The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology.

Genetic screens are powerful tools to identify the genes required for a given biological process. However, for technical reasons, comprehensive screens have been restricted to very few model organisms. Therefore, although deep sequencing is revealing the genes of ever more insect species, the functional studies predominantly focus on candidate genes previously identified in Drosophila, which is biasing research towards conserved gene functions. RNAi screens in other organisms promise to reduce this bias. Here we present the results of the iBeetle screen, a large-scale, unbiased RNAi screen in the red flour beetle, Tribolium castaneum, which identifies gene functions in embryonic and postembryonic development, physiology and cell biology. The utility of Tribolium as a screening platform is demonstrated by the identification of genes involved in insect epithelial adhesion. This work transcends the restrictions of the candidate gene approach and opens fields of research not accessible in Drosophila.

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.

Large-scale insertional mutagenesis of a coleopteran stored grain pest, the red flour beetle Tribolium castaneum, identifies embryonic lethal mutations and enhancer traps.

BACKGROUND: Given its sequenced genome and efficient systemic RNA interference response, the red flour beetle Tribolium castaneum is a model organism well suited for reverse genetics. Even so, there is a pressing need for forward genetic analysis to escape the bias inherent in candidate gene approaches. RESULTS: To produce easy-to-maintain insertional mutations and to obtain fluorescent marker lines to aid phenotypic analysis, we undertook a large-scale transposon mutagenesis screen. In this screen, we produced more than 6,500 new piggyBac insertions. Of these, 421 proved to be recessive lethal, 75 were semi-lethal, and eight indicated recessive sterility, while 505 showed new enhancer-trap patterns. Insertion junctions were determined for 403 lines and often appeared to be located within transcription units. Insertion sites appeared to be randomly distributed throughout the genome, with the exception of a preference for reinsertion near the donor site. CONCLUSION: A large collection of enhancer-trap and embryonic lethal beetle lines has been made available to the research community and will foster investigations into diverse fields of insect biology, pest control, and evolution. Because the genetic elements used in this screen are species-nonspecific, and because the crossing scheme does not depend on balancer chromosomes, the methods presented herein should be broadly applicable for many insect species.

Germ-cell cluster formation in the telotrophic meroistic ovary of Tribolium castaneum (Coleoptera, Polyphaga, Tenebrionidae) and its implication on insect phylogeny.

Tribolium castaneum has telotrophic meroistic ovarioles of the Polyphaga type. During larval stages, germ cells multiply in a first mitotic cycle forming many small, irregularly branched germ-cell clusters which colonize between the anterior and posterior somatic tissues in each ovariole. Because germ-cell multiplication is accompanied by cluster splitting, we assume a very low number of germ cells per ovariole at the beginning of ovariole development. In the late larval and early pupal stages, we found programmed cell death of germ-cell clusters that are located in anterior and middle regions of the ovarioles. Only those clusters survive that rest on posterior somatic tissue. The germ cells that are in direct contact with posterior somatic cells transform into morphologically distinct pro-oocytes. Intercellular bridges interconnecting pro-oocytes are located posteriorly and are filled with fusomes that regularly fuse to form polyfusomes. Intercellular bridges connecting pro-oocytes to pro-nurse cells are always positioned anteriorly and contain small fusomal plugs. During pupal stages, a second wave of metasynchronous mitoses is initiated by the pro-oocytes, leading to linear subclusters with few bifurcations. We assume that the pro-oocytes together with posterior somatic cells build the center of determination and differentiation of germ cells throughout the larval, pupal, and adult stages. The early developmental pattern of germ-cell multiplication is highly similar to the events known from the telotrophic ovary of the Sialis type. We conclude that among the common ancestors of Neuropterida and Coleoptera, a telotrophic meroistic ovary of the Sialis type evolved, which still exists in Sialidae, Raphidioptera, and a myxophagan Coleoptera family, the Hydroscaphidae. Consequently, the telotrophic ovary of the Polyphaga type evolved from the Sialis type.

The larval development of the telotrophic meroistic ovary in the bug Dysdercus intermedius (Heteroptera, Pyrrhocoridae).

Bug ovaries are of the telotrophic meroistic type. Nurse cells are restricted to the anterior tropharium and are in syncytial connection with the oocytes via the acellular trophic core region into which cytoplasmic projections of oocytes and nurse cells open. The origin of intercellular connections in bug ovaries is not well understood. In order to elucidate the cellular processes underlying the emergence of the syncytium, we analysed the development of the ovary of Dysdercus intermedius throughout the five larval instars. Up to the third instar, the germ cell population of an ovariole anlage forms a single, tight rosette. In the center of the rosette, phosphotyrosine containing proteins and f-actin accumulate. This center is filled with fusomal cytoplasm and closely interdigitating cell membranes known as the membrane labyrinth. With the molt to the fourth instar germ cells enhance their mitotic activity considerably. As a rule, germ cells divide asynchronously. Simultaneously, the membrane labyrinth expands and establishes a central column within the growing tropharium. In the fifth instar the membrane labyrinth retracts to an apical position, where it is maintained even in ovarioles of adult females. The former membrane labyrinth in middle and posterior regions of the tropharium is replaced by the central core to which nurse cells and oocytes are syncytially connected. Germ cells in the most anterior part of the tropharium, i.e. those in close proximity to the membrane labyrinth remain proliferative. The posterior-most germ cells enter meiosis and become oocytes. The majority of the ovarioles' germ cells, located in between these two populations, endopolyploidize and function as nurse cells. We conclude that the extensive multiplication of germ cells and their syncytial assembly during larval development is achieved by incomplete cytokineses followed by massive membrane production. Membranes are degraded as soon as the trophic core develops. For comparative reasons, we also undertook a cursory examination of early germ cell development in Dysdercus intermedius males. All results were compatible with the known basic patterns of early insect spermatogenesis. Germ cells run through mitotic and meiotic divisions in synchronous clusters emerging from incomplete cytokineses. During the division phase, the germ cells of an individual cluster are connected by a polyfusome rich in f-actin.