Immunohistochemical survey of the gut endocrine cells in the common tree shrew (Tupaia belangeri).

Regional distribution and relative frequency of endocrine cells in the gastrointestinal tract of the common tree shrew (Tupaia belangeri) were studied immunohistochemically. Ten types of immunoreactive endocrine cells were localized in the gastric mucosa, i.e., chromogranin-, serotonin-, gastrin-, somatostatin-, bovine pancreatic polypeptide (BPP)-, enteroglucagon-, pancreatic glucagon-, peptide tyrosine tyrosine (PYY)-, motilin-, and substance P (SP)-immunoreactive (IR) cells. In the intestine, 13 types of immunoreactive cells were observed, i.e., chromogranin-, serotonin-, somatostatin-, gastrin-, BPP-, enteroglucagon-, PYY-, secretin-, cholecystokinin (CCK)-, gastric inhibitory peptide (GIP)-, motilin-, neurotensin-, and SP-IR cells. The regional distribution and relative frequency of the cell types varied along the gastrointestinal tract. Basically, the types, distribution, and relative frequency of the gut endocrine cells were similar to those reported in other mammalian species. However, some characteristic findings were noted in the present study: (1) the considerably large number of gastrin-IR cells in the pyloric region; (2) numerous serotonin-IR cells in the stomach; (3) appreciable number of BPP-IR cells in the transitional region of the stomach; and (4) wide distribution of PYY- and motilin-IR cells in the gut.     

Genetic and molecular analysis of the restoration of fertility ( Rf ) genes for Ogura male-sterility from a Japanese wild radish ( Raphanus sativus var. hortensis f. raphanistroides Makino)

Ogura male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. In this study, in order to gain better understanding of the variation and evolution of the restoration of the fertility (Rf) gene for Ogura male-sterile cytoplasm, the nucleotide sequence of the orf687 homologue in the Japanese wild radish (Raphanus sativus var. hortensis f. raphanistroides Makino) was analyzed using an F₂ population made with a cross between a Japanese wild radish plant containing the Rf gene and ‘Uchiki-Gensuke’ (a maintainer of Ogura-male sterility). Segregation of male-fertile/-sterile plants in the F₂ generation suggested that another unidentified Rf gene unlinked to orf687 exists in the Japanese wild radish. The genotype of orf687 was determined for each F₂ plant by Southern hybridization with an orf687 gene probe, mismatch-specific endonuclease digestion of PCR products, and direct sequencing of a PCR product. Genotyping revealed that some fertility-restored plants are homozygotic for the ‘Uchiki-Gensuke’ type orf687 allele, supporting the idea that another gene different from orf687 also functions as an Rf gene for Ogura male-sterility. Protein analysis using an antibody raised against the Ogura-specific ORF138 protein suggests a mechanism of fertility restoration by the unidentified Rf similar to that by orf687. Sequence analysis of orf687 from a Japanese wild radish plant and ‘Uchiki-Gensuke’ revealed that both orf687 regions encode a mitochondrially-targeted protein consisting of 687 amino acids with 16 PPR motifs. Comparison of the deduced amino acid sequences with those of the known orf687 sequences from ‘Yuan hong’ and ‘Kosena’ containing Rf and recessive one (rf), respectively, showed that three unique amino acid replacements are present in ORF687 of the Japanese wild radish. Two of the three replacements, that from lysine to isoleucine at position 232 and from asparagine to asparate at position 240, confer negative charges to the protein. Since the Rf of ‘Yuan hong’ was reported to have a unique replacement that confers a negative charge to ORF687 (from asparagine to aspartate at position 170), it is proposed that the amino acid replacements conferring a negative charge to ORF687 are important for determining the status of the Rf/rf gene.     

Effect of dietary high amount of calcium and phosphorous on reducing the prevalence of morphological deformities in GH-transgenic Nile tilapia

To satisfy the world’s growing need for animal protein, it is important to develop or select fish species which are suitable for optimal cultivation in closed recirculating aquaculture systems (CRASs) as an environment-friendly aquaculture technology for providing safe and secure aquaculture food products. We have developed an ‘all fish’ growth-hormone (GH)-transgenic Nile tilapia Oreochromis niloticus (GHTi) which appears to be a predominant candidate for CRAS since it has been found to exhibit two important characteristics, namely, a shorter rearing duration to a market size and lowered N and P discharge load. However, these fish have undergone morphological changes, resulting in a short body which is less acceptable to consumers as an edible fish. One possible solution to reducing the prevalence of such morphological deformities is to determine the association between changes in the nutritional conditions of these fish. To this end, we first calculated the ideal total calcium (Ca) and phosphorous (P) intake (g/kg), ideal daily Ca and P intake and retention (g/kg/day) and ideal daily feeding rate (%) for the fourth filial (F₄) offspring of GHTi. Our hypothesis was that GHTi would achieve the same total retention of Ca (14.6 g/kg) or P (7.69 g/kg) during a 69-day shorter rearing period as their production-matched pedigree common domestic strain (CTi). We estimated that when GHTi were fed conventional commercial diets to satiety, those with a body weight (BW) of more than approximately 230 g could not retain sufficient Ca, while those with a BW of more than approximately 429 g could not retain sufficient P. We then attempted to reduce the prevalence of morphological deformities in GHTi via dietary compound regulation by supplying a high amount of dietary Ca [6.82 % in the diet with Ca supplementation (Ca-S)] or P [4.75 % in the diet with P supplementation (P-S)]. Both the Ca-S and P-S diet improved mineral retention, while those fed on a Ca-S diet showed a lower prevalence of deformities, close to the size-matched CTi. These results suggest that the requirements of dietary Ca and P were significantly higher in GHTi than in CTi and, consequently, that the conventional levels of Ca and P in commercial diets for CTi cannot meet the mineral requirement of GHTi. We therefore propose to develop an optimal feed to meet the altered nutritional requirements of GH-transgenic Nile tilapia for growth-promoting optimization. This may become prerequisite for rearing this GHTi line in CRASs.     

Phylogeography based on the nuclear ribosomal DNA internal transcribed spacer region of native Miscanthus sinensis (Poaceae) populations in Japan

Nucleotide sequence variations were investigated with respect to the geographical distribution patterns of Miscanthus sinensis populations that were sampled from 26 Japanese national parks and three populations of the Ryukyu Islands. Twelve homozygous sequences in the nuclear ribosomal DNA internal transcribed spacer region were detected. The populations of M. sinensis in mainland Japan mainly were composed of a monophyletic group with a symapomorphic character, whereas those in the Ryukyu Islands included a polyphyletic group. Only an internal transcribed spacer haplotype with a plesiomorphic character was found in both mainland Japan and the Ryukyu Islands. Thus, no clear geographical isolation was observed in this species. These facts might be caused by the ability of M. sinensis as a pioneer plant to have a high migration potential and high gene flow by outcrossing. On the basis of the results of this study (nuclear ribosomal DNA) and the previous study (chloroplast DNA), a phylogeographical history of M. sinensis was presumed where the ancestral and derived lineages were distributed in the southern and northern regions of Japan, respectively, without strict geographical isolation.     

Effects of nutrient enrichment,irradiance control,and boiling on the color of the brown alga <Emphasis Type="Italic">Undaria pinnatifida</Emphasis>

The color of Undaria pinnatifida after boiling is an important factor determining its marketable value. Our previous study showed that decreased nutrients and elevated irradiance resulted in increases of lightness and yellowness (i.e., discoloration) of this alga. However, little is known about the optimal levels of nutrients and irradiance required to decrease these color values and the combined effects of these factors and boiling. We conducted two culture experiments to test the effects of nutrients (non-enriched and 1.25, 5, and 25% PESI enriched treatments), irradiance (0, 10, 30, and 180 µmol m^?2 s^?1), and boiling on lightness L*, redness a*, and yellowness b* of this alga. L* and b* did not differ between non-enriched and 1.25–5% PESI treatments, but were lower in the 25% PESI treatment. L* and b* were lowest at 0–10 µmol m^?2 s^?1, although negative growth occurred at 0 µmol m^?2 s^?1. Decreased irradiance had a positive or little effect on a* before boiling, but had a negative effect after boiling. These results suggest that around 25% PESI and 10 µmol m^?2 s^?1 were the optimal levels to decrease the three color values of this species after boiling.     

Biochemical changes in oyster tissues and hemolymph during long-term air exposure

The Pacific oyster Crassostrea gigas is a sessile bivalve that inhabits the intertidal zone and therefore frequently exposed to air during the tidal cycle. It is highly adaptive to hypoxic conditions. We have studied the physiological state of oysters during long-term exposure to air. The oysters became hypoxic when exposed to air or hypoxic seawater. The 50% lethal time of oysters exposed to air at 4, 15 and 20°C was 47.8, 15.9 and 12.2 days, respectively. The hemolymph pH decreased by day 3; however, it showed a slight increase by day 5 at both 4 and 20°C. The adenylate energy charge (AEC) values decreased rapidly on the first day of air exposure in the adductor muscle, mantle, gill and body trunk, and these decreases were accompanied by decreases in ATP concentrations and increases in AMP concentrations. The AEC values in all of the tissues had fallen to below 30% by day 50 of air exposure at 4°C. These data suggest that the energy state of oysters deteriorates rapidly with air exposure. Consequently, AEC values may be useful indices of the physiological state of the oyster during long-term exposure to air.     

Effect of cadmium binding form on rabbit aminolevulinate dehydratase in high molecular weight fraction of rainbow trout liver

Enzyme inhibition owing to cadmium (Cd) accumulation in the soluble high molecular weight fraction (HMF) of the liver from rainbow trout was investigated by measuring the inhibition of aminolevulinate dehydratase (ALAD). Factors affecting ALAD, which reacts specifically to metal, were examined before measuring ALAD activity. Although enzyme inhibition by Cd in the HMF was confirmed in the excretion 2 (E2) group fed Chinese parsley (CP), it was weaker than that in the excretion 1 (E1, control) group, probably due to the chelation of Cd by some substances contained in CP. Based on the absorption spectrum, the proportion of Cd bound to cysteine thiol radicals in the HMF of the E2 group was greater than that of E1. The HMF might detoxify harmful metals to some extent because it contains relatively higher levels of cysteine residues than metallothionein (MT) and shows comparatively weak enzyme inhibition. The results of the present study demonstrated that, in addition to MT, HMF is involved in the detoxification of harmful heavy metals in fish.     

Immunological effects of glucan and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> GG,a probiotic bacterium,on Nile tilapia <Emphasis Type="Italic">Oreochromis niloticus</Emphasis> intestine with oral <Emphasis Type="Italic">Aeromonas</Emphasis> challenges

The present study histologically examined the effects of glucan-containing and Lactobacillus rhamnosus GG (LGG)-containing diets on intestinal damages inflicted on Nile tilapia by Aeromonas challenges. Tilapia were fed control, glucan, and LGG diets for 2 weeks and were subsequently challenged with Aeromonas. The intestines were then histologically examined at 1, 7, 14, and 21 days post-infection. Mortality following the challenge was lower for the fish fed the glucan and LGG diets. The intestines of these groups also showed increased inflammatory cell infiltration and reduced intestinal damage from Aeromonas. Moreover, inflammatory cell infiltration occurred more rapidly in the glucan-fed than in the LGG-fed fish following the challenge. Before the challenge, the dominant mucous cell was the acid type in all the tests. After the challenge, the main mucus cell type in the proximal intestine of the glucan-fed fish shifted to AB-PAS double-staining cells, while in the LGG-fed fish, it remained the acid type throughout the test period, and the number of double-staining cells was smaller than in the control fish after the challenge. Thus, the different mucous cell and inflammatory cell responses show that glucan and LGG might have different immunostimulative effects, although they both reduced the intestinal damage following Aeromonas challenges.