Distribution of Anopheles species
Eight locations with active foci of malaria  were visited to collect anopheline mosquitoes. A total of 1320 adult individuals of seven Anopheles species were collected and identified by a taxonomic key (Fig. 2): Anopheles (Nyssorhynchus) albimanus Wiedemann, An. (Nys.) darlingi Root, An. (Anopheles) vestitipennis Dyar & Knab, An. (An.) crucians Wiedemann, An. (An.) pseudopunctipennis Theobald, An. (An.) punctimacula s.l. Dyar & Knab, and An. (Kerteszia) neivai Howard, Dyar & Knab (Table 2). More morphological details of the vouchers can be observed in the project “CIGAN Bionomy of Anopheles sp. in Honduras” of the BOLD database.
Most specimens were identified as Anopheles albimanus (74.02%), An. crucians (10%), An. vestitipennis (7.2%), and An. darlingi (6.97%). The remaining 3 species accounted for less than 1% of the total. Anopheles albimanus was found in all locations except Sonaguera (Colón). The highest species richness (n = 5) was found in La Ceiba (Atlántida) followed by Kaukira (Gracias a Dios) (n=4). Moreover, five other localities reported only one to three species. Anopheles crucians was only found in Gracias a Dios. The greatest mosquito abundance was obtained in Gracias a Dios (33.8%), Atlántida (31.67%), and Comayagua (22.34%) (Fig. 3). An. darlingi was only present in Atlántida and Colón.
A total of 160 COI sequences and 122 ITS2 sequences were obtained for six out of seven Anopheles species. No sequences of An. neivai were obtained for either of the two markers. A second set of primers for COI (Fig.1) was able to produce 5 sequences of An. neivai and 10 sequences of four other species: An. albimanus, An. darlingi, An. punctimacula, and An. vestitipennis.
All COI and ITS2 sequences were deposited in the BOLD system database and the following BINs were assigned: CIGAN001-19 to CIGAN067-19, CIGAN068-20 to CIGAN178-20. These sequences were also deposited in GenBank under the following accession numbers: MT033921 – MT034050, MT040803 – MT040831, MT048394 – MT048399, MT049952 – MT049958, MT053086, MT062520, MT066404, MN998028 – MN998149.
The COI intra- and inter-specific percentage of identity for the six species were non-overlapping, averaging 99.04% (98.35 to 100.0) and 88.52% (86.51 to 91.60), respectively. Inter-specific pairwise genetic distances greater than 3% support the “barcoding gap” between the Anopheles species reported in this study.
COI sequences were analysed with the NCBI BLAST tool in order to confirm the morphological identification of the species. Anopheles albimanus, An. darlingi, An. pseudopunctipennis, and An. punctimacula were correctly identified by BLAST with identity percentages of 95.6% to 99.7%. Sequences of An. crucians, An. vestitipennis, and An. neivai could not be identified by BLAST due to the absence of sequences of these species in the databases, making them the first COI sequences reported for the three species in GenBank. All species were correctly identified by ITS2 with identity percentages of 99.63% to 100% with the exception of An. vestitipennis, whose sequences were not available in the databases. This is also the first report of ITS2 sequences for An. vestitipennis. In summary, the morphological identification coincided with the molecular identification of both markers for the species with sequences previously reported in the databases.
Nucleotide diversityand haplotypes
Intraspecific variation was calculated for both markers. COI showed a higher level of polymorphism than ITS2. According to COI, the species with the highest nucleotide diversity was Anopheles crucians (π = 0.05), followed by An. vestitipennis (π = 0.03) (Table 3). The species with the lowest diversity was Anopheles darlingi. Anopheles albimanus revealed a high number of haplotypes (n=55). Anopheles pseudopunctipennis showed the highest proportion of COI haplotypes with respect to the number of sequences analysed (11/11) and An.darlingi revealed the lowest haplotype index (3/16). ITS2 showed a low number of haplotypes (1 - 4) in all species (Table 3) (Fig.4).
Three analyses were performed to infer phylogenetic relationships between sequences. The first analysis included all the sequences of each marker for six Anopheles species. Both dendrograms (COI and ITS2) showed that the species clearly separated into clades (Fig.5).
The second analysis included sequences of An. albimanus classified according to geographic region. Phylogenetic relationships based on COI sequences showed only one separate cluster that included 11 out of 14 sequences of mosquitoes collected in Gracias a Dios. The other sequences were not clustered (Additional file 1: Figure S1). ITS2 sequences did not reveal any clustering according to geographical origin. This analysis was not performed for other Anopheles species due to the low intraspecific variation.
The third phylogenetic analysis included the COI sequences of five species obtained in this study (An. albimanus, An. darlingi, An. pseudopunctipennis, An. punctimacula, and An. neivai) together with analogous sequences available in GenBank in order to understand the relationships between individuals from Honduras with mosquitoes from other countries in the Neotropical region. The same analysis was performed separately with the ITS2 sequences of five species from Honduras (An. albimanus, An. darlingi, An. pseudopunctipennis, An. punctimacula, and An. neivai) and sequences from specimens of other countries.
The phylogenetic tree of An. albimanus included 12 COI sequences of mosquitoes from Colombia and 103 sequences of mosquitoes from Honduras, however the sequences of Colombia clustered together with the majority of sequences from Honduras. Eleven sequences of mosquitoes captured in Gracias a Dios formed a well-supported clade (Fig.6a). For An. darlingi 16 sequences from Honduras, 6 sequences from Colombia, 5 sequences from Brazil, and 4 sequences from Peru were analysed. According to this analysis the population was divided into two clusters, one including all the sequences of Honduras, and another with the sequences of South America (Fig.6b).
In addition, 12 sequences of An. pseudopunctipennis from Honduras and nine sequences from Colombia were analysed. For the analysis of An. punctimacula, seven sequences from Brazil, 14 sequences from Colombia and one sequence from Honduras were included. No clusters were detected for both species (Fig.6c, 6d). The analysis for An. neivai included three sequences from French Guiana, six sequences from Colombia, and five sequences from Honduras. The specimens of the three countries showed a defined separation according to geographical origin (Fig.6e).
The phylogenetic analysis of the ITS2 sequences included a total of eight countries of the Americas, including Honduras, Colombia, Brazil, French Guiana, Panama, Nicaragua, Ecuador and Belize. None of the trees could demonstrate separation of populations based on geographic origin (Additional file 2: Figure S2).