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Revista do Instituto de Medicina Tropical de São Paulo

On-line version ISSN 1678-9946

Rev. Inst. Med. trop. S. Paulo vol.54 no.6 São Paulo Nov./Dec. 2012 



Low genetic diversity in Wolbachia-Infected Culex quinquefasciatus (Diptera: Culicidae) from Brazil and Argentina


Baixa diversidade genética em Culex quinquefasciatus (Diptera: Culicidae) infectado por Wolbachia do Brasil e Argentina



Sirlei Antunes MoraisI; Fábio de AlmeidaII,III; Lincoln SuesdekII,III; Mauro Toledo MarrelliI

IDepartamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, SP, Brazil
IISeção Parasitologia, Instituto Butantan, São Paulo, SP, Brazil
IIIBiologia da Relação Patógeno-Hospedeiro, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil





Culex quinquefasciatus is a vector of human pathogens, including filarial nematodes and several viruses. Although its epidemiological relevance is known to vary across geographical regions, an understanding of its population genetic structure is still incipient. In light of this, we evaluated the genetic diversity of Cx. quinquefasciatus and Cx. pipiens x Cx. quinquefasciatus hybrids collected from nine localities in Brazil and one site in Argentina. We used mitochondrial genes cox1 and nd4, along with the coxA and wsp genes of the maternally-inherited Wolbachia endosymbiont. The nd4 fragment was invariant between samples, whilst cox1 exhibited four haplotypes that separated two types of Cx. quinquefasciatus, one clustered in southern Brazil. Low sequence diversity was generally observed, being discussed. Both Brazilian and Argentinian mosquitoes were infected with a single Wolbachia strain. As reported in previous studies with these populations, cox1 and nd4 diversity is not congruent with the population structure revealed by nuclear markers or alar morphology. Future Cx. quinquefasciatus research should, if possible, evaluate mtDNA diversity in light of other markers.

Keywords: Culex quinquefasciatus; Genetic diversity; Mitochondrial markers; Wolbachia.


Culex quinquefasciatus é vetor de patógenos humanos, incluindo nematódeos filarídeos e vários vírus. Embora a sua relevância epidemiológica varie entre as diferentes regiões geográficas, o conhecimento da estrutura genética da população é ainda incipiente. Em vista disso, foram avaliados os níveis de diversidade genética de Cx. quinquefasciatus e de híbridos Cx. quinquefasciatus x Cx. pipiens de nove cidades do Brasil e em La Plata, na Argentina. Para os testes foram utilizados fragmentos dos genes mitocondriais cox1 e nd4, juntamente com coxA e wsp do endossimbionte Wolbachia, herdado maternalmente. O fragmento nd4 não apresentou variação entre as amostras, e o cox1 exibiu quatro haplótipos que separaram dois tipos de Cx. quinquefasciatus, com um deles agrupado no sul do Brasil. Os dados de sequência mostraram baixa diversidade, sendo esta discutida. Ambas as amostras de mosquitos brasileiros e argentinos estão infectados com uma única cepa de Wolbachia. A diversidade apresentada por nd4 e cox1 não é congruente com a estrutura da população revelada por marcadores nucleares e morfologia alar de estudos anteriores com estas mesmas populações. Pesquisas com Cx. quinquefasciatus devem, se possível, avaliar a diversidade por DNA mitocondrial na luz de outros marcadores.




Although Culex quinquefasciatus Say (Diptera: Culicidae) is a vector of human pathogens, including agents for filariasis and several arboviruses, its epidemiological importance varies considerably among regions14. In equatorial-tropical regions, the species is reported as the primary vector of Wuchereria bancrofti in the transmission cycle of lymphatic filariasis36. In subtropical and temperate urban areas, it is implicated as the primary vector of West Nile Virus23 and other arboviruses11.

Species of the Culex pipiens complex share morphological similarities and generally proliferate in human settlements, with Cx. quinquefasciatus adapted to tropical and subtropical areas and Cx. pipiens to temperate regions. Their ranges overlap in intermediate areas, resulting in genetic introgression and hybridization10. In Brazil, Cx. quinquefasciatus has an expansive distribution, including in almost all major cities28. Hybrids of quinquefasciatus/pipiens occur in Uruguay and central Argentina, whilst Cx. pipiens occupies regions southward into Argentina4,30.

Because they are vectors of both urban and rural diseases, members of the pipiens subgroup have been targets of population control programs worldwide, with anthropophilic species killed by contact with chemical or biological reagents19. However, these populations often adapt resistance to these measures, with the selected organisms eventually expanding into different biogeographic regions.

In addition to pressure from control initiatives, these mosquitoes are often infected by endosymbionts such as Wolbachia pipientis rickettsies, which are associated with cytoplasmatic incompatibility (CI)3. Under CI dynamics, crosses between infected males with non-infected females produce eggs with decreased viability. Progenies originating from infected females and either infected or non-infected males are normally fertile. However, research has shown that offspring of individuals infected with different Wolbachia strains may be infertile24. Consequently, infected females have a reproductive advantage that leads to the expansion of infection in populations.

Recently, ALMEIDA2 showed that infection by a single Wolbachia strain was ubiquitous in all Cx. quinquefasciatus samples tested from São Paulo City, Brazil. Among the pipiens subgroup, insecticide-resistant populations tend to be infected with higher Wolbachia densities12. It has been posited that a low efficiency of Wolbachia control may be related to mosquito immunity factors. In view of this, the manipulation of Wolbachia infections has generated enthusiasm in the field of vector control21.

Despite its epidemiological relevance, population genetics data for Cx. quinquefasciatus are still sparse. We thus sought to analyze the genetic diversity of Cx. quinquefasciatus populations from urban areas of Brazil and central Argentina using cytochrome c oxidase subunit I (cox1) and NADH dehydrogenase subunit 4 (nd4) and these populations' patterns of Wolbachia pipientis infection through analysis of the Wolbachia surface protein (wsp) and cytochrome c oxidase subunit I (coxA).



Mosquitoes. Adult mosquitoes were collected by aspiration32 near residual-water channels during February and March 2008 in the Brazilian municipalities of Teresina (5°S), Recife (8°S), Rio Branco (9°S), Pariquera-Açu (24°S), Pelotas (31°S), Chapecó (27°S), Pontes e Lacerda (15°S), Santa Vitória do Palmar (33°S) and São Paulo (23°S), and in the Argentinian city of La Plata (34°S) (Fig. 1). Samples were stored in individual tubes on silica gel until processing. Thirty specimens from each locality (15 males and 15 females) were identified following taxonomic keys15 and subsequently analyzed genetically.



DNA extraction and amplification of mitochondrial genes. Genomic DNA was isolated from individual mosquitoes using DNeasy® Blood & Tissue kits (Qiagen) following the manufacturer's instructions. DNA was eluted to a final volume of 100 µL and stored at -20 °C until polymerase chain reaction (PCR). Internal primers were designed to shotgun-amplify the complete 1260-bp cox1 amplicon: two smaller fragments (~700-bp) were assembled following amplification using primers pairs Fly1027 + coxF1 (5'-TTT GAG CTC ATC ATA TAT TTA-3') and UEA341 + coxR2 (5'-GCT CGT GTA TCA ACA TCT-3'). Primers ND4+/ND4-16 were used to amplify the nd4 gene.

PCR was performed in a final volume of 50 µL, which included 2 mM MgCl2, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.5 mM of each primer, 0.2 mM dNTP mix, 1U Taq DNA polymerase (Invitrogen), and 5-10 ng genomic DNA. The thermocycler program was configured for an initial denaturation step at 94 °C five min, followed by 35 amplification cycles (94 °C/60 s, 50 °C/60 s, and 72 °C/60 s) and a final elongation at 72 °C for 10 min.

Amplification of Wolbachia genes (wsp and coxA). Mosquito samples from Teresina, Rio Branco, Santa Vitória and La Plata were tested for Wolbachia using the total DNA preparations employed for PCR above. The PCR conditions described in ZHOU et al.42 and SANOGO et al.38 were used to amplify wsp. Samples from each mosquito population were then submitted to PCR-amplification of bacterial cytochrome c oxidase (coxA) to detect population/strain polymorphism using primers and conditions described in BALDO et al.6.

DNA sequencing and sequence analyses. Amplified fragments were sequenced using BigDye TM Terminator 3.1 (Applied Biosystems), following the manufacturer's recommendations and directly sequenced on an ABI prism®3100. Sequences were analyzed using Chromas (Technelysium Pty), aligned with Clustalw2 (EBI) and edited with the software Geneious Pro 5.5.7 (Biomatters) and BioEdit 7.0.9 (Ibis Biosciences). Structural fragments were evaluated using ORF Finder (NCBI) and protein-structure predictions were developed initially through PSIPRED and then queried in the InterPro databases (NCBI). Pairwise, similarity data and genetic distance were calculated with Geneious Pro 5.5.7. Statistical data, such as haplotypes diversity (Hd) and nucleotides diversity per site (p) were tabulated from DnaSP v5.



Mosquito samples. Culex quinquefasciatus were caught almost exclusively in each mosquito collection. Samples were killed and preliminarily identified in the field, stored in individual tubes with silica gel (100 adult females and 100 adult males) and transported to the laboratory of the Faculdade de Saúde Pública USP, in São Paulo. At least two adult male and female vouchers from each locality were mounted and deposited in the Coleção de Referência da Faculdade de Saúde Pública USP, under access numbers E-13706 to E-13729. An equal number of females and males (N = 30) from each locality were separated for molecular analyses.

Mitochondrial molecular markers. Amplification of the nd4 gene produced fragments of 321-bp (after primer trimming) (GenBank accession number GQ255653). This fragment showed high A+T content (72.6%) and 100% sequence identity among samples. The 1260-bp cox1 fragment was comprised of four haplotypes (H1, H2, H3 and H4), whose geographical distribution is shown in Figure 1. The sequences were deposited in GenBank, under accession numbers: GQ255650 (H1), GQ255651 (H2), GQ255649 (H3) and GQ255648 (H4).

The H1 haplotype was that with the highest frequency (76.6%), followed by H2 (22.6%) and H3 and H4 (0.4%). Haplotypes H3 and H4 each contained one mutation in a single mosquito sample. To confirm the singleton status of these haplotypes, we repeated PCR and sequencing from the same genomic DNA. The alignment of the four haplotypes showed 99.7% genetic identity among geographic samples. Genetic diversity of the cox1 fragment was low: haplotype diversity (Hd = 0.636) and nucleotide diversity per site (p = 0.00091). The H2 haplotype was restricted to southern Brazil (Santa Vitória and Pelotas), but was also detected in a mosquito sample from Chapecó City.

Genic products. Prediction analyses confirmed the proteins cytochrome c oxidase 1 (COX1 EC1.9.3.1) and NADH dehydrogenase 4 (NADH4 EC1.6.5.3) as products of the cox1 and nd4 fragments, respectively. Analyses showed that the cox1 transition (C®T) detected in a La Plata sample (H4) results in modification of the primary protein structure throughout the domain region. This amino acid modification did not show identity with other cox1 enzymes. Other transitions in haplotypes H1, H2 and H3 did not produce changes in the primary protein structure. Pairwise comparisons of cox1 resulted in 100% identity with Cx. pipiens and Cx. quinquefasciatus from other regions of the world, indicating homology and conserved functional domain.

Wolbachia infection. The Wolbachia pipientis wsp and coxA amplicon sizes were 504-bp (61% A+T) and 478-bp (62.6% A+T), respectively. All sequences for both genes were identical in all samples analyzed and have been deposited under GenBank under accession numbers HM563687 (wsp) and HM563686 (coxA). In comparison to the Wolbachia genome available from GenBank (accession number AM999887), the wsp sequence was 100% similar to the homologous region in the Wolbachia wPip strain, an endosymbiont of Cx. quinquefasciatus. The coxA sequence was also 100% similar to the homologous region of cytochrome c oxidase subunit 1 of the wPip strain.



Comparative analyzes with the partial nd4 of Cx. quinquefasciatus showed 100% identity with mosquitoes from South Africa (AY793692) and Thailand (AY793692), and a one-base substitution in comparison to sequences from Riverside USA (AY793693). Although the complete Cx. quinquefasciatus nd4 sequence is 1343-bp long (HQ724617), studies commonly use the primer pair used here (nd4+/nd4-) to produce an amplicon of approximately 350-bp located in the gene's central portion. This region has been widely used in others culicid mosquitoes in studies of genetic diversity, systematics and phylogeny, being that the Cx. quinquefasciatus nd4 sequences yielded diversity indices higher for both Anopheles31 and Aedes,26 genera (Hd = 0.895; p = 0.0127). The low variation seen in the nd4 gene highlights its conservation and tendency toward homoplasmy in Cx. quinquefasciatus.

The variation seen in the four Cx. quinquefasciatus cox1 haplotypes is incongruent with the results from the ace2 intron and for mosquitoes of hybrid origin (with Cx. pipiens) found by MORAIS et al.30, potentially because ace2 mutates slower than cox120. This suggests that mitochondrial DNA does not distinguish taxa within the pipiens subgroup. Sequences of cox1 show both intra and inter-specific polymorphism, indicating that taxonomic diagnoses should include nuclear markers to avoid overestimates of diversity in Cx. quinquefasciatus-pipiens populations. However, mitochondrial markers identified different Cx. quinquefasciatus genetic types, including the Brazilian groupings.

The H1 haplotype is broadly distributed throughout the Brazilian tropics and in La Plata. It shares identity with the Cx. quinquefasciatus of tropical India (GenBank acc. no. FN395201). The H2 haplotype forms a population group, with a latitudinal range between 31 and 33°S in Brazil. It shares identity with Cx. pipiens cox1 sequences from Ohio (DQ360492), Cx. pipiens (FN395187) and Cx. p. f. molestus (FN395179) from Russia, Cx. p. pallens (FN395203) from Japan and Cx. quinquefasciatus (DQ181446) from Puerto Rico.

The H2 haplotype is found predominantly in southern Brazil. Its restriction to this region may be a consequence of founder effects. The low frequency of H2 in Chapecó suggests that this city may represent the limits of haplotype's northern distribution. However, the La Plata population, which is genetically confirmed as a hybrid30, contains individuals sharing nd4 and cox1 genes with pure Cx. quinquefasciatus elsewhere. We suggest that reproductive isolation is incomplete within the pipiens subgroup and that this affects the taxonomic resolution usually provided by mitochondrial genes, including gene cox1 barcode.

The differences between haplotypes H3 and H4 are insufficient to ascertain their relevance to intra-specific divergence or their relationship with mosquitoes from other parts of the world. Single mutations were also found in cox1 sequences by COOK et al.9 and reflect the polymorphic character of this gene.

Prediction tests showed that the four-base substitutions detected in cox1 are transitions. Three of these are synonymous and two form population groupings. Transitions and synonymous substitutions were also found by NAVAJAS et al.33 in other insects cox1. According to those authors, changes in the base composition of mitochondrial genes occur most frequently through synonymous transitions, without detectable effects on gene functioning. However, the sequences enabled the identification of divergence and gene flow dynamics in biogeographically distinct populations.

The low diversity of Cx. quinquefasciatus and Cx. pipiens mitochondrial genes has been reported by GUILLEMAUD et al.17. HASAN et al.18 also found low diversity (Hd = 0.502 and p = 0.0007) in the Cx. quinquefasciatus cox2 of populations from central Bangladesh, potentially a result of a recent common mitochondrial ancestor. Such data may also be explained by the recent and rapid expansion of these species (assisted by human migration and population growth) and successive bottlenecks caused by control measures in urban areas13.

Culex quinquefasciatus populations have been significantly impacted by control programs in Brazil, particularly in northern and northeastern equatorial regions, where the species is the primary vector of lymphatic filariasis and dirofilariasis1. The regions' low altitude, hot and humid climate and constant thermal amplitude facilitate both the development of the heartworm pathogen and the hematophagy of Culex vectors throughout the year25.

Unlike, vector control programs are less intense in southern Brazil due to the humid subtropical climate and high peaks of thermal amplitude. Although Culex populations in these areas have shown resistance to thermal shock, they suffer reduced numbers of over-wintering individuals and lower metabolic activity during cold months37. Although no cases of heartworm have been identified from southern Brazil, there are records of encephalitis and arbovirosis8. In Argentina, members of the pipiens complex are implicated as vectors of arboviruses, such as Saint Louis Encephalitis11.

Due to the epidemiological importance of Culex, vector-control programs have been organized in urban and rural areas for decades. These have involved countless chemical insecticides such as pyrethroids, Dichloro-diphenyltrichloroethane (DDT) and their derivades5. Organophosphates, Bacillus thuringiensis7 and Bacillus sphaericus29 larvicides have also been used. The exposure of mosquito populations to insecticides often confers resistance40 and the expansion of resistant individuals can explain population-level selective sweeps34.

On another view, Brazilian Cx. quinquefasciatus populations share identical nd4 sequences to those of RASGON et al.35 in North America (GenBank acc. no. AY793688) and parts of Asia (AY793691), but are from 5 to 6% divergent from South African populations (AY793694). These authors suggest that mitochondrial diversity may be related to Wolbachia infection: the American and Asian samples, which are infected, have low diversity, whilst the uninfected populations of South Africa have higher mitochondrial variation. All Culex samples are apparently infected by a single Wolbachia strain because of the invariant wsp and coxA sequences. This appears to be wPip, with which the Wolbachia infections herein share sequence similarity and similar hosts: Cx. quinquefasciatus and Cx. pipiens.

The fact that, despite being geographically scattered, all populations sampled herein share the same Wolbachia strain may be explained by host-endosymbiont specificity, as pointed out by WERREN et al.39. The genetic homogeneity observed among Wolbachia samples may also indicate that this endosymbiont only recently infected these Culex populations.

Culex quinquefasciatus appears to possess two mitochondrial types in Brazil. This fact should be taken into consideration in investigations of disease distribution and in aspects of blood-hosts in those locations. Recent studies suggest that feeding preferences may be influenced by genetic factors22.



To Dr. Almério de Castro Gomes (Faculdade de Saúde Pública, Universidade de São Paulo, Brazil) for providing mosquito samples from Teresina. Gustavo Rossi (Centro de Estudios Parasitologicos y de Vectores, La Plata, Argentina) for providing specimens from La Plata. We also thank Ministério da Saúde - FUNASA and Secretaria Estadual de Saúde, municipalities of Pelotas and Rio Branco for helping us during the field works. This investigation was supported by FAPESP (grants 05/50225-2 and 06/02622-5). S.A.M. is fellow of FAPESP (06/57272-9). F.A is fellow of CNPq (142766/2008-9).



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Correspondence to:
Mauro Toledo Marrelli, Departamento de Epidemiologia, Faculdade de Saúde Pública, Universidade de São Paulo
Av. Dr. Arnaldo 715, 01246-904 São Paulo, SP, Brasil
Tel: +55-11-30617922.

Received: 10 October 2011
Accepted: 4 June 2012

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