NHA1 is a cation/proton antiporter essential for the water-conserving functions of the rectal complex in Tribolium castaneum.

More than half of all extant metazoan species on earth are insects. The evolutionary success of insects is linked with their ability to osmoregulate, suggesting that they have evolved unique physiological mechanisms to maintain water balance. In beetles (Coleoptera)-the largest group of insects-a specialized rectal ("cryptonephridial") complex has evolved that recovers water from the rectum destined for excretion and recycles it back to the body. However, the molecular mechanisms underpinning the remarkable water-conserving functions of this system are unknown. Here, we introduce a transcriptomic resource, BeetleAtlas.org, for the exceptionally desiccation-tolerant red flour beetle Tribolium castaneum, and demonstrate its utility by identifying a cation/H+ antiporter (NHA1) that is enriched and functionally significant in the Tribolium rectal complex. NHA1 localizes exclusively to a specialized cell type, the leptophragmata, in the distal region of the Malpighian tubules associated with the rectal complex. Computational modeling and electrophysiological characterization in Xenopus oocytes show that NHA1 acts as an electroneutral K+/H+ antiporter. Furthermore, genetic silencing of Nha1 dramatically increases excretory water loss and reduces organismal survival during desiccation stress, implying that NHA1 activity is essential for maintaining systemic water balance. Finally, we show that Tiptop, a conserved transcription factor, regulates NHA1 expression in leptophragmata and controls leptophragmata maturation, illuminating the developmental mechanism that establishes the functions of this cell. Together, our work provides insights into the molecular architecture underpinning the function of one of the most powerful water-conserving mechanisms in nature, the beetle rectal complex.

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.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Shaking hands is a homeodomain transcription factor that controls axon outgrowth of central complex neurons in the insect model Tribolium.

Gene regulatory mechanisms that specify subtype identity of central complex (CX) neurons are the subject of intense investigation. The CX is a compartment within the brain common to all insect species and functions as a 'command center' that directs motor actions. It is made up of several thousand neurons, with more than 60 morphologically distinct identities. Accordingly, transcriptional programs must effect the specification of at least as many neuronal subtypes. We demonstrate a role for the transcription factor Shaking hands (Skh) in the specification of embryonic CX neurons in Tribolium. The developmental dynamics of skh expression are characteristic of terminal selectors of subtype identity. In the embryonic brain, skh expression is restricted to a subset of neurons, many of which survive to adulthood and contribute to the mature CX. skh expression is maintained throughout the lifetime in at least some CX neurons. skh knockdown results in axon outgrowth defects, thus preventing the formation of an embryonic CX primordium. The previously unstudied Drosophila skh shows a similar embryonic expression pattern, suggesting that subtype specification of CX neurons may be conserved.

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.

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.

Role of odorant binding protein C12 in the response of Tribolium castaneum to chemical agents.

Tribolium castaneum is a worldwide pest of stored grain that mainly damages flour, and not only causes serious loss of flour quality but also leads to deterioration of flour quality. Chemical detection plays a key role in insect behavior, and the role of odorant-binding proteins (OBPs) in insect chemical detection has been widely studied. OBPs can interact with small molecule compounds and thereby modulate variation in insecticide susceptibility in insects. In this study, a total of 65 small molecule compounds are selected to investigate the bound effect with TcOBP C12. The molecular docking results showed that ß-caryophyllene, (-)-catechin, butylated hydroxytoluene, diphenyl phthalate and quercetin were the top five compounds, with docking binding energies of -6.11, -5.25, -5.09, -5.05, and - 5.03 Kcal/mol, respectively. Molecular dynamics analysis indicated that odorant binding protein C12 (TcOBP C12) exhibited high binding affinity to all five tested chemical ligands, evidenced by fluorescence quenching assay in vitro. In addition, the contact toxicity assay results suggested that these chemical agents caused a dose-dependent increase in mortality rate for T. castaneum adults. The TcOBP C12 gene was upregulated >2 times after a 24-h exposure, indicating that OBP C12 may play an important role for T. castaneum in response to these chemical agents. In conclusion, our results provide a theoretical basis for future insecticide experiments and pest management.

Analyses of ecdysteroid transporters in the fat body of Tribolium castaneum.

The control of insect moulting and metamorphosis involves ecdysteroids that orchestrate the execution of developmental genetic programs by binding to dimeric hormone receptors consisting of the ecdysone receptor (EcR) and ultraspiracle (USP). In insects, the main ecdysteroids comprise ecdysone (E), which is synthesized in the prothoracic gland and secreted into the haemolymph, and 20-hydroxyecdysone (20E), which is considered the active form by binding to the nuclear receptor of the target cell. While biosynthesis of ecdysteroids has been studied in detail in different insects, the transport systems involved in guiding these steroid hormones across cellular membranes have just recently begun to be studied. By analysing RNAi phenotypes in the red flour beetle, Tribolium castaneum, we have identified three transporter genes, TcABCG-8A, TcABCG-4D and TcOATP4-C1, whose silencing results in phenotypes similar to that observed when the ecdysone receptor gene TcEcRA is silenced, that is, abortive moulting and abnormal development of adult compound eyes during the larval stage. The genes of all three transporters are expressed at higher levels in the larval fat body of T. castaneum. We analysed potential functions of these transporters by combining RNAi and mass spectrometry. However, the analysis of gene functions is challenged by mutual RNAi effects indicating interdependent gene regulation. Based on our findings, we propose that TcABCG-8A, TcABCG-4D and TcOATP4-C1 participate in the ecdysteroid transport in fat body cells, which are involved in E → 20E conversion catalysed by the P450 enzyme TcShade.

A novel GSTe2 involved in metamorphosis by regulating 20E signal pathway in Tribolium castaneum.

Insect-specific epsilon glutathion S-transferases (GSTs) are a class of multifunctional GST superfamily, which play important roles in detoxification of xenobiotic substances. Most research on GSTs has focused on insecticide detoxification and resistance, with little research on other physiological functions. Here, we identified and cloned the novel GSTe2 from Tribolium castaneum (TcGSTe2). Recombinant TcGSTe2 protein was successfully overexpressed in Escherichia coli and purified with affinity purification, which had high ability to catalyze the conjugation of reduced glutathione with 1-chloro-2,4-dinitrobenzene (CDNB). The expression level of TcGSTe2 was significantly decreased after exposure with four insecticides, phoxim, λ-cyhalothrin, dichlorvos, and carbofuran, in larval stage. Interestingly, RNA interference knockdown of TcGSTe2 caused metamorphosis deficiency in larval and pupal stages by inhibiting the 20E signal pathway. Furthermore, exogenous 20E injection partially rescued this metamorphosis deficiency and also increased the expression levels of 20E downstream response genes. This study illustrated TcGSTe2 plays an important role at metamorphosis beside the insecticide detoxification and resistance in T. castaneum.

Diet supplementation with egg yolk powder fattens the beetle Tribolium castaneum.

Insects have become essential models in studying human metabolic diseases, mainly due to their low maintenance cost and available tools. Both mutations and modified diets induce metabolic states similar to human obesity and diabetes. Here, we explore the effect of a high-calorie, high-fat diet on the metabolism of the beetle Tribolium castaneum. Supplementation of the wheat flour diet with powdered egg yolk for 3 weeks increased the total triacylglycerol and accelerated larval development. In addition, this diet increased the triacylglycerol levels of adult beetles. However, this egg yolk supplementation did not alter the larvae's total glucose levels or lipogenic capacity and ATP citrate lyase activity. The diet also did not change the expression profile of several lipid and carbohydrate metabolism genes and insulin-like peptides. Thus, we conclude that the diet supplemented with egg yolk induces increased fat without causing diabetes phenotypes, as seen in other hypercaloric diets in insects.

A new neuropeptide insect parathyroid hormone iPTH in the red flour beetle Tribolium castaneum.

In the postgenomics era, comparative genomics have advanced the understanding of evolutionary processes of neuropeptidergic signaling systems. The evolutionary origin of many neuropeptidergic signaling systems can be traced date back to early metazoan evolution based on the conserved sequences. Insect parathyroid hormone receptor (iPTHR) was previously described as an ortholog of vertebrate PTHR that has a well-known function in controlling bone remodeling. However, there was no sequence homologous to PTH sequence in insect genomes, leaving the iPTHR as an orphan receptor. Here, we identified the authentic ligand insect PTH (iPTH) for the iPTHR. The taxonomic distribution of iPTHR, which is lacking in Diptera and Lepidoptera, provided a lead for identifying the authentic ligand. We found that a previously described orphan ligand known as PXXXamide (where X is any amino acid) described in the cuttlefish Sepia officinalis has a similar taxonomic distribution pattern as iPTHR. Tests of this peptide, iPTH, in functional reporter assays confirmed the interaction of the ligand-receptor pair. Study of a model beetle, Tribolium castaneum, was used to investigate the function of the iPTH signaling system by RNA interference followed by RNA sequencing and phenotyping. The results suggested that the iPTH system is likely involved in the regulation of cuticle formation that culminates with a phenotype of defects in wing exoskeleton maturation at the time of adult eclosion. Moreover, RNAi of iPTHRs also led to significant reductions in egg numbers and hatching rates after parental RNAi.

Identification and Characterization of Development-Related microRNAs in the Red Flour Beetle, Tribolium castaneum.

MicroRNAs (miRNAs) play important roles in insect growth and development, but they were poorly studied in insects. In this study, a total of 883 miRNAs were detected from the early embryo (EE), late larva (LL), early pupa (EP), late pupa (LP), and early adult (EA) of Tribolium castaneum by microarray assay. Further analysis identified 179 differentially expressed unique miRNAs (DEmiRNAs) during these developmental stages. Of the DEmiRNAs, 102 DEmiRNAs exhibited stage-specific expression patterns during development, including 53 specifically highly expressed miRNAs and 20 lowly expressed miRNAs in EE, 19 highly expressed miRNAs in LL, 5 weakly expressed miRNAs in EP, and 5 abundantly expressed miRNAs in EA. These miRNAs were predicted to target 747, 265, 472, 234, and 121 genes, respectively. GO enrichment analysis indicates that the targets were enriched by protein phosphorylation, calcium ion binding, sequence-specific DNA binding transcription factor activity, and cytoplasm. An RNA interference-mediated knockdown of the DEmiRNAs tca-miR-6-3p, tca-miR-9a-3p, tca-miR-9d-3p, tca-miR-11-3p, and tca-miR-13a-3p led to defects in metamorphosis and wing development of T. castaneum. This study has completed the identification and characterization of development-related miRNAs in T. castaneum, and will enable us to investigate their roles in the growth and development of insect.

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Response of xenobiotic biodegradation and metabolic genes in Tribolium castaneum following eugenol exposure.

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

MiR-3017b contributes to metamorphosis by targeting sarco/endoplasmic reticulum Ca2+ ATPase in Tribolium castaneum.

In recent years, increasing numbers of microRNAs (miRNAs) have been reported to regulate insect metamorphosis. One thousand, one hundred fifty-four miRNAs have been previously identified from Tribolium castaneum by high-throughput sequencing; however, little is known about which miRNAs can participate in metamorphosis, leaving the role of miRNAs in regulating the underlying mechanism elusive. Here, we report the participation of miR-3017b in the metamorphosis of T. castaneum. Temporal profiles revealed that miR-3017b was highly expressed at the late larval stage, but significantly decreased at the early pupal stage. Overexpression of miR-3017b caused larval to pupal to adult metamorphosis arrested. Dual-luciferase reporter assay and miRNA-mRNA interaction assay illustrated that miR-3017b interacts with the coding sequence of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) and suppresses its expression. Knockdown of SERCA caused metamorphosis arrested, similar to that observed in miR-3017b overexpression beetles. Further functional mechanism analyses revealed that 20-hydroxyecdysone application downregulates miR-3017b and up-regulates SERCA expression. The expression level of downstream genes in the 20E pathway was disrupted after overexpressing miR-3017 and the knockdown of SERCA. These results provided evidence miR-3017b-SERCA contributes to metamorphosis by regulating the 20E pathway in T. castaneum. It could advance our understanding of the coordination of 20E and miRNA regulation in insect metamorphosis.

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity.

Telomerase, a unique reverse transcriptase that specifically extends the ends of linear chromosomes, is up-regulated in the vast majority of cancer cells. Here, we show that an indole nucleotide analog, 5-methylcarboxyl-indolyl-2'-deoxyriboside 5'-triphosphate (5-MeCITP), functions as an inhibitor of telomerase activity. The crystal structure of 5-MeCITP bound to the Tribolium castaneum telomerase reverse transcriptase reveals an atypical interaction, in which the nucleobase is flipped in the active site. In this orientation, the methoxy group of 5-MeCITP extends out of the canonical active site to interact with a telomerase-specific hydrophobic pocket formed by motifs 1 and 2 in the fingers domain and T-motif in the RNA-binding domain of the telomerase reverse transcriptase. In vitro data show that 5-MeCITP inhibits telomerase with a similar potency as the clinically administered nucleoside analog reverse transcriptase inhibitor azidothymidine (AZT). In addition, cell-based studies show that treatment with the cell-permeable nucleoside counterpart of 5-MeCITP leads to telomere shortening in telomerase-positive cancer cells, while resulting in significantly lower cytotoxic effects in telomerase-negative cell lines when compared with AZT treatment.

The Mitochondrial Phosphatase PTPMT1 is Required for the Proper Growth Rate in the Red Flour Beetle, Tribolium castaneum.

Protein tyrosine phosphatase, mitochondrial 1 (PTPMT1) is a mitochondrial phosphatase that is highly conserved in animals. Functional analyses using knockout animals have revealed a variety of physiological roles of PTPMT1 in vertebrates and insects. However, because of the high lethality of knockout in these animals, the roles of PTPMT1 in the later postembryonic development remain relatively obscure. In the present study, using the RNA interference technique, we analyzed PTPMT1 functions in later larval stages of the red flour beetle, Tribolium castaneum. PTPMT1 was expressed in both anterior and posterior parts of the body constitutively without obvious fluctuations from the middle larval instar through pupation. The PTPMT1-knockdown larvae injected with PTPMT1 double-stranded RNA at the middle instar showed a prolonged larval period, which was mainly caused by an extra larval molt. On the other hand, the increase in adult body length was subtle in the PTPMT1-knockdown T. castaneum, and the head capsule width was smaller than that of the control animals at the same larval instar. The expression levels of genes encoded by the mitochondrial genome were reduced in PTPMT1-knockdown larvae, indicating that PTPMT1 plays an important role in mitochondrial function in T. castaneum, like in other species. By contrast, the expression levels of a juvenile hormone (JH)-biosynthetic gene and a JH-signaling gene were rather increased in the PTPMT1-knockdown larvae, which may have been caused indirectly by the reduction of larval growth rate. Altogether, these findings indicate that PTPMT1 is required for the proper growth rate via some mitochondrial physiological role in T. castaneum larvae.

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.

Amplification refractory mutation system based real-time PCR (ARMS-qPCR) for rapid resistance characterization of Tribolium castaneum to phosphine.

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.

Effects of defect action level of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) fragments on quality of wheat flour.

BACKGROUND: Tribolium castaneum (Herbst) is one of the most important secondary storage pests of all types of flour and flour-based products. The present study focuses on the fragment producing behaviour of T. castaneum in wheat flour during storage and its effect on the quality parameters and defect action level (DAL) of fragments. The US Food and Drug Administration has set a DAL of 75 insect fragments in 50 g of flour. Box-Behnken design was used to optimize the storage conditions (storage period in days and temperature in degrees Celsius) and insect density (numbers) to keep insect fragments below the DAL. RESULTS: Optimization results indicated that the presence of single number of adult of T. castaneum is enough to cross the DAL of insect fragments within a storage period of 21 days at a storage temperature of 30 °C. Insect fragments cause perceptible changes in the quality of wheat flour. When sample attained DAL of T. castaneum fragments in wheat flour,the various quality parameters were analysed in that moisture content of wheat flour was 10.8 ± 0.26%, total colour change was 2.052 (ΔE value), T. castaneum progeny emergence was 19.66 ± 1, uric acid was 1.8 ± 0.16 g kg-1 and microbial count was 7.34 ± 0.5 cfu g-1 . CONCLUSIONS: Results from the present study indicate that the presence of even a single adult of stored pest in wheat flour should not be ignored. It is mandatory to determine the threshold level and frequent sampling is required to achieve zero tolerance of stored product insects in food commodities. © 2021 Society of Chemical Industry.