Mosquitoes rely on their gut microbiota for development

Field studies indicate adult mosquitoes (Culicidae) host low diversity communities of bacteria that vary greatly among individuals and species. In contrast, it remains unclear how adult mosquitoes acquire their microbiome, what influences community structure, and whether the microbiome is important for survival. Here, we used pyrosequencing of 16S rRNA to characterize the bacterial communities of three mosquito species reared under identical conditions. Two of these species, Aedes aegypti and Anopheles gambiae, are anautogenous and must blood‐feed to produce eggs, while one, Georgecraigius atropalpus, is autogenous and produces eggs without blood feeding. Each mosquito species contained a low diversity community comprised primarily of aerobic bacteria acquired from the aquatic habitat in which larvae developed. Our results suggested that the communities in Ae. aegypti and An. gambiae larvae share more similarities with one another than with G. atropalpus. Studies with Ae. aegypti also strongly suggested that adults transstadially acquired several members of the larval bacterial community, but only four genera of bacteria present in blood fed females were detected on eggs. Functional assays showed that axenic larvae of each species failed to develop beyond the first instar. Experiments with Ae. aegypti indicated several members of the microbial community and Escherichia coli successfully colonized axenic larvae and rescued development. Overall, our results provide new insights about the acquisition and structure of bacterial communities in mosquitoes. They also indicate that three mosquito species spanning the breadth of the Culicidae depend on their gut microbiome for development.     

Human skin microbiota and their volatiles as odour baits for the malaria mosquito Anopheles gambiae s.s

Host seeking by the malaria mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae) is mainly guided by volatile chemicals present in human odours. The skin microbiota plays an important role in the production of these volatiles, and skin bacteria grown on agar plates attract An. gambiae s.s. in the laboratory. In this study, the attractiveness of volatiles produced by human skin bacteria to An. gambiae s.s. was tested in laboratory, semi‐field, and field experiments to assess these effects in increasing environmental complexity. A synthetic blend of 10 compounds identified in the headspace of skin bacteria was also tested for its attractiveness. Carbon dioxide significantly increased mosquito catches of traps baited with microbial volatiles in the semi‐field experiments and was therefore added to the field traps. Traps baited with skin bacteria caught significantly more An. gambiae s.s. than control traps, both in the laboratory and semi‐field experiments. Traps baited with the synthetic blend caught more mosquitoes than control traps in the laboratory experiments, but not in the semi‐field experiments. Although bacterial volatiles increased mosquito catches in the field study, trapping several mosquito vector species, these effects were not significant for An. gambiae s.l. It is concluded that volatiles from skin bacteria affect mosquito behaviour under laboratory and semi‐field conditions and, after fine tuning, have the potential to be developed as odour baits for mosquitoes.     

Comparative analysis of midgut bacterial communities in three aedine mosquito species from dengue‐endemic and non‐endemic areas of Rajasthan,India

Dengue viruses are transmitted to humans through the bites of infected female aedine mosquitoes. Differences in the composition and structure of bacterial communities in the midguts of mosquitoes may affect the vector's ability to transmit the disease. To investigate and analyse the role of midgut bacterial communities in viral transmission, midgut bacteria from three species, namely Stegomyia aegypti (= Aedes aegypti), Fredwardsius vittatus (= Aedes vittatus) and Stegomyia albopicta (= Aedes albopictus) (all: Diptera: Culicidae), from dengue‐endemic and non‐endemic areas of Rajasthan, India were compared. Construction and analyses of six 16S rRNA gene libraries indicated that Serratia spp.‐related phylotypes dominated all clone libraries of the three mosquito species from areas in which dengue is not endemic. In dengue‐endemic areas, phylotypes related to Aeromonas, Enhydrobacter spp. and uncultivated bacterium dominated the clone libraries of S. aegypti, F. vittatus and S. albopicta, respectively. Diversity indices analysis and real‐time TaqMan polymerase chain reaction assays showed bacterial diversity and abundance in the midguts of S. aegypti to be higher than in the other two species. Significant differences observed among midgut bacterial communities of the three mosquito species from areas in which dengue is and is not endemic, respectively, may be related to the vectorial capacity of mosquitoes to carry dengue viruses and, hence, to the prevalence of disease in some areas.     

Assessing the efficacy of candidate mosquito repellents against the background of an attractive source that mimics a human host

Mosquito repellents are used around the globe to protect against nuisance biting and disease‐transmitting mosquitoes. Recently, there has been renewed interest in the development of repellents as tools to control the transmission of mosquito‐borne diseases. We present a new bioassay for the accurate assessment of candidate repellent compounds, using a synthetic odour that mimics the odour blend released by human skin. Using DEET (N,N‐diethyl‐meta‐toluamide) and PMD (p‐menthane‐3,8‐diol) as reference compounds, nine candidate repellents were tested, of which five showed significant repellency to the malaria mosquito Anopheles gambiae sensu stricto (Diptera: Culicidae). These included: 2‐nonanone; 6‐methyl‐5‐hepten‐2‐one; linalool; δ‐decalactone, and δ‐undecalactone. The lactones were also tested on the yellow fever mosquito Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae), against which they showed similar degrees of repellency. We conclude that the lactones are highly promising repellents, particularly because these compounds are pleasant‐smelling, natural products that are also present in human food sources.     

Variation in host preferences of malaria mosquitoes is mediated by skin bacterial volatiles

The host preferences of the anthropophilic mosquito species in the Anopheles gambiae complex (Diptera: Culicidae) are mediated by skin bacterial volatiles. However, it is not known whether these mosquitoes respond differentially to skin bacterial volatiles from non‐human host species. In this study, the responses of two malaria mosquito species in the An. gambiae complex, Anopheles gambiae s.s. (hereafter, An. gambiae) and Anopheles arabiensis, with different host preferences, to volatiles released from skin bacteria were tested. Skin bacteria collected from human, cow and chicken skin significantly increased trap catches; traps containing bacteria collected from human skin caught the highest proportions of An. gambiae and An. arabiensis. Traps with bacteria of human origin caught a significantly higher proportion of An. gambiae than of An. arabiensis, whereas bacterial volatiles from the chicken attracted significantly higher numbers of An. arabiensis than of An. gambiae. Additionally, An. gambiae showed a specialized response to volatiles from four specific bacteria, whereas An. arabiensis responded equally to all species of bacteria tested. Skin bacterial volatiles may therefore play important roles in guiding mosquitoes with different host preferences. The identification of these bacterial volatiles can contribute to the development of an odour blend that attracts mosquitoes with different host preferences.     

Culex torrentium mosquitoes from Germany are negative for Wolbachia

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high‐throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia‐induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.     

Detection of Orthobunyavirus in mosquitoes collected in Argentina

Bunyamwera virus (BUNV) (Bunyaviridae, genus Orthobunyavirus, serogroup Bunyamwera) is considered an emerging pathogen for humans and animals in American countries. The CbaAr‐426 strain of BUNV was recovered from mosquitoes Ochlerotatus albifasciatus (Diptera: Culicidae) collected in Córdoba province (Argentina), where serological studies detected high seroprevalences in humans and animals. Molecular detection of Orthobunyavirus was performed in mosquitoes collected in Córdoba province. Seventeen mosquito pools of Oc. albifasciatus, Ochlerotatus scapularis and Culex quinquefasciatus (Diptera: Culicidae) showed positive results; four of these positive pools, all of Oc. scapularis, were sequenced. All amplicons grouped with BUNV in the Bunyamwera serogroup. The findings highlight the circulation of BUNV in Córdoba province and represent the first report of BUNV‐infected Oc. scapularis mosquitoes in Argentina.     

Evaluation of a temperate climate mosquito,Ochlerotatus detritus (=Aedes detritus), as a potential vector of Japanese encephalitis virus

The U.K. has not yet experienced a confirmed outbreak of mosquito‐borne virus transmission to people or livestock despite numerous autochthonous epizootic and human outbreaks of mosquito‐borne diseases on the European mainland. Indeed, whether or not British mosquitoes are competent to transmit arboviruses has not been established. Therefore, the competence of a local (temperate) British mosquito species, Ochlerotatus detritus (=Aedes detritus) (Diptera: Culicidae) for transmission of a member of the genus Flavivirus, Japanese encephalitis virus (JEV) as a model for mosquito‐borne virus transmission was assessed. The JEV competence in a laboratory strain of Culex quinquefasciatus (Diptera: Culicidae), a previously incriminated JEV vector, was also evaluated as a positive control. Ochlerotatus detritus adults were reared from field‐collected juvenile stages. In oral infection bioassays, adult females developed disseminated infections and were able to transmit virus as determined by the isolation of virus in saliva secretions. When pooled at 7–21 days post‐infection, 13% and 25% of O. detritus were able to transmit JEV when held at 23 °C and 28 °C, respectively. Similar results were obtained for C. quinquefasciatus. To our knowledge, this study is the first to demonstrate that a British mosquito species, O. detritus, is a potential vector of an exotic flavivirus.     

Mosquito vector‐associated microbiota: Metabarcoding bacteria and eukaryotic symbionts across habitat types in Thailand endemic for dengue and other arthropod‐borne diseases

Vector‐borne diseases are a major health burden, yet factors affecting their spread are only partially understood. For example, microbial symbionts can impact mosquito reproduction, survival, and vectorial capacity, and hence affect disease transmission. Nonetheless, current knowledge of mosquito‐associated microbial communities is limited. To characterize the bacterial and eukaryotic microbial communities of multiple vector species collected from different habitat types in disease endemic areas, we employed next‐generation 454 pyrosequencing of 16S and 18S rRNA amplicon libraries, also known as metabarcoding. We investigated pooled whole adult mosquitoes of three medically important vectors, Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, collected from different habitats across central Thailand where we previously characterized mosquito diversity. Our results indicate that diversity within the mosquito microbiota is low, with the majority of microbes assigned to one or a few taxa. Two of the most common eukaryotic and bacterial genera recovered (Ascogregarina and Wolbachia, respectively) are known mosquito endosymbionts with potentially parasitic and long evolutionary relationships with their hosts. Patterns of microbial composition and diversity appeared to differ by both vector species and habitat for a given species, although high variability between samples suggests a strong stochastic element to microbiota assembly. In general, our findings suggest that multiple factors, such as habitat condition and mosquito species identity, may influence overall microbial community composition, and thus provide a basis for further investigations into the interactions between vectors, their microbial communities, and human‐impacted landscapes that may ultimately affect vector‐borne disease risk.     

Modified veranda‐trap hut for improved evaluation of vector control interventions

Experimental huts with veranda traps have been used in Tanzania since 1963 for the study of residual insecticides for use with insecticide‐treated nets and indoor residual spraying. Mosquitoes are allowed unrestricted entry through the eaves to facilitate the collection of an estimable proportion of mosquitoes that attempt to exit through the eave gaps, which are left open on two sides of the hut. This study was designed to validate the use of eave baffles to funnel entry and to prevent mosquito escape, and to determine biting times of Anopheles arabiensis (Patton) (Diptera: Culicidae). Anopheles arabiensis and Culex quinquefasciatus (Say) (Diptera: Culicidae) were released into the room at 20.30 hours and collected the following morning from veranda traps, window traps and the room. Centers for Disease Control light traps hung overnight next to volunteers were emptied every 2 h to determine peak biting times. A total of 55% of An. arabiensis were trapped before 22.30 hours and the highest peak in ‘biting’ was recorded during 18.30–20.30 hours. Of the released An. arabiensis that exited into veranda traps, 7% were captured in veranda traps entered through baffles and 93% were captured in traps entered through unmodified eaves. When veranda screens were left open to allow for escape outdoors, recapture rates were 68% for huts with eave baffles and 39% for huts with unmodified eaves. The comparison of open eaves with baffled eaves validated the assumption that in huts of the traditional non‐baffled design, 50% of mosquitoes escape through open eaves. Eave baffles succeeded in reducing the potential for mosquito exit and produced more precise estimates of effect.     

Assessing natural infection with Zika virus in the southern house mosquito,Culex quinquefasciatus,during 2016 in Puerto Rico

The epidemic of Zika in the Western hemisphere has led to intense investigations of all species important in the transmission of Zika virus (ZikV), including putative mosquito vectors. Although evidence points to Stegomyia (= Aedes) (Diptera: Culicidae) mosquitoes as the primary vectors in nature among humans, there remains the possibility that other common mosquito species may be implicated in the rapid spread of the virus. Herein, field‐caught Culex quinquefasciatus (Diptera: Culicidae) collected during June 2016 in different neighbourhoods in San Juan, Puerto Rico were examined for the presence of natural infection with ZikV. Stegomyia aegypti (= Aedes aegypti) from the same locations were also analysed. None of the Cx. quinquefasciatus tested showed natural infection for ZikV, whereas S. aegypti tested positive at seven sites. The present results suggest that Cx. quinquefasciatus was not involved in the transmission of ZikV in San Juan, Puerto Rico in 2016.     

Mosquitoes and other aquatic insects in fallow field biotopes and rice paddy fields

Fallow field biotopes that develop from abandoned rice fields are man‐made wetlands that provide new habitats for various aquatic animals. Although consideration of such biotopes generally focuses on their positive aspects, this study evaluated the negative aspects of establishing fallow field biotopes with regard to mosquito breeding sites. To determine whether fallow field biotopes become breeding habitats for vector mosquitoes, we evaluated mosquito fauna in fallow field biotopes and adjacent rice fields. We found larvae of Anopheles lesteri, Anopheles sinensis and Culex tritaeniorhynchus (all: Diptera: Culicidae) in the biotopes. Although abundances of mosquito larvae in the biotopes and rice fields were statistically similar, mosquito abundances in rice fields increased dramatically in August when the water level reduced after the rainy season. The abundance and variety of the mosquitoes' natural predators were greater in biotopes than in rice fields because the former are a permanent and stable aquatic environment. A generalized linear mixed model showed a negative effect of predator diversity on mosquito larvae abundance in both habitats. Although fallow field biotopes become breeding habitats for vector mosquitoes, establishing biotopes from fallow fields in order to protect various aquatic animals, including mosquito insect predators, may help to control mosquito breeding.     

Culicoides biting midges and mosquito fauna at three dog and cat shelters in rural and periurban areas in Northern Spain

The diversity and abundance of Culicoides (Diptera: Ceratopogonidae) and mosquitoes (Diptera: Culicidae) were studied in three animal protection centres (APCs) in Northern Spain between 1 July and 31 October 2018. Four miniature suction CDC light traps (two UV and two standard incandescent bulb traps, both types baited and non‐baited with CO₂) were placed in each APC to compare their efficiency in the collection of these Diptera groups. A total of 1176 biting midges (14 species), 224 mosquitoes (8 species) and 1 black fly were collected and identified by both morphological and molecular approaches. The Culicoides obsoletus complex (C. obsoletus/C. scoticus) accounted for 58.2% of the total collection within the Ceratopogonidae family, whereas Culex pipiens/Cx. torrentium comprised 76.8% of the Culicidae. The input of CO₂ in light traps proved largely ineffective in improving the collections of both Diptera groups. UV‐light traps were 7.8 and 2.2 times more effective than incandescent light traps in trapping Culicoides and mosquitoes, respectively. Seasonal dynamics differed between both Diptera taxa but captures of both taxa were significantly larger at the beginning of the summer. The epidemiological relevance of the most prevalent species is also discussed.     

Insecticide susceptibility status in Mexican populations of Stegomyia aegypti (= Aedes aegypti): a nationwide assessment

In Mexico, mosquito vector‐borne diseases are of public health concern as a result of their impact on human morbidity and mortality. The use of insecticides against adult mosquitoes is one of the most common ways of controlling mosquito population densities. However, the use of these compounds has resulted in the development of insecticide resistance. The aim of this study was to estimate susceptibility to six pyrethroids, two carbamates and two organophosphates in Mexican populations of Stegomyia aegypti (Linnaeus, 1762) (= Aedes aegypti) (Diptera: Culicidae) mosquitoes. Bottle insecticide susceptibility tests, with 1 h exposure, were performed on adult mosquitoes from 75 localities across 28 states. At 30 min of exposure, the proportion of fallen mosquitoes was recorded. After 60 min of exposure, mosquitoes were recovered in non‐treated containers and mortality was determined at 24 h after the set‐up of the experiment. In general, the carbamate insecticides represented the most effective group in terms of the proportion of mosquitoes fallen at 30 min (72–100%) and 24‐h mortality (97–100%). High and widespread resistance to pyrethroids Types I and II and, to a lesser extent, to organophosphates was observed. Insecticide susceptibility among and within states was highly variable.     

Population structure of the mosquito Aedes aegypti (Stegomyia aegypti) in Pakistan

Eleven microsatellite markers were used to determine the genetic population structure and spread of Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) in Pakistan using mosquitoes collected from 13 different cities. There is a single genetic cluster of Ae. aegypti in Pakistan with a pattern of isolation by distance within the population. The low level of isolation by distance suggests the long‐range passive dispersal of this mosquito, which may be facilitated by the tyre trade in Pakistan. A decrease in genetic diversity from south to north suggests a recent spread of this mosquito from Karachi. A strong negative correlation between genetic distance and the quality of road connections shows that populations in cities connected by better road networks are less differentiated, which suggests the human‐aided passive dispersal of Ae. aegypti in Pakistan. Dispersal on a large spatial scale may facilitate the strategy of introducing transgenic Ae. aegypti or intracellular bacteria such as Wolbachia to control the spread of dengue disease in Pakistan, but it also emphasizes the need for simple measures to control container breeding sites.     

An updated checklist of the Culicidae (Diptera) of Morocco,with notes on species of historical and current medical importance

An updated checklist of the mosquito species (Diptera: Culicidae) recorded in Morocco from 1916 to 2016 is provided, including synonyms and synonymous usage for each species. Forty‐three species belonging to seven genera are recorded so far: Anopheles (9), Aedes (12) Coquillettidia (2), Culex (12), Culiseta (5), Orthopodomyia (1) and Uranotaenia (2). Traditional and equivalent names in the polyphyletic concept of Aedes are provided for the aedine species. The historical importance and current potential threat of mosquitoes to human health in Morocco is reviewed.     

Mosquito and West Nile virus surveillance in northeast Montana,U.S.A., 2005 and 2006

Mosquito and West Nile virus (WNV) surveillance was conducted on a national wildlife refuge in northeast Montana in 2005 and 2006, during which outbreaks of WNV in a colony of American white pelicans (Pelecanus erythrorhynchos Gmelin) (Pelecaniformes: Pelecanidae) resulted in juvenile mortality rates of ～ 31%. Both years, floodwater species Ochlerotatus dorsalis (Meigen) (Diptera: Culicidae), Aedes vexans (Meigen) (Diptera: Culicidae) and Ochlerotatus flavescens (Muller) (Diptera: Culicidae) comprised 78% of the total collection and heightened host‐seeking activity was observed from mid‐June to mid‐July. Culex tarsalis Coquillett (Diptera: Culicidae) was most active from mid‐July to mid‐August and comprised 18% of the collection in 2005 and 20% in 2006. However, fewer than 10% of the Cx. tarsalis females collected in 2006 were obtained adjacent to the pelicans' nesting grounds. Minimum infection rates per 1000 Cx. tarsalis tested for WNV were 1.36 in 2005 and 1.41 in 2006. All pools in which WNV was detected in 2006 were composed of females collected 10 km from the nesting grounds. Substantial juvenile pelican mortality in 2006 despite reductions in the population of the primary vector and in mosquito infection rates near the colony suggests that the methods used to detect the introduction of WNV were too coarse and that amplification of the virus within the colony may reflect causes other than mosquito infection.     

Combined sewage overflow accelerates immature development and increases body size in the urban mosquito Culex quinquefasciatus

In urban landscapes the crowding of humans and their waste products may alleviate intra‐specific interactions of common mosquitoes. Here, we present the results of a semi‐natural experiment addressing the effects of water from a sewage overflow stream on density dependent fitness components and phenotypic traits of a common tropical and subtropical urban mosquito, Culex quinquefasciatus Say (Diptera: Culicidae). This semi‐natural experiment was designed to quantify the relative importance of density dependence, weather forces and water quality on larval mortality, sex ratio and size at adult emergence. Results showed that mortality hazards were independent of larval density, decreased in sewage overflow water and increased with minimum temperatures. Under all rearing conditions adult mosquito size decreased with density. Mosquitoes from sewage overflow water emerged faster, were bigger and had an increased ratio of females to males. All these traits may contribute to the regulation of mosquito populations.     

Forty‐two compounds in eleven essential oils elicit antennal responses from Aedes aegypti

Essential oils of various plants can be effective at repelling mosquitoes. The repellent properties are often ascribed to their dominant constituents. Our objective was to analyse several essential oils by coupled gas chromatographic‐electroantennographic detection (GC‐EAD) on the premise that those compounds that are detected by the antennae of the yellow fever mosquito, Aedes aegypti L. (Diptera: Culicidae), are candidate repellents even though they may be minor constituents and thus be overlooked in GC‐mass spectrometric analyses of essential oils. In the essential oils of catnip, cinnamon, citronella, cumin, eucalyptus, geranium, ginger, melissa, peppermint, rosemary, and thyme, 42 components induced antennal responses, most commonly β‐caryophyllene, linalool, 1,8‐cineole, geraniol, and geranial. Some of these 42 components are known insect repellents, indicating that GC‐EAD screening of essential oils is a viable analytical technique to detect quantitatively minor constituents, which could be potent repellents when tested at an appropriate dose.