Memórias do Instituto Oswaldo Cruz Fundação Oswaldo Cruz, Fiocruz ISSN: 1678-8060 EISSN: 1678-8060 Vol. 89, Num. 1, 1994, pp. 111-112

Mem Inst Oswaldo Cruz, Rio de Janeiro, 
Vol. 89(1): 111-112, jan./mar. 1994

RESEARCH NOTE

Trypomastigotes in Cultures of Blastocrithidia culicis (Novy, MacNeal & Torrey, 1907) (Kinetoplastida:Trypanosomatidae)

Departamento de Protozoologia, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

Received 1 June 1993, Accepted 6 January 1994

Key words: trypomastigotes - Blastocrithidia culicis

Code Number: OC94018
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Arthropod trypanosomatids of the genus Blastocrithidia Laird, 1959 typically present the epimastigote stage, being largely accepted that their development never goes beyond this, although some species display amastigote stages as flagellar cysts (M Laird 1959 Can J Zool 37: 749-752, CA Hoare & FG Wallace 1966, Nature 212: 1385-1386, K Vickerman 1976, p. 1-34. In DHR Lumsden & DA Evans (eds), Biology of the Kinetoplastida vol. I, Academic Press, New York). Among the species of the genus, Blastocrithidia culicis (Novy, MacNeal & Torrey, 1907) has been much studied on account of two related features: its easy cultivation and the presence of bacterium-like symbionts in its cytoplasm (K-P Chang 1975 J Protozool 22: 271-276). In our laboratory, a strain of this species (ATCC 30268) isolated from Aedes vexans by FG Wallace & A Johnson (1961 J Insect Path 3: 75-80) and supplied by Dr EP Camargo (Sao Paulo University, SP, Brazil) has been routinely maintained in the Yeager's LIT medium (EP Camargo 1964 Rev Inst Med trop Sao Paulo 6: 93-100), either at room temperature or constantly at 27.3 C. On examining Giemsa-stained smears of these cultures, a trypomastigote stage was found in several opportunities, from the 8th to the 21st cultivation days. Such trypomastigotes occurred at rates ranging from 0.8 to 18.7% (more commonly 2-7%), being considerably smaller than the epimastigotes and generally presenting an inconspicuous undulating membrane and a short flagellum (see Figs). It has been proved that such trypomastigotes in fact belong to B. culicis by cloning the parasite culture in a flow cytometer (Coulter's EPICS 751), since they were found in eleven randomly selected clones. Trypomastigotes were also seen in cultures of an aposymbiotic strain of this parasite (ATCC 30257), suggesting that their occurrence is unrelated to the symbiont presence.

fig.1: Blastocrithidia culicis culture forms (symbiont-bearing strain). Giemsa stain after HCl treatment according to ALM Carvalho (1973 Rev Pat Trop 2: 223-274). Fig. 1: epimastigote and trypomastigote side-to-side for comparison. Figs 2-3: transitional forms between epi- and trypomastigote. Figs 4-8: trypomastigotes. All photomicrographs at same magnification (scale bar 10 um).

As far as I know, the presence of trypomastigotes in B. culicis cultures had not been previously demonstrated. Early descriptions of this species did not include this stage (FG Novy et al. 1907 J Infect Dis 4: 223-276, D Mezincesco 1908 C R Soc Biol 64: 975-976, Wallace & Johnson loc. cit., FG Wallace 1966 Exp Parasitol 18: 124-193). Other reports in culicids of morphological types capable of being identified as epi and trypomastigotes (sometimes both in the same insect) (A Laveran & G Franchini 1920 Bull Soc Path Exot 13: 138-147, A Missiroli 1930 Riv Malariol 9: 111-119) cannot be surely assigned to B. culicis mainly accounting that, in such cases, no symbiont-like organelles (the so called "diplosome" typical of this species) were mentioned and mixed infections were also possible, furthermore regarding that culicids can support the development and multiplication of trypanosomes from different vertebrates (JE Bardsley & R Harmsen 1973 Adv Parasitol 11: 1-73, JR Baker 1976 p. 131-174. In Lumsden & Evans (eds) loc. cit.).

At present, the meaning of such finding has not been determined, but taking into account the ability of B. culicis to defferentiate into trypomastigote and its isolation from bloodsucking insects (Novy et al. loc. cit., Mezincesco loc. cit.), the possibility of its being a Trypanosoma species is one that should be considered. This hypothesis had been already advanced by HM Woodcock (1914 Zool Anz 44: 26-33) and even verified by Novy et al. (loc. cit.) and Mezincesco (loc. cit.), both obtaining negative results throughout inoculations into several vertebrates. However, these experiments cannot be considered conclusive, since these authors did not report the presence of trypomastigotes in the inoculum (the possible infective forms) and could have employed unsusceptible hosts in their experiments. Moreover, as several Trypanosoma species only produce scanty parasitemia, their presence cannot be easily evidenced, accounting for negative results (CA Hoare 1972 The trypanosomes of mammals. A zoological monograph. Blackwell Sc. Publ., Oxford and Edinburg, V Apanius 1991 Parasitol Today 7: 87-90). Then, studies will be undertaken to verify the infectivity to different vertebrates of B. culicis cultures presenting trypomastigotes. Furthermore, the occurrence of this stage in experimentally infected culicids will be investigated.

It must me emphasized that the possibility of B. culicis being exclusively an insect parasite has not been rejected. This being true, the diagnosis of the genus Blastocrithidia proposed by Laird (loc. cit.) should be reviewed according to Wallace (loc. cit.) that also allowed the occurrence of individuals with postnuclear kinetoplast. However, the species presenting trypomastigotes included by Wallace (loc. cit.) in the genus Blastocrithidia (B. anophelis and B. pessoai) were also found in culicids and likewise the possibility of they being stages of some trypanosome cannot be ruled out. This view had been already advanced by Missiroli (loc. cit.) in the case of "Crithidia" anophelis (= B. anophelis).

Trypomastigotes in monoxenous trypanosomatids were also reported in the genus Rhynchoidomonas Patton, 1910, a poorly known group of parasites of the Malpighian tubules and intestine of nonhematophagous Diptera (review by Wallace loc. cit.). Despite the presence of trypomastigotes in cultures of B. culicis, with the data now at hand, it is not possible to establish any relationship between it and the Rhynchoidomonas species.

Acknowledgements: to Mrs Celeste da Silva Freitas de Souza for her assistance, to Dr Alvaro Bertho for operating the flow cytometer and to Dr Ortrud Monika Barth for allowing the photomicrographs to be taken at her laboratory.

Copyright 1994 Memorias do Instituto Oswaldo Cruz.

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