Our study is the first to investigate the genetic variability of Ae. aegypti populations in municipalities of Mato Grosso. We observed a genetic variation in COI, ND4, and ND5 mtDNA sequences between specimens per analyzed location, with inter- and intrapopulational genetic distance. These genes accumulate base substitutions in the mitochondrial genome most rapidly33. In this study, when populations analyzed together and by gene, the distance found unlikely represents populational fragmentation nor events that could significantly distance a representative sample of individuals.
Birungi and Munstermann26 investigated Ae. albopictus in Brazil and the United States and stated that the effect of genetic drift is more pronounced in mtDNA than in nuclear loci. The present study results showed variability in the inter- and intrapopulational genetic distance for the Ae. aegypti populations analyzed, notably for the ND5 gene in between municipalities' populations. Ae. aegypti specimen analysis confirmed that these markers are essential in population sampling.
The dispersal of mosquitoes over long distances only occurs passively (eggs and adults transported), which may explain the diversity of some haplotypes in different locations. The shortest genetic distance between genes was found for ND4 in specimens captured in CP, with an exception in one sample only. Ponce et al.10 compared Ecuadorian populations of Ae. aegypti from 17 sites and revealed the presence of only two haplotypes. The variations detected between ND5 gene sequences in the municipalities may indicate a genetic structure in Ae. aegypti resulting from several factors, including extinction and recolonization events, genetic drift, and geographical differentiation.
Interpopulational genetic distance values for the COI gene showed low variability in the municipalities. However, population genetic analyzes of Ae. aegypti performed by Lv et al.34 identified a relatively high degree of polymorphism in the COI and ND4 sequences in eight populations, which were divided into eleven haplotypes. In studies using ND4 primer by Scarpassa et al.35, the polymorphism values were higher for the nucleotides, the same as what we found, probably because in Ae. aegypti this gene is under most mutations in many samples. In some studies, distance isolation may not be significant, indicating that genetic distance is not always linked to geographic distance (r=-0.1216 and p = 0.75536).
The genetic differentiation study of 15 populations from Maranhão37, based on the mitochondrial marker ND4, found 15 haplotypes among the polymorphic sites. It revealed that most of the variation (58.47%) was found within populations. In our study, fewer haplotypes variations occurred using ND5 and confirmed the total separation per marker applied. Of the 71 distinct haplotypes, ND4 represented 41% of the variation, COI with 35%, and ND5 with 24%. Few haplotypes were evidenced with a high mutation rate or variation about the others, within the same gene. The ND4 gene had the smallest interpopulation genetic distance among the analyzed specimens, especially in the municipality of Cuiabá.
Regarding the four municipalities analyzed in this study, the populations had specimens that were distant because of the ND5 gene, as shown by the genetic diversity values (h = 0.702; p = 0.015) and the FST value of 0.329, which indicated more considerable intrapopulational variation. Highly significant genetic distances can suggest speciation events. We identified a difference in the interindividual genetic distance values, notably for the ND5 gene from the populations captured in the four municipalities. Thus, our results show a robust hypothesis that new morphospecies is adapted to local environmental conditions, although morphological data is still lacking to support this assumption.
Specimens with the most considerable genetic distance may originate from distinct lineages from areas adjacent to the capture site or reflect the natural selection. They might also result from passive transport by human movement with vehicles and cargo38. Barbosa et al.39 mention that the passive dispersion of vectors found mainly in tires started due to the intense tire trade between the municipalities of the regions where the first outbreaks occurred (in the state of São Paulo), with subsequent dissemination. Thus, tires can become a severe problem for public health.
The species populations from other microhabitats introduced into adjacent areas may share polymorphisms within the same and between different populations. However, the local gene flow can also be stopped due to geographical barriers; thus, no new characteristics are shared40. In the present study, we found no significant barriers between populations with the highest genetic distance and intrapopulational variation.
The ordering using dendrograms with the highest genetic distance indicates the dissimilarity between some loci, as found for the ND4 gene in CP in the interpopulational analyses. Population-extrinsic factors, including breeding habitat elimination, may contribute to the low variability at some points, with some dendrogram similarity per analyzed gene. Eleven ND4 gene and 10 ND5 gene specimens formed a distinct group in the alignment of 28 individuals in the municipality of CB. Such formation confers distance and genetic variability between the specimens within the populations analyzed. Similarly, this observation was also present in the other investigated cities, and the ND5 gene presented variability between populations, which may be related to old and repeated introductions of the species into different habitats. This statement is supported by Seixas et al.3 in an article on the colonization and populational diversity of Ae. aegypti on Madeira Island, Portugal.
In the municipality of CB, we statistically recorded the highest intra-populational variability for the mitochondrial COI gene. With analyses of this gene, Van de Vossemberg et al.21 identified specimens in different groups formed in dendrograms. These results are in keeping with those from this study, which can imply intraspecific variation between Ae. aegypti specimens. According to Seixas et al.3, different specimens analyzed based on COI exhibit greater vectorial competence than other populations with a single haplotype. The higher the genetic variability in these mosquitoes, the greater likelihood they will disperse viruses and other disease-causing parasites.
Factorial ANOVA results for the investigated genes of each Ae. aegypti specimen showed the differences derived from variations between populations, leading to a genetic distance within each municipality. In general, the populations' genetic profile varies by place throughout the distribution of a species. These differences can arise from random occurrences, including the genetic composition of specimens that disperse and create a new population14.
Population-intrinsic changes can suggest the influence of genetic distance, with the consequent genetic drift of rare or restricted genes to a single population or geographically close populations, as the municipalities of CB and VG. More considerable genetic distances between specimens for the ND5 gene in these municipalities can be associated with multiple introductions linked to different strains, as their habitats have high movement of people, passive dispersion patterns, and infestation control activities.
Based on nucleotide differences and sequencing, given the nitrogenous base proportionality, molecular markers did not show differences between the municipalities through the Kruskal-Wallis Test (analysis performed using the joint gene sequences). The differences were present only in the base frequency between the encoded genes. The polymorphism among Ae. aegypti population was greater for the ND4 gene, with a greater number of mutations and nucleotide diversity, according to the results of the haplotype network such as the template proposed by Bandelt et al. (1999) and Rozas et al. (1999). Certain specimens of Ae. aegypti may be more similar to each other when using COI and ND5 primers.
Scarpassa et al.35 researched in fourteen locations in Brazil, including the Cuiabá city, using mitochondrial gene Cytochrome Oxidase I (COI) to examine gene flow among 163 mosquitoes from 14 cities. Phylogenetic analysis identified two clades in genetic variability. They recorded two types of haplotypes and found a significant polymorphism among other loci. These authors' analysis revealed two strains separated by 8 fixed mutations, suggesting that Ae. aegypti populations likely came from eastern and western Africa, with evidence of multiple introductions.
Our findings complement those results of earlier studies and have significant implications for understanding how these mosquitoes behave under distinct environments and, under human interventions. Therefore, the monitoring of Ae. aegypti species is essential for preventing and controlling vector-borne infectious diseases and, governments need to designate effective control measures, how mentioned by Lv et al34. Our principal limitation was, less than 20% of de DNA samples had been amplified using the primer COI of distinct populations (see material and methods) and, the authors suggest another's primers need to be designed for Brazilian populations of Ae. aegypti.
In conclusion, this pioneering study conducted in the state of Mato Grosso shows the intrapopulation diversity of Ae. aegypti in each municipality investigated. The monitoring with ovitraps and genetic research of Ae. aegypti aims to identify genetic variations to boost actions to prevent diseases transmitted by this species. Genetic flow and dispersion estimates can support the government's measures that are essential to effectively control and fight the vector.