DEcEMBER 1991 Gpr, Drprusror ANAr.vsrsor Bloortuonls GEL DIFFUSIONANALYSIS OF ANOPHELESBLOODMEALS FROM 12 MALARIOUS STUDY VILLAGES OF ORISSA STATE. INDIA R. T. COLLINS,I M. V. V. L. NARASIMHAM,' K. B. DHAL' AND B. P. MUKHEzuEE' ABSTRACT. In Orissa State, India, the double gel diffusion technique was used to analyze97,405 bloodmealsof all fed anophelinesthat were caught during standardizedmonthly surveysin 12 malarious study villages,from 1982through 1988.Anoph.eles culicifaci.es contributed the highest number of smears from the 19 Anophelcsspecies recovered. It was observedthat a pronouncedpredilection to take mixed bloodmealsattenuates the vector potential of the speciesconcerned. Consequently, prevalences based "pure" only upon (unmixed) primate bloodmealsprovide the most accurate way to assessthe intensity of feeding contact that actually occurs between a given speciesand man. By this method, the ranking order is Anophelesfluuintilis, An. culicifaci.esand An. annulnris (N); a sequencewhich concurs with current knowledgeon the vector status of malaria mosquitoesin Orissa. INTRODUCTION upgrade entomology, so 3 field studies were es- tablished in Orissa, with primary objectives to Anopheles sundaicus Rodenwaldt was de- incriminate or reincriminatevector speciesand scribed as a coastal vector of malaria by Senior to study larval and adult bionomics,particularly White (1937)and asa vectorin the Chilika Lake to improve control strategies.By the time a areaof Orissaby Covelland Singh (1942).Sub- double gel diffusion (DGD) mosquito bloodmeal sequently, early DDT malaria sprays appear to identification systemwas developed,the 3 study have eliminated An. sundaicusfrom coastal Or- teams already had been conducting weekly rou- issa,but it is still found in seashoreareas north tine field collections for more than a year, so it and south of the state. Further observationsby required very little extra effort for the teams to Senior White and Das (1938) in the Jeypore make blood smears and record data on all fed Hills of southwestern Orissa revealed,that An. specimensbeing caught. The smear workloads fluuiatilis James,An. minimus Theobald and An. were far lessthan the number the Bhubaneswar uaruna Iyengar are primary vectors, while An. Iaboratory could processand costsinvolved were jeyporiensis Jamesis a secondaryvector. Anoph- relatively negligible. Consequently, bloodmeal el.esannularis Van der Wulp was revealedas the determinations were incorporated as a special main, if not the only, vector in the plains of adjunct to the baseline studies and were carried Orissaby Panigrahi(1942) and SeniorWhite et out as a routine procedure,like making identi- al. (1943). Anophelesculicifacies Giles was not fications, evaluating mosquito midguts or dis- regardedas a malaria vector in Orissa by these secting salivary glands, ovaries, etc. Our objec- early workers. It was first incriminated in 1980 tives were to explore the parametersof the DGD by our workersin Dhenkanaldistrict of Orissa, method under field conditions and to contribute and thereafter several additional positive An. detailed data on adult feeding bionomics to the culicifaci.eswere recoveredby our study teams in study team objectives. Sambalpur,Mayurbhanj and Phulbani districts, Orissa.These teams also proved that An. fluuia- MATERIALS AND METIIODS tilis is a perennial transmitter by the dissection for sporozoitesevery month of the year. In a Identical. standardized collections were con- reviewof Orissaanophelines, Guha et al. (1981) ducted by 3 Entomology Field Investigation reported 21 speciesin rural Orissa,but they said Units (FIUs) in 3 physiographically distinct no reports were available from Dhenkanal, areasof Orissa State, India (Fig. 1). Each FIU Phulbani, Puri, Bolangir or Kalahandi districts, used the same work schedule in each of its 4 thus showing that entomologicaldata were very study villages, where blood smears were made scanty up to 1980.The National Malaria Erad- on strips of Whatman no. 1 filter-paper from oll ication Program recogrrizedthe pressingneed to fed anophelines collected. A separate filter-pa- per was used for each species,each collection biotope, each village and each year. A single r 95 K Street,Turners Falls,MA 01376,USA. filter-paper held up to 25 smears.For all-night, 2Directorate of the National Malaria Eradication 10-min per hour collections,each smear was Programme/National Institute of CommunicableDis- labeled to show the day and hour of collection. eases,22 Sham Nath Marg (Alipore Road), Delhi, Regardingindoor-resting collections,code num- India 110054. bers linked each smear to its routine mosquito 596 JouRr.rlr,or rnr AvrRIcnNMoseurro Cor.lrnoL Assocrerron VoL.7,No.4 ORISSA STATE, INDIA 1:10,OOO GARJHAT HILLS FIU = Field Investigation Unit * = Sludy Villages Fig. 1. Map of Orissa State showing the major physiographic categories, the location of Field Investigation Units and their respective study villages (from Das Gupta 19??, plate 41). collectionform, on which the housecode num- analyze the bloodmeals of all fed females col- ber,type ofresting surface,abdominal condition lectedin areasof stablemalaria, where periodic and the estimated height from the floor, were high-endemictransmission is known to be ac- recordedfor eachmosquito caught. Smearswere tually autochthonous to those villages which kept in sealed plastic bags and stored inside havebeen selected for study.If it is not possible screw-capplastic containerswhich were held in to collect mosquitoesevery month of the year, as cool a place as possible,until being sent then surveysshouldbe conductedat least during (about every 3 or 4 months) to the PfCPt Zone months when peaks of transmission occur. If II EntomologyGel Diffusion Laboratoryat Bhu- all major biotopescannot be sampled,then col- baneswar,Orissa State. This laboratorywas es- lectionsshould be focusedon human-biasedbio- tablishedaccording to a manual by Collins and topes, such as human dwellings; and whenever Bane (1988),and smearswere processed accord- substantial numbers of humans are observed ing to the methodologyoutlined in this manual. sleepingoutdoors, collections should include all Generalguidelines collecting srnears:The for fed found best way to assessArwph.el.es feeding risk is to anophelines resting in the immediate vicinity of the sleepers.The collectionsmen- tioned are designedto distinguish which species " are feeding most extensively on humans during Plasrnodium falciparum Containment Programme: periodsof intense transmission,thus exposing a project funded by the Swedish International Devel- opment Agency (SIDA), which supplernents the Na- the most likely vector species.An analysis of tional Malaria Eradication Programme through their bloodmeals reveals the proportion of the WHO/SEARO. total fed specimens (fed density) that contain DECEMBER1991 Gpl DrrrusroNANer,vsrs or Br-ooorr,rnels Table 1. Gel diffusionresults on anophelinesof Orissa,India, from 1982through 1988,rated by anthropophilicindex (A.I.), human bloodprevalence (H.B.P.) and "pure" human bloodprevalence (P.H.B.P.) Anthropophilicindex Human blood Pure human blood (A.I.) prevalence (H.B.P.) prevaience(P.H.B.P.) l. An. fluuintilis' l. An. fluuiatilis" L. An. fluuiatilis" 2. An. theobaldi 2. An. culicifacies^ 2. An. culicifacies^ 3. An. paLlidw 3. An. uagus 3. An. annularis(N)b 4. An. uaruna" 4. An. annularis(N)b 4. An. uagus 5. An. annularis(N)D 5. An. hyrcanus group 5. An. subpictus 6. An. jeyporiensis' 6. An. subpictus 6. An. hyrcanus grortp 7. An. culicifacies" 7. An. pallidus 7. An. paLlidus 8. An. uogus 8. An. theobaldi 8. An. theobaldi 9. An, aconitus 9. An. uaruna" 9. An. uaruna' 10. An. macuLatus 10. An. jamesi 10. An. jamesi ll. An. hyrcanus group ll. An. aconitu^s I7. An. aconittts 12. An. jamesi 12. An. maculatus 12. An. macuLatus 13. An. barbirostris 13. An. barbirostris 73. An. barbirostris 14. An. subpictus 14. An. tessellntw 14. An. tessellatus 15. An. tessellatus 15. An. jeyporiensis" 75. An. jeyporiensis" 16. An. splendidus 16. An. splendidus 76. An. splendid.us 17. An. annularis(A) 17. An. annulnris(A) 17. An. annulnris(A) u Proven vectors of study villages. b Suspectedvector of study villages. " Local vectors of Jeypore hills, accordingto previous studies;do not appear to be vectors in study villages. human blood, and from these a further refine- haviorpatterns. However, blood prevalences ap- ment may be made by considering only the pear to be more accurate than either of these 2 "pure" (unmixed) feedings.Such data clearly indices. confirms and quantifies the degreeof man-mos- The human blood prevalence(H.B.P.) is sim- quito contact occurring when the survey was ilar to the H.B.I. except that it refers to the maoe. proportion of the total smearscontaining human However, besidesfeeding behavior and popu- blood (mixed and unmixed) from oll species lation density, there are additional characteris- combined, which are comprised by a particular tics that enhancevector competence,such as species. short gonotrophic cycles, high levels of mos- The pure human bloodprevalence (P.H.B.P.) quito-parasite compatibility and prolonged lon- is similar, except that only smearsthat contain gevity. "pure" (unmixed) human blood are compiled. Such blood consists of one or many feedingson RESULTS human blood, upon one or severalhuman hosts, but the total number of human feedingsis cryp- Definition of terms and interpretation of feed- tic. The focusof the P.H.B.P. on unmixed feed- ing behnuior: In this paper, the anthropophilic ings, especially those from