基于UPLC-Q-TOF/MS代谢组学探究虹鳟鳃对热应激的生理反应

全球气温日趋升高导致的水温上升可引起虹鳟(Oncorhynchus mykiss)代谢紊乱,为解析其在热应激下的代谢变化特征,研究基于UPLC-Q-TOF/MS代谢组学技术,探究了虹鳟鳃靶器官在高温暴露(20℃和24℃)及恢复到初始温度(14℃)下的代谢生理反应。研究结果表明,与对照组相比,在20℃、24℃高温组和14℃恢复组中分别鉴定出128、130和108种差异显著代谢物。在高温暴露下,亚油酸、花生四烯酸等与脂质代谢相关的代谢物及还原型辅酶Ⅱ(NADPH)、谷胱甘肽(GSH)、谷胱甘肽二硫化物(GSSG)等与细胞氧化还原状态相关的代谢物均发生显著改变。富集分析表明这些代谢物主要涉及虹鳟鳃的甘油磷脂代谢、鞘脂代谢、亚油酸代谢、花生四烯酸代谢、磷酸戊糖途径和谷胱甘肽代谢等代谢通路,但在温度恢复到14℃后,除鞘脂代谢外,其他代谢途径均未恢复至正常状态。上述结果表明,热激导致了虹鳟鳃靶器官的脂质代谢紊乱,可能导致鳃细胞膜的结构和功能的损伤,诱使鳃细胞出现炎症反应,并产生免疫应答。同时,虹鳟鳃细胞通过磷酸戊糖途径产生的NADPH来调节谷胱甘肽代谢中GSH/GSSG比值以提高细胞的抗氧化能力来...     

Changes in the mucous cells of the gills, buccal cavity and epidermis of the nine-spined stickleback, Pungitius pungitius L., induced by transferring the fish to sea water

A histochemical analysis of mucous cells in the gills, buccal cavity and epidermis of the nine-spined stickleback, Pungitius pungitius L., using alcian blue and PAS, showed that most cells contained more than one type of glycoprotein. In all regions, the cell content of sialylated glycoproteins increased, and of sulphated glycoproteins decreased during adaptation to sea water. The histochemical properties of the cuticle-secreting cells in the epidermis were less easy to study, but it seems that their content of neutral glycoproteins increased and their content of acid glycoproteins decreased, during adaptation to sea water. Mucous cells were generally less common in seawater-adapted animals, though in the epidermis of fish kept in sea water for 21 days, there were as many as in the freshwater controls. Sulphated glycoproteins may be more effective than others in freshwater osmoregulation for they are highly charged and could attract cations strongly. They may also help to combat bacterial and fungal infections, and perhaps these are less likely when laboratory fish are transferred to sea water.     

The distribution and function of mucous cells and their secretions in the alimentary tract of Arrhamphus sclerolepis krefftii

The mucosa of the mouth, pharynx, oesophagus and rectum of Arrhamphus sclerolepis krefftii contain saccular mucous cells and the lining of the intestinal mucosa contains goblet mucous cells. Saccular mucous cells in the buccal epithelium are present in relatively low densities and contain acidic and neutral glycoprotein-secreting cells in an approximately 1:1 ratio. The saccular mucous cells in the mucosa of the pharynx, oesophagus and rectum are abundant and contain acidic glycoprotein which consists principally of sialomucin with traces of sulphomucin distributed around the periphery of the mucous vacuoles. Goblet cells in the intestinal mucosa contain neutral glycoprotein. Mechanically digested plant material within the lumen of the gut is bound by a sheath of acidic glycoprotein which is in contact with the intestinal mucosa. From these observations and with information on the known properties of acidic glycoproteins, a novel mechanism for the involvement of mucus in the extraction of nutrients from plant material mechanically digested by fish is proposed.     

Morphochemical analysis of mucosubstances in some epithelial tissues of the eel (Anguilla japonica).

In attempts to gain insight into the nature of mucosubstances in the eel epithelial tissues, the epidermis and epithelia lining the gill and intestine of the fish have been reacted for a series of light microscopic stainings of mucosaccharide and protein histochemistry under normal and experimental conditions. The present morphochemical analyses indicate that (1) the mucosubstances of the epidermis are found within two well differentiated cell types, goblet and clavate cells which elaborate a neuraminic acid containing mucosaccharide with vicinal hydroxyl, sulfate and carboxyl groupings and a glycoprotein respectively and (2) the mucosubstances of the gill and intestinal epithelia are found within goblet cells which elaborate a mucosaccharide with histochemical properties comparable to those of mucosaccharide within the epidermal goblet cells. The histochemical characteristics of all these mucosubstances have been briefly discussed in relation to the activities of the epithelial tissues in the eel.     

Surface secretions of the skin of Blennius (Lipophrys) pholis L.

Mucus is secreted to the surface of the body and fin webs of Blennius pholis by superficial epithelial cells and by goblet cells. Some goblet cells secrete sulphated acid glycoproteins, others produce a mucus which is neutral or mixed in its reactions. The superficial epithelial cells of these areas secrete sulphated acid glycoproteins, seen by electron microscopy as electron-lucent or moderately lucent vesicles; this secretion is not normally visible external to the skin in transmission electron microscope (TEM) sections. These cells do not react to the bromphenol blue test for proteins. Over part of the surface of the pelvic fins and the distal parts of the rays of the pectoral fins, the skin contains no goblet cells and bears a thick external secretion, or cuticle, containing protein and glycoprotein which is mainly neutral in reaction, although some cells at the edges of the region secrete weakly sulphated or non-sulphated acidic glycoprotein. The protein content of the columnar superficial epithelial cells of these regions correlates with the fibrous nature of the secreted cuticular layer as seen by TEM; the columnar cells are characterized by extensive ribosomal endoplasmic reticulum and vesicles which stain darkly with phosphotungstic acid, less so with uranyl acetate. The distal part of the cell, containing these vesicles, reacts positively to the PAS stain. In some places the borders of the zones with fibrous cuticle are characterized by cuboidal superficial epithelial cells which give a strong positive reaction to alcian blue at pH 1.0, indicating the presence of sulphated acid glycoproteins, but also react positively to the bromphenol blue test for proteins.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Sulphur, thiols, and disulphides in the fish epidermis, with remarks on keratinization

Energy dispersive x-ray (EDX) analysis and qualitative and quantitative histochemistry were applied to study the distribution and contents of sulphur, thiols and disulphides in the epidermis of the river lamprey Lampetra fluviatilis , the lesser spotted dogfish Scyliorhinus canicula and the brown trout Salmo trutta fario . Thiols generally reacted weakly throughout the entire epidermis, whereas disulphide reactions were more distinct and differentiated. In the river lamprey, the concentrations of -S-S- groups clearly increased in the developing mucous cells from the stratum basale to the stratum superficiale; skein cells and granular cells reacted negatively to weakly. In the lesser spotted dogfish, amounts of disulphides appeared at moderate concentrations, and only goblet cells displayed a strong reaction. In the brown trout, filament cells showed low concentrations or weak reactions of disulphides, goblet cells and the most outer superficial cells stained strongly. Sulphur distribution and contents generally supported the histochemical observations in normal epidermis cells (absolute sulphur contents: 41–59 mM), only the brown trout showed high amounts of sulphur in the stratum basale (81 mM). The findings corroborate the view that there is an inverse correlation between keratinization and mucous secretion in normal fish epidermis. The sometimes distinct contents of disulphides in the outer mucous layer indicate that this system could endure higher mechanical stress than predictable from its large amounts of neutral glycoproteins.     

Transfection efficiency and cytotoxicity of cationic liposomes in primary cultures of rainbow trout (Oncorhynchus mykiss) gill cells

Immunisation of fish by immersion has been applied for inactivated, whole cell bacterins, where the gill epithelial cells are considered as one of the prime uptake sites. Antigen entry is a critical factor for delivery of vaccine antigens through the immersion route, also for DNA vaccines, and delivery systems like cationic liposomes may enhance uptake. In this study, the aim was to examine the efficiency of cationic liposomes as a means to transfect primary cultures of rainbow trout gill cells with plasmids encoding viral or reporter proteins. Furthermore, the effects of the concentration and composition of liposomes/lipoplex on the viability of the cells were evaluated. Transfection of the gill cells was possible with both plasmids following transfection with lipoplexes of a neutral charge. Low concentrations and neutral/negatively charged formulations were favourable with respect to the toxicity of the formulations. Given that the mucous barrier covering the gills is overcome, this system might be useful for the priming of the local immunity in the fish gills.     

Histochemical analysis of glycoproteins in the secretory cells in the gill epithelium of a catfish, Rita rita (Siluriformes, Bagridae)

Glycoproteins (GPs) were visualised histochemically in the secretory cells – the mucous goblet cells (the type A and the type B), the serous goblet cells, the club cells and the epithelial cells in the gill epithelium of Rita rita. The type A mucous goblet cells, the type B mucous goblet cells and the epithelial cells elaborate GPs with oxidizable vicinal diols and GPs with sialic acid residue without O-acyl substitution. In addition, GPs with O-sulphate esters are elaborated by the type A and GPs with O-acyl sugars by the type B mucous goblet cells. GPs are absent in the serous goblet cells and are with oxidizable vicinal diols in low moieties in the club cells. The analysis of the results elucidates interesting differences in the composition and concentration of GPs in the mucus elaborated by the epithelium of the gill arches and the gill rakers; and the gill filaments and the secondary lamellae indicating the potential importance of the glycoproteins at these locations. GPs elaborated on the surfaces of the gill arches and the gill rakers could be associated to assist in feeding activities and on the surfaces of the gill filaments and the secondary lamellae in the respiratory activity.     

Histochemical studies of mucosubstances in the epidermis of a nonscaly teleost Mystus (Mystus) vittatus Bl. (Pisces-Bagridae)

The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.     

Studies on the skin of plaice (Pleuronectes platessa L.)

A histological, histochemical and ultrastructural examination of the skin of wild and cultured plaice was carried out, using fish from each year class from 0+ to 4+. The skin was shown to be similar in general structure to that of other teleosts but a previously undescribed cell, designated the Eosinophilic Granular Cell, a dendritic secretory cell found throughout the basal layers of the epidermis, is described. It was fixed only by formalin or dichromate, and contained numerous acidophilic granules. Melanin-bearing macrophages were observed migrating through the epithelium, but no DOPA or tyrosinase positive cells were observed by the methods used. Mast cells were very common in the dermis but were only demonstrable by special techniques. The melanophore and guano-phore systems are described and although no melanophores or melanocytes were found in the unpigmented areas of partially pigmented hatchery-reared fish, the integrity of the guanophore system was complete in such fish.     

The viscosity and glycoprotein biochemistry of salmonid mucus varies with species,salinity and the presence of amoebic gill disease

Fish mucus has previously been reported to change in appearance and composition among species and in response to changes in salinity and disease status. This study reports on the mucus viscosity and glycoprotein biochemistry of Atlantic salmon (Salmo salar L.), brown trout (Salmo trutta L.) and rainbow trout (Oncorhynchus mykiss Walbaum) in freshwater and seawater, both naïve to and affected by amoebic gill disease (AGD). Cutaneous mucus viscosity was measured over a range of shear rates (11.5, 23, 46 and 115 s^–1), and non-Newtonian behaviour was demonstrated for all three species. Mucus viscosity was significantly greater in seawater than in freshwater for all species, and significantly lower in AGD-affected Atlantic salmon and brown trout. Mucus glucose, total protein and osmolality data indicated that differences in viscosity due to salinity were mostly attributed to changes in mucus hydration, while differences due to disease were mostly attributed to changes in mucus composition. Trends in gill mucus cell histochemistry included shifts in glycoproteins from neutral mucins in freshwater to acidic mucins in seawater, and shifts towards neutral mucins, with an increase in mucus cell numbers, in response to AGD. Results suggested that Atlantic salmon and brown trout are more similar to one another in their mucus profile than to rainbow trout. Atlantic salmon and brown trout both exhibited a whole-body mucus response to AGD, whereas rainbow trout exhibited only a local gill response. Findings hold implications for fish physiology and pathology, and indicate that future fish-disease management strategies should be species and condition specific.Communicated by I.D. HumeThe word mucus has been used in its noun form throughout the paper for clarity

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An erratum to this article can be found at .     

Histochemical studies of oxidoreductases, acid carbohydrates and glycoproteins in the epidermis of the electric eel Electrophorus electricus

The activities of various oxidative enzymes and the presence of acid carbohydrates and acid glycoproteins in the epithelial cells and the mucous goblet cells of the epidermis of the Teleost Electrophorus electricus have been determined by means of a series of selected histochemical techniques of light microscopy. The enzyme activities show a distribution pattern confined mainly to basal cell layers and outer cell layers with a comparatively lower gradient in the transitional cell layers. A mixture of sialic acids of both N-acylated and O-acylated types is found in the mucous goblet cells and the functional significance of mucous production is related to the first line of defense against pathogenic colonization. The higher incidence of various oxidoreductases distributed throughout the entire epidermis is correlated with their key role which can play in the processes of cell differentiation and cell multiplication except for those regarding keratin formation which is not produced in the epidermis of most fish.     

Histochemistry of glycoconjugates in mucous cells of Salmo trutta uninfected and naturally parasitized with intestinal helminths

Mucus secreted onto the surface of the intestine forms a physical barrier to invading parasites so that a possible attachment of helminths to the surface is prevented and their expulsion by peristalsis facilitated. In mammals, intestinal parasites induce hyperplasia and hypertrophy of intestine goblet cells and provoke changes in the mucus composition. In fish, this topic has received less attention. In the present investigation, histochemical methods were employed to compose intestinal mucous cell numbers and their glycoconjugate composition were compared by uninfected brown trout Salmo trutta and in S. trutta parasitized with Cyathocephalus truncatus or Pomphorhynchus laevis. When P. laevis was present in the intestine of the brown trout, the total mucous cell number, and the number of mucous cells containing acid or mixed glycoconjugates were significantly enhanced. No significant change in the total mucous cell number was detected in the intestine of fish parasitized with C. truncatus in comparison with uninfected brown trout. A significant increase was observed in the number of both acid (especially sulphated) and mixed glycoconjugates containing mucous cells as well as a significant decrease in the number of neutral glycoconjugates containing mucous cells. When intestinal helminths were present, the thickness of the adherent mucous gel increased. In a limited number of other fish species, the occurrence of gill and intestinal parasites has been reported to increase the mucosal glycoconjugate secretions. Our study is the first quantitative report on the effects of intestinal helminths on the density of mucous cells and mucus composition in a fish species.     

Immunogenicity of a recombinant infectious hematopoietic necrosis virus glycoprotein produced in insect cells.

A recombinant infectious hematopoietic necrosis virus (IHNV) glycoprotein (G protein), produced in Spodoptera frugiperda (Sf9) cells following infection with a baculovirus vector containing the full-length (1.6 kb) glycoprotein gene, provided very limited protection in rainbow trout Oncorhynchus mykiss challenged with IHNV. Fish were injected intraperitoneally (i.p.) with Sf9 cells grown at 20 degrees C (RecGlow) or 27 degrees C (RecGhigh) expressing the glycoprotein gene. Various antigen (Ag) preparations were administered to adult rainbow trout or rainbow trout fry. Sera collected from adult fish were evaluated for IHNV neutralization activity by a complement-dependent neutralization assay. Anti-IHNV neutralizing activity was observed in sera, but the percent of fish responding was significantly lower (p < 0.05) in comparison to fish immunized with a low virulence strain of IHNV (LV-IHNV). A small number of fish immunized with RecGlow or RecGhigh possessed IHNV G protein specific antibodies (Abs) in their serum. Cumulative mortality (CM) of rainbow trout fry (mean weight, 1 g) vaccinated by i.p. injection of freeze/thawed Sf9 cells producing RecGlow was 18% in initial trials following IHNV challenge. This level of protection was significant (p < 0.05) but was not long lasting, and neutralizing Abs were not detected in pooled serum samples. When trout fry (mean weight, 0.6 g) were vaccinated with supernatant collected from sonicated Sf9 cells, Sf9 cells producing RecGlow, or Sf9 cells producing RecGhigh, CM averaged 46%. Protection was enhanced over negative controls, but not the positive controls (2% CM), suggesting that in the first trial soluble cellular proteins may have provided some level of non-specific protection, regardless of recombinant protein expression. Although some immunity was elicited in fish, and RecGlow provided short-term protection from IHNV, Ab-mediated protection could not be demonstrated. The results suggest that recombinant G proteins produced in insect cells lack the immunogenicity associated with vaccination of fish with an attenuated strain of IHNV.     

Development of lamellar epithelial hyperplasia in gills of pantothenic acid-deficient rainbow trout, Salmo gairdneri Richardson

Two semisynthetic diets differing in the levels of pantothenic acid (PA) supplementation (0 or 40 mg kg⁻¹ diet) were fed to rainbow trout, Salmo gairdneri Richardson, initially weighing 0.7 g fish⁻¹. Each diet was fed to two tanks of fish and, at the start of the feeding trial, each tank contained 200 animals. The experiment was conducted for 28 days during which time, every 2 days, gill tissue was sampled and food consumption was determined. Lamellar hyperplasia was first detected in the gills of deficient fish on the 12th day sample, while reduced feed intake was first manifest at 16 days. Hyperplasia first appeared in the distal regions of the gill filaments, but the lesion rapidly progressed in a proximal direction and 35 of 40 fish examined on days 22–28 exhibited hyperplasia on more than 75% of the filament surface. Gill lamellar hyperplasia is a sensitive indicator of PA deficiency in the rainbow trout. Moreover, the lesion is specific to PA deficiency since its developmental pattern differs histologically from the lamellar hyperplasia of the non-nutritional gill diseases.     

The relationship between nitrogen output and changes in mucous cell function in the amphibious teleost Blennius pholis L. during aerial exposure

The numbers of mucous cells in the epidermis of the head and body and in the surface of the gill arch of the amphibious blenny, Blennius pholis L., were estimated on fish immersed in sea water and after 4 h aerial exposure. During emersion there appeared to be a considerable reduction in the frequency of epithelial mucous cells in the areas studied, although counts for the head epidermis were somewhat variable. A concomitant decline in the number of cells thought to be actively secreting was also recorded in tissue samples from both the head and gills, while in the body epidermis the potential for mucus-secretion was maintained close to the levels observed in immersed fish.
Histochemical studies revealed epidermal mucous cells containing either sialylated acid mucopolysaccharides or neutral mucins, or a mixture of these, in both the head and the body, whereas in the gill arch epithelia there were, in addition, cells containing sulphated acid mucopolysaccharides. After emersion, a disproportionate loss of cells containing neutral and sialylated mucus from the gill epithelia resulted in an increase in the proportion of secreting cells staining positively for sulphated acid mucopolysaccharides.
The results of this study are discussed in relation to nitrogenous excretion during aerial exposure.     

Identification of a molecular marker for type A spermatogonia by microarray analysis using gonadal cells from pvasa-GFP transgenic rainbow trout (Oncorhynchus mykiss)

The spermatogonia of fish can be classified as being either undifferentiated type A spermatogonia or differentiated type B spermatogonia. Although type A spermatogonia, which contain spermatogonial stem cells, have been demonstrated to be a suitable material for germ cell transplantation, no molecular markers for distinguishing between type A and type B spermatogonia in fish have been developed to date. We therefore sought to develop a molecular marker for type A spermatogonia in rainbow trout. Using GFP-dependent flow cytometry (FCM), enriched fractions of type A and type B spermatogonia, testicular somatic cells, and primordial germ cells were prepared from rainbow trout possessing the green fluorescent protein (GFP) gene driven by trout vasa regulatory regions (pvasa-GFP rainbow trout). The gene-expression profiles of each cell fraction were then compared with a microarray containing cDNAs representing 16,006 genes from several salmonid species. Genes exhibiting high expression for type A spermatogonia relative to above-mentioned other types of gonadal cells were identified and subjected to RT-PCR and quatitative PCR analysis. Since only the rainbow trout notch1 homologue showed significantly high expression in the type A spermatogonia-enriched fraction, we propose that notch1 may be a useful molecular marker for type A spermatogonia. The combination of GFP-dependent FCM and microarray analysis of pvasa-GFP rainbow trout can therefore be applied to the identification of potentially useful molecular markers of germ cells in fish.