Rapid temperature‐dependent wound closure following adipose fin clipping of Atlantic salmon Salmo salar L

Three groups of Atlantic salmon were kept at a constant temperature of 4, 10 and 14 °C. The adipose fins were removed; six fish/group were sampled at 11 subsequent time points post‐clipping. Samples were prepared for histopathological examination to study the course of re‐epithelization. A score sheet was developed to assess the regeneration of epidermal and dermal cell types. Wounds were covered by a thin epidermal layer between 4 and 6 h post‐clipping at 10 and 14 °C. In contrast, wound closure was completed between 6 and 12 h in fish held at a constant temperature of 4 °C. By 18 h post‐clipping, superficial cells, cuboidal cells, prismatic basal cells and mucous cells were discernible in all temperature groups, rapidly progressing towards normal epidermal structure and thickness. Within the observation period, only minor regeneration was found in the dermal layers. A positive correlation between water temperature and healing rates was established for the epidermis. The rapid wound closure rate, epidermal normalization and the absence of inflammatory reaction signs suggest that adipose fin clipping under anaesthesia constitutes a minimally invasive method that may be used to mark large numbers of salmon presmolts without compromising fish welfare.     

Wound‐healing potential of curcumin in the carp,Labeo rohita

In this investigation, the effect of dietary administration of curcumin on the healing of skin wound in fish, Labeo rohita, has been reported. Fish were divided into three groups: control group (fish without skin wound), sham group (fish with skin wound without curcumin treatment) and curcumin‐treated group (fish with skin wound and subjected to dietary administration of 1% curcumin). Experiments were conducted for 30 days to assess the healing of skin wounds at different time intervals using scanning electron microscopy, histology, and mucopolysaccharide and enzyme histochemistry. In the curcumin‐treated group, healing of skin wounds was found to be enhanced than in the sham group as indicated by early restoration of morphology of the surface layer of epithelial cells; the density of the mucous goblet cells; the density of club cells in epidermal layer; and early granular tissue formation, collagen deposition and tissue remodelling in dermal layer. Furthermore, peroxidase and catalase enzyme activity showed increased endogenous defence system in the curcumin‐treated group compared with the sham group. It could be concluded that dietary administration of curcumin is beneficial in rapid healing of skin wounds in fish. Early healing of wounds could be considered to prevent the invasion of pathogens and to maintain the integrity of the surrounding tissue.     

Epidermal proteases of the Japanese eel

A fluorescent-sensitive assay was used to demonstrate the protease activity in the dorsal skin of Japanese eel (Anguilla japonica). Two distinct extracts were separately prepared from skin mucus and epidermal cell layers, with no mutual contamination. The epidermal extract was sensitive to various substrates, whereas there was no, or only marginal, susceptibility to the same substrates for the mucous extract. Optimum hydrolysis pHs of the epidermal extract was variable and below pH 7.0, and the optimum hydrolysis temperatures were between 40 and 50 °C. In addition, Tos-Phe-Ch₂Cl, chymostatin, CdCl₂, CuCl₂, HgCl₂ and ZnCl₂ inhibited protease activities to different extents. Several other reagents specifically affected the protease activities, and their induced effects were useful for the identification of epidermal proteases. The findings indicate that a proteolytic factor, exhibiting various enzymological specificities, is retained within epidermal cell layers of Japanese eel. This factor is composed of 4 distinct proteases, such as cathepsins L and B-like proteases, a serine protease and an aminopeptidase.     

养殖水体邻苯二甲酸二乙酯暴露对凡纳滨对虾壳膜几丁质酶的影响

邻苯二甲酸二乙酯(DEP)是常用的塑化剂,也是环境广泛存在的内分泌干扰物。采用酶促反应动力学法及实时荧光定量PCR检测分析养殖水体DEP暴露对凡纳滨对虾(Litopenaeus vannamei)壳膜几丁质酶的比活力和酶系列基因mRNA表达量的影响。结果显示不同浓度组的DEP暴露对壳膜几丁质酶比活力的影响不具有显著性;10μg/L DEP暴露120 h后,对虾壳膜几丁质酶系的LV-CHT2、LV-CHT3、LV-CHT7等三个基因的mRNA表达量均被显著抑制,但随着暴露时间延长,这种显著性差异转化成非显著性;而20μg/L DEP胁迫对LV-CHT2、LV-CHT3、LV-CHT7等三个基因的mRNA表达量没有显著性影响,不同浓度组的DEP暴露不同时间后对LVCHT5、LV-CHT6等两个基因的mRNA表达量也均不具有显著性影响。     

养殖水体邻苯二甲酸二乙酯暴露对凡纳滨对虾壳膜几丁质酶的影响

邻苯二甲酸二乙酯(DEP)是常用的塑化剂,也是环境广泛存在的内分泌干扰物。采用酶促反应动力学法及实时荧光定量PCR检测分析养殖水体DEP暴露对凡纳滨对虾(Litopenaeus vannamei)壳膜几丁质酶的比活力和酶系列基因mRNA表达量的影响。结果显示不同浓度组的DEP暴露对壳膜几丁质酶比活力的影响不具有显著性;10μg/L DEP暴露120 h后,对虾壳膜几丁质酶系的LV-CHT2、LV-CHT3、LV-CHT7等三个基因的mRNA表达量均被显著抑制,但随着暴露时间延长,这种显著性差异转化成非显著性;而20μg/L DEP胁迫对LVCHT2、LV-CHT3、LV-CHT7等三个基因的mRNA表达量没有显著性影响,不同浓度组的DEP暴露不同时间后对LV-CHT5、LV-CHT6两个基因的mRNA表达量也均不具有显著性影响。     

Lysis of pathogenic bacteria by epidermal cathepsins L and B in the Japanese eel

Cysteine proteases, cathepsins L and B, in Japanese eel epidermis are suspected bacteriolysins against aquatic pathogens. This work examines the lysis of three species of gram-negative pathogenic bacteria by the eel epidermal extract according to the assay method for cysteine proteases. The optimum pH for the lysis of the three species of bacteria were commonly determined to be 5.0, where both of eel epidermal cathepsins L and B caused active proteolysis. Four kinds of specific inhibitors for cysteine proteases strongly inhibited these catheptic proteolyses and bacteriolyses. The activities for the lysis of three types of bacteria were at a similar level, but the effects on the bacterium which infects predominantly eel skin largely varied with individual fish. These results suggest that epidermal cathepsins L and B are responsible for lysis of any pathogenic bacteria in the nonspecific defense of Japanese eel.     

Tissue localization of Aeromonas salmonicida in Atlantic salmon, Salmo salar L., following experimental challenge

Three groups of Atlantic salmon, Salmo salar L., were exposed to live, colony-forming, radiolabelled Aeromonas salmonicida bacteria in a bath challenge: (1) fish with artificial wounds; (2) fish with a reduced epidermal mucus layer caused by removal of the mucus layer on two occasions by a swabbing procedure; and (3) a control group of untreated fish. Fish were killed 2, 6 and 24 h after challenge, and radioactivity (cpm g^–1) was measured in the blood, mucus, skin, wound area, gills, anterior kidney, posterior kidney, spleen, midgut and hindgut. The highest levels of radioactivity were measured in the wound areas and in the gills. There was a significant positive correlation between the levels of radioactivity in the gills and blood, and between the mucus and skin at 2 h post-challenge. Two hours after the bath challenge, live A. salmonicida bacteria were found in the blood of fish in the 'swabbed' and 'artificial wound' groups, and not in the control group. Twenty-four hours after the bath challenge, the kidney of fish from all groups contained viable bacteria, whereas the blood was negative.     

Organic components from scallop shell increase expression of keratinocyte growth factor in human skin fibroblast

We observed previously that scallop shells contain organic components that have various useful applications for skin. The organic components from scallop shell (scallop shell extract) enhance the turnover rate of the rat skin epidermal layer and increase the rate of recovery of UVB-induced injury to rat dorsal skin. We investigated the effect of scallop shell extract on the expression of keratinocyte growth factor (KGF) from human skin fibroblasts because it has been reported that KGF promotes wound healing and reduces the generation of UVB-induced free radicals in skin. Scallop shell extract increased the mRNA expression level of KGF in skin fibroblasts and the secretion of KGF from the fibroblasts at the protein level. These results suggest that the scallop shell extract may promote recovery from UVB-induced injury in skin through an increase in the secretion of KGF from the fibroblast cell.     

Characterization of the pattern of cytokeratin proteins in the epidermal cells of loach,Misgurnus anguillicaudatus (Cyprinformes)

The pattern of cytokeratin proteins in the epidermal cells of loach was studied by immunotechniques and partial separation of the epidermal cells. Two monoclonal antibodies, namely 8F7 and 1C45, against the cytokeratin proteins of the loach epidermis were prepared. these two monoclonal antibodies exhibit distinctive results in immunohistochemical staining. The 8F7 monoclonal antibody stains mainly with the epithelial cells, while the 1C45 monoclonal antibody stains specifically with the club cells. The pattern of cytokeratin proteins in the club cells and the epithelial cells of various epidermal layers was further determined by partial separation of these cells. Immunoblotting analysis of the cell fractions confirms the cytokeratin proteins to be differentially expressed in the club cells and the epithelial cells. However, the cytokeratin proteins expressed in the epithelial cells of the basal, middle and outer layers are same. The results indicate that differentiation of the epithelial cells seems limited during their translocation from basal to upper layers, but in those cells that do differentiate into club cells, the cytokeratin pattern changes.     

河蟹盖虫病的防治研究

对成蟹养殖期,附着于成蟹体表的盖虫进行了形态学观察及其分类学鉴定,并做了不同药物的防治实验,其结果表明,７．５ｍｇ／ＬＺｎＳｏ４、６ｍｇ／Ｌ－１２ｍｇ／Ｌ蟹安、０．３ｍｇ／Ｌ孔雀石及３０ｍｇ／Ｌ福尔马林对盖虫有较强的杀伤效果。     

Studies on Herpesvirus scophthalmi infection of turbot Scophthalmus maximus (L.): Histopathological observations

Abstract. The histological features of the disease caused by infection of turbot by Herpesvirus scophthalmi are described. Infection of epidermal cells of skin and gill resulted in the formation of giant cells showing a variety of nuclear and cytoplasmic changes associated with the replication of the virus. The effects of the condition together with possible control measures are discussed.     

Imidazole‐dicarboxylic Acid Nickel Complex as a Novel Grafting Agent for Pearl Oyster,Pinctada martensii

In the pearl cultivation industry, the implantation of the nucleus is a critical step in the production process. The surgical insertion of the nucleus and the mantle results in a wound and stress that affect not only the survival chances of the pearl oysters but also the quality of the pearls produced. This study proposes a synthesized imidazole‐dicarboxylic acid nickel complex that may hold potential in assisting the wound healing and nacre formation processes. The acute toxicity of the complex to pearl oysters, Pinctada martensii, was examined in a dose–response study over a 90‐h exposure. The safe concentrations of the nickel complex determined using probit analysis were 0.916 mg/L and 0.471 mg/L for 72‐h and 96‐h treatments, respectively. The reliability of the regression was verified using the chi‐square test. The alkaline phosphatase (ALP) activity in different regions of the treated and control oysters, along with relevant blood biochemical indices, were measured over a 90‐d period to predict the potential effects of the nickel complex on wound healing and nacre formation. A significant increase in ALP activity in the mantle, together with elevated blood levels of Mg (II) and globulin, and reduced blood levels of Zn (II) and blood urea nitrogen were observed. These results indicate that the prepared nickel complex may hold promise in helping wound healing post mantle implantation, promote the secretion of nacre, and enhance the immunity of the oysters, thus ultimately improving the yield and quality of the pearls.     

Histopathology of culture‐associated skin erosions and papillary hyperplasia of Atlantic halibut,Hippoglossus hippoglossus (L.)

Atlantic halibut, Hippoglossus hippoglossus (L.), that were cultured in tanks with a smooth bottom (gel‐coated fibreglass) substrate developed papillary hyperplasia and skin erosions on the blind (ventral) side. No viruses, bacteria or parasites were observed in any sections of affected skin. Comparison of microscopic pathology with that of skin from normal, wild‐caught halibut showed severe epidermal proliferation with foci of severe mucous cell hyperplasia. Both epidermal thickness and mucous cell density were significantly greater in fish held on a smooth substrate compared to skin of healthy fish. Spongiosis was present, especially at the base of the papillary, hyperplastic epidermis, and there was a chronic inflammatory infiltrate in the scale pockets composed of lymphocytes, histiocytes and erythrocytes. Skin erosions had various degrees of epidermal loss, in some cases to the basement membrane. A predisposing factor for the epidermal sloughing may have been related to the spongiosis, and the vacuolated, degenerated basal cells. When affected halibut were cultured for an additional 28, 62 or 97 days on a sand substrate, which is conducive to skin lesion healing, there was no apparent change in epidermal thickness over time. However, mucous cell density significantly increased from day 0 to 97 during the healing process.     

Epidermal response of the Japanese eel to environmental stress

Nonspecific responses of Japanese eels to environmental stress were monitored by assaying various lytic activities in eel epidermal extract. In fish maintained at 10 and 30 °C for up to 10 days, epidermal proteolytic activities due to serine protease and aminopeptidase and hemolytic activity varied within a 2-fold value range. Other proteolytic activities, due to cathepsins B and L, in the fish at 30 °C increased for up to 8 days and were 3.4 and 2.9-fold over those in fish maintained at 10 °C, respectively. This was accompanied by a 3.0-fold increase in bacteriolytic activity. Other forms of stress were exerted on the fishes by immersing them in a suspension of Flavobacterium columnare or giving them intraperitoneal injections of Edwardsiella tarda over 72 h. Although serine protease and aminopeptidase activities and hemolytic activity in the fishes exposed to F. columnare changed marginally, and were similar to those in the control fish, cathepsins B and L activities in the infected fishes increased more than 1.5-fold over their initial values over a 48 h period, along with a 4.5-fold increase in bacteriolytic activity. No marked change was detected in any of the lytic activities of the fishes exposed to E. tarda. These findings indicate that epidermal cathepsins B and L probably participate in bacteriolysis associated with Japanese eel skin and that their activities are elicited by environmental stimuli and may be an important nonspecific response of eels. Abbreviations: Cbz – carbobenzoxy; MCA – 4-methylcoumaryl-7-amide.     

不同个体大小和环境因子对瘤背石磺水中耗氧率及皮肤特性的影响

本研究设定了3种不同规格,以及温度(15℃、20℃、25℃和30℃)、盐度(5、15、25、35和45)和pH(7.5、7.8、8.1和8.4)3种主要环境因子,通过测定瘤背石磺(Onchidium struma)耗氧率和呼吸器官组织结构的变化研究了其对瘤背石磺呼吸的影响。结果显示,个体较小的瘤背石磺的耗氧率显著高于个体较大的瘤背石磺,且温度、盐度和pH对瘤背石磺的呼吸均有显著影响(P0.05)。在低于25℃时,瘤背石磺的耗氧率和黏液腺长短径随温度的升高而增大,并在25℃时出现峰值;高于25℃后,耗氧率和黏液腺长短径随之减小;表皮厚度的变化趋势与其相反;每0.1 cm~2皮肤所含颗粒腺数目及长短径变化规律不明显。盐度组各项指标的变化规律或趋势与温度组相似,峰值出现在盐度15。pH实验组中,耗氧率和黏液腺长短径随着pH的增加而增加,表皮厚度和颗粒腺长短径却在减小,每0.1 cm~2皮肤所含颗粒腺数目先增大,在pH 8.1时达到最高值,随后急剧减少。随着时间的增加,各影响因子对瘤背石磺的呼吸强度均减小。由此得出,在水下,环境因子对瘤背石磺皮肤呼吸具有显著影响,在25℃、盐度15和pH 8.1时,呼吸代谢良好;而在15℃、盐度45和pH 7.5时,呼吸代谢受到严重胁迫,这为深入揭示瘤背石磺呼吸机制、物种保护和人工增养殖奠定基础。     

病原菌感染对虾体血淋巴组分影响的初步研究

本项实验通过虾体肌肉注射病原菌和人工创伤后用病原菌浸浴虾体后,用聚丙烯酰胺凝胶电泳法测定其对虾体血淋巴蛋白组分的影响。结果表明:病原菌可使血淋巴蛋白组分的分子量和含量增大或减小,从而使虾体表现出活力下降或死亡。     

Studies on the pathology of ulcerative dermal necrosis (UDN) in Swedish salmon, Salmo salar L., and sea trout, Salmo trutta L., populations

Abstract. Continuous visual observations of the signs of development of UDN in fish kept in aquaria were correlated with light microscopical, transmission and scanning electron microscopical results. The initial signs of the disease are circles of pathologically-changed epidermis. The tight junctions of the squamous cells within these mucus-free areas disintegrate resulting in the loss of the protective function of the zonula occludens. Subsequently, the intercellular spaces dilate and communicate with the exterior. Necrosis of the epidermal cells due to hydromineral disturbances occur simultaneously with fungal infections and marked responses of the melanophores. It is suggested that the fungal infections are triggered by metabolites of the necrotic epidermal cells. The epidermal cells are shed and the fungus determines the further course of the disease which terminates in large ulcers covered with fungal hyphae. No substantial evidence for the presence of a virus could be recorded at any stage of the disease. It is concluded that the disease is a squames and the fungus appears as an opportunist which causes the eventual death of the fish.     

Pathogenesis and autoradiographic studies of the epidermis of salmonids infested with Ichtyobodo necator (Henneguy, 1883)

Abstract. The sequential pathology of Ichtyobodo necator infestations of the skin of 0+ and 1+ salmon and rainbow trout was studied. Areas of greatest shelter from water currents were found to be most commonly infested and no parasites were found attached to the epidermis on the head of the fish. The parasite caused hyperplasia of the malphigian cells and exhaustion of the goblet cells below infestations, followed by spongiosis of the underlying epidermis. The epidermal plaque then sloughed off leaving a single layer of cells attached to the basement membrane. Cell kinetic studies showed that I. necator caused the cells immediately below infestations to divide, a markedly different pattern from that of normal teleost epidermal cell proliferation. The possibility that the parasite secretes some form of digestive enzyme is postulated. In areas where sloughing had occurred, the remaining malphigian cells were seen to be in the process of division.     

Effects of dietary taurine levels on epidermal thickness and scale loss in red sea bream,Pagrus major

Taurine is often added to artificial fish diets to compensate for a reduction in fish meal (FM). However, the taurine content of FM‐based diets is typically lower than in diets consisting of raw fish, even in diets where FM is the only protein source. We evaluated the effects of dietary taurine in FM‐based diets on epidermal thickness and scale detachability in red sea bream Pagrus major. We compared the effect of diets containing 0% (control), 0.3% (Tau‐0.3%), 0.6% (Tau‐0.6%) and 1.0% (Tau‐1.0%) taurine. Red sea bream (average body weight, 39 g) were fed these diets for 7 weeks. Taurine supplementation had no effect on growth, feed intake, feeding efficiency, or survival. However, the epidermal thickness was higher in fish in the Tau‐0.6% and Tau‐1.0% groups than in the control and Tau‐0.3% groups. Similarly, scale loss was significantly higher in the control group than in the Tau‐0.6% and Tau‐1.0% groups. Our results suggest that supplementation with >0.6% taurine (1.0% in diet) improves skin condition.