Novel Inhibitor of Glutamate-Cysteine Ligase Catalytic Subunit against Tribolium castaneum: High-Throughout Virtual Screening, Molecular Docking and Dynamics Simulation, and Bioassay.

The enzyme glutamate-cysteine ligase catalytic subunit (Gclc) is a rate-limiting enzyme in the biosynthesis of glutathione that is involved in antioxidant defense, detoxification of xenobiotics, and/or its metabolites and regulates the cell cycle and immune function. Therefore, Gclc presents an appealing target for the development of novel insecticides. In this study, we conducted high-throughput virtual screening from the ZINC20 database and identified three compounds with high binding affinity to the Tribolium castaneum Gclc (TcGclc). Ultimately, we selected ZINC000032992384 due to its superior stability and lowest binding energy, as determined through molecular dynamics simulations. Bioassay results revealed that the IC50 value of ZINC000032992384 was 19.70 µM lower than that of BSO (49.67 µM). Furthermore, the larval mortality in the ZINC000032992384 treated group was 63.8%, significantly higher than that of the controls (29.1% in the dichlorvos group and 6.4% in the acetone group). This study provides novel insights for the development of a Gclc-targeted inhibitor as a potent insecticide based on the interaction between receptors and ligands.

Characterization of double-stranded RNA and its silencing efficiency for insects using hybrid deep-learning framework.

RNA interference (RNAi) technology is widely used in the biological prevention and control of terrestrial insects. One of the main factors with the application of RNAi in insects is the difference in RNAi efficiency, which may vary not only in different insects, but also in different genes of the same insect, and even in different double-stranded RNAs (dsRNAs) of the same gene. This work focuses on the last question and establishes a bioinformatics software that can help researchers screen for the most efficient dsRNA targeting target genes. Among insects, the red flour beetle (Tribolium castaneum) is known to be one of the most sensitive to RNAi. From iBeetle-Base, we extracted 12 027 efficient dsRNA sequences with a lethality rate of ≥20% or with experimentation-induced phenotypic changes and processed these data to correspond to specific silence efficiency. Based on the first complied novel benchmark dataset, we specifically designed a deep neural network to identify and characterize efficient dsRNA for RNAi in insects. The dna2vec word embedding model was trained to extract distributed feature representations, and three powerful modules, namely convolutional neural network, bidirectional long short-term memory network, and self-attention mechanism, were integrated to form our predictor model to characterize the extracted dsRNAs and their silencing efficiencies for T. castaneum. Our model dsRNAPredictor showed reliable performance in multiple independent tests based on different species, including both T. castaneum and Aedes aegypti. This indicates that dsRNAPredictor can facilitate prescreening for designing high-efficiency dsRNA targeting target genes of insects in advance.

A new trypsin inhibitor from Centrosema plumieri effective against digestive proteases from Tribolium castaneum, an eco-friendly alternative.

Tribolium castaneum, also known as the red flour beetle, is a polyphagous pest that seriously damages agricultural products, including stored and processed grains. Researchers have aimed to discover alternative pest control mechanisms that are less harmful to the ecosystem than those currently used. We conduct the purification and characterization of a protease inhibitor from C. plumieri seeds and an in vitro evaluation of its insecticidal potential against the insect pest T. castaneum. The trypsin inhibitor was isolated from C. plumieri seeds in a single-step DEAE-Sepharose column chromatography and had a molecular mass of 50 kDA. When analyzed for interaction with different proteolytic enzymes, the inhibitor exhibited specificity against trypsin and no activity against other serine proteases such as chymotrypsin and elastase-2. The isolated inhibitor was able to inhibit digestive enzymes of T. castaneum from extracts of the intestine of this insect. Therefore, we conclude that the new protease inhibitor, specific in tryptic inhibition, of protein nature from the seeds of C. plumieri was effective in inhibiting the digestive enzymes of T. castaneum and is a promising candidate in the ecological control of pests.

Density-mediated foraging behavioral responses of Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae).

Tribolium castaneum and Rhyzopertha dominica are cosmopolitan, destructive postharvest pests. Although research has investigated how high densities of T. castaneum affect attraction to the aggregation pheromone by conspecifics, research into the behavioral response of both species to food cues after high density exposure has been lacking despite its importance to foraging ecology. Our goal was to manipulate and observe the effects of crowding on the behavioral response of both species to common food and pheromonal stimuli and to determine how the headspace emission patterns from grain differed under increasing densities. Densities of colonies for both species was altered (10-500 adults) on a fixed quantity of food (10 g of flour or whole wheat), then the behavioral response to common food and pheromonal cues was evaluated in a wind tunnel and release-recapture experiment, while volatiles were examined through gas chromatography coupled with mass spectrometry. Importantly, at least for T. castaneum, crowded conditions attenuate attraction to food-based stimuli, but not pheromonal stimuli. Crowding seemed to have no effect on R. dominica attraction to food and pheromonal stimuli in the wind tunnel, but exposure to high density cues did elicit 2.1-3.8-fold higher captures in traps. The relative composition and abundance of headspace volatiles emitted varied significantly with different densities of beetles and was also species-specific. Overall, our results have implications for expanding our understanding of the foraging ecology of two economically important pests.

Diatomaceous earth as insecticide: physiological and morphological evidence of its underlying mechanism.

BACKGROUND: Wheat grain containers or silos can be perfect habitats for insects, which generate large economic losses to grain production. Natural alternatives to synthetic insecticides have grown in popularity because of health, economic and ecological issues. Diatomaceous earth is a natural compound that has an insecticide effect by enhancing an insect's dehydration with no toxicity on mammals including humans. The aim of this study is to confirm the effect of diatomaceous earth as an insecticide for the wheat grain pest, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae) and demonstrate its underlying mechanisms as an insecticide by open-flow respirometry and scanning electron microscopy. RESULTS: Survival bioassays of T. castaneum revealed a dose-dependent insecticide effect of diatomaceous earth. Gravimetric measurements showed that 2 days exposure to diatomaceous earth produces a significant increase of mass loss. Open-flow respirometry measurements showed an increase of total water emission rate on insects due to an increase of both, respiratory and cuticular water loss. Our study revealed that diatomaceous earth produces an increase of insect's cuticle permeability, which is responsible for elevated cuticular water loss. Scanning electron microscopy images provided visual evidence of the lipid absorbent properties of diatomaceous earth particles, and showed a tendency for higher, although not significant, damaged area of the cuticle's surface from diatomaceous earth treated insects compared to control ones. CONCLUSION: With state-of-the art techniques like open-flow respirometry and scanning electron microscopy, we demonstrated the underlying mechanism of diatomaceous earth as an insecticide and provided new cues for understanding the properties of the cuticle and its ecological importance. © 2024 Society of Chemical Industry.

The impact of UV-C radiation on the sugar metabolism of the red flour beetle Tribolium castaneum herbst (coleoptera; tenebrionidae).

PURPOSE: Ultraviolet-C (UV-C) is known to induce morphological abnormality in various parts of the red flour beetle, Tribolium castaneum, including its wings, antennae, eyes, legs, and reproductive organs. However, little is known about the effects of UV-C on T. castaneum's sugar content and enzyme activity. MATERIAL AND METHODS: We investigated the concentrations of glucose and trehalose as well as changes in trehalase activity in different developmental stages following UV-C radiation at different exposure periods (1, 2, 4, 8, 16, 32, and 64 min). In addition, the larval mortality and body weight were examined. RESULTS: A reduction in glucose content was recorded in 10-, 15- and 20-day-old larvae and trehalase enzyme activity was recorded in 5- and 10-day-old larvae, whereas an increase in trehalose content was found in adults irradiated with UV-C. In addition, UV-C radiation for 1-64 min caused larval mortality on the first and subsequent days post-irradiation. Moreover, UV-C irradiated larvae exhibited lower body weight, which aligned with the reduction of trehalase activity and glucose content from days 1-6 post-exposure, and the degree of these reductions corresponded to the exposure times. CONCLUSION: UV-C affected sugar content through the reduction of trehalase activity, and glucose declination may cause mortality in T. castaneum; however, further research is needed to provide a better understanding of the impact of UV-C on sugar metabolism.

Gut Bacteria Promote Phosphine Susceptibility of Tribolium castaneum by Aggravating Oxidative Stress and Fitness Costs.

Knowledge about resistance mechanisms can provide ideas for pesticide resistance management. Although several studies have unveiled the positive or negative impacts of gut microbes on host pesticide resistance, minimal research is available regarding the association between gut microbes and host phosphine resistance. To explore the influence of gut bacteria on host phosphine susceptibility and its molecular basis, mortality, fitness, redox responses, and immune responses of adult Tribolium castaneum were determined when it was challenged by phosphine exposure and/or gut bacteria inoculation. Five cultivable gut bacteria were excised from a population of phosphine-resistant T. castaneum. Among them, only Enterococcus sp. inoculation significantly promoted host susceptibility to phosphine, while inoculation of any other gut bacteria had no significant effect on host phosphine susceptibility. Furthermore, when T. castaneum was exposed to phosphine, Enterococcus sp. inoculation decreased the female fecundity, promoted host oxidative stress, and suppressed the expression and activity of host superoxide dismutase, catalase, and peroxidase. In the absence of phosphine, Enterococcus sp. inoculation also elicited overactive immune responses in T. castaneum, including the immune deficiency and Toll signaling pathways and the dual oxidase-reactive oxygen species system. These results indicate that Enterococcus sp. likely promotes host phosphine susceptibility by aggravating oxidative stress and fitness costs.

Zinc Oxide Nanoparticles Biosynthesized by Eriobotrya japonica Leaf Extract: Characterization, Insecticidal and Antibacterial Properties.

Zinc oxide nanoparticles (ZnO-NPs) have gained significant attention in nanotechnology due to their unique properties and potential applications in various fields, including insecticidal and antibacterial activities. The ZnO-NPs were biosynthesized by Eriobotrya japonica leaf extract and characterized by various techniques such as UV-visible (UV-vis) spectrophotometer, X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and zeta potential analysis. The results of SEM revealed that NPs were irregular and spherical-shaped, with a diameter between 5 and 27 nm. Meanwhile, DLS supported that the measured size distributions were 202.8 and 94.7 nm at 11.1° and 90.0°, respectively, which supported the polydisperse nature of NPs, and the corresponding zeta potential was -20.4 mV. The insecticidal activity of the produced ZnO-NPs was determined against the adult stage of coleopteran pests, Sitophilus oryzae (Linnaeus) (Curculionidae) and Tribolium castaneum (Herbst) (Tenebrionidae). The LC50 values of ZnO-NPs against adults of S. oryzae and T. castaneum at 24 h of exposure were 7125.35 and 5642.65 µg/mL, respectively, whereas the LC90 values were 121,824.56 and 66,825.76 µg/mL, respectively. Moreover, the biosynthesized nanoparticles exhibited antibacterial activity against three potato bacterial pathogens, and the size of the inhibition zone was concentration-dependent. The data showed that the inhibition zone size increased with an increase in the concentration of nanoparticles for all bacterial isolates tested. The highest inhibition zone was observed for Ralstonia solanacearum at a concentration of 5 µg/mL, followed by Pectobacterium atrosepticum and P. carotovorum. Eventually, ZnO-NPs could be successfully used as an influential agent in pest management programs against stored-product pests and potato bacterial diseases.

N6-adenosine (m6A) mRNA methylation is required for Tribolium castaneum development and reproduction.

Gene expression is regulated at various levels, including post-transcriptional mRNA modifications, where m6A methylation is the most common modification of mRNA. The m6A methylation regulates multiple stages of mRNA processing, including splicing, export, decay, and translation. How m6A modification is involved in insect development is not well known. We used the red flour beetle, Tribolium castaneum, as a model insect to identify the role of m6A modification in insect development. RNA interference (RNAi)-mediated knockdown of genes coding for m6A writers (m6A methyltransferase complex, depositing m6A to mRNA) and readers (YTH-domain proteins, recognizing and executing the function of m6A) was conducted. Knockdown of most writers during the larval stage caused a failure of ecdysis during eclosion. The loss of m6A machinery sterilized both females and males by interfering with the functioning of reproductive systems. Females treated with dsMettl3, the main m6A methyltransferase, laid significantly fewer and reduced-size eggs than the control insects. In addition, the embryonic development in eggs laid by dsMettl3 injected females was terminated in the early stages. Knockdown studies also showed that the cytosol m6A reader, YTHDF, is likely responsible for executing the function of m6A modifications during insect development. These data suggest that m6A modifications are critical for T. castaneum development and reproduction.

Immediate and delayed movement of resistant and susceptible adults of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) after short exposures to phosphine.

BACKGROUND: During the last decade, the evaluation of certain behavioral attributes has been utilized as an indicator of resistance to phosphine. In this context, an underappreciated challenge may be the development of behavioral traits that are related with resistance to phosphine such as the movement to refugia and recovery of stored product insects after short exposures. Thus, the aim of the current study was to track the movement of phosphine-resistant and -susceptible adults of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), which is a major pest of stored products, after brief exposures to phosphine. Exposures were followed for extended intervals to assess the recovery patterns and how those patterns are related to known resistance to phosphine. A video-tracking procedure coupled with Ethovision software was used to assess movement after exposure. RESULTS: Overall, we found baseline movement was less for phosphine-resistant T. castaneum, suggesting resistance comes at a considerable fitness cost. In the presence of phosphine (1000 or 3000 ppm), there was a much greater reduction in movement for phosphine-susceptible than phosphine-resistant T. castaneum adults immediately after brief 5-min exposures. Twenty-four hours later, these effects were more variable and less apparent, regardless of the susceptibility level. CONCLUSIONS: The initial knockdown associated with successful fumigation may just be a temporary state whereafter insects shortly resume movement and may be able to seek out refugia from phosphine, thereby promoting the development of resistance. Our results strengthen a growing consensus that it is the speed to knockdown that truly matters, with quick knockdown indicating slow recovery and a reduced likelihood for the occurrence of resistance. © 2023 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Transcriptomic exploration of the Coleopteran wings reveals insight into the evolution of novel structures associated with the beetle elytron.

The acquisition of novel traits is central to organismal evolution, yet the molecular mechanisms underlying this process are elusive. The beetle forewings (elytra) are evolutionarily modified to serve as a protective shield, providing a unique opportunity to study these mechanisms. In the past, the orthologs of genes within the wing gene network from Drosophila studies served as the starting point when studying the evolution of elytra (candidate genes). Although effective, candidate gene lists are finite and only explore genes conserved across species. To go beyond candidate genes, we used RNA sequencing and explored the wing transcriptomes of two Coleopteran species, the red flour beetle (Tribolium castaneum) and the Japanese stag beetle (Dorcus hopei). Our analysis revealed sets of genes enriched in Tribolium elytra (57 genes) and genes unique to the hindwings, which possess more "typical" insect wing morphologies (29 genes). Over a third of the hindwing-enriched genes were "candidate genes" whose functions were previously analyzed in Tribolium, demonstrating the robustness of our sequencing. Although the overlap was limited, transcriptomic comparison between the beetle species found a common set of genes, including key wing genes, enriched in either elytra or hindwings. Our RNA interference analysis for elytron-enriched genes in Tribolium uncovered novel genes with roles in forming various aspects of morphology that are unique to elytra, such as pigmentation, hardening, sensory development, and vein formation. Our analyses deepen our understanding of how gene network evolution facilitated the emergence of the elytron, a unique structure critical to the evolutionary success of beetles.

Acute toxicity of Zanthoxylum bungeanum against two stored product insects and synergistic interactions between two major compounds limonene and linalool.

In order to find and develop new botanical pesticides against storage pests, components of the essential oil (EO) from Zanthoxylum bungeanum were identified by GC-MS and their insecticidal activity against the stored product pests were studied. The EO was obtained by steam distillation. Results showed that EO was rich in limonene (23.67), linalool (21.76) and linalyl anthranilate (10.87). In contact assays, linalool exhibited strongest toxicity to red flour beetle adult (LD50 = 17.06 µg/adult) and larvae (LD50 = 16.42 µg/larvae), and linalool was the most active one against the Lasioderma serricorne (LD50 = 15.36 µg/larvae). Then limonene and linalool showed different levels of fumigant activities against the two insect species. Synergism effect existed in the proportion of contact assays against Tribolium castaneum adults, and additive was observed in the proportion of 7:1 against T. castaneum larvae. This work provides important information for the development and utilization of Z. bungeanum and suggests that the EO of Z. bungeanum has the potential to serve as bio-insecticides for controlling pest damage in stored products.

Mobility of Phosphine-Susceptible and -Resistant Rhyzopertha dominica (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) After Exposure to Controlled Release Materials With Existing and Novel Active Ingredients.

There is interest in developing controlled release materials (CRMs) with novel modes of action to improve resistance management. Long-lasting insecticide-incorporated netting (LLIN) with deltamethrin has been effectively used against stored-product pests. Here, we evaluated the efficacy of different CRMs (LLIN or packaging) with each of four active ingredients (AI) (deltamethrin, permethrin, indoxacarb, and dinotefuran) and compared them to control CRMs in reducing movement and increasing mortality of phosphine-susceptible and -resistant Rhyzopertha dominica and Tribolium castaneum. Adults were exposed for 0.5, 2, or 60 min, and movement was assessed immediately or after 24, or 168 h using video-tracking and Ethovision software. We recorded total distance and velocity traveled by adults. Finally, we tested higher rates of each AI on surrogate netting material (e.g., standardized-sized cheesecloth) and varied exposure time to obtain median lethal time (LT50) for each compound and susceptibility. Exposure to LLIN with deltamethrin significantly reduced the movement of both species compared to the other CRMs regardless of their susceptibility to phosphine. Deltamethrin was the most effective AI for both species, while dinotefuran and indoxacarb were the least effective for R. dominica and T. castaneum adults, respectively. Most AIs resulted in appreciable and approximately equivalent mortality at higher concentrations among phosphine-susceptible and -resistant strains. Our results demonstrate that CRMs can be an additional approach to combat phosphine-resistant populations of stored product insects around food facilities. Other compounds such as permethrin, dinotefuran, and indoxacarb are also effective against phosphine-resistant populations of these key stored product insects except indoxacarb for T. castaneum.

A Biomass Pyrolysis Oil as a Novel Insect Growth Regulator Mimic for a Variety of Stored Product Beetles.

As fumigants face increasing regulatory restrictions, resistance, and consumer pushback, it is vital to expand the integrated pest management (IPM) chemical toolkit for stored products. The production of biomass derived insecticides (e.g., bio-oil fraction) from byproducts of biofuel production may be a promising alternative source of chemistries for controlling stored product insects. These potential insecticidal bio-oils were fractionated based on boiling points (ranging from 115 to 230°C in one series and 245-250°C in another). Fractions were analyzed using GC-MS, and were found to be unique in composition. The lethality of these fractions was tested on Tribolium castaneum, Tribolium confusum, and Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae). Fractions were tested at concentrations ranging from 5-260 mg/ml to screen for efficacy against adults for durations of 2-8 hr sprayed on concrete arenas. In addition, a separate assay evaluated adult emergence of larvae after 6 wk with supplemental food in arenas, while repellency was evaluated against four stored product insect species in a laminar wind tunnel. A greenhouse gas (GHG) emissions life cycle assessment was also performed, which found the use of the bio-oil fraction could reduce GHG emissions associated with the insecticide supply chain by 25-61% relative to a fossil-fuel based insecticide or pyrethroid. While adults were largely unaffected, we found that larval emergence was significantly suppressed compared to controls by roughly half or more. We also determined that there was minimal repellency to most fractions by most species. We conclude that the use of bio-oil fractions is a climate-friendly choice that may support IPM programs.

Demographic responses of Tribolium castaneum (Coleoptera: Tenebrionidae) to different temperatures in soft wheat flour.

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the most dangerous insects of a wide spectrum of stored products around the globe. The population growth of this species is affected by temperature. However, there are no data on comparative demographic parameters (i.e., net reproductive rate, intrinsic rate of increase, finite rate of increase, mean generation time and doubling time) in different temperatures, parameters that allow the in-depth exploration of its survival, mortality and reproduction patterns. This study evaluated egg-to-adult development, adult mortality and female fecundity on white soft wheat flour at 20, 25, 30 and 32.5 °C. The net reproductive rate increased from 0.08 females/female at 20 °C to 11.77 females/female at 25 °C and 102.07 females/female at 30 °C, followed by a decrease to 10.73 females/female at 32.5 °C. The lowest values of the intrinsic rate of increase and the finite rate of increase were observed at 20 °C (- 0.0105 females/female/day and 0.9895, respectively) and the highest at 30 °C (0.0348 females/female/day and 1.0354, respectively). While the mean generation time did not differ significantly between 20 and 25 °C (249.9 and 225.5 days, respectively), this decreased to 132.8 and 115.1 days at 30 and 32.5 °C, respectively. The value of the doubling time was negative at 20 °C (- 67.5 days), increased to 19.9, 34.0 and 63.9 days at 30, 32.5 and 25 °C, respectively. Using the non-linear Briere model, the lower threshold for T. castaneum population increase was estimated to be 22.2 °C, the upper threshold at 33.2 °C, and the temperature for maximum growth rate was 30.1 °C. Survival analysis indicated that temperature also affected the mortality risk of T. castaneum. The mean survival time increased from 112.1 days at 20 °C to 462.4 days at 25 °C, followed by a decrease to 206.5 and 64.5 days at 30 and 32.5 °C, respectively. We expect these results to be useful for the prediction of the population growth, the potential expansion and consequently management of T. castaneum.

Nuclear receptor HR96 up-regulates cytochrome P450 for insecticide detoxification in Tribolium castaneum.

BACKGROUND: Red flour beetle, Tribolium castaneum (T. castaneum), is a major agricultural pest that causes significant damage to stored grains and products. Although hormone receptor 96 (HR96) is known to be the single ortholog corresponding to mammalian constitutive androstane receptor and pregnane X receptor, the structural features of Tribolium HR96 (TcHR96) and its role in insecticide-mediated transcription control of cytochrome P450 enzyme genes in T. castaneum have not been elucidated yet. RESULTS: We cloned full-length complementary DNA encoding TcHR96 and revealed the role of TcHR96 in transcriptional control of cytochrome P450 enzyme genes. Interestingly, genome-wide transcriptome analysis of HR96-deficient beetles using RNA sequencing showed a positive correlation between TcHR96 and gene transcription of metabolizing enzymes involved in phase I detoxification processes. Moreover, TcHR96 overexpression significantly increased the promoter activity of genes encoding phase I P450 enzymes such as CYP4Q4, CYP4G7, CYP4BR3, and CYP345A1. Chromatin immunoprecipitation analysis showed that TcHR96 could directly bind to the promoter of gene encoding CYP345A1, an enzyme for metabolizing insecticides in T. castaneum. Furthermore, imidacloprid, a neonicotinoid insecticide, significantly increased gene expression of phase I P450 enzymes in old larvae of T. castaneum, which were reversed by TcHR96 knockdown. Finally, TcHR96 knockdown significantly decreased the resistance of old larvae to imidacloprid concomitant with reduction of imidacloprid-mediated phase I P450 enzyme gene expression. CONCLUSION: TcHR96 plays a major role in transcriptional control of P450 enzyme for imidacloprid detoxification. Controlling TcHR96 might facilitate the regulation of insecticide tolerance in T. castaneum, thus providing a promising new strategy to manage pest beetle populations. © 2021 Society of Chemical Industry.

Valorization Potential of a Novel Bacterial Strain, Bacillus altitudinis RSP75, towards Lignocellulose Bioconversion: An Assessment of Symbiotic Bacteria from the Stored Grain Pest, Tribolium castaneum.

Bioconversion of lignocellulose into renewable energy and commodity products faces a major obstacle of inefficient saccharification due to its recalcitrant structure. In nature, lignocellulose is efficiently degraded by some insects, including termites and beetles, potentially due to the contribution from symbiotic gut bacteria. To this end, the presented investigation reports the isolation and characterization of cellulolytic bacteria from the gut system of red flour beetle, Tribolium castaneum. Out of the 15 isolated bacteria, strain RSP75 showed the highest cellulolytic activities by forming a clearance zone of 28 mm in diameter with a hydrolytic capacity of ~4.7. The MALDI-TOF biotyping and 16S rRNA gene sequencing revealed that the strain RSP75 belongs to Bacillus altitudinis. Among the tested enzymes, B. altitudinis RSP75 showed maximum activity of 63.2 IU/mL extract for xylanase followed by ß-glucosidase (47.1 ± 3 IU/mL extract) which were manifold higher than previously reported activities. The highest substrate degradation was achieved with wheat husk and corn cob powder which accounted for 69.2% and 54.5%, respectively. The scanning electron microscopy showed adhesion of the bacterial cells with the substrate which was further substantiated by FTIR analysis that depicted the absence of the characteristic cellulose bands at wave numbers 1247, 1375, and 1735 cm-1 due to hydrolysis by the bacterium. Furthermore, B. altitudinis RSP75 showed co-culturing competence with Saccharomyces cerevisiae for bioethanol production from lignocellulose as revealed by GC-MS analysis. The overall observations signify the gut of T. castaneum as a unique and impressive reservoir to prospect for lignocellulose-degrading bacteria that can have many biotechnological applications, including biofuels and biorefinery.

Knockdown of a ß-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a ß-adrenergic-like octopamine receptor (TcOctß2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctß2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

A Molecular Approach for the Detection and Quantification of Tribolium castaneum (Herbst) Infestation in Stored Wheat Flour.

RESEARCH BACKGROUND: The presence of insect fragments is one of the major constrains in stored food commodities and it causes considerable loss in the quality of the produce. The management of the pest is viewed as a huge challenge in foodprocessingindustry. Conventionally, the detection of Tribolium castenaum in the food processing industry is carried out by acid hydrolysis and staining methods that are time consuming and lack precision. EXPERIMENTAL APPROACH: Considering the importance of a quick and effective method, a quantitative polymerase chain reaction (qPCR)-based approach was developed and elucidated in this study. The mitochondrial cytochrome oxidase I (mtCOI) gene was identified as a target due to its abundance in the pest. Specific primers were designed against the target gene by Primer Premier software and amplified in a qPCR. RESULTS AND CONCLUSIONS: This method is capable of detecting all the ontogenic stages of T. castaneum in stored wheat flour. Earlier experiments had demonstrated that about 20 µg of DNA can be obtained from 2.2 mg of insects. To quantify the infestation levels, the cycle threshold (Ct) values obtained from known samples were subjected to regression analysis and expressed as adult equivalents. In the unknown samples, the infestation was calculated as 1.74 and 0.046 adult insects in 5 g of wheat flour. The maximum permissible limit of insect fragments in flour is 75 insect fragments or approx. 3 adults per 50 g of flour as per the US Food and Drug Administration (FDA). Hence, by adopting this new method, it is possible for the warehouse operators to arrive at a decision to proceed with efficient management practices where wheat flour is stored. Also, this method can be ratified by government agencies associated with international business to ascertain whether the wheat flour meets the standards set by the respective country before subjecting to foreign trade. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study is the first of its kind in the detection and quantification of T. castaneum in milled products. So far, only conventional methods have been employed to assess the presence of the pests and manual counting of fragments are practiced to quantify the infestation levels. The developed qPCR method is faster, reliable and can be employed in milling industries, bakery industries, food processing plants and foreign trade units for critical detection and quantification of T. castaneum pest infestation.

Detection of Phosphine Resistance in Field Populations of Four Key Stored-Grain Insect Pests in Pakistan.

In Pakistan, the control of stored-product insect pests mainly relies on the use of phosphine gas along with other control tactics. The aim of this study was to determine the level of phosphine resistance among ten differently located populations of the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae), the granary weevil, Sitophilus granarius (L.) (Coleoptera: Curculionidae), the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and the khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae). Laboratory-susceptible populations of all insect species were also considered in the experiments. Concentration-response bioassays were conducted for each species. All of the tested populations (10 out of 10) of each species were found to be resistant to phosphine, but varied in their level of resistance. Probit analysis estimated LC50 at 2.85, 1.90, 2.54 and 2.01 ppm for laboratory-susceptible populations of R. dominica, S. granarius, T. castaneum and T. granarium, respectively. Against R. dominica, the highest and lowest resistance levels were observed in the Rahim Yar Khan (LC50 at 360.90 ppm) and Rawalpindi (LC50 at 210.98 ppm) populations, respectively. These resistant populations were 126.67- and 74.02-fold more resistant than the laboratory population. The Multan and Lahore populations of S. granarius exhibited the maximum (LC50 at 122.81 ppm) and minimum (LC50 at 45.96 ppm) resistance levels, respectively, i.e., they were 64.63- and 24.18-fold more resistant than the laboratory population. The Layyah population of T. castaneum showed the maximum resistance level (LC50 at 305.89 ppm) while the lowest was observed in the Lahore population (LC50 at 186.52 ppm), corresponding to 120.42- and 73.43-fold more resistant than the laboratory population, respectively. Regarding T. granarium, the Layyah population showed the maximum resistance level (LC50 at 169.99 ppm) while the Lahore population showed the minimum resistance (LC50 at 74.50 ppm), i.e., they were 84.57- and 37.06-fold more resistant than the laboratory population, respectively. Overall, R. dominica presented the highest resistance level, followed by T. castaneum, T. granarium and S. granarius. The current study suggests that the application of phosphine may not be an adequate control strategy for the management of the above tested insect pests in Pakistan.