Journal of the American Mosquito Control Association, 18(4):359-363' 2OOz Copyright @ 2002 by the American Mosquito Control Association' Inc' SCIENTIFIC NOTE COLONIZATION OF ANOPHELES MACULAZUS FROM CENTRAL JAVA, INDONESIA' MICHAEL J. BANGS,' TOTO SOELARTO,3 BARODJI,3 BIMO P WICAKSANA'AND DAMAR TRI BOEWONO3 ABSTRACT, The routine colonization of Anopheles maculatus, a reputed malaria vector from Central Java, is described. The strain is free mating and long lived in the laboratory. This species will readily bloodfeed on small rodents and artificial membrane systems. Either natural or controlled temperatures, humidity, and lighting provide acceptable conditions for continuous rearing. A simple larval diet incorporating a l0:4 powdered mixture of a.i"a beef and rice hulls proved acceptable. Using a variety of simple tools and procedures, this colony strain appears readily adaptable to rearing under most laboratory conditions. This appears to be the first report of continuous colonization using a free-mating sffain of An. maculatus. Using this simple, relatively inexpensive method of mass colonization adds to the short list of acceptable laboratory populations used in the routine production of human-infecting plasmodia. KEY WORDS Anopheles maculatus, Central Java, colonization, larval diet, malaria vector, Indonesia Anop he Ie s (Ce ll ia) maculat us Theobald belongs nificantly divergent in phylogenetic terms from oth- to the Theobaldi group of the Neocellia series, er members of the complex and may represent one which also includes Anopheles karwari (James) and or more separate species awaiting formal descrip- Anopheles theobaldi Giles (Subbarao 1998). The tion (Rongnoparut, personal communication). For An. maculatus species complex is considered an purposes of this article, the Central Java strain will "spe- important malaria vector assemblage over certain be referred to as An. maculatus [sensu stricto, parts of its wide Oriental distribution, namely cies B"l (Subbarao 1998). southem Thailand, western Indonesia, peninsular In nature, An. maculatus occupies a wide range Malaysia, and the Philippines (Reid 1968). It is of- of shaded to partially sunlit larval habitats, includ- ten reported as an important malaria vector in hilly ing small rock pools and ground depressions. In areas of Central Java Province and southern Su- Central Java, this malaria vector is closely associ- matra (Mardihusodo 1989, Takken and Knols ated with the hill regions' numerous small steams, 1990). In contrast, because of its more zoophilic which serve as a primary breeding habitat. This behavior and normally low human-biting densities, species prefers water that has collected in drying this species is of little or no medical importance on streambeds and seepages or along the margins of other islands of the Indonesian archipelago-Kali- slow-running creeks and small garden irrigation mantan. Sulawesi. and eastward to Timor Island ditches. Generally, clear fresh water with no or lim- (Hoedojo 1989). Eight species have been formally ited amounts of emergent and floating vegetation recognized in tllreAn. maculatus complex based on are prefered oviposition sites. Little else is defini- morphologic and cytogenetic studies of polytene tively known about the bionomics and adult behav- chromosomes (Rattanarithikul and Green 1986, ior of this species in Java. Rattanarithikul and Harbach 1990). Recent DNA The reputed importance of An. maculatus as a evidence indicates the complex may likely contain disseminator of malaria prompted an investigation (Rong- more members awaiting formal description into the selection and colonization of this species status of At?. noparut et a]. 1999). The taxonomic to facilitate scientific studies on insecticide resis- known; however, maculatus in Indonesia is not tance, vector capacity/competence, and life history the Javan populations are sig- there is evidence that by ensuring a reliable and continuous supply of mosquitoes. Previous attempts at continuous colo- An. maculatus appear limited, based on ' The opinions and assertions contained herein are those nization of (Jay- of the authors and are not to be construed as official or only 2 published accounts from Sri Lanka reflecting the views of the U.S. Naval Service or the In- ewickrema 1952) and peninsular Malaysia (Ow donesian Ministry of Health. Send reprint requests to the Yang et al. 1963). Jayewickrema's brief description Publications Office, American Embassy Jakarta, U.S. Na- did not mention mating conditions or egg output val Medical Research Unit No. 2, FPO AP 95620-8132. for this species, while Ow Yang et al. (1963) were 'U.S. Naval Medical Research Unit No. 2, Kompleks able to maintain a colony only by induced artificial P2M/LitBangKes, Jl. Percertakan Negara No. 29, Jakarta, mating procedures. Soon afterward, An. maculatus 10560 Indonesia. I Disease Vector and Reservoir Research Unit was obtained by the Centers for Disease Control (BPVRP), Jl. Hasanudin 123, PO Box 100, Salatiga, Cen- (CDC) and Prevention (Atlanta, GA) from the In- tral Java, 507 l3 lndonesia. stitute for Medical Research (Kuala Lumpur, Ma- 359 JounNar oF THE AMERIcIN Mosquno CoNrnor_ Assocr,qrroN Vol. 18,No.4 laysia) for use in a series of investigations on in- with 4-cm-wide filter paper strips placed along the fectivity and plasmodium transmission of differe n1 inner walls to prevent stranding and drying of eggs. species (Collins and strains et al. 1976, 1980, After hatching, approximately 400 first-stage larvae 1986). The CDC colony still had the distinct dis- were transferred into larger enamelware or plastic advantage of requiring artificial mating for its prop- rearing pans (35 X 24 x. 5 cm) containing approx- agation (Chin et al. 1966). In 1999, a colonv ofAn. imately 2 liters of clean, nonchlorinated water from maculatus form B was established in Thailand, but either well or bottled sources. Finely powdered lar- again, colony maintenance has required artificiat val food was spread evenly onto the water surface (Chareonviriyaphap, mating personal communica- twice daily, beginning with approximately 50 mg tion). The materials and methods described herein divided on day I and increasing by 50 mg incre- for maintaining a continuous breeding stock were mentally to 350 mg by day 7 and beyond as needed. developed using mostly local, inexpensive products Larval food was decreased as late fourth instars and and standard handling procedures. The only signif- pupae developed. In general, daily food require- icant items purchased from outside of Indonesia ments can vary by rearing pan and careful obser- were the collapsible aluminum adult cages (Bio- vation was necessary to adjust amounts to avoid Quip, Gardena, CA, and American Biological Sup- over- or underfeeding. The amount of food was ad- ply Co., Gainesville, FL). General collection, rear- justed to the developmental stage and numbers pre- ing, and handling methods for anopheline sent. Several different food combinations for larvae mosquitoes were based on extractions from stan- were tried (Barodji et al. 1985). A mixture (10:4) dard insectary methodology and procedures (Co- of low-fat (lean), dried, powdered beef and finely htzzr 1964, Gahan 1966, Foster 1980, Gerberg et ground rice hulls (bekatul) as previously described al- 1994). with the colonization of Anopheles aconitus Doe- ln t994, natural bloodfed An. maculat rs were nitz and Anopheles barbirostris Van der Wulp (Bar- obtained from outdoor and animal shelter resting odji et al. 1985, Soelarto et al. 1995) was found the collections in Desa (village) Hargotirto, Subdistrict most acceptable food for all instars. The ingredients Kokap (07"49'64"5; 110'06'07'E, elevation -775 ft are readily available at relatively low cost com- above sea level) within the Special Administrative pared with many other food mixtures that use com- Province of Yogyakarta in central Java. This site is mercially refined products like liver powder, pow- located in the Menoreh Hills of Kulonprogo Dis- dered brewer's yeast, finely ground food formulated trict, one of the most malaria-endemic areas in for laboratory animals, aquarium fish, dog food, or Java. This strain was first established in the Disease dog biscuits, to name a few. This beef/bekatul mix- Vector and Reservoir Research Unit, Salatiga, Cen- ture also helps reduce rearing maintenance because tral Java, within a spacious rearing room provided many larval foods (e.9., commercial dog food) are with ample natural light from windows and ceiling more likely to create unfavorable conditions (e.g., skylights. Temperature and humidity in the facility scum, bacterial growth) in the aquatic medium be- were not controlled and the mosquitoes were sub- cause of their excessive fat content. To further re- ject to prevailing tropical ambient temperatures, hu- duce scum formation on the water surface, posi- -f + midity (27 6oC; 75 l5%o relarive humidity), tioned electric fans provided a light flow of air over and lighting conditions. This strain later was adapt- the pans. Direct low-volume aeration through the ed under more controlled conditions of temoerature water using small electric aquarium pumps was also and humidity with a natural 12 h light:li h dark found acceptable. In some instances. water temper- regimen. ature was regulated at a constant 28-30"C by plac- In Salatiga, individual bloodfed females derived ing rearing pans on flat silicone rubber-coated eiec- from field samples were placed in paper cups for trical heating strips (Cole-Parmer, Vernon Hills, oviposition. Each cup (200-ml volume) was lined IL). Fresh water was added to the rearing pans daily with filter paper and filled one third with nonchlor- to replace loss from evaporation. inated water. The tops of the cups were covered Pupation normally occurs between days 8 and with a fine mesh synthetic screen and females were 10, sometimes sooner' depending on the mean wa- provided with a lOVo sucrose solution soaked into ter temperature (-28-30"C). Pupae were trans- cotton wool. Eggs were freely laid on the water ferred daily using a hand pipette from rearing pans surface and allowed to hatch, occurring within 2-3 into plastic cups (100 ml) containing clean water.