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Double-stranded RNA (dsRNA) can trigger RNA interference (RNAi) and lead to directed silencing of specific genes. This natural defense mechanism and RNA-based products have been explored for their potential as a sustainable and ecofriendly alternative for pest control of species of agricultural importance and disease vectors. Yet, further research, development of new products and possible applications require a cost-efficient production of dsRNA. In vivo transcription of dsRNA in bacterial cells has been widely used as a versatile and inducible system for production of dsRNA combined with a purification step required to extract the dsRNA. Here, we optimized an acidic phenol-based protocol for extraction of bacterially produced dsRNA at low cost and good yield. In this protocol, bacterial cells are efficiently lysed, with no viable bacterial cells present in the downstream steps of the purification. Furthermore, we performed a comparative dsRNA quality and yield assessment of our optimized protocol and other protocols available in the literature and confirmed the cost-efficiency of our optimized protocol by comparing the cost of extraction and yields of each extraction method.

Genetic sexing strains (GSS) are an important tool in support of sterile insect technique (SIT) applications against insect pests and disease vectors. The yet unknown temperature-sensitive lethal (tsl) gene and the recently identified white pupae (wp) gene have been used as selectable markers in the most successful GSS developed so far, the Ceratitis capitata (medfly) VIENNA 8 GSS. The molecular identification of the tsl gene may open the way for its use as a marker for the development of GSS in other insect pests and disease vectors of SIT importance. Prior studies have already shown that the tsl gene is located on the right arm of chromosome 5, between the wp and Zw loci (tsl genomic region). In the present study, we used genomic, transcriptomic, bioinformatic, and cytogenetic approaches to characterize and analyze this genomic region in wild-type and tsl mutant medfly strains. Our results suggested the presence of 561 genes, with 322 of them carrying SNPs and/or insertion-deletion (indel) mutations in the tsl genomic region. Furthermore, comparative transcriptomic analysis indicated the presence of 32 differentially expressed genes, and bioinformatic analysis revealed the presence of 33 orthologs with a described heat-sensitive phenotype of Drosophila melanogaster in this region. These data can be used in functional genetic studies to identify the tsl gene(s) and the causal mutation(s) responsible for the temperature-sensitive lethal phenotype in medfly, and potentially additional genes causing a similar phenotype.

Selective inhibition of eukaryotic initiation factor 4A (eIF4A), an RNA helicase, has been proposed as a strategy to fight pathogens. Plant-derived rocaglates exhibit some of the highest specificities among eIF4A inhibitors. Sensitivity to rocaglates is determined by key amino acid (aa) residues mediating reversible clamping of the eIF4A:RNA complex. To date, no comprehensive assessment of eIF4A sensitivity to rocaglates across the eukaryotic tree of life has been performed to determine their anti-pathogenic potential.We performed an in silico analysis of the substitution patterns of six aa residues in eIF4A1 critical to rocaglate binding (human positions 158, 159, 163, 192, 195, 199), uncovering 35 pattern variants among 365 eIF4As sequenced to date. In silico molecular docking analysis of the eIF4A:RNA:rocaglate complexes of the 35 variants, modeled in a human eIF4A environment, and in vitro thermal shift assays with recombinantly expressed human eIF4A mutants, representing select natural and artificial variants, revealed that sensitivity to a natural or one of two synthetic rocaglates—silvestrol, CR-1-31-B, or zotatifin—was associated with lower inferred binding energies and higher melting temperature shifts. Helicase activities were comparable across variants and independent of sensitivity to rocaglates.In vitro testing with silvestrol validated predicted resistance based on position 163 substitutions in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii.Our analysis shows resistance to rocaglates emerging in disparate eukaryotic clades pointing to resistance being a selective neutral trait except in rocaglate-producing Aglaia plants and their fungal parasite Ophiocordyceps. The analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens including Galleria mellonella, Ustilago maydis, Babesia ovata, and Cryptosporidium sp., with rocaglates. Finally, combined docking and thermal shift analyses might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens.Author SummaryIn the ongoing search for novel ways to fight non-viral and non-bacterial pathogens, targeting translation—the universal process of protein synthesis—to inhibit growth and cell proliferation has emerged as an attractive strategy. Here, we focused on the potential of rocaglates, a group of plant-derived compounds, to inhibit an early step in translation mediated by a RNA helicase called eIF4A. We performed a comprehensive analysis of eIF4A sequence variants to determine their potential sensitivities to rocaglates, especially in pathogens of prokaryotic, fungal, or animal origin. We complemented this in silico analysis with enzyme-based in vitro and whole pathogen in vivo experiments to confirm the sensitivity or resistance to rocaglates of specific variants of eIF4A. Our analysis provides the first comprehensive picture of rocaglate sensitivity among pathogens and establishes targeting important insect, plant, animal, and human pathogens such as wax moth larvae, a major parasite of honey bees, corn smut, a widely distributed fungal disease, Babesia, a livestock parasite that causes anemia and babesiois, and Cryptosporidium, the causative organism of cryptosporidiosis in humans, with rocaglates as a viable anti-pathogen strategy.

Native to Southeast Asia, the spotted wing drosophila (SWD), Drosophila suzukii Matsumura, rapidly invaded America and Europe in the past 20 years. As a crop pest of soft-skinned fruits with a wide range of host plants, it threatens the fruit industry worldwide, causing enormous economic losses. To control this invasive pest species, an understanding of its population dynamics and structure is necessary. Here, we report the population genetics and development of SWD in Germany from 2017–19 using microsatellite markers over 11 different sample sites. It is the first study that examines SWD’s genetic changes over 3 years compared to multiple international SWD laboratory strains. Results show that SWD populations in Germany are highly homogenous without differences between populations or years, which indicates that populations are well adapted, migrate freely, and multiple invasions from outside Germany either did not take place or are negligible. Such high genetic variability and migration between populations could allow for a fast establishment of the pest species. This is especially problematic with regard to the ongoing spread of this invasive species and could bear a potential for developing pesticide resistance, which could increase the impact of the SWD further in the future.

Apoptosis is a fundamental process for the elimination of damaged or unwanted cells, and is a key aspect of development. It is triggered by pro-apoptotic genes responding to the intrinsic pathway that senses cell stress or the extrinsic pathway that responds to signals from other cells. The disruption of these genes can therefore lead to developmental defects and disease. Pro-apoptotic genes have been studied in detail in the fruit fly Drosophila melanogaster, a widely-used developmental model. However, little is known about the corresponding genes in its relative D. suzukii, a pest of soft fruit crops that originates from Asia but is now an invasive species in many other regions. The characterization of D. suzukii pro-apoptotic genes could lead to the development of transgenic sexing strains for pest management. Here, we describe the isolation and characterization of the pro-apoptotic genes reaper (Dsrpr), head involution defective (Dshid) and grim (Dsgrim) from a laboratory strain of D. suzukii. We determined their expression profiles during development, revealing that all three genes are expressed throughout development but Dsrpr is expressed most strongly, especially at the pupal stage. Functional analysis was carried out by expressing single genes or pairs (linked by a 2A peptide) in S2 cell death assays, indicating that Dsgrim and Dshid are more potent pro-apoptotic genes than Dsrpr, and the lethality can be significantly enhanced by co-expression of two genes. Therefore, the binary or multiple expression of different pro-apoptotic genes can be considered to build an efficient transgenic sexing system in D. suzukii.

Olfaction plays an essential role in insect behavior such as host location, foraging, mating, and oviposition. The odorant receptor co-receptor (Orco) is an obligatory odorant receptor and indispensable in odor perception. Here, we characterized the Orco gene from the oriental fruit fly, Bactrocera dorsalis (Hendel), a notorious agriculture pest. The olfactory deficiency mutants were generated by editing the BdorOrco gene using the CRISPR/Cas9 system. Electroantennograms (EAG) and olfactory preference assays confirmed that BdorOrco

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. However, irradiation in normal atmospheric conditions can be damaging for males, because irradiation generates substantial biological oxidative stress that, combined with domestication and mass-rearing conditions, may reduce sterile male sexual competitiveness and quality. In this study, biological oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate their role in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited only a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

Reproductive sterility is the basis of the sterile insect technique (SIT) and essential for its success in the field. Numerous factors that influence dose–response in insects have been identified. However, historically the radiation dose administered has been considered a constant. Efforts aiming to standardize protocols for mosquito irradiation found that, despite carefully controlling many variable factors, there was still an unknown element responsible for differences in expected sterility levels of insects irradiated with the same dose and handling protocols. Thus, together with previous inconclusive investigations, the question arose whether dose really equals dose in terms of biological response, no matter the rate at which the dose is administered. Interestingly, the dose rate effects studied in human nuclear medicine indicated that dose rate could alter dose–response in mammalian cells. Here, we conducted experiments to better understand the interaction of dose and dose rate to assess the effects in irradiated mosquitoes. Our findings suggest that not only does dose rate alter irradiation-induced effects, but that the interaction is not linear and may change with dose. We speculate that the recombination of reactive oxygen species (ROS) in treatments with moderate to high dose rates might minimize indirect radiation-induced effects in mosquitoes and decrease sterility levels, unless dose along with its direct effects is increased. Together with further studies to identify an optimum match of dose and dose rate, these results could assist in the development of improved methods for the production of high-quality sterile mosquitoes to enhance the efficiency of SIT programs.

The Sterile Insect Technique (SIT) is a successful autocidal control method that uses ionizing radiation to sterilize insects. Unfortunately, irradiation in normal atmospheric conditions can be damaging for males, because it generates substantial oxidative stress that, combined with mass-rearing conditions, may reduce their sexual competitiveness and quality. In this study, oxidative stress and antioxidant capacity were experimentally manipulated in Anastrepha suspensa using a combination of low-oxygen conditions and transgenic overexpression of mitochondrial superoxide dismutase (SOD2) to evaluate the role of oxidative stress and cellular antioxidants in the sexual behavior and quality of irradiated males. Our results showed that SOD2 overexpression enhances irradiated insect quality and improves male competitiveness in leks. However, the improvements in mating performance were modest, as normoxia-irradiated SOD2 males exhibited a 22% improvement in mating success compared to normoxia-irradiated wild type males. Additionally, SOD2 overexpression did not synergistically improve the mating success of males irradiated in either hypoxia or severe hypoxia. Short-term hypoxic and severe-hypoxic conditioning hormesis, per se, increased antioxidant capacity and enhanced sexual competitiveness of irradiated males relative to non-irradiated males in leks. Our study provides valuable new information that antioxidant enzymes, particularly SOD2, have potential to improve the quality and lekking performance of sterile males used in SIT programs.

The spotted-wing Drosophila (Drosophila suzukii Matsumura) is native to eastern Asia, but has become a global threat to fruit production. In recent years, CRISPR/Cas9 targeting was established in this species allowing for functional genomic and genetic control studies. Here, we report the generation and characterization of Cas9-expressing strains of D. suzukii. Five independent transgenic lines were generated using a piggyBac construct containing the EGFP fluorescent marker gene and the Cas9 gene under the control of the D. melanogaster heat shock protein 70 promoter and 3’UTR. Heat-shock (HS) treated embryos were analyzed by reverse transcriptase PCR, revealing strong heat inducibility of the transgenic Cas9 expression. By injecting gRNA targeting EGFP into one selected line, 50.0% of G0 flies showed mosaic loss-of-fluorescence phenotype, and 45.5% of G0 flies produced G1 mutants without HS. Such somatic and germline mutagenesis rates were increased to 95.4% and 85.7%, respectively, by applying a HS. Parental flies receiving HS resulted in high inheritance of the mutation (92%) in their progeny. Additionally, targeting the endogenous gene yellow led to the lack of pigmentation and male lethality. We discuss the potential use of these efficient and temperature-dependent Cas9-expressing strains for the genetic studies in D. suzukii.

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of novel white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of the wp− mutations suggest that this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

The Spotted-wing Drosophila (Drosophila suzukii) is a devastating invasive pest of fruit crops. In D. melanogaster, the white (w) gene was associated with pigmentation and mating behavior, which are also important aspects to understand the invasion biology as well as to develop control strategies for D. suzukii. Here, we show that the generation of D. suzukii white-eyed mutants by CRISPR/Cas9 mutagenesis of the w gene resulted in the complete failure of copulation when w− males were individually paired with w− females in small circular arenas (diameter 0.7 cm) for 24 h. Further analysis showed that the mating defect was associated with w− males and could not be rectified by two years of inbreeding by crossing sibling w− females with w+ males, dim red illumination, male–female sexual training, changing to large arenas (diameter 3.5 cm), or different sex ratios. Profound pigmentation deficiency was detected in the compound eyes, ocelli, Malpighian tubules and testis sheaths in the w− flies. Specifically, testis imaging showed that w− males failed to deposit any pigments into pigment cells of the testis sheath, and produced smaller sperms and less seminal fluid compared to those from wildtype males. Together these observations suggest that the w gene plays an essential role in the regulation of sexual behavior and reproduction in D. suzukii. The similarities and differences in w gene function between D. suzukii and D. melanogaster in the context of pigmentation and mating behavior are discussed.

Vector control programs based on population reduction by matings with mass-released sterile insects require the release of only male mosquitoes, as the release of females, even if sterile, would increase the number of biting and potentially disease-transmitting individuals. While small-scale releases demonstrated the applicability of sterile males releases to control the yellow fever mosquito Aedes aegypti, large-scale programs for mosquitoes are currently prevented by the lack of efficient sexing systems in any of the vector species.Different approaches of sexing are pursued, including classical genetic and mechanical methods of sex separation. Another strategy is the development of transgenic sexing systems. Such systems already exist in other insect pests. Genome modification tools could be used to apply similar strategies to mosquitoes. Three major tools to modify mosquito genomes are currently used: transposable elements, site-specific recombination systems, and genome editing via TALEN or CRISPR/Cas. All three can serve the purpose of developing sexing systems and vector control strains in mosquitoes in two ways: first, via their use in basic research. A better understanding of mosquito biology, including the sex-determining pathways and the involved genes can greatly facilitate the development of sexing strains. Moreover, basic research can help to identify other regulatory elements and genes potentially useful for the construction of transgenic sexing systems. Second, these genome modification tools can be used to apply the gained knowledge to build and test mosquito sexing strains for vector control.

The spotted-wing drosophila or cherry vinegar fly (Drosophila suzukii) is native to Asia but has invaded other continents since 2008 and has spread throughout Europe. The females have a serrated ovipositor allowing them to penetrate the skins of intact ripening fruits to deposit their eggs, and the developing larvae rapidly destroy the fruits close to harvest. Drosophila suzukii has a rapid life cycle and the larvae develop well beneath the fruit surface. This means that the use of pesticides is problematic and often not effective, first due to their restricted use close to harvest to protect consumers, and second because the larvae are deep enough inside the fruit to avoid contact. There are currently no effective and environmentally sustainable pest control methods for this species, resulting in extensive damage to fruit crops. Here, we review the current status of D. suzukii as a fruit crop pest and discuss the feasibility of current pesticide-free control methods. We also consider the potential of new technologies as a basis for the urgently needed specific and long-term control of this species.

The spotted wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is an invasive pest of stone fruits and berries currently without effective control management. The sterile insect technique (SIT) is an environmentally friendly and effective pest control method that releases sterile males to mate with wild females resulting in the suppression or eradication of targeted pest populations. New molecular technologies and genome editing methods are paving the way to bring this technology to invasive species like D. suzukii. The CRISPR/Cas technology is emerging as a versatile option for efficient tailored gene manipulation. Components of this system, the guide RNA (gRNA) and Cas9 protein, can be delivered into the organism in the form of DNA, RNA, or protein. We report CRISPR/Cas-mediated site-specific white gene editing using purified Cas9 protein delivered directly into D. suzukii embryos. Mutant flies with 13, 3, and 1 nt deletions within the cutting site of the Cas9 protein were retrieved. Moreover, the two independent G1 deletion events (13 and 3 nt) derived from the same G0 female. Our results show that the recombinant Cas9 protein could be a method of choice for generating heritable gene modifications in D. suzukii.

BackgroundThe spotted-wing Drosophila (Drosophila suzukii) is a widespread invasive pest that causes severe economic damage to fruit crops. The early development ofD. suzukiiis 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.ResultsHere, we report the isolation of theD. suzukiicellularization genesnullo, serendipity-α, bottleneckandslow-as-molassesfrom 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 intopiggyBacvectors and compared in vitro for the promoter activities. TheDsnullopromoter showed the highest activity in the cell culture assays usingD. melanogasterS2 cells.ConclusionsThe similarities in the gene coding and 5′ flanking sequence as well as in the expression pattern of the four cellularization genes betweenD. melanogasterandD. 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 theDsnullopromoter could be a suitable candidate, all reported promoters here are subject to further in vivo analyses before constructing potential pest control systems.

The sterile insect technique (SIT) is an environmentally friendly pest control method involving sterilization of mass reared insects and their release into the field to suppress insect populations. It has been a valuable tool for insect pest management for several decades. Besides the classical genetic approaches in use, transgenic systems have been established that have or could be transferred to the agricultural pest species Drosophila suzukii to improve the efficiency of population suppression such as through the production and release of only male flies. For male-only strains, conditional gene expression systems are required to inhibit female lethality due to expression of a lethal gene. Practically, such a strain with an “off-system” can be reared in a bisexual way in the mass rearing facility with food supplement and create male-only populations for field release by activating female lethality through removal of the supplement. In this chapter, we discuss conditional expression systems that have been developed in the past and their potential for D. suzukii control. Methods include the Tet-Off and Tet-On, Erythromycin-Off, Biotin-On, Vanillic acid regulated, Phloretin-Off, Bile acid-Off, and the Quinic acid systems for expression control. In addition, systems that work on stimuli based on light and temperature are discussed.

BackgroundRadiation induced sterility is the basis of the Sterile Insect Technique, by which a target insect pest population is suppressed by releasing artificially reared sterile males of the pest species in overflooding numbers over a target site. In order for the sterile males to be of high biological quality, effective standard irradiation protocols are required. Following studies investigating the effects of mosquito pupae irradiation in waterversusin air, there is a need to investigate the oxy-regulatory behavior of mosquito pupae in water to better understand the consequences of irradiation in hypoxic versus normoxic conditions.MethodsPupae ofAedes aegypti,Ae. albopictus, andAnopheles arabiensiswere submerged in water inside air-tight 2 ml glass vials at a density of 100 pupae/ml and the dissolved oxygen (DO) levels in the water were measured and plotted over time. In addition, male pupae ofAe. aegypti(aged 40–44 h),Ae. albopictus(aged 40–44 h) andAn. arabiensis(aged 20–24 h) were irradiated in a gammacell220 at increasing doses in either hypoxic (water with 2content) or normoxic (in air) conditions. The males were then mated to virgin females and resulting eggs were checked for induced sterility.ResultsAll three species depleted the water of DO to levels under 0.5% within 30 minutes, withAn. arabiensisconsuming oxygen the fastest at under 10 minutes. Following irradiation, the protective effect of hypoxia was observed across species and doses (P An. arabiensis.ConclusionsThe consumption of dissolved oxygen by pupae submerged in water was significantly different between species, indicating that their oxy-regulatory capacity seems to have possibly evolved according to their preferred breeding site characteristics. This needs to be considered when sterilizing male mosquitoes at pupal stage in water. Depending on species, their DO consumption rates and their density, irradiation doses needed to achieve full sterility may vary significantly. Further assessments are required to ascertain optimal conditions in terms of ambient atmosphere during pupal irradiation to produce competitive sterile males, and temperature and density dependent effects are expected.

International audience; Gene Drives are regarded as future tools with a high potential for population control. Due to their inherent ability to overcome the rules of Mendelian inheritance, gene drives (GD) may spread genes rapidly through populations of sexually reproducing organisms. A release of organisms carrying a GD would constitute a paradigm shift in the handling of genetically modified organisms because gene drive organisms (GDO) are designed to drive their transgenes into wild populations and thereby increase the number of GDOs. The rapid development in this field and its focus on wild populations demand a prospective risk assessment with a focus on exposure related aspects. Presently, it is unclear how adequate risk management could be guaranteed to limit the spread of GDs in time and space, in order to avoid potential adverse effects in socio-ecological systems. The recent workshop on the “Evaluation of Spatial and Temporal Control of Gene Drives” hosted by the Institute of Safety/Security and Risk Sciences (ISR) in Vienna aimed at gaining some insight into the potential population dynamic behavior of GDs and appropriate measures of control. Scientists from France, Germany, England, and the USA discussed both topics in this meeting on April 4–5, 2019. This article summarizes results of the workshop.

In many species, courtship displays are reliable signals of male quality, and current hypotheses suggest that these displays allow females to choose males with high cellular function. Environmental stressors generate excess reactive oxygen species (ROS) that impair cellular function, and thus antioxidant pathways that remove ROS are probably critical for preserving complex sexual behaviours. Here, we test the hypothesis that enhanced antioxidant activity in mitochondria preserves mating performance following oxidative stress. Using a transgenic approach, we directly manipulated mitochondrial antioxidant activity in the Caribbean fruit fly,Anastrepha suspensa,a lek-mating species with elaborate sexual displays and intense sexual selection that is also a model for sterile insect technique programmes. We generated seven transgenic lines that overexpress mitochondrial superoxide dismutase (MnSOD). Radiation is a severe oxidative stressor used to induce sterility for sterile insect programmes. After radiation treatment, two lines with intermediate MnSOD overexpression showed enhanced mating performance relative to wild-type males. These improvements in mating corresponded with reduced oxidative damage to lipids, demonstrating that MnSOD overexpression protects flies from oxidative stress at the cellular level. For lines with improved mating performance, overexpression also preserved locomotor activity, as indicated by a laboratory climbing assay. Our results show a clear link between oxidative stress, antioxidant capacity and male performance. Our work has implications for fundamentally understanding the role of antioxidants in sexual selection, and shows promise for using transgenic approaches to enhance the field performance of insects released for area-wide pest management strategies and improving performance of biological control agents in general.

The Drosophila melanogaster β2-tubulin gene (Dm-β2t) controls the function of microtubules in the testis and sperm, and has been evaluated for use in biocontrol strategies based on the sterile insect technique, including sexing and the induction of male sterility. The spotted-wing Drosophila (Drosophila suzukii) is native to eastern Asia but has spread globally as an invasive pest of fruit crops, so biocontrol strategies are urgently required for this species. We therefore isolated the β2tubulin ortholog Ds-β2t from the USA laboratory strain of D. suzukii and confirmed the presence of functional motifs by aligning orthologs from multiple insects. The developmental expression profile of Ds-β2t was determined by RT-PCR using gene-specific primers and was similar to that of Dm-β2t. We then isolated the Ds-β2t promoter and used it to generate transgenic strains expressing a testis-specific fluorescent protein starting from the thirdinstar larvae. Efficient sexing was achieved based on fluorescence detection, and the transgenic males showed a similar survival rate to wild-type males. Fluorescence imaging and PCR were also used to confirm the insemination of wild-type females by transgenic males. We therefore confirm that D. suzukii strains expressing fluorescent markers under the control of the Ds-β2t promoter can be used for sexing and the confirmation of mating, and we discuss the wider potential of the Ds-β2t promoter in the context of genetic control strategies for D. suzukii.

The sterile insect technique (SIT) involves the mass release of sterile males to suppress insect pest populations. SIT has been improved for larval pests by the development of strains for female-specific tetracycline-suppressible (Tet-off) embryonic lethal systems for male-only populations. Here we describe the extension of this approach to the Mexican fruit fly, Anastrepha ludens, using a Tet-off driver construct with the Tet-transactivator (tTA) under embryo-specific Anastrepha suspensa serendipity α (As-sry-α) promoter regulation. In the absence of tetracycline, tTA acts upon a Tet-response element linked to the pro-apoptotic cell death gene lethal effector, head involuation defective (hid), from A. ludens (Alhid(Ala2) ) that contains a sex-specific intron splicing cassette, resulting in female-specific expression of the lethal effector. Parental adults double-homozygous for the driver/effector vectors were expected to yield male-only progeny when reared on Tet-free diet, but a complete lack of oviposited eggs resulted for each of the three strains tested. Ovary dissection revealed nonvitellogenic oocytes in all strains that was reversible by feeding females tetracycline for 5 days after eclosion, resulting in male-only adults in one strain. Presumably the sry-α promoter exhibits prezygotic maternal expression as well as zygotic embryonic expression in A. ludens, resulting in a Tet-off sterility effect in addition to female-specific lethality.

Polycistronic 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.

Drosophila suzukii is a significant pest of stone and small fruits. The genome of this species has been sequenced and manipulated by transposon-mediated transformation and CRISPR/Cas9 gene editing. These technologies open a variety of possibilities for functional genomics and genetic modifications that might improve biologically based population control strategies. Both of these approaches, however, would benefit from genome targeting that would avoid position effects and insertional mutations associated with random transposon vector insertions, and the limited DNA fragment insertion size allowed by gene editing. Here, we describe an efficient recombinase-mediated cassette exchange (RMCE) system for D. suzukii in which heterospecific lox recombination sites were integrated into the genome by transposon-mediated transformation and subsequently targeted for double recombination by a donor vector in the presence of Cre recombinase. Three loxN/lox2272 landing site lines have previously been created in D. suzukii, and quantitative PCR determined that polyubiquitin-regulated enhanced green fluorescent protein expression is least susceptible to position effect suppression in the 443_M26m1 line. We presume that RMCE target sites may also be inserted more specifically into the genome by homology-directed repair gene editing, thereby avoiding position effects and mutations, while eliminating restrictions on the size of donor constructs for subsequent insertion.

The Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing enormous economic damage in horticultural systems. A successful and environment-friendly control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. However, the SIT is not readily applicable to each pest species. While transgenic approaches hold great promise to improve critical aspects of the SIT to transfer it to new species, they are suspect to strict or even prohibitive legislation regarding the release of genetically modified (GM) organisms. In contrast, specific mutations created via CRISPR-Cas genome editing are not regulated as GM in the US, and might thus allow creating optimal strains for SIT. Here, we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring. Based on the achieved efficiency, this approach could also be used to introduce mutations which do not produce a screenable phenotype and identify positive mutants with a reasonable workload. Furthermore, CRISPR-Cas HDR would allow to recreate mutations formerly identified in classical mutagenesis screens and to transfer them to related species to establish new (SIT-like) pest control systems. Considering the potential that CRISPR-induced alterations in organisms could be classified as non-GM in additional countries, such new strains could potentially be used for pest control applications without the need to struggle with GMO directives.

Site-specific genome modification (SSM) is an important tool for mosquito functional genomics and comparative gene expression studies, which contribute to a better understanding of mosquito biology and are thus a key to finding new strategies to eliminate vector-borne diseases. Moreover, it allows for the creation of advanced transgenic strains for vector control programs. SSM circumvents the drawbacks of transposon-mediated transgenesis, where random transgene integration into the host genome results in insertional mutagenesis and variable position effects. We applied the Cre/lox recombinase-mediated cassette exchange (RMCE) system to Aedes aegypti, the vector of dengue, chikungunya, and Zika viruses. In this context we created four target site lines for RMCE and evaluated their fitness costs. Cre-RMCE is functional in a two-step mechanism and with good efficiency in Ae. aegypti. The advantages of Cre-RMCE over existing site-specific modification systems for Ae. aegypti, phiC31-RMCE and CRISPR, originate in the preservation of the recombination sites, which 1) allows successive modifications and rapid expansion or adaptation of existing systems by repeated targeting of the same site; and 2) provides reversibility, thus allowing the excision of undesired sequences. Thereby, Cre-RMCE complements existing genomic modification tools, adding flexibility and versatility to vector genome targeting.