BACKGROUND: Despite their complex life-cycles involving various types of hosts and free-living stages, digenean trematodes are becoming recurrent model systems. The infection and penetration strategy of the larval stages, i.e. cercariae, into the fish host is poorly understood and information regarding their entry portals is not well-known for most species. Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Digenea, Strigeidae) uses the gilthead seabream (Sparus aurata L.), an important marine fish in Mediterranean aquaculture, as a second intermediate host, where they encyst in the brain as metacercariae. Labelling the cercariae with in vivo fluorescent dyes helped us to track their entry into the fish, revealing the penetration pattern that C. longicollis uses to infect S. aurata. METHODS: Two different fluorescent dyes were used: carboxyfluorescein diacetate succinimidyl ester (CFSE) and Hoechst 33342 (NB). Three ascending concentrations of each dye were tested to detect any effect on labelled cercarial performance, by recording their survival for the first 5 h post-labelling (hpl) and 24 hpl, as well as their activity for 5 hpl. Labelled cercariae were used to track the penetration points into fish, and cercarial infectivity and later encystment were analysed by recording brain-encysted metacercariae in fish infected with labelled and control cercariae after 20 days of infection. RESULTS: Although the different dye concentrations showed diverse effects on both survival and activity, intermediate doses of CFSE did not show any short-term effect on survival, permitting a brighter and longer recognition of cercariae on the host body surface. Therefore, CFSE helped to determine the penetration points of C. longicollis into the fish, denoting their aggregation on the head, eye and gills region, as well as on the dorsal fin and the lower side. Only CFSE-labelled cercariae showed a decreased number of encysted metacercariae when compared to control. CONCLUSIONS: Our study suggests that CFSE is an adequate labelling method for short-term in vivo studies, whereas NB would better suit in vivo studies on long-term performance. Cardiocephaloides longicollis cercariae seem to be attracted to areas near to the brain or those that are likely to be connected to migration routes to neuronal canals.

• Klíčová slova
• Cardiocephaloides longicollis, Cercarial penetration pattern, Cercarial survival and activity, Digenea, Metacercarial encystment,
• MeSH
• benzimidazoly MeSH
• cerkárie MeSH
• fluoresceiny MeSH
• fluorescenční barviva * MeSH
• infekce červy třídy Trematoda přenos   veterinární   MeSH
• interakce hostitele a parazita * MeSH
• larva MeSH
• metacerkárie MeSH
• mořan zlatý parazitologie   MeSH
• nemoci ryb parazitologie   MeSH
• stadia vývoje MeSH
• sukcinimidy MeSH
• Trematoda růst a vývoj   fyziologie   MeSH
• vodní hospodářství MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 5-(6)-carboxyfluorescein diacetate succinimidyl ester MeSH Prohlížeč
• benzimidazoly MeSH
• bisbenzimide ethoxide trihydrochloride MeSH Prohlížeč
• fluoresceiny MeSH
• fluorescenční barviva * MeSH
• sukcinimidy MeSH

During a regular veterinary inspection of fishes from Lake Balaton, Hungary, echinostomatid metacercariae (Digenea), with collar spines characteristic of species of the genera Petasiger Dietz, 1909 and Paryphostomum Dietz, 1909, were found in the lateral line scales of a roach Rutilus rutilus (Linnaeus), an apparently unique site. In a subsequent examination of 586 fishes from 20 different species, similar infections were found in 11 species. The infection was virtually restricted to the lateral line scales, other scales being infected only incidentally. These encysted metacercariae had 27 collar spines, including eight larger angle spines and 19 smaller dorsal spines arranged in two rows. Two types of metacercarial cyst were found. One type had a cyst diameter of 138-171 µm × 105-120 µm and three central dorsal spines that were larger than the remainder and tended to resemble the angle spines. The second type of metacercarial cyst had a diameter of 128-157 µm × 105-115 µm and all 19 dorsal spines of the metacercaria were of a similar size. ITS sequences of the second type of metacercaria exhibited a 100% similarity to sequences of two adult Petasiger phalacrocoracis (Yamaguti, 1939) specimens collected from the gut of Phalacrocorax carbo (Linnaeus) in Hungary and to P. phalacrocoracis deposited in the GenBank database. Sequences obtained from two metacercariae of the first type showed a 2.8-2.9 % difference from sequences of the second type of metacercaria and from those of adult specimens of P. phalacrocoracis from cormorants. Based on these results, the second type metacercaria is considered to be a larval stage of P. phalacrocoracis, but the identity of the first type is uncertain. The unusual location of these metacercariae in the lateral line scales is discussed in relation to their transmission.

• Klíčová slova
• Cyprinidae, Kis-Balaton Reservoir, Lake Balaton, metacercaria, molecular identification,
• Publikační typ
• časopisecké články MeSH

Parasites, especially brain-encysting trematodes, can have an impact on host behaviour, facilitating the transmission to next host and completion of the life cycle, but insufficient research has been done on whether specific brain regions are targeted. Using Cardiocephaloides longicollis as a laboratory model, the precise distribution of metacercariae in experimentally-infected, wild and farmed fish was mapped. The brain regions targeted by this parasite were explored, also from a histologic perspective, and potential pathogenic effects were evaluated. Experimental infections allowed to reproduce the natural infection intensity of C. longicollis, with four times higher infection intensity at the higher dose (150 vs 50 cercariae). The observed metacercarial distribution, similar among all fish groups, may reflect a trematode species-specific pattern: metacercariae occur with highest density in the optic lobe area (primarily infecting the periventricular gray zone of optic tectum) and the medulla oblongata, whereas other areas such as the olfactory lobes and cerebellar lobes may be occupied when the more frequently invaded parts of the brain were crowded. Mono- and multicysts (i.e. formed either with a single metacercaria, or with 2-25 metacercariae encapsulated together) may be formed depending on the aggregation and timing of metacercariae arrival, with minor host inflammatory response. Larvae of C. longicollis colonizing specific brain areas may have an effect on the functions associated with these areas, which are generally related to sensory and motor functions, but are also related to other host fitness traits such as school maintenance or recognition of predators. The detailed information on the extent and distribution of C. longicollis in fish encephalon sets the ground to understand the effects of brain parasites on fish, but further investigation to establish if C. longicollis, through purely mechanical damage (e.g., occupation, pressure and displacement), has an actual impact on host behaviour remains to be tested under controlled experimental conditions.

• Klíčová slova
• Brain-encysting, Cardiocephaloides longicollis, Histology, Microhabitat selection, Trematoda,
• Publikační typ
• časopisecké články MeSH