Osamu Kurata

INTRODUCTION: Temporal elevation of water temperature positively affects immune activity and disease resistance in poikilothermic teleost fish. The ayu, Plecoglossus altivelis, an important fish species for Japanese freshwater fisheries, is usually produced under higher water temperatures than the natural conditions to facilitate rapid growth. However, it has been reported that rearing fish at higher water temperatures inhibits the development of the thymus, suggesting that resistance to infectious diseases is reduced in ayu reared at higher water temperatures. Here, we show that decreased resistance to bacterial cold-water disease and excessive inflammatory responses occurred in ayu reared at 22°C compared with those reared at lower temperatures. METHODS: Ayu larvae were reared at 12°C, 15°C and 22°C for 77 days and fed 3% of their body weight. Thymus index and condition factor was calculated after the fish rearing. Then, ayu reared at the different temperatures were challenged with Flavobacterium psychrophilum and the fish were sampled for histopathology and gene expression analyses. Further, the fish were vaccinated with formalin-killed F. psychrophilum and continuously reared at the three different water temperatures. Serum antibody titer was determined by ELISA and cumulative mortality in each group was recorded after the bacterial challenge. RESULTS: Ayu reared at 22°C showed a significantly lower thymus index and higher condition factor than those reared at lower temperatures. Infiltrated leukocytes and many melanin pigments were frequently observed in the adipose tissues and spleens of ayu reared at 22°C, respectively, but not in those reared at 12°C. The gene expression levels of inflammatory cytokines such as IL-1β, IL-8 and TNFα in the spleen were significantly higher in the 22°C group than in the 12°C group. The cumulative survival rate after challenge with Flavobacterium psychrophilum was 51.7%, 40.0% and 13.3% in the 12°C, 15°C and 22°C groups, respectively. The relative percent survival values of vaccinated fish reared at 15°C and 22°C groups were lower than those reared at 12°C. Moreover, the specific antibody titer of the vaccinated fish was the lowest in the 22°C group and the highest in the 12°C group. DISCUSSION: These results suggest that rearing the fish under high water temperature causes excessive inflammatory responses similar to metabolic inflammation in human obesity, resulting in a decrease of disease resistance. In addition, thymic involution induced by higher water temperature probably leads the poor response to vaccination. The present study provides insights into the physiological and immunological changes of fish under global warming.

The efficacy of Oxilink-SP, a food-additive-based disinfectant used in the environmental control of livestock pathogens, to prevent Saprolegnia infection of salmonid eggs was evaluated. ​In the laboratory, Oxilink-SP showed fungicidal activity against 5 species of Saprolegnia after immersion of rainbow trout Oncorhynchus mykiss eggs with Saprolegnia mycelia in a 10,000-fold dilution of Oxilink-SP for 30 min. ​In salmonid fish hatcheries, following immersion in a 5,000-fold dilution of Oxilink-SP for 30 min per day, hyphal growth on eggs of rainbow trout and white-spotted char Salvelinus leucomaenis was inhibited. ​This was not the case in masu salmon Oncorhynchus masou which may have been affected by water-borne molds of taxa other than Saprolegnia. ​Although rainbow trout eggs were slightly affected by treatment with Oxilink-SP in eyed-egg developmental stages and hatching, white-spotted char and masu salmon eggs were not affected. ​Furthermore, this treatment did not affect the incidence of fry deformity in all tested salmonid fish eggs. ​These results indicated that the disinfectant can prevent the spread of Saprolegnia species on salmonid eggs in hatcheries. ​We intend to use Oxilink-SP to safely manage fish eggs.

The temperature of water used to rear juvenile stages of ayu affects the development of the thymus. However, subsequent differences in the thymus in adults after rearing juveniles at different temperatures are unclear. We analyzed thymus development of ayu grown at a constant water temperature of 15 degrees C from juveniles reared at different water temperatures. Juvenile ayu weighed at 0.6 g and 1.4 g were reared at different temperatures (10 degrees C, 12 degrees C, 15 degrees C, 18 degrees C and 22 degrees C) for 64 days and 54 days, respectively, and then held at 15 degrees C for analysis of the thymus development. Thymus volume was measured by computed tomography and thymus development of experimental fish was evaluated as the ratio of thymus volume to body length. The thymus volume ratios of juveniles immediately after rearing at lower water temperatures (10 degrees C, 12 degrees C) were higher than in those reared at the higher temperatures (18 degrees C, 22 degrees C). These levels of thymus volume ratio were preserved in grown ayu continuously reared at 15 degrees C. After the summer solstice, the thymus began to atrophy and no longer exhibited differences among the experimental groups. Thus the thymus formed at the juvenile stage retains its morphological properties until the summer solstice.