Comparison of histological responses and tissue damage expansion between resistant and susceptible Pinus thunbergii infected with pine wood nematode Bursaphelenchus xylophilus

Pine wilt disease caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has been epidemic and has had disastrous impacts on pine forests and forest ecosystems in eastern Asia. Many pine species in this area are susceptible to this disease. Pinus thunbergii is particularly susceptible. In Japan, tree breeders have selected surviving trees from severely damaged forests as resistant candidates, and have finally established several resistant varieties of P. thunbergii. However, this breeding procedure requires much time and effort due to the lack of physiological and phenotypical information about resistance. To investigate the resistance mechanisms of selected P. thunbergii, we compared histochemical responses, tissue damage expansion, and PWN distribution in resistant and susceptible clones of P. thunbergii after PWN inoculation. The results suggested that the mechanisms of resistance are as follows: damage expansion in the cortex, cambium, and xylem axial resin canals are retarded in resistant trees soon after inoculation, probably due to the induction of wall protein-based defenses. Suppression of PWN reproduction was particularly caused by inhibition of damage expansion in the cambium. The slow expansion of damage in each tissue provides time for the host to complete the biosynthesis of lignin in the walls of cells that surround the damaged regions. This lignification of cell walls is assumed to effectively inhibit the migration and reproduction of the PWNs. The mechanism of initial damage retardation is presumed to be a key for resistance.     

Isoflurane anaesthesia in four sea lions (Otaria byronia and Zalophus californianus)

OBJECTIVE: To (1) evaluate a purpose-built chamber for inducing isoflurane anaesthesia in sea lions and (2) assess isoflurane as an anaesthetic for dental surgery in these species. STUDY DESIGN: Prospective case study. ANIMALS: Four sea lions, aged between 5 and 12 years and weighing 74-110 kg, with dental disease. METHODS: Sea lions were restrained in a custom-built acrylic chamber into which 5% isoflurane (vapourizer setting) was delivered in O(2) (30 L minute(-1)) from three anaesthetic machines. When the animals were recumbent, the chamber was opened and induction completed using a face mask. Anaesthesia was maintained with 1.5-3.0% isoflurane (vapourizer setting) for 10-15 minutes and after tracheal intubation, was maintained with 1.0-3.0% isoflurane (end-tidal) for 41-255 minutes, using a partial rebreathing system with CO(2) absorption. RESULTS: During induction, the sea lions attempted to support their weight on their thoracic limbs before slipping into sternal or lateral recumbency. Sea lions underwent either root canal surgery, 'crowning' or canine tooth extraction. The animals were completely unresponsive during these operations and apnoea was never encountered at any point during anaesthesia. After surgery, the sea lions were placed into transport cages and their tracheae extubated once they began to move their heads. Thereafter, recovery from anaesthesia was smooth and no signs of distress were observed. Animals were able to raise their forequarters using their flippers within 1 hour of the operation and were returned to the aquarium about 2 hours after surgery. CONCLUSIONS AND CLINICAL RELEVANCE: The purpose-built induction chamber is safe for inducing isoflurane anaesthesia in spontaneously breathing sea lions and isoflurane is an effective anaesthetic in this species.     

Association between the planktonic larval and benthic stages of Manila clam <Emphasis Type="Italic">Ruditapes philippinarum</Emphasis> in eastern Hokkaido,Japan

The association between planktonic larval and benthic stages of Manila clam Ruditapes philippinarum and factors determining successful recruitment were studied in Akkeshi-ko estuary and Akkeshi Bay, Hokkaido, northern Japan. The field study showed that planktonic larvae were localized mainly in the bay after dispersal from the estuary and recruitment to the estuarine fishing grounds was not always successful. A numerical analysis suggested that estuarine circulation transported tracers, used to simulate planktonic larva, from the estuary to the bay but the number of returning tracers varied depending on the river discharge and wind direction. The planktonic larvae were affected by unsuitable conditions for survival in the estuary, such as high turbidity. We conclude that most larvae develop within the bay, where conditions are more saline and less turbid, and that although return to the estuary is essential for recruitment, it is not necessarily related to the abundance of larvae.     

In situ observation of pinewood nematode in wood

In pine wilt disease, xylem dysfunction occurs in relation to nematode migration and proliferation in host tissue, but the detection accuracy of pinewood nematode (PWN), Bursaphelenchus xylophilus, in pine stem tissue remains unclear. This study describes the use of cryo-scanning electron microscopy (cryo-SEM) and fluorescein-conjugated wheat germ agglutinin (F-WGA) staining to detect PWN. After PWN invasion, the frequency of surface fluorescence in PWN increased in pine stems from the day of inoculation to 3 weeks after inoculation. However, the fluorescence frequency decreased significantly during the advanced disease stage after 5 weeks. Thus, detecting PWN based on fluorescent staining of the nematode body surface coat protein can be misleading when used to examine the correlation between the development of disease symptoms and the nematode population. In contrast, all cut body segments were fluorescent, and their fluorescent components were common in pine-stem cross sections, regardless of the timing after inoculation. In addition, PWN were observed under cryo-SEM only in empty resin canals and this distribution was confirmed by F-WGA staining of PWN cut in a cross section. Thus, PWN detection based on fluorescent staining of surface coat proteins by F-WGA was not reliable in longitudinal sections of pine stems because of changes in nematode stainability during disease progression. To detect PWN in infected plants, we concluded that a combination of both methods is most effective.     

Rapid semi-quantification of triacylglycerols,phosphatidylcholines, and free fatty acids in the rice bran of one grain

We developed a microplate assay method for determining the contents of triacylglycerols(TAGs), phosphatidylcholines(PCs), and free fatty acids(FFAs) in the rice bran of one grain using enzymatic reactions. In this method, enzymes from commercially available kits were used. Optimum reaction conditions were established. It was found that Nonidet P-40 was the optimal among the three surfactants used(Triton X-100, Tween 40, and Nonidet P-40) when lipid was dissolved in a reaction solution. Using this method, it was possible to quantify TAGs, PCs, and FFAs in concentration ranges of 7–150, 5–70, and 8–200 mg L~(–1), respectively. Furthermore, when the TAG contents in the rice bran were measured, the values closely corresponded to those obtained by extracting from large amounts of rice bran. However, sufficient data on the PC and FFA contents in rice bran are not available for valid comparisons. Although this method can accurately quantify the TAG contents in the rice bran of one grain, the accuracy of the PC and FFA contents has not been verified. Hence, future study is necessary.     

Nodavirus associated with pathological changes in adult spotted coralgroupers (Plectropomus maculatus) in Thailand with viral nervous necrosis

The present study characterized viral nervous necrosis in sea cage-reared adult spotted coralgroupers (Plectropomus maculatus). Histopathological study showed extensive vacuolation and neuronal necrosis of the olfactory bulb and the optic lobe of the forebrain and the inner and outer nuclear layer of retina. Mild necrosis was observed in the spinal cord. Homogeneous intranuclear inclusion bodies were noted in the hyperplastic and hypertrophic glandular epithelial cells of the swim bladder suggesting viral etiology. Etiological diagnosis of VNN was confirmed by RT-PCR, immunohistochemistry and in situ hybridization. The immunohistochemistry and in situ hybridization gave strongly positive staining in the same area of the infected cells of the brain, spinal cord and retina correlating with histopathological changes. No positive reaction was detectable in the affected gas glandular epithelium and other organs, confirming the consistent neurotropism of this nodavirus. Nodavirus was mainly detected in the olfactory bulb of the brain. The result suggests nasal transmission was the major route of infection.