Cultivation of the Insect Cycle of Plasmodia

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In vitro cultivation of the entire mosquito phase of Plasmodium relictum is now possible by steps, starting cultures of the parasite taken at various stages of development from the mosquito Culex tarsalis. Development of any individual culture, however, persists for only about 5 days. Sporozoites, produced in vitro from oocysts, are infective to canaries. This result offers additional evidence that sojourn in the salivary glands is not necessary for the sporozoites to become infective.

No post-zygotic development has been observed in cultures of the parasite initiated with freshly drawn blood from an infected bird, when stomachs, tissues, or whole body extract from uninfected mosquitoes are added to cultures. Similarly, when dissected out from the infective blood meal of the mosquito, these early post-zygotic stages do not establish themselves on uninfected mosquito stomachs which have been introduced into the culture.

Complex media, containing chick serum and chick embryo extract, are better for the development of oocysts in vitro than simple media. However, the presence of chick embryonic cells, mosquito tissue, mosquito whole body extract, or material from other biological sources does not appreciably improve the medium.

High O2 tension or the complete lack of O2 is inhibitory to the development of oocysts in vitro. The most suitable redox tension for these cultures is near neutrality, but a pH of 7.9 seems most favorable for the in vitro production of motile sporozoites.

Antibiotics also inhibit, to some degree, the development of oocysts as well as the contractility of the mosquito stomach. Streptomycin in normal saline, when given in successive injections to infected mosquitoes for only a short period of time, retards oocyst growth and causes a higher rate of mortality in the mosquitoes than injections of normal saline alone. Micro-organisms isolated from the midguts of both sexes of adult C. tarsalis have been found primarily insensitive to penicillin. This antibiotic has therefore been excluded from our media.

In starved but otherwise untreated mosquitoes, the parasite completes its normal development at the usual rate in the few surviving mosquitoes. Such nutritional stress is apparently more harmful to the mosquito than to the parasite.

The normal development of P. relictum in C. tarsalis is accelerated as temperature increases between 18° and 31°C. At 35°C, oocysts either fail to mature or the sporozoites produced do not enter the salivary glands, rendering the mosquito non-infective. Once the sporozoites have entered the salivary glands at room temperature, exposure of the mosquito to 35°C has no ill effect on them. The very early development of the parasite in the mosquito is irreversibly arrested when infected mosquitoes are kept at temperatures lower than 10°C, for extended periods of time.

The in vitro development of oocysts is also accelerated as temperature increases within the range of 18° to 31°C; but at any temperature in this range, the final degree of development is the same.

There seems to be no appreciable development of the parasite in mosquito abdominal sections placed in the chorio-allantoic cavity of chick or pigeon embryos incubated at 31°C. Similarly, no oocyst development takes place when the mosquito abdomen is transplanted to the hemocoel of a Tenebrio larva or pupa.

We have shown, for the first time, that chicks, ducklings, and pigeons are susceptible to sporozoite infection of P. relictum. Several natural cycles of infection through pigeons and C. tarsalis have resulted in no abatement of infectiousness of this parasite for either host.

Author Notes

Department of Zoology, University of California, Los Angeles, California.