Survival of ovarian somatic cells during sex change in the protogynous wrasse, Halichoeres trimaculatus

The three-spot wrasse (Halichoeres trimaculatus), which inhabits the coral reefs of Okinawa, changes sex from female to male. Sex change in this species is controlled by a social system. Oocytes disappear completely from the ovary, and male germ cells and somatic cells comprising testicular tissue arise a new during the sex change process. However, little is known of the fate and origin of the gonadal tissue-forming cells during sex change. In particular, the fate of ovarian somatic cells has not been determined, although the ovarian tissue regresses histologically. To approach this question, we analyzed apoptosis and cell proliferation in the sex-changing gonads. Unexpectedly, we found that few apoptotic somatic cells were present during sex change, suggesting that ovarian somatic cells might survive during the regression of the ovarian tissue. On the other hand, cell proliferation was detected in many granulosa cells surrounding the degenerating oocytes, a few epithelial cells covering ovigerous lamella and a few somatic cells associated with gonial germ cells at an early stage of sex change. Then, we found that proliferative ovarian somatic cells remained in the gonads late in the sex change process. Based on these results, we concluded that some functional somatic cells of the ovary are reused as testicular somatic cells during the gonadal sex change in the three-spot wrasse.     

吉富罗非鱼sIgM基因多克隆抗体的制备及组织定位分析

为研究sIgM在吉富罗非鱼体内的组织分布及其在亚细胞水平上的定位,本实验利用纯化的SIGM融合蛋白,按照常规方法免疫新西兰大白兔,制备了兔抗吉富罗非鱼SIGM多克隆抗体;并对吉富罗非鱼肠、脾脏及鳃组织进行了免疫组织化学分析以及胶体金免疫电镜分析。结果显示,ELISA检测所获得的抗血清效价为1∶256 000,该血清能与SIGM融合蛋白发生特异性免疫反应。在肠和鳃组织中,阳性信号存在于富含黏液细胞的上皮细胞层表面,在杯状细胞和黏液细胞中则不存在;在脾脏组织中,阳性信号存在于特定的细胞中。免疫电镜结果显示SIGM主要存在于肠上皮细胞膜附近,并且在肠组织上皮细胞膜表面的微绒毛处含量较多;在鳃上皮组织中,SIGM主要存在于包围鳃丝和鳃小片的上皮细胞及部分红细胞内;而在脾脏组织淋巴细胞内部高尔基体的分泌小泡处存在大量的胶体金颗粒。研究表明,sIgM在鱼体黏膜免疫中具有重要作用,这为探讨鱼类sIgM的特性及功能奠定了基础,并丰富了鱼类黏膜免疫的研究内容。     

Localization of major yolk protein in the digestive tract of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis>

In the present study, we examined the localization of the major yolk protein (MYP) in the intestine of the sea urchin Strongylocentrotus intermedius. First, partial MYP complementary DNA was isolated from the sea urchin intestine. The expression level of MYP messenger RNA (mRNA) along the sea urchin digestive tract is highest in the intestine, so we performed in situ hybridization and immunohistochemical analysis using this tissue. No MYP mRNA was detected in the luminal epithelium, connective tissue, muscle tissue, or coelomic epithelium by in situ hybridization analysis. Positive immunohistochemical staining was observed in the luminal epithelium, inner epithelium and connective tissue, the signal being strongest in the latter. We conclude that MYP synthesized in the inner epithelial cells is moved to and stored in connective tissue and the luminal epithelium, before being secreted into the body cavity and the inner digestive cavity of the sea urchin.     

Scp3 expression in relation to the ovarian differentiation in the protogynous hermaphroditic ricefield eel <Emphasis Type="Italic">Monopterus albus</Emphasis>

Synaptonemal complex protein 3 (Scp3), which is encoded by scp3, is a meiotic marker commonly used to trace the timing of gonadal differentiation in vertebrates. In the present study, the ricefield eel scp3 cDNA was cloned, and a fragment encoding amino acids 49 to 244 was overexpressed. The recombinant Scp3 polypeptide was purified and used to generate a rabbit anti-Scp3 polyclonal antiserum. In adult ricefield eels, scp3 mRNA was predominantly detected in the gonads and faintly detected in discrete brain areas. In the gonads, Scp3 immunoreactivities were shown to be localized to the germ cells, including meiotic primary growth oocytes, spermatocytes, and pre-meiotic spermatogonia. During early ovarian differentiation, immunoreactive Scp3 was not detected in the gonads of ricefield eels at 6 days post-hatching (dph) but was found to be abundantly localized in the cytoplasm of some oogonia at 7 dph, coinciding with the appearance of the ovarian cavity and ovarian differentiation. At 14 dph, strong Scp3 immunostaining was detected on one side of the nucleus with a distinct polarity in some germ cells, presumably at the leptotene stage. Consistent with these results, the expression of scp3 mRNA was faintly detected in the gonads of ricefield eels at 6 dph, increased at 8 dph, and then remained relatively high thereafter. Taken together, these results suggest that the appearance of immunoreactive Scp3 in oogonia could be a marker for early ovarian differentiation in ricefield eels. The translocation of the Scp3 protein from the cytoplasm to the nucleus in the oogonium of ricefield eels appears to be a controlled process that warrants further study.     

香鱼背部脂肪腔的形态特征、显微结构及内容物成分分析

香鱼背部有一较为特殊的脂肪腔,但迄今尚未对脂肪腔形态、结构和成分进行研究。本实验采用常规方法对其进行了形态解剖、显微结构观察及内容物成分分析。结果显示,脂肪腔开始于头部上枕骨,止于脂鳍下方,呈长梭形,横切面倒三角形;脂肪腔质量[(1.95±0.86)g]占鱼体总质量[(39.31±5.12)g]的4.96%,长度[(9.17±0.82)cm]占鱼体全长[(17.23±0.72)cm]的53.22%;脂肪腔内有上下腺体、肌肉组织、未分化的间充质细胞、脂肪细胞以及分布其中的微血管;内容物的主要成分为粗脂肪(49.10%)、水(42.4%)、粗蛋白(6.8%)和灰分(1.13%);脂肪酸占内容物总量的34.266%,包含5种饱和脂肪酸和6种不饱和脂肪酸,其中,含量最高的为棕榈酸(11.800%),最低的为月桂酸(0.022%);氨基酸在脂肪腔中总含量为7.1%,其中含量最高的为谷氨酸(1.18%),最低的是蛋氨酸(0.10%)。     

凡纳滨对虾白便综合征的组织病理学观察

本文比较观察了不同规格的健康和患白便综合征凡纳滨对虾Litopenaeus vannamei肝胰腺、中肠、后肠、后盲囊等的组织结构。组织病理显示:白便综合征凡纳滨对虾肠道严重病变,肠腔空,环肌两侧增生大量成纤维细胞,这些增生细胞取代了肠上皮,而不见正常柱状上皮细胞;肝胰腺有不同程度和性质的病变,如腺管萎缩、崩解、坏死等。凡纳滨对虾患白便综合征的组织病变演变过程为:最初肠上皮基膜下出现一圈增生细胞,肠上皮柱状细胞脱离基膜;增生细胞持续增生增多,肠上皮崩解脱落至肠腔,增生细胞完全取代肠上皮;增生细胞不断增生,增生细胞层逐渐变厚,随着时间的推移,增生细胞层近腔端的一圈增生细胞坏死,出现一圈棕黄色物质,最终坏死细胞连同棕黄色物质脱落至肠腔。脱落至肠腔的腺管细胞、上皮细胞、增生细胞排出体外即为肉眼可见的白便。     

中华鳖淋巴心的组织学研究

采用解剖、石蜡切片和电镜制片方法,对中结鳖淋巴心的解剖部位、大小、显微结构和内容物的超微结构进行了观察。结果表明,中华鳖有３对淋巴心,位于肺部两侧的体腔外,通过结缔组织与背甲相连。测量和统计检验结果显示,其长径和短径均与背甲长呈直线正相关,性别间无显著差异。淋巴心呈椭球形,中央有１个腔,由淋巴心壁向内突出数条环形嵴状瓣膜。淋巴心壁的组织结构可分为４层,由内向外依次为主方上皮层、致密结缔组织层、横纹     

Evaluation of the effect of commercially available plant and animal protein sources in diets for Atlantic salmon (Salmo salar L.): digestive and metabolic investigations

The present study investigated the effect of various alternative diet ingredients partially replacing fishmeal (FM) on digestive and metabolic parameters in Atlantic salmon (Salmo salar L.) post-smolts (initial body mass 305 ± 69 g) following 12 weeks of feeding. Experimental diets containing 20 % extracted sunflower (ESF), pea protein concentrate (PPC), soy protein concentrate (SPC), feather meal (FeM) and poultry by-product (PBY) were compared to a reference diet containing FM as the main protein source. For the different intestinal compartments trypsin, lipase, bile salts, dry matter and chyme-associated leucine aminopeptidase (LAP) were measured from the content and LAP was measured in the tissue. Selected metabolites were measured in plasma samples. In general, use of plant proteins resulted in low C-LAP activity, low plasma cholesterol and high plasma magnesium. The plasma levels of cholesterol and Mg reflecting were most likely reflections of the composition of the diet, while the LAP activity in chyme may indicate lower epithelial cell turnover. Other responses varied depending on the plant protein source. Results from the animal protein substitution also varied both between diets and compartments; however, both materials increased lipase activity in DI. FeM resulted in a significant increase in both total and specific LAP activities suggesting an attempt to increase the digestive capacity in response to low digestibility of the diet while PBY showed very little difference from the FM-fed control fish. The present trial indicates that 20 % PPC, SPC and PBY can partially replace FM in diets for Atlantic salmon. The qualities of ESF and FeM used in this trial show little promise as FM replacement at 20 % inclusion level.     

饲料中添加柠檬黄对鲫肝、肠组织结构、抗氧化指标及肠道菌群的影响

为研究合成食用色素柠檬黄对水生生物的影响，本研究以初始体重为（35.0±1.5）g的鲫为研究对象，探究了不同剂量（0、1.4、5.5和10 mg/kg bwt/day）柠檬黄摄入对鲫肝、肠组织结构，抗氧化性能以及肠道菌群的影响。结果显示 ，与对照组相比，1.4 mg/kg bwt/day柠檬黄的摄入即可导致肠上皮细胞空泡化，肠绒毛变短；5.5 mg/kg bwt/day的摄入量引起肠绒毛断裂，而10 mg/kg bwt/day处理组病理现象更严重；肝脏组织学显示，1.4 mg/kg bwt/day柠檬黄摄入导致肝细胞细胞核萎缩、变形；5.5 mg/kg bwt/day的摄入量引起肝细胞核溶解，细胞空泡化增多；10 mg/kg bwt/day导致细胞界限模糊不清，细胞变形，空泡化加剧，肝血窦不可见，肝小叶结构模糊；1.4 mg/kg bwt/day柠檬黄摄入即可引起血清与肝脏中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）以及谷胱甘肽（GSH-Px）活性下降，丙二醛（MDA）含量升高；肠道内容物测序结果表明，柠檬黄的摄入引起鲫肠道微生物Chao1、Shannon和Simpson等多样性指数变化，一些有益菌（芽孢杆菌和梭状芽胞杆菌）数量明显减少，而一些致病微生物（如蛭弧菌和希瓦氏菌）的数量明显增加。 以上结果表明，柠檬黄的摄入会导致鲫肝、肠组织结构造成损伤，并对其血清抗氧化指标及肠道菌群造成不同程度的影响。本研究结果有助于进一步了解柠檬黄在水生生物体内的毒性机制，为水生态环境污染及水质检测提供科学依据。     

Changes of vitellogenin and Lipase in captive Sterlet sturgeon <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> females during previtellogenesis to early atresia

Plasma chemistry, lipid metabolism and vitellogenin gene expression of captive Sterlet sturgeon Acipenser ruthenus were studied in different maturity stages. A total of 32 fish were sampled, and maturity stages were identified on the basis of histological criteria and direct observation. Females were classified to four groups: previtellogenic, vitellogenic, post-vitellogenic, and atresia. Blood, gonad and liver tissue samples were taken through non-lethal biopsy. Our results showed that plasma levels of glucose, cholesterol, triacylglycerol, high-density lipoprotein, low-density lipoprotein, very low-density lipoprotein, calcium, phosphorus, alkaline phosphatase activity, albumin and total protein increased during ovarian development and were highest at post-vitellogenic stage. The lowest amounts in atresia stage demonstrate that lipid and energy imbalance was related to reabsorption and digestion of the yolk. These results suggested that the VLDL was the main plasma lipoprotein component of Sterlet. We determined that lipoprotein lipase and hepatic lipase activity increased during vitellogenesis process which suggested the role of lipase enzymes in regulating blood lipid metabolism. RT-PCR analysis indicates that Vitellogenin (VTG) mRNA could be detected both in livers and ovaries of female Sterlet. Throughout the study, the expression level of VTG gene showed an increase both in ovaries and in livers reaching its peak at late vitellogenesis stage. This strongly indicated a relation between VTG mRNA and ovarian development.     

Is the turbot,Scophthalmus maximus (L.), intestine a portal of entry for the fish pathogen Vibrio anguillarum?

The role of the intestinal tract in Vibrio anguillarum infection of turbot, Scophthalmus maximus (L.), fingerlings was investigated in two in vivo models and the possible mechanisms involved were studied in vitro. Viable V. anguillarum cells were detected in spleens from more than 50% of the fish administered the pathogen orally or rectally, suggesting that the intestinal tract is a portal of entry for V anguillarum. In transmission electron micrographs, V. anguillarum- like cells were seen close to the rectal epithelium, suggesting penetration of the mucus layer, but no epithelial cell penetration or endocytosis was evident. Attachment to intact turbot intestines was investigated, and 80% or more of the bacterial cells still remained attached after serial washings. A significantly higher number of cells attached to rectal segments than to the other intestinal segments. In vitro, V. anguillarum cells did not adhere specifically to intestinal mucus, but rather accumulated close to intestinal mucus interfaces and subsequently penetrated them. It is proposed that the intestinal ttact of tutbot is a portal of entry for V. anguillarum and that the cells penetrate the intestinal mucus overlaying the epithelial cells.     

The sexually dimorphic adipose fin is an androgen target tissue in the brown trout (Salmo trutta fario)

An investigation has been described on the relationship of body length, age and sex with adipose fin length and the number of androgen receptor (AR)-containing cells in the adipose fin as a secondary sexual characteristic for brown trout (Salmo trutta fario). Firstly, body and adipose fin lengths of 2- to 5-year-old brown trout were measured. Thereafter, these fish were killed by decapitation, then their sexes were determined, and adipose fins were excised. The cellular bases of AR binding activities in the adipose fins were analyzed with an antibody against human/rat AR peptide. Immunocytochemistry and western blotting techniques were performed with this antibody. Analysis of morphological measurements indicated that body length and age had a linear relationship with adipose fin length. The coefficients of determination for the body length and age were 0.92 and 0.85 in the male fish and 0.76 and 0.73 in the female fish against the adipose fin length, respectively. At 2 years of age, cells in the adipose fin did not exhibit AR immunoreactivity. However, AR-immunopositive cells were abundant in the adipose fin of 3- to 5-year-old fish. Moreover, the number of AR-immunopositive cells was significantly (P < 0.05) high in males and increased with age. These observations indicate that the adipose fin in the brown trout is a probable target for androgen action and that tissue function or development may to some extent be androgen dependent. In addition, it is likely that such an effect will be mediated by specific androgen receptors.     

Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon,Salmo salar L., with soybean meal‐induced enteritis

Extracted soybean meal (SBM) in the diet for Atlantic salmon, Salmo salar L., causes an inflammatory response in the distal intestine. The morphological changes of the epithelial cells and a characterization of the inflammatory cell infiltrate of the distal intestinal mucosa were studied using a panel of enzyme and immunohistochemical markers. The salmon (average body weight 927 g) used in the study were fed either a fishmeal‐based diet (control diet) or a diet in which 30% of the fishmeal protein was replaced with SBM protein (SBM diet). In salmon fed SBM, there were markedly reduced enzyme reactivities in the distal intestinal epithelial cells, both in the brush border [5′‐nucleotidase (5′N), Mg2+‐ATPase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP)] and in the intracellular structures [alkaline and acid phosphatase, non‐specific esterase (NSE) and alanine aminopeptidase (AAP)]. There appeared to be an increased presence of cells of monocytic lineage, including macrophages, as well as neutrophilic granulocytes and immunoglobulin (Ig) M in the lamina propria of the SBM‐fed fish. The mid intestine showed little response to the diet. The results suggest that toxic/antigenic component(s) of SBM affect the differentiation of the distal intestinal epithelial cells and may help explain the reduced nutrient digestibilities previously reported in salmonids fed extracted SBM.     

Immunofluorescence screening of Renibacterium salmoninarum in the tissues and eggs of farmed chinook salmon spawners

A simple and rapid on site-test method was used to screen farmed chinook salmon (Oncorhynchus tschawytscha) broodstock for bacterial kidney disease (BKD) at the time of spawning. Kidney tissue and ovarian fluid smears were examined, using the indirect fluorescent antibody technique (IFAT). To check the validity of our field screening procedure, intra-ovum fluid, taken from random samples of 300 surface-sterile, fertilized eggs, was examined in the laboratory for the presence or absence of Renibacterium salmoninarum, using the IFAT. Prior to sampling, eggs were incubated at 15°C for 40 days in enrichment KDM-2 broth. R. salmoninarum cells were found in one out of 10 samples of pooled contents of eggs from fish showing no R. salmoninarum in the kidney tissue (these fish were assumed to have R. salmoninarum-free ovarian fluid); three out of 10 pooled samples from fish in which R. salmoninarum was detected in the kidney but not the ovarian fluid; all of the 10 pooled samples from fish with R. salmoninarum in both the kidney tissue and the ovarian fluid. The number of R. salmoninarum in the pooled egg contents was low (2–23 per 100 fields). Using the IFAT technique it was not possible to determine whether the BKD-bacteria in the eggs were alive or dead. Methods of BKD treatment and screening are discussed as is the validity of using these techniques to prevent vertical transmission of BKD in farmed salmon.     

Localization of Zn-binding protein in the digestive tract tissue of common carp

ABSTRACT: The concentrations of Zn and sulfhydryl (SH) groups in the digestive tract tissue of common carp and some aquatic animals were studied. It was found that Zn and bound SH groups could be used as indicators for detecting the Zn-binding protein in the digestive tract tissue of common carp. The digestive tract tissue of the fish underwent subcellular fractionation, and it was found that the nuclei/cell debris fraction contained most of the DNA (85%), Na⁺/K⁺-ATPase (82%), organic phosphate (90%) and the Zn-binding protein (79%), but only part of the 5'-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the digestive tract tissue of common carp was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that treatment with collagenase type IV could release more than 50% of the Zn-binding protein, Na⁺/K⁺-ATPase and organic phosphate from collagen. Sections of digestive tract tissue of common carp were stained for Zn. It was observed that Zn can be found mainly on the edge of the epithelial layer, and everywhere in the 'membrane-like' portion of the submucosal and muscular layers. It is proposed that most of the Zn-binding protein in the digestive tract tissue of common carp is located on the basolateral plasma membranes of the epithelial cells and on the surrounding muscle cells that are attached to the collagen type IV of basal laminae.     

Light and electron microscopic studies on Epieimeria anguillae (Léger & Hollande, 1922), a coccidium parasitizing the European eel, Anguilla anguilla L.

Abstract. The foregut of eels naturally infected by Epieimeria anguillae (Léger & Hollande, 1922) was studied by light and electron microscopy. It has been established that this parasite, which develops in a characteristic location on the surface of epithelial cells, and was classified on this basis by Dyková & Lom (1981) as a member of the genus Epieimeria , undergoes intracellular merogony and gamogony similarly to other eimerians; however, its sporogony takes place outside the fish or intercellularly. The trophozoites and merogonic and gamogonic stages each develop in a para-sitophorous vacuole which is half embedded in the epithelial cell and protrudes into the intestinal lumen. The parasitophorous vacuote is surrounded by a single membrane; however, towards the intestinal lumen it is covered also by the cell membrane. In its location, Epieimeria anguillae resembles cryptosporidia, but differs from the latter significantly in its relationship with the host cell.     

饲料脂肪水平对吉富罗非鱼体脂沉积及脂肪酸组成的影响

研究了饲料脂肪水平对吉富罗非鱼(Genetic Improvement of Farmed Tilapia,GIFT)的部分形体指标、肌肉肝脏及腹腔脂肪组织的脂肪沉积及脂肪酸组成的影响,同时探讨了鱼体对饲料脂肪酸的吸收利用能力.实验设置饲料脂肪水平分别为1.73%,3.71%,5.69%,7.67%,9.64%,16.55%共6个梯度组,每组3个重复,饲养90 d.结果显示:5.69%,7.67%及9.64%脂肪组吉富罗非鱼肥满度较高1.73%和16.55%脂肪组的肝体指数显著高于其他实验组(P<0.05);除了1.73%脂肪组,其他各组中饲料脂肪水平越高,鱼体的脏体指数越高.7.67%脂肪组吉富罗非鱼肌肉脂肪含量显著高于3.71%组(P<0.05),同时显著低于16.55%组(P<0.05),但是其肝脏中脂肪含量与其他各组差异均不显著(P>0.05);饲料脂肪水平越高,鱼体的脂肪含量越高,同时不饱和脂肪酸在总脂肪酸中的比例越高.结果表明,饲料脂肪水平影响吉富罗非鱼的部分形体指标,尤其对肝脏形态的影响较为明显.饲料中过多的脂肪容易在肌肉和肝脏组织中沉积,同时鱼体的脂肪含量和脂肪酸组成能够反映饲料的脂肪水平和脂肪酸组成.本研究旨在为饲料脂肪水平的合理设置及脂肪营养对脂肪代谢的调控提供参考.     

Cytochrome P450 2AA molecular clone,expression pattern,and different regulation by fish oil and lard oil in diets of grass carp (<Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>)

Cytochrome P450 enzymes (CYP enzymes) catalyze important metabolic reactions of exogenous and endogenous substrates, including fatty acid. In this study, we cloned the complete CDS of the cytochrome P450 2AA (CYP2AA) gene from the grass carp (Ctenopharyngodon idella) for the first time. CYP2AA consisted of 1500 bp, which encoded a predicted protein of 499 amino acids. The identities of CYP2AA between C. idella and zebrafish were 86%. It consists of the conserved heme-binding motif FXXGXXXCXG. Quantitative real-time PCR analysis indicated that CYP2AA mRNA in C. idella was highly expressed in liver and adipose tissue. The effects of fish oil and lard oil in diets on expression of CYP2AA mRNA in vivo were also investigated. The fish oil (FO) group exhibited significantly higher CYP2AA expression in adipose tissue than the lard oil (LO) group (P?<?0.01), whereas the mRNA expression of CYP2AA was not notably different in liver. It suggested that the high abundance of CYP2AA mRNA expression in adipose tissue could be induced by fish oil. Our findings provided molecular characterization and expression profile of CYP2AA, and enhanced our understanding of CYP2AA in fish lipid metabolism.