Infections of Plasmodium knowlesi in the rhesus monkey were shown to be most infectious to mosquitoes 1 and 2 days prior to death. Higher numbers of oocysts per gut increased the percentage of mosquitoes with infected salivary glands, but did not appear to affect the intensity of the gland infections. Mosquitoes which were fed on a monkey which did not require antimalarial treatment were infected in 4 separate waves during a 50-day period. Transmission to rhesus monkeys was obtained by mosquito bite on 33 occasions with prepatent periods of 5 to 9 days and a median of 7 days. Twelve transmissions obtained by the intrahepatic inoculation of sporozoites resulted in prepatent periods of from 5 to 6 days. Plasmodium knowlesi is one of the monkey malaria parasites which has been used extensively as a laboratory model for studies of pathology, physiology, immunity, and drug response. Some of these studies were limited by the fact that sporozoite transmission was difficult to obtain; this problem appeared solved, however, when it was found that the mosquito Anopheles balabacensis balabacensis was not only readily susceptible to infection, but could also transmit the infection by bite (Collins et al., 1967). Since that time, we have investigated this mosquito further with regard to experimental transmission of P. knowlesi. Reported here are the results of these studies to determine the usefulness of this and other species of Anopheles as reliable experimental vectors of this parasite. MATERIALS AND METHODS Two strains of Plasmodium knowlesi were used in the studies. The H strain was isolated from a naturally acquired infection in man, the origin probably being the Temerloh district of peninsular Malaysia (Chin et al., 1965). The Hackeri strain was isolated from an A. hackeri mosquito collected near the Selangor coast, also in peninsular Malaysia (Wharton and Eyles, 1961). The strains have been maintained since their isolation either in the frozen state or by periodic passage in Macaca mulatta monkeys. Macaca mulatta monkeys from northern India were used throughout the study. Prior blood film and serologic examinations indicated that they were free of malarial infection. Received for publication 23 March 1971. 961 The A. b. balabacensis, A. freeborni, A. quadrimaculatus, A. stephensi, and A. maculatus mosquitoes have been maintained in our laboratory for several to many years (Collins et al., 1967). The A. atroparvus mosquito was obtained from the London School of Hygiene and Tropical Medicine. For the experimental feedings, 3to 6-day-old test mosquitoes were caged in pint ice cream cartons and allowed to feed on restrained infected M. mulatta monkeys. After feeding, all mosquitoes were held in a constant temperature incubator at 25 + 1 C and fed 5% Karo solution daily on a cellulose pledget. Beginning at 6 and continuing through 10 days after feeding, sample mosquitoes were removed from the cages, dissected in physiologic saline, and examined for the presence of oocysts. Examination of salivary glands for sporozoites commenced on day 9. The intensity of the gland infections was graded 1+ to 4+ as previously described (Collins et al., 1966). For the transmission attempts, mosquitoes were caged and allowed to bite individually on restrained monkeys. Immediately after biting, mosquitoes were dissected and the salivary glands examined for the presence of sporozoites. In some instances, the salivary glands were dissected into 10% monkey serum-saline solution, crushed under a cover slip, and then aspirated into a tuberculin syringe. Such suspensions of sporozoites were then introduced, at laparotomy, directly into the liver. The remaining bodies of the mosquitoes were triturated in a mortar and pestle with monkey serum-saline solution, centrifuged lightly, and the supernatant injected intravenously into the animal. Peripheral blood smears were made daily beginning 4 days after exposure to infection. Blood films, prepared by the Earle-Perez technique, were stained with Geimsa stain and the parasitemias recorded per mm3 of blood. Gametocyte counts were made from the thin film preparation and recorded per 500 WBC. This content downloaded from 157.55.39.195 on Tue, 26 Apr 2016 07:02:35 UTC All use subject to http://about.jstor.org/terms 962 THE JOURNAL OF PARASITOLOGY, VOL. 57, NO. 5, OCTOBER 1971 TABLE I. Results of 54 feedings of Anopheles b. balabacensis mosquitoes on 34 Macaca mulatta monkeys infected with Plasmodium knowlesi. Days prior to death -4 -3 -2 -1 0 Parasites/mm3 (X 1,000) Range 1.3* 1-97 7-760 86-1,600 600-2,360 Mean 1.3* 33 220 600 1,550 Mosq. lots pos./lots fed 1/1 5/12 14/18 12/18 2/4 Per cent lots pos. 100 41.7 77.8 67.7 50 Mean per cent infection 40 17.7 24.7 21.9 12.5 Mean GIIt 40 1,066 1,569 1,822 385 * Only one feeding. t Average number of oocysts per 100 guts.