鲫卵黄脂磷蛋白的纯化鉴定及其夹心ELISA的建立

鲫(Carassius carassius)卵黄原蛋白(vitellogenin,Vtg)是检测水体环境雌激素活性常用的生物标志物。本研究利用凝胶过滤结合离子交换层析与选择性沉淀结合离子交换层析2种方法,从鲫卵巢匀浆液中纯化得到了卵黄脂磷蛋白(lipovitellin,Lv),经鉴定该蛋白含有糖、磷、脂基团,天然分子量约为521 kD,SDS变性电泳显示分子量为117 kD和103 kD的2个亚基。Western blot结果显示,金鱼(Carassius auratus)Lv抗体和斑马鱼(Danio rerio)Lv抗体都能与鲫Lv发生很好的交叉反应。利用纯化的鲫Lv与金鱼Lv抗体和斑马鱼Lv抗体建立了2种夹心ELISA,发现金鱼Lv和鲫Lv的结合曲线基本重合,并且利用鲫Lv与金鱼Lv抗体建立的夹心ELISA工作范围为15.6~1000 ng/mL,检出限约为6.8 ng/mL,显著低于利用斑马鱼Lv抗体建立的夹心ELISA,结合此前研究者建立的鲫Vtg竞争ELISA,为鲫Vtg指标的测定提供了可靠方法。     

The development of a radioimmunoassay for carp,Cyprinus carpio,vitellogenin

The development of an easily — performed and robust radioimmunoassay (RIA) to carp, Cyprinus carpio, vitellogenin (c-VTG) is described. Purified c-VTG was iodinated using Iodogen. The resulting c-VTG label was useful for up to 60 days. High titre antibodies were raised in rabbits to the purified c-VTG. The practical operating range of the c-VTG RIA was between 2 and 200 ng. ml^-1. VTG was detected in plasma from all female mirror carp investigated, and all plasmas diluted parallel to the standard. The plasma VTG level in female carp increased concomitantly with the GSI; levels increasing from the ng. ml^-1 level in juveniles to a maximum of 1 mg. ml^-1 in fully mature females. VTG level was a far more sensitive index of the degree of sexual development than was GSI. In males, blood levels of VTG were always undetectable. Vitellogenic plasma from all subspecies of C. carpio (e.g. mirror, common, Koi) diluted parallel to the standard, as did blood from most other female cyprinids, such as the roach (Rutilis rutilis), bream (Abramis brama), and dace (Leuciscus leuciscus), but not all. These results suggest that the structure of VTG is highly conserved within this family. However, plasma from vitellogenic salmonids did not cross-react in the RIA. The relationship between plasma VTG and calcium levels was studied in both carp and rainbow trout. In rainbow trout it was found that plasma calcium levels do not rise above basal levels until the VTG level exceeds about 1 mg. ml^-1, and therefore in this species it is only useful as an indicator of the degree of ovarian development in the later stages of the reproductive cycle. In the carp, however, and probably other cyprinids, blood VTG levels do not appear to naturally exceed about 1 mg.ml^-1, and plasma calcium levels are not suitable as an indirect measure of the VTG level.     

Anoxia survival in common carp and crucian carp is related to high zinc concentration in tissues

ABSTRACT:   Ten lots of common carp and six lots of crucian carp (each lot of 100 fish) were treated under anoxia for five days at water temperatures of 25–31°C. The average per group mortality of common carp was 17%, but none of the 600 crucian carp died. The digestive tract tissues of the common carp that died had significantly lower zinc concentrations than those that survived (59 ± 41 vs 142 ± 60 μg/[g fresh tissue], P  < 0.001). The digestive tract tissues of the crucian carp had mean zinc concentrations of 652 ± 458 μg/(g fresh tissue). One lot of common carp that had low tolerance for anoxia was fed a high zinc diet (2000 mg zinc/kg diet) for 1, 2 or 6 months and then subjected to 5 days anoxia. The survival rates of those fed the high zinc diet 1 and 2 months increased from 0 to 50%, respectively; all of fish that had fed a high zinc diet for 6 months survived. Thus, anoxia survival in common carp and crucian carp is closely related to the high concentrations of zinc in their tissues.     

Distribution of the introduced cyprinid herpesvirus 3 in a wild population of common carp, Cyprinus carpio L.

Cyprinid herpesvirus 3 (CyHV-3), which causes a lethal disease in common carp, Cyprinus carpio L., and koi, C. carpio koi , first occurred in Lake Biwa, Japan in 2004. To elucidate distribution of CyHV-3 in a wild common carp population, we conducted a PCR survey of CyHV-3 among such fish in Lake Biwa in 2006. Only 6% (1/18) of the common carp smaller than 300 mm were positive with PCR, whereas 31% (18/58) of fish larger than 300 mm were positive. To evaluate their past exposure to CyHV-3 infection based on the presence of antibodies, we also measured the levels of serum anti-CyHV-3 antibodies in the carp, using an enzyme-linked immunosorbent assay. None (0/26) of the fish smaller than 300 mm was positive for the antibodies, whereas 54% (33/61) of fish larger than 300 mm were positive. Of the antibody-positive individuals, 44% (14/32) were also positive by PCR strongly suggesting that wild common carp that survived infection become CyHV-3 carriers. Five individuals were positive by PCR but negative for antibodies indicating that their infection with CyHV-3 had occurred recently. These results suggest that transmission of CyHV-3 from carriers to naïve common carp is still occurring in Lake Biwa.     

Body depth variation in crucian carp Carassius carassius: an experimental individual-based study

Abstract –  Induced morphological defences, where individuals develop morphological structures as a response to signals from sympatric predators, are common among invertebrates. Such defences have also been reported in a freshwater fish, the crucian carp Carassius carassius . In crucian carp, the presence of piscivorous fish presumably induces the development of a deep body as a defence mechanism. We tested how the growth rate (in body depth, length and mass) of individually tagged crucian carp responds to pike Esox lucius odours and control water. In general, crucian carp exposed to pike odour grew better (both in length and mass) than control fish. The treatment crucian carp also developed deeper bodies than control crucian carp. Further, there was a strong positive relationship between individual growth rate and the rate of change in body depth and this relationship did not differ between control and treatment fish.     

Prey fish selection by Far Eastern catfish <Emphasis Type="Italic">Silurus asotus</Emphasis> and largemouth bass <Emphasis Type="Italic">Micropterus salmoides</Emphasis>

ABSTRACT:   Prey fish selection by Far Eastern catfish and largemouth bass were examined using bluegill, Japanese dace and crucian carp as food fish. In both aquaria (1.2 m × 0.45 m) and ponds (2.8 m × 1.2 m), bluegill was not preyed on by catfish and bass more than dace and crucian carp. In aquaria, there was no significant difference in the consumption of dace and crucian carp between catfish and bass, but in ponds, catfish preyed on dace and crucian carp more and less than bass, respectively. In the case that only bluegill and catfish were introduced in ponds, catfish consumed 4–15 g of bluegill per day. The introduction of catfish into ponds and lakes for the purpose of eradicating bluegill is considered appropriate for areas with few native fish species.     

天然滁州鲫氨基酸成分分析与营养价值评价

应用生化分析方法对滁州鲫鱼中氨基酸成分进行了定量分析,并对氨基酸含量进行营养分析评价。结果显示：滁州鲫鱼中氨基酸总量为86.35%（占干重）,其中8种必需氨基酸和2种半必需氨基酸总量为36.76%,4种呈味氨基酸含量为29.29%,必须氨基酸总量和呈味氨基酸含量均高于草鱼、鲢、鳙鱼等鱼类。表明滁州鲫较之其他鱼类是一种营养价值高,肉质鲜美的鱼类。     

几种鲤、鲫鱼腹膜色素细胞的初步观察

通过普通鲫、普通鲤、红鲫、荷包红鲤、水晶彩鲫和锦鲤的腹膜脏层和腹膜壁层色素细胞观察,腹膜脏层分布有黑色素细胞,其中普通鲫致密完整,普通鲤和红鲫密集均匀,水晶彩鲫和锦鲤缺失,荷包红鲤完全缺失;腹膜壁层分布有鸟粪素细胞,除水晶彩鲫缺失,其它品种致密完整。     

Seasonality of serum levels of vitellogenin in male Japanese whiting, Sillago japonica, reared under natural temperature and photoperiod

ABSTRACT:   Because blood vitellogenin (Vg) has been considered a biomarker for environmental estrogens, the basal levels of Vg and 17β-estradiol (E₂) were determined in male Japanese whiting reared under natural conditions. Serum levels of Vg and E₂ were measured and gonadal development was assessed by gonadosomatic index (GSI) and histological observation in 8–10 male fish at monthly intervals throughout the annual reproductive cycle. Serum E₂ was <60 pg/mL throughout the study period. In contrast, serum Vg exhibited seasonal changes: serum levels of Vg gradually increased from April to May (mean 63 ± 13 ng/mL and 124 ± 48 ng/mL in April and May, respectively), and then reached a peak value (mean 352 ± 68 ng/mL) in June. Thereafter, serum Vg gradually decreased, reaching undetectable levels (<50 ng/mL) in October. Serum levels of Vg tended to increase in the male fish in which the GSI was >1%. Histological observation revealed that testes in such male fish were in active spermatogenesis and then all of the testes of male fish in which serum Vg decreased to ND levels were regressed. These results suggest that Vg productive potency (sensitivity to estrogens) may increase in the spermatogenic stage, resulting in production of Vg in response to very low levels of natural or xenobiotic estrogens.     

Genetic characterization of the progeny of a pair of the tetraploid silver crucian carp Carassius auratus langsdorfii

Silver crucian carp Carassius auratus langsdorfii comprises a diploid-polyploid complex in wild Japanese populations. Bisexually reproducing diploids are sympatrically distributed with gynogenetically developing triploids and tetraploids. Triploid and tetraploid males are very rare among Japanese silver crucian carp due to their gynogenetic reproduction. We examined the genetic characteristics of progeny that arose in a tank by natural spawning of a tetraploid silver crucian carp pair. The ploidy status of 120 samples randomly collected from these progeny was determined to be tetraploid by DNA content flow cytometry. DNA fingerprints from a random amplified polymorphic DNA assay indicated that almost all the progeny examined had genotypes identical to the maternal tetraploid female with no paternally derived fragments. Selected specimens’ cytogenetic analyses revealed that the progeny examined had tetraploid chromosome numbers, categorized into 40 metacentric, 80 submetacentric, and 80 subtelocentric or telocentric chromosomes, which were arranged into quartets and six supernumerary microchromosomes. Fluorescence in situ hybridization signals were detected in four homologous chromosomes in all analyzed metaphases prepared from diploid goldfish specimens. Contrary, tetraploid silver crucian carp gave eight rDNA signals. These results suggest that gynogenetic development in eggs spawned by tetraploid females should be triggered by tetraploid males’ homospecific sperm.     

Resource use of crucian carp along a lake productivity gradient is related to body size,predation risk,and resource competition

Generalist fish species can feed on a wide resource spectrum and across trophic levels depending on resource availability and trophic interactions. Crucian carp (Carassius carassius) represents a good candidate species to investigate variation in the trophic ecology of generalist fish as it can be found in highly variable fish communities and its resource use is well documented. In this study, we explored the trophic ecology of crucian carp at the individual and population levels using stable isotope and gut content analysis. We tested if trophic resource use varied according to lake productivity, predation risk, intra- and interspecific competition, or individual fish size. We found that crucian carp resource preference was highly variable among and within lakes. In predator-free lakes, small crucian carp occurred in high densities, showed increased interindividual specialisation, and relied mainly on pelagic zooplankton. In presence of predators, large crucian carp occurred in low densities and included greater proportions of benthic macroinvertebrates in their diet. This shift in resource use was further favoured in productive, shallow lakes where littoral prey were probably abundant. Resource partitioning was an important factor determining crucian carp niche use, as fish had higher trophic position in absence of other cyprinids. Crucian carp showed highly dynamic resource use and food preferences in response to variable environmental conditions. Overlooking complex diet preferences of generalist fish may lead to an oversimplification of freshwater community dynamics.     

利用鲫鱼监测水环境中汞污染的研究

为科学利用鱼类等水生动物监测并评价水环境中汞污染的程度,试验在松花江、黑龙江、乌苏里江等河流的特定区域设置5个采样点,先监测对应水环境中汞含量,然后在禁渔期前采集监测区域内的鲫鱼(Carassius auratus),测定体重和肌肉中的汞含量.结果发现,同一水域中鲫鱼肌肉汞含量与其体重明显正相关(r>0.765),当鱼体大小不统一时,可用线性回归方法拟合计算相同规格的鲫鱼肌肉汞含量,经拟合计算的同一规格的鲫鱼肌肉汞含量与其栖息水环境中汞的污染水平极显著正相关(p<0.01).利用线性回归方法拟合计算的鲫鱼肌肉汞含量可以定量评价和比较水环境受汞污染的程度和大小.     

辛基酚对鲤的雌激素效应

研究了辛基酚对鲤的雌激素效应。经10、50、100、300和500μg/L辛基酚暴露32d后,鲤存活率和肥满度与对照组无差异;性腺指数变化明显,雌鱼性腺指数随暴露剂量的增大而增大,雄鱼性腺指数随暴露剂量的增大而减小;50μg/L及以上暴露组与对照组差异显著(P<0.05)。辛基酚能诱导鲤雄鱼产生卵黄蛋白原,暴露剂量为10μg/L组有部分雄鱼肝脏匀浆和血液中检出卵黄蛋白原,50、100和300μg/L组卵黄蛋白原含量随辛基酚浓度的增大而显著增大,500μg/L组卵黄蛋白原含量低于300μg/L组、高于100μg/L组,均为极显著差异(P<0.01)。     

Balancing protein ingredients in carp feeds to limit dischargeof phosphorus and nitrogen into water bodies

ABSTRACT:   Through a series of experiments, it has been demonstrated successfullythat an appropriate choice of ingredients is primary for achievinga reduction in phosphorus (P) and nitrogen (N) loading from carpculture. In this final study, four multi-ingredient diets were examined.In addition to the basal ingredients (fish meal 15%, corngluten meal 8%, defatted soybean meal 10%, blood meal3–5%), three of the test diets (PF, MM, and SP)contained 6% each of either poultry feather meal, meatmeal or soy protein concentrate, respectively, and the fourth diet(PMS) had each of the three aforementioned ingredients includedat 3%. These diets had crude protein < 35%,digestible energy > 3.5 kcal/g,available P between 0.65 and 0.66% and total P rangingfrom 1.26 to 1.36%. The diets were fed to juvenile carpduring a 12-week feeding trial. The highest growth was obtained forfish fed the MM diet. The absorption and retention rates of P werehigher for the PF diet, whereas these values for N did not showtreatment-related differences. The total discharges per ton fish productionwere estimated to range between 7.8 and 9.2 kg for P andbetween 39.1 and 40.7 kg for N. These were lower than thefigures for Lake Kasumigaura, a main carp culture site, indicatingthat proper balancing of protein ingredients aids in efficientlylowering emissions from aquaculture.     

The effect of feeding commercial diets on the development of juvenile crucian carp (Carassius carassius,L.). Part 1: Skeletal deformations

The crucian carp (Carassius carassius, L.) is a cyprinid freshwater species, whose wild‐living populations have decreased in recent years due to an increasing competition of invasive species. Several initiatives were launched, attempting to reintroduce this fish back into its native open waters, which requires the use of crucian carp fry grown under controlled conditions. However, the use of popular commercial diets results in severe developmental abnormalities. The aim of this study was to analyse the impact of feeding natural feed (Chironomidae sp. larvae) or two popular commercial diets on the skeletal development of crucian carp juveniles. Whole‐mount staining and histological methods were used in the study. Skeletal tissues of fish fed both commercial diets were demineralized and displayed a variety of deformities, such as abnormal vertebral spines and vertebral fusion, as well as a new type of deformation, which involved the action of dorsal fin pterygiophores upon the spine. Finally, a novel analytical method was proposed, which utilizes LA‐ICP‐MS for the estimation of bone mineralization. The study proved that basic commercial diets are inadequate for the rearing of crucian carp juveniles.     

青海湖裸鲤不同繁殖群体繁殖特性的比较研究

2017年7月~2018年6月,在青海湖的洱海、大湖、黑马河口、泉吉河口、沙柳河口和布哈河口6处采样点,共采集青海湖裸鲤(Gymnocypris przewalskii)繁殖群体样本295尾,对不同繁殖群体的繁殖特性进行比较研究。结果显示,除泉吉河口的性比(♀∶♂)<1(即0.82∶1)外,其余5个繁殖群体雌性个体数量均大于雄性(♀∶♂>1);6个繁殖群体的所有样本中,雌性的生物学最小型大于雄性。其中,雌性群体(布哈河口)的生物学最小型的年龄、全长和体重分别为5龄、157 mm和42.5 g,雄性(沙柳河口)为4龄、124 mm和24.5 g;6个繁殖群体的雌性性成熟系数(GSI)显著高于雄性(P<0.05),沙柳河口雌性GSI(19.67±1.36)%显著高于洱海(10.28±1.36)%和大湖(13.35±1.24)%(P<0.05),而不同繁殖群体雄性GSI间差异不显著(P>0.05);黑马河口平均绝对繁殖力Ft[(3338.18±324.25)粒]和相对繁殖力Fr[(21.88±2.10)粒/g]分别与沙柳河口的Ft[(10699.40±613.91)粒]和Fr[(82.68±4.63)粒/g]存在显著差异(P<0.05)。以上研究结果表明,青海湖裸鲤的繁殖特性在不同群体之间已出现分化趋势或已经分化。本研究可为进一步了解青海湖裸鲤的种群结构以及制定合理的渔业资源管理措施提供基础数据,特别是增殖放流策略的优化。     

Growth and reproduction of threatened native crucian carp Carassius carassius in small ponds of Epping Forest,south‐east England

• 1. The crucian carp Carassius carassius is a species of cyprinid fish native to south‐east England, but few studies exist on its growth and reproduction in England, and the species is threatened by introductions of its Asiatic congener, goldfish Carassius auratus. To increase knowledge of the crucian carp as a means of aiding its conservation, the present study assesses the growth (back‐calculated length at age, body condition) and reproduction (fecundity, egg size, length and age at maturity) of crucian carp in small ponds of Epping Forest (north‐east London, England).
• 2. Evaluation of growth and reproduction data with published data for populations from northern Europe (i.e. latitude ?50°) suggest that growth is very variable, even within the same area, and that data on reproductive indices, in particular length and age at maturity, are scarce. In England, the length at age trajectory of crucian carp living in sympatry with feral goldfish Carassius auratus did not differ significantly from allopatric crucian carp populations, but crucian carp body condition and relative fecundity (eggs per body weight) were highest, and mean age and standard length at maturity were lowest in sympatry with goldfish.
• 3. These data suggest that somatic growth and reproductive output may be maximized in crucian carp when confronted by coexistence with feral goldfish. However, the potential impact of goldfish introductions on crucian carp growth and reproduction requires further study, involving a much larger number of crucian carp populations, both in allopatry and sympatry with feral goldfish populations.

© Crown copyright 2009. Reproduced with the permission of Her Majesty's stationery Office. Published by John Wiley & Sons, Ltd.     

A study of feral carp, Cyprinus carpio L., exposed to Microcystis aeruginosa at Lake Mokoan, Australia, and possible implications for fish health

Feral carp, Cyprinus carpio L., were collected at bimonthly intervals for 12 months from Lake Wellington, where there was no evidence of significant growth of Microcystis aeruginosa . Various blood parameters of these carp were measured to ascertain the normal range values for feral carp in this region. A similar study was conducted at Lake Mokoan, where M. aeruginosa formed a visible scum that contained detectable levels of microcystins for 4 consecutive months. Carp were taken from Lake Mokoan each month, and the histology and blood biochemistry of the fish were examined for indications of microcystin toxicity. During February, March and April, 66% of the carp from Lake Mokoan had livers with widespread hepatocyte atrophy, and 37% of the fish had gills with pinpoint necrosis, epithelial ballooning, folded lamellar tips and exfoliation of the lamellar epithelium. Compared to carp from Lake Wellington, the serum aspartate aminotransferase activity and the serum bilirubin concentrations of carp from Lake Mokoan were elevated during January, February, March and April. The data were consistent with impaired hepatocyte function in some carp from Lake Mokoan. Serum sodium and chloride concentrations in carp from Lake Mokoan were lower than those for carp at Lake Wellington from December to April, indicating that most carp from Lake Mokoan had difficulty maintaining cation-anion homeostasis during this period. Nevertheless, carp at Lake Mokoan always appeared healthy, and no mortalities were reported.     

Deficient melanin production contributes to the absence of melanophores in early development of red carp

Red carp and red crucian carp are ornamental fish with a red body color. Unlike in red crucian carp, no melanophores are observed in red carp embryos or larvae. To explore the roles of the mitfa gene in body color formation in red carp, we investigated the structural characteristics and physicochemical properties of the mitfa gene in 16 kinds of fish. The mitfa amino acid sequence similarity between red carp and red crucian carp was 95.6%, and this was 91.5% similar between carp and zebrafish. Compared with red crucian carp, red carp showed lower tyrp1 messenger ribonucleic acid (mRNA) expression but similar mitfa mRNA expression in the body pigment stage of the embryo. Moreover, mitfa⁺ cells as well as melanocytes could be observed in cultured embryo cells derived from red carp and red crucian carp. Our data show that the absence of melanophores in red carp is not the result of mitfa gene deletion or mutation, increasing our understanding of the molecular and genetic mechanisms of coloration in cyprinid fish.     

A non‐classical biomanipulation experiment in Gonghu Bay of Lake Taihu: control of Microcystis blooms using silver and bighead carp

A non‐classical biomanipulation experiment was carried out in Gonghu Bay of Lake Taihu in 2009. Silver and bighead carp were stocked in a large fish enclosure to control cyanobacterial blooms. Water quality, plankton abundance, and the intracellular and extracellular microcystins (MCs) in lake water were investigated monthly in 2009. The concentrations of nitrogen nutrients were significantly lower in the fish enclosure than in the surrounding lake, while phosphorus (especially total phosphorus) concentration was higher in fish enclosure. During the blooming period, Cyanophyta contributed to more than 90% of the phytoplankton in the surrounding lake, whereas it represented only 40–80% in the fish enclosure. The phytoplankton and crustacean zooplankton biomasses and the zooplankton/phytoplankton ratios were all significantly lower in the fish enclosure than in the lake. This result suggested that silver and bighead carp can effectively suppress the phytoplankton biomass with the initial stocking density of 7.5 g m^?3 for silver carp and 1.1 g m^?3 for bighead carp, despite a simultaneous decrease in the grazing pressure of the zooplankton on the phytoplankton. During the blooming period, the intracellular and extracellular MCs in the fish enclosure were reduced by 93.8% and 69.8% compared with the surrounding lake. MCs content varied from 0.34 to 18.8 ng (mean 4.8 ng) MC‐LReqg^?1 wet weight in the muscle sample of silver and bighead carp in the experimental enclosure, which suggested that these fish were safe to consume for human. However, the long‐term effects of MCs on aquatic ecosystem and on public health cannot be overlooked.