INTRODUCTION

Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) is the vector transmitting several arboviruses and is responsible for the circulation of several serotypes of dengue virus in sympatry with chikungunya and zika viruses in the Americas ¹. Currently, these viruses, particularly dengue, have had a great impact on public health and on the loss of working hours. This loss translates into 70% of hospitalization costs and costs for treatment and subsidies that can reach up to 80% of the total cost ¹. In the last quarter of 2020, 60,925 cases of dengue were reported in Paraguay, mostly in the capital district (Asunción) and the Central department ².

Ae. aegypti females, which exercise hematophagy for oviposition, show increasing anthropophilic habits, associating their high population densities to the presence of water reservoirs in artificial or natural containers such as leaf axils, bamboo internodes and other phytotelmata plants (they accumulate water in their structures), which may be present in the home or peridomicile ³.

The population variability of Ae. aegypti is susceptible to climatic changes, mobility of human groups and selection by insecticides; knowledge of these factors contributes to the understanding of its population dynamics ^{4 - 6}.

An alternative for the study of genetic analysis is the use of the molecular technique called random amplification of polymorphic DNA by PCR or Random Amplified Polymorphic DNA (RAPD), useful for the search of random repetitive sequences in genomic regions of high genetic variability, within and between populations, with a minimum amount of DNA and without a previous target sequence ⁷. The study of natural mosquito populations allows the determination of genetic variations existing in different geographical areas and provides information about gene flow events in geographically separated populations, providing data for surveillance and control programs in areas of high human dispersion and high trade in order to implement specific surveillance measures.

This study aimed to study the genetic diversity of Ae. aegypti, in populations of four departments of the cross-border axis constituted between the departments of Central, Cordillera, Caaguazú and Alto Paraná, Paraguay. The vectors were captured between 2018 and 2019, in localities with reports of dengue cases.

MATERIALS AND METHODS

RESULTS

The collection provided 1500 to 2000 eggs per locality, from which we obtained the adults that provided the genetic material. The RAPD technique revealed the amplification of 40 bands (between 300 and 1700 bp), with primers H3 and B03, four of which corresponded to conserved fragments of 700 to 1200 bp for primer H3 and 450 to 1200 bp for primer B03; these were present in all individuals of the populations under study (Figure 1).

Figure 1. A) B) Band amplification patterns by random amplification of polymorphic DNA by PCR (RAPD-PCR) for the study of variability in natural populations of Aedes aegypti from the Central-Alto Paraná cross-border axis in Paraguay. Profiles of bands amplified with primer H3. White arrows indicate common band profiles corresponding to approximately 700 and 1200 bp (Lanes: 1-2 Hernandarias, 3-4 Caaguazú, 5-6 Piribebuy, 7-8 San Lorenzo, 9-10, Rockefeller [standard strain]), NC: negative control, M: molecular weight marker/100 bp DNA Ladder from Promega®. Profiles of bands amplified with primer B03. The white arrows indicate the common band profiles corresponding to approximately 450 and 1200 bp (lanes: 1-2 Piribebuy, 3-4 Hernandarias, 5-7 Caaguazú), NC: negative control, M: molecular weight marker/200bpb Hyperladder I Bioline®. 

The analysis of the matrix of presence and absence of bands allowed us to estimate the mean values of Ht (total gene diversity) and Hs (gene diversity relative to the population), with which we attributed greater genetic diversity to that found between populations (interpopulation) and not within populations. The mean value obtained for the Gst=0.1231 index suggested moderate genetic differentiation ¹⁸ and the mean value of Nm inferred effective gene migration (Table 2), in line with the average of 15 polymorphic loci found in all the populations studied. The populations of Hernandarias and San Lorenzo showed the same percentage of intraspecific polymorphism (58.3%), while the populations of Caaguazú and the Rockefeller reference strain showed a percentage of intraspecific polymorphism of 62.5%. The highest percentage was found in the Piribebuy mosquito population (66.6%) (Table 3).

The relationships between mosquito populations according to locality of origin, including the Rockefeller reference strain, revealed the existence of two groups (clusters), independently of the ecoregions; one group of the Ae. aegypti populations from Hernandarias, Piribebuy and another with the populations of San Lorenzo, Caaguazú and the Rockefeller reference strain. At the terminal point of the cluster, the Caaguazú populations separated from the rest of the populations from other regions of the axis and the San Lorenzo populations separated from those of the reference strain (Figure 2).

Figure 2 Dendrogram based on Nei’s genetic distance using the unpaired method of analysis of the arithmetic mean (UPGMA), Nei, 1978, constructed by analysis of band frequencies by random amplification of polymorphic DNA by PCR (RAPD-PCR), for Ae. aegypti populations from four localities in the Eastern Region of Paraguay and the reference Rockefeller strain (Pop Gene software). 

The model of actual and potential spatial distribution of Ae. aegypti based on 19 bioclimatic variables (DIVA-GIS and MAXENT) confirmed the existence of suitable areas for vector establishment in 70% of the regions of the cross-border axis. The map shows the corridor between the ecoregions of the Humid Chaco (San Lorenzo-Central and Piribebuy-Cordillera) and the Atlantic Forest (Coronel Oviedo-Caaguazú and Hernandarias-Alto Paraná). The gray scale (0 to 1) indicates the probability of presence of the species (Figure 3).

Figure 3 Areas of suitability for actual and potential spatial distribution of Ae. aegypti populations in the Central-Upper Paraná cross-border axis of Paraguay according to bioclimatic variables (DIVA-GIS and MAXENT; 10 percentile cut-off threshold). In light gray; the Humid Chaco ecoregion (Central and Cordillera) and in dark gray; the Atlantic Forest (Caaguazú and Alto Paraná). 

The MAXENT model revealed that the bioclimatic variables suitable for the presence of Ae. aegypti were weighted 33.3% for precipitation in the warmest quarter (mean 184 mm/month), 13.2% for precipitation in the wettest month (mean 75 mm/month), and 13% for average temperature in the driest quarter (mean 26°C). The data were taken from the OGIMET Meteorological Information Service (https://www.ogimet.com).

The seasonality of temperature and isothermality followed each other in importance (approximately 7.4%), for the presence of the species. In other words, in the sampling areas, the seasonality of temperature was diverse according to the ecoregions, being 6 °C below the maximum temperature for the Humid Chaco and between 4 and 5 °C below the maximum temperature for the Atlantic Forest (OGIMET). On the other hand, the DIVA-GIS model revealed the presence of polar geographic points or “hot spots of presence” (black color) at the extremes of the cross-border axis, these being Asunción (near the border with Argentina) and Alto Paraná (near the border with Brazil) (Figure 3).

DISCUSSION

Our results showed that the natural populations of Ae. aegypti collected in the Central-Alto Paraná cross-border axis in Paraguay had moderate genetic diversity indices, which seems to indicate that, along the cross-border axis, the population would be genetically stabilizing with a gene flow greater than 1 and medium weighting of the effect of migrations between localities.

Genetic diversity is conditioned, among other factors, by human inclusion or by the effects of natural environmental changes on the population dynamics of organisms ¹⁹, and allows the evaluation of the conditions of disease vectors, invasive species or the existence of improved and genetically modified varieties, as well as the intra- and inter-population gene flow of agents that have effects on public health ²⁰.

These results are similar to the diversity indexes obtained by RAPD-PCR in six Brazilian states for Ae. aegypti, in which the Ht index was 0.390 with 27 polymorphic loci ²¹ and with 60 polymorphic loci, the Ht index was 0.388 ²²; analogous results were reported in Mexico with 60 polymorphic loci, where the Ht index=0.34 ¹² and in Puerto Rico this index was Ht= 0.35 with 57 polymorphic loci ²³. These values revealed indices of moderate heterogeneity among the studied populations, in compliance with the classification criteria ¹⁸. The study populations showed moderate differentiation (Gst=0.107 for primer H3, and Gst=0.138 for primer B03) not dependent on geographic region. The short flight distance covered by the insect and the very low Gst indices suggest passive or forced dispersal between neighboring localities, favored by land transport and modulated by human commercial activity, as well as by accidental transport of eggs in artificial reservoirs; in this sense, some researchers have shown that there is a significant correlation between gene flow in Ae. aegypti and passive human transport ²⁴. This type of migration would also explain the reduced variation between distant populations, which probably have not had enough evolutionary time to diverge, and their similarity results from the inertia of gene frequency over time ^{21 , 25 , 26}.

Regarding the segregation cluster of the populations, constructed based on Nei’s genetic distance, we observed two similar groups, each with populations from both ecoregions and congruent with moderate genetic differentiation, with the Caaguazú population being the most divergent from the other populations. The similarity of the Ae. aegypti mosquito populations of San Lorenzo and the Rockefeller reference strain, which is highly inbred and laboratory-adapted, resembles artificial laboratory selection (Rockefeller strain) with the domiciliation of wild Ae. aegypti in highly anthropized regions (San Lorenzo), in this sense, under both conditions there is optimal growth, availability and frequency of blood intake, conferring “docility” to the behavior in space-time that intervenes in the selection processes ²⁷.

The model of actual and potential spatial distribution of Ae. aegypti based on 19 bioclimatic variables (DIVA-GIS and MAXENT) confirmed the existence of suitable areas for the establishment of the vector in 70% of the regions of the cross-border axis (Argentina-Paraguay-Brazil), with the determining variables being, in order of importance, precipitation in the warmest quarter, precipitation in the wettest month, average temperature in the driest quarter, seasonality of temperature and isothermality.

The peri-urban areas near Coronel Oviedo (Caaguazú) do not appear to be suitable for establishment of the vector, probably because of the large distances between urbanized areas and access routes, the low population density and the high mobility of Amerindian family groups in the area, which would make it difficult for the vector to establish itself perennially, even when climatic conditions are favorable. In other countries, the distribution of Ae. aegypti has been reported in indigenous communities, where there are endemic areas of virus transmission and health emergencies related to dengue; however, in Paraguay, there are very few records of dengue in native populations ²⁸.

A limitation of this study is that the RAPD-PCR marker requires rigorous standardization to reduce the variation caused by the components of the reaction, and its dominant nature implies indirect estimates of genetic diversity. However, its simple, rapid and less expensive methodology allows the detection of polymorphisms without sequencing or characterizing the genome of interest by analyzing several loci at the same time.

This study is the first to address a genetic study with natural populations of Ae. aegypti in Paraguay, which allowed us to approximate what would occur in a road axis of great commercial importance (land connection between Asunción, capital of Paraguay, and the border with Brazil, Alto Paraná), where Ae. aegypti is established; these dynamics should be monitored, as these are regions of high anthropic activity with tourist and commercial areas that establish entry and exit points between countries, thus favoring the dissemination of the transmitting agent, depending on the appearance of cases and the selection of subpopulations resistant to chemical control.

This study showed that there is moderate genetic diversity in Ae. aegypti populations from areas with records of dengue cases located in the cross-border road corridor linking the Central and Alto Paraná departments of Paraguay. Interpopulation diversity was greater than intrapopulation diversity, which leads us to assume that there are frequent but moderate migrations of the vector, conditioned by suitable bioclimatic factors that contribute to this differentiation. As a recommendation, we it is considered necessary to include other markers, such as mitochondrial genes that would allow us to continue with the assignment of haplotypes through reference tests such as the sequencing of the genome of Ae. aegypti in Paraguay, with the aim of better understanding its biodiversity and/or local migration routes to stratify risk areas in order to define better vector control strategies and methods to ensure adequate entomological surveillance.