Occurrence and distribution of larval and juvenile Japanese flounder Paralichthys olivaceus at sandy beaches in eastern Hiuchi-Nada, central Seto Inland Sea, Japan

ABSTRACT:   The occurrence and distribution of larval and juvenile Japanese flounder Paralichthys olivaceus and major demersal fish from May to September were examined at seven sandy beaches at the central Seto Inland Sea. The flounder appeared from late May to August and were mainly collected at a depth of 2–5 m. Settlement of larval flounder was seen from late May to late June. A total of 6412 individuals of 17 species representing 12 families were collected in the study area. The dominant fish species were Favonigobius gymnauchen (56.0%), Tarphops oligolepis (8.1%), Rudarius ercodes (7.8%), P. olivaceus (7.3%), Repomucenus spp. (7.0%) and Heteromycteris japonica (3.3%). The catches of flounder differed among beaches and months. The number of flounder in June at Ohama Beach, where the most flounder were collected, was 22.6 individuals per haul (400 m²). The density of the flounder correlated negatively to that of F. gymnauchen .     

Growth Characteristics of a Neurotoxin-Producing Chloromonad Fibrocapsa japonica (Raphidophyceae)

The effects of temperature, salinity, irradiance and pH on the growth of Fibrocapsa japonica (Raphidophyceae) were examined to determine how environmental factors affect the distribution of this species. Optimal growth was observed at temperatures of 15–25 C, salinities of 25–35 parts per thousand (ppt), irradiances of 60–140 μmol quanta/m² per s and pH between 7.5–8.5. Growth did not occur at temperatures below 10 C or above 30 C nor at salinities below 15 ppt. Fibrocapsa japonica grew when subjected to irradiances of 20–180 μmol quanta/m² per s, and could tolerate a pH range of 6.5–8.5.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

A 32-kDa tyrosine-phosphorylated protein shows a protease-dependent increase in dead boar spermatozoa

Boar sperm TyrP32 is a 32-kDa tyrosine-phosphorylated protein that increases during the capacitation and acrosome reaction and during cryocapacitation. However, it is still unclear whether the increase in TyrP32 is an event that is limited to the process of sperm fertilization, including cryocapacitation. The aims of the present study were to demonstrate that TyrP32 is increased in dead spermatozoa after freeze-thawing without a cryoprotectant and to find the causal factors for this increase. Washed spermatozoa were resuspended in a salt solution and then frozen. The frozen samples were rapidly thawed in a warm water bath and then used for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)/Western blotting to detect TyrP32, SDS-PAGE/silver staining of sperm proteins and staining of acrosomal contents with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA). In the samples before freezing, TyrP32 was barely detectable, and the distribution of the acrosomal contents was normal in most spermatozoa. One cycle of freeze-thawing induced an increase in TyrP32, a decrease in major sperm proteins and disorder in the acrosomal contents. However, the addition of a protease inhibitor (APMSF, 1 mM) suppressed the increase in TyrP32 and the decrease in the major sperm proteins, although it did not have any influence on the disorder in the acrosomal contents. Additionally, the spermatozoa did not exhibit any flagellar movement after freeze-thawing, which showed that almost all of them were dead. These results indicate that TyrP32 can show a protease-dependent increase in dead spermatozoa after freeze-thawing without a cryoprotectant even though the dead spermatozoa do not undergo cryocapacitation.     

Development and evaluation of a rapid enzyme-immunoassay system for measurement of the urinary concentration of estrone-3-glucuronide in a female giant panda (Ailuropoda melanoleuca)

To detect estrus for reproductive management, and to determine the relationship between urinary estrogen and estrous behavior, in a female giant panda, we developed and evaluated a rapid enzyme immunoassay (EIA) system for urinary Estrone-3-glucuronide (E1G) using commercial reagents. The developed EIA system took only around 3 hours, including all procedures to obtain a result. It indicated good reproducibility (intra-assay CV of 5.16%, interassay CV of 15.4%) and sensitivity (lowest standard concentration was 0.0156 ng/ml) for measurement of the urinary concentrations of E1G in the giant panda. There was a positive correlation (r=0.934) with the data for estrone (E1) in the same samples, as measured by radioimmunoassay (RIA) performed in a commercial laboratory. The changes in the E1G concentrations were almost synchronous with the changes in E1 assayed by RIA in urine collected during 4 consecutive estrous seasons. The dynamics of urinary E1G measured by this system highly correlated with the occurrence of the presenting estrous behavior in the giant panda. The above results indicate that this assay system may be normally, rapidly and practically used for measurement of the urinary concentration of E1G in the giant panda.     

Isolation and Sequence of a Gene, celD Xo Involved in Cellobiose Utilization in Xanthomonas oryzae pv. oryzae

A genomic library of Xanthomonas oryzae pv. oryzae (X. o. pv. oryzae) T7174 was screened for 4-methylumbelliferyl β-D-glucoside-hydrolyzing (MUGase) activity. In subcloning of one of the MUGase-positive clones, an approximately 4.2-kb SacI-SphI fragment conferred not only MUGase activity but also 4-methylumbelliferyl β-D-cellobioside-hydrolyzing (MUCase) activity. Sequence analysis showed that the fragment contained an ORF of 2951 bp. The conceptual ORF product was significantly homologous with 1,4-β-D-glucan glucohydrolase D (CELD) from Pseudomonas fluorescens subsp. cellulosa, and was named CELD_Xo. Cell fractionation experiments suggested that CELD_Xo is localized in the cell-envelope fraction. We constructed a CELD_Xo-deficient mutant (74ΔCELD) from X. o. pv. oryzae. Little MUCase activity was detected in the cell-envelope fraction prepared from the mutant. The mutant 74ΔCELD did not grow in synthetic medium containing cellobiose as the sole sugar source. On the other hand, growth in rice leaves and pathogenicity of the mutant and the parental strain did not differ. These results suggested that CELD_Xo is involved in cellobiose utilization of X. o. pv. oryzae but that the gene is not required for bacterial growth in rice leaves. Received 16 February 2001/ Accepted in revised form 11 April 2001     

Collie eye anomaly in Hokkaido dogs: case study

Objective To describe a Hokkaido dog, one of the traditional Japanese breeds that was affected by Collie eye anomaly (CEA), and to report the genotype of this dog and the Hokkaido dog allelic frequency of the CEA‐associated mutation. Case A nine‐month‐old intact female Hokkaido dog without any obvious visual disturbance was diagnosed ophthalmoscopically with CEA. Severe choroidal hypoplasia was observed in the bilateral temporal area adjacent to the optic nerve head, appearing as whitish areas. Therefore, the dog was suspected of possessing the CEA‐associated mutation that was previously reported as an intronic 7.8‐kilo base deletion in the canine NHEJ1 gene. Procedures SYBR Green‐based real‐time PCR with a melting curve analysis, conventional PCR with agarose gel electrophoresis, and direct DNA sequencing were carried out to determine the genotype of the dog. Furthermore, a preliminary genotyping survey was carried out in 17 Hokkaido dogs from three kennels using the real‐time PCR method, and the pedigree relationships were analyzed using their pedigree papers. Results The Hokkaido dog affected by CEA was proven to possess the CEA‐associated mutation. Of these 17 Hokkaido dogs, 12 dogs were heterozygous carriers and five dogs were affected by this mutation. The preliminary genotyping survey and pedigree analysis demonstrated that the allelic frequency of the CEA‐associated mutation is very high in Hokkaido dogs. Conclusion These data suggest that the Hokkaido breed is highly susceptible to CEA because of the known CEA‐associated mutation much like the Collie‐related breeds.     

Dermocystid infection in Japanese fire-bellied newt,Cynops pyrrhogaster

Here, we report details of a new infectious disease in wild-caught Japanese fire-bellied newts (Cynops pyrrhogaster), a Near Threatened species. Skin lesions consisting of numerous masses were found in the animals near Lake Biwa, Shiga Prefecture, Japan. The gross appearance of the skin lesions showed blister-, cyst-, and/or tumor-like morphology. Various sizes of skin lesions were observed on their entire body surface. Histologically, spherical basophilic cysts, including numerous spores, were observed in the dermis layer. Ultrastructural analysis indicated the presence of main bodies of flagellated zoospores within the spores. While 18s rRNA gene sequencing indicated that the skin lesions were due to dermocystid infection. To our knowledge, this is the first report of dermocystid infection in this amphibian in Japan. Further studies are needed to prevent epidemics and to establish diagnostic and treatment methods.     

Enzyme-linked immunosorbent assay for total sennosides using anti-sennside A and anti-sennoside B monoclonal antibodies

Total sennosides concentration is a very important factor when rhubarb and senna will be used as crude drugs. However, one-step analytical technique for total sennosides has not been reported except HPLC. An enzyme-linked immunosorbent assay (ELISA) for total sennosides concentration by using the combination of anti-sennoside A (SA) and anti-sennoside B (SB) monoclonal antibodies (MAbs) in a single assay has been investigated. Total sennosides concentration in rhubarb and senna samples determined by newly developed assay system showed good agreement with those analyzed by ELISA using anti-SA MAb and anti-SB MAb, respectively.