Recycling of water for irrigation: Persistence of enteroviruses in sewage effluent and natural waters receiving the effluent

A virological analyses of a sewage treatment plant which provided chlorinated, activated sludge treated sewage effluent to irrigate a complete two-year crop of sugarcane was made. The raw, the activated sludge treated and the chlorinated sewage effluent, as well as streams and a harbor receiving sewage effluents were concentrated by either the polymer two-phase, PE-60, Al(OH)₃, protamine sulfate, or cellulose membrane method and assayed for human enteric viruses. Viruses were recovered from 100% (11/11) of the raw sewages tested at concentrations ranging from 27 to 19 000 PFU l^?1 while 76% (13/17) of the activated sludge treated effluent was positive at concentrations ranging from 7 to 5222 PFU l^?1. After chlorination, 58% (31/53) of the samples was positive for virus at concentrations ranging from 2 to 750 PFU l^?1. Human enteroviruses were also isolated from shallow flowing streams at distances up to 3 mi (5 km) from the closest known sewage effluent discharge point and from a harbor approximately 0.5 mi (0.8 km) from the point of sewage discharge entering the harbor. The viruses most often isolated were echovirus 7, coxsackievirus B-4, B-5 and poliovirus 1, 2, and 3. These results indicate that although activated sludge treatment plus chlorination remove approximately 90% of the virus from the raw sewage, the final treated sewage effluent, which is normally discharged into a stream and in this experimental study to irrigate sugarcane, still contains a significant concentration of infectious viruses. Furthermore, the recovery of enteroviruses from waterways at points distant from the sewage treatment plants indicates that sewage-borne viruses persist in natural water environment. The significance of enteric viruses in waters accessible to the public and used for irrigation purposes remains to be determined.     

Improved malonaldehyde assay using headspace solid-phase microextraction and its application to the measurement of the antioxidant activity of phytochemicals

A modified malonaldehyde (MA) assay for antioxidant activity, which involves derivatization and headspace solid-phase microextraction (HS-SPME) was developed and validated. The recovery of MA as 1-methylpyrazole (product of MA and N-methylhydrazine) from a headspace of an aqueous solution containing MA, buffer, surfactant, and cod liver oil using HS-SPME with a PDMS/DVB fiber was 91.3 +/- 3.38%. MA was analyzed by a gas chromatograph with a nitrogen-phosphorus detector, and its detection limit was 0.0103 nmol/mL. The antioxidant activities of natural compounds were determined as the percentage inhibition of MA formed from cod liver oil oxidized by Fenton's reagents in the above aqueous solution. Sesamol inhibited MA formation most (86.1%), followed by eugenol (84.4%), capsaicin (80.7%), ethylvanillin (45.3%), and vanillin (31.6%) at a level of 50 microg/mL. This method did not require any organic solvents and is a simple, fast, and a highly sensitive method for MA determination.     

Localization of the motoneurons innervating the hindlimb muscles in the spinal cord of the domestic fowl

The origins of the motor nerve fibers supplying the individual hindlimb muscles were elucidated in the fowl by the retrograde degeneration method. Chromatolytic cells were seen in the ipsilateral lamina 9 of the lumbosacral enlargement. Motoneurons innervating the individual hindlimb muscles were localized in the characteristic position of lamina 9. Both hip and thigh muscles are supplied by motoneurons of almost all the columns in lumbar segments and by those of dorsolateral columns in sacral segments, while the shank and foot muscles are supplied by motoneurons of ventromedial columns in sacral segments. The origins of the dorsal cord of the lumbosacral plexus are situated laterally in the lamina 9 and those of the ventral cord are located medially in it.     

Habitat use and movement patterns of small (age‐0) juvenile Pacific bluefin tuna (Thunnus orientalis) relative to the Kuroshio

The habitat use of Pacific bluefin tuna (Thunnus orientalis; PBF) in nursery waters off the southern coast of Japan was investigated using archival tags over a 3 year study period (2012–2015), and the data were used to examine the free‐ranging habitat preferences of PBF and the relationship between their horizontal movements and the path of the Kuroshio off the Pacific coast of Japan. The path of the Kuroshio fluctuated seasonally, leading to changes in water temperature that strongly influenced the habitat use of small PBF (2–3 months after hatching). Most PBF were present in coastal waters inshore of the path of the current, and their habitat use changed in response to the distance of the current from the coast. The Kuroshio typically flowed along the coast from summer to autumn, and PBF remained in the coastal waters off Kochi Prefecture during this period. In contrast, PBF quickly moved eastward in winter when the current moved away from the coast. Throughout the winter and spring, the area of habitat use extended widely from the eastern end of the southern coast of Japan (the Boso Peninsula) to the offshore Kuroshio‐Oyashio transition region. These findings suggest that the seasonal habitat use and movement behavior of juvenile PBF are influenced by the distance of the Kuroshio axis from the coast, and the ultimate drivers are likely variations in oceanographic conditions and prey availability along the southern coast of Japan.     

Long‐term changes in recruitment of age‐0 Pacific bluefin tuna (Thunnus orientalis) and environmental conditions around Japan

Recruitment of age‐0 Pacific bluefin tuna (Thunnus orientalis) from 1952 to 2014 was examined by a sequential regime shift detection method. The regime shifts in recruitment were detected in 1957, 1972, 1980, 1994 and 2009. The durations of regime shift ranged from 8–15 years and averaged 13.0 years. In both the total (1952–2014) and data rich (1980–2014) periods, negative relationships were found between recruitment and the Pacific Decadal Oscillation in autumn, and positive relationships were found between recruitment and sea surface temperature (SST) anomalies in the northern part of the East China Sea, in the southwestern part of the Sea of Japan, and in the waters off Shikoku and Tokai in summer and autumn. The 1994 and 2009 regime shifts in recruitment occurred in the same years as shifts in SST anomalies in the northern part of the East China Sea in summer. These results suggest that the ocean conditions in the northern part of the East China Sea are closely related to recruitment of Pacific bluefin tuna, and that the warmer conditions result in higher recruitment of the species.     

Establishment of a new cell line susceptible to Cyprinid herpesvirus 3 (CyHV‐3) and possible latency of CyHV‐3 by temperature shift in the cells

A new cell line named CCF‐K104 predominantly consisting of fibroblastic cells showed optimal growth at temperatures from 25 °C to 30 °C. Serial morphological changes in the cells induced by Cyprinid herpesvirus 3 (CyHV‐3) included cytoplasmic vacuolar formation, cell rounding and detachment. Mature virions were purified from CyHV‐3‐infected CCF‐K104 cells by sucrose gradient ultracentrifugation and had a typical herpesvirus structure on electron microscopy. Infectious CyHV‐3 was produced stably in CCF‐K104 cells over 30 viral passages. Our findings showed that CCF‐K104 is a useful cell line for isolation and productive replication of CyHV‐3. A temperature shift from 25 °C to 15 °C or 35 °C did not allow serial morphological changes as observed at 25 °C for 14 days. Under the same conditions, real‐time PCR showed that CyHV‐3 was present with low viral DNA loads, suggesting that CyHV‐3 may establish latent infection in CCF‐K104 cells. Amplification of the left and right terminal repeat sequences of the CyHV‐3 genome arranged in a head‐to‐tail manner was detected by nested PCR following an upshift in temperature from 25 °C to 35 °C. The PCR results suggested that the circular genome may represent a latent form of CyHV‐3.     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Genetic contribution of parents in sex determination of honmoroko Gnathopogon caerulescens

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (<50 %). One second-generation brood of gynogenetic diploids consisted wholly of females, but others produced some males. In crosses with a control diploid female, four males from a high-percentage male brood of gynogenetic diploids produced offspring with a balanced sex ratio. Sib-mating between a gynogenetic female and three gynogenetic males from the brood produced predominantly male progeny. These results suggest that there are at least four possible genotypes: genotypic female (XX), phenotypic female carrying a silent Y chromosome, genotypic male (XY), and genotypic supermale (YY). These inferences suggest that this fish has an XY system but a relatively high proportion of females possess a mutated, silent Y chromosome which does not lead to testis formation.     

Effects on Maize Starch Properties of Heat-treatment with Water–ethanol Mixtures

Maize starch (1 part) was heated at 120 °C for 20 min in water–ethanol mixtures (5 volumes), in which the water content varied over the range 0–70%, and the product solvent-exchanged with acetone. On treatment with mixtures containing 40–60% water, the swelling power and solubility at 97 °C decreased and the differences between these values measured at 25 and 97 °C narrowed. The properties of starch treated with a mixture containing 30% water resembled those of heat–moisture treated starch. The values of ΔH and ΔS for swelling were greatly decreased by treatment in mixtures with a water content greater than 20–40% (0·39–0·63 mole fraction). Furthermore, the digestibility ratio ofalpha-amylase (endo-type) to glucoamylase (exo-type) was at a maximum after treatment with a mixture containing 30% water. These findings suggest that rearrangements between the amylose and amylopectin in starch granules are occurring after treatment with mixtures containing more than 30% water. These changes in the starch granule are enhanced when the treatment temperature is increased to 100–130 °C.