Depending on different apoptosis pathways, the effector Cscaspase-3 in Chilo suppressalis exposed to temperature and parasitic stress was induced.

Apoptosis is a form of programmed cell death (PCD) that is largely triggered by caspases through both the mitochondria-dependent and mitochondria-independent pathways. The rice stem borer, Chilo suppressalis, serves as an economically important pest of rice, which is often suffered by temperature and parasitic stress under natural conditions. In the present study, effector Cscaspase-3 encoding caspase was obtained from the rice pest Chilo suppressalis. CsCaspase-3 possesses p20 and p10 subunits, two active sites, four substrate-binding sites, and two cleavage motifs. Real-time quantitative PCR showed that Cscaspase-3 was expressed at maximal levels in hemocytes; furthermore, transcription was most highly in female adults. Expression of Cscaspase-3 was induced by hot and cold temperatures, with the highest expression at 39 °C. Cscaspase-3 expression was also significantly induced at 10 h, 2 d, 5 d, and 7 d of parasitism. Flow cytometry results showed that both temperature and parasitism trigger apoptosis, but only parasitism induces apoptosis via the mitochondrial apoptosis pathway in C. suppressalis. RNAi-mediated silencing of Cscaspase-3 expression reduced C. suppressalis survival at -3 °C. This study provides a foundation for further studies of caspases in insects during biotic and abiotic stress.

Chilo suppressalis heat shock proteins are regulated by heat shock factor 1 during heat stress.

Heat shock factor 1 (HSF1) functions to maintain cellular and organismal homeostasis by regulating the expression of target genes, including those encoding heat shock proteins (HSPs). In the present study, the gene encoding HSF1 was cloned from the rice pest Chilo suppressalis, and designated Cshsf1. The deduced protein product, CsHSF1, contained conserved domains typical of the HSF1 family, including a DNA-binding domain, two hydrophobic heptad repeat domains, and a C-terminal transactivation domain. Real-time quantitative PCR showed that Cshsf1 was highly expressed in hemocytes. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsf1 was most highly expressed in male adults. RNAi-mediated silencing of Cshsf1 expression reduced C. suppressalis survival at high temperatures. To investigate the regulatory interactions between Cshsf1 and Cshsps, the promoters and expression patterns of 18 identified Cshsps in C. suppressalis were analysed; four types of heat shock elements (HSEs) were identified in promoter regions including canonical, tail-tail, head-head, and step/gap. The expression of Cshsp19.0, Cshsp21.7B, Cshsp60, Cshsp70 and Cshsp90 was positively regulated by Cshsf1; however, Cshsp22.8, Cshsp702, Cshsp705 and Cshsp706 gene expression was not altered. This study provides a foundation for future studies of HSF1 in insects during thermal stress.

Characterization and functional analysis of Cshsp19.0 encoding a small heat shock protein in Chilo suppressalis (Walker).

Small heat shock proteins (sHSPs) function as ATP-independent chaperones that preserve cellular proteostasis under stressful conditions. In this study, Cshsp19.0, which encodes a new small heat shock protein, was isolated and characterized from Chilo suppressalis (Walker) to better understand the contribution of sHSPs to insect development and stress tolerance. The full-length Cshsp19.0 cDNA was 697 bp and encoded a 19.0 kDa protein with an isoelectric point of 5.95. Phylogenetic analysis and amino acid alignments indicated that Cshsp19.0 is a member of the sHSP family. Cshsp19.0 was expressed at maximal levels in foreguts and showed the least amount of expression in fat bodies. Expression analysis in different developmental stages of C. suppressalis revealed that Cshsp19.0 was most highly expressed in 1st instar larvae. Furthermore, Cshsp19.0 was upregulated when insects were exposed to heat and cold stress for a 2-h period. There were significant differences in the male and female pupae in response to humidity; Cshsp19.0 expression increased in male pupae as RH increased, whereas the inverse pattern was observed in female pupae. Larvae exhibited a lower rate of survival when Cshsp19.0 was silenced by a nanomaterial-promoted RNAi method. The results confirm that Cshsp19.0 functions to increase environmental stress tolerance and regulates physiological activities in C. suppressalis.

Transcriptomic analysis of pre-diapause larvae of Chilo suppressalis (Walker) (Lepidoptera: Pyralidae) in natural populations.

Chilo suppressalis Walker is a devastating pest of rice in Asia and exhibits facultative diapause in the larval stage. Most prior experiments on diapausing and non-diapausing C. suppressalis were conducted in the laboratory. In this study, transcriptome analyses were performed on pre-diapausing larvae collected from field populations of C. suppressalis and compared to laboratory populations. Among 2674 differentially expressed genes (DEGs), 32 DEGs related to pre-diapause and 239 universally expressed genes were screened; these were primarily enriched in "neuroactive ligand-receptor interaction", "lysosome" and "glycerolipid metabolism" in KEGG pathway analysis. With respect to clusters of orthologous genes (COG), DEGs were assigned to "posttranslational modification, protein turnover, chaperones", "carbohydrate transport and metabolism", and "secondary metabolite biosynthesis, transport and catabolism" categories. Further analysis also revealed that a key "circadian clock-controlled protein" gene is sensitive to photoperiod and significantly decreased during the pre-diapause phase. Genes encoding two small heat shock proteins, hsp21.4 and hsp27.2, were significantly expressed on August 15 as compared to three other sampling times in August 2018. Eight DEGs were randomly chosen and evaluated by real-time quantitative PCR (RT-qPCR) to validate the accuracy of the transcriptome data. The expression of six DEGs (gene-evm_000752, gene-evm_006486, gene-evm_008626, gene-evm_002485, gene-evm_011981 and Chilo_suppressalis_newGene_18103) showed significant same patterns of differential expression in both the RT-qPCR and RNA-Seq analyses. This study increases our understanding of the complex physiological and molecular mechanisms involved in C. suppressalis at the pre-diapause phase.

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.

Altered immunity in crowded Mythimna separata is mediated by octopamine and dopamine.

Similar to pathogenic infection, high population density alters insects' prophylactic immunity. Density-dependent prophylaxis has been reported in many polyphenic insects, but the regulatory mechanism underlying this phenomenon remains unclear. The biogenic monoamines are known to play critical roles in mediating insect immune responses. In the current study, the immune capacity and the levels of three biogenic monoamines were investigated in the polyphenic larvae of Mythimna separata, reared at the densities of 1, 2, 5, 10, and 30 larvae per 650-mL jar. Concomitant with the increased phenoloxidase (PO) activity and total haemocyte count in the larvae at high densities (5, 10, 30 larvae/jar), the octopamine level was also increased. In contrast, the dopamine level was decreased, and the 5-hydroxytryptamine level was not significantly affected. Injection of octopamine induced significant increases in the total haemocyte count and PO activity. Conversely, epinastine, a specific antagonist of octopamine, decreased the total haemocyte count and PO activity. Another octopamine antagonist, phentolamine, inhibited the activity of PO and lysozymes. In addition, injection of dopamine induced a significant increase in PO activity and decreased the total haemocyte count and lysozyme activity. These results suggested that both octopamine and dopamine mediate the increases in total haemocyte count and PO activity in the crowded larvae.