Environmental DNA metabarcoding reveals distinct fish assemblages supported by seagrass (Zostera marina and Zostera pacifica) beds in different geographic settings in Southern California.

Seagrass beds are disappearing at a record pace despite their known value to our oceans and coastal communities. Simultaneously, our coastlines are under the constant pressure of climate change which is impacting their chemical, physical and biological characteristics. It is thus pertinent to evaluate and record habitat use so we can understand how these different environments contribute to local biodiversity. This study evaluates the assemblages of fish found at five Zostera beds in Southern California using environmental DNA (eDNA) metabarcoding. eDNA is a powerful biodiversity monitoring tool that offers key advantages to conventional monitoring. Results from our eDNA study found 78 species of fish that inhabit these five beds around Southern California representing embayment, open coastal mainland and open coastal island settings. While each bed had the same average number of species found throughout the year, the composition of these fish assemblages was strongly site dependent. There were 35 fish that were found at both open coast and embayment seagrass beds, while embayment seagrass sites had 20 unique fish and open coast sites had 23 unique fish. These results demonstrate that seagrass fish assemblages are heterogenous based on their geographic positioning and that marine managers must take this into account for holistic conservation and restoration efforts.

Plumbing the depths with environmental DNA (eDNA): Metabarcoding reveals biodiversity zonation at 45-60 m on mesophotic coral reefs.

Mesophotic coral ecosystems (MCEs) are tropical reefs found at depths of ~30-150 m, below the region most heavily impacted by heat stress and other disturbances. Hence, MCEs may serve as potential refugia for threatened shallow reefs, but they also harbour depth-endemic fauna distinct from shallow reefs. Previous studies have characterized biodiversity patterns along depth gradients, but focussed primarily on conspicuous taxa (fishes, corals, etc.). Environmental DNA (eDNA) metabarcoding offers a more holistic approach to assess biodiversity patterns across the tree of life. Here, we use three metabarcoding assays targeting fishes (16S rRNA), eukaryotes (18S rDNA) and metazoans (COI) to assess biodiversity change from the surface to ~90 m depth across 15-m intervals at three sites within the Hawaiian Archipelago. We observed significant community differences between most depth zones, with distinct zonation centred at 45-60 m for eukaryotes and metazoans, but not for fishes. This finding may be attributable to the higher mobility of reef fishes, although methodological limitations are likely a contributing factor. The possibility for MCEs to serve as refugia is not excluded for fishes, but invertebrate communities >45 m are distinct, indicating limited connectivity for the majority of reef fauna. This study provides a new approach for surveying biodiversity on MCEs, revealing patterns in a much broader context than the limited-taxon studies that comprise the bulk of our present knowledge.

Hypothalamus-pituitary-interrenal (HPI) axis signaling in Atlantic sturgeon (Acipenser oxyrinchus) and sterlet (Acipenser ruthenus).

In vertebrates, the hypothalamic-pituitaryadrenal/interrenal (HPA/HPI) axis is a highly conserved endocrine axis that regulates glucocorticoid production via signaling by corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Once activated by ACTH, Gs protein-coupled melanocortin 2 receptors (Mc2r) present in corticosteroidogenic cells stimulate expression of steroidogenic acute regulatory protein (Star), which initiates steroid biosynthesis. In the present study, we examined the tissue distribution of genes involved in HPI axis signaling and steroidogenesis in the Atlantic sturgeon (Acipenser oxyrinchus) and provided the first functional characterization of Mc2r in sturgeon. Mc2r of A. oxyrinchus and the sterlet sturgeon (Acipenser ruthenus) are co-dependent on interaction with the melanocortin receptor accessory protein 1 (Mrap1) and highly selective for human (h) ACTH over other melanocortin ligands. A. oxyrinchus expresses key genes involved in HPI axis signaling in a tissue-specific manner that is indicative of the presence of a complete HPI axis in sturgeon. Importantly, we co-localized mc2r, mrap1, and star mRNA expression to the head kidney, indicating that this is possibly a site of ACTH-mediated corticosteroidogenesis in sturgeon. Our results are discussed in the context of other studies on the HPI axis of basal bony vertebrates, which, when taken together, demonstrate a need to better resolve the evolution of HPI axis signaling in vertebrates.

Molecular Characterization, Tissue Distribution Profile, and Nutritional Regulation of acsl Gene Family in Golden Pompano (Trachinotus ovatus).

Long chain acyl-coA synthase (acsl) family genes activate the conversion of long chain fatty acids into acyl-coA to regulate fatty acid metabolism. However, the evolutionary characteristics, tissue expression and nutritional regulation of the acsl gene family are poorly understood in fish. The present study investigated the molecular characterization, tissue expression and nutritional regulation of the acsl gene family in golden pompano (Trachinotus ovatus). The results showed that the coding regions of acsl1, acsl3, acsl4, acsl5 and acsl6 cDNA were 2091 bp, 2142 bp, 2136 bp, 1977 bp and 2007 bp, encoding 697, 714, 712, 659 and 669 amino acids, respectively. Five acsl isoforms divided into two branches, namely, acsl1, acsl5 and acsl6, as well as acsl3 and acsl4. The tissue expression distribution of acsl genes showed that acsl1 and acsl3 are widely expressed in the detected tissues, while acsl4, acsl5 and acsl6 are mainly expressed in the brain. Compared to the fish fed with lard oil diets, the fish fed with soybean oil exhibited high muscular C18 PUFA contents and acsl1 and acsl3 mRNA levels, as well as low muscular SFA contents and acsl4 mRNA levels. High muscular n-3 LC-PUFA contents, and acsl3, acsl4 and acsl6 mRNA levels were observed in the fish fed with fish oil diets compared with those of fish fed with lard oil or soybean oil diets. High n-3 LC-PUFA levels and DHA contents, as well as the acsl3, acsl4 and acsl6 mRNA levels were exhibited in the muscle of fish fed diets with high dietary n-3 LC-PUFA levels. Additionally, the muscular acsl3, acsl4 and acsl6 mRNA expression levels, n-3 LC-PUFA and DHA levels were significantly up-regulated by the increase of dietary DHA proportions. Collectively, the positive relationship among dietary fatty acids, muscular fatty acids and acsl mRNA, indicated that T. ovatus Acsl1 and Acsl3 are beneficial for the C18 PUFA enrichment, and Acsl3, Acsl4 and Acsl6 are for n-3 LC-PUFA and DHA enrichment. The acquisition of fish Acsl potential function in the present study will play the foundation for ameliorating the fatty acids nutrition in farmed fish products.

The effects of dietary supplementation of α-lipoic acid on the growth performance, antioxidant capacity, immune response, and disease resistance of northern snakehead, Channa argus.

The study was the first time to explore the positive effects of α-LA on growth performance, antioxidant capability, immunity, and disease resistance of northern snakehead (Channa argus). Five hundred and forty northern snakehead fish (initial body weight: 8.74 ± 0.12 g (mean ± SE)) were randomly allocated into six groups with three replicates each. Six diets supplemented with α-LA at doses of 0 (CON), 300 (LA300), 600 (LA600), 900 (LA900), 1200 (LA1200), and 1500 (LA1500) mg/kg were fed to northern snakehead for 8 weeks. The results demonstrated that, when compared with the control group, optimal dietary α-LA increased the weight gain (WG), protein efficiency ratio (PER), and specific growth rate (SGR) and reduced the feed conversion ratio (FCR) of the fish (P < 0.05). Also, optimal dietary α-LA enhanced the immune-related parameters and antioxidant enzyme parameters levels in the head kidney, spleen, and liver of northern snakehead (P < 0.05). Dietary α-LA upregulated the mRNA expression levels of anti-inflammatory cytokines (il10 and tgfß) and antioxidant related genes (gst, gsh-px, gr and Cu/Zn sod), down-regulated the pro-inflammatory cytokines (il1ß, il8, il12 and tnfα) mRNA levels in the liver, spleen and head kidney of the northern snakehead (P < 0.05). The above results demonstrated that optimal dietary α-LA showed enhancement effects on the growth, antioxidant and anti-inflammatory capability, and immune response of northern snakehead. The survival rates in all α-LA treatments were significantly raised after the challenge with Aeromonas veronii (P < 0.05). Based on the quadratic regression analysis of WG, GSH-Px, LYS, and il1ß, the optimal dietary α-LA levels were estimated to be 737.0, 775.0, 890.0, and 916.7 mg/kg, respectively. Considering the overall responses in growth performance, antioxidant status, immune response, and inflammatory factors, the recommended dose of α-LA in the diet of fish is 737.0-916.7 mg/kg.

Analyzing the Hypothalamus/Pituitary/Interrenal axis of the neopterygian fish, Lepisosteus oculatus: Co-localization of MC2R, MC5R, MRAP1, and MRAP2 in interrenal cells.

RT-PCR analysis indicated that steroidogenic tissues are located along the length of the kidney of the neopterygian fish, Lepisosteus oculatus (spotted gar; g). However, RT-PCR analysis of the distribution of mc2r mRNA and mrap1 mRNA, critical components of the gar hypothalamus/pituitary/interrenal (HPI) axis, was only associated with the anterior and medial regions of the kidney. Steroidogenic cells were designated as interrenal cells that possess star mRNA (in situ hybridization) and lipid vesicles (histological analysis) within the kidney. RT-PCR also detected mc5r mRNA along the length of the tissues associated with the kidney. In situ hybridization analysis of the putative interrenal cells revealed co-expression of mc2r, and mc5r mRNAs in the same steroidogenic cells. Co-expression of gar Mc2r (gMc2r) and Mrap1 (gMrap1) in Chinese Hamster Ovary (CHO) cells stimulated with ACTH(1-24) resulted in activation with an EC50 value of 1.0 × 10-11M +/- 4.6 × 10-11); whereas stimulation of CHO cells co-expressed with gar Mc5r (gMc5r) and gMrap1 and stimulated with ACTH(1-24) resulted in an EC50 value that was 3 orders of magnitude lower (2.1 × 10-8 M +/- 3.5 × 10-9). Interesting, when CHO cells were co-transfected with gMc2r, gMc5r, and gMrap1 there was a decline in activation as measured by the Vmax values for CHO cells stimulated with either ACTH(1-24) or α-MSH. These results suggest that some interaction may occur between gMc2r and gMc5r when both receptors are expressed in the same cells. Phylogenetic and selection pressure analyses of vertebrate mc2r and mc5r genes concluded that the two genes are evolving at different rates after duplication from a proposed common ancestral gene.

Adaptations and Diversity of Antarctic Fishes: A Genomic Perspective.

Antarctic notothenioid fishes are the classic example of vertebrate adaptive radiation in a marine environment. Notothenioids diversified from a single common ancestor ∼22 Mya to between 120 and 140 species today, and they represent ∼90% of fish biomass on the continental shelf of Antarctica. As they diversified in the cold Southern Ocean, notothenioids evolved numerous traits, including osteopenia, anemia, cardiomegaly, dyslipidemia, and aglomerular kidneys, that are beneficial or tolerated in their environment but are pathological in humans. Thus, notothenioids are models for understanding adaptive radiations, physiological and biochemical adaptations to extreme environments, and genetic mechanisms of human disease. Since 2014, 16 notothenioid genomes have been published, which enable a first-pass holistic analysis of the notothenioid radiation and the genetic underpinnings of novel notothenioid traits. Here, we review the notothenioid radiation from a genomic perspective and integrate our insights with recent observations from other fish radiations.

Identification, tissue distribution, and anorexigenic effect of amylin in Siberian sturgeon (Acipenser baeri).

Amylin is a 37-amino acid polypeptide that has been found to be involved in feeding regulation in some mammals, birds, and goldfish. We cloned amylin of Siberian sturgeon and detected its distribution pattern in 15 tissues. The expression levels in the periprandial period (pre-and post-feeding), the changes in the food intake, and the expression levels of related appetite factors after the intraperitoneal injection of amylin were detected. The expression of amylin was found to be the highest in the hypothalamus. Compared with 1 h pre-feeding, the expression levels of amylin in the hypothalamus and duodenum were increased significantly 1 h post-feeding. Compared with the control group (saline), intraperitoneal injection of 50 ng/g, 100 ng/g, and 200 ng/g of amylin significantly inhibited food intake at 1 h post injection, but not at 3 h and 6 h. The injection of 50 ng/g, 100 ng/g, and 200 ng/g amylin significantly inhibited the cumulative feed. After 1 h of 50 ng/g amylin injection, the levels of MC4R and somatostatin in the hypothalamus increased significantly, while the levels of amylin and NPY decreased significantly. The levels of CCK in the valvular intestine were increased significantly. Insulin in the duodenum was also increased significantly, but there was no significant change in ghrelin in the duodenum. These results show that amylin inhibits feeding in Siberian sturgeon by down-regulating the appetite-stimulating factor NPY and up-regulating the appetite-suppressing factors somatostatin, MC4R, CCK, and insulin. This study provides a theoretical basis for studying the feeding function and action mechanisms of amylin in Siberian sturgeon.

Environmental DNA preserved in marine sediment for detecting jellyfish blooms after a tsunami.

Environmental DNA (eDNA) can be a powerful tool for detecting the distribution and abundance of target species. This study aimed to test the longevity of eDNA in marine sediment through a tank experiment and to use this information to reconstruct past faunal occurrence. In the tank experiment, juvenile jack mackerel (Trachurus japonicus) were kept in flow-through tanks with marine sediment for two weeks. Water and sediment samples from the tanks were collected after the removal of fish. In the field trial, sediment cores were collected in Moune Bay, northeast Japan, where unusual blooms of jellyfish (Aurelia sp.) occurred after a tsunami. The samples were analyzed by layers to detect the eDNA of jellyfish. The tank experiment revealed that after fish were removed, eDNA was not present in the water the next day, or subsequently, whereas eDNA was detectable in the sediment for 12 months. In the sediment core samples, jellyfish eDNA was detected at high concentrations above the layer with the highest content of polycyclic aromatic hydrocarbons, reflecting tsunami-induced oil spills. Thus, marine sediment eDNA preserves a record of target species for at least one year and can be used to reconstruct past faunal occurrence.

Genotoxicity detection of oil-containing drill cuttings by Comet assay based on a demersal marine fish Mugilogobius chulae.

An enormous amount of oil-containing drill cuttings have been produced by the marine oil and gas industry. The environmental impacts of discharged drilling waste have been extensively studied. However, there is still an urgent need to develop alternative methods to identify the genotoxicity of untreated and treated drill waste in a timely manner before it is discharged. In this study, we developed a relatively rapid, sensitive, and accurate genotoxicity-detection method using Comet assay and the marine benthic goby Mugilogobius chulae. This goby is sensitive to a standard toxicant mitomycin C (MMC). The optimal exposure period for genotoxicity detection using M. chulae was determined. Three genotoxic indices (tail length (TL), tail DNA content (TD), and tail moment (TM)) were used to assess the effectiveness of high-temperature treatment of oil-contaminated waste. Untreated oil-containing drill cuttings exhibited the highest genotoxicity to goby cells. Genotoxicity was dramatically reduced after thermal treatment of drill cuttings at 350 °C and 500 °C. TD and TM exhibited significant correlation with the concentration of total petroleum hydrocarbons (TPHs)/total polycyclic aromatic hydrocarbons (PAHs) according to Pearson and Mantel correlation analyses (P values were <0.05). Using redundancy analysis (RDA) and variation partition analysis (VPA), the genotoxic effects of the drill cuttings were ascribed to total alkanes and specific groups of PAHs. In conclusion, this newly established biological model has the potential to be widely used to detect the genetic damage of untreated or treated oil-containing drill cuttings discharged into the marine environment.

Comparative genomic analysis of human GLI2 locus using slowly evolving fish revealed the ancestral gnathostome set of early developmental enhancers.

BACKGROUND: The zinc finger-containing transcription factor Gli2, is a key mediator of Hedgehog (Hh) signaling and participates in embryonic patterning of various organs including the central nervous system (CNS) and limbs. Abnormal expression of Gli2 can impede the transcription of Hh target genes through disruption of proper balance between Gli2 and Gli3 functions. Therefore, delineation of enhancers that are required for complementary roles of Glis would allow the interrogation of those pathogenic variants that cause gene dysregulation, and a corresponding abnormal phenotype. Previously, we reported tissue-specific enhancers for Gli family including Gli2 through direct tetrapod-teleost comparisons. RESULTS: Here, we employed the sequence alignments of slowly evolving spotted gar and elephant shark and have identified six novel conserved noncoding elements in human GLI2 containing locus. Zebrafish-based transgenic assays revealed that combined action of these autonomous CNEs reflects many aspects of Gli2 specific endogenous transcriptional activity, including CNS and pectoral fins. CONCLUSION: Taken together with our previous findings, this study suggests that Hh-signaling controlled deployment of Gli2 activity in embryonic patterning arose in the common ancestor of gnathostomes. These GLI2 specific cis-regulatory modules will help to identify DNA variants that probably reside outside of coding intervals and are associated with congenital anomalies.

The reproductive regulation of LPXRFa and its receptor in the hypothalamo-pituitary-gonadal axis of the spotted scat (Scatophagus argus).

Gonadotropin-inhibitory hormone (GnIH) plays a critical role in regulating gonadotropin-releasing hormone (GnRH), gonadotropin hormone (GtH), and steroidogenesis. The Lpxrfa (the piscine ortholog of GnIH) system has been found to regulate fish reproduction. To gain insight into the role of Lpxrfa in the regulation of spotted scat (Scatophagus argus) reproduction, spotted scat Lpxrfa (ssLpxrfa), and its receptor (ssLpxrfa-r) were cloned and analyzed. Tissue distribution and expression patterns at the hypothalamo-pituitary-gonadal axis (HPG axis) of sslpxrfa and sslpxrfa-r mRNA were also investigated during gonadal development of spotted scat. The open reading frame (ORF) of the sslpxrfa was 606 bp encoding 201 amino acids and includes a putative signal peptide and two mature ssLpxrfa peptides with LPXRFamide motif at their C-terminus. The sslpxrfa-r ORF was 1449 bp encoding 482 amino acids and contracted a seven-hydrophobic transmembrane (TM) domain structure. The tissue distribution showe d that the sslpxrfa was highly expressed in hypothalami, gill, and the gonads. In addition, sslpxrfa-r was highly expressed in hypothalami, pituitaries, and the gonads. Quantitative real-time polymerase chain reaction (qPCR) revealed that sslpxrfa had the highest expression in the hypothalami and pituitaries, and the lowest expression in the gonads in stage V. During gonadal development, the expression of sslpxrfa-r was gradually increased in the hypothalami but reduced in the gonads. However, no obvious trend was observed in the pituitaries. The expression of sslpxrfa and sslpxrfa-r decreased significantly after injection with 17ß-estradiol (E2). However, the expression of both sslpxrfa and sslpxrfa-r was not changed after injection with 17α-methyltestosterone(17α-MT) in the hypothalami. In addition, no changes were observed in the expression of fshß and lhß in the pituitaries after injecting ssLpxrfa-1. However, ssLpxrfa-2 could downregulate the expression of sbgnrh and fshß in the hypothalami and pituitaries, respectively. Taken together, these findings suggested that ssLpxrfa may participate in E2 feedback in reproduction and regulate the reproductive axis of spotted scat.

Urban stormwater and crude oil injury pathways converge on the developing heart of a shore-spawning marine forage fish.

Understanding how aquatic organisms respond to complex chemical mixtures remains one of the foremost challenges in modern ecotoxicology. Although oil spills are typically high-profile disasters that release hundreds or thousands of chemicals into the environment, there is growing evidence for a common adverse outcome pathway (AOP) for the vulnerable embryos and larvae of fish species that spawn in oiled habitats. Molecular initiating events involve the disruption of excitation-contraction coupling in individual cardiomyocytes, which then dysregulate the form and function of the embryonic heart. Phenanthrenes and other three-ring (tricyclic) polycyclic aromatic hydrocarbons (PAHs) are key drivers for this developmental cardiotoxicity and are also relatively enriched in land-based urban runoff. Similar to oil spills, stormwater discharged from roadways and other high-traffic impervious surfaces contains myriad contaminants, many of which are uncharacterized in terms of their chemical identity and toxicity to aquatic organisms. Nevertheless, given the exceptional sensitivity of the developing heart to tricyclic PAHs and the ubiquitous presence of these compounds in road runoff, cardiotoxicity may also be a dominant aspect of the stormwater-induced injury phenotype in fish early life stages. Here we assessed the effects of traffic-related runoff on the embryos and early larvae of Pacific herring (Clupea pallasii), a marine forage fish that spawns along the coastline of western North America. We used the well-characterized central features of the oil toxicity AOP for herring embryos as benchmarks for a detailed analysis of embryolarval cardiotoxicity across a dilution gradient ranging from 12 to 50% stormwater diluted in clean seawater. These injury indicators included measures of circulatory function, ventricular area, heart chamber looping, and the contractility of both the atrium and the ventricle. We also determined tissue concentrations of phenanthrenes and other PAHs in herring embryos. We find that tricyclic PAHs are readily bioavailable during cardiogenesis, and that stormwater-induced toxicity is in many respects indistinguishable from canonical crude oil toxicity. Given the chemical complexity of urban runoff, non-tricyclic PAH-mediated mechanisms of developmental toxicity in fish remain likely. However, from the standpoint of managing wild herring populations, our results suggest that stormwater-driven threats to individual survival (both near-term and delayed mortality) can be understood from decades of past research on crude oil toxicity. Moreover, Pacific herring embryos are promising sentinels for water quality monitoring in nearshore marine habitats, as in situand sensitive indicators of both toxic runoff and the effectiveness of pollution reduction efforts such as green stormwater infrastructure.

Identification, tissue distribution, periprandial expression, and anorexigenic effect of spexin in Siberian sturgeon, Acipenser baeri.

Spexin (Spx), an endogenous peptide, is considered to be a neuropeptide. In a few fish and mammals, it has been proved to play a role in the regulation of animal feeding. However, the possible mechanisms of spexin regulating food intake are mostly blurry in vertebrates including Siberian sturgeon. In this study, firstly, the coding sequence of spexin cDNA was cloned and sequenced in Siberian sturgeon. Then, we detected that spexin mRNA was widely expressed in the hypothalamus, gastrointestinal tract, and liver, with the highest expression in the hypothalamus. The expression of spexin mRNA in the hypothalamus was significantly increased after food intake. At 1 h, 3 h, and 6 h after injection, the food intake in the spexin group (0.10, 0.30, and 0.90 µg/g BW) was significantly lower than that in the saline group. Moreover, compared with the saline group, the mRNA expression of anorectic nucb2, cart, ucn3, and pyy in the hypothalamus was significantly upregulated and orectic npy was significantly downregulated at 1 h after spexin injection; in the stomach, the mRNA expression of nucb2 and pyy was significantly upregulated. All in all, these results provide evidence for the anorexic effect of spexin on Siberian sturgeon.

Characterization of two kcnk3 genes in rabbitfish (Siganus canaliculatus): Molecular cloning, distribution patterns and their potential roles in fatty acids metabolism and osmoregulation.

KCNK3 is a two-pore-domain (K2P) potassium channel involved in maintaining ion homeostasis, mediating thermogenesis, controlling breath and modulating electrical membrane potential. Although the functions of this channel have been widely described in mammals, its roles in fishes are still rarely known. Here, we identified two kcnk3 genes from the euryhaline rabbitfish (Siganus canaliculatus), and their roles related to fatty acids metabolism and osmoregulation were investigated. The open reading frames of kcnk3a and kcnk3b were 1203 and 1176 bp in length, encoding 400 and 391 amino acids respectively. Multiple sequences alignment and phylogenetic analysis revealed that the two isotypes of kcnk3 were extensively presented in fishes. Quantitative real-time PCRs indicated that both genes were widely distributed in examined tissues but showed different patterns. kcnk3a primary distributed in adipose, eye, heart, and spleen tissues, while kcnk3b was mainly detectable in heart, kidney, muscle and spleen tissues. In vivo experiments showed that fish fed diets with fish oil as dietary lipid (rich in long chain polyunsaturated fatty acids, LC-PUFA) induced higher mRNA expression levels of kcnk3 genes in comparison with fish fed with plant oil diet at two different salinity environments (32 and 15‰). Meanwhile, the expression levels of kcnk3 genes were higher in seawater (32‰) than that in brackish water (15‰) when fishes were fed with both types of feeds. In vitro experiments with rabbitfish hepatocytes showed that LC-PUFA significantly improved hepatic kcnk3a expression level compared with treatment of linolenic acid. These results suggest that two kcnk3 genes are widely existed and they might be functionally related to fatty acids metabolism and osmoregulation in the rabbitfish.

Comparing the efficiency of open and enclosed filtration systems in environmental DNA quantification for fish and jellyfish.

Water sampling and filtration of environmental DNA (eDNA) analysis have been performed by several different methods, and each method may yield a different species composition or eDNA concentration. Here, we investigated the eDNA of seawater samples directly collected by SCUBA to compare two widely used filtration methods: open filtration with a glass filter (GF/F) and enclosed filtration (Sterivex). We referred to biomass based on visual observation data collected simultaneously to clarify the difference between organism groups. Water samples were collected at two points in the Sea of Japan in May, September and December 2018. The respective samples were filtered through GF/F and Sterivex for eDNA extraction. We quantified the eDNA concentration of five fish and two cnidarian species by quantitative polymerase chain reaction (qPCR) using species-specific primers/probe sets. A strong correlation of eDNA concentration was obtained between GF/F and Sterivex; the intercepts and slopes of the linear regression lines were slightly different in fish and jellyfish. The amount of eDNA detected using the GF/F filtration method was higher than that detected using Sterivex when the eDNA concentration was high; the opposite trend was observed when the eDNA concentration was relatively low. The concentration of eDNA correlated with visually estimated biomass; eDNA concentration per biomass in jellyfish was approximately 700 times greater than that in fish. We conclude that GF/F provides an advantage in collecting a large amount of eDNA, whereas Sterivex offers superior eDNA sensitivity. Both filtration methods are effective in estimating the spatiotemporal biomass size of target marine species.

Dissecting the Interaction Deficiency of a Cartilaginous Fish Digestive Lipase with Pancreatic Colipase: Biochemical and Structural Insights.

A full-length cDNA encoding digestive lipase (SmDL) was cloned from the pancreas of the smooth-hound (Mustelus mustelus). The obtained cDNA was 1350 bp long encoding 451 amino acids. The deduced amino acid sequence has high similarity with known pancreatic lipases. Catalytic triad and disulphide bond positions are also conserved. According to the established phylogeny, the SmDL was grouped with those of tuna and Sparidae lipases into one fish digestive lipase cluster. The recently purified enzyme shows no dependence for bile salts and colipase. For this, the residue-level interactions between lipase-colipase are yet to be clearly understood. The structural model of the SmDL was built, and several dissimilarities were noticed when analyzing the SmDL amino acids corresponding to those involved in HPL binding to colipase. Interestingly, the C-terminal domain of SmDL which holds the colipase shows a significant role for colipase interaction. This is apt to prevent the interaction between fish lipase and the pancreatic colipase which and can provide more explanation on the fact that the classical colipase is unable to activate the SmDL.

Melanocortin-4 receptor regulation of reproductive function in black rockfish (Sebastes schlegelii).

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor with multiple functions in mammals. However, the functions of MC4R in fish have not been investigated extensively. The purpose of this study was to determine potential regulation of reproduction by the MC4R. We cloned the black rockfish MC4R and analyzed its tissue distribution and function. The results showed that black rockfish mc4r cDNA consisted of 981 nucleotides encoding a protein of 326 amino acids. The quantitative PCR data showed that mc4r mRNA was primarily expressed in the brain, gonad, stomach and intestine. In the brain, mc4r was found to be primarily located in the hypothalamus. Both α-MSH and ß-MSH increased gnih expression and decreased sgnrh and cgnrh expression (P < 0.05). α-MSH and ß-MSH had opposite effects on kisspeptin expression. In contrast, α-MSH and ß-MSH increased the expression of cyp11, cyp19, 3ß-hsd and star. In summary, our study shows that MC4R in black rockfish might regulate reproductive function and that the effects of α-MSH and ß-MSH might differ.

Influence of Olive Extracts on the Expression of Genes Involved in Lipid Metabolism in Medaka Fish.

Aims. To assess the possible effect of polyphenol-rich olive extracts on lipid metabolism in medaka fish by quantifying the expression of lipogenic and lipolytic genes. Materials and methods. Adult medaka fish were maintained in tanks for five days with five extracts at 0.01% in water, causing obesity through a diet rich in carbohydrates, with a control group maintained in water with a normal diet. The extracts contained polyphenols ranging between 7 and 116 mg/g (oleuropein, hydroxytyrosol) with an antioxidant power of 2-13 mmol of 2,4,6-tri(2-pyridyl)-1,3,5-triazine/100 g. After five days, the fish were sacrificed and the hepatic mRNA and its complementary DNA were extracted by reverse transcription. Complementary DNAs were quantified for three lipolytic and three lipogenic genes by real-time PCR. The relative gene expression was calculated from the amplification curves in reference to the control group. Results. The expression of genes involved in lipolysis, including peroxisome proliferator-activated receptor-±, acyl-CoA oxidase 1, and carnitine palmitoyltransferase 1, were clearly decreased in fish subjected to an obesogenic diet, and this situation could not be reversed in fish maintained with polyphenol-rich extracts. In contrast, lipogenic fatty acid synthase, acetyl-CoA carboxylase 1, and sterol regulatory element-binding protein 1 genes increased considerably with the obesogenic diet and reverted to the normal state with the olive extracts. The effect was not dependent on the total polyphenol content, the specific oleuropein or hydroxytyrosol concentration, or the antioxidant power, suggesting a synergistic effect. Conclusion. Olive polyphenols, acting as anti-lipogenic agents, have a positive effect on lipid metabolism, but their mechanism in each gene is different according to the extract, which supports synergistic mechanisms with the different proportions of polyphenols and accompanying phytochemicals in each extract.