Relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes

To study the relationship between resistance to pine wilt disease and the migration or proliferation of pine wood nematodes (PWN) (Bursaphelenchus xylophilus), we conducted experiments using clonally-propagated Japanese black pines (Pinus thunbergii) with pre-evaluated individual resistance levels. Bark including the cortical resin canals—one of the main migration pathways of PWN—was removed by girdling, but neither the migration of PWN nor symptom development of pine wilt disease were inhibited by this treatment in non-resistant clones. Histological observations showed no significant differences in the lumen area or the number of cortical- and xylem- axial resin canals between resistant and susceptible clone groups from a half-sib family. A bioassay using methanol extracts from resistant and susceptible clones showed that extracts from both clones showed similar attractant effects to PWN, but neither had repellent effects. The resistant clones were multi-inoculated with PWN into three split points to mimic migration in the stem. The proportion of damaged plants was not significantly different from that in single-inoculated plants (control). In this experiment, the number of PWN detected from partially-damaged plants was much higher than that from non-damaged plants. An inoculation test using stem cuttings showed that the population of PWN increased in susceptible cuttings at 1–20 days after inoculation (dai), while it remained unchanged or gradually decreased in resistant cuttings. These findings suggested that the factors contributing to resistance were associated with inhibiting the proliferation of PWN, rather than inhibiting their migration.     

Promotion of initial swimbladder inflation in Pacific bluefin tuna,Thunnus orientalis (Temminck and Schlegel), larvae

Initial swimbladder inflation (ISI) of Pacific bluefin tuna (PBT), Thunnus orientalis, larvae was studied to increase the survival of cultured larvae. Experiment 1 was conducted to explore promotion and inhibition of ISI under different water surface conditions; including the use of surface skimmer to remove autogenous surface substances (SS), covering the water surface with liquid‐paraffin‐layer (LP) and oil film (OF), and a control (non‐treatment, NT). Significantly higher inflation frequency was observed in SS (62.2%) than NT (11.9%), LP (2.7%) and OF (3.9%). This indicates that ISI in PBT larvae can be promoted by removal of surface substances on rearing water which inhibit larval air gulping. Experiment 2 aimed to elucidate proper day of larval age to start skimming for promoting ISI with four different periods of oil film removal: from 3 to 8 (SF3D), 4 to 8 (SF4D), 5 to 8 (SF5D), 6 to 8 (SF6D) days‐post‐hatch (dph). Significant improvement in ISI frequency was observed in SF3D (80.2%) but the frequency was very poor in SF4D, SF5D, and SF6D (17.8–7.5%). This implies the need of oil film removal without missing a narrow window, 1 day of 3 dph, to promote ISI in practical PBT larviculture.     

Optimal periods of night‐time lighting in the sea cage culture of Pacific bluefin tuna,Thunnus orientalis,juvenile (Temminck and Schlegel)

We have previously reported that night‐time lighting prevents the mass death of Pacific bluefin tuna (PBT) juvenile due to collision and/or contact with the walls of sea cages, immediately after transfer to the cages, and that night‐time lighting does not negatively impact fish stress levels. However, the period of night‐time lighting should be limited to minimize negative impacts on the surrounding environment and aid management. Therefore, we investigated the optimal period of night‐time lighting by evaluating the whole‐body cortisol and glucose levels as stress parameters, growth performance and survival of PBT juvenile in four cages with different periods of night‐time lighting (i.e. unlit, 4‐day, 8‐day and 12‐day lighting). The results showed that almost all fish were died 1 day after transfer to the unlit cage. In comparison, the other groups (4‐day, 8‐day and 12‐day lighting) had high survival rates (92.5–96.0%) without significant difference. However, in the 4‐day‐lighting group, an obvious stress response was recorded on day 5, and growth performance was significantly lower. In the 8‐day‐lighting group, whole‐body cortisol levels were slightly elevated on day 9; however, significantly elevation was not recorded on day 12. These results indicate that the recommended lighting period of night‐time lighting in sea cages is 8–12 days.     

Near-infrared spectroscopic sensing system for on-line milk quality assessment in a milking robot

A near-infrared (NIR) spectroscopic sensing system was constructed on an experimental basis. This system enabled NIR spectra of raw milk to be obtained in an automatic milking system (milking robot system) over a wavelength range of 600–1050 nm. Calibration models for determining three major milk constituents (fat, protein and lactose), somatic cell count (SCC) and milk urea nitrogen (MUN) of unhomogenized milk were developed, and the precision and accuracy of the models were validated. The coefficient of determination (r²) and standard error of prediction (SEP) of the validation set for fat were 0.95 and 0.25%, respectively. The values of r² and SEP for lactose were 0.83 and 0.26%, those for protein were 0.72 and 0.15%, those for SCC were 0.68 and 0.28 log SCC/mL, and those for MUN were 0.53 and 1.50 mg/dL, respectively. These results indicate that the NIR spectroscopic system can be used to assess milk quality in real-time in an automatic milking system. The system can provide dairy farmers with information on milk quality and physiological condition of an individual cow and, therefore, give them feedback control for optimizing dairy farm management. By using the system, dairy farmers will be able to produce high-quality milk and precision dairy farming will be realized.     

Oocyte maturation and active motility of spermatozoa are triggered by retinoic acid in pen shell <Emphasis Type="Italic">Atrina pectinata</Emphasis>

For recovery of the declining population of pen shells in the wild, the production of pen shell juveniles for transplantation or aquaculture is underway in Japan. For more stable juvenile production, artificial fertilization methods for pen shells are needed, but methods to induce oocyte maturation (meiosis resumption) used in other bivalves, which make oocytes fertilizable, were ineffective for pen shells. Here, we report evidence showing that retinoic acid (RA) has strong activity in inducing oocyte maturation and activating sperm motility in pen shells. Treatment of fully developed oocytes with 1.0 μM all-trans-RA (at-RA) induced germinal vesicle breakdown, a typical morphological sign of oocyte maturation, but 1.0 μM at-retinol and at-retinal, 2 mM ammonia, and 1.0 μM serotonin were ineffective. Treatment with at-RA for 30 min was sufficient for oocyte maturation and was more potent than its isomers, 9-cis- and 13-cis-RA. Parallel results were obtained for sperm motility activation. Oocyte responsiveness to at-RA increased during the final stage of ovary development. Artificial fertilization was successful only with the oocytes treated with at-RA, and fertilized eggs developed to D-shaped (veliger) larvae without apparent morphological abnormalities. These results indicate the possible application of RA for the artificial fertilization of pen shells.     

Effect of different microbubble generation methods on growth of Japanese mustard spinach

To provide the necessary oxygen for plant growth, the effectiveness of different generating methods of microbubbles (MB), a gas-water circulation type or a pressurization type (pressurization treatment), was investigated by comparing the growth characteristics and morphological observation of Brassica campestris L. var. Komatsuna Matsum. The plant growth in the pressurization treatment were significantly less than those in the circulation treatment at 3 weeks after planting. In plants grown in the pressurization treatment, epidermal cell abrasion and plasmolysis, indicators of cell death, were observed in root tip cells at 3 weeks after planting. The growth inhibition of plant in the pressurization treatment is suggested to be related to the oxidization of Fe in the nutrient solution under super-saturated dissolved oxygen conditions, the generation of hydroxyl radicals from MB, the oxidization of the root tip cell membrane, and osmotic stress to the roots.     

Variations in structural,biochemical, and physiological traits of photosynthesis and resource use efficiency in Amaranthus species (NAD-ME-type C4)

C₄ plants show higher photosynthetic capacity and productivity than C₃ plants owing to a CO₂-concentrating mechanism in leaves, which reduces photorespiration. However, which traits regulate the photosynthetic capacity of C₄ plants remains unclear. We investigated structural, biochemical, and physiological traits associated with photosynthesis and resource use efficiency in 20 accessions of 12 species of Amaranthus, NAD-malic enzyme-type C₄ dicots. Net photosynthetic rate (P_N) ranged from 19.7 to 40.5 μmol m^?2 s^?1. P_N was positively correlated with stomatal conductance and nitrogen and chlorophyll contents of leaves and was weakly positively correlated with specific leaf weight. P_N was also positively correlated with the activity of the C₃ enzyme ribulose-1,5-bisphoshate carboxylase/oxygenase, but not with the activities of the C₄ enzymes phosphoenolpyruvate carboxylase and NAD-malic enzyme. Structural traits of leaves (stomatal density, guard cell length, leaf thickness, interveinal distance, sizes of mesophyll and bundle sheath cells and the area ratio between these cells) were not significantly correlated with P_N. These data suggest that some of the biochemical and physiological traits are involved in interspecific P_N variation, whereas structural traits are not directly involved. Photosynthetic nitrogen use efficiency ranged between 260 and 458 μmol mol^?1 N s^?1. Photosynthetic water use efficiency ranged between 5.6 and 10.4 mmol mol^?1. When these data were compared with previously published data of C₄ grasses, it is suggested that common mechanisms may determine the variations in resource use efficiency in grasses and this dicot group.     

Morphological observations on frozen and thawed Muscovy spermatozoa

1. Fresh Muscovy drake spermatozoa were examined using a scanning electron microscope (SEM). The average lengths of the segments were: acrosome 1·8 μm, nucleus 10‐9 μm, midpiece 3·6 μm and flagellum (exclusive of midpiece) 71 μm.

2. Under the light microscope, the incidence of abnormal spermatozoa in Muscovy semen subjected to freezing and thawing (almost all with crooked necks) was about 5% higher than that in diluted unfrozen semen.

3. In thawed semen, various abnormalities of the acrosome were observed under the SEM. It seemed that the most radical change was the complete separation of the acrosome from the apical part of the nucleus.

4. The incidence of abnormal acrosomes was increased more than 20% by freezing and thawing.

5. These results suggest that low fertility in thawed semen may be related to increases in the proportion of spermatozoa with crooked necks and acrosomal damage.

     

Studies of the factors causing abnormal acrosomes and crooked-necks in fowl spermatozoa during freezing and thawing

In order to investigate the factors causing crooked-necked spermatozoa (CNS) or those with abnormal acrosomes during freezing and thawing, fowl spermatozoa in NaCl or glucose solutions containing 92 ml glycerol/l were examined using a scanning electron microscope before and after freezing and thawing. The incidence of CNS in NaCl solution significantly increased after freezing and thawing, but not in glucose solution. The acrosomal damage caused by freezing and thawing was considerable in both solutions, and the incidence of damage in glucose solution was significantly higher than that in NaCl solution. In neither solution was there a significant difference between the incidence of acrosomal damage in CNS and in non-CNS. The ratios of incidences of abnormal acrosome after, versus before, freezing were higher in non-CNS than in CNS. It appears from these results that the factors during freezing and thawing which cause CNS may differ from those causing acrosomal damage.