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Cuticle is not only critical for protecting insects from noxious stimuli but is also involved in a variety of metabolic activities. Cuticular proteins (CPs) affect cuticle structure and mechanical properties during insect growth, reproduction, and environmental adaptation. Here, we describe the identification and characterization of a member of the RR‐1 subfamily of CPs with an R&R consensus (CPR) in Tribolium castaneum (TcCPR69). Although it was previously reported to be highly expressed in the wings, we found that knocking down TcCPR69 by RNA interference (RNAi) did not cause obvious wing abnormalities but markedly disrupted the growth and metamorphosis of beetles with 100% cumulative mortality; additionally, the chitin content of the pharate adult was decreased and the new abdominal cuticle was significantly thinner before molting. TcCPR69 showed chitin‐binding ability and the expression levels of key genes involved in chitin metabolism (trehalase [TcTRE], chitin synthase [TcCHSA and TcCHSB], and chitinase [TcCHT5 and TcCHT10]) were also decreased by TcCPR69 knockdown. TcCPR69 gene expression peaked shortly after molting and was increased 2.61 fold at 12 h after 20‐hydroxyecdysone (20E) injection. This was reversed by RNAi of the ecdysone‐related genes ecdysone receptor (TcECR) and fushi tarazu transcription factor 1 (TcFTZ‐F1). These results indicate that TcCPR69 is positively regulated by 20E signaling to contribute to cuticle formation and maintain chitin accumulation during the growth and metamorphosis of beetles. [ABSTRACT FROM AUTHOR]

In recent years, increasing numbers of microRNAs (miRNAs) have been reported to regulate insect metamorphosis. One thousand, one hundred fifty‐four miRNAs have been previously identified from Tribolium castaneum by high‐throughput sequencing; however, little is known about which miRNAs can participate in metamorphosis, leaving the role of miRNAs in regulating the underlying mechanism elusive. Here, we report the participation of miR‐3017b in the metamorphosis of T. castaneum. Temporal profiles revealed that miR‐3017b was highly expressed at the late larval stage, but significantly decreased at the early pupal stage. Overexpression of miR‐3017b caused larval to pupal to adult metamorphosis arrested. Dual‐luciferase reporter assay and miRNA–mRNA interaction assay illustrated that miR‐3017b interacts with the coding sequence of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and suppresses its expression. Knockdown of SERCA caused metamorphosis arrested, similar to that observed in miR‐3017b overexpression beetles. Further functional mechanism analyses revealed that 20‐hydroxyecdysone application downregulates miR‐3017b and up‐regulates SERCA expression. The expression level of downstream genes in the 20E pathway was disrupted after overexpressing miR‐3017 and the knockdown of SERCA. These results provided evidence miR‐3017b‐SERCA contributes to metamorphosis by regulating the 20E pathway in T. castaneum. It could advance our understanding of the coordination of 20E and miRNA regulation in insect metamorphosis. [ABSTRACT FROM AUTHOR]

In the postgenomics era, comparative genomics have advanced the understanding of evolutionary processes of neuropeptidergic signaling systems. The evolutionary origin of many neuropeptidergic signaling systems can be traced date back to early metazoan evolution based on the conserved sequences. Insect parathyroid hormone receptor (iPTHR) was previously described as an ortholog of vertebrate PTHR that has a well-known function in controlling bone remodeling. However, there was no sequence homologous to PTH sequence in insect genomes, leaving the iPTHR as an orphan receptor. Here, we identified the authentic ligand insect PTH (iPTH) for the iPTHR. The taxonomic distribution of iPTHR, which is lacking in Diptera and Lepidoptera, provided a lead for identifying the authentic ligand. We found that a previously described orphan ligand known as PXXXamide (where X is any amino acid) described in the cuttlefish Sepia officinalis has a similar taxonomic distribution pattern as iPTHR. Tests of this peptide, iPTH, in functional reporter assays confirmed the interaction of the ligand-receptor pair. Study of a model beetle, Tribolium castaneum, was used to investigate the function of the iPTH signaling system by RNA interference followed by RNA sequencing and phenotyping. The results suggested that the iPTH system is likely involved in the regulation of cuticle formation that culminates with a phenotype of defects in wing exoskeleton maturation at the time of adult eclosion. Moreover, RNAi of iPTHRs also led to significant reductions in egg numbers and hatching rates after parental RNAi. Author summary: Vertebrate parathyroid hormone (PTH) and its receptors have been extensively studied with respect to their function in bone remodeling and calcium metabolism. Insect parathyroid hormone receptors (iPTHRs) have been previously described as counterparts of vertebrate PTHRs, however, they are still orphan receptors for which the authentic ligands and biological functions remain unknown. We describe an insect form of parathyroid hormone (iPTH) by analyzing its interactions with iPTHRs. Identification of this new insect peptidergic system proved that the PTH system is an ancestral signaling system dating back to the evolutionary time before the divergence of protostomes and deuterostomes. We also investigated the functions of the iPTH system in a model beetle Tribolium castaneum by using RNA interference. RNA interference of iPTHR resulted in defects in wing exoskeleton maturation and fecundity. Based on the differential gene expression patterns and the phenotype induced by RNAi, we propose that the iPTH system is likely involved in the regulation of exoskeletal cuticle formation and fecundity in insects. [ABSTRACT FROM AUTHOR]

Methyl-CpG binding domain proteins (MBD) can specifically bind to methylated CpG sites and play important roles in epigenetic gene regulation. Here, we identified and functionally characterized the MBD protein in Tribolium castaneum. T. castaneum genome encodes only one MBD protein: TcMBD2/3. RNA interference targeting this gene at different developmental stages caused lethal phenotypes including metamorphosis deficiency in larvae and pupae, gastrointestinal system problems and fecundity deficiency in adult. Moreover, Tcmbd2/3 knockdown adult showed progressive reduced locomoter activity, a typical neurodegeneration phenotype. This is a common feature of DNA methylation in mammals and has not been found in other insects. However, band shift assays demonstrated that TcMBD2/3 could not bind to methylated DNA, indicating the essential roles of TcMBD2/3 is independent of DNA methylation. Our study provides Tcmbd2/3 plays important roles in T. castaneum and gives new insights into the potential mechanism of action of MBD proteins in insect. • A single methyl-CpG binding domain protein TcMBD2/3 was identified in Tribolium castaneum. • TcMBD2/3 is essential for metamorphosis, gastrointestinal system and reproductive activity in T. castaneum. • TcMBD2/3 plays important roles in neurogenesis of adult. • TcMBD2/3 lost the methylated DNA binding activity. [ABSTRACT FROM AUTHOR]

BACKGROUND: Many insect cytochrome P450 proteins (CYPs) are involved in the metabolic detoxification of exogenous compounds such as plant toxins and insecticides. Tribolium castaneum, the red flour beetle, is a major agricultural pest that damages stored grains and cereal products. With the completion of the sequencing of its genome, two T. castaneum species‐specific CYP genes, CYP4BN6, and CYP6BQ11, were identified. However, it is unknown whether the functions of most CYPs are shared by TcCYP4BN6 and TcCYP6BQ11, and the upstream regulatory mechanism of these two CYPs remains elusive. RESULTS: QRT‐PCR analysis indicated that TcCYP4BN6 and TcCYP6BQ11 were both most highly expressed at the late pupal stage and were mainly observed in the head and gut, respectively, of adults. Moreover, the transcripts of these two CYPs were significantly induced by dichlorvos and carbofuran, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to these two insecticides. Intriguingly, knockdown of the latrophilin (lph) gene, which has been reported to be related to the insecticide susceptibility, reduced the expression of TcCYP4BN6 and TcCYP6BQ11 after insecticide treatment, suggesting that these two CYP genes are regulated by lph to participate in insecticide susceptibility in T. castaneum. CONCLUSION: These results shed new light on the function and mechanism of CYP genes associated with insecticide susceptibility and could facilitate research on appropriate and sustainable pest control management. © 2019 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Small heat shock proteins (sHSPs) are important modulators of insect survival. Previous research revealed that there is only one orthologous cluster of shsps in insects. Here, we identified another novel orthologous cluster of shsps in insects by comparative analysis. Multiple stress experiments and function investigation of Tchsp21.8a belonging to this orthologous cluster and seven species-specific shsps were performed in the stored-grain pest Tribolium castaneum. The results indicated that expression of Tchsp21.8a showed weak responses to different stresses. However, expressions of most species-specific shsps exhibited hyper-responses to heat stress, and expressions of all species-specific shsps displayed diverse responses during other stresses to protect beetles in a cooperative manner. Additionally, Tchsp21.8a and species-specific Tcshsp19.7 played important roles in the development of T. castaneum , and all Tcshsps had a certain impact on the fecundity. Our work created a comprehensive reliable scaffold of insect shsps that can further provide instructive insights to pest bio-control • Firstly identified another novel orthologous cluster of sHSPs in insects. • Two shsps were identified in Acrythociphon pisum and one new shsp was found in Anopheles gambiae. • Novel orthologous Tchsp21.8a in Tribolium castaneum had typical features of orthologous shsps. • Most species-specific Tcshsps were related to anti-heat stress by hundreds-fold up-regulation. • Species-specific Tcshsps did not respond to cold but had different responses under other stresses. [ABSTRACT FROM AUTHOR]

Latrophilin (LPH) is an adhesion G protein-coupled receptor (aGPCR) that participates in multiple essential physiological processes. Our previous studies have shown that lph is not only indispensable for the development and reproduction of red flour beetles (Tribolium castaneum), but also for their resistance against dichlorvos or carbofuran insecticides. However, the regulatory mechanism of lph -mediated insecticide susceptibility remains unclear. Here, we revealed that knockdown of lph in beetles resulted in opposing changes in two chemoreception genes, chemosensory protein 10 (CSP10) and odorant - binding protein C01 (OBPC01), in which the expression of TcCSP10 was downregulated, whereas the expression of TcOBPC01 was upregulated. TcCSP10 and TcOBPC01 were expressed at the highest levels in early pupal and late larval stages, respectively. High levels of expression of both these genes were observed in the heads (without antennae) of adults. TcCSP10 and TcOBPC01 were significantly induced by dichlorvos or carbofuran between 12 and 72 h (hrs) after exposure, suggesting that they are likely associated with increasing the binding affinity of insecticides, leading to a decrease in sensitivity to the insecticides. Moreover, once these two genes were knocked down, the susceptibility of the beetles to dichlorvos or carbofuran was enhanced. Additionally, RNA interference (RNAi) targeting of lph followed by exposure to dichlorvos or carbofuran also caused the opposing expression levels of TcCSP10 and TcOBPC01 compared to the expression levels of wild-type larvae treated with insecticides alone. All these results indicate that lph is involved in insecticide susceptibility through positively regulating TcCSP10 ; and the susceptibility could also further partially compensated for through the negative regulation of TcOBPC01 when lph was knockdown in the red flour beetle. Our studies shed new light on the molecular regulatory mechanisms of lph related to insecticide susceptibility. lph was involved in insecticide susceptibility through positively regulating TcCSP10 , and it could further partially compensate for insecticide susceptibility by negatively regulating TcOBPC01 when lph was knockdown in Tribolium castaneum. Unlabelled Image • TcCSP10 was down-regulated and TcOBPC01 was up-regulated after knockdown of Tclph. • TcCSP10 and TcOBPC01 were significantly induced by insecticides of dichlorvos or carbofuran. • RNAi of TcCSP10 and TcOBPC01 increased the susceptibilities of larval to insecticides. • Tclph related to insecticide susceptibility via regulating TcCSP10 and partially compensated by TcOBPC01. [ABSTRACT FROM AUTHOR]

Glutathione S-transferases (GSTs) are a large group of enzymes having both detoxification roles conferring insecticide resistance and specialist metabolic functions. Tribolium castaneum GST Delta 1 ( TcGSTd1 ) has been found playing crucial role in insecticide resistance and biological processes in insect species. However, the regulatory system of TcGSTd1 has still rarely been known. Comparing the transcriptome profile of RNAi treated larvae (ds- TcGSTd1 ) and control larvae of T. canstaneum by using RNA-sequencing, we obtained 14,284,085 sequence reads aligned with 13,275 genes. And 512 differentially expressed genes (DEGs) were identified from ds- TcGSTd1 treated group. Est/CCE , CYP , MRPs were significantly down-regulated in ds -TcGSTd1 group when compared with control group, which illustrated that they cooperated with TcGSTd1 to reduce the activity of cellular metabolism system. While, SNO was up-regulated in ds- TcGSTd1 insects suggested it may also involve in detoxifying alkaloid of insect metabolism system. These results established that TcGSTd1 not only acts as a vital gene for phase II cellular detoxification but also participates in phase 0, I, and III cellular detoxification by cooperating with CSPs , OBPs , CYP9 , ESTB1, CCE6 , MRPs and other detoxification genes. Knockdown of TcGSTd1 also suppressed several genes encoding antioxidant enzymes, e.g. CuZnSOD , Duox , Prx , HPX , CPO , and MCORP . Suggested that they may modulate the function of TcGSTd1 on lifespan, immune, development and reproduction. All these results shed the new insights into the regulatory mechanism of TcGSTd1 involved in insect physiology and could further facilitate the research of suitable and sustainable managements for the pest control. [ABSTRACT FROM AUTHOR]

• Chat knockdown severely affected larval development and pupal eclosion, but vacht RNAi only disrupted pupal eclosion. • Parental RNAi of chat or vacht would reduce egg production and completely inhibite egg hatchability in T. castaneum. • Reduction of chat or vacht decreased the resistance to carbofuran and dichlorvos. Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are essential enzymes for synthesizing and transporting acetylcholine (ACh). But their functions in metamorphosis, reproduction, and the insecticide susceptibility were poorly understood in the insects. To address these issues, we identified the orthologues of chat and vacht in Tribolium castaneum. Spatiotemporal expression profiling showed Chat has the highest expression at the early adult stage, while vacht shows peak expression at the early larval stage. Both of them were highly expressed at the head of late adult. RNA interference (RNAi) of chat and vacht both led to a decrease in ACh content at the late larval stage. It is observed that chat knockdown severely affected larval development and pupal eclosion, but vacht RNAi only disrupted pupal eclosion. Further, parental RNAi of chat or vacht led to 35 % or 30 % reduction in fecundity, respectively, and knockdown of them completely inhibited egg hatchability. Further analysis has confirmed that both the reduction in fecundity and hatchability caused through the maternal specificity in T. castaneum. Moreover, the transcript levels of chat and vacht were elevated after carbofuran or dichlorvos treatment. Reduction of chat or vacht decreased the resistance to carbofuran and dichlorvos. This study provides the evidence for chat and vacht not only involved in development and reproduction of insects but also could as the potential targets of insecticides. [ABSTRACT FROM AUTHOR]

[Display omitted] • New orthologous cluster of eclosion hormone-like (EHL) was identified in Arthropoda. • 20E up-regulated the expression of TcEHL during pupal-adult development. • RNAi of TcEHL caused lethal phenotypes with un-tanning adult cuticle in T. castaneum. • RNAi of TcEHL affected ecdysis behavior and tanning pathway genes in T. castaneum. Eclosion hormone (EH) is a neurohormone that plays a key role in the regulation of insect pre-ecdysis behavior at the end of each molt. Previous research has reported more than one EH gene was found in certain insects, with their functions and mechanisms still unclear. Here, aside from the classical EH gene orthologous group, we characterized another novel orthologous cluster of eclosion hormone-like (EHL) genes in Arthropoda and investigated the roles of EHL during development in Tribolium castaneum. T. castaneum EHL (TcEHL) shows high expression levels during pupal − adult development, which also positively responded to 20‐hydroxyecdysone (20E) treatment as well as RNA interference (RNAi) of ECR (20E nuclear receptor). Knockdown of TcEHL prevented the tanning of the adult cuticle and caused lethal phenotypes. Further analysis indicated that knockdown of TcEHL could upregulate expression levels of the classical TcEH , and downregulate the ecdysis behavior cascade genes, as well as tanning pathway enzymes. This suggests a critical role for TcEHL in adult eclosion and cuticle tanning. In addition, our data indicated that TcEHL is responsible for the female reproduction process. Taken together, these results suggest that TcEHL has specific roles in adult cuticle tanning during the post-eclosion process and female reproduction. They also suggest that EHL gene is the ancestral copy for the EH family and it is functionally shuffled by synfunctionalization. [ABSTRACT FROM AUTHOR]