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.     

Polymorphism of mitochondrial DNA and infection with symbiotic cytoplasmic bacterium <Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in mosquitoes of the <Emphasis Type="Italic">Culex pipiens</Emphasis> (Diptera,Culicidae) complex from Russia

A total of 208 mosquitoes of the Culex pipiens complex from 15 basement and terrestrial populations collected in different regions of the European part of Russia and Siberia were examined by genetic methods. Among these, two major mitotypes, M and P, were identified. These mitotypes differed by six substitutions in the 246-bp mitochondrial DNA cytochrome oxidase I gene fragment examined. Urban basement mosquitoes C. pipiens (form molestus) were characterized by the presence of mitotype M and infection with the endosymbiotic bacteria of the genus Wolbachia. Mosquitoes of the C. pipiens complex inhabiting opened biotopes harbored mitotype P, or its variety, mitotype P1, and were not infected with Wolbachia. Thus, in natural conditions marked linkage disequilibrium between cytoplasmic elements, mitochondrial DNA and Wolbachia, can be observed. Similarity of mitotypes in form molestus mosquito from different geographical localities favors the hypothesis on the common ancestry of urban mosquitoes.Translated from Genetika, Vol. 41, No. 3, 2005, pp. 320–325.Original Russian Text Copyright © 2005 by Shaikevich, Vinogradova, Platonov, Karan, Zakharov.     

Evidence of natural Wolbachia infections and molecular identification of field populations of Culex pipiens complex (Diptera: Culicidae) mosquitoes in western Turkey

Establishing reliable risk projection information about the distribution pattern of members of the Culex pipiens complex is of particular interest, as these mosquitoes are competent vectors for certain disease‐causing pathogens. Wolbachia, a maternally inherited bacterial symbiont, are distributed in various arthropod species and can induce cytoplasmic incompatibility, i.e., reduced egg hatch, in certain crosses. It is being considered as a tool for population control of mosquito disease vectors. The Aegean region is characterized by highly populated, rural, and agricultural areas and is also on the route of the migratory birds. In this study, a fragment of the 658 bp of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which includes the barcode region, was employed to differentiate Cx. pipiens complex species found in this region. Moreover, for the first time, the prevalence of Wolbachia endobacteria in these natural populations was examined using PCR amplification of a specific wsp gene. Our results revealed a widespread (more than 90%, n=121) presence of the highly efficient West Nile virus vector Cx. quinquefasciatus in the region. We also found that Wolbachia infection is widespread; the average prevalence was 62% in populations throughout the region. This study provided valuable information about the composition of Cx. pipiens complex mosquitoes and the prevalence of Wolbachia infection in these populations in the Aegean region. This information will be helpful in tracking mosquito‐borne diseases and designing and implementing Wolbachia‐based control strategies in the region.     

Impacts of fungal entomopathogens on survival and immune responses of Aedes albopictus and Culex pipiens mosquitoes in the context of native Wolbachia infections

Microbial control of mosquitoes via the use of symbiotic or pathogenic microbes, such as Wolbachia and entomopathogenic fungi, are promising alternatives to synthetic insecticides to tackle the rapid increase in insecticide resistance and vector-borne disease outbreaks. This study evaluated the susceptibility and host responses of two important mosquito vectors, Ae. albopictus and Cx. pipiens, that naturally carry Wolbachia, to infections by entomopathogenic fungi. Our study indicated that while Wolbachia presence did not provide a protective advantage against entomopathogenic fungal infection, it nevertheless influenced the bacterial / fungal load and the expression of select anti-microbial effectors and phenoloxidase cascade genes in mosquitoes. Furthermore, although host responses from Ae. albopictus and Cx. pipiens were mostly similar, we observed contrasting phenotypes with regards to susceptibility and immune responses to fungal entomopathogenic infection in these two mosquitoes. This study provides new insights into the intricate multipartite interaction between the mosquito host, its native symbiont and pathogenic microbes that might be employed to control mosquito populations.     

Ankyrin repeat domain-encoding genes in the wPip strain of Wolbachia from the Culex pipiens group

Background  

Wolbachia are obligate endosymbiotic bacteria maternally transmitted through the egg cytoplasm that are responsible for several reproductive disorders in their insect hosts, such as cytoplasmic incompatibility (CI) in infected mosquitoes. Species in the Culex pipiens complex display an unusually high number of Wolbachia-induced crossing types, and based on present data, only the wPip strain is present.     

Vector competence of northern and southern European Culex pipiens pipiens mosquitoes for West Nile virus across a gradient of temperatures

In Europe, West Nile virus (WNV) outbreaks have been limited to southern and central European countries. However, competent mosquito vectors and susceptible bird hosts are present in northern Europe. Differences in temperature and vector competence of mosquito populations may explain the absence of WNV outbreaks in northern Europe. The aim of the present study was to directly compare vector competence of northern and southern European Culex pipiens (Cx. p.) pipiens mosquitoes for WNV across a gradient of temperatures. WNV infection and transmission rates were determined for two Cx. p. pipiens populations originating from The Netherlands and Italy, respectively. Mosquitoes were orally exposed by providing an infectious bloodmeal, or by injecting WNV (lineage 2) in the thorax, followed by 14‐day incubation at 18, 23, or 28 °C. No differences in infection or transmission rates were found between the Cx. p. pipiens populations with both infection methods, but WNV transmission rates were significantly higher at temperatures above 18 °C. The absence of WNV outbreaks in northern Europe cannot be explained by differences in vector competence between Cx. p. pipiens populations originating from northern and southern Europe. This study suggests that low temperature is a key limiting factor for WNV transmission.     

Wolbachia infection in West Nile Virus vectors of northwest Iran

Mosquito-borne viral diseases are serious health problems in many countries. Various methods have been used for controlling the vectors of these diseases. Among symbiotic bacteria, the members of the genus Wolbachia are the most ubiquitous symbionts of arthropods and play key roles in their host biological characteristics with various effects on their hosts. The identification of these bacteria in Iranian mosquitoes is limited to a few studies. The current study was carried out to determine (1) the Wolbachia infection of probable arbovirus vectors (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), (2) the Wolbachia strain(s) infecting the mosquitoes, and (3) the geographical distribution of the Wolbachia strain(s) in the northwest of Iran. Eight species including Ae. caspius, Anopheles hyrcanus, An. maculipennis, Cx. hortensis, Cx. modestus, Cx. pipiens, Cx. theileri, and Cs. longiareolata were identified, amongst which Ae. caspius with 63.1% and An. hyrcanus with 0.3% were the most and the least abundant species, respectively. The results of semi-nested PCR using Wolbachia surface protein (wsp) fragment assays showed that Wolbachia infection was present in three out of the four above mentioned arboviral vector species (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), where the highest infection rate was seen in Cx. pipiens. The infection rates of mosquitoes with Wolbachia in the species of Cx. pipiens, Cs. longiareolata, Cx. theileri, and Ae. caspius were 96.9%, 11.5%, 5.2% and 0%, respectively.

     

Stable coexistence of incompatible Wolbachia along a narrow contact zone in mosquito field populations

In arthropods, the intracellular bacteria Wolbachia often induce cytoplasmic incompatibility (CI) between sperm and egg, which causes conditional embryonic death and promotes the spatial spread of Wolbachia infections into host populations. The ability of Wolbachia to spread in natural populations through CI has attracted attention for using these bacteria in vector‐borne disease control. The dynamics of incompatible Wolbachia infections have been deeply investigated theoretically, whereas in natural populations, there are only few examples described, especially among incompatible infected hosts. Here, we have surveyed the distribution of two molecular Wolbachia strains (wPip11 and wPip31) infecting the mosquito Culex pipiens in Tunisia. We delineated a clear spatial structure of both infections, with a sharp contact zone separating their distribution areas. Crossing experiments with isofemale lines from different localities showed three crossing types: wPip11‐infected males always sterilize wPip31‐infected females; however, while most wPip31‐infected males were compatible with wPip11‐infected females, a few completely sterilize them. The wPip11 strain was thus expected to spread, but temporal dynamics over 7 years of monitoring shows the stability of the contact zone. We examined which factors may contribute to the observed stability, both theoretically and empirically. Population cage experiments, field samples and modelling did not support significant impacts of local adaptation or assortative mating on the stability of wPip infection structure. By contrast, low dispersal probability and metapopulation dynamics in the host Cx. pipiens probably play major roles. This study highlights the need of understanding CI dynamics in natural populations to design effective and sustainable Wolbachia‐based control strategies.     

Wolbachia induces male-specific mortality in the mosquito Culex pipiens (LIN strain)

Background

Wolbachia are maternally inherited endosymbionts that infect a diverse range of invertebrates, including insects, arachnids, crustaceans and filarial nematodes. Wolbachia are responsible for causing diverse reproductive alterations in their invertebrate hosts that maximize their transmission to the next generation. Evolutionary theory suggests that due to maternal inheritance, Wolbachia should evolve toward mutualism in infected females, but strict maternal inheritance means there is no corresponding force to select for Wolbachia strains that are mutualistic in males.

Methodology/Principal findings

Using cohort life-table analysis, we demonstrate that in the mosquito Culex pipiens (LIN strain), Wolbachia-infected females show no fitness costs due to infection. However, Wolbachia induces up to a 30% reduction in male lifespan.

Conclusions/significance

These results indicate that the Wolbachia infection of the Culex pipiens LIN strain is virulent in a sex-specific manner. Under laboratory situations where mosquitoes generally mate at young ages, Wolbachia strains that reduce male survival could evolve by drift because increased mortality in older males is not a significant selective force.     

Wolbachia increases susceptibility to Plasmodium infection in a natural system

Current views about the impact of Wolbachia on Plasmodium infections are almost entirely based on data regarding artificially transfected mosquitoes. This work has shown that Wolbachia reduces the intensity of Plasmodium infections in mosquitoes, raising the exciting possibility of using Wolbachia to control or limit the spread of malaria. Whether natural Wolbachia infections have the same parasite-inhibiting properties is not yet clear. Wolbachia–mosquito combinations with a long evolutionary history are, however, key for understanding what may happen with Wolbachia-transfected mosquitoes after several generations of coevolution. We investigate this issue using an entirely natural mosquito–Wolbachia–Plasmodium combination. In contrast to most previous studies, which have been centred on the quantification of the midgut stages of Plasmodium, we obtain a measurement of parasitaemia that relates directly to transmission by following infections to the salivary gland stages. We show that Wolbachia increases the susceptibility of Culex pipiens mosquitoes to Plasmodium relictum, significantly increasing the prevalence of salivary gland stage infections. This effect is independent of the density of Wolbachia in the mosquito. These results suggest that naturally Wolbachia-infected mosquitoes may, in fact, be better vectors of malaria than Wolbachia-free ones.     

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus,Anopheles sinensis,Armigeres subalbatus,Culex pipiens and Cx. tritaeniorhynchus in China

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.     

The Potential Use of Wolbachia-Based Mosquito Biocontrol Strategies for Japanese Encephalitis

Japanese encephalitis virus (JEV) is a zoonotic pathogen transmitted by the infectious bite of Culex mosquitoes. The virus causes the development of the disease Japanese encephalitis (JE) in a small proportion of those infected, predominantly affecting children in eastern and southern Asia. Annual JE incidence estimates range from 50,000–175,000, with 25%–30% of cases resulting in mortality. It is estimated that 3 billion people live in countries in which JEV is endemic. The virus exists in an enzootic transmission cycle, with mosquitoes transmitting JEV between birds as reservoir hosts and pigs as amplifying hosts. Zoonotic infection occurs as a result of spillover events from the main transmission cycle. The reservoir avian hosts include cattle egrets, pond herons, and other species of water birds belonging to the family Ardeidae. Irrigated rice fields provide an ideal breeding ground for mosquitoes and attract migratory birds, maintaining the transmission of JEV. Although multiple vaccines have been developed for JEV, they are expensive and require multiple doses to maintain efficacy and immunity. As humans are a “dead-end” host for the virus, vaccination of the human population is unlikely to result in eradication. Therefore, vector control of the principal mosquito vector, Culex tritaeniorhynchus, represents a more promising strategy for reducing transmission. Current vector control strategies include intermittent irrigation of rice fields and space spraying of insecticides during outbreaks. However, Cx. Tritaeniorhynchus is subject to heavy exposure to pesticides in rice fields, and as a result, insecticide resistance has developed. In recent years, significant advancements have been made in the potential use of the bacterial endosymbiont Wolbachia for mosquito biocontrol. The successful transinfection of Wolbachia strains from Drosophila flies to Aedes (Stegomyia) mosquitoes has resulted in the generation of “dengue-refractory” mosquito lines. The successful establishment of Wolbachia in wild Aedes aegypti populations has recently been demonstrated, and open releases in dengue-endemic countries are ongoing. This review outlines the current control methods for JEV in addition to highlighting the potential use of Wolbachia-based biocontrol strategies to impact transmission. JEV and dengue virus are both members of the Flavivirus genus, and the successful establishment of Drosophila Wolbachia strains in Cx. Tritaeniorhynchus, as the principal vector of JEV, is predicted to significantly impact JEV transmission.     

West Nile virus cluster analysis and vertical transmission in Culex pipiens complex mosquitoes in Sacramento and Yolo Counties,California, 2011

West Nile virus (WNV) is now endemic in California, with annual transmission documented by the statewide surveillance system. Although much is known about the horizontal avian‐mosquito transmission cycle, less is known about vertical transmission under field conditions, which may supplement virus amplification during summer and provide a mechanism to infect overwintering female mosquitoes during fall. The current study identified clusters of WNV‐infected mosquitoes in Sacramento and Yolo Counties, CA, during late summer 2011 and tested field‐captured ovipositing female mosquitoes and their progeny for WNV RNA to estimate the frequency of vertical transmission. Space‐time clustering of WNV‐positive Culex pipiens complex pools was detected in the northern Elk Grove area of Sacramento County between July 18 and September 18, 2011 (5.22 km radius; p<0.001 and RR=7.80). Vertical transmission by WNV‐infected females to egg rafts was 50% and to larvae was 40%. The estimated minimal filial infection rate from WNV‐positive, ovipositing females was 2.0 infected females/1,000. The potential contribution of vertical transmission to WNV maintenance and amplification are discussed.     

Laboratory demonstration of the vertical transmission of Rift Valley fever virus by Culex tarsalis mosquitoes

Rift Valley fever virus (RVFV) is a mosquito-transmitted virus with proven ability to emerge into naïve geographic areas. Limited field evidence suggests that RVFV is transmitted vertically from parent mosquito to offspring, but until now this mechanism has not been confirmed in the laboratory. Furthermore, this transmission mechanism has allowed for the prediction of RVFV epizootics based on rainfall patterns collected from satellite information. However, in spite of the relevance to the initiation of epizootic events, laboratory confirmation of vertical transmission has remained an elusive research aim for thirty-five years. Herein we present preliminary evidence of the vertical transmission of RVFV by Culex tarsalis mosquitoes after oral exposure to RVFV. Progeny from three successive gonotrophic cycles were reared to adults, with infectious RVFV confirmed in each developmental stage. Virus was detected in ovarian tissues of parental mosquitoes 7 days after imbibing an infectious bloodmeal. Infection was confirmed in progeny as early as the first gonotrophic cycle, with infection rates ranging from 2.0–10.0%. Virus titers among progeny were low, which may indicate a host mechanism suppressing replication.     

Coevolution of symbiotic bacteria Wolbachia and host mtDNA in Russian populations of the Culex pipiens mosquito complex

The mitochondrial DNA phylogenies of closely related forms of mosquitoes from the Culex pipiens complex and of strains of the endosymbiotic bacteria Wolbachia pipientis were compared. Based on the cytochrome oxidase subunit I gene polymorphism, six mitochondrial haplotypes and four W. pipientis groups were discovered in mosquitoes from geographically remote populations. A strict correlation between the COI type and the type of W. pipientis proves the stable coinheritance and distribution of both cytoplasmic components in the examined mosquito populations and suggests either the absence or rarity of horizontal transfer of the symbionts in the Culex pipiens complex.     

Blood‐feeding ecology of mosquitoes in zoos

To determine if the unique host assemblages in zoos influence blood‐feeding by mosquitoes (Diptera: Culicidae), a sampling programme was conducted in Greenville and Riverbanks Zoos, South Carolina, U.S.A., from April 2009 to October 2010. A total of 4355 female mosquitoes of 14 species were collected, of which 106 individuals of nine species were blood‐fed. The most common taxa were Aedes albopictus (Skuse), Aedes triseriatus (Say), Anopheles punctipennis (Say), Culex erraticus (Dyar & Knab), Culex pipiens complex (L.) and Culex restuans (Theobald). Molecular analyses (cytochrome b) of bloodmeals revealed that mosquitoes fed on captive animals, humans and wildlife, and took mixed bloodmeals. Host species included one amphibian, 16 birds, 10 mammals (including humans) and two reptiles. Minimum dispersal distances after feeding on captive hosts ranged from 15.5 m to 327.0 m. Mosquito–host associations generally conformed to previous accounts, indicating that mosquito behaviour inside zoos reflects that outside zoos. However, novel variation in host use, including new, exotic host records, warrants further investigation. Zoos, thus, can be used as experiment environments in which to study mosquito behaviour, and the findings extrapolated to non‐zoo areas, while providing medical and veterinary benefits to zoo animals, employees and patrons.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

A correlated response of a parasite’s virulence and life cycle to selection on its host’s life history

We demonstrate a correlated response of the virulence and the mode of transmission of the microsporidian parasite Edhazardia aedis to selection on the age at pupation of its host, the mosquito Aedes aegypti. We selected three lines of mosquitoes each for early or late pupation and exposed the larvae after zero, two and four generations of selection to a low and a high concentration of the parasite’s spores. Before selection the parasites induced a similar level of mortality in the six lines; after four generations of selection mortality was higher in the mosquitoes selected for late pupation than in those selected for early pupation. Overall, parasite-induced mortality was positively correlated with the mean age at pupation of the matching uninfected line. When they died, mosquitoes selected for early pupation harboured mostly binucleate spores, which are responsible for vertical transmission. Mosquitoes selected for late pupation were more likely to harbour uninucleate spores, which are responsible for horizontal transmission. The parasite enhanced this tendency for horizontal transmission by prolonging the larval period in the lines selected for late pupation, but not in the ones selected for early pupation. These results suggest that the genetic basis of the mosquito’s age at pupation helps to determine the parasite’s mode of transmission: parasites in rapidly developing mosquitoes are benign and transmit vertically, while parasites in slowly developing mosquitoes are virulent and transmit horizontally. Thus, as the host’s life history evolves, the parasite’s performance changes, because the host’s evolution changes the environment in which the parasite develops.