IN VITRO CULTURE OF THE MOSQUITO PHASE OF AVIAN MALARIA.

The development of a successful technique for the cultivation of the sporogonic cycle of Plasmodium relicturn is described. By overlapping successive stages, it has been possible to grow P. relictum in vitro from the stage of the gametocytes to that of infective sporozoites. Oocysts will develop about as well when separated from the stomach wall as when attached to it, and sporozoites reach an infective stage in culture in the complete absence of the mosquito's salivary glands. The course of adaptation to changes in temperature in the invertebrate as contrasted to that in the vertebrate host was followed. The insect phase does not develop satisfactorily in vitro much above the concentration of oxygen found in air. The possibility of correlating the biochemical requirements of the parasite with the biochemical constitution of different species of mosquito is pointed out. The topic which I have chosen to discuss in this Presidential Address will cover mainly investigations that have been carried on in our own laboratories. Very little work has been done elsewhere in attempting to cultivate the sporogonic or mosquito phase of the malaria parasite, perhaps because other investigators had a better appreciation than myself of the difficulties, technical and otherwise, connected with this method of approach, or else they may have doubted if even successful results could be applied to an understanding of the in vivo relationships of host and parasite. Nevertheless, it seemed to me that successful cultivation of the mosquito phase, which is extracellular, would offer a clearer understanding of the relationship between host and parasite than would be possible from the cultivation of the intracellular or erythrocytic phase. For at the beginning of our studies, the paraReceived for publication 11 November 1963. * Presidential Address, American Society of Parasitologists, Thursday, 7 November 1963, 11:00 AM, Illinois Room, LaSalle Hotel, Chicago, Illinois. site had not yet been cultivated apart from the red blood cell and hence had not been actually separated from the host tissue. Cultivation of the plasmodium in media of known constitution, even if the latter were not completely synthetic, would indicate through appropriate alterations in the composition of the media what was the nature of the dependence of the parasite upon the mosquito host. In other words, what biochemical systems had been altered or lost by the parasite so that it had come to rely upon the host to provide the essential substances or reactions needed for the completion of its life cycle? If the plasmodium from one particular species of mosquito could be grown successfully in a medium of known composition, it might be possible to determine with relative ease if failure of the parasite to develop in closely related species of mosquitoes had a biochemical basis. Another result of the in vitro cultivation of the mosquito phase would be the opportunity of comparing the biochemical dependence of NUMBER 1 This content downloaded from 157.55. 39.221 on Sun, 06 Nov 2016 05:09:20 UTC All use subject to http://about.jstor. org/terms 4 THE JOURNAL OF PARASITOLOGY, VOL. 50, NO. 1, FEBRUARY 1964 the parasite in a warm-blooded vertebrate with that in the poikilothermous insect. I propose to follow, at least briefly, the history of the development of these investigations in our laboratory and to summarize the progress that has been achieved so far. Finally, I shall discuss the implications of our results, most of which were not even suspected when the problem was initiated. As is true of most research today, these investigations were carried on with the help of many collaborators. I wish to acknowledge particularly the assistance of Dr. E. W. Clark and of Dr. Jowett Chao. For various reasons, principally the availability of a highly infectible vector as well as the ease of maintaining a plasmodium in a then readily obtainable vertebrate host, we chose to work with the mosquito, Culex tarsalis, and the avian parasite, Plasmodium relictum, which we have carried ordinarily in the canary. Our first experiments were merely attempts to see if easily visible stages of the plasmodium in the mosquito, namely the 5to 7-day oocysts, could be kept alive in vitro for a period of time approximating that required for their maturation in the living mosquito. The first medium used was a relatively simple one which had been employed by Trager (1938) for growing an insect virus in the reproductive tissue of mosquitoes. Despite a considerable loss of cultures, due to bacterial or fungal contamination, some of the oocysts attached to the mosquito's stomach remained alive in vitro for as long as 6 days, although neither growth nor development could be demonstrated in them (Ball, 1947). After these initially favorable results, we then attempted to improve the culture conditions in order to provide a more suitable environment for the oocyst. It was realized that the initial medium was woefully inadequate for the growth of a parasitic protozoan, and possibly also for the persistence of the tissues of the mosquito stomach. Consequently, we began to employ, with some modifications, the more complex media of known composition perfected by Kidder and Dewey (1945) for Tetrahymena, and by Anfinsen, Geiman, McKee, Ormsbee, and Ball (1946) for the erythrocytic phase of P. knowlesi. Using the knowledge that the mosquito stomach had relatively few bacteria when empty of food, and the finding that unfed mosquitoes, although rarely living for as long as 48 hr at 26 C, would survive well for at least this period of time at 18 C, we were able to obtain much better control of bacterial contamination. We merely starved the mosquitoes for 48 hr at 18 C. We could then add, as a further precaution, very low concentrations of antibiotics. The result of the use of these more nutritionally complete media, together with the better control of contaminants, was a considerable extension of the period of survival in vitro of 5to 7-day-old oocysts on the mosquito stomach. However, there was still no indication of any growth of this stage of the parasite (Ball, 1948). Since it was noted in the preparations where the oocysts were being maintained for these longer periods that the cells of the mosquito stomach had completely degenerated, leaving only a fibrous supporting framework, it seemed logical to investigate the possibility that the failure of the oocysts to develop might be due to loss of materials supplied to them by the host tissue. Since the media available at that time for maintaining insect tissue a few days in vitro proved entirely unsuitable for keeping whole mosquito stomachs viable for more than a day or so, we then attempted to analyze chemically the bodies of various species of mosquitoes, including Culex tarsalis. Our aim was to find out if it was possible to devise a medium which would resemble more closely the hemocoel fluid of the mosquito host and at the same time would not be detrimental