Screens in fly and beetle reveal vastly divergent gene sets required for developmental processes

Abstract

Background Most of the known genes required for developmental processes have been identified by genetic screens in a few well-studied model organisms, which have been considered representative of related species, and informative—to some degree—for human biology. The fruit fly Drosophila melanogaster is a prime model for insect genetics, and while conservation of many gene functions has been observed among bilaterian animals, a plethora of data show evolutionary divergence of gene function among more closely-related groups, such as within the insects. A quantification of conservation versus divergence of gene functions has been missing, without which it is unclear how representative data from model systems actually are. Results Here, we systematically compare the gene sets required for a number of homologous but divergent developmental processes between fly and beetle in order to quantify the difference of the gene sets. To that end, we expanded our RNAi screen in the red flour beetle Tribolium castaneum to cover more than half of the protein-coding genes. Then we compared the gene sets required for four different developmental processes between beetle and fly. We found that around 50% of the gene functions were identified in the screens of both species while for the rest, phenotypes were revealed only in fly (~ 10%) or beetle (~ 40%) reflecting both technical and biological differences. Accordingly, we were able to annotate novel developmental GO terms for 96 genes studied in this work. With this work, we publish the final dataset for the pupal injection screen of the iBeetle screen reaching a coverage of 87% (13,020 genes). Conclusions We conclude that the gene sets required for a homologous process diverge more than widely believed. Hence, the insights gained in flies may be less representative for insects or protostomes than previously thought, and work in complementary model systems is required to gain a comprehensive picture. The RNAi screening resources developed in this project, the expanding transgenic toolkit, and our large-scale functional data make T. castaneum an excellent model system in that endeavor.

The red flour beetle T. castaneum: elaborate genetic toolkit and unbiased large scale RNAi screening to study insect biology and evolution

“…This divergence probably reflects both biological divergence and technical differences in the screening approaches. These data underline the necessity of additional model systems even for well-studied processes [ 29 ]. Increasingly, T. castaneum is also used to study basic biological questions for which other model systems are less well suited.…”

“…An outstanding strength of T. castaneum is the ease of large- or mid-scale RNAi screens. As part of the iBeetle screen [ 28 , 29 ], a genome-wide collection of templates was produced, based on which dsRNAs can be commercially ordered, obviating the need for cloning (Eupheria Biotech, Dresden). This resource, together with the ease of microinjection (students learn the procedure within a day), enables mid or large scale RNAi screens to search for novel gene functions or to test gene lists derived from omics analyses.…”

Phenotypic screen and transcriptomics approach complement each other in functional genomics of defensive stink gland physiology

“…4 ). This indicates that a large, first-pass, multipurpose screen carried out by multiple screeners with diverse interests such as iBeetle [ 37 , 38 ] can only serve as a starting point for the identification of genes acting in a certain biological process not studied by genetic means before.

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“…In the 1st phase of the iBeetle screen, which covered 5300 genes, potential gland phenotypes were analyzed in the ‘larval screen’ part, in which penultimate larvae (L6) of a specific cross [ 37 ] were injected with 1 µg/µl double-stranded RNA of the respective iBeetle fragment ( https://ibeetle-base.uni-goettingen.de/ ) [ 38 , 39 ]. The glands were then inspected at the adult stage 38 to 41 days after injection for aberrant gland phenotypes.…”

“…The functional genetic tools in T. castaneum include forward genetics based on insertional mutagenesis [ 29 ], transgene-based miss-expression systems [ 30 , 31 ], a fully annotated genome sequence [ 32 – 34 ], as well as systemic RNAi [ 35 , 36 ]. The efficient use of RNAi in this beetle has allowed the development of this approach into a forward genetics application to perform an unbiased genome-wide large-scale phenotypic screen (iBeetle screen) to identify gene functions in embryonic and postembryonic development as well as cell biology and physiology [ 37 , 38 ]. In this screen, knock-down beetles and their progeny were systematically analyzed for phenotypic changes including their stink glands and methodically documented in iBeetle-Base [ 39 , 40 ].…”