Time series variations in protein expression profiles in field Chattonella marina cells were investigated during a HAB occurred in the inner part of Ariake Sea, Japan (5-14 September, 2012).This study aimed to gather information on the molecular mechanisms underlying the physiological responses in field C. marina population during HAB.Proteomic analysis showed that the abundance of ~37% protein spots (40 out of 108 detected from 2-DE gel images) significantly varied with the sampling date.Significant decreases in the abundances of proteins involved in photosystem II (LHCP 4), electron transfer chain (Cyt c553), Calvin cycle (GAPDH), and chloroplast antioxidant system (2-Cys Prx) were observed as the bloom progressed, suggesting the efficiencies of those photosynthetic pathways declined during the bloom.In addition, the abundances of the above proteins showed significant positive correlations with the F v /F m ratio and growth rate of C. marina and with DIN concentrations (except LHCP 4).Our findings suggested that declined expressions of those photosynthesis-related proteins presented some molecular foundation of the decreases in F v /F m ratio and growth rate of C. marina during the bloom, and also provided insight into mechanistic links between the external/internal factors and physiological responses of C. marina that may ultimately dictate the ecology of the bloom.
Selection of suitable reference genes is important for relative quantification in quantitative PCR (qPCR). We investigated the expression stability of candidate reference genes for qPCR in the raphidophyte Chattonella marina at different irradiance, temperature, and oxidative stress conditions, and in different growth phases. Nine candidate reference genes (18S rRNA, cytochrome c oxidase subunit 2, glyceraldehyde-3-phosphate dehydrogenase, actin, alpha-tubulin, beta-tubulin, calmodulin, 60S ribosomal protein L18, and elongation factor) were selected and used for qPCR analysis. After qPCR analysis, gene expression stabilities were evaluated using four frequently used algorithms (geNorm, NormFinder, BestKeeper and ΔCt). A comprehensive evaluation, based on statistical analysis, revealed that calmodulin constantly ranked in the top three at all conditions and growth phases. The gene expression profile of peroxiredoxin, a known antioxidant enzyme, was analysed in C. marina grown at different temperatures (10, 20, and 30 °C) to confirm the applicability of the high-ranked reference genes to relative quantification. The 2-Cys peroxiredoxin gene expression profile using the top-three ranked genes (18S rRNA, alpha-tubulin, and calmodulin) and the second-lowest ranked gene (elongation factor) showed a temperature-dependent increase in expression levels. However, there was no significant difference in the lowest ranked gene, cytochrome c oxidase subunit 2, at the three temperatures. This result showed that evaluation of the candidate reference genes using the four algorithms was valid, and indicates the importance of reference gene selection for relative qPCR in C. marina.
Microplastic (MP) pollution and the related impacts on aquatic species have drawn worldwide attention. However, knowledge of the kinetic profiles of MPs in fish remains fragmentary. In this study, we conducted exposure and depuration tests of the following fluorescent-labeled MPs: polyethylene (PE; sphere with 200 or 20 µm diameter) and polystyrene (PS; sphere with 20 or 2 µm diameter) using juvenile Japanese medaka (Oryzias latipes). The distribution and concentration of MPs in medaka were directly determined in-situ after tissue transparency. During the 14-day exposure, MPs was mainly detected in the gastrointestinal tract, while some MPs at the size of ≤ 20 µm were located in the area of the gills and head. The bioconcentration factor (BCF; L/kg) for MPs in medaka was estimated as 74.4 (200 µm PE), 25.7 (20 µm PE), 16.8 (20 µm PS), and 139.9 (2 µm PS). Within the first five days of depuration, MPs were exponentially eliminated from the fish body, but 2 µm PS-MPs could be still detected in the gastrointestinal tract at the end of the 10-day depuration phase. Our results suggest that MPs 2 µm in diameter may pose ecological risks to aquatic species due to their relatively higher BCF and the potential for long-term persistence in the body.
Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.
We conducted exposure tests using three small fish species, medaka (Oryzias latipes), guppy (Poecilia reticulata), and white cloud mountain minnow (Tanichthys albonubes). The fish were exposed to nominal concentrations of test chemicals (potassium cyanide [KCN; 1 and 5 mg/L], sodium cyanide [NaCN; 1 and 5 mg/L], and aldicarb [5 mg/L]) for one hour. We used two cameras for tracking the fish behavior in three dimensional (3D) data. Then the 3D data were analyzed for fish behavior such as, swimming speed and surfacing behavior. Swimming speed and position of the three fish species were affected by KCN, NaCN, and aldicarb exposure test. Three fish species showed the different behavioral patterns after the exposure of test chemicals. Although the patterns of effect on behavioral parameters differed among the test species and toxic chemicals, we could find the significant effects on both the frequency of high swimming speed and duration time of surfacing behavior in test fish. We concluded that three fish species are useful species for monitoring the water quality by their behavioral changes.
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