| Insect biorefinery by black soldier fly larvae（BSFL）,Hermetia illucens,has emerged as an innovative technique for the valorization of food waste.However,despite BSFL being an attractive natural source of antimicrobial proteins（AMP）,there is a scarcity of research on the antimicrobial activity and transcriptome expression of AMP derived from BSFL following waste treatment.In particular,there are few studies on the inhibitory effect and mechanism of AMP on Vibrio parahaemolyticus（VP）,a disease in aquaculture.This study explored the inhibition effect of AMP on VP from the following two aspects.On the one hand,food waste was used as the treatment object,and C/N ratio regulation was used as the main regulation method to study the yield,extraction method,in vitro and in vivo antibacterial activity of AMP under different C/N（21:1-10:1）conditions,as well as the expression pattern of AMP gene family at transcriptomic level.The results showed that both acidic and alkaline methods could extract antimicrobial peptides,but the activity of AMP extracted by acidic method was stronger.Different C/N conditions（21:1-10:1）had no significant effect on the yield of AMP,but had significant effect on the waste reduction rate and insect yield.The C/N21:1-16:1 conditions was better than the C/N14:1-10:1 conditions.Based on the results of half inhibitory concentration(IC50),minimum inhibitory concentration（MIC）and zone diameter（DIZ）,it was found that C/N16:1 AMP was superior to other C/N conditions,and had significant ability to inhibit VP.In vivo bacteriotic experiments of model animal zebrafish showed that C/N16:1 AMP had similar bacteriotic activity to sulfamethoxine antibiotic,and could increase the 96 h survival rate of zebrafish infected with VP from 0%to39%.Transcriptome analysis and comparison of C/N16:1 and C/N21:1（blank）showed that lysozyme gene was the gene with the highest expression abundance in the AMP gene family.C/N16:1 significantly up-regulated the expression of 12 genes out of 51 lysozyme genes,so it showed significantly strong antimicrobial activity of AMP.On the other hand,study used strain as the research object which were specific induced by black soldier fly AMP,through the pure culture strains induced black soldier fly to produce AMP,and through the comparison of AMP in vitro bacteriostasis ability,find specific induction of bacteria species,further aided the transcriptome sequencing,showed the strain to induce expression characteristics of AMP gene family,to reveal the mechanism of non-pathogenic bacteria inducing the expression of AMP against VP in black soldier fly.The results showed that there was no significant difference in antibacterial activity of AMP expressed by black soldier fly induced by acupuncture,soaking and feeding,and soaking method became the further method to induce the expression of antimicrobial peptides because of its simple process.Among the 10 pure cultured strains,4 strains were found to be significant strong inducible effect,which were Bacillus subtilis（BS）,Rummeliibacillus stabekisii（RS）,Lactobacillus plantarum（LP）and Streptococcus thermophilus（ST）,and one strain with significant weak inducible effect was Enterococcus faecalis（EF）.In the comparison of the transcriptome analysis and gene expression pathway analysis found that different strains induced expression of AMP gene families show significant features,including BS and ST significantly increased the expression of the lysozyme gene families,LP,RS and VP significantly improved the expression of cecropin and defensin gene families,while EF down-regulated the expression of many lysozyme at the same time.This indicated that the expression abundance of lysozyme,cecropin and defensin was significantly positively correlated with antibacterial activity of AMP.Over all,This study is of great significance to reveal the action mechanism and specific expression of BSFL AMPs,and also lays a foundation for promoting the application of BSFL AMPs in the prevention and control of aquatic pathogens. |
