Establishment of medium for laboratory cultivation and maintenance of Fredericella sultana for in vivo experiments with Tetracapsuloides bryosalmonae (Myxozoa)

The freshwater bryozoan Fredericella sultana (Blumenbach) is the most common invertebrate host of the myxozoan parasite Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease in salmonid fish. Culture media play an important role in hatching of statoblasts and maintaining clean bryozoan colonies for Malacosporea research. We developed a novel culture medium, Bryozoan Medium C (BMC), for the cultivation and maintenance of F. sultana under laboratory conditions. Statoblasts of F. sultana were successfully hatched to produce transparent‐walled, specific pathogen‐free (SPF) colonies that were maintained >12 months in BMC at pH 6.65. Tetracapsuloides bryosalmonae was successfully transmitted from infected brown trout, Salmo trutta L., to newly hatched F. sultana colonies in BMC, then from the infected bryozoan to SPF brown trout. This study demonstrated the utility of BMC (pH 6.65) for hatching statoblasts, long‐term cultivation of clean and transparent bryozoan colonies and maintenance of the Tetracapsuloides bryosalmonae life cycle in the laboratory for molecular genetic research and other studies such as host–parasiteinteraction.     

A survey of the distribution of the PKD‐parasite Tetracapsuloides bryosalmonae (Cnidaria: Myxozoa: Malacosporea) in salmonids in Norwegian rivers – additional information gleaned from formerly collected fish

The malacosporean Tetracapsuloides bryosalmonae was detected in kidneys from Atlantic salmon parr in 64 of 91 sampled Norwegian rivers. Using real‐time PCR, this parasite was found to be present in Atlantic salmon parr in rivers along the whole coast, from the northernmost and southernmost areas of the country. In addition, T. bryosalmonae was found in kidneys from brown trout parr in 17 of 19 sampled rivers in south‐east Norway, and in Arctic charr sampled in the River Risfjordelva, located at the northernmost edge of the European mainland. In conclusion, T. bryosalmonae has a widespread distribution in salmonids in Norwegian watercourses. Proliferative kidney disease (PKD) caused by T. bryosalmonae and PKD‐induced mortality has been observed in salmonids in several Norwegian rivers and it can be speculated that more PKD outbreaks will occur as a result of climate change.     

Descriptions, development and pathogenicity of myxozoan (Myxozoa: Myxosporea) parasites of juvenile cyprinids (Pisces: Cyprinidae)

Approximately 5000 young of the year (0+) cyprinids comprising roach, chub, dace, minnow, bleak, bream, barbel and gudgeon were examined histologically for the presence of myxozoan infections. Thirteen myxozoans were identified to species, the majority being Myxobolus spp. In addition, two species of Myxidium and of Sphaerospora were recorded. All organs were examined, with the majority of infections being found in the gills, musculature and kidney. However, isolated spores were occasionally found in other tissues. Whilst roach contained the highest number of myxozoan species, it was chub that showed the greatest host response to sporogonic forms. Data are provided on spore morphology, pathogenic responses and tissue and host specificity of the myxozoans recorded.     

The life cycle of Chloromyxum auratum (Myxozoa) from goldfish, Carassius auratus (L.), involves an antonactinomyxon actinospore

The myxozoan parasite Chloromyxum auratum Hallett, Atkinson, Holt, Banner & Bartholomew, 2006 , was shown experimentally to have a two‐host life cycle which involved a previously undescribed antonactinomyxon actinospore stage. Myxospores obtained from gall bladders of naturally infected feral goldfish, Carassius auratus (L.), were used to infect samples of mixed species of oligochaete worms obtained from the same locality as the fish: Fern Ridge Dam, Oregon, USA. After some 110 days post‐exposure, actinospores were detected from the water above the oligochaetes. The 18S rDNA sequence of these actinospores was identical to the original myxospores. Spore release was sporadic, of low intensity and short duration, which confounded efforts to identify the host oligochaete species and infect naïve fish. This is the first life cycle that incorporates an actinospore of the collective group Antonactinomyxon, and the first life cycle demonstrated in the laboratory for a species of Chloromyxum.     

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.     

Real-time PCR detection of Parvicapsula pseudobranchicola (Myxozoa: Myxosporea) in wild salmonids in Norway

The myxozoan genus Parvicapsula contains 14 species infecting fish, some of which are known to cause severe disease in farmed and wild salmonids. Parvicapsula pseudobranchicola infections were first reported from seawater-reared Atlantic salmon, Salmo salar, in Norway in 2002 and have since then been an increasing problem. The present study describes a Taqman real-time PCR assay for specific detection of P. pseudobranchicola. The Taqman assay targets the 18S rRNA gene of P. pseudobranchicola and is able to detect as few as ten copies of the target sequence. Using the described assay, P. pseudobranchicola was detected in both farmed and wild salmonids, indicating that wild Atlantic salmon, sea trout, Salmo trutta, and Arctic char, Salvelinus alpinus, may be natural hosts of the parasite. Parvicapsula pseudobranchicola was found in samples from wild salmonids in the far south and the far north of Norway, displaying a wide geographic range of the parasite. Farmed salmonids showed P. pseudobranchicola infection levels many folds higher than that observed for wild sea trout, indicating that farmed Atlantic salmon are subjected to an elevated infection pressure compared with wild salmonids.     

Dynamics of experimental production of Thelohanellus hovorkai (Myxozoa: Myxosporea) in fish and oligochaete alternate hosts

The dynamics of development and production of Thelohanellus hovorkai (Myxozoa) were examined to investigate factors inducing haemorrhagic thelohanellosis in carp, Cyprinus carpio L. Fresh actinospores of T. hovorkai were harvested from the oligochaete alternate host, Branchiura sowerbyi, and used for infection experiments with myxosporean-free carp. Visualization of actinospores by fluorescent labelling revealed that sporoplasms penetrated the gill filaments of carp immersed in an actinospore suspension as early as 30 min post-exposure (PE). Plasmodia of T. hovorkai developed in the connective tissues of various organs and matured 3-5 weeks PE; dispersion of myxospores from degenerate plasmodia occurred 5-7 weeks PE. Challenges with a high dose of actinospores (4.5 x 10(6) spores per fish) resulted in the onset of disease, which was more easily achieved by the oral intubation of actinospores than by immersion in an actinospore suspension. Actinosporean-free B. sowerbyi were exposed to different densities of myxospores (10(4)-10(6) spores per oligochaete) and subsequently reared at different temperatures (15, 20, 25 degrees C). At 20 and 25 degrees C, actinospore releases were first detected 40-43 days PE, with multiple peaks of release (max. 7 x 10(5) actinospores day(-1)) during the next 60 days. We concluded that the developmental cycle of T. hovorkai was completed within 3-5 months at 20-25 degrees C, and that the ingestion of large numbers of actinospores orally, possibly by feeding on infected oligochaetes, resulted in a disease condition in carp.     

The susceptibility of diverse species of cultured oligochaetes to the fish parasite Myxobolus pseudodispar Gorbunova (Myxozoa)

This study provides detailed information on the invertebrate hosts of Myxobolus pseudodispar (Myxozoa) and explores the susceptibility range of several species and analyses the relevance of the species composition of an oligochaete population. Our findings demonstrate that the oligochaete host range of M. pseudodispar is similarly wide as the number of vertebrate host species. Besides Tubifex tubifex and Limnodrilus hoffmeisteri, Psammoryctides barbatus and Psammoryctides moravicus were also found to be susceptible invertebrate hosts. The genetic characterization of the mitochondrial 16S rDNA of T. tubifex sensu lato revealed that lineages I, II and III are susceptible to M. pseudodispar, whereas T. tubifex lineage VI seems to be non‐susceptible. T. tubifex lineage V and L. hoffmeisteri specimens were positive in a M. pseudodispar‐specific PCR, but in most cases, the release of mature actinospores could not be detected. Hence, these non‐susceptible oligochaetes likely serve as `biological filters` as they remove myxospores from the sediment without producing actinospores. Together with the phylogenetic analysis of the susceptible and non‐susceptible oligochaete hosts on the basis of mt 16S rDNA sequences, the route of the development of M. pseudodispar in the oligochaete hosts was tracked by in situ hybridization. According to our findings, the gut epithelia seem to be a portal of entry of the sporoplasms, where the development of the parasite also takes place. The basal lamina seems to be involved in the migration of the parasite, and the worm’s cellular immune response is activated by the infection.     

Myxobolus erythrophthalmi sp. n. and Myxobolus shaharomae sp. n. (Myxozoa: Myxobolidae) from the internal organs of rudd, Scardinius erythrophthalmus (L.), and bleak, Alburnus alburnus (L.)

During a survey of myxosporean parasites of cyprinid fish in Hungary, infections caused by unknown Myxobolus spp. were found in the internal organs of rudd, Scardinius erythrophthalmus, and bleak, Alburnus alburnus . Small plasmodia developed in blood vessels of the kidney, liver, testes and intestinal wall. The parasites were studied on the basis of spore morphology and by histological and molecular methods. In most cases, plasmodia were surrounded by host tissue without a host reaction; however, in advanced cases, a connective tissue capsule was seen around plasmodia. Spores collected from the two fish species differed from each other and from the known Myxobolus spp. both in their morphology and 18S rDNA sequences. The two species, described as M. erythrophthalmi sp. n. from rudd and M. shaharomae sp. n. from bleak, are characterized by a specific histotropism to blood vessels, while the organ specificity involves the kidney and for the latter species, most internal organs.     

<Emphasis Type="Italic">Myxobolus nagaraensis</Emphasis> n. sp. (Myxozoa: Myxosporea) causes abdominal distension of freshwater goby <Emphasis Type="Italic">Rhinogobius</Emphasis> sp. OR type from the Nagara River

ABSTRACT:   A new myxosporean parasite was found in the body cavity and caudal peduncle of the freshwater goby Rhinogobius sp. Orange type (OR) collected from the Nagara River, Gifu Prefecture, Japan. Infected fish exhibited substantial swelling of the abdomen caused by large parasitic cysts approximately 10 mm in size, formed in the visceral cavity. The cyst was a compacted aggregate of several smaller cysts, similar to a bunch of grapes in appearance. Histological examination showed that plasmodia developed within the renal capsule, and finally occupied the visceral cavity. Spores were ovoid with an attenuated anterior end. Sutural ridges were conspicuous with several folds on the edge. Average spore size was 11.9 (10.5–13.5) μm long, 9.0 (8.0–10.0) μm wide, and 6.5 (6.0–7.0) μm thick. Two equal polar capsules were 5.5 (4.5–6 0) μm long and 3.0 (2.5–4.0) μm wide. Partial small subunit ribosomal DNA sequences of the myxosporean were distinct from those of other myxozoan species in GENBANK. A new species name, Myxobolus nagaraensis , is proposed for this parasite.     

不同地理群体菲律宾蛤仔壳形态对体重性状的影响研究

采集我国南北沿海9个地理群体的菲律宾蛤仔(Ruditapes philippinarum),利用通径分析等方法评估各群体形态性状对活体重和软体重的影响。结果显示,各群体壳长、壳高、壳宽和壳厚4 个形态性状对菲律宾蛤仔活体重和软体重的影响存在显著差异。除壳厚外,其他形态性状与活体重和软体重呈显著相关(P < 0.05)。通径分析和决定系数分析显示,大多数群体的壳宽对活体重和软体重的直接作用最大。2个形态比例参数(壳宽/壳长和壳高/壳长)最大值出现在山东莱州群体(0.49和0.74),而最小值出现在大连东港群体(0.42和0.67)。壳厚的变异系数最大(22.74),而壳高的变异系数最小(9.47)。通过检验偏回归系数的显著性,建立了各群体形态性状对软体重的最优回归方程。聚类分析表明,菲律宾蛤仔不同群体未出现明显的地域分布特征,而呈现出不规律的南北交替聚类现象。本研究查明了不同地理群体菲律宾蛤仔壳形态性状对体重性状的影响,为菲律宾蛤仔地理群体的形态判别、种质资源分析和遗传育种研究等提供了重要科学依据。     

不同的水煮条件对花刺参体壁形态结构和蛋白溶失的影响

研究了花刺参( Stichopus monotuberculatus) 体壁在不同水煮温度( 50 ℃、60 ℃、70 ℃、80 ℃和90 ℃) 和时间( 10 min、20 min 和30 min) 条件下的失重率、蛋白溶失率、横截面形态结构、新鲜体壁含水量和最大转变温度的变化。结果显示, 新鲜花刺参体壁含水量约为93. 67% ; 随着温度和时间的递增, 体壁水煮于50 ～60 ℃开始收缩失重, 失重率逐渐升高; 从70 ～80 ℃开始蛋白溶失率逐渐升高; 新鲜体壁最大转变温度为73. 5 ℃, 与海参体壁收缩和蛋白溶失发生温度相一致。采用扫描电子显微镜观察体壁横截面形态结构发现, 体壁胶原纤维网络密度从70 ℃开始显著升高, 并出现空洞。因此, 花刺参的水煮条件应为70 ℃加热20 ～30 min, 或80 ℃加热不超过10 min。     

Henneguya pseudorhinogobii n. sp. (Myxozoa: Myxosporea) parasitizing the gills of the freshwater goby Rhinogobius sp. OR from the Nagara River and redescription of Henneguyarhinogobii

Two types of infection with myxosporean parasites, which were different in cyst size, were found from the gill of Rhinogobius sp. OR collected from the Nagara River, Gifu Prefecture, Japan. A myxosporean forming large-type cysts in the gill filament was morphologically identified as Henneguya rhinogobii. Another myxosporean forming small-type cysts was found to be parasitic in the gill lamella. Average spore sizes of the latter myxosporean were 15.8 (range 14.2–17.8) μm in length, 5.3 (4.7–5.8) μm in width, 6.5 (5.9–7.6) μm in polar capsule length and 34.9 (25.3–42.9) μm in caudal appendage length, which were almost identical to those of H. rhinogobii. However, the two were distinguishable by the location in the gill (intrafilamental or intralamellar) and by the difference in the sequence of SSU rDNA (about 96% similarity). We propose the myxosporean forming intralamellar small-type cysts as Henneguya pseudorhinogobii n. sp. and redescribe H. rhinogobii by morphological and molecular studies.     

Commercial Bacillus probiotic supplementation of rainbow trout (Oncorhynchys mykiss) and brown trout (Salmo trutta): growth,immune responses and intestinal morphology

Probiotic administration is associated with the enhancement of host resistance to environmental and nutritional stressors, improving survival and growth rates. This study was carried out to evaluate the effects of dietary supplementation of Bacillus subtilis and Bacillus cereus toyoi on growth performance, innate immune responses and gut morphology of two trout species feeding a commercial diet high in soybean meal. A commercial probiotic (4.2 × 10⁹ CFU g^?1 of additive) was supplemented to the experimental diets at 0% (control), 0.03% (P₁; 6 × 10³ CFU g^?1 of diet) or 0.06% (P₂; 1.5 × 10⁶ CFU g^?1 of diet) and fed to brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) for 9 and 20 weeks respectively. Rainbow trout showed significantly better growth performance than brown trout, regardless of the dietary treatment. No effect of dietary probiotic supplementation was detected on growth performance, body composition or innate immune parameters (plasma lysozyme, alternative complement and peroxidase activities). In both species, after 9 weeks, intestinal lamina propria and submucosa were widened, with increased presence of inflammatory cells, significantly higher in groups fed probiotics. This inflammatory process, with villi and enterocytes noticeably damaged compared with the control group, was more pronounced in brown trout. Under the current trial conditions, the B. subtilis + B. cereus toyoi had no positive impact in either trout species, on the contrary a harmful effect was observed.     

Molecular characterization and confocal laser scanning microscopic study of Pygidiopsis macrostomum (Trematoda: Heterophyidae) parasites of guppies Poecilia vivipara

Pygidiopsis macrostomum and Ascocotyle (Phagicola) pindoramensis (Digenea: Heterophyidae) parasitize guppies as intermediate hosts and, respectively, fish‐eating mammals or birds as definitive hosts. Heterophyids have zoonotic potential, and molecular studies associated with morphological and ecological aspects have helped to clarify their taxonomy and phylogeny. Poecilia vivipara naturally parasitized by metacercariae of both species (100% prevalence) exhibit no external signs of parasitism. In this work, four new sequences of P. macrostomum (18S rDNA, 28S rDNA and ITS2 rDNA) and one new sequence of A. (P.) pindoramensis (mtDNA cox‐1) are presented. Phylogeny reconstructions linked P. macrostomum to other heterophyids, but the separation of the Heterophyidae and Opisthorchiidae remains unclear. Additionally, we used indirect immunocytochemistry and the phalloidin‐fluorescence techniques allied with confocal laser scanning microscopy to describe muscular and neuronal structures of P. macrostomum. A complex arrangement of muscular fibres is associated with the tegument, suckers, gut and reproductive system. Radial fibres around the ventral sucker are thick, branched and extend to the body wall. High‐resolution confocal imaging revealed a typical digenean muscular arrangement and important heterophyid morphological traits. These data will support future control measures to reduce the parasitism in guppies reared in fish farming systems, especially for aquarium and experimental purposes.     

The effects of constant and oscillating temperature on embryonic development and early larval morphology in longfin yellowtail (Seriola rivoliana Valenciennes)

Constant and oscillating egg incubation temperatures on embryonic development and early larval morphology were studied in longfin yellowtail (Seriola rivoliana Valenciennes). We investigated the effects of constant temperatures from 16 to 32°C on embryo development and larval morphology at hatch, and whether oscillating temperature during embryogenesis could lead to larval morphological variations. After hatching, larval morphology and development during yolk sac (YS) utilization were examined in larvae at constant temperatures and larvae at 25°C that had oscillating temperature during egg incubation. Hatching rates were > 75%, only decreasing to ~ 50% at 30°C. At constant temperatures, the largest larvae occurred at 22 and 24°C. The oscillating temperature did not affect the timing of embryo development but resulted in larger and smaller larvae with a smaller and bigger YS, respectively, with a similar hatching time. Therefore, a growth response occurred in embryos during a window of development before hatching, depending on the adaptive response to temperature (spawn‐specific). After hatching, most of the YS was absorbed within 24 hr in all treatments, and the growth of the larval head was a priority with an optimal development at 26°C. There was compensatory growth in smaller larvae resulting in similar sizes after YS utilization, but larvae showed variations in body structure that could be important in further aquaculture research.     

橄榄蛏蚌血细胞形态及吞噬能力的初步研究

对橄榄蛏蚌(Solenaia oleivora)血细胞的形态特征和吞噬作用进行了初步研究。基于瑞氏染色后血细胞的形态、颜色、大小等特征,可将橄榄蛏蚌的血细胞分为大颗粒细胞、小颗粒细胞、透明细胞、类淋巴细胞共4种类型。透明细胞的直径最大,小颗粒细胞次之,类淋巴细胞最小;透明细胞的胞核直径最大,小颗粒细胞次之,大颗粒细胞最小。类淋巴细胞的核质比最大(0.67),其他3种细胞的核质比为0.37～0.48。4种血细胞所占比例以小颗粒细胞最高(43.6%),透明细胞次之(36.6%),类淋巴细胞最低(7.0%)。橄榄蛏蚌平均血细胞浓度为(4.21±1.51)×105个/mL,个体的血细胞浓度与体重(或壳长)不相关(P>0.05),不同体重组(或壳长组)的平均血细胞浓度无显著差异(P>0.05)。在37℃体外吞噬试验中,橄榄蛏蚌血细胞对金黄色葡萄球菌的吞噬比例为(43.6±7.0)%,吞噬指数为(0.743±0.145)。4种血细胞中,小颗粒细胞的吞噬比例最大(0.288±0.061),类淋巴细胞不具吞噬能力。     

舟山近海黄姑鱼胚胎发育及前期仔鱼形态的观察

通过对黄姑鱼胚胎发育全过程的连续观察,描述了黄姑鱼胚胎发育的特征、发育速度及前期仔鱼的形态特征等。观察结果表明,黄姑鱼的胚胎发育与其它硬骨鱼浮性卵鱼类基本相似,属端黄卵,盘状分裂。在水温22.0~22.5℃、盐度26.5的条件下,黄姑鱼受精卵经24h15m in孵出仔鱼。初孵仔鱼全长1.75±0.09mm,孵出后约50h,卵黄囊完全被吸收。     

Habitat utilization and vertical movements of white marlin (Tetrapturus albidus) released from commercial and recreational fishing gears in the western North Atlantic Ocean: inferences from short duration pop-up archival satellite tags

Researchers have applied numerous techniques to improve billfish stock assessments, including habitat‐based models that incorporate behavioral and oceanographic parameters to standardize historical catch‐per‐unit‐effort time‐series data. These methods have allowed researchers to account for significant changes in the depths of pelagic longline (PLL) gear deployments over time. This study presents habitat‐use data recovered from high‐resolution 5‐ and 10‐day pop‐up satellite archival tags (PSATs) attached to 47 surviving white marlin released from commercial and recreational fishing gears offshore of the U.S. East Coast, the northern Caribbean, and Venezuela between 2002 and 2004. Data recovered from transmitting tags indicated that white marlin spent nearly half of their time associated with warm, near‐surface waters (<10 m). All fish displayed frequent short duration (mean: 39.8 min) vertical excursions from surface waters to depths averaging 51 m. Qualitative and multivariate classifications of data from completely transmitted movements of surviving white marlin revealed two major types of descents: one pattern was characterized by deep ‘V’‐shaped excursions of relatively short duration (mean: 23.4 min) while the other featured descents that were more broadly ‘U’‐shaped and confined to a specific depth range for an extended period of time (mean: 75.8 min). Based on the frequency, persistence, and patterns of these vertical movements, white marlin appear to direct a considerable proportion of foraging effort well below surface waters, a behavior that may account for relatively high catch rates of white marlin on some deep‐set PLL deployments.