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.

Dengue and chikungunya virus loads in the mosquito Aedes aegypti are determined by distinct genetic architectures.

Aedes aegypti is the primary vector of the arboviruses dengue (DENV) and chikungunya (CHIKV). These viruses exhibit key differences in their vector interactions, the latter moving more quicky through the mosquito and triggering fewer standard antiviral pathways. As the global footprint of CHIKV continues to expand, we seek to better understand the mosquito's natural response to CHIKV-both to compare it to DENV:vector coevolutionary history and to identify potential targets in the mosquito for genetic modification. We used a modified full-sibling design to estimate the contribution of mosquito genetic variation to viral loads of both DENV and CHIKV. Heritabilities were significant, but higher for DENV (40%) than CHIKV (18%). Interestingly, there was no genetic correlation between DENV and CHIKV loads between siblings. These data suggest Ae. aegypti mosquitoes respond to the two viruses using distinct genetic mechanisms. We also examined genome-wide patterns of gene expression between High and Low CHIKV families representing the phenotypic extremes of viral load. Using RNAseq, we identified only two loci that consistently differentiated High and Low families: a long non-coding RNA that has been identified in mosquito screens post-infection and a distant member of a family of Salivary Gland Specific (SGS) genes. Interestingly, the latter gene is also associated with horizontal gene transfer between mosquitoes and the endosymbiotic bacterium Wolbachia. This work is the first to link the SGS gene to a mosquito phenotype. Understanding the molecular details of how this gene contributes to viral control in mosquitoes may, therefore, also shed light on its role in Wolbachia.

The immune deficiency and c-Jun N-terminal kinase pathways drive the functional integration of the immune and circulatory systems of mosquitoes.

The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial haemocytes, destroy pathogens in the areas of the body that experience the swiftest haemolymph (blood) flow. An infection recruits additional periostial haemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial haemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNA sequencing in the African malaria mosquito, Anopheles gambiae, and discovered that an infection upregulates multiple components of the immune deficiency (IMD) and c-Jun N-terminal kinase (JNK) pathways in the heart with periostial haemocytes. This upregulation is greater in the heart with periostial haemocytes than in the circulating haemocytes or the entire abdomen. RNA interference-based knockdown then showed that the IMD and JNK pathways drive periostial haemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating that these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones.

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.

Artificial Selection Finds New Hypotheses for the Mechanism of Wolbachia-Mediated Dengue Blocking in Mosquitoes.

Wolbachia is an intracellular bacterium that blocks virus replication in insects and has been introduced into the mosquito, Aedes aegypti for the biocontrol of arboviruses including dengue, Zika, and chikungunya. Despite ongoing research, the mechanism of Wolbachia-mediated virus blocking remains unclear. We recently used experimental evolution to reveal that Wolbachia-mediated dengue blocking could be selected upon in the A. aegypti host and showed evidence that strong levels of blocking could be maintained by natural selection. In this study, we investigate the genetic variation associated with blocking and use these analyses to generate testable hypotheses surrounding the mechanism of Wolbachia-mediated dengue blocking. From our results, we hypothesize that Wolbachia may block virus replication by increasing the regeneration rate of mosquito cells via the Notch signaling pathway. We also propose that Wolbachia modulates the host's transcriptional pausing pathway either to prime the host's anti-viral response or to directly inhibit viral replication.

Nitric oxide produced by periostial hemocytes modulates the bacterial infection-induced reduction of the mosquito heart rate.

The circulatory and immune systems of mosquitoes are functionally integrated. An infection induces the migration of hemocytes to the dorsal vessel, and specifically, to the regions surrounding the ostia of the heart. These periostial hemocytes phagocytose pathogens in the areas of the hemocoel that experience the highest hemolymph flow. Here, we investigated whether a bacterial infection affects cardiac rhythmicity in the African malaria mosquito, Anopheles gambiae We discovered that infection with Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, but not Micrococcus luteus, reduces the mosquito heart rate and alters the proportional directionality of heart contractions. Infection does not alter the expression of genes encoding crustacean cardioactive peptide (CCAP), FMRFamide, corazonin, neuropeptide F or short neuropeptide F, indicating that they do not drive the cardiac phenotype. Infection upregulates the transcription of two superoxide dismutase (SOD) genes, catalase and a glutathione peroxidase, but dramatically induces upregulation of nitric oxide synthase (NOS) in both the heart and hemocytes. Within the heart, nitric oxide synthase is produced by periostial hemocytes, and chemically inhibiting the production of nitric oxide using l-NAME reverses the infection-induced cardiac phenotype. Finally, infection induces the upregulation of two lysozyme genes in the heart and other tissues, and treating mosquitoes with lysozyme reduces the heart rate in a manner reminiscent of the infection phenotype. These data demonstrate an exciting new facet of the integration between the immune and circulatory systems of insects, whereby a hemocyte-produced factor with immune activity, namely nitric oxide, modulates heart physiology.

Selection on Aedes aegypti alters Wolbachia-mediated dengue virus blocking and fitness.

The dengue, Zika and chikungunya viruses are transmitted by the mosquito Aedes aegypti and pose a substantial threat to global public health. Current vaccines and mosquito control strategies have limited efficacy, so novel interventions are needed1,2. Wolbachia are bacteria that inhabit insect cells and have been found to reduce viral infection-a phenotype that is referred to as viral 'blocking'3. Although not naturally found in A. aegypti4, Wolbachia were stably introduced into this mosquito in 20114,5 and were shown to reduce the transmission potential of dengue, Zika and chikungunya6,7. Subsequent field trials showed Wolbachia's ability to spread through A. aegypti populations and reduce the local incidence of dengue fever8. Despite these successes, the evolutionary stability of viral blocking is unknown. Here, we utilized artificial selection to reveal genetic variation in the mosquito that affects Wolbachia-mediated dengue blocking. We found that mosquitoes exhibiting weaker blocking also have reduced fitness, suggesting the potential for natural selection to maintain blocking. We also identified A. aegypti genes that affect blocking strength, shedding light on a possible mechanism for the trait. These results will inform the use of Wolbachia as biocontrol agents against mosquito-borne viruses and direct further research into measuring and improving their efficacy.

Expanding the canon: Non-classical mosquito genes at the interface of arboviral infection.

Mosquito transmitted viruses cause significant morbidity and mortality in human populations. Despite the use of insecticides and other measures of vector control, arboviral diseases are on the rise. One potential solution for limiting disease transmission to humans is to render mosquitoes refractory to viral infection through genetic modification. Substantial research effort in Drosophila, Aedes and Anopheles has helped to define the major innate immune pathways, including Toll, IMD, Jak/Stat and RNAi, however we still have an incomplete picture of the mosquito antiviral response. Transcriptional profiles of virus-infected insects reveal a much wider range of pathways activated by the process of infection. Within these lists of genes are unexplored mosquito candidates of viral defense. Wolbachia species are endosymbiotic bacteria that naturally limit arboviral infection in mosquitoes. Our understanding of the Wolbachia-mediated viral blocking mechanism is poor, but it does not appear to operate via the classical immune pathways. Herein, we reviewed the transcriptomic response of mosquitoes to multiple viral species and put forth consensus gene types/families outside the immune canon whose expression responds to infection, including cytoskeleton and cellular trafficking, the heat shock response, cytochromes P450, cell proliferation, chitin and small RNAs. We then examine emerging evidence for their functional role in viral resistance in diverse insect and mammalian hosts and their potential role in Wolbachia-mediated viral blocking. These candidate gene families offer novel avenues for research into the nature of insect viral defense.

Mosquito Hemocytes Associate With Circulatory Structures That Support Intracardiac Retrograde Hemolymph Flow.

A powerful immune system protects mosquitoes from pathogens and influences their ability to transmit disease. The mosquito's immune and circulatory systems are functionally integrated, whereby intense immune processes occur in areas of high hemolymph flow. The primary circulatory organ of mosquitoes is the dorsal vessel, which consists of a thoracic aorta and an abdominal heart. In adults of the African malaria mosquito, Anopheles gambiae, the heart periodically alternates contraction direction, resulting in intracardiac hemolymph flowing toward the head (anterograde) and toward the posterior of the abdomen (retrograde). During anterograde contractions, hemolymph enters the dorsal vessel through ostia located in abdominal segments 2-7, and exits through an excurrent opening located in the head. During retrograde contractions, hemolymph enters the dorsal vessel through ostia located at the thoraco-abdominal junction, and exits through posterior excurrent openings located in the eighth abdominal segment. The ostia in abdominal segments 2 to 7-which function in anterograde intracardiac flow-are sites of intense immune activity, as a subset of hemocytes, called periostial hemocytes, respond to infection by aggregating, phagocytosing, and killing pathogens. Here, we assessed whether hemocytes are present and active at two sites important for retrograde intracardiac hemolymph flow: the thoraco-abdominal ostia and the posterior excurrent openings of the heart. We detected sessile hemocytes around both of these structures, and these hemocytes readily engage in phagocytosis. However, they are few in number and a bacterial infection does not induce the aggregation of additional hemocytes at these locations. Finally, we describe the process of hemocyte attachment and detachment to regions of the dorsal vessel involved in intracardiac retrograde flow.

Structural and functional characterization of the contractile aorta and associated hemocytes of the mosquito Anopheles gambiae.

The primary pump of the circulatory system of insects is a dorsal vessel that traverses the length of the insect. The anterior portion, located in the head, neck and thorax, is the aorta, and the posterior portion, located in the abdomen, is the heart. Here, we characterize the structure and function of the aorta and conical chamber of the mosquito, Anopheles gambiae The aorta begins in the head with an excurrent opening located above the dorsal pharyngeal plate and ends at the thoraco-abdominal junction where it joins the conical chamber of the heart. The aorta lacks ostia, and based on the diameter of the vessel as well as the density and helical orientation of muscle, consists of three regions: the anterior aorta, the bulbous chamber, and the posterior aorta. The aorta contracts in the anterograde direction, but these contractions are independent of heart contractions and do not play a major role in hemolymph propulsion. Intravital imaging of the venous channels, the first abdominal segment and the neck revealed that hemolymph only travels through the aorta in the anterograde direction, and does so only during periods of anterograde heart flow. Furthermore, hemolymph only enters the thoraco-abdominal ostia of the conical chamber when the heart contracts in the retrograde direction, propelling this hemolymph to the posterior of the body. Finally, very few hemocytes associate with the aorta, and unlike what is seen in the periostial regions of the heart, infection does not induce the aggregation of hemocytes on the aorta.

An alternative pathway to eusociality: Exploring the molecular and functional basis of fortress defense.

Some animals express a form of eusociality known as "fortress defense," in which defense rather than brood care is the primary social act. Aphids are small plant-feeding insects, but like termites, some species express division of labor and castes of aggressive juvenile "soldiers." What is the functional basis of fortress defense eusociality in aphids? Previous work showed that the acquisition of venoms might be a key innovation in aphid social evolution. We show that the lethality of aphid soldiers derives in part from the induction of exaggerated immune responses in insects they attack. Comparisons between closely related social and nonsocial species identified a number of secreted effector molecules that are candidates for immune modulation, including a convergently recruited protease described in unrelated aphid species with venom-like functions. These results suggest that aphids are capable of antagonizing conserved features of the insect immune response, and provide new insights into the mechanisms underlying the evolution of fortress defense eusociality in aphids.

Mosquito hemocytes preferentially aggregate and phagocytose pathogens in the periostial regions of the heart that experience the most hemolymph flow.

When a mosquito acquires an infection in the hemocoel, dedicated immune cells called hemocytes aggregate around the valves of the heart. These sessile hemocytes are called periostial hemocytes. In the present study we scrutinized the immune response mounted by the periostial hemocytes of the malaria mosquito, Anopheles gambiae, against bacterial pathogens, and tested the relationship between periostial hemocyte aggregation, immune activity, and hemolymph flow. Initially, we quantified the process of periostial hemocyte aggregation and found that hemocytes migrate to the periostial regions in response to infection with Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus (all infections tested). Then, we investigated whether the periostial hemocytes are evenly distributed along the six periostial regions of the heart, and found that they preferentially aggregate in the periostial regions of the mid-abdominal segments (4, 5 and 6). This distribution perfectly correlates with the spatial distribution of phagocytic activity along the surface of the heart, and to a lesser extent, with the distribution of melanin deposits. Finally, experiments measuring circulatory physiology found that the majority of hemolymph enters the heart through the ostia located in the periostial regions of abdominal segments 4, 5, and 6. These data show that periostial hemocytes aggregate on the surface of the heart in response to diverse foreign stimuli, and that both hemocytes and immune activity preferentially occur in the regions that experience the swiftest hemolymph flow. Thus, these data show that two major organ systems - the immune and circulatory systems - interact to control infections.