养殖条石鲷消化道形态构造及组织学观察

应用解剖学和光学显微镜检术研究了养殖条石鲷（Oplegnathus fasciatus）消化道的形态学和组织学特性。条石鲷消化道包括口咽腔、食道、胃、幽门盲囊和肠。条石鲷口咽腔较大,颌齿愈合成鹦鹉喙嘴状,舌半椭圆形。食道较短,显微组织可分为4层：粘膜层、粘膜下层、肌肉层和浆膜层,上皮含有极少量杯状细胞和黏液分泌细胞。胃膨大,呈V形,和食道一样分为4层,粘膜层表皮下含有发达的胃腺。小肠组织分为3层：粘膜层、肌肉层和浆膜层。直肠组织结构类似于小肠。小肠和直肠上皮中均分布有较多的杯状细胞。文章对养殖条石鲷消化道的形态学和组织学特征与其食性的适应关系作了探讨。     

南海区驯养条石鲷亲鱼的初次性成熟和产卵

2008年4月初在广东省饶平县对2006年5月人工孵化和培育的2龄条石鲷（ Oplegnathus fasciatus）成熟亲鱼进行激素诱导,研究和观察条石鲷亲鱼初次性成熟的繁殖生物学。结果显示,在南海区全人工养殖的条石鲷亲鱼初次性成熟年龄为2龄,成熟亲鱼的最小型为全长245 mm、体质量610 g,最大个体为全长300 mm、体质量1450 g;产卵季节为4月10日~7月15日,产卵盛期为4月中旬至6月下旬;为升温产卵型鱼类,产卵温度为20.0~28.8℃,适宜的产卵温度为20.7~27.6℃;雌、雄亲鱼发育同步,个体大小和成熟年龄差别不大;雌鱼属于一年一次分批产卵类型,产卵期超过3个月;24尾初次性成熟雌性亲鱼的总产卵量为3180.0×104粒,日最高产卵量为341.5×104粒;其产卵量和受精卵质量与水温的变化关系密切,受精卵的浮卵率随温度的变化而变化,在水温相对衡定时,浮卵率相对稳定并维持在80%~95%的较高水平;受精卵平均卵径为（0.860±0.023）mm,油球径为（0.191±0.009）mm。     

Studies on epizootic iridovirus infection among red sea bream,Pagrus major (Temminck & Schlegel), cultured in Taiwan

Since 1993, an epizootic viral disease has occurred in net-cage cultured red sea bream, Pagrus major (Temminck & Schlegel), in Peng-hu Island located on the south-western coast of Taiwan. The diseased fish exhibited abnormal swimming and were lethargic, but few visible external signs were observed. The cumulative mortality because of the disease sometimes reached 50-90% over 2 months. Histopathogical studies of the affected fish showed enlarged basophilic cells in the gill, kidney, heart, liver and spleen. These necrotic cells were Feulgen-positive and stained blue using Giemsa. Transmission electron microscopy revealed icosahedral virions in the cytoplasm of the necrotic cells. The viral particles consisted of a central nucleocapsid (75-80 nm) and envelope, and were 120-150 nm in diameter. These results suggest that the virus belongs to the Iridoviridae. Using polymerase chain reaction (PCR), approximately 570 bp fragments were produced from the viral DNA using as a template 1-F and 1-R primers derived from red seabream iridovirus (RSIV) from red sea bream in Japan. Similar results were also obtained using nested-PCR with different primer sets (1-F, 2-R and 2-F, 1-R). Although the size and some features of epizootics of this virus differed from RSIV in Japan, it shows close genetic affinities with the latter and it is suggested that RSIV has been introduced to Taiwan.     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

Establishment and characterization of a fibroblast cell line derived from the dorsal fin of red sea bream,Pagrus major (Temminck & Schlegel)

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF‐2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 °C in Leibovitz L‐15 medium with 10% foetal bovine serum. Propagation of RSBF‐2 cells was serum dependent and exhibited low plating efficiency (<1.7%). Aside from long‐term cryopreservation, the cells could also be kept at 4 °C for 72 days. The distribution of the chromosome number was 38–98 with a mode of 48. The RSBF‐2 cell line was susceptible to red sea bream iridovirus but only produced a few rounded and refractory cells. Virus‐inoculated RSBF‐2 cells were then subcultured to generate a persistently infected cell line. RSBF‐2 was also very sensitive to the extracellular products of Photobacterium damselae ssp. piscicida and produced significant fluorescent signals after transfection with pEGFP‐C3. Analysis of mitochondrial cytochrome b gene sequences revealed 99% identity between the cell line and Pagrus major.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Effects of dietary water content on meal size, daily food intake, digestion and growth in turbot, Scophthalmus maximus (L.)

When fed once daily with wet squid, turbot (30–50 g) accustomed to dry pellets require many days to increase intake to meet their feed requirement (≈ 10 mg dry matter g⁻¹ bw meal⁻¹). Adaptation takes 1–2 days if several daily feedings are given. With dried squid, they ingest about 20% of the wet squid bulk because the stomach contents expand when moisturised. In contrast, turbot eat enough wet squid to fill most of the available stomach volume (≈ 7.6 mL 100 g⁻¹ bw). When presented in gelatine capsules, food water content is masked and does not affect the volume ingested. Moistening the contents shortens the delay before gastric emptying starts to one-third (0.6 h) compared with dry food (1.9 h). Daily dry-matter intake increased when dry contents were moistened but only if two or more meals were offered per day. Turbot adapt their digestion to supply water for dry diets but this may add extra metabolic costs. When offered 20 mg dry matter g bw⁻¹ day⁻¹, divided into four equal meals, turbot grew faster and more efficiently with moist than with dry squid. Protein, energy and dry-matter digestibilities were also enhanced. The increased daily protein absorption did not increase ammonia release, indicating that the extra protein was used for somatic growth.     

Effects of bicarbonate ions and pH on acquisition and maintenance of potential for motility in ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), spermatozoa

The effects of extracellular ions on the acquisition and maintenance of the potential for motility in the spermatozoa of the ayu, Plecoglossus altivelis Temminck et Schlegel (Osmeridae), were examined. Testicular spermatozoa and milt spermatozoa were obtained from fully matured males and diluted with buffered solution (BS, 20 mM HEPES–NaOH, pH 7.5). Testicular spermatozoa showed a significantly low rate of motility (0.8 ± 0.4%), whereas milt spermatozoa showed a high rate (89.4 ± 2.1%). The spermatozoa were incubated with various isotonic media for 2 h, diluted with BS, and changes in the rates of motility were then compared. When incubated for 2 h with artificial seminal plasma (ASP), corresponding in terms of ionic constituents to seminal plasma buffered at pH 8.0, both spermatozoa showed a high rate of motility. Testicular spermatozoa acquired and milt spermatozoa maintained the potential for motility in response to the HCO₃^– ion concentrations (between 0 and 20 mM) in the ASP. The differences in the pH of the ASP had a significant effect on the acquisition and maintenance of the potential for motility, and spermatozoa showed the highest rate of motility with the ASP at pH 8.0 and 8.5. These results suggest that the quality of milt in the ayu can be regulated by controlling the concentration of bicarbonate and the pH of the incubating media.     

Ultrasonic immunization of sea bream, Pagrus major (Temminck & Schlegel), with a mixed vaccine against Vibrio alginolyticus and V. anguillarum

In order to clarify the effectiveness of ultrasonication on vaccine delivery, juvenile sea bream, Pagrus major , were treated with eight different ultrasonic methods. A mixed vaccine against Vibrio alginolyticus and V. anguillarum was used to immunize the fish . The intensity and frequency of the ultrasound were 280 mW cm^–2 and 35 kHz, respectively. The ultrasonic methods included continuous or pulsed ultrasound for 3 min, and continuous or pulsed ultrasound for 3 min before and/or after immersion for 3 min. Of all the eight ultrasonic methods tested, `pulsed ultrasound followed by immersion' and `immersion, pulsed ultrasound, and followed by immersion again' provided the best protection, which were comparable with protection of fish immunized by intraperitoneal injection. Moreover, the convenience of applying these two ultrasonic methods for immunization was comparable with the immersion method and was much better than intraperitoneal injection. If 2 × 10⁸ CFU mL^–1 of this mixed vaccine was used for vaccination repeatedly five times by ultrasonic methods, it could still produce good protection for the immunized sea bream. Therefore, the ultrasonic method is an effective and practical approach for fish vaccination on a large scale.     

Stimulation of growth performance without causing stress response in young red sea bream, Pagrus major (Temminck & Schlegel), by photoperiod manipulation

Three photoperiods (12L:12D, 16L:8D and 24L:0D) were used to investigate the growth performance and stress response in red sea bream, Pagrus major (body weight 200–400 g). Fish were fed a commercial diet to apparent satiation, two times a day for 8 weeks. Fish exposed to a 24L:0D photoperiod showed a significantly higher weight gain (%) than those exposed to other photoperiods (P<0.05). The best specific growth rate and feed conversion efficiency were achieved at 24L:0D and 16L:8D, without significant differences among them. Although fish exposed to 16L:8D showed a significantly higher plasma level of cortisol than those exposed to other photoperiods in the third week, there was no major variation in the cortisol level among the treatments either at the sixth week or at the end of this study. There were no significant differences either in the haematocrit level or the plasma levels of glucose, total cholesterol and total protein among the treatments during this study. The results revealed that the growth performance of red sea bream reared from 200 to 400 g can be stimulated significantly using a continuous (24L:0D) photoperiod without any measurable significant stress response in fish.     

Growth and production performance of threatened snakehead fish,Channa striatus (Bloch), at different stocking densities in earthen ponds

Production trials of threatened snakehead fish (Channa striatus) were carried out under different stocking densities in earthen ponds of Bangladesh. The average weight and length of the fingerlings during stocking was 17.63 ± 1.23 g and 13.21 ± 0.52 cm. Fingerlings were stocked at 5000 ha^?1 in treatment‐1 (T₁), 6250 ha^?1 in treatment‐2 (T₂) and 7500 ha^?1 in treatment‐3 (T₃) respectively. Fish in all the experimental ponds were fed with supplementary feed comprising of fish meal (30%) and mustard oilcake (70%) at the rate of 3–6% of estimated body weight two times per day. In addition, trash fish were supplied at the rate of 2–3% of the estimated biomass on each alternate day. In situ water quality parameters of the pond were within the suitable range for fish culture. The growth and survival of fingerlings were significantly higher in T₁ than in T₂ and T₃. The food conversion ratio was significantly lower (P<0.05) in T₁ than in T₂ and T₃. The estimated gross and net production of fish was higher in T₁, followed by T₂ and T₃. Overall, the highest growth, survival and production were obtained from T₁. Therefore, it could be concluded that of 5000 fingerlings ha^?1 is the most suitable stocking density for culturing C. striatus under a monoculture system in the earthen ponds for better production.     

Perkinsus sp. infestation in carpet-shell clams, Ruditapes decussatus (L), along the Portuguese coast. Results from a 2-year survey

This study characterises Perkinsus atlanticus disease prevalence and intensity within the economically important beds of the clam Ruditapes decussatus along the Iberian Atlantic coast. Samples were collected from five different sites along the Portuguese coast (Ria Formosa, Ria do Alvor, V.N. Milfontes, Aveiro, Lagoa de Obidos) and in Galicia (Ria de Arosa) between winter 2000/2001 and 2002/2003. The infection level was evaluated by the Ray Fluid Thioglycollate medium (RFTM) method using the body burden assay. In addition, the measure of the condition index (CI) (percentage between the edible part and the total weight of the clams) was conducted to investigate a possible correlation between this parameter and the intensity of the infection. However, no clear relationship between infection intensity and condition index (CI) could be determined although a significant decline in CI was found for heavily infected clams. Results of a 2-year survey demonstrate the presence of Perkinsus sp. in all sampling sites albeit with different intensities. Sites where human interference was minimal showed the lowest levels of infection. There were no significant differences in Perkinsus sp. infection intensity between samples collected in winter and summer, in contrast with several studies describing higher intensities at the end of the summer. Nevertheless, major differences were observed from year to year and site to site, indicating that factors other than those responsible for seasonal climatic variations might affect the prevalence and the intensity of Perkinsus sp. infection.     

Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue

A nodavirus was isolated from diseased yellow grouper, Epinephelus awoara , larvae cultured in southern Taiwan. The histopathology and RT–PCR results confirmed that it was a fish nodavirus; its coat protein gene sequence was similar to that of red spotted grouper nervous necrosis virus (RGNNV) and it is named yellow grouper nervous necrosis virus (YGNNV). A new nodavirus-susceptible cell line, grouper brain (GB) was established and characterized from the brain tissue of yellow grouper. The GB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum at temperatures between 24 and 32 °C, and have been subcultured more than 80 times, becoming a continuous cell line. The GB cell line consists of fibroblast-like cells and some epithelioid cells. The cell line yielded titres of YGNNV up to 10^8.5 TCID₅₀ mL^–1. The GB cells effectively replicated the virus at 28 °C, which could be purified to homogeneity by caesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 25–30 nm in diameter. The cytoplasm of infected cells was filled with aggregates of virus particles. These results indicate that the GB cell line is a significant tool for the study of fish nodaviruses.     

Development and characterization of a fibroblastic‐like cell line from caudal fin of the red‐line torpedo,Puntius denisonii (Day) (Teleostei: Cyprinidae)

A fibroblastic‐like cell line was established from the ornamental fish, red‐line torpedo (Puntius denisonii). The red‐line torpedo fin (RTF) cell line is being maintained in Leibovitz's L‐15 medium supplemented with 10% fetal bovine serum (FBS) for over 1 year at 28 °C on a continuous basis in normal atmosphere. The growth rate of RTF cells increased as the FBS proportion increased from 5% to 20% at 28 °C with optimum growth at the concentrations of 10% FBS. The morphology of RTF cell was predominantly fibroblastic like. Propagation of these cell lines was serum dependent, with a low plating efficiency (<15%). Karyotyping analysis of RTF cells at the 25th passage indicated that the modal chromosome number was 2n=50. The cell line was cryopreserved in liquid nitrogen at ?196 °C and could be recovered from storage after 6 months with good cell viability. Polymerase chain reaction amplification of a fragment of two mitochondrial genes, 16S rRNA and CO1, confirmed the identity of these cell lines with those reported from this animal species, confirming that the cell lines originated from P. denisonii. The bacterial extracellular products from Vibrio cholerae MTCC3904 and Aeromonas hydrophila were found to be toxic to RTF. The cell lines were not susceptible to viral nervous necrosis virus, a marine fish virus.     

Lesions associated with plerocerci (Platyhelminthes: Cestoda: Trypanorhyncha) in the gastric wall of a cownose ray, Rhinoptera bonasus (Mitchill), (Myliobatiformes: Rhinopteridae) from the northern Gulf of Mexico

We describe lesions associated with a seemingly intense infection of trypanorhynch plerocerci (Platyhelminthes: Cestoda: Trypanorhyncha) in the gastric wall of a female cownose ray, Rhinoptera bonasus (Myliobatiformes: Rhinopteridae) captured in the northern Gulf of Mexico. Grossly, the multitude of encapsulated, encysted plerocerci imparted a bumpy and cobbled appearance to the serosa of the stomach, and none was observed in any other tissue during routine parasitological necropsy. Histologically, the plerocerci were associated with severe intramural granulomatous gastritis, vascular ectasia and mesothelial polyposis with the exclusion of the mucosa. To our knowledge, this is the first published case study documenting platyhelminth-associated histopathological changes in the gastrointestinal tract of R. bonasus as well as that of the efficacy of immunocytochemical markers for smooth muscle actin, Factor VIII, S-100, and proliferating cell nuclear antigen in Myliobatiformes. It also may serve as a potential primer for much needed ecological investigations regarding the potential role of elasmobranchs as intermediate or 'paratenic' hosts in the life cycles of trypanorhynch cestodes.     

Pancreas disease (PD) in sea‐reared Atlantic salmon,Salmo salar L., in Norway; a prospective,longitudinal study of disease development and agreement between diagnostic test results

A prospective longitudinal study was performed on three cages at each of three Norwegian Atlantic salmon seawater sites that experienced outbreaks of pancreas disease (PD). Once salmonid alphavirus (SAV) ribonucleic acid (RNA) was detected by real‐time RT‐PCR (Rt RT‐PCR) at a site, it became detected in all studied cages and was persistently found until the end of the study period up to 19 months after first detection. SAV‐specific antibodies were detected at all sites until the end of the study period and were also found at a high prevalence in broodfish at the time of stripping. No evidence of increased viral activity was detected in these broodfish. One site tested negative over several months prior to the first detection of SAV by Rt RT‐PCR and SAV‐specific antibody, which occurred 1 month prior to clinical manifestations of PD. Moribund fish or thin fish/runts that were sampled after the first PD diagnosis had almost twice the risk of testing positive by one or more diagnostic tests compared to that of randomly selected apparently healthy individuals. This paper describes the first detailed investigation of the disease development of PD at site and cage level in Norway, as well as an assessment of the performance and agreement of the commonly used diagnostic tests.     

Incidence of white muscle disease, a viral like disease associated with mortalities in hatchery-reared postlarvae of the giant freshwater prawn Macrobrachium rosenbergii (De Man) from the south-east coast of India

Incidence of post‐larval mortalities of 30–100% was reported from commercial freshwater prawn, Macrobrachium rosenbergii (De Man) hatcheries in Andhra Pradesh and Tamil Nadu (south‐eastern states of India) since 2001. Infected postlarvae (PL) exhibited clinical symptoms with lethargy, anorexia and whitening of abdominal muscles and the disease was identified as white muscle disease (WMD). The waterborne infection of WMD was induced in the laboratory by exposing uninfected and healthy M. rosenbergii PL to the filtered muscle homogenates of the naturally infected PL, resulting in mortality that reached 99% within 10 days post infection. Histopathological examination of the infected animals revealed highly necrotic musculature. Degenerated muscle areas showed aggregations of melanized nuclei, many of which looked like inclusion bodies. Bacteriological examination of affected PL showed the presence of Staphylococcus spp. as a predominant organism, while laboratory challenge of healthy PL with this bacterial isolate did not reproduce WMD.