MEM INST OSWALDO CRUZ, RIO DE JANEIRO, 95(6) Nov/Dez 2000
PAGES: 839-842 DOI: Full paper
Differences in Brazilian Strains of Schistosoma mansoni Evaluated by Means of Morphometric Analysis of Cercariae of Both Sexes

José Roberto Machado-Silva, Cléber Hooper da Silva ++, Mário José dos Santos PereiraI, Regina Maria Figueiredo de Oliveira, Roberto Magalhães PintoII ++, Delir Corrêa GomesII + ++

Departamento de Patologia e Laboratórios, Faculdade de Ciências Médicas
IDepartamento de Anatomia Humana, Instituto de Biologia Roberto Alcântara Gomes, Uerj, Rio de Janeiro, RJ, Brasil
IILaboratório de Helmintos Parasitos de Vertebrados, Instituto Oswaldo Cruz, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brasil

Abstract

Morphometrics of Brazilian strains (BH, SJ and CMO) of Schistosoma mansoni cercariae were obtained with a computerized image analyzer (IMAGE PRO PLUS, MEDIA CYBERNETICS), considering the following characters: body area, tail, furcae, oral and ventral suckers and distance between them. For statistical analysis, the variance test (one-way Anova) was applied and significant differences of p 0.05 were considered. All morphometric values in the BH strain were significantly higher (p 0.05) than in the others. Lower values were obtained in females of SJ strain for all charaters, excepting the body area. Only this character showed to be significantly different in males and females of the three strains. pecimens of both sexes in the BH and SJ strains showed significant differences regarding all characters. It was observed that this morphometric analysis permits the characterization of strains and also the sex identification in S. mansoni cercariae. Due to its feasibility, this method can be applied as a tool in laboratories devoid of more complex equipment.

Based on the phenotypic characteristics of adult worms of both sexes, it was demonstrated that the Schistosoma mansoni Sambon, 1907 strains present morphometric differences (Saoud 1966, Coles & Thurston 1970, Magalhães & Carvalho 1973, Paraense & Corrêa 1981, Soliman et al. 1984, Machado-Silva et al. 1994, 1995, Neves et al. 1998). Moreover, S. mansoni strains can be identified by the chetotaxic study of the sensorial argentophilic papillae of cercariae (Bayssade-Dufour 1977, 1979, Cassone et al. 1979, Pino et al. 1988, 1997). Nevertheless, there are few reports on the morphometric analysis of non-sensorial structures in the cercariae aiming at taxonomic approaches (Zanotti-Magalhães et al. 1993). In this paper, data on male and female cercariae of different strains of S. mansoni are presented and discussed, since these larval forms show morphometric differences that also permit the identification of strains.

 

MATERIALS AND METHODS

Strains - All strains (BH _ Belo Horizonte, Minas Gerais; SJ _ São José dos Campos, São Paulo; CMO _ Ceará-Mirim, Rio Grande do Norte) are from Brazil and their maintenance under laboratory conditions was reported elsewhere (Machado-Silva et al. 1995).

Obtainment of single-sex cercariae - Miracidia were isolated from faeces of Swiss Webster (SW) albino mice experimentally infected with S. mansoni cercariae from each strain. One hundred and ninety-one specimens of Biomphalaria glabrata Say, 1819 and the same number of specimens of B. tenagophila Orbigny, 1835, measuring 2-4 mm in diameter, were infected with one miracidium each, of the referred strains. All infections were sympatric. Thirty-five days post-infection, snails were individually exposed to artificial light and those positive for cercariae were isolated. Shed cercariae from each snail were utilized in the infection of albino mice and for staining procedures.

Two 7-day-old SW mice (Mus musculus Lin-naeus, 1758) were percutaneously infected, with 50 cercariae each, shed by two positive snails/strain, respectively. Remaining cercariae from each snail were preserved for study.

Infected animals were sacrificed 30 days later in an ether chamber. Adult worms (males or females) were recovered from the portal and mesenteric venous system in Petri dishes containing a 0.85% NaCl solution. Sex of worms was confirmed under a stereoscope microscope and specimens were associated to the source of infecting cercariae. Samples of S. mansoni cercariae of the BH strain were previously identified regarding the sex and were supplied by the staff of the Department of Malacology, Oswaldo Cruz Institute.

Preparation of cercariae - Cercariae were fixed (10% formaldehyde solution), stained (carmine + ethanol 70o GL), dehydrated (ethanol 70o-100o GL) and clarified (phenol). Each step was performed using conical tubes with the cercarial solution, centrifuged at 1,000 g for 5 min. Whole mounts were obtained with two drops of the sedimented stained and clarified cercariae on slides under coverslip and preserved in a 1:1 Canada balsam and beechwood creosote solution.

Morphometric analysis of cercariae - A computerized image analyzer system (IMAGE PRO PLUS, MEDIA CYBERNETICS) was utilized. Areas of body (a), tail (b), furcae (c), oral (d) and ventral (e) suckers and the distance between them (f) were investigated (Figure). Fifty to 54 cercariae of each sex/strain were analyzed. Measurements were in mm2, except for the shorter distance between suckers (mm).

Statistical analysis - For statistical analysis, the variance test (one-way Anova) was applied, and significant differences of p< 0.05 were considered.

 

RESULTS

Sixteen snails (8.3%) were positive for S. mansoni cercariae of the CMO strain and 12 (6.2%) for the SJ. Groups of snails infected with miracidia of either the CMO or the SJ strains, were those that presented more specimens shedding male cercariae: 10 in the CMO and 7 in the SJ.

After comparison of strains, it was observed that all morphometric values were higher for males of the BH strain. Males of CMO strain showed higher values than those of SJ, except for the distance between suckers that was the same for both strains (Table I).

By means of statistical analysis of these data it was verified that the strains BH and SJ presented significant differences (p<0.05) when compared to one another, regarding all characters, whereas in the comparison of BH with CMO, differences were not observed in the oral sucker of both sexes and in the ventral sucker of females (Table II). Comparison of male specimens of CMO and SJ strains showed that there are no significant differences in morphometric values related to tail, furcae and ventral sucker areas and to the distance between suckers. Females of the BH strain also presented the highest morphometric values, except for the area of suckers that increased in the CMO strain (Table I).

Excluding the body area, lower values were those obtained with females of the SJ strain. Generally, females presented greater dimensions than those of the males; nevertheless, this did not occur for body area, furcae and oral sucker (CMO strain), furcal area (SJ strain) and ventral sucker (BH and SJ strains) (Table I).

All female characters showed significant differences (p<0.05) when comparing BH x SJ and SJ x CMO strains. Nevertheless, between BH and CMO strains these differences were not observed in the areas of the suckers (Table II). Males and females of the BH strain presented significant differences (p<0.05) related to all characters (Table III).

 

DISCUSSION

Traditionally, sex identification in cercariae has been achieved by means of their innoculation in laboratory animals and further recovery of adult worms 3-4 weeks later; nevertheless, even with this methodology, sex of adult worms is not always identified, for according to previous experiments, even during this interval, some worms do not reach sexual maturity (Paraense & Malheiros-Santos 1949).

Taking into account the asynchronism regarding maturation (Barbosa et al. 1978) worms may not show, after 3-4 weeks reliable morphological differences to induce sex identification.

Moreover, this procedure is slow, expensive and should not be applied in situations that require a fast and reliable sexual identification of larval S. mansoni. Although with the employment of molecular biology techniques this identification is easily obtained (Barral et al. 1993, Mc Cutchan et al. 1994), their access may be restricted, due to the necessity of a special equipment (Pino et al. 1988).

Few are the data on morphological analysis aiming at sex identification in S. mansoni cercariae. Pino et al. (1988, 1997)verified that this can be achieved by means of chetotaxic studies.

During the present investigation it was observed that morphometric differences in larval specimens have similarities with those referred for adult worms. Some reports indicate that the BH strain shows bigger dimensions than the SJ (Magalhães & Carvalho 1973, Paraense & Corrêa 1981, Zannoti-Magalhães et al. 1993, Machado-Silva et al.1995); this same situation occurs in the comparison between CMO and SJ strains (Machado-Silva et al. 1995). The present findings demonstrate that significant differences are not restricted to the dimensions of the body and tail as previously referred (Zanotti-Magalhães et al. 1993), but are also observed when other characters are analyzed (Table I).

Taking this into account, the present approach aims at the establishment of a simple, practical and reliable method, based on morphological evaluation, to properly identify the sex and the Brazilian strains of S. mansoni cercariae, just after their shedding from infected snails.

 

ACKNOWLEDGEMENTS

To Heloisa Diniz, Laboratório de Produção e Tratamento de Imagens, Departamento de Ensino, for technical assistance regarding figures and Departamento de Malacologia, Instituto Oswaldo Cruz, for the supply of a sample of cercariae.

 

REFERENCES

Barbosa MA, Pellegrino J, Coelho PMZ, Sampaio IBM 1978. Quantitative aspects of the migration and evolutive asynchronism of Schistosoma mansoni in mice. Rev Inst Med Trop São Paulo 20: 121-132.

Barral V, This P, Imbert-Establet D, Combes C, Delseny M 1993. Genetic variability and the evolution of theSchistosoma mansoni genome analysed by using random amplified polymorphic DNA markers. Mol Biochem Parasitol 59: 211-222.

Bayssade-Dufour C 1977. Variation chétotaxique chez les cercaires de Schistosoma mansoni, agent de la bilharziose intestinale en corrêlation avec l'hôte vertébré du parasite. C R Acad Sc Paris 285 Série D: 1511-1513.

Bayssade-Dufour C 1979. Variations du systeme sensoriel de la cercarie de Schistosoma mansoni. Intérét éventuel en épidémiologie. Ann Parasitol 54: 593-614.

Cassone J, Albaret J, Jourdane J 1979. L'indice cercarien de Schistosoma mansoni. Donnés expérimentales et épidémiologiques. Ann Parasitol 56: 381-394.

Coles GC, Thurston JP 1970. Testes number in East African Schistosoma mansoniJ Helminthol 44: 69-73.

Machado-Silva JR, Galvão C, Oliveira RMF, Presgrave OAF, Gomes DC 1995. Schistosoma mansoni Sambon, 1907: comparative morphological studies of some Brazilian strains. Rev Inst Med Trop São Paulo 37: 441-447.

Machado-Silva JR, Galvão C, Presgrave OAF, Rey L, Gomes DC 1994. Host-induced morphological changes ofSchistosoma mansoni Sambon, 1907 male worms. Mem Inst Oswaldo Cruz 89: 411-416.

Magalhães LA, Carvalho JF 1973. Estudo morfológico de Schistosoma mansoni pertencentes a linhagens de Belo Horizonte (MG) e de São José dos Campos (SP). Rev Saúde Públ (São Paulo) 7: 289-294.

Mc Cutchan T, Simpson A, Mulins J 1994. Differentiation of schistosomes by species, strain and sex using cloned DNA markers. Proc Natl Acad Sci USA 81: 889-893.

Neves RH, Pereira MJS, Oliveira RMF, Gomes DC, Machado-Silva JR 1998. Schistosoma mansoni Sambon, 1907: morphometrics differences between adult worms from sympatric rodent and human isolates. Mem Inst Oswaldo Cruz 93 (Suppl. I): 309-312.

Paraense WL, Corrêa LR 1981. Observations on two biological races of Schistosoma mansoniMem Inst Oswaldo Cruz 76: 287-291.

Paraense WL, Malheiros-Santos J 1949. O sexo do Schistosoma mansoni nas infestações produzidas por cercárias de um único molusco. Mem Inst Oswaldo Cruz 47: 35-62.

Pino LA, Morales G, Matinella L 1997. Cercaria chaetotaxy of two Venezuelan strains of Schistosoma mansoni. Bol Chil Parasitol 52: 61-66.

Pino LA, Morales G, Alarcon de Noya B, Noya O 1988. La quetotaxia cercariana en la diferenciación de sexos deSchistosoma mansoni. Mem Inst Oswaldo Cruz 83: 367-374.

Saoud MFA 1966. On the infra-specific variations of the male sexual glands of Schistosoma mansoniJ Helminthol 40: 385-394.

Soliman GN, Mansour NS, El-Fassal FM 1984. On the infra specific variations in the frequency of supernumerary testes in Schistosoma mansoniAm J Trop Med Hyg 31: 57-82.

Zanotti-Magalhães EM, Magalhães LA, Ueta MT 1993. Morphometrics of the adult worm and larvae of twoSchistosoma mansoni strains maintained in Mus musculus. Abstracts 4th International Symposium on Schistosomiasis, Rio de Janeiro, p. 212.

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