Cadmium exposure of rainbow trout, Salmo gairdneri Richardson: effects on immune functions

Differential leucocyte counts, phagocytosis, humoral antibody response and the in vitro blasto-genetic response to mitogens (lipopolysaccharide and Concanavalin A) and to an antigen ( Vibrio anguillarum ) were studied in rainbow trout exposed to 0,0.7 or 3.6 μg Cd 1⁻¹ for 12 weeks.
Although the fish did not exhibit any clinical or histological changes, cadmium exposure was found to affect two of the immune parameters measured. The cellular response of fish immunized with V. anguillarum to the homologous antigen was significantly lower for splenocytes obtained from fish exposed to cadmium for 9 weeks (3.6 μg Cd 1⁻¹ group) than for splenocytes obtained from non-exposed fish. Conversely, the humoral antibody response to V. anguillarum O-antigen was higher in the 3.6 μg Cd 1⁻¹ group than in the non-exposed group. Protective immunity of fish vaccinated against V. anguillarum was equally as good in the cadmium-exposed group as in the non-exposed group. No cadmium-induced changes in differential leucocyte counts or in the proportions of phagocytic cells were observed.     

Effects of implanted dummy transmitters on mortality, growth and tissue reaction in rainbow trout, Salmo gairdneri Richardson

For the first time, workers in the U.K. with a Home Office licence may surgically implant transmitters into wild fish in order to track them. A laboratory study was designed to investigate the effects of intraperitoneally implanted transmitters on rainbow trout. No significant difference in mortality or growth occurred between control, sham-implanted and implanted groups of fish over a 7-month period. Transmitters became encapsulated by connective tissue, the composition of which is described. Three fish expelled transmitters via the body wall without subsequent mortality or morbidity. Histological evidence suggests pressure necrosis of the body wall adjacent to the tag to be the mechanism of expulsion. With good surgical procedure, intraperitoneal implantation appears to provide a suitable technique for attaching transmitters to fish, and may be preferable to existing methods for many fish.     

Biochemical polymorphisms in muscle and liver extracts and in the serum of the rainbow trout Salmo gairdneri

The frequency of polymorphic phenotypes determined by starch gel electrophoresis from six enzyme systems was investigated on 664 rainbow trout (stock I) originating equally from six full-sib families. The enzyme systems studied were CA, AKP, G-6-PD, SOD, AEP, and 6-PGD.
On material deriving from six parental matings totalling 212 offspring (stock II) the mode of inheritance of the first four enzymes (AEP and 6-PGD were not polymorphic) and the additional systems IDH, PGM, Alb, and Psta, not analysed in stock I, were investigated.
The G-6-PD system showed no polymorphism in the family material. The CA, PGM, Alb, and Psta systems were easily identifiable. Their mode of inheritance with two alleles each can be considered as proven. For SOD three alleles, in four out of six possible progeny types, were found, for which the postulated mode of inheritance was confirmed.
For IDH the mode of inheritance found by Allendorff & Utter (1973) was confirmed. This pattern shows two disomic gene loci, one of which is monomorphic, while the other carries four different alleles.
The number of alleles and their mode of inheritance for the AEP system, which was not clearly identifiable, could not be elucidated.     

Allelic differences in initial expression of paternal alleles at an isocitrate dehydrogenase locus in rainbow trout (Salmo gairdneri)

We examined a temporal series of embryos from 14 full-sib families of rainbow trout with starch gel electrophoresis to determine the time of initial detection of enzyme produced by ldh-3. Maternal enzyme was detected in unfertilized eggs, whereas paternal alleles showed evidence of initial expression after gastrulation and epiboly. Two alleles, 40 and 71, were expressed synchronously several days before the 114 allele. Measurement of enzyme activity by spectrophotometric analysis and serial dilution supported these observations. The degree of delay of expression of the 114 allele between families was coupled with other estimates of developmental rate. These data suggest the existence of allelic variation at a cis-acting genetic element controlling the pattern of ontogenetic expression of structural alleles at Idh-3.     

The saccus dorsalis of the rainbow trout,Salmo gairdneri Richardson

The saccus dorsalis of the brain of the rainbow trout, Salmo gairdneri Richardson, has been investigated by means of histological, cytochemical, enzyme-cytochemical, electron microscopical autoradiographical techniques. The saccus dorsalis is a rostro-dorsal evagination of the diencephalic roof, and consists of a partly folded epithelial wall separating the cerebrospinal fluid from the meningeal matrix fluid. The well-developed vascular system around the epithelial wall, consisting of capillaries with different diameters, seems to be part of the pineal vascular system. No structures were found that may be involved in a possible mechanical or nervous blood flow control. The single-layered epithelium consists of highly specialized cells of one specific type. These cells are mainly characterized by infolded basal membranes, long microvilli of a peculiar shape, non-folded lateral membranes bordering intercellular spaces, apical concentrations of elongate and cup-shaped macromitochondria, a basally located rough endoplasmic reticulum, an apically situated smooth endoplasmic reticulum and apical concentrations of micropinocytotic vesicles. Morphological evidence is presented of a multiple function of these cells: (1) fluid secretion, (2) extrusion of low molecular weight organic substances into the ventricular system, (3) uptake of high molecular weight substances, and (4) uptake of low molecular weight organic substances (aminergic neurotransmitters [GABA]) from the cerebrospinal fluid. The significance of light and dark cells is discussed. Indications of a possible innervation of the saccus dorsalis epithelial cells were not observed. The functional significance of the saccus dorsalis (possible analogue of the choroid plexus?) is discussed.     

The influence of dietary and waterborne zinc on heat-stable metal ligands in rainbow trout, Salmo gairdneri Richardson: quantification by 109Cd radioassay and evaluation of the assay

The ¹⁰⁹Cd binding assay of Eaton & Toal was critically evaluated and then used to assess the induction of cytosolic metal-binding ligands in rainbow trout exposed to Zn in the diet and/or in the water for 16 weeks. With purified rabbit Cd-Zn metallothionein (MT), ¹⁰⁹Cd binding and total Cd recovery in the assay were linear up to 5 μg of protein; gel chromatography revealed a single peak. With heat-denatured extracts of gill, liver and intestine from control and Cd- and Zn-injected trout, ¹⁰⁹Cd binding was generally linear with sample size. Gel chromatography demonstrated that ¹⁰⁹Cd was bound by a protein with the same apparent weight as MT (∼ 11000 daltons), but significant binding occurred also at three other regions [molecular weight (mol wt) >70 000, 30000 and <3000]. In the dietary/waterborne Zn exposure, induced ¹⁰⁹Cd-binding activity occurred not in the MT peak but in the low mol wt peak (< 3000). Activity in the gill rose in response to both dietary and waterborne Zn, but the liver did not respond. The maximum five-fold elevation in the gill was primarily a waterborne effect. In the intestine, the maximum rise was 25-fold due to both factors. The thresholds for induction were > 39 μg Zn^| in water, and > 90 mg kg ^| in the diet, but only when waterborne Zn was also high. There was no correlation between ¹⁰⁹Cd binding and acid soluble thiol levels, which tended to decline at higher Zn exposures.     

Immunocytochemical localization of hormone-producing cells within the pancreatic islets of the rainbow trout (Salmo gairdneri)

Summary A histological study of the pancreatic islets in rainbow trout, Salmo gairdneri, was undertaken in which polypeptide hormone-producing cells were localized, using immunocytochemical staining techniques. Four different celltypes were identified in this manner. These were the insulin, somatostatin, pancreatic polypeptide and glucagon/gastric inhibitory polypeptide (GIP) cells. The glucagon/GIP cell was designated thus as antisera to both hormones crossreacted with a common population of cells. A fifth cell-type, commonly referred to as a clear cell, was also identified although its secretory product is as yet undetermined. These functional cell types were compared to the standard tinctorial properties of pancreatic endocrine cells. The relationships of the various cell types with each other was also observed.     

Nutrient digestibility and growth responses of rainbow trout (Salmo gairdneri) fed different levels of cassava and rice

Seven groups of fingerling rainbow trout (Salmo gairdneri) were fed for 10 weeks on 0%, 10%, 20% and 30% of cassava or rice in isonitrogenous diets.Optimum growth and food utilisation was at 20% dietary cassava. High fibre content of the control diet did not suppress protein digestibility in this group. Rather, at all levels, protein digestibility was good and remained between 84.4% and 87.5%. However, in the control group, carbohydrate digestibility was very poor. The cassava diet which had the highest digestible energy as carbohydrate produced the best growth performance, food utilization and protein sparing. At the levels studied, the dietary carbohydrates produced no hyperglycaemic effect on the fish. There was no evidence of drastic adverse effects on the tissue and liver composition of the fish receiving these carbohydrates.     

Ultrastructural and histochemical study of the gallbladder epithelia of rainbow trout and tench

The simple, high columnar gallbladder epithclia of the rainbow trout (Salmo gairdneri, Rich.) and the tench (Tinca linca, L.) are composed of light and dark cells. Within the dark cells two types must be distinguished: less hydrated, but normal epithelial cells and degenerating cells. The latter seem to be eliminated from the epithelium through phagocytosis by monocytes and macrophages, which possibly correspond with the ‘basal regenerative cells’ of earlier investigations. Since they are mainly found near the basement membrane, an hypothesis is put forth that they do not enter the gallbladder lumen, but migrate back into the subepithelial connective tissue after phagocytosis. Light and less hydrated dark cells are specialized for absorption of water and dissolved bile salts and for secretion of mucus, which mainly consists of acid glycoproteins. Interspecific differences are seen with regard to both functions. So, in the tench only single microvilli arc developed apically, whereas a true brush border is seen in the rainbow trout. Thus, a higher absorptive rate can be assumed in the rainbow trout. Or, with respect to mucus secretion, in the rainbow trout the granular endoplasmic reticulum predominates, whereas in the tench the smooth endoplasmic reticulum, often associated with glycogen and even forming ‘glycogen bodies’, is better developed. In the rainbow trout fibrillar granules of highly condensed acid glycoproteins are seen to be secreted. In the tench only vesicles of flocculent content occur. Perhaps, these morphological and subsequent metabolic differences may be related to the various feeding habits of the predatory rainbow trout and the omnivorous tench. Seasonal variations as expected especially in the tench after winter rest could not be established.     

Esterases and phosphatases in the leucocytes of rainbow trout, Salmo gairdneri Richardson

A preliminary report is made on the distribution of acid phosphatases and acid esterases in the cells of rainbow trout. A modified technique for the cytochemical demonstration of acid esterases is given, resulting in clearer visualization. Lymphocytes undergoing transformation in the presence of phytohaemagglutinin show a parallel decrease in enzyme localization for acid esterase and acid phosphatase as transformation in culture proceeds.     

Exercise training and the stress response in rainbow trout, Salmo gairdneri Richardson

Plasma levels of catecholamines, cortisol, and glucose were monitored in rainbow trout during a 6-week forced swimming exercise programme. Compared to resting non-exercised controls, resting trained fish had lower levels of epinephrine, norephinephrine, cortisol, and glucose during the last 3 weeks of training. Initially, trained fish that were swimming had higher levels of epinephrine than resting trained fish. After 2 weeks of exercise, swimming did not significantly elevate epinephrine levels in trained fish. Glucose levels were consistently greater in swimming fish than in resting fish. At the end of the training period, exercised trout had lower (15–20%) oxygen consumption rates while resting or swimming than unexercised fish.
After a 5-month forced swimming exercise programme plasma levels of catecholamines and glucose were monitored in trained and untrained cannulated rainbow trout after 2 min of mild agitation. Trained fish showed an immediate (within 1 min) increase in the levels of epinephrine, but not norepinephrine and a delayed (within 15 min) increase in the levels of plasma glucose. Epinephrine levels returned to pre-stress levels within 15 min. Untrained fish had no significant increase in the plasma levels of norepinephrine, epinephrine, or glucose.     

Ultrastructural changes in the coronet cells of the saccus vasculosus from rainbow trout,Salmo gairdneri (Richardson), kept in sea water

Summary The coronet cells of saccus vasculosus of fresh-water living and sea-water adapted rainbow trout were studied with the electron microscope, with special regard to changes in the latter group. Only quantitative differences were observed, namely a raised number of mitochondria in the apical region and the head and also a concentration of the agranular endoplasmic reticulum with a higher amount of electron-dense material and vesicles around the Golgi saccules. Together, these findings suggest a secretory function for the coronet cell. A supposed transport of vesicles from the head region of the coronet cell out into the globules is suggested. Interrelation between primary and secondary vesicles is discussed.This work was supported by grants from the Royal Physiographical Society of Lund and the Faculty of Natural Sciences at the University of Lund.—I am greatly indebted to Mrs. Lena Svenre and Miss Inger Norling for excellent technical assistance.     

Induction of antibody-producing cells in rainbow trout, Salmo gairdneri Richardson; by flush exposure

Splenic antibody-producing cells were produced by rainbow trout that had been exposed to O-antigens extracted from Yersinia ruckeri and Aeromonas salmonicida by adding the concentrated antigen preparation directly into the water of the tank holding the fish for a flush exposure. This method was compared with the proven techniques of exposure: intraperitoneal injection or a 2 minute immersion of the fish in the antigen preparation. Dosage experiments showed that the production of antibody-producing cells was induced by the immersion of trout for 2 minutes in water with 5.0 μg/ml-1 (or more) with the Y. ruckeri O-antigen, or 500 μg ml-1 (or more) of the A. salmonicida O-antigen. Similar differences were evident when the respective antigens were added directly to the water.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

The immune response of wild rainbow trout, Salmo gairdneri Richardson, to naturally acquired plerocercoid infections of Diphyllobothrium dendriticum (Nitzsch, 1824) and D. ditremum (Creplin, 1825)

Natural infections of rainbow trout with two species of Diphyllobothrium result in a host inflammatory response encapsulating the plerocercoid. The encapsulating cyst, observed by light and electron microscopy, comprises leucocytes, fibroblasts and collagenous connective tissue and is infiltrated with a blood vascular network. An indirect immunofluorescence technique and enzyme-linked immunosorbant assay (ELISA) have shown that specific antibodies are elicited by the fish host to these Diphyllobothrium spp. These antibodies have been semi-quantitatively measured by ELISA and correlated with worm burdens in individual fish.     

Characterization of sugar moieties and oligosaccharide sequences in the distal intestinal epithelium of the rainbow trout by means of lectin histochemistry

Lectin histochemical study was performed on the distal intestinal mucosa of the rainbow trout. A panel of 25 biotinylated leetins for detection of sugar residues and oligosaccharide sequences of glycoconjugates on Bouin's solution-fixed paraffin embedded sections were used as probes. The avidin-biotin-peroxidase complex (ABC) was used to visualize the binding sites of lectins. A rich supply of carbohydrates was detected in this gut mucosal region. Marked differences in lectin labelling of goblet cells and enterocytes (absorptive cells) probably reflect various functional activities. The results are compared with those (scarce) obtained from other teleosts. Species differences in carbohydrate residue composition may be related to various feeding habits. The roles of posterior intestinal glycoconjugates in host-parasite interaction, macromolecular ingestion and immunological implications are discussed.     

Structure and function of the paraphysis cerebri in the rainbow trout,Salmo gairdneri Richardson

Summary The paraphysis cerebri of adult Salmo gairdneri is represented by a differentiated part of the pars impar telencephali of the telencephalic roof. It consists of a vaulted epithelial sheet, which displays only a few rostral evaginations and separates the cerebrospinal fluid (CSF) from the meningeal interstitial fluid. The fenestrated, sinusoidal portal system surrounding the paraphyseal epithelium appears to be part of a complex vascular bed of the dorsal telencephalic and diencephalic area. Myelinated and unmyelinated nerve fibers observed in the vicinity of the paraphyseal epithelium fail to make synaptic contact with paraphyseal cells. The single-layered epithelium is composed of characteristic, rather small, optically dense, cuboidal and cylindrical cells, apically mutually attached by junctional complexes including zonulae occludentes.These paraphyseal cells execute a high energetic and a moderate synthetic metabolism as indicated by ultrastructural, cytochemical and enzyme-cytochemical observations. Morphological evidence is presented for a multiple function of these cells in the regulation of the CSF: 1) water and solute elaboration into the ventricular system, 2) restricted uptake of high molecular weight organic substances from the CSF, 3) restricted uptake of low molecular weight substances from the CSF, but apparently not of GABA and of biogenic amines, 4) the formation and pinching-off of blebs as expression of a physiological mechanism not yet elucidated. The possible relationship between the level of development of the paraphysis cerebri and the sensitivity of animals to hydro-mineral metabolism is discussed.     

Effects of sampling on blood parameters in the rainbow trout, Salmo gairdneri Richardson

The effects of sampling and short-term storage of blood samples on the blood parameters of rainbow trout were evaluated by comparing values obtained from resting fish sampled via cannulae and from fish sampled by cardiac puncture. Sampling stress causes an increase in haematocrit value and a decrease in mean cellular haemoglobin concentration, indicating red cell swelling. These changes are aggravated by consecutive storage of samples. Also, blood ionic balance is affected. The K⁺ concentration of plasma increases and plasma Cl⁻ concentration decreases owing to sampling. During storage the plasma K⁺ concentration decreases far below resting levels in samples taken by cardiac puncture, whereas plasma Cl⁻ level returns to pre-stress levels. Owing to the sampling- and storage-induced changes in blood parameters, care must be taken to ensure that similar sampling procedure is employed throughout to allow reliable comparisons between groups to be made.     

Seasonal and diurnal rhythms of thyroidal status in the rainbow trout, Salmo gairdneri Richardson

The concentrations of thyroxine (T₄) and triiodothyronine (T₃) in the blood plasma of rainbow trout ( Salmo gairdneri Richardson) at intervals throughout the year have been measured by a chemically specific gas-liquid chromatographic (GLC) method. Mean hormone levels showed a seasonal variation, maximal levels of both hormones occurring in winter and minimal concentrations in mid summer. An apparent secondary maximum in mean T₄ and T₃ concentrations was observed in spring. (T₄)/(T₃) ratios have been found to be highest in winter and lowest in summer. A radioimmunoassay procedure, validated by GLC analyses, revealed the presence of a diurnal rhythm in serum T₄ levels of trout sampled in September and in April.