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Female mosquitoes are reproductively obligate bloodfeeders which feed 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 tenets of medical entomology. Here, we show that male Culex tarsalis and Aedes aegypti mosquitoes will readily take blood from a membrane feeder when reared under dehydration conditions with no toxic effects. Mosquitoes with impaired humidity detection do not increase their bloodfeeding rates when dehydrated compared to wild-type controls. While conventionally reared males ignore a human host, dehydrated males are attracted to and attempt to probe, with some success, although they cannot access host capillaries. However, they will take blood from a vertebrate host wound. When fed a blood meal containing West Nile virus, male mosquitoes can become infected with and orally transmit the pathogen at rates and titers equivalent to females. Finally, vertebrate DNA, likely from blood, was detected in wild-caught specimens of male Culex quinquefasciatus mosquitoes from Texas. These data suggest that under some circumstances male mosquitoes may be able to probe and/or ingest blood and transmit pathogens to vertebrate hosts, and that their role in maintaining pathogen transmission cycles should be re-examined.

Competing Interests: Competing interests statement:The author declares no competing interest.

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 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 C. tarsalis mosquitoes. 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. In C. 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, in C. 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.

Multiple Wolbachia strains can block pathogen infection, replication and/or transmission in Aedes aegypti mosquitoes under both laboratory and field conditions. However, Wolbachia effects 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 which Wolbachia governs pathogen transmission phenotypes; rather, the genetics of the host can significantly modulate Wolbachia-mediated pathogen blocking. Specifically, previous work linked variation in Wolbachia pathogen 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 both Wolbachia and virus levels, using dengue virus (DENV; Flaviviridae) and Mayaro virus (MAYV; Togaviridae). Wolbachia titres were significantly elevated in αMan2 knockout (KO) mosquitoes, but there were complex interactions with virus infection and replication. In Wolbachia-uninfected mosquitoes, the αMan2 KO mutation was associated with decreased DENV titres, but in a Wolbachia-infected background, the αMan2 KO mutation significantly increased virus titres. In contrast, the αMan2 KO mutation significantly increased MAYV replication in Wolbachia-uninfected mosquitoes and did not affect Wolbachia-mediated virus blocking. These results demonstrate that αMan2 modulates arbovirus infection in A. aegypti mosquitoes in a pathogen- and Wolbachia-specific manner, and that Wolbachia-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 of Wolbachia-based strategies to control vector-borne pathogens., (© 2024 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Mushroom cultivation vastly improves the yield of mushrooms under optimized, controlled conditions, but may be susceptible to opportunistic colonization by pest species that can establish themselves, as well as the pathogens and pests they may transmit. Here, we describe our investigation into the bacterial communities of adult Lycoriella ingenua (Diptera: Sciaridae) and Megaselia halterata (Diptera: Phoridae) collected from button mushroom ( Agaricus bisporus ) production houses in Pennsylvania. We collected adult flies and sequenced the hypervariable v4 region of the bacterial 16S rRNA using the Illumina MiSeq. The most abundant bacterial genus detected in both species was Wolbachia , but phylogenetic analysis revealed that the infections are from different clades. Future studies include the characterization of Wolbachia infections on fly behavior and biology, comparison of microbial diversity of fly species colonizing wild mushrooms, and other microbiota that may contribute to the success of certain pest fly species.

Survival through periods of drought is critical for mosquitoes to reside in semi-arid regions with humans, but water sources may be limited. Previous studies have shown that dehydrated mosquitoes will increase blood feeding propensity, but how this would occur over extended dry periods is unknown. 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 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. The increased blood feeding during the vitellogenic stage and higher survival during dry periods are predicted to increase pathogen transmission and explain the elevated levels of specific arbovirus cases during dry conditions. 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.

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.

Background: 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., Methods: 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 °C) and hot (32 °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., Results: 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 time points (7 vs. 14 days post infection) for Mayaro. 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., Conclusions: 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. However, more studies are necessary to clarify the role of co-infection at different temperature regimes, including under more natural temperature settings., (© 2024. The Author(s).)

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 Author(s) 2023. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Importance: Relative humidity (RH) is an environmental variable that affects mosquito physiology and can impact pathogen transmission. Low RH can induce dehydration in mosquitoes, leading to alterations in physiological and behavioral responses such as blood-feeding and host-seeking behavior. We evaluated the effects of a temporal drop in RH (RH shock) on mortality and Mayaro virus vector competence in Ae. aegypti . While dehydration induced by humidity shock did not impact virus infection, we detected a significant effect of dehydration on mosquito mortality and blood-feeding frequency, which could significantly impact transmission dynamics., Competing Interests: The authors declare no conflict of interest.

Understanding the ecological and evolutionary processes that drive host-pathogen interactions is critical for combating epidemics and conserving species. The Varroa destructor mite 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 despite Varroa infestations, 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.

Ticks are important vectors of pathogenic viruses, bacteria, and protozoans to humans, wildlife, and domestic animals. Due to their life cycles, ticks face significant challenges related to water homeostasis. When blood-feeding, they must excrete water and ions, but when off-host (for stretches lasting several months), they must conserve water to avoid desiccation. Aquaporins (AQPs), a family of membrane-bound water channels, are key players in osmoregulation in many animals but remain poorly characterized in ticks. Here, we bioinformatically identified AQP-like genes from the deer tick Ixodes scapularis and used phylogenetic approaches to map the evolution of the aquaporin gene family in arthropods. Most arachnid AQP-like sequences (including those of I. scapularis) formed a monophyletic group clustered within aquaglycerolporins (GLPs) from bacteria to vertebrates. This gene family is absent from insects, revealing divergent evolutionary paths for AQPs in different hematophagous arthropods. Next, we sequenced the full-length cDNA of I. scapularis aquaporin 1 (IsAQP1) and expressed it heterologously in Xenopus oocytes to functionally characterize its permeability to water and solutes. Additionally, we examined IsAQP1 expression across different life stages and adult female organs. We found IsAQP1 is an efficient water channel with high expression in salivary glands prior to feeding, suggesting it plays a role in osmoregulation before or during blood feeding. Its functional properties are unique: unlike most GLPs, IsAQP1 has low glycerol permeability, and unlike most AQPs, it is insensitive to mercury. Together, our results suggest IsAQP1 plays an important role in tick water balance physiology and that it may hold promise as a target of novel vector control efforts., (© 2023 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

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., Competing Interests: The authors declare no conflict of interest.

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Globalization and climate change have contributed to the simultaneous increase and spread of arboviral diseases. Cocirculation of several arboviruses in the same geographic region provides an impetus to study the impacts of multiple concurrent infections within an individual vector mosquito. Here, we describe coinfection and superinfection with the Mayaro virus (Togaviridae, Alphavirus ) and Zika virus (Flaviviridae, Flavivirus ) in vertebrate and mosquito cells, as well as Aedes aegypti adult mosquitoes, to understand the interaction dynamics of these pathogens and effects on viral infection, dissemination, and transmission. Aedes aegypti mosquitoes were able to be infected with and transmit both pathogens simultaneously. However, whereas Mayaro virus was largely unaffected by coinfection, it had a negative impact on infection and dissemination rates for Zika virus compared to single infection scenarios. Superinfection of Mayaro virus atop a previous Zika virus infection resulted in increased Mayaro virus infection rates. At the cellular level, we found that mosquito and vertebrate cells were also capable of being simultaneously infected with both pathogens. Similar to our findings in vivo , Mayaro virus negatively affected Zika virus replication in vertebrate cells, displaying complete blocking under certain conditions. Viral interference did not occur in mosquito cells. IMPORTANCE Epidemiological and clinical studies indicate that multiple arboviruses are cocirculating in human populations, leading to some individuals carrying more than one arbovirus at the same time. In turn, mosquitoes can become infected with multiple pathogens simultaneously (coinfection) or sequentially (superinfection). Coinfection and superinfection can have synergistic, neutral, or antagonistic effects on viral infection dynamics and ultimately have impacts on human health. Here we investigate the interaction between Zika virus and Mayaro virus, two emerging mosquito-borne pathogens currently circulating together in Latin America and the Caribbean. We find a major mosquito vector of these viruses-Aedes aegypti-can carry and transmit both arboviruses at the same time. Our findings emphasize the importance of considering co- and superinfection dynamics during vector-pathogen interaction studies, surveillance programs, and risk assessment efforts in epidemic areas.

Anopheles mosquitoes are the principal vectors for malaria and lymphatic filariasis, and evidence for arboviral transmission under laboratory and natural contexts has been demonstrated. Vector management approaches require an understanding of the ecological, epidemiological, and biological contexts of the species in question, and increased interest in gene drive systems for vector control applications has resulted in an increased need for genome assemblies from understudied mosquito vector species. In this study, we present novel genome assemblies for Anopheles crucians, Anopheles freeborni, Anopheles albimanus, and Anopheles quadrimaculatus and examine the evolutionary relationship between these species. We identified 790 shared single-copy orthologs between the newly sequenced genomes and created a phylogeny using 673 of the orthologs, identifying 289 orthologs with evidence for positive selection on at least 1 branch of the phylogeny. Gene ontology terms such as calcium ion signaling, histone binding, and protein acetylation identified as being biased in the set of selected genes. These novel genome sequences will be useful in developing our understanding of the diverse biological traits that drive vectorial capacity in anophelines., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Despite its ecological flexibility and geographical co-occurrence with human pathogens, little is known about the ability of Anopheles albimanus to transmit arboviruses. To address this gap, we challenged An. albimanus females with four alphaviruses and one flavivirus and monitored the progression of infections. We found this species is an efficient vector of the alphaviruses Mayaro virus, O'nyong-nyong virus, and Sindbis virus, although the latter two do not currently exist in its habitat range. An. albimanus was able to become infected with Chikungunya virus, but virus dissemination was rare (indicating the presence of a midgut escape barrier), and no mosquito transmitted. Mayaro virus rapidly established disseminated infections in An. albimanus females and was detected in the saliva of a substantial proportion of infected mosquitoes. Consistent with previous work in other anophelines, we find that An. albimanus is refractory to infection with flaviviruses, a phenotype that did not depend on midgut-specific barriers. Our work demonstrates that An. albimanus may be a vector of neglected emerging human pathogens and adds to recent evidence that anophelines are competent vectors for diverse arboviruses.

Mayaro virus (MAYV) is an emerging New World alphavirus (genus Alphavirus , family Togaviridae ) that causes acute multiphasic febrile illness, skin rash, polyarthritis and occasional severe clinical phenotypes. The virus lifecycle alternates between invertebrate and vertebrate hosts. Here we characterize the replication features, cell entry, lifecycle and virus-related cell pathology of MAYV using vertebrate and invertebrate in vitro models. Electron-dense clathrin-coated pits in infected cells and reduced viral production in the presence of dynasore, ammonium chloride and bafilomycin indicate that viral entry occurs through pH-dependent endocytosis. Increase in FITC-dextran uptake (an indicator of macropinocytosis) in MAYV-infected cells, and dose-dependent infection inhibition by 5-(N-ethyl-N-isopropyl) amiloride (a macropinocytosis inhibitor), indicated that macropinocytosis is an additional entry mechanism of MAYV in vertebrate cells. Acutely infected vertebrate and invertebrate cells formed cytoplasmic or membrane-associated extracytoplasmic replication complexes. Mosquito cells showed modified hybrid cytoplasmic vesicles that supported virus replication, nucleocapsid production and maturation. Mature virus particles were released from cells by both exocytosis and budding from the cell membrane. MAYV replication was cytopathic and associated with induction of apoptosis by the intrinsic pathway, and later by the extrinsic pathway in infected vertebrate cells. Given that MAYV is expanding its geographical existence as a potential public health problem, this study lays the foundation for biological understanding that will be valuable for therapeutic and preventive interventions.

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint shows a number of proteases regulated at 2 and 7 dpi, potentially representative of Toll or melanization pathway activation, and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: GLH is affiliated to the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford. GLH is based at LSTM.

Background: Anopheles gambiae densovirus (AgDNV) is an insect-specific, single-stranded DNA virus that infects An. gambiae sensu stricto (s.s.), the major mosquito species responsible for transmitting malaria parasites throughout sub-Saharan Africa. AgDNV is a benign virus that is very specific to its mosquito host and therefore has the potential to serve as a vector control tool via paratransgenesis (genetic modification of mosquito symbionts) to limit transmission of human pathogens. Prior to being engineered into a control tool, the natural transmission dynamics of AgDNV between An. gambiae mosquitoes needs to be fully understood. Additionally, improved knowledge of AgDNV infection in male mosquitoes is needed. In the study presented here, we examined the tissue tropism of AgDNV in the male reproductive tract and investigated both venereal and vertical transmission dynamics of the virus., Methods: Anopheles gambiae s.s. adult males were infected with AgDNV via microinjection, and reproductive tissues were collected and assayed for AgDNV using qPCR. Next, uninfected females were introduced to AgDNV-infected or control males and, after several nights of mating, both the spermatheca and female carcass were assessed for venereally transmitted AgDNV. Finally, F1 offspring of this cross were collected and assayed to quantify vertical transmission of the virus., Results: AgDNV infected the reproductive tract of male mosquitoes, including the testes and male accessory glands, without affecting mating rates. AgDNV-infected males venereally transmitted the virus to females, and these venereally infected females developed disseminated infection throughout the body. However, AgDNV was not vertically transmitted to the F1 offspring of this cross., Conclusions: Infected male releases could be an effective strategy to introduce AgDNV-based paratransgenic tools into naïve populations of An. gambiae s.s. females., (© 2022. The Author(s).)

One approach to control dengue virus transmission is the symbiont Wolbachia, which limits viral infection in mosquitoes. Despite plans for its widespread use in Aedes aegypti, Wolbachia's mode of action remains poorly understood. Many studies suggest that the mechanism is likely multifaceted, involving aspects of immunity, cellular stress and nutritional competition. A previous study from our group used artificial selection to identify a new mosquito candidate gene related to viral blocking; alpha-mannosidase-2a (alpha-Mann-2a) with a predicted role in protein glycosylation. Protein glycosylation pathways tend to be involved in complex host-viral interactions; however, the function of alpha-mannosidases has not been described in mosquito-virus interactions. We examined alpha-Mann-2a expression in response to virus and Wolbachia infections and whether reduced gene expression, caused by RNA interference, affected viral loads. We show that dengue virus (DENV) infection affects the expression of alpha-Mann-2a in a tissue- and time-dependent manner, whereas Wolbachia infection had no effect. In the midgut, DENV prevalence increased following knockdown of alpha-Mann-2a expression in Wolbachia-free mosquitoes, suggesting that alpha-Mann-2a interferes with infection. Expression knockdown had the same effect on the togavirus chikungunya virus, indicating that alpha-Mann-2a may have broad antivirus effects in the midgut. Interestingly, we were unable to knockdown the expression in Wolbachia-infected mosquitoes. We also provide evidence that alpha-Mann-2a may affect the transcriptional level of another gene predicted to be involved in viral blocking and cell adhesion; cadherin87a. These data support the hypothesis that glycosylation and adhesion pathways may broadly be involved in viral infection in Ae. aegypti., (© 2022 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of Royal Entomological Society.)

Despite their capacity to acquire and pass on an array of debilitating pathogens, research on ticks has lagged behind other arthropod vectors, such as mosquitoes, largely because of challenges in applying available genetic and molecular tools. CRISPR-Cas9 is transforming non-model organism research; however, successful gene editing has not yet been reported in ticks. Technical challenges for injecting tick embryos to attempt gene editing have further slowed research progress. Currently, no embryo injection protocol exists for any chelicerate species, including ticks. Herein, we report a successful embryo injection protocol for the black-legged tick, Ixodes scapularis , and the use of this protocol for genome editing with CRISPR-Cas9. We also demonstrate that the ReMOT Control technique could be successfully used to generate genome mutations outside Insecta. Our results provide innovative tools to the tick research community that are essential for advancing our understanding of the molecular mechanisms governing pathogen transmission by tick vectors and the underlying biology of host-vector-pathogen interactions., Competing Interests: JLR, DCR, and CCH have filed for patent protection on the ReMOT Control technology. No other authors have any competing interests., (© 2022 The Authors.)

Understanding how vectors alter the interactions between viruses and their hosts is a fundamental question in virology and disease ecology. In honey bees, transmission of deformed wing virus (DWV) by parasitic Varroa mites has been associated with elevated disease and host mortality, and Varroa transmission has been hypothesized to lead to increased viral titres or select for more virulent variants. Here, we mimicked Varroa transmission by serially passaging a mixed population of two DWV variants, A and B, by injection through in vitro reared honey bee pupae and tracking these viral populations through five passages. The DWV-A and DWV-B variant proportions shifted dynamically through passaging, with DWV-B outcompeting DWV-A after one passage, but levels of both variants becoming equivalent by Passage 5. Sequencing analysis revealed a dominant, recombinant DWV-B strain (DWV-A derived 5' IRES region with the rest of the genome DWV-B), with low nucleotide diversity that decreased through passaging. DWV-A populations had higher nucleotide diversity compared to DWV-B, but this also decreased through passaging. Selection signatures were found across functional regions of the DWV-A and DWV-B genomes, including amino acid mutations in the putative capsid protein region. Simulated vector transmission differentially impacted two closely related viral variants which could influence viral interactions with the host, demonstrating surprising plasticity in vector-host-viral dynamics.

Mosquito bites transmit a number of pathogens via salivary droplets deposited during blood-feeding, resulting in potentially fatal diseases. Little is known about the genomic content of these nanodroplets, including the transmission dynamics of live pathogens. Here we introduce Vectorchip, a low-cost, scalable microfluidic platform enabling high-throughput molecular interrogation of individual mosquito bites. We introduce an ultra-thin PDMS membrane which acts as a biting interface to arrays of micro-wells. Freely-behaving mosquitoes deposit saliva droplets by biting into these micro-wells. By modulating membrane thickness, we observe species-dependent differences in mosquito biting capacity, utilizable for selective sample collection. We demonstrate RT-PCR and focus-forming assays on-chip to detect mosquito DNA, Zika virus RNA, as well as quantify infectious Mayaro virus particles transmitted from single mosquito bites. The Vectorchip presents a promising approach for single-bite-resolution laboratory and field characterization of vector-pathogen communities, and could serve as a powerful early warning sentinel for mosquito-borne diseases., (© 2021. The Author(s).)

Mosquito-borne diseases, such as dengue and malaria, pose significant global health burdens. Unfortunately, current control methods based on insecticides and environmental maintenance have fallen short of eliminating the disease burden. Scalable, deployable, genetic-based solutions are sought to reduce the transmission risk of these diseases. Pathogen-blocking Wolbachia bacteria, or genome engineering-based mosquito control strategies including gene drives have been developed to address these problems, both requiring the release of modified mosquitoes into the environment. Here, we review the latest developments, notable similarities, and critical distinctions between these promising technologies and discuss their future applications for mosquito-borne disease control., (© 2021. The Author(s).)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel human respiratory viral infection that has rapidly progressed into a pandemic, causing significant morbidity and mortality. Blood clotting disorders and acute respiratory failure have surfaced as the major complications among the severe cases of coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 infection. Remarkably, more than 70% of deaths related to COVID-19 are attributed to clotting-associated complications such as pulmonary embolism, strokes and multi-organ failure. These vascular complications have been confirmed by autopsy. This study summarizes the current understanding and explains the possible mechanisms of the blood clotting disorder, emphasizing the role of (1) hypoxia-related activation of coagulation factors like tissue factor, a significant player in triggering coagulation cascade, (2) cytokine storm and activation of neutrophils and the release of neutrophil extracellular traps and (3) immobility and ICU related risk factors., (© 2020 John Wiley & Sons Ltd.)

Mosquito-borne diseases are responsible for millions of human deaths every year, posing a massive burden on global public health. Mosquitoes transmit a variety of bacteria, parasites and viruses. Mosquito control efforts such as insecticide spraying can reduce mosquito populations, but they must be sustained in order to have long term impacts, can result in the evolution of insecticide resistance, are costly, and can have adverse human and environmental effects. Technological advances have allowed genetic manipulation of mosquitoes, including generation of those that are still susceptible to insecticides, which has greatly increased the number of mosquito strains and lines available to the scientific research community. This generates an associated challenge, because rearing and maintaining unique mosquito lines requires time, money and facilities, and long-term maintenance can lead to adaptation to specific laboratory conditions, resulting in mosquito lines that are distinct from their wild-type counterparts. Additionally, continuous rearing of transgenic lines can lead to loss of genetic markers, genes and/or phenotypes. Cryopreservation of valuable mosquito lines could help circumvent these limitations and allow researchers to reduce the cost of rearing multiple lines simultaneously, maintain low passage number transgenic mosquitoes, and bank lines not currently being used. Additionally, mosquito cryopreservation could allow researchers to access the same mosquito lines, limiting the impact of unique laboratory or field conditions. Successful cryopreservation of mosquitoes would expand the field of mosquito research and could ultimately lead to advances that would reduce the burden of mosquito-borne diseases, possibly through rear-and-release strategies to overcome mosquito insecticide resistance. Cryopreservation techniques have been developed for some insect groups, including but not limited to fruit flies, silkworms and other moth species, and honeybees. Recent advances within the cryopreservation field, along with success with other insects suggest that cryopreservation of mosquitoes may be a feasible method for preserving valuable scientific and public health resources. In this review, we will provide an overview of basic mosquito biology, the current state of and advances within insect cryopreservation, and a proposed approach toward cryopreservation of Anopheles stephensi mosquitoes., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Herbivore-induced plant volatiles (HIPVs) are widely recognized as an ecologically important defensive response of plants against herbivory. Although the induction of this 'cry for help' has been well documented, only a few studies have investigated the inhibition of HIPVs by herbivores and little is known about whether herbivores have evolved mechanisms to inhibit the release of HIPVs. To examine the role of herbivore effectors in modulating HIPVs and stomatal dynamics, we conducted series of experiments combining pharmacological, surgical, genetic (CRISPR-Cas9) and chemical (GC-MS analysis) approaches. We show that the salivary enzyme, glucose oxidase (GOX), secreted by the caterpillar Helicoverpa zea on leaves, causes stomatal closure in tomato (Solanum lycopersicum) within 5 min, and in both tomato and soybean (Glycine max) for at least 48 h. GOX also inhibits the emission of several HIPVs during feeding by H. zea, including (Z)-3-hexenol, (Z)-jasmone and (Z)-3-hexenyl acetate, which are important airborne signals in plant defenses. Our findings highlight a potential adaptive strategy where an insect herbivore inhibits plant airborne defenses during feeding by exploiting the association between stomatal dynamics and HIPV emission., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses., (© 2021 Ma et al.; Published by Cold Spring Harbor Laboratory Press.)

The mosquito, Culex tarsalis, is a key vector in the western United States due to its role in transmission of zoonotic arboviruses that affect human health. Extensive research has been conducted on Cx. tarsalis ecology, feeding behavior, vector competence, autogeny, diapause, genetics, and insecticide resistance. Population genetic analyses in the western U.S. have identified at least three genetic clusters that are geographically distinct. However, in-depth genetic studies have been hindered by the lack of a reference genome. In this study, we present the first whole-genome assembly of this mosquito species (CtarK1) based on PacBio HiFi reads from high-molecular-weight DNA extracted from a single male. The CtarK1 assembly is 790 Mb with an N50 of 58 kb, which is 27% larger than Culex quinquefasciatus (578 Mb). This difference appears to be mostly composed of transposable elements. To annotate CtarK1, we used a previously assembled Cx. tarsalis transcriptome and approximately 17,456 protein genes from Cx. quinquefasciatus (N = 17,456). Genome completeness was assessed using the Benchmarking Universal Single-Copy Orthologs (BUSCO) tool, which identified 84.8% of the 2799 Dipteran BUSCO genes. Using a Bayesian phylogeny based on mitochondrial genomes, we place Cx. tarsalis in the context of other mosquito species and estimate the divergence between Cx. tarsalis and Cx. quinquefasciatus to be between 15.8 and 22.2 million years ago (MYA). Important next steps from this work include characterizing the genetic basis of diapause and sex determination in Culex mosquitoes., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the Drosophila model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions. Herein we propose that genetic engineering methods will be essential to uncover the full functionality of PIWI/piRNA biology in mosquitoes and that extending the applications of genetic engineering on other aspects of mosquito biology will grant access to a much larger pool of knowledge in disease vectors that is just out of reach. We discuss motivations for and impediments to expanding the utility of genetic engineering to study the underlying biology and disease transmission and describe specific areas where efforts can be placed to achieve the full potential for genetic engineering in basic biology in mosquito vectors. Such efforts will generate a refreshed intellectual source of novel approaches to disease control and strong support for the effective use of approaches currently in development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Macias, Palatini, Bonizzoni and Rasgon.)

The jewel wasp, Nasonia vitripennis, has become an efficient model system to study epigenetics of haplo-diploid sex determination, B-chromosome biology, host-symbiont interactions, speciation, and venom synthesis. Despite the availability of several molecular tools, including CRISPR/Cas9, functional genetic studies are still limited in this organism. The major limitation of applying CRISPR/Cas9 technology in N. vitripennis stems from the challenges of embryonic microinjections. Injections of embryos are particularly difficult in this organism and in general in many parasitoid wasps, due to small embryo size and the requirement of a host pupa for embryonic development. To address these challenges, Cas9 ribonucleoprotein complex delivery into female ovaries by adult injection, rather than embryonic microinjection, was optimized, resulting in both somatic and heritable germline edits. The injection procedures were optimized in pupae and female wasps using either ReMOT Control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules). These methods are shown to be effective alternatives to embryo injection, enabling site-specific and heritable germline mutations.

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., (© 2020 Royal Entomological Society.)

Insect epithelial cells contain cellular extensions such as bristles, hairs, and scales. These cellular extensions are homologous structures that differ in morphology and function. They contain actin bundles that dictate their cellular morphology. While the organization, function, and identity of the major actin-bundling proteins in bristles and hairs are known, this information on scales is unknown. In this study, we characterized the development of scales and the role of actin bundles in the mosquito, Aedes aegypti. We show that scales undergo drastic morphological changes during development, from a cylindrical to flat shape with longer membrane invagination. Scale actin-bundle distribution changes from the symmetrical organization of actin bundles located throughout the bristle membrane to an asymmetrical organization. By chemically inhibiting actin polymerization and by knocking out the forked gene in the mosquito (Ae-Forked; a known actin-bundling protein) by CRISPR-Cas9 gene editing, we showed that actin bundles are required for shaping bristle, hair, and scale morphology. We demonstrated that actin bundles and Ae-Forked are required for bristle elongation, but not for that of scales. In scales, actin bundles are required for width formation. In summary, our results reveal, for the first time, the developmental process of mosquito scale formation and also the role of actin bundles and actin-bundle proteins in scale morphogenesis. Moreover, our results reveal that although scale and bristle are thought to be homologous structures, actin bundles have a differential requirement in shaping mosquito scales compared to bristles.

Background: Recent studies demonstrate that insect-specific viruses can influence the ability of their mosquito hosts to become infected with and transmit arboviruses of medical and veterinary importance. The aim of this study was to evaluate the interactions between Anopheles gambiae densovirus (AgDNV) (Parvoviridae) (a benign insect-specific virus that infects An. gambiae mosquitoes) and Mayaro virus (MAYV) (Togaviridae) (an emerging human pathogen that can be transmitted by An. gambiae) in both insect cell culture and mosquitoes., Methods: For in vitro studies, An. gambiae Mos55 cells infected or uninfected with AgDNV were infected with MAYV. For in vivo studies, An. gambiae mosquitoes were injected intrathoracically with AgDNV and 4 days later orally infected with MAYV. Mosquitoes were dissected 10 days after MAYV infection, and MAYV titers in the body, legs and saliva samples quantified using focus-forming assay., Results: MAYV virus replication was reduced 10-100-fold in An. gambiae Mos55 cells infected with AgDNV. In mosquitoes, there was a significant negative correlation between AgDNV and MAYV body titers 10 days post-blood meal., Conclusions: AgDNV infection was associated with reduced production of MAYV in cell culture, and reduced body titers of MAYV in An. gambiae mosquitoes. As densovirus infections are common in natural mosquito populations, these data suggest that they may affect the epidemiology of viruses of medical importance.