Rapid discrimination of two cryptic species within Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae) by PCR–RFLP

Brontispa longissima (Coleoptera: Chrysomelidae) is a serious invasive pest of coconut palm (Cocos nucifera) supposedly originating from Indonesia and New Guinea. It has recently invaded Southeast and East Asia, where it has caused serious damage to coconut plants. Brontispa longissima as currently defined contains two cryptic species: we herein referred to one as the “Asian clade”, which is distributed over a wide area, including Asia and the Pacific region; and we referred to the other one as the “Pacific clade”, which is found in a limited area in the Pacific region. We developed a PCR–RFLP method for differentiating the two clades. Digestion of the PCR product of a 1,014-bp region within the mitochondrial cytochrome oxidase I gene (COI) with BslI, HpyCH4III, or NlaIV resulted in clade-specific patterns as estimated by the sequence data. We applied the method to specimens newly obtained from various locations to investigate the geographical distribution of B. longissima. Although B. longissima collected from Samoa in April 2003 had been placed in the Pacific clade, specimens collected from the same island in April 2010 were placed in the Asian clade, suggesting that the predominant clade may have been changing from the former to the latter. On Timor, specimens included both clades in apparently segregated habitats.     

Malignant Peritoneal Mesothelioma with a Sarcomatoid Growth Pattern and Signet-Ring-Like Structure in a Female F344 Rat

We report a biphasic malignant mesothelioma in an aged female F344/DuCrlCrlj rat. Macroscopically, multiple pale brown nodules were observed in the abdominal cavity with retention of bloody ascites. Histopathologically, the tumor cells spread over the peritoneum and formed masses on the surface and underlying adipose tissues. The tumor cells dominantly proliferated in a solid, nodular or nest-like pattern with modest amount of fibrillar connective tissues, which contained hyaluronan. The tumor consisted of ovoid, polygonal or spindle-shaped cells that possessed eosinophilic cytoplasms including glycogen; some tumor cells showed a signet-ring-like structure. Multinucleated cells and mitosis were found frequently, and direct invasion to intra-abdominal organs and intravascular metastasis to the liver were observed. Immunohistochemically, keratin and mesothelin were strongly positive in most of tumor cells, while vimentin was mainly positive in spindle-shaped cells. Podoplanin was also positive, particularly in the cell membrane of tumor cells. Electron microscopically, tumor cells showed an intercellular desmosome-like structure, basement membrane and microvillus. We diagnosed the case as a malignant peritoneal mesothelioma with a sarcomatoid growth pattern and signet-ring-like structure.     

Effect of different organic amendments on the resistance and resilience of the organic matter decomposing ability of soil and the role of aggregated soil structure

The effect of organic amendment on the resistance and resilience of the organic matter decomposing activity was compared between soils amended with compost and with chemical fertilizers. The impact of metam sodium disinfection on cellulose-decomposing activity and on the number of nematodes in three types of soils was periodically measured. In an andosol, cellulose-decomposing activity was significantly suppressed by soil disinfection only in the chemically fertilized soil (CF-soil) and not in the soils to which cow manure compost and okara (the residue in tofu production)/coffee compost was added. In a brown lowland soil, cellulose-decomposing activity was significantly suppressed by soil disinfection in the CF-soil, but not in the soils to which higher amounts of cow manure compost and pig manure compost had been added. In a red-yellow soil, cellulose-decomposing activity was significantly suppressed by soil disinfection in all soils, but its resilience was higher in the soils to which cow manure compost or coffee compost was added compared with the CF-soil. Total numbers of nematodes were markedly decreased by soil disinfection in all soils. These results may suggest that the resistance and resilience of cellulose-decomposing activity against soil disinfection were enhanced by organic amendments, while disinfection had fatal effects on soil nematodes. In most of the organically amended soils, the mean weight diameters of aggregates were larger compared with the CF-soils, suggesting that highly structured soil pore networks may provide shelters for the soil microbes responsible for cellulose decomposition against disinfection. This hypothesis was supported by the result that the resistance of cellulose-decomposing activity against soil disinfection decreased when the soil structure was destroyed by grinding in a mortal and pestle.     

Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.     

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.