Cryopreservation of male and female gonial cells by vitrification in the critically endangered cyprinid honmoroko <Emphasis Type="Italic">Gnathopogon caerulescens</Emphasis>

We investigated the feasibility of cryopreservation of spermatogonia and oogonia in the critically endangered cyprinid honmoroko Gnathopogon caerulescens using slow-cooling (freezing) and rapid-cooling (vitrification) methods. Initially, we examined the testicular cell toxicities and glass-forming properties of the five cryoprotectants: ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG), and 1,3-butylene glycol (BG), and we determined cryoprotectant concentrations that are suitable for freezing and vitrification solutions, respectively. Subsequently, we prepared the freezing solutions of EG, GC, DMSO, PG, and BG at 3, 2, 3, 2, and 2 M and vitrification solutions at 7, 6, 5, 5, and 4 M, respectively. Following the cryopreservation of the testicular cells mainly containing early-stage spermatogenic cells (e.g., spermatogonia and primary spermatocytes), cells were cultured for 7 days and immunochemically stained against germ cell marker protein Vasa. Areas occupied by Vasa-positive cells indicated that vitrification led to better survival of germ cells than the freezing method, and the best result was obtained with 5 M PG, about 50% recovery of germ cells following vitrification. In the case of ovarian cells containing oogonia and stage I, II, and IIIa oocytes, vitrification with 5 M DMSO resulted the best survival of oogonia, with equivalent cell numbers to those cultured without vitrification. The present data suggest that male and female gonial cells of the endangered species G. caerulescens can be efficiently cryopreserved using suitable cryoprotectants for spermatogonia and oogonia, respectively.     

Long-term ecological impacts of clear-fell logging on tree species diversity in a subtropical forest,southern Japan

Defining the spatial arrangement and length of the cutting cycle in a logged area is crucial for reconciling potential conflicts between timber yields and maintenance of ecosystem services in natural forests. In this study, we investigated long-term impacts of clear-fell logging on timber production and tree species diversity in a subtropical forest on the Ryukyu Islands, using an individual-based simulation model. We assumed six logging scenarios defined by combinations of forest type and regeneration processes, which acted as surrogates for spatial scales of clear-fell logging. These scenarios were simulated under cutting cycles ranging from 20 to 150 years. Short-cutting cycles resulted in dominance by the sprouting species Castanopsis sieboldii. The compositional shift was accelerated by the lack of seed dispersal from surrounding forest areas. The simulations demonstrated that a sustainable logging regime maintaining both yield and tree species diversity requires a cutting cycle longer than 50 years. The simulation results also suggest that the trade-off between the recovery of tree species diversity and timber production is favored more in stands surrounded by mature forest than in isolated stands or stands surrounded by immature forest. Ecological risk assessments based on model simulations provide an alternative to current forest management practices that rely on empirical knowledge.     

Keratoconus in a cynomolgus monkey

In a seven-year-old male cynomolgus monkey, erythema of the upper eyelid and forehead and corneal opacity, edema and conical protrusion in the eye were observed. At necropsy, ophthalmological and serological examinations revealed binocular corneal opacity and conical protrusion and a high IgE level, respectively. Thinning of the epithelium and stroma of the cornea were noted histopathologically. At the center of the corneal epithelium, the number of epithelial cells was reduced, their cytoplasm was poorer and the basal cells were flatter than at the periphery. Bowman's membrane was folded with partial loss or breakage. Collagen fibers were compacted or disarranged, and the keratocytes were increased in the stroma, with focal pyknosis or loss of the endothelium and folding of Descemet's membrane. Electron microscopical examination revealed atrophy of the corneal epithelial basal cells. This is the first report of a case of keratoconus in a cynomolgus monkey.     

Analysis of genetic and biological characters of Japanese potato strains of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>

We assessed the geographic distribution, biovar, phylotype, DNA fingerprints (rep-PCR), and/or endoglucanase sequence of potato bacterial wilt pathogen, Ralstonia solanacearum (Rs), in Japan. Rs has been isolated from potato fields in southwestern, warm, temperate regions. Of the 188 isolates, 74 belonged to biovar N2 (39%), 44 to biovar 3 (24%), and 70 to biovar 4 (37%). Biovars N2 and 4 strains were widely distributed, from northern (Hokkaido) to southern (Okinawa) Japan. Based on the results of multiplex-PCR analysis, every potato strains belonged to either phylotype I or IV. Phylotype I comprised both biovars 3 and 4 strains. On the other hand, phylotype IV included biovar N2 strains. None of the strains belonged to phylotype II or III or biovar 1 or 2. Phylogenetic analysis based on DNA fingerprints and endoglucanase gene sequences clarified the genetic diversity of the Japanese potato strains and the close genetic relationship between the Japanese strains and the Asian strains in phylotypes I and IV.     

Bioeconomic assessment of size separators in Pacific saury fishery

In the middle of the 1990s, Pacific saury fishery vessels began to install “size separators” to selectively land large-size class fish with a higher price. Contrary to expectations, this resulted in removal of the separators in 2006 because fishers believed the separators had contributed to price collapses in the 2000s. The intent of this paper is to investigate the effects of separators on both the fishery economy and stock of Pacific saury by simulating population and economic models under a single framework. For this purpose, we developed (1) an age-structured population dynamics model with stochasticity, and (2) an economic model spanning both price and inventory dynamics with stochasticity, in which each set of model parameters were estimated on the basis of time series data. In a 10-year simulation, the harvest quota was set constant from 20,000 to 400,000 t at intervals of 20,000 t, and the effects of separators were incorporated by controlling the catchability of 0-year-old fish. We found that separators increase the expected yield and reduce the deficit risk for harvests of 140,000 t and smaller. However, separators have the opposite effects for harvests of 160,000 t and larger.     

Direct estimation of stock abundance of Pacific saury <Emphasis Type="Italic">Cololabis saira</Emphasis> in the northwestern Pacific Ocean using a mid-water trawl

Drifting gill-nets were for a long time the main research gear for investigating the distribution of Pacific saury Cololabis saira. However, it has proven to be difficult to estimate the absolute abundance of saury using drifting gill-nets due to a lack of knowledge on the survey areas swept by these gill-nets. A mid-water trawl stock abundance estimation has the advantage of being able to estimate the absolute stock abundance before the commercial fishing season begins, whereas the conventional stock abundance estimation based on catch per unit efforts of commercial fisheries must be done after the commercial fishing season has ended. To confirm the practicality of a mid-water trawl to estimate the stock abundance of saury, we conducted a comparative survey in the northwestern Pacific Ocean using both a mid-water trawl and drifting gill-nets. We found that the geographic distribution pattern of catch per unit effort for both the mid-water trawl and driftnets was similar. Using the area-swept method and data on the fishing efficiency of the mid-water trawl obtained in a previous study, we were able to estimate the stock abundance plus confidence limits within the research areas. Our results suggest that the size composition of saury sampled by the mid-water trawl can be considered to approximate the actual size composition of saury.     

An application of digital imagery analysis to understand the effect of N application on light interception,radiation use efficiency,and grain yield of maize under various agro-environments in Northern Mozambique

Light-based analysis is a fundamental approach to quantify the effects of factors determining crop growth in a given environment. The objectives of this study are to confirm the applicability of a digital imagery technique to extract green leaf areas for estimating light interception (LI) of maize canopy and to understand the effect of fertilizer application on the LI and radiation use efficiency (RUE) of maize under various agro-environments in Northern Mozambique. A locally recommended variety, Matuba, was grown in a single season with three different N application rates (0, 30, and 80 kgN ha^?1) at one hot/dry low-elevation site, two hot/humid mid-elevation sites, and one cool/humid high-elevation site. Repeated measurements with quantum sensors revealed that the digital imagery is applicable to estimate the LI of maize except for leaf-senescing period close to maturity. The N application demonstrated profitable yield increases with agronomic nitrogen use efficiencies (kg grain yield per kg N input) of 20.6–35.3 kg kg^?1 except for the low-elevation site with severe drought stress. In the mid-elevation sites, the yield increases were mostly explained by the improvement of RUE while the effect on LI was small because the vegetative growth was naturally vigorous under high temperatures irrespective of N inputs. At the high-elevation site, the N application improved its stagnant initial canopy development and increased both RUE and LI. The simple and inexpensive imagery technique should be useful to identify physiological basis of maize responses to fertilizer application and its interaction with regional environment even under poorly equipped regions in the tropics.     

Morphological analysis of cell distribution and network structure via gap junctions in swine corneal stroma

Because of corneal transplantation limitations, there is a need for cornea-specific regenerative medicine. The development of such regenerative medicine has been delayed because of the complex and unique structure of the corneal stroma. Few studies have explored the corneal stroma cell distribution and cell types in vivo. This study investigated regional differences in morphological characteristics and distributions of corneal keratocytes and immunocompetent cells in the corneal stroma to clarify their functions and structural characteristics. The porcine eyeballs were subjected to light microscopy, transmission electron microscopy, scanning electron microscopy, and immunofluorescence staining analyses. Corneal cells were primarily located in the limbus, rather than the center of the cornea; the long keratocyte diameter was largest on the epithelial side of the corneal limbus, while the short diameter was largest on the endothelial side of the central cornea. Moreover, there were significantly more corneal cells on the epithelial side than on the endothelial side in both the central and limbus areas. Gap junctions between cells in the corneal stroma were present on the surfaces of cytoplasmic processes. Many cytoplasmic processes were scattered throughout the corneal stroma; they were connected both vertically and horizontally, forming an intercellular network. Additionally, immunocompetent cells on the epithelial side suggested to participate in this network via gap junctions. The morphology of keratocytes and immunocompetent cells on the epithelial side suggests that they play important roles in corneal homeostasis.     

High genetic stability of TM1 and TM2 strains of subtype B feline immunodeficiency virus in long-term infection

To know the genetic changes of feline immunodeficiency virus (FIV) in long-term infection in cats, we inoculated three specific pathogen-free cats with FIV isolates and determined a partial env sequence covering the V3-V5 region. In 2 cats infected with subtype B strains TM1 and TM2, only one amino acid change in region V3 was observed at 9 years post infection (y.p.i.), and no nucleotide substitutions were observed between 9 and 10 y.p.i., indicating that these strains are genetically stable. On the other hand, in a cat infected with subtype A strain Petaluma at 8.7 y.p.i., 3 nucleotide insertions (one amino acid insertion) in region V5, and 1 synonymous nucleotide substitution and 2 non-synonymous nucleotide substitutions in region V5, were observed.