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The manuscript provides an overview of the mid-term results of the citizen science campaign activities conducted within the Life CONCEPTU MARIS project (LIFE20 NAT/IT/001371) whose aim is to improve the conservation status of cetaceans and pelagic sea turtles by addressing information gaps, setting up an internationally agreed-upon approach for surveillance, and identifying appropriate conservation measures. It fosters a cooperative effort by engaging the scientific community, stakeholders, policy makers and citizens in a common effort to support biodiversity.

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.

Microplastics’ presence in the pelagic environment is still largely unknown due to the difficulty of sampling in this part of the ocean. In this study, we quantify microplastics’ exposure in a pelagic lanternfish species from the western Mediterranean, Lampanyctus crocodilus (Risso 1810), which occupies an intermediate position in the marine food web. L. crocodilus were captured in the Ibiza Channel by a trawling vessel and microplastics were extracted by digestion of their gastrointestinal systems. Almost half of the analysed lanternfish contained microplastics, mostly blue and black fibres (40.9% and 34.66%, respectively). In fishes with at least one microplastic, the median was 3 MPs/fish (CI 95% = 3.46–6.8), similar to other studies performed in other fish species in the area. Biometric parameters of fish, such as total length and body condition, were not correlated with the number of microplastics. Data presented here contribute to quantifying the severity of microplastic pollution in the pelagic environment and in a wild, non-commercial species.

Metastrongyloid lungworms from the family Pseudaliidae infect the lungs and cranial sinuses of cetaceans worldwide, except Stenuroides herpestis, which exhibits a striking terrestrial association with the Egyptian mongoose, Herpestes ichneumon. Previous phylogenies of the Metastrongyloidea that included some (2–7) marine species of the Pseudaliidae confirmed that these species are closely related, but also grouped species of Parafilaroides (family Filaroididae) between the members of the Pseudaliidae. In this study we extracted DNA from representatives of all six genera of the Pseudaliidae and amplified the second internal transcribed spacer (ITS2) and cytochrome c oxidase subunit I (cox1) genes in order to investigate the concept of the Pseudaliidae as a monophyletic unit. Three species of Parafilaroides were also included in the analysis. Maximum Likelihood and Bayesian Inference analyses of the concatenated genes resulted in a well-supported clade including the marine pseudaliids, S. herpestis and Parafilaroides spp. These findings validate the status of S. herpestis as a pseudaliid species and support the inclusion of Parafilaroides in the Pseudaliidae. Although males of Parafilaroides spp. lack a copulatory bursa, this trait itself is highly variable in the Pseudaliidae, which include abursate species. Furthermore, life cycles seem to be closely similar between both taxa. When all the available phylogenetic data on the Metastrongyloidea were mapped of onto a phylogeny of the Laurasiatheria, there was a strong suggestion that the Pseudaliidae may have descended from ancestors infecting terrestrial carnivores, with odontocetes being colonized in the marine realm as a result of a host-switching event from pinnipeds through shared fish prey. The origin of the association between S. herpestis and mongooses remains uncertain.

Temporal and geographical changes in the infections of intestinal helminths of striped dolphins, Stenella coeruleoalba were investigated in waters off the Spanish Mediterranean coast based on a sample of 233 dolphins stranded during 1982-2016. The influence of host-related factors (length and sex) and seasonality was also examined. Four helminth species had a prevalence > 5%, including adults of three cestodes, i.e, Trigonocotyle globicephalae (prevalence: 5.2%), Tetrabothrius forsteri (94.4%) and Strobilocephalus triangularis (18%); and juveniles of an acanthocephalan of the genus Bolbosoma (15.9%), identified as B. capitatum using molecular techniques. One immature specimen of Diphyllobothrium sp. (Cestoda) was also found in a single dolphin, and two juveniles of Bolbosoma balaenae in two dolphins. Trigonocotyle globicephalae seems to have low specificity for striped dolphins and was only found sporadically. Tetrabothrius forsteri and S. triangularis experienced a slight vs. a strong decrease, followed by a recovery, in infection levels throughout the study period, which are compatible with a reduction in the striped dolphin population caused by the Dolphin Morbillivirus outbreak in 1990. Infections of B. capitatum, a parasite typical from pilot whales, suddenly increased in 1990, then vanished. This rapid increase and following decrease are puzzling and can hardly be explained by a single factor. Infections of any helminth species were apparently uncorrelated to geographic origin, dolphin sex or season, but parasite load decreased with dolphin length in the case of T. forsteri and S. triangularis. To our knowledge, this study represents the first investigation of multi-decadal changes in cetacean parasites.

Routine monitoring of monogenean loads is a crucial step for infection management in aquaculture facilities. This analysis is usually performed from partial gill analyses and thus, knowledge of parasite distribution and size can be helpful to optimise diagnosis, estimate infection levels and improve management procedures. Gill location preferences and intraspecific size variations relative to parasite age, host factors and parasite abundance are herein determined for the monogenean Sparicotyle chrysophrii, one of the most challenging pathogens in gilthead sea bream aquaculture. Present findings on parasite location show that the life-history stages of S. chrysophrii have distinct location preferences. Analysis of intraspecific size variations of adult stages of S. chrysophrii revealed that the size of most morphological features of S. chrysophrii increased significantly with parasite age and host size. Positive and significant relationships between the size of the posteriormost clamp and host size pointed out the adaptation of the parasite to the gilthead sea bream size from the initial settlement. Host size also seems to restrict the development of parasites after maturity as the oldest specimens were exclusively found in mid-large fish (>18 cm). By contrast, no clear patterns were observed for the relationship between parasite size and location or abundance. Post-maturity size variations of S. chrysophrii should be considered for parasite identification and the clamp pair number should be fixed for accurate taxonomic diagnosis. Based on the site location of S. chrysophrii, the dorsal sections or the outermost holobranches would be suitable for adult detection while the analysis of the central and dorsal sections of the outermost holobranches may be a better option for finding most of the adult, juvenile or post-larval stages.

Abstract One of the most promising aquaculture species is the Atlantic bluefin tuna (Thunnus thynnus) with high market value; disease control is crucial to prevent and reduce mortality and monetary losses. Microsporidia (Fungi) are a potential source of damage to bluefin tuna aquaculture. A new microsporidian species is described from farmed bluefin tunas from the Spanish Mediterranean. This new pathogen is described in a juvenile associated with a highly severe pathology of the visceral cavity. Whitish xenomas from this microsporidian species were mostly located at the caecal mass and ranged from 0.2 to 7.5 mm. Light and transmission electron microscopy of the spores revealed mature spores with an average size of 2.2 × 3.9 μm in size and a polar filament with 13–14 coils arranged in one single layer. Phylogenetic analysis clustered this species with the Glugea spp. clade. The morphological characteristics and molecular comparison confirm that this is a novel microsporidian species, Glugea thunni. The direct life-cycle and the severe pathologies observed makes this parasite a hard risk for bluefin tuna cultures.

The Cetacean Migration Corridor is an important marine protected area for cetacean species in the Western Mediterranean, and part of its waters constitute the main fishing grounds for the Valencia region (East Spain) fishing fleet. Here we aim to assess the interactions between all active fisheries operating in the waters of the Valencia region and the cetacean species inhabiting this area. A total of 282 face-to-face interviews (51.37% of the total fleet) to fishers were held on 20 ports at the study area to gather information about cetacean by-catch and all types of interactions between fisheries and cetaceans. The interviewed bottom trawlers (n = 148 boats) reported a monthly by-catch rate (C) of 0.01 dolphins per vessel. Bottom trawling vessels operating at neritic zones only reported by-catch of common bottlenose dolphin (C = 0.009), while those fishing at oceanic zones by-caught striped dolphin (C = 0.006) and common bottlenose dolphin (C = 0.003). The interviewed artisanal fishers (n = 114 boats), the second most important fishery in the study area, only reported one dolphin by-catch event, but 90 of these boats communicated continued negative interactions with the common bottlenose dolphin, causing gear damage and catch take. The interviewed vessels gave an estimation of their annual economic loss produced by this interaction (mean ± SD: 2,998.10 ± 2095.02 € per boat). Other fisheries operating in the Valencia region were purse-seiners (n = 15 boats) and pelagic longlines, a fishery that has almost disappeared in the study area (n = 5 boats). The first one had the highest estimated dolphin by-catch rate (C= 0.04) and mainly interacted with common bottlenose dolphin, but also with striped dolphin. The information gathered from interviews was complimented with long-term stranding data (1990–2020). Despite the fact that only 7.80% of all recorded stranded cetaceans showed fisheries interaction signs, 26.72% of the common bottlenose dolphins recorded showed evidence of this interaction, thus confirming that it is the cetacean species most affected by fisheries in the area. Records of stranded striped dolphin (6.45%) also showed evidence of fisheries interaction. Based on both dolphin by-catch and gear damage, management plans are needed in the area to ensure cetacean conservation and also fisheries sustainability in waters inside and around marine protected areas at the Spain’s Mediterranean.

Abstract Background Current data about Pseudaliidae show contrasting patterns of host specificity between congeneric species. We investigated how both contact and compatibility between hosts and parasites contributed to the patterns of lungworm infection observed in a community of five species of cetaceans in the western Mediterranean. Methods The lungs of 119 striped dolphins Stenella coeruleoalba, 18 bottlenose dolphins Tursiops truncatus, 7 Risso’s dolphins Grampus griseus, 7 long-finned pilot whales Globicephala melas, and 6 common dolphins Delphinus delphis were analysed for lungworms. Parasites were identified by morphology and analysis of ITS2 sequences using both maximum likelihood and Bayesian inference methods. Body length was used as a proxy for lungworm species fitness in different hosts and compared with Kruskal-Wallis tests. Infection parameters were compared between cetacean species using Fisher’s exact tests and Kruskal-Wallis tests. Phylogenetic specificity was explored by collating the overall lungworm species prevalence values in hosts from previous surveys in various localities. To explore the relative importance of vertical and horizontal transmission, Spearman’s rank correlation was used to look for an association between host size and lungworm burden. A Mantel test was used to explore the association between lungworm species similarity and prey overlap using dietary data. Results Halocercus delphini had higher infection levels in striped dolphins and common dolphins; Stenurus ovatus had higher infection levels in bottlenose dolphins; and Stenurus globicephalae had higher infection levels in long-finned pilot whales. These results are congruent with findings on a global scale. Morphometric comparison showed that the larger nematodes were found in the same host species that had the highest parasite burden. Lungworms were found in neonatal striped dolphins and a Risso’s dolphin, and there was a weak but significant correlation between host size and parasite burden in striped dolphins and bottlenose dolphins. There was also a weak but significant association between prey overlap and lungworm species similarity. Conclusions Data indicate that phylogenetic specificity has an important role in governing host–parasite associations, as indicated by the higher infection levels and larger nematode size in certain hosts. However, diet can also influence infection patterns in these preferred hosts and contribute to less severe infections in other hosts.

Abstract Background Microcotyle erythrini van Beneden & Hesse, 1863 (Platyhelminthes: Monogenea) and other closely related species of the genus are often considered as cryptic. Records in hosts other than the type-host with no species confirmation by molecular analyses have contributed to this situation. Methods Gill parasites of five sparid fishes, Boops boops (L.), Pagellus erythrinus (L.), P. acarne (Risso), Dentex dentex (L.) and Pagrus pagrus (L.), from the Western Mediterranean off Spain were collected. Specimens of Microcotyle spp. were characterised both molecularly and morphologically. Partial fragments (domains D1-D3) of the 28S rRNA gene and the cytochrome c oxidase subunit 1 (cox1) gene were amplified and used for molecular identification and phylogenetic reconstruction. Principal components analysis was used to look for patterns of morphological separation. Results Molecular analyses confirmed the identity of three species: M. erythrini ex P. erythrinus and Pa. pagrus; M. isyebi Bouguerche, Gey, Justine & Tazerouti, 2019 ex B. boops; and a species new to science described herein, M. whittingtoni n. sp. ex D. dentex. The specific morphological traits and confirmed hosts (P. erythrinus and Pa. pagrus) are delimited here in order to avoid misidentifications of M. erythrini (sensu stricto). Microcotyle erythrini (s.s.) is mostly differentiated by the shape of its haptor, which is also longer than in the other congeners. New morphological and molecular data are provided for M. isyebi from the Spanish Mediterranean enlarging the data on its geographical range. Microcotyle whittingtoni n. sp. is described from D. dentex and distinguished from the remaining currently recognised species of the genus by the number and robustness of the clamps. Conclusions New diagnostic morphological traits useful to differentiate Microcotyle spp. are suggested: (i) haptor dimensions including lobes; (ii) the thickness of the clamps; (iii) the size and shape of spines of the genital atrium; (iv) the extension of the posterior extremities of vitelline fields; and (v) the shape of egg filaments. The use of new morphological approaches may allow considering these species of Microcotyle as being pseudocryptic. The use of representative undamaged specimens that have been genetically confirmed as conspecific is considered crucial to avoid abnormally wide morphological ranges that prevent species differentiation.

Abstract 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.

Abstract Background Functional inference on the attachment of acanthocephalans has generally been drawn directly from morphology. However, performance of structures is often non-intuitive and context-dependent, thus performance analysis should be included whenever possible to improve functional interpretation. In acanthocephalans, performance analysis of attachment is available only for Acanthocephalus ranae, a species that solely relies on the proboscis to attach. Here we compare body morphology and muscle arrangement in 13 species of Corynosoma, which use their spiny body as a fundamental holdfast. A basic performance analysis using live cystacanths of two representative species is also provided. Methods Adults of 13 Corynosoma spp. were obtained from 11 marine mammal species. Specimens were cut and carefully cleaned to examine muscle arrangement through light and scanning electron microscopy. Live cystacanths of C. australe and C. cetaceum were selected for performance analysis. Video records of evagination-invagination cycles of the proboscis were obtained and analysed with a video editor. Results The basic arrangement of proboscis retractors, trunk circular and longitudinal muscles, neck retractors and receptacle retractors, was conserved in all Corynosoma species. Interspecific variability was found in the relative development of disk muscles: minimum in C. enhydri, maximum in C. cetaceum; the distal insertion of the ventral neck retractor: ventro-lateral in C. cetaceum, C. hamannni and C. pseudohamanni and ventral in the other species; and the distal insertion of the receptacle retractors: more proximal in species with a longer hindtrunk. Performance analysis indicated striking similarities to that described for A. ranae except that (i) the foretrunk bends ventrally during the evagination-invagination cycles of the proboscis; (ii) disk muscles can flatten the tip of the foretrunk regardless of these cycles; and (iii) the receptacle bends ventrally and is driven to the hindtrunk by coordinated action of receptacle retractors. Conclusions Species of Corynosoma are able to use up to six holfast mechanisms. Attachment relies on a similar performance to that described for A. ranae. However, structural ventral bending of an inflated, spiny foretrunk, with a parallel re-arrangement of foretrunk muscles, have generated unexpected novel functions that make attachment extremely effective in species of Corynosoma. Interspecific variability in trunk shape and muscle arrangement grossly correlates with the rheological conditions each species experiences in their microhabitats within the gut of marine mammals.

Abstract Background Evidence concerning the environmental influence on monogenean transmission and infection processes is widely accepted, although only the effects of a limited number of abiotic factors on particular monogenean species have been explored. The current context of climate change calls for further research both on this subject, and also that concerning monogenean hosts, especially in aquaculture. Methods In this study, four experiments were used to assess the response of the infective stages of Sparicotyle chrysophrii, a pathogenic monogenean from gilthead sea bream (Sparus aurata) cultures in the Mediterranean, to variations of temperature (from 10 °C to 30 °C), pH (7.0 and 7.9), photoperiod (LD 12:12, LD 0:24 and LD 24:0) and salinity (from 27 ppt to 47 ppt). Results Thermal variations cause the strongest responses among the infective stages of S. chrysophrii, which reduced development and survival times as temperature increased. The optimal thermal range for maximum hatching success was found between 14 and 22 °C, whereas temperatures of 10 and 30 °C probably represent biological thermal limits. Reductions of development time and hatching rates were recorded at the lowest pH level, but hatching success remained above 50%, suggesting a certain degree of tolerance to slight pH variations. Photoperiod acts as an environmental cue synchronising the circadian hatching rhythm of S. chrysophrii with the first four hours of darkness. Response to a wide range of salinities was negligible, suggesting a high tolerance to variations of this abiotic factor. Conclusions Larval development and hatching of S. chrysophrii are modulated according to environmental factors, mainly temperature, thus parasite-host coordination and successful infections are enhanced. Therefore, abiotic factors should be broadly considered to design treatments against this monogenean. The high tolerance to the predicted environmental variations over the next century reported for gilthead sea bream and herein exposed for S. chrysophrii suggests that neither will be notably affected by climate change in the western Mediterranean region.

Abstract Background At present, much research effort has been devoted to investigate overall (“average”) responses of parasite populations to specific factors, e.g. density-dependence in fecundity or mortality. However, studies on parasite populations usually pay little attention to individual variation (“inequality”) in reproductive success. A previous study on the acanthocephalan Corynosoma cetaceum in franciscana dolphins, Pontoporia blainvillei, revealed no overall intensity-dependent, or microhabitat effects, on mass and fecundity of worms. In this study, we investigated whether the same factors could influence mass inequalities for this species of acanthocephalan. Methods A total of 10,138 specimens of C. cetaceum were collected from 10 franciscanas accidentally caught in Buenos Aires Province between 1988 and 1990. To investigate mass inequalities, all the specimens were sexed, and females were classified according to their developmental stage and weighed. Additionally, the relationship between biomass and fecundity (estimated as the number of acanthors) was investigated for some females. Inequalities in fecundity and biomass were assessed using standard methods, i.e. the Lorenz curve and the Gini coefficient (G). Results We found a modest, but highly significant linear relationship between mass and fecundity. The G was very low (0.314) compared with that from other helminth species. G values were significantly lower in gravid females, which presumably exhibit a slow rate of growth. Also, G values significantly increased with total intensity, but only for gravid females, and the effect was more predictable considering only the intensity of gravid females. Conclusions Apparently, high intensity infections increase inequality of reproducing females without producing “crowding” effects. Although several processes could generate this pattern, gravid females, at higher intensities, expanded their distribution and occupied gut chambers with contrasting environmental conditions, which might result in greater variability in body size. The observed inequalities are not expected to strongly influence the population genetics of C. cetaceum, but they reveal subtle individual effects beyond an overall population impact.

Abstract Background Accacoelium contortum (Rudolphi, 1819) Monticelli, 1893 is a frequent but poorly known trematode found on gills, pharynx and digestive tract of the ocean sunfish Mola mola (L.). Although the morphology of A. contortum agrees with that of a typical endoparasitic trematode, with two relatively small suckers and no large holdfasts, this parasite is normally ectoparasitic. The main objective of this paper is to explore this peculiar host-parasite relationship. Methods A total of 106 ocean sunfish were examined for the presence of A. contortum. The oropharyngeal chamber (gills and pharynx) and the digestive tract were analysed. As the previous descriptions of this species seem to be based on contracted specimens, for the morphological study the parasites were killed using two methods: with hot 70 % ethanol (with relaxed bodies) and with 70 % ethanol at room temperature (with contracted bodies). For histological studies, samples from fresh fish with parasitised left gills, pharynx and digestive tract were fixed in buffered 10 % formalin. For molecular studies the 18S, 28S and ITS-2 sequences were provided and compared with the available data in GenBank®. Results New information on the morphology of A. contortum and on the parasite-related response and pathological alterations in the host are given. New diagnostic traits for some structures are provided: e.g. tegumental papillae of the forebody with apical digitiform swellings and mouth surrounded by a circum-oral crown of simple papillae. The length of the ventral sucker peduncle and the position of the vitellarium were found to be associated with the contraction degree of the specimen. Immature individuals of this species are described for the first time. An intense proliferative inflammatory response of host gill and pharynx epithelium at the host-parasite interface was detected and parasites became partially covered by overgrowths of host tissues. Conclusions The induction of prominent histological alterations associated with A. contortum seems to be an adaptation to the external environment, an unusual location for trematodes.

Species of Balaenophilus are the only harpacticoid copepods that exhibit a widespread, obligate association with vertebrates, i.e., B. unisetus with whales and B. manatorum with marine turtles and manatees. In the western Mediterranean, juveniles of the loggerhead sea turtle, Caretta caretta are the only available hosts for B. manatorum, which has been found occurring at high prevalence (>80%) on them. A key question is how these epibionts are transmitted from host to host. We investigated this issue based on experiments with live specimens of B. manatorum that were cultured with turtle skin. Specimens were obtained from head-started hatchlings of C. caretta from the western Mediterranean. Hatched nauplii crawled only on rough substrates and lacked the ability to swim. Only copepodites IV and V, and adults, were able to perform directional swimming. Legs 2, 3 and 4 played a major role in swimming and were only well-developed in these stages. Nauplii reared in wells with turtle skin readily fed on this item. Late copepodites and adults also fed on turtle skin but did not consume other potential food items such as fish skin, baleen plates or planktonic algae. Evidences suggest that the transmission of B. manatorum should rely on hosts' bodily contacts and/or swimming of late developmental stages between spatially close hosts. The possibility of long-ranged dispersal is unlikely for two reasons. First, all developmental stages seem to depend on turtle skin as a food resource. Second, the average clutch size of ovigerous females was small (< 70 eggs) for free-living phases to successfully contact turtles that occur at very low densities (< 0.6 turtles·km-2) in the western Mediterranean. The high prevalence of B. manatorum in loggerhead turtles in this area raises the question whether these turtles have contacts, or tend to closely aggregate, more than is currently believed.

Trophically-transmitted parasites are regularly exposed to potential new hosts through food web interactions. Successful colonization, or switching, to novel hosts, occur readily when 'donor' and 'target' hosts are phylogenetically related, whereas switching between distantly related hosts is rare and may result from stochastic factors (i.e. rare favourable mutations). This study investigates a host-switching event between a marine acanthocephalan specific to pinnipeds that is apparently able to reproduce in Magellanic penguins Spheniscus magellanicus from Brazil. Detailed analysis of morphological and morphometrical data from acanthocephalans from penguins indicates that they belong to Corynosoma australe Johnston, 1937. Partial fragments of the 28S rRNA and mitochondrial cox1 genes were amplified from isolates from penguins and two pinniped species (i.e. South American sea lion Otaria flavescens and South American fur seal Arctocephalus australis) to confirm this identification. Infection parameters clearly differ between penguins and the two pinniped species, which were significantly lower in S. magellanicus. The sex ratio of C. australe also differed between penguins and pinnipeds; in S. magellanicus was strongly biased against males, while in pinnipeds it was close to 1:1. Females of C. australe from O. flavescens were smaller than those from S. magellanicus and A. australis. However, fecundity (i.e. the proportion of fully developed eggs) was lower and more variable in females collected from S. magellanicus. At first glance, the occurrence of reproductive individuals of C. australe in Magellanic penguins could be interpreted as an adaptive colonization of a novel avian host through favourable mutations. However, it could also be considered, perhaps more likely, as an example of ecological fitting through the use of a plesimorphic (host) resource, since the ancestors of Corynosoma infected aquatic birds.

We studied the phylogeography and historical demography of the most generalist digenean from cetaceans, Pholeter gastrophilus, exploring the effects of isolation by distance, ecological barriers and hosts' dispersal ability on the population structure of this parasite. The ITS2 rDNA, and the mitochondrial COI and ND1 from 68 individual parasites were analysed. Worms were collected from seven oceanic and coastal cetacean species from the south western Atlantic (SWA), central eastern Atlantic, north eastern Atlantic (NEA), and Mediterranean Sea. Pholeter gastrophilus was considered a single lineage because reciprocal monophyly was not detected in the ML cladogram of all individuals, and sequence variability was

Abstract Background Trematode communities often consist of different species exploiting the same host population, with two or more trematodes sometimes co-occuring in the same host. A commonly used diagnostic method to detect larval trematode infections in snails has been based on cercarial shedding, though it is often criticized as inaccurate. In the present study we compare infection prevalences determined by cercarial emission with those determined, for the first time, by molecular methods, allowing us to quantify the underestimation of single and double infections based on cercarial emission. We thus developed a duplex PCR for two host-parasite systems, to specifically differentiate between single and double infections. The Ebro samples include two morphologically similar opecoelids, whereas the Otago samples include two morphologically different larval trematodes. Methods Snails were screened for infections by incubating them individually to induce cercarial emission, thus determining infection following the “classical” detection method. Snail tissue was then removed and fixed for the duplex PCR. After obtaining ITS rDNA sequences, four species-specific primers were designed for each snail-trematode system, and duplex PCR prevalence was determined for each sample. Results from both methods were statistically compared using the McNemar’s Chi-squared test and Cohen’s Kappa Statistic for agreement between outcomes. Results Overall infection prevalences determined by duplex PCR were consistently and substantially higher than those based on cercarial shedding: among Ebro samples, between 17.9% and 60.1% more snails were found infected using the molecular method, whereas in the Otago samples, the difference was between 9.9% and 20.6%. Kappa values generally indicated a fair to substantial agreement between both detection methods, showing a lower agreement for the Ebro samples. Conclusions We demonstrate that molecular detection of single and double infections by duplex PCR strongly outcompetes the classical method. Detection failure is most likely due to immature and covert infections, however, the higher incidence of misidentified double infections in the Ebro samples arises from morphological similarity of closely-related species. The higher accuracy of the duplex PCR method also adds to our understanding of community structure of larval trematodes in snail hosts, by providing a clearer assessment of the importance of interspecific interactions within the host.

The diet of Risso’s dolphin in the Mediterranean is described based on 15 animals stranded between 40°25’N 00°32’W and 37°35’N 00°45’E from April 1987 to January 2003. The prey were mainly oceanic cephalopods. Pelagic octopods, especially A. argo, were the most abundant (mean = 41.28%; SD±34.32). Species belonging to the families Ommastrephidae, Histioteuthidae and Onychoteuthidae were also frequent components of its diet. The bathymetric distribution of cephalopods shows that Risso’s dolphin preferentially feeds on the middle slope (600 to 800 m depth) in the Mediterranean.

We report on a juvenile Kemp´s ridley sea turtle, Lepidochelys kempii, captured alive by fishermen in the waters off Alicante (western Mediterranean, Spain) on October 15th 2001. The analysis of a 460 base pair fragment of the mt DNA control region revealed a perfect match with haplotype D previously described in this species. This is the second report of L. kempii in the Mediterranean basin.

Free, amoeboid movement of organisms within media as well as substrate-dependent cellular crawling processes of cells and organisms require an actin cytoskeleton. This system is also involved in the cytokinetic processes of all eukaryotic cells. Myxozoan parasites are known for the disease they cause in economical important fishes. Usually, their pathology is related to rapid proliferation in the host. However, the sequences of their development are still poorly understood, especially with regard to pre-sporogonic proliferation mechanisms. The present work employs light microscopy (LM), electron microscopy (SEM, TEM) and confocal laser scanning microscopy (CLSM) in combination with specific stains (Nile Red, DAPI, Phalloidin), to study the three-dimensional morphology, motility, ultrastructure and cellular composition of Ceratomyxa puntazzi, a myxozoan inhabiting the bile of the sharpsnout seabream.Our results demonstrate the occurrence of two C. puntazzi developmental cycles in the bile, i.e. pre-sporogonic proliferation including frequent budding as well as sporogony, resulting in the formation of durable spore stages and we provide unique details on the ultrastructure and the developmental sequence of bile inhabiting myxozoans. The present study describes, for the first time, the cellular components and mechanisms involved in the motility of myxozoan proliferative stages, and reveals how the same elements are implicated in the processes of budding and cytokinesis in the Myxozoa. We demonstrate that F-actin rich cytoskeletal elements polarize at one end of the parasites and in the filopodia which are rapidly de novo created and re-absorbed, thus facilitating unidirectional parasite motility in the bile. We furthermore discover the myxozoan mechanism of budding as an active, polarization process of cytokinesis, which is independent from a contractile ring and thus differs from the mechanism, generally observed in eurkaryotic cells. We hereby demonstrate that CLSM is a powerful tool for myxozoan research with a great potential for exploitation, and we strongly recommend its future use in combination with in vivo stains.