Comparative Genetics Using Three mtDNA Markers in Aedes aegypti (Linnaeus) Populations from Four Municipalities in the Satate of Mato Grosso, Brazil.


 Aedes aegypti mosquito has spread throughout the tropical and subtropical world and is currently the primary species responsible for transmitting dengue, urban yellow fever, Chikungunya, and Zika virus. This study aimed to investigate the inter- and intrapopulational genetic variability of Aedes aegypti through mitochondrial DNA, COI, ND4, and ND5 molecular markers in four municipalities in Mato Grosso. We used the Geneious software to build dendrograms for differentiating populations from each municipality. The interpopulational genetic distance obtained from sequence analysis showed a difference within populations through groups' formation in the ordering. Besides, we identified a difference in the interindividual genetic distance values, notably for the ND5 gene from the populations captured in the four municipalities. We recorded the smallest interindividual genetic distance within populations for populations from Chapada dos Guimarães. Extrinsic factors, including breeding habitat removal, can contribute to decreasing variability. Consequently, the dendrogram showed some similarities. Ovitrap monitoring, vector elimination, and genetic flow investigation stimulate actions to prevent transmitted diseases and support essential effective measures to control and fight Ae. aegypti.


Introduction
Aedes (Stegomyia) aegypti mosquito (Linnaeus, 1762), a mosquito native to Africa 1 , has spread throughout tropical and subtropical areas due to anthropogenic activities. Today, Ae. aegypti is responsible for transmitting dengue, yellow fever, Chikungunya, and Zika virus infection. Dengue is currently one of the most frequent diseases in Brazil 2 . Its annual variations in reporting and expansion is directly related to several factors, such as favorable environment, new serotype circulation, human population movements, and vector local infestation level. For this reason, Ae. aegypti adaptation to urban environments favors anthropic long-distance dispersion, supported by the eggs' resistance to desiccation and human host 3 .
An alternative monitoring method of Ae. aegypti is the detection and quanti cation of eggs deposited in oviposition traps installed in the home environment, which allows the identi cation of areas with the presence of mosquitoes and the analysis of the spatial and temporal distribution of its population 4 . An essential tool in this vector's study is the capture employing ovitraps to control and monitor populations by obtaining a signi cant number of specimens. This tool provides information on the presence and population density in different environments that can be estimated using samples, based on genetic methods 5 . Population diagnosis of Ae. aegypti through ovitraps allows checking the presence of mosquitoes throughout the year by attracting the pregnant female to oviposition 6 . A study in China, which employed this capture method, showed that vector control is critical and sometimes the only effective way to block or decrease dengue transmission 7 . Infestations can increase according to higher temperatures, humidity 8 , and disordered urbanization.
The genetic diversity of some South American populations have been assessed using a wide range of genetic markers. Mitochondrial DNA (mtDNA) genes are widely used for the identi cation of genetic variants, dispersal patterns, phylogeny, and population dynamic studies of Ae. Aegypti 9,10 . Besides the populational quanti cation of Ae. Aegypti, the literature reports the use of markers, including mitochondrial DNA 4,11 , especially the Cytochrome Oxidase I (COI) gene, widely used in culicid research, to analyze its genetic variability 12 . Molecular techniques, including the polymerase chain reaction (PCR), can contribute to the understanding of vector-human relationships and also enable the genetic study of populations 13 and their diagnostic investigation in different biological samples 14 .
The evaluation of genetic diversity on a smaller scale, such as in cities, allows us to verify viral dispersion. Ae. aegypti genetic diversity analysis might be applied to know the structure of the populations to comprehend their dynamics, providing data that might lead to new control measures 15 .
Several genetic mechanisms are known to generate variability within and between populations, whose differences can arise from random occurrences, including the genetic composition of specimens that disperse and create new populations 16 . In addition, there may be changes in allele frequencies resulting from occasional breeding in small 17 or large populations, whose effect can be underestimated in short periods. Differences between populations under different environmental conditions for survival and reproduction (adaptive value 18 ) can accumulate and result in the development of a new species 19 .
Mitochondrial DNA (mtDNA) is an excellent target for molecular analysis due to the absence of introns, haploid inheritance, and because it contains only genes associated with mitochondrial functions. mtDNA is used in population genetic analysis 20 and traces a species' evolutionary history 21 . Interestingly, repair mechanisms for this molecule have not yet been described, which likely facilitates the accumulation of mutations and, consequently, the detection of intraspeci c variations 21 . Base substitutions, mtDNA deletions, and tRNA-encoding gene translocations cause changes in gene order between phylogenetically related organisms 22 .
Given the above, studies on the genetic variability of mosquitoes transmitting prominent urban viruses are critical in warmer regions with favorable environments for Ae. aegypti proliferation, as in the state of Mato Grosso. Therefore, this research aimed to investigate the inter-and intra-populational genetic variability of this vector in four municipalities in Mato Grosso by using three mitochondrial DNA markers: COI, ND4, and ND5.

Results
Genetic Distance with Dendrograms.
Of the 400 specimens identi ed as Ae. aegypti, 169 underwent PCR using COI, ND4, and ND5 primers. However, due to the low annealing capacity, the ampli cations occurred in about 30% for COI primer, which had the lowest annealing rate ( Table 1). The ND5 primer presented the best genetic variability results.
Through the Geneious application, we ordered the three primers to identify the different populations in each municipality. The dendrograms ordered the specimens' distance between the municipalities (interpopulational, Fig. 1) and within each municipality (intrapopulational, Fig. 2). We also observed that the obtained sequences had interindividual differences within populations, especially when considering each municipality's sampling location. The most distant specimens indicate that there is a genetically variation, which may arise from a transitory individual into this population. In the interpopulation analysis of the trees generated, we veri ed distances that only one specimen from the municipality of CB was signi cantly distant from the others. This nding was based on observations from the COI mitochondrial gene (Fig. 1). Similarly, one sample based on this gene in the municipality of VG. Lastly, for CP and SA municipalities, we did not observe any signi cant genetic distance among the evaluated specimens. However, intrapopulation analysis of these three genes together (Fig. 2) showed a signi cant distance, especially in VG municipality with COI gene.
Genetic Distance by Analysis of Variance -ANOVA between Factors.
In CP, the ND4 gene presented the lowest interpopulational genetic distance among the analyzed specimens (Fig. 4A). Comparatively and signi cantly (F 6, 97 =5.4826, p = 0.00006), we considered the sequence of the three primers together between the four municipalities ( Fig. 4B), and the municipality with the shortest distance was also CP.
We considered intrapopulational differences in the genetic distance for each gene (Fig. 5). The most signi cant intrapopulational genetic distance was recorded for the COI gene from specimens captured in the municipality of CB (F 2, 25 = 34562; p = 0.0000). The recording in the VG municipality was signi cant, with low variability for all primers (F 2, 26 =34.069; p = 0.000). In CP (F 2, 25 =1169.2; p = 0.000), the ND5 gene reached the highest genetic distance. We observed a trend of less variability in the ND5 gene in the intrapopulational genetic distance of specimens from the municipality of SA (F 2, 21 =19.911; p = 0.000).
Gene polymorphisms and DNA sequencing.
We sequenced the segments of the mitochondrial DNA COI, ND4, and ND5 genes using the Sanger method, whose factorial ANOVA results derived from nitrogenous bases sequencing for each gene. Genetic diversity values were high (h = 0.702; p = 0.015) for the four municipalities. Such analysis allowed us to identify the greatest genetic diversity, which indicated that the variation occurred within populations, with an FST value of 0.329.
Haplotipic analysis map using Median Joining.
Based on genes COI, ND4, and ND5 a network haplotype of Ae. aegypti populations from Baixada Cuiabana has complemented this research (Fig. 6). Under this, COI was represented with a total number of mutations of S/Eta: 54, the variance of haplotype diversity: 0,01009, and nucleotide diversity, Pi: 0,02972. For ND4, the number of polymorphic (segregating) sites was S: 48, the total number of mutations Eta: 61 and, the variance of Haplotype diversity: 0,00358; the Nucleotide diversity had the Pi: 0,03916. In this same analysis for the ND5 gene, the number of polymorphic (segregating) sites was S: 19, the total number of mutations was Eta: 19 and, the variance of haplotype diversity: 0,00210 with nucleotide diversity, Pi: 0,00396.

Discussion
Our study is the rst 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 rapidly 33 . In this study, when populations analyzed together and by gene, the distance found unlikely represents populational fragmentation nor events that could signi cantly distance a representative sample of individuals.
Birungi and Munstermann 26 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 con rmed 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 identi ed 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 signi cant, indicating that genetic distance is not always linked to geographic distance (r=-0.1216 and p = 0.755 36 ).
The genetic differentiation study of 15 populations from Maranhão 37 , 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 con rmed 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 signi cant genetic distances can suggest speciation events. We identi ed 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 re ect the natural selection. They might also result from passive transport by human movement with vehicles and cargo 38 . 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 rst 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 ow can also be stopped due to geographical barriers; thus, no new characteristics are shared 40 . In the present study, we found no signi cant 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 identi ed specimens in different groups formed in dendrograms. These results are in keeping with those from this study, which can imply intraspeci c 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 pro le 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 population 14 .
Population-intrinsic changes can suggest the in uence 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 ow among 163 mosquitoes from 14 cities. Phylogenetic analysis identi ed two clades in genetic variability. They recorded two types of haplotypes and found a signi cant polymorphism among other loci. These authors' analysis revealed two strains separated by 8 xed mutations, suggesting that Ae. aegypti populations likely came from eastern and western Africa, with evidence of multiple introductions.
Our ndings complement those results of earlier studies and have signi cant 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 al 34 . Our principal limitation was, less than 20% of de DNA samples had been ampli ed 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 ow and dispersion estimates can support the government's measures that are essential to effectively control and ght the vector.

Material And Methods
Sampling and data.
This study was carried out in four municipalities in the state of Mato Grosso: Cuiabá (CB), Várzea Grande (VG), Chapada dos Guimarães (CP), and Santo Antônio do Leverger (SA) (Fig. 7). We selected such regions due to their higher human population movement (CB and VG), distinct geographical position and altitude (SA, at about 100 m, and CP, at about 700 meters above sea level). The number of adults resulting from the development of eggs in the laboratory are shown in table 2.
This was a descriptive observational study with a quantitative and qualitative approach. Data were collected with the aid of ovitraps during December/2015 ( ooding), February/2016 ( ood), June/2016 (drought), and November/2016 ( ood) to investigate the number (n) of copies of Ae. aegypti in the four municipalities (Table 2). We applied factorial ANOVA considering the interindividual genetic distance data based on dendrogram results per municipality. We considered gene factors (COI, ND4, and ND5) as independent variables and genetic distance as the dependent variable (factorial). The factors 'genes' and 'municipalities' were analyzed jointly using two-way ANOVA together with nitrogenous base proportionality. We used Ae. albopictus as an external group and identi ed signi cant differences between each primer for the two vector species (F 1, 119 = 32,962; p = 0.000). Haplotypic diversity calculation was performed using the  Dendrogram for intrapopulational analysis based on the results for COI, ND4, and ND5 genes from Ae.
aegypti populations recorded in the four municipalities. We used Ae. albopictus as an outside group, using the results for each primer.   Haplotypes network map showing the threes primers, COI in yellow color, ND4 in blue and, ND5 in green color. The outgroup of Ae albopictus is shown in red. Some haplotypes per each gene were signi cantly segregated.