Culex Species Research Articles

Prevalence of Wuchereria bancrofti Infection in Mosquitoes from Pangani District, Northeastern Tanzania

Background: Wuchereria bancrofti is the most widely distributed of the 3 nematodes known to cause lymphatic filariasis, the other 2 being Brugia malayi and Brugia timori. Anopheles gambiae and Anopheles funestus are the main vectors. However, the relative contributions of mosquito vectors to disease burden and infectivity are becoming increasingly important in coastal East Africa, and this is particularly true in the urban and semiurban areas of Pangani District, Tanzania. Methods: Mosquitoes were sampled from 5 randomly selected villages of Pangani District, namely, Bweni, Madanga, Meka, Msaraza, and Pangani West. Sampling of mosquitoes was done using standard Centers for Disease Control light traps with incandescent light bulbs. The presence of W. bancrofti in mosquitoes was determined via polymerase chain reaction (PCR) assays using NV1 and NV2 primers, and PoolScreen 2 software was used to determine the estimated rate of W. bancrofti infection in mosquitoes. Results: A total of 951 mosquitoes were collected, of which 99.36% were Culex quinquefasciatus, 0.32% were Anopheles gambiae, and 0.32% other Culex species. The estimated rate of W. bancrofti infection among these mosquitoes was 3.3%. Conclusion: This was the first study employing the use of PoolScreen PCR to detect W. bancrofti circulating in mosquito vectors in Pangani District, northeastern Tanzania. The presence of W. bancrofti infection suggests the possibility of infected humans in the area. The high abundance of Cx. quinquefasciatus calls for integrated mosquito control interventions to minimise the risk of W. bancrofti transmission to humans. Further research is required to gain an in-depth understanding of the W. bancrofti larval stages in mosquitoes, their drug sensitivity and susceptibility profiles, and their fecundity.

Increased Biting Rate of Insecticide-Resistant Culex Mosquitoes and Community Adherence to IRS for Malaria Control in Urban Malabo, Bioko Island, Equatorial Guinea.

Sustaining high levels of indoor residual spraying (IRS) coverage (≥85%) for community protection against malaria remains a challenge for IRS campaigns. We examined biting rates and insecticide resistance in Culex species and Anopheles gambiae s.l., and their potential effect on community adherence to IRS. The average IRS coverage in urban Malabo between 2015 and 2017 remained at 80%. Culex biting rate increased 6.0-fold (P < 0.001) between 2014 and 2017, reaching 8.08 bites per person per night, whereas that of An. gambiae s.l. remained steady at around 0.68. Although An. gambiae s.l. was susceptible to carbamates and organophosphates insecticides, Culex spp. were phenotypically resistant to all four main classes of WHO-recommended IRS insecticides. Similarly, the residual activity of the organophosphate insecticide used since 2017, ACTELLIC 300CS, was 8 mo for An. gambiae s.l., but was almost absent against Culex for 2 mo post-spray. A survey conducted in 2018 within urban Malabo indicated that 77.0% of respondents related IRS as means of protection against mosquito bites, but only 3.2% knew that only Anopheles mosquitoes transmit malaria. Therefore, the increasing biting rates of culicines in urban Malabo, and their resistance to all IRS insecticides, is raising concern that a growing number of people may refuse to participate in IRS as result of its perceived failure in controlling mosquitoes. Although this is not yet the case on Bioko Island, communication strategies need refining to sensitize communities about the effectiveness of IRS in controlling malaria vectors in the midst of insecticide resistance in nonmalaria vector mosquitoes.

Rift Valley fever: An open-source transmission dynamics simulation model.

Rift Valley fever (RVF) is one of the major viral zoonoses in Africa, affecting humans and several domestic animal species. The epidemics in eastern Africa occur in a 5-15 year cycle coinciding with abnormally high rainfall generally associated to the warm phase of the El Niño event. However, recently, evidence has been gathered of inter-epidemic transmission. An open-source, easily applicable, accessible and modifiable model was built to simulate the transmission dynamics of RVF. The model was calibrated using data collected in the Kilombero Valley in Tanzania with people and cattle as host species and Ædes mcintoshi, Æ. ægypti and two Culex species as vectors. Simulations were run over a period of 27 years using standard parameter values derived from two previous studies in this region. Our model predicts low-level transmission of RVF, which is in line with epidemiological studies in this area. Emphasis in our simulation was put on both the dynamics and composition of vector populations in three ecological zones, in order to elucidate the respective roles played by different vector species: the model output did indicate the necessity of Culex involvement and also indicated that vertical transmission in Ædes mcintoshi may be underestimated. This model, being built with open-source software and with an easy-to-use interface, can be adapted by researchers and control program managers to their specific needs by plugging in new parameters relevant to their situation and locality.

Effectiveness of landmark- and semi-landmark-based geometric morphometric to identify four species of Culex mosquitoes in Thailand.

Objective:This research aims to study the effectiveness of landmark- and semi-landmark-based geometric morphometric (LMSL) in the identification of four Culex vectors, namely, C. quinquefasciatus, C. visnui, C. sitiens, and C. whitmorei in Thailand and also compared the potential between LMSL and the landmark-based geometric morphometric method (LM).Materials and Methods:The overall size of the wing sample was estimated by the centroid size. Shape variables were computed as principal components of the “partial warp” calculated after generalized procrustes analysis of raw coordinates. Discriminant analysis of the canonical variables performed to explore the shape dissimilarity between Culex species has been shown as a factor map and to calculate the Mahalanobis distance. Size and shape differences based on pairwise Mahalanobis distances were tested using non-parametric methods (1,000 cycles) with Bonferroni correction at a p-value of <0.05.Results:A total of 120 individuals were used that were divided into 30 individuals per Culex species. The mean CS of C. sitiens had the largest wings followed by C. visnui, C. quinquefasciatus, and C. whitmorei in LM and LMSM. The patterns of statistical difference in CS of both methods were similar and wing shapes among Culex species were different based on a comparison of pairwise Mahalanobis distances (p < 0.05) in both methods. For the cross-validated reclassification test, LM provided Culex species separation ranging from 54% to 84% and 51% to 93% for LMSM.Conclusion:Thus, LMSM is another option to use for the identification in mosquito vectors that have a curved line on the wing specific to the species.

Identification of a novel alphavirus related to the encephalitis complexes circulating in southern Brazil

ABSTRACT In early 2017, an outbreak caused by an unknown and supposedly viral agent in the Marilena region of southern Brazil was investigated. Since the etiological agent causing the outbreak was not identified from human samples, mosquitoes from this region were collected. Three out of 121 mosquito pools collected from the region tested positive for alphavirus in molecular tests. Next generation sequencing results revealed the presence of a novel alphavirus, tentatively named here as Caainguá virus (CAAV). DNA barcoding analyses indicated that different species of Culex are hosts for CAAV. This new virus was basal to the New World encephalitic alphaviruses in a comprehensive and robust phylogenetic approach using complete genomes. Viral particles were observed in the cytosol and inside of intracellular compartments of cells in mosquito-derived cell cultures. Despite being noninfectious in vertebrate derived cell cultures, primary culturing of CAAV in human mononuclear cells suggests monocytes and lymphocytes as CAAV targets. However, the epidemiological link of CAAV on the human outbreak should be further explored.

Effect of Temperature on Biological Parameters of the West Nile Virus Vector Culex pipiens form 'molestus' (Diptera: Culicidae) in Greece: Constant vs Fluctuating Temperatures.

Studying the biology of Culex species is crucial to understanding their role in arbovirus transmission and for the development of efficient control strategies. Assessments of survival, development, adult longevity, fecundity and egg hatching of Culex pipiens form 'molestus' (Forsskål), were conducted, under nine constant and fluctuating temperatures ranging from 15 to 35 ± 0.5°C. Higher survival rates were observed at constant temperature of 25°C as well as fluctuating with the same mean. Complete mortality occurred at 35°C in both constant and fluctuating temperature regimes. Development rate from egg to adult increased between 15 and 32.5°C, in a linear fashion. Adult longevity ranged from 1.4 d at 32.5°C to 73.5 d at 15°C. Females lived significantly longer compared to males at all temperature regimes with the exception of constant 32.5°C where adult longevity was similar between males and females. Fecundity was higher at moderate constant and fluctuating temperatures compared to high temperatures, where females laid a significantly smaller number of eggs. Likewise, egg hatching was significantly lower at the highest tested temperature regimes compared to low and moderate ones. The lowest developmental thresholds of the species in different developmental stages ranged between 11.17 and 11.95°C at constant temperatures and between 11.09 and 12.74°C at fluctuating ones. Differences between constant and fluctuating temperatures were observed concerning developmental time, fecundity, and male adult longevity at the two lowest tested temperatures, highlighting the importance of testing also fluctuating temperatures that simulate field conditions.

Diverse novel resident Wolbachia strains in Culicine mosquitoes from Madagascar

Wolbachia endosymbiotic bacteria are widespread throughout insect species and Wolbachia transinfected in Aedes mosquito species has formed the basis for biocontrol programs as Wolbachia strains inhibit arboviral replication and can spread through populations. Resident strains in wild Culicine mosquito populations (the vectors of most arboviruses) requires further investigation given resident strains can also affect arboviral transmission. As Madagascar has a large diversity of both Culicine species and has had recent arboviral outbreaks, an entomology survey was undertaken, in five ecologically diverse sites, to determine the Wolbachia prevalence. We detected diverse novel resident Wolbachia strains within the Aedeomyia, Culex, Ficalbia, Mansonia and Uranotaenia genera. Wolbachia prevalence rates and strain characterisation through Sanger sequencing with multilocus sequence typing (MLST) and phylogenetic analysis revealed significant diversity and we detected co-infections with the environmentally acquired bacteria Asaia. Mosquitoes were screened for major arboviruses to investigate if any evidence could be provided for their potential role in transmission and we report the presence of Rift Valley fever virus in three Culex species: Culex tritaeniorhynchus, Culex antennatus and Culex decens. The implications of the presence of resident Wolbachia strains are discussed and how the discovery of novel strains can be utilized for applications in the development of biocontrol strategies.

High Resistance to Bacillus sphaericus and Susceptibility to Other Common Pesticides in Culex pipiens (Diptera: Culicidae) from Salt Lake City, UT.

Biorational mosquito larvicides based on microbial organisms and insect growth regulators (IGRs) have played a vital role in integrated mosquito control, particularly since the invasion of West Nile virus to the United States in 1999. Products that are formulated with technical powder of the bacterium, Bacillus sphaericus Neide (recently Lysinibacillus sphaericus Meyer and Neide), are among the ones that have been extensively applied to combat Culex and other mosquito species. Due to the simplicity of the binary toxins, resistance to this pesticide in laboratory and field populations of Culex pipiens L. complex has occurred globally since 1994. A Cx. pipiens population with a high level of resistance to B. sphaericus (VectoLex WDG) was identified in Salt Lake City, UT, in September 2016. The resistance ratios in this population were 20,780.0- and 23,926.9-fold at LC50 and LC90, respectively, when compared with a susceptible population of a laboratory reference colony of the same species. This B. sphaericus-resistant population remained mostly susceptible to other commonly used pesticides to control arthropods of public health and urban significance, including ones based on microbial organisms (Bacillus thuringiensis subsp. israelensis, spinosad, spinetoram, abamectin), IGRs (pyriproxyfen, methoprene, diflubenzuron, novaluron), organophosphate (temephos), neonicotinoid (imidacloprid), phenylpyrazole (fipronil), oxadiazine (indoxacarb), and pyrethroid (permethrin). Results are discussed according to the modes of action of the pesticides tested, and suggestions are made to manage B. sphaericus-resistant mosquito populations.

Culex tarsalis is a competent vector species for Cache Valley virus

BackgroundCache Valley virus (CVV) is a mosquito-borne orthobunyavirus endemic in North America. The virus is an important agricultural pathogen leading to abortion and embryonic lethality in ruminant species, especially sheep. The importance of CVV in human public health has recently increased because of the report of severe neurotropic diseases. However, mosquito species responsible for transmission of the virus to humans remain to be determined. In this study, vector competence of three Culex species mosquitoes of public health importance, Culex pipiens, Cx. tarsalis and Cx. quinquefasciatus, was determined in order to identify potential bridge vector species responsible for the transmission of CVV from viremic vertebrate hosts to humans.ResultsVariation of susceptibility to CVV was observed among selected Culex species mosquitoes tested in this study. Per os infection resulted in the establishment of infection and dissemination in Culex tarsalis, whereas Cx. pipiens and Cx. quinquefasciatus were highly refractory to CVV. Detection of viral RNA in saliva collected from infected Cx. tarsalis provided evidence supporting its role as a competent vector.ConclusionsOur study provided further understanding of the transmission cycles of CVV and identifies Cx. tarsalis as a competent vector.

First record of Culex (Culex) bidens (Diptera: Culicidae) in Colombia: Taxonomic and epidemiological implications

Arbovirus transmission cycles must be studied locally since both vectors and hosts vary in different regions. Colombia has a highly diverse mosquito fauna. Culex (Culex) bidens is reported here for the first time in Colombia. Because Cx. bidens Dyar and Knab and Cx. declarator Dyar and Knab share a close taxonomic history and because it is difficult to differentiate between them, a morphological and molecular comparison was performed. The male genitalia of three specimens of Cx. bidens from Colombia were mounted on microscope slides and morphologically compared with the male genitalia of Cx. declarator also from Colombia. In Cx. bidens, the individual teeth of the lateral plate are long, straight, laterally directed and sharply pointed; in Cx. declarator these teeth are robust, curved, with convex margins and bluntly rounded. Moreover, DNA was extracted from the same specimens and a fragment of the cytochrome c oxidase subunit I mitochondrial gene was amplified and sequenced. Neither Cx. bidens nor Cx. declarator were clustered in the Neighbour-joining topology, with K2P interspecific divergence between 0.15–1.45%. The circulation of Eastern Equine Encephalitis Virus in Colombia was reported since 1957 and Cx. bidens was suspected to be the vector of this virus during an epizootic in Argentina in 1988. Hybridization between species of the subgenus Culex has been demonstrated, hence the degree of reproductive isolation between Cx. bidens and Cx. declarator should be investigated, as well as their taxonomic status, because they only can be discriminated by a single male genitalic feature and not by nuclear or mitochondrial markers.

Vertebrate Hosts ofAedes aegypti,Aedes albopictus, andCulex quinquefasciatus(Diptera: Culicidae) as Potential Vectors of Zika Virus in Florida

Zika virus (ZIKV), once considered an obscure pathogen, spread rapidly from 2014 to 2016 to become an internationally notifiable condition of major public health concern. The relative importance of various Culex and Aedes species mosquitoes (Diptera: Culicidae) in ZIKV transmission is a topic of debate. Quantifying host use is important in determining the vectorial capacity of a mosquito species for transmitting ZIKV in nature. In the United States, few data are available on host use of Aedes aegypti L. (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae), confirmed and suspected vectors of ZIKV, respectively. Here, we report results of bloodmeal analysis to quantify host use of confirmed (Ae. aegypti) and suspected (Ae. albopictus and Culex quinquefasciatus Say (Diptera: Culicidae)) vectors of ZIKV in two Florida counties. At an auto salvage yard in Indian River County, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus fed mainly on humans, taking 90.2, 90.8, and 78.6% of bloodmeals from humans, respectively. At a residential area in Martin County, Ae. aegypti, Ae. albopictus took 61.5 and 66.7% of bloodmeals from humans, higher than Cx. quinquefasciatus (11.1%). Patterns of host use suggest that Ae. aegypti and Ae. albopictus are the most likely vectors of ZIKV in Florida and that Cx. quinquefasciatus would likely play a lesser role in ZIKV transmission in Florida. However, the relative importance of the three species in ZIKV transmission is likely location and population specific. Detailed studies quantifying other parameters of vectorial capacity, including vector competence, are needed in order to determine the actual role for each species in ZIKV transmission.

Vector competence of Aedes aegypti, Culex tarsalis, and Culex quinquefasciatus from California for Zika virus.

Zika virus (ZIKV) has emerged since 2013 as a significant global human health threat following outbreaks in the Pacific Islands and rapid spread throughout South and Central America. Severe congenital and neurological sequelae have been linked to ZIKV infections. Assessing the ability of common mosquito species to transmit ZIKV and characterizing variation in mosquito transmission of different ZIKV strains is important for estimating regional outbreak potential and for prioritizing local mosquito control strategies for Aedes and Culex species. In this study, we evaluated the laboratory vector competence of Aedes aegypti, Culex quinquefasciatus, and Culex tarsalis that originated in areas of California where ZIKV cases in travelers since 2015 were frequent. We compared infection, dissemination, and transmission rates by measuring ZIKV RNA levels in cohorts of mosquitoes that ingested blood meals from type I interferon-deficient mice infected with either a Puerto Rican ZIKV strain from 2015 (PR15), a Brazilian ZIKV strain from 2015 (BR15), or an ancestral Asian-lineage Malaysian ZIKV strain from 1966 (MA66). With PR15, Cx. quinquefasciatus was refractory to infection (0%, N = 42) and Cx. tarsalis was infected at 4% (N = 46). No ZIKV RNA was detected in saliva from either Culex species 14 or 21 days post feeding (dpf). In contrast, Ae. aegypti developed infection rates of 85% (PR15; N = 46), 90% (BR15; N = 20), and 81% (MA66; N = 85) 14 or 15 dpf. Although MA66-infected Ae. aegypti showed higher levels of ZIKV RNA in mosquito bodies and legs, transmission rates were not significantly different across virus strains (P = 0.13, Fisher’s exact test). To confirm infectivity and measure the transmitted ZIKV dose, we enumerated infectious ZIKV in Ae. aegypti saliva using Vero cell plaque assays. The expectorated plaque forming units PFU varied by viral strain: MA66-infected expectorated 13±4 PFU (mean±SE, N = 13) compared to 29±6 PFU for PR15-infected (N = 13) and 35±8 PFU for BR15-infected (N = 6; ANOVA, df = 2, F = 3.8, P = 0.035). These laboratory vector competence results support an emerging consensus that Cx. tarsalis and Cx. quinquefasciatus are not vectors of ZIKV. These results also indicate that Ae. aegypti from California are efficient laboratory vectors of ancestral and contemporary Asian lineage ZIKV.

Evaluation of resting traps to examine the behaviour and ecology of mosquito vectors in an area of rapidly changing land use in Sabah, Malaysian Borneo

BackgroundWidespread deforestation occurring in the tropics is hypothesized to impact the transmission of vector-borne diseases (VBD). Predicting how environmental changes will impact VBD transmission is dependent on understanding the ecology and behaviour of potential vector species outside of domestic settings. However there are few reliable sampling tools for measuring the habitat preference and host choice of mosquito vectors; with almost none suitable for sampling recently blood-fed, resting mosquitoes. This study evaluated the use of two mosquito traps: the resting bucket (RB) and sticky resting bucket (SRB) traps relative to CDC backpack aspiration (CDC) for sampling mosquitoes resting in a range of habitats representing a gradient of deforestation. Eight habitats were selected for sampling around two villages in Kudat District, Malaysian Borneo, to reflect the range of habitats available to mosquitoes in and around human dwellings, and nearby forest habitats where reservoir hosts are present: secondary forest (edge, interior and canopy); plantations (palm and rubber); and human settlements (inside, under and around houses).ResultsOver 31 days, 2243 mosquitoes were collected in 5748 discrete collections. Nine mosquito genera were sampled with Aedes and Culex species being present in all habitats and most abundant. RB and CDC backpack aspiration were most efficient for sampling Culex whereas CDC backpack aspiration and SRB were most efficient for Aedes. Most Aedes identified to species level were Ae. albopictus (91%), with their abundance being highest in forest edge habitats. In contrast, Culex were most abundant under houses. Most blood-fed mosquitoes (76%) were found in human settlements; with humans and chickens being the only blood source.ConclusionsRB and SRB traps proved capable of sampling mosquitoes resting in all sampled habitats. However, sampling efficiency was generally low (c.0.1 per trap per day), necessitating traps to be deployed in high numbers for mosquito detection. None of the traps were effective for sampling zoonotic malaria vectors; however, SRB collected relatively higher numbers of the dengue vector Ae. albopictus. The higher abundance of mosquitoes in forest edge habitats indicates the potential value of these traps for investigating sylvatic dengue transmission. This study has demonstrated the merits in application of simple resting traps for characterising mosquito vector resting behaviour outside of the home.

Toxicological Response of &lt;i&gt;Poecilia reticulata, Hyla species&lt;/i&gt; and &lt;i&gt;Culex species&lt;/i&gt; to Leachates from Olusosun Landfill, Lagos State, Nigeria

The effect of leachate samples from Olushosun dumpsite in Ojota, Lagos on the toxicological response of Poecilia reticulata , Hyla and Culex species was investigated using acute and chronic toxicity tests. Physicochemical parameters were analysed in the leachate prior to toxicological assessment against test animals. Computed results showed that pH 7.62 while the acute toxicity assay of leachate sample against test animals revealed a 96hr LC 50 of 71.53ml/L, 58.34ml/L and 60.643ml/L for Poecilia, Hyla and Culex species respectively. On the basis of the 96hr LC 50 values, the leachate was found to be more toxic against Hyla than Poecilia . The exposure of the Poecilia and Hyla species to sub-lethal concentrations (1/10th of 96hrs LC 50 ) of leachate revealed varied biochemical responses over a 28 day period. The activity of Catalase, Superoxide Dismutase (SOD), Malondialdehyde (MDA) and Glutathione-S-Transferase (GST) showed significant increase (p<0.05) when compared to control in Hyla while the activity of GSH, SOD and CAT showed significant increase (p<0.05) when compared to control in Poecilia sp. Results from this study showed that leachate can induce adverse environmental effect on organisms. The acute and chronic toxicological response of the test animals could be incorporated into risk assessment of dumpsite leachate, design and management of landfill sites.

Mosquito traps for urban surveillance: collection efficacy and potential for use by citizen scientists.

Mosquito-borne diseases are a pervasive public health problem on a global scale, and effective management of them requires well-designed surveillance programs for both vectors and pathogens. Mosquito traps are a common component of such programs, and their reach can be expanded by engaging citizen scientists. In this study in a southern Australian city, we compared the mosquito collection efficacy of two types of traps and assessed their suitability for use in citizen science programs. BG Sentinels and BG Gravid Aedes Trap (BG-GAT) traps both collected Aedes and Culex species in similar proportions, albeit with the former collecting approximately nine times as many mosquitoes. However, BG Sentinels have a greater per unit cost than BG-GATs and are restricted to deployment near power outlets. Importantly, despite being devised for collection of Aedes (Stegomyia) dengue vectors (such as Aedes aegypti), both traps can be effectively used in temperate climates for collection of a range of mosquito species. These traps could conceivably be used in citizen science programs to enhance the reach of surveillance at reduced cost.

Diversity, distribution, abundance, and feeding pattern of tropical ornithophilic mosquitoes.

Bird-biting mosquitoes act as bridge vectors of diverse pathogens of emerging infectious diseases. In this study, we report for the first time the abundance, diversity, distribution, and feeding pattern of bird-biting mosquitoes on an island where avifaunal diversity is rich. Monthly mosquito collections were done at six different habitats in three different climatic zones using bird-baited traps over a year. Collected mosquitoes were identified using morphological and molecular tools. A total of 2,655 bird-biting mosquitoes of eight genera and 25 species were identified. Of these, 52% were Culex species, which represents 35% of the Culex species in the country. The most abundant species were Culex sitiens, Cx. pseudovishnui, Cx. nigropunctatus and Cx. quinquefasciatus, whereas the latter two were common to all habitats. The highest abundance was reported in lowland forests (49.6%), while it was lowest in highland forests (22.3%). Highest species similarity was reported from highland forests. Seasonal variations of the most abundant species were significantly different in selected habitats (p< 0.05). Two distinct biting peaks were identified, from 06:00 to 21:00 and 22:00 to 02:00. The biting nature of identified ornithophilic mosquitoes suggests the potential vector status of these mosquitoes.

Vector competence of certain Culex and Aedes mosquitoes for the Chittoor virus, the Indian variant of the Batai virus.

Chittoor virus (CHITV), a mosquito-borne bunyavirus (Orthobunyavirus: Bunyaviridae) isolated in India, has been found to be antigenically close to the Batai virus (BATV), which has a wide distribution across Asia, Europe, and Africa. The latter virus causes influenza-like illness in humans and mild illness in sheep and goats. BATV has been involved in genetic reassortment with other bunyaviruses, generating novel genome combinations and causing severe clinical manifestations including hemorrhagic fever. Conversely, CHITV has never been associated with any major outbreaks in India, although neutralizing antibodies have been detected in humans and domestic animals. Repeated isolations and seroprevalence have prompted us to determine the vector competence of three important mosquito species, viz., Culex quinquefasciatus, Culex tritaeniorhynchus, and Aedes aegypti, for CHITV. The three mosquito species replicated CHITV to titers of 6.3, 5.0, and 5.2 log10 TCID50/mL, respectively, and maintained the virus for substantial periods. Both of the Culex species demonstrated vector competence, while A. aegypti did not. Horizontal transmission to infant mice was also demonstrated by both Culex species. Active circulation of the virus and the availability of both susceptible hosts and competent vector mosquitoes pose a serious threat to public health should there be a reassortment.

Molecular phylogeny of Culex subgenus Melanoconion (Diptera: Culicidae) based on nuclear and mitochondrial protein-coding genes.

The subgenus Melanoconion of the mosquito genus Culex is taxonomically diverse and is widely distributed in the Neotropical Region, with 10 species occurring in the Nearctic Region. Species of this subgenus pose a taxonomical challenge because morphological identification is based largely on anatomical characters of the male genitalia. We addressed the monophyly of the Spissipes and Melanoconion Sections of the subgenus Melanoconion and some of the informal groups in each section. Our sample taxa included 97 specimens representing 43 species, from which we analysed fragments of two single-copy nuclear genes (CAD, HB) and one mitochondrial gene (COI). Phylogenetic relationships within the subgenus are presented based on results of maximum-likelihood and Bayesian analyses using a multi-locus matrix of DNA sequences. We show a molecular phylogeny of Melanoconion in which both sections were recovered as monophyletic groups. The monophyly of the Atratus and Pilosus groups was confirmed. Within each section, other monophyletic groups were recovered highlighting the potential need for future nomenclature rearrangement. The phylogenetic signal contained in nuclear genes, when analysed together, was more informative than each gene analysed separately, corroborating monophyly of Melanoconion relative to Culex (Culex) species included in the analyses, the Melanoconion and Spissipes Sections and some species groups. Our results provide new information for the classification of the subgenus and additional data that can be used to improve species identification when a more representative taxon sampling is available.

Japanese encephalitis: the vectors, ecology and potential for expansion.

Japanese encephalitis (JE) is a viral disease predominantly located in South East Asia and commonly associated with transmission between amplifying hosts, such as pigs, and the mosquito Culex tritaeniorhynchus, where human infection represents a dead end in the life cycle of the virus. The expansion of JE beyond an Asiatic confine is dependent on a multitude of complex factors that stem back to genetic subtype variation. A complex interplay of the genetic variation and vector competencies combine with variables such as geography, climate change and urbanization. Our understanding of JE is still at an early stage with long-term longitudinal vector surveillance necessary to better understand the dynamics of JE transmission and to characterize the role of potential secondary vectors such as Cx. pipiens and Cx. bitaeniorhynchus. The authors review the vectors indicated in transmission and the ecological, genetic and anthropological factors that affect the disease's range and epidemiology. Monitoring for the presence of JE virus in mosquitoes in general can be used to estimate levels of potential JE exposure, intensity of viral activity and genetic variation of JEV throughout surveyed areas. Increased surveillance and diagnosis of viral encephalitis caused by genotype 5 JE virus is required in particular, with the expansion in epidemiology and disease prevalence in new geographic areas an issue of great concern. Additional studies that measure the impact of vectors (e.g. bionomics and vector competence) in the transmission of JEV and that incorporate environmental factors (e.g. weekly rainfall) are needed to define the roles of Culex species in the viral pathogenesis during outbreak and non-outbreak years.

DNA barcoding of morphologically characterized mosquitoes belonging to\xa0the subfamily Culicinae from Sri Lanka

BackgroundVectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization.ResultsAccording to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity.ConclusionsThe current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.