Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I: 213-216, 1999 213 Biological Factors Involving Life Cycle in the Invertebrate Vector, prolixus ES Garcia/+, MS Gonzalez*, P Azambuja

Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900, Rio de Janeiro, RJ, Brasil * Departamento de Biologia Geral, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brasil

Key words: Trypanosoma cruzi - life cycle - invertebrate host

Trypanosoma cruzi, the ethiologic agent of some mechanisms involved in the interaction T. (Chagas 1909), is transmitted cruzi-triatomine and also link insect factors through triatomine vectors during the meal on the with the success or the failure of strains/clones of invertebrate host. The parasite displays quite dis- parasites to establish the infection in the digestive tinct morphological and functional forms, alternat- tract of the vector. ing between replicative stages (epimastigotes in the TRYPANOSOMA CRUZI AND TRIATOMINE FACTORS vector midgut and amastigotes in vertebrate cells) and infective, non-dividing forms (metacyclic The T. cruzi-triatomine host interaction is com- trypomastigotes in the vector, which are deposited plex and mediated by several parasite and insect together with feces and urine, and bloodstream factors (Garcia & Azambuja 1991, 1996, Gonzalez trypomastigotes in mammals) (for revision see et al. 1998a). The parasite multiplies and differen- Brener 1973, Zeledon 1974, Garcia & Azambuja tiates in the triatomine gut. If bloodstream 1991, 1996, Gonzalez et al. 1998a). trypomastigotes are ingested by the vector, the first Because of the importance of Chagas disease transformation into epimastigotes occurs in the in Latin America a great effort has been devoted stomach and initiates few hours after parasite in- to develop an experimental model that could imi- gestion. The rate of differentiation and develop- tate basic features on T. cruzi life cycle in the in- ment of the parasite in the vector gut depends on vertebrate vector. Thus, we developed in our labo- the strains/clones of parasites and vector species ratory the Rhodnius prolixus model to study sev- (Garcia et al. 1984a). The transformation of eral parameters related to the high degree of inter- epimastigotes to metacyclic trypomastigotes occurs action between T. cruzi and invertebrate vector. in the entire gut but predominantly in the rectum Basically, this model implies in feeding of larvae (Brener 1973, Zeledon 1974, Garcia & Azambuja and adults of R. prolixus, through a special mem- 1991, 1996). Similarly, the metacyclogenesis of brane feeding apparatus, on blood containing dif- different T. cruzi strains/clones are related to the ferent strains/clones of T. cruzi and, at different nature of the parasite and its susceptibility to a intervals, determination of the number of parasite triatomine species (Garcia et al. 1986a). in the gut, urine and feces of the vector (see Garcia The establishment of T. cruzi infection in the & Azambuja 1991, 1996, Gonzalez et al. 1998a, gut of the invertebrate host is affected by several 1999). Herein, we will describe experiments, us- insect factors. Between these factors include a crop ing this useful parasite-vector model, to elucidate lytic factor which was purified, characterized as a basic peptide which lysed the erythrocyte mem- brane, releasing free hemoglobin for digestion (Azambuja et al. 1983). The crop hemolytic factor lysed many strains/clones of parasites, with differ- ent lysis kinetics (Azambuja et al. 1989a, Mello et This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), al. 1996). For instance, in vitro experiments with Escola Brasil-Argentina de Biotecnologia (MCT), epimastigotes of different T. cruzi strains/clones Programa de Apoio à Pesquisa Estratégica em Saúde demonstrated that some flagellates are more resis- (Papes, Fiocruz), and the ProBal project from Capes/ tant than others to the action of this lysing agent DAAD. The authors are fellowships from CNPq. (Azambuja et al. 1989a,b). Thus, Y strain of T. cruzi Corresponding author Fax: +55-21-590.3495. E-mail: is more susceptible to the incubation with this fac- [email protected] tor than Dm28c clone of this parasite. In vivo ex- Received 9 June 1999 periments confirmed these findings, i.e., Y strain Accepted 9 August 1999 of T. cruzi has low levels of development in 214 Biological Factors Involving T. cruzi • ES Garcia et al.

Rhodnius and Dm28c clone of this parasite devel- al. 1987). Therefore, we postulated that ops quite well in the vector (Garcia & Azambuja azadirachtin diminishes the production of 1991). Also, lectins are factors involved in the es- prothoracicotropic hormone (PTTH, brain hor- tablishment of the T. cruzi infection in the insect mone) that stimulates the production of vector. Mello et al. (1996) demonstrated that in- ecdysteroids rather than affects directly ecdysteroid fectivity in the gut is more complex correlating with synthesis in the PGs. Garcia et al. (1990), using agglutination of the parasite strain/clone by the gut head-transplant techniques and measuring the extract. Thus, DM28c clone of T. cruzi is aggluti- ecdysteroid levels in the hemolymph, confirmed nated and achieves a high infectivity levels while this hypothesis. They demonstrated that heads Y strain of the parasite, in contrast, is not aggluti- obtained from treated with azadirachtin nated and fails to develop in the vector gut. These transplanted into control headless insects decrease data indicate that gut lectins and crop hemolytic the ecdysteroid levels in the hemolymph. In con- factor can be important determinants of infectivity verse experiments, heads taken from control in- in the parasite-triatomine vector interaction. Sev- sects implanted into brainless azadirachtin-treated eral aspects on this subject are discussed by insects enhance the levels of these hormones in Azambuja et al. (present Symposium). the hemolymph (Garcia et al. 1990). Thus, they Fraidenraich et al. (1993) isolated peptides from suggested that larvae of R. prolixus treated with the hindgut of Triatoma infestans resulting from azadirachtin decreases the synthesis and release of the digestion of a component of the αD-globin chain PTTH, consequently affecting ecdysteroid produc- of chicken blood that are capable to induce the tion in the PGs (Garcia et al. 1990). All these facts metacyclogenesis of T. cruzi in vitro. Later, Garcia imply that the insect is not able to ecdysis to the et al. (1995) demonstrated that differentiation of next instar (Garcia et al. 1984b, 1986b, 1987). epimastigotes into metacyclic trypomastigotes in On the other hand, Nogueira et al. (1997) dem- the insect’s intestine is obtained if either hemoglo- onstrated that azadirachtin causes drastic changes bin or peptides corresponding to residues 30-49 in the epithelial cell organization of the midgut, and 35-73 of αD-globin chain are added to an in- which, after a blood meal, is normally composed fective plasma diet. They postulated that globin of epithelial cells and an associated membrane fragments, released by proteolytic enzymes attack- named perimicrovillar or extracellular membrane. ing hemoglobin, modulate the dynamics of T. cruzi These modifications included: clumps of the mi- transformation from epimastigotes into metacyclic crovilli, disorganization of the extracellular mem- trypomastigotes in the vector’s gut. brane layers, and alteration in the organization of TRYPANOSOMA CRUZI AND TRIATOMINE NEU- the basal portion of the epithelial cells. According ROENDOCRINE INTERACTION to Garcia et al. (1991) these midgut alterations are due to extensive changes in the neuroendocrine Garcia and Rembold (1984) and Garcia et al. system induced by azadirachtin. Moreover, (1984b) demonstrated that azadirachtin, an insect Gonzalez et al. (1998b) demonstrated, using de- growth inhibitor, blocks the ecdysis in R. prolixus. capitation, head transplantation, and ecdysone Along with the inhibitory effect on moulting, therapy, that insects decapitated before HCP azadirachtin also prolongs the duration in the stage present the same midgut alterations observed in followed by death (Garcia et al. 1984b, 1990). azadirachtin-treated insects. Also, they showed that Garcia et al. (1986b, 1987) described that the sen- head transplantation and ecdysone therapy partially sitive period to azadirachtin in R. prolixus is coin- reverse the decapitation effect on the development cident with the head critical period (HCP), i.e., the of the extracellular membranes. Thus, Gonzalez et period that the insect´s brain is essential for induc- al. (1998b) postulated that a brain factor (possibly ing the moulting (for details see Garcia et al. 1990). PTTH) may be a factor responsible for the midgut Clearly, azadirachtin decreases the ecdysteroid lev- cell organization in Rhodnius. els in the hemolymph (Garcia et al. 1986b, 1987) Furthermore, it was postulated that azadirachtin and blocks synthesis of the new cuticle, which is can affect not only the triatomine but also T. cruzi dependent on the ecdysteroid hormones (Garcia et development. Garcia’s group demonstrated that al. 1986a). As a consequence of these facts ecdys- azadirachtin applied at different intervals before, ial stasis as induced by azadirachtin is reversed by during, or after T. cruzi infection, reduces the num- treatment of R. prolixus larvae with ecdysone ber of parasites in the gut (Garcia et al. 1989a,b). (Garcia & Rembold 1984). Interestingly, in vitro A simple dose of azadirachtin is able of prevent- experiments indicated that only high concentration ing infection or re-infection of the vector by the of azadirachtin directly affects the production of parasites for long time (Gonzalez & Garcia 1992). ecdysteroids by the prothoracic glands in vitro After feeding R. prolixus with blood infected with (PGs) but physiological dose does not (Garcia et T. cruzi clone Dm28c without azadirachtin, the Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 94, Suppl. I, 1999 215 number of parasites increases 15 fold within three ria and trypanosomes, p. 270-276. In D Borovsky & weeks of infection, but in the azadirachtin fed in- A Spielman (eds), Host Regulated Developmental sects the infection drops within the same period to Mechanisms in Vector , Vero Beach, Fl. 14%, and after 30 days no parasites can be detected Brener Z 1973. Biology of Trypanosoma cruzi. Annu (Garcia et al. 1989a,b). In addition, to the total loss Rev Microbiol 27: 347-382. Chagas C 1909. Nova tripanomíase humana. Estudos of the ingested parasites, a later infection with T. sobre a morfologia e o ciclo evolutivo do Schizotry- cruzi is inhibited for up to 120 days at least panum cruzi, n. gen., n. sp., agente etiológico de nova (Gonzalez & Garcia 1992). Finally, a direct toxic entidade mórbida do homem. Mem Inst Oswaldo effect on the parasite can be nearly excluded, Cruz 1: 159-218. becasue T. cruzi cultivated in LIT-medium are not Fraidenraich D, Pena C, Isola E, Lammel E, Coso O, affected by annaddition of azadirachtin. T. cruzi Díaz Anel A, Pongor S, Torres HN, Flawiá MM also develops normally if blood is incubated with 1993. Stimulation of Trypanosoma cruzi azadirachtin and then injected into a mouse (Garcia adenylcyclase by alpha globin fragments from Tri- et al. 1989a,b). atoma infestans hindgut: effect of differentiation of Taken together, the effect of azadirachtin, de- epimastigote to trypomastigote forms. Proc Nat Acad Sci USA 90: 10140-10144. capitation, head transplantation and ecdysone Garcia ES, Azambuja P 1991. Development and inter- therapy on the gut organization and the action of actions of Trypanosoma cruzi within the insect vec- azadirachtin on the development of T. cruzi in the tor. Parasitol Today 8: 240-244. gut, we suggested that the changes induced on the Garcia ES, Azambuja P 1996. Infection of Triatomines gut epithelium by blood feeding may be respon- with Trypanosoma cruzi, p. 143-155. In JM sible for the establishment of T. cruzi infection in Crampton, CB Beard & Louis K (eds), The Molecu- the insect vector. Recently, Gonzalez et al. (1999) lar Biology of Insect Disease Vectors: a Methods explored the techniques of decapitation and head Manual, Chapman & Hall, London. transplantation on the development of T. cruzi in Garcia ES, Rembold H 1984. Effects of azadirachtin on R. prolixus. They demonstrated that decapitation ecdysis of Rhodnius prolixus. J Insect Physiol 30: 939-941. as well as azadirachtin treatment induce a dimin- Garcia ES, Azambuja P, Forster H, Rembold H 1984b. ishing of the parasite infection. In converse experi- Feeding and molt inhibition by azadirachtins A, B ments, head transplantation and ecdysone therapy and 7-acetyl-azadirachtin in Rhodnius prolixus reestablish the development of T. cruzi infection nymphs. Z Naturforsch 39c: 1155-1158. in headless and azadirachtin-treated larvae. They Garcia ES, Feder D, Gomes JEPL, Azambuja P 1987. also observed a direct relationship between the Effects of precocene and azadirachtin in Rhodnius development of extracellular membrane layers in prolixus: some data on development and reproduc- the gut and the growth of T. cruzi (Gonzalez et al. tion. Mem Inst Oswaldo Cruz 87: 67-73. 1999). They postulated that a neurohormone, pos- Garcia ES, Gonzalez MS, Azambuja P, Rembold H sibly PTTH, has an important role in maintaining, 1989a. Chagas disease and its insect vector. Effect of azadirachtin A on the interaction of a triatomine directly or indirectly, the ultrastructural organiza- host (Rhodnius prolixus) and its parasite (Trypano- tion of the R. prolixus gut, and that the extracellu- soma cruzi). Z Naturforsch 44c: 317-322. lar membranes are involved in the establishment Garcia ES, Gonzalez MS, Rembold H 1989b. Chagas and development of T. cruzi in the gut of this vec- disease and its insect vector. Curing effect of tor. 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