In vitro Culture of the Vector Phase of Snake Hemogregarines in Mosquito Cell Lines

Abstract

Invertebrate stages of 2 species of reptilian hemogregarines belonging to the genus Hepatozoon have been grown in cultures of Grace's cell line of Aedes aegypti. Uninucleate oocysts, removed from the hemocoels of Culex tarsalis 6 days after the mosquitoes have bitten, develop to produce infective sporozoites. Three-day-old oocysts reach the sporocyst stage but do not develop further. Although various other kinds of coccidia such as the Eimeriina and the Haemosporina have been grown in culture, in some instances for the entire life cycle, there seems to be no report of the successful cultivation of any hemogregarine. Ball, Chao, and Telford (1969) maintained motile gametocytes or vermicules of an Hepatozoon from Boa constrictor for 16 days in a medium designed for cultivation of the insect phase of avian plasmodia (Ball, 1964). Although the gametocytes often associated in pairs or small clumps, no further development occurred. We now report the development in cultures of mosquito cell lines of the invertebrate phase of two species of Hepatozoon from uninucleate oocysts to infective sporozoites. MATERIALS AND METHODS One species of Hepatozoon was originally present in the blood of a Boa constrictor taken in the vicinity of Colima, Mexico. This may be H. fusifex (Ball, Chao, and Telford, 1969) or a possible second species in this host with a high infectivity for the mosquito, Culex tarsalis. The question of the number of species of Hepatozoon in the infected boas we have been investigating was discussed in the paper by Ball, Chao, and Telford (1969) and is still unresolved. The other species of Hepatozoon was H. rarefaciens isolated from the blood of the indigo snake, Drymarchon corais, also from the area of Colima, Mexico (Ball, Chao, and Telford, 1967). It is possible to transfer both species of Hepatozoon to clean gopher snakes, Pituophis melanoleucus catenifer, by feeding them infected C. tarsalis (Chao and Ball, 1969; Oda, Chao, and Ball, 1971). Since gopher snakes are easier to obtain and to handle than either B. constrictor or D. corais, we maintain both species of Hepatozoon in gopher snakes. Specimens of C. tarsalis which had Received for publication 1 June 1971. * Aided by Research Grants 00087 NIH, AID; 14,587 NSF, and 254-UCLA, Zoology. bitten infected gopher snakes were dissected under sterile conditions at various times after biting, and the oocysts in the hemocoel transferred to either Grace's (Grace, 1966) or Singh's (Singh, 1967) cell line of Aedes aegypti. These cell lines were carried respectively in either Grace's or Singh's medium containing Cynthia hemolymph. The cultures were maintained in small petri dishes (Chao and Ball, 1964) and kept at 22 to 23 C. Results were the same whether or not the medium was changed during the course of a single experiment.