Induction, Purification And Activity Analysis Of Antimicrobial Substance From Black Soldier Fly, Hermetia Illucens (L.) Larvae Hemolymph

Although further study is still needed to understand the detailed defense mechanisms of insects, it is widely accepted that their innate immune system constitutes of humoral immunity, and cellular immunityeffectively destroy the intruding microorganisms by disrupting membranes, interfering with metabolism, and targeting cytoplasmic components of the target cell. (Boman& Hultmark,1987; Bulet et al.,1999; Hoffmann, 1995; Hoffmann et al.,1996; Lavine& Strand,2002). The lysozyme and antimicrobial peptides/proteins are the main immune factors in the innate immune system.The lysozyme was the first substance found with antimicrobial activity in insect, also the unique action mode of antimicrobial peptides/proteins of insects make themselves excellent research system for studying innate immunity and to solve the urgent problem of antibacterial resistance.Up to now, in more than one million insect species described and characterized, only about 170 antimicrobial peptides/polypeptides from various insect species have been clearly characterized (Bulet et al.,1999; Saito et al.,2004)let alone a few million more undiscovered insects. More novel antimicrobial substances are expected to be characterized and identified in insect (Thomas,2005).Black soldier fly is an ideal insect to use for such experiments because it commonly colonizes and oviposits on rotting carcasses where pathogens thrive. Its ability of reducing manure volume as well as the ability to suppress pathogenic microorganisms such as E.coli O157:H7 and Salmonella enterica serotype Enteritidis (ME 18) populations were also discovered and characterized by researchers (Erickson et al.2004; liu qiaolin et al.2008). These researches indicated the possibility of the insect’s ability in generation of antimicrobial peptides.Up to now, no attempt was made before to characterize the ability of generation of antimicrobial substances in this insect. In this study, we focused on black soldier fly. We chose to use black soldier fly for study since it has not been examined before for its immune system and antimicrobial substances in spite that they already demonstrated their potentials as their strong ability in colonizing composting material where pathogens thrive. The objective of our study was seek to investigate the putative antimicrobial substances generated in the insect’s hemolymph, in addition, we also looked into if black soldier fly could accelerate the inactivation of bacteria while growing in swine manure.Here we report for the first time that artificial injection of bacteria can induce an immune response with in black soldier fly larvae with increased antibacterial activity in hemolymph. We found that experimental injection of bacteria introduced to last larval stage produced hemolymph exhibiting greater antibacterial activity compared with hemolymph immunized and collected at other life stages.To obtain the natural antibacterial substances from black soldier fly larvae,crude hemolymph was first subjected to conventional chromatography and then further RP-HPLC analysis Investigations revealed that at least several fractions of antibacterial peptide repertoire with low molecular weight (<20kDa) present within the bacteria challenged larval hemolymph,active against the Gram-positive S.aureus,and gram negative bacteria E.coli and S.enteritis,and plant pathogen Cotton Wilt Fusarium(Fusarium oxysporium f. sp. Vasinfectum). The purified hemolymph was confirmed to be highly stable under a certain pH range. Two of the purified component were measured by MALDI-TOFF,one of them was proved to be lysozyme-like peptide/protein by MALDI-TOFF,and the other active component was probably a new kind of peptide/proten, considering its low match with protein bank in NCBInr.However, the results are still reserved until further analysis.This is the first report describing the attempt made to characterize the antibacterial activity in black soldier fly immune hemolymph. Considering the activity against organisms typically associated with clinical infection, it may be a source of novel antibiotic-like compounds that may be used for infection control.Further studies will address the purity and sequence of the purified fractions. The study in the generation of antimicrobial substances in the insect may contribute considerably to the understanding of the immune response in the black soldier fly.