Comparative analysis of C-type lectin domain proteins in the ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae)

Insects have a large family of C-type lectins involved in cell adhesion, pathogen recognition and activation of immune responses. In this study, 32 transcripts encoding C-type lectin domain proteins (CTLDPs) were identified from the Thitarodes xiaojinensis transcriptome. According to their domain structures, six CTLDPs with one carbohydraterecognition domain (CRD) were classified into the CTL-S subfamily. The other 23 CTLDPs with two CRDs were grouped into the immulectin (IML) subfamily. The remaining three with extra regulatory domains were sorted into the CTL-X subfamily. Phylogenetic analysis showed that CTL-S and CTL-X members from different insects could form orthologous groups. In contrast, no T. xiaojinensis IML orthologues were found in other insects. Remarkable lineage-specific expansion in this subfamily was observed reflecting that these CTLDPs, as important receptors, have evolved diversified members in response to a variety of microbes. Prediction of binding ligands revealed that T. xiaojinensis, a coldadapted species, conserved the ability of CRDs to combine with Ca^2+ to keep its receptors from freezing. Comparative analysis of induction of CTLDP genes after different immune challenges indicated that IMLs might play critical roles in immune defenses. This study examined T. xiaojinensis CTLDPs and provides a basis for further studies of their characteristics.

Metabolomics reveals the key role of oxygen metabolism in heat susceptibility of an alpine-dwelling ghost moth, Thitarodes xiaojinensis (Lepidoptera: Hepialidae)

Ghost moths inhabiting the alpine meadows of the Tibetan Plateau are cold? adapted stenothermal organisms that are susceptible to heat (dead within 7 days at 27 °C exposure). Exploring the metabolic basis of their heat susceptibility would extend our understanding of the thermal biology of alpine-dwelling invertebrates. Here, gas chromatography-mass spectrometry-based metabolomics was combined with physiological and transcriptional approaches to determine the metabolic mechanisms of heat susceptibility in Thitarodes xiaojinensis larvae. The metabolomics results showed that 27 °C heat stress impaired the Krebs cycle and lipolysis in T. xiaojinensis larvae, as demonstrated by the accumulation of intermediary metabolites. In addition, carbohydrate reserves were highly and exclusively consumed, and an anaerobic product, lactate, accumulated. This evidence suggested a strong reliance on glycolysis to anaerobically generate energy. The respiration rate and enzymatic activity test results indicated a deficiency in O2 metabolism;in addition, the Krebs cycle capacity was not decreased, and the metabolic flux through aerobic pathways was limited. These findings were further supported by the occurrence of hypoxia symptoms in midgut mitochondria (vacuolation and swelling) and increased transcription of hypoxia-induced factor 1-α.Overall, heat stress caused O2 limitation and depressed the overall intensity of aerobic metabolism in ghost moths, and less efficient anaerobic glycolysis was activated to sustain their energy supply. As carbohydrates were depleted, the energy supply became deficient. Our study presents a comprehensive metabolic explanation for the heat susceptibility of ghost moths and reveals the relationship between O2 metabolism and heat susceptibility in these larvae.

Effects of methamidophos and deltamethrin on in vitro protein phosphorylation in Monochamus alternatus

Monochamus alternatus Hope （Coleoptera： Cerambycidae） is not only a serious pest insect to pine trees but also the main vector of pine wood nemadote Bursaphelenchus xylophilus, which causes pine wilt disease. To explore the insecticidal mechanism of insecticides to M. alternatus, we chose methamidophos and deltamethrin as the representatives of two groups of insecticides （organophosphates and pyrethroids）, which are widely used for pest control in China and investigated their effects on phosphorylation of proteins from the insect. Phosphorylation of proteins from the insect fat body and head was determined by in vitro 32P-labelling. In the fat body, deltamethrin obviously reduced basal phosphorylation levels of proteins at 111, 95, 77, and 44 kDa, but enhanced the basal phosphorylation level of a protein at 138 kDa. However, in the presence of calmodulin but not cyclic adenosine monophosphate （cAMP）, deltamethrin increased phosphorylation of the protein at 111 kDa. In the head, deltamethrin inhibited basal phosphorylation levels of proteins at 113, 98, and 51 kDa, but potentiated phosphorylation of a protein at 167 kDa activated by cAMP. Methamidophos inhibited phosphorylation of a protein at 44 kDa in the fat body. Although methamidophos did not impact basal phosphorylation levels of any proteins in the head, it inhibited calcium/calmodulin （Ca^2＋CaM）-stimulated phosphorylation of a protein at 51 kDa. Together, our data indicate that methamidophos and deltamethrin altered phosphorylation levels of various proteins in the head and fat body of the pine insect and these two kinds of insecticides acted on the proteins that can be phosphorylated in the tissues respectively, which is possibly related to their toxicity.