Fish stem cell cultures

Stem cells have the potential for self-renewal and differentiation. First stem cell cultures were derived 30 years ago from early developing mouse embryos. These are pluripotent embryonic stem (ES) cells. Efforts towards ES cell derivation have been attempted in other mammalian and non-mammalian species. Work with stem cell culture in fish started 20 years ago. Laboratory fish species, in particular zebrafish and medaka, have been the focus of research towards stem cell cultures. Medaka is the second organism that generated ES cells and the first that gave rise to a spermatogonial stem cell line capable of test-tube sperm production. Most recently, the first haploid stem cells capable of producing whole animals have also been generated from medaka. ES-like cells have been reported also in zebrafish and several marine species. Attempts for germline transmission of ES cell cultures and gene targeting have been reported in zebrafish. Recent years have witnessed the progress in markers and procedures for ES cell characterization. These include the identification of fish homologs/paralogs of mammalian pluripotency genes and parameters for optimal chimera formation. In addition, fish germ cell cultures and transplantation have attracted considerable interest for germline transmission and surrogate production. Haploid ES cell nuclear transfer has proven in medaka the feasibility of semi-cloning as a novel assisted reproductive technology. In this special issue on "Fish Stem Cells and Nuclear Transfer", we will focus our review on medaka to illustrate the current status and perspective of fish stem cells in research and application. We will also mention semi-cloning as a new development to conventional nuclear transfer.     

Fish embryo cell cultures for derivation of stem cells and transgenic chimeras.

It has been demonstrated in mammalian systems that techniques using embryonal stem cells provide advantages over conventional injection of DNA into embryos for generation of transgenic animals. We employed cell culture approaches in an attempt to develop this technology for fish transgenesis. Using a trout embryo-derived mitogenic preparation in a specialized culture medium, we initiated replication of zebrafish blastula-derived cell cultures and expressed marker genes introduced into the cells by plasmid transfection. Reintroduction of cells from the cultures into blastula-stage embryos indicated that the cultured cells survived and may contribute to the developing organism.     

Distribution Characteristics and Risk Assessments of PAHs in Fish from Lake Taihu,China

The concentrations of PAHs in four species of fish (Common carp, Crucian carp, Bighead carp, and Topmouth culter) from Lake Taihu were tested, and the human health risks of PAHs by fish consumption were evaluated. Results showed that concentrations of PAHs in fish from Lake Taihu were 52.5–247.6 ng/g wet weight (ww), and the BaP equivalent concentrations of total PAHs (B[a]P_eq) were 0.2–0.6 ng/g ww, which were less than the screening value of 2.6 ng/g wet for human consumption. The concentration sequences of PAHs in fish from Lake Taihu from high to low were Bighead carp > Crucian carp > Common carp > Topmouth culter. The human health risk level of PAHs by fish consumption was 5.8 ± 2.5 × 10^?6, which was less than the maximum acceptable risk level of 1 × 10^?5 for human health set by the U.S. Environmental Protection Agency. The tissue residue guideline (TRG) of PAHs for protecting aquatic wildlife was 1.3 mg/kg diet ww, which was higher than the concentrations of PAHs in fish from Lake Taihu. The results indicated that fish consumption from Lake Taihu would not cause health risk or harmful effects on wildlife that consume aquatic biota.     

乳状鱼蛋白的营养评价

乳状鱼蛋白是以鱼类为原料采用酶水解方法制作的一种鱼蛋白制品。以低值淡水鱼鲢鱼整鱼为原料制作的乳状 鱼蛋白,其营养成分和氨基酸组成具有如下特点:蛋白质含量丰富,脂肪含量较高(质量分数分别为18.4%和7.1%),其组成 中不饱和脂肪酸质量分数约占70%,且EPA和DHA的总量达到12.1%;此外,铁、锌含量较高,质量分数分别为30.7mg/k 和42.2mg/kg。组成蛋白质的各必需氨基酸除甲硫氨酸和胱氨酸外,其他均接近或超过FAO/WHO模式或全蛋蛋白。由大鼠 生长试验结果表明,其蛋白质的PER和NPR均高于酪蛋白,因此可以认为乳状鱼蛋白是食品加工的良好的蛋白质源。     

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.     

Fish gut content from biological collections as a tool for long-term environmental impact studies

Most of the world’s fish fauna is suffering from different types of human impacts and new conservation tools are required. The fish diet analysis is a tool that has been used to evaluate degradation processes of aquatic environments, however, few long-term studies are performed by several reasons (e.g., lack of funding, opportunity). Our aim was to test whether the fish gut content from biological collections can be used for comparisons with current data and, consequently, be used as a tool for long-term environmental impact studies. We compared the gut content of fish preserved for fifteen years in a biological collection with recently sampled fish, considering the factors size of the specimen, preservation time and preservation form. We did not find differences in the gut content percentage of preservation between fish size classes and preservation time. However, we found differences between preservation form, in which the fish fixed in formalin kept the digestive content preserved while the fish preserved directly in alcohol did not. Thus, we encourage the use of fish gut content from biological collections fixed in formalin for long-term ecological studies. Our findings may help elucidate some long-term effects of human impacts on fish fauna.     

Fish scales as Biosensors for Catecholamines

Certain fish scales contain specialized cells (chromatophores) with pigment granulas, which can be dispersed or aggregated in the cells. The degree of aggregation is determined by a transmitter substance, nonadrenaline, released by the sympathetic nerve endings in the skin. Isolated scales from, for example, cuckoo wrasse (Labrus ossifagus) retain a large sensitivity to externally applied nonadrenaline (or more generally catecholamines) for several weeks. The degree of aggregation in isolated scales can be measured objectively by simple photometric techniques. We demonstrate in this paper how fish scales can be used to monitor catecholamine levels in human blood plasma. A discussion of other potential biosensor applications of this intact biological receptor-effector system is also given.     

Xenotransplantation of Human Adipose-Derived Stem Cells in Zebrafish Embryos

Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.     

Studies on Pattern of Producing Transgenic Fish I.Production and Detection of All fish TRansgenic Common Carp With cMT sGH Gene

转基因鱼工作的开展被用于水产科学基础与应用研究的各个领域。应用方面的一项重要研究就是利用转生长激素基因来提高鱼的增长速度。需要解决两个问题：一是转基因鱼所使用的基因元件;二是外源基因的高整和率与高表达。已有许多报道认为：不同种动物的生长激素不一定相互促进生长。我们通过显微注射,将鲤鱼金属硫蛋白启动子（ｃＭＴ）与大马哈鱼生长激素基因（ｓＧＨ）的融合基因（ｃＭＴｓＧＨ）导入鲤鱼单细胞后期的早期胚胎,构建了全鱼转基因鲤鱼。通过斑点杂交、Ｓｏｕｔｈｅｒｎｂｌｏｔ结合ＰＣＲＳｏｕｔｈｅｒｎｂｌｏｔ,对外源ｓＧＨ的整和进行了精确的检测和分析。实验选取性成熟鲤鱼,收集卵子和精子,湿法受精获得受精卵。经过基因注射,孵化后的鱼苗放入水族箱。待鱼苗平游,提取总ＤＮＡ进行检测。以ＰｓｔＩ酶切质粒ｐｃＭＴｃＧＨ（Ｆｉｇ．４）分离３．４ＫｂｓＧＨ片段,用随机引物标记,作为杂交探针。同时,设计并合成ｓＧＨ基因的ＰＣＲ特异引物。Ｆｉｇ．１的斑点杂交结果与Ｆｉｇ．２的ＰＣＲＳｏｕｔｈｅｒｎ结果相比较（Ｔａｂｌｅ１）,说明斑点杂交存在着高的假阳性。而ＰＣＲＳｏｕｔｈｅｒｎ将ＰＣＲ的快速、方便与Ｓｏｕｔｈｅｒｎ的准确性相结合,排除了Ｐ     

海南万泉河中发现外来有害种——马那瓜丽体鱼

2011年在海南琼海万泉河发现外来鱼种马那瓜丽体鱼(Parachromis managuensis)。该种为凶猛的肉食性鱼类,已在野外建立种群,可能会对本土鱼类产生不利影响。建议主管部门禁止人为放生。     

海洋野生鱼酶解提取鱼油的工艺分析

海洋野生鱼与养殖鱼比较, 其鱼油中含更多的二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）、脂溶性维生素等活性成分。为提高海洋野生鱼的利用价值, 以野生小带鱼为原料进行酶法提油工艺研究。分析了不同的温度, 时间, pH值等影响因素下的提取、萃取以及离心效果, 以响应面法确定了最佳的酶解工艺条件: 液固比为6、pH7.3、酶量1000 u/g原料、搅拌速度200 r/min、45oC酶解90 min; 最优萃取条件: 萃取剂100 mL(每20 g鱼糜原料)、pH4.0、40oC萃取25 min; 离心条件: 离心速度3000 r/min (1865 g)、离心时间10 min。上述工艺条件下提油率为79.90%。改进了传统的鱼油提取工艺, 在活性成分保护上有较大改善。     

Mangroves Enhance Reef Fish Abundance at the Caribbean Regional Scale

Several studies conducted at the scale of islands, or small sections of continental coastlines, have suggested that mangrove habitats serve to enhance fish abundances on coral reefs, mainly by providing nursery grounds for several ontogenetically-migrating species. However, evidence of such enhancement at a regional scale has not been reported, and recently, some researchers have questioned the mangrove-reef subsidy effect. In the present study, using two different regression approaches, we pursued two questions related to mangrove-reef connectivity at the Caribbean regional scale: (1) Are reef fish abundances limited by mangrove forest area?; and (2) Are mean reef fish abundances proportional to mangrove forest area after taking human population density and latitude into account? Specifically, we tested for Caribbean-wide mangrove forest area effects on the abundances of 12 reef fishes that have been previously characterized as “mangrove-dependent”. Analyzed were data from an ongoing, long-term (20-year) citizen-scientist fish monitoring program; coastal human population censuses; and several wetland forest information sources. Quantile regression results supported the notion that mangrove forest area limits the abundance of eight of the 12 fishes examined. Linear mixed-effects regression results, which considered potential human (fishing and habitat degradation) and latitudinal influences, suggested that average reef fish densities of at least six of the 12 focal fishes were directly proportional to mangrove forest area. Recent work questioning the mangrove-reef fish subsidy effect likely reflects a failure to: (1) focus analyses on species that use mangroves as nurseries, (2) consider more than the mean fish abundance response to mangrove forest extent; and/or (3) quantitatively account for potentially confounding human impacts, such as fishing pressure and habitat degradation. Our study is the first to demonstrate at a large regional scale (i.e., the Wider Caribbean) that greater mangrove forest size generally functions to increase the densities on neighboring reefs of those fishes that use these shallow, vegetated habitats as nurseries.     

Fish plasminogen activators: their identification and characterization

Immunoblots of proteins extracted from the skin of a small viviparous fish (Xiphophorus) showed that a monoclonal antibody against human urokinase recognizes multiple molecular weight species of antigens. The immunoaffinity-purified antigens had serine-protease activity for the hydrolysis of a chromogenic substrate and could convert human plasminogen to plasmin in a manner similar to that for human urokinase in vitro. Two antigens with apparent molecular weights of 55 and 50 kilodaltons that had been purified on a fibrin-Celite column were separable on SDS-polyacrylamide gels and were characterized as major plasminogen activators on fibrin-agar indicator plates. The 125I-tryptic peptide maps of both antigens were similar to that of human urokinase; therefore, the fish activators and human urokinase are structurally and functionally related.     

Overview of Fish Consumption Rates in the United States

Contaminants that accumulate in fish may pose health risks and have raised concerns among consumers. Contaminated finfish and shellfish are possible routes of human exposure to toxic chemicals. Fish advisories are established to protect local populations from the consumption of contaminated commercial and non-commercially caught fish. Children, women of childbearing age, and subsistence fishermen or other highly exposed individuals who for socioeconomic or cultural reasons consume more fish than the general population are among the populations of concern from the ingestion of contaminated fish. Estimating exposure to a toxic chemical among fish-consuming populations requires knowledge about intake rates of fish and shellfish. Data on fish consumption rates come from various sources, including national, state, and local government studies. Although these data have their limitations, they serve as a source for deriving fish consumption rates that may be used by exposure assessors. Data on specific populations of concern are critical. Studies presented here show that ethnicity, age, and geographical region play an important role in fishing behavior and consumption. Results from studies on Native American populations show that this ethnic group eats fish at a higher rate than recreational anglers. This article summarizes data on fish consumption for various populations and provides a framework for evaluating data from these studies for exposure assessment applications.     

Human Health Risks of Selenium-Contaminated Fish: A Case Study for Risk Assessment of Essential Elements

A screening level human health risk assessment (HHRA) was applied to evaluate the human health implications of consuming selenium found in fish tissues collected downstream of coal mines in southeastern British Columbia, Canada. The study evaluated the potential for adverse human health effects associated with selenium, and considered known and potential benefits of selenium and fish ingestion. The results indicated that risks of selenosis due to consumption of selenium-contaminated fish in the region are negligible. Conclusions were strengthened by consideration of the potential benefits of selenium to human health, including: selenium essentiality for maintenance of good health; potential cancer prevention properties due to its role as an antioxidant; potential benefits for cardiovascular health; and other positive health benefits. The findings indicated that some aspects of the traditional framework for HHRA (e.g., application of safety factors to “err on the side of safety”) are inappropriate for the assessment of selenium-contaminated fish. Due to both deficiency and toxicity in the selenium dose-response relationship, application of compounding conservatism in risk assessment may lead to recommended intakes of fish that are contrary to the public health interest. The need for balancing risk types, for incorporating positive responses in risk assessments, and the linkage to the precautionary principle, are discussed.     

Asymmetrical Development of the Gonads in the Embryos and Fry of the Fish, Oryzias celebensis

The development of the gonad in Oryzias celebensis was studied by light and electron microscopy. The path of PGC-migration, the time of the sex-differentiation, and the pattern of germ cell proliferation were almost identical to those in O. latipes. The most conspicuous difference was in the distribution of germ cells after migration. The gonadal anlage in O. celebensis developed only on the right side of the dorsal mesentery, although PGCs were stituated on the both sides of the embryos before migration. The testis retained its unilateral condition throughout development and acquired a unilobed shape. In the female, the right presumptive ovary developed over the mesentery, and the ovary became bilobed. Thereafter, right and left parts of the presumptive ovary fused to develop into a single ovary in the adult fish. The situation appears to be comparable to the gonadal asymmetry in birds, but the present observations suggest that the developmental processes of the asymmetrical gonads in this fish are quite different from those in birds.     

海洋野生鱼酶解提取鱼油的工艺分析

海洋野生鱼与养殖鱼比较, 其鱼油中含更多的二十碳五烯酸（EPA）、二十二碳六烯酸（DHA）、脂溶性维生素等活性成分。为提高海洋野生鱼的利用价值, 以野生小带鱼为原料进行酶法提油工艺研究。分析了不同的温度, 时间, pH值等影响因素下的提取、萃取以及离心效果, 以响应面法确定了最佳的酶解工艺条件: 液固比为6、pH7.3、酶量1000 u/g原料、搅拌速度200 r/min、45oC酶解90 min; 最优萃取条件: 萃取剂100 mL(每20 g鱼糜原料)、pH4.0、40oC萃取25 min; 离心条件: 离心速度3000 r/min (1865 g)、离心时间10 min。上述工艺条件下提油率为79.90%。改进了传统的鱼油提取工艺, 在活性成分保护上有较大改善。     

Fish remains from the Neolithic site of El Mirador cave (Atapuerca,Spain): Seasonality and resource management

Ichthyoarchaeological analyses of the freshwater fish remains from levels 19 and 20 in El Mirador cave (Atapuerca, Spain) have been conducted. Fish were always present as a source of animal protein, although their importance in the human diet was not fully exploited by people during the Neolithic on the Iberian Peninsula. Two principal goals are treated here: a taxonomic study of the fish remains and a characterization of the exploitation of this resource. The results show that the human community of El Mirador cave practiced fishing, and that fish was part of their diet and social life.