248 Jounuu oF THEArunRrceN Mosqurro Cournol AssocrATroN VoL.4,No.3 HOST FEEDING PATTERNS OF ANOPHELES CULICIFACIES SPECIESA AND B HEMA JOSHI, K. VASANTHA, SARALA K. SUBBARAO lro V. p. SHARMA Malarin ResearchCentre (ICMR),2Z-Shatn Nath Marg, Dethi_I10 054, India ABSTRACT. Countercurrent immunoelectrophoresiswas used to assaybloodmeals to determine the hostspecificity of Anophelesculicifarics species-Aand B, collected from areas in Delhi, Uttar piadesh and Bihar. Results indicated the-predominantly zoophagicnature of speciesA and B with " r.t"tiullv higher degreeof anthropophagy for speciesA. Furth-er,the human blood index was found to be relatei to the proportion of human and cattle population in an area. This study is significant because,of the two speciesonly speciesA was incriminated as the vector of malaria in t[ese arlas. INTRODUCTION and districts in Uttar Pradesh and Bihar, (areas in northern India). In northern India speciesA Anopheles culicifacies sensu lato has a wide and B are sympatric (Subbarao et al. 1983, distribution extending from Iran in the west 1988c). through India to Thailand in the east. It is also found in southern China and Nepal in the north and Sri Lanka in the south. It is an important MATERIALS AND METHODS vector of malaria in India and severalneighbor- ing countries.This speciesis predominantly zoo- Indoor resting An. culicifacieswere collected phagic and becomesanthropophagic when the with suction tubes from the following areasdur- cattle population is low (Bhatia and Krishnan ing 1985-87: Gopalpura, a periurban locality in 1961,Jambulingam et al. 1984).Anthropophagic Delhi; 18 villages in Ghaziabad, 9 in Buland- indices ranging between 2 and 80% have been shahr and 8 in Jaunpur and Ballia districts in reported for this speciesin India (Ramachandra Uttar Pradeshand 3 villages in Saran district in Rao 1984). With discovery of 4 sibling species Bihar. Ghaziabadand Bulandshahr are in west- [A, B, C and D, in the taxon An. culicifacbs ern Uttar Pradesh whereasBallia and Jaunpur (Green and Miles, 1980, Subbarao et al. 1983, are situated in the easternpart ofUttar Pradesh. Vasanthaet al.,unpublished data)1, the question The proceduresfollowed in the collection of arose as to whether the differences observedin An. culicifaciesare similar to those describedin the rate of anthropophagy of this taxon are Subbarao et al. (1988a). Anopheles culicifaci.es related to the biological variations existing was collected during August 1985-June 1986 in among the sibling species.The differences ob- Gopalpura, during January 1985-June 1987 in served in the feeding preferencesin An. m,acu- Ghaziabad,in July, August and November 1985 lipennis Meigen populations from different re- and March, July, August, September and No- gions in Europe was one of the factors which led vember 1986 in Bulandshahr and in Jaunpur, to the concept of the possible existance of sev- Ballia and Saran in May, August and November eral biological specieswithin a morphologically 1985and March 1986.Anopheles culicifacies col- similar anopheline speciesin Europe (Hackett lected from the field are brought to the labora- L937).Anopheles gambiae sensu strictu and An. tory for processing.After mild etherization half- arabiewis Patton, two membersof the An. gam- gravid female mosquitoes having Cristophers' bioe complex were also reported to have differ- stage III ovaries were separated. Only half- encesin the anthropophagicindex; An. gambiae gravid females were used as it is only at this s.s. was more anthropophagic (Coluzzi et al. stagewell developedpolytene chromosomesare 1975,Mollineaux and Gramiccia 1980).A pre- present which are suitable for reading the band- Iiminary study carried out in southern India on ing pattern. From each half-gravid mosquito, the feeding preferencesof speciesA and B of ovaries were removed and stored in a glass vial the An. culicifaciescomplex revealed speciesA containing 1:3 glacial acetic acid and methanol to be more anthropophagic than speciesB (Su- fixative. From the same mosquito blood from guna et al. 1983).The sibling speciesof the An. the gut was smeared on Whatman No. 1 filter culicifacies complex have specific distribution paper for blood meal assay and the rest of the patterns (Subbarao1984), variations in the sea- mosquito was stored in a microfuge tube for sonal prevalence(Subbarao et al. 1987),differ- sporozoite detection [results of which are pub- ential responseto DDT (Subbaraoet al. 1988a) lished in Subbaraoet al. (1988b)1.Each mos- and susceptibility to malaria parasite infection quito, its blood smear and the ovary were given (Subbarao et al. 1988b). This study examines the same number. the blood meals of speciesA and B from Delhi, Ovaries were processedfor polytene chromo- Snprprr4srn 1988 Att. cuttapecns SrBLrNc Sppcrps: Hosr FEEDING 249 Table 1. Bloodmeal analysis of Anophnlcsculicifacies sibling species. Jaunpur Delhi Ghaziabad Bulandshahr Saran Areas & Ballia Species Hosts Percentagepositive Human 1.3 0.0 2.2 0.8 3.5 0.6 0.0 0.3 0.0 0.0 Bovine 98.5 100.0 94.0 97.0 90.0 96.0 100.0 99.0 0.0 100.0 Mixed 0.2 0.0 2.2 0.3 4.0 2.0 0.0 0.0 0.0 0.0 Not detected 0.6 0.0 1.6 1.5 2.5 1.8 0.0 0.6 0.0 0.0 Total samples 466 4345 382 833 493 332 tested Table 2. Monthly variations in human blood index ratio with distilled water and 0.375 gm calcium (HBI) of speciesA and B of Anoph.elcsculicifacies in Iactate per liter buffer was added. district Ghaziabad. Glass plates of 14 cm x 8.5 cm size coated SpeciesA SpeciesB with 0.2% agarosewere layered with 0.97o aga- rose. The procedure used for coating and layer- No. No. ing was that from Collins et al. (1986). Wells Montht tested HBI tested HBI were punched 5 mm apart on the agarose gel January I 0 00 with the help of a 1.5 mm diameter punch. February 0 0 00 Agarosepieces from the wells were removedwith March 0 0 00 a syringe needle. April 130 0 10 From blood smears5 mm diameter discs were May 5r4 0.01 50 punched and soakedin the wells of a microtiter June 390 0.04 50 July 572 0.11 150 plate containing 50 pl of 0.65% normal saline. August 607 0.08 330 Smearswere allowed to soak for 4 hours. Even September 315 0.06 56 0.02 when soaked for 2 hours satisfactory results October DIY 0.03 107 0.02 were obtained. The antisera used in the assay November 437 0.04 100 0 were obtained from the Serologistand Chemical December 1q 0 00 Examiner to the Government of India. Calcutta. I Samples collected in 1985 and 1986 were pooled. The antisera were subjectedto blind tests using known bloodmeal samplesand various dilutions some preparation following the procedure of before they were used in the assays.On each Greenand Hunt (1980).Polybene chromosomes agarosegel plate one blood sample of human, were examined to identify the sibling species. one of cow/buffalo and one of rabbit were as- Diagnostic inversion genotypes used for the sayedalong with the test samples.On the human identification of .sibling specieswere: SpeciesA antisera plate, mosquitoesfed on cow and rabbit - X +" +b 2 +e' +h' and speciesB - Xab 29' served as negative controls and on the bovine +h' (Subbaraoet al. 1983). antisera plate, rabbit and human samplesserved Bloodmeal samples were tested by counter as negative controls. Mosquitoes fed on human current immunoelectrophoresis(CCIE) (Bray et and cow blood served as positive controls on al. 1984) and gel diffusion (Collins et al. 1986) their respective plates, Antigen extracts were techniquesusing agarosegels. Although both the loadedon the cathodic wells while antisera were techniquesproduced equally sensitive results in charged on anodic wells. Electrophoresis was detecting the antigen up to 40 hours after the performed at a constant voltage of 150 volts for bloodmeal,CCIE was quicker as the electropho- 30 min. at room temperature. After the comple- retic run was completed within 25-30 minutes tion of the run, the glass plate was taken out and precipitin arcs were sharper. Therefore, from the electrophoreticchamber and immersed counter current immunoelectrophoresis was in 0.9Vonormal saline.After 10-15 min., results used for assaying the samples. Details of the were read against a black background over an technique are given below. X-ray viewer. Barbitone buffer powder (0.5 M, pH 8.6) was obtained from Ms. Centron, India and 0.5 M RESULTS AND DISCUSSION stock solution wasprepared. For gel buffer, stock solutionwas diluted in 1:12.5ratio with distilled Results of An. culicifacles sibling species A water and calcium lactate was addedto obtain a and B specimensshowing positive reactions to final concentration of 0.5 gm/liter. For electrode human and bovine antisera are given in Table (tank) buffer, stock solution was diluted in 1:5 1. A small percentageof samples did not react 250 JounNel oF THEAurnrclu Mosqurro Cournol AssocrerroN VoL.4,No.3 with either of the sera tested. These mosquitoes has been incriminated as the primary vector, might have taken blood of other animals for whereas in Jaunpur, Ballia and Saran malaria which the antisera tested were not specific. Re- is almost absent (Subbaraoet al. 1988b,1988c). sults showed that both speciesA and B were SpeciesA had a relatively high sporozoite rate predominantly zoophagic as only a small pro- in August (1.09%) and September(2.07%) in portion showed positivity against human anti- comparison to other months (Subbarao et al. sera. Comparative chi-squarevalues for the ob- 1988b).During the other months it was always served numbers of human. bovine and mixed less than one. The proportions of human blood positive antigens between speciesA and B were feeding observedfor speciesA and speciesB in 8.8 (P < 0.05) in Ghaziabad and LG.27(P < this study are in accordance with the earlier 0.001)in Bulandshahr.The human blood index reports for An.