Castaneum Larvae Research Articles

Efficiency of Bacillus thuringiensis strains and their Cry proteins against the Red Flour Beetle, Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

Bacillus thuringiensis (Bt) is one of the used bioagents in insect pest control. Its toxicity is largely due to the insecticide endotoxins (crystalline (Cry) proteins) that act selectively on insects and nematodes. The efficiency of 20 of the most common Coleopteran-specific Cry proteins of Bt strains was tested against third instar-larvae of the red flour beetle Tribolium castaeum (Herbst.) (Coleoptera: Tenebrionidae). The primary screening results revealed that 11 Cry proteins (Cry8Ea, Cry8Fa, Cry1Ba, Cry8Ca, Cry1Fb, Cry1Ea, Cry1Ca, Cry55Aa, Cry9Da, Cry1Da, and Cry1Ia) were not toxic at all, 4 Cry proteins (Cry1Aa, Cry14Aa, Cry8Aa, and Cry7Ab) did not cause mortality but caused significant inhibition of growth, and 5 Cry proteins (Cry3Aa, Cry37Aa, Cry22Aa, Cry51Aa, and Cry3Ba) were toxic to T. castaneum larvae. The active 5 Cry proteins were used in the subsequent experiments. Five concentrations, being 0.25, 0.5, 1.0, 1.5, and 2.0 g Cry protein/10 g diet were used against the third instar larvae, and their mortalities were estimated. The LC50 values of Cry3Aa, Cry37Aa, Cry22Aa, Cry51Aa, and Cry3Ba were 0.46, 0.77, 1.25, 1.45, and 1.60 g/10 g, respectively. While the LT50 values of the same Cry proteins (for the concentration 2 g/10 g diet) were 1.50, 1.93, 2.29, 2.23, and 4.22 days, respectively. The results indicated that Cry3Aa was the most active one against T. castaneum larvae. The results of the sublethal study showed that the application of LC30 value of the active 5 Cry proteins reduced total eggs laid daily per female within 2 weeks, where, the percent decrease in egg numbers were 50.55, 38.56, 31.31, 23.20, and, 18.10% for Cry3Aa, Cry37Aa, Cry22Aa, Cry51Aa, and Cry3Ba, respectively. In addition, the hatchability rate of eggs, the larval, and pupal durations of T. castaneum decreased, while the pre-ovipositional period was prolonged. Furthermore, the larvae fed on a diet containing LC30 concentrations of Cry protein showed lower glycogen and lipid rates and generally lower protein content than the control larvae. When T. castaneum larvae were treated by Cry proteins, the level of digestive enzymes found in the midgut was decreased. The present findings indicated that Bt strains/Cry proteins had significant potential for controlling T. castaneum

Transcript-Level Analysis in Combination with Real-Time PCR Elucidates Heat Adaptation Mechanism of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) Larvae.

Tribolium castaneum (Herbst) ranks as one of the most prevalent insects in food processing and storage facilities worldwide. Heat treatment has been revisited to disinfest food processing and storage facilities due to increasingly strict regulation on chemicals. The effect of acclimation of T. castaneum larvae to sublethal high temperatures of 36 and 42℃ for 10 h on their heat adaptation was investigated, and transcript-level analysis combinating with real-time PCR (RT-qPCR) was applied for elucidating the heat adaptation mechanism of T. castaneum larvae. Short-term sublethal high temperature acclimation could greatly enhance the thermal adaptability in T. castaneum larvae. In total, 575, 875, and 1017 differentially expressed genes (DEGs) were, respectively, determined in comparisons between the 28 and 36℃ treatments, the 28 and 42℃ treatments, and the 36 and 42℃ treatments. Fifty-three and 96 genes were commonly up- and down-regulated in both the 36 and 42℃ treatments relative to 28℃, respectively. The results of RT-qPCR analysis further confirmed the RNA-seq analysis. The current results are in favor of enhancing the insecticidal effectiveness of extreme high temperature treatment and elucidating the heat adaptation mechanism in T. castaneum larvae.

The legacy of larval infection on immunological dynamics over metamorphosis

Insect metamorphosis promotes the exploration of different ecological niches, as well as exposure to different parasites, across life stages. Adaptation should favour immune responses that are tailored to specific microbial threats, with the potential for metamorphosis to decouple the underlying genetic or physiological basis of immune responses in each stage. However, we do not have a good understanding of how early-life exposure to parasites influences immune responses in subsequent life stages. Is there a developmental legacy of larval infection in holometabolous insect hosts? To address this question, we exposed flour beetle (Tribolium castaneum) larvae to a protozoan parasite that inhabits the midgut of larvae and adults despite clearance during metamorphosis. We quantified the expression of relevant immune genes in the gut and whole body of exposed and unexposed individuals during the larval, pupal and adult stages. Our results suggest that parasite exposure induces the differential expression of several immune genes in the larval stage that persist into subsequent stages. We also demonstrate that immune gene expression covariance is partially decoupled among tissues and life stages. These results suggest that larval infection can leave a lasting imprint on immune phenotypes, with implications for the evolution of metamorphosis and immune systems.This article is part of the theme issue ‘The evolution of complete metamorphosis'.

Digital gene expression profiling in larvae of Tribolium castaneum at different periods post UV-B exposure

UV-B radiation is an important environmental factor. Exposure to excess UV-B radiation can cause serious effects on the development, survival, and reproduction of different organisms. Plants and animals have developed many different strategies to cope with UV-B-induced damage, but the physiological response of insects to UV-B remains unclear. In the present study, the red flour beetle Tribolium castaneum (Herbst) was used to assess the stress response of UV-B. The underlying molecular mechanisms were explored using RNA sequencing. We investigated the transcriptomic profile of T. castaneum larvae at 4 and 24 h after treatment with UV-B radiation via digital gene expression analysis. The 310 and 996 differentially expressed genes were detected at 4 and 24 h, respectively. Then the biological functions and associated metabolic processes of these genes were determined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. The reliability of the data was verified using qRT-PCR. The results indicated that several differentially expressed genes are involved in antioxidation, DNA repair, protein folding, carbon flux diversion, and the extracellular matrix to protect against UV-B-induced damage. This study will increase our understanding of the molecular mechanism underlying insect response to UV-B radiation.

Latrophilin mediates insecticides susceptibility and fecundity through two carboxylesterases, esterase4 and esterase6, in Tribolium castaneum.

Latrophilin (LPH) is known as an adhesion G-protein-coupled receptor which involved in multiple physiological processes in organisms. Previous studies showed that lph not only involved the susceptibility to anticholinesterase insecticides but also affected fecundity in Tribolium castaneum. However, its regulatory mechanisms in these biological processes are still not clear. Here, we identified two potential downstream carboxylesterase (cce) genes of Tclph, esterase4 and esterase6, and further characterized their interactions with Tclph. After treatment of T. castaneum larvae with carbofuran or dichlorvos insecticides, the transcript levels of Tcest4 and Tcest6 were significantly induced from 12 to 72 h. RNAi against Tcest4 or Tcest6 led to the higher mortality compared with the controls after the insecticides treatment, suggesting that these two genes play a vital role in detoxification of insecticides in T. castaneum. Furthermore, with insecticides exposure to Tclph knockdown beetles, the expression of Tcest4 was upregulated but Tcest6 was downregulated, indicating that beetles existed a compensatory response against the insecticides. Additionally, RNAi of Tcest6 resulted in 43% reductions in female egg laying and completely inhibited egg hatching, which showed the similar phenotype as that of Tclph knockdown. These results indicated that Tclph affected fecundity by positively regulating Tcest6 expression. Our findings will provide a new insight into the molecular mechanisms of Tclph involved in physiological functions in T. castaneum.

Effects of 20-hydroxyecdysone on the development and morphology of the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

The red flour beetle, Tribolium castaneum, is a pest of stored products. It is also regarded as a model species for studying development, genetics, biology, physiology and biochemistry. Recently, it has become a model for use in RNA interference experiments. 20-hydroxyecdysone (20E) is involved in insect metamorphosis and its role in organ development in T. castaneum are based on hormonal treatment in conjunction with RNAi. However, information on the biological, morphological and physiological effects of 20E on T. castaneum is still limited. This study reveals the responses of T. castaneum larvae to injections with various doses of 20E (100, 200, 300, 400 and 500 ng / insect). The results show that larvae injected with 20E reached the prepupal, pupal and adult stages earlier than the control group. Different degrees of morphological change were observed in nine traits, including the appearance of pupal prothetelic organs in the larvae. Moreover, an injection of a high dose of 20E reduced the body weights of the resulting insects at each stage, as well as the length and width of elytra. The enzymatic activity of α-amylase in the resulting adults also decreased significantly. This indicates that injection of 20E caused precocious metamorphosis in T. castaneum by inducing changes in morphology and α-amylase activity, and the optimal concentrations that induce such phenomena were in the range of 100-200 ng / insect. Further investigations are needed to examine the roles of 20E in the regulation of α-amylase in T. castaneum.

A Systematic Study of RNAi Effects and dsRNA Stability in Tribolium castaneum and Acyrthosiphon pisum, Following Injection and Ingestion of Analogous dsRNAs.

RNA interference (RNAi) effects in insects are highly variable and may be largely dependent upon the stability of introduced double-stranded RNAs to digestion by nucleases. Here, we report a systematic comparison of RNAi effects in susceptible red flour beetle (Tribolium castaneum) and recalcitrant pea aphid (Acyrthosiphon pisum) following delivery of dsRNAs of identical length targeting expression of V-type ATPase subunit E (VTE) and inhibitor of apoptosis (IAP) genes. Injection and ingestion of VTE and IAP dsRNAs resulted in up to 100% mortality of T. castaneum larvae and sustained suppression (>80%) of transcript levels. In A. pisum, injection of VTE but not IAP dsRNA resulted in up to 65% mortality and transient suppression (ca. 40%) of VTE transcript levels. Feeding aphids on VTE dsRNA reduced growth and fecundity although no evidence for gene suppression was obtained. Rapid degradation of dsRNAs by aphid salivary, haemolymph and gut nucleases contrasted with stability in T. castaneum larvae where it appears that exo-nuclease activity is responsible for relatively slow digestion of dsRNAs. This is the first study to directly compare RNAi effects and dsRNA stability in receptive and refractory insect species and provides further evidence that dsRNA susceptibility to nucleases is a key factor in determining RNAi efficiency.

Toxicities of Eucalyptus dives Oil, 3-Carvomenthenone, and Its Analogues against Stored-Product Insects

The insecticidal toxicities of essential oils and other active components extracted from Eucalyptus dives leaves (as well as structural analogues) were studied against stored-product insects, Plodia interpunctella and Tribolium castaneum. 3-Carvomenthenone was purified from E. dives oil, and the structures were elucidated by electron ionization mass spectra, 1H-nuclear magnetic resonance (NMR), 13C-NMR, heteronuclear multiple quantum coherence, 1H-1H correlation spectroscopy, and distortionless enhancement by polarization transfer NMR. Using the fumigant method against P. interpunctella larvae and adults, cyclohexenone exhibited the strongest toxicity (50% lethal dose [LD50] against larvae and adults, 2.45 and 3.63 μg/cm3), followed by methylcyclohexenone (2.95 and 4.24 μg/cm3), seudenone (3.02 and 4.44 μg/cm3), and 3-carvomenthenone (52.4 and 68.7 μg/cm3). Using the contact method, cyclohexenone (LD50 against larvae and adults, 17.25 and 19.35 μg/cm2) exhibited the most potent toxicities against T. castaneum larvae and adults, followed by methylcyclohexenone, seudenone, and 3-carvomenthenone. No functional radical on the backbone (2-cyclohexen-1-one) was more toxic than other chemicals. Structure-activity relationships between 3-carvomenthenone analogues and toxicities indicated that the toxicity of 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone might be conferred through the dermal organs of T. castaneum and P. interpunctella. This study indicates that E. dives oil, 3-carvomenthenone, cyclohexenone, methylcyclohexenone, and seudenone have potential as natural agents to control stored-product insects.

CREB-binding protein plays key roles in juvenile hormone action in the red flour beetle, Tribolium Castaneum

Juvenile hormones (JH) and ecdysteroids regulate many biological and metabolic processes. CREB-binding protein (CBP) is a transcriptional co-regulator with histone acetyltransferase (HAT) activity. Therefore, CBP is involved in activation of many transcription factors that regulate expression of genes associated with postembryonic development in insects. However, the function of CBP in JH action in insects is not well understood. Hence, we studied the role of CBP in JH action in the red flour beetle, Tribolium castaneum and the Tribolium cell line. CBP knockdown caused a decrease in JH induction of genes, Kr-h1, 4EBP and G13402 in T. castaneum larvae, adults and TcA cells whereas, Trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor] induced the expression of these JH-response genes. Western blot analysis with specific antibodies revealed the requirement of CBP for the acetylation of H3K18 and H3K27 in both T. castaneum and TcA cells. Chromatin immunoprecipitation (Chip) assays showed the importance of CBP-mediated acetylation of H3K27 for JH induction of Kr-h1, 4EBP, and G13402 in TcA cells. These data suggest that CBP plays an important role in JH action in the model insect, T.castaneum.

Susceptibility of selected stored product insects to a combination treatment of pyriproxyfen and novaluron

There are many published studies of either insect growth regulators (IGR) or chitinase inhibitors applied directly to larvae of stored product insects or incorporated into their diets, but few studies evaluating efficacy of IGRs or chitinase inhibitors applied alone or in combination with a surface for residual control. Tests were conducted to evaluate susceptibility of Tribolium castaneum (Herbst), the red flour beetle, Trogoderma variabile (Ballion), the warehouse beetle, and Dermestes maculatus (DeGeer), the hide beetle, to Tekko® Pro which contained the IGR pyriproxyfen and the chitinase inhibitor novaluron as active ingredients. Efficacy was assessed by adult emergence of exposed immatures, an index based on development of those exposed immatures, and progeny production of exposed adults. Concrete arenas were treated with the label rate of the insecticide applied to a surface, and bioassays were conducted at 0–16 weeks post-treatment. No exposed T. castaneum eggs or larvae reached the adult stage and index values for exposed eggs and larvae remained near the minimum. Adult emergence of T. variable from eggs or larvae did not exceed 25% for the first 8 weeks of the test, but at the end of the test at 16 weeks adult emergence was 44 and 71%, respectively, for eggs and larvae. No eggs or larvae of D. maculatus emerged as adults, but excessive cannibalization in untreated controls occurred throughout the test. Results show that Tekko® Pro will give residual control of T. castaneum and T. variabile, and could replace older conventional neurotoxic insecticides for management of stored product insects.

Influence of acclimation to sublethal temperature on heat tolerance of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) exposed to 50°C.

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is a serious pest of stored agricultural products and one of the most common insects found in grain storage and food processing facilities. Heat treatment has been revisited to control stored-product insects as a potential alternative to methyl bromide for disinfesting mills and food-processing facilities. The influence of acclimation of T. castaneum adults, pupae, larvae, and eggs to sublethal temperatures of 36, and 42°C on their subsequent susceptibility to lethal temperature of 50°C was respectively investigated. The acclimation of T. castaneum eggs, larvae, pupae, and adults to 36, and 42°C significantly decreased their subsequent susceptibility to lethal high temperature of 50°C. The influence of acclimation to 42°C was significantly greater than that of acclimation to 36°C. The most influential acclimation times at 42°C for mortality of T. castaneum eggs, larvae, pupae, and adults were 15, 5, 5, and 5 h, respectively, and their corresponding mortality were 41.24, 5.59, 20.19, and 4.48%, compared to 100% mortality of T. castaneum eggs, larvae, pupae, and adults without acclimation when exposed to 50°C for 35 min, respectively. The present results have important implications for developing successful heat treatment protocols to control T. castaneum, improving disinfestation effectiveness of heat treatment and understanding insect response to high temperatures.

Probiotic Enterococcus mundtii Isolate Protects the Model Insect Tribolium castaneum against Bacillus thuringiensis.

Enterococcus mundtii strains isolated from the larval feces of the Mediterranean flour moth Ephestia kuehniella show antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria. The in vitro probiotic characterization of one isolate revealed a high auto-aggregation score, a hydrophilic cell surface, tolerance for low pH, no hemolytic activity, and susceptibility to all tested antibiotics. We used the red flour beetle Tribolium castaneum, an established model organism, for the in vivo characterization of one probiotic E. mundtii isolate from E. kuehniella larvae. Tribolium castaneum larvae were fed orally with the probiotic isolate or the corresponding supernatant and then infected with either the entomopathogen Bacillus thuringiensis or Pseudomonas entomophila. Larvae exposed to the isolate or the supernatant showed increased survival following infection with B. thuringiensis but not P. entomophila. Heat treatment or treatment with proteinase K reduced the probiotic effect of the supernatant. However, the increased resistance attracts a fitness penalty manifested as a shorter lifespan and reduced fertility. T. castaneum has, pending on further research, the potential as an alternative model for the pre-screening of probiotics.

FoxO mediates the timing of pupation through regulating ecdysteroid biosynthesis in the red flour beetle, Tribolium castaneum

The steroid hormone 20-hydroxyecdysone (20E), the major developmental hormone in insects, controls all the developmental transitions including ecdysis and metamorphosis. In our study with last larval stages of the red flour beetle, Tribolium castaneum, dsRNA-mediated gene silencing of Forkhead box protein O (FoxO) resulted in reduced food intake and larval mass and this agreed with a reduction in the expression of insulin signaling-related genes (insulin-like peptides 2, 3, 4, and chico). Interestingly, we also observed a significant delay in the moment of the pupation and these FoxO-silenced larvae then turned brown at the middle pupal stage followed by death. The observed delay of pupation concurred with a significant delay in 20E titer in dsFoxO-injected larvae and this in turn agreed with a significant delay in expression of prothoracicotropic hormone (ptth) that is a gene stimulating ecdysteroid biosynthesis, and of spook (spo) that is one of the early Halloween genes involved in ecdysteroid biosynthesis. In addition, there was also a delayed expression of the ecdysteroid response gene hormone receptor 3 (HR3). In an attempt to rescue the effects by dsFoxO, injection of 20E into T. castaneum larvae stimulated the expression of HR3 and induced one extra larval-larval molt, confirming the responsiveness for ecdysteroid signaling in dsFoxO-injected larvae. The observations of this project suggest that FoxO is a player in the timing of pupation via the regulating of ecdysteroid biosynthesis, together with the regulation of both insulin signaling and nutrition.

Walking Responses of Tribolium castaneum (Coleoptera: Tenebrionidae) to Its Aggregation Pheromone and Odors of Wheat Infestations.

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a worldwide pest of stored grains. Using "Y"-tube olfactometry we studied the response of T. castaneum to odors from simulated wheat infestations containing conspecifics, and infestations containing the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae), and the granary weevil Sitophilus granarius (L.) (Coleoptera: Curculionidae). Tribolium castaneum larvae were significantly attracted to odors from all three test species. Tribolium castaneum adults were attracted to grains infested by R. dominica and flour infested by T. castaneum but repelled from grains infested by S. granarius. Further behavioral analysis with pheromones showed that T. castaneum were significantly attracted to their aggregation pheromone, dimethyldecanal (DMD), but not to the R. dominica aggregation pheromone, a mixture of dominicalure 1 and 2. Female T. castaneum adults were attracted to ∼50-fold less DMD than larvae and 100-fold less than male adults, suggesting they are more sensitive to DMD. This study improves our understanding of T. castaneum behaviors to infested grain volatile compounds and pheromones, and may help develop new control methods for grain pest species.

Toxicities of active constituent isolated from Thymus vulgaris flowers and its structural derivatives against Tribolium castaneum (Herbst)

Insecticidal activity of 2-isopropyl-5-methylphenol isolated from Thymus vulgaris oil and its derivatives against Tribolium castaneum adults and larvae were evaluated using contact and fumigant bioassays. To elucidate the structure–activity relationship, 2-isopropyl-5-methylphenol and its derivatives were determined. Based on the LD50 values, 2-isopropylphenol was most toxic to T. castaneum adults and larvae, followed by 4-isopropylphenol, 2-methylphenol, 3-methylphenol, and phenol in contact and fumigant bioassays. These results indicate that the introduction of a functional group such as methyl and isopropyl group into the phenol skeleton has an important influence on contact and fumigant toxicities. Furthermore, 2-isopropyl-5-methylphenol and its derivatives could be used as an alternative to control stored-grain insects.

UVB Radiation Delays Tribolium castaneum Metamorphosis by Influencing Ecdysteroid Metabolism.

Ultraviolet B (UVB) radiation is an important environmental factor. It is generally known that UVB exhibits high genotoxicity due to causing DNA damage, potentially leading to skin carcinogenesis and aging in mammals. However, little is known about the effects of UVB on the development and metamorphosis of insects, which are the most abundant terrestrial animals. In the present study, we performed dose-response analyses of the effects UVB irradiation on Tribolium castaneum metamorphosis, assessed the function of the T. castaneum prothoracicotropic hormone gene (Trcptth), and analyzed ecdysteroid pathway gene expression profile and ecdysterone titers post-UVB irradiation. The results showed that UVB not only caused death of T. castaneum larvae, but also delayed larval-pupal metamorphosis and reduced the size and emergence rate of pupae. In addition, we verified the function of Trcptth, which is responsible for regulating metamorphosis. It was also found that the expression profiles of Trcptth as well as ecdysteroidogenesis and response genes were influenced by UVB radiation. Therefore, a disturbance pulse of ecdysteroid may be involved in delaying development under exposure to irradiation. To our knowledge, this is the first report indicating that UVB can influence the metamorphosis of insects. This study will contribute to a better understanding of the impact of UVB on signaling mechanisms in insect metamorphosis.

Microbiota Plays a Role in Oral Immune Priming in Tribolium castaneum.

Animals are inhabited by a diverse community of microorganisms. The relevance of such microbiota is increasingly being recognized across a broad spectrum of species, ranging from sponges to primates, revealing various beneficial roles that microbes can play. The red flour beetle Tribolium castaneum represents a well-established experimental model organism for studying questions in ecology and evolution, however, the relevance of its microbial community is still largely unknown. T. castaneum larvae orally exposed to bacterial components of the entomopathogen Bacillus thuringiensis bv. tenebrionis showed increased survival upon a subsequent challenge with spores of this bacterium. To investigate whether T. castaneum microbiota plays a role in this phenomenon, we established a protocol for raising microbe-free larvae and subsequently tested whether they differ in their ability to mount such a priming response. Here we demonstrate that larvae with significantly lowered microbial loads, show decreased survival upon secondary challenge with B. thuringiensis bv. tenebrionis spores, compared to animals that were allowed to regain their microbiota before priming. Although the exact mechanism of oral immune priming is unclear, we here suggest that microbiota plays a crucial role in oral immune priming in this species.

Susceptibility of two maize grain flour to infestation by the rust- red flour beetle (Tribolium castaneum Herbst)

The susceptibility of white and yellow maize grain flour to Tribolium castaneum was evaluated at ambient temperature and relative humidity in the laboratory were 33.6±0.21 0C and 35.8±0.13% RH. Different quantities of the two varieties of maize grain flour was infested with 5 adults T. castaneum in the ratio of 3:2. The result showed that at 20 days after infestation, there were significant differences in the number of T. castaneum larvae in the different quantities of the yellow maize. Moreover, the number of T. castaneum pupae in the yellow and while maize grain flour was statistically different from each other at 66 days after infestation. However, the results at 66 days after infestation showed that there were significantly differences in the mean number of T. castaneum adults emerged from the different quantities of yellow and white maize grain flour.

Fragments of Tenebrio molitor cadherin enhance Cry3Aa toxicity for the red flour beetle, Tribolium castaneum (Herbst)

AbstractBacillus thuringiensis crystalline (Cry) proteins are highly toxic to a wide range of insect pests, but some species resist their action. This is true for many economically important beetles, including stored product pests, such as Tribolium castaneum. In this article, we show that the susceptibility of T. castaneum larvae to natural as well as to a recombinant Cry3Aa‐type toxin, applied in the diet, is enhanced by supplementing the diet with recombinant fragments of Tenebrio molitor cadherin; Cry toxin‐binding sites occur in several cadherin repeats (CR). In our study, we used the toxin‐binding region CRtb, which represents a substantial part of the repeat CR12‐MPED (membrane‐proximal extracellular domain). CRtb and CR12‐MPED consistently increased Cry3Aa toxicity. This synergistic effect occured at diverse mass ratios between the toxin and the cadherin fragments, suggesting that optimal ratios can be found. In our 6‐week‐long assay with T. castaneum, we achieved mortality of up to 96.6% with toxin concentration 30 μg/g. Cadherin fragments CR11 and CR9‐11 elicited small and diverse effects that require further analysis.

Tribolium castaneum immune defense genes are differentially expressed in response to Bacillus thuringiensis toxins sharing common receptor molecules and exhibiting disparate toxicity

In Tribolium castaneum larvae we have demonstrated by RNA interference knockdown that the Bacillus thuringiensis Cry3Ba toxin receptors Cadherin-like and Sodium solute symporter proteins are also functional receptors of the less active Cry3Aa toxin. Differences in susceptibility to B. thuringiensis infection might not only rely on toxin–receptor interaction but also on host defense mechanisms. We compared the expression of the immune related genes encoding Apolipophorin-III and two antimicrobial peptides, Defensin3 and Defensin2 after B. thuringiensis challenge. All three genes were up-regulated following Cry3Ba spore–crystal intoxication whereas only Defensins gene expression was induced upon Cry3Aa spore–crystal treatment, evidencing a possible association between host immune response and larval susceptibility to B. thuringiensis. We assessed the antimicrobial activity spectra of T. castaneum defensins peptide fragments and found that a peptide fragment of Defensin3 was effective against the human microbial pathogens, Escherichia coli, Staphylococcus aureus and Candida albicans, being S. aureus the most susceptible one.