Scoliosis and kyphosis in blue-spotted and marbled flathead fish associated with a Myxobolus acanthogobii-like parasite.

Spinal deformities in finfish have the potential to impact aquaculture industries and wild populations by increasing morbidity, mortality, and reducing growth rates. Myxobolus acanthogobii has been implicated in causing scoliosis and lordosis in various aquatic species in Japan. We investigated 4 cases of spinal deformity in 2 flathead (Platycephalus) species that were submitted to the Elizabeth Macarthur Agricultural Institute (EMAI) in New South Wales (NSW), Australia, between 2015 and 2021. Flathead are commercially significant species that are popular among Australian consumers, and are also sought-after species targeted by recreational fishers. Gross deformities are concerning to the community and may impact the quality and quantity of specimens available for consumption. Three blue-spotted flathead (P. caeruleopunctatus) and one marbled flathead (P. marmoratus) were submitted, all with marked scoliosis and kyphosis; 1-2-mm cysts were present on the dorsum of the brain, most often over the optic lobe or cerebellum. Cytology and differential interference microscopy of cyst material revealed numerous oval spores, x̄ 14 ± SD 0.75 µm × x̄ 11.5 ± SD 0.70 µm, with 2 pyriform polar capsules, the morphology of which is consistent with a Myxobolus sp. PCR assay and 18S rDNA sequencing of the cyst material identified a Myxobolus sp. with 96% identity to M. acanthogobii. The identification of this Myxobolus sp. confirms the presence of parasites with the potential to cause spinal deformity in significant aquatic species in NSW waterways.

Integrative taxonomy of Serrasentis gibsoni n. sp. (Acanthocephala: Isthmosacanthidae) from flatfishes in the Gulf of Mexico.

The Isthmosacanthidae acanthocephalan species of the genus Serrasentis are parasites of marine teleosts and an elasmobranch. In this study, Serrasentis gibsoni n. sp. is described from the intestines of four flatfish species (Paralichthyidae), namely Ancyclopsetta quadrocellata, Cyclopsetta chittendeni, Syacium gunteri, and S. papillosum from 10 oceanic sites in the Gulf of Mexico (GoM). Twenty sequences of the 'barcoding' region of cytochrome C oxidase subunit I gene were obtained from 20 adults of Serrasentis gibsoni n. sp. Additionally, five sequences of the barcoding region were obtained from five adults of rhadinorhynchid Gorgorhynchus lepidus from C. chittendeni, S. papillosum and one species of Haemulidae, Haemulom aurolineatum, from five oceanic sites from the GoM. Two phylogenetic approaches were followed: Bayesian inference and maximum likelihood. In both phylogenetic reconstructions, the sequences of Serrasentis gibsoni n. sp. were recovered as a monophyletic group within the genus Serrasentis and placed as a sister group to G. lepidus. However, due to the lack of molecular data for species of the Isthmosacanthidae and Rhadinorhynchidea, these phylogenetic inferences must be taken with caution. Serrasentis gibsoni n. sp. is the first species of Serrasentis described from Paralichthyidae flatfish species from marine waters of the Americas and from the GoM. Based on the barcoding data set analyzed, Serrasentis gibsoni n. sp. appears to have high intraspecific genetic variation; thus, it is necessary to continue exploring the genetic diversity of this species to infer its intraspecific evolutionary patterns.

From Argentine Abyssal Plain to farmed turbot in Spain: A ubiquitous amoeba species Vannella robusta sp. nov. (Amoebozoa, Vannellida).

A strain with the characters of the genus Vannella was isolated from the water layer immediately above the deep-sea sediment collected in the south-western Atlantic Ocean, ca. 4.6 km deep. Small-subunit ribosomal RNA (SSU rRNA) and cytochrome c oxidase (Cox1) gene phylogenetic analyses showed that the new strain branches within the clade of previously isolated unnamed Vannella strains from different marine fish and invertebrate hosts. Although the SSU rRNA gene sequences of these strains show variability within 2% of all nucleotide positions without any regular pattern, the available Cox1 gene sequences from within this clade are identical. Given the morphological homogeneity of the revealed clade, all of its strains can be assigned under the same species name, and the variation of their SSU rRNA is comparable to its intragenomic variation, as shown by molecular cloning of the PCR amplicons. High variability of the SSU rRNA gene sequences within and between independently isolated morphologically identical strains in combination with highly conserved Cox1 gene sequences may be a feature in some clades of Vannella, but is not a general rule for this genus, as SSU rRNA genes conserved between different morphospecies occur in several other clades within Vannella.

Emendation of the genus Neoheterobothrium and a proposal of a new genus Paraheterobothrium (Monogenea: Diclidophoridae) for five species of diclidophorids from Pleuronectiform fishes.

Neoheterobothrium chilense González, Oliva & Acuña, 2002 is redescribed based on newly collected specimens from the type host, Hippoglossina macrops and type locality (Coquimbo, Northern Chile). Type specimens of Neoheterobothrium hippoglossini Piasecki, Wierzbicka & Kempter, 2000 and Choricotyle exilis Crane, 1972 are also redescribed. These three diclidophorid monogeneans infecting pleuronectiform flatfishes have pre-ovarian seminal receptacle, ootype inside the semicircle of ovary and elongated pharynx, differentiated from the most similar Orbocotyle Euzet & Suriano, 1975 in that members of the latter genus possess post-ovarian seminal receptacle, post-ovarian ootype and cylindrical or pyriform pharynx. Paraheterobothrium n. g. is proposed for P. chilense n. comb. (type), P. hippoglossini n. comb., P. exilis n. comb., P. papillosum n. comb. and P. syacii n. comb. A redescription of Neoheterobothrium affine (Linton, 1898), the type species of the genus, based on voucher specimens confirmed differences from the new genus in the presence of lamellate plate in the anterior jaw of clamps, absence of seminal receptacle and ovoid pharynx. The genus Neoheterobothrium was emended and N. cynoscioni (MacCallum, 1917) and N. mcdonaldi Payne, 1987 were removed from the genus. Neoheterobothrium now comprises N. affine (type), N. insulare Oliva & Luque, 1995, N. hirame Ogawa, 1999, and N. paralichthyi Suriano & Labriola, 1999, all of them parasites of flatfishes of the genus Paralichthys. Molecular analyses with the internal transcribed spacer 1 distinguished the clade for Paraheterobothrium spp. (P. chilense n. comb. and P. papillosum n. comb.) and Neoheterobothrium spp. (N. hirame, N. affine and N. paralichthyi), supporting this taxonomic emendation.

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi.

Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

A new species of Neoheterobothrium Price, 1943 (Monogenea, Diclidophoridae) from Syacium papillosum (Linnaeus) (Pleuronectiformes, Paralichthyidae) in the Yucatan Shelf, with notes on the validity of the subfamilies in the Diclidophoridae.

Neoheterobothrium papillosum n. sp. (Monogenea: Diclidophoridae) is described based on specimens collected from the gills of the dusky flounder Syacium papillosum (Linnaeus) (Paralichthyidae) in the Yucatan Shelf. The new species can be differentiated from other congeneric species by the following characteristics: (1) number of hooks in the genital atrium (8 hooks), (2) number of testes (20-34), (3) pharynx size, (4) peduncle length, and (5) egg size. Neoheterobothrium papillosum n. sp. resembles Neoheterobothrium syacii; however, detailed morphological analysis allowed for their separation into two different species. Sequences of the nuclear ribosomal DNA partial 28S and ITS1 were obtained and compared with available sequences of other species and genera of the family Diclidophoridae from GenBank. A phylogenetic analysis was conducted, including 33 sequences, 18 of which represented 17 species and 11 genera of the family Diclidophoridae. The phylogenetic tree showed that Diclidophoridae is a monophyletic family and species of Diclidophora were grouped as the sister group of Neoheterobothrium papillosum n. sp. Our phylogenetic analyses were consistent with Mamaev's hypothesis, who divided Diclidophoridae into Diclidophorinae and Choricotylinae and concluded that Neoheterobothrium was a genus belonging to Diclidophorinae.

Identification and Molecular Characterization of Superoxide Dismutases Isolated From A Scuticociliate Parasite: Physiological Role in Oxidative Stress.

Philasterides dicentrarchi is a free-living microaerophilic scuticociliate that can become a facultative parasite and cause a serious parasitic disease in farmed fish. Both the free-living and parasitic forms of this scuticociliate are exposed to oxidative stress associated with environmental factors and the host immune system. The reactive oxygen species (ROS) generated by the host are neutralized by the ciliate by means of antioxidant defences. In this study we aimed to identify metalloenzymes with superoxide dismutase (SOD) activity capable of inactivating the superoxide anion (•O2-) generated during induction of oxidative stress. P. dicentrarchi possesses the three characteristic types of SOD isoenzymes in eukaryotes: copper/zinc-SOD, manganese-SOD and iron-SOD. The Cu/Zn-SOD isoenzymes comprise three types of homodimeric proteins (CSD1-3) of molecular weight (MW) 34-44 kDa and with very different AA sequences. All Cu/Zn-SODs are sensitive to NaCN, located in the cytosol and in the alveolar sacs, and one of them (CSD2) is extracellular. Mn- and Fe-SOD transcripts encode homodimeric proteins (MSD and FSD, respectively) in their native state: a) MSD (MW 50 kDa) is insensitive to H2O2 and NaN3 and is located in the mitochondria; and b) FSD (MW 60 kDa) is sensitive to H2O2, NaN3 and the polyphenol trans-resveratrol and is located extracellularly. Expression of SOD isoenzymes increases when •O2- is induced by ultraviolet (UV) irradiation, and the increase is proportional to the dose of energy applied, indicating that these enzymes are actively involved in cellular protection against oxidative stress.

Unique ultrastructural characteristics of the tegument of the digenean blood fluke Aporocotyle simplex Odhner, 1900 (Digenea: Aporocotylidae), a parasite of flatfishes.

This paper includes the first transmission electron microscopical (TEM) study of the tegument of a member of the basal digenean family Aporocotylidae. Scanning electron microscopical investigations of the fish blood fluke Aporocotyle simplex show that each boss on the lateral body surface bears 12-15 simple, uniform spines which extend from 0.5-2.7 µm above the surface of the boss. TEM observations revealed that these spines reach deep beneath the distal cytoplasm of the tegument for much of their length (9-12 µm) and are surrounded by a complex of diagonal muscles in each boss. This is the first record of any digenean with so-called 'sunken' spines. The results suggest that aporocotylid spines arise from within the sarcoplasm of the boss diagonal muscles. The sunken cell bodies (perikarya) of the tegument are connected to the distal cytoplasm via ducts (specialised processes lined by microtubules); this in contrast to other digeneans studied, where they are connected via non-specialised cytoplasmic processes. Within the distal cytoplasm, the tegumental ducts of A. simplex are surrounded by invaginations of the basal membrane and release their cytoplasmic inclusions into the distal cytoplasm. These apparently unique morphological features of the tegument, especially the deep origin of the spines, may represent useful characteristics for understanding aporocotylid relationships, especially in view of the known variation in the spine patterns of aporocotylids.

Ultrastructural organisation of the spermatozoon of Allopodocotyle tunisiensis Derbel and Neifar, 2009 (Digenea, Opecoelidae), an intestinal parasite of Solea aegyptiaca Chabanaud, 1927 (Teleostei, Soleidae).

The ultrastructure of the spermatozoon of Allopodocotyle tunisiensis (Digenea, Opecoelidae), an intestinal parasite of Solea aegyptiaca (Teleostei, Soleidae), is described by transmission electron microscopy (TEM). The mature spermatozoon is a filiform cell that exhibits two axonemes of different length with the 9+'1' pattern of trepaxonematan Platyhelminthes. In the anterior spermatozoon extremity, cortical microtubules are absent. They appear after the disappearance of an anterior electron-dense material, being initially in a continuous and submembranous layer. They surround only partially the sperm cell. Later, these cortical microtubules are distributed into two bundles. Additionally, the spermatozoon of A. tunisiensis shows two mitochondria, a nucleus, an external ornamentation of the plasma membrane, spine-like bodies, and a large amount of glycogen granules. According to the location of the external ornamentation, A. tunisiensis presents a Quilichini et al.'s type 2 spermatozoon. With respect to the posterior extremity, the sperm cell of A. tunisiensis corresponds to the Quilichini et al.'s opecoelid type. The morphology of the first mitochondrion with a U-shaped posterior extremity is described for the first time in a digenean spermatozoon.

Genetic differences in host infectivity affect disease spread and survival in epidemics.

Survival during an epidemic is partly determined by host genetics. While quantitative genetic studies typically consider survival as an indicator for disease resistance (an individual's propensity to avoid becoming infected or diseased), mortality rates of populations undergoing an epidemic are also affected by endurance (the propensity of diseased individual to survive the infection) and infectivity (i.e. the propensity of an infected individual to transmit disease). Few studies have demonstrated genetic variation in disease endurance, and no study has demonstrated genetic variation in host infectivity, despite strong evidence for considerable phenotypic variation in this trait. Here we propose an experimental design and statistical models for estimating genetic diversity in all three host traits. Using an infection model in fish we provide, for the first time, direct evidence for genetic variation in host infectivity, in addition to variation in resistance and endurance. We also demonstrate how genetic differences in these three traits contribute to survival. Our results imply that animals can evolve different disease response types affecting epidemic survival rates, with important implications for understanding and controlling epidemics.

Revealing trophic transmission pathways of marine tapeworms.

Parasites are important components of natural systems, and among their various roles, parasites strongly influence the flow of energy between and within food webs. Over 1000 tapeworm species are known to parasitise elasmobranchs, although full life cycles are resolved for fewer than 10 of them. The lack in resolution stems from the inability to distinguish larval from adult stages using morphology alone. Molecular elucidation of trophic transmission pathways is the next step in understanding the role of hosts and parasites within food webs. We investigated the parasite assemblage of New Zealand's rough skate, Zearaja nasuta. Skates and their prey items (obtained from the skates' stomachs) were dissected for the recovery of adult and larval tapeworms, respectively. A fragment of the 28S rDNA region was amplified for worm specimens with the aim to confirm species identity of parasites within rough skates and to uncover trophic transmission pathways that exploit the predation links between rough skates and their prey. We identified seven species of tapeworms from four tapeworm orders. Four trophic transmission pathways were resolved between three prey items from skates stomachs and skates, and one pathway between larval tapeworm sequence from a New Zealand sole and skate, i.e. a genetic match was found between larval tapeworms in prey and adult worms in skates. We report the first case of an adult trypanorhynch parasitising rough skate. These findings contribute to our limited understanding of cestode life cycles as well as providing insights into the importance of predator-prey relationships for parasite transmission.

The coagulation system helps control infection caused by the ciliate parasite Philasterides dicentrarchi in the turbot Scophthalmus maximus (L.).

Many studies have shown that coagulation systems play an important role in the defence against pathogens in invertebrates and vertebrates. In vertebrates, particularly in mammals, it has been established that the coagulation system participates in the entrapment of pathogens and activation of the early immune response. However, functional studies investigating the importance of the fish coagulation system in host defence against pathogens are scarce. In the present study, injection of turbot (Scopthalamus maximus) with the pathogenic ciliate Philasterides dicentrarchi led to the formation of macroscopic intraperitoneal clots in the fish. The clots contained abundant, immobilized ciliates, many of which were lysed. We demonstrated that the plasma clots immobilize and kill the ciliates in vitro. To test the importance of plasma clotting in ciliate killing, we inhibited the process by adding a tetrapeptide known to inhibit fibrinogen/thrombin clotting in mammals. Plasma tended to kill P. dicentrarchi slightly faster when clotting was inhibited by the tetrapeptide, although the total mortality of ciliates was similar. We also found that kaolin, a particulate activator of the intrinsic pathway in mammals, accelerates plasma clotting in turbot. In addition, PMA-stimulated neutrophils, living ciliates and several ciliate components such as cilia, proteases and DNA also displayed procoagulant activity in vitro. Injection of fish with the ciliates generated the massive release of neutrophils to the peritoneal cavity, with formation of large aggregates in those fish with live ciliates in the peritoneum. We observed, by SEM, numerous fibrin-like fibres in the peritoneal exudate, many of which were associated with peritoneal leukocytes and ciliates. Expression of the CD18/CD11b gene, an integrin associated with cell adhesion and the induction of fibrin formation, was upregulated in the peritoneal leukocytes. In conclusion, the findings of the present study show that P. dicentrarchi induces the formation of plasma clots and that the fish coagulation system may play an important role in immobilizing and killing this parasite.

A new species of Steringotrema Odhner, 1911 (Trematoda: Fellodistomidae) from the New Zealand sole Peltorhamphus novaezeelandiae Günther off Kaka point in the Catlins, South Island, New Zealand.

As a part of a comprehensive survey of macroparasites of commercially exploited fish species off the coast of Otago, New Zealand, the parasite fauna of the New Zealand sole Peltorhamphus novaezeelandiae Günther was recently studied. Steringotrema robertpoulini n. sp. is described from this host and compared with known species of Steringotrema Odhner, 1911. The new species is readily distinguished from all of its congeners, except for S. divergens (Rudolphi, 1809) Odhner, 1911, by having the follicular vitellarium divided in four zones rather than two, and can be differentiated from S. divergens mainly by the posterior extent of the intestinal caeca in the hindbody, as well as by host association and geographical distribution. DNA sequences of the 28S ribosomal gene were generated and phylogenetic analyses were undertaken using maximum likelihood and Bayesian inference to assess the phylogenetic position of the new species within the family Fellodistomidae Nicoll, 1909. Analyses included the available sequences for 14 species of the family distributed among eight genera, along with nine species of other members of the order Plagiorchiida La Rue, 1957 as outgroups. The resulting topology shows that the new species of Steringotrema is nested as the sister species of Steringophorus dorsolineatus (Reimer, 1985) Bray, 1995. However, low nodal support indicates that relationships among these species are not fully resolved and require further revision and denser taxon sampling for more detailed molecular work. More information is required to draw further conclusions about the taxonomic status of the genera Steringotrema and Steringophorus Odhner, 1905.

Protocol for cryopreservation of the turbot parasite Philasterides dicentrarchi (Ciliophora, Scuticociliatia).

Philasterides dicentrarchi is a free-living marine ciliate that can become an endoparasite that causes a severe disease called scuticociliatosis in cultured fish. Long-term maintenance of this scuticociliate in the laboratory is currently only possible by subculture, with periodic passage in fish to maintain the virulence of the isolates. In this study, we developed and optimized a cryopreservation protocol similar to that used for the long-term storage of scuticociliates of the genus Miamiensis. The cryogenic medium comprised ATCC medium 1651 and a combination of 11% dimethylsulfoxide and 5% glycerol. We have verified that the most important factor ensuring the efficiency of the cryopreservation procedure is the growth phase of the culture, and that ciliates should be cryopreserved at the stationary phase (around the sixth day of culture). The cryopreservation protocol described here can be used for all strains of P. dicentrarchi as well as commercial strains of Miamiensis and enables the virulence of the strains to be maintained. Finally, this cryopreservation protocol has been shown to be more effective than others routinely applied to scuticociliates, yielding a higher survival rate with a lower initial concentration of ciliates. The results obtained indicate that the cropreservation protocol enables the long-term storage of scuticociliate parasites while maintaining the virulence of the isolates. The protocol is therefore suitable for use in vaccine production and related studies.

A new species of Furcohaptor Bijukumar & Kearn, 1996 (Monogenea: Diplectanidae) parasitic on Cynoglossus robustus Günther (Pleuronectiformes: Cynoglossidae) in the Seto Inland Sea, Japan, with comments on its systematic position and an amended generic diagnosis of Furcohaptor.

Furcohaptor brevis n. sp. is described from the gills of Cynoglossus robustus Günther caught in the Seto Inland Sea off Hiroshima Prefecture, Japan. Although Furcohaptor Bijukumar & Kearn, 1996 has been assigned to the Ancyrocephalinae Bychowsky, 1937 in the Dactylogyridae Bychowsky, 1933, this genus is transferred to the Diplectaninae Monticelli, 1903 in the Diplectanidae Monticelli, 1903 based on both morphological and molecular data. An amended generic diagnosis is provided.

First Freshwater Bothriocephalidean (Cestoda) from Tropical South America, Closely Related to African Taxa.

Regobothrium microhamulinum n. gen. and n. sp. (Bothriocephalidea: Bothriocephalidae) is proposed to accommodate a new cestode from flatfish Catathyridium jenynsii (Chabanaud, 1922) (Pleuronectiformes: Acharidae; type host) and another 3 freshwater fishes of the orders Characiformes, Cyprinodontiformes, and Siluriformes in the Neotropical Region. The new genus is placed in the Bothriocephalidae because it possesses medioventral uterine and mediodorsal genital pores and a follicular vitellarium. Regobothrium n. gen. is characterized by possessing a tiny, slightly subovate scolex narrower than the strobila, with an apical disc armed with 2 semicircles of 15-17 tiny hooks in each and an acraspedote strobila. Regobothrium n. gen. differs from all bothriocephalid cestodes that have a scolex armed with hooks by their small size (maximum length less than 20 µm) and a triangular shape with the basal part (handle or basal plate) shorter than the distal coniform part (blade). In the other hooked bothriocephalids, hooks have a longer handle than a blade. Regobothrium microhamulinum n. gen. and n. sp. is the third bothriocephalidean cestode described from freshwater teleosts in South America but the first out of Patagonia. Molecular phylogenetics consider Regobothrium as a member of a lineage consisting of (up to now exclusively) freshwater bothriocephalids from the Ethiopian biogeographic region, thus indicating Gondwanan relationship.

Differential effects of exposure to parasites and bacteria on stress response in turbot Scophthalmus maximus simultaneously stressed by low water depth.

The stress response of turbot Scophthalmus maximus was evaluated in fish maintained 8 days under different water depths, normal (NWD, 30 cm depth, total water volume 40 l) or low (LWD, 5 cm depth, total water volume 10 l), in the additional presence of infection-infestation of two pathogens of this species. This was caused by intraperitoneal injection of sublethal doses of the bacterium Aeromonas salmonicida subsp. salmonicida or the parasite Philasterides dicentrarchi (Ciliophora:Scuticociliatida). The LWD conditions were stressful for fish, causing increased levels of cortisol in plasma, decreased levels of glycogen in liver and nicotinamide adenine dinucleotide phosphate (NADP) and increased activities of G6Pase and GSase. The presence of bacteria or parasites in fish under NWD resulted in increased cortisol levels in plasma whereas in liver, changes were of minor importance including decreased levels of lactate and GSase activity. The simultaneous presence of bacteria and parasites in fish under NWD resulted a sharp increase in the levels of cortisol in plasma and decreased levels of glucose. Decreased levels of glycogen and lactate and activities of GSase and glutathione reductase (GR), as well as increased activities of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH) and levels of nicotinamide adenine dinucleotide phosphate (NADPH) occurred in the same fish in liver. Finally, the presence of pathogens in S. maximus under stressful conditions elicited by LWD resulted in synergistic actions of both type of stressors in cortisol levels. In liver, the presence of bacteria or parasites induced a synergistic action on several variables such as decreased activities of G6Pase and GSase as well as increased levels of NADP and NADPH and increased activities of GPase, G6PDH and 6PGDH.

Morphological variability, ultrastructure and molecular characterisation of Hysterothylacium reliquens (Norris & Overstreet, 1975) (Nematoda: Raphidascarididae) from the oriental sole Brachirus orientalis (Bloch & Schneider) (Pleuronectiformes: Soleidae).

Hysterothylacium reliquens (Norris & Overstreet, 1975), a common ascaridoid nematode parasitic in the marine fishes in the Indian and Atlantic Oceans, has been reported to exhibit remarkable morphological variability in the lengths of body, oesophagus and spicules, and the number and arrangement of postcloacal papillae. In order to determine whether H. reliquens with a broad range of morphological variability in the above-mentioned respects, represents a complex of sibling species or a single species, and to evaluate if the specimens of H. reliquens collected from the Indian and Atlantic Oceans are the same species, numbers of H. reliquens collected from the oriental sole Brachirus orientalis (Bloch & Schneider) (Pleuronectiformes: Soleidae) in the Arabian Gulf (off Iraq) were characterised using molecular approaches by sequencing and analysing the ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS)] and mitochondrial [cytochrome c oxidase subunit 2 (cox2)] target regions, respectively. The results of molecular analysis strongly supported that: (i) the broad ranges of morphological variability in the above-mentioned respects in the newly collected specimens of H. reliquens, should be considered as intraspecific variation and the nematode material collected from B. orientalis in the Arabian Gulf represented a single species; (ii) the specimens of H. reliquens collected from the Indian and Atlantic Oceans belong to the same species, but they possibly represent different geographical populations. Moreover, the detailed morphology of the labial papillae, lateral alae, excretory pore, distal end of spicules, midventral precloacal papilla, phasmids and caudal papillae was revealed by SEM for the first time. The molecular and morphological data of H. reliquens obtained herein contribute to a more accurate diagnosis of this little known ascaridoid nematode.

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.

Enteromyxum leei (Myxosporea: Bivalvulida) as the cause of myxosporean emaciation disease of farmed olive flounders (Paralichthys olivaceus) and a turbot (Scophthalmus maximus) on Jeju Island, Korea.

A life-threatening emaciation disease of unknown cause(s) is affecting the farming of olive flounders (Paralichthys olivaceus) and turbots (Scophthalmus maximus) on Jeju Island, Korea. As this is one of the major industries in the region, it is of great concern to local farmers trying to develop successful and sustainable aquaculture. We examined 16 olive flounders and one turbot cultured at three farms located in the southern part of Jeju Island, which manifested moderate to severe emaciation such as thinning of the body with notable appearance of bony ridges of the skull on heads. Fresh mucosal scrapings of the intestinal mucosa contained many myxosporean vegetative stages at various developments but not fully grown spores. Histological examination of gastrointestinal and other visceral organs revealed striking changes in the intestinal mucosa such as detachment and loss of the epithelium due to intensive parasitism of the myxosporean vegetative stages, accompanied by considerable leukocyte infiltration in the lamina propria, and at the final stage villus atrophy with no epithelial lining. Specific polymerase chain reaction using a pair of primers targeting a fragment of the 18S ribosomal RNA gene (rDNA) of Enteromyxum leei, a known pathogen causing myxosporean emaciation disease in a variety of cultured fish in Mediterranean countries and Japan, amplified 433-bp products in almost all diseased fish samples, particularly the gastrointestinal tract. Nearly the whole length of the 18S rDNA, 1672-bp long excluding primer-aligning sequences, of the present Korean isolate was comparable to those of E. leei isolates from Japan and Europe, particularly those from the former region. Taking the heavy load of various developmental stages of E. leei in the gastrointestinal mucosa into account, we ascribe the emaciation disease of the fish examined in the present study to this well-known myxosporean species and not to another unknown pathogen(s).