Actividad repelente e insecticida de cuatro aceites esenciales de plantas recolectadas en Chocó-Colombia contra Tribolium castaneum / Repellent and insecticidal activity of four essential oils from plants recolleted in Chocó-Colombia against Tribolium castaneum

This study aimed to determine the repellent and insecticidal activity of four essential oils (EOs) from plants collected in the Chocó rain forest, Colombia, against T. castaneum . Conventional hydrodistillation was used to obtain the EOs. The repellent and insecticidal activities were evaluated by the preference area and gas dispersion methods, espectively. Statistical differences (p<0.05) were determined by applying a student's t-test. EOs of Siparuna guianensis, S. conica, Piper marginatum, and Nectandra acutifolia showed excellent repellent properties as the main findings, highlighting S. conicaEO with 84% repellency (1-hµL/cm2), while P. marginatum showed to be bioactive to the dose of 500 µL/mL (72 h), inducing mortality of 100% of the exposed population. In conclusion, the results evidenced the repellent properties of the EOs evaluated against T. castaneum , which allows us to conclude that these plant species are potential natural sources producing bio-repellents that contribute to the integrated control of T. castaneum.

Se evaluaron cuatro aceites esenciales (AEs) de plantas recolectadas en la selva pluvial del Chocó, Colombia, para determinar su actividad repelente e insecticida contra T. castaneum. Los AEs fueron obtenidos por hidrodestilación convencional. Las actividades repelentes e insecticidas se evaluaron por los métodos de área de preferencia y dispersión de gas, respectivamente. Las diferencias significativas (p<0,05) fueron determinadas aplicando una prueba t de student. Los AEs de Siparuna guianensis, S. conica, Piper marginatum y Nectandra acutifolia mostraron excelentes propiedades repelentes, destacando el AE de S. conicacon un 84% de repelencia (1µL/cm2), mientras que el AE de P. marginatummostró ser bioactivo a la dosis de 500 µL/mL (72 h) al inducir la mortalidad del 100% de la población expuesta. Se concluye que estas especies de plantas son fuentes naturales potencialmente viables para la producción de biorepelentes que contribuyan en el control integrado de T. castaneum.

Boron compounds are effective on Tribolium castaneum (Coleoptera: Tenebrionidae): Reduced lipogenesis and induced body weight loss.

In the current study, we investigated the insecticidal efficacy of two borates, disodium octaborate tetrahydrate (Etidot-67) and calcium metaborate (CMB) via surface application or diet delivery on the red flour beetle, Tribolium castaneum (Herbst, 1797) (Coleoptera: Tenebrionidae). The application method did not change the boron-related mortality, but CMB was more effective than Etidot-67. At the highest dose, it took around 13 days to reach the highest mortality (≥98.1%) for CMB, while it was 19 days for Etidot-67 (≥95.8%). Both boron compounds led to a significant reduction in triglyceride levels in parallel to the downregulation of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), the two primary genes involved in de novo lipogenesis, while they also induced body weight loss. In conclusion, the current study indicated the insecticidal potential of boron compounds but CMB is more promising and more effective in controlling T. castaneum, while lipogenesis is inhibited and weight loss is induced by boron compounds.

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.

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.

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.

Physicochemical comparison of chitin characteristics in three major stored-product beetle pests: Implications for biofumigant toxicity.

The present study investigates the chitin properties of stored-product insect pests and their association with the fumigant toxicity of garlic essential oil. Chitin isolates of Callosobruchus maculatus, Sitophilus oryzae, and Tribolium castaneum adults were characterized using FT-IR, XRD, EA, SEM-EDS, and NMR techniques. Fumigant toxicity assay was performed under airtight condition in glass vial. The S. oryzae contains highest chitin content (19 %), followed by T. castaneum (10 %) and C. maculatus (8 %). The degree of crystallinity was lower in C. maculatus (67.13 %) than in S. oryzae (77.05 %) and T. castaneum (76.56 %). Morphologically, C. maculatus chitin displayed a flat lamellar surface with pores, while S. oryzae and T. castaneum exhibited densely arranged microfibrils based surfaces. Fumigant toxicity assays revealed varied susceptibility levels, C. maculatus exhibited higher susceptibility (0.27 µL/L air of LC50) compared to S. oryzae and T. castaneum (14.35 and 3.74 µL/L air of LC50, respectively) to garlic essential oil. The higher chitin content, greater crystallinity, and densely arranged structures in S. oryzae might contribute to its tolerance towards fumigant. Additionally, physico-chemical properties and penetration potentiality of the bioactive constituents might be linked to the toxicity in insects. Understanding these relations can enrich knowledge of chitin's role in fumigant toxicity mechanism.

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.

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.

Chemical Composition, Toxicity, and Repellency of Essential Oils from Three Hedychium Species Against Stored-Product Insects.

Stored products are constantly infested by insects, so finding eco-friendly bioinsecticides for insect management is important. The work aimed to assess the insecticidal and repellent activity of essential oil (EO) from Hedychium glabrum S. Q. Tong, Hedychium coronarium Koen., and Hedychium yunnanense Gagnep. against Tribolium castaneum, Lasioderma serricorne, and Liposcelis bostrychophila. Results showed that 88 chemical components were identified in the extracted Hedychium EOs, indicating that they exhibited diversity in components. According to principal component analysis (PCA), the composition of the EO from the H. yunnanense stem and leaf (EOHYSL) was significantly different from other EOs due to the different organs and species. The biological activity also varied continuously with plant species and organs. Only the EO of H. yunnanense (EOHY) showed strong fumigant toxicity. While in the contact tests, EOHGR showed the strongest toxicity effect on L. bostrychophila, with a LC50 value of 71.76 µg/cm2, which was closest to the positive control (Pyrethrin). All EOs had remarkable repellent activities against the three target insects, and repellency increased with concentration. According to the results of the comprehensive score, EOHY had the highest potential, which ranged from 0.7999 to 0.8689. Thus, Hedychium EOs possess potential biorational traits to be biological insecticides.

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

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.

Functional importance of groups I and II chitinases, CHT5 and CHT10, in turnover of chitinous cuticle during embryo hatching and post-embryonic molting in the red flour beetle, Tribolium castaneum.

Chitinases (CHT) comprise a large gene family in insects and have been classified into at least eleven subgroups. Many studies involving RNA interference (RNAi) have demonstrated that depletion of group I (CHT5s) and group II (CHT10s) CHT transcripts causes lethal molting arrest in several insect species including the red flour beetle, Tribolium castaneum, presumably due to failure of degradation of chitin in their old cuticle. In this study we investigated the functions of CHT5 and CHT10 in turnover of chitinous cuticle in T. castaneum during embryonic and post-embryonic molting stages. RNAi and transmission electron microscopic (TEM) analyses indicate that CHT10 is required for cuticular chitin degradation at each molting period analyzed, while CHT5 is essential for pupal-adult molting only. We further analyzed the functions of these genes during embryogenesis in T. castaneum. Real-time qPCR analysis revealed that peak expression of CHT10 occurred prior to that of CHT5 during embryonic development as has been observed at post-embryonic molting periods in several other insect species. With immunogold-labeling TEM analysis using a fluorescein isothiocyanate-conjugated chitin-binding domain protein (FITC-CBD) probe, chitin was detected in the serosal cuticle but not in any other regions of the eggshell including the chorion and vitelline membrane layers. Injection of double-stranded RNA (dsRNA) for CHT5 (dsCHT5), CHT10 (dsCHT10) or their co-injection (dsCHT5/10) into mature adult females had no effect on their fecundity and the resulting embryos developed normally inside the egg. There were no obvious differences in the morphology of the outer chorion, inner chorion and vitelline membrane among eggs from these dsRNA-treated females. However, unlike dsCHT5 eggs, dsCHT10 and dsCHT5/10 eggs exhibited failure of turnover of the serosal cuticle in which the horizontal chitinous laminae remained intact, resulting in lethal embryo hatching defects. These results indicate that group I CHT5 is essential for pupal-adult molting, whereas group II CHT10 plays an essential role in cuticular chitin degradation in T. castaneum during both embryonic hatching and all of the post-embryonic molts. CHT10 can serve in place of CHT5 in chitin degradation, except during the pupal-adult molt when both enzymes are indispensable to complete eclosion.

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.

Extraction, Chemical Composition and Insecticidal Activities of Lantana camara Linn. Leaf Essential Oils against Tribolium castaneum, Lasioderma serricorne and Callosobruchus chinensis.

Storage pests and the food spoilage they cause are problems of great concern. Using essential oil obtained from different plants as an insecticide against these storage pests can be considered an environmentally friendly pest management option. Lantana camara Linn. (family Verbenaceae) is a flowering species, and is also a noxious weed that can proliferate well in nearly all geographical habitats. A biopesticide derived from the essential oil extracted from this plant can offer an effective solution for controlling storage pests. The goal of this study is to extract and analyse the chemical composition of essential oil obtained from L. camara leaves, and assess its effectiveness as a bioactive substance against three storage pests: Tribolium castaneum, Lasioderma serricorne, and Callosobruchus chinensis. The yield of essential oil extracted from L. camara leaves was about 0.24 ± 0.014%. By employing the GC-MS technique, the major phytochemicals contained in L. camara leaf essential oil were identified as caryophyllene (69.96%), isoledene (12%), and ɑ-copaene (4.11%). The essential oil exhibited excellent fumigant toxicity (LC50 of 16.70 mg/L air for T. castaneum, 4.141 mg/L air for L. serricorne and 6.245 mg/L air for C. chinensis at 24 h), contact toxicity (LC50 of 8.93 mg/cm2 for T. castaneum, 4.82 mg/cm2 for L. serricorne and 6.24 mg/cm2 for C. chinensis after 24 h) along with effective repellent activity towards the test insects. In addition, the oil showed no significant phytotoxicity on the germination of paddy seeds. This presents the potential to utilize a weed in developing a biopesticide for effectively managing stored product insects because of its strong bioactivity.

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.

Effect of α-cypermethrin and pirimiphos-methyl on wing morphology of Tribolium castaneum (Herbst) and T. confusum Jacquelin du Val: a comparative study.

Tribolium castaneum (Herbst) and Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) are widespread and serious pests of stored products. Various insecticides are applied aiming to effectively manage both species. Here, two insecticides are tested, the pyrethroid α-cypermethrin and the organophosphate pirimiphos-methyl, hypothesizing that they can lead to morphological changes in the certain body parts of the adult offspring of treated T. castaneum and T. confusum parental female adults. For this purpose, the geometric morphometric method to the elytra and hindwings was applied. Both males and females were included in the analysis. The results showed that adult individuals of T. confusum showed higher tolerance to both insecticides compared to T. castaneum adults. This finding is reflected in analyses of both pairs of wings in T. confusum where changes in shape were negligible. The hindwings of T. castaneum experienced deformations to both insecticides. More significant changes in wing shape were observed in the α-cypermethrin treatment compared to pirimiphos-methyl. In the case of T. castaneum, even the shortest exposure to insecticides (5 min) is enough to provoke shape changes in the hindwings. Deformities in offspring, caused after treatment of their parents with insecticides, could moderate the frequency of insecticidal applications in storages.

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.

Population dynamics of Tribolium castaneum (Coleoptera: Tenebrionidae) in wheat and in wheat mixed with cracked wheat held in different types of containers.

The population dynamics of Tribolium castaneum (Herbst), red flour beetle, was studied at 30 °C using long vertical columns (LVCs) (150 mm diameter and 1,020 mm long) and shallow containers (SCs) (460 mm long, 660 mm wide, and 150 mm high), containing 14 kg of whole wheat or a diet made of whole wheat and cracked wheat in 19:1 ratio by mass. The moisture content of the wheat or mixed diet was 14.5% (wb). Every 4 wk and up to 24 wk the live and dead adults were counted in the SCs or in each section of the LVCs. Each LVC was separated into 10 equal sections before removing grain from the LVC. After counting, the grains were incubated at 30 °C and 70% RH for 4 wk, and emerged adults after re-incubation were counted as offspring. The adults and offspring were mainly concentrated in the top section of the LVCs, which could be due to higher mortality in the lower sections and preference of T. castaneum for the surface of grain bulk. The diet influenced the population, and the insects developed better in the cracked wheat-based diet. Greater surface area of the container increased the multiplication and/ or survival of T. castaneum and insects inside SCs with larger surface area and with cracked wheat-based diet, had quicker population increase rate and larger carrying capacity than LVCs.

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.