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Alpha‐ketoglutarate‐dependent dioxygenase ALKB homologue 4 (ALKBH4) is a member of the Fe (II) and 2‐oxoglutarate‐dependent ALKB homologue family that plays important roles in epigenetic regulation by alkyl lesions removal in mammals. However, the roles of ALKBH4 in insects are not clear. Here, TcALKBH4 was cloned and functionally characterised in Tribolium castaneum. Temporal expression revealed that TcALKBH4 was highly expressed in early embryos and early pupae. Spatial expression showed that TcALKBH4 was highly expressed in the adult testis, and followed by the ovary. RNA interference targeting TcALKBH4 at different developmental stages in T. castaneum led to apparent phenotypes including the failure of development in larvae, the reduction of food intake and the deficiency of fertility in adult. However, further dot blot analyses showed that TcALKBH4 RNAi does not seem to influence 6 mA levels in vivo. qRT‐PCR was used to further explore the underlying molecular mechanisms; the result showed that TcALKBH4 mediates the development of larvae possibly through 20E signalling pathway, and the fertility of female and male adult might be regulated by the expression of vitellogenesis and JH signalling pathway, respectively. Altogether, these findings will provide new insights into the potential function of ALKBH4 in insects. [ABSTRACT FROM AUTHOR]

Background: Insect cytochrome P450 monooxygenases (P450s) play a crucial role in phytochemical metabolism and tolerance. Three P450 genes (TcCYP4Q3, TcCYP4Q5, and TcCYP4Q7) are associated with the response of eugenol in Tribolium castaneum. However, the responding mechanisms of these P450 genes to eugenol remain unknown. Results: Here, spatiotemporal expression profiling revealed that TcCYP4Q3 and TcCYP4Q5 were most highly expressed in late adult, while TcCYP4Q7 was predominantly expressed in late larva; and all of these three P450 genes were mainly expressed in the fat body of larvae. Furthermore, the expressions of these three P450 genes were significantly up‐regulated after exposure to eugenol, and depletion of them enhanced the susceptibility of beetles to eugenol. Interestingly, RNA interference (RNAi) against the CncC gene, a transcription factor of CncC signaling pathway associated with regulation of insect P450s in response to phytochemicals, reduced the transcripts of these three P450 genes following exposure to eugenol. Investigation of CncC signaling pathway showed that this pathway could be activated by eugenol. Conclusion: Altogether, the results indicate that these three P450 genes are regulated by CncC signaling pathway to participate in the susceptibility of Tribolium castaneum to phytochemicals. These findings will aid implications for the development of novel therapeutics to control pest. © 2022 Society of Chemical Industry. [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]

Eugenol, a plant-derived component possessing small side effects, has an insecticidal activity to Tribolium castaneum; however, the underlying molecular mechanisms of eugenol acting on T. castaneum are currently unclear. Here, a nerve conduction carboxylesterase and a detoxifying glutathione S-transferase were significantly inhibited after eugenol exposure, resulting in the paralysis or death of beetles. Then, RNA-sequencing of eugenol-exposed and control samples identified 362 differentially expressed genes (DEGs), containing 206 up-regulated and 156 down-regulated genes. RNA-seq data were validated further by qRT-PCR. GO analysis revealed that DEGs were associated with 1308 GO terms of which the most enriched GO terms were catalytic activity, and integral component of membrane; KEGG pathway analysis showed that these DEGs were distributed in 151 different pathways, of which some pathways associated with metabolism of xenobiotics or drug were significantly enriched, which indicated that eugenol most likely disturbed the processes of metabolism, and detoxication. Moreover, several DEGs including Hexokinase type 2, Isocitrate dehydrogenase, and Cytochrome b-related protein, might participate in the respiratory metabolism of eugenol-exposed beetles. Some DEGs encoding CYP, UGT, GST, OBP, CSP, and ABC transporter were involved in the xenobiotic or drug metabolism pathway, which suggested that these genes of T. castaneum participated in the response to eugenol exposure. Additionally, TcOBPC11/ TcGSTs7, detected by qRT-PCR and RNA-interference against these genes, significantly increased the mortality of eugenol-treated T. castaneum, providing further evidence for the involvement of OBP/GST in eugenol metabolic detoxification in T. castaneum. These results aid eugenol insecticidal mechanisms and provide the basis of insect control. [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]

C-type lectin S group (CTL-S) plays a variety of roles in invertebrate including pathogen recognition and activation of immune responses. Previous studies have shown that CTL-S subfamily of Tribolium castaneum is mainly divided into two clades, of which only TcCTL1 was separately located in one clade. However, it remains unclear whether TcCTL1 occurs the differentiation of function. Therefore, the CTL-S TcCTL1 gene was cloned and characterized from T. castaneum. Functional analysis revealed that TcCTL1 could recognize and agglutinate pathogens, as well as activate immune signaling pathways to participate in immune response, which was consistent with our previously reported for TcCTL5 and TcCTL6. Differently, RNAi of TcCTL1 discovered that the egg produced by ds TcCTL1- treated adult could not hatch into larvae. Further DAPI-straining embryo indicated that the process of embryogenesis in ds TcCTL1 -treated beetle was defeated, implying that TcCTL1 is required for embryogenesis in T. castaneum except for immune response. These results will aid implications for the understanding of CTL-S in invertebrate. • First report of TcCTL1 involved in embryogenesis except for immune response. [ABSTRACT FROM AUTHOR]