Decreased resistance to bacterial cold-water disease and excessive inflammatory response in ayu (Plecoglossus altivelis) reared at high water temperature.

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-1ß, 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.

MODELING OF TEMPORAL AND VERTICAL CHANGES IN 137Cs CONCENTRATION IN LAKE ONUMA.

According to the observed data of the activity concentration of $^{137}$Cs in Lake Onuma on Mount. Akagi, located in Gunma Prefecture, the decrease of the activity concentration slowed down since 2 years after the accident of Fukushima Daiichi Nuclear Power Plant. In the present study, we applied the time fractional diffusion equation to reproduce long-term changes in the time series of the activity concentration. In addition, we also reproduced the vertical distribution of the activity concentration with the parameter values, estimated by the time series data. We found that the model fit the observed data well for both time series and vertical distribution.

Radiocesium concentrations in great cormorants (Phalacrocorax carbo) between 2011 and 2012 after the Fukushima Dai-ichi Nuclear Power Plant accident.

Radiocesium concentrations were measured in the pectoralis muscles of great cormorant (n = 36) collected from Gunma Prefecture between November 2011 and July 2012. The 137Cs contamination levels in great cormorant were consistent with the 137Cs deposition levels on ground soil and 137Cs concentrations in freshwater fishes. The Tag of great cormorants were comparable with those of other birds except for copper pheasant.

Long-term prediction of [Formula: see text]Cs in Lake Onuma on Mt. Akagi after the Fukushima accident using fractional diffusion model.

The Fukushima Daiichi Nuclear Power Plant accident also contaminates lakes in Japan. Especially in closed lakes, there is a problem of prolonged low-level [Formula: see text]Cs contamination because the activity concentration of [Formula: see text]Cs declines sharply immediately after the accident, but then begins to decrease slowly. In this paper, we derived a long-term prediction formula based on the fractional diffusion model (FDM) for the temporal variation in [Formula: see text]Cs activity concentrations of the water in Lake Onuma on Mt. Akagi, one of the closed lakes, and of pond smelt (Hypomesus nipponensis), a typical fish species inhabiting in the lake. The formula reproduced well the measured [Formula: see text]Cs activity concentration of the lake water and pond smelt for 5.4 years after the accident. Next, we performed long-term prediction for 10,000 days using this formula and compared it with the prediction results of the two-component decay function model (TDM), which is the most common model. The results suggest that the FDM prediction will lead to a longer period of contamination with low-level [Formula: see text]Cs than the TDM prediction.

Immune responses induced by oil-adjuvanted inactivated vaccine against Flavobacterium psychrophilum in ayu Plecoglossus altivelis.

Oil-adjuvant formulated formalin killed cells of Flavobacterium psychrophilum (FKC + Adj) is strongly effective against bacterial cold-water disease (BCWD) in ayu Plecoglossus altivelis. In this study, we aimed to understand mechanisms underlying the strong protection by the vaccine in ayu. Antibody titer of FKC + Adj and formalin-killed cells (FKC) group was significantly higher than those of modified cytophaga broth injected (MCY) group and MCY with the adjuvant (MCY + Adj) group. The highest antibody titer was observed in FKC + Adj group. Granulomatous inflammation without lymphocyte cuff was observed in the spleen and trunk kidney of FKC + Adj and MCY + Adj group, while the size of the granuloma was bigger in FKC + Adj than in MCY + Adj group. Gene expression level for IL-8 was significantly up-regulated in FKC + Adj group at 4 weeks after the vaccination. In contrast, IL-10 gene expression level was significantly suppressed in FKC + Adj at 4 weeks after the vaccination. F. psychrophilum was almost cleared in the spleen and trunk kidney of FKC + Adj group within 2 days after the challenge. Fluorescent immunohistochemistry showed that a lot of bacterial signals were detected in the spleen and trunk kidney of challenged fish in MCY, FKC and MCY + Adj group. However, the fluorescent signal was not detected in the organs of FKC + Adj group after the challenge. These data suggest that F. psychrophilum is immediately cleared in FKC + Adj vaccinated fish and both specific antibody and activation of phagocytes are essential to clear F. psychrophilum in ayu.

Radiocesium dynamics in the aquatic ecosystem of Lake Onuma on Mt. Akagi following the Fukushima Dai-ichi Nuclear Power Plant accident.

Understanding ecosystem dynamics of radionuclides is necessary to ensure effective management for food safety. The Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident on March 11, 2011 released large amounts of radiocesium (134Cs and 137Cs) and contaminated the environment across eastern Japan. In this study, we aimed to elucidate the temporal dynamics of 137Cs in the aquatic ecosystem of Lake Onuma on Mt. Akagi. The effective ecological half-life (Teff) of 137Cs in fishes, western waterweed (Elodea nuttallii), seston (phytoplankton and zooplankton), and lake water was estimated using survey data of 137Cs concentration collected from 2011 to 2016, and single- and two-component decay function models (SDM and TDM, respectively). The decay processes of 137Cs concentrations in wakasagi (Hypomesus nipponensis), pale chub (Zacco platypus), phytoplankton, and total 137Cs concentrations of the water column (WC) in the lake were well suited by the TDMs. The Teff in the fast component of the TDMs in these samples ranged from 0.49 to 0.74years. The Teff in the slow component of the TDMs could converge towards the physical half-life of 137Cs. Nearly five and a half years after the FDNPP accident, we concluded that 137Cs concentrations approached a state of dynamic equilibrium between some aquatic organisms (wakasagi, pale chub, and phytoplankton) and the environment (lake water). However, the decay processes of 137Cs concentrations in Japanese dace (Tribolodon hakonensis), western waterweed, zooplankton, and particulate- and dissolved-forms in the WC were better predicted for the SDM. The total 137Cs concentrations in inflowing river and spring waters were one to two orders of magnitude lower than lake water under normal flow conditions. However, particulate 137Cs contamination level in the river water was high after heavy rains. Overall, 137Cs contamination levels have significantly decreased in Lake Onuma, but monitoring surveys should be continued for further understanding of the reduction processes.

Maturation-associated changes in the non-specific immune response against Flavobacterium psychrophilum in Ayu Plecoglossus altivelis.

In this study, we investigated maturation-associated changes in non-specific immune responses of ayu against Flavobacterium psychrophilum. The gonadosomatic index was minimum on 16 June, began to increase on 17 July, and reached the maximum value during August. The highest phagocytic rate (16.3%) was observed on 16 June, which decreased significantly to 5.6% on 26 August. The number of viable bacteria after the serum treatment was highest during August, suggesting that bactericidal activity of the serum decreased along with the sexual maturation. Gene expression levels of interleukin-8, and tumor necrosis factor-α in the spleen did not change significantly during this period, whereas the level of suppressor of cytokine signaling (SOCS)3 was significantly higher on 26 August than that on 16 July (p < 0.05). These results suggest that phagocytic activity of trunk kidney leukocytes and serum bactericidal activity against F. psychrophilum decreased with sexual maturation, and that SOCS3 may be related to the decrease in non-specific immune activity in ayu.

Fractionation of radiocesium in soil, sediments, and aquatic organisms in Lake Onuma of Mt. Akagi, Gunma Prefecture using sequential extraction.

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident has resulted in the contamination of the environment in Gunma Prefecture with radioisotope cesium (radio-Cs, 134Cs and 137Cs). Concentrations of radio-Cs >500Bqkg-1 were found in wakasagi (Hypomesus nipponensis) in Lake Onuma at the top of Mount (Mt.) Akagi in August 2011. To explain the mechanism of this contamination, monitoring studies have been conducted around Lake Onuma by measuring radio-Cs concentrations in samples of fish, aquatic plants, plankton, lake water, lake sediments, and surrounding soil. The leachability of radio-Cs was evaluated using sequential extraction by Tessier et al. The total concentration of radio-Cs in Lake Onuma ecosystems decreased gradually with time. In the brown forest soil, radio-Cs concentrations of 2000 to 6000Bqkg-1 were detected. The abundance ratio of the easy-elution form (exchangeable and carbonate forms) in the samples was <10%. The concentrations in phytoplankton samples were 3-6 times higher than those in wakasagi samples. The ratios of easy-elution forms increased by the rank in the food chain; 37% in phytoplankton, 78% in zooplankton, and 97% in wakasagi. It is likely that the lower ratio of the easy-elution form in phytoplankton is related to the adsorption of radio-Cs on suspended substances in the lake, as suggested by the analyses of aluminum and titanium in the phytoplankton, zooplankton, and wakasagi samples. The high concentrations of radio-Cs in wakasagi would be related also to the characteristics of closed mountain lakes.

Protective efficacies and immune responses induced by recombinant HCD, atpD and gdhA against bacterial cold-water disease in ayu (Plecoglossus altivelis).

Protective efficacies of three antigenic proteins (3-hydroxyacyl-CoA dehydrogenase (HCD), ATP synthase beta subunit (atpD), and glutamate dehydrogenase (gdhA)) against Flavobacterium psychrophilum were investigated in ayu (Plecoglossus altivelis). Recombinant proteins of HCD, atpD, and gdhA were expressed in Escherichia coli BL21 cells. Ayu were then vaccinated with inactivated cells via the intraperitoneal route. Compared with the empty BL21- and PBS-injected groups, the vaccinated group had a significantly longer survival time after challenge with F. psychrophilum. The antibody titers against each recombinant protein were significantly higher in serum from vaccinated fish, compared with serum from control fish. Results of indirect immunofluorescence assays using serum indicated that the HCD, atpD, and gdhA proteins are located on the surface of F. psychrophilum. These results suggest that these three surface proteins are protective antigens and are good candidates for development of vaccines against bacterial cold-water disease in ayu.

Antigenic proteins of Flavobacterium psychrophilum recognized by ayu Plecoglossus altivelis antisera.

Flavobacterium psychrophilum is the causative agent of bacterial coldwater disease (BCWD) in ayu Plecoglossus altivelis altivelis and is responsible for substantial economic losses in ayu culture in Japan. To develop effective vaccines for the disease, we identified antigenic proteins of F. psychrophilum using immunoglobulin from ayu that had recovered from BCWD. The whole protein extracted from the bacterium was separated using 2-dimensional polyacrylamide gel electrophoresis and was transferred to a polyvinylidene fluoride membrane. Subsequently, antigenic proteins of the bacterium were detected using western blotting and ayu antisera against F. psychrophilum. Each protein spot showing antigenicity was subjected to tandem mass spectrometry (MS/MS) analysis using a MALDI-QIT-TOF mass spectrometer. Protein identification based on the MS/MS data was performed using the genome database for F. psychrophilum JIP02/86, and the subcellular localization for each identified protein was predicted with web-based tools (LipoP and PSORTb). In total, 62 antigenic proteins were identified: of these, 46 were putative cytoplasmic proteins (e.g. elongation factor Tu and heat shock protein 90). The remaining 21 proteins were identified as putative membrane-bound or secreted proteins and are potential vaccine candidates. These proteins include OmpA, Omp 121, M13 family metallopeptidase, and M48 family metalloprotease.

Detection and sequence analysis of DNA polymerase and major envelope protein genes in koi herpesviruses derived from Cyprinus carpio in Gunma prefecture, Japan.

Koi herpesvirus (KHV), which is believed to be an emerging virus, causes fatal diseases in carps. Since the 1990s, the presence of KHV has been confirmed in several countries. In Japan, from 2003 to 2004, large outbreaks of KHV infection in farmed carps resulted in the death of numerous fishes. From April to May 2004, we collected 43 dead or dying carps exhibiting typical symptoms of KHV infection in Gunma prefecture. To conduct a molecular epidemiologic study of KHV in our prefecture, we amplified DNA polymerase and the major envelope protein genes of KHV derived from carp gills using newly designed primers. We also performed sequence analysis of both genes of KHV. Sensitivity of our PCR method for amplification of DNA polymerase and the major envelope protein genes of KHV was 3 x 10(2) (100 fg) and 3 x 10(3) (1000 fg) copies of KHV genome, respectively. We detected both DNA polymerase and major envelope protein genes in 37 of 43 carps (86%). No mutation was found in both the genes sequenced from 11 strains, which included two foreign strains and one domestic strain. The results suggested that KHV strains derived from carps in our prefecture were closely related genetically to the other KHV strains.