Biology subjects

Rasgon, J. L.

Publications and source records attributed to Rasgon, J. L..

At least 19 recordsLinked to original sources

bioRxiv2026

Infectious transgenesis in arthropods

In the last decade, there has been an explosion in the development of in vivo germline genetic editing technologies in arthropods, first pioneered by the Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) method. While ReMOT Control and related technologies (DIPA-CRISPR and SYNCAS) have shown great utility for gene knock-out in many different species, consistent gene insertion has been elusive, limiting widespread adoption. We developed a novel system based on the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) to deliver heritable CRISPR knock-out and knock-in constructs to the arthropod germline by oral feeding. We call this method "Germline Engineering by Viral Transduction" ("GEValT"). With GEValT, newly-eclosed female and male adult arthropods are fed on virus in sugar (and/or blood) and allowed to breed, resulting in easy generation of genetically modified offspring. GeValT is easy to use and has the potential to revolutionize gene editing methodology for researchers working across diverse arthropod species.

molecular biology↗

bioRxiv2025

Hologenomic structure of bacterial and fungal community composition in the West Nile virus vector Culex tarsalis

BackgroundMicrobiota play a crucial role in determining the ability for arthropod disease vectors to transmit pathogens. Microbial community structure can be heavily influenced by microbe-microbe interactions, host genetics and environmental factors. Here, we characterize the host population genetic structure, and bacterial and fungal communities in natural populations of the West Nile virus mosquito vector Culex tarsalis. Mosquitoes were collected and analyzed across the species range of the mosquito in the United States, where we used PoolRADSeq to quantify population genetic structure. Microbial community composition was characterized using bacterial 16S rRNA gene sequencing (V3-V4 region) and fungal ITS sequencing (ITS1 region). ResultsPoolRADSeq identified four broad genetic clusters of mosquito populations, which corresponded to previous clusters identified by microsatellite analysis and RADSeq on individual mosquitoes. Microbiome diversity grouped mosquito populations into three broad clusters, with each cluster distinctively represented by diagnostic abundant bacteria (Ralstonia, Pseudomonas, or Zymobacter/Providencia, respectively). Clustering for fungal taxa was less pronounced. Geographic distance between populations was positively correlated with microbiome community dissimilarity, and multiple environmental factors were significantly correlated with microbial species richness and diversity. ConclusionsThese results suggest that bacterial and fungal communities are geographically structured in Cx. tarsalis, interact with important environmental factors, and are partially correlated with host genetic structure. As microbiota can affect the ability for mosquitoes to transmit pathogens, understanding the factors underpinning microbiome variation across space and time has important implications for the spread of vector-borne pathogens such as WNV.

ecology↗

bioRxiv2025

In situ architecture of the endosymbiont Wolbachia pipientis

Hidden within host cells, the endosymbiont Wolbachia pipientis is the most prevalent bacterial infection in the animal kingdom. Scientific breakthroughs over the past century yielded fundamental mechanisms by which Wolbachia controls arthropod reproduction to shape dynamic ecological and evolutionary trajectories. However, the structure and spatial organization of symbiont machineries that underpin intracellular colonization and orchestrate maternal inheritance remain unknown. Here, we used cryo-electron tomography to directly image the nanoscale architecture of bacterial tools deployed for host manipulation and germline transmission. We discovered that Wolbachia assembles multiple structures at the host-endosymbiont interface including a filamentous ladder-like framework hypothesized to serve as a specialized motility mechanism that enables bacterial translocation to specific host cell compartments during embryogenesis and somatic tissue dissemination. In addition, we present the first in situ structure of the Rickettsiales vir homolog type IV secretion system (rvh T4SS). We provide evidence that the rvh T4SS nanomachine exhibits architectural similarities to the pED208-encoded T4SS apparatus including the biogenesis of rigid conjugative pili extending hundreds of nanometers beyond the bacterial cell surface. Coupled with integrative structural modeling, we demonstrate that in contrast to canonical T4SS architectures, the -proteobacterial T4SS outer membrane complex assembles a periplasmic baseplate structure predicted to comprise VirB9 oligomers complexed with cognate VirB10 subunits that form extended antennae projections surrounding the translocation channel pore. Collectively, these studies provide an unprecedented view into Wolbachia structural cell biology and unveil the molecular blueprints for architectural paradigms that reinforce ancient host-microbe symbioses.

microbiology↗

bioRxiv2025

Effect of prolonged dehydration stress on the vector competence of Aedes aegypti for Mayaro virus

Aedes aegypti is a competent vector for a variety of mosquito-borne viruses including Zika, chikungunya, Mayaro, yellow fever, and dengue, which cause debilitating diseases in animals and humans. It is highly invasive and is widely distributed across Asia, Europe, Oceania, and the Americas. Climatic factors such as relative humidity (RH) can have substantial effects on mosquito biological characteristics and the dynamics of pathogen spread. Low RH leads to dehydration in mosquitoes causing modifications in behavioral and physiological responses pertaining to pathogen spread, such as host-seeking behavior and blood-feeding patterns. Here, we evaluated the effects of prolonged dehydration stress on Mayaro virus infection and vector competence in Ae. aegypti mosquitoes. Our findings suggest that prolonged dehydration stress following Mayaro virus infection alters viral dissemination dynamics in mosquitoes, indicating that humidity may modulate intra-vector viral dissemination potentially impacting vector competence and pathogen spread. The previously observed effects of higher feeding and altered survival and our current observations on altered vector competence suggest that the impact of dehydration on viral transmission is expected to be complex and will be crucial to understanding the dynamic disease patterns of mosquito-borne viruses across diverse climatic conditions.

microbiology↗

bioRxiv2025

Virome profiling of Culex tarsalis through small RNA-seq: A challenge of suboptimal samples

Viral infections in mosquitoes trigger the RNA interference (RNAi) pathway, a key antiviral defense mechanism that generates virus-derived small RNAs (vsRNAs). Given the natural enrichment of vsRNAs during infection and their stability, small RNA sequencing (sRNA-seq) has emerged as a powerful tool for virome characterization. Culex tarsalis is a widely distributed mosquito species in North America and is an important vector of West Nile virus (WNV). Previous studies have shown that co-infection with insect-specific viruses (ISVs) can modulate WNV replication in Cx. tarsalis, highlighting the importance of characterizing the virome of this species. Here, we investigated the virome of Cx. tarsalis populations across 5 states of the Midwestern United States using sRNA-seq. We analyzed samples from 17 geographic locations which were collected under suboptimal field conditions during the COVID-19 pandemic, presenting challenges related to sample integrity. Despite these challenges, sRNA-seq proved to be a reliable method for virome analysis. We identified seven viruses associated with Cx. tarsalis, along with their respective sRNA (siRNA and piRNA) profiles. These findings not only deepen our understanding of ISVs, but also demonstrate the utility of sRNA-seq in non-ideal situations, enabling the collection and analysis of samples under real-world surveillance scenarios.

microbiology↗

bioRxiv2024

Revisiting the paradigm of anhematophagy in male mosquitoes

Most female mosquitoes are reproductively obligate feeders on vertebrate blood to obtain nutrients required for egg production (driving transmission of vector-borne pathogens in the process), and which rely on plant sugars for their non-reproductive energy requirements. Male mosquitoes, on the other hand, are thought to rely exclusively on plant sugars for their energetic needs; indeed, this dichotomy is one of the central paradigms of medical entomology. Here, we provide multiple lines of evidence challenging this assumption. We show that when reared under dehydration/starvation conditions, male mosquitoes will readily take blood from a membrane feeder while CRISPR mutants with impaired humidity detection do not increase their bloodfeeding rates. For multiple mosquito species, dehydrated males are attracted to human hosts and attempt to probe. We also observed several instances where dehydrated probing males were able to successfully pierce vertebrate host skin and acquire blood from a vertebrate host. When fed a blood meal containing West Nile virus, male Cx. tarsalis mosquitoes became infected with and orally transmitted the pathogen at rates and titers equivalent to females. Finally, we collected wild, putatively blood-fed male mosquitoes from Texas, USA and Mallorca, Spain, and identified vertebrate DNA in these samples (canine and human, respectively). These data suggest that under specific circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts. These results are a paradigm shift in our understanding of male mosquito biology and suggest they may be more directly involved in pathogen transmission cycles than previously recognized.

animal behavior and cognition↗

bioRxiv2024

Expression of endogenous Anopheles gambiae microRNAs using an Anopheles gambiae densovirus (AgDNV) intronic expression system

Anopheles gambiae densovirus (AgDNV) is a highly species-specific parvovirus that reaches high titers in adult Anopheles gambiae mosquitoes with few transcriptomic effects and no 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 enhanced green fluorescent protein (EGFP) expressing plasmid that can be used to co-transfect cells to generate infectious recombinant transducing AgDNV virions. Generated virions infect the An. gambiae midgut, 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. Considering 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 evaluated, and the impact of miRNA expression on putative miRNA targets was measured in vitro and in vivo. While the intron was correctly spliced in cells and mosquitoes, miRNA delivery resulted in inconsistent changes to miRNA and target gene transcript levels--possibly due to organ-specific miRNA expression or miRNA-target gene sequence mismatch. However, with optimization this viral vector and developed intron have potential as an expression tool within An. gambiae mosquitoes or cells.

microbiology↗

bioRxiv2024

Multiple bouts of blood feeding in mosquitoes allow prolonged survival and are predicted to increase viral transmission during drought

Survival through periods of drought is critical for mosquitoes to reside in semi-arid regions with humans. Dry conditions increase blood feeding propensity in mosquitoes, but it is unknown if dehydration-induced bloodmeals increase feeding beyond what is necessary for reproduction. Following a bloodmeal, prolonged exposure to dry conditions increased secondary blood feeding in mosquitoes by nearly two-fold, and chronic blood feeding allowed mosquitoes to survive up to twenty days without access to water sources. This refeeding did not alter the number of eggs generated, suggesting this refeeding is for hydration and nutrient replenishment. Exposure to desiccating conditions following a bloodmeal resulted in increased activity, decreased sleep levels, and prompted a return of CO2 sensing before egg deposition. Increased blood feeding during the vitellogenic stage and higher survival during dry periods are predicted to increase pathogen transmission, allowing for a rapid rebound in mosquito populations when more favorable conditions return. This explains the elevated levels of specific arbovirus cases in association with periodic dry conditions and warrants further consideration as climate change progresses. Overall, these results solidify our understanding of the role of dry periods on mosquito blood feeding and how mosquito dehydration contributes to vectorial capacity and disease transmission dynamics Significance statementBouts of dehydration yield substantial changes to insects physiology and behavior. Mosquitoes are exceptionally prone to dehydration due to high water loss rates, but few integrative studies have examined the comprehensive impact of drought conditions on mosquitoes. Here, we demonstrate that dry conditions lead to multiple blood feeding events, allowing mosquitoes to survive dry periods. This repeated blood feeding is associated with higher activity and an early return of attraction to vertebrate hosts. Increased dry season survival and more frequent blood feeding are predicted to yield higher transmission of mosquito-borne viruses. This suggests that a higher prevalence of drought associated with climate change will have varying impacts on mosquito-borne diseases.

physiology↗

bioRxiv2023

In Silico Characterization of Intracellular Localization Signals and Structural Features of Mosquito Densovirus (MDV) Viral Proteins

As entomopathogenic viruses, mosquito densoviruses (MDVs) are widely studied for their potential as biocontrol agents and molecular laboratory tools for mosquito manipulation. The nucleus of the mosquito cell is the site for MDV genome replication and capsid assembly, however the nuclear localization signals (NLSs) and nuclear export signals (NES) for MDV proteins have not yet been identified. We carried out an in silico analysis to identify putative NLSs and NESs in the viral proteins of densoviruses that infect diverse mosquito genera (Aedes, Anopheles, and Culex) and identified putative phosphorylation and glycosylation sites on these proteins. These analyses lead to a more comprehensive understanding of how MDVs are transported into and out of the nucleus and lay the foundation for the potential use of densoviruses in mosquito control and basic research. Data summaryData used in this article were obtained from the GenBank database using accession numbers AYH52680, AYH52678, AYH52679, ABX83665, ABX83663, ABX83664, ABU95013, ABU95011, ABU95012, and AXQ04861. Impact statementMosquito densoviruses (MDVs) are of interest as mosquito biocontrol agents and as laboratory research tools. The trafficking of viral proteins to the cell nucleus is a critical step in viral replication. We used in silico approaches to identify putative nuclear localization signals and nuclear export signals for MDVs that infect the three major genera of pathogen-transmitting mosquitoes (Aedes, Anopheles, and Culex). These analyses lead a more comprehensive understanding of how MDVs are transported into and out of the nucleus and lay the foundation for the potential use of densoviruses in mosquito control and basic research.

microbiology↗

bioRxiv2023

NS2B-D55E and NS2B-E65D Variations are Responsible for Differences in NS2B-NS3 Protease Activities Between Japanese Encephalitis Virus Genotype I and III in Fluorogenic Peptide Model

Japanese Encephalitis Virus (JEV) NS2B-NS3 is a protein complex composed of NS3 proteases and a NS2B cofactor. The N-terminal protease domain (180 residues) of NS3 (NS3(pro)) interacts directly with a central 40-amino acid hydrophilic domain of NS2B (NS2B(H)) to form an active serine protease. In this study, the recombinant NS2B(H)-NS3(pro) proteases were prepared in E. coli and used to compare the enzymatic activity between genotype I (GI) and III (GIII) NS2B-NS3 proteases. The GI NS2B(H)-NS3(pro) was able to cleave the sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions that were identical to the sites proteolytically processed by GIII NS2B(H)-NS3(pro). Analysis of the enzymatic activity of recombinant NS2B(H)-NS3(pro) proteases using a model of fluorogenic peptide substrate revealed that the proteolytical processing activity of GIII NS2B(H)-NS3(pro) was significantly higher than that of GI NS2B(H)-NS3(pro). There were eight amino acid variations between GI and GIII NS2B(H)-NS3(pro), which may be responsible for the difference in enzymatic activities between GI and GIII proteases. Therefore, recombinant mutants were generated by exchanging NS2B(H) and NS3(pro) domains between GI and GIII NS2B(H)-NS3(pro) and subjected to protease activity analysis. Substitution of NS2B(H) significantly altered the protease activities, as compared to the parental NS2B(H)-NS3(pro), suggesting that NS2B(H) played an essential role in regulation of NS3(pro) protease activity. To further identify the amino acids responsible for the difference in protease activities, multiple substitution mutants including the individual and combined mutations at the variant residue 55 and 65 of NS2B(H) were generated and subjected to protease activity analysis. Replacement of NS2B-55 and NS2B-65 of GI to GIII significantly increased the enzymatic activity of GI NS2B(H)-NS3(pro) protease, whereas mutation of NS2B-55 and NS2B-65 of GIII to GI remarkably reduced the enzymatic activity of GIII NS2B(H)-NS3(pro) protease. Overall, these data demonstrated that NS2B-55 and NS2B-65 variations in hydrophilic domain of NS2B co-contributed to the difference in NS2B(H)-NS3(pro) protease activities between GI and GIII. These observations gain an insight into the role of NS2B in regulation of NS3 protease activities, which is useful for understanding the replication of JEV GI and GIII viruses.

microbiology↗

bioRxiv2023

Gene editing in the Chagas disease vector Rhodnius prolixus by Cas9-mediated ReMOT Control

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. Despite the broad use of double-stranded RNA interference for the knockdown of gene function in R. prolixus, transgenesis and gene editing protocols are still lacking. Here we tested Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and direct parental injection of CRISPR (DIPA-CRISPR) for the maternal delivery of CRISPR/Cas9 elements to developing R. prolixus oocytes, and strategies for the identification of insertions/deletions (indels) in target loci of resulting gene-edited G0 nymphs. We demonstrate successful ReMOT Control-mediated gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using the ovary-targeting BtKV ligand. These results provide proof-of-concepts for generating somatic mutations in R. prolixus and potentially for generating germline edited lines in triatomines. Our studies also suggest that optimal strategies for recovery of mutations include performing multiple gRNA injections and the use of visible phenotypes such as those displayed in the Rp-scarlet, Rp-white and Rp-yellow loci for future Co-CRISPR experiments. These results will lay the foundation for gene editing protocols for triatomines and could lead to the development of novel control strategies for vectors of Chagas disease. Author SummaryRhodnius prolixus is an insect vector of the protozoan Trypanossoma cruzi, causative agent of debilitating Chagas disease. To fight the spread of the disease, it has been suggested that biological control of the insect should be attempted. Gene editing by the novel CRISPR methodology holds great promise in this sense, as it enables to target almost any gene in the genome for mutagenesis, thus allowing the control of insect physiology and reproduction. Here we have tested protocols for the delivery of CRISPR reagents as an attempt to enable genome editing of the vector. Our results show that maternal delivery of CRISPR by the ReMOT Control method is efficient for mutating eye and cuticle color genes in the resulting nymphs, generating edited animals with red eyes, white eyes or a yellow cuticle. This is the first report of gene editing in a vector of Chagas disease and should lay the basis to produce modified animals either unable to carry the T. cruzi parasite or to reproduce.

molecular biology↗

bioRxiv2023

Wolbachia-induced inhibition of O'nyong nyong virus in Anopheles mosquitoes is mediated by Toll signaling and modulated by cholesterol

Enhanced host immunity and competition for metabolic resources are two main competing hypotheses for the mechanism of Wolbachia-mediated pathogen inhibition in arthropods. Using an Anopheles mosquito - somatic Wolbachia infection - Onyong nyong virus (ONNV) model, we demonstrate that the mechanism underpinning Wolbachia-mediated virus inhibition is up-regulation of the Toll innate immune pathway. However, the viral inhibitory properties of Wolbachia were abolished by cholesterol supplementation. This result was due to Wolbachia-dependent cholesterol-mediated suppression of Toll signaling rather than competition for cholesterol between Wolbachia and virus. The inhibitory effect of cholesterol was specific to Wolbachia-infected Anopheles mosquitoes and cells. These data indicate that both Wolbachia and cholesterol influence Toll immune signaling in Anopheles mosquitoes in a complex manner and provide a functional link between the host immunity and metabolic competition hypotheses for explaining Wolbachia-mediated pathogen interference in mosquitoes. In addition, these results provide a mechanistic understanding of the mode of action of Wolbachia-induced pathogen blocking in Anophelines, which is critical to evaluate the long-term efficacy of control strategies for malaria and Anopheles-transmitted arboviruses. HIGHLIGHTSO_LIWolbachia inhibits Onyong nyong virus (ONNV) in Anopheles mosquitoes. C_LIO_LIEnhanced Toll signaling is responsible for Wolbachia-induced interference of ONNV. C_LIO_LICholesterol suppresses Toll signaling to modulate Wolbachia-induced ONNV interference. C_LI

microbiology↗

bioRxiv2023

Eilat virus (EILV) causes superinfection exclusion against West NILE virus (WNV) in a strain specific manner in Culex tarsalis mosquitoes

West Nile virus (WNV) is the leading cause of mosquito-borne illness in the United States. 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 vector Culex tarsalis is 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 and Culex tarsalis mosquitoes. The titers 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 multiplicity of infections (MOIs) tested in our study. The titers of WN02-1956 at both MOIs 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 titers. However, EILV had no effect on the attachment of WN02-1956 or internalization of either WNV strain under superinfection conditions. In Cx. tarsalis, EILV did not affect the infection rate of either WNV strain at either timepoint. However, in mosquitoes, EILV enhanced NY99 infection titers at 3 days post superinfection, but this effect disappeared at 7 days post superinfection. In contrast, WN02-1956 infection titers 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, in Cx. 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. AUTHOR SUMMARYWest Nile virus (WNV) is the main cause of mosquito-borne disease in the United States. In the absence of a human vaccine or WNV-specific antivirals, vector control is the key strategy to reduce WNV prevalence and transmission. The WNV mosquito vector, Culex tarsalis, is a competent host for the insect-specific virus Eilat virus (EILV). EILV and WNV potentially interact within the mosquito host, and EILV can be used as a safe tool to target WNV in mosquitoes. Here, we characterize the ability of EILV to cause superinfection exclusion (SIE) against two strains of WNV--WN02-1956 and NY99--in C6/36 cells and Cx. tarsalis mosquitoes. EILV suppressed both superinfecting WNV strains in C6/36 cells. However, in mosquitoes, EILV enhanced NY99 whole-body titers at 3 days post superinfection and suppressed WN02-1956 whole-body titers at 7 days post superinfection. Vector competence measures, including infection, dissemination, and transmission rates and transmission efficacy, as well as leg and saliva titers of both superinfecting WNV strains, were not affected by EILV at both timepoints. Our data show the importance of not only validating SIE in mosquito vectors but also testing multiple strains of viruses to determine the safety of this strategy as a control tool.

microbiology↗

bioRxiv2023

Temperature affects viral kinetics and vectorial capacity of Aedes aegypti mosquitoes co-infected with Mayaro and Dengue viruses

Increasing global temperatures and unpredictable climatic extremes have contributed to the spread of vector-borne diseases. The mosquito Aedes aegypti is the main vector of multiple arboviruses that negatively impact human health, mostly in low socioeconomic areas of the world. Co-circulation and co-infection of these viruses in humans have been increasingly reported; however, how vectors contribute to this alarming trend remains unclear. Here, we examine single and co-infection of Mayaro virus (-D strain, Alphavirus) and dengue virus (serotype 2, Flavivirus) in Ae. aegypti adults and cell lines at two constant temperatures, moderate (27{degrees}C) and hot (32{degrees}C), to quantify vector competence and the effect of temperature on infection, dissemination and transmission, including on the degree of interaction between the two viruses. Both viruses were primarily affected by temperature but there was a partial interaction with co-infection. Dengue virus quickly replicates in adult mosquitoes, with a tendency for higher titers in co-infected mosquitoes at both temperatures and mosquito mortality was more severe at higher temperatures in all conditions. For dengue, and to a lesser extent Mayaro, vector competence and vectorial capacity were higher at hotter temperature in co- vs single infections and was more evident at earlier timepoints (7 vs 14 days post infection). The temperature-dependent phenotype was confirmed in vitro by faster cellular infection and initial replication at higher temperatures for dengue but not for Mayaro virus. Our study suggests that contrasting kinetics of the two viruses could be related to their intrinsic thermal requirements, where alphaviruses thrive better at lower temperatures compared to flaviviruses, but further studies are necessary to clarify the role of co-infection at different and variable temperature regimes. Author summaryGlobal warming is having devastating consequences for the environment, and a cause of concern is the increase in local abundance and geographic range of mosquitoes and the associated viruses they transmit. This study explores how temperature affects the mosquitos ability to survive and potentially spread two viruses, Mayaro and dengue, in single or co-infections. We found that Mayaro virus was not clearly affected by temperature or the presence of dengue infection. In contrast, dengue virus showed higher infection and potential for higher transmission in mosquitoes kept at high temperatures, and this trend was stronger in co-infections compared to single infections. Mosquito survival consistently decreased at high temperatures. We hypothesize the differences observed for dengue virus are due to the faster growth and viral activity in the mosquito at hotter temperatures, a pattern not observed for Mayaro virus. More studies under different temperature regimes are needed to clarify the role of co-infection.

microbiology↗

bioRxiv2023

Dehydration stress and Mayaro virus vector competence in Aedes aegypti

The mosquito Aedes aegypti is a competent vector of multiple pathogens including dengue, Zika, yellow fever, chikungunya, and Mayaro viruses. Ae. aegypti is highly invasive and is currently present in the Americas, Oceania, Asia, and Europe, but its distribution and the pathogens it transmits are expected to change due to climate change. Relative humidity is an environmental variable that affects mosquito biology and distribution and can differ between location, habitat, and season, with mosquitoes facing significant variation in relative humidity during their lifespan. Low relative humidity can induce dehydration in mosquitoes, leading to alterations in physiological and behavioral responses relevant for pathogen transmission such as bloodfeeding and host-seeking behavior. In this study, we evaluated the short and long-term effects of dehydration stress on mortality and Mayaro virus vector competence in Ae. aegypti. Our results show that exposure to dehydration does not impact viral titers, nor infection, dissemination and transmission rates, in mosquitoes infected with Mayaro virus. However, we detected a significant effect of dehydration on mosquito mortality and blood feeding frequency regardless of infection status. The previously observed effects of higher feeding during dehydration and the current observation of altered survival along with no impact on vector competence suggest that the impact of dehydration on viral transmission in mosquitoes will likely be complex.

microbiology↗

bioRxiv2023

CRISPR/Cas9-mediated mutagenesis of the Asian Citrus Psyllid, Diaphorina citri

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 via 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 (ReMOT Control) for 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 towards advancing D. citri research in preparation for future genetic-based systems for controlling HLB.

bioengineering↗

bioRxiv2023

Sindbis virus is suppressed in the yellow fever mosquito Aedes aegypti by ATG-6/Beclin-1 mediated activation of autophagy

Autophagy is a critical modulator of pathogen invasion response in vertebrates and invertebrates. However, how it affects mosquito-borne viral pathogens that significantly burden public health remains underexplored. To address this gap, we use a genetic approach to activate macroautophagy/autophagy in the yellow fever mosquito (Aedes aegypti), infected with a recombinant Sindbis virus (SINV) expressing an autophagy activator. We first demonstrate a 17- amino acid peptide derived from the Ae. aegypti autophagy-related protein 6 (ATG-6/beclin-1-like protein) is sufficient to induce autophagy in C6/36 mosquito cells, as marked by lipidation of ATG- 8 and puncta formation. Next, we engineered a recombinant SINV expressing this bioactive beclin- 1-like peptide and used it to infect and induce autophagy in adult mosquitoes. We find that modulation of autophagy using this recombinant SINV negatively regulated production of infectious viruses. The results from this study improve our understanding of the role of autophagy in arboviruses in invertebrate hosts and also highlight the potential for the autophagy pathway to be exploited for arboviral control.

microbiology↗

bioRxiv2023

Variable effects of Wolbachia on alphavirus infection in Aedes aegypti

2.Wolbachia pipientis (=Wolbachia) has promise as a tool to suppress virus transmission by Aedes aegypti mosquitoes. However, Wolbachia can have variable effects on mosquito-borne viruses. This variation remains poorly characterized, yet the multimodal effects of Wolbachia on diverse pathogens could have important implications for public health. Here, we examine the effects of somatic infection with two strains of Wolbachia (wAlbB and wMel) on the alphaviruses Sindbis virus (SINV), Onyong-nyong virus (ONNV), and Mayaro virus (MAYV) in Ae. aegypti. We found variable effects of Wolbachia including enhancement and suppression of viral infections, with some effects depending on Wolbachia strain. Both wAlbB- and wMel-infected mosquitoes showed enhancement of SINV infection rates one week post-infection, with wAlbB-infected mosquitoes also having higher viral titers than controls. Infection rates with ONNV were low across all treatments and no significant effects of Wolbachia were observed. The effects of Wolbachia on MAYV infections were strikingly strain-specific; wMel strongly blocked MAYV infections and suppressed viral titers, while wAlbB did not influence MAYV infection. The variable effects of Wolbachia on vector competence underscore the importance of further research into how this bacterium impacts the virome of wild mosquitoes including the emergent human pathogens they transmit. Impact statementIn recent years, wild populations of Aedes aegypti mosquitoes have been deliberately infected with Wolbachia--a bacterium that helps to curb the spread of some pathogens including dengue virus. But how does Wolbachia affect the ability of mosquitoes to become infected with and spread the many different viruses they encounter in nature? Here, we characterize the effects of Wolbachia on three alphaviruses that cause illness in humans-- Sindbis virus, Onyong-nyong virus, and Mayaro virus. We find Wolbachia has variable effects on these pathogens, including significant enhancement of Sindbis virus infections. Our research has important implications for the design of vector control strategies, and suggests further research is needed to understand how Wolbachia shapes the replication and transmission of diverse viruses. 3. Data SummaryAll data in the study are available in the Figures and supplementary material.

microbiology↗