Francisca I. Cavaleiro

Morphology, ultrastructure, genetics, and morphometrics of Diplostomum sp. (Digenea: Diplostomidae) metacercariae infecting the European flounder, Platichthys flesus (L.) (Teleostei: Pleuronectidae), off the northwest coast of Portugal

The morphology, ultrastructure, genetics, and morphometrics of a species of Diplostomum von Nordmann 1832 (Digenea: Diplostomidae), isolated from the European flounder (Platichthys flesus (L.)) caught off the northwest coast of Portugal, are characterized. The metacercarial stage was found unencysted in the lens capsule of the eye. Light microscopical observations revealed the existence of some variability in specimen shape and size, with two morphotypes, referred to as “round” and “long”, being apparent. Scanning electron microscopy revealed a smooth, unarmed tegument, with the lappet region being the most irregular and porose. Both the oral and ventral suckers were provided with a series of papillae, which presented very distinctive ultrastructural features and were particularly conspicuous in the case of the ventral sucker. The two morphotypes detected were found to have 100% genetic correspondence in the 18S + ITS1 + 5.8S region of the rDNA. Since the genetic data for this metacercaria differed from those of the species of Diplostomum available in GenBank, a description of a new genotype (accession number GQ370809) is provided. The molecular phylogenetic analyses, in conjunction with principal components and cluster analyses based on morphometric data, revealed the existence of consistent differences between the Diplostomum sp. metacercariae from flounder compared with Diplostomum spathaceum, Diplostomum mergi, Diplostomum pseudospathaceum, and Diplostomum paracaudum. The latter of these species was found to be the most similar to the present material. Our results do not support an evolutionary separation of the European and North American species of Diplostomum.

Seasonality of metazoan ectoparasites in marine European flounder Platichthys flesus (Teleostei: Pleuronectidae)

Seasonal occurrence of metazoan ectoparasites is described for the first time in marine European flounder, Platichthys flesus (L.). The parasitofauna, in this study monitored during 1 year, was found to be similar to that previously recorded for flounder. Moreover, specimens of Caligus sp. Müller, 1785 and Lepeophtheirus pectoralis (Copepoda: Caligidae), Acanthochondria cornuta (Copepoda: Chondracanthidae), Holobomolochus confusus (Copepoda: Bomolochidae) and Nerocila orbignyi (Isopoda: Cymothoidae), and also, a praniza larva (Isopoda: Gnathiidae), were isolated. From these, L. pectoralis and A. cornuta were the dominant parasites in all samples of flounder, while Caligus sp., H. confusus, N. orbignyi and the gnathiid praniza seemed to infect the flounder only occasionally. As far as the seasonality of infections is concerned, it differed considerably from that described for estuarine environments. Indeed, both prevalence and abundance of L. pectoralis and A. cornuta reached significant peaks in the summer, whereas the literature identifies the autumn as the season of maximum infection on estuarine flounder. Thus, the former period seems more favourable for the occurrence of epizooties of L. pectoralis and A. cornuta in flounder culturing systems running on seawater and operated in the studied or similar environments.

Egg number-egg size: an important trade-off in parasite life history strategies.

Parasites produce from just a few to many eggs of variable size, but our understanding of the factors driving variation in these two life history traits at the intraspecific level is still very fragmentary. This study evaluates the importance of performing multilevel analyses on egg number and egg size, while characterising parasite life history strategies. A total of 120 ovigerous females of Octopicola superba (Copepoda: Octopicolidae) (one sample (n=30) per season) were characterised with respect to different body dimensions (total length; genital somite length) and measures of reproductive effort (fecundity; mean egg diameter; total reproductive effort; mean egg sac length). While endoparasites are suggested to follow both an r- and K-strategy simultaneously, the evidence found in this and other studies suggests that environmental conditions force ectoparasites into one of the two alternatives. The positive and negative skewness of the distributions of fecundity and mean egg diameter, respectively, suggest that O. superba is mainly a K-strategist (i.e. produces a relatively small number of large, well provisioned eggs). Significant sample differences were recorded concomitantly for all body dimensions and measures of reproductive effort, while a general linear model detected a significant influence of season*parasite total length in both egg number and size. This evidence suggests adaptive phenotypic plasticity in body dimensions and size-mediated changes in egg production. Seasonal changes in partitioning of resources between egg number and size resulted in significant differences in egg sac length but not in total reproductive effort. Evidence for a trade-off between egg number and size was found while controlling for a potential confounding effect of parasite total length. However, this trade-off became apparent only at high fecundity levels, suggesting a state of physiological exhaustion.

Site selection of Acanthochondria cornuta (Copepoda: Chondracanthidae) in Platichthys flesus (Teleostei: Pleuronectidae)

Acanthochondria cornuta (Copepoda: Chondracanthidae) (N=4841; prevalence: 80·0%; mean±s.d. [range] intensity: 28·8±24·0 [1-110] parasites) infected the branchial chambers of the European flounder, Platichthys flesus (L.), (N=210) according to an established spatial pattern. This was independent of host size. Higher intensities resulted, most frequently, in higher numbers of infection sites, probably due to increased intraspecific competition. Preferential infection of the ocular side was supported by the recorded abundance data and reflected, probably, the fishs bottom-dwelling behaviour. As the parasite develops from one stage into another, it seems to migrate towards different sites: the copepodites and pre-adult females occurred, mainly, in the holobranchs; the adults preferred the internal wall (non-gravid/post-gravid females; adult males) or the pseudobranchs (gravid females). The ventilating water current along with the blood supply are suggested as 2 major factors in determining parasite spatial distribution within the chamber. Parasite crowding in a restricted and narrow space of the posterior region of the internal wall was recorded frequently and resembled that previously reported for the plaice. Differences to other host-parasite systems previously studied should relate with the anatomy of the respiratory apparatus. Bigamous females are reported for the first time.

Impact of amoeba and scuticociliatidia infections on the aquaculture European sea bass (Dicentrarchus labrax L.) in Portugal.

In this work, a survey of sea bass, Dicentrarchus labrax, for amoebae and scuticociliatidia infections was carried out to evaluate their effects on the aquaculture of this fish species. The study was conducted in two different fish farms, one using seawater and the other brackish water. Infection with parasitic amoebae was found to be fairly high (prevalence: 43-73%), being more frequent in sea bass from the brackish water system. Although it was never found to cause outbreaks of disease or mortality in the surveyed fish, amoebic gill disease (AGD) histopathological signs, i.e., hyperplasia, secondary lamellae fusion and cavity formation (interlamellar vesicles), were observed in fish manifesting no macroscopic lesions. Furthermore, some evidence was found that amoebae affects the fishs general state of health and growth rate. These results indicate that cautious and detailed surveys to detect this sort of infection, and thus carefully plan its control, are fully justified. Compared with amoebic infection, the prevalence of scuticociliatosis was found to be low (7-13%). No outbreaks of disease or mortality were ever recorded, even when scuticociliatidia was present in turbot raised in the same water system, leading to serious outbreaks of disease and mortalities in that species. This suggests that sea bass is far more resistant than turbot to such infections, and if this is the case, the former fish may be a good farming alternative when scuticociliatidia is present.

Survey of the Metazoan Ectoparasites of the European Flounder Platichthys flesus (Linnaeus, 1758) along the North-Central Portuguese Coast

A survey was undertaken to identify metazoan ectoparasite species on the European flounder, Platichthys flesus (Linnaeus, 1758), in 4 different locations off the north-central Portuguese coast. Parasites of 7 different taxa were found: Caligus diaphanus, Caligus sp., and Lepeophtheirus pectoralis (Copepoda: Caligidae); Acanthochondria cornuta (Copepoda: Chondracanthidae); Holobomolochus confusus (Copepoda: Bomolochidae); Nerocila orbignyi (Isopoda: Cymothoidae); and praniza larvae (Isopoda: Gnathiidae). Lernaeocera branchialis, a common European flounder parasite in the North and Baltic Seas, was not observed among the surveyed fish. Caligus diaphanus, Caligus sp., and Nerocila orbignyi are new host records. The high prevalence and intensity values recorded for L. pectoralis and A. cornuta suggest that both parasite species are common to the European flounder along the north-central Portuguese coast. In contrast, infection levels with respect to the other parasite taxa were, in most cases, comparatively lower, thereby indicating that they only occur occasionally among flounders in the surveyed area.

Revisiting the octopicolid copepods (Octopicolidae: Octopicola Humes, 1957): comparative morphology and an updated key to species

A review of the present state of knowledge on the octopicolid copepods (Octopicolidae: Octopicola Humes, 1957) is presented. Characteristic morphological features are illustrated with scanning electron micrographs of Octopicola superba superba Humes, 1957. Comparative morphology analysis led to the conclusion that there is sufficient evidence to justify raising the two subspecies of O. superba to full species rank. A new identification key for the four species of Octopicola Humes, 1957, i.e. O. superba Humes, 1957, O. antillensis Stock, Humes & Gooding, 1963, O. stocki Humes, 1963 and O. regalis Humes, 1974, is proposed after evaluation of the morphological characters which vary more markedly between them. Among other characters, these species differ in the ornamentation of the third antennal segment, maxilla and male maxilliped. They are further distinguished by a combination of several character states concerning the fifth pedigerous somite.

Tetractinomyxon stages genetically consistent with Sphaerospora dicentrarchi (Myxozoa: Sphaerosporidae) found in Capitella sp. (Polychaeta: Capitellidae) suggest potential role of marine polychaetes in parasite's life cycle.

Known life cycles of myxosporean parasites have two hosts, but very few life cycles have been disclosed, especially in the marine environment. Sphaerospora dicentrarchi Sitjà-Bobadilla and Álvarez-Pellitero, 1992 is a systemic parasite from the European seabass, Dicentrarchus labrax (Linnaeus, 1758), a highly valuable commercial fish. It affects its health, leading to aquaculture production losses. During 2013 and 2014, an actinospore survey was conducted in a total of 5942 annelids collected from a fish farm in Algarve and from the Aveiro Estuary, in Portugal. A new tetractinomyxon actinospore was found in a capitellid polychaete, belonging to the genera Capitella collected at the fish farm. The tetractinomyxons were pyriform measuring 11·1 ± 0·7 µm in length and 7·2 ± 0·4 µm in width, and presented three rounded polar capsules measuring 2·4 ± 0·3 µm in diameter. The molecular analysis of the 18S rRNA gene sequences from the tetractinomyxons revealed a similarity of 100% with the DNA sequences deposited in the GenBank from S. dicentrarchi myxospores collected from the European seabass and the spotted seabass in the same fish farm and 99·9% similarity with the DNA sequence obtained from the myxospores found infecting the European seabass in the Aveiro Estuary. Therefore, the new tetractinomyxons are inferred to represent the actinospore phase of the S. dicentrarchi life cycle.

Syndesmis aethopharynx Westervelt & Kozloff, 1990 (Rhabdocoela: Umagillidae): a revisitation supported by scanning electron microscopy and molecular analyses

Species of Syndesmis Francois, 1886 are rhabdocoel platyhelminths typically found in echinoids. Our knowledge of this group is based on old and insufficient studies, generally representing light microscopy-based species descriptions. Syndesmis aethopharynx Westervelt & Kozloff, 1990 is an understudied endosymbiont of Paracentrotus lividus (Lamarck), which is likely to have been confused with the type-species, i.e. Syndesmis echinorum François, 1886, in the literature. In this work, S. aethopharynx is revisited based on new data on surface morphology and phylogeny and basic ecological data are provided. Scanning electron microscopy analysis revealed that the whole ventral region of the worm is equipped with cilia, which supports the assumption that the unciliated epidermal area reported for some species of umagillids, likewise endosymbiotic in echinoderms, is an apomorphy. Following the results of the molecular phylogenetic analysis, species of Syndesmis are closely-related to symbionts of other echinoderms, i.e. holothurians, and like them, may have evolved from some free-living or symbiotic Provorticidae ancestor. Syndesmis spp. may stand for a key group in studying the evolution of feeding strategies in rhabdocoels, as their phylogenetic position is between intestinal and coelomic symbionts, and since both the digestive tube and perivisceral fluid were recorded as sites of infection. The infection levels were low, likely reflecting the aggregated distribution of the host and the fragile nature of the symbiont.

The reproductive effort of Lepeophtheirus pectoralis (Copepoda: Caligidae): insights into the egg production strategy of parasitic copepods

The reproductive effort of Lepeophtheirus pectoralis (Müller O. F., 1776), a caligid copepod, which is commonly found infecting the European flounder, Platichthys flesus (Linnaeus, 1758), is studied in detail for the first time. Seasonal variation in body dimensions and reproductive effort are analysed. Data for 120 ovigerous females, 30 from each season of the year, were considered in the analyses. Females were larger and produced a larger number of smaller eggs in winter, than during the summer. The relationship between egg number and egg size is similar to that recorded for other copepods exploiting fish hosts. Much of the recorded variation was also similar to that reported for a copepod parasitic on an invertebrate host, which suggests the possibility of a general trend in copepod reproduction. Overall, our results provide further support for the hypothesis that there is an alternation of summer and winter generations.