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Tribolium castaneum is a member of the most species-rich eukaryotic order, a powerful model organism for the study of generalized insect development, and an important pest of stored agricultural products. We describe its genome sequence here. This omnivorous beetle has evolved the ability to interact with a diverse chemical environment, as shown by large expansions in odorant and gustatory receptors, as well as P450 and other detoxification enzymes. Development in Tribolium is more representative of other insects than is Drosophila, a fact reflected in gene content and function. For example, Tribolium has retained more ancestral genes involved in cellcell communication than Drosophila, some being expressed in the growth zone crucial for axial elongation in short-germ development. Systemic RNA interference in T. castaneum functions differently from that in Caenorhabditis elegans, but nevertheless offers similar power for the elucidation of gene function and identification of targets for selective insect control.

Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) has developed extensive resistance to the fumigant phosphine. Knowledge of the resistance mechanisms offers insight into resistance management. Although several studies have highlighted the positive or negative impacts of symbiotic microbiota on host pesticide resistance, little is known about the association between gut symbionts and host phosphine resistance. To reveal the effect of the gut bacterium, Enterococcus faecalis (Andrewes & Horder) (Lactobacillales: Enterococcaceae), on host phosphine resistance and its underlying mechanism, we investigated mortality, fitness, redox responses, and immune responses of adult T. castaneum when challenged with E. faecalis inoculation and/or phosphine exposure. When T. castaneum was exposed to phosphine, E. faecalis inoculation decreased its survival and female fecundity and aggravated its oxidative stress. Furthermore, E. faecalis inoculation suppressed the expression and activity of superoxide dismutase, catalase, and peroxidase in phosphine-exposed T. castaneum. Enterococcus faecalis inoculation also triggered excessive host immune responses, including the immune deficiency signaling pathway and the dual oxidase-reactive oxygen species system. These findings suggest that E. faecalis likely modulates host phosphine resistance by interfering with the redox system. This provides information for examining the symbiotic function in the insect-microorganism relationship and new avenues for pesticide resistance management.

Background The ATP-binding cassette (ABC) transporters belong to a large superfamily of proteins that have important physiological functions in all living organisms. Most are integral membrane proteins that transport a broad spectrum of substrates across lipid membranes. In insects, ABC transporters are of special interest because of their role in insecticide resistance. Results We have identified 73 ABC transporter genes in the genome of T. castaneum , which group into eight subfamilies (ABCA-H). This coleopteran ABC family is significantly larger than those reported for insects in other taxonomic groups. Phylogenetic analysis revealed that this increase is due to gene expansion within a single clade of subfamily ABCC. We performed an RNA interference (RNAi) screen to study the function of ABC transporters during development. In ten cases, injection of double-stranded RNA (dsRNA) into larvae caused developmental phenotypes, which included growth arrest and localized melanization, eye pigmentation defects, abnormal cuticle formation, egg-laying and egg-hatching defects, and mortality due to abortive molting and desiccation. Some of the ABC transporters we studied in closer detail to examine their role in lipid, ecdysteroid and eye pigment transport. Conclusions The results from our study provide new insights into the physiological function of ABC transporters in T. castaneum, and may help to establish new target sites for insect control.

1. Parasitized females in mammals, fish and birds can enhance the immune defence of their offspring by transferring specific antibodies for the embryo. Likewise, social insect mothers transfer immunity despite the fact that invertebrates lack antibodies. 2. Female trans-generational immune priming is consistent with parental investment theory, because mothers invest more into rearing their offspring than fathers. However, when immune priming is not directly linked to parental care, as is often the case in insects that abandon their eggs after oviposition, both sexes might benefit from protecting their offspring. 3. Using the red flour beetle, Tribolium castaneum, we show that after parental exposure to heat-killed bacteria, trans-generational immune priming occurs through fathers as well as mothers. 4. This novel finding challenges the traditional view that males provide only genes to their offspring in species without paternal care, and raises the possibility of a division of tasks with respect to immune protection between parents.

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.

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Drosophila larvae and adults possess a potent innate immune response, but the response of Drosophila eggs is poor. In contrast to Drosophila, eggs of the beetle Tribolium are protected by a serosa, an extraembryonic epithelium that is present in all insects except higher flies. In this study, we test a possible immune function of this frontier epithelium using Tc-zen1 RNAi-mediated deletion. First, we show that bacteria propagate twice as fast in serosa-less eggs. Then, we compare the complete transcriptomes of wild-type, control RNAi, and Tc-zen1 RNAi eggs before and after sterile or septic injury. Infection induces genes involved in Toll and IMD-signaling, melanisation, production of reactive oxygen species and antimicrobial peptides in wild-type eggs but not in serosa-less eggs. Finally, we demonstrate constitutive and induced immune gene expression in the serosal epithelium using in situ hybridization. We conclude that the serosa provides insect eggs with a full-range innate immune response. Insects are among the most numerous and diverse creatures on Earth, and over a million different species of insects have been described. Insects have a hard exoskeleton that protects their segmented bodies, and adult insects and their young are also well protected from pathogens. To fight off infection by bacteria or viruses, these creatures release antimicrobial molecules in the fluid that bathes their internal organs. Insects can also mount a localized immune response that kills off invading microbes. Most of what scientists have learned about the insect immune system has come from studying fruit flies. While much of the knowledge gained has been applicable to other insects, there is an important exception—fruit fly eggs are incredibly vulnerable to infection. Eggs from other insects are far better protected. In some species, the mother insect protects her eggs either through scrupulous care or by coating them with her own antimicrobial fluids. However, it was unclear if insect eggs could also defend themselves and counter an infection with a strong immune response. To better understand the immune response in insect eggs, Jacobs et al. studied the eggs of red flour beetles. These beetles are common agricultural pests that eat stored grains and are often studied by scientists in the laboratory. The beetle eggs share a trait with all other insect eggs that is missing from fruit flies and some other flies; the beetle eggs have an extra layer—called the serosa—that envelops the yolk and the developing embryo. To test whether this extra layer provides immune protection for the egg, Jacobs et al. used a technique called RNA interference to prevent the formation of the serosa. Beetle eggs either with or without a serosa were then pricked with a bacteria-covered object, and Jacobs et al. observed that the bacteria grew twice as fast in the eggs lacking a serosa compared with the eggs that had a serosa. Next, Jacobs et al. examined gene expression in response to the infection in the eggs. Over 500 genes that are expressed after an infection were identified, and of these genes, 481 were only expressed in eggs with a serosa. Three of these genes, including two that encode antimicrobial molecules, were looked at in more detail, and found to be only expressed within the serosa, indicating that the serosa is the most likely source of the egg's immune response. Importantly, Jacobs et al. found that eggs with a serosa produce the same immune system response as adult insects and concluded that most insect eggs are far from defenseless and are capable of fending off infection.

All immature animals undergo remarkable morphological and physiological changes to become mature adults. In winged insects, metamorphic changes either are limited to a few tissues (hemimetaboly) or involve a complete reorganization of most tissues and organs (holometaboly). Despite the differences, the genetic switch between immature and adult forms in both types of insects relies on the disappearance of the antimetamorphic juvenile hormone (JH) and the transcription factors Krüppel-homolog 1 (Kr-h1) and Broad-Complex (BR-C) during the last juvenile instar. Here, we show that the transcription factor E93 is the key determinant that promotes adult metamorphosis in both hemimetabolous and holometabolous insects, thus acting as the universal adult specifier. In the hemimetabolous insect Blattella germanica , BgE93 is highly expressed in metamorphic tissues, and RNA interference (RNAi)-mediated knockdown of BgE93 in the nymphal stage prevented the nymphal–adult transition, inducing endless reiteration of nymphal development, even in the absence of JH. We also find that BgE93 down-regulated BgKr-h1 and BgBR-C expression during the last nymphal instar of B. germanica , a key step necessary for proper adult differentiation. This essential role of E93 is conserved in holometabolous insects as TcE93 RNAi in Tribolium castaneum prevented pupal–adult transition and produced a supernumerary second pupa. In this beetle, TcE93 also represses expression of TcKr-h1 and TcBR-C during the pupal stage. Similar results were obtained in the more derived holometabolous insect Drosophila melanogaster , suggesting that winged insects use the same regulatory mechanism to promote adult metamorphosis. This study provides an important insight into the understanding of the molecular basis of adult metamorphosis.

Vertebrate segmentation relies on a mechanism characterized by oscillating gene expression. Whether this mechanism is used by other segmented animals has been controversial. Rigorous proof of cyclic expression during arthropod segmentation has been lacking. We find that the segmentation gene odd-skipped (Tc-odd) oscillates with a two-segment periodicity in the beetle Tribolium castaneum. By bisecting embryos and culturing the two halves over different time intervals, we demonstrate that Tc-odd cycles with a period of about 95 minutes at 30°C. Using live imaging and cell tracking in green fluorescent protein-expressing embryos, we can exclude that cell movements explain this dynamic expression. Our results show that molecular oscillators represent a common feature of segmentation in divergent animals and help reconcile the contrasting paradigms of insect and vertebrate segmentation.