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Tribolium castaneum is a worldwide pest of stored grain that mainly damages flour, and not only causes serious loss of flour quality but also leads to deterioration of flour quality. Chemical detection plays a key role in insect behavior, and the role of odorant-binding proteins (OBPs) in insect chemical detection has been widely studied. OBPs can interact with small molecule compounds and thereby modulate variation in insecticide susceptibility in insects. In this study, a total of 65 small molecule compounds are selected to investigate the bound effect with TcOBP C12. The molecular docking results showed that β-caryophyllene, (-)-catechin, butylated hydroxytoluene, diphenyl phthalate and quercetin were the top five compounds, with docking binding energies of -6.11, -5.25, -5.09, -5.05, and - 5.03 Kcal/mol, respectively. Molecular dynamics analysis indicated that odorant binding protein C12 (TcOBP C12) exhibited high binding affinity to all five tested chemical ligands, evidenced by fluorescence quenching assay in vitro. In addition, the contact toxicity assay results suggested that these chemical agents caused a dose-dependent increase in mortality rate for T. castaneum adults. The TcOBP C12 gene was upregulated >2 times after a 24-h exposure, indicating that OBP C12 may play an important role for T. castaneum in response to these chemical agents. In conclusion, our results provide a theoretical basis for future insecticide experiments and pest management., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Terpinen-4-ol has high fumigating activity to stored-grain pests including Tribolium confusum. To understand the detoxification of terpinen-4-ol in insects, proteomic analysis was performed to identify related proteins and pathways in response to terpinen-4-ol fumigation in T. confusum. By using isobaric tags for relative and absolute quantitation (iTRAQ)-based strategy, 4,618 proteins were obtained from T. confusum adults in the present study. Comparative proteomic analysis showed that 148 proteins were upregulated and 137 proteins were downregulated in beetles under the LC 50 of terpinen-4-ol treatment for 24 hr. According to functional classifications, differentially expressed proteins (DEPs) were enriched in xenobiotic metabolism pathways. In the detoxification pathway, the levels of 25 cytochrome P450s, 5 glutathione S-transferases, and 2 uridine diphosphate (UDP)-glucuronosyltransferases were changed, most of which were upregulated in T. confusum exposed to terpinen-4-ol. The results indicated that terpinen-4-ol was potentially metabolized and detoxified by enzymes like P450s in T. confusum., (© 2019 Wiley Periodicals, Inc.)

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., (© 2019 Society of Chemical Industry.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Potassium ion channels play a critical role in the generation of electrical signals and thus provide potential targets for control of insect pests by RNA interference., Results: Genes encoding the small conductance calcium-activated potassium channel (SK) and the voltage-gated potassium channel (SH) were knocked down in Tribolium castaneum by injection and oral delivery of dsRNA (dsTcSK and dsTcSH, respectively). Irrespective of the delivery mechanism a dose-dependent effect was observed for knockdown (KD) of gene expression and insect mortality for both genes. Larvae fed a 400 ng dsRNA mg -1 diet showed significant gene (P < 0.05) knockdown (98% and 83%) for SK and SH, respectively, with corresponding mortalities of 100% and 98% after 7 days. When injected (248.4 ng larva -1 ), gene KD was 99% and 98% for SK and SH, causing 100% and 73.4% mortality, respectively. All developmental stages tested (larvae, early- and late-stage pupae and adults) showed an RNAi-sensitive response for both genes. LC50 values were lower for SK than SH, irrespective of delivery method, demonstrating that the knockdown of SK had a greater effect on larval mortality. Biosafety studies using adult honeybee Apis mellifera showed that there were no significant differences either in expression levels or mortality of honeybees orally dosed with dsTcSK and dsTcSH compared to control-fed bees. Similarly, there was no significant difference in the titre of deformed wing virus, used as a measure of immune suppression, between experimental and control bees., Conclusion: This study demonstrates the potential of using RNAi targeting neural receptors as a technology for the control of T. castaneum. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Latrophilin (LPH) is known as an adhesion G-protein-coupled receptor which involved in multiple physiological processes in organisms. Previous studies showed that lph not only involved the susceptibility to anticholinesterase insecticides but also affected fecundity in Tribolium castaneum. However, its regulatory mechanisms in these biological processes are still not clear. Here, we identified two potential downstream carboxylesterase (cce) genes of Tclph, esterase4 and esterase6, and further characterized their interactions with Tclph. After treatment of T. castaneum larvae with carbofuran or dichlorvos insecticides, the transcript levels of Tcest4 and Tcest6 were significantly induced from 12 to 72 h. RNAi against Tcest4 or Tcest6 led to the higher mortality compared with the controls after the insecticides treatment, suggesting that these two genes play a vital role in detoxification of insecticides in T. castaneum. Furthermore, with insecticides exposure to Tclph knockdown beetles, the expression of Tcest4 was upregulated but Tcest6 was downregulated, indicating that beetles existed a compensatory response against the insecticides. Additionally, RNAi of Tcest6 resulted in 43% reductions in female egg laying and completely inhibited egg hatching, which showed the similar phenotype as that of Tclph knockdown. These results indicated that Tclph affected fecundity by positively regulating Tcest6 expression. Our findings will provide a new insight into the molecular mechanisms of Tclph involved in physiological functions in T. castaneum.

PA1b (Pea Albumin 1, subunit b) peptide is an entomotoxin, extracted from Legume seeds, with a lethal activity towards several insect pests, such as mosquitoes, some aphids and cereal weevils. This toxin acts by binding to the subunits c and e of the plasma membrane H + -ATPase (V-ATPase) in the insect midgut. In this study, two cereal weevils, the sensitive Sitophilus oryzae strain WAA42, the resistance Sitophilus oryzae strain ISOR3 and the insensitive red flour beetle Tribolium castaneum, were used in biochemical and histological experiments to demonstrate that a PA1b/V-ATPase interaction triggers the apoptosis mechanism, resulting in insect death. Upon intoxication with PA1b, apoptotic bodies are formed in the cells of the insect midgut. In addition, caspase-3 enzyme activity occurs in the midgut of sensitive weevils after intoxication with active PA1b, but not in the midgut of resistant weevils. These biochemical data were confirmed by immuno-histochemical detection of the caspase-3 active form in the midgut of sensitive weevils. Immuno-labelling experiments also revealed that the caspase-3 active form and V-ATPase are close-localized in the insect midgut. The results concerning this unique peptidic V-ATPase inhibitor pave the way for the utilization of PA1b as a promising, more selective and eco-friendly insecticide.

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A bioinformatics search of the genome of the red flour beetle, Tribolium castaneum, resulted in the identification of two genes encoding proteins closely related to UDP-N-acetylglucosamine pyrophosphorylases (UAPs), which provide the activated precursor, UDP-N-acetylglucosamine, for the synthesis of chitin, glycoproteins and glycosylphosphoinositide (GPI) anchors of some membrane proteins as well as for the modification of other substrates. This is in contrast to other arthropods whose genomes have been completely sequenced, all of which have only a single copy of this gene. The two T. castaneum UAP genes, TcUAP1 and TcUAP2, share both nucleotide and amino acid sequence identities of about 60%. RT-PCR analysis revealed that the two genes differ in their developmental and tissue-specific patterns of expression. RNA interference (RNAi) indicated roles for TcUAP1 and TcUAP2 at the molt and intermolt stages, respectively: RNAi for TcUAP1 resulted in specific arrest at the larval-larval, larval-pupal or pupal-adult molts, depending on time of injection of double-stranded RNAs, whereas RNAi for TcUAP2 prevented larval growth or resulted in pupal paralysis. Analysis of elytral cuticle indicated loss of structural integrity and chitin staining after RNAi for TcUAP1, but not after RNAi for TcUAP2. Loss of peritrophic matrix (PM)-associated chitin was also observed following RNAi for TcUAP1, but not after RNAi for TcUAP2. Down-regulation of transcripts for either TcUAP gene at the mature adult stage resulted in cessation of oviposition in females, as well as fat body depletion and eventual death in both sexes. These results demonstrate that both TcUAP genes are critical for beetle development and survival, but that only TcUAP1 is clearly associated with synthesis of cuticular or PM chitin. However, both of these genes appear to have additional critical role(s) unrelated to chitin synthesis, presumably in the glycosylation of proteins and/or secondary metabolites., (Published by Elsevier Ltd.)

Juvenile hormone (JH) prevents ecdysone-induced metamorphosis in insects. However, our knowledge of the molecular mechanisms of JH action is still fragmented. Krüppel homolog 1 (Kr-h1) is a JH-inducible transcription factor in Drosophila melanogaster (Minakuchi, C., Zhou, X., Riddiford, L.M., 2008b. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech. Dev. 125, 91-105). Analysis of expression of the homologous gene (TcKr-h1) in the beetle Tribolium castaneum showed that its transcript was continuously present in the larval stage but absent in the pupal stage. Artificial suppression of JH biosynthesis in the larval stage caused a precocious larval-pupal transition and a down-regulation of TcKr-h1 mRNA. RNAi-mediated knockdown of TcKr-h1 in the larval stage induced a precocious larval-pupal transition. In the early pupal stage, treatment with an exogenous JH mimic (JHM) caused formation of a second pupa, and a rapid and large induction of TcKr-h1 transcription. JHM-induced formation of a second pupa was counteracted by the knockdown of TcKr-h1. RNAi experiments in combination with JHM treatment demonstrated that in the larval stage TcKr-h1 works downstream of the putative JH receptor Methoprene-tolerant (TcMet), and in the pupal stage it works downstream of TcMet and upstream of the pupal specifier broad (Tcbr). Therefore, TcKr-h1 is an early JH-response gene that mediates JH action linking TcMet and Tcbr.

The evolution of complete metamorphosis in insects is a key innovation that has led to the successful diversification of holometabolous insects, yet the origin of the pupa remains an enigma. Here, we analyzed the expression of the pupal specifier gene broad (br), and the effect on br of isoform-specific, double-stranded RNA-mediated silencing, in a basal holometabolous insect, the beetle Tribolium castaneum. All five isoforms are weakly expressed during the penultimate instar and highly expressed during the prepupal period of the final instar. Application of hydroprene, a juvenile hormone analog, during the penultimate instar caused a repeat of the penultimate br expression patterns, and the formation of supernumerary larvae. Use of dsRNA against the br core region, or against a pair of either the br-Z2 or br-Z3 isoform with the br-Z1 or br-Z4 isoform, produced mobile animals with well-differentiated adult-like appendages, but which retained larval-like urogomphi and epidermis. Disruption of either the br-Z2 or the br-Z3 isoform caused the formation of shorter wings. Disruption of both br-Z1 and br-Z4 caused the appearance of pupal traits in the adults, but disruption of br-Z5 had no morphological effect. Our findings show that the br isoform functions are broadly conserved within the Holometabola and suggest that evolution of br isoform expression may have played an important role in the evolution of the pupa in holometabolous insects.