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Yogi K.,Okinawa Institute of Science and Technology | Yogi K.,Okinawa Prefectural Institute of Health and Environment | Oshiro N.,Okinawa Prefectural Institute of Health and Environment | Inafuku Y.,Okinawa Prefectural Institute of Health and Environment | And 2 more authors.
Analytical Chemistry | Year: 2011

Toxin profiles of representative ciguatera species caught at different locations of Japan were investigated in fish flesh by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Identification and quantification of 16 toxins were facilitated by the use of 14 reference toxins prepared by either synthesis or isolation from natural sources and the previous LC-MS data thereof. Sodium adduct ions [M + Na] + were used as parent and product ions. Distinct regional differences were unveiled: ciguatoxin-1B type toxins were found in snappers and groupers from Okinawa, ciguatoxin-3C type toxins were found in a spotted knifejaw, Oplegnathus punctatus, from Miyazaki located 730 km north of Okinawa, and both types of toxins were found in a red snapper, Lutjanus bohar, from Minamitorishima (Marcus) Island. Twelve toxins were identified in a dinoflagellate, Gambierdiscus toxicus, collected as the primary toxin source in French Polynesia. Occurrence of M-seco-toxins in fish and oxidized toxins in the dinoflagellate was confirmed for the first time. The present LC-MS/MS method is rapid, specific, and accurate. It not only outperforms the currently employed mouse bioassays but also enables the study of the toxin dynamics during the food chain transmission. © 2011 American Chemical Society.


Ishihara Y.,Tohoku University | Lee N.,Tohoku University | Oshiro N.,Okinawa Prefectural Institute of Health and Environment | Matsuoka S.,University of Tokyo | And 3 more authors.
Chemical Communications | Year: 2010

Ciguatoxins, the principal causative toxins of ciguatera seafood poisoning, are potent neurotoxic polycyclic ethers. We report herein the total synthesis of a 10-membered F-ring analogue of 51-hydroxyCTX3C, which constitutes the first example of an F-ring modified ciguatoxin that exhibits potent cytotoxicity as well as mouse acute toxicity. © The Royal Society of Chemistry 2010.


Igawa T.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | Sumida M.,Hiroshima University
Heredity | Year: 2013

Isolation by distance and landscape connectivity are fundamental factors underlying speciation and evolution. To understand how landscapes affect gene flow and shape population structures, island species provide intrinsic study objects. We investigated the effects of landscapes on the population structure of the endangered frog species, Odorrana ishikawae and O. splendida, which each inhabit an island in southwest Japan. This was done by examining population structure, gene flow and demographic history of each species by analyzing 12 microsatellite loci and exploring causal environmental factors through ecological niche modeling (ENM) and the cost-distance approach. Our results revealed that the limited gene flow and multiple-population structure in O. splendida and the single-population structure in O. ishikawae were maintained after divergence of the species through ancient vicariance between islands. We found that genetic distance correlated with geographic distance between populations of both species. Our landscape genetic analysis revealed that the connectivity of suitable habitats influences gene flow and leads to the formation of specific population structures. In particular, different degrees of topographical complexity between islands are the major determining factor for shaping contrasting population structures of two species. In conclusion, our results illustrate the diversification mechanism of organisms through the interaction with space and environment. Our results also present an ENM approach for identifying the key factors affecting demographic history and population structures of target species, especially endangered species. © 2013 Macmillan Publishers Limited All rights reserved.


Kurabayashi A.,Hiroshima University | Nishitani T.,Hiroshima University | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | Oumi S.,Section of Agriculture and Forest | Sumida M.,Hiroshima University
Genes and Genetic Systems | Year: 2012

Crocodile newts, which constitute the genera Echinotriton and Tylototriton, are known as living fossils, and these genera comprise many endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered taxa, we determined the complete nucleotide sequences of the mt genomes of the Japanese crocodile newt Echinotriton andersoni and Himalayan crocodile newt Tylototriton verrucosus. Although the control region (CR) is known as the most variable mtDNA region in many animal taxa, the CRs of crocodile newts are highly conservative. Rather, the genes of NADH dehydrogenase subunits and ATPase subunit 6 were found to have high sequence divergences and to be usable for population genetics studies. To estimate the inter-population divergence ages of E. andersoni endemic to the Ryukyu Islands, we performed molecular dating analysis using whole and partial mt genomic data. The estimated divergence ages of the inter-island individuals are older than the paleogeographic segmentation ages of the islands, suggesting that the lineage splits of E. andersoni populations were not caused by vicariant events. Our phylogenetic analysis with partial mt sequence data also suggests the existence of at least two more undescribed species in the genus Tylototriton. We also found unusual repeat sequences containing the 3′ region of cytochrome apoenzyme b gene, whole tRNA-Thr gene, and a noncoding region (the T-P noncoding region characteristic in caudate mtDNAs) from T. verrucosus mtDNA. Similar repeat sequences were found in two other Tylototriton species. The Tylototriton taxa with the repeats become a monophyletic group, indicating a single origin of the repeat sequences. The intra-and inter-specific comparisons of the repeat sequences suggest the occurrences of homologous recombination-based concerted evolution among the repeat sequences.


Kakehashi R.,Hiroshima University | Kurabayashi A.,Hiroshima University | Oumi S.,Section of Agriculture and Forest | Katsuren S.,Okinawa Prefectural Institute of Health and Environment | And 3 more authors.
Genes and Genetic Systems | Year: 2013

Genus Babina is a member of Ranidae, a large family of frogs, currently comprising 10 species. Three of them are listed as endangered species. To identify mitochondrial (mt) genes suitable for future population genetic analyses for endangered species, we determined the complete nucleotide sequences of the mt genomes of 3 endangered Japanese Babina frogs, B. holsti, B. okinavana, and B. subaspera and 1 ranid frog Lithobates catesbeianus. The genes of NADH dehydrogenase subunit 5 (nad5) and the control region (CR) were found to have high sequence divergences and to be usable for population genetics studies. At present, no consensus on the phylogenetic position of genus Babina has been reached. To resolve this problem, we performed molecular phylogenetic analyses with the largest dataset used to date (11,345 bp from 2 ribosomal RNA- and 13 proteinencoding genes) in studies dealing with Babina phylogeny. These analyses revealed monophyly of Babina and Odorrana. It is well known that mt gene rearrangements of animals can provide usable phylogenetic information. Thus, we also compared the mt gene arrangements among Babina species and other related genera. Of the surveyed species, only L. catesbeianus manifested typical neobatrachian-type mt gene organization. In the B. okinavana, an additional pseudogene of tRNA-His (trnH) was observed in the CR downstream region. Furthermore, in the B. holsti and B. subaspera, the trnH/nad5 block was translocated from its typical position to the CR downstream region, and the translocated trnH became a pseudogene. The position of the trnH pseudogene is consistent with the translocated trnH position reported in Odorrana. Consequently, the trnH rearrangement seems to be a common ancestry characteristic (synapomorphy) of Babina and Odorrana. Based on the "duplication and deletion" gene rearrangement model, a single genomic duplication event can explain the order of derived mt genes found in Babina and Odorrana.


Blanco A.C.,Tokyo Institute of Technology | Blanco A.C.,University of the Philippines at Diliman | Nadaoka K.,Tokyo Institute of Technology | Yamamoto T.,Tokyo Institute of Technology | Kinjo K.,Okinawa Prefectural Institute of Health and Environment
Hydrological Processes | Year: 2010

Excessive terrestrial nutrient loadings adversely impact coral reefs by primarily enhancing growth of macroalgae, potentially leading to a phase-shift phenomenon. Hydrological processes and other spatial and temporal factors affecting nutrient discharge must be examined to be able to formulate effective measures for reducing nutrient export to adjacent reefs. During storm events and baseflow periods, water samples were obtained from the tropical Todoroki River, which drains an intensively agricultural watershed into Shiraho coral reef. In situ nutrient analyzers were deployed for 6 months to hourly measure dissolved nutrient (NO3 --N and PO4 3--P) concentrations. Total phosphorus (TP) and suspended solid concentration (TSS) were increased by higher rainfall intensity (r = 0·94, p < 0·01) and river discharge Q (r = 0 88, p < 0 01). In contrast, NO3 --N concentration tends to decrease drastically (e.g. from 3 to 1 mg l-1) during flood events. When base flow starts to dominate afterwards, NO3 --N manifested an increasing trend, but decreases when baseflow discharge becomes low. This counter-clockwise hysteresis for NO3 --N highlights the significant influence of groundwater discharge. N delivery can therefore be considered a persistent process compared to sediment and P discharge, which are highly episodic in nature. Based on GIS analysis, nutrient concentration along the Todoroki River was largely affected by the percentage of sugarcane/bare areas and bedrock type. The spatial distribution of N concentration in the river reflects the considerable influence of subsurface geology-higher N levels in limestone-dominated areas. P concentrations were directly related to the total length of artificial drainage, which enhances sediment transport. The use of high-resolution monitoring data coupled with GIS-based spatial analysis therefore enabled the clarification of control factors and the difference in the spatio-temporal discharge characteristics between N and P. Thus, although erosion-reduction schemes would reduce P discharge, other approaches (e.g. minimize fertilizer) are needed to reduce N discharge. Copyright © 2010 John Wiley & Sons, Ltd.


Kyan H.,Okinawa Prefectural Institute of Health and Environment | Taira M.,Okinawa Prefectural Institute of Health and Environment | Yamamoto A.,Okinawa Prefectural Institute of Health and Environment | Inaba C.,Okinawa Prefectural Institute of Health and Environment | Zakimi S.,Okinawa Prefectural Institute of Animal Health
Japanese Journal of Infectious Diseases | Year: 2012

Toxoplasma gondii genotypes were isolated and characterized from cephalic muscle samples collected from 24 goats slaughtered at an abattoir in Okinawa between 2008 and 2009. Of the 24 samples assayed using latex agglutination, 18 were seropositive, 2 were pseudo-positive, and 4 were seronegative against T. gondii antibodies. The samples were then inoculated into laboratory mice to isolate the parasite. Among the isolated samples, 13 (72.2% of the 18 seropositive strains in the latex agglutination assay) were seropositive, 1 (50%) was pseudo-positive, and none were seronegative. However, after being frozen and stored at -20°C, all samples were found to be T. gondii-free. Of the 14 isolates of the GRA6 genotype, 6 were of type I, 7 were of type II, and 1 was of type III; the genotype distribution ratio was similar to that of T. gondii strains isolated from locally raised pigs. Moreover, no sulfonamide-tolerant dhps gene mutant of T. gondii was detected.


Nishimura M.,Okinawa Prefectural Institute of Health and Environment
International Journal of Tropical Medicine | Year: 2011

Practical cheap tools are required to protect the inhabitants of the Okinawa and Amami islands, Japan against encounters and bites of the venomous pit viper snake Habu (Protobothrops flavoviridis) in their houses and the surrounding environment. In this study, the researchers conducted laboratory and field experiments to identify the most effective net settings (i.e., mesh size and short supported nets versus long unsupported nets) for capturing Habu. We found that short net-traps of approximately 1 min length with 25 mm mesh were the most effective with Habu being captured at a rate of > 1 snake per 10m net length per year in the field. This tool which is cheap and easy to maintain, entangles few non-target animals and applicable to reduce risk of other venomous snake species permanently in residential and farming areas. © Medwell Journals, 2011.


Aird S.D.,Okinawa Institute of Science and Technology | Watanabe Y.,Okinawa Institute of Science and Technology | Villar-Briones A.,Okinawa Institute of Science and Technology | Roy M.C.,Okinawa Institute of Science and Technology | And 2 more authors.
BMC Genomics | Year: 2013

Background: Advances in DNA sequencing and proteomics have facilitated quantitative comparisons of snake venom composition. Most studies have employed one approach or the other. Here, both Illumina cDNA sequencing and LC/MS were used to compare the transcriptomes and proteomes of two pit vipers, Protobothrops flavoviridis and Ovophis okinavensis, which differ greatly in their biology. Results: Sequencing of venom gland cDNA produced 104,830 transcripts. The Protobothrops transcriptome contained transcripts for 103 venom-related proteins, while the Ovophis transcriptome contained 95. In both, transcript abundances spanned six orders of magnitude. Mass spectrometry identified peptides from 100% of transcripts that occurred at higher than contaminant (e.g. human keratin) levels, including a number of proteins never before sequenced from snakes. These transcriptomes reveal fundamentally different envenomation strategies. Adult Protobothrops venom promotes hemorrhage, hypotension, incoagulable blood, and prey digestion, consistent with mammalian predation. Ovophis venom composition is less readily interpreted, owing to insufficient pharmacological data for venom serine and metalloproteases, which comprise more than 97.3% of Ovophis transcripts, but only 38.0% of Protobothrops transcripts. Ovophis venom apparently represents a hybrid strategy optimized for frogs and small mammals. Conclusions: This study illustrates the power of cDNA sequencing combined with MS profiling. The former quantifies transcript composition, allowing detection of novel proteins, but cannot indicate which proteins are actually secreted, as does MS. We show, for the first time, that transcript and peptide abundances are correlated. This means that MS can be used for quantitative, non-invasive venom profiling, which will be beneficial for studies of endangered species. © 2013 Aird et al.; licensee BioMed Central Ltd.


Toshino S.,Kitasato University | Miyake H.,Kitasato University | Iwanaga S.,Okinawa Prefectural Institute of Health and Environment
Plankton and Benthos Research | Year: 2014

The small box jellyfish Copula sivickisi was collected from the Ryukyu Archipelago, southern Japan, in June 2011, in order to observe its early life history, including polyp formation and metamorphosis. Fertilization occurred internally. Fertilized eggs were packed in an embryo strand with nematocysts and released into the water from the female’s manubrium. Blastulae developed into planulae bearing about 30 larval ocelli within two days, and then settled and metamorphosed into primary polyps. Primary polyps developed into adult polyps within 40 days, and both polyp stages could actively detach to change location by creeping on the central part of their body. Adult polyps formed cysts at temperatures below 20°C, which when favorable conditions retuned (above 28°C), excysted from the capsules within a week. Budding occurred in adult polyps, and buds were released two days after the commencement of budding. Metamorphosis of a whole polyp into a single medusa occurred within 10 days. Juvenile medusae were distinguished from those of other cubozoans by the pattern of nematocyst warts on the exumbrella and the adhesive pads on the apex. The developmental features of C. sivickisi resemble most closely those of T. cystophora among the cubozoans. The similarities in all early life stages of both species support recent molecular results. © The Plankton Society of Japan.

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