In this study, 288 anophelines from five departments of Honduras (Gracias a Dios, El Paraíso, Comayagua, Cortés, and Bay Islands) were identified through morphology and molecular biology. The relative abundance of mosquitoes is also described and the presence of Plasmodia in the head/thorax of mosquitoes is reported. The results described here build upon a recent study published in 2020 [6], which included the taxonomic and molecular identification of 1,320 Anopheles mosquitoes collected in five departments of Honduras (Gracias a Dios, El Paraíso, Comayagua, Atlántida, and Colón).
The majority (73%) of the anophelines were collected in the department of Gracias a Dios, which comprises a region known as La Moskitia, shared with Nicaragua, and endemic for malaria. La Moskitia exhibits unique ecological and socio-cultural characteristics. This region is separated from the rest of the country by the Río Plátano biosphere reserve, with a population of more than 98,000 people distributed in almost 17,000 km2 [29]. Fishing and hunting are the main activities. Access to health services is inadequate, and communication routes outside the region are almost non-existent. This region currently contributes 98% of malaria cases − 195 out of 199 cases reported until epidemiological week number 5 of 2022 (Personal communication, Honduras Ministry of Health). The humid tropical ecosystem explains the great abundance of anophelines collected in the area.
In total, most of the specimens (80%) were identified as An. albimanus. This species was present in the five departments despite the ecological differences among the study sites. Anopheles albimanus has been described as the dominant species in most regions of Mesoamerica and northern South America [4]. This finding is also consistent with a previous study conducted in Honduras where 74% of anophelines identified were An. albimanus [6]. Anopheles albimanus was also found to be a dominant anopheline in other countries in Mesoamerica and northern South America. A study where 22,000 larvae of 13 species of anophelines were collected in 19 states of Mexico revealed that An. albimanus and An. pseudopunctipennis were the two most abundant species [30]. These two species were also found to be the most abundant and widely distributed anophelines along the Pacific coast of Mexico [31]. Two recent studies conducted among indigenous communities in Panama identified between 43% and 98% of mosquitoes as An. albimanus [32, 33]. In Colombia, a retrospective descriptive study showed that An. albimanus, An. nuneztovari s.l., and An. darlingi were the main vectors in receptive areas for malaria [34], and a study assessing the potential distribution of the three main malaria vectors in Colombia determined that An. albimanus had the greatest niche breadth mainly in coastal areas [35].
The second species reported in this study and considered a dominant vector of malaria [4] was An. pseudopunctipennis, which was recorded only in Comayagua. The remaining 17.4% of the collected mosquitoes belong to the following six species: An. crucians, An. argyritarsis, An. neomaculipalpus, An. vestitipennis, An. apicimacula, and An. punctimacula. Three of these species were not described in the 2020 study (An. argyritarsis, An. apicimacula, An. neomaculipalpus) [6], and are not considered dominant malaria vectors [3, 36]. A notable difference from the 2020 study is the decrease in the proportion of two species collected in Gracias a Dios. Anopheles vestitipennis went from 49.4–4.7% and An. crucians went from 29.5–6.2% [6]. However, the differences in the relative abundance of these species could be influenced by the dates and collection sites. The low proportions of uncommon Anopheles species found in this study and the previous study suggest that malaria control in Honduras should continue to focus on the most abundant An. albimanus and An. pseudopunctipennis. No An. darlingi or An. neivai specimens were collected in this study, which could be related to the limited geographical distribution of these species in Honduras, having been reported in the department of Atlántida [6].
Anopheles argyritarsis was collected in Puerto Lempira, Gracias a Dios, and in San José de Comayagua. Although this species is widely distributed in the Neotropics [37, 38, 39, 40], its potential as a malaria vector is controversial with evidence for insufficient, or non-existent vectorial capacity [39, 41]. Anopheles apicimacula and An. neomaculipalpus were collected only in Gracias a Dios. Both species have been widely reported in Mesoamerica [42, 43, 44] and South America [45, 46, 47, 48, 49, 50, 51, 52, 53, 54], and have been historically reported by the Honduran health authorities (unpublished data), but natural infections with Plasmodium sporozoites have only been detected in An. neomaculipalpus [54].
In addition to taxonomic identification, a partial segment of the COI gene was amplified and sequenced to confirm the identity of some specimens and decipher intraspecies genetic variability. All morphologically identified individuals were molecularly confirmed by barcoding. These results confirm the usefulness of the mitochondrial genome as a genetic marker [1, 19], especially when cryptic species or complexes of species difficult to distinguish solely by their phenotype coexist in the same area [55, 56]. In this study, the molecular identification of specimens collected in Roatán (Bay Islands), located more than 68 km from the mainland, was performed for the first time. All mosquitoes collected on the island were identified as An. albimanus and the phylogenetic analyses revealed no geographic region-based clustering, suggesting genetic flow between both populations. This result supports those of Molina-Cruz et al [57] after analyzing a large population of mosquitoes from the Caribbean, Central America, and South America using microsatellites. These authors demonstrated little genetic variation among the populations of northern Central America and weak isolation by distance. However, it has been suggested that there might be some barrier to gene flow [57], or contemporary isolation by distance in the isthmus [58] between the populations of An. albimanus from northern Central America and those of Panama and South America.
Herein, we report the first COI sequences for An. argyritarsis, An. neomaculipalpus, and An. apicimacula from Honduras. When comparing the sequences obtained here with homologous sequences of An. argyritarsis, a clear separation was found between the populations of Brazil/Colombia and the populations of Honduras/Mexico. A similar pattern was observed in the An. neomaculipalpus cladogram, in which the sequences from Colombia and Honduras are separated. Although the number of sequences is small, it is possible to speculate that there is geographic isolation between the Central American and South American populations. Further analyses including a greater number of individuals and the use of more robust molecular markers such as microsatellites could help decipher the evolutionary relationship of this species in the Neotropics. For An. apicimacula, no geographical separation was observed between the sequences from Honduras and those from Colombia even though the low percentage of identity yielded by the BLAST tool (93.5%) with respect to the sequences previously deposited in GenBank. According to the “barcoding gap” hypothesis, pairwise genetic differences greater than 3% are suggestive of separation between two species. Recent studies have described two geographically isolated lineages of An. apicimacula in Colombia [45, 47], which supports the existence of an Apicimacula species Complex that would encompass several species, including the specimens from Honduras. This question should be studied further in the future to clarify the taxonomy of this species.
Finally, no moaquitoes tested were positive for Plasmodium DNA by conventional mt cox1 gene PCR, which has been proven to be more sensitive than the CSP antigen detection by ELISA [59]. Results coincide with the absence of parasite infections in 36 engorged female Anopheles collected from Gracias a Dios, Honduras, in 2019 [8]. Several authors have reported mosquitoes infected by Plasmodium spp, especially in highly endemic regions such as Africa [60, 61, 62, 63]. Similar studies in South America have also detected infected mosquitoes in Colombia, Brazil, or Venezuela [46, 50, 54], but with infection rates of less than 1% in direct relation to a lower prevalence of malaria compared to Africa.