Effect of Cullural Conditions on the Growth of Agrostemma githago Cells in Suspension Culture and the Concomitant Production of an Anti-Plant Virus Substance

An extract of cultured Agroxieinma githago L. cells was found to show potent inhibitory activity against plans virus infection. The effects of cultural conditions on the growth of the cell suspension and on the production of the inhibitor were examined. Since the production of substance was dependent on growth. experiments were made to improve growth. The optimum temperature was 26 to 30°C and optimum pH of the medium before autoclaving was between 5 and 7. In a medium of higher osmotic pressure, the water content of the cultured cells was lowered markedly. The growth rate in a small volume of the medium was higher than that in a larger volume at an early stage of the cultivation, but it was not changed by different inoculum sizes. The cells required thiamine and 2,4-D for growth but no other vitamins or growth regulators. The optimum level of 2,4-D was 0.1 mg/l. Higher sucrose concentration in the medium gave higher production of cell mass and of the inhibitor. However, 3% of sucrose was selected as the most economical concentration. For normal cell growth, the presence of both NH₄NO₃ and KNO₃ as nitrogen sources was required. The use of a single nitrogen source caused a long lag period or inhibition of the cell growth. KH₂PO₄ stimulated the growth when in was used in the level of 2.5 to 5 mM. The cell adhesion on the surface of the fermentor sometimes causes trouble in a large-scale cultivation. It was found that reducing the Ca²⁺ level in the medium prevented the cell adhesion and foaming remarkably. Based on the results obtained, a modified medium was established which was excellent for shortening the culture period and for efficient production of the anti-plant virus inhibitor.     

Effects of temperature,photoperiod and soil humidity on induction of pseudopupal diapause in the bean blister beetle Epicauta gorhami

Larvae of the bean blister beetle Epicauta gorhami Marseul (Coleoptera: Meloidae) feed on grasshopper eggs in soil and undergo hypermetamorphosis. This beetle undergoes larval diapause in the fifth instar as a pseudopupa, a form characteristic of hypermetamorphosis in meloid beetles. The effects of temperature, photoperiod and soil humidity on larval development of E. gorhami are examined in a population in Miyazaki, Japan, using egg pods of Locusta migratoria L. as food. At lower temperatures (20 and 22.5 °C), all larvae become pseudopupae, regardless of the photoperiod. By contrast, at higher temperatures (27.5 and 30 °C), almost all larvae pupate at the end of the fourth instar, again regardless of the photoperiod. A long‐day photoperiodic response occurs only at an intermediate temperature (25 °C): under an LD 12 : 12 h photocycle, all larvae enter diapause, although the diapause incidence tends to decrease as the day length becomes longer. Pseudopupae are immobile and remain in diapause for ≥120 days when they are kept under the same conditions, except that diapause terminates within a relatively short time at 30 °C. Although lower soil humidity retards post‐feeding development, soil humidity has no effect on the diapause incidence. On the basis of the short developmental period and diapause avoidance under summer conditions, it is suggested that this beetle partially produces two generations a year in southwestern Japan.     

How can river-estuarine planktonic copepods survive river floods?

Distribution of brackish-water zooplankton was surveyed in the Chikugo River estuary, Japan, at 3-h intervals for 24h, including several hours when the river was heavily flooded because of rainfall and discharge from an upstream dam. The populations of the dominant copepods Pseudodiaptomus inopinus and Sinocalanus sinensis were concentrated in the mixing zone of the salt wedge before river flooding. During the period of flooding, they were almost completely swept out from the river, but examination of water trapped by a sediment grab sampler revealed that adults of Pseudodiaptomus inopinus were aggregated immediately above the bottom of the submerged channel outside the river mouth. After the flood, aggregations of both species appeared again in the river together with the salt wedge, and the proportion of adults in the aggregations became significantly higher than before. These results suggest that immature copepods suffer population losses through river flooding, but that the copepods can survive such flooding by means of both behavioral and reproductive mechanisms; that is, adult copepods maintain their position on the bottom of submerged channels outside the river mouth during a river flood and replace the population loss through reproduction after the flood. Channels outside river mouths are thus regarded as important topographic refuges for river-estuarine zooplankton to survive a flood.     

Characteristics of Sleep and Wakefulness inWild-Derived Inbred Mice

Genetic variations in the wild-derived inbred mouse strains are more diverse than that of classical laboratory inbred mouse strains, including C57BL/6J (B6). The sleep/wake and monoamine properties of six wild-derived inbred mouse strains (PGN2, NJL, BLG2, KJR, MSM, HMI) were characterized and compared with those of B6 mice. All examined mice were nocturnal and had a polyphasic sleep pattern with a “main sleep period” identified during the light period. However, there were three sleep/wake phenotypic differences between the wild-derived mouse strains and B6 strain. First, the amount of sleep during the dark phase was comparable with that of B6 mice. However, the amount of sleep during the light phase was more varied among strains, in particular, NJL and HMI had significantly less sleep compared with that of B6 mice. Second, PGN2, NJL, BLG2, and KJR mice showed a “highly awake period” (in which the hourly total sleep time was <10%) immediately after the onset of the dark period, which was not seen in B6 mice. Third, relative to that of B6 mice, PGN2 and KJR mice showed longer duration of wakefulness episodes during the 12-h dark phase. Differences in whole brain noradrenaline, dopamine, and 5-hydroxy-tryptamine contents between the wild-derived mouse strains and B6 strain were also found. These identified phenotypes might be potentially under strong genetic control. Hence, wild-derived inbred mice could be useful for identifying the genetic factors underlying the regulation of sleep and wakefulness.     

Ancient genome‐wide admixture extends beyond the current hybrid zone between Macaca fascicularis and M. mulatta

Macaca fascicularis and Macaca mulatta are two of the most commonly used laboratory macaques, yet their genetic differences at a genome‐wide level remain unclear. We analysed the multilocus DNA sequence data of 54 autosomal loci obtained from M. fascicularis samples from three different geographic origins and M. mulatta samples of Burmese origin. M. fascicularis shows high nucleotide diversity, four to five times higher than humans, and a strong geographic population structure between Indonesian‐Malaysian and Philippine macaques. The pattern of divergence and polymorphism between M. fascicularis and M. mulatta shows a footprint of genetic exchange not only within their current hybrid zone but also across a wider range for more than 1 million years. However, genetic admixture may not be a random event in the genome. Whereas randomly selected genic and intergenic regions have the same evolutionary dynamics between the species, some cytochrome oxidase P450 (CYP) genes (major chemical metabolizing genes and potential target genes for local adaptation) have a significantly larger species divergence than other genes. By surveying CYP3A5 gene sequences of more than a hundred macaques, we identified three nonsynonymous single nucleotide polymorphisms that were highly differentiated between the macaques. The mosaic pattern of species divergence in the genomes may be a consequence of genetic differentiation under ecological adaptation and may be a salient feature in the genomes of nascent species under parapatry.     

In Situ Detection of Immunocompetent Cells in Murine B16 Melanoma Locally Treated With Interleukin-2 or Microwaval Hyperthermia

Fidler's B16-F10 melanoma was locally treated either with recombinant interleukin-2 (rIL-2) or microwaval hyperthermia, and immunological responses of the host to the melanoma after each treatment were investigated by immunofluorescent staining of the tissue. It was found that the local injection of rIL-2 either into the base of the tumor or into the upper part of the tumor directly caused infiltration of mainly NK cells and macrophages in the interstitials and/or in the tumor nests. T cells were also observed but the extent of infiltration was less in both treatments. Local microwaval hyperthermia of melanoma at 42°C for 30 min or at 43°C for 15 min also caused infiltration of NK cells and macrophages. Positive staining of the melanoma tissue with anti-ICAM-1 antibody after hyperthermia was seen in the interstitials adjacent to melanoma nests containing necrotic melanoma cells caused by hyperthermia. Based on the results, the rationality of combination of hyperthermia with local injection of rIL-2 will be discussed.