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Anopheles culicifacies is the primary rural vector of malaria in India and ... Sites were selected on the basis as they represent the tribal belt along the streams of ...
International Journal of Malaria Research and Reviews www.resjournals.org/IJMR ISSN: 2346-7266 Vol. 1(5): 47-53, December, 2013

Characteristics of the larval breeding sites of Anopheles culicifacies sibling species in Madhya Pradesh, India *Tyagi Varun1, Sharma Ajay Kumar1, Yadav Ruchi1, Agrawal O.P.2, , Sukumaran Devanathan1 Vijay Veer3 1

Vector Management Division, Defence Research and Development Establishment (DRDE), Jhansi Road, Gwalior – 474002, M.P., India 2 School of Studies in Zoology, Jiwaji University, Gwalior – 474011, M.P., India 3 Defence Research Laboratory, Tezpur- 784001, Assam., India * E-mail Correspondence: [email protected], Tel. +917512390143, 8889636345 Abstract Malaria being the major public health problem in India and Madhya Pradesh constitute as one of the major state endemic to malaria. Anopheles culicifacies is the primary rural vector of malaria in India and is a complex of five morphologically indistinguishable sibling species A, B, C, D and E with varying biological characteristics. Mosquito breeding generally occurs in a wide range of habitats with different types of water. Selected physico- chemical characteristics of larval habitats of An. culicifacies were investigated in the present study. The samples were collected during August - October from different malaria endemic areas of Madhya Pradesh, India. An. culicifacies immature stages were found from 11 collection sites. The sibling species were identified using AS-PCR techniques. The sibling species A was found from Khandwa; B from Jabalpur, Chindwada; C from Hoshangabad only; D from Narsinghpur and Khandwa. Species A and D were found to breed in same site but different habitat. It was observed that maximum numbers of larvae were found in irrigation tanks and river side water bodies. Water parameters of larval habitats as temperature, dissolved O2, pH, conductivity, total dissolved solids, alkalinity, ammonia nitrogen, nitrate nitrogen, calcium, chloride, magnesium and total phosphate were measured. Data was examined by using descriptive statistical analysis. This study provides information on mosquito ecology in relation to breeding habitats. Further research on seasonal variations in population with more detailed analysis of water chemistry is suggested. Key Words: Anopheles culicifacies, Sibling species, Physico- chemical characteristics, Larval habitats

INTRODUCTION Anopheles culicifacies is the major malaria vector in India which contributes to the transmission of about 65% of the total malaria cases in India (Subbarao, 1988; Sharma, 1998). It is a complex of five isomorphic species which are provisionally designated as species A, B, C, D and E (Green et al., 1980) with varying biological characteristics. Irrigation tanks, wells and streams are the major breeding habitats for An. culicifacies (Tiwari et al., 2001). Madhya Pradesh (M. P.) along with other states like Orrisa, Jharkhand, Chhattisgarh and West Bengal contributes more than 60% of reported (confirmed) malaria cases in India. Madhya Pradesh is situated in the central part of India with an area of 308 thousand km 2 of which forest cover 76,429 km 2 (about 25% of the total land area). Malaria is complex in M.P. because of vast tracts of forest with tribal settlement (20% of state population) (Anon 1997–2006, Singh et al., 2004). The reasons for such a high diseases prevalence in M. P. is mainly due to locations of the villages in the deep forest

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Table 1. List of collection sites for sibling species of An. Culicifacies mosquito along with their longitude and latitude Locality Jabalpur Chindwada Hoshangabad Narsinghpur Khandwa

Code JBP CHW HSB NSP KNW

Longitude and Latitude 0 0 (23 10’ N, 79 59’ E) 0 0 (22 03’ N, 78 59’ E) 0 (22 46’ N, 770 45’ E) 0 0 (22 57’ N, 79 15’ E) 0 (21 49’ N, 760 22’ E)

and is characterized by rocky undulation interspersed with ravines and foothills. Another reason is the innumerable streams which flow into the river, Narmada. These streams flow continuously and provide ample breeding sites covered with dense aquatic vegetation for production of number of anophelines particularly An. culicifacies (Neeru Singh, 2006). Various chemical properties of the larval habitat related to vegetation, ranging from pH, optimum temperature, concentration of ammonia, nitrate and sulphate have been found to affect larval development and survival (Mutero et al., 2004). Presence of different vector species and their population accelerate the disease transmission. Therefore bioecological studies are helpful in order to locate risk areas for malaria vectors. Very few studies to date have investigated breeding water quality parameters in relation to sibling species occurrence and abundance. The physical and chemical nature of the water probably determines the selection of breeding sites. The present study investigated the occurance of immature stages of An. culicifacies sibling species and their association with water parameters.

MATERIALS AND METHODS Study area Sites were selected on the basis as they represent the tribal belt along the streams of Narmada River and also show high incidence of malaria (Sharma, 2012; Singh, 2006 and 2009) including - Jabalpur (Barela, Bijora village, Bargi Dam), Chindwada (Chikhla village), Hoshangabad (Padav village), Narsinghpur (Chinki, Bhalpani, Sherpipariya, Gotegaon villages) and Khandwa (Narmada Nagar and Chikdhaliya) (Table. 1, Figure 1). Larval sampling Larvae samples were collected from selected breeding sites in the different localities of Madhya Pradesh region from where malaria cases are reported. Larvae were collected with the help of dippers (cap. 250 ml). Ten dips per sq m of breeding habitat surface area were taken. Identification of mosquito Collected larval samples were reared in laboratory up to adult stage in wooden cages (30 x 30 x 30 inches) and were provided cotton soaked with 10% sugar solution. An. culicifacies mosquitoes were separated on the basis of their morphology and sitting posture and identified by using standard keys (Christopher, 1933; Wattal and Kalra, 1961; Das et al. 1990; Nagpal and Sharma 1995). Each representative sample was pinned as a voucher specimen and kept in laboratory as a reference collection. The adults emerged from identified samples (An. culicifacies) were used for further identification of sibling species using allele specific polymerase chain reaction (AS-PCR) (Goswami, et al., 2006).

Water Sampling and water quality parameters Water samples were collected in plastic containers. Selected positive samples for An. culicifacies were evaluated for water quality parameters. All parameters were tested on site at the time of collection. Temperature, pH, conductivity, total dissolved solids (TDS), and dissolved oxygen (DO) were measured using the M90 multi-probe meter (MettlerToledo AG, CH 8603, Schwerzenbach, Switzerland). Alkalinity, ammonia nitrogen, nitrate nitrogen, calcium, magnesium, ferrous iron, phosphate and turbidity were measured using LaMotte water analysis test kit.

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Figure 1. Collection of Larvae of An. culicifacies from Different Field Location of Madhya Pradesh, India

Analysis of Data Data obtained from analyzed water samples were computed by using Sigma Stats software. Descriptive analysis was done to calculate the mean values of water parameters. To establish the association between mosquito abundance and individual physico- chemical parameters, Spearman Correlation analysis was done. RESULTS Total 12 samples from 11 sites were found to be positive for Anopheles culicifacies larvae (Table 2). Species A in irrigation tank and D in rain water pond were found in Chikdhaliya villege, Khandwa. Total 4 samples of species B were collected from Jabalpur (3 samples) and Chhindwada (1 sample). C was found in temporary pool of rain water from Hoshangabad district only, whereas maximum number of larvae of species D was collected from Narsinghpur and Khandwa district. Species B and species D were found to be most abundant as compared to other species (D> B> C> A), among all the samples collected (Figure 2). It was observed during survey that large number of breeding sites with high larval density were found in irrigation tanks and around the river side (Figure 3). All the positive samples were analyzed for physico-chemical parameters. Mean values of tested water quality parameters are given in Table 3. Spearman correlation analysis between the vector abundance and 12 parameters of larval water showed that An. culicifacies was strongly and positively associated with Dissolved Oxygen (DO; r = 0.618, p < 0.05). Maximum occurance of larvae was also associated with pH range 6-7 (pH; r = -0.49, p < 0.05).

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Table 2. Larval density of An. culicifacies collected from breeding sites Locality (District) Barela (Jabalpur) Bijora (Jabalpur) Bargi Dam (Jabalpur) Chikhla (Chindwada) Padav (Hoshangabad) Chinki (Narsinghpur) Bhalpani (Narsinghpur) Sherpipariya (Narsinghpur) Gotegaon (Narsinghpur) Narmada Nagar (Khandwa) Chikdhaliya (Khandwa)

Type of breeding habitat Irrigation Tank Rock pool Riverbed pool Irrigation Tank Temporary pool River Margins Riverbed pool Rock pool Temporary pool Irrigation Tank Rain water Collection Irrigation tank

Larval density (10 dips/m2) 38 62 75 53 39 81 56 43 54 80 67 15

Species found B B B B C D D D D D D A

Figure 2. Percent larval density of An. culicifacies sibling species collected from different sites

DISCUSSION Water is an important component of ecosystem and its quality in the breeding site is an important determinant of whether or not the female mosquitoes will lay their eggs and the resulting immature stages will successfully complete their development to the adult stage (Piyaratne et al., 2005). Results of study revealed that An. culicifacies mosquito breeds in wide range of habitats. Irrigation tanks and river side water bodies were identified as potential habitats for the development of An.culicifacies. Tiwari et al. (2001) investigated 50 villages of Arsikere Taluk, district Hassan, Karnataka and reported that irrigation tanks, wells and streams are the major breeding habitats for An. culicifacies. It had earlier been reported that the population of An. culicifacies of Shri Lanka is very sensitive to minute qualitative change in water, as it prefers to breed in clear, open stagnant water pools (Carter, 1930). In the present investigation species B and D were found to be most abundant than A and C. The density of the sibling species and their distribution pattern at a given time is depends upon several biological characteristics viz. vectorial competence, seasonal prevalence (Subbarao et al., 1987), response to insecticides (Raghavendra et al., 1992) and host preferences (Joshi et al., 1988). In riverine villages of Gujarat, high density of An. culicifacies has been observed from January to April and it remains moderate to low for rest of the year (Bhatt et al., 1991).

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Table 3. Range of measured physico-chemical parameters of the larval breeding sites (n=12) Analyzed Parameters o Temperature( C) Dissolved oxygen (ppm) pH Conductivity (us/cm) Total dissolved solids (mg/l) Alkalinity (ppm) Ammonia nitrogen (ppm) Nitrate nitrogen (ppm) Calcium (ppm) Magnesium (ppm) Chloride (ppm) Total phosphate (ppm)

Minimum 27.0 3.90 6.17 120.0 1.10 72.0 0.0 0.01 50.0 338.8 64.0 0.02

Maximum 31.50 7.10 8.80 748.0 4.86 653.0 16.30 0.86 1070 3816.2 349.0 3.0

Mean 28.51 5.49 7.61 423.89 2.64 247.60 3.840 0.20 289.73 1195.2 175.94 0.88

Standered Error 0.41 0.38 0.26 79.76 0.37 53.44 1.63 0.08 94.68 329.4 30.99 0.32

Figure 3. Distribution of An. culicifacies larvae in different water bodies

In this present study the highest density of larval samples were collected from Jabalpur and Narsinghpur districts. These localities are situated in close proximity of riverine zones and with human settlements may be the reason of highest larval density. An. culicifacies preffered irrigation tanks, rock pools, temporary pools and rain water collections for breeding, since these water bodies contain suitable physico-chemical properties such as calcium, magnesium sulphate, nitrate, phosphate and dissolved solids as nutrient compositions. Various other chemical properties of the larval habitat in relation to vegetation, optimum pH and temperature, concentration of ammonia, nitrate and sulphate have been reported to affect larval development and survival (Pal, 1945; Mutero et al., 2004). In the present investigation it was observed that An. culicifacies abundance was significantly associated with dissolved oxygen and pH value. Maximum occurance of larvae was found from the water body of higher OD value and pH value in the range of 6-7. Geller et al. (2000) reported that under laboratory conditions, An. gambiae carries out normal development when pH varies as much as from 4.0 to 7.8 as long as there is sufficient phytoplankton and zooplankton for it to consume. Still and clear water with suitable pH, temperature and nutrient composition has been found to encourage breeding in Anopheles species (Okorie et al., 1978; Okogun, 2005). This study did not find any significant association with other water parameters. An. culicifacies s.l. naturally breeds in clear water (Mehta, 1934) and when given an opportunity to select waters with

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different amounts of free ammonia and ammonium carbonate, lays eggs indiscriminately, even in water containing 6.6 ppm of saline ammonia (Barik et al. 2009). Concentration of salinity of water up to 12 ppm does not affect the larvae of An. culicifacies s.l. (Rao, 1984). The result suggests that abundance of An. culicifacies complex is associated with various environmental and physicochemical parameters of breeding sites. The study producing some information which is useful for the development and evaluation of effective malaria control strategies and vector surveillance programmes in Madhya Pradesh. All the five sibling species of An. culicifacies are found in India, and the work on the water quality parameters in relation to sibling species occurrence and abundance is lacking. Studies are needed to explore potential differences in habitat requirements. ACKNOWLEDGEMENT The authors are thankful to Prof. (Dr.) M. P. Kaushik, Outstanding Scientist and Director, DRDE, Gwalior, Madhya Pradesh, India for interest and providing all necessary facility to conduct this research work. Sincere thanks also due to the scientists and supportive staff of Vector Management Division for their kind cooperation for carrying out the above work. REFERENCES Anon: State wise malaria situation during 1997-2006. National Vector Borne Disease Control Programme. 22- Sham Nath Marg Delhi-110054. Barik TK., Sahu B and Swain V (2009). A review on Anopheles culicifacies: from bionomics to control with special reference to Indian subcontinent. Acta Trop., 109: 87-97. Bhatt RM., Sharma RC., Gautam AS and Gupta, DK (1991). Seasonal prevalence of anophelines in Kheda district, Gujarat. Indian J. Malariol., 28: 9–18. Carter HF (1930). Further observations on the transmission of malaria by anopheline mosquitoes in Ceylon. Ceylon J. Sci., 2: 159–176. Christophers SR (1933) The fauna of British India including Ceylon and Burma, Diptera, Vol. IV. Family Culicidae, Tribe Anopheline, Taylor and Francis, London, p 371. Das BP (1990) Morphological difference between sibling species of An. culicifacies: A preliminary report. Mosquito – Borne diseases Bulletin., 7: 131-133. Geller N., Graczyk TK., Patz JA and Vittor AY (2000). Effects of environmental changes on emerging parasitic diseases. Int. J. Parasito., pp 1495-1405. Goswami G., Singh OP., Nanda N., Raghavendra K., Gakhar SK and Subbarao SK (2006). Identification of all members of the Anopheles culicifacies complex using allele-specific polymerase chain reaction assays. Am. J. Trop. Med. Hyg., 75: 454–460. Green CA and Miles SJ (1980). Chromosomal evidence for sibling species of the malaria vector Anopheles (Cellia) culicifacies Giles. J. Trop. Med. Hyg., 83: 75–78. Joshi H., Vasantha K., Subbarao SK and Sharma VP (1988). Host feeding patterns of Anophelese culicifacies species A and B. J. Am. Mosq Contr. Assoc., 4: 248–251. Mehta DR (1934). Effect of saline and free ammonia on the oviposition of Anopheles culicifacies and Anopheles subpictus (rossi). Rec. Mal. Surv. India., 4: 411–420. Mutero CM., Nga’ang’a PN., Wekoyela P., Githure J and Konradsen F (2004). Ammonium sulphate fertilizer increases larval populations of Anopheles arabiensis and culicine mosquitoes in rice fields. Acta. Trop., 89: 187-192. Nagpal BN and Sharma VP (1995). Indian Anophelines. Oxford andIBH Publishing Co. Pvt. Ltd., Mohan Primlani, N .Delhi, pp.1-416. Neeru Singh (2006) Tribal Malaria an Update on Changing Epidemiology. Proceedings of National symposium on Tribal Health, RMRCT, ICMR, Jabalpur, 19-20: pp 47-54. Okorie TG (1978). The breeding site preferences of mosquitoes in Ibadan, Nigeria. Niger. J. Entomol., 1: 71-80. Okogun GRA (2005). Life table Analysis of Anopheles malaria vectors: generational mortality as tool in mosquito vector abundance and control studies. J. Vector Borne Dis., 42: 45-53. Pal R (1945). On the bionomics of Anopheles culicifacies Giles. Part II. The ecology of immature stages. J. Mal. Inst. Ind. 6: 53–74. Piyaratne MK., Amerasinghea FP., Amerasinghea PH and Konradsen F (2005). Physico-chemical characteristics of Anopheles culicifacies and Anopheles varuna breeding water in a dry zone stream in Sri Lanka. J. Vector Borne Dis., 42: 61–67.

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