Dataset Information

Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

ABSTRACT:

Background

The invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing.

Results

We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established ?C31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform ?C31-Recombinase Mediated Cassette Exchange (?C31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express ?C31 integrase maternally to generate self-docking lines for ?C31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity ? to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (?2t) promoter/enhancer.

Conclusion

Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using ?C31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The ?C31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12863-020-00940-5.

SUBMITTER: Ahmed H

PROVIDER: S-EPMC7747376 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The invasive fruit pest Drosophila suzukii was reported for the first time in Europe and the USA in 2008 and has spread since then. The adoption of type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) as a tool for genome manipulation provides new ways to develop novel biotechnologically-based pest control approaches. Stage or tissue-specifically expressed genes are of particular importance in the field of insect biotechnology. The enhancer/promoter of the spermatogenesis-specific beta-2-tubulin (β2t) gene was used to drive the expression of fluorescent proteins or effector molecules in testes of agricultural pests and disease vectors for sexing, monitoring, and reproductive biology studies. Here, we demonstrate an improvement to CRISPR/Cas-based genome editing in D. suzukii and establish a sperm-marking system. RESULTS:To improve genome editing, we isolated and tested the D. suzukii endogenous promoters of the small nuclear RNA gene U6 to drive the expression of a guide RNA and the Ds heat shock protein 70 promoter to express Cas9. For comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. We demonstrate the homology-dependent repair (HDR)-based genome editing efficiency by applying a previously established transgenic line that expresses DsRed ubiquitously as a target platform. In addition, we isolated the Ds_β2t gene and used its promoter to drive the expression of a red fluorescence protein in the sperm. A transgenic sperm-marking strain was then established by the improved HDR-based genome editing. CONCLUSION:The deployment of the endogenous promoters of the D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, enabled the effective application of helper plasmid co-injections instead of preformed ribonucleoproteins used in previous reports for HDR-based genome editing. The sperm-marking system should help to monitor the success of pest control campaigns in the context of the Sterile Insect Technique and provides a tool for basic research in reproductive biology of this invasive pest. Furthermore, the promoter of the β2t gene can be used in developing novel transgenic pest control approaches and the CRISPR/Cas9 system as an additional tool for the modification of previously established transgenes.

Project description:BackgroundDrosophila suzukii differs from other melanogaster group members in their proclivity for laying eggs in fresh fruit rather than in fermenting fruits. Olfaction and gustation play a critical role during insect niche formation, and these senses are largely mediated by two important receptor families: olfactory and gustatory receptors (Ors and Grs). Earlier work from our laboratory has revealed how the olfactory landscape of D. suzukii is dominated by volatiles derived from its unique niche. Signaling and reception evolve in synchrony, since the interaction of ligands and receptors together mediate the chemosensory behavior. Here, we manually annotated the Ors and Grs in D. suzukii and two close relatives, D. biarmipes and D. takahashii, and compared these repertoires to those in other melanogaster group drosophilids to identify candidate chemoreceptors associated with D. suzukii's unusual niche utilization.ResultsOur comprehensive annotations of the chemosensory genomes in three species, and comparative analysis with other melanogaster group members provide insights into the evolution of chemosensation in the pestiferous D. suzukii. We annotated a total of 71 Or genes in D. suzukii, with nine of those being pseudogenes (12.7 %). Alternative splicing of two genes brings the total to 62 genes encoding 66 Ors. Duplications of Or23a and Or67a expanded D. suzukii's Or repertoire, while pseudogenization of Or74a, Or85a, and Or98b reduced the number of functional Ors to roughly the same as other annotated species in the melanogaster group. Seventy-one intact Gr genes and three pseudogenes were annotated in D. suzukii. Alternative splicing in three genes brings the total number of Grs to 81. We identified signatures of positive selection in two Ors and three Grs at nodes leading to D. suzukii, while three copies in the largest expanded Or lineage, Or67a, also showed signs of positive selection at the external nodes.ConclusionOur analysis of D. suzukii's chemoreceptor repertoires in the context of nine melanogaster group drosophilids, including two of its closest relatives (D. biarmipes and D. takahashii), revealed several candidate receptors associated with the adaptation of D. suzukii to its unique ecological niche.

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