Historical isolates of dolphin morbillivirus: origin and new data.

Dolphin morbillivirus (DMV) was isolated in striped dolphins Stenella coeruleoalba from the Mediterranean Sea stranded along the coast of Spain during a lethal epidemic that killed thousands of individuals in 1990-1992. Though some of these isolates (MUC, 16A and the reference strain) have been extensively characterised, details on their origin were not reported in the literature, and records for these isolates are often difficult to trace and are, sometimes, erroneous. Here, we provide unpublished biological and histopathological data for these isolates, summarize the literature on their characterization and make suggestions for future studies.

Mapping a brain parasite: occurrence and spatial distribution in fish encephalon.

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.

Molecular phylogeny of the Pseudaliidae (Nematoda) and the origin of associations between lungworms and marine mammals.

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.

Parasite communities in a marine fish indicate ecological recovery from the impacts of the Prestige oil-spill 12-13 years after the disaster.

The Prestige oil-spill (2002) is one of the major marine accidents resulting in contamination of virtually all types of marine habitat along c.900 km of coastline in the North-East Atlantic. Environmental pollution affects parasite populations and communities, both directly and through effects on intermediate and definitive hosts. However, the effects of oil-spills on shelf benthic communities are poorly known. This study addressed the hypothesis of recovery of parasite communities in a marine sparid teleost, the bogue Boops boops (L.) (Teleostei: Sparidae), as indicators of environmental pollution and its effects on benthic/pelagic invertebrate communities in an impacted area off the Galician coast, Spain, 12-13 years after the Prestige oil-spill. Novel data for the metazoan parasite communities collected during 2014-2015 were analysed in association with two unique datasets, one comprising baseline data collected in 2001, one year before the Prestige oil-spill, and one comprising data collected 3-4 years post-spill (2005-2006). Using the taxonomically consistent data on parasites in a series of fish seasonal samples, we found significant differences between the two seasonal post-spill datasets taken at a 9-year time interval (3-4 years and 12-13 years post-spill) in most community metrics and infection parameters of the common species. This was in sharp contrast with the few differences between the long-term post-spill dataset of 2014-2015 and the pre-spill dataset of 2001. Multivariate community similarity analyses confirmed that these differences reflected in the substantial differentiation of parasite community composition and structure of the post-spill communities and the significant homogenisation of communities sampled 12-13 years post-spill and baseline communities. Overall, the novel analyses demonstrated a long-term directional trend in parasite community succession towards ecological recovery irrespective of the natural seasonal variability. These findings suggest longer-term oil-spill impacts on shelf benthic/pelagic invertebrate communities lasting over 10 years.

From development to taxonomy: the case of Sciaenacotyle pancerii (Monogenea: Microcotylidae) in the Mediterranean meagre.

The microcotylid Sciaenacotyle pancerii is a pathogenic monogenean infecting Argyrosomus regius, a candidate for species diversification in the Mediterranean aquaculture. Life-history stages of S. pancerii commonly co-occur in field infections, but to date, morphological data have only been provided for oncomiracidia and adults although identifying life-history stages can be useful in infection management. A total of 114 specimens of S. pancerii were analysed to characterize the developmental events and to assess morphological and morphometric variations before and after maturity. The post-larval development of S. pancerii is characterized by: expansion and bifurcation of the gut, loss of the larval haptor, protandrous development of the genitalia and vitellaria formation. The size variability of larval hooks, hamuli and germanium of S. pancerii is firstly reported and dimensional ranges of parasite body, haptor, testes, posteriormost clamps and eggs are widened. The size of most of the diagnostic features of S. pancerii significantly increases after parasite maturity and therefore, only those specimens with more than 116 clamps should be considered for minimising development-related variability in size. The high number of clamps, their fast development and the asymmetry in their size and arrangement suggest that S. pancerii may use a mixed attachment strategy between the closely related microcotylids and heteraxinids. This combination of features may be host related and linked to the gill structure of the sciaenid fish and the phylogenetic position of the genus Sciaenacotyle; distant from other microcotylids while close to heteraxinid species.

A severe microsporidian disease in cultured Atlantic Bluefin Tuna (Thunnus thynnus).

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.

Cardicola mediterraneus n. sp. (Trematoda, Aporocotylidae): a new species infecting the gilthead seabream, Sparus aurata L., from the Western Mediterranean Sea.

The genus Cardicola Short, 1953 has the highest number of species within the family Aporocotylidae (Trematoda: Digenea). Five Cardicola species have been reported to date in the Mediterranean Sea, one of them in the gilthead seabream Sparus aurata L. Analyses of infected S. aurata recovered from cultured fish off Sardinia (Italy) and from wild and cultured fish off the Levantine coast (Southeastern Spain) have revealed the presence of two species identified as Cardicola aurata Holzer, Montero, Repullés, Nolan, Sitjà-Bobadilla, Álvarez-Pellitero, Zarza and Raga, 2008 and Cardicola mediterraneus n. sp.. New morphological and molecular data are provided for both species. Features of C. aurata specimens differ slightly from those of the original description of the species, the most important differences being the longer extension of the metraterm and the central and posterior position of the female genital pore. Cardicola mediterraneus n. sp. can be easily distinguished from other Cardicola species by two unique specific characters: (i) the very unequal posterior caeca length and (ii) the shape of the testis, deeply notched at the anterior extremity. Cardicola spp. from sparids occupy a basal phylogenetic position respect the other congeneric species. The genus Cardicola has a complex taxonomy and shows high intrageneric differences for both 28S and ITS2 rDNAs, similar to the intergeneric differences among other aporocotylid genera, suggesting that it could be split. The presence of two Cardicola species could hamper treatment design and application; thus, data discriminating species herein reported can improve the infection management in fish farms.

Determinants of lungworm specificity in five cetacean species in the western Mediterranean.

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.

Causes of cetacean stranding and death on the Catalonian coast (western Mediterranean Sea), 2012-2019.

The causes of cetacean stranding and death along the Catalan coast between 2012 and 2019 were systematically investigated. Necropsies and detailed pathological investigations were performed on 89 well-preserved stranded cetaceans, including 72 striped dolphins Stenella coeruleoalba, 9 Risso's dolphins Grampus griseus, 5 bottlenose dolphins Tursiops truncatus, 1 common dolphin Delphinus delphis, 1 Cuvier's beaked whale Ziphius cavirostris and 1 fin whale Balaenoptera physalus. The cause of death was determined for 89.9% of the stranded cetaceans. Fisheries interaction was the most frequent cause of death in striped dolphins (27.8%) and bottlenose dolphins (60%). Cetacean morbillivirus (CeMV) was detected on the Catalan coast from 2016 to 2017, causing systemic disease and death in 8 of the 72 (11.1%) striped dolphins. Chronic CeMV infection of the central nervous system was observed from 2018-2019 in a further 5 striped dolphins. Thus, acute and chronic CeMV disease caused mortality in 18% of striped dolphins and 14.6% of all 89 cetaceans. Brucella ceti was isolated in 6 striped dolphins and 1 bottlenose dolphin with typical brucellosis lesions and in 1 striped dolphin with systemic CeMV. Sinusitis due to severe infestation by the nematode parasite Crassicauda grampicola caused the death of 4 out of 6 adult Risso's dolphins. Maternal separation, in some cases complicated with septicemia, was a frequent cause of death in 13 of 14 calves. Other less common causes of death were encephalomalacia of unknown origin, septicemia, peritonitis due to gastric perforation by parasites and hepatitis caused by Sarcocystis spp.

Using Boops boops (osteichthyes) to assess microplastic ingestion in the Mediterranean Sea.

This study assesses microplastic ingestion in Boops boops at different geographical areas in the Mediterranean Sea. A total of 884 fish were caught at 20 coastal sites in Spain, France, Italy and Greece and analyzed using a common methodological protocol. Microplastics were found in 46.8% of the sampled fish, with an average number of items per individual of 1.17 ± 0.07. Filaments were the predominant shape type, while polyethylene and polypropylene were indicated by FTIR as the most common polymer types of ingested microplastics. The frequency of occurrence, as well as the abundance and proportion of types (size, shape, color and polymer) of ingested microplastics, varied among geographical areas. The spatial heterogeneity of the abundance of ingested microplastics was mainly related to the degree of coastal anthropogenic pressure at the sampling sites. Our findings further support the suitability of B. boops as bioindicator of microplastic pollution in the Mediterranean Sea.

The taxonomic status of Skrjabinalius guevarai Gallego & Selva, 1979 (Nematoda: Pseudaliidae) and the synonymy of Skrjabinalius Delyamure, 1942 and Halocercus Baylis & Daubney, 1925.

Within the cetacean lungworm family Pseudaliidae Raillet & Henry, 1909, the distinction between the two genera of the subfamily Halocercinae Delamure, 1952, i.e. Halocercus Baylis & Daubney, 1925 and Skrjabinalius Delyamure, 1942, is principally based on the structure and shape of the male copulatory bursa. In species of Halocercus, the bursa is unlobed, whereas in the two species included in Skrjabinalius, S. cryptocephalus Delyamure, 1942 and Skrjabinalius guevarai Gallego & Selva, 1979, the bursa is described as clearly lobed. During a parasitological analysis of cetaceans from the Spanish Mediterranean, we collected a number of specimens of S. guevarai with variable levels of bursal lobulation, including individuals with unlobed bursae. Examination of voucher specimens of the type-species of Halocercus, H. delphini Baylis & Daubney, 1925, collected from cetaceans in the North-East Atlantic revealed the same variations in bursal shape, and the same arrangement of bursal rays and papillae. A morphometric comparison did not reveal substantial differences between both species. Moreover, Maximum Likelihood and Bayesian Inference analyses of the second internal transcribed spacer (ITS2) sequences of 11 specimens of S. guevarai and one of H. delphini grouped both species together, regardless of bursal shape, in a highly supported clade within the Pseudaliidae. Accordingly, we consider S. guevarai as a junior synonym of H. delphini. The great variability found in bursal lobulation in the type-species of Halocercus invalidates the use of this trait as a genus-level diagnostic character and, therefore, Skrjabinalius should also be considered synonymous with Halocercus.

Cranial crassicaudiasis in two coastal dolphin species from South Africa is predominantly a disease of immature individuals.

Crassicauda spp. (Nematoda) infest the cranial sinuses of several odontocetes, causing diagnostic trabecular osteolytic lesions. We examined skulls of 77 Indian Ocean humpback dolphins Sousa plumbea and 69 Indo-Pacific bottlenose dolphins Tursiops aduncus, caught in bather-protecting nets off KwaZulu-Natal (KZN) from 1970-2017, and skulls of 6 S. plumbea stranded along the southern Cape coast in South Africa from 1963-2002. Prevalence of cranial crassicaudiasis was evaluated according to sex and cranial maturity. Overall, prevalence in S. plumbea and T. aduncus taken off KZN was 13 and 31.9%, respectively. Parasitosis variably affected 1 or more cranial bones (frontal, pterygoid, maxillary and sphenoid). No significant difference was found by gender for either species, allowing sexes to be pooled. However, there was a significant difference in lesion prevalence by age, with immature T. aduncus 4.6 times more likely affected than adults, while for S. plumbea, the difference was 6.5-fold. As severe osteolytic lesions are unlikely to heal without trace, we propose that infection is more likely to have a fatal outcome for immature dolphins, possibly because of incomplete bone development, lower immune competence in clearing parasites or an over-exuberant inflammatory response in concert with parasitic enzymatic erosion. Cranial osteolysis was not observed in mature males (18 S. plumbea, 21 T. aduncus), suggesting potential cohort-linked immune-mediated resistance to infestation. Crassicauda spp. may play a role in the natural mortality of S. plumbea and T. aduncus, but the pathogenesis and population level impact remain unknown.

Sex-specific impact of inbreeding on pathogen load in the striped dolphin.

The impact of inbreeding on fitness has been widely studied and provides consequential inference about adaptive potential and the impact on survival for reduced and fragmented natural populations. Correlations between heterozygosity and fitness are common in the literature, but they rarely inform about the likely mechanisms. Here, we investigate a pathology with a clear impact on health in striped dolphin hosts (a nematode infection that compromises lung function). Dolphins varied with respect to their parasite burden of this highly pathogenic lung nematode (Skrjabinalius guevarai). Genetic diversity revealed by high-resolution restriction-associated DNA (43 018 RADseq single nucleotide polymorphisms) analyses showed a clear association between heterozygosity and pathogen load, but only for female dolphins, for which the more heterozygous individuals had lower Sk. guevarai burden. One locus identified by RADseq was a strong outlier in association with parasite load (heterozygous in all uninfected females, homozygous for 94% of infected females), found in an intron of the citron rho-interacting serine/threonine kinase locus (associated with milk production in mammals). Allelic variation at the Class II major histocompatability complex DQB locus was also assessed and found to be associated with both regional variation and with pathogen load. Both sex specificity and the identification of associating functional loci provide insight into the mechanisms by which more inbred individuals may be more susceptible to the infection of this parasite. This provides important insight towards our understanding of the impact of inbreeding in natural populations, relevant to both evolutionary and practical conservation considerations.

Considerations on the taxonomy and morphology of Microcotyle spp.: redescription of M. erythrini van Beneden & Hesse, 1863 (sensu stricto) (Monogenea: Microcotylidae) and the description of a new species from Dentex dentex (L.) (Teleostei: Sparidae).

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.

Transmission and Predictors of Burden of Lungworms of the Striped Dolphin (Stenella coeruleoalba) in the Western Mediterranean.

Pseudaliid lungworms infect the lungs and sinuses of cetaceans. Information on the life cycle and epidemiology of pseudaliids is very scarce and mostly concerns species that infect coastal or inshore cetaceans. Available evidence indicates that some pseudaliids are vertically transmitted to the host, whereas others are acquired via infected prey. We documented pseudaliid infections in an oceanic cetacean, the striped dolphin (Stenella coeruleoalba) in the western Mediterranean, and investigated the possibilities of vertical vs. horizontal transmission and the potential influence of host body size, sex, and season on infection levels. We found two species of lungworm in 87 dolphins that stranded along the Spanish Mediterranean coast between 1987 and 2018. One or two larvae of Stenurus ovatus were found in three adult dolphins. Larger numbers of larvae and adults of Skrjabinalius guevarai were collected in 51 dolphins, including unweaned calves. These observations suggested that Skrjabinalius guevarai could be vertically transmitted. The abundance of Skrjabinalius guevarai increased significantly with host size, which suggested that it could be trophically transmitted, as well, with larger hosts consuming more infected prey. Infection levels peaked in spring, outside of the calving season, which is likely a reflection of a seasonal shift in dolphin diet. In summary, results indicate that Skrjabinalius guevarai was capable of both vertical and horizontal transmission, but future research should be directed at clarifying the potential mechanics behind transmission and intermediate hosts.

In vivo fluorescent cercariae reveal the entry portals of Cardiocephaloides longicollis (Rudolphi, 1819) Dubois, 1982 (Strigeidae) into the gilthead seabream Sparus aurata L.

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.

Determining suitable fish to monitor plastic ingestion trends in the Mediterranean Sea.

The presence of marine litter is a complex, yet persistent, threat to the health and biodiversity of the marine environment, and plastic is the most abundant, and ubiquitous type of marine litter. To monitor the level of plastic waste in an area, and the prospect of it entering the food chain, bioindicator species are used extensively throughout Northern European Seas, however due to their distribution ranges many are not applicable to the Mediterranean Sea. Guidance published for the Marine Strategy Framework Directive suggests that the contents of fish stomachs may be analyzed to determine trends of marine plastic ingestion. In order to equate transnational trends in marine plastic ingestion, the use of standardized fish species that widely occur throughout the basin is favoured, however for the Mediterranean Sea, specific species are not listed. Here we propose a methodology to assess how effective Mediterranean fish species, that are known to have ingested marine plastic, are as bioindicators. A new Bioindicator Index (BI) was established by incorporating several parameters considered important for bioindicators. These parameters included species distribution throughout the Mediterranean basin, several life history traits, the commercial value of each species, and the occurrence of marine litter in their gut contents. By collecting existing data for Mediterranean fish, ranked scores were assigned to each trait and an average value (BI value) was calculated for each species. Based on their habitat preferences, Engraulis encrasicolus (pelagic), Boops boops (benthopelagic), three species of Myctophidae (Hygophum benoiti, Myctophum punctatum and Electrona risso) (mesopelagic), Mullus barbatus barbartus (demersal) and Chelidonichthys lucerna (benthic), were identified as currently, the most suitable fish for monitoring the ingestion of marine plastics throughout the Mediterranean basin. The use of standardized indicator species will ensure coherence in the reporting of marine litter ingestion trends throughout the Mediterranean Sea.

Neither Diplectanum nor specific: a dramatic twist to the taxonomic framework of Diplectanum (Monogenea: Diplectanidae).

The taxonomy of the genus Diplectanum has been exclusively based on morphological features, with 28 nominal species parasitic on perciform fishes recognised. We used molecular data, to our knowledge for the first time, to evaluate the taxonomic framework of Diplectanum, infer the relationships amongst species attributed to this genus, re-assess the degree of host specificity and explore the population genetic structure of Diplectanum spp. parasitising Mediterranean sciaenids, which are potential target fish species for aquaculture diversification in the region. A minimum of 10 specimens of Diplectanum spp. were sequenced per host species (Argyrosomus regius, Sciaena umbra, Umbrina canariensis and Umbrina cirrosa) and locality (Burriana, Sant Carles de la Ràpita and Santa Pola (Spain)) together with five individuals of the type species Diplectanum aequans. Sequences of partial 28S rDNA and internal transcribed spacer region of Diplectanum spp. were analysed together with those from other Diplectanidae spp. in GenBank using Bayesian inference and maximum likelihood phylogenetic methods. Population genetic analyses were performed using cytochrome c oxidase subunit I gene sequences for a diplectanid species with a wide distribution across host species and localities. Results showed that Diplectanum was not monophyletic, nor were the specimens infecting sciaenids. Instead they formed two separate clades, 26.1-28.6% divergent for the internal transcribed spacer and 13.2-16.9% for the 28S region from D. aequans. Altogether, our results suggest that these specimens represent two distinct new genera from Diplectanum and five putative species with low host specificity. It is likely that morphological variability has led to the description of more species than were detected by molecular methods. In contrast to other monogeneans, Diplectaninae gen. spp. are chiefly generalists. Nonetheless, intraspecific genetic divergence in the internal transcribed spacer region of Diplectaninae gen. spp., and population genetic analyses of one presumed generalist species, Diplectaninae gen. sp. 1.2, showed significant variation between subpopulations living on different hosts. The intraspecific genetic structure by host also suggests different cross-infection potential amongst sciaenid species.

Morphology, performance and attachment function in Corynosoma spp. (Acanthocephala).

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.

Come rain or come shine: environmental effects on the infective stages of Sparicotyle chrysophrii, a key pathogen in Mediterranean aquaculture.

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.