Comparison of morphological and DNA-based techniques for stomach content analyses in juvenile chum salmon <Emphasis Type="Italic">Oncorhynchus keta</Emphasis>: a case study on diet richness of juvenile fishes

To identify the stomach contents of marine organisms, morphological observations are commonly conducted. However, the results obtained by this traditional method are frequently biased, as it is difficult to detect partially digested, soft-bodied organisms. To resolve this, recent studies have used DNA-based (DNA barcoding) analyses to examine the diet breadth of marine organisms. Here, we compared the prey richness of juvenile fishes using morphological observations and DNA-based analyses, with a focus on juvenile chum salmon Oncorhynchus keta. A higher number of prey taxa were successfully identified using DNA-based analyses than morphological observations. However, we also noticed several shortcomings of the DNA-based analyses, as reported in other diet-analysis studies that used molecular techniques. For example, the degree of digestion among prey taxa might have resulted in differential sensitivity to DNA detection. Additionally, several prey taxa could not be precisely identified, as the sequence data for some of the targeted organismal groups are unavailable in public gene databases. Remarkably, it is also possible that DNA-based analyses detected secondary prey, and therefore, the richness of prey taxa was likely overestimated. Thus, dietary analyses of juvenile fishes need to be carefully conducted, considering the respective advantages and disadvantages of DNA-based and morphological techniques.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

Lipopolysaccharide induces expression of collagen VI in the rat lung

The involvement of the lung during the septic systemic inflammatory response elicited by administration of lipopolysaccharide (LPS) was investigated. Eight-week-old male Sprague–Dawley rats were injected i.p. with 15 mg/kg LPS. After 24 h, the lungs were excised to evaluate the cellular responses to LPS. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) analysis revealed that type VI collagen (ColVI) was extremely upregulated during sepsis in the rat lung within the first 24 h of LPS administration. Upregulation of ColVI protein and its mRNA was demonstrated by Western blot analysis, real time PCR, and immunohistochemistry. To the best of our knowledge, this is the first report demonstrating the activation of ColVI in the rat lung at the early stage of systemic inflammation. Activation of ColVI might be involved in sepsis-mediated lung fibrosis at an early stage.     

Persimmon breeding in Japan for pollination-constant non-astringent (PCNA) type with marker-assisted selection

Oriental persimmon (Diospyros kaki) originated in Eastern Asia, and many indigenous cultivars have been developed in China, Japan, and Korea. These cultivars are classified into four groups based on their natural astringency loss on the tree and seed formation: pollination-constant non-astringent (PCNA), pollination-variant non-astringent (PVNA), pollination-constant astringent (PCA), and pollination-variant astringent (PVA). PCNA is the most desirable type because the fruit can be eaten without any postharvest treatment; therefore, one of the goals of our persimmon breeding programs is to release superior PCNA cultivars. The PCNA genotype is recessive to the other three non-PCNA genotypes, and PCNA-type F₁ offspring are obtained exclusively from crosses among PCNA genotypes. Moreover, the number of superior PCNA cross-parents have been limited. In the late 1980s, inbreeding depression became obvious, especially in terms of fruit size, tree vigor, and productivity. To mitigate the inbreeding, a backcross program using PCNA [(non-PCNA × PCNA) × PCNA] was started in 1990. This process, however, was inefficient because only 15% of the offspring were PCNA, and all offspring had to be grown to the fruiting stage. Therefore, molecular markers linked to the PCNA locus were developed for discriminating PCNA offspring. A molecular marker linked to Chinese PCNA has also been developed.     

Effect of water temperature on the feeding activity and the resultant mercury levels in the muscle of cultured bluefin tuna Thunnus orientalis (Temminck and Schlegel)

Tuna muscle often contains high levels of mercury, and fish samples with mercury concentrations ten times higher than the specified safety standards have been reported. Here, we report on the relationship between water temperature and the concentration of mercury in the tail muscle tissue of cultured bluefin tuna Thunnus orientalis. The fish used in this study were cultured at Fisheries Laboratory of Kinki University (Amami Experimental Station, Kagoshima, Japan). One hundred fish weighing 26.2–89.4 kg were selected for analysis between February 2007 and January 2008. Water temperature during rearing ranged from 21 to 29 °C. The total mercury levels were measured using the reduction vaporizing atomic absorption method after acid digestion. Body weight increased approximately 1.5 times that observed in a previous study, despite feeding activity either being the same or less than that observed previously. The average mercury concentration in white muscle was 0.353 mg kg^?1, remaining almost constant and independent of body growth. Unlike previous studies, seasonality was not observed in this study. Based on these findings, water temperatures within a certain range were considered to stabilize feeding activity and increase feeding efficiency. Consequently, water temperature is considered to have a moderating effect on seasonal fluctuations in muscle mercury concentrations in cultured bluefin tuna.     

Protective effect of floor cover against soil erosion on steep slopes forested with <Emphasis Type="Italic">Chamaecyparis obtusa</Emphasis> (hinoki) and other species

 We evaluated the protective effects of floor cover against soil erosion in three types of forest located on steep slopes under a humid climate: 22- and 34-year-old Chamaecyparis obtusa (hinoki), 34-year-old Cryptomeria japonica (sugi), and 62-year-old Pinus densiflora (red pine) stands. We measured sediment transport rates (sediment mass passing through one meter of contour width per millimeter of rainfall), using sediment traps, before and after removing floor cover. Raindrop splash erosion was dominant in the experimental stands. Floor cover percentage (FCP) during the preremoval stage varied from 50% to 100% among the four stands, and sediment transport rates ranged from 0.0079 to 1.7 g m⁻¹ mm⁻¹. The rates increased to 1.5–5.6 g m⁻¹ mm⁻¹ immediately after removing floor cover, and remained high throughout the experiment. The presence of physical cover near the ground has a crucial effect on sediment transport on forested slopes. The protective effect ratio (the ratio of the sediment transport rate in a control plot to that in the removal plot) in a young hinoki stand, in which the FCP decreased markedly, was 0.3 at most, which is close to the rate for bare ground. The protective effect ratio in the red pine stand was ≤0.003. We concluded that the protective effect of floor cover in undisturbed forests in Japan differs by over two orders of magnitude, based on comparisons with previous studies. Received: March 11, 2002 / Accepted: August 16, 2002 Present address: Department of Forest Site Environment, Forestry and Forest Products Research Institute, Ibaraki 305-8687 Japan Tel. +81-298-73-3211; Fax +81-298-74-3720 e-mail: miura@affrc.go.jp Present address: Department of Forest Site Environment, Forestry and Forest Products Research Institute, Ibaraki 305-8687 Japan Tel. +81-298-73-3211; Fax +81-298-74-3720 e-mail: miura@affrc.go.jp Acknowledgments This study was supported by the Research Council of the Ministry of Agriculture, Forestry, and Fisheries, of Japan. We thank H. Ujihara, S. Ujihara, and M. Ogasawara in Otoyo, Kochi, who provided the experimental stands used in this study. We also thank K. Hirai, S. Kuramoto, E. Kodani, and the rest of the staff at the Shikoku Research Center, Forestry and Forest Products Research Institute, for their help in conducting the experiments. Correspondence to:S. Miura     

Absolute configuration of arylglycerol-β-aryl ethers obtained by asymmetric reduction of the correspondingα-ketonic compound with intactFusarium solani cells

When (±)--oxo-guaiacylglycerol--(vanillic acid) ether (1) is degraded byFusarium solani M-13-1, the-ketone is initially reduced to giveerythro andthreo guaiacylglycerol--(vanillic acid) ethers (2), arylglycerol--aryl ethers, both of which are enantiomerically pure. The absolute configuration in each2 was determined by Mosher's method; the products were converted to,-di-(R)--methoxy--trifluoromethylphenylacetates (MTPA esters) (3) oferythro (-)- andthreo (+)-veratrylglycerol--(methyl vanillate) ethers (3), whose¹H nuclear magnetic resonance (NMR) spectra were examined and compared with those of four di-(R)-MTPA ester (3) diastereomers from chemically synthesizederythro (±)-3 andthreo (±)-3. To assign the- and-MTPA-OCH₃ peaks, the¹H NMR scans of several compounds that have substructures of 3 and their 3,4,5-trimethoxyphenyl analogues were examined. When a racemic alcohol reacts with (R)-MTPA to give a pair of (R)-MTPA ester diastereomers, the value was defined as the absolute value of the difference in the¹H chemical shifts of the peak between the diastereomers. It was found that the values of-MTPA-OCH₃ were larger than those of-MTPA-OCH₃ owing to a shielding effect of the veratryl ring located on the-MTPA-OCH₃, and that the-MTPA-OCH₃ peaks in the 3,4,5-trimethoxyphenyl compounds shifted downfield relative to those in the veratryl compounds. On the basis of the¹h NMR data of (R)-MTPA esters, the absolute configuration of the four chemically prepared diastereomers (3) were determined. The catabolicerythro 3 [fromerythro (-)-3] andthreo 3 [fromthreo (+)-3] were identical to (R, S, R)-erythro 3 and (R, S, S)- threo 3, respectively. An hydrogen species in the fungal reduction would attack the-ketone fromre-face of both (R)-1 and (S)-1, givingerythro (S, R)-2 andthreo (S, S)-2, respectively.Part of this paper was presented at the 33rd Lignin Symposium, Tsukuba, November 1988