Microinjection of the antifreeze protein type III (AFPIII) in turbot (Scophthalmus maximus) embryos: Toxicity and protein distribution

Fish embryo cryopreservation has not been achieved. Different methods and alternative cryoprotective agents (CPAs) should be explored in order to succeed in this purpose. Antifreeze proteins (AFPs) are naturally expressed in sub-arctic fish species, and they inhibit the growth of ice crystals as well as recrystallization during thawing. Therefore, their introduction into embryos can be highly beneficial for vitrification purposes. In this study, AFP type III was introduced into turbot embryos, by microinjection into the yolk sac and the perivitelline space at F stage (tail bud).Toxicity and distribution of protein in microinjected embryos were established before testing the protein effect on embryo cryopreservation. AFP-FITC distribution within the embryo was analyzed by confocal microscopy at 5 min and 24 h after microinjection in F stage embryos. To test the sensitivity of microinjected embryos to CPAs, embryos were subjected to a protocol for the incorporation of a vitrifying solution that was specially designed for turbot embryos. Hatching rates after CPA incorporation were determined. Results indicate that embryos at late developmental stages are more resilient to microinjection, with embryo survival rates between 60 and 82%. Confocal microscopic images demonstrated that the protein was homogeneously distributed within the microinjected embryo compartment, but did not enter any other compartment. On the other hand, microinjected embryos successfully surmounted their incubation in the CPAs. This study explores new alternatives for cryopreservation suggesting the use of natural cryoprotectants (AFPs) in the protection of intra-embryo compartments, which are usually unprotected with the conventional cryopreservation protocols for fish embryos.     

Susceptibility of marine fish species to a megalocytivirus, turbot iridovirus, isolated from turbot, Psetta maximus (L.)

Turbot iridovirus (TBIV), a member of the genus Megalocytivirus in the family Iridoviridae, was isolated from diseased turbot, Psetta maximus (L.), in Korea in 2003. In this study, experimental infection of turbot, Japanese flounder, Paralichthys olivaceus (Temminck & Schlegel), and rock bream, Oplegnathus fasciatus (Temminck & Schlegel), with TBIV was performed to evaluate the viral susceptibility of these fish species. After virus exposure, the mortalities of turbot reared at 22 and 25 degrees C were 60% and 100%, respectively, suggesting that TBIV is the causative agent of the mass mortality of turbot that occurred in Korea in 2003. Moreover, TBIV was detected in Japanese flounder and rock bream by polymerase chain reaction after experimental infection (26 days post-inoculation) despite no viral pathogenicity in these fish, suggesting that these two fish species are also susceptible to the virus. It is possible that horizontal transmission of TBIV occurs among these three fish species because turbot is routinely cultured with Japanese flounder and rock bream in Korea.     

大菱鲆受精卵显微注射技术的建立

本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。     

Comparative composition and shelf-life of fillets of wild and cultured turbot (Scophthalmus maximus) and Atlantic halibut (Hippoglossus hippoglossus)

Turbot and Atlantic halibut are highly valued fish species. However,very little is known about fillet shelf-life characteristics associated withboth species. Thus, fillet -tocopherol content and proximate compositionof wild turbot (1.5 kg) and Atlantic halibut (1.1 kg)caught off the south coast of Ireland and the north-west coast of Iceland,respectively, were investigated. In addition, the susceptibility of fillets, storedunder retail conditions, to lipid oxidation and colour change was studied.Proximate composition analysis showed that turbot had significantly highermoisture (P < 0.001) and lower protein (P < 0.001) contents compared toAtlantic halibut. Atlantic halibut incorporated significantly higher (P <0.001) levels of -tocopherol into fillets than turbot. Over 14 days ofstorage on ice, fillets from Atlantic halibut exhibited significantly lower (P =0.020) levels of lipid oxidation than those of turbot. However, malondialdehyde(MDA) concentrations were generally very low, never exceeding 0.6 gg^–1 fillet. Turbot maintained a significantly higher (P< 0.001) pH over the storage period. The lightness (L^* values) offillets from both species increased over 14 days of storage, but wassignificantly higher (P < 0.001) in Atlantic halibut than in turbot. Turbotdeveloped a relatively intense yellow colour during storage (decrease in hueangle and increase in b^* values), whereas this was not the case forAtlantic halibut. The results of this study demonstrate that fillets of wildAtlantic halibut stored on ice, were less prone to lipid oxidation anddiscolouration than those of wild turbot. However, quality changes in turbotwere very small showing that both fish have tremendous shelf-life capacities interms of lipid oxidation. These findings are considered in the context of knownmaterial for farmed fish.     

应用高通量测序技术分析大菱鲆幼鱼肠道及其养殖环境的微生物群落结构

采用基于Illumina测序平台的高通量测序技术,对大菱鲆(Scophthalmus maximus)幼鱼肠道及其养殖水体、生物饵料中细菌种类及丰度进行研究。测序结果显示,养殖水体、生物饵料和大菱鲆幼鱼肠道等19个样品共获得有效序列547621条,可聚类于3771个可分类操作单元(OTUs),归属于养殖水体、生物饵料、健康幼鱼和发病幼鱼的操作分类单元(OTU)个数分别为3038、1090、87和777,其中,健康幼鱼与生物饵料、健康幼鱼与养殖水体特有的OTU个数分别为57和0,发病幼鱼与生物饵料、发病幼鱼与养殖水体特有的OTU个数分别为481和31。表明幼鱼肠道微生物多样性与生物饵料密切相关。根据细菌注释结果,拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)在大菱鲆幼鱼肠道中占优势地位,其中,健康幼鱼肠道微生物共聚类为8个门,发病幼鱼的肠道微生物可聚类为19个门。与健康幼鱼相比,发病幼鱼肠道门水平上的3种主要优势菌群落结构出现失衡。此外,对各样品中丰度最高的100位OTU分析显示,幼鱼肠道优势菌种类与生物饵料中的优势菌种类密切相关,而每个发病幼鱼肠道优势菌种类具有一定的独立性。本研究旨在为大菱鲆健康养殖和微生态调控提供实验依据。     

Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration

The structure of the chorion and plasma membranes of gastrula‐stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5–2.5 μm in thickness and to consist of three layers, electron‐dense outer and innermost layers (0.2–0.3 and 1.0–1.6 μm in thickness respectively) separated by an electron‐lucent middle layer (0.3–0.6 μm in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula‐stage embryos, and FE‐SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE‐SEM and TEM studies reveal details of the syncytial layer (1–4 μm thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.     

A production season of turbot larvae Scophthalmus maximus (Linnaeus, 1758) reared on copepods in a Danish (56°N) semi‐intensive outdoor system

Turbot were reared from yolk sack larvae to juvenile in an outdoor semi‐intensive system. Three production cycles were monitored from May to September. A pelagic food chain was established with phytoplankton, copepods and turbot larvae. Abiotic and biotic parameters of lower trophic levels together with turbot larval survival, development, prey electivity and growth were monitored. A decreasing larval survival from 18.4% in May to 13.6% in July and just 7.0% in September was observed. The overall phytoplankton and copepod abundance decreased during the productive season. The turbot larval growth showed significant differences between larvae below (isometric) and above (allometric) 7 mm. Larval fish gut content showed no differences with available prey between production cycles. Therefore, it appears that the available prey concentration is governing their growth in this outdoor system. First‐feeding turbot larvae exhibited active selection for nauplii whereas developed larvae switched to copepodites and adult copepods. Although developing turbot larva exhibited active selection towards copepod size classes, there was no evidence of selective feeding on either of the two dominant copepod species. The turbot larvae's prey ingestion was modelled together with the standing stock of copepod biomass. The model results indicated that the estimated need for daily ingestion exceeded the standing stock of copepods. Hence, the initially established food web was unable to sustain the added turbot larvae with starvation as a consequence. We therefore suggest several solutions to circumvent starvation in the semi‐intensive system.     

Experimental infection of turbot, Scophthalmus maximus (L.), by Moritella viscosa, vaccination effort and vaccine-induced side-effects

Moritella viscosa is the causative agent of winter ulcers in farmed salmonids and Atlantic cod in countries around the North Atlantic. The bacterium has also been isolated from various marine fish species. Bacterial diseases have been a limiting factor in farming of turbot, but M. viscosa has not so far been isolated. In this study, turbot was shown to be sensitive to M. viscosa infection in experimental challenges. Pathological changes in infected turbot were comparable with those previously described for winter ulcers in salmon. A multivalent commercial salmon vaccine, containing M. viscosa as one of five antigens and a mineral oil adjuvant, did not protect turbot against challenge 13 weeks post-vaccination. Weight gain of vaccinated turbot compared with controls was not reduced 7 weeks post-vaccination. Vaccination did not induce a specific anti-M. viscosa response, while elevated anti-M. viscosa antibody levels were detected both in vaccinated and unvaccinated fish 5 weeks post-challenge. The vaccine did, however, induce an antibody response against Aeromonas salmonicida, another vaccine component. Minor intra-abdominal adhesions were detected in vaccinated fish and fish injected with a mineral oil adjuvant. The measurement of various innate humoral immune parameters did not reveal significant differences between vaccinated and control groups.     

大菱鲆"温室大棚+深井海水"工厂化养殖模式

大菱鲆Turbot(Scophthalmus maximus）的引进一方面为我国北方沿海工厂化养鱼增加了一个新的品种，另一方面又创立了“温室大棚＋深井海水”工厂化养殖的新模式，大大丰富了工厂化养鱼的内容，有效的推动了我国海水养殖“第四次浪潮”的形成与发展。本文综述了大菱鲆温室大棚式工厂化养殖模式的养殖技术，以促进大菱鲆养殖业的健康、持续、稳定发展。     

Performance of outdoor seawater treatment systems for recirculation in an intensive turbot (Scophthalmus maximus) farm

Water treatment systems are mandatory in recirculating aquaculture facilities facing existing regulations, but data on system efficiency, especially for marine species, are scarce. The present work aimed at contributing to the evaluation of the effluent characteristics and the performance of a combined outdoor biological and non-biological treatment system in an intensive turbot (Scophthalmus maximus) farm, operating under different hydraulic regimes. A preliminary study on the biofilter bacterial populations was also undertaken. Changes in effluent characteristics with pumping, season of the year and fish biomass were observed. The treatment system showed performance instability under the conditions assayed (outdoors, changeable recycle rates). Maximum removal of solids was observed in winter, with microscreen or biological filtration (up to 60%) and nitrite removal (40–98%) was achieved with ozonation. Reduction in ammonium levels was higher in summer, either mechanically (74%) or biologically (33%). Phosphate removal was higher in winter with both systems (37 and 60%, respectively). Compliance with Portuguese discharge standards was achieved. For improvements in the treatment loop, further studies on biofilter bacteria under outdoor conditions are needed, and biological denitrification is encouraged. This revised version was published online in August 2006 with corrections to the Cover Date.     

The effect of ammonia on the growth of juvenile Dover sole, Solea solea (L.) and turbot, Scophthalmus maximus (L.)

The growth of sole and turbot was followed in groups of fish exposed to constant unionised ammonia concentrations at pH levels both at and below that of normal sea water. Low pH levels were produced by the addition of carbon dioxide. Results for both species showed evidence for a threshold level of unionised ammonia below which little or no effect on growth was evident. This level was determined to be 0.066 mg N/l for sole and 0.11 mg N/l for turbot at 16°C, 34‰ salinity. The different pH conditions did not have any marked effect on these threshold levels.Above the threshold levels growth appeared to be depressed in a linear manner with increased unionised ammonia. The rate at which growth was depressed was greatest at the low pH levels for both species. The level at which growth ceased thus decreased from 0.77 to 0.38 mg N/l for sole and from 0.9 to 0.3 mg N/l for turbot, over the pH range from 7.9 to 6.9.The absence of disease and gill damage in the experimental populations is discussed. It is suggested that this and the tolerance shown by the fish, which is higher than is generally accepted for fish in high density culture, may be due to the clean environment in which the fish were held while under test.     

Ocular bacterial signatures of exophthalmic disease in farmed turbot (Scophthalmus maximus)

As is the case at other sites in the body (e.g. the gut, skin and mouth), the ocular microbiota plays a crucial role in their host, as disturbances of the composition and function of the ocular microbiota are known to be associated with ocular disorders. Exophthalmic disease (ED) is a significant cause of high mortality in fish species, including farmed turbot (Scophthalmus maximus). However, the relationship between alterations in the ocular microbiota and ED in turbot is unclear. In this work, we collected turbot samples from farmed ponds with ED and healthy samples to understand changes in the ocular microbiota of turbot suffering from ED. We compared the structural and metabolic differences of ocular bacterial communities from farmed turbot with exophthalmic disease and those of healthy controls. Besides less microbial diversity found in turbot with ED regarding the control group, we also found that Aeromonas was the dominant bacteria both in controls and ED samples, but the abundance of Aeromonas was significantly greater in ED individuals. Moreover, the results of correlation test further suggest that Aeromonas overgrowth was correlated with the progress of the disease and shifts in ocular microbiota functional pathways in turbot. These findings emphasize that an increased abundance of Aeromonas serves as an ocular bacterial signature associated with ED in turbot, which provide basic information useful for diagnoses, prevention and treatment of ocular diseases occurring in cultured fish.     

Experimental infection of turbot, Psetta maxima (L.), with strains of viral haemorrhagic septicaemia virus isolated from wild and farmed marine fish

The susceptibility of turbot, Psetta maxima, to infection with two strains of viral haemorrhagic septicaemia virus (VHSV) obtained from wild Greenland halibut, Reinhardtius hippoglossoides, and from farmed turbot was examined. A marine VHSV strain known to be highly pathogenic for turbot was also utilized for comparative purposes. Fish were infected by intra-peritoneal (i.p.), immersion or cohabitation, and maintained at two different temperatures (8 and 15 degrees C). Infection trials showed that the three VHSV isolates were pathogenic for turbot fingerlings by i.p. injection at both temperatures, with high levels of mortality. Virus was recovered from most pools of dead fish i.p. challenged, but not from surviving fish. Although clinical signs were not induced following waterborne exposure, viral growth was obtained from some pools of surviving fish challenged by immersion with strain GH40 from Greenland halibut, which indicates that the virus can survive in sea water and infect other fish via horizontal transmission. Furthermore, although low, the clinical signs and mortality observed in fish cohabitating with turbot challenged with strain GH40 confirms horizontal transmission and indicates that the passage through fish increases the virulence of this strain for turbot. These findings indicate that Greenland halibut, as other wild fish, may play an important role in the epizootiology of VHSV and suggest a potential risk for the turbot farming industry.     

Feeding at different illumination levels in larvae of three marine teleost species: cod, Gadus morhua L., plaice, Pleuronectes platessa L., and turbot, Scophthalmus maximus (L.)

Abstract. Optimal illumination levels for feeding vary in different fish larvae according to feeding strategy, prey type and habitat. Determining optimal illumination conditions is important in aquaculture, as illumination seems to be the dominating factor affecting the distribution of many pelagic fish larvae. Cod, Gadus morhua L., plaice, Pleuronectes platessa L., and turbot, Scophthalmus maximus (L.), have all been considered for aquaculture, and have different vertical positioning strategies. Three parallel groups of larvae of each of these species were exposed to planktonic feed organisms at seven illumination levels ranging from darkness to 10 800 lux. The number of ingested organisms was counted. Cod had maximal feed uptake at 1·0 lux, and higher uptake at 0·1 lux than at 87 lux. Plaice reached a threshold level at 87 lux, and turbot at 860 lux. Cod larvae, which are slowly searching dusk/ dawn predators, preferred very low illumination levels. Plaice larvae, being constant swimmers with a vertically orientated behaviour, fed equally well over a wide illumination range. The near-surface-feeding turbot larvae preferred high illumination levels.     

复合有益菌剂对工厂化大菱鲆育苗水净化效果研究

On the basis of purifying effect of single strain probiotic bacteria on water of intensive turbot breeding, the purifying effect of multistrain bacteria product was studied. The results of experiments showed that the free multistrain mixed bacteria had better purifying effect on turbot breeding water than that of any singlestrain bacteria. this phenomenon can be explained with the principle of “ biopump”. The immobilized multistrain probiotics product, biofilm, also showed excellent purifying effect on wate r of turbot breeding. The removal rates of COD and ammonia in 5 days were up to 73.1% and 80.0%, respectively. The concentration of nitrite kept low, which indicated most ammonia had been transformed into nitrate and accumulation of harmful intermediate productnitrite was decreased. This paper supplied theoretical and technical foundation for saving sea water through biopurification of wastewater from marine fish culture.     

Effects of size grading on growth and survival of juvenile turbot at two temperatures

Juvenile turbot were size graded into three size groups (mean initial size): Small (3.4 g), medium (7.0 g) and large (10.5 g), and additional fish were held in ungraded (6.6 g) groups. Subgroups (n = 396) of fish were tagged and individual growth rates and social interactions within different size categories were studied in fish reared at 13 and 19 °C. Size grading of juvenile turbot did not improve growth. Specific growth rates (SGR) were mainly size-related, and no differences in SGR or mortality between the experimental groups at both tem-peratures were found. A higher level of social interactions was indicated amongst medium-sized fish than amongst those in the smallest and largest categories. Excess feeding may have been important factors in reducing aggression, so that the growth of the smallest individuals was not suppressed by the larger individuals in the present study. Grading seems to be an unnecessary operation to improve the growth and survival of juvenile turbot (5- 150 g). However, as it was mainly the smallest individuals in each group that died, grading of very small turbot (2-5 g) can be recommended.     

Price and production trends in the marine fish aquaculture in Spain

The Spanish aquaculture sector has undergone a rapid and continuous growth becoming one of the biggest European producers. A wide range of species are farmed and, among them, the sea bass, sea bream and turbot make a significant contribution to the total of the Spanish fish farming output. In this paper, we analyse and predict the medium-term trend of the production and price level for the three leading species previously mentioned. The results obtained show a significant increase in the production of sea bream, sea bass and turbot. The results show also a slight decrease in the sea bream average price, a stable trends for the sea bass and increasing prices for the turbot.     

Efficacy of furunculosis vaccines in turbot, Scophthalmus maximus (L.): evaluation of immersion, oral and injection delivery

The commercial furunculosis vaccine Aquavac Furovac 5 and an autogenous vaccine, based on the challenge strain, induced immune protection in turbot, Scophthalmus maximus (L.), as shown in challenge tests 120 days post-immunization by injection (relative percentage of survival, RPS = 72-99%). This protective effect lasted for at least 6 months post-immunization at appreciable levels (RPS = 50-52%). Neither the autogenous vaccine nor the commercial vaccine was able to induce significant levels of protection against Aeromonas salmonicida in turbot when administered by immersion. Antibody levels were high or moderate in fish vaccinated by injection with the different vaccines and very low in fish vaccinated by immersion. The field results show that delivering an oral boost after the primary vaccination by injection did not enhance protection of turbot against furunculosis and that water-based (autogenous vaccine) and oil adjuvanted (Alpha Ject 1200) vaccines administered by injection conferred similar levels of protection (RPS > 80%) in turbot.     

黄、渤海区不同生态类型鱼卵卵膜和受精孔区亚显微形态

黄、渤海区分布的硬骨鱼类鱼卵几乎涵盖了所有鱼卵生态类型。本研究借助扫描电镜观察技术对近年来黄、渤海渔业资源监测调查期间采集的15种不同生态类型鱼卵卵膜和受精孔区亚显微形态特征进行观察,以阐明其亚显微结构并总结提炼可用于鱼卵形态分类的特征,同时将相应鱼种产卵场环境因子引入到卵膜形态特征的认识中,以揭示其生境适应性。结果显示不同生态类型或不同鱼种卵膜形态不尽相同,浮性鱼卵和口含鱼卵卵膜较薄,沉性鱼卵和具卵膜丝鱼卵卵膜较厚;浮性鱼卵卵膜表面壁孔密度、卵膜外表面呈现的蚀刻或特殊结构以及受精孔形状类型,沉性鱼卵卵膜黏膜层表面结构、受精孔形状类型,具卵膜丝鱼卵卵膜丝数量、着生位置及受精孔形状类型,口含鱼卵受精孔区形状及周边附着丝数量等均可用于鱼卵形态分类。鱼卵卵膜和受精孔区亚显微结构差异与鱼种系统分类地位、产卵场环境和地理分布区相关联。鱼卵卵膜表面结构可以作为区分鱼类科或属的性状,卵膜壁孔密度、表面呈现的蚀刻或奇异结构是鱼卵形态分类的重要依据;受精孔结构则一般具有物种特异性。卵膜结构与厚度可反映鱼卵对不同生境的适应性,而卵膜表面的许多特殊结构和装饰被认为是鱼卵应对产卵场环境的保护和适应性策略。本研究将为黄、渤海区硬骨鱼类早期生活史阶段个体发生和鱼卵形态分类研究积累基础资料。