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.     

Characterization and functional analysis of hsp21.8b: An orthologous small heat shock protein gene in Tribolium castaneum

Small heat shock proteins (sHSPs) act as molecular chaperones and are widely distributed in all kinds of organisms. Comparative analysis revealed that an orthologous shsp was present during insect evolution. Here, hsp21.8b, one insect orthologous shsp, had been identified in Tribolium castaneum. Quantitative real‐time PCR illustrated that Tchsp21.8b was expressed in all developmental stages, along with the lowest expression at early embryonic stage and relative high expression at other stages especially in late eggs and late pupae. In the adult period, Tchsp21.8b exhibited the highest expression level in central nervous system and followed in elytron, epidermis, ovary and fat body. Moreover, it was upregulated 3.39‐fold in response to enhanced heat stress (45°C) for 4 hr but not to cold stress (4°C) and was upregulated by 1.73‐ to 1.94‐fold under ultraviolet (UV) exposure during 4–6 hr. It was also downregulated by 20.8%–41.8% under starvation in 3 days and had a “down‐up‐down” trend under the pathogen stresses. Larval RNA interference of Tchsp21.8b caused 40.6% insects mortality and reduced the oviposition amount by 66.0% and only 21.0% of the ds‐Tchsp21.8b eggs could hatch into larvae. These results suggested that as an orthologous shsp, Tchsp21.8b not only plays important roles in the growth, development and fecundity of T. castaneum but with the competence to resist the environment stresses, although the response is relatively weak compared to other hsps. Results from this study also uncovered the functions of the orthologous shsp in the development and anti‐stresses ability of T. castanuem. It provided more scientific evidence for revealing the physiological mechanisms of shsps of the insects and enhanced the capabilities to control different pests.     

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism

Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects. In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum. Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc‐sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild‐type beetles. Tripolyphosphate was able to increase the F‐ATPase activity in wild‐type and Tc‐sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum.     

Larval RNAi in <Emphasis Type="Italic">Tribolium</Emphasis> (Coleoptera) for analyzing adult development

We report here on the use of RNA interference (RNAi) to create pupal and adult loss-of-function phenotypes in the red flour beetle, Tribolium castaneum, by injection of double-stranded RNA (dsRNA) into late instar larvae (we refer to this method as larval RNAi). RNAi is well-established as a useful method to mimic loss-of-function phenotypes in many organisms including insects. However, with a few exceptions (such as in the fruit fly Drosophila melanogaster), RNAi analysis has usually been limited to studies of embryogenesis. Here we demonstrate that injection of green fluorescent protein (GFP) dsRNA into the larval body cavity can inhibit GFP expression beginning shortly after injection and continuing through pupal and adult stages. RNAi analysis of the Tc-achaete-scute-homolog (Tc-ASH) revealed that larval RNAi can induce morphological defects in adult beetles, and also that larval RNAi affects the entire body rather than being localized near the site of injection. The larval RNAi technique will be useful to analyze gene functions in post-embryonic development, giving us the opportunity to study the molecular basis of adult morphological diversity in various organisms.Edited by D. Tautz     

Identification and characterization of novel ER-based hsp90 gene in the red flour beetle,Tribolium castaneum

Heat-shock protein 90 (HSP90) is a highly conserved molecular chaperone found in all species except for Archaea, which is required not only for stress tolerance but also for normal development. Recently, it was reported that HSP83, one member of the cytosolic HSP90 family, contributes to oogenesis and responds to heat resistance in Tribolium castaneum. Here, a novel ER-based HSP90 gene, Tchsp90, has been identified in T. castaneum. Phylogenetic analysis showed that hsp90s and hsp83s evolved separately from a common ancestor but that hsp90s originated earlier. Quantitative real-time polymerase chain reaction illustrated that Tchsp90 is expressed in all developmental stages and is highly expressed at early pupa and late adult stages. Tchsp90 was upregulated in response to heat stress but not to cold stress. Laval RNAi revealed that Tchsp90 is important for larval/pupal development. Meanwhile, parental RNAi indicated that it completely inhibited female fecundity and partially inhibited male fertility once Tchsp90 was knocked down and that it will further shorten the lifespan of T. castaneum. These results suggest that Tchsp90 is essential for development, lifespan, and reproduction in T. castaneum in addition to its response to heat stress.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0487-y) contains supplementary material, which is available to authorized users.     

Larval RNA Interference in the Red Flour Beetle,Tribolium castaneum

The red flour beetle, Tribolium castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle’s body cavity.In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.     

Tcmof REGULATES LARVAL/PUPAL DEVELOPMENT AND FEMALE FECUNDITY IN RED FLOUR BEETLE,Tribolium castaneum

Males absent on the first (MOF) was originally identified as an essential component of the X chromosome dosage compensation system in Drosophila melanogaster, and is also a member of the MYST family of histone acetyltransferases. MOF has been extensively studied in D. melanogaster and mammals. However, whether MOF is involved in dosage compensation and/or other vital functions for newly emerging model insects such as Tribolium castaneum, is unclear. We cloned the mof from T. castaneum, named Tcmof. Phylogenetic analysis revealed that mof is highly conserved in eukaryotes but lost in birds. qPCR showed that Tcmof was most highly expressed in the early embryo stage and equally expressed in males and females. Treating larvae with ds‐Tcmof led 79.1% of the insects to arrest during its eclosion; the remaining insects died either in the larval stage or immediately following eclosion. Treating pupae with the same construct eliminated the fertility of T. castaneum. This effect was rescued by reciprocal crosses with wild‐type females, but not males. We infer that the mof gene is essential for larval/pupal development and female fertility in T. castaneum.     

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.     

Limitations of RNAi of α6 nicotinic acetylcholine receptor subunits for assessing the in vivo sensitivity to spinosad

Abstract Spinosad is a widely used insecticide that exerts its toxic effect primarily through interactions with the nicotinic acetylcholine receptor. The α6 nicotinic acetylcholine receptor subunit is involved in spinosad toxicity as demonstrated by the high levels of resistance observed in strains lacking α6. RNAi was performed against the Dα6 nicotinic acetylcholine receptor subunit in Drosophila melanogaster using the Gal4‐UAS system to examine if RNAi would yield results similar to those of Dα6 null mutants. These Dα6‐deficient flies were subject to spinosad contact bioassays to evaluate the role of the Dα6 nicotinic acetylcholine receptor subunit on spinosad sensitivity. The expression of Dα6 was reduced 60%–75% as verified by quantitative polymerase chain reaction. However, there was no change in spinosad sensitivity in D. melanogaster. We repeated RNAi experiments in Tribolium castaneum using injection of dsRNA for Tcasα6. RNAi of Tcasα6 did not result in changes in spinosad sensitivity, similar to results obtained with D. melanogaster. The lack of change in spinosad sensitivity in both D. melanogaster and T. castaneum using two routes of dsRNA administration shows that RNAi may not provide adequate conditions to study the role of nicotinic acetylcholine receptor subunits on insecticide sensitivity due to the inability to completely eliminate expression of the α6 subunit in both species. Potential causes for the lack of change in spinosad sensitivity are discussed.     

Glycogen and Glucose Metabolism Are Essential for Early Embryonic Development of the Red Flour Beetle Tribolium castaneum

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.