Immunometabolic crosstalk in Aedes fluviatilis Wolbachia pipientis symbiosis.

Wolbachia pipientis is a maternally transmitted symbiotic bacterium that mainly colonizes arthropods, potentially affecting different aspects of the host's physiology, e.g. reproduction, immunity, and metabolism. It has been shown that Wolbachia modulates glycogen metabolism in mosquito Aedes fluviatilis (Ae. fluviatilis). Glycogen synthesis is controlled by the enzyme GSK3, which is also involved in immune responses in both vertebrate and invertebrate organisms. Here we investigated the mechanisms behind immune changes mediated by GSK3ß in the symbiosis between Ae. fluviatilis and Wolbachia pipientis using a GSK3ß inhibitor or RNAi-mediated gene silencing. GSK3ß inhibition or knockdown increased glycogen content and Wolbachia population, together with a reduction in Relish2 (REL2) and gambicin transcripts. Furthermore, knockdown of REL2 or Caspar revealed that the Imd pathway acts to control Wolbachia numbers in the host. In conclusion, we describe for the first time the involvement of GSK3ß in Ae. fluviatillis immune response, acting to control the Wolbachia endosymbiotic population.

Exploring dose-response relationships in Aedes aegypti survival upon bacteria and arbovirus infection.

A detailed understanding of how host fitness changes in response to variations in microbe density (an ecological measure of disease tolerance) is an important aim of infection biology. Here, we applied dose-response curves to study Aedes aegypti survival upon exposure to different microbes. We challenged female mosquitoes with Listeria monocytogenes, a model bacterial pathogen, Dengue 4 virus and Zika virus, two medically relevant arboviruses, to understand the distribution of mosquito survival following microbe exposure. By correlating microbe loads and host health, we found that a blood meal promotes disease tolerance in our systemic bacterial infection model and that mosquitoes orally infected with bacteria had an enhanced defensive capacity than insects infected through injection. We also showed that Aedes aegypti displays a higher survival profile following arbovirus infection when compared to bacterial infections. Here, we applied a framework for investigating microbe-induced mosquito mortality and details how the lifespan of Aedes aegypti varies with different inoculum sizes of bacteria and arboviruses.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme-induced peroxidase.

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

The Aedes aegypti peritrophic matrix controls arbovirus vector competence through HPx1, a heme–induced peroxidase

Aedes aegypti mosquitoes are the main vectors of arboviruses. The peritrophic matrix (PM) is an extracellular layer that surrounds the blood bolus. It acts as an immune barrier that prevents direct contact of bacteria with midgut epithelial cells during blood digestion. Here, we describe a heme-dependent peroxidase, hereafter referred to as heme peroxidase 1 (HPx1). HPx1 promotes PM assembly and antioxidant ability, modulating vector competence. Mechanistically, the heme presence in a blood meal induces HPx1 transcriptional activation mediated by the E75 transcription factor. HPx1 knockdown increases midgut reactive oxygen species (ROS) production by the DUOX NADPH oxidase. Elevated ROS levels reduce microbiota growth while enhancing epithelial mitosis, a response to tissue damage. However, simultaneous HPx1 and DUOX silencing was not able to rescue bacterial population growth, as explained by increased expression of antimicrobial peptides (AMPs), which occurred only after double knockdown. This result revealed hierarchical activation of ROS and AMPs to control microbiota. HPx1 knockdown produced a 100-fold decrease in Zika and dengue 2 midgut infection, demonstrating the essential role of the mosquito PM in the modulation of arbovirus vector competence. Our data show that the PM connects blood digestion to midgut immunological sensing of the microbiota and viral infections.

Hemocyte differentiation to the megacyte lineage enhances mosquito immunity against Plasmodium.

Activation of Toll signaling in Anopheles gambiae by silencing Cactus, a suppressor of this pathway, enhances local release of hemocyte-derived microvesicles (HdMv), promoting activation of the mosquito complement-like system, which eliminates Plasmodium ookinetes. We uncovered the mechanism of this immune enhancement. Cactus silencing triggers a Rel1-mediated differentiation of granulocytes to the megacyte lineage, a new subpopulation of giant cells, resulting in a dramatic increase in the proportion of circulating megacytes. Megacytes are very plastic cells that are massively recruited to the basal midgut surface in response to Plasmodium infection. We show that Toll signaling modulates hemocyte differentiation and that megacyte recruitment to the midgut greatly enhances mosquito immunity against Plasmodium.

Malaria causes hundreds of thousands of deaths each year. This devastating disease is caused by Plasmodium parasites, which are transmitted to people through female Anopheles gambiae mosquitos. Mosquitos become infected with Plasmodium when they ingest blood containing these malaria-causing parasites. However, Plasmodium must avoid the mosquito immune system to survive and spread. The mosquito immune system is made up of several types of immune cells, including cells known as granulocytes. Granulocytes can further develop into additional cell subtypes, such as megacytes and antimicrobial granulocytes, but it is not clear how these types of cells work to protect mosquitos against infections. In the mosquitos that transmit malaria, a cell signaling pathway called Toll helps control immune responses to disease-causing microbes, such as Plasmodium. When Toll signaling is strongly triggered in mosquitos, Plasmodium infection is eliminated because immune cell responses are enhanced ­ which results in lower levels of transmission to humans. But what is the underlying mechanism through which high levels of Toll signaling eradicate Plasmodium infection? To find out, Barletta et al. collected cell samples from A. gambiae mosquitos and analyzed what happened when Toll signaling was strongly activated. They observed a large increase in the proportion of megacytes in these mosquitos (from 2% to 80% of all granulocytes). Toll signaling also caused megacytes to become bigger, cluster together, and have higher plasticity ­ meaning they could adopt different shapes. Barletta et al. used microscopy to show that these megacytes were releasing large mitochondria-like structures and membrane vesicles , which may be the trigger activating the mosquito's immune system. In live mosquitos, megacytes move towards the area of the Plasmodium infection and release microvesicles. These microvesicles are known to activate a part of the the mosquito's immune system called the complement-like system, destroying the parasites and preventing mosquito infection and disease transmission. These findings show how strong Toll signaling triggers the mosquito immune system to eliminate Plasmodium infections. Understanding how the mosquito immune system tackles Plasmodium infection may help reveal ways to reduce or block transmission.

Zika virus infection drives epigenetic modulation of immunity by the histone acetyltransferase CBP of Aedes aegypti.

Epigenetic mechanisms are responsible for a wide range of biological phenomena in insects, controlling embryonic development, growth, aging and nutrition. Despite this, the role of epigenetics in shaping insect-pathogen interactions has received little attention. Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we explored the role of the Aedes aegypti histone acetyltransferase CBP (AaCBP) after infection with Zika virus (ZIKV), focusing on the two main immune tissues, the midgut and fat body. We showed that the expression and activity of AaCBP could be positively modulated by blood meal and ZIKV infection. Nevertheless, Zika-infected mosquitoes that were silenced for AaCBP revealed a significant reduction in the acetylation of H3K27 (CBP target marker), followed by downmodulation of the expression of immune genes, higher titers of ZIKV and lower survival rates. Importantly, in Zika-infected mosquitoes that were treated with sodium butyrate, a histone deacetylase inhibitor, their capacity to fight virus infection was rescued. Our data point to a direct correlation among histone hyperacetylation by AaCBP, upregulation of antimicrobial peptide genes and increased survival of Zika-infected-A. aegypti.

Non-immune Traits Triggered by Blood Intake Impact Vectorial Competence.

Blood-feeding arthropods are considered an enormous public health threat. They are vectors of a plethora of infectious agents that cause potentially fatal diseases like Malaria, Dengue fever, Leishmaniasis, and Lyme disease. These vectors shine due to their own physiological idiosyncrasies, but one biological aspect brings them all together: the requirement of blood intake for development and reproduction. It is through blood-feeding that they acquire pathogens and during blood digestion that they summon a collection of multisystemic events critical for vector competence. The literature is focused on how classical immune pathways (Toll, IMD, and JAK/Stat) are elicited throughout the course of vector infection. Still, they are not the sole determinants of host permissiveness. The dramatic changes that are the hallmark of the insect physiology after a blood meal intake are the landscape where a successful infection takes place. Dominant processes that occur in response to a blood meal are not canonical immunological traits yet are critical in establishing vector competence. These include hormonal circuitries and reproductive physiology, midgut permeability barriers, midgut homeostasis, energy metabolism, and proteolytic activity. On the other hand, the parasites themselves have a role in the outcome of these blood triggered physiological events, consistently using them in their favor. Here, to enlighten the knowledge on vector-pathogen interaction beyond the immune pathways, we will explore different aspects of the vector physiology, discussing how they give support to these long-dated host-parasite relationships.

Prostaglandins regulate humoral immune responses in Aedes aegypti.

Prostaglandins (PGs) are immuno-active lipids that mediate the immune response in invertebrates and vertebrates. In insects, PGs play a role on different physiological processes such as reproduction, ion transport and regulation of cellular immunity. However, it is unclear whether PGs play a role in invertebrate's humoral immunity, and, if so, which immune signaling pathways would be modulated by PGs. Here, we show that Aedes aegypti gut microbiota and Gram-negative bacteria challenge induces prostaglandin production sensitive to an irreversible inhibitor of the vertebrate cyclooxygenase, acetylsalicylic acid (ASA). ASA treatment reduced PG synthesis and is associated with decreased expression of components of the Toll and IMD immune pathways, thereby rendering mosquitoes more susceptible to both bacterial and viral infections. We also shown that a cytosolic phospholipase (PLAc), one of the upstream regulators of PG synthesis, is induced by the microbiota in the midgut after blood feeding. The knockdown of the PLAc decreased prostaglandin production and enhanced the replication of Dengue in the midgut. We conclude that in Ae. aegypti, PGs control the amplitude of the immune response to guarantee an efficient pathogen clearance.

Unlike Zika, Chikungunya virus interferes in the viability of Aedes aegypti eggs, regardless of females' age.

Chikungunya and Zika are arboviruses transmitted by the mosquito Aedes aegypti. Mosquito fecundity and egg viability are important parameters of vectorial capacity. Here we aim to understand, comparatively, the effects of Chikungunya virus (CHIKV) and Zika virus (ZIKV) infections on the fecundity and fertility of young and old Aedes aegypti females. Using artificial infection blood feeding experiments we observed that both CHIKV and ZIKV do not alter the number of eggs laid when compared to uninfected females, although the egg fertility significantly decreases in both young and old CHIKV-infected females. There is an upward trend of null females (infertile females) from 2.1% in young to 6.8% in old ZIKV-infected females. Together, our data revealed that CHIKV and ZIKV affects differently Ae. aegypti physiology, that may be related to different viral spread in nature.

Natural infection by the protozoan Leptomonas wallacei impacts the morphology, physiology, reproduction, and lifespan of the insect Oncopeltus fasciatus.

Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth.

Non-canonical transcriptional regulation of heme oxygenase in Aedes aegypti.

Heme oxygenase (HO) is a ubiquitous enzyme responsible for heme breakdown, which yields carbon monoxide (CO), biliverdin (BV) and ferrous ion. Here we show that the Aedes aegypti heme oxygenase gene (AeHO - AAEL008136) is expressed in different developmental stages and tissues. AeHO expression increases after a blood meal in the midgut, and its maximal transcription levels overlaps with the maximal rate of the further modified A. aegypti biglutaminyl-biliverdin (AeBV) pigment production. HO is a classical component of stress response in eukaryotic cells, being activated under oxidative stress or increased heme levels. Indeed, the final product of HO activity in the mosquito midgut, AeBV, exerts a protective antioxidant activity. AeHO, however, does not seem to be under a classical redox-sensitive transcriptional regulation, being unresponsive to heme itself, and even down regulated when insects face a pro-oxidant insult. In contrast, AeHO gene expression responds to nutrient sensing mechanisms, through the target of rapamycin (TOR) pathway. This unusual transcriptional control of AeHO, together with the antioxidant properties of AeBV, suggests that heme degradation by HO, in addition to its important role in protection of Aedes aegypti against heme exposure, also acts as a digestive feature, being an essential adaptation to blood feeding.

Regulation of midgut cell proliferation impacts Aedes aegypti susceptibility to dengue virus.

Aedes aegypti is the vector of some of the most important vector-borne diseases like dengue, chikungunya, zika and yellow fever, affecting millions of people worldwide. The cellular processes that follow a blood meal in the mosquito midgut are directly associated with pathogen transmission. We studied the homeostatic response of the midgut against oxidative stress, as well as bacterial and dengue virus (DENV) infections, focusing on the proliferative ability of the intestinal stem cells (ISC). Inhibition of the peritrophic matrix (PM) formation led to an increase in reactive oxygen species (ROS) production by the epithelial cells in response to contact with the resident microbiota, suggesting that maintenance of low levels of ROS in the intestinal lumen is key to keep ISCs division in balance. We show that dengue virus infection induces midgut cell division in both DENV susceptible (Rockefeller) and refractory (Orlando) mosquito strains. However, the susceptible strain delays the activation of the regeneration process compared with the refractory strain. Impairment of the Delta/Notch signaling, by silencing the Notch ligand Delta using RNAi, significantly increased the susceptibility of the refractory strains to DENV infection of the midgut. We propose that this cell replenishment is essential to control viral infection in the mosquito. Our study demonstrates that the intestinal epithelium of the blood fed mosquito is able to respond and defend against different challenges, including virus infection. In addition, we provide unprecedented evidence that the activation of a cellular regenerative program in the midgut is important for the determination of the mosquito vectorial competence.

The redox-sensing gene Nrf2 affects intestinal homeostasis, insecticide resistance, and Zika virus susceptibility in the mosquito Aedes aegypti.

Production and degradation of reactive oxygen species (ROS) are extensively regulated to ensure proper cellular responses to various environmental stimuli and stresses. Moreover, physiologically generated ROS function as secondary messengers that can influence tissue homeostasis. The cap'n'collar transcription factor known as nuclear factor erythroid-derived factor 2 (Nrf2) coordinates an evolutionarily conserved transcriptional activation pathway that mediates antioxidant and detoxification responses in many animal species, including insects and mammals. Here, we show that Nrf2-mediated signaling affects embryo survival, midgut homeostasis, and redox biology in Aedes aegypti, a mosquito species vector of dengue, Zika, and other disease-causing viruses. We observed that AeNrf2 silencing increases ROS levels and stimulates intestinal stem cell proliferation. Because ROS production is a major aspect of innate immunity in mosquito gut, we found that a decrease in Nrf2 signaling results in reduced microbiota growth and Zika virus infection. Moreover, we provide evidence that AeNrf2 signaling also controls transcriptional adaptation of A. aegypti to insecticide challenge. Therefore, we conclude that Nrf2-mediated response regulates assorted gene clusters in A. aegypti that determine cellular and midgut redox balance, affecting overall xenobiotic resistance and vectorial adaptation of the mosquito.

Amino acid metabolic signaling influences Aedes aegypti midgut microbiome variability.

The mosquito midgut microbiota has been shown to influence vector competence for multiple human pathogens. The microbiota is highly variable in the field, and the sources of this variability are not well understood, which limits our ability to understand or predict its effects on pathogen transmission. In this work, we report significant variation in female adult midgut bacterial load between strains of A. aegypti which vary in their susceptibility to dengue virus. Composition of the midgut microbiome was similar overall between the strains, with 81-92% of reads coming from the same five bacterial families, though we did detect differences in the presence of some bacterial families including Flavobacteriaceae and Entobacteriaceae. We conducted transcriptomic analysis on the two mosquito strains that showed the greatest difference in bacterial load, and found that they differ in transcript abundance of many genes implicated in amino acid metabolism, in particular the branched chain amino acid degradation pathway. We then silenced this pathway by targeting multiple genes using RNA interference, which resulted in strain-specific bacterial proliferation, thereby eliminating the difference in midgut bacterial load between the strains. This suggests that the branched chain amino acid (BCAA) degradation pathway controls midgut bacterial load, though the mechanism underlying this remains unclear. Overall, our results indicate that amino acid metabolism can act to influence the midgut microbiota. Moreover, they suggest that genetic or physiological variation in BCAA degradation pathway activity may in part explain midgut microbiota variation in the field.

Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika.

BACKGROUND: Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. METHODOLOGY/PRINCIPAL FINDINGS: We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). CONCLUSION/SIGNIFICANCE: Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections.

Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti.

BACKGROUND: Aedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication. RESULTS: Here, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads. CONCLUSION: Together, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.

The use of a chemically defined artificial diet as a tool to study Aedes aegypti physiology.

Aedes aegypti mosquitoes obtain from vertebrate blood nutrients that are essential to oogenesis, such as proteins and lipids. As with all insects, mosquitoes do not synthesize cholesterol but take it from the diet. Here, we used a chemically defined artificial diet, hereafter referred to as Substitute Blood Meal (SBM), that was supplemented with cholesterol to test the nutritional role of cholesterol. SBM-fed and blood-fed mosquitoes were compared regarding several aspects of the insect physiology that are influenced by a blood meal, including egg laying, peritrophic matrix formation, gut microbiota proliferation, generation of reactive oxygen species (ROS) and expression of antioxidant genes, such as catalase and ferritin. Our results show that SBM induced a physiological response that was very similar to a regular blood meal. Depending on the nutritional life history of the mosquito since the larval stage, the presence of cholesterol in the diet increased egg development, suggesting that the teneral reserves of cholesterol in the newly hatched female are determinant of reproductive performance. We propose here the use of SBM as a tool to study other aspects of the physiology of mosquitoes, including their interaction with microbiota and pathogens.

Heme Signaling Impacts Global Gene Expression, Immunity and Dengue Virus Infectivity in Aedes aegypti.

Blood-feeding mosquitoes are exposed to high levels of heme, the product of hemoglobin degradation. Heme is a pro-oxidant that influences a variety of cellular processes. We performed a global analysis of heme-regulated Aedes aegypti (yellow fever mosquito) transcriptional changes to better understand influence on mosquito physiology at the molecular level. We observed an iron- and reactive oxygen species (ROS)-independent signaling induced by heme that comprised genes related to redox metabolism. By modulating the abundance of these transcripts, heme possibly acts as a danger signaling molecule. Furthermore, heme triggered critical changes in the expression of energy metabolism and immune response genes, altering the susceptibility towards bacteria and dengue virus. These findings seem to have implications on the adaptation of mosquitoes to hematophagy and consequently on their ability to transmit diseases. Altogether, these results may also contribute to the understanding of heme cell biology in eukaryotic cells.