Alteration of hemoglobin ß gene expression in mucosal tissues of Japanese flounder, Paralichthys olivaceus, in response to heat stress, Edwardsiella piscicida infection, and immunostimulants administration.

Hemoglobin beta (Hbß) is a heme-binding protein capable of oxygen delivery. The oligopeptides derived from Hbß in fish mucus are active against a variety of gram-negative bacteria and protozoa. To gain information on the physiological and immunological roles of Hbß in the mucosal tissues of fish, we analyzed changes in Hbß gene expression levels in the epidermis, gills, and intestine of Japanese flounder, Paralichthys olivaceus, in response to heat stress, Edwardsiella piscicida infection, and trial feeding of immunostimulants, high-concentration ascorbic acid (AsA) or lactoferrin (LF). The results of quantitative real-time PCR showed that expression of the Hbß gene in the gills decreased markedly when exposed to heat stress, whereas that in the epidermis exhibited an increase 3h after infection with E. piscicida. Seven days after starting to feed either immunostimulant, epidermal Hbß gene expression in all AsA or LF dose groups was significantly higher than in the control group. The results of in situ hybridization showed that the abundance and intensity of the stained cells in the epidermis and in the gills were consistent with the expression levels of Hbß gene obtained from the infection and immunosuppressant experiments and the heat stress experiment, respectively. Our results suggest that mucosal Hbß gene expression is closely related to physiological and immunological status and could be a useful indicator for monitoring condition of fish health.

Enhancement of immune proteins expression in skin mucus of Japanese flounder Paralicthys olivaceus upon feeding a diet supplemented with high concentration of ascorbic acid.

To search immune defense proteins in skin mucus of Japanese flounder fed with a diet containing high concentration of ascorbic acid, we carried out 2D-PAGE and compared the resolved pattern of proteins between control group that fed commercial diet and ascorbic acid supplemented group (AsA group) fed a diet supplemented with high concentration of ascorbic acid (2,000 mg/kg) for 7 days. The results revealed that there were many proteins exhibited distinct increase in AsA group. Among them, 6 regions that showed a dramatic elevation were chosen for protein identification using LC-MS/MS analysis and Mascot database search. Six proteins were identified, i.e. serotransferrin (Sero), transferrin (Trans), warm temperature acclimation-related 65 kDa protein (Wap65), complement component c3 (C3), hemoglobin beta-A chain (Hbß) and apolipoprotein A-1 (Apo). Quantitative RT-PCR analysis showed that the mRNA level of Hbß in epidermis of AsA group gave much higher increase (11.6 folds) than control group; the levels of Sero/Trans, Wap65, C3 and Apo showed no apparent difference between the two groups. The mRNA levels of wap65 and c3 in the liver and Apo in the kidney of AsA group exhibited significant increase in comparison to control group. In the case of secreted immunoglobulin M (IgM) and lysozyme (lyz), no difference of the mRNA levels of IgM in epidermis, gill, kidney, spleen and intestine, and lyz in epidermis, gill, spleen and intestine, was observed. The results of in situ hybridization confirmed the elevation of Hbß mRNA level in the epidermis tissue of AsA group. Our present study provided additional evidence showing the effectiveness of AsA in activating innate immune defense system in skin mucosal tissue of fish.

Super-Protective Child-Rearing by Japanese Bess Beetles, Cylindrocaulus patalis: Adults Provide Their Larvae with Chewed and Predigested Wood.

Beetles of the family Passalidae (Coleoptera: Scarabaeoidea) are termed subsocial. The insects inhabit rotten wood as family groups consisting of the parents and their offspring. The Japanese species Cylindrocaulus patalis has the lowest fecundity among passalids because siblicide occurs among the first-instar larvae; accordingly, parental care toward the survived larva is the highest among Passalidae. To clarify the nutritional relationships between the parents and their offspring, we investigated their ability to digest three types of polysaccharides that are components of wood (cellulose and ß-1,4-xylan) and fungal cell walls (ß-1,3-glucan). Although carboxymethyl-cellulase activity was barely detectable, ß-xylosidase, ß-glucosidase, ß-1,4-xylanase and ß-1,3-glucanase activities were clearly detected in both adults and larvae. Because the activities of enzymes that digest ß-1,3-glucan were much higher than those for degrading ß-1,4-xylan, in both adults and larvae, it is concluded that they are mainly fungivorous. Furthermore, these digestive enzymatic activities in second- and third-instar larvae were much lower than they were in adults. Although all larval instars grew rapidly when fed chewed wood by their parents, larvae ceased growing and died when fed only artificially ground wood meals. We conclude that the larvae are assumed to be provided with chewed predigested wood in which ß-1,3-glucan is degraded by parental enzymes.

Glycolytic Activities in the Larval Digestive Tract of Trypoxylus dichotomus (Coleoptera: Scarabaeidae).

The larvae of the Japanese horned beetle, Trypoxylus dichotomus (Coleoptera: Scarabaeidae: Dynastinae), are an example of a saprophage insect. Generally, Scarabaeid larvae, such as T. dichotomus, eat dead plant matter that has been broken down by fungi, such as Basidiomycota. It is thought that ß-1,3-glucan, a constituent polysaccharide in microbes, is abundant in decayed plant matter. Studies of the degradation mechanism of ß-1,3-glucan under these circumstances are lacking. In the current study, we sought to clarify the relationship between the capacity to degrade polysaccharides and the food habits of the larvae. The total activities and optimum pH levels of several polysaccharide-degrading enzymes from the larvae were investigated. The foregut, midgut and hindgut of final instar larvae were used. Enzymatic activities were detected against five polysaccharides (soluble starch, ß-1,4-xylan, ß-1,3-glucan, pectin and carboxymethyl cellulose) and four glycosides (p-nitrophenyl (PNP)-ß-N-acetylglucosaminide, PNP-ß-mannoside, PNP-ß-glucoside and PNP-ß-xyloside). Our results indicate that the digestive tract of the larvae is equipped with a full enzymatic system for degrading ß-1,3-glucan and ß-1,4-xylan to monomers. This finding elucidates the role of the polysaccharide-digesting enzymes in the larvae, and it is suggested that the larvae use these enzymes to enact their decomposition ability in the forest environment.

Antiviral protection mechanisms mediated by ginbuna crucian carp interferon gamma isoforms 1 and 2 through two distinct interferon gamma-receptors.

Fish genomes possess three type II interferon (IFN) genes, ifnγ1, ifnγ2 and ifnγ-related (ifnγrel). The IFNγ-dependent STAT signalling pathway found in humans and mice had not been characterized in fish previously. To identify the antiviral functions and signalling pathways of the type II IFN system in fish, we purified the ifnγ1, ifnγ2 and ifnγrel proteins of ginbuna crucian carp expressed in bacteria and found them to elicit high antiviral activities against crucian carp hematopoietic necrosis virus. We also cloned two distinct ifnγ receptor alpha chain (ifngr1) isoforms, 1 and 2, and stably expressed them in HeLa cells by transfecting the cells with ifngr1-1 or ifngr1-2 cDNA. When receptor transfectants were treated with the ligands in a one-ligand-one-receptor manner (ifnγ1 and ifngr1-2 or ifnγ2 and ifngr1-1), the stat1 protein was phosphorylated at both serine-727 and tyrosine-701 residues. Gel shift mobility analysis and reporter assay clearly showed that the specific ligand-receptor interaction resulted in the binding of the stat1 protein to the GAS element and enhanced transcription. Therefore, the actions of ifnγ1 and ifnγ2 were found to be mediated by a specific receptor for each signalling pathway via a stat1-dependent mechanism.

Structural analysis of an acidic, fatty acid ester-bonded extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4.

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides, which are assumed to play an important role in the hydrocarbon tolerance of this bacterium. The strain produced two acidic extracellular polysaccharides, FR1 and FR2, and the latter showed emulsifying activity toward clove oil, whereas the former did not. FR2 was composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1, and contained 2.9% (w/w) stearic acid and 4.3% (w/w) palmitic acid attached via ester bonds. Therefore, we designated FR2 as a PR4 fatty acid-containing extracellular polysaccharide or FACEPS. The chemical structure of the PR4 FACEPS polysaccharide chain was determined by 1D (1)H and (13)C NMR spectroscopies as well as by 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The sugar chain of PR4 FACEPS was shown to consist of tetrasaccharide repeating units having the following structure: [structure: see text].

Structural analysis of mucoidan, an acidic extracellular polysaccharide produced by a pristane-assimilating marine bacterium, Rhodococcus erythropolis PR4.

Rhodococcus erythropolis PR4 is a marine bacterium that can degrade various alkanes including pristane, a C(19) branched alkane. This strain produces a large quantity of extracellular polysaccharides (EPS), which are assumed to play an important role in the hydrocarbon tolerance of R. erythropolis PR4. The strain produced an acidic EPS, mucoidan, together with a fatty acid-containing EPS, PR4 FACEPS. The chemical structure of the mucoidan was determined using (1)H and (13)C NMR spectroscopy and by conducting 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The mucoidan was shown to consist of a pentasaccharide repeating unit with the following structure: [structure: see text].

Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2.

A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).

Structural analysis of an extracellular polysaccharide produced by a benzene tolerant bacterium, Rhodococcus sp. 33.

Rhodococcus sp. 33 can tolerate and efficiently degrade various concentrations of benzene, one of the most toxic and prevailing environmental pollutants. This strain produces a large quantity of extracellular polysaccharide (33 EPS), which plays an important role in the benzene tolerance in Rhodococcus sp. 33, especially by helping the cells to survive an initial challenge with benzene. This EPS has been reported to be composed of D-galactose, D-glucose, D-mannose, D-glucuronic acid, and pyruvic acid at a molar ratio of 1:1:1:1:1. To understand the protective effect of 33 EPS, we determined its chemical structure by using 1H and 13C NMR spectroscopy including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: [structure: see text].