A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum

Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.     

The fish assemblage on a coralligenous shallow shelf off the Mediterranean coast of northern Israel

The fish assemblage on the shallow coralligenous shelf (16-30m depth) off Haifa, Israel was sampled using trammel nets throughout a period of 1 year. Complementary data were obtained via underwater censuses of fish on an artificial reef established later in the sampling area. Fortythree species of fish were sampled by trammel nets, 79% of which were observed also during the underwater censuses. Although fish of Red Sea origin constituted only 11.6% of the species composition in the net samples, they contributed 46.2% of the fish abundance and 40.6% of the biomass in these samples. This was supported by the finding that species of Red Sea origin contributed 64% of the abundance of large fish counted on the artificial reef. Siganus luridas, S. rivulatus and Sargocentron rubrum are the main contributors in number and biomass among fish of Red Sea origin. It is suggested that the biogenic rocky bottom of this area contains several components which are similar to biogenic habitats favoured by these benthic species in the Red Sea; this may explain the high abundance of these species in the studied area.     

Biochemical responses of fish exposed to a harmful dinoflagellate Cochlodinium polykrikoides

To elucidate the ichthyotoxic mechanisms of a harmful dinoflagellate Cochlodinium polykrikoides, biochemical responses of fish exposed to blooms were investigated. Particularly, based on our finding that oxidative damages of gill were associated with fish mortality (J. Plankton Res. 21 (1999) 2105-2115), dysfunction of ion-transporting enzymes and secretion of gill mucus of fish exposed to this bloom species were examined. The susceptibilities of several fishes to C. polykrikoides were different; the active pelagic fishes such as black scraper Thamnaconus septentrionalis, red sea bream Pagrus major, beakperch Oplegnathus fasciatus and seaperch Malakichthys wakiyae, were more vulnerable than the benthic fishes, flounder Paralichthys olivaceus and rockfish Sebastes inermis. In addition, the higher the algal cell density, the higher the fish mortality. When the test fishes were exposed to C. polykrikoides of 5000 cells ml(-1), the transport-related enzymes, carbonic anhydrase and Na(+)/K(+)-ATPase activities were significantly decreased. The activity of carbonic anhydrase was decreased with increasing algal cell density and exposure time. The quantity of total polysaccharide in gill mucus is higher in the fish exposed to C. polykrikoides than in the control fish; the magnitudes were higher in the pelagic fishes than that of benthic fishes. Moreover, a drop of blood pH and oxygen partial pressure (pO(2)) was also observed in red sea bream and flounder subjected to C. polykrikoides. These results suggest that the inactivation of gill transport-related enzymes activities, the fall in blood pO(2) and abnormal secretion of gill mucus by the C. polykrikoides may be one of the principal causes of fish kill.     

The determinants of species richness of a relatively young coral-reef ichthyofauna

Aim Unique topographic features left the Red Sea and its north‐eastern extension into the Gulf of Aqaba practically devoid of coral‐reef‐based organisms during the last glacial maximum. The current ichthyofauna in these two ‘regions’ thus represents the product of relatively recent colonization by species found in the Arabian Sea, which adjoins the Red Sea at its southern tip. We used this system to test why some marine species seemingly fail to extend their geographic range, thereby generating spatial heterogeneity in biodiversity. Location The Arabian Sea, Red Sea, and the Gulf of Aqaba. Methods A list of coral‐reef‐associated fish species, belonging to the 10 most speciose families, was compiled for each region using published sources. The data were analysed (major axis regression, randomization tests) for taxonomic and body‐size‐dependent biases in colonization probabilities. A simple probabilistic model was used to examine the potential contribution of local (within‐region) extinctions to determining species composition in the Red Sea. Results Of the 462 reef‐associated species that inhabit the Arabian Sea, 69% have crossed successfully into the Red Sea; of these, 55% have crossed into the Gulf of Aqaba. A species’ probability of being found in either ‘target’ was independent of presumed innate differences, i.e. ecological correlates of taxonomic affiliation and body size. Similarly, local extinctions were found unlikely to have been of consequence over the past several thousand years. Main conclusions Present‐day differences in the species richness of reef‐associated fish species among the Arabian Sea, Red Sea and Gulf of Aqaba appear to be the product of external, non‐selective constraints on colonization. The random nature of the colonization process is suggestive of ecological redundancy among coral‐reef fish species. Importantly, the study places a time frame on the processes that determine spatial patterns of biodiversity in reef fish.     

Molecular cloning of mRNA from toad granular gland secretion and lyophilized skin: identification of Bo8--a novel prokineticin from Bombina orientalis

Prokineticins are small (approximately 8 kDa), biologically active secretory proteins whose primary structures have been highly conserved throughout the Animal Kingdom. Representatives have been identified in the defensive skin secretions of several amphibians reflecting the immense structural/functional diversity of polypeptides in such. Here we describe the identification of a prokineticin homolog (designated Bo8) from the skin secretion of the Oriental fire-bellied toad (Bombina orientalis). Full primary structural characterization was achieved using a combination of direct Edman microsequencing, mass spectrometry and cloning of encoding skin cDNA. The latter approach employed a recently described technique that we developed for the cloning of secretory peptide cDNAs from lyophilized skin secretion, and this was further extended to employ lyophilized skin as the starting material for cDNA library construction. The Bo8 precursor was found to consist of an open-reading frame of 96 amino acid residues consisting of a putative 19-residue signal peptide followed by a single 77-residue prokineticin (Mr=7990 Da). Amino acid substitutions in skin prokineticins from the skin secretions of bombinid toads are confined to discrete sites affording the necessary information for structure/activity studies and analog design.     

The skin of fish as a transport epithelium: a review

The primary function of fish skin is to act as a barrier. It provides protection against physical damage and assists with the maintenance of homoeostasis by minimising exchange between the animal and the environment. However in some fish, the skin may play a more active physiological role. This is particularly true in species that inhabit specialised environmental niches (e.g. amphibious and air-breathing fish such as the lungfish), those with physiological characteristics that may subvert the need for the integument as a barrier (e.g. the osmoconforming hagfish), and/or fish with anatomical modifications of the epidermis (e.g. reduced epithelial thickness). Using examples from different fish groups (e.g. hagfishes, elasmobranchs and teleosts), the importance of fish skin as a transport epithelium for gases, ions, nitrogenous waste products, and nutrients was reviewed. The role of the skin in larval fish was also examined, with early life stages often utilising the skin as a surrogate gill, prior to the development of a functional branchial epithelium.     

Hyperparasitism of trichodinid ciliates on monogenean gill flukes of two marine fish

Two unusual cases of hyperparasitism of trichodinid ciliates on monogenean gill flukes are described from southern Israel (Red Sea). The first case occurred in cultured European sea bass Dicentrarchus labrax infected by Diplectanum aequans, while the second was observed in a feral devil firefish Pterois miles infected by Haliotrema sp. In both cases, the trichodinids heavily co-infested the host fish gills. The flukes were completely coated by the ciliates, which gave them a cobblestone appearance, but no damage to their tegument was apparent. Both cases are most likely a result of accidental hyperparasitism, brought about by perturbed environmental conditions.     

Evoxymetopon moricheni,a new cutlassfish from the northern Red Sea (Teleostei: Trichiuridae)

Evoxymetopon moricheni n. sp. is described from the northern Gulf of Aqaba, Red Sea, on the basis of a single specimen. It is characterised within the genus by a convex upper head profile; the posterior confluence of the frontal ridges slightly elevated at the nape, resulting in the presence of a slight sagittal crest; eye 5.2 times in head length; the lower hind margin of opercle slightly pointed in its lower half; the pelvic fins reduced to a scale-like structure with two soft rays on the posterior process of the basipterygium, its origin below the eighth dorsal-fin element; the first anal spine single, oval, scale like, and originating a short distance behind the vent (opposite the 34th dorsal-fin soft ray), anterior anal-fin soft rays minute and embedded, barely penetrating the skin and not visible externally, while the posterior fin supporting the rays enlarged and visible externally; approximately 17 anal-fin rays externally visible; approximately 83 dorsal-fin elements, with first spine not elongate, shorter than second; 12 pectoral-fin rays (ii + 6 + iv), with shorter anterior (fourth ray shortest) and longer posterior rays extending above the lateral line; 10 upper and 13 lower gill rakers of the first gill arch; head and body silver white, with a blackish margin anteriorly on snout and head, continuing along the anterior half of the dorsal-fin base. The new finding represents a new record of the genus from the Red Sea.     

Increasing southern invasion enhances congruence between endemic and exotic Mediterranean fish fauna

Species movements in relation with global warming may increase the spatial overlap between exotic and endemic species, which is a critical issue for the conservation of biodiversity. The Mediterranean Sea, which is a receptacle for exotic species while being a hotspot for endemism, provides exceptional material for a case study. The aim of our study was to quantify (i) the increasing invasion from southern fish exotic species (Red Sea and Atlantic Ocean) that the Mediterranean biota is experiencing and (ii) the spatial overlap between exotic and endemic Mediterranean fish fauna following the northward movement of exotic species within the Mediterranean Sea in the context of global warming. The historical invasion dynamic of exotic fish species and the sea surface temperature series were reconstructed from 1810 to 2006 in order to estimate the correlation between invasion rate and climate. The geographical distributions of exotic and endemic fish richness before and after the period of global warming were used to assess the dynamic of spatial congruence. The results revealed (i) an acceleration of successful introductions from the Red Sea and (ii) the introduction of Atlantic species from lower latitudes in correlation with the increasing temperature of the Mediterranean Sea. We also showed an increasing overlap between the spatial distributions of endemic and exotic species richness. Taken together, our results suggest that endemic fish species are facing a growing number of exotic species because the Mediterranean Sea is acting as a catchment basin for southern species.     

Characterization of ichthyocidal activity of Pfiesteria piscicida: dependence on the dinospore cell density

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.     

The Japanese threadfin bream Nemipterus japonicus, a new Indo‐Pacific fish in the Mediterranean Sea

A specimen of the Japanese threadfin bream Nemipterus japonicus was recorded for the first time from the Mediterranean Sea. The presence of this Indo‐Pacific fish in the Mediterranean is evidently due to migration from the Red Sea via the Suez Canal.     

A new parasitic copepod (Cyclopoida: Bomolochidae) from a ponyfish (Leiognathidae) caught in Egyptian Mediterranean waters,with a review of hosts and key to species of Nothobomolochus

A new bomolochid copepod belonging to the genus Nothobomolochus Vervoort, 1962 is described from a Red Sea fish species, a ponyfish of the family Leiognathidae that has become established in the Eastern Mediterranean. The new species, N. leiognathicola n. sp., is based on material obtained from the gill chamber of the Red Sea immigrant ponyfish Leiognathus klunzingeri (Steindachner), caught in Egyptian waters off the Alexandria coast at Abuqir. A second new species, N. monodi n. sp., is established to accommodate some material previously described as N. denticulatus (Bassett-Smith, 1898), from the host Hemiramphus far Forsskål. A review of host records reveals that Nothobomolochus species utilise hosts representing five different orders, but are most commonly found on beloniform, clupeiform and perciform fishes. A newly constructed key to the 37 valid species of Nothobomolochus is presented.     

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI-sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation-a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.     

Free radical production by the red tide alga, Chattonella antiqua.

Summary The red tide alga,Chattonella antiqua, was found to show a strong chemiluminescence, using luminol as the reagent, when exposed to ultraviolet irradiation. This luminescence was completely inhibited by ascorbate or catalase, suggesting that hydrogen peroxide was generated by the plankton. Red tide cells exposed to fish gill mucus from young yellowtail resulted in the release of a large number of mucocysts and a weak luminosity, and showed a strong reduction of cytochromec in the medium. Therefore, the discharge of mucocysts from the red tide, induced by the presence of gill mucus, may be accompanied by the release of active oxygen species. The active oxygen may be involved in depolymerization of mucus glycoproteins from the gill lamellae.     

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.     

No reduction in neutral variability of mitochondrial and nuclear genes for a Lessepsian migrant, Upeneus moluccensis

Using nuclear (introns) and mitochondrial markers, two populations of the Lessepsian fish Upeneus moluccensis sampled from its original (Red Sea) and new habitat (Mediterranean Sea) were compared. No significant genetic difference was observed hence the passage through the Suez Canal does not seem to have affected the genetic diversity at the presumably neutral loci studied.     

Enhanced Aeromonas salmonicida bacterin uptake and side effects caused by low frequency sonophoresis in rainbow trout (Oncorhynchus mykiss)

Low frequency sonophoresis (LFS) has been recognized as one of the most advanced technologies in transdermal delivery of substances, due to the modification of the stratum corneum lipid bilayer, in focal skin applications in mammals. Based on these findings, LFS has been suggested as a potential technology to be used for enhancement in immersion fish vaccination. In contrast to mammals where LFS is applied to discrete regions of the skin, in fish the whole individual needs to be exposed for practical purposes. The current study evaluated the impact of LFS at 37 kHz on the uptake of an Aeromonas salmonicida bacterin and side effects of the treatment in rainbow trout. Quantitative real time PCR (qPCR) and immunohistochemistry were used to examine the bacterin uptake into skin and gill tissue. Side effects were assessed by behavioural examination, histology and blood serum analysis. The sonication intensity of 171 mW/cm² was enough for increasing skin permeability, but caused heavy erratic swimming and gill haemorrhages. Sonication intensities as low as 105 mW/cm² did not modify skin permeability and enhanced the bacterin uptake into the gill tissue by factor 15 compared to conventional immersion. Following sonication, the gill permeability for the bacterin decreased after 20 min and 120 min by factor 3 and 2, respectively. However, during sonication, erratic swimming of the fish raised some concerns. Further reduction of the sonication intensity to 57 mW/cm² did not induce erratic swimming, and the bacterin uptake into the gill tissue was still increased by factor 3. In addition, a decreasing albumin–globulin ratio in the serum of the rainbow trout within 40 min revealed that LFS leads to an inflammatory response. Consequently, based on both increased bacterin uptake and the inflammatory response, low intensity LFS has the potential to enhance vaccine immunity without significant side effects.     

The Mediterranean Sea as a ‘cul‐de‐sac’ for endemic fishes facing climate change

The Mediterranean Sea is a hotspot of biodiversity, and climate warming is expected to have a significant influence on its endemic fish species. However, no previous studies have predicted whether fish species will experience geographic range extensions or contractions as a consequence of warming. Here, we projected the potential future climatic niches of 75 Mediterranean Sea endemic fish species based on a global warming scenario implemented with the Mediterranean model OPAMED8 and a multimodel inference, which included uncertainty. By 2070–2099, the average surface temperature of the Mediterranean Sea was projected to warm by 3.1 °C. Projections for 2041–2060 are that 25 species would qualify for the International Union for the Conservation of Nature and Natural Resources (IUCN) Red List, and six species would become extinct. By 2070–2099, 45 species were expected to qualify for the IUCN Red List whereas 14 were expected to become extinct. By the middle of the 21st century, the coldest areas of the Mediterranean Sea (Adriatic Sea and Gulf of Lion) would act as a refuge for cold‐water species, but by the end of the century, those areas were projected to become a ‘cul‐de‐sac’ that would drive those species towards extinction. In addition, the range size of endemic species was projected to undergo extensive fragmentation, which is a potentially aggravating factor. Since a majority of endemic fishes are specialists, regarding substratum and diet, we may expect a reduced ability to track projected climatic niches. As a whole, 25% of the Mediterranean Sea continental shelf was predicted to experience a total modification of endemic species assemblages by the end of the 21st century. This expected turnover rate could be mitigated by marine protected areas or accelerated by fishing pressure or competition from exotic fishes. It remains a challenge to predict how these assemblage modifications might affect ecosystem function.     

Mast cell responses to Ergasilus (Copepoda), a gill ectoparasite of sea bream

Immunocytochemical, light microscopy and ultrastructural studies were conducted on gill of sea bream, Sparus aurata L., naturally parasitized with the important parasitic copepod Ergasilus sp. to assess pathology and cellular responses. Thirty-seven S. aurata were examined from a fish farm; 26 (70%) were parasitized, with infection intensity ranging from 3 to 55 parasites per fish. Hosts were divided into two groups, lightly infected fish (<15 parasites per fish) and heavily infected fish (>15 parasites per fish). In histological sections, the copepod encircled gill lamellae with its second antennae, compressed the epithelium, provoked hyperplasia and hemorrhage, occluded arteries and often caused lamellar disruption. Fusion of the secondary lamellae due to epithelial hyperplasia was common in all infected fish; heavily infected fish showed more intense branchial inflammation. In both healthy and infected fish, mast cells (MCs) were free within the connective tissue inside and outside the blood vessels of the primary lamellae and made close contact with vascular endothelial cells, mucous cells and rodlet cells (RCs). MCs were irregular in shape with a cytoplasm filled by numerous electron-dense, membrane-bound granules. Immunostaining of primary and secondary gill filaments with an antibody against the antimicrobial peptide (AMP) piscidin 3 (anti-piscidin 3 antibody, anti-HAGR) revealed a subpopulation of MCs that were positive. These MCs were more abundant in gills of heavily infected fish than in either lightly infected or uninfected fish (ANOVA, P<0.05). Our report documents the response of gill to ectoparasite infection and provides further evidence that mast cells and their AMPs may play a role in responding to branchial ectoparasite infections.