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.

A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species.

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

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.

A C-type lectin (CTL2) mediated both humoral and cellular immunity against bacterial infection in Tribolium castaneum.

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.

Silencing gram-negative bacteria binding protein 1 decreases the immunity of Tribolium castaneum against bacteria.

Gram-negative bacteria binding proteins (GNBPs) have the ability to recognize molecular patterns associated with microbial pathogens (PAMPs), leading to the activation of immune responses downstream. In the genome of Tribolium castaneum, three GNBP genes have been identified; however, their immunological roles remain unexplored. In our study, a GNBP1, designated as TcGNBP1, were identified from the cDNA library of T. castaneum. The coding sequence of TcGNBP1 consisted of 1137 bps and resulted in the synthesis of a protein comprising 378 amino acids. This protein encompasses a signal peptide, a low-complexity region, and a glycoside hydrolase 16 domain. TcGNBP1 was strongly expressed in early adult stages, and mainly distributed in hemolymph and gut. Upon being challenged with Escherichia coli or Staphylococcus aureus, the transcript levels of TcGNBP1 were significantly changed at different time points. Through molecular docking and ELISA analysis, it was observed that TcGNBP1 has the ability to interact with lipopolysaccharides, peptidoglycan, and ß-1, 3-glucan. Based on these findings, it was further discovered that recombinant TcGNBP1 can directly bind to five different bacteria in a Ca2+-dependent manner. After knockdown of TcGNBP1 with RNA interference, expression of antimicrobial peptide genes and prophenoloxidase (proPO) activity were suppressed, the susceptibility of T. castaneum to E. coli or S. aureus infection was enhanced, leading to low survival rate. These results suggest a regulatory mechanism of TcGNBP1 in innate immunity of T. castaneum and provide a potential molecular target for dsRNA-based insect pest management.

scRNA-seq Reveals Novel Genetic Pathways and Sex Chromosome Regulation in Tribolium Spermatogenesis.

Spermatogenesis is critical to sexual reproduction yet evolves rapidly in many organisms. High-throughput single-cell transcriptomics promises unparalleled insight into this important process but understanding can be impeded in nonmodel systems by a lack of known genes that can reliably demarcate biologically meaningful cell populations. Tribolium castaneum, the red flour beetle, lacks known markers for spermatogenesis found in insect species like Drosophila melanogaster. Using single-cell sequencing data collected from adult beetle testes, we implement a strategy for elucidating biologically meaningful cell populations by using transient expression stage identification markers, weighted principal component clustering, and SNP-based haploid/diploid phasing. We identify populations that correspond to observable points in sperm differentiation and find species specific markers for each stage. Our results indicate that molecular pathways underlying spermatogenesis in Coleoptera are substantially diverged from those in Diptera. We also show that most genes on the X chromosome experience meiotic sex chromosome inactivation. Temporal expression of Drosophila MSL complex homologs coupled with spatial analysis of potential chromatin entry sites further suggests that the dosage compensation machinery may mediate escape from meiotic sex chromosome inactivation and postmeiotic reactivation of the X chromosome.

Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle).

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/ß conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

Comparative transcriptomic and metabolomics analysis of modified atmosphere responses in Tribolium castaneum (Coleoptera: Tenebrionidae).

Modified atmosphere is effective in controlling Tribolium castaneum Herbst, but it has adaptations. Comprehending the potential mechanism of resistance to T. castaneum in a modified atmosphere will help advance related management methods. This study conducted a comparative transcriptomic and metabolomic analysis to understand the physiological mechanism of T. castaneum in adapting to CO2 stress. Results showed that there were a large number of differentially expressed genes (DEGs) in T. castaneum treated with different concentrations of CO2. Gene ontology (GO) analysis revealed significant enrichment of DEGs mainly in binding, catalytic activity, cell, membrane, membrane part, protein-containing complex, biological regulation, and cellular and metabolic process. Kyoto Encyclopedia of Genes and Genomes analysis showed that different treatments had different effects on the metabolic pathways of T. castaneum. DEGs induced by 25% CO2 were involved in arginine and proline metabolism, and 50% air + 50% CO2 treatment affected most kinds of metabolic pathways, mainly the signal transduction pathway, including PI3K-Akt signaling pathway, AMPK signaling pathway, neurotrophin signaling pathway, insulin signaling pathway, and thyroid hormone signaling. Ribosome and DNA replication were enriched under high CO2 stress (75% and 95%). The metabolomics revealed that different concentrations of CO2 treatments might inhibit the growth of T. castaneum through acidosis, or they may adapt to anoxic conditions through histamine and N-acetylhistamine. Multiple analyses have shown significant changes in histamine and N-acetylhistamine levels, as well as their associated genes, with increasing CO2 concentration. In conclusion, this study comprehensively revealed the molecular mechanism of T. castaneum responding to CO2 stress and provided the basis for an effectively modified atmosphere in the T. castaneum.

Mapping the functional form of the trade-off between infection resistance and reproductive fitness under dysregulated immune signaling.

Immune responses benefit organismal fitness by clearing parasites but also exact costs associated with immunopathology and energetic investment. Hosts manage these costs by tightly regulating the induction of immune signaling to curtail excessive responses and restore homeostasis. Despite the theoretical importance of turning off the immune response to mitigate these costs, experimentally connecting variation in the negative regulation of immune responses to organismal fitness remains a frontier in evolutionary immunology. In this study, we used a dose-response approach to manipulate the RNAi-mediated knockdown efficiency of cactus (IκBα), a central regulator of Toll pathway signal transduction in flour beetles (Tribolium castaneum). By titrating cactus activity across four distinct levels, we derived the shape of the relationship between immune response investment and traits associated with host fitness, including infection susceptibility, lifespan, fecundity, body mass, and gut homeostasis. Cactus knock-down increased the overall magnitude of inducible immune responses and delayed their resolution in a dsRNA dose-dependent manner, promoting survival and resistance following bacterial infection. However, these benefits were counterbalanced by dsRNA dose-dependent costs to lifespan, fecundity, body mass, and gut integrity. Our results allowed us to move beyond the qualitative identification of a trade-off between immune investment and fitness to actually derive its functional form. This approach paves the way to quantitatively compare the evolution and impact of distinct regulatory elements on life-history trade-offs and fitness, filling a crucial gap in our conceptual and theoretical models of immune signaling network evolution and the maintenance of natural variation in immune systems.

The BTB transcription factor, Abrupt, acts cooperatively with Chronologically inappropriate morphogenesis (Chinmo) to repress metamorphosis and promotes leg regeneration.

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.

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.

Transcriptomic comparison between populations selected for higher and lower mobility in the red flour beetle Tribolium castaneum.

Movement is an important behavior observed in a wide range of taxa. Previous studies have examined genes controlling movement using wing polymorphic insects and genes controlling wing size. However, few studies have investigated genes controlling movement activity rather than morphological traits. In the present study, we conducted RNA sequencing using populations with higher (WL) and lower (WS) mobility established by artificial selection in the red flour beetle Tribolium castaneum and compared gene expression levels between selected populations with two replicate lines. As a result, we found significant differences between the selected populations in 677 genes expressed in one replicate line and 1198 genes expressed in another replicate line, of which 311 genes were common to the two replicate lines. Furthermore, quantitative PCR focusing on 6 of these genes revealed that neuropeptide F receptor gene (NpF) was significantly more highly expressed in the WL population than in the WS population, which was common to the two replicate lines. We discuss differences in genes controlling movement between walking activity and wing polymorphism.

Prediction and analysis of cis-regulatory elements in Dorsal and Ventral patterning genes of Tribolium castaneum and its comparison with Drosophila melanogaster.

Insect embryonic development and morphology are characterized by their anterior-posterior and dorsal-ventral (DV) patterning. In Drosophila embryos, DV patterning is mediated by a dorsal protein gradient which activates twist and snail proteins, the important regulators of DV patterning. To activate or repress gene expression, some regulatory proteins bind in clusters to their target gene at sites known as cis-regulatory elements or enhancers. To understand how variations in gene expression in different lineages might lead to different phenotypes, it is necessary to understand enhancers and their evolution. Drosophila melanogaster has been widely studied to understand the interactions between transcription factors and the transcription factor binding sites. Tribolium castaneum is an upcoming model animal which is catching the interest of biologists and the research on the enhancer mechanisms in the insect's axes patterning is still in infancy. Therefore, the current study was designed to compare the enhancers of DV patterning in the two insect species. The sequences of ten proteins involved in DV patterning of D. melanogaster were obtained from Flybase. The protein sequences of T. castaneum orthologous to those obtained from D. melanogaster were acquired from NCBI BLAST, and these were then converted to DNA sequences which were modified by adding 20 kb sequences both upstream and downstream to the gene. These modified sequences were used for further analysis. Bioinformatics tools (Cluster-Buster and MCAST) were used to search for clusters of binding sites (enhancers) in the modified DV genes. The results obtained showed that the transcription factors in Drosophila melanogaster and Tribolium castaneum are nearly identical; however, the number of binding sites varies between the two species, indicating transcription factor binding site evolution, as predicted by two different computational tools. It was observed that dorsal, twist, snail, zelda, and Supressor of Hairless are the transcription factors responsible for the regulation of DV patterning in the two insect species.

A life-history allele of large effect shortens developmental time in a wild insect population.

Developmental time is a key life-history trait with large effects on Darwinian fitness. In many insects, developmental time is currently under strong selection to minimize ecological mismatches in seasonal timing induced by climate change. The genetic basis of responses to such selection, however, is poorly understood. To address this problem, we set up a long-term evolve-and-resequence experiment in the beetle Tribolium castaneum and selected replicate, outbred populations for fast or slow embryonic development. The response to this selection was substantial and embryonic developmental timing of the selection lines started to diverge during dorsal closure. Pooled whole-genome resequencing, gene expression analysis and an RNAi screen pinpoint a 222 bp deletion containing binding sites for Broad and Tramtrack upstream of the ecdysone degrading enzyme Cyp18a1 as a main target of selection. Using CRISPR/Cas9 to reconstruct this allele in the homogenous genetic background of a laboratory strain, we unravel how this single deletion advances the embryonic ecdysone peak inducing dorsal closure and show that this allele accelerates larval development but causes a trade-off with fecundity. Our study uncovers a life-history allele of large effect and reveals the evolvability of developmental time in a natural insect population.

Development, application and evaluation of three novel TaqMan qPCR assays for phosphine resistance monitoring in major stored product pests Tribolium castaneum and Rhyzopertha dominica.

BACKROUND: Stored product protection from insect pests relies heavily on the use of phosphine. The most serious drawback of phosphine is the development of resistance in major stored product insects worldwide, including the red flour beetle, Tribolium castaneum (Herbst) and the lesser grain borer, Rhyzopertha dominica (F.). Two genetic loci are responsible for phosphine resistance: the rph1 (S349G mutation in the cyt-b5-r homolog) in T. castaneum and the rph2 (P45/49S mutation in the dihydrolipoamide dehydrogenase (dld) gene) in T. castaneum and R. dominica. RESULTS: In this study, we have developed and applied high-throughput, practical and specific molecular diagnostics (TaqMan qPCR) for monitoring mutations S349G, P45S and P49S. In our pilot monitoring application, we have included phosphine-resistant and susceptible populations from different parts of the world (USA, Australia, Brazil) and European strains from Greece and Serbia. Our results for the resistant T. castaneum showed a P45S mutant allele frequency (MAF) of 100% and 75.0% in the populations from Serbia and Brazil, respectively. Regarding the susceptible T. castaneum, P45S was detected in Greece (MAF = 62.5%) and was absent in Australia (MAF = 0.0%). Additionally, the S349G mutation was found to be fixed in all resistant populations, while it was also detected in susceptible ones (frequencies: 65.0% and 100.0%). The only case where both mutations were fixed (100%) was a resistant population from Serbia. In R. dominica, the P49S mutation was found only in the two resistant R. dominica populations from Serbia and Greece (50.0% and 100%) and was absent from the susceptible one from Greece; thus, P49S seems to be a satisfactory indicator for monitoring phosphine resistance. CONCLUSIONS: Our P49S detection assay in R. dominica seems to be a viable option in this direction, yet its utilization needs additional large-scale confirmatory work. The identification of additional resistance markers also should be prioritized. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Transcriptomic landscape of the interaction between the entomopathogenic fungus Beauveria bassiana and its tolerant host Tribolium castaneum revealed by dual RNA-seq.

Entomopathogenic fungi such as Beauveria bassiana are the only insect pathogens able to start the infection process by penetrating through the host cuticle. However, some insects try to avoid fungal infection by embedding their cuticle with antifungal compounds. This is the case of the red flour beetle Tribolium castaneum, which generates economical loss of great significance in stored product environments worldwide. In this study, T. castaneum adults were fed during different time periods (from 3 to 72 h) on B. bassiana conidia-covered corn kernels. The progression of fungal infection was monitored using the dual RNA-seq technique to reconstruct the temporal transcriptomic profile and to perform gene enrichment analyses in both interacting organisms. After mapping the total reads with the B. bassiana genome, 904 genes were identified during this process. The more expressed fungal genes were related to carbon catabolite repression, cation binding, peptidase inhibition, redox processes, and stress response. Several immune-related genes from Toll, IMD, and JNK pathways, as well as genes related to chitin modification, were found to be differentially expressed in fungus-exposed T. castaneum. This study represents the first dual transcriptomic approach to help understand the interaction between the entomopathogenic fungus B. bassiana and its tolerant host T. castaneum.

Next generation marker-based vector concepts for rapid and unambiguous identification of single and double homozygous transgenic organisms.

For diploid model organisms, the actual transgenesis processes require subsequent periods of transgene management, which are challenging in emerging model organisms due to the lack of suitable methodology. We used the red flour beetle Tribolium castaneum, a stored-grain pest, to perform a comprehensive functional evaluation of our AClashOfStrings (ACOS) and the combined AGameOfClones/AClashOfStrings (AGOC/ACOS) vector concepts, which use four clearly distinguishable markers to provide full visual control over up to two independent transgenes. We achieved comprehensive statistical validation of our approach by systematically creating seventeen novel single and double homozygous sublines intended for fluorescence live imaging, including several sublines in which the microtubule cytoskeleton is labeled. During the mating procedures, we genotyped more than 20,000 individuals in less than 80 working hours, which corresponds to about 10 to 15 s per individual. We also confirm the functionality of our combined concept in two double transgene special cases, i.e. integration of both transgenes in close proximity on the same chromosome and integration of one transgene on the X allosome. Finally, we discuss our vector concepts regarding performance, genotyping accuracy, throughput, resource saving potential, fluorescent protein choice, modularity, adaptation to other diploid model organisms and expansion capability.

Identification of odorant receptors of Tribolium confusum in response to limonene repellent activity.

Tribolium confusum is an important storage pest showing significant resistance to various chemical pesticides, development of botanical pesticides is an effective strategy to resolve above problem and decrease utilization of chemical pesticides. Present study attempts to explore the molecular mechanism about the repellent activity of limonene. When treatment concentration of limonene was 200.00 µg/cm2, the repellent level remained at grade V after 24 hours. Our study showed that limonene could be distinguished by T. confusum antenna with a maximal electroantennography test value of 0.90 mV. Simultaneously, 88 upregulated and 98 downregulated genes were sequenced in limonene-repellent T. confusum, and RT-qPCR analysis showed that four down-regulated and one up-regulated OR genes play an important role in the response to limonene. The repellent rate was decreased by 22.13% mediated with a knockdown of dsTconOR93, while the EAG value of the female and male adults was reduced to 0.26 mV (49.06%) and 0.20 mV (54.05%), respectively. In conclusion, limonene had a strong repellent activity against T. confusum and TconOR93 gene was determined to be a major effector in perception of limonene. This study provides a basis for the development of limonene as a novel botanical pesticide for the control to storage pests, which will reduce the utilization of chemical pesticides and postpone the development of resistance.

Latrophilin, an adhesion GPCR with galactose-binding lectin domain involved in the innate immune response of Tribolium castaneum.

Latrophilin is a member of adhesion GPCRs involved in various physiological pro1cesses. The extracellular fragment of Tribolium castaneum Latrophilin (TcLph) contains a galactose-binding lectin (GBL) domain. However, the biological function of GBL domain remains mysterious. Here, we initially studied the role of TcLph in recognizing pathogens through its GBL domain and then triggering immune defense in invertebrates. Results showed that GBL domain was highly conserved, and its predicted 3D structure was similar to rhamnose-binding lectin domain of mouse Latrophilin-1 with a unique α/ß fold and two long loops. Molecular docking and ELISA results revealed the GBL domain can bind to D-galactose, L-rhamnose, lipopolysaccharide and peptidoglycan. The recombinant extracellular segment of TcLph and the recombinant GBL exhibited strong agglutinating and binding activities to all tested bacteria in a Ca2+-dependent manner. Moreover, TcLph was markedly induced after infection by Escherichia coli or Staphylococcus aureus, while its silencing exacerbated bacterial loads and larvae mortality. TcLph-deficient larvae significantly decreased the transcription levels of antimicrobial peptides and prophenoloxidase activating system-related genes, leading to a significant reduction in phenoloxidase activity. It indicated that TcLph functioned as a pattern recognition receptor in pathogen recognition and activated immune responses to eliminate invasive microbes, suggesting a potential target for insecticides.

Insight into the molecular mechanism of phosphine toxicity provided by functional analysis of cytochrome b5 fatty acid desaturase and dihydrolipoamide dehydrogenase in the red flour beetle, Tribolium castaneum.

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.