Alpha-COPI coatomer protein is required for rough endoplasmic reticulum whorl formation in mosquito midgut epithelial cells

Background

One of the early events in midgut epithelial cells of Aedes aegypti mosquitoes is the dynamic reorganization of rough endoplasmic reticulum (RER) whorl structures coincident with the onset of blood meal digestion. Based on our previous studies showing that feeding on an amino acid meal induces TOR signaling in Ae. aegypti, we used proteomics and RNAi to functionally identify midgut epithelial cell proteins that contribute to RER whorl formation.

Methodology/Principal Findings

Adult female Ae. aegypti mosquitoes were maintained on sugar alone (unfed), or fed an amino acid meal, and then midgut epithelial cells were analyzed by electron microscopy and protein biochemistry. The size and number of RER whorls in midgut epithelial cells were found to decrease significantly after feeding, and several KDEL-containing proteins were shown to have altered expression levels. LC-MS/MS mass spectrometry was used to analyze midgut microsomal proteins isolated from unfed and amino acid fed mosquitoes, and of the 127 proteins identified, 8 were chosen as candidate whorl forming proteins. Three candidate proteins were COPI coatomer subunits (alpha, beta, beta''), all of which appeared to be present at higher levels in microsomal fractions from unfed mosquitoes. Using RNAi to knockdown alpha-COPI expression, electron microscopy revealed that both the size and number of RER whorls were dramatically reduced in unfed mosquitoes, and moreover, that extended regions of swollen RER were prevalent in fed mosquitoes. Lastly, while a deficiency in alpha-COPI had no effect on early trypsin protein synthesis or secretion 3 hr post blood meal (PBM), expression of late phase proteases at 24 hr PBM was completely blocked.

Conclusions

alpha-COPI was found to be required for the formation of RER whorls in midgut epithelial cells of unfed Aa. aegypti mosquitoes, as well as for the expression of late phase midgut proteases.     

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles (P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.     

A novel multiple membrane blood‐feeding system for investigating and maintaining Aedes aegypti and Aedes albopictus mosquitoes

A novel multiple membrane blood‐feeding system for mosquitoes has been developed for the study and routine maintenance of Aedes aegypti L. and Aedes albopictus Skuse that require a meal of vertebrate blood to produce eggs. This blood‐feeding system uses cattle collagen sausage‐casing membrane to facilitate feeding. The efficiency of this blood‐feeding system was compared to a live mice blood source. We observed that Ae. aegypti that fed on pig whole blood had 89.7% (w/o ATP) and 90.7% (w/ ATP) blood‐feeding rates, which were not significantly different from the mice‐fed ones (98.0%). Ae. albopictus fed on pig whole blood (w/ ATP) had a success rate of 84.4%, which was significantly different from the mice‐fed mosquitoes (51.1%). The feeding rates did not differ between sausage‐casing membrane and Parafilm‐M®. The survival rate, fecundity, pupation, and pupal emergence rates of Aedes females fed on pig whole blood were not significantly different from the mice‐fed ones. The artificial blood feeder can be applied to replace live animals as blood sources. Considering that this simple, inexpensive, convenient, and efficient feeding device can be built with common laboratory materials for research on Aedes mosquitoes.     

Effect of body size and sugar meals on oviposition of the yellow fever mosquito,Aedes aegypti (Diptera: Culicidae)

The effects of dietary sugar and body size on the oviposition of Ae. aegypti were studied under laboratory conditions. In female mosquitoes provided with sugar, the start of maximum fecundity was significantly delayed and the oviposition period was longer than in females provided with water. The peak of oviposition was also delayed in sugar‐fed females. Large females oviposited more eggs per day than small females at maximum fecundity and during eight days of observations. Large females also visited significantly more water‐containing cups in their cages per day than small females at maximum fecundity. During the eight days of observations, large females and sugar‐fed females visited more water‐containing cups in their cages than water‐fed small females. Both large females and sugar‐fed females oviposited their eggs at sites higher above the water line than water‐fed small females. These results suggested that large and sugar‐fed female Ae. aegypti mosquitoes had more energy reserves and oviposited their eggs at higher sites, which would lead to a time lag in hatching.     

COPI-mediated blood meal digestion in vector mosquitoes is independent of midgut ARF-GEF and ARF-GAP regulatory activities

We have previously shown that defects in COPI coatomer proteins cause 80% mortality in blood fed Aedes aegypti mosquitoes by 96 h post-feeding. In this study we show that similar deficiencies in COPII and clathrin mediated vesicle transport do not disrupt blood meal digestion and are not lethal, even though both COPII and clathrin functions are required for ovarian development. Since COPI vesicle transport is controlled in mammalian cells by upstream G proteins and associated regulatory factors, we investigated the function of the orthologous ADP-ribosylation factor 1 (ARF1) and ARF4 proteins in mosquito tissues. We found that both ARF1 and ARF4 function upstream of COPI vesicle transport in blood fed mosquitoes given that an ARF1/ARF4 double deficiency is required to phenocopy the feeding-induced mortality observed in COPI coatomer deficient mosquitoes. Small molecule inhibitors of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are often transitory, and therefore, we investigated the role of five Ae. aegypti ARF-GEF and ARF-GAP proteins in blood meal digestion using RNA interference. Surprisingly, we found that ARF-GEF and ARF-GAP functions are not required for blood meal digestion, even though both vitellogenesis and ovarian development in Ae. aegypti are dependent on GBF1 (ARF-GEF) and GAP1/GAP2 (ARF-GAPs) proteins. Moreover, deficiencies in orthologous COPI regulating genes in Anopheles stephensi mosquitoes had similar phenotypes, indicating conserved functions in these two mosquito species. We propose that based on the need for rapid initiation of protein digestion and peritrophic membrane formation, COPI vesicle transport in midgut epithelial cells is not dependent on ARF-GEF and ARF-GAP regulatory proteins to mediate vesicle recycling within the first 48 h post-feeding.     

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.     

Wolbachia infection in Aedes aegypti mosquitoes alters blood meal excretion and delays oviposition without affecting trypsin activity

Blood feeding in Aedes aegypti is essential for reproduction, but also permits the mosquito to act as a vector for key human pathogens such as the Zika and dengue viruses. Wolbachia pipientis is an endosymbiotic bacterium that can manipulate the biology of Aedes aegypti mosquitoes, making them less competent hosts for many pathogens. Yet while Wolbachia affects other aspects of host physiology, it is unclear whether it influences physiological processes associated with blood meal digestion. To that end, we examined the effects of wMel Wolbachia infection in Ae. aegypti, on survival post-blood feeding, blood meal excretion, rate of oviposition, expression levels of key genes involved in oogenesis, and activity levels of trypsin blood digestion enzymes. We observed that wMel infection altered the rate and duration of blood meal excretion, delayed the onset of oviposition and was associated with a greater number of eggs being laid later. wMel-infected Ae. aegypti also had lower levels of key yolk protein precursor genes necessary for oogenesis. However, all of these effects occurred without a change in trypsin activity. These results suggest that Wolbachia infection may disrupt normal metabolic processes associated with blood feeding and reproduction in Ae. aegypti.     

Effects of blood meal sources on the biological characteristics of Aedes aegypti and Culex pipiens (Diptera: Culicidae)

Mosquitoes transmit many diseases to humans and animals e.g., malaria, yellow fever, dengue, filariasis and encephalitis. The fundamental target of this search was to study the effect of three different blood meal sources (human; rabbit and pigeon) on some biological and behavioral properties of Aedes aegypti and Culex pipiens. The results have assured that the females of the mosquito Ae. aegypti that were fed on human blood meal has registered the highest feeding activity from feeding on the blood meal whereas the females of the other mosquito Cx. pipiens have shown the highest feeding activity after being fed on pigeons when compared with its feeding on other factors. The results have shown non-significant variation in the average time necessary to digest the blood meal on both mosquito species Ae. aegypti and Cx. pipiens that were fed on vertebrate hosts under laboratory conditions. Furthermore, results assured that the difference in blood meal sources has yielded distinct variation in the reproductive capacity and efficiency of both female mosquitoes under investigation where both species Ae. aegypti and Cx. pipiens already fed on human blood meal have yielded a pronounced distinctive increase in egg production (oviposition) when compared with females that were fed on pigeon or rabbit blood meal respectively. Moreover, feeding of the female mosquitoes under lab conditions on different blood meal sources did not affect the level of the hatching eggs that were laid by both mosquito females.     

Water channel activity of putative aquaporin‐6 present in Aedes aegypti

Aquaporins (AQPs) are integral membrane channels that facilitate the bidirectional transport of water and sometimes other small solutes across biological membranes. AQPs are important in mediating environmental adaptations in mosquitoes and are considered as a novel target for the development of effective insecticides against mosquitoes. Here, we expressed Aedes aegypti AQP6 ( AaAQP6) in human embryonic kidney (HEK) 293 cells and analyzed the water permeability by a conventional swelling assay, that is, a real‐time change in cell size corresponding to the cell swelling induced by hyposmotic solution. The swelling assay revealed that AaAQP6 is a mercury‐sensitive water channel. Gene expression studies showed that AaAQP6 is highly expressed in the pupae than other developmental stages. Heterologous expression of AaAQP6 in HEK cell was mainly observed intracellularly suggesting AaAQP6 possibly could be a subcellular water channel and may play an osmoregulatory function in the pupae of A. aegypti.     

Efficiency of fungus‐impregnated black cloths combined with Imidacloprid for the control of adult Aedes aegypti (Diptera: Culicidae)

Entomopathogenic fungi are potential candidates for use in integrated vector management. However, efficient delivery systems for these fungi need to be investigated. It is known that adult mosquitoes are attracted to dark surfaces, and therefore, black cotton cloths impregnated with Metarhizium anisopliae alone or in combination with the insecticide imidacloprid (IMI) were tested under laboratory conditions. Black cloths impregnated with fungus were also tested in large‐cage trials under natural extradomicile conditions. Blood‐fed Rockefeller and wild‐type strain Aedes aegypti had higher levels of survival when compared with sucrose‐fed counterparts following exposure to fungus‐impregnated cloths. However, when blood‐fed A. aegypti were exposed to a combination of M. anisopliae + IMI, the survival rates were statistically equal to those of sucrose‐fed females. Large‐cage trials showed significant decreases in A. aegypti survival following a minimum 12 h exposure of the mosquitoes to fungus‐impregnated cloths. Increased exposure times results in further reductions in survival. The synergism between M. anisopliae and IMI resulted in reduced survival rates independent of feeding regime under laboratory conditions. Fungus‐impregnated cloths tested under simulated field conditions, considered to be unfavourable for fungal infection, resulted in significant reductions in adult A. aegypti survival. We are currently testing the combined use of fungi and insecticides against blood‐fed insects under simulated field conditions.

Significance and Impact of the Study

The use of fungus‐impregnated cotton cloths is a promising point source application method for the control of adult Aedes aegypti, and this strategy could be incorporated into an integrated vector management programme aiming to reduce the incidence of dengue fever.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti

BackgroundAedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results.Methodology/Principal findingsTo resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection.Conclusions/SignificanceMating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.     

Understanding the mechanism of Chikungunya virus vector competence in three species of mosquitoes

Chikungunya virus (CHIKV) is primarily transmitted by Aedes spp. mosquitoes. The present study investigated vector competence for CHIKV in Aedes aegypti and Aedes albopictus mosquitoes found in Madurai, South India. The role of receptor proteins on midguts contributing to permissiveness of CHIKV to Aedes spp. mosquitoes was also undertaken. Mosquitoes were orally infected with CHIKV DRDE‐06. Infection of midguts and dissemination to heads was confirmed by immunofluorescence assay at different time points. A plaque assay was performed from mosquito homogenates at different time points to study CHIKV replication. Presence of putative CHIKV receptor proteins on mosquito midgut epithelial cells was detected by virus overlay protein binding assay (VOPBA). The identity of these proteins was established using mass spectrometry. CHIKV infection of Ae. aegypti and Ae. albopictus midguts and dissemination to heads was observed to be similar. A plaque assay performed with infected mosquito homogenates revealed that CHIKV replication dynamics was similar in Aedes sp. mosquitoes until 28 days post infection. VOPBA performed with mosquito midgut membrane proteins revealed that prohibitin could serve as a putative CHIKV receptor on Aedes mosquito midguts, whereas an absence of CHIKV binding protein/s on Culex quinquefasciatus midguts can partially explain the non‐permissiveness of these mosquitoes to infection.     

Evidence for hormonal control of diuresis after a blood meal in the mosquito Aedes aegypti

In previous studies we have presented evidence for the role of peptides, isolated from heads of the mosquito Aedes aegypti, in stimulating fluid secretion by isolated Malpighian tubules. In the present study we conducted experiments to investigate whether these peptides are involved in hormone-mediated diuresis after a blood meal. In vivo experiments showed that the head was required to maintain diuresis after the blood meal. Whereas feeding on blood triggered a prompt diuresis in the intact mosquito, subsequent decapitation caused a gradual, not an abrupt, decline in urine excretion rate. Hemolymph collected from mosquitoes fed blood significantly stimulated fluid secretion in vitro by isolated Malpighian tubules, whereas hemolymph from unfed or blood-fed decapitated mosquitoes did not. These results indicate that a diuretic factor was released into the hemolymph after a blood meal. This factor was not present in the hemolymph of decapitated females. We identified the head as a source of diuretic factors. Peptides isolated from a head extract by high-performance liquid chromatography, when injected into the hemocoel of blood-fed decapitated mosquitoes, triggered diuresis in vivo and also stimulated fluid secretion in isolated Malpighian tubules. These studies support the hypothesis that the head is a storage site for diuretic peptides that may be released after a blood meal to control diuresis.