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.

Revealing the Role of CYP346 Family Genes on Phosphine Resistance in Indian Populations of Tribolium castaneum.

BACKGROUND: Phosphine resistance in Tribolium castaneum challenges grain storage. This study investigates the impact of cytochrome P450 (CYP) enzymes and CYP346 family genes on phosphine resistance in Indian Tribolium castaneum populations. METHODS: Seven field populations of T. castaneum were compared with Lab- susceptible population for their resistance to phosphine. The levels of cytochrome P450 enzyme and expression of certain CYP346 family genes were tracked in these populations. RESULTS: The highly resistant Patiala population showed significantly increased CYP450 activity (11.26 ± 0.14 nmol/min/mg protein, 7.41-fold higher) compared to the lab-susceptible population (1.52 ± 0.09 nmol/min/mg protein) when assayed using 8 mM p-nitroanisole as the substrate. The mRNA expression was measured relative to the standard gene RPS18 and revealed significant upregulation of CYP346B1 and CYP346B3 in highly resistant populations Moga and Patiala (CYP346B1: 12.09 ± 2.19 to 21.74 ± 3.82; CYP346B3: 59.097 ± 10.265 to 50.148 ± 8.272). Patiala's CYP346B1 exhibited an impressive 685.76-fold change, and Moga's CYP346B3 showed a 361.893-fold change compared to lab-susceptible. Linear regression confirmed robust fits for each gene (R2: 0.693 to 0.756). Principal component analysis (PCA) demonstrated a strong positive correlation between CYP346 genes expression; and cytochrome P450 activity. Patiala, Moga, and Hapur populations showed conformity, associating higher resistance with increased P450 activity and CYP346 gene expression. Cluster analysis highlighted a potential correlation between CYP346B1, CYP346B2, and CYP346B3 and P450 activity, with Patiala and Moga clustering together. CONCLUSIONS: Variability in CYP346B1 and CYP346B3 in strong resistance populations may contribute to adaptation and resistance mechanisms. The study provides insights into specific CYP346 family genes associated with phosphine resistance, emphasizing the intricate interaction between CYP450 detoxifying enzymes, CYP346 family genes, and resistance mechanisms. The upregulation of CYP346 genes suggests a survival advantage for T. castaneum against phosphine, diminishing phosphine's efficacy as a pest control measure.

The Genome Organization of 5S rRNA Genes in the Model Organism Tribolium castaneum and Its Sibling Species Tribolium freemani.

5S ribosomal DNAs (rDNAs) are arranged in tandem and are often under-represented in genome assemblies. In the present study, we performed a global and in-depth analysis of the 5S rDNAs in the model insect Tribolium castaneum and its closely related species Tribolium freemani. To accomplish this goal, we used our recently published genome assemblies based on Nanopore and PacBio long-read sequencing. Although these closely related species share the 5S rRNA gene sequence with high homology, they show a different organization of the 5S rDNA locus. Analysis of 5S rDNA arrays in T. castaneum revealed a typical tandemly repeated organization characterized by repeat units consisting of the 121 bp long 5S rRNA gene and the 71 bp long nontranscribed spacer (NTS). In contrast, T. freemani showed a much more complex organization of 5S rDNA arrays characterized by two patterns. The first is based on the association of 5S rRNA gene with arrays of a satellite DNA, representing the NTS sequence of the 5S rDNA genes in T. freemani. The second, more complex type is characterized by a somewhat less frequent occurrence of the 5S rRNA gene and its association with longer satellite DNA arrays that are regularly interrupted by Jockey-like retrotransposons. This organization, in which the ribosomal gene is associated with two completely different repetitive elements such as satellite DNAs and retrotransposons, suggests that the 5S rRNA gene, regardless of its crucial function in the genome, could be a subject of extremely dynamic genomic rearrangements.

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.

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle.

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Insecticidal activity of Thymus pallescens de Noë and Cymbogon citratus essential oils against Sitophilus zeamais and Tribolium castaneum.

The thrust of the study was to determine the chemical composition of the essential oils extracted from Thymus pallescens de Noé and Cymbogon citratus Stapf. as well as to evaluate their efficacy in controlling Sitophilus zeamais Motschulsky and Tribolium castaneum (Herbst) in either single or combined populations. Carvacrol (56.04%) and geraniol (20.86%) were identified as the major constituents of T. pallescens and C. citratus respectively. The tested essential oils showed pronounced insecticidal activity against the pest species in relation with the applied doses. T. pallescens EO had the highest efficacy and S. zeamais was found to be more susceptible to both individual and combined treatments. With reference to the contact and fumigation assessments, T. pallescens EO effectuated corrected mortality rates ranging from 42.5-100% to 25-100% in S. zeamais with corresponding lethal concentration (LC50) values of 17.7 µl/ml and 15µL/L air respectively. Whereas, the T. pallescens EO exhibited corrected mortality rates of 42.5-100% and 20-100% with corresponding LC50 values of 18.1 µl/ml and 15.5 µL/L air against T. castaneum in contact and fumigation assessments, respectively. The corrected mortality rates increased for both insect species when using combination treatments, with significant increases in the LC50 values, ranging from 8.59 to 49.9% for both pest species. Analysis of energy biomarkers in the treated insects indicate significantly increased protein and carbohydrate contents and decreased lipids levels. The study therefore demonstrated the bio-insecticidal toxicity of the EOs from T. pallescens and C. citratus against two important maize post-harvest pests, concurrently revealing significant positive and negative insecticidal activity gradients in relation to single or combined populations.

Bioactivities of essential oil extracted from Elsholtzia densa Benth. And its main components against Tribolium castaneum eggs and pupae.

This study aimed to develop a relatively natural and safe botanical insecticide for controlling the storage pest Tribolium castaneum in the egg and pupal stages. It examined how Elsholtzia densa Benth. essential oil (EO) and its primary components, ß-caryophyllene and limonene, affected T. castaneum eggs and pupae through contact and fumigation. Among th, the contact activities of ß-caryophyllene against T. castaneum eggs and pupae are LD50 (median lethal dose, 50%) = 0.156 mg/cm2 and ED50 (median effective dose, 50%) = 16.35 mg/pupa respectively. The study also investigated the effect of ß-caryophyllene and limonene on T. castaneum eggs and pupae through synergistic contact and fumigation. When the mixing ratio of ß-caryophyllene and limonene was 7:1, the LD50 value of contact activity against T. castaneum eggs was reduced to 0.100 mg/cm2, displaying an obvious synergistic effect. Experiments were conducted to investigate the antitoxic effect of ß-caryophyllene on T. castaneum eggs and pupae, as well as its effects on the enzymatic activity of acetylcholinesterase, succinate dehydrogenase, glutathione S-transferase and carboxylesterase in T. castaneum pupae. Finally, the molecular docking techniques were employed to confirm the aforementioned effects on enzyme function. The findings of this study might help improve storage pest control with T. castaneum and create eco-friendly insecticides using E. densa EO, ß-caryophyllene, and limonene.

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.

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.

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

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

Insecticidal Activities and Phenological Variations of Characteristic Component from Michelia yunnanensis.

Volatile secondary metabolites of plants interact with environments heavily. In this work, characteristic components of Michelia yunnanensis essential oils (EOs) were isolated, purified and identified by column chromatography, GC-MS and NMR. Leaves of M. yunnanensis were collected monthly and extracted for EOs to investigate chemical and insecticidal activity variations as well as potential influencing environments. Different organs were employed to reveal distribution strategies of characteristic components. Results of insecticidal activities showed that all EOs samples exerted stronger contact activity to Lasioderma serricorne, but repellent effect was more efficient on Tribolium castaneum. One oxygenated sesquiterpene was isolated from EOs, basically it could be confirmed as (+)-cyclocolorenone (1). It exerted contact toxicity to L. serricorne (LD 50 = 28.8 µg/adult). Chemical analysis showed that M. yunnanensis leaves in reproductive period would produce and accumulate more 1 than in vegetative period. Moreover, reproductive organs (flowers and fruits) contained more 1 than vegetative organs (leaves and twigs). Partial correlation analysis indicated that temperature-related elements positively correlated with the relative content of 1.

Influence of molecular descriptors of plant volatilomics on fumigant action against the three major stored product beetle pests.

Plant volatilomics such as essential oils (EOs) and volatile phytochemicals (PCs) are known as potential natural sources for the development of biofumigants as an alternative to conventional fumigant pesticides. This present work was aimed to evaluate the fumigant toxic effect of five selected EOs (cinnamon, garlic, lemon, orange, and peppermint) and PCs (citronellol, limonene, linalool, piperitone, and terpineol) against the Callosobruchus maculatus, Sitophilus oryzae, and Tribolium castaneum adults. Furthermore, for the estimation of the relationship between molecular descriptors and fumigant toxicity of plant volatiles, quantitative structural activity relationship (QSAR) models were developed using principal component analysis and multiple linear regression. Amongst the tested EOs, garlic EO was found to be the most toxic fumigant. The PCs toxicity analysis revealed that terpineol, limonene, linalool, and piperitone as potential fumigants to C. maculatus (< 20 µL/L air of LC50), limonene and piperitone as potential fumigants to T. castaneum (14.35 and 154.11 µL/L air of LC50, respectively), and linalool and piperitone as potential fumigants to S. oryzae (192.27 and 69.10 µL/L air of LC50, respectively). QSAR analysis demonstrated the role of various molecular descriptors of EOs and PCs on the fumigant toxicity in insect pest species. In specific, dipole and Randic index influence the toxicity in C. maculatus, molecular weight and maximal projection area influence the toxicity in S. oryzae, and boiling point and Dreiding energy influence the toxicity in T. castaneum. The present findings may provide insight of a new strategy to select effective EOs and/or PCs against stored product insect pests.

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

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

Increasing protein identifications in bottom-up proteomics of T. castaneum - Exploiting synergies of protein biochemistry and bioinformatics.

Depending on the respective research question, LC-MS/MS based bottom-up proteomics poses challenges from the initial biological sample all the way to data evaluation. The focus of this study was to investigate the influence of sample preparation techniques and data analysis parameters on protein identification in Tribolium castaneum by applying free software proteomics platform Max Quant. Multidimensional protein extraction strategies in combination with electrophoretic or chromatographic off-line protein pre-fractionation were applied to enhance the spectrum of isolated proteins from T. castaneum and reduce the effect of co-elution and ion suppression effects during nano-LC-MS/MS measurements of peptides. For comprehensive data analysis, MaxQuant was used for protein identification and R for data evaluation. A wide range of parameters were evaluated to gain reproducible, reliable, and significant protein identifications. A simple phosphate buffer, pH 8, containing protease and phosphatase inhibitor cocktail and application of gentle extraction conditions were used as a first extraction step for T.castaneum proteins. Furthermore, a two-dimensional extraction procedure in combination with electrophoretic pre-fractionation of extracted proteins and subsequent in-gel digest resulted in almost 100% increase of identified proteins when compared to chromatographic fractionation as well as one-pot-analysis. The additionally identified proteins could be assigned to new molecular functions or cell compartments, emphasizing the positive effect of extended sample preparation in bottom-up proteomics. Besides the number of peptides during post-processing, MaxQuant's Match between Runs exhibited a crucial effect on the number of identified proteins. A maximum relative standard deviation of 2% must be considered for the data analysis. Our work with Tribolium castaneum larvae demonstrates that sometimes - depending on matrix and research question - more complex and time-consuming sample preparation can be advantageous for isolation and identification of additional proteins in bottom-up proteomics.

Life history correlations and trade-offs resulting from selection for dispersal in Tribolium castaneum.

Dispersal is an important facet of the life history of many organisms and is, therefore, subject to selective pressure but does not evolve in isolation. Across nature, there are examples of dispersal syndromes and life history strategies in which suites of traits coevolve and covary with dispersal in combinations that serve to maximize fitness in a given ecological context. The red rust flour beetle, Tribolium castaneum, is a model organism and globally significant post-harvest pest that relies on dispersal to reach new patches of ephemeral habitat. Dispersal behaviour in Tribolium has a strong genetic basis. However, a robust understanding of the relationship between dispersal and other life-history components, which could elucidate evolutionary processes and allow pest managers to control their spread and reduce the impact of infestation, is currently lacking. Here, we use highly replicated lines of T. castaneum previously artificially selected for divergent small-scale dispersal propensity to robustly test several important life history components: reproductive strategy, development time, and longevity. As predicted, we find that a suite of important changes as a result of our selection on dispersal: high dispersal propensity is associated with a lower number of longer mating attempts by males, lower investment in early life reproduction by females, slower development of later-laid offspring, and longer female life span. These findings indicate that correlated intraspecific variation in dispersal and related traits may represent alternative life history strategies in T. castaneum. We therefore suggest that pest management efforts to mitigate the species' agro-economic impact should consider the eco-evolutionary dynamics within multiple life histories. The benefits of doing so could be felt both through improved targeting of efforts to reduce spread and also in forecasting how the selection pressures applied through pest management are likely to affect pest evolution.

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.

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

C-type lectins (CTLs) play essential roles in humoral and cellular immune responses of invertebrates. Previous studies have demonstrated the involvement of CTLs in the humoral immunity of Tribolium castaneum, a worldwide pest in stored products. However, the function of CTLs in cellular immunity remains unclear. Here, we identified a CTL gene located on chromosome X and designated it as CTL2 (TcCTL2) from T. castaneum. It encodes a protein of 305 amino acids with a secretion signal peptide and a carbohydrate-recognition domain. TcCTL2 was mainly expressed in the early pupae and primarily distributed in the hemocytes in the late larvae. It was significantly upregulated after larvae were infected with Escherichia coli or Staphylococcus aureus, while knockdown of TcCTL2 exacerbates larval mortality and bacterial colonization after infection. The purified recombinant TcCTL2 (rTcCTL2) can bind to pathogen-associated molecular patterns and microbes and promote hemocyte-mediated encapsulation, melanization and phagocytosis in vitro. rTcCTL2 also induced bacterial agglutination in a Ca2+-dependent manner. Knockdown of TcCTL2 drastically suppressed encapsulation, melanization, and phagocytosis. Furthermore, silencing of TcCTL2 followed by bacterial infection significantly decreased the expression of transcription factors in Toll and IMD pathways, antimicrobial peptides, and prophenoloxidases and phenoloxidase activity. These results unveiled that TcCTL2 mediates both humoral and cellular immunity to promote bacterial clearance and protect T. castaneum from infectious microbes, which will deepen the understanding of the interaction between CTLs and innate immunity in T. castaneum and permit the optimization of pest control strategies by a combination of RNAi technology and bacterial infection.

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

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

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.

Immigration delays but does not prevent adaptation following environmental change: experimental evidence.

In today's rapidly changing world, it is critical to examine how animal populations will respond to severe environmental change. Following events such as pollution or deforestation that cause populations to decline, extinction will occur unless populations can adapt in response to natural selection, a process called evolutionary rescue. Theory predicts that immigration can delay extinction and provide novel genetic material that can prevent inbreeding depression and facilitate adaptation. However, when potential source populations have not experienced the new environment before (i.e., are naive), immigration can counteract selection and constrain adaptation. This study evaluated the effects of immigration of naive individuals on evolutionary rescue using the red flour beetle, Tribolium castaneum, as a model system. Small populations were exposed to a challenging environment, and 3 immigration rates (0, 1, or 5 migrants per generation) were implemented with migrants from a benign environment. Following an initial decline in population size across all treatments, populations receiving no immigration gained a higher growth rate one generation earlier than those with immigration, illustrating the constraining effects of immigration on adaptation. After 7 generations, a reciprocal transplant experiment found evidence for adaptation regardless of immigration rate. Thus, while the immigration of naive individuals briefly delayed adaptation, it did not increase extinction risk or prevent adaptation following environmental change.