Abundance, Parity, and Japanese Encephalitis Virus Infection of Mosquitoes (Diptera: Culicidae) in Sepang District, Malaysia INDRA VYTHILINGAM, KAZUMASA ODA, 1 S. MAHADEVAN, GHANI ABDULLAH, CHAN SENG THIM, CHOO CHOON HONG, 2 B. VIJAYAMALAR,2 MANGALAM SINNIAH, 2 AND AKIRA IGARASHI3 Division of Medical Entomology, Institute for Medical Research, Jalan Paliang 50588, Kuala Lumpur, Malaysia J. Med Entomol. 34(3): 257-262 (1997) ABSTRACT A 2-yr study of Japanese encephalitis (JE) virus in Sepang District, Selangor, Malaysia, was carried out to identify the mosquito vectors and to determine their seasonal abundance, parity, and infection rates. In total, 81,889 mosquitoes belonging to 9 genera and >50 species were identified from CDC trap collections augmented with dry ice during 1992 and 1993. Culex tritaeniurhynchus Giles and Culex gelidus Giles were the most abundant species, and both increased in numbers with increases in rainfall. Overall, 45 JE virus isolations were made from 7 species—Cx. tritaeniorhynchus (24), Cx. gelidus (12), Culex fuscocephala Theobald (2), Aedes hutleri Theobald (4), Culex quinquefasciatus Say (1), Aedes lineatopennis Ludlow (1), and Aedes (Cancraedes) sp. (1). Based on elevated abundance and JE infection rates, Cx. tritaeniorhynchus appears to be the most important vector of JE virus in Sepang. KEY WORDS Culex spp., Aedes spp., Japanese encephalitis virus, seasonal abundance, age grading, pig farm (JE) virus was isolated in Malaysia for the 1st time in 1952 (Paterson et al. 1952). The presence of JE virus in peninsular Malaysia was confirmed by Pond et al. (1954) and McCnimb (1955) and in Sarawak by Simpson et al. (1970, 1974). In addition to equines, JE virus infected a variety of domestic animals including pigs, bo\ines, dogs, goats, and sheep (Pond et al. 1954) and mosquitoes including Culex tritaeniorhynchus Giles, Culex gelidus Giles, and Aedes curtipes Edwards (Simpson et al. 1974). Currently, JE is endemic and occurs in almost every state in Malaysia; most cases have been reported from Penang, Perak, Selangor, and Johore in West Malaysia and Sarawak in East Malaysia (Sinniah 1989). Contributory factors in these states may be high human population densities and pig farming (Sinniah 1989). There is no definite seasonal pattern of human infection and JE cases have been detected all year round (Fang et al. 1980). Because most research was carried out decades ago, the current study was carried out to elucidate the current JE virus—vector situation. During this study, a case of JE was reported from Sabak Bernam, 80 km north of Kuala Lumpur. Studies were JAPANESE ENCEPHALITIS 'Formerly of IMR-JICA Research Project on Tropical Diseases, Institute for Medical Research, Kuala Lumpur, Malaysia. -Division of Virology, Institute for Medical Research, Kuala Lumpur, Malaysia. 'Departmen • t of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan. carried out there and have been published (Vythilingam et al. 1995). Entomological studies were carried out in Sepang between January 1992 and December 1993. The main objectives were to determine the seasonal abundance and JE infection rates of mosquito vector populations. Materials and Methods Study Area. The study area is situated in Sungai Pelek, Sepang District, 80 km south of Kuala Lumpur. Pig farms were situated adjacent to the town and were surrounded by oil palm culture; other domestic animals present were dogs, goats, chickens, and ducks. Mosquito Collections. In total, 12 CDC battery-operated light traps augmented with 300 g of dry ice were operated between 1800 and 0700 hours during 3 nights each month at fixed sites in or near pig sties. Mosquitoes were immobilized by placing them on dry ice, sorted according to species, counted and pooled into lots of 50, then placed in liquid nitrogen for transport to the laboratory for virus isolation. Blood-fed mosquitoes were not tested. Nulliparity Rates. From January to December 1993, representative specimens of Cx. tritaeniorhynchus and Cx. gelidus were examined for parity by ovarian dissection (Detinova 1962). Careful ex- 0022-2585/97/0257-0262$02.00/0 © 1997 Entomological Society of America 258 Vol. 34, no. 3 JOURNAL OF MEDICAL ENTOMOLOGY 250 Cx. tritaeniorhynchus H~ Cx. gelidus LJ Rainfall 0 J F M A M J J A S O N D J F M A M J J A S O N D | 92 | 93 | Fig. 1. Abundance of Cx. tritaeniorhynchus and Cx. gelidus and total rainfall per month in Sepang, 1992-1993. animation identified dilations on the follicular pedicel of multiple follicles per ovary. Virus Isolation. Each pool of 50 mosquitoes was triturated with 2 ml of 2% fetal calf serum in Eagle's medium in Earle's saline with nonessential amino acids (MEM) and was centrifuged at 3,000 X g for 15 min at 4°C. The supernatant was passed through a 0.2-fim filter and was inoculated into Aedes albopictus (Skuse) C6/36 cells (Igarashi 1978). Infected C6/36 cells were harvested after incubation at 28°C for 7 d. Detection of intracellular viral antigen was carried out by peroxidaseantiperoxidase (PAP) staining as described by Vythilingam et al. (1995). Positive PAP tests were confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR) using JE virus-specific primers (Vythilingam et al. 1995). to December, whereas in 1993, peak rainfall occurred in November. Cx. tritaeniorhynchus was most abundant during May and December 1992 and September-December 1993 (Fig. 1). Analysis of variance (ANOVA) showed that abundance varied significantly among months (F = 30.07; df = 1, 46; P < 0.001). Culex gelidus was most abundant in May and October 1992 and June, July, and October 1993 (Fig. 1). Abundance varied significantly among months (F = 7.34; df = 1, 46; P < 0.005). Cx. fuscocephala showed peaks in SeptemberOctober 1992, May-July, and September-November 1993 (Fig. 2). Cx. fuscocephala comprised the 3rd highest collection over the 2-yr period and mean catch fluctuated significantly over the months (P < 0.005). In some months Culex sitiens Wiedemann, Cx. quinquefasciatus Say, and Aedes butleri Theobald were more abundant than Cx. Results fuscocephala (Fig. 2) Ae. butleri showed peaks in Culex tritaeniorhynchus was the most abundant species, comprising 60% of the total collected, fol- January, May, and November 1992 and Septemlowed in decreasing order by Cx. gelidus (20%), ber-December 1993. The abundance of Cx. triCulex fuscocephala Theobald (5.8%), Culex sitiens taeniorhynchus was correlated strongly with rainWiedemann (3.5%), Culex quinquefasciatus Say fall (r = 0.72, df = 22, P < 0.001), whereas the (3.0%) and Aedes butleri Theobald (3.6%). Collec- abundance of Cx. gelidus varied independently of tively, these species comprised >90% of the mos- rainfall (r = 0.29, df = 22, P = 0.162). quitoes collected during most months. Population age structure is summarized for Cx. In 1992, the highest rainfall occurred in May, tritaeniorhynchus and Cx. gelidus in Table 1. Popfollowed by another wet period from September ulations of Cx. tritaeniorhynchus and Cx. gelidus May 1997 259 VYTHILINGAM ET AL.: JE VIRUS INFECTION IN MALAYSIAN MOSQUITOES Cx.fuscocephala " Cx. sitiens Cx. quinquefasciatus "•" Ae. butleri ^Rainfall 0 J F M A M J J A S O N D J F M A M J J A S O N D 92 | 93 Fig. 2. Abundance of Culex spp. and Ae. butleri in Sepang, 1992-1993. were >50% parous during 8 of 12 mo. Decreased isolates were obtained from 7 species, 24 of them parity in November corresponded to greater abun- from Cx. tritaeniorhtjnchus. Other isolates were dance caused by greater rainfall and probably in- from Cx. gelidus, Cx. fuscocephala, Cx. quinquecreased local larval production. fasciatus, Ae. butleri, Ae. lineatopennis, and Aedes In total, 66,755 mosquitoes were tested in 1,336 (Cancraedes) sp. January 1993 had the greatest pools for JE virus (Table 2). Of these, 54.7% were number of positive pools (23.4%), followed by SepCx. tritaeniorhtjnchus, 16.8% Cx. gelidus, and tember (12.6%) (Table 3). 4.5% Cx. fuscocephala. In 1992, only 3 pools of Monthly minimum JE infection rates and abunmosquitoes were positive for JE, whereas in 1993, dance of Cx. tritaeniorhynchus are shown in Fig. 42 pools were positive by PAP staining and all ex- 3. When the seasonal abundance of Cx. tritaeniocept 2 were con firmed by PCR (Table 2). The 45 rhynchus was low, the minimum field infection Table 1. Age structure and parous rates for Cx. tritaeniorhynchus 1993 and Cx. gelidus collected in Sepang during Cx. tritaeniorhynchus Jan. Feb. Mar. April May June July Aug. Sept. Oct. Nov. Dec. Cx. gelidus Dilations Months Dilations 0 1 2 >2 74 17 15 40 38 56 51 55 49 58 112 42 48 17 26 27 47 23 69 57 67 54 46 46 5 7 9 9 14 9 25 6 10 8 3 20 4 3 0 1 0 3 0 0 0 3 0 2 43.5 61.4 70.0 48.1 61.6 38.5 64.8 53.4 61.1 52.8 30.4 61.8 0 1 2 >2 64 28 2 4 16 75 58 33 60 53 70 33 31 23 3 7 27 37 64 62 38 54 34 45 12 5 3 4 6 9 16 15 6 12 1 13 5 1 0 0 2 ] 1 1 1 0 0 2 42.9 50.9 75.0 73.3 68.6 38.5 58.3 70.3 42.9 55.5 33.3 64.5 JOURNAL OF MEDICAL ENTOMOLOGY 260 Vol. 34, no. 3 Table 2. JE virus isolations from mosquitoes collected in Sepang during 1992 and 1993 1992 Species Pools tested (no. 9 9) Cx. tritaeniorhijnchtis Cx. gelidus Cx. fiiscocejjhala Cx. sitiens Cx. quinquefasciatus Cx. hitacniorhtjnchus Cx. nigropunctatus Cx. vishnui Culex sp. Ae. albopicttis Ae. btitleri Ac. (Cancraedes) sp. Ae. lineatopennis Ac. vexans Aedes sp. At: subalbatus Annigcres sp. Uranotaenia sp. Ma. unifonnis Coq. crassipes Total 296 (14,800) 78 (3,900) 16 (800) 19 (950) 17 (850) 0 0 1 (50) 1 (43) 7 (345) 40 (2,000) 13 (645) 0 3 (148) 2 (95) 29 (1,450) 5 (250) 3 (150) 1 (50) 0 531 (26,526) 1993 Pools positive 0 2 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 3 Pools tested (no. 9 9) 435(21,750) 146 (7,300) 60 (3,000) 39 (1,950) 31 (1,550) 1 (50) 1 (45) 1 (46) — 3 (150) 39 (1,950) 6 (300) 6 (300) 7 2 14 0 2 11 1 (350) (100) (700) (100) (550) (38) 805 (40,229) Pools positive 24° 10 2 0 1 0 0 0 — 0 4 0 1 0 0 0 0 0 0 0 42 " Two pools were negative by PCR. rate and parity rates generally were high (Fig. 3; Table 1). Discussion Culex tritaeniorhynchus was the most common mosquito collected from the pig-farming area of Sepang. Isolations of JE have come mainly from this species in Sarawak (Macdonald et al. 1965, 1967; Simpson et al. 1970) and elsewhere in Malaysia (McCrumb 1955, Vythilingam et al. 1995). Cx. tritaeniorhynchus also was one of the predominant mosquitoes in ricefield ecosystems (Macdonald et al. 1965, 1967; Hill et al. 1969; Vythilingam et al. 1993). Table 3. Pools of mosquitoes tested and virus isolated by months during 1 9 9 2 - 1 9 9 3 1993 1992 Month Jan. Feb. Mar. April May June July Aug. Sept. Oct. Nov. Dec. Total a M No. pools tested — 54 19 29 125 53 40 22 34 51 36 68 531 Negative by PCR. pools l positive) — 0 0 1 (3.3) 0 0 0 0 0 2 (3.9) 0 0 3 No. pools tested 46 20 7 30 46 35 92 45 114 120 166 84 805 No. pools positive (% positive) 11(23.4) 2 (8.3) 0 0 2 (4.2) 2 (6.1) 4 (4.3) 2 (4.5) 12(12.6) 5 (3.7) 2 (1.4)° 0 42 In addition to Cx. tritaeniorhynchus, Cx. gelidus also was abundant in this area. The abundance of Cx. tritaeniorhynchus fluctuated as a function of rainfall. An increase in the numbers of Cx. fuscocephala, Cx. sitiens, and Ae. butleri also was observed during the northeast monsoon (SeptemberDecember). In contrast, Cx. quinquefasciatus abundance decreased with heavy rainfall, probably because the breeding habitats were washed away. The distribution of the different mosquito species was in close agreement with those described by other workers in endemic areas of JE (Macdonald et al. 1965, 1967; Hill et al. 1969; Vythilingam et al. 1993). Parity rates for both Cx. tritaeniorhynchus and Cx. gelidus were low during the month of November, thus supporting the hypothesis that an increased emergence was occurring in both species. In January, there were more mosquitoes with >2 dilations, which showed that the chances of being both infected and infectious was greater during this period. This could be one of the reasons why January had the highest percentage of infected mosquito pools. In addition to Cx. tritaeniorhynchus and Cx. gelidus, JE also was isolated from Cx. fuscocephala, Cx. quinquefasciatus, Ae. butleri, Ae. lineatopennis, and Ae. (Cancraedes) sp. Virus isolations have been reported from Cx. fuscocephala and Cx. quinquefasciatus elsewhere (Wang et al. 1962, Gould et al. 1974, Banerjee et al. 1977); however, these are the 1st isolations from Ae. butleri, Ae. (Cancraedes) sp., and Ae. lineatopennis in peninsular Malaysia. In Sarawak, JE virus has been isolated from Ae. curtipes Edwards (Simpson et al. 1970) and in Selangor it has been isolated from a mixed pool of May 1997 261 VYTHILINCAM ET AL.: JE VIRUS INFECTION IN MALAYSIAN MOSQUITOES 250 3 I Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 1993 0 Fig. 3. Minimum field infection rate of Japanese encephalitis virus infection and abundance of Cx. tritaeniorhynrlms. Ac. biitleri and Ae. albopictus (Vythilingam et al. 1995). Because only unfed mosquitoes were tested, the virus must have been from infected mosquito tissues. Further research must be carried out to compare the vector competence of these species. As for the temporal pattern of JE isolation from Cx. tritaeniorhynchiis, it was difficult to explain why the minimum field infection rate was low when the mosquito population was high. In November, a small percentage of the mosquitoes were parous; this could be one of the reasons for the low minimum field infection rate. Simpson et al. (1974) also reported that infection rates were low when population abundance was high. Other contributing factors may be the high proportion of antibody-positive swine which cannot function as virus amplifiers. Although JE virus has been isolated frequently from mosquitoes especially in 1993, there seem to be no reports of JE clinical cases from this area of Malaysia. One possible reason is that most adult Malaysians possess neutralizing antibody to the virus (Marchette 1967). Another reason could be that the cases are not diagnosed or that there is an under-reporting of JE cases. Serosurveys should be carried out to determine inapparent infection rates in this endemic area. Acknowledgments We thank the director, Institute for Medical Research, for his permission to publish this article. We also thank the staff of the Entomology Division for technical assistance in the field. This project was funded by IRPA-IMR 92-6, IMR 94-26, and JICA. References Cited Banerjee, K., P. K. Deshmukh, M. A. Ilkal, and V. Dhanda. 1977. Experimental transmission of Japanese encephalitis virus through Anopheles tessellatus and Culex fatigans mosquitoes. Ind. J. Med. Res. 65: 746-752. Detinova, T. S. 1962. Age grouping methods in Diptera of medical importance with special reference to some vectors of malaria. W.H.O. Monogr. Ser. 47. Fang, R., Hsu, and T. W. Lim. 1980. Investigation of a suspected outbreak of Japanese encephalitis in Pulau Langkawi. Malays. J. Pathol. 3: 23-30. Could, D. J., R. Edelman, R. A. Grossman, A. Nisalak, and M. F. Sullivan. 1974. Study of Japanese encephalitis virus in Chiangmai valley, Thailand IV. Vector studies. Am. J. Epidemiol. 100: 49-56. Hill, M. N., M.G.R. Varma, S. Mahadevan, and P. D. Meers. 1969. Arbovirus infections in Sarawak: observations on mosquitoes in the premonsoon period, September to December 1966. J. Med. Entomol. 6: 398-406. 262 JOURNAL OF MEDICAL ENTOMOLOGY [garashi, A. 1978. Isolation of Singh's Aedes albopictus cell clone sensitive to dengue and chikungunya viruses. J. Gen. Virol. 40: 531-544. Macdonald, W. W., C.E.G. Smith, P. S. Dawson, A. GanapathipiUai, and S. Mahadevan. 1967. Arbovirus infections in Sarawak: further observations on mosquitoes. J. Med. Entomol. 4: 146-157. Macdonald, W. W., C.E.G. Smith, and H. E. Web. 1965. Arbovirus infections in Sarawak: observations on the mosquitoes. J. Med. Entomol. 1: 335-347. Marchette, N. 1967. Arbovirus of Malaya. Med. J. Malaya 21: 388-389. McCrumb, F. R. 1955. Japanese encephalitis in Federation of Malaya and neighbouring countries, pp. 95— 96. Annual report. Institute for Medical Research Malaya, Kuala Lumpur, Malaysia. Paterson, P. Y., H. L. Ley, Jr, C. L. Wisseman, Jr., W. L. Pond, J. E. Smadel, F. H. Diercks, H.D.G. Hetherington, P.H.A. Sneath, D. H. Witherington, and W. E. Lancaster. 1952. Japanese encephalitis in Malaya. I. Isolation of virus and serologic evidence of human and equine infections. Am. J. Hyg. 56: 320— 330. Pond, W. L., S. B. Russ, W. F. Lancaster, J. R. Audy, and J. E. Smadel. 1954. Japanese encephalitis in Malaya. II. Distribution of neutralizing antibodies in man and animals. Am. J. Hyg. 59: 17-25. Simpson, D.I.H., C.E.G. Smith, E. W. Bowen, G. S. Platt, H. Way, D. McMahon, W. F. Bright, N. M. Vol. 34, no. 3 Hill, S. Mahadevan, and W. W. Macdonald. 1970. Arbovirus infections in Sarawak: virus isolations from mosquitoes. Ann. Trop. Med. Parasitol. 64: 137-151. Simpson, D.I.H., E. T. Bowen, H. J. Way, G. S. Platt, M. N. Hill, S. Kamath, T. W. Lim, P.J.E. BendeU, and O.H.U. Heathcote. 1974. Arbovirus infections in Sarawak October 1968-February 1970: Japanese encephalitis virus isolations from mosquitoes. Ann. Trop. Med. Parasitol. 68: 393-404. Shmiah, M. 1989. A review of Japanese-B virus encephalitis in Malaysia. Southeast Asian J. Trop. Med. Public Health 20: 581-585. Vythilingam, I., K. Inder Singh, S. Mahadevan, M. S. Zaridah, K. K. Ong, and M. H. Zainul Abidin. 1993. Studies on Japanese encephalitis vector mosquitoes in Selangor, Malaysia. J. Am. Mosq. Control Assoc. 9: 467-469. Vythilingam, I., K. Oda, T. K. Chew, S. Maliadevan, B. Vijayamalar, K. Morita, H. Tsucliie, and A. Igarashi. 1995. Isolation of Japanese encephalitis virus from mosquitoes collected in Sabak Bernam, Selangor, Malaysia in 1992. J. Am. Mosq. Control Assoc. 11: 94-98. Wang, S. P., J. P. Grayston, and S.M.K. Hu. 1962. Encephalitis on Taiwan. III. Virus isolations from mosquitoes. Am. J. Trop. Med. Hyg. 11: 141-148. Received for publication 19 March 1996; accepted 26 September 1996.