Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(4): 39-49, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2389 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Şuţan, N.A., Bărbuceanu, M., Bărbuceanu, D., & Deliu, I. (2023). Cytogenotoxic and antimicrobial effects of Nezara viridula (L.) (Hemip- tera: Heteroptera: Pentatomidae) alco- holic extracts. Caryologia 76(4): 39-49. doi: 10.36253/caryologia-2389 Received: November 18, 2023 Accepted: February 08, 2024 Published: March 14, 2024 Copyright: © 2023 Şuţan, N.A., Bărbuceanu, M., Bărbuceanu, D., & Deliu, I. This is an open access, peer- reviewed article published by Firenze University Press (http://www.fupress. com/caryologia) and 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 rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. ORCID NAŞ: 0000-0001-7459-628X MB: 0000-0003-4387-7884 Cytogenotoxic and antimicrobial effects of Nezara viridula (L.) (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts Nicoleta Anca Şuţan1, Mircea Bărbuceanu2, Daniela Bărbuceanu1,*, Ionica Deliu1 1 Department of Natural Sciences, National University of Science and Technology, POLITEHNICA Bucharest - Pitesti Universitary Center, Romania 2 Department of Environmental Engineering and Engineering Applied Sciences, National University of Science and Technology POLITEHNICA Bucharest - Pitesti Universitary Center, Romania *Corresponding author. E-mail: daniela.barbuceanu@upb.ro Abstract. Due to their multifunctionality and the numerous fields of applicability, insects are extensively studied today for both their biomedical and nutritional prop- erties. In the current study the cytogenotoxic and antimicrobial potential of etha- nol and methanol extracts of Nezara viridula (Linnaeus 1758) was evaluated using the Allium test, respectively the disk diffusion test. A mitostimulatory effect of the extracts of N. viridula and a variation of the cytogenotoxic activity of the extracts in a gender-dependent manner was noticed. As well, significant variations of the mitot- ic index were determined through the type of solvent used and the concentration of the extracts. High frequency chromosomal aberrations and mitotic abnormalities were recorded with high concentration ethanolic extracts. Following the testing of four standard bacterial strains and two standard yeast strains, a slightly antimicrobial activ- ity was observed when compared to control. The use of invasive species in such studies opens up new perspectives on the potential of organisms considered harmful. Keywords: insect, gender, extracts, bioactivity, mitotic index. INTRODUCTION Throughout time, despite their numbers and their significant therapeutic properties, insects had a minor role in traditional medicine and healthcare practices or in the synthesis of modern drugs as compared to plants. In tra- ditional medicine the use of insect species has been recorded in regions of eastern Asia (e.g. China, India, Korea, and Japan), Africa, South and Central America (Costa-Neto 2002; Figueirêdo et al. 2015; Meyer-Rochow 2017; Bair- agi 2019; Zhang et al. 2023; Yong et al. 2023). According to Feng et al. (2009), in China, over 100 insect species have been used for their medicinal potential since ancient times. Namba et al. (1988) showed that 54 types of crude drugs derived from insects are men- http://www.fupress.com/caryologia https://doi.org/10.36253/caryologia-2389 https://doi.org/10.36253/caryologia-2389 http://www.fupress.com/caryologia http://www.fupress.com/caryologia https://orcid.org/0000-0001-7459-628X https://orcid.org/0000-0003-4387-7884 mailto:daniela.barbuceanu@upb.ro 40 Nicoleta Anca Şuţan et al. tioned in a Chinese manuscript from the beginning of the 7th century. Stink bugs are among the insect species mentioned by traditional medicine. Aspongopus chinen- sis (Hemiptera: Pentatomidae), common in China and known in traditional medicine for its analgesic effects and for its role in the treatment of nephropathy, was investigated recently for its antitumor properties (Luo et al. 2012; Tan et al. 2019). Syrup, powder, wax, oils, and tea obtained from the eggs, larvae or adults of the insects from the families Formicidae, Belastomatidae, Termitidae, Cicadidae, Gryllotalpidae, Asilidae, Pompi- lidae, Pentatomidae, etc. are used in north-east of Brazil for therapeutic purposes (Costa-Neto 2002). In the European culture, the use of insects as a source of food or for therapeutic purposes is rarely mentioned (Ulicsni et al. 2016). Cantharidin, known in European traditional medicine especially for its high toxicity to human body, has anti-tumor properties (Rauh et al. 2007). Maggot therapy, simple and effec- tive, has been used in Europe in the treatment of chronic wounds, such as diabetic food wounds or postoperative infections (Sherman et al. 2000). The use of the products provided by Apis mellifera, such as honey, venom, royal jelly, and propolis, was discussed in recent, comprehen- sive reviews (Pasupuleti et al. 2017; Wehbe et al. 2019). The reviews by Zhou et al. (2005) and Park and Kim (2010) systematized the notable applications of chi- tin and its derivatives, which due to their biocompat- ibility and non-toxic nature were thoroughly studied to document their biological and biomedical properties. Furthermore, an increasing number of authors have observed the progress made during the last decades in the treatment of different conditions through the use of compounds obtained from insects and other arthropods. They support the development of insect-based biotech- nologies and biotesting using insects in order to obtain new products for modern medicine (Ratcliffe et al. 2014; Ejiofor 2016; Seabrooks and Hu 2017). Ratcliffe et al. (2011) recommended the use of insects as models in the study of the immune response to human pathogens. Moreover, in a world facing an alarming increase in bacterial and fungal resistance to antimicrobials, the identification of new substances or complex mixtures with antimicrobial properties has become a priority. For instance, strains of Staphylococcus aureus have a great variety in their resistance to antibiotics, often through hor- izontal gene transfer of genetic elements (Foster 2017), so new therapeutic solutions are required. Candida albicans and C. parapsilosis are human pathogens and also a nor- mal commensal; the frequency of C. parapsilosis infections is higher in immunocompromised patients (Trofa et al. 2008) and they need an alternative solution for treatment. The green stink bug N. viridula presents a remark- able polyphagia, with over 150 plant species identified as hosts; however, they prefer leguminous and brassi- caceous plants and they cause serious damage to these plants (Oho and Kiritani 1960; Panizzi et al. 2000; Panizzi 2004). The very efficient secretory / defensive system developed by this species was probably one of the most important factors that led to their worldwide range expansion. A series of research studies was focused on the composition and the role of secretions in stink bugs (Gilby and Waterhouse 1965; Aldrich et al. 1978; Lock- wood and Story 1987; Borges and Aldrich 1992; Pavis et al. 1994; Sturaro et al. 1994). Starting from the wide distribution of the species N. viridula and the fact that it is an invasive species with specific defensive secretions, in the current paper we set out to investigate the cytogenotoxic and antimicrobial effects of alcoholic extracts of N. viridula as a platform for further applications. From our knowledge, this is the first paper to present the bioactivity of alcoholic extracts of N. viridula. MATERIALS AND METHODS Preparation of insect extracts The biological material consisted of larvae and adults of N. viridula collected in the period July-Octo- ber from cucumbers of the Cornichon variety from an organic culture located in the Cotmeana Plateau in southern Romania, from site N 44.98482°, E 024.72828°, altitude 348 m a.s.l. The larvae were reared in labora- tory conditions until the emergence of adults; they were fed with organic cucumber leaves. The two genders were separated considering the phenotypic differences and the biological material was kept in the freezer at -18 °C until the preparation of extracts. The alcoholic extracts of N. viridula were obtained by grinding and macerating 5 g of each gender, females and males respectively, in 100 ml ethyl alcohol 96° and methyl alcohol 96°, respectively, for 48 hours, at room temperature (18-20 °C). The extracts were filtered using Whatman no. 1 filter paper. Evaluation of genotoxic activity of male and female extracts The cytogenotoxic effects of the N. viridula extracts were evaluated with the Allium test. The bulbs of A. cepa (a local variety) with a diameter of 3-4 cm were taken from a private farm. No phytosanitary treatments were applied to obtain the bulbs. Before use they were macro- 41Cytogenotoxic and antimicrobial effects of Nezara viridula L. (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts scopically inspected to be free from pests. Root primor- dia were carefully exposed and immediately afterwards the bulbs were suspended in 30 ml containers with the discoid stem in contact with distilled water for 48 h. The treatment with ethanol and methanol female and male extracts of N. viridula with concentrations of 5%, 15% and 25% (Table 1) was applied for the next 48 h. The negative sample was represented by the onion roots obtained by suspending the bulbs with the discoid stem in contact with distilled water for 96 hours. Three identi- cal samples were prepared for each specific sample con- figuration. Rhizogenesis was stimulated by keeping the containers in the dark at room temperature (20-22 °C). After 96 hours of semi-static exposure (Rank 2003), the roots with a length of 5-10 mm were cut from the base with a sharp razor blade and immersed into Fram- er’s fixative (absolute ethanol: glacial acetic acid, 3:1 v/v), over the night, at 4 °C, to preserve cell integrity. The fixed roots were hydrolysed in HCl 1N, at 60 °C, for 15 minutes by partial dissolution of pectic substances and stained with orcein-acetic solution 1%, for 15 minutes, at 60 °C. Microscopic slides were obtained through the squash technique. To prevent the quick drying of the microscopic slides the edges of the cover slips were sealed with nail varnish (Grant 1982). Microscopic slides were analysed using an Olympus CX 31 microscope at a magnification of 400× (ocular - objective 10×40). The representative images of the dif- ferent phases of mitosis, as well as of the chromosomal aberrations were captured using Color View I CCD digi- tal camera. For each experimental sample we analysed approx- imately 3 000 cells in different phases of the cell cycle. The mitotic index (MI) was determined as the percent- age ratio of the total number of mitotic cells to the total number of cells examined in the microscopic prepara- tion. The frequency of mitotic phases was determined by calculating the percentage ratio of the number of cells in a certain mitotic phase (prophase, metaphase, anaphase or telophase) to the total number of cells examined in the microscopic slides. Evaluation of antimicrobial effect of insect extracts To estimate the antimicrobial effect of insect extracts we used four standard bacterial strains (both Gram positive and Gram negative): Staphylococcus aureus ATCC 25923, Streptococcus pyogenes A Group ATCC 19615, Bacillus subtilis subsp. spizizenii ISM 68/53 (equivalent ATCC 6633) and Escherichia coli ATCC 25922. Two standard yeast strains of Candida albicans ATCC 10231 and C. parapsilosis ATCC 22019 were also used. In vitro assessment of antimicrobial effects of N. viridula extracts were performed by disk diffusion test (Ma et al. 2019; Balouiri et al. 2016). Specific culture medium was used to test the sensitivity of standard bac- terial strains: Mueller Hinton agar (MHA) for S. aureus, B. subtilis and E. coli strains, Mueller Hinton agar sup- plemented with 5% for S. pyogenes strain and Sabouraud agar for Candida strains (Graso Biotech). The bacterial or yeast suspensions (0.5 McFarland) were inoculated on the sterile medium, then sterile filter paper disks (6 mm Ø) were placed onto medium surface. Each paper disk was impregnated with 10 µL of undilut- ed insect extracts. Ethanol 96° (E) and methanol 99.8% (M) were used as negative controls. Gentamicin 10 µg per disk (Tody Laboratories) and Fluconazole 25 µg per disk (Oxoid) were used as positive controls to test bacte- ria (ATB), and yeasts (AM), respectively. The experimen- tal variants were coded according to Table 1. After 18-20 h at appropriate temperature (37 °C), the diameter of inhibition zones was measured and the average of those three values was compared. The yeast strains were incu- bated for 48 h to reveal the sensitivity to the extracts. Statistical analysis Each experimental variant comprised 3 trials. For the processing and valorisation of the data we used the statistical analysis program SPSS for Windows (Statis- tical Package for Social Science), version 20.0 (2010), applying the One-Way ANOVA model, and the Dun- can’s test for multiple comparison, respectively. The sig- nificance of the differences between the effects of the variables or the interaction between them, for which the calculated F had significant values at a level of con- fidence of 95%, was noted in small letters. The relation- ship between an interval variable and a categorical vari- able was determined by Eta correlation ratio. The results are presented as mean ± SE for n=3 bulbs/ sample. Table 1. Encoding of the samples. Encoding Male/Female (M/F) Ethanol/Methanol (Et/Met) NEM 5% / 15% / 25% M Et NMM 5% / 15% / 25% M Met NEF 5% / 15% / 25% F Et NMF 5% / 15% / 25% F Met 42 Nicoleta Anca Şuţan et al. RESULTS Cytogenotoxic activity of male and female extracts of N. viridula After 48 hours from the exposure to the alcoholic extracts, the cells in different mitotic phases were evalu- ated using the optical microscope; results are shown in Figure 1 as percentage of cells in mitosis. The MI deter- mined for the control sample (5.96%) was not signifi- cantly different from the MIs calculated for the samples defined by the concentration 5% of the extracts of N. viridula. Similarly, compared to the control, the extracts of N. viridula with a concentration of 15% produced an insignificant increase of the MI, except for NEF 15%. However, increasing the concentration of the extracts to 25% was associated with a significant increase of MI in meristematic root cells, irrespective of the solvent used. Regarding the correlation between the dependent and the nominal variables, the Eta coefficient of 0.638 indicates that 63% of the IM variation may be attrib- uted to the independent variable (concentration), while the Eta coefficient of 0.09 indicates that there is a very slight positive correlation between gender and IM, with only 0.09% of the IM variation being attributed to gen- der (Table 2). Figure 2 shows the results regarding the distribu- tion of the phases of mitotic division in the root meris- tem cells of A. cepa exposed to the action of ethanol and methanol female and male extracts of N. viridula. In the control roots the values of the indices were of 75.4% for the prophase, 11.2% for the metaphase, 4.7% for the anaphase, and 8.6% for the telophase. Keeping the roots for 48 hours in ethanol and methanol extracts of N. viridula with a concentration of 5% led to a signifi- cant increase in the percentage of prophases associated with a significant decrease of metaphases, except for the sample NMM 5%. The prophase index with a high value was associated with decreased or zero metaphase index. A significant increased metaphase index was noticed for NMM 5%, 15% and 25%. Compared with the control, the highest values of the anaphase index were recorded in the experimental samples NEF 15%, NEM 25% and NMM 25% (Figure 2). The genotoxic effects of the alcoholic extracts of N. viridula were assessed by registering the chromo- somal aberrations in the meristematic root cells of A. cepa (Table 3). Sticky chromosomes, anaphase bridges, Figure 1. Influence of ethanol and methanol female and male extracts of N. viridula on the mitotic index in the meristematic root cells of A. cepa (a, b, c, d, e: interpretation of significant differences using Duncan’s test, p<0.05). Table 2. Influence of nominal variables on the mitotic index in the root meristem cells of A. cepa (interpretation of the correlation between the dependent and the categorical variables using the Eta coefficient). Directional measures Value Nominal by Interval Eta Concentration Dependent 1.000 IM Dependent 0.638 Gender Dependent 0.957 IM Dependent 0.098 43Cytogenotoxic and antimicrobial effects of Nezara viridula L. (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts C-mitoses, vagrant and laggard chromosomes, poly- ploidy, fragments of chromosomes, but also some mitotic anomalies such as micronuclei, binucleate cells or nucleoplasmic bridges (Figure 3) were observed with a variable frequency in root tip cells. The incubation of onion roots in extracts of N. viridula did not produce significant differences in the total frequency of chromo- somal and mitotic aberrations, except for NEF 25%, the sample with the highest frequency. Of the interphase anomalies, micronuclei were observed in the experimental samples defined by extracts at 15% concentration, and the nucleoplasmic bridges were more frequently identified in the cells treated with extracts of N. viridula female, irrespective of the sol- vent. Laggards, stickies, anaphase bridges and multipolar anaphases were predominant in the tested samples. The frequency of sticky chromosomes was ranging between 20.06% in the sample NMM 5% and 90.47% in the sam- ple NEF 15%, and the anaphase bridges varied between 5.55% in the NMM 5% and 91.67% in NMF 15%. Antimicrobial activity of male and female extracts of N. viridula The antimicrobial effect of N. viridula extracts is presented in Table 4. The largest zones of growth inhibition were observed for Candida albicans (between 11.33 mm and 15 mm) and C. parapsilosis (between 10.66 mm and 12.66 mm), under the action of ethanol extracts. While the same inhibition zone was induced by extracts and negative control in C. albicans, C. parapsilosis was sensi- tive to ethanol extracts. The inhibitory effect produced by insect extracts on B. subtilis had almost the same value with the one for C. parapsilosis. Both ethanol and methanol extracts had a greater impact against bacteria than negative controls (an exception occurred for methanol extracts from females of N. viridula). With smaller values for the diameter of the inhibition zones than B. subtilis, but big- ger than the negative controls, the E. coli strains demon- strated a sensitivity to insect extracts. However, S. aureus, in correlation with its well- known resistance to many antimicrobial substances (Foster 2017), and S. pyogenes, revealed the smallest zones of growth inhibition. The S. aureus strain had a slight sensitivity to the methanol extract of males of N. viridula, and S. pyogenes demonstrated a slight sensitivi- ty to the ethanol extract of males of N. viridula; the oth- er values were smaller than those to negative controls. Certain bacteria were influenced by insect extracts, depending on the gender of insects. For instance, for Figure 2. Influence of alcoholic extracts of N. viridula on the distribution of the phases of mitotic division in the root meristem cells of A. cepa (a, b, c, d, e, f, g, h: interpretation of significant differences, using Duncan’s test, p<0.05). 44 Nicoleta Anca Şuţan et al. B. subtilis the inhibition zones had higher diameters by the ethanol extracts from females of N. viridula and the methanol extracts of males of N. viridula. At the same Ta bl e 3. F re qu en cy o f c hr om os om al a be rr at io ns a nd m ito tic a no m al ie s in du ce d by th e ac tio n of a lc oh ol ic e xt ra ct s of N . v iri du la in th e ro ot m er ist em s of A . c ep a (a , b , c , d , e , f , g : in te rp re ta tio n of si gn ifi ca nt d iff er en ce s u sin g D un ca n’s te st , p <0 .0 5) . Ex po su re va ria nt s M ic ro - nu cl eu s Bi nu cl ea te ce lls N uc le op la sm ic br isg es Va gr an t fo rm s C -m ito ze St ic ky ch ro m os om es A np ha se br id ge s M ul tip ol ar an ap ha se Te lo ph as e br id ge s La gg ar ds fo rm s O th er s To ta l C on tr ol - 0. 2± 0. 03 ab - - - 35 .7 4± 12 .1 6a bc 14 .0 9± 5. 9d e 0. 32 ±0 .3 2b - 0. 32 ±0 .3 2b 0. 41 ±0 .4 1a 1. 26 ±0 .4 1b N EF 5 % 0. 65 ±0 .2 1a 0. 22 ±0 .0 5a b 0. 33 ±0 .3 3b - - - - 0. 27 ±0 .2 7b 2. 08 ±2 .0 8 ab 0. 69 ±0 .1 2a 0. 17 ±0 .0 6a 2, 5± 0. 32 b N EF 1 5% - 0. 45 ±0 .4 5a 16 .6 6± 9. 62 a - - 90 .4 7± 4. 76 a - - - - 0. 14 ±0 .1 a 3. 32 ±0 .7 6b N EF 2 5% - - 3. 45 ±1 .0 3b - - 77 .5 ±1 1. 46 ab 76 .3 9± 6. 06 ab c - - - 0. 03 ±0 .0 3a 9. 48 ±2 .3 9a N EM 5 % 0. 96 ±0 .2 2a 0. 18 ±0 .0 6a b - - - - - 0. 33 ±0 .3 3b - 0. 33 ±0 .3 3 b 0. 64 ±0 .2 7a 2. 03 ±0 .5 1b N EM 1 5% - - - 1. 75 ±1 .7 5a - 85 .6 7± 7. 69 ab 77 .7 8± 14 .7 ab 0. 06 ±0 .0 6c - 1. 45 ±1 .4 5 a - 1. 43 ±0 .2 6b N EM 2 5% - - - - - 86 .7 5± 6. 88 ab 78 .7 1± 7a b - - - - 2. 46 ±0 .2 2b N M F 5% 0. 81 ±0 .4 5a - 0. 04 ±0 .0 4b - - 73 .8 1± 14 .4 8a b 35 .5 5± 19 .3 7c d 0. 11 ±0 .1 1c - 0. 11 ±0 .1 1b 0. 05 ±0 .0 3a 1. 57 ±0 .3 5b N M F 15 % - 0. 04 ±0 .0 4a b 0. 1± 0. 1b - - 61 .5 2± 6. 24 ab c 91 .6 7± 8. 33 a 0. 96 ±0 .5 3a - 0. 96 ±0 .5 3 b - 2. 55 ±0 .4 3b N M F 25 % - 0. 08 ±0 .0 4a b 0. 69 ±0 .3 5b - - 37 .6 1± 1. 81 ab c 41 .6 6± 12 .7 3c d 0. 19 ±0 .0 9c - 0. 19 ±0 .0 9b 0. 03 ±0 .0 3a 1. 74 ±0 .3 7b N M M 5 % 0. 15 ±0 .0 6b 0. 03 ±0 .0 3a b 0. 18 ±0 .1 4b 2. 22 ±2 .2 2a 68 .5 3± 11 .8 8a 20 .0 6± 0. 77 bc 5. 55 ±5 .5 5e 0. 1± 0. 06 c 9. 52 ±9 .5 2 a 2. 56 ±2 .5 6a 4. 76 ±4 .7 6a 3. 41 ±0 .7 b N M M 1 5% - - - - 1. 19 ±1 .1 9b 65 .2 8± 63 .6 5a bc 61 .1 1± 13 .8 9a bc - - - - 2. 21 ±0 .4 8b N M M 2 5% - - - - 6. 95 ±3 .5 6b 64 .6 8± 4. 57 ab c 50 .5 3± 3. 68 bc - - - - 1. 57 ±0 .1 3b Figure 3. Chromosomal and mitotic aberrations identified in the root tips of A. cepa exposed to the extracts of N. viridula: (a) sticky chromosomes – NEM 15%; (b) anaphase bridges – NEM 25%; (c) C-Mitosis – NMM 15%; (d) giant cell – NEF 25%; (e) vagrants – NEM 15%; (f) micronucleus – NMF 15%; (g) binucle- ate – NEM 5%; (h) polyploidy – NMM 15%; (i) telophase bridge and chromosome fragment – NEF 25%; (j) laggard – NMF 15%; (k) multipolar telophase – NMM 15%; (l) star polar anaphase – NEM25%; (m) multipolar anaphase – NMM 15%; (n) nucleoplas- mic bridges – NEF 5%; (o); (p) apoptotic bodies – NMF5%. a b c d Figure 4. The inhibition zones induced by alcoholic extracts of Nezara viridula (L.): (a) Gram negative bacteria - bacillus; (b) Gram positive bacteria - bacillus; (c) Gram positive bacteria - coccus; (d) Eukariotic microorganism - yeast. 45Cytogenotoxic and antimicrobial effects of Nezara viridula L. (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts time, Escherichia coli were equally inhibited by the extracts from males or females of N. viridula. All the values obtained for the growth inhibition zones under the action of insect extracts were smaller than the values for positive controls (either antibiotic or antifungal agents). DISCUSSIONS Statistical analysis of the data revealed differences and significant differences of the cytogenotoxic end- points under research. Aldrich et al. (1978) found that the repugnant defensive secretion from both males and females of N. viridula contains (E)-2-hexenal, hexanal, 1-hexanol, and n-tridecane. Although the above authors found the n-tridecane content was three times more than males, in our study mitosis progression was influenced by gender only by ethanolic samples which induced the highest and the lowest MI. One of the main mechanisms involved in cytotoxicity and genotoxicity is the overpro- duction of reactive oxygen species (ROS) that can induce reversible and irreversible changes in proteins and cause DNA damage (Zhu et al. 2013; Choudhury et al. 2016; Tanaka and Hadwiger 2017). In this context, it is worth mentioning that aldehydes, such as hexanal can act as secondary messengers of oxidative stress controlling cell proliferation, cell differentiation and cell death (Barerra et al. 2008; Barrera 2012). The cytotoxicity of extracts, which was dependent on concentration, could be attrib- uted to epoxides (Marshall and Caldwell 1996), which are the main constituents of pheromones in the males of N. viridula (Brézot et al. 1994). The cytotoxic effect of the N. viridula extracts was manifested through an increase in prophase frequency and a decrease in metaphase frequency, suggesting that cells underwent mitosis, but were arrested during pro- phase. Inhibition of an early mitotic stage could be due to the alteration of the chromosome condensation mech- anism or the inhibition of the microtubule assembly mechanism, which leads to prometaphase arrest (Oliva et al. 2002). NMM, irrespective of their concentration, induced a significant metaphase arrest in root tip cells suggesting the disturbed spindle function, which pro- duced C-mitosis and polyploidy (Figure 3). Chromosomal aberrations and micronuclei are bio- markers of genotoxicity and chromosomal instability determined by mutagenic agents (Bonciu et al. 2018). As complex mixtures, the extracts act as clastogenic agents inducing the formation of ana-telophase bridges and micronuclei, but also as aneugenic agents by inducing delays, adherence and multipolarity (Leme and Marin- Morales 2009). However, analysing the total frequency of chromosomal and mitotic aberrations it was noticed that NEF 25% induced the highest genotoxicity. The ana- lytical characterization of the extracts could add a clue regarding the high genotoxicity, especially for the etha- nol female extracts of N. viridula. Until now, literature analysis provides scarce information on the cytogeno- toxic effects of extracts obtained from insects, in terms of investigating their therapeutic potential. A series of studies noticed the absence of cytogenotoxic effects in the case of extracts obtained from edible insect species, including Zonocerus variegatus, Oryctes boas (Memiş et al. 2013), Onitis spp., Caelifera spp. and Gryllotalpa spp. (Koc et al 2014), Locusta migratoria (Turkez et al. 2014), confirming the safety of their consumption by humans. Several recent papers presented the results of research on the genotoxic effects of extracts obtained from other invertebrate animals. Jayathilake and Jayewardena (2021) investigated with the Allium test aqueous extracts from the sea cucumber, Bohadschia vitiensis, known for cer- tain biological activities and found reduced genotoxic effects consisting of 0.1-0.2% chromosomal aberrations Table 4. The diameter of inhibition zones induced by alcoholic extracts of N. viridula. Experimental variants Inhibition zone (mm) S. aureus Inhibition zone (mm) S. pyogenes Inhibition zone (mm) B. subtilis Inhibition zone (mm) E. coli Inhibition zone (mm) C. parapsilosis Inhibition zone (mm) C. albicans NEM 8.66 9.33 12.66 11.66 12.66 15 NEF 8.66 8.66 13.33 11.66 12.33 15 NMM 9.33 7.33 11.66 9 10.66 12 NMF 7.66 7.66 9 9 11.33 11.33 Ethanol (E) 8.66 9 11.66 10 12 15 Methanol (M) 8.66 8 9 8 11.33 14 Gentamicin 10 µg (ATB) 25 28 31 25 - - Fluconazole 25 µg (AM) - - - - 37 25 46 Nicoleta Anca Şuţan et al. including chromosomal bridges, c-mitosis, chromosomal breaks, and vagrants. By using the Allium bioassay, the mitodepressive effect of the marine sponge extracts Luf- fariella herdmani was highlighted, the results obtained suggesting the antitumor potential of the substances contained (Kuruppuarachchi et al. 2023). In our study, the antimicrobial effect of insect extracts was noted especially for ethanol extracts of N. viridula against those microorganisms which usually are opportunistic pathogens. The bacteria and yeast with higher pathogenic prop- erties (S. aureus, S. pyogenes and C. albicans) are less sensitive to the action of extracts, although other stud- ies emphasized that Methicillin-resistant S. aureus can be inhibited by extracts from insects used in traditional Chinese medicine (Ma et al. 2019) or that Candida albi- cans growth can be inhibited by peptides from super- meal worm, Zophobas morio (Fabricius) (Faruck et al. 2017). Some studies found the antimicrobial activity of cer- tain edible insects to their microbiota, particularly in the case of antimicrobial peptides that can be used for devel- oping new drugs against multidrug-resistant pathogens (Mudalungu et al. 2021). Antimicrobial peptides (AMPs) from insect sources were also used to reduce biofilm associated S. aureus and E. coli; the authors found out the AMPs combined with antibiotics may be a better alternative than antibiotics alone (Sahoo et al. 2021). The effect of ethanol as a negative control was mostly higher than methanol and the diameter of inhi- bition zones was the largest for positive control (anti- biotic or antifungal agent). Neither bacteria nor yeasts were affected by insect extracts more than by standard antimicrobials (Figure 4). The results revealed some dif- ferences between extracts according to the gender of insects, but no obvious correspondence was established. CONCLUSION The mitotic index recorded after the exposure of the onion roots to the action of alcoholic extracts of N. viridula indicated specific and significant variations in relation to solvent and dilution. The ethanolic extracts determined the widest variation of the mitotic index, regardless of the tested concentration, while the metha- nolic extracts had significant mitostimulatory effects only in higher concentrations. The lower concentra- tion extracts were associated with a blocking of cells in prophase, while the extracts obtained from N. viridula males determined the increase in the frequency of meta- phases. The antimicrobial effect of alcoholic extracts from N. viridula was obvious against bacterial strains Escheri- chia coli and Bacillus subtilis; Staphylococcus aureus and Streptococcus pyogenes presented only a slight sensitiv- ity to the insect extracts. The differences between male or female insect extracts regarding their antimicrobial activity were unsteady, requiring further investigation into the production and specific application of insect extracts. FUNDING This work was supported by a grant of the Roma- nian Ministry of Research, Innovation and Digitiza- tion, CNCS–UEFISCDI, project number PN-III-P4-ID- PCE-2020-0620, within PNCDI III. REFERENCES Aldrich JR, Blum MS, Lloyd HA, Fales HM. 1978. Pen- tatomid natural products. Chemistry and morphol- ogy of the III–IV dorsal abdominal glands of adults. J Chem Ecol. 4(2):161-172. https://doi.org/10.1007/ BF00988052. Bairagi SH. 2019. Insects with potential medici- nal significance: a review. Biomed J Sci & Tech Res. 16(3):12024-12027. https://doi.org/10.26717/ BJSTR.2019.16.002849 Barrera G, Pizzimenti S, Dianzani MU. 2008. Lipid per- oxidation: control of cell proliferation, cell differenti- ation and cell death. Mol. Aspects Med. 29(1-2):1–8. https://doi.org/10.1016/j.mam.2007.09.012. Barrera G. 2012. Oxidative stress and lipid per- oxidation products in cancer progression and therapy. ISRN Oncol. 2012:137289. https://doi. org/10.5402/2012/137289. Bonciu E, Firbas P, Fontanetti CS, Wusheng J, Karaismailoğlu MC, Liu D, Menicucci F, Pesnya DS, Popescu A, Romanovsky AV, Schiff S, Ślusarczyk J, de Souza CP, Srivastava A, Sutan A, Papini A. 2018. An evaluation for the standardization of the Allium cepa test as cytotoxicity and genotoxicity assay. Caryolo- gia. 71(3):191–209. https://doi.org/10.1080/00087114. 2018.1503496. Borges M, Aldrich J.R. 1992. Instar-specific defensive secretions of stink bugs (Heteroptera: Pentatomidae). Experientia. 48(9):893–896. https://doi.org/10.1007/ BF02118429. Brézot P, Malosse C, Mori K, Renou M. 1994. Bisabolene epoxides in sex pheromone in Nezara viridula  (L.) https://doi.org/10.1007/BF00988052 https://doi.org/10.1007/BF00988052 https://doi.org/10.26717/BJSTR.2019.16.002849 https://doi.org/10.26717/BJSTR.2019.16.002849 https://doi.org/10.1016/j.mam.2007.09.012 https://doi.org/10.5402/2012/137289 https://doi.org/10.5402/2012/137289 https://doi.org/10.1080/00087114.2018.1503496 https://doi.org/10.1080/00087114.2018.1503496 https://doi.org/10.1007/BF02118429 https://doi.org/10.1007/BF02118429 47Cytogenotoxic and antimicrobial effects of Nezara viridula L. (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts (Heteroptera: Pentatomidae): Role of cis  isomer and relation to specificity of pheromone. J Chem Ecol. 20(12):3133-3147. https://doi.org/10.1007/ BF02033716. Choudhury FK, Rivero RM, Blumwald E, Mittler R. 2016. Reactive oxygen species, abiotic stress and stress combination. Plant J. 90(5):856-867. https://doi. org/10.1111/tpj.13299. Costa-Neto EM. 2002. The use of insects in folk medi- cine in the State of Bahia, Northeastern Brazil, with notes on insects reported elsewhere in Brazilian folk medicine. Hum Ecol. 30(2):245-263. https://doi. org/10.1023/A:1015696830997. Ejiofor AO. 2016. Insect Biotechnology. In: Raman C, Goldsmith M, Agunbiade T. (eds) Short Views on Insect Genomics and Proteomics. Entomology in Focus. 4:185-210. https://doi.org/10.1007/978-3-319- 24244-6_8. Faruck MO, Yusof F, Chowdhury S. 2017. Effect of extraction process parameters on antifungal peptides from Supermeal worm, Zophobas morio (Fabricius). IFRJ. 24(Suppl):463-467. Feng Y, Zhao M., He Z., Chen Z., Sun L. (2009). Research and utilization of medicinal insects in China.  Ento- mol. Res.  39  (5):313-316. https://doi.org/10.1111/ j.1748-5967.2009.00236.x. Figueirêdo RECR, Vasconcellos A, Policarpo IS, Alves RRN. 2015. Edible and medicinal termites: a global overview.  J Ethnobiol Ethnomed. 11(29):1-7. https:// doi.org/10.1186/s13002-015-0016-4. Foster T. 2017. Antibiotic resistance in Staphylococ- cus aureus. Current status and future prospects. FEMS Microbiol. Rev. 41(3):430-449. https://doi. org/10.1093/femsre/fux007. Gilby AR, Waterhouse DF. 1965. The composition of the scent of the green vegetable bug, Nezara viridu- la. Proc. R. Soc. Lond. B. 162:105-120. https://doi. org/10.1098/rspb.1965.0027. Grant WF. 1982. Chromosome aberration assay in Alli- um. A report of the United States Environmental Protection Agency Gene Toxicity Program. Mutat Res. 99:273-291. https://doi.org/10.1016/0165- 1110(82)90046-X. Jayathilake N, Jayewardena U. 2021. Genotoxic potential of aqueous extract: A sea cucumber species, Bohad- schia vitiensis using genotoxicity model Allium cepa. Adv Pharm J. 6(1):9-14. https://doi.org/10.31024/ apj.2021.6.1.2. Koc K, Incekara U, Turkez H. 2014. Biomonitoring of the genotoxic effects and oxidative potentials of com- mercial edible dung beetles (Onitis sp.), grasshopper (Caelifera sp.) and mole crickets (Gryllotalpa sp.) in vitro. Toxicol Ind Health. 30(8):683-689. https://doi. org/10.1177/0748233712457451. Kuruppuarachchi SU, Jayawardena UA, Gunathilake VK. 2023. Use of the Allium cepa Model to Assess the Cytogenotoxicity of Luffariella herdmani Marine Sponge Extract. ATLA Altern Lab Anim. 51(3):175- 187. https://doi.org/10.1177/02611929231171943. Leme DM, Marin-Morales MA. 2009. Allium cepa test in environmental monitoring: A review on its applica- tion. Mutat Res. 682:71–81. https://doi.org/10.1016/j. mrrev.2009.06.002. Lockwood J, Story R. 1987. Defensive Secretion of the Southern Green Stink Bug (Hemiptera: Pentato- midae) as an Alarm Pheromone. Ann. Entomol. Soc. Am. 80(5):686–691. https://doi.org/10.1093/ AESA/80.5.686. Luo XH, Wang XZ, Jiang HL, Yang JL, Crews P, Valeri- ote FA, Wua QX. 2012. The biosynthetic products of chinese insect medicine, Aspongopus chinensis. Fitoterapia. 83(4):754–758. https://doi.org/10.1016/j. fitote.2012.03.002. Ma G, Wu L, Shao F, Zhang C, Wan H. 2019. Anti- microbial Activity of 11 Insects Extracts Against Multi Drug Resistant (MDR) Strains of Bacte- ria and Fungus. IOP Conf. Ser.: Earth Environ. Sci. 252(2):022132. https://doi.org/10.1088/1755- 1315/252/2/022132. Marshall AD, Caldwell J. 1996. Lack of influence of mod- ulators of epoxide metabolism on the genotoxicity of  tans-anethole in freshly isolated rat hepatocytes assessed with the unscheduled DNA synthesis assay. FCT. 34(4):337-345. https://doi.org/10.1016/0278- 6915(96)00109-3. Memiş E, Türkez H, Incekara Ü, Banjo AD, Fasunwon BT, Toğar B. 2013. In vitro biomonitoring of the genotoxic and oxidative potentials of two commonly eaten insects in southwestern Nigeria. Toxicol Ind Health. 29(1):52-59. doi:10.1177/0748233712446721. Meyer-Rochow VB. 2017. Therapeutic arthropods and other, largely terrestrial, folk-medicinally impor- tant invertebrates: A comparative survey and review. J Ethnobiol Ethnomed. 13(1):9. https://doi. org/10.1186/s13002-017-0136-0. Mudalungu CM, Tanga CM, Kelemu S, Torto B. 2021. An Overview of Antimicrobial Compounds from Afri- can Edible Insects and Their Associated Microbiota. Antibiotics. 10(6):621. https://doi.org/10.3390/ antibi- otics10060621. Namba T, Ma YH, Inagaki K. 1988. Insect-derived crude drugs in the Chinese Song dynasty. J Ethnopharma- col. 24(2-3):247-85. https://doi.org/10.1016/0378- 8741(88)90157-2. https://doi.org/10.1007/BF02033716 https://doi.org/10.1007/BF02033716 https://doi.org/10.1111/tpj.13299 https://doi.org/10.1111/tpj.13299 https://doi.org/10.1023/A https://doi.org/10.1023/A https://doi.org/10.1007/978-3-319-24244-6_8 https://doi.org/10.1007/978-3-319-24244-6_8 https://doi.org/10.1111/j.1748-5967.2009.00236.x https://doi.org/10.1111/j.1748-5967.2009.00236.x https://doi.org/10.1186/s13002-015-0016-4 https://doi.org/10.1186/s13002-015-0016-4 https://doi.org/10.1093/femsre/fux007 https://doi.org/10.1093/femsre/fux007 https://doi.org/10.1098/rspb.1965.0027 https://doi.org/10.1098/rspb.1965.0027 https://doi.org/10.1016/0165-1110(82)90046-X https://doi.org/10.1016/0165-1110(82)90046-X https://doi.org/10.31024/apj.2021.6.1.2 https://doi.org/10.31024/apj.2021.6.1.2 https://doi.org/10.1177/0748233712457451 https://doi.org/10.1177/0748233712457451 https://doi.org/10.1177/02611929231171943 https://doi.org/10.1016/j.mrrev.2009.06.002 https://doi.org/10.1016/j.mrrev.2009.06.002 https://doi.org/10.1093/AESA/80.5.686 https://doi.org/10.1093/AESA/80.5.686 https://doi.org/10.1016/j.fitote.2012.03.002 https://doi.org/10.1016/j.fitote.2012.03.002 https://doi.org/10.1088/1755-1315/252/2/022132 https://doi.org/10.1088/1755-1315/252/2/022132 https://doi.org/10.1016/0278-6915(96)00109-3 https://doi.org/10.1016/0278-6915(96)00109-3 https://doi.org/10.1186/s13002-017-0136-0 https://doi.org/10.1186/s13002-017-0136-0 https://doi.org/10.3390/ https://doi.org/10.1016/0378-8741(88)90157-2 https://doi.org/10.1016/0378-8741(88)90157-2 48 Nicoleta Anca Şuţan et al. Oho N, Kiritani K. 1960. Bionomics and control of the southern green stink bug. Shokubutsu Boeki [Plant Protect.]. 14:237–241. Oliva A, Moraes RM, Watson SB, Duke SO, Dayan FE. 2002. Aryltetralin lignans inhibit plant growth by affecting the formation of mitotic microtubular organizing centers. Pestic Biochem Physiol. 72(1):45- 54. https://doi.org/10.1006/pest.2002.2582. Panizzi AR. 2004. Southern Green Stink Bug, Nezara vir- idula (L.) (Hemiptera: Heteroptera: Pentatomidae). In: Capinera JL editor. Encyclopedia of Entomol- ogy. Springer, Dordrecht (The Netherlands): Klu- wer Academic Publishers; p. 2058-2059. https://doi. org/10.1007/0-306-48380-7_3989. Panizzi AR, McPherson JE, James DG, Javahery M, McPherson RM. 2000. Stink bugs (Pentatomidae). In: Schaefer CW, Panizzi AR, editors. Heteroptera of Economic Importance. Boca Raton: CRC Press USA; p. 421-474. Park BK, Kim MM. 2010. Applications of chitin and its derivatives in biological medicine. Int J Mol Sci. 11:5152-5164. https://doi.org/10.3390/ijms11125152. Pasupuleti VR, Sammugam L, Ramesh N, Gan SH. 2017. Honey, propolis, and royal jelly: a comprehensive review of their biological actions and health ben- efits. Oxid Med Cell Longev. p. 1259510. https://doi. org/10.1155/2017/1259510. Pavis C, Malosse C, Ducrot PH, Descoins C. 1994. Dorsal abdominal glands in nymphs of southern green stink bug, Nezara viridula (L.) (Heteroptera: Pentatomi- dae): Chemistry of secretions of five instars and role of E)-4-oxo-2-decenal, compound specific to first instars. J Chem Ecol. 20(9):2213-2227. https://doi. org/10.1007/BF02033198. Rank J. 2003. The method of Allium anaphase-telophase chromosomal aberration assay. Ekologija 1:38-42. Ratcliffe N, Azambuja P, Mello CB. 2014. Recent advances in developing insect natural products as potential modern day medicines. Evid Based Com- plement Alternat Med. 2014:904958. https://doi. org/10.1155/2014/904958. Ratcliffe NA, Mello CB, Garcia ES, Butt TM, Azam- buja P. 2011. Insect natural products and processes: new treatments for human disease. Insect Biochem Mol Biol. 41(10):747-769. https://doi.org/10.1016/j. ibmb.2011.05.007. Rauh R, Khal S, Boechzelt H, Bauer R, Kaina B, Efferth T. 2007. Molecular biology of chantaridin in cancer cells. Chin Med. 2:8. https://doi.org/10.1186/1749- 8546-2-8. Sahoo A, Swain SS, Behera A, Sahoo G, Mahapatra PK, Panda SK. 2021. Antimicrobial Peptides Derived from Insects Offer a Novel Therapeutic Option to Combat Biofilm: A Review. Front Microbiol. 12:661195. htt- ps://doi.org/10.3389/fmicb.2021.661195. Seabrooks L, Hu L. 2017. Insects: an underrepresented resource for the discovery of biologically active nat- ural products. Acta Pharm Sin B. 7(4):409–426. htt- ps://doi.org/10.1016/j.apsb.2017.05.001. Sherman RA, Hall MJR, Thomas S. 2000. Medicinal Mag- gots: an ancient remedy for some contemporary afflictions. Annu Rev Entomol. 45:55-81. https://doi. org/10.1146/annurev.ento.45.1.55. Sturaro A, Parvoli G, Doretti L. 1994. A simple and fast sampling method for the characterization of volatile compounds released by Nezara viridula. Chroma- tographia. 39(1/2):103-106. https://doi.org/10.1007/ BF02320467. Tan J., Tian Y., Cai R., Yi T., Jin D., Guo J. 2019. Antipro- liferative and proapoptotic effects of a protein com- ponent purified from aspongopus chinensis Dallas on cancer cells in vitro and in vivo. Evid Based Com- plement Alternat Med. 2019. 8934794. https://doi. org/10.1155/2019/8934794 Tanaka K, Hadwiger LA. 2017. Nonhost resistance: Reac- tive oxygen species (ROS) signal causes DNA damage prior to the induction of PR genes and disease resist- ance in pea tissue. Physiol Mol Plant Pathol. 98:18-24. Trofa D, Gácser A, Nosanchuk J. 2008. Candida parapsi- losis, an Emerging Fungal Pathogen. Clin Microbiol Rev. 21(4):606-625. doi: 10.1128/CMR.00013-08. Turkez H, Incekara U, Güner A, Aydın E, Dirican E, Togar B. (2014). The cytogenetic effects of the aque- ous extracts of migratory locust (Locusta migrato- ria L.) in vitro. Toxicol Ind Health. 30(3):233-237. doi:10.1177/0748233712452610. Ulicsni V, Svanberg I, Molnár Z. 2016. Folk knowledge of invertebrates in Central Europe - folk taxonomy, nomenclature, medicinal and other uses, folklore, and nature conservation. J Ethnobiol Ethnomed. 12(1):1- 47. https://doi.org/10.1186/s13002-016-0118-7. Wehbe R, Frangieh J, Rima M, El Obeid D, Sabatier J-M, Fajloun Z. 2019. Bee venom: overview of main com- pounds and bioactivities for therapeutic interests. Molecules. 24(16):2997. https://doi.org/10.3390/mol- ecules24162997. Yong HI, Kim TK, Cha JY, Lee JH, Kang MC, Jung S, Choi YS. 2023. Effects of edible insect extracts on the antioxidant, physiochemical, and microbial properties of Tteokgalbi during refrigerated storage. Food Biosci. 52:102377. https://doi.org/10.1016/j. fbio.2023.102377. Zhang E, Ji X, Ouyang F, Lei Y, Deng S, Rong H, Deng X, Shen H. 2023. A minireview of the medicinal and https://doi.org/10.1006/pest.2002.2582 https://doi.org/10.1007/0-306-48380-7_3989 https://doi.org/10.1007/0-306-48380-7_3989 https://doi.org/10.3390/ijms11125152 https://doi.org/10.1155/2017/1259510 https://doi.org/10.1155/2017/1259510 https://doi.org/10.1007/BF02033198 https://doi.org/10.1007/BF02033198 https://doi.org/10.1155/2014/904958 https://doi.org/10.1155/2014/904958 https://doi.org/10.1016/j.ibmb.2011.05.007 https://doi.org/10.1016/j.ibmb.2011.05.007 https://doi.org/10.1186/1749-8546-2-8 https://doi.org/10.1186/1749-8546-2-8 https://doi.org/10.3389/fmicb.2021.661195 https://doi.org/10.3389/fmicb.2021.661195 https://doi.org/10.1016/j.apsb.2017.05.001 https://doi.org/10.1016/j.apsb.2017.05.001 https://doi.org/10.1146/annurev.ento.45.1.55 https://doi.org/10.1146/annurev.ento.45.1.55 https://doi.org/10.1007/BF02320467 https://doi.org/10.1007/BF02320467 https://doi.org/10.1155/2019/8934794 https://doi.org/10.1155/2019/8934794 https://doi.org/10.1186/s13002-016-0118-7 https://doi.org/10.3390/molecules24162997 https://doi.org/10.3390/molecules24162997 https://doi.org/10.1016/j.fbio.2023.102377 https://doi.org/10.1016/j.fbio.2023.102377 49Cytogenotoxic and antimicrobial effects of Nezara viridula L. (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts edible insects from the traditional Chinese medicine (TCM).  Front  Pharmacol.  14:1125600. https://doi. org/10.3389/fphar.2023.1125600. Zhu X, Hondroulis E, Liu W, Li Y. 2013. Biosensing approaches for rapid genotoxicity and cytotoxicity assays upon nanomaterial exposure. Small. 9(9-10): 1821-1830. https://doi.org/10.1002/smll.201201593. https://doi.org/10.3389/fphar.2023.1125600 https://doi.org/10.3389/fphar.2023.1125600 https://doi.org/10.1002/smll.201201593 First cytogenetic study of the Somphong’s rasbora (Trigonostigma somphongsi) (Perciformes, Cyprinidae), a critically endangered species in Thailand Surachest Aiumsumang1, Chavalit Vidthayanon2, Sitthi Kulabtong3, Alongklod Tanomtong4, Sumalee Phimphan1,* Evaluation of the evolutionary process within Populus caspica species from Hyrcanian forests by karyotype analysis Fereshteh Asadi-Corom1,*, Farhad Asadi2, Hossein Mirzaie-Nodoushan1 Chromosomal and genome size variations in Opium poppy (Papaver somniferum L.) from Afghanistan Sayed Zia Rasekh, Ghasem Karimzadeh* Karyological analyses in several Algerian populations of six species of the genus Vicia L. (Fabaceae) Zahia Sebkhi1,4, Rachida Issolah1,*, Nabila Melzi2, Hassina Benmouhoub3, Mohamed Mefti4 Cytogenotoxic and antimicrobial effects of Nezara viridula (L.) (Hemiptera: Heteroptera: Pentatomidae) alcoholic extracts Nicoleta Anca Şuţan1, Mircea Bărbuceanu2, Daniela Bărbuceanu1,*, Ionica Deliu1 Apogamous Isoetes coromandelina L.f. (Isoetaceae) with asynaptic meiosis V. Irudayaraj1,2, A. Benniamin3,*, S. Arokia Raj1 Evaluation of the antigenotoxic potential of fresh bovine whey in onion meristematic roots exposed to Quizalofop-P-tefuryl Florica Colă1, Elena Bonciu1,*, Mugurel Colă1, Nicoleta Anca Șuțan2