The endangered Amami rabbit (Pentalagus furnessi), endemic to two Japanese subtropical islands, has a habit of egesting faeces on streambanks in their habitats. We hypothesized that rabbit faeces transported into streams are consumed by aquatic animals. Underwater behavioural observations using time-lapse photography showed that decapod crustaceans foraged on rabbit faeces introduced experimentally into a forest stream on Amami-Oshima Island. From in situ samples, stable isotopic analysis showed mean contributions of rabbit faeces to diets of benthic macroinvertebrates ranged from 12% to 66%. This study suggests that rabbit faeces form an allochthonous resource unique to this insular stream ecosystem.

Attached algae are important primary producers in river ecosystems that are foraged by benthic macroinvertebrates and grazing fishes. The ayu Plecoglossus altivelis, which is an algae-feeding fish that feeds on attached algae. Fine particulate organic matter (FPOM) is generated when P. altivelis use their comb-like teeth to graze algae off the surface of stones, and dislodge them into streams. Therefore, in the present study, we investigated how P. altivelis feeding on algae contributes to the production of algae-derived FPOM. We subsequently evaluated the produced FPOM to determine whether it affected the origin of the macroinvertebrate food supply. To investigate P. altivelis density, the biomass of primary producers, and the food use fraction of consumers, field surveys were conducted in the Kamo-gawa River, Japan, in the summer season from 2018 to 2020. Carbon and nitrogen stable isotope ratios of macroinvertebrates and food sources were measured to estimate the composition of FPOM and the contribution of different food sources to primary consumers. Our results showed that algal biomass decreased at sites with a high density of P. altivelis, where the proportion of algae-derived FPOM increased. The algal contribution to macroinvertebrates increased at sites with high algae-derived FPOM. Furthermore, structural equation modelling revealed that the feeding behaviour of P. altivelis contributed to macroinvertebrate food utilisation via the production of algae-derived FPOM. These results suggest that P. altivelis is a keystone species that not only produces algae-derived FPOM but also improves the food supply to macroinvertebrates and promotes river ecosystem dynamics.

The Amami rabbit (Pentalagus furnessi), endemic to the Amami-Oshima and Tokunoshima Islands, is an endangered species that faces habitat disturbance. This study used fecal DNA metabarcoding to analyze the dietary preferences of Amami rabbits. Fecal samples from six rabbits were collected and analyzed for plant DNA. The results revealed the presence of 85 operational taxonomic units (OTUs) representing different plant species, with individual rabbits consuming 17-38 plant species. The dietary composition varied significantly among individuals, with a notable preference for ferns in some cases. Diet diversity, assessed using Simpson's Diversity Index, ranged from 0.328 to 0.889. This study highlights the importance of a diverse plant diet for Amami rabbits and underscores the need for habitat preservation to ensure a stable food supply. Conservation efforts must focus on maintaining a unique subtropical moist forest ecosystem and mitigating human-wildlife conflicts to promote sustainable coexistence.

The Mekong River in Cambodia is inhabited by approximately ten species of freshwater pufferfish of the family Tetraodontidae. However, limited information is available regarding the toxin profile of these fish. In this study, to obtain sufficient information on the intra-body distribution of toxins in Cambodian freshwater pufferfish, one species of Pao freshwater pufferfish (Pao sp. A) were collected from Phnom Penh (PNH) in November 2019 (n = 23) and from Kratie (KTI) in March 2023 (n = 21). Toxin analyses of these samples revealed that individuals from both regions possessed saxitoxins (STXs) and contained no tetrodotoxin. Pao sp. A specimens from PNH possessed high STXs levels in the skin, muscle, liver, and gonads, whereas those from KTI showed relatively low levels of STXs concentrated in the skin. STXs amount in the ovaries in PNH individuals increased exponentially with increasing the gonadosomatic index (GSI). We also clarified that STXs concentration in the skin became increasingly higher as KTI individuals grew. These results suggest that differences in living regions may affect the intra-body distribution of STXs in Pao freshwater pufferfish, with maturation and/or growth contributing as well.

This study revealed the previously unappreciated consumption of terrestrial plant matter by Ryukyu-ayu Plecoglossus altivelis ryukyuensis, enabled by psbA-based DNA metabarcoding. Although ferns are a minor dietary component, their presence indicates the facultative consumption of terrestrial macrophytes. In terms of the origin of this material, we hypothesize that they ingest drifting material. Eustigmatophytes, a dominant dietary element, have not been previously reported in the diets of ayu Plecoglossus altivelis altivelis. Their presence, along with that of terrestrial ferns, suggests a terrestrial origin. Individual Ryukyu-ayu exhibit a dietary dichotomy: some primarily consume eustigmatophytes, ferns, and seed plants (drift-feeding), while others consume diatoms (grazing). This shift may be correlated to resource availability and competition. Downstream sites with reduced algal productivity and competition from grazing gobies favor drift-feeding in Ryukyu-ayu. A positive correlation existed between dietary diversity and individual conditions in Ryukyu-ayu, indicating that diverse diets would promote better growth. Drift-feeding represents an adaptive foraging tactic within the island's unique ecosystem, characterized by low riverine algal productivity and frequent rainfall, leading to a substantial influx of terrestrial particulate organic matter. In conclusion, Ryukyu-ayu exhibits dietary flexibility and adaptation to the dynamic island river food web. This study highlights the importance of diverse food resources and interspecific relationships, such as the Amami rabbit-Ryukyu-ayu connection, for effective Ryukyu-ayu conservation.

The central-marginal hypothesis predicts that genetic diversity decreases and genetic differentiation increases toward the species distribution margin. We analyzed the mitochondrial DNA control region of ayu (Plecoglossus altivelis) collected from different localities, including central and marginal populations, to test the hypothesis. The results show that the ayu populations at southern marginal habitats (Tien Yen River in Vietnam and Yakugachi River in Amami-oshima Island) displayed low genetic diversity (Haplotype diversity, H = 0.52 and 0.67; Nucleotide diversity, Pi = 0.01 and 0.006, respectively) and significantly differed from the central populations (p < 0.001), consistent with the central-marginal hypothesis. However, ayu populations at the center of their distribution range (Shinano River in Honsyu and Hamoji River in Sado Island) and the northern marginal habitats (Yoichi River in Hokkaido and Aonae River in Okushiri Island) exhibited high genetic diversity (H = 1 for all rivers; Pi = 0.024–0.029). The genetic differences between the populations at the center and the northern marginal habitats were insignificant (p > 0.05). This study showed that in ayu, the central-marginal hypothesis is valid in the southern direction but not in the northern direction. Urgent conservation is needed for the genetically distinct population in the degraded southern habitat with severely low genetic diversity.

The preference of females for male body size was examined in an experimental environment where direct interference between males was eliminated. In this environment, females preferred to spawn with larger males. To understand the preference of females for larger males, paternal care and egg defence abilities and paternal cannibalism intensity were further tested with respect to the body size of males. The dead egg rate for an egg mass during the paternal care period was not related to the body size of the paternal males. However, larger males could defend their egg masses more successfully against nest intruders than smaller ones. During the paternal egg care period, paternal males often consumed eggs in their nests. In particular, smaller males compared to females caused paternal brood cannibalism. High egg defence ability and low paternal cannibalism intensity are the most plausible reasons for females to choose larger males.

The paddy field is a man-made habitat and is subjected to various agronomic practices but only for a temporary period, in line with the stages of paddy planting. We hypothesised that the spatial scale (types of habitats within a paddy field) and temporal scale (the stages of paddy planting) affect fish diversity and assemblages. We sampled the fish from four different habitats within the paddy field from three different stages of paddy planting, for two seasons of paddy cultivation. There were 26 species of freshwater fishes recorded from 16 families. Generally, the diversity values of fishes across the stages and habitats fluctuate for both seasons. Both spatial and temporal scales have a significant effect on fish diversity (p < 0.05). Fish assemblages later were also separated according to the habitats and stages in the paddy field via nMDS analysis. The results suggest that spatial and temporal effects are important drivers for the changes in fish diversity in an urban environment as well as maintaining the ecosystem balance in man-made habitats.

Grazing fish play a role of controlling autochthonous primary production and converting it to secondary production by consuming benthic algae. Moreover, they will also provide autochthonous particulate organic matter (POM) for freshwater ecosystems by dislodging the benthic algal matrix while feeding. However, it has not been confirmed whether grazing fish induce POM production during their feeding on benthic algae. To verify this POM production during grazing by fish, short-term (90 min) feeding experiments were conducted by introducing grazing fish Plecoglossus altivelis into indoor aquaria with benthic algal mats. We found that the fish can generate POM by feeding on benthic algae. Although POM was generated in the aquaria where the fish had not grazed on the benthic algal mats at all, the concentration of POM in the aquaria increased with the frequency of the acts of scraping benthic algae by the fish. Furthermore, we observed that the fish egested faeces which corresponded to about 44% on average of the ingested benthic organic matter. In some cases, the fish generated POM that corresponded to 88% of the removed (dislodged and ingested) benthic organic matter through their feeding and defecation. This study suggested that grazing fish have a function of providing autochthonous POM to freshwater ecosystems both via dislodging benthic algae during feeding and through egestion of consumed benthic algae.

亜熱帯性気候である奄美大島の河川において,流下微粒状有機物(SFPOM)の生産経路は未だ不明な点が多い.そこで本研究では,炭素・窒素安定同位体比を用いて河川内のSFPOMの起源と生産経路を推定することを目的とした.アマミノクロウサギの生息地域では,SFPOMに対する糞の寄与率が39%を示した.また,付着藻類量が多い地点でリュウキュウアユの個体数密度も高まったが,SFPOM中の藻類現存量は0.5mgL-1に留まり,リュウキュウアユの個体数密度とSFPOM中の藻類現存量との間には有意な相関関係が認められなかった.本研究の結果から,河川生態系内においてアマミノクロウサギは陸上有機物をSFPOM起源物質へ変換して供給する役割を担っていることが示唆された.また,リュウキュウアユは藻類由来SFPOMの生産に対する寄与が現状では低いことが示唆された.

The ayu, Plecoglossus altivelis, with an amphidromous life migrating between the sea and streams, is a commercially important fish for the inland fisheries of Japan. Despite intensive transplantation, seedlings originating from non-native population landlocked in the Biwa Lake have been less contributable to stock recovery. In fact, no apparent evidence of introgression from the released fish to the native population has been obtained. We hypothesized that the parental property from the landlocked strain would give some disadvantages to the offspring against saline osmotic pressure during their early life in the sea. The acute performance of newly hatched larvae in 180

Stream-dwelling white-spotted charr, Salvelinus leucomaenis, populations tend to have unique color and spot patterns in different regions and may even display stream-specific patterns. An extreme edge of such diversity, found in individuals with atypical body color patterns (so-called nagaremon-type charr, a rare morphotype of Salvelinus leucomaenis [hereafter termed nagaremon-charr]), sympatrically occurring with normal-charr, has been reported from only six small isolated populations in Japan. Based on morphological and ecological perspectives, nagaremon-charr has been considered as an intraspecific color variant of white-spotted charr, although the genetic status of nagaremon-charr has not been determined. In this study, genetic diversity and population structure of the nagaremon-charr in a tributary of the Ane River (Lake Biwa system) were investigated through microsatellite and mtDNA analyses. Nagaremon-charr and sympatric normal-charr in the tributary shared the mtDNA haplotypes and were assigned to the same cluster in the STRUCTURE analysis and discriminant analysis of principal components (DAPC). These results suggested that nagaremon-charr in the Ane River is an intra-populational specific color variant of white-spotted charr. Above a waterfall, nagaremon-charr specimens exhibited extremely reduced genetic diversity, indicating that genetic drift may account for the fixation of the nagaremon-morphotype. Normal-charr below the waterfall clustered separately from hatchery-reared charr, indicative of native status of the former. Thus, both nagaremon-charr and normal-charr in the entire Ane River tributary should be conserved.

Although river floods and spates have a major influence on fish populations, few studies have examined the selective pressure on individuals and traits that favour their survival. In this study, the factors that potentially affect the survival of an amphidromous fish species, ayu Plecoglossus altivelis altivelis (Temminck & Schlegel), during a major spate were examined. Using otolith analyses, ayu in the Takahashi River, Okayama, Japan, were compared before (April; n = 50) and after (September; n = 32) a spate with regard to their respective hatch-date distributions, standard body length and age at upstream migration, age at metamorphosis, and larval and juvenile growth rates. When comparing juvenile traits, surviving adult ayu grew faster than individuals collected before the spate. These findings suggest that juvenile growth rate is a key factor for enhancing subsequent survival and highlight the importance of conserving suitable nursery habitats to enhance fish survival during future floods.

The Ryukyu-ayu population on Okinawa-jima Island went extinct suddenly in the late 1970s, but the cause remains unclear. Reports say that genetic diversity was not responsible, so we investigated food shortage as a probable driver. We examined pre-extinction specimens to look for territorial individuals, which have unique morphological characteristics and occur only in food-affluent conditions. We found that 42.1% (n = 45) of individuals possessed longer dorsal fins than non-territorial fish. Combining this with another key characteristic (blackened membrane at posterior dorsal fin ray), we found that territorial fish probably constituted 35.5% which fell in the range of the expected proportion for territorial fish in the nominotypical subspecies and higher than that observed in the extant population on Amami-oshima Island. These findings imply that the food resources in the riverine environments of Okinawa-jima Island before the extinction event might be sufficient for Ryukyu-ayu to establish feeding territories. Therefore, food shortage did not cause this population extinction.

The present study tested the hypothesis that whether limited genetic diversity was the main factor that led the Ryukyu-ayu population on Okinawa-jima Island to extinction in the late 1970s. The usability of fluctuating asymmetry (FA) in reflecting genetic diversity was tested using two extant populations of Ryukyu-ayu on Amami-oshima Island, then used to estimate genetic diversity for the extinct population which only formalin-preserved specimens are available. The results showed that higher FA indices were associated with lower genetic diversity in the two extant populations, and the extinct population on Okinawa-jima Island had lower FA indices than the two extant populations on Amami-oshima Island. This finding suggests that the genetic diversity of the extinct population was not low enough as to cause its extinction.

Asexual vertebrates are rare and at risk of extinction due to their restricted adaptability through the loss of genetic recombination. We explore the mechanisms behind the generation and maintenance of genetic diversity in triploid asexual (gynogenetic) Carassius auratus fish, which is widespread in East Asian fresh waters and exhibits one of the most extensive distribution among asexual vertebrates despite its dependence on host sperm. Our analyses of genetic composition using dozens of genetic markers and genome-wide transcriptome sequencing uncover admixed genetic composition of Japanese asexual triploid Carassius consisting of both the diverged Japanese and Eurasian alleles, suggesting the involvement of Eurasian lineages in its origin. However, coexisting sexual diploid relatives and asexual triploids in Japan show regional genetic similarity in both mitochondrial and nuclear markers. These results are attributed to a unique unidirectional gene flow from diploids to sympatric triploids, with the involvement of occasional sexual reproduction. Additionally, the asexual triploid shows a weaker population structure than the sexual diploid, and multiple triploid lineages coexist in most Japanese rivers. The generated diversity via repeated interploidy gene flow as well as an increased establishment of immigrants is assumed to offset the cost of asexual reproduction and might contribute to the successful broad distribution of this asexual vertebrate.

The placement of artificial structures in coastal areas can adversely impact the life histories of larval and juvenile fish by reducing suitable nursery areas. However, little is known about the responses of them to such habitat loss. The ayu Plecoglossus altivelis altivelis is an amphidromous fish that spends its early ontogenetic stages in suitable nurseries, the surf zone, within 2 km from river mouths. To evaluate the possible impact of habitat loss on larval and juvenile ayu, we collected 50 juveniles from the Takahashi River, Okayama, Japan, on 19 April 2018, where more than 95% of the coastal outlet area is covered with artificial structures. Otolith Sr/Ca- and increment analyses were conducted to examine the habitat use and growth rates of each individual. We also collected nine larvae on 27 December 2018 from a semi-natural surf zone, which is located 5 km from the Takahashi River mouth, in order to evaluate their growth rate. The 50 juveniles spent approximately one-third of their marine life stage in estuaries with brackish water, suggesting that the estuaries are the only nursery grounds remaining in this area. Among the 50 juveniles, specimens that experienced lower salinities showed higher growth rates and larger body sizes in the larval stage (1-80 days old). However, a large variation in growth rates was also observed among the specimens that grew in estuaries, probably owing to environmental instability in the area. While the difference was not statistically significant, the nine larvae from the semi-natural surf zone showed higher growth rates than the 50 juveniles from the Takahashi River. Our results indicate that the estuaries can be an alternative habitat, but that they might not be a better habitat than the regular surf zone habitat.

Two subspecies of ayu, Plecoglossus altivelis altivelis (nominotypical subspecies) and P. a. ryukyuensis (Ryukyu-ayu), differ morphologically, behaviorally, and genetically. Specimens from nine populations comprising both subspecies were investigated to examine whether their meristic characteristics follow a widely observed geographical tendency known as Jordan’s rule. Seven out of the eleven characteristics increased in frequency with latitude, following Jordan’s rule. Contrarily to the expectation from Jordan’s rule, however, Ryukyu-ayu had more anal fin rays than nominotypical subspecies in the temperate zone, suggesting genetic divergence underlying this characteristic between these two subspecies. The two subtropical populations showed a reversal of the geographic cline in the number of scales and dorsal fin rays, indicating these do not share a gene pool. The present study, without using genetic indicator, remains disputable to consider genetic status within Ryukyu-ayu further.

The establishment of fluvial fish populations from anadromous populations by natural or artificial barriers obstructing migration is a good research subject to study life history plasticity. Biwa salmon, Oncorhynchus masou subsp., a salmonid fish endemic to the Lake Biwa water system, exhibit life history variation (e.g., mature stream-resident males) in addition to a typical lacustrine life history type, indicating potential adaptations of life histories in response to emergence of barriers. Currently, fluvial populations that are morphologically similar to both stream-resident Biwa salmon and amago, the fluvial red-spotted masu salmon, Oncorhynchus masou ishikawae , are found upstream of dams which were constructed in the inflowing rivers of Lake Biwa. However, it is unknown whether they are Biwa salmon or amago. To explore that, the genetic characteristics of nine fluvial populations were investigated through AFLP and mtDNA analyses. Bayesian admixture analysis based on the AFLP data revealed that three fluvial populations were admixed populations between Biwa salmon and amago. In addition, a Biwa salmon mtDNA haplotype was detected in some individuals from three populations. However, no genetically pure fluvial populations of Biwa salmon were found, indicating no life history plasticity in this subspecies, and thus hybridization with amago boosted the ability of this subspecies to establish fluvial populations. Nevertheless, other scenarios, such as hybridization after establishment of fluvial populations of Biwa salmon, are also possible. The latter hypothesis could be supported by the fact that amago did not inhabit the river before emergence of barriers. However, a significant genetic population structure was found only in amago, suggesting that this subspecies is native to the Lake Biwa water system. But the possibility that multiple sources of amago have been released into rivers cannot be excluded. Therefore, further studies on the relationships between amago populations in the upper reaches of the Lake Biwa water system and other populations in the surrounding areas of the Lake Biwa water system are needed to clarify the origins of the admixed populations.

We examined the effects of interstitial space between stream substrate particles on the colonization of aquatic organisms using three types of substrates (gravel, a cobble, and a cobble on gravel) in a riffle and pool of a temperate stream. Significantly greater abundance, wet weight, diversity ( H ′), taxonomic richness, and evenness of aquatic organisms were found in the riffle than in the pool, and the interstitial space substrate (i.e., a cobble on gravel) had significantly greater abundance, wet weight, and taxonomic richness of aquatic organisms than did the cobble substrate. Of the 13 families observed in the experiments, larval net-spinning caddisfly (Hydropsychidae) dominated the riffle in terms of the abundance and wet weight; chironomid larvae dominated both the riffle and the pool in terms of abundance. Simple main effect tests indicated significant effects of substrate on the abundance and wet weight of larval caddisfly in the riffle, and post hoc tests on substrate in each habitat indicated that the abundance and wet weight of larval caddisfly on interstitial space substrate were significantly greater than those on cobble substrate in the riffle. Our results suggest the importance of interstitial space between stream substrates in riffles to ensure higher colonization rates of aquatic organisms such as larval net-spinning caddisflies characterized as filter feeders.