Why does only Athalia Japonica enter summer diapause among three sympatric Athalia sawflies feeding on crucifers?

This report assesses the primary factor for the evolution of summer diapause of the three species of sawfly, Athalia japonica, A. rosae and A. infumata that feed on cruciferous plants and coexist in the same area. A. japonica has two discrete spring and autumn generations, but A. rosae and A. infumata 5–6 generations. Only A. japonica enters summer diapause in response to the long daylengths in spring. Although these three sawflies usually feed on the same cultivated crucifers, they differ markedly in the utilization of wild crucifers. They oviposit only on young leaves. A. japonica mainly uses Cardamine plants which sprout in spring and autumn. A. rosae and A. infumata primarily use hosts with new leaves all the year round, i.e. cultivated crucifers and Rorippa indica, respectively. The thermal threshold for development is lower in A. japonica than in the other two species. The low heat tolerance of A. japonica is adapted only to cool shady habitats where Cardamine grows. Presumably, summer diapause of A. japonica is adaptation to the deterioration of the primary host plants rather than unfavorable climatic conditions. This interpretation is supported by the movement patterns of the three Athalia sawflies, alternative means to escape from deteriorated habitat conditions.     

Detection and quantitative analysis of zoospores of Pythium porphyrae, causative organism of red rot disease in Porphyra, by competitive PCR

The detection and quantitative analysis of Pythium porphyraezoospores was performed by PCR using PP-1 and PP-2 primers specific tothe internal transcribed spacer region of P. porphyrae. To estimatethe amount of fungal zoospores of P. porphyrae, an internal standardplasmid (pPPISC) containing a modified DNA fragment was constructed. Both ends of this fragment were complementary to the PCR primers. Amplification using primers PP-1 and PP-2 produced DNA fragments ofapproximately 700 and 400 bp from the target DNA of P. porphyraezoospores and from the pPPISC, respectively. To perform quantitativePCR, known quantities of pPPISC were added to reaction mixturescontaining the experimental DNAs extracted from zoospores. After aco-amplification reaction, the two different sized PCR products wereseparated by agarose gel electrophoresis and visualized by ethidium bromidestaining. The number of zoospores was estimated by comparing thefluorescence intensities of the PCR products using a charge-coupled deviceimage analyzer. The results show that competitive PCR using P.porphyrae specific primers and competitor pPPISC are useful tools for thequantitative analysis of P. porphyrae zoospores in seawater from Porphyra cultivation farms.     

Immunoreactive atrial natriuretic peptide in the fish heart and blood plasma examined by electron microscopy,immunohistochemistry and radioimmunoassay

Summary The immunoreactivity of atrial natriuretic peptide and ultrastructure of cardiocytes were examined in 5 species each of freshwater and seawater teleosts, as well as in 2 species each of elasmobranchs and cyclostomes. Immunoreactivity was strong in the atria of Cyprinus carpio, Anguilla japonica and Conger myriaster, rather weak in atria of Channa maculata, Lepomis macrochirus, Salmo gairdneri, Oplegnathus fasciatus and Eptatretus burgeri, very weak in atria of Pagrus major, Trachurus japonicus and Triakis scyllia, and not detectable in atria of Hexagrammos otakii, Narke japonica and Lampetra japonica. The immunoreactivity of the atrial cardiocytes was generally stronger in freshwater than seawater fish. Ventricular immunoreactivity was detected only in 7 species, always being weaker than that observed in the atrium. Ultrastructurally, however, secretory granules were found in atria and ventricles of all species examined, being more frequent in the former than the latter. By radioimmunoassay, immunoreactive ANP was detected in the extracts of blood plasma and both atrial and ventricular tissues of all species examined. There were no statistically significant differences in the values between freshwater and seawater species.     

A Quick Method for Species Identification of Japanese Eel (<Emphasis Type="Italic">Anguilla japonica</Emphasis>) Using Real-Time PCR: An Onboard Application for Use During Sampling Surveys

To compensate for the limited number of morphological characteristics of fish eggs and larvae, we established a convenient and robust method of species identification for eggs of the Japanese eel (Anguilla japonica) using a real-time polymerase chain reaction (PCR) that can be performed onboard research ships at sea. A total of about 1.2 kbp of the mitochondrial 16S ribosomal RNA gene sequences from all species of Anguilla and 3 other anguilliform species were compared to design specific primer pairs and a probe for A. japonica. This real-time PCR amplification was conducted for a total of 44 specimens including A. japonica, A. marmorata, A. bicolor pacifica, and 6 other anguilliform species. Immediate PCR amplification was only observed in A. japonica. We then tested this method under onboard conditions and obtained the same result as had been produced in the laboratory. These results suggest that real-time PCR can be a powerful tool for detecting Japanese eel eggs and newly hatched larvae immediately after onboard sampling during research cruises and will allow targeted sampling efforts to occur rapidly in response to any positive onboard identification of the eggs and larvae of this species.     

Arbuscular mycorrhizal fungi in roots of non-photosynthetic plants, <Emphasis Type="Italic">Sciaphila japonica</Emphasis> and <Emphasis Type="Italic">Sciaphila tosaensis</Emphasis> (Triuridaceae)

The mycorrhizal fungi in the roots of achlorophyllous Sciaphila japonica and S. tosaensis (Triuridaceae) were identified by molecular methods. The habitats of S. japonica were in a tree plantation of Japanese cypress, Chamaecyparis obtusa, and bamboo forests, and those of S. tosaensis were in a camellia forest and a bamboo forest. In the root cortical cells of both plants, aseptate hyphal coils were observed, which suggested the Paris-type arbuscular mycorrhiza (AM). A phylogenetic analysis based on a partial sequence of an AM fungal nuclear small subunit ribosomal RNA gene showed that the fungal DNA sequences of S. japonica were separated into three closely related clades. Those of S. tosaensis were separated into two clades, which were also closely related to each other. The AM fungi of S. japonica and S. tosaensis were completely separated in the phylogenetic tree even among those found in the same habitat, which suggests the high specificities in the plant-fungal partnerships. All the detected AM fungi in these plants belonged to Glomus-group A. Even though the habitats are in quite common environments, both plant species are known as endangered species in Japan. Such a definite specificity in AM symbioses seems to restrict the distribution of the myco-heterotrophic plants.     

Mitochondrial DNA Variation Follows a Geographic Pattern in Japanese Beech Species

The amount and distribution of mitochondrial (mt) DNA restriction fragment length polymorphism was determined among individual tree samples of two Japanese beech species, Fagus crenata and F.japonica. Individual plants were collected from 16 F. crenata populations throughout the range of the species, and from three F. japonica populations. We detected enough variation to characterize eleven and three chondriome types in F. crenata and F.japonica, respectively. The grouping of beech chondriome types based upon the cladistic analysis of mtDNA polymorphism allowed us to recognize the apparent geographical patterns of mtDIMA diversity: the resulting three main groups occupied distinct geographic areas. This geographic differentiation is likely to reflect the history of the Japanese beech forests after the last glacial period of the Pleistocene. In addition, the mtDNA polymorphism encountered within F. crenata encompassed all the variation observed in F.japonica. Our result suggests the need for re-evaluation of their phylogenetic relationships.     

Real-Time Polymerase Chain Reaction Assays for Rapid Detection and Quantification of <Emphasis Type="Italic">Noctiluca scintillans</Emphasis> Zoospore

Application and availability of real-time polymerase chain reaction (PCR) assay to detect and quantify the Noctiluca scintillans zoospore were investigated seasonally. Specific primer set for N. scintillans 18S rDNA was designed and applied to real-time PCR assay using the serial dilutions of N. scintillans zoospores. The real-time PCR assays with Ns63F and Ns260R primers were applied to sea water samples collected weekly in Manazuru Port of Sagami Bay, Japan from April 2005 to June 2006. We developed effective DNA preparation steps for collecting the template DNA of N. scintillans zoospore: size fraction and filter concentration of the water samples, fixation with Lugol solution, cell lysis, and purification. This method is useful for the monitoring of the zoospores of N. scintillans, and can also be used for other small and physiologically fragile planktonic cell. Variation in the density of zoospore was successfully detected in the field samples. The peak density of N. scintillans zoospore was observed to occur just before or at the same time as the peak of the vegetative cells. Moreover, zoospores were detected in seawater even when the vegetative cells were not observed. The presence of zoospore was found all year round in the present study. In this regards, this information is essential for the study of the life cycle and seasonal variation of N. scintillans in the coastal waters.     

Effect of salinity on seed germination and seedling growth of the halophyte Suaeda japonica Makino

Seed germination and seedling growth of the annual halophyte species Suaeda japonica Makino were investigated in response to variable salinity of sediment pore water. The germination percentage of S. japonica’s soft brown seeds, which are dominant among dimorphic seeds, decreased with an increase in salinity, although germination was still observed at 1200‐mM NaCl concentration. The germination percentage and germination speed observed in April were higher than those observed in December when treated with sediment water with 400–1200 mM of NaCl concentrations. These data suggest that S. japonica seedlings could be established on sediments that experience high temperatures. Germination recovery of S. japonica seeds transferred from 600‐mM NaCl containing sediment (seawater equivalent) was lowest among 0–1200‐mM NaCl treatments, implying the low tolerance of seawater conditions of S. japonica seeds. Seeds germinated in 900‐ to 1200‐mM NaCl medium showed poor growth, but survived, in hypersaline conditions, and exhibited improvement in growth upon transfer to lower salinity.     

Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro

Pythium myriotylum is the causal organism of Cocoyam Root Rot Disease (CRRD). Significant numbers of zoospores were induced within 1.5 h in cultures in Petri dishes containing P. myriotylum soaked in 0.01 M Ca⁺⁺ and sterile deionized distilled water. Soaking solutions # 2 and # 3 inhibited the production of zoospores of P. myriotylum. This may be due to the delay in maturation of sporangia and the release of zoospores when the soaking solutions contain sucrose. Significant necrosis of detached cocoyam plantlet roots in 100 ml beakers confirmed the infection of zoospores of two `local white' cocoyam genotypes. Detached `yellow' cocoyam roots in 100 ml beakers of genotype RO3015 resisted infection of P. myriotylum with no necrosis of the inoculated roots, which may indicate resistance. This provides a quick and reliable pathogenicity test of P. myriotylum on susceptible cocoyam detached roots. Necrosis of inoculated detached cocoyam roots could be reliably used to screen cocoyam germplasm for resistance to P. myriotylum.     

Sika deer presence affects the host–parasite interface of a Japanese land leech

Since the 1990s, increasing populations of a blood feeding land leech (Haemadipsa japonica) have become a serious issue in several Japanese prefectures, and it may be caused by the increases in sika deer (Cervus nippon) populations seen over the last quarter of the century. Therefore, this study aimed to reveal the host animal species of H. japonica using iDNA (vertebrate DNA isolated from invertebrates) and to test the hypothesis that the increasingly widespread distribution of sika deer results in increased H. japonica populations through changes to the host–parasite interface. We amplified mitochondrial DNA 16S ribosome RNA fragments from iDNA isolated from the blood clots of H. japonica collected across Japan. We identified 17 host animal species, including four orders of Mammalia (Carnivora, Artiodactyla, Rodentia, and Lagomorpha) and two orders of Amphibia (Caudata and Anura). The sika deer was the dominant host species of H. japonica. Additionally, the host animal species composition of H. japonica differed according to the presence or absence of sika deer. In the sites where sika deer were not found, Anura (frog) species were the most commonly identified hosts of H. japonica. These results suggest that the increases in H. japonica populations might have occurred via a change in host preference to sika deer. This change might be driven by the increases in sika deer populations and subsequent increase in the frequency that H. japonica uses the sika deer as easy prey, as well as by sika deer providing more reproductive energy per blood meal than blood meal from frog species. The present study suggests that a more widespread distribution of sika deer resulted in an increase in H. japonica through a change in the host–parasite interface. Therefore, management that focuses on decreasing sika deer populations would likely be an effective method for the reduction of H. japonica populations.     

Contrastive seasonal changes in ecophysiological traits of leaves of two perennial Polygonaceae herb species differing in leaf longevity and altitudinal distribution

To identify the leaf characteristics associated with the difference in altitudinal distribution of Polygonaceae plants, we investigated the seasonal changes in leaf characteristics of co-occurring Aconogonum weyrichii and Reynoutria japonica at 2250 m a.s.l. on Mt. Fuji. Aconogonum weyrichii is distributed in the alpine region and R. japonica from the lowlands to the highlands. At the beginning of the growing period, leaves of A. weyrichii had larger amounts of nitrogen, Rubisco and higher photosynthetic capacity than R. japonica. The relationship between the amounts of nitrogen and Rubisco did not significantly differ between the species, but a higher Rubisco activity per unit Rubisco protein content was found in A. weyrichii, and was thought to be responsible for the high photosynthetic capacity of this species in summer. Activity of ascorbate peroxidase, a key enzyme of the hydrogen peroxide scavenging system against oxidative stress under chilling and light conditions, increased significantly in R. japonica late in the growing period, but did not significantly change in A. weyrichii. In A. weyrichii, rapid maturation of leaves and a high photosynthetic capacity are thought to be adaptive features for the short growing season in alpine regions. On the other hand, the slower accumulation of nitrogen and Rubisco during the early stage of the growing period in R. japonica resulted in insufficient utilization of the growing period for photosynthetic production and is thought to be one of the factors restricting the upper limit of its distribution.     

A novel method for efficient and abundant production of Phytophthora brassicae zoospores on Brussels sprout leaf discs

Background  

Phytophthora species are notorious oomycete pathogens that cause diseases on a wide range of plants. Our understanding how these pathogens are able to infect their host plants will benefit greatly from information obtained from model systems representative for plant-Phytophthora interactions. One attractive model system is the interaction between Arabidopsis and Phytophthora brassicae. Under laboratory conditions, Arabidopsis can be easily infected with mycelial plugs as inoculum. In the disease cycle, however, sporangia or zoospores are the infectious propagules. Since the current P. brassicae zoospore isolation methods are generally regarded as inefficient, we aimed at developing an alternative method for obtaining high concentrations of P. brassicae zoospores.     

Host–parasite interactions and host species susceptibility of the marine oomycete parasite, Olpidiopsis sp., from Korea that infects red algae

Porphyra farms in Korea occasionally suffer from Olpidiopsis infection. As Porphyra farming proceeds from October to March, this obligatory biotrophic parasite may need an alternative host to survive during other months of the year. To find a possible alternative summer host, we collected algae from Wando, Korea, where extensive Porphyra plantations are located, and discovered an oomycete assignable to the genus Olpidiopsis from Heterosiphonia pulchra. Host susceptibility tests showed that this oomycete could also infect Heterosiphonia japonica, Dasya sp., Dasysiphonia chejuensis, and also blades of Porphyra tenera. The minimum incubation time for this Olpidiopsis sp. to infect its hosts was approximately 4 h. Zoosporangia matured in 2 days and biflagellate zoospores were released. Free zoospores remained infective in seawater for up to 7 days. The infection of Olpidiopsis sp. to H. japonica was cell-type specific and extended rhizoid-like apical cells of determinate branches were preferentially infected. FITC-conjugated lectin staining showed specific binding of concanavalin A (ConA) to extended rhizoid-like apical cells. Attachment of Olpidiopsis sp. zoospores to the host cells was inhibited by α-mannosidase. Monosaccharide inhibition experiments showed that d(+)-mannose, complementary to the lectin ConA, could also block the infection, suggesting a lectin–carbohydrate interaction during host–parasite recognition.     

Movement patterns of threeAthalia sawflies in relation to the spatio-temporal distributions of their habitats

I compared the differences in the movement intensity of three species of sympatric Athalia sawflies, A. japonica, A. rosae and A. infumata feeding on cruciferous plants. Mark-release-recapture census was conducted to estimate movement distance, sex ratio and age composition of adult sawflies. In addition, the sex ratio of newlyemerged adults was examined by rearing field-collected larvae until adult emergence. Age composition and longevity of adults were estimated experimentally. The movement intensity was evaluated mostly with the indirect information thus obtained. Females moved more actively than males in all three sawflies. A. japonica females of all age classes moved actively in spring and autumn, but in summer they disappeared. Also, A. rosae females of all age classes moved actively in spring and autumn. In summer, in contrast with A. japonica, A. rosae females moved most actively among the three species in all seasons. A. infumata females, in particular the young females, moved most actively among the three species, except A rosae in summer. The movement patterns of the three sawflies were deduced in relation to the spatio-temporal distributions of their habitats. In spring and autumn, when host plants were abundant, A. japonica and A. rosae females were dispersed among the host patches within the census are. In summer, however, when host plants were scarce, A. japonica entered diapause, whereas A. rosae migrated to neighboring areas. On the other hand, A. infumata, in particular young famale, innately dispersed to seek for temporary host plants throughout the census seasons.     

Seasonal Pattern of Reproduction Of Hizikia Fusiformis (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China

The maturation pattern of sexual reproduction in Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeaophyta) was examined in 2003 at Yunao Bay, Nanao Island, Shantou, China. Maturation began in mid-April (seawater temperature 19–21 ^∘C), reached the peak in mid-May (maturation rate ca. 70%, and seawater temperature 23.5–25 ^∘C) and finished in late-June (seawater temperature 27.5–30 ^∘C). The Hizikia plants continued to gain the length from the beginning of maturation season to reach a maximum mean length of 34.8 cm in mid-May, after which the mean length was reduced drastically due to the senescence and rupture of the larger plants in size. The major portion of the mature plants belonged to the larger plants between April and May, but to the smaller ones in June. It is suggested that the plant must achieve a critical size before reproductive maturation occurred. There was a positive relationship between the number of receptacles (NR), as well as the reproductive allocation (RA), and the plant size of Hizikia population, with the recorded maximum values of NR and RA being 1220 and 64.3% respectively, for a single plant.     

How have the alpine dwarf plants in Yakushima been miniaturized? A comparative study of two alpine dwarf species in Yakushima, Blechnum niponicum (Blechnaceae) and Lysimachia japonica (Primulaceae)

Many plant species are miniaturized in the alpine region in Yakushima, Japan. To examine how these alpine dwarf plants are different from their related lowland ones of the same species, we analyzed two phylogenetically distinct species cytologically, genetically and morphologically: one is a fern species, Blechnum niponicum, and the other is an angiosperm species, Lysimachia japonica. The analysis shows that the alpine dwarf and the lowland plants in each of these species do not differ in chromosome number or genetic constitution. The organ-level comparison between the alpine dwarf and lowland plants of B. niponicum shows that the fertile leaf size correlates closely with the sterile one. By contrast, the flower size does not correlate with the leaf size in L. japonica. At the cell level, the leaf size of the alpine dwarf plants of B. niponicum consists of a smaller number of epidermal cells than that of the lowland plants of this species. On the other hand, the smaller leaf size of the alpine dwarf plants of L. japonica depends on both the smaller number and the smaller size of the epidermal cells. We conclude that plant dwarfism in Yakushima shows variation at both the organ and cell levels.     

Physical map of chloroplast DNA in sugi,Cryptomeria japonica

Summary To investigate the evolution of conifer species, we constructed a physical map of the chloroplast DNA of sugi, Cryptomeria japonica, with four restriction endonucleases, PstI, SalI, SacI and XhoI. The chloroplast genome of C. japonica was found to be a circular molecule with a total size of approximately 133 kb. This molecule lacked an inverted repeat. Twenty genes were localized on the physical map of C. japonica cpDNA by Southern hybridization. The chloroplast genome structure of C. japonica showed considerable rearrangements of the standard genome type found in vascular plants and differed markedly from that of tobacco. The difference was explicable by one deletion and five inversions. The chloroplast genome of C. japonica differed too from that of the genus Pinus which also lacks one of the inverted repeats. The results indicate that the conifer group originated monophyletically from an ancient lineage, and diverged independently after loss of an inverted repeat structure.     

UNUSUAL PHENOMENA IN THE LIFE HISTORIES OF FLORIDEAE IN CULTURE1

Several Florideae grown in natural seawater media under defined laboratory conditions have interesting and unusual life histories. Antithamnion occidentale males of one generation produced tetraspores that gave rise to nonsporangiate males. The functional females of A. pygmaeum developed spermatangia and tetrasporangia; the tetraspores formed new females. Antithamnion defectum tetrasporophytes of one generation bore spermatangia in addition to tetrasporangia; the tetraspores gave rise to typical gametophytes. Tetraspores from successive generations of Callitham-nion sp. developed into tetrasporophytes and males but no females were produced. Functional female gametophytes of Platythamnion sp. bore abortive tetrasporangia. Field-collected plants of two species of Fauchea produced tetraspores that yielded additional sporangiate plants: those of F. pygmaea being bispo-rangiate and tetrasporangiate, and those of F. lacini-ata being strictly tetrasporangiate. Male plants of Pleonosporium vancouverianum from a running seawater table bore spermatangia and polysporangia when collected. The same plants in unialgal culture produced only spermatangia.     

Duration of freezing necessary to damage the leaves of Fallopia japonica (Houtt.) Ronse Decraene

Fallopia japonica (Houtt.) Ronse Decraene is an invasive plant species that introduces economic, social, and environmental stresses. After observing frost damage to F. japonica plants in the field, we exposed leaves of F. japonica and a native species (Acer saccharum Marshall) to freezing temperatures in the laboratory and compared their net photosynthetic rate to that of fresh leaves. In both species, the net photosynthetic rate of leaves frozen for 0.5 h or for 1 h were not significantly different from each other but were both significantly less than that of fresh leaves. Fresh leaves of F. japonica had a higher net photosynthetic rate than those of A. saccharum, but the relationship was reversed in all freezing treatments. Frozen leaves of F. japonica contained microscopically visible frost lenses, which revealed the mechanism of the damage. These results quantify how quickly F. japonica is damaged by freezing conditions and suggest that minimum vernal temperatures may limit its range expansion.     

Two closely related species of Nipponaphis (Hemiptera: Aphididae) that migrate between Distylium racemosum and Machilus trees in Japan

Nipponaphis loochooensis Sorin, 1996 and N. machilicola (Shinji, 1941) form fig‐shaped or globular galls on Distylium racemosum in southern Japan. Nipponaphis machilicola migrates to the lauraceous evergreen Machilus thunbergii. Nipponaphis loochooensis has also been supposed to migrate to M. thunbergii, but its secondary‐host generation has not been found in the field to date. Through sampling Nipponaphis aphids from trees of M. japonica in addition to M. thunbergii, and sequencing their mitochondrial DNA, we found that the two species form colonies on twigs of both Machilus species, and that the two at times colonize on the same trees and even form mixed colonies. Only N. loochooensis forms colonies on leaves of M. japonica, but neither species colonizes on leaves of M. thunbergii. The secondary‐host generations of the two species could be clearly discriminated from each other, based on morphology. It was confirmed by examining the type specimens of Nipponaphis amamiana Takahashi, 1962 that the name is a junior synonym of N. machilicola.