¹ Instituto de Investigaciones Biomédicas "Dr. Francisco J. Triana Alonso", Facultad de Ciencias de la Salud, Universidad de Carabobo, Sede Aragua, Maracay, Venezuela.

² Centro de Estudios de Enfermedades Endémicas y Salud Ambiental (CEEESA), S.A. Instituto de Altos Estudios "Dr. Arnoldo Gabaldon" (IAE/MPPS), Maracay, Venezuela

*Author of correspondence: flormhq@gmail.com

SUMMARY

The mosquito Aedes aegypti is the main Dengue vector in Latin America. This study investigated the genetic structure of this vector using samples collected in Venezuela, Colombia, Peru, Mexico, Argentina, Puerto Rico, and French Guiana. We examined the distribution of a 246-basepair region of the NADH dehydrogenase subunit 4 mitochondrial gene among a total of 369 Ae. aegypti from all the populations. This gene was amplified by the polymerase chain reaction and tested for variation using single strand conformation polymorphism analysis. Twelve haplotypes were detected among all the countries sampled and grouped into two clades. Significant differentiation was detected among the populations studied and these were not genetically isolated by distance.

Keywords: Dengue, Aedes aegypti, ND4, haplotypes, differentiation, genetic.

RESUMEN

Palabras clave: Dengue, Aedes aegypti, ND4, haplotipos, diferenciación, genética.

Recibido el 01/11/2015 Aceptado el 11/03/2016

INTRODUCCIÓN

Dengue is the most important viral disease transmitted to human by Aedes aegypti mosquitoes in Venezuela and other Latin American countries, and its incidence and prevalence are rising annually (San Martin et al., 2010). Aedes aegypti is the principal vector for Dengue viruses and it is able to maintain the four serotypes in an urban transmission cycle. In addition, this mosquito is also a vector for other viruses like Chikungunya and Zika which were responsible for a serious outbreaks of those disease in the Americas recently (Vorou, 2016; WHO, 2014).

Aedes aegypti populations may differ in biting behaviour, vectorial capacity, and other characteristics of epidemiological importance which may be detected through molecular markers (Tabachnick, 1991, Getis et al., 2003, Harrington et al., 2005). In addition, these markers could be used to determine the relatedness of geographic populations and associate this information with vector movements. This will help to analyze the risk of disease transmission (Getis et al., 2003, Liotta et al., 2005, Bracco et al., 2007).

Single strand conformation polymorphism (SSCP) analysis offers a simple, sensitive, and cheap method of detecting DNA polymorphisms, therefore, SSCP has become one of the most used means for determining genetic variation (Orita et al., 1989). SSCP is based on the principle that changes in the nucleotide sequences of a singlestrand DNA molecule alter its three-dimensional conformation. Point mutations could thereby change strands migration through polyacrylamide gels. SSCP detected 99% of base changes in 100 to 300 bp DNA fragments and 89% of point mutations in 300-450 bp molecules (Sunnucks et al., 2000).

The understanding of genetic variability, population structure, and migration dynamics of the mosquito Ae. aegypti is a major issue because of the risk associated with invasive new mosquito strains in a region that could cause the emergence and spread of Dengue (Manguin & Boete, 2011). The persistent increase in air and sea traffic between American countries increases the probability of introducing new Ae. aegypti strains into the region. These vector invasions are particularly accentuated in regions where vector surveillance is poor for lack of routine entomological surveys.

The aim of this study was to investigate the population genetic structure of Ae. aegypti from Colombia, Peru, Mexico, Argentina, Puerto Rico and French Guiana, using ND4 mitochondrial DNA markers and compared these sequences with the haplotype sequences obtained from Venezuela in a previous work (Herrera et al., 2006).

Mitochondrial gene amplification - A 246-bp region of the ND4 gene was amplified using the oligonucleotides and reaction conditions of Herrera et al. (2006) but with a high annealing temperature (55°C) to limit the amplification of Nuclear Mitochondrial Elements (NUMTs) which are present in the Ae. aegypti genome (Black & Bernhardt, 2009). The PCR amplifications were carried out in 50 μL reaction volumes using 1 μL of template DNA in a PTC-100 thermal cycler (MJ Research, Inc., Watertown, MA). Negative controls (all reagents except template) were run to detect possible contamination. The amplified products were visualized by electrophoresis in 2% agarose gels stained with ethidium bromide.

SSCP analysis - The PCR product (10 μL: 40-50 ng) was mixed with 8 μL of loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, and 0.05% xylene cyanol), centrifuged and heated to 95ºC for 10 min on a thermal cycler, then transferred immediately into ice. Samples were loaded onto 27 × 20 cm, 1 mm thick, 7% polyacrylamide gels. Gels were run at 4ºC for 20 h at a constant 8 milliamps and silver stained to visualize DNA fragments (Black & DuTeau, 1997). ND4 PCR products from 3-6 individuals of each haplotype were sequenced in both directions using the PCR primers. Sequencing reactions were performed with ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit on an Applied Biosystem Model ABI 3130XL.

Statistical analysis of mitochondrial haplotype frequencies- Analysis of molecular variance (AMOVA) (Excoffier et al., 1992) was conducted on the resulting haplotypes within and among regions using Arlequin versión 3.1 (Excoffier et al., 2005). The significance of the variance components was computed using a non-parametric permutation test (Excoffier et al., 1992). The DNA sequences were aligned using the Clustal W software package (Thompson et al., 1994). The nucleotide sequence and the frequency of each haplotype for each population were analyzed using DnaSP version 5.10 (Librado & Rozas, 2009). The number of polymorphic sites, the average number of nucleotide differences (k) (Tajima, 1983), the nucleotide diversity (π1) and the nucleotide diversity with the Jukes and Cantor correction (π2) (Nei 1987) were estimated. Effective migration rates (Nm) were calculated from FST. Transformed FST /(1- FST) were regressed on the natural logarithm of pairwise geographic distances among populations to test for isolation by distance (Slatkin, 1993). The Mantel test was performed using FORTRAN program MANTEL (William C. Black IV, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO). Genetic distance matrices were used to construct a cladogram among all populations by means of unweighted pair-group method with arithmetic averaging analysis in the NEIGHBOR procedure of PHYLIP 3.63 (Felsentein, 2004).

Phylogenetic relationships among haplotypes-MEGA (version 4.0) was used to perform phylogenetic analyses using maximum parsimony (Tamura et al., 2007). A bootstrap analysis with 1000 replications was done to assess the consistency with which the dataset supported the resolved phylogenies. ND4 homologous regions of Anopheles darlingi and Ae. albopictus were used as outgroups.

RESULTS

A total of six samples, representing 324 wild specimens of Ae. aegypti collected in Colombia, Peru, Mexico, Argentina, Puerto Rico and French Guiana (Table I) were analyzed. Twelve haplotypes were detected by SSCP analysis and sequencing. The haplotype sequences were aligned with ND4 sequences from Venezuela (Herrera et al., 2006). All these haplotypes sequences, including haplotypes from Venezuela, were denoted from A-L and are already registered in the GenBank (KR349202-KR349213). The frecuencies of these haplotypes in each country, including the previous reported in Venezuela, are shown in Table II.

The haplotype A was shared among Peru, Mexico, Puerto Rico, French Guiana and Venezuela populations. Except Mexican population, its frequency was > 40%. Haplotype H was the most frequent in the Colombia (96.22%) and Argentina (90.74%) populations. Haplotype J was present in the Mexico and French Guiana populations and its frequency varied from 98.14 to 32.07 % respectively. Haplotype L was found only in the population from Puerto Rico at a high frecuency (57.89%). The rest of the haplotypes (I and K) occurred at low frequencies.

The haplotype sequences were also aligned with other ND4 sequences registered in the GenBank. The haplotypes HH from Colombia and Argentina and HI from Colombia have identical sequences to the ones reported in Brazil for haplotypes H1 and H2, respectively (Sousa & Scaparssa, 2009). Several haplotypes from the coast of northeastern Mexico (Gorrochotegui-Escalante et al., 2000) have similarities to the haplotypes reported in this work: haplotype-9b is 99% similar to the haplotype HJ from Mexico and French Guiana, haplotype 3a-3c is 98% analogous to HK from Argentina, haplotype-6b corresponds to 99% of HL from Puerto Rico and haplotype-10a is 99% similar to the haplotype HA found in Peru, Mexico, Puerto Rico, French Guiana and Venezuela. HA is also 97% analogous to one haplotype detected in Kenya (Burugu et al., 2008).

Geographical analysis of variation in ND4 haplotype frequencies was conducted by AMOVA (Table III). Most of the variation (70.6%) arose among populations while 29.5% arose among mosquitoes in populations. The average FST was 0.705 indicating a very high genetic structure among populations. In addition, the effect of the distance on levels of gene flow was estimated by regressing linearized FST values on geographic distance. This analysis indicated that significant correlation was not detected between genetic and geographic distances neither among all populations nor among populations in any particular subgroup of these countries (data not shown).

The number of polymorphic sites and the diversity indices for each population and for all mosquitoes are listed in Table IV. The majority of populations had similar numbers of polymorphic sites (5-7) except for Peru (0), Argentina (1) and Venezuela (10). In relation to values for the mean number of nucleotide differences (k) and nucleotide diversity (p), populations from Venezuela, French Guiana and Puerto Rico had higher values of k and π, in which values were at least 8-18 times higher than the k and π from Argentina, Mexican and Colombian populations. The values of k and π from the Peruvian population were 0. Tajima's D test gave a statistically significant positive result for the total sample (Table IV).

Phylogenetic analysis, based upon use as outgroups of one related subgenus Stegomyia species (Aedes albopictus) and other species of the genus Anopheles (An. darlingi), provided a well-supported phylogeny with two maternal lineages (Fig 3). The Clade I contained all the haplotypes found in the total population while the Clade II missed haplotypes from Argentina and Peruvian populations. The Clade I divided into clearly three sub-branches with 100% bootstrap support while Clade II divided into two branches with ≥ 76% support. In addition, both clades contained haplotypes that have been found independently in several studies from America (Brazil, Mexico, and Peru), Asia (Thailand, Myanmar, Cambodia) and Africa (Kenya).

DISCUSSION

Aedes aegypti populations from different countries were genetically differentiated. This suggests a restricted level of interchange of genes among populations. This genetic differentiation did not arise by geographic distance. Similar results were reported in Mexico (Gorrochotegui-Escalante et al., 2000), Thailand (Bosio et al., 2005), Peru (Costa da Silva et al., 2005) and Brazil (Da Costa-Ribeiro et al., 2007, Twerdochlib et al., 2012). The existence of geographic barriers (Caribbean Sea and the Andean Mountain Range) among populations might account for our findings.

Another possibility that could explain the results may be genetic drift arising from a strong reduction in the effective population size in collections caused by insecticide applications (Sharma et al., 2009). The presence of only few major haplotypes, unique haplotypes or haplotypes with very small frequencies in some countries could arise due to major population bottlenecks caused by insecticide treatment. A similar hyphotesis has been proposed by Gonçalves da Silva et al. (2012).

Contrary to these results, some authors have found isolation by distance among samples from Brazil and other countries (Monteiro et al., 2014). They have suggested that in Brazil first ocurred a complete eradication of Ae. aegypti (late 50's) followed by re-colonization (in the 1970's), probably from mosquitoes from neighboring countries like Venezuela. It could be argued that in this time span the lineages that may have initially re-invaded Brazil are now exchanging genes and perhaps merging.

The genetic diversity found in this work is high and comparable to another similar study (Bracco et al., 2007). This caused a very high genetic structure with large FST (~ 1.0) values among populations. Similarly, Ayres et al. (2004) have reported that Brazilian populations of Ae. aegypti showed high levels of genetic differentiation in areas most frequently treated with chemical insecticides. Other investigators suggested that insecticide pressure is probably the major cause of genetic diversity in Ae. aegypti from highly populated urban areas (Paupy et al., 2003, Ocampo & Wesson, 2004, Twerdochlib et al., 2012). The use of insecticides has also facilitated the appearance of insecticide resistance in Ae. aegypti (Bisset et al., 2001, Rodríguez et al., 2001, Álvarez et al.,. 2008, Mourya et al., 2015). This phenomenom appears to be happening in these populations as the statistically significant Tajima's D values were positive for all populations, indicating that there are haplotypes under positive selection (Tajima, 1983).

Phylogenetic analysis of haplotype sequences demonstrated two lineages. These did not contain haplotypes from a particular region since Asian, African and American haplotypes were found in both lineages. The haplotypes with greater frequencies (HA and HH) in the total population were located only in the lineage I. At least one of them was present in all subpopulations and together they constituted over 50% of the total haplotypes. Thus, HA was found in 5 of 7 populations and, except for Mexico, its frecuency varied from 42.1 to 100%. It can be argued, therefore, that this lineage is the oldest. Probably HA and HH have spread to all subpopulations through migration of individuals that are best adapted to environmental changes (insecticide pressure) which has allowed their high frequencies (Sousa & Scarpassa, 2009). The presence of two phylogenetic lineages that subdivide the Ae. aegypti populations is consistent with earlier research (Gorrochotegui-Escalante et al., 2002, Bosio et al., 2005, Herrera et al., 2006, Bracco et al., 2007, Paduan & Ribolla, 2008, Sousa & Scarpassa, 2009, Fraga et al., 2013, Moore et al., 2013).

Populations from Puerto Rico, French Guiana and Venezuela had at least two haplotypes with frecuencies ≥ 25%, therefore their nucleotide diversity (p ) and the mean number of nucleotide differences (k) were higher than the corresponding ones to the others populations with only one dominat haplotype ≥ 90,7%. In addition, all the populations with the highest level of genetic diversity contained HA in a major frecuency which strengthen the idea that HA is an ancient haplotype with a nucleotide differentiation already fixed.

The genetic variability found in these american countries (AC) was higher than those from Thailand (T) (k~1,6 and p ~2,9 times) and the island of São Luís in the Brazilian state of Maranhão (BM) (k~2 and p ~3 times) with an equal number of polymorphic sites (S =15) (Bosio et al., 2005, Fraga et al., 2013). It could be explained because AC have more haplotypes than T (12 vs 7) and than BM (12 vs 10). On the contrary, other comparison with populations from Brazilian Amazon (BA) resulted in πAC > p BA (2 times) while k value and haplotype number were in a similar range. This could mean that the more frequent BA haplotypes are more similar to each other than those of AC (Sousa & Scaparssa, 2009). The amount of polymorphic sites varied among the populations: 7-10 for Venezuela, México and French Guiana, 5-6 for Colombia and Puerto Rico, 1 for Argentina and 0 for Perú. This last result arose because Peru had only one haplotype. At the same time, it can also be noted that even though existed a striking difference between the populations of Colombia and Argentina (the first had 5 times more polymorphic sites than the second), both populations had a common dominant haplotype (Colombia 96.22%, Argentina 90.74%). This result agrees with the observed distribution of Colombia (HH and HI) and Argentina (HH and HK) haplotypes in different and same lineages respectively. This means that the nucleotide divergence of Argentina haplotypes that share a common ancestor, is smaller than those of Colombia.

In addition, the data presented here could have important implications for Dengue, Zika or Chikungunya transmission since the genetic variability in Ae. aegypti from these countries may cause differences in their susceptibility to these viruses. A correlation between population differentiation and heterogenous patterns of vector competence in Ae. aegypti has been suggested for other authors (Tran et al., 1999, Vazeille-Falcoz et al., 1999, 2001, Paupy et al., 2003, Black WC et al., 2002, García-Franco et al., 2002, Lourenço-de-Oliveira et al., 2004, Ocampo & Wesson, 2004). A population genetic analysis and an evaluation of susceptibility to Dengue 2 virus were conducted among Ae. aegypti samples from different states of Brazil (Laurenço-de-Oliveira et al., 2004). It was demonstrated that Brazilian Ae. aegypti were genetically differentiated within most of the regions, and that their infection rates towards DENV2 were heterogenous. Other study has found a correlation between genetic distances of Ae. aegypti populations and their infections rates (GarcíaFranco et al., 2002). Recently, it was observed considerable variation in vector competence within and between Brazilian populations analyzed from short spatial and temporal collections (Gonçalves et al., 2014).

This study suggests that the vector control programme from these countries may induce recurrent emergence of insecticide resistant populations. Moreover, the rapid fixation of haplotypes conferring insecticide resistance could take place; the resistant populations may be spread at a larger scale as the migration of resistant mosquitoes could be facilitated over the one of susceptible insects. Ae. aegypti resistant to insecticides represent a great problem in Dengue, Chikungunya or Zika control. The next step could be to look for insecticide resistance in the studied populations to confirm the involvement of selection effects in the actual genetic structure of their Ae. aegypti populations.

ACKNOWLEDGEMENTS

To Walter Almirón (Argentina), Mario Henry Rodríguez (Mexico), Isabelle Dusfour (French Guiana), Manuel Amador (Puerto Rico), Palmira Ventosilla (Peru), Claudia Romero and Carlos Andrés Morales (Colombia) for kindly giving us the mosquito samples; to the staff of the Centro de Estudios de Enfermedades Endémicas y Salud Ambiental, Instituto de Altos Estudios "Dr. Arnoldo Gabaldon"

for mosquito rearings; to Johanny Ruiz for helping in preliminary experiments and to José Rivero for technical assistance on sequencing the samples.

Financial support: Fondo Nacional de Ciencia, Tecnología e Innovación, Venezuela. Project Nº 2008000911-1.

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