The Timing of the Asexual Cycles of Plasmodium lophurae and of P. cathemerium

Abstract

Previous work had shown that with malaria parasites (such as Plasmodium knowlesi, P. cynomolgi, and P. cathemerium) which have a synchronous asexual cycle of 24 or 48 hr, the gametocytes are mature and infective to mosquitoes only for a short period (6 to 12 hr), and the synchronous asexual cycle is timed to ensure that this short-lived infective phase of the gametocytes coincides with the time at which the vector mosquitoes suck blood, i.e., at night; furthermore the synchronous asexual cycle is oriented by the cycle of the host's body temperature. In the present paper we have studied P. lophurae in which the asexual cycle is usually asynchronous and not a multiple of 24 hr. (Note: the present examples of P. lophurae form a somewhat unusual organism, because they are derived from a single isolation made in 1938 and maintained for 30 years since then by syringe passage.) We have found that the asexual cycle of P. lophurae takes 33 to 50 hr (mean, 40 hr). It bears no relation to day/night but it can be synchronized by blood passage at appropriate regular intervals; the cycle is then oriented to the hour of inoculation (so that the timing is a laboratory artifact, with no significance for natural infections). When P. lophurae is studied in chick embryos, the influence of a 24-hr temperature cycle on the asexual cycle is slight or negligible. (By contrast, the asexual cycle of P. cathemerium in duck embryos readily orients itself to a 24-hr temperature cycle in the environment.) The relation of the asexual cycle of P. lophurae to the behavior of its gametocytes cannot now be investigated because our strain of P. lophurae no longer shows gametocytes. This paper reports investigations on the asexual cycles of Plasmodium lophurae and P. cathemerium with particular reference to the degree of synchronicity and to their timing by stimuli supplied by the host. The work forms part of a larger program on the asexual cycles of P. knowlesi, P. cynomolgi, and P. cathemerium which has been reported in detail elsewhere (Hawking et al., 1968). P. lophurae was initially included in this study because its development in chick embryos seemed to facilitate study of the relation of parasite development to host temperature cycles (since the temperature of incubated eggs can be controlled easily). As will be described below, however, it was found that the asexual cycle of P. lophurae is normally not synchronous (except under certain artificial conditions) and that it is probably not very sensitive to entraining influences from the host. The study with embryos was then transferred to P. cathemerium, in which parasite the asexual cycle is more synchronous and is oriented to the cycle of its host. MATERIALS AND METHODS The strain of P. lophurae was kindly supplied by Dr. W. Trager and was described by Siddiqui and Trager (1966). It was maintained by intraReceived for publication 18 April 1969. peritoneal blood inoculation into young ducklings (and sometimes chickens) usually at weekly intervals. (As will be described later, the interval of subinoculation greatly influences the synchronicity.) P. cathemerium (German) was an old laboratory strain kindly supplied by Dr. 0. Dann (see Hawking et al., 1968). All experimental ducks and chickens were kept in a windowless animal room with light on at 07.00 and light off at 23.00. (Times in current British time for that season of the year.) Technique of inoculating and sampling embryos All manipulations (apart from cutting the shell) were performed in a sterile warm flattopped perspex box 45 cm long, 33 cm deep, and 20 cm high. At each end there was access through an opening 11 cm square (covered by a door when not in use). The floor contained a warm plate thermostatically controlled at 37 C. The egg was placed on its side in a small holder in the middle of the box and operations were viewed through a stereo-dissecting microscope (10 X) (with a built-in lamp) mounted above and outside the box. The embryos had been incubated 10 days when used. The inoculations were made intravenously by the standard technique. Briefly, the egg was candled and a suitable vein was marked on the shell. A piece approximately 1 cm square was cut from the shell with a dental drill and abrasive disk. The egg was then transferred to the sterile box described above, the surface was swabbed with alcohol, and the piece of shell was lifted off. The underlying membrane was then removed to expose the vein, great care being taken not to damage any underlying blood vessels. The injec-