Sporogony and Experimental Transmission of Plasmodium mexicanum

Abstract

Plasmodium mexicanum oocysts developed on the stomach wall of Lutzomyia vexatrix occidentis. Pigment was visible in the oocysts until obscured by sporulation. Sporoblasts formed as islands in the oocyst. Sporozoites were routinely obtained from the hemocoel and produced infection in laboratoryreared lizards. In one laboratory-infected lizard, peak schizogony occurred in the late evening at a 4to 5-day interval. This article describes the sporogony of Plasmodium mexicanum and the experimental transfer of infection to laboratory-reared lizards with sporozoites from Lutzomyia vexatrix occidentis (Fig. 1). Phlebotomine sandflies are apparently the natural vectors of lizard malaria in California. The three known species of California sandflies breed in ground squirrel burrows and feed on a variety of cold-blooded vertebrates (Chaniotis and Anderson, 1968). Western fence lizards, Sceloporus occidentalis, commonly use the rodent burrows as refuges. Lizards in areas where sandflies occur are frequently infected with malaria (Ayala, 1970a; Jordan, 1970). The complete extrinsic cycle of P. mexicanum was followed in two species of sandflies: L. v. occidentis and L. stewarti (Ayala and Lee, 1970). MATERIALS AND METHODS The difficulty and effort required to raise sandflies in the laboratory has been cited by numerous workers (e.g., Hertig, 1964). Only wild L. v. occidentis were used in these studies. They were collected from the Twin Sisters Recreation Ranch, Solano County, where surveys indicated that lizard malaria did not occur (Ayala, 1970b). Collection methods using battery-powered light traps and burrow funnel traps were described by Chaniotis and Anderson (1968). In the laboratory, the sandflies were lightly anesthetized with carbon dioxide and transferred to wooden boxes containing clay pots of water covered with fine-mesh cloth screen. The flies were maintained at room temperature (24 to 26 C) or in chambers under controlled temperature and Received for publication 5 November 1970. *Current address: Departamento de Microbiologia, Facultad de Medicina, Universidad del Valle, Apartado aereo 2188, Call, Colombia, South America. humidity. Good survival was obtained only when the humidity was kept above 75%. Lizards were confined in small wire containers or cricket cages and placed with the flies. After feeding, female sandflies were kept in plastic tubes with a cloth screen top and a bottom layer of plaster of paris. The plaster was kept moist by placing the tubes in shallow trays of water. Rinsing the vials beforehand with Mycostatin? (Squibb & Sons, New York) helped control fungus growth. Fresh strawberries or raisins were placed on the screen top for a food source; if they became moldy they were easily removed without excessively contaminating the tubes. For examination, an insect was dipped in a dilute detergent solution, rinsed, and dissected in sterile cold-blooded Ringer's solution. The intestine was removed posteriorly from the abdomen as described by Chaniotis and Anderson (1968), or anteriorly by severing the terminal abdominal segments and pulling on the head with a fine dissecting needle. After examination under a compound microscope, the entire midgut and sporozoites in the surrounding fluid were taken up in a syringe and inoculated intraperitoneally into experimental lizard hosts. Hatchling lizards were raised from eggs in the laboratory using a modification of the incubation technique described by Zweifel (1961). The eggs were collected shortly after they were laid since thin-shelled reptile eggs soon desiccate in a dry terrarium. They were placed on the surface of a mixture of sterile sand and moist wood shavings in a closed plastic bag. Little further care was required. A light was occasionally directed toward the bag to increase the temperature (otherwise kept at room temperature). The hatchlings emerged 28 to 34 days later and were fed small mcalworms, fruit flies, and termites. Wild yearling lizards were collected on the northern abutment of the Golden Gate Bridge, where sandflies were not found and were assumed not to occur because of the severe terrain. No malaria was found in 24 adult lizards examined from the same site. Donor lizards came from Stark Creek in Riverside County and Calaveras reservoir in Santa Clara County. Lizards were kept in long tin cages (100 by 8 by 40 cm) with a 100-watt light bulb at one end providing a