><, engy refaat rashed2 >< dalia essam eissa3 ><  abstract the recent pandemic that has hit the world has affected humanity in all aspects of life. since the outbreak of this worldwide epidemic, a huge amount of data has been generated. in this article, we have provided a new simplified insight into the 2019 coronavirus disease (covid -19) using the pareto principle to highlight the main contributors to morbidity and mortality. a time series database of confirmed cumulative cases and deaths for all countries was processed from the humanitarian data exchange website provided by the united nations office for the coordination of humanitarian affairs. more than 85% of the incidents recorded worldwide were from the amro, euro, and searo who regions, and the united states, russia, and india were found to account for the largest proportion of cases and deaths in these affected areas. the application of pareto analysis is useful in finding focus groups for further study and modeling. keywords: covid-19, morbidity, mortality, pareto principle, who introduction the new coronavirus disease (covid -19), which has been causing a worldwide pandemic for about two years, has affected every aspect of human life, and the world has changed significantly since then [1]. a large amount of data has been compiled showing morbidity and mortality rates as cumulative numbers of cases and deaths on a daily basis for each affected country and/or area [2]. the database was processed based on the world health organization classification (who) and then subdivided according to the daily records of the political region. implementation of the pareto principle on big data of covid-19 morbidities and mortalities before assessing the distribution of covid -19 deaths and morbidities, a brief look at the distribution of the world population based on the census should be taken to find a match with the reported cases and deaths according to the 60/40 and 80/20 rule using minitab version 17.0.1. this software was implemented as it has been used in other research. [3-6]. the asian population accounts for about 60% of the world population, followed by africa and europe (figure 1a ) with a cumulative total of over 85% of the world population. however, the covid -19 cases and deaths show a different descending order, with the american regional office (amro) zone ranking first, followed by the european regional office (euro) and the southeastern regional office (searo) with 0.40, 0.32, and 0.17 cases and 0.48, 0.30, and 0.12 deaths, respectively, as a proportion of the world population (figure 1b and 1c ). contribution of countries by cumulative cases and deaths in who regions an examination of contributing countries and/or areas with cumulative covid -19 mortality and morbidity rates based on the who classification revealed that few regions are particularly affected. the main contributing countries in the amro region were, in descending order of cases, the united states of america (usa), brazil, argentina, and colombia, with a cumulative share of more than 80%. for deaths, the same order was maintained for the first two countries, followed by mexico and pero with a cumulative contribution of about 82% [7]. highlights in bioscience page 1 of 4 august 2022|volume 5 https://doi.org/10.36462/h.biosci.202204 https://creativecommons.org/licenses/by/4.0/ mostafaessameissa@yahoo.com https://orcid.org/0000-0003-3562-5935 engyrefaat@yahoo.com https://orcid.org/0000-0002-6593-378x daliaessameissa@yahoo.co.uk https://orcid.org/0000-00002-6340-8973 http://bioscience.highlightsin.org/ eissa et al., 2022 implementation of the pareto principle to global coronavirus disease rates both the usa and brazil had a cumulative contribution of more than 50% of the total number of people affected in the amro region, as shown in figure 2a and 2b . another area who which made a relatively small contribution was studied. the west pacific regional office (wpro) showed that the philippines accounted for about one-third of the cumulative daily morbidity and mortality rates. however, the order varied for the other nations that followed, as japan and malaysia showed an alternating order in cases and deaths. interestingly, the most populous country in the world, from which the first epidemic cases originated that triggered the global outbreak, contributed only marginally (≈ 11% in deaths and fourth in cumulative rate) because of rapid and rigorous government and public health interventions [8]. a detailed visualization of the sequence of records is shown in fig. 3 figure 2c and 2d . figure 1. pareto chart showing major continents in population census (a) and who main contributors in morbidities (b) and mortalities (c). a look at a third important region, searo, showed that india had the highest number of deaths and morbidities at daily rates in the who region. this is evident in figure 2e and 2f with a proportion of over 70%. adding indonesia and india to the cumulative deaths gives a proportion of about 0.90 from the searo region. another marginally affected who area, the eastern mediterranean regional office (emro), showed a different pattern, where only the first country was strongly affected by covid -19, followed by a group of seven and six figure 2. pareto chart showing major countries in mortalities and morbidities from covid-19 from amro (a and b), wpro (c and d) and searo ( e and f), respectively. countries showing relatively small variations among them. for the cumulative case rate, the descending order was the islamic republic of iran, iraq, pakistan, morocco, jordan, the united arab emirates (uae), saudi arabia, and lebanon, with contribution factors of approximately 0.32, 0.13, 0.09, 0.06, 0.06, 0.05, 0.05, and 0.04, respectively (figure 3). for the cumulative mortality rate, the descending order was iran, pakistan, iraq, egypt, tunisia, morocco, and saudi arabia with percentage contributions of 42.1, 9.7, 8.8, 7.1, 6.7, 4.8, and 3.9%, respectively (figure 3a and 3b). data from the african regional office (afro) which is also a minority show similar behavior to emro, but with a greater contrast between the first country affected and the other nations that follow, as shown in figure 3c and 3d. moreover, the reported cumulative cases show a broader range of joint contributions. south africa alone accounted for approximately 50% and 60% of the total cumulative cases and deaths, respectively. in terms of morbidity and mortality for the remaining countries, the order was as follows: ethiopia (6.0%), kenya (4.2%), nigeria (4.1%), algeria (3.0 %), zambia (3.6%), ghana (2.5%), mozambique (2.1%), botswana highlights in bioscience page 2 of 4 august 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al., 2022 implementation of the pareto principle to global coronavirus disease rates (2.1%), namibia (1.8%) and zimbabwe (1.8%) and algeria (4.4 %), ethiopia (4.0%), kenya (3.3%), zimbabwe (2.4%), nigeria (2.3%) and zambia (2.1%), respectively. euro lands showed a unique pattern in figure 3e and 3f . while this region was considered one of the three main zones affected by daily death and disease rates from covid -19, much less variation was shown with broader contributing countries, resulting in an observable low declining ramp in the pareto diagram. thus, 15 and 12 nations shared the top contributing countries (80%) in morbidity and mortality data, respectively. the first two countries (the russian federation and the united kingdom (uk)) accounted for only about one-fifth and one-quarter, respectively, of the cumulative cases and deaths in the data set. the descending list of remaining countries in the third and fourth morbidity categories included france (with about the same contribution as the united kingdom), turkey, spain, italy, germany, poland, ukraine, the netherlands, the czech republic, belgium, and romania, with contribution ratios of 0.10, 0.09, 0.07, 0.07, 0.06, 0.04, 0.04, 0.03, 0.03, and 0.02, respectively. the descending order of the remaining nations for the fourth three cumulative deaths was as follows: italy, france, spain, germany, poland, turkey, ukraine, and romania, with proportions of 0.11, 0.09, 0.08, 0.07, 0.05, 0.04, 0.04, and 0.03, respectively. figure 3. pareto chart showing major countries in mortalities and morbidities from covid-19 from emro (a and b), afro (c and d) , euro (e and f). findings and limitations the previously analyzed report is time-bound, as updating the dataset with new inputs may show some discrepancies [9, 10]. in particular, the distribution of covid -19 across the globe seemed to be independent of the climatic nature of the regions, as seen in figure 4 , and showed a wide geographic dispersion within different temperature and humidity ranges. nevertheless, table 1 showed that countries near the equator had a higher proportion of daily cumulative morbidity and mortality rates than those farther away, where amro and searo countries had about 45% and 43% of cumulative morbidity and mortality rates, respectively. figure 4. political map showing the distribution of more than 60% of morbidities and mortalities around the globe. since other researchers have concluded that hot and humid climates can halt the spread of the disease to some degree, this suggests that other factors played a greater role in these political regions, including but not limited to public health policy and agency actions, public perception and awareness of outbreak control measures, general health status of citizens, racial factors, extent and rate of vaccination, and population size and density, in addition to political and economic conditions [1114]. another important factor that should not be underestimated concerns the government and public health officials, as well as the routine system for recording, monitoring, and tracking affected individuals, as this could affect the reliability and quality of the derived databases. highlights in bioscience page 3 of 4 august 2022|volume 5 http://bioscience.highlightsin.org/ eissa et al., 2022 implementation of the pareto principle to global coronavirus disease rates table 1. major countries in the global distribution of morbidities and mortalities cases by who regions. who region a regional cumulative * rate contribution (%) morbidity country cases contribution (%) * mortality country death contribution (%) * cases deaths amro b 40.1 48.2 usa 19.93 usa 16.39 brazil 9.38 brazil 11.86 argentina 2.13 mexico 6.15 searo c 16.8 11.6 india 13.73 india 8.41 euro d 31.8 30.0 russia 3.50 russia 3.90 uk 3.18 uk 3.60 france 3.18 italy 3.30 turkey 2.86 france 2.70 spain 2.23 spain 2.40 italy 2.23 germany 2.10 a world health organization , b american regional office, c southeast asia regional office, d european regional office, * based on cumulative daily record. conclusion in the world of big data, it will be useful to analyze a large amount of information quickly, easily and effectively to derive a useful conclusion. from the cumulative daily cases and deaths in the coronavirus pandemic database, a focus study group could be identified using pareto analysis. these segregated countries will be subjected to further quantitative study using a modeling approach for morbidity and mortality that could serve as the basis for further outbreak investigations. abbreviations afro: african regional office amro: american regional office covid-19: coronavirus disease of 2019 emro: eastern mediterranean region euro: european regional office searo: southeast asia regional office who: world health organization wpro: west pacific regional office references 1. keshky e, el sayed m, basyouni ss, al sabban am. getting through covid-19: the pandemics impact on the psychology of sustainability, quality of life, and the global economya systematic review. frontiers in psychology. 2020:3188. 2. ocha services. novel coronavirus (covid-19) cases data [internet]. humanitarian data exchange. 2022 [cited 30 march 2022]. available from: https://data.humdata.org/dataset /novel -coronavirus-2019-ncov-cases 3. eissa m. avian schistosomes impact on public health: a longterm disease observation. annals of medical research. 2019;(0):12. 4. essa m, rashed e. inventory digital management using statistical process control analysis in healthcare industry. journal of business in the digital age. 2020;3(2):123-8. 5. whitehead a. 60/40 is the new neutral pareto ratio [internet]. seekingalpha. 2022 [cited 30 march 2022]. available from: https://seekingalpha.com/article/ 4198408-60-40-is-newneutral-pareto-ratio 6. mitra a. fundamentals of quality control and improvement. john wiley & sons; 2016 may 2. 7. pan american health organization /world health organization. epidemiological update: coronavirus disease (covid-19). 27 september 2021, washington, d.c.: paho/who; 2021 8. yu x, li n, dong y. observation on chinas strategies to prevent the resurgence of the covid-19 epidemic. risk management and healthcare policy. 2021;14:2011. 9. altakarli ns. chinas response to the covid-19 outbreak: a model for epidemic preparedness and management. dubai medical journal. 2020;3(2):44-9. 10. an agency of the european union. covid-19 situation update worldwide, as of week 11 updated 24 march 2022 [internet]. european centre for disease prevention and control. 2022 [cited 31 march 2022]. available from: https://www.ecdc.europa.eu/en/ geographical-distribution-2019-ncov-cases 11. mecenas p, bastos rt, vallinoto ac, normando d. effects of temperature and humidity on the spread of covid-19: a systematic review. plos one. 2020 sep 18;15(9):e0238339. 12. bukhari q, jameel y. will coronavirus pandemic diminish by summer?. available at ssrn 3556998. 2020 mar 17. 13. ma y, pei s, shaman j, dubrow r, chen k. role of meteorological factors in the transmission of sars-cov-2 in the united states. nature communications. 2021 jun 14;12(1):1-9. 14. ganegoda nc, wijaya kp, amadi m, erandi kk, aldila d. interrelationship between daily covid-19 cases and average temperature as well as relative humidity in germany. scientific reports. 2021 may 28;11(1):1-6. highlights in bioscience page 4 of 4 august 2022|volume 5 https://data.humdata.org/dataset/novel-coronavirus-2019-ncov-cases https://data.humdata.org/dataset/novel-coronavirus-2019-ncov-cases https://seekingalpha.com/article/4198408-60-40-is-new-neutral-pareto-ratio https://seekingalpha.com/article/4198408-60-40-is-new-neutral-pareto-ratio https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncov-cases https://www.ecdc.europa.eu/en/geographical-distribution-2019-ncov-cases http://bioscience.highlightsin.org/ abstract introduction implementation of the pareto principle on big data of covid-19 morbidities and mortalities contribution of countries by cumulative cases and deaths in who regions findings and limitations conclusion abbreviations references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202305 research article open access 1 laboratory of ecology, biology and physiology of aquatic organisms, tunis faculty of science, university of tunis el manar2092 tunistunisia. 2 university of carthage, higher institute of fisheries and aquaculture of bizerte (ispab), errimel, b.p.15. 7080, bizerte, tunisia. 3 laboratory of fisheries sciences, national institute of marine sciences and technologies (instm), 28 rue du 2 mars 1934, salammbô 2025, tunis, tunisia. * to whom correspondence should be addressed: ferielghribi@yahoo.fr editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): khaled h. radwan, agricultural genetic engineering research institute (ageri), agricultural research center (arc), giza, egypt. ahmed e. nassar, department of biotechnology, faculty of agriculture, mansoura university, dakahlia, egypt. received: march 29, 2023 accepted: november 10, 2023 published: november 22, 2023 citation: ghribi f, chetoui i, bejaoui s, belhassen d, trabelsi w, fouzai c, mili s, soudani n. comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis. 2023 nov 22;6:bs202305 copyright: © 2023 ghribi et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis feriel ghribi*,1 ><, imene chetoui 1 >< , safa bejaoui 1,2 >< , dalya belhassen 1 ><, wafa trabelsi 1 ><, chaima fouzai 1 >< , sami mili 2,3 ><, nejla soudani 1 >< abstract the blue swimming crab portunus segnis has a great economic interest in local and foreign fishing markets and is in high demand by consumers. the aim of the present work was to evaluate the change in total lipid (tl), fatty acids composition and nutritional quality indices (nqi) of the meat of blue swimming crab portunus segnis after freezing at -18◦c for 15, 30, 60 and 90 days. the comparison of fresh and frozen crabs showed that freezing had significant effects on the nutritional quality of this marine product. lipid peroxidation was enhanced during the freezing process. total lipid content decreased significantly as a function of days of storage, especially from 30 days. a significant change was also observed in the fatty acid composition of frozen crab meat. during the freezing process, saturated fatty acids (sfa) increased significantly, while polyunsaturated fatty acids (pufa) and monounsaturated fatty acids (mufa) decreased. we can conclude that storage of blue crab p. segnis at -18◦c was not efficient enough for long preservation, as it has a strong effect on the deterioration of the nutritional quality of the meat over time. frozen crabs should preferably be consumed within 15 days of storage. our research targets both domestic and international consumers of this crab, with a specific focus on restaurants and hotels that incorporate this item into their menus. we recommend that consumers of this product exercise increased caution regarding the advantages and drawbacks associated with the freezing techniques employed. keywords: portunus segnis, frozen storage, lipid content, fatty acids profile, lipid peroxidation, nutritional quality introduction in the mediterranean sea, crustaceans are the second most abundant taxon of confirmed nonnative species [1]. in tunisian waters, a total of 163 alien species have been reported, with crustaceans dominating with a 24% share of reported alien species [2]. crabs are common in all regions of the world and are found in freshwater, brackish water, and marine habitats. however, other species are also found on land, where they can live hundreds of kilometers from aquatic habitats [3]. crabs occupy a variety of trophic niches; they also play an important ecological role in aquatic ecosystems [4]. the blue swimming crab, formerly called p. pelagicus [5], is one of the first lessepsian invaders. in tunisian waters, the occurrence of p. segnis was first reported in 2014 by [6] in shallow, sandy areas with seagrass beds and algae. since 2015, the number of p. segnis individuals in the gulf of gabes has greatly increased [7] and includes much larger areas on the southeastern coasts of tunisia. p. segnis is in high demand by consumers and is regularly sold at all local fish markets. crustaceans are recommended for human consumption because of their beneficial and healthy properties [8]. they are rich in polyunsaturated fatty acids such as epa (ecosapentaenoic fatty acid) and dha (decosahexaenoic fatty acid). epa and dha are very useful for human health due to their anti-inflammatory, antithrombotic, and antiarrhythmic properties. in recent years, the increasing consumption of seafood has led to an improvement in preservation methods to obtain products of high nutritional quality. highlights in bioscience page 1 of 8 november 2023|volume 6 https://doi.org/10.36462/h.biosci.202305 https://creativecommons.org/licenses/by/4.0/ mailto:ferielghribi@yahoo.fr https://orcid.org/0000-0001-9350-7510 mailto:chetouiimene@gmail.com https://orcid.org/0000-0002-2259-5397 mailto:safa.bejaoui@fst.utm.tn https://orcid.org/0000-0002-7946-2763 mailto:dalya.belhassen@gmail.com https://orcid.org/0009-0003-5518-1435 mailto:wafa.trabelsi@etudiant-fst.utm.tn https://orcid.org/0000-0002-1114-4080 mailto:fouzai.chaima93@gmail.com https://orcid.org/0000-0001-7588-1859 mailto:sami_mili@yahoo.fr https://orcid.org/0000-0002-2625-8064 mailto:nejla.soudani@tunet.tn http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis freezing is considered the most suitable and effective method to preserve food quality [9]. this method is most suitable for long-term preservation of seafood because it preserves the organoleptic properties of the food well [10]. however, the formation of ice crystals under freezing conditions could lead to cell cracking and tissue destruction [11]. fishery products are indeed subject to natural postmortem degradation due to endogenous and exogenous reactions [12]. this phenomenon is largely dependent on storage conditions, which further promote oxidation reactions. due to the lack of information on the effects of freezing on blue swimming crab meat, lipid content, fatty acid composition, nqi, and lipid peroxidation markers in p. segnis after freezing at -18◦c for 15, 30, 60, and 90 days were studied in this work. material and methods sampling site although the gulf of gabes is considered one of the most productive fishing areas in tunisia, it is subject to numerous anthropogenic impacts that affect its biodiversity [13]. in the present study, our samples were collected in the commercial port of "ghannouch" in the gulf of gabes (34o 05’ 37”n, 10o 26’ 13”e) (figure 1). figure 1. sampling site location of sample collection in the gulf of gabes. preparation of the samples fifty adult specimens were collected in january 2018 to study the effects of freezing on the blue crab portunus segnis. the crabs ranged in length from 41.75 to 62.9 mm, width from 85.8 to 130 mm, and total weight from 29.3 to 114.1 g. the samples were immediately brought to the laboratory in a cool box. then, the crabs were well cleaned, packed in plastic bags, labeled, and frozen at -18oc for 15, 30, 60, and 90 days. for each period, the meat of ten whole animals was homogenized using a grinder (ultraturrax®) and prepared for lipid and fatty acid content analysis. one batch of 10 animals was analyzed fresh. lipid content total lipid content in fresh and frozen tissue samples was determined according to the method of folch et al. (1957) using a mixture of chloroform-methanol (2v/1v) and 0.01% butylhydroxytoluene (bht). the volume of chloroform-methanol extraction solvent is proportional to the weight of the organ: 30 ml of extraction solvent for 1 g of the tissue. elimination of proteins was performed after the addition of a 15% nacl solution. after centrifugation at 3000 rpm for 15 min, the organic phase was stored in previously weighed flasks. fatty acid profile fatty acid methyl esters (fame) were determined by gas chromatography (hp 6890 gc). qualitative identification of fatty acids (fa) was performed using a standard "pufa3" chromatogram of menhaden oil from supelco injected under the same chromatographic conditions. the determination of a fatty acid (x) is done by comparing its retention time with the time indicated in the "pufa3" chromatogram. the areas of the peaks are proportional to the amounts of the corresponding fatty acids. in our study, the fatty acids in percent (%) were calculated from the areas of the peaks of the chromatogram and the internal standard (c19:0 belefonte pa. usa. crm47885), the amount of which is known in µg. nutritional quality indices (nqi) the nqi of the fresh and frozen samples were determined using the indices mentioned by [14; 15; 16; 17; 18] and summarized in table 1. lipid peroxidation peroxide value (pv) pv was estimated based on the aocs (1989) method. results were expressed in ml g-1 and calculated according to equation (e1): pv = (sample titration−blank titration)×nthiosulfate×1000 sample weight (e1) thiobarbituric acid reactive substances (tbars) tbars were determined according to the aocs method (1989), and results were expressed in mg kg-1. statistical analysis data were presented as means ± standard deviations (sd) of 10 replicates for lipids, fa, and lipid peroxidation markers and highlights in bioscience page 2 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis table 1. nutritional quality indices (nqi) references index calculation marques et al. 2010 [14] n-3 n-6 marques et al. 2010 [14] pufa sfa unusan 2007 [15] epa + dha ulbricht and southgate 1991[16] ai = (4×c14:0)+c16:0+c18:0 σmufa+σ pufa n−6+σ pufa n−3 ulbricht and southgate 1991 [16] ti = c14:0+c16:0+c18:0 0.50×mufa+0.5× pufa n−6+3× pufa n−3+ pufa n−3 pufa n−6 rodriguez et al. 2007 [17] pi = c20:5n−3+c22:6n−3 c16:0 fernandez et al. 2014 [18] h/h = c18:1n−9+c18:2n−6+c20:4n−6+c18:3n−3+c20:5n−3+c22:5n−3+c22:6n−3 c14:0+c16:0 analyzed with statistica 8 software (stat-soft inc). homogeneity and normality of variables were tested with the shapiro test. significant differences were found at the 5% level. results were checked with one-way analysis anova and post-hoc tukey and kruskall-wallis test. to show possible correlations between biochemical parameters of raw and frozen crabs, principal component analysis (pca) was performed. results lipid content total lipid content in fresh blue crab was estimated to be 41.85 ± 4.33 mg/g (figure 2). a significant decrease in lipid content was observed in the flesh of crabs frozen at −18◦c after 30, 60, and 90 days of storage (27.28, 17.08, and 14.57 mg/g weight, respectively). figure 2. lipid content of raw and frozen blue crab during freezing (−18◦c) fa composition the fa profile of fresh and frozen crabs, expressed as a percentage of the total identified fa, was presented in table 2. for fresh crabs, pufa accounted for 33.60% of the total fa, followed by sfa (20.49%) and mufa (15.78%). palmitic acid (c16:0; 6.28%) and arachidic acid (c20:0; 5.82%) dominated sfas in fresh crab meat. pufa were dominated by eicosenoic acid (c20:1; 6.26%), oleic acid (c18:1; 2.14%), and palmitoleic acid (c16:1; 2.87%), while alpha-linolenic acid (c18:3n−3; 7.82%), docosahexaenoic acid (dha; 6.23%), eicosapentaenoic acid (epa; 4.58%), and eicosatetraenoic acid (c20:4n−3; 3.63%) were the most abundant fas in pufa (table 2). in frozen crabs, pufa and mufa significantly decreased (p < 0.05), while sfa increased. in sfa, both c16:0 and c18:0 fatty acids significantly increased by 60.03% and 158.30%, respectively, after 90 days of freezing (p < 0.05). in contrast, pufa significantly decreased after 90 days of storage (3.44%). this decrease was influenced by the decrease in major fa such as c16:1 and c18:1 (0.28% and 0.63%, respectively). frozen crabs showed a significant decrease in n-3 pufa after storage. the levels of omega-3 (pufa n-3) and omega-6 fatty acids (pufa n-6) in the fresh and frozen crabs are shown in table 2. dha and epa reached their lowest levels of 0.61% and 0.56%, respectively, after 90 days of storage compared to the fresh samples (6.23% and 4.58%, respectively). in contrast, n-6 pufas in frozen crabs showed a different variation with a significant increase (p < 0.05) during the freezing process and reached their high values after 90 days of storage (16.19%) compared to fresh samples (2.92%). highlights in bioscience page 3 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis table 2. fatty acid profile of fresh and frozen crabs for 15, 30, 60 and 90 days. fatty acids fresh 15 days 30 days 60 days 90 days c14:0 1.67±0.28 2.38±0.36* 3.13±0.09*** 4.50±0.22*** 5.23±0.07*** c15:0 1.04±0.13 0.74±0.39*** 2.12±0.05*** 2.93±0.46*** 4.17±0.06*** c16:0 6.28±0.25 7.74±0.61** 8.74±0.75*** 9.18±0.51*** 10.05±0.58** c17:0 1.86±0.24 1.26±0.29 1.56±0.24*** 4.05±0.50*** 5.62±0.30*** c18:0 2.14±0.32 2.91±0.91 3.16±0.13* 5.02±0.50*** 7.00±0.71*** c20:0 5.82±0.63 5.89±0.59 6.46±0.28 8.10±0.31*** 9.65±0.45*** c22:0 1.41±0.06 1.62±0.48* 2.95±0.36*** 5.54±0.97*** 8.59±0.20*** c14:1 1.61±0.13 1.30±0.01** 0.87±0.06*** 0.64±0.12*** 0.16±0.03*** c15:1 0.57±0.38 0.66±0.22 0.23±0.03 0.18±0.01 0.14±0.05 c16:1 2.87±0.07 2.66±0.29 1.04±0.14*** 0.85±0.06*** 0.28±0.04*** c18:1 2.14±0.32 1.76±0.10 1.31±0.29* 0.87±0.09*** 0.63±0.09*** c20:1 6.26±0.94 5.83±0.68 4.24±0.43*** 2.78±1.05*** 1.64±0.25*** c22:1 2.33±0.57 2.22±0.27** 1.89±0.54 1.30±0.17*** 0.59±0.06*** c16:2n-4 3.17±1.58 0.33±0.20** 0.49±0.05*** 0.25±0.02* 0.09±0.01*** c16:3n-4 1.39±0.31 1.54±0.40 0.47±0.15*** 0.16±0.02*** 0.11±0.01*** c18:2n-6 0.64±0.05 0.86±0.03** 0.91±0.08*** 1.76±0.10* 2.68±0.29*** c18:3n-4 1.82±0.74 0.97±0.08* 0.54±0.15* 0.48±0.05*** 0.14±0.02*** c18:3n-3 7.82±0.26 7.48±0.47** 5.95±0.71*** 2.56±0.36*** 0.27±0.06*** c20:3n-6 1.71±0.09 2.22±0.15** 3.96±0.29* 5.28±0.97*** 7.05±0.06*** c20:4n-6 0.57±0.03 1.56±0.30** 2.52±0.61*** 4.96±0.18*** 6.46±0.33*** c20:3n-3 2.06±0.01 2.52±0.30* 2.77±0.42 1.22±0.09*** 0.56±0.01*** c20:4n-3 3.63±0.45 3.61±0.36 2.26±0.12* 1.98±0.30*** 0.61±0.06*** c20:5n-3 4.58±0.51 4.87±0.04 2.52±0.61*** 1.52±0.74*** 0.61±0.06*** c22:6n-3 6.23±0.38 5.51±0.48* 3.24±0.32*** 1.98±0.12*** 0.56±0.06*** pufa n-3 24.31±0.07 23.99±1.09 16.57±0.11*** 9.26±1.08*** 3.40±0.16*** pufa n-6 2.92±0.17 4.64±0.34*** 8.47±0.33*** 12.00±0.92*** 16.19±0.28*** values are presented by mean ± sd of ten replicates. values in a row that does not share the same letter(s) are significantly different at p < 0.05. nutritional quality indices the nqis are listed in table 3. after freezing, a significant decrease in the n-3/n-6 ratio was observed (table 3). this ratio decreased by 97.48% after 90 days of storage. the pufa/sfa ratio decreased significantly during freezing (p < 0.05). similarly, epa + dha decreased significantly during storage and reached its lowest value after 90 days (1.17) compared to fresh crabs (10.81). the polyene index (pi), used as a marker of oxidative rancidity of crab meat, also decreased during freezing (p < 0.05) (table 3). however, the indices of atherogenicity (ai) and thrombogenicity (ti) significantly increased by 385.33% and 818.66%, respectively, in frozen crab after 90 days of storage. hypercholesterol ratio (h/h) decreased significantly (p < 0.05) by 59.73% in frozen crabs after 90 days compared to raw crabs (table 3). lipid peroxidation the results of lipid peroxidation are shown in figure 3. this phenomenon increased significantly during the storage period, as tbars and pv levels reached their highest values after 90 days of storage compared to fresh crabs (figure 3). figure 3. lipid peroxydation indices in raw and frozen crabs. highlights in bioscience page 4 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis table 3. nutritional quality indices of fresh and frozen crab’s meat after 15, 30, 60 and 90 days of frozen storage. indices fresh 15 days 30 days 60 days 90 days n−3 n−6 8.35±0.51 5.18±0.28*** 0.95±0.05*** 0.78±0.11*** 0.21±0.01*** pufa s fa 1.64±0.02 1.33±0.14 0.95±0.05*** 0.56±0.03*** 0.40±0.01*** epa+dha 10.81±0.13 10.38±0.44*** 5.76±0.64*** 3.50±0.77*** 1.17±0.03*** pi 1.72±0.09 1.35±0.16*** 0.66±0.03*** 0.38±0.10*** 0.12±0.01*** ai 0.36±0.01 0.47±0.07*** 0.71±0.03*** 1.16±0.04*** 1.65±0.06*** ti 0.12±0.01 0.15±0.02*** 0.25±0.01*** 0.50±0.03*** 1.10±0.08*** h h 2.98±0.10 2.42±0.31*** 1.82±0.18*** 1.39±0.15*** 1.20±0.05*** values are presented by mean ± sd of ten replicates. values in a row that does not share the same letter(s) are significantly different at p < 0.05. figure 4. pca analysis pca analysis showing separation between fresh and frozen blue crab groups principal component analysis (pca) the pca revealed a two-dimensional pattern that explained 85.3% of the total variance, including factor 1 (71.7%) and factor 2 (13.6%), and mainly showed the difference between raw and frozen individuals (figure 4). the fresh crabs were characterized by higher contents and amounts of tl, mufa, pufa, pufa/sfa, epa + dha, n-3/n-6, and h/h. the group of 15 days frozen crabs was the closest to the raw group. the 90-day frozen crab group had high values for sfa, pufa n-6, tbars, pv, ai, and ti (figure 4). pca analysis performed for all measured parameters highlighted the clear separation between the experimental groups during the freezing process. discussion crab species are considered a good source of lipids, proteins, essential elements, and vitamins beneficial for maintaining human health [19]. however, inadequate preservation of seafood could result in a significant loss of their nutritional quality. freezing is the most popular form of food preservation (e.g., chicken, fish, beef, etc.). this process is known to preserve food quality by reducing the deterioration of color, flavor, and texture over time and limiting the degradation process caused by microorganism activity [20]. in the present study, total lipids (tl) in blue crab meat decreased significantly over time during storage at −18◦c. this lipid degradation could be responsible for the shortening of the shelf life of the marine product due to the oxidation of unsaturated fatty acids (ufa). however, benjakul et al., [21] reported that catalysts released from denatured or destroyed muscle cells of marine products can accelerate lipid oxidation. the current results are consistent with the work of [22] on the mussel (perna canaliculus) from new zealand. the variations in lipid content in frozen samples could be related to lipid oxidation. our results are in agreement with the work of [23], who confirmed that the decrease in tl content in the meat of frozen catfish was due to oxidation. [24] reported that the lipid content in the muscle of four fish species (mugilidae, sparidae, sciaenidae, and platycephalidae) decreased significantly during the freezing process. the predominant fatty acids (fa) in fresh crab meat were palmitic acid, oleic acid, epa, and dha fatty acids. when the crab meat was frozen at −18◦c, the fa composition changed in their proportions; sfa increased, while mufa and pufa decreased significantly. similar results on the changes of pufa, mufa, and sfa during freezing were observed in fish [10]. the reduction in pufa content is likely due to oxidative reactions that occurred during the freezing process. it should be noted that the high degree of unsaturation favors the oxidation of pufas [25]. the n-3 pufas such as epa and dha were significantly reduced. in contrast, arachidonic acid (aa), which is known as a pro-inflammatory lipid compound, increased upon freezing. the results showed that the polyene index (pi) and the sum of dha + epa decreased due to the reduction of epa and dha, reflecting the degradation of pufa, as previously reported by [26] in frozen hake. the n-3/n-6 index is considered a good standard for comparing fish oil quality [27]. a ratio of 1:1 or 1:5 could be beneficial for human health [28]. our results showed that the n-3/n-6 ratio decreased after freezing, indicating a significant loss in the nutritional quality of the crab meat, but it was higher than the values recommended by the world health organization (who) up to 60 days of storage [29]. the same trend was observed for pufa/sfa ratio. the reduction in pufa resulted in a significant decrease in this index, confirming that frozen crabs are no longer a good source of pufa. however, up to a storage period of 60 days, this ratio remained higher (0.45) than the minimum value recommended by hmso (1994) for a healthy human diet. similar results were observed in frozen fish at −18◦c [30]. in this study, the h/h ratio decreased significantly, indicating a deterioration in meat quality, as higher values are considered healthier for humans [31]. atherogenic (ai) and thrombogenic (ti) indices are commonly used to determine how healthy the fat content of the food in question is [32]. these indices increased, possibly due to the loss of beneficial fa and the enhancement of atherogenic fa. others reported similar results in rainbow trout frozen at −15◦c for 3 months [33]. the observed changes in the main fa showed that they were highlights in bioscience page 5 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis involved in the oxidation process. the freezing temperature used was able to significantly increase pv and tbars levels in frozen crab meat. the appearance of radicals and the formation of hydroperoxides can lead to oxidative changes [34]. the oxidation of lipids in seafood can produce a rancid taste and odor that compromises the nutritional value and safety of seafood through the formation of primary oxidation products that rapidly convert to secondary products [35]. the increase in pv, which is commonly used as an index to quantify the amount of hydroperoxide indicating oxidative deterioration [36], showed the development of rancidity, off-flavor, color, and nutritional deterioration in frozen crab meat. our results confirm previous work on frozen fish and shrimp [37]. the higher increase in tbars, known as secondary lipid oxidation products, during frozen storage suggests a high rate of lipid oxidation and decay. similar results were observed in frozen silver catfish [38] and pink salmon [39]. from the above results, we can conclude that prolonged storage at −18◦c leads to a loss of nutritional quality of crab meat due to the formation of oxidation products and contributes to the acceleration of lipid degradation. according to this study, shorter storage of no more than 15 days at −18◦c is strongly recommended when crabs are frozen at this temperature. conclusion the freeze had significant effects on the biochemical composition of the crabs. major changes were observed in the fa profile and nutritional quality indices (nqi) of frozen crabs. as with other seafood, the nutritional quality of crab species can be affected by frozen storage. the deterioration of flesh quality of frozen crabs increased with the duration of freezing, as shown by the elevated peroxide value (pv) and thiobarbituric acid reactive substances (tbars) values. it can be concluded that blue crabs stored at −18◦c are still a good source of fatty acids, especially omega-3, after a reasonable period of time. our study concerns local consumers of this crab and, in particular, restaurants and hotels that use this product on their menus. we recommend that consumers of this product gain a deeper understanding of the potential hazards and advantages related to the freezing methods employed. 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alarcón-rojo ad, ortega-gutiérrez já, holguín-licón c, corral-flores g, et al. comparison of fatty acid content of fresh and frozen fillets of rainbow trout (oncorhynchus mykiss walbaum). brazilian archives of biology and technology. 2014;57:103-9. 34. dragoev s, kiosev d, danchev s, joncheva i, genov n. study on the oxidative processes in frozen fish. bulgarian journal of agricultural science (bulgaria). 1998;4(1). 35. choe e, min db. mechanisms and factors for edible oil oxidation. comprehensive reviews in food science and food safety. 2006;5:169-86. 36. wang n, ma t, yu x, xu l, zhang r. determination of peroxide values of edible oils by ultraviolet spectrometric method. food analytical methods. 2016;9:1412-7. 37. shi j, lei y, shen h, hong h, yu x, zhu b, et al. effect of glazing and rosemary (rosmarinus officinalis) extract on highlights in bioscience page 7 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ ghribi et al., 2023 comparison of lipid content and fatty acid profile of fresh and frozen blue swimming crab portunus segnis preservation of mud shrimp (solenocera melantho) during frozen storage. food chemistry. 2018. 38. daniel a, ferreira l, klein b, ruviaro a, quatrin a, parodi t, et al. oxidative stability during frozen storage of fillets from silver catfish (rhamdia quelen) sedated with the essential oil of aloysia triphylla during transport. ciência rural. 2016;46:560-6. 39. sathivel s, liu q, huang j, prinyawiwatkul w. the influence of chitosan glazing on the quality of skinless pink salmon (oncorhynchus gorbuscha) fillets during frozen storage. journal of food engineering. 2007;83:366-73. highlights in bioscience page 8 of 8 november 2023|volume 6 http://bioscience.highlightsin.org/ abstract introduction material and methods sampling site preparation of the samples lipid content fatty acid profile nutritional quality indices (nqi) lipid peroxidation statistical analysis results lipid content fa composition nutritional quality indices lipid peroxidation principal component analysis (pca) discussion conclusion highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202504 research article open access 1 laboratory of ecology, biology and physiology of aquatic organisms, department of biology, faculty of science of tunis, university of tunis el manar, 2092 tunis, tunisia. 2 basic sciences departement, physiology and functional explorations section, faculty of medicine of tunis, tunisia. * to whom correspondence should be addressed: safa.bejaoui@fst.utm.tn editor: muhammad m. adeel arthritis clinical immunology program oklahoma medical research foundation, oklahoma city, united states. reviewer(s): sami mili university of carthage, higher institute of fisheries and aquaculture of bizerte (ispab), tunisia. chetoui imene laboratory of ecology, biology and physiology of aquatic organisms, tunis faculty of science, university of tunis el manar, tunis-, tunisia. received: march 13, 2025 accepted: july 02, 2025 published: july 20, 2025 citation: bejaoui s, ghribi f, belhassen d, trabelsi w, baati r, soudani n. projected climate change scenarios and their effects on the nutritional quality of p. segnis muscle: macromolecular and fatty acid transformations. 2025 july 20;8:bs202504 copyright: © 2025 bejaoui s et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this work was supported by the laboratory of ecology, biology and physiology of aquatic organisms (ur18es41), faculty of sciences, university of tunis el manar. competing interests: the authors declare that they have no competing interests. projected climate change scenarios and their effects on the nutritional quality of p. segnis muscle: macromolecular and fatty acid transformations safa bejaoui*1 ><�, feriel ghribi1 ><�, dalya belhassen1 ><�, wafa trabelsi1 >< �, rym baati 2 ><�, nejla soudani1,2 ><� abstract this work presents the effects of increasing temperature on the physiological changes of portunus segnis in natural environments (in situ) and under controlled conditions (in vivo). following the increase in temperature from 19oc to 30oc and 40oc, physicochemical analyses showed that the percentages of oxygen consumption increased and the quantities of dissolved oxygen decreased. under the effect of natural and controlled thermal stresses (19oc, 30oc and 40oc), the water and ash contents in the muscles of blue crabs decreased with increasing temperature. for protein, glycogen and lipid contents, a decrease was observed with increasing water temperature. saturated fatty acids (sfas) significantly increased compared to polyunsaturated fatty acids (pufas), mainly eicosapentaenoic acid (epa) and docosahexaenoic acid (dha), which gradually decreased with increasing temperature. these results highlight the need to closely monitor climate change, particularly temperature increases, due to their potential impact on the structure and stability of biological macromolecules. such alteration can compromise essential physiological functions of aquatic organisms. these findings open new avenues for future research aimed at better understanding adaptation and resilience mechanisms to thermal stress in a warming climate. keywords: portunus segnis, globl warming, temperature, macro-molecules, fatty acids introduction the world’s oceans, representing nearly 71% of the earth’s surface, are experiencing the effects of pollution, eutrophication, and over-exploitation of their resources [1]. moreover, this marine environment plays a significant role in human society, as over 50% of the global population lives within 60 km of the coast. on the other hand, oceans and seas constitute the largest source of biodiversity on the planet [2]. many human activities such as fishing, tourism, and aquaculture depend on marine biodiversity and the overall health of seas and oceans, but these activities alter the chemical composition of aquatic ecosystems [3]. it is highly probable, according to the commonly accepted understanding by the intergovernmental panel on climate change, that the accelerating global temperature rise since the late 1970s is a result of the increased concentration of greenhouse gases in the atmosphere, such as carbon dioxide and methane, which have continued to rise [4]. the global co2 emissions level increased from 30.4 gigatonnes in 2010 to 33.3 gigatonnes in 2019. the increase in emissions has led to a widespread reduction of the cryosphere (areas of the planet where water is frozen), a continuous rise in ocean temperatures, a decrease in ocean ph and oxygen levels, changes in currents, and an increase in extreme events such as heatwaves [5]. in addition to direct issues related to pollution, eutrophication, over-exploitation, and the invasion of exotic species, the rise in carbon dioxide concentration and temperature poses a risk of causing major modifications to biodiversity, structure, and functioning of marine ecosystems [6]. climate change poses a growing threat to marine ecosystems, directly affecting species distribution, physiology, and population dynamics. rising seawater temperatures disrupt the thermal tolerance limits of many species, leading to geographical shifts toward the poles or to greater depths. the gradual rise in highlights in bioscience page 1 of 8 july 2025|volume 8 https://doi.org/10.36462/h.biosci.202504 https://creativecommons.org/licenses/by/4.0/ mailto:safa.bejaoui@fst.utm.tn https://orcid.org/0000-0002-7946-2763 mailto:feriel.ghribi@fst.utm.tn https://orcid.org/0000-0001-9350-7510 mailto:dalyabelhassen@gmail.com https://orcid.org/0009-0003-5518-1435 mailto:wafatrabelsi@gmail.com https://orcid.org/0000-0002-1114-4080 mailto:rym.baati@fmt.utm.tn https://orcid.org/0000-0003-0138-1434 mailto:nejlasoudani@gmail.com https://orcid.org/0000-0002-7652-9678 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions ocean temperature is altering the environmental conditions essential for the survival of marine organisms, causing range shifts toward colder areas, mainly toward the poles or at depth [7; 8]. climate change is also influencing the physiology of ectothermic species, reducing their metabolic performance, growth, reproduction, and survival [9]. increasingly frequent extreme marine heat events, such as ocean heatwaves, are leading to mass mortalities and local population collapses [10; 11]. these rapid thermal changes often exceed the adaptive capacities of species, particularly those with low mobility or a narrow ecological niche, increasing the risk of local or global extinction. it is urgent to place these systems under surveillance in order to detect, better understand, and anticipate changes in biological and ecological systems in the face of global climate change. climate warming is detected in numerous functional units of the earth’s system. the signature of warming is identified in the ocean and the terrestrial and aquatic biosphere. global temperatures have risen by 0.76oc between the periods 1850-1899 and 2001-2005 [4]. this temperature increase has mainly affected the oceans, which have absorbed 84% of the heat added to the climate system over the last four decades [12]. the increase in heat stored by the ocean has contributed to thermal expansion by 25% since the 1950s [13]. the warming projected by ocean-atmosphere general circulation models varies between 1.1oc (scenario b1, rapid introduction of efficient and clean technologies) and 6.4oc (scenario a1fi, intensive consumption of fossil carbon) by the end of this century [4]. indeed, the exposure of aquatic organisms to higher temperatures could also reduce the metabolic rate, decreasing physiological energy costs and providing short-term tolerance above the critical temperature. among the numerous species affected by this phenomenon, crustaceans are considered significant fisheries in many countries, and the influence of climate change poses a serious environmental and economic threat. global climate change is perhaps the most concerning anthropogenic impact currently, not only for the ocean but also for the entire planet. these manifestations will directly act through the effects of temperature on marine organisms. however, this disturbance will also indirectly influence marine ecosystems through its impact on regional climate and hydrology (atmospheric oscillation and ocean currents). organisms’ responses will be differential, and abrupt and unexpected ecosystem balance changes are to be feared. despite existing uncertainties, our level of knowledge is sufficient to urge policies to reduce greenhouse gas emissions into the atmosphere and also to consider the other consequences of human activities that go against sustainable development. furthermore, temperature and climate changes are considered the most important abiotic factors for the biogeographical distribution, abundance, and communities of marine fish. in aquatic environments, several studies also show a change in fish communities in response to climate changes. in fact, an increase in the temperature of the living environment, beyond a tolerance limit that varies by species, leads to the onset of physiological stress, figure 1. experimental design for p. segnis exposure to increased temperature especially pronounced when the exposure duration to this temperature is long, and the species are already closer to their upper thermal tolerance limit. if these stresses occur regularly, they can lead either to changes in geographical distribution, modifications in the life cycle, and in situ adaptations to new conditions, or, in sessile or less mobile forms, to significant mortality accompanied by epizootics and the substitution of affected species by others more resistant [14] therefore, the set of physico-chemical changes induced by climate change leads to the evolution of distribution areas, phenology, migratory movements, abundance, and interspecific interactions for many marine, freshwater, and terrestrial species [7]. these modifications, of course, have repercussions on biodiversity and can significantly alter the appearance of underwater landscapes. for this reason, we designed our study to determine the effect of temperature on the physiology of blue crabs (portunus segnis). this current work focuses on the effect of increased temperature on the biochemical composition of p. segnis. the main objective is to analyze macromolecules (proteins, lipids, and glycogens) as well as the fatty acid composition and the nutritional quality of blue crab meat under controlled conditions. materials and methods samples and experimental design the individuals of blue crabs were collected from the ghar el melh lagoon during the summer season. once collected, the specimens were immediately transported to the laboratory of the higher institute of fisheries and aquaculture of bizerte (ispab) in a ventilated cooler. the specimens were acclimatized for three days in a 200 l cylindro-conical basin, in a closed circuit, containing filtered natural seawater, under continuous aeration and under strictly controlled physicochemical conditions: temperature (19oc), oxygen content (62 µ g/l), salinity (35 psu), and photoperiod (12 h/12 h), reproducing those of the sampling area. after three days of acclimatization, the crabs were divided highlights in bioscience page 2 of 8 july 2025|volume 8 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions into three tanks and subjected to different temperatures. for the control group, specimens were exposed to an ambient temperature of 19.81 ±0.14oc. in the second tank, the temperature was raised to 30.18 ±0.36oc using a thermostat. regarding the third tank, two thermostats were installed to increase the temperature to 40.03 ±0.56oc. the temperature gradually increased to reach the desired values after approximately 2 hours of thermostat installation. during the one week of the experiment, the physicochemical parameters of the water, such as temperature and dissolved oxygen levels, were measured daily (every 24 hours) using a multimeter figure 1. crabs dissection and muscles preparation after exposure to increasing temperature, each individual from the crabs sampled from the natural environment and the controlled condition was weighed (170.04 ±16.51 g), measured using a caliper (60.44 ±6.51 mm), and subsequently dissected with a scalpel. the muscles from each batch were carefully extracted using forceps and weighed with a precision electronic balance. the choice of the organ taken from individuals is based on the fact that muscles are the edible tissues for living beings. table 1. proximate composition of p. segnis muscle under increasing temperature parameter 19oc 30oc 40oc ash (%) 20.23 ±1.25a 19.98 ±1.45a 19.52 ±1.00a moisture (%) 80.00 ±2.82a 82.66 ±3.26a 80.66 ±3.26a protein (mg/g) 66.08 ±6.62a 62.49 ±7.90a 10.75 ±1.46c lipid (mg/g) 11.14 ±2.35a 11.82 ±2.56a 1.97 ±0.48c glycogen (mg/g) 2.03 ±0.25a 1.77 ±0.22b 0.37 ±0.07c note: values are expressed as mean ±standard deviation. different superscript letters (a, b, c) in the same row indicate significant differences between temperature conditions at the 0.5% level. determination of water, ash, and proximate composition content using an electronic balance, 0.5 g of crab muscle was weighed to determine the initial wet weight. after drying in an oven at a temperature of 105°c for 24 hours, the samples were weighed again to determine the dry weight. to determine the ash content in crab muscles, the aoac method was employed [15]. in brief, approximately 0.5 g of muscles were weighed and then dried at a temperature of 400°c for 24 hours. lipid analysis was performed according to the method of [16], using vanillin solution. glycogen extraction was performed according to the enzymatic method of [17], with glucose solution (100 mg/l). protein determination figure 2. temperature measurement in aquariums subjected to different temperatures note: the difference between c1 and c2, c3 is significant at 0.5%. figure 3. measurement of dissolved oxygen in aquariums subjected to different temperatures was carried out according to the method of [18], using bovine serum albumin as a standard. fatty acid composition analysis lipid extraction is a method for isolating these compounds using organic solvents, including a mixture of chloroform and methanol, according to the technique described by [19]. this process consists of grinding 0.5 g of flesh in a mortar with 15 ml of this mixture (2:1, v/v). in order to facilitate phase separation, 1 ml of sodium chloride (nacl 15%) is then added to each sample. the homogenate obtained is subjected to centrifugation at 4000 rpm for 15 minutes, thus allowing the separation of the chloroform phase (containing the lipids) from the aqueous phase. the lower phase, containing the lipid extract, is then carefully recovered using a pasteur pipette. from this extract, 20 µl are collected and placed in test tubes, to which 1 ml of hexane, 500 µl of an internal standard (c19:0), 500 µl of sodium methylate, 200 µl of sulfuric acid and 1.5 ml of sodium chloride are added. after methylation, the tubes are centrifuged under the same conditions, then the upper phase is recovered and stored highlights in bioscience page 3 of 8 july 2025|volume 8 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions figure 4. oxygen consumption by p.segnis in aquariums subjected to different temperatures at −30 ◦c, according to the method of [20]. the identification of fatty acids is based on the comparison of their retention times with those of a reference mixture of methyl esters (supelco pufa-3). the analysis of the chromatographic peaks is carried out using hp chemstation software, and the relative amount of each fatty acid is expressed as a percentage of total fatty acids. the calculation of the percentage of each group of fatty acids (fa) was carried out according to the method of [21], using the following formula: %fa = (faa × 100) / (total fa), where %fa corresponds to the relative surface area of a fatty acid (faa) expressed as a percentage of the total surface area of the fatty acids detected. statistical analysis the results of the biochemical parameters are expressed as means ± standard error (sd). statistical analysis of the data was performed using r software (version 4.2.2), which was also used to generate the illustrative graphs. the normality of the distribution was checked by the shapiro–wilk test. a one-way analysis of variance (anova) was then performed, followed by the tukey test to identify significant differences between the means of the control and experimental groups. the significance threshold was set at 0.05. finally, a correlation matrix was established to assess the significance of the pearson correlation coefficients. results temperature and oxygen variation during the experiment during the experiment, the temperature of the ponds was measured every 24 hours figure 2. the control pond maintained a stable temperature of 19.81 ± 0.14 oc, whereas the ponds exposed to elevated temperatures reached significantly higher values of 30.18 ± 0.36 oc and 40.03 ± 0.56 oc, respectively (p < 0.001). dissolved oxygen levels followed a similar pattern figure 3, with the control pond showing 10.8 mg/l, while the figure 5. correlation matrix illustrates the relationships between essential fatty acids and the biochemical composition of p. segnis subjected to different temperatures ponds at 30 oc and 40 oc recorded 10.18 mg/l and 9.8 mg/l, respectively. furthermore, oxygen consumption increased with prolonged exposure to higher temperatures figure 4. a significant rise in oxygen consumption was observed in the tanks at 30 oc and 40 oc compared to the control at 19 oc, with consumption doubling in all tanks within the first 24 hours. water, ash, protein, lipid, and glycogen content as shown in table 1, water and ash contents exhibited similar trends across all temperature groups, with no statistically significant differences detected. in contrast, protein content varied significantly, with higher values observed in crabs exposed to 19 oc (66.08 mg/g) and 30 oc (62.49 mg/g), while a markedly lower protein concentration was recorded at 40 oc (10.75 mg/g) (p < 0.001). lipid analysis revealed that crabs exposed to 40 oc had the lowest lipid levels (1.9 mg/g), whereas significantly higher concentrations were measured in crabs from the 19 oc and 30 oc groups (p < 0.001). similarly, glycogen content was highest in the control group at 19 oc (2.03 mg/g), followed by the 30 oc group (1.77 mg/g), and was significantly reduced in crabs exposed to 40 oc (0.37 mg/g) (p < 0.001). fatty acid composition table 2 presents the fatty acid composition of p. segnis muscles exposed to temperatures of 19 oc, 30 oc, and 40 oc. our results show that crabs exposed to 30 oc and 40 oc had significantly higher contents of saturated fatty acids (sfa), reaching 43.64 ± 2.64% and 75.50 ± 1.15%, respectively, compared to the control group at 19 oc (p < 0.001). this increase is mainly attributed to the rise in the major fatty acids c16:0 and c18:0, which reached 33.71 ± 0.007% and 5.843 ± 0.013% at 30 oc, and 63.82 ± 1.28% and 9.66 ± 0.02% at 40 oc, respectively. highlights in bioscience page 4 of 8 july 2025|volume 8 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions figure 6. principal component analysis (pca), performed using two factors (f1 = 57.4% and f2 = 24.4%), allowed us to examine the variations of biochemical parameters (ash, humidity, lipids, proteins, carbohydrates) and essential fatty acids in the muscle of p. segnis subjected to different temperatures exposure to higher temperatures also resulted in a significant reduction in monounsaturated fatty acids (mufas) in crabs exposed to 30 oc compared to those maintained at 19 oc and 40 oc (p < 0.01). crabs exposed to 40 oc showed the lowest concentrations of c16:1 and c20:1 and the highest concentration of c18:1 compared to the 19 oc and 30 oc groups (p < 0.05). polyunsaturated fatty acids (pufas) varied significantly between temperature groups. the control group (19 oc) exhibited a high pufa content (80.53%), which was significantly greater than those observed at 30 oc and 40 oc (p < 0.001). a similar trend was found for omega-3 and omega-6 fatty acids, with higher percentages in the muscles of crabs maintained at 19 oc compared to those exposed to 30 oc and 40 oc (p < 0.05). this was confirmed by the high concentrations of docosahexaenoic acid (dha, 39.99%) and eicosapentaenoic acid (epa, 31.65%) in the 19 oc group, which significantly decreased in the muscles of crabs at higher temperatures (p < 0.01). finally, arachidonic acid (ara) showed an opposite trend, with significantly lower concentrations in crabs exposed to 19 oc compared to those at 30 oc and 40 oc (p < 0.001). correlation and principal component analysis the pearson correlation coefficient matrix highlights the relationships between the levels of biochemical composition and the main fatty acids present in p. segnis muscle under different temperature exposures. a significant positive correlation (p < 0.05) was observed between essential fatty acids, proteins, and glycogen, with coefficients greater than 0.62. conversely, monounsaturated fatty acids (mufas) showed a negative correlation with glycogen (r = −0.54), lipids (r = −0.66), water content (r = −0.36), and ash content (r = −0.46). furthermore, an increase in arachidonic acid (ara) and saturated fatty acid (sfa) levels appeared to be associated with a reduction in essential fatty acids, as evidenced by a strong negative correlation (r < −0.85), also illustrated in figure 5. each cell of the matrix represents the pearson correlation coefficient (r) for a pair of parameters. positive correlations (0 < r < 1.0) are shown in blue, while negative correlations (−1.0 < r < 0) are shown in red. the intensity of the colors corresponds to the absolute values of the correlation coefficients. statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001; values not statistically significant (p > 0.05) are represented by a checkbox symbol. principal component analysis (pca) biplot was employed to analyze the variation in measured parameters in p. segnis muscle samples exposed to different temperatures figure 6. the first two principal components explained 81.8% of the total variance, with pc1 contributing 53.6% and pc2 26.6%. this analysis revealed a clear separation between the groups of crabs subjected to 30 oc and 40 oc compared to those exposed to 19 oc. the 19 oc group was associated with high levels of lipids, glycogen, dha, pufa omega-3, and epa. in contrast, the 30 oc and 40 oc groups showed strong associations with elevated sfa, ara, ash, omega-6, and moisture contents. discussions climate change, which occurs over time through natural variability, is also exacerbated by human activity. these activities, responsible for the increase of greenhouse gases in the atmosphere, have led to an increase in water temperatures. the impact of climate change represents a serious environmental threat to fisheries, especially to economically exploited species, such as crabs. indeed, temperature, as a major abiotic factor, significantly influences the biochemical, metabolic and physiological processes of these species [22]. studies have shown that natural temperature variation can induce alterations in the fish carassius auratus [23], indicating that the increase in temperature generates molecular stress and increased energy demand, probably explaining the decrease in the organism’s energy reserves. in addition, it has been reported that increased temperature causes molecular disruptions in proteins, lipids, and fatty acids in fish [24]. with this knowledge, an in vivo study was conducted to assess the effects of increased temperature on the physiology and biochemical composition of ectothermic organisms. analyses of the biochemical composition of muscles, after exposure to elevated temperatures, revealed a decrease in protein and lipid contents compared to the control group. similar results were reported for the blue swimmer crab, p. pelagicus, where an elevated temperature of 30 °c affected the protein content [25]. similarly, it was shown that increased temperature alters lipids in carassius auratus [26]. these decreases can be explained by the energy production resulting from the complete oxidation of highlights in bioscience page 5 of 8 july 2025|volume 8 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions table 2. fatty acid composition of p. segnis muscle under increasing temperature fatty acid 19oc 30oc 40oc c16:0 5.760 ±0.106 33.712 ±0.007*** 63.829 ±1.287***### c18:0 1.001 ±0.044 5.843 ±0.013*** 9.661 ±0.022*## c20:0 3.136 ±0.069 4.090 ±0.000** 1.019 ±0.148***### sfa 9.898 ±0.220 43.645 ±2.639*** 74.509 ±1.159***# c16:1 4.649 ±0.052 0.239 ±0.004*** 1.198 ±0.154***## c18:1 0.073 ±0.014 2.380 ±0.002*** 9.661 ±0.022***### c20:1 4.841 ±0.059 3.860 ±0.001*** 1.019 ±0.148***# mufa 9.563 ±0.126 6.479 ±0.768** 11.878 ±0.279**## c18:3n3 0.759 ±0.046 0.796 ±0.001 2.186 ±0.156**## c20:5n3 31.657 ±0.180 26.800 ±0.003*** 5.456 ±0.056***### c22:6n3 39.995 ±0.362 15.810 ±0.003*** 0.993 ±0.162***# omega-3 72.411 ±0.496 43.406 ±2.031*** 8.635 ±0.375***# c18:2n6 5.472 ±0.052 4.510 ±0.002*** 1.688 ±0.161***## c18:3n6 1.994 ±0.101 0.492 ±0.002*** 0.241 ±0.197** c20:4n6 0.658 ±0.004 1.462 ±0.000*** 2.972 ±0.144**## omega-6 8.125 ±0.149 6.464 ±1.009** 4.901 ±0.503** pufa 80.537 ś 0.346 49.871 ś 2.756*** 12.938 ś 0.879***# note: results are presented as means ±standard deviation. significant differences in saturated fatty acids between: 19 oc vs 30 oc and 19 oc vs 40 oc are denoted by: p < 0.001 ***, and between 30 oc vs 40 oc by: p < 0.05. for monounsaturated fatty acids, significant differences between: 19 oc vs 30 oc and 19 oc vs 40 oc are denoted by: p < 0.01 **, and the difference between 30 oc vs 40 oc is indicated by: p < 0.05 #. regarding polyunsaturated fatty acids, significant differences between: 19 oc vs 30 oc and 19 oc vs 40 oc are noted by: p < 0.001 ***, and between 30 oc vs 40 oc by: p < 0.05 #. macromolecules in response to temperature variations. concerning glycogen contents, a highly significant decrease was observed with increasing water temperature. the study by [27], on glycogen concentrations and agonistic behavior of swimming crab (portunus trituberculatus) subjected to temperatures of 16 °c, 24 °c and 32 °c, shows that glycogen concentrations decrease as temperature increases. higher temperatures enhance ros production and consequently increase the risk of lipid peroxidation [28]. this hypothesis was observed in our work through the alteration of fatty acid composition of p. segnis subjected to different temperatures. fatty acid analyses revealed that in response to heat stress, saturated and monounsaturated fatty acids significantly increase, while polyunsaturated fatty acids decrease in the muscles of crabs exposed to high temperatures. heat stress is well established as a factor with physiological repercussions on poikilothermic organisms, eliciting a series of adaptive cellular responses. among these, one of the most notable is the modification of membrane lipid composition, particularly through fa remodeling. this process involves changes in the proportions of sfa and ufa fatty acids, as well as changes in carbon chain length, with an increased predominance of sfa, or longer chains, in response to temperature elevation, to counterbalance the increase in membrane fluidity. for example, alterations in polyunsaturated fatty acids, mainly dha and epa, were observed in the gastropod lymnaea [29]. similarly, in trematomus bernacchii, increased temperature induced lipid remodeling, with a decrease in c18:3n-3 and c22:6n-3 fatty acids [30]. similar results have been reported in different bivalves where warming caused marked modifications in their fatty acid profiles illustrated by the depletion of essential fa such as dha and epa [31]. in our study, the observed changes in fatty acid profiles indicate an adjustment in lipid metabolism. although significant changes were noted in the proportions of some fatty acids, the strong alterations observed in our analysis suggest that p. segnis is relatively affected by an increase in temperature of 30 and 40 °c and manages to deteriorate its lipid composition, indicating its high thermal sensitivity. in general, the multivariate analysis, particularly the princihighlights in bioscience page 6 of 8 july 2025|volume 8 http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions pal component analysis (pca) performed on the entire dataset, clearly highlighted distinct patterns associated with increasing thermal stress. the pca revealed a clear clustering of the physiological responses of portunus segnis across different temperature treatments in vivo, consistently separating the effects of low (19 °c), moderate (30 °c), and high (40 °c) temperatures. this separation underscores the substantial impact of temperature on key biochemical parameters such as protein, lipid, glycogen, and fatty acid profiles. the discriminative power of pca not only confirms the robustness of the observed trends but also emphasizes the high sensitivity of this species to thermal fluctuations. the results obtained clearly indicate that proteins and lipids play a crucial role in the metabolic mechanisms that enable organisms to cope with thermal stress, while changes in fatty acid compositionparticularly the decrease in polyunsaturated fatty acids such as epa and dhacan be considered reliable indicators of thermal variation in aquatic organisms. this study confirms that global warming significantly affects the physiological state and biochemical composition of p. segnis under both natural and experimental temperature conditions. from a practical perspective, these findings provide valuable insights for aquaculture stakeholders, including breeders and shellfish farmers, by identifying physiological thresholds and stress markers essential for monitoring the health and viability of crustacean stocks. furthermore, this work supports the development of adaptive and sustainable management strategies in response to climate-induced thermal stress, thus contributing to the long-term resilience and productivity of benthic resources. conclusions this study investigates the effects of elevated temperatures on the physiological and biochemical responses of the blue crab (portunus segnis) under in vivo conditions, following a three-day exposure to thermal stress. the analysis focused on key biochemical parameters in muscle tissue, including moisture, ash, protein, lipid, glycogen, and fatty acid composition. the results yielded several noteworthy findings. after 72 hours of exposure to high temperatures, no significant differences in water and ash content were observed compared to the control group. however, a marked decrease in glycogen, protein, and lipid levels was recorded with increasing temperature, indicating altered energy metabolism. in terms of fatty acid composition, a significant rise in saturated and monounsaturated fatty acids was detected, while polyunsaturated fatty acids, particularly those essential for maintaining membrane fluidity, decreased substantially. physicochemical measurements also showed a reduction in dissolved oxygen levels in the rearing tanks, alongside an increase in oxygen consumption, pointing to elevated metabolic demand and thermal stress. overall, these findings enhance our understanding of the physiological and biochemical responses of p. segnis to thermal variation and underscore the potential impacts of global warming on aquatic organisms, particularly regarding biochemical degradation and metabolic disruption. reference 1. halpern b, frazier m, potapenko j, casey k, koenig k, longo c, et al. spatial and temporal changes in cumulative human impacts on the worlds ocean. nature communications. 2015;6(7615):1000-38. 2. palmer cp. marine biodiversity and ecosystems underpin a healthy planet and social well-being; 2017. 3. weatherdon lv, magnan ak, rogers ad, sumaila ur, cheung wwl. observed and projected impacts of climate change on marine fisheries, aquaculture, coastal tourism, and human health: an update. frontiers in marine science. 2016;3:48. 4. masson-delmotte v, zhai p, pörtner h, roberts d, skea j, shukla p, et al. giec bilan 2007 des changements climatiques. contribution des groupes de travail i, ii et iii au quatrième rapport dévaluation du groupe dexperts intergouvernemental sur lévolution du climat. suisse: giec genève; 2007. 5. intergovernmental panel on climate change (ipcc). ipcc fourth assessment report: synthesis report; 2007. accessed: 2025-07-15. https://www.ipcc.ch/report/ ar4/syr/. 6. verma ak, rout pr, lee e, bhunia p, bae j, surampalli ry, et al. sustainability: fundamentals and applications. in: biodiversity and sustainability. wiley; 2020. . 7. poloczanska es, et al. responses of marine organisms to climate change across oceans. frontiers in marine science. 2016;3:62. 8. cheung wwl, pauly d. impacts of climate change on marine biodiversity and fisheries. nature reviews earth & environment. 2023;4(3):160-73. 9. pörtner ho, et al.. scientific outcome of the ipcc ar6 working group ii: climate change 2022 impacts, adaptation and vulnerability; 2021. https://www.ipcc.ch/ report/ar6/wg2/. 10. smale da, et al. marine heatwaves threaten global biodiversity and the provision of ecosystem services. nature climate change. 2019;9(4):306-12. 11. laufkötter c, et al. projected impacts of ocean warming on marine species biomass and ecosystem structure. global change biology. 2020;26(2):1305-18. 12. cheng l, abraham j, trenberth ke, et al. upper ocean temperatures hit record high in 2020. advances in atmospheric sciences. 2021;38:523-30. highlights in bioscience page 7 of 8 july 2025|volume 8 https://www.ipcc.ch/report/ar4/syr/ https://www.ipcc.ch/report/ar4/syr/ https://www.ipcc.ch/report/ar6/wg2/ https://www.ipcc.ch/report/ar6/wg2/ http://bioscience.highlightsin.org/ bejaoui s et al., 2025 change in p.segnis quality under warming conditions 13. cheng l, von schuckmann k, abraham jp, et al. past and future ocean warming. nature reviews earth and environment. 2022;3:776-94. 14. intergovernmental panel on climate change (ipcc). climate change 2022: impacts, adaptation and vulnerability. contribution of working group ii to the sixth assessment report of the ipcc; 2022. accessed: 2025-07-15. https://www.ipcc.ch/report/ar6/wg2. 15. aoac. official methods of analysis. association of official analytical chemists; 1990. food and nutrition sciences, 14(7), 1364–1374. 16. frings c, fendley t, dunn r, queen c. improved determination of total serum lipids by the sulfo-phospho-vanillin reaction. clinical chemistry. 1972;18(7):673-4. 17. dubois m, gilles k, hamilton j, rebers p, smith f. colorimetric method for determination of sugars and related substances. analytical chemistry. 1956;28(3):350-6. 18. lowry oh, rosebrough nj, farr al, randall rj. protein measurement with the folin phenol reagent. journal of biological chemistry. 1951;193:265-75. 19. folch j, lees m, stanley ghs. a simple method for the isolation and purification of total lipids from animal tissues. journal of biological chemistry. 1957;226:497-509. 20. cecchi g, basini s, castano c. méthanolyse rapide des huiles en solvant. note de laboratoire. revue française des corps gras. 1985;4:163-4. 21. visentainer jv, claus t, santos jr oo, chiavelli lur, maruyama sa. analytical aspects of the flame ionization detection in comparison with mass spectrometry with emphasis on fatty acids and their esters. in: guo x, editor. advances in gas chromatography. intechopen; 2013. p. 224. 22. ren x, wang q, shao h, xu y, liu p, li j. effects of low temperature on shrimp and crab physiology, behavior, and growth: a review. frontiers in marine science. 2021;8:746177. 23. sokolova im. energy-limited tolerance to stress as a conceptual framework to integrate the effects of multiple stressors. integrative and comparative biology. 2013;53(4):597-608. 24. fadhlaoui m, couture p. combined effects of temperature and metal exposure on the fatty acid composition of cell membranes, antioxidant enzyme activities and lipid peroxidation in yellow perch (perca flavescens). aquatic toxicology. 2016. s0166445x1630251x. 25. sugumar v, vasu p. effect of temperature on the biochemical constituents of the blue swimmer crab portunus pelagicus. world applied sciences journal. 2013;28(3):382-91. 26. bagnyukova tv, lushchak ov, storey kb, lushchak vi. oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23řc. journal of thermal biology. 2007;32(4):227-34. 27. xianpeng s, yunfei s, dapeng l, fang w, jingjing l, boshan z. agonistic behaviour and energy metabolism of bold and shy swimming crabs portunus trituberculatus. journal of experimental biology. 2019;222(3):188706. 28. bejaoui s, trabelsi w, chetoui i, ghribi f, fouzai c, soltani a, et al. assessment of trace elements uptake on the invasive crab portunus segnis hepatopancreas using a multivariate biochemical approach. humanities and social sciences. 2021;1(1):131-51. 29. fadhlaoui m, lavoie i. effects of temperature and glyphosate on fatty acid composition, antioxidant capacity, and lipid peroxidation in the gastropod lymnaea sp. water. 2021;13(8):1039. 30. truzzi c, illuminati s, antonucci m, scarponi g, annibaldi a. heat shock influences the fatty acid composition of the muscle of the antarctic fish trematomus bernacchii. marine environmental research. 2018;139:122-8. 31. pernet f, tremblay r, comeau l, guderley h. temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids. journal of experimental biology. 2007;210(17):2999-3014. highlights in bioscience page 8 of 8 july 2025|volume 8 https://www.ipcc.ch/report/ar6/wg2 http://bioscience.highlightsin.org/ abstract introduction materials and methods samples and experimental design crabs dissection and muscles preparation determination of water, ash, and proximate composition content fatty acid composition analysis statistical analysis results temperature and oxygen variation during the experiment water, ash, protein, lipid, and glycogen content fatty acid composition correlation and principal component analysis discussions conclusions highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202403 research article open access 1 department of biotechnology, mandalay technological university, mandalay, myanmar. * to whom correspondence should be addressed: tinmyatmyatsoe1977@gmail.com editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), cairo, egypt. reviewer(s): morad m. mokhtar, university mohammed vi polytechnic, benguerir, morocco.. mohamed hassan mubarak, plant production department (crop breeding branch), faculty of environmental agricultural sciences, arish university, north sinai, egypt. received: march 19, 2024 accepted: june 9, 2024 published: june 25, 2024 citation: soe tmm, thinn nw, zaw nm. study on biosorption of lead (ii) and manganese (ii) from aqueous solutions using sodium alginate and pleurotus ostreatus (oyster mushroom) beads. 2024 jun 25;7:bs202403 copyright: © 2024 soe et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. study on biosorption of lead (ii) and manganese (ii) from aqueous solutions using sodium alginate and pleurotus ostreatus (oyster mushroom) beads tin myat myat soe1 ><�, ngu wah thinn1 >< �, nyein min zaw1 ><� abstract the present study investigated the development of beads from alginate and mushroom powder for the removal of lead (pb) and manganese (mn) from water. batch biosorption experiments were conducted, varying ph and the composition of alginate and mushroom powder. results indicated that the combination of alginate and mushrooms achieved over 86.8% reduction in pb and 65.9% reduction in mn concentrations, with higher reductions observed for both metals at ph 5. these findings suggest that the beads effectively reduced pb and mn concentrations, with the mushroom content playing a significant role in their efficacy. the analysis of the ftir spectrum showed that the uptake of metal ions by mushrooms involves interactions of ions with hydroxyl, carboxyl, and amide groups. this study underscored the potential applications of these beads in addressing heavy metal pollution in water sources. by providing a sustainable and effective method for heavy metal removal, the use of alginate and mushroom-based beads could offer a valuable solution for environmental remediation efforts. keywords: aas, biosorption, ftir, heavy metal, mushroom, alginate bead introduction heavy metals are naturally occurring elements in the earth’s crust since its formation, posing serious risks to human health and the environment. groundwater and surface water contamination by heavy metals is a global concern, particularly in low-income or developing countries. most heavy metals are non-biodegradable and tend to accumulate in living organisms [1; 2]. in myanmar, heavy metal pollution has been reported in various regions, including mandalay, where population growth has exacerbated environmental challenges. studies have identified the presence of calcium (ca), copper (cu), magnesium (mg), mercury (hg), arsenic (as), manganese (mn), lead (pb), iron (fe), and zinc (zn) in water samples, with concentrations sometimes exceeding who standards [1; 2; 3; 4; 5]. the removal or reduction of these heavy metals is crucial for human health and environmental protection. researchers have investigated various techniques, including physical, chemical, and biological methods, to address this issue. while these methods have shown promise, challenges such as high costs, sludge production, and technical constraints remain [6]. water, essential for all life forms, supports plants, animals, and human life. however, heavy metal contamination in groundwater is a serious issue globally. in myanmar, mandalay has seen a rise in population, leading to increased studies on heavy metals since 2014. samples from regions like aung-myay-tharzan, chan-ayetharzan, chan mya-tharzi, mahar-aung-myay, pyi-gyi-tagon, and amarapura townships have shown levels of calcium (ca), copper (cu), magnesium (mg), mercury (hg), arsenic (as), manganese (mn), lead (pb), iron (fe), and zinc (zn). lead is particularly toxic to every organ and system in the human body, with the maximum acceptable lead limits in water set at 0.01 mg/l (10 ţg/l) by the world health organization [4; 7]. similarly, excessive manganese (mn) in the environment can cause health issues. the who suggests a concentration of manganese in drinking water should be less than 0.05 mg/l [3; 6; 8]. highlights in bioscience page 1 of 9 june 2024|volume 7 https://doi.org/10.36462/h.biosci.202403 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:tinmyatmyatsoe1977@gmail.com https://orcid.org/0009-0008-1827-898x mailto:nguwahthin1983@gmail.com https://orcid.org/0009-0004-0058-6168 mailto:ngyein2006@gmail.com https://orcid.org/0009-0008-5693-5274 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads in 2015, it was reported that the concentrations of manganese and other metals were found unsafe in many wells in myanmar [5]. in a study conducted by maw et al. in 2020, manganese was found to exceed permissible limits in 3 out of 6 townships, and lead in 1 out of 6 townships in the mandalay region [7]. recent studies have explored biosorption and adsorption methods for heavy metal removal, utilizing materials like chitosan, bentonite, zeolite, and alginate as biosorbents. alginate, extracted from brown marine algae and bacterial sources, has shown promise for heavy metal removal. its high surface area, biocompatibility, and abundance make it an attractive option. alginate works by forming complexes with metal ions, effectively sequestering them from aqueous solutions. studies have demonstrated its effectiveness in removing various heavy metals, including lead, copper, cadmium, and nickel. alginate-based adsorbents are relatively easy to prepare and can be tailored to specific metal removal requirements, making them a promising candidate for future research in heavy metal remediation [1; 9]. mushrooms, including pleurotus ostreatus, have shown excellent binding properties and tolerance to metals and adverse environments, such as various ph and temperature conditions [10; 11]. oyster mushrooms, such as pleurotus ostreatus, are environmentally friendly and can decompose organic or lignocellulosic wastes within 34 weeks [12]. pleurotus ostreatus mushroom, in particular, has shown promise in absorbing heavy metals from its surroundings. in combination with other materials, such as alginate, mushrooms have demonstrated synergistic effects, enhancing heavy metal removal efficiency. this study aims to reduce heavy metal concentrations using a biosorption method with pleurotus ostreatus mushroom as an inexpensive biosorbent, mixed with sodium alginate to form beads for its low cost, ease of use, and excellent removal efficiency. the objective of this work is to evaluate the effect of pleurotus ostreatus powder and alginate mixture beads on removing lead and manganese from aqueous solutions. materials and methods instrumentation the atomic absorption spectrophotometer (aa-7000, shimadzu, japan) with an air-acetylene flame system was utilized for precise metal determination. it is capable of measuring heavy metals and other elements at parts per billion (ppb or µg/l) concentrations with minimal sample volumes. a shaker (sk 2000, thermo fisher, usa) was used for mixing solutions, while a ph meter (serial no. 600719039029) was employed to measure acidity or alkalinity in water-based suspensions. glassware was sterilized and dried using an air oven (sh-do-100fg, serial no. 200530do-100fg, korea). an analytical balance (ay120, serial no. d432712541, shimadzu, japan) was used for measuring object mass or chemical weight. biomass spectra were analyzed using a fourier transform infrared spectrophotometer (ftir) (irtracer-100, shimadzu, usa) with spectral scanning from 500 to 3500 cm−1 to identify functional groups responsible for heavy metal adsorption and provide information on oxidation state, bonding, and morphology of dispersed clusters of heavy metal ions. a digital hotplate stirrer (hotplate stirrer, hsd-330, korea) was used to homogenize alginate and mushroom powder for bead formation. chemicals and reagents sodium alginate (c6h7o6na), anhydrous calcium chloride (cacl2), lead (ii) acetate, manganese (ii) solution, hydrogen peroxide (h2o2), nitric acid (hno3), and hydrochloric acid (hcl) were purchased as analytical-grade chemicals from wellestablished scientific supply stores in myanmar. collection and preparation of mushroom powders the fresh mushrooms (pleurotus ostreatus) samples were purchased from the local market in kyaukse, myanmar. the mushroom samples were thoroughly cleaned with tissue paper to remove dust, cut into pieces with a knife, and dried at room temperature for 3 days. after drying, they were ground with a blender and stored in plastic bags at room temperature for later use. preparation of bead formation all glassware (tubes, conical flasks, beakers, etc.) was soaked in hno3 for several hours in acidic water (10% nitric acid) to remove any residual organic contaminants and oxidize any trace metal ions present on the surface before undergoing a regular washing procedure with deionized (di) water. then, they were dried in an oven at 60řc for 24 hours. based on preliminary experiments assessing the structural integrity of formulated beads, concentrations of 4% and 6% for both sodium alginate and mushroom powders were selected to investigate their effects on bead efficacy for heavy metal removal. firstly, sodium alginate (4% and 6%) was added to 100 ml of di water and stirred with a magnetic stirrer at 60řc for 35 minutes. mushroom powders (4% and 6%) were then added to the sodium alginate solutions and stirred with a magnetic stirrer for 35 minutes. different concentrations of sodium alginate and mushroom powders were used to investigate their effects on bead properties and efficacy for heavy metal removal. this approach helps understand the influence of these parameters on bead performance, providing insights for potential optimization of bead synthesis. after cooling, the solution was dropped using a 60 ml cc syringe without a needle into 1m calcium chloride solution and left for 5 hours to solidify. fresh bead diameters ranged from 2 to 4.5 mm. beads were washed with di water and dried at room temperature overnight. blank samples were included to account for background contamination. figure 1 illustrates the bead formation process. preparation of lead (ii) and manganese (ii) solutions lead (ii) acetate and manganese (ii) solution were purchased from the local market in myanmar. pb (ii) and mn (ii) solutions were prepared separately by dissolving 100 ppm of pb (ii) and mn (ii) in deionized water. the solutions were stirred with a magnetic stirrer for 30 minutes. highlights in bioscience page 2 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads (a) (b) (c) (d) figure 1. bead formation of (a) 4% alginate and 4% mushroom powders, (b) 4% alginate and 6% mushroom powders, (c) 6% alginate and 4% mushroom powders, and (d) 6% alginate and 6% mushroom powders. ph adjustment the original ph of pb (ii) and mn (ii) solutions was adjusted using 1 m naoh solution and 1 m hcl solution to achieve ph 3 and ph 5. after adjusting the ph, the mixture of alginate and mushroom powder beads was added to the pb (ii) and mn (ii) solutions. biosorption experiments a bead dosage of 2 g was added to flasks containing 20 ml of lead (ii) and mn (ii)-bearing solutions adjusted to ph 3 and ph 5. flasks were shaken at 100 rpm at room temperature for 45 minutes, during which the equilibrium stage of pb (ii) concentration removal was reached [11]. samples were filtered using cellulose membrane papers. the filtrate of lead (ii) was digested using closed digestion (microwave), and that of mn (ii) using the open digestion method. metal concentrations in the filtrate were determined by aas (aa-7000) for each treatment in duplicate. ftir characterization dried metal-loaded and metal-free (control) beads were separately prepared for ftir analysis. firstly, the dried bead sample was ground with a mortar and pestle to a fine powder. equal amounts (2.0 mg) from each sample were taken and mixed with approximately 200.0 mg of potassium bromide (kbr). each potassium bromide-treated control and sample were thoroughly mixed with a mortar and pestle and pressed under vacuum into pellets, which were then analyzed using ftir. statistical analysis statistical analysis was conducted in r (version 4.3.0) using several packages. data were read from an excel file using the read_excel function from the readxl package. variable renaming was performed using the str_c function from the stringr package to ensure consistent naming conventions. for data manipulation and variable coding, the mutate function from the dplyr package was utilized. linear regression analysis was conducted using the lm function from the stats package to model the relationship between treatment variables and the reduction percentage of pb and mn. data visualization was performed using the ggplot2 and ggpattern packages to create plots illustrating the relationships between variables. the data were trimmed to only include treatment variables and calculated as the percent reduction relative to the initial concentrations of pb and mn. a 2k factorial design was employed, with lower values coded as -1 and higher values as 1 for all three treatment variables, allowing evaluation of main effects (individual variables) and interaction effects (combined effects of variables) on the sorption capacity of the biosorbent. separate regression models were conducted for each metal (pb and mn) to assess the impact of ph, alginate concentration, and mushroom powder concentration on the reduction percentage. the coefficients of the regression models were interpreted to determine the magnitude and direction of the effects of each variable. additionally, p-values were calculated to assess the statistical significance of each variable’s effect on the reduction percentage. results and discussions the reduction of lead across all ph, alginate, and mushroom powder combinations, an average reduction of 82.1% of pb (ii) was achieved, with the maximum reduction reaching 86.8% at 4% alginate, 6% mushroom powder, and ph 5, indicating the effectiveness of the biosorbent across various conditions in figure 2:a. the effect of ph was stronger at ph 5 compared to ph 3 in pb (ii) reduction, aligning with findings by [8], who observed increased biosorption of pb (ii) at ph 5 due to increased negative charge on the biosorbent surface. the regression analysis of pb (ii) reduction is shown in table 1 along with the respective probability values. the residual error for the model was 1.889, the multiple r-squared was 0.7514, and the adjusted r-squared was 0.5857. the f-statistic of the model for pb (ii) reduction was 4.535 on 6 and 9 degrees of freedom and the probability value of the model was 0.02164. although the model was significantly better than the default model, the adjusted r-squared value showed that the model explained only over half of the variations in the experiment. the analysis showed that the mushroom had a significant positive effect on the reduction of pb (ii) with a p-value of 0.00276. this might mean that the higher the percentage of mushroom in the beads, the better the removal would be. our findings are consistent with [13], who found that increased mushroom biomass enhanced pb (ii) biosorption because increasing the biomass dosage progressively increases the adsorption sites for the metal ions [13]. however, the mentioned fact is entangled with the interaction effect of mushrooms and alginate. the interaction graph was depicted in figure 2:b showing that 4% alginate inclusion in the highlights in bioscience page 3 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads table 1. linear regression analysis of the effect of mushroom, alginate, and ph on pb reduction estimates t-value pr(>|t|) intercept 82.1177 173.861 < 2e-16 *** ph 1.0054 2.129 0.06216. alginate -0.5710 -1.209 0.25750 mushroom 1.9265 4.079 0.00276 ** ph:alginate 0.3775 0.799 0.44473 ph:mushroom 0.2902 0.614 0.55416 alginate:mushroom -0.8914 -1.887 0.09173 signif. codes: 0 ’***’ 0.001 ’**’ 0.01 ’*’ 0.05 ’.’ 0.1 ’ ’ 1. residual standard error: 1.889 on 9 degrees of freedom. multiple r-squared: 0.7514, adjusted r-squared: 0.5857. f-statistic: 4.535 on 6 and 9 df, p-value: 0.0216 beads increased the chance of removing the metal at the higher percentage of mushroom powder in the beads. the change in ph effect in the range of 3 and 5 also showed a positive term for reducing the pb (ii) ions with a p-value of 0.06216. it has been known that ph has a strong influence on metal ion removal. the appearance of the weak effect in this experiment might be due to the fact that the range chosen was narrow and its effect showed a low value. the reduction of manganese similar to pb (ii) reduction, all combinations of ph, alginate, and mushroom showed an average reduction of 59.2% of mn (ii) from the mixture, with the maximum reduction of 65.9% observed at 4% alginate, 6% mushroom powder, and ph 5 (figure 2:c). the effect of ph was more pronounced at ph 5 than at ph 3 in the removal of mn (ii), consistent with findings by [14] regarding optimal biosorption at ph above 5. the mushroom showed the most distinct effect on the reduction of mn (ii). in general, the overall graph for both pb (ii) and mn (ii) reduction experiment showed that the inclusion of mushroom increased the percentage of the metals removed from the tested mixture (figure 2:d). table 2 shows the regression analysis for the mn (ii) reduction from the mixture. the linear model had a residual error of 1.224. the multiple r-squared for the model was 0.9515, and the adjusted r-squared was 0.909. f-statistic for the model was 22.4 on 7 and 8 degrees of freedom, and the model was significantly better than the default model with a p-value less than 0.001. the adjusted r-squared value indicated that the model explained more than 90% of the variation of the data in the experiment. the regression estimates for most of the variables and their interaction effects showed significant terms. the estimate for the mushroom effect on the mn (ii) reduction was distinct, with a magnitude of 2.4624 and a p-value of 4.18e-05, and it had a positive effect, meaning the more mushroom inclusion in the bead might increase the reduction percentage in removing mn (ii) from the mixture. [15] also revealed a marked increase in the removal of mn (ii) by increasing of biosorbent mass of aspergillus spp.. however, this interpretation might not be complete because there was an interaction term among all three variables with a magnitude of -1.7290 and a p-value of 0.0005. the analysis of interaction was visualized in figure 2:d. while the effect of mushroom on mn (ii) reduction increased at both ph values of 3 and 5 in 4% alginate, its effect showed a higher degree of slope at ph 5. the mushroom effect in 6% alginate seemed lower than in 4% at both ph values, although the effect at ph 5 showed a higher reduction percentage. table 2. linear regression analysis of the effect of mushroom, alginate, and ph on mn reduction estimates t-value pr(>|t|) intercept 59.1987 193.511 5.69e-16 *** ph 0.7100 2.321 0.048852 * alginate -1.1167 -3.650 0.006492 ** mushroom 2.4624 8.049 4.18e-05 *** ph:alginate 1.1577 3.784 0.005355 ** ph:mushroom 0.2896 0.947 0.371461 alginate:mushroom -1.5648 -5.115 0.000913 *** ph:alginate:mushroom -1.7290 -5.652 0.000480 *** signif. codes: 0 ’***’ 0.001 ’**’ 0.01 ’*’ 0.05 ’.’ 0.1 ’ ’ . residual standard error: 1.224 on 8 degrees of freedom. multiple r-squared: 0.9515, adjusted r-squared: 0.909. f-statistic: 22.4 on 7 and 8 df, p-value: 0.0001179 the mn (ii) reduction model appears to be more robust, with a higher adjusted r-squared and more significant predictors, especially the three-way interaction term. mushroom is consistently a significant positive predictor for both mn (ii) and pb (ii) reduction, suggesting its importance in promoting metal reduction. these results prompt further investigation into the specific mechanisms by which mushroom, ph, alginate, and their interactions influence mn (ii) and pb (ii) reduction. the significance of the three-way interaction in the mn (ii) reduction model underscores the complexity of these relationships, necessitating a nuanced understanding of the variables involved. replication studies with a larger sample size and under varying environmental conditions, such as temperature, initial concentration, and contact time, are essential to validate these findings and enhance their reliability and generalizability. while this study focused on ph, alginate, and mushroom powder, exploring alternative explanations could highlights in bioscience page 4 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads figure 2. (a) the effect of mushroom, alginate, and ph on pb (ii) reduction. (b) interaction effect between mushroom and alginate on pb (ii) reduction. (c) the effect of mushroom, alginate, and ph on mn reduction. (d) interaction effect between mushroom and alginate on mn (ii) reduction. strengthen future analysis. factors like organic matter or other ions in water might interact with these variables, affecting adsorption. metal ion properties, such as oxidation state, could also interact with treatment variables. investigating these could enhance our understanding and improve biosorption material design. ftir characterization the characteristic spectra of alginate and mushroom bead exhibit several notable features: a broad band centered at approximately 3313 cm-1 corresponding to hydroxyl group stretching, low-intensity bands around 2926 cm-1 attributed to ch2 groups, a distinct peak at 2364 cm-1 representing stretching of the (cn) group, a peak at 1612 cm-1 indicating c=o stretching vibration, peaks at 1533 cm-1 and 1450 cm-1 representing asymmetric and symmetric stretching modes, respectively, of carboxylate salt groups (-coona), and various vibrations between 11381093 cm-1 associated with glycoside bonds in the polysaccharide (co-c stretching). ftir spectra before and after adsorption are shown in figure 3 and table 3. after the adsorption of pb (ii) ions on a mixture of alginate and mushroom bead, notable spectral changes are observed: there is a shift of nearly 20 cm-1 towards lower wavenumbers in the peak assigned to c-h stretching vibration. the peak associated with o-h stretching mode at over 3300 cm-1 disappears. two peaks related to c=o stretching vibration and c=c stretching mode shift towards higher wavenumbers by approximately 20 cm-1. the c=n vibration mode experiences a downward shift of 10 cm-1. a new peak is observed at 1344 cm-1, attributed to c-h bending. peaks at 1081 cm-1 and 1045 cm-1 correspond highlights in bioscience page 5 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads table 3. position of the main peaks in ftir spectra of sodium alginate and mushroom bead before adsorption, after adsorption of pb (ii), and mn (ii) wave length before adsorption (cm−1) wave length after adsorption (cm−1) assignment pb (ii) mn (ii) 3313 3435 o-h stretching 2926 2902 2924 asymmetric ch3 and ch2 stretching 2364 2394 (cn) stretching 1612 1637 1639 c=o stretching 1533 1550 c=n stretching 1450 1423 1385 c-h bending 1138 1344 1269 c-n stretching 1093 1081 1153 c-n stretching 756 1045 1045 c-h bending 657 1022 893 c-metal stretching 935 819 to c-o stretching vibrations in carbohydrates and polysaccharides, potentially indicating changes due to metal-ion interactions. furthermore, the peak at 1022 cm-1 could be associated with co or c-c stretching vibrations in the context of carbohydrates or polysaccharides, suggesting possible alterations post-pb (ii) adsorption. after the adsorption of mn (ii) ions on a mixture of alginate and mushroom bead, distinct changes are observed in the spectrum. the o-h stretching vibration peak shifts to a nearly 10 cm-1 higher wavelength compared to the pure bead spectrum. however, the c-h stretching mode peak remains at the same wavelength as in the pure bead spectrum. the stretching mode peak of the (cn) group disappears. the c-h bending mode experiences a significant shift of 60 cm-1 to lower wavelengths compared to the pure bead ir spectrum. similarly, peaks related to c-o stretching, c-n stretching, and c-h bending vibration modes also shift to higher wavelengths. these shifts or alterations in the peaks post-mn (ii) adsorption likely signify interactions between mn (ii) ions and the functional groups present in alginate and mushrooms. the infrared (ir) spectra of alginate and mushroom beads, along with the alterations observed following the adsorption of pb (ii) and mn (ii) ions onto the mixture of alginate and mushroom beads, offer insights into changes in the molecular composition induced by metal-ion interactions. the observed shifts and variations in peak intensities post-metal ion adsorption (pb (ii) and mn (ii)) on the alginate and mushroom bead mixture imply modifications in the bead’s molecular structure due to interactions with the metal ions. these modifications potentially denote the coordination of metal ions with specific functional groups present in alginate and mushrooms. such alterations provide crucial insights into the adsorption mechanism and the nature of interactions between metal ions and the components of the beads. [1; 2; 12]. various methods for removing heavy metals, including precipitation, filtration, ion exchange, carbon adsorption, evaporation, membrane technology, reverse osmosis pre-concentration, redox reactions, electrowinning, chelation, wastewater coagulation, and electrochemical processes, have been demonstrated to be ineffectual according to studies by ali et al. (2019) and taha et al. (2023) [16; 17]. under metal stress, a notable decline in protein and carbohydrate content was observed, as evidenced by a reduction in the intensity of absorption bands, particularly within the 1800 to 800 cm-1 regions. this region is known to be characteristic of proteins and carbohydrates, as highlighted in studies by dumas and miller (2003), walkers et al. (2004), and yee et al. (2004) [18; 19; 20]. future research should investigate the impact of contact time and explore pre-treatment methods for the mushroom powder to further optimize lead and manganese removal efficiency. comparison of different adsorbents for pb (ii) and mn (ii) removal efficiency the removal efficiency of pb and mn by various adsorbents in recent studies is summarized in table 4. the alginate + mushroom powder beads in this work exhibited a pb removal efficiency of 86.8% and a mn removal efficiency of 65.9%. while these results demonstrate the competitive nature of the alginate +mushroom powder beads in terms of pb removal efficiency, they also indicate a slightly lower efficiency for mn removal compared to some other adsorbents. further optimization efforts could potentially lead to an enhancement in the effectiveness of the beads, thereby improving their performance in removing pb and mn from water. highlights in bioscience page 6 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads figure 3. ftir spectrum of (a) pure beads, (b) biomass after pb (ii) adsorption, and (c) biomass after mn (ii) adsorption practical implications the high reduction percentages observed for both lead (pb) and manganese (mn) highlight the potential of these biosorbent materials in treating contaminated water sources. implementing such biosorption techniques could offer a cost-effective and environmentally friendly approach to reducing heavy metal pollution in water bodies. furthermore, understanding the factors that influence the efficiency of these biosorbents, such as ph, alginate, and mushroom powder concentrations, can inform the design of optimized remediation strategies tailored to specific environmental conditions. this research provides a foundation for further studies and the development of practical applications for the sustainable management of heavy metal contamination in aquatic environments. conclusions the analyses of mn (ii) and pb (ii) reduction dynamics reveal relationships among key variables in biological metal reduction processes. in mn (ii) reduction, the model demonstrates a good fit (adjusted r-squared: 0.909), emphasizing the role of mushroom in promoting mn (ii) reduction. the negative impact of alginate hints at potential inhibitory effects, while the three-way interaction (ph, alginate, and mushroom) underscores the complexity of their contributions. further research should explore the biochemical pathways underlying these effects, enhancing our understanding of mn (ii) reduction. in pb (ii) reduction, the model, though statistically significant (f-statistic: 4.535, p-value: table 4. comparison of pb and mn removal efficiency (re) of different adsorbents. biosorbent re (%) pb mn aguapé 63.00 [21] moringa oleifera leaves 97.00 [22] azadirachta indica (neem leaves) 93.50 [23] raw tea factory waste 94.07 [24] modified tea factory waste 97.73 [24] sugarcane bagasse 89.31 [25] milled olive stones 94.50 [26] dried biomass microalgae aphanothece sp 99.90 [27] waste fungal biomass 87.00 [28] waste fungal biomass with pre-treatment 93.00 [28] moringa oleifera seed 92.00 [29] modified sugarcane bagasse biochar 65.80 [30] date palm biochar 73.20 [31] polyvinyl alcohol/sodium alginate 72.34 [32] alginate + mushroom powder beads 86.80 65.90 this work 0.02164), exhibits a slightly lower goodness of fit (adjusted rsquared: 0.5857). mushroom remains a significant positive predictor, aligning with its role in mn (ii) reduction. the marginal significance of ph and the non-significant effect of alginate suggest a nuanced relationship with pb (ii) reduction. ftir results suggested carboxyl, hydroxyl, and amide were the potential functional groups that may be involved in the mn (ii) biosorption. in conclusion, the mixture of mushrooms (pleurotus ostreatus) and alginate beads has been more effective for the removal of pb (ii) than mn (ii) from the aqueous solution. future research could focus on replicating the study with larger sample sizes and in different environmental conditions to validate the findings. additionally, exploring the long-term stability and reusability of the beads in real-world applications would provide valuable insights into their practicality for environmental remediation. moreover, to further elucidate the mechanisms underlying the observed effects of mushroom, ph, and alginate interactions on metal reduction, future investigations could employ advanced analytical techniques, such as surface characterization or scanning electron microscopy. reference 1. kamarudzamana na, chayb ct, amira a, taliba as. biosorption of mn (ii) ions from aqueous solution by pleurotus spent mushroom compost in a fixed-bed column. social and behavioral sciences. 2015;195:2709-16. 2. talbi a, kemper k, minnatullah k, foster s, tuinhof a. an highlights in bioscience page 7 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads overview of current operational responses to the arsenic issue in south and east asia. washington, dc: world bank; 2005. 3. world health organization. guidelines for drinking-water quality. 3rd ed. geneva: world health organization; 2008. cited june 2017. accessed december 2018. available from: https://www.who.int/water_sanitation_ health/dwq/fulltext.pdf. 4. world health organization. lead in drinking-water. background document for preparation of who guidelines for drinking-water quality. geneva: world health organization; 2011. who/sde/wsh/03.04/9/rev/1. 5. bacquart t, frisbie s, mitchell e, grigg l, cole c, small c, et al. multiple inorganic toxic substances contaminating the groundwater of myingyan township, myanmar: arsenic, manganese, fluoride, iron, and uranium. elsevier bv. 2015;517. available from: http://dx.doi.org/10. 1016/j.scitotenv.2015.02.038. 6. chen g, viengvilay k, yu w, mao t, qu zz, liang b, et al. effect of different modification methods on the adsorption of manganese by biochar from rice straw, coconut shell, and bamboo. acs. 2023. 7. maw a, phyu m, aung pk, mar nm, khin kk, khaing so, et al. approach to assessment of heavy metals contamination in drinking water, mandalay region, myanmar. iop conference series: earth and environmental science. 2020. 8. united states environmental protection agency. secondary drinking water regulations: guidance for nuisance chemicals. washington, dc, usa; 2013. available from: https: //www.epa.gov. 9. xiangliang p, jianlong w, daoyong z. biosorption of pb (ii) by pleurotus ostreatus immobilized in calcium alginate gel. process biochemistry. 2005;40:2799-803. 10. abbas sh, ismail im, mostafa tm, sulaymon ah. biosorption of heavy metals: a review. j chemical sci technol. 2014;3(4):74-102. available from: https://www. researchgate.net/publication/266795209. 11. qazilbash aa. isolation and characterization of heavy metal tolerant biota from industrially polluted soils and their role in bioremediation [doctoral dissertation]. quaid-i-azam university islamabad; 2004. 12. khan mw, ali ma, khan na, muhammad aslam khan ma, abdul rehman a, jav n. effect of different levels of lime and ph on mycelial growth and production efficiency of oyster mushroom (pleurotus spp). pak j bot. 2013;45(1):297-302. 13. kariuki z, kiptoo j, onyancha d. biosorption studies of lead and copper using rogers mushroom biomass ’lepiota hystrix’. south african journal of chemical engineering. 2017;23. 14. fadel m, hassanein nm, elshafei mm, mostafa ah, ahmed ma, khater hm. biosorption of manganese from groundwater by biomass of saccharomyces cerevisiae. hbrc journal. 2017;13(1):106-13. 15. mahmoud a, massoud m, abdel-motaal f, el-zayat s. tolerance and biosorption of manganese, iron and aluminium by five aspergillus species isolated from freshwater. catrina: the international journal of environmental sciences. 2017;16:61-9. 16. taha a, hussien w, gouda sa. bioremediation of heavy metals in wastewaters: a concise review. egypt j aquat biol fish. 2023;27(1):143-66. 17. ali h, khan e, ilahi i. environmental chemistry and ecotoxicology of hazardous heavy metals: environmental persistence, toxicity, and bioaccumulation. j chem. 2019:1-14. 18. dumas p, miller l. the use of synchrotron infrared microspectroscopy in biological and biomedical investigations. vib spec. 2003;32:3-21. 19. wolkers wf, oliver ae, tablin f, crowe jh. a fourier transform infrared spectroscopy study of sugar glasses. carb res. 2004;339:1077-85. 20. yee n, benning lg, phoenix vr, ferris fg. characterization of metal-cyanobacteria sorption reactions: a combined macroscopic and infrared spectroscopic investigation. 2004. 21. de freitas f, battirola ld, arruda r. assessment of the cu(ii) and pb(ii) removal efficiency of aqueous solutions by the dry biomass aguapé: kinetics of adsorption. environ monit assess. 2019;191:751. 22. imran m, anwar k, akram m, shah gm, ahmad i, samad shah n, et al. biosorption of pb(ii) from contaminated water onto moringa oleifera biomass: kinetics and equilibrium studies. international journal of phytoremediation. 2019;21(8):777-89. 23. elkhaleefa a, ali ih, brima ei, shigidi i, elhag ab, karama b. evaluation of the adsorption efficiency on the removal of lead (ii) ions from aqueous solutions using azadirachta indica leaves as an adsorbent. processes. 2021;9(3):559. 24. nuholu y, ekmekyapar kul z, kul s. pb (ii) biosorption from the aqueous solutions by raw and modified tea factory waste (tfw). int j environ sci technol. 2021;18:2975-86. highlights in bioscience page 8 of 9 june 2024|volume 7 https://www.who.int/water_sanitation_health/dwq/fulltext.pdf https://www.who.int/water_sanitation_health/dwq/fulltext.pdf http://dx.doi.org/10.1016/j.scitotenv.2015.02.038 http://dx.doi.org/10.1016/j.scitotenv.2015.02.038 https://www.epa.gov https://www.epa.gov https://www.researchgate.net/publication/266795209 https://www.researchgate.net/publication/266795209 http://bioscience.highlightsin.org/ soe et al., 2024 biosorption of lead and manganese from water using alginate-mushroom beads 25. ezeonuegbu ba, machido da, whong cmz, japhet ws, alexiou a, elazab st, et al. agricultural waste of sugarcane bagasse as efficient adsorbent for lead and nickel removal from untreated wastewater: biosorption, equilibrium isotherms, kinetics and desorption studies. biotechnology reports. 2021;30:e00614. 26. amar mb, walha k, salvadó v. evaluation of olive stones for cd(ii), cu(ii), pb(ii) and cr(vi) biosorption from aqueous solution: equilibrium and kinetics. int j environ res. 2020;14:193-204. 27. keryanti k, mulyono ews. determination of optimum condition of lead (pb) biosorption using dried biomass microalgae aphanothece sp. periodica polytechnica chemical engineering. 2021;65(1):116-23. 28. rozman u, kalíková g, marolt g, skalar t, gajnar gotvajn a. potential of waste fungal biomass for lead and cadmium removal: characterization, biosorption kinetic and isotherm studies. environmental technology & innovation. 2020;18:100742. 29. alghamdi a, rajan kp, thomas sp. comprehensive evaluation of moringa oleifera seed as a low-cost adsorbent for removal of manganese (mn) from aqueous solutions. case studies in chemical and environmental engineering. 2024;9:100635. 30. de castro ae, penido es, souza tf, camargos jb, lobato rlm, ribeiro-soares j, et al. biochars from modified sugarcane bagasse for manganese removal from mining effluents. journal of environmental chemical engineering. 2023;11:110761. 31. fseha yh, sizirici b, yildiz i. manganese and nitrate removal from groundwater using date palm biochar: application for drinking water. environmental advances. 2022;8:100237. 32. chang q, ali a, su j, wen q, bai y, gao z, et al. efficient removal of nitrate, manganese, and tetracycline by a polyvinyl alcohol/sodium alginate with sponge cube immobilized bioreactor. bioresource technology. 2021;331:125065. highlights in bioscience page 9 of 9 june 2024|volume 7 http://bioscience.highlightsin.org/ abstract introduction materials and methods instrumentation chemicals and reagents collection and preparation of mushroom powders preparation of bead formation preparation of lead (ii) and manganese (ii) solutions ph adjustment biosorption experiments ftir characterization statistical analysis results and discussions the reduction of lead the reduction of manganese ftir characterization comparison of different adsorbents for pb (ii) and mn (ii) removal efficiency practical implications conclusions highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20209 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 8 review article open access 1biofuel research laboratory, department of microbiology, school of life sciences, central university of tamil nadu, thiruvarur, tamil nadu 610 005, india. 2department of microbiology, prist, institution deemed to university, thanjavur – 613 403, india. 3biomass refining lab, department of renewable energy engineering, tamil nadu agricultural university, coimbatore 641 003, tamil nadu, india. contacts of authors * to whom correspondence should be addressed: suchitra rakesh citation: rakesh s, kumar j t, sri b, jothibasu k, karthikeyan s. 2020. sustainable cost-effective microalgae harvesting strategies for the production of biofuel and oleochemicals. highlights in bioscience volume 3. article id 20209. dio:10.36462/ h.biosci.20209 received: may 30, 2020 accepted: july 3, 2020 published: july 7, 2020 copyright: © 2020 rakesh et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: this work is financially supported by the serb dst project (eeq/2018/001463) competing interests: the authors declare that they have no competing interests. sustainable cost-effective microalgae harvesting strategies for the production of biofuel and oleochemicals suchitra rakesh 1 *, jayakumar tharunkumar 1 , bhavya sri 1 , karuppaiyan jothibasu 2 and subburamu karthikeyan 3 abstract microalgae have been explored for sustainable production of biofuel and chemicals. microalgae is promising feed stock for the production of several oleochemicals. it has the ability to utilize a variety of low cost feed stocks, accumulated large quantities of lipids and variety of value added products in their biomass. one of the major obstacles associated with the conversion of algae into value-added products is harvesting. the harvesting of algae is the most problematic area due to its low sedimentation rate, low biomass concentration, and high capital costs. harvesting of algae is carried out by different physical, chemical, mechanical, biological, and electrolytic methods such as sedimentation, centrifugation, microstraining, dissolved air flotation, electrolytic flotation, chemical flocculation, bioflocculation, autoflocculation, filtration. this review highlights the various methods of microalgae harvesting with advantages and future perspective of sustainable and cost-effective harvesting of microalgae. keywords: microalgae, harvesting, autoflocculation, sedimentation, bioflocculation. introduction algae are simple aquatic photosynthetic microorganisms that live in saline and freshwater environments. sunlight plays a key role in the harvesting of the algae. they can grow at any place where enough sunlight is found. apart from sunlight, the chemical composition of algae includes proteins, carbohydrates, lipids, and nucleic acids. algae are known as thirdgeneration biofuels due to their higher biomass production capacity compared to other cerealbased crops [1]. they are one of the important sources of food, animal feed, medicinal products, and most important oil for fuels [2]. cost-effective production of microalgae biomass for value added products like biodiesel is usually restricted due to poor efficiency in essential processes like cultivation, harvesting and lipid extraction [3]. harvesting of microalgae involves two consecutive steps i.e., bulk harvesting and thickening. bulk harvesting involves flocculation, floatation, and sedimentation, in which solid biomass is separated from suspensions. whereas, thickening involves filtration, centrifugation [4]. the present paper foccuses on recent techniques involved in harvesting microalgae and to assess their technical, economical and application potention. rakesh et al., 2020 sustainable cost-effective microalgae harvesting strategies highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 algal metabolites algae like macrocystis, laminaria, and ascophyllum produce alginic acids. alginate has the potential to make a highly viscous solution and chelating properties, so they are widely used in the pharma and food industries. alginates in textile industries are used for sizing cotton yarn [5]. agar derived from macroalgae is used in making desserts, jellies, candies, frozen foods, etc. due to its semi-solid nature and jellying properties. agarose, as one of the byproducts of algae, is used significantly in molecular biology and many biomedical fields for the production of tablets, capsules, and anticoagulants [6]. many of the algae are used for aquaculture feed like nannochloropsis, chlorella, tetraselmis, phaeodactylum, pavlova, etc [7]. for hundreds of years, microalgae have been used as nutrient supplements as it is a rich source of carbohydrates, protein, enzymes, vitamins, and minerals. there are a variety of pigments produced by microalgae like chlorophyll, phycobiliproteins, carotenoids, β-carotene, astaxanthin, lutein, canthaxanthin [8]. vitamins like biotin, pantothenate, riboflavin, folic acid, nicotinic acid are produced by microalgae [9]. harvesting of microalgae harvesting of algae is a two-step process that includes cultivation and separation of the algae. the separation of the microalgae can be of mechanical, chemical, electrical, and biological methods. harvesting by using cost-effective procedures is the most difficult phase in algal biofuel production [4]. the techniques applied for microalgae harvesting are filtration, gravity sedimentation, centrifugation, microstraining, floatation, flocculation, etc., [10-11] and sometimes a combination of one or two methods. an ideal harvesting method must be effective for more algal species, which gives a higher biomass concentration with minimal costs, energy, and maintenance. centrifugation centrifugation is the ideal process for harvesting of the microalgae because of its quickest and effective procedure to remove particles from the algal suspension without any additive agents. during the process of centrifugation, large shear forces are exerted and lyse the algal cells releasing algal oil into the medium. later to which the oil recovery has been done [2]. chlorella vulgaris shows a very high recovery of biomass and oil yield when subjected to the centrifugation process [12]. pahl et al. [13] have examined various centrifuges like disk stack centrifuge, tubular centrifuge, nozzle-type centrifuge, decanters, perforated and imperforated basket centrifuges, hydrocyclones for microalgae separation. disc stack centrifuges are exclusively used in industrial high-value product recovery from algae but recorded poor energy return as more energy is exhausted than produced [14]. decanter centrifuge is highly efficient but involves high energy consumption, whereas hydrocyclone centrifuges can be used with less maintenance but due to its high energy and capital cost, restricted its use for large scale purposes [15-16]. gravity sedimentation in gravity sedimentation, the gravitational forces separate the solids and liquids from one another depending on their size and density. the factors affecting sedimentation are particle size and density, ph, temperature, the intensity of light, and aging of the cells [17]. the rate of sedimentation is directly proportional to the difference in densities of the particles. if there is a high difference in densities between solids and liquids, the sedimentation rate is faster and in case of less difference, the sedimentation rate is slower [15; 18]. sedimentation requires low cost and less energy. sedimentation is mostly species-specific, slow separation, and low final concentration. the chlorella sp. cultured at ph=10.5 and sedimentation time of 12 h showed effective harvesting from 1.01g/l initial concentration to 3.95g/l [19]. the centrifugation method is not appropriate for the cost-effective harvesting of microalgae like chlorella sp. due to its small cell size. the application of a combination of various harvesting techniques is necessary for enhanced recovery efficiency. a study suggests that the granulation of the biomass obtained from microalgae (chlorella sp. cells) via gravity sedimentation with filamentous microalgae is a possible solution for effective recovery [20]. microstraining in microstraining, drum rotates slowly in the partially submerged condition in the trough of suspended algal cells. a mesh act as a screen that captures large particles of algae, whereas microalgae still pass via mesh and were not able to be harvested [21]. it is a simple cost-effective method, but due to low harvesting efficiency, it is not desired much. filtration the problems associated with the use of the filtration process are the small size of the algal cells, their shape [spherical], and the gelatinous and other extracellular materials of the algal cells. these results in the poor filtration causing plugging of the filters [22]. the particles in the suspension are introduced onto the screen of different sizes, the particles either pass through the filters or retained on the screen depending upon their size [23]. filtration techniques can be classified into microfiltration, macrofiltration, ultrafiltration, and reverse osmosis (figure 1). figure 1: the filteration technique for microalgae harvesting. rakesh et al., 2020 sustainable cost-effective microalgae harvesting strategies highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 many types of filters and membranes with different sizes are available for easy filtration. this method is suitable for only large algal cells; smaller cells may clog in the filter pores [24]. filtration is the process of separation of microalgal biomass from the broth medium by passing the suspension through the permeable membrane [25]. membrane filtration technology for harvesting microalgae comprises osmotic and pressure-driven processes. the pressure-driven membrane process involves hydraulic pressure to force biomass passage via the permeable membrane for solids separation from liquid [26]. common membrane filter designs include microfiltration (0.1-10µm), macro filtration (10µm), ultrafiltration (0.01-0.1 µm), nanofiltration (0.001-0.01µm), tangential flow filtration, vacuum filtration, dead-end and cross-flow filtration based on the size of the filter [27-28]. osmotic pressure is used to separate the microalgal biomass and liquid medium in osmotically driven membranes; this includes reverse and forward osmosis. however, reverse osmosis has lower water flux, reverse draw solute flux, and fouling process compared to forward osmosis [29-30]. the criteria for the selection of membrane material involve characteristics of species, biomass concentration, hydrophilicity, hydrophobicity, surface charge, flow parameters, etc. the typical materials used for polymer preparation of membrane are polyethersulfone polyvinylpyrrolidone, polyvinyl chloride, cellulose acetate, polyvinylidene fluoride, polyacrylonitrile, polyethersulfone, polyamide, etc [26; 28]. recent studies have reported the use of ceramic-based material for membrane in the form of a diatomite dynamic membrane (ddm) [31]. one of the studies has reported that a mixture of sand, starch, and kaoline can be used for the novel ceramic membrane production for microalgae concentration and can overcome difficulties like high shrinkage, low porosity, high thickness, poor flexibility, small pore size, etc [32]. other membrane developments include electromembrane [33], steel-use-stainless membrane coated with selective polymers, etc [34]. research on the application of membrane processes for microalgae harvesting is mostly restricted to lab-scale and lab-scale performance does not guarantee the same performance on a large scale too. hence, a study on a larger scale membranebased harvesting of microalgae is pre-requisite to focus on the near future [26]. floatation in this process, tiny air bubbles are introduced which in turn transport the solid particles suspended in the system to the surface. later, the floating solid particles from the surface can be removed by skimming or other methods. dissolved air floatation: high pressure-recycled water which is saturated with dissolved air is introduced into the chamber. this creates the release of microbubbles, and they get attached to the suspended algal particles causing them to float on the surface [35]. electrolytic floatation involves the formation of gas bubbles by electrolysis [15]. it is more rapid than the sedimentation method. this method is species-specific and requires high capital and operational costs. flotation involves gravity separation via gas bubbled through a microalgal suspension, due to which soil particles get attached to gaseous molecules and can be skimmed off from the surface [36]. flotation processes are classified based on bubble generation methods, these involve dispersed air flotation, dissolved air flotation, dispersed ozone flotation, electrolytic flotation, jet flotation, etc [37]. dissolved air flotation involves the use of bubbles of mean size 40 μm with range 10 to 100 μm, under high pressure with air dissolved in water and later atmospheric pressure is released in the unit. the air bubbles aggregate are released with the microalgae particles and float on the surface which is later skimmed off [36; 38]. dissolved air floatation of chlorella sorokiniana after coagulation with four different coagulants: aluminum sulfate, ferric chloride, tanfloc, and zetag showed the maximum efficiency of 98.4, 94.5, 95.4, and 96.7% at 8 cm·min −1 , respectively [39]. in dispersed air flotation method bubbles ranging from 700-1500 µm size are produced by continuous passage of air stream via high-velocity mechanical fomenter. this process requires less energy; whereas the equipment cost is relatively quite high. the recovery percentage of microalgal cells has been raised to 90% with surfactants by increasing the hydrophobicity of the microalgae cells and this will facilitate the attachment of bubbles to microalgae cells [37]. dispersed air flotation of chlorella saccharophila with cationic trimethyl-ammonium bromide (ctab) added as a surfactant was investigated as an effective harvesting technique for to be used for the production of bio-diesel [40]. in the electrolytic floatation process, the gas bubbles are produced by electrolysis of water. the investigation of electrolytic floatation of microalgae dunaliella salina showed similar harvesting efficiency as well as energy consumption while a graphite plate was used as the anode, and different materials and forms of cathodes (stainless steel plate, perforated stainless steel plate and graphite plate) [41].dispersed ozone flotation (dof) uses ozone gas instead of atmospheric air to produce charged bubbles that promote the flotation of microalgal cells on the surface. ozone oxidizes the organic contents of the effluent and also improves the quality of water by lowering the turbidity [25; 28]. however, the dispersed ozone flotation technique is not desired for large scale purposes, due to contamination issues and high operating prices [42]. flocculation flocculation is the aggregation of the microalgal cells to enhance the size of the particle that eases the sedimentation and recovery of the microalgae. the settling rakesh et al., 2020 sustainable cost-effective microalgae harvesting strategies highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 velocity and the concentration factor are the key parameters of the flocculation process. usually, the negative charge present on the surface of the microalgal cells prevents its aggregation in the suspension [43]. this negative charge can be neutralized by adding flocculating agents to initiate flocculation in the microalgal cells. the flocculating agents should be non-toxic, inexpensive, easily available, and effective at low concentrations [44]. multivalent metal salts ferric chloride [fecl3], aluminium sulfate [al (so4)3, alum], and ferric sulfate [fe (so4)3] are the multivalent metal salts used to initiate flocculation in the microalgal cell suspension. it is the phenomena where positively charged ions are used to neutralize or reduce the negative charge on the surface of the algae to form flocs [45-46]. the addition of these compounds may negatively affect the recycling of the medium and also the quality of the product. the metal salts possess a positive charge to interact with the negative charge present on the algal surface and result in floc formation [43]. the study compared and evaluated harvesting microalgae arthrospira maxima through flocculation by the addition of cacl2 as a flocculant and/or ph increase above 10 using naoh showed an effective harvesting technique [47]. biodegradable organic flocculation biopolymers like guar gum, starch, alginic acid, and chitosan are used as organic flocculants for microalgae harvesting. these biopolymers may not contaminate the algal suspensions [48-49]. chitosan has been shown to be an effective biopolymer for harvesting microalgae without any toxic effect on the harvested algae. bioflocculation bioflocculation is the harvesting of microalgae without any addition of flocculating agents. bioflocculation involves other microorganisms having the capability of flocculation in the medium, as shown in figure 2. figure 2: bioflocculation process in microalgae harvesting. the flocculating fungi or bacteria or algae are harvested along with the non-flocculating microalgae in the medium. extracellular polymeric substances secreted by organisms such as bacteria or fungi or algae act as bioflocculants. these bioflocculating agents can increase the growth rate of the algae in the system [44; 50]. the flocculating microorganisms do not require any special cultivation conditions and their presence don’t interfere with the downstream process of algal lipids. bthe study showed that the co-flocculation of citrobacter freundii and chlorella pyrenoidosa (bacteria: microalgae ratio was 1.6:1) showed the maximum flocculation efficiency of 97.45% and mucor circinelloides and chlorella pyrenoidosa (microalgae: fungi ratio was 333:1) showed the maximum flocculation efficiency of 92.08% [51]. autoflocculation few autoflocculating microalgae can form flocs without any addition of flocculating agents. autoflocculation can be defined as the interaction between the surface molecules of the algae and the surrounding medium or interaction between the microalgae among themselves. the algal cells aggregate and form into larger flocs. the flocs induce the sedimentation of the algae [52]. the microalgae produce extracellular polymeric substances [eps], which plays a key role in the autoflocculation. when this eps production reaches its maximum level, the microalgae tends to undergo flocculation by settling down itself. autofloculation is largely dependent on algal species and ph manipulation [53]. chlorococcum sp. at ph 12 has been reported with a harvesting efficiency of 94% [50]. in the case of scenedesmus obliquus, autoflocculation efficiency was improved from 10.4 to 33.2%, when the ph increased from 7 to 10 [54]. a study on auto-flocculation of ettlia sp. driven by polysaccharides had a positive impact on harvesting efficiency of 91 ± 2.7%; whereas, autoflocculation of chlorella sp. driven by polysaccharides had a negative impact on harvesting efficiency of 51 ± 1.3%. this study revealed that autoflocculation is speciesdependent and for each species harvesting protocol will vary [55]. electrolytic flocculation in electrolytic flocculation, electrodes are placed in the culture, and the current is supplied to run across the electrodes. the negatively charged algal cells migrate toward the positive charge of the electrode causing aggregation [23]. this method is more effective than chemical flocculation [56]. the study evaluated the harvesting of scenedesmus sp. using electro-coagulation– flocculation (ecf) showed the effective harvesting efficiency (>99%) under optimal conditions [57]. thus, this technique could be well-suited harvesting technique for biofuel production. polymeric flocculation in polymeric flocculation, long-chain polymers are used to bind the algal cells by bridging the gaps between the cells, causing aggregation and settling. polymers with a positive charge like polyethylene amine and polyacrylamides bind with negatively charged cells of rakesh et al., 2020 sustainable cost-effective microalgae harvesting strategies highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 microalgae in the suspension and result in floc formation [18]. the harvesting of freshwater microalgae viz., chlorella vulgaris and marine algae viz., phaeodactylum tricornutum using a cationic polyacrylamide flocculant, showed 100% and 90% biomass recovery, respectively. the optimal flocculant concentration for chlorella and phaeodactylum was 18.9 and 13.7 mg/g of dry algal biomass, respectively [58]. physical flocculation sometimes the flocculating agents may contaminate the algal suspension; to avoid such problems physical forces were induced. one such example is sonication. in this process, high-frequency sound waves are introduced into the algal suspensionthe cells charge negative and will move to the anode where they get neutralized and form into aggregates. always high-frequency sound waves should be used, as low-frequency sound waves may cause rupture of algal cells [59]. magnetic separation using nanoparticles magnetic separation using nanoparticles has recently gained attention for harvesting microalgae due to its potential over traditional harvesting techniques [60]. in this technique, magnetic nanoparticles are tagged to the suspended microalgal cells and are recovered from the culture broth via external magnetic field [42]. microalgal harvesting is carried out by two types of magnetic nanoparticles i.e., naked and surface functionalized. in naked magnetic nanoparticles, a wide range of microalgal strains is recovered based on its specific surface area, biocompatibility, super-paramagnetism, etc [61-62]. in the case of surface-functionalized magnetite, there are two strategies for tagging polyelectrolyte, "attached-to" and "immobilized" strategy. in the "attached-to" approach, the surface of microalgal cells is coated with a polymer binder that helps attach with the magnetic particles. in the case of "immobilized-on" strategy surface of the uncoated magnetic particles is functionalized with a polyelectrolyte that aids the binding with the algal cells [63]. the harvesting efficiency of microalgae can be improved by surface coating of magnetic nanoparticles with various polymers, surfactants, etc [64]. a recent study has confirmed that the harvesting efficiency of scenedesmus sp is improved when different fe3o4 -based nanoparticles are used as adsorbents. cetyl-trimethyl-ammonium bromidecoated fe3o4 nanoparticles, polyethyleneimine coated fe3o4 nanoparticles, amino-propyl-tri-ethoxy-silane functionalized fe3o4 nanoparticles. all the synthesized magnetite-based nanoparticles showed high potential efficiency for microalgae harvesting [60]. concluding remarks various methods for microalgae harvesting were reviewed. centrifugation is a rapid and effective method that required high capital and operational costs. other methods like floatation, filtration techniques, and other electrochemical techniques require high capital cost. autoflocculation and bioflocculation are found to be inexpensive and effective dewatering techniques for algal harvesting. autoflocculationhas a high sedimentation rate without any addition of the flocculants. the autoflocculation can be enhanced by a high aeration rate, co2 concentration, and nitrogen levels. bioflocculation is also an efficient, ecofriendly, and cost-effective algal harvesting method (table 1). table 1: various microalgal harvesting technique advantages and disadvantages. sl. no. harvesting method advantages disadvantages references 1 centrifugation  rapid and efficient cell harvesting  handles most of the microalgae cells  high operational cost [2] 2 sedimentation  reduces cost and energy  mostly used for non-motile algal cells  species specific  low separation rate [15, 17, 18] 3 filtration  filters and membranes of different sizes are available which makes the process of harvesting easy and rapid  species dependent  mostly suitable for large algal cells  fowling and clogging becomes the main problem when using filtration as the harvesting method [22-24] 4 floatation  rapid when compared with sedimentation  species specific  high capital and operational costs [15, 35] 5 chemical flocculation  low cost  simple and rapid harvesting  no energy requirements are needed  removal of chemical flocculants  sometimes the chemicals are toxic to the algae [43, 45, 46] 4 bioflocculation  eco-friendly and cost effective algal dewatering technique.  no energy inputs are required  the bioflocculants [bacteria/fungi/algae] used in the flocculation sometimes causes contamination [44, 50] 5 autoflocculation  effective  high sedimentation rate with no energy inputs  changes in cellular composition [44, 52] 6 magnetic separation  short time period for microalgal harvesting.  reusability of the culture medium  nanoparticles are costly.  quire special recycling eruipment [63] rakesh et al., 2020 sustainable cost-effective microalgae harvesting strategies highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 acknowledgements this work is financially supported by the serb dst project (eeq/2018/001463) references 1. sharma p, sharma n. industrial and biotechnological applications of algae: a review. journal of advances in plant biology. 2017 aug 10;1(1):01. 2. grima em, belarbi eh, fernández fa, medina ar, chisti y. recovery of microalgal biomass and metabolites: process options and economics. biotechnology advances. 2003 jan 1;20(7-8):491-515. 3. rakesh s, dhar dw, prasanna r, saxena ak, saha s, shukla m, sharma k. cell disruption methods for improving lipid extraction efficiency in unicellular 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nanoparticles grafted with amino-riched dendrimer as magnetic flocculant for efficient harvesting of oleaginous microalgae. chemical engineering journal. 2016 aug 1;297: 304-14. highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 1 of 10 citation: nassar a.e., mousa k.h., madbouly a.a., ibrahim s.d. and alsamman a.m. (2018). identification of genes for wheat fungal resistance using bioinformatics techniques. highlights in bioscience, volume 1. article id 20185, dio:10.36462/ h.biosci.20185 received: september 5, 2018 accepted: october 25, 2018 published: november 10, 2018 copyright:© 2018 nassar et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests identification of genes for wheat fungal resistance using bioinformatics techniques ahmed e. nassar 1 , khaled h. mousa 1 , ahmed a. madbouly 2 , shafik d. ibrahim 3 and alsamman m. alsamman 3 * 1 genetics department, faculty of agriculture, mansoura university. 2 plant tissue culture department, date palm research institute, agricultural research institute 3 mggm lab., agricultural genetic engineering research institute, agricultural research center, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg abstract for the majority of world populations, wheat (triticum aestivum l.) would be the first essential and economic cereal grain crop. pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. in addition the introduction for restriction fragment length polymorphism (rflp) for such genes in the new primer database. approximately 138 sequences of dna were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. the ncbi blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. the total number of nucleotides was 48385, where the smallest dna sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. the wheat chromosomes 3d, and 4b have the highest number of sequences (9) followed by chromosomes 3b (7) and 3a(6), where wheat genomes a, b and d have 30, 35 and 27 genes, respectively. five different amino acids motifs have been revealed among studied wheat amino acid sequences. the gene annotation tools used to infer studied amino acid gene annotation. amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (stk), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. the proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics. keywords: wheat, pathogens, pathways, fungal, phylogenetic, rflp. research article open access nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 2 of 10 introduction for the majority of world populations, wheat (triticum aestivum l.) would be the first essential and economic cereal grain crop. its 29 percent share shows the world economy's significance of wheat production from world's 730 million hectares of crops cultivated lands. depending on fao figures for 2017, this percentage is comparable to 218 million hectares of wheat region (1). in most of the world, wheat is an key ingredient of human diet. worldwide, 735 million tons of wheat were produced in 2015/2016, worth around us$ 145 billion (2). wheat accounts for almost 55% of the carbohydrates and 20% of the world's food calorie intake (3). in egypt, the significance of wheat crops returns to its strategic importance in egyptian dietary commodities as it offers more than one-third of egyptian consumers ' daily calorie intake and 45% of their daily total protein intake (4). wheat is egypt's largest winter grain and is cultivated across the nile delta and along the nile valley length. wheat is usually planted after summer maize, cotton, or rice crops in early november and collected in late april or early may. germination happens after planting at 19 to 22 weeks (5). while egypt's wheat productivity has increased over the past few years, wheat production provides only 45% of its yearly domestic demand. egypt is still one of the largest importing wheat countries. wheat imports were approximately 9.8 million tons in 2011, costing approximately us$ 3.2 billion (6). pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development (7). a key component for trying to meet this challenge is proper management of fungal diseases that may be accountable for yield losses of 15–20 percent per year. the rusts, blotches and head blight/scab are among the major wheat diseases that make a contribution to these losses(2). the rusts, blotches and head blight/scab are among the major wheat diseases that currently contribute to these losses. there are three diseases of wheat rust, namely stem, stripe and leaf rust, all caused by members of the basidiomycete family, genus puccinia, named p. graminis f. sp. tritici, p. striiformis f. sp. tritici (pst) and p. triticina (pt), respectively (2). multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. the resistance is attributable to the additive impacts of multiple resistance metabolites and proteins generated through a network of multiple plant r gene structures. plants recognize pathogenic elicitors or receptors and then stimulate downstream genes to ultimately produce resistance metabolites and proteins that suppress plant pathogen progression (8). following the view of pathogens and microbes, reactive oxygen species (ros) were consistently identified to accumulate in the plant and over the years, ros was postulated as an essential part of the plant's defense response (9). the connection between genetic variability identified using distinct molecular marker assays and genes responsible for morphological and physiological characteristics could be identified through sophisticated bioinformatics analysis tools (10–12). these tools could be used to identify and reveal new genes in wheat related to resistance to fungal diseases. our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. in addition the introduction for restriction fragment length polymorphism (rflp) for such genes in the new primer database. materials and methods anti fungal gene sequences have been downloaded from ncbi database (13). we have downloaded 3845 antifungal protein sequences from ncbi. the draft genome sequence of wheat has been downloaded from ensemble database (14). the local ncbi blast package (15) was used to build sequence databases using the wheat draft genome and blast all anti-fungal genes with tblastn against wheat database. the online ncbi blast tblatn was used to annotate sequences recovered from previous step. meme suite was used to discover amino acid motifs in the sequence (16). megax program was used to construct phylogenetic analysis through maximum likelihood algorithm (17). clustalo (18) tool was used to construct sequence similarity matrix through multiple sequence alignment. clust-vis was applied to create principal component analysis (pca) plots and heatmaps depicting genes similarities. pepstat (19) was used through in-home per scrips to assess proteins chemical and physical characteristics. the perl script restrictiondigest was used to detect restriction enzymes recognition sites in dna sequences (20). batchprimer3 online tool was used for designing pcr primers could be used to target studied wheat genes (21). result and discussion chromosomal distribution of wheat anti-fungal genes approximately 138 sequences of dna were recovered from the wheat genome by aligning 3845 antifungal amino acids through tblastn tool. the ncbi blastn nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 3 of 10 online tool used to detect sequences with functional genes, where 92 genes have been detected (supplementary file 1). the total number of nucleotides was 48385, where the smallest dna sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. the wheat chromosomes 3d, and 4b have the highest number of sequences (9) followed by chromosomes 3b (7) and 3a(6), where wheat genomes a, b and d have 30 , 35 and 27 genes, respectively (figure 1). . figure 1 : the circos diagram for the position of predicated anti-fungal genes and their proposed gene description, where the inner connections represent similar genes on separate chromosomes with sequence similarity over 75%. wheat anti-fungal proteins chemical and physical properties the proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics (figure 2 and table 1). the total molecular weight (mw) of studied fungal amino acid sequences was 1913 kda with an average of 20 kda, where 1a:551827789-551828139 has the minimum mw (10.6) and 5d:59043196-59044206 generated the maximum mw (71.911.68) extinction coefficients are a measure of how much light the protein can be measured at a certain wavelength from the extinction factor. approximation of this component is needed to be able to track the protein within the spectrophotometer. it is necessary to know the amino acid content to assess the molar extinction coefficient of the protein (22). table 1: the highest and lowest proteins characteristics and their corresponding proteins. protein characteristic total mean min. min. protein max. max. protein a280 molar extinction coefficients cystine bridges 2261000 24576.09 1740 3b 716295639-716296412 104570 5d 59043196-59044206 a280 molar extinction coefficients reduced 2214500 24070.65 1490 3b 716295639-716296412 103820 5d 59043196-59044206 improbability of expression in inclusion bodies 65.124 0.794 0.504 7d 45432639-45432947 0.977 3d 462838207-462838962 average residue weight 9829.348 106.841 98.498 7a 164401571-164402272 116.631 3d 550382548-550383990 isoelectric point 588.9429 6.402 4.0084 1a 20888904-20889590 10.3971 7b 40654848-40655408 molecular weight 1913642.24 20800.46 10658.89 1a 551827789-551828139 71911.68 5d 59043196-59044206 non-polar 5471.072 59.468 48.81 3a 719941535-719942353 69.919 5b 461292418-461292831 polar 3728.357 40.526 30.081 5b 461292418-461292831 51.19 3a 719941535-719942353 nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 4 of 10 protein characteristic total mean min. min. protein max. max. protein residues 17857 194.098 101 7d 45432639-45432947 644 5d 59043196-59044206 small 5629.919 61.195 40.488 6a 610941935-610943086 77.027 5b 518810105-518810797 tiny 3673.776 39.932 20.27 2b 634173118-634173630 54.054 5b 518810105-518810797 acidic 858.259 9.329 5.172 3a 20936815-20937576 17.593 4b 41828978-41829304 aliphatic 2702.584 29.376 19.048 5a 14266438-14266881 41.27 3d 8508332-8509102 aromatic 952.739 10.356 3.604 3b 716295639-716296412 16.292 4d 29673605-29674213 basic 932.965 10.141 4.082 7d 634805112-634805630 19.672 3b 176321011-176321379 charge 24.5 0.266 -9.5 3b 690358283-690358846 22 7b 40654848-40655408 charged 1791.221 19.47 10.204 7d 634805112-634805630 32.258 3d 550382548-550383990 the a280 molar extinction coefficients cystine bridges (a280-mecc ) and reduced (a280-mecr) are two different measurements of extinction coefficient, where salt bridges are important motifs of the tertiary protein structure and are mostly correlated with the structural influence force which maintains the stability of the protein. commonly found on the solvent system and particularly vulnerable to solvent-solute interactions typically with water as well as other cosolvents (23). the minimum scores of a280-mecc and a280-mecr were 1740 and 1490, generated by 3b:716295639716296412, while the highest scores were 104570 and 103820, revealed by 5d:59043196-59044206 with a mean of 24576 and 24070, respectively. expression improbability in bodies of inclusion (ieib) is a form of measurement of solubility. for instance, recombinant protein in escherichia coli could be expressed either as soluble throughout the cytosol or as insoluble in the bodies of inclusion (24). the ieib of wheat amino acids showed a mean of 0.794, where it ranges from 0.504 (7d:45432639-45432947) to 0.977 (3d: 462838207-462838962). figure 2: the protein chemical composition and physical properties for the predicated anti-fungal genes. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 5 of 10 the average residue weight (arw) stands for the average collective weight for all amino acid sequences according to its length. the total arw for all amino acid sequences was 9829.3 da, where 7a:164401571-164402272 revealed the minimum arw (98.498 da). 3d:550382548550383990 generated the maximum (116.631 da) and the mean was 106.841 da. the isoelectric point (ip) is the ph level at which protein's net charge is positive and is associated with its production of amino acids and protein conformation (25). the collective wheat fungal genes amino acids was 588.9 with a mean of 6.402, where it ranges from 4 (1a:20888904-20889590) to 10.4 (7b:40654848-40655408). in addition, a protein's folded structure becomes thermodynamically less desirable because it decreases the protein's disorder or entropy. nonpolar (water hating) side chains tend to push themselves inside a protein whereas side chains of polar (water loving) prefer to put themselves outside of the molecule (26). the non-polar and polar amino acid scores were ranged from 48.81 and 30.081 (3a:719941535-719942353 and 5b:461292418-461292831) to 69.919 and 51.19 (5b:461292418-461292831 and 3a:719941535-719942353), respectively. acidic amino acids have an acidic group in side chain while basic amino acids have some basic group in side chain. acidic amino acids have low pka while basic amino acids have high pka. the score of acidic and basic amino acids range from 5.172 and 4.082 (3a:20936815-20937576 and 7d:634805112-634805630 ) to 17.593 and 19.672 (4b:41828978-41829304 and 3b:176321011-176321379 ). the studied fungal amino acids charges ranged from -9.5 (3b:690358283-690358846) to 22 (7b:40654848-40655408) (figure 2 and table 1). detection of protein domain and phylogenetic analysis a motif for the protein sequence is a brief pattern that nature retains. for proteins, a motif can refer to an enzyme's active site or a structural unit required for proper protein folding. therefore, sequence motifs are among molecular evolution's basic functional components (16). five different amino acids motifs have been revealed among studied wheat amino acid sequences (motif-1: yglaqctpdl, motif-2: cylrysnknf, motif-3: pnrvwgitwc, motif-4: ydvs vvdgfn, and motif-5: npgdcgqclq) (figure 3 and 4). figure 3: the identified protein domains / motifs in predicated wheat genes. the motif-1 and motif-3 has a match to elme000249 in motif database with p-value of 9.64e-04. this motif is a trfh domain docking motifs wich are coordinating the telomeres with other proteins. not only do they form homodimers with their trfh domains, they also provide specific protein-binding surfaces for interaction (27). trfhs and other proteins have been proposed to have important implications for the biology and evolution of telomeres. in general, this offers a basis for recognizing and controlling the hierarchical assembly and stoichiometry of telomere subunits during the cell cycle, cell division and senescence (28). motif-2 has an elme000084 match which is ligands of phosphotyrosine bound by domains sh2. src homology 2 (sh2) domains identify tiny patterns comprising a residue of phosphorylated tyrosine. up to four positions after the ptyr was found mainly to assess additional specificity. in plants sh2 dominates new signaling scenarios and participates in metazoan signal transduction, serving as key mediators of controlled protein-protein interactions with tyrosinephosphorylated substrates (29, 30). moreover, the database motif of elme000377 was highly similar to motif-4, this motif codes for pex14 ligand motif, which belongs to peroxisomes. peroxisomes are subcellular organelles present in eukaryotes that are singlemembrane spherical. peroxisomes relate together with glyoxysomes found in plants and glycosomes found in trypanosomes to microbody group of organelles. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 6 of 10 figure 4: the phylogenetic tree developed using multiple sequence alignment of predicted anti-fungal genes as compared to the expected positions of protein domains/motifs. nassar at al., 2018 identification of genes for wheat fungal resistance highlights in bioscience doi:10.36462/h.biosci.20185 november 2018 | volume1 page 7 of 10 import into the peroxisome of peroxisomal matrix enzyme proteins (pts1 cargo) includes the identification of the pts1 cargo by the cytosol pex5 receptor, the docking of the pts1-pex5 complex at the peroxisomal membrane, and the translocation of the pts1 cargo through the peroxisomal membrane into the matrix. this is preceded by recycling the pex5 receptor back into the cytosol for another round of export of pts1 stock. imported into peroxisome are unfolded, folded oligomeric or cofactor-bound proteins (31). additionally, motif-5 was with high similarity with elme000159, which is mapk phosphorylation site. cascades of mitogen-activated protein kinase (mapk) are highly conserved downstream signaling modules of receptors/sensors that transform extracellular stimuli in eukaryotes into intracellular responses. plant mapk cascades play crucial functions against pathogen invasion in signaling plant defense (32). the ncbi-blastx tool used to infer studied amino acid gene annotation. amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (stk), thaumatin, and cysteine-rich repeats representing respectively, 2, 9, 8, 19, 23 genes, in addition to 31 hypothetical genes (figure 1). lectins are non-immune proteins which attach carbohydrates directly and reversibly. the lectin's biochemical functions are really diverse. a common theme arising from the identified functions of many plant and animal lectins is their involvement in communicating with other organisms whether symbiosis or defense, as effectors or regulators (33). new types of nucleocytoplasmic plant lectins have been described and defined over the past decade, especially lectins expressed within the nucleus and the cytoplasm of plant cells, much as part of a particular plant response when exposed to different stressors or shifting environmental conditions (34). lectins include proteins containing at least one non-catalytic domain which helps them to selectively identify and reversibly attach to different glycans that are either freely available or are member of glycoproteins or glycolipids. plants release an overwhelming number of highly complex lectins with various molecular structures and attaching specificities to endogenous (plant) glycans as well as exogenous (non-plant) glycans (35). the role of plant lectins in plant defense againt different pathogens including fungi have been reported in previous researches (36,37). there are several receptor-like kinases among the plant proteins proposed to engage in immunity pathways (38). for example, multiple kinase proteins have been recorded for wheat resistance to fungal diseases, which provides temperature-dependent resistance to wheat stripe rust (39), and fungal resistance in arabidopsis (40). in this regards , various thaumatin-like proteins have been reported to generate tolerance to fungal pathogens arabidopsis (41), potato (42) and grape (43). moreover, cysteine-rich proteins have been informed to confer resistance for fungal diseases such as maize fungal pathogens (44) and legume modules (45-47). using maximum likelihood analysis phylogenetic analysis was effective in differentiating between anti-fungal wheat proteins according to their protein domains. four classes grouped the phylogenetic tree. group a comprises only motif-3 domain protein fragments, group b has motif-3, motif-4 and motif-5 domain proteins. in addition some group c contains genes with motif-1, motif-2 and motif-5 domains, and eventually group d contains genes with all protein domains (figure 4). pcr primers and restriction enzymes analysis about 85 pcr primers pairs have been designed to target most of the predicted anti-fungal genes (supplementary file 2). the pcr predicated product size ranged from 300 bp to 708 pb, where the gc content ranges from 36.4 to 66.7% and the primers annealing temperature ranges from 58.05 oc to 62.55 oc. a total of 40 different restriction enzymes (re) were used to identify potential locations for future rflp tests for genetic polymorphism inside predicted anti-fungal wheat genes. figure (5) demonstrates the site redundancy ratio for re identification within genes. figure (5) shows that some re such as banii, nlaiii and baegi have high probability to produce fragment length polymorphism isnide most of predicated anti-fungal genes. conclusion predicting using available fungal resistance genes in the public database was very helpful and suggested that several wheat genes could be used to restrict genetic research of genes that hold the key to fungal resistance in wheat. most of the identified protein domains clarify the genetic structure of anti-fungal genes and suggest a potential role for the mapk gene family in such pathways. providing restriction enzyme information and gene-specific pcr primers could be useful to 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alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20214 highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 8 research article open access 1 department of animal science, cross river university of technology, faculty of agriculture and forestry obubra campus, cross river state, nigeria. 2 department of forestry and wildlife, kogi state university, anyigba, nigeria contacts of authors * to whom correspondence should be addressed: peter. noah gboshe citation: gboshe pn and osarenakhue es (2020). the effect of concentrated feed supplementation regimes on the nutrients digestibility of grasscutters (thryonomys swinderianus) fed pennisetum purpureum as basal feed. highlights in bioscience volume 3. article id 20214. dio:10.36462/ h.biosci.20214 received: may 26, 2020 accepted: july 30, 2020 published: august 13, 2020 copyright: © 2020 gboshe and osarenakhue. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. the effect of concentrated feed supplementation regimes on the nutrients digestibility of grasscutters (thryonomys swinderianus) fed pennisetum purpureum as basal feed. peter. noah gboshe1* and ebiloma stella osarenakhue2 abstract the study was designed to determine the effect of concentrated feed regimes on nutrients digestibility of grasscutters (thryonomys swinderianus) fed pennisetum purpureum as basal feed. a total of fifty grower grasscutters were randomly assigned to 5 treatment groups of 10 grasscutters each with 5 replicates, two animals serving as a replicate. treatment was 1, 2, 3, 4, and 5 for 1, 3, 5, 7, and 9%, respectively, of concentrated feed served at their weekly live weight. parameters measured included apparent coefficient of digestibility and percentage digestible nutrients. the results obtained showed that the coefficient of digestibility showed a significant difference in all the nutrients evaluated. percentage digestible nutrients and total digestible nutrients were also significantly affected. the high coefficient of digestibility of the various nutrients and total digestible nutrients are indications that adequate nutrients were available for growth and maintenance of the grasscutters. the findings of this study suggested that supplementation of forage with formulated concentrate can be an aid in the digestion of nutrients by grasscutters for their growth performance while in captivity. the digestibility of nutrients when concentrate supplement and elephant grass were combined, particularly 5% concentrate supplement was not affected. based on the results, it was, therefore, recommended that it should be used in improving the growth performance of grasscutters. keywords: concentrate, supplementation regimes, nutrient digestibility, grasscutters, pennisetum purpureum. introduction animal protein production in nigeria from domestic livestock has been steadily falling by approximately 1.4% annually, while the human population has been increasing at an annual rate of 3.3% [1]. so, indeed, there is need to attempt to bridge the animal protein gap for nigerians to attain the fao recommended daily animal protein intake that is deficient in the diets of an average nigerian and also try reducing the under-five death that is usually experienced as a result of malnutrition. this can be achieved by the provision of animal protein knowing its functions in the health status in the human body [2,3]. unfortunately, conventional and regular sources of animal protein supply in the country such as beef, pork, goat meat and poultry are getting out of the reach of the common populace due to economic down-turn. in an attempt to reduce this situation therefore, we must think of animals from the wild with acceptable potentials and one of such animals is grasscutter (thryonomys swinderianus), a microlivestock. mbah [4] have suggested that an area where effort may be rewarded in terms of meeting the animal protein requirement of nigerians is the contribution of wild animal. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20214 https://doi.org/10.36462/h.biosci.20214 http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 he added that, the shortage of animal protein in the third world countries can be ameliorated by improving the existing conservation programme of wildlife particularly the conservation and domestication of rodents that are tractable, prolific and widely accepted to the public for consumption. again, that, captive rearing of rodents and other species of manageable sizes in cages and enclosures might augment the bush meat supply from the wild which can lead to a reduction of hunting pressure on the wild game and rodent grasscutter could be the rodent of choice. it was observed a gradual shift from the production and consumption of conventional animal protein sources (cattle, sheep, swine, goat, poultry) to a class of livestock referred to as ‘mini-livestock’, ‘micro‘unconventional livestock’ [5]. with the shortage of animal protein, nutritionists are now interested in prolific, good converters of feed to flesh and short production cycle animals [6]. the grasscutter meat is favourite and it accounts for the greater proportion of bush meat sold in most part of the continent of africa particularly west africa [7]. the major problem in the production of monogastric animals, such as poultry and pigs, emanates from the high costs of ingredients such as cereal grains and soybeans, which form 50-80% of formulated feeds [8]. their high cost emanates from the fact that, they feature prominently in human diets in the developing world but grasscutters do not belong solely in that group. akinloye, [9] and opara and fagbemi [10] reported that, proper feeding is the major factor that determines the health of any animal that captive reared grasscutters are fed with both forages and concentrates but the proportion of concentrate must be in low quantity to avoid digestive problems hence the need to search for a concentrate-feeding regime that can improve the performance of grasscutters. diseases are not very common problem in grasscutters [11]. they added that, the severity of these diseases is influenced by the nutritional status of the animals, especially in the dry season when feed is inadequate in quantity. backyard rearing of grasscutter in rural or urban settlement has the capacity to provide adequate animal protein of an average family year round [12]. in addition, its management requirements are low-cost. it feeds on a wide range of feedstuffs, such as grasses, legumes, to tuber crops such as sweet potatoes and cassava and other industrial by-products [12]. asibey [13] earlier reported that grasscutter meat has a protein content of 24.7%, and a low fat content of 1.2% makes it better than beef with a protein content of 19.6% and fat content of 6.6% and pork with a protein content of 19.4% and fat content of 13.4%. the grasscutter’s gastro-intestinal tract is adapted to handle high fibre materials because of the presence of a large caecum which houses some microbes that help in the digestion of cellulose. the presence of cellulolytic bacteria in the caecum of the grasscutter has been shown by [14]. animals with this system are referred to as hind-gut fermenters, meaning that feed is broken down by bacteria at the end of the digestive system. the microbial activity of the caecum is of great importance for the processes of digestion and nutrient utilization from fibrous material [15]. grasscutter exhibit some features of the ruminant by consuming large quantities of forages (leaves and stems) and fermenting it in the gut chamber that harbours microbes and b-vitamins are useful by-product of the process. the difference is that whereas, for the ruminants the rumen is at the beginning of the gut and the products of fermentation are absorbed from the rumen and lower gut, the caecum is towards the end of the gut, and the grasscutter has to re-ingest fermentation products through the mouth, that is, they also practice caecotrophy such as the rabbit. the fermentation of fibre in the guts of herbivorous results in the production of volatile fatty acids (vfas) [16] which is absorbed across the epithelial membrane. vfas account for much of the metabolizable energy supply to the animals [17]. karikari and nyemeasem [18] reported that increase in the dietary components of crude fibre (cf) acid detergent fibre (adf) and neutral detergent fibre (ndf) decreased digestibility and daily weight gain in grasscutters. in another study with grasscutters, van zyl et al [19] found that high dietary fibre reduced the digestibility of dry matter, protein and fat while the digestibility of fibre component (ndf, adf and hemicellulose and cellulose) was comparable to that of grasscutters on the low fibre diet. elephant grass is rather low in energy and protein, due to its high cell wall content [20]. elephant grass is very sensitive to climatic conditions, maturity and regrowth days, with a decrease in crude protein content, an increase in fibre, a decrease in dry matter and cell wall, as measured by in situ degradability and in vivo digestibility [21, 22, 23]. since at the moment, no baseline nutrient requirements and no particular feeding regime for concentrate have been established that can aid for nutrients digestion for optimal performance. therefore, the objective of this research was to evaluate the concentrate-feeding regime on the nutrients digestibility of grasscutters using elephant grass as basal feed. materials and methods experimental location the study was carried out in obubra, cross river state, nigeria. this is located between longitude 80-90 e and latitude 60-70 n of the equator. the mean annual rainfall of the area ranges from 500 to 1070 mm with warm weather and ambient temperature of about 200c-300c [24]. obubra is located along the banks of the cross river in the southern guinea agro-ecological zone of nigeria. obubra is about 159 km from calabar, the state capital of cross river state of nigeria. http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 experimental animals and design a total of fifty weaned grasscutters between the ages of 3-4 months obtained from a local farmer in ibadan were used for the digestibility trial. the grasscutters were put into groups of similar body weights and were randomly assigned to five treatments feeding regimes in a completely randomized design. the grasscutters were randomly assigned to 5 treatment groups of 10 grasscutters each with 5 replicates with two animals serving as a replicate. the animals were given elephant grass as basal feed ad libitum and formulated concentrate supplemented at a feeding regime of 1, 3, 5, 7, and 9% for t1, t2, t3, t4 and t5 respectively of their weekly live body weight throughout the period of the experiment. housing and experimental procedure the grasscutters were individually housed in clearly and properly-labelled concrete cells measuring 90 x 75 x 40 cm (length x width x height). there was an opening only at the top in order to eliminate cross-ventilation and prevent the adverse effect of cold because grasscutters are highly susceptible to pneumonia. the top was partly covered to create a darken area meant for hiding which is their habit. each cell was provided with a feeder and a drinker. the cemented cells were constructed in a well-ventilated cement block walled house roofed with asbestos sheets to protect the animals from bad environmental conditions such as rainfall and cold conditions. on the introduction into the cells, the animals were provided with antistress agents in drinking water. they were also dewormed and given a dosage of 5 gm each of coccidiostat. elephant grass (pennisetum purpureum), were cut and allowed to wilt for about 12 hours, weighed and fed daily as a basal diet. water and elephant grass were supplied ad libitum. the animals were weighed weekly throughout the period of 20 weeks of the study. all cells were cleaned daily in order to ensure an adequate level of sanitation. experimental diet a concentrate supplement diet was formulated to contain approximately 18% crude protein and a metabolizable energy of 2961.47 kcal/kg (table 1) which was used at different feeding regimes of 1, 3, 5, 7 and 9% of their weekly body live weight and fed with wilted elephant grass as basal feed. chemical assay the grasses were harvested around the study area wilted for about 18 hours chopped to pieces, milled and analysed for proximate chemical composition (to include gross energy) according to the procedure outlined by [25] while the fibre obtained was characterized to its component fractions according to the procedures outlined by [26]. digestibility trial at the end of the feeding trial, one grasscutter per replicate with the live weight approximately their treatment average live weight, were selected and used for the digestibility trial. faecal collection lasted for seven (7) days, during this period the grasscutters were fed about 75% of weight of their feed intake per day since the level of feed intake increases the rate of passage of the digest from the git and thus reduces the digestibility of nutrients [27]. faecal collections were obtain after sorting out other contaminants. before the commencement of faecal collection the grasscutters were deprived of feed for 18 hours to ensure that the faecal collection corresponded to the feed offered. the fresh faeces from each replicate were weighed and oven-dried at 800c for 24 hours, the oven-dried faeces per replicate were bulked, thoroughly mixed together and milled. samples of the ground faeces were stored in airtight sample bottled properly labelled and taken for chemical analysis. table 1. composition of concentrate feed and calculated values of experimental diets ingredients % maize 58.41 soybean meal 27.59 rice offal 10 bone meal 3 vitamin-min-premix* 0.5 common salt 0.5 total 100 calculated nutrients composition. crude protein 18 metabolizable energy (/kcal/kg) 2961.47 crude fibre (%) 6.97 calcium (%) 1.16 phosphorus (%) 0.89 * each 1 kg of vitamin/mineral premix manufactured by beauts co. inc. man, u.s.a., contains vitamin a 220,000, vitamin d 66,000, vitamin e 44, 014; vitamin k 88 mg; vitamin b 12; 0.76 mg; niacin 1122 mg, calcium 27%, phosphorus 10%, iron 0.6%, zinc 0.35%, manganese 0.25%, copper 0.06%; iodine 0.002%, cobalt 26 ppm, selenium 4 pp. me = metabolizable energy. statistical analysis data collected were subjected to analysis of variance (anova) using mini-tab version 16.0 [28], where means were significant, there were separated at 5% significant level of probability using the least significant difference according to the procedures contained in the software. results and discussion proximate composition of the concentrate feed supplement and elephant grass the result of the proximate composition of the formulated diet and elephant grass used for the study are presented in table 2. the crude protein content of the experimental diet analysed (17.8%) was similar to the http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 calculated value (18%) and met the crude protein requirements of growing grasscutters (18%) [29]. elephant grass has low protein content of 8.18%, high fibre content of 27.19% and ether extract content of 1.18%, thus necessitating the use of formulated concentrate which may be suitable to satisfy the nutrient requirements of a growing grasscutter especially when supplemented with concentrate [9, 10]. the results of proximate analysis is similar to that of onyeanusi et al. [30] and wogar et al [31] with some variations may be due to soil nutrients or period of harvest since these factors among others are known to affect the nutrients availability of forages. characterization of the crude fibre content of elephant grass the characterization of the crude fibre content of elephant grass presented in table 3 showed that, it contained 39.91% cellulose, 20.82% hemicellulose, 66.95% neutral detergent fibre (ndf), 41.91% acid detergent fibre (adf) and 35.10% acid detergent lignin (adl). characterization of the crude fibre of the elephant grass showed that, cellulose content of 39.9% recorded in this study was slightly different from 45.60% reported by [32] but this fell within the range of 35-48% reported by [33, 34]. the hemicellulose content of 20.82% was similar to 20.78% reported by ogunjobi, et al. [35]. the neutral detergent fibre (ndf) of 66.95% fell within the minimum and maximum range of 54.1-79.9% reported by egbunogie and bamikole [33], mohammed et al [34] but slightly higher than 63.65% reported by ogunjobi et al [35]. the acid detergent fibre (adf) of 41.91% was similar to 40.5% reported by (tangendjaja et al. [32] and also fell within the range 29.5-52.9% reported by egbunuogie and bamikole [33], islam et al [34]. the acid detergent lignin (adl) of 35.10% corresponds to 35% reported by ogunjobi et al. [35] but differed from the value range of 27-59% reported by egbunuogie and bamikole [33, mohammed et al. [34]. these variations in the fibre characterization in this study and these authors may be due to the season or age of harvest of the elephant grass. it is known that lignification of forages depends on the seasons and particularly the age because the cell wall gets more lignified as the plant matures [36]. apparent coefficient of digestibility of grasscutter fed elephant grass with different levels of concentrate supplement regimes the nutrient digestibility of grasscutter fed elephant grass with different levels of concentrate supplement regimes is shown in table 3. the high coefficient of digestibility of crude protein of the diets in this study (68.53 to 82.94%) is an indication that dietary fibre levels of 5% and 31% concentrate and elephant grass, respectively, did not adversely affect the digestibility of crude protein. the crude fibre levels present in this study did not impair the utilization of other nutrients, especially crude protein. this animal being a pseudo-ruminant was able to handle the fibre content of the elephant grass. it is known that, fibre in the diet of monogastric impair the utilization of other nutrients, especially crude protein utilization [37]. table 3. characterization of the crude fibre fractions of elephant grass (pennisetum purpureum). constituents proportion (%) neutral detergent fibre (ndf) 66.95 cellulose 39.91 hemicellulose 20.82 acid detergent fibre (adf) 41.91 acid detergent lignin (adl) 35.1 elephant grass have acid detergent fibre adf content of (35.10%) and thus, elephant grass dietary crude fibre may not reduce digestibility of cf and other nutrients in grasscutter since this species of animal digestive system has been adapted to this type of grass with this cf level in the wild. this explains why the crude protein digestibility was very high in t1 in attempt to meet their nutrient requirements since the nutrients were low in grasses. adegbola and okonkwo [38] reported that there is a negative correlation coefficient between crude fibre and nutrients digestibility, an indication that high crude fibre in the diet depresses nutrient digestibility. the levels of fibre inclusion in this study may be within the acceptable range for grasscutter, thus the digestibility of crude protein was high. table 2: proximate composition of elephant grass and concentrate supplement. the high and comparable coefficient of digestibility of crude fibre in all the feeding regime treatments (67.46 to 85.95%) may indicate that the elephant grass dietary fibre contains more digestible carbohydrates than indigestible. the acid detergent lignin present in elephant grass fibre is 35.10% and may be low enough to be handled by grasscutters. the digestibility of crude fibre has earlier been shown to be modified by the degree of lignification of the dietary fibre rather than the fibre level, and this shows differences in feed consumption [39]. the high coefficient of digestibility of non-lignified materials has been previously reported adegbola and okonkwo [38]. fibres from different sources could vary in digestibility depending on the proportions of cellulose, hemicelluloses and lignin. karikari and constituents dry matter crude protein crude fibre ether extract ash nfe me/kcal/kg elephant grass % composition 34.3 9.25 31 1.17 9.28 49.3 2187.17 concentrate % composition 86.98 17.85 5.07 3.2 10.12 50.74 2720.92 http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 nyameasem [18] reported that concentrate of high neutral detergent fibre (ndf) (55%) content may not be able to support rapid growth of captive reared grasscutters. they added that increase in the dietary component of crude fibre, acid detergent fibre (adf) and ndf decreases digestibility and daily gain in grasscutters. in another study with grasscutters, van zyl et al. [19] reported that high dietary fibre reduced the digestibility of dry matter, protein and fat while digestibility of fibre component, ndf, adf, hemicelluloses and cellulose was comparable to that of grasscutters on a low fibre diet. the coefficient of digestibility of crude fat, (27.65 to 72.69%) observed in this study is similar to the value 64.40% reported by ogunjobi et al. [35]. the high coefficient of digestibility of fat in t1 by the grasscutters in this study can be attributed to the acceptable range of crude fibre in that treatment which did not contain high amount of lignin even though they ate more forage, thus nutrients utilization and availability at the tissue level were not depressed, it was just the quantity of nutrients in the diet that may not be sufficient. the coefficient of digestibility of ash in this study ranged from 65.01 to 84.47% which was highest at t1 but similar at t5, t7 and t9. the concentrate supplement was formulated to supply balanced nutrients to the growing grasscutters. the coefficient of digestibility of ash can be attributed to increase in retention time of the food in the caecum since it was given alongside with elephant grass increased the availability of nutrients by increasing the period of exposure of food to the digestive enzymes and absorptive surfaces, thus, increasing digestibility and absorption of nutrients. underwood [40] had earlier on reported that, diets with adequate mineral supplementation seldom vary in their ash digestibility. the nitrogen-free extract (nfe) in the experimental feeding regime diets which represents the readily available carbohydrates recorded a relatively low coefficient of digestibility of 25.10 to 56.41%. however, this implies that, this concentrate feeding regime and elephant grass as basal feed can be reported not to have relatively adverse effect on the readily available carbohydrate, since the coefficient of digestibility of nfe of the treatments were within the range of at least (50.02%). the dry matter coefficient of digestibility, which varied from 75.65 to 82.05% was high and more that 66 to 68% obi et al. [41] when they carried out a study to evaluate the nutritive potentials of four conventional forages fed to growing grasscutters. the high coefficient of digestibility of dry matter of the grasscutters in this study probably means that crude fibre level in the experimental diet did not induce excessive production of mucin. excessive mucin increases the viscosity of the digesta within the gastro intestinal tract which adversely affects the digestibility of dry matter (dm) [42]. in the absence of this adverse effect of high fibrous diets, the digestibility of dry matter increases. the negative correlation coefficient between crude fibre and dry matter digestibility values reported by adegbola and okonkwo [38] did not apply, which implies that the crude fibre levels in the diet may be within the acceptable and tolerable range for the grasscutters. it was observed that, digestible energy which represents the proportion of gross energy which does not appear in the faeces was found to be very high across the treatments (91.82 to 95.38 kcal/kg). the proximate analysis of concentrate supplement and elephant grass presented in table 4 reveals that the concentrate supplement and elephant grass have fibre ratio of (5.07:31.00%) respectively that can be handled by grasscutters being pseudo-ruminant with large caecum that occupies about 40% of the abdominal cavity [43]. crude fibre is feed diluents and reduces the caloric content (digestible energy) of feed, but that was not the case in this study as seen by this very high digestibility of energy. table 4. mean apparent coefficient of digestibility of grasscutter fed elephant grass with different concentrate feeding regimes. a, b, c, and d means within rows with similar superscripts are not significantly different (p>0.05), sem= standard error of mean, nfe = nitrogen-free extractives. effects of concentrate supplement regimes on percentage digestible nutrients and total digestible nutrients (tdn) of grasscutter the effect of concentrate supplement regimes on percentage digestible nutrients and total digestible nutrients (tdn) of grasscutters is presented in table 5. the feeding regimes affected significantly (p<0.05) all digestible nutrients evaluated in this study. it was observed that digestible crude protein (dcp) of t5 t7 and t9 were similar (p>0.05) while t3, t5 and t7 did not also differ (p>0.05) but differed with that of t1. however, these significant differences cannot be linked to the different feeding regimes since a particular sequence was not followed. the digestible crude fibre (dcf) also followed the same sequence of nutrients t1 t3 t5 t7 t9 sem dry matter (%) 82.05a 75.65b 68.92c 69.77c 67.46c 1.68 crude protein (%) 82.94a 77.52a 68.09b 70.66b 66.53b 1.92 crude fibre (%) 85.95a 74.84b 70.51bc 75.05b 67.46c 1.62 ether extract (%) 72.69a 53.59b 35.16cd 41.94c 27.65d 3.13 ash (%) 84.47a 77.23b 65.01d 71.66c 67.05cd 1.54 nitrogen-free extractive (%) 56.41a 43.01b 29.80c 28.37c 25.10c 3.95 energy 95.38a 94.09b 91.82c 91.82c 93.10b 0.41 http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 significant such as the (dcp) which may suggest that the crude fibre was at the tolerable level that nutrients could be digestible by grasscutters as seen in the digestible crude protein level. the digestible ether extract (dee) was also observed to be significant but no particular sequence was followed. the digestible nitrogen-free extract (dnfe) was significant (p<0.05). it was also observed that t5, t7 and t9 did not differ significantly (p>0.05). the total digestible nutrient (tdn) is a figure used to indicate the relative energy value of a feed to an animal. it is influenced by the percentage dry matter since water doses do not contribute to the energy value of feeds. the dry matter of feed has to be digestible for feed to have a tdn value [42]. the dry matter digestibility value obtained in this study 75.65 to 82.05% is higher than values by obi et al. [41]. an indication that, the crude fibre content of the diet did not interfere with nutrients availability at the tissue level and adequate nutrients were available for growth and maintenance. the different feeding regimes (1 to 9%) of concentrate supplement using elephant grass as basal feed did not adversely affect the digestible nutrients. the percentage digestible energy (pde) was observed to be significantly affected (p<0.05) and the highest value observed was in t9 though did not differ (p>0.05) significantly with t5 and t7. the lowest was observed in t1 which suggests that t1 with 1% level of concentrate supplement was not enough for energy supply for the experimental group of the grasscutters which reflected in them having the lowest total weight gains of 667.50g as was observed in the growth performance. conclusion the findings of this study suggest that supplementation of forage with formulated concentrate can be an aid in the digestion of nutrients by grasscutters for their growth performance while in captivity. the digestibility of nutrients when concentrate supplement and elephant grass are combined particularly 5% concentrate supplement were not affected. it was therefore recommended that, it should be used in improving the growth performance of grasscutters. table 5. effects of concentrate supplement-feeding regimes on percentage digestible nutrients and total digestible nutrient (tdn) of grasscutter. nutrients t1 t3 t5 t7 t9 sem digestible crude protein 5.14c 5.84b 6.27ab 6.46ab 6.73a 0.22 digestible crude fibre 7.11b 7.50b 8.88a 9.13a 9.04a 0.29 digestible ether extract 0.73a 0.64b 0.50c 0.62b 0.44d 0.02 digestible nitrogen-free extractive 14.55a 13.19ab 11.64bc 10.76c 10.23c 0.65 total digestible nutrient 28.44a 27.98ab 27.92b 27.73b 26.99c 0.17 energy. 1075.60c 1290.70bc 1575.80a 1520.70ab 1716.60a 79.15 a, b, c, and d means within rows with similar superscripts are not significantly different (p>0.05), sem= standard error of mean. references 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march; 2007.p. 369-372. 32. tangendjaja b, rahardjo yc, lowry jb. leucaena leaf meal in the diet of growing rabbits: evaluation and effect of a low-mimosine treatment. anim feed sci technol.. 1990 may 1;29(1-2):63-72. 33. egbunuogie bo, bamikole ma. nutrient content variability in three forms of elephant grass (pennisetum purpureum) preservation. niger j agric food environ. 2014;10(1):48-52. 34. islam mr, saha ck, sarker nr, jalil ma, hasanuzzaman m. effect of variety on proportion of botanical fractions and nutritive value of different napiergrass (pennisetum purpureum) and relationship between botanical fractions and nutritive value. asian austral j anim.2003;16 (6): 837-842 35. ogunjobi ja, adu bw, jayeola ob. growth performance of captive-bred juvenile male grasscutters (tyonomys swinderianus temminck 1827) fed two common grasses in nigeria. international journal of agriscience. 2014;4(2):119-26. 36. minson dj. the chemical composition and nutritive value of tropical grasses. in: skerman pj, cameroon dg, riveros f (eds), tropical grasses, p.172–180. rome (italy): food and agriculture organization of the united nations;1990. 37. hon fm, oluremi oi, anugwa fo. the effect of dried sweet orange (citrus sinensis) fruit pulp meal on the growth performance of rabbits. pak j nutr. 2009;8(8):1150-5. 38. adegbola ta, okonkwo jc. nutrient intake, digestibility and growth rate of rabbits fed varying levels of cassava leaf meal. niger j anim prod. 2002;29(1):21-6. 39. diaz af, perez alm, perez hm. digestibility and energy retention by young rabbits fed different levels of intake. annales de zootechnie . 1999;48:289–295. 40. underwood ej, suttle nf. 3rd ed. the mineral nutrition of livestock. wallingford (england): cabi international publishing; 1999. 41. obi oo, omole aj, ajasin fo, tewe oo. nutritive potentials of four conventional forages fed to growing http://bioscience.highlightsin.org/ gboshe and osarenakhue 2020 the effect of concentrate feed supplementation regimes on grasscutters highlights in bioscience august 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 8 grass-cutter (thryonomys swinderianus). livest. res. rural. 2008;20(11):179. 42. choct m, annison g. anti‐nutritive effect of wheat pentosans in broiler chickens: roles of viscosity and gut microflora. br poult sci. 1992 sep 1;33(4):821-34. 43. olomu jm, ezieshi ve, orheruata am. grasscutter production in nigeria: principles and practice. benin city(nigeria): jachem publ. 2003. http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20210 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 12 research article open access 1university of tunis el manar, faculty of sciences of tunis, laboratory of ecology, biology and physiology of aquatic organisms, 2092 tunis, tunisia contacts of authors * to whom correspondence should be addressed: imene chetoui citation: chetoui i, bejaoui s, ghribi f, el cafsi m (2020). annual variation of the biochemical composition, energy reserves and physiological indices in mactra stultorum tissues from the tunisian coasts. highlights in bioscience volume 3. article id 20210. dio:10.36462/ h.biosci.20210 received: may 23, 2020 accepted: july 9, 2020 published: july 12, 2020 copyright: © 2020 chetoui et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. annual variation of the biochemical composition, energy reserves and physiological indices in mactra stultorum tissues from the tunisian coasts imene chetoui 1 *, safa bejaoui 1 , feriel ghribi 1 and m'hamed el cafsi 1 abstract the present study assessed the consequences of environmental changes on the biochemical quality and the physiological condition of the whole body and others organs mactra stultorum collected from the north coast of tunisia. significant variations in the biochemical components, pe and ci have shown between seasons. the depletion of glycogen in all tissues was recorded during spawning periods (late spring and summer). lipid accumulation in gonad–visceral mass during the early and late gametogenesis stages was associated with the increase in ci and gsi. however, decreases in lipid and protein contents were recorded in all the tested organs during summer when the animals are in the spawning period, suggesting that clams could accumulate proteins and lipids. regarding the environmental factors, a negative and a significant correlation was recorded between glycogen, lipid contents and water temperature. based on these results and as m. stultorum is appreciated by the consumers, we suggest that the harvesting of m. stultorum should be concentrated on february to june when the whole body was characterized by an important amount of the biochemical composition when the seafood is at its highest nutritive value as compared to august to january. because of its high nutritive value especially it’s richness by some important and essentials fatty acids, m. stultorum seems to be considered as an important commercial species in many countries and in tunisia particularly. we suggest that harvesting of m. stultorum should be concentrated on the period when the seafood is at its highest nutritive value. survivability rates from malignancy. the greatest variations in the mortality ratios existed within the european countries. keywords: mactra stultorum, seasonal variations, biochemical composition, percentage edibility, condition index and gonado-somatic index. introduction over the last few decades, mollusk bivalves became a high nutritious food source in the human diet. they are considered highly nutritional with interesting dietetic properties because of their valuable biochemical constituents such as proteins, glycogen, lipids, fatty acids and minerals. research on the biochemical and energy reserves of bivalve species was launched in the 19 th century. several authors have studied the seasonal variation of the biochemical compounds of bivalves, the storage cycle of their energy reserves relative to the reproduction of the species and the environmental fluctuations of the environment [1-3]. https://creativecommons.org/licenses/by/4.0/ chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 12 in bivalves, glycogen constitutes the most prominent reserve, especially during the processes of gametogenesis [4, 5]. glycogen is a good supplying energy demand during the formation of gametes [6]. due to their high caloric content, lipids serve as energy storage under critical nutritional conditions [7]. they play an important role in metabolic pathways in the formation of gametes and are considered as the major reserve of oocytes [8, 9]. protein, one of the abundant biochemical components in bivalves, can be serving also serve as an energy reserve during gametogenesis when other reserves are exhausted [10]. certain exogenous factors, such as the temperature of the water and the availability of food in the surrounding environment as well as some endogenous conditions such as the reproductive cycle play a key role in the variation of the physiological fitness of many marine organisms [11, 12]. so it has been demonstrated that there is an important and broad relationship between the percentage edibility, the condition index and organic content. additionally, the condition index, which supported as the health index, considered as a good index to determine the evolution of the physiological processes in relation to the reproduction, growth, mortality, parasitic and other contagions factors [13, 14]. the bivalve mactra stultorum is a benthic species inhabiting sandy beaches of the lower infra-littoral zone. it is widely distributed along the north and south coasts of the mediterranean sea, the black sea, senegal and europe [15]. in tunisia, it has been reported from the north coasts and from the south in the gulf of gabes [16]. because of its high nutritive value especially it’s richness by some important and essentials fatty acids such as eicosapentanoic (epa, 20:5n-3) and docosahexanoic (dha, 22:6n-3) acids [17], m. stultorum seems to be considered as an important commercial species in many countries such as in manche (atlantic coast) [18]. recently, the reproductive cycle of m. stultorum has been determined by chetoui et al. [19]. the assessment of histological studies indicated that its reproductive cycle is synchronous for both sexes. the gametogenesis process is initiated in january. the maturation phase is extended from april to july and the partial spawning started in spring to reach its peak in late summer, indicating a prolonged reproductive activity. to the best of our knowledge, the biochemical composition of m. stultorum from the mediterranean coasts in relation to various endogenous (reproductive cycle…etc) and exogenous conditions (temperature, salinity, food accessibility…etc) have not yet been studied. the goal of the present work is to investigate the annual dynamic of the biochemical composition (including macromolecules and glycogen) of the whole soft tissue and different organs of m. stultorum collected from northern tunisian coasts and to assess its percentage of edibility (pe) and the condition index (ci) in relation to the total reproductive period and environmental parameters. materials and methods sampling site and collection specimens of mactra stultorum were collected during 12 months (from june 2008 until may 2009) from the sandy beach of station kalaat el andalous located at the gulf of tunis (37°03’73’’n and 10°10’67’’e). the current northsouth is reduced in this range through advanced sidi ali mekki. thus, these waters are usually calm and the bottom consists mainly of fine sand deposited on the front of the delta plain of oued medjerda (figure 1). figure 1. maps of sampling area in tunisia (the black full star in the gulf of tunis shows the sampling site: kalaât el andalous). kalaât el andaous lake of medgerda gulf of tunis chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 12 environmental parameters were measured monthly. surface seawater temperature and salinity were carried out in situ depth of 30cm using a mercury thermometer and a salinometer, respectively. chlorophyll a was extracted using whatman gf/f filters with 90% methanol in the laboratory and concentration was evaluated according to the protocol of aminot and chaussepied [20]. samples of m. stultorum were transferred to the laboratory for acclimation in filtered seawater in order to eliminate all types of nutrients from their digestive glands. after 72 h, clams of similar size (30 and 57mm) were selected for experimentation. the total wight and shell dry weight of all m. stultorum were analyzed using 0.001 g electronic precision balance. the biometric measurements such as shell length (sl), height (sh) and width (sw) were measured with a digital caliper (0.1mm). experimental analysis after sacrifice, separate organs such as mantle (mt), adductor muscle (am), foot (ft), gonad-visceral mass (gv) and rest (re) were dissected for the first group and for the second group we selected the body whole. all selected animals were kept in a freezer at -30 ° c for biochemical analysis. the gonad and visceral mass are taken as a single organ (gv) due to the difficulty of their separation. for biochemical analysis, each month six replicates were used to produce the means with 6–8 m. stultorum pooled in each replicate. total protein content was determined by the method of lowry et al. [21] using the bovine serum albumin (bsa) as a standard range. samples were homogenized in a tris-hcl buffer (20 mm; ph=7.4), then, centrifuged at 10.000 × g for 20 min (4°c). supernatants were stored in eppendorf tubes at -80°c for subsequent analyses. a volume of 10 µl supernatants was dissolved in 400 µl of water solution to which we added a 2ml of a mixture composed of three solutions: sodium carbonate hydrate dissolved in a solution of naoh (0.1n), copper sulfate dissolved in water and sodium hydrate dissolved in water. to the last mixture we added 200 µl of folin reagent, then, incubated for 30 min at the darkness. the proteins were quantified using the spectrometric method at 540 nm. the total glycogens were determined by the enzymatic potocol of dubois et al. [22]. the glycogen was released from the tissues by heating 0.5 g of fresh tissue in the presence of 2 ml of alkaline solution (koh, 300 g / l) for 2 min in a water bath at 100 ° c. glycogen was precipitated by the addition of ethanol 95°c. this precipitate was recovered after cooling the tubes in ice and centrifugation at 3000 rpm for 10 min. it was dissolved in 10 ml of water. then, 1 ml of the last glycogen solution was hydrolyzed by a hydrochloric acid hcl (1.2mol / l) in a bath at 100°c for 2 h. then, this hydrolyzate was neutralized by the addition of sodium hydroxide solution naoh (0.4 n) until the color changes to pink. finally, the glucose oxidase reagent was added to these last mixtures, which were subsequently incubated in a bath at 37 °c for 15 min. the optical density was measured at 520 nm. total lipids (tl) were extracted using the method of folch et al. [23]. the flesh was crushed with a solution of chloroform: methanol (2:1, v:v) including 0.01% butylhydroxy-toluene (bht) as an antioxidant. the extraction volume was estimated by 30 ml of extraction solvent per g of tissue. the mixture of the extract volume and 2 ml of nacl solution (15%) were centrifuged at 3000 x g for 15 min. the chloroform phase was recovered and evaporated to dryness by rotavapor® r-300. finally, the residue was diluted in 2 ml of chloroform solution. the levels of total lipid, glycogen and protein are expressed as mg / g dry weight (mg/g dw). the percentage edibility (pe), the condition index (ci) and the gonado-somatic index (gsi) were assessed monthly from may 2008 to jun 2009. a total of 30 clams were selected each month for the analysis of each index. the flesh and shells of each clam were dried in an oven at 60°c for up to 72 h to determine the constant dry tissue and shell weight. for each index, 30 individuals were collected each month. according to deslou-paoli et al. [24], the ci was calculated as equation 1 and the gsi was expressed by equation 2: equation 1: ci= (dry weight of the soft parts/dry weight of shell) *100; equation 2: gsi= (dry weight of the gonad-visceral mass/dry weight of shell) *100. the percentage edibility (pe) was calculated using anibal et al. method [25] and was estimated using equation 3: equation 3: pe= (wet meat weight (g)/total wet weight including shell (g)) *100. statistical analysis data analysis was performed using the software statistica version 5.0. the biochemical composition of each separate tissue and whole body was compared between months. normality was assessed for all datasets using the shapiro-wilcoxon test. the significant differences between variables were analyzed using one-way analysis of variance (anova) followed by posthoc tukey's test (p<0.05). when the conditions for anova were not satisfied, nonparametric kruskal–wallis's test was used (p<0.05). the differences between samples were deemed to be significant at p<0.05. pearson correlation matrix and principal component analysis (pca) were used to exhibit the association between biochemical, biological indices, percentage ediblity and environmental factors among the studied periods results environmental parameters the temperature of the seawater varied between 8°c in january to 28 °c in july and august. salinity was highest (38.2 psu) in july, dropping to <35psu from december to chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 12 march with a minimum value noted in january (31 psu). this decrease in salinity is generally due to the relatively high pluviometry (84.14mm in january) and to discharge from the river medjerda. chlorophyll a values ranged from 1.28 mg/l (november) to 6.35mg/l (april) (figure 2). figure 2. physicochemical parameters of kalaât el andalous during the sampling period (jun 2008–may 2009). biometric analyses the biometric data (shell length, height and width) are elucidated in table 1. the shell length (sl) ranged from 32.68 to 47.82 mm; the shell height (sl) ranged from 25.72 to 39.83mm, the shell width (sw) varied from 12.88 to 25.58 mm and the shell dry weight ranged from 3.57 to 10.5g. table1: biometric measurements of mactra stultorum during one year. month sl (mm) sh (mm) sw (mm) sdw (g) mean sd mean sd mean sd mean sd j(08) 40.15 6.39 33.81 7.67 20.94 4.35 5.69 1.7 j(08) 47.52 4.04 39.41 4.81 25.22 2.68 10.5 2.43 a(08) 46.25 3.16 38.09 3.84 24.52 2.2 8.73 2.21 s(08) 47.97 2.84 39.2 3.44 25.48 2.1 9.16 2.31 o(08) 43.55 4.83 36.3 5.9 23.54 3.9 8.08 2.26 n(08) 40.95 3.68 33.69 4.88 21.83 4.02 5.31 1.99 d(08) 36.82 3.32 30.8 3.86 19.32 2.38 3.57 1.41 j(09) 32.68 7.04 25.72 7.79 12.88 5.64 6.34 2.5 f(09) 46.71 5.64 39.35 6.59 25.56 3.78 8.18 1.74 m(09) 45.49 4.56 37.99 5.63 24.31 3.22 7.03 2.47 a(09) 46.97 3.49 39.83 6.22 25.58 3.8 9.78 2.07 m(09) 40.54 8.08 33.63 9.1 19.8 7.02 9.46 2.85 results are given as mean ± sd. n=50 individuals each month. sl: shell length; sh: shell height; sw: shell width and sdw: shell dry weight. biochemical composition seasonal variations in the protein, glycogen and total lipid contents of different body parts (foot, gonad-visceral mass, mantle, adductor muscle and rest) are shown in (table 2). the biochemical constituents showed a significant variation between seasons and tissues (p<0.001). in all tissues, the highest protein content (235.±8.42 mg/g dw) was noted in the foot, while, the minimal amount of protein (31.2±3.4 mg/g dw) was noted in the adductor muscle. using to hsd tukey’s tests (f=3.35, p = 0.001), significant differences were recorded for a protein of foot which varied from 140 to 235 mg/g dw (p <0.05). it marked by tow peaks in september (220.78±10.20 mg/g dw) and december (175.07±11.53 mg/g dw).in the gonad-visceral mass, the seasonal evolution of protein has shown a significant variation throughout the year. the minimum level was observed in july (54.18±19.97 mg/g dw), then; increased significantly to reach two peaks in september (231.84±23.13mg/g dw) and april (250.94±22.60 mg/g dw). for the mantle, the level was relatively stable from june to december, then it dropped in january (39.67 ± 9.38 mg/g dw) and enhanced in march (126.84 ± 14.48mg/g dw). the protein level in the adductor muscle remained stable between 120 and 130mg /g dw throughout the study year except october (31.17 ± 8.38 mg/g dw) and february (60.41 ± 9.40 mg/g dw) in which protein level decreased significantly. the protein content in the rest part was comprised between 24.3± 0.37 mg/g dw in january to 144.9± 1.35 mg/g dw in march. the gradient of the glycogen content in m stultorum body parts has shown that the ft tissue has the high level, followed by those in the tissues from the am, mt and gv. in contrast, lower glycogen content was recorded in the re tissues. the glycogen contents varied similarly in the four examined tissues (foot, gonadvisceral mass, mantle, adductor muscle). they were lower from july to september (<20 mg/g dw) and peaked in april, and then declined considerably in may. the lowest values of glycogen in the rest part (re) were detected during autumn and winter in particular in november and january with (3.54±1.17mg/g dw) and (4.94±1.28 mg/g dw). however, similar to other parts of the body, the quantity of glycogen peaked in april (17.64±3.9 mg/g dw) one-way anova analysis (hsd tukey’s tests, f=6.51, p = 0) indicated that glycogen levels in all examined tissues showed significant monthly variations. total lipids contents are unevenly distributed between tissues of m. stultorum and varied significantly (p<0.05) between seasons except for foot, which varied from 14 to 22 mg/g. in the gonad-visceral mass, tl content started at (50.38± 8.58 mg/g dw) in june and decreased in august (25.16± 8.78 mg/g dw). then, it gradually increased in autumn and stabilized in winter. chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 12 table 2. seasonal variations in the contents of total protein, glycogen and lipids (mg/g dw) of the body parts and whole body of clam mactra stultorum. total protein contents in mg/g dry weight tissue j(08) j(08) a(08) s(08) o(08) n(08) d(08) j(09) f(09) m(09) a(09) m(09) ft 150±9.48be 142±8.65 ab 152.1±9.80 bc 221±12 f 170±8.07de 147±11.2abc 175.1±10.8 e 138±10.1ab 130.4±11.41 a 142.3±7.828 ab 162.9±12.62dca 235±8.42 f gv 112±10.1b 54.2±9.99 a 164±10.77c 232±11.6 e 117±11.1 bd 97±10.9 b 157±10.94 c 146±12.3 cd 95.2±11.36b 177.8±10.21 c 250.9±11.3 c 172±10.9 c mt 96.8±6.14 d 83.4±8.55 be 70.82±13.65 b 77.2±9.66 b 79.6±6.35 b 81±10.6 b 77.82±5.7 b 39.7±5.0 a 96.22±7.27 ad 126.8±5.35 f 97.92±8.47 de 97.6±10. ce am 136±8.21 f 137±7.57 f 142.5±6.63 f 126±3.7 ed 31.2±3.4 a 73.9±3.8c 135±8.19 ef 138±9.7 f 60.43±6.36 b 70.6±6.6 bc 121.4±6.9 d 134±4.58 ef re 93.4±3.08 c 83.8±4.01c 79.76±1.075 c 97.9±2.3 d 53.9±4.08 b 57.5±7.87 b 98.11±1.71 d 24.3±0.37 a 84.37±5.52 c 144.9±1.35 f 121.2±1.38 e 97.2±2.1 d wb 114±4.89 c 97.4±9.2 b 114.8±3.9 c 142±9.2e 84.2±6.7 a 85.8±4.5a 123.5±5.56d 85.2±2.52 a 91.84±6.16ab 134.6±3.11 f 145.9±2.98e 139±2.11 ef total glycogen contents in mg/g dry weight tissue j(08) j(08) a(08) s(08) o(08) n(08) d(08) j(09) f(09) m(09) a(09) m(09) ft 14.6±1.96 cd 10.06±1.5 acd 6.45±1.56 ab 5.15±1 a 15.4±1.49 d 11.5±1.64 bca 28.84±2 ef 26.8±1.41 eg 29.7±2.82 ef 33.4±1.17 fh 35.5±3 h 23±2.4g gv 9.04±2.93 bc 6.22±1.06 b 3.47±1.68 a 4.18±1.02 ab 11.88±1 cd 16.56±1.89 de 20.05±2.96 eg 14.84±1.02 df 18.5±1.9 ef 23.81±1.1 g 35.46±1.2 h 8.53±0.81 ab mt 14.11±1.57 c 9.51±1.05 b 8.37±0.75 a 7.55±2.03 b 8.86±1.83 b 10.45±1.51 bc 22.37±1.4 d 12±1.31 bc 10.25±1.28 c 24.78±2.21d 27.71±1.5 e 20.54±1.34 d am 12.71±0.92 b 10.54±1.1 a 6.03±1.25 a 7.01±1.76 a 10.81±1.61 ab 19.84±2.36 ab 17.20±0.82 bc 19.55±1.35 cd 21.61±2.09 cd 33.8±1.9 e 34.55±1.5 e 22.47±2.3d re 7.07±1.17 c 10.8±1.78 d 7.71±1.44 c 6.24±0.6b 5.77±1.43b 3.54±1.17a 8.94±2.3 f 4.94±1.28b 13.69±3.8 e 17.64±3.9 f 10.94±2.5 d 5.21±1.3 b wb 10.8±3.97 be 10±2.91 be 6.12±2.54 a 5.73±2.44 a 11.6±3.71 cd 12.7±4.57 cd 21.05±5.41f 13.8±4.67 d 18.65±5.86e 25.19±5.91g 27.34±5.42g 15.6±3.7d total lipid contents in mg/g dry weight tissue j(08) j(08) a(08) s(08) o(08) n(08) d(08) j(09) f(09) m(09) a(09) m(09) ft 14.7±0.93 16.4±1.67 16.8±1.80 19±0.93 17.2±2.03 18.9±1.15 20.89±1.95 21.4±1.14 20.13±2.08 21.12±1.13 22.62±1.9 20.2±2.7 gv 50.4±17.8 e 45.2±12.6 d 25.17±8.78 a 28.9±8.58 a 43.7±5.25 cd 43.8±5.15 cd 40.58±5.64 c 43.9±16.5 cd 34.47±12.02 b 57.07±11.42f 72.46±14.64 h 64±7.8 g mt 36.6±7.8 b 32.1±4.57 acd 30.84±8.5 ac 35.1±6.42 bd 30.9±3.44 a 35.5±8.63 bd 35.56±9.87 bd 36.1±6.13 bd 28.27±8.89 a 35.35±3.3b 41.25±3.25 e 283.23 a am 13.8±2.4ab 14.6±4.97 b 9.82±2.02 a 24.1±7.82 c 25.4±3.51 c 30.8±4.68d 32.86±8.16e 28±3.43 cde 26.06±5.64 c 16.51±3.63 b 15.25±5.52b 13.6±2.3 ab re 55.5±4.38 f 44.9±4.94 d 31.64±3.47cb 34.3±3.63 cb 24.2±3.09 b 55.6±3.94 f 58.33±3.57 f 24.3±2.6 b 28.24±3.352 b 39.73±4.48 d 25.22±3.7 b 20.3±3.3a wb 37.8±8.2 c 33±7.61 c 24.32±6.12 a 29.3±6.93 b 30.9±4.56 b 40.1±6.44 d 41.09±7.2 d 28.8±6.99 b 28.07±7.55 b 34.92±7.2 c 33.67±6.86 c 27.7±5.4 b results are given as mean ± sd. n=8 replicates each month. ft: foot. gv: gonad-visceral mass. mt: mantle. am: adductor muscle. re: rest. wb: whole body. different letters indicate significant monthly difference for each organ (hsd tukey’s tests. p < 0.05). chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 12 tl in the gonad-visceral mass reached a maximum value during april (72.45 ± 5.73 mg/g dw). in mantle, tl content presented low values in october (30.86 ± 3.44 mg/g dw) and february (28.27± 8.89mg/g dw). for adductor muscle, the content of lipids seems to be low from jun to august and from march to may (below 20mg/g dw). however, the high values were observed in autumn and winter in order of 25 and 32 mg/g dm, respectively. in the rest part the tl contents fluctuated between 20.3 mg/g dw and 58 mg/g dw throughout the study year. one-way anova analysis (hsd tukey’s tests, f=18.11, p = 0) indicated that the tl contents in gv, am, mt and re showed significant monthly variations. seasonal variations in the content of proteins, total lipids and glycogen of the m. stultorum whole body are shown in (table 2) expressed as mg per g of dry weight. protein content was the major biochemical compound and underwent significant variations during the experimental period (hsd tukey’s tests, f =146.42, p = 0). it was in minimum value (84.2±6.7 mg/g dw) in october and reached a maximum (145.9±2.98mg/g dw) in april. the total lipid contents, which are the second major biochemical constituents in the whole body, it were comprised between 24 and 37 mg/g dw during the experimental period. glycogen content varied in a similar way to that observed in gv. it was in a lowest value during august and september and increased gradually during winter and spring to reach a maximum in april. this compound has demonstrated significant changes among month (hsd tukey’s tests, f =270.20, p = 0). condition index (ci) and gonado-somatic (gsi) index the ci and gsi indexes showed a synchronous evolution. lowest values of ci (4.43 ± 1.24%) and of gsi (1.26±0.32%) were recorded in august (08). however, high percent of ci and gsi were observed in april with values of (14.41± 2.09%) and (3.97±0.52%) respectively. these indexes have shown significant differences among months (anova, (p< 0.05) (figure 3). figure 3: seasonal variation of the condition index (ci) and gonado-somatic index (gsi) of mactra stultorum throughout the study period.results are given as mean ± sd, n=30 replicates each month. different letters indicate significant monthly difference for each index (hsd tukey’s tests, p < 0.05). percentage edibility (pe) during the reproductive cycle, the percentage of edibility showed a significant fluctuation from 25%60%, with a sharp enhancement in the percentage edibility during april (59.64±1.93%). during the summer, autumn and winter, it decreased considerably, with the lowest of the edibility percent (25.34±0.71%) being noted in august (figure 3). the percentage edibility has shown significant differences among months (anova, (p< 0.05) (figure 4). figure 4: seasonal variation of percentage edibility (%) in mactra stultorum throughout the study period. results are given as mean ± sd, n=30 replicates each month. different letters indicate significant monthly difference for pe (hsd tukey’s tests, p < 0.05). statistical analyses the interaction between biochemical compositions in each separate tissue and in the whole body of m. stultorum (1); environmental parameters (water temperature, salinity and chlorophyll a) (2); reproductive indices of m. stultorum (ci and gsi) (3), percentage edibility (4) and seasons (5) was tested using principal components analysis (pca) and a pearson correlation (figure 5 and table 3). results of pca allowed us to retain the first two factorial axes that explain 57.32% of the total variance. factor 1 displayed 41.30% of the total variance, defined by ci; gsi; pe and different biochemical’s compounds in each organ and whole body except the lipwb; lipma; lipre; promt; proft, which characterized factor 2 (14.5%). in addition, the environmental parameters (t °c, s, and chlo a) described the factor 2 (figure 5a). analyze of biochemical’s composition reveled significant seasonal fluctuations marked by two phases the first one is the storage phase and the second is the depletion of reserves. during late winter and spring, glycogen was accumulated in high quantities in all tissues and whole body, which was positively and highly correlated with ci and gsi (table 3). chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 12 figure 5. principal analysis component (pca) represented by two factors f1 and f2 and produced by the biochemical parameters and reproductive indices of mactra stultorum and the environmental condition. projection of the variables on the factor-plane (1-2) (a) and projection of the cases on the factor-plane (1-2) (b) pro: protein, gly: glycogen, lip: total lipid, ft : foot , gv: gonad-visceral mass, mt: mantle, am : adductor muscle, re: rest, bw: whole body, ci: condition index, gsi: gonado-somatic index, t°c: water temperature, chlo a: chlorophyll a, s: salinity. this reserve seems to be lower during summer when the temperature is higher which presented a negative correlation with glygv and glyft. the total lipid in gv, mt, ft increased significantly especially during spring when the ci, gsi and pe are higher (positive correlation with lipgv). while the am deplete their lipid reserves during this same period and when the water temperature increased (negative correlation).in other hand the position of lipwb in the middle of the two factors of pca can be explained by the insignificant correlation with all parameters except with lipre. the major biochemical compound in all tissues, proteins significantly fluctuated with seasons and varied with tissues. additionally, the protein in the gonad-visceral mass was negatively correlated with chlorophyll a, especially during autumn and winter when ci and chlo a values were lower (p <0.05). pe showed a positive and high correlation with the accumulation of protein and carbohydrate (glycogen) in the tissue of m. stultorum and with ci and gsi when increased. only the accumulation of lipid in gv is positively correlated with pe index of physiological fitness. the salinity did not correlate with the biochemical compound of m. stultorum. a synergy affirmed by a positive correlation was observed between seasonal evolutions of glycogen contents of body parts and those of total lipid levels in foot and gonad-visceral mass of m. stultorum. nevertheless, the changes in protein contents of m. stultorum did not demonstrate a significant correlation with the biochemical compounds (lipid and glycogen) except for prore. the projection of individuals (each sample from each month) on the same factorial plan (1:2) showed that the different cases could be clustered into three groups. it has been demonstrated that are an interactions between groups 1 and 2, which each individuals accumulated high levels of biochemical’s compounds in each tissue during winter and spring corresponding to gametogenetic and maturity stages and they are characterized by a high level of pe. the third group represents the animals how depleted their reserve when environmental parameters such as sea water temperature and chlorophyll a increased signaling to spawning period (figure 5b). discussion this study provides new data about the seasonal evolution of the biochemical composition on the surf clam m. stultorum from tunisian coasts. previous studies on marine invertebrates such as bivalves showed that the changes in their biochemical composition are mostly affected by endogenous factors (the reproductive cycle) and exogenous factors (environmental conditions) [1, 26]. glycogen is considered as the major energy reserve in bivalves. it may be used concurrently as an energy source for growth and also stored in specific cells as an energetic reserve during gametogenesis process when the energy demands are high [27, 28]. in our study, the glycogen content in all tissues and the whole body of m. stultorum was lower during summer corresponding with the spawning phase. this compound increased gradually during winter and during spring and reached a maximum in april coinciding with the maturation of gonads. however, in may, they decreased considerably when 40% of the populations are partially spawned. the seasonal variation of glycogen seems to be related to the reproductive cycle of m. stultorum, as mentioned previously by chetoui et al. [19], which was also confirmed by a positive correlation with ci and gsi physiological indices. chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 8 of 12 table 3: correlation matrix of the biochemical parameters and reproductive indices of mactra stultorum and the environmental condition. proft progv promt proam prore glyft glygv glymt glyam glyre lipft lipgv lipmt lipam lipre prowb glywb lipwb ci gsi pe t (°c) chlo a progv ns promt ns ns proam ns ns ns prore ns ns 0,85 ns glyft ns ns ns ns ns glygv ns ns ns ns ns 0,85 glymt ns ns ns ns 0,66 0,86 0,82 glyam ns ns ns ns ns 0,87 0,88 0,88 glyre ns ns ns ns 0,63 0,59 ns ns ns lipft ns ns ns ns ns 0,78 0,73 0,69 0,76 ns lipgv ns ns ns ns ns 0,61 0,62 0,80 0,77 ns ns lipmt ns ns ns ns ns ns ns ns ns ns ns ns lipam ns ns ns ns ns ns ns ns ns ns ns ns ns lipre ns ns ns ns ns ns ns ns ns ns ns ns ns ns prowb 0,60 0,81 ns ns 0,81 ns ns ns ns ns ns ns ns ns -0,09 glywb ns ns ns ns ns 0,94 0,93 0,93 0,93 0,65 0,75 0,68 ns ns -0,06 ns lipwb ns ns ns ns ns ns ns ns ns ns ns ns ns ns 0,83 ns ns ci ns ns ns ns ns 0,82 0,74 0,80 0,91 ns 0,70 0,82 ns ns -0,41 ns 0,79 ns gsi ns ns ns ns ns 0,71 0,66 0,77 0,87 ns ns 0,88 ns ns -0,35 ns 0,72 ns 0,97 pe ns ns 0,71 ns 0,61 0,76 0,77 0,80 0,83 ns ns 0,73 ns ns ns ns 0,83 0.75 0,84 0,85 t (°c) ns ns ns ns ns -0,69 -0,61 ns ns ns -0,81 ns ns -0,72 0,04 ns ns ns ns ns ns chlo a ns ns ns ns ns ns ns ns ns ns ns ns ns ns -0,48 ns ns ns ns ns ns ns s ns ns ns ns ns ns ns ns ns ns ns ns ns ns 0,20 ns ns ns ns ns ns 0,60 ns correlation coefficients statistically significant (p <0.05); ns: not significant (p <0.05); the positive correlation is presented with yellow color and the negative one is presented with red color. pro: protein, gly: glycogen, lip: total lipid, ft : foot , gv: gonad-visceral mass, mt: mantle, am : adductor muscle, re: rest, bw: whole body, ci: condition index, gsi: gonado-somatic index, t°c: water temperature, chlo a: chlorophyll a, s: salinity. chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 9 of 12 we suggested that the accumulation of glycogen at the end of the cycle (autumn) serves as energy for the start of a new reproductive cycle in winter and support the essential metabolic maintenance during this season food scarcity. likewise, during gametogenesis development (in winter) and the maturation phase (in spring), the storage of glycogen can be used as precursors for lipid synthesis needed for the formation of gametes for mollusks [6] and as an immediate available energy source during spawning period [29, 30]. similar results were recorded for m. veneriformis [31]. however, some authors demonstrated that glycogen levels in m. chinensis and r. decussatus decreased considerably during the sexual maturity process [11, 32]. the biochemical composition of bivalve seems to be affected by many environmental parameters such as temperature, salinity and food accessibility [33]. a negative correlation between glycogen content in m. stultorum and the water temperature can be related to the direct or indirect effects of the energy reserves fluctuations (storage and exploitation) [34]. however, the accumulation of glycogen in gonad-visceral mass of m. stultorum, especially during march and april, could be explained by its positive correlation with food availability expressed by the peak of the chlorophyll a [31]. for that reason, the glycogen content represents a good characteristic of nutritional conditions [35]. our results showed that protein was the major compound in all organs and whole body of m. stultorum. in the gonad-visceral mass of m. stultorum, the content of protein was higher during ripening (march and april) and during spawning (july and august). by contrast, this compound in other organs (ft, am and mt) decreased during the same periods. from another point, the whole body seems to accumulate this organic constituent during the same periods. it should be noted that probably m. stultorum used protein in the mantle and foot as a source of energy during the ripening phase and partially the spawned and those of adductor muscles during spawning phase. furthermore, the glycogen quantity of these muscular organs decreased considerably when gametes are released. our result can be interpreted by the main role of protein to serve as an energy reserve in adult bivalves in periods of reduced glycogen levels [9, 10] and particularly to support the end of gametogenesis [3, 30]. the negative correlation between chlorophyll a and protein level in gonad-visceral mass of m. stultorum during autumn and winter suggested that the deficiency of food availability (low levels of chlorophyll a) has reduced the protein levels [9]. unuma et al. [32] and qi et al. [36] have suggested that the lower level of protein during the same period was interpreted by its role as a nutrient source for the proliferation and differentiation of cell germ in the gametogenesis process (in particularly during spermatogenesis of m. chinensis and pseudocentrotus depressus). the total lipid in m. stultorum gonad-visceral mass was higher than in the other organs, suggesting that gv is the major lipid storage site [31, 32]. based on the reproductive cycle of m. stultorum, the lipid content of the gonad-visceral mass presented a positive correlation with ci and gsi. they begin to accumulate progressively during the earlier and late developmental stages, reached a high level when the gametes mature during the ripening stage (march and april) and finally decreased considerably when the spawning occurred from may to august. it is necessary to mention that the total lipid accumulated in gv has been considered as good indicators of oocyte value and larva viability (1). in this context, it may be admitted as a good index of gonad maturity [34]. our results are in concordance with those found for m. chinensis, ruditapes philippinarum and scapharca broughtonii [27, 32]. although mantle and adductor muscles were not really considered as lipid organs storage. in the present study, the lipid reserves stocked in mantle decreased during spawning stage. nevertheless, those of adductor muscle declined dramatically during the ripening and spawning periods. these decreases indicate that the conversion of lipids accumulated in these tissues could be used to provide the necessary energy for the gametogenesis process of m. stultorum. however, the total lipid of foot did not show any changes throughout the year which was affirmed by a positive correlation with ci and a negative correlation with water temperature. it can be suggested that the lipid located in this tissue did not contribute to the energy conversion utilization process of m. stultorum. comparable results have been observed in m. veneriformis during sexual maturity and spawning periods [31]. however, dissimilar results were also described by qiaozhen and qi, [32] and qi et al. [31]. with regards to the annual changes in total lipid of whole body, it showed significant alterations with seasons. bayne, [37] has described two reproductive patterns of marine bivalves based on the relationship between energy storage and gonad development cycles. in a conservative pattern, gametogenesis used the energy stored in various organs in forms of lipids and glycogen. however, in an opportunistic pattern, gametogenesis occurs using the energy supplied by the nutrients when the food in water is abundant. therefore, we can suggest that m. stultorum adopts an intermediate pattern closer to m. veneriformis [31]. they used at the beginning of their reproductive cycle during the winter the energy reserves stored previously. nevertheless, during the spring season, they exploited at the same time the energy stored and energy provided by food availability. several studies have demonstrated that pe and ci are the main tools for the commercialization of bivalves, which varied seasonally and geographically, depending generally on exogenous and endogenous conditions such as food accessibility and the gametogenic cycle, respectively [11, 38]. chetoui et al., 2020 annual dynamic of the biochemical compositions on m. stultorum highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 10 of 12 the percentage edibility showed significant differences among seasons. it seems to be higher during early summer (08) and during spring (09) which corresponds to the beginning of the gametogenetic cycle. in this period the animals accumulate biochemical’s constituents for their gamete development when food in water is available and/ or abundant. therefore, this index of physiological fitness was positively correlated firstly with accumulation of protein and carbohydrate (glycogen) in all tissues of m. stultorum; secondly with the ci and gsi indexes and thistly with lipid contents in gv. while, during late summer, autumn and early winter, it decreased considerably, with the lowest value recorded in august. some decline in pe coincided with spawning and inactive periods. m. stultorum during the last periods has depleted their reserve during the spawning process. our results are in agreement with those in clams r. decussatus and paphia malabarica [25, 39]. moreover, ci is considered as a good tool widely used to measure the physiological state and to identify the spawning seasons of bivalves [13]. this indirect method of analysis [40] is necessarily confirmed by histological sectioning and microscopic evaluation of the gonads [41]. our data of seasonal variation of ci in m. stultorum during the period (jun 2008 to may 2009) was studied and confirmed by the determination of different stages of the gametogenic cycle of m. stultorum [19]. according to chetoui et al. [19], ci increased from winter to spring. these seasons represent the start of gametogenic development and ripe periods for both sexes [19]. however, the decrease of ci coincides with spawning and inactive stages [19]. ci was positively correlated with other reproductive index as gonad-somatic index which was specific for the assessment of physiological state of gonad. in the mediterranean sea, mainly in tunisia, bivalves are considered as a rich food source and a healthy proportionate diet [3, 42]. since, they are known for their nutritious and essential energy reserves (e.g., protein, vitamins and minerals). in addition, bivalves are the best source of polyunsaturated fatty acids n-3 family (pufa (n-3)) mainly the two essentials fatty acids epa and dha which play crucial roles in human nutrition and health [43].these elements present a dissimilar organization and concentration depending on the bivalve tissues that can be commonly associated with external climate fluctuations. the variation of the nutritional value could be assessed using percentage of edibility and the condition index; however, information about them is crucial for the cultivation and harvesting strategy of bivalves [44]. in other hand, not only the biochemical compounds, pe and ci have influences on the flesh quality during cultivation and harvesting process. the supplement information’s about the feeding strategy and the quality of the food in aquaculture are crucial for to keep the good nutritional value of the animals flesh as well for human consumption as for manufacturing animal foodstuff. consequently, all these data allow adopting a good management of the breeding cost of bivalves. conclusion in conclusion, this study reported, at the first time, the assessment of the biochemical composition in several organs and the whole body of m. stultorum in relation to the environmental conditions and the reproductive cycle. a decrease of glycogen levels in all tissues during the spawning periods (from may to august) suggesting it important role as energy in the gametogenesis process. lipid and protein varied significantly and played an important role as fuel for gametogenesis when stored glycogen was exhausted and to support the basic metabolic maintenance cost in periods of food insufficiency. additionally, the best nutritive season for m. stultorum was noted during the beginning of gametogenic cycle and ripe periods (from february to jun) associated with the highest contents of the biochemical composition, percentage of edibility and condition index and when the food availability was abundant. however, from july to january, it is not advisable to consume it due to its lowest nutritional value represented by low levels of the organic constituents and the percentage edibility. based on these results, we suggest that the harvesting of m. stultorum should be concentrated on the period when the seafood is at its highest nutritive value. the percentage of edibility and the condition index are very important tools in physiological studies providing valuable information required for assessing larvae development and promoting successful restocking actions in aquaculture management. further studies of other aquaculture parameters such as food quality and pathological tests seem to be necessary in order to avoid disease and increase the larva growth and bivalve’s quality. acknowledgments this study was supported by the tunis university of sciences and the research laboratory. references 1. massapina c, joaquin s, matias d, devauchelle n. oocyte and embryo quality in crassostrea gigas (portuguese strain) during a spawning period in algarve, south portugal. aquat living resour.1999; 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condition index and percentage edibility of the short neck clam paphia malabarica (chemnitz) from estuarine regions of ratnagiri, west coast of india. aquac res. 2009;40: 69-73. 40. rahim aa, idris mh, kamal ah. wong sk, arshad a. analysis of condition index in polymesoda expansa (mousson 1849). pjbs.2012;15: 629-634. 41. adjei-boateng d, wilson, jg. body condition and gametogenic cycle of galatea paradoxa (mollusca: bivalvia) in the volta river estuary, ghana. estuar coastal shelfish sci. 2011. 42. ghribi f, boussoufa d., aouini f, bejaoui s, chetoui i, rabeh i, el cafsi m. seasonal variation of biochemical composition of noah's ark shells (arca noae l. 1758) in a tunisian coastal lagoon in relation to its reproductive cycle and environmental conditions. aquat living resour.2018; 3:1-15. 43. shanmugam a, palpandi c, sambasivam s. some valuable fatty acids exposed from wedge clam donax cuneatus (linnaeus). afjr.2007; 1(2): 014-018. 44. okumus i, stirling hp. seasonal variations in the meat weight, condition index and biochemical composition of mussels (mytilus edulis l.) in suspended culture in two scottish sea lochs. aquaculture.1998; 159: 249-261. highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20208 highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 1 of 8 research article open access 1 national center for radiation research and technology, cairo, egypt 2 independent phd researcher and candidate, faculty of pharmacy, cairo university, egypt contacts of authors * to whom correspondence should be addressed: mostafa essam eissa citation: rashed er, eissa me (2020). longterm quantitative assessment of women survivability from cancer: a unique descriptive analysis. highlights in bioscience volume 3. article id 20208. dio:10.36462/ h.biosci.20208 received: june 4, 2020 accepted: july 3, 2020 published: july 5, 2020 copyright: © 2020 rashed and eissa. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. long-term quantitative assessment of women survivability from cancer: a unique descriptive analysis engy refaat rashed 1 , mostafa essam eissa* 2 abstract statistical process control (spc) methodologies are a set of statistical methods and techniques that were initially designed for industrial processes but could be adopted for non-industrial applications. the current prospective study aimed to provide a unique quantitative investigation of an epidemiological disease using the spc program platform. the selected case herein was a long-term monitoring record of the yearly cancer mortality rates in women worldwide. multidimensional segregation of the dataset into subgroups was conducted to visualize the clustering pattern based on nations (42 countries as boxplot), time and the gaussian mixture model (two-interfering bell-shaped distributions approach). the trend of death rates versus the elapsed years would demonstrate a moderately negative correlation with the time following the theory of splines. construction of control chart based on the fitted weibull distribution showed a gradual steady improvement in survivability rates from malignancy. the greatest variations in the mortality ratios existed within the european countries. keywords: cancer, gaussian mixture model, mortality rate, weibull. introduction malignancy is one of the most devastating diseases that affect humanity in the modern era of civilization. it threatens human lifestyle quality, productivity, financial and economic status of society and country, in addition to morbidity, disability and mortality [1]. notably, women's health and well-being are severely impacted by the rising challenge of cancer disease in the community, which hinders them from serving their crucial role in any rising and developed nations. the burden of cancer among women is high in both high income countries (hics) and low middle income countries (lmics), although the distribution of most common cancers differs. this burden is predicted to extend as populations grow and age and because the prevalence of cancer risk factors increases in some countries, especially in lmics. the costs of cancer are considerable and even catastrophic in hics and lmics alike. however, this burden of disease, loss of life, and economic hardship is not inevitable [2]. all of the foremost common cancers among women worldwide, including lung, breast, cervix, liver, and colorectum, have known means of prevention and/or early detection, which may be used to cut back incidence and mortality. furthermore, carcinoma and cervical cancer, two of the highest four cancers in women worldwide have several proven prevention measures [3]. these two cancers combined represent about 20% of all cancer deaths among women [4]. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 8 many of those deaths may be prevented through effective tobacco control, vaccination, and screening activities. there are varieties of effective cancer control measures available to countries of all resource levels [4]. many of those measures are extremely cost-effective given the lives saved for the cost of the intervention, especially within the case of vaccination. to stop cancer within the future, countries must prioritize policies to cut back known cancer risk factors and make prevention accessible to any or all. for those that have cancer today, effective treatments and palliative care are also needed. additionally, to those needs, cancer surveillance and research for the prevention and treatment are indispensable for the setting of cancer control priorities and for determining the foremost effective interventions and coverings in an exceedingly given context [4]. for lmics, all of those activities may require support and commitment from the worldwide community. while malignant diseases are holding and exhaustive and comprehensive databases that were collected from extensive surveys and gathering centers from national and international organizations, it is the interpretation of the results that would derive useful conclusions and insight through record’s pattern [5, 6]. the application of statistical process control (spc) methodologies was historically started at the end of the first quarter of the 20 th century in the industrial field the monitoring, control and improvement of the manufacturing processes as well as product quality by walter a. shewhart at bell laboratories [7, 8]. in addition to the wide industrial applications, spc techniques have been applied even in the improvement of service fields. moreover, spc techniques were found to be useful in other non-industrial fields for assessment, evaluation and investigation of specific inspection properties or events [9, 10]. some interesting fields of implementation include but not limited to environmental monitoring (em), microbiological water quality, surgical site infection, epidemiological diseases and outbreaks [11-15]. the previous studies embraced control charts, pareto diagrams, box-and-whisker plots, distribution identification, correlation analysis and fitted line curves, in addition to other conventional analysis tools such as descriptive statistics. while epidemiological diseases constitute a continuous threat that strike humanity, it would be useful to develop a quantitative descriptive tool that describes the progression and behavior of these diseases. one of the important indicators that could provide insight into cancer progression is the mortality ratio of the patients that could be found in the database of national and international health organizations. the application of statistical tools and process control methodologies was sought as a unique metric means in the evaluation and comparison of the epidemiological status that could support in the decision-making and cancer management and control. accordingly, the present analysis might reveal new horizons for researchers to a simple, fast and useful way to study other diseases, outbreaks and pandemics. materials and methods spc perspective of long-term global trend of cancer in women cancer mortality rates records of different countries were obtained as a downloadable database file from the websites: https://www.cancer.gov/ and https://www.who.int/ [16, 17] and women data were extracted from the excel file by filtering and arranged chronologically (table s1). three different computer programs were used in data processing combined as statistical software platform viz prism graphpad v6.01, minitab v17.1.0 and excel built-in xlstat v2014.05.03 [18-21]. the prism graphpad v6.01 commercial scientific software was used for the preliminary data description followed by defining the significance estimation of the change of the yearly mortality rates with time. the excel built-in xlstat v2014.05.03, which is a statistical suite add-in, was used for drawing of gaussian mixture model (gmm), which is a probabilistic model to spot typically disseminated subpopulations inside a general populace. blend models, as a rule, do not require prior knowledge of datasets subordination to the subgroup, permitting the model to become familiar with the subpopulations spontaneously [22]. thus, it will be important in this case for viewing the overall data-clustering pattern for the record from 1960 to 2017. on the other hand, minitab as a statistical tool package was assigned for fitting line modeling, box plot creation, histogram drawing and data trending using process behavior chart [23]. two-dimensional data segregation was done based on gmm and individual countries found in the record to identify clustering pattern of data. construction of the mean (x-bar)-variation ( -s) trending chart was executed according to the distribution identification scheme in minitab. shewhart charts show mean of the death rate and the standard deviation along with the event window i.e. upper control limit (ucl) and lower control limit (lcl). results and discussion descriptive statistics and correlation output a preliminary examination of the database for the basic statistical analysis yielded a result in table 1 showing the coinciding countries with the annual values. data are mildly positive or right-skewed [24]. failure to comply with normality distribution was followed by the screening of the closest possible spreading pattern that might fit the record. a weibull distribution hypothesis could not be rejected as a suitable fit for the raw dataset based on the andersondarling test (ad). https://en.wikipedia.org/wiki/walter_a._shewhart https://en.wikipedia.org/wiki/bell_laboratories rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 8 table 1: descriptive statistics of the cancer mortality rates showing the number of deaths per 100000 individuals at ci * 95% and the corresponding countries numerically to the parameters value using prism graphpad v6.01 column statistics mortality rates (per 100000) concerned department minimum 93.30 tur 2009 25% percentile 159.1 zaf 2005 median 183.8 aus 1963, fin 1984, pol 1985 75% percentile 205.4 gbr 1962, fin 1964, chl 1991, nld 1997 maximum 286.4 isl 1960 10% percentile 140.9 grc 1983 90% percentile 227.4 lux 1984, cze 1996 arithmetic mean 183.2 swe 1987 std. deviation 32.85 na std. error of mean 0.7461 na lower 95% ci of mean 181.8 aus 1975, fin 1980, cri 1990 upper 95% ci of mean 184.7 swe 1984, svn 1988, nor 2003, nzl 2010, cze 2012, gbr 2016 lower 95% ci of median 181.8 aus 1975 upper 95% ci of median 185.4 swe 1983 and 1988, ita 1990 shapiro-wilk normality test w 0.9973 p value 0.0017 na data passing normality test (alpha=0.05) no coefficient of variation 17.93% geometric mean 180.2 svk 2006, isl 2010 lower 95% ci of geo. mean 178.7 pol 2011 upper 95% ci of geo. mean 181.7 est 1990 method of outlier detection: rout ε (q = 10.00%) no aberrant record na skewness 0.04484 na kurtosis -0.3441 na *ci = confidence interval ; na = not applicable; ε robust regression and outlier removal no aberrant values could be detected through the overall record. thus, mortality rates showed apparent concatenation in the values with a range of 193.1 deaths/100000 cases with half and four-fifths of data record resides within a range of 46.3 and 86.5 deaths/100000 cases, respectively, suggesting extensive tailing. the mean and the median were almost coincided with the geometric mean very close to the average confirming that there is no extreme value(s) distant from the group collectively [25]. however, the arithmetic mean is slightly greater than that of the geometric mean as expected for the basic rule in mathematics [26]. this gap is reflected similarly on the confidence intervals (cis) which showed the corresponding shift too. the relative standard deviation (rsd) showed significant dispersion of data around the mean of about 18%. in addition, kurtosis value suggests data broadening with shoulders that is a variation from the normal bellshaped spreading if the normal distribution is expected, a sign of possible mixed intervening patterns [24]. this would probably be the result of the impact of annual and regional variations that was reflected in the total record of women cancer mortality. table 2 demonstrates the moderately negative correlation between the women mortality rates and the time in years [27, 28]. accordingly, there was an improvement of women survivability with time passing, which might be partially attributed to the development of effective detection and control measures for the containment of cancer epidemiology [29]. dataset clustering approaches: a multidimensional analysis data stratification using gmm analysis estimated two possible mixed bell-shaped spreading, indicating a global overlapping distribution of mortality rates among the studied nations in figure 1. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 8 table 2: non-parametric correlation of the mortality rates from malignant diseases versus time (in years) at ci 95% with two-tailed p-value using prism graphpad v6.01 spearman (r) time versus death rate interpretation r -0.4110 95% ci * -0.4484 to -0.3721 p (two-tailed) < 0.0001 p value summary **** true or asymptotic p value calculation ** asymptotic significance at α = 0.05 yes number of paired xy 1938 *ci = confidence interval; **refer to the calculation method of pvalue using either the exact or approximation to the actual distribution; ****p ≤ 0.0001 cumulative distribution function (cdf) and q-q plots showed the convenience of this assumption. the first and the second distributions were called (φ phi) and (ψ psi) with contribution proportions 0.57 and 0.43, respectively (figure 1). however, there was no clustering tendency that could show any heterogeneity according to the normalized entropy criterion (nec), which was greater than one with the selected the integrated completed likelihood (icl) criterion with the standard algorithm (em) expectation maximization [30]. the means and covariances of φ and ψ for malignancy mortality rates in women were 222.7, 241.0 and 1892.4, 909.9, respectively. figure 1: dataset segregation, based on gaussian mixture model (gmm) approach, showing two mixed distribution pattern with low (green) and high (pink) rates of mortality from malignancy, generated using xlstat v2014.05.03. the geographically based data segregation shows that, ascendingly, turkey, the republic of korea, brazil, mexico and greece demonstrated the lowest average mortality rate values could be demonstrated in figure 2. while countries with the ascending order from the lowest order of survivability to the highest means were denmark, hungary, ireland, czechia, gbr and netherlands (figure 2). figure 2: dataset segregation, based on who geographical distribution by countries shown as box plot, indicating the means, data spreading and the aberrant annual mortality rates records generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 5 of 8 the highest mortality rates from cancer occurred primarily in the eur region with the domination of economically rich nations suggesting that the overall survivability of women from cancer depends on other factors that seemed to be important to be controlled in addition to the improvement of the healthcare quality. this assumption might be partially in agreement with previous analysis provided by some researchers that had linked the prevalence of some types of cancer in the high-income countries with some adverse lifestyles such as smoking, obesity and alcohol consumption [31, 32]. nevertheless, it should be noted that beforehand rigorous control, awareness and actions taken about cancer detection and diagnosis associated with controlled monitoring might be one of the important reasons for the comprehensiveness of data records of high-income and developed nations if compared with those from the relatively lower income-countries. this hypothesis may be a factor that contributed to a possible underestimation of the actual overall estimation of the mortality rates in the developing and low-income countries. the time-based data segregation shows that, europe who region was the most versatile with divergent mortality rates between nations, as it is rich with country data, in contrast to the african area that includes only south africa. this could also be seen on the extend of the outlier figures (denoted by asterisks) above and below the box plot graphs in figure 3. south africa data record only started in 1993, which is later than the other three regions by 33 years and reaching its peak in 1999 (figure 3). the annual spreading of death cases rates for amr and wpr was less than that of eur. however, the distribution of wpr values was generally more compact than that of amr, where greater pervasion could be observed between annual records. the general profile can be seen in figure 3 with the yearly steady and almost regular decline in death rates started with amr in late 1980s, eur and wpr at almost 1995. figure 3: dataset segregation based on who geographical distribution by time order shown as box plot, indicating data spreading and the aberrant mortality rates records generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 6 of 8 fitted line of yearly average death rates from cancer for the affected women populations versus time dataset pattern showed a steady decline in the global mortality rates from cancer after the initial high plateau that could be demonstrated by the quadratic relation with significant correlation (figure 4). the regression equation was expressed through polynomial regression analysis of average mortality rates from cancer for women versus the time (in years). log10 (y) = 10245 + 6222 log10 (t)944.5 log10 (t)2…eq.(1) where: y and t are the years and death rates from cancer worldwide were recorded for women, respectively, with s = 0.00577903, r-sq and r-sq(adj) = 97.0%. figure 4 illustrates this relation between death rates and years graphically. figure 4: polynomial regression analysis graph showing quadratic equation modeling, confidence interval (ci) and prediction interval (pi) along with r 2 and the standard error of the regression (s), generated using minitab v17.1.0. trending of global women survivability from malignancy using process-behavior chart in figure 5, histogram showed a pattern of dual hump that could be explained from gmm model. boxplot graph is indicative of the pattern for the studied who regions (figure 5). afr demonstrated the lowest mean and spreading due to the inclusion of zaf only for relatively a short period from the region, in contrast of eur with upper and lower aberrant records from some countries in the european region from island (1960) and turkey (2009, 2010 and 2011). the survivability rate in wpr is better that amr. analysis of the distribution fitting showed the convenience of the weibull distribution based on the probability plot examination as could be observed in figure 5. plots the method mean (xbar chart) and method standard deviation (s chart) over time for variable information in subgroups (figure 5). this dual type of control chart is extensively used to observe the steadiness of procedures in lots of fields. both the xbar and s charts are displayed together due to the need to interpret each chart to decide if the trend is stable [32]. the procedure variation in s chart was almost stable within the control limits and tendency toward lower annual variation since 1979. the xbar chart in figure 5 showed initially intermittent excursions in the first few years followed by a successive shift in the annual mortality means leading to a declining curve with the improvement of survivability over time until exceeding the lower bound, which is a desirable outcome [33]. the last record of 2017 was incomplete during data registration that may explain the apparently sudden rise in mortality ratio due to the inclusion of few countries with high values in the process-behavior chart of figure 5. figure 5: spc analysis tools, (control chart, histogram and box plot) of the global cancer mortality rates in women with weibull probability fitting plot, showing the overall trend of the annual death rates as -s control chart and the pattern of who regions as box and whisker plot, generated using minitab v17.1.0. rashed and eissa 2020 long-term quantitative assessment of women survivability from cancer highlights in bioscience july 2020| volume 3 http://bioscience.highlightsin.org/ page 7 of 8 conclusion spc methodologies could provide a unique insight into the characteristics and patterns of epidemiological diseases. quantitative assessment of mortality and/or morbidity levels may be a useful indispensable tool in management and future decision-making. further studies are crucial to access the absolute number of deaths since using the ratio record alone may not reflect the real progression of the disease. for instance, the annual increase in the number of cancer patients may counterbalance the efforts to control deaths and the actual yearly mortality number could be a rising challenge to the efforts of cancer healthcare sector due to exacerbation of cancer epidemiology. in addition, the accuracy of the investigation and analysis is largely dependent on the impartialness and comprehensiveness of the database. however, cancer researchers could adopt similar techniques to track different types of malignancies in their countries over time to access the changes in the epidemiological pattern and the effectiveness of the control measures. the application of spc methodologies would be encouraged to be projected in future studies of other diseases such as coronavirus disease (covid-19) global cases. supplementary files the supplementary material for this article can be found online at: https://doi.org/10.36462/h.biosci.20208 supplementary table s1: 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https://sciencing.com/differences-arithmetic-geometric-mean-6009565.html https://sciencing.com/differences-arithmetic-geometric-mean-6009565.html https://www.sciencedaily.com/ highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202108 research article open access 1 electronic engineering department, federal university of rio de janeiro, rio de janeiro, brazil. 2 department of electrical engineering, hakim sabzevari university, sabzevar, iran. 3 city university health sciences center, federal university of rio de janeiro, biochemistry, rio de janeiro, brazil. 4 department of electrical engineering, azad university, sabzevar, iran. contacts of authors * to whom correspondence should be addressed: h.r.khezri2@gmail.com received: march 23, 2021 accepted: june 15, 2021 published: august 3, 2021 citation: khezri h, farzaneh m, ghasemishahrestani z, moghadam ap . ann-based diagnosis method for skin cancers using dermoscopic images. 2021 aug 3;4:bs202108 copyright: © 2021 khezri et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. ann-based diagnosis method for skin cancers using dermoscopic images hamidreza khezri*1 >< , mojtaba farzaneh2 >< , zeinab ghasemishahrestani3 ><, ali pakizeh moghadam4 >< abstract melanoma is one of the most dangerous skin cancers in the world. it accounts for 55% of all deaths associated with skin cancer. researchers believe that skin cancer increases the risk of other cancers if not diagnosed early. therefore, prompt and timely diagnosis of this disease is very important for the successful treatment of the patient. this system can detect melanoma lethal carcinoma from other skin lesions without the need for surgery, with a low cost, accuracy of about 98.88% and specificity 99%. in this article, a new, intelligent and accurate software (delphi) system has been used to diagnose melanoma skin cancer. to detect malignant melanoma, the abcdt rule, asymmetry (a), boundary (b), color (c), diameter (d) and textural variation (t) of the lesion are calculated and finally, an artificial neural network (ann) is used to obtain an accurate result. the ann with multi-layer perceptron (mlp) contains the five extraction characteristics (abcdt) of lesions is used as inputs, two hidden layers, and two outputs. very good results were obtained using this method. it was observed that for a dataset of 180 dermoscopic lesion images including 80 malignant melanomas, 20 benign melanomas and 80 nevus lesions. due to its automatic recognition and ability to be installed on a computer, this system can be very useful for dermatologists as well as the general public. keywords: melanoma skin cancer, abcdt rule, feature extraction, artificial neural network introduction unfortunately, skin cancer has become very popular these days. melanoma is caused by genetic mutations in the pigment-producing cells called melanocytes [1]. melanoma affects mostly both sexes of the caucasian population [2], and the prognosis of the disease becomes very poor at the metastatic stage [3-4]. there are no effective treatments for metastatic melanoma [5], and has grown rapidly over the past 30 years [6]. according to clinical definitions, malignant lesions are not regularly [6]. figure figure 1 shows the symmetric and asymmetric lesions. diagnosis of melanoma in the early stages of the disease can certainly prevent the death of patients. usually for two reasons skin lesions turn from benign to malignant: first, lack of attention to the skin lesions on body surface. second, high similarity of skin lesions features and inaccessibility to a dermatologist. for example, figure 2 shows two very similar skin lesions, malignant melanoma and clark’s nevus, which is a benign skin lesion. melanoma is the most common skin disease that can lead to death [7], which often begins with malignant pigment cell tumors that cause more than 70% of deaths in patients with skin cancer [8-9]. it should be noted that if skin cancer is not diagnosed in the early stages, it can affect different parts of the body, including the liver, bones, lungs and brain, and makes the treatment process very difficult and complicated. the need for an automatic and accurate device for reduce unnecessary biopsy and rapid detection is quite clear. therefore, a method for early detection of melanoma is very useful and valuable [10-11] so that dermatologists can use this system to diagnose skin lesions with high accuracy. in fact, melanoma is evaluated by clinical imaging. dermoscopy is often used to assess melanoma lesions, a non-invasive type of image analysis. a new approach is shown here, which examines the skin lesion image by a trained neural network to analyze if it is benign or malignant. the article is arranged as follows. highlights in bioscience page 1 of 7 august 2021|volume 4 https://doi.org/10.36462/h.biosci.202108 https://creativecommons.org/licenses/by/4.0/ h.r.khezri2@gmail.com https://orcid.org/0000-0002-5839-3690 mojtaba.farzaneh@gmail.com https://orcid.org/0000-0002-6037-6486 farzaneh.ghasemi.sha@gmail.com https://orcid.org/0000-0002-2482-4296 a.pakizehmoghadam@gmail.com http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 1. a:symmetric and b: asymmetric melanoma lesions. figure 2. clark nevus. the second section describes previous work on melanoma analysis in skin images. in the third section, the new contribution to this article describes the analysis, feature extraction, classification, ann, details, tests and evaluations. section iv presents the conclusions. as a basic step towards computer-aided skin cancers, automatic diagnosis, and image analysis have often been studied in the literature [12-19]. in the last few years, many studies have been done to detect melanoma from skin images with an accuracy of 70% to 96%. telemedicine techniques have been studied as a source for the diagnosis of skin lesions. compared to physicians in-person diagnoses (face to face) and telemedicine diagnoses (remote detection), tests on skin diseases have shown that the use of telecommunications technology diagnosis (teledermatology) is more effective and safer. these techniques include the benefits of easy access, low cost, and quick and accurate access to treatment results [20-22]. melanocytic cutaneous lesions have been reported to be the deadliest among the three skin cancer outbreaks and the second most common among adults aged 15 to 29 years [2]. melanoma is less common in asia, africa and latin america than in australia, europe, north america and new zealand. melanomas sometimes change in appearance, including changes in size, irregular edges, and discoloration, itching or fracture of the skin [23]. in fact, melanoma can rarely occur in the mouth, intestines, or eyes, but is most commonly found on the skin. it is common in men and women in the back and legs, respectively [24]. the automatic detection of asymmetry in digital images was proposed by the stolz technique based on abcd rule [25]. a study on the asymmetry using imaging techniques to identify melanoma skin lesions was presented by ravichandran et al [26]. to date, many researchers are working on image processing, visual techniques, and various melanoma parameters such as size, shape, asymmetry, border, color, and diameter to detect skin cancer [27-31]. one of the known methods is the abcd rule. the algorithm for detection is divided into four steps: asymmetry, border, color, and diameter. abcd is a fast learning, calculation and a reliable way to diagnose melanoma [32-34]. lesion irregularity, borders, colors and diameters can be analyzed and calculated by dividing the image of the lesion into sub-images and extracting the properties of each image [35]. finally, melanoma could be detected by a simple threshold for the values obtained from the extracted features (lesion irregularity, borders, colors and diameter) [36]. these features are used as inputs to the first layer of the ann [37-38]. we’ve developed the novel abcdt rule by improve the abcd, for automatic diagnosis of skin cancer with greater accuracy and precision. in other words, in the current study, the extraction of features is done based on the abcdt rule in dermoscopy. materials and methods this article aims to develop a new, intelligent and accurate software system for skin cancer diagnosis using neural network and abcdt rule. the input of the device is images of skin lesions. this system with pre-processing, abcdt rule, and separation, extract appropriate features from the image. to get the total dermoscopic score (tds), for each of the "asymmetries, boundary, colors, diameters and texture changes", a coefficient is determined by which the tds can be calculated. in other words, to obtain tds (table 1, the score of each "abcdt" is multiplied by a specific weight factor. finally, ushighlights in bioscience page 2 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images ing nn as a smart medical decision-making system based on tds, the type of lesion is determined to be melanoma or benign. in other words, an nn has been used to implement the new automated classification of melanocytic lesions. table 1. the proposed diagnostic criteria. diagnostic criteria score weight factor asymmetric (a) 0-5 1.3 border (b) 0-8 0.5 color variation (c) 1-6 1 diameter (d) 0-1 0.1 textural variation (t) 1-10 0.9 in this work, briefly, after the detection and elimination of noise and hair on the image, abcdt rule, bayes learning algorithm, and the neural network method (feed forward back propagation) were used to detect the lesion is classified as benign or malignant. as shown in figures 3 and 3, the architecture of the skin cancer smart system used in this study consists of the five steps of pre-processing, segmentation, feature extraction, classification and diagnosis as follows: • pre-processing: it involves filtering and contrast enhancement techniques using the retinex algorithm. resize pictures, to analyze and compare data with the bank, all images are transformed into the same size. this size is 450×350 selected. • blurring and segmentation: the purpose of blurring is to reduce noise. if we use edge detection algorithms for highresolution images, we will find many results that we are not interested in. conversely, if we blur the images too much, we will lose data. so, we have to find the amount of blurring we want to use without destroying the desired edges. there are various techniques to achieve blurred effects, but gaussian blur is used with a factor of 2 to remove the noise and the hairs on the skin. so, first, the image is converted to binary format. using filters, detect and eliminate hair and noise from the image. then, the lesion image is completely separated from the background. in other words, the exact location of the lesion on the image is determined by calculating the threshold and statistical characteristics. • feature extraction: abcdt rule examines the characteristics of a lesion. these properties include asymmetry, border, color, diameter, and textural variation. this rule is a development of the known abcd rule [39], commonly used to diagnose melanoma from images. the extracted properties are fed to the first layer of the nn. properties used to characterize the asymmetry of the lesion the asymmetric feature of the lesion is one of the important features in diagnosis. natural moles are usually symmetrical. asymmetry is usually calculated in two ways: entropy and bifold. to calculate the asymmetry score, each lesion is examined by two 90-degree axes, and the ann determines its score. if the lesion is properly symmetrical on both axes, this score is 0, and if it is only on one axis, it will be 2.5. in case of asymmetry in both axes, the score is 5. finally, the asymmetry score must be multiplied by 1.3 as the weight factor. features used for irregular characterization at the lesion border uneven or irregularly shaped margins increase the likelihood of some kind of skin lesion. to calculate boundary irregularities, the lesion is divided into eight sections. if the entire border of the lesion has a severe incision, it is given a maximum score. otherwise the minimum score of 0 is given. the minimum and maximum score of b are defined 0 and 8, respectively with the 0.5 weight factor. features used to characterize lesion color variation color properties are calculated between six colors, and each color represents 1 point. properties used for diameter according to our information, if the lesion diameter is larger than 0.6 mm, the risk of cancer is higher. diameter with a weight coefficient of 0.1 is measured by converting the total number of pixels in the largest diameter to millimeters (mm). characteristics used to characterize the textural variation of the lesion since healthy skin is reddish, and skin lesions have more textural variation and lower pixel intensity than healthy areas of the skin. for this purpose, first, a low pass filter was used to normalize the color and then extracted the correct features [40]. the textural variation values are considered from 1 to 10 with a weight factor of 0.9. classification the different features extracted from the lesion surface have different weights. the weight of each group was drawn based on the experience of dermatologists. table 1 shows the importance of each group. the total dermoscopy score (tds) can be calculated using eq. (1). t ds = 1.3 × a + 0.5 × b + 1 ×c + 0.1 × d + 0.9 × t (1) where: a, b, c, d, and t scores are for the asymmetry feature, the border irregularity, the color feature, the diameter size feature, and the textural variation, respectively. highlights in bioscience page 3 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 3. a flowchart illustrating the proposed machine learning system for detecting skin cancers using dermoscopic images. figure 4. clark nevus. diagnosis if individual scores of asymmetry, boundary, color, diameter, and textural variation are multiplied by weightfactor of 1.3, 0.5, 1, 0.1, and 0.9, respectively, a precise distinction can be made between benign and malignant melanocytic lesions. the tds greater than 5.40 means a cancerous lesion, otherwise, it is benign. results and discussion all of these steps and the final decision (benign or malignant) are performed by a trained neural network. various classifications have been performed by researchers for the anns [43-46]. in this work, the ann with a multi-layer perceptron (mlp) utilizes a supervised learning technique and includes features extracted as inputs to the input layer with two hidden layers containing 10 and 7 neurons for each layer (figure 5). the mlp uses a region-oriented hybrid algorithm, a method called elliptical symmetry to determine asymmetry, a gaussian smoothing to measure boundary irregularities, and a threshold method for the lesion segment. therefore, the system designed to diagnose melanoma uses five features of the lesion. the images used in this work were taken from the international skin imaging collaboration (isic) [41]. out of this dataset, 180 dermoscopic lesion images including 80 malignant melanomas, 20 benign melanomas and 80 nevus lesions were extracted and preprocessed for this research. in several studies [42-44] they used abcd rule to detect and analyze pictures, whereas in our study we added one more factor (abcdt) to increase the efficiency and get the better result. we found out the t factor has a huge impact on increasing accuracy. highlights in bioscience page 4 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic images figure 5. the neural network diagram of the proposed mlp model. given the weight factor (0.9) and values of the (0-10) change intended for t, the number obtained from t has a great effect on the result, and this is the most important difference between this work and other similar tasks. according to the accuracy (acc) and specificity (spec) obtained and compared with pennisi et al. [47], fan et al. [48], jahanifar et al. [49] and sreelatha et al. [50] can be claimed that the average performance of the technique used in this study is better than previous techniques (table 2). table 2. the average performance evaluation metrics (%). algorithm acc spec pennisi [47] 89.40 97.10 fan [48] 93.60 jahanifar [49] 97.90 98.20 sreelatha [50] 98.64 99.22 proposed mlp 98.88 99.00 the proposed method for the diagnosis of melanoma skin cancer by abcdt method revealed 98.88% accuracy and specificity 99%. this approach is safe, accessible, effective, noninvasive and based on the principles of telemedicine with high accuracy and reasonable price. with this software system, people can make an early diagnosis of their skin lesions without referring to a physician and specialists can use it as an intelligent, fast and accurate assistant. in summary, the abcdt rule, bayes learning algorithm, and neural network method were used to detect the type of carcinogenic or non-cancerous lesions. according to tds, it is a clear fact that the three features of asymmetry, color, and textural variation of the lesion are crucial in the diagnosis of melanoma from benign lesions. a tds value above 5.40 indicates melanoma. in abcdt method, all steps perform by a fully automated neural network. references 1. tolleson wh. human melanocyte biology, toxicology, and pathology. journal of environmental science and health part c. 2005 jul 1;23(2):105-61. 2. ferlay j, soerjomataram i, dikshit r, eser s, mathers c, rebelo m, parkin dm, forman d, bray f. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. international journal of cancer. 2015 mar 1;136(5):e359-86. 3. bombelli fb, webster ca, moncrieffm, sherwood v. the scope of nanoparticle therapies for future metastatic melanoma treatment. the lancet oncology. 2014 jan 1; 15(1):e22-32. highlights in bioscience page 5 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ khezri et al., 2021 ann-based diagnosis method for skin cancers using dermoscopic 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math. methods biomed. image anal, 2012. 39. aswin rb, jaleel ja, salim s. implementation of ann classifier using matlab for skin cancer detection. international journal of computer science and mobile computing. 2013 dec;1002:8794. 40. messadi m, cherifi h, bessaid a. segmentation and abcd rule extraction for skin tumors classification. arxiv preprint arxiv :2106.04372. 2021 jun 8. 41. santiago-montero r, asael d, hernandez g. border and asymmetry measuring of skin lesion for diagnostic of melanoma using a perimeter ratio. asian j comput sci and inf technol. 2016;6(2). 42. jaleel ja, salim s, aswin rb. artificial neural network based detection of skin cancer. international journal of advanced research in electrical, electronics and instrumentation engineering. 2012 sep;1(3). 43. ahmed k, jesmin t, rahman mz. early prevention and detection of skin cancer risk using data mining. international journal of computer applications. 2013 jan 1;62(4). 44. thirumavalavann s, jayaraman s. ann based computer aided diagnosis and classification of skin cancers. power and computing technologies. 2017;12(4).pp1137-1142. 45. alam fi, faruqui ru. optimized calculations of haralick texture features. european journal of scientific research. 2011 mar 1;50(4):543-53. 46. codella nc, gutman d, celebi me, helba b, marchetti ma, dusza sw, kalloo a, liopyris k, mishra n, kittler h, halpern a. skin lesion analysis toward melanoma detection: a challenge at the 2017 international symposium on biomedical imaging (isbi), hosted by the international skin imaging collaboration (isic). in2018 ieee 15th international symposium on biomedical imaging (isbi 2018) 2018 apr 4 (pp. 168-172). ieee. 47. pennisi a, bloisi dd, nardi d, giampetruzzi ar, mondino c, facchiano a. skin lesion image segmentation using delaunay triangulation for melanoma detection. computerized medical imaging and graphics. 2016 sep 1;52:89-103. 48. fan h, xie f, li y, jiang z, liu j. automatic segmentation of dermoscopy images using saliency combined with otsu threshold. computers in biology and medicine. 2017 jun 1;85:75-85. 49. jahanifar m, tajeddin nz, asl bm, gooya a. supervised saliency map driven segmentation of lesions in dermoscopic images. ieee journal of biomedical and health informatics. 2018 may 22; 23(2):509-18. 50. sreelatha t, subramanyam mv, prasad mg. early detection of skin cancer using melanoma segmentation technique. journal of medical systems. 2019 jul;43(7):1-7. highlights in bioscience page 7 of 7 august 2021|volume 4 http://bioscience.highlightsin.org/ abstract introduction materials and methods properties used to characterize the asymmetry of the lesion features used for irregular characterization at the lesion border features used to characterize lesion color variation properties used for diameter characteristics used to characterize the textural variation of the lesion classification diagnosis results and discussion references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202103 research article open access 1 natubhai v. patel college of pure and applied sciences , sardar patel university, vallabh vidyanagar, anand, india. contacts of authors * to whom correspondence should be addressed: yachana jha received: august 23, 2020 accepted: january 15, 2021 published: january 25, 2021 citation: jha y . macrophytes as a potential tool for crop production by providing nutrient as well as protection against common phyto pathogen. 2021 jan 25;4:bs202103 copyright: © 2021 jha. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. macrophytes as a potential tool for crop production by providing nutrient as well as protection against common phyto pathogen yachana jha*1 abstract applications of synthetic chemical fertilizers and pesticides lead to several environmental hazards, causing damages to entire ecosystem. to reduce damage caused by such chemical inputs in agriculture and environment required a serious attention for replacement of chemicals input with eco-friendly options. in this study decaying macrophytes were selected as an option for organic agriculture, by analyzing its ability to provide important mineral nutrient to the maize crop grown in low nutrient soil as well as for providing resistant towards many common phyto-pathogens to enhance yield. the results of the study showed that decaying macrophytes have high concentration of stored important mineral nutrient in their body mass, which get released in the soil during its decay and to be used by the maize plant. the decaying macrophytes leaf extract have considerable amount of phenolic and flavonoids also having antimicrobial activity. the antimicrobial activity of the leaf extract has been analyzed against the common phyto-pathogen pseudomonas aeruginosa , and s. aureus by agar disc method and the formation of clear zone indicate its potential as bio-control agent. so under intensive agricultural practices, application of such biological waste is of particular importance for enhancing soil fertility without chemical input, to ensure sustainable agriculture. keywords: macrophytes, phytochemicals, phenolic, flavonoids, antimicrobial activity, mineral nutrient. introduction aquatic plants are adapted to live in aquatic environments and are referred to as hydrophytes or macrophytes. these plants require special adaptations for living and can only grow in water or soil that is perfectly saturated with water. aquatic plants generate one of the most productive ecosystems of the world and are essential life supporting systems, which provide a wide array of benefits to the aquatic life and human kind. however, these ecosystems are today’s fast declining and rapidly deteriorating ecosystems in many parts of the world. ignoring the economic and ecological significance of aquatic plants is often regarded menace [1]. due to continuous pollution of water bodies and urbanization resulted in eutrophication, which disturbed the aquatic ecosystem. so to maintain the aquatic ecosystem, cleaning of water bodies and dumping of macrophytes is a global problem. global climate change causes a series of extremities like frost, salinity or drought, which adversely affect the plant nutrient dynamics as well as world food requirement. the availability of nutrients in the soil and its acquisition, assimilation, distribution within the plants or in the crop fields are directly disturbed by climatic stress factors [2]. low soil nutrient has deleterious effect on nutrient dynamics within the plant root as well as in shoot, including transportation of nutrient to reproductive structures via xylem and phloem, or mobilization of nutrient from senescing leaves. water fluxes, assimilatory activities, and the redistribution of inorganic nutrients are also affected by environmental stress. macrophytes have the ability to accumulate large amount of water and nutrient in their leaves, can serve as a good option for the plant nutrient under stress/low soil nutrient condition. macrophyte decomposition is important for carbon and nutrient cycling in lake ecosystems or in the low nutrient soil [3]. highlights in bioscience page 1 of 5 january 2021|volume 4 https://doi.org/10.36462/h.biosci.202103 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production it act as a important source of essential nutrient like phosphorus, carbon, potassium, zinc and nitrogen in the form of ammonia etc., due to the ability of macrophyte plant to absorbs and accumulate these nutrients from soils or water in their body, which ultimately disposes during its decomposition in the ecosystem. such nutrient rich macrophytes plants can be used efficiently in organic agriculture/bio-composting that can be applied to obtain required mineral nutrient effectively and successfully for the proper growth of plant, results in increased plant growth and yield. as bio-composting is an economically and ecologically important necessity in resent agriculture, which in return increases the soil quality and crop yield. biological agents are nowadays drawing attention because of its increased organic efficiency in terms of physical and chemical characteristics of the soil [4]. in nature, plants species grow together and interact with each other by inhibiting or stimulating the growth and development through various interactions. a special form of competition among plants is known as allelopathy, also shown by aquatic plants. such interaction is a biological phenomenon in which an organism produces many bio-chemical compounds that influence the growth, and reproduction of other organisms. such interactions are primarily based on the synthesis and release of secondary metabolites, known as allelochemicals or allelotoxins [5]. such allelochemicals initiate a wide array of biochemical reactions and induce several biological changes, which inhibit the growth of target pathogenic organism, which is not possible solely by physical or chemical agents. the macrophytes also have ability to overcome stress arise due to multiple symbiotic or pathogenic interaction. the aim of the work is analyze the effect of decaying macrophytes on the plant nutrient status, growth and its ability for the production of antibiotics and related secondary metabolites, to protect host maize plant against the potential phyto-pathogens. materials and methods aquatic plants collection and preparation of samples macrophyte plants were collected from nearby lake and ponds from anand-tarapur district of gujarat. it was immediately packed in polyethylene bags to prevent the degradation of bioactive compounds of the collected plant samples and was brought to the laboratory for further study. the healthy and disease free plants were used for further study. the collected leaf samples were thoroughly washed with water to eradicate surface dust and foreign materials and used for sample preparation. the washed leaves were properly dried at room temperature for 7 days under shade and were ground into a powder with the help of grinder. estimation of mineral nutrient concentrations of soil, hydrophytes and maize leaves the physio-chemical property of the soil, hydrophytes and maize leaves were analyzed by saturated water extraction method in sicart lab. the leaves powder sample were digested for 6 hours in nitric-perchloric acid (5:3) and used to estimate the concentration of phosphorus by colorimetric. similarly the concentration of nitrogen was estimated after the kjeldahl digestion by colorimetry and 2g of plant material were digested in mixture of sulphuric acid, nitric acid and perchloric acid (tri-acid) in the ratio of 9:3:1 and were used to determine the concentration of n, p, k, zn and ca. the aliquots of digested filtered plant material were analyzed on digital flame photometry by using specific filter. preparation of leaf extract the leaf extract was prepared by soaking 10g of leaves material powder in 50 ml of distilled water for 24 hours under shaking condition at 130-140 rpm at room temperature. after that the extract was filtered from eight layers of muslin cloth and filtrate was collected in petri-dish. the obtained filtered extracts were dried at room temperature in petri-dish, and was scraped, transferred to tubes for further use. similarly, plant material was re-extracted with 50 ml of ethyl acetate, and methanol also. finally obtained extracts were dried under reduced pressure and were stored in a refrigerator at 4 °c until use. qualitative analysis of phyto-chemical screening the phyto-chemical tests to detect the presence of alkaloid, saponins, quinones, steroids, phenols, flavonoide, tannins, coumarines and reducteurs compound were performed. the visual observations like formation of a precipitate or change in color after the addition of specific reagents were used to determine the presence of specific phyto-chemical in the plant extract. quantitative analysis on phytochemicals constituents total phenolic content the total phenolic content of leaf extracts was measured using colorimetric folin–ciocalteu method [6]. the leaf extract (1ml) was mixed with 5ml of distilled water and 250 μl of 1n folin-ciocalteau reagent. the mixture was covered and allowed it to stand for 3min at 25°c. in this mixture, 1ml of saturated na2co3 and 1ml of distilled water were added. the mixture was incubated for 1 hour at 25°c for color development and measured at 725 nm using spectrophotometer. standard graph was prepared by using different concentration of phenol crystals. total flavonoid content the colorimetric assay was used to estimate total flavonoid content in presence of aluminum chloride [7]. in 10 ml volumetric flask 1ml of leaf extract, 4 ml of distilled water and 0.3 ml of 5% nano2 was added. after proper mixing for 5 minutes 0.3 ml of 10% alcl3 and 2 ml of 1 m naoh was added in it. finally 3.7 ml of distilled water was added to made up total 10 ml volume. the obtained mixed solution was used estimate total flavonoid content by taking absorbance at 510 nm against blank and calculated by the help of standard graph of gallic acid. phyto-pathogenic microorganism two most common plant pathogenic microorganisms pseudomonas aeruginosa and s. aureus has been selected to analyze highlights in bioscience page 2 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production the antibacterial potential of the leaf extract. these isolates were obtained from a biotechnology research lab of biosciences department sp university. antimicrobial activity assay the agar disc diffusion method was used for this analysis. the culture suspension was spread all over plate by placing 0.1ml of culture at center of nutrient agar plate and spreading uniformly with sterile spreader. then, 6 mm in diameter filter paper discs containing the different types (water, ethyl acetate, and methanol) of leaf extract at a concentration of 100µl were used with distill water as control. and 4 filter paper discs were placed on the nutrient agar medium surface for the diffusion of leaf extract in the nutrient agar. the petri discs were then incubated in incubator at 37 °c for 24 h, plates were observed for zone of inhibition by measuring the colony diameter. a 100 % dmso was used as control. effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves for this analysis, the maize seedlings were planted in the field with low soil nutrient supplemented with decaying macrophytes without chemical fertilizer for 5 weeks. after 5 weeks plant sample were used determined the foliar phosphorus and nitrogen content by colorimeter, after digesting the plant sample with nitric-perchloric acid (5:3) for 6h kjeldahl digestion respectively. the foliar potassium, calcium and zinc content were estimated by digesting 1 g of plant sample with tri-acid mixture, which contain mixture of sulphuric acid, nitric acid and perchloric acid in the ratio of 9:3:1. the filtrates of digested material were used for the further analysis by using specific filter on digital flame photometry. effect of decaying macrophytes on plant growth parameters macrophytes were collected from the polluted water bodies and dumped at a site to initiate its decaying process. after 3 days of collection 10 cm thick bed was prepared on the crop field and was masked by 5 cm soil layer in a low soil nutrient field. after 10 days maize seed were sown in this field to analyze the effect of decaying macrophytes on plant growth parameters. then seedlings were collected carefully with root after 35 days of sowing to measure the shoot and root length and dry weight. the plants were dried in an oven at 80°c for 72 hrs for dry weight. statistical analysis all experiments described were performed three times independently, with measurements taken from three (for all other measurements) or ten (for dry weight measurements) different plants for each treatment in each of the three independent experiments. one-way analysis of variance (anova) test was used to analyze all data and mean (of three independent experiments) was compared at 5 % level of significance. results and discussion soil hardly ever supports crop production at their full potential yield due to lack of sufficient nutrient. so use of chemical fertilizers is a regular mechanism to achieve the yield potential. but low use of chemical fertilizers may be results in reduced yield and excessive application pollute the environment including water bodies, if applied without prior information about the nutrient content of the soil. at the same time pollution of water bodies resulted in overgrowth of macrophytes having harmful affect on water life, so such macrophytes are problem for water bodies. therefore use of such macrophytes as a source of nutrient in low nutrient soil status is the best option to overcome the problem, as it accumulates extra nutrient and water in their bodies. so in the present study, the macrophytes have been used as a source of supplement nutrient in the low nutrient soil in place of chemical fertilizer. so the initial major mineral nutrients (n, p, k, zn and ca) has been analyzed in the soil used for maize cultivation, control maize leaves and decaying macrophytes leaves and result of the study, showed significant variation in the initial mineral nutrient concentration in these three. mineral nutrient concentration was considerably high in decaying macrophyte as shown in table 1, in compare to control maize leaves as well as soil under consideration. the decomposition of macrophytes release higher concentration of phosphorus and nitrogen and its assimilation was also higher in plants as reported by lu et al. [8]. the synthesis of the phenolic acids, flavonoids, quinones, flavonols, phenols, coumarins and tannins like aromatic secondary metabolites in plant takes place to acquire resistant against the adverse environmental stress [9]. and in the present study, such compounds present in significantly higher amount in macrophytes leaves. phytochemical constituents of the macrophytes showed presence of different phytochemical constituents as alkaloid, saponins, cardiac glycosides, steroids, phenols and flavon -oides (table 2), but there was considerable variation in its constituents in fresh and decaying macrophytes. phenolics having broad spectrum biochemical activity like antimicrobial, antitumors, antioxidant and also has the ability to alter the expression of related gene in plant under stress. the majority of the antioxidant activities in plants have been carried out by phenolics, which is a largest group of phytochemical [10]. flavonoids are type of phenolic compounds naturally produced in the plant either in free or as glycosides have numerous biological functions like protein kinase inhibition, antioxidants, antimicrobial and mitochondrial adhesion inhibition etc.with the development of resistant towards the pesticides, there is requirement for ecologically sustainable natural alternatives or plants derivates as a choice to enhanced crop production. plant induced phyto-chemicals as well as plant extracts have antimicrobial properties against different phytopathogen including bacteria. plants interact with its changing surrounding environment efficiently due to production of secondary metabolites, which helps in acquiring tolerance towards harsh environment for sustainable adaptation. biotic stress in plants resulted in retarded highlights in bioscience page 3 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production table 1. concentration of minerals nutrient concentration in soil, hydrophytes and maize leaves (n=5). s.no sample n (mgkg-1) p (mgkg-1) k (mgkg-1) zn (mgkg-1) ca (mg kg-1) 1 soil 19.5±0.12 952.1±1.12 26246±0.11 285.4±1.27 11782±0.16 2 decaying hydrophytes 32.6±1.3 1372.4±2.11 58362±0.23 429.2±1.32 14467±1.37 3 maize leaves 27.9±1.21 1068.2±0.16 31583±0.21 363.3±2.01 12947±0.13 values are mean of three replications (p≤0.05; lsd test). . table 2. qualitative analysis of phytochemicals in fresh (a) and decaying (b) macrophytes. phytochemicals a b alkaloid + + saponins + + quinones steroids + phenols + + flavonoides + + tannins + coumarines + + reducteurs + +: present and : absent. plant growth with enhanced secondary metabolites production as phenolic compounds to defend biotic stress [11]. phenolic and flavonoids metabolites of plants are organic compounds having aromatic rings with one or many hydroxyl groups. in this study, the phytochemicals constitutes as total phenolic and total flavonoid contents were quantitatively analyzed in the macrophyte and result showed that, the total phenolics content was higher in decaying macrophyte, while total flavonoids content was higher in fresh macrophyte (table 3), and play major role in attaining maximum yield potential by providing nitrogenous product in normal state as well as in low soil nutrient. also presence of significant amount of phenolic and flavonoids provide protection against wide range of phytopathogen. table 3. quantitative analysis of phytochemicals in in fresh (a) and decaying (b) macrophytes. phytochemicals a b total phenolic content (mg/g of extract) 3.34±0.01 4.76±0.12 total flavonoid content (mg/g of extract) 6.83±0.02 5.37±0.02 for each parameter, values are mean of three replications (p≤ 0.05; lsd test). the use of synthetic pesticides is a quick and effective tool to manage plant diseases. but nonstop and arbitrary use of such synthetic pesticides in the crop field has several deleterious effects on human health and ecosystem, due to its incorporation in food chain and residual toxicity. use of synthetic pesticides attributes to long degradation periods, elevated acute toxicity, and gathering in the food chain. so the use of biological agents is an economically viable and safe option, to reduce the use of chemicals application. in the present study, the antibacterial activity of the decaying macrophytes showed significant effect on the both pseudomonas aeruginosa and s. aureus potential phytopathogenic bacteria observed during agar disc diffusion method, by formation of clear zone around the disc having different leave extract (figure 1). figure 1. effect of macrophytes plant extract (water, ethyl acetate, and methanol extract) with distill water as control on the bacterial pathogen on nutrient agar plate as zone of inhibition (zoi). where a for pseudomonas aeruginosa and b for s. aureus. plant growth is a complex process and the plant production depends on availability of sunlight, chemical energy, carbon source, mineral nutrients and water from the soil. many mineral nutrients in proper proportion are necessary for plant growth to attain its full strength yield. the plant having limited above ground resources like sunlight, and carbon source, then plant enhance the biomass allocation to the shoots and when there is limited below ground resources like mineral nutrients and water, then plant enhance the biomass allocation to the roots resulted in shunted growth according to functional balance theory[12]. so for proper plant growth the main nutrients required are potassium, phosphorus and nitrogen. these primary nutrients are accountable for limiting crop growth and among them nitrogen, is most intensively used element. nitrogen is often unavailable in the correct form to be utilized by the plant. in the present study, concentration of important mineral nutrients was analyzed in maize leaves grown in different condition. in this study, the nutrient content of the maize leaves has been assayed and result showed that concentration of mineral nutrient was significantly high in the maize leaves grown in low soil nutrient state supplemented with decaying macrophytes (table 4). so, use of decaying macrophytes as under soil bed has ability to enhance the nutrient content of maize for its the growth and development in low nutrient soil. such organic agriculture plays an important role in minimizing the use of inorganic fertilizers highlights in bioscience page 4 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience jha, 2021 macrophytes as a potential tool for crop production table 4. effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves (n=5). s.no sample n (mgkg-1) p (mgkg-1) k (mgkg-1) zn (mgkg-1) ca (mg kg-1) 1 maize in low nutrient soil 26.3±1.02 2383.1±1.02 73246±0.21 724.4±1.04 13782±1.43 2 maize in low nutrient soil + decaying macrophytes 32.6±0.11 3272.4±0.78 78362±0.64 729.2±2.11 14467±0.87 3 maize in low nutrient soil + chemical fertilizer 28.9±0.21 2668.2±0.65 76583±0.73 733.3±1.87 13947±1.23 values are mean of three replications (p≤ 0.05; lsd test). and consequently minimizes potential pollution and improves soil quality. it is important to develop new applications which are sustainable both agronomically and economically, as well as environmentally to produce safe food for human consumption. so the use of biological based compounds of plant extracts is considered successful and environment friendly option for sustainable agriculture. references 1. reitsema re, meire p and schoelynck j. the future of freshwater macrophytes in a changing world: dissolved organic carbon quantity and quality and its interactions with macrophytes. front. plant sci.2018, 9: 629. 2. etienne p, diquelou s, prudent m, salon c, maillard a, ourry a. macro and micronutrient storage in plants and their remobilization when facing scarcity: the case of drought. agriculture. 2018, 8: 14. 3. pravaiz ahmad l, bhardwaj ak, shah kw. macrophyteas powerful natural tools for for water quality improvement. research journal of botany. 2014, 9:24-30. 4. vidya s, girish l. water hyacinth as a green manure for organic farming. international journal of research in applied natural and social sciences 2014, 2(6):65-72. 5. zandalinas si, sales c, beltrán j, gómez-cadenas a, arbona v. activation of secondary metabolism in citrus plants is associated to sensitivity to combined drought and high temperatures. front plant sci. 2017, 7:1954. 6. salawu so, ola-salawu bb, ogundare ao, akindahunsi aa. antimicrobial activities of phenolic containing extracts of some tropical vegetables. afr j pharm pharmacol 2011, 5: 486-492. 7. mathur r, vijayvergia r. determination of total flavonoid and phenol content in mimusopselengi linn. int j pharm sci res. 2017, 8(12): 5282-85. 8. lu j, bunn se, burford ma. nutrient release and uptake by littoral macrophytes during water level fluctuations. sci total environ. 2018, 622-623:29-40. 9. jha y, subramanian rb, sahoo s. antifungal potential of fenugreek coriander, mint, spinach herbs extracts against aspergillus niger and pseudomonas aeruginosa phyto-pathogenic fungi. allelopathy journal 2014, 34 (2): 325-334. 10. okpuzar j, ogbunugafor h, kareem gk, igwo-ezikpe mn. in vitro investigation of antioxidant phenolics compounds in extract of sennealata. res j phytochem. 2009, 3:68–76. 11. compean kl, ynalvez ra. antimicrobial activity of the plant secondary metabolites; a review. j med plant res 2014, 8: 204213. 12. hendrik p , karl jn, peter b, reich j, oleksyn p, poot liesje m. biomass allocation to leaves, stems and roots: meta-analyses of inter specific variation and environmental control. new phytol. 2012, 193:30–50. highlights in bioscience page 5 of 5 january 2021|volume 4 http://highlightsin.org/index.php/bioscience abstract introduction materials and methods aquatic plants collection and preparation of samples estimation of mineral nutrient concentrations of soil, hydrophytes and maize leaves preparation of leaf extract qualitative analysis of phyto-chemical screening quantitative analysis on phytochemicals constituents phyto-pathogenic microorganism effect of decaying macrophytes on minerals concentration under low soil nutrient condition in maize leaves effect of decaying macrophytes on plant growth parameters statistical analysis results and discussion references highlights in bioscience; highlights in bioscience issn: 2682-4043 doi:10.36462/h.biosci.20194 highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 1 of 11 research article open access 1 mggm lab., agricultural genetic engineering researchinstitute, agricultural research center, egypt. 2 genetics department, faculty of agriculture, mansoura university. 3 faculty of pharmacy, october 6 university, giza, egypt. 4 department of biodiversity and crop improvement, international center for agriculture research in the dry areas (icarda), giza, egypt. * to whom correspondence should be addressed: smahmoud@ageri.sci.eg citation: alsamman a.m, mousa k.h., nassar a.e., shereif g.a., habib p.t. and ibrahim s.d. (2019). genome-wide identification and comprehensive study of chickpea anti-fungal genes. highlights in bioscience, volume 2. article id 20194, dio:10.36462/ h.biosci.20194 received: september 15, 2019 accepted: october 20, 2019 published: november 10, 2019 copyright: © 2019 alsamman et al. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests genome-wide identification and comprehensive study of anti-fungal genes in chickpea alsamman m. alsamman * 1 , khaled h. mousa 2 , ahmed e. nassar 2 , ghada a. shereif 3 , peter t. habib 4 and shafik d. ibrahim 1 abstract chickpea is an important crop that delivers nutritious food to the increasing global community and it will become increasingly popular as a result of climate change. our objective was to use comprehensive data analysis to locate and identify candidate genes for fungal disease resistance. we used a comprehensive bioinformatics pipeline of sequence alignment, phylogenetic analysis, protein chemical and physical properties assessment and domain structure classification. in order to study gene evolution and genetic diversity, we compared these genes with known anti-fungal genes in different species of plants. a total of 19721 protein sequences belonging to 187 plant species have been downloaded from public databases, including the entire chickpea genome. we have successfully identified 23 potential anti-fungal genes in 10 different chromosomes and genomic scaffolds using sequence alignment and gene annotation. ca2 and ca6 have the highest number of genes followed by ca3 and ca4. anti-fungal chickpea proteins have been identified as cysteine-rich (10), thaumatin (6), pathogenesis (4) and plasmodesmata (3) proteins. analysis of the chemical and physical correlation of anti-fungal proteins revealed a high correlation between different aspects of anti-fungal proteins. five different pattern patterns have been detected in the anti-fungal chickpea proteins identified, including domain families associated with fungal resistance. the maximum likelihood of phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as seen in their protein patterns / domains. keywords: chickpea, fungal resistance, phylogenetic analysis, protein domain, protein property. introduction chickpea (cicer arietinum) is an important crop that delivers nutritious food to the increasing global community and it will become increasingly popular as a result of climate change. production ranked third following beans with an average annual yield of over 11.5 million tons, where india has the highest share. land allocated to chickpea has dramatically increased and is now reported at 14.56 million hectares. more than 2.3 million tons of chickpea reach world markets every year to fund the requirements of countries unable to satisfy domestic consumption (1). alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 2 of 11 chickpea yield is significantly affected by both abiotic as well as biotic stress susceptibility (2,3). fungal infections have been shown to destructive effects on the chickpea production compared to various diseases triggered by a wide range of pathogens. within numerous fungal diseases, the most prevalent foliar and root infections are the diseases caused by ascochyta rabiei (ascochyta blight) and fusarium oxysporum (fusarium wilt), respectively, which cause serious crop yield decline (4). the genome of chickpea (2n = 2x = 16) is estimated to be 738.09 mb in length, where different cultivars whole genomes have been introduced for publicly use with more than 73% of the genomic content has been successfully sequenced. the chickpea gene pool is estimated to be about 29,000 genes, and about half (49.41%) of the chickpea genome consists of transposable elements and unclassified repetitions. on the other hand, in species-specific groups, 4,468 chickpea genes occur, these groups may arise through structural rearrangements, as happens among the disease resistance genes of nucleotide-binding site leucine-rich repeat (nbs-lrr). in this regard, it has been stated that the chickpea genome has been observed as having 187 disease resistance gene homologs (rghs) (5,6). the continuing breakthroughs in genome sequencing and genome-wide association studies have unlocked the ability to scan genomic content of chickpea for genes to control their resistance to multiple infections of the fungal disease. several predicted genes have been reported to have been statistically associated with chickpea fungal resistance, including nbs-lrr receptor-like kinase, wall-associated kinase, zinc finger protein, and serine / threonine protein kinase (7). in addition, it has been reported that chickpea resistance to some fungal diseases, such as ascochyta blight, may be linked to a number of motif families, such as at-hook motif containing nuclear localized (ahl) (8). in addition, bioinformatics methods have been accelerated over the last few years and several genomic and molecular databases have been established (9). such repositories could be used as a central stone in the quest for anti-fungal resistance in different plant species and in the analysis of their specific and special molecular structure (10,11). such bioinformatics tools have been used to study several gene families in chickpea, these families are considerably important in the plant defense system and essential membrane proteins (12,13). genome-wide characterization of anti-fungal genes in chickpea may enable researchers and breeders to overcome different fungal infections and develop new cultivars with high tolerance and better yield. our objective was to use comprehensive data analysis to locate and identify candidate genes for fungal disease resistance. we used a comprehensive bioinformatics pipeline of sequence alignment, phylogenetic analysis, protein chemical and physical properties assessment and domain structure classification. in order to study gene evolution and genetic diversity, we compared these genes with known anti-fungal genes in different species of plants. materials and methods genomic sequences related to anti-fungal resistance have been downloaded from the ncbi database. (14). a total of 19721 sequences of proteins belonging to 187 species of plants were downloaded from ncbi. the chickpea genome sequence was downloaded from the http:/www.cicer.info database (5). using the chickpea genome, the local blast+ (15) kit was used to create sequence database and align all anti-fungal amino acids with tblastn against the chickpea database. the ncbi tblastn online tool was used to annotate recovered sequences from the previous step. meme suite (16) was used to explore patterns of amino acids in chickpea anti-fungal genes. the megax program was used to perform a phylogenetic analysis using a maximum likelihood algorithm(17). in order to assess the chemical and physical characteristics of the amino acids, the pepstat program (18) was used through in-home perl scripts. these chemical properties are, a280 molar extinction coefficients cystine bridges (a280-meccb), a280 molar extinction coefficients reduced (a280-mecr), acidic (ac), aliphatic (aph), aromatic(ar), average residue weight (arw), basic (bs), charge (chr), charged (chrd), improbability of expression in inclusion bodies (ieeb), isoelectric point (ip), molecular weight (mw), non-polar (np), polar (po), residues (re), small (s), tiny (t). the itol online tool was used to visualize phylogenetic trees combined with information on amino acids (19)⁠ . the statistical correlation analysis (pvalue<0.01) was conducted using r packages (20). the circos package was used for displaying the genomic location of genes (21). results and discussion identification of chickpea anti-fungal genes identifying anti-fungal genes in chickpea could provide a useful resource for plant breeding programs by narrowing the pool of targeted genes. we have successfully identified 23 potential anti-fungal genes on 10 different chromosomes and genomic scaffolds (file s1). the total number of amino acids was 7077, ranging from 147 to 866, with an average of 307.7 amino acids. chromosomes ca2 and ca6 have the highest number of genes (4 genes) followed by ca3 and ca4 (3 genes) (figure 1 and table 1). in this regard, the entire genome re-sequencing of chickpea was used to identify 12 chromosomal regions associated with resistance to ascochyta blight, all of which alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 3 of 11 are located on ca4 (7). in addition, 8 quantitative trait loci (qtls) were identified on chromosomes ca2, ca3, ca4, ca5 and ca6 for the resistance of the same disease (22). the chemical and physical properties of chickpea anti-fungal proteins the chemical properties of the chickpea anti-fungal proteins were assessed across 17 different chemical and physical features of the amino acids. the total amino acid mw was 765.4, ranging from 16.0 kda (ca af17) to 97.7 kda (ca af9) with an average of 33.3 kda (figure 2 and table 2). by studying anti-fungal proteins in wheat total mw 1913 kda with an average of 20 kda (10). the amino acids charge ranges from -25 to (ca_af13) to 14 (ca_af9) (figure 2 and table 2).the extinction coefficient is a measure of how much light at a given wavelength a chemical element attenuates. calculating the content of the amino acid is necessary in order to determine the protein's molar extinction coefficient (23). the a280 molar extinction coefficients reduced (a280-mecr) and a280 molar extinction coefficients cystine bridges (a280-meccb) are two separate extinction coefficient measures, where salt bridges are essential motifs of the tertiary protein structure and are mostly associated with the molecular influence force that maintains the protein's stability (24). the a280-mecr and a280-meccb total values are 836030, 864530 m-1cm-1, ranging from 10430 and 11555 m-1cm-1 (ca af11) to 87560 and 90560 m-1cm-1 (ca af9), respectively (figure 2 and table 2). in some anti-fungal wheat proteins, the a280-mecc and a280-mecr minimum scores were recorded as 1740 and 1490, with the highest scores being 104570 and 103820 respectively (10). improbability of expression in inclusion bodies (ieeb) is a type of solubility measurement. in escherichia coli, for example, recombinant protein can be produced either as insoluble in the bodies of inclusion or soluble throughout the cytosol (25). the total ieeb of chickpea anti-fungal protein was 18.33 ranging from 0 (ca_af11) to 0.972 (ca_af16) with a mean of 0.797 (figure 2 and table 2). the ieib of anti-fungal amino acids revealed an average of 0.794 by examining wheat anti-fungal proteins, ranging from 0.504 to 0.977 (10). the average collective weight as per their length for all amino acid sequences is measure though the average residue weight (arw). table 1: the chromosomal location and gene definition for identified anti-fungal genes in chickpea genome. gene code chr. start end gene name gene code chr. start end gene name ca_af1 chr1 13449810 13450553 cysteine-rich ca_af13 chr5 15134097 15134777 pathogenesis-related ca_af2 chr1 39499412 39500080 thaumatin ca_af14 chr5 64978467 64979294 cysteine-rich ca_af3 chr2 15365865 15366560 plasmodesmata-located ca_af15 chr6 11037122 11037844 pathogenesis-related ca_af4 chr2 15365877 15366521 plasmodesmata-located ca_af16 chr6 12421759 12422469 thaumatin ca_af5 chr2 22066674 22068239 cysteine-rich ca_af17 chr6 13070688 13071419 cysteine-rich ca_af6 chr2 29604091 29604798 thaumatin ca_af18 chr6 65201755 65202474 plasmodesmata-located ca_af7 chr3 39833663 39834379 cysteine-rich ca_af19 chr7 24258666 24259331 thaumatin ca_af8 chr3 51987009 51987668 cysteine-rich ca_af20 chr8 18540320 18541342 cysteine-rich ca_af9 chr3 59460263 59461033 cysteine-rich ca_af21 chr8 5747704 5748729 thaumatin ca_af10 chr4 2480078 2480725 cysteine-rich ca_af22 scaffold0585 6485 7156 thaumatin ca_af11 chr4 4601812 4602510 cysteine-rich ca_af23 scaffold4365 250412 251062 pathogenesis-related ca_af12 chr4 56994913 56995608 pathogenesis-related alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 4 of 11 figure 1 : the genomic location and suggested definition of predicated anti-fungal genes, where the width of the internal links indicates similarity percentage of gene sequences. the total arw was 2478.4 da, where ca af13 and ca af14 have the minimum and maximum values of 101.8 da and 116.7 da, with an average of 107.7 da, respectively. the ieib of anti-fungal amino acids revealed an average of 0.794, ranging from 0.504 to 0.977, through examining wheat anti-fungal proteins (10). the isoelectric point (ip) is the ph level with which the net charge of the protein is positive, and is correlated with amino acid composition and protein structure (26). the ip of chickpea anti-fungal proteins range from 3.9 (ca_af13) to 8.9 (ca_af19) with a mean of 6 (figure 2 and table 2). thus, it revealed a collective ip in wheat anti-fungal proteins with an average of 6,402, ranging from 4 to 10.4 in wheat (10). in addition, the folded structure of a protein becomes less desirable in terms of thermodynamics because it decreases the protein disorder or entropy, where non-polar chains tend to squeeze inside the protein while polar chains push outside the molecule (27). the non-polar (np) values ranges from 49.2 (ca_af20) to 62.1 (ca_af16) with an average of 56 (figure 2 and table 2). the non-polar and polar amino acid scores ranged from 48.81 and 30.081 to 69.919 and 51.19 respectively, in wheat anti-fungal proteins (10). basic amino acids have a certain basic group within the chain whereas acidic amino acids have an acidic group within the chain. basic amino acids have high pka while acidic amino acids have low pka. the count of basic and acidic amino acids range from 9.011 and 9.359 to (ca_af13 and ca_af16) to 13.613 and 16.915 (ca_af19 and ca_af11) (figure 2 and table 2). alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 5 of 11 figure 2: the chemical and physical properties of identified anti-fungal proteins in chickpea. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 6 of 11 table 2: chemical properties of identified anti-fungal proteins in chickpea. protein a280-meccb a280-mecr ac aph ar arw bs chr chrd ieeb ip mw np po re s t ca_af1 54470 52720 11.364 24.621 10.606 109.947 11.364 -2 22.727 0.82 5.8518 58051.77 51.894 48.106 528 55.303 33.712 ca_af2 29420 28420 7.207 21.622 10.811 104.536 8.108 2 15.315 0.951 7.5063 23206.95 56.306 43.694 222 69.369 47.748 ca_af3 40365 38740 10 26 9 105.494 8 -8 18 0.77 4.5636 42197.5 60.5 39.5 400 63 42.5 ca_af4 20120 19370 9.953 25.118 9.479 104.952 9.005 -3 18.957 0.787 4.929 22144.77 58.768 41.232 211 63.981 44.076 ca_af5 59135 57760 8.802 22.005 13.447 113.33 10.024 2 18.826 0.845 6.899 46352.15 49.633 50.367 409 54.279 32.763 ca_af6 40295 39420 7.623 24.215 11.659 109.223 9.417 3.5 17.04 0.949 7.7696 24356.69 57.399 42.601 223 61.435 34.978 ca_af7 12545 11920 10.596 25.166 8.609 106.551 10.596 0 21.192 0.818 6.208 16089.2 56.954 43.046 151 58.278 38.411 ca_af8 31830 30830 10.638 26.596 11.702 108.504 9.574 -5.5 20.213 0.74 4.941 30598.04 53.191 46.809 282 57.092 39.362 ca_af9 90560 87560 10.162 26.328 12.009 112.861 12.009 14 22.171 0.793 7.9722 97737.52 53.118 46.882 866 51.27 30.947 ca_af10 34155 32780 8.122 22.843 12.183 107.791 8.629 0 16.751 0.841 6.4334 42469.67 54.315 45.685 394 58.376 41.117 ca_af11 11555 10430 16.915 22.886 10.448 113.048 8.955 -17 25.871 0 4.082 22722.56 49.751 50.249 201 51.244 32.338 ca_af12 36410 35410 8.072 23.767 11.211 102.971 6.726 -4 14.798 0.843 4.6572 22962.57 61.883 38.117 223 70.404 48.879 ca_af13 58965 56840 9.545 20.455 10.909 101.798 4.545 -25 14.091 0.756 3.9557 44790.92 59.091 40.909 440 71.818 50.909 ca_af14 40545 39420 9.17 21.834 13.1 116.75 12.227 6 21.397 0.817 7.9878 26735.85 53.712 46.288 229 48.908 27.074 ca_af15 32400 31400 10 20.476 10.952 103.8 4.762 -11 14.762 0.7 3.9687 21797.95 59.048 40.952 210 69.048 51.905 ca_af16 33890 32890 5.023 23.288 12.785 106.665 10.502 10.5 15.525 0.972 8.4799 23359.67 62.1 37.9 219 63.927 41.553 ca_af17 18045 17420 10.884 25.17 10.204 108.53 9.524 -2 20.408 0.613 4.8281 15953.84 52.381 47.619 147 59.864 42.857 ca_af18 43345 41720 9.596 27.273 10.606 106.838 8.586 -4 18.182 0.814 4.8941 42307.82 60.606 39.394 396 62.121 39.899 ca_af19 23210 22460 7.33 19.895 9.424 107.128 13.613 11.5 20.942 0.949 8.9491 20461.39 58.115 41.885 191 61.78 39.267 ca_af20 29630 28880 8.333 25.794 9.524 111.225 9.127 1.5 17.46 0.856 7.2867 28028.72 49.206 50.794 252 58.333 34.524 ca_af21 32400 31400 7.895 20.175 11.842 104.278 6.14 -4 14.035 0.927 4.542 23775.41 57.895 42.105 228 67.982 49.561 ca_af22 61820 59820 9.417 18.834 10.762 105.318 6.726 -13 16.143 0.844 4.3753 46971.95 55.605 44.395 446 68.61 43.498 ca_af23 29420 28420 8.612 20.096 11.962 106.915 9.091 1 17.703 0.925 7.1537 22345.18 57.416 42.584 209 65.55 42.584 alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 7 of 11 on the other hand, in addition to the chickpea genes retrieved through this study, we have studied the chemical and physical properties of 1216 anti-fungal proteins identified in different plant species (figure 3 and table s2). the protein mw range from 21 kda (silene latifolia) to 97.7 kda (cicer arietinum) while the protein charge range from -25 (cicer arietinum) to 21 (rosa chinensis) (figure 3 and table s2). the minimum arw was 99.74 kda (striga asiatica) and the maximum 116.75 kda (cicer arietinum) (figure 3 and table s2). the a280-mecr and a280-meccb range from 30193 and 31133 to 87560 and 90560 m-1cm-1, respectively (figure 3 and table s2). the charged amino acids range from 8.108 (cephalotus follicularis) to 26.941 (populus trichocarpa) (figure 3 and table s2). figure 3 : the phylogenetic tree developed using multiple sequence alignment of chickpea anti-fungal proteins and other species, where its chemical and physical properties are plotted. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 8 of 11 figure 4 :statistical correlation between different chemical and physical properties of anti-fungal proteins; (a) the heatmap of inter-correlation matrix and (b) the correlation networks, where pvalue > 0.01 and r 2 > 0.3. phylogenetic analysis and domain identification of anti-fungal proteins the phylogenetic tree constructed using 1216 anti-fungal proteins of 187 was clustered into 11 clusters (figure 3). the anti-fungal chickpea proteins identified are cysteine-rich (10), thaumatin (6), pathogenesis-related (4) and plasmodesmata (3) proteins (figure 3). a protein sequence motif is a brief pattern that is preserved by nature. for proteins, a motif may relate to the active site of an enzyme or to a functional unit required to properly fold proteins. hence, sequence motifs are among the basic functional components of molecular evolution (16). five specific motifs for amino acids were discovered among the sequences of identified anti-fungal proteins of chickpea alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 9 of 11 (figure 5). the maximum likelihood phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as illustrated by their protein motifs / domains. these motifs are motif1 (elme000385), motif2 (elme000094), motif3 (elme000321), motif4 (elme000003), and motif5 (elme000287) (figure 5). motif1 is very similar to elme000385 (pvalue of 1.60e-03), which functions as mtr4-air2-interaction site. this domain distinguishes the tramp complex, which engages in the nucleus with exosome-mediated degradation of abnormal rnase. oligo-adenylated tails are introduced to substrates of abnormal rna by air2 and mtr4 , thus highlighting them for degradation (28). motif2 has a high significance similarity with integrin binding sites (elme000094) with a pvalue of 1.16e-05. integrins are cell surface receptors which are responsible for cell migration, cell adhesion to extracellular matrix, and cell adhesion to cells (29)⁠. motif3 is similar to caspase cleavage motif (elme000321) with a pvalue of 5.46e-03. proteases caspases-3 and-7 play a major role in programmed cell apoptosis, and non-apoptotic caspases include involvement in immune response (30). similarly, pattern 5 is similar to iap-binding pattern (ibm): (elme000287) that distinguishes apoptosis protein inhibitor (iap) which exhibits several immune functions, mitosis regulation, tnf-receptor signal transduction, and many more (31)⁠. finally, motif4 was in high similar to ww domain ligands (elme000003) motif, which are small but widespread domains are found in various regulatory circumstances (16). the maximum likelihood of phylogenetic analysis was successful in distinguishing between anti-fungal chickpea proteins as seen in their protein patterns/domains. where it cluster chickpea genes into 4 clusters (figure 6). figure 5: the domains/motifs found by the meme tool in chickpea predicated anti-fungal proteins. alsamman et al., 2019 comprehensive study of chickpea anti-fungal genes highlights in bioscience november 2019 | volume 2 http://bioscience.highlightsin.org/ page 10 of 11 figure 6: the phylogenetic analysis of chickpea anti-fungal proteins, where the protein motif structures are shown as detected using meme tool. conclusion it was very helpful to predict anti-fungal resistance genes using publicly available repositories and indicated that several chickpea genes could be used to limit genetic research of genes that hold the key to fungal resistance in chickpea. we have successfully identified 23 potential anti-fungal genes on 10 different chromosomes and genomic scaffolds. a high number of anti-fungal chickpea proteins are cysteine-rich (20), thaumatin (9), and pathogenesis-related (8), which could indicate the importance of these gene classes in chickpea resistance to fungal. in addition, the chemical and physical analysis shed light on the uniqueness and consistency of these proteins, where several of these parameters could be used in future research to identify anti-fungal genes in different plant species. moreover, the domain identification analysis identified several potential anti-fungal protein domains such as tramp complex and caspase cleavage motifs. supplementary files the supplementary material for this article can be found online at: https://doi.org/10.36462/h.biosci.20194 supplementary file 1: the amino acid sequences of anti-fungal genes identified in chickpea. supplementary file 2: the physical and chemical properties of some anti-fungal proteins in plant species. references 1. merga b, haji j. economic importance of chickpea: production, value, and world trade. cogent food agric. 2019;5(1):1615718. 2. ahmed sm, alsamman am, mubarak mh, badawy ma, kord ma, momtaz oa, et al. dowsing for salinity tolerance related genes in chickpea through genome wide association and in silico pcr analysis. biorxiv. 2019;519744. 3. hamwieh a, imtiaz m, malhotra rs. multi-environment qtl analyses for drought-related traits in a recombinant inbred population of chickpea (cicer arientinum l.). theor appl genet. 2013;126(4):1025–38. 4. kukreja s, salaria n, thakur k, goutam u. fungal disease management in chickpea: current status and future prospects. in: fungi and their role in sustainable development: current perspectives. springer; 2018. p. 293–309. 5. edwards d. 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be addressed: ibrahim mustafa citation: roan zaid and ibrahim mustafa (2020). the distribution of the abo and rh blood groups among different populations in the mena region: a review. highlights in bioscience volume 3. article id 20221. dio:10.36462/h.biosci.20221 received: june 13, 2020 accepted: august 18, 2020 published: september 10, 2020 copyright: © 2020 zaid and mustafa. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. the distribution of the abo and rh blood groups among different populations in the mena region: a review roan zaid and ibrahim mustafa* abstract abo and rh (rhesus) blood grouping is one of the most widely available laboratory tests that could prevent possibly deadly mismatches following blood transfusion and organ transplantation. the distribution of the blood group antigens varies between different populations and ethnic groups. our objective was to showcase these variations within the middle east and north africa (mena) region and to outline the relationship between blood type and disease risk association to determine the current findings and outline possible future study areas. in healthy blood donors, blood group o was found to consistently be the most prevalent blood group and ab the least prevalent blood group except in turkey where a is the most prevalent and, in the united arab emirates (uae), where b is the least prevalent blood type. keywords: abo blood groups, rh blood groups, blood donors introduction according to the international society of blood transfusion (isbt), there are 33 blood group systems that represent more than 30 antigens as of date [1, 2]. the four common blood group antigens (a, b, ab and o) of the abo system, along with the d (rhesus/rh) and kell antigens are the most important in blood transfusions and organ transplantation and are commonly tested due to their clinical significance [3]. the abo gene codes for glycosyltransferases that transfer complex oligosaccharide antigens to the h antigen on the surface of red blood cells (rbcs). n-acetylgalactosamine and galactose are transferred in the case of a and b blood groups, respectively. individuals with the o blood group do not produce the glycosyltransferase due to a single point deletion (guanine-258) and, as a result, lack these antigens on the surface of their rbcs [4, 5]. the distribution of the abo/rh blood groups among different populations within the mena region as well as some the diseases that are associated with these blood groups will be addressed in this review. the distribution of the abo blood groups in the mena region ever since the discovery of the abo blood groups by landsteiner in 1900, the studies on human population genetics were on the rise [6]. blood group typing entails finding the antigenic properties of rbcs. these antigenic properties are found to vary between different racial/ethnic groups. population and prevalence studies are therefore important to help elucidate the distribution of the abo/rh blood groups and to help prevent possibly deadly mismatches before blood transfusions or organ transplantation [7]. table 1 shows the distribution of the abo and rh blood groups among different populations in the mena region. https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ http://bioscience.highlightsin.org/ https://doi.org/10.36462/h.biosci.20221 https://doi.org/10.36462/h.biosci.20221 http://bioscience.highlightsin.org/ zaid and mustafa, 2020 the distribution of the abo and rh blood groups in the mena region highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 2 of 4 in healthy blood donors, blood group o was consistently found to be the most prevalent and ab the least prevalent blood group. turkey and the uae were the only exception where a was found to be the most prevalent in turkey and b was the least prevalent in the uae. blood group antigens and disease association abo blood groups have been statistically associated with many diseases including diabetes, venous thromboembolism, ovarian cancer, hepatocellular carcinoma, lung cancer, pancreatic cancer and other gastrointestinal cancers [15, 18-22]. however, reports have been variable and inconsistent between studies. some of these diseases and their associated blood group antigens will be addressed. venous thromboembolism and blood group antigens one of the most described associations between non-o blood groups (a, b, and ab) and other diseases is that of venous thromboembolism (vte). multiple studies have established a relationship between the abo blood groups and its influence on normal haemostasis [19, 23]. this relationship is attributed to the expression of the abo antigens on the surface of other human cells, including cells of the epithelium, vascular endothelium and platelets [24]. in addition, the blood group antigens largely determine the plasma levels of the von willebrand factor (vwf) where vwf levels are reported to be 25% higher in non-o blood groups [23, 25-27]. elevated vwf would prompt platelet adhesion as well as maintain the coagulation factor ⅷ (fⅷ) in the plasma increases the risk of vte [24]. it is also important to note that individuals of the a2 blood groups are reported to have low levels of the vwf and fⅷ. the risk of developing vte in these individuals is said to be lower when compared to the other blood groups (a1, b and ab) [19]. in concordance with this phenomenon, higher risk of myocardial infraction and ischemic strokes were also previously described in individuals with blood group a and ab [28]. table 1. distribution of the abo and rh blood groups among different populations within the mena region. type 2 diabetes mellitus and blood group antigens a study done in qatar revealed that type 2 diabetes mellitus (t2dm) patients are significantly more likely to be of blood type b as compared to healthy individuals (25.7% vs. 20.4%; p < 0.001) (table 1). in the same study, blood group o was significantly higher in healthy individuals than in t2dm patients (38.5% vs. 45.4%; p < 0.001) [10]. a study done in kingdom of saudi arabia (ksa) found a 52.2% decrease (2.1 fold reduction) in rh (-) blood groups in t2dm female patients as compared to healthy controls (4.21% vs. 8.8%; p = 0.043) [12]. similar findings were reported for male t2dm patients in ksa where males had a 3.4 fold reduction in rh (-) blood group in comparison to healthy controls (3.13% vs. 10.6%; p = 0.028) [13]. blood group b was higher in both male and female t2dm patients in ksa, however, these findings were not statistically significant [12, 13]. in iraq, it was reported that blood group o individuals had significantly higher levels of total cholesterol, glucose and blood pressure in comparison to other blood groups (p < 0.01). these parameters showed a decreasing trend starting from group o to a to b and then ab [22]. although all the reported results were of high significance in all populations, it is clear that these findings cannot be extrapolated to other populations due to the conflicting reports. these variations between populations could be explained by the multifactorial nature of t2dm. cancer and blood group antigens multiple studies consistently report that individuals with the o blood group have reduced risk of developing pancreatic cancer than non-o individuals [4, 20, 29, 30]. one study reported that the risk of pancreatic cancer increases with each addition of non-o allele. in this study, individuals with ao and aa genotypes had odds ratios (ors) of 1.33 and 1.66 respectively when compared with individuals of the oo genotype. whereas bo and bb individuals had ors of 1.45 and 2.42, respectively [30]. it was also recently reported that the genetic variant of the abo gene, rs505922 (t allele), which is said to confer protective properties against pancreatic cancer, is in complete linkage disequilibrium (r2= 1.0) with the guanine-258 deletion that encodes the o allele [4, 31]. this variant entails a single nucleotide polymorphism in the first intron of the abo gene. comparable findings were reported for hepatocellular carcinoma (hcc) where blood group o was found to improve the disease’s prognosis after hepatectomy [21]. country population studied sample size a (%) b (%) ab (%) o (%) rh+ (%) rh(%) ref. iraq blood donors 5,000 32.6 22.8 7.6 37.0 91.3 8.7 [8] pakistan blood donors 3679 31.87 21.71 12.22 34.2 88.86 11.14 [9] qatar blood donors t2dm patients 1,650 1,633 27.6 29 20.4 25.7 6.5 6.8 45.4 38.5 [10] [10] saudi arabia blood donors male blood donors t2dm female patients t2dm male patients 57,396 944 214 128 26.0 33.4 24.8 (a+) 22.6 (a+) 18.0 6.0 27.1 (b+) 30.5 (b+) 4.0 3.8 7.0 (ab+) 4.7 (ab+) 51.0 56.8 36.9 (o+) 39.1 (o+) 92.0 92.8 95.8 96.88 8.0 7.2 4.21 3.13 [11] [7] [12] [13] sudan blood donors 3,087 19.14 14.0 66.8 74.4 25.6 [14] turkey blood donors blood donors lung cancer patients 3,022,883 86,797 2,044 42.2 44 43.9 16.4 16.2 17.3 7.5 6.5 8.1 33.9 33.3 30.7 87.7 88.2 86.2 12.3 11.8 13.8 [15] [16] [15] united arab emirates blood donors 661 24.0 22.8 31 48.4 91 8.9 [17] http://bioscience.highlightsin.org/ zaid and mustafa, 2020 the distribution of the abo and rh blood groups in the mena region highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 3 of 4 studies conducted by wu et al., revealed the mean overall survival (os) of hcc after hepatectomy to be 55, 39, 34 and 34 months for blood groups o, a, b and ab, respectively. the overall hazard ratio for non-group o individulas in this study was 1.485 (95% ci, 1.204–1.830; p < 0.001) [21]. studies done by urun et al. in turkey found that having o and rhblood decreased the risk of lung cancer by 14% and 13% respectively [15]. however, another study, also done in turkey, showed no correlation between blood groups and lung cancer status of patients [32]. finally, zhang et al. compared findings for 89 studies with 30 different cancer types. for these studies, the pooled or for a blood groups vs. non a blood groups was 1.12 (95%ci: 1.09-1.16) showing a significantly increased risk of blood group a. in the other hand, individuals with the o blood group had a significantly reduced risk of cancer compared to other blood groups with an or of 0.84 (95%ci: 0.80-0.88) [33]. in all the reported cases, the exact mechanism by which blood group o causes a risk reduction is not fully understood. however, many possible explanations were suggested. for instance, the intracellular adhesion molecule-1 (sicam-1) levels were found to be higher in group o individuals [34]. it is possible that the reduced levels of sicam-1 promote tumor metastasis in non-group o individuals [35]. furthermore, the single nucleotide polymorphism (snp) of the abo gene (rs505922) was found to be associated with higher levels of tumor necrosis factor (tnf) levels. [31] these findings could explain the relationship between specific blood groups and carcinogenesis [36]. conclusion in conclusion, an understanding of blood group data between different populations is important for effective management of blood bank inventory and for more efficient transfusion practices. the present study collected data from different populations of the mena region to reveal that blood group o is the most common blood group type, while ab is the least prevalent. some of the diseases associated with specific blood groups were also addressed. however, more studies are required to fully understand the reported associations and to address the inconsistencies between studies. author contributions im conceived and planned the manuscript writing. rz took the lead in writing the manuscript with equal contribution. all authors provided critical feedback and helped shape the research, analysis and manuscript. references 1.logdberg l, reid me, lamont re, zelinski t. human blood group genes 2004: chromosomal locations and cloning strategies. transfus med rev. 2005;19(1):45-57. 2.logdberg l, reid me, zelinski t. human blood group genes 2010: chromosomal locations and cloning strategies revisited. transfus med rev. 2011;25(1):36-46. 3.basu d, datta ss, montemayor c, bhattacharya p, mukherjee k, flegel wa. abo, rhesus, and kell antigens, alleles, and haplotypes in west bengal, india. transfus med hemother. 2018;45(1):62-6. 4.amundadottir l, kraft p, stolzenberg-solomon rz, fuchs cs, petersen gm, arslan aa, et al. genome-wide association study identifies variants in the abo locus associated with susceptibility to pancreatic cancer. nature genetics. 2009;41(9):986. 5.el-zawahri mm, luqmani ya. molecular genotyping and frequencies of a 1, a 2, b, o 1 and o 2 alleles of the abo blood group system in a kuwaiti population. international journal of hematology. 2008;87(3):303-9. 6.landsteiner k. uber agglutinationserscheinungen normalen menschlichen blutes. wien klin wshr. 1901;14:1132-4. 7.sarhan ma, saleh ka, bin-dajem sm. distribution of abo blood groups and rhesus factor in southwest saudi arabia. saudi med j. 2009;30(1):116-9. 8.getta ha, amin ss, khoshnaw n, muhammad ba. distribution of red cell antigens according to abo, rh and other rare blood group systems in kurdish ethnicity. iraqi journal of hematology. 2016;5(1):55. 9.ahmad t, nadeem m, khan fu, uddin s, maqsood-ur-rehman m, anwar n. incidence of hbv, hcv and hiv among blood donors from peshawar kpk, pakistan. population. 2017;6:19. 10.bener a, yousafzai mt. the distribution of the abo blood groups among diabetes mellitus patients in qatar. nigerian journal of clinical practice. 2014;17(5):565-8. 11.bashwari la, al-mulhim aa, ahmad ms, ahmed ma. frequency of abo blood groups in the eastern region of saudi arabia. saudi med j. 2001;22(11):1008-12. 12.farshori mp, al-muzaini ay, al-wakid ih, ibrahim ika, alshammari af, alduejieman m, et al. frequency of distribution of abo and rhesus (rh) blood group antigens in the female type 2 diabetes mellitus (t2dm) patients in hail region of saudi arabia. integrative obesity and diabetes. 2017;3(1):1-6. 13.farshori mpq, al-wakid ih, ibrahim ika, alshammari af, alduejieman m, almhanaa am, et al. distribution of abo and rhesus (rh) blood group antigens in male type 2 diabetes mellitus patients in hail region of saudi arabia: high incidences of diabetes mellitus in males with b+ blood type. integr obesity diabetes. 2016;2:233-8. 14.khalil i, phrykian s, farr a. blood group distribution in sudan. gene geography: a computerized bulletin on human gene frequencies. 1989;3(1):7-10. 15.urun y, utkan g, cangir ak, oksuzoglu ob, ozdemir n, oztuna dg, et al. association of abo blood group and risk of lung cancer in a multicenter study in turkey. asian pac j cancer prev. 2013;14(5):2801-3. 16.torun ya, kaynar lg, karakükcü ç, yay m, kurnaz f, mutlu h, et al. abo and rh blood group distribution in kayseri province, turkey. turkish journal of hematology. 2012;29(1):97. 17.taha jy. rh antigen and phenotype frequency in kalba region, uae. bahrain medical bulletin. 2012;158 (690):1-3. http://bioscience.highlightsin.org/ zaid and mustafa, 2020 the distribution of the abo and rh blood groups in the mena region highlights in bioscience september 2020| volume 3 http://bioscience.highlightsin.org/ page 4 of 4 18.cozzi gd, levinson rt, toole h, snyder m-r, deng a, crispens ma, et al. blood type, abo genetic variants, and ovarian cancer survival. plos one. 2017;12(4):e0175119. 19.trégouët d-a, heath s, saut n, biron-andreani c, schved j-f, pernod g, et al. common susceptibility alleles are unlikely to contribute as strongly as the fv and abo loci to vte risk: results from a gwas approach. blood. 2009;113(21):5298-303. 20.greer jb, yazer mh, raval js, barmada mm, brand re, whitcomb dc. significant association between abo blood group and pancreatic cancer. world journal of gastroenterology: wjg. 2010;16(44):5588. 21.wu t, ma x-a, wang g-q, li q, li m-j, guo j-y, et al. abo blood type correlates with survival in hepatocellular carcinoma following hepatectomy. scientific reports. 2017;7(1):4412. 22.jassim w. association of abo blood group in iraqis with hypercholesterolaemia, hypertension and diabetes mellitus. 2012. 23.jenkins pv, o’donnell js. abo blood group determines plasma von willebrand factor levels: a biologic function after all? transfusion. 2006;46(10):1836-44. 24.zhou s, welsby i. is abo blood group truly a risk factor for thrombosis and adverse outcomes? world journal of cardiology. 2014;6(9):985. 25.franchini m, capra f, targher g, montagnana m, lippi g. relationship between abo blood group and von willebrand factor levels: from biology to clinical implications. thrombosis journal. 2007;5(1):14. 26.franchini m, makris m. non-o blood group: an important genetic risk factor for venous thromboembolism. blood transfusion. 2013;11(2):164. 27.kamphuisen pw, eikenboom jc, bertina rm. elevated factor viii levels and the risk of thrombosis. arteriosclerosis, thrombosis, and vascular biology. 2001;21(5):731-8. 28.wiggins kl, smith nl, glazer nl, rosendaal fr, heckbert sr, psaty bm, et al. abo genotype and risk of thrombotic events and hemorrhagic stroke. j thromb haemost. 2009;7(2):263-9. 29.wolpin bm, chan at, hartge p, chanock sj, kraft p, hunter dj, et al. abo blood group and the risk of pancreatic cancer. journal of the national cancer institute. 2009;101(6):424-31. 30.wolpin bm, kraft p, gross m, helzlsouer k, bueno-de-mesquita hb, steplowski e, et al. pancreatic cancer risk and abo blood group alleles: results from the pancreatic cancer cohort consortium. cancer research. 2010:0008-5472. can-09-2993. 31.melzer d, perry jr, hernandez d, corsi a-m, stevens k, rafferty i, et al. a genome-wide association study identifies protein quantitative trait loci (pqtls). plos genetics. 2008;4(5):e1000072. 32.oguz a, unal d, tasdemir a, karahan s, aykas f, mutlu h, et al. lack of any association between blood groups and lung cancer, independent of histology. asian pacific journal of cancer prevention. 2013;14(1):453-6. 33.zhang b-l, he n, huang y-b, song f-j, chen k-x. abo blood groups and risk of cancer: a systematic review and meta-analysis. asian pac j cancer prev. 2014;15 (11):4643-50. 34.witkowska am, borawska mh. soluble intercellular adhesion molecule‐1 (sicam‐1): an overview. european cytokine network. 2004;15(2):91-8. 35.kobayashi h, boelte kc, lin pc. endothelial cell adhesion molecules and cancer progression. current medicinal chemistry. 2007;14(4):377-86. 36.liumbruno gm, franchini m. hemostasis, cancer, and abo blood group: the most recent evidence of association. journal of thrombosis and thrombolysis. 2014;38(2):160-6. http://bioscience.highlightsin.org/ highlights in bioscience; highlights in bioscience highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 1 of 7 citation: girish k. and dubey s. (2018). higher organism’s molecular databases: an overvie. highlights in bioscience, volume 1.article id 20184, dio:10.36462/ h.biosci.20184 received: march 5, 2018 accepted: april 28, 2018 published: may 10, 2018 copyright:© 2018 girish and dubey. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper funding: the authors have no support or funding to report. eukaryotic molecular biology databases: an overview kumar girish 1 and singh dubey 2 1 department of biotechnology, indian agricultural research institute, india. 2 department of biology, agricultural university, india abstract a biological database is a big, structured body of continuous information, generally connected with computerized software intended to update, query, and recover information elements deposited within the framework. a straightforward database could be a single folder comprising several data, each carrying the same number of data. such famous databases are genbank from the national center for biotechnology information, swissprot from the swiss institute of bioinformatics and pir from the protein information resource. biological databases are bibliotheques of life science data, gathered from science studies, published literature, high-performance experimental technology, and computational analysis. here we brefly described some recently published molecular databases. keywords: biological database, computational analysis, bioinformatics, software. introduction bioinformatics is a steadily increasing area of studies fueled by the need to handle and evaluate the vast amounts of information produced by omics techniques. decades of studies have produced several known multi-omics tools, including genome, proteome and transcriptome (1–3) with the sole objective of understanding every part of biology. here we brefly described recent published database such as phenomicdb, aagmarker, cscd, cr2cancer, activedriverdb, amtdb, hedd, diseaseenhancer, dreambase, dashr, msdd, cancersysdb, mirdip, medreaders, linkedomics, pancan-meqtl, pitdb, qprimerdb, seecancer, unilectin3d, translatomedb, tiss-gdb, and tc3a table (1). review article open access eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 2 of 7 table 1. list of some recently published database. database name link reference database name link reference phenomicdb http://www.phenomicdb.de kahraman et al. (4) mirdip http://ophid.utoronto.ca /mirdip/ tokar et al. (16) aagmarker http://bioinfo.wilmer.jhu.edu/ aagmarker/ pan et al. (5) medreaders http://medreader.org/ wang et al. (17) cscd http://gb.whu.edu.cn/cscd xia et al. (6) linkedomics http://www.linkedomics.org vasaikar et al. (18) cr2cancer http://cis.hku.hk/cr2cancer ru et al. (7) pancan-meqtl http://bioinfo.life.hust.edu.cn/ pancan-meqtl/ gong et al. (19) activedriverdb https://www.activedriverdb.org krassowski et al. (8) pitdb http://pitdb.org saha et al. (20) amtdb https://amtdb.org ehler et al. (9) qprimerdb http://biodb.swu.edu.cn/ qprimerdb lu et al. (21) hedd http://zdzlab.einstein.yu.edu/1/ hedd.php wang et al. (10) seecancer http://biocc.hrbmu.edu.cn/ seecancer zhang et al. (22) diseaseenhancer http://biocc.hrbmu.edu.cn/ diseaseenhancer/ zhang et al. (11) unilectin3d https://www.unilectin.eu/ unilectin3d mariethoz et al. (23) dreambase http://rna.sysu.edu.cn/dreambase zheng et al. (12) translatomedb http://www.translatomedb .net/ liu et al. (24) dashr http://lisanwanglab.org/dashrv2 kuksa et al. (13) tiss-gdb http://zhaobioinfo.org/ tissgdb kim et al. (25) msdd http://tdb.ccmb.res.in/msdb yue et al. (14) tc3a http://tc3a.org feng et al. (26) cancersysdb https://cancersys.uni-koeln.de krempel et al. (15) phenomicdb by combining government genotype/phenotype information from a broad spectrum of model organisms and modern humans, kahraman et al. (4) built a multi-species genotype / phenotype repository. they collected these riches of information into a single embedded database through coarse-grained semiconductor mapping of phenotypic information areas, including prevalent gene codes (ncbi gene) and the use of related orthology. phenomicdb enables researchers to concurrently compare and browse recognized phenotypes for a specified gene or a collection of genes from distinct species with its use-case-oriented interface. it can be found at http://www.phenomicdb.de. aagmarker pan et al. (5) has collected many released raw information sets on proteome microarrays acquired from serum profiling assays and offers a toolbox for mining these information. aagmarker's present edition includes 854 serum samples with 136 092 proteins. for 12 diseases, such as alzheimer's disease, bechet's disease, and parkinson's disease, a total of 7803 (4470 non-redundant) candidate auto virus biomarkers were recognized and gathered. in order to quantitatively evaluate these biomarkers, seven statistical parameters were implemented. users can use fundamental search, sophisticated search and browse to collect, analyze and compare the datasets. these biomarkers can also be downloaded in terms of disease. the aagmarker can be found at http://bioinfo.wilmer.jhu.edu/aagmarker/. cscd xia et al. (6) gathered 228 complete samples of rna or polya(-) rna-seq from both cancer and ordinary cell lines and found 272,152 circr-nas specific to cancer. in ordinary samples only a total of 950 962 circrnas were recognized, and in both tumor and ordinary samples 170 909 circrnas were detected, that could be even farther used as non-tumor context. a cancer-specific circrna databases http://gb.whu. edu.cn/cscd were built. they anticipated the microrna response element locations and rna binding protein positions for each circrna in order to comprehend the functional impacts of circrnas. in addition, prospective open reading frames were anticipated to show translatable circrnas. they also identified the splicing occurrences in linear transcripts of each circrna to comprehend the correlation between linear splicing and back splicing. as the first extensive circrna database specific to cancer. cr2cancer ru et al. (7) introduced cr2cancer, an extensive database for crs in human cancer annotation and visualization built through high-throughput data analysis and mining literature. for more than 400 crs across various kinds of cancer, they gathered and incorporated genomic, transcriptomic, proteomic, clinical and functional data. they also constructed various kinds of cr-related relationships, including dependent and autonomous cancer type relationships. in addition, about 6000 items of aberrant molecular alterations and cr interactions were manually curated from 5007 publications in the development of cancer. cr2cancer offers an easy-to-use online interface to navigate, http://www.phenomicdb.de/ http://bioinfo.wilmer.jhu.edu/%20aagmarker/ http://bioinfo.wilmer.jhu.edu/%20aagmarker/ http://medreader.org/ http://gb.whu.edu.cn/cscd http://www.linkedomics.org/ http://cis.hku.hk/cr2cancer http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ https://www.activedriverdb.org/ http://pitdb.org/ https://amtdb.org/ http://biodb.swu.edu.cn/%20qprimerdb http://biodb.swu.edu.cn/%20qprimerdb http://zdzlab.einstein.yu.edu/1/%20hedd.php http://zdzlab.einstein.yu.edu/1/%20hedd.php http://biocc.hrbmu.edu.cn/%20seecancer http://biocc.hrbmu.edu.cn/%20seecancer http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ https://www.unilectin.eu/%20unilectin3d https://www.unilectin.eu/%20unilectin3d http://rna.sysu.edu.cn/dreambase http://lisanwanglab.org/dashrv2 http://zhaobioinfo.org/%20tissgdb http://zhaobioinfo.org/%20tissgdb http://tdb.ccmb.res.in/msdb http://tc3a.org/ https://cancersys.uni-koeln.de/ http://www.phenomicdb.de/ http://bioinfo.wilmer.jhu.edu/aagmarker/ eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 3 of 7 search and download interesting information conveniently. http://cis.hku.hk/cr2cancer, is readily accessible. activedriverdb activedriverdb is a comprehensive database of human proteogenomics which annotate disorder mutations and variants of the population via the ptm lens. more than 385,000 released ptm locations with 3.6 million replacements from the cancer genome atlas (tcga), the clinvar disease gene database, and human genome mapping projects were incorporated (8). the database involves sitespecific protein interaction networks, upstream enzymes like kinases, and those enzyme-targeted drugs. also, by evaluating advantages and losses of kinase-bound gene motifs, anticipated network-rewiring effect of mutations. for researching ptm-associated mutations, activedriverdb offers comprehensive visualization, processing, navigating, and search opportunities. users can interactively submit mutation datasets and use the coding tool in pipelines. integrative assessment of mutations and ptms, as illustrated by case studies of tp53, brca2 and vhl, can assist decipher molecular processes of disease and phenotypes. it can be found at https://www.activedriverdb.org. amtdb amtdb, the first database of ancient human mitochondrial genomes, is presented by ehler et al. (9). release edition includes 1107 hand-cured ancient samples, freely available for download, along with personal descriptors including geographic place, radiocarbon dating, and association to archeological culture. an interactive map for sample position visualization is also available in the database. on https://amtdb.org, amtdb is a main platform for ancestral population genetic research. hedd wang et al. (10) founded the human enhancer disease database (hedd) to promote enhancers research and their prospective functions in complicated human disorders. for some 2,8 million human enhancers recognized by en-code, fantom5 and roadmap, hedd presently offers extensive genomic data with disease connection results based on enhancer-gene and gene-disease links. it also offers webbased analytical tools to conceptualize enhancer networks and rank enhancers in a particular gene network given a collection of targeted genes. at http://zdzlab.einstein.yu.edu/ 1/hedd.php, hedd is readily available. diseaseenhancer zhang et al. (11) developed diseaseenhancer for disease-associated enhancers, a manually curated database. as of july 2017, in 143 human illnesses, diseaseenhancer involves 847 disease-associated enhancers. database attributes include fundamental data about enhancers; varieties of diseases; related variations of enhancers and their phenotypes mediated. they also include a website function for exporting any request outcomes to a folder and downloading the complete database. diseaseenhancer offers scientists with a successful opportunity for knowing enhancer deregulation in pathogenesis of disease and identifying new biomarkers for diagnosis and treatment of disorder. you can easily access diseaseenhancer at http://biocc.hrbmu.edu.cn/ diseaseenhancer/. dreambase zheng et al. (12) established dreambase to enable research into dna alteration, rna regulation and protein interacting from multidimensional high-throughput sequencing information of probable expressed pseudogenes. based on a series of data sets chip-seq and dnase-seq, genome-wide binding patterns of different transcriptionassociated variables were recognized around pseudo-gene loci. by incorporating some 18 000 rna-seq information, they evaluated pseudogene expression profiles and examined their modelsof co-expressionin 32 cancers and 31 ordinary tissues with their parent genes.they revealed complicated post-transcription regulation networks comprising 275 micrornas and 1201 pseudo-genes by connecting binding sites with microrna. they researched transcriptome-wide relationships based on 458 clip-seq datasets between rna binding enzymes (rbps) and pseudo-genes. they also linked 1039 rna alteration locations to 635 pseudo-genes in combination with epi-transcriptome sequencing information. this database would provide perspectives into pseudo-genes ' transcription regulating, expression, features and mechanisms as well as their functions in biological procedures and illnesses. at http://rna.sysu.edu.cn/dreambase, dreambase is readily available. dashr kuksa et al. (13) created a comprehensive catalog of annotation, expression, handling, conservation, tissue specificity and other biological characteristics for all human sncrna genes and mature products obtained from all main rna groups. the dashr (small human non-coding rnas database) database is the first to incorporate human sncrna gene as well as mature product profiles from numerous rna-seq protocols. for both grch38/hg38 and grch37/hg19 assemblies, 185 tissue / cell forms and sncrna annotations and > 800 curated tests from encode and geo / sra are incorporated into dashr. in addition, dashr is the first to comprise both recognized and novel http://cis.hku.hk/cr2cancer https://www.activedriverdb.org/ https://amtdb.org/ http://zdzlab.einstein.yu.edu/%201/hedd.php http://zdzlab.einstein.yu.edu/%201/hedd.php http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://biocc.hrbmu.edu.cn/%20diseaseenhancer/ http://rna.sysu.edu.cn/dreambase eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 4 of 7 sncrna loci detected by unsupervised segmentation, earlier unnoted. dashr also contains > 3,200,000 annotations for non-small genes of rna and other genomic characteristics. in addition, an improved user interface, integrated experiment-by-locus table display, sncrna locus sorting and biological feature processing is introduced. on http:// lisanwanglab.org/dashrv2, dashr is readily accessible. msdd yue et al. (14) have been described as msdd, presently documenting 525 associations among 182 human mirnas, 197 snps, 153 genes, and 164 human illnesses through an analysis of over 2000 articles released. each organization includes data on mirnas, snps, mirna target genes and disorder designations, snp places and allies, mirna dysfunctional model, experimental methods, a short functional description, initial reference and extra annotation. msdd offers a user-friendly interface for browsing, retrieving, downloading and submitting new information conveniently. msdd there is free access to http://tdb.ccmb. res.in/msdb. cancersysdb the cancer systems biology database (cancersysdb) was created by krempel et al. (15), a tool for extremely flexible cancer queries and data analysis across various data types and various research. a cancersysdb primary openly accessible instance can be used to achieve extremely versatile queries across various kinds of information as shown in extremely appropriate instances of use. they also show how the cancersysdb can be used in the study network the cancer genome atlas (tcga) to classify predictive cancer based on all-exome information from 9091 individuals. cancersysdb at https://cancersys.uni-koeln.de, it is publicly accessible. mirdip tokar et al. (16) provided mirdip, providing practically 152 million predictions of human microrna targets collected across 30 various resources. they also incorporated an interdisciplinary score, statistically implied from the predictions obtained and appointed to each unique microrna-target activity to provide a unified measure of trust. they show that integration of predictions throughout various assets does not accumulate predictive bias towards biological procedures or pathways. mirdip is accessible free of charge at http://ophid.utoronto.ca/mirdip/. medreaders a total of 731 tfs that could attach to methylated dna sequences in human and mouse research mentioned in the literature were individually selected by wang et al. (17). six human cell lines and one mouse cell line derived from the encode and geo database were used in silico approaches to anticipate methylated and un-methylated patterns of 292 tfs by incorporating whole genome bisulfite sequencing (wgbs) and chip-seq data. the database of medreaders provides an extensive resource for further research and experiment plans to guide. the portal can be found at http://medreader.org/. linkedomics the database of linkedomics (18) includes information on multi-omics and clinical information for 32 kinds of cancer and a total of 11,158 clients from the tcga initiative. it is also the first multi-omics repository to incorporate worldwide proteomics information from chosen tcga tumor samples based on mass spectrometry (ms) produced by the clinical proteomic tumor analysis consortium (cptac). linked omics has over a billion information points in total., to enable a extensive assessment of these data, the web application linked omics developed three analytical modules. the link finder module enables for flexible discovery of connections between a molecular or clinical trait of concern and all other characteristics, offering a chance to evaluate and visualize connections for each cancer cohort between billions of attribute pairs. they show that linked omics offers biologists and clinicians with a distinctive platform for accessing, analyzing and comparing multi-omics cancer information within and across tumor kinds. on http://www.linkedomics.org, linked omics is readily accessible. pancan-meqtl gong et al. (19) provided pancan-meqtl, a repository that integrates genome-wide genotype and dna methylation information to provide meqtls across 23 cancer kinds from the cancer genome atlas. in maximum, 8,028,964 cis-meqtls and 965,050 trans-meqtls were recognized. overall patient survival rates are correlated with 23,432 meqtls. in addition, 2,214 458 meqtls were recognized that overlap with identified loci recognized by genome-wide correlation studies. pancan-meqtl offers an easy-to-use web interface http://bioinfo.life.hust.edu.cn/ pancan-meqtl/ for browsing, scanning and accessing of interesting information. pitdb pitdb (20) is a free repository of translated genomic elements (tges) found in pit (transcriptomics-informed proteomics) studies. in pit, both rna-seq transcriptomics as well as proteomic mass spectrometry are used to evaluate a https://cancersys.uni-koeln.de/ http://ophid.utoronto.ca/mirdip/ http://medreader.org/ http://www.linkedomics.org/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ http://bioinfo.life.hust.edu.cn/%20pancan-meqtl/ eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 5 of 7 sample. transcripts constructed from rna-seq reads are being used to develop a library of sample-specific amino acid sequences that are searched for the obtained mass spectra, allowing the detection of any tge, not just those in canonical proteome databases. pitdb includes more than 74 000 separate tges from four species at the moment of writing, backed by more than 600 000 matches in the peptide spectrum. the database, available at http://pitdb.org, offers guiding evidence for each tge, often from multiple studies and an indicator of trust in the assessment and form of the tge, varying from recognized protein to multiple protein component kinds including different splice isoforms, to a putative novel molecule. qprimerdb lu et al. (21) established the qprimerdb repository based on the design of an automatic gene-specific qpcr primer and the verification workflow depending on thermodynamics. the qprimerdb database is the most comprehensive qpcr primary database currently available, with a web front-end that provides gene-specific and precomputed primary pairs across 147 major organisms including human, mouse, zebra fish, yeast, thale cress, rice, and corn. this database provides 3331426 of the best priming pairs for each gene, based on priming pair coverage, and 47760359 alternative gene-specific priming pairs, that can be easily downloaded in batch format. for qpcr priming pairs for 66 randomly chosen genes, the specificity and effectiveness was validated in six distinct species through qpcr assays and gel electrophoresis and it is open to the public via http://biodb.swu.edu.cn/qprimerdb. seecancer zhang et al. (22) has constructed a database on seecancer, which serves to show an extensive developmental stage of cancer-specific somatic occurrences and their temporal orders. by manually curing more than 10 000 published papers, 1231 developmental stage-specific genomic occurrences and 5772 temporal orders containing 82 human cancers and 23 tissue origins were gathered and recorded in the seecancer database. each entry includes the somatic occurrence, the developmental phase, the sort of cancer, the identification strategy and the appropriate evidence. seecancer offers an easy-to-use tool for browsing, finding and downloading developmental stage-specific somatic occurrences and temporal associations in multiple cancers.increasing regard to the evolution of cancer genome, the required data in seecancer has facilitated the knowledge of cancer etiology and the growth of developmental therapy, and has helped clinicians to find biomarkers for tumor progression surveillance. it is freely available via http://biocc.hrbmu.edu.cn/seecancer. unilectin3d mariethoz et al. (23) has created a unilectin3d database, a curated database that classifies source and fold lectins, cross-links to literature, many glycoscience databases and functional information such as recognized specificity. the database offers thorough data on lectins, their linked glycan ligands as well as their relationships using the protein–ligand interaction profiler (plip) server. special attention was paid to the depiction of linked glycan ligands using easy graphical depiction and statistical format of crosslinking to other glycol scientific databases.they designed the layout of the database structure and navigation instruments to account for all species, and the search for oligosaccharide epitopes complexed inside selected binding regions. unilectin3d is available link https://www.unilectin.eu/ unilectin3d. translatomedb liu et al. (24) has developed an extensive translatomedb database that offers a set and embedded assessment of published and client-generated translatom sequencing information. the present edition involves 2453 ribo-seq, 10 rnc-seq and 1394 associated mrna-seq datasets for 13 species. in relation to dataset sets, the database emphasizes the evaluation tasks. all datasets were evaluated using a unified, reliable, precise and experimentally verifiable pipeline centered on the fanse3 mapping algorithm and edger for dge analysis. translatomedb also enables clients to upload their own datasets and use the same unified pipeline to evaluate their information. accessible at http://www.translatomedb.net/. tissgdb kim et al. (25) constructed tiss-gdb (tissue specific gene database for cancer) by collecting and manually curated 2461 tissue-specific genes (tissgenes) across 22 tissue classes corresponding to 28 cancers of the cancer genome atlas (tcga) from three different tissue-specific gene cell assets: the human protein atlas (hpa), the tissuespecific gene expression and regulation (tiger) and the genoty map. these 2461 tissgenes also conducted gene expression, somatic mutation, and prognostic marker-based analyzes of 28 kinds of cancer using tcga information. tissgdb sets out seven types of annotations: tissgeneexp,tissgene summary, tissgenemut, and tissgenemirna. it is freely via http://zhaobioinfo.org/tissgdb. http://pitdb.org/ http://biodb.swu.edu.cn/qprimerdb http://biocc.hrbmu.edu.cn/seecancer https://www.unilectin.eu/%20unilectin3d https://www.unilectin.eu/%20unilectin3d http://www.translatomedb.net/ http://zhaobioinfo.org/tissgdb eukaryotic molecular biology databases highlights in bioscience doi:10.36462/h.biosci.20184 may 2018 | volume1 page 6 of 7 tc3a feng et al. (26) proved that cfim25-mediated 3 utr reducing through apa encourages in vitro and in vivo development of glioblastoma tumors and further underscores its importance for tumorgenesis. cancer 3 utr atlas (tc3a), an extensive apa use tool for 10,537 tumors across 32 kinds of cancer, is reported. these apa occurrences are possibly novel prognostic biomarkers and may uncover novel processes for the regulation of cancer-causing genes. tc3a is constructed on top of the current cbioportal de facto standard. a big group of current cbioportal consumers and clinical researchers will therefore discover tc3a acquainted and immediately available at http://tc3a.org. referances 1. mokhtar mm, adawy ss, el-assal se-ds, hussein eha. genic and intergenic ssr database generation, snps determination and pathway annotations, in date palm (phoenix dactylifera l.). plos one. public library of science; 2016;11(7):e0159268. 2. adawy ss, mokhtar mm, alsamman am, sakr mm. development of annotated est-ssr database in olive (olea europaea). int j sci res. 2015;4(9):1063–73. 3. zhang y, zhang x, wang y, shen s. de novo assembly of transcriptome and development of novel est-ssr markers in rhododendron rex lévl . through illumina sequencing. 2017;8(september):1–12. 4. kahraman a, avramov a, nashev lg. phenomicdb : a multi-species genotype / phenotype database for comparative phenomics. bioinformatics. 2018; 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2018;46(october 2017):113–20. 11. zhang g, shi j, zhu s, lan y, xu l, yuan h, et al. diseaseenhancer : a resource of human diseaseassociated enhancer catalog. nucleic acids res. oxford university press; 2018;46(october 2017):78–84. 12. zheng l, zhou k, liu s, zhang d, wang z, chen z, et al. dreambase : dna modification , rna regulation and protein binding of expressed pseudogenes in human health and disease. nucleic acids res. oxford university press; 2018;46(october 2017):85–91. 13. kuksa pp, amlie-wolf a, katanić ž, valo, wang l, leung yy. dashr 2 . 0 : integrated database of human small non-coding rna genes and mature prod ucts. bioinformatics. 2018;(august). 14. yue m, zhou d, zhi h, wang p, zhang y, gao y, et al. msdd : a manually curated database of experimentally supported associations among mirnas , snps and human diseases. nucleic acids res. oxford university press; 2018;46(november 2017):181–5. 15. krempel r, kulkarni p, yim a, lang u, habermann b, frommolt p. integrative analysis and machine learning on cancer genomics data using the cancer systems biology database ( cancersysdb ). bmc bioinformatics. bmc bioinformatics; 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2018;46:1–7. 20. saha s, chatzimichali ea, matthews da, bessant c. pitdb : a database of translated genomic elements. nucleic acids res. oxford university press; 2018;46(october 2017):1223–8. 21. lu k, li t, he j, chang w, zhang r, liu m, et al. qprimerdb : a thermodynamics-based gene-specific qpcr primer database for 147 organisms. nucleic acids res. oxford university press; 2018;46(august 2017):1229–36. 22. zhang h, luo s, zhang x, liao j, quan f, zhao e, et al. seecancer : a resource for somatic events in evolution of cancer genome. nucleic acids res. oxford university press; 2018;46(october 2017):1018–26. 23. mariethoz j, salentin s, robin x, imberty a, schroeder m, perez s. unilectin3d , a database of carbohydrate binding proteins with curated information on 3d structures and interacting ligands franc. nucleic acids res. 2018;46:1–9. 24. liu w, xiang l, zheng t, jin j, zhang g. translatomedb : a comprehensive database and cloudbased analysis platform for translatome sequencing data. nucleic acids res. oxford university press; 2018;46(november 2017):206–12. 25. kim p, park a, han g, sun h, jia p, zhao z. tissgdb : tissue-specific gene database in cancer. nucleic acids res. oxford university press; 2018;46(september 2017):1031–8. 26. feng x, li l, wagner ej, li w. tc3a : the cancer 3 utr atlas. nucleic acids res. oxford university press; 2018;46(october 2017):1027–30. highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202203 research article open access 1 biotechnology department, science faculty, helwan university, helwan, egypt. 2 biotechnology department, agriculture faculty, al-azhar university, cairo, egypt. * to whom correspondence should be addressed: saifeldeenmib99@gmail.com editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): jean legeay, université de lorraine, inra, umr iam interactions arbres-microorganismes, f54000 nancy, france. vivek chaudhary, motilal nehru national institute of technology, allahabad, prayagraj, uttar pradesh 211004, india. received: september 20, 2022 accepted: december 3, 2022 published: december 15, 2022 citation: ibrahim ms, ibrahim sm. identifying biotic stress-associated molecular markers in wheat using differential gene expression and machine learning techniques. 2022 dec 15;5:bs202203 copyright: © 2022 ibrahim and ibrahim. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. identifying biotic stress-associated molecular markers in wheat using differential gene expression and machine learning techniques manar s. ibrahim1 ><, saifeldeen m. ibrahim*2 >< abstract wheat is an important crop for global food security and a key crop for many developing countries. thanks to next-generation sequencing (ngs) technologies, researchers can analyze the transcriptome of wheat and reveal differentially expressed genes (degs) responsible for essential agronomic traits and biotic stress tolerance. in addition, machine learning (ml) methods have opened new avenues to detect patterns in expression data and make predictions or decisions based on these patterns. we used both techniques to identify potential molecular markers in wheat associated with biotic stress in six gene expression studies conducted to investigate powdery mildew, blast fungus, rust, fly larval infection, greenbug aphid, and stagonospora nodorum infections. a total of 24,152 threshold genes were collected from different studies, with the highest threshold being 7580 genes and the lowest being 1073 genes. the study identified several genes that were differentially expressed in all comparisons and genes that were present in only one data set. the study also discussed the possible role of certain genes in plant resistance. the ta-tlp, hbp1, wrky, ppo, and glucan endo-1,3-beta-glucosidase genes were selected by the interpretable model-agnostic explanation algorithm as the most important genes known to play a significant role in resistance to biotic stress. our results support the application of ml analysis in plant genomics and can help increase agricultural efficiency and production, leading to higher yields and more sustainable farming practices. keywords: wheat, gene expression, machine learning, biotic stress, disease introduction agriculture is the primary source of food production on a global scale. in the new era, sustainable agriculture is used to preserve or improve food quality while protecting the environment [1]. wheat (triticum aestivum) is a member of the poaceae family and is considered the oldest and most commonly cultivated crop [2], with an annual cultivation area of 217 million hectares. it is the most commonly grown crop in the world [3]. also, it is one of three primary cereal crops with the greatest impact on global food security, second only to maize (zea mays) and third only to rice [4]. most experts assume that wheat was the first crop to be produced between 10,000 and 8,000 b.c. [2], and this is considered evidence of the importance of wheat from ancient times. wheat is one of the most essential crops for ensuring global food security [5]. considering the position of wheat in the world trade, wheat accounts for over 50% of the worldwide grain trade and 30% of the global grain yield [6]. and for its role in nutrition, wheat is a staple crop in more than 40 nations worldwide, feeding 82% to 85% of the world’s population [6]. according to the estimations, the annual wheat production is expected to reach 750.4 million metric tonnes in the next few years. africa would consume 76.5 mmt of wheat, with imported grain accounting for 48.3 mmt, or 63.3 percent of total consumption [7]. powerful yet user-friendly bioinformatics tools are required by the expanding field of gene expression profiling in order to facilitate systems-level data comprehension [8]. studies on transcriptomics have a significant potential for unique and exploratory research aimed at new insights into molecular pathways [9]. highlights in bioscience page 1 of 11 december 2022|volume 5 https://doi.org/10.36462/h.biosci.202203 https://creativecommons.org/licenses/by/4.0/ manar.samir249@science.helwan.edu.eg https://orcid.org/0000-0003-0143-2596 saifeldeenmib99@gmail.com https://orcid.org/0000-0002-6593-378x http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat next-generation sequencing (ngs) technologies greatly improve studies on genome-wide mrna expression data. in comparison to microarray technology, ngs provides higher resolution data and more precise transcript level measurements for gene expression studies [10]. by studying the wheat transcriptome, it is possible to identify the differentially expressed genes, genomic annotations, regulatory elements, molecular markers, and expression quantitative trait loci (eqtls), as well as their sequence variants, which are responsible for significant characteristics [11]. with the advancement of next-generation sequencing technology, the genetic diversity of wheat has recently been studied using a high-throughput, low-cost molecular technique [12].the significant genetic variation seen in the primary, secondary, and tertiary wheat gene pools provides the starting point for the breeding of nutrient-rich wheat genotypes [13]. a variety of molecular markers have been used to study the genetic diversity of wheat [12] to measure biological population divergence’s magnitude and each component character’s proportionate contribution to the overall divergence [14]. machine learning (ml) is a rapidly growing field of computer science that has the potential to revolutionize many industries, including plant science. machine learning algorithms use statistical techniques to identify patterns in data and make predictions or decisions based on those patterns. this makes them well-suited to solving complex problems that are difficult for humans to tackle, such as analyzing vast amounts of data from experiments or monitoring the health of crops. in plant science, machine learning can be used to predict the best time to plant crops, optimize irrigation systems, identify pests and diseases, and even breed new varieties of plants. this could help improve the efficiency and productivity of agriculture, leading to better yields and more sustainable farming practices. overall, the use of machine learning in plant science has the potential to significantly advance the field and improve our understanding of plants. consequently, in this study, the aim of our investigation is to perform expression profiling analysis of wheat to identify unknown molecular markers. we aim to use bioinformatics tools to ;(a) study the gene expression of wheat under various environmental and developmental conditions. (b) study the genetic diversity of these genes across the wheat genome. (c) identify important genes and their interactions. (d) use machine learning methods to identify genes associated with wheat response to biotic stresses. by doing so, we hope to gain a better understanding of the genetic basis of wheat response to biotic stresses, which could ultimately lead to the development of more resilient and productive varieties of wheat. this is important because biotic stresses, such as pests and diseases, can cause significant yield losses and can threaten the global food supply. by identifying molecular markers that are associated with wheat response to biotic stresses, we can improve our ability to breed new varieties of wheat that are better able to withstand these stresses. this could help to improve the efficiency and productivity of agriculture, leading to better yields and more sustainable farming practices. methodology data acquisition the gene expression omnibus (geo) database, first established in 2005 [15], has continued to be a vital resource for the global scientific community. with over 20 years of experience, geo remains the most widely used public repository for highthroughput gene expression and other functional genomics data sets. this valuable resource allows researchers to quickly and easily access a wide range of raw and processed data, along with detailed experimental descriptions, at no cost. the convenience and accessibility of geo make it an invaluable tool for advancing scientific research. in this study, geo has been used to download the data of six different experiments on wheat for further analysis. these experiments have been conducted under different biotec stresses. such as the response to powdery mildew infection, the response to blast fungus magnaporthe, the resistance pathways of transgenic wheat lines, the response to a hessian fly larval attack, the response to greenbug aphid feeding, and the response to the necrotrophic effector sntox3. all gse accessions are provided with the experiment name of each accession in (table 1). differential gene expression analysis with the help of the r/bioconductor and limma package, the online statistical tool geo2r [16] was used to analyze the raw six gene expression data sets of wheat under different biotic stresses from geo datasets [17] (table1). the samples have been clustered into two groups (control and infected / treatment). differential expression genes were analyzed using r packages to create a volcano plot highlighting all significant genes and a heatmap of the top differentially expressed genes based on each group in all samples according to log2fc (fold change) ≥ 1 and adjusted p-value < 0.05 as the threshold for degs. whereby up-regulated degs were considered if the logfc (fold change) ≥ 1 and down-regulated degs were considered if the logfc ≤ -1. a volcano plot shows statistical significance (p value) versus magnitude of change (fold change). it enables quick visual identification of genes with large fold changes that are also statistically significant. these may be the most biologically significant genes [18]. first, a volcano plot was generated using the r package (ggplot2) after reading the data and filtering it according to the adjusted p-value and logfc (blue: down-regulated, red: upregulated). second, a heatmap of the top differentially expressed genes in the rna-seq data set was then generated. to do this, we need to extract the differentially expressed genes from the de results file. the heatmap for the gene expression data was generated using r package (pheatmap) after filtering the top differential gene expression from the data. then, a venn diagram was used to show all the interrelationships of the six wheat experiments. the venn diagram of the overlapping degs was output highlights in bioscience page 2 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat by the interactivenn website [19]. protein-protein interaction and and functional enrichment analyses the string database was utilised for protein-protein interaction analysis [20] to evaluate the link between genes associated with wheat gene expression. the string database was chosen because this database seeks to integrate all known and predicted relationships between proteins, including functional and physical interactions [20]. all information about go, annotated keywords, and protein domains for each gse dataset has been provided in (table 2) machine learning model the results of the differential gene expression study of putative biotic-associated gene biomarkers were employed as attribute values for machine learning (ml) analysis. we retrieved gene expression data from genes that were strongly expressed in gene expression. their expression data were utilised as training and validation sets for the ml analysis. the gene expression data were adjusted before to the ml analysis. the sklearn and lime0.2.0.1 python libraries were used to run the "extra-tree regressor" and local interpretable model-agnostic explanations algorithms. results and discussion identification of degs a total of 24,152 threshold genes were collected from different studies. the highest threshold of genes was collected by gse27320, with 7580 genes, while gse31760 had the lowest threshold of genes, with only 1073 genes. the volcano plot indicates the upregulated (red color) and downregulated (blue color) genes in triticum aestivum samples. the horizontal axis represents the fold change (log2fc), and the vertical axis represents the adjusted p-values. a total of 3701 upregulated and 3879 downregulated degs were identified from the gse27320 dataset, while 241 upregulated and 832 downregulated degs were identified from the gse31760 dataset. gse32151 yielded 3533 upregulated and 5138 downregulated degs, gse34445 yielded 658 upregulated and 1442 downregulated degs, gse45995 yielded 2054 upregulated and 1459 downregulated degs, and gse59723 yielded 74 upregulated and 1141 downregulated degs. the volcano plot of each gene expression profile data is shown in (figure 1). users can use heatmaps to examine the expression of a subset of genes. this can provide helpful insight into the expression of various groups and samples without losing sight of the larger study or losing clarity when evaluating patterns averaged over hundreds of genes at once [18]. according to the adjusted pvalue ranking, a heatmap is created for the top de genes.the heatmap clusters the samples into control (blue) and infected / treatment (red) groups. red indicates samples with relatively high gene expression, while blue indicates samples with relatively low gene expression. genes with intermediate expression levels are represented by lighter tones and white. a dendrogram hierarchical clustering has been used to reorder the samples and genes (figure 2). venn diagrams of the degs between the integrated six geo data sets are shown in (figure 2). the number in each circle indicates the quantity of differentially expressed genes across the various comparisons. the overlapping number refers to genes that are differently expressed across all comparisons, while the non-overlapping number refers to genes that are unique to each sample. genes were identified as being upor down-regulated. the gse32151 and gse45995 data sets shared 12 genes, while the gse45995 and gse59723 data sets shared 6 genes (pdi2, pepc, gstf5, gstf5:1, and ltp9.2). according to the study of guo-tian liu et al.[21], gstf5 is a member of the (gsts) family, which is responsible for catalysing the conjugation of the reduced form of glutathione to a number of electrophilic substrates, they also reported that gsts contribute to resistance against powdery mildew. the gse31760, gse45995, and gse59723 data sets shared 3 genes (rgp, pr4, and pox3). the relationship between methyl jasmonate (meja) and the expression profiles of nine pathogenesisrelated protein genes (pr genes) was examined in the zongbiao duan et al. [22] study to determine the interactive role in powdery mildew resistance. this investigation revealed that pr4 is one of the pathogenesis-related protein genes (pr genes), and the expressions of pr4 and other pr genes were most significantly activated by meja and showed a significant resistance to powdery mildew. the gse32151 and gse59723 datasets shared 1 gene (xip-r1). xip-r1 is one of the xylanase inhibitors. r.-j. sun et al. [23], xylanase inhibitors (xis) are plant cell wall proteins found mostly in monocots that limit microbial xylanases’ hemicellulose degrading ability. silvio tundo et al. [24]. the gse34445 and gse59723 data sets shared 4 genes (wrky45, cht2, wrky, and ccd1), and the gse31760 and gse59723 shared 2 genes (taaos and tamdr1). the allene oxide synthase (taaos) gene has been identified as being engaged in the ja signalling system, which increases plant resistance to fusarium head blight (fhb) [25], tamdr1 has also been identified as a fusarium head blight resistance gene [26]. the gse27320 and gse31760 shared 2 genes (ald and psbger4), and the gse27320 and gse32151 data sets shared 2 genes ( hsp16.9-3lc2 and s276 ) . the gse32151 and gse34445 data sets shared 1 gene (taaml15), and the gse27320 and gse34445 data sets shared 3 genes (ss1, wpi6, and gstf6b). the gse34445 and gse45995 data sets shared 4 genes (taglb2b, taakt1, tamrp2, and omet). pratiksha singh et al. [27] reported that taglb2b showed a significant response to erysiphe graminis and fusarium graminearum. the gse27320, gse31760, and gse34445 data sets shared 1 gene (hsp101c) which has an important role in heat tolerance in hexaploid wheat [28]. the gse27320 and gse45995 data sets shared 13 genes (tra2, tra2:1, tac23, zip1, tagi1, psbp, ctpa, wcor726, pre-fbpase, lgul, gapn, 6-feh, and lgul:1). in a study by yu liu et al. to examine the response of psbp to highlights in bioscience page 3 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat table 1. information for the six geo datasets for wheat. unique identifier for the dataset within geo (accession), full name of the experiment (experiment name), shortened version of the experiment name (short name), ans the abbreviated version of the experiment name (abbrev). accession experiment name short name abbrev gse27320 expression data in wheat (t. aestivum l.) near isogenic lines in response to powdery mildew infection wheat responses to powdery mildew wrpm gse31760 transcription profiling wheat responses to adapted and non-adapted isolates of the blast fungus, magnaporthe wheat responses to blast fungus wrbf gse32151 lr1-mediated leaf rust resistance pathways of transgenic wheat lines mediated leaf rust resistance mlrr gse34445 expression data from wheat following hessian fly larval attack. wheat following fly larval wffl gse45995 transcriptomics of induced defense responses to greenbug aphid feeding in near isogenic wheat lines wheat responses to greenbug aphid wrga gse59723 transcriptional analyses of wheat responses to the necrotrophic effector sntox3 wheat responses to sntox3 wrst table 2. the sring database table provides information about go, annotated keywords, and protein domains for each gse dataset. gse category id description fdr gse27320 molecular function go:0005200 structural constituent of cytoskeleton 0.0046 cellular component go:0110165 cellular anatomical entity 2.86e-24 kegg pathways map04145 phagosome 0.0343 annotated keywords kw-0963 cytoplasm 0.00025 kw-0206 cytoskeleton 0.0015 kw-0493 microtubule 0.0016 kw-0342 gtp-binding 0.0065 kw-0809 transit peptide 0.0256 protein domains pf03953 tubulin c-terminal domain 0.00055 pf00091 tubulin/ftsz family, gtpase domain 0.0012 protein features ipr000217 tubulin 2.43e-05 ipr002453 beta tubulin 2.43e-05 ipr003008 tubulin/ftsz, gtpase domain 2.43e-05 ipr008280 tubulin/ftsz, c-terminal 2.43e-05 ipr013838 beta tubulin, autoregulation binding site 2.43e-05 ipr017975 tubulin, conserved site 2.43e-05 ipr018316 tubulin/ftsz, 2-layer sandwich domain 2.43e-05 ipr023123 tubulin, c-terminal 2.43e-05 ipr037103 tubulin/ftsz, c-terminal superfamily 2.43e-05 ipr036525 tubulin/ftsz, gtpase domain superfamily 2.71e-05 ipr000877 proteinase inhibitor i12, bowman-birk 0.0046 protein domains sm00269 bowman-birk type proteinase inhibitor 0.004 gse31760 cellular component go:0110165 cellular anatomical entity 0.001 gse32151 cellular component go:0110165 cellular anatomical entity 6.46e-37 annotated keywords kw-0325 glycoprotein 3.38e-05 kw-1015 disulfide bond 0.00015 kw-0732 signal 0.00024 kw-0809 transit peptide 0.0023 kw-0963 cytoplasm 0.0023 kw-0326 glycosidase 0.0028 kw-0378 hydrolase 0.0062 kw-0676 redox-active center 0.0206 kw-0119 carbohydrate metabolism 0.0217 kw-0624 polysaccharide degradation 0.0386 protein features ipr000877 proteinase inhibitor i12, bowman-birk 0.00055 protein domains sm00269 bowman-birk type proteinase inhibitor 4.17e-05 gse34445 cellular component go:0110165 cellular anatomical entity 1.42e-07 gse45995 cellular component go:0110165 cellular anatomical entity 1.53e-19 subcellular localization gocc:0005576 extracellular region 0.0056 annotated keywords kw-0809 transit peptide 0.0011 kw-0150 chloroplast 0.0022 kw-0732 signal 0.0039 kw-0676 redox-active center 0.0081 kw-0597 phosphoprotein 0.0241 kw-0963 cytoplasm 0.0338 protein domains sm00269 bowman-birk type proteinase inhibitor 0.004 gse59723 cellular component go:0110165 cellular anatomical entity 1.75e-08 annotated keywords kw-1015 disulfide bond 0.00015 kw-0732 signal 0.00097 kw-0325 glycoprotein 0.0088 kw-0326 glycosidase 0.0214 protein features ipr000877 proteinase inhibitor i12, bowman-birk 0.0199 protein domains sm00269 bowman-birk type proteinase inhibitor 0.0016 table 3. the results of the machine learning model represent the most significant genes with their corresponding values. accsession marker p/n value gene.symbol gse27320 ta.85.1.s1 positive 28944.5 psbp ta.1842.1.s1_a positive 309.3 wpeamt ta.1848.2.s1 positive 4463.8 pip1:2 ta.2907.1.s1 positive 4340.4 loc543101 ta.10.1.s1_a positive 47759.5 loc543334 taaffx.120000.1.s1 positive 465.7 tlk1 ta.10.2.s1 positive 63930.8 loc543334 ta.21350.2.s1 positive 216.2 wrsi5-1 ta.21348.1.s1 positive 292.1 loc543233 ta.23758.1.s1 positive 265.9 wcor518 gse31760 ta.28.1.s1 negative 16942 loc543330 ta.27762.1.s1 negative 3687.7 ta-tlp ta.24501.1.s1 negative 2852.3 loc543292 ta.2788.1.a1 negative 768.65 1-sst ta.82.1.s1 negative 8065.5 loc543287 taaffx.115935.1.s1 negative 375.52 loc543157 ta.87.1.s1 negative 654.58 psbger4 ta.24254.3.s1 negative 333.75 loc606342 ta.25053.1.s1 negative 5220.3 loc542887 ta.22673.1.s1_s negative 14994 loc543498 gse32151 ta.9320.1.s1 negative 11.7 ccoamt ta.9320.1.s1 negative 11.81 ccoamt ta.9402.1.s1 negative 8.97 loc542918 ta.8629.1.a1 negative 5.9 wltp1 taaffx.39351.2.a1 negative 9.64 hrbp1-1 ta.9402.1.s1 negative 8.92 loc542918 ta.24806.1.s1 negative 10.79 wlip19 ta.28700.1.s1 negative 4.55 chs ta.2907.1.s1 negative 11.76 loc543101 ta.28734.1.s1 negative 15.08 tagrp2 gse34445 ta.8614.1.s1 negative 46.58 wrky45 ta.27312.1.s1 negative 63.75 amt1 ta.4050.1.s1 negative 136.86 loc100037581 ta.203.1.s1 negative 86.99 loc543416 ta.1058.1.s1 negative 11290.8 samdc1 ta.192.1.s1 negative 79 loc543235 ta.4678.2.s1 negative 61.95 wrky71 ta.217.1.s1 negative 1033.27 loc543244 ta.1058.3.s1 negative 15078.5 samdc1 taaffx.129134.2.s1 negative 281.44 a2b gse45995 ta.28.1.s1 negative 37166.6 loc543330 ta.9226.1.s1 negative 41349.6 pr4 ta.2784.1.a1 negative 42724.1 chi 1 ta.14183.1.s1 negative 304.86 loc543077 ta.21342.1.s1 negative 34995.6 chi 3 ta.22871.1.s1_s negative 13472.6 gamma-tip taaffx.39351.2.a1 negative 947.06 hrbp1-1 ta.27762.1.s1 negative 31690.6 ta-tlp ta.278.1.s1 negative 41378.7 loc543422 ta.278.1.s1 negative 41179.1 loc543422 gse59723 ta.5428.1.s1 negative 7.41 act3 ta.8228.1.s1 negative 4.69 loc543097 taaffx.128418.102.s1 negative 2.53 ltp9.2 ta.706.1.s1_s negative 7.5 g6pdh ta.81.1.s1 negative 6.86 pepc ta.24501.1.s1 negative 10.2 loc543292 ta.4725.1.s1 negative 5.08 wrky53-b ta.27762.1.s1 negative 7.64 ta-tlp ta.234.1.s1 negative 4.48 lct1 taaffx.115935.1.s1 negative 5.11 loc543157 highlights in bioscience page 4 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat figure 1. visualization of degs volcano plots.the representations are as follows: x-axis, log2fc; y-axis, -log10 of an adjusted p-value. highlights in bioscience page 5 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat figure 2. heatmap and venn diagram of the top de genes data highlights in bioscience page 6 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat figure 3. ppi networks show the interaction of degs. highlights in bioscience page 7 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat figure 4. machine learning results and venn diagrams of the markers, genes, and gene titles that were shared between gses. highlights in bioscience page 8 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat wheat dwarf virus (wdv), they noticed that the psbp gene was down-regulated under wdv infection [29]. the gse27320, gse45995, and gse59723 data sets shared 3 genes (pox4, wrsi5-1, and was-2). the gse27320, gse31760, gse45995, and gse59723 data sets shared 2 genes (ta-tlp and chi 3). the gse27320, gse32151, and gse45995 data sets shared 14 genes, while the gse27320, gse32151, gse45995 , and gse59723 data sets shared 5 genes (taxi-iii, taxi-iii:1, wali6, taxi-iv, and pox2). the gse27320, gse34445, and gse45995 data sets shared 1 gene (wcab) which was highly significant in the response to stripe rust in wheat [30]. the gse27320, gse31760, gse34445, and gse45995 data sets shared 1 gene (rbcl), in an investigation by ceyda ozfidan -konakci et al., the rbcl gene was observed at higher transcription levels in the chloroplasts of wheat as a response to hydrogen sulphide (h2s) and nitric oxide (no) alleviate cobalt toxicity. the gse31760, gse34445, and gse45995 data sets shared 1 gene (prx), and the gse27320, gse34445, and gse59723 data sets shared 1 gene (fkbp77) which has a high expression under heat stress [31]. the gse27320, gse32151, gse34445, and gse59723 data sets shared 1 gene (taxi-i). gse32151, gse34445, gse45995, and gse59723 data sets shared 3 genes (xipi:1, tamyb7, and xipi). the tamyb7 is one of the myb family, which is considered to have a significant role in resisting stripe rust [32]. gse27320, gse34445, gse45995, and gse59723 data sets shared 1 gene (wrn2). gse31760, gse34445, gse45995, and gse59723 data sets shared 1 gene (s85). the gse31760, gse32151, gse34445, and gse59723 data sets shared 1 gene (wck-1), wck-1 expression is reported to be induced by the fungal elicitor calcium ionophore a23187 as well as drought [33]. gse31760, gse34445, and gse59723 data sets shared 2 genes (cht4 and gstu2), cht-4 expression was substantially faster in the resistant cultivar than in the susceptible one, in the response to fusarium graminearum in wheat [34]. gse32151, gse45995, and gse59723 data sets shared 4 genes (tamyb7:2, wali5, chi 2, and tamyb7:1), guozhang kang et al. [35] discovered that freezing stress increased the expression of the wali5 protein gene. gse31760, gse32151, gse45995, and gse59723 data sets shared 2 genes (wrky53-b and chi 1). the gse31760, gse32151, and gse45995 data sets shared 1 gene (dbp), and the gse27320, gse32151, and gse34445 data sets shared 2 genes (amt1 and amt1:1). ppi network construction protein-protein interaction (ppi) networks were created using the string tool. six modules were identified in this constructed network, which was made up of 115 nodes and 56 edges. the top significant module was ges32151, which was composed of 44 nodes and 30 edges, and had a ppi enrichment pvalue of 0.0565. the lowest module was gse34445, which was made up of only 11 nodes, 1 edge, and had a ppi enrichment p-value of 0.174 (figure 3). machine learning the machine learning model identified 53 markers, 48 genes, and 46 gene titles, as shown in (table 3). also see (figure 4). each gse accession consisted of ten markers. only gse27320 had positive values, ranging from 216.2 to 63930.8. the rest of the gses had negative values, as the table shows. some of the gses shared the same markers, genes, and gene titles. according to the venn diagram (figure 4) of the markers, gse32151 and gse45995 shared taaffx.39351.2.a1. gse31760, gse45995, and gse59723 shared ta.27762.1.s1. gse31760 and gse59723 shared ta.24501.1.s1 and taaffx.115935.1.s1. gse27320 and gse32151 shared ta.2907.1.s1. gse31760 and gse45995 shared ta.28.1.s1. for genes, gse32151 and gse45995 shared hrbp1-1. gse31760 , gse45995 , and gse59723 shared ta-tlp. gse31760 and gse59723 shared loc543292 and loc543157. gse27320 and gse32151 shared loc543101. gse31760 and gse45995 shared loc543330. for gene titles, gse32151 and gse45995 shared harpin binding protein 1. gse31760, gse45995, and gse59723 shared thaumatin-like protein. gse34445 and gse59723 shared wrky transcription factor. gse31760 and gse59723 shared polyphenol oxidase. gse27320 and gse32151 shared methylmalonate semialdehyde dehydrogenase. gse31760 and gse45995 shared glucan endo-1 and 3-beta-d-glucosidase. danielle et al. [36] mention in their study that the ta-tlp gene is a member of pathogenesis-related proteins and has an important role in plant resistance to powdery mildew. another study by zhi-hui he et al. [37] reported that harpin-binding protein 1 is thought to be involved in plant disease resistance and drought resilience because it is responsible for plastid glutamine synthase (gs). it was shown to be down-regulated in wheat’s single seed descent line 10 (ssdl 10). furthermore, thaumatinlike proteins (tlps), as reported by weibo sun et al. [38], play roles in plant resistance to pathogen stress by acting as a positive factor in transgenic poplars with enhanced resistance to spots disease. for wrky transcription factors, patel p et al [39] mentioned in their study that wrky transcription factors are thought to have a role in contributing to the level of thermotolerance. it was reported [39] that polyphenol oxidase (ppo) has a role in plant resistance to osmotic stress-tolerant bacteria. they found that bacterized seedlings showed a slight improvement in wheat roots and shoots, and the polyphenol oxidase was greater in the roots and shoots. hegedus et al. [40] reported that the overexpression of glucan endo-1,3-beta-glucosidase has been linked to a variety of physiological and developmental processes, as well as resistance to biotic and abiotic stress. this suggests that these genes may play an important role in plant resistance to various stresses and diseases. highlights in bioscience page 9 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ ibrahim and ibrahim, 2022 identifying biotic stress associated molecular markers in wheat reference 1. gimenez e, salinas m, manzano-agugliaro f. worldwide research on plant defense against biotic stresses as improvement for sustainable agriculture. sustainability. 2018;10(2):391. 2. igrejas g, branlard g. the importance of wheat. in: wheat quality for improving processing and human health. springer; 2020. p. 1-7. 3. erenstein o, jaleta m, mottaleb ka, sonder k, donovan j, braun hj. global trends in wheat production, consumption and trade. in: wheat improvement. springer; 2022. p. 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transformation as efficient tools to fight against fungal diseases. journal of agricultural science and technology a. 2015;5(3):153-61. 37. he zh, li hw, shen y, li zs, mi h. comparative analysis of the chloroplast proteomes of a wheat (triticum aestivum l.) single seed descent line and its parents. journal of plant physiology. 2013;170(13):1139-47. 38. sun w, zhou y, movahedi a, wei h, zhuge q. thaumatinlike protein(pe-tlp)acts as a positive factor in transgenic poplars enhanced resistance to spots disease. physiological and molecular plant pathology. 2020;112:101512. 39. patel p, patil t, maiti s, paul d, amaresan n. screening of osmotic stress-tolerant bacteria for plant growth promotion in wheat (triticum aestivum l.) and brinjal (solanum melongena l.) under drought conditions. letters in applied microbiology. 2022;75(5):1286-92. 40. hegedus g, kutasy b, kiniczky m, decsi k, juhász á, nagy á, et al.. liposomal formulation of botanical extracts may enhance yield triggering pr genes and phenylpropanoid pathway in barley (hordeum vulgare). plants 2022, 11, 2969. s note: mdpi stays neutral with regard to jurisdictional claims in published ; 2022. highlights in bioscience page 11 of 11 december 2022|volume 5 http://bioscience.highlightsin.org/ abstract introduction methodology data acquisition differential gene expression analysis results and discussion identification of degs ppi network construction machine learning highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202505 research article open access 1 department of biotechnology research, kyaukse, ministry of science and technology, myanmar * to whom correspondence should be addressed: sakurasan2007@gmail.com editor: muhammad m. adeel arthritis clinical immunology program oklahoma medical research foundation, oklahoma city, united states. reviewer(s): alsayed alsoudy, mohammed vi polytechnic university, ben guerir, morocco. wan-atirah azemin, school of biological sciences, universiti sains malaysia, minden, pulau pinang, malaysia. received: march 25, 2025 accepted: june 27, 2025 published: july 26, 2025 citation: yu ss, marr kt, mon ww, aye ks. molecular identification and screening the antagonistic and cellulolytic activities of native strain bacillus subtilis isolated from myanmar marine sediment. 2025 july 26;8:bs202505 copyright: © 2025 yu ss et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: this work was supported by the department of biotechnology research department, kyaukse, ministry of science and technology, myanmar, by the department of plant medicine, kyungpook national university, daegu, south korea, and by the international scholar exchange fellowship program, chey institute of advanced studies. competing interests: the authors declare that they have no competing interests. molecular identification and screening the antagonistic and cellulolytic activities of native strain bacillus subtilis isolated from myanmar marine sediment san san yu*1 ><�, khin thae marr1 >< �, wai wai mon1 ><�, khin swe aye1 >< abstract in order to combat plant diseases and produce profitable agricultural crops, biocontrol agents derived from bacteria offer sustainable and eco-friendly options. numerous fungal diseases have been successfully treated by some advantageous strains of bacillus subtilis. the bacterial strain used in this study, designated s1-10, was isolated from a marine sediment sample. the antifungal qualities of the isolate were evaluated against eight out of ten plant-pathogenic fungi. the highest cellulolytic activity of the isolate was observed after four days of incubation. the association of the strain with bacillus subtilis was confirmed by phylogenetic analysis based on the sequences of the gyrb and 16s rrna regions. the ncbi has received strain s1-10, which has been assigned accession codes or708648 for 16s and pv007904 for gyrb. keywords: bacillus subtilis, marine sediment, antifungal effect, cellulolytic activity, phylogenetic analysis introduction marine sediments represent one of the largest habitats on our planet. their distinctive ecological characteristics, such as elevated salinity, high pressure, and low oxygen levels, can trigger the activation of previously dormant genes in marine microbes. this process leads to the emergence of unique microbes, enzymes, bioactive compounds, and specialized metabolic pathways that have adapted to these specific environmental conditions. the microorganisms sourced from marine sediments and their bioactive metabolites hold significant importance and present promising opportunities for commercial development in sectors such as food, pharmaceuticals, chemicals, agriculture, environmental protection, and human nutrition and health [1]. the unique living conditions found in marine environments compared to terrestrial ones make marine microorganisms highly promising candidates for producing innovative natural compounds [2]. marine bacillus isolates represent a diverse collection of bacteria, exhibiting both phylogenetic and phenotypic variations within the broader microbial world. these bacteria are widely distributed throughout the marine environment and possess remarkable resilience, thriving in challenging conditions like high temperatures, pressure, salinity, and ph levels. typically, bacillus strains necessitate ample nutrients and space to achieve their maximum growth rates, a competitive advantage they utilize against other organisms. the vastness of the ocean, with its constant dilution, compels marine organisms to develop powerful bioactive compounds for survival, either to outcompete rivals or to defend against smaller predators [3]. numerous bioactive compounds, such as cyclic peptides, glycopeptides, lipopeptides, and bacteriocins, are produced by bacillus species, which are well known for their wide spectrum of antibacterial properties [4]. chemical pesticides are currently the main tool used by agricultural producers to control or prevent crop diseases. the accumulation of these pesticide residues in food, soil, and aquatic environments poses significant threats to human health, the environment, and ecosystems [5]. as a result, there is a growing demand from consumers and environmental advocates to substitute chemical pesticides with natural, eco-friendly microorganisms that have innovative modes of action for the sustainable cultivation of crops [6]. in nature, biomass rich in lignocellulose and agricultural residues is widely available as a source of carbon. utilizing microorganisms or their enzymes for the biological highlights in bioscience page 1 of 5 july 2025|volume 8 https://doi.org/10.36462/h.biosci.202505 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:sakurasan2007@gmail.com https://orcid.org/0000-0003-2465-2866 mailto: khinthaemarrmm@gmail.com https://orcid.org/0009-0005-9920-9385 mailto:thetmin.wai333@gmail.com https://orcid.org/0009-0006-1088-936x mailto:khinsweaye.mdy@gmail.com http://bioscience.highlightsin.org/ yu ss et al., 2025 molecular identification and screening the antagonistic and cellulolytic activities figure 1. colony and microscopic morphology (100×) of (s1–10) isolate on lb (luria-bertani) and n/a (nutrient agar) pretreatment of lignocellulosic biomass provides an environmentally friendly method for processing biomass. the use of cellulases and hemicellulases to hydrolyze lignocellulosic biomass results in the formation of monomeric sugars that can be easily fermented by microorganisms [7]. numerous studies have shown that both bacterial and fungal species can produce cellulolytic enzymes. bacterial species, particularly those from the bacillus genus, have been predominantly utilized in industrial applications due to their ability to secrete a variety of hydrolytic enzymes [8]. the cellulolytic enzymes derived from b. subtilis have been extensively researched because of their significant production capacity and usefulness in biomass valorization [9]. bacillus subtilis is a catalase-positive, rod-shaped bacterium that is gram-positive. like other members of the bacillus genus, it can produce an endospore that enables it to endure extreme conditions such as high temperatures and dryness. b. subtilis is regarded as the most extensively studied model organism for investigating bacterial chromosome replication and cell differentiation among gram-positive bacteria [10]. additionally, it is frequently utilized as an industrial cell factory for synthesizing vitamins, inositol, acetoin, hyaluronan, and various other chemicals. research in agriculture has shown that incorporating a suitable amount of b. subtilis can significantly enhance the humus and carbon levels in compost, thereby improving soil quality and supporting crop growth [11]. myanmar’s economy is largely based on agriculture, making the agricultural sector crucial for business. thus, enhancing agricultural output, ensuring food safety, and improving soil quality are fundamental strategies. the agricultural inputs currently in use are primarily chemical in nature. poor management practices on farms and the incorrect application of agrochemicals have led figure 2. detection of cellulase and optimum growth condition to soil degradation and environmental harm. this study aims to isolate and identify bacillus subtilis from marine sediment and to explore the effective antagonistic and cellulolytic properties of this bacterial isolate for use as a biofungicide and in composting. materials and methods sample collection and strain isolation strain s1-10 was isolated from a sample of wet sediment taken from the sea near panga village, located in thanbyuzayat township, mawlamyine district, mon state, myanmar (15° 54′ 18′′ n, 97° 43′ 31′′ e). ten milliliters of a 0.85% saline solution were used to suspend one gram of the marine sediment, which was then shaken for half an hour at 30°c. after settling for two hours, the sample solution was serially diluted (10−1 to 10−4). using these dilutions, 100 µl was plated onto a 3% naclenriched nutritional agar medium and cultured for three days at 30°c [12]. repeated streaking on the same medium allowed for the selection and purification of the colonies that emerged. for future research, the pure culture of strain s1-10 was stored at −80°c in a 20% (v/v) glycerol solution after being regularly maintained and incubated at 30°c. screening of antagonistic activity and cellulolytic activity plant pathogenic fungi were generously supplied by professor dr. hee-young jung from the department of plant medicine at kyungpook national university in daegu, south korea. the dual culture technique was employed to assess the efficacy of a bacillus strain against these fungi, utilizing petri dishes measuring (90 × 15 mm) filled with potato dextrose agar (pda). in this method, a 6 mm diameter circle containing a suspension of conidia and mycelium, serving as the pathogen inoculum, was placed at the center of the dish. the antagonist was positioned 3 cm away from the center. each experiment was conducted in triplicate, with control fungal colonies not exposed to the antagonists. the inoculated petri dishes were incubated at 30oc for 14 days. after this incubation period, the inhibition zone between the phytopathogen and antagonist colonies was observed on the petri dish [13]. highlights in bioscience page 2 of 5 july 2025|volume 8 http://bioscience.highlightsin.org/ yu ss et al., 2025 molecular identification and screening the antagonistic and cellulolytic activities the assessment of cellulolytic activity was conducted using lb medium supplemented with 1% cellulose powder. colonies of the isolate were inoculated onto this medium and incubated at room temperature for 48 hours. following incubation, the plate was treated with 0.1% congo red for a duration of 15 to 20 minutes, then rinsed with 1 m nacl for an additional 15 to 20 minutes. the presence of a clear zone surrounding the colony was noted, indicating cellulase activity [14]. this transparent area indicates the cellulolytic activity of the isolate. according to miller gl’s 1959 description, the 3,5-dinitrosalicylic acid (dns) method was used for the quantitative analysis of cellulase. by adding 3 ml of dns reagent—which is made up of 1 g dinitrosalicylic acid, 16 g naoh, 300 g potassium sodium tartrate, and distilled water to a final volume of 1 l—to the 1 ml reaction mixture, the enzymatic reaction was stopped. the optical density of this mixture was then measured at 540 nm after it had been boiled for five minutes in capped glass tubes and cooled in cold water. d-glucose-based calibration curves were used to measure the cellulase activity. analysis of morphology and physiology a variety of agar media, such as nutrient agar (na; difco), marine agar 2216 (ma; difco), tryptic soy agar (tsa; difco), and luria bertani agar (lb; difco), were used to assess the growth of the isolated bacterial strain s1-10. on lb agar, the temperature range for growth was determined at 4, 10, 15, 20, 25, 30, 37, 42, and 45 °c. furthermore, after a 7-day incubation period, the ph range for growth was evaluated on lb agar at ph levels ranging from 4.0 to 10.0, in increments of 1.0 ph unit. to adjust the ph of the medium, 1 m naoh and hcl were used. additionally, growth was observed on nutrient agar after 7 days in the presence of nacl concentrations ranging from 1.0% to 10.0% (w/v), with 1.0% increments. phylogenetic analysis the higene genomic dna prep kit (biofact, daejeon, korea) was used to extract the genomic dna of strain s1-10 from 24-hour-old cultures grown on nutrient medium. the methodology developed by weisburg et al. was followed in the polymerase chain reaction (pcr) procedure used in this study to amplify the 16s rrna region. primers 9f (5’-gag ttt gat cct ggc tca g-3’) and 1512r (5’-acg gct acc ttg tta cga ctt-3’) were used in this investigation [15]. for phylogenetic analysis and species identification within the bacillus group, the dna gyrase b subunit gene, gyrb, was selected for examination. dna sequencing was conducted using degenerate primers for gyrb, specifically gyrb-f (5-gaa gtc atc atg acc gtt ctg-3) and gyrb-r (5-agc agg gta cgg atg tgc gag-3) [16]. the pcr product cleaning reagent exosap-it (thermo fisher scientific, waltham, ma, usa) was used to purify the amplified pcr products. the full sequence of the 16s rrna gene was assembled using seqman software (dnastar, madison, wi, usa). manual adjustment of gaps and terminal ends of the alignment was figure 3. a neighbor-joining phylogenetic tree constructed from 16s rrna gene sequences illustrates the evolutionary relationships of strain s1–10 and related members of the bacillaceae family. nodes with bootstrap support values exceeding 50% are marked. the strain that was isolated is highlighted in bold. heynadriskxia sporothermodurans m215 served as the outgroup. the scale bar represents 0.0050 substitutions per nucleotide position. performed with bioedit version 5.0.6 to ensure accuracy and completeness of the gene sequences. identification of the phylogenetically closest sequences and calculation of pairwise sequence similarity values were conducted using the ezbiocloud server and blast searches targeting the 16s rrna gene. sequences of the 16s rrna gene from related taxa were retrieved from genbank, and multiple alignments were carried out using the clustalw software [17]. a phylogenetic tree was constructed using the neighbor-joining method implemented in mega 7 software [18]. evolutionary distances were calculated using kimuras two-parameter model, and the reliability of the tree topology was assessed through bootstrap analysis with 1,000 replicates. results and discussions analysis of morphology and physiology colonies of the s1-10 strain grown on nutrient agar supplemented with 3% nacl appeared flat, white, and were approximately 3–5 mm in diameter after three days of incubation at 30 °c (figure 1). significant growth was observed on nutrient agar, marine agar, tsa, and lb agar. the s1-10 strain demonstrated the ability to grow in nacl concentrations ranging from 1% to 10%, within a ph range of 4 to 10, and at temperatures between 15 °c and 45 °c. optimal growth conditions were determined to be 3% nacl, ph 7, and 30 °c. in this research, a new strain of bacillus subtilis, named s1-10, was extracted from highlights in bioscience page 3 of 5 july 2025|volume 8 http://bioscience.highlightsin.org/ yu ss et al., 2025 molecular identification and screening the antagonistic and cellulolytic activities figure 4. neighbor-joining phylogenetic tree constructed from gyrb gene sequences, illustrating the phylogenetic placements of strain s1–10 alongside the bacillus subtilis group. bootstrap support values exceeding 70% are shown at the nodes. the isolate is indicated in bold. heynadriskxia sporothermodurans m215 served as an outgroup. bar, 0.050 replacements for each nucleotide position. marine sediment in myanmar and evaluated for its antagonistic characteristics against several phytopathogenic fungi. the s1-10 strain of b. subtilis showed notable antagonistic effects on sclerotinia sclerotium, botrytis cinerea, fusarium tricinctum, rosellinia necatrix, cytospora sp., colletotrichum aenigma, and colletotrichum fructicola. it demonstrated minimal inhibition of the plant pathogenic fungus diaporthe eres and showed no inhibitory effect on the development of sclerotium rolfsii and botryosphaeria sinensis. these findings are consistent with earlier research by[19], which reported growth suppression in various phytopathogenic fungi, with the exception of sclerotium rolfsii and botryosphaeria sinensis. moreover,[20] noted that the effect of bacillus subtilis bs-1 cell-free supernatant (cfs) on botryosphaeria dothidea was due to the stimulation of oxidative damage in the mycelial cells (figure s1). the degradation of cellulose by the cellulase enzyme is vital, as many agricultural byproducts include cellulose, which is a key element of plant cell walls. this cellulose can be transformed into glucose, which acts as a key raw ingredient for alcohol manufacturing [21]. the fermentation process has been employed for a long time, as it increases nutritional value while minimizing anti-nutritional elements, toxins, and levels of contamination [22]. a successful method for waste management includes the enzymatic hydrolysis of lignocellulosic waste, where fermentation produces reducing sugars that can be utilized for creating desired metabolites or biofuels [23]. in this research, the peak activity of cellulase enzyme production and the optimum growth conditions for the bacillus subtilis isolate were observed after four days of incubation (figure 2). phylogenetic analysis the 16s rrna gene of strain s1-10 is 793 base pairs long. its sequence shows a 100% match with several species, including bacillus subtilis, bacillus halotolerans, bacillus amyloliquefaciens, bacillus safensis, bacillus velezensis, bacillus mojavensis, and bacillus stercoris. a phylogenetic tree built using the neighbor-joining method indicates that strain s1-10 is grouped on the same branch as bacillus subtilis, bacillus stercoris, bacillus tequilensis, and colidifontibacillus erzurumensis (figure 3). therefore, the gyrb gene, which encodes the b subunit of dna gyrase, was selected as an alternative marker for phylogenetic analysis and species identification within the bacillus group. the ncbi blast results showed that the isolate shares 100% identity with bacillus subtilis. a phylogenetic tree constructed using the neighbor-joining method again places strain s1-10 alongside bacillus subtilis strains. similar clustering patterns were also observed in trees generated using the maximum likelihood and maximum parsimony methods (figure 4). conclusions the indigenous bacillus strain found in the marine sediment of myanmar was classified as bacillus subtilis through gene analysis of 16s rrna and gyrb. the isolated strain exhibited antagonistic effects on the mycelial growth of the plant pathogenic fungi sclerotinia sclerotiorum, botrytis cinerea, fusarium tricinctum, rosellinia necatrix, cytospora sp., colletotrichum aenigma, and colletotrichum fructicola. it demonstrated weak inhibition against the plant pathogenic fungus diaporthe eres and showed no inhibition on the growth of sclerotium rolfsii and botryosphaeria sinensis. the bacillus subtilis s1-10 isolate may be used as an effective strain for composting agricultural waste due to its cellulolytic and antagonistic properties. supplementary figure s1: screening the antifungal activity of isolate (s110) by showing fungal growth inhibition compared with the control and treatment plates. red box indicates the isolate (s110). reference 1. yao h, liu s, liu t, ren d, yang q, zhou z, et al. screening of marine sediment-derived microorganisms and their bioactive metabolites: a review. world journal of microbiology and biotechnology. 2023;39(7):172. 2. voser tm, campbell md, carroll ar. how different are marine microbial natural products compared to their terrestrial counterparts? natural product reports. 2022;39(1):7-19. 3. sayem sa, manzo e, ciavatta l, tramice a, cordone a, zanfardino a, et al. anti-biofilm activity of an exopolysachighlights in bioscience page 4 of 5 july 2025|volume 8 http://bioscience.highlightsin.org/ yu ss et al., 2025 molecular identification and screening the antagonistic and cellulolytic activities charide from a sponge-associated strain of bacillus licheniformis. microbial cell factories. 2011;10:1-12. 4. elshaghabee fm, rokana n, gulhane rd, sharma c, panwar h. bacillus as potential probiotics: status, concerns, and future perspectives. frontiers in microbiology. 2017;8:1490. 5. damalas ca, eleftherohorinos ig. pesticide exposure, safety issues, and risk assessment indicators. international journal of environmental research and public health. 2011;8(5):140219. 6. islam mt. potentials for biological control of plant diseases by lysobacter spp., with special reference to strain sb-k88. bacteria in agrobiology: plant growth responses. 2011:33563. 7. jin x, song j, ma j, liu gq. thermostable β-xylosidase from aspergillus fumigatus: purification, characterization and potential application in lignocellulose bioethanol production. renewable energy. 2020;155:1425-31. 8. alokika v, kumar v, singh b. biochemical characteristics of a novel ethanol-tolerant xylanase from bacillus subtilis subsp. subtilis jjbs250 and its applicability in saccharification of rice straw. biomass conversion and biorefinery. 2021:1-13. 9. deka d, bhargavi p, sharma a, goyal d, jawed m, goyal a. enhancement of cellulase activity from a new strain of bacillus subtilis by medium optimization and analysis with various cellulosic substrates. enzyme research. 2011;2011(1):151656. 10. errington j, aart ltvd. microbe profile: bacillus subtilis: model organism for cellular development, and industrial workhorse. microbiology. 2020;166(5):425-7. 11. duan m, zhang y, zhou b, qin z, wu j, wang q, et al. effects of bacillus subtilis on carbon components and microbial functional metabolism during cow manure–straw composting. bioresource technology. 2020;303:122868. 12. ntozonke n, okaiyeto k, okoli as, olaniran ao, nwodo uu, okoh ai. a marine bacterium, bacillus sp. isolated from the sediment samples of algoa bay in south africa produces a polysaccharide-bioflocculant. international journal of environmental research and public health. 2017;14(10):1149. 13. aquino-martínez jg, vázquez-garcía lm, reyes-reyes bg. biocontrol in vitro e in vivo de fusarium oxysporum schlecht. f. sp. dianthi (prill. y delacr.) snyder y hans. con hongos antagonistas nativos de la zona florícola de villa guerrero, estado de méxico. revista mexicana de fitopatología. 2008;26(2):127-37. 14. hendricks cw, doyle jd, hugley b. a new solid medium for enumerating cellulose-utilizing bacteria in soil. applied and environmental microbiology. 1995;61(5):2016-9. 15. weisburg wg, barns sm, pelletier da, lane dj. 16s ribosomal dna amplification for phylogenetic study. journal of bacteriology. 1991;173(2):697-703. 16. munir s, li y, he p, he p, he p, cui w, et al. bacillus subtilis l1-21 possible assessment of inhibitory mechanism against phytopathogens and colonization in different plant hosts. pak j agric sci. 2018;55(4):996-1002. 17. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. the clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acids research. 1997;25(24):4876-82. 18. kumar s, stecher g, tamura k. mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. molecular biology and evolution. 2016;33(7):1870-4. used to assess evolutionary distances via kimuras two-parameter model and bootstrap analysis (1,000 replicates). 19. nalisha i, muskhazli m, nor farizan t. production of bioactive compounds by bacillus subtilis against sclerotium rolfsii. malaysian journal of microbiology. 2006;2(2):19-23. 20. fan y, liu k, lu r, gao j, song w, zhu h, et al. cell-free supernatant of bacillus subtilis reduces kiwifruit rot caused by botryosphaeria dothidea through inducing oxidative stress in the pathogen. journal of fungi. 2023;9(1):127. 21. qu y, zhu m, liu k, bao x, lin j. studies on cellulosic ethanol production for sustainable supply of liquid fuel in china. journal of biotechnology. 2006;1(11):1235-40. 22. evans e, musa a, abubakar y, mainuna b. nigerian indigenous fermented foods: processes and prospects. in: mycotoxin and food safety in developing countries. intechopen; 2013. . 23. singhania rr, sukumaran rk, pillai a, prema p, szakacs g, pandey a. solid state fermentation of lignocellulosic substrates for cellulase production by trichoderma reesei nrrl 11460. indian journal of biotechnology. 2006;5:3326. highlights in bioscience page 5 of 5 july 2025|volume 8 http://bioscience.highlightsin.org/ abstract introduction materials and methods sample collection and strain isolation screening of antagonistic activity and cellulolytic activity analysis of morphology and physiology phylogenetic analysis results and discussions analysis of morphology and physiology phylogenetic analysis conclusions supplementary highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202404 research article open access 1 department of biotechnology research, microbiology laboratory, kyaukse, myanmar. 2 department of zoology, mandalay university, mandalay, myanmar. * to whom correspondence should be addressed: khinthaemarrmm@gmail.com editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), cairo, egypt. reviewer(s): abdulaziz ascandari, chemical and biochemical sciences-green process engineering, university mohammed vi polytechnic, benguerir, morocco. jean legeay, université de lorraine, inrae, umr iam interactions arbres-microorganismes, france. received: june 4, 2024 accepted: october 11, 2024 published: december 25, 2024 citation: marr kt, wilbur nd, yu ss, ei sl. evaluation on multi-trait activities of azotobacter spp. from various water samples in mandalay environments. 2024 dec. 25;7:bs202404 copyright: © 2024 marr et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. evaluation on multi-trait activities of azotobacter spp. from various water samples in mandalay environments khin thae marr1 >< �, san san yu1 >< �, naw dolly wilbur2 >< �, shun lai ei1 >< � abstract fourteen water samples were collected from different locations of mandalay and kyaukse township. among them, eight bacterial isolates named a1 to a8 were nominated as azotobacter according to their colonial morphology, microscopic morphology, and pigment production. according to the biochemical characteristics and sequence analysis of isolated bacteria, they were azotobacter chroococcum, azotobacter vinelandii, and azotobacter beijerinckii. the phosphate solubilizing of these isolates was observed from 2.3 to 2.6 si, and a4 had the highest solubility index. the potassium decomposing of these isolates was observed 2.5 to 3.8 si, and a6 and a8 had the highest k-decomposing activity. the zinc solubilizing of these isolates was observed at 2.3 to 3.5 si, and a3 had the highest zinc solubilizing index. screening methods showed that all eight strains have nitrogen fixing activity and indole acetic acid (iaa) producing activity. the antagonistic activity of all isolated strains was also found against pythium sp. and fusarium oxysporum. the isolated azotobacter sp. can be used as biofertilizer in the agriculture sector, which can increase crop yields and enhance soil fertility according to their plant growth promoting activities. keywords: isolation, characterization, 16s rrna gene, plant growth, azotobacter, biofertilizer. introduction azotobacteraceae are heterotrophic gram-negative bacteria and a group of aerobic. the main characteristic of azotobacteraceae is the ability to fix nitrogen in the natural atmosphere, which is vital in the nitrogen cycle. azotobacter species are generally spherical on lb medium or oval on glucose nitrogen free medium and motile (g-nfmm) [1]. azotobacter can live as cysts and produce enormous amounts of capsular slime. they can grow on any medium that has a suitable ph and an organic carbon source [2]. azotobacter produces blue white fluorescent, green, and yellow green pigments [3]. the optimum temperature range of azotobacter is between 20 and 30 oc, and the optimum ph of azotobacter is 6.5 in neutral to alkaline soil [4]. they can be found in the soil of a number of crop plants and vegetative plants [5]. azotobacter species is the preferred organism because they are non-pathogenic and can stay as cysts in soil for many years [6]. beijerinck studied azotobacter chroococcum isolated from the rhizosphere in holland in 1901, and it was the first species. azotobacter agilis was also studied by beijerinck [7]. lipman found azotobacter vinelandii in 1903 and azotobacter beijerinckii in 1904 from the rhizosphere [8]. azotobacter vinelandii has the ability to produce a yellow-green pigment that gives fluorescence under ultraviolet light. in alkaline soils, marine sediments, and marsh water, azotobacter can be generally found [9]. biofertilizers, also known as organic preparations or bioinoculants, contain microorganisms that promote nitrogen fixation, phosphate solubilizing, and potassium decomposing for plant growth [10]. azotobacter, azospirillum, and rhizobium are beneficial microbes for crop production to use as biofertilizers [11; 12]. nowadays, many countries use biofertilizers that give advantages to economic activity. the chemical fertilizers threaten environmental pollution and human health. the research was designed to isolate azotobacter sp. from various water samples, to characterize biochemical tests, identify the isolates by 16s rrna sequencing analysis, and study the multi-trait activities of isolated bacteria. highlights in bioscience page 1 of 8 december 2024|volume 7 https://doi.org/10.36462/h.biosci.202404 https://creativecommons.org/licenses/by/4.0/ mailto:khinthaemarrmm@gmail.com https://orcid.org/0009-0005-9920-9385 mailto:sakurasan2007@gmail.com https://orcid.org/0000-0003-2465-2866 mailto:2929852582@qq.com https://orcid.org/0009-0004-4759-6638 mailto:shunlai.ei@gmail.com https://orcid.org/0000-0003-2444-2439 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. materials and methods study area and sample collection the bottles of water samples were collected from different places in mandalay environs and kyaukse township, myanmar (figure 1). the water samples were taken from 5 to 12 cm of water depth. the plastic bottle (1 liter) was used to collect water samples. then, the water bottles were carried to the department of biotechnology research to be studied. the samples were named s1 to s14 (table 1). the strains isolated from these samples were named a1 to a8. their sampling sites and different locations of bacterial isolates were shown in table 2. table 1. water samples collected from different locations in mandalay and kyaukse township. no. samples collection site township 1 s1* ayeyarwady river jetty3 mandalay 2 s2 kandawgyi lake site1 mandalay 3 s3* kandawgyi lake site2 mandalay 4 s4* kandawgyi lake site3 mandalay 5 s5* fish pond in mahar muni image mandalay 6 s6* taw-dwin lake kyaukse 7 s7* tone-long (southern fish lake) kyaukse 8 s8* tone-lone (northern fish-lake) kyaukse 9 s9 sunye loch site1 kyaukse 10 s10 sunye loch site 2 kyaukse 11 s11 sunye loch site3 kyaukse 12 s12* sunye loch site 4 kyaukse 13 s13 thamote canal kyaukse 14 s14 zawgyi river kyaukse isolation of bacteria to isolate bacteria, 5 ml of water was added to the sterile test tube, and 5 ml of 0.9% nacl solution was mixed with it. the tubes with the water sample were kept standing for about 30 minutes, and 100 µl of water was spread on g-nfmm [13]. after inverting the gnfmm plates, they were incubated at 35 oc for 48 hours. the bacterial colonies that appeared on g-nfmm were purified by subculturing and incubated at 35 oc for 2 days. the shapes and sizes of isolated bacteria were characterized by colonial and microscopic morphology. microscopic morphology was checked by using gram staining method [14]. the single isolated colony was stained and examined with a drop of oil immersion under a microscope at 100 magnifications to check purity. biochemical characteristics of isolated bacteria the isolated bacteria were characterized by using some biochemical tests. biochemical characteristics of selected bacteria were studied according to bergey's manual of determinative bacteriology [15]. these tests include the motility test, the catalase test, the starch hydrolysis test, the citrate utilization test, the methyl red test, the voges-proskauer test, the indole test, and the triple sugar iron agar test. figure 1. location map of study area, mandalay environments, and kyaukse township. (source: myanmar information management unit and google earth pro) molecular identification of bacterial isolates the purified bacterial isolates were sent to a dna sequencing service, eurofins company in germany, and the sequence data of individual isolates were queried using blast in the national center for biotechnology information (ncbi) and genbank databases. sequence alignment results showed the maximum identities of each isolate and their relevant accession number. muscle was used to align sequences that were identified, and a phylogenetic tree was constructed based on the neighbor joining (nj) and the number of differences method algorithms using the mega 11 software. to acquire accession numbers, the nucleotide sequences of isolated azotobacter species were then sent to genbank. for further application, all isolates were maintained at the microbiology laboratory at the department of biotechnology research. screening on nitrogen fixing activity by indophenol method the bacterial strains were injected in the bottles that contain g-nfmm broth and incubated for one week at 35oc. then 1 ml of sample was centrifuged at 10000 rpm for 20 minutes. ammonium concentration was measured by indophenol method by taking the supernatant. firstly, 0.5 ml of edta solution was added to supernatant. then 0.5 ml of p-buffer was added. after adding 2.5 ml of nitroprusside reagent, 2.5 ml of hypochlorite solution was added to the supernatant in a small bottle. the reaction mixture was placed at room temperature for 3 hours and ammonium was estimated by the color of the sample. highlights in bioscience page 2 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. table 2. bacteria isolates from different locations in mandalay environments and kyaukse township. no. samples strain collection site ph temperature 1 s1 a1 96°03 21.745" e and 21°58 13.576" n 7.8 25°c 2 s3 a2 96°03 21.709"e and 21°57 07.664" n 7.3 25°c 3 s4 a3 96°03 21.470" e and 21°56 49.277"n 7.4 25°c 4 s5 a4 96°04 48.758" e and 21°57 09.017"n 7.6 25°c 5 s6 a5 96°07 44.652" e and 21°32 32.639" n 8.2 25°c 6 s7 a6 96°07 34.727" e and 21°33 40.007"n 8.4 25°c 7 s8 a7 96°07 34.588" e and 21°33" 40.333" n 8.3 25°c 8 s12 a8 96°13 28.758" e and 21°40 36.516" n 8.2 25°c in this method, 0 mg/ml of ammonium concentration will show yellow color, and 3 mg/ml ammonium concentration will show green color. screening on phosphate solubilizing activity to determine the phosphate solubilizing activity of isolated bacteria, national botanical research institute phosphate (nbrip) media was used. the single colony of bacteria was placed on nbrip media. after five days of incubation at 35oc, the colony showed a zone of clearance around it. the halo-zone diameter was recorded by the phosphate solubilizing index [16]. the solubilizing index (si) can be determined using the following formula: s i = colony diameter + halozone colony diameter screening on potassium decomposing activity to determine the decomposing of potassium, the eight bacterial strains were tested on k decomposing medium by plating method. pure colonies were spotted on k medium and incubated at 35oc. after five days, the clear zone around the colony was observed, and the diameters of the clear zone were recorded [17]. the solubilizing index (si) was calculated using the same formula as the phosphate solubilizing activity. screening on zinc solubilizing activity to determine the solubilizing of zinc, zinc solubilizing medium was used to test the eight bacterial strains. the single colony of bacteria was dotted on medium at 35oc. after incubating them for five days, clear zones around the colonies were recorded [18]. the si was measured and calculated using the above formula. screening on iaa producing activity the production of iaa by the isolated strains was determined calorimetrically using salkowski's reagent mixture [35% perchloric acid (50 ml); 0.5 m fecl3 (1 ml)]. to test iaa quality, isolated strains were aseptically cultured in g-nfmm plates containing 0.5 mg l-tryptophan per ml, and then incubated at 35oc for 3 days. after incubating the plates, 2 ml of salkowski's reagent and one drop of orthophosphoric acid were added into the plate medium at room temperature. the color turned pink, which indicated iaa production [19; 20]. table 3. biochemical characterization of isolates biochemical test a1 a2 a3 a4 a5 a6 a7 a8 c gram staining catalase + + + + + + + + starch hydrolysis citrate utilization + + + + + + + + motility + + + + + + + + methyl red + + + + voges-proskauer + + + + + + + + indole + tsi agar glucose + + + + + + + + lactose + + + + + + + + sucrose + + + + + + + + gas h2s + + + + solubility index phosphate 2.5 2.5 2.3 2.6 2.3 2.3 2.3 2.3 0 potassium 2.5 3 3 3 3.5 3.8 3.5 3.8 0 zinc 2.8 2.3 3.5 2.3 2.3 2.3 2.3 3 0 screening on antagonistic activity to determine the antagonistic activity, a total of eight bacterial isolates were tested with pythium sp. and fusarium oxysporum on potato dextrose agar (pda) medium by plating method. dual cultures (bacteria and fungus) were spotted separately on opposite sides on potato dextrose agar medium and incubated at 25oc. after three weeks of incubation, an antagonistic effect was observed. the zone diameter of inhibition on fungal growth was determined and recorded. results study area and sample collection collection sites and township of s1 to s14 samples are shown in table 1, and the results of strains a1 to a8 from 14 samples highlights in bioscience page 3 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. figure 2. colonial and microscopic morphology of isolated strains. are shown in table 2. the samples showed a ph range of 7-9 and a temperature of 25°c. isolation and biochemical characterization of bacteria the purified bacteria were observed for their colonial morphology and microscopic morphology such as color, shape and size (figure 2). the isolated colonies were checked by gram staining method under microscope that revealed and characterized as azotobacter species. they were pale-yellow and mucoid according to their morphology and colony sizes were 2 to 4 mm. according to gram straining, they were gram negative, cocci and their sizes were 2 to 4 µm. the isolated bacteria, a1 to a8 were identified by biochemical characterization tests shown in table 3. identification of azotobacter spp. by 16s rrna gene sequencing detection of 16s rrna genes of eight isolated bacteria by gel electrophoresis was shown in figure s1. the isolated strains belonged to the genus azotobacter according to molecular analysis. the strain a1 showed 100% identity with azotobacter chroococcum in ncbi blast search while the strain a2 showed 99.77% identity with the azotobacter chroococcum, respectively. the strain a5 revealed 100% similarity with azotobacter vinelandii strain iam 15004 (nr 041039.1) (figure 3). the 16s rrna gene sequences of a6 showed 99.52% similarity with azotobacter vinelandii, a7 showed 98.87% with azotobacter beijerinckii, figure 3. phylogentic tree of 16s rrna gene sequences of the bacterial eight isolated strain from water samples. a8 showed 99.58% with azotobacter chroococcum and a3 exhibited 99.50% with azotobacter chroococcum, respectively. the accession numbers were mt658735 for a1, mt658739 for a3, mt658736 for a5, mt658737 for a6 and mt658738 for a7, respectively, after submitting to the genbank. the closely related sequences were aligned the number of differences method by using mega 11 and the neighbor-joining tree was created [21]. the bootstrap (1000 replication) was used for a statistical support for the nodes in phylogenetic tree constructed with the 16s rrna similarities (%). figure 3 showed that the phylogenetic tree analysis involved 26 nucleotide sequences was united through muscle using mega 11 software. bootstrap values below 50% are not indicated, fewer than 5% alignment gaps. a total of 757 positions in the final data were involved. screening on nitrogen fixing activity by indophenol method when the activity of isolated strains that fix nitrogen was screened by culturing in the g-nfmm broth, all isolates changed the color (figure 4). the potential of nitrogen fixation was indicated by their color changing of all isolates. screening on phosphate solubilizing activity a1, a2, a4 gave higher halo zone diameter than a5 in nbrip media. strain a3, a5, a6, a7, and a8 showed the same clear zone in phosphate solubilizing activity test (figure 5). screening on potassium decomposing activity in k-decomposing test, strain a6, a8 exhibited 3.8 mm clear zone and the strains a5, a7 showed the same 3.5 mm clear zone. highlights in bioscience page 4 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. figure 4. screening of azotobacter species nitrogen fixing activity. figure 5. screening on phosphate solubilizing activity of azotobacter. strains a2, a3, a4 showed 3 mm clear zone strain a1 showed 2.5 mm clear zone (figure 6). screening on zinc solubilizing activity all strains showed clear zone around their colonies on zinc agar plates after 5 days of incubation at temperature 35°c. among eight strains, a3 showed the maximum clear zone, and followed by a8 and a1. strains a2, a4, a5, a6, and a7 showed the same clear zone (figure 7). screening on iaa producing activity iaa producing activities of all isolated strains were screened by using gnfmm containing 0.5 mg/ml l-tryptophan and incubated at 35oc for 3 days. after incubating the plates, salkowski's reagent was added into the culture plates. iaa producing activity was indicated by the development of pink color (figure 8). screening on antagonistic activity the antifungal activity of isolated strains a1 to a8 showed good antifungal activity against pythrium sp. and did not show figure 6. screening on zinc solubilizing activity of azotobacter. figure 7. screening on potassium decomposing activity of azotobacter. distinct activity against fusarium sp. after 3 weeks incubation (figure s2). discussion azotobacter can be found in the rhizosphere of vegetables and plantation crops [22]. all plants need nitrogen, phosphate, potassium and zinc for its growth. however, plants can absorb ammonia converted from nitrogen. azotobacter is proficient to convert nitrogen into ammonia, making it readily available for plant absorption [23]. azotobacter is beneficial nitrogen fixer. azotobacter bio-fertilizer benefits in agriculture to integrate nitrogen management. rhizobium, azotobacter, and azospirillum enrich nitrogen nutrition in n-deficient soils [24]. after the death of the azotobacter cell, cell proteins are mineralized in soil and contribute to the nitrogen accessibility of crop plants [25]. gibberellic acid and iaa produced by azotobacter encourage plant growth development. antimicrobial compounds produced by some species of azotobacter are able to inhibit plant pathogens and prevent economic losses [26]. in this research, eight isolated azotobacter strains were obtained from 14 water samples. the bacteria were characterized by 16s rrna gene sequencing method to know exactly about the genus of azotobacter sp. they were a. chroococcum, a. beijerinckii,and a. vinelandii. eight strains were cultured in g-nfm medium containing btb as indicator and showed changing of the color from green to blue that was an indication of nitrogen fixing activity. the isolated strains a1 to a8 showed nitrogen-fixing activity on the nitrogenfree medium. all eight strains showed phosphate solubilization activity on nbrip medium. the widest clear zone diameter was found in a. chroococcum of all isolated strains in phosphate solubilization. to convert insoluble phosphorus to an accessible form, orthophosphate, the capacity of some microorganisms is an important trait in a pgpr for increasing crop yields. azotobacter can also convert insoluble phosphate to orthophosphate that is soluble for plants to absorb [27]. insoluble inorganic phosphate can be absorbed by the roots of plants with the help of bacteria that can solubilize phosphate [28]. eight isolates showed the clear zone around the colonies in potassium decomposition. a. vinelandii a6 and a. chroococcum a8 showed the highest potassium solubilizing activity among eight strains. the potassium is available in four forms i.e., k solution, k as mineral, k as transferable metal and k as non-exchangeable metal. mineral k held tightly on the surface of clay. non-exchangeable highlights in bioscience page 5 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. figure 8. indole acetic acid (iaa) producing activity of azotobacter sp. k is a primary factor in determining soil and tightly bound in the crystal lattice. exchangeable k is loosely bound water in the soil. soluble k could be measured in a water extract of soil [29; 30]. zinc solubilizing bacteria showing clear halo zone around their colonies were found in all eight strains. azotobacter chroococcum a3 showed the widest clear zone among the eight strains. a very slight portion of total zinc is present as soluble form in soil solution. the majority of zinc in soil is found in insoluble compounds and minerals [31]. one of the important hormones which promote plant growth is iaa. a positive correlation was detected between tryptophan concentrations and iaa production. all strains produced iaa in screening of iaa production, using tryptophan as carrier. fusarium oxysporum and pythium fungi were found to cause severe diseases in various crops [32]. pythium fungi are water molds, and their spores attack living plants, causing issues such as seedling decay, root and crown rot, and leaf blight [33; 34]. fusarium oxysporum is a fungal pathogen that infect banana, tomato, many other vegetables and ornamentals [35; 36]. in the present study, all strains noticeably showed fungal growth inhibition against the tested pathogen (pythium). the antifungal activity of a. chroococcum a1, a3 and a4, a. vinelandii a5 and a6, a. beijerinckii a7 were occurred against pythium sp. a. chroococcum a2 and a8 were fair antifungal activity against pythium sp. after 3 weeks. a. chroococcum a4 and a8, the other strains were observed as moderate antifungal activity. as for antifungal activity against fusarium oxysporum, all isolated strains exhibited minimal antagonistic activity, as indicated by the small zone areas. according to the data, eight isolated strains showed good antifungal activity against the pythrium sp. although they have no good activity against fusarium sp. azotobacter can be used as a bio-fertilizer without any significant limitations or challenges, except that it should not be used in conjunction with chemical pesticides and herbicides. the optimum conditions such as ph and temperature of isolated bacteria should be explored for their optimum growth and plant growth promoting activities. as for future research, isolated azotobacter strains would be used in combination with other bio-fertilizers and would be used as antifungal and bio-fertilizers in agricultural fields. biofertilizers in myanmar using azotobacter have already been used in agriculture [37]. azotobacter can fix nearly 20 kg n/ha per year to use as a substitute for fertilizer in crop production [38; 39]. therefore, it can reduce the cost for mineral fertilizers. it has economic impact and safety in farmers. conclusion eight bacterial isolates were obtained and identified as azotobacter vinelandii, azotobacter beijerinckii and azotobacter chroococcum. their existence of spherical form is about 2 µm and that of oval form is about 3-4 µm. their shapes were ranging from cocci which occurred single, in pairs or irregular clusters and some were arranged in chains of variable lengths. they were motile, gram negative and cysts formed. all eight strains showed nitrogen fixing, phosphate solubilizing, potassium decomposing, zinc solubilizing and iaa producing activities. they also have antifungal activity against pythium and fusarium oxysporum fungi. therefore, azotobacter strains from this study could be used to develop bio-fertilizer which can enhance soil fertility due to their plant growth promoting activities. acknowledgement we are greatly indebted to dr. thant zin, professor, head of the department of zoology, university of mandalay, ministry of education for allowing us to conduct the research. our special thanks to dr. aye aye khaing, director general, department of biotechnology research, ministry of science and technology for allowing us to conduct the study in the laboratory. thanks to dr. tin mar lynn and all the researchers of microbiology laboratory, department of biotechnology research, kyaukse for their kind help and sharing their experiences. supplementary figure s1: detection of 16s rrna gene from isolated bacteria by gel electrophoresis. figure s2: screening on antagonistic activity of azotobacter against a. pythium sp. and b. fusarium sp. reference 1. jnawali a, ojha r, marahatta s. role of azotobacter in soil fertility and sustainability–a review. advances in plants and agricultural research. 2015;2(6):1-5. 2. ramadhan zk, issa fa. the screening azotobacter spp., bioavailability from four ecological systems in zakho town, kurdistam region iraq. science journal of university of zakho. 2022;10(4). 3. kyaw ep, soe mm, yu zklss, lynn tm. study on plant growth promoting activities of azotobacter isolates for sustainable agriculture in myanmar. journal of biotechnology and bioresources. 2019;1(5):1-6. 4. dar sa, bhat ra, dervash ma, dar za, dar gh. azotobacter as biofertilizer for sustainable soil and plant health under saline environmental conditions. microbiota and biofertilizers: a sustainable continuum for plant and soil health. 2021:231-54. highlights in bioscience page 6 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. 5. wani sa, chand s, ali t. potential use of azotobacter chroococcum in crop production: an overview. current agricultural research journal. 2013;1(1):35-8. 6. tariq m, jameel f, ijaz u, abdullah m, rashid k. biofertilizer microorganisms accompanying pathogenic attributes: a potential threat. physiology and molecular biology of plants. 2022;28(1):77-90. 7. mrkovacki n, milic v. use of azotobacter chroococcum as potentially useful in agricultural application. annals of microbiology. 2001;51(2):145-58. 8. patil sv, mohite bv, patil cd, koli sh, borase hp, patil vs. azotobacter. in: beneficial microbes in agro-ecology. elsevier; 2020. p. 397-426. 9. becking jh. the family azotobacteraceae. in: the prokaryotes: a handbook on habitats, isolation, and identification of bacteria. springer; 1981. p. 795-817. 10. chakraborty t, akhtar n. biofertilizers: prospects and challenges for future. in: biofertilizers: study and impact; 2021. p. 575-90. 11. chakraborty t, akhtar n. biofertilizers: characteristic features and applications. in: biofertilizers: study and impact; 2021. p. 429-89. 12. selvakumar g, lenin m, thamizhiniyan p, ravimycin t. response of biofertilizers on the growth and yield of blackgram (vigna mungo l.). recent research in science and technology. 2009;1(4). 13. latt zk, san sy, kyaw ep, lynn tm, nwe mt, mon ww, et al. using cellulolytic nitrogen fixing bacterium, azomonas agilis for effective degradation of agricultural residues. the open microbiology journal. 2018;12:154. 14. smith ac, hussey ma. gram stain: gram-positive rods. american society for microbiology. 2005. 15. buchanan re, gibbons ne. bergey’s manual of determinative bacteriology; 1974. 16. kalayu g. phosphate solubilizing 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microbes for sustainable crop production; 2017. p. 87-106. 27. rodríguez h, fraga r, gonzalez t, bashan y. genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria. plant and soil. 2006;287:15-21. 28. perez e, sulbaran m, ball mm, yarzabal la. isolation and characterization of mineral phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the southeastern venezuelan region. soil biology and biochemistry. 2007;39(11):2905-14. 29. mouhamad r, alsaede a, iqbal m. behavior of potassium in soil: a mini review. chemistry international. 2016;2(1):5869. 30. zakaria aab. growth optimization of potassium solubilizing bacteria isolated from biofertilizer. faculty of chemical & natural resources engineering, university malaysia pahang; 2009. highlights in bioscience page 7 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ marr et al., 2024 evaluation on multi-trait activities of azotobacter spp. 31. sadeghzadeh b. a review of zinc nutrition and plant breeding. journal of soil science and plant nutrition. 2013;13(4):905-27. 32. horst rk. field manual of diseases on fruits and vegetables. springer; 2013. 33. kaya aa, gültekin hc, karakaya a. fungi and water molds found on woody plants grown in the forest nurseries of the marmara region. turkish journal of forestry. 2019;20(4):324-32. 34. duniway j. water relations of water molds. annual review of phytopathology. 1979;17(1):431-60. 35. kang s, demers j, del mar jimenez-gasco m, rep m. fusarium oxysporum. in: genomics of plant-associated fungi and oomycetes: dicot pathogens; 2014. p. 99-119. 36. ortoneda m, guarro j, madrid mp, caracuel z, roncero mig, mayayo e, et al. fusarium oxysporum as a multihost model for the genetic dissection of fungal virulence in plants and mammals. infection and immunity. 2004;72(3):1760-6. 37. lynn tm, win hs, kyaw ep, latt zk, yu s. characterization of phosphate solubilizing and potassium decomposing strains and study on their effects on tomato cultivation. international journal of innovation and applied studies. 2013;3(4):959-66. 38. kizilkaya r. nitrogen fixation capacity of azotobacter spp. strains isolated from soils in different ecosystems and relationship between them and the microbiological properties of soils. journal of environmental biology. 2009;30(1):73-82. 39. hajnal-jafari t, latkovi d, ðuri s, mrkovaki n, najdenovska o. the use of azotobacter in organic maize production; 2012. highlights in bioscience page 8 of 8 december 2024|volume 7 http://bioscience.highlightsin.org/ abstract introduction materials and methods study area and sample collection isolation of bacteria biochemical characteristics of isolated bacteria molecular identification of bacterial isolates screening on nitrogen fixing activity by indophenol method screening on phosphate solubilizing activity screening on potassium decomposing activity screening on zinc solubilizing activity screening on iaa producing activity screening on antagonistic activity results study area and sample collection isolation and biochemical characterization of bacteria identification of azotobacter spp. by 16s rrna gene sequencing screening on nitrogen fixing activity by indophenol method screening on phosphate solubilizing activity screening on potassium decomposing activity screening on zinc solubilizing activity screening on iaa producing activity screening on antagonistic activity discussion conclusion supplementary highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202202 research article open access 1 international center for agricultural research in the dry areas (icarda), cairo, egypt. 2 genetics department, faculty of agriculture, mansoura university, mansoura, egypt. * to whom correspondence should be addressed: khaledhelmy444@gmail.com editor: hatem zayed, college of health and sciences, qatar university, doha, qatar. reviewer(s): santosh k maurya, molecular signaling & drug discovery laboratory, department of biochemistry, central university of punjab, bathinda, punjab, india. akhilesh maurya, indian institute of information technology allahabad, devghat, jhalwa, prayagraj, uttar pradesh 211015, india. received: may 1, 2022 accepted: july 1, 2022 published: july 15, 2022 citation: mousa kh, nassar ae . identification of hub genes and potential molecular mechanisms associated with inflammatory bowel diseases using meta-analysis of gene expression data. 2022 july 15;5:bs202202 copyright: © 2022 mousa and nassar. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. identification of hub genes and potential molecular mechanisms associated with inflammatory bowel diseases using meta-analysis of gene expression data khaled h. mousa*1,2 >< `, ahmed e. nassar1,2 >< ` abstract inflammatory bowel diseases (ibds), which primarily include crohn’s disease (cd) and ulcerative colitis (uc), are chronic recurrent diseases of the gastrointestinal tract with increasing prevalence and incidence worldwide. in this study, we aimed to identify key factor genes that control the progression of inflammatory bowel disease, identify common and unique nodal genes, examine gene-protein interactions, assess current advances in the published literature on inflammatory bowel disease, and examine the impact of various biological pathways. gene expression profiles were obtained from the gene expression omnibus (geo) database. we performed gene expression analysis to identify deferentially expressed genes. subsequently, go and kegg pathway enrichment analyzes and proteinprotein interaction network analyzes (ppi) of degs were performed. text mining was used to examine the frequency of genes in the published ibd literature. four geo databases (gse156044, gse159751, gse159008, and gse102746) were downloaded from geo databases. a total of 368 degs were identified. the results of go term analysis showed that degs were mainly involved in the activity of cytokine receptors, integral components of the plasma membrane, and cytokine-mediated signaling. kegg pathway analysis showed that degs were mainly enriched in bile secretion, mineral absorption, and cytokine-cytokine receptor interaction. the results of ppi analysis showed that about 10 genes were the key genes for the occurrence of ced. text mining revealed the existence of 399 genes associated with ced. our results suggest a possible link between ced and other diseases such as triple negative breast cancer (tnbc) and lung adenocarcinoma (luad), and provide new insights into the mechanisms of inflammatory bowel disease and new treatment targets. keywords: inflammatory bowel diseases, deferentially expressed genes, hub genes, text mining introduction inflammatory bowel disease (ibd), which includes crohn’s disease (cd) and ulcerative colitis (uc), is a chronic inflammatory disease of the gastrointestinal tract that is becoming increasingly common worldwide [1]. inflammatory bowel diseases (ibds) are the most common gastrointestinal (gi) diseases, affecting 0.3% to 0.5% of the world population [2]. ulcerative colitis (cd) is confined to the mucosa of the colon, always involving the rectum, and may continuously extend to more proximal parts of the intestine [3]. crohn’s disease (cd), on the other hand, is a transmural, progressive inflammatory disease that can affect any part of the gastro-intestinal tract (gi). as a result, complications such as strictures, fistulas, and abscesses are more common in cd [4]. patients with these conditions have symptoms such as bloating, abdominal pain, and altered bowel habits (diarrhea and/or constipation), and approximately 40% of ibd patients have an irritable bowel irritable bowel syndrome (ibs)-like symptoms [5]. ibd affects both children and adults, with 15-20% of patients diagnosed in childhood [6]. inflammatory bowel disease is caused by a variety of agents, including genetic, immune system, microbial, and environmental factors [7]. highlights in bioscience page 1 of 8 july 2022|volume 5 https://doi.org/10.36462/h.biosci.202202 https://creativecommons.org/licenses/by/4.0/ khaledhelmy444@gmail.com hindu8446@gmail.com http://bioscience.highlightsin.org/ mousa and nassar, 2022 hub genes and potential molecular mechanisms associated with inflammatory bowel diseases malnutrition is a common problem inibd patients [8]. malnutrition inibd patients is multi-factorial and has been associated with malabsorption, caloric restriction, pharmacological treatment, loss of nutrients in the gastrointestinal tract, and increased energy consumption [9]. inflammatory bowel disease (ibd) is a lifelong condition that has no cure. patients suffering from ibd may present with symptoms of common mental disorders such as anxiety and depression [10]. the prevalence of cd and uc can be attributed to a variety of factors, including geographic location, poor nutrition, genetics, and an ineffective immune response [11]. studies of familial clustering and twin pairs have provided evidence that genetic factors contribute to the development of ibd. studies of discordant twin pairs with ibd have pointed to some genetically independent factors underlying pathogenesis [12]. genetic research on inflammatory bowel disease (ibd) has identified genes and pathways in inflammatory pathology. [13]. most available ibd therapies suppress the immune system, which increases the risk of infections and some cancers and does not benefit all patients [14]. to reduce the prevalence of ibd, the ibd community should focus on prevention of the disease [15]. differential gene expression (dge) analysis is one of the most common applications of rna sequencing (rna-seq) data. this method can be used to elucidate differentially expressed genes under two or more conditions, and it is used in many applications for the analysis of rna-seq data. [16]. using differential gene expression, nodal genes have been identified in alzheimer’s disease (ad) [17], gastric cancer (gc) [18], breast cancer (bc) [19], tuberculosis (tb) [20], lung adenocarcinoma [21], colon cancer (crc) [22]. in this study, we used bioinformatics approaches to identify differentially expressed genes (degs) between normal and inflamed tissues. these degs were also analyzed, including go and kegg enrichment analysis of degs, construction of protein-protein interaction networks (ppi), identification of key genes associated with ced, and identification of genes common to crohn’s disease (cd) and ulcerative colitis (uc). the biological functions and key signaling pathways of these degs are discussed. the major genes discovered in previous work were reviewed. materials and methods gene expression data we downloaded the original gene expression profiles gse156044, gse159751, gse159008 and gse102746 from the national center for biotechnology information (ncbi) gene expression omnibus (geo) (https://www.ncbi.nlm.nih.gov/geo/) [23]. a total of 40 normal tissues (control) and 31 inflamed tissues (patients) were downloaded. we excluded 27 inflamed tissues in gse159008. we selected samples of normal tissues and inflamed tissues of different types of inflammatory bowel disease only. identification of degs raw data were subjected to differentially gene expression analysis using the r package limma. gene expression was analyzed with the package limma in r and displayed as a heat map. genes with p.val 0.05 were considered as deferentially expressed. gene enrichment analysis gene set enrichment (gse) is the most effective method for determining the underlying biological functions of various genes or proteins [24]. to determine the functions of overlapping degs, go functional and kegg pathway enrichment of proteins encoded by candidate genes were analyzed and these genes were annotated using the enrichr online analysis database (available online: https://maayanlab.cloud/enrichr/). enrichr is a gene set search engine that can be used to query hundreds of thousands of annotated gene sets. this platform provides several methods for calculating gene set enrichment, and the results are visualized in several interactive ways. [25]. ppi network construction and analysis protein-protein interactions was assessed using the database string (search tool for the retrieval of interacting genes; http://string-db.org/cgi/input.pl). using the string database and the cytoscape programme, a ppi network of degs was produced (version 3.9.0). additionally, the cytoscape program is built on a network. each node represents a gene, protein, or other biological molecule, and the connections between nodes represent how these biological molecules interact. this model can be used to determine how proteins encoded by degs interact with one another and with other proteins in pathways in inflammatory bowel diseases (ibds). text-mining the importance of text mining in service management is growing with the expansion of access to big data via digital platforms that enable such services [26]. text mining tools are commonly used to extract information about disease-related genes, proteins, molecular interactions, and signaling pathways [27]. previous research has documented the use of these tools in studying regulatory mechanisms for various types of diseases, including inflammatory bowel disease [28]. in the current study, we obtained ibd-related abstracts from the pubmed database by searching for ’inflammatory bowel disease’ using the esearch tool and downloading these data using the efetch tool, which is responsible for retrieving records in the desired format [29]. this tool is provided by entrez direct (edirect) software [30]. we counted genes associated with ibds from the published literature using python, and these genes were illustrated using the r package. these genes had been studied extensively in the context of ibd. results identification of degs a total of 93, 361, 907, and 1123 degs were identified from gse156044, gse159008, gse102746, and gse159751, respectively, according to differential gene expression analysis. highlights in bioscience page 2 of 8 july 2022|volume 5 http://bioscience.highlightsin.org/ mousa and nassar, 2022 hub genes and potential molecular mechanisms associated with inflammatory bowel diseases the heat map of degs expression is shown in figure 1. among the 2484 genes with adj.p.val ≤ 0.04, 368 degs were identified, including 180 upregulated genes and 188 downregulated genes. biological annotation of degs in inflammatory bowel disease was performed using the enrichr online database. bars of the graph sorted by p-value ranking. figure 3 shows the ten most enriched pathways from go and kegg analyzes for enrichment of degs. go analysis of degs was divided into three functional groups, including molecular function (mf), cellular component (cc), and biological process (bp). at mf, these degs were significantly enriched in cytokine receptor activity, metal ion binding, cxcr chemokine receptor binding, icosatetraenoic acid binding, and arachidonic acid binding (figure 3c). at cc, degs were enriched in the integral component of the plasma membrane, the lumen of secretory granules, the lumen of intercellular organelles, the rna polymerase iii complex, and the alpha-beta t-cell receptor (figure 3b). at bp, degs were enriched in cytokine-mediated signaling, positive regulation of telomerase rna localization in the cajal body, neutrophil degranulation, neutrophil activation in immune response, and regulation of telomerase rna localization in the cajal body (figure 3a). in kegg signaling pathways, degs were enriched mainly in bile secretion, mineral absorption, cytokine-cytokine receptor interaction, alanine, aspartate, and glutamate metabolism, and jak-stat signaling (figure 3d). these significantly enriched go terms and kegg signaling pathways will help us better understand the key molecules involved in the progression of ibd. ppi network construction and analysis genes associated with inflammatory bowel disease were subjected to ppi analysis. the ppi network grouped genes based on their interaction activity to discover genes associated with the studied diseases, as shown in figure 4. as a result, we obtained 42 genes with significant interactions, 10 of which were associated with ibd. these genes were polr2h, lcn2, timp1, cxcl1, muc1, csf3r, s100a9, spi1, cftr, and fcgr3b. text mining we examined 42192 abstracts and obtained the 399 most frequent genes in the published literature associated with ced (figure 5). using the online analysis tool shinygo. gene ontology enrichment analysis was performed according to biological processes (bp), cellular components (cc), and molecular functions (mf). in addition, the cutoff value for screening pathways and significant functionality was set at p 0.05. table 1 shows the ten most enriched pathways of the go enrichment analysis. kegg pathway enrichment analysis was also performed using g:profiler. a venn diagram was used to classify the similarities and differences between the degs of the four datasets and the ibd-related genes identified by text mining (two genes). figure 1. heat maps of gene expression data. the gene expression profiles of the studied ibds, (a) gse99816(crohn’s disease), (b) gse156044(crohn’s disease), (c) gse159008(ulcerative colitis), (d) gse102746 (ulcerative colitis) you can add references of every experiment. figure 2. venn digram, similarities and differences between four datasets and 399 most frequent genes in previous publications related to ibd, identified by text mining. highlights in bioscience page 3 of 8 july 2022|volume 5 http://bioscience.highlightsin.org/ mousa and nassar, 2022 hub genes and potential molecular mechanisms associated with inflammatory bowel diseases figure 3. go molecular function and kegg pathways analysis of degs. red bars represent the number of degs. here only show the top 10 pathways: (a) biological processes (bp); (b) cellular components (cc); (c) molecular function (mf); (d) kegg pathway. discussion inflammatory bowel disease (ibd) is a chronic inflammatory disease of the gastrointestinal tract [31]. ulcerative colitis and crohn’s disease are the main forms of inflammatory bowel disease [32]. inflammatory bowel disease is a long-term chronic condition, with approximately 20% of ibd patients progressing to colorectal cancer [33]. intestinal fibrosis is a common complication of inflammatory bowel disease, which is usually the result of chronic inflammation [34]. genetic studies have identified more than 200 loci that regulate ibd risk [35]. in patients with ibd, alterations in various immune cells and neuroimmune signaling pathways have been identified in the lamina propria [36]. therefore, it is important to investigate the molecular mechanisms of inflammation and inflammatory bowel disease development. bioinformatics tools provide extensive technical support for the processing and analysis of gene expression profiles. as a result, bioinformatics analysis has been widely used in recent years to identify novel therapeutic targets and diagnostic markers [37]. in this study, four geo datasets related to ibds, gse156044, gse159008, gse102746 and gse159751, were examined using the limma package in r statistical software and 368 degs were identified. the results of go analysis, which included mf, cc, and bp, showed that these degs were mainly enriched in cytokine receptor activity, extracellular response, and cellular response to chemical stimuli. kegg pathway enrichment analysis also showed that these degs were significantly enriched in bile secretion, cytokine-cytokine receptor interaction, mineral absorption, retinol metabolism, and jak-stat pathways. this gene set enrichment analysis provides insights into the molecular mechanism of ibd progression. we used cytoscape to create a ppi network according to degs and identified 42 genes with significant interaction, 10 of which are associated with ibd. they are polr2h, lcn2, timp1, cxcl1, muc1, csf3r, s100a9, spi1, cftr, and fcgr3b. polr2h is overexpressed in organoids of rectal tumors. polr2h is the gene encoding the common h subunit of rna polymerases i, ii and iii [38]. lcn2 is secreted into the intestinal lumen at high levels and has a significant impact on controlling both the composition of the gut microbiome and host inflammation [39]. short-term consumption of a high-fat diet (hfd) significantly increases intestinal lcn2 expression and secretion into the intestinal lumen. lcn2 deficiency accelerates the development of hfd-induced intestinal inflammation [40]. timp1 plays an important role in promoting tumorigenesis [41] and acts as a prognostic biomarker for ulcerative colitis-related colorectal cancer [42]. cxcl1 enhances myeloid cell-mediated immunosuppression during acute colitis and has been linked to tumorigenesis in various tumor types [43]. muc1 is a heterodimeric protein that enhances the response to inflammation. expression of muc1 is associated with activation of inflammatory pathways and development of colitis [44]. csf3r is an important regulator of proliferation and differentiation of myeloid cells. mutations in the csf3r gene have been found in patients with chronic neutrophilic leukemia (cnl) and acute myeloid leukemia (aml) [45]. recent research has shown that mutations in the csf3r gene play an oncogenic role in the development of hematologic malignancies [46]. s100a9 is a myeloid-related protein involved in inflammatory processes and various carcinogens [47]. spi1 is a gene cluster containing 39 genes encoding t3ss-1. spi1 genes are responsible for host cell invasion and host inflammatory response. t3ss-1 encoded by spi1 provides effector proteins required for intestinal invasion and the development of enteritis [48]. cftr is a protein located at the apical membrane of epithelial cells and has been associated with cystic fibrosis disease. cystic fibrosis is a potentially fatal disease that causes severe damage to the lungs and digestive system [49]. fcy receptors (fcyrs), are expressed by six genes, one of which is fcgr3b [50]. fcgr3b increases the risk of inflammatory diseases such as systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) [51]. text mining is concerned with obtaining information about biological entities such as genes, proteins, and phenotypes, or more broadly, biological pathways [52]. text mining was used to extract genes associated with ced from the pubmed database. we obtained the 399 most frequent genes in the published literature associated with ced. comparison between the degs of the four datasets and the text mining results revealed 21 overlapping genes that had been intensively studied. by examining the ppi network of these 21 genes, we discovered four genes that interact highly with each other (ptprc, lcn2, cxcl1, and s100a9 ). these genes suggest that there may be a potential link between them and other diseases. in our study, the results of text mining highlights in bioscience page 4 of 8 july 2022|volume 5 http://bioscience.highlightsin.org/ mousa and nassar, 2022 hub genes and potential molecular mechanisms associated with inflammatory bowel diseases figure 4. ppi network of significant genes associated with ibd diseases. (a) protein-protein interaction network of degs. (b) clustering of genes according to their interaction activity. figure 5. text mining of genes associated with ibds in published literature. 399 most frequent genes in previous publications related to ibd, identified by text mining. highlights in bioscience page 5 of 8 july 2022|volume 5 http://bioscience.highlightsin.org/ mousa and nassar, 2022 hub genes and potential molecular mechanisms associated with inflammatory bowel diseases partially agreed with the results of deg analysis, but they also provided some unique conclusions. ptprc was associated with triple negative breast cancer (tnbc) [53]. ptprc may be a potential prognostic marker in lung adenocarcinoma (luad), which could affect the immune function of t cells and other immune cells by participating in the regulation of tumor microenvironment (tme) immune activity [54]. lcn2 is an acute-phase response protein that is upregulated in cns disease or injury and serves as a regulatory enhancer in neuroinflammation [55]. during lymphangiogenesis, cxcl1 enables tumor cells to migrate into lymphatic vessels, leading to lymph node metastasis [56]. a growing body of research suggests that s100a9, also known as myeloid-related proteins 8 and 14, plays an important role in inflammation and inflammation-related tissue damage [57]. s100a9 has been reported to be regulated by pathogen infection and to play a role in the progression of various cancers, including hepatitis b virus (hbv)-related hepatocellular carcinoma (hcc) [58]. in summary, the aim of this study was to describe ibd-related gene expression and systematically investigate the pathways and networks of ibd-related genes using bioinformatics to reveal beneficial targets and pathways in the pathogenesis of ibd. references 1. john gubatan, steven levitte, akshar patel, tatiana balabanis, mike t wei, and sidhartha r sinha. artificial intelligence applications in inflammatory bowel disease: emerging technologies and future directions. world journal of 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introduction materials and methods gene expression data identification of degs gene enrichment analysis ppi network construction and analysis text-mining results identification of degs ppi network construction and analysis text mining discussion references highlights in bioscience issn:2682-4043 doi:10.36462/h.biosci.202304 review article open access 1 department of microbiology, central university of tamil nadu, tamil nadu, india. * to whom correspondence should be addressed: rkaushik@cutn.ac.in editor: alsamman m. alsamman, international center for agricultural research in the dry areas (icarda), giza, egypt. reviewer(s): suleiman aminu, department of biochemistry, ahmadu bello university, zaria, nigeria. abdulaziz ascandari, chemical and biochemical sciences-green process engineering, university mohammed vi polytechnic, benguerir, morocco. received: october 10, 2023 accepted: december 24, 2023 published: december 30, 2023 citation: mary as, patil mm, kundu g, rajaram k. machine learning mediated advanced phage and antimicrobial therapy a futuristic approach. 2023 dec 30;6:bs202304 copyright: © 2023 mary et al.. this is an open access article distributed under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. data availability statement: all relevant data are within the paper and supplementary materials. funding: the authors have no support or funding to report. competing interests: the authors declare that they have no competing interests. machine learning mediated advanced phage and antimicrobial therapy a futuristic approach aarcha shanmugha mary 1 >< , manali manik patil 1 >< , godhuli kundu 1 >< , kaushik rajaram *,1 ><� abstract the emergence of antimicrobial resistance (amr) has overwhelmed the contemporary curatives and have turned into one of the major challenges in the biomedical sector. with increasing deaths being associated with amr every year; early detection of pathogens and development of novel drugs and alternative therapies, have all become ad hoc in diagnosis, prognosis and patient survival. bacteriophage therapy remains a viable strategy to counteract amr, yet unduly restrained by phage resistance. phage infection is a natural phenomenon and can be widely manipulated in vitro using advanced techniques including the crispr/cas systems which renders phage therapy an upper hand in comparison to conventional drugs. phage identification, host range detection, determination of phage-receptor binding efficiency, adsorption rate, phage genome analysis are crucial stages in phage selection and phage cocktail preparation and moreover pivotal in flourishing phage therapy. the ascent of translational research and omics has allowed the development of quick, reliable and precise strategies for phage-based diagnosis and treatment techniques. however, in vitro evaluation of amr and phage factors as well as storing, processing and analyzing large laboratory data outputs are expensive, time-consuming and labor-intensive. machine learning (ml) is a utilitarian strategy to organize, store, analyze data sets and more importantly allows prediction of certain features by recognizing patterns in the data sets. with the huge number of research been carried out around the globe and enormous data sets being published and stored in databases, ml can utilize the available data to perform and guide in developing alternative therapeutics. several ml based tools have been developed to predict resistance in host, phage grouping for cocktail preparation, resistance and lysogenic genes detection, phage genomic evaluation and to understand phage-host interactions. ml also allows the in silico analysis of large samples (drug/phage) and reduces sample size for in vitro evaluation thereby reducing overall costs, time and labor. the present review summarizes the available ml algorithms and corresponding databases used in amr and phage research. it also emphasizes the status quo of antimicrobial and phage resistance in the healthcare sector and analyses the role of ml in analyzing biological databases in order to predict possible phage/drug-host interaction patterns, phage susceptibility, suitability of phage strains for therapy and recommends the most efficient drug combinations and treatment strategies. keywords: antimicrobial, amr, bacteriophage, resistance, machine learning, database, phage, bacteria, pathogen, infection background an alarming hike in morbidity and mortality due to bacterial infections have been reported worldwide in the past decade [1]. the centre for disease control and prevention and the world health organisation have arbitrated in this global health threat, and have identified and listed the priority pathogens entitled with the acronym eskape, and have implemented strategies to combat pan drug-resistant (pdr), multidrug-resistant (mdr) and extensively drug-resistant (xdr) bacteria [2; 3]. the clinical and laboratory standards institute (clsi) has disclosed the ineffectiveness and failure of antibiotics against eskape pathogens overtime [4]. highlights in bioscience page 1 of 18 december 2023|volume 6 https://doi.org/10.36462/h.biosci.202304 https://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/ mailto:aarchaachu3939@gmail.com mailto:manalipatil85801@gmail.com mailto:gksonai03@gmail.com mailto:rkaushik@cutn.ac.in http://orcid.org/0000-0002-1830-4083 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach development of alternative drugs is on the horizon and in 2019, mulani et al., catalogued the emerging approaches like antimicrobial peptides, antibiotic combination therapies, photodynamic light therapy, bacteriophage therapy, nanoparticles and phytochemicals to combat antimicrobial resistance (amr) [4]. additionally, state-of-the-art treatment strategies like crispr/ cas9 and bacterial vaccines have also been surfaced and are under exploratory research and development [5]. bacteriophage are ubiquitous and are present alongside the host and are pivotal in regulating bacterial populations; thereby maintaining microbial balance. phage therapy has been largely neglected during the antibiotic era; however, with the emergence of drug-resistant bacteria it has regained its lost glory. on one hand, bacteriophage remains a logical and sustainable remedy for amr, whereas bacterial evolution and fitness in presence of phage allows the persistent cells/mutants to grow and propagate [6]. bacteriophage resistance can also be associated to bacterial abortive mechanisms, receptor mutations, restrictionmodification (rm) systems and crispr-cas systems [7]. while bacteriophage resistance is a backlash for phage based treatment regimen, certain clinical implications incurred due to phage resistance are of therapeutic importance. in a clinical setting, phage resistance does not only result in treatment failure but can also aid cross-resistance across other phage and antibiotics [7]. in 2018, wright et al., demonstrated the modular network of cross-resistance in spontaneous mutants of p. aeruginosa against 27 distinct phage; and emphasized the role of cross-resistance network based prediction of mutation frequency and phage combinations for successful therapy [8]. screening and identifying putative phage for effective host inhibition is yet another major challenge where conventional methods can turn out to be unreliable, time-consuming and labour-intensive. melo et al., in 2022, evinced the use of flow cytometry for fast, reliable and highthroughput phage screening [9]. genomic analysis associated to receptor synthesis of p. aeruginosa phage (k8) resistant-mutants by pan et al., in 2016, revealed the absence of o-antigen (serve as receptor) in mutants [10]. similarly, innumerable research has documented the genes associated to the receptors modification in host [11; 12; 13], receptor binding proteins (rbp) [14; 15], antimicrobial genes (args) [16; 17], cross-resistance network [8], phage-host interaction [18; 19], endotoxins [20; 21], lysins [22; 23; 24; 25], pharmacokinetics [26; 27], whole genome data [28; 29; 30], gene expression data [31; 32] and structural information [33; 34] all of which are paramount information in establishing phage therapy. the advancements in research strategies, subsequent outputs and innumerable data has been largely controlled by computational intervention and developments in bioinformatics [35]. even when bioinformatics provides large range of data storage, indexing, analysis and data retrieval; recent developments in machine learning including deep learning and artificial neural network (ann) have allowed the unprecedented prediction by analysing massive data sets [36]. biological databases or repositories are key prerequisites in machine learning and are classified into primary, secondary and composite, and typically reserve molecular sequences (genbank, ddbj, pir), structures (pdb), metabolic pathways (kegg, metacyc), enzyme structure and interactions (brenda), molecules (pubchem), microarray gene expression data (geo), taxonomic data (catalogue of life), disease data (omim), model organisms (rgd, flybase) and bibliographic data (pubmed) [37]. machine learning algorithms learns from these data sets by analysing and identifying patterns; and this is particularly useful in prediction, classification, feature identification/selection and clustering [38]. ml algorithms have remarkable applications in biomedical research including disease diagnosis, drug discovery and development, personalized medicine, medical image analysis, electronic health record analysis and supreme role in analysing raw data in genomics and proteomics [39]. several concerns arise in using machine learning and deep learning for biological data analysis, including ethical integrity and analysis of noise data along with selected features [40]. this review, elaborates the use of machine learning in devising efficient therapeutic strategies to combat amr as well as its applications in exploratory and basic research. it also briefs the potential of phage therapy and corresponding databases and ml algorithms to aid in the development of phage based curatives and strategies to overcome phage resistance by predicting bacterial susceptibility, phage host range and infectivity. this review also summarizes currently used ml algorithms and databases for research and medical purposes. phage therapy the antibiotic pipeline has shown seldom growth with very few novel compounds being commercialized in the recent years [41]. it is safe to say that the ’post-antibiotic era’ is approaching, and the development of effective alternative therapies is essential. the discovery of the mighty penicillin and the subsequent development of other broad-spectrum antibiotics marked the beginning of the golden age of antibiotics, and as a result, the then-used alternatives like phage and other therapeutics have been sidelined since the 1940s [42]. lytic phages are potential bactericidal agents and have been successfully used in clinical practices. the phage-bacterial interactions are commonly observed to be obligate lytic (virulent), pseudolysogenic, lysogenic, and chronic [43]. lytic phages are bacterial viruses that replicate immediately after entering the host cell and release the progenies; on the other hand, lysogenic phages are viruses that integrate the phage genome with the bacterial genome for generations and can resume the lytic life cycle [44]. pseudolysogeny occurs when the host cells are deprived of nutrients, and the phage genome neither enters the lytic nor lysogenic cycle but stays inactive [45]. chronic life cycle is also known as the carrier state where the progeny is released through the host cell membrane without rupturing or damaging the cell, resulting in a long-term infection [44]. while lytic phages are the only bacterial viruses that could be translated for therapy, lysogenic conversion of phage in bacteria has allowed the acquisition of undesirable genes. for highlights in bioscience page 2 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach instance, corynebacterium diphtheria and enterohaemorrhagic escherichia coli acquired toxin-producing genes diphtheria toxin (siphovirus β-phage) and shiga toxin (lambdoid phage) respectively from the integrated phage genome [46; 47]. besides the type of phage life cycle, the optimal bactericidal efficacy of phage is also dependent on factors like adsorption rate, latency period, multiplicity of infection (moi), and burst size. however, bacterial features including the host outer membrane (lps, capsule, peptidoglycan), receptors, presence or absence of flagella or pili can alter the fate of phage infectivity. xuan et al., through their experiment in 2022, proved that quorum sensing upregulated the synthesis of lipopolysaccharides (typically for biofilm production) which are receptors for phage adsorption, thereby promoting phage infection [42]. phage-encoded enzymes like depolymerases are capable of degrading the glycan protective layer in bacteria to establish infection [48]. at a clinical setting, the use of phage cocktails and lysins are also potential strategies to ensure bacterial growth suppression. in 2019, aslam et al., reported the use of bacteriophage cocktails for treatment against p. aeruginosa and burkholderia dolosa infection in lung transplant recipients [49]. similarly, independent studies have evaluated the use of ct-pa (p. aeruginosa cocktail), ab-sa01 and nov012 (staphylococcus aureus cocktail) for treatment against chronic rhinosinusitis [50; 51; 52]. uyttebroek et al., in 2021, also summarized the use of bacteriophage against recalcitrant chronic rhinosinusitis due to s. aureus and p. aeruginosa colonization and biofilm formation [53]. a recent study in 20 patients positive for mycobacterium infection treated with intravenous phage administration, 11 patients showed a favorable response, and phage neutralizing antibodies were found in 8 patients [54]. topical phage administration is a reliable strategy to ensure maximum phage stability, with minimal immune responses. pp1131 , phage cocktail used against p. aeruginosa burn wound infections, was found to reduce the bacterial load, but at a significantly lower rate in comparison to the standard of care treatment. the drop in the phage performance was linked to the loss of titer during manufacturing where the participants received low phage concentrations than intended [55]. fagoma (spanish network of bacteriophages and transducing elements) in its regard to bacteriophage therapy advised the use of phage-based therapy only for patients infected with mdr pathogens or in case of antibiotic hypersensitivity and infection in antibiotic reluctant areas such as the prosthetics [56]. even with a vast number of in vivo and clinical studies, commercialization of phage-based therapy has been under scrutiny due to the heterogeneity in the outcome and also due to the lack of unreported adverse effects. from the stage of selection of phages (single phage/cocktails), mode of administration to the treatment duration, no standards have been established till date. onsea et al., in 2021, devised a multidisciplinary strategy to overcome aforementioned hurdles in establishing phage therapy as a standard of care treatment [57]. consecutive reports have been updated on the status of phage therapy in germany from the 1930s till date along with the present challenges in phage production and commercialization on a large scale [58]. phage-based therapeutic product development should ensure high quality, efficacy, and safety for clinical usage and, moreover, should be gmp-certified [59]. another widely accepted aspect of phage application in clinical practice is the adjunct use of phage with antibiotic. liu et al., in 2020, evaluated the phage-antibiotic interactions (antagonism, synergy, additive) by analyzing the stoichiometry and among different classes of antibiotics. through specific real-time readout synograph’, they concluded that the mechanism of phageantibiotic synergy is antibiotic class dependent, and the synergistic activity can be suppressed by bacterial growth conditions [60]. the resensitization of antibiotic-resistant strains during phage-antibiotic combination therapy is attributed to the trade-off costs during bacterial fitness. wang et al., in 2021, demonstrated the use of colistin-phage (phab24) combinations against acinetobacter baumannii, and reported that phage phab24 was capable of eliminating both colistin-sensitive and resistant strains along with increased sensitivity of phage-resistant mutants to colistin [61]. emerging proofs are indicative of successful phage therapy, whereas resistance is a significant factor in establishing the same. antimicrobial and phage resistance the resurrection of phage therapy and augmentation of antibiotics have shown potential in developing efficient bactericidal therapeutics; whereas, phage and antibiotic/drug resistance prevails as a major concern. bacteria attain antimicrobial resistance by employing one or more mechanisms that may involve either drug alteration/inactivation, overcoming intracellular drug accumulation, modification of the drug binding sites, efflux pumps, or by forming biofilms, figure 1 [62]. eskape pathogens also acquire novel resistance mechanisms which are not part of their natural intrinsic defense methods. enterococcus faecium produces penicillin-binding-protein 5 (pbp5) that provides protection against β-lactam drugs (penicillin, ampicillin, and cephalosporins). e. faecium also resists the combined doses of aminoglycosides and β-lactam/glycopeptides by chromosomal aac (6’)-i enzyme [63]. the clonal complex 17 (cc17) strains of e. faecium are assigned to be responsible for hospitalacquired e. faecium infection and contain virulence and resistance genes conferring protection against a series of antibiotics, including ampicillin and quinolones [64]. similarly, s. aureus have attained resistance against penicillinase-resistant drugs like methicillin, cloxacillin, and oxacillin (mrsa strains) but remain susceptible to glycopeptides. resistance towards β-lactam drugs is conferred by plasmid-encoded blaz gene as well as through pbps (meca & mecc) [65]. last resort drugs like colistin and tigecycline are used for esbl (extended-spectrum β-lactamases) and carbapenemase-producing klebsiella pneumoniae infections; whereas, colistin resistance is also reported (mcr gene) giving rise to untreatable pan-drug infections [66]. highlights in bioscience page 3 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach in regard to acinetobacter baumannii and p. aeruginosa, the intrinsic defense is quite intriguing with impermeable outer membrane and enhanced efflux pumps. β-lactamases and pbp confer protection against β-lactam and carbapenem drugs in most cases [67]. in p. aeruginosa, oprd loss is associated with imipenem resistance [68]. antibiogram is one of the most commonly used cost-effective in vitro resistance detection strategies. other phenotypic methods include mic detection (micro-dilution method), breakpoint agar method, biochemical tests, immunographic tests (carba-5, resist-4), electrochemical test (byg test), motility test, staining, etc. microscan walk assay, bd phoenix, and vittek 2 are examples of semi-automated mic determination approaches [69]. bacterial strain identification and differentiation are carried out using maldi-tof analysis and strain-specific tests like mgp (methyl-α-d-glucopyranoside) test (e. faecium), slide agglutination test ( s. aureus), modified hodge test, and carbapenemase inhibition method ( p. aeruginosa & a. baumannii), etc. genotypic characterization is ad hoc and mostly targets the detection of genes including superoxide dismutase (soda), vana, vanb (glycopeptides resistance genes), meca, mecc (methicillin resistance genes), blakpc, blandm, blaimp, blavim, blaoxa-48 (carbapenem resistance), mcr (colistin resistance), apha6 , arma (amikacin resistance) through lamp, microarray, real-time pcr, and whole genome sequencing (wgs) [69]. bacteriophage resistance is a complex yet a natural evolutionary process in bacteria. nevertheless, the implications of phage resistance have more arena of challenges than antimicrobial resistance. phage resistance is associated with trade-off /fitness costs that can alter the genotypic and phenotypic characteristics of the bacterial strain. studies have reported reduced virulence, enhanced cross-resistance, altered antibiogram, changes in biofilm formation, permeability variations, and several other modifications during phage exposure [70]. hesse et al., in 2020, evaluated 57 phage-resistant mutants ( k. pneumoniae) and reported that each mutant had distinct genes involving in mutation but with similar function associated with assembly or synthesis of surface receptors which ultimately affected phage adsorption in mutants [71]. phage-resistant upec (uropathogenic e. coli) strains exhibited alterations in lps (lipopolysaccharide) and were confirmed via wgs [72]. garb et al., studied the defense-associated sirtuin (dsr) proteins in 2022, and found that sir2 (n-terminal sirtuin) domain acts as nad+ depletors and helps bacteria to abort phage adsorption and propagation [73]. one of the major findings by laure et al., in 2022, on trade-off costs in salmonella typhimurium was the increased β-lactamase activity of mutants obtained after phage and phage +ampicillin treatment [74]. li et al., in 2022, investigated multiple mutant strains of p. aeruginosa and revealed major mutations in pilt and pilb (type iv pili) genes and chromosomal deletions of approximately 294 kb involving galu (utp-glucose-1-phosphate uridylyltransferase) and hmga (homogentisate 1,2-dioxygenase) [75]. enhanced dna exonuclease activity through overexpression of mpr gene in mycobacterium smegmatis was proved to confer resistance against phage [76]. owen et al., in 2021, identified bsta-phage-defense proteins encoded by prophage that inhibit exogenous lytic phage infections [77]. similarly, charity et al., in 2022, correlated mtmii prophage genome integration in salmonella typhimurium to cause increased fitness and drug resistance [78]. aforementioned studies are rather a glimpse at the ongoing research to unravel phage-host interactions and resistance mechanisms. a compelling need to develop sophisticated strategies to control and allow the development of prediction models prevails. the data produced through these studies could be put in use to manifest a much clearer picture for the development of phage and other antimicrobial therapeutics. machine learning machine learning (ml) is a scale-up strategy in refining and developing artificial intelligence (ai). contradictory to symbolic ai; ml focuses on learning from datasets and develops an algorithm that could be novel with a distinct understanding of certain features and their respective weights [79]. in biomedical research, computational methodologies have failed in analyzing enormous datasets and ml comes in handy where a labeled/unlabeled dataset is used to train, validate, and test algorithms. interpretation using ml becomes more systematic and allows classification, clustering, and, more importantly, prediction [80]. supervised, semi-supervised, unsupervised, and reinforcement learning are the major learning methods used in ml. in layman’s terms, ml algorithms are fed with raw data, data that are labeled (input/output, cause/effect), or with unlabeled data; either way, ml identifies hidden patterns and allows classification/regression (supervised) grouping/clustering (unsupervised) [81]. supervised learning uses a labeled dataset with identified features and targets, and unsupervised datasets use unlabeled (raw) data where the algorithm determines the best features and target [82]. regression and classification are two common tasks in supervised learning. on the other hand, association, clustering, and anomaly detection are tasks in unsupervised ml. reinforcement learning is a trial-and-error-based learning strategy and is regarded as the best attempt at modeling human-like learning experience [83]. support vector machine (svm), linear regression, logistic regression, naïve bayes classifier (nb), ann, k-nearest neighbor (knn), random forest, and decision trees are ml algorithms that are used to understand the relationship among the features and outcome/target [84]. while linear regression (univariate/multivariate) uses a linear line to describe the relationship, logistic regression predicts a sigmoidal relationship between features and the probability of an outcome. decision trees classify the data based on features, beginning from a root node and partitioning into decision and terminal nodes. random forest is an ensemble producing several decision trees [83]. deep learning is a subset of ml, where hidden neural layers perform learning and decision-making more efficiently without other intervention [85]. stochastic models are more approachable in the case of highlights in bioscience page 4 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach figure 1. antibiotic and phage resistance mechanisms in bacteria. a) intrinsic phage defence involves production of thick outer membrane and receptor modifications. after the phage genome enters the bacterial cell restriction modification (rm) system or crispr cas system degrades or inactivates the genome and avoids further transcription, translation and phage assembly. b) antibiotic or drug resistance mechanisms involves enzymatically inactivating the drugs, drug modification and target site alteration. intrinsic mechanisms involve biofilm production, reduced drug uptake by altering membrane permeability and enhanced efflux pumps. randomness and ambiguity in data. in 2021, goodswen et al., reviewed the uses of ml in biology, which included disease diagnosis, predicting outbreaks/vaccine candidate/drug targets, drug resistance identification, and microbial interactions [86]. with the assistance of some reliable methodological strategies, it will be more realistic to model the global pan-genome. the expectation-maximization (em) algorithm is the multitude approach for determining maximum a posteriori estimates (map) or maximum likelihood estimates (mle) for undocumented models in statistical analysis [87]. reboredo et al., in 2021, have updated the trends in the usage of ml in drug discovery; the authors also emphasized on the economic advantages of ml in detecting active compounds and thereby reducing the cost and effort in processing large sample sizes in pre-clinical and clinical studies [88]. convolutional neural network (cnn) in ml is reportedly efficient in analyzing images (mri, ct, pet scans) with significant application in nuclear medicine [89]. with unlimited application in the biomedical sector along with novel developments, ml has become a reliable tool. among several other applications, ml in amr and development of phage therapy is a growing area of interest. machine learning and antimicrobial resistance computation of data ensures greater accuracy in analysis, which is the foremost requirement for data analysis in the biomedical sector. several infectious diseases are frequently being treated with antimicrobial drugs; however, antimicrobial resistance in bacteria has become a generic reason for treatment failure. developments in machine learning (ml) have allowed the detection and prediction-based applications in antimicrobial resistance [90]. distinct tools for determining the antimicrobial resistance genes and virulence genes based on ml are currently available (figure 2). a specific approach of ml in amr is by analyzing the suitable synergistic drugs for the development of possible combinatorial therapies. indigo (inferring drug interactions using chemogenomics and orthology) uses the best-fitted synergistic drugs for the treatment of intra-abdominal infection using gentamycin -ampicillin developed using model predictions [91]. another refined model that is a bit different in application is the magenta (metabolism and genomics-based tailoring of antibiotic regimens), which facilitates the treatment of biofilm using combinatorial rifampicin dosage [92]. determination of antimicrobial resistance using ml is now being used in various highlights in bioscience page 5 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach figure 2. machine learning algorithm utilized antibiotic and/or virulence gene databases to predict antimicrobial resistance, alternative therapies and its corresponding side effects. the machine learning algorithms can be used to develop personalized medicine and also can predict the synergistic effects of certain combinatorial therapies. sectors, including the first line of intensive care. ml is not only limited to a targeted approach but also narrows down the frequent use of multiple antibiotics under the same diseased conditions [93]. for medical professionals, ml has now become the key predictor for antimicrobial resistance, where ml-based tools are taken into consideration for allowing the screening of the bestsuited antibiotics for the pathogens. recursive partitioning is applicable for the prediction-based analysis of esbl production from klebsiella sp. and e. coli [94]. modified recursive partitioning models are found to be advantageous for developing the logistic regression models [95]. xgboost, one of the openly accessible platforms for ml-based algorithms, has guided the prediction-based analysis of antibiotic resistance for some of the gram-negative bacterial species [96]. the predicted performance by xgboost is better than other risk assessment tools; however, the prediction model is a bit selective and applicable to limited antibiotics. the ml system can also be futuristic for determining the extent of antibiotic resistance during the culture collection. ml would capture the empowerment for the upcoming advancement of antimicrobials determination. ml enables the development of personalized medicinal approaches to cut down frequent uses of antibiotics. the random forest classifier, one of the most supervised ml algorithms, is allocated to accelerate the screening of mdr bacteria among the patients in icu [97]. the associated empirical data of the ml-based algorithm can also be implemented for local antimicrobial susceptibility assessment. some of the icu antimicrobial susceptibility-based ml algorithms are multilayer perceptron and j48 [98]. as discussed earlier, the principles of ml-based systems are i.) being trained from the existing datasets (supervised) and ii.) analyzing or giving output based on unlabeled data (unsupervised). information regarding the underlying drug interactions has to be made available for ml to learn and develop algorithms for predicting the best combinatorial treatment approach. primarily, certain attributes like changes in gene expression, response to the drugs, etc., need to be characterized before computerized for decision-making [99]. based on the available datasets of drug information like chemical structure and function, the algorithms are trained to determine the combinatorial therapy. among multiple algorithms, cosyne (combination synergy estimation) governs the direct structural analysis for an individual drug candidate to analyze the overall combinatorial therapy. basically, to have knowledge of every aspect of the combinatorial compounds, there should be prior data for the drug and the target as well [100]. with the cosyne approach, the level of application can be extended towards diversified combinatorial drug designing against highlights in bioscience page 6 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach drug-resistant e. coli and also against malarial parasite plasmodium [99]. through consistent records, the ml algorithms were able to predict drugs to counteract antimicrobial resistance in mycobacterium and salmonella [101]. the predictability is wellcurated to reframe the ml so that antibiotic resistance can lay the foundation for the model-based prediction. the extent of genetic variations and drug resistance can be supervised using machine learning algorithms. the drug resistance predictability is done so far in accordance with ic50 indicators. the various searches are based upon the retrieval of different sequence databases. tuberculosis, caused by m. tuberculosis, is one of the infectious diseases which could be eradicated using antibiotics, but due to the dosage’s dysfunctionality and random uses of antibiotics, it concludes with a highlighted remark towards the development of mdr tuberculosis [102]. administration of fluoroquinolones is currently considered, but the usage could lead to after effects due to reported toxicity. ml predicts novel drugs, mechanistic performance of the drugs, microarray data construction and analysis, side effect prediction, etc. based upon the existing known dataset, ml-based algorithms can display the futuristic blind prediction. the performance based on the suggestive prediction can confer the right indication towards clarifying the single nucleotide variance followed by the mutation within the genome of mycobacterium tuberculosis. sequenced based algorithms and structure-based simulations employ the right directive approach to analyze the genomic variation, so that suitability for selecting the target gene can be obtained. deep learning is efficient in predictable analysis to distinctively diagnose the inherent antimicrobial peptide (amp) to combat antimicrobial resistance. deep learning was able to evaluate amps as antimicrobial agents and predicted that amp can surpass mdr in carbapenemase-producing e. coli. deep learning is also capable of simulating the newer versions of amps from the existing peptides [103]. with access to different databases like args, a number of deep learning models have been recruited so far to deliberately screen the best-hit searches for antimicrobial genes. a summary of the ml tools for antimicrobial resistance and its associated factors is depicted in table 1. machine learning in phage therapy machine learning has arisen as a promising strategy in phage therapy prediction, intending to discern the most effective bacteriophage that can selectively target distinct bacterial strains. currently, various techniques exist to measure bacteriophage-host interactions experimentally, such as phagefish-clem [113], microfluidic digital multiplex pcr [114], flow cytometry [115], agar overlay assay [116], rna-sequencing [117], spot test, and efficiency of plating [118]. while these methods are highly accurate, they are costly, labor-intensive, time-consuming, and can turn out to be inconclusive. to overcome these limitations, researchers have developed high-precision computational methods for predicting phage-host interactions. the initial phase in phage therapy is the selection of suitable phage, characterized by three indicators that include 1. presence of temperate markers 2. presence of anti-microbial genes and 3. presence of virulence gene. ml basically uses the whole phage genome and corresponding proteome analysis, and from the sequence similarity and analysis of the conserved domains, determines whether the phage undergoes lytic or lysogenic cycle. a recent approach has been developed to determine the suitability of phage for therapeutic purposes based on an online single-step predictor tool. this predictor tool utilizes the protein features such as integrase, cro/ci repressor protein, anti-repressor proteins, immunity repressors, etc., along with abricat tools for determining the antibacterial and virulence genes. apart from ml, other computational tools are also in use to identify host range and host-phage interactions. the alignment-free and alignment-based techniques are widely used tools, where the sequence homology and sequence similarity among host and phage are read by computational tools to predict its host range [119]. blast is a classic example of an alignmentbased method. while the alignment-based method (e.g., phirbo) is the most reliable strategy with the maximum prediction accuracy, certain factors like the prediction of multiple related hosts, spurious alignment, and artifacts lead to comparable false results. alignment-free tools use similarity in the sequence composition of codons, oligonucleotide frequencies, etc. this tool comes in handy where the host and phage lack sequence homology, and thus alignment-based tools are not suitable. ml develops algorithms based on dataset creation (phage genomes and its corresponding protein sequences), feature generation (custom scripts), and validation. computational evaluation precedes other methods by allowing the comparison in a large dataset, especially advantageous for non-cultivable bacteria and the availability of limited host strains. hostphinder examines phage genome sequences to predict the bacterial host of phage [120], virhostmatcher measures crispr sequences and alignment-free similarity to predict virus-host interaction [121], wish outperforms virhostmatcher at various taxonomic levels and predicts the host range of bacteriophages through genome sequences [122], machine learning takes into consideration the measurable properties referred to as features’. nucleotide sequence is a characteristic feature and is used by ml algorithms like prokaryotic virus host predictor (php) and host taxon predictor (htp) for phage-host interaction prediction. the absolute relative oligonucleotide frequencies and a gaussian model are used to predict the host in htp and php, respectively. based on the virus-host associations, ilmf-vh and lmfh-vh integrate the virus and host similarity network [123], [124]. leite’s method uses a one-class learning method to predict the host-viral interaction at the bacterial strain level [125], spacepharer (crispr spacer phagehost pair finder) predicts bacterial and viral interactions at the protein level by comparing spacers and phage [126]. on the other hand, virsorter uses phage-host interaction signals to predict the phage-host interaction [127], and predphi (predicting phagehost interactions) predicts the prokaryote-phage interaction by sequence data [128]. phagetb leverhighlights in bioscience page 7 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach table 1. methods, tools & algorithms, model organisms, genes, databases, applications, and references method tools & algorithms used model organism genes database applications reference pan-genome construction, random forest scoary, prodigal, cd-hit, glimmer3, naive-bayes classifier e. coli amr genes (from core and accessory gene clusters) patric, card analyses the accessory part of the genome to predict args [104] wgs, antibiogram k-mer, random forest, xgboost s. aureus scc mec genes patric predicts antimicrobial phenotype resistance [105] colony screening, rna sequencing, dna sequencing, snp calling, pangenome analysis and indel calling, multilocus sequence typing msa2vcf, illumina hiseq 2500, mafft, samtools, bamtools, bcftools p. aeruginosa pa14 gene ucbpppa14, mlst database ml identified biomarkers assess amr profiles through a molecular test system [106] phylogenomics, and genome sequencing mafft, raxml-ng, t-rex, treetime e. coli malt gene, manz bimat software studies the phage-bacterial coevolution dynamics (molecular and ecological mechanisms) [107] oxford nano-pore technologies, single nucleotide real time (smrt) rebase, decipher, clustal omega cutibacterium acnes mob genes, res gene rebase, ncbi genome database, pfam database, crispr-cas finder restriction-methylation and host protective mechanisms in c. acnes strains [108] twitching motility assays, swimming motility assay. drosophila melanogaster virulence assays. secreted enzyme assay. antimicrobial resistance assay. shearing assays. microb express kit, agilent 2100 bioanalyzer, bedtools software v2.16.2. p. aeruginosa morons clc genomics workbench software v5.1 bacterial and phage symbiotic interaction, bacterial adaptation in various selective pressures [109] mathematical model using an arbitrium-like communication system in a serial passage set up. matlab r2017b bacillus github small peptide mediated signal communication (phagephage),phage life cycle prediction. [110] antimicrobial susceptibility testing, statistical analysis of multidrug resistance, association set mining sentry, apriori s. aureus amr genes analysis of multidrug resistance in staphylococcus aureus [111] serotyping, antibiotic susceptibility testing, set-covering machine, cmy-2 locus analysis python libraries, sistr, iqtree, prodigal v2.6.3, kover v2.0.0, bwa-mem salmonella enterica amr genes plasmidfinder database, diamond v0.8.36 amr genomic characterization of non-typhoidal salmonella serovars to train prediction models for amr phenotypes. [112] highlights in bioscience page 8 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach ages accurately identifies hosts for bacteriophages using genomic sequences [129]. these user-friendly tools enable the study of phage interactions, identifying potential phages that can specifically target pathogenic bacteria, leaving beneficial bacteria intact. machine learning-based approaches for phage therapy prediction have shown great promise in identifying effective bacteriophages for targeted treatment of bacterial infections. these methods offer faster and more cost-effective alternatives to traditional experimental techniques. however, challenges in data availability, model selection, evaluation metrics, interpretability, and user-friendliness must be addressed for their successful implementation in clinical settings [130]. additionally, tools like phacts (phage classification tool set) and bacphlip (bacteriophage lifestyle predictor) are widely used for determining phage lifestyle by annotating proteomes and classifying lifestyles based on conserved protein domains [131], [132]. bacteriophagehostprediction is one other ml which considers more features, including genomic sequences, protein sequences, protein secondary structures, and physiochemical properties. pheri identifies bacterial host from phage sequence through annotated protein sequence clusters. phageleads are ml that focuses on predicting the lifestyle of phage (lytic or temperate) using protein features of temperate markers. the quick prediction times of phageleads make it a valuable tool for efficiently identifying phages suitable for combating antibiotic resistance, and its ability to detect resistance and virulence genes further enhances its utility [133]. incorporating protein biological feature spaces may further enhance the functional similarities predictions [134]. several databases like card [135], shortbred ar [136], megares [137], and vfdb [138] aid in detecting these genes. by overcoming these challenges, machine learning-based phage therapy prediction holds significant potential in combating antibiotic resistance and improving treatment outcomes for bacterial infections. after the development of ml, the usability is determined by several factors such as operating system restriction, automation, and reproducibility. php, hostphinder are web-based prediction tools that are also available, which do not require a specific os. table 2 summarizes the ml algorithms developed for phage research. databases in antimicrobial resistance and phage research in silico approaches for omics studies have become more widely available, which has in turn made it feasible to precisely recognize and catalog determinants in the case of antibiotic resistance and its associated genes. amr archives, and software applications tools have been constructed for wgs-ast based on the available databases, which includes the comprehensive antibiotic resistance database (card), resfinder and its companion database pointfinder, arg-annot, and many more (table 3) [139]. the card offers an informatics paradigm for the notation and assessment of resistomes through integrating the antibiotic resistance ontology (aro) [140]. card is a database on the molecular basis of antimicrobial resistance that is ontology-focused. card has the potential to serve as both reference material and software instruments and resources for directing amr investigation, particularly for arg details and other findings from genomic information and metagenomic facts. this is made possible through the combination of an extensive modulated concept of aro, with arg [141]. the resistance gene identifier (rgi), anticipates amr from genome-wide facts and data using the bioinformatics prediction approaches and coordinated in card, and is a sophisticated strategy [142]. amrfinderplus, a tool of ncbi, analyzes protein annotations and/or gathered nucleotide sequence to find amr genes, resistancelinked point mutations, and particular subclasses of genes. the pathogen detection operations make use of amrfinderplus, and these data are shown in ncbi’s isolate browser [143]. amrfinderplus makes use of the carefully selected hidden markov models and reference gene database from the ncbi. the ncbi’s pathogen detection project incorporates the output of amrfinderplus to quickly group and locate associated pathogenic genetic patterns residing in food, environment, and people with illnesses. microbigg-e (genomic data) together with the amrfinderplus; findings are provided in a more thorough manner, with extra data such as strain names and source of isolates [144]. in amrfinderplus the outcomes are provided for download by the users in two interfaces graphics [139]. each unique isolate has a synopsis of its antimicrobial resistance, stress-related responses, and virulence gene sequence generated in the isolates browser, which may also be retrieved for more research. challenges and opportunities the use of machine learning in phage therapy faces several limitations and challenges. the future of health care system will be influenced by the intervention of ai and ml from organization to personalized precision care. the input of ml will also positively impact the diagnosis systems. one major obstacle is the need for diverse and unbiased datasets to train predictive models. current datasets often suffer from data imbalance, leading to biased predictions and limited generalization to other bacterial species. enriching a dataset with a broader range of phage-bacterium pairs involving different species and strains is essential for improving model accuracy and applicability [147]. another limitation lies in the reliance on outdated databases to extract informative features that affect model relevance and performance. incorporating up-to-date and comprehensive databases is crucial for generating more reliable predictions. additionally, using deep learning models while achieving high accuracy presents challenges in model interpretability. these models are often considered "black boxes," creating challenges for users to comprehend the underlying decision-making process [152]. balancing accuracy and interpretability is crucial for user trust and the real-world application of the models. the computational module for predicting phage lifestyle faces the limitation of uncerhighlights in bioscience page 9 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach table 2. machine learning based tools to aid bacteriophage research. machine learning predictors purpose of ml tools used major data sources algorithms predicting protein efficiency applications references gradient boosting classifier (gbc) predict phage virion protein (pvps) using phage protein sequences. protlearn adaboost classifier (abc), virion proteins 80 & 83 % accuracy for training and independent dataset. discovery of holins and other phage-driven proteins, endolysins, and exopolysaccharides identification of phage virion proteins (pvps) [145] phageleads determines the presence of temperate markers, antimicrobial resistance virulence genes abricate tool,bacphlip plasmid finder integrases, cro/ci repressor proteins, immunity repressors accuracy of 96.5% predicts the lifestyle of phagedetects lysogenic protein and temperate markers [146] phacts predicts the interaction of phage-bacterium hostphinder,k-nearest neighbors (k-nn), random forests (rf)], support vector machines (svm) , and artificial neural networks(ann) genbank, phagesdb.org, genemarks, domine, genemarks, pfam hmm, hmmer api receptor-binding proteins 86% to 90% accuracy predicts phage-host interaction [147] phagetb, blasthost, blastphage , crisprpred . predicts phage-host interactions virhostmatcher-net xgboost, multi-layer perceptron accuracy of 67.993.5% identifying hosts, assessing phage-host interactions, aids candidate phage therapy [129] acrnet anti-crispr analysis raptorx,esm-1b,possum, pacrispr, acranker,deepacr, hidden markov model (hmm) , meme anti-crisprdb, pacrispr, acrs database, uniprot,uniparc, alphafold acr protein,anti-crispr proteins. determining anti crispr from the large-scale protein database [148] predphi identification of phage-host interactions from sequence data k-means clustering phagesdb ,genbank, 81.00% development of personalized treatment for bacterial infections. [149] cryo-electron tomography (cryo-et) phage based therapy against k. pneumoniae strains. chimerax,hmmer,hhpred,phyre2 capsid and tail fibers of phage. receptor-binding proteins, protein gp118,protein gp119 imod,rosettafold functional insight into kp24 adaptation to variable surfaces of capsulated bacterial pathogens [150] pred-bvp-unb identification of bvps within a huge volume of proteins matlab tool,adboost, knn, universal protein resource bacteriophage virion proteins 92.54% & 83.06% accuracy on benchmark and independent datasets designing antibacterial drugs expedite discovery of bvp [151] highlights in bioscience page 10 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach table 3. list of accessible databases for antimicrobial / phage resistant genes, receptor sequences, genomic sequences and snps. databases type of input sequence format of the input sequence purpose developed/ maintained by supporting tools/ db link of the website comprehensive antibiotic resistance database (card) nucleotide, amino acid fasta snps, validated and curated reference sequencing details and molecular foundation of antimicrobial resistance ncbi rgi https://card.mcmaster.ca /home amrfinder nucleotide, amino acid fasta, gff identifies amr genes & resistance-associated point mutations ncbi https://www.ncbi.nlm.ni h.gov/pathogens/antimic robialresistance/amrfinder/ pathosystems resource integration centre (patric) nucleotide, amino acid fasta large database of infectious bacterial genomic information brcs by niaid http://www.patricbrc.org megares nucleotide fastq a metagenomics dataset consisting of amr genes that are identified, characterized and evaluated. amr++ (bioinformatics pipeline), card, arg-annot, lahey https://megares.meglab. org/ resfinder nucleotide fasta, fastq imports assembled contigs, data sequences or completed genomes and detects amr genes. web-based portal and python script https://cge.cbs.dtu.dk/ser vices/resfinder/ food and environment associated anti-microbial resistance database (feamr db) nonclinical test results as well as dietary and surroundings related amr data worldwide antimicrobial research lab, department of biotechnology, university of mumbai card, ndaro, megares, ncbi https://feamrudbtamrlab.mu.ac.in/ national databases of antibiotic resistant organisms (ndaro) a co-operative, centrally controlled, cross-agency center where researchers may obtain amr data to enable real-time pathogen monitoring. ncbi amrfinder functional antibiotic resistant metagenomic element database (farmedb) nucleotide, amino acid fasta it explores functional metagenomics antibiotic resistant genetic variables, serves as an opportunity for investigating ar in the majority of bacteria that are difficult to culture in lab. it serves as a repository for globally available antibiotic resistance linked genome sequences, predicted amino acid sequences, regulatory factors, jumping genes, and predicted peptides associated with antibiotic resistant genes. university of washington http://staff.washington.e du/jwallace/farme/index. html lrefinder nucleotide fasta, fastq offers information on the genes and alterations that cause enterococci to become tolerant to linezolid. centre for genomic epidemiology https://cge.cbs.dtu.dk/ser vices/lre-finder/ galileo amr nucleotide fasta offers quick and precise labelling of genes associated with amr for any dna fragment of gram-negative bacteria. arc bio mara, rac https://galileoamr.arcbio .com/mara/ arg-miner gathersandaccessallof thedata fromvariousargresources. itoffers proofofargs,specificallyplasmids,viruses,orprophages, thatcanbe carriedbymges. ardb, argannot, megares, card, ndaro, resfinder, uniprot, patric https://bench.cs.vt.edu/a rgminer/#/home shortbred amino acid fasta facilitates the highly selective characterization of target protein families and amr genes in shotgun metagomics sequence reading information. the huttenhower lab, department of biostatistics, havard t.h. chan school of public health ardb, card http://huttenhower.sph.h arvard.edu/shortbred comprehensive βlactamase molecular annotation resource (cbmar) nucleotide, amino acid fasta the approach groups beta-lactamases into classes and then subsequently subgroups them based on factors such as gene location, phylogenetic relationships, active site, parent fingerprints, mutational characteristics, antibiotic resistance characteristics, blocker vulnerability, and nucleotide diversity. lahey, pdb, uniprot, genebank, laced, arbd, http://proteininformatics .org/mkumar/lactamased b/ deeparg nucleotide, amino acid fasta, fastq it makes highly confident predictions about args from quick reads and complete gene length sequences based on metagenomic study of environmental sources. ardb, card, uniprot https://bench.cs.vt.edu/d eeparg integrall nucleotide fasta gathers and arranges the integrons' data https://integrall.bio.ua. ppt/? phage receptor database (phred) fastq, scf it gathers bacterial receptors that are necessary for phage-host identification and interacting associations. bio-conversion databank foundation the actinobacteriophage database it sequences, identities, defines and characterizes mycobacteriophages. department of biologicals sciences at the university of pitsburg https://phagesdb.org/ beta-lactamase database (bldb) lahey clinic database nucleotide it collects architectural and biochemical characteristics, along with sequencing data, for every known bl. it describes the genetic factors that give resistance to betalactam substances. part of bacterial antimicrobial resistance reference gene database, ncbi pdb http://bldb.eu http://www.lahey.org/st udies/ antibacterial biocide and metal resistance genes database (bacmet) nucleotide, amino acid fasta it aims to target genetic factor that provide resistance to metalbased substances and biocidal compounds. http://bacmet.biomedici ne.gu.se/ highlights in bioscience page 11 of 18 december 2023|volume 6 http://bioscience.highlightsin.org/ mary et al., 2023 machine learning mediated advanced phage and antimicrobial therapy a futuristic approach tain predictions owing to the variability from random sampling during classification. continuously expanding the phage lifestyle database to include more known phage lifestyles will enhance prediction precision and sensitivity [153]. furthermore, achieving high-precision rates for complex classification schemes, such as host strains and phage families, remains challenging [132]. addressing these challenges requires refining the methodology and exploring novel classification methods. it is necessary to update the tool with new features and experimentally validated data to improve its accuracy and reliability [154]. the insufficient data on ssdna and rna phage is a significant limitation attributed to experimental constraints and resulted in the lack of respective genomic data in databases. extensive research on phage clades and its diversity can bring much revolution in the collection of genetic information and databases [155; 156]. furthermore, predicting phage-host relationships in complex microbial environments remains challenging because current methods often demand large amounts of homogeneous data. developing more data-efficient techniques, such as similarity networks and machine learning, is crucial for accurate predictions [152]. despite advances in computational methods, identification of phage-host interactions remains a central challenge for effective phage therapy. the application of deep learning techniques has shown promise, but their lack of interpretability hinders user understanding. improving model interpretability while maintaining predictive performance is necessary for practical implementation. integrating sequence similarity information and exploring novel features for phage-host interaction pairs could enhance model robustness and accuracy [129]. in conclusion, addressing the limitations and challenges of using machine learning in phage therapy, including dataset bias, outdated databases, model interpretability, and the need for continuous updates, will be pivotal in developing personalized therapies against antibiotic-resistant bacterial infections and in advancing the field of phage therapy. acknowledgements i extend my sincere gratitude to the central university of tamil nadu for its profound support for the successful completion of the manuscript. reference 1. mancuso g, midiri a, gerace e, biondo c. bacterial antibiotic resistance: the most critical pathogens. pathogens. 2021 oct;10(10):1310. 2. basak s, singh p, rajurkar m. multidrug resistant and extensively drug resistant bacteria: a study. j pathog. 2016;2016:4065603. 3. magiorakos ap, et al. multidrug-resistant, extensively drugresistant and pandrug-resistant bacteria: an international expert proposal for interim standard 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phage therapy databases in antimicrobial resistance and phage research challenges and opportunities acknowledgements