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The reported pathogenicity of the monogenean Gyrodactylus salaris Malmberg, 1957 on Atlantic Salmo salar in Norway has necessitated a national survey of salmonid sites throughout the British Isles to determine which species of the genus Gyrodactylus are resident on these hosts in British waters. Eight morphotypes were identified following examination of the opisthaptoral sclerites at both the light and scanning electron microscope level. Gyrodactylus truttae Glaser, 1974 was found on wild brown trout Salmo trutta in Scotland, England and Wales with an uncharacteristic morphotype, having long, thin marginal hooks. Two morphotypes of Gyrodactylus derjavini Mikailov, 1975 were recorded for the first time from Britain. G. caledoniensis n. sp. from Scottish S. salar is erected, based on differences in the shape and size of the marginal hooks. Two possible new species from English Salvelinus alpinus and a possible new species from Welsh S. salar and Scottish O. mykiss are described.

The elemental compositions of the hamuli, marginal hooks and ventral bars were examined following their release from the opisthaptor of the monogenean parasites Gyrodactylus salaris, G. caledoniensis and G. colemanensis. Their compositions were determined using X-ray elemental analysis on an analytical scanning electron microscope, and the variation between each species investigated. Hamuli and marginal hooks exhibited a major peak for sulphur, whilst the ventral bar had high peaks of both sulphur and calcium. The high sulphur content and structure of the hamuli suggest they are composed of a keratin-like protein, while the nature of the ventral bar is less clear. The hamuli of G. colemanensis were differentiated from those of G. caledoniensis by virtue of their calcium fraction. Low levels of several other elements, including vanadium, were also detected in some sclerites. The composition of the hooks of Gyrodactylus and their relationship to the sclerites of monopisthocotyleans and polyopisthocotyleans is commented upon.

Histopathological and ultrastructural investigations were conducted on 36 tench, Tinca tinca (L.), from Lake Trasimeno (Italy). The gills, intestine, liver, spleen, kidney and heart of 21 individuals were found to harbour an extensive infection of larvae of an unidentified digenean trematode. The eyes, gonads, swim bladder and muscles were uninfected. The parasites in each tissue type were embedded in a granulomatous proliferation of tissue, forming a reactive fibroconnective capsule around each larva. Most of the encysted larvae were metacercariae, in a degenerative state, but on occasion some cercariae were found. Many of the granulomas were either necrotic or had a calcified core. Within the granuloma of each, the occurrence of granulocytes, macrophages, rodlet cells and pigment-bearing macrophage aggregates was observed. Hearts bore the highest parasitic infection. Whilst the presence of metacercariae within the intestine was found positioned between the submucosa and muscle layers, metacercariae in the liver were commonly found encysted on its surface where the hepatocytes in close contact with the granuloma were observed to have electron-lucent vesicles within their cytoplasm. Metacercariae encysting adjacent to the cartilaginous rods of gill filaments were seen to elicit a proliferation of the cartilage from the perichondrium. Rodlet cells, neutrophils and mast cells were frequently observed in close proximity to, and within, infected gill capillaries. Given the degenerated state of most granulomas, a morphology-based identification of the enclosed digeneans was not possible.

Marine cultured meagre, Argyrosomus regius Asso, in central and western Greece were affected by an outbreak of systemic granulomatous disease subsequently demonstrated to be nocardiosis. The fish were originally imported as juveniles from hatcheries in France and Italy and on-grown in Greece, the latter also providing broodstock for a small number of local Greek hatcheries for the production of second-generation juveniles. The disease in cage reared fish had been present throughout the year, particularly in the 1+ and 2+ year old fish with a low to variable morbidity and 1-4% total mortality. Multiple lesions were visible externally on the skin of affected fish, with severe ulcerations and necrosis. Internally, multifocal yellowish-white nodules, 0.1-0.5 cm in diameter, were visible on the surface of several internal organs. Histopathology revealed systemic granulomatous inflammation. Fite-Faraco staining clearly demonstrated the presence of Nocardia-like organisms which were Gram-positive, long, rod to beaded filamentous bacteria. Nocardia genus-specific 16s RNA primers NG1 and NG2 were used to generate a 600 bp fragment recovered from affected tissue, confirming the diagnosis of Nocardia spp. To our knowledge, this is the first report of nocardiosis in meagre.

The management and control of parasitic infections in aquaculture are a constant challenge, highly complicated by the current limited availability of efficacious licensed products; a situation exacerbated by the development of resistance to anti-parasitic drugs in parasite populations. In addition, parasite control in aquaculture requires a keen awareness of environmental, water quality and host parameters, and is subject to the constraints of economics and the requirement for aquaculture sustainability and environmental protection. Here, we look at some of the factors underlying general decision-making as it applies to drug treatment, including parasite identification, choice of chemical, strategy and dosage. Trigger-points, and the rationale underpinning these, are also considered.

A total of 37 European eels, Anguilla anguilla, collected from Lake Piediluco, Central Italy, and measuring 35 to 75.5 cm in total length (mean±1 SD, 56.41 ± 10.89 cm) were examined, and their acanthocephalan infections assessed. Thirty-two (86.49%) eels were infected with Acanthocephalus rhinensis (mean±1 SD, 67.38 ± 65.16; range, 1–350), a species that, purportedly, can be discriminated on the basis of a characteristic band of orange–brown pigmentation encircling the anterior end of the trunk. This feature, however, was not seen on any of the A. rhinensis specimens that were removed, either attached to the gut wall or free within the gut lumen, from infected eels. Approximately 40% of the eels were coinfected with the dracunculid swimbladder nematode Anguillicoloides crassus, while a single eel was also coinfected with eight specimens of a second acanthocephalan, Dentitruncus truttae. From the stomachs of two eels, 109 intact and partially digested specimens of amphipod Echinogammarus tibaldii (Pinkster & Stock 1970) were recovered, 16 (14.6%) of these were infected with one to two cystacanths of A. rhinensis per host. From a sample of 850 E. tibaldii taken from the peripheral lakeside vegetation, 102 (12%; sex ratio, 1:1) gammarids were infected with one to two A. rhinensis cystacanths. Unparasitised ovigerous female E. tibaldii specimens had significantly higher numbers of eggs in their brood pouches compared with their infected counterparts (t-test, P

Background Myxosporeans are known from aquatic annelids but parasitism of platyhelminths by myxosporeans has not been widely reported. Hyperparasitism of gill monogeneans by Myxidium giardi has been reported from the European eel and Myxidium-like hyperparasites have also been observed during studies of gill monogeneans from Malaysia and Japan. The present study aimed to collect new hyperparasite material from Malaysia for morphological and molecular descriptions. In addition, PCR screening of host fish was undertaken to determine whether they are also hosts for the myxosporean. Results Heavy myxosporean infections were observed in monogeneans from two out of 14 fish and were detected from a further five fish using specific PCRs and pooled monogenean DNA. Positive DNA isolates were sequenced and were from a single species of myxosporean. Myxospore morphology was consistent with Myxidium with histozoic development in the parenchymal tissues of the monogenean. Simultaneous infections in the fish could not be confirmed microscopically; however, identical myxosporean DNA could be amplified from kidney, spleen and intestinal tract tissues using the specific PCR. Small subunit (SSU) rDNA for the myxosporean was amplified and was found to be most similar (92%) to that of another hyperparasitic myxosporean from a gill monogenean from Japan and to numerous multivalvulidan myxosporeans from the genus Kudoa (89-91%). Phylogenetic analyses placed the hyperparasite sequence basally to clades containing Kudoa, Unicapsula and Sphaerospora. Conclusions The myxosporean infecting the gill monogenean, Diplectanocotyla gracilis, from the Indo-Pacific tarpon, Megalops cyprinoides, is described as a new species, Myxidium incomptavermi, based on a histozoic development in the monogenean host and its phylogenetic placement. We have demonstrated for the first time that a myxosporean hyperparasite of gill monogeneans is detectable in the fish host. However, myxospores could not be isolated from the fish and confirmation was by PCR alone. The relationship between the myxosporean infection in gill monogeneans and the presence of parasitic DNA in fish is not yet fully understood. Nonetheless, myxospores with a Myxidium-like morphology, two of which we have shown to be phylogenetically related, have now been reported to develop in three different gill monogeneans, indicating that myxosporeans are true parasites of monogeneans.

SUMMARYInfection by the ciliate protozoanIchthyophthirius multifiliisFouquet, 1876 causes significant economic losses in freshwater aquaculture worldwide. Following the ban on the use of malachite green for treating food fish, there has been extensive research aimed at identifying suitable replacements. In this paper we critically assess drug and non-drug interventions, which have been tested for use or have been employed against this parasite and evaluate possibilities for their application in farm systems. Current treatments include the administration of formaldehyde, sodium chloride (salt), copper sulphate and potassium permanganate. However, purportedly more environmentally friendly drugs such as humic acid, potassium ferrate (VI), bronopol and the peracetic acid-based products have recently been tested and represent promising alternatives. Further investigation, is required to optimize the treatments and to establish precise protocols in order to minimize the quantity of drug employed whilst ensuring the most efficacious performance. At the same time, there needs to be a greater emphasis placed on the non-drug aspects of management strategies, including the use of non-chemical interventions focusing on the removal of free-swimming stages and tomocysts ofI. multifiliisfrom farm culture systems. Use of such strategies provides the hope of more environmentally friendly alternatives for the control ofI. multifiliisinfections.

Background The high transmission potential of species belonging to the monogenean parasite genus Gyrodactylus, coupled with their high fecundity, allows them to rapidly colonise new hosts and to increase in number. One gyrodactylid, Gyrodactylus salaris Malmberg, 1957, has been responsible for devastation of Altantic salmon (Salmo salar L.) populations in a number of Norwegian rivers. Current methods of eradicating G. salaris from river systems centre around the use of non-specific biocides, such as rotenone and aluminium sulphate. Although transmission routes in gyrodactylids have been studied extensively, the behaviour of individual parasites has received little attention. Specimens of Gyrodactylus gasterostei Gläser, 1974 and G. arcuatus Bychowsky, 1933, were collected from the skin of their host, the three-spined stickleback (Gasterosteus aculeatus L.), and permitted to attach to the substrate. The movements of individual parasites were recorded and analysed. Results The behaviour patterns of the two species were similar and parasites were more active in red light and darkness than in white light. Four octopaminergic compounds were tested and all four inhibited the movements of parasites. Treatment ultimately led to death at low concentrations (0.2 μM), although prolonged exposure was necessary in some instances. Conclusions Octopaminergic compounds may affect the parasite's ability to locate and remain on its host and these or related compounds might provide alternative or supplementary treatments for the control of G. salaris infections. With more research there is potential for use of octopaminergic compounds, which have minimal effects on the host or its environment, as parasite-specific treatments against G. salaris infections.

Gyrodactylus salmonis (Yin et Sproston, 1948) isolates collected from feral rainbow trout, Oncorhynchus mykiss (Walbaum) in Veracruz, southeastern Mexico are described. Morphological and molecular variation of these isolates to G. salmonis collected in Canada and the U.S.A. is characterised. Morphologically, the marginal hook sickles of Mexican isolates of G. salmonis closely resemble those of Canadian specimens - their shaft and hook regions align closely with one another; only features of the sickle base and a prominent bridge to the toe permit their separation. The 18S sequence determined from the Mexican specimens was identical to two variable regions of SSU rDNA obtained from a Canadian population of G. salmonis. Internal transcribed spacer (ITS) regions (spanning ITS1, 5.8S and ITS2) of Mexican isolates of G. salmonis are identical to ITS sequences of an American population of G. salmonis and to Gyrodactylus salvelini Kuusela, Zietara et Lumme, 2008 from Finland. Analyses of the ribosomal RNA gene of Mexican isolates of G. salmonis show 98-99% similarity to those of Gyrodactylus gobiensis Glaser, 1974, Gyrodactylus salaris Malmberg, 1957, and Gyrodactylus rutilensis Glaser, 1974. Mexican and American isolates of G. salmonis are 98% identical, as assessed by sequencing the mitochondrial cox1 gene. Oncorhynchus mykiss is one of the most widely-dispersed fish species in the world and has been shown to be an important vector for parasite/disease transmission. Considering that Mexican isolates of G. salmonis were collected well outside the native distribution range of all salmonid fish, we discuss the possibility that the parasites were translocated with their host through the aquacultural trade. In addition, this study includes a morphological review of Gyrodactylus species collected from rainbow trout and from other salmonid fish of the genus Oncorhynchus which occur throughout North America.

BACKGROUND: Infections of the ciliate protozoan Ichthyophthirius multifiliis Fouquet, 1876, can cause heavy mortalities in freshwater aquaculture systems. Following the ban of malachite green on fish for human consumption, currently available chemical therapies are only partially efficacious even when repeatedly applied. There is, therefore, an urgent necessity to identify more efficient and environmentally friendly control strategies. In the present study, the use of a substrate algae feeder leopard pleco, Glyptoperichthys gibbiceps, as a biological agent to control I. multifiliis infections on blue tilapia, Oreochromis aureus, reared in a warm-water, experimental tank-based system is explored. RESULTS: The free-swimming protomont stage of I. multifiliis demonstrated selective settlement behaviour towards biofilm-covered substrates. Following a controlled exposure, the number of I. multifiliis trophonts establishing on naive blue tilapia, O. aureus, was 4.9 ± 0.4 when reared with G. gibbiceps, significantly lower than when reared in isolation (13.3 ± 0.9; ANOVA, P < 0.001). CONCLUSION: This study demonstrates for the first time the potential of G. gibbiceps as a biological control to reduce I. multifiliis on commercially valuable fish stocks in warm-water systems. This likely originated from the grazing activity of G. gibbiceps on the biofilm layer upon which the multiplicative stage of I. multifiliis was shown preferentially to settle. Therapeutic strategies against pathogenic protozoan species like I. multifiliis could greatly benefit from incorporating, where appropriate, a biological control targeting the external multiplicative life stages of the parasite, potentially reducing the current dependence on chemical interventions. Copyright © 2012 Society of Chemical Industry

Background Historically, non-native species of Gambusia (Poeciliidae) have been used to control larval stages of the Asian tiger mosquito, Stegomyia albopicta Reinert, Harbach et Kitching, 2004 throughout Italy. The potential utility of indigenous populations of Aphanius fasciatus (Valenciennes) (Teleostei: Cyprinodontidae) as an appropriate alternative biological control is currently being explored. A sub-sample of ten fish collected from Cervia Saline, Italy (salinity 65 ppt; 30°C) to assess their reproductive capability in captivity, harboured a moderate infection of Gyrodactylus von Nordmann, 1832 (Platyhelminthes, Monogenea). A subsequent morphological and molecular study identified this as being a new species. Results Gyrodactylus salinae n. sp. is described from the skin, fins and gills of A. fasciatus. Light and scanning electron microscopical (SEM) examination of the opisthaptoral armature and their comparison with all other recorded species suggested morphological similarities to Gyrodactylus rugiensoides Huyse et Volckaert, 2002 from Pomatoschistus minutus (Pallas). Features of the ventral bar, however, permit its discrimination from G. rugiensoides. Sequencing of the nuclear ribosomal DNA internal transcribed spacers 1 and 2 and the 5.8S rRNA gene and a comparison with all species listed in GenBank confirmed they are unique and represent a new species (most similar to Gyrodactylus anguillae Ergens, 1960, 8.3% pair-wise distance based on 5.8S+ITS2). This represents the first species of Gyrodactylus to be described from Aphanius and, to date, has the longest ITS1 (774 bp) sequenced from any Gyrodactylus. Additional sampling of Cervia Saline throughout the year, found G. salinae n. sp. to persist in conditions ranging from 35 ppt and 5°C in December to 65 ppt and 30°C in July, while in captivity a low level of infection was present, even in freshwater conditions (0 ppt). Conclusions The ability of G. salinae n. sp. to tolerate a wide range of salinities and temperatures shows its potential to readily adapt to several environmental conditions. These findings, together with the fact that A. fasciatus is a protected species and is considered as a biological control organism, necessitate further studies on the ecology and virulence of G. salinae n. sp.

Larvae of the cyclophyllidean tapewormsParadilepis scolecina(Rudolphi, 1819),Neogryporhynchus cheilancristrotus(Wedl, 1855) andValipora campylancristrota(Wedl, 1855), are described from British freshwater fish. The morphometrics of the rostellar hooks, infection characteristics and host ranges of these parasites from fisheries in England and Wales are presented. Difficulties in the detection, handling and identification of these tapeworms are highlighted, and may in part explain the paucity of records from Britain. Tissue digestion was shown to be a useful technique for the examination of these parasites, providing clear and consistent preparations of the rostellar hooks for measurement. The pathological changes caused byP. scolecinato the liver of wild tench,Tinca tinca, are detailed for the first time. Tapeworms located in the hepatic parenchyma and pancreatic tissues caused little pathological damage and invoked only mild inflammatory responses. The small size of these tapeworms and their encapsulation within host tissues appear to limit the severity of pathology, compared with parasites that insert their rostellum during attachment.

Gyrodactylus longipes n. sp. (Monogenea, Gyrodactylidae) is described from the gills of farmed juvenile gilthead seabream ( Sparus aurata L.) from two sites located in Italy and Bosnia–Herzegovina and represents the second species of Gyrodactylus to be described from S. aurata. Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 was the first gyrodactylid to be described from S. aurata , from populations cultured in Albania and Croatia. In the current study, G. longipes was found in a mixed infection with G. orecchiae on fish maintained in Latina Province, Italy, thus extending the reported distribution of the latter throughout the Mediterranean. The morphology of the opisthaptoral hard parts of G. longipes is compared to those of G . orecchiae , using light and scanning electron microscopy. Gyrodactylus longipes is characterised by having larger, elongated ventral bar processes and long, triangular-shaped toe region to their marginal hook sickles which, by comparison, are rhomboid in G. orecchiae . The marginal hook sickles of G. longipes are almost double the size of G. orecchiae which allows for their rapid discrimination from each other in mixed infections. A comparison of the DNA sequence of the ribosomal internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) of G. longipes with the corresponding sequence from G. orecchiae and with those available in GenBank, supports the separate species status of G. longipes . Part of this study necessitated an overview of the existing Gyrodactylus fauna from Italy and Bosnia–Herzegovina; a summary from each country is provided here to assist future investigations.

Three new species of Gyrodactylus are described from two species of Oreochromis (Cichlidae): Gyrodactylus hildae sp. nov. from the Nile tilapia, Oreochromis niloticus niloticus, and from an unconfirmed cichlid in Ethiopia; Gyrodactylus ulinganisus sp. nov. from a South African population of Mozambique tilapia, Oreochromis mossambicus; and, Gyrodactylus yacatli sp. nov. from O. n. niloticus reared in Mexico. The hamuli and marginal hooks of G. hildae sp. nov. and G. yacatli sp. nov. differ notably from G. cichlidarum, a species commonly found on O. n. niloticus. The hooks of G. ulinganisus sp. nov., however, are morphologically similar to those of G. cichlidarum, but the two species were found to differ by 42 nucleotide substitutions (24 within the 342 bp long ITS1; 18 within the 303 bp long ITS2) and by 1 insertion/deletion. This study confirms that Nile and Mozambique tilapia harbour a number of different species of Gyrodactylus, with G. cichlidarum being the most frequently encountered and being associated with mortalities of juvenile O. n. niloticus. This study discusses the host specificity of gyrodactylids on commercial cichlid species and the potential repercussions of their movement on stocks of fish into new environments where cichlids are already present.

A new genus and species of Gyrodactylidae, Ieredactylus rivuli gen. et sp. nov. (Platyhelminthes, Monogenea), is described from the skin of Hart’s Rivulus (Rivulus hartii Boulenger), a cyprinodontiform fish collected from streams of the Caroni and Oropouche drainages and the Pitch Lake in Trinidad (prevalence all localities: 16.7–94.6%; mean parasite intensity 1–9 parasites/fish; range 1–34) with the holotype originating from a tributary of the Aripo River. This viviparous monogenean is distinctive from other genera of Gyrodactylidae by its split ventral bar membrane, the shape of its male copulatory organ, the presence of two conical accessory pieces associated with the hamulus root and two differently shaped marginal hook sickles. Its unique rDNA sequence shows the closest ITS2 similarity (70%) to Gyrodactyloides andriaschewii Bychowsky et Poljansky, 1953. The presence of I. rivuli gen. et sp. nov. in the Pitch Lake indicates an adaptation to extreme environmental conditions such as high temperatures and hydrocarbons and adverse pH. Guppies may potentially serve as temporary hosts. The parasite displays distinct behaviours, including a characteristic ‘swimming-like’ movement. The ecology and phylogeny of I. rivuli gen. et sp. nov. is discussed in relation to the diversity of other gyrodactylids in Trinidad.

Techniques for the preparation of haptoral sclerites of Gyrodactylus species for scanning electron microscopy are described. Enzymatic digestion using pepsin allows fresh, frozen and alcohol-fixed material to be examined, but some sclerites, such as the dorsal and ventral bars, are lost. Sonication of fresh and frozen material retained these accessory bars, including the ventral bar membrane, the guard or sickle filament loop on the marginal hooks and even on some occasions ligaments associated with the sclerites. The sclerites of Gyrodactylus spp. from Gasterosteus aculeatus (L.) (three-spined stickleback), Oncorhynchus mykiss (Walbaum) (rainbow trout) and Salvelinus alpinus (L.) (charr) are described from SEM observations following the use of these preparation techniques, and the articulation between the hamuli and bars is commented upon.

Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.

Sea lice are copepod ectoparasites that constitute a major barrier to the sustainability and economic viability of marine finfish aquaculture operations worldwide. In particular, the salmon louse, Lepeophtheirus salmonis , poses a considerable problem for salmoniculture in the northern hemisphere. The free-swimming nauplii and infective copepodids of L. salmonis are lecithotrophic, subsisting principally on maternally-derived lipid reserves. However, the lipids and fatty acids of sea lice have been sparsely studied and therefore the present project aimed to investigate the lipid and fatty acid composition of sea lice of the genus Lepeophtheirus obtained from a variety of fish hosts. Total lipid was extracted from eggs and adult female L. salmonis obtained from both wild and farmed Atlantic salmon ( Salmo salar L . ) sampled at two time points, in the mid 1990s and in 2009. In addition, L. salmonis from wild sea trout ( Salmo trutta L . ) and L. hippoglossi from wild Atlantic halibut ( Hippoglossus hippoglossus L . ) were sampled and analyzed. The lipids of both females and egg strings of Lepeophtheirus were characterized by triacylglycerol (TAG) as the major neutral (storage) lipid with phosphatidylcholine and phosphatidylethanolamine as the major polar (membrane) lipids. The major fatty acids were 22:6 n −3 (DHA), 18:1 n −9 and 16:0, with lesser amounts of 20:5 n −3, 22:5 n −3 and 18:0. L. salmonis sourced from farmed salmon was characterized by higher levels of 18:2 n −6 and 18:3 n −3 than lice from wild salmon. Egg strings had higher levels of TAG and lower DHA compared to females, whereas L. hippoglossi had lower levels of TAG and higher DHA than L. salmonis . The results demonstrate that the fatty acid compositions of lice obtained from wild and farmed salmon differ and that changes to the lipid and fatty acid composition of feeds for farmed salmon influence the louse compositions.

Gyrodactylus infections in intensively-reared populations of Nile tilapia, Oreochromis niloticus niloticus, have been associated world-wide with high mortalities of juvenile fish. In this study, 26 populations of Gyrodactylus parasitising either O. n. niloticus or Mozambique tilapia, Oreochromis mossambicus, were sampled from fourteen countries and compared with type material of Gyrodactylus cichlidarum Paperna, 1968, Gyrodactylus niloticus (syn. of G. cichlidarum) and Gyrodactylus shariffi Cone, Arthur et Bondad-Reantaso, 1995. Representative specimens from each population were bisected, each half being used for morphological and molecular analyses. Principal component analyses (PCA) identified five distinct clusters: (1) a cluster representing G. cichlidarum collected from O. n. niloticus from 13 countries; (2) the G. shariffi paratype; (3) three specimens with pronounced ventral bar processes collected from two populations of Mexican O. n. niloticus (Gyrodactylus sp. 1); (4) four specimens collected from an Ethiopian population nominally identified as O. n. niloticus (Gyrodactylus sp. 2); (5) nine gyrodactylids from South African O. mossambicus (Gyrodactylus sp. 3). Molecular analyses comparing the sequence of the ribosomal transcribed spacer regions (ITS 1 and 2) and the 5.8S gene from the non-hook bearing half of worms representative for each population and for each cluster of parasites, confirmed the presence of G. cichlidarum in most samples analysed. Molecular data also confirmed that the DNA sequence of Gyrodactylus sp. 2 and Gyrodactylus sp. 3 (the morphologically-cryptic group of South African specimens from O. mossambicus) differed from that of G. cichlidarum and therefore represent new species; no sequences were obtained from Gyrodactylus sp. 1. The current study demonstrates that G. cichlidarum is the dominant species infecting O. n. niloticus, being found in 13 of the 15 countries sampled.

The histopathology, ultrastructure, and immunohistochemistry of the alimentary canal of flounder Platichthys flesus (L.), naturally infected with the nematode Anisakis simplex s.l. (Rudolphi 1809) from the River Forth (Scotland), were investigated and described. Eight of the 16 flounders were infected with A. simplex s.l. larvae (L3); parasites were encapsulated by serosa on the external surface of the host's digestive tract (intensity of infection 1-8 parasites per host), although nematode larvae were found encysted under the peritoneal visceral serosa of the host spleen and liver and, occasionally, in the liver parenchyma (intensity of infection 3-10 parasites per host). In all sites, different structural elements were recognized within the capsule surrounding larvae. Among the epithelial cells of the intestine of 5 flounders with larvae encysted on external surface of the gut, the presence of several rodlet cells (RCs) was observed. Furthermore, often the occurrence of macrophage aggregates (MAs) was noticed in infected liver and spleen, mainly around the parasite larvae. Eight neuropeptide antisera were tested with immunohistochemistry methods on gut sections of 4 P. flesus infected with extraintestinal nematodes. Sections from the gut of 5 uninfected flounder were used for comparative purposes. In the tunica mucosa of parasitized P. flesus, several endocrine epithelial cells were immunoreactive to anti-CCK-39 (cholecystokinin 39) and -NPY (neuropeptide Y) sera; furthermore, in the myenteric plexus, a high number of neurons were immunoreactive to antibombesin, -galanin, and several to -NPY and -PHI (peptide histidine isoleucine) sera.

This review concerns the parasitic marine copepod Lernaeocera branchialis (L., 1767) and provides an overview of current knowledge concerning its biology and host-parasite interactions. The large size and distinctive appearance of the metamorphosed adult female stage, coupled with the wide exploitation and commercial importance of its final gadoid hosts, means that this species has long been recognised in the scientific literature. The fact that the Atlantic cod, Gadus morhua L., is one of its key host species, and has itself had a major impact on the social and economic development of many countries bordering the North Atlantic for more than 10 centuries is also a factor in its widespread recognition. L. branchialis is recognised as a pathogen that could have major effects on the aquaculture industry and with gadoid (especially cod) farming expanding in several North Atlantic countries, there is considerable potential for this parasite to become a serious problem for commercial mariculture. The main subject areas covered are the parasite's taxonomy; the life history of the parasite including its life cycle, reproduction and host associations; parasite physiology; parasite seasonality and distribution; and the pathogenic effects of the parasite on its host.

A recent infestation of Gyrodactylus cichlidarum Paperna, 1968 on yolk sac fry of Nile tilapia, Oreochromis niloticus niloticus (L.), in an isolated aquarium system in the U.K. resulted in high mortalities and provided an opportunity to study this species in greater detail. A tentative identification was made using the measurements and drawings of the ventral bar and hamuli provided in the original description; however, details on the morphology of the marginal hooks were lacking. A comparison of the gyrodactylid material collected from O. n. niloticus with the holotype of G. cichlidarum, the only known available specimen, from Mango tilapia, Sarotherodon galilaeus galilaeus (L.), confirmed its identity. Proteolytic digestion and image analysis of the opisthaptoral hard parts were used to obtain tissue-free, accurate measurements as part of a complete revised description of G. cichlidarumn. Further, a comparison of G. cichlidarum from both hosts with the holotype and several paratypes of Gyrodactylus niloticus Cone, Arthur et Bondad-Reantaso, 1995 cited as parasitizing captive stocks of Nile tilapia in the Philippines revealed the two species to be synonymous. An 803 bp fragment of the ribosomal internal transcribed spacers 1 and 2 and the 5.8S was obtained and is provided with the revised description. This is the first DNA sequence from a Gyrodactylus species originating from the African continent. The sequence is very divergent from other species in the genus and only the 5.8S sequence places it unambiguously in the genus Gyrodactylus. In addition to G. cichlidarum, two specimens of another morphological similar species of Gyrodactylus were also found on the UK held stock of O. n. niloticus. These latter specimens, Gyrodacrylus sp., differed from G. cichlidarum in having a longer hamulus point with a smaller hamulus aperture and possessing marginal hook sickles that had a shorter shaft with a longer point giving the sickles a more rounded, closed appearance.

The mortality of juvenile European sea bass, Dicentrarchus labrax (L.), in the spring of the last 5 years in the northern coast of the Adriatic Sea has been attributed to heavy infections of the gill monogenean Diplectanum aequans (Wagener 1857) Diesing 1858. The histopathological examination of 38 sets of gills from hosts measuring 16.46 +/- 0.26 cm in total length (mean+/-S.E.) and weighing 45.98 +/- 2.37 g (mean+/-S.E.) were conducted using light and transmission electron microscopy. Twenty-eight (73.6%) D. labrax specimens were infected (34.61 +/- 4.42, mean intensity+/-S.E.; 5-100, range) with the majority of D. aequans attaching to the median and apical portions of the primary gill filaments. The sites of attachment were marked by the common presence of haemorrhages and a white mucoid exudate. In histological sections, the opisthaptors of the parasites were observed to penetrate deeply, lying in close proximity to the basal membrane of primary lamella where they induced a hyperplastic response. Disruption and fusion of the secondary lamellae were common in all infected specimens with several individuals also exhibiting a marked erosion and inflammation of the epithelium of the primary and secondary lamellae. In infected fish, cellular changes in the epithelium underlying the bodies of worms were noted typified by an elevation in the number of mucous and rodlet cells and a reduction in the number of chloride cells.

The spatial distribution and histopathological changes induced by metacercariae of the digenean trematode Diplostomum phoxini (Faust, 1918) in the brains of European minnows Phoxinus phoxinus (L.) from the River Endrick, Scotland, were studied by light and electron microscopy. Post-mortem examination of a sample of 34 minnows revealed that 50% (n = 17) of the population was infected with 13.7 +/- 2.6 (mean +/- SE; range 1 to 38) metacercariae per infected host. Serial histological sections of the infected minnow brains revealed that the metacercariae were unevenly distributed throughout the brain, with aggregations occurring in the cerebellum, the medulla oblongata and the optic lobes. In fish with highest intensities of infection, over 40% of the cerebellar area and about 30% of the medulla oblongata area were occupied by larvae. Metacercariae disrupt the integrity of brain tissue, with individuals being found in small pockets surrounded by cellular debris. Metacercariae were rarely encountered on the surface of the brain. Electron microscopic examination of infection sites revealed that the granular layer surrounding metacercariae was necrotic, exhibited nuclear degradation and was marked by vacuolation of the cytoplasm. Rodlet cells, the only inflammatory cell types recorded in this study, were found only in parasitized brains and in close proximity to the teguments of metacercariae. It is hypothesised that secretions released from the teguments of metacercariae are a counter response to protect the metacercariae from the fish brain's cellular defence mechanisms.

This review concerns the parasitic marine copepod Lernaeocera branchialis (L., 1767) and provides an overview of current knowledge concerning its biology and host-parasite interactions. The large size and distinctive appearance of the metamorphosed adult female stage, coupled with the wide exploitation and commercial importance of its final gadoid hosts, means that this species has long been recognised in the scientific literature. The fact that the Atlantic cod, Gadus morhua L., is one of its key host species, and has itself had a major impact on the social and economic development of many countries bordering the North Atlantic for more than 10 centuries is also a factor in its widespread recognition. L. branchialis is recognised as a pathogen that could have major effects on the aquaculture industry and with gadoid (especially cod) farming expanding in several North Atlantic countries, there is considerable potential for this parasite to become a serious problem for commercial mariculture. The main subject areas covered are the parasite's taxonomy; the life history of the parasite including its life cycle, reproduction and host associations; parasite physiology; parasite seasonality and distribution; and the pathogenic effects of the parasite on its host.

A recent infestation of Gyrodactylus cichlidarum Paperna, 1968 on yolk sac fry of Nile tilapia, Oreochromis niloticus niloticus (L.), in an isolated aquarium system in the UK resulted in high mortalities and provided an opportunity to study this species in greater detail. A tentative identification was made using the measurements and drawings of the ventral bar and hamuli provided in the original description; however, details on the morphology of the marginal hooks were lacking. A comparison of the gyrodactylid material collected from O. n. niloticus with the holotype of G. cichlidarum, the only known available specimen, from Mango tilapia, Sarotherodon galilaeus galilaeus (L.), confirmed its identity. Proteolytic digestion and image analysis of the opisthaptoral hard parts were used to obtain tissue-free, accurate measurements as part of a complete revised description of G. cichlidarum. Further, a comparison of G. cichlidarum from both hosts with the holotype and several paratypes of Gyrodactylus niloticus Cone, Arthur et Bondad-Reantaso, 1995 cited as parasitizing captive stocks of Nile tilapia in the Philippines revealed the two species to be synonymous. An 803 bp fragment of the ribosomal internal transcribed spacers 1 and 2 and the 5.8S was obtained and is provided with the revised description. This is the first DNA sequence from a Gyrodactylus species originating from the African continent. The sequence is very divergent from other species in the genus and only the 5.8S sequence places it unambiguously in the genus Gyrodactylus. In addition to G. cichlidarum, two specimens of another morphological similar spe- cies of Gyrodactylus were also found on the UK held stock of O. n. niloticus. These latter specimens, Gyrodactylus sp., differed from G. cichlidarum in having a longer hamulus point with a smaller hamulus aperture and possessing marginal hook sickles that had a shorter shaft with a longer point giving the sickles a more rounded, closed appearance.

The Omani abalone, Haliotis mariae, is the only species of abalone found in Omani waters. Given the rarity of this species and the high price it can fetch on the market (US$ 150 kg-1 dry weight), the wild abalone fishery in the Dhofar region has been regarded as a valuable income source for the past decade. The present study was undertaken set to investigate the mortality of abalone held at the Mirbat Research Center, through bacteriological and histopathological based investigations and challenge tests. Only the adult wild abalone that had been kept for a year in the hatchery, visually, appeared to be clear of disease symptoms. Infected individuals typically were swollen around the mouth, had fluid tinged with blood, bubbles in the intestines, and, very weak adhesive strength. The foot area (muscle) of diseased animals was noticeably very soft and individuals that were seen lying upside down on the bottom of the tank subsequently died. On dissection, the intestinal organs released bubbles and a foul smelling odour. Identification of the isolated bacteria using various identification methods indicated that individuals were infected with Staphylococcus sciuri. Histopathology of infected individuals revealed spongiosis of the tissues with evident bacterial infection. Neither of these histopathological conditions were seen in healthy abalone. The study concludes that the bacterium Staphylococcus sciuri may be the likely cause of abalone mortalities.

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.

Four species of the genus Eubothrium (E. crassum, E. fragile, E. rugosum and E. salvelini) were subjected to morphometric comparison. Discriminant analysis was conducted utilising 17 characters measured on the scolex and strobila of 101 specimens. Univariate statistics were first used to detect features that were useful for separating individual Eubothrium species and two different host populations of E. salvelini. Subsequent multivariate discriminant analysis, combining all the measured variables, made it possible to separate all four species. A comparison of the four taxa revealed that (1) E. fragile is the most distinct species, possessing a much smaller scolex than the other congeners, and its similarity with the other marine species E. crassum is not proven; (2) the two freshwater taxa, E. rugosum and E. salvelini are the most similar; (3) the characters most suitable for species differentiation are the length of the scolex, the width of the apical disc, the width of the neck and its area, the width of eggs and the number of testes; (4) the width of the apical disc was confirmed to be the most stable character at the intraspecific level (within E. salvelini host populations) and is therefore considered to be a trait of the highest discriminative power in the subset of four Eubothrium species.

Light and scanning electron microscopical examinations of nematode samples collected from the stomachs and intestines of catfishes Trichomycterus spp. (Trichomycteridae, Siluriformes) from three streams in Colombia revealed the presence of two species, Dentinema trichomycteri n. g., n. sp. (Cosmocercidae) (type-host Trichomycterus sp.) and Procamallanus (Spirocamallanus) chimusensis Freitas & Ibanez, 1968 (Camallanidae). The new, monotypic genus Dentinema is characterised mainly by the presence of a triangular mouth surrounded by three poorly developed lips, four submedian cephalic papillae, three conical teeth in the well-developed buccal cavity, an elongate oesophageal isthmus which is clearly separated from the corpus, precloacal oblique muscle bands, and by the absence of a ventral sucker. P. chimusensis, recorded from Colombia for the first time, is redescribed from specimens collected from Trichomycterus chapmani (Eigenmann) (a new host record) and Trichomycterus sp.; new observations show that this species belongs to a small group of Procamallanus spp. exhibiting a distinct sexual diversity in the structure of the buccal capsule. P. pexatus Pinto, Fabio, Noronha & Rolas, 1976 is synonymised with P. chimusensis.

Gyrodactylus thymalli Žitňan, 1960 and G. salaris Malmberg, 1957 have an indistinguishable ribosomal internal transcribed spacer (ITS) DNA sequence, but exhibit surprisingly high levels of intra- and interspecific sequence variation of the mitochondrial cytochrome oxidase I (CO1) gene. To test whether different populations of these reportedly very similar species could be discriminated using morphometric methods, we examined the morphometry of four different populations representing different mitochondrial clades. Twenty five point-to-point measurements, including five new characters of the attachment hooks, were recorded from three Norwegian laboratory populations (G. salaris from the Rivers Lierelva and Rauma, and G. thymalli from the River Rena), and from one wild population of G. thymalli from the River Test, UK. The Norwegian populations were kept under identical environmental conditions to control for the influence of temperature on the haptoral attachment hooks. Data were subsequently subjected to univariate and linear stepwise discriminant analyses. The model generated by the linear stepwise discriminant analysis used 18 of the 25 original variables, the first two roots accounting for 96.6% of the total variation between specimens. The hamulus shaft length accounts for 66.7% of the overall correct classification efficiency. Based on morphometry, all specimens were assigned to the correct species. Apart from three specimens of G. salaris from the River Lierelva population which were misclassified as belonging to the G. salaris Rauma population, all specimens were assigned to the correct population. Thus, populations of Gyrodactylus identified by mtDNA can also be discriminated using morphometric landmark distances.

The total diversity of the monogenean genus Gyrodactylus is evaluated. There are 409 potentially valid species names within the genus, recorded from c. 400 host species. Five species have been placed within Fundulotrema and an additional 51 Gyrodactylus species names represent synonyms, nomina nuda or have been reassigned to other non-viviparous monogenean genera. While the majority of Gyrodactylus species (59%) are recorded from single hosts, some have a much broader broad range.

Immunohistochemical and histopathological studies were conducted on a population of 3-spined sticklebacks Gasterosteus aculeatus (L.) from Loch Airthrey (Stirling, Scotland) naturally infected with the microsporean Glugea anomala (Moniez 1887). Of the 55 host specimens that were examined, 16 (29.09%) were infected, the intensity of infection ranging from 1 to 4 xenomas per fish, which were principally located within the central portion of the body lateral flank musculature. All 32 G. anomala xenomas examined were mature, their diameter ranging from 936 to 2232 µm, and their walls of presented a laminar structure. Subcutaneously situated xenomas protruded from the fish body surface, whilst xenomas encountered within the intestine were seen to cause distortion. Light and electron microscopical observations confirmed a host cellular reaction around the xenoma, seen by the presence of eosinophile granule cells (EGCs), and some neutrophils. The occurrences of rodlet cells among the intestinal epithelial cells, and in close proximity to the xenoma wall, were observed in certain specimens. Outside the xenoma wall, macrophage aggregates (MAs) were commonly encountered. Within the xenoma wall, the presence of eosinophile granular cells immunoreactive to the anti-serotonin serum was also recorded. Further immunohistochemical tests revealed that a high number of nerve fibres running along the white lateral muscle fibres were immunoreactive to bombesin-, galanin-, and leu-enkephalin-antisera. Nerve fibres containing bombesin- and leu-enkephalin-like substances were also observed in the connective inflammatory tissue around the protozoan cyst, while neurons in the spinal ganglia were immunoreactive to met-enkephalin, and serotonin antisera. The control for the specificity of immunohistochemical reactions was performed using preabsorption tests of each antiserum with the corresponding antigen, and no immunoreactiv- ity was noticed. The data presented are discussed in relation to the occurrence of G. anomala, which alters the pattern of nerve fibres present in the host. Specifically, the protozoan induces a response in the stickleback nervous system, the reaction of which is revealed through the application of immuno- histochemical techniques.

Gyrodactylus salaris Malmberg, 1957 is a major pathogen of wild Salmo salar L. parr populations in Norway, and its delimitation from non-pathogenic species is important. The present study was undertaken to test the power of chaetotaxy to differentiate between three populations belonging to both the same and different clades (as stated by mtDNA) of G. salaris, in addition to three different species of gyrodactylids (G. salaris, G. thymalli and G. caledoniensis). The gyrodactylids were processed for chaetotaxy in situ and a maximum of 50 specimens per collection site were used to construct a generalised map over the sensilla. The sensilla were found in all populations to be symmetrically distributed around the median longitudinal axis, according to a formula of 7 dorsal (34 sensilla) and 8 ventral (44 sensilla) clusters on each side of the median line. The three Norwegian populations of G. salaris were found identical, as were the population of G. thymalli. The specimens of G. caledoniensis from Scotland, however, were found to differ from the Norwegian species G. salaris and G. thymalli by the position of one sensillum in two of the clusters. A comparison of the sensillum pattern of laboratory maintained G. salaris (River Lierelva) with results obtained ten years earlier, questions the temporal stability of the chaetotaxy pattern. The present results indicate that chaetotaxy can be used to discriminate between certain Gyrodactylus spp. but not generally.

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal RNA gene of Pomphorhynchus laevis (Zoega in Müller, 1776) (Acanthocephala) isolated from various fish species across Central and Southern Europe were compared with those of P. lucyi Williams and Rogers, 1984 collected from the largemouth bass Micropterus salmonoides Boulenger from the USA. The nucleotide sequences of ITS regions of P. laevis from minnows Phoxinus phoxinus (L.) and chub Leuciscus cephalus (L.) from two distant localities in the Slovak Republic were found to be 100% identical. The ITS-1 and ITS-2 of P. laevis from chub from the Czech Republic and Italy were also mutually identical, but significantly different from Slovak worms (88.7% identity for ITS-1, 91.3% identity for ITS-2). A fifth sample collected from Barbus tyberinus Bonaparte from Italy was very similar to the sympatric Italian isolate from chub, possessing four nucleotide substitutions in ITS-1 (98.4% identity). The ITS rDNA sequences of P. lucyi differed significantly from those of P. laevis; the values of identity were 51.8-56.1% for ITS-1 and 63.1-65.3% for ITS-2, and were significantly higher than the range of P. laevis within-species variability. The results based on the ITS sequences confirmed the occurrence of strains in P. laevis from Continental Europe which are well defined by molecules but reveal only slight differences in their morphology.

OZER, AHMET/0000-0002-2890-6766; Shinn, Andrew/0000-0002-5434-2685 WOS: 000180503200004 PubMed: 12641199 A total of 28,387 oligochaetes belonging to the families Tubificidae, Lumbriculidae, Naididae and Enchytraeidae were examined at regular intervals for actinosporean infections from October 1996 to August 1998 from a freshwater salmon farm in Nor-them Scotland. A total of 21 types of actinosporeans belonging to seven collective groups synactinomyxon (three types), aurantiactinomyxon (four types), echinactinomyxon (five types), raabeia (six types), triactinomyxon (one type), neoactinomyxum (one type) and siedleckiella (one type) were found. Synactinomyxon type 1, echinactinomyxon type 1 and raabeia type 4 were most abundant. The overall infection prevalence of oligochactes was 2.9%. Aurantiactinomyxon, synactinomyxon and neoactinomyxum were most common in summer and autumn. Raabeia was most common in spring and summer and echinactinomyxon in winter and spring, Siedleckiella was found only in spring and triactinomyxon in all seasons except winter. A positive relationship between water temperature and the number of actinosporean types released was observed. Most actinosporean types were found in only one host species.

The cuticle of the chalimus II stage of Lepeophtheirus salmonis (Copepoda: Caligidae) comprised a four-layered epicuticle with a pronounced fuzzy coat which was separated from the outer and inner procuticles by a layer of transitional procuticle. The cuticle is underlain by a single-layered epidermis which overlies integumental glands and chromatophores. The structure of this cuticle is very similar to that described for free-living copepods and does not display the modifications associated with more highly transformed parasitic species.

The chaetotaxy of argentophilic structures on three species of the monogenean genus Gyrodactylus was investigated in an attempt to distinguish species of this genus. Maps were prepared for Gyrodactylus salaris from Scandinavia and compared with two native species of Gyrodactylus parasitising salmonids in Britain, namely Gyrodactylus derjavini and Gyrodactylus truttae. The maps were subsequently refined and analysed for zones of homology and differentiation. The results demonstrate that G. salaris can be readily distinguished by this technique, which is, therefore, of great potential value in the identification of this notifiable pathogen. The key aggregations of sensilla discriminating G. salaris are, ventrally, the antero-ventral set, the medio-lateral set and the postero-lateral set, and, dorsally, the postero-dorsal set.

Gyrodactylus gemini n. sp. (Monogenea, Gyrodactylidae) is described from the surface of the body and fins of the fish Semaprochilodus taeniurus (Steindachner) imported into Britain from the Venezuelan Amazon. The new species differs from other species of the genus, including those described from South and Central America, by having: (i) stout hamuli with straight shafts and diverging roots; (ii) marginal hooks with the sickle length larger than the width; (iii) a dorsal bar without a medial constriction; (iv) a rectangular ventral bar with short processes; (v) a triangular ventral bar membrane; and, most obviously, (vi) at least two generations which can develop two embryos simultaneously. This is the first known species of the genus Gyrodactylus from the Venezuelan Amazon and the first record of the subgenus Gyrodactylus (Gyrodactylus) from South America.

Background Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata. Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan. Results Phylogenetic analyses of small subunit ribosomal DNA (SSU rDNA) sequence data from Atlantic cod X-cells show that they are a related parasite that occupies a basal position to the clade containing other X-cell parasites. The X-cell parasite causing epidermal pseudotumours in P. obscurus is the same parasite that causes pseudotumours in H. dubius. Direct, fish to fish, transmission of the X-cell parasites used in this study, via oral feeding or injection, was not achieved. Non-amoeboid X-cells are contained within discrete sac-like structures that are loosely attached to epidermal pseudotumours in flatfish; these X-cells are able to tolerate exposure to seawater. A sensitive nested PCR assay was developed for the sub clinical detection of both parasites and to assist in future life cycle studies. PCR revealed that the parasite in P. obscurus was detectable in non-pseudotumourous areas of fish that had pseudotumours present in other areas of the body. Conclusions The inability to successfully transmit both parasites in this study suggests that either host detachment combined with a period of independent development or an alternate host is required to complete the life cycle for X-cell parasites. Phylogenetic analyses of SSU rDNA confirm a monophyletic grouping for all sequenced X-cell parasites, but do not robustly support their placement within any established protist phylum. Analysis of SSU rDNA from X-cells in Japanese flatfish reveals that the same parasite can infect more than one species of fish.

In species of the monogenean helminth Gyrodactylus, the opisthaptor is the main organ of attachment to the host. The opisthaptor comprises two large centrally positioned hooks or hamuli and sixteen peripherally distributed marginal hooks. This paper describes the functional morphology and the mechanism and sequence of attachment in this species. Information on the attachment process was gathered from observations of live gyrodactylids, from transmission electron microscopy, from scanning electron microscopy of skeletal elements, and by histochemical and X-ray elemental analysis of hook chemical composition. The marginal hooks provide the principal force of attachment whilst the hamuli are not actively employed in the process of attachment. Instead, the hamuli provide a system preventing accidental dislodgement and assist the action of the marginal hooks. Attachment is achieved by the alternating action of two systems of muscles attached respectively to the hamuli and to the marginal hooks. Relaxation or contraction of the muscles connected to the hamuli manoeuvres the hamuli over the extremities of the accessory ventral bar and allows them to pivot around their longitudinal axis, effectively raising or lowering the opisthaptoral dome. Under reduced opisthaptoral tension, the independent gaffing activity of the marginal hooks ensures a secure attachment to the host's epidermis. Repositioning of the hamuli then raises the opisthaptoral dome to tension the peripheral marginal hooks. The sequence of attachment is complete when all the muscles associated with the hooks are in a state of relaxation but are held securely and under tension by the surrounding, stretched, opisthaptoral dome.

The internal transcribed spacers (ITS-1 and ITS-2) of the ribosomal DNA (rDNA) of fish cestodes of the genus Eubothrium were sequenced. The ITS sequences of Eubothrium rugosum were determined and compared with previously analysed congeneric species, Eubothrium crassum and Eubothrium salvelini. The ITS-1 sequences of E. rugosum and E. crassum were 535 bp long, the length of E. salvelini ITS-1 was 536 bp. The ITS-2 region was found to be 403 bp in E. rugosum and E. crassum, and 401-402 bp in E. salvelini. The ITS-1 region of E. rugosum was closer to E. salvelini (identity 98.9-99.1%) than to E. crassum (97.9-98.1%), while the sequence similarity within the ITS-2 region was almost identical (97.5-98.0% for E. crassum; 97.7-98.3% for E. salvelini). Several restriction enzymes were found to be suitable for the differentiation of the three Eubothrium species by PCR-RFLP. The intraspecific sequence variation of E. crassum from different fish hosts (freshwater and marine) and European regions was very low, 0.2% for ITS-1 and 0.5% for ITS-2. Analysis of the ITS sequences of specimens from rainbow trout from three localities in Scotland revealed that both E. crassum and E. salvelini are present in this fish host.

Active Shape Models (ASM) are applied to the attachment hooks of several species of Gyrodactylus, including the notifiable pathogen G. salaris, to classify each species to their true species type. ASM is used as a feature extraction tool to select information from hook images that can be used as input data into trained classifiers. Linear (i.e. LDA and KNN) and non-linear (i.e. MLP and SVM) models are used to classify Gyrodactylus species. Species of Gyrodactylus, ectoparasitic monogenetic flukes of fish, are difficult to discriminate and identify on morphology alone and their speciation currently requires taxonomic expertise. The current exercise sets out to confidently classify species, which in this example includes a species which is notifiable pathogen of Atlantic salmon, to their true class with a high degree of accuracy. The findings from the current exercise demonstrates that data subsequently imported into a K-NN classifier, outperforms several other methods of classification (i.e. LDA, MLP and SVM) that were assessed, with an average classification accuracy of 98.75%.

Morphometric data were collected from scanning electron micrographs of released hamuli and marginal hooks collected from five species and three morphotypes of Gyrodactylus parasitising salmonids in the UK and Canada. Five new parameters for measurement from the hamulus are described: shaft-point length, hamulus angle, hamulus width, shaft area and the root area; and three from the marginal hook: sickle aperture, toe length and the marginal hook area. Univariate statistics were used to select seven of nine useful metrical hamulus features and seven of eight marginal hook features. Of the hamulus features, the shaft-point length discriminated all Gyrodactylus salaris populations from all other Gyrodactylus populations and the hamulus angle discriminated only the Swedish G. salaris populations from those gyrodactylids on British salmonids. The marginal hook features, the sickle length, sickle aperture, total length and shaft length of G. salaris were significantly larger than those of all other gyrodactylids included within the study. Of the British fauna, it was found that G. derjavini from Welsh salmon was significantly smaller than all other British collections of Gyrodactylus in this study, having smaller hamuli point lengths, total lengths and shaft lengths. Separate principal components analyses were performed on the variables selected for the hamuli and for the marginal hooks. The principal component analysis (PCA) analysis demonstrated that individual structures from either the hamulus or the marginal hook, significantly discriminate every specimen of G. salaris from all other gyrodactylid specimens included within the analysis. The discrimination of G. derjavini from G. truttae by this method, was however, not possible and the description of additional morphometric parameters may be necessary before this separation can be achieved.