| Density-dependent prophylaxis(DDP)has been widely reported in many kinds of insect.However,the mechanism of DDP is still unclear.Antimicrobial peptides(AMPs)are important parts of insect innate immunity system,but there has been no reports related with AMPs in DDP.Our previous results have been showed that there existed the DDP in Mythimna separata larvae.The antimicrobial activity of the haemolymph and the transcription level of AMPs(Ceropin C and Defensin)genes of larvae that reared at high density(10 larvae per jar)were significantly higher than the larvae reared at low density(1 larva per jar).It has been found that bursicon plays an important role in the regulation of AMPs of newly emerged adults.We hypothesized that bursicon may be play an important role in regulating prophylactic immunity of crowded larvae of M.separata.In this study,the cDNA sequence of bursicon genes in M.separata(Mybursa and Mybursβ)was cloned,and transcriptional levels of Mybursa and Mybursβ genes in different developmental stages and tissues of larvae were analyzed.And the transcriptional expression relationship between the bursicon gene and AMPs genes was studied in high and low density larvae.Furthermore,the expression pattern of AMPs genes and the antimicrobial ability of larvae were further studied respectively by RNA interference bursicon genes and injection of recombinant bursicon protein.The results will provide an important theoretical basis for revealing the mechanism of insect DDP,and have important theoretical significance for understanding the role of bursicon in larval immune regulation.The full-length cDNA sequences of Mybursa and Mybursβ genes were cloned from the transcriptome data of M.separata larvae.The open reading frame(ORF)of Mybursa is 438bp,encoding 145 amino acid residues.Sequences alignment results showed that the sequences similarity between Mybursa and the homologous genes of Spodoptera exigua/Spodoptera litura was the highest,and it was 84.8%.The ORF of Myburβ was 420 bp,encoding 139 amino acid residues.The sequences similarity between Myburβ and the homologous genes of Helicoverpa armigera was the highest,and it was 87.1%.At the same time,11 cysteine residues and C-X-G-X-C structures,which are typical features of bursicon protein,were found in the amino acid sequences of Myburα and Myburβ.The transcriptional level of bursicon gene at different developmental stage of M.separata was detected.The results showed that the exepression level of Mybursa was the highest in adults,followed by pupae and egg,and the lowest in the larvae.The expression level of Mybursβ was consistent with that of Mybursa at different developmental stages.The results of the transcription levels of Mybursa and Mybursβ genes in different tissues showed that Mybursa was the highest in the ventral nerve cord,followed by the brain,and low in the fat body,hemolymph,epidermis,malpighian tube and gut.The expression pattern of Mybursβ gene was similar with that of Mybursa in different tissues.The results of the expression levels of bursicon gene and AMPs Ceropin C(CeC)and Defensin(Def)genes of M.separata larvae reared at high(10 larvae per jar)and low density(1 larva per jar)showed that the expression level of bursicon gene in the 4th and 5th instar larvae reared at high density was significantly higher than that of the larvae reared at low density.Expression patterns of AMPs CeC and Def genes in the larvae reared at high and low density were similar with that of bursicon gene of the larvae,and the expression of AMPs genes was delayed 24h than that of bursicon genes.The effects of RNA interference with bursicon gene in M.separata on the expression of AMPs genes showed that,high density 4th instar larvae injected with burα-dsRNA or burβ-dsRNA,the expression level of bursicon gene was significantly down-regulated from 12h to 72h,and the expression levels of CeC and Def genes were significantly down-regulated.The survival rate of the larvae infected by Bacillus thuringiensis(Bt)was detected after RNAi with Mybursicon genes and MyAMPs genes,respectively.The results showed that after the 4th instar larvae were injected with burα-dsRNA(or burβ-dsRNA or CeC-dsRNA or Def-dsRNA),the survival rate of the larvae was decreased significantly.The effect of recombinant bursicon protein on the expression of AMPs showed that the expression levels of MyCeC and MyDef genes were significantly induced in the 4th instar larvae injected of recombinant Mybursα(or Mybursβ)protein.In conclusion,Mybursicon gene plays an important role in the regulation of antimicrobial peptides in larval stage,and the differences in the expression of bursicon and antimicrobial peptides in the larvae with high and low density were the main reasons for the changes in the disease resistance of the larvae. |
