Competitive Sperm-Marked Beetles for Monitoring Approaches in Genetic Biocontrol and Studies in Reproductive Biology.

Sperm marking provides a key tool for reproductive biology studies, but it also represents a valuable monitoring tool for genetic pest control strategies such as the sterile insect technique. Sperm-marked lines can be generated by introducing transgenes that mediate the expression of fluorescent proteins during spermatogenesis. The homozygous lines established by transgenesis approaches are going through a genetic bottleneck that can lead to reduced fitness. Transgenic SIT approaches have mostly focused on Dipteran and Lepidopteran pests so far. With this study, we provide sperm-marked lines for the Coleopteran pest model organism, the red flour beetle Tribolium castaneum, based on the ß2-tubulin promoter/enhancer driving red (DsRed) or green (EGFP) fluorescence. The obtained lines are reasonably competitive and were thus used for our studies on reproductive biology, confirming the phenomenon of 'last-male sperm precedence' and that the spermathecae are deployed for long-term sperm storage, enabling the use of sperm from first mating events even after secondary mating events for a long period of time. The homozygosity and competitiveness of the lines will enable future studies to analyze the controlled process of sperm movement into the long-term storage organ as part of a post-mating cryptic female choice mechanism of this extremely promiscuous species.

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

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 φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α 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 (ß2t) 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 φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-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.

The Red Flour Beetle as Model for Comparative Neural Development: Genome Editing to Mark Neural Cells in Tribolium Brain Development.

With CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) scientists working with Tribolium castaneum can now generate transgenic lines with site-specific insertions at their region of interest. We present two methods to generate in vivo imaging lines suitable for marking subsets of neurons with fluorescent proteins. The first method relies on homologous recombination and uses a 2A peptide to create a bicistronic mRNA. In such lines, the target and the marker proteins are not fused but produced at equal amounts. This work-intensive method is compared with creating gene-specific enhancer traps that do not rely on homologous recombination. These are faster to generate but reflect the expression of the target gene less precisely. Which method to choose, strongly depends on the aims of each research project and in turn impacts of how neural cells and their development are marked. We describe the necessary steps from designing constructs and guide RNAs to embryonic injection and making homozygous stocks.

Improvement and use of CRISPR/Cas9 to engineer a sperm-marking strain for the invasive fruit pest Drosophila suzukii.

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 (ß2t) 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_ß2t 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 ß2t 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.

Consequences of resistance evolution in a Cas9-based sex conversion-suppression gene drive for insect pest management.

The use of a site-specific homing-based gene drive for insect pest control has long been discussed, but the easy design of such systems has become possible only with the recent establishment of CRISPR/Cas9 technology. In this respect, novel targets for insect pest management are provided by new discoveries regarding sex determination. Here, we present a model for a suppression gene drive designed to cause an all-male population collapse in an agricultural pest insect. To evaluate the molecular details of such a sex conversion-based suppression gene drive experimentally, we implemented this strategy in Drosophila melanogaster to serve as a safe model organism. We generated a Cas9-based homing gene-drive element targeting the transformer gene and showed its high efficiency for sex conversion from females to males. However, nonhomologous end joining increased the rate of mutagenesis at the target site, which resulted in the emergence of drive-resistant alleles and therefore curbed the gene drive. This confirms previous studies that simple homing CRISPR/Cas9 gene-drive designs will be ineffective. Nevertheless, by performing population dynamics simulations using the parameters we obtained in D. melanogaster and by adjusting the model for the agricultural pest Ceratitis capitata, we were able to identify adequate modifications that could be successfully applied for the management of wild Mediterranean fruit fly populations using our proposed sex conversion-based suppression gene-drive strategy.

Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species.

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.