The effects of Farinocystis tribolii on the growth and development of the flour beetle Tribolium castaneum

Farinocystis tribolii multiplied only in the fatbody of Tribolium castaneum larvae. In the advanced stages of infection, the fatbody was destroyed and the albuminoid granules were completely depleted. Larvae in late stages of infection were “C” shaped. Diseased pupae were flat and straight with the head and mouth parts not completely flexed with the pupal body. Infected pupae failed to develop into adults. Heavily infected adults were distended. Larvae of third, fourth, and fifth instars, although succumbing to the infection ultimately, continued to live even after the healthy larvae of the same age had entered pupation, indicating a prolongation of larval period. Molting was either affected or delayed in infected insects. F. tribolii infection induced a juvenile hormonal effect producing larval-pupal and pupal-adult intermediate forms. Ether extract of spores of F. tribolii when applied on pupae of T. castaneum and final instar nymphs of Dysdercus cingulatus produced adultoids.     

The development of PCR-based markers for molecular sex identification in the model insect species Tribolium castaneum

The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of stored grain and cereal crops. It is also an important model species in genetic, ecological, and evolutionary research. The majority of its genome was recently sequenced and published. However, the genomic sequence of the small Y-chromosome is still undetermined, which hinders the development of molecular sex identification methods. Traditional methods for sexing adult forms of Tribolium beetles are troublesome. Therefore, a method for molecular sex identification in the red flour beetle was developed. One sex-linked randomly amplified polymorphic DNA marker was converted into a sequence-characterized amplified region (SCAR). The SCAR was aligned with the T. castaneum reference whole-genome sequence and fully matched a fragment of a single contig of unknown genomic location. The novelty of the method is that the fragment consists of shorter DNA fragments that are also present at other locations around the genome, but the particular order of these fragments within the sequenced region appeared to be Y-specific and this property was utilized for marker development. A set of three primers for multiplex PCR reaction was designed resulting in amplification of different length Y-specific and not-Y-specific (control) DNA fragments in a single PCR, which allows to distinguish males from females. The primers successfully sexed pre-sexed pupae and adult beetles from six laboratory strains, showing that the order of the repeated fragments is conserved in the species and is not strain-specific.     

Two orthodenticle-related genes in the short-germ beetle Tribolium castaneum

 To investigate the molecular basis of head evolution, we searched for genes related to the Drosophila orthodenticle (otd) homeobox gene in the short-germ beetle Tribolium castaneum. Unexpectedly, we found that there are two otd-related genes in Tribolium, with predicted homeodomains highly similar to that of the single Drosophila gene. One of the two genes (Tc otd-1) is more related in both amino acid sequence and expression pattern to fruitfly otd. Tc otd-1 is expressed in a broad anterior stripe in the blastoderm embryo, suggesting a role in early head segmentation similar to that of the Drosophila gene. The second gene (Tc otd-2) is more similar in sequence to the otd-related genes isolated from different vertebrate species (the Otx gene family). Tc otd-2 is not transcribed in the blastoderm, but is expressed later in more limited subsets of cells in the anterior brain. Both Tribolium genes and the Drosophila gene are, unlike the vertebrate genes, also expressed at the developing ventral midline of the embryo. Our results are consistent with the idea that an otd/Otx gene specified anterior head structures in the last ancestor common to arthropods and vertebrates. Within the arthropod lineage, we propose that this gene acquired a function in cells at the developing midline prior to the duplication that generated the two Tribolium genes. Received: 16 February 1996 / Accepted: 1 March 1996 Edited by C. Desplan     

Heat shock-mediated misexpression of genes in the beetle Tribolium castaneum

Insect gene function has mainly been studied in the fruit fly Drosophila melanogaster because in this species many techniques and resources are available for gene knock down and the ectopic activation of gene function. However, in order to study biological aspects that are not represented by the Drosophila model, and in order to test to what degree gene functions are conserved within insects and what changes in gene function accompanied the evolution of novel traits, the establishment of respective tools in other insect species is required. While gene knock down can be induced by RNA interference in many insects, methods to misexpress genes are much less developed. In order to allow misexpression of genes in a timely controlled manner in the red flour beetle Tribolium castaneum, we have established a heat shock-mediated misexpression system. We show that endogenous heat shock elements perform better than artificial heat shock elements derived from vertebrates. We carefully determine the optimal conditions for heat shock and define a core promoter for use in future constructs. Finally, using this system, we study the effects of misexpressing the head patterning gene Tc-orthodenticle1 (Tc-otd1), We show that Tc-otd1 suppresses Tc-wingless (Tc-wg) in the trunk and to some degree in the head.     

Correlated effects of the response to conditioned medium in the flour beetle,Tribolium castaneum

Dispersal rates were measured in lines selected for high and low response of egg production to conditioned media, and responsivness of egg production was measured in lines selected for high and low dispersal. A positive correlation was found between these two traits, each of which had previously been found to have a simple genetic basis. It is suggested that in Tribolium castaneum the sensitivity to environmental conditions is mediated through a Sensor gene, which can activate either response, according to circumstances.     

Pair-rule and gap gene mutants in the flour beetle Tribolium castaneum

 Early pattern formation in the Drosophila embryo occurs in a syncytial blastoderm where communication between nuclei is unimpeded by cell walls. During the development of other insects, similar gene expression patterns are generated in a cellular environment. In Tribolium, for instance, pair-rule stripes are transiently expressed near the posterior end of the growing germ band. To elucidate how pattern formation in such a situation deviates from that of Drosophila, functional data about the genes involved are essential. In a genetic screen for Tribolium mutants affecting the larval cuticle pattern, we isolated 4 mutants (from a total of 30) which disrupt segmentation in the thorax and abdomen. Two of these mutants display clear pair-rule phenotypes. This demonstrates that not only the expression, but also the function of pair-rule genes in this short-germ insect is in principle similar to Drosophila. The other two mutants appear to identify gap genes. They provide the first evidence for the involvement of gap genes in abdominal segmentation of short-germ embryos. However, significant differences between the phenotypes of these mutants and those of known Drosophila gap mutants exist which indicates that evolutionary changes occurred in either the regulation or action of these genes. Received: 8 May 1998 / Accepted: 17 June 1998     

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.     

First evidence of DNA methylation in insect Tribolium castaneum

DNA methylation has been studied in many eukaryotic organisms, in particular vertebrates, and was implicated in developmental and phenotypic variations. Little is known about the role of DNA methylation in invertebrates, although insects are considered as excellent models for studying the evolution of DNA methylation. In the red flour beetle, Tribolium castaneum (Tenebrionidae, Coleoptera), no evidence of DNA methylation has been found till now. In this paper, a cytosine methylation in Tribolium castaneum embryos was detected by methylation sensitive restriction endonucleases and immuno-dot blot assay. DNA methylation in embryos is followed by a global demethylation in larvae, pupae and adults. DNA demethylation seems to proceed actively through 5-hydroxymethylcytosine, most probably by the action of TET enzyme. Bisulfite sequencing of a highly abundant satellite DNA located in pericentromeric heterochromatin revealed similar profile of cytosine methylation in adults and embryos. Cytosine methylation was not only restricted to CpG sites but was found at CpA, CpT and CpC sites. In addition, complete cytosine demethylation of heterochromatic satellite DNA was induced by heat stress. The results reveal existence of DNA methylation cycling in T. castaneum ranging from strong overall cytosine methylation in embryos to a weak DNA methylation in other developmental stages. Nevertheless, DNA methylation is preserved within heterochromatin during development, indicating its role in heterochromatin formation and maintenance. It is, however, strongly affected by heat stress, suggesting a role for DNA methylation in heterochromatin structure modulation during heat stress response.     

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.     

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.     

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.     

Toxicity of the insect growth regulator diflubenzuron to the rove beetle Aleochara bilineata, a parasitoid and predator of the cabbage maggot Delia radicum

The insect growth regulator Dimilin^®, common name: diflubenzuron, applied at concentrations effective against the cabbage maggot Delia radicum L., did not affect the hatching of eggs of the staphylinid predator and parasite, Aleochara bilineata Gyllenhal. Applied externally in distilled water, Dimilin was non-toxic to first instar larvae, to larval instars developing within host puparia, and to adults. When Dimilin was dissolved in dimethylsulfoxide, then applied to the exoskeleton of host puparia, emergence of A. bilineata adults was suppressed. The conventional insecticide Furadan, at a concentration (0.001% in distilled water) equal to the lowest concentration of Dimilin used, was toxic to both eggs and larvae of A. bilineata.

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Résumé Lorsqu' appliqué dans des concentrations efficaces contre la mouche du chou Delia radicum L. (0.1, 0.01, 0.001% p/v dans de l'eau distillée), le Dimilin (diflubenzuron), un régulateur de croissance des insectes, n'a pas affecté l'éclosion des oeufs de Aleochara bilineata Gyllenhal, un staphylin prédateur et parasite. En application externe, à la plus forte concentration (0.1% p/v dans de l'eau distillée), cet inhibiteur de synthèse de la chitine s'est avéré non toxique pour les larves du premier stade, les stades larvaires qui se développent à l'intérieur du puparium de l'hôte, et les adultes. Le traitement au Dimilin n'a pas affecté la fertilité des staphylins adultes. Cependant, lorsque le diflubenzuron était dissous dans du diméthylsulfoxyde et appliqué sur l'exosquelette du puparium de l'hôte, l'émergence des adultes de A. bilineata était éliminée. L'insecticide conventionnel Furadan (carbofurane), à une concentration égale à la plus faible concentration de Dimilin utilisée (0.001% p/v dans de l'eau distillée), s'est avéré toxique à la fois pour les oeufs et pour les larves de A. bilineata.

     

The EU mutant of the flour beetle,Tribolium castaneum herbst. Environmental and genetic effects on penetrance

The mutation eu in the flour beetle Tribolium castaneum, causes an easily detectable morphological change in the larva and the pupa. The character shows incomplete penetrance at 30°C and almost full penetrance at 25°C. Penetrance at 30°C appears to be partly under genetic control.     

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.