N(alpha)-acetyltransferase 40-mediated histone acetylation plays an important role in ecdysone regulation of metamorphosis in the red flour beetle, Tribolium castaneum.

Histone acetylation, a crucial epigenetic modification, is governed by histone acetyltransferases (HATs), that regulate many biological processes. Functions of HATs in insects are not well understood. We identified 27 HATs and determined their functions using RNA interference (RNAi) in the model insect, Tribolium castaneum. Among HATs studied, N-alpha-acetyltransferase 40 (NAA40) knockdown caused a severe phenotype of arrested larval development. The steroid hormone, ecdysone induced NAA40 expression through its receptor, EcR (ecdysone receptor). Interestingly, ecdysone-induced NAA40 regulates EcR expression. NAA40 acetylates histone H4 protein, associated with the promoters of ecdysone response genes: EcR, E74, E75, and HR3, and causes an increase in their expression. In the absence of ecdysone and NAA40, histone H4 methylation by arginine methyltransferase 1 (ART1) suppressed the above genes. However, elevated ecdysone levels at the end of the larval period induced NAA40, promoting histone H4 acetylation and increasing the expression of ecdysone response genes. NAA40 is also required for EcR, and steroid-receptor co-activator (SRC) mediated induction of E74, E75, and HR3. These findings highlight the key role of ecdysone-induced NAA40-mediated histone acetylation in the regulation of metamorphosis.

The zona pellucida protein piopio regulates the metamorphosis and reproduction in Tribolium castaneum.

The zona pellucida domain protein piopio (Pio) was only reported to mediate the adhesion of the apical epithelial surface and the overlying apical extracellular matrix in Drosophila melanogaster, but the developmental roles of Pio were poorly understood in insects. To address this issue, we comprehensively analyzed the function of Pio in Tribolium castaneum. Phylogenetic analysis indicated that pio exhibited one-to-one orthologous relationship among insects. T. castaneum pio had a 1236-bp ORF and contained eight exons. During development pio was abundantly expressed from larva to adult and lowly expressed at the late stage of embryo and adult, while it had more transcripts in the head, epidermis, and gut but fewer in the fat body of late-stage larvae. Knockdown of pio inhibited the pupation, eclosion, and reproduction of T. castaneum. The expression of vitellogenin 1 (Vg1), Vg2, and Vg receptor (VgR) largely decreased in pio-silenced female adults. Silencing pio increased the 20-hydroxyecdysone titer by upregulating phm and spo expression but decreased the juvenile hormone (JH) titer through downregulating JHAMT3 and promoting JHE, JHEH-r4, and JHDK transcription. These results suggested that Pio might regulate the metamorphosis and reproduction via modulating the ecdysone and JH metabolism in T. castaneum. This study found the novel roles of pio in insect metamorphosis and reproduction, and provided the new insights for analyzing other zona pellucida proteins functions in insects.

Arpc2 integrates ecdysone and juvenile hormone metabolism to influence metamorphosis and reproduction in Tribolium castaneum.

BACKGROUND: Actin-related protein 2/3 complex regulates actin polymerization and the formation of branched actin networks. However, the function and evolutionary relationship of this complex subunit 2 (Arpc2) has been poorly understood in insects. RESULTS: To address these issues, we performed comprehensive analysis of Arpc2 in Tribolium castaneum. Phylogenetic analysis revealed that Arpc2 was originated from one ancestral gene in animals but evolved independently between vertebrates and insects after species differentiation. T. castaneum Arpc2 has a 906-bp coding sequence and consists of 4 exons. Arpc2 transcripts were abundantly detected in embryos and pupae but less so in larvae and adults, while it had high expression in the gut, fat body and head but low expression in the epidermis of late-stage larvae. Knockdown of it at the late larval stage inhibited the pupation and resulted in arrested larvae. Silencing it in 1-day pupae impaired eclosion, which caused adult wings to fail to close. Injection of Arpc2 dsRNAs into 5-day pupae made adults have smaller testis and ovary and could not lay eggs. The expression of vitellogenin 1 (Vg1), Vg2 and Vg receptor (VgR) was downregulated after knocking down Arpc2 5 days post-adult emergence. Arpc2 silencing reduced 20-hydroxyecdysone titer by affecting the enzymes of its biosynthesis and catabolism but increased juvenile biosynthesis via upregulating JHAMT3 expression. CONCLUSION: Our results indicate that Arpc2 is associated with the metamorphosis and reproduction by integrating ecdysone and juvenile hormone metabolism in T. castaneum. This study provides theoretical basis for developing Arpc2 as a potential RNA interference target for pest control. © 2024 Society of Chemical Industry.

The glutathione biosynthesis is involved in metamorphosis, antioxidant function, and insecticide resistance in Tribolium castaneum.

BACKGROUND: Reduced glutathione (GSH) synthesis is vital for redox homeostasis, cell-cycle regulation and apoptosis, and immune function. The glutamate-cysteine ligase catalytic subunit (Gclc) is the first and rate-limiting enzyme in GSH synthesis, suggesting the potential use of Gclc as a pesticide target. However, the functional characterization of Gclc, especially its contribution in metamorphosis, antioxidant status and insecticide resistance, is unclear in Tribolium castaneum. RESULTS: In this study, we identified and cloned Gclc from T. castaneum (TcGclc) and found that its expression began to increase significantly from the late larvae (LL) stage (3.491 ± 0.490-fold). Furthermore, RNA interference-mediated knockdown of TcGclc resulted in three types of aberration (100% total aberration rate) caused by the downregulation of genes related to the 20-hydroxyecdysone (20E) pathway. This deficiency was partially rescued by exogenous 20E treatment (53.1% ± 3.2%), but not by antioxidant. Moreover, in the TcGclc knockdown group, GSH content was decreased to 62.3%, and total antioxidant capacity, glutathione peroxidase and total superoxide dismutase activities were reduced by 14.6%, 83.6%, and 82.3%, respectively. In addition, treatment with different insecticides upregulated expression of TcGclc significantly compared with a control group during the late larval stage (P < 0.01). CONCLUSION: Our results indicate that TcGclc has an extensive role in metamorphosis, antioxidant function and insecticide resistance in T. castaneum, thereby expanding our understanding of GSH functions and providing a scientific basis for pest control. © 2024 Society of Chemical Industry.

TGFß/activin-dependent activation of Torso controls the timing of the metamorphic transition in the red flour beetle Tribolium castaneum.

Understanding the mechanisms governing body size attainment during animal development is of paramount importance in biology. In insects, a crucial phase in determining body size occurs at the larva-pupa transition, marking the end of the larval growth period. Central to this process is the attainment of the threshold size (TS), a critical developmental checkpoint that must be reached before the larva can undergo metamorphosis. However, the intricate molecular mechanisms by which the TS orchestrates this transition remain poor understood. In this study, we investigate the role of the interaction between the Torso and TGFß/activin signaling pathways in regulating metamorphic timing in the red flour beetle, Tribolium castaneum. Our results show that Torso signaling is required specifically during the last larval instar and that its activation is mediated not only by the prothoracicotropic hormone (Tc-Ptth) but also by Trunk (Tc-Trk), another ligand of the Tc-Torso receptor. Interestingly, we show that while Tc-Torso activation by Tc-Ptth determines the onset of metamorphosis, Tc-Trk promotes growth during the last larval stage. In addition, we found that the expression of Tc-torso correlates with the attainment of the TS and the decay of juvenile hormone (JH) levels, at the onset of the last larval instar. Notably, our data reveal that activation of TGFß/activin signaling pathway at the TS is responsible for repressing the JH synthesis and inducing Tc-torso expression, initiating metamorphosis. Altogether, these findings shed light on the pivotal involvement of the Ptth/Trunk/Torso and TGFß/activin signaling pathways as critical regulatory components orchestrating the TS-driven metamorphic initiation, offering valuable insights into the mechanisms underlying body size determination in insects.

Developmental O-glycan profile analysis shows pentasaccharide mucin-type O-glycans are linked with pupation of Tribolium castaneum.

Eukaryotic cells can decorate their proteins with carbohydrate structures or glycans, significantly affecting the properties and activities of these proteins. Despite the importance of protein glycosylation in numerous biological processes, our knowledge of this modification in insects is far from complete. While N-glycosylation is the most studied, the study of O-glycans in insects is still very fragmentary and these studies are limited to a specific developmental stage or a specific tissue. In this article, matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) technology was used to analyze the O-glycan profile for the different developmental stages of egg, larva, pupa, and adult of the red flour beetle Tribolium castaneum, an important insect model and pest worldwide. The results on the O-glycan profile showed that the mucin-type glycans dominate the O-glycome of the red flour beetle. Interestingly, some of the more complex mucin-type O-glycans, such as a tetra- (O-GalNAcGalGlcAGalNAc) and pentasaccharide O-glycan (O-GalNAc(GalGlcA)GalNAcGlcA), were highly abundant during the pupa stage, the intermediate stage between larval and adult stage in holometabolous insects, demonstrating that insect metamorphosis is accompanied with a change in the insect O-glycan profile. Together with the N-glycan profile, the current data are a foundation to better understand the role of protein glycosylation in the development of insects.

Antifungal roles of adult-specific cuticular protein genes of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite structure that can further be divided into a few sub-structural layers. Its large moiety comprises a lattice of chitin fibrils and structural proteins, both of which are stabilized by covalent bonding among them. The cuticle covers the whole surface of insect body, and thus has long been suggested for the involvement in defense against entomopathogens, especially entomopathogenic fungi that infect percutaneously. We have been addressing this issue in the past few years and have so far demonstrated experimentally that chitin synthase 1, laccase2 as well as benzoquinone synthesis-related genes of Tribolium castaneum have indispensable roles in the antifungal host defense. In the present study we focused on another major component of the insect cuticular integument, structural cuticular proteins. We chose three genes coding for adult-specific cuticular proteins, namely CPR4, CPR18 and CPR27, and examined their roles in forming immunologically sound adult cuticular integuments. Analyses of developmental expression revealed that the three genes showed high level expression in the pupal stage. These results are consistent with their proposed roles in constructing cuticle of adult beetles. The RNA interference-mediated gene knockdown was employed to silence these genes, and the administration of double strand RNAs in pupae resulted in the adults with malformed elytra. The single knockdown of the three genes attenuated somewhat the defense of the resulting adult beetles against Beauveria bassiana and Metarhizium anisopliae, but statistical analyses indicated no significant differences from controls. In contrast, the double or triple knockdown mutant beetles displayed a drastic disruption of the host defense against the two entomopathogenic fungal species irrespective of the combination of targeted cuticular protein genes, demonstrating the important roles of the three cuticular protein genes in conferring robust antifungal properties on the adult cuticle. Scanning electron microscopic observation revealed that the germination of conidia attached on the adult body surface was still suppressed after the gene knockdown as in the case of wild-type beetles, suggesting that the weakened antifungal phenotypes resulted from the combined knockdown of the adult-specific cuticular protein genes could not be accounted for by the disfunction of secretion/retention of fungistatic benzoquinone derivatives.

Activation of EGFR signaling by Tc-Vein and Tc-Spitz regulates the metamorphic transition in the red flour beetle Tribolium castaneum.

Animal development relies on a sequence of specific stages that allow the formation of adult structures with a determined size. In general, juvenile stages are dedicated mainly to growth, whereas last stages are devoted predominantly to the maturation of adult structures. In holometabolous insects, metamorphosis marks the end of the growth period as the animals stops feeding and initiate the final differentiation of the tissues. This transition is controlled by the steroid hormone ecdysone produced in the prothoracic gland. In Drosophila melanogaster different signals have been shown to regulate the production of ecdysone, such as PTTH/Torso, TGFß and Egfr signaling. However, to which extent the roles of these signals are conserved remains unknown. Here, we study the role of Egfr signaling in post-embryonic development of the basal holometabolous beetle Tribolium castaneum. We show that Tc-Egfr and Tc-pointed are required to induced a proper larval-pupal transition through the control of the expression of ecdysone biosynthetic genes. Furthermore, we identified an additional Tc-Egfr ligand in the Tribolium genome, the neuregulin-like protein Tc-Vein (Tc-Vn), which contributes to induce larval-pupal transition together with Tc-Spitz (Tc-Spi). Interestingly, we found that in addition to the redundant role in the control of pupa formation, each ligand possesses different functions in organ morphogenesis. Whereas Tc-Spi acts as the main ligand in urogomphi and gin traps, Tc-Vn is required in wings and elytra. Altogether, our findings show that in Tribolium, post-embryonic Tc-Egfr signaling activation depends on the presence of two ligands and that its role in metamorphic transition is conserved in holometabolous insects.

High-temperature shock consequences on the red flour beetle (Tribolium castaneum) and the rice weevil (Sitophilus oryzae).

Temperature shocks have profound effects on biological and physiological functions at all levels of organization. However, the recovery periods from these shocks and their subsequent impacts remain unknown. Herein, our study investigated the effect of short temperature stress on survival, dormancy recovery time, nutritional indices, life traits and development rate for T. castaneum (larvae and adults) and S. oryzae adults. The results showed significant effects on survival rates of T. castaneum (larvae and adults) and S. oryzae adults. When both insects had been exposed to high-temperature shock, survival rates decreased with higher temperatures and longer periods of exposure. Furthermore, recovery times varied between and within the insect species, as prolonged exposure to thermal shocks increased recovery periods. Moreover, dormancy time resulting from the high-temperature shocks significantly affected food deterrence and food intake, regardless of the stage of development, species, exposure periods and temperature-exposure conditions. Subsequently, differences in body growth rates and food consumption rates are an appropriate indicator of differences in food conversion rates under high-temperature shocks, regardless of the species and developmental stages. On the other hand, our results indicated that as high-temperature shocks increased, the total development period increased of T. castaneum. Likewise, the pupal stage increased with increasing high-temperature shocks, and the larval stage decreased with increasing thermal shocks and increasing the periods of exposure. In summary, our study showed the importance of dormancy recovery time and its subsequent effects for improving disinfestation effectiveness of heat treatment, and understanding insect response to high temperatures.

Cuticular Hydrocarbon Trails Released by Host Larvae Lose their Kairomonal Activity for Parasitoids by Solidification.

Successful host search by parasitic wasps is often mediated by host-associated chemical cues. The ectoparasitoid Holepyris sylvanidis is known to follow chemical trails released by host larvae of the confused flour beetle, Tribolium confusum, for short-range host location. Although the hexane-extractable trails consist of stable, long-chain cuticular hydrocarbons (CHCs) with low volatility, the kairomonal activity of a trail is lost two days after release. Here, we studied whether this loss of kairomonal activity is due to changes in the chemical trail composition induced by microbial activity. We chemically analyzed trails consisting of hexane extracts of T. confusum larvae after different time intervals past deposition under sterile and non-sterile conditions. GC-MS analyses revealed that the qualitative and quantitative pattern of the long-chain CHCs of larval trails did not significantly change over time, neither under non-sterile nor sterile conditions. Hence, our results show that the loss of kairomonal activity of host trails is not due to microbially induced changes of the CHC pattern of a trail. Interestingly, the kairomonal activity of trails consisting of host larval CHC extracts was recoverable after two days by applying hexane to them. After hexane evaporation, the parasitoids followed the reactivated host trails as they followed freshly laid ones. Cryo-scanning electron microscopy showed that the trails gradually formed filament-shaped microstructures within two days. This self-assemblage of CHCs was reversible by hexane application. Our study suggests that the long-chain CHCs of a host trail slowly undergo solidification by a self-assembling process, which reduces the accessibility of CHCs to the parasitoid's receptors as such that the trail is no longer eliciting trail-following behavior.

Developing a Hazomalania voyronii Essential Oil Nanoemulsion for the Eco-Friendly Management of Tribolium confusum, Tribolium castaneum and Tenebrio molitor Larvae and Adults on Stored Wheat.

Most insecticides commonly used in storage facilities are synthetic, an issue that generates concerns about food safety and public health. Therefore, the development of eco-friendly pest management tools is urgently needed. In the present study, a 6% (w/w) Hazomalania voyronii essential oil-based nanoemulsion (HvNE) was developed and evaluated for managing Tribolium confusum, T. castaneum, and Tenebrio molitor, as an eco-friendly wheat protectant. Larval and adult mortality was evaluated after 4, 8, and 16 h, and 1, 2, 3, 4, 5, 6, and 7 days, testing two HvNE concentrations (500 ppm and 1000 ppm). T. confusum and T. castaneum adults and T. molitor larvae were tolerant to both concentrations of the HvNE, reaching 13.0%, 18.7%, and 10.3% mortality, respectively, at 1000 ppm after 7 days of exposure. However, testing HvNE at 1000 ppm, the mortality of T. confusum and T. castaneum larvae and T. molitor adults 7 days post-exposure reached 92.1%, 97.4%, and 100.0%, respectively. Overall, the HvNE can be considered as an effective adulticide or larvicide, depending on the target species. Our results highlight the potential of H. voyronii essential oil for developing green nanoinsecticides to be used in real-world conditions against key stored-product pests.

Assessment of the potential of non-thermal atmospheric pressure plasma discharge and microwave energy against Tribolium castaneum and Trogoderma granarium.

This study was carried out to investigate the efficacy of the non-thermal atmospheric pressure plasma produced with dielectric barrier discharge (APPD) using air as a processing gas and microwave energy to control Tribolium castaneum and Trogoderma granarium adults and larvae in wheat grains. Insects' mortality was found to be power and time-dependent. The results indicated that non-thermal APPD and the microwave have enough insecticidal effect on the target pests. From the bioassay, LT50's and LT90's levels were estimated, T. granarium larvae appeared more tolerant to non-thermal APPD and the microwave energy than adults 7 days post-exposure. The germination percentage of wheat grains increased as the time of exposure to the non-thermal APPD increased. On the contrary, the germination percentage of wheat grains decreased as the time of exposure to the microwave increased. In addition, changes in antioxidant enzyme activities, catalase (CAT), glutathione S-transferase (GST) and peroxidase, in adults and larvae were examined after 24 h post-treatment to non-thermal APPD at 15.9 W power level, which caused 50% mortality. The activity of CAT, GST and lipid peroxide in the treated larvae showed a significant increase post-exposure to the non-thermal APPD at 15.9 W power level. On the other hand, no significant change in GSH-Px activity was observed. Reductions in the level of glutathione (GSH) and protein content occurred in treated larvae in comparison with the control.

Functional analysis of sense organ specification in the Tribolium castaneum larva reveals divergent mechanisms in insects.

Insects and other arthropods utilise external sensory structures for mechanosensory, olfactory, and gustatory reception. These sense organs have characteristic shapes related to their function, and in many cases are distributed in a fixed pattern so that they are identifiable individually. In Drosophila melanogaster, the identity of sense organs is regulated by specific combinations of transcription factors. In other arthropods, however, sense organ subtypes cannot be linked to the same code of gene expression. This raises the questions of how sense organ diversity has evolved and whether the principles underlying subtype identity in D. melanogaster are representative of other insects. Here, we provide evidence that such principles cannot be generalised, and suggest that sensory organ diversification followed the recruitment of sensory genes to distinct sensory organ specification mechanism. RESULTS: We analysed sense organ development in a nondipteran insect, the flour beetle Tribolium castaneum, by gene expression and RNA interference studies. We show that in contrast to D. melanogaster, T. castaneum sense organs cannot be categorised based on the expression or their requirement for individual or combinations of conserved sense organ transcription factors such as cut and pox neuro, or members of the Achaete-Scute (Tc ASH, Tc asense), Atonal (Tc atonal, Tc cato, Tc amos), and neurogenin families (Tc tap). Rather, our observations support an evolutionary scenario whereby these sensory genes are required for the specification of sense organ precursors and the development and differentiation of sensory cell types in diverse external sensilla which do not fall into specific morphological and functional classes. CONCLUSIONS: Based on our findings and past research, we present an evolutionary scenario suggesting that sense organ subtype identity has evolved by recruitment of a flexible sensory gene network to the different sense organ specification processes. A dominant role of these genes in subtype identity has evolved as a secondary effect of the function of these genes in individual or subsets of sense organs, probably modulated by positional cues.

Disruption of the cytochrome P450 CYP6BQ7 gene reduces tolerance to plant toxicants in the red flour beetle, Tribolium castaneum.

In insects, the cytochrome P450 CYP6B family plays key roles in the detoxification of toxic plant substances. However, the function of CYP6 family genes in degrading plant toxicants in Tribolium castaneum, an extremely destructive global storage pest, have yet to be elucidated. In this study, a T. castaneum CYP gene, TcCYP6BQ7, was characterized. TcCYP6BQ7 expression was significantly induced after exposure to essential oil of the plant Artemisia vulgaris (EOAV). Spatiotemporal expression profiling revealed that TcCYP6BQ7 expression was higher in larval and adult stages of T. castaneum than in other developmental stages, and that TcCYP6BQ7 was predominantly expressed in the brain and hemolymph from the late larval stage. TcCYP6BQ7 silencing by RNA interference increased larvae mortality in response to EOAV from 49.67% to 71.67%, suggesting that this gene is associated with plant toxicant detoxification. Combined results from this study indicate that the CYP6 family gene TcCYP6BQ7 likely plays a pivotal role in influencing the susceptibility of T. castaneum to plant toxicants. These findings may have implications for the development of novel therapeutics to control this agriculturally important pest.

The red flour beetle Tribolium castaneum: A model for host-microbiome interactions.

A large body of ongoing research focuses on understanding the mechanisms and processes underlying host-microbiome interactions, and predicting their ecological and evolutionary outcomes. To draw general conclusions about such interactions and understand how they are established, we must synthesize information from a diverse set of species. We analysed the microbiome of an important insect model-the red flour beetle Tribolium castaneum-which is a widespread generalist pest of stored cereals. The beetles complete their entire life cycle in flour, which thus serves multiple functions: habitat, food, and a source of microbes. We determined key factors that shape the T. castaneum microbiome, established protocols to manipulate it, and tested its consequences for host fitness. We show that the T. castaneum microbiome is derived from flour-acquired microbes, and varies as a function of (flour) resource and beetle density. Beetles gain multiple fitness benefits from their microbiome, such as higher fecundity, egg survival, and lifespan; and reduced cannibalism. In contrast, the microbiome has a limited effect on development rate, and does not enhance pathogen resistance. Importantly, the benefits are derived only from microbes in the ancestral resource (wheat flour), and not from novel resources such as finger millet, sorghum, and corn. Notably, the microbiome is not essential for beetle survival and development under any of the tested conditions. Thus, the red flour beetle is a tractable model system to understand the ecology, evolution and mechanisms of host-microbiome interactions, while closely mimicking the host species' natural niche.

Unexpected mutual regulation underlies paralogue functional diversification and promotes epithelial tissue maturation in Tribolium.

Insect Hox3/zen genes represent an evolutionary hotspot for changes in function and copy number. Single orthologues are required either for early specification or late morphogenesis of the extraembryonic tissues, which protect the embryo. The tandemly duplicated zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We dissect the paralogues' expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) throughout embryogenesis. We identify an unexpected role of Tc-Zen2 in repression of Tc-zen1, generating a negative feedback loop that promotes developmental progression. Tc-Zen2 regulation is dynamic, including within co-expressed multigene loci. We also show that extraembryonic development is the major event within the transcriptional landscape of late embryogenesis and provide a global molecular characterization of the extraembryonic serosal tissue. Altogether, we propose that paralogue mutual regulation arose through multiple instances of zen subfunctionalization, leading to their complementary extant roles.

Functional characterization of a special dicistronic transcription unit encoding histone methyltransferase su(var)3-9 and translation regulator eIF2γ in Tribolium castaneum.

Operons are rare in eukaryotes, where they often allow concerted expression of functionally related genes. While a dicistronic transcription unit encoding two unrelated genes, the suppressor of position-effect variegation su(var)3-9 and the gamma subunit of eukaryotic translation initiation factor 2 (eIF2γ) has been found in insecta, and its significance is not well understood. Here, we analyzed the evolutionary history of this transcription unit in arthropods and its functions by using model Coleoptera insect Tribolium castaneum. In T. castaneum, Tcsu(var)3-9 fused into the 80 N-terminal amino acids of TceIF2γ, the transcription of these two genes are resolved by alternative splicing. Phylogenetic analysis supports the natural gene fusion of su(var)3-9 and eIF2γ occurred in the ancestral line of winged insects and silverfish, but with frequent re-fission during the evolution of insects. Functional analysis by using RNAi for these two genes revealed that gene fusion did not invoke novel functions for the gene products. As a histone methyltransferase, Tcsu(var)3-9 is primarily responsible for H3K9 di-, and tri-methylation and plays important roles in metamorphosis and embryogenesis in T. castaneum. While TceIF2γ plays essential roles in T. castaneum by positively regulating protein translation mediated ecdysteroid biosynthesis. The vulnerability of the gene fusion and totally different role of su(var)3-9 and eIF2γ in T. castaneum confirm this gene fusion is a non-selected, constructive neutral evolution event in insect. Moreover, the positive relationship between protein translation and ecdysteroid biosynthesis gives new insights into correlations between translation regulation and hormonal signaling.

Mechanism of threshold size assessment: Metamorphosis is triggered by the TGF-beta/Activin ligand Myoglianin.

Although the mechanisms that control growth are now well understood, the mechanism by which animals assess their body size remains one of the great puzzles in biology. The final larval instar of holometabolous insects, after which growth stops and metamorphosis begins, is specified by a threshold size. We investigated the mechanism of threshold size assessment in the tobacco hornworm, Manduca sexta. The threshold size was found to change depending on the amount of exposure to poor nutrient conditions whereas hypoxia treatment consistently led to a lower threshold size. Under these various conditions, the mass of the muscles plus integuments was correlated with the threshold size. Furthermore, the expression of myoglianin (myo) increased at the threshold size in both M. sexta and Tribolium castaneum. Knockdown of myo in T. castaneum led to larvae that underwent supernumerary larval molts and stayed in the larval stage permanently even after passing the threshold size. We propose that increasing levels of Myo produced by the growing tissues allow larvae to assess their body size and trigger metamorphosis at the threshold size.

Dendrimer-coated carbon nanotubes deliver dsRNA and increase the efficacy of gene knockdown in the red flour beetle Tribolium castaneum.

In this study, the use of dendrimer-coated carbon nanotubes (CNTs) as a delivery vehicle for dsRNA was assessed in Tribolium castaneum. Exposure to low dosages of polyamidoamine dendrimer carbon nanotubes (PAMAM-CNTs) did not affect T. castaneum larval mortality. Expression of key apoptotic factors, Dronc (Tc12580), Dredd (Tcn-like, Tc014026) and Buffy, (Tcinhib apop1), which can act as toxicity indicators, were not altered in T. castaneum larvae following injection of PAMAM-CNTs. The level of knockdown of two target genes, α-tubulin and mitochondrial RNA polymerase (mtpol), were significantly increased when larvae were injected with double-stranded RNA bound to CNTs (PAMAM-CNT-dsRNA), compared to those injected with target dsRNA alone. PAMAM-CNTs were visualised in cellular vacuoles and in the cell nucleus. Increase occurrence of a blistered wing phenotype was found in a subset of PAMAM-CNT-dsRNAαtub injected larvae, relative to the level seen in larvae injected with naked dsRNAαtub alone. These results suggest that the use of functionalised CNTs for dsRNA delivery could increase the efficacy of RNA interference in insect pest species.

Evaluation of inhibitor of apoptosis genes as targets for RNAi-mediated control of insect pests.

Apoptosis has been widely studied from mammals to insects. Inhibitor of apoptosis (IAP) protein is a negative regulator of apoptosis. Recent studies suggest that iap genes could be excellent targets for RNA interference (RNAi)-mediated control of insect pests. However, not much is known about iap genes in one of the well-known insect model species, Tribolium castaneum. The orthologues of five iap genes were identified in T. castaneum by searching its genome at NCBI (https://www.ncbi.nlm.nih.gov/) and UniProt (https://www.uniprot.org/) databases using Drosophila melanogaster and Aedes aegypti IAP protein sequences as queries. RNAi assays were performed in T. castaneum cell line (TcA) and larvae. The knockdown of iap1 gene induced a distinct apoptotic phenotype in TcA cells and induced 91% mortality in T. castaneum larvae. Whereas, knockdown of iap5 resulted in a decrease in cell proliferation in TcA cells and developmental defects in T. castaneum larvae which led to 100% mortality. Knockdown of the other three iap genes identified did not cause a significant effect on cells or insects. These data increase our understanding of iap genes in insects and provide opportunities for developing iap1 and iap5 as targets for RNAi-based insect pest control.