Structural insights into the Aedes aegypti aquaporins and aquaglyceroporins – an in silico study

There has been a fair bit of understanding on the structure–function relationship of Aquaporins (AQPs) from plants and vertebrates obtained from available X-ray crystallography data. However, there is a lacuna in understanding the structure of AQPs from sanguinivorous insects like the mosquito where it plays a crucial role in survival. In this study, we have built homology models for the Aedes aegypti AQPs, identified key channel lining residues and compared the structure and sequence with orthodox AQPs. Although Ar/R filter residues of AaAQP1 were exactly similar to orthodox AQPs, AaAQP2 has a substitution at LE1position possibly making it less efficient in high capacity water transport. The huge difference in the selectivity filter region of AaAQP3 suggests a different transport property for this channel. The changes observed in the H5 position of the filter of AaAQP4 and AaAQP5 may explain the presence of a larger pore aperture to permit the passage of larger solute molecules. AaAQP6 possesses a completely hydrophobic filter like that in mammalian super aquaporins. The identified key residues are pivotal in understanding the mechanism of action and gating of these channels.     

Blood‐feeding and immunogenic Aedes aegypti saliva proteins

Mosquito‐transmitted pathogens pass through the insect's midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar‐fed and blood‐fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar‐ or blood‐based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar‐fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up‐regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2‐DE profiles of immunogenic Ae. aegypti SG BF‐related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.     

Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation

Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black mMexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P_f) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.Key words: aquaporin, suspension cultured cells, hetero-oligomerization, maize, plasma membrane intrinsic protein, protein interaction, water movement     

1,3‐propanediol binds deep inside the channel to inhibit water permeation through aquaporins

Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all‐atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3‐propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro‐homeostasis indirectly by altering the osmotic gradients.     

Wolbachia enhances insect‐specific flavivirus infection in Aedes aegypti mosquitoes

Mosquitoes transmit a diverse group of human flaviviruses including West Nile, dengue, yellow fever, and Zika viruses. Mosquitoes are also naturally infected with insect‐specific flaviviruses (ISFs), a subgroup of the family not capable of infecting vertebrates. Although ISFs are not medically important, they are capable of altering the mosquito's susceptibility to flaviviruses and may alter host fitness. Wolbachia is an endosymbiotic bacterium of insects that when present in mosquitoes limits the replication of co‐infecting pathogens, including flaviviruses. Artificially created Wolbachia‐infected Aedes aegypti mosquitoes are being released into the wild in a series of trials around the globe with the hope of interrupting dengue and Zika virus transmission from mosquitoes to humans. Our work investigated the effect of Wolbachia on ISF infection in wild‐caught Ae. aegypti mosquitoes from field release zones. All field mosquitoes were screened for the presence of ISFs using general degenerate flavivirus primers and their PCR amplicons sequenced. ISFs were found to be common and widely distributed in Ae. aegypti populations. Field mosquitoes consistently had higher ISF infection rates and viral loads compared to laboratory colony material indicating that environmental conditions may modulate ISF infection in Ae. aegypti. Surprisingly, higher ISF infection rates and loads were found in Wolbachia‐infected mosquitoes compared to the Wolbachia‐free mosquitoes. Our findings demonstrate that the symbiont is capable of manipulating the mosquito virome and that Wolbachia‐mediated viral inhibition is not universal for flaviviruses. This may have implications for the Wolbachia‐based DENV control strategy if ISFs confer fitness effects or alter mosquito susceptibility to other flaviviruses.     

Vector competence and innate immune responses to dengue virus infection in selected laboratory and field‐collected Stegomyia aegypti (= Aedes aegypti)

Control of dengue virus (DenV) transmission, primarily based on strategies to reduce populations of the principle vector Stegomya aegypti (= Aedes aegypti) (Diptera: Culicidae), is difficult to sustain over time. Other potential strategies aim to manipulate characteristics such as vector competence (VC), the innate capacity of the vector to transmit the virus. Previous studies have identified genetic factors, including differential expression of apoptosis‐related genes, associated with the refractory and susceptible phenotypes in selected strains of S. aegypti from Cali, Colombia. The present study was designed to evaluate the variability of VC in selected strains against different DenV serotypes and to determine whether field‐collected mosquitoes respond similarly to selected laboratory strains in terms of enhanced or reduced expression of apoptosis‐related genes. Vector competence differed between strains, but did not differ in response to different DenV serotypes. Differences in VC were observed among mosquitoes collected from different localities in Cali. The overexpression of the pro‐apoptosis genes, caspase 16 and Aedronc, was conserved in field‐collected refractory mosquitoes and the selected laboratory refractory strain. The results suggest that the apoptosis response is conserved among all refractory mosquitoes to inhibit the development of all DenV serotypes.     

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.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.     

Multi‐scale analysis of the associations among egg,larval and pupal surveys and the presence and abundance of adult female Aedes aegypti (Stegomyia aegypti) in the city of Merida,Mexico

Despite decades of research, there is still no agreement on which indices of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) presence and abundance better quantify entomological risk for dengue. This study reports the results of a multi‐scale, cross‐sectional entomological survey carried out in 1160 households in the city of Merida, Mexico to establish: (a) the correlation between levels of Ae. aegypti presence and abundance detected with aspirators and ovitraps; (b) which immature and egg indices correlate with the presence and abundance of Ae. aegypti females, and (c) the correlations amongst traditional Aedes indices and their modifications for pupae at the household level and within medium‐sized geographic areas used for vector surveillance. Our analyses show that ovitrap positivity was significantly associated with indoor adult Ae. aegypti presence [odds ratio (OR) = 1.50; P = 0.03], that the presence of pupae is associated with adult presence at the household level (OR = 2.27; P = 0.001), that classic Aedes indices are informative only when they account for pupae, and that window screens provide a significant level of protection against peridomestic Ae. aegypti (OR = 0.59; P = 0.02). Results reinforce the potential of using both positive collections in outdoor ovitraps and the presence of pupae as sensitive indicators of indoor adult female presence.     

Wolbachia pipientis occurs in Aedes aegypti populations in New Mexico and Florida,USA

The mosquitoes Aedes aegypti (L.) and Ae. albopictus Skuse are the major vectors of dengue, Zika, yellow fever, and chikungunya viruses worldwide. Wolbachia, an endosymbiotic bacterium present in many insects, is being utilized in novel vector control strategies to manipulate mosquito life history and vector competence to curb virus transmission. Earlier studies have found that Wolbachia is commonly detected in Ae. albopictus but rarely detected in Ae. aegypti. In this study, we used a two‐step PCR assay to detect Wolbachia in wild‐collected samples of Ae. aegypti. The PCR products were sequenced to validate amplicons and identify Wolbachia strains. A loop‐mediated isothermal amplification (LAMP) assay was developed and used for detecting Wolbachia in selected mosquito specimens as well. We found Wolbachia in 85/148 (57.4%) wild Ae. aegypti specimens from various cities in New Mexico, and in 2/46 (4.3%) from St. Augustine, Florida. Wolbachia was not detected in 94 samples of Ae. aegypti from Deer Park, Harris County, Texas. Wolbachia detected in Ae. aegypti from both New Mexico and Florida was the wAlbB strain of Wolbachia pipientis. A Wolbachia‐positive colony of Ae. aegypti was established from pupae collected in Las Cruces, New Mexico, in 2018. The infected females of this strain transmitted Wolbachia to their progeny when crossed with males of Rockefeller strain of Ae. aegypti, which does not carry Wolbachia. In contrast, none of the progeny of Las Cruces males mated to Rockefeller females were infected with Wolbachia.     

Pre‐treatment of Stegomyia aegypti mosquitoes with a sublethal dose of imidacloprid impairs behavioural avoidance induced by lemon oil and DEET

The present study was conducted to determine whether imidacloprid can impair the avoidance behaviour of the mosquito Stegomyia aegypti. Laboratory investigations using a T‐maze apparatus showed that St. aegypti mosquitoes present long term avoidance behaviour when they are exposed to repetitive trials with lemon oil and DEET. The present study tested the effect of a sublethal dose of imidacloprid on the avoidance behaviour of St. aegypti mosquitoes over a 48 h period. Data suggest that 0.5 ng of imidacloprid/mosquito reduces the avoidance behaviour of mosquitoes exposed to lemon oil, on the first day of exposure, after the second trial; whereas imidacloprid affected DEET repellency only the first day of exposure, after the second trial. Imidacloprid was toxic against St. aegypti mosquitoes, and at sublethal doses was able to impair the repellency induced by lemon oil and DEET. The present data were consistent with the finding that St. aegypti mosquitoes exhibit long term avoidance behaviour, and treatment of mosquitoes with a sublethal dose of imidacloprid under DEET application can affect the repellency of DEET against St. aegypti.     

The potential of Metarhizium anisopliae and Beauveria bassiana isolates for the control of Aedes aegypti (Diptera: Culicidae) larvae

Fungal isolates were screened against Aedes aegypti larvae. Exposure of larvae to conidial suspensions resulted in 6–90% mortality. An inoculum persistence assay using one of the most virulent isolates showed an approximate half-life of 10 days in water containers. Not all larvae surviving to form pupae resulted in adult emergence.     

Evaluation of collection methods for Culex quinquefasciatus,Aedes aegypti,and Aedes simpsoni in northeastern Tanzania

In East Africa, significant morbidity and mortality are caused by infections spread by Culex quinquefasciatus and Aedes aegypti. Sticky traps have been shown to be effective tools for sampling populations of Aedes mosquitoes and have been found to catch Cx. quinquefasciatus. Thus, they could potentially be used to sample populations of this species. This study compared Sticky ovitraps (SO) and MosquiTraps (MQT) with the CDC Gravid trap (CDC‐GT) for collection of Culex and Aedes mosquito populations in Tanzania. A follow‐up experiment was carried out using traps set for a 24‐h period to accommodate the oviposition habits of Aedes aegypti and Ae. simpsoni s.l. mosquitoes. The results showed that the CDC‐GT caught significantly more Cx. quinquefasciatus and Ae. aegypti than the SO or MQT, but there was no significant difference in the number of mosquitoes caught between the two sticky traps or of Ae. simpsoni s.l. caught among the three trap types. The results suggest that CDC‐GTs are the most appropriate in sampling of Cx. quinquefasciatus. Although CDC‐GTs collected more Ae. aegypti than the sticky traps, the simplicity and cost benefit of sticky traps facilitates large scale studies. All three trap types should be considered for monitoring Aedes mosquitoes.