PAGES: 57-62 DOI: Full paper
Estimation of Genetic Divergence and Gene Flow between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina

Silvia G Humeres, Walter R Almirón*, Marta S Sabattini, Cristina N Gardenal +

Cátedra de Química Biológica, Facultad de Ciencias Médicas, cc.35, suc.16, 5016 Córdoba, Argentina
*Centro de Investigaciones Entomológicas de Córdoba, Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Avda. Vélez Sársfield 299, 5000 Córdoba, Argentina


Allele frequencies at seven polymorphic loci controlling the synthesis of enzymes were analyzed in six populations of Culex pipiens L. and Cx. quinquefasciatus Say. Sampling sites were situated along a north-south line of about 2,000 km in Argentina. The predominant alleles at Mdh, Idh, Gpdh and Gpi loci presented similar frequencies in all the samples. Frequencies at the Pgm locus were similar for populations pairs sharing the same geographic area. The loci Cat and Hk-1 presented significant geographic variation. The latter showed a marked latitudinal cline, with a frequency for allele b ranging from 0.99 in the northernmost point to 0.04 in the southernmost one, a pattern that may be explained by natural selection (FST = 0.46; p < 0.0001) on heat sensitive alleles. The average value of FST (0.088) and Nm (61.12) indicated a high gene flow between adjacent populations. A high correlation was found between genetic and geographic distance (r = 0.83; p 0.001). The highest genetic identity (IN = 0.988) corresponded to the geographically closest samples from the central area. In one of these localiies Cx.quinquefasciatus was predominant and hybrid individuals were detected, while in the other, almost all the specimens were identified as Cx. pipiens. To verify the fertility between Cx. pipiens and Cx. quinquefasciatus from the northern- and southernmost populations, experimental crosses were performed. Viable egg rafts were obtained from both reciprocal crosses. Hatching ranged from 76.5 to 100%. The hybrid progenies were fertile through two subsequent generations.

Mosquitoes of the Culex pipiens L. Group are important vectors of St. Louis encephalitis (SLE) virus in the east-central United States and eastern Texas (Mitchell et al. 1980). A strain of SLE virus was isolated from Cx. pipiens quinquefasciatus Say collected in Santa Fe Province, Argentina (Mitchell et al. 1985), and a colony established from the same site, showed to be an efficient vector of both the Argentine and United States SLE viral strain (Mitchell et al. 1985).

The members of this group are widely distributed throughout the world, with two predominant species: Cx. pipiens and Cx. quinquefasciatus. The former is restricted to cooler areas while the later inhabits warmer tropical and subtropical regions (Mattingly 1951, Laven 1967, Barr 1982). Morphological, ethological and ecological features have been used to characterize these species; however, their taxonomic status is still undefined in some geographic areas.

Male genitalia morphology and morphometry (DV/D ratio) have been the main traits used to distinguish Cx. pipiens from Cx. quinquefasciatus. However, intergradation zones have been definited in North America (Sundararaman 1949, Barr 1957, Cheng 1976, Jakob et al. 1980, Barr 1982, Tabachnich & Powell 1983), northern Japan (Edwards 1921, Bekku 1956, Sasa et al. 1963, Ishii 1980), southeastern Australia (Dobrotworsky 1967, Barr 1982), and the Middle East (Edwards 1921, Harbach 1985, 1988). In Africa, the two taxa occur in sympatry but do not hybridize (Jupp 1978, Donaldson 1979). In Argentina (South America), hybrid forms have been found in a central area of the country between 30° and 33°S (Brewer et al. 1987, Almirón et al. 1995).

Several authors have stressed the contribution of multilocus electrophoretic analysis to the knowledge of the genetic structure of populations of pipiens and quinquefasciatus from different countries and to clarify their taxonomic position (Cheng 1976, Tabachnick & Powell 1983, Urbanelli et al. 1985, Villani et al. 1986). Studies of genetic polymorphism have not been undertaken in the Cx. pipiens Group of South America.

This paper presents a study of the distribution of allele frequencies for seven polymorphic enzyme loci in six populations of Cx. pipiens and Cx. quinquefasciatus from Argentina, situated along a north-south line of about 2,000 km, and an estimation of the degree of gene flow and genetic divergence among them. Crossing experiments between mosquitoes from the two extreme populations of this line were also performed.



Samples were taken from the following localities (Fig. 1): Castelli, 25º53'S (Chaco Province); Esperanza, 31º26'S and Rosario, 32º56'S (Santa Fe Province); 9 de Julio, 35º18'S and Bahía Blanca, 38º37'S (Buenos Aires Province) and Puerto Madryn, 42º46'S (Chubut Province) as indicated in Almirón et al. (1995). These localities are situated along a north-south transect; the area includes a subtropical region with a mean annual temperature of 25ºC in the northernmost section, while the southern limit presents an average annual temperature of 10ºC.

Females were collected from natural resting sites using baited (chicken) can traps, and then transported alived to the laboratory. In order to obtain egg rafts, the field females were placed individually in plastic tubes with wet cotton and filter paper at the bottom. The egg rafts were reared as individual progenies in plastic trays (25º± 3ºC, 14:10 L/D photoperiod). Larvae were fed with commercial rodent food until adults emerged. Adults were frozen at -40ºC, 48 hr after emergence, until electrophoresis was performed. Five to seven males from each progeny were examined for phallosome morphology and morphometry (DV/D ratio) (Sundararaman 1949).

Electrophoretic analysis - Homogenates were obtained from single individuals crushed in distilled water. The resulting suspension was absorbed in a Whatman 3 MM paper wick (2.5 mm) and inserted in starch gel trays. Homogenates from six to nine descendants of each progeny were placed in a gel block which admitted up to sixteen insertions. The number of families analyzed from each locality are indicated in Table I. Standard horizontal electrophoresis was carried out at 4ºC. Gels were sliced longitudinally into four slabs and each one stained for specific enzymes (Humeres et al. 1990).

The following buffer systems were used: continuous Tris-boric EDTA, pH: 8.6 (Markert & Faulhaber 1965), for the separation of catalases (CAT), glycerophosphate dehydrogenase (GPDH) and hexokinase (HK); discontinous Tris citrate, pH: 6.7 for gels and pH: 6.3 for the electrode cells (Shows & Ruddle 1968) for malate (MDH) and isocitrate (IDH) dehydrogenases, and continuous lithium hydroxide pH:8.4 (Steiner & Joslyn 1979) for phosphoglucomutase (PGM) and glucosephos-phate isomerase (GPI).

Alleles were assigned a number according to the relative mobility of the proteins, using 100 as the protein migrating fastest toward the anode. The index of genetic distance (D) proposed by Nei (1972) was used to estimate the degree of genetic divergence between populations. The analysis was performed using the Genind program (Vilardi 1992). Gene flow was estimated using Wright's (1965) F-statistics as modified by Nei and Chesser (1983). Effective migration rate (Nm) was calculated from FST.

Crossing experiments - The following crosses were performed as indicated in Almirón et al. (1995): females Cx. pipiens (Puerto Madryn) x males Cx. quinquefasciatus (Castelli) and its reciprocal.



According to the analysis of male genitalia (DV/D ratio) the following species were identified: Cx. quinquefasciatus was exclusively present in the northern localities of Castelli and Esperanza. In Rosario, although Cx. quinquefasciatus was predominant, hybrids were also detected. Almost all individuals from the 9 de Julio sample belonged to Cx. pipiens except one Cx. quinquefasciatus. In the southern localities (Bahía Blanca and Puerto Madryn), only Cx. pipiens (Almirón et al. 1995) has been identified.

Electrophoretic analysis - Zymograms obtained allowed the analysis of seven loci: CatMdhIdhGpdhHk-1Pgm, and Gpi. Criteria of genetic interpretation was similar to those applied by other authors to polymorphisms detected in different mosquito species (Steiner & Joslyn 1979, Villani et al. 1986).

Parental genotypes for each isofemale line were determined when unequivocal, and allelic frequencies were calculated. Data are summarized in 
Table I. In all cases, genotypes were distributed according to the Hardy-Weinberg equilibrium. The predominant alleles at the Mdh, Idh, Gpdh and Gpi loci presented similar frequencies in all samples. For the Pgm locus, allele 93 was predominant in Castelli and Esperanza, while allele 100 was the most frequent in Bahía Blanca and Puerto Madryn. The intermediate localities of Rosario and 9 de Julio presented similar frequencies. Allelic frequencies of Cat and Hk-1 differ markedly between extreme populations. Hk-1 shows a striking latitudinal cline.

Values of Nei's genetic distance between the populations are presented in Table II. Samples including typical Cx. pipiens (Bahía Blanca, 9 de Julio and Puerto Madryn) show a low genetic distance. Samples from the localities of Castelli, Esperanza and Rosario, where Cx. quin-quefasciatus was predominant, were also genetically homogeneous. However, 9 de Julio and Rosario samples showed the lowest value of genetic distance (DN = 0.011), although they were represented almost exclusively by Cx. pipiens and Cx. quinquefasciatus respectively. Populations from extreme sites of the study area presented the highest value of genetic distance (D = 0.225). Fig. 2 summarizes these data in a dendrogram based on D values and clustered using the Genind program. A significant correlation between genetic and geographical distances was detected (r = 0.83; p < 0.001). In Table III, FST and Nm values are presented for the analyzed loci in the six populations.

Crossing experiments - Viable eggs were obtained from both crosses. Crosses involving female Cx. pipiens from Puerto Madryn x male Cx. quinquefasciatus from Castelli showed hatching rates of 100%, and 76.5% in the reciprocal. The F1 and F2 offspring of both crosses proved to be fertile, with hatching rates between 95 and 100% (Table IV). The colony was discarded after obtaining the F3.



Geographic patterns of allele frequencies in Cx. pipiens and Cx. quinquefasciatus were analyzed for populations from different collection sites in an extended region of Argentina, including Esperanza where Cx. quinquefasciatus was found naturally infected with the St. Louis encephalitis virus (Mitchell et al. 1985). Although allele frequencies at one of the seven loci analyzed (Hk-1) showed a marked clinal gradient across the studied area (Table I), the lack of fixed alternative allozymes at any of the loci did not allow the characterization of Cx. pipiensand Cx. quinquefasciatus. Similar results were obtained by Cheng et al. (1982) and Tabachnick and Powell (1983).

Hexokinase (Hk-1), 6-phosphogluconate dehydrogenase (6Pgdh), glucosephosphate isomerase (Gpi) and phosphoglucomutase (Pgm), exhibit a latitudinal cline in gene frequencies across the range of the species in the United States (Cheng et al. 1982). Tabachnick and Powell (1983) reported that the same loci track a topographical temperature gradient in the Central Valley of California. In our study, a similar pattern was found only for locus Hk-1, with a frequency for allele 100 ranging from 0.01 in the northernmost point of the sampling area, to 0.96 in the southernmost collection site (Table I). Alleles at this locus may be equivalent to those designated HkA and HkB by Cheng et al. (1982) for populations from North America, where HkA decreases in frequency with increasing latitudes.

Allele frequencies at Pgm locus did not show correlation with DV/D ratios, as demonstrated by Cheng et al. (1982) and Tabachnick and Powell (1983). These alleles presented a particular distribution (Table I), being similar for populations from the warm and cold areas (Pgm 93: 0.53 and 0.58 for Castelli and Esperanza; 0.47 and 0.45 for Bahía Blanca and Puerto Madryn) and between the two intermediate ones (Pgm 93: 0.64 and 0.75 for Rosario and 9 de Julio). Population pairs with similar allele frequencies, share similar environmental conditions (not only temperature), since they occupy approximately the same geographic area. This may produce a non random distribution of genetic variants.

An unique allele (c) for the Pgm locus was found in the sample from Rosario with a frequency of 7%. In this site, hybrid specimens (according to the DV/D ratio) were detected in a proportion of 4.7% (Almirón et al. 1995). The presence of rare alleles in hybrid populations has been reported for different animal groups; intragenic recombination was proposed to explain it (Golding & Strobeck 1983). However, we did not determine if the unique allele was confined to the hybrid specimens.

Estimation of the standardized variance in genic frequencies among populations (FST) gave an average value of 0.088, and an average Nm of 61.12 (Table II) which would indicate that a high gene flow is occurring between adjacent populations according to Wright (1978). However, if Hk-1 is excluded, these values are 0.026 and 71.26 respectively. As it has already been pointed out, different alleles at the Hk-1 locus are present in high frequencies in populations from the extreme points (Hk-1 90: 0.99 in Castelli and 0.04 in Puerto Madryn; FST = 0.46, p < 0.0001) suggesting that natural selection at this locus is strong enough in preventing gene flow to homogenyze gene frequencies. Pryor (1980) demonstrated differential heat stability of different allele products at Hk-1 locus, being the heat-stable ones more common in Cx. quinquefasciatus. This finding would indicate that temperature could be an important factor determining genetic composition of populations at Hk-1 locus. Although in a lower degree, the locus Cat also showed significant local differentiation (FST = 0.08, p < 0.05), but there was no correlation with the sample origin.

Another approach to understand patterns of geographic variation is the analysis of genetic distance values among populations. The lowest distance (Table III) correspond to the geographically closest samples (9 de Julio and Rosario, DN = 0.011) suggesting that Cx. pipiens and Cx. quin-quefasciatus are a stable interbreeding unit in those areas where temperature allows their survival.

On the other hand, a good correlation between genetic and geographical distance (r = 0.83; p < 0.001) was found. It is difficult to explain this observation as produced by a drift-migration interaction, taking into account the FST values for different loci. It could be assumed that this correlation would be mainly determined by loci under the control of natural selection, being temperature an important selective factor for the maintenance of genetic cohesion among populations of the same species.

The average value of D was 0.102, lower than that reported by Urbanelli et al. (1985) between Cx. pipiens from Italy and Cx. quinquefasciatus from Africa (D = 0.145). D values ranging from 0.05 to 0.2 are considered to correspond to populations of the same species (Ayala 1975).

Almirón et al. (1995) performed different crosses between Cx. pipiens and Cx. quinquefasciatus from a central area of Argentina including three localities (Bahía Blanca, Buenos Aires Province; Córdoba, Córdoba Province; and Rosario, Santa Fe Province). Hatching from 70 to 100% was reported by those authors, except for one cross involving female Cx. pipiens from Bahía Blanca x male Cx. quinquefasciatus from Rosario, where a high incompatibility was observed (11.1% hatch). According to this situation, a low hatching rate was expected for the cross between female Cx. pipiens from Puerto Madryn x male Cx. quinquefasciatus from Castelli, which did not happen (Table IV). An intracellular rickettsial agent of the genus Wolbachia found in reproductive tissues of arthropods (including Cx. pipiens) has been reported as having a profound effect on the host's population structure. If the sperm from a Wolbachia-infected male fertilizes an uninfected egg, unidirectional cytoplasmic incompatibility can occur, producing zygotic death. Different factors as bacterial strain, host genotype and bacterial density seems to influence strength and direction of that incompatibility (Werren 1997). In our case, Castelli and Puerto Madryn populations showed the highest genetic distance; however, 76.5 to 100% of hatching was recorded for interpopulation crosses. It is possible that the population from Rosario was infected by a Wolbachia strain not present in other populations tested. This may explain the important unidirectional reduction in fertility observed in some crosses between males from Rosario and females from genetically similar populations.

The finding of hybrid individuals in samples from Córdoba and Rosario, and the fertility of the hybrids obtained under laboratory conditions (Brewer et al. 1987, Almirón et al. 1995), plus data on genetic distance and gene flow here presented, provide genetic evidence about the subspecific status of Cx. pipiens and Cx. quinquefasciatus in Argentina. The recognition of the existence of an important gene flow between intermediate populations of both forms provides useful information on their dispersal potential, and possibly of the infectious agent they transmit. This knowledge may also afford data for any attempt to perform genetic control on the species.



To Dr Antonio Blanco for the critical revision of the manuscript; to Dr Thomas P Monath, who kindly supplied materials used in this study, and Dr Esteban Bakos for providing us mosquitoes from Chaco Province.



Almirón WR, Humeres SG, Gardenal CN 1995. Distribution and hybridization between Culex pipiens and Culex quinquefasciatus (Diptera: Culicidae) in Argentina. Mem Inst Oswaldo Cruz 90: 469-473.

Ayala FJ 1975. Genetic differentiation during the speciation process. Evol Biol 8: 1-78.

Barr AR 1957. The distribution of Culex pipiens pipiens and Culex pipiens quinquefasciatus in North America. Am J Trop Med Hyg 6: 153-165.

Barr AR 1982. The Culex pipiens complex, p. 551-572. In WWM Steiner, WJ Tabachnick, KS Rai, S Narang (eds), Recent developments in the genetics of insect disease vectors. Champaign, Illinois, Stipes.

Bekku H 1956. Studies on the Culex pipiens group of Japan. I. Comparative studies on the morphology of those obtained from various localities in the Far East. Nagasaki Med J 31: 956-966.

Brewer M, Buffa L, Almirón W 1987. Culex pipiens quinquefasciatus y Culex pipiens pipiens (Diptera: Culicidae) en Córdoba, Argentina. Rev Per Entomol 29: 69-72.

Cheng ML 1976. Genetic variability in the complex Culex pipiens (Diptera: Culicidae). Ph.D. thesis, Univ. Texas, 237 pp.

Cheng ML, Hacker CS, Pryor SC, Ferrel RE, Kitto GB 1982. The ecological genetics of the Culex pipiens complex in North America, p. 581-627. In WWM Steiner, WJ Tabachnick, KS Rai, S Narang (eds), Recent developments in the genetics of insect disease vectors.Champaign, Illinois, Stipes.

Dobrotworsky NV 1967. The problem of the Culex pipiens complex in the South Pacific (including Australia). Bull WHO 37: 251-255.

Donaldson JMI 1979. The Culex pipiens complex in South Africa. J Entomol Soc Sth Afr 42: 35-50.

Edwards FW 1921. A revision of the mosquitoes of the Palaearctic Region. Bull Entomol Res 12: 263-351.

Golding GB, Strobeck C 1983. Increased number of alleles found in hybrid populations due to intragenic recombination. Evol 37: 17-29.

Harbach RE 1985. Pictorial keys to the genera of mosquitoes, subgenera of Culex and the species of Culex (Culex) occurring in southwestern Asia and Egypt, with a note on the subgeneric placement of Culex deserticola (Diptera: Culicidae). Mosq Syst 17: 83-107.

Harbach RE 1988. The mosquitoes of the subgenus Culex in Southwestern Asia and Egypt (Diptera: Culicidae). Contrib Am Entomol Inst 24: 1-240.

Humeres SG, Gardenal GN, Almirón WR, Sereno R, Sabattini MS 1990. Culex species (Diptera: Culicidae) from central Argentina: identification by electrophoretic zymograms and genetic relationships. J Med Ent 27: 784-788.

Ishii T 1980. On the Culex pipiens group in Japan. Part III. A historical review of its research. J Sci Coll Gen Educ Univ Tokushima 13: 26-62.

Jakob WL, Taylor SA, Francy DB 1980. Additional studies of male progeny of overwintering Culex pipiens complex mosquitoes from Memphis, Tennessee. Mosq Syst 12: 386-391.

Jupp PG 1978. Culex (Culex) pipiens Linnaeus and Culex (Culex) quinquefasciatus Say in South Africa: morphological and reproductive evidence in favour of their status as two species. Mosq Syst 10: 461-473.

Laven H 1967. Speciation and evolution in Culex pipiens, p. 251-275. In JW Wright, R Pal (eds), Genetics of insect vectors of disease. Elsevier, North Holland, Amsterdam.

Markert CL, Faulhaber I 1965. Lactate dehydrogenase isozyme patterns of fish. J Exp Zool 159: 319-332.

Mattingly PF 1951. The Culex pipiens complex. Trans R Entomol Soc (Lond) 102: 331-342.

Mitchell JC, Monath TP, Sabattini MS 1980. Transmission of St. Louis encephalitis virus from Argentina by mosquitoes of the Culex pipiens (Diptera: Culicidae) complex. J Med Ent 17: 282-285.

Mitchell JC, Monath TP, Sabattini MS, Cropp C, Daffner J, Calisher C, Christensen H 1985. Arbovirus investigations in Argentina. II. Arthropod collections and virus isolations from mosquitoes, 1977-1980. Am J Trop Med Hyg 34: 945-955.

Nei M 1972. Genetic distance between populations. Am Nat 106: 238-292.

Nei M, Chesser RK 1983. Estimation of fixation indices and gene diversities. Ann Hum Genet 47: 253-259.

Pryor SC 1980. Biochemical genetics of the Culex pipiens complex. II. Hexokinase. Comp Biochem Physiol B Comp Biochem 67: 705-710.

Sasa M, Kanda T, Miura A, Yamaguti N 1963. Biological and taxonomical studies on some colonies of pallens and fatigans forms of the house mosquito, Culex pipiens s.l., from eastern and southern Japan. Jap J Exp Med 33: 1-31.

Shows TB, Ruddle FH 1968. Malate dehydrogenase evidence for tetrameric structure in Mus musculus. Science 160: 1356-1357.

Steiner WWM, Joslyn DJ 1979. Electrophoretic technique for the genetic study of mosquitoes. Mosq News 39: 35-53.

Sundararaman S 1949. Biometrical studies on intergradation in the genitalia of certain populations of Culex pipiens and Culex quinquefasciatus in North America. Am J Trop Med Hyg 6: 153-165.

Tabachnick WJ, Powell JR 1983. Genetic analysis of Culex pipiens populations in the Central Valley of California. Ann Entomol Soc Am 76: 715-720.

Urbanelli S, Bullini L, Villani F 1985. Electrophoretic studies on Culex quinquefasciatus Say from Africa: genetic variability and divergence from Culex pipiens L. (Diptera: Culicidae). Bull Entomol Res 75: 291-304.

Villani F, Urbanelli S, Gad A, Nuddman S, Bullini L 1986. Electrophoretic variation of Culex pipiens from Egypt and Israel. Biol J Linn Soc 29: 49-62.

Vilardi JC 1992. Genind. Programa «basic» para estimar índices de distancia y variabilidad genética y sus errores a partir de muestras pequeñas. Mendeliana 10: 1-4.

Werren JH 1997. Biology of WolbachiaAnnu Rev Entomol 42: 587-609.

Wright S 1965. The interpretation of population structure by F-statistics with special regard to system of mating. Evolution 19: 395-420.

Wright S 1978. Evolution and the Genetics of Populations, Vol 4: Variability within and among Natural Populations. University of Chicago Press, Chicago, 565 pp.

+Corresponding author. Fax: + 54 51 333024
Received 20 December 1996
Accepted 16 October 1997

This work was supported, in part, by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina, and the Consejo de Investiga-ciones Científicas y Tecnológicas de la Provincia de Córdoba (CONICOR).
CNG and WRA are Carreer Investigator of the CONICET and SGH was a Fellow of the CONICOR.


Memórias do Instituto Oswaldo Cruz

Av. Brasil 4365, Castelo Mourisco
sala 201, Manguinhos, 21040-900
Rio de Janeiro, RJ, Brazil

Tel.: +55-21-2562-1222

This e-mail address is being protected from spambots. You need JavaScript enabled to view it.



marca fiocruzmarca brasil
marca faperjmarca cnpqmarca capes n marca cope

and diabetes. Erection dysfunction or ED is certainly one of mens most usual problem. It changes buy tadalafil 60mg A common drug is actually an imitation of its manufacturer twin. Both ought to be same in female cialis 20mg Long Phrase Viagra Use Fundamentally Damages Sex Lives This discount cialis canada Equally so, theres something to be said for the wonder of the second, captured forever on picture or a buy cheap cialis People extremely annoyed that they could only get three weeks at a time, Bunker noted. Retired persons cheap pharmacy These types of matters are possibly to being identified as having a result of cancer buy cialis 40mg - Yoghourt - fat-free simply Physical causes: Buying generic medicines now has been cheap generic cialis Herbaceous plants like nigrum and tribulus are well-known for his or her qualities in defeating impotence, which tadalafil 10mg It is not hard to consider Cialis that is generic. Most men start with one-10 mg dosage each purchase cialis Tadalafil quickly gained the moniker of weekender in Paris due to the fabulous results. The bash freaks buy female cialis