Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982

Born-Torrijos, Ana, van Beest, Gabrielle S., Merella, Paolo, Garippa, Giovanni, Raga, Juan Antonio & Montero, Francisco E., 2023, Mapping a brain parasite: occurrence and spatial distribution in fish encephalon, International Journal for Parasitology: Parasites and Wildlife 21, pp. 22-32 : 26-29

3.3. Mono- and multicysts of Cardiocephaloides longicollis

Cercariae of C. longicollis were found in the brain by 6 dpi with a similar morphology to that of free-swimming cercariae ( Fig. 3 ). However, by 21 dpi the parasite increased considerably in size and the tegumental structures changed to resemble metacercariae rather than free-

26

living cercariae, but lacking a visible cyst. Larvae found at 8 and 15 mpi had the normal morphology of encysted metacercariae that had developed a cyst in the brain. These were surrounded by a wall, and filled with a viscous glycocalyx-like material that maintained the position of the parasite within the cyst. Nevertheless, the host surface showed little disruption in response to the parasite.

Two types of cyst of C. longicollis metacercariae were observed in the brain of captured and experimentally-infected fish: (i) monocyst in which a single metacercaria was wrapped by a capsule ( Fig. 4 B ), or (ii) multicyst in which 2 to 25 metacercariae were encapsulated together ( Fig. 4 I ). Whereas the first type had a single wall in direct contact with the host, the multicyst may have a common capsule surrounding all metacercariae, or each metacercariae may produce its own capsule as a single parasite, likely resulting in metacercariae with more than one capsule. The composition of the capsule walls, both inner and outer, was identified by TEM and was composed mostly of host cells in necrosis and filamentous material possibly originated from the glycocalyx between the tegument of the parasite and the capsule ( Fig. 4 C, G ). The capsule was produced by the parasite so that the material accumulated in the inner layers of the capsule wall ( Fig. 4 C–D ), where the necrotic cells were probably produced by the host tissue when it came into contact with the parasite. Thus, the necrotic cells appeared to accumulate on the

27

outer layers of the capsule of monocysts and also in the outermost capsule of a multicyst ( Fig. 4 A–E ). However, the composition of the wall in each individual capsule within a multicyst was different, showing cells in necrosis on both sides of the wall ( Fig. 4 F–M ). In this case, the degraded material was not only in the outer, but also in the inner layers of the capsule wall, resulting in a thick but loose capsule wall ( Fig. 4 L, M ) that likely merged with other inner wall capsules close to it ( Fig. 4 K ). Although the immune response was likely to be very low, TEM revealed

28

that the capsule wall was characterised by the presence of fibrocytes that were flattened in the inner layers of the wall. In addition, the host tissue in contact with the cyst showed the occasional presence of macrophages ( Fig. 4 E ).

Figures

Fig. 3. Occupation of the fish brain by Cardiocephaloides longicollis in fresh (A, B) and histological samples (C–F). Cardiocephaloides longicollis metacercariae within (A) the PGZ and (B) the medulla oblongata in experimentally-infected fish one month after infection. Asterisks indicate the position of metacercariae. Cardiocephaloides longicollis metacercariae are found at 6 dpi in the tectal ventricle (C), and as they grow (D, 21 dpi; E, 8 mpi; F, 15 mpi) they occupy larger part of the tectal ventricle, and also the PGZ. The representations of brains indicate the sections and positions (yellow square) where metacercariae have been found. Legend: TeO striped, cerebellum in dots and Mo squared. ICL, inferior cerebellar lobe; Mo, medulla oblongata; PGZ, periventricular gray zone of optic tectum; TeO, tectum opticum; TV, tectal ventricle. Scale bars: A = 300 μm; B = 450 μm; C–F = 200 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4. Transmission electron micrographs showing the tegument and capsule walls of metacercariae of Cardiocephaloides longicollis. A and F illustrate the capsule wall of monocyst and multicyst metacercariae; B and I represent diagrams of monocyst and multicyst metacercariae showing the location of the following TEM micrographs. C – E Longitudinal section through the capsule wall and tegument of a monocyst. G, H, J-M Longitudinal section through the inner capsule wall and tegument of a multicyst metacercaria. D, E, J-M Detail of necrotic material accumulated on the capsule wall surrounding the metacercaria. K, Detail of inner capsule walls merging together within a multicyst. CW, capsule wall; F, fibrocyte; Gx, glycocalyx; GxF, glycocalyx filaments; ICW, inner capsule wall; M, metacercaria; M1- M3 number of metacercaria in a multicyst; MA, macrophage; Mt, metacercarial tegument; N, nucleus; NC, necrotic cells. Head arrows indicate glycocalyx filaments, asterisks (*) outside of the cyst, (**) inside of the cyst, (***) inside of the cyst when encysted with more than one capsule wall. Scale bars: D, E = 1 μm; C, G, H, J, L, M = 5 μm; K = 10 μm.