Background
According to the 2015 World Malaria Report [
1], 17 % of malaria cases reported in the Americas region were from Colombia, where malaria transmission exhibits an endemic/epidemic pattern that maintains unstable endemic transmission levels throughout the country [
2]. A decreasing trend in clinical malaria cases has been reported over the past 14 years in Colombia, falling from 144,432 in 2000 [
2] to 40,768 in 2014, with a more than 75 % decrease in the incidence of microscopically confirmed malaria during this period of time [
1]. Despite this, malaria remains one of the foremost public health concerns in the western region of Colombia where more than 85 % of malaria cases were reported from 2010 to 2015 [
3].
The last review of the geographical distribution of
Anopheles species in Colombia was published over 4 years ago and mentioned the presence of 40–47
Anopheles species including those belonging to species complexes [
4]. Many of the records included in this review took into account data collected more than 50 years ago by the Malaria Eradication Service (Servicio de Erradicacion de la Malaria, SEM).
Environmental, social, economic, and demographic changes have occurred since that time which may have substantially influenced the observed distribution of
Anopheles species in Colombia. In addition, it is necessary to differentiate the members of the
Anopheles species complexes, which can now be achieved using several molecular tools [
5]. A clear example is the recent analysis, based on the sequencing of the mitochondrial DNA marker, Cytochrome
c Oxidase I (COI); which showed the presence of two species of the
Anopheles (Nyssorhynchus) albitarsis complex in Colombia:
An. (Nys.) albitarsis F, in the departments of Meta, Norte de Santander and Vichada; and
An. (Nys.) albitarsis I, in departments of Antioquia and Norte de Santander [
6]. Based on sequences of molecular markers, COI, and internal transcribed spacer 2 (ITS 2), analysis showed that there are three species of the complex
oswaldoi-
konderi in Colombia:
Anopheles (Nys.) oswaldoi A in department of Amazonas;
An. (Nys.) oswaldoi B in departments of Antioquia, Caqueta, Meta, Norte de Santander and Putumayo; and
Anopheles sp. nr. konderi in department of Caqueta [
7]. However, there are still gaps in knowledge about the distribution of
Anopheles mosquitoes in Colombia, as well as the presence of
Anopheles species of other species complexes [
8].
Of
Anopheles species reported in Colombia, 12 have been implicated in malaria transmission. The main malaria vectors are
Anopheles (Nys.) darlingi,
Anopheles (Nys.) nuneztovari s.l., and
Anopheles (Nys.) albimanus [
9]. Other species are referred to as vectors of local importance in some areas, or are suspected of being associated with malaria transmission, such as:
Anopheles (An.) pseudopunctipennis s.l.;
Anopheles (An.) punctimacula s.l.;
Anopheles (An.) calderoni;
Anopheles (An.) neomaculipalpus;
Anopheles (Kerteszia) pholidotus;
Anopheles (Ker.) neivai s.l.;
Anopheles (Nys.) rangeli;
Anopheles (Nys.) benarrochi B; and
An. (
Nys.)
oswaldoi s.l. [
9‐
14].
Studies of malaria vector species in Colombia have usually been performed in just a few localities and usually with the aim to clarify taxonomic identifications or to understand the biology, ecology, and the role in malaria transmission of the species involved. This is the first study to associate spatial vector distributions with intensity of malaria transmission and parasite prevalence. The aim of this entomological study is to describe the relationship between Anopheles species distribution patterns and malaria incidence in the highest malaria transmission region of Colombia.
Discussion
During this study, using PCR–RFLP-ITS 2 and morphological characters, 13 formally named Anopheles species were confirmed in the endemic malaria region of Colombia, however, the NJ, ML, and BI analysis using COI barcode sequences genes revealed the presence of four new mitochondrial lineages. One new lineage of An. albimanus, called An. albimanus B in the Boldsystem databases, was found in the South Pacific coast. One new lineage of An. pseudopunctipennis s.l., was found in the Northwest and the South Pacific coast. One new lineage for An. neivai (An. neivai
nr. An. neivai 4) was found in South Pacific coast and one new lineage for An. apicimacula that include sequences from Northwest and South Pacific coast regions.
Regarding the association of
Anopheles species with malaria distribution, the department of Cordoba, located in the Northwest region of Colombia, contributes with 18 % of malaria cases reported annually in the country [
2]. This region was the study area with the highest number of
Anopheles species, ten out of the 13 species identified. An important aspect of the current study is that in this area three primary malaria vectors were registered:
An. nuneztovari s.l. (=
An. nuneztovari C)
, An. darlingi and
An. albimanus (northwest lineage). This may partly explain the local high malaria burden. Both
An. nuneztovari s.l. and
An. darlingi are anthropophilic species with an important biting activity in evening early hours [
29‐
38]. During this period, people are still active and unprotected by LLINs, the main measure used for the control of malaria vectors in this region. Interestingly, the results show significant differences in relation to the predominant species in adult stage and immature stage. Predominant species in adults was
An. nuneztovari C (85 % of collections), whereas
An. nuneztovari C and
An. triannulatus s.l. were collected in similar proportions in immature stages. Differences in
Anopheles species composition in adult and immature stages may be explained by the presence of cows that provides blood sources for the zoophilic species like
An. triannulatus s.l. [
39‐
41].
With respect to another
Anopheles species registered in the Northwest region, in this study, the presence of
An. albitaris I (n = 5) was confirmed. This species had been reported in the same region by Gutierrez et al. [
42] as
An. albitarsis near janconae; however, afterwards it was confirmed by Ruiz et al. [
6] as
An. albitaris I. According to the results of this study, the distribution of
An. albitaris I was extended to others localities in the Northwest region. The malaria vector status of
An. albitaris I is unknown in South America [
6]. Additionally, morphological characters and COI sequence analysis confirmed the presence of other
Anopheles species in low densities:
An. pseudopunctipennis s.l. (northwest lineage) (n = 19),
An. punctimacula s.l. (n = 16),
An. apicimacula (northwest lineage) (n = 1),
An. argyritarsis (n = 1), and,
An. neomaculipalpus (n = 1).
In relation to the role that
Anopheles species play in malaria transmission in the Northwest region, three primary vectors were registered:
An. nuneztovari C
, An. darlingi and
An. albimanus. In this study, two specimens of
An. nuneztovari C were found infected with
P. falciparum; however, previous studies conducted in the same region reported
An. nuneztovari s.l. as infected with
P. vivax VK247. The taxonomic determination of species within this species complex was not clarified in these studies [
34,
43]. It is likely that the species corresponded to
An. nuneztovari C from the results found in the present study. Other species reported as positive for
Plasmodium infection were
An.
darlingi infected with
P. vivax VK247 [
34,
43,
44], and
An. triannulatus s.l. infected with
P. vivax VK247 [
43]; however, the role of the latter species in the Northwest region is not yet clear. Other
Anopheles species registered in Northwest region,
An. pseudopunctipennis s.l.,
An. punctimacula s.l., and,
An. neomaculipalpus are considered malaria vector in America [
45‐
47], however, in this region their importance in the sustaining of malaria transmission is unknown.
During the past ten years, in Buenaventura (department of Valle del Cauca), API fluctuated between nine and 167 cases/1000 inhabitants and in ten municipalities of department of Nariño, with the highest malaria transmission, this index ranged between 22 and 100 cases/1000 inhabitants, with a prevalence of
P. vivax inland, along the Cali-Buenaventura road, and
P. falciparum in the Pacific plain in the South Pacific region. In this region, seven
Anopheles species were identified. The most abundant
Anopheles species in the area along the Cali-Buenaventura road were
An. nuneztovari C and
An. pseudopunctipennis s.l. (Pacific coast lineage).
Anopheles neivai s.l. was identified with evidence of two lineages (
An. neivai and
An. neivai
nr.
An. neivai 4). In the Pacific plain area,
An. albimanus B was the most important species along the coast; while inland
An. calderoni,
An. neivai, and
An. apicimacula (Northwest-Pacific coast and only Pacific coast lineages) were confirmed. Different composition in adults and larval stages of
Anopheles species were observed in the South Pacific coast. As evidenced in the maps (Fig.
3), in coastal localities
An. albimanus B and
An. neivai were registered in adult state, whereas inland
An. nuneztovari C,
An. pseudopunctipennis and
An. calderoni were registered.
Anopheles neivai were collected in adult but not in immature states in the coastal towns. This can be explained by the difficulty of sampling in the water that is accumulated in the axils of bromeliads leaves,
An. neivai s.l. larval habitat [
48‐
51]. Sampling of bromeliads could only be carried out in three localities where the species was present in the adult stage. In general, these Colombian Pacific coastal localities are surrounded by mangroves, which host several species of bromeliads.
In the study sites of the South Pacific coast, two specimens were found infected with
Plasmodium species:
An. albimanus B from the Pacific plain region was infected with
P. falciparum; and
An. nuneztovari C from the Cali-Buenaventura road was infected with
P. vivax VK247. In various studies carried out in recent years in the same region,
An. albimanus was reported infected with
P. falciparum [
52] and with
P. vivax VK210 [
52,
53]. Additionally, in this region another two
Anopheles species have been reported infected with
Plasmodium:
Anopheles neivai infected with
P. vivax VK210,
P. vivax VK 247 and
P. falciparum [
52,
53], and
An. calderoni infected with
P. vivax VK210 and
P. falciparum [
14,
44]. The previous results provides evidence to confirm the status as malaria vectors of:
An. nuneztovari C,
An. albimanus B, An
. neivai, and
An. calderoni in the Southwest of Colombia, due to their presence and wide distribution in a region with active malaria transmission, anthropophilic behaviour, and detection of natural infection with
Plasmodium species [
14,
44,
53‐
55].
Although this study included data about Anopheles species composition at locality level, the relationship between the presence of Anopheles species and the specific locality burden of malaria was difficult to determine, due mainly to difficulties in the reporting system. Although, in Colombia it is mandatory to report malaria cases by locality of origin, it is common to find the locality origin of the infection mixed up with the locality where the patient lives, or with the locality in which the diagnosis was made. Also, patients could refuse to give information about the locality in which they probably got the infection. These situations make the analysis by localities difficult. For this reason, the relation between Anopheles species composition and malaria prevalence is presented at municipality level.
Anopheles species with two lineages identified in this study were:
Anopheles albimanus,
An. pseudopunctipennis s.l.,
An. neivai s.l., and
An. apicimacula. Some considerations about taxonomic status are put forward here:
Anopheles albimanus shows two lineages: Northwest,
An. albimanus and Pacific coast,
An. albimanus B, each lineage with different geographical distribution. The differences between
An. albimanus populations of the Caribbean regions (North of Colombia) and the Pacific coast had previously been reported by Narang et al. [
56] using allozyme variability and chromosomal analysis to characterize
An. albimanus populations collected in 11 localities from Colombia (north and Pacific coast), and were confirmed by Gutierrez et al. [
57], who defined two distinctive groups corresponding to haplotypes from the Caribbean and Pacific coast regions by analysis of COI and microsatellite. However, the reported data of hybridization and backcrosses that included North and South Pacific coast populations of
An. albimanus, showed that hybrid females and males were fertile and had normal ovaries and testes, indicating the absence of cryptic speciation for this species in Colombia [
56]. It is necessary to carry out studies in vector competence to identify differences in infection susceptibility by strains of
Plasmodium, because both lineages are present in regions with different malaria prevalence.
Similarly, evidence to support two lineages of
An. pseudopunctipennis s.l. with different geographical distribution was found: one linage conformed by specimens collected in the Northwest region and the other by specimens collected from the Southwest in the Pacific region. Several studies have attempted to evaluate the taxonomic status of
An. pseudopunctipennis s.l. Estrada-Franco et al. [
58] used isoenzyme electrophoresis and rDNA restriction fragment length polymorphisms (RFLPs) and Coetzee et al. [
59] used cross-mating experiments, suggested that this species is actually a complex of three species:
An. pseudopunctipennis A, represented by specimens from central Mexico;
An. pseudopunctipennis B, represented by specimens from the Andes of Peru and Bolivia [
58]; and
An. pseudopunctipennis C, represented by specimens collected on the island of Grenada in the Lesser Antilles [
59]. However, based on the evidence of isozyme analyses of 42 populations of this species collected in ten countries in North, Central, and South America, Manguin et al. [
60] suggested that
An. pseudopunctipennis s.l. is a single species with three geographical populations represented in: (1). North America (United States and Mexico) and Guatemala; (2). Belize and South America (Colombia, Ecuador, Peru, Chile, and Argentina); and (3). Grenada Island. Additionally, Manguin et al. [
60] suggested that the first and second geographic population converge in Southern Mexico and Northern Central America on the border between Belize and Guatemala. However, it should be noted that in the study by Manguin et al. [
60], the samples included for analysis from Colombia came from the South of the country with no representation of specimens of this species from the North, which would probably be more like those found in Central America: a hypothesis that should be tested.
Anopheles neivai s.l. was another species that showed two lineages; however, in contrast to An. albimanus and An. pseudopunctipennis s.l., these lineages were found in the same region. The limitation of this finding is that all specimens identified by morphological characters as An. neivai s.l., in localities where the second lineage (An. neivai nr. An. neivai 4) is present, were not sequenced and the lineage An. neivai were confirmed in the same area, leaving untested the sympatry of the two lineages in the South Pacific coast region.
Regarding
An. apicimacula, the findings partially support the results reported by Gomez et al. [
61] who inferred the existence of two lineages, Atlantic coast and Pacific coast. The results of the present study show two lineages with a BVS >95, the first included the COI sequence of specimens collected exclusively in Pacific coast, and the second included the sequence generated for specimens collected in the Northwest and the South Pacific coast. In the same sequence analysis, a third lineage was observed with a BVS = 100, which only included sequences generated by Gomez et al. [
61] using specimens collected in the Northwest region, which were grouped in the original study as
An. apicimacula Caribbean lineage (Fig.
1). These results suggest that is necessary perform an analysis that includes more COI sequences of Northwest region to clarify because the second lineage include the Northwest and South Pacific coast sequences and confirm the different populations of
An. apicimacula present in Colombia.
This study provides evidence about the richness of Anopheles species found in malaria transmission regions in Colombia and establishes that several species incriminated as malaria vectors are sympatric. This condition is an important aspect to consider designing control strategies, given the fact that species can exhibit different biting behaviours that could diminish the effectiveness of traditional control measures when applied to large scale.