Marsha L. Landolt

The relationship between diet and the immune response of fish

Abstract Diseases exert heavy economic losses in fish cultural facilities due to mortality, morbidity, poor product quality and costs associated with chemotherapy. Aquaculturists are, therefore, interested in developing cost-effective management strategies that can either prevent the outbreak or reduce the severity of epizootics. One management strategy currently under exploration is nutritional modification. This paper reviews investigations that have been conducted to evaluate the possibility of enhancing the immune response of fish through dietary supplementation with vitamins, minerals and other nutrients. Alterations in constitutive as well as inducible defence mechanisms are examined, as are changes in cell-mediated and humoral immune responses. Although results of the various investigations are variable, this field of inquiry offers promise for disease management.

Cytotoxicity/genotoxicity: The application of cell culture techniques to the measurement of marine sediment pollution

Abstract Fish cell cultures were used to determine whether in vitro aquatic animal cell culture systems were capable of detecting pollution in the marine environment. Cells derived from rainbow trout gonad (RTG-2) and bluegill fry tissues (BF-2) were used as model cell systems to measure cytotoxicity and genotoxicity following exposure to Puget Sound sediment extracts, benzo(a)pyrene and MNNG. Sediment was collected from several sites within Puget Sound known to be contaminated with compounds such as polycyclic aromatic hydrocarbons, polychlorinated biphenyls, chlorinated hydrocarbons and heavy metals. Each of the sediment samples was extracted with organic solvents and added to cultures of the two model cell systems in DMSO. Following exposure the cultures were evaluated for cell death, mitotic inhibition, stimulatory effects, and chromosomal damage. These cell cultures responded to the sediment extracts much as they did to known mutagenic/carcinogenic chemicals which were used as model compounds. Those sediments known to be contaminated, based on chemical analysis and historical use patterns were also found to be qualitatively and quantitatively more toxic than were sediments from areas which were relatively unaffected by human activity. The results show that in vitro cell systems are capable of detecting pollution in the marine environment and have the potential of being powerful tools in aquatic toxicology in conjunction with whole animals or as a method of screening materials prior to in vivo testing.

Benzo(a)pyrene-induced morphologic and developmental abnormalities in rainbow trout

To determine if the widespread environmental mutagen, benzo(a)pyrene (BaP), was embryotoxic or teratogenic in the rainbow trout (Salmo gairdneri R.), newly fertilized eggs were reared on sand experimentally coated with 1 to 500 ppm BaP. The system produced relatively constant aqueous BaP concentrations ranging from 0.08 to 2.99 ppb, levels comparable with those found in polluted rivers. Rainbow trout alevins reared in 2.40 ppb aqueous BaP for 36 days accumulated an average of 12.34 ppm BaP, and autoradiographic examination revealed accumulation of14C-benzo(a)pyrene primarily in the yolk sac and in developing neural and ocular tissues. Although no differences in either survival or hatching success were found between control and BaP-treated eggs, exposure to BaP did alter the length of the hatching process. Morphological abnormalities were significantly increased in BaP-treated alevins compared to controls at aqueous exposures of 0.21, 2.40, and 2.99 ppb. Insufficient yolk sacs, lack of body pigment, kyphosis, and abnormalities or absence of the eyes were among the anomalies present in alevins exposed to BaP.

In vitro toxicity of eight mutagens/carcinogens for three fish ceil lines

Recent interest in and concern for the quality of the environment has prompted a great deal of research into methods of measuring and assessing changes in it. One problem of major interest is that of increasing amounts of mutagenic/carcinogenic chemicals generated by modern society and released into marine and freshwater ecosystems (KRAYBILL et ai. 1977). Numerous techniques involving whole animal testing for cancer, cell culture for mutation and cytogenetic changes and bacterial mutagenicity have been devised to assay specific chemicals and inspect unknown mixtures (KILBEY et ai. 1977). Little, however, has been done to determine the effects of foreign chemicals on aquatic organisms (DIAMOND AND CLARK 1970).

Mapping the neutralizing epitopes on the glycoprotein of infectious haematopoietic necrosis virus, a fish rhabdovirus.

Twelve neutralizing monoclonal antibodies (MAbs) against the fish rhabdovirus, infectious haematopoietic necrosis virus (IHNV), were used to select 20 MAb escape mutants. The nucleotide sequence of the entire glycoprotein (G) gene was determined for six mutants representing differing cross-neutralization patterns and each had a single nucleotide change leading to a single amino acid substitution within one of three regions of the protein. These data were used to design nested PCR primers to amplify portions of the G gene of the 14 remaining mutants. When the PCR products from these mutants were sequenced, they also had single nucleotide substitutions coding for amino acid substitutions at the same, or nearby, locations. Of the 20 mutants for which all or part of the glycoprotein gene was sequenced, two MAbs selected mutants with substitutions at amino acids 230-231 (antigenic site I) and the remaining MAbs selected mutants with substitutions at amino acids 272-276 (antigenic site II). Two MAbs that selected mutants mapping to amino acids 272-276, selected other mutants that mapped to amino acids 78-81, raising the possibility that this portion of the N terminus of the protein was part of a discontinuous epitope defining antigenic site II. CLUSTAL alignment of the glycoproteins of rabies virus, vesicular stomatitis virus and IHNV revealed similarities in the location of the neutralizing epitopes and a high degree of conservation among cysteine residues, indicating that the glycoproteins of three different genera of animal rhabdoviruses may share a similar three-dimensional structure in spite of extensive sequence divergence.

Effects of benzo(a)pyrene on early development of flatfish

The ontogenetic effects of the environmental carcinogen benzo(a)pyrene (BAP) on three species of larval flatfish were investigated using concentrations (from 0.10 to 4.2 ppb) which were comparable to levels found in polluted harbors. BAP-treated sand sole (Psettichthys melanostichus) eggs displayed a significant decline in hatching success and a significantly higher incidence of developmental anomalies than did control eggs. Flathead sole (Hippoglossoides elassodon) eggs exposed to a single dose of a water-soluble BAP-bovine serum albumin complex demonstrated evidence of toxie injury with pycnotic nuclei present in the integument and, more commonly, in ocular and neural tissues. An increased incidence of morphological anomalies in English sole (Parophyrs vetulus) eggs and larvae exposed to BAP was not detected.

Histologic and skeletal abnormalities in benzo(a)pyrene-treated rainbow trout alevins

Histological and skeletal examinations were performed on rainbow trout (Salmo gairdneri Richardson) alevins reared in 0.00, 0.08, 0.21, 0.39, 1.48, 2.40 or 2.99 ng/ml aqueous benzo(a)pyrene (BaP). Nuclear pycnosis and karyorrhexis were most common in neuroectodermal and mesodermal derivatives and in liver of BaP-treated alevins. Microphthalmia was observed in 17% of the test fish and was frequently associated with a patent optic fissure. Depressed mitotic rates in the retina and brain (but not liver) were observed in alevins reared in 0.21 to 1.48 ng/ml aqueous BaP. Test alevins had a significantly higher incidence of skeletal malformations in the skull and vertebral column, and abnormalities of vertebral arcualia often corresponded to areas of kyphoscoliotic flexures.

Effect of dietary vitamin E and selenium on growth, survival and the prevalence of Renibacterium salmoninarum infection in chinook salmon (Oncorhynchus tshawytscha)

Abstract Groups of juvenile spring chinook salmon naturally infected with Renibacterium salmoninarum, the causative agent of bacterial kidney disease, were fed diets containing different levels of vitamin E and selenium for 214 days in fresh water and 110 days in seawater. The fish were fed vitamin E at concentrations of either 53±3 mg (designated e) or 299±9 mg (designated E) α-tocopheryl acetate equivalence/kg dry diet in combination with sodium selenite to give selenium concentrations of either 0.038±0.008 mg (designated s) or 2.49±0.15 mg (designated S)/kg dry diet. No mortality occurred in the group fed the S E diet, whereas mortality was 3% in the groups fed the s E and S e diets, and 31% in the group fed the s e diet. At the end of the experiment, weight gain and hematocrit values were significantly greater in those fish fed the E diets compared with those fed the e diets, whereas the hepato-somatic index was significantly higher in fish fed the e diets. Glutathione peroxidase activity in blood plasma was significantly higher in fish fed the S diets compared with those fed the s diets. No definite effect of dietary vitamin E and selenium on the prevalence and severity of natural R. salmoninarum infections was demonstrated.

Cellular reaction to injury in the anthozoan Anthopleura elegantissima

Abstract Small thermal injuries were used to examine the ability of Anthopleura elegantissima (Cnidaria: Anthozoa) to establish a successful inflammatory response. Experimental animals were seen to react by an initial influx of phagocytes derived from resident amoebocytes. Within 72 hr, a zone of repair formed which contained distinctly atypical cells morphologically suited for the production and secretion of unknown substances. At all times the wound remained infection free and was rapidly repaired by the passive influx of cells from the surrounding epithelium as well as progressive replacement of lost tissue by the mesogleal repair zone.

Uptake of benzo[a]pyrene by gonadal tissue of flatfish (family Pleuronectidae) and its effects on subsequent egg development.

Accumulation of benzo[a]pyrene (BaP) by sexually mature flatfish gonad, its transfer to developing gametes, and its subsequent effects on developing embryos were studied. Thin-layer chromatography revealed both unmetabolized BaP and polar metabolites in the ovary, wolffian ducts, oocytes, and semen of English sole 24 h after ip injection with 200 microCi [3H]BaP. Concentrations of BaP and its metabolites were 3-11 times higher in oocytes and semen than in gonadal tissue. Fertilized eggs from flathead sole that had been fed 4.0 mg BaP 5 h before spawning demonstrated a significantly lower (p less than 0.001) hatching success (11.9%) than eggs from control fish (56.6%). Morphological abnormalities were found in only 1.6% of control embryos but in 5.6% of embryos from treated females.