Artificial Blood Meals Research Articles

Experimental study of the susceptibility of a European Aedes albopictus strain to dengue virus under a simulated Mediterranean temperature regime.

Dengue virus (DENV) has re-emerged in Europe driven by the geographic expansion of the mosquito species Aedes albopictus and Aedes aegypti (Diptera: Culicidae) and the introduction of the virus by viraemic travellers. In the present study, the vector competence (VC) of Ae.albopictus collected in Catalonia (northeast Spain) was evaluated for two different DENV strains, DENV-1 and DENV-2, the serotypes responsible for all outbreaks of dengue that have occurred in Europe. Mosquitoes were reared under environmental conditions mimicking the mean temperature and humidity recorded in July on the Mediterranean coast of Catalonia. Mosquitoes were fed on an artificial infectious bloodmeal and, at 14 days post-exposure, infection, disseminated infection and transmission rates (IR, DIR, TR) and transmission efficiency (TE) were determined by testing the virus in the body, legs and saliva. The tested Ae.albopictus strain was found to be susceptible to both DENV-1 and DENV-2 and to be able to transmit DENV-1. This is the first time that the VC of Ae.albopictus for DENV has been tested in Europe in this specific context (i.e. mimicking the Mediterranean temperature and humidity recorded in Catalonia in July). This study confirms the potential of Ae.albopictus to start autochthonous DENV transmission cycles in the Mediterranean basin.

The Effect of SkitoSnack, an Artificial Blood Meal Replacement, on Aedes aegypti Life History Traits and Gut Microbiota

Public health research and vector control frequently require the rearing of large numbers of vector mosquitoes. All target vector mosquito species are anautogenous, meaning that females require vertebrate blood for egg production. Vertebrate blood, however, is costly, with a short shelf life. To overcome these constraints, we have developed SkitoSnack, an artificial blood meal replacement for the mosquito Aedes aegypti, the vector of dengue, Zika and chikungunya virus. SkitoSnack contains bovine serum albumin and hemoglobin as protein source as well as egg yolk and a bicarbonate buffer. SkitoSnack-raised females had comparable life history traits as blood-raised females. Mosquitoes reared from SkitoSnack-fed females had similar levels of infection and dissemination when orally challenged with dengue virus type 2 (DENV-2) and significantly lower infection with DENV-4. When SkitoSnack was used as a vehicle for DENV-2 delivery, blood-raised and SkitoSnack-raised females were equally susceptible. The midgut microbiota differed significantly between mosquitoes fed on SkitoSnack and mosquitoes fed on blood. By rearing 20 generations of Aedes exclusively on SkitoSnack, we have proven that this artificial diet can replace blood in mosquito mass rearing.

Competence of mosquitoes native to the United Kingdom to support replication and transmission of Rift Valley fever virus

BackgroundRift Valley fever phlebovirus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and livestock. It is endemic in Africa and spread to the Arabian Peninsula in 2000 raising concerns it could emerge in Europe. The ability of temperate mosquitoes from the United Kingdom (UK) to support replication and transmission of RVFV is unknown.MethodsIn this study, two colonised lines of Culex pipiens, wild-caught Aedes detritus and Ae. rusticus from the UK were infected with pathogenic strains of RVFV to assess their vector competence. Mosquitoes were offered artificial blood-meals containing 106 or 107 plaque forming units (PFU)/ml RVFV, simulating natural peak viraemia in young ruminants, and maintained at 20 °C or 25 °C for up to 21 days. Bodies, legs and saliva were collected and tested for the presence of viral RNA and infectious virus to determine the infection, dissemination and transmission potential.ResultsAcross temperatures, doses and strains the average infection, dissemination and transmission rates were: 35, 13 and 5% (n = 91) for Cx. pipiens (Caldbeck); 23, 14 and 5% (n = 138) for Cx. pipiens (Brookwood); 36, 28 and 7% (n = 118) for Ae. detritus. However, despite 35% (n = 20) being susceptible to infection, Ae. rusticus did not transmit RVFV. Survival of Aedes species was negatively affected by maintenance at 25 °C compared to the more representative peak average British summer temperature of 20 °C. Increased mortality was also observed with some species infected with 107 PFU/ml compared to 106 PFU/ml.ConclusionsIt can be concluded that temperate mosquito species present in the UK demonstrate a transmission potential for RVFV in the laboratory but, even at high temperatures, this occurred at low efficiency.

Correction: Dengue virus serotype 2 infection alters midgut and carcass gene expression in the Asian tiger mosquito, Aedes albopictus.

[This corrects the article DOI: 10.1371/journal.pone.0171345.].

Differential outcomes of Zika virus infection in Aedes aegypti orally challenged with infectious blood meals and infectious protein meals.

BackgroundInfection of mosquitoes is an essential step for the transmission of mosquito-borne arboviruses in nature. Engorgement of infectious blood meals from viremic infected vertebrate hosts allows the entry of viruses and initiates infection of midgut epithelial cells. Historically, the infection process of arboviruses in mosquitoes has been studied through the engorgement of mosquitoes from viremic laboratory animals or from artificial feeders containing blood mixed with viruses harvested from cell cultures. The latter approach using so-called artificial blood meals is more frequently used since it is readily optimized to maximize viral titer, negates the use of animals and can be used with viruses for which there are no small animal models. Use of artificial blood meals has enabled numerous studies on mosquito infections with a wide variety of viruses; however, as described here, with suitable modification it can also be used to study the interplay between infection, specific blood components, and physiological consequences associated with blood engorgement. For hematophagous female mosquitoes, blood is the primary nutritional source supporting all physiological process including egg development, and also influences neurological processes and behaviors such as host-seeking. Interactions between these blood-driven vector biological processes and arbovirus infection that is mediated via blood engorgement have not yet been specifically studied. This is in part because presentation of virus in whole blood inevitably induces enzymatic digestion processes, hormone driven oogenesis, and other biological changes. In this study, the infection process of Zika virus (ZIKV) in Aedes aegypti was characterized by oral exposure via viral suspension meals within minimally bovine serum albumin complemented medium or within whole blood. The use of bovine serum albumin in infectious meals provides an opportunity to evaluate the role of serum albumin during the process of flavivirus infection in mosquitoes.MethodsInfectious whole blood meals and infectious bovine serum albumin meals containing ZIKV were orally presented to two different groups of Ae. aegypti through membrane feeding. At 7 and 14 days post infection, infectious viruses were detected and viral dissemination from gut to other mosquito tissues was analyzed in orally challenged mosquitoes with 50% tissue culture infectious dose method on Vero76 cells.Results/ConclusionsZika virus infection was significantly impaired among mosquitoes orally challenged with infectious protein meals as compared to infectious whole blood meals. These results indicate the importance of the blood meal in the infection process of arboviruses in mosquitoes. It provides the basis for future studies to identify critical components in the blood of vertebrate hosts that facilitate arbovirus infection in mosquitoes.

Variation in Aedes aegypti Mosquito Competence for Zika Virus Transmission.

To test whether Zika virus has adapted for more efficient transmission by Aedes aegypti mosquitoes, leading to recent urban outbreaks, we fed mosquitoes from Brazil, the Dominican Republic, and the United States artificial blood meals containing 1 of 3 Zika virus strains (Senegal, Cambodia, Mexico) and monitored infection, dissemination, and virus in saliva. Contrary to our hypothesis, Cambodia and Mexica strains were less infectious than the Senegal strain. Only mosquitoes from the Dominican Republic transmitted the Cambodia and Mexica strains. However, blood meals from viremic mice were more infectious than artificial blood meals of comparable doses; the Cambodia strain was not transmitted by mosquitoes from Brazil after artificial blood meals, whereas 61% transmission occurred after a murine blood meal (saliva titers up to 4 log10 infectious units/collection). Although regional origins of vector populations and virus strain influence transmission efficiency, Ae. aegypti mosquitoes appear to be competent vectors of Zika virus in several regions of the Americas.

Efficacy of Blood Sources and Artificial Blood Feeding Methods in Rearing of Aedes aegypti (Diptera: Culicidae) for Sterile Insect Technique and Incompatible Insect Technique Approaches in Sri Lanka

Introduction Selection of the artificial membrane feeding technique and blood meal source has been recognized as key considerations in mass rearing of vectors. Methodology Artificial membrane feeding techniques, namely, glass plate, metal plate, and Hemotek membrane feeding method, and three blood sources (human, cattle, and chicken) were evaluated based on feeding rates, fecundity, and hatching rates of Aedes aegypti. Significance in the variations among blood feeding was investigated by one-way ANOVA, cluster analysis of variance (ANOSIM), and principal coordinates (PCO) analysis. Results Feeding rates of Ae. aegypti significantly differed among the membrane feeding techniques as suggested by one-way ANOVA (p < 0.05). The metal plate method was identified as the most efficient and cost-effective feeding technique. Blood feeding rate of Ae. aegypti was higher with human blood followed by cattle and chicken blood, respectively. However, no significant difference was observed from the mosquitoes fed with cattle and human blood, in terms of fecundity, oviposition rate, and fertility as suggested by one-way ANOVA (p > 0.05). Conclusions Metal plate method could be recommended as the most effective membrane feeding technique for mass rearing of Ae. aegypti, due to its high feeding rate and cost effectiveness. Cattle blood could be recommended for mass rearing Ae. aegypti.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25°C as well as 18°C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25°C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18°C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.

Analysis of CHIKV in Mosquitoes Infected via Artificial Blood Meal.

Having a mechanism to assess the transmission dynamics of a vector-borne virus is one critical component of understanding the life cycle of these viruses. Laboratory infection systems using artificial blood meals is one valuable approach for monitoring the progress of virus in its mosquito host and evaluating potential points for interruption of the cycle for control purposes. Here, we describe an artificial blood meal system with Chikungunya virus (CHIKV) and the processing of mosquito tissues and saliva to understand the movement and time course of virus infection in the invertebrate host.

Use of Centrifugal Filter Devices to Concentrate Dengue Virus in Mosquito per os Infection Experiments.

Dengue virus (DENV) is an arbovirus transmitted to humans by the bite of infected Aedes mosquitoes. Experimental per os infection of mosquitoes with DENV is usually a preliminary step in virus/vector studies but it requires being able to prepare artificial blood-meals with high virus titers. We report here the convenient use of centrifugal filter devices to quickly concentrate DENV particles in cell-culture supernatants. The median viral titer in concentrated-supernatants was 8.50 log10 TCID50/mL. By using these DENV concentrated-supernatants to prepare infectious blood-meals in Aedes aegypti per os infection experiments, we obtained a mean mosquito-infection rate of 94%. We also evaluated the use of centrifugal filter devices to recover DENV particles from non-infectious blood-meals presented to infected mosquitoes through a feeding membrane to collect their saliva.

Genetic determinants of differential oral infection phenotypes of West Nile and St. Louis encephalitis viruses in Culex spp. mosquitoes.

St. Louis encephalitis virus (SLEV) has shown greater susceptibility to oral infectivity than West Nile virus (WNV) in Culex mosquitoes. To identify the viral genetic elements that modulate these disparate phenotypes, structural chimeras (WNV-pre-membrane [prM] and envelope [E] proteins [prME]/SLEV.IC (infectious clone) and SLEV-prME/WNV.IC) were constructed in which two of the structural proteins, the prM and E, were interchanged between viruses. Oral dose-response assessment with the chimeric/parental WNV and SLEV was performed to characterize the infection phenotypes in Culex mosquitoes by artificial blood meals. The median infectious dose required to infect 50% of Cx. quinquefasciatus with WNV was indistinguishable from that of the SLEV-prME/WNV.IC chimeric virus. Similarly, SLEV and WNV-prME/SLEV.IC virus exhibited an indistinguishable oral dose-response relationship in Cx. quinquefasciatus. Infection rates for WNV.IC and SLEV-prME/WNV.IC were significantly lower than SLEV.IC and WNV-prME/SLEV.IC infection rates. These results indicated that WNV and SLEV oral infectivities are not mediated by genetic differences within the prM and E proteins.

Relative quantification of the effect of blood meal iron on protein expression in the ovaries of Aedes aegypti (778.8)

Diseases transmitted by mosquitoes are estimated to kill more than 2 million people annually. The vector for dengue, Aedes aegypti (yellow fever mosquito), is found in the desert Southwest of the continental United States. One approach to limiting the spread of disease is to reduce the numbers of mosquito vectors. Our area of research is iron metabolism. Our long term goal is to use molecular techniques to alter the expression of iron related proteins in mosquitoes to reduce mosquito fecundity and survival. Our hypothesis is that female mosquitoes require iron in the blood meal as an essential nutrient to aid in egg development. Although the iron is necessary for fecundity it is also highly oxidative and toxic in biological systems unless the mineral is strictly regulated to mitigate its oxidative potential. We are currently analyzing tissue‐specific, iron regulated proteins of the mosquito ovary 24 h post‐blood meal fed on artificial blood meal diets supplemented with no iron or low iron, or a porcine blood meal (high iron). Measurement of the blood meal iron effect on ovarian protein expression was carried out using Tandem Mass Tags (TMT) which permits simultaneous identification and relative quantification of proteins during LTQ Velos Orbitrap LC‐MS/MS analysis. This research will contribute to our understanding of mosquito development in general, as well as ovarian iron metabolism.Grant Funding Source: Supported by funds from the Arizona Ag Experiment Station and CALS

Vector competence of Aedes albopictus and Aedes aegypti (Diptera: Culicidae) for DEN2-43 and New Guinea C virus strains of dengue 2 virus

The vector competence of Aedes albopictus and Aedes aegypti with regard to DEN2-43 and New Guinea C (NGC) virus strains of Dengue 2 viruses was assessed and compared. The infection and dissemination rate and distribution of DEN2-43 antigens in orally infected Ae. albopictus was investigated using the reverse transcription polymerase chain reaction and an indirect immunofluorescence assay. To better understand the initial infection, dissemination and transmission of these viral strains in vector mosquitoes, Ae. albopoictus and Ae. aegypti were fed an artificial blood meal containing either the DEN2-43 or NGC strain. There was no significant difference in the infection and dissemination rates of DEN2-43 and NGC virus strains in Ae. albopictus, however, Ae. aegypti was more susceptible to infection by NGC than DEN2-43 vrius strain. Ae. albopictus mosquitoes infected with the NGC strain developed a higher percentage of midgut infections than those infected with the DEN2-43 strain (t=2.893, df=7, P=0.024). Approximately 26.7% of midgut samples were positive for the NGC antigen 5 days after infection, and 80% of mosquitoes had infected midgets after 15 days. The NGC antigen first became evident in mosquito salivary glands on Day 5, and 40% of mosquitoes had infected salivary by Day 9. In contrast, the DEN2-43 antigen first became evident in salivary glands on Day 7. The infection rate of NGC and DEN2-43 virus strains in salivary glands were similar. These results indicate that Ae. albopictus and Ae. aegypti are moderately competent vectors for the DEN2-43 virus, which could provide basic data for the epidemiology study of dengue fever in China.

Altered Response to DEET Repellent After Infection of Aedes aegypti (Diptera: Culicidae) With Sindbis Virus

To determine whether a Sindbis virus (family Togaviridae, genus Alphavirus, SINV) infection in Aedes aegypti (L.) (Diptera: Culicidae) affected its response to the repellent DEET, we orally exposed Ae. aegypti to an artificial bloodmeal containing SINV or diluent and then allowed to feed on a 10% sucrose suspension containing 3% DEET. When tested seven or more days after the initial bloodmeal, although none of the diluent-exposed mosquitoes fed on the DEET-sucrose suspension, at least 60% of the SINV-exposed mosquitoes fed on the suspension. When legs from the SINV-exposed mosquitoes were tested to determine dissemination status, 89% of those that fed on the DEET-sucrose suspension contained virus. In contrast, only 34% of the nonfeeders had a disseminated infection. When offered a choice between sucrose with or without DEET, a significantly higher percentage of the SINV-exposed mosquitoes than the control mosquitoes fed on the sucrose containing 3% DEET. Together, these results indicate that mosquitoes with a disseminated SINV infection may be less responsive to DEET than uninfected mosquitoes. Therefore, repellent use may be less effective in deterring infected mosquitoes from biting than previously believed.

Experimental Studies on Comparison of the Potential Vector Competence of Four Species of &lt;I&gt;Culex&lt;/I&gt; Mosquitoes in China to Transmit West Nile Virus

To assess the risk that indigenous mosquitoes in China are capable of transmitting and sustaining West Nile virus (WNV), four important Culex mosquito species, Culex tritaeniorhynchus, Culex modestus, Culex pipiens pallens, and Culex pipiens quinquefasciatus, were allowed to feed on the artificial infectious blood meal with WNV dose of 10(6.8) plaque-forming unit/ml and tested approximately 2 wk later to determine infection and transmission rates. The results indicated that four Culex mosquitoes were competent laboratory vectors of WNV. The infection rates and transmission rates were statistical differences among different species of mosquito (chi2 = 20.620, P = 0.000; chi2 = 15.020, P = 0.005, respectively). The highest infection rate and transmission rate were obtained with Cx. tritaeniorhynchus (87.5 and 74.2%, respectively).

Experimental Studies on Comparison of the Potential Vector Competence of Four Species ofCulexMosquitoes in China to Transmit West Nile Virus : Table 1

To assess the risk that indigenous mosquitoes in China are capable of transmitting and sustaining West Nile virus (WNV), four important Culex mosquito species, Culex tritaeniorhynchus, Culex modestus, Culex pipiens pallens, and Culex pipiens quinquefasciatus, were allowed to feed on the artificial infectious blood meal with WNV dose of 106.8 plaque-forming unit/ml and tested ≈2 wk later to determine infection and transmission rates. The results indicated that four Culex mosquitoes were competent laboratory vectors of WNV. The infection rates and transmission rates were statistical differences among different species of mosquito (χ2 = 20.620, P = 0.000; χ2 = 15.020, P = 0.005, respectively). The highest infection rate and transmission rate were obtained with Cx. tritaeniorhynchus (87.5 and 74.2%, respectively).

The impact of larval and adult dietary restriction on lifespan, reproduction and growth in the mosquito Aedes aegypti

Dietary restriction extends lifespan in many organisms, but little is known about how it affects hematophagous arthropods. We demonstrated that diet restriction during either larval or adult stages extends Aedes aegypti lifespan. A. aegypti females fed either single or no blood meals survived 30–40% longer than those given weekly blood meals. However, mosquitoes given weekly blood meals produced far more eggs. To minimize reproduction's impact on lifespan, adult mosquitoes were fed artificial blood meals containing < 10% of the protein in normal human blood, minimizing egg production. A. aegypti fed artificial blood meals containing 25 mg/ml of BSA had significantly shorter lifespans than those fed either 10 or 5 mg/ml. To assess the impact of larval dietary restriction on adult lifespan, we maintained larval A . aegypti on 2X, 1X (normal diet), 0.5X or 0.25X diets. Adult mosquitoes fed 0.5X and 0.25X larval diets survived significantly longer than those fed the 2X larval diet regardless of adult diet. In summary, dietary restriction during both larval and adult stages extends lifespan. This diet-mediated lifespan extension has important consequences for understanding how dietary restriction regulates lifespan and disease transmission.

Identification and characterization of a novel ferritin subunit from Aedes aegypti

Mosquitoes consume and absorb significant amount of iron from a blood meal. Ferritin is a protein that is used to store iron. Iron is necessary for egg development in mosquitoes. We wanted to examine the proteome of ovaries from Aedes aegypti (yellow fever mosquito) that were fed an artificial blood meal with or without hemoglobin. The number of proteins identified in ovaries from mosquitoes that were fed an artificial blood meal with or without hemoglobin was 48 and 13 respectively. A previously uncharacterized glycosylated ferritin light chain homologue (G‐LCH) was found in ovaries of mosquitoes that were fed hemoglobin rich diet. Primers were designed specifically for G‐LCH to perform PCR and real‐time PCR. G‐LCH ferritin was cloned and sequenced. Real‐time PCR showed that eggs, larvae and pupae have similar expression levels of G‐LCH. Males and 72h post blood fed (PBF) females have higher expression levels of this message. Real‐time PCR will be performed on tissues of 24h and 72h PBF mosquitoes. The 5’‐UTR region of G‐LCH mRNA will be examined using rapid amplification of cDNA ends to determine if stem loop moieties are present. Characterization of G‐LCH subunit will give further insights into the unique regulation of iron metabolism in mosquitoes. Research support was from NIH(GM056812), the Agricultural Experiment Station and CALS at the University of Arizona.

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.

Susceptibility of &lt;I&gt;Ae. aegypti&lt;/I&gt; (Diptera: Culicidae) to Infection with Epidemic (Subtype IC) and Enzootic (Subtypes ID, IIIC, IIID) Venezuelan Equine Encephalitis Complex Alphaviruses

To test the hypothesis that enzootic and epidemic Venezuelan equine encephalitis (VEE) complex alphaviruses can infect and be transmitted by Ae. aegypti, we conducted a series of experimental infection studies. One set of experiments tested the susceptibility of geographic strains of Ae. aegypti from Peru and Texas (U.S.A.) for epidemic (subtype IC) and enzootic (subtype ID) strains from Colombia/Venezuela, whereas the second set of experiments tested the susceptibility of Ae. aegypti from Iquitos, Peru, to enzootic VEE complex strains (subtypes ID, IIIC, and IIID) isolated in the same region, at different infectious doses. Experimental infections using artificial bloodmeals suggested that Ae. aegypti mosquitoes, particularly the strain from Iquitos, Peru, is moderately to highly susceptible to all of these VEE complex alphaviruses. The occurrence of enzootic VEE complex viruses circulating endemically in Iquitos suggests the possibility of a dengue-like transmission cycle among humans in tropical cities.