Natural malaria infection in anophelines vectors and their incrimination in local malaria transmission in Darién, Panama

Background More than 85% of the malaria cases in Panama occur in poor, rural and indigenous regions like Darien Province. Vector diversity, infection rate and spatial distribution are important entomological parameters of malaria transmission dynamics. Their understanding is crucial for the development of effective disease control strategies. The objective of this study was to determine the composition of Anopheles species, their natural infection rate and their geographic distribution to better understand the malaria transmission dynamics in Darién, Panama. Methods Anophelines mosquitoes were captured during the rainy and dry season of 2016. We selected five communities where adult anophelines were collected using CDC light-traps, and through protective human-baited traps. Detection of natural infection and Plasmodium genotype were detected via nested PCR through the amplification of ssrRNA and the circumsporozoite protein gene (csp), respectively. Results A total of 1,063 mosquitoes were collected mosquitoes were collected for the detection of natural infection with Plasmodium spp. Nine Anophelines species were identified, with the predominant species being: An. (Nys.) darlingi (45.0%) and An. (Nys.) albimanus (42.6%). Natural infection in An. (Nys.) albimanus with P. vivax was detected in one mosquito pool from the community Pueblo Tortuga (0.6%), three from Marraganti (1.7%), two from Bajo Chiquito (1.1%) and three pools from Alto Playona 3 (1.7%). For An. (Nys.) darlingi mosquitoes, we detected seven positive pools from the community Bajo Chiquito (4.0%), two pools from Marraganti (1.1%) and two pools from Alto Playona (1.1%). The P. vivax allelic variant VK210 was detected in infected mosquitoes. Conclusion The results from this study provide new information on the transmission dynamics associated with anophelines vectors in the Darién region. This is the first report of natural P. vivax infection in An. (Nys.) darlingi and its incrimination as a potential malaria vector in this region of Panama. Additional studies are necessary to expand our knowledge and determine crucial parameters in malaria transmission in Darién, which in turn will aid the National Malaria Program in attaining an adequate malaria control strategy towards malaria elimination.

Introduction conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned; the USDA is an equal opportunity employer. This does not alter our adherence to PLOS ONE policies on sharing data and materials.
of Latin America and is known to readily colonize habitats with diverse ecological characteristics. Furthermore, depending on the environment, this mosquito species displays a range of behaviors: anthropophily, opportunism, endo-exophagy, and endo-exophily. It also frequently colonizes anthropogenic sites and is susceptible to P. vivax and P. falciparum [9,10].
The determination of natural Plasmodium infection in anophelines mosquitoes is an important step to discern the main malaria vector species that exists in a region [11]. In Panama, experimental infections with human blood infected with circulating gametocytes, have found that seven Anopheles species are susceptible to infection with either P. vivax or P. falciparum. Nevertheless, only four of these species have been found to be naturally infected in the wild with Plasmodium spp. oocysts and/or sporozoites [12][13][14][15][16]. Currently there are 26 Anopheles species described in Panama [17][18][19]. The species An. (Ano.) malefactor, was also included by Wilkerson and Strickman [19] following a systematic revision differentiating this species from its synonym An. (Ano.) punctimacula s.l. [18]. Additionally, a recent report described the presence of An. (Nys.) darlingi in two communities in Darién province located at the eastern end of the country bordering with Colombia [20].
Social, environmental, economic, demographic and climatic changes can significantly influence the distribution of anophelines in endemic areas [21][22][23]. Although some species have been incriminated as local or regional vectors in neighboring countries [24], their importance as malaria vectors in Panama has yet to be determined. Additional entomological studies evaluating the vectorial capacity of other anophelines species are needed to determine their epidemiological importance in each of the Panamanian endemic regions. Equally important is the frequent monitoring of entomological parameters such as abundance, composition and natural infection with Plasmodium. Information gathered in these areas will contribute to the improvement of regional and local vector control strategies that can lead to malaria elimination in Panama. In this regard the objectives of this entomological study were to determine the species composition, spatial distribution, detection of natural Plasmodium infection and genotype, and their association with dominant mosquito species in Darién. This study provides new information on malaria transmission dynamics and has great public health significance allowing a more adequate selection of vector control interventions in this endemic region of Panama.

Description of the study area
The collection of adult Anopheles mosquitoes was conducted in the indigenous comarcas Emberá-Wounaan and Wargandi, located in Darién. The term comarca refers to an administrative region within Panama and it is assigned to a given indigenous population. The eastern side of Panama, where Darién is located, has seen 61.5% of national malaria cases in the last years [5]. The climate of this region is tropical, with high humidity like the rest of Panama, and it is influenced by the intertropical convergence zone [25]. Darién has two well defined seasons with moderate high temperature and humidity: the dry season (January-April) and the rainy season (May-December). The average annual precipitation ranges from 1,700 to 2,000 mm, with May and November being the rainiest months. The temperature does not present significant variations, ranging from 25.6 to 27.1˚C. This region registers a relative humidity of 84% and it ranges from 75% (March, dry season) to 93% (November, rainy season) [26]. According to the system of biosphere classifications by Holdridge, Darién belongs to a tropical rainforest [27].

Study design and site selection
Site selection was conducted in conjunction with technical personnel from the National Malaria Program (NMP) of the Ministry of Health (known as MINSA in Spanish). The selected sites were indigenous communities that presented active malaria transmission: Marraganti, Bajo Chiquito, Pueblo Tortuga and Alto Playona, located in the comarca Embera-Wounaan, and Morti in the indigenous comarca Guna of Wargandi (Fig 1 and Table 1). All the communities were characterized by being located near a river that are used as the main communication means with other nearby communities, surrounded by an ecology of lowlands, creeping vegetation and secondary forest. Most households in this area presented wooden walls, tin roofs and a few were protected from insect entrance. Selection of communities was based on the following criteria: (1) its entomological importance with regards to vectors, (2) its epidemiological importance according to the transmission rate and (3) accessibility and safety to visit the community, as the presence of irregular armed groups and drug traffickers is frequent in certain areas from Darién.
Comarca: The term comarca refers to an administrative region within Panama and it is assigned to a given indigenous population. We designed a cross-sectional study to identify the species of Anopheles vectors present in the selected communities and to detect natural Plasmodium infection and genotype in these vectors. Mosquitoes were collected via two methods

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in each of the selected communities where malaria cases were registered: protective humanbaited capture (PHBC), which consists of exposing only one limb and capturing the mosquito upon landing before the bite begins [24]; and CDC light traps (LTs). Each locality represented a sampling site, and for each selected location mosquito samplings were conducted for nine months; twice during the dry season (March and April) and twice during the rainy season (May to November) of 2016. The NMP provided the following epidemiological data: geographical location of the malaria cases, clinical characteristics of the disease, date of the first cases, affected and exposed population, socioeconomic information, basic sanitation services, health service characteristics, demographic information and prevention and control interventions carried out in the region. This information was important to select the study sites, based on the communities that registered higher malaria transmissions in recent years.

Collection of adult mosquitoes
To collect adult mosquitoes, four houses that presented malaria or were near to a mosquito breeding sites were selected. Mosquito collection in each of the sites were conducted at different dates but within the same dry and rainy season. Mosquito collections with PHBC were carried out in the five selected communities during four consecutive nights in the intra and peridomicile areas simultaneously once a month, for nine months including the dry (March and April) and rainy seasons (May to November) of 2016. To determine the human biting rate per night (HBRs), the PHBC consisted in simultaneously having one-person intradomicile and another in the peridomicile per night in the four households within each locality. Mosquito collections were conducted during the 18:00 and 22:00 hours, given that previous reports have indicated that this is the period of greater mosquito biting activity [28,29]. Collections were made during the first 50 min of every hour. During this period, HBRs were calculated, such as the biting/human/night (BHN) rate during the period from 18:00 p.m. to 22:00 p.m. [30]. The biting rate was obtained from the total number of mosquitoes captured divided by the number of collectors and the total hours of captures [31]. Mosquito collections were conducted by experienced technical personnel from the NMP and the Medical Entomology Department from the Gorgas Memorial Institute for Health Studies, trained in the capture of malaria mosquito vectors. To reduce bias during collections, personnel were rotated at specific periods every hour. The technical biosafety recommendations from WHO [32] were followed to minimize the risk of malaria infection in the field team.
Mosquito collections using LTs were also conducted during four consecutive nights simultaneously to the PHBC collections. The (LTs) were located at the height of 1.5 m above the

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Natural malaria infection in anophelines vectors in Darié n, Panama ground during the hours of 18:00 and 6:00 [33,34]. Neither octanol nor carbon dioxide (CO 2 ) were used as mosquito attractant. Traps were distributed near houses (different from the homes where PHBC collections were made) and mosquito breeding sites (extradomicile) to a distance about 25 m. Collections with PHBC were used to measure the intensity of the anophelines bite, while the LTs were used to improve the opportunity to collect a greater variety of species of [35,36]. Additional parameters such as temperature and relative humidity were recorded during the periods of mosquito collection using a data logger (EXTECH instru-ments1 RHT10, No. 11092261). Rainfall data were obtained from a meteorological station in the locality of Yaviza, near the collection sites.

Taxonomic determination
All biological material collected was placed in previously labeled containers and preserved in liquid nitrogen (Thermo Scientific™). Each container had information such as locality, type of collection (intra, peridomicile and LTs) and date. Samples were sent to the Department of Medical Entomology at the Gorgas Institute for its identification. Mosquito identification to the species level was conducted using taxonomical keys for adult mosquitoes [19,37] and the Reference Collection from the Gorgas Memorial Institute for Health Studies.
To confirm the identity of Anopheles species in Plasmodium positive pools, the mitochondrial COI gene was analyzed using primers UEA3 (5-0 TATAGCATTCCCACGAATAAATAA-3 0 ) and UEA10 (5-0 TCCAATGCACTAATCTGCCATATTA-3 0 ) and PCR conditions as previously described [20,38,39]. The PCR-amplified products from these samples were excised from agarose gel and purified using the Qiaquick gel extraction Kit (Qiagen, CA, USA) following the manufacturer's instructions. DNA sequencing of both strands was carried out using the same primers with an ABI Prism 3500 XL130 sequencer (Applied Byosistems, Foster City, CA, USA). Resulting sequences were edited using Sequencher software. The DNA sequences were compared with Anopheles COI sequences available in the GenBank by performing a BLAST search from the National Center for Biotechnology Information Database (http://www.ncbi.nlm.nih. gov/BLAST/).

Detection of Plasmodium spp. genotype and natural infection in Anopheles spp. mosquitoes
Adult female mosquitoes were grouped per species in anophelines pools. Each pool was made of two to five female mosquitoes of the same species, according to the date and site of collection, and type of capture (PHBC or LTs). It has been described that it is possible to detect infectivity in mixtures of mosquitoes; thus, complete mosquitoes of the same species were processed [40][41][42][43][44]. Each pool was placed in an Eppendorf tube, where they were first macerated in a solution of 180 μl of 1X PBS. Next, DNA extraction was performed following the protocol from Qiagen DNeasy Blood & Tissue (Qiagen, USA). The isolated DNA was stored at -20˚C until further use.
We used a nested PCR that amplifies the small subunit ribosomal RNA (ssrRNA) genes to confirm the presence of natural infection of Plasmodium spp. in pools of Anopheles spp. mosquitoes, following a slightly modified methodology proposed by Snounou et al. [45]. Two genus-specific primers rPLU5 (5 0 -CCT GTT GTT GCC TTA AAC TTC-3 0 ) and rPLU6 (5 0 -TTA AAA TTG TTG CAG TTA AAA CG-3 0 ), are used for the first cycle of amplification. An aliquot of the product obtained is used for a second amplification cycle, in which each parasite species is detected separately using species specific primers. In the second PCR reaction only specific primers for P. falciparum (205 pb): rFAL1 (5 0 -TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT-3 0 ), rFAL2 (5-0 ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3 0 ) and P. vivax (120 pb) rV1V1 (5 0 -CGC TTC TAG CTT AAT CCA CAT AAC TGA TAC-3 0 ), rV1V2 (5 0 -ACT TCC AAG CCG AAG CAA AGA AAG TCC TTA-3 0 ) were included, because the other Plasmodium human species (P. ovale and P. malarie) have not been reported in Panama in more the four decades. All mixture and amplification conditions were as described previously [45].
The circumsporozoite protein gene (csp) was used as marker to genotype Plasmodium vivax in positive pools of natural infected An. (Nys.) darlingi and An. (Nys.) albimanus. A nested PCR amplification method was used to amplify the csp gene (700 to 800bp) following previously reported protocols with some minor modifications [46]. The primers were as follows: VCS-OF (ATGTAGATCTGTCCAAGGCCATAAA), VCS-OR (TAATTGAATAATGCTAG GACTAACAATATG) as primary primers and VCS-NF (GCAGAACCAAAAAATCCACGTGA AAATAAG), VCS-NR (CCAACGGTAGCTCTAACTTTATCTAGGTAT) as a nested primer. All mixture and amplification conditions were as described previously [46]. The nested PCR products were electrophoresed on a 1.5% agarose gel and then directly sequenced in both directions using an ABI Prism 3500 XL sequencer (Applied Byosistems, Foster City, CA, USA). The sequences were edited and aligned with Sequencher 4.1.4, and Phylogenetic trees were made using the Molecular Evolutionary Genetics Analysis (MEGA) 7.0.

Data analysis
The abundance of each species of mosquito per community was expressed as a percentage, obtained from the number of each species of mosquito captured in each community among the total of the species captured in all communities. The percentage of Plasmodium infected Anopheles was assessed by the number of positive Anopheles specimens of a given species (np) out of the total analyzed by community (nt) by 100 [IR = (np/nt) × 100]. The natural infection rate (IR) was also estimated by the method of pooled prevalence for variable pool size and perfect tests using the pooled prevalence calculator (https://epitools.ausvet.com.au/ppvariablepoolsize). In addition, the confidence interval (CI 95%) was calculated to indicate the reliability of the estimated value. The BHN activity was registered directly from PHBC. Hourly data from all collections were grouped and the total number of bites per hour was obtained for each species by intradomiciliary and peridomiciliary behavior. Mosquito species mean density for Anopheles species with more than five specimens per locality, was calculated as the geometric mean of the number of mosquitoes captured per person each night.

Ethical considerations
The methodology used to collect the mosquitoes was approved by the Technical and Institutional Ethics Committee of the NMP of the Ministry of Health (MINSA) (Note No. 25/DCV/ DGS/MINSA/2016). Samples and human data were not used.

Biting activity
The collections yielded 1,063 female adult mosquitoes, from which we were able to identify nine species ( Table 2) (Figs 4 and 5). Observations made in the nine months of this study, allowed to establish that An. (Nys.) darlingi and An. (Nys.) albimanus were present throughout the year with endophagic and exophagic behavior, given that they were captured in the intradomicile and in the peridomicilio (Fig 5).
Collections with PHBC resulted more effective with respect to the number of adult mosquitoes collected (n = 1,024, 96.3%), in comparison to the number of mosquitoes collected with LTs (n = 39, 3.7%). The majority of mosquitoes were collected during May, when the rainy season begins, and October (n = 173, 16.3%) and November (n = 208, 19.6%); which traditionally are the rainiest months of the year (Table 4). Collections during the dry season months registered a lower number of captured adult mosquitoes, March (n = 47, 4.4%) and April (n = 18, 1.7%). The studied communities that registered the major percentage of captured mosquitoes were Bajo Chiquito (n = 589, 55.4%) and Marraganti (n = 345, 32.5%), followed by Pueblo Tortuga (n = 64, 6.0%) and Alto Playona (n = 52, 4.9%). In turn, the localities in which a larger number of mosquito species was collected were Bajo Chiquito with seven species, followed by Marraganti and Alto Playona with five species, respectively.

Detection and genotype of natural Plasmodium spp. infection in Anopheles spp.
A total of 574 the 1,063 adult females collected were selected and processed according to the quantity, mosquito species, collecting date, capture type (PHBC or LTs) and study site for the detection and genotype of Plasmodium natural infections by PCR. The samples were grouped in 148 anophelines pools per species of varying size (between 1 and 5 individuals). The contribution of all the species selected for this test and number of the pools analyzed were as follow:    From the positive pool samples, it was possible to genotype P. vivax in seven of them, four from An. (Nys.) darlingi and three from An. (Nys.) albimanus by sequencing the csp gene marker. Amplified products were in the range between 700 to 800 bp and their sequence analysis determined that they were homologous to the VK210 variant, grouping together with other isolates from Central America (Fig 6). Sequences from this study are available at the GenBank database under the accession numbers: MN66917, MN66918, MN66919, MN66920, MN66921, MN66922, MN66923 (Fig 7).

Biting activity
The diversity of Anopheles mosquitoes in Panama is favored by a vast variety of habitats and environmental conditions that support the development, dispersion and persistence of these mosquito populations. The abundance and human feeding preference are among the main characteristics that define a mosquito species as an effective mosquito vector [47]. Nevertheless, the composition and frequency of mosquito species vary between sampling sites and seasons, making it even more important the need for a constant monitoring to better understand these parameters [47,48]. Our data shows that An. (Nys.) albimanus was the dominant species

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Natural malaria infection in anophelines vectors in Darié n, Panama in four out of the five sampled sites. This species is described as predominantly exophagic, with preference for animals and with hematophagy occurring during the entire night [48]. In turn, An. (Nys.) darlingi was the species that registered the major number of captured mosquitoes and was dominant in one of the collection sites (Bajo Chiquito). Anopheles (Nys.) darlingi is considered one of the most effective primary vectors due to its high anthropophilic behavior, high abundance in certain areas, susceptibility to infection by several Plasmodium species and plastic behavior [49]. This species can also adapt to diverse habitats, including habitats developed because of human activities [50,51]. Our study showed that both mosquito species (An. (Nys.) darlingi and An. (Nys.) albimanus) had the greatest density and biting activity between 18:30 and 19:30 hours, diminishing towards midnight as described in previous studies [22,28,51]. A study in Colombia also observed that the greatest intradomicile and peridomicile biting activity of An. (Nys.) darlingi was between 18:00 and 19:00 hours [24,52], similarly to our data and those described in French Guyana and Brazil [46]. Furthermore, studies conducted in Barú municipality, a locality in Panama bordering Costa Rica, demonstrated that An. (Nys.) albimanus had greater abundance and biting frequency than An. (Ano.) punctimacula s.l. between 18:30 and 19:30 hours [29]. Overall, our entomological data from the intradomicile and peridomicile collections suggest that there is higher transmission risk between 18:00 and 22:00 hours, when the anophelines population is higher. Therefore, considering the abundance and high Plasmodium natural infection of these two species in most collecting sites, our data is highly suggestive that An. (Nys.) darlingi and An. (Nys.) albimanus are acting as the main malaria vectors in this area. Nevertheless, other malaria vector species found in this study and also known to be present in other neotropical countries, such as An. (Ano.) pseudopunctipennis s.l., An (Nys.) oswaldoi s.l., An. (Ano.) punctimacula s.l. and An. (Ano.) triannulatus s.l. [28,53], could also be implicated in the local transmission.
The intra and peridomicile PHBC rates for both An. (Nys.) darlingi and An. (Nys.) albimanus were similar, without significant differences in the biting activity percentages, confirming the exophagic and endophagic behavior of these species [54]. While the PHBC for both species was respectively 0.20 to 0.50 bites per night, other studies have found more variable biting

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rates for An. (Nys.) darlingi [55,56]. The low density recorded in these two species may be due to variations in temperature, rainfall, human activity, proximity and availability of breeding sites and other environmental factors [57,58]. It could also be due to the collection period during the study, thus it was not possible to establish if these populations present a greater activity at other hours of the night, as it has been described in other populations of the region [24]. For instance, rates of 0.1-15.1 BHN [22] and 2.2-55.5 BHN [54] were observed in Colombia and 53.8 to 837.7 BHN in the Brazilian amazon [59]. Also, studies conducted with An. (Nys.) albimanus in eight communities during a malaria outbreak in Panama displayed a PHBC of 1.9 to 30.9 BHN [29]. A previous study conducted in 31 endemic sites in Panama found that the BHN oscillated between 2.4 and 10.2 BHN [8]. Further studies conducted in the endemic regions of the Colombian Pacific found that An. (Nys.) albimanus had the highest biting activity between 17:00 and 19:00 hours, reaching a PHBC of 38.4 bites/night [60]. In general, our results indicated a lower BHN rate compared to reports conducted in other Neotropical countries. It is thus necessary to conduct prolonged studies to estimate the densities and understand the biting behavior of this mosquito species in the different Panamanian regions.
Our monthly mosquito collections indicated that the greatest anophelines density was obtained in the months of May, October and November (rainy season), with predominance of An. (Nys.) darlingi and An. (Nys.) albimanus. The lowest densities for both species were observed in March (dry season). Several studies have described patterns of seasonal fluctuation in the abundance and frequency of anophelines vectors, showing greater densities during the rainy months and lower densities during the dry months [61,62]. Few studies have demonstrated no correlation between the levels of precipitation and the density of anophelines mosquitoes [41,58]. On the other hand, it has also been reported that an increase in density can be observed on the transition periods between the dry and rainy seasons [63,64]. However, it has been proposed that the changes in vector density not only depends on the aforementioned factors but rather it responds to an interaction between the availability of breeding sites, the levels of water sources and other environmental variables [48].
In this study, captures with PHBC were more effective compared to LTs. In general, catches with LTs outdoors are less efficient compared to PHBC. They are used as a supplement to captures with PHBC, with the purpose of having a greater opportunity and probability of capturing a larger number of anophelines species. For instance, in a study carried out in Córdoba, Colombia, only six species of Anopheles were captured intradomicile and in the peridomicile with LTs. This number of captured species was small considering that there are 20 species recorded in that region out of a total of 47 species registered in Colombia. In addition, trap catches require that the traps do not harm captured mosquitoes [54,65]. Therefore, captures with PHBC have become the most widely used in malaria studies [43,66,67].

Detection of natural Plasmodium infection in Anopheles spp.
The communities that yielded P. vivax positive mosquito pools also registered active malaria transmission during our study, with the highest number of cases observed during the transition period between the rainy season and the dry season [5]. The dynamics of malaria epidemics are strongly influenced by climate. In particular, at the geographical limits of its distribution, malaria transmission is driven by environmental factors changes as temperature, rain and humidity [68].
Interestingly, our study allowed us to detect for the first time in the country the natural P. vivax infection of An. (Nys.) darlingi, and to suggest its potential incrimination as a malaria vector in this region of Panama. At the same time, our results showed that two other species historically considered to be important malaria vectors, An. (Ano.) punctimacula s.l. and An.
(Ano.) pseudopunctipennis s.l., were not infected Plasmodium. Thus, their epidemiological relevance as vectors could not be confirmed for this region. This could have been due to the low number of individuals of both species captured during this study.
The genus Anopheles is present around the world with about 465 species, of which 41 species are important malaria vectors [69]. Although different mosquito species can be involved in malaria transmission in different regions [58,70,71], little is known about the contribution of each species in malaria prevalence in heterogenic environments. This is especially true given that each species has unique developmental, ecological and behavioral characteristics. A study conducted in Brazil indicated that this scenario becomes more complex with the presence of three different malaria parasite species [72,73] in the same geographical region. Anopheles (Nys.) albimanus and An. (Ano.) punctimacula s.l. are considered as the primary and secondary malaria vectors, respectively in Panama. Anopheles (Nys.) albimanus is the most prevalent in the endemic regions [8]. A recent study reported the detection, via PCR, of natural P. vivax infections in An. (Nys.) albimanus mosquitoes collected from the communities of Achutupo and Playon Chico, both in the indigenous comarca of Guna Yala [5]. In addition, other studies have detected P. vivax natural infections in An. (Nys.) albimanus (via ELISA tests) and in An. (Ano.) punctimacula s. l. (through PCR assays) collected in Bocas del Toro province [74]. In another study An. (Nys.) albimanus mosquitoes was found naturally infected with P. vivax in samples collected in the community of Ipeti Guna, located in the Madungandi comarca [44].
The determination of natural Plasmodium infection of anophelines mosquitoes is an important component in the assessment of different mosquito species as malaria vectors [11].

Plasmodium spp. genotyping
Genetic studies of circulating malaria parasite populations in humans and vectors reveal critical information about the epidemiology and dynamics of disease transmission and offer tools to support control and elimination efforts [77]. In the present study, we only identified the P. vivax allelic variant VK210 naturally infecting An. (Nys.) albimanus and An. (Nys.) darlingi mosquitoes circulating in the studied areas. It is possible, however, the lack of detection of the VK247 variant might be due the low number of samples analyzed, as this variant has been described circulating in malaria endemic communities near the border with Colombia close to the ones studied [78].
There are studies related to the diversity in infectivity of allelic variants and the susceptibility of mosquito species to different allelic variants, which may explain the detection patterns at the species level, where An. (Nys.) albimanus seems to be susceptible to VK210 and VK247 infection [52]. However, other studies conducted in anophelines mosquitoes from Brazilian Amazon, observed the distribution of P. vivax VK247 changed over time in the main malaria vectors on the Brazilian Amazon. Anopheles (Nys.) darlingi was abundant in certain localities while An. (Nys.) albitarsis s.l. in anothers, which highlights the importance of entomological studies for the control of human malaria [79]. Investigations carried out in Mexico considered that An. (Nys.) albimanus is more susceptible to VK210 infection and An. (Ano.) pseudopunctipennis was more susceptible to VK47 [80].
Studies on genotyping parasite populations have the power to reveal key information about the epidemiology and dynamics of malaria transmission, with the potential to offer tools to support control and elimination efforts [81]. In future studies, is also critical to evaluate the epidemiologic role on malaria transmission of other anophelines species previously described in the Darien region such as An. (Nys.) triannulattus s.l., An. (Nys.) oswaldoi s.l., An. (Ano.) pseudopunctipennis s.l., An. (Ano.) punctimacula s.l., and An. (Ano.) malefactor. Additional molecular studies of malaria allelic variants circulating in Panama at the local and regional are necessary to expand our current knowledge, determine parameters that affect malaria transmission dynamics, and to develop novel malaria control strategies [82,83]. Previously in An. (Nys.) albimanus collected from 2006-2007 in Bocas del Toro, Panama, nine pools were detected naturally infected with P. vivax by an ELISA test (three pools with the VK210 variant and six with the VK247 variant) [74].
An important limitation of this study was that it was not possible to carry out salivary gland dissections for the detection of P. vivax sporozoites in An. (Nys.) albimanus and An. (Nys.) darlingi. Therefore, we were not able to corroborate and confirm the PCR results that suggested the incrimination of An. (Nys.) albimanus and An. (Nys.) darlingi in malaria transmission at the local level. However, it should be noted that An. (Nys.) Darlingi has been sufficiently proved for its great vector capacity through various studies, classifying it as the most efficient malaria-transmitting species in the Americas.
In conclusion, this study provides important new information on the transmission dynamics associated with mosquito vectors in the Darien region. The data show that the most abundant and distributed species were An. (Nys.) albimanus and An. (Nys.) darlingi, which were also found coexisting in the same geographical area. importance has yet to be determined. In addition, results of this research add important new entomological information that should be considered in transmission dynamic studies, and in surveillance/control strategies in the Darien region. Specifically, at the local level, these findings provide a new geographical range for mosquitoes, some of which could be acting as local vectors. Our findings highlight the need for additional studies to expand our knowledge on the behavior, spatial/temporal distribution, and malaria transmission dynamics by An. (