Abstract
Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. However, current SIT methods are not directly transferable to many important pest insect species due to the lack of genetic sexing strains. Genome editing is revolutionizing the way we conduct genetics in insects, including in Tribolium castaneum, an important genetic model and agricultural pest. We identified orthologues of β2Tubulin, Rad50-ATPase and enolase in T. castaneum. Using RT-PCR, we confirmed that these genes are predominantly expressed in the testis. PiggyBac-based transformation of T. castaneum cis-regulatory regions derived from Tc-β2t, Tc-rad50 or Tc-eno resulted in EGFP expression specifically in the T. castaneum testis. Additionally, we determined that each of these regulatory regions regulates EGFP expression in different cell types of the male gonad. Cis-regulatory regions from Tc-β2t produced EGFP expression throughout spermatogenesis and also in mature sperms; Tc-rad50 resulted in expression only in the haploid spermatid, while Tc-eno expressed EGFP in late spermatogenesis. In summary, the regulatory cis-regions characterized in this study are not only suited to study male gonadal function but could be used for development of transgenic sexing strains that produce one sex in pest control strategies.
Highlights
Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects
We report on the development of transgenic strains expressing a fluorescent marker in the male gonads of T. castaneum, a model for coleopterans and an important pest of stored grain
In this report we show that cis-regulatory regions derived from three different T. castaneum genes were capable of controlling transgene expression specific to the testes
Summary
Genetics-based pest management processes, including the sterile insect technique, are an effective method for the control of some pest insects. A transgenic sperm-marking strain was established by HDR-based genome editing in the pest Drosophila suzuki[7] The development of such a “genetic-sexing” strain (GSS) is an alternative approach for improving the efficiency of SIT that could facilitate the mass scale separation of males and females for new pest species, including c oleopterans[8]. In this study we describe the identification and use of cis-regulatory regions derived from three genes (β2Tubulin, rad[50] and enolase) expressed predominantly in the testis of T. castaneum. These DNA cisregulatory regions enable testes-specific EGFP expression when introduced into the T. castaneum germline via piggyBac-mediated transformation. Uni-sex and tissue specific gene expression is vital for the development of novel pest insect control approaches and for experiments plan to enhance or improve our knowledge of insect molecular biology
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