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 indoor-resting, hu- index (A.I.) is the percentageof blood smears man (AM) collectionsseems to provide from a single Anophelesspecies, which is posi- dwelling tive for human blood. The A.I. usually combines the most accurate meansof assessingman-mos- quito data from several different biotopes, and these contact in Orissa. may be strongly biased toward one host or an- Reui.ewof data on feeding behauior: Table 2 shows the general feeding proclivities for the other. Quite often the biotopes involved are not named or describedat all by the compiler, so the anopheline fauna of Orissa. From 1982through A.I. should be regarded with caution and used 1988,a total of97,405blood smears,collected in for generalcomparisons only. 15 biotopes from 19 Anoph.elcsspecies, were The human blood index (H.B.I.) is the pro- processedagainst human and cow antisera. Of portion of blood smearsfrom a singleAnopheles these,94,371 smears were positive for primate species,which is positive for human blood and or bovine blood, and only 3.1% were negative. which was collected in a particular biotope Among the speciesin Table I, An. annularis which is identified by location and time of day. appearsas 2 forms: An. annularis (N) is an "normal" Such smears may include the mixed blood of 2 arbitrary name coined locally for spec- "abnor- or even more types of hosts, as long as one of imens and An. annularis (A) designates them was human. The H.B.I. is more precise mal" winter forms, marked by additional dark than the A.I. for assessingspecific feeding be- bands on the tarsi or palpi, that appear in about 598 JouRNer,or rnn Arr,rrnrcANMoseurro Cournol AssocrerroN VoL. 7, No. 4
L0% of the population at Mayurbhanj District, acteristically registersa much higher Human from Novemberto March. Anophelinesin Table Blood Index (H.B.I.) than any other species. 1 are listed in descending order, according to Unsurprisingly, collections involving human total blood smearscollected. Anophcles culicifa- dwellingsare stronglybiased and yield the high- cies, An. vagus,An. subpictus Grassi and An. est H.B.I.s regardlessof species.In comparing hyrcanus group n have relatively high fed-den- various biotopes, the ease of conducting the sities, while that of An. annulnris (N) is inter- collection involved, the total number caught and mediate.All other fed-densitiesare considerably the magnitudeof the H.B.I. for eachspecies are lowet. Anophelesfluuiatilis, the most egregious primary considerations.In general,indoor-rest- vector in the study villages, ranked only 7th in ing, human dwelling (AM) collectionsperform fed-densityprevalence. Species contributing less best, except when large numbers of people sleep than 1,000 smearsare probably too low in fed- outside. However, indoor-resting, cattle-shed density to be regardedas dangerousin the study (AM) collectionsoffer a promising alternative villages. The data for human positives show under certain conditionsbecause they are con- details on smearspositive only for primate blood siderably easier to carry out, achieve higher ("pure") and thosecontaining both primate and catch-rates (except for An. fluuiatilis), are rela- bovine blood. Table 2 includes 3 assessment tively unaffected by outdoor sleeping activities methods for ranking speciesaccording to their and they often register H.B.Ls that are ade- anthropophagictendencies. Table 1 simply lists quately high to reveal man-mosquito contact, the Arnph.el.esspecies in ranking order by each especially when high levels of transmission or ofthe 3 methods,and it indicatesthe proven or epidemicsoccur. suspectedvectors of the study villages. Accord- ing to Table 1, an appraisal based on pure hu- man bloodprevalences (P.H.B.P.) seems to pro- DISCUSSION vide the most accurateassessment method. Bachground information on double gel dlffu- Table 3 contains results of the 3 paramount sion; Wharton (1953) and Boreham (1975)at- species,from catchesin eachof 15 biotopes.The tested that identifying the hosts of hematopha- catch-rates of one biotope usually cannot be gous arthropods from bloodmeals determines compared directly with those ofanother, because whether an anopheline speciesmay be an effi- the collecting time and effort expendedusually cient vector of malaria.Tempelis (1975) named variesfrom one biotope to another. For example, the precipitin test as the basic serologicaltool all-night, 10-min indoor-wall collections soon for identifying bloodmeals,but Najera (1974) proved to be unfeasible because villagers ob- pointed out that its use has been limited by high jected to being disturbed hourly throughout the costs, which were estimated by the World night. Consequently,these collections had to be Health Organization (WHO 1985) to be U.S. discontinued after a trial period lasting only a $4.00 per blood smear. However, in 1982 an few months. In addition, the experimental hut adaptation of the double gel diffusion (DGD) and human dwelling, space-spray collections technique was developedby Collins et al. (1986) were implemented only in FIU 3, Phulbani. specifically for processing anopheline blood- Even for the same type of collection, such as meals. This provided a simple, rapid, sensitive, indoor-resting, twice as many human dwellings reliable and inexpensive method, whereby one were surveyed as were cattle-sheds or mixed person can test over 300 smears a day against dwellings,because the former are more strongly human and cow antisera, for under %0 (U.S. biasedto reveal man-mosquito contact. $0.005)per blood smear.In 1987this DGD was Indoor-resting,cattle-shed (AM) collectionis comparedwith other available blood identifica- the most productive technique for all species, tion techniques in an interlaboratory trial, and exceptAn. Due to its very pronounced fluuiatilis. the DGD provided the best results (Pant et al. anthropophagicbehavior, An. usually fluuiatilis 1987).If the DGD specificationsfor preparing is caught in highest numbers by indoor-resting, the substrateand cutting wells are followed very human dwelling (AM) collections,where it char- scrupulously,and precisetiters at 1:20,000of human and bovine antisera are used, acutely n sensitive reactions result, revealingmany mixed A key forAnophelesof the Cuttack Region (Wattal feedings that would not be detected by other and Kalra 1961)was used for routine identifications. serological techniques. Since mixed feedings Although An. hyrcanus appearsin this key, this spe- give visible cies is not known to occur in India and specimens testimony that all bovine feedings labeledas An. hyrcanus representclosely related spe- are noninfective for human malaria, any predi- cies of the Anopheleshyrcanus group. In Orissa, An. lection for a given species such as Anopheles nigerrimus Giles is probably the commonest member vogusDonit4 to take a high proportion of mixed ofthe group (RamachandraRao 1984). feedingsattenuates its vector potential. Because GEL DTFFUSIoTqANnLvsIs or Br,ooouplr,s
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Most Cornmentson gel diffusion data: One of the cryptic multiple feedingsprobably occur within most difficult aspects of interpreting blood a single biotope containing several individuals smearresults is trying to evaluatethe etiological of a singletype. This is supportedby Washino "It importanceof patent mixed bloodmeals,by es- and Tempelis(1983), who state: is reasonable timating the number of cryptic multiple part- to assume that a high proportion of multiple meals that may have been taken on human meals will be cryptic in a habitat containing hosts. Our gel diffusion results can reveal only predominantly one host (e.g.,chicken house)." unmixed bovine, unmixed primate or mixed bo- This assumption also applies to cattle-shedsor vine-primate bloodmeals.A mixed bloodmeal human dwellings, etc., where severalindividuals consists of a multiple meal, which is defined by of the sametype inhabit the samestructure. The Boreham and Garrett-Jones (1973) as any proximity of many available hosts precludesthe bloodmeal resulting from 2 or more feeds, the needfor additional appetential flights, so if feed- last of which has been taken before the first feed ing is interrupted, only very short successive has been digestedsufficiently to prevent identi- consumatory flights from one host to another fication of its origin. In this same article the within the biotope are necessaryfor the mos- authors provide a manner of computing an index quito to become fully engorged. Such cryptic on the proportion of multiple meals (including feedingswould not be revealedby gel diffusion cryptic meals) in a samPle. results. Previously, before bloodmeal analysis was One weaknessof most bloodmeal identifica- being done, it was generally assumedthat hu- tion techniques is an insufficient sensitivity to man dwellings were so highly biased to provide revealmixed feedings.Each patent mixed blood- mosquitoeswhich fed on human blood that each meal provides proof that one or more feedings specimen caught in a house was presumed to taken on bovine were noninfective for human represent a contact between that mosquito and malaria. It seemsquite plausible that any pro- man. However,Table 3 showsvery clearly that pensity for a speciesto switch readily from one this is not true, especiallyfor An. culicifaciesano type of host to another type seriously dilutes its (N), only 6.5and An. annularis which showthat vectorpotential. For example,as shownin Table re- ll.6%, respectively,of the fed specimens 2, An. uaguswas caught in very high fed-density covered from the indoor-resting, human dwell- and it showedrelatively high evidenceoffeeding (AM) primate blood. ing collectionscontained on primate blood. However,677oof its human providedsmears Only An. fluuiatilisconsistently positives actually were of mixed primate and percentage(807o in Table 3) that showinga high bovine origin. Of course,all bovine feedingswere positive for primate blood. AII other were harmless.Perhaps this attenuatedfeeding be- Anopheles smears from this very strongly hu- havior helps explain why An. uogusis not rec- man-biased biotope characteristically ranged ognizedas a primary or a secondaryvector. from only abott 257o down to l0% ot even less, , The tendency to take multiple meals could in positivity for primate blood. Consequently, possibly be genetically defined, but it seems irrespective of the place where mosquitoes are more probable that it is either an expressionof captured,it is essentialthat their blood be ana- preferredfeeding, or (evenmore likely) is caused Iyzed before even a rudimentary understanding feeding due to anti-mosquito ac- of their actual feeding behavior can be ascer- by interrupted (aggressiveor evasive)taken by the in- tained. tions preferredmultiple Anopheline deportment: According to tended host. An exampleof feeding and Weitz (1959), Bidlingmayer (1985), a resting mosquito em- feedingis providedby Smith gambiae in- barks upon an appetential (searching) flight in who observedthat Anopheles Giles terrupted its feedingto seekout a preferred host, responseto a physiologicalstimulus, such as the "subsidiary" need for a bloodmeal. During the flight her if the initial feedingwas madeon a appropriate senseorgans, whether olfactory, vis- host (animals other than man and cattle, the ual, thermal, auditory or humidity receptors, preferredhosts). Interrupted feeding due to anti- indicate the presenceof her objective. The mo- mosquito behavior by several ciconiiform birds ment such a cue is encountered,the appetential is describedin detail by Edman and Kale (1971) flight ends and the target or consumatory flight and by Webber and Edman (1972). It seems begins. The consumatory flight is direct and reasonablethat the majority of appetential and brief, since visual and biochemical cues do not consumatoryflights end in a biotope frequented operate over long distances.Therefore, it is by the host upon which the mosquito feeds, likely that most patent mixed bloodmealsin- although they may seekharborage elsewhere. 602 JouRNel oF THEAuonrceN Mosqutto CotttRol AssocIATIoN VoL. 7, No. 4 For example, in many malarious villages An. ACKNOWLEDGMENTS is caught in relatively high numbers fluuiatilis Malaria only in pre-dawn collections inside human The authors thank all of the National Eradication Program, World Health Organiza- dwellings. For this biotope, An. shows fluuiatilis tion and Orissa State Health Authorities, who a very high H.B.I. (0.80) in Table 3, and only encouragedus and expresseda keen interest in 3% of these human-positives involved mixed this work. We are grateful to all FIU entomology feedings.Any interrupted feeding of An. fluuia- staff for collecting blood smears over a pro- tilis on a human inside a housemost Iikely would longedperiod under very diffrcult and hazardous result in a second feeding, same either on the conditions. The assistanceand support provided person or upon another nearby human within by Mrs. Bernardita V. Collins also is greatly the same house. Since humans are fairly excit- appreciated. able hosts, before a mosquito becomesfully en- gorged,several interrupted part-meals may oc- REFERENCES CITED cur. If an infective mosquito takes a series of Bidlingrnayer,W. L. 1985.The measurementof adult part-meals that involve several uninfected per- mosquito population changes-someconsidera- sons,many new malaria casesmay be generated. tions.J. Am.Mosq. Control Assoc. 1:328-348. The dangerposed by this type of feeding activity Boreham,P. F. L. 1975.Some applications of blood is clear and may explain why An. fluuiatilis is meal identifications in relation to the epidemiology such an effective vector, even in very low popu- of vector-borne tropical diseases. Am. J. Trop. Med. lation density. Hyg. 78:83-91. Boreham, P. F. L. and C. Garrett-Jones. 1973. Preva- The extreme contrast between feeding habits lence of mixed blood meals and double feeding in a of An. fluuiatilis andAn. uogusis clearly depicted malaria vector (Anopheles sacharoui) Favre. Bull. in a graph of bloodmeal results on anophelines W.H.O.48:605-614. of Orissa by Collins et al. (1989).This graph Collins,R. T. and Y. H. Bang.1988. Manual on the takes into account species fed-density, H.B.I. establishment of a gel diffusion laboratory to iden- "pure" and (unmixed) human bloodmeals. tify the bloodmealsof haematophagousarthropods. AnopheLes is in the upper right corner WHO/SEARO SEA/VBC/38. fluuiatilis Collins,R. T., G. K. Sharma and K. graph, B. Dhal. 1989. of the indicating very dangerousfeeding Gel diffusion determinations on feeding behavior behavior, while An. uagrusis near the lower left patterns of anophelinesin Orissa State, India. Ind. corner, showingrelatively harmlessfeeding hab- J. Commun.Dis. 21:1-23. its. Collins,R. T., B. K. Dash, R. S. Agarwalaand K. B. Significanceof the study: The comprehensive Dhal. 1986. An adaptation of the GeI Diffusion data garnered in this study engenderedconfi- technique for identifying the source of mosquito bloodmeals.Ind. J. Malariol. 23:81-89. dencewhen selectingpriorities and sample sizes Covell,G. and P. Singh. 1942.Malaria in the coastal of bloodmeal collections in subsequentstudies, belt of Orissa.J. Malaria Inst. Ind. 4:457-493. accordingto the location, season,species, collec- Das Gupta, S. P. 1977.India physiographicregions. tion methods and major biotopes involved. In National atlas of India, Plate 41. 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