Role of Artificial Containers as Breeding Sites for Anopheline Mosquitoes in Malaria Hypoendemic Areas of Rural Bandarban, Bangladesh: Evidence from a Baseline Survey Mohammad Shafiul Alam1*, Sumit Chakma1, H M Al-Amin1, Rubayet Elahi1, Abu Naser Mohon1, Wasif Ali Khan1, Rashidul Haque1, Gregory E. Glass2, David A. Sack2, David J. Sullivan2 and Douglas E. Norris2 1ICDDR,B, GPO Box 128, Dhaka 1000, Bangladesh 2Johns Hopkins Malaria Research Institute, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205, USA Presentation no. 210 Materials and Methods Abstract Within the framework of our Mapping Malaria Epidemiology of Bandarban project, a survey was conducted for breeding habitats of vector mosquitoes during September-October 2011. The survey was carried out in seven of the 24 study clusters in two unions (Kuhalong and Rajbila), based on malaria incidence. The survey team screened mosquito larvae from natural reservoirs irrespective to their size and artificial or natural containers where natural water (e.g. rain) can stand for few days following standard protocols. Twenty larval habitats from each selected cluster were surveyed. A total of 3,696 immature mosquitoes (larvae and/or pupae) representing five mosquito genera were recorded from 122 habitats. The collection was dominated by Culex spp. (n = 2082; 46 single and 28 mixed occupancy) followed by Aedes spp. (n = 1469; 36 single and 14 mixed occupancy). Immature stages of Anopheles species (n = 128) were collected from 25 habitats, nine of which were single occupancy whereas, 14 habitats were shared with Culex spp and the remaining two with Aedes species. Anopheline larvae were reared to adult before identification and seven species were recorded. Median temperature and PH of Anopheline larval habitat were 35°C (IQR: 32.5°C -36.0°C) and 7.4(IQR: 7.05-7.85) respectively. Rice fields have been implicated as the most preferred breeding site for Anopheles spp. Anopheline breeding sites also include puddle, irrigation canal, animal hoof print, artificial container and livestock wallow. Among the collected anophelines, An. kochi and An. vagus were found in containers (abundant plastic buckets and cement tanks respectively). An. vagus is considered an important malaria vector in Bangladesh. However, the preferences of An. vagus to artificial container needs further attention and should consider an alarming sign to malaria control in Bangladesh. Mosquito larval survey was carried out in seven clusters (four from Kuhalong and three from Rajbila) These clusters were selected based on malaria incidence Two teams consist of two members each sampled one cluster in a given day The team screened Anopheles larvae from natural reservoir irrespective to their size and artificial or natural containers where natural water (e.g. rain) can stand for few days Twenty collections of different habitats from each selected cluster were surveyed. Thus, a total of 140 collections (of different habitats) were done during the period Mosquito larvae and pupae were sampled using standard dipping techniques In case of small reservoir (animals hoof/foot print, leaves, pit etc) maximum amount of water was sampled using serological pipette All larval samples were passed through a mesh sieve and placed with water into labeled plastic container and were taken to the laboratory placed at the Bandarban field office for further processing Larvae were reared to adult. Standard taxonomic keys were used for identification Searching Mosquito Larvae by the side of a Rice field Sampling Mosquito Larvae from Animal hoof print Sampling Mosquito Larvae from Bamboo pole Rearing Anopheles Larvae in cages at Bandarban Field office Results Among the collection, mosquito larvae (and/or pupae) of any species was found in 122 habitats represented by immature of five mosquito genera The collection was dominated by Culex spp. (46 single and 28 mixed occupancy) followed by Aedes spp. (36 single and 14 mixed occupancy) Immature stages of Anopheles species were collected from 25 habitats, nine of which were single occupancy whereas, 14 habitats were shared with Culex spp and remaining two with Aedes species (Table 1) A total of 3,696 different immature stages of mosquitoes were collected More than half of those were from Culex species (2082) followed by Aedes (1469) and Anopheles (128) Seven species Anopheles mosquitoes were identified Most frequent mosquito breeding site was bamboo pole which was found to harbor all five genera of mosquitoes Bamboo is used as a fence in most of the tribal houses which temporarily reserves rainy water and served as a breeding site for mosquitoes preferably for Culex spp. and Aedes spp. However, An. jamesii and An. vagus were found to share bamboo pole as their breeding sites either with Culex spp. or Aedes spp Rice field has been implicated as the most preferred breeding site for Anopheles spp. Other Anopheline breeding sites include puddle, irrigation canal, animal hoof print, artificial container and livestock wallow (table 2) Larvae of An. kochi were collected from artificial containers (abundant plastic bucket) which is unusual for Anopheles mosquitoes. In another occasion larvae of An. vagus were collected from a type of large artificial container Median temperature and PH of Anopheline larval habitat (water body) were 35°C (IQR: 32.5°C-36.0°C) and 7.4(IQR: 7.05-7.85) respectively Sampling Mosquito Larvae contained water from a large artificial container (Cement tank) Sampling Mosquito Larvae from puddle Table 1. Distribution of Mosquito species according to larval habitats  Larval habitat Anopheles Aedes Culex Armigeres An+Ae An+Cx Ae+Cx Cx+Tx Ae+Cx+Ar Lake/Pond Margin - Rice Field + Puddle Irrigation Canal Sluggish Stream Animal Hoof Print Large Artificial Container Small Artificial Container Tree Hole Livestock Wallow Bamboo hole Others Malaria is a major health burden in this remote, mountainous south-eastern region of Bangladesh bordering India and Myanmar Malaria in Bangladesh is mainly caused by two Plasmodium species: Plasmodium falciparum and Plasmodium vivax Out of 35 anopheline species reported in Bangladesh, 16 have shown evidence to have some role in transmitting human malaria parasites Among those, An. aconitus, An. annularis An. baimaii, An. jeyporiensis, An. minimus, An. nivipes, An. philippinensis, An. sundicus and An. vagus are vectors A total of 25 Anopheles spp. have been reported Anopheles breeding ecology is an important component to formulate effective control strategies Unfortunately, there are very few reports available on Anopheles breeding habitats in Bangladesh Introduction Table 2. Anopheles larvae identified up to species level according to study unions Species Rajibila Kuhalong Total An. baimaii 1 An. barbirostris 16 An. karwari 5 An. kochi 10 14 24 An. jamesii 11 An. philippinensis-nivipes 4 An. vagus 51 7 58 Unidentified 3 86 42 128 Map of study area showing the Larval habitats of different Anopheles mosquitoes This entomological investigation was carried out as a part of a collaborative research project between ICDDR,B and Johns Hopkins Malaria Research Institute entitled “ Mapping malaria epidemiology in Bangladesh”. The main study includes: A Demographic Surveillance System (DSS) which includes KPC survey GIS with coordinates of all houses and other key features Active surveillance from a random sample of persons Passive surveillance of malaria cases as and when they are identified Mosquito vector surveillance Data management system which integrates all data Objectives for this Mosquito breeding sites investigations were: To record the preferred breeding habitats of different Anopheles species To observe the distribution of breeding sites maintained by dominant Anopheles species To correlate the malaria epidemiology with the larval abundance in both peak and off peak transmission season Objectives Table 3. List of larval habitats of Anopheles species in Bandarban Species Larval Habitat An. baimaii Puddle An. barbirostris Irrigation canal, puddle, Rice field An. jamesii Bamboo hole, Irrigation canal, Puddle An. karwari An. kochi Small artificial container, livestock wallow, puddle, rice field An. philippinensis/nivipes Rice field An. vagus Animal hoof print, large artificial container, Bamboo hole, Puddle Conclusion Bandarban has high diversity in anopheline species with a wide range of larval sites Rice field has been implicated as the most preferred breeding place for Anopheles spp. Anophelines found to breed in the artificial containers, though they are not usual nor expected breeding habitat More detailed longitudinal data is required to reveal larval habitats of the major vector species present in CHT in order to design effective vector control strategies *For correspondence: Mohammad Shafiul Alam, email: shafiul@icddrb.org