The nervous and prenervous roles of serotonin in Echinococcus spp.

Serotonin (5-hydroxytryptamine, 5-HT) is an important neuroactive and morphogenetic molecule in several metazoan phyla, including flatworms. Serotoninergic nervous system studies are incomplete and 5-HT function/s are unknown in Echinococcus spp., the flatworm parasites that cause hydatid disease. In the present work, we searched for genes of the serotoninergic pathway and performed immunocytochemical and functional analyses of 5-HT in Echinococcus spp. Bioinformatic analysis using the recently available Echinococcus multilocularis and Echinococcus granulosus genomes suggests the presence of genes encoding enzymes, receptors and transporters participating in 5-HT synthesis, sensing and transport in these parasites. However, some components of the pathway could not be identified, suggesting loss or divergence of parasite homologous genes. The serotoninergic neuroanatomy study performed by confocal scanning laser microscopy on different E. granulosus stages showed an increasing level of complexity when the protoscolex develops towards the adult stage and a progressive diminution when the parasite develops towards the metacestode stage. The role of 5-HT as a neurotransmitter in E. granulosus was evaluated by determining the effect of this substance on protoscolex motility. The addition of 5-HT to protoscoleces induced a significant increase in motility for short time periods. Preincubation with 100 µM citalopram, a known 5-HT transporter inhibitor, abolished the 5-HT-induced increase in motility, indicating that the effect could be mediated by a 5-HT transporter. Incubation of protoscoleces with 5-HT for time periods of several days induced a progressive differentiation towards the metacestode stage. The results indicate that 5-HT could have nervous and prenervous roles during Echinococcus spp. development. Taking into account the important roles of 5-HT in parasite biology and the divergence of 5-HT pathway genes with respect to human counterparts, the serotoninergic system could be considered as an amenable drug target against hydatid disease.

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes.

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.