Functional characterization of cytochrome P450s associated with pyrethroid resistance in the olive fruit fly Bactrocera oleae.

Resistance to pyrethroid insecticides has evolved in Bactrocera oleae populations in Greece, threatening the efficacy of control interventions based on this insecticide class. Here we report the collection of populations from Crete, with resistance levels reaching up to 132-folds, compared to susceptible laboratory strains and show that pyrethroid resistance is substantially suppressed by the PBO synergist, suggesting the involvement of detoxification enzymes. To identify specific candidate genes implicated in resistance, we performed comparative transcriptomic analysis, between the pyrethroid resistant populations from Crete and the susceptible laboratory strains, using both whole bodies and Malpighian tubules. Several genes were found differentially transcribed between resistant and susceptible flies in each comparison, with P450s being among the most highly over-expressed detoxification genes in pyrethroid resistant populations. Four of the over-expressed P450s (Cyp6A61, Cyp6G6, Cyp4P6 and Cyp6G28) were recombinantly expressed in Escherichia coli and in vitro metabolism assays revealed that CYP6A61 is capable of metabolizing alpha-cypermethrin, while CYP6G6, CYP4P6 and CYP6G28 are capable of metabolizing deltamethrin. No metabolism of neonicotinoid insecticides was recorded. We further silenced CYP6G6 in vivo, via RNAi, which led to a small, but significant increase in deltamethrin toxicity. The study provides valuable information towards the development of molecular diagnostics and evidence-based insecticide resistance management strategies.

Dynamic interactions between the symbiont Candidatus Erwinia dacicola and its olive fruit fly host Bactrocera oleae.

The olive fruit fly, Bactrocera oleae, the most serious pest of olives, requires the endosymbiotic bacterium Candidatus Erwinia dacicola in order to complete its development in unripe green olives. Hence, a better understanding of the symbiosis of Ca. E. dacicola and its insect host may lead to new strategies for B. oleae control. The relative abundance of bacteria during the fly life cycle comparing black and green olives was estimated by real time quantitative PCR revealing significant fluctuations during development in black olives with a peak of the bacteria in the second instar larvae. By microscopy analysis of larvae, we show that the bacteria reside extracellularly in the gastric caeca. During the transition to late third instar larvae, the bacteria were discharged into the midgut concomitant with a change in caeca size and morphology due to the contraction of the muscles surrounding the caeca. A similar alteration was also observed in a laboratory strain devoid of bacteria. To further investigate the symbiotic interaction and the change in caeca morphology a comparative transcriptomics analysis was undertaken. Samples of dissected caeca from second and third instar larvae collected from the field as well as second instar larvae from a laboratory strain devoid of symbionts showed significant changes in transcript expression. This highlighted genes associated with the developmental changes revealed by the microscopic analysis as well as responses to microorganisms.

Stably inherited transfer of the bacterial symbiont Candidatus Erwinia dacicola from wild olive fruit flies Bactrocera oleae to a laboratory strain.

The olive fruit fly, Bactrocera oleae, the most serious pest of olives, requires the endosymbiotic bacteria Candidatus Erwinia dacicola in order to complete its development in unripe green olives. Hence a better understanding of the symbiosis of Ca. E. dacicola and its insect host may lead to new strategies for reduction of B. oleae and thus minimize its economic impact on olive production. Studies of this symbiosis are hampered as the bacterium cannot be grown in vitro and the established B. oleae laboratory populations, raised on artificial diets, are devoid of this bacterium. Here, we sought to develop a method to transfer the bacteria from wild samples to laboratory populations. We tested several strategies. Cohabitation of flies from the field with the laboratory line did not result in a stable transfer of bacteria. We provided the bacteria directly to the egg and also in the food of the larvae but neither approach was successful. However, a robust method for transfer of Ca. E. dacicola from wild larvae or adults to uninfected flies by transplantation to females was established. Single female lines were set up and the bacteria were successfully transmitted for at least three generations. These results open up the possibilities to study the interaction between the symbiont and the host under controlled conditions, in view of both understanding the molecular underpinnings of an exciting, unique in nature symbiotic relationship, as well as developing novel, innovative control approaches.

Targeted mutagenesis using CRISPR-Cas9 in the chelicerate herbivore Tetranychus urticae.

The use of CRISPR-Cas9 has revolutionized functional genetic work in many organisms, including more and more insect species. However, successful gene editing or genetic transformation has not yet been reported for chelicerates, the second largest group of terrestrial animals. Within this group, some mite and tick species are economically very important for agriculture and human health, and the availability of a gene-editing tool would be a significant advancement for the field. Here, we report on the use of CRISPR-Cas9 in the spider mite Tetranychus urticae. The ovary of virgin adult females was injected with a mix of Cas9 and sgRNAs targeting the phytoene desaturase gene. Natural mutants of this laterally transferred gene have previously shown an easy-to-score albino phenotype. Albino sons of injected virgin females were mated with wild-type females, and two independent transformed lines where created and further characterized. Albinism inherited as a recessive monogenic trait. Sequencing of the complete target-gene of both lines revealed two different lesions at expected locations near the PAM site in the target-gene. Both lines did not genetically complement each other in dedicated crosses, nor when crossed to a reference albino strain with a known genetic defect in the same gene. In conclusion, two independent mutagenesis events were induced in the spider mite T. urticae using CRISPR-Cas9, hereby providing proof-of-concept that CRISPR-Cas9 can be used to create gene knockouts in mites.

Development of efficient RNAi in Nezara viridula for use in insecticide target discovery.

Stink bugs are an emerging pest in many regions of the world but their molecular biology is still poorly understood. While several transcriptomes are available, the lack of validated gene manipulation tools like RNA interference (RNAi) in species such as the southern green stinkbug Nezara viridula precludes the characterization of individual genes in vivo. Such tools are particularly useful in performing high-throughput screens to search for essential genes that can be prioritized as potential insecticide targets. Here, we developed and optimized an efficient RNAi in N. viridula for use in insecticide target discovery and beyond. The visible marker Sex combs reduced and the essential gene Actin were used to verify the usability and efficiency of RNAi by microinjection at both the adult and nymphal stages, respectively, with nymphal approach presenting significant advantages. Following validation, RNAi was then used to measure lethality following the knockdown (KD) of two genes that are known insecticide targets, Chitin synthase, and Acetyl-CoA carboxylase. The KD of each gene resulted in >75% corrected mortality. These results indicate that RNAi is an effective tool in N. viridula and set a benchmark to evaluate potential targets in future RNAi screens aimed at insecticide target discovery.

Medfly-Wolbachia symbiosis: genotype x genotype interactions determine host's life history traits under mass rearing conditions.

BACKGROUND: Wolbachia pipientis is a widespread, obligatory intracellular and maternally inherited bacterium, that induces a wide range of reproductive alterations to its hosts. Cytoplasmic Incompatibility (CI) is causing embryonic lethality, the most common of them. Despite that Wolbachia-borne sterility has been proposed as an environmental friendly pest control method (Incompatible Insect Technique, IIT) since 1970s, the fact that Wolbachia modifies important fitness components of its hosts sets severe barriers to IIT implementation. Mass rearing of Mediterranean fruit fly, Ceratitis capitata (medfly), is highly optimized given that this pest is a model species regarding the implementation of another sterility based pest control method, the Sterile Insect Technique (SIT). We used the medfly-Wolbachia symbiotic association, as a model system, to study the effect of two different Wolbachia strains, on the life history traits of 2 C. capitata lines with different genomic background. RESULTS: Wolbachia effects are regulated by both C. capitata genetic background and the Wolbachia strain. Wolbachia infection reduces fertility rates in both C. capitata genetic backgrounds and shortens the pre-pupa developmental duration in the GSS strain. On the other hand, regardless of the strain of Wolbachia (wCer2, wCer4) infection does not affect either the sex ratio or the longevity of adults. wCer4 infection imposed a reduction in females' fecundity but wCer2 did not. Male mating competitiveness, adults flight ability and longevity under water and food deprivation were affected by both the genetic background of medfly and the strain of Wolbachia (genotype by genotype interaction). CONCLUSION: Wolbachia infection could alter important life history traits of mass-reared C. capitata lines and therefore the response of each genotype on the Wolbachia infection should be considered toward ensuring the productivity of the Wolbachia-infected insects under mass-rearing conditions.

The Role of Insulators in Transgene Transvection in Drosophila.

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented in Drosophila where homologs are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus in Drosophila and have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologs. Homotypic trans-interactions between four different insulators can support transvection: the gypsy insulator (GI), Wari, Fab-8 and 1A2; GI and Fab-8 are more effective than Wari or 1A2 We show that, in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homolog pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair in cis, and it is further suppressed by cis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The identity of enhancers and promoters in the vicinity of a trans-interacting insulator pair is also important, indicative of complex insulator-enhancer-promoter interactions.

Resistance mutation conserved between insects and mites unravels the benzoylurea insecticide mode of action on chitin biosynthesis.

Despite the major role of chitin biosynthesis inhibitors such as benzoylureas (BPUs) in the control of pests in agricultural and public health for almost four decades, their molecular mode of action (MoA) has in most cases remained elusive. BPUs interfere with chitin biosynthesis and were thought to interact with sulfonylurea receptors that mediate chitin vesicle transport. Here, we uncover a mutation (I1042M) in the chitin synthase 1 (CHS1) gene of BPU-resistant Plutella xylostella at the same position as the I1017F mutation reported in spider mites that confers etoxazole resistance. Using a genome-editing CRISPR/Cas9 approach coupled with homology-directed repair (HDR) in Drosophila melanogaster, we introduced both substitutions (I1056M/F) in the corresponding fly CHS1 gene (kkv). Homozygous lines bearing either of these mutations were highly resistant to etoxazole and all tested BPUs, as well as buprofezin-an important hemipteran chitin biosynthesis inhibitor. This provides compelling evidence that BPUs, etoxazole, and buprofezin share in fact the same molecular MoA and directly interact with CHS. This finding has immediate effects on resistance management strategies of major agricultural pests but also on mosquito vectors of serious human diseases such as Dengue and Zika, as diflubenzuron, the standard BPU, is one of the few effective larvicides in use. The study elaborates on how genome editing can directly, rapidly, and convincingly elucidate the MoA of bioactive molecules, especially when target sites are complex and hard to reconstitute in vitro.

The bacterial metabolite 2-aminoacetophenone promotes association of pathogenic bacteria with flies.

Bacteria contaminate insects and secrete metabolites that may affect insect behaviour and potentially fitness through unknown mechanisms. Here we show that the 'grape-like' odorant 2-aminoacetophenone (2AA), secreted by Pseudomonas aeruginosa (a ubiquitous opportunistic human pathogen), facilitates attraction to food for several fly species including Musca domestica, Ceratitis capitata and Drosophila melanogaster. Constant feeding on 2AA increases the level of long-term colonization of the flies' intestine by P. aeruginosa. Odour perception is necessary for enhanced attraction to food containing 2AA, and expression in the Drosophila olfactory organs of odorant receptors Or49b and Or10a potentiates, while expression of Or85a inhibits, preference for 2AA. Our study shows that 2AA lures the flies to the bacterial source and increases the extent of colonization of the fly intestine by the bacteria that produce it, as a means to facilitate bacterial dissemination to new locations.

Essential roles of Da transactivation domains in neurogenesis and in E(spl)-mediated repression.

E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. We have characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and have evaluated their roles in promoting peripheral neurogenesis. In this context, Da heterodimerizes with proneural proteins, such as Scute (Sc), which is dynamically expressed and also contributes a TAD. We found that either one of the Da TADs in the Da/Sc complex is sufficient to promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its transcriptional activation role, the Da AD1 domain serves as an interaction platform for E(spl) proteins, bHLH-Orange family repressors which antagonize Da/Sc function. We show that the E(spl) Orange domain is needed for this interaction and strongly contributes to the antiproneural activity of E(spl) proteins. We present a mechanistic model on the interplay of these bHLH factors in the context of neural fate assignment.

Multiple rescue factors within a Wolbachia strain.

Wolbachia-induced cytoplasmic incompatibility (CI) is expressed when infected males are crossed with either uninfected females or females infected with Wolbachia of different CI specificity. In diploid insects, CI results in embryonic mortality, apparently due to the the loss of the paternal set of chromosomes, usually during the first mitotic division. The molecular basis of CI has not been determined yet; however, several lines of evidence suggest that Wolbachia exhibits two distinct sex-dependent functions: in males, Wolbachia somehow "imprints" the paternal chromosomes during spermatogenesis (mod function), whereas in females, the presence of the same Wolbachia strain(s) is able to restore embryonic viability (resc function). On the basis of the ability of Wolbachia to induce the modification and/or rescue functions in a given host, each bacterial strain can be classified as belonging in one of the four following categories: mod(+) resc(+), mod(-) resc(+), mod(-) resc(-), and mod(+) resc(-). A so-called "suicide" mod(+) resc(-) strain has not been found in nature yet. Here, a combination of embryonic cytoplasmic injections and introgression experiments was used to transfer nine evolutionary, distantly related Wolbachia strains (wYak, wTei, wSan, wRi, wMel, wHa, wAu, wNo, and wMa) into the same host background, that of Drosophila simulans (STCP strain), a highly permissive host for CI expression. We initially characterized the modification and rescue properties of the Wolbachia strains wYak, wTei, and wSan, naturally present in the yakuba complex, upon their transfer into D. simulans. Confocal microscopy and multilocus sequencing typing (MLST) analysis were also employed for the evaluation of the CI properties. We also tested the compatibility relationships of wYak, wTei, and wSan with all other Wolbachia infections. So far, the cytoplasmic incompatibility properties of different Wolbachia variants are explained assuming a single pair of modification and rescue factors specific to each variant. This study shows that a given Wolbachia variant can possess multiple rescue determinants corresponding to different CI systems. In addition, our results: (a) suggest that wTei appears to behave in D. simulans as a suicide mod(+) resc(-) strain, (b) unravel unique CI properties, and (c) provide a framework to understand the diversity and the evolution of new CI-compatibility types.

Stable expression of human growth hormone over 50 generations in transgenic insect larvae.

Developments in insect transgenesis using transposons combined with available mass rearing technology for insects such as the Medfly, Ceratitis capitata, provide opportunity for the production of protein for industrial, agricultural and healthcare purposes on a very large scale. In this study, we report the germ-line transformation and expression of a cDNA encoding human growth hormone (hGH) in transgenic Drosophila using the Minos transposon. Production and secretion of a bioactive hGH into the haemolymph of transgenic larvae was demonstrated by immunoblot analysis, ELISA and a proliferation bioassay. Stable expression of hGH was observed over 50 generations. The results indicate that mass reared transgenic diptera with a rapid period of larval growth could provide cost effective production systems for the manufacture of therapeutic and other high value proteins.

Variations of the 3' protruding ends in synthetic short interfering RNA (siRNA) tested by microinjection in Drosophila embryos.

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.

High frequencies of Minos transposon mobilization are obtained in insects by using in vitro synthesized mRNA as a source of transposase.

One of the most frequently encountered problems in transposon-mediated transgenesis is low transformation frequency, often resulting from difficulty in expressing from injected plasmid DNA constructs adequate levels of transposase in embryos. Capped RNA corresponding to the spliced transcript of the Minos transposable element has been synthesized in vitro and shown to be an effective source of transposase protein for Minos transposon mobilization. Transposase produced by this mRNA is shown to catalyze excision of a Minos transposon from plasmid DNA in Medfly embryos. When injected into Drosophila or Medfly embryos, transposase mRNA leads to a several-fold increase in transformation efficiencies compared with injected plasmids expressing transposase. Also, frequent mobilization of a Minos transposon from the X chromosome into autosomes was demonstrated after injections of Minos transposase mRNA into pre-blastoderm Drosophila embryos. The high rates of transposition achieved with transposase mRNA suggest that this is a powerful system for genetic applications in Drosophila and other insects.