Okinawan Waters Research Articles

Identification of Fish Species Involved with Ciguatera Food Poisoning in Okinawan Waters by Using PCR-RFLP analysis

Ciguatera fish poisoning (CFP), known as a seafood-borne disease, is caused by consumption of fish contaminated with ciguatoxins in tropical and subtropical sea. The ciguatera fishes, Variola louti, Lutjanus monostigma and L. bohar have an absolute majority in the Ryukyu Archipelago, southwestern Japan. We developed the cluster analysis of phylogenetic tree by using mitochondrial (mt) DNA 16S rRNA sequences of V. louti, L. monostigma and L. bohar and differentiate them from morphologically similar species (L. fulviflamma, L. russellii, L. argentimaculatus, Plectropomus leopardus and V. albimarginata) in our previous study. The fish were acquired from the coastal waters of the Ryukyu Archipelago, and a polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker of the mtDNA 16S rRNA region was used, employing the restriction enzymes BmgT120 I, Dde I, and SnaB I, to identify the fish species responsible for CFP. These results showed that a PCR-RFLP marker can be obtained more easily than a nucleotide sequence.

Briastecholide M, a New Polyoxygenated Briarane from Briareum stechei.

A formerly unpublicized briarane diterpenoid, briastecholide M (1), and its established analogue, brianodin B (2), were purified from Briareum stechei, an octocoral collected from Okinawan waters. Using spectroscopic methods, the structure of 1 was established. Functional study showed that 1 can reducing the release of inducible nitric oxide synthase (iNOS) but enhancing cyclooxygenase-2 (COX-2) protein expression.

日本沿岸産イシガキダイのシガトキシン分析

Ciguatera fish poisoning (CFP) is recognized as the most frequent seafood poisoning due to the consumption of fish containing the principal toxins, ciguatoxins (CTXs). In Japan, CFP events have been reported annually from Okinawa and Amami Islands, locating subtropical regions. In addition, there have been reported several outbreaks due to consumption of the fish caught from the Pacific coast of the Mainland and they were often caused by the matured spotted knifejaw, Oplegnathus punctatus. As part of our research on CFP in Japan, we investigated CTXs analysis by LC-MS/MS on 176 individuals of O. punctatus (weight: 100-6,350 g, standard length: 13-60 cm) from the coast of the Mainland (Honshu, Shikoku, and Kyushu), Amami, Okinawa, and Ogasawara (Bonin) Islands. CTXs were detected from only two specimens collected from Okinawa. Total CTXs levels of the two specimens were at 0.014 and 0.040 μg/kg, respectively, exceeding FDA guidance level at 0.01 μg CTX1B equivalent/kg. However, they might be little risk of CFP because consuming over 1.5 kg of flesh is needed to develop intoxication. The toxins consisted of CTX1B analogs including CTX1B, 52-epi-54-deoxyCTX1B, CTX4A, and CTX4B, and no CTX3C analogs, supporting the finding that ciguatoxic fishes in Okinawan Waters containing only CTX1B analogs.

Identification of fish species involved with ciguatera fish poisoning in Okinawan waters by using phylogenetic analysis

Identification of fish species involved with ciguatera fish poisoning in Okinawan waters by using phylogenetic analysis

Briastecholides G–J, new polyoxygenated briaranes from the octocoral Briareum stechei

Chemical composition screening of octocoral Briareum stechei, collected from Okinawan waters, led to the isolation of 10 briarane-type diterpenoids, including four previously-undescribed metabolites, briastecholides G–J (1–4), in addition to six known analogues, brianodin A (5), briarlides G (6) and P (7), and violides G, O, and P (8–10). The structures of 1–10 were elucidated by spectroscopic methods. Briastecholide D (1) was found to possess an unprecedented 10/6/5/5-fused tetracyclic scaffold. Furthermore, the stereochemistry of brianodin A (5) was elucidated directly by the single-crystal X-ray diffraction analysis for the first time in this study. Briaranes 2 and 5–8 reduced the release of iNOS to 17.01, 37.95, 28.76, 1.21 and 26.66%, respectively, at a concentration of 10 μM.

Barnacle Epibiosis on Sea Turtles in Korea: A West Pacific Region With Low Occurrence and Intensity of Chelonibia testudinaria (Cirripedia: Chelonibiidae)

Loggerhead and green turtles inhabit all oceans except the polar regions. External surfaces of sea turtles are often colonized by epibiotic chelonibiid barnacles. Barnacle taxonomy studies in Korea began in 1985, but until present, no turtle barnacles were recorded. This suggests that either the diversity and frequency of occurrence of turtle barnacles in Korean waters are low or the turtle barnacles have been understudied. This study complies with data collected over 6 years of sea turtle stranding events in Korea (2015–2020). We examined the diversity, frequency, and intensity of turtle barnacle occurrence. Of the 55 recorded strandings, loggerhead turtles were the most common (58%), followed by green turtles (33%). Only one species of barnacle, Chelonibia testudinaria, was found on both loggerhead and green turtles. The frequency of barnacle occurrence on loggerhead turtles was 28%, with an intensity of 2.4 ± 2.7 barnacles per turtle. Notably, 11% of green turtles had barnacles, with an average of one individual per turtle. The frequency and intensity of barnacle occurrence on green turtles analyzed in this study were five times lower than that on green turtle populations in Okinawan, Bornean, and Australian waters in the Indo-Pacific. Based on these new data and the available literature, we speculated that the barnacle larval pools in cold, high-latitude Korean waters are smaller than those occurring in other locations in the Indo-Pacific. The frequency and intensity of occurrence of barnacles on loggerhead turtles in Korea fall within the range recorded in other Indo-Pacific locations. The longer migratory routes of loggerhead turtles allow them to pass through different larval pools in the Indo-Pacific water, exposing them to higher barnacle abundances.

Briarane-Related Diterpenoids from Octocoral Briareum stechei.

A known polyoxygenated briarane, briaexcavatolide P (1), was isolated from a Formosan octocoral Briareum stechei. Moreover, the same species B. stechei, collected from Okinawan waters, yielded three chlorine-containing briaranes, including two new compounds, briastecholides B (2) and C (3) as well as a known analogue, briarenol R (4). The structures of 1–4 were established using spectroscopic methods. In addition, briarane 1 demonstrated anti-inflammatory activity in lipo-polysaccharide-induced RAW 264.7 mouse macrophage cells by suppressing the expression of inducible nitric oxide synthase (iNOS) protein.

Addition of the Sea Snake, Hydrophis stokesii (Reptilia: Squamata: Elapidae), to the Herpetofauna of Japan

Hydrophis stokesii was collected in the coastal water of Okinawajima Island on 30 March 2021. This is the first record of the species from Japanese waters, extending its known range of occurrence toward east-northeast by more than 900 km. The snake was an adult female, 1,545 mm in snout-vent length, 203 mm in tail length, and 3,360 g in body mass. It had 11 well-developed yolked follicles in ovaries and a half-digested spiny puffer fish (Diodon sp.) in its stomach. This individual may possibly represent a stable population of H. stokesii in the Okinawan waters.

Diel patterns in singing activity of humpback whales in a winter breeding area in Okinawan (Ryukyuan) waters

AbstractMale humpback whales produce complex sounds known as songs during their breeding season. Previous studies have shown diel patterns of song in their breeding areas, but there had been no similar studies in the breeding area around Okinawa, Japan. To study diel patterns of song and the behavior of humpback whales in Okinawa, we conducted 24 hr recording with a fixed hydrophone in 2007, and vessel‐based sighting surveys during 2014–2017. Song was monitored for 15 days, with peaks at sunrise and around 2200. Singing activity declined significantly between sunrise and sunset, then increased until 2200. Activity levels at night were higher and more stable than during the day. During 278 days of sighting surveys, 2,551 whales in 1,382 groups were observed. 79 individuals were confirmed as singers, all of which were lone whales. In six cases, singing individuals stopped singing before joining a group or began singing after leaving a group. Previous studies have shown that group size of humpback whales increases through the day. Considering the results from our study and the former studies, the decrease in singing activity as the day progresses may be a result of aggregation increasing, thus reducing the number of lone singers during the day.

Apparent Low Densities of Small Cetaceans in Okinawa May Be Due to Uncontrolled Local Hunting

From October 2011 to May 2012, we conducted the first marine mammal surveys of Okinawan waters, using line-transect vessel surveys and passive acoustic monitoring (PAM). We obtained 913 km of visual survey effort, and 1,039 cumulative hours of PAM data from around the island. Only a single on-effort sighting was made (of two common bottlenose dolphins [Tursiops truncatus]) during visual surveys, and acoustic detection rates were generally low, with the exception of humpback whale (Megaptera novaeangliae) observations during their winter/spring breeding season. The apparent low density of at least some marine mammals observed in Okinawan waters may be due to the depletion of coastal populations. We believe this could result from many decades of heavy and largely-uncontrolled hunting of small cetaceans in waters around the island.

Reproductive traits of deep-sea snappers (Lutjanidae): Implication for Okinawan bottomfish fisheries management

We examined the reproductive cycle, sexuality, and length at 50% maturity (L50) of the deep-sea snappers Etelis coruscans, Paracaesio caerulea, Pristipomoides filamentosus, and Pr. sieboldii from Okinawan waters based on gonadal histology. The overall sex ratios of E. coruscans and Pr. sieboldii were significantly female-biased, whereas those of Pa. caerulea and Pr. filamentosus were not significantly sex-biased. The spawning season was estimated to occur from May to November for E. coruscans, from April to September for Pa. caerulea, and from March to October for Pr. filamentosus and Pr. sieboldii. Their initial reproductive activity coincided with an increase in sunlight duration. Of the four species examined, E. coruscans and Pr. filamentosus indicated significant peaks of spawning around a specific lunar phase. We observed the presence of multiple oocyte maturation stages with postovulatory follicles (POFs) during the spawning season of each species. Transitional gonads were not observed in these four snappers. The L50 of the females and males were 67.1 cm fork length (FL) and 37.6 cm FL for E. coruscans; 34.8 cm FL and 26.1 cm FL for Pa. caerulea; 35.7 cm FL and ≥20.0 cm FL for Pr. filamentosus; and 24.6 cm FL and ≥24.0 cm FL for Pr. sieboldii, respectively. Thus, the reproductive traits of E. coruscans, Pa. caerulea, Pr. filamentosus and Pr. sieboldii are characterized by a long spawning season and iteroparity. The E. coruscans and Pa. caerulea show relatively slow maturation compared to the two species of Pristipomoides. The implications of our results vis-à-vis the conservation and management of Okinawan bottom fisheries are discussed.

Genetic differentiation of the soft shore barnacle Fistulobalanus albicostatus (Cirripedia: Thoracica: Balanomorpha) in the West Pacific

AbstractThis study examined the phylogeography of the barnacle Fistulobalanus albicostatus, which inhabits mangroves and estuarine shores in the West Pacific. Differentiation in the mitochondrial cytochrome c oxidase subunit I (COI) and 12S ribosomal RNA (12S) genes of 401 specimens of F. albicostatus was examined in samples from 16 locations in the West Pacific, ranging from Honshu to Southern China. Our results revealed that F. albicostatus comprises two major clades exhibiting a COI divergence ranging from 1.25% to 2.8%. Clade A demonstrated the widest distribution, ranging from Japan to China, and was divided into three subclades occurring in the South China Sea (A1), Okinawa (A2), and Honshu, Korea and Qingdao (A3). Clade B was determined to be endemic to Okinawa; i.e. two endemic lineages occur in this island. Thus, F. albicostatus resembles several inter‐tidal species in having clades that are endemic to Okinawan waters. Nevertheless, in contrast to the rocky inter‐tidal barnacles Tetraclita spp. and Chthamalus malayensis, F. albicostatus was not found to be separated into continental and oceanic populations, but instead is divided into northern and southern clades, probably because of the Yangtze River discharge, which limits gene flow between the northern and southern populations.

Distribution and Local Movement of Humpback Whales in Okinawan Waters Depend on Sex and Reproductive Status.

The distribution and local movement patterns of humpback whales in waters off the west coast of Okinawa Island, southwest Japan, were investigated using line transect and photo-identification methodologies. Line transect surveys were conducted from 2011 to 2014 and photo-identification survey from 2006 to 2012. During the surveys, humpback whales aggregated in the areas around Ie and Kerama Islands, and tended to travel along the inshore coast of Okinawa Island when they move locally between those two sites. A total of 496 humpback whales of the known sex were photo-identified (322 males, 75 females and 99 females with a calf). Of these, 24.8% were confirmed moving locally between the sites of Ie and Kerama Islands within the same season. Frequency rates of the local movement for males, females and females with a calf were 41.9, 25.0, and 15.1%, respectively; the frequency of local movement for males was significantly higher than that for females and females with a calf. These results indicate that male humpback whales tend to move more actively between the local breeding sites as compared to females and females with a calf. We speculate that the males search for more opportunities to mate, whereas females with a calf tend to remain in the same areas to nurse their calves. These findings extend our knowledge of the habitat use and reproductive ecology of humpback whales in Okinawan waters, which remain poorly understood.

3. 沖縄地区のフグの毒性

3. 沖縄地区のフグの毒性

Peak Mating and Breeding Period of the Humpback Whale <br/>—(<i>Megaptera novaeangliae</i>) in Okinawa Island, Japan

Although detailed knowledge on breeding ecology of humpback whales is required for developing effective and sustainable whale watching programs in breeding areas, the breeding ecology of this species is still poorly understood. Hence, we examine the migratory timing of humpback whales in Okinawa that is one of their breeding ground, distinguishing the reproductive status (male, female, or female with a calf), group compositions (singleton, pair, or whales more than three) and group types (singer or competitive group) in order to assess the peak period of breeding activities. A total of 1192 days of photo-identification surveys were conducted from 1991 to 2012 and a total of 7366 humpback whales were sighted during the surveys. Among them, 1284 whales were sex-determined (848 males, 147 females and 289 females with a calf), 1138 singletons, 1416 pairs and 710 groups of more than three whales were observed. Females without calves tended to occur from late January to late February, which was the beginning of the breeding season and male-female pairs were observed most frequently during this period. The peak occurrence of competitive group which was considered a mating-related behavior group, formed by females and males, was also observed during this period. These results indicated that humpback whales peak mating period in Okinawa occurred between late January and late February. Females with a calf tended to increase from mid-February toward the end of the breeding season maintaining a high sighting per unit effort (SPUE) value in late March. We, therefore, suggested that the peak time of birthing and newborn care was probably that period in Okinawa. These findings extended our knowledge on the reproductive ecology of humpback whales in Okinawan waters.

Determination of toxins involved in ciguatera fish poisoning in the Pacific by LC/MS.

Ciguatera fish poisoning is the most extensive and difficult to control of the seafood poisonings. To facilitate monitoring of fish toxicity, toxin profiles were investigated by an LC/MS/MS method using 14 reference toxins on eight representative species of fish collected in four different areas of the Pacific. Snappers and groupers from Okinawa contained ciguatoxin-1B (CTX1B) and two deoxy congeners at variable but species-specific ratios, while red snapper, Lutjanus bohar, from Minamitorishima, and amberjack, Seriola dumerili, from Hawaii, contained both CTX1B-type and CTX3C-type toxins. Spotted knifejaw, Oplegnathus punctatus, from Okinawan waters, contained mainly CTX4A and CTX4B, but the same species caught at Miyazaki was contaminated primarily with the CTX3C-type toxins. Otherwise, the toxin profiles were consistently species-specific in fish collected from various locations around Okinawa over 20 years. The LC/MS/MS and mouse bioassay results agreed well, indicating the LC/MS/MS method is a promising alternative to the mouse bioassay. Pure CTX1B and CTX3C were prepared for use in future LC/MS/MS analysis.

Morphology and molecular characterization of the epiphytic dinoflagellate Amphidinium massartii, isolated from the temperate waters off Jeju Island, Korea

Amphidinium massartii Biecheler is an epiphytic and toxic dinoflagellate. Prior to the present study, A. massartii has been reported in the waters off the Mediterranean, Australian, USA, and Canadian coasts. We isolated Amphidinium cells from the coastal waters of Jeju Island, Korea and their morphology and rDNA sequences indicated that they were A. massartii. Herein, we report for the first time the occurrence of A. massartii in the waters of the temperate region in the northwestern Pacific Ocean. The large subunit (LSU) rDNA sequences of the Korean strains were 0.7% different from those of an Australian strain of A. massartii CS-259, the closest species, but were 4.1-5.8% different from those of the other Australian strains and the USA strains of A. massartii and from those of Amphidinium sp. HG115 that was isolated from subtropical Okinawan waters. In phylogenetic trees based on LSU, internal transcribed spacer, small subunit rDNA, and cytochrome b sequences, the Korean strains belonged to the A. massartii clade, which was clearly divergent from the A. carterae clade. The morphology of the Korean A. massartii strains was similar to that of the originally described French strain and recently described Australian strain. However, we report for the first time here that scales were observed on the surface of the flagella. In conclusion, the Korean A. massartii strains have unique rDNA sequences, even though they have a very similar morphology to that of previously reported strains. This report extends the known range of this dinoflagellate to the temperate waters of the northwestern Pacific Ocean.

Structure of the Loggerhead Turtle Assemblage in Okinawan Waters Estimated from Variation in Body Size and Blood Profile

In order to estimate the structure of the loggerhead turtle assemblage in Okinawan waters, we measured the straight carapace length (SCL) and sexed 35 loggerhead turtles captured by set net between 2007 and 2011. Sexual maturity in females was determined on the basis of plasma triglyceride, cholesterol, total protein, and calcium concentrations. Of the 35 captured loggerhead turtles, 18 turtles were identified as males based on their elongated tails, while 16 turtles were identified as females in the process of vitellogenesis based on highly elevated blood plasma levels. Only one turtle with low plasma measurements was of indeterminate sex. We suggest that blood plasma measurements may serve as a useful indicator of female maturity. SCL means were 865 ± 48 mm (mean ± SD) for all turtles, 865±47 mm for males, and 871±52 mm for females; the turtle of unknown gender had an SCL of 791 mm. The loggerhead turtle assemblage in Okinawan waters was found to be larger in SCL and higher in the frequency of mature individuals than those in conspecific assemblages in other areas of Japanese waters so far studied. We suggest that the loggerhead turtles migrating to Okinawan waters are primarily in active reproductive states in preparation for mating and nesting.

Molecular ecological study of Siganus spinus and S. guttatus from Okinawan waters based on mitochondrial DNA control region sequences

The mitochondrial DNA (mtDNA) control region was sequenced to examine the genetic variability and gene flow of juvenile Siganus spinus and S. guttatus. In total, 461 nucleotide sequences were obtained from 69 specimens of juvenile S. spinus from Okinawa Island and Ishigaki Island, in which 17 variable sites and 22 haplotypes were identified. Haplotype diversity (h) was high (0.9244 in Okinawa and 0.8984 in Ishigaki), whereas nucleotide diversity (π) was low (0.0063 in Okinawa and 0.0059 in Ishigaki). The two populations were not genetically distinct. Siganus guttatus, which do not form large schools at recruitment, in contrast S. spinus, were also analyzed by studying 152 individuals collected off Okinawa Island, Miyako Island, and Ishigaki Island. Of 476 nucleotide sequences, 50 were variable, and 42 haplotypes were identified. Genetic variability values were high (h = 0.8766 and π = 0.0151 in Okinawa; h = 0.9640 and π = 0.0192 in Miyako; h = 0.9161 and π = 0.0199 in Ishigaki). The Okinawa population was genetically isolated from the Miyako and Ishigaki populations. As a result, genetic diversity was high for each of these siganid populations despite their being target species for fisheries; however, the degree of inter-population gene flow was higher for S. spinus than S. guttatus, suggesting that these species exhibit different dispersal strategies.

Histological observations of seasonal reproductive and lunar-related spawning cycles in the female honeycomb grouper Epinephelus merra in Okinawan waters

Seasonal reproductive and lunar-related spawning cycles of the female honeycomb grouper Epinephelus merra inhabiting Okinawan waters were examined by histological observation of ovaries. Gonadosomatic index (GSI) increased beginning in May and peaked in June. Histological observations revealed that many oocytes laden with yolk were present in the ovaries from May to August. From September to October, ovaries were occupied by immature oocytes. These results suggest that the reproductive season of E. merra lasts for 4 months from May through August in Okinawan waters. When the fish were collected according to the lunar cycle, GSI increased with the approach of the full moon. Oocytes at various development stages were observed from the first quarter to the full moon. Fresh ovulatory follicles were present in the ovaries around the last quarter moon. These results suggest that E. merra has a lunar spawning cycle and spawns between the full moon and the last quarter moon. Ovarian features of the fish collected around the last quarter moon were different among individuals; some fish had many oocytes at the tertiary yolk stage in the ovaries, while the ovaries of the others were occupied by the oocytes at the peri-nucleolus and the oil droplet stages. This observation suggests that a minor release of eggs occurs in this species before or after a major spawning lunar day. Oocytes at the migratory nucleus and the maturation stages were not observed in any ovarian samples. This may mean that maturation of oocytes is related to the spawning behavior of this species and makes rapid progress in the process of aggregation at the spawning sites.