Nutrition can play a major role in the responses of organisms to both infectious disease and environmental pollutants. A great deal of research has been directed toward studying the effects of macronutrient (protein, lipid, carbohydrate) deficiencies as well as deficiencies of various vitamins and minerals on disease resistance and toxicity of various contaminants in mammals. It has been shown that nutritional status has a significant effect on various nonspecific disease resistance factors, the specific immune response and the hepatic enzyme systems necessary for xenobiotic metabolism. However, relatively little information of this type is available for fish

The present study was undertaken to assess the effects of three diets on both hepatic xenobiotic-metabolizing enzymes and disease resistance mechanisms in channel catfish. Two commercially available feeds and a laboratory-prepared "control" diet were compared.

The results obtained are important in the practical sense of keeping fish alive healthy in culture situations. The differential responses indicate a significant effect of diet on the ability to resist infectious disease. Since enzyme systems examined are also responsible for the metabolism of many drugs, disease treatment may also be significantly affected by diet. The results also emphasize the need for standard diets in aquatic toxicological research. In addition, many investigators involved in basic fish immunology research use commercially available diets and should be aware of the differing responses which may be observed.

Hepatic Xenobiotic-Metabolizing Enzymes

Commercial feeds were donated by Rangen, Inc., Buhl, Idaho and Gold Kist, Inc., Atlanta, Georgia. All diets were frozen at -20°C to minimize nutrient losses. Daily rations were removed from the freezer one day prior to feeding and thawed at 40C overnight. The laboratory diet was prepared according to the National Research Council's guidelines for experimental warmwater finfish diets. Fish in each group were fed 2.0-2.5% body weight per day depending on water temperature and size. Fish were maintained in 850 liter fiberglass tanks in a 90% reuse system. Water temperatures varied from 23-27°C.

Fish were maintained on the experimental diets for three months, during which time all fish appeared healthy and no differential mortalities were observed. However, there was a significant difference in growth (Table 1).

Table 1. Final body weights of channel catfish fed three different diets. Data are Means ± SD for 40 fish. The three values differed significantly (p < 0.05) from one another. The mean initial weight of the fish was 60.7g.

The effects of diet on both control (uninduced) enzyme activities as well as PCB-induced activities were measured. Eighteen fish from each dietary group were injected intraperitoneally with either 2 ml corn oil/kg body weight (controls) or 50 mg Aroclor 1254/kg body weight dissolved in corn oil. Previous work in our laboratory with channel catfish had shown 50 mg/kg produced enzyme induction without mortality, within 4 days. Hence, fish were sampled 4 days later, livers removed, microsomal and cytosolic fractions prepared and stored at -800C until analyzed.

Both Phase I and Phase II enzyme activities were measured. The Phase I enzymes are microsomal, oxidative enzymes such as cytochrome P-450-dependent monoxygenases (MO's). 0-dealkylation of 7-ethoxyresorufin (EROD) and 7-ethoxycoumarin (ECOD) by MO's was measured. Phase II enzymes are conjugating enzymes such as UDP-glucuronosyltransferase (UDPGT). Microsomal UDPGT activity was assayed using the substrate 4-methylumbelliferone.

The results of the microsomal enzyme assays are presented in Table 2. Diet significantly affected both control enzyme activities as well as the inducibility of a number of the enzymes. Fish maintained on the Goldkist diet had significantly lower control levels of EROD activity. Although fish maintained on the Rangen feed were lower than those fed the laboratory diet, they were not significantly different. The same trend - Laboratory highest, Rangen intermediate and Goldkist lowest - was observed in the ECOD activity. Control activities of UDPGT were not significantly different among the dietary treatments.

Perhaps the most significant effects of diet were in the differential responses when fish were exposed to PCB's. Catfish fed the Goldkist feed did not show significant induction of ECOD or UDPGT. Although EROD activity was induced in fish fed the Goldkist diet, the induction was to a lesser degree than that observed in the other dietary groups. Treatment with Aroclor 1254 increased EROD activity 4.4, 4.7 and 3.4 fold in the laboratory, Rangen and Goldkist, respectively.

Table 2. Effects of diet and treatment with corn oil (control) or Aroclor 1254 on two MO activities and UDPGT activity in channel catfish. Data are expressed as X ± SD for nine groups of two pooled livers. MO activities are given as pmol of resorufin (EROD) or 7-hydroxycoumarin (ECOD) formed/min, mg microsomal protein^-1 and UDPCT activity is given as nmol 4- methylumbelliferone conjugated min^-1 mg microsomal protein.^-1

Disease Resistance Factors

The humoral immune response to Edwardsiella ictaluri and the phagocytic ability of head kidney macrophages were compared in fish from the same groups used to assess liver enzyme activities. Fish were injected with 2 mg/kg body weight formalin-killed E. ictaluri. Two weeks later fish were bled and head kidneys removed for isolation of macrophages. The same trends observed in the enzyme assays were seen in both macrophage phagocytic ability (nonspecific disease resistance) and circulating antibody (specific immune response). Fish maintained on the laboratory diet had the highest activity, Rangen intermediate and Golkist the lowest (Table 3).

Table 3. Phagocytosis and Immune Response of Channel Catfish Maintained on Three Diets for Four Months. Values are Means ± SD. Numbers Followed by the Same Letter are Not Significantly Different. Numbers in parentheses Indicate Sample Size.

Due to the responses observed in these fish we decided to feed three additional groups of fish the same diets for six weeks, evaluate nonspecific resistance factor, again immunize fish and evaluate both macrophage function and the specific humoral response. The data for these experiments is presented in Tables 4 and 5. Again similar trends were noted, however in many cases the differences were not significant.

Table 4. Disease Resistance Factors of Channel Catfish Maintained on Experimental Diets for Six Weeks. Sample Size was 12 and Values are Means ± SD. Numbers Followed by the Same Letter are Not Significantly Different.

Table 5. Disease Resistance Factors of Immunized Channel Catfish Maintained on Experimental Diets Six Weeks Prior to Immunization. Sample Size was 12 and Values are Means + SD. Numbers Followed by the Same Letter are Not Significantly Different.

The differences observed in this study indicate the significant effects diet can have on both disease resistance and toxicity of environmental contaminants. To our knowledge, this is the first report of variations in the activities of xenobiotic-metabolizing enzyme systems of fish fed different commercial or commercial versus synthetic diets. Because the composition of the diets used was somewhat different, it is not possible to make conclusive statements concerning underlying mechanisms. A number of nutritional factors were analyzed in the three diets and some of these are presented in Table 6.

Table 6. Levels of Selected Nutritional Factors. Analyses were done by Woodson-Tenent Laboratories, Inc., Gainesville, Georgia.