三聚氰胺对花鲈的急性毒性实验研究

本文以花鲈(Lateolabrax japonicus)为实验对象,采用接触、腹腔注射及口服三种致毒方式,进行了非蛋白氮物质-三聚氰胺对花鲈的急性毒性实验研究。口服急性毒性实验中,通过在饲料中添加不同浓度的三聚氰胺（0、500、2000、5000及10000 mg/kg）,进行了21d的花鲈养殖实验。实验结果表明：三聚氰胺溶解度较低,其水溶液没有表现出急性毒性,LC50 > 3500 mg/L;而在腹腔注射致毒方式下,三聚氰胺对花鲈的半致死剂量LD50 = 890.07 mg/kg•w;LD5095%可信限为：778.63－1017.45 mg/kg•w;在口服急性毒性实验中,10000 mg/kg组三聚氰胺降低了花鲈的摄食与生长(p<0.05),饲料系数显著升高 (p<0.05);三聚氰胺对花鲈的存活率、肥满度、肝体比及脏体比均无显著影响 (p>0.05)。饲料中添加三聚氰胺没有显著影响花鲈血清的丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总蛋白、葡萄糖、胆固醇、甘油三酯及尿素氮 (p>0.05),但显著影响了花鲈血清的碱性磷酸酶活性,10000 mg/kg组的碱性磷酸酶活性显著高于其他各处理组 (p<0.05),其他各组间无显著差异(p>0.05)。三聚氰胺对花鲈21d的最大未观察到有害作用剂量（NOAEL, no-observed-adverse-effect-level）为131.99 mg/kg•w•d。     

Isolation and characterization of 22 polymorphic microsatellite DNA markers of Japanese sea bass (Laterolabrax japonicus)

Japanese sea bass (Laterolabrax japonicus) inhabits the coast of East Asia and is cage‐cultured currently in China as well. Twenty‐two microsatellite DNA markers were developed and used to type 35 individuals collected along the Chinese coast. The number of alleles per locus ranged from three to 23. The expected heterozygosity ranged from 0.111 to 0.938, while the observed heterozygosity ranged from 0.114 to 0.914. All 22 loci are neutral and independent of each other; two deviated significantly from Hardy–Weinberg equilibrium. These microsatellite DNA markers are moderately informative, which will certainly facilitate the management and exploitation of the genetic resource of L. japonicus.     

饥饿对于鲈肌肉、肝脏和血清主要生化组成的影响

于 2 2 93± 2 15℃条件下 ,在室外水泥池 (3m× 2m× 1m)中对正常鲈 (2 85 2 6± 6 5 4 g)和患脂肪肝病鲈 (4 6 4 71± 5 4 2 2g)进行为期 9周的饥饿处理。分别在实验开始后第 0周、 3周、 5周、 7周和 9周取样 ,以观察饥饿对于鲈内脏相对重量、肌肉肝脏和血清主要生化指标的影响。研究表明 ,鲈对饥饿耐受能力较强 ,在饥饿时首先快速动用肠系膜脂肪和肌肉脂肪作为能量供应 ,而在整个饥饿阶段则主要以肌肉蛋白质作为能量来源 ,肝脏中能源物质在饥饿中并无明显减少 ,故不是鲈饥饿时的主要供能物质。饥饿时 ,肌肉和肝脏中的水分和脂肪含量呈现负相关 ,尤其在肝脏中表现明显。鲈血清中脂肪酶、甘油三酯、胆固醇、低密度脂蛋白和高密度脂蛋白在饥饿中表现出周期性和阶段性的变化 ,其中正常鲈表现出有规律的波浪状图形 ,而脂肪肝病鲈则表现出山峰状图形 ,说明脂肪肝病鲈代谢机制不如正常鲈灵敏 ,9周的饥饿并不能减轻或消除鲈的脂肪肝病.     

Recruitment in Japanese sea bass,Lateolabrax japonicas (Cuvier, 1828): effects of timing on spawning and larval quality and quantity

This study aimed to elucidate the causes of variability in larval survival and juvenile abundance (recruitment) within and among cohorts of Japanese sea bass (JSB; Lateolabrax japonicus), a winter‐spawning temperate coastal marine fish. Larvae and settled individuals (settlers) belonging to four cohorts were collected from Tango Bay (the Sea of Japan coast) during eight sampling cruises in 2007 and 2008. Larvae were sampled in January and February each year using an ichthyoplankton net, and settlers were collected in February and March each year using a beam trawl. Age of individual larva and settlers was determined and growth history was back‐calculated from otolith microstructure, and the hatch date distribution was computed. Temperature, daily growth rate, size‐at‐age, hatch date, and density data of larvae and settlers allowed elucidating the effects of the timing of spawning and larval quantity and quality (growth rate and body size) on larval survival and recruitment within and among cohorts of JSB. Results showed that cohorts that hatched earlier in the season had higher quantity of larvae, experienced higher mean temperatures and survived better than cohorts hatched later. Recruitment variability among cohorts is determined largely by the initial quantity of larvae, as this explained >97% of the variability in recruitment among cohorts. Within cohorts, larger hatched larvae grew faster than their smaller conspecifics, and the bigger and faster growing larvae survived and settled. Results from this study suggest the following scenarios for recruitment of JSB: (i) earlier spawning in the season promotes larval survival since earlier cohorts are likely to encounter a better temperature and perhaps food conditions, and therefore recruit better than later cohorts; (ii) the initial quantity of larvae appears to be an important determinant of recruitment variability among cohorts; and (iii) the size‐ and growth‐related mechanisms operating during the larval phase appear to start at the time of the hatch.     

Isolation and characterization of polymorphic microsatellite loci from black sea bass (Centropristis striata) and cross-species amplification

Black sea bass (Centropristis striata) is an economically important serranid species. A number of 39 microsatellite loci were isolated from two enriched genomic library of C. striata. Eleven of these loci were polymorphic in a test population with alleles per locus ranging from 3 to 8, and observed and expected heterozygosities per locus from 0.26 to 1.00 and from 0.61 to 0.84, respectively. Four loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium was found between pairs of loci. Cross-species amplification of these polymorphic microsatellite loci was performed in additional two related species. These polymorphic microsatellite loci would be useful for investigating genetic resource of C. striata and other related species.     

Differential population structuring and demographic history of two closely related fish species, Japanese sea bass (Lateolabrax japonicus) and spotted sea bass (Lateolabrax maculatus) in Northwestern Pacific

The Quaternary cold periods in the Northwestern Pacific are thought to have heavily influenced the amount and distribution of intraspecific genetic variation in marine fishes. To estimate the demographic history and genetic structure of Lateolabrax maculatus and L. japonicus in the Northwestern Pacific, 256 individuals were sampled from 19 localities throughout the distribution range of the two species. Mitochondrial DNA variation was analyzed using DNA sequence data from the cytochrome b gene and control region. Nucleotide diversity was much higher in L. japonicus (0.030) than in L. maculatus (0.012). The demographic history of the two species was examined using neutrality tests and mismatch distribution analyses and results indicated Pleistocene population expansion in both species. Estimates of population expansion time suggested earlier population expansion in L. japonicus than in L. maculatus. Molecular variance analyses showed differential genetic structuring for these two closely related species. The results indicated that L. japonicus is panmictic throughout its range. In contrast, populations of L. maculatus showed statistically significant levels of genetic structuring. Pattern of isolation by distance was observed in L. maculatus, suggesting that L. maculatus is in genetic equilibrium. In contrast, L. japonicus did not exhibit isolation by distance.     

Nutritional condition, starvation status and growth of early juvenile Japanese sea bass (Lateolabrax japonicus) related to prey distribution and feeding in the nursery ground

The nutritional condition and protein growth rates of Japanese temperate bass larvae and juveniles were studied in relation to prey distribution and feeding habits in the nursery grounds in Chikugo estuary, Ariake Sea, Japan. Samples were collected from a wide spatial area covering the nursery grounds of the fish in March and April 2003. Food habits of the fish were analyzed by examining the gut contents. Fish condition was evaluated by using RNA/DNA ratio and other nucleic acid-based indices and protein growth rates. The nucleic acid contents in individually frozen larvae and juveniles were quantified by fluorometric method. Two distinguished feeding patterns, determined by the distribution of prey copepods, were identified along the nursery ground. The first pattern showed the dependency of the fish on the calanoid copepod Sinocalanus sinensis, which was the single dominant prey in low saline upper river areas and the second pattern involved a multi-species dietary habit mainly dominated by Acartia omorii, Oithona davisae and Paracalanus parvus. Values of RNA, DNA, total protein, growth rates and for all the nucleic acid-based indices were higher in upstream areas than in the downstream areas. The proportion of the starving fish was higher in the downstream areas than in the upstream areas. Condition of juvenile sea bass was not equal throughout the nursery grounds; fish in the upper river were in better condition than those in the lower estuary. We speculated that utilization of S. sinensis, which appears a suitable prey item and provide a better foraging environment in the upstream nursery ground, is one of the key factors for early survival and growth of Japanese temperate bass larvae and juveniles in the Chikugo estuary.     

Distribution and dietary relationships of the Japanese temperate bass Lateolabrax japonicus juveniles with two contrasting copepod assemblages in estuarine nursery grounds in the Ariake Sea, Japan

Early juvenile Japanese temperate bass Lateolabrax japonicus samples were collected during four cruises to study the spatial and temporal patterns of distribution and feeding habits of the fish in Chikugo estuary, Ariake Sea, Japan. Gut contents were studied by separating, identifying and counting the prey organisms. Plankton samples were collected during each cruise to study the numerical abundance of copepods in the water. Copepod dry biomass and gut content dry mass were estimated. Juveniles were distributed over wide estuarine areas in salinities ranging from as low as 0·37 to as high as 28·81. Considerable spatial and temporal variations were observed in the copepod distribution in the environment and in the food habits of the fish. Two distinctly different copepod assemblages were identified along the estuary: one in the upper river, dominated by a single species Sinocalanus sinensis, and the other a multi‐specific assemblage in the lower estuary dominated by common coastal copepods such as Acartia omorii, Oithona davisae, Paracalanus parvus and Calanus sinicus. The gut content composition corresponded strongly with the copepod composition in the environment, i.e. a single species (S. sinensis) dietary habit at the upper river and a multi‐specific dietary habit dominated by the common coastal copepods in the lower estuary. Ivlev's electivity index showed that the fish strongly preferred larger copepods and avoided smaller ones. Higher dry biomass of copepods in the water as well as higher dry masses of the gut contents were recorded in the low‐to‐medium saline upper river areas, indicating that these areas are of particular importance as nursery grounds for the juvenile Japanese temperate bass. It was speculated that ascending to the upstream nursery areas to utilize S. sinensis, which is the single dominant copepod in these areas, is one of the most important survival strategies of the Japanese temperate bass juveniles in the Chikugo estuary.     

Gene flow at major transitional areas in sea bass (Dicentrarchus labrax) and the possible emergence of a hybrid swarm

The population genetic structure of sea bass (Dicentrarchus labrax) along a transect from the Atlantic Ocean (AO) to the Eastern Mediterranean (EM) Sea differs from that of most other marine taxa in this area. Three populations (AO, Western Mediterranean [WM], EM) are recognized today, which were originally two allopatric populations. How two ancestral genetic units have evolved into three distinct units has not been addressed yet. Therefore, to investigate mechanisms that lead to the emergence of the central WM population, its current status, and its connectivity with the two parental populations, we applied 20 nuclear loci that were either gene associated or gene independent. Results confirmed the existence of three distinct gene pools, with higher differentiation at two transitional areas, the Almeria‐Oran Front (AOF) and of the Siculo‐Tunisian Strait (STS), than within any population. Significant linkage disequilibrium and heterozygote excess indicated that the STS is probably another tension zone, as already described for the AOF. Neutrality tests fail to reveal marker loci that could be driven by selection within or among metapopulations, except for locus DLA0068. Collectively, results support that the central WM population arose by trapping two tensions zones at distinct geographic locations of limited connectivity. Population assignment further revealed that WM individuals were more introgressed than individuals from the other two metapopulations. This suggests that this population might result from hybrid swarming, and was or is still seeded by genes received through the filter of each tension zone.     

Combined effects of physicochemical variables (pH and salinity) on sperm motility: characterization of sperm motility in European sea bass Dicentrarchus labrax

Gamete activation in fish is an important step in terms of artificial fertilization of oocytes, cryopreservation studies and other experimental manipulations. Salinity and pH differences in activation media affect to sperm motility and fertilizing ability. These experiments were therefore designed to investigate the combined effects of pH (range 5.0–9.0) and salinity (20, 30, 37, and 45‰) of activation media on sperm motility of European sea bass Dicentrarchus labrax. The best results were obtained at salinity 37‰ and a pH of 9.0. Our results also demonstrated that non-progressive motility at salinity 45‰ was observed in the range of 5.0–9.0 pH. In conclusion, spermatozoa can be motile at a wide range of pH and salinity values although the percent of motile spermatozoa and motility duration are negatively affected by low pH values.     

Microsatellite polymorphism and population subdivision in natural populations of European sea bass Dicentrarchus labrax (Linnaeus, 1758)

Polymorphism of microsatellite markers was used to study the genetic variability and structure in natural populations of European sea bass Dicentrarchus labrax. The data consisted of six microsatellite loci analysed for 172 individuals from three samples collected in the Golfe-du-Lion (France) and one sample collected in the Golfo-de-Valencia (Spain). Our goals were (i) to assess the level of genetic variability as revealed by these markers, (ii) to estimate the genetic differentiation among natural populations within a restricted area, and (iii) to evaluate how microsatellite loci fit the predictions of the two most widely used mutation models (the infinite allele model and the stepwise mutation model). As expected, our results indicate that the genetic polymorphism is very high when compared with previously used genetic markers, the mean expected heterozygosity per locus ranging between 0.69 and 0.93. We also found that all loci but one fitted the infinite allele model better. Using this model as a lower limit, we could extrapolate from the observed diversity effective population sizes on the order of 35 000 individuals. Our results also suggest that there may be a slight genetic differentiation between the two gulfs (F_ST= 0.007, P < 0.05), indicating that the corresponding populations are likely to be dynamically independent. This finding for a species with high dispersal abilities, if confirmed, has important beatings on fish-stock assessment.     

The novel use of pop‐off satellite tags (PSATs) to investigate the migratory behaviour of European sea bass Dicentrarchus labrax

A total of 12 adult European sea bass Dicentrarchus labrax were tagged with pop‐off satellite archival tags (PSAT) in Irish coastal waters and in offshore waters in the north‐east Celtic Sea between 2015 and 2016. Archived data were successfully recovered from five of the 12 tags deployed, three from fish released in inshore Irish waters and two from fish released offshore in the eastern Celtic Sea. All three fish tagged in inshore waters were found to undertake migrations into the open ocean coinciding with the spawning period. These fish also exhibited fidelity to inshore sites post‐migration, returning to the same general location (within c. 73 km, which is roughly the predicted mean accuracy of the method) of their original release site. Although the number of tracks obtained here was limited, some degree of aggregation between inshore and offshore tagged fish in the eastern Celtic Sea was noted during the expected spawning period suggesting PSATs can provide new information on specific spawning locations of European sea bass.     

Characterization of the estrogenic response to genistein in Japanese medaka (Oryzias latipes)

This study was designed to determine the estrogenic effect of the phytoestrogen genistein on several measures of endocrine function in adult Japanese medaka (Oryzias latipes) relative to 17-beta-estradiol. Adult animals of both sexes were exposed to 75, 750 and 30,000 ng/fish (average fish weight equals 0.26 g) of genistein by i.p. injection, with a positive control group treated with 300 ng/fish of 17-beta-estradiol, while a negative control group received a vehicle-only (corn oil) injection. Content of vitellogenin, the yolk glycoprotein made in the liver in response to estradiol stimulation, was measured using Western blots. Circulating estradiol and testosterone levels were measured using a steroid-enzyme immunosorbant assay. The ability of ovaries and testes to synthesize and release estradiol and testosterone was determined by ex vivo incubation of gonads with 25-hydroxycholesterol. Vitellogenin, while induced by 17-beta-estradiol, was not increased in the liver of individuals treated with genistein. In females, genistein treatment at 750 and 30,000 ng increased the estradiol production of ovaries more than the 17-beta-estradiol treatment. In males, genistein treatment resulted in decreased testosterone production from ex vivo testis and a comparable reduction in circulating testosterone level. The changes in vitellogenin, circulating steroids and ex vivo steroidogenesis in medaka in response to genistein are similar to that of 17-beta-estradiol. However, some endpoints are more sensitive to estradiol treatment (vitellogenin), while others are more sensitive to genistein (male testosterone and ovarian estrogenesis).     

Monitoring the reproductive status of Japanese monkeys (Macaca fuscata) by measurement of the steroid hormones in fecal samples

The menstrual cycles as well as the pregnancy in female Japanese monkeys (Macaca fuscata) were monitored by measuring the fecal estradiol concentrations and relative amounts of fecal progesterone. Steroids from fecal samples were extracted by using a previously developed simplified two-step method and then measured by radioimmunoassay. We successfully demonstrated that the two-step method is effective and convenient for monitoring the reproductive status of Japanese monkeys.     

Assessment of growth characteristics,the survival rate and body composition of Asian sea bass Lates calcarifer (Bloch, 1790) under different feeding rates in closed aquaculture system

One of the essential factors to be addressed in the development of aquaculture is the feeding regime. This study was investigated to assess the effects of feeding rate on growth performance, feed utilization, chemical body composition survival rate, cannibalism and morphological indices of Asian Seabass, Lates calcarifer. Intended for the trial, one hundred forty sea bass individuals with an average weight 5.47 ± 0.11 g were randomly distributed in 4 concrete tanks (914 cm × 183 cm 122 cm) each; length × width × depth) and volume 18,399 L, for total 68 days. The fishes were fed with a pelleted diet containing 46% crude protein for different feeding groups designated as (T1, T2, T3 and T4). The feed was supplied with a rate of (T1) 3%, (T2) 4%, (T3) 6%, and (T4) 9% of fish biomass per day and feeding frequency were maintained three times per day to all the groups. At the end of the trial water physicochemical parameters was in acceptable range for Asian sea bass growth. The average daily weight gain (g), weight gain (g) and specific growth rate (%) was significantly higher (p < 0.05) in T3 and T4 as compared to T1 and T2 group. The poorest feed conversion ratio was recorded in T1 group with 3% biomass per day. The cannibalism rate was significantly (p < 0.05) higher in T1 (3%) compared to T3 and T4 treatment. The morphological indices, condition factors (CF), viscerosomatic index and hepatosomatic index (HSI) was significantly higher in T4 group as compared to other treatments. The protein, moisture and ash contents of the whole biomass of the Asian sea bass were not significantly influenced by feeding rate. The fat levels in the fish bodies increased significantly (p < 0.05) with increasing feeding ratio. The phenomenal regression indicates that 6.5% feeding rate per day is optimum for best growth performance, survival and minimum cannibalism rate for Asian sea bass in captivity. The outcome of the finding will help in promotion for not only the coastal aquaculture in Pakistan abut also elsewhere.     

Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

Molecular cloning of protamine-2 and expression with aging in Japanese monkey (<Emphasis Type="Italic">Macaca fuscata</Emphasis>)

In order to isolate genes relating spermatogenesis with postnatal development and aging, we have attempted to obtain genes showing differences in expression in testis of Japanese monkeys (Macaca fuscata) by means of differential display PCR, and we have cloned, sequenced and characterized protamine-2 (PRM2) of Japanese monkey. The predicted open reading frame encoded a protein of 103 amino acid residues, most of which are common to those of other mammals. Northern analysis revealed that the PRM2 gene is expressed at adult and aged stages, but not at the juvenile stage. In situ hybridization revealed that the PRM2 gene is expressed mainly in late spermatids and its expressional potential is decreased from adult to aged stages. It suggests that PRM2 in spermiogenesis is mediated by the age of the animal.     

Molecular characterization of expressed DQA and DQB genes in the California sea lion ( Zalophus californianus)

To date, there are no published MHC sequences from the California sea lion (Zalophus californianus), a thriving species that, by feeding high on the marine food web, could be a sentinel for disturbances in marine and coastal ecosystems. In this study, degenerate primers and RACE technology were used to amplify near-full-length (MhcZaca- DQB) and full-length (MhcZaca- DQA) expressed class II MHC gene products from the peripheral blood mononuclear cells of two California sea lions in rehabilitation. Five unique Zaca- DQA sequences and eight unique Zaca- DQB sequences, all encoding functional proteins, were identified in the two animals, indicating the presence of multiple DQ- loci in this species. An additional three Zaca- DQB sequences containing features compatible with pseudogenes or null alleles were also identified. Despite the identification of multiple DQA and DQB sequences, the degree of heterogeneity between them was extremely low. To confirm the limited degree of Zaca-DQ nucleotide variation between individuals, we used denaturing gradient gel electrophoresis to examine putative peptide binding region sequences from the peripheral blood leukocyte-derived RNAs of 19 wild-caught California sea lions from physically distinct populations. The pattern of Zaca-DQ sequence migration was identical between individuals and independent of geographical region. This apparent Zaca-DQ sequence identity between sea lions was confirmed by direct sequencing of individual bands. In combination, these findings raise important questions regarding immunogenetic diversity within this thriving species, and should prompt further research into the existence of a highly polymorphic sea lion class II MHC molecule with sequence features that support traditional peptide binding functions.