Transgenic line for characterizing GABA-receptor expression to study the neural basis of olfaction in the yellow-fever mosquito.

The mosquito Aedes aegypti is an important vector of diseases including dengue, Zika, chikungunya, and yellow fever. Olfaction is a critical modality for mosquitoes enabling them to locate hosts, sources of nectar, and sites for oviposition. GABA is an essential neurotransmitter in olfactory processing in the insect brain, including the primary olfactory center, the antennal lobe. Previous work with Ae. aegypti has suggested that antennal lobe inhibition via GABA may be involved in the processing of odors. However, little is known about GABA receptor expression in the mosquito brain, or how they may be involved in odor attraction. In this context, generating mutants that target the mosquito's olfactory responses, and particularly the GABAergic system, is essential to achieve a better understanding of these diverse processes and olfactory coding in these disease vectors. Here we demonstrate the potential of a transgenic line using the QF2 transcription factor, GABA-B1QF2-ECFP, as a new neurogenetic tool to investigate the neural basis of olfaction in Ae. aegypti. Our results show that the gene insertion has a moderate impact on mosquito fitness. Moreover, the line presented here was crossed with a QUAS reporter line expressing the green fluorescent protein and used to determine the location of the metabotropic GABA-B1 receptor expression. We find high receptor expression in the antennal lobes, especially the cell bodies surrounding the antennal lobes. In the mushroom bodies, receptor expression was high in the Kenyon cells, but had low expression in the mushroom body lobes. Behavioral experiments testing the fruit odor attractants showed that the mutants lost their behavioral attraction. Together, these results show that the GABA-B1QF2-ECFP line provides a new tool to characterize GABAergic systems in the mosquito nervous system.

Expansion of the genetic toolbox for manipulation of the global crop pest Drosophila suzukii: Isolation and assessment of eye colour mutant strains.

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), commonly called spotted wing Drosophila, is an important agricultural pest recognised worldwide. D. suzukii is a pest of soft-skinned fruits as females can lay eggs in ripening fruit before harvest. While strains for genetic biocontrol of D. suzukii have been made, the development of transgenic D. suzukii strains and their further screening remain a challenge partly due to the lack of phenotypically trackable genetic-markers, such as those widely used with the model genetic organism D. melanogaster. Here, we have used CRISPR/Cas9 to introduce heritable mutations in the eye colour genes white, cinnabar and sepia, which are located on the X, second and third chromosomes, respectively. Strains were obtained, which were homozygous for a single mutation. Genotyping of the established strains showed insertion and/or deletions (indels) at the targeted sites. A strain homozygous for mutations in cinnabar and sepia showed a pale-yellow eye colour at eclosion but darkened to a sepia colour after a week. The fecundity and fertility of some of the cinnabar and sepia strains were comparable with the wild type. Although white mutant males were previously reported to be sterile, we found that sterility is not fully penetrant and we have been able to maintain white-eyed strains for over a year. The cinnabar, sepia and white mutant strains developed in this study should facilitate future genetic studies in D. suzukii and the development of strains for genetic control of this pest.

CRISPR/Cas9-based split homing gene drive targeting doublesex for population suppression of the global fruit pest Drosophila suzukii.

Genetic-based methods offer environmentally friendly species-specific approaches for control of insect pests. One method, CRISPR homing gene drive that target genes essential for development, could provide very efficient and cost-effective control. While significant progress has been made in developing homing gene drives for mosquito disease vectors, little progress has been made with agricultural insect pests. Here, we report the development and evaluation of split homing drives that target the doublesex (dsx) gene in Drosophila suzukii, an invasive pest of soft-skinned fruits. The drive component, consisting of dsx single guide RNA and DsRed genes, was introduced into the female-specific exon of dsx, which is essential for function in females but not males. However, in most strains, hemizygous females were sterile and produced the male dsx transcript. With a modified homing drive that included an optimal splice acceptor site, hemizygous females from each of the four independent lines were fertile. High transmission rates of the DsRed gene (94 to 99%) were observed with a line that expressed Cas9 with two nuclear localization sequences from the D. suzukii nanos promoter. Mutant alleles of dsx with small in-frame deletions near the Cas9 cut site were not functional and thus would not provide resistance to drive. Finally, mathematical modeling showed that the strains could be used for suppression of lab cage populations of D. suzukii with repeated releases at relatively low release ratios (1:4). Our results indicate that the split CRISPR homing gene drive strains could potentially provide an effective means for control of D. suzukii populations.

Development of transgenic corn planthopper Peregrinus maidis that express the tetracycline transactivator.

The corn planthopper, Peregrinus maidis, is a vector of several maize viruses and is consequently a significant agricultural pest in many tropical and subtropical regions. As P. maidis has developed resistance to insecticides, the aim of this study was to develop transgenic P. maidis strains that could be used for future genetic biocontrol programs. To facilitate the identification of transgenic P. maidis, we isolated and characterized the promoters for the P. maidis ubiquitin-like and profilin genes. Transient expression assays with P. maidis embryos showed that both promoters were active. Transgenic lines were established using piggyBac vectors and fluorescent protein marker genes. The lines carried an auto-regulated tetracycline transactivator (tTA) gene, which has been widely used to establish conditional lethal strains in other insect species. The transgenic lines showed low levels of tTA expression but were viable on diet with or without doxycycline, which inhibits the binding of tTA to DNA. We discuss possible modifications to the tTA overexpression system that could lead to the successful development of conditional lethal strains. To our knowledge, this is the first report of a transgenic Hemiptera. The approach we have taken could potentially be applied to other Hemiptera and, for P. maidis, the technology will facilitate future functional genomics studies.