Aedes (Georgecraigius) epactius from Zacatecas and Chihuahua Mexico: New Geographical Distribution and Altitude Records

Simple Summary

In this work, we report a new distribution for the Western Rock Pool mosquito, Aedes epactius, in the state of Zacatecas, Mexico. Additionally, we document its occurrence at 2300 m above sea level in the state of Chihuahua and at nearly 2600 m above sea level in Zacatecas. These records highlight the remarkable dispersal plasticity of the Western Rock Pool mosquito in the Americas, demonstrating its ability to adapt to a wide range of altitudes, from coastal areas with warm temperatures to high-altitude regions with considerably lower average temperatures.

Abstract

Adults and immatures of Aedes epactius were collected in July and December 2022 at sites of high elevation in the states of Chihuahua (2300 masl) and Zacatecas (2182 and 2595 masl), Mexico, respectively. Mosquitoes were identified morphologically and sequenced for a DNA barcode of the cytochrome c oxidase I (COX1). This is the first distributional record of Ae. epactius in Zacatecas and provides evidence of the highest altitude in the Americas, including Mexico. The geographical distribution of Ae. epactius in Mexico was reviewed, and the COX1 analysis, using phylogenetic Bayesian analysis to confirm species identification, was performed.

1. Introduction

The mosquito Aedes (Georgecraigius) epactius Dyar & Knab, commonly known as the Western Rock Pool mosquito, has not been conclusively incriminated as an arbovirus vector [1]. However, laboratory studies have demonstrated its potential as a competent vector for the Jamestown Canyon virus [2] and its ability to vertically transmit the St. Louis encephalitis virus [3]. Zuñiga et al. (2023) [4] documented WNV-RNA-positive Ae. epactius mosquitoes in the municipalities of Casas Grandes (1488 masl), Nuevo Casas Grandes (1460 masl), Praxedis G. Guerrero (1085 masl), Guadalupe (1093 masl), and Ciudad Juárez (1230 masl), in the state of Chihuahua, northwest Mexico. Previously, Correa-Morales et al. (2019) [5] found Ae. epactius mosquitoes in both urban and semi-urban areas of Mexico, with some pools testing positive for Zika virus (ZIKV) RNA. In the Americas, the geographic distribution of Ae. epactius is widespread, with records in Costa Rica, El Salvador, Guatemala, Honduras, Panama, and the U.S. [5,6,7]. In Mexico, extensive studies on mosquito fauna have been documented, as well as their distribution across multiple regions, including the states of northwest Mexico, the Gulf of Mexico region, the South Pacific, and the Neovolcanic Axis [8,9,10]. Notably, Ae. epactius exhibits high plasticity, allowing it to tolerate cold climates at high altitudes [8,9,10,11,12]. The highest previously published elevation record for Ae. epactius in Mexico was 2417 masl, where mosquitoes were collected across various elevations and climates in the states of Veracruz and Puebla [9]. These studies underscore the importance of accurately estimating the spatial distribution of Ae. epactius and registering precise morphological identification in both immature and adult stages during surveillance operations targeting Aedes aegypti and Ae. albopictus in Mexico [10,11]. Given the importance of routine surveillance for other Culicidae species incriminated as vectors in Mexico, this research communication reports two significant findings: (1) a new altitude record for Ae. epactius in the Americas, including Mexico; and (2) a new distribution record for this species in Zacatecas state, Mexico.

2. Materials and Methods

This study is part of a broader investigation into the surveillance of arboviruses in dipteran species of medical and veterinary importance in Mexico. In July and December 2022, resting adults and immature stages of mosquitoes were collected in the states of Chihuahua and Zacatecas. Adults were caught using a buccal aspirator, while immatures were collected using a 50 mL plastic pipette. Immatures were kept in Whirl-pak^® plastic bags (Nasco Co., Fort Atkinson, WI, USA) and were reared in individual plastic tubes until imago emergence. Resting adults and recently emerged imagos were killed using chloroform vapors, dried and preserved in sterile 1.5 mL polypropylene tubes, and then kept at −20 °C until processing for morphological identification and molecular analysis. Mosquitoes were identified using an Olympus SZ61 stereomicroscope (Olympus, Tokyo, Japan), and the identification keys of Clark-Gil and Darsie (1983) [13] and Darsie and Ward (2005) [14] were used. Photographs of the key characters were taken using an Olympus SZ61 stereomicroscope with a 55MPixel 4K microscope camera.

For molecular analysis, mosquitoes from each collection site were processed for DNA barcoding and phylogenetic reconstruction to confirm their identification. Briefly, the mitochondrial 5′ cytochrome oxidase region (COX1) was sequenced using the established protocols of Hernández-Triana, L.M., et al. [15]. Overall, COX1 sequences were matched in GenBank using nucleotide BLAST. Then, at least one COX1 reference sequence of some representative species of the subgenera Aedimorphus, Aztecaedes, Georgecraigius, Howardina, Jarnellius, Kompia, Lewnielsenius, and Ochlerotatus, belonging to the genus Aedes, was downloaded from GenBank. Overall sequences were aligned using MUSCLE algorithms and were then compared with our sequences from Chihuahua and Zacatecas by a Bayesian phylogenetic reconstruction analysis. The phylogenetic tree was inferred using the Bayesian algorithm. Sequences were modeled by the general time-reversible (GTR) substitution model, along with Gamma + Invariant sites as the heterogeneity model, using BEAUti software v10.5.0. Then, the tree priors shared by all tree models was computed by the Yule speciation model using a random starting tree, and the MCMC chain was run for 10⁸ generations to ensure convergence. Subsequently, the BEAST file was processed to build the phylogenetic tree. The tree was built using the TreeAnnotator software (v1.10) in the BEAST package with a Burnin (as states) parameter of 10⁵. The tree generated was edited using the FigTree program (v1.4.4) in the BEAST package. An outgroup of the Lutzomyiz crusiata COX1 gene was used as the root tree. Finally, an intraspecific genetic distance analysis using the nucleotide substitution model Kimura 2-parameter (K2P) was performed between the sequences of Ae. epactius from Chihuahua and Zacatecas and compared with GenBank reference sequences of Ae. epactius reported in Mexico, with the aim of verifying low-genetic variability and corroborating that they belong to the same species.

3. Results

Ae. epactius was found at high-altitude sites in Aguatachi, a municipality of Uruachi, in the state of Chihuahua (27°54′37″ N; 108°10′14″ W), at 2300 masl. Ae. epactius was also found in Zacatecas city (22°46′41″ N; 102°33′46″ W), in a municipality of Zacatecas at 2595 masl, and in Fresnillo city, municipality of Fresnillo (23°10′53″ N; 102°51′46″ W) at 2182 masl, both in the state of Zacatecas (Figure 1). According to the Institute of Statistics and Geography of Mexico (INEGI, in Spanish, 2024) [16], Uruachi is a municipality of Chihuahua, located in the Sierra Madre Occidental (SMO), with an average altitude of 2639 masl, reaching a maximum of 2900 masl, and a minimum of 200 masl; the climate is temperate–subhumid, with average temperatures ranging from 14 °C to 16 °C. The cities of Zacatecas and Fresnillo are situated in a region where the SMO and the Sierra Madre Oriental intersect, resulting in one of the highest elevations in Mexico. In Zacatecas municipality, the average altitude is 2256 masl and ranges from 2800 to 2100 m. In Fresnillo municipality, the average altitude is 2121 masl, with a maximum of 2900 m and a minimum of 1900 m. In both municipalities, the climate is predominantly temperate, ranging from 10 °C to 19 °C.

In the Aguatachi community, seven imagos were obtained from 10 to 15 larvae collected from a tire on the roadside. In Zacatecas City, three adults were found resting indoors in a public restroom. Despite our efforts to locate the breeding site and collect larvae for characterization, we were unsuccessful. In Fresnillo city, 14 imagos were obtained from at least 20 larvae found in a water tank located in the municipal cemetery. Figure 2 shows the Ae. epactius female adult morphology and the breeding sites where their immatures were found.

The COX1 sequences obtained were deposited in GenBank (accession numbers: Urique, Chihuahua: PQ288531 and PQ288532; Fresnillo city, Zacatecas: PQ288533; and Zacatecas city, Zacatecas: PQ288534). Overall, the COX1 sequences from Chihuahua and Zacatecas were compared with those in GenBank using nucleotide BLAST, confirming a strong match with Ae. Epactius, with 98% query coverage and 94.75–99.85% identity. The estimation of intraspecific genetic distances between Ae. epactius from Chihuahua and Zacatecas, with those from other regions of Mexico, was 0.018 ± 0.001. Phylogenetic analysis confirmed that the genetic relationships of the sequences from Ae. epactius in Chihuahua and Zacatecas, when compared with reference sequences from GenBank, were grouped distinctly within the Ae. epactius species cluster (Figure 3).

4. Discussion

The present communication research supplements existing knowledge on the altitudinal distribution of Ae. epactius in Mexico, previously documented by Lozano-Fuentes et al. (2014) [9]. It also provides the first record of the Mexican endemic mosquito species Ae. epactius in the state of Zacatecas, Mexico, where it was found at an elevation of 2595 masl, recording the highest altitude at which Ae. epactius has been recorded in the Americas, including Mexico, to date.

Lozano-Fuentes et al. (2014) [9] conducted a systematic investigation of Ae. epactius across a broad altitudinal and climatic gradient, ranging from near-sea level in Veracruz to high elevations across Veracruz to Puebla, Mexico. The highest altitude where Ae. epactius was recorded in Puebla city (19°02′24″ N; 98°11′31″ W), at 2133 masl, where 530 specimens were collected from diverse breeding sites, including discarded containers such as bottles, cans, and plastic bags, as well as larger items like washing machines, refrigerators, tires, buckets, flowerpots, cement troughs, large clay jars, and drums. These records indicate the distribution of Ae. epactius at high altitudes in the template–cold climates in Mexico, highlight the species’ remarkable ability to adapt to extreme environmental conditions.

Ae. epactius is a species that competes within its niche with other mosquito species. For example, the documented coexistence of Ae. epactius with Ae. aegypti, an invasive species from Africa, suggests potential competition for ecological niches in similar habitats during their immature stages [11,12]. Additionally, fluctuations in the population dynamics of both species have been observed in response to seasonal changes. For instance, in September—a month characterized by variable temperatures ranging from warm to cold—Ae. epactius demonstrates higher densities compared to Ae. aegypti. This adaptation to high elevations and cold–temperate climates may have significant implications for vector ecology and the epidemiology of mosquito-borne diseases, influencing mosquito community structures, habitat competition for abiotic resources, predation dynamics, species distribution, and disease transmission [18]. Furthermore, this study presents a new record of the geographic distribution of Ae. epactius in Mexico, including Zacatecas, among the states where this species has been documented. Although Ae. epactius is not recognized as a major vector of arboviruses, its presence in Zacatecas could have important epidemiological implications. Specifically, Ae. epactius may play a ‘protective role’ by potentially limiting the spread of invasive species such as Ae. aegypti or/and Ae. albopictus [18].

Finally, DNA barcoding based on the analysis of the COI gene is an important tool for species identification and is widely regarded as a key component of integrative taxonomy. It utilizes genetic information to complement and enhance traditional morphological data, improving the accuracy and resolution of species identification [19]. The Ae. epactius sequences obtained from Chihuahua and Zacatecas fit precisely with reference sequences within the clade of the subgenus Georgecraigius (Figure 3). The calculated low intrageneric distance (less than 2%) indicates that mosquito species in these regions are distributed relatively uniformly, without forming complex clusters, and that the genetic variability within the population is consistent [20].

5. Conclusions

This communication research presents the first confirmed distributional record of Ae. epactius in Zacatecas and establishes a new highest altitude record for this species in the Americas, including Mexico, suggesting the importance of vector surveillance in regions of high altitude in Mexico. From a wider perspective, generating additional COI gene sequences from various biogeographic regions of Mexico would be of great importance for studying the biology of Ae. epactius. This would enable more robust biogeographic analyses and help determine whether multiple lineages of Ae. epactius exist in Mexico. Such research is crucial, as a deeper understanding of Ae. epactius’ genetic variability could uncover evolutionary patterns, local adaptations, and potential ecological and behavioral differences, all of which could contribute to more effective control and management strategies for this species.