INTRODUCTION

Malaria is a major public health problem in DistrictRamgarh of Jharkhand state, India. The malaria ende-micity in this area is primarily due to prevalence of threemalaria vector species, viz. Anopheles culicifacies, An.fluviatilis and An. annularis1. In spite of well-known vec-torial status of these species in the district, no adequateinformation on ecological factors such as breeding andresting habitats as well as environmental factors such astemperature, humidity and rainfall directly affecting vec-tor population and disease transmission are available. Pre-vious studies suggest the predominant role of ecologicaland environmental conditions on availability of suitablebreeding sites, which play important role in malaria epi-demiology2–4. The land transformation due to deforesta-tion causes vast ecological changes in the area, whichdirectly affect breeding habitats of anophelines, their adult

population structure and density, and finally the dynam-ics of disease transmission5–7. The District Ramgarh hasalso been facing extensive deforestation due to exces-sive anthropogenic activities such as construction ofdams, roads, industry, coal mining and agricultural landdevelopment over the years, leading to the formation ofnew and diversified breeding habitats. As compared toadult anophelines, the immatures are relatively immo-bile and occupy small breeding area8–9, therefore, site-specific vector control strategies could be developed byunderstanding the vectors’ breeding ecology, their dis-tribution and seasonal prevalence.

The previous studies also suggest that rainfall pro-vides ample breeding sites for anophelines to lay theireggs and the temperature plays important role in larvaldevelopment and survival which takes about 10 days tometamorphosize from an egg to adult stage at an opti-mum temperature of 28°C10. In view of above, this study

*Authors made equal contribution.

J Vector Borne Dis 53, December 2016, pp. 327–334

Diversity of breeding habitats of anophelines (Diptera: Culicidae) inRamgarh district, Jharkhand, India

Siddharth Pandey1*, M.K. Das2* & Ramesh C. Dhiman3

1National Institute of Malaria Research, GECH Project site-Ranchi; 2National Institute of Malaria Research, Field Unit, Ranchi, Jharkhand;3National Institute of Malaria Research, New Delhi, India

ABSTRACT

Background & objectives: The Ramgarh district of Jharkhand state, India is highly malarious owing to abundanceof different malaria vector species, namely Anopheles culicifacies, An. fluviatilis and An. annularis. In spite ofhigh prevalence of malaria vectors in Ramgarh, their larval ecology and climatic conditions affecting malariadynamics have never been studied. Therefore, the objective of this study was to identify the diversity of potentialbreeding habitats and breeding preferences of anopheline vectors in the Ramgarh district.

Methods: Anopheles immature collection was carried out at potential aquatic habitats in Ramgarh and Gola sitesusing the standard dipper on fortnightly basis from August 2012 to July 2013. The immatures were reared tilladult emergence and further identified using standard keys. Temperature of outdoor and water bodies was recordedthrough temperature data loggers, and rainfall through standard rain gauges installed at each site.

Results: A total of 6495 immature specimens representing 17 Anopheles species including three malaria vectors,viz. An. culicifacies, An. fluviatilis and An. annularis were collected from 11 types of breeding habitats. Thehighly preferred breeding habitats of vector anophelines were river bed pools, rivulets, wells, ponds, river margins,ditches and irrigation channels. Larval abundance of vector species showed site-specific variation with temperatureand rainfall patterns throughout the year. The Shannon-Weiner diversity index ranged from 0.19 to 1.94 at Ramgarhsite and 0.16 to 1.76 at Gola site.

Interpretation & conclusion: The study revealed that malaria vector species have been adapted to breed in a widerange of water bodies. The regular monitoring of such specific vector breeding sites under changing ecologicaland environmental conditions will be useful in guiding larval control operations selectively for effective vector/malaria control.

Keywords Anophelines; breeding habitats; Jharkhand; larvae; malaria; Ramgarh; temperature

J Vector Borne Dis 53, December 2016328

was conducted in District Ramgarh to investigate thediversity of breeding habitats, composition of malariavector and non-vector species therein and most preferredbreeding habitats for vector species. The effect ofrainfall and ambient and water body temperature wasalso studied to know their effect on density of anophelinelarvae in different breeding habitats to formulateeffective larval control strategy for curbing malariatransmission.

MATERIAL & METHODS

Study areaThe District Ramgarh (latitude 23°38', longitude

85°34' in Jharkhand, India covers an area of 1360.08 km2,including 487.93 km2 forest area with 949,159 population(Census 2011). The survey of immature anophelines wascarried out during August 2012 to July 2013 in two pri-mary health centres (PHCs), viz. Ramgarh and Gola withlow and high malaria incidence, respectively. In each PHC,four villages were selected for conducting the study. Thegeographical coordinates of each village along with its as-sociated aquatic habitats were recorded using global po-sitioning system. The study villages are situated in the for-ested terrain and characterized with both permanent andtemporary mosquito breeding habitats such as perennialstreams, rivers, rivulets, ponds, wells, large drains etc.congenial for mosquitoes breeding.

Larval collection, storage and identificationImmature anophelines were sampled from water bod-

ies in the vicinity of 1 km of each village in morninghours between 0900 and 1100 hrs using standard dippers(250 ml capacity) on fortnightly basis as per the WHO(1975) manual on practical entomology in malaria11. Col-lection of anopheline larvae and pupae was carried outfrom different breeding habitats (Fig. 1), i.e. ponds, pools,rivulets, river margins, river bed pools, ditches, pits, wells,irrigation channels, rice-fields and stray fields. About2–20 dips were taken from each breeding habitat depend-ing upon to its size. The sampled larvae and pupae weretransferred to plastic containers (500 ml capacity) usinga pipette. All the containers were properly labeled,brought to laboratory, kept at room temperature (25°C)with the help of thermostat for rearing till adult emer-gence and further identified to species using standardkeys12–17. Larval densities were calculated as the propor-tion of total number of larvae and pupae per dip for eachbreeding site.

Larval/pupal density =

Meteorological data collectionTo record daily ambient air temperature (since

August 2012 to July 2013) at every four hour interval,HOBO data loggers (Australia) were installed inStevenson screens (H.L. Scientific Industries, Ambala)fixed in open space at each site. The four-hourly tem-perature of aquatic habitat was recorded using water tem-perature data loggers by submerging the device in an iden-tified breeding habitat found positive for mosquito larvalbreeding site. The four-hourly generated temperature datawere computed to daily and monthly maximum, mini-mum, and average temperature using MsExcel 2010. Toobserve the spatial variation in rainfall, one rain gauge(tipping bucket) was installed at each site. The rainfallwas recorded on daily basis.

Statistical analysisTo assess anophelines community structure in each

breeding habitat, the Shannon diversity index (H') andspecies evenness (E) were measured. Shannon diversityis the quantitative measure of species diversity in a com-munity which takes into account the species abundanceand evenness of the species present in a habitat. How-ever, evenness is the measure of species closeness in termsof their number in the habitat, which is expressed be-tween 0 and 1 being 0 as complete dominance and 1 ascomplete evenness of the species.

Relative density (D), Shannon index and evennessof anopheline species were calculated using MsExcel2010. Relative density of each anopheline species wasexpressed as its percentage in the whole sample18 as:

Where, D is relative density, I is number of individu-

Fig. 1. Different breeding habitats (from left to right — River, pond,ditch, pond & well, rivulet, and river) of anopheline speciesat Ramgarh district, Jharkhand.

Pandey et al: Anophelines breeding habitats in Jharkhand India 329

als of a species, and N is total number of species. Basedon values of D, each anopheline species was categorizedas satellite (D <1%), subdominant (1 <D <5%) and domi-nant (D >5%) species19. The diversity of anophelinespecies was calculated as Shannon-Weiner diversityindex (H') following Shannon method20 :

Where, s is the number of species in the sample, pi isrelative abundance of ith species (ni/N), N is total num-ber of individuals of all kinds, ni is number of individu-als of ith species and ln is natural log. The species even-ness (E)20 swas calculated as:

Where, H' is Shannon-Weiner diversity index, ln (S)is the natural logarithm of species richness. Species rich-ness is the total number of species present in the area.

RESULTS

Anopheline larval surveillanceThe anopheline larval abundance varied spatially and

temporally at Ramgarh and Gola sites. A total of 6495immature anophelines were collected from 11 types ofbreeding habitats during the study period (Table 1). Oftotal immatures collected, 17 Anopheles species, viz. An.culicifacies, An. fluviatilis, An. annularis, An. pallidus,An. theobaldi, An. vagus, An. subpictus, An. nigerrimus,An. sergentii, An. splendidus, An. barbirostris, An.tessellatus, An. jamesii, An. maculatus, An. jeyporiensis,An. crawfordi and An. aconitus were identified (Tables 2and 3). Among these, An. culicifacies, An. fluviatilis andAn. annularis have already been reported as malaria vec-tor species in this region1. The categorization ofanopheline species on the basis of relative density [asdominant (>5%), subdominant (<5 and >1%) and satel-lite species (<1%)] revealed that An. culicifacies (32.7%)was most dominant species at Ramgarh site followed byAn. fluviatilis (25.1%), An. annularis (12.8%), An.subpictus (8.4%), An. theobaldi (5.5%), An. splendidus(5.3%), while An. barbirostris, An. crawfordi, An.pallidus and An. vagus belonged to subdominant species(Table 4). Under satellite category, An. nigerrimus, An.sergentii, An. tessellatus, An. jamesii, An. maculatus andAn. jeyporiensis were identified.

However, at Gola site, An. annularis (26.4%)emerged as most dominant species followed by An.fluviatilis (19.8%), An. subpictus (15.4%), An.

culicifacies (15%), An. splendidus (7.8%) andAn. crawfordi (5.2%) (Table 4). Likewise, An. pallidus,An. barbirostris, An. jeyporiensis and An. vagus werecategorized as subdominant species and An. theobaldi,An. jamesii, An. sergentii and An. aconitus as satellitespecies.

Anopheline larval ecology/Breeding habitatsDuring the survey, both permanent and temporary

breeding habitats were identified in the study area. A to-tal of 11 types of predominant breeding habitats, viz.ponds, river bed pools, rice-fields, rivulets, river mar-gins, stray fields, borrow pits, ditches, irrigation chan-nels, wells and seepages were recorded from bothRamgarh and Gola sites (Tables 2 and 3). At Ramgarhsite, the permanent water bodies, i.e. river bed pools(RBP) (28%), rivulets (22%), ponds (14%) and wells(11%) were main breeding source suggesting that RBPsand rivulets were highly productive for anopheline mos-quitoes. However, temporary breeding habitats, i.e. strayfields (7%) and rice-fields (7%) were also found withanopheline breeding.

Likewise, at Gola, both permanent and temporary

Table 1. Total number of anopheline immatures collected fromdifferent breeding habitats and died during rearing from Ramgarh

and Gola sites from August 2012 to July 2013

Breeding habitats No. of larvae and No. of larvaepupae collected and pupae died

RamgarhRiver bed pools 952 537Rivulets 762 394Ponds 479 228Wells 375 166Stray fields 225 107Rice-fields 221 148River margins 142 78Ditches 133 72Irrigation channels 76 30Burrow pits 31 29

Total 3396 1789

GolaPonds 1383 645Ditches 422 245RBPs 380 182Stray fields 342 163Wells 250 136Rice-fields 203 142Irrigation channels 64 30River margins 34 9Seepages 21 9

Total 3099 1561

J Vector Borne Dis 53, December 2016330Ta

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13 (7

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53 (5

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2 (1

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(13.

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(26.

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(3.6

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(7.1

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(0.6

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(11.

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(0.9

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4 (3

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1 (0

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00

00

10 (1

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An. s

ubpi

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20 (5

7.1)

36 (3

2.7)

51 (1

1.3)

31 (2

7.9)

02

(3.3

)0

2 (1

3.3)

014

2 (1

5.4)

An. s

erge

ntii

00

00

01

(1.6

)0

00

1 (0

.1)

An. s

plen

didu

s0

25 (2

2.7)

14 (3

.1)

14 (1

2.6)

9 (7

.8)

1 (1

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00

9 (1

00)

72 (7

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An. b

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ris

00

12 (2

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01

(0.9

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(16.

4)0

00

23 (2

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An. j

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1 (0

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02

(1.8

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00

00

3 (0

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An. j

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sis

03

(2.7

)13

(2.9

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00

00

016

(1.7

)An

. cra

wfo

rdi

02

(1.8

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(5.3

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(0.9

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(0.9

)9

(14.

8)11

(61.

1)0

048

(5.2

)An

. aco

nitu

s0

00

1 (0

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00

00

01

(0.1

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Tota

l35

110

450

111

115

6118

159

924

Valu

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par

enth

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com

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Pandey et al: Anophelines breeding habitats in Jharkhand India 331

breeding habitats were recorded for anopheline species.The maximum anopheline producing habitats were ponds(45%) followed by ditches (14%), RBP (12%), stray fields(11%) and wells (8%) (Table 3), indicating pond as ma-jor contributor to anopheline prevalence at this site.

Malaria vectors’ breeding preferencesThe results revealed that malaria vector species are

adapted to breed in a wide range of water bodies. Basedon their emergence, the highly preferred seven breedinghabitats of An. culicifacies, An. fluviatilis and An.annularis were RBPs, rivulets, wells, ponds, rivermargins, ditches and irrigation channels (Tables 2 and3). At Ramgarh site, An. culicifacies showed maximumpreference for RBPs (68.3%) followed by rivulets(38.6%), rice-fields (23.8%), wells (17.8%) and rivermargins (14.6%) for its breeding (Table 2), whereas, An.fluviatilis maximally preferred river margins (70.7%)followed by wells (57.6%) and irrigation channels (50%)as its ideal breeding site. The other breeding habitats, i.e.rivulets, RBPs, ponds and stray fields were alsoassociated with An. fluviatilis breeding. In contrast,An. annularis showed its highest preference for ponds(35.4%) followed by ditches (31.4%) and other habitats(Table 2).

At Gola site, An. culicifacies preferred mostly twobreeding habitats, i.e. RBPs (67%) and river margins

(40%) (Table 3). The other habitats were less preferred.However, An. fluviatilis mostly preferred wells (52.5%)followed by river margins (46.7%), irrigation channels(38.9%) and ditches (29.7%) (Table 3). Anophelesannularis immatures were more commonly found inponds (44.9%) than other breeding habitats (Table 3).

The highest H' in stray fields (1.94) at Ramgarh sitedepicted highest anopheline diversity (Table 5). Com-paratively, other breeding habitats, i.e. ponds (1.89), rivu-lets (1.7), irrigation channels (1.61), and rice-fields (1.6)exhibited less anopheline diversity. The species E in strayfields (0.49), irrigation channels (0.48), rice-fields andditches (0.43) (Table 5) indicated that the individuals inthe community are not distributed more equitably amongthese habitats. However, E values near 0 for RBP (0.19)and wells (0.29) clearly indicated the dominance of someanopheline species and their unequal proportion inrespective habitats. At Gola, the Shannon diversityindex (H') was highest in stray fields (1.76), ponds (1.75)and ditches (1.7) (Table 5). The individuals of anophelinespecies in the community were distributed less equitablyamong these habitats. There was high dominance of fewanophelines over other species in irrigation channels(0.16), RBPs (0.24) and ponds (0.29) (Table 5).

Meteorological data revealed that average daily airand water body temperatures were highly fluctuating ateach sites of Ramgarh and Gola.The daily outdoor tem-

Table 5. Species diversity index and evenness at Ramgarh and Gola study sites during August 2012 to July 2013

Rice- Stray Ditches RBPs Wells Ponds Irrigation River Rivulet/fields fields channels margins Seepages

RamgarhShannon diversity index (H') 1.6 1.94 1.52 1.08 1.37 1.89 1.61 0.96 1.7Evenness (E) 0.43 0.49 0.43 0.19 0.29 0.37 0.48 0.26 0.31

GolaShannon diversity index (H') 1.14 1.76 1.7 1.14 1.48 1.75 0.67 0.99 0+

Evenness (E) 0.32 0.37 0.36 0.24 0.31 0.29 0.16 0.34 0+

Table 4. Anopheline species categorization based on their relative density (%) at Ramgarh and Gola sites during August 2012 to July 2013

RamgarhRelative density 32.66 25.13 12.76 1.51 5.53 1.21 8.44 0.20 0.50 5.33 2.41 0.30 0.10 0.80 0.70 2.41 0Density class D D D SD D SD D S S D SD S S S S SD S

GolaRelative density 15.04 19.81 26.41 3.90 0.65 1.08 15.37 0 0.11 7.79 2.49 0 0.32 0 1.73 5.19 0.11Density class D D D SD S SD D S S D SD S S S SD D S

D—Dominant, SD—Sub-dominant, S—Satellite species.

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J Vector Borne Dis 53, December 2016332

perature of Ramgarh site ranged from 11.75–37.07°C andthe water body temperature ranged from 14.29–30.7°Cduring August 2012 to July 2013 (Fig. 2a). The averagedaily outdoor temperature was lower than average dailywater body temperature from October to February monthsand higher from March to July months. The maximumoutdoor temperature was 6.37°C higher and the minimumoutdoor temperature was 2.54°C lower than water bodytemperature. However, at Gola site, the outdoor tempera-ture ranged from 12.87–36.5°C (Fig. 2b). Comparatively,the monthly outdoor temperature of Gola site was re-corded approximately 2°C higher than Ramgarh fromOctober to April months, the trend was reversed after-wards. Likewise, rainfall pattern also varied at each site(Figs 2a and b). There was substantial variation inmonthly rainfall in most of months. The maximummonthly rainfall was 470.8 mm at Ramgarh, whereas385.4 mm at Gola site in the month of August.

DISCUSSION

The survey of immature anopheline breeding habi-

tats at Ramgarh district, Jharkhand revealed that therewere ample and conducive sites for anopheline mosqui-toes to breed and survive throughout the year. The riverbed pools and rivulets with a constant flow of water wereimportant vector breeding habitats at both Ramgarh andGola sites. Similar observations have been reported byKusumawathie et al21 for anopheline breeding. Inaddition, the role of ponds, wells, rice-fields, ditches, strayfields with stagnant water conditions22–23 can not be ig-nored in anopheline larval productivity as those contrib-uted significantly to anopheline breeding.

During this study, An. culicifacies, An. fluviatilis andAn. annularis, the known malaria vectors of DistrictRamgarh, were identified in wide range of breeding habi-tats. At Ramgarh site, An. culicifacies, the most domi-nant species showed highest breeding preference for RBPand rivulets, which might have served predominant ovi-position sites for this species. Similar findings were alsorecorded in earlier studies21–23. Besides, irrigation chan-nels, wells, seepages, pits, ditches, rice-fields have alsobeen observed as preferred breeding habitats of An.culicifacies24–25. Though, An. fluviatilis was earlier con-

Fig.2: Average daily outdoor temperature (°C), aquatic habitat temperature (°C) and rainfall (mm) at Ramgarh (a), and Gola (b) study sitesduring August 2012 to July 2013.

Pandey et al: Anophelines breeding habitats in Jharkhand India 333

sidered to breed only in clean and slow flowing water, inthe present study it preferred wells and rivulets whichindicated its adaptation to changing climatic conditions.Earlier studies also reported its occurrence in slowsflowing streams, river margins, irrigation channels26,wells and thick vegetation27 as observed in the presentstudy. It was observed that An. annularis breed in alltype of habitats; however, its major breeding was recordedin ponds, rivulets, and ditches associated with algalgrowth and grassy margins. Similar findings were re-corded by Yadav et al28 and Sahu et al29. The ability ofAn. culicifacies, An. fluviatilis and An. annularis to breedexcessively in RBPs, wells and ponds, respectively posesa particular problem for this region. Increased breedingof anopheline vectors in such local water bodies may in-fluence malaria transmission in the region. Therefore,critical surveillance and subsequent management ofbreeding habitats is prerequisite for effective vector con-trol in this area.

Meteorological data of Ramgarh and Gola sites re-vealed temperature variation in the outdoor condition inspite of being located in the same district, which mightdelay or fasten the development of immature anophelinesinto adults. Earlier studies also suggested the higher wa-ter body temperature in typical mosquito breeding sitesthan that of atmospheric air, with a general difference of4°C30, as observed in present study. Therefore, higherwater temperature might shorten the larval developmenttime, and hence increase population growth rate31. In ad-dition, rainfall plays an important role in increasing tem-porary breeding habitats, which provides the medium fordevelopment of aquatic stages of anophelines, influenc-ing vector population and malaria prevalence32. Previ-ous studies also suggested that anopheline population dy-namics are not directly influenced by rainfall, but by thelevel of the stable reservoirs, which offer a constant op-portunity for female anophelines to lay their eggs33.

CONCLUSION

The study revealed that a vast number of productiveAnopheles breeding sites in the area of Ramgarhdistrict, Jharkhand state, India are responsible for greaterabundance of malaria vectors, which have been adaptedto wide range of breeding habitats under changingecological and meteorological conditions. The river bedpools, river margins, wells and ponds were the majorbreeding sites for anopheline vector species in thisregion. Besides ecological conditions, the prevailingoutdoor temperature, water body temperature and rain-fall also affected larval density. Therefore, the selective

larval control methods should also be considered inthis area.

ACKNOWLEDGEMENTS

The authors acknowledge the Director, NationalInstitute of Malaria Research, New Delhi, for providingopportunity to carry out this study and Indian Council ofMedical Research, New Delhi for providing financialsupport through ‘Global environmental change and healthtask force project’. The authors also thank field and labo-ratory staff, Messrs Saroj Kumar Das and Chandan Guptafor their assistance during the course of study.

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Correspondence to: Dr R.C. Dhiman, Scientist ‘G’, National Institute of Malaria Research (ICMR), Sector 8, Dwarka, New Delhi–110 077,India.E-mail: r.c.dhiman@gmail.com

Received: 26 February 2016 Accepted in revised form: 5 September 2016