Authors

Discussion

The trematode parasite Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Digenea, Strigeidae) is widespread in marine ecosystems along European coastlines, especially in the Mediterranean. Its metacercariae parasitise, among other fishes, the gilt-head seabream (Sparus aurata L.), one of the most important marine species in Mediterranean aquaculture, with prevalence up to 54 %. Cardiocephaloides longicollis has a three-host life cycle. The cercariae penetrate the skin and migrate into the fish brain, where they encyst as metacercariae. The cysts are supposed to cause significant alterations in fish behaviour, possibly increasing their transmission to the definitive host.

For the study of the infectivity and penetration pattern of C. longicollis in fish second intermediate host, different experimental assays were performed. First, the effects of two in vivo fluorescent dyes on survival and activity of labelled cercariae were tested. Thereafter, an experimentally designated dose of one of the dyes helped to determine the penetration points of C. longicollis into S. aurata. Finally, the effect of both dyes on the infectivity capacity of labelled cercariae was tested.

Two different fluorescent dyes were tested: (1) 5(6)-carboxyfluorescein N-hydroxysuccinimidyl ester (CFSE), which stains intracellular amines in life cells and concentrates in the acetabular gland of the cercariae, and (2) Hoechst 33342 (NucBlue), which specifically stains DNA, i.e. the nuclei of life or fixed cells. Three different ascending concentrations were tested for each dye (CFSE at 20 µM, 50 µM and 100 µM, and NucBlue at1 drop/ml, 2 drops/ml and 3 drops/ml), and their effect on cercarial activity was recorded during 24 hours, showing no differences between concentrations and unlabelled cercariae. Additionally, intermediate doses of both dyes were selected based on labelling efficacy over time, to test the effect of dyes on cercarial activity and survival during the first five hours post labelling (hpl), i.e. the time during which cercariae should be highly efficient during penetration.

Intermediate doses of CFSE enabled a quick and accurate location of the cercariae on the host body surface without affecting cercarial survival. Results suggest a centralisation of the larvae around the facial areas of the fish. Furthermore, in order to evaluate the post-labelling infectivity of the larval trematodes, labelled and control cercariae were used to infect S. aurata. The water where the fish were infected was analysed to count the tails as cercariae lose them when penetrating the host. Additionally, 20 days post infection, fish brains were examined for metacercariae, showing no significant differences between the number of metacercariae developed from NB-labelled and control cercariae. In contrast, the number of metacercariae developed from CFSE-labelled cercariae was significantly lower compared to the control treatment.

In conclusion, the in vivo fluorescent dye CFSE appears to be the most adequate labelling treatment in experimental assays studying the infectivity capacity and penetration strategy of cercariae of C. longicollis.

This study was supported by projects MSM200961706 (Czech Academy of Sciences), European Centre of IchthyoParasitology, Centre of excellence program of the Czech Science Foundation; project No. P505/12/G112, AGL2015-68405-R (MINECO/FEDER, UE), Prometeo/2015/018 and Revidpaqua ISIC/2012/003 of the Valencian Regional Government.