Contrasting riverine distribution and habitat use of the Japanese eel, Anguilla japonica, and the giant mottled eel, Anguilla marmorata, in a sympatric river.

Electrofishing and visual observations revealed contrasting riverine distribution and habitat use of the Japanese eel, Anguilla japonica, and the giant mottled eel, Anguilla marmorata, in a relatively gentle-slope river of Southern Kyushu, Japan. A. japonica was distributed in the lower and upper reaches. A. marmorata inhabited the middle reaches with relatively fast water velocity, heterogeneous water depth, coarse substrata and no adjacent paddy fields. This may have important conservation implications because a decrease in environmental diversity and/or river connectivity could result in the disappearance of A. marmorata habitats.

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.

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.

Whole-Genome Sequencing of 84 Japanese Eels Reveals Evidence against Panmixia and Support for Sympatric Speciation.

The Japanese eel (Anguilla japonica), European eel (Anguilla anguilla), and American eel (Anguilla rostrata) are migratory, catadromous, temperate zone fish sharing several common life cycle features. The population genetics of panmixia in these eel species has already been investigated. Our extensive population genetics analysis was based on 1400 Gb of whole-genome sequence (WGS) data from 84 eels. It demonstrated that a Japanese eel group from the Kuma River differed from other populations of the same species. Even after removing the potential adapted/selected single nucleotide polymorphism (SNP) data, and with very small differences (fixation index [Fst] = 0.01), we obtained results consistently indicating that panmixia does not occur in Japanese eels. The life cycle of the Japanese eel is well-established and the Kuma River is in the center of its habitat. Nevertheless, simple reproductive isolation is not the probable cause of non-panmixia in this species. We propose that the combination of spawning area subdivision, philopatry, and habitat preference/avoidance accounts for the non-panmixia in the Japanese eel population. We named this hypothesis the "reproductive isolation like subset mapping" (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Variation of Lipids and Fatty Acids of the Japanese Freshwater Eel, Anguilla japonica, during Spawning Migration.

The lipid and fatty acid composition of the muscle of the wild Japanese freshwater eel, Anguilla japonica, was analyzed between the initial and terminal stages of spawning migration to clarify the relationship between lipid physiology and maturation. Triacylglycerols were the only major component in the initial-phase eels, which contained high levels of lipids, while comparatively low triacylglycerol levels were observed in terminal-phase eels (Mariana silvers) at spawning area. Significant levels of plasmalogens were found in its phosphatidylethanolamine, different from other common fish species, which have their little levels. The major fatty acids in A. japonica depot triacylglycerols were 14:0, 16:0, 18:0, 16:1n-7, 18:1n-7, 18:1n-9, and 18:2n-6. Noticeable levels of 20:4n-6, EPA, 22:5n-3, and DHA were also found in initial-phase sample TAG at the yellow and initial silver stages. High 18:2n-6 levels in all A. japonica lipids were similar to those in other common freshwater fishes. In all A. japonica tissue phospholipids, high levels of n-6 and n-3 PUFA, such as 20:4n-6, EPA, 22:5n-3, and DHA, were observed except for the matured terminal female sample. High n-6 PUFA levels in terminal-phase samples caught at the spawning area suggest that A. japonica maintains and uses initial fatty acids from inland waters without feeding during long spawning migrations. The post-spawning sample, containing low levels of 20:4n-6 and DHA with unusually high levels of its degradation products (18:3n-6, 20:2n-6, and 18:4n-3), indicates that A. japonica may finally use its most important PUFA as energy for spawning before ending its life.

Light-sensitive vertical migration of the Japanese eel Anguilla japonica revealed by real-time tracking and its utilization for geolocation.

Short-time tracking (one to eight days) of the Japanese eel (Anguilla japonica) using ultrasonic transmitter was performed in the tropical-subtropical area adjacent to the spawning area and temperate area off the Japanese Archipelago. Of 16 eels (11 wild and five farmed) used, 10 wild eels displayed clear diel vertical migration (DVM) from the beginning, while the other five farmed eels tracked for 19 to 66 hours did not. During daytime, a significantly positive correlation between migration depth and light intensity recorded on the vessel was observed in the 10 wild eels, indicating that the eels were sensitive to sunlight even at the middle to lower mesopelagic zone (500 to 800 m). During nighttime, the eel migration depth was observed to be associated with the phase, rising and setting of the moon, indicating that the eels were sensitive to moonlight at the upper mesopelagic zone (<300 m). Two of 10 wild eels were in the yellow stage but shared similar DVM with the silver stage eels. Swimbladders of three silver stage eels were punctured before releasing, but very little effect on DVM was observed. The eels very punctually initiated descent upon nautical dawn and ascent upon sunset, enabling us to determine local times for sunrise and sunset, and hence this behavior may be used for geolocating eels. In fact, estimated positions of eels based on the depth trajectory data were comparable or even better than those obtained by light-based archival tag in other fish species.

Spawning sites of the Japanese eel in relation to oceanographic structure and the West Mariana Ridge.

The Japanese eel, Anguilla japonica, spawns within the North Equatorial Current that bifurcates into both northward and southward flows in its westward region, so its spawning location and larval transport dynamics seem important for understanding fluctuations in its recruitment to East Asia. Intensive research efforts determined that Japanese eels spawn along the western side of the West Mariana Ridge during new moon periods, where all oceanic life history stages have been collected, including eggs and spawning adults. However, how the eels decide where to form spawning aggregations is unknown because spawning appears to have occurred at various latitudes. A salinity front formed from tropical rainfall was hypothesized to determine the latitude of its spawning locations, but an exact spawning site was only found once by collecting eggs in May 2009. This study reports on the collections of Japanese eel eggs and preleptocephali during three new moon periods in June 2011 and May and June 2012 at locations indicating that the distribution of lower salinity surface water or salinity fronts influence the latitude of spawning sites along the ridge. A distinct salinity front may concentrate spawning south of the front on the western side of the seamount ridge. It was also suggested that eels may spawn at various latitudes within low-salinity water when the salinity fronts appeared unclear. Eel eggs were distributed within the 150-180 m layer near the top of the thermocline, indicating shallow spawning depths. Using these landmarks for latitude (salinity front), longitude (seamount ridge), and depth (top of the thermocline) to guide the formation of spawning aggregations could facilitate finding mates and help synchronize their spawning.

Efficiency of peptide nucleic acid-directed PCR clamping and its application in the investigation of natural diets of the Japanese eel leptocephali.

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.

Offshore spawning of Conger myriaster in the western North Pacific: evidence for convergent migration strategies of anguilliform eels in the Atlantic and Pacific.

The spawning area of the common Japanese conger, Conger myriaster, had remained unknown because spawning adults or its newly hatched larvae were never collected. Using genetic identification, we determined that C. myriaster spawns far offshore in the western North Pacific, just west of the spawning area of the Japanese eel, Anguilla japonica. In June 2008, six newly hatched C. myriaster larvae, 5.6-6.9 mm, were collected at the eastern edge of where many small unidentified Conger leptocephali (7-20 mm) were collected previously. The offshore spawning location of C. myriaster is analogous to that of the American conger eel, Conger oceanicus, and the American eel, Anguilla rostrata, in the Sargasso Sea, suggesting that convergent evolution of large-scale reproductive migration strategies in both anguillid and conger eels has occurred in the north Atlantic and Pacific subtropical gyres. The realization that two anguillids, A. rostrata and A. japonica, and two congers, C. oceanicus and C. myriaster, have evolved almost identical migration strategies in widely separated ocean basins suggests that natural selection for larval survival and recruitment success has resulted in long offshore spawning migrations in two phylogenetically distant taxa of anguilliform eels.

Oceanic spawning ecology of freshwater eels in the western North Pacific.

The natural reproductive ecology of freshwater eels remained a mystery even after some of their offshore spawning areas were discovered approximately 100 years ago. In this study, we investigate the spawning ecology of freshwater eels for the first time using collections of eggs, larvae and spawning-condition adults of two species in their shared spawning area in the Pacific. Ovaries of female Japanese eel and giant mottled eel adults were polycyclic, suggesting that freshwater eels can spawn more than once during a spawning season. The first collection of Japanese eel eggs near the West Mariana Ridge where adults and newly hatched larvae were also caught shows that spawning occurs during new moon periods throughout the spawning season. The depths where adults and newly hatched larvae were captured indicate that spawning occurs in shallower layers of 150-200 m and not at great depths. This type of spawning may reduce predation and facilitate reproductive success.