Mem Inst Oswaldo Cruz, Rio de Janeiro, 109(3) May 2014
Anthropophilic Anopheles species composition and malaria in Tierradentro, Córdoba, Colombia
1Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University, Mainz, Germany
2Departamento de Salud Pública, Universidad Nacional de Colombia, Bogotá, Colombia
Malaria is still a primary health problem in Colombia. The locality of Tierradentro is situated in the municipality of Montelíbano, Córdoba, in the northwest of Colombia, and has one of the highest annual parasite index of malaria nationwide. However, the vectors involved in malaria transmission in this locality have not yet been identified. In this study, the local anthropophilic Anopheles composition and natural infectivity with Plasmodium were investigated. In August 2009, 927 female Anopheles mosquitoes were collected in eight localities using the human landing catch method and identified based on their morphology. Cryptic species were determined by restriction fragment length polymorphism-internal transcribed spacer (ITS)2 molecular analysis. Eight species Anopheles nuneztovari s.l. (92.8%), Anopheles darlingi (5.1%), Anopheles triannulatus s.l. (1.8%), Anopheles pseudopunctipennis s.l. (0.2%), Anopheles punctimacula s.l. (0.2%), Anopheles apicimacula (0.1%), Anopheles albimanus (0.1%) and Anopheles rangeli (0.1%) were identified and species identity was confirmed by ITS2 sequencing. This is the first report of An. albimanus, An. rangeli and An. apicimacula in Tierradentro. Natural infectivity with Plasmodium was determined by ELISA. None of the mosquitoes was infectious for Plasmodium. An. nuneztovari s.l. was the predominant species and is considered the primary malaria vector; An. darlingi and An. triannulatus s.l. could serve as secondary vectors.
Colombia is among the 31 high-malaria-burden countries (Aregawi et al. 2009); although the total number of cases decreased by 45% from 2003 (125,064) to 2012 (56,175), malaria remains a major health problem. In 2012, the departments of Antioquia and Córdoba, situated in the northwest of the country, reported 53.1% of the total cases [National System for Public Health Surveillance (SIVIGILA)]. Recent studies of the anthropophilic Anopheles composition and natural mosquito infectivity with Plasmodium in the municipality of Montelíbano have led to the identification of four Anopheles species: Anopheles nuneztovari s.l., Anopheles darlingi, Anopheles oswaldoi s.l. and Anopheles punctimacula s.l. Of those, An. nuneztovari s.l. (99.4%) was the most abundant species in the municipality. An. nuneztovari s.l. was also found to be infected with Plasmodium vivax [infectivity rate (IR) = 0.489%] and is therefore considered the main malaria vector in the municipality (Gutiérrez et al. 2009). Naranjo-Diaz et al. (2013) analysed species diversity in the neighbouring municipalities of Tierralta and Puerto Libertador and identified six species: An. nuneztovari s.l., An. darlingi, Anopheles triannulatus s.l., Anopheles pseudopunctipennis s.l., An. punctimacula s.l. and Anopheles argyritarsis, of which An. nuneztovari s.l. (IR = 0.05-0.10) and An. triannulatus s.l. (IR = 1.52) were found to be infective for P. vivax VK247 and An. darlingi (IR = 0.09) for P. vivax VK210. Tierradentro is one of the localities of Montelíbano that has a high malaria incidence. However, the previous entomological studies were performed in the urban sector only due to serious disturbances affecting law, order and security.
This study aimed to investigate urban and rural mosquito populations and their natural Plasmodium infectivity to determine malaria vectors in the locality of Tierradentro, Montelíbano. Eight species (complexes) of the subgenera Nyssorhynchus and Anopheles were identified: An. nuneztovari s.l., An. darlingi, An. triannulatus s.l., An. pseudopunctipennis s.l., An. punctimacula s.l., Anopheles apicimacula, Anopheles albimanus and Anopheles rangeli. Species identity was confirmed by restriction fragment length polymorphism-internal transcribed spacer (RFLP-ITS)2 analysis, ITS2 sequencing and National Center for Biotechnology Information (NCBI) BLAST search. None of the mosquitoes was found to be infectious for Plasmodium (IR< 0.1%).
The locality of Tierradentro is situated in the northwest of Colombia in the south of Córdoba in the municipality of Montelíbano (Supplementary data). The urban centre is located along the San Jorge River at 55 m above mean sea level (07º48'50''N 75º52'40''W). The tropical climate is characterised by a monthly mean temperature of 25.6-27.1ºC (SIPLAN 2008). The average annual precipitation is 2,386 mm and ranges from 350 mm in August to 20 mm in January (Hydrometrical Station Cuba Hda). The region is mountainous and the vegetation consists of natural forests, gallery forests, stubble, pastures and cropland. The total population of Tierradentro is 6,447, with 77.75% inhabiting the urban sector and 22.25% the rural regions (SIPLAN 2008). From 2003-2009, Montelíbano reported 6.29% of the nationwide malaria cases (664,489); 42,039 cases were registered in the municipality, of which 77% were caused by P. vivax, 22% were caused by Plasmodium falciparum and 1% were caused by infection with both parasites. The annual parasite index for Montelíbano was estimated at 114/1,000 in 2007. The region is classified as endemic for malaria, with a high and constant transmission throughout the year (SIVIGILA).
In August 2009, during the rainy season, human landing catches were performed in the urban centre [Claret (CLA)] and seven rural localities of Tierradentro with distinct environmental conditions: Parcela Belén (BEL), San Antonio (SAN), Bocas de San Cipriano, Bocas de San Mateo (BSM), Vallecito (VAL) and Parque Paramillo, Venado (VEN) and Santa Isabel (ISA). The walking distance between the collection sites was 1-8 h. Female Anopheles mosquitoes were captured during one-five nights in each locality depending on the weather conditions, with one-three baits per site (inclusive the main investigator and local volunteers), indoors and peridomestically from 06:00-10:00 pm sacrificed hourly, individualised in perforated tubes and stored in plastic bags supplied with silica gel until they were transported to the laboratory where they were stored at 20ºC. The mosquitoes were identified based on their morphological characteristics (Carrejo & González 2007). However, Anopheles species belonging to the Oswaldoi Group may be misidentified due to intra and interspecific variations in morphology. Therefore, the identity of cryptic species (An. nuneztovari s.l., An. oswaldoi s.l., An. rangeli) was confirmed by polymerase chain reaction amplification of the ITS2 region directly from a mosquito leg and detection of an RFLP within ITS2 with Alu I restriction enzyme digestion (Zapata et al. 2007, Cienfuegos et al. 2008). The ITS2 amplicon from one-three specimens of each species was purified with ExoSAP-IT and sequenced by StarSEQ GmbH (Mainz, Germany); an NCBI GenBank database search using the BLAST algorithm was performed. All specimens were tested for P. falciparum and P. vivax infectivity by ELISA (Wirtz et al. 1987), as instructed in the ELISA kit protocol distributed by the Centers for Disease Control and Disease (Atlanta, GA, USA).
A total of 927 Anopheles mosquitoes were collected in Tierradentro in August 2009; 30.7% (285) in the urban centre (suburb CLA) and 69.3% in the rural sector (BSM: 182, BSC: 165, VAL: 163, BEL: 66, SAN: 40, VEN: 21, ISA: 11). Based on morphological characteristics and RFLP-ITS2 and ITS2 sequencing, eight Anopheles species belonging to the subgenera Nyssorhynchus and Anopheles were identified: An. nuneztovari s.l. (92.4%, KF436936), An. darlingi (5.1%, KF436940), An. triannulatus s.l. (1.8%, KF436938), An. pseudopunctipennis s.l. (0.2%, KF436937), An. punctimacula s.l. (0.2%, KF436941), An. albimanus (0.1%, KF436939), An. apicimacula (0.1%, KF436935) and An. rangeli (0.1%). The species identity of 43 specimens remained unclear. NCBI BLAST identification resulted in 99% sequence identity for An. nuneztovari B/C (Fritz et al. 1994, Sierra et al. 2004, Marrelli et al. 2005), An. darlingi (Marrelli et al. 2005), An. pseudopunctipennis s.l. (Miller et al. 1997, M Herrera et al., unpublished observations), An. triannulatus of the lineage NW (Rosero et al. 2012, Moreno et al. 2013), An. punctimacula s.s. [Cienfuegos & Correa (GU477275) (Loaiza et al. 2013)], An. albimanus (L78065) (Cienfuegos et al. 2011) and An. apicimacula (Loaiza et al. 2013). The species composition differed in each locality. An. nuneztovari s.l. was the most abundant species in all localities [CLA (198/201, 98.5%), BEL (64/67, 95.5%), BSC (131/164, 79.9%), BSM (171/183, 93.4%), SAN (36/40, 90%), VAL (144/160, 90%), VEN (19/21, 90.5%) and ISA (10/11, 90.9%)], due to its adaptability to variable larval habitats, particularly artificial ones (Tadei et al. 1998, Tadei & Thatcher 2000). In accordance with former studies, this species is considered the primary malaria vector (Gutiérrez et al. 2009). An. darlingi was largely collected in BSC (20/163, 12.3%) and VAL (14/160, 8.6%), with a few specimens from CLA (2/210, 1%), BEL (1/67, 1.5%), SAN (1/40, 2.5%) and BSM (3/183, 1.6%). BSC and VAL are located in partly deforested flat areas that constitute perfect breeding sites for this species (Hiwat & Bretas 2011). An. triannulatus s.l. showed no restriction to specific localities and was encountered in BSM (8/183, 4.4%), SAN (2/40, 5%), BSC (2/163, 1.2%), CLA (1/201, 0.5%), BEL (1/67, 1.5%) and VAL (1/160, 0.6%). As a habitat generalist, this species shows a wide distribution across Latin America, with no environmental constraints and is considered a regionally important malaria vector (McKeon et al. 2013). Although the An. darlingi and An. triannulatus s.l. density was low and locally restricted, these species could constitute regional secondary malaria vectors due to their high IR (> 1.5) (Gutiérrez et al. 2009). An. apicimacula (VEN: 1/21, 4.8%), An. pseudopunctipennis s.l. (VEN: 1/21, 4.8%; ISA: 1/11, 9.1%) and An. punctimacula s.l. (SAN: 2/40, 5%) were found in low numbers in dispersed villages and solitary farms in forested mountainous areas with little human activity and natural small water bodies. The latter two species have historically been collected in small numbers and have never been found to be infected with Plasmodium. Nevertheless, they are widely distributed and are considered secondary vectors of local importance in some regions. However, these species are not of epidemiological relevance in Colombia (Olano et al. 2001, Gutiérrez et al. 2008).
We for the first time registered specimens of An. albimanus, An. rangeli and An. apicimacula in Montelíbano. The increase in species diversity for the region may have resulted from the inclusion of urban and rural collection sites with different ecological conditions. An. albimanus is commonly associated with the low coastal regions of the Caribbean and Pacific in Colombia, where it is a primary malaria vector. An. albimanus may also be found at higher elevations (Montoya-Lerma et al. 2011) and has previously been reported for the neighbouring municipality of Tierralta (Gutiérrez et al. 2009). An. rangeli has been considered to be a main local malaria vector in the department of Putumayo, in the south of Colombia (Quiñones et al. 2006), and recent studies provide evidence for Anopheles benarrochi B being mainly responsible for Plasmodium transmission in that region (Orjuela et al. 2013). Little is known about the distribution and taxonomic status of An. apicimacula and its epidemiologic importance for malaria transmission.
Constant epidemiological surveillance is essential because human activities, climate change and evolutionary processes might have an impact on the geographical and temporal distribution, susceptibility and efficiency of the vectors and the lifecycles of the pathogens (Gould & Higgs 2009). The high Anopheles density in the urban centre and the villages along roads is most likely the result of artificial water bodies (road ditches, fish breeding pools, drinking troughs), which provide adequate larval habitats for Anopheles, particularly An. nuneztovari s.l. (Rodríguez et al. 2010). Structural interventions for avoiding stagnant water near settlements will likely significantly reduce malaria transmission and cases.
Further studies should focus on breeding and crossing experiments to clarify the taxonomic status of the Anopheles species complexes. Contemporary studies have focused on applying molecular markers for species identification; however, in spite of their utility for analysing population structures, evolutionary events and speciation tendencies, such studies do not provide conclusive results regarding species differentiation. Furthermore, the presence of other arthropod-borne pathogens, e.g., arboviruses and possible synergies between different pathogen life cycles should be extensively studied to acquire comprehensive, integrated knowledge on the transmission cycles of infectious diseases.
To the community of Tierradentro, to Patricia Gutiérrez and Viviana Cerón, National Institute of Health, Bogotá, and Martha Irene Castro and John Jairo González, Departmental Health Secretariat of Córdoba, Montería, for their support during field work, to San Isidro Foundation, for access to the Zonal Planning System Community, Alto San Jorge, to Dr Adrian Luty, Radbound University, Nijmegen, Centre for Molecular Life Sciences, for providing P. falciparum infected Anopheles, and to all the collaborating members of the Medical Entomology Laboratory, National University of Colombia, and to the Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University Mainz.
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