JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to larva weight, pupa weight, pupa width and adult weight in Tribolium castaneum. Seven diverse inbred lines and the F₁. produced by crossing the two tester lines were examined for indications of epistasis. Larva weight was the only trait for which no significant epistasis was detected. There was significant epistasis for pupa weight in three of the inbred lines; for pupa width in four of the inbred lines; for adult weight in five of the inbred lines. Only one inbred line and the F₁ line failed to exhibit significant epistasis for any trait. Each inbred line had a unique pattern of epistasis, suggesting that a number of different loci were contributing to the detected epistasis.This paper (No. 76-5-158) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.     

Epistatic contributions to quantitative traits in Tribolium castaneum

Summary Triple-testcross experiments were used to analyze epistatic contributions to % hatchability of eggs, age of pupation, number of eggs laid in 24-hour period, and survival from hatching to day 35. Seven diverse inbred lines and the F₁ produced by crossing the two tester lines were examined for the presence of epistasis. There was evidence of epistasis for each of the 4 traits in at least one of the 8 lines tested. Epistasis was a major source of variation in survival in all of the lines tested.     

Individual and group selection for productivity in Tribolium castaneum

Summary The usefulness of a non-random group selection method in the improvement of a character influenced by interactions among individuals has been experimentally tested in a population of Tribolium castaneum. The selected trait —number of adults produced in a fixed period of time —showed a clear increase after 11 generations of selection. This increase is related to a reduction in developmental time, and it was specific for the population structure in which selection was applied, vanishing when this structure was altered.     

Mutational variance for pupa weight in Tribolium castaneum

Summary Directional selection for heavier pupa weight in Tribolium castaneum was practiced for 18 generations in two replicates of an inbred line, each separately maintained in small population cages for more than 90 generations. Mutational variance was estimated in two ways, based on Hill's (1982a) prediction equation for response to directional selection where an equilibrium state between effective population size and variation created by new mutation is assumed. Estimates of mutational variance based on response to selection in a selected population and from a sire-offspring regression analysis in an unselected control population were in strong agreement within each replicate population. Significant differences between the two replicates were observed. Estimates of the ratio of mutational variance to environmental variance ranged from 0.0002 to 0.0012, depending upon the assumptions made about effective population sizes maintained in the two replicate lines. Estimates of realized heritability from the 18 generations of selection were 0.23±0.02 and 0.12±0.02 in the two replicates. The results support the hypothesis that mutation may have played a significant role in supplying useful genetic variation for long-continuing response to selection for this trait in experiments reported earlier.     

The genetic basis of egg lay response to conditioned medium in the flour beetle,Tribolium castaneum

Summary The rate of egg laying in Tribolium castaneum is affected by the quality of the environment and can thus serve not only as a component of fitness of the individual, but also as an adaptation to the long-term survival of the population. In an attempt to determine whether it is an independent genetic character, selection for high and low rates of response was carried jut in beetles from three wild populations. When tests were done on virgin females, and responsiveness, corrected for scale effects, was used as the criterion for selection, separation between the lines was achieved in one generation. It is suggested that in natural populations of Tribolium, the responsiveness of the rate of egg laying to the quality of the environment is controlled by a small number of genes.     

Artificial selection for 18-day pupa weight and opposing simulated naturel selection in Tribolium castaneum

Summary The effect of simulated opposing natural selection on the response to mass selection for 18-day pupa weight of Tribolium castaneum was studied for 10 generations of selection. Natural selection was simulated in replicated treatment lines by imposing a negative relationship between mid-parent genetic value for pupa weight and fertility. Responses to selection and realized heritabilities were smaller (P < 0.05 and P < 0.10, respectively) for the treatment lines than for control lines under selection for pupa weight only. One treatment, line E3G1, reached an intermediate selection plateau by generation 10, and responded linearly to 4 generations of artificial selection after natural selection had been discontinued. Possible explanations for the different behaviors of the replicate lines E3G1 and E3G2 were also discussed.     

Tribolium Wnts: evidence for a larger repertoire in insects with overlapping expression patterns that suggest multiple redundant functions in embryogenesis

Wingless (wg)/Wnt family genes encode secreted glycoproteins that function as signalling molecules in the development of vertebrates as well as invertebrates. In a survey of Wnt family genes in the newly sequenced Tribolium genome, we found a total of nine Wnt genes. In addition to wg or Wnt1, Tribolium contains orthologs of the vertebrate Wnt5-7 and Wnt9-11 genes. As in Drosophila, Wnt1, Wnt6 and Wnt10 are clustered in the genome. Comparative genomics indicates that Wnt9 is also a conserved member of this cluster in several insects for which genome sequence is available. One of the Tribolium Wnt genes appears to be a member of the WntA family, members of which have been identified in Anopheles and other invertebrates but not in Drosophila or vertebrates. Careful phylogenetic examination suggests an Apis Wnt gene, previously identified as a Wnt4 homolog, is also a member of the WntA family. The ninth Tribolium Wnt gene is related to the diverged Drosophila WntD gene, both of which phylogenetically group with Wnt8 genes. Some of the Tribolium Wnt genes display multiple overlapping expression patterns, suggesting that they may be functionally redundant in segmentation, brain, appendage and hindgut development. In contrast, the unique expression patterns of Wnt5, Wnt7 and Wnt11 in developing appendages likely indicate novel functions.     

Expression of esterases during ontogenesis of the flour beetle Tribolium castaneum (tenebrionidae; coleoptera)

Two electrophoretically fast-migrating, nonspecific esterases were detected in two strains of the flour beetle Tribolium castaneum and designated F (fast) and S (slow) according to their relative migration distances. Both isozymes are associated with the alimentary canal and display ontogenetic changes. Their activity is very low in the egg stage, increases in the larva, and declines dramatically in the pharate pupa and pupa. The overall activity in the pupal stage is low, yet increases gradually throughout this period. In the adult, the activity of the esterases rises sharply. The larval and adult F and S isozymes were found to hydrolyze - and -naphthylacetate and -naphthylpropionate with almost equal capacity. -Naphthyl laurate was cleaved by the F enzyme of both larvae and adults. The F and S were insensitive to inhibitors of arylesterases and cholinesterases and were markedly inhibited by the organophosphate diisopropylphosphorofluoridate (DFP) and could be classified as carboxylesterases. Differential sensitivities of larval and adult esterases to urea and heat treatment as well as to DFP may indicate the expression of different genes during metamorphosis.     

Sequence and phylogenetic analysis of the complete mitochondrial genome of the flour beetle Tribolium castanaeum

We describe the first complete mitochondrial genome sequence from a representative of the insect order Coleoptera, the flour beetle Tribolium castaneum. The 15,881 bp long Tribolium mitochondrial genome encodes 13 putative proteins, two ribosomal RNAs and 22 tRNAs canonical for animal mitochondrial genomes. Their arrangement is identical to that in Drosophila melanogaster, which is considered ancestral for insects and crustaceans (Boore et al., 1998; Hwang, et al., 2001a). Nucleotide composition, amino acid composition, and codon usage fall within the range of values observed in other insect mitochondrial genomes. Most notable features are the use of TCT as tRNA(Ser(AGN)) anticodon instead of GCT, which is used in most other arthropod species, and the relative scarcity of special sequence motifs in the 1431 bp long control region. Phylogenetic analysis confirmed resolving power in the conserved regions of the mitochondrial proteome regarding diversification events, which predate the emergence of pterygote insects, while little resolution was obtained at the level of basal perygote diversification. The partition of faster evolving amino acid sites harbored strong support for joining Lepidoptera with Diptera, which is consistent with a monophyletic Mecopterida.     

Directional and stabilizing selection for developmental time and correlated response in reproductive fitness in Tribolium castaneum

Summary Directional and stabilizing selection for developmental time (DT) were done for seven generations in replicated lines of Tribolium castaneum. There were no significant differences between sexes or among replicates in means or coefficients of variation. For directional selection, there were significant responses in both directions, measured as deviation from control, viz. -0.35±0.15 day per generation for Fast (F) and 0.73±0.15 day for Slow (S). The unselected control (C) and the stabilizing selection (I) lines were similar, with average response per generation not significantly different from zero. — Phenotypic variation, from the first generation, was larger in the S line than in the other three lines. The I line showed a significant decrease in phenotypic variation, due mainly to a decrease in genetic variance. The realized heritability was 0.219±0.045 for F and 0.324±0.036 for S, the difference being highly significant. — Correlated response in reproductive fitness (number of pupae produced) was significant only for S (r_p=-0.88 and r_{G realized}=-0.79). Regression of the correlated response on DT in this line was-19.28±4.77 pupae per day (phenotypic) and -28.77±10.06 pupae per day (genetic).     

Molecular analysis of nutritional and hormonal regulation of female reproduction in the red flour beetle, Tribolium castaneum

Female reproduction includes maturation of oocytes and the synthesis of yolk proteins (vitellogenin, Vg) in the fat body and their deposition into the oocytes. Our recent studies showed that juvenile hormone (JH) regulates Vg synthesis and 20-hydroxyecdysone (20E) regulates oocyte maturation in the red flour beetle (Tribolium castaneum). Here, we report on the role of nutritional signaling on vitellogenesis and oogenesis. Comparison of gene expression between fed and starved beetles by microarray analysis showed the up-regulation of genes involved in energy homeostasis and down-regulation of genes involved in egg production in the starved beetles. The RNA interference (RNAi) aided knock-down in the expression of genes involved in insulin and TOR signaling pathways showed that both these signaling pathways play key roles in Vg synthesis and oocyte maturation. Starvation of female beetles resulted in a block in Vg synthesis but not in the progression of primary oocyte development to the resting stage. Feeding after starvation induced Vg synthesis and the progression of primary oocytes from the resting stage to the mature stage. However, in the beetles where JH or 20E synthesis or action was blocked by RNAi, both Vg synthesis and oocyte maturation were affected suggesting that both these hormones (JH and 20E) and nutritional signaling and their cross-talk regulate vitellogenesis and oogenesis.     

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.     

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.     

FGF signalling controls anterior extraembryonic and embryonic fate in the beetle Tribolium

Fibroblast growth factor (FGF) signalling plays a key role in early embryonic development and cell migration in vertebrates and in invertebrates. To gain novel insights into FGF signalling in an arthropod, we characterized the fgf1b ortholog in the beetle Tribolium that is not represented in the Drosophila genome.     

The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum

Axis patterning and appendage development have been well studied in Drosophila melanogaster, a species in which both limb and segment morphogenesis are derived. In Drosophila, positional information from genes important in anteroposterior and dorsoventral axis formation, including wingless (wg) and decapentaplegic (dpp), is required for allocating and patterning the appendage primordia. We used RNA interference to characterize the functions of wg and dpp in the red flour beetle, Tribolium castaneum, which retains more ancestral modes of limb and segment morphogenesis. We also characterized the expression of potential targets of the WG and DPP signaling pathways in these embryos. Tribolium embryos in which dpp had been downregulated had defects in the dorsalmost body wall, but did not appear to have been globally repatterned and had normal appendages. Downregulation of wg led to the loss of segment boundaries, gnathal and thoracic appendages, and lateral head lobes, and to changes in the expression of dpp, Distal-less, and Engrailed. The functions of wg varied along both the anteroposterior and dorsoventral axes of the embryo. Phylogenetic comparisons indicate that the role of WNT signaling in segment boundary formation is evolutionarily old, but that its role in appendage allocation originated in the common ancestor of holometabolous insects.