Fruitless RNAi knockdown in males interferes with copulation success in Schistocerca gregaria

Research Group Functional Genomics and Proteomics, K.U. Leuven, Naamsestraat 59, Leuven, Belgium.
Insect biochemistry and molecular biology (Impact Factor: 3.45). 02/2011; 41(5):340-7. DOI: 10.1016/j.ibmb.2011.01.012
Source: PubMed


In Drosophila melanogaster, the male-specific splice isoform of the fruitless gene (Fru(M)) codes for a set of transcription factors that are involved in the regulation of male courtship and copulation. Fru(M) is expressed in an interconnected neuronal circuit containing central and sensory neurons as well as motor neurons. A partial sequence from the Schistocerca gregaria fru-gene from an EST database allowed quantitative real time analysis of fru-expression in adult locusts, and revealed the highest expression in the testes, accessory glands as well as the brain (and optic lobes). Starting fru specific RNAi knockdown in the third and fourth nymphal stage resulted in a significantly lower cumulative copulation frequency of the RNAi-treated animals compared to controls after 3 h of observation. In addition, the testes of RNAi-treated males weigh less. Analysis of the egg pods resulting from a successful copulation event revealed that egg pods from females that mated with an RNAi-treated male were smaller and contained less fertilized eggs compared to egg pods from females who mated with control males. Starting injections in the fifth nymphal stage showed the complete opposite for the cumulative copulation frequency and testes weight. We conclude that already in the early nymphal phases of male desert locusts, fruitless starts to play an important role in the regulation of successful copulation in the adult. The RNAi treatment in the male has also its effects on fertility and fecundity. It remains unknown whether this effect is coming from aberrant courtship behaviour or from an altered composition of the sperm or seminal fluids.

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    • "Substitutions occurring within alternatively spliced genes are both more localised (mainly in those exons being alternatively spliced) and less conservative than those in genes that have been duplicated (Talavera et al., 2007). The gene fruitless (fru) is an alternatively spliced transcription factor that has been identified in a broad range of insect groups (Salvemini et al., 2010), including Orthoptera (Ustinova and Mayer, 2006; Boerjan et al., 2011), Blattodea (Clynen et al., 2011), Hymenoptera (Bertossa et al., 2009) and Diptera (Ryner et al., 1996; Gailey et al., 2006; Salvemini et al., 2009; Sobrinho and de Brito, 2010; Salvemini et al., 2013). fru is a pleiotropic gene with at least two major functions: one that controls male sexual behaviour and another that is essential for viability in both sexes. "
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    ABSTRACT: There has been much debate concerning whether cis-regulatory or coding changes are more likely to produce evolutionary innovation or adaptation in gene function, but an additional complication is that some genes can dramatically diverge through alternative splicing, increasing the diversity of gene function within a locus. The fruitless gene is a major transcription factor with a wide range of pleiotropic functions, including a fundamental conserved role in sexual differentiation, species-specific morphology and an important influence on male sexual behaviour. Here, we examine the structure of fruitless in multiple species of Drosophila, and determine the patterns of selective constraint acting across the coding region. We found that the pattern of selection, estimated from the ratio of non-synonymous to synonymous substitutions, varied considerably across the gene, with most regions of the gene evolutionarily conserved but with several regions showing evidence of divergence as a result of positive selection. The regions that showed evidence of positive selection were found to be localised to relatively consistent regions across multiple speciation events, and are associated with alternative splicing. Alternative splicing may thus provide a route to gene diversification in key regulatory loci.Heredity advance online publication, 23 October 2013; doi:10.1038/hdy.2013.106.
    Heredity 10/2013; 112(3). DOI:10.1038/hdy.2013.106 · 3.81 Impact Factor
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    • "Unfortunately, none of the tested transgenic lines effectively suppressed Md-fru (data not shown). On the other hand, downregulation of the respective fru homolog in the hemimetabolous species, Blatetella germanica and Schistocerca gregaria, both of which are responsive to systemic effects of dsRNA silencing, have been shown to disrupt proper male behavior suggesting that fru plays an important and widely conserved role in male behavior [45], [46], [47]. However, it is not known whether the fru homologs in these species are direct targets of the sex-determining cascade and whether of not these homologs are specifically expressed in the CNS. "
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    ABSTRACT: In Drosophila melanogaster, genes of the sex-determination hierarchy orchestrate the development and differentiation of sex-specific tissues, establishing sex-specific physiology and neural circuitry. One of these sex-determination genes, fruitless (fru), plays a key role in the formation of neural circuits underlying Drosophila male courtship behavior. Conservation of fru gene structure and sex-specific expression has been found in several insect orders, though it is still to be determined whether a male courtship role for the gene is employed in these species due to the lack of mutants and homologous experimental evidence. We have isolated the fru ortholog (Md-fru) from the common housefly, Musca domestica, and show the gene's conserved genomic structure. We demonstrate that male-specific Md-fru transcripts arise by conserved mechanisms of sex-specific splicing. Here we show that Md-fru, is similarly involved in controlling male courtship behavior. A male courtship behavioral function for Md-fru was revealed by the behavioral and neuroanatomical analyses of a hypomorphic allele, Md-tra(man) , which specifically disrupted the expression of Md-fru in males, leading to severely impaired male courtship behavior. In line with a role in nervous system development, we found that expression of Md-fru was confined to neural tissues in the brain, most prominently in optic neuropil and in peripheral sensory organs. We propose that, like in Drosophila, overt sexual differentiation of the housefly depends on a sex-determining pathway that bifurcates downstream of the Md-tra gene to coordinate dimorphic development of non-neuronal tissues mediated by Md-dsx with that of neuronal tissues largely mediated by Md-fru.
    PLoS ONE 04/2013; 8(4):e62476. DOI:10.1371/journal.pone.0062476 · 3.23 Impact Factor
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    • "In orthopteran insects, as various grasshoppers of Chorthippus spp. [35], the desert locust Schistocerca gregaria [36] and the cockroach Blatella germanica [37], fru orthologues were isolated but no sex-specific transcripts were detected by RT-PCR analysis. In spite of this, fru nymphal RNAi knockdown experiments revealed that in S. gregaria and B. germanica fru orthologues play important roles respectively in the regulation of successful copulation in the adult male [36] and in male sexual behaviour [37]. "
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    ABSTRACT: In Drosophila melanogaster the doublesex (dsx) and fruitless (fru) regulatory genes act at the bottom of the somatic sex determination pathway. Both are regulated via alternative splicing by an upstream female-specific TRA/TRA-2 complex, recognizing a common cis element. dsx controls somatic sexual differentiation of non-neural as well as of neural tissues. fru, on the other hand, expresses male-specific functions only in neural system where it is required to built the neural circuits underlying proper courtship behaviour. In the mosquito Aedes aegypti sex determination is different from Drosophila. The key male determiner M, which is located on one of a pair of homomorphic sex chromosomes, controls sex-specific splicing of the mosquito dsx orthologue. In this study we report the genomic organization and expression of the fru homologue in Ae. aegypti (Aeafru). We found that it is sex-specifically spliced suggesting that it is also under the control of the sex determination pathway. Comparative analyses between the Aeafru and Anopheles gambiae fru (Angfru) genomic loci revealed partial conservation of exon organization and extensive divergence of intron lengths. We find that Aeadsx and Aeafru share novel cis splicing regulatory elements conserved in the alternatively spliced regions. We propose that in Aedes aegypti sex-specific splicing of dsx and fru is most likely under the control of splicing regulatory factors which are different from TRA and TRA-2 found in other dipteran insects and discuss the potential use of fru and dsx for developing new genetic strategies in vector control.
    PLoS ONE 02/2013; 8(2):e48554. DOI:10.1371/journal.pone.0048554 · 3.23 Impact Factor
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