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.

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.

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.

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.

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.

A new species of Tinrovia Mamaev, 1987 (Monogenea: Microcotylidae) from the deep-sea fish Notacanthus bonaparte Risso (Notacanthiformes: Notacanthidae) in the Western Mediterranean and the North East Atlantic.

A new microcotylid, Tinrovia mamaevi n. sp. (Monogenea: Polyopisthocotylea), is described from the gills of Notacanthus bonaparte Risso (Notacanthiformes: Notacanthidae), sampled in the Western Mediterranean and North East Atlantic. This species is allocated to the subfamily Syncoelicotylinae Mamaev & Zubchenko, 1978 due to the possession of a symmetrical haptor with two separate frills. The clamps in T. mamaevi n. sp. are of the "microcotylid" type, arranged in two distinct lateral haptoral frills; the genital atrium and the copulatory organ are armed and the vaginal pore is unarmed. The new species differs from the type- and only species of the genus, T. papiliocauda Mamaev, 1987, in having a shorter and narrower haptor with a smaller number of clamps. Clamps are also smaller in the new species, testes are more numerous, the genital atrium is smaller, divided into two lateral lobes (instead of five) with a smaller number of spines and the eggs have a short and a long filament (instead of two short filaments). The host species and locality of T. mamaevi n. sp. also differ as T. papiliocauda which was recorded in Notacanthus sexspinis Richardson from the South Pacific.

Accacoelium contortum (Trematoda: Accacoeliidae) a trematode living as a monogenean: morphological and pathological implications.

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.

Dichelyne (Cucullanellus) romani n. sp. (Nematoda: Cucullanidae) in notacanthid fishes from the Northeast Atlantic and Western Mediterranean.

A new cucullanid, Dichelyne (Cucullanellus) romani n. sp. (Nematoda: Cucullanidae), is described from the digestive tract of two notacanthid fishes, Notacanthus chemnitzii Bloch and N. bonaparte Risso (Notacanthiformes: Notacanthidae), from the Northeast Atlantic and Western Mediterranean. The presence of a precloacal sucker and ten pairs of caudal papillae in males allocates it to the subgenus Cucullanellus Törnquist, 1931. The new species differs from other members of this subgenus in its larger body size, smaller spicule/body length ratio, the distribution of caudal papillae, and the position of deirids and excretory pore. Both notacanthid fishes act as definitive hosts with slightly larger nematode specimens detected in N. chemnitzii. A wide intraspecific variability was found in the distribution of caudal papillae, and in some specimens the position of deirids, excretory pore and length of intestinal caecum also varied. A complete list of all assigned species of the subgenus Cucullanellus is presented and discussed as there are several uncertainties regarding the validity and synonymy of some species. This is the first species of Dichelyne Jägerskiöld, 1902 in a notacanthid fish and one of the only two records in deep-sea fish species.

A revision of the genus Neogrubea Dillon & Hargis, 1968 (Monogenea: Mazocraeidae): new morphological and molecular data from off the Patagonian coast of Argentina.

The genus Neogrubea Dillon & Hargis, 1968 (syn. Asymmetria Suriano, 1975) is revised based on examination of type- and voucher material, and from new specimens collected from the gills of Seriolella porosa Guichenot and Stromateus brasiliensis Fowler from off Patagonia, Argentina. Morphological comparisons based on light and scanning electron microscopy and molecular data (partial SSU and LSU rDNA sequences) of the monogeneans from off Patagonia suggest that N. seriolellae Dillon & Hargis, 1968 (syns N. stromateae Gibson, 1976, A. asymmetria Suriano, 1975 and A. platensis Rey & Meneses, 1985) is currently the only species of the genus. Neogrubea soni Evdokimova, 1969 is considered a species inquirenda. An emended diagnosis of Neogrubea is presented, and new host and locality records for N. seriolellae are given in detail. Morphological characters of the members of the mazocraeid subfamily Grubeinae Price, 1961 are also discussed.

Parasite communities in the red mullet, Mullus barbatus L., respond to small-scale variation in the levels of polychlorinated biphenyls in the Western Mediterranean.

We examined parasite populations and communities in the Mediterranean sentinel fish species, Mullus barbatus, sampled at a small-scale PCB gradient at the shelf sediments off Catalonian coasts of the Western Mediterranean. A recurrent feature at both the population and community level was the differentiation of the samples along the increasing PCB levels simultaneously registered in the sediments. Both directly transmitted ectoparasites and endoparasites with complex life-cycles transmitted via food chains exhibited a decrease in abundance with the increase in PCB levels. Parasite numerical responses translated into significant differences in infracommunity structure with decreasing predictability associated with increasing PCB levels. The abundance of two species, the specialist Opecoeloides furcatus and the generalist nematode Hysterothylacium fabri, contributed substantially to the observed dissimilarity between infracommunity samples along the gradient. The observed parasite responses to moderate levels of pollution were simultaneously validated by both chemical monitoring and effect biomarkers.

A new species of Ascocotyle (Trematoda: Heterophyidae) from the South American sea lion, Otaria flavescens, off Patagonia, Argentina.

We describe a new heterophyid species, Ascocotyle (Ascocotyle) patagoniensis n. sp., based on specimens collected from the intestines of the South American sea lion Otaria flavescens from Patagonia (Argentina). Ascocotyle (A.) patagoniensis n. sp. is distinguished from the other species of the subgenus by the number of circumoral spines, which are arranged in 2 rows of 18 to 23. The new species also differs from the other species in having a gonotyl without papillae. The specimens exhibited the widest seminal receptacle described for a species of this subgenus. Species of the subgenus Ascocotyle usually infect fish-eating birds or mammals in freshwater or brackish habitats. Ascocotyle (A.) patagoniensis n. sp. is the first species of the subgenus described from a marine mammal. However, no metacercariae of Ascocotyle spp. were found in 542 marine teleosts from 20 species collected in the same locality. The life cycle of the marine species from the Ascocotyle -complex infecting pinnipeds remains elusive.

Skoulekia meningialis n. gen., n. sp. (Digenea: Aporocotylidae Odhner, 1912) a parasite surrounding the brain of the Mediterranean common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) (Teleostei: Sparidae): description, molecular phylogeny, habitat and pathology.

This study describes a new aporocotylid genus and species, Skoulekia meningialis n. gen., n. sp. which was detected in the ectomeningeal veins surrounding the optic lobes of the brain of the common two-banded seabream Diplodus vulgaris (Geoffroy Saint-Hilaire, 1817) from the Gulf of Valencia (Mediterranean Sea). A detailed morphological description of S. meningialis is provided, including drawings, measurements and scanning electron microscopy images as well as a phylogenetic study of S. meningialis and closely related taxa using DNA sequence data obtained from whole ITS2 and partial 18S and 28S rDNA regions. Morphology as well as molecular phylogeny strongly support the erection of a new genus and demonstrate its close relationship with the genera Psettarium (Goto & Ozaki, 1930) and Pearsonellum Overstreet & Køie, 1989. Skoulekia is the second aporocotylid genus described in the Sparidae, a family including economical important fishes. In contrast to the majority of the aporocotylids, which inhabit the heart or the blood vessels of the gills, S. meningialis inhabits the ectomeningeal veins surrounding the optic lobes. Eggs were found trapped within the gill vessels. Normally, blood fluke pathology is almost exclusively related to the eggs. However, in the case of S. meningialis, main histopathological alterations were related to the adult blood flukes which were found to cause mild localised meningitis.

Diclidophora merlangi (Monogenea: Diclidophoridae) on Atlantic cod, Gadus morhua.

Diclidophora merlangi (Kuhn, in Nordmann, 1832), as species specific to whiting, Merlangius merlangus (Linnaeus, 1758), is reported and described for the first time on the gills of Atlantic cod Gadus morhua (Linnaeus, 1758). Of the about 1,200 cod examined in this article, only 2 specimens of D. merlangi occurred on 2 fish, suggesting that they represented accidental infections. The 2 specimens showed specific traits of D. merlangi, but their size was considerably smaller than that of D. merlangi on whiting. Principal-component analyses showed that the morphology of the specimens on cod was more similar to that of D. merlangi than those of other congeneric species from the North Atlantic, additionally supporting their assignment to D. merlangi. Quantitative data documenting the potential reproductive consequences of D. merlangi occurring on cod is also provided. The testes and germaria of the specimens on cod were noticeably smaller than those of D. merlangi on whiting, but the number of testes was similar and spermatozoa were observed in the sperm duct. By contrast, the aspect and smaller size of the oocytes suggested that D. merlangi on cod could not produce viable ova, although 1 specimen exhibited an egg with normal sized and shaped capsule.