Effects of adding Bacillus subtilis natto NTU-18 in paste feed on growth, intestinal morphology, gastrointestinal microbiota diversity, immunity, and disease resistance of Anguilla japonica glass eels.

Japanese eel, Anguilla japonica, holds significant importance in Taiwanese aquaculture. With the intensification of eel farming, the impact of Edwardsiella tarda has become increasingly severe. Consequently, the abusive use of antibiotics has risen. Bacillus subtilis natto NTU-18, a strain of Bacillus with a high survival rate in feed processing, plays a crucial role in promoting intestinal health through competitive rejection, enhancing immune responses against bacterial pathogens, and improving intestinal health by modulating gastrointestinal microbiota to produce beneficial metabolites of mice and grass carp, Ctenopharyngodon idella. This study investigated the effects of different proportions (control, 0.25 %, 0.5 %, 1 %, and 2 %) of B. subtilis natto NTU-18 added to paste feed on the growth performance, intestinal morphology, and microbiota, expression of immune-related genes, and resistance to E. tarda in Japanese glass eel. The results indicated that the growth performance of all groups with B. subtilis natto NTU-18 added was significantly higher than that of the control group and did not impact the villi morphology. The expression of immune-related genes in the kidney, specifically HSP70 and SOD, was significantly higher from 0.5 % and above than the control; however, no significant differences were observed in CAT, POD, and HSP90. In the liver, significant differences were found in HSP70 and IgM above 0.25 % compared to the control group, with no significant differences in SOD, CAT, POD, and HSP90 among all groups. Additionally, intestinal microbiota analysis revealed that the 2 % additional group had significantly lower diversity than other groups, with Cetobacterium as the dominant species. The challenge test observed that the survival rates of the 0.5 % and 1 % groups were significantly higher. This research suggests that adding 0.5 % and 1 % of B. subtilis natto NTU-18 to the diet is beneficial for Japanese glass eel's immunity, growth performance, and disease resistance.

Effects of Different LED Light Spectra on Growth and Immunity of the Japanese Eel (Anguilla japonica) and Giant Mottled Eel (A. marmorata).

Indoor recirculating aquaculture systems make light control possible and enable the usage of specific coloured lights to promote the growth and immunity of aquaculture species. Five different LED wavelengths (white light [460 nm], red light [622 nm], green light [517 nm], blue light [467 nm], and the dark) were used in this study to evaluate growth and immunity in the glass eel stage of two high-valued anguillid species, Japanese eel (Anguilla japonica) and giant mottled eel (A. marmorata). There were no significant differences in growth of the Japanese eel among the groups after 12 weeks of feeding (p > 0.05); the survival rate of each group was over 95%. The giant mottled eel showed better growth in total length and body weight in the red light and dark groups (p < 0.05). Expression levels of immune-related genes were not significantly different between each group of the Japanese eel and the giant mottled eel (p > 0.05). The growth of the Japanese glass eel was not significantly sensitive to different LED wavelengths, while the giant mottled glass eel showed better growth under red light and dark environments. Neither eel species showed significant differences in innate immunity under different LED wavelengths.

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.

A method for the process of collagen modified polyester from fish scales waste.

In this study, we introduced a novel polymerization method of polyester using collagen peptides derived from fish scale waste. After the extraction process of collagen peptide from fish scales, putting collagen peptide, ethylene glycol and Benzenedicarboxylic acid into a container, and mixing them to form a mixture; heating the mixture for executing an esterification reaction, to product esters and water; heating the esters, and stirring the esters via a mixer; in a specific period, decreasing the pressure in the container for executing a polycondensation reaction; decreasing the pressure in the container to a second pressure, and stirring the esters via the mixer, to produce a collagen modified polyester. Collagen peptides are rich in glycine, proline, and hydroxyproline, and by forming a triple helix structure, such as that of the copolyester, gain better hydrophilicity, antistaticity, and ductility. As a result, the produced collagen modified polyester fiber keeps the characteristics of the traditional polyethylene terephthalate fibers including strength, durability, and resistance to wrinkle and shrink. However, the supramolecular collagen modified polyester containing animal collagen peptides has naturally a soft touch and champagne-like color. Consequently, it can be used as a suitable material for skin-friendly functional clothes with or without additional dying. In brief,•This study introduces a novel method for collagen modified polyester.•Upcycled fish scale waste brings the sustainable benefits of circular economy.•Collagen modified polyester provides a new direction for future technological development in the textile industry.

North Equatorial Current and Kuroshio velocity variations affect body length and distribution of the Japanese eel Anguilla japonica in Taiwan and Japan.

The larval stage of Japanese eel travels a substantial distance over a long duration through the North Equatorial Current (NEC) and the Kuroshio, and the spawning behavior of mature eels leads to monthly arrival waves in eastern Taiwan between November and February. The total length (TL) of the glass eel relates to its larval duration and age; therefore, the TL can indicate the larval duration. The monthly mean TLs of eels along eastern Taiwan from 2010 to 2021 were used to estimate the batch age, and the recruitment patterns and relative abundances were compared. The TLs of glass eels followed a normal distribution, and the estimated ages were highly correlated with their mean TLs. Early recruit TLs were significantly greater than those of late recruits. The mean tracer drift time was longer in early recruitment months (November-December) than in later dates (February-March). The recruitment lag between Taiwan and Japan was approximately 1-1.5 months, with relative more abundance in Taiwan for the early recruits and in Japan for the late recruits. Speculated cohorts followed the main streams of the NEC and Kuroshio, and the monthly velocity changes of these currents could affect the mean TLs as well as the distribution patterns of Japanese glass eels in Taiwan and Japan.

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.

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.

Stable species boundaries despite ten million years of hybridization in tropical eels.

Genomic evidence is increasingly underpinning that hybridization between taxa is commonplace, challenging our views on the mechanisms that maintain their boundaries. Here, we focus on seven catadromous eel species (genus Anguilla) and use genome-wide sequence data from more than 450 individuals sampled across the tropical Indo-Pacific, morphological information, and three newly assembled draft genomes to compare contemporary patterns of hybridization with signatures of past introgression across a time-calibrated phylogeny. We show that the seven species have remained distinct for up to 10 million years and find that the current frequencies of hybridization across species pairs contrast with genomic signatures of past introgression. Based on near-complete asymmetry in the directionality of hybridization and decreasing frequencies of later-generation hybrids, we suggest cytonuclear incompatibilities, hybrid breakdown, and purifying selection as mechanisms that can support species cohesion even when hybridization has been pervasive throughout the evolutionary history of clades.

Two HEPN domains dictate CRISPR RNA maturation and target cleavage in Cas13d.

Cas13d, the type VI-D CRISPR-Cas effector, is an RNA-guided ribonuclease that has been repurposed to edit RNA in a programmable manner. Here we report the detailed structural and functional analysis of the uncultured Ruminococcus sp. Cas13d (UrCas13d)-crRNA complex. Two hydrated Mg2+ ions aid in stabilizing the conformation of the crRNA repeat region. Sequestration of divalent metal ions does not alter pre-crRNA processing, but abolishes target cleavage by UrCas13d. Notably, the pre-crRNA processing is executed by the HEPN-2 domain. Furthermore, both the structure and sequence of the nucleotides U(-8)-C(-1) within the repeat region are indispensable for target cleavage, and are specifically recognized by UrCas13d. Moreover, correct base pairings within two separate spacer regions (an internal and a 3'-end region) are essential for target cleavage. These findings provide a framework for the development of Cas13d into a tool for a wide range of applications.

The genome-wide landscape of small insertion and deletion mutations in Monopterus albus.

Insertion and deletion (indel) mutations, which can trigger single nucleotide substitutions on the flanking regions of genes, may generate abundant materials for disease defense, reproduction, species survival and evolution. However, genetic and evolutionary mechanisms of indels remain elusive. We establish a comparative genome-transcriptome-alignment approach for a large-scale identification of indels in Monopterus population. Over 2000 indels in 1738 indel genes, including 1-21 bp deletions and 1-15 bp insertions, were detected. Each indel gene had ∼1.1 deletions/insertions, and 2-4 alleles in population. Frequencies of deletions were prominently higher than those of insertions on both genome and population levels. Most of the indels led to in frame mutations with multiples of three and majorly occurred in non-domain regions, indicating functional constraint or tolerance of the indels. All indel genes showed higher expression levels than non-indel genes during sex reversal. Slide window analysis of global expression levels in gonads showed a significant positive correlation with indel density in the genome. Moreover, indel genes were evolutionarily conserved and evolved slowly compared to non-indel genes. Notably, population genetic structure of indels revealed divergent evolution of Monopterus population, as bottleneck effect of biogeographic isolation by Taiwan Strait, China.

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.

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.

Effect of ENSO events on larval and juvenile duration and transport of Japanese eel (Anguilla japonica).

Spawning ground of Japanese eel (Anguilla japonica) is located near the West Mariana Ridge seamount. The species travels through the North Equatorial Current (NEC) and then enters the Kuroshio Current (KC) on the migration toward East Asian growth habitats. Therefore, El Niño-Southern Oscillation (ENSO) events serve as the potentially important drivers of interannual variability across the equatorial Pacific. Because the NEC bifurcation and salinity profiles are related to ENSO events, we investigated the influence of locations of the NEC bifurcation and salinity front on the success of larval entry to the KC by numerically modeling particle transport in ocean currents from 1972 to 2013 and possible effects on the size of glass eels at continental recruitment and, via otolithometry on the duration of larval migration. Circulation and hydrography used for particle tracking were obtained from the results of the Model for Interdisciplinary Research on Climate (MIROC) high-resolution forecasting experiment. Our results demonstrated that during El Niño years, (1) the southward movement of the salinity front might cause the larvae to experience slower currents and (2) the northward movement of the NEC bifurcation might broaden the separation between their spawning ground and NEC bifurcation, thus prolonging the time needed for the larvae to enter the KC from their spawning ground, because of which the duration of entrainment in the water column and body size increase when eels reach estuarine waters. In addition, this might cause more water to flow into the Mindanao Current (MC), leading to a decline in the rate at which larvae get entrained into the KC.

Physical and biological roles of mesoscale eddies in Japanese eel larvae dispersal in the western North Pacific Ocean.

The physical and biological roles of mesoscale eddies in Japanese eel larvae dispersal are investigated using a three-dimensional (3D) particle-tracking method, with a focus on the Subtropical Counter Current eddies of the western North Pacific Ocean. Virtual eel larvae (v-larvae) movements depends on the 3D ocean currents and active swimming behavior, including vertical swimming (diel vertical migration), horizontal directional swimming toward settlement habitat, and horizontal swimming toward available food. V-larvae are able to remain in eddies passively due to mesoscale eddy nonlinearity and/or actively due to attraction to rich food supplies. Thus, both physical trapping and biological attraction to food contribute to the retention of v-larvae in eddies. Physical trapping dominates the retention of v-larvae whose swimming speeds are slower than the eddy propagation speed, whereas biological food attraction prevails in the retention of v-larvae swimming faster than eddy propagation. Food availability differs between warm (anti-cyclonic) and cold (cyclonic) eddies, with the latter providing a richer food supply. Fish larvae that are retained for longer durations in cold eddies (shorter durations in warm eddies) are able to obtain more food and potentially grow faster, which enhances survival rates.

Seaward Migration Routes of Indigenous Eels, Anguilla japonica, A. marmorata, and A. bicolor pacifica, via Satellite Tags.

Shih-Chong Chen, Ching-Rong Chang, and Yu-San Han (2018) The spawning migration of the Japanese eel, Anguilla japonica, along the western Mariana Ridge is considered one of the longest animal migrations. To establish an appropriate conservation policy for this species, a complete life cycle study is required, but the migratory routes of anguillid eels remain poorly documented. Anguilla japonica, A. marmorata, and A. bicolor pacifica are three common eel species in Taiwan. The present study used pop-up archival transmitting tags to track their movements during the oceanic phase of their migration. Twenty silver eels (farmed and wild) were tagged and released near the Gueishan and Liuchiu Islands. All three species in the Gueishan Island group were entrained in the Kuroshio Current, and pop-up tag data showed that, after 23 days, the farthest distance travelled was approximately 1,500 km, to the southern waters of Yakushima, Japan. In comparison, eels released near Liuchiu Island moved in the opposite direction, which suggests that they were influenced by the recirculation in the southwestern waters of Taiwan. The migration speed of eels was estimated to be between 12.9 km/d and 65.2 km/d. Several eels exhibited diel vertical migration behavior, moving into deeper water during the daytime and ascending into shallower waters at night. The farmed eels exhibited similar patterns of diel vertical migration to wild eels. The results of this study suggest that eels from Taiwan travel through the Kuroshio Current during their early migration, irrespective of species and source. Of importance, farmed eels appeared to behave similar to wild ones following artificial release. This information provides important rationale for releasing cultivated eels for resource enrichment programs.

A combination mode of climate variability responsible for extremely poor recruitment of the Japanese eel (Anguilla japonica).

Satellite data and assimilation products are used to investigate fluctuations in the catch of Japanese eel (Anguilla japonica) in eastern Asian countries. It has been reported that the salinity front has extended farther south, which has shifted the eel's spawning grounds to a lower latitude, resulting in smaller eel catches in 1983, 1992, and 1998. This study demonstrates that interannual variability in the eel catch is strongly correlated with the combination mode (C-mode), but not with the El Niño-Southern Oscillation. These eels continue to spawn within the North Equatorial Current (NEC), but the salinity front shifts south during a canonical El Niño. On the other hand, the spawning grounds accompanied by the salinity front extend farther south during the C-mode of climate variability, and eel larvae fail to join the nursery in the NEC, resulting in extremely poor recruitment in East Asia. We propose an appropriate sea surface temperature index to project Japanese eel larval catch.

Batch-like Arrival Waves of Glass Eels of Anguilla japonica in Offshore Waters of Taiwan.

Yu-San Han, Chau-Ron Wu, and Yoshiyuki Iizuka (2016) The larval stage of Anguilla japonica includes a long dispersal time over a long distance. In theory, the larvae should be distributed evenly throughout their transportation route when using both the NEC and Kuroshio, but the hypothesized new moon spawning of mature eels should lead to recruited glass eels exhibiting batch-like arrival waves, with a one-month-long cycle. However, environmental disturbances could mask the expected batch-like waves of glass eel recruitment. Thus, this phenomenon is best observed in glass eels collected from offshore waters, which are closer to the spawning site and less disturbed by these environmental factors. The offshore area of Yilan, Taiwan, is a suitable place to observe the arrival dynamics of the A. japonica glass eel. In this area, batch-like waves of glass eel arrival of A. japonica were observed, with peaks occurring between the last quarter and first quarter lunar periods, with a near one-month periodicity. No arrival peaks were found during the full moon period, suggesting that the glass eels exhibit light-avoidance behavior. Furthermore, all of the batches of arrivals were in the early pigmentation stage and similar in age (around 150-160 days), suggesting that they are likely a new arrival cohort. The tracer simulation showed that the mean tracer drift time, from the presumed spawning site to Yilan, was 155 ± 19.8 days. The observed batch-like arrival waves of glass eels in the offshore waters of Taiwan support the "New Moon Hypothesis," which suggests that there is synchronized spawning behavior of the eels during the new moon period.

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.

Determining anti-betanodavirus compounds through a GF-1 cell-based screening platform.

Betanodavirus is a highly contagious pathogen, responsible for severe losses incurred in the aquaculture industry. Currently, there are no commercially available antivirals against the virulence observed during very early stages of fish larvae development. Therefore, we developed a novel GF-1 (grouper fin cell) cell viability-based screening assay to facilitate the discovery of an anti-betanodavirus agent. The assay conditions were optimized and the robustness of the assay was confirmed by a Z' factor value ranging from 0.7 to 0.94. After screening a library of 2000 small molecule compounds, 43 compounds with a virus inhibition capacity of ⩾55% were identified. A cytochrome P450 inhibitor, proadifen hydrochloride, was validated with an EC50 value of 6.48µM and a CC50 value of 20.63µM. This compound inhibited the amplification of viral RNA by 99.68% 5days post-infection. Surprisingly, we found that 18 of 43 compounds act as neurotransmitter agents. These findings indicate a novel way of investigating the infection mechanism of betanodavirus, and suggest potential candidates for an anti-betanodavirus drug.

Zinc finger protein 219-like (ZNF219L) and Sox9a regulate synuclein-γ2 (sncgb) expression in the developing notochord of zebrafish.

Zebrafish synuclein-γ2 (sncgb) has been reported to be expressed specifically in the notochord. However, the mechanism by which the sncgb gene promoter is regulated has not been described. In this paper, we demonstrate that Zinc finger protein 219-like (ZNF219L) and sox9a are involved in the regulation of sncgb gene expression. Furthermore, we observed that over-expression of both ZNF219L and Sox9a resulted in increased sncgb expression. In addition, ZNF219L is physically associated with Sox9a, and simultaneous morpholino knockdown of znf219L and sox9a caused a synergistic decrease of sncgb expression in the notochord. Taken together, our results reveal that coordination of ZNF219L with Sox9a is involved in the regulation of notochord-specific expression of sncgb.