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 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.

A mosquito small RNA genomics resource reveals dynamic evolution and host responses to viruses and transposons.

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.

Dehydration stress and Mayaro virus vector competence in Aedes aegypti.

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.

Culex tarsalis Is a Competent Host of the Insect-Specific Alphavirus Eilat Virus (EILV).

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.

In Vitro and In Vivo Coinfection and Superinfection Dynamics of Mayaro and Zika Viruses in Mosquito and Vertebrate Backgrounds.

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.

Alpha-mannosidase-2 modulates arbovirus infection in a pathogen- and Wolbachia-specific manner in Aedes aegypti mosquitoes.

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.

Signatures of adaptive decreased virulence of deformed wing virus in an isolated population of wild honeybees (Apis mellifera).

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.

Function and evolution of the aquaporin IsAQP1 in the Lyme disease vector Ixodes scapularis.

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.

Characterization of Mayaro virus (strain BeAn343102) biology in vertebrate and invertebrate cellular backgrounds.

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.

Assessing Aedes aegypti candidate genes during viral infection and Wolbachia-mediated pathogen blocking.

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.

Clotting disorder in severe acute respiratory syndrome coronavirus 2.

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.

Simulated vector transmission differentially influences dynamics of two viral variants of deformed wing virus in honey bees (Apis mellifera).

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.

Silencing the alarm: an insect salivary enzyme closes plant stomata and inhibits volatile release.

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.

Towards a method for cryopreservation of mosquito vectors of human pathogens.

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.

Aquaglyceroporin function in the malaria mosquito Anopheles gambiae.

BACKGROUND INFORMATION: Anopheles gambiae is the major mosquito vector for Plasmodium falciparum malaria in sub-Saharan Africa, where it survives in stressful climates. Aquaporin water channels are expressed in all life forms, where they provide environmental adaptation by conferring rapid trans-cellular movement of water (classical aquaporins) or water plus glycerol (aquaglyceroporins). Here, we report an aquaglyceroporin homolog in A. gambiae, AgAQP3 (A. gambiae aquaglyceroporin 3). RESULTS: Despite atypical pore-lining amino acids, AgAQP3 is permeated by water, glycerol and urea, and is not significantly inhibited by 1 mM HgCl2 . AgAQP3 is expressed more heavily in male mosquitoes, yet adult female A. gambiae abundantly express AgAQP3 in Malpighian tubules and gut where large amounts of fluid exchange occur during blood meal digestion, water and nutrient absorption and waste secretion. Reducing expression of AgAQP3 by RNA interference reduces median mosquito survival at 39°C. After an infectious blood meal, mosquitoes with depleted AgAQP3 expression exhibit fewer P. falciparum oocysts in the midgut compared to control mosquitoes. CONCLUSIONS: Our studies reveal critical contributions of AgAQP3 to A. gambiae heat tolerance and P. falciparum development in vivo. SIGNIFICANCE: This study indicates that AgAQP3 may be a major factor explaining why A. gambiae is an important malaria vector mosquito in sub-Saharan Africa, and may be a potential target for novel malaria control strategies.

Native microbiome impedes vertical transmission of Wolbachia in Anopheles mosquitoes.

Over evolutionary time, Wolbachia has been repeatedly transferred between host species contributing to the widespread distribution of the symbiont in arthropods. For novel infections to be maintained, Wolbachia must infect the female germ line after being acquired by horizontal transfer. Although mechanistic examples of horizontal transfer exist, there is a poor understanding of factors that lead to successful vertical maintenance of the acquired infection. Using Anopheles mosquitoes (which are naturally uninfected by Wolbachia) we demonstrate that the native mosquito microbiota is a major barrier to vertical transmission of a horizontally acquired Wolbachia infection. After injection into adult Anopheles gambiae, some strains of Wolbachia invade the germ line, but are poorly transmitted to the next generation. In Anopheles stephensi, Wolbachia infection elicited massive blood meal-induced mortality, preventing development of progeny. Manipulation of the mosquito microbiota by antibiotic treatment resulted in perfect maternal transmission at significantly elevated titers of the wAlbB Wolbachia strain in A. gambiae, and alleviated blood meal-induced mortality in A. stephensi enabling production of Wolbachia-infected offspring. Microbiome analysis using high-throughput sequencing identified that the bacterium Asaia was significantly reduced by antibiotic treatment in both mosquito species. Supplementation of an antibiotic-resistant mutant of Asaia to antibiotic-treated mosquitoes completely inhibited Wolbachia transmission and partly contributed to blood meal-induced mortality. These data suggest that the components of the native mosquito microbiota can impede Wolbachia transmission in Anopheles. Incompatibility between the microbiota and Wolbachia may in part explain why some hosts are uninfected by this endosymbiont in nature.

Impact of trehalose transporter knockdown on Anopheles gambiae stress adaptation and susceptibility to Plasmodium falciparum infection.

Anopheles gambiae is a major vector mosquito for Plasmodium falciparum, the deadly pathogen causing most human malaria in sub-Saharan Africa. Synthesized in the fat body, trehalose is the predominant sugar in mosquito hemolymph. It not only provides energy but also protects the mosquito against desiccation and heat stresses. Trehalose enters the mosquito hemolymph by the trehalose transporter AgTreT1. In adult female A. gambiae, AgTreT1 is predominantly expressed in the fat body. We found that AgTreT1 expression is induced by environmental stresses such as low humidity or elevated temperature. AgTreT1 RNA silencing reduces the hemolymph trehalose concentration by 40%, and the mosquitoes succumb sooner after exposure to desiccation or heat. After an infectious blood meal, AgTreT1 RNA silencing reduces the number of P. falciparum oocysts in the mosquito midgut by over 70% compared with mock-injected mosquitoes. These data reveal important roles for AgTreT1 in stress adaptation and malaria pathogen development in a major vector mosquito. Thus, AgTreT1 may be a potential target for malaria vector control.

Aquaporin water channel AgAQP1 in the malaria vector mosquito Anopheles gambiae during blood feeding and humidity adaptation.

Altered patterns of malaria endemicity reflect, in part, changes in feeding behavior and climate adaptation of mosquito vectors. Aquaporin (AQP) water channels are found throughout nature and confer high-capacity water flow through cell membranes. The genome of the major malaria vector mosquito Anopheles gambiae contains at least seven putative AQP sequences. Anticipating that transmembrane water movements are important during the life cycle of A. gambiae, we identified and characterized the A. gambiae aquaporin 1 (AgAQP1) protein that is homologous to AQPs known in humans, Drosophila, and sap-sucking insects. When expressed in Xenopus laevis oocytes, AgAQP1 transports water but not glycerol. Similar to mammalian AQPs, water permeation of AgAQP1 is inhibited by HgCl(2) and tetraethylammonium, with Tyr185 conferring tetraethylammonium sensitivity. AgAQP1 is more highly expressed in adult female A. gambiae mosquitoes than in males. Expression is high in gut, ovaries, and Malpighian tubules where immunofluorescence microscopy reveals that AgAQP1 resides in stellate cells but not principal cells. AgAQP1 expression is up-regulated in fat body and ovary by blood feeding but not by sugar feeding, and it is reduced by exposure to a dehydrating environment (42% relative humidity). RNA interference reduces AgAQP1 mRNA and protein levels. In a desiccating environment (<20% relative humidity), mosquitoes with reduced AgAQP1 protein survive significantly longer than controls. These studies support a role for AgAQP1 in water homeostasis during blood feeding and humidity adaptation of A. gambiae, a major mosquito vector of human malaria in sub-Saharan Africa.