Description and phylogeny of Ceratomyxa anko sp. n. and Zschokkella lophii sp. n. from the Japanese anglerfish, Lophius litulon (Jordan)

Two new species of myxozoans from the Japanese anglerfish, Lophius litulon, are described using myxospore morphology and small subunit rDNA sequences. Ceratomyxa anko sp. n. is a parasite of the gall bladder and had a prevalence of 57%. Mature spores of C. anko sp. n. are arcuate to crescent shaped with valves tapering to rounded tips. A prominent sutural line runs centrally between the round adjacent polar capsules containing the polar filament coiled two to three times. Spore measurements: length 10.8 (9.7-11.9) microm, width 41.9 (36.9-47.2) microm, polar capsule diameter 4.6 (4.1-5.3) microm. Ceratomyxa anko sp. n. can be distinguished from other Ceratomyxa spp. due to its spore dimensions and shape. Zschokkella lophii sp. n. is a parasite of the urinary bladder and had a prevalence of 70%. Mature spores are ellipsoidal to semicircular with bluntly pointed ends. The sutural line is curved or sinuous and the valves have no discernable surface ornamentation. Two almost spherical polar capsules are located separately in the ends of the spore, opening in almost opposite directions and contain the polar filament with five coils. Spore measurements: length 20.1 (16.8-24.0) microm, width 14.9 (12.7-16.8) microm, polar capsule diameter 5.1 (3.6-5.8) microm. Zschokkella lophii sp. n. can be distinguished from other Zschokkella spp. due to the terminal opening of the polar capsules within the spores and the site of infection within the host fish. In the phylogenetic analyses, C. anko sp. n. grouped with other members of the same genus forming a monophyly. Zschokkella lophii sp. n. forms a discrete clade with another Zschokkella sp. that infects the urinary bladder of marine fish. This grouping forms a sister clade to one containing members of the genus Parvicapsula, all of which are parasites of the urinary system in marine fish.     

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian,Loma morhua

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial‐scale cultivation approaches. This study details the establishment of a larval cell line (GML‐5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML‐5 has survived over 100 passages in 8 years of culture. The line remains active and viable between 8 and 21°C in Leibovitz‐15 (L‐15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno‐positive results for vimentin, α‐smooth muscle actin, collagen I and S‐100 proteins, while being desmin‐negative. GML‐5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod‐specific Loma morhua. Using GML‐5, spore germination and proliferation of L. morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A. algerae, which required no cultural modifications. Loma morhua‐associated xenoma‐like structures were observed 2 weeks postexposure. This in vitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.     

Modes of transmission of Glugea plecoglossi (Microspora) via the skin and digestive tract in an experimental infection model using rainbow trout, Oncorhynchus mykiss (Walbaum)

Glugea plecoglossi (Microspora) is a significant cause of economic loss in cultured ayu, Plecoglossus altivelis, in Japan, due to the unsightly appearance of infected fish harbouring xenomas in the body cavity. Modes of transmission of G. plecoglossi via the skin and digestive tract were studied in an experimental infection model using rainbow trout, Oncorhynchus mykiss. Combined with Uvitex 2B and in situ hybridization (ISH) assays, the early development of G. plecoglossi was successfully traced. Following a bath exposure of fish Uvitex 2B-labelled G. plecoglossi spores were observed to attach to microscopic injuries (trypan blue-positive sites) of fish skin, after which ISH-positive sporoplasms were found to invade the epidermis as early as 5 min post-infection (PI), migrating rapidly to the subdermis. It was also shown that G. plecoglossi entering via the skin does not spread into the internal organs but develops into subdermal xenomas. After rainbow trout were exposed to G. plecoglossi spores by oral intubation, spores germinated in the intestinal lumen, followed by penetration of sporoplasms into the gut mucosal epithelium 5 min PI. In vitro trials determining stimulation factors (fish mucus, changes in pH, digestive enzymes) for the extrusion of the polar tube were inconclusive. The present study indicates that skin wounds and the gut epithelium can be portals of entry of G. plecoglossi and that natural infection in fish seems to occur perorally rather than via the skin.     

The transmission potential of Loma salmonae (Microspora) in the rainbow trout, Oncorhynchus mykiss (Walbaum), is dependent upon the method and timing of exposure

The ability of a parasite to transmit from one fish to another is important in the dissemination of disease. Groups of 25 naive rainbow trout (RBT), Oncorhynchus mykiss (Walbaum), were exposed to Loma salmonae by feeding on the viscera (gills, hearts and spleens) from L. salmonae -infected donor RBT (DRBT) or by cohabitation with infected DRBT. Exposure occurred 3, 7, 11 and 15 weeks after the DRBT were infected. All naive RBT were examined 7 weeks post-exposure (PE) to the DRBT. Naive RBT, exposed to DRBT at week 3 PE, by feeding on viscera or by cohabitation, failed to develop visible branchial xenomas. Cohabiting naive RBT with DRBT, at week 7 PE and week 11 PE, resulted in the development of branchial xenomas. Xenomas failed to develop in naive RBT exposed via cohabitation to week 15 PE DRBT. Naive RBT, exposed by feeding on the viscera of DRBT at week 7 PE, week 11 PE and week 15 PE, developed branchial xenomas. The transmission potential of viscera from L. salmonae -infected DRBT at week 15 and week 20 PE was also examined. Naive RBT, fed with viscera free of visible branchial xenomas, from DRBT at week 15 PE and week 20 PE, developed branchial xenomas by week 7 PE. A polymerase chain reaction (PCR) was used to detect L. salmonae DNA from the water and sediments of a tank of L. salmonae -infected RBT at week 7 PE. The method and timing of exposure of naive fish to L. salmonae -infected fish are important in disease transmission and may be useful in predicting and preventing disease outbreaks in aquaculture.     

Morphological characterization and notes on the life cycle of a newly discovered variant of Loma salmonae (Putz, Hoffman & Dunbar) from a natural infection of chinook salmon, Oncorhynchus tshawytscha (Walbaum)

Two variants of Loma salmonae occur in net-pen reared chinook salmon, Oncorhynchus tshawytscha. The typical variant (OA) has a host specificity for salmonids of the genus Oncorhynchus whereas the atypical variant (SV) has a host specificity for brook trout, Salvelinus fontinalis, and in this study, the ultrastructure of the two are compared. In fish at 8 weeks post-exposure xenomas of the SV variant have a very high proportion of mature spores compared with other developmental stages, while in xenomas of the OA variant there are fewer spores and many other developmental stages. Spores of the SV variant had up to 20 turns of their polar tube whereas those of the OA variant only had 17. Furthermore, the spores of the SV variant were significantly larger than those of the OA variant. The sporophorous vesicle for both variants appears to form around a sporogonial plasmodia, which results in many spores developing within the vesicle.     

The parasite–host interface in the zonetail butterfly ray,Gymnura zonura (Bleeker), infected with Hexacanalis folifer (Cestoda: Lecanicephalidea)

A wild‐caught specimen of the zonetail butterfly ray, Gymnura zonura (Bleeker), harboured numerous specimens of Hexacanalis folifer Cielocha & Jensen, 2011 (Systematic Parasitology, 79, 1–16; Cestoda: Lecanicephalidea) within its spiral intestine. The cestodes were primarily attached in single rows along the base of mucosal folds, each associated with a nodular mucosal thickening. Microscopically, the scolex was embedded within the submucosa and muscularis; the attachment sites were marked by ulceration and necro‐proliferative inflammation demarcating the parasite from normal host tissues. Physical attachment of the cestode was restricted to the anterior portion of its scolex where presumed tegumental secretions from the apical organ contributed to a cementing intermediate layer blending with necrotic host cells. The presence of tegumental differentiation between the apical organ and the scolex proper, associated with presumed different roles in attachment, correlated with ultrastructural observations of the surface modifications on the scolex. Despite the locally severe pathological change, insignificant morbidity owing to this particular host–parasite relationship is suggested.     

Effects of tributyltin and hypoxia on the progression of Perkinsus marinus infections and host defence mechanisms in oyster, Crassostrea virginica (Gmelin)

Oysters, Crassostrea virginica (Gmelin), naturally infected by the protozoan parasite Perkinsus marinus in the field were exposed for 6 weeks to tributyltin (TBT), hypoxia, or to both stressors simultaneously. The TBT-exposed oysters continuously bioaccumulated TBT, reaching about 4 mg kg^??1 dry weight by 6 weeks; hypoxic oysters were exposed to water containing an average dissolved oxygen level of about 3 mg L^??1. Untreated control oysters suffered about 30% cumulative mortality by 6 weeks as a result of the progression of their P. marinus infections. The TBT treatment alone produced no additional mortality; however, cumulative mortality in hypoxic oysters was elevated. Mortality among oysters receiving both TBT and hypoxia significantly exceeded that caused by either stressor alone, suggesting a synergistic effect. In an attempt to identify immunotoxicological mechanisms underlying stress-related augmentation of P. marinus infections, defence-related immune functions were measured at 3 and 6 weeks in control and treated oysters. In general, the total number of haemocytes increased as the infections progressed, and the TBT and hypoxic treatments also caused significant additional increments in some samples. However, oxygen-dependent (reactive oxygen species) and oxygen-independent (lysozyme) antimicrobial host defence mechanisms appeared to be largely unaffected by TBT and/or hypoxia. This may be explained by the death of those oysters with marked immunological lesions prior to sampling or by the actual lack of treatment effects.     

高碳水化合物水平日粮对异育银鲫生长、生理、免疫和肝脏超微结构的影响

为了探讨异育银鲫对日粮中高碳水化合物的利用,选用168尾异育银鲫,分成2组,一组为正常对照组,投喂35%碳水化合物(35%CHO)水平日粮;另一组为高糖试验组,投喂50%碳水化合物(50%CHO)水平日粮每组设3个重复。在控温的循环水系统中饲养10周后,测定鱼体生长指标、肌肉成分、肝糖元、血液生化指标和免疫指标。结果表明,高糖组(50%CHO)显著(P<0.05)降低了特定生长率、增重率,但是对饵料系数、肝体比、脏体比、鱼体肌肉成分等没有显著(P>0.05)影响;两组异育银鲫血浆总蛋白、甘油三酯、总胆固醇、血糖和肝糖原含量都没有显著差异(P>0.05);高糖组(50%CHO)肝脏丙二醛含量显著升高(P<0.05)、天门冬氨酸转氨酶显著降低(P<0.05),而肝脏超氧化物歧化酶、总抗氧化能力、丙氨酸转氨酶和碱性磷酸酶都没有显著变化(P>0.05)。试验还对两组异育银鲫肝细胞进行超微结构观察,发现高糖饲料引起了异育银鲫肝细胞中糖原和脂肪的沉积,并对细胞和细胞器的结构产生一定损伤。从本试验结果来看,异育银鲫可以耐受50%饲料糖含量,但是长期摄食50%碳水化合物日粮对其生长不利,并会引起一定程度肝脏功能和结构的损伤。     

Gill pathology caused by infestations of adult and preadult Dissonus manteri Kabata (Copepoda: Dissonidae) on coral trout, Plectropomus leopardus (Lacépède), (Serranidae)

Adult and preadult Dissonus manteri attached to the gills of Plectropomus leopardus cause significant pathology in the form of large hyperplastic nodules on the afferent (leading) edges of gill filaments. Nodules result from the dual actions of parasite attachment and feeding. The host response is characterized by severe epithelial hyperplasia, supplemented by fibroplasia and inflammation. Parasites attach close to the gill arch near the base of filaments. They have little effect on gill vasculature as the maxillipeds penetrate the filament superficial to the efferent filament artery and do not interfere with the blood vessels of the secondary lamellae. Tissue proliferation is limited to the wide portion of filament 'edge' epithelium in the proximal third and also does not extend to the secondary lamellae. Nodules are most numerous towards the ends of hemibranchs and are generally absent from the central regions. Leading hemibranchs bear significantly more nodules than their trailing counterparts. Of the total number of nodules, 20.5% are located on the pseudobranchs. Distribution patterns are considered to be primarily the result of D. manteri avoiding strong water currents, although this cannot explain the difference between numbers on leading and trailing hemibranchs.     

Morphological characterization of the tunic in the edible ascidian, Halocynthia roretzi (Drasche), with remarks on 'soft tunic syndrome' in aquaculture

'Soft tunic syndrome' is a serious problem in the aquaculture of the edible ascidian, Halocynthia roretzi (Drasche), and often leads to mass mortality. Here, we describe the tunic morphology of intact and diseased ascidians to reveal structural differences between them. Morphologically, diseased tunics are not very different from intact tunics, although the former are thinner and softer than the latter. While several types of cells are distributed in the tunic, the cell types and their cytomorphologies were almost identical in both groups. As bacterial/protozoan cells were not found in either intact or diseased tunics, they are not the direct cause of soft tunic syndrome. The most remarkable difference was in the bundles of tunic fibres that compose the tunic matrix; in intact tunics, the thick bundles interlace to form a firm matrix, whereas in soft tunics, the tunic fibres do not form thick bundles. Furthermore, areas of low fibre density were found in diseased tunics. Therefore, soft tunic syndrome probably causes inhibition of bundle formation and degradation of tunic bundles, creating areas of low fibre density, although the causes remain unknown.     

Culture of the mysid, Mysidopsis almyra (Bowman), (Crustacea: Mysidacea) in a static water system: effects of density and temperature on production, survival and growth

The effects of animal density and water temperature on the culture of the mysid, Mysidopsis almyra (Bowman), in a static water system were evaluated. An initial set of experiments tested the effects of mysid density on production. Densities of 25, 37.5, 50, 100 and 200 mysids L^–1 were placed in trays with 20 L of sea water. Temperatures were maintained at 26 ± 2 °C. A second set of experiments was conducted in the same system at three different temperatures (18 ± 1, 22 ± 1 and 26 ± 2 °C) using a mysid density of 50 mysids L^–1 (1000 mysids tray^–1). All experiments had a duration of 30 days. The mysids in all trials were cultured at 20 ± 2‰ salinity and fed Artemia nauplii enriched with marine fatty acids. There was a positive correlation between production and mysid densities up to populations of 100 mysids L^–1; maximum production was 273 ± 99 hatchlings day^–1. At a population density of 200 mysids L^–1, high mortality and low production were recorded 4 days after the start of the experiment. The experiments testing different temperatures showed that mysid production was higher at 22 ± 1 °C, although this result was not significant (P > 0.05). Growth rates and hatchling survival after 7 days were significantly higher (P < 0.05) at 26 ± 2 °C compared to survival and growth at 18 or 22 °C.     

An immunohistochemical study on endocrine cells in the neuroendocrine system of the digestive tract of milkfish Chanos chanos (Forsskal, 1775)

In this study, several kinds of endocrine cells which produce neuropeptides and hormones have been identified and located in the digestive tract of milkfish (Chanos chanos) by the immunohistochemical technique. The oesophagus, stomach (cardiacus, fundic and pyloric regions), pyloric caeca, intestine (anterior, middle and posterior regions) and rectum were divided from the digestive tract of milkfish. The distribution and frequency of the endocrine cells produced these neuropeptides and hormones including gastrin, cholecystokinin, leu‐enkephalin, neuropeptide Y and somatostatin were described in these segments. Endocrine cells containing all neuropeptides and hormones immunoreactivities were present in the anterior intestine region, but no reaction was found in oesophagus. Only a few L‐ENK‐ and SST‐immunoreactive cells were detected in the pyloric caeca. Large numbers of SST‐immunoreactive cells were observed in the stomach with no immunoreactivities to other neuropeptides and hormones. Two different types of endocrine cells were founded in the intestine and pyloric caeca. These results indicate that the anterior intestine region may be the main region of food digestion and absorption, and the neuropeptides and hormones regulate the food intake process by different ways in milkfish.     

A review of host finding behaviour in the parasitic sea louse, Lepeophtheirus salmonis (Caligidae: Copepoda)

Ectoparasitic sea lice are the most important parasite problem to date for the salmon farming industry in the northern and southern hemispheres. An understanding of host location in the specialist species, Lepeophtheirus salmonis , the most important louse species in the North Atlantic, is now being realized using behavioural in vitro and in vivo bioassays coupled with chemical analysis of fish conditioned waters. Both physical and chemical cues are important in host location. Responses of sea lice to physical cues such as light and salinity may enable them to gather in areas where host fish are likely to be found. Mechanoreception is an important sensory modality in host location and acts by switching on specific behaviours that enable landing on a fish. Chemoreception plays a defining role in host location and recognition. The detection of host kairomones switches on 'host search' behavioural patterns and also induces landing responses whereas non-host kairomones fail to induce attraction or significant landing behaviour. Semiochemicals derived from salmon and also non-host fish have been identified, and may prove useful for the development of integrated pest management strategies, by the introduction of odour traps for monitoring lice numbers, and by the use of stimulo-deterrent diversionary (push:pull) strategies in their control.     

Effects of starvation on RNA : DNA ratio, glycogen content, and C : N ratio in columellar muscle of the Japanese turban shell Turbo (Batillus) cornutus (Gastropoda)

ABSTRACT: The validity of using the RNA : DNA ratio, glycogen content, and C : N ratio in the columellar muscle as indicators of the nutritional condition in the Japanese turban shell Turbo cornutus was examined. These biochemical indices were compared between fed and starved animals. The fed animals were fed brown algae to satiation for 108 days as a control, whereas the starved animals were not fed for the first 75 days and then fed for next 33 days. All three indices declined during the starvation period, and the values for the starved group were significantly lower than those for the fed group during days 20–75. At day 108, after the re-feeding period, the indices for the starved group were found to have increased. Among the indices, only the RNA : DNA ratio for the starved males responded rapidly to starved conditions and became significantly lower even at day 4, and only the RNA : DNA ratio for the starved males and females recovered to those levels of the fed animals at day 108. The results indicated that the RNA : DNA ratio is the most rapid indicator of nutritional stress among the three indices. However, it is recommended that glycogen content and C : N ratio be used in addition to the RNA : DNA ratio for monitoring the health of T. cornutus as the RNA : DNA ratio showed large variations.     

饥饿和再投喂对日本黄姑鱼代谢率和消化器官组织学的影响

采用封闭式呼吸仪测定日本黄姑鱼(8.14±1.07 g)在饥饿和恢复投喂过程中的耗氧率;用石蜡切片法观察和分析饥饿和再投喂对日本黄姑鱼消化器官形态结构和组织学的影响。结果表明:随着饥饿时间的延长,其耗氧率逐渐降低;恢复投喂之后,其耗氧率开始逐步上升。从组织结构看,食道无明显变化,而胃、肠和肝胰脏则随着饥饿时间的延长出现不同程度的损伤;饥饿5 d组无明显变化,而饥饿15 d组变化明显,表现为胃腺细胞收缩,皱壁和上皮细胞高度减少,分泌颗粒减少,肠胃绒毛退化,肝组织致密,肝细胞内脂滴减少,胰腺泡缩小,排列不规则;饥饿10 d组介于饥饿5 d组和饥饿15 d组之间,再投喂各主要结构均有恢复。     

Changes in the neuromodulators of the diffuse endocrine system of the alimentary canal of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), naturally infected with Eubothrium crassum (Cestoda)

A histopathological and immunohistochemical study on the intestines of 45 specimens of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), from Loch Awe, Scotland, revealed a number of cellular deviations in individuals naturally infected with the pseudophyllidean cestode Eubothrium crassum (Bloch, 1779). Twenty-five individuals (55.5%) were infected with an average worm burden of 18.84 +/- 4.06 (mean +/- SE) cestodes per host (range, 2-80 worms; total 471 worms). The cestodes, measuring an average 8.23 +/- 1.10 cm (mean +/- SE; range, 5.3-13.0 cm) in length, were found attached by their scolices to the mucosal lining of the distal portion of the pyloric caeca. Within the caeca, the strobila evoked a mild catarrhal enteritis, namely an enhanced mucus production with epithelial cellular desquamation, a leucocytic infiltration of the lamina propria-submucosa and vacuolization of the intestinal epithelial cells. Eosinophilic granular cells of the stratum granulosum exhibited granular depletion, while within the catarrh, the presence of a high number of rodlet cells was noticed. Immunohistochemically, the occurrence of E. crassum caused a significant reduction in the number of bombesin-, gastrin-releasing peptide and glucagon-like immunoreactive endocrine cells, but an increase in the relative densities of endocrine cells containing cholecystokinin-8- and gastrin-like substances. There were, however, no significant differences in the number of endocrine cells that were immunoreactive to secretin, neuropeptide Y and peptide histidine-isoleucine antisera in the digestive tracts of either the infected or non-infected O. mykiss.