克氏原螯虾消化道内分泌细胞的鉴别与定位

目的 对克氏原螯虾（Procambarus clarkii）消化道的嗜银细胞及5种内分泌细胞进行鉴别与定位。方法应用整块组织Grimelius银染法和过氧化物酶标记的链霉亲和素（SP法）免疫组织化学技术结合生物统计学分析。结果嗜银细胞在克氏原螫虾消化道除幽门胃外的各段均有分布,位于消化道上皮细胞间及结缔组织中。五羟色胺（5-HT）细胞在除幽门胃外的消化道各段均有分布。生长抑素（SS）细胞仅在食道和后肠中有分布。胃泌素（Gas）细胞分布于除幽门胃和中肠外的消化道各段。胰高血糖素（Glu）细胞在除幽门胃外的整个消化道均有分布,在食道和贲门胃中最多。胰多肽（PP）细胞仅在肠道中有较多分布。结论克氏原螫虾消化道中存在多种内分泌细胞,它们的分布情况与其它甲壳动物存在一定的共性,然而也有其一定的特异性。     

Utilizing stomach content and faecal DNA analysis techniques to assess the feeding behaviour of largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus

In this study, the feeding behaviour of the non‐native invasive predatory fishes largemouth bass Micropterus salmoides and bluegill Lepomis macrochirus was studied in the Ezura River, a northern tributary of Lake Biwa, Japan. Prey composition was estimated based on visual examination of stomach contents and faecal DNA analysis to determine feeding habits of these predatory fishes. Stomach content analysis showed that native fishes (e.g. ayu Plecoglossus altivelis and gobies Rhinogobius spp.) and shrimps (e.g. Palaemon paucidens) were the major prey items for M. salmoides, while snails, larval Chironomidae and submerged macrophytes were the dominant prey items of L. macrochirus. Micropterus salmoides tended to select larger fish in the case of crucian carp Carassius spp., but smaller fishes in the case of P. altivelis and Rhinogobius spp. Faecal DNA analyses revealed prey compositions similar to those identified in predator stomach contents, and identified additional prey species not detected in stomach content inspection. This study demonstrated that both stomach content inspection and DNA‐based analysis bear several inherent shortcomings and advantages. The former method is straightforward, although identification of species can be inaccurate or impossible, whereas the latter method allows for accurate species identification, but cannot distinguish prey size or stage. Hence, integration of morphology‐based and DNA‐based methods can provide more reliable estimates of foraging habits of predatory fishes.     

鱼类Fc受体研究

Fc受体是免疫细胞表面一种重要受体分子,通过与免疫球蛋白Fc段结合触发多种生物学功能,是联系体液免疫和细胞免疫的桥梁。部分硬骨鱼中已经发现了Fc受体,在斑马鱼、斑点又尾鲴和鲤鱼中都克隆到了Fc受体的γ亚基,在鲨鱼和大西洋鲑中证明有能够与免疫球蛋白结合的Fc受体存在,并在斑点叉尾鲴、河豚和虹鳟中存在着类似α亚基的Fc受体。对鱼类Fc受体的发现和研究必将为了解鱼类的免疫机制及免疫进化提供重要的资料。     

Immunofluorescence study of the formation of evolutionary stable lens proteins in chick embryos]

In the lens of fishes (carp, spiny dogfish) beta-crystallins were identified which were characteristic also of reptiles, amphibians, birds and mammals (evolutionary stable beta-crystallins). The dynamics of the formation of such beta-crystallins in 5--14 days old chick embryos was studied by the indirect immunofluorescence method with antisera to fish lens. These proteins are reliably indentified first at the lens sections from 7--8days old chick embryos. At all stages under study these beta-crystallins are localized mainly in the epithelial cells and practically not found in the lens fibers. They were, however, found in the fibrous (central) part of developing lens as well by the method of immunoelectrophoresis.     

Epitopes associated with mature spores not recognized on Kudoa thyrsites from recently infected Atlantic salmon smolts

Atlantic salmon Salmo salar skeletal muscle was examined for Kudoa thyrsites by polymerase chain reaction (PCR) and positive fish were further examined by in situ hybridization (ISH) and immunohistochemistry (IHC). The infection was detected in 42% of salmon by PCR following a 60 d exposure to infective seawater at a temperature of 10 degrees C (= 600 degree-days, degreeD). The parasite was detected by ISH in skeletal and cardiac muscle but not in gill, kidney, spleen, liver, stomach, intestine, pyloric caeca and skin. None of 4 monoclonal antibodies (2F4, 4H2, 1H2, 3E8) raised against mature K. thyrsites spores reacted with the stages identified by ISH following a 600 degreeD exposure, but they did react with ISH-identified stages following a 1600 degreeD exposure. In contrast, a polyclonal antibody reacted with K. thyrsites stages in salmon with both 600 and 1600 degreeD exposures, suggesting that the parasite observed in 600 degreeD infections represents an antigenically distinct developmental stage of K. thyrsites.     

Parasites of wrasse (Labridae) in inshore waters of west Scotland stocked as cleaner fish of sea lice (Caligidae) on farmed Atlantic salmon

The prevalence, abundance and intensity of protistan and metazoan parasites were examined in the labrid fishes Ctenolabrus rupestris, Centrolabrus exoletus, Crenilabrus melops, Labrus bergylta and L. mixtus in west Scotland. The 22 parasite species identified do not appear to be a major threat to farmed Atlantic salmon. Few mortalities of wrasse stocked on salmon farms were due to resident parasites or to transfers from salmon.     

Comparative immunohistochemical study of Carassius RFamide localization in teleost guts in different salinity habitats

Carassius RFamide (C-RFa) is a peptide, isolated originally from the brain of Japanese crucian carp and sharing homologies with mammalian prolactin-releasing peptides. From the physiological aspect, it is known that C-RFa has contraction-promoting action on fish intestines, but its localization in peripheral tissues is unknown. We observed the localization of C-RFa in teleost guts using an immunohistochemical technique. C-RFa-like immunoreactive (irC-RFa) sites were observed in not only the smooth muscle cells in the longitudinal muscle layer, but also in both Auerbach's and Meissner's nerve plexus in the stomach, pyloric ceca and intestine. In epithelial mucous cells, irC-RFa sites were observed in the surface mucous cells in the stomach in freshwater fish (FW), and in the goblet cells of the apical sites in the villi of the pyloric ceca and intestine in all fish. In the stomach, irC-RFa sites were found in the fundic glands of the body regions in seawater (SW) and brackish water (BW) fish, but not in FW fish. This study confirmed that one of the functions of C-RFa is the smooth muscle contraction of the longitudinal muscle layer in digestive organs. We suggest that C-RFa may have functional roles in both central and peripheral neurotransmission. In addition, it appears that the difference in C-RFa localization of SW, BW, and FW fish reflects the adaptation of the stomach function to different salinity habitats.     

鲢,鳙,银鲫和团头鲂肠道内分泌细胞中3种肽激素的免疫组化研究

应用ＡＢＣ免疫组化技术,用抑胃多肽、５－羟色胺和内啡肽３种哺乳动物抗血清对鲢、鳙、银鲫和团头鲂的肠粘膜中内分泌细胞进行了检测。结果４种鱼的肠粘膜中均有抑胃多肽免疫反应内分泌细胞存在,但未发现５－羟色胺和内啡肽的免疫反应。抑胃多肽免疫反应内分泌细胞主要分布在前肠前段,并单个地散布在肠褶顶部上皮细胞与杯状细胞之间,多呈长梭形。本文比较了不同食性鱼类和其他动物肠道抑胃多肽免疫反应内分泌细胞的分布规律,讨论了用５－羟色胺和内啡肽免疫染色的结果。     

江豚(Neophocaena asiaeorientalis)的消化器官 Ⅰ.舌、食管、胃

江豚是属于鼠海豚科(Phocoenidae)的一种小型齿鲸。我国沿海以及大的江河、湖泊中均有分布。1926年秉志曾报道过江豚某些消化器官的大体结构。以后Howell(1927)和Pilleri等(1972)也分别描述了其胃的构造,可是都缺少组织学的观察,对江豚的舌、肠和胰等的叙述极简短。此外,Arvy等(1972)、Yamasaki等(1976)和Komatsu等(1980)对江豚的舌进行过不同程度的研究。     

Sortase inhibitor phenyl vinyl sulfone inhibits Renibacterium salmoninarum adherence and invasion of host cells

Renibacterium salmoninarum, the causative agent of bacterial kidney disease in salmonid fishes, is a Gram-positive diplococcobacillus belonging to the family Micrococcaceae. Analysis of the genome sequence of the bacterium demonstrated the presence of a sortase homolog (srtD), a gene specifying an enzyme found in Gram-positive bacteria and required for covalent anchoring of cell surface proteins. Interference of sortase activity is being examined as a target for therapeutic prevention of infection by several pathogenic Gram-positive bacterial species. In silico analysis identified 8 open reading frames containing sortase recognition motifs, suggesting these proteins are translocated to the bacterial cell wall. The sortase and potential sortase substrate genes are transcribed in R. salmoninarum, suggesting they encode functional proteins. Treatment of R. salmoninarum with phenyl vinyl sulfone (PVS) significantly reduced bacterial adherence to Chinook salmon fibronectin. In addition, the ability of the PVS-treated bacteria to adhere to Chinook salmon embryo cells (CHSE-214) in vitro was dramatically reduced compared to that of untreated bacteria. More importantly, PVS-treated bacteria were unable to invade and replicate within CHSE-214 cells (demonstrated by an intracellular growth assay and by light microscopy). When treated with PVS, R. salmoninarum was not cytopathic to CHSE-214 cells, whereas untreated bacteria produced cytopathology within a few days. These findings clearly show that PVS, a small molecule drug and a known sortase inhibitor, can interfere with the ability of R. salmoninarum to adhere and colonize fish cells, with a corresponding decrease in virulence.     

喂养不同饵料对匙吻鲟和鳙消化道微生物区系的影响

为了解消化道的菌群结构及影响因素, 用PCR-DGGE技术和序列分析调查投喂鲜活饵料(BI-L)和配合饲料(BI-C)的鳙肠道菌群, 和投喂鲜活饵料(PS-L, PI-L)和配合饲料(PS-C, PI-C)的匙吻鲟胃和肠道菌群。实验共回收测序16条DGGE条带, 系统发育分析显示鳙和匙吻鲟所有待测样本的优势菌为厚壁菌门(31.25%, 5条)。其他属于-变形菌纲(-Proteobacteria)(25%, 4条)、拟杆菌门(Bacteroidetes)(12.5%, 2条)、梭杆菌门(Fusobacteria)(12.5%, 2条)、蓝藻门(Cyanobacteria)(6.25%, 1条)、放线菌门(Actinobacteria)(6.25%, 1条)、-变形菌门(-Proteobacteria)(6.25%, 1条)。不同饵料喂养的匙吻鲟肠道菌群具有较高的相似性(49%), 不同饵料喂养的鳙肠道菌群也具有较高的相似性(59%), 说明这些生态位中存在着更稳定成型的微生物群落。然而, 两组不同饵料喂养的匙吻鲟胃样本菌群之间存在相当大的差异, 表明匙吻鲟胃部菌群可能更易受到饵料的影响。     

草鱼生肌因子5基因的克隆及其组织表达谱分析

目的:获得草鱼生肌因子5(Myf-5)基因序列,分析其在草鱼不同组织和不同发育阶段中的表达规律。方法:根据鲤鱼Myf-5基因序列设计引物,用草鱼肌肉组织总RNA,经RT-PCR扩增其Myf-5基因序列;利用半定量RT-PCR分析草鱼Myf-5基因在草鱼不同组织和不同发育阶段的mRNA表达特性。结果:获得了草鱼Myf-5基因开放读框序列723 bp,GenBank登录号为GU290227;该基因编码由240个氨基酸残基组成的蛋白,具有MyoD家族基因的典型性碱性螺旋-环-螺旋(bHLH)结构,其氨基酸序列与斑马鱼、鲤鱼、虹鳟、大西洋鲑等的同源性较高(74%~97%),与哺乳动物和禽类如人、小鼠、大鼠、猪、牛和鸡的同源性较低(56%~60%);在草鱼红肌、白肌、肝胰脏、肾脏、脑和肠中均检测到Myf-5基因的表达,红肌、白肌和脑组织中Myf-5基因mRNA的表达量显著高于其他组织(P<0.05);草鱼Myf-5基因的表达随着其生长发育呈下降趋势,在较大规格试验鱼(500 g)中的表达显著低于其他2种规格(50~60 g、120~130 g)的试验鱼(P<0.05)。结论:获得了草鱼Myf-5基因序列,其在红肌、白肌和脑组织中的表达量显著高于其他组织,并随生长发育呈下降趋势,为研究Myf-5在草鱼肌肉发育过程中的作用提供了基础资料。     

Cholecystokinin mRNA in Atlantic herring, Clupea harengus--molecular cloning, characterization, and distribution in the digestive tract during the early life stages

The mRNA of the peptide hormone cholecystokinin (CCK) was isolated from juvenile Atlantic herring, Clupea harengus, by RT-PCR. The open reading frame encodes a 137 amino acid-long precursor protein. The peptide sequence of herring CCK-8, DYMGWMDF, is identical to that of higher vertebrates and elasmobranchs, and contains methionine in the sixth position from the C-terminus, which has not been reported previously in teleosts. Expression analysis by in situ hybridization shows that positive endocrine-like cells were mainly located in the pyloric caeca and to a less extent in the rectum of the juvenile. A few positive cells were also found in the pyloric portion of the stomach and the intestine. CCK cells were present in all the larvae examined from the day of hatching onwards. Although the CCK cells were scattered throughout the whole midgut, no signals were detected in either the foregut or the hindgut. Since herring larvae have a straight gut, the distribution pattern of CCK cells seems to be reflected in the anatomy of the gut.     

Histamine in endocrine cells in the stomach. A survey of several species using a panel of histamine antibodies

Antibodies to histamine were used to examine the localization of the amine in cells of the stomach and upper small intestine of a great variety of species, including cartilaginous and bony fish, amphibia, reptiles (lizard), birds (chicken) and a large number of mammals. In all species gastric histamine was localized in endocrine cells (invariably found in the epithelium) and mast cells (usually with an extra-epithelial localization). The endocrine cells were identified as such by immunostaining with antibodies to chromogranin A and the mast cells were identified by toluidine blue staining. Histamine-immunoreactive endocrine cells were found almost exclusively in the acid-producing part of the stomach; only rarely were such cells observed in the pyloric gland area. They were fairly numerous in the gastric mucosa of the two subclasses of fish as well as in the amphibia and reptile species studied. Here, the majority of the histamine-immunoreactive endocrine cells seemed to have contact with the gastric lumen (open type cells) and were located in the surface epithelium (certain fish only) or together with mucous neck cells at the bottom of the pits. In the chicken, histamine-immunoreactive endocrine cells were numerous and located peripherally in the deep compound glands. They were without contact with the lumen (closed type) and had long basal extensions ("paracrine" appearance), running close to the base of the oxyntic-peptic cells. In mammals, the number of histamine-immunoreactive endocrine cells in the stomach varied greatly. They were particularly numerous in the rat and notably few in the dog, monkey and man. In all mammals, the histamine-immunoreactive endocrine cells were of the closed type and located basally in the oxyntic glands. They often had a "paracrine" appearance with long basal processes. Histamine-storing mast cells, finally, were few in both subclasses of fish as well as in the amphibian species and in the lizard. They were fairly numerous in chicken proventriculus (beneath the surface epithelium), few in the oxyntic mucosa of mouse, rat and hamster, moderate in number in hedgehog, guinea-pig, rabbit, pig and monkey, and numerous in cat, dog and man.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of a gonadotropin-releasing hormone antagonist on gonadotropin levels in Masu salmon and Sockeye salmon

The salmon gonadotropin-releasing hormone (sGnRH) is considered to be involved in gonadal maturation via gonadotropin (GTH) secretion in salmonid fishes. However, there is no direct evidence for endogenous sGnRH-stimulated GTH secretion in salmonids. In this study, to clarify whether endogenous sGnRH stimulates GTH secretion, we examined the effects of the mammalian GnRH (mGnRH) antagonist [Ac-Delta(3)-Pro(1), 4FD-Phe(2), D-Trp(3,6)]-mGnRH on luteinizing hormone (LH) levels in 0-year-old masu salmon Oncorhynchus masou and sockeye salmon Oncorhynchus nerka. First, the effects of the GnRH antagonist on LH release were examined in 0-year-old precocious male masu salmon. GnRH antagonist treatment for 3 hr significantly inhibited an increase in plasma LH levels that was artificially induced by exogenous sGnRH administration, indicating that the GnRH antagonist is effective in inhibiting LH release from the pituitary. Subsequently, we examined the effect of the GnRH antagonist on LH synthesis in 0-year-old immature sockeye salmon that were pretreated with exogenous testosterone for 42 days to increase the pituitary LH contents; the testosterone treatment did not affect the plasma LH levels. GnRH antagonist treatment slightly but significantly inhibited an increase in the testosterone-stimulated pituitary LH content levels. However, no significant differences in the plasma LH levels were observed between the GnRH antagonist-treated and control groups. These results suggest that endogenous sGnRH is involved in LH secretion in salmonid fishes.     

Growth and morphological changes in the stomach of newborn pigs during the first three days after birth.

Growth and morphological changes in the stomach of newborn pigs were examined during the first 3 days after birth. The stomach grew disproportionately faster than the body as a whole during this period. The growth was due to hyperplasia and hypertrophy during the first day and mainly to hyperplasia thereafter as gastric DNA content increased progressively after birth, and the protein:DNA and RNA:DNA ratios increased only on the first day. Histological and morphometric analyses revealed that the growth was more pronounced in the gastric body region than in the cardiac and pyloric regions, and more pronounced in the mucosal layer than in other layers. The percentage of mucosal volume occupied by parietal cells (volume density) and the number of parietal cells per unit volume of gastric mucosa (numerical density) increased significantly 3 days after birth in the cardiac and body regions, but not in the pyloric region, of the stomach. The observed morphological changes coincide with the known pattern of functional maturation during the immediate postnatal period. It is suggested that a high level of circulating gastrin and oral ingestion of milk-derived growth factors in the newborn pig contribute to these changes.     

Cells with radiating vesicles in different fish species

A study was made of the ultrastructure of cells with radiating vesicles from the head kidney of different fish species: Atlantic salmon Salmo salar L., carp Cyprinus carpio L., roach Rutilus rutilus (L.), bream Abramis brama (L.), high-body pickarel Spicara staris (L.), long-striped wrasse Symphodus tinca (L.), and from spleen of the rainbow trout Salmo gairdneri Richardson, and the tilapia Sarotherodon mossambicus Peters. A disposition of vesicles around centrioles was characteristics of all the investigated cells. Some differences were marked in the fine structure of vesicles.     

Microsatellite markers in common carp (Cyprinus carpio L.)

Microsatellite markers of the poly (CA) type in common carp ( Cyprinus carpio L.) are described. Clones containing a (CA) repeat were isolated from a common carp genomic library and sequenced. The number of repeats found was high compared to mammals but comparable with other teleost fishes. Classification of the repeats (perfect, imperfect and compound) are compared with the Atlantic cod ( Gadus morhua L.), rainbow trout ( Oncorhynchus mykiss ), and Atlantic salmon ( Salmo salar L.). A total of 41 primer sets were designed and tested for polymorphism on a test panel of eight animals (derived from outbred lines, inbred lines and gynogenetic clones). Thirty-two markers were found to be polymorphic. The heterozygosity in the outbred animals was 60·4%, 51·1% in the inbred animals and 0% in the gynogenetic clones. The average number of alleles among the eight animals was 4·7 per marker. Six markers (18·8%) gave an additional polymorphic amplification product besides the polymorphic amplification product in the expected size range. The possibility that these loci are tetraploid is discussed. The polymorphic loci described for common carp will be valuable as genetic markers for use in population, breeding, and evolutionary studies.     

Non-specific carboxylic esterase activity in the digestive tract of the perch, Perca fluviatilis L.

The distribution of non-specific carboxylic esterases (Ec 3.1.1) in the digestive tract of perch, Perca fluviatilis L., was investigated histochemically using 1-naphthyl acetate as the substrate. Strong enzymatic activity was present in the gastric glands and surface cells of the stomach, intestinal mucosa of the pyloric caeca, upper and middle intestine, pancreas (exocrine cells) and liver. The enzymatic activity in the lower intestine and rectum was weak. The activity was not demonstrated in the oesophagus or pyloric sphincter. In the intestine, the activity was localized in the columnar cells especially in the supranuclear cytoplasm. The enzymatic activity demonstrated in the digestive tract of perch using 1 -naphthyl acetate represents combined esterolytic and lipoproteolytic activity.