Cryopreservation of sperm from the gudgeon, Microphysogobio rapidus (Cyprinidae): Effects of cryoprotectant, diluents, and dilution ratio.

We investigated methods for cryopreserving sperm from the endangered gudgeon, Microphysogobio rapidus, by examining the effects of cryoprotective agent (CPA) concentration, diluent, and dilution ratio on post-thaw sperm quality. The quality of frozen sperm was evaluated in terms of motility and kinematic parameters, viability, DNA damage, and fertilization rate. We evaluated methanol, glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol as CPAs. Sperm motility, velocity, and viability were significantly higher when methanol was used as the CPA (p < 0.05). The diluents tested were Ringer's solution, Kurokura's Extender, Common Carp Sperm Extender (CCSE), and buffered sperm motility-inhibiting saline solution (BSMIS); post-thaw motility was highest when Ringer's solution was used as the diluent. Next, various quantities of methanol were combined with Ringer's solution to identify the optimal dose of methanol. The dilution ratios tested ranged from 1:1 to 1:7. Cryopreserved sperm was thawed at 20 °C for 15 s. The use of 10% methanol with Ringer's solution at a dilution ratio of 1:5 resulted in the highest post-thaw sperm motility, viability, and velocity including VAP, VCL, and VSL. Post-thaw sperm showed significantly greater DNA damage than the control (fresh sperm) (p < 0.05). The fertilization rate was highest with fresh sperm (p < 0.05), followed by sperm frozen with 10% methanol + Ringer's solution. We recommend that the best way to preserve sperm in the studied species is to use a combination of Ringer's solution and 10% methanol at a 1:5 dilution ratio. Our findings will facilitate the artificial fertilization of M. rapidus.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

Background: As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. Methods: We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. Results: The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3 °C, and that of D. japonicus was 77.7-78.0 °C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Conclusion: Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

Characterization of the complete mitogenome of the endangered freshwater fish Gobiobotia naktongensis from the Geum River in South Korea: evidence of stream connection with the Paleo-Huanghe.

BACKGROUND: The freshwater fish Gobiobotia naktongensis (Teleostei, Cypriniformes, and Gobionidae) is an endangered class I species whose population size has been greatly reduced. OBJECTIVE: To successfully protect and restore the highly endangered freshwater fish G. naktongensis from the Geum River in South Korea. METHODS: The mitogenome was characterized using the primer walking method with phylogenetic relationships. RESULTS: The complete mitogenome of G. naktongensis Geum River was 16,607 bp, comprising 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA (tRNA) genes. Seventeen substitutions were found by comparing the tRNA regions between G. naktongensis Geum and Nakdong Rivers and G. pappenheimi; most were specific to G. naktongensis Nakdong River, with changes in their secondary structures. The comparison between G. naktongensis Geum River and G. pappenheimi revealed differences in the lengths of the D-loop and two tRNAs (tRNAArg and tRNATrp) and the secondary structures in the TΨC-arm of tRNAHis. In the phylogenetic tree, G. naktongensis Geum River did not cluster with its conspecific specimen from the Nakdong River in South Korea, but showed the closest relationship to G. pappenheimi in mainland China. CONCLUSIONS: Our results support the existence of the Paleo-Huanghe River connecting the Korean peninsula and mainland China, suggesting that G. naktongensis in the Geum River should be treated as a different evolutionarily significant unit separated from that in the Nakdong River. The complete mitogenome of G. naktongensis Geum River provides essential baseline data to establish strategies for its conservation and restoration.

Trophic Ecology of Endangered Gold-Spotted Pond Frog in Ecological Wetland Park and Rice Paddy Habitats.

The gold-spotted pond frog (Pelophylax chosenicus) is an endangered amphibian species in South Korea. In order to obtain ecological information regarding the gold-spotted pond frog's habitat environment and biological interactions, we applied stable isotope analysis to quantify the ecological niche space (ENS) of frogs including black-spotted pond frogs (P. nigromaculatus) and bullfrogs (Lithobates catesbeianus) within the food web of two different habitats-an ecological wetland park and a rice paddy. The gold-spotted pond frog population exhibited a broader ENS in the ecological wetland park than in the rice paddy. According to the carbon stable isotope ratios, gold-spotted pond frogs mainly fed on insects, regardless of habitat type. However, the results comparing the range of both carbon and nitrogen stable isotopes showed that gold-spotted pond frogs living in the rice paddy showed limited feeding behavior, while those living in the ecological wetland park fed on various food sources located in more varied trophic positions. Although the ENS of the gold-spotted pond frog was generally less likely to be overlapped by that of other frog species, it was predicted to overlap with a high probability of 87.3% in the ecological wetland park. Nevertheless, gold-spotted pond frogs in the ecological wetland park were not significantly affected by the prey competition with competitive species by feeding on other prey for which other species' preference was low. Since these results show that a habitats' food diversity has an effect on securing the ENS of gold-spotted pond frogs and prey competition, we recommend that the establishment of a food environment that considers the feeding behavior of gold-spotted pond frogs is important for the sustainable preservation of gold-spotted pond frogs and their settlement in alternative habitats.

Movement Patterns and Diel Activity of Anguilla japonica in the Middle Part of a Large River in South Korea.

To investigate movement patterns and diel activities of Anguilla japonica in the freshwater ecosystem, we applied acoustic telemetry on A. japonica in the Geum River, a large river in South Korea. The acoustic tags were attached on 19 individuals of A. japonica (12 with a depth sensor) in May and October 2015 and tracked at approximately 100-km sections from an estuary barrage by 20 automatic listening stations. Only four individuals showed longitudinal movement (mean, 5.2 km), and others were detected by the receivers at release sites; therefore, A. japonica showed high site fidelity. We did not identify seaward migration during the study period (May to November). Conversely, A. japonica showed active diel movement. The number of detections (p = 0.002) and movement distance (p = 0.004) were higher at night-time (18:00-06:00). As most individuals were actively moving at nighttime, we confirmed that A. japonica is nocturnal, although few individuals also showed daytime movement. Although the population and habitats of A. japonica have been decreasing simultaneously, the East-Asian countries are still severely exploiting rivers and streams to use water resources, and result in habitat simplification generated. Therefore, these results contribute to effective A. japonica management regarding habitat and population conservation and restoration.

Responses of fish assemblage structure to large-scale weir construction in riverine ecosystems.

Worldwide, increasing amounts of dams and weirs have been established in rivers in recent decades, often with drastic effects on their ecosystems. Between late 2009 and 2011, 16 large-scale dams were built in the main channels of the four largest rivers in South Korea, eight of these along the main channel of Nakdong River (300 km, 520 km in total). We studied the effect of these constructions on the fish community in the riparian zone based on intensive fish field surveys conducted in the littoral zone during 2007-2017, analysis of fishermen's catch data and molecular analysis of the diet of the keystone species, largemouth bass (Micropterus salmoides). Our results, based on RDA and GLM analyses, showed that environmental characteristics and fish species composition changed significantly with dam construction. Total fish abundance and biomass decreased after the start of the weir construction and fish richness decreased with time. The abundance and biomass of exotic fish decreased during construction but recovered afterwards, leading to higher proportions of exotics. Dominance and Shannon indices did not change significantly from before to after construction, while the Evenness index showed a significant decreasing trend. A comparison of the diet composition of largemouth bass showed decreasing genetic variation after construction. The changes in Functional Feeding Group (FFG) of the benthic communities at the study sites did not coincide with FFG changes in the prey items of largemouth bass, indicating a transition in feeding mode from before to after construction. In conclusion, the endemic and native fish species were most sensitive and showed lower resilience to disturbance by the large-scale dam construction than the translocated and exotic species, and the lake-like ecosystems after construction markedly improved the competitive capacity of these exotic fish over the native and endemic species in the riparian zone of the river.