Identification of Sexually Dimorphic Genes in Pectoral Fin as Molecular Markers for Assessing the Sex of Japanese Silver Eels (Anguilla japonica).

The Japanese eel (Anguilla japonica) is an important species in East Asian aquaculture. However, the production of seedlings for this purpose still depends on natural resources, as the commercial production of glass eels is not yet possible. Confusion about the sex of silver eels is one of the factors affecting the success rate of artificial maturation. This study sought to devise a harmless method to precisely assess the sex of silver eels. Partial pectoral fins were collected from females and males and the total RNA was extracted for transcriptomic analysis to identify sexually dimorphic genes as molecular markers for sex typing. An online database was constructed to integrate the annotations of transcripts and perform comparative transcriptome analysis. This analysis identified a total of 29 candidate sexually dimorphic genes. Ten were selected for a real-time quantitative polymerase chain reaction (RT-qPCR) to validate the transcriptomic data and evaluate their feasibility as markers. The transcriptomic analysis and RT-qPCR data implicated three potential markers (LOC111853410, kera, and dcn) in sex typing. The expression of LOC111853410 was higher in females than in males. In contrast, the expression of kera and dcn was higher in males than in females. The ΔCT values of three markers were analyzed to determine their inferred thresholds, which can be used to determine the sex of Japanese eels. The results suggested that if a silver eel had a pectoral fin with the pectoral fin having the ΔCT of LOC111853410 < 11.3, the ΔCT of kera > 11.4, or the ΔCT of dcn > 6.5 can be assessed it could be assessed as female. Males could be assessed by the ΔCT of LOC111853410 > 11.3, the ΔCT of kera < 11.4, or the ΔCT of dcn < 6.5 in their pectoral fins. The molecular functions of these markers and the biological significance of their differential expression require further exploration.

Estimations of Riverine Distribution, Abundance, and Biomass of Anguillid Eels in Japan and Taiwan Using Environmental DNA Analysis.

Although populations of anguillid eels have declined remarkably in recent decades, monitoring data on the spatial and temporal variation in their dynamics are often limited, particularly for tropical eel species. As there are often sympatries of multiple eel species in tropical rivers, identifying eel species based solely on morphological characteristics is challenging. Basin-scale surveys were conducted in rivers of southern Japan and northern Taiwan to investigate (1) whether the spatial distribution, abundance, and biomass of the tropical eel species, the giant mottled eel (Anguilla marmorata), can be monitored in rivers by comparing the results obtained from environmental DNA (eDNA) analysis with data from electrofishing and (2) the riverine distribution of the sympatric A. marmorata and the temperate eel species, the Japanese eel (Anguilla japonica), in this region using eDNA analysis. Although we found an much lower abundance of A. marmorata in the study region, we identified the eDNA of the species from all of the study sites (21 sites) where it was collected by electrofishing, in addition to 22 further study sites where it was not collected directly. This indicates that eDNA analysis has a greater sensitivity for detecting A. marmorata, making it a powerful tool for monitoring the spatial distribution of the species in rivers. We found a significant positive relationship between eDNA concentration and both the abundance and biomass of A. marmorata, and eDNA concentration seemed to better reflect the abundance of the species than did biomass. eDNA of both A. japonica and A. marmorata was identified from almost all rivers, indicating the sympatry of these species in this region, although the degree of sympatry differed between rivers. Though the eDNA concentration of A. japonica decreased significantly with increasing distance from the river mouth, no significant relationship was found for A. marmorata. This study is the first to demonstrate the potential usefulness of eDNA analysis for estimating the spatial distribution, abundance, and biomass of tropical eels in rivers and to further apply this method to investigate sympatry among anguillid species. eDNA analysis can help in obtaining data on the population dynamics of tropical eels, providing invaluable information for managing these species.

Habitat Partitioning and its Possible Genetic Background Between Two Sympatrically Distributed Eel Species in Taiwan.

The geographical distributions of the Japanese eel (Anguilla japonica) and Giant-mottled eel (A. marmorata) overlap in many regions in East Asia and therefore suffer from interspecific competition in the same rivers. After a long period of adaptation, the Japanese eel and Giant-mottled eel may exhibit habitat partitioning in the rivers to diminish the interspecific competition between them. In this study, we conducted a field investigation in the Fengshan River in Taiwan to survey the habitat distributions of the Japanese eel and Giant-mottled eel throughout a river. Moreover, we investigated whether their habitat distributions are related to their swimming and upstream migration. Thus, the mRNA expression levels of several candidate genes that may be associated with the swimming and upstream migration of eel were examined in the glass eels of the Japanese eel and Giant-mottled eel. Field investigation indicated that the Japanese eel mainly inhabited the lower and middle reaches of the Fengshan River, but the Giant- mottled eel was distributed over the middle to upper reaches. The mRNA expression levels of fMYH, dio2, gria3, and neurod1 were higher in the Giant-mottled eel than in the Japanese eel, implying that Giant- mottled eels might have better swimming bursts and more active upstream migration than Japanese eels. These results suggest that there is a habitat partition at which these two eel species coexist in a river, and their habitat distributions may be linked to their swimming bursts and upstream migration. Determining the habitat distributions of freshwater eels is important for developing applicable plans for eel conservation and resource management.

De Novo Assembly of the Whole Transcriptome of the Wild Embryo, Preleptocephalus, Leptocephalus, and Glass Eel of Anguilla japonica and Deciphering the Digestive and Absorptive Capacities during Early Development.

Natural stocks of Japanese eel (Anguilla japonica) have decreased drastically because of overfishing, habitat destruction, and changes in the ocean environment over the past few decades. However, to date, artificial mass production of glass eels is far from reality because of the lack of appropriate feed for the eel larvae. In this study, wild glass eel, leptocephali, preleptocephali, and embryos were collected to conduct RNA-seq. Approximately 279 million reads were generated and assembled into 224,043 transcripts. The transcript levels of genes coding for digestive enzymes and nutrient transporters were investigated to estimate the capacities for nutrient digestion and absorption during early development. The results showed that the transcript levels of protein digestion enzymes were higher than those of carbohydrate and lipid digestion enzymes in the preleptocephali and leptocephali, and the transcript levels of amino acid transporters were also higher than those of glucose and fructose transporters and the cholesterol transporter. In addition, the transcript levels of glucose and fructose transporters were significantly raising in the leptocephali. Moreover, the transcript levels of protein, carbohydrate, and lipid digestion enzymes were balanced in the glass eel, but the transcript levels of amino acid transporters were higher than those of glucose and cholesterol transporters. These findings implied that preleptocephali and leptocephali prefer high-protein food, and the nutritional requirements of monosaccharides and lipids for the eel larvae vary with growth. An online database (http://molas.iis.sinica.edu.tw/jpeel/) that will provide the sequences and the annotated results of assembled transcripts was established for the eel research community.

Electrospun microcrimped fibers with nonlinear mechanical properties enhance ligament fibroblast phenotype.

Fiber structure and order greatly impact the mechanical behavior of fibrous materials. In biological tissues, the nonlinear mechanics of fibrous scaffolds contribute to the functionality of the material. The nonlinear mechanical properties of the wavy structure (crimp) in collagen allow tissue flexibility while preventing over-extension. A number of approaches have tried to recreate this complex mechanical functionality. We generated microcrimped fibers by briefly heating electrospun parallel fibers over the glass transition temperature or by ethanol treatment. The crimp structure is similar to those of collagen fibers found in native aorta, intestines, or ligaments. Using poly-L-lactic acid fibers, we demonstrated that the bulk materials exhibit changed stress-strain behaviors with a significant increase in the toe region in correlation to the degree of crimp, similar to those observed in collagenous tissues. In addition to mimicking the stress-strain behavior of biological tissues, the microcrimped fibers are instructive in cell morphology and promote ligament phenotypic gene expression. This effect can be further enhanced by dynamic tensile loading, a physiological perturbation in vivo. This rapid and economical approach for microcrimped fiber production provides an accessible platform to study structure-function relationships and a novel functional scaffold for tissue engineering and cell mechanobiology studies.

Study of sodium ion selective electrodes and differential structures with anodized indium tin oxide.

The objective of this work is the study and characterization of anodized indium tin oxide (anodized-ITO) as a sodium ion selective electrode and differential structures including a sodium-selective-membrane/anodized-ITO as sensor 1, an anodized-ITO membrane as the contrast sensor 2, and an ITO as the reference electrode. Anodized-ITO was fabricated by anodic oxidation at room temperature, a low cost and simple manufacture process that makes it easy to control the variation in film resistance. The anodized-ITO based on EGFET structure has good linear pH sensitivity, approximately 54.44 mV/pH from pH 2 to pH 12. The proposed sodium electrodes prepared by PVC-COOH, DOS embedding colloid, and complex Na-TFBD and ionophore B12C4, show good sensitivity at 52.48 mV/decade for 10(-4) M to 1 M, and 29.96 mV/decade for 10(-7) M to 10(-4) M. The sodium sensitivity of the differential sodium-sensing device is 58.65 mV/decade between 10(-4) M and 1 M, with a corresponding linearity of 0.998; and 19.17 mV/decade between 10(-5) M and 10(-4) M.