Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection.

Suppressive subtractive hybridization (SSH) was employed to identify differentially expressed genes in ayu (Plecoglossus altivelis) associated with Listonella anguillarum infection. 800 random clones were selected from forward and reverse subtractive libraries and 787 were successfully sequenced. After assembling, 105 contigs and 414 singletons were finally obtained, some of which were immune-related genes. A real-time quantitative PCR (RT-qPCR) analysis of the expression patterns of 28 transcripts showed that the false-positive rate was approximately 7.1%. Furthermore, Wap65-2 was overexpressed in Escherichia coli, purified and used for antiserum preparation. Western blot analysis revealed that serum Wap65-2 of ayu significantly increased after bacterial infection, suggesting that it was a positive acute-phase protein (APP).

Proteomic analysis on the alteration of protein expression in gills of ayu (Plecoglossus altivelis) associated with salinity change.

Gill is the primary osmoregulatory organ for euryhaline fish to acclimate salinity change. The effect of salinity on gill proteome in ayu, Plecoglossus altivelis, was investigated by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Eight of eighteen altered proteins were successfully identified. They are involved in osmoregulation, cytoskeleton, energy metabolism, and stress response. Our results showed that vinculin, echinoderm microtubule-associated protein like protein 1, pyruvate kinase, betaine-homocysteine methyltransferase (BHMT), transaldolase, glyceraldehyde 3-phosphate dehydrogenase, and heat shock protein 70 (HSP70) were down-regulated, whereas cofilin was up-regulated when ayu transferred from fresh water (FW) to brackish water (BW). Partial cDNA sequences of BHMT, HSP70, Na(+)/K(+) ATPase (NKA) alpha-subunit and 18S rRNA genes were subsequently determined and used for 2-DE data verification by real-time PCR. Gill BHMT and HSP70 mRNAs decreased significantly in BW-transferred ayu, while NKA alpha-subunit mRNA had no significant change. It was suggested that cell volume-regulatory response, especially the protection by the BHMT/betaine system might play an important role in ayu acclimation to salinity change.

An interaction between a C-type lectin receptor and leukocyte cell-derived chemotaxin 2 of ayu, Plecoglossus altivelis.

Yeast two-hybrid (Y2H) screens were used to test for interactions between leukocyte cell-derived chemotaxin 2 (LECT2) and a liver cDNA expression library of ayu, Plecoglossus altivelis. Of the 9 independent interacting clones identified, 2 were identical and closely related to C-type lectin receptor (CLR) genes of fish, while the other 7 were partial sequences from transferrin genes. Ayu CLR (aCLR) showed similarity to immune-relevant mammalian receptors in terms of amino acid sequence and overall organization within the C-type lectin-like domain (CTLD). The aCLR transcript was detected with the highest levels in the head kidney and peripheral blood leukocytes (PBLs), and more weakly in the heart, liver and gill. The interaction between aCLR and ayu LECT2 (aLECT2) was confirmed by in vitro co-immunoprecipitation of the two proteins. This interaction may be responsible for the "neutrophil-chemotactic" characteristic of LECT2. Y2H assays using different parts of the two proteins showed that the CTLD part of aCLR was involved in the interaction with mature aLECT2, and the contact structure of CTLD was essential for the interaction. The identification of this CLR/LECT2 interaction sheds light on the mechanism of serum LECT2 changes resulting in cellular immune responses.