seasonal distribution, parity, resting, host-seeking behavior and association of malarial parasites...

RESEARCH PAPER

Seasonal distribution, parity, resting, host-seekingbehavior and association of malarial parasites ofAnopheles stephensi Liston in Kolkata, West BengalAnupam GHOSH1, Samir MANDAL2 and Goutam CHANDRA2

1 Department of Zoology, Bankura Christian College, Bankura, West Bengal, India2 Mosquito and Microbiology Research Unit, Department of Zoology, Burdwan University, West Bengal, India

Correspondence

Anupam Ghosh, Department of Zoology,Bankura Christian College, Bankura, WestBengal, India.Email: [email protected]

Received 15 September 2009;accepted 3 November 2009.

doi: 10.1111/j.1748-5967.2009.00262.x

Abstract

Indoor resting and human-landing mosquito collections were conducted at selectedlocalities in Kolkata, India to determine resting and host-seeking behavior, nightbiting activity, seasonal distributions and malaria infection rates. During a two-yearstudy (2006–2007), 5123 and 1716 female mosquitoes were captured in indoor-resting and human-landing collections, respectively, from two types of residences(brick built rooms, temporary huts). Regression analysis demonstrated that theabundance of indoor resting An. stephensi was positively correlated with ambienttemperature and relative humidity. The average duration of the gonotrophic cyclefor laboratory-reared An. stephensi was about 4 days. Average proportion of parousAn. stephensi, daily survival and daily mortality rates were 46%, 82% and 18%,respectively. Plasmodium vivax sporozoite infections were detected in the salivaryglands of two wild-caught An. stephensi (sporozoite rate 2.2%) and one An.annularis (sporozoite rate 1.5%). No P. falciparum infections were detected.Oocyst infections were observed in one An. annularis mosquito (oocyst rate 1.5%).

Key words: Anopheles stephensi, host-seeking behavior, Kolkata, resting behavior,seasonal abundance.

Introduction

Malaria is a serious scourge on humanity, causing significanthuman mortality and morbidity along with great financiallosses. Every year about 300–500 million people are esti-mated to be affected by malaria. Malaria further threatens2400 million people (about 40% of the world’s population)and kills nearly three million people annually (WHO 2005;Yadav et al. 2007).

Malaria is the most important cause of morbidity andmortality in India with approximately 2–3 million new casesannually and 658 deaths recorded in 1998. According to thereport of the Planning Commission, 1999–2000 and theNational Anti Malaria Programme, Government of India, atotal of 2 452 653 (P. vivax 1 404 737, P. falciparum1 047 916) malaria cases were reported in India in 2000

(Sharma 2003). The high numbers of malaria cases in Indiaare, in part, the result of insecticide resistance, pronouncedexophilic vector behavior, malaria drug resistance andunder-funded medical and vector control resources, in addi-tion to poverty and poor housing. Urban and peri-urbanmalaria poses a significant public health problem in manysmall villages and large metropolitan cities. The principalvector of urban malaria in India is Anopheles stephensiListon, first incriminated as a malaria vector in Kolkata in1943 (Siddons 1943). Urban malaria is largely attributed tolow economic thresholds that have resulted in the rapid andhaphazard expansion of cities, breakdown in municipal rulesand regulations (particularly building codes), lack of exten-sion of health services into peri-urban areas, inadequatepiped water supplies, storage of water in cisterns and openoverhead tanks, disuse or scarce use of wells, aggregation of

Entomological Research 40 (2010) 46–54

© 2010 The AuthorsJournal compilation © 2010 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

mailto:[email protected]

migrant labor force, inadequate community participation,and population movement (Kumar 1997; Singh et al. 2004).

Before combating malaria in a locality, it is essential toidentify the ecological parameters that affect longevity andpopulation dynamics of vector mosquitoes (Samuel 1926;Hati 1979). Entomological surveillance is used to identifychanges in the geographical and seasonal distributions, iden-tify density of vectors, evaluate vector control programs andfacilitate appropriate and timely decisions regarding inter-ventions. These data also may serve to identify areas of highdensity or periods of vector population increases (Elkhalifaet al. 2006). Thus, assessment of the vector density anddiversity of anopheline mosquito surveillance providesessential information for monitoring the potential formalaria transmission in Kolkata, as well as formulatingvector control strategies. The purpose of this study was todetermine the resting and host-seeking behaviors (human–mosquito contact and preferential biting sites on humanbody) and seasonal prevalence of An. stephensi and othermosquitoes in an urban environment. The seasonalgonotrophic cycle of An. stephensi was assessed to deter-mine age composition and to determine the potential thatmalaria of An. stephensi can be used to develop, implementand evaluate local government malaria control programs.

Materials and methods

Study area

The present study was carried out in Kolkata, West Bengal,the largest metropolitan city of India, situated on the easternbank of the Hugli River, about 154 km (96 miles) upstreamfrom the Bay of Bengal (Fig. 1).

Collection of indoor resting mosquitoes

Indoor resting collections for all species of mosquitoes wereconducted from a total of 48 residences (24 brick builtrooms, 24 temporary huts or jhoopries) from eight localities(Sinthi, Sealdah, Kasba, College Street, Garia, Maniktala,Jadavpur, Rajarhat) in Kolkata from January 2006 toDecember 2007. Selected brick built rooms were one storywith concrete roofs, or tile sheds. Temporary huts wereconstructed of bamboo, wood, mattresses, corrugated tin,tiles, tarpaulins and asbestos. The areas were selected basedupon authenticated previous reports of malaria endemicity inKolkata. Resting mosquitoes were collected for two hoursfrom 05.00 to 07.00 hours as previously described by De andChandra (1994), WHO (1962, 1964), Holstein (1954) andHati (1986). Mosquitoes were collected for 10 min eachfrom six rooms (three brick built rooms, three jhoopries) fortwo of the eight collection sites, followed by weekly collec-tions at two additional sites until all eight sites were sur-

veyed, with a total of 48 rooms at eight localities surveyed assuggested by De and Chandra (1994). During the study, 192worker-hours were employed for the indoor mosquito col-lections. A 20-mL test-tube was brought perpendicularlyover the resting mosquito and the mouth of the tube closedwith a piece of cotton wool that was pushed towards thebottom of the tube with sufficient space provided to allowthe mosquito to move freely. This was repeated until therewere five or six mosquitoes in the long test tubes, eachisolated from the others by cotton wool. After each collec-tion, the mosquitoes were brought to the laboratory andfemale mosquitoes held separately in 30 cm ¥ 30 cm ¥30 cm cloth cages until they were immobilized with petro-leum ether and identified to species according to standardtaxonomic keys and catalogues (Christophers 1933; Nagpal& Sharma 1995). The temperature and relative humidity alsowere recorded during each collection.

Human landing collections

To determine the host-seeking behavior of An. stephensi,two brick built rooms and one temporary hut with twowindows and a door were selected and fixed in each of eightlocalities where indoor-resting collections were done (i.e. 24human shelters were selected in the study area). Humanlanding collections were conducted once a month fromJanuary 2006 to December 2007 (24 times), by placing thehuman bait in each shelter consecutively, using male volun-teers (aged 20–45 years) wearing only shorts (human bait) orfull covering (collectors). Indoor human bait was stationedin a room with two windows and a door, while outdoorhuman bait was located 45 m from the indoor collectionsites. During the study, the windows were kept open but thedoor remained closed. Hourly human landing mosquito col-lections were made by six collectors, two during each of the4-hr shifts using test tubes and flashlights (Chandra & Hati1993). The volunteers laid on cots 45 cm above the groundfor 12 h (18.00–06.00 h), one indoor and one outdoor whilethe collectors wore full-sleeve aprons, full pants, gloves anda hat to protect them from biting mosquitoes. Collectorswere rotated nightly to reduce collector bias. Mosquitoesthat landed on the volunteers were collected by placing a testtube over it, inserting a cotton plug into the mouth of thetest tube and pushing the mosquito towards the bottom ofthe tube as previously described for resting mosquitoes. Thecollected mosquitoes were divided into groups labeled“head” (for mosquitoes landing on volunteers’ head andneck), “body” (torso), “upper extremity” (hands, arms) or“lower extremity” (thighs, legs, feet), and stored in indi-vidual tubes with silica gel and later transferred to the sepa-rate cloth cages in the laboratory. The collected mosquitoeswere brought to the laboratory for identification and to deter-mine mosquito species composition and malaria (sporozoite

Anopheles stephensi in Kolkata

47Entomological Research 40 (2010) 46–54© 2010 The Authors. Journal compilation © 2010 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

Figure 1 City map of Kolkata.

A. Ghosh et al.

48 Entomological Research 40 (2010) 46–54© 2010 The Authors. Journal compilation © 2010 The Entomological Society of Korea and Blackwell Publishing Asia Pty Ltd

and oocyst) infection rates. In total 576 worker-hours wereemployed (288 indoor, 288 outdoors). For comparison, thenumber of collected mosquitoes was divided by the percent-age of total body surface area [head 9%, upper extremities18%, body 36%, lower extremities 36%, genitalia 1%] (Kyle& Smith 1984). Data were analyzed for each of the three-hour intervals as described by Chandra (1995).

Physiological age and gonotrophic

cycle determination

Age composition of An. stephensi mosquitoes was deter-mined on the basis of the number of ovariolar dilatations(Polovodova 1949). Ovaries of 70 An. stephensi mosqui-toes, which were collected during indoor-resting andlanding collections, were examined. Daily survival anddaily mortality rates were calculated as described byDavidson (1954) and Service (1976), respectively. Pre-sumptive mortality between two successive parous mosqui-toes was calculated by the formula of Gillies and Wilkes(1965). The gonotrophic cycles (time between successiveblood meals and oviposition) of 12 field-collected andlaboratory-reared An. stephensi mosquitoes were alsodetermined [four mosquitoes each in summer (temperaturerange 28.3–44.6°C), winter (11.2–25.4°C), rainy season(21.3–38.3°C)]. Determination of the gonotrophic cyclewas made by maintaining mosquitoes on a 10% glucosesolution provided on a saturated cotton pad in a Petri dishfor three days. On day four, the glucose solution wasremoved and on day five, the starved mosquitoes were pro-vided blood meals to engorgement on human volunteers,placed in a screened cage (30 cm ¥ 30 cm ¥ 30 cm) andprovided a 10% glucose solution. A 500-mL beaker filledto 2.5 cm from the top with water and a piece of rectan-gular filter paper (10 cm ¥ 4 cm) was placed on the innerwall of the beaker with a small piece of cork placed tofreely float on the water surface where females oviposited.Times between blood meal and oviposition were recordedand the duration of the gonotrophic cycle determinedaccording to WHO (1975).

Detection of malaria infections

All female Anopheles mosquitoes captured at indoor restingand human landing collections (604 An. subpictus, 161 An.annularis, 70 An. vagus, 202 An. barbirostris, 81 An.stephensi; except for 12 An. stephensi mosquitoes that wereused in physiological age determination) were dissected foroocysts and sporozoites in the salivary glands as describedby WHO (1975). Only malaria sporozoite infections wereidentified to species by enzyme-linked immunosorbentassay (ELISA) (Chatterjee & Chandra 2000).

Statistical analysis

A two-tailed Student’s t-test was performed during the quad-rant analysis and to compare the relative abundance of mos-quitoes for the two different habitats (brick built rooms,jhoopries). Correlations were done between quantitativevariables (e.g. atmospheric temperature, humidity) and thenumber of adult mosquitoes using conventional linear cor-relation; regression equations were estimated at P < 0.05significance level. A single-factor analysis of variance analy-sis (anova) was used to determine biting site preference orlocation-wise variation for human landing collections. Asingle-factor anova also was carried out to establish thedifference between various parous states of mosquitoes.

Results

During the two-year study, 5123 and 1716 female mosqui-toes belonging to 11 species and 5 genera were captured inresting and human landing collections, respectively, fromhuman habitations (jhoopries, brick built rooms) (Table 1).Culex quinquefasciatus Say (49.4%) was the most fre-quently collected species for indoor-resting collections,while An. vagus Donitz (0.5%) was the least frequentlycollected. In total 93 An. stephensi were collected [indoorresting 59 (1.2%), human landing 34 (2.0%)] (Table 1). Thetotal number of indoor resting mosquitoes were significantlydifferent in jhoopries than brick built rooms (t = 3.7, P <0.001 against table value of 1.8).The average per-worker-hour density of indoor-resting mosquitoes for all species was26.7 (11.2, brick built rooms; 15.5, jhoopries) and 0.3 (0.2,brick built rooms; 0.4 jhoopries) for An. stephensi. Monthlydistributions of An. stephensi were highest during July (15,25.4%) and lowest in October (1, 1.7%) (Fig. 2). There wereno significant differences in the number of An. stephensicaptured in human habitations between 2006 and 2007 (two-tailed t = 0.067, P = 0.033). Regression equation analysisshowed that the abundance of indoor-resting female An.stephensi mosquitoes (Y) was positively correlated withtemperature and humidity (Table 2).

During the two-year human landing collections, 354female anopheline mosquitoes were collected comprisingfive species, with the highest biting density recorded duringthe rainy season (Table 3). Although more An. stephensiwere collected from the lower extremities, when consideringthe unit surface area of the human body, the proportioncaptured was highest (0.6) on the upper extremities (Fig. 3).The biting sites of human body and the location (indoor/outdoor) of human landing collections were not significantlydifferent (P = 0.076) (Table 4). Significant variation wasfound between total indoor and outdoor human landing col-lections (t = 4.062, P < 0.001). Significant differences wereobserved in indoor and outdoor human landing collections



between 18.00–21.00 h and 24.00–03.00 h (t = 2.66, P <0.001 and t = 2.31, P < 0.001, respectively) and 24.00–03.00hours and 03.00–06.00 hours (t = 4.03, P < 0.001 and t =2.39, P < 0.001, respectively), while no significant differ-

ences were observed between indoor and outdoor collectionsconducted at 21.00–24.00 h and 24.00–03.00 h (t = 0.712,P > 0.001 and t = 0.364, P > 0.001) (Table 5). The results ofthe two-way anova of month and quadrant-wise variations

Table 1 Total number of female mosquitoes captured in indoor resting and human landing (indoor and outdoor) collections, Kolkata, WestBengal, 2006–07

Species

Total number (%)†

Total(%)Indoorresting

Percent of indoor restingto total mosquitoes

Human landingcollectionBrick built rooms Jhoopries

An. stephensi 59 (1.1) 0.3 0.5 34 (2.0) 93 (1.4)An. subpictus 448 (8.7) 2.4 4.2 156 (9.1) 604 (8.8)An. annularis 68 (1.3) 0.3 0.7 93 (5.4) 161 (2.4)An. vagus 24 (0.5) 0.2 0.2 46 (2.7) 70 (1.0)An. barbirostris 177 (3.5) 1.1 1.5 25 (1.5) 202 (3.0)Ae. aegypti 252 (4.9) 1.6 2.0 77 (4.5) 329 (4.8)Ae. albopictus 164 (3.2) 2.4 1.4 69 (4.0) 233 (3.4)Cx. vishnui 463 (9.0) 1.9 4.8 102 (5.9) 565 (8.3)Cx. quinquefasciatus 2529 (49.4) 17.6 19.4 842 (49.1) 3371 (49.3)Ar. subalbatus 568 (11.1) 3.4 4.9 178 (10.4) 746 (10.9)Ma. indiana 371 (7.2) 1.7 3.7 94 (5.5) 465 (6.8)Total 5123 32.9 43.3 1716 6839 (100)

†Percent of mosquitoes (number of mosquitoes collected for each collection type/total number of mosquitoes collected for each collection type).

Figure 2 Monthly number of Anophelesstephensi, Anopheles annularis and totalAnopheles captured in indoor resting atKolkata, West Bengal, 2006–2007.

0

10

20

30

40

50

60

70

80

Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Months

Nu

mb

er o

f m

osq

uit

oes

2006 An. stephensi

2006 An. annularis

2006 TotalAnopheles mosquitoes

2007 An. stephensi

2007 An. annularis

2007 TotalAnopheles mosquitoes

A. Ghosh et al.


Figure 3 Proportion (number of biting mosquitoes/ unit body surface area) of An. stephensi captured in indoor and outdoor human landingcollections at Kolkata, West Bengal, 2006–2007. The numbers at the top of the bar graphs indicate the total number of mosquitoes collectedfrom each body part.

Table 2 Statistical analysis of the relation-ship between total collected mosquitoes(indoor resting and host-seeking) and tem-perature and humidity

Environmentalparameters

Regression equations R2 values

2006 2007 2006 2007

Temperature Y = -2.94 + 0.20X Y = -3.38 + 0.23X 0.42 0.54Humidity Y = -11.37 + 0.19X Y = -18.59 + 0.29X 0.48 0.62

X, mean monthly temperature/humidity; R, correlation coefficient; Y, mosquito abundance.

Table 3 Indoor and outdoor human landing rates of anopheline mosquitoes, Kolkata, West Bengal, 2006–07

Species

Number (%) mosquitoes captured†

Ratioindoor/outdoor‡

Meannumber/worker-hour§Indoor Outdoor

An. stephensi 23 (67.6) 11 (32.4) 2.07 0.06An. subpictus 94 (60.3) 62 (39.7) 1.52 0.27An. annularis 56 (60.2) 37 (39.8) 1.51 0.16An. barbirostris 15 (60.0) 10 (40) 1.50 0.09An. vagus 27 (58.7) 19 (41.3) 1.42 0.08Total 215 (60.7) 139 (39.3) 1.60 0.13

†Percent mosquitoes collected for indoor and outdoor collections.‡Ratio of mosquitoes collected indoors/number of mosquitoes collected outdoors.§Total number of mosquitoes collected indoors and outdoors/number of hours of collection.



in human landing collections were not significantly different(F-values 4.49 and 13.77, respectively; P = 1.75). The meanduration of each gonotrophic cycle for all seasons was fourdays (96 h) for An. stephensi held at ambient temperatures,

while the mean duration during the rainy season was 4.1days (98 h, range 94–101 h); winter season, 4.3 days (102 h,range 100–106 h); and summer season, 3.3 days (88 h, range80–97 h). Seasonal overall parity, daily survival and mortal-ity rates and presumptive mortality between two successiveparous states are shown in Table 6. In the present study, themean proportion of parous females was 46.0%, the dailysurvival rate was 82.0% and daily mortality rate, 18.0%. Theresults of single-factor anova demonstrated significant dif-ferences between various parous states (F = 9.626, P < 0.01).Plasmodium vivax sporozoites were detected in the salivaryglands of two An. stephensi (sporozoite rate 2.2%) and oneAn. annularis (sporozoite rate 1.5%). All the other species ofAnopheles dissected were negative for sporozoites. One

Table 4 Single-factor ANOVA analysis with biting sites and locationsof human bait

Source of variation SS df MS F P-value

Between biting sites 18 1 18 4.59 0.076Between indoor and outdoor

locations23.5 6 3.92 1.21 0.012

MS, Mean square; SS, Sums of square.

Table 5 Monthly number of Anophelesstephensi captured nightly at 3-hr intervalsfor indoor (24) and outdoor (24) collectionsKolkata, West Bengal, 2006–07

Month

Period of collection (h)

Total18.00–21.00 21.00–24.00 24.00–03.00 03.00–06.00

In Out In Out In Out In Out In Out

January 0 0 0 0 0 0 0 0 0 0February 0 0 0 0 0 0 0 0 0 0March 0 0 1 0 0 0 0 0 1 0April 0 0 0 1 1 0 0 0 1 1May 1 0 0 1 2 0 0 0 3 1June 0 0 2 1 1 0 0 0 3 1July 1 0 1 1 1 1 0 0 3 2August 0 0 3 1 1 1 0 0 4 2September 0 0 1 0 2 1 0 0 3 1October 0 0 1 0 1 1 0 0 2 1November 0 0 0 0 2 1 0 0 2 1December 0 0 0 0 1 1 0 0 1 1Total 2 0 9 5 12 6 0 0 23 11

In, indoor; Out, outdoor.

Table 6 Seasonal proportion of parous, daily survival and mortality rates and presumptive mortality of natural population of Anophelesstephensi, Kolkata, West Bengal, 2006–07

SeasonsNo. of mosquitoes

dissected

Follicular stage of developmentParity(%)

DailyMortality Rate

DailySurvival RateNP P1 P2 P3 P4

Summer 2003 15 10 3 1 1 0 0.33 0.76 24Rainy 2003 15 7 2 1 2 3 0.53 0.85 15Winter 2003 5 2 1 1 1 0 0.6 0.88 12Summer 2004 15 9 1 3 1 1 0.4 0.80 20Rainy 2004 15 8 2 1 2 2 0.47 0.83 17Winter 2004 5 2 1 2 0 0 0.6 0.88 12Total 70 38 10 9 7 6 0.46Percentage 54.3 14.3 12.9 10.0 8.6Presumptive mortality rate (%) 73.7 10.0 22.2 14.3

NP, nulliparous stage; P, parous stage.

A. Ghosh et al.


midgut infection with oocysts was detected in one An. annu-laris (oocyst infection rate 1.5%).

Discussion

Both field and laboratory studies that address the socialstructure, vector species, density, infection rates, parity ratesand survival are necessary to understand the dynamics ofmalaria transmission and human risks (Samuel 1926).Vector density and anthropophilic behavior are two primarycomponents that affect the epidemiology of malaria, basedon the degree of human–vector contact and intensity ofmalaria transmission. Similar to other studies, the relativelyhigh population densities of An. stephensi were similar inbrick built rooms and jhoopries (Mahadev et al. 1978;Tandon & Tandon 1994).

The direct collection of mosquitoes coming to bitehumans provides important epidemiological informationand it can be measured with a reasonable degree of accuracyif properly planned and carried out (Pant & Pull 1980).Nightly collections demonstrated that the number of host-seeking An. stephensi were significantly higher for indoorcollections, suggesting a high degree of endophilic behavior.Similar to other observations, An. stephensi populationswere highest during the rainy and summer seasons andreflect higher numbers of malaria cases during these periods(Reuben & Panicker 1979). These seasonal variations werelikely due to increased numbers of breeding sites, whichwere greater during the rainy and summer seasons than thosein winter. While the higher numbers of An. stephensi wereattracted to the lower extremities, based on surface area, theupper extremities had a greater proportion of landing mos-quitoes. The cause of such preference is not clearly under-stood, but may be related to specific body odors.

The study of the age determination of anopheline popu-lations is important to understand the population dynamicsand the epidemiology and transmission of pathogens. UnlikeHati (1997), who indicated the mean duration of the annualaverage gonotrophic cycle was 72 h, our studies showed amean duration of gonotrophic cycle of 96 h, which is likelyrelated to ambient temperature in which mosquitoes werereared. Further study with an increase in sample size issuggested to reach a conclusion about this. While parityrates of our studies were limited, the results were similar toHati (1997), with the highest proportion of parous An.stephensi females during the rainy season, followed by thewinter and summer seasons. These observations demon-strate the need to intensify mosquito control programsduring the rainy season to decrease the proportion of parousand sporozoite infected mosquitoes.

Transmission of malaria is determined by factors such asvector abundance, biting rates, variations in seasonal andgeographic densities, endophagous or exophagous behavior,

vector longevity and fecundity, social factors (e.g. housingconditions, levels of poverty), environmental factors (e.g.temperature, precipitation, humidity) and vector controlmeasures. For a considerable period, malaria has beenendemic in Kolkata, but the observed density of the principalvector (An. stephensi) demonstrated a decreasing trend,showing the need to identify other potential vectors in thestudy area. According to Bangs et al. (2002), detection ofadvanced-stage sporozoites in mosquitoes provides compel-ling evidence to incriminate a particular vector species.Among the other species of Anopheles mosquitoes, An.annularis was established as a potential vector of malaria indifferent parts of India (Mahapatra et al. 2006; Dash et al.2008) including West Bengal (Ghosh et al. 1985). Duringthe present investigation, detection of both sporozoite andoocyst infections in An. annularis indicates its role as asecondary malaria vector in the study area. Further studieson age composition and blood meal analysis of An. annu-laris are needed to confirm its role as a secondary vector.

Acknowledgements

We are grateful to the male volunteers who participated inhuman landing collections and all the people of selectedlocalities of Kolkata in which the study was performed. Weare also thankful to Sri Anindya Sen (Department of English,Bankura Christian College) for revising the manuscript.

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