Seperation of bioactive compounds from Haemolymph of scarab beetle Scarabaeus sacer (Coleoptera: Scarabaeidae) by GC-MS and determination of its antimicrobial activity
Nancy Taha Mohamed1, Doaa Hassan Abdelsalam1, Ahmed Salem El-Ebiarie1, Mahmoud Elaasser2
1Zoology & Entomology Department, Faculty of Science, Helwan University
2Regional Centre of Mycology and Biotechnology, Al-Azhar University
*Corresponding author: Doaa Hassan Abdelsalam, Zoology & Entomology Department, Faculty of Science, Helwan University, Egypt.
Received: 20 November 2021; Accepted: 02 December 2021; Published: 07 December 2021
Citation: Nancy Taha Mohamed, Doaa Hassan Abdelsalam, Ahmed Salem El-Ebiarie, Mahmoud Elaasser. Seperation of bioactive compounds from Haemolymph of scarab beetle Scarabaeus sacer (Coleoptera: Scarabaeidae) by GC-MS and determination of its antimicrobial activity. International Journal of Applied Biology and Pharmaceutical Technology 12 (2021): 461-480.View / Download Pdf Share at Facebook
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.
Haemolymph, GS-MS, gram negative bacteria, gram positive bacteria, antimicrobial activity
Haemolymph articles, GS-MS articles, gram negative bacteria articles, gram positive bacteria articles, antimicrobial activity articles
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 . 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  thereby, reducing the greenhouse gas fluxes . It also improves plant growth and grain production . Scarabeus sacer is considered a species of genus Scarabaeus, occurs in coastal dunes and marshes around theMediterranean Basin. It can be found across North Africa, Southern Europe and parts of Asia (Afghanistan, Corsica, Cyprus, France, Iran, Israel,Italy,Morocco,Sardinia,Sicily,SudanandSyria). Scarabaeus sacer is aspeciesofdung beetlebelonging to the familyScarabaeidae .
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 . 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 .
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 . 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 . 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 .
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 . 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 . 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 .
Nowadays, the using of GC-MS technique is important in analyzing and separation the compounds found in plants extracts  and also the haemolymph of arthropods. This will be a new method for discovering a future drug 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.
2. Material and Methodology
2.1 Collection of Insects
Scarab beetle was collected from Baltim in theKafr El Sheikh Governorate, in thenorth coastofEgypt.
2.2 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.
2.3 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.
2.4 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 . 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 microorganisms. Gentamycin and ketoconazole (Sigma Aldrich, USA) were used as standard antibacterial and antifungal drugs at 30 and 50ug/ml, respectively.
2.4.1 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 .
3. 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 mg/ml against E. coli, 1250 mg/ml against Enterobacter cloacae, Bacillus subtilis and 10000 mg/ml against Staphylococcus aureus respectively (Table 3).
Table 1: Bioactive compounds in haemolymph of scarab beetle separating by GC-MS
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 . 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 . 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 . Most of these compounds exhibit biological activities such as anticancer, antiviral, antioxidant, and anti-inflammatories .
Phytol isomer is a diterpenes identified in the haemolymph of adult S. sacer. phytol is a bioactive compound that has a potent anticancer activity . It also serve as a chemical attractant for parasitoids, according to research on these species: Lucilia sericata , Leptinotarsa decemlineata . 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  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 . Also the hydrocarbons used to distinguish between the male and female of Sarcophaga species . Alkanes were also marked in the surface lipids of Liposcelis bostrychophila, Cryptolestes ferrugineus  and Laelius utilis . Larvae of potato beetle contain hydrocarbons of high molecular weight, particularly tetrapentacontane (C54H 110), pentapentacontane (C55H 112) and heptapentacontane (C57H 116) .
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 . Hexadecanoic acid methyl ester is also known as palmitic acid ester and efficiently used as an antioxidant, pesticide, anti-androgenic, nematicide, flavoring agent, hypocholesterolemic, and lubricant . Also hexadeconic acid was the major fatty acid in Sargassum granuliferum seaweed which prevents the biofilm forming bacteria .
Esters with even longer C22, C24, C42 and C46 carbon chains were determined in Aleurotithius timberlakei . 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 . Fatty acids C18:1, C18:2 and C18:3 were estimated in the larvae of Drosophila melanogaster, Musca domestica and Galleria mellonella . Fatty acids C16 – C18 have also been determined in the surface lipids of Cryptolestes ferrugineus and Liposcelis bostrychophila insects . Cholest-5-en-3-ol, 24- propylidene have been detected in the methyl extract of Sargassum crassifolium [67 and  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 . Also ketones are separated from Tessaratoma papillosa . The relationships of these analyzed compounds in insects play vital roles as they can be transmitters of information and signals  and also serve as pheromones . 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 
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. , 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-  and Dibutyl phthalate act as ectoparasiticide .
Table 2: Antimicrobial activity (as a mean zone of inhibition) of the haemolymph of adult scarab beetle
Table 3: The antimicrobial activity as Minimum Inhibitory concentration (MIC) in mg/ml of the tested micro-organisms. The test was done using the diffusion agar technique
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 . Contrary to our results, the whole body extract of housefly maggots show no activity against E. coli and exhibit antifungal activity . While  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 , the European bumble bee, Bombus pascuorum  and Tenebrio molitor larvae . On the other hand, some other insects revealed activity only against Gram-positive bacteria as Aedes aegypti , Chironomus plumosus  and Anopheles gambiae .
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) which act as a natural antibiotic . 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 . 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 . The presence of microorganisms invading insects causes the fat body to rapidly synthesize AMPs, which are then released into the hemolymph . Previous research shows that each insect species produces a distinct antimicrobial peptide that acts against specific microorganisms . 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 . 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 . AMPs have pharmacological properties such as low molecular weight, high water solubility, broad-spectrum antimicrobial activity, and low levels of cytotoxicity .  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 , dung beetles , Red Palm Weevil , pupae of the giant silk moths .
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 . 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 .
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.
Great thanks and appreciation for the Mycology Center, Al-Azhar University for their cooperation.
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