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Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species.

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.

Animals↗

Molecular phylogeny of the Vomerifer and Pedroi Groups in the Spissipes Section of the subgenus Culex (Melanoconion).

Members of the New World mosquito subgenus Melanoconion of the genus Culex are important vectors of many alphaviruses including eastern and Venezuelan equine encephalitis viruses (VEEV). We investigated the phylogenetic relationships among nine putative species of the Vomerifer and Pedroi Groups of the Neotropical Spissipes Section by sequencing the internal transcribed spacer 2 (ITS-2) region of ribosomal DNA and using phylogenetic analyses. Results demonstrated that, within the Spissipes Section, the Vomerifer and Pedroi Groups are monophyletic sister groups. The clade comprised by Culex adamesi and Culex ribeirensis showed a sister group relationship to the group consisting of Culex pedroi sequences. The monophyly of the Vomerifer Group corroborated previous suggestions that it is a natural group. However, our topology showed that there are two well-supported, divergent groups within a major clade consisting of Cx. pedroi sequences, suggesting the possibility of a cryptic Cx. pedroi-like species. This finding could have important epidemiological implications for VEEV transmission in Central and South America.

Alphavirus↗

Culex population dynamics and West Nile virus transmission in east-central Illinois.

Temporal changes in the abundance Culex restuans and Culex pipiens were monitored in east-central Illinois for over a decade using infusion-baited oviposition traps. The 2 species typically exhibited a seasonal shift in relative abundance with a mean crossover date (when the proportion of egg rafts from both species is equal) of August 10 or 11, depending on leap year, with a 95% confidence interval of +/- 10.7 days. The date of crossover was linearly related to the date of last spring frost and occurred on average about 123 days after the last spring frost. Despite the predictability of crossover, the weekly pattern in the proportion of Cx. pipiens before and after crossover varied considerably, even between years with similar crossover dates. After West Nile virus became established in our area, we found that transmission based on Culex from gravid traps did not increase until Cx. pipiens abundance increased in oviposition traps. Infection rates peaked within the half-month period after crossover. The peak in Cx. pipiens abundance in oviposition traps during this 3-year period was between the 2nd half of August and the end of September. A higher magnitude of transmission in 2002 coincided with warmer temperatures during July and August and an extended period in which the 2 Culex species were in relatively equal abundance.

Animals↗

Experimental transovarial transmission of St. Louis encephalitis virus by Culex and Aedes mosquitoes.

Colonized and field-collected female Culex tarsalis, infected with St. Louis encephalitis, (SLE) virus by intrathoracic inoculation or by feeding on a viremic host, transmitted virus to their F1 adult and/or larval progeny when reared at 18(+/- 1) degree C but not when reared at 27(+/- 1) degrees C. The minimal infection rates (MIR) for different populations of Cx. tarsalis ranged from 1:32 to less than 1:250 (mean = 1:121) for larval progeny and from 1:32 to less than 1:1, 989 (mean = 1:1,571) for adult progeny. SLE virus also was transmitted transovarially by colonized and field-collected populations of Culex pipiens (mean MIRs = 1:340 and 1:1,815 for larval and adult progeny, respectively) and by a field population of Culex quinquefasciatus (MIR = 1:500 and less than 1:246 for larval and adult progeny, respectively), but not by colonized strains of Cx. quinquefasciatus and Culex peus. SLE virus was not recovered in tests on 5,522 Cx. tarsalis and 4,798 Cx. quinquefasciatus that were collected as larvae or pupae from field sites in Southern California and reared to adults at 18 degrees C in the laboratory. Transovarial transmission of SLE virus by Aedes epactius was confirmed and extended to a closely related species, Aedes atropalpus. Efforts to demonstrate transovarial transmission of SLE virus by Aedes melanimon, Aedes sierrensis, and Aedes triseriatus were unsuccessful. Aedes dorsalis, Cx. peus, and Toxorhynchites amboinensis were equally sensitive hosts for viral isolation when inoculated with suspensions of larvae transovarially infected with SLE virus.

Aedes↗

A polymerase chain reaction based method for the detection of Culex quinquefasciatus (Diptera: culicidae).

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus Dönitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.

Animals↗

The abundance and seasonal distribution of Culex mosquitoes in Iowa during 1995-97.

The abundance and seasonal distribution of Culex mosquitoes were monitored by measuring oviposition activity during the summers of 1995, 1996, and 1997. Five species of culicine mosquitoes laid egg rafts in ovitraps. Egg rafts of 4 Culex species-Cx. restuans (54.98%), Cx. pipiens (25.41%), Cx. salinarius (12.18%), and Cx. tarsalis (0.14%)-constituted more than 92% of the total egg rafts collected. Culiseta inornata (0.1%) was the only other species to lay viable egg rafts in the ovitraps. A small percentage (7.19%) of egg rafts did not hatch; thus identification was not possible. Compared with New Jersey light trap data in a nearby area, the abundance of Cx. tarsalis and Cs. inornata was markedly underestimated with ovitraps. These data may also reflect differences in the specific location of the 2 trap sites rather than attractiveness of ovitraps for Cx. tarsalis and Cs. inornata. In general, Cx. restuans oviposition activity began in late May, and it was the dominant Culex species through June. After July 1, the number of egg rafts laid by Cx. restuans decreased continually until the end of the summer. Culex pipiens and Cx. salinarius oviposition began in early June and increased gradually during the summer. Over the course of a summer, there were about twice as many Cx. pipiens as there were Cx. salinarius. A crossover in the number of Cx. pipiens and Cx. restuans egg rafts occurred in late July or early August, depending on the year. By late August each year, Cx. pipiens was the most abundant species. At this time, Cx. restuans and Cx. salinarius populations were similar, but each was about half of the Cx. pipiens population. Differences in the abundance of all 3 species could not be explained by changes in ambient temperature (both minimum and maximum) or relative humidity, either within or among years.

Animals↗

Molecular and electron microscopic identification of Wolbachia in Culex pipiens complex populations from the Upper Rhine Valley, Germany, and Cebu City, Philippines.

We detected Wolbachia infections in Culex pipiens complex mosquitoes from the Upper Rhine Valley, Germany, and Cebu City, Philippines, with the use of polymerase chain reaction (PCR) amplification of the 16S rDNA of the bacteria and further confirmation by electron microscopy. Species of 5 different autogenous and anautogenous populations of Culex pipiens from Germany and 1 population of Culex quinquefasciatus from the Philippines were used in this study. Larvae were sampled from different localities and reared to the adult stage for further processing. Whole adult males and the heads of adult females were processed by PCR, whereas the abdomens of females were used for electron microscopic study when an infection was detected by PCR. All Culex populations assayed by PCR showed infection of Wolbachia at rates between 10 and 100%. Females from different populations exhibited higher infection rates than did the males, and, likewise, Cx. pipiens populations that were autogenous showed higher infection rates of Wolbachia than did anautogenous individuals. In Cx. quinquefasciatus populations, males were infected at 33.33% and females, only 10%. We studied the ultrastructure of Wolbachia in the ovaries of species belonging to Cx. pipiens complex by means of transmission electron microscopy. The bacteria exhibited typical morphology for Wolbachia with 3 enveloping membranes.

Animals↗

The relative attractiveness of carbon dioxide and octenol in CDC- and EVS-type light traps for sampling the mosquitoes Aedes aegypti (L.), Aedes polynesiensis Marks, and Culex quinquefasciatus say in Moorea, French Polynesia.

Two dominant day-biting pests and vector species on the island of Moorea in French Polynesia are Aedes (Stegomyia) aegypti (L.) and Aedes (Stegomyia) polynesiensis Marks, major vectors of dengue viruses and Wuchereria bancrofti, respectively. Their surveillance is hindered by a relative lack of attraction to light traps, necessitating the undesirable use of human bait collections with the inherent risks of pathogen transmission. The effectiveness of CDC- and EVS-type light traps baited with olfactory attractants was evaluated for these two Aedes species and the nocturnal Culex (Culex) quinquefasciatus Say in three sites in urban and semi-rural environments on Moorea in October/November 2003. Firstly, four CDC-type traps with light only, light with octenol, light with carbon dioxide (dry ice), and light with octenol plus carbon dioxide were operated continuously over four days with daily rotation to compensate for position effects. Secondly, two CDC- and two EVS-type traps with carbon dioxide or carbon dioxide plus octenol were operated continuously over four days with similar rotation. Variation was found in the numbers of the three species collected at the different sites, reflecting the relative availability of their preferred larval habitats. With the CDC traps in the first trial, the addition of octenol to the light did not significantly increase the collection of any species, the addition of carbon dioxide did significantly increase collection of all three species, while the addition of octenol to the light plus carbon dioxide did not significantly increase the collections further. In the second trial, there was no significant difference in the mean number of Ae. aegypti or Ae. polynesiensis collected in either EVS or CDC traps when baited with carbon dioxide or with octenol added. For Cx. quinquefasciatus, the supplementation with octenol made no significant difference with EVS traps but resulted in significantly reduced collections in CDC traps. Overall, neither trap, however baited, provided large samples when compared with landing/ biting collections at human bait. Only two other species were collected, Culex (Culex) roseni Belkin and Aedes (Aedimorphus) nocturnus (Theobald), the latter being a first record for the island of Moorea and for French Polynesia.

Aedes↗

[Seasonal variation of three Culex species (Diptera: Culicidae) and its parasites and pathogens in Punta Lara, Buenos Aires, Argentina].

The seasonality of immatures, imagoes and pathogens of Culex dolosus Lynch Arribálzaga, Culex intrincatus Brethes and Culex maxi Dyar from Punta Lara (Buenos Aires province, Argentina) was monitored weekly (larvae and pupae) or fortnightly (adults), from 1989 to 1991. Culex dolosus was present during all months. The larvae were parasitized by Geotrichum candidum Link ex Person, Smittium morbosum var. rioplatensis López Lastra, Coelomomyces sp., Achlya sp, Amblyospora dolosi García & Becnel and Strelkovimermis spiculatus Poinar & Camino. Conidia of Hyphomycetes, filarial worms of Onchocercidae and S. spiculatus were detected in adults. Cx. intrincatus was present from January to May; the immatures were parasitized by G. candidum, S. morbosum, Achlya sp. and Amblyospora sp. Larvae of Cx. maxi were collected from January to April, and adults in all months except for June and July. S. morbosum was the only parasite found in this mosquito.

Animals↗

Molecular comparisons of the Culex pipiens (L.) complex esterase gene amplicons.

The amplification of carboxylesterase genes is a mechanism of organophosphate resistance in Culex mosquitoes. Amplified carboxylesterase genes from an insecticide resistant Culex pipiens strain collected in Cyprus were analysed and compared to other Culex amplified carboxylesterase alleles. A 12 kb section of genomic DNA containing two gene loci coding for carboxylesterase alleles A5 and B5 was cloned and sequenced. A comparison between this amplicon and one from a strain with co-amplified carboxylesterase alleles A2 and B2 revealed a number of differences. The intergenic spacer was 3.7 kb in length in the A5-B5 amplicon (2.7 kb in A2-B2) and contained putative Juan and transposable elements upstream of B5. A fragment of a gene with high homology to aldehyde oxidase was also present immediately downstream of A5. The comparison revealed no differences that would explain the successful spread of the A2-B2 amplicon worldwide whilst the A5-B5 amplicon is restricted to the Mediterranean.

Aldehyde Oxidase↗

Genetic characterization of a new insect flavivirus isolated from Culex pipiens mosquito in Japan.

We found a new flavivirus that is widespread in Culex pipiens and other Culex mosquitoes in Japan. The virus isolate, named Culex flavivirus (CxFV), multiplied only in mosquito cell lines producing a moderate cytopathic effect, but did not grow in mammalian cells. The CxFV genome is single-stranded RNA, 10,834 nt in length and containing a single open reading frame encoding a polyprotein of 3362 aa with 5' and 3' untranslated regions (UTRs) of 91 and 657 nt, respectively. Phylogenetic analyses revealed that CxFV is closely related to the insect flaviviruses associated with Aedes mosquitoes, Cell fusing agent (CFA) and Kamiti River virus (KRV). The 3' UTR of CxFV contains four tandem repeats, which have sequence similarities to the two direct repeats in the CFA and KRV 3' UTRs. These results suggest that CxFV may be a new group of insect flaviviruses.

Aedes↗

The receptor of Bacillus sphaericus binary toxin in Culex pipiens (Diptera: Culicidae) midgut: molecular cloning and expression.

Culex pipiens larval midgut is the primary target of the binary toxin (Bin) present in parasporal inclusions of Bacillus sphaericus. Cpm1, a 60-kDa protein purified from brush border membranes, has been proposed as the receptor of the Bin toxin in the midgut epithelial cells of mosquitoes. We have cloned and characterized the corresponding cDNA from midgut of Culex pipiens larvae. The open reading frame predicted a 580 amino-acid protein with a putative signal peptide at the N-terminus and a putative GPI-anchoring signal at the C-terminus. The amino acid sequence of the cloned Cpm1 exhibited 39-43% identities with insect maltases (alpha-glucosidases and alpha-amylases). Recombinant Cpm1 expressed in E. coli specifically bound to the Bin toxin and had a significant alpha-glucosidase activity but no alpha-amylase activity. These results support the view that Cpm1 is an alpha-glucosidase expressed in Culex midgut where it constitutes the receptor for the Bin toxin. To date, this is the first component involved in the mosquitocidal activity of the Bacillus sphaericus Bin toxin to be characterized. Its identification provides a key step to elucidate the mode of action of the Bin toxin and the mechanisms of resistance developed against it by some mosquito strains.

Amino Acid Sequence↗

Molecular characterization of the amplified aldehyde oxidase from insecticide resistant Culex quinquefasciatus.

Primary structural information including the complete nucleotide sequence of the first insect aldehyde oxidase (AO) was obtained from the common house mosquito Culex quinquefasciatus (Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO. The Culex AO sequence contains a molybdopterin cofactor binding domain and two iron-sulfur centres. A comparison of the partial sequences of AO from insecticide resistant and susceptible strains of C. quinquefasciatus shows two distinct alleles of this enzyme, one of which is amplified in the insecticide resistant strain on a 30-kb DNA amplicon alongside two resistance-associated esterases. The amplified AO gene results in elevated AO activity in all life stages, but activity is highest in 3rd instar larvae. The elevated enzyme can be seen as a separate band on polyacrylamide gel electrophoresis. The role of AO in xenobiotic oxidation in mammals and the partial inhibition of elevated AO activity by a range of insecticides in Culex, suggest that this AO may play a role in insecticide resistance.

3' Untranslated Regions↗

Observations on Culex quinquefasciatus Say in relation to transmission of filariasis due to subperiodic Wuchereria bancrofti in Samoa.

Observations on Culex quinquefasciatus Say in Samoa during a study of the epidemiology and control of subperiodic bancroftian filariasis are reported. The man-biting rate of C. quinquefasciatus was comparable with that of Aedes (Finlaya) samoanus Gruenberg in one indicator village and lower in another. The house-frequenting behaviour of the two species was similar. Culex quinquefasciatus was active throughout the night with peak biting during midnight hours, and its survivorship, estimated by two methods, was higher than those of the main vectors, A. (Stegomyia) polynesiensis Marks and A. samoanus. Culex quinquefasciatus showed low susceptibility to subperiodic Wuchereria bancrofti, compared with the two main vectors, in laboratory feeding experiments on volunteers with three levels of microfilaraemia. This agrees with the observed very low natural infection rate, suggesting that C. quinquefasciatus is an inefficient vector of subperiodic W. bancrofti in Samoa.

Animals↗

Culex species (Diptera: Culicidae) from central Argentina: identification by electrophoretic zymograms and genetic relationships.

Starch gel electrophoretic zymograms were used for the taxonomic separation of mosquitoes of the genus Culex, possible vectors of encephalitis in Argentina. Catalase, hexokinase, malic enzyme, isocitrate and malate dehydrogenases, and leucine aminopeptidase were species markers that allowed the clear identification of individual Culex apicinus Philippi, Cx. bidens Dyar, Cx. brethesi Dyar, Cx. declarator Dyar and Knab, Cx. dolosus Arribalzaga, Cx. maxi Dyar, Cx. quinquefasciatus Say, Cx. saltanensis Dyar, and Cx. tatoi Casal & García. Intraspecific polymorphism was revealed but did not preclude species separation. On the basis of allele frequencies, an index of genetic distance between species was calculated. Genetic affinities determined by electrophoretic data agreed with taxonomic affinities ascertained by conventional systematics. Thus, electrophoretic zymograms proved to be a powerful tool for the study of the genus Culex in Argentina.

Alleles↗

Interrupted blood feeding by Culex (Diptera:Culicidae) in relation to individual host tolerance to mosquito attack.

Field studies at Delta Marsh (1991) and Winnipeg (1993), MB, and Vero Beach (1992), FL, examined variation among individual quail hosts in terms of the blood-feeding success by mosquitoes that fed on them. Culex tarsalis Coquillett was the predominant species collected in box traps baited with 2 quail at Delta Marsh in 1991, Culex nigripalpus Theobald was the predominant species collected at Vero Beach in 1992, and both Cx. tarsalis and culex restuans Theobald were collected in approximately equal numbers at Winnipeg in 1993. In 48 of 70 samples, blood feeding was skewed significantly from an even distribution between the 2 quail in each trap. In 5 of 13 samples of Cx. tarsalis collected in 1991, incomplete feeding (proportion of blood meals < or = 1/2 full) was from 2 to 8 times more likely on one bird relative to the other. In 7 of 13 samples of Cx. nigripalpus collected in 1992, incomplete feeding was from 2 to > 10 times more likely on one bird relative to the other. Incomplete feeding was from 2 to > 10 times more likely on one bird relative to the other is 7 of 11 mixed Cx. restuans/Cx. tarsalis samples collected in 1993. The probability that a given quail would interrupt a feeding mosquito was correlated negatively with the probability of the same quail being fed on. The proportion of detectable multiple meals in a given trap night was correlated negatively with the degree to which the distribution of blood meals in each trap night was skewed away from 0.5 on each bird. Individual quail varied significantly in terms of the blood feeding success of mosquitoes that fed on them.

Animals↗

Contrasting levels of variability between cytoplasmic genomes and incompatibility types in the mosquito Culex pipiens.

Reproductive incompatibilities called cytoplasmic incompatibilities are known to affect a large number of arthropod species and are mediated by Wolbachia, a maternally transmitted microorganism. The crossing relationships between strains of potential hosts define their incompatibility types and it is generally assumed that differences between strains of Wolbachia induce different crossing types. Among all the described host species, the mosquito, Culex pipiens, displays the greatest variability of cytoplasmic incompatibility crossing types. We analysed mitochondrial and bacterial DNA variability in Culex pipiens in order to investigate some possible causes of incompatibility crossing type variability. We sequenced fragments of the ftsZ gene, and the A + T-rich control region of the mtDNA. We also sequenced the second subunit of the mitochondrial cytochrome oxidase (COII) gene, in Culex pipiens and a closely related species, C. torrentium, in order to verify the usefulness of the A + T-rich region for the present purposes. No variability was found in the Wolbachia ftsZ gene fragment, and very limited variation of the mitochondrial marker whatever the compatibility type or the origin of the host. A low variability was found in the A + T-rich region and comparison of divergence of the A + T-rich region and COII gene between C. pipiens and C. torrentium did not reveal any special constraints affecting this region. In contrast to observations in other host species, variability of incompatibility crossing types is not due to multiple infections by distantly related Wolbachia strains.

Animals↗

Overwintering of Culex mosquitoes in Sweden and their potential as reservoirs of human pathogens.

1. Culex pipiens L. and Cx torrentium Martini are suspected enzootic vectors in Sweden of Sindbis virus causing Ockelbo disease in man. To estimate their potential as virus reservoirs over the winter, mosquitoes were collected from August to May from a cellar in a rural area, central Sweden. The mosquitoes were dissected and examined for blood content, gonotrophic stage, parity and insemination, and tested for fructose. 2. Cx pipiens, Cx torrentium and Cx territans Walker constituted 94% of 887 females caught. A few Culiseta annulata (Schrank), Cs. alaskaensis (Ludlow) and An. maculipennis Meigen s.l. were also collected. 3. Data suggested that all blood-fed, parous Culex females disappeared during the winter and that only non-bloodfed, nulliparous females survived. 4. Fructose, detected in some of these females, and other plant sugars may be the main energy sources for winter survival. 5. Unless transovarially or venerally infected, Culex females are presumably of no or very limited importance in rural, central Sweden as winter reservoirs for blood-borne pathogens of vertebrates.

Animals↗