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Abstract BackgroundAnopheles gambiaedensovirus (AgDNV) is a highly species-specific parvovirus that reaches high titers in adultAnopheles gambiaemosquitoes with few transcriptomic effects and minimal significant fitness effects. Given these characteristics, AgDNV has been proposed as a viral vector for basic research and mosquito control. Previous work created an AgDNV co-expression system with a wild-type AgDNV helper plasmid and a transducing plasmid expressing enhanced green fluorescent protein (EGFP) that can be used to co-transfect cells to generate infectious recombinant transducing AgDNV virions. Generated virions infect theAn. gambiaemidgut, fat body, and ovaries, yet this viral vector system is limited in the size of transgenes that can be expressed due to capsid packaging limitations. MethodsConsidering these size constraints, we created an artificial intron within the EGFP gene of the transducing construct that can express small pieces of genetic material such as microRNAs (miRNAs), microRNA sponges, or other small sequences. Placement of this intron in EGFP created a fluorescent reporter such that incorrect splicing produces a frameshift mutation in EGFP and an early stop codon, whereas correct splicing results in normal EGFP expression and co-transcription of the intronic genetic cargo. A selection of miRNAs with predicted or demonstrated importance in mosquito immunity and reproduction with expression localized to the fat body or ovaries were chosen as intronic cargo. Construct expression and splicing was evaluated, and the impact of miRNA expression on putative miRNA targets was measuredin vitroandin vivo. ResultsThe created intron was correctly spliced in cells and mosquitoes; however, miRNA delivery resulted in inconsistent changes to miRNA and predicted target gene transcript levels—possibly due to organ-specific miRNA expression or inaccurate putative target predictions leading to miRNA–target gene sequence mismatch. ConclusionsAlthough our results on target gene expression were inconsistent, with optimization this viral vector and developed intron have potential as an expression tool withinAn. gambiaemosquitoes or cell lines. Graphical Abstract 

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the USA. There are currently no human vaccines or therapies available for WNV, and vector control is the primary strategy used to control WNV transmission. The WNV vectorCulex tarsalisis also a competent host for the insect-specific virus (ISV) Eilat virus (EILV). ISVs such as EILV can interact with and cause superinfection exclusion (SIE) against human pathogenic viruses in their shared mosquito host, altering vector competence for these pathogenic viruses. The ability to cause SIE and their host restriction make ISVs a potentially safe tool to target mosquito-borne pathogenic viruses. In the present study, we tested whether EILV causes SIE against WNV in mosquito C6/36 cells andC. tarsalismosquitoes. The titres of both WNV strains – WN02-1956 and NY99 – were suppressed by EILV in C6/36 cells as early as 48–72 h post-superinfection at both m.o.i. values tested in our study. The titres of WN02-1956 at both m.o.i. values remained suppressed in C6/36 cells, whereas those of NY99 showed some recovery towards the final timepoint. The mechanism of SIE remains unknown, but EILV was found to interfere with NY99 attachment in C6/36 cells, potentially contributing to the suppression of NY99 titres. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. InC. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes,EILV enhanced NY99 infection titres at 3 days post-superinfection, but this effect disappeared at 7 days post-superinfection. In contrast, WN02-1956 infection titres were suppressed by EILV at 7 days post-superinfection. The dissemination and transmission of both WNV strains were not affected by superinfection with EILV at either timepoint. Overall, EILV caused SIE against both WNV strains in C6/36 cells; however, inC. tarsalis, SIE caused by EILV was strain specific potentially owing to differences in the rate of depletion of shared resources by the individual WNV strains. 

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. However, transgenesis and gene editing protocols to advance the field are still lacking. Here, we tested protocols for the maternal delivery of CRISPR-Cas9 (clustered regularly spaced palindromic repeats/Cas-9 associated) elements to developing R. prolixus oocytes and strategies for the identification of insertions and deletions (indels) in target loci of resulting gene-edited generation zero (G0) nymphs. We demonstrate successful gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) with the ovary-targeting BtKV ligand. These results provide proof of concepts for generating somatic mutations in R. prolixus and potentially for generating germ line-edited lines in triatomines, laying the foundation for gene editing protocols that could lead to the development of novel control strategies for vectors of Chagas disease. 

Understanding the ecological and evolutionary processes that drive host–pathogen interactions is critical for combating epidemics and conserving species. TheVarroa destructormite and deformed wing virus (DWV) are two synergistic threats to Western honeybee (Apis mellifera) populations across the globe. Distinct honeybee populations have been found to self-sustain despiteVarroainfestations, including colonies within the Arnot Forest outside Ithaca, NY, USA. We hypothesized that in these bee populations, DWV has been selected to produce an avirulent infection phenotype, allowing for the persistence of both host and disease-causing agents. To investigate this, we assessed the titre of viruses in bees from the Arnot Forest and managed apiaries, and assessed genomic variation and virulence differences between DWV isolates. Across groups, we found viral abundance was similar, but DWV genotypes were distinct. We also found that infections with isolates from the Arnot Forest resulted in higher survival and lower rates of symptomatic deformed wings, compared to analogous isolates from managed colonies, providing preliminary evidence to support the hypothesis of adaptive decreased viral virulence. Overall, this multi-level investigation of virus genotype and phenotype indicates that host ecological context can be a significant driver of viral evolution and host–pathogen interactions in honeybees. 

Synopsis In the past 20 years, sequencing technologies have led to easy access to genomic data from nonmodel organisms in all biological realms. Insect genetic manipulation, however, continues to be a challenge due to various factors, including technical and cost-related issues. Traditional techniques such as microinjection of gene-editing vectors into early stage embryos have been used for arthropod transgenesis and the discovery of Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR–Cas) technologies allowed for targeted mutagenesis and the creation of knockouts or knock-ins in arthropods. Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) acts as an alternative to embryonic microinjections, which require expensive equipment and extensive hands-on training. ReMOT Control’s main advantage is its ease of use coupled with the ability to hypothetically target any vitellogenic species, as injections are administered to the egg-laying adult rather than embryos. After its initial application in the mosquito Aedes aegypti, ReMOT Control has successfully produced mutants not only for mosquitoes but for multiple arthropod species from diverse orders, such as ticks, mites, wasps, beetles, and true bugs, and is being extended to crustaceans, demonstrating the versatility of the technique. In this review, we discuss the current state of ReMOT Control from its proof-of-concept to the advances and challenges in the application across species after 5 years since its development, including novel extensions of the technique such as direct parental (DIPA)-CRISPR. 

The most devastating disease affecting the global citrus industry is Huanglongbing (HLB), caused by the pathogen Candidatus Liberibacter asiaticus. HLB is primarily spread by the insect vector Diaphorina citri (Asian Citrus Psyllid). To counteract the rapid spread of HLB by D. citri, traditional vector control strategies such as insecticide sprays, the release of natural predators, and mass introductions of natural parasitoids are used. However, these methods alone have not managed to contain the spread of disease. To further expand the available tools for D. citri control through generating specific modifications of the D. citri genome, we have developed protocols for CRISPR-Cas9-based genetic modification. Until now, genome editing in D. citri has been challenging due to the general fragility and size of D. citri eggs. Here we present optimized methods for collecting and preparing eggs to introduce the Cas9 ribonucleoprotein (RNP) into early embryos and alternative methods of injecting RNP into the hemocoel of adult females for ovarian transduction. Using these methods, we have generated visible somatic mutations, indicating their suitability for gene editing in D. citri. These methods represent the first steps toward advancing D. citri research in preparation for future genetic-based systems for controlling HLB. 

ABSTRACT Eilat virus (EILV) is an insect-specific alphavirus that has the potential to be developed into a tool to combat mosquito-borne pathogens. However, its mosquito host range and transmission routes are not well understood. Here, we fill this gap by investigating EILV’s host competence and tissue tropism in five mosquito species: Aedes aegypti , Culex tarsalis , Anopheles gambiae , Anopheles stephensi , and Anopheles albimanus . Of the tested species, C. tarsalis was the most competent host for EILV. The virus was found in C. tarsalis ovaries, but no vertical or venereal transmission was observed. Culex tarsalis also transmitted EILV via saliva, suggesting the potential for horizontal transmission between an unknown vertebrate or invertebrate host. We found that reptile (turtle and snake) cell lines were not competent for EILV infection. We tested a potential invertebrate host ( Manduca sexta caterpillars) but found they were not susceptible to EILV infection. Together, our results suggest that EILV could be developed as a tool to target pathogenic viruses that use Culex tarsalis as a vector. Our work sheds light on the infection and transmission dynamics of a poorly understood insect-specific virus and reveals it may infect a broader range of mosquito species than previously recognized. IMPORTANCE The recent discovery of insect-specific alphaviruses presents opportunities both to study the biology of virus host range and to develop them into tools against pathogenic arboviruses. Here, we characterize the host range and transmission of Eilat virus in five mosquito species. We find that Culex tarsalis —a vector of harmful human pathogens, including West Nile virus—is a competent host of Eilat virus. However, how this virus is transmitted between mosquitoes remains unclear. We find that Eilat virus infects the tissues necessary for both vertical and horizontal transmission—a crucial step in discerning how Eilat virus maintains itself in nature. 

Abstract MultipleWolbachiastrains can block pathogen infection, replication and/or transmission inAedes aegyptimosquitoes under both laboratory and field conditions. However,Wolbachiaeffects on pathogens can be highly variable across systems and the factors governing this variability are not well understood. It is increasingly clear that the mosquito host is not a passive player in whichWolbachiagoverns pathogen transmission phenotypes; rather, the genetics of the host can significantly modulateWolbachia‐mediated pathogen blocking. Specifically, previous work linked variation inWolbachiapathogen blocking to polymorphisms in the mosquito alpha‐mannosidase‐2 (αMan2) gene. Here we use CRISPR‐Cas9 mutagenesis to functionally test this association. We developed αMan2 knockouts and examined effects on bothWolbachiaand virus levels, using dengue virus (DENV;Flaviviridae) and Mayaro virus (MAYV;Togaviridae).Wolbachiatitres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. InWolbachia‐uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in aWolbachia‐infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication inWolbachia‐uninfected mosquitoes and did not affectWolbachia‐mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection inA. aegyptimosquitoes in a pathogen‐ andWolbachia‐specific manner, and thatWolbachia‐mediated pathogen blocking is a complex phenotype dependent on the mosquito host genotype and the pathogen. These results have a significant impact for the design and use ofWolbachia‐based strategies to control vector‐borne pathogens. 

Abstract MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression during important biological processes including development and pathogen defense in most living organisms. Presently, no miRNAs have been identified in the mosquito Culex tarsalis (Diptera: Culicidae), one of the most important vectors of West Nile virus (WNV) in North America. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate a repertoire of miRNAs in Cx. tarsalis mosquitoes. Using bioinformatic approaches we analyzed small RNA sequences from the Cx. tarsalis CT embryonic cell line to discover orthologs for 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Culex quinquefasciatus, miR-184 was found to be the most abundant miRNA in Cx. tarsalis. We also identified 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both the CT cell line and in adult Cx. tarsalis mosquitoes using RT–qPCR and sequencing. These results will open new avenues of research into the role of miRNAs in Cx. tarsalis biology, including development, metabolism, immunity, and pathogen infection.