Detection of Rift Valley Fever Virus in Aedes (Aedimorphus) durbanensis, South Africa.

Rift Valley fever virus (RVFV) is a mosquito-borne, zoonotic phlebovirus-causing disease in domestic ruminants and humans in Africa, the Arabian Peninsula and some Indian Ocean islands. Outbreaks, characterized by abortion storms and a high morbidity rate in newborn animals, occur after heavy and prolonged rainfalls favouring the breeding of mosquitoes. However, the identity of the important mosquito vectors of RVFV is poorly known in most areas. Mosquitoes collected in the Ndumo area of tropical north-eastern KwaZulu-Natal (KZN), South Africa, were tested for RVFV nucleic acid using RT-PCR. The virus was detected in a single pool of unfed Aedes (Aedimorphus) durbanensis, indicating that this seasonally abundant mosquito species could serve as a vector in this area of endemic RVFV circulation. Phylogenetic analysis indicated the identified virus is closely related to two isolates from the earliest outbreaks, which occurred in central South Africa more than 60 years ago, indicating long-term endemicity in the region. Further research is required to understand the eco-epidemiology of RVFV and the vectors responsible for its circulation in the eastern tropical coastal region of southern Africa.

The mosquito fauna of the western region of Spain with emphasis on ecological factors and the characterization of Culex pipiens forms.

This study updates the diversity, distribution, and seasonal trends of mosquitoes in a western region of Spain, assesses ecological determinants of Culex pipiens s.l., and determines form composition of Cx. pipiens s.s. POPULATIONS: A total of 1,495 mosquitoes of 16 species was collected during 2012-2013, of which Cx. pipiens s.l. and Cx. theileri were the most abundant. Five new records for An. maculipennis s.s., Orthopodomyia pulcripalpis, Aedes (Ochlerotatus) punctor, Cx. europaeus, and Cx. modestus were found for this region. Cx. pipiens density varied across weather and habitat patterns, correlating positively with high temperatures and with a preference for urbanized areas and rural areas within a proximity of ovine farms. Moreover, molecular identification by CQ11FL was performed in 467 Cx. pipiens s.s., detecting both pipiens (66%) and molestus (8.4%) forms coexisting in different habitats (urban, peri-urban and rural) aboveground with a high degree of hybridization (25.7%). The abundance of Cx. pipiens in urban areas and farms, with the presence of hybrids, may increase their capacity to act as bridge vectors for the transmission of arboviral infections. These data will be helpful for further implementation of entomological programs focused on risk assessment for arboviruses or other mosquito-borne pathogens.

Culex pipiens as a potential vector for transmission of Dirofilaria immitis and other unclassified Filarioidea in Southwest Spain.

Dirofilaria immitis is one of the most frequently detected mosquito-transmitted zoonotic filarioid nematode in mammals in Europe, being canine dirofilariosis a major animal health problem, endemic in the Mediterranean area. This study, focused on Southwest Spain, in order to bring new insights into (i) the epidemiology of Dirofilaria spp., (ii) the species of Culicid vectors possibly involved in their transmission and (iii) the genetic variability of those potential vectors. A total of 881 adult female mosquitoes from 11 different species, were captured during 2012-2013, and detection of filarioid DNA was attempted by PCR using specific primers (ITS-2 and COI), followed by DNA sequencing. In a single Culex pipiens specimen D. immitis DNA was detected both in the head-thorax and abdomen sections. Filarioid nematode DNA was also detected in eight additional Cx. pipiens specimens also in both the thorax and the abdomen, but analysis of sequence data did not allow unambiguous assignment of any of the obtained sequences to a previously defined species. All Cx. pipiens with filarioid DNA were individually analysed by CQ11 to discriminate between pipiens, molestus, and hybrid forms. Besides, rDNA ITS-2 sequence analysis revealed the presence of haplotype H1 and H2 of Cx. pipiens. To our knowledge this study revealed, for the first time in Spain, the occurrence of likely mature infection of D. immitis in Cx. pipiens, as well as with other yet uncharacterized nematodes, supporting its role as a potential vector of these filarids.

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures.

BACKGROUND: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. RESULTS: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. CONCLUSION: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats.

Negeviruses found in multiple species of mosquitoes from southern Portugal: Isolation, genetic diversity, and replication in insect cell culture.

In this report, an RT-PCR approach based on the use of degenerate primers allowed the identification of negeviruses in four different species of mosquitoes (Ochlerotatus caspius, Culex pipiens, Cx. theileri and Cx. univittatus) collected in southern Portugal. The genomes of two of these viruses, sequenced to full completion, were shown to encode all the proteins encoded by previously described negeviruses. One of these viruses induces exuberant cytopathic effect in insect cell culture, with no obvious signs of apoptosis induction, replicating very rapidly and allowing for the detection of viral genomes in the infected culture supernatant as soon as 4h post-infection. This virus was also shown to use a dsRNA intermediate, which was found to be fully formed and active 3h after infection. Phylogenetic analysis of two products encoded by the viral ORF1 placed both viruses among Negev virus cluster, in the recently proposed Nelorpivirus taxon.

Distribution and hybridization of Culex pipiens forms in Greece during the West Nile virus outbreak of 2010.

In 2010, an outbreak of West Nile virus (WNV) infections occurred in the region of Thessaloniki, Central Macedonia, in northern Greece. During this period, Culex pipiens sensu stricto mosquitoes were found infected by WNV lineage 2. Cx. pipiens s.s. presents two distinct biological forms, denoted molestus and pipiens. Hybrids between the two forms may potentiate the accidental transmission of WNV to humans. We have genetically characterized the form composition of Cx. pipiens s.s. samples collected during the outbreak from the region of Thessaloniki, where WNV cases occurred, and from the region Schinias-Marathonas, with no reported cases at the time. Information on bird fauna was also obtained for the two regions. Application of the CQ11FL diagnostic marker revealed a 350 bp variant of the pipiens-specific allele. Sympatric pipiens and molestus populations were detected in Thessaloniki, whereas Schinias-Marathonas presented a more genetically homogenous molestus population. A pattern of asymmetric introgression between molestus and pipiens was also observed in Thessaloniki. The presence of hybrids between molestus and pipiens forms suggests a greater receptivity of the Thessaloniki region for the establishment of WNV zoonotic cycles. However, the Schinias-Marathonas region also displayed characteristics to sustain WNV transmission cycles. These observations highlight the importance of maintaining active surveillance systems in selected regions geographically located within the range of major migratory bird flyways.

The Culex pipiens complex in continental Portugal: distribution and genetic structure.

Portugal is a southern European country that displays favorable ecological conditions for the establishment of West Nile virus (WNV) transmission cycles. Competent mosquito vector species are present throughout the country. Among the species with reported cases of WNV isolation in Portugal, Culex pipiens is the most ubiquitous and abundant mosquito. This species exhibits two biological forms with differences in host preferences. The molestus form has a greater tendency to feed upon humans and other mammals whereas the pipiens form prefers avian hosts. In northern latitudes, both forms are physically separated, with molestus occupying underground habitats and pipiens being found aboveground. However, the warmer climatic conditions of southern regions such as Portugal may favor the sympatric occurrence of both forms hence promoting interform hybridization. Genetic introgression between molestus and pipiens forms may result in a higher propensity for admixed populations to serve as bridge-vectors of WNV between humans and birds. Here we revise our present knowledge on the distribution, role in WNV transmission and genetic structure of the Cx. pipiens complex in continental Portugal. We focus on recent findings of sympatric molestus and pipiens populations that display considerable levels of hybridization and discuss the epidemiological repercussions of this occurrence.

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal.

BACKGROUND: Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group. RESULTS: Autogeny and stenogamy was evaluated in the F1 progeny of 145 individual Cx. pipiens females. Bayesian clustering analysis based on the genotypes of 13 microsatellites revealed two distinct genetic clusters that were highly correlated with the alternative traits that define pipiens and molestus. Admixture analysis yielded hybrid rate estimates of 8-10%. Higher proportions of admixture were observed in pipiens individuals suggesting that more molestus genes are being introgressed into the pipiens form than the opposite. CONCLUSION: Both physiological/behavioural and genetic data provide evidence for the sympatric occurrence of molestus and pipiens forms of Cx. pipiens in the study area. In spite of the significant genetic differentiation between forms, hybridisation occurs at considerable levels. The observed pattern of asymmetric introgression probably relates to the different mating strategies adopted by each form. Furthermore, the differential introgression of molestus genes into the pipiens form may induce a more opportunistic biting behaviour in the latter thus potentiating its capacity to act as a bridge-vector for the transmission of arboviral infections.