scholarly journals Behavioral manipulation of Drosophila suzukii for pest control: high attraction to yeast enhances insecticide efficacy when applied on leaves

2021 ◽  
Author(s):  
Guillermo Rehermann ◽  
Urban Spitaler ◽  
Karolina Sahle ◽  
Carlo S. Cossu ◽  
Lorenz Delle Donne ◽  
...  
Keyword(s):  
Pest Control ◽  
Drosophila Suzukii ◽  
Behavioral Manipulation ◽  
Insecticide Efficacy ◽  
High Attraction

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

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Hassan M. M. Ahmed ◽  
Luisa Hildebrand ◽  
Ernst A. Wimmer
Keyword(s):  
Genome Editing ◽  
Reproductive Biology ◽  
Pest Control ◽  
Basic Research ◽  
Invasive Pest ◽  
Agricultural Pests ◽  
Drosophila Suzukii ◽  
Helper Plasmid ◽  
Additional Tool

Abstract 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.

scholarly journals Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

2019 ◽  
Author(s):  
Hassan M. M. Ahmed ◽  
Luisa Hildebrand ◽  
Ernst A. Wimmer
Keyword(s):  
Genome Editing ◽  
Reproductive Biology ◽  
Pest Control ◽  
Basic Research ◽  
Invasive Pest ◽  
Agricultural Pests ◽  
Drosophila Suzukii ◽  
Helper Plasmid ◽  
Additional Tool

Abstract 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 diseases 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. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

Conditional Expression Systems for Drosophila suzukii Pest Control

2020 ◽  
pp. 195-215
Author(s):  
Syeda A. Jaffri ◽  
Ying Yan ◽  
Maxwell J. Scott ◽  
Marc F. Schetelig
Keyword(s):  
Pest Control ◽  
Expression Systems ◽  
Drosophila Suzukii ◽  
Conditional Expression

scholarly journals Identification and characterization of four Drosophila suzukii cellularization genes and their promoters

BMC Genetics ◽  
2020 ◽  
Vol 21 (S2) ◽  
Author(s):  
Ying Yan ◽  
Syeda A. Jaffri ◽  
Jonas Schwirz ◽  
Carl Stein ◽  
Marc F. Schetelig
Keyword(s):  
Control Systems ◽  
Pest Control ◽  
Embryonic Lethality ◽  
Invasive Pest ◽  
Economic Damage ◽  
Lethal Gene ◽  
Gene Promoters ◽  
Flanking Sequence ◽  
Drosophila Suzukii ◽  
Blastoderm Stage

Abstract Background The spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development of D. suzukii is similar to that of other Drosophilids, but the roles of individual genes must be confirmed experimentally. Cellularization genes coordinate the onset of cell division as soon as the invagination of membranes starts around the nuclei in the syncytial blastoderm. The promoters of these genes have been used in genetic pest-control systems to express transgenes that confer embryonic lethality. Such systems could be helpful in sterile insect technique applications to ensure that sterility (bi-sex embryonic lethality) or sexing (female-specific embryonic lethality) can be achieved during mass rearing. The activity of cellularization gene promoters during embryogenesis controls the timing and dose of the lethal gene product. Results Here, we report the isolation of the D. suzukii cellularization genes nullo, serendipity-α, bottleneck and slow-as-molasses from a laboratory strain. Conserved motifs were identified by comparing the encoded proteins with orthologs from other Drosophilids. Expression profiling confirmed that all four are zygotic genes that are strongly expressed at the early blastoderm stage. The 5′ flanking regions from these cellularization genes were isolated, incorporated into piggyBac vectors and compared in vitro for the promoter activities. The Dsnullo promoter showed the highest activity in the cell culture assays using D. melanogaster S2 cells. Conclusions The similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes between D. melanogaster and D. suzukii, suggest that conserved functions may be involved in both species. The high expression level at the early blastoderm stage of the four cellularization genes were confirmed, thus their promoters can be considered in embryonic lethality systems. While the Dsnullo promoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

scholarly journals Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

2019 ◽  
Author(s):  
Hassan M. M. Ahmed ◽  
Luisa Hildebrand ◽  
Ernst A. Wimmer
Keyword(s):  
Genome Editing ◽  
Reproductive Biology ◽  
Pest Control ◽  
Fluorescent Proteins ◽  
Basic Research ◽  
Invasive Pest ◽  
Agricultural Pests ◽  
Drosophila Suzukii ◽  
Additional Tool

Abstract Background: The invasive fruit pest Drosophila suzukii has been reported for the first time in Europe and 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 has opened the doors for researchers to develop novel biotechnologically based pest control approaches. Stage and or tissue specific 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 diseases vectors for sexing, monitoring and reproductive biology studies. Here, we demonstrate an improvement in the efficiency of 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. As a comparison, we used recombinant Cas9 protein and in vitro transcribed gRNA as a preformed ribonucleoprotein. To evaluate the homology-dependent repair (HDR)-based genome editing efficiency, we used as a target platform a previously established transgenic line that expresses DsRed ubiquitously. In addition, we isolated 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 use of the endogenous promoters of D. suzukii U6 and hsp70 genes to drive the expression of gRNA and Cas9, respectively, showed improved efficiency of gene editing compared to previous reports. 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. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

scholarly journals Behavioral Manipulation for Pest Control

Insects ◽  
2021 ◽  
Vol 12 (4) ◽  
pp. 287
Author(s):  
Valerio Mazzoni ◽  
Gianfranco Anfora
Keyword(s):  
Pest Control ◽  
Dramatic Reduction ◽  
Ecological Intensification ◽  
Behavioral Manipulation

Pest control is moving towards a dramatic reduction in pesticide-based approaches in favor of more eco-friendly strategies characterized by the promotion of ecological intensification of agriculture and reduction of human inputs (especially pesticides) [...]

Bicistronic expression and differential localization of proteins in insect cells and Drosophila suzukii using picornaviral 2A peptides

2019 ◽  
Author(s):  
Jonas Schwirz ◽  
Ying Yan ◽  
Zdenek Franta ◽  
Marc F. Schetelig
Keyword(s):  
Cell Lines ◽  
Nuclear Localization ◽  
Pest Control ◽  
Nuclear Localization Signal ◽  
Expression Systems ◽  
Drosophila Suzukii ◽  
Pest Insect ◽  
Localization Signal ◽  
Comparable Activity

AbstractPolycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells) and in vivo in the pest insect Drosophila suzukii. We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. Two of the 2A peptides were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the EGFP-CAAX protein could be bred to homozygosity whereas the DsRed-CAAX construct was lethal in the homozygous state. Our results therefore demonstrate that four different 2A constructs and two novel targeting motifs are functional in D. suzukii, and that DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains.HighlightsFour picornaviral 2A peptides have been studied for their self-cleaving ability in cell lines and in vivo in the pest insect Drosophila suzukii.All tested 2A peptides showed comparable activity that resulted in the production of independent upstream and downstream proteins.The proteins co-expressed by 2A peptides were either directed to the cell nucleus by a C-terminal nuclear localization signal (NLS), or to the cell membrane by a poly-lysine/CAAX membrane anchor.The combination of optimized membrane localization signals fused to DsRed generated an intrinsically lethal phenotype, which can be used to develop novel pest control strains.

scholarly journals Improvement and Use of CRISPR/Cas9 to Engineer a Sperm-marking Strain for the Invasive Fruit Pest Drosophila suzukii

2019 ◽  
Author(s):  
Hassan M. M. Ahmed ◽  
Luisa Hildebrand ◽  
Ernst A. Wimmer
Keyword(s):  
Genome Editing ◽  
Reproductive Biology ◽  
Pest Control ◽  
Basic Research ◽  
Invasive Pest ◽  
Agricultural Pests ◽  
Drosophila Suzukii ◽  
Helper Plasmid ◽  
Additional Tool

Abstract 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 diseases 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. The CRISPR/Cas9 system can be used as an additional tool for the modification of previously established transgenes.

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