Trypanosoma Cruzi: Kinetics of Metacyclogenesis in Adult and Nymphal Panstrongylus Megistus

EXPERIMENTAL PARASITOL.OGY 59, 23 1-23X (1985)

Trypanosoma cruzi: Kinetics of Metacyclogenesis in Adult and Nymphal Panstrongylus megistus

JOSEPH PIESMAN Department of Tropical Public Health, Harvard School of Public Health, 665 Huntington Avenue, Boston, Massachusetts 02115, U.S.A.

AND

ITALOA. SHERLOCK

Centro de Pesquisas Goncalo Monk, Rua Waldemar Falcao 121, Brota.r, 40.000 Salvador, Bahia, Brazil

(Accepted for publication 6 November 1984)

PIESMAN, J., AND SHERLOCK, I. A. 1985. Trypanosoma cruzi: Kinetics of metacyclogenesis in adult and nymphal Panstrongylus megistus. Experimental Parasitology 59, 231-238. Adult triatomine insects, Panstrongylus megistus, naturally infected with the protozoa, Trypanosoma cruzi, contained significantly more metacyclic trypomastigote forms in their digestive tracts than did P. megistus nymphs. Metacyclics were scarce in all stages of triatomines fed once on infected mammalian hosts and subsequently starved. In contrast, triatomines fed repeatedly on infected mammalian hosts developed prolific T. cruzi infec- tions. Repeatedly fed adults contained 14 x more metacyclics than similarly fed nymphs. Nutritional factors appear to play a key role in regulating metacyclogenesis. Control cam- paigns designed to alter the age structure of populations of triatomines must take notice of

the high transmission potential of the adult stage. Q 1985 Academic PIW, IX. INDEX DESCRIPTORS: Trypanosoma cruzi; Protozoa, parasitic; Hemoflagellate; Chagas’ disease; Metacyclogenesis; Nutritional factors; Trypomastigotes; Panstrongylus megistus; Triatomine; Insect; Guinea pig.

descriptive (Brack 1968; Brener 1972; Zel- edon et al. 1984). Attempts have been made The development of Trypanosoma cruzi to measure the number of metacyclic try- within triatomine vectors has long been pomastigotes (= metacyclics) in triato- known. Brumpt (1912) originally recog- mines (Mshelbwala and Ormerod 1973; nized that the stage infectious to verte- Neal and McHardy 1977; Urdancta-Mo- brates, metacyclic trypomastigotes, is rales and Rueda 1977; Perlowagora-Szum- present in the hindgut of the insect. More- lewicz and Muller 1982), but information over, he realized that transmission oc- concerning the kinetics of metacycloge- curred on contact with contaminated feces. nesis in vivo is lacking. Dias (1934) later confirmed these findings. Torres (1915) was the first to note that The process by which T. cruzi transforms infection rates of T. cruzi in naturally in- into the metacyclic stage, “metacyclogen- fected adult triatomines exceeded those of esis” (Zeledon et al. 1977), has been quan- nymphal triatomines. Others have con- tified in vitro (Fernandes et al. 1969; Pan firmed this observation (Minter 1978; 1978a; Sher et al. 1983; Ucros et aZ. 1983). Zarate et al. 1980). However, the density In contrast, studies of metacyclogenesis of trypanosomes in naturally infected adult within triatomines have been principally and nymphal triatomines has not been com-

231 0014.4894BS $3.00 Copyright 0 19R5 by Academic Preqc, Inc. Ail rights of reproduction in any form reserved. 232 PIESMANAND SHERLOCK pared. Wood (1960) observed that fecal tween the tips of fine dissecting forceps. The fecal droplets of laboratory-infected adult Tria- suspension was withdrawn for measurement in a lOO- toma protacta contained more metacyclic mm3 Wiretrol pipet (Drummond Scientific Co. Broomall, PA, USA) and graduated into 50 equal di- trypomastigotes than similar droplets from visions, and the volume was measured to the nearest nymphal T. protracta. In this study, we de- 2 mm3 prior to transfer to another well. A 15-mm3 termined the density of metacyclic trypo- sample of feces was then transferred to a microscope mastigotes present in different stages of slide and examined under phase-contrast at 400 x naturally infected Panstrongylus megistus. magnification. If flagellates were detected, the density of flagellates (no. flagellates/ml fecal solution) was de- In addition, we examined the factors which termined with a hemocytometer. If the parasite sus- control metacyclogenesis in nymphal and pension was dense, a preliminary 1:10 dilution in phos- adult triatomines. phate-buffered saline was made before counting. If only a few parasites were present, a 15-mm3 sample was placed on a microscopic slide under a 22 x 22- MATERIALS AND METHODS mm coverslip and the total number of flagellates was The CA 31 strain of Trypanosoma cruzi, used to determined. We determined the number of parasites experimentally infect triatomines, was isolated from present in each bug as follows: Number of flagellates/ four infected Panstrongylus megistus collected from milliliter fecal solution X volume fecal solution = an infested house in Castro Alves, Bahia, Brazil, in Number of flagellates per bug. In order to test the May, 1982. A pooled fecal sample from these triato- accuracy of the assay, we added known quantities of mines was inoculated intraperitoneally into a guinea cultured trypanosomes (lo3 to 106) to diluted bug pig. For routine maintenance of the strain, parasites feces. The number of parasites detected in the assay were passaged alternately between guinea pigs and was generally slightly less than the known quantity triatomines at l-month intervals. Triatomines were al- added (mean = -6% in 40 trials). lowed to feed on guinea pigs containing l-10 trypo- Two methods were used to quantitate the motile par- mastigotes/mm3 blood. Guinea pigs were inoculated asite stages present in triatomine digestive tracts. Epi- intraperitoneally with the pooled feces of four to six mastigotes were distinguished from metacyclic try- infected P. megistus containing about lo6 flagellates. pomastigotes on the basis of motility in fresh 15-mm3 P. megistus were obtained from colonies maintained fecal samples, viewed with a phase-contrast objective at the Centro de Pesquisas Goncalo Moniz, Salvador, at 400x magnification. In addition, a lo-mm3 fecal Bahia, Brazil, for up to 10 years. Colonies were main- sample was spread on a slide, dried, and stained with tained by weekly feedings on chickens. These colonies Wright-Giemsa stain as previously described (Pan originate from P. megistus collected in the state of 1978a). Stained slides were examined at 1000x mag- Bahia, Brazil. nification. Epimastigotes were distinguished from Triatomines were starved for 15-30 days after metacyclic trypomastigotes on the basis of mor- molting before being fed on infected guinea pigs that phology (Hoare and Wallace 1966). Nonmotile amas- earlier had been inoculated intraperitoneally with lo6 tigotes or “sphaeromastigotes” were not counted. The flagellates harvested from P. megistus feces. Four to phase-contrast and Giemsa stain methods were ap- five weeks after inoculation, bugs were fed on animals plied to fecal samples collected from 50 naturally in- with parasitemias varying between 2.0 and 6.6 trypo- fected P. megistus. All preparations were examined mastigotes/mm3 blood. Parasitemia in hosts was de- for 20 min or until 200 parasites had been counted. termined by counting the number of trypanosomes in The mean proportion of metacyclics was similar in both 5 mm3 of blood placed onto a microscopic slide and methods (phase-contrast, 32.9%; Giemsa stain, 28.6%; examined under a phase-contrast objective at 400x r = 0.94, P < 0.001). Thereafter, only stained prepa- magnification. Bugs in plastic cups were held in con- rations were examined. tact with shaved guinea pig skin for 1 hr during each Triatomines were collected from 247 houses in experimental feeding. Infected triatomines were held Castro Alves, Bahia, Brazil, where Chagas’ disease is at room temperature (25-30 C.; 70-90% relative hu- endemic (Mott et al. 1976, 1978; Piesman et al. 1983). midity). P. megistus is the only triatomine species commonly We determined the number of flagellates present in infesting houses. Bugs were collected from the walls individual triatomine digestive tracts using the tech- and beds of infested houses without pyrethrum appli- niques of Miles et al. (1975). A 150-mm3drop of phos- cation. Captured triatomines were taken to the labo- phate-buffered saline was placed into 500-mm3 wells ratory, maintained at room temperature, and examined of plastic microtiter plates (Sterlin Co., Surrey, En- for evidence of infection within 6 days after collection. gland, UK). The entire contents of a digestive tract Triatomine collections were made between August, was placed in a well and thoroughly macerated be- 1981, and July, 1982. Trypanosoma cruzi: KINETICS OF METACYCLOGENESIS 233

TABLE I nificantly greater than in nymphs (21%) (x2 Prevalence of Tiypanosoma cruzi Infection = 59.8, P < 0.001). According to Stage in Nymphal and Adult Panstrongylus megistus Collected in Castro Alves, We determined the number of flagellates Bahia, Brazil and metacyclic trypomastigotes in 228 naturally infected P. megistus (Fig. 1). The Percentage No. of bugs overall number of flagellates and meta- Stage examined infected cyclics per insect increased with age; adults had significantly more flagellates (t = 9.02, 1 110 2.1 P < 0.001) and metacyclics (t = 9.24, P < 2 144 8.3 3 196 21.4 0.001) than did nymphs. Since adults are 4 171 26.9 generally larger than nymphs, we weighed 5 228 31.6 all triatomines (to the nearest 0.1 mg) be- M” 99 44.4 fore examination and computed the density F” 127 47.2 of flagellates and metacyclics per milligram a Male. insect body weight. While flagellate density b Female. per milligram was only slightly greater in adults than in nymphs, the metacyclic density was significantly (t = 8.06, P < 0.001) RESULTS higher in adults. Moreover, a significantly A total of 1075 Panstrongylus megistus greater proportion of parasites in adult bugs were collected from 247 houses in Castro were in the metacyclic stage (X = 37.5%, Alves. About 25% were infected (Table I). SD = 23.6) as compared to parasites in The proportion infected was related to age, nymphs (X = 18.7%, SD = 22.3) (t = 5.89, being greatest in adult females (47%) and P < 0.001). least in first stage nymphs (3%). The prev- The flagellate and metacyclic density in alence of infection in adults (46%) was sig- individual bugs of different stages, each fed

(N) 13) (I I) (40) (38) (491 (32) (55) (141) (67) Stage of Triotomine

FIG. 1. Numbers of Trypanosoma cruzi flagellates in naturally infected Panstrongylus megistus captured in houses in Castro Alves, Bahia, Brazil. Light bars represent mean number of flagellates. Dark bars represent mean number of metacyclic trypomastigote forms (i SE). 234 PIESMAN AND SHERLOCK once on a Trypanosoma cruzi-infected this experiment. The ingestion pattern in guinea pig and starved for 1 month, was adults and nymphs differed markedly. measured (Fig. 2). There was no difference During the 8 weeks of feedings, adults in- in the number of flagellates among the vari- gested more blood (X = 118.9 mg per week) ous stages. Adults had slightly higher meta- than did fourth-stage nymphs (Z = 57.2 cyclic densities overall, but third-stage mg). Moreover, the amount of blood di- nymphs contained more metacyclics than gested from one week to the next was much did adults. Thus, metacyclogenesis in greater in adults (X = 88.8% of prior weeks starved adults and nymphs was similar. bloodmeal) than in nymphs (67.5%). Con- In a subsequent experiment, the effect of sequently, the mean number of trypano- multiple infectious feedings was evaluated. somes ingested by adults per week (358.2) Fifty adult female and SOfourth-stage nym- was more than twice that ingested by phal P. megistus were allowed to feed on nymphs (167.5). infected guinea pigs at weekly intervals The pattern of metacyclogenesis in adult over a 2-month period. At the end of this and nymphal triatomines was examined. period, surviving triatomines were exam- The number of flagellates and meta- ined. Flagellate density was similar in fe- cyclics in 12 adults and 12 nymphs fed males and fourth-stage nymphs (Table II). weekly on infected guinea pigs was deter- However, significantly more metacyclics mined at 2-week intervals. While there were present (t = 5.47, P < 0.001) in adults were no metacyclics present in nymphs at and the density of metacyclics per mg was 2 weeks after initial bloodmeal, metacyclics greater in adults (t = 4.64, P < 0.001). The were present in adults (Fig. 3). The density mean proportion of parasites in the meta- of metacyclics increased rapidly from cyclic stage was 29.6% (t 16.1 SD) in Weeks 2 to 4 in adults. In contrast, the den- adults, as opposed to 11.6% (2 10.4 SD) in sity of metacyclics rose gradually in nymphs (t = 6.07, P < 0.001). nymphs during Weeks 2 to 8. Adults always Individual triatomines were weighed be- contained more metacyclics. fore and after each weekly feeding during The resistance of adult and nymphal P.

llympils AdYllS Slage of Triatomine FIG. 2. Numbers of Trypanosoma cruzi in the triatomine, Panstrongylus megistus, fed once on an infected guinea pig. Triatomines were examined 1 month after feeding. A total of 2.5infected triatomines of each stage were examined. Flagellate and metacyclic density represented as in Fig. 1. TYypanosoma cruzi: KINETICS OF METACYCLOGENESIS 235

TABLE II Number of T~JXUXWJ~U cruzi Flagellates and Metacyclics in Punstrongylus megistus Examined Two Months after Initial Feeding No. No. No. No. flagellates metacyclics No. flagellates metacyclics per mg per m Stage bugs per bug per bug body wt body wt F” 41b 5.75 f 0.36” 5.15 rt 0.47 3.27 -c 0.36 2.67 1 0.48 4 46’ 5.38 I 0.49 4.01 + 1.33 3.13 i 0.55 2.01 t 0.81 Note. Triatomines fed on infected guinea pigs at weekly intervals. a Female. b Fifty triatomines of each stage initiated experiment; only bugs surviving 2 months were examined. c TI i SD of Log,, (x + 1). megistus to starvation was tested. We held containing 25 bugs of approximately equal 20 adult females and 20 fourth-stage weight (76-86 mg). Group 1 was fed on in- nymphs without feeding immediately after fected guinea pigs weekly for 8 weeks. molting. Bugs were observed daily and the Group 2 fed on infected guinea pigs weekly mortality was recorded. Nymphs were for 4 weeks and then fed during the four more resistant to starvation. Ninety-five subsequent weeks on noninfected guinea percent of adults died after 40 days of star- pigs. Group 3 fed the first week on an in- vation and all died by 50 days. In contrast, fected guinea pig and was then fed on non- only 5% of nymphs died by 40 days, 50% infected guinea pigs for 7 weeks. Group 4 mortality was reached by 120 days, and fed the first week on an infected guinea pig 100% mortality at 150 days. and then was starved during 7 weeks. All We examined the influence of feeding re- nymphs were examined 2 months after ini- gimes on metacyclogenesis. Fourth-stage tial bloodmeal. Group 1 contained the nymphs were divided up into four groups greatest density of flagellates and meta-

2 4 6 8 Weeks Weeks FIG. 3. Timing of differentiation to the metacyclic trypomastigote stage during Tq~panosorna cruzi infection in Punstrongylus megistus. Fourth-stage nymphs and adult females fed weekly on infected guinea pigs. Twelve nymphs and twelve adult P. megistus were examined at biweekly intervals. Closed circles represent means of female adults, open circles represent means of fourth-stage nymphs (i SE). 236 PIESMAN AND SHERLOCK

cyclics (Table III). However, there was little difference between Groups 1, 2, and 3. Only Group 4 contained markedly fewer flagellates’ and metacyclics. Group 3 contained significantly more metacyclics than Group 4 (t = 5.33, P < 0.001). The provi- sion of a bloodmeal, albeit noninfectious, was enough to stimulate metacyclogenesis in Group 3, while little metacyclogenesis occurred in the starved Group 4.

DISCUSSION The ability to undergo metacyclogenesis may be both an inherent quality of partic- ular Trypanosoma cruzi strains (Crane and Dvorak 1982; Garcia and Dvorak 1982) and the response to stimuli provided by triatomine organs or extracts (Wood and Sou- za 1976; Lanar 1979; Isola et al. 1981). Or- ganic acids have been considered to be im- portant in controlling metacyclogenesis (Fernandes et al. 1969). However, the addition of organic acids did not trigger metacyclogenesis in vitro (Zeledon et al. 1977; Pan et al. 1978a). Moreover, antibodies (Sher and Snary 1982) as well as lectins (Pereira et al. 1981) have been implicated in the control of metacyclogenesis. How- ever, the effect of these agents within triatomines has not been established. Metacy- clogenesis may take place only after re- peated multiplication of epimastigotes (Pan 1978a, b). Perhaps, nutritional factors control metacyclogenesis (Sher et al. 1983; Ucros et al. 1983). In our study, starved triatomines did not produce large numbers of metacyclic trypomastigotes. Multiple bloodmeals could have provided the stim- ulus for epimastigote division and subsequent differentiation to metacyclics. In adult triatomines, the rapid rate of digestion may speed up melacyclogenesis. Wood (1960) suggested that contamination with feces from adult triatomines car- ried greater risk of acquiring T. cvuzi than did contamination with feces from nymphal triatomines, since nymphal feces contained few metacyclics. However, he found that Trypanosoma cvuzi: KINETICS OF METACYCLOGENESIS 237 nymphs defecate more frequently than ce parasite. Bulletin de la Societe de Pathologie ex- adults, making contact with nymphal feces otique 5, 360-367. more likely. Piesman and Sherlock (1983) CRANE, M. S. J., AND DVORAK, J. A. 1982. Trypano- soma cruzi: Spontaneous transformation by a Y observed that adult Panstrongylus me- strain variant in liquid medium. Experimental Par- gistus produced more feces than first- asitology 54, 87-92. to fourth-stage nymphs, but fifth-stage DIAS, E. 1934. Estudos sobre o Schizotrypanum cruzi. nymphs produced the largest volume of Memorias do Institute Oswaldo Cruz 28, l-110. feces. In our present study, we found nat- FERNANDES,J. F., CASTELLANI, O., AND KIMURA, E. 1969. Physiological events in the course of the urally infected adult P. megistus contain growth and differentiation of Trypanosoma cruzi. 9.8 x more metacyclics than fifth-stage Genetics 61 (Suppl.), 214-225. nymphs. The increased metacyclic density GARCIA, E. S., AND DVORAK, J. A. 1982. Growth and in adults probably presents a greater risk of development of two Trypanosoma cruzi clones in transmission than does the slightly greater the arthropod Dipetalogaster maximus. American Journal of Tropical Medicine and Hygiene 31, 2.59- volume of feces produced by fifth-stage 262. nymphs. HOARE, C. A., AND WALLACE, F. G. 1966. Develop- Attempts to control triatomines have uti- ment stages of trypanosomatid flagellates: A new lized insect growth regulators, i.e., juvenile terminology. Nature (London) 212, 1385- 1386. hormone (Pinchin et al. 1978) and preco- ISOLA, E. L. D., LAMMEL, E. M., KATZIN, V. J., AND GONCALEZ CAPPA, S. M. 1981. Influence of organ cene (Tarrant and Cupp 1978). In the short extracts of Triatoma infestans on differentiation of run, such manipulations may increase the Trypanosoma cruzi. Journal of Parasitology 67, proportion of triatomines in the adult stage. 53-58. A triatomine population so structured may LANAR; D. E., 1979. Growth and differentiation of increase the potential for transmission of Trypanosoma cruzi cultivated with a Triatoma in- Trypanosoma crud. festans embryo cell line. Journal of Protozoology 26, 457-462. MILES, M. A., PATTERSON,J. W., MARSDEN, P. D., ACKNOWLEDGMENTS AND MINTER, D. M. 1975. A comparison of Rhod- The Harvard component, under the direction of niusprolixus, Triatoma infestans and Panstrongylus Thomas H. Weller, is supported by the Wellcome Trust megistus in the xenodiagnosis of a chronic Trypa- and U.S. National Institutes of Health Grant AI16305- nosoma (Schizotrypanum) cruzi infection in a rhesus 05; its collaborative activities in Brazil are under the monkey (Macaca mullatta). Transactions of the aegis of the Pan American Health Organization. Field Royal Society of Tropical Medicine and Hygiene 69, work was assisted by Sr. Antonio C. Batista, Sr. An- 311-382. tonio C. Santos, and Sr. Tome S. Oliveiro. Laboratory MINTER, D. M. 1978. Triatomine bugs and the house- support was provided by Celia Lima and Maria C.B. hold ecology of Chagas’ disease. In “Medical En- Soares. Facilities at the Faculdade de Medicina were tomology Centenary Symposium Proceedings,” pp. provided by the Federal University of Bahia, Brazil. 85-93. 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