Migration Behavior of Anguilla celebesensis Silver Eels within their Tomini Bay Spawning Area.

The tropical Celebes eel, Anguilla celebesensis, has a short migration between its spawning and growth habitats. Its spawning areas were hypothesized to be in Tomini Bay and the Celebes Sea after collecting their small leptocephali. However, there is no information about the silver eel oceanic spawning migration behavior of A. celebesensis. To better understand their short-distance spawning migration behavior, four large female silver eels (Eel 1-4) were equipped with pop-up satellite archival tags (PSATs) and released near the mouth of the Poso River in Tomini Bay of Sulawesi Island on 22 February (Eel 1-3) and 11 March 2010 (Eel 4). All PSATs ascended in Tomini Bay and transmitted their data. Eel 3 and 4 provided clear records of consistent diel vertical migration (DVM: eight days-Eel 3, 13 days-Eel 4) with daytime dives to mean depths of 444.7 m (Eel 3) and 539.0 m (Eel 4), where mean temperatures were 9.1°C (Eel 3) and 7.7°C (Eel 4), and nighttime ascents to mean depths of 132.8 m (Eel 3) and 112.4 m (Eel 4), where mean temperatures were 20.6°C (Eel 3) and 23.4°C (Eel 4). Eel 3 and 4 started to dive to deeper water around nautical dawn and swam up to shallower water around sunset. During nighttime, both eels swam in deeper and colder water during nights with moonlight than during nights without moonlight, and there was a negative linear relationship between experienced water temperatures with the moon in the sky and the lunar age for the eels. The A. celebesensis daily rhythm of DVM behaviors was similar to spawning-migration DVM behaviors of other anguillid species. Essential life history characteristics of A. celebesensis appear to be a short migration between freshwater growth habitat and ocean spawning habitat, and high GSI values with advanced gonadal development in downstream-migrating silver eels.

Novel tissue thickening around the notochord sheath found in deformed Japanese eel Anguilla japonica leptocephali.

Even though reared leptocephalus larvae of the Japanese eel Anguilla japonica have a high incidence of notochord deformities (>60%), the cause is unknown. We performed histological examinations of the notochord and associated organs in reared larvae to better understand the process causing notochord deformation in eel larvae. In deformed larvae, unknown tissue thickening was discovered near the notochord sheath. Azan staining revealed that these tissue thickenings are most likely collagen fibers within fibrous connective tissue. This was almost identical to the connective tissue found in the primordium of the vertebral body around the notochord sheath in properly metamorphosing larvae. Furthermore, the amount of the thyroid hormone triiodothyronine (T3) was significantly higher in deformed larvae than in normal larvae, indicating that notochord deformity is probably linked to metamorphosis despite the immature stage of growth. We suggest that the aberrant growth of connective tissue surrounding the notochord sheath induced by incomplete metamorphosis causes deformities in eel larvae. The reason why deformed larvae have greater thyroid hormone levels is still unknown. It is important to assess how environmental and dietary factors affect the thyroid hormone levels of eel larvae raised in captivity.

Preliminary insight into parental contributions to Japanese eel (Anguilla japonica) preleptocephali spawned on different nights.

Parentage sibship-inference analyses were conducted using mtDNA sequencing and six microsatellite genotypes of 182 Japanese eel preleptocephali that were collected from one net-tow near the West Mariana Ridge in May 2014. At least 328 parents were involved in producing the 182 preleptocephali, and several parents may have spawned a few times during 3 days of a spawning period. Half-sibs suggested that a few parents mated with 1-3 partners, indicating that the Japanese eel can form spawning aggregations in which several parents mate with each other in the ocean.

Capacity for freshwater acclimation and differences in the transcription of ion transporter genes underlying different migratory life histories of Takifugu fish.

The genus Takifugu is a group of approximately 20 species of puffer fishes living in a wide range of salinity environments around East Asian countries. This group presents a broad spectrum of evolutionary stages adapted to anadromy as a result of speciation that occurred a short time (2-5 million years) ago on an evolutionary timescale. This group thus can be considered as a model for studying the evolutionary mechanisms of anadromy. We firstly conducted a transfer experiment from seawater to low-salinity waters on five Takifugu species: two anadromous species T. obscurus and T. ocellatus, two euryhaline wanderer marine species T. rubripes and T. niphobles, and a strictly marine species T. snyderi, and confirmed that the capacity for acclimation to hypotonic environments was associated with their life history strategies. Next, transcriptomes of the gill and intestine of these species in hypotonic condition were compared to those under hypertonic condition for each species using RNA-Sequencing so as to determine possible candidate transporters playing an important role on freshwater adaptation. As this analysis suggested that cftr, encoding an important ion transporter for seawater acclimation in the gill, and ncc, encoding a transporter that is suggested to play important osmoregulatory roles in the intestine, are important candidates, their expression was validated by quantitative real-time PCR analysis. Expression of cftr was downregulated in the gills of the four euryhaline species under the hypotonic condition, but no change was detected in the gill of stenohaline T. snyderi, which may be one reason for the poor hypotonic acclimation capacity of T. snyderi. Expression of ncc was clearly upregulated in the intestines of the two anadromous species under the hypotonic condition, but not in other three species. Different ion transporter expression patterns between the five species indicate that the transcriptional regulation of cftr in the gill and ncc in the intestine may be important for the improvement of hypotonic acclimation capacity and evolution of anadromy in the Takifugu species.

Distribution of anguillid leptocephali and possible spawning areas in the South Pacific Ocean.

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 mor-phometric 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.

New clues on the Atlantic eels spawning behavior and area: the Mid-Atlantic Ridge hypothesis.

The Sargasso Sea has long been considered as the only spawning area for Atlantic eels, despite the absence of direct observations. The present study raises a novel scenario, deviating from Schmidt's dogma, begins with a review of historical and recent observations that were combined to build up a global theory on spawning ecology and migration behavior of Atlantic eels. From this, it is argued that a favorable spawning area could be located eastward of Sargasso Sea at the intersection between the Mid-Atlantic Ridge and the oceanic fronts. Ocean circulation models combined with 3D particle-tracking method confirmed that spawning at this specific area would result in larval distribution fitting the field observation. This study explores the hypothesis that leptocephali are able to swim and orientate to reach their specific growth areas. It proposes a novel framework about spawning ecology, based on orientation, navigation and meeting cues of silver eels to the spawning area. Together this framework may serve as a stepping-stone for solving the long-lasting mystery of eel reproduction which first came out 2,400 years ago and promotes the understanding of oceanic migration and reproduction of marine organisms.

Morphological, ecological and physiological characteristics of downstream-migrating and non-migrating Pacific bicolor eels Anguilla bicolor pacifica.

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.

Ghrelin and food acquisition in wild and cultured Japanese eel (Anguilla japonica).

To clarify the relationships between growth, endocrine status and habitat characteristics in Japanese eel (Anguilla japonica), plasma and stomach mRNA levels of ghrelin were examined in wild eels captured in the river and the bay, and in cultured eels during and after experimental fasting. Wild juvenile eels captured in freshwater habitats within the river showed significantly higher plasma and stomach mRNA levels of ghrelin than did fish obtained from brackish-water habitats within the bay. In cultured eels experimentally fasted for 4 weeks, plasma and stomach mRNA levels of ghrelin increased. After refeeding, the both parameters returned to the levels observed in continuously feeding control fish. In pigmented elvers, 2 months of feed restriction resulted in a significant increase in whole-body ghrelin mRNA. It is suggested that interaction between ghrelin and feeding is related to their habitats through differential food acquisition in fresh and brackish water environments.

Estimation of the spawning time of Japanese eels in the open ocean.

To understand the spawning ecology of the Japanese eel, the spawning time of this species was estimated based on measurements of the ascending speed of eggs and previously obtained data. Two types of water temperature parameters were calculated assuming an arbitrary spawning time. The 'incubation temperature' of 53 eggs collected in the spawning area was estimated based on the developmental stage of each egg and experimentally determined relationships between water temperature and incubation duration. The 'experienced temperature' of eggs ascending in the water column after spawning was estimated based on an ascending egg speed of 3.69 m/h and spawning depth of 230 m determined from a pop-up satellite archival tag release experiment on silver eels conducted in the same area. The incubation and experienced temperatures of the eggs coincided only at 20:20-22:30 h, 3 days prior to the new moon. This period is only a few hours after the diel vertical migration of Japanese eels in the evening, when adults move up from a depth of ~800 m (approximately 5 °C) to shallower waters of 200-250 m depth (approximately 20 °C). Our findings will facilitate improvements in aquaculture techniques and the detection of eel spawning events in the open ocean.

Freshwater entry behaviour of a non-migratory stenohaline marine fish Takifugu snyderi.

We conducted salinity choice trials with the stenohaline marine species Takifugu snyderi to test their freshwater (FW) entry frequency in relation to starvation. The fish preferred to enter non-natal FW rather than remain in seawater. No relationship was detected between starvation and FW entry behaviour. Our results provide new empirical evidence of a stenohaline fish entering a non-natal osmotic environment. Further research on the entry of stenohaline species such as this one into lethal environments may help determine if this might help promote the evolution of diadromous life histories.

Reproductive physiological characteristics of tropical Celebes eels Anguilla celebesensis in relation to downstream migration and ovarian development.

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, 17ß-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.

Evaluation of downward movements of Japanese eel Anguilla japonica inhabiting brackish water areas.

This study evaluated the size and age distributions and otolith microchemistry of the Japanese eel Anguilla japonica in freshwater and brackish water areas in the Aki and Tsuchikawa rivers for 1 year, and in brackish water areas in the Asahi River for 3 years to understand the movements of Japanese eels between continental habitats of different salinity after recruitment (n = 759). For all three rivers, the total length (LT ) and age distributions were consistent; yellow eels captured in the upper brackish water (Aki River: 353.5 ± 77.4 mm and 3.0 ± 0.8 years; Tsuchikawa River: 287.7 ± 87.3 mm and 3.7 ± 1.3 years; Asahi River: 418.2 ± 112.1 mm and 4.2 ± 1.7 years) were smaller and younger than not only those in the fresh water of the two rivers but also those in the lowest brackish water sampling areas (Aki River: 436.0 ± 71.6 mm and 3.8 ± 1.1 years; Tsuchikawa River: 370.9 ± 121.7 mm and 4.9 ± 2.3 years; Asahi River: 558.5 ± 85.9 mm and 5.7 ± 1.7 years). In the Asahi River, these tendencies were found throughout the 3 years. Otolith analysis indicated that the majority of the eels captured in the lowest brackish water areas had moved down from upstream. These results suggest that Japanese eels inhabiting saline water generally move from the upper estuary as they grow. The upper estuary can be an important area for the management of this species because these eels spend their early continental growth life there.

Changes in PRL Gene Expression During Upstream Movement of the Japanese Eel, Anguilla japonica.

Changes in mRNA levels of prolactin (PRL) during upstream movement were examined in juvenile Japanese eels, Anguilla japonica. Glass eels and elvers were collected from 2007 to 2009 near the entrance of Hamana Lake, and in a small inflowing stream, the Egawa River. Quantification of mRNA was performed by real-time PCR and expressed as whole-body content. PRL mRNA levels of glass eels caught in the coastal zone and tidal area were low. Eels that moved downward in the tidal zone and migrated upstream to enter into freshwater showed increased levels of PRL mRNA. These changes suggest the importance of up-regulation of PRL gene expression in juvenile eels during their upstream movement from seawater to fresh water, particularly in relation to hyperosmoregulation.

Influence of ocean circulation and the Kuroshio large meander on the 2018 Japanese eel recruitment season.

Japanese eel (Anguilla japonica) recruitment to Japan was very low during the early 2017-2018 recruitment season when most glass eels are usually caught, but catches increased in the late recruitment season when recruitment usually decreases. Concurrently, the Kuroshio meander south of Japan had formed again after the previous event ended in 2005. The role of the large meander and ocean circulation features such as the North Equatorial Current (NEC) in the unusual 2017-2018 Japanese eel recruitment timing-pattern was investigated using a three-dimensional particle tracking model that simulated swimming behaviors of virtual larvae (v-larvae) in addition to their drift in ocean currents. Four recruitment seasons were selected for when the Kuroshio large meander was present (2004-2005, 2017-2018) or absent (2009-2010, 2015-2016), and when NEC was shifted north (2004-2005, 2015-2016) or south (2009-2010, 2017-2018). The simulated recruitment timing-patterns were similar to the actual recent-year recruitment, with no early recruitment period v-larvae arrival to southern Japan and increased late period recruitment occurring. Rather than being related to the presence of the Kuroshio large meander, the late arrival appeared to be caused by a southward shifted, weak North Equatorial Current near the spawning area, a longer Subtropical Countercurrent eddy region retention time, and a weak Kuroshio during the early migration and recruitment period of those years. In the late recruitment period, the Kuroshio was stronger than other selected years near the East China Sea and south of Japan and v-larvae were transported more rapidly. The Kuroshio large meander may influence local eel recruitment in Japan, and the recirculation formed by the large meander could potentially enhance recruitment to the Tokai region. Oriented (northwestward) swimming v-larvae were less affected by the large meander, and showed higher recruitment success than those using along-current swimming. Although the Kuroshio large meander did not seem to be responsible for the unusual recruitment pattern in 2018, how Japanese eel larvae and glass eels actually cross out of the Kuroshio and reach coastal waters in Japan remains to be explored.

New PCR primers for metabarcoding environmental DNA from freshwater eels, genus Anguilla.

Freshwater eels of the genus Anguilla comprise 16 species that include three subspecies and are characterized by their unique catadromous life cycles. Their life histories and nocturnal life styles make it difficult to observe them in freshwater and marine habitats. To investigate their distribution and ecology in aquatic environments, we developed new PCR primers for metabarcoding environmental DNA (eDNA) from Anguilla. The new primers (MiEel) were designed for two conserved regions of the mitochondrial ATP6 gene, which amplify a variable region with sufficient interspecific variations ranging from five to 22 nucleotide differences (one to three nucleotide differences between three subspecies pairs). We confirmed the versatility of the MiEel primers for all freshwater eels using tissue DNA extracts when analyzed separately. The metabarcoding combined with the MiEel primers using mock communities enabled simultaneous detection of Anguilla at the species level. Analysis of eDNA samples from aquarium tanks, a controlled pond and natural rivers demonstrated that the MiEel metabarcoding could successfully detect the correct Anguilla species from water samples. These results suggested that eDNA metabarcoding with MiEel primers would be useful for non-invasively monitoring the presence of the endangered anguillid eels in aquatic environments where sampling surveys are difficult.

Spawning by the European eel across 2000 km of the Sargasso Sea.

It has been known for about a century that European eels have a unique life history that includes offshore spawning in the Sargasso Sea about 5000-7000 km away from their juvenile and adult habitats in Europe and northern Africa. Recently hatched eel larvae were historically collected during Danish, German and American surveys in specific areas in the southern Sargasso Sea. During a 31 day period of March and April 2014, Danish and German research ships sampled for European eel larvae along 15 alternating transects of stations across the Sargasso Sea. The collection of recently hatched eel larvae (≤12 mm) from 70° W and eastward to 50° W showed that the European eel had been spawning across a 2000 km wide region of the North Atlantic Ocean. Historical collections made from 1921 to 2007 showed that small larvae had also previously been collected in this wide longitudinal zone, showing that the spatial extent of spawning has not diminished in recent decades, irrespective of the dramatic decline in recruitment. The use of such a wide spawning area may be related to variations in the onset of the silver eel spawning migration, individual differences in their long-term swimming ability, or aspects of larval drift.

Release of eDNA by different life history stages and during spawning activities of laboratory-reared Japanese eels for interpretation of oceanic survey data.

To assist in detection of offshore spawning activities of the Japanese eel Anguilla japonica and facilitate interpretation of results of environmental DNA (eDNA) analysis in their spawning area, we examined the eDNA concentration released by each life history stage of artificially reared Japanese eels in the laboratory using quantitative real-time PCR (qPCR). We also compared eDNA concentrations between before and after artificially induced spawning activities. eDNA was not detected from three 30 L seawater tanks containing each single fertilized egg, but eDNA was found from other tanks each containing single individuals of larval stages (preleptocephalus and leptocephalus), juvenile stages (glass eel, elver and yellow eel) or adult stage (silver eel). The eDNA concentrations increased in the life history stages, showed a significant difference among all stages, and were positively correlated with the total length and wet weight. Moreover, the eDNA concentration after spawning was 10-200 times higher than that before spawning, which indicated that the spawning events in the ocean would produce relatively high eDNA concentration. These results in the laboratory suggested that eDNA analysis appears to be an effective method for assisting oceanic surveys to estimate the presence and spawning events of the Japanese eel in the spawning area.

Mucosal galectin genes in all freshwater eels of the genus Anguilla.

In this study, we determined the genomic DNA sequences of the mucosal galectin-encoding genes from all 19 species and subspecies of the genus Anguilla. The nucleotide sequences of the galectin genes were c. 2.3-2.5 kb long and the organisation of their four exons and three introns was conserved in all species. An unusual sequence was found in the fourth exon of Anguilla reinhardtii, resulting in a unique deduced amino-acid sequence at the C-terminus. All six amino-acid residues important for ß-galactoside binding were conserved in three species, while one residue (R73 ) was substituted to K73 in the other 16 species-subspecies, including Anguilla marmorata. However, this substitution did not appear to affect the sugar-binding ability of galectins because the galectin of A. marmorata was previously shown to bind to lactose. We also discuss the molecular evolution of galectins among Anguilla spp. and the homologues previously identified in Conger myriaster.

Effect of larval swimming in the western North Pacific subtropical gyre on the recruitment success of the Japanese eel.

The possible effect of directional larval swimming on the recruitment success of the Japanese eel, Anguilla japonica, was examined with a three-dimensional particle-tracking ocean circulation model using horizontal northwestward swimming and diel vertical migration (DVM). Four separate experiments included virtual larvae (v-larvae) movement from the spawning area over 290 days (total migration) and 160 days (stage A), from the STCC eddy region in 70 days (stage B), and from the origin of the Kuroshio in 60 days (stage C) to evaluate the effect of directional swimming and DVM compared to simple drifting. Passive or random swimming were not the most effective strategies for larvae dispersing from the spawning area because most v-larvae remained south of 20°N without entering the Kuroshio. Northwestward swimming resulted in wider dispersion and a better chance of successful recruitment, with v-larvae becoming widely distributed in the STCC eddy zone, arriving at the east coast of the Philippines (stage A), escaping the STCC eddy area and reaching the Kuroshio (stage B), and crossing the Kuroshio into the East China Sea shelf (stage C). DVM slightly shortened the migration period due to faster shallow layer ocean currents during nighttime. The NEC transported non-swimming v-larvae westward to the Kuroshio and occasionally northward into the Subtropical Countercurrent (STCC) area where eddies transported v-larvae westward into the Kuroshio, but less than with swimming. Directional swimming increased recruitment success, northwestward swimming was more effective than other directions, and a slower swimming speed was still better than no/random swimming in sensitivity tests. The present study demonstrated a first view of the possibility that Japanese eel larvae might be able to use a strategy of single-direction swimming to increase arrival at their recruitment areas.

Growth potential can affect timing of maturity in a long-lived semelparous fish.

Many diadromous fishes such as salmon and eels that move between freshwater and the ocean have evolved semelparous reproductive strategies, but both groups display considerable plasticity in characteristics. Factors such as population density and growth, predation risk or reproduction cost have been found to influence timing of maturation. We investigated the relationship between female size at maturity and individual growth trajectories of the long-lived semelparous European eel, Anguilla anguilla A Bayesian model was applied to 338 individual growth trajectories of maturing migration-stage female silver eels from France, Ireland, the Netherlands and Hungary. The results clearly showed that when growth rates declined, the onset of maturation was triggered, and the eels left their growth habitats and migrated to the spawning area. Therefore, female eels tended to attain larger body size when the growth conditions were good enough to risk spending extra time in their growth habitats. This flexible maturation strategy is likely related to the ability to use diverse habitats with widely ranging growth and survival potentials in the catadromous life-history across its wide species range.