Studies on the promastigote morphotypes of Leishmania mexicana and L. panamensis

The purpose of this study was to investigate the occurrence of different developmental forms of leishmania promastigotes during growth in vitro and attempt to correlate these with forms that occur in different localities in the gut of infected sandflies. One aim was to identify and characterise biochemical differences between the promastigotes and so provide insights into the adaptations of the promastigotes to the different environments encountered in the sandfly. The study was initiated with L. panamensis to enable investigation of the hindgut forms, stages which occur in only a few Leishmania species. A comparative approach was adopted later, however, and involved L. mexicana, a species which does not undergo hindgut development. Several promastigote forms were distinguished on morphological grounds and were investigated biochemically in order to ascertain stage-specific features. Since amastigotes of L. mexicana were readily available pure and in reasonable quantities, these were also included in my studies in an attempt to identify biochemical differences between the sandfly and mammalian stages of the parasite. Three morphologically distinct promastigote forms were identified in in vitro cultures of L. panamensis and L. mexicana. These forms morphologically resembled promastigotes that occur at different localities in and at different times after infection of, the sandfly host. Morphotype 1 promastigotes, which are similar in appearance to those found in the blood meal, occur primarily in early-log phase cultures in vitro. Morphotype 2 promastigotes have longer and more slender cell bodies than the rounded morphotype 1 forms and occur in large numbers in mid- to late-log phase in vitro cultures. They appear to resemble multiplicative mid-gut forms of L mexicana. L. panamensis promastigotes undergo a different developmental route in the sandfly and multiply as spatulate-shaped cells in the hindgut rather than the midgut. The morphotype 2 forms of this species may be equivalent to these developmental forms. Stationary phase cultures are less homogeneous, in terms of cell morphology, and contain a mixture of morphotype 2 and morphotype 3 promastigotes. The latter appear to be the same as the infective metacyclic forms that are thought to be responsible for transmission of infection from the sandfly to a mammalian host. The three promastigote morphotypes were found to differ metabolically. Morphotype 1 promastigotes, of both species, consumed larger quantities of amino acids during short term incubations in simple media than morphotypes 2 and 3. L. panamensis morphotype 2 stages consumed smaller quantities of some of the amino acids than stationary phase cultures containing morphotype 3 promastigotes. The opposite pattern was observed with L. mexicana promastigotes. All three promastigote morphotypes of L. panamensis released alanine during the incubation while morphotype 1 promastigotes of L mexicana consumed large quantities of this amino acid. In contrast morphotypes 2 and 3 of L. mexicana consumed and released negligible amounts of alanine. When each form was incubated with amino acids and glucose, amino acid utilisation differed only slightly from when amino acids were the sole exogenous substrates. The main difference with promastigotes of L. panamensis was that alanine release was greater when glucose was available. Morphotypes 2 and 3 produced alanine under both conditions but in larger quantities when glucose was present. L mexicana promastigotes behaved differently. In the absence of glucose, alanine was consumed by morphotype 1 promastigotes but not by morphotypes 2 and 3, the concentration not changing during the incubations. (Abstract shortened by ProQuest.)