Occurrence and effect of trematode metacercariae in two endangered killifishes from Greece

We report digeneans (Diplostomidae, Crassiphialinae) in the endangered freshwater fishes Valencia letourneuxi and Valencia robertae, endemics of Western Greece. Digenean metacercariae occurred in two forms in the abdominal cavity, excysted and encysted, the latter attached to the gonads, liver and alimentary tract. Parasites were, using morphological and molecular techniques, identified as two representatives of Crassiphialinae, specifically part of the Posthodiplostomum-Ornithodiplostomum clade. The spatial, seasonal, and age class variation in parasite prevalence was examined. Autumn parasite prevalence varied between the six populations sampled (18.2 to 100%). Seasonal prevalence at the two sites sampled quadannually peaked in autumn and reached its lowest value in spring; prevalence increased with size to 100% in young adult fish. We did not find a correlation between prevalence and host sex. Overall parasites’ weight averaged 0.64% of the host’s, while parasite weight increased with host weight. A comparison of relative condition and hepatosomatic and gonadosomatic indices of infected and metacercariae-free specimens showed that infection did not have a significant effect on host body condition and reproduction. Regarding the parasite’s life cycle, planorbid gastropods are proposed as potential first intermediate hosts in view of the host’s diet and occurrence data of molluscs in the ecosystem. This is the first record of a diplostomid digenean in valenciid fishes and of representatives of the Posthodiplostomum-Ornithodiplostomum clade in a native Greek freshwater fish. Our findings are discussed in conjunction to fish conservation interventions, since parasites may contribute to the decline of endangered species.This research was conducted in the frame of a wider study on the ecology, biology, genetics, and diet of V. letourneuxi undertaken by scientists of HCMR and was partially funded by the European Union of Aquarium Curators (EUAC) in the frame of the project "Rapid Assessment of the Status of Valencia letourneuxi, the Greek Killifish" (2005-2009). This research received support from the SYNTHESYS Project (http://www.synthesys.info/) (GB-TAF-2984) which is financed by European Community Research Infrastructure Action under the FP7 Integrating Activities Programme. N.K. and M.P.M.V. are supported by the Czech Science Foundation [P505/12/G112 (ECIP)]. The authors wish to thank Andrea Waeschenbach and D. Tim J. Littlewood for the advice regarding the molecular work, Rod A. Bray for the advice regarding parasite identification, the staff of the Wolfson Wellcome Biomedical Laboratories at the Natural History Museum for their technical support, Nicholas Koutsikos for producing the map with the sampling sites' location, and Marcelo Kovacic for curatorial services. For this study, HCMR had secured all necessary permits for fish collection from the Greek Ministry of Environment, Energy and Climate Change (permit numbers 97429/4350 and 85404/130).digenea; parasite prevalence; host body condition; planorbidae; platyhelminthes; valenciida

Anthelmintic Potential of Thymoquinone and Curcumin on Fasciola gigantica

Fasciolosis an economically important global disease of ruminants in the temperate and tropical regions, caused by Fasciola hepatica and F. gigantica, respectively, also poses a potential zoonotic threat. In India alone it causes huge losses to stakeholders. Anthelmintics including triclabendazole have been used to control this menace but the emerging resistance against the available compounds necessitates identification of novel and alternative therapeutic measures involving plant derived natural compounds for their anthelmintic potential. Thymoquinone (T) and curcumin (C), the active ingredients of Nigella sativa and Curcuma longa respectively have been used as antiparasitic agents but the information on their flukicidal effect is very limited. Adult flukes of F. gigantica were in vitro exposed to different concentrations of thymoquinone and curcumin separately for 3h at 37+ 1°C. A significant (p<0.05) reduction in the worm motility at 60 μM concentration of both T and C was observed though all the worms remained alive after 3h exposure, whereas the effect on egg shedding was statistically insignificant. Pronounced tegumental disruptions and erosion of spines in the posterior region and around the acetabulum was evident. A significant (p<0.05) decrease in glutathione-S-transferase and superoxide dismutase activity and reduced glutathione (GSH) level was observed, while protein carbonylation increased differentially. A significant inhibition of CathepsinL (CatL) gene expression in thymoquinone treated worms was also evident. Further, in silico molecular docking of T and C with CatL revealed a stronger interaction of curcumin with the involvement of higher number of amino acids as compared to thymoquinone that could be more effective in inhibiting the antioxidant enzymes of F. gigantica. It is concluded that both the compounds understudy will decrease the detoxification ability of F. gigantica, while inhibition of CatL will significantly affect their virulence potential. Thus, both thymoquinone and curcumin appeared to be promising anthelmintic compounds for further investigations