CONTACT : Nancy Taha Mohamed nancyt0000@yahoo.com 98 Abstract This study aimed to investigate the bioactive compounds in the haemolymph of scarab beetle Scarabaeus sacer by using the Gas chromatography–mass spectrometry (GS-MS) analysis. The identification of the bioactive compounds is based on peak area, retention time, molecular formula and molecular weight. There are 129 compounds are detected in the haemolymph of scarab beetle and 43 of them were reported to have a bioactivity. The most analyzed bioactive compounds are alcohols, steroids, fatty acids and terpenoids. The current study also test the antimicrobial activity of scarab beetle haemolymph against gram-negative bacteria (Escherichia coli, Enterobacter cloacae, gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), and fungi (Aspergillus fumigatus and Candida albicans). The haemolymph has highest antibacterial activity against gram negative bacteria Enterobacter cloacae, Escherichia coli respectively and against gram-positive bacteria Bacillus subtilis, Staphylococcus aureus respectively. No antifungal activity has been detected. ISSN : 2580-2410 eISSN : 2580-2119 Separation of bioactive compounds from Haemolymph of scarab beetle Scarabaeus sacer (Coleoptera: Scarabaeidae) by GC-MS and determination of its antimicrobial activity Nancy Taha Mohamed 1*, Doaa Hassan Abdelsalam 1, Ahmed Salem El-Ebiarie 1, Mahmoud Elaasser 2 1 Zoology & Entomology Department, Faculty of Science, Helwan University. 2 Regional centre of Mycology and Biotechnology, Al-Azhar University. Introduction Coleoptera is one of the largest order of insects with about 370,000 insect species described worldwide. The family Scarabaeidae encompasses over 30.000 species of beetles worldwide; they are often called scarabs. Dung beetles are a major insect group (Coleoptera: Scarabaeidae) distributed globally except Antarctica with a high number of diversity comprising approximately 6,200 species and nearly 267 genera (Tarasov and Génier, 2015). These species are coprophagous in nature which live freely in soil and mostly feed on both wet and dry dung materials of herbivorous mammals. The undigested excreta of mammals are utilized as food and nesting material throughout their life cycle, hence, they possess many ecologically beneficial functions. The dung beetles play a vital role in nutrient recycling by decaying organic matter and developing soil aeration (Manning et al., 2016) thereby, reducing the greenhouse gas fluxes (Slade et al., 2016). It also improves plant growth and grain production (Koyama et al., 2003). Scarabeus sacer is considered a species OPEN ACCESS International Journal of Applied Biology Keyword Haemolymph, GS-MS, gram negative bacteria, gram positive bacteria, antimicrobial activity. Article History Received 11 November 2021 Accepted 30 December 2021 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. International Journal of Applied Biology, 5(2), 2021 99 of genus Scarabaeus, occurs in coastal dunes and marshes around the Mediterranean Basin. It can be found across north Africa, Southern Europe and parts of Asia (Afghanistan, Corsica, Cyprus, France, Iran, Israel, Italy, Morocco, Sardinia, Sicily, Sudan and Syria). Scarabaeus sacer is a species of dung beetle belonging to the family Scarabaeidae (Long, 1836) Insects are known for their ability to resist infection. They protect themselves against bacterial infection by secreting a battery of antimicrobial peptides (AMP) into the hemolymph. Hemolymph, also known as the insect blood, is a clear fluid, with or without yellow or greenish pigmentation. It constitutes 16-40% of the body weight of certain insects. The volume and component of hemolymph are vary in different types of insects and their developmental stages. It spends much of its time flowing freely within body cavities where it makes direct contact with all internal tissues and organs. Therefore the circulation would help to transport the AMP to its target site (Kurata, 2006). In insects, AMPs / polypeptides are manufactured mainly in a fat body (similar to mammalian liver) and are released into hemolymph where they play a vital role in innate immune systems and host defense mechanisms, and having a broad spectrum of activity against both gram + ve and gram -ve bacteria and against fungi (Januszanis et al., 2013) However, misuse of antibiotics intake has caused many problems, such as the appearance of antibiotic-resistant bacteria, weakening of disease resistance in livestock, and ecosystem pollution (Looft et al., 2012). Insects exhibit innate immune systems that produce potent AMPs to protect them from pathogen invasion, and these AMPs are viewed as strong natural antibiotic applicants (Kalsy et al., 2020). The insect innate immune system is categorized into cellular and humoral immunity. Cellular immunity involves the phagocytosis of bacteria, fungi, and protozoa, and nodule formation and encapsulation, while humoral immunity involves the secretions of proteins and peptides produced in fat and blood cells to hemolymph in response to infection (Wu et al., 2018). AMPs secreted by the humoral immune response are classified according to their structure and amino acid sequence into cecropins, defensins, proline-rich peptides, glycine- rich peptides, and lysozymes and are found in various insect orders including Coleoptera, Diptera, Hymenoptera, and Lepidoptera (Michael Zasloff, 2002). Melittin is familiar AMP contained in bee venom and its antimicrobial activity was observed greatly in methicillin - resistant Staphylococcus aureus (MRSA) and Gram-positive and Gram-negative bacteria (Pashaei et al., 2019). AMPs are small molecules that vary in size, ranging from 10 to 100 amino acid residues and are produced by all living organisms. The rich diversity of insects makes them rich sources of AMPs. The black soldier fly Hermetia illucens L. (Diptera: Stratiomyidae), particularly, able to live in hostile environments rich in microbial colonies, making it one of the most promising sources of AMPs (Moretta et al., 2020). Nowadays, the using of GC-MS technique is important in analyzing and separation the compounds found in plants extracts (Eva de Rijke et al., 2006)and also the haemolymph of arthropods. This will be a new method for discovering a future drugs to be used in traditional medicine system. In this article, the haemolymph of scarab beetle was analyzed by GC-MS and resulting in many bioactive compounds. Materials and Methods Collection of Insects Scarab beetle was collected from Baltim in the Kafr El Sheikh Governorate, in the north coast of Egypt. https://en.wikipedia.org/wiki/Scarabaeus https://en.wikipedia.org/wiki/Mediterranean_Basin https://en.wikipedia.org/wiki/Italy https://en.wikipedia.org/wiki/Morocco https://en.wikipedia.org/wiki/Sardinia https://en.wikipedia.org/wiki/Sicily https://en.wikipedia.org/wiki/Sudan https://en.wikipedia.org/wiki/Syria https://en.wikipedia.org/wiki/Species https://en.wikipedia.org/wiki/Dung_beetle https://en.wikipedia.org/wiki/Scarabaeidae https://en.wikipedia.org/wiki/Kafr_El_Sheikh_Governorate https://en.wikipedia.org/wiki/North_coast_of_Egypt https://en.wikipedia.org/wiki/Egypt International Journal of Applied Biology, 5(2), 2021 100 Withdrawing of Hemolymph Scarab beetle body surface cleaned with 70% alcohol. Then, in order to collect haemolymph, hind pair legs were cut from coxa, and haemolymph fluid was extracted with a capillary tube placed into micro tubes containing EDTA. Haemolymph was centrifuged at 10000 × g for 10 minutes and the supernatant was collected for the antimicrobial testing and stored in 4°C. Gas Chromatography–Mass Spectrometry (GC-MS) Analysis Haemolymph was collected and 30 milligrams were homogenized in 1ml methanol centrifuged at 4500 rpm for 10 minutes, the supernatant was taken to GC-MS. The chemical composition of samples was performed using Trace GC1310-ISQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG–5MS (30 m x 0.25 mm x 0.25 µm film thickness). The column oven temperature was initially held at 50 C and then increased by 5°C /min to 230°C for 2 min. increased to the final temperature 290°C by 30°C /min and hold for 2 min. The injector and MS transfer line temperatures were kept at 250, 260°C respectively; Helium was used as a carrier gas at a constant flow rate of 1 ml/min. The solvent delay was 3 min and diluted samples of 1 µl were injected automatically using Auto - sampler AS1300 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–1000 in full scan mode. The ion source temperature was set at 200 °C. The components were identified by comparison of their retention times and mass spectra with those of the WILEY 09 and NIST 11 mass spectral databases. Antimicrobial Activity Assay: The antimicrobial activity was investigated in the haemolymph against microorganisms. All microbial strains were provided from the culture collection of the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Cairo, Egypt. The antimicrobial profile was tested against two Gram‐positive bacterial species (Bacillus subtilis, Staphylococcus aureus), two Gram-negative bacterial species (Escherichia coli, Enterobacter cloacae) and two fungi (Aspergillus fumigatus and Candida albicans) using a modified well diffusion method. Briefly, 100 μl of the test bacteria/or fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/ml for bacteria or 105 cells/mL for fungi (Ibrahim et al., 2014). One hundred μl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained and tested for susceptibility by well diffusion method on Mueller‐Hinton and Sabaroud agar (Clinical and Laboratory Standards Institute, 2012.) One hundred µL of each sample (at 10 mg/ml) was added to each well (10 mm diameter holes cut in the agar gel). The plates were incubated for 24-48 h at 37 °C (for bacteria and yeast) and for 48 h at 28 °C (for filamentous fungi). After incubation, the microorganism's growth was observed. The resulting inhibition zone diameters were measured in millimeters and used as a criterion for antimicrobial activity. If an organism is placed on the agar, it will not grow in the area around the well if it is susceptible to the chemical. This area of no growth around the disc is known as a "Zone of inhibition" or "Clear zone". The size of the clear zone is proportional to the inhibitory action of the compound under investigation. Solvent controls (DMSO) were included in every experiment as negative controls. DMSO was used for dissolving the tested compounds and showed no inhibition zones, confirming that it has no influence on growth of the tested International Journal of Applied Biology, 5(2), 2021 101 microorganisms. Gentamycin and ketoconazole (Sigma Aldrich, USA) were used as standard antibacterial and antifungal drugs at 30 and 50ug/ml, respectively. MIC Determination The tested extract was screened in vitro for their antibacterial and antifungal activities at a different concentration to determine the lowest concentration inhibiting the growth of the organism that recorded as the MIC (Ibrahim et al., 2014). Results and Discussion In the present article, the separation of compounds in the haemolymph of scarab beetle by using GC-MS analysis gas separation technique resulting in 43 bioactive compounds as shown in table (1). The identification of the bioactive compounds is based on peak area, retention time, molecular formula and molecular weight. In this study, the antibacterial effectiveness of the haemolymph against gram -V bacteria (Escherichia coli, Enterobacter cloacae, against gram +V bacteria (Staphylococcus aureus, Bacillus subtilis) as well as antifungal activity (Aspergillus fumigatus, Candida albicans) is investigated. Antibacterial activity against Bacillus subtilis was first, followed by Enterobacter cloacae, Escherichia coli and Staphylococcus aureus respectively. No antifungal activity has been investigated yet (Table 2). The minimum inhibitory concentration (MIC) is measured for the tested sample and it was 2500 g/ml against E. coli, 1250 g/ml against Enterobacter cloacae, Bacillus subtilis and 10000 g/ml against Staphylococcus aureus respectively (Table 3). Table 1. Bioactive compounds in haemolymph of scarab beetle separating by GC-MS No. Compound Name Molecular Formula Chemical nature MWT Area % RT Bioactivity 1 Trichloromethane CHCl3 Trihalomethane 118 3.57 4.06 Anti-virus, anti-cancer, anti-mutagenic, anti- allergic and anti-ulcer (Ali et al., 2015) 2 Tert-Hexadecanethiol C16H34S Thio-Alcohol 258 0.11 5.16 Enzyme activators (Rajendran et al., 2017) 3 Ethanol, 2 octadecyloxy- C20H42O2 Alcohol 314 0.11 5.16 Antimicrobial activity (Sudhandra Karthi, 2016) 4 1-Heptatriacotanol C37H76O Alcohol 536 0.48 39.87 Anticancer, antineoplastic and anti-HIV (Kala and Ammani, 2017) 5 1,4-Benzenediol, 2-(1,1- dimethylethyl)-5-(2- propenyl)- C13H18O2 Alcohol 206 2.66 25.14 Anticancer, Antioxidant activity and pesticidies (Swamy et al., 2017) 6 E,E,Z-1,3,12- Nonadecatriene- 5,14-diol C19H34O2 Alcohol 294 0.40 39.20 Antimicrobial activity (Hadi et al., 2016) 7 3,7,11,15- Tetramethyl-2- hexadecen-1-ol C20H40O Terpene alcohol 296 6.43 30.04 Antimicrobial (Ponmathi Sujatha et al., 2017) Precursor of synthetic forms of vitamin E and vitamin K1 (Devakumar et al., 2017) International Journal of Applied Biology, 5(2), 2021 102 8 Dotriacontane C32H66 Saturated hydrocarbons 450 0.11 5.16 Antimicrobial, antifungal, anti-inflammatory, cytotoxic activity (Harris, 1992) 9 Isochiapin B C19H22O6 Terpenoids 346 0.32 10.83 Anti-insect Antitumor agent (Elsharkawy, 2016) 10 14-á-H-Pregna C21H36 Steroids 288 0.32 10.83 defense chemical and Diabetic retinopathy prevention (Durak and Kalender, 2007) 11 Cycloheptasiloxane, tetradecamethyl- C14H42O7Si7 Organo- Silicone compound 518 1.69 15.25 Antimicrobial, Antiseptic, Hair Conditioning Agent, Skin- Conditioning Agent- Emollient and Solvent (Mary and Giri, 2018) 12 4H-1-Benzopyran-4 one, 2-(3,4- dimethoxyphenyl)-3, 5-dihydroxy-7- methoxy- C18H16O7 Ketonic comound 344 0.38 17.48 Antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (Albergoni et al., 1980) 13 Cyclooctasiloxane, hexadecamethyl- C16H48O8Si8 Alkanes 592 2.84 19.68 Antimicrobial (Al Bratty et al., 2020) 14 Cyclododecasiloxane, tetracosamethyl C24H72O12Si12 Alkanes 888 2.84 19.68 Hepatoprotective, antispasmotic, antirheumatic (Al Bratty et al., 2020) 15 9,12- Octadecadienoic acid (Z,Z)-, 2,3- bis [(trimethysilyl)oxy]pr opyl ester C27H54O4Si2 Linoleic acid ester 498 0.74 21.58 Anti-inflamation (Rani and Kapoor, 2019) 16 4H-1-Benzopyran-4- one, 2-(3,4- dimethoxyphenyl)-3, 5-dihydroxy-7- meyhoxy- C18H16O7 Phenolic compound 344 0.28 25.36 Antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (Huang and Irwin, 2006) 17 9,12,15- Octadecatrienoic acid ,2,3- bis[(Trimethylsilyl)ox y ]propyl ester, (Z,Z,Z)- C27H52O4Si2 Alpha linoleic acid 496 0.28 25.36 anti-oxidant, anti-diabetic, and anti-inflammatory (Rajendran et al., 2017) 18 Eicosamethyl,cyclode casiloxane C20H60O10Si10 Organohetero- silane 740 2.59 27.05 Prevent degenerative diseases (Budayatin et al., 2021) 19 Silcone oil N/A polysiloxane compounds 0 2.59 27.05 Toxic for bed- bug(insecticide) (Zha et al., 2018) 20 1H-Purin-6- amine,[(2- flourophenyl)methyl] C12H10FN5 Amino compounds 243 2.59 27.05 Anti-oxidant (Budayatin et al., 2021) 21 Neophytadiene C20H38 Alkenes 278 6.43 30.04 analgesic, antipyretic, anti- inflammatory, International Journal of Applied Biology, 5(2), 2021 103 antimicrobial, and antioxidant compound (Venkata raman et al., 2012) 22 2,6,10-Trimethyl,14- ethylene-14- pentadecene C20H38 Alkenes 278 6.43 30.04 Antiproliferative activity (Devakumar et al., 2017) 23 Phytol Isomer C20H40O Diterpene 296 0.29 30.93 antimicrobial, antioxidant, and anticancer activities (Rani and Kapoor, 2019) 24 7-Methyl-Z- tetradecen-1-ol acetate C17H32O2 Acetate ester 268 0.64 31.36 Anticancer, anti- inflammatory, hepatoprotective (Hameed et al., 2015) 25 2-Dodecen-1-yl(- )succinic anhydride C16H26O3 SuccinicAcid anhydride 266 0.13 33.83 Antineoplastic agents, Antioxidants, Antimicrobial (Jatin and Sonawani, 2016) 26 Hexadecanoic acid, methyl ester C17H34O Palmitic acid ester 270 2.09 34.34 Anti-oxidant, decrease blood cholesterol, anti- inflammatory (Hema et al., 2015) 27 Pentadecanoic acid, 14-methyl-, methyl ester C17H34O Ester 270 2.09 34.34 Antimicrobial, antifungal (Beschi et al., 2021) 28 Dasycarpidan-1- methanol, (Ester) C20H26N2O2 Ester 326 0.74 21.58 Antimicrobial (Rani and Kapoor, 2019) 29 Dibutyl phthalate C16H22O4 Benzoic acid ester 278 0.98 38.03 Use in cosmotics (Mary Ann Liebert, 1985), ectoparasiticide (Prabhu et al., 2018) 30 Phthalic acid, butyl undecyl ester C23H36O4 Ester 376 0.98 38.03 Antimicrobial Antibacterial & Anti-inflammatory (Al-Gara’Awi et al., 2019) 31 Phthalic acid, butyl tetradecyl ester C26H42O4 Ester 418 0.98 38.03 Antimicrobial activity (Bekele et al., 2016) 32 11,14-Eicosadienoic acid, methyl ester C21H38O2 Ester 322 0.40 39.20 Anti-inflammatory, anti- oxidant, anti-arthritic, anti- coronary. (Chinnasamy P.S et al., 2018) 33 1,2- Benzenedicarboxylic acid, butyl decyl ester C22H34O4 Ester 362 5.04 39.35 Antimicrobial activity (Shoge et al., 2016) 34 Diisooctyl phthalate C24H38O4 Phthalic acid derivative (Ester) 390 1.80 50.09 Antimicrobial activity (Ali Shafaghat, 2012) 35 9,12,15- Octadecatrienoic acid, 2,3- dihydroxypropyl ester, (Z,Z,Z)- C21H36O4 Linoleic acid ester 352 0.87 52.61 Analgesic, Antipyretic, Anticonvulsant, Antiseptic (Srivastava et al., 2015) 36 9,12,15- Octadecatrienoic acid, 2-phenyl-1,3- dioxan-5-yl ester C28H40O4 Phenolic ester 440 0.46 58.55 Antimicrobial & anti- inflammatory (Kadhim et al., 2017) 37 Cholest-5-en-3-ol, 24- propylidene-, (3á)- C30H50O Fatty acid 426 6.09 63.51 Antibacterial activity (Hussein et al., 2017) 38 Cis-13-Eicosenoic acid C20H38O2 Fatty acid 310 0.08 33.78 Anti-inflammatory activity (Sosa et al., 2016) International Journal of Applied Biology, 5(2), 2021 104 39 Ppropiolic acid, 3-(1- hydroxy-2-isopropyl- 5 methylcyclohexyl)- C13H20O3 Fatty acid 224 1.72 37.54 Anti-angiogenic activity against solid tumor growth (Hussein et al., 2016) 40 2-Nonadecanone 2,4- dinitrophenylhydrazi ne C25H42N4O4 Nitrogen compound 462 0.36 50.66 Antimicrobial activity (Muthulakshmi et al., 2012) 41 Ethyl iso-allocholate C26H44O5 Steroid derivative 436 0.48 39.87 Antimicrobial,Antioxidant, anti-inflammatory & anti- arthritic antiasthmatic (Sheela and Uthayakumari, 2013) 42 1,2-15,16- Diepoxyhexadecane C16H30O2 Epoxide 254 0.40 39.20 Antitumor, anti- inflammatory (Hameed et al., 2016) 43 Milbemycin B, 6,28- anhydro-15-chloro- 25-isopropy l-13- dehydro-5-O- demethyl-4-methyl C33H47ClO7 ------ 590 0.33 58.77 Anti MRSA (Vilas and Amit, 2015) The GC-Ms analysis on the haemolymph of Scarabeus sacer revealed presence of some bioactive compounds such as alcohols, terpenoids, ketones, phenolic compounds, alkanes, alkenes, amino-compounds, Fatty acids and steroids. Alcohols were discovered to have antimicrobial activity (Gołêbiowski et al., 2012). Isochiapin B, 3, 7, 11, 15-Tetramethyl- 2-hexadecen-1-ol and Phytol Isomer are terpenoids present in haemolymph of S. sacer. Terpenoid compounds (Phorbol, Isochiapin B, stigmasterol acetate, and b-sitosterol) were detected in essential oil of Achillea fragmmentissma that well known for their biological activities as anti-insect and anti-tumor agents (Elsharkawy, 2016). Terpenes are bioactive compounds detected in Ulva fasciata, U. lactuca and Corallina mediterranea seaweeds extract and steroids were detected in the extracts of U. fasciata, and Amphiroa anceps seaweeds (Mofeed et al., 2021). Most of these compounds exhibit biological activities such as anticancer, antiviral, antioxidant, and anti-inflammatories (Jiang et al., 2017). Phytol isomer is a diterpenes identified in the haemolymph of adult S. sacer. phytol is a bioactive compound that has a potent anticancer activity (Sheeja et al., 2016). It also serve as a chemical attractant for parasitoids, according to research on these species: Lucilia sericata (Gobiowski et al., 2012c), Leptinotarsa decemlineata (Nelson et al., 2003). Alkanes can help distinguish organisms by acting as a chemical signal (Lockey 1988). Alkanes were also marked in the surface lipids of Liposcelis bostrychophila, Cryptolestes ferrugineus (Howard and Lord 2003) and Laelius utilis (Howard, 1992). Dotriacontane is saturated hydrocarbons present in haemolymph of scarab beetle and reported to has antimicrobial, antifungal, anti - inflamatory and cytotoxic activity (Harris, 1992). Also the hydrocarbons used to distinguish between the male and female of Sarcophaga species (Moore et al., 2021). Alkanes were also marked in the surface lipids of Liposcelis bostrychophila, Cryptolestes ferrugineus (Howard and Lord, 2003) and Laelius utilis (Howard R. w, 1992). Larvae of potato beetle contain hydrocarbons of high molecular weight, particularly tetrapentacontane (C54H 110), pentapentacontane (C55H 112) and heptapentacontane (C57H 116) (Ardenne et al., 1965). Pentadecanoic acid, 9,12,15-octadecatrienoic acid, hexadecanoic acid methyl ester were detected in the haemolymph of adult S. sacer. Pentadecanoic acid and 9,12,15- octadecatrienoic acid reported to have anti-inflammatory, antimicrobial, antioxidants, and antiproliferative activity (Rani and Kapoor, 2019). Hexadecanoic acid methyl ester is also known as palmitic acid ester and efficiently used as an antioxidant, pesticide, anti - International Journal of Applied Biology, 5(2), 2021 105 androgenic, nematicide, flavoring agent, hypocholesterolemic, and lubricant (Karthikeyan and Sudan, 2017). Also hexadeconic acid was the major fatty acid in Sargassum granuliferum seaweed which prevents the biofilm forming bacteria (Bakar et al., 2017). Esters with even longer C22, C24, C42 and C46 carbon chains were determined in Aleurotithius timberlakei (Nelson et al., 1997). In the haemolymph of scarab beetle, fatty acids such as – Cis-13-eicosenoic acid, Cholest-5-en-3-ol, 24-propylidene- (3á), and Ppropiolic acid, 3-(1-hydroxy-2-isopropyl-5 methylcyclohexyl) were detected. In case of Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), the presence of C6, C9, C10, C12, C14, C16 and C18 fatty acids were found. Fatty acids C18 and C20 are found also in Bombyx mori and Blatella germanica (Barlow J. S., 1964). Fatty acids C18:1, C18:2 and C18:3 were estimated in the larvae of Drosophila melanogaster, Musca domestica and Galleria mellonella (Barlow J. S., 1964). Fatty acids C16 – C18 have also been determined in the surface lipids of Cryptolestes ferrugineus and Liposcelis bostrychophila insects (Howard and Lord, 2003). Cholest-5-en-3-ol, 24- propylidene have been detected in the methyl extract of Sargassum crassifolium (Albratty et al., 2021) and (Erwan Plouguerné et al., 2006) seperated Cholest-5-en-3-ol, 24- propylidene from the red alga Grateloupia turuturu. In the adult scarab beetle haemolymph, there is a ketonic compounds such as 4H-1- Benzopyran-4 one, 2-(3, 4-dimethoxyphenyl)-3,5-dihydroxy-7-methoxy that act as Antioxidant, antimicrobial, cancer enzyme inhibitors in pharmaceutical, cosmetics, and food industries (Albergoni et al., 1980). Also ketones are separated from Tessaratoma papillosa (Zhang et al., 2009). The relationships of these analyzed compounds in insects play vital roles as they can be transmitters of information and signals (Taylor et al., 2012)and also serve as pheromones (Noguez et al., 2013). Ethyl iso-allocholate is a steroid derivative compounds detected in the haemolymph of adult scarab beetle and in the black fruit of Pistacia lentiscus, this steroid compound has antimicrobial, anti-inflammatory, anticancer, antiasthma and diuretic activities (Daffodil D Almeida et al., 2012) Silcone oil, milbemycin B, 6, 28-anhydro-15-chloro-25-isopropyl-13-dehydro-5-O- demethyl-4-met-, and Dibutyl phthalate were detected in the haemolymph of adult scarab beetle. Zha et al., 2018, reported that silicone oil in the bed bug is cytotoxic and has an insecticidal activity that can kill insects by physical mean that affecting on tracheal system causing asphyxiation of insects. Anti MRSA activity was reported for milbemycin B, 6, 28- anhydro-15-chloro-25-isopropyl-13-dehydro-5-O-demethyl-4-met- (Vilas and Amit , 2015) and Dibutyl phthalate act as ectoparasiticide (Prabhu et al., 2018). Table 2. Antimicrobial activity (as a mean zone of inhibition) of the haemolymph of adult scarab beetle Sample Tested microorganisms Heamolyph Control FUNGI Ketoconazole Aspergillus fumigatus NA 17 Candida albicans NA 20 Gram Positive Bacteria: Gentamycin Staphylococcus aureus 8 24 http://en.wikipedia.org/wiki/Leaf_beetle International Journal of Applied Biology, 5(2), 2021 106 Bacillus subtilis RCMB 015 (1) 15 26 Gram Negatvie Bacteria: Gentamycin Escherichia coli 10 30 Enterobacter cloacae 14 27 *NA: No activity. Table 3. The antimicrobial activity as Minimum Inhibitory concentration (MIC) in g/ml of the tested microorganisms. The test was done using the diffusion agar technique Sample Tested microorganisms Heamolyph Control Gram Positive Bacteria: Gentamycin Staphylococcus aureus 10000 3.9 Bacillus subtilis 1250 1.95 Gram Negatvie Bacteria: Gentamycin Escherichia coli 2500 1.95 Enterobacter cloacae 1250 3.9 *NA: No activity. The present works approved that the haemolymph of S. sacer possesses antibacterial activity against gram –negative bacteria (Escherichia coli, Enterobacter cloacae) and against gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis). No antifungal activity had been investigated against Aspergillus fumigatus and Candida albicans. There are many works hassling to our work; the methanol extract of oriental hornet Vespa orientalis and Zophobas mori (Coleoptera:Tenebrionidae) larva show antibacterial activity against E. coli and no antifungal activity (HASSAN et al., 2015). Contrary to our results, the whole body extract of housefly maggots show no activity against E. coli and exhibit antifungal activity (Meylaers et al., 2004). While (Hou et al., 2007) documented that the extract of the housefly maggots have higher activity against Gram- positive bacteria than Gram negative bacteria and had not antifungal activity yet. Most of insect extracts show antibacterial activity against Gram-positive and Gram- negative bacteria, the silk worm Bombyx mori (Seiichi Hara and Yamakawa, 1995), the European bumble bee, Bombus pascuorum (Rees JA et al., 1997) and Tenebrio molitor larvae (Lee et al., 1998). On the other hand, some other insects revealed activity only against Gram-positive bacteria as Aedes aegypti (Lowenberger et al., 1995), Chironomus plumosus (Lauth et al., 1998) and Anopheles gambiae (Vizioli et al., 2001). Synthetic antibiotics and antimicrobials have contributed to public health and stimulated the growth of livestock. Conversely, overuse and abuse of antibiotics and antimicrobial drugs may causes drug-resistant bacteria, which threaten public and livestock health. Several studies reported that insects manufacture antimicrobial peptides (AMPs) International Journal of Applied Biology, 5(2), 2021 107 which act as a natural antibiotic (Vetterli et al., 2018). Insects not only perform different roles in the environment, but also host a variety of community of microorganisms. The complicated cellular and humoral mechanisms include the innate immune system of an insect (Kanost et al., 2004). The cellular mechanism is rely on phagocytosis process which is activated by enzymes and invading microorganisms then encapsulated by the hemolymph. Moreover, The humoral response is represented in the production of broad-spectrum antimicrobial peptides (AMPs), reactive oxygen or nitrogen intermediates, and complex enzymatic cascades that help to regulate hemolymph coagulation or melanization (Ahmed MH Ali et al., 2020). The presence of microorganisms invading insects causes the fat body to rapidly synthesize AMPs, which are then released into the hemolymph (Hoffmann and Reichhart, 2002). Previous research shows that each insect species produces a distinct antimicrobial peptide that acts against specific microorganisms (Yi et al., 2014). On the other hand, in order to enhance the insect’s defense system against other pathogens, some of the peptides are expressed simultaneously, encouraging synergism (Rahnamaeian et al., 2015). As such, AMPs have a specific modes of action, such as altering the electrochemical gradient at the membrane, producing reactive oxygen/nitrogen species (ROS/RNS) that cause cell death, inhibiting protein synthesis, and permeabilizing the cell membrane (Thevissen et al., 2004). AMPs have pharmacological properties such as low molecular weight, high water solubility, broad-spectrum antimicrobial activity, and low levels of cytotoxicity (Lei et al., 2019). (Turillazzi et al., 2004) reported that the antibacterial activity in salivary secretions of Polistes dominulus larvae inhibits growth of Gram positive Bacillus subtilis and Gram negative E. coli. There are number of studies that have tested ability of the insect extracts against pathogenic bacteria, especially antimicrobial peptides extracted from various insects maggots (Guo et al., 2007), dung beetles (Mohtar et al., 2014), Red Palm Weevil (Chernysh et al., 2015), pupae of the giant silk moths (Sewify et al., 2017). Contrary to our study on the haemolymph of adult S. sacer, the non-induced hemolymph of dung beetle, Onthophagus taurus did not show inhibitory activity against any of the bacterial strains and fungus. It does not mean that peptides are absent but it may be present in smaller quantity so that no visible action in in-vitro studies is detected (Patil and Kumar, 2013). But the immune induced hemolymph exhibits activity against all tested bacteria and no activity against fungus. Therefore, the peptide is active against prokaryotes and doesn’t affect the fungus which is a eukaryote. Many studies on insect species assert that bacteria injected into the haemocoel stimulate the synthesis of number of peptides and proteins which are active singly or in concert against the invaders and are secreted into the hemolymph (Gillespie JP et al., 1997). Conclusions On conclusion, antimicrobial activity of haemolymph of adult S. sacer may be due to presence of the previous bioactive compounds which separated by the GC-MS technique. Future studies are necessary to purify the compounds with antimicrobial activity and investigate their antitumor effect against different cell lines. Acknowledgment Great thanks and appreciation for the Mycology Center, Al-Azhar University for their cooperation. International Journal of Applied Biology, 5(2), 2021 108 References Ahmed MH Ali, Abdur Rauf, Emad M Abdallah, 2020. Insects as producers of antimicrobial polypeptides: A short review. GSC Biol. Pharm. Sci. 12, 102–107. https://doi.org/10.30574/gscbps.2020.12.3.0281. Al-Gara’Awi, N.I., Abu-Serag, N.A., Alee Shaheed, K.A., Bahadly, Z.K. Al, 2019. Analysis of bioactive phytochemical compound of ( Cyperus alternifolius L.) by using gas chromatography-mass spectrometry. IOP Conf. Ser. Mater. Sci. Eng. 571. https://doi.org/10.1088/1757-899X/571/1/012047. Al Bratty, M., Makeen, H.A., Alhazmi, H.A., Alhazmi, H.A., Syame, S.M., Syame, S.M., Abdalla, A.N., Abdalla, A.N., Homeida, H.E., Sultana, S., Ahsan, W., Khalid, A., Khalid, A., 2020. Phytochemical, Cytotoxic, and Antimicrobial Evaluation of the Fruits of Miswak Plant, Salvadora persica L. J. Chem. 2020. https://doi.org/10.1155/2020/4521951. Albergoni, V., E, P., O, C., 1980. esponse to heavy metals in organisms-I. Excretion and accumulation of physiological and non physiological metals in Euglena gracilis. Comp Biochem Physiol C Comp Pharmacol 67, 121–127. Albratty, M., Bajawi, A.A.M., Marei, T.M.H., Shamsher Alam, M., Alhazmi, H.A., Najmi, A., ur Rehman, Z., Sivagurunathan Moni, S., 2021. Spectral analysis and antibacterial potential of bioactive principles of Sargassum crassifolium J. Agardh from Red sea of Jazan origin. Saudi J. Biol. Sci. 28, 5745–5753. https://doi.org/10.1016/j.sjbs.2021.06.017. Ali, H.A.M., Imad, H.H., Salah, A.I., 2015. Analysis of bioactive chemical components of two medicinal plants (Coriandrum sativum and Melia azedarach) leaves using gas chromatography-mass spectrometry (GC-MS). African J. Biotechnol. 14, 2812–2830. https://doi.org/10.5897/ajb2015.14956. Ali Shafaghat, 2012. Phytochemical and antimicrobial activities of Lavandula officinalis leaves and stems against some pathogenic microorganisms. J. Med. Plants Res. 6, 455–460. https://doi.org/10.5897/jmpr11.1166. Ardenne, M. von, Osske, G., Schreiber, K., Steinfelder, K., R Tümmler, 1965. No Title. J. Insect Physiol. 11, 1365. Bakar, K., Mohamad, H., Latip, J., Tan, H.S., Herng, G.M., 2017. Fatty acids compositions of Sargassum granuliferum and Dictyota dichotoma and their anti-fouling activities. J. Sustain. Sci. Manag. 12, 8–16. Barlow J. S., 1964. Fatty acids in some insect and spider fats. Can. J. Biochem. 42, 1365– 1374. Bekele, D., Tekie, H., Asfaw, Z., Petros, B., 2016. Bioactive Chemical Constituents from the Leaf of Oreosyce africana Hook.f (Cucurbitaceae) with Mosquitocidal Activities against Adult Anopheles arabiensis, the Principal Malaria Vector in Ethiopia. J. Fertil. Pestic. 7, 1–8. https://doi.org/10.4172/2471-2728.1000159. International Journal of Applied Biology, 5(2), 2021 109 Beschi, D.A., Appavoo, M.R., Wilsy, J.I., 2021. GC-MS analysis, collected from Kavalkinaru area, Tirunelveli District, Tamil Nadu, India. Eur. J. Mol. & Clin. Med. 7, 4287– 4292. Budayatin, Waluyo, J., Wahyuni, D., Dafik, 2021. Antibacterial effects of Pheretima javanica extract and bioactive chemical analysis using Gas Chromatography Mass Spectrum. J. Phys. Conf. Ser. 1751. https://doi.org/10.1088/1742-6596/1751/1/012055. Chernysh, S., Gordya, N., Suborova, T., 2015. Insect antimicrobial peptide complexes prevent resistance development in bacteria. PLoS One 10. https://doi.org/10.1371/journal.pone.0130788. Chinnasamy P.S, Parimala, S., Kandhasamy, M., 2018. Phytochemical Evaluation of Seed and Fruit Pulp EXTRACTS OF PASSIFLORA FOETIDA L. P. 7, 1924–1932. https://doi.org/10.20959/wjpr20187-11770. Daffodil D Almeida, Mohan, V.R., Uthayakumari, F.K., 2012. GC-MS determination of bioactive compounds of Curculigo orchioides gaertn. Sci. Res. Report. 2, 198–201. Devakumar, J., Keerthana, V., Sudha, S.S., 2017. Identification of bioactive compounds by gas chromatography-mass spectrometry analysis of Syzygium jambos (L.) collected from Western Ghats region Coimbatore, Tamil Nadu. Asian J. Pharm. Clin. Res. 10, 364–369. https://doi.org/10.22159/ajpcr.2017.v10i1.15508. Durak, D., Kalender, Y., 2007. Fine structure and chemical analysis of the metathoracic scent gland of Eurygaster maura (Linnaeus, 1758) (Heteroptera: Scutelleridae). Folia Biol. (Praha). 55, 133–141. https://doi.org/10.3409/173491607781492551. Elsharkawy, E., 2016. Anti-inflammatory activity and chemical compositions of essential oil of Achillea fragmmentissma. Natl. J. Physiol. Pharm. Pharmacol. 6, 258–262. https://doi.org/10.5455/njppp.2016.6.23022016130. Erwan Plouguerné, Kikuchi, H., Oshima, Y., Deslandes, E., Stiger-Pouvreau, V., 2006. Isolation of Cholest-5-en-3-ol formate from the red alga Grateloupia turuturu Yamada and its chemotaxonomic significance. Biochem. Syst. Ecol. 34, 714–717. https://doi.org/10.1016/j.bse.2006.04.003. Eva de Rijke, Out, P., Niessen, W.M.A., Ariese, F., Gooijer, C., Brinkman, U.A.T., 2006. Analytical separation and detection methods for flavonoids. J. Chromatogr. 11, 31– 63. Gillespie JP, MR, K., T, T., 1997. Biological mediators of insect immunity. Annu Rev Entomol. 42, 611–643. https://doi.org/10.1146/annurev.ento.42.1.611. Gołêbiowski, M., Dawgul, M., Kamysz, W., Boguœ, M.I., Wieloch, W., Włóka, E., Paszkiewicz, M., Przybysz, E., Stepnowski, P., 2012. Antimicrobial activity of alcohols from Musca domestica. J. Exp. Biol. 215, 3419–3428. https://doi.org/10.1242/jeb.073155. Guo, G., Wu, J., Fu, P., Qin, R., Zhang, Y., Song, Z., 2007. Isolation and purification of International Journal of Applied Biology, 5(2), 2021 110 antibacterial peptides from the larvae secretion of housefly and the characteristics. Chinese J. Parasitol. Parasit. Dis. 25, 87–92. Hadi, M.Y., Mohammed, G.J., Hameed, I.H., 2016. Analysis of bioactive chemical compounds of Nigella sativa using gas chromatography-mass spectrometry. J. Pharmacogn. Phyther. 8, 8–24. https://doi.org/10.5897/JPP2015.0364. Hameed, I.H., Altameme, H.J., Idan, S.A., 2016. Artemisia annua: Biochemical products analysis of methanolic aerial parts extract and anti-microbial capacity. Res. J. Pharm. Biol. Chem. Sci. 7, 1843–1868. Hameed, I.H., Hussein, H.J., Kareem, M.A., Hamad, N.S., 2015. Identification of five newly described bioactive chemical compounds in Methanolic extract of Mentha viridis by using gas chromatography – mass spectrometry (GC-MS). J. Pharmacogn. Phyther. 7, 107–125. https://doi.org/10.5897/JPP2015.0349. Harris, E.D., 1992. Symposium : Regulation of Antioxidant Enzymes Copper as a Cofactor and Regulator of Copper , Zinc Superoxide Dismutase12 636–640. https://doi.org/10.1093/jn/122.suppl. HASSAN, M.I., MOHAMED, A.F., AMER, M.S., KOTB M. HAMMAD, MAHBOUB, M.T., 2015. Antimicrobial Activity of Three Insect Species, Crude Extracts Againts Certain Microbial Agents. Al-Azhar Bull. Sci. 26, 19–24. https://doi.org/10.21608/absb.2015.23779. Hema, R., Kumaravel, S., AlagusundaramAgardh, K., 2015. GC - MS determination of bioactive components of Gracilaria dura. J. Am. Sci. 5, 100–105. Hoffmann, J.A., Reichhart, J.M., 2002. Drosophila innate immunity: An evolutionary perspective. Nat. Immunol. 3, 121–126. https://doi.org/10.1038/ni0202-121. Hou, L., Le, Y.S., Zhai, P., Guowei Le, 2007. Antibacterial activity and in vitro antitumor activity of the extract of the larvae of the housefly, Musca domestica. J. Ethnopharmacol. 111, 227–231. Howard R. w, 1992. Comparative analysis of cuticular hydrocarbons from the ectoparasitoids Cephalonomia waterstoni and Laelius utilis (Hymenoptera: Bethylidae) and their respective hosts, Cryptolestes ferrugineus (Coleoptera: Cucujidae) and Trogoderma variabile (Coleoptera: Ann. Entomol. Soc. Am. 85, 317– 325. Howard, R.W., Lord, J.C., 2003. Cuticular lipids of the booklouse, Liposcelis bostrychophila: Hydrocarbons, aldehydes, fatty acids, and fatty acid amides. J. Chem. Ecol. 29, 615– 627. https://doi.org/10.1023/A:1022806922246. Huang, D., Irwin, G., 2006. Computational intelligence: international conference on intelligent computing. Springer-Verlag 16–19. Hussein, H.J., Hameed, I.H., Hadi, M.Y., 2017. Using gas chromatography-mass spectrometry International Journal of Applied Biology, 5(2), 2021 111 (GC-MS) technique for analysis of bioactive compounds of methanolic leaves extract of Lepidium sativum. Res. J. Pharm. Technol. 10, 3981–3989. https://doi.org/10.5958/0974-360X.2017.00723.5. Hussein, H.M., Hameed, I.H., Ibraheem, O.A., 2016. Antimicrobial activity and spectral chemical analysis of methanolic leaves extract of adiantum capillus-veneris using GC-MS and FT-IR spectroscopy. Int. J. Pharmacogn. Phytochem. Res. 8, 369–385. Ibrahim, H., Eldehna, W., Abdel-Aziz, H., Elaasser, M., Abdel-Aziz, M., 2014. improvement of antibacterial activity of some sulfa drugs through linkage to certain phthalazin- 1(2H)-one scaffolds. Eur. J. Med. Chem. 85, 480. Januszanis, B., Stączek, S., Zdybicka -Barabas, A., Bądziul, D., Jakubowicz-Gil, J., Langner, E., Rzeski, W., Cytryńska, M., 2013. The effect of Galleria mellonella polypeptides on human brain glioblastoma multiforme cell line – a preliminary study / Badania wstępne wpływu polipeptydów hemolimfy Galleria mellonella na komórki ludzkiego glejaka wielopostaciowego. Ann. UMCS, Biol. 67. https://doi.org/10.2478/v10067- 012-0020-1. Jatin, R.R., Sonawani, P., 2016. Determination of Bioactive Components of Cynodon dactylon by GC-MS Analysis & i t ’ s In Vitro Antimicrobial Activity © Sakun Publishing House ( SPH ): IJPLS. Int. J. Pharm. LIFE Sci. (Int. 7, 4880–4885. Jiang, L., Wang, W., He, Q., Wu, Y., Lu, Z., Sun, J., Liu, Z., Shao, Y., Wang, A., 2017. Oleic acid induces apoptosis and autophagy in the treatment of Tongue Squamous cell carcinomas. Sci. Rep. 7, 1–11. https://doi.org/10.1038/s41598-017-11842-5. Kadhim, M.J., Al-Rubaye, A.F., Hameed, I.H., 2017. Determination of Bioactive Compounds of Methanolic Extract of Vitis vinifera Using GC-MS. Int. J. Toxicol. Pharmacol. Res. 9, 113–126. https://doi.org/10.25258/ijtpr.v9i02.9047. Kala, S.C., Ammani, K., 2017. GC–MS analysis of biologically active compounds in Canthium parviflorum Lam. leaf and callus extracts. Ijcrgg 10, 1039–1058. Kalsy, M., Tonk, M., Hardt, M., Dobrindt, U., Zdybicka-Barabas, A., Cytrynska, M., Vilcinskas, A., Mukherjee, K., 2020. The insect antimicrobial peptide cecropin A disrupts uropathogenic Escherichia coli biofilms. npj Biofilms Microbiomes 6. https://doi.org/10.1038/s41522-020-0116-3. Kanost, M.R., Jiang, H., Yu, X.Q., 2004. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol. Rev. 198, 97–105. https://doi.org/10.1111/j.0105- 2896.2004.0121.x. Karthikeyan, A.V.P., Sudan, I., 2017. Gc-Ms Profile of in Vivo and in Vitro Shoots of Cleome Gynandra L. Int. J. Pharm. Pharm. Sci. 9, 21. https://doi.org/10.22159/ijpps.2017v9i11.17351. Koyama, M., Iwata, R., Yamane, A., Katase, T., Ueda, S., 2003. Nutrient intake in the third instar larvae of Anomala cuprea and Protaetia orientalis submarmorea (Coleoptera: International Journal of Applied Biology, 5(2), 2021 112 Scarabaeidae) from a mixture of cow dung and wood chips: Results from stable isotope analyses of nitrogen and carbon. Appl. Entomol. Zool. 38, 305–311. https://doi.org/10.1303/aez.2003.305. Kurata, S., 2006. Intra- and extracellular recognition of pathogens and activation of innate immunity. Yakugaku Zasshi 126, 1213–1218. https://doi.org/10.1248/yakushi.126.1213. Lauth, X., Nesin, A., Briand, J., Roussee, J., C. Hetru, 1998. solation, characterization and chemical synthesis of a new insect defensin from Chironomus plumosus (Diptera). insect Biochem Mol Biol. 28, 1059–1066. https://doi.org/10.1016/s0965- 1748(98)00101-5. Lee, K.H., Hong, S.Y., Oh, J.E., Kwon, M., Yoon, J.H., Lee, J., Lee, B.L., 1998. of Tenebrio molitor 105, 99–105. Lei, J., Sun, L.C., Huang, S., Zhu, C., Li, P., He, J., Mackey, V., Coy, D.H., He, Q.Y., 2019. The antimicrobial peptides and their potential clinical applications. Am. J. Transl. Res. 11, 3919–3931. Long, G., 1836. No Title The British Museum: Egyptian Antiquities, Volume 2 Library of entertaining knowledge The British Museum: Egyptian Antiquities, George Long. Charles Knight, 1836, new york. Looft, T., Johnson, T.A., Allen, H.K., Bayles, D.O., Alt, D.P., Stedtfeld, R.D., Sul, W.J., Stedtfeld, T.M., Chai, B., Cole, J.R., Hashsham, S.A., Tiedje, J.M., Stanton, T.B., 2012. In-feed antibiotic effects on the swine intestinal microbiome. Proc. Natl. Acad. Sci. U. S. A. 109, 1691–1696. https://doi.org/10.1073/pnas.1120238109. Lowenberger, C., Bulet, P., Charlet M, Hetru, C., Hodgeman B, Christensen BM, JA, H., 1995. Insect immunity: isolation of three novel inducible antibacterial defensins from the vector mosquito, Aedes aegypti. Insect Biochem Mol Biol. 25, 867–873. https://doi.org/10.1016/0965-1748(95)00043-u. Manning, P., Slade, E.M., Beynon, S.A., Lewis, O.T., 2016. Functionally rich dung beetle assemblages are required to provide multiple ecosystem services. Agric. Ecosyst. Environ. 218, 87–94. https://doi.org/10.1016/j.agee.2015.11.007. Mary, A.P.F., Giri, S., 2018. World Journal of Pharmaceutical Research GC-MS ANALYSIS OF BIOACTIVE COMPOUNDS OF 7, 1045–1056. https://doi.org/10.20959/wjpr20181- 10540. Mary Ann Liebert, 1985. Final Report on the Safety Assessment of Dibutyl Phthalate, Dimethyl Phthalate, and Diethyl Phthalate. IOURNAL Am. Coll. Toxicol. 4, 267–303. Meylaers, K., Clynen, E., Daloze, D., Deloof, A., Schoofs, L., 2004. dentification of 1- lysophosphatidylethanolamine (C(16:1)) as an antimicrobial compound in the housefly, Musca domestica. Insect Biochem. Mol. Biol. 34, 43–49. https://doi.org/10.1016/j.ibmb.2003.09.001. International Journal of Applied Biology, 5(2), 2021 113 Michael Zasloff, 2002. Antimicrobial peptides of multicellularorganisms. Nature 415, 389– 395. Mofeed, J., Deyab, M., Sabry, A.E.-N., Ward, F., 2021. In Vitro Anticancer Activity of Five Marine Seaweeds Extract From Egypt Against Human Breast and Colon Cancer Cell Lines. Res. Sq. 1, 1–15. Mohtar, J.A., Yusof, F., Ali, N.M.H., 2014. Screening of novel acidified solvents for maximal antimicrobial peptide extraction from Zophobas morio fabricius. Adv. Environ. Biol. 8, 803–809. Moore, H.E., Hall, M.J.R., Drijfhout, F.P., Cody, R.B., Whitmore, D., 2021. Cuticular hydrocarbons for identifying Sarcophagidae (Diptera). Sci. Rep. 11, 1–11. https://doi.org/10.1038/s41598-021-87221-y. Moretta, A., Salvia, R., Scieuzo, C., Di Somma, A., Vogel, H., Pucci, P., Sgambato, A., Wolff, M., Falabella, P., 2020. A bioinformatic study of antimicrobial peptides identified in the Black Soldier Fly (BSF) Hermetia illucens (Diptera: Stratiomyidae). Sci. Rep. 10, 1–15. https://doi.org/10.1038/s41598-020-74017-9. Muthulakshmi, A., Jothibai Margret, R., Mohan, V.R., 2012. GC-MS analysis of bioactive components of Feronia elephantum Correa (Rutaceae). J. Appl. Pharm. Sci. 2, 69– 74. Nelson, D.R., Walker, G.P., Buukner, J.S., Fatland, C.L., 1997. Composition of the wax particles and surface wax of adult whiteflies: Aleuroplatus coronata, Aleurothrixus floccosus, Aleurotithius timberlakei, Dialeurodes citiri, Dialeurodes citrifolii, and Parabemisia myricae. Comp. Biochem. Physiol 117B, 241–251. Noguez, J.H., Conner, E.S., Zhou, Y., Ciche, T.A., Ragains, J.R., Butcher, R.A., 2013. NIH Public Access 7, 961–966. https://doi.org/10.1021/cb300056q.A. Pashaei, F., Bevalian, P., Akbari, R., Pooshang Bagheri, K., 2019. Single dose eradication of extensively drug resistant Acinetobacter spp. In a mouse model of burn infection by melittin antimicrobial peptide. Microb. Pathog. 127, 60–69. https://doi.org/10.1016/j.micpath.2018.11.055. Patil, H.. V., Kumar, B.. S., 2013. Isolation and partial purification of antimicrobial peptides/ proteins from dung beetle, Onthophagus Taurus immune hemolymph. J. Microbiol. Biotechnol. Food Sci. 2, 2414–2418. Ponmathi Sujatha, A., Michael Evanjaline, R., Muthukumarasamy, S., Mohan, V., 2017. Determination of bioactive components of barleria courtallica nees (Acanthaceae) by gas chromatography–mass spectrometry analysis. Asian J. Pharm. Clin. Res. https://doi.org/10.22159/ajpcr.2017.v10i6.18035. Prabhu, V., Devi, K.V., Priya, M.K., 2018. Gc-Ms Analysis of Bioactive Compounds Present in the Petroleum Ether , Chloroform and Methanol Extract of Ixora Coccinea ’ S Flower and in-Vitro Cytotoxic Activity of. Int. J. Res. Anal. Rev. 5, 801–807. International Journal of Applied Biology, 5(2), 2021 114 Rahnamaeian, M., Cytryńska, M., Zdybicka-Barabas, A., Dobslaff, K., Wiesner, J., Twyman, R.M., Zuchner, T., Sadd, B.M., Regoes, R.R., Schmid-Hempel, P., Vilcinskas, A., 2015. Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria. Proc. R. Soc. B Biol. Sci. 282. https://doi.org/10.1098/rspb.2015.0293. Rajendran, P., dasan, R.B., esh, K.S., mar, K., 2017. GC-MS Analysis of Phyto-Components in Raw and Treated Sugarcane Juice. Int. J. Curr. Microbiol. Appl. Sci. 6, 51–61. https://doi.org/10.20546/ijcmas.2017.607.007. RANI, J., KAPOOR, M., 2019. Gas Chromatography-Mass Spectrometric Analysis and Identification of Bioactive Constituents of Catharanthus Roseus and Its Antioxidant Activity. Asian J. Pharm. Clin. Res. 12, 461–465. https://doi.org/10.22159/ajpcr.2019.v12i3.30865. Rees JA, M, M., P., B., 1997. Novel antibacterial peptides isolated from a European bumblebee, Bombus pascuorum (Hymenoptera, Apoidea). Insect Biochem Mol Biol 27, 413–422. https://doi.org/10.1016/s0965-1748(97)00013-1. Seiichi Hara, Yamakawa, M., 1995. A novel antibacterial peptide family isolated from the silkworm, Bombyx mori. Biochem. J. 310, 651–656. Sewify, G.H., Hamada, H.M., Alhadrami, H.A., 2017. In Vitro Evaluation of Antimicrobial Activity of Alimentary Canal Extracts from the Red Palm Weevil, Rhynchophorus ferrugineus Olivier Larvae. Biomed Res. Int. 2017. https://doi.org/10.1155/2017/8564601. Sheeja, L., Lakshmi, D., Bharadwaj, S., Sajidha Parveen, K., 2016. Anticancer activity of phytol purified from Gracilaria edulis against human breast cancer cell line (MCF-7). Int J Curr Sci 19, 36–46. Sheela, D., Uthayakumari, F., 2013. GC-MS ANALYSIS OF BIOACTIVE CONSTITUENTS FROM COASTAL SAND DUNE TAXON – SESUVIUM PORTULACASTRUM (L.). Biosci. Discov. 4, 47–53. Shoge, M., Garba, S., Labaran, S., 2016. Antimicrobial Activities of 1,2-benzenedicarboxylic acid , butyldecyl ester isolated from the seeds and pods of Acacia nilotica Linn. Basic Res. J. Microbiol. 3, 08–11. Slade, E.M., Riutta, T., Roslin, T., Tuomisto, H.L., 2016. The role of dung beetles in reducing greenhouse gas emissions from cattle farming. Sci. Rep. 6. https://doi.org/10.1038/srep18140. Sosa, A.A., Bagi, S.H., Hameed, I.H., 2016. Analysis of bioactive chemical compounds of Euphorbia lathyrus using gas chromatography-mass spectrometry and fourier- transform infrared spectroscopy. J. Pharmacogn. Phyther. 8, 109–126. https://doi.org/10.5897/JPP2015.0371. Srivastava, R., Mukerjee, A., Verma, A., 2015. GC-MS analysis of phytocomponents in, PET International Journal of Applied Biology, 5(2), 2021 115 ether fraction of wrightia tinctoria seed. Pharmacogn. J. 7, 249–253. https://doi.org/10.5530/pj.2015.4.7. Sudhandra Karthi1, B.S. and A.J.A.H., 2016. Journal of Chemical , Biological and Physical Sciences Efficacy of Methanolic Extract of a Marine Ascidian , Lissoclinum bistratum for Antimicrobial Activity. Swamy, M.K., Arumugam, G., Kaur, R., Ghasemzadeh, A., Yusoff, M.M., Sinniah, U.R., 2017. GC-MS Based Metabolite Profiling, Antioxidant and Antimicrobial Properties of Different Solvent Extracts of Malaysian Plectranthus amboinicus Leaves. Evidence- based Complement. Altern. Med. 2017. https://doi.org/10.1155/2017/1517683. Tarasov, S., Génier, F., 2015. Innovative bayesian and parsimony phylogeny of dung beetles (coleoptera, scarabaeidae, scarabaeinae) enhanced by ontology-based partitioning of morphological characters. PLoS One 10. https://doi.org/10.1371/journal.pone.0116671. Taylor, R., Romaine, I., Liu, C., Murthi, P., Jones, P., Waterson, A., Sulikowski, G., Zwiebel, L., 2012. Structure-Activity Relationship of a Broad-Spectrum Insect Odorant Receptor Agonist. ACS Chem. Biol. 7, 1647–1652. Thevissen, K., Warnecke, D.C., François, I.E.J.A., Leipelt, M., Heinz, E., Ott, C., Zähringer, U., Thomma, B.P.H.J., Ferket, K.K.A., Cammue, B.P.A., 2004. Defensins from Insects and Plants Interact with Fungal Glucosylceramides. J. Biol. Chem. 279, 3900–3905. https://doi.org/10.1074/jbc.M311165200. Turillazzi, S., Perito, B., Pazzagli, L., Pantera, B., Gorfer, S., Tancredi, M., 2004. Antibacterial activity of larval saliva of the European paper wasp Polistes dominulus (Hymenoptera, Vespidae). Insectes Soc. 51, 339–341. https://doi.org/10.1007/s00040-004-0751-3. Venkata raman, B., M, P.S., LA, S., B, N.R., A, N.V.K., M, S., TM, R., 2012. Academic Sciences Asian Journal of Pharmaceutical and Clinical Research. Asian J. Pharm. Clin. Res. 5, 99–106. Vetterli, S.U., Zerbe, K., Müller, M., Urfer, M., Mondal, M., Wang, S.Y., Moehle, K., Zerbe, O., Vitale, A., Pessi, G., Eberl, L., Wollscheid, B., Robinson, J.A., 2018. Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport in Escherichia coli. Sci. Adv. 4. https://doi.org/10.1126/sciadv.aau2634. Vilas, A.K., Amit, H.M., 2015. Bioassay-guided fractional isolation and identification of anti- mrsa compounds by gc-ms from tridax procumbens. World J. Pharm. Pharm. Sci. 4, 1022–1031. Vizioli, J., Richman, A., Uttenweiler-Joseph, S., Blass, C., Bulet, P., 2001. he defensin peptide of the malaria vector mosquito Anopheles gambiae: antimicrobial activities and expression in adult mosquitoes. Insect Biochem Mol Biol. 31, 241-248. https://doi.org/10.1016/s0965-1748(00)00143-0. International Journal of Applied Biology, 5(2), 2021 116 Wu, Q., Patočka, J., Kuča, K., 2018. Insect antimicrobial peptides, a mini review. Toxins (Basel). 10, 1–17. https://doi.org/10.3390/toxins10110461. Yi, H.Y., Chowdhury, M., Huang, Y.D., Yu, X.Q., 2014. Insect antimicrobial peptides and their applications. Appl. Microbiol. Biotechnol. 98, 5807–5822. https://doi.org/10.1007/s00253-014-5792-6. Zha, C., Wang, C., Li, A., 2018. Toxicities of selected essential oils, silicone oils, and paraffin oil against the common bed bug (hemiptera: Cimicidae). J. Econ. Entomol. 111, 170–177. https://doi.org/10.1093/jee/tox285. Zhang, Z.-M., Wu, W.-W., Li, G.-K., 2009. Study of the Alarming Volatile Characteristics of Tessaratoma papillosa Using SPME-GC-MS. J. Chromatogr. Sci. 47, 291–295.