PCR effectiveness for sexing Schistosoma mansoni cercariae: application for sexing clonal cercarial populations

Abstract

Schistosoma mansoni is a digenean parasite known for its importance as a human pathogen. Compared with the other genera belonging to the digenean families, the Schistosoma genus is characterized by having acquired gonochorism from hermaphroditic ancestors [1]. Sex is genetically determined in the egg [2] and female schistosome is heterogametic (ZW) such as lepidoptera, reptiles and birds. A marked sexual dimorphism exists between male and female worms in the definitive host. However, no clear evidence for sexual dimorphism exists in the free larval stages (miracidia or cercariae) and in the intramolluscan stages (sporocysts). Morphological, histological and molecular methods have been developed in order to discriminate between male and female schistosome larvae. Differences in the number and pattern of the argentophilic papillae (chaetotaxy) were observed between male and female cercariae [3]. Histological studies were based on the fact that the female exhibits heterochromatin of the W chromosome in the interphase nuclei. Staining methods were applied on cercariae [4] and C-banding on miracidia, cercariae and sporocysts of S. mansoni [5]. Since 10 years, molecular methods have emerged in order to sex schistosome larvae and are always based on the heterosexual female-specific W chromosome. Three DNA repeated sequences were isolated and characterized from female W chromosome, D9 [6], W1 [7] and W2 [8], whose genomic sizes are ,respectively, 339, 476 and 715 bp. The W1 repeated sequence was shown to be female-specific in each stage of the life cycle in a Puerto Rican strain of the parasite [9]. These authors used a PCR technique with W1-specific primers on extracted DNA. The female specificity was confirmed by Southern blot. However, Grevelding [10] contested the female specificity of W sequences. This author showed, using extracted adult DNA, that the femalespecific W1 sequence may occur in both genders of a Liberian strain, but reminds female-specific in a Puerto Rican strain of S. mansoni. The variability of the W gender-specificity was studied for many strains by Quack et al. [11]. This study revealed that W1 and W2 elements exist in both genders in numerous strains, Liberian, Old Kenya, New Kenya, Senegal, Campinas and an isolate of Puerto Rican strain. But, the Old Brazilian strain remained W1 and W2 female-specific. The presence of W sequences in males comes from genomic instability, probably originating from DNA recombinations during sporocyst development [12]. The PCR technique commonly uses extracted DNA. However, a PCR technique without DNA extraction (direct PCR) was developed for individual S. mansoni cercariae [13]. This paper aims to test the effectiveness of the direct PCR on cercariae from a W1 female-specific strain and to propose an optimized protocol in order to determine the sex of mollusc infection. * Corresponding author. Tel: +33-4-68662050; fax: +33-468662281. E-mail address: mone@univ-perp.fr (H. Mone).