Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.     

Identification of microsatellite markers in the red flour beetle,Tribolium castaneum

We isolated and characterized microsatellite markers for the red flour beetle Tribolium castaneum, an important model species for studies in various areas of evolutionary biology and ecology. A microsatellite‐enriched genomic library was constructed and screened with single stranded oligonucleotide probes [(CCT)₁₇, (AAT)₁₇ and (CAG)₁₇]. Forty‐five primer pairs were designed of which 19 pairs produced successful amplification. Polymorphism screening involved beetles from five beetle strains and revealed 15 polymorphic and four monomorphic markers. The development of polymorphic microsatellite markers will facilitate future ecological and genetic studies involving T. castaneum beetles.     

Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum

As invertebrates lack the molecular machinery employed by the vertebrate adaptive immune system, it was thought that they consequently lack the ability to produce lasting and specific immunity. However, in recent years, it has been demonstrated that the immune defence of invertebrates is by far more complicated and specific than previously envisioned. Lasting immunity following an initial exposure that proves protection on a secondary exposure has been shown in several species of invertebrates. This phenomenon has become known as immune priming. In the cases where it is explicitly tested, this priming can also be highly specific. In this study, we used survival assays to test for specific priming of resistance in the red flour beetle, Tribolium castaneum, using bacteria of different degrees of relatedness. Our results suggest an unexpected degree of specificity that even allows for differentiation between different strains of the same bacterium. However, our findings also demonstrate that specific priming of resistance in insects may not be ubiquitous across all bacteria.     

Characterization and functional analysis of hsp18.3 gene in the red flour beetle,Tribolium castaneum

Small heat shock proteins (sHSPs) are diverse and mainly function as molecular chaperones to protect organisms and cells from various stresses. In this study, hsp 18.3, one Tribolium castaneum species-specific shsp、has been identified. Quantitative real-time polymerase chain reaction illustrated that Tchsp 18.3 is expressed in all developmental stages, and is highly expressed at early pupal and late adult stages, while it is highly expressed in ovary and fat body at the adult period. Moreover, it was up-regulated 4532 ± 396-fold in response to enhanced heat stress but not to cold stress;meanwhile the lifespan of adults in ds-Tchsp 18.3 group reduced by 15.8% from control group under starvation. Laval RNA interference (RNAi) of Tchsp 18.3 caused 86.1%±4.5% arrested pupal eclosion and revealed that Tchsp 18.3 played an important role in insect development. In addition, parental RNAi of Tchsp 18.3 reduced the oviposition amount by 94.7%. These results suggest that Tchsp 18.3 is not only essential for the resistance to heat and starvation stress, but also is critical for normal development and reproduction in T. castaneum.     

Insulin receptor regulates food intake through sulfakinin signaling in the red flour beetle,Tribolium castaneum

Insects obtain energy and nutrients via feeding to support growth and development. The insulin signaling pathway is involved in the regulation of feeding; however, the underlying mechanisms are not fully understood. Here, we show that insulin signaling regulates food intake via crosstalk with neuropeptide sulfakinin in the red flour beetle, Tribolium castaneum. Silencing of the insulin receptor (InR) decreased the food intake in the penultimate and final instar stages, leading to a decrease of weight gain and mortality during larval-pupal metamorphosis. Interestingly, the knockdown of InR co-occurred with an increased expression of sulfakinin (sk), a gene encoding neuropeptide SK functioning as a satiety signal. In parallel, double silencing of sk and InR eliminated the inhibitory effect on food intake as induced by silencing of InR and the larvae died as prepupae. In conclusion, this study shows, for the first time, that the insulin/InR signaling regulates food intake through the sulfakinin signaling pathway in the larval stages of this important model and pest insect, indicating a novel target for pest control.     

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. Here, we study the metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum, using RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, we tested the prediction that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, we tested hypotheses for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. Our data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles.     

Female influence over offspring paternity in the red flour beetle Tribolium castaneum

In animals having internal fertilization, both sexes can potentially influence the post-copulatory processes of sperm transfer, sperm storage and sperm use for fertilization. In this experiment, we investigated whether Tribolium castaneum females can influence male paternity success following consecutive matings with two different males. We compared second male paternity success (P2) between females exposed to carbon dioxide (CO2) and control females kept in air, in both cases for 30 min between two matings. CO2 exposure inhibits muscular activity and has previously been shown to decrease sperm storage by T. castaneum females. Females exposed to CO2 after their first mating showed significantly higher P2 than control females during the later portion of a one-month oviposition period. These results are consistent with reduced storage of first male sperm by CO2-exposed females. Also, T. castaneum females showed considerable variation in spermathecal morphology, and P2 decreased with increasing spermathecal tubule volume. These results demonstrate that T. castaneum females can influence male paternity success, and suggest that differential sperm storage may be an important mechanism of post-copulatory female choice.     

Juvenile hormone regulation of vitellogenin synthesis in the red flour beetle,Tribolium castaneum

To elucidate the endocrine regulation of vitellogenin (Vg) synthesis in the red flour beetle, Tribolium castaneum, the titers of juvenile hormone (JH) and ecdysteroids in the whole body of female beetles were measured and compared with Vg mRNA levels. Juvenile hormone levels remained high while the ecdysteroid levels declined steadily during 1–5 days post adult emergence (PAE). The Vg mRNA levels began to increase by the end of 3rd day PAE and peaked by the 4th–5th day PAE. Gene expression profiling by microarray and quantitative real-time PCR analyses of RNA isolated from 1 to 5 days PAE beetles revealed that the genes coding for proteins involved in JH biosynthesis and action, but not those involved in 20-hydroxyecdysone (20E) biosynthesis and action had similar expression patterns as the genes coding for Vg. RNA interference (RNAi)-aided knock-down in the expression of these genes showed that both JH and 20E were required for Vg gene expression. However, Vg mRNA was induced by the application of JH III but not by the injection of 20E into the previtellogenic females. These data suggest that JH is required for Vg synthesis in the fat body and 20E influences Vg synthesis through its action on oocyte maturation.     

Roles of transient receptor potential channels in eclosion and movement in the red flour beetle Tribolium castaneum

Transient receptor potential channels (TRPs) are a family of cation channels involved in various sensory mechanisms in Drosophila melanogaster, including mechanosensing. Phylogenetic analysis of mechanosensory TRPs in seven members of the TRPV, TRPN and TRPA subfamilies reveals a unique TcTrpA5 in Tribolium castaneum that is likely lost in D. melanogaster. Because mechanosensors are implicated in various key physiology, we investigate the roles of eight candidate mechanosensory TRPs in survival, walking behaviour and tonic immobility in T. castaneum using the RNA interference technique. Double‐stranded RNA treatment of nompC (dsNompC) and trpA5 (dsTrpA5) results in eclosion failure, causing 93% mortality of beetles subjected to each of these treatments. The beetles that survive the dsNompC treatment show defects during sclerotization of the elytra. Adult beetles treated with dsNanchung and dsInactive show defects in the folding of the joint between the femur and tibia segments of the hind legs, resulting in abnormal walking behaviour and reduced walking speed. Regarding tonic immobility induced by mechanical stimulations on the ventral surface, knockout responses (death feignings) are significantly extended with dsNanchung, dsInactive and dsWaterwitch treatments. These data suggest that both nompC and trpA5 are required for adult eclosion. The functions of nanchung and inactive are involved in the folding the hind leg segments. In addition, nanchung, inactive, and waterwitch are likely the receptors involved in recovery from tonic immobility.     

Functions of duplicated genes encoding CCAP receptors in the red flour beetle, Tribolium castaneum

Crustacean cardioactive peptide (CCAP) is a nonapeptide originally isolated from the shore crab, Carcinus maenas, based on its cardioacceleratory activity. This peptide is highly conserved in insects and other arthropods. In insects CCAP also has an essential role in ecdysis behavior. We previously identified two homologous genes, ccapr-1 and ccapr-2, encoding putative CCAP receptors in the red flour beetle, Tribolium castaneum. In contrast, some insects, including Drosophila melanogaster, carry only one gene encoding a CCAP receptor. Phylogenetic analysis of putative CCAP receptor orthologs reveals a number of independent gene duplications in several insect lineages. In this study, we confirmed that CCAP activates both putative T. castaneum receptors in a heterologous expression system. RNA interference (RNAi) of ccapr-1 and ccapr-2 revealed that ccapr-2 is essential for eclosion behavior in T. castaneum, while RNAi for ccapr-1 did not result in any abnormal phenotype. In vivo cardioacceleratory activity of exogenously applied CCAP was abolished by RNAi of ccapr-2, but not by that of ccapr-1. Thus, only ccapr-2 mediates the cardioacceleratory function, ccapr-1 having apparently lost both functions for eclosion behavior and for cardioacceleration since the recent gene duplication event.     

Juvenile hormone regulates vitellogenin gene expression through insulin-like peptide signaling pathway in the red flour beetle, Tribolium castaneum

Our recent studies identified juvenile hormone (JH) and nutrition as the two key signals that regulate vitellogenin (Vg) gene expression in the red flour beetle, Tribolium castaneum. Juvenile hormone regulation of Vg synthesis has been known for a long time in several insects, but the mechanism of JH action is not known. Experiments were conducted to determine the mechanism of action of these two signals in regulation of Vg gene expression. Injection of bovine insulin or FOXO double-stranded RNA into the previtellogenic, starved, or JH-deficient female adults increased Vg mRNA and protein levels, thereby implicating the pivotal role for insulin-like peptide signaling in the regulation of Vg gene expression and possible cross-talk between JH and insulin-like peptide signaling pathways. Reduction in JH synthesis or its action by RNAi-mediated silencing of genes coding for acid methyltransferase or methoprene-tolerant decreased expression of genes coding for insulin-like peptides (ILPs) and influenced FOXO subcellular localization, resulting in the down-regulation of Vg gene expression. Furthermore, JH application to previtellogenic female beetles induced the expression of genes coding for ILP2 and ILP3, and induced Vg gene expression. FOXO protein expressed in baculovirus system binds to FOXO response element present in the Vg gene promoter. These data suggest that JH functions through insulin-like peptide signaling pathway to regulate Vg gene expression.     

AFLP-based genetic linkage map for the red flour beetle (Tribolium castaneum)

The red flour beetle (Tribolium castaneum) is a major pest of stored grain and grain products and a popular model species for a variety of ecological, evolutionary, and developmental biology studies. Development of a linkage map based on reproducible and highly polymorphic molecular markers would greatly facilitate research in these disciplines. We have developed a genetic linkage map using 269 amplified fragment length polymorphism (AFLP) markers. Ten previously known random amplified polymorphic DNA (RAPD) markers were used as anchor markers for linkage group assignment. The linkage map was constructed through genotyping two independent F(2) segregating populations with 48 AFLP primer combinations. Each primer combination generated an average of 4.6 AFLP markers eligible for linkage mapping. The length of the integrated map is 573 cM, giving an average marker resolution of 2.0 cM and an average physical distance per genetic distance of 350 kb/cM. A cluster of loci on linkage group 3 exhibited significant segregation distortion. We have also identified six X-linked and two Y-linked markers. Five mapped AFLP fragments were sequenced and converted to sequence-tagged site (STS) markers.     

Ecdysteroid regulation of ovarian growth and oocyte maturation in the red flour beetle,Tribolium castaneum

Previous studies from our laboratory showed the involvement of juvenile hormone (JH) and ecdysteroid signaling in the regulation of female reproduction in the red flour beetle, Tribolium castaneum. JH regulates vitellogenin (Vg) synthesis in the fat body but the role of ecdysteroid signaling is not known. Here, we report on ecdysteroid regulation of ovarian growth and oocyte maturation. Microarray analysis of RNA isolated from ovaries showed the up-regulation of several genes coding for proteins involved in ecdysteroid signaling on the 4th day after female adult eclosion. The functional analyses of genes coding for proteins involved in ecdysteroid and JH signaling pathways by RNA interference (RNAi) revealed that ecdysteroids but not JH regulate ovarian growth and primary oocyte maturation. Ultrastructural studies showed the temporal sequences of key events in oogenesis including the development of primary oocytes, the differentiation and development of follicle epithelial cells, and the formation of intercellular spaces to facilitate uptake of Vg protein. RNAi studies showed that ecdysone receptor (EcR) and ultraspiracle (USP) are required for the ovarian growth, primary oocyte maturation and the growth and migration of the follicle cells. These studies suggest important roles for ecdysteroids in the regulation of oocyte maturation in the beetle ovaries.     

冰核真菌削弱赤拟谷盗抗寒力的初步研究

赤拟谷盗Tribolium castaneum是不耐结冰的害虫,在冬季它通过降低过冷却点以避免结冰造成的致命伤害。冰核活性细菌能显著提高昆虫的过冷却点,使之在较高的零下温度发生结冰。试验证明冰核活性真菌也能显著提高赤拟谷盗的过冷却点。对照组平均过冷却点为-14.9℃。用10 g/L的冰核真菌制剂喷洒虫体,风干后测定,平均过冷却点提高到-4.8℃。用0.1 g/L处理后至少在7天内过冷却点保持较高。这些结果表明冰核真菌可能成为一种在冬季使用的、防治不耐结冰害虫的促冻杀虫剂。