Field trap test for bioassay of synthetic (1<Emphasis Type="Italic">S</Emphasis>,4<Emphasis Type="Italic">R</Emphasis>)-4-isopropyl-1-methyl-2-cyclohexen-1-ol as an aggregation pheromone of <Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Coleoptera: Platipodidae)

An ambrosia beetle, Platypus quercivorus, is a vector of Japanese oak wilt, which causes massive mortality of oak trees in Japan. Previous studies have identified (1S,4R)-4-isopropyl-1-methyl-2-cyclohexen-1-ol ((−)-IMCH) as the major component of the aggregation pheromone of P. quercivorus. We tested the ability of synthetic (−)-IMCH to attract P. quercivorus adults. The synthetic compound attracted 14.4 times as many P. quercivorus, of both sexes, as control traps. Control and (−)-IMCH-baited traps both captured 3.32 times as many males as females, probably because of the nature of Lindgren funnel traps and of male-biased emergence sex ratio early in the season of adult flights. (−)-IMCH was proved to be a major component of the aggregation pheromone of P. quercivorus.     

Chicken peripheral blood CD3+CD4-CD8- cells are regulated by endocrine and nerve systems

The existence of CD3(+)CD4(-)CD8(-) T cells in thymus and spleen has already been known. However, because of the presence of large amounts of thrombocytes in peripheral blood (PB), the proportion of CD3(+)CD4(-)CD8(-) T cells in PB has yet to be investigated. Therefore, the proportion of peripheral T cell-subsets was investigated in 6-week-old chickens. The percentage of CD3(+) cells, CD4(+) cells, CD8 alpha(+) cells, CD8 beta(+), and CD3(+)CD4(-)CD8(-) cells was 76%, 41%, 14%, 5%, and 15%, respectively. The proportion of CD3(+)CD4(-)CD8(-) cells in PB increased during egg-laying periods and in chickens treated with an analog of estrogen, while it decreased with age and in response to restraint stress. All of the CD3(+)CD4(-)CD8(-) cells expressed TCR1, and did not have NK activity. CD3(+)CD4(-)CD8(-) cells represent about 60% of peripheral TCR1(+) cells. These findings indicate that the proportion of CD3(+)CD4(-)CD8(-) cells is regulated by the endocrine and nerve systems.     

‘巨峰’葡萄主梢和副梢花器官与种子发育的研究

 ‘巨峰’葡萄主梢花粉粒大于副梢, 花粉染色率与副梢差异不显著, 发芽率低于副梢, 但花粉发芽率不影响种子的形成。主梢小花雌蕊在减数分裂后形成的功能大孢子小于副梢。同时开花前主梢胚囊的发育状况优于副梢, 开花期正常胚囊率主梢也明显低于副梢。因此, 开花前后主梢异常胚囊的出现是导致受精发生障碍并使结籽率低下的原因。     

Reduction of transport stress of ayu by obligated schooling

We investigated stress responses of ayu Plecoglossus altivelis under the hypothesis that overcrowding during transport can be a stressor and the stress predisposes fish to disease by decline in immunocompetence. The extent of stress response was determined under different fish densities. As inferred from the serum concentration of cortisol 1 day after the treatment, the density at more than 23 kg/m³, which was within the range of normal transporting procedures, acutely evoked intense stress responses. To examine a possible way to reduce the stress response of fish under crowding conditions, we compared two fish groups established in water tanks with circulating (whirling) and turbulent currents at the same density. The cortisol levels 1 day after the treatment were 2.5-fold higher in turbulent current than in whirling current. Fish in whirling current formed a school, while those in turbulent current aggregated with each other. Schooling of fish induced by water flow may reduce their stress responses during transport. A transport system with a directional water flow should keep fish in good condition without reducing the number of fish contained.     

Molecular phylogenetic relationship of Tetraodon pufferfish based on mitochondrial DNA analysis

Pufferfishes belonging to the genus Tetraodon form one of the largest groups in the family Tetraodontidae. This group consists of more than 20 species distributed across freshwater, as well as brackish and coastal waters, of Africa and Southeast Asia. However, their phylogenetic and evolutionary relationships are still ambiguous. In the present study, mitochondrial genes encoding 16S rRNA and cytochrome b were sequenced for 17 Tetraodon species collected from various areas mentioned above. Supermatrix analysis based on the complete sequences of the two genes from Tetraodon species was performed using a tree with 50 tetraodontid species (including 7 Tetraodon species) as a backbone. The obtained phylogenetic tree classified Tetraodon species into three groups, correlating well with their habitats, such as Asian freshwater, Asian brackish water, and African freshwater. We also showed that salinity tolerance of Asian freshwater species T. cochinchinensis was apparently lower than that of Asian brackish water species T. nigroviridis, suggesting that the speciation of Tetraodon species in the Asian freshwater group was caused by the molecular evolution associated with osmotic regulation.     

Biological and chemical stability of garlic-derived allicin

This study verifies the instability of garlic ( Allium sativum L.)-derived allyl 2-propenylthiosulfinate (allicin) in various aqueous and ethanolic solutions as well as in vegetable oil through chemical and biological analyses performed simultaneously. Crushed fresh garlic cloves generated antibacterial activity and chemically detectable allicin, a major antibacterial principle, and both declined on a daily basis in aqueous and ethanolic solutions at room temperature, showing biological and chemical half-lives of about 6 and 11 days, respectively. Allicin was more stable in 20% alcohol than in water, but surprisingly unstable in vegetable oil, with an activity half-life 0.8 h, as estimated from its antibacterial activity toward Escherichia coli, and a chemical half-life of 3.1 h, based on chromatographic quantification. In alcoholic and aqueous extracts, the biological half-life of allicin tended to be longer than the chemical one, suggesting the occurrence of bioactive compounds other than allicin in the extracts.     

Targeted Knock-in of a Fluorescent Protein Gene into the Chicken Vasa Homolog Locus of Chicken Primordial Germ Cells using CRIS-PITCh Method

In chickens, primordial germ cells (PGCs) are effective targets for advanced genome editing, including gene knock-in. Although a long-term culture system has been established for chicken PGCs, it is necessary to select a gene-editing tool that is efficient and precise for editing the PGC genome while maintaining its ability to contribute to the reproductive system. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) and CRISPR-mediated precise integration into the target chromosome (CRIS-PITCh) methods are superior as the donor vector is easier to construct, has high genome editing efficiency, and does not select target cells, compared to the homologous recombination method, which has been conventionally used to generate knock-in chickens. In this study, we engineered knock-in chicken PGCs by integrating a fluorescent protein gene cassette as a fusion protein into the chicken vasa homolog (CVH) locus of chicken PGCs using the CRIS-PITCh method. The knock-in PGCs expressed the fluorescent protein in vitro and in vivo, facilitating the tracking of PGCs. Furthermore, we characterized the efficiency of engineering double knock-in cell lines. Knock-in cell clones were obtained by limiting dilution, and the efficiency of engineering double knock-in cell lines was confirmed by genotyping. We found that 82% of the analyzed clones were successfully knocked-in into both alleles. We suggest that the production of model chicken from the knock-in PGCs can contribute to various studies, such as the elucidation of the fate of germ cells and sex determination in chicken.     

Purification and characterization of feline ghrelin and its possible role

Ghrelin, a novel 28-amino acid peptide with an n-octanoyl modification at Ser3, has been isolated from rat and human stomach as an endogenous ligand for the growth hormone secretagogue receptor. Here, we purified feline ghrelin and examined its possible physiological role in cats. The major active form of feline ghrelin is a 28-amino acid peptide octanoylated (C8:0) at Ser3; except for one amino acid residue replacement, this structure is identical to those of rat and human ghrelins. However, much structural divergence in peptide length and fatty acid modification was observed in feline ghrelin: peptides consisting of 27 or 26 amino acids lacking Gln14 and/or Arg28 were found, and the third serine residue was modified by octanoic acid (C8:0), decanoic acid (10:0), or unsaturated fatty acids (C8:1, C10:1 and C10:2). In agreement with the structural divergence, two kinds of cDNA with different lengths were isolated. Administration of synthetic rat ghrelin increased plasma growth hormone levels in cats, with a potency similar to that in rat or human. Plasma levels of ghrelin in cats increased approximately 2.5-fold after fasting. The present study indicates the existence of structural divergence in feline ghrelin and suggests that, as in other animals, ghrelin may play important roles in GH release and feeding in cats.     

Distribution of inclusion bodies in tissues from 100 dogs infected with canine distemper virus

One hundred dogs that were positive for canine distemper virus antigen and inclusion bodies in the tonsils were examined for the distribution of inclusion bodies in various tissues. Inclusion bodies were found in the lungs (70 dogs), brains (20 dogs), urinary bladders (73 dogs), stomachs (78 dogs), spleens (77 dogs), and lymph nodes (81 dogs) of the dogs. Based on these results, the tonsils may be the most suitable tissue for detection of inclusion bodies in canine distemper.