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Many insects undergo the process of metamorphosis when larval precursor cells begin to differentiate to create the adult body. The larval precursor cells retain stem cell-like properties and contribute to the regenerative ability of larval appendages. Here we demonstrate that two Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (BTB) domain transcription factors, Chronologically inappropriate morphogenesis (Chinmo) and Abrupt (Ab), act cooperatively to repress metamorphosis in the flour beetle, Tribolium castaneum. Knockdown of chinmo led to precocious development of pupal legs and antennae. We show that although topical application of juvenile hormone (JH) prevents the decrease in chinmo expression in the final instar, chinmo and JH act in distinct pathways. Another gene encoding the BTB domain transcription factor, Ab, was also necessary for the suppression of broad (br) expression in T. castaneum in a chinmo RNAi background, and simultaneous knockdown of ab and chinmo led to the precocious onset of metamorphosis. Furthermore, knockdown of ab led to the loss of regenerative potential of larval legs independently of br. In contrast, chinmo knockdown larvae exhibited pupal leg regeneration when a larval leg was ablated. Taken together, our results show that both ab and chinmo are necessary for the maintenance of the larval tissue identity and, apart from its role in repressing br , ab acts as a crucial regulator of larval leg regeneration. Our findings indicate that BTB domain proteins interact in a complex manner to regulate larval and pupal tissue homeostasis. [Display omitted] • Chinmo RNAi triggers precocious pupal leg and antenna development in Tribolium castaneum. • Chinmo and juvenile hormone (JH) act in distinct pathways. • Double knockdown of Chinmo and abrupt (ab) leads to precocious metamorphosis. • Chinmo and Ab repress br expression. • Ab, but not Chinmo, is necessary for larval leg regeneration. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs), as a large family of pattern recognition receptors (PRRs), have been reported to be involved in bacterial infection, but the role of CTLs in development has been poorly understood in insects. The orthologues of Tribolium castaneum CTL9 (TcCTL9) have been identified among insects, but its functions were currently unclear. Therefore, we performed functional analysis of TcCTL9 in this study. Our results indicated that TcCTL9 could bind to bacteria through lipopolysaccharide and peptidoglycan, and agglutinate Gram-positive and Gram-positive bacteria in a Ca2+-dependent manner. Silencing TcCTL9 reduced the immune resistance to Staphylococcus aureus and Escherichia coli , decreased the expression of antimicrobial peptides and prophenoloxidase, and inhibited the phenoloxidase activity. These data suggested that TcCTL9 functioned in the immune response via the Toll and IMD pathways and prophenoloxidase system. During development, TcCTL9 had high expression in the periods of egg to larva and pupa to adult, and knockdown of TcCTL9 suppressed the metamorphosis, egg production and hatchability, and ovary development through ecdysone and juvenile hormone pathways in T. castaneum. This study comprehensively clarified the functions of TcCTL9 orthologues in insects and provided the theoretical basis for developing novel targets of pesticides. [Display omitted] • TcCTL9 could bind to bacteria via LPS and PGN. • TcCTL9 agglutinated the bacteria in a Ca2+ dependent manner. • Silencing TcCTL9 reduced the immune response via the Toll, IMD and proPO system. • TcCTL9 RNAi blocked the metamorphosis and reproduction by ecdysone and JH pathways. [ABSTRACT FROM AUTHOR]

Phosphine (PH 3) fumigation is widely used to control insect pests in stored products globally. However, intensive PH 3 use has led to the emergence of significant resistance in target insects. To address this issue, this study investigated PH 3 resistance mechanisms by conducting both qualitative and quantitative proteomic analyses on the whole proteome of a PH 3 -resistant Tribolium castaneum strain (AUS-07) using LC-MS/MS. Besides, proteins from both strains were separated in 1D-PAGE, and qualitatively analyzed using LC-MS/MS after in-gel digestion. Differentially expressed proteins (DEPs) with cut-off values (4-fold expression difference and p < 0.05) were selected, and 107 proteins were identified in the AUS-07 strain. Among them, several proteins involved in oxidative phosphorylation were notably upregulated in response to PH 3 exposure. Upregulation of Complex I and III in the electron transport chain of the AUS-07 strain may lead to the excessive generation of reactive oxygen species (ROS) in the form of superoxide, which can damage Fe-S cluster-containing proteins such as cytochrome P450s (CYP450s). Upregulation of detoxifying enzymes, such as CYP450s and glutathione S -transferases (GSTs), was observed, likely to repair superoxide-induced damage on CYP450s as well as quenching superoxide. Upregulation of aldose reductases involved in polyol pathways and downregulation of the trehalose transporter were observed, suggesting that PH 3 -resistance may be linked to diapause-like physiological adaptations, including quiescence. Further studies are essential to quantify polyol levels in the AUS-07 strain and to conduct other molecular analyses to validate the roles of identified DEPs in PH 3 resistance. Altogether, our findings suggest a new control strategy to stored product insect pests by other type of fumigant such as ethyl formate with different molecular structure. • A PH 3 -resistant strain (AUS07) of Tribolium castaneum was used for proteome study. • A total of 2338 proteins were quantitatively and qualitatively analyzed using LC-MS/MS. • Total 135 proteins were identified with the cut-off values as p -value < 0.05 and 4-fold difference. • Proteins related to xenobiotic metabolism were highly upregulated in AUS07. • AUS07 highly expressed diapause-related proteins including aldose reductase. [ABSTRACT FROM AUTHOR]

The glutamate-gated chloride channels (GluCls) are widely existed in the neural and nonneural tissues of invertebrate. In addition to play important roles in signal transduction, the GluCls also showed multiple physiological functions in insects such as participate in the juvenile hormone synthesis. In the present study, the potential roles of TcGluCl in growth and development of the red flour beetle Tribolium castaneum were explored. Knockdown of TcGluCl showed no effects on the survivability, weight growth, final pupation rate, eclosion and fecundity of T. castaneum , whereas resulted in the significant premature pupation of larvae. Inhibition of TcGluCl expression significantly changed the levels of juvenile hormone and ecdysone as well as the expressions of hormone biosynthetic genes. The increased ecdysone level and decreased juvenile hormone level were observed at the late stage of dsGluCl-treated larvae. Knockdown of TcGluCl significantly reduced the expressions of TcSTIM1 and TcOrai1 , which were the primary proteins in store-operated calcium entry (SOCE) mediated Ca2+ influx mechanism. Whilst the L-glutamic acid treatment led to the increased TcOrai1 expression in T. castaneum. These findings suggested that knockdown of TcGluCl increased the ecdysone level and contributed to the premature pupation of larvae, which might be due to the reduced Ca2+ influx caused by the decreased expressions of TcSTIM1 and TcOrai1. These studies provide novel insights on the function of GluCls in insects. [Display omitted] • Knockdown of TcGluCl showed no effects on survivability, growth, pupation rate, eclosion and fecundity of T. castaneum. • Inhibition of TcGluCl expression increased ecdysone level and resulted in the premature pupation of larvae. • Knockdown of TcGluCl reduced the expressions of TcSTIM1 and TcOrai1. [ABSTRACT FROM AUTHOR]

[Display omitted] • • A major new insect transcriptomics web resource for Tribolium castaneum. • • View RNAseq data for adult and larval tissues, and different embryonic stages. • • Query by gene, tissue and function, or retrieve genes with similar patterns of expression. • • Use both Drosophila and Tribolium gene identifiers for searches. • • Gene results link out to tissue expression tables of beetle paralogues and fly homologues. The red flour beetle Tribolium castaneum has emerged as a powerful model in insect functional genomics. However, a major limitation in the field is the lack of a detailed spatio-temporal view of the genetic signatures underpinning the function of distinct tissues and life stages. Here, we present an ontogenetic and tissue-specific web-based resource for Tribolium transcriptomics: BeetleAtlas (https://www.beetleatlas.org). This web application provides access to a database populated with quantitative expression data for nine adult and seven larval tissues, as well as for four embryonic stages of Tribolium. BeetleAtlas allows one to search for individual Tribolium genes to obtain values of both total gene expression and enrichment in different tissues, together with data for individual isoforms. To facilitate cross-species studies, one can also use Drosophila melanogaster gene identifiers to search for related Tribolium genes. For retrieved genes there are options to identify and display the tissue expression of related Tribolium genes or homologous Drosophila genes. Five additional search modes are available to find genes conforming to any of the following criteria: exhibiting high expression in a particular tissue; showing significant differences in expression between larva and adult; having a peak of expression at a specific stage of embryonic development; belonging to a particular functional category; and displaying a pattern of tissue expression similar to that of a query gene. We illustrate how the different feaures of BeetleAtlas can be used to illuminate our understanding of the genetic mechanisms underpinning the biology of what is the largest animal group on earth. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) play a pivotal role in the regulation of insect immunity and growth, making them potential molecular targets for RNA interference (RNAi)-mediated pest control. Although multiple CTLs have been identified in the genomes of various insects, their specific functions and underlying molecular mechanisms remain unclear. In the present study, a novel CTL, Tcctl13 with a single CRD, was identified in Tribolium castaneum. Tcctl13 is expressed in diverse immune-related tissues and developmental stages, with a notable increase in its expression upon exposure to lipopolysaccharides (LPS) and peptidoglycan (PGN). Molecular docking and enzyme-linked immunosorbent assay (ELISA) analyses revealed that TcCTL13 possesses the ability interacted with LPS and PGN. The binding and agglutinating activities of recombinant TcCTL13 (rTcCTL13) were demonstrated against both gram-negative and positive bacteria. After using RNAi to silence Tcctl13 , the expression of the eight antimicrobial peptide (AMP) genes was significantly reduced. In addition, knocking down Tcctl13 during the early larval or pupal stage hindered, the normal metamorphosis process in T. castaneum , ultimately leading to the demise of all beetles. Further research showed that Tcctl13 and nine AMPs were significantly downregulation after 20-Hydroxyecdysone (20E) injection. Instead, the up-regulation of Tcctl13 and six AMPs was observed following interference with the 20E receptor (ecdysone receptor, EcR), indicating that the function of Tcctl13 is regulated by 20E in T. castaneum. Collectively, these findings suggest that Tcctl13 plays a role in the regulation of innate immunity and development in T. castaneum , offering a promising molecular target for managing insect pests using RNAi-based approaches. [Display omitted] • Tcctl13 contributed to T. castaneum 's innate immunity by agglutinating bacteria and regulating the expression of AMPs. • Repressing Tcctl13 hindered normal metamorphosis in T. castaneum , ultimately causing the demise of all beetles. • The function of Tcctl13 was regulated by 20E in T. castaneum. [ABSTRACT FROM AUTHOR]

While pair-rule patterning has been observed in most insects examined, the orthologs of Drosophila pair-rule genes have shown divergent roles in insect segmentation. In the beetle Tribolium castaneum , while odd-skipped (Tc-odd) was expressed as a series of pair-rule stripes, RNAi-mediated knockdown of Tc-odd (Tc-odd RNAi) resulted in severely truncated, almost asegmental phenotypes rather than the classical pair-rule phenotypes observed in germbands and larval cuticles. However, considering that most segments arise later in germband stages of Tribolium development, the roles of Tc-odd in segmentation of growing germbands could not be analyzed properly in the truncated Tc-odd RNAi germbands. Here, we investigated the segmentation function of Tc-odd in germband stages of Tribolium development by analyzing Tc-odd RNAi embryos that resumed germband extension. In the larval cuticles of Tc-odd RNAi embryos, normal mandibular and maxillary and loss of the labial segments were consistent in the head, whereas a broad range of segmentation defects including loss or fusion of thoracic and/or abdominal segments was observed in the trunk. Interestingly, a group of Tc-odd RNAi germbands showed pair-rule-like defects in the segmental stripes of the segment-polarity genes, engrailed , hedgehog , or wingless , in the abdominal regions. While the pair-rule genes even-skipped , runt , odd , and paired were misregulated in the growing Tc-odd RNAi germbands, paired expression required for odd-numbered segment formation was largely abolished, which might cause the pair-rule-like defects. Taken together, these findings suggest that Tc-odd can function as a pair-rule gene in the germband stages of Tribolium development. • Intermediate knockdown of odd causes pair-rule-like defects in the segmental stripes of en / wg in Tribolium germbands. • In odd RNAi germbands, a series of the primary stripes of eve and run arises but does not resolve into secondary stripes. • In odd RNAi germbands, the prd expression required for odd-numbered segment formation is reduced significantly. • Prd expression of Odd might be a key genetic basis of pair-rule patterning in Tribolium. [ABSTRACT FROM AUTHOR]

The pair-rule gene even-skipped (eve) is essential for insect segmentation, yet its function varies among insect clades. While loss of eve results in typical pair-rule phenotypes in Drosophila , knock-down of eve orthologs shows segmental, gap-like, or asegmental phenotypes in non- Drosophila insects. In Tribolium, knock-down of the eve ortholog (Tc-eve) resulted in a graded phenotypic series ranging from strong to weak, the most informative of which was intermediate phenotypes. The strong knock-down embryos displayed asegmental phenotypes and severely disorganized germ bands which have prevented determination of Tc-eve function in later stages. In order to understand the segmentation function of Tc-eve during later germ band elongation stages, we analyzed intermediate Tc-eve RNAi embryos in which germ band elongation was less affected. Most intermediate Tc-eve RNAi germ bands displayed segmentation defects with a double segmental periodicity in the abdomen. In these intermediate embryos, Tc-engrailed (Tc-en) stripes were ectopically expanded into large bands with a double segmental periodicity, while the remaining Tc-en stripes between the expanded Tc-en stripes were absent or barely formed. The expanded Tc-en stripes seemed to be activated by primary Tc-eve stripes and Tc-paired , both of which failed to resolve into secondary segmental stripes. The absence of Tc-en stripes appeared to be a consequence of the absence of the secondary stripes of Tc-runt that were required for the activation of Tc-en stripes. These results suggest that Tc-eve functions as a pair-rule gene at least in the germ band stages of Tribolium development. • Injection of eve dsRNA causes pair-rule-like defects in Tribolium germ bands. • Primary eve stripes form in knockdown embryos but fail to resolve into segmental stripes. • Abnormal eve stripes lead to an expansion of en stripes with a double segmental periodicity. • Secondary stripes of the pair rule gene run are not maintained in the germ bands of knockdown embryos. • Absence of run stripes leads to a reduced number of en stripes. [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]

Unlike vertebrate animals, invertebrates lack lymphocytes and therefore have historically been believed not to develop immune memory. A few studies have reported evidence of immune priming in insects; however, these studies lack the molecular mechanism and proposed it might be different among taxa. Since lncRNAs are known to regulate the immune response, we identified 10,120 lncRNAs in Tribolium castaneum genome-wide followed by transcriptome analysis of primed and unprimed larvae of different infectious status. A shift in lncRNA expression between Btt primed larvae and other treatment groups provides evidence of immune memory response. A few "priming" lncRNAs (n = 9) were uniquely regulated in Btt primed larvae. Evidence suggests these lncRNAs are likely controlling immune priming in Tribolium by regulating expression of genes involved in proteasomal machinery, Notch system, zinc metabolism, and methyltransferase activity, which are necessary to modulate phagocytosis. Our results support a conserved immune priming mechanism in a macrophage-dependent manner. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum , a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus , while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection. [Display omitted] • Microbes remarkably induce TcCTL2 as a PRR to bind to and agglutinate them. • TcCTL2 regulated Toll and IMD signaling pathways and the prophenoloxidase cascade. • TcCTL2 enhanced hemocytic encapsulation, melanization and phagocytosis. [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]

Functions of myosin heavy chain (myosin) family genes are poorly understood in some insects. To address this, we determined the expression and function of myosin family genes in Tribolium castaneum. TcMyo15 is predominantly expressed in early embryos, late larvae and early adults, but TcMyo7B transcripts significantly increased in late larvae. TcMyo20 transcripts are abundant in pre-adults, whereas TcMhc1 is highly expressed in post-embryonic stages. TcMhc2 shows peak expression in late pupae. TcMyo9 transcripts reached their highest levels in late pupae. TcMyo15 , TcMyo7B and TcMhc2 are abundantly expressed in the adult epidermis, gut and testis, respectively. TcMyo9 and TcMyo20 are highly expressed in the epidermis, fat body and ovary, and TcMhc1 exhibits high mRNA levels in the epidermis and accessory gland. TcMyo20 RNAi reduced wing and leg size, fertility and egg hatchability. TcMyo9 knockdown completely inhibited eclosion and fecundity, but TcMyo15 or TcMhc1 silencing only impaired eclosion. TcMhc2 RNAi affected pupation and wing development. This study suggests myosin genes as potential targets of pesticides due to their critical roles in insect development and reproduction. • TcMyo20 regulates wing and leg growth, fertility and egg hatchability in T. castaneum. • Tcmyo15 or Tcmhc1 is required for adult eclosion in T. castaneum. • TcMyo9 has essential roles in eclosion and fecundity. • Knockdown of TcMhc2 impairs pupation and adult wing development. [ABSTRACT FROM AUTHOR]

Abstract UV-B radiation is an important environmental factor. Exposure to excess UV-B radiation can cause serious effects on the development, survival, and reproduction of different organisms. Plants and animals have developed many different strategies to cope with UV-B-induced damage, but the physiological response of insects to UV-B remains unclear. In the present study, the red flour beetle Tribolium castaneum (Herbst) was used to assess the stress response of UV-B. The underlying molecular mechanisms were explored using RNA sequencing. We investigated the transcriptomic profile of T. castaneum larvae at 4 and 24 h after treatment with UV-B radiation via digital gene expression analysis. The 310 and 996 differentially expressed genes were detected at 4 and 24 h, respectively. Then the biological functions and associated metabolic processes of these genes were determined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The reliability of the data was verified using qRT-PCR. The results indicated that several differentially expressed genes are involved in antioxidation, DNA repair, protein folding, carbon flux diversion, and the extracellular matrix to protect against UV-B-induced damage. This study will increase our understanding of the molecular mechanism underlying insect response to UV-B radiation. Graphical abstract fx1 Highlights • Transcriptome responses of Tribolium castaneum larvae exposed to UV-B were examined. • The data provided new evidence for the physiological response to radiation in insects. • The study illustrated the defense strategy against UV-B in Tribolium castaneum. • The data provided new evidence of physiological response to radiation in insects. [ABSTRACT FROM AUTHOR]

Abstract Many organisms both undergo dramatic morphological changes during post-embryonic development and also regenerate lost structures, but the roles of epigenetic regulators in such processes are only beginning to be understood. In the present study, the functions of two histone modifiers were examined during metamorphosis and larval limb regeneration in the red flour beetle Tribolium castaneum. Polycomb (Pc), a member of Polycomb repressive complex 1 (PRC1), and Enhancer of zeste (E(z)), a member of Polycomb repressive complex 2 (PRC2), were silenced in larvae using RNA interference. In the absence of Pc, the head appendages of adults transformed into a leg-like morphology, and the legs and wings assumed a metathoracic identity, indicating that Pc acts to specify proper segmental identity. Similarly, silencing of E(z) led to homeotic transformation of legs and wings. Additional defects were also observed in limb patterning as well as eye morphogenesis, indicating that PcG proteins play critical roles in imaginal precursor cells. In addition, larval legs and antennae failed to re-differentiate when either Pc or E(z) was knocked down, indicating that histone modification is necessary for proper blastema growth and differentiation. These findings indicate that PcG proteins play extensive roles in postembryonic plasticity of imaginal precursor cells. Graphical abstract Image 1 Highlights • Knockdowns of Pc and E(z) lead to homeotic transformations during metamorphosis. • Pc and E(z) are necessary for re-differentiation of larval legs and antennae. • PcG proteins play extensive roles in postembryonic plasticity of imaginal cells. [ABSTRACT FROM AUTHOR]

Abstract In this work, the essential oils (EO) were extracted from seven typical Chinese herbs, and their repellent and contact toxicities against Tribolium castaneum adults (red flour beetles) were evaluated. The experimental results showed that the above EOs presented the various levels of repellent and contact toxicities. The EOs extracted from A. lancea and A argyi of the Compositae (Asteraceae) family presented obvious repellent effects (Repellency Percentage > 90% at 3.15 nL/cm2 after 4 h exposure) and strong contact toxicity with LD 50 values of 5.78 and 3.09 μg/adult respectively. Based on literature researches and screening results, the EO from A. lancea was analyzed by GC-MS and chosen for further identification of bioactive components. Altogether 59 chemical components were identified and 17 of them were recognized as sesquiterpene compounds, accounting for 57.8% of the total weight of the EO. From the identified sesquiterpenes, three individual compounds (β -eudesmol, hinesol, valencene) were selected for the laboratory bioassays of the toxicity against red flour beetles. It was found that all the three compounds expressed some repellent effects. Although β -eudesmol (31.2%) and hinesol (5.1%) were identified as main constituents and had been considered to be symbolic characteristics of high medicinal value, valencene (0.3%) showed strong repellent property which could be comparable to that of DEET (N, N‑diethyl‑3‑methylbenzamide), a powerful commercial pesticides, and it had best toxicity with LD 50 values of 3.25 (μg/adult) in the contact test. This work may provide toxicity evidence of seven common herbs against red flour beetles, add the information for the development and comprehensive utilization of A. lancea , and will contribute to the application of grain preservation. Graphical abstract fx1 Highlights • Seven EOs from common herbs were found to be potential resource to prevent Tribolium castaneum. • Atractylodes argyi and A. lancea (Compositae family) had high repellent and contact effects. • The toxicity of the three sesquiterpenoids from A. lancea were evaluated. • A micro active substance called valencene from A. lancea were found. [ABSTRACT FROM AUTHOR]

There has been a considerable growth in interest to use RNA interference (RNAi) as a novel insect pest management strategy in the past 10 years. However, there has been virtually no information on insect population variations in response to double-stranded RNA (dsRNA) molecules. The objective of this study was to generate baseline susceptibilities of the red flour beetle ( Tribolium castaneum ) to an insecticidal dsRNA targeting vacuolar H + -ATPase subunit E gene (ds vATPaseE ), and correlate the susceptibility data with sequence and expression variations of the target gene ( vATPaseE ), expression variations of the RNAi core genes, and overall genetic differences among a laboratory strain and seven geographical field populations of T. castaneum collected in China. Our results showed limited variations in the LD 50 values of ds vATPaseE , which ranged from 0.10 to 0.29 ng/larva among the laboratory strain and the seven field populations. Considering the overlapping of the 95% confidence intervals of their LD 50 values, there were no significant differences among the laboratory strain and field populations. We also found limited sequence polymorphisms and low frequencies of the polymorphisms of vATPaseE , and limited variations (<2-fold) of the endogenous expression of vATPaseE among the laboratory strain and field populations. However, we found considerable genetic variations among the individuals within each field population for most of eight loci and moderate to large genetic variations among the field populations. These results demonstrated that although the genetic variabilities were considerable among these field populations, the efficiency of RNAi targeting vATPaseE was highly consistent in T. castaneum . Our study provides work frames of resistance risk assessment for RNAi-based insect pest management programs. [ABSTRACT FROM AUTHOR]

The existence of widespread male same-sex sexual behaviour (SSB) is puzzling: why does evolution allow costly homosexual activity to exist, when reproductive fitness is primarily achieved through heterosexual matings? Here, we used experimental evolution to understand why SSB occurs in the flour beetle Tribolium castaneum . By varying the adult operational sex ratio across 82–106 generations, we created divergent evolutionary regimes that selected for or against SSB depending upon its function. Male-biased (90:10 M:F) regimes generated strong selection on males from intrasexual competition, and demanded improved ability to locate and identify female mates. By contrast, Female-biased regimes (10:90 M:F) generated weak male–male competition, and relaxed selection on mate-searching abilities in males. If male SSB functions through sexually selected male–male competition, it should be more evident within Male-biased regimes, where reproductive competition is nine times greater, than in the Female-biased regimes. By contrast, if SSB exists due to inaccurate mate choice, it should be reduced in Male-biased regimes, where males experience stronger selection for improved mate finding and discrimination abilities than in the Female-biased regime, where most potential mating targets are female. Following these divergent evolutionary regimes, we measured male engagement in SSB through choice experiments simultaneously presenting female and male mating targets. Males from both regimes showed similar overall levels of mating activity. However, there were significant differences in levels of SSB between the two regimes: males that evolved through male-biased operational sex ratios located, mounted and mated more frequently with the female targets. By contrast, males from female-biased selection histories mated less frequently with females, exhibiting almost random choice between male and female targets in their first mating attempt. Following experimental evolution, we therefore conclude that SSB does not function through sexually selected male–male competition, but instead occurs because males fail to perfectly discriminate females as mates. [ABSTRACT FROM AUTHOR]

Tribolium castaneum (Herbst) is a worldwide grain storage pest controlled by chemical control methods of phosphine fumigation, which results in many hazards, damages human health, makes pests resistant to pesticides, and pollutes the environment. In recent years, the popularity of botanical insecticides has continued to rise, and plant essential oils (EO) are considered potential alternatives for developing insecticides. In the current study, we selected the Chamaecyparis obtusa EO to determine its insecticidal effects and component analysis on T. castaneum. Through gas chromatography-ion mobility spectrometry (GC-IMS) technology, cedrol was the most obvious compound in the signal peak of the volatile components detected in the C. obtusa EO. The results of the bioassay showed that the C. obtusa EO had certain contact activity against T. castaneum , and the LD 50 was 52.54 μg/adult. At three concentrations (0.41,1.62, 2.83 uL/cm2), the repellent rates of C. obtusa EO agains t T. castaneum were all above 80% at 15, 30, 60, and 120 min, respectively, indicating that the repellent effect was strong. Meanwhile, the C. obtusa EO exhibited fumigant toxicity against T. castaneum with LC 50 values of 7.09 μg/L air. In addition, C. obtusa EO significantly increased the activity of AChE, CarE, POD, CAT, T-SOD, and chitinase in T. castaneum. Finally, the mechanism of C. obtusa EO on T. castaneum adults was explored based on transcriptome sequencing. We found that the DEGs focused on the chitin metabolic process and some aging genes in T. castaneum. Therefore, C. obtusa EO could be used as potential eco-friendly candidates for stored grain pest management. A simple model showing that C. obtusa EO control T. castaneum. C. obtusa EO had obvious repellent activity, fumigant toxicity and contact activity against T.castaneum. On the other hand, the main volatile component of C. obtusa EO were analyzed though GC-IMS. In addition, C. obtusa EO treatment significantly increased AChE, CarE, POD, CAT, T-SOD, and chitinase activities in T.castaneum. Moreover, GO enrichment analysis showed that the DEGs were primarily focused on growth of insect cuticle and aging genes in T. castaneum. [Display omitted] • The insecticidal activity of C. obtusa EO against T. castaneum is notable. • C. obtusa EO induced the increase of T.castaneum enzyme activity. • T. castaneum CP gene upregulated by C. obtusa EO treatment. • A reference for the study of the insecticidal mechanism of C. obtusa EO [ABSTRACT FROM AUTHOR]

We previously reported that the moderate knockdown of chitin synthase 1 gene of the model beetle Tribolium castaneum impairs the host defense against entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae , which infect host insects via the direct penetration of cuticular integuments (Hayakawa et al., 2017). In this study, we focused on the antifungal roles of laccase2 ( Lac2 ) as well as yellow-e ( Y-e ) genes, both of which are shown to be important to the establishment of stable cuticular structures in this beetle species. The expression profiles of the two genes somewhat resembled each other, peaking in late prepupae and mid to late pupae, while the transcript levels of Lac2 were higher than Y-e throughout. The knockdown of Lac2 gene at the prepupal and pupal peaks with relatively small amounts of dsRNA resulted in pupae with a lighter color and adults with a lighter color and dimpled/wrinkled elytra, respectively. Meanwhile, similar gene knockdown of Y-e but with 10 times more dsRNA compared to Lac2 resulted in pupae having a normal appearance and adults with a darker color. We conducted fungal infection assays with B. bassiana and M. anisopliae using these knockdown animals. The knockdown of Y-e gene had no or limited effects in both pupae and adults in terms of the antifungal host defense. Similarly, the knockdown of Lac2 gene did not change significantly the defense phenotypes of the resulting pupae. By sharp contrast, the host defense of the adult beetles against the two fungal species was almost totally destroyed by the moderate knockdown of Lac2 gene, suggesting its indispensable role in antifungal host defense presumably through the construction of sound cuticles of the adults. Finally, we investigated the maturation of host defense against fungal infection in the Lac2 knockdown adults and found that while the day 10 adults were still susceptible to M. anisopliae infection with some delay of death in comparison with day 1 adults, they exhibited complete refractoriness to B. bassiana . [ABSTRACT FROM AUTHOR]

To date, although some microRNAs (miRNAs) have been discovered in the holometabolism insect Tribolium castaneum , large numbers of miRNAs still require investigation. Knocking down Dicer-1 ( Dcr-1 ) and Argonaute-1 ( Ago-1 ) in late larvae impaired miRNA synthesis, affected the juvenile hormone pathway by up-regulating Methoprene-tolerant ( Met ) and Krüppel-homolog1 ( Kr-h1 ) transcript levels, and resulted in a series of defects in T. castaneum development and metamorphosis. Thus, high-throughput Illumina/Solexa sequencing was performed with a mixed sample of eight key developmental stages of T. castaneum . In total, 1154 unique miRNAs were discovered containing 274 conserved miRNAs belong to 68 miRNA families, 108 known candidate miRNAs and 772 novel miRNAs. Genome locus analysis showed that miRNA clusters are more abundant in T. castaneum than other species. The results indicated that RNAi of Dcr-1 and Ago-1 in T. castaneum resulted in miRNA-induced metamorphosis defects. Furthermore, large numbers of novel miRNAs were discovered in T. castaneum and localized to T. castaneum genome loci. [ABSTRACT FROM AUTHOR]

Phosphine is the dominant chemical used in postharvest pest control. Widespread and highly frequent use of phosphine has been selected for pest insects, including Tribolium castaneum , which is highly resistant. Lipid peroxidation and reactive oxygen species (ROS) are two major factors determining phosphine toxicity; however, the mechanisms of production of these two factors in phosphine toxicity are still unknown. Here, we first determined the time course of phosphine-induced lipid peroxidation and ROS production in T. castaneum. Our results showed that lipid peroxidation occurs before ROS in the process of phosphine toxicity, and fumigated beetles with higher resistance levels were associated with weaker activity on lipid peroxidation and ROS. A significant decline in lipid peroxidation was observed in fumigated individuals after knockdown of cytochrome b5 fatty acid desaturase (Cyt-b5-r) via RNA interference (RNAi), indicating that Cyt-b5-r is critical for triggering phosphine-induced lipid peroxidation. Moreover, significant decreases in both ROS and mortality were detected in fumigated T. castaneum adults fed melatonin for 7 days, an inhibitor of lipid peroxidation. Cyt-b5-r RNAi also inhibited ROS production and mortality in phosphine-treated beetles. Meanwhile, a significant decrease in ROS production (68.4%) was detected in dihydrolipoamide dehydrogenase (DLD) knockdown individuals with phenotypes susceptible to phosphine, suggesting that lipid peroxidation initiates ROS with the expression of DLD. However, a significant increase in ROS (122.1%) was detected in the DLD knockdown beetles with strongly resistant phenotypes, indicating that the DLD -involved pathway may not be the only mechanism of ROS generation in phosphine toxicity and the existence of a moonlighting role in downregulating ROS in strongly resistant T. castaneum. [Display omitted] • Lipid peroxidation happens before ROS in phosphine-fumigated T. castaneum. • Cyt-b5-r is critical to trigger phosphine-induced lipid peroxidation. • Lipid peroxidation initials ROS with the expression of DLD in susceptible beetles. • DLD may downregulate ROS in strongly phosphine-resistant individuals. [ABSTRACT FROM AUTHOR]

The importance of the insect cuticle as a primary protective barrier against entomopathogens has long been noted. In the present study, we addressed this issue by utilizing an experimental infection system composed of the model beetle T. castaneum and two entomopathogenic fungal species, Beauveria bassiana and Metarhizium anisopliae . The pupae were relatively susceptible to these fungi by the natural route of infection, with some refractoriness developed with age, while the adults exhibited much higher refractoriness. Whereas M. anisopliae exhibited seemingly higher infectivity to the pupae compared to B. bassiana when the natural conidium infection was employed, direct inoculation of cultured hyphal body cells into the hemocoel was found highly and equally virulent in the pupae for the both fungal species. These results collectively suggest an important role of the cuticular integument in antifungal host defense, and we subsequently conducted the knockdown of chitin synthase 1 gene ( CHS1 ). We targeted the prepupal and mid-pupal peaks of its expression respectively by using injection of the dsRNA at very low dosages to avoid lethality. The resulting pupae looked normal, but the adults showed a mild phenotype with dimpled/wrinkled elytra. The CHS1 gene knockdown compromised significantly host defense against the fungal infection via the natural route, except the configuration of knockdown pupae and M. anisopliae , suggesting an indispensable role of CHS1 . [ABSTRACT FROM AUTHOR]

Transitioning between life stages involves significant changes to the physiology, structural morphology, biochemistry and behaviour of an organism. Eclosion, metamorphosis and the onset of sexual maturity have consequences for the life history evolution of an organism by initiating reproductive and dispersalrelated behaviours that are both energetically costly and directly related to fitness. Animal movement, particularly dispersal when sexually mature, is critical for mate location, controlling population density and promoting gene flow. Here we examined changes in dispersal-related and physiological traits during a significant transitional period in red flour beetles, Tribolium castaneum. We measured the metabolic rate, spontaneous activity, body size and movement behaviour traits of individuals of known age and sex. Traits were compared between immature and sexually mature adults as well as during early adult life when there is a strong tendency to disperse and reproduce. Spontaneous activity, movement speed and metabolic rate were distinctly reduced in immature adults prior to the onset of sexual maturity, and immature individuals moved more intermittently than mature ones. We found that these traits increased with age following eclosion, up to a relatively stable mean once sexual maturity was attained. The reduced metabolic expenditure found in immature individuals was attributable to a reduced energy demand due to relative inactivity, which we hypothesize to be a protective mechanism from conspecifics while the cuticle is undergoing sclerotization. Understanding the precise developmental trajectories of behavioural and physiological traits allows us to interpret the trait syndromes that underlie dispersal and their evolution. [ABSTRACT FROM AUTHOR]

We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle’s pathways have broader specificity in terms of microbial activation than that of Drosophila . To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part. [ABSTRACT FROM AUTHOR]

In this study, we characterized prophenoloxidase (proPO, (PPO)) genes of Tribolium castaneum and examined their involvement in antimicrobial host defense. Amino acid sequence comparison with well-characterized PPO proteins from other insect species suggested that T. castaneum PPO genes encoded functional proenzymes, with crucial sequence motifs being conserved. Developmental kinetics of the mRNA of two PPO genes, PPO1 and PPO2 in the pupal stage were different to each other. The PPO1 mRNA levels consistently decreased during pupal development while that of PPO2 peaked at mid-pupal stage. The two mRNAs also exhibited distinct responses upon immune challenges with heat-killed model microbes. The PPO1 mRNA stayed nearly unchanged by 6 h post challenge, and was somewhat elevated at 24 h. In contrast, the PPO2 mRNA significantly decreased at 3, 6 and 24 h post challenge. These trends exhibited by respective PPO genes were consistent irrespective of the microbial species used as elicitors. Finally, we investigated the involvement of T. castaneum PPO genes in antimicrobial host defense by utilizing RNA interference-mediated gene silencing. Survival assays demonstrated that double knockdown of PPO genes, which was accompanied by weakened hemolymph PO activities, significantly impaired the host defense against Bacillus subtilis . By contrast, the knockdown did not influence the induction of any of the T. castaneum antimicrobial peptide genes that were studied here, except for one belonging to the gene group that shows very weak or negligible microbial induction. PPO knockdown as well weakened host defense against Beauveria bassiana moderately but significantly depending on the combination of infection methods and targeted genes. Our results indicated that the PPO genes represented constituents of both antibacterial and antifungal host defense of T. castaneum . [ABSTRACT FROM AUTHOR]

The red flour beetle Tribolium castaneum is a destructive insect pest of stored food and feed products, and a model organism for development, evolutionary biology and immunity. The insect innate immune system includes antimicrobial peptides (AMPs) with a wide spectrum of targets including viruses, bacteria, fungi and parasites. Defensins are an evolutionarily-conserved class of AMPs and a potential new source of antimicrobial agents. In this context, we report the antimicrobial activity, phylogenetic and structural properties of three T. castaneum defensins (Def1, Def2 and Def3) and their relevance in the immunity of T. castaneum against bacterial pathogens. All three recombinant defensins showed bactericidal activity against Micrococcus luteus and Bacillus thuringiensis serovar tolworthi , but only Def1 and Def2 showed a bacteriostatic effect against Staphylococcus epidermidis . None of the defensins showed activity against the Gram-negative bacteria Escherichia coli and Pseudomonas entomophila or against the yeast Saccharomyces cerevisiae . All three defensins were transcriptionally upregulated following a bacterial challenge, suggesting a key role in the immunity of T. castaneum against bacterial pathogens. Phylogenetic analysis showed that defensins from T. castaneum , mealworms, Udo longhorn beetle and houseflies cluster within a well-defined clade of insect defensins. We conclude that T. castaneum defensins are primarily active against Gram-positive bacteria and that other AMPs may play a more prominent role against Gram-negative species. [ABSTRACT FROM AUTHOR]

The evolution of imaginal cells, or stem cell-like cells, contributed to the spectacular diversification of holometabolous insects, which undergo complete metamorphosis. The proliferation and differentiation of these imaginal cells is under the control of juvenile hormone (JH), but which patterning genes respond to JH is currently unknown. Here, the role of Hedgehog (Hh) signaling in the development of imaginal cells was investigated. RNA interference-mediated knockdown of the components of the Hh signaling pathway showed that Hh is required for the proliferation of polymorphic and imaginal cells in Tribolium castaneum . Hh was also necessary for the regeneration of larval appendages. In contrast, knockdown of Hh signaling antagonists, patched and costal 2 led to the overgrowth and precocious maturation of structures derived from imaginal cells and the occasional appearance of ectopic appendages from the head epidermis. In addition, JH suppressed the expression of hh both in vivo and in vitro . Our findings suggest that imaginal cells are created and maintained by modulating Hh signaling. Thus, Hh signaling may have played a critical role during the evolution of complete metamorphosis. [ABSTRACT FROM AUTHOR]

Yellow protein (dopachrome conversion enzyme, DCE) is involved in the melanin biosynthetic pathway that significantly accelerates pigmentation reactions in insects. Recent studies have suggested that the insect yellow genes represent a rapidly evolving gene family generating functionally diverse paralogs, but the exact physiological functions of several yellow genes are still not understood. To study the function(s) of one of the yellow genes, yellow-e ( TcY-e ), in the red flour beetle, Tribolium castaneum , we performed real-time PCR to analyze its developmental and tissue-specific expression, and utilized immunohistochemistry to identify the localization of the TcY-e protein in adult cuticle. Injection of double-stranded RNA for TcY-e (ds TcY-e ) into late instar larvae had no effect on larval–pupal molting or pupal development. The pupal cuticle, including that lining the setae, gin traps and urogomphi, underwent normal tanning. Adult cuticle tanning including that of the head, mandibles and legs viewed through the translucent pupal cuticle was initiated on schedule (pupal days 4–5), indicating that TcY-e is not required for pupal or pharate adult cuticle pigmentation in T. castaneum . The subsequent pupal–adult molt, however, was adversely affected. Although pupal cuticle apolysis and slippage were evident, some of the adults (~25%) were unable to shed their exuvium and died entrapped in their pupal cuticle. In addition, the resulting adults rapidly became highly desiccated. Interestingly, both the failure of the pupal–adult molt and desiccation-induced mortality were prevented by maintaining the ds TcY-e -treated insects at 100% relative humidity (rh). However, when the high humidity-rescued adults were removed from 100% rh and transferred to 50% rh, they rapidly dehydrated and died, whereas untreated beetles thrived throughout development at 50% rh. We also observed that the body color of the high humidity-rescued ds TcY-e -adults was slightly darker than that of control animals. These results support the hypothesis that TcY-e has a role not only in normal body pigmentation in T. castaneum adults but also has a vital waterproofing function. [ABSTRACT FROM AUTHOR]

Resistance of Tribolium castaneum to phosphine is related to point mutations in DNA code corresponding to amino acid changes associated with a core metabolic enzyme dihydrolipoamide dehydrogenase (DLD), but the mutation patterns vary among different resistant populations. Thus, there is a great need to develop a cost-effective method to detect core mutations in T. castaneum , which would be the key factor to understand the molecular basis of phosphine resistance. Amplification refractory mutation system-based quantitative Real-Time PCR (ARMS-qPCR) is an ideal method that can rapidly detect point mutations. Here, the P45S and G131D mutations existed in the DLD of T. castaneum selected from strong Chinese resistance phenotypes, and the DLD P45S mutation, which represents a strong phosphine resistance allele, was confirmed as the most abundant mutation to determine strong resistance genotypes. Our study found that 85 out of 120 beetles carried the P45S resistance allele, including 51 homozygous and 34 heterozygous individuals. Moreover, there was a strong linear relationship (R2 = 0.917) between the resistance ratio and the resistance allele frequency among the strongly resistant populations. Our data showed that the ARMS-qPCR method that we developed could rapidly determine strong resistance phenotypes of T. castaneum to phosphine by detecting the DLD P45S mutation. These results not only provide a detailed example for developing an ARMS-qPCR-based method to characterize pesticide resistance, but also support further elucidation of the molecular basis of phosphine resistance. [Display omitted] • The variants of DLD were identified firstly in Chinese T. castaneum populations. • The P45S was the ubiquitous mutation in strongly phosphine-resistant populations. • An ARMS-qPCR method based on P45S can rapidly characterize phosphine resistance. • The phosphine resistance of nine Chinese T. castaneum populations was investigated. [ABSTRACT FROM AUTHOR]

Many cases of insecticide resistance in insect pests give resulting no-cost strains that retain the resistance genes even in the absence of the toxic stressor. Malathion (rac- diethyl 2-[(dimethoxyphosphorothioyl)sulfanyl]succinate) has been widely used against the red flour beetle, Tribolium castaneum Herbst. in stored products although no longer used. Malathion specific resistance in this pest is long lasting and widely distributed. A malathion resistant strain was challenged with a range of stressors including starvation, hyperoxia, malathion and a pathogen to determine the antioxidant responses and changes to some lifecycle parameters. Adult life span of the malathion-specific resistant strain of T. castaneum was significantly shorter than that of the susceptible. Starvation and/or high oxygen reduced adult life span of both strains. Starving, with and without 100% oxygen, gave longer lifespan for the resistant strain, but for oxygen alone there was a small extension. Under oxygen the proportional survival of the resistant strain to the adult stage was significantly higher, for both larvae and pupae, than the susceptible. The resistant strain when stressed with malathion and oxygen significantly increased catalase activity, but the susceptible did not. The resistant strain stressed with Paranosema whitei infection had significantly higher survival compared to the susceptible, and with low mortality. The malathion resistant strain of T. castaneum showed greater vigour than the susceptible in oxidative stress situations and especially where stressors were combined. The induction of the antioxidant enzyme catalase could have helped the resistant strain to withstand oxidative stresses, including insecticidal and importantly those from pathogens. These adaptations, in the absence of insecticide, seem to support the increased immunity of the insecticide resistant host to pathogens seen in other insect species, such as mosquitoes. By increasing the responses to a range of stressors the resistant strain could be considered as having enhanced fitness, compared to the susceptible. [Display omitted] • Adult longevity of malathion-specific resistant strain shorter than the susceptible. • Starvation and/or 100% oxygen reduced adult life span of both strains, less for the resistant. • Under oxygen the survival of the resistant strain to the adult stage was significantly higher. • Resistant strain stressed with malathion and oxygen induced catalase, but not the susceptible. • Resistant strain stressed with Paranosema whitei infection had low mortality. [ABSTRACT FROM AUTHOR]

Highlights: [•] Bt challenge induced ApoLp-III mRNA up-regulation in Tc larvae. [•] ApoLp-III is involved in Tc defense against Bt. [•] ApoLp-III activates the prophenoloxidase cascade in Tc immune response to Bt. [ABSTRACT FROM AUTHOR]

Highlights: [•] Sulfakinin and sulfakinin receptor genes of Tribolium castaneum were cloned and proteins conserved in their active regions. [•] qPCR demonstrated expression in over different developmental stages and high expression in the head. [•] RNAi experiments with dssk and dsskr caused an increase of food intake. [•] Injection of a SK analog in larval stages reduced food intake. [ABSTRACT FROM AUTHOR]

Abstract: The flour beetle, Tribolium castaneum, is an intermediate host for the tapeworm Hymenolepis diminuta and has become an important genetic model to explore immune responses to parasite infection in insect hosts. The present study examined the immune responses to tapeworm infection in resistant (TIW1) and susceptible (cSM) strains of the red flour beetle, T. castaneum, using real-time quantitative reverse transcription PCR on 29 immunity-related genes that exhibit antimicrobial properties. Thirteen of the 29 genes showed constitutive differences in expression between the two strains. Fourteen to fifteen of the 29 genes exhibited significant differences in transcription levels when beetles were challenged with tapeworm parasite in the resistant and susceptible strains. Nine genes (GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2 and serpin29) in cSM and 13 genes (lysozyme2, proPO1, GNBP3, cSPH2, lysozyme4, defensin1, PGRP-SA, defensin2, coleoptericin1, attacin2, proPO2/3, PGRP-LE and PGRP-SB) in TIW1 were up-regulated by infections or showed parasite infection-induced expression. Seven genes (attacin2, coleoptericin1, defensin1, defensin2, lysozyme2, PGRP-SA and PGRP-SB) were more than 10 folds higher in the resistant TIW1 strain than in the susceptible cSM strain after exposure to tapeworm parasites. This study demonstrated the effects of genetic background, the transcription profile to parasite infection, and identified the immunity-related genes that were significantly regulated by the infection of tapeworms in Tribolium beetles. [Copyright &y& Elsevier]

Abstract: Glutathione S-transferases are important detoxification enzymes involved in insecticide resistance. Sequencing the Tribolium castaneum genome provides an opportunity to investigate the structure, function, and evolution of GSTs on a genome-wide scale. Thirty-six putative cytosolic GSTs and 5 microsomal GSTs have been identified in T. castaneum. Furthermore, 40, 35, 13, 23, and 32 GSTs have been discovered the other insects, Drosophila, Anopheles, Apis, Bombyx, and Acyrthosiphon, respectively. Phylogenetic analyses reveal that insect-specific GSTs, Epsilon and Delta, are the largest species-specific expanded GSTs. In T. castaneum, most GSTs are tandemly arranged in three chromosomes. Particularly, Epsilon GSTs have an inverted long-fragment duplication in the genome. Other four widely distributed classes are highly conserved in all species. Given that GSTs specially expanded in Tribolium castaneum, these genes might help to resist poisonous chemical environments and produce resistance to kinds of different insecticides. [Copyright &y& Elsevier]

Abstract: The evolution of a mechanism to generate a proximal–distal axis perpendicular to the anterior–posterior body axis was a key event in arthropod evolution and facilitated the formation of multi-functional limbs. The study of proximodistal limb patterning in extant arthropods can provide insight into the origin and evolution of the proximal–distal axis. In Drosophila melanogaster, proximal–distal patterning is mainly organized by Wg/Dpp signaling. Egfr signaling is also involved, but is restricted to late stages and distal leg parts (tarsus and pretarsus). Here we study the role of Epidermal growth factor receptor (Egfr), spitz (spi), and pointed (pnt) in leg development in the beetle Tribolium castaneum. We show that Egfr signaling has a more complex role in T. castaneum than in D. melanogaster and is not only required in the distal leg, but is also involved in formation of the medial leg (including femur and tibia). Egfr and spi are required for the regulation of clawless (cll), Distal-less (Dll) and dachshund (dac), and RNAi leads to thickened and fused leg segments. Intriguingly, regulation of Dll and dac by Egfr signaling appears functionally separate from its role in cll regulation, because it is not mediated by the transcription factor Pnt. This suggests that Egfr signaling has a dual role with separate mediators in proximodistal axis patterning. While the regulation of distal factors like cll is evolutionarily conserved, regulation of Dll and dac appears to function in parallel or redundant with Wg/Dpp signaling, thus providing a possible explanation why this role is less evolutionarily conserved in the insects. [Copyright &y& Elsevier]

Abstract: The α-amylase inhibitor from corms of Colocasia collected from Bhota village of Hamirpur district, Himachal Pradesh was purified to 17.21 folds with 61.61% recovery using ammonium sulfate precipitation, gel filtration chromatography (sephadex G-200) and ion exchange chromatography (DEAE-sephadex). A single band of the purified inhibitor was obtained by Native-PAGE. SDS–PAGE revealed the purified inhibitor to be a monomer with molecular weight of 13,900 daltons. The nature of inhibition was found to be of non-competitive type as determined by Lineweaver–Burk plot and a K i value of 0.54nmole was obtained by Dixon’s plot. The inhibitor was found to be heat stable and retained 81.50% activity at 70°C temperature. Inhibitor was found to have pH optima of 6.9. The purified inhibitor was found to have inhibitory activity against α-amylases extracted from the larvae of Callosobruchus chinensis, Tribolium castaneum, Corcyra cephalonica and midgut α-amylase of Spodoptera littoralis. 100% larval mortality of C. cephalonica was observed when fed on wheat flour mixed with 0.0036% (w/w) of purified inhibitor. Purified α-amylase inhibitor was found to inhibit the activity of human salivary α-amylase. It also had inhibitory activity against potato α-amylases and reduced sugar content in treated potato slices. The purified inhibitor was found to be a glycoprotein. In the present study, the ability of the inhibitor to inhibit insect amylases highlights its possible role in pest resistance and post harvest decay of crop plants. Inhibitory activity of α-amylase inhibitor against mammalian amylases could suggest its potential in treatment of diabetes and cure of nutritional problems, which result in obesity. [Copyright &y& Elsevier]

Abstract: A viral histone H4 is encoded in a polydnavirus called Cotesia plutellae bracovirus (CpBV), which is symbiotic to an endoparasitoid wasp, C. plutellae. Compared to general histone H4s, the viral H4 possesses an extra N-terminal tail containing 38 amino acid residues, which has been presumed to control host gene expression in an epigenetic mode. To analyze the epigenetic control activity of CpBV-H4 on expression of immune-associated genes, it was transiently expressed in larvae of Tribolium castaneum that had been annotated in the immune genes from a full genome sequence. Subsequent alteration of gene expression pattern was compared with that of its mutant form deleting N-terminal tail (truncated CpBV-H4). In response to bacterial challenge, T. castaneum induces expression of 13 antimicrobial peptide (AMP) genes. When CpBV-H4 was expressed, the larvae failed to express 12 inducible AMP genes. By contrast, when truncated CpBV-H4 was transiently expressed, all AMP genes were expressed. Hemocyte nodule formation was significantly impaired by expression of CpBV-H4, in which expressions of tyrosine hydroxylase and dihydroxyphenylalanine decarboxylase were suppressed. However, expression of truncated CpBV-H4 did not give any significant adverse effect on the cellular immunity. The immunosuppression of CpBV-H4 was further supported by its activity of enhancing bacterial pathogenicity of an entomopathogenic bacterium, Xenorhabdus nematophila, against larvae transiently expressing CpBV-H4. These results suggest that CpBV-H4 suppresses both humoral and cellular immune responses of T. castaneum by altering a normal epigenetic control of immune-associated gene expression. [Copyright &y& Elsevier]

Abstract: In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning. [ABSTRACT FROM AUTHOR]

Abstract: The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as “tritocerebral segment.” In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment. [ABSTRACT FROM AUTHOR]

Abstract: Bursicon is an insect neuropeptide hormone that is secreted from the central nervous system into the hemolymph and initiates cuticle tanning. The receptor for bursicon is encoded by the rickets (rk) gene and belongs to the G protein-coupled receptor (GPCR) superfamily. The bursicon and its receptor regulate cuticle tanning as well as wing expansion after adult eclosion. However, the molecular action of bursicon signaling remains unclear. We utilized RNA interference (RNAi) and microarray to study the function of the bursicon receptor (Tcrk) in the model insect, Tribolium castaneum. The data included here showed that in addition to cuticle tanning and wing expansion reported previously, Tcrk is also required for development and expansion of integumentary structures and adult eclosion. Using custom microarrays, we identified 24 genes that are differentially expressed between Tcrk RNAi and control insects. Knockdown in the expression of one of these genes, TC004091, resulted in the arrest of adult eclosion. Identification of genes that are involved in bursicon receptor mediated biological processes will provide tools for future studies on mechanisms of bursicon action. [ABSTRACT FROM AUTHOR]

Metabolism of insecticides is an energy-consuming process. As an important component of the intracellular energy buffering system, arginine kinase (AK) plays an important role in insect cellular energy homeostasis and environmental stress response, but the involvement of AKs in the response to chemical stressors (insecticides) remains largely unknown. In this study, using Tribolium castaneum as a model organism, we found that deltamethrin treatment significantly upregulated the expression of TcAK1 and TcAK2 and decreased the whole body ATP content. The knockdown of TcAK1 or TcAK2 significantly enhances the deltamethrin-induced ATP depletion and increase the susceptibility of T. castaneum to deltamethrin. In addition, pretreatment with two AK inhibitors, rutin and quercetin, significantly decreased the lifespan of beetles treated with deltamethrin. These results suggest that AKs might be involved in detoxification of insecticides by regulating cellular energy balance. [Display omitted] • Exposure of Tribolium castaneum to deltamethrin led to ATP depletion and upregulation of TcAK1 and TcAK2. • RNAi of TcAK1 and TcAK2 increased the susceptibility of T. castaneum to deltamethrin. • Rutin and quercetin act as synergists to deltamethrin in T. castaneum. [ABSTRACT FROM AUTHOR]

Tribolium castaneum is an agricultural and stored pest found throughout the world. The cytochrome P450 genes of T. castaneum can encode various detoxification enzymes and catabolize heterologous substances, conferring tolerance to insecticides. Herein, we describe the identification of a P450 gene (CYP9Z6) from T. castaneum and investigated its expression profile and potential role in the detoxification of terpinen-4-ol. TcCYP9Z6 expression was significantly induced after exposure to terpinen-4-ol, and RNA-mediated silencing of TcCYP9Z6 increased terpinen-4-ol-induced larval mortality from 47.75% to 63.92%, showing that TcCYP9Z6 is closely related to the detoxification of terpinen-4-ol. The developmental expression profile revealed that TcCYP9Z6 was mainly expressed in late adults and late larvae. Tissue expression profiling revealed that the highest TcCYP9Z6 expression occurred in the head, in both the adult and the larval tissues, followed by the gut in larvae and the antennae in adults. These developmental stages and tissues with high TcCYP9Z6 expression are closely related to the detoxification of heterologous substances. These results indicated that TcCYP9Z6 may play a pivotal role in the detoxification of terpinen-4-ol, which provides support for using TcCYP9Z6 a potential gene for the RNAi-mediated prevention and control of T. castaneum. [Display omitted] • Expression of TcCYP9Z6 gene was induced with terpinen-4-ol. • Knockdown of TcCYP9Z6 increases sensitivity of T. castaneum to terpinen-4-ol. • The CYP9Z6 of T. castaneum is related to its detoxification. [ABSTRACT FROM AUTHOR]

Abstract: Antenna structure varies widely among insects, in contrast to the well-conserved structure of legs. The adult capitate antenna of the red flour beetle, Tribolium castaneum, is composed of eleven articles, organized into four distinct morphological regions (scape, pedicel, funicle and club). Here, we report the use of RNA interference to examine the functions of 21 genes during antenna metamorphosis in T. castaneum. Genes with conserved functions relative to the developmental model species Drosophila melanogaster include Distal-less and EGF signaling (antennal growth), spineless (determination of antennal identity) and the Notch signaling pathway (antennal growth, joint formation, and sensory bristle development). However, the functions of many genes differed from those predicted from the Drosophila model. In addition to a conserved gap phenotype, depletion of dachshund transformed funicle articles toward club-like identity. Depletion of Distal-less or homothorax did not cause antenna-to-leg transformation. Lim1 was required only for development of the scape-pedicle joint. Depletion of odd-skipped-related genes led to the loss of the entire funicle, while spalt, rotund, spineless, and dachshund affected smaller regions. Growth and joint formation were linked developmentally in the funicle, but not in the club. Joint formation within the club required bric-a-brac, aristaless, apterous, and pdm. Gene functions are discussed in terms of a model of antenna development in T. castaneum. This model provides a contrast to knowledge of antenna development in D. melanogaster, insight into the likely ancestral mode of antenna development, and a framework for considering diverse antenna morphologies. [Copyright &y& Elsevier]

Abstract: The dramatic transformation from a larva to an adult must be accompanied by a coordinated activity of genes and hormones that enable an orchestrated transformation from larval to pupal/adult tissues. The maintenance of larval appendages and their subsequent transformation to appendages in holometabolous insects remains elusive at the developmental genetic level. Here the role of a key appendage patterning gene Distal-less (Dll) was examined in mid- to late-larval stages of the flour beetle, Tribolium castaneum. During late larval development, Dll was expressed in appendages in a similar manner as previously reported for the tobacco hornworm, Manduca sexta. Removal of this late Dll expression resulted in disruption of adult appendage patterning. Intriguingly, earlier removal resulted in dramatic loss of structural integrity and identity of larval appendages. A large amount of variability in appendage morphology was observed following Dll dsRNA injection, unlike larvae injected with dachshund dsRNA. These Dll dsRNA-injected larvae underwent numerous supernumerary molts, which could be terminated with injection of either JH methyltransferase or Methoprene-tolerant dsRNA. Apparently, the partial dedifferentiation of the appendages in these larvae acts to maintain high JH and, hence, prevents metamorphosis. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Bioluminescence in beetles is dependent upon the enzyme luciferase. It has been hypothesised luciferase evolved from a fatty acyl-CoA synthetase gene deriving a novel bioluminescent function (neofunctionalization) after a gene duplication event. We evaluated this hypothesis within a phylogenetic framework using independent evidence obtained from the genome of Tribolium castaneum, published luciferase genes and novel luciferase and luciferase-like sequences. This phylogenetic study provides evidence for a large gene family of luciferase and luciferase-like paralogues in bioluminescent and non-bioluminescent beetles. All luciferase sequences formed a clade supporting a protoluciferase existing prior to the divergence of the Lampyridae, Elateridae and Phengodidae (Elateroidea). Multiple luciferase genes were identified from members of the Photurinae and the Luciolinae indicating complex gene duplication events within lampyrid genomes. The majority of luciferase residues were identified to be under purifying selection as opposed to positive selection. We conclude that beetle luciferase may have arisen from a process of subfunctionalization as opposed to neofunctionalization early on in the evolution of the Elateroidea. [Copyright &y& Elsevier]

Abstract: Segment formation in the long germ insect Drosophila is dominated by overlapping gap gene domains in the syncytial blastoderm. In the short germ beetle Tribolium castaneum abdominal segments arise from a cellular growth zone, implying different patterning mechanisms. We describe here the single Tribolium ortholog of the Drosophila genes knirps and knirps-related (called Tc-knirps). Tc-knirps expression is conserved during head patterning and at later stages. However, posterior Tc-knirps expression in the ectoderm is limited to a stripe in A1, instead of a broad abdominal domain covering segment primordia A2–A5 as in Drosophila. Tc-knirps RNAi yields only mild defects in the abdomen, at a position posterior to the abdominal Tc-knirps domain. In addition, Tc-knirps RNAi larvae lack the antennal and mandibular segments. These defects are much more severe than the head defects caused by combined inactivation of Dm-knirps and Dm-knirps-related. Our findings support the notion that the role of gap gene homologs in abdominal segmentation differs fundamentally in long and short germ insects. Moreover, the pivotal role of Tc-knirps in the head suggests an ancestral role for knirps as head patterning gene. Based on this RNAi analysis, Tc-knirps functions neither in the head nor the abdomen as a canonical gap gene. [Copyright &y& Elsevier]

A prey animal has the alternative of fleeing or feigning death to survive when it encounters predators. We found that fleeing by an artificial threat, locomotion and feigning death are pleiotropically correlated with a genetic factor related to a biogenic amine in the red flour beetle, Tribolium castaneum. Walking distance of adults was significantly lower in strains artificially selected for longer (L strains) than shorter duration (S strains) of death-feigning. Crosses showed that S-strain adults were dominant in the frequency and duration of death-feigning and locomotor activity compared to those of L strains, suggesting that death-feigning and activity have the same genetic basis. S-strain adults fled, but L-strain adults feigned death, when they encounter artificial threat. Not only adults that were directly selected for the duration of death-feigning, but also the larvae of L strains frequently showed tonic immobility, when they were dropped onto the ground: the larvae of S strains showed this behaviour less often. This suggests that chemical modulators of behaviour present in these insects before and after metamorphosis control both general locomotor activity and death-feigning. Brain levels of the candidate neuromodulator dopamine were, in fact, found to be significantly higher in S strains compared to L strains in the two selection replications. Thus, we suggest that two alternative behaviours related to antipredator strategies, fleeing or feigning death, are associated with the pleiotropic effects of a neuroactive substance in T. castaneum. [Copyright &y& Elsevier]

Abstract: In the Drosophila segmentation hierarchy, periodic expression of pair-rule genes translates gradients of regional information from maternal and gap genes into the segmental expression of segment polarity genes. In Tribolium, homologs of almost all the eight canonical Drosophila pair-rule genes are expressed in pair-rule domains, but only five have pair-rule functions. even-skipped, runt and odd-skipped act as primary pair-rule genes, while the functions of paired (prd) and sloppy-paired (slp) are secondary. Since secondary pair-rule genes directly regulate segment polarity genes in Drosophila, we analyzed Tc-prd and Tc-slp to determine the extent to which this paradigm is conserved in Tribolium. We found that the role of prd is conserved between Drosophila and Tribolium; it is required in both insects to activate engrailed in odd-numbered parasegments and wingless (wg) in even-numbered parasegments. Similarly, slp is required to activate wg in alternate parasegments and to maintain the remaining wg stripes in both insects. However, the parasegmental register for Tc-slp is opposite that of Drosophila slp1. Thus, while prd is functionally conserved, the fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. [Copyright &y& Elsevier]