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Selection acting across environmental gradients, such as latitudes, can cause spatial structuring of genomic variants even within panmictic populations. In this study, we focused on the within-generation latitudinal selection between northernmost and southernmost individuals of the North Pacific population of a tropical eel Anguilla marmorata, which shares its northernmost distribution with a temperate eel Anguilla japonica. Whole-genome sequencing data indicated that the northernmost and southernmost individuals of A. marmorata belong to a single panmictic population, as suggested by previous studies. On the contrary, parts of genomic regions across multiple chromosomes exhibited significant genetic differentiation between the northernmost and southernmost individuals, and in these genomic regions, the genotypes of the northernmost individuals were similar to those of A. japonica. These findings suggested within-generation latitudinal selection of A. marmorata, which might have led to genetic closeness between northernmost A. marmorata and A. japonica.

Eight silver-phase male Indo-Pacific eels (giant mottled eels) Anguilla marmorata were collected from three small rivers on subtropical Amami-Oshima Island, Japan, and their morphological and gonadal-histological characteristics were examined. The total length, body mass and age of the silver eels were 543.8 ± 49.3 (496-659) mm, 376.0 ± 116.0 (282-660) g and 14.6 ± 2.3 (11-19) years old, respectively. The silvering-related characteristics (eye index, pectoral-fin index, gut-somatic index) and reproductive characteristics (gonadosomatic index, histological stage) of the silver eels were significantly advanced compared to those of yellow eels, which is similar to other anguillid species.

Our understanding of maternal control of development in vertebrates remains incomplete. In this study, we investigated levels of maternal transcripts in good and poor quality eggs from artificially matured Japanese eel, using RNA-Seq and quantitative polymerase chain reaction (qPCR), to identify candidate maternal transcripts related to development. De novo assembly or mapping of reads to the eel draft genome yielded 619,029 contigs and 85,906 transcripts, respectively; normalized read counts to these assemblies were calculated using reads (RPKM) or fragments (FPKM) per kilobase of transcript per million mapped reads. In silico screening identified 1,594 contigs and 150 transcripts with lower RPKM or FPKM in poor than in good quality eggs, 245 contigs, and 85 transcripts of which could be annotated by BLASTx, respectively. From selected contigs or transcripts, six genes (dnajb4, gnpat, card14, pdp1, fcgbp, ttn) had significantly lower messenger RNA levels in poor than in good quality eggs by qPCR. Multiple regression analysis showed that five genes (gnpat, b4galnt1, acsl6, rtkn, trim24) significantly correlated with hatchability. Taken together, 10 genes were identified as candidate maternal transcripts, regulating development in Japanese eel. Our results contribute to understanding the molecular basis for maternal control of development in vertebrates.

The unique semi-enclosed Indian Ocean basin includes large Mascarene Plateau banks, offshore coral-reef islands, seasonal equatorial current jets, and cross-basin westward South Equatorial Current (SEC) flow, making it interesting for studying long larval-duration eel larvae (leptocephali) and regional eel biodiversity. Three surveys for leptocephali (in 2003, 2006, 2010) included sampling west of the Mascarene Plateau (west), a major survey and other stations off Sumatra and Java (east), and 2 cross-basin transects across the SEC. The highest numbers of leptocephali species were observed along Sumatra (2003: ~143 species; 2006: 72 species) and south of Java (2010: 69), with intermediate numbers being collected in the western Indian Ocean (2006: 71; 2010: 53) compared to low numbers in the hydrographically variable offshore zones (2006, 2010: 3–27). The larger continental shelf areas along Sumatra including the Mentawai Islands provide more coral reef and other habitats for species such as congrid, muraenid, ophichthid, and chlopsid eels compared to the Mascarene Plateau banks. Some larvae in these areas get transported offshore, but the majority of offshore larvae were of Nemichthyidae and Serrivomeridae mesopelagic eels that were spawning across the basin. Habitat differences between the southern Mascarene Plateau and Sumatra and southern Indonesia along the edge of the high biodiversity Coral Triangle likely explain the higher biodiversity of eel larvae observed along the western side of the basin, which for the Congridae and Ophichthidae included more species than observed previously within the central Indonesian Seas. In addition to local spawning, seasonal currents likely transport larger larvae towards Sumatra from the north or west and larvae may enter the basin from the Indonesian Throughflow in the east, but it is unknown if equatorial jets or the SEC can transport larvae across the whole basin.

This is the first study describing the morphological, ecological and physiological characteristics of two downstream-migrating and two non-migrating female Pacific bicolor eels, Anguilla bicolor pacifica. The total length and age of the downstream-migrating eels were 1005 mm and 10 years and 1110 mm and 11 years old, respectively, and those of the non-migrating eels were 892 mm and 8 years and 805 mm and 9 years, respectively. Silvering-related characteristics (silvering index, eye index, pectoral-fin index, gut-somatic index and swimbladder-somatic index) and reproductive physiological characteristics (gonado-somatic index, follicle diameter, oocyte stage, transcription of gonadotropins and concentration of sex steroids) of the migrating eels were more advanced than those of the non-migrating eels.

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

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.

The mechanisms that govern sex differentiation in sturgeon are poorly understood. This study aimed to examine sexual dimorphic expression of foxl2 and dmrt1 in Amur sturgeon Acipenser schrenckii during molecular and morphological sex differentiation. Histologically, ovaries were first observed at 9 months after hatching (mah), reflected in an invaginated gonadal epithelium and the presence of germ cells just below the epithelium, whereas gonads with smooth epithelium and germ cells in the stroma were characterized as testis. Some fish showed undifferentiated gonads until 16 mah, but all sampled fish had morphologically differentiated sex by 19 mah. The full-length cDNAs of foxl2 and two types of dmrt1, dmrt1a and dmrt1b, of Amur sturgeon were isolated. The 2 types of dmrt1 cDNA had an identical structure from the 5′-UTR to the Y-rich domain, whereas the sequence downstream from this domain showed no homology and is considered to have resulted from alternative splicing. Foxl2 was principally expressed in the ovary and the 2 types of dmrt1 in the testis of 7-year-old Amur sturgeon, and much less so in various somatic tissues. Foxl2 expression was sexually dimorphic in morphologically differentiated gonads and was similarly dichotomous in the undifferentiated gonads of fish at 9 mah. In contrast, sexually dimorphic expression of dmrt1a and dmrt1b was not observed in gonads of the juvenile Amur sturgeon, but these genes showed testis-dominant expression in the adults. We suggest that foxl2 expression is closely related to ovarian differentiation and that two types of dmrt1 play a role in testis development and/or sperm formation rather than testicular differentiation. Additionally, we suggest that mRNA levels of foxl2 are an appropriate marker for sexing of sturgeon at an early developmental stage.

We investigated the source of spontaneous polyploidization in the critically endangered Acipenser mikadoi. Fourteen sib progeny of A. mikadoi and 11 hybrids between an A. mikadoi female and a Huso dauricus male, all showing atypically high ploidy, were analysed. Parent assignment based on five highly polymorphic microsatellite markers confirmed spontaneous duplication of maternal chromosome sets via retention of the second polar body to be the source of spontaneous polyploidization. To our knowledge, this provides the first evidence of the maternal origin of spontaneous polyploidization in A. mikadoi. Factorial correspondence analysis of the multilocus microsatellite genotypes placed the parent fish of spontaneous polyploids in clearly delineated clusters of A. mikadoi and H. dauricus, and parent fish had mitochondrial control region haplotypes corresponding to their presumed species. Thus, parent fish were confirmed to be of pure genetic origin, and hybridization did not promote the observed spontaneous polyploidization.

International audience; Seven South Pacific anguillid eel species live from New Guinea to French Polynesia, but their spawning areas and life histories are mostly unknown despite previous sampling surveys. A July–October 2016 research cruise was conducted to study the spawning areas and times, and larval distributions of South Pacific anguillid eels, which included a short 155°E station-line northeast of New Guinea and five long transects (5–25°S, 160°E–140°W) crossing the South Equatorial (SEC) and other currents. This survey collected nearly 4000 anguilliform leptocephali at 179 stations using an Isaacs-Kidd Midwater Trawl accompanied by 104 CTD casts. Based on morphometric observations and DNA sequencing, 74 anguillid leptocephali were collected, which in the southern areas included 29 larvae of six species: Anguilla bicolor pacifica, A. marmorata, A. australis, A. reinhardtii, A. megastoma, and A. obscura (all anguillid species of the region were caught except A. dieffenbachii). Small A. australis (9.0–16.8 mm) and A. reinhardtii (12.4, 12.5 mm) leptocephali were collected south of the Solomon Islands, other A. australis (10.8–12.0 mm) larvae were caught northwest of Fiji along with an A. obscura (20.0 mm) larva, and an A. marmorata (7.8 mm) larva was collected near Samoa. Considering collection sites, larval ages from otolith analysis, and westward SEC drift, multiple spawning locations occurred from south of the Solomon Islands and the Fiji area (16–20 days old larvae) to near Samoa (19 days old larva) during June and July in areas where high-salinity Subtropical Underwater (STUW, ~150 m depth) and the warm, low-salinity surface Fresh Pool were present. Five long hydrographic sections showed the strong Fresh Pool in the west and the STUW formation area in the east.

Downstream-migrating (n = 64) and non-migrating (n = 21) female Celebes eels Anguilla celebesensis were captured from the Poso Lake-River system on Sulawesi Island, Indonesia, and their reproductive physiological characteristics were examined. A histological observation of the ovaries revealed that most non-migrating eels were at the perinucleolus (43%) or oil-droplet (48%) stage, whereas most migrating eels were at the early vitellogenic (36%) or midvitellogenic (61%) stage. Transcript levels of gonadotropin genes (fshb, lhb) in the pituitary gland and concentrations of sex steroids [11-ketotestosterone (11-KT), testosterone, 17β-oestradiol (E2 )] in blood plasma of migrating eels were significantly higher than those of non-migrating eels. The fshb messenger (m)RNA levels were lower in perinucleolus and oil-droplet stages and then significantly increased in the early vitellogenic stage. The lhb mRNA levels in vitellogenic-stage eels were significantly higher than those in perinucleolus- and oil-droplet-stage eels. The 11-KT levels of eels at the oil-droplet and vitellogenic stages were significantly higher than those of eels at the perinucleolus stage. The E2 levels at the vitellogenic stage were significantly higher than those at the perinucleolus and oil-droplet stages. These dynamics of the reproductive hormones represented the physiological background of oogenesis in A. celebesensis that has remarkably well-developed oocytes just before downstream migration.

The Indonesian Seas are at the center of the Coral Triangle, which has the highest marine biodiversity in the world, and the region is under threat from climate change. Freshwater habitats in the region have a high number of anguillid eels compared to other regions of the world, but it is more difficult to capture marine eels to assess their biodiversity. Catches of leptocephali from 5 internationally collaborative surveys for eel larvae (leptocephali) in the Coral Triangle have collected about 126-169 species of larvae, which indicates that the Coral Triangle region likely has the highest marine eel biodiversity in the world based on comparisons to similar larval surveys in the Indian, Pacific, and Atlantic oceans (29-107 species). These marine eel species inhabit a wide range of benthic and pelagic habitats, but how they might be affected by climate changes such as ocean warming has not been considered. Anguillid eels in the Coral Triangle region could be affected mainly by changes in rainfall patterns that could affect their freshwater growth stage or their reproductive maturation patterns and migration. Effects on marine eels would depend on the types of habitats where they live, with the least impacts occurring for deep benthic or pelagic species. Marine eels that live in shallow habitats would be most affected if warming seas and coral bleaching reduce the types of prey species they depend on. Based on their possible association with coral reef habitats, eels of the families Muraenidae and Chlopsidae appear to the most likely types of eels to be impacted by changes in community structure resulting from coral bleaching. All leptocephali species live in the ocean surface layer where they feed on marine snow, so warmer ocean temperatures might reduce the amount or quality of marine snow that is available, resulting in lower larval survival rates. Further studies on eel biodiversity and habitat use will provide more insight into the possible loss of endemic species in the Coral Triangle due to climate change, but presently it is unclear how many species of eels may be directly affected by climate change.

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.

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.

When and where marine eels spawn is poorly known even though species such as those of the family Congridae, Muraenidae and Ophichthidae can be caught in continental shelf habitats. The congrid genus Ariosoma includes small continental shelf eel species whose life histories are not yet known. Mature male and female eels of Ariosoma meeki were observed and captured on 17 August 2009 at the surface at night in the western side of the Kuroshio Current in the East China Sea close to new moon, while they were swimming slowly at the surface and exhibiting apparent reproduction-related behaviour. One male and one sex-unidentified eel (seemingly a male based on body shape) were observed to be chasing one larger female with their heads located near her urogenital pore area. The gonads of the female (540 mm) and the male (410 mm) that were caught by a long-handled dip net were in reproductive condition, because some eggs or seminal fluid were released during handling of the two specimens and high gonad-somatic index (GSI) values of 53 in the female and 20 in the male were recorded. This is one of the few cases in which fully ripe reproductive-condition marine eels have been observed or collected and it provides rare information about the spawning location and timing of this eel species.

To describe the histological characteristics of the oocyte chorion in wild adult and artificially matured Japanese eels, we investigated changes in chorion thickness during artificially induced oogenesis and compared the chorion thickness and ultrastructure between wild and artificially matured eels. In artificially maturing eels, the chorion thickness and volume increased significantly with increasing follicle diameter, peaking at approximately 450 A mu m; beyond this point, the chorion thinned significantly, whereas there were no significant changes in volume. A significant positive correlation was observed between the number of salmon pituitary extract (SPE) injections and chorion thickness. In wild post-spawning adult eels, chorion thickness varied among individuals, and two had chorions that were significantly thinner than those of artificially matured eels. Ultrastructural examination revealed electron-dense layers in the chorions of wild post-spawning adult eels, as was seen in artificially matured eels. This result is inconsistent with our hypothesis that the formation of an electron-dense layer is unique to artificially maturing eels due to repeated SPE injections. These results suggest that the formation cycle of the chorion might be affected by SPE injections in artificially maturing eels, whereas that of wild eels might be synchronized with behavioral and/or environmental fluctuations that occur during the oceanic spawning migration.

Jun Aoyama, Sam Wouthuyzen, Michael J. Miller, Hagi Y. Sugeha, Mari Kuroki, Shun Watanabe, Augy Syahailatua, Fadly Y. Tantu, Seishi Hagihara, Triyanto, Tsuguo Otake, and Katsumi Tsukamoto (2018) Sulawesi Island of north-central Indonesia is located in a region where at least 6 species of tropical anguillid eels are present, but the reproductive ecology and biodiversity of these eels in each area of the Indonesian archipelago remains poorly understood. Some information about these species was obtained from collections of their leptocephalus larvae made during several times of the year and from year-round collections of their recruitment-stage glass eels at a few locations. A sampling survey of anguillid leptocephali was conducted in March 2010 in both the Celebes Sea and Tomini Bay of Sulawesi Island to learn about the biodiversity and reproductive ecology of the eels in the region. Twenty-eight anguillid leptocephali were collected at 13 different stations, with genetic identification indicating that 3 species of eels had spawned in the two areas. Larvae were more abundant in the Celebes Sea (N = 21; 16.0-52.1 mm TL) than in Tomini Bay (N = 7; 9.6-54.8 mm). The abundant 16-21 mm size-class of Anguilla bornensis in the Celebes Sea indicated that species had recently spawned there, and spawning had also occurred in Tomini Bay by A. celebesensis (17.4 mm). These data and previous life history information suggest that A. celebesensis may have two spawning seasons in the Celebes Sea, but only one main spawning season in Tomini Bay. Anguilla borneensis may spawn at several times of the year in the Celebes Sea. Anguilla marmorata and A. biocolor pacifica spawn outside the Indonesian Seas, with A. marmorata recruiting in large numbers in the Sulawesi Island region during much of the year. Other spawning locations of A. celebesensis and A. interioris likely exist in Indonesian waters. Therefore, further research is needed to understand the reproductive ecologies and biodiversity of the tropical anguillid eels in each region of Indonesia in relation to geographic and climatic factors.

Summary The establishment of a sexing technique at an early developmental stage is an important issue in sturgeon aquaculture, yet the mechanism of sex differentiation in sturgeons remains poorly understood. This study aimed to identify genes involved in gonadal sex differentiation and to investigate sexually dimorphic gene expression in undifferentiated gonads. RNA-seq analyses using next-generation sequencers were carried out on undifferentiated gonads of five 9-month-old juvenile Russian sturgeon Acipenser gueldenstaedtii. A total of 45 686 832 (8498 Mb) and 79 743 269 (7887 Mb) quality-controlled reads were obtained using Ion PGM and HiSeq 2000 sequencers, respectively, and were assembled into 338 648 contigs (N50: 532 b). tBLASTx analyses identified 26 transcripts potentially involved in gonadal differentiation. Read-mapping analyses were performed to obtain relative transcript levels (RPKM values). Multivariate analyses of the RPKM values of five transcripts (gsdf, dmrt1a, dmrt1b, foxl2 and cyp19a1a) showed that the five individuals could be separated into two distinct groups. One group, comprising two individuals, had increased levels of foxl2, hsd17b1 and cyp19a1a, suggesting differentiation into females. Alternatively, levels of gsdf were higher in the other three individuals, suggesting differentiation into males. We describe the levels of 26 transcripts potentially involved in gonadal differentiation in undifferentiated sturgeon gonads and suggest that levels of gsdf, foxl2, hsd17b1 and cyp19a1a are appropriate sexing markers for sturgeon at an early developmental stage.

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.

The maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (CR/20β-HSD) was reported to convert 17α-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20β-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20β-hydroxysteroid dehydrogenase (20β-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and ∼12 million reads were obtained, which yielded 71,062 contigs of100 bp. tBlastx analysis identified 1 contig (#f103496) as similar to 17β-hydroxysteroid dehydrogenase type 12 (hsd17β12); however, because the full-length #f103496 sequence was different from hsd17β12, it was termed hsd17β12-like (hsd17β12l). We found that mammalian cells transfected with full-length hsd17β12l exhibited considerable 20β-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17β12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocyte maturation. The levels of hsd17β12l mRNA were also considerably increased in granulosa layers in which 20β-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17β12l, not CR/20β-HSD, is the 20β-HSD responsible for DHP production by granulosa cells in masu salmon during final oocyte maturation.

Eggs of the Japanese eel Anguilla japonica collected in the western North Pacific were identified by onboard species-specific polymerase chain reaction (PCR) and DNA nucleotide sequencing after the cruise. Fish eggs of various species were collected by large plankton net tows at 12 stations along the southern part of the West Mariana Ridge on 19–25 May 2009. A total of 43 fish eggs were distinguished morphologically as possibly being of A. japonica. Thirty-one of those were analyzed by PCR, which included 15 eggs collected at 12°50–55′N, 141°15–20′E (in 5 tows) that showed positive results. The 16S ribosomal RNA (rRNA) gene sequences of eggs determined after the cruise indicated that 31 A. japonica eggs had been collected. The remaining eggs were of mesopelagic eel species (Serrivomeridae and Derichthyidae), or unidentified species. The morphological characteristics of the A. japonica eggs were consistent with those of artificially spawned eggs, except they had a slightly larger diameter. The egg diameter range did not overlap with those of mesopelagic eels of the Serrivomeridae, which often spawn in the same area as A. japonica. These results suggest that egg diameter and embryo shape can be used to morphologically identify naturally spawned A. japonica eggs.

The distribution of all larval stages of the Japanese eel, Anguilla japonica, were examined using historical catch records and original data in the western North Pacific (WNP) to evaluate existing information about the larval distribution and migration of this species. A total of 148 preleptocephali, 2547 leptocephali, 6 metamorphosing larvae, and 21 glass eels were collected during 37 cruises over a 52-year period (1956–2007). Sampling effort was spatio-temporally biased in latitude/longitude among seasons with sampling effort being concentrated near the western margin of the subtropical gyre near Taiwan in the winter season and extensive effort occurring near the spawning area to the east near the seamount chain of the West Mariana Ridge in summer during the spawning season. The distribution of preleptocephali (4.2–8.7 mm) was limited to a narrow area around 14°N, 142°E just west of the southern part of the seamount chain, while leptocephali (7.7–62.0 mm) were widely distributed at increasing size westward in the North Equatorial Current (NEC) to the region east of Taiwan. Metamorphosing larvae (52.7–61.2 mm) were collected only in the area 21–26°N, 121–129°E to the east of Taiwan, while glass eels (51.3–61.2 mm) occurred only within or west of the Kuroshio. These distributions suggest that leptocephali begin to metamorphose within or just east of the Kuroshio, then after completion of metamorphosis the glass eels detrain from the current and migrate inshore. The relationship between catch date and body size of leptocephali suggested that the spawning season is from April to August, but further sampling is needed to eliminate possible effects of sampling bias. This analysis is consistent with the existing hypothesis that Japanese eel larvae born near the West Mariana Ridge are transported westward in the NEC and then transfer to the Kuroshio to recruit to East Asia, although more sampling effort is needed for later stage larvae in the NEC bifurcation region to help understand the larval migration in relation to the possible impacts of ocean–atmosphere changes.

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.

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.

Estradiol-17β (E2) and maturation-inducing hormone (MIH) are two steroid hormones produced in the teleost ovary that are required for vitellogenic growth and final oocyte maturation and ovulation. During this transition, the main steroid hormone produced in the ovary shifts from estrogens to progestogens. In the commercially important Japanese eel (Anguilla japonica), the MIH 17α,20β-dihydroxy-4-pregnen-3-one (DHP) is generated from its precursor by P450c17, which has both 17α-hydroxylase and C17-20 lyase activities. In order to elucidate the regulatory mechanism underlying the steroidogenic shift from E2 to DHP and the mechanistic basis for the failure of this shift in artificially matured eels, the cDNA for cyp17a2—which encodes P450c17-II—was isolated from the ovary of wild, mature Japanese eel and characterized, and the expression patterns of cyp17a1 and cyp17a2 during induced ovarian development were investigated in cultured eel ovaries. Five cDNAs (types I–V) encoding P450c17-II were identified that had minor sequence variations. HEK293T cells transfected with all but type II P450c17-II converted exogenous progesterone to 17α-hydroxyprogesterone (17α-P), providing evidence for 17α-hydroxylase activity; however, a failure to convert 17α-P to androstenedione indicated that C17-20 lyase activity was absent. Cyp17a2 mRNA was expressed mainly in the head kidney, ovary, and testis, and quantitative PCR analysis demonstrated that expression in the ovary increased during induced vitellogenesis and oocyte maturation/ovulation. In contrast, P450c17-I showed both 17α-hydroxylase and C17-20 lyase activities, and cyp17a1 expression increased until the mid-vitellogenic stage and remained high thereafter. Considering the high level of cyp17a2 transcript in the eel ovary at the migratory nucleus stage together with our previous report demonstrating that eel ovaries have strong 17α-P-to-DHP conversion activity, the failure of artificially maturing eels to produce the maturation-inducing DHP may be explained by a deficiency in 17α-P production due to the persistence of cyp17a1 expression after the completion of vitellogenesis.

The maturation-inducing hormone 17α,20β-dihydroxy-4-pregnen-3-one (DHP) was first identified in the amago salmon. Although carbonyl reductase-like 20β-hydroxysteroid dehydrogenase (CR/20β-HSD) was reported to convert 17α-hydroxyprogesterone (17OHP) to DHP in rainbow trout, we previously found that CR/20β-HSD messenger RNA (mRNA) was not upregulated in stimulated granulosa cells from masu salmon, which suggested that DHP is synthesized by a different enzyme. Accordingly, the current study aimed to identify the specific 20β-hydroxysteroid dehydrogenase (20β-HSD) responsible for DHP production by granulosa cells during final oocyte maturation in masu salmon. RNA sequencing was performed on granulosa layers that were isolated from ovarian follicles at 1 month before ovulation and incubated with or without forskolin, which was used to mimic luteinizing hormone, and ∼12 million reads were obtained, which yielded 71,062 contigs of >100 bp. tBlastx analysis identified 1 contig (#f103496) as similar to 17β-hydroxysteroid dehydrogenase type 12 (hsd17β12); however, because the full-length #f103496 sequence was different from hsd17β12, it was termed hsd17β12-like (hsd17β12l). We found that mammalian cells transfected with full-length hsd17β12l exhibited considerable 20β-HSD activity, as indicated by efficient conversion of exogenous 17OHP to DHP. In addition, we found that hsd17β12l mRNA levels were consistently low in follicles during vitellogenic growth; however, the levels increased significantly during final oocyte maturation. The levels of hsd17β12l mRNA were also considerably increased in granulosa layers in which 20β-HSD activity was induced by salmon pituitary extract. Therefore, we suggest that hsd17β12l, not CR/20β-HSD, is the 20β-HSD responsible for DHP production by granulosa cells in masu salmon during final oocyte maturation.

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.

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 donor's 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.