Morphological and molecular characterization of Chloromyxum dasyatidis n. sp. (Myxosporea: Chloromyxidae) in the common stingray Dasyatis pastinaca (Linnaeus) from Tunisian waters (Central Mediterranean Sea).

A new species of Chloromyxum Mingazzini, 1890, C. dasyatidis n. sp., is described from the gallbladder of the elasmobranch fish Dasyatis pastinaca (Linnaeus) from the Mediterranean coast off Tunisia. Mature myxospores were subspherical measuring 13.0 ± 0.3 (12.5-13.9) µm in length, 11.3 ± 0.5 (10.2-12.2) µm in width, and 11.8 ± 0.5 (11.4-12.7) µm in thickness. Four pyriform polar capsules 4.4 ± 0.3 (4.0-4.9) µm long and 3.5 ± 0.3 (3.0-3.8) µm wide. Valves were ornamented by 5 to 7 elevated surface ridges and displayed short caudal filaments. Chloromyxum dasyatidis n. sp. had an overall prevalence of infection of 38.5%, with significant seasonal variation, being more prevalent during summer (60.0%) and autumn (70.8%). Phylogenetically, this species grouped within the Chloromyxum sensu stricto lineage, forming a small clade together with the genetically more similar species C. acuminatum and C. myolibati, both from stingray hosts.

Myxidium tunisiensis n. sp. (Myxosporea: Myxidiidae) infecting the rough skate Raja radula Delaroche, 1908 (Rajiformes: Rajidae) from North East Tunisia.

A new marine myxosporean, Myxidium tunisiensis n. sp. (Myxosporea: Myxidiidae), is described infecting the gallbladder of the rough skate Raja radula Delaroche, 1908 (Chondrichthyes: Rajidae) harvested from the Bay of Bizerte, Tunisia. Observed stages of the parasite were floating free in the bile. Plasmodia, disporic or polysporic, contain disporic pansporoblasts measuring 23.0-27.2 µm in maximum diameter and 21.0-25.5 µm in minimum diameter. Mature myxospores, which are elongated, typically fusiform measured 18.6 ± 1.7 (17.2-22.0) µm in length, 8.6 ± 1.2 (7.2-11.2) µm in width and 8.2 ± 0.9 (6.4-10.1) in thickness. Myxospore valves are symmetrical with 9-10 longitudinal striations. Two polar capsules, pyriform, were equal in size, measuring 5.8 ± 0.4 (5.2-6.7) µm in length and 3.1 ± 0.2 (3.0-3.2) µm in width, with 5-6 polar filament turns. The infection was prevalent throughout the year with the highest prevalence in autumn (81.8%) and the lowest in winter (32.3%). The seasonal prevalence of M. tunisiensis n. sp. in the Bay of Bizerte appeared to fluctuate according to seawater temperature. The morphological and morphometrical differences with all Myxidium spp. described so far, complemented by molecular data, prove clearly that the present species is a new member of the genus Myxidium. The SSU rDNA phylogenetic analyses revealed that Myxidium tunisiensis n. sp. clustered within the oligochaete-gallbladder clade in a basal position to the marine subclade of Myxidium baueri and Myxidium coryphaenoideum. While the new species branching pattern is stable in the ML and the BI analyses, it changed in the MP analysis. This is the first molecular study of a Myxidium species infecting an elasmobranch host inhabiting the Mediterranean Sea.

A new species of nematode parasite, Cucullanus tunisiensis sp. nov. (Nematoda: Cucullanidae) from Epinephelus aeneus (Perciformes: Serranidae) off Tunisia.

Cucullanus tunisiensis sp. nov., (Nematoda: Cucullanidae), collected from the intestine of the white grouper Epinephelus aeneus from waters off the coast of Tunisia is described based on light and scanning electron microscopic observations. The new species is characterized by the presence of lateral alae, ventral sucker, long unequal spicules (left spicule 2474-2789 µm long, right spicule 2357-2518 µm long). This is the sixth nominal species of the genus Cucullanus Müller, 1777 and the first representative of this genus infecting fishes of Serranidae family reported from Tunisian waters.

Description of two new species of Ceratomyxa Thélohan, 1892 (Cnidaria: Myxosporea) infecting the gallbladder of Epinephelinae fishes from Tunisian waters using morphological and molecular data.

Ceratomyxa marginati n. sp. and C. aenei n. sp. are two new coelozoic myxosporean species infecting the gallbladder of the dusky grouper (Epinephelus marginatus) and the white grouper (Epinephelus aeneus), respectively. These two ceratomyxids were described using morphological characteristics and molecular analysis of the SSU rDNA. Ceratomyxa marginati n. sp. exhibits disporic plasmodia measuring 12-14 µm long and 11-12 µm wide and mature myxospores which are slightly crescent-shaped, measuring 6.0 ± 0.3 (5.6-6.8) µm in length and 12.9 ± 0.9 (11.5-14.0) µm in thickness. Plasmodia of C. aenei n. sp. were disporic and ellipsoidal and measured 28-32 µm in length and 19-22 µm in width. Mature myxospores of C. aenei n. sp. were elongated with unequal shell valves and measured 7.4 ± 0.6 (6.9-9.0) µm in length and 26.9 ± 2.4 (23.2-30.0) µm in thickness. Based on the SSU rDNA sequences, Ceratomyxa marginati n. sp. and C. aenei n. sp. are distinct from all other Ceratomyxa sequences available in GenBank. Phylogenetic analysis reveals that the two new species branched together within a clade with other Ceratomyxa species from different host families and different geographical localities with maximum support (100%).

First description of Chloromyxum squali Gleeson and Adlard, 2012 (Myxozoa) in the Mediterranean Sea in a new host Squalus blainville (Chondrichthyes: Squalidae): morphological, ultrastructural and phylogenetic data.

Chloromyxum squali Gleeson and Adlard, 2012 originally described from Squalus acanthias Linnaeus, 1758 is reported for the first time from the gallbladder of Squalus blainville (Risso, 1827) caught from the eastern coast of Tunisia. In the present study, this chloromyxid was described based on morphological and ultrastructural features combined with molecular analysis of 18S rDNA. Young plasmodia were found attached to the gallbladder, while mature plasmodia and myxospores were observed floating free in the bile. Mature plasmodia were polysporic, subspherical in shape, measured 97.8 ± 0.5 µm long and 63.4 ± 0.4 µm wide. Mature myxospores were ovoid with a pointed anterior end, measuring 10.2 ± 0.5 µm long and 8.3 ± 0.5 µm wide. Two asymmetrical shell valves adhered together along an S-shaped suture line. Each valve has 5-7 elevated surface ridges parallel to suture line. A bundle of long caudal filaments extended from the basal end of shell valves. Four pyriform polar capsules equal in size, measuring 3.1 ± 0.4 µm long and 2.5 ± 0.3 µm wide, were situated at the same level in the anterior pole of the myxospore, each with a polar filament coiled in 7-8 turns. Pairwise comparisons among the SSU rDNA sequences revealed significant similarity between Chloromyxum squali infecting S. acanthias with the sequence obtained in this study. Phylogenetic analysis revealed that C. squali clustered in the clade of Chloromyxum species infecting the gallbladder of marine Chondrichthyes. Chloromyxum squali showed a seasonal variation of prevalence with significantly higher prevalence noted in summer and in autumn and absence of infection in winter.

Redescription and molecular characterization of two species of Pauciconfibula (Monogenea, Microcotylidae) from trachinid fishes in the Mediterranean Sea.

Many Pauciconfibula spp. have a long and complicated taxonomic history. The remaining unsolved taxonomic confusion in this genus is impelled by the host range and status of Pauciconfibula spp. from trachinid fishes: Pauciconfibula trachini and Pauciconfibula draconis, from Trachinus radiatus and Trachinus draco (Trachinidae), respectively. Pauciconfibula trachini was reported on Trachinus draco, type host of Pauciconfibula draconis suggesting thus a stenoxenic specificity for the former monogenean and the occurrence of two congeneric polyopisthocotyleans on a single host. Moreover, the validity of Pauciconfibula draconis was repeatedly questioned by several authors, unjustified synonymy between the two species was proposed, and the delimitations between the two species remained unsolved. Original descriptions were also incomplete and poorly illustrated. In this study, we provide a detailed illustrated redescription of both species based on newly collected specimens of Pauciconfibula trachini and Pauciconfibula draconis collected from their type hosts from off three Mediterranean localities: Algeria, Tunisia, and Italy. Integrative taxonomy using COI sequences was applied to resolve the delimitation between Pauciconfibula trachini and P. draconis. This study provides the first DNA barcoding for members of this genus.

Zschokkella epinepheli n. sp. (Myxosporea: Myxidiidae) infecting the gallbladder of the white grouper Epinephelus aeneus (Serranidae) from Tunisian waters.

A new coelozoic myxosporean species, Zschokkella epinepheli n. sp., collected from the gallbladder of the white grouper Epinephelus aeneus (Perciformes: Serranidae) from the bay of Bizerte, Tunisia, is described based on morphological and molecular characteristics. Myxospores and plasmodia were observed floating free in the bile. Mature plasmodia were polysporic and subspherical in shape, measuring 85.0-94.0 µm long and 70.0-82.0 µm wide. Mature myxospores were ovoid in valvular view, measuring 10.0 ± 1.7 (8.0-11.0) µm in length and 7.0 ± 0.3 (6.6-7.5) µm in width. Polar capsules were pyriform and equal in size, measuring 3.0 ± 0.2 (2.8-3.6) µm in length and 2.3 ± 0.3 (1.8-2.7) µm in width. Myxospore valves had 12-14 longitudinal striations. Based on the small subunit rDNA, the new species Z. epinepheli n. sp. differs from all other Zschokkella species for which there is a DNA sequence deposited in GenBank. Phylogenetic analysis revealed that Z. epinepheli n. sp. clustered in the marine subclade of Zschokkella species within the biliary tract IV clade. This is the first report of a Zschokkella species from the gallbladder of an epinephelin fishes.

Morphological and molecular identification of Hysterothylacium larvae (Nematoda: Raphidascarididae) in marine fish from Tunisian Mediterranean coasts.

The taxonomy of Hysterothylacium genus in Mediterranean waters remains incomplete and unresolved. The aim of the current study was to investigate the morphological and molecular identification of selected species of Hysterothylacium larvae in marine fish from the Tunisian Mediterranean coasts. A total of 192 marine fish samples were examined. In total, thirty-seven third-stage larvae of Hysterothylacium were morphologically identified as Hysterothylacium type V. In the present study, representatives of this type from the Mediterranean Sea were genetically characterized for the first time by sequencing the rDNA ITS (ITS1-5.8S-ITS2) regions and mtDNA cox2 gene. This study represents the first report of Hysterothylacium type V from the Mediterranean Sea. We also report Mullus barbatus, M. surmuletus, and Pagellus erythrinus as new hosts for this larval type. Based upon molecular and phylogenetic analyses considering the rDNA ITS regions, the Hysterothylacium type V described here was classified as a new genotype, named Genotype B. The valid genetic data of the described Hysterothylacium type V in the present study can be used to establish the phylogenetic relationships among Hysterothylacium species from the Mediterranean Sea and worldwide for future research.

Morphology, seasonality and molecular characterization of Ceratomyxa draconis n. sp. parasite of Trachinus draco (L.) from the Bay of Bizerte, Tunisia.

The specimens of Trachinus draco collected from the Bay of Bizerte were found to be infected with a new Ceratomyxa species described as Ceratomyxa draconis n. sp. The sequence of small subunit ribosomal RNA gene obtained in this study differs from other Ceratomyxa sequences available in GenBank. Mature spores of this species were elongated and crescent-shaped in sutural view, measuring 7.4 ± 0.77 (6.4-8.0) µm in thickness and 30.8 ± 1.65 (28.8-32.8) µm in width. The polar capsules were spherical, equal in size, and measuring 3.3 ± 0.2 (3.6-4.0) µm in diameter. The Ceratomyxa draconis n. sp. showed a clearly seasonal variation of prevalence with highest prevalence noted during summer months.

Ceratomyxa mennani n. sp. (Myxosporea: Bivalvulida) parasitizing the gallbladder of the dusky grouper Epinephelus marginatus (Serranidae) from Tunisian waters.

Ceratomyxa mennani n. sp. is a new coelozoic Ceratomyxa species found in the gallbladder of Epinephelus marginatus from the Gulf of Tunis, Tunisia. Mature plasmodia were disporic, ovoid in shape measuring 9-12 µm in width and 11-14 µm in length. Mature myxospores were slightly crescent-shaped with almost straight posterior margin, measuring 5.8 ± 0.2 (5.4-6.1) µm in length and 12.7 ± 0.3 (11.9-13.0) µm in thickness. The two valves were unequal with rounded ends. Polar capsules were spherical, equal in size with 2.1 ± 0.2 (1.9-2.6) µm in diameter. The binucleated sporoplasm filled the entire cavity of the myxospore. Molecular analysis of SSU rDNA sequences indicated that C. mennani n. sp. was distinct from all other Ceratomyxa sequences in GenBank. Phylogenetic analysis revealed that C. mennani n. sp. clustered with Ceratomyxa species infecting Epinephelinae fishes. Seasonal prevalence of infection over one year was significantly higher in winter and the lowest in autumn. This is the third report of Ceratomyxa species infecting the gallbladder of Epinephelus marginatus from Tunisia and the first study to include molecular data.

Ceratomyxa gouletti n. sp. (Myxosporea: Ceratomyxidae), a parasite of the red scorpionfish Scorpaena scrofa (L.) from Tunisian waters.

Ceratomyxa gouletti n. sp. is a new parasite described from the gallbladder of the red scorpionfish Scorpaena scrofa (Scorpaeniformes: Scorpaenidae) collected from La Goulette in the Northeast of Tunisia. The parasite develops disporic plasmodia, elliptical to ovoid in shape, measuring up to 33.1 ± 3.6 (28.0-38.2) µm in length and 12.5 ± 2.1 (11.3-14.0) µm in width. Mature spores are elongated transversely and crescent-shaped with rounded ends and unequal shell valves, measuring 8.1 ± 1.27 (7.2-9.0) µm in length and 32.0 ± 2.5 (27.0-38.2) µm in thickness. Sutural line is straight and visible between valves. Polar capsules are subspherical with 3.15 ± 0.63 (2.7-3.6) µm long and 1.9 ± 0.14 (1.8-2.0) µm wide. Posterior spore angle is slightly concave 160°-175°. Molecular analysis based on the small subunit 18S rDNA sequence shows that C. gouletti n. sp. is different from all other ceratomyxid species DNA sequences in GenBank. Phylogenetic trees clustered the new species with long-branching Ceratomyxa species, and it was closely related to the species Ceratomyxa longipes from two gadid fish hosts with 89% bootstrap support.

Scanning electron microscopy study of Strabax monstrosus von Nordmann, 1864 (Copepoda: Chondracanthidae) from Tunisian waters and histopathological study of its effects on host.

Strabax monstrosus von Nordmann, 1864 (Copepoda: Chondracanthidae) is an ectoparasite and it was recently recorded from small red scorpionfish, Scorpaena notata Rafinesque, and red scorpionfish, Scorpaena scrofa L., collected from Tunisian coastal waters. Copepod females were found deeply embedded in the branchial cavity reaching the cardiac region with their mouth-parts. Observations of females and males using scanning electron microscopy showed a number of morphological features that have been previously overlooked or incompletely presented. In this study, we report for the first time the fine structures of appendages, mouthparts, external genital organs (nuptial organs), and other structures of male and female Strabax monstrosus. Histopathological study showed lesions in the cardiac region of the host fish which is the target site of the parasite. The presently reported finding is the first record of Strabax monstrosus from Scorpaena notata and Scorpaena scrofa collected from Tunisian waters.

Ultrastructural observations on the development of Ceratomyxa aegyptiaca (Myxozoa: Bivalvulida) infecting Solea aegyptiaca (Pleuronectiformes: Soleidae) from Tunisian coastal lagoon.

Ultrastructural description of Ceratomyxa aegyptiaca Yemmen, Marton, Eszterbauer and Bahri, 2012 infecting the gallbladder of Solea aegyptiaca Chabanaud, 1927 from a tunisian north-east costal lagoon, was presented in this study. The primary cell was attached to the gallbladder epithelium and presented at one side cytoplasmic projections corresponding to pinocytotic invaginations. Netherless, early sporogonic stages development was carried with contact to the epithelial cells of gallbladder. Immature spores were identified in early sporoblasts by their valvogenic, capsulogenic and binucleated sporoplasmic cells. Capsulogenesis was asynchronous. Each capsulogenic cell presented a large condensed nucleus and a capsular primordium that extended into an external tube. The eversion and coiling of the external tube gave rise to the polar filament, which displayed six turns and an apical plug.

Morphological description and phylogeny of Ceratomyxa scorpaeni n. sp. (Myxosporea: Ceratomyxidae) infecting the gallbladder of Scorpaena porcus (L.) (Scorpaeniformes: Scorpaenidae) from the bay of Bizerte in Tunisia.

A new marine myxosporean species Ceratomyxa scorpaeni n. sp. is described using morphological characteristics and small subunit ribosomal DNA (rDNA) sequence data. The parasite was found infecting the gallbladder of Scorpaena porcus Linnaeus, 1758 collected from the Bay of Bizerte. Mature plasmodia were disporous free floating in the bile. Spores were transversely elongated and crescent-shaped with unequal shell valves and measured 7.6 ± 0.2 (7.2-8.0) µm in length and 32.8 ± 0.3 (21.6-44.0) µm in thickness. Two sub-spherical polar capsules equal in size, situated at the same level and measured 3.0 ± 0.2 (3.0-3.2) µm in length and 2.4 ± 0.3 (2.3-3.3) µm in width. The infection with C. scorpaeni n. sp. was prevalent throughout the year with a maximum prevalence of infection during spring and summer. Molecular analysis of the SSU rRNA gene confirmed the parasite as a new member of the genus Ceratomyxa and showed close relationship with the species of Ceratomyxa infecting the gallbladders of Sparidae from the Mediterranean Sea. This is the first report of Ceratomyxa species from the gallbladder of S. porcus in Tunisian coastal waters.

Morphology, seasonality and phylogeny of Zschokkella trachini n. sp. (Myxozoa, Myxosporea) infecting the gallbladder of greater weever Trachinus draco (L.) from Tunisian waters.

We describe a new myxosporean species, Zschokkella trachini n. sp., infecting the gallbladder of greater weever Trachinus draco Linnaeus 1758 from Tunisia. This is the first record of Zschokkella species in T. draco. Plasmodia were polysporic producing six to eight mature spores; they were attached to the gallbladder epithelium or free floating in the bile. Mature spores were sub-ovoid in the frontal view, measured 15.2 ± 0.6 (14.4-16.0) µm in length and 9.8 ± 0.7 (9.0-10.8) µm in width. Two equal spherical polar capsules 4.0 ± 0.4 (3.6-4.5) µm in diameter, were located separately at the spore's extremities. The prevalence of infection ranged from 23.5 to 87.7 %. Morphological data and molecular analysis of the small subunit rDNA gene identified this parasite as a new species of Zschokkella. Neighbour joining clustered the parasite in a sub-clade containing other Zschokkella species parasiting the gallbladder of marine fish hosts, located within the coelozoic clade of the major freshwater clade. This is the second Zschokkella species reported from Tunisia.

Ceratomyxa aegyptiaca n. sp. (Myxozoa: Myxosporea) from the gall-bladder of Solea aegyptiaca Chabanaud (Pleuronectiformes: Soleidae) in a Tunisian coastal lagoon.

A new marine myxosporean species, Ceratomyxa aegyptiaca n. sp. is described from the gall-bladder of Solea aegyptiaca Chabanaud collected from the Ghar El Melh Lagoon in northeastern Tunisia. Mature spores are elongate and crescent-shaped, measuring 8-11 µm in length and 48-58 µm in width. The polar capsules are spherical, 3.2-4 µm in diameter and equal in size. Trophozoites are polysporous and float free in the bile or are attached on the epithelium of the gall-bladder. Morphological data and molecular analysis based on 18S rDNA sequences are provided. The 18S rDNA of C. aegyptiaca is readily distinguishable from that of other myxozoan species, as the genetically most similar myxozoan parasite, C. seriolae Yokoyama & Fukuda, 2001 (AB530265) collected from Seriola quinqueradiata Temminck & Schlegel in Japanese waters, shares with it only 67.5% identical nucleotides over a 1,680-bp long fragment of 18S rDNA.

Protozoan and myxozoan infections in wild gilthead seabream (Sparus aurata L.) from North Lake of Tunis, Tunisia.

A total of 150 gilthead seabream Sparus aurata L., from North Lake of Tunis, Tunisia, were studied for protozoan and myxozoan parasites. The parasitological survey revealed the presence of ectoparasites (Amyloodinium ocellatum Brown, 1931, Trichodina lepsii Lom, 1962 on the gills) and endoparasites (Ceratomyxa sparusaurati Sitjà-Bobadilla, Palenzuela et Alvarez-Pellitero, 1995 infecting the gallbladder, and Eimeria sparis Sitjà-Bobadilla, Palenzuela et Alvarez-Pellitero, 1996 parasitizing the intestine). This is the first record of Amyloodinium ocellatum, Trichodina lepsii, Ceratomyxa sparusaurati, and Eimeria sparis in S. aurata from Tunisian waters. Data on prevalence and intensity of infection are provided. A comparison of the present species with previously described species in cultured gilthead seabream from other Mediterranean countries is also presented. In this study Trichodina lepsii is identified for the first time in Sparus aurata. A taxonomic description of this species based on silver nitrate method is provided.

Henneguya tunisiensis n. sp. (Myxosporea: Bivalvulida), a new gill parasite of Symphodus tinca (L.) (Teleostei: Labridae) off Tunisia.

Henneguya tunisiensis n. sp., a new myxosporean, is described from the gill-arches of the East Atlantic peacock wrasse Symphodus tinca (L.) collected from off the Kerkennah Islands, Tunisia. It is characterised by the presence of elongate white plasmodia of 1-1.5 x 1.5-2 mm in size. The mature spores are rounded in frontal view and have two identical polar capsules and two caudal appendages which taper considerably at the end. Both light and electron microscopical data show that this species differs in several morphological features from all previously described Henneguya spp. A molecular analysis, based on 18S rDNA sequence data, indicates that H. tunisiensis n. sp. is readily distinguishable from other myxozoan DNA sequences in GenBank. Phylogenetically, the new species is placed in the marine Henneguya clade, which is a sister group of marine Myxobolus spp. from perciform fishes in Tunisian waters.

Morphological and phylogenetic studies of marine Myxobolus spp. from mullet in Ichkeul Lake, Tunisia.

Six species of the genus Myxobolus (Myxozoa) from the marine environment were collected from two species of mullet (Mugil cephalus and Liza ramada) in Ichkeul Lake, Tunisia. Four of these species were described previously (Myxobolus bizerti, Myxobolus ichkeulensis, Myxobolus spinacurvatura, and Myxobolus episquamalis) and two (Myxobolus exiguus and Myxobolus muelleri) are redescribed. The small subunit ribosomal (18S rDNA) sequences of these six myxozoans were obtained and compared to traditional criteria used in the identification and taxonomy of myxozoan species (such as spore morphology, host specificity, and tissue tropism). A distance analysis of 1,600-1,700 base pairs of the 18S rDNA of the six species indicates that they formed a monophyletic group separate from Myxobolus spp. found as parasites of freshwater fish. The sequence analyses also confirm that these morphologically different Myxobolus spp. that infect mullet represent different species. Lastly, M. exiguus and M. muelleri, which were found in the same host, exhibit clear differences in spore morphology but sequencing of two different regions of the 18S rDNA show they are closely related. These results demonstrate the utility of DNA sequence data in providing more detailed relationships among the Myxobolus species based upon existing morphological taxonomic approaches. We suggest that future descriptions of Myxobolus spp. provide both careful spore descriptions as part of the traditional criteria but also 18S rDNA sequence data that will aid in situations where morphological details may be absent or misleading.