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Reappearance of Anopheles minimus in Singhbum hills of East-Central India
Acta Tropica 96 (2005) 31–35
Reappearance of Anopheles minimus in Singhbum hills of East-Central India P. Jambulingam ∗ , S.S. Sahu, A. Manonmani Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, Pondicherry 605006, India Received 16 July 2004; received in revised form 31 March 2005; accepted 3 May 2005 Available online 10 August 2005
Abstract Anopheles minimus, an important malaria vector of South East Asia, has reappeared in the Singhbum hills, East-Central India where deforestation and DDT residual spraying had reportedly eliminated it during the Malaria Eradication Programme. The species reported has been identified as sibling species A of the An. minimus complex. An. minimus is susceptible to both deltamethrin and DDT. The study shows that the environmental conditions in this region still favour the existence of the species and one of the possible reasons for its reappearance may be the scaling down of residual insecticide spraying in the area. © 2005 Elsevier B.V. All rights reserved. Keywords: Anopheles minimus; Malaria vector; Reappearance; Susceptibility, Orissa, India
1. Introduction Anopheles minimus is widely distributed in the oriental region and is known to occur in India, Nepal, Bangladesh, Sri Lanka, Thailand, Myanmar, Malaysia, Indonesia, southern China, Hong Kong, Taiwan and the Ryukyu Islands (Rao, 1984). Throughout its range of distribution, it was an important malaria vector. In India, it was one of the primary vectors of malaria all along the foothills of the Himalayas extending from the Terai region of Uttar Pradesh to Assam and the neighbouring eastern region. In East-Central India (Jeypore ∗ Corresponding author. Tel.: +91 413 2279157; fax: +91 413 2272041. E-mail address: [email protected] (P. Jambulingam).
0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.05.013
and the Singhbhum hills), sporozoite rates ranging from 4.3 to 15.4% were reported in this species (Rao, 1984). The introduction of DDT in 1958 for malaria control and the ecological changes due to extensive deforestation caused several changes in its distribution. The An. minimus population declined in numbers or completely disappeared from the Terai region of Uttar Pradesh, East-Central India and also in large parts of Assam (Kalra, 1991). While several studies conducted in the 1970s and 1980s have confirmed its continuous presence in West Bengal and the North Eastern states as the major vector (Nandi et al., 2000; Kamal and Das, 2001; Prakash et al., 2000; Dev et al., 2001), the species was not any more encountered in other regions (Rao, 1984; Dash et al., 1984; Gunasekaran et al., 1989; Collins et al., 1990; Kalra, 1991; Sharma,
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P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
2002). Recently, Das et al. (2000) recorded An. minimus in the Rajamahal range in Bihar (Northern India). We report in this communication, the reappearance of An. minimus in the Singhbum hills, East-Central India, where this species has been reported to be absent for nearly 45 years after the launching of the Malaria Eradication Programme.
2. Materials and methods 2.1. Study area The study was carried out in Keonjhar district (latitude 20◦ 11 to 20◦ 10 N, longitude 85◦ 11 to 86◦ 22 ) of Orissa state during 2001–2003. The district is divided into 13 administrative blocks or Primary Health Centre areas. It is spread over an area of 8303 km2 of which about two-third is traversed by the Singhbum hill range with 30–40% of the area under forest cover. A population of approximately 1.5 million (according to 2001 census) is distributed in 2118 villages. Tribes constitute about 44.5% of the population. The climate is characterized by a hot summer (March–June), a rainy (July–September) and a cool season (October–February). The minimum temperature ranges from 8 ◦ C in December to 32 ◦ C in May and the maximum temperature ranges from 19 ◦ C in December to 42 ◦ C in May. Malaria has been endemic in this district and the majority (>95%) of the cases are caused by Plasmodium falciparum. During 1997–2002, malaria incidence in the district has shown an increasing trend with the annual parasite incidence (API) ranging from 31.8 to 36.3. There were 119 malaria deaths in the district in 2000 and 82 in 2001 (compared to 442 and 305, respectively, in Orissa state). The number of deaths in Keonjhar in 2000 accounted for about 20% of the total number of malaria deaths (522) recorded in India. Since 1958, the district has been under DDT residual spraying. Under the modified plan of operation implemented in 1977, the district continued to receive DDT residual spraying, since in all the areas, >2 API were recorded. From 1981 onwards, the areas receiving DDT spray started reducing depending on the availability of DDT stock and funds for spray operations. The population targeted for protection using DDT spray ranged from 14 to 84% during 1981–1990
and from 12 to 66% during 1991–2000. During the latter period, only seven first rounds and three second rounds of spraying were carried out in the district. Priorities were given to the areas, where cerebral malaria and malarial deaths were reported. In view of persistent transmission, the district has been included under the Enhanced Malaria Control Programme in 1998 and from 2001 onwards, pyrethroids (lambdacyhalolthrin, alphacypermethrin or cyfluthrin) have been used for residual spraying in place of DDT in 11 of the 13 PHC areas (where API > 10). Since malaria incidence peaks during July–August and November–December, two rounds of spraying have been carried out during May–June and September–October, respectively. During the last 3 years (2001–2003), the population protected varied from 9 to 84%. 2.2. Entomological collections The first entomological survey of this series was made in August 2001 in Keonjhar district of Orissa state. An. minimus was recorded during the survey along with the other known malaria vector of the area, Anopheles fluviatilis. The presence of An. minimus prompted the performance of four more surveys during 2003–2004, one in the summer (March–April 2003), one in the rainy season (August–September 2003) and two in the cold season (October 2003 and February 2004). Surveys were made in five hilltop villages, viz. Doyonala, Dhanakunia Sahi, Lohanda, Mundasahi and Ulupuri, randomly selected from the Banspal, Basudevpur and Joda PHC areas, respectively, where a high incidence of malaria was recorded during 1999–2001. Day time indoor resting adult anophelines were collected using aspirators and torch lights between 06:00 and 07:00 h in nine human dwellings and three cattle sheds selected randomly in each village. A modified version of the CDC light trap (Gunasekaran et al., 1994) was installed in two human dwellings and two cattle sheds in a village (one trap in one structure) from 18:00 to 06:00 h. Man-landing collections were made between 18:00 and 21:00 h at three catching stations in each village. The anophelines collected were identified morphologically according to species and the species composition was recorded. Females of An. fluviatilis, An. minimus and Anopheles culicifacies were dissected for gut and gland infection with Plasmodium.
P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
2.3. Susceptibility test Susceptibility of An. minimus to DDT and deltamethrin (pyrethroid) was determined using WHO kits (WHO, 1975). Insecticide papers used for the tests were prepared at Vector Control Research Centre (VCRC) following standard procedures. Fully fed wild caught females were exposed to DDT (4%) and deltamethrin (0.05%) impregnated papers for 1 h in triplicates and mortality was recorded after 24 h of holding. Two controls were maintained simultaneously. Since no mortality was observed in controls, the treated mortality was not corrected. 2.4. DNA sequence In view of similarities and overlapping morphological characters occurring among the species of the An. minimus group causing considerable taxonomic controversies and erroneous reports on the distribution of An. minimus (Reid, 1968; Harrison, 1980), species identification of An. minimus was confirmed using a molecular method. A few specimens of An. minimus and the other anophelines species were kept in Eppendorf tubes, dried for 4–5 h at 90 ◦ C and sent to the VCRC laboratory for molecular identification. DNA was extracted from the samples, the ITS2 and the D3 regions of the ribosomal DNA were amplified (Manonmani et al., 2001; Sharpe et al., 1999) and sequenced. The ITS2 sequences of the samples were compared with the ITS2 sequences of An. minimus (Acc. Nos. AF194495–194506), An. fluviatilis (Acc. Nos. AF167298–167299), Anopheles aconitus or Anopheles varuna (Acc. Nos. AF194489–194494). 3. Results and discussion The collections comprised a total of 3858 anophelines belonging to 18 species, including An. fluviatilis (24.4%), An. culicifacies (6.3%) and An. minimus (3.5%). In total, 135 An. minimus were collected, 115 from hand catches in human dwellings and 20 from man-landing catches. While 98 were obtained during the rainy season (96 caught during the survey in 2001), 37 were collected during the cold season. In the summer survey, this species was not encounterd. Of the 100 An. fluviatilis dissected, one was found with Plasmodium sporozoites, but out of 24 An. minimus dissected,
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none harboured sporozoites. Compared to the survey in 2001, vector densities were low during the subsequent surveys (2003–2004), probably due to intensive indoor residual spraying. An. minimus showed 100% (45/45) mortality when exposed to deltamethrin impregnated paper and 86% (18/21) mortality to DDT paper. According to WHO (1998) criterion, An. minimus is susceptible to both deltamethrin and DDT. DNA sequencing of four adult specimens showed that they were species A of the An. minimus complex. The D3 sequences were identical to those deposited by Chen et al. (2002) for the same species of this complex (Acc. No. AJ459420). An. minimus from Assam, India, incriminated with sporozoite rates ranging from 2.3 to 3.3% has also been identified as species A (Subbarao, 1998). In many countries, vectors that were believed to have been almost eliminated or having completely disappeared from areas brought under the expanded programme of residual spraying with DDT have reemerged with the cessation of house spraying (Roberts et al., 2002). In South Africa, An. funestus, a morphologically similar species to An. minimus disappeared in the 1950s with DDT spraying and 4 years after a switch over to pyrethroid spraying in 1990s, it reappeared and found to be pyrethroid resistant (Hargreaves et al., 2000). An. minimus, once the major malaria vector in Hainan Island and eliminated through indoor residual spray, has again been incriminated as a vector of local malaria outbreaks in recent years (Wu et al., 1993). In India, many changes have taken place in Anopheles species composition, distribution and importance as malaria vectors following the introduction of DDT spraying countrywide. An. minimus and An. sundaicus were notably affected (Kalra, 1991). Anopheles sundaicus was a major malaria vector in the eastern and western coasts of India and in the Andaman and Nicobar Islands. DDT spraying eliminated the species from the mainland and its distribution in India is now limited to the Andaman and Nicobar Islands. An. minimus, once a major malaria vector in Uttar PradeshTerai, Singhbhum hills and Jeypore hills and responsible for hyperendemic malaria, had been eliminated from these areas and its distribution narrowed to the North Eastern states. Similarly, diminution of the population was reported in Nepal and China (Rao, 1984; Sharma, 2002).
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P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
Surveys conducted in Jeypore hill ranges and in the present study area in 1980s and 1990s did not record the presence of An. minimus (Dash et al., 1984; Collins et al., 1990; Gunasekaran et al., 1989). Besides DDT residual spraying, ecological changes due to extensive deforestation were considered responsible for the elimination of this species from these areas. The present study confirms the reappearance of An. minimus in Singhbum hills after a period of about 45 years after the launching of the malaria eradication programme. In this survey, An. minimus was collected from villages situated in hill ranges and surrounded by forest. Analysis of meteorological data recorded for the past 5 decades indicates no significant change of the climatic conditions in this region. However, comparison of recent data on forested areas in different districts of Orissa state shows that deforestation has been more extensive in the southern districts of Orissa (Jeypore hills) than in the Northern districts (Singhbhum hills). The Koraput district located in southern Orissa has a forest cover of only 16% of the total area while Keonjhar district still has 40% of the total area covered with forest (Anonymous, 1999). Overall, DDT residual spraying for malaria control in India declined in quantity as well as in the quality of applications in the 1990s, resulting in a resurgence of malaria in parts of the country (Sharma and Mehrotra, 1986). Therefore, it is clear that the environmental conditions in this region still favour or support An. minimus and once the pressure imposed by the large scale DDT residual spraying was withdrawn, the vector has started re-establishing its presence. Unlike An. funestus in South Africa, An. minimus is found to be highly susceptible to pyrethroids. Since malaria incidence in this area is showing an increasing trend in recent years, further studies are needed to understand the extent of distribution of An. minimus, its bionomics and the role in malaria transmission in this region.
Acknowledgements We thank Dr. P.K. Das, Director, Vector Control Research Centre, Pondicherry, for his keen interest and encouragement for this survey. The co-operation extended by the technical staff of VCRC field station, Malkangiri is gratefully acknowledged. We are grateful to the District Administration and the Health
Department Keonjhar for all the co-operation and help extended during the surveys. References Anonymous, 1999. Forest Survey of India. State of Forest Department, Dehradun. Chen, B., Harbach, R.E., Butlin, R.K., 2002. Molecular and morphological studies on the Anopheles minimus group of mosquitoes in southern China: taxonomic review, distribution and malaria vector status. Med. Vet. Entomol. 16 (3), 253–265. Collins, R.T., Beljaev, A.E., Pattanayak, S., Agarwal, R.S., 1990. Studies on malaria transmission in Orissa state, India 1981 through 1986. Part II: observation on the Anopheles fauna. J. Commun. Dis. 22 (3), 191–204. Das, N.G., Bhuyan, M., Das, S.C., 2000. Entomological and epidemiological studies on malaria in Rajmahal range, Bihar. Indian J. Malariol. 37 (3–4), 88–96. Dash, A.P., Behura, B.K., Roy, J.R., 1984. On the distribution of anopheline mosquitoes in Orissa. India J. Zool. Soc. India 36 (1 & 2), 1–14. Dev, V., Ansari, M.A., Hira, C.R., Barman, K., 2001. An outbreak of Plasmodium falciparum malaria due to Anopheles minimus in central Assam, India. Indian J. Malariol. 38 (1–2), 32–38. Gunasekaran, K., Sahu, S.S., Parida, S.K., Sadanandane, C., Jambulingam, P., Das, P.K., 1989. Anopheline fauna of Koraput district, Orissa state with particular reference to transmission of malaria. Indian J. Med. Res. 89, 340–343. Gunasekaran, K., Jambulingam, P., Sadanandane, C., Sahu, S.S., Das, P.K., 1994. Reliability of light trap sampling for Anopheles fluviatilis, a vector of malaria. Acta Trop. 58, 1–11. Hargreaves, K., Koekemoer, L.L., Brooke, B.D., Hunt, R.H., Mthembu, J., Coetzee, M., 2000. Anopheles funestus resistant to pyrethroid insecticides in South Africa. Med. Vet. Entomol. 14, 181–189. Harrison, B.A., 1980. Medical Entomology Studies-XIII. The myzomyia series of Anopheles (Cellia) in Thailand, with emphasis on intra-interspecific variations (Diptera: Culicidae). Contrib. Am. Entomol. Inst. 17, 1–195. Kalra, N.L., 1991. Forest Malaria Vectors in India: Ecological Characteristics and Epidemiological Implications. Forest Malaria in Southeast Asia, New Delhi, pp. 93–114. Kamal, S., Das, S.C., 2001. Epidemiological observations on malaria in some parts of Darrang district, Assam. Indian J. Malariol. 38 (1–2), 25–31. Manonmani, A., Townson, H., Adeniran, T., Jambulingam, P., Sahu, S., Vijayakumar, T., 2001. rDNA-ITS2 polymerase chain reaction assay for the sibling species of Anopheles fluviatilis. Acta Trop. 78, 3–9. Nandi, J., Kaul, S.M., Sharma, S.N., Lal, S., 2000. Anthropophily of Anophelines in Duars of West Bengal and other regions of India. J. Commun. Dis. 32 (2), 95–99. Prakash, A., Bhattacharyya, D.R., Mohapatra, P.K., Mahanta, J., 2000. Mosquito fauna and malaria vectors in Jairampur, district Changlang, Arunachal Pradesh. Indian J. Malariol. 37 (3–4), 74–81.
P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35 Rao, T.R., 1984. The anophelines of India. Malaria Research Centre (ICMR), p. 518. Reid, J.A., 1968. Anopheline mosquitoes of Malaya and Borneo. Stud. Inst. Med. Res. Malaya 31, 1–520. Roberts, D.R., Penny, Masuoka, Andrew, Y. Au., 2002. Determinants of malaria in the Americas. In: Casman, E.A., Dowlatabadi, H. (Eds.), The Contextual Determinants of Malaria. RFF Press, WA, pp. 35–58 (Part 2, Chapter 4). Sharma, V.P., 2002. Determinants of malaria in South Asia. In: Casman, E.A., Dowlatabadi, H. (Eds.), The Contextual Determinants of Malaria. RFF Press, WA, pp. 110–132 (Part 2, Chapter 8). Sharma, V.P., Mehrotra, K.N., 1986. Malaria resurgence in India: a critical study. Soc. Sci. Med. 22, 835–845. Sharpe, R.G., Hims, M.M., Harbach, R.E., Butlin, R.K., 1999. PCRbased methods for identification of species of the Anopheles minimus group: allele-specific amplification and single-strand
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conformation polymorphism. Med. Vet. Entomol. 13, 265– 273. Subbarao, S.K., 1998. Anopheline Species Complexes in South-East Asia. Technical Publications of WHO, SEARO, No. 18, New Delhi. World Health Organization, 1975. Manual on Practical Entomology in Malaria. Part II: Methods and Techniques. World Health Organization, Geneva, pp. 141–147. World Health Organization, 1998. Test Procedures for Insecticide Resistance Monitoring in Malaria Vectors, Bio-Efficacy and Persistence of Insecticides on Treated Surfaces. Document WHO/CDS/CPC/MAL/98.12. World Health Organization, Geneva. Wu, K.C., et al., 1993. Studies on distribution and behaviour of Anopheles minimus and its role of malaria transmission in Hainan province at present. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 11 (2), 120–123.
Reappearance of Anopheles minimus in Singhbum hills of East-Central India P. Jambulingam ∗ , S.S. Sahu, A. Manonmani Vector Control Research Centre (ICMR), Medical Complex, Indira Nagar, Pondicherry 605006, India Received 16 July 2004; received in revised form 31 March 2005; accepted 3 May 2005 Available online 10 August 2005
Abstract Anopheles minimus, an important malaria vector of South East Asia, has reappeared in the Singhbum hills, East-Central India where deforestation and DDT residual spraying had reportedly eliminated it during the Malaria Eradication Programme. The species reported has been identified as sibling species A of the An. minimus complex. An. minimus is susceptible to both deltamethrin and DDT. The study shows that the environmental conditions in this region still favour the existence of the species and one of the possible reasons for its reappearance may be the scaling down of residual insecticide spraying in the area. © 2005 Elsevier B.V. All rights reserved. Keywords: Anopheles minimus; Malaria vector; Reappearance; Susceptibility, Orissa, India
1. Introduction Anopheles minimus is widely distributed in the oriental region and is known to occur in India, Nepal, Bangladesh, Sri Lanka, Thailand, Myanmar, Malaysia, Indonesia, southern China, Hong Kong, Taiwan and the Ryukyu Islands (Rao, 1984). Throughout its range of distribution, it was an important malaria vector. In India, it was one of the primary vectors of malaria all along the foothills of the Himalayas extending from the Terai region of Uttar Pradesh to Assam and the neighbouring eastern region. In East-Central India (Jeypore ∗ Corresponding author. Tel.: +91 413 2279157; fax: +91 413 2272041. E-mail address: [email protected] (P. Jambulingam).
0001-706X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.actatropica.2005.05.013
and the Singhbhum hills), sporozoite rates ranging from 4.3 to 15.4% were reported in this species (Rao, 1984). The introduction of DDT in 1958 for malaria control and the ecological changes due to extensive deforestation caused several changes in its distribution. The An. minimus population declined in numbers or completely disappeared from the Terai region of Uttar Pradesh, East-Central India and also in large parts of Assam (Kalra, 1991). While several studies conducted in the 1970s and 1980s have confirmed its continuous presence in West Bengal and the North Eastern states as the major vector (Nandi et al., 2000; Kamal and Das, 2001; Prakash et al., 2000; Dev et al., 2001), the species was not any more encountered in other regions (Rao, 1984; Dash et al., 1984; Gunasekaran et al., 1989; Collins et al., 1990; Kalra, 1991; Sharma,
32
P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
2002). Recently, Das et al. (2000) recorded An. minimus in the Rajamahal range in Bihar (Northern India). We report in this communication, the reappearance of An. minimus in the Singhbum hills, East-Central India, where this species has been reported to be absent for nearly 45 years after the launching of the Malaria Eradication Programme.
2. Materials and methods 2.1. Study area The study was carried out in Keonjhar district (latitude 20◦ 11 to 20◦ 10 N, longitude 85◦ 11 to 86◦ 22 ) of Orissa state during 2001–2003. The district is divided into 13 administrative blocks or Primary Health Centre areas. It is spread over an area of 8303 km2 of which about two-third is traversed by the Singhbum hill range with 30–40% of the area under forest cover. A population of approximately 1.5 million (according to 2001 census) is distributed in 2118 villages. Tribes constitute about 44.5% of the population. The climate is characterized by a hot summer (March–June), a rainy (July–September) and a cool season (October–February). The minimum temperature ranges from 8 ◦ C in December to 32 ◦ C in May and the maximum temperature ranges from 19 ◦ C in December to 42 ◦ C in May. Malaria has been endemic in this district and the majority (>95%) of the cases are caused by Plasmodium falciparum. During 1997–2002, malaria incidence in the district has shown an increasing trend with the annual parasite incidence (API) ranging from 31.8 to 36.3. There were 119 malaria deaths in the district in 2000 and 82 in 2001 (compared to 442 and 305, respectively, in Orissa state). The number of deaths in Keonjhar in 2000 accounted for about 20% of the total number of malaria deaths (522) recorded in India. Since 1958, the district has been under DDT residual spraying. Under the modified plan of operation implemented in 1977, the district continued to receive DDT residual spraying, since in all the areas, >2 API were recorded. From 1981 onwards, the areas receiving DDT spray started reducing depending on the availability of DDT stock and funds for spray operations. The population targeted for protection using DDT spray ranged from 14 to 84% during 1981–1990
and from 12 to 66% during 1991–2000. During the latter period, only seven first rounds and three second rounds of spraying were carried out in the district. Priorities were given to the areas, where cerebral malaria and malarial deaths were reported. In view of persistent transmission, the district has been included under the Enhanced Malaria Control Programme in 1998 and from 2001 onwards, pyrethroids (lambdacyhalolthrin, alphacypermethrin or cyfluthrin) have been used for residual spraying in place of DDT in 11 of the 13 PHC areas (where API > 10). Since malaria incidence peaks during July–August and November–December, two rounds of spraying have been carried out during May–June and September–October, respectively. During the last 3 years (2001–2003), the population protected varied from 9 to 84%. 2.2. Entomological collections The first entomological survey of this series was made in August 2001 in Keonjhar district of Orissa state. An. minimus was recorded during the survey along with the other known malaria vector of the area, Anopheles fluviatilis. The presence of An. minimus prompted the performance of four more surveys during 2003–2004, one in the summer (March–April 2003), one in the rainy season (August–September 2003) and two in the cold season (October 2003 and February 2004). Surveys were made in five hilltop villages, viz. Doyonala, Dhanakunia Sahi, Lohanda, Mundasahi and Ulupuri, randomly selected from the Banspal, Basudevpur and Joda PHC areas, respectively, where a high incidence of malaria was recorded during 1999–2001. Day time indoor resting adult anophelines were collected using aspirators and torch lights between 06:00 and 07:00 h in nine human dwellings and three cattle sheds selected randomly in each village. A modified version of the CDC light trap (Gunasekaran et al., 1994) was installed in two human dwellings and two cattle sheds in a village (one trap in one structure) from 18:00 to 06:00 h. Man-landing collections were made between 18:00 and 21:00 h at three catching stations in each village. The anophelines collected were identified morphologically according to species and the species composition was recorded. Females of An. fluviatilis, An. minimus and Anopheles culicifacies were dissected for gut and gland infection with Plasmodium.
P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
2.3. Susceptibility test Susceptibility of An. minimus to DDT and deltamethrin (pyrethroid) was determined using WHO kits (WHO, 1975). Insecticide papers used for the tests were prepared at Vector Control Research Centre (VCRC) following standard procedures. Fully fed wild caught females were exposed to DDT (4%) and deltamethrin (0.05%) impregnated papers for 1 h in triplicates and mortality was recorded after 24 h of holding. Two controls were maintained simultaneously. Since no mortality was observed in controls, the treated mortality was not corrected. 2.4. DNA sequence In view of similarities and overlapping morphological characters occurring among the species of the An. minimus group causing considerable taxonomic controversies and erroneous reports on the distribution of An. minimus (Reid, 1968; Harrison, 1980), species identification of An. minimus was confirmed using a molecular method. A few specimens of An. minimus and the other anophelines species were kept in Eppendorf tubes, dried for 4–5 h at 90 ◦ C and sent to the VCRC laboratory for molecular identification. DNA was extracted from the samples, the ITS2 and the D3 regions of the ribosomal DNA were amplified (Manonmani et al., 2001; Sharpe et al., 1999) and sequenced. The ITS2 sequences of the samples were compared with the ITS2 sequences of An. minimus (Acc. Nos. AF194495–194506), An. fluviatilis (Acc. Nos. AF167298–167299), Anopheles aconitus or Anopheles varuna (Acc. Nos. AF194489–194494). 3. Results and discussion The collections comprised a total of 3858 anophelines belonging to 18 species, including An. fluviatilis (24.4%), An. culicifacies (6.3%) and An. minimus (3.5%). In total, 135 An. minimus were collected, 115 from hand catches in human dwellings and 20 from man-landing catches. While 98 were obtained during the rainy season (96 caught during the survey in 2001), 37 were collected during the cold season. In the summer survey, this species was not encounterd. Of the 100 An. fluviatilis dissected, one was found with Plasmodium sporozoites, but out of 24 An. minimus dissected,
33
none harboured sporozoites. Compared to the survey in 2001, vector densities were low during the subsequent surveys (2003–2004), probably due to intensive indoor residual spraying. An. minimus showed 100% (45/45) mortality when exposed to deltamethrin impregnated paper and 86% (18/21) mortality to DDT paper. According to WHO (1998) criterion, An. minimus is susceptible to both deltamethrin and DDT. DNA sequencing of four adult specimens showed that they were species A of the An. minimus complex. The D3 sequences were identical to those deposited by Chen et al. (2002) for the same species of this complex (Acc. No. AJ459420). An. minimus from Assam, India, incriminated with sporozoite rates ranging from 2.3 to 3.3% has also been identified as species A (Subbarao, 1998). In many countries, vectors that were believed to have been almost eliminated or having completely disappeared from areas brought under the expanded programme of residual spraying with DDT have reemerged with the cessation of house spraying (Roberts et al., 2002). In South Africa, An. funestus, a morphologically similar species to An. minimus disappeared in the 1950s with DDT spraying and 4 years after a switch over to pyrethroid spraying in 1990s, it reappeared and found to be pyrethroid resistant (Hargreaves et al., 2000). An. minimus, once the major malaria vector in Hainan Island and eliminated through indoor residual spray, has again been incriminated as a vector of local malaria outbreaks in recent years (Wu et al., 1993). In India, many changes have taken place in Anopheles species composition, distribution and importance as malaria vectors following the introduction of DDT spraying countrywide. An. minimus and An. sundaicus were notably affected (Kalra, 1991). Anopheles sundaicus was a major malaria vector in the eastern and western coasts of India and in the Andaman and Nicobar Islands. DDT spraying eliminated the species from the mainland and its distribution in India is now limited to the Andaman and Nicobar Islands. An. minimus, once a major malaria vector in Uttar PradeshTerai, Singhbhum hills and Jeypore hills and responsible for hyperendemic malaria, had been eliminated from these areas and its distribution narrowed to the North Eastern states. Similarly, diminution of the population was reported in Nepal and China (Rao, 1984; Sharma, 2002).
34
P. Jambulingam et al. / Acta Tropica 96 (2005) 31–35
Surveys conducted in Jeypore hill ranges and in the present study area in 1980s and 1990s did not record the presence of An. minimus (Dash et al., 1984; Collins et al., 1990; Gunasekaran et al., 1989). Besides DDT residual spraying, ecological changes due to extensive deforestation were considered responsible for the elimination of this species from these areas. The present study confirms the reappearance of An. minimus in Singhbum hills after a period of about 45 years after the launching of the malaria eradication programme. In this survey, An. minimus was collected from villages situated in hill ranges and surrounded by forest. Analysis of meteorological data recorded for the past 5 decades indicates no significant change of the climatic conditions in this region. However, comparison of recent data on forested areas in different districts of Orissa state shows that deforestation has been more extensive in the southern districts of Orissa (Jeypore hills) than in the Northern districts (Singhbhum hills). The Koraput district located in southern Orissa has a forest cover of only 16% of the total area while Keonjhar district still has 40% of the total area covered with forest (Anonymous, 1999). Overall, DDT residual spraying for malaria control in India declined in quantity as well as in the quality of applications in the 1990s, resulting in a resurgence of malaria in parts of the country (Sharma and Mehrotra, 1986). Therefore, it is clear that the environmental conditions in this region still favour or support An. minimus and once the pressure imposed by the large scale DDT residual spraying was withdrawn, the vector has started re-establishing its presence. Unlike An. funestus in South Africa, An. minimus is found to be highly susceptible to pyrethroids. Since malaria incidence in this area is showing an increasing trend in recent years, further studies are needed to understand the extent of distribution of An. minimus, its bionomics and the role in malaria transmission in this region.
Acknowledgements We thank Dr. P.K. Das, Director, Vector Control Research Centre, Pondicherry, for his keen interest and encouragement for this survey. The co-operation extended by the technical staff of VCRC field station, Malkangiri is gratefully acknowledged. We are grateful to the District Administration and the Health
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