Malaria Journal BioMed Central Research Open Access The resting sites and blood-meal sources of Anopheles minimus in Taiwan Mei-Chun Chang1, Hwa-Jen Teng*1, Chen-Fu Chen1, Yung-Chen Chen1 and Chian-Ren Jeng2 Address: 1Research and Diagnostic Center, Centers for Disease Control, Taiwan and 2Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan Email: Mei-Chun Chang - [email protected]; Hwa-Jen Teng* - [email protected]; Chen-Fu Chen - [email protected]; Yung- Chen Chen - [email protected]; Chian-Ren Jeng - [email protected] * Corresponding author Published: 9 June 2008 Received: 27 February 2008 Accepted: 9 June 2008 Malaria Journal 2008, 7:105 doi:10.1186/1475-2875-7-105 This article is available from: http://www.malariajournal.com/content/7/1/105 © 2008 Chang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: The WHO declared Taiwan free from malaria in 1965, but in 2003 the reporting of two introduced cases in a rural area suggested a possible local transmission of this disease. Therefore, understanding the resting sites and the blood sources of Anopheles minimus is crucial in order to provide information for implementing vector control strategies. Methods: During a two-year survey, mosquitoes were collected in houses and their surrounding areas and at the bank of larval habitats by backpack aspirators in 17 villages in rural areas of southern and eastern Taiwan for 1 hr. On the same day, blacklight traps were hung downward overnight. Blood-fed mosquito samples were analysed by PCR. Results: Of the 195 total households surveyed by backpack aspirators, no Anopheles adults were collected inside the houses, while a single Anopheles minimus and a single Anopheles maculatus were collected outside of the houses. On the same day, 23 An. minimus, two An. maculatus, two Anopheles ludlowae, two Anopheles sinensis, and one Anopheles tessellatus were collected along the bank of larval habitats. In blacklight traps hung outside of the houses in the villages, 69 An. minimus, 62 An. ludlowae, 31 An. sinensis, and 19 An. maculatus were collected. In larval habitats, 98 An. ludlowae, 64 An. minimus, 49 An. sinensis, and 14 An. maculatus were collected. Of a total of 10 blood-fed samples, An. minimus fed on four animals including bovine (60%), dogs (20%), pig (10%), and non-chicken avian (10%). Conclusion: Anopheles minimus, an opportunist feeder in Taiwan, was not collected inside the houses, but was found outside of the houses in villages and surrounding larval habitats. Therefore, an outdoor transmission of malaria is likely to occur and, thus, the bed nets, which are favoured for controlling the late biting of An. minimus, should be a very efficient and effective method for those local residents who sleep outdoors. Additionally, space spray of insecticides for Anopheles at night, as well as residual spray inside animal huts and selective larval habitats, are also helpful to control female adults. Page 1 of 8 (page number not for citation purposes) Malaria Journal 2008, 7:105 http://www.malariajournal.com/content/7/1/105 Background of mosquito transmission. Since the eradication of Malaria is documented to have been prevalent throughout malaria in Taiwan, environmental and housing condi- much of Taiwan in the19th and 20th centuries. The maxi- tions have largely changed. Data on the resting sites and mum estimated case number was 1.2 million in 1952 [1]. blood-fed hosts of the malaria vectors need to be updated In November 1946, the International Health Division of in order to provide information for implementing vector the Rockefeller Foundation, in collaboration with the Tai- control strategies. The objective of this study is to under- wanese government, established a Malaria Research stand the resting sites and blood-meal sources of An. min- Center in southern Taiwan in order to instigate a series of imus in Taiwan. antimalaria measures. Among these actions, a four-year island-wide malaria control programme was launched in Methods 1951. The principal control measure was indoor residual Resting site study and mosquito collection house spraying with DDT (0.5–2.0 g of active ingredient The survey villages (in southern and eastern Taiwan) were per m2), which was conducted in conjunction with the chosen based on a large number of An. minimus adults col- larviciding of streams with DDT and automatic flushing lected by light traps during the same year or the previous of streams. As a result of the success of these actions and year (Figure 1). Based on the surveys conducted by local patient treatments, the World Health Organization health bureaus, the average density (± SD) of An. minimus (WHO) declared Taiwan to be free from malaria on at the study villages comparing with that of all villages December 4, 1965. Furthermore, after 1973, almost all of examined were 1.80/night/trap (± 8.56) in the study vil- the reported cases of malaria in Taiwan (22–83 cases per lages and 0.32/night/trap (± 3.18) in all villages exam- year) were imported [1-3]. ined. From April to September in 2005 and 2006, two to three villages were surveyed each month. On each visit, a Among the 15 Anopheline species that are found in Tai- larval survey by 14-cm-diameter dippers was conducted wan, Anopheles minimus is regarded as the principal along the bank of streams and ditches around or in the malaria vector [4]. This species (A) is the malaria vector in surveyed village. A section of a larval habitat was chosen the Oriental Region and its morphology is similar to its in the morning based on the collection of An. minimus lar- two sibling species, C and E [5,6]. Recently, species C was vae or, at least, other Anopheles larvae. Two teams collected resolved as Anopheles harrisoni by comparing DNA mosquito adults along the bank and its surroundings for sequence data [7]. Based on DNA analysis of the D3 1 hr during the period between 10:00 and 12:00. Each region of the 28S gene of ribosomal DNA, samples col- team included two individuals with one modified CDC lected from Taiwan are identical to species A [8,9]. Species backpack aspirator (Model 1412, John W. Hock Com- A has now formally been recognized as An. minimus s.s. pany, Gainesville, Florida) and one sweeping net. During [10]. More thorough study on molecular identification of the period between 15:00 and 17:00, the same 1 hr collec- An. minimus is on going to include samples collected from tion was also conducted in human dwellings, including wider areas. Anopheles minimus is able to change its host inside the houses and their surroundings. The mean preference based on host availability and is known as an number (± SD) of houses sampled per visit was 9.75 (± opportunist feeder [11,12]. The vector status of the sec- 1.77). Screen conditions for each surveyed house were ondary species, Anopheles sinensis, was questioned by the recorded. On the same day, one updraft blacklight (UV) misidentification of one slide from a 1947–1949 study trap (Model 1312, John W. Hock Company, Gainesville, [4], on which the crithidial flagellates had been misiden- Florida) with dry ice was set up downward outside of the tified as malaria sporozoites. Of the other 14 Anopheline houses and the larval habitat, (separately) overnight. All species in Taiwan, Anopheles maculatus, Anopheles ludlowae, collected mosquitoes were stored in a dry ice box and Anopheles tessellatus, Anopheles jeyporiensis and Anopheles brought back to the laboratory for species identification. annularis have been implicated in malaria transmission in Blood-fed mosquitoes were kept at -20°C for blood meal other countries; however, they are not considered to be identification. Additional mosquitoes were collected in potential vectors of malaria in Taiwan [4]. animal huts (including pigs, buffalos, and horses) by aspi- rators or blacklight traps outside of the villages in order to In addition to the imported malaria found in Taiwan, increase the blood-fed mosquito sample. induced malaria also occurred in 1980 (1 case), 1995 (6 cases with 66.67% mortality), and 1997 (1 case) [2]. In Blood-meal identification August 2003, two cases of locally transmitted malaria (the Blood-fed mosquitoes were processed individually using first since the eradication of malaria in Taiwan) occurred the PCR method to identify the blood-meal source. in a rural area of Taitung County. However, only the pres- Genomic DNA from blood-fed mosquitoes was extracted ence of an imported Plasmodium carrier, the existence of using the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, competent female vectors (An. minimus), and the high- Germany) and the protocol described by the manufac- risk behaviour of sleeping outdoors suggest the possibility turer was followed. The same DNA extraction procedure Page 2 of 8 (page number not for citation purposes) Malaria Journal 2008, 7:105 http://www.malariajournal.com/content/7/1/105 1.5 mM MgCl2, 2.5 mM dNTP Mix, 1 μM of each primer, 5 units/μl of Tag DNA polymerase (TAKARA BIO Inc., Shiga, Japan), and 1 μl of DNA product. The sequences of the primers for the 10 animals used in the PCR are listed in Table 1. Two additional order-specific primers (mam- malian and avian) were included to detect other possible hosts as well. Reactions began with an incubation at 94°C for 2 min, followed by 35 cycles consisting of 94°C at 30 sec, 54–70°C at 30 sec (detailed temperature for each primer listed in Table 1), and 72°C at 30 sec.