Bursicon mediates antimicrobial peptide gene expression to enhance crowded larval prophylactic immunity in the oriental armyworm, Mythimna separata.

It has been reported that a high population density alters insect prophylactic immunity. Bursicon plays a key role in the prophylactic immunity of newly emerged adults. In this paper, full-length cDNAs encoding the alpha and beta subunits of bursicon in Mythimna separata larvae (Msburs α and Msburs ß) were identified. The cDNAs of Msburs α and Msburs ß contain open reading frames (ORFs) encoding 145- and 139-amino acid residue proteins, respectively. Multiple alignment sequences and phylogenetic analysis indicated that Msbursicons (Msburs α and Msburs ß) are orthologous to bursicons in other lepidopterans. The Msbursicons were expressed throughout all developmental states with higher relative expression during the egg, pupae, and adult stages. Msbursicons (Msburs α and Msburs ß) were highly expressed in the ventral nerve cord and brain relative to other tested tissues. Msbursicon expression of larvae subject to high-density treatment (10 larvae per jar) was significantly increased compared with that of the larvae subject to low-density treatment (1 larva per jar) in the whole fourth and fifth instar stages. The trend in the expression of the antimicrobial peptide (AMP) genes cecropin C and defensin in the test stage was accorded and delayed with increased expression of bursicons. Silencing Msburs α (or Msburs ß) expression by dsRNA injection in larvae subject to high-density treatment significantly decreased the expression levels of the cecropin C and defensin genes. Recombinant Msbursicon homodimers significantly induced the expression of the cecropin C and defensin genes. There was a notable decrease in the survival rate of the Msburs α (or Msburs ß or Mscecropin C or Msdefensin) knockdown larvae infected by Beauveria thuringiensis. Our findings provide the first insights into how larval density mediates AMP gene expression, which subsequently affects the prophylactic immunity of insects under high-density conditions.

Effects of Larval Density on Plutella xylostella Resistance to Granulosis Virus.

It has been reported that some phase-polyphenic insects from high-density conditions are more resistant to pathogens than those from low-density conditions. This phenomenon is termed "density-dependent prophylaxis" (DDP). However, whether non phase-polyphenic insects exhibit DDP has rarely been elucidated. The diamondback moth (DBM), Plutella xylostella, one of the most destructive insect pests affecting cruciferous crops, is non phase-polyphenic. In this study, the resistance of DBM larvae to P. xylostella granulosis virus (Plxy GV) and their immune response to the virus when reared at densities of 1, 2, 5, 10, 15, and 20 larvae per Petri dish were investigated under laboratory conditions. Compared with larvae reared at lower densities, larvae reared at moderate density showed a significantly higher survival rate, but the survival rate significantly decreased with further increases in rearing density. Furthermore, the phenoloxidase, lysozyme and antibacterial activity and total hemocyte count in the hemolymph of the larvae, regardless of whether they were challenged with the virus, from different larval densities corresponded to the observed differences in resistance to Plxy GV. These results demonstrated that P. xylostella larvae exhibited DDP within a certain limited density. This study may help to elucidate the biocontrol effect of different density populations of P. xylostella by granulosis virus and guide improvements in future management strategy.

Immunological regulation by a ß-adrenergic-like octopamine receptor gene in crowded larvae of the oriental Armyworm, Mythmina separata.

Recent reports demonstrate that octopamine plays an important immunological role in crowded larvae of the Oriental Armyworm, Mythmina separata. We identified an octopamine receptor, the ß-adrenergic-like gene (designated MsOctß2R), with a 1191 bp open reading frame that encodes 396 amino acids and contains seven conserved hydrophobic transmembrane domains. Multiple sequence alignments and a phylogenetic analysis indicated that MsOctß2R was orthologous to Octß2R that is present in other lepidopterans. MsOctß2R was expressed throughout all developmental stages with higher relative expression during the fourth instar and adult stages. MsOctß2R was highly expressed in the ventral nerve cord and the fat body relative to other examined tissues. Elevated MsOctß2R expression was observed in larvae that were under higher-density conditions (7 and 10 larvae per jar). Silencing MsOctß2R expression via dsRNA injections in larvae from higher-density conditions significantly decreased phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival rates against Beauveria bassiana infections (54.06%, 9.91%, 36.22%, and 23.53%, respectively) when compared with control larvae. These results suggest that high-density conditions might alter prophylactic immunity in larvae by regulating the MsOctß2R gene in M. separara and provide new insights into density-dependent prophylaxis in insects.

The Role of Tyramine ß-Hydroxylase in Density Dependent Immunityof Oriental Armyworm (Mythmina separata) Larva.

High population density alters insect prophylactic immunity, with density-dependent prophylaxis (DDP) being reported in many polyphonic insects. However, the molecular mechanism for DDP remains unclear. In current study, the role of tyramine ß-hydroxylase (Tßh) in the immune response of M. separata larvae that were subject to different rearing densities conditions was investigated. The tyramine ß-hydroxylase activity of larvae from high density treatments (10 and 30 larvae per jar) was significantly higher than that of the larvae from low density treatments (one, two, and five larvae/jar). A tyramine ß-hydroxylase (designated MsTßh) containing a 1779 bp open reading frame was identified. Multiple sequence alignment and phylogenetic analysis indicated that MsTßh was orthologous to the Tßh that was found in other lepidopterans. Elevated MsTßh expression was observed in larvae under high density (10 larvae per jar). Silencing MsTßh expression by the injection of dsRNA in larvae from the high density treatment produced a 25.1% reduction in octopamine levels, while at the same time, there was a significant decrease in phenoloxidase (PO) and lysozyme activity, total haemocyte counts, and survival against Beauveria infection 56.6%, 88.5%, 82.0%, and 55.8%, respectively, when compared to control larvae. Our findings provide the first insights into how MsTßh mediates the octopamine level, which in turn modulates the immune response of larvae under different population densities.

Transcriptomic insight into antimicrobial peptide factors involved in the prophylactic immunity of crowded Mythimna separata larvae.

Similar to pathogenic infection, a high population density alters insect prophylactic immunity. Antimicrobial peptides (AMPs) are known to play critical roles in an insect's humoral immune response to microbial infection. We applied RNA sequencing to investigate differential gene expression levels in fat body and hemocyte samples from larvae reared in high- (10 larvae per jar) and low-density (1 larva per jar) conditions; the samples exhibited density-dependent prophylaxis. A number of AMP molecule-related proteins were annotated for the first time from 145,439 assembled unigenes from M. separata larvae. The transcript levels of AMP molecules such as gloverin-, defensin-, cecropin-, lebocin- and attacin-related unigenes were increased with the prophylactic immunity of high-density larvae. The pattern recognition receptor peptidoglycan recognition protein (PGRP), a key protein in the synthesis of AMPs in IMD- and Toll pathway-related unigenes, was also upregulated in the larvae from the high-density group. The resultant transcriptomic database was validated by the transcript levels of four selected AMP genes quantified from the high- and low-density larval groups with quantitative real-time PCR. The antimicrobial activity against gram-positive Staphylococcus aureus and Bacillus subtilis and gram-negative Edwardsiella ictaluri and Vibrio anguillarum in the hemolymph of larvae from the high-density group was significantly higher than that of larvae from the low-density group. Our findings provide the first insight into the role of AMP genes in the mechanisms of density-dependent prophylaxis in M. separata and provide new insight into the control of M. separata with biopesticides.