Trypanosomes of the species Trypanosoma brucei reproduce primarily by binary fission, but the frequency of enzyme electrophoretic variants in natural populations of T. brucei has provided indirect evidence for the existence of a sexual cycle. These studies, coupled with studies of restriction fragment length polymorphisms of genes encoding glycolytic enzymes, have also provided evidence for T. brucei being diploid. Here we report direct evidence of gene exchange between two different clones of trypanosomes after mixed infection and full cyclical development in the tsetse fly vector.

Although much is known about factors which determine infection rates of salivarian trypanosomes (subgenera Nannomonas, Duttonella and Tryanozoon) in the tsetse fly Glossina, it is not clear why infection rates of Trypanozoon are high in mammalian hosts but low in wild-caught Glossina and why trypanosomiasis occurs where Glossina is not readily detectable. We report here that the feeding behaviour of trypanosome-infected Glossina differed from that of uninfected control flies. Infected flies probed more frequently and fed more voraciously. We describe a specific relationship between trypanosomes and the mechanoreceptors responsible for detecting the rate of blood flow, and show how infection affects that rate in the labrum. We suggest that the observed differences in feeding behaviour result from impaired function of the labral mechanoreceptors in infected Glossina.

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.

Genetic exchange may occur between two particular Trypanosoma brucei clones simultaneously transmitted by the same tsetse fly. We report here that this exchange takes place in the fly, through nuclear fusion. The resulting hybrids appear to be sub‐tetraploid, some particular DNA sequences from one of the parental stocks being lost before enough cloned hybrid trypanosomes could be harvested for DNA analysis. A further reduction of the DNA content of these hybrids occurs gradually upon growth and yields near diploid value in a major part of the population. This mode of hybrid generation is different from the fusion of haploid gametes, which is thought to occur normally upon inoculation of metacyclic trypanosomes in their mammalian host. In this respect, the sub‐tetraploid hybrids appear to undergo meiosis in the fly, generating sub‐diploid metacyclic forms, then fusion in the mammalian blood.

A series of new in vitro systems for the cultivation of bloodstream forms of Trypanosoma (Trypanozoon) brucei brucei, T. (T.) b. rhodesiense, and T. (T.) b. gambiense was developed. The standard system consists of a feeder layer of fibroblast-like cells derived from embryos of New Zealand White rabbits (REF( or a mountain vole, Microtus montanus (MEF), with HEPES-buffered Minimum Essential Medium (MEM), with Earle's salts, supplemented with 15% inactivated rabbit serum. These two and other feeder layers were cross-checked with different sera to test for growth support of bloodstream forms of the three trypanosome subspecies studied. Cultures could be initiated with bloodstream forms from mammalian hosts or from cryopreserved stabilates. Metacyclic forms from infected Glossina m. morsitans could also be sued as inoculum; they transformed within 6 h to bloodstream forms. Maintenance of cultures and growth properties are described in detail. Experiments were undertaken to confirm that the cultivated bloodstream forms still possess some fo the characteristic features of pleomorphic bloodstream populations. Cultivated bloodstream forms were always infective for mice, and a surface coat could be demonstrated by electron microscopy. They could also be cyclically transmitted through tsetse flies, and the metacyclic forms from these flies could be brought back into culture. In vitro cloning with single bloodstream forms and metacyclic forms could be achieve with high cloning efficiency. The consumption of glucose and the production of pyruvate and lactate were determined.