Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein.

CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster-derived peptide 'P2C' fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.

Potential biological control of Erwinia tracheiphila by internal alimentary canal interactions in Acalymma vittatum with Pseudomonas fluorescens.

AIMS: We aim to determine if Pseudomonas fluorescens is a viable biological control for Erwinia tracheiphila within the insect vector, Acalymma vittatum. METHODS AND RESULTS: Pseudomonas fluorescens secreted fluorescein and inhibited growth of E. tracheiphila in disc diffusion assays. To determine if this antagonism was conserved within the insect vector, we performed in vivo assays by orally injecting beetles with bacterial treatments and fluorescent in situ hybridization to determine bacterial presence within the alimentary canal. CONCLUSIONS: Pseudomonas fluorescens inhibited the growth of E. tracheiphila on a nutrient-limiting medium. In situ experiments demonstrated that P. fluorescens is maintained within the alimentary canal of the beetle for at least 4 days, and co-occurred with E. tracheiphila. When beetles were first presented with Pseudomonas and then challenged with E. tracheiphila, E. tracheiphila was not recovered via FISH after 4 days. These data suggest that P. fluorescens has potential as a biological control agent to limit E. tracheiphila within the insect vector. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a novel approach for controlling E. tracheiphila that has the potential to decrease reliance on insecticides, providing a safer environment for pollinators and growers.

20-hydroxyecdysone mediates non-canonical regulation of mosquito vitellogenins through alternative splicing.

Vitellogenesis is one of the most well-studied physiological processes in mosquitoes. Expression of mosquito vitellogenin genes is classically described as being restricted to female adult reproduction. We report premature vitellogenin transcript expression in three vector mosquitoes: Culex tarsalis, Aedes aegypti and Anopheles gambiae. Vitellogenins expressed during non-reproductive stages are alternatively spliced to retain their first intron and encode premature termination codons. We show that intron retention results in transcript degradation by translation-dependent nonsense-mediated mRNA decay. This is probably an example of regulated unproductive splicing and translation (RUST), a mechanism known to regulate gene expression in numerous organisms but which has never been described in mosquitoes. We demonstrate that the hormone 20-hydroxyecdysone (20E) is responsible for regulating post-transcriptional splicing of vitellogenin. After exposure of previtellogenic fat bodies to 20E, vitellogenin expression switches from a non-productive intron-retaining transcript to a spliced protein-coding transcript. This effect is independent of factors classically known to influence transcription, such as juvenile hormone-mediated competence and amino acid signalling through the target of rapamycin pathway. Non-canonical regulation of vitellogenesis through RUST is a novel role for the multifunctional hormone 20E, and may have important implications for general patterns of gene regulation in mosquitoes.

Transinfection: a method to investigate Wolbachia-host interactions and control arthropod-borne disease.

The bacterial endosymbiont Wolbachia manipulates arthropod host biology in numerous ways, including sex ratio distortion and differential offspring survival. These bacteria infect a vast array of arthropods, some of which pose serious agricultural and human health threats. Wolbachia-mediated phenotypes such as cytoplasmic incompatibility and/or pathogen interference can be used for vector and disease control; however, many medically important vectors and important agricultural species are uninfected or are infected with strains of Wolbachia that do not elicit phenotypes desirable for disease or pest control. The ability to transfer strains of Wolbachia into new hosts (transinfection) can create novel Wolbachia-host associations. Transinfection has two primary benefits. First, Wolbachia-host interactions can be examined to tease apart the influence of the host and bacteria on phenotypes. Second, desirable phenotypes induced by Wolbachia in a particular insect can be transferred to another recipient host. This can allow the manipulation of insect populations that transmit pathogens or detrimentally affect agriculture. As such, transinfection is a valuable tool to explore Wolbachia biology and control arthropod-borne disease. The present review summarizes what is currently known about Wolbachia transinfection methods and applications. We also provide a comprehensive list of published successful and unsuccessful Wolbachia transinfection attempts.

Vitellogenin gene expression in autogenous Culex tarsalis.

Autogeny, the ability of a mosquito to mature an initial batch of eggs without blood feeding, is an alternative reproductive strategy with important implications for vector-borne disease transmission. Regulation of the major yolk protein (vitellogenin; Vg) genes during bloodmeal-induced oogenesis is well studied, but little is known about regulation of vitellogenesis in autogenous mosquitoes. We characterized the expression of four vitellogenin genes (Vg1a, Vg1b, Vg2a and Vg2b) in an autogenous strain of the West Nile Virus vector, Culex tarsalis. All vitellogenin genes were expressed during autogenous reproduction and following a bloodmeal, although the intensity and duration of expression varied amongst genes. Quantitative PCR analysis of vitellogenin transcription during autogeny revealed a similar temporal pattern to known vitellogenin expression profiles in anautogenous Aedes aegypti. Vitellogenin transcript, primarily produced from the Vg1b gene, was also detected in the larval and pupal stages of development, but no detectable vitellogenin protein was produced during this time period.

An initial linkage map of the West Nile Virus vector Culex tarsalis.

We have constructed the first genetic linkage map for the North American arboviral vector Culex tarsalis. 120 F(2) offspring from a cross between two colonies were genotyped using 25 microsatellites and six inter-simple sequence repeat (ISSR) markers. We resolved four linkage groups which likely correspond to two full-length chromosomes and two arms of the final chromosome. The longest linkage group contains the sex locus and corresponds to chromosome 3. Recombination rates around the sex locus were dramatically higher in females compared to males. The majority of microsatellite loci share sequence identity with regions of the Culex quinquefasciatus genome, whose assembly should aid in anchoring linkage groups to physical chromosomes. This map will aid in identification of loci involved with variable phenotypes in C. tarsalis including WNV susceptibility.

Using fluorescently labelled M13-tailed primers to isolate 45 novel microsatellite loci from the arboviral vector Culex tarsalis.

Culex tarsalis Coquillett (Diptera: Culicidae) is a highly efficient arbovirus vector. Spatial and temporal heterogeneity have been observed in Cx tarsalis for phenotypic traits including autogeny, virus susceptibility and host preference. Genetic differences between populations may in part explain these observations. Using the M13-tailed primer method, we identified 45 novel polymorphic microsatellite markers from microsatellite-enriched Cx tarsalis genomic libraries. The M13-tailed primer method was more efficient in identifying useful loci than traditional screening by acrylamide gel electrophoresis. These markers will be useful for investigating genetic questions in this important vector mosquito.

Transposable element insertion location bias and the dynamics of gene drive in mosquito populations.

Some vector-borne disease control strategies using transgenic mosquitoes require transgene spread to high frequency in populations. Transposable elements (TEs) are DNA sequences that replicate and transpose within the genomes of other organisms and may therefore be represented in the next generation in higher frequencies than predicted by Mendelian segregation. This over-representation has allowed some TEs to spread through natural populations. Transgenes incorporated within a TE sequence are expected to be driven into populations as long as there is a positive balance between fitness costs and over-representation. Models have been used to examine parameters that affect this balance but did not take into account biased insertion of TEs to linked sites in the genome. A simulation model was created to examine the impact of insertion bias on TE spread in mosquito populations. TEs that induce no fitness costs are predicted to increase in frequency over a wide range of parameter values but spread is slower for lower levels of transposition and non-local movement. If TEs are costly, high proportions of local movement can slow or halt spread. To function as a robust transgene drive mechanism a TE should replicate and transpose > 10%/insert/generation, induce < 1% fitness cost/insert, and move preferentially to unlinked sites in the genome.