Seishi Hagihara

Sympatric spawning of Anguilla marmorata and Anguilla japonica in the western North Pacific Ocean

Extensive collections were made of the larvae of the temperate Japanese eel Anguilla japonica and the tropical giant mottled eel Anguilla marmorata in an overlapping area of the North Equatorial Current region of the western North Pacific Ocean. Collections of 189 A. marmorata and > 2500 A. japonica larvae during nine surveys from 1991 to 2007 showed that these two anguillid eels have similar spawning areas just west of the southern West Mariana Ridge. In July to August 2006 and August 2007, morphologically and genetically identified A. marmorata preleptocephali were mainly collected between 14.5-15 degrees N and 142-142.5 degrees E, where A. japonica preleptocephali were also caught in some of the same net tows. Fewer A. marmorata preleptocephali, however, were collected (n = 31) compared to those of A. japonica (n = c. 165), and fewer small larvae of A. marmorata were collected per tow than A. japonica (n = 1-10 and 1-294, respectively), suggesting relatively smaller spawning aggregations of A. marmorata. The distribution of preleptocephali and small larvae was wider in longitude in A. marmorata (131- 143 degrees E) than in A. japonica (137-143 degrees E), while the latitudinal range was almost the same (12-17 degrees N). Although spawning by these two species overlaps both spatially and temporally, the tropical eels of the North Pacific population of A. marmorata probably have a much longer spawning season with fewer spawners, at least in summer, and recruit to a much wider latitudinal range of growth habitats.

Morphological and physiological changes of female tropical eels, Anguilla celebesensis and Anguilla marmorata, in relation to downstream migration

The morphological and physiological characteristics of migrating and non-migrating female tropical eels, Anguilla celebesensis and Anguilla marmorata were examined in relation to their downstream migration on central Sulawesi Island, Indonesia. Migrating eels (64 A. celebesensis and 37 A. marmorata) were obtained from weirs set near the outlet area of Poso Lake and non-migrating eels (21 A. celebesensis and 21 A. marmorata) were sampled by set-lines and eel pots in Poso Lake, its inlet rivers, and in the La River system during February 2009 to October 2010. In both species, values of eye index, pectoral-fin length index, gonado-somatic index (I(G)), hepato-somatic index, swimbladder-somatic index and cardio-somatic index of migrating eels were significantly higher than those of non-migrating eels and the gut-somatic index values of the migrating eels were significantly lower than that of non-migrating eels. When silvering stages of eels were classified by the silvering index for Anguilla japonica, in A. celebesensis, all non-migrating eels were Y1 stage and the migrating eels consisted of Y2, S1 and S2 stages eels. In A. marmorata, the non-migrating eels consisted of Y1 and Y2 eels, and the migrating eels consisted of Y2 and S1 eels, but there were no S2 eels. Results of principal component analysis (PCA) of morphological and physiological variables suggested that these characteristics changed drastically between the Y1 and Y2 stages in A. celebesensis, while A. marmorata showed a gradual change with silvering, which differs from the temperate species A. japonica. The mean ±S.D. I(G) value of migrating A. celebesensis (6.9 ± 1.8, 3.3-11.4) was very high and that of A. marmorata (3.1 ± 0.8, 1.8-5.7) was comparatively low. The very different rates of maturation that were found between these two species provide support for the hypothesis that the reproductive characteristics of silver eels can reflect their migration scale.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donors gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

Genetic population structure and morphological characters of Japanese psychrolutids of genus Malacocottus (Scorpaeniformes: Psychrolutidae)

The genetic population structure and the diagnostic characters of Malacocottus gibber from the Japan Sea and Malacocottus zonurus from the Okhotsk Sea and the northwestern Pacific were compared. Analysis of the nucleotide sequences of the mitochondrial control region revealed no genetic differences between the populations of M. gibber and M. zonurus, even though most individuals of both the species were found to be morphologically distinct. Most of the Malacocottus gibber specimens had the typical morphological characters of this species, namely the absence of an accessory spine on the preopercle of both sides and the absence of modified body scales above the lateral line. All the specimens of M. zonurus had accessory spines on both sides, and most of them had modified body scales. The results of this study suggest that M. gibber should be treated as a subspecies or a synonym of M. zonurus. The nested clade analysis and the analysis of molecular variance (AMOVA) showed that the Japanese Malacocottus fishes are genetically homogenous over their geographical range. The mismatch distribution of the Japanese Malacocottus fishes indicated that a sudden population expansion had occurred recently. The contrast in phylogeographic structures between the Malacocottus fish and the zoarcid Bothrocara hollandi—the most dominant deep-sea demersal fish in the Japan Sea—might be attributed to the differences in the depths of the habitats and larval ecology between these two fishes.

Nuclear DNA markers for identification of Beluga and Sterlet sturgeons and their interspecific Bester hybrid

Sturgeons (Acipenseriformes) are among the most endangered species in the world due to fragmentation and destruction of their natural habitats and to overexploitation, mainly for highly priced caviar. This has led to the development of sturgeon culture, originally for reintroduction, but more recently for caviar production. In both cases, accurate species identification is essential. We report a new tool for accurate identification of Huso huso and Acipenser ruthenus based on nuclear DNA markers. We employed ddRAD sequencing to identify species-specific nucleotide variants, which served as specific binding sites for diagnostic primers. The primers allowed identification of Huso huso and Acipenser ruthenus as well as their discrimination from A. baerii, A. schrenckii, A. gueldenstaedtii, A. stellatus, A. persicus, A. mikadoi, A. transmontanus, and H. dauricus and identification of A. ruthenus and H. huso hybrids with these species, except hybrid between A. ruthenus and A. stellatus. The species-specific primers also allowed identification of bester (H. huso × A. ruthenus), the most commercially exploited sturgeon hybrid. The tool, based on simple PCR and gel electrophoresis, is rapid, inexpensive, and reproducible. It will contribute to conservation of remaining wild populations of A. ruthenus and H. huso, as well as to traceability of their products.

Anguilla huangi Teng, Lin, and Tzeng, 2009, is a junior synonym of Anguilla luzonensis Watanabe, Aoyama, and Tsukamoto, 2009

Anguilla luzonensis and A. huangi were each described in 2009 using eels obtained from northern Luzon Island. We examined the taxonomic status of these two groups of eels using morphological and molecular genetic characters. There were no significant differences in two vertebrae counts between eels of A. luzonensis and A. huangi. Mitochondrial 16S ribosomal RNA and cytochrome b genes sequences were obtained and compared among 28 specimens of A. luzonensis, the holotypes of A. luzonensis and A. huangi, and one specimen of the other 15 anguillid species. The specimens of A. luzonensis exhibited almost identical sequences, including the holotype, with only a few site differences, and the genetic difference between the holotypes of A. luzonensis and A. huangi was within the range of differences of specimens of A. luzonensis. The other anguillid species were genetically very different from A. luzonensis and A. huangi, although A. interioris is a closely related species. It is clear that A. luzonensis and A. huangi are the same species, and according to the principle of priority in zoological nomenclature, A. luzonensis Watanabe, Aoyama, and Tsukamoto, 2009 is the valid species name, and A. huangi Teng, Lin, and Tzeng, 2009 is a junior synonym of A. luzonensis.

Age and growth of migrating tropical eels, Anguilla celebesensis and Anguilla marmorata : AGE AND GROWTH OF MIGRATING TROPICAL EELS

The age and growth of migrating tropical eels, Anguilla celebesensis and Anguilla marmorata from central Sulawesi, Indonesia, were examined. Migrating eels (63 A. celebesensis and 38 A. marmorata) were obtained from weirs near the Poso Lake outlet and non-migrating eels (35 A. celebesensis and 119 A. marmorata) were captured by baited hooks, eel pots, scoop net and electro-fishing in the Poso River system, Laa River system, Baluga River, Tongku River and Padapu River from February 2009 to October 2010. In both species, the proportion of eels with opaque otolith edges showed a single peak in July, suggesting that one annulus (a pair of translucent and opaque zones) was formed each year in their otoliths. Mean ± s.d. and range of total length (LT ) and age was 785·2 ± 114·9 (585-1083) mm and 7·5 ± 1·6 (5-11) years in migrating female A. celebesensis and 1132·2 ± 173·7 (800-1630) mm and 11·6 ± 3·3 (7-23) years in A. marmorata. The age of migrating female eels was negatively correlated with annual growth rate, 100·7 ± 17·2 (68·1-145·0) mm year-1 in A. celebesensis and 97·9 ± 19·3 (66·6-131·6) mm year-1 in A. marmorata, but there was no significant correlation between the LT and annual growth rate in either species. The annual growth rates of these female tropical eels were typically higher than those of temperate anguillid species, suggesting a latitudinal cline in growth rate in the genus Anguilla reflecting the environmental conditions of their growth habitat.

Characterization of type I and II procollagen α1chain in Amur sturgeon (Acipenser schrenckii) and comparison of their gene expression

To characterize type I and II collagen in the Amur sturgeon at the molecular level, mRNAs encoding the proα chain of both types of collagen were cloned and sequenced. Full sequences of both were obtained, and the molecular phylogeny based on the deduced amino acid sequence indicated that the correct sequences of the target genes were obtained. Analyses of primary structure of the proα chains revealed that type I and II collagen share the basic structure of the proα chain of fibril collagen, but have different characteristics, especially in residues related to thermal stability. In the triple helical domain, Gly-Pro-Pro sequence stabilizing the tripeptide unit was more frequent in type II than in type I, and Gly-Gly, which likely decline in thermal stability, was more frequent in type I than in type II. These results suggested that the denaturation temperature of type II would be remarkably higher than type I. The spatial pattern of gene expression was analyzed by quantitative real-time PCR, which showed that relatively ubiquitous type I gene and strongly skewed distribution of type II gene, which highly expressed only in vertebra, snout cartilage, and notochord. This pattern was similar to the distribution pattern of each collagen protein detected by previous biochemical analyses using Amur and Bester sturgeons. The present study is the first report of the cloning of the full-length cDNAs for both of type I and type II collagen in the Amur sturgeon, and is the first comparative analysis of type I and II collagens in a sturgeon species at the molecular level. The results provide basic and general information on collagens in sturgeons.

Interspecific and sexual differences in riverine distribution of tropical eels Anguilla spp.

A total of 261 individuals of the four tropical eel species, Anguilla celebesensis, Anguilla marmorata, Anguilla bicolor pacifica and Anguilla interioris, were collected from 12 locations around Sulawesi Island, Indonesia, to gain knowledge about the riverine distribution of tropical eels. Anguilla marmorata was predominant in the lower reaches of Poso River (94·4% of total eel catch in the sampling area), Poso Lake (93·3%), three small inlet rivers of Tomini Bay (100%) and Laa River (92·3%). Anguilla celebesensis occurred frequently in the inlet rivers of Poso Lake (63·5%). Anguilla bicolor pacifica and Anguilla interioris were rare (1.5 and 0.4%, respectively). Otolith Sr:Ca ratio electron-probe micro analysis (EPMA) for individual migratory histories revealed that 15 A. celebesensis caught in Poso Lake and its inlet rivers were categorized into 14 river eels (Sr:Ca < 2·5) showing upstream migration seemingly at their elver stage and only one sea eel (Sr:Ca ≥ 6·0) that stayed in the marine habitat for the majority of its life after recruiting to Sulawesi Island before its late upstream migration. In A. marmorata, 19 examined eels from Poso Lake and its inlet rivers were all river eels, while 17 eels from the lower reaches of Poso River were two river eels, six sea eels and nine estuarine eels (2·5 ≤ Sr:Ca < 6·0) that mostly lived in the brackish water. The sex ratio of A. celebesensis was highly skewed towards a dominance of females (99%). In A. marmorata, females were predominant in Poso Lake (95·2%), its inlet rivers (94·7%) and Laa River (100%), while males were more frequent in the lower reaches of Poso River (76·5%) and small inlet rivers of Tomini Bay (94·1%). These results indicate that the riverine distribution pattern of tropical eels differs among species and between sexes.

Genetic characterization of Amur sturgeon Acipenser schrenckii and its hybrid caught around Hokkaido

Genetic characterization was performed in five individuals of wild Amur sturgeon Acipenser schrenckii, and/or its presumed hybrid caught around Hokkaido, using a mitochondrial DNA (mtDNA) marker and two markers of nuclear DNA (nDNA). Genetic analyses indicated that two of the five fish had the mtDNA haplotype of Kaluga, Huso dauricus, whereas the nDNA markers indicated signs for both A. schrenckii and H. dauricus genotypes, referring to a hybrid origin. The other three fish were plausibly pure A. schrenckii. The results indicated the importance of combined usage of mtDNA and nDNA markers for correct species identification in sturgeon.