The present study provides an overview of the structures linked to fish host finding, recognition, and invasion of one of the most commonly occurring morphotypes among trematodes, furcocercariae. For this, we use free-swimming cercariae of the strigeid Cardiocephaloides longicollis (Rudolphi 1819) Dubois, 1982. Their elongated cercarial body and bifurcated tail are covered by a tegument with an irregular surface, showing numerous folds arranged in different directions and a typical syncytial organization. Both the body and the bifurcated tail are covered with short spines, rose-thorn shaped, as well as four types of sensory papillae, distinguished by the presence or absence of a cilium, its length, and their position on the cercarial body. These papillae are especially important for free-living stages that rely on external stimuli to locate and adhere to the host. A specialized anterior organ is located at the anterior part of the cercariae and is encircled by a triangle-shaped group of enlarged pre-oral spines followed by a transverse row of enlarged post-oral spines that, together with the sensory papillae, allow active finding, recognition, and penetration into fish. The ventral sucker, covered with inner-oriented spines, sensory papillae, and cilia, helps during this process. The cercariae of C. longicollis possess three types of gland cells (a head gland and two types of penetration glands), each containing different types of secretory granules that play a role in host invasion. The protonephridial excretory system consists of an excretory bladder, a system of collecting tubules, flame cells, and two excretory pores in the middle of each furcae, which serve to control osmoregulation in their marine environment, as well as to eliminate metabolic waste. Together with the four types of sensory endings, the central ganglion forms the nervous system. Our results add novel information on the ultrastructure of strigeid furcocercariae, being essential to interpret these data in relation of their functional role to better understand the transmission and penetration strategies that cercariae display to infect their fish hosts.

• Keywords
• Free-swimming larvae, TEM, Transmission strategy, Trematode parasite,
• MeSH
• Cercaria MeSH
• Microscopy, Electron, Scanning MeSH
• Fishes MeSH
• Trematoda * ultrastructure   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

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.

• Keywords
• Cardiocephaloides longicollis, Cercarial penetration pattern, Cercarial survival and activity, Digenea, Metacercarial encystment,
• MeSH
• Benzimidazoles MeSH
• Cercaria MeSH
• Fluoresceins MeSH
• Fluorescent Dyes * MeSH
• Trematode Infections transmission   veterinary   MeSH
• Host-Parasite Interactions * MeSH
• Larva MeSH
• Metacercariae MeSH
• Sea Bream parasitology   MeSH
• Fish Diseases parasitology   MeSH
• Life Cycle Stages MeSH
• Succinimides MeSH
• Trematoda growth & development   physiology   MeSH
• Aquaculture MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 5-(6)-carboxyfluorescein diacetate succinimidyl ester MeSH Browser
• Benzimidazoles MeSH
• bisbenzimide ethoxide trihydrochloride MeSH Browser
• Fluoresceins MeSH
• Fluorescent Dyes * MeSH
• Succinimides MeSH