Observations on Nocturnal Biting Activity and Host Preference of Anophelines Collected in Southern Thailandi

Journal of the American Mosquito Control Association, l2(l):52_57, 1996

OBSERVATIONS ON NOCTURNAL BITING ACTIVITY AND HOST PREFERENCE OF ANOPHELINES COLLECTED IN SOUTHERN THAILANDI

RAMPA RATTANARITHIKUL.,3 EUI KONISHI3 INo KENNETH J. LINTHICUM,

ABSTRACT. Over a l3-month period, 5,127 adrlt female mosquitoes were collected on human bait during multiple collecting periods between 1800 and 06O0 h in 5 villages in southern Thailand. There were marked differences in the biting activity ofdifferent species at each ofthe villages studied. Anopheles maculatus and, Anopheles sawadwongporni were collected most often at dusk or in the first hours of darkness between 1800 and 2100 h. Although specimens of Anopheles dirus were collected consistently between 1900 and 0400 h, peak collections were made between 2000 and 2300 h. Anopheles minimus was collected consistently throughout the night without a clearly discernible peak. Of more than 2,40O Anopheles mosquitoes collected in CDC light traps, 133 specimens (5.4Vo) contained blood, nearly all of which was identified by enzymeJinked immunosorbent assay (ELISA) to be of bovine origin. Ten specimens contained blood from more than one host species.

INTRODUCTION to villages in Phetchaburi, Prachaup Khiri Khan, and Chumphon provinces in southern Thailand Although Anopheles dirus Peyton and Harri- near the Myanmar border. Details concerning son and Anopheles minimus Theobald are con- climate, geography, and human and animal pop- sidered to be the principal vectors of malaria ulations at the collection sites are presented in throughout much of Thailand (Rosenberg et al. an accompanying paper (Rattanarithikul et al. 1990), little is known about the ecology of an- 1996b). At these collection sites, sunset and sun- opheline malaria vectors in peninsular Thailand. rise ranged from 1750 to 1850 h and 0555 to This area is particularly important ecologically 0650 h, respectively, throughout the year (Tech- because it serves as a crossroads for faunal dis- nical Support Sub-Division, Forecast Division persal from the Indian, Chinese, and Malaysian- 1995). Indonesian geographic regions (Harrison 1980). Mosquito collections: To determine the time Knowledge of the biting habits of anopheline of bloodfeeding activity, man-biting collections mosquitoes associated with malarious areas is of were conducted outdoors 2-3 m away from a in establishing the vector potential of importance house for one or 2 nights per month over a 13- a species and in understanding the epidemiology month period. A landing-biting collection for a malaria. Furthermore, detailed information on of night consisted of 12 cycles of 5O min collection feeding and host-seeking behavior of mos- the and 10 min rest. A pair of collectors worked quitoes can be used to improve the design of between 1800 and 24OOh and a second pair be- strategies and to improve repellent or avoidance tween 24OO and O60O h. At the end of each l-h control projects. The purpose of this malaria collection cycle, mosquitoes collected in vials determine the bloodfeeding activity study was to were placed in a paper cup covered with netting, anophelines seeking human blood patterns of labeled with the time of collection, and stored mosquito host preference patterns and to identify alive in cool boxes. found in an endemic malarious area in species To determine host preference patterns, blood- Thailand along the Myanmar border. of southern fed mosquitoes were removed from light trap collections. These were made once or twice each MATERIALS AND METHODS month for 13 months using COr-baited CDC light traps. Light traps were placed overnight un- Five collection sites were located Study area: der trees (2 m above ground) 10-20 m from a Palao-U, Wangpao, Salui, and Pha- in Thasalao, dwelling. All Anopheles mosquitoes were identified to species using adult keys to anopheline rThe views of the authors do not purport to reflect mosquitoes in Thailand (Rattanarithikul and the positions of the Department of the Army or the Green 1986, Rattanarithikul and Panthusiri Department of Defense. 1994\. 2 Department of Entomology, U.S. Army Medical Blood meal identification' Host blood in en- Med- Component-Armed Forces Research Institute of gorged mosquitoes collected from light traps Sciences, AFRIMS, Rajvithi Road, Bangkok ical was identified using a single sandwich enzyme- 1040O (within the USA use USAMC-AFRIMS, APO linked immunosorbent assay (ELISA) (Konishi AP 96546). that were 3 Department of Medical Zoology, Kobe University and Yamanishi 1984). All specimens School of Medicine, Kobe 650, Japan. thought to contain blood, based on their appear-

52 M,lncn 1996 BrrNo BeHlvroR oF ANoPHELrrvBsIN THnu-nNo 53

Table l. Nocturnal biting activity of Anopheles species collected at human bait in the village of Palao-U, Prachaup Khiri Khan Province, Thailand.

Percentage of total number of specimens collected for each species An' An. sawad- barbi- An. An. An. An. wong- An. rostris hyrcanus Time dirus maculatus minimus porni nivipes gp.t gp.' (hour) (n:3r) (n = 144) (n : 1,113) (n: 387) (n -- 3) (n : r2r) (n : 56)

1800 0 30.0 4.O 44.O JJ.J 53.7 55.4 1900 6.5 -tt-J 10.8 22.5 66.7 16.5 8.9 2000 16.0 r9.5 15.0 12.7 o 2.5 8.9 2loo 22.5 4.3 ll.l 8.5 0 4.1 3.6 2200 19.4 3.5 to.7 2.8 0 1.7 t.8 2300 9.7 1.3 9.2 0.5 0 2.5 0 2400 3.2 1.3 6.6 2.1 0 t.7 0 oloo 9.7 1.3 tt.7 0.5 0 2.5 1.8 0200 6.5 0 7.4 1.0 0 J.J 3.6 0300 6.5 0 4.5 0.8 0 1.7 0 0400 0 0 3.4 0.5 0 0.8 7.1 0500 0 1.3 5.6 4.1 0 9.0 8.9

I Includes An. barbirostis nd. An. campestis. 2 Includes An- nigerimus, An- nitidus, An. peditaeniatus, and A. sinensis.

ance, were triturated in a microvial. Specimens RESULTS that had no detectable color of blood after trit- uration were not tested. Whole bodies of mos- We collected 5,127 adult female Anopheles quitoes were triturated in I ml of ELISA diluent mosquitoes, consisting of 17 species, on human bait. The nocturnal (phosphate-buffered saline containing 57o horse biting activity of the 7 most common anopheline species collected at human serum and 0.057o Tween 20). Antisera used as bait in Palao-U is shown in Table 1. Anopheles capture antibodies included goat anti-cat IgG, maculatus Theobald and Anopheles sawadwong- rabbit anti-bovine IgG, rabbit anti-chicken IgG, porni Rattanarithikul and Green were collected rabbit anti-human IgG (Cappel Laboratories Co. more often between 1800 and 2000 h than dur- Ltd., Durham, NC), rabbit anti-dog IgG (Miles- ing all other time periods (chi-square, P < 0.01). Yeda, Ltd., Rehovot, Israel), and rabbit anti-por- Although An. dirus was collected between 1900 cine IgG (Yagai Co. Ltd., Tokyo). The IgG frac- and 0300 h, peak collections were made between tions of these antisera, except the rabbit anti- 2OO0 and 2300 h. Biting activity during this pe- human IgG, were conjugated to horseradish per- riod was higher than during other periods (chi- oxidase (Sigma Chemical Co.) by the method square, P < O.0l). Anopheles minimus was col- described by Wilson and Nakane (1978) to pre- lected throughout the night without a clearly dis- pare detection antibodies. The horseradish per- cernible peak; however, more specimens were oxidase conjugate of anti-human IgG was pur- collected during the first half of the night (1800- chased from Kirkegaard & Perry Laboratories, 23OO h) than during the second half of the Inc., Gaithersburg, MD. Positive and negative night (2400--O5OOh) (chi-square, P < O.0l). All controls prepared were as a l:100 dilution ofone Anopheles nivipes (Theobald) and the majority homologous serum and 4 heterologous sera, re- of Anopheles barbirostris Van der Wulp and spectively. Microtiter plates were sensitized with Anopheles campestis Reid (here referred to as the capture antibody, blocked with bovine serum the An. barbirostris group) and Anopheles craw- albumin, incubated with the test sample, fol- fordi Reid, Anopheles nigrerrimus Glles, Anoph- lowed by the detection antibody, and visualized eles nitidus Harrison, Scanlon, and Reid, Anoph- with o-phenylene diamine in the presence of hy- eles pediataeniatus (Leicester), and Anopheles drogen peroxide. Samples that exhibited absor- sinensis Wiedemann (here referred to as the An. bance values at 495 nm greater than 3 SD above hyrcanus group) were collected before 2000 h. the mean of the negative controls were consid- There were no significant differences between ered positive. months in the time of peak feeding for any spe- 54 JounNel oF THE AMERTcIN Mosqurro CoNr.nol AssocrenoN VoL. 12,No. I

Table 2. Nocturnal biting activity of 4 potential Anopheles malaria vectors at human bait in Thasalao (T), Wangpao (W), Salui (S), and Phato (P) villages in southern Thailand.

Percentage of total number of specimens collected for each species An. dirus An. maculatus TimeTWS P T WSP (hour) (n:2O) (n = 0) (n: 5) (n:28) (n: l) (n : 5O) (4: 35) (n: 106) 1800 10.0 0 0 7.1 0 14.0 2.9 9.4 1900 0 0 0 3.6 0 20.o 34.2 32.1 2000 10.0 0 0 14.3 0 26.O 20.o 27.4 2100 10.0 0 0 25.O 0 24.O 22.9 5.7 2200 50.0 0 40.0 17.8 0 12.O 14.2 5.7 2300 20.o o 20.o t4.3 100.0 2.O 2.9 0.9 2400 0 0 0 lo.7 0 0 2.9 0.9 0100 0 0 0 3.6 0 0 o 3.8 0200 0 0 40.0 3.6 0 0 0 1.9 0300 0 0 0 0 0 0 o 2.8 M00 0 0 0 0 0 0 0 t.9 0500 0 0 0 0 0 2.0 0 7.5

cies in Palao-U except for An. minimus. During (Rosenberg et al. l99O). Because An. dirus com- May there were significantly more An. minimus prises a species complex involving at least 5 dis- collected after 24OOh than before (chi-square, P tinct species in Thailand that can be differenti- < 0.01). ated only by using either cytogenetic (Baimai et The nocturnal biting activity of 4 potential al. 1988) or DNA probe (Panyim et al. 1988) malaria vectors at Wangpao, Salui, Phato, and techniques, it is possible that the species studied Thasalao villages in southern Thailand is shown in these reports may represent different taxa. in Table 2. In general, the results are similar to Baimai et al. (1988) reported that An. dirus spe' observations in Palao-U with a few exceptions. cies A, B, C, and D each have distinct times of More An. maculatus were found biting between biting during the night. The biting activity pat- l8O0 and 1900 h in Palao-U than in each of the tern we observed was not consistent with that of other villages. More An. dirus, An. maculatus, any of the An. dinrs species reported by Baimai and An. minimus were collected after 24OO h in et al. (1988). We did not confirm the identity of Phato than in Wangpao, Salui, and Thasalao. the An. dirus collected; however, species A, C, Of 2,489 female mosquitoes collected in light and D have been found within the geographic traps, 133 (5.3Eo)specimens composed of I 1 an- range of the present study (Baimai 1988). opheline species had evidence of blood in the In Palao-U, more than 85Vo of all An. macu' abdomen. The blood meals of all I I species Iatus were collected before 2100 h. This early were identified (Table 3) and rr:,orc than 96Eo biting activity pattern was similar to that previ- contained bovine blood. Six specimens (4.5Voof ously reported elsewhere in Thailand (Scanlon the total tested) of Anopheles aconitus Doenitz, and Sandhinand 1965, Wilkinson et al. 797O, An. minimus and An. barbirostris group con- Harbach et al. 1987). Also, similar to what has tained human blood. Ten specimens (7.5Vo), been observed elsewhere in Thailand (Scanlon composed of An. aconitus, An. minimus, and An. and Sandhinand 1965, Harbach et al. 1987)' An. barbirostris group, contained blood from more minimus fed throughout the night without a dis- than one species of vertebrate host. tinct peak. We observed that in Palao-U villagers hunt and trap in the forest after sunset and that fam- DISCUSSION ilies remain active in the village until at least The time of peak biting in the village of Pa- 2100 h. The early evening biting activity ob- lao-U for An. dirus is similar to that reported for served for An. dirus and An. maculatus in each specimens collected (as Anop hele s balabacensis of the villages in peninsular Thailand suggests Baisas) in central Thailand (Wilkinson et al. that there would be considerable human expo- nor- 1970) but is in contrast to reports from southeast sure to these species before bed nets would Thailand that described the peak feeding activity mally serve as a barrier to biting. Conversely, either between 24OO and 0300 h (Scanlon and the extended biting activity of An. minimrs sug- ex- Sandhinand 1965) or between 22OO and 0100 h sests that the use of bed nets may reduce 55 Mancu 1996 Br.rtNc Begnvrop or ANopHEI-rNpsIN TneL,cNo

^fable 2. Extended

Percentage of total number of specimens collected for each species An. minimus An. sawadwongporni T w SPTWSP (n:74) (n: 67) (n: 534) (n: 1,977) (n : 5) (n: 12) (n: l) (n: 2) 1.4 13.4 2.3 o.2 0 16.7 0 0 5.4 14.9 lo.7 4.5 20.o 8.3 loo.0 loo.0 13.5 10.5 l 1.8 7.6 20.o 16.7 0 0 18.9 9.0 l7.o 7.O 0 16.7 0 0 36.5 2t.o 12.8 lo.7 40.0 25.O 0 0 24.3 r7.9 10.9 to.7 20.o 8.3 0 0 0 3.0 4.9 8.5 0 0 0 o 0 3.0 6.9 13.8 0 0 0 0 o 1.3 6.2 11.6 0 0 0 0 0 3.0 4.9 8.9 0 0 0 0 0 0 2.8 5.9 0 0 0 o o 3.0 8.8 10.6 0 8.3 0 o

posure to more than TOVoof host-seeking mos- sporozoites in the hemolymph, suggesting pos- quitoes of this species. Rattanarithikul et al. sible infection of the salivary glands. (1996a) previously found that the head and tho- All specimens tested for blood meal analysis rax of l.4Vo of An. dirus, O.7Voof An. maculatus, were collected with light traps. This method and O,3Voof An. minimas collected in peninsular probably introduced a bias toward exophilic spe- Thailand contained P lasmodium falc ip arum cir- cies. It is important to realize that bloodfed spec- cumsporozoite (CS) antigen. In Palao-U, An. sa- imens represented only abott 5Vo of the total wadwongporni, An. nivipes, An. barbirostris specimens collected in light traps. No bloodfed group, and An. hyrcanus group, which all exhib- An. dirus were found. ited early biting activity peaks, were also found More than 96Vo of specimens containing to contain P. falciparum CS protein (Rattanari- blood had fed on cattle, although light traps thikul et al.1996a). The detection ofCS antigen were placed 1O-20 m away from cattle. Fur- in the head and thorax indicates the presence of thermore, humans and dogs were more numer-

Table 3. Bloodfeeding patterns of 17 Anopheles species collected in CDC light traps in southern Thailand.

No. mosquitoes with blood in abdomen Human/ Human/ Cattle/ Cattle/ Cattle/ Human Cattle Pig dog cattle piC dog dog/cat Species blood blood blood blood blood blood blood blood An. aconitus l3 l-22 An. culicifacies I '7 An. jamesii t_ An. kochi I An. maculatus 3 An. minimus a 46 An. sawadwongporni 3 An. tessellatus J An. varuna 1 An. barbirostris gp.l 29 An. hyrcanus gp.2 t2 ,, Total r19 2143tl ' IncludesAn. barbirostrisand An. campestris. ' lncludes An. nigerrimus, An. nitidus, An. peditaeniatus, and An. sinensis JounNel oF THE AMERTcIN Mosquno CoNrnor_ AssocrnrroN Vor-. 12, No. 1 ous in the villages than were cattle. In restins ACI(NOWLEDGMENTS collections in India, Roy et al. (1991) ."pott"i We thank Ronald Rosenberg, Thkeo Matsu- that the major species of Anopheles fed predom- mura, and Thkeshi Kurihara for support and crit- inantly on cattle. It is possible that a higher per- icism of the study; Chumnong Noikamol, Som- centage of bovine feeders are attracted to light. porn Chanaimongkol, Pradith Mahaphibul, and However, in studies in which specimens were Suda Rattanawong for field and laboratory as- collected in light traps placed adjacent to areas sistance; and Somprathana Kiewlongya, Pra- where cattle were the predominant large verte- chong Panthusiri, and Scott W. Gordon for as- brate, the proportion of mosquitoes of various sistance in preparation of the manuscript. This genera and species containing bovine blood nev- study was supported in part by the Japan Society er exceeded 857o (Linthicum et al. 1985, Gordon for Promotion of Science Ronpaku Program. et al. 1991). Anopheles minimus accounted for the most bloodfed specimens. Bovine blood meals were REFERENCES CITED most numerous irrespective of collection site. Baimai, V. 1988. Population cytogenetics of the ma- Anopheles minimus also accounted for the great- Iaria vector Anopheles leucosphyrus group. South- est number of human blood meals. The only oth- east Asian J. Tiop. Med. Public Health 19:667-68O. er human blood meals were found in An. acon- Baimai, V., U. Kijchalao, P Sawadwongporn and C. A. Green. 1988. Geographic distribution and biting itus and An. barbirostrls group. Anopheles acon- behaviour of four species of the Anopheles dirus itus is a rice field-breeding mosquito and is an complex (Diptera: Culicidae) in Thailand. Southeast important vector of malaria in southwestern Asian J. Tiop. Med. Public Health 19:151-161. Asia, although it is not considered to be an im- Boreham, P E L. and C. Garrett-Jones. 1973. Preva- portant vector in Thailand (Rao 1984). lence of mixed blood meals and double feeding in Multiple mosquito feeding has been reported a malaria vector (Anopheles sacharovi Favre). Bull. previously (Edman and Downe 1964, Boreham W.H.O. 48:605-614. and Garrett-Jones 1973). This is an important Burkot, T, R., P. M. Graves, R. Paru and M. Lagog. 1988. Mixed blood feeding by the malaria vectors factor involved in the transmission of infectious in the Anopheles punctulatus complex (Diptera: Cu- present indicated agents. Although our study licidae). J. Med. Entomol. 25:2O5-213. that the rate of multiple feeding ranged from Edman, J. D. and A. E. R. Downe. 1964. Host-blood 7.77a in An. minimus to 22Vo in An. aconitus, sources and multiple-feeding habits of mosquitoes bloodfed specimens represented less than 6.07o in Kansas. Mosq. News 24:154-160. of all specimens collected in light traps. Edman Gordon, S. W, R. E Tammariello, K. J. Linthicum, R. and Downe (1964) reported the incidence of A. Wirtz and J. P. Digoutte. 1991. Feeding pattems multiple feeding ranging from 9.7 to 6l.8%o in of mosquitoes collected in the Senegal River basin. Assoc.T:424432. culicine species in Kansas. With the ELISA test J. Am. Mosq. Control Harbach, R. E., J. B. Gingrich and L. W. Pang. 1987. used in the present study, multiple feeding was Some entomological observations on malaria trans- detected only when mosquitoes fed on different mission in a remote village in northwestern Thai- species of animals. Actual multiple feeding rates land. J. Am. Mosq. Control Assoc. 3:296-301. are probably higher than test results indicate be- Harrison, B. A. 1980. Medical entomology studies- cause multiple blood meals may occur on the XIII. The Myzomyia series of Anopheles (Cellia) in same species of host (e.g., human-human). Es- Thailand, with emphasis on intra-interspecific vari- timates of multiple bloodfeeding using histolog- ations (Diptera: Culicidae). Contrib. Am. Entomol. ical techniques have been developed to detect Inst. (Ann Arbor) l7(4):1-195. E. and H. Yamanishi. 1984. Estimation of multiple blood meals during a single gonotroph- Konishi, blood meal size of Aedes albopictus (Diptera: Cu- from the same species of host (Romoser ic cycle licidae) using enzyme-linked immunosorbent assay. et al. 1989). Studies in Thailand using these J. Med. Entomol. 2l:506-513. techniques found that rr'ost Aedes aegypti Linthicum, K. J., H. G. A. Kaburia, E G. Davies and (Linn.) collected had fed twice in each gono- K. J. Lindqvist. 1985. A blood meal analysis of trophic cycle (Scott et al. 1993). Species with a engorged mosquitoes found in Rift Valley fever ep- greater likelihood of taking multiple blood meals izootics areas in Kenya. J. Am. Mosq. Control As- may have a higher vectorial capacity by increas- soc. l:93-95. V. Baimai. 1988. ing the chances of acquiring and transmitting a Panyim, S., S. Yasothornsrikul and Species-specific DNA sequences from the Anophe- disease agent (Boreham and Garrett-Jones les dirus complex-a potential for efficient identi- It is also possible that multiple feeding 1973). fications of isomorphic species, pp. 193-2O2. In: M. reduces the potential of a mosquito to serve as W. Service. (ed.). Biosystematics of haematophagus an efficient malaria vector by reducing the like- insects. Syst. Assoc. Spec. Vol. 37. lihood of obtaining enough gametocytes to be- Rao, T, R. 1984. Ahe Anopheles of lndia. Malaria Re- come infected and by diminishing the number of search Centre, Indian Council of Medical Research, sporozoites inoculated (Burkot et al. 1988). New Delhi. Mrncs 1996 BrrrNc BeuevIoR oF ANoPHELINT'SIN THeILeNp 57

Rattanarithikul, R. and C. A. Green. 1986. Formal Roy, A., M. A. Ansari and V. P Sharma. l99l' Feed- recognition of the species of the Anopheles macu- ing behavior patterns of anophelines from Uttar Pra- latus grotp (Diptera: Culicidae) occurring in Thai- desh and Gujarat states of India' J. Am. Mosq. Con- land, including the descriptions of two new species trol Assoc. 7:ll-15. and a preliminary key to females. Mosq. Syst. l8: Scanlon, J. E. and J. Sandhinand. 1965. The distri- 246-278. bution and biology of Anopheles balabacensis in Rattanarithikul, R. and P. Panthusiri. 1994. Illustrated Thailand (Diptera: Culicidae). J. Med. Entomol. 2: keys to the medically important mosquitoes of Thai- 61-69. land. Southeast Asian J. Trop. Med. Public Health Scott. T. W., G. G. Clark, L. H. Lorenz, P H. Amera- 25(Suppl.): 1-66. singhe, P Reiter and J. D. Edman. 1993. Detection Rattanarithikul. R.. E. Konishi and K. J. Linthicum. of multiple blood feeding in Aedes aegypti (Dipterul 1996a. Detection of Plasmodium vivax and Plas' Culicidae) during a single gonotrophic cycle using modium falciparzrz circumsporozoite antigen in an- a histologic technique- J. Med. Entomol. 3O:94-99. opheline mosquitoes collected in southern Thailand. Technical Support Sub-Division, Forecast Division. Am. J. Tiop. Med. Hyg. (in press) 1995. Weather almanac. Thailand Meteorological Rattanarithikul, R., K. J. Linthicum and E. Konishi. Department, Bangkok, Thailand. 1996b. Seasonal abundance and parity rates of Wilkinson. R. N.. T A. Miller and S. Esah. 1970. An- Anopheles species in southern Thailand. J. Am. thropophilic mosquitoes in central Thailand, with Mosq. Control Assoc. l2:75-83. notes on Anopheles balabacensis Baisas and malar- Romoser, W. S., J. D. Edman, L. H. Lorenz and T, W. ia. Mosq. News 30:146-148. Scott. 1989. Histological pa.rameters useful in the Wilson, M. B. and P K. Nakane. 1978. Recent de- identification of multiple bloodmeals in mosquitoes. velopments in the periodate method of conjugating Am. J- Trop. Med. Hyg. 4l:737-742. horseradish peroxidase (HRPO) to antibodies, pp. Rosenberg, R., R. G. Andre and L. Sornchit. 1990. 215-224. 1n.' W. Knapp, K. Holular and G. Wick Highly efficient dry season transmission of malaria (eds.). Immunofluorescence and related staining in Thailand. Trans. R. Soc. Tiop. Med. Hyg. 84:22* techniques. Elsevier/North Holland Biomedical 28. Press, Amsterdam.