Quantification of eggs and sperm in the Black-lip pearl oyster Pinctada margaritifera using an enzyme-linked immunosorbent assay (ELISA)

We have developed immunological probes to quantify eggs and sperm of the Black-lip pearl (BLP) oyster Pinctada margaritifera. The western blot assay revealed that the polyclonal antibodies developed in this study specifically recognized only egg and sperm proteins. These polyclonal antibodies also showed high sensitivities to the antigens, detecting 0.31–10 μg/ml of the egg or sperm proteins in an indirect enzyme-linked immunosorbent assay (ELISA). Accordingly, we used an indirect ELISA to quantify the eggs and sperm in BLP oysters collected in December 2009 from Weno Island in Micronesia and in May 2010 from Tahiti. The gonad somatic index (GSI), a ratio of gonad weight to somatic tissue weight, of the females collected from Weno Island ranged from 3.6 to 18.4 %, while the GSI of the females collected from Tahiti ranged from 5.6 to 12.6 %. Similarly, the GSI of the male BLP oysters from Weno Island ranged from 0.8 to 8.5 %, while the GSI of the males from Tahiti ranged from 4.8 to 7.5 %. These results lead to the conclusion that the immunological probes developed in this study can be successfully applied to quantify BLP oyster gonadal tissues and that these probes can be used in studies of BLP oyster reproductive ecology based on their sensitivity, rapidity, and affordability.     

Effects of the nitrile group substitution on the gas separation properties of aromatic polyamide membranes

The effects of nitrile group substitution onto aromatic polyamide backbone on the gas permeability and permselectivity of the polymers are examined. The gas permeability of aromatic polyamides increase with increasing the content of nitrile group substitution, whereas the permselectivity decreases with increasing the nitrile group contents. The effects of chain linrearity on the permeability and permselectivity are also examined. The non-linearity of the polymers increases the permeability. These behaviors are interpreted in terms of chain packing and crystallinity of the aromatic polyamides.     

Effects of various wavelengths of light on physiological stress and non-specific immune responses in black rockfish <Emphasis Type="Italic">Sebastes schlegelii</Emphasis> subjected to water temperature change

We investigated the effects of light-emitting diodes (LEDs) on physiological stress and immunity in black rockfish Sebastes schlegelii subjected to water temperature change [20 (normal temperature) → 14 → 20 °C] using progressive stepwise decline [experiment (Exp.) I; 2 °C/day] and direct decline (Exp. II; immediate exposure) in water temperature. We measured stress-related parameters such as cortisol, glucose, heat shock protein 70 and Na⁺/K⁺-ATPase, and immune parameters such as immunoglobulin M and lysozyme. We confirmed nuclear DNA damage using a comet assay. Physiological stress indicators were significantly higher, immune parameters were significantly lower, and the amount of nuclear DNA damage was higher after exposure to low temperatures, but these effects were mitigated by exposure to green or blue LEDs. The direct decline in temperature induced more physiological stress and nuclear DNA damage than the progressive stepwise change. Green and blue LEDs aid recovery from physiological damage in fish.     

Evaluation of different <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> extracts as feed additives for growth and immunity of Japanese flounder <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

We evaluated the effects of diets incorporating the red algae Pyropia yezoensis, prepared by several different extraction methods, on the growth of juvenile Japanese flounder Paralichthys olivaceus. We assessed growth performance, as well as the levels of amino acids, fatty acids, insulin-like growth factor I (IGF-I) and interleukins (ILs). Four experimental diets were developed based on different methods of processing P. yezoensis. A commercial feed, laver powder (P), high-pressure heat extraction of laver (HPHE) and acid hydrolysis extraction of laver were used as the experimental diets. Three experimental replicates were established for each diet (40 fish/group, body weight 123.7 ± 1.1 g), and the fish were fed for 6 weeks. We found no significant differences in weight gain, specific growth rate or feeding efficiency among the groups (P > 0.05); however, the fish fed HPHE had the greatest growth performance. Fish fed the laver extracts exhibited the highest protein efficiency ratio compared with the control and P groups. The experimental groups fed P. yezoensis extracts had significantly higher levels of IGF-I (P < 0.05) than those of the control group. High levels of IL-2 were found in the P and HPHE groups, IL-12 in the HPHE group, and IL-6 in all experimental groups. Therefore, these results suggest that a P. yezoensis extract improved the growth performance and immunity of Japanese flounder. In particular, the high-pressure heating process was a useful extraction method for preparing a P. yezoensis extract, which had beneficial effects as a dietary supplement in Japanese flounder.     

In vitro monoamine oxidase A and B inhibitory activity and molecular docking simulations of fucoxanthin

Fucoxanthin is the primary carotenoid found in edible seaweeds, such as Eisenia bicyclis, Undaria pinnatifida, and others. The aim of this study was to determine the effectiveness of fucoxanthin against Parkinson’s disease (PD) by inhibiting monoamine oxidase (MAO) A and B because MAO inhibitors are used in early management of PD. A sensitive enzyme-based chemiluminescent assay and kinetics methods were used to investigate the enzyme inhibitory activities and mode of inhibition. A molecular docking simulation was performed to clarify the binding characteristics of fucoxanthin to hMAO-A (2z5x) and hMAO-B (2v5z). Our results suggest that fucoxanthin shows significant inhibitory activity against hMAO-A and hMAO-B with IC₅₀ values of 197.41 ± 2.20 and 211.12 ± 1.17 μM, respectively. Selegiline was used as the positive control (IC₅₀: 10.54 ± 1.25 and 0.128 ± 0.01 μM for hMAO-A and hMAO-B, respectively). The enzyme-based kinetics results demonstrated that fucoxanthin inhibited both hMAOs in a reversible competitive manner. The molecular docking simulation predicted that fucoxanthin exhibits higher binding affinity towards hMAO-A and hMAO-B through hydrogen bonding and hydrophobic interactions. These findings suggest that fucoxanthin is a reversible competitive hMAO inhibitor that could be used to manage PD.     

Tunic morphology and viral surveillance in diseased Korean ascidians: soft tunic syndrome in the edible ascidian,Halocynthia roretzi (Drasche), in aquaculture

‘Soft tunic syndrome’ causes mass mortality in the edible ascidian Halocynthia roretzi in Korean and Japanese aquaculture. In histopathological comparison, there were no specific differences between diseased specimens from Korea and Japan, indicating that soft tunic syndrome occurring in Korea and Japan is the same disease. No bacterial or protozoan cells were microscopically detected in either healthy or diseased tunics suggesting they are not the direct causes of soft tunic syndrome. Attempts were made to isolate virus from affected ascidians taking into account temperature conditions in which soft tunic syndrome is most prevalent in the field. However, no viruses were isolated from diseased or non‐diseased specimens using chinook salmon embryo (CHSE‐214), flounder fin (FFN) or epithelioma papillosum cyprini (EPC) cell lines.     

Acute responses of gill mitochondria-rich cells in Mozambique tilapia Oreochromis mossambicus following transfer from normal freshwater to deionized freshwater

Acute responses of gill mitochondria-rich (MR) cells to direct transfer from freshwater to deionized freshwater were examined in Mozambique tilapia. Scanning electron microscopic observations showed that apical openings of MR cells were morphologically classified into a small apical pit, a convex apical surface, and a concave apical surface. Following transfer from normal freshwater to deionized freshwater, the sizes of convex and concave apical surfaces were significantly increased at 24 and 6 h, respectively. Meanwhile, whole-mount immunocytochemistry revealed that freshwater-type, ion-absorbing MR cells were functionally classified into MR cells with apical Na⁺/Cl^? cotransporter (NCC) and those with apical Na⁺/H⁺ exchanger 3 (NHE3). On transfer to deionized freshwater, the intensity and size of apical-NHE3 immunoreaction were increased at 6 h, whereas the apical-NCC immunoreaction became enlarged and intense at 24 h. Considering that convex and concave MR cells are activated forms of apical-NCC cells and apical-NHE3 cells, respectively, our findings indicate that preexisting concave MR cells with apical NHE3 enlarge their apical surfaces as an acute response to exposure to deionized freshwater, followed by activation of convex MR cells with apical NCC.     

Coincidence of virulence shifts and population genetic changes of Pseudoperonospora cubensis in the Czech Republic

Pseudoperonospora cubensis is an oomycete pathogen causing downy mildew disease on a variety of Cucurbitaceae, and has recently re‐emerged as a destructive disease on crops in this family, mainly on cucumber and squash. Multilocus sequence analysis (MLSA) of four mitochondrial and two nuclear DNA regions was used to detect changes in the genetic structure of P. cubensis populations occurring in the Czech Republic that might be associated with recently reported shifts in virulence. The analysed sample set contains 67 P. cubensis isolates collected from 1995 to 2012 in the Czech Republic and some other European countries. Sequence analyses revealed differences and changes in the genetic backgrounds of P. cubensis isolates. While all isolates sampled before 2009 exhibited the genotype of the subspecies of Clade II and were collected from cucumber, all samples collected from other hosts belonged to Clade I (P. cubensis sensu stricto) or were sampled from 2009 onwards. In addition, 67·16% of all post‐2009 isolates from Clade II had two heterozygous positions in their nrITS sequence, which suggests sexual reproduction and/or a mutational origin. Thus, the results indicate that, apart from the rise in prevalence of Clade I, the change in the genetic structure of P. cubensis populations may be linked with a hybridization or, less likely, a mutation event that rendered strains able to infect a broader spectrum of host species.     

Preparation of plastic and biopolymer multilayer films by plasma source ion implantation

The plasma source ion implantation (PSII) technique was used to improve the adhesion between linear low-density polyethylene (LLDPE) and biopolymer. LLDPE was treated with the PSII using O(2) or CF(4) gas to modify its surface. After modification, chitosan or corn zein was used for coating on LLDPE. Wettability of the LLDPE surface was evaluated with a contact angle meter by the sessile drop method. X-ray photoelectron spectroscopy (XPS) was used to analyze the LLDPE surface. Before and after treatment, in the case of LLDPE treated with O(2) PSII, oxygen-containing functional groups were formed on the implanted surface. In the CF(4) PSII treated LLDPE, it was observed that the fluorine concentration on the surface of LLDPE remarkably increased and hydrophobic groups were formed by chemical reaction. Bilayer films coated with chitosan or corn zein showed 10 times lower oxygen permeability. Tensile strength of multilayer films was decreased a little compared with that of LLDPE. The plastic and biopolymer multilayer films have potential for food packaging application because of their O(2) gas barrier property and easy recyclability of the multilayer film.