Culex Theileri Research Articles

Effect of blood meal digestion and DNA extraction protocol on the success of blood meal source determination in the malaria vector Anopheles atroparvus

BackgroundHost identification is an essential step in studies on the transmission dynamics of vector-borne disease. Nowadays, molecular tools allow the identification of vertebrate hosts to the species level. However, the proportion of successful identifications is variable and may be affected by the quality of the samples and the laboratory protocols. Here, the effect of two of these factors, namely the digestion status of mosquito blood meal and the DNA extraction procedure, on the success of host identification by amplification and sequencing of a fragment of the cytochrome oxidase 1 gene were tested.MethodsMosquitoes collected both outdoors and indoors during 2012 in southern Spain were identified to species level and their blood meal digestion status recorded using the Sella score, a visual estimation of the digestion status of mosquito blood meals. Each mosquito was assigned randomly to one of two DNA extraction procedures: the quick and cheap HotSHOT procedure or the QIAGEN DNeasy Blood and Tissue® kit and their hosts identified by a molecular method.ResultsThree hundred and forty-seven blood-fed mosquitoes belonging to Anopheles atroparvus (n=171), Culex perexiguus (n=84), Culex pipiens (n=43), Culex theileri (n=39), Culex modestus (n=5), Ochlerotatus caspius (n=4), Culiseta sp. (n=1) were included in this study. Overall, hosts were identified from 234 blood meals compromising at least 25 species including mammals, birds and a single reptile. The success of host identification was lower in mosquitoes with an advanced stage of blood meal digestion and for blood meals extracted using the HotSHOT procedure.ConclusionsThe success of host identification decreases with the advanced stage of mosquito blood meal digestion, from 84.5% for recent blood meals to 25.0% for more digested ones. Using the QIAGEN kit, the identification success improved by 17.6%, with larger increases at more advanced stages of blood meal digestion. Availability of blood-fed females used to be very limited for studies of vector ecology, and these results may help to increase the efficiency of blood meal analyses. In addition, results obtained in this study clearly support that the potential malaria vector An. atroparvus feeds on animals located outdoors but use human-made shelters for resting after feeding.

Molecular Characterization of Culex Theileri from Canary Islands, Spain, a Potential Vector of Dirofilaria Immitis

Dirofilaria immitis is the causal agent of heartworm diseases and of human pulmonary dirofilariosis. The infection is transmitted by several species of culicid mosquitoes that are frequently able to bite both animal reservoirs and humans. Canary Islands (Spain) constitute a well documented endemic area of canine dirofilariosis in which the mosquito species involved in the transmission of D. immitis are not known. The objectives of the present work were the identification of vectors of this parasite in Canary Islands and their molecular characterization. A total of 1219 female mosquitoes were captured. The most abundant species was Culex theileri (52.26%) followed by Cx. pipiens (35.44%), Anopheles cinereus hispaniola (6.23%), Culiseta longiareolata (5.74%), and Culex laticintus (0.33%). PCR was applied for the detection of larval D. immitis DNA in mosquitoes. D. immitis DNA was observed in the abdomen of one Cx. theileri female: 0.082% of the entire mosquito population and 0.17% in Cx. theileri. A molecular identification of Cx. theileri, the potential mosquito vector of dirofilariosis in this zoonotic focus in Canary Islands of Spain, has been made for first time based on sequences of the 18S rRNA gene, the second internal transcribed spacer (ITS2) of ribosomal DNA and the barcode region of the cytochrome c oxidase I (cox1) gene of mitochondrial DNA, allowing a broad mosquito molecular basis for future populations genetic analyses of this vector species. Parasitological and entomological molecular results suggest that Cx. theileri is a potential natural vector of D. immitis in Canary Islands.

Introduction to the Special Issue on Epidemiology and Ecology of Parasitic Diseases

Human African Trypanosomiasis (HAT), commonly known as sleeping sickness is a vector-borne parasitic infection caused by protozoa of the genus Trypanosoma. It can be fatal if left untreated. HAT is found in sub-Saharan Africa, but is restricted to the range of its tsetse vector. In recent decades, the foci of HAT infections have changed and it has been suggested that climate change and changes to the habitats of the tsetse vectors have influenced the observed changes. The paper by Tongue et al. clearly demonstrates the effects of habitat change on the transmission of African trypanosomiasis. Certain subspecies of Glossina have adjusted to the decrease in natural habitats by exhibiting peri-domestic behaviors that allow them to live in urban and suburban habitats near humans. The increase in urban and suburban habitats for subspecies of the tsetse vector Glossina has reduced the effectiveness of efforts to control, much less eliminate, transmission of the causative species Trypanosoma brucei rhodesiense (causes the acute form of the disease) and Trypanosoma brucei gambiense (causes the chronic from of the disease). This study provides a cautionary tale about the influence of habitat changes and climate change on the future of HAT transmission (and other pathogens), assuming these changes continue to occur. In the next study, we move west off of the continent of Africa to consider a species of mosquito in the genusCulex and its possible role in the transmission of Dirofilaria immitis, the primary causative agent of heartworm disease. Although the Canary Islands are a known endemic focus of Dirofilaria immitis, the mosquito vector on the island has, heretofore, not been identified. This article by Morchon Garcia et al. provides convincing evidence that they found the mosquito species that serves as the vector of D. immitis on the islands. Five potential vector species of mosquitoes were collected on the islands. Using molecular biological techniques, the authors identified one species of mosquito, Culex theileri, which was infected with D. immitis. Recent studies in Iran [4] and Spain [5] suggest that C. theileri is a competent vector species. In addition, comparing analyses of the mtDNA in the cox1 gene between C. theileri populations in the Canary Islands and Iran suggest that the two populations are molecularly similar. Although the results of this study are supportive of the role of C. theileri in the transmission of D. immitis in the Canary Islands, the authors caution that additional studies are required to conclusively support their suggestions. Our next study takes us to Asia and consideration of the status of Visceral Leishmaniasis (VL) in Iran. In his review, Mohebali combines his personal experience with numerous (>3,000) cases of leishmaniasis in Iran with results obtained from a search of databases from 1996 through 2010. The author’s results suggest that canids serve as the primary reservoir hosts of infection of humans a result found in previous studies [6]. Several species of Phlebotomus serve the insect vectors in Iran. In Iran, VL appears to be a disease of the young as children 12 years of age and younger made up 92.8% of the reported cases of VL during the time span under review. Mohebali found that the direct agglutination test was an effective means of detecting antiLeishmania antibodies and recommended it as a surveillance tool. The author concluded that VL is a serious zoonotic threat in Iran with sporadic prevalence in most geographic zones while being endemic in northwestern and southern Iran. Finally, we go further east and north to Mongolia where Papageorgiou et al. examined the epidemiology of tick-borne pathogens in two of the country’s provinces (aimags). Livestock were investigated using serology and PCR to identify exposure and infection

Host-Feeding Pattern of <I>Culex theileri</I> (Diptera: Culicidae), Potential Vector of <I>Dirofilaria immitis</I> in the Canary Islands, Spain

To identify the host range of potential vectors of Dirofilaria immitis Leidy, the causal agent of canine dirofilariasis, we studied the bloodmeal origin of mosquitoes trapped on two of the Canary Islands, Gran Canaria and Tenerife, where this disease is considered hyperendemic. On Gran Canaria, mosquitoes were captured using Centers for Disease Control and Prevention (CDC) traps (outdoors) and resting in a bathroom (indoors). Only CDC traps were used to capture mosquitoes in Tenerife. The species captured in decreasing order of abundance were Culex theileri Theobald, Culex pipiens L., Culiseta longiareolata Macquart, Anopheles atroparvus van Thiel, and Anopheles cinereus Theobald. The origins of bloodmeals were identified for 121 Cx. theileri and 4 Cx. pipiens after amplification and sequencing of a fragment of the vertebrate cytochrome oxidase I (COI) gene. Cx. theileri fed on goats, sheep, dogs, cattle, cats, humans, and chickens, and Cx. pipiens fed on goats and chickens. A lower success of bloodmeal identification was obtained in mosquitoes captured resting indoors than outdoors in CDC traps, probably because of a longer time period between feeding and capture. Although most Cx. theileri fed on ruminants, this species also fed on different mammal species susceptible to dirofiliarasis, including humans, suggesting it could play a role on parasite transmission.

Identification and Characterization of Single Nucleotide Polymorphisms (SNPs) inCulex theileri(Diptera: Culicidae)

Culex theileri Theobald (Diptera: Culicidae) is one of the most common mosquito species in northeastern Turkey and serves as a vector for various zoonotic diseases including West Nile virus. Although there have been some studies on the ecology of Cx. theileri, very little genetic data has been made available. We successfully sequenced 11 gene fragments from Cx. theileri specimens collected from the northeastern part of Turkey. On average, we found a Single nucleotide polymorphism every 45 bp. Transitions outnumbered transversions, at a ratio of 2:1. This is the first report of genetic polymorphisms in Cx. theileri and Single nucleotide polymorphism discovered from this study can be used to investigate population structure and gene-environmental interactions.

Host-Feeding Patterns ofCulex pipiensand Other Potential Mosquito Vectors (Diptera: Culicidae) of West Nile Virus (Flaviviridae) Collected in Portugal

The host blood-feeding patterns of mosquito vectors affects the likelihood of human exposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus, WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquito species, including Culex pipiens L., considered the primary vector of WNV to humans. The sources of bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010 in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipiens was the most common species collected and successfully analyzed (n = 135/78). In addition, blood-fed females of the following species were analyzed: Ochlerotatus caspius Pallas (n = 20), Culex theileri Theobald (n = 16), Anopheles maculipennis s.l. Meigen (n = 10), Culiseta longiareolata Macquart (n = 7), Aedes aegypti L. (n = 6), Culex perexiguus Theobald (n = 3), Culiseta annulata Schrank (n = 3), and Ochlerotatus detritus Haliday (n = 3). The Cx. pipiens mosquitoes fed predominantly on birds (n = 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood was identified in 18 specimens (18/78, 23.1%). No significant differences were found between the host-feeding patterns of blood-fed Cx. pipiens collected in residential and nonresidential habitats. The occurrence of human derived blood meals and the presence of a mix avian-human bloodmeal accordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiens may play a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission. In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and the potential consequence on feeding behavior and WNV transmission are discussed.

Genetic characterization of an insect-specific flavivirus isolated from Culex theileri mosquitoes collected in southern Portugal

We describe the full genetic characterization of an insect-specific flavivirus (ISF) from Culex theileri (Theobald) mosquitoes collected in Portugal. This represents the first isolation and full characterization of an ISF from Portuguese mosquitoes. The virus, designated CTFV, for Culex theileri flavivirus, was isolated in the C6/36 Stegomyia albopicta (=Aedes albopictus) cell line, and failed to replicate in vertebrate (Vero) cells in common with other ISFs. The CTFV genome encodes a single polyprotein with 3357 residues showing all the features expected for those of flaviviruses. Phylogenetic analyses based on all ISF sequences available to date, place CTFV among Culex-associated flaviviruses, grouping with recently published NS5 partial sequences documented from mosquitoes collected in the Iberian Peninsula, and with Quang Binh virus (isolated in Vietnam) as a close relative. No CTFV sequences were found integrated in their host's genome using a range of specific PCR primers designed to the prM/E, NS3, and NS5 region.

Species Composition and WNV Screening of Mosquitoes from Lagoons in a Wetland Area of the Algarve, Portugal.

The aim of this study was to evaluate mosquito abundance, species diversity, larval and adult population dynamics in seven lagoons integrated in the wetland coastal system of the Algarve, Portugal, in the summer of 2007, as well as the screening of these for West Nile virus (WNV). WNV has been isolated from mosquitoes in this region, in the summer of 2004, next to the putative area of infection of two linked human WN cases. Adult mosquitoes were collected with CDC traps baited with CO2, and potential breeding sites were surveyed for immature stages. Morphological identification of 1,432 adult mosquitoes and 85 larvae revealed the presence of 10 species: Anopheles atroparvus, Anopheles algeriensis, Coquillettidia richiardii, Culex modestus, Culex pipiens, Culex theileri, Culex univittatus, Culiseta longiareolata, Aedes caspius, and Aedes detritus. Adult mosquito peak densities were recorded in July, contrasting with null larval breeding in the same month in the surveyed biotopes. Most abundant species were C. pipiens (52%), C. theileri (29%), and A. caspius (11%). Lagoon Salgados and Quinta das Salinas, exhibited the highest similarity of culicid fauna, despite being most distant from each other, Female mosquitoes (1,249 specimens) screened by RT-PCR, did not reveal WNV products. However, previous detection of WNV activity in this area, susceptible to re-introductions, demands for continued vigilance.

Mosquito Surveys andWest Nile VirusScreening in Two Different Areas of Southern Portugal, 2004–2007

Longitudinal mosquito surveys were carried out in southern Portugal from 2004 to 2007, in a wetland area (Comporta, District of Setúbal) and around the perimeter of a dam irrigation plant that created the largest artificial lake in Europe, 250 km(2) (Alqueva, Districts of Evora and Beja). Our aim was to study the diversity, abundance, and seasonal dynamics of mosquitoes, comparing these two different areas, to screen mosquitoes for West Nile Virus (WNV), an arboviral agent already detected in Portugal, because these areas are populated with abundant avian fauna. Monthly collections of adult mosquitoes were carried out by Centers for Disease Control light-traps with CO(2) and by indoor resting collections. Mosquitoes were identified and screened for arboviruses by reverse transcriptase (RT)-polymerase chain reaction directed toward amplification of a 217-bp fragment of the NS5 gene. Mosquito peak densities were observed in July-August in Comporta and May-June, with a plateau in July-October, in Alqueva. However, densities were far higher in Comporta area (220,821 specimens) than in Alqueva area (9442 specimens), with a clear difference in species distribution, as in Comporta the predominant species was Culex theileri (85%), followed by Aedes caspius (6%), Anopheles atroparvus (4%), and Culex pipiens sensu latu (s.l.) (3%), whereas in Alqueva the predominant species was Cx. pipiens s.l. (56%), followed by An. atroparvus (18%), Cx. theileri (14%), and Culiseta longiareolata (9%). Female mosquitoes (8842 in 175 pools) of the species Ae. caspius, An. atroparvus, Culex mimeticus, Cx. pipiens Sensu latu (s.l.), Cx. theileri, and Culex univittatus were screened and found to be negative for WNV genomic RNA. Although there was no detection of WNV sequences in mosquitoes, vigilance should continue as the circulation of virus has been previously detected more than once in Portugal, in humans, animals, and mosquitoes, and in other surrounding Mediterranean countries.

Biting Activity of Mosquito Species (Diptera: Culicidae) in the Turkey-Armenia Border Area, Ararat Valley, Turkey

During nine consecutive nights in July 2007 (from 18:15-05:45 h), mosquitoes landing-biting on humans were collected outdoors and indoors at the Turkey-Armenia border. A total of 1005 females were collected consisting of nine species. The dominant species was Aedes dorsalis (Meigen) (47.5% of total catch), followed by Anopheles hyrcanus (Pallas) (22.9%), Culex theileri (Theobald) (9.3%),Ae. vexans (Meigen) (6.6%), Ae. caspius (Pallas) (4.9%),Anopheles maculipennis s.l. (Meigen) (3.1%), Culex territans (Walker) (2.8%), Coquillettidia richiardii (Ficalbi) (1.6%), and Cx. pipiens L. (1.5%). The biting rate outdoors (15.1 mosquitoes/human/h) was greater than indoors (3.4 mosquitoes/human/h). The landing-biting of Ae. dorsalis peaked at dusk (19:15-19:45 h) and dawn (04:15-04:45 h). Ae. vexans activity increased soon after dark (20:15-20:45 h) and reached a peak at dawn (04:15-04:45 h). Maximum biting activity of An. hyrcanus and Cx. theileri occurred during the first sampling interval after dusk (20:15-20:45 h). A large number of An. maculipennis s.l. adults were collected during the second half of the night. We believe that these findings will contribute to decisions on the timing of mosquito control in Ararat Valley.

Distribution of ‘promoter’ sandflies associated with incidence of classic Kaposi's sarcoma

The patchy geographical distributions of classic Kaposi's sarcoma (KS) and human herpesvirus type 8 (HHV-8), better known as Kaposi's sarcoma-associated herpesvirus (KSHV) remain unexplained. It has been proposed that certain species of bloodsucking insects ('promoter arthropods') promote the reactivation of HHV-8/KSHV and facilitate both HHV-8/KSHV transmission and KS development. This hypothesis was tested by sampling the presence and density of human-biting Diptera with CDC light traps in two areas of Sardinia with contrasting incidence rates of classic KS. In total, 11,030 specimens (99.9% sandflies and 0.1% mosquitoes) belonging to 10 species were collected from 40 rural sites. Five of these species are considered to be possible promoter arthropods because of the irritation their bites cause: Phlebotomus perniciosus Newstead; Phlebotomus perfiliewi Parrot (Diptera: Psychodidae); Aedes berlandi Seguy; Culiseta annulata (Schrank) and Culex theileri Theobald (Diptera: Culicidae). Five species are probable 'non-promoters' because their bites are not particularly irritating: Culiseta longiareolata (Macquart); Culex pipiens s.l.; Anopheles algeriensis Theobald; Anopheles maculipennis s.l., and Anopheles plumbeus Stephens. A significant correlation was found between the geographical distribution of promoter arthropods and incidence rates of KS (Spearman's r = 0.59,P < 0.01). Promoter arthropods were more likely to be caught in areas with cutaneous leishmaniasis and a past high prevalence of malaria, and in areas of limestone, acid volcanic soil and cereal cultivation. The study supports the association between promoter arthropods and classic KS, which may explain the geographic variability of KS and HHV-8/KSHV, and highlights the links with a number of variables previously associated with the incidence of KS.

The Mosquito Fauna of the Republic of Cyprus: A Revised List

The Cyprus Public Health Service has regularly conducted mosquito surveillance in the Republic of Cyprus over the past 10 years. Twenty-three species belonging to 6 genera and 10 subgenera have been recorded to date, including species documented from earlier surveys. As a result of this program, new mosquito species for Cyprus have been recorded, including Anopheles marteri, Culex theileri, Cx. impudicus, Culiseta subochrea, and Uranotaenia unguiculata. Importantly, mosquito species previously considered eradicated have reemerged (An. sacharovi). Monitoring and identification of mosquito species is an important component of the Public Heath Service's commitment to protecting the health of residents and preventing the spread of vector-borne diseases.

Initial and Residual Activity of VectoBac® 12 AS, VectoBac® WDG, and VectoLex® WDG for Control of Mosquitoes in Ararat Valley, Turkey

Two formulations, VectoBac 12 AS and VectoBac WDG, of Bacillus thuringiensis israelensis (Bti) and one formulation, VectoLex WDG, of Bacillus sphaericus were tested against Anopheles maculipennis, Culex pipiens, Culex theileri, Aedes caspius, and Aedes dorsalis larvae in drainage canals, a flooded plain, and a drainage well in the Igdir Plain of Arafat Valley, Turkey. VectoBac 12 AS applied at 0.5 and 1 liter/ha to a drainage canal provided complete control of Cx. theileri and 80% to 98% control of An. maculipennis. VectoLex WDG at 400 g/ha provided 100% larval control of Cx. pipiens and Cx. theileri in polluted drainage canals. High larval density of Ae. caspius decreased the efficacy of VectoBac 12 AS and VectoBac WDG in the flooded plain. Residual effects of Bti formulations lasted only a few days in the breeding areas. Residual activity of VectoLex WDG was negatively influenced by water depth, organic pollution, and sunlight and positively influenced by low water velocity and emergent vegetation.

Natural Infection of Culex theileri (Diptera: Culicidae) with Dirofilaria immitis (Nematoda: Filarioidea) on Madeira Island, Portugal

Field and laboratory studies were performed to verify whether Culex theileri Theobald functions as a natural vector of Dirofilaria immitis (Leidy) on Madeira Island, Portugal. CO2-baited light traps (EVS traps) were use to sample mosquitoes monthly basis between February 2002 and February 2003 in the area of Quebradas (Funchal). Three mosquito species were captured, including 58 Culex pipiens L., 790 Cx. theileri, and three Culiseta longiareolata (Macquart). Only C. theileri tested positive for D. immitis. The presence of this filarial worm was detected by direct observation, infectivity assay dissection technique, and polymerase chain reaction methods. Infected mosquitoes were recovered in October and December 2002 and January 2003. These data provide evidence that Cx. theileri could be the main vector of D. immitis in Funchal, Madeira.

The ecology of West Nile virus in South Africa and the occurrence of outbreaks in humans.

This paper reviews studies done on West Nile virus (WNV) in South Africa, mainly between 1962 and 1980 on the temperate inland plateau (Highveld and Karoo). The virus is maintained in an enzootic transmission cycle between feral birds and the ornithophilic mosquito Culex univittatus. About 30 avian species have been shown to be involved without mortality. Humans, and other mammals, although they may have antibodies, are considered blind-alleys in the transmission cycle except perhaps some dogs. Cx. univittatus also transfers infection to humans, almost invariably causing only a mild illness. Its usually low anthropophilism may explain why annual human infection on the Highveld is limited to sporadic cases. Besides multiple isolations from field collections of Cx. univittatus, this mosquito is both highly susceptible to the virus and an efficient transmitter. Culex theileri is a minor vector. In the summer of 1974 there was a large epidemic in the dry Karoo after unusual rains: there were many human cases, the infection rate in Cx. univittatus was 39.0/1000, and postepidemic immune rates in humans and birds were high. In 1984 there was an epizootic in Gauteng Province in the Highveld with an infection rate in Cx. univittatus reaching 9.6/1000 and more human infections than usual. The much lower immune rates in the KwaZulu-Natal coastal lowlands than on the plateau and the single isolation from Cx. neavei, which replaces Cx. univittatus in the lowlands, are explained by the low susceptibility of Cx. neavei to the virus. Genetic relatedness of isolates from different countries showed two lineages, with one lineage comprising only African isolates, including 25 South African strains, which had a sequence homology of 86.3-100%. This suggests that the viral enzooticity does not depend on annual importation of virus in migrant birds.

Establishment and Characterization of a Cell Line from the Mosquito Culex (Culex) theileri (Diptera: Culicidae) and Its Susceptibility to Infection with Arboviruses

The mosquito Culex (Culex) theileri Theobald is probably the major vector of Rift Valley fever (RVF) virus during epizootics on the inland plateau of southern Africa. A cell line was established from the tissues of this mosquito species to facilitate laboratory studies on RVF and other arboviruses. After temperature pretreatment, egg rafts from a laboratory colony were disinfected, mechanically disrupted, and grown in preconditioned Eagles minimum essential medium supplemented with 10% fetal calf serum. The cells were characterized by their protein content, karyology, and their susceptibility to three arboviruses. Although high infectivity titers were obtained, no cytopathic effects were produced by these viruses. The cell line was shown to be free from latent viruses. These results show that this cell line may be employed fruitfully in arbovirus studies.

Isolation of Germiston virus, a hitherto unknown agent, from culicine mosquitoes, and a report of infection in two laboratory workers.

Summary 1. Two strains of filterable virus were isolated from culicine mosquitoes collected at Germiston, Union of South Africa. One was derived from a mixed pool of 5 Culex theileri and 12 C. rubinotus and the other from a pool of 11 C. rubinotus. Results of tests reported herein indicate that the two strains are antigenically indistinguishable and the name Germiston virus has been applied in reference to the locality of origin. 2. Germiston virus is related to but not identical with Bunyamwera virus. With immune sera obtained after inoculation of laboratory animals, antigenic differences between the two viruses were easily demonstrated by HI and CF tests, less easily by neutralization test. 3. Mice immunized with Bunyamwera were not significantly protected when challenged with Germiston virus. 4. Two clinical cases of Germiston virus infection occurred in laboratory workers. Even though a significant level of Bunyamwera virus neutralizing antibody was present in sera of one of the individuals within only a few hours after onset and also in a specimen taken approximately 7 months previously, infection with Germiston virus was not prevented. The implications of this observation and results of other tests on sera from this case obtained at various time intervals are discussed. 5. Observations are presented which lead to the suspicion that birds may have had a role in infecting mosquitoes with Germiston virus.