中华鳖胃肠胰内分泌细胞的鉴定与定位

应用过氧化物酶标记的链霉亲和素（Streptavidin-peroxidase简称S－P法）免疫组织化学技术，用抗胰高血糖素、胃泌素、生长抑素、5－羟色胺抗体对中华鳖（Trionyx simensis）的胃、肠、胰内分泌细胞进行研究，结果表明：5－HT细胞在消化道各段均有分布，以小肠密度最高，胃、大肠、直肠其次，食管最少。Som细胞在胃底部、幽门部和小肠各段密度较高，胃贲门部、大肠和直肠密度较低。Gas细胞分布于胃体部、幽门部和小肠各段；胃贲门部、大肠和直肠未检出Gas细胞。Glu细胞在消化道各段均未检出，在胰腺中均鉴别出Glu细胞和Som细胞。     

牙鲆生长抑素基因的表达及细胞定位

采用RACE和荧光定量PCR技术克隆牙鲆生长抑素(SS)基因cDNA序列并且分析了其在仔鱼期的表达规律,同时通过免疫细胞化学染色,对牙鲆胃肠道和脑垂体等器官的生长抑素分泌细胞进行定位。最终获取包含完整编码区的牙鲆SS基因cDNA序列,其编码区长为381 bp,推测编码的蛋白质为126个氨基酸,其前23个氨基酸为信号肽,分子量为14.3 ku,理论等电点为8.33。牙鲆仔鱼不同发育时期的SS基因表达量比较结果表明,出膜后仔鱼体内SS基因表达不断增加,到变态发育期变化不大,变态后期又开始显著增加。免疫组织化学染色结果表明,SS细胞存在于胃、胰腺和脑垂体中。在胃中,生长抑素分泌细胞主要分布于胃上皮层;在胰腺中,生长抑素分泌细胞零散地分布,血管周围较多;在脑垂体中细胞密度高,集中在腺垂体的周边区域。     

野生与养殖黄鳍鲷消化道中粘液细胞的类型及分布

采用常规石蜡组织切片及AB-PAS的染色方法对野生和养殖黄鳍鲷（Sparus latus）消化道中粘液细胞的类型及分布情况进行了比较观察研究。黄鳍鲷粘液细胞在消化道各部位均有分布,不同部位粘液细胞的类型和分布均有差异。食道中以Ⅰ、Ⅱ型粘液细胞为主;贲门部和胃体部只有Ⅰ、Ⅱ型粘液细胞分布,幽门部只有Ⅰ型粘液细胞;幽门盲囊、肠道及直肠都是Ⅱ型粘液细胞最多。野生与养殖黄鳍鲷粘液细胞的区别在于消化道的相同部位养殖鱼比野生鱼粘液细胞数量更多。根据研究结果推测粘液细胞的类型和分布与鱼的生活环境及食物相关,也可能与消化道中各种菌群的分布有关。     

平鲷消化系统形态学、组织学及组织化学研究

对平鲷(Rhabdosargus sarba)消化系统结构进行观察,采用HE和AB-PAS染色方法分别从组织学和组织化学方面进行研究.结果表明,平鲷口咽腔宽大,具不同类型发达齿;食道粗短,内表面分布纵行粘膜褶;胃分为贲门、胃旨囊和幽门,贲门胃小凹和胃腺最多,胃盲囊厚度最大;肠道具丰富粘膜褶;幽门与前肠交界处有幽门盲囊8～10个.肝小叶分界不明显,管状结构丰富.胰腺腺泡和胰岛分布于肝脏内大静脉周围,腺泡细胞染色深蓝色.根据AB-PAS染色结果将消化道粘液细胞分为4型,Ⅰ型为红色,Ⅱ型为蓝色,Ⅲ型为紫红色,Ⅳ型为蓝紫色.胃部町见极少量Ⅱ型及Ⅲ型粘液细胞,其余部位均以Ⅱ型粘液细胞为主.结果表明,粘液细胞消化系统不同部位的分布密度与对应部位消化功能相关.     

6 种胃肠激素样内分泌细胞在细鳞鲑消化道的定位

采用免疫细胞化学ABC方法,选择5-羟色胺(5-hydroxytryptamine,5-HT)、生长抑素(somatostatin,SS)、胰多肽(pancreatic polypeptide,PP)、胃泌素(gastrin,GAS)和P物质(substance P,SP)6种特异性哺乳类胃肠激素抗血清,对不同年龄段(1龄、2龄和3龄)细鳞鲑(Brachymystax lenok)的消化道内分泌细胞进行了免疫细胞化学定位研究.结果表明,仅在食管、胃贲门、胃体和胃幽门检出有5-HT、SS和PP阳性细胞的分布(除1龄鱼食道),且这3种内分泌细胞均大量定位于胃部;在前肠、中肠、后肠和直肠中均未检测到这3种内分泌细胞的阳性反应.在3个年龄段的细鳞鲑胃肠各部位均未检测到GAS、GLU和SP阳性细胞.细鳞鲑的5-HT、SS和PP与其他有胃鱼类的内分泌细胞一样,可分为2种类型,即开放型和闭合型,这类细胞主要通过腔分泌和旁分泌两种方式释放激素.5-HT、SS和PP这3种细胞在1龄幼鱼消化道内就已经发育成熟,其对胃肠道活动的调节作用已经与成鱼没有差别,细胞的分布密度随着细鳞鲑的年龄增长不断增加.本研究揭示了不同生长阶段细鳞鲑消化道中这6种胃肠激素内分泌细胞的发育特征,并阐明了这些胃肠激素细胞在细鳞鲑幼鱼消化道的分布、形态以及生理作用.     

[07]5种胃肠激素样内分泌细胞在斜带石斑鱼稚鱼期消化道中的定位

应用免疫细胞化学SABC方法,用5-羟色胺(5-Hydroxytryptamine,5-HT)、生长抑素(Sonmtostatin,Som)、P物质(substance P,SP)、胃泌素(Gastrin,GAS)和神经肽Y(Neuropeptide,NPY)5种特异性哺乳类胃肠激素抗血清对斜带石斑鱼(Epinephelus coioides)稚鱼期的消化道进行免疫细胞化学定位.结果显示,5-HT-和Som-样免疫活性细胞定位在斜带石斑鱼稚鱼的胃各部,包括胃贲门部、胃体和胃幽门部;SP-、GAS-样免疫活性细胞则在胃和肠均存在,其中SP-样细胞定位在胃体部和胃幽门部以及整个肠道,GAS-样细胞则从胃幽门部至肠道各段均存在;NPY-样免疫活性细胞仅在肠道中被检测到.根据这5种胃肠激素样内分泌细胞的形态、染色强度及其分布,可判断其在稚鱼期已经发育成熟,已像成鱼胃肠激素内分泌细胞一样,调节消化道的运动和消化液的分泌.本研究结果提示,斜带石斑鱼稚鱼死亡的原因可能与其胃肠激素样内分泌细胞的功能没有直接关系.     

黄鳝胃肠的生长抑素分泌细胞及超微结构

在肠卷石蜡法切片上，用ABC免疫组织化学方法，显示消化管全长的生长抑素分泌细胞（又称D细胞），结果只在胃体部显现，主要散在分布于胃腺中，个别出现在粘膜上皮。消化道内分泌细胞根据基部有否胞质突起，可分为4种类型，在黄鳝胃中均有，其中Ⅲ型数量最多，I、Ⅳ型次之，而Ⅱ型数量最少。由于产生生长抑素的D细胞在消化道内分泌细胞中作用多并且类型复杂，故黄鳝胃还是一个十分重要且复杂的内分泌器官。应用透射电镜，对黄鳝的胃肠作超薄切片观察显示在胃腺上皮细胞内含大量的微管泡系和一定量的酶原颗粒，表明其兼有泌酸及产生酶原的功能，属泌酸胃酶细胞。在肠上皮细胞游离面，含有大量微绒毛，但幼鳝比成鳝的长、密且整齐，提示幼鳝的肠上皮比成鳝有更强的吸收能力。     

日本鳗鲡胃肠胰内分泌细胞的鉴别与定位

应用过氧化物酶标记的链霉亲和素(streptavidin-peroxidase简称S-P法)免疫组织化学技术，用抗5-羟色胺、生长抑素、胃泌素、β-内啡肽、胰高血糖素抗体对日本鳗鲡的胃肠胰内分泌细胞进行研究。结果表明：日本鳗鲡胃肠道各段均存在5-HT和Som免疫活性内分泌细胞。Gas细胞分布于胃体部、幽门部和前肠、中肠；胃贲门部和后肠未检出Gas细胞。β-内啡肽、Glu细胞在胃肠道各段均未检出。在胰腺中均鉴别出Glu细胞和Som细胞。     

斑鳠消化系统组织结构及适应性特征研究

为了解斑鳠(Mystus guttatus)消化系统组织结构特征,采用形态解剖和组织学方法对斑鳠消化系统进行研究。结果表明,斑鳠为一种杂食性偏肉食性鱼类,解剖观察其消化道结构包括口咽腔、食道、胃、肠及肛门,肝脏和胰脏为其主要的外消化腺。组织学观察发现口咽腔上皮组织中有较为丰富味蕾和黏液细胞;食道粗而短,肌层发达,且含有丰富的黏液细胞;胃贲门部和盲囊部,体壁较厚,胃壁内侧有丰富的黏膜褶皱,黏膜层为单层柱状上皮,分布大量的分泌胃酸和胃蛋白酶原的管状腺体,胃幽门部内层为环肌层,外层为纵肌层;斑鳠肠道系数为(0.89±0.11),肠道分为前肠、中肠和后肠三部分,肠腔内有丰富黏膜褶皱,褶皱高度从前向后逐渐降低。肠黏膜上皮肠粘膜表层为单层柱状上皮,在黏膜上皮分布大量的黏液细胞。肝脏与胰脏分离,肝脏分为两叶,胰脏弥散分布于肠系膜上。斑鳠消化系统结构特征与其喜好摄食甲壳类动物密切相关。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.