Effects of dietary supplementation of Gracilaria lemaneiformis-derived sulfated polysaccharides on the growth, antioxidant capacity, and innate immunity of rabbitfish (Siganus canaliculatus).

The dietary supplementation of red seaweed-derived polysaccharides has been shown to be beneficial to fish and shellfish aquaculture. However, the function of red seaweed (Gracilaria lemaneiformis)-extracted polysaccharide (GLP) on the health status of rabbitfish (Siganus canaliculatus) is still unknown. This study explored the influences of GLP on growth performance, antioxidant activity, and immunity of rabbitfish. Herein, the fish were fed commercial pelleted feed incorporated with the diverse amount of GLP: 0 (control), 0.10 (GLP0.10), and 0.15 g kg-1 (GLP0.15) for 60 days. The results demonstrated that dietary GLP0.15 significantly elevated FBW and WG, while feed utilization efficiency improved (reduced feed conversion ratio and increased protein efficiency ratio) upon GLP0.10 treatment, regarding the control (P < 0.05). Also, dietary administration of GLP0.15 suggestively improved the serum acid phosphatase and lysozyme activity as well as hepatic total antioxidant capacity, catalase, and superoxide dismutase activity. In contrast, GLP0.15decreased the serum alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and malonaldehyde activity when compared to the control (P<0.05). Moreover, the lipase (36.08 and 16.46 U/mgprot in GLP0.10 and GLP0.15, respectively) and amylase (0.43 and 0.23 U/mgprot in GLP0.10 and GLP0.15, respectively) activity recorded the maximum values than the control (8.61 and 0.13 U/mgprot, respectively).Further, the intestinal morphometry was developed (such as increased villus length, width, and area) in the fish fed with a GLP-supplemented diet compared to the control. The KEGG pathway analysis unveiled that several differentially expressed genes (DEGs) in control vs. GLP0.10 and control vs. GLP0.15 were associated with metabolic or immune-associated pathways like antigen processing and presentation, phagosome, complement and coagulation cascades, and platelet activation. The DEGs, namely C3, f5, fgb, MHC1, and cfb, were evaluated in control vs. GLP0.10 and C3 and MHC1 in control vs. GLP0.15, suggesting their possible contributions to GLP-regulated immunity. Additionally, the cumulative mortality of rabbitfish after the Vibrio parahaemolyticus challenge was lower in both GLP0.10 (8.88%) and GLP0.15 (11.11%) than in control (33.33%) (P<0.05). Thus, these findings direct the potential use of GLP as an immunostimulant and growth promoter in rabbitfish aquaculture.

Maximization of fitness by phenological and phenotypic plasticity in range expanding rabbitfishes (Siganidae).

Global warming is modifying the phenology, life-history traits and biogeography of species around the world. Evidence of these effects have increased over recent decades; however, we still have a poor understanding of the possible outcomes of their interplay across global climatic gradients, hindering our ability to accurately predict the consequences of climate change in populations and ecosystems. We examined the effect that changes in biogeography can have on the life-history traits of two of the most successful range-extending fish species in the world: the tropical rabbitfishes Siganus fuscescens and Siganus rivulatus. Both species have established abundant populations at higher latitudes in the northern and southern hemispheres and have been identified as important ecological engineers with the potential to alter the community structure of seaweed forests (Laminariales and Fucales) in temperate regions. Life-history trait information from across their global distribution was compiled from the published literature and meta-analyses were conducted to assess changes in (i) the onset and duration of reproductive periods, (ii) size at maturity, (iii) fecundity, (iv) growth rates, (v) maximum body sizes and (vi) longevity in populations at the leading edge of range expansion in relation to sea surface temperature and primary productivity (a common proxy for nutritional resource levels). Populations at highest latitudes had shortened their reproductive periods and reduced growth rates, taking longer to reach sexual maturity and maximum sizes, but compensated this with higher fecundity per length class and longer lifespans than populations in warmer environments. Low primary productivity and temperature in the Mediterranean Sea resulted in lower growth rates and body sizes for S. rivulatus, but also lower length at maturity, increasing life-time reproductive output. The results suggest that plasticity in the phenology and life-history traits of range-expanding species would be important to enhance their fitness in high latitude environments, facilitating their persistence and possible further poleward expansions. Quantifying the magnitude and direction of these responses can improve our understanding and ability to forecast species redistributions and its repercussions in the functioning of temperate ecosystems.

Sp1 is Involved in Vertebrate LC-PUFA Biosynthesis by Upregulating the Expression of Liver Desaturase and Elongase Genes.

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability for the biosynthesis of long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all the catalytic enzymes including two fatty acyl desaturase 2 (Δ4 Fads2 and Δ6/Δ5 Fads2) and two elongases (Elovl4 and Elovl5) have been identified, providing a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in fish. Stimulatory protein 1 (Sp1) has been speculated to be a vital transcription factor in determining the promoter activity of Fads-like genes in fish, however its regulatory effects on gene expression and LC-PUFA biosynthesis have not been demonstrated. Bioinformatic analysis predicted potential Sp1 binding sites in the promoters of the rabbitfish Δ6/Δ5 fads2 and elovl5, but not in Δ4 fads2 promoter. Here we cloned full-length cDNA of the rabbitfish sp1 gene, which encoded a putative protein of 701 amino acids, and was expressed in all tissues studied with highest levels in gill and eyes. The dual luciferase reporter assay in HepG2 line cells demonstrated the importance of the Sp1 binding site for the promoter activities of both Δ6/Δ5 fads2 and elovl5. Moreover, the electrophoretic mobility shift assay confirmed the direct interaction of Sp1 with the two promoters. Insertion of the Sp1 binding site of Δ6/Δ5 fads2 promoter into the corresponding region of the Δ4 fads2 promoter significantly increased activity of the latter. In the Siganus canaliculatus hepatocyte line (SCHL) cells, mRNA levels of Δ6/Δ5 fads2 and elovl5 were positively correlated with the expression of sp1 when sp1 was overexpressed or knocked-down by RNAi or antagonist (mithramycin) treatment. Moreover, overexpression of sp1 also led to a higher conversion of 18:2n-6 to 18:3n-6, 18:2n-6 to 20:2n-6, and 18:3n-3 to 20:3n-3, which related to the functions of Δ6/Δ5 Fads2 and Elovl5, respectively. These results indicated that Sp1 is involved in the transcriptional regulation of LC-PUFA biosynthesis by directly targeting Δ6/Δ5 fads2 and elovl5 in rabbitfish, which is the first report of Sp1 involvement in the regulation of LC-PUFA biosynthesis in vertebrates.

Hnf4α Is Involved in LC-PUFA Biosynthesis by Up-Regulating Gene Transcription of Elongase in Marine Teleost Siganus canaliculatus.

The rabbitfish Siganus canaliculatus is the first marine teleost shown to be able to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors catalyzed by two fatty acyl desaturases (fad) including Δ4 Fad and Δ6/Δ5 Fad as well as two elongases (Elovl4 and Elovl5). Previously, hepatocyte nuclear factor 4α (Hnf4α) was demonstrated to be predominant in the transcriptional regulation of two fads. To clarify the regulatory mechanisms involved in rabbitfish lipogenesis, the present study focused on the regulatory role of Hnf4α to elovl5 expression and LC-PUFA biosynthesis. Bioinformatics analysis predicted two potential Hnf4α elements in elovl5 promoter, one binding site was confirmed to interact with Hnf4α by gel shift assays. Moreover, overexpression of hnf4α caused a remarkable increase both in elovl5 promoter activity and mRNA contents, while knock-down of hnf4α in S. canaliculatus hepatocyte line (SCHL) resulted in a significant decrease of elovl5 gene expression. Meanwhile, hnf4α overexpression enhanced LC-PUFA biosynthesis in SCHL cell, and intraperitoneal injection to rabbitfish juveniles with Hnf4α agonists (Alverine and Benfluorex) increased the expression of hnf4α, elvol5 and Δ4 fad, coupled with an increased proportion of total LC-PUFA in liver. The results demonstrated that Hnf4α is involved in LC-PUFA biosynthesis by up-regulating the transcription of the elovl5 gene in rabbitfish, which is the first report of Hnf4α as a transcription factor of the elovl5 gene in vertebrates.

Hnf4α is involved in the regulation of vertebrate LC-PUFA biosynthesis: insights into the regulatory role of Hnf4α on expression of liver fatty acyl desaturases in the marine teleost Siganus canaliculatus.

Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis is an important metabolic pathway in vertebrates, especially fish, considering they are the major source of n-3 LC-PUFA in the human diet. However, most fish have only limited capability for biosynthesis of LC-PUFA. The rabbitfish (Siganus canaliculatus) is able to synthesize LC-PUFA as it has all the key enzyme activities required including Δ6Δ5 Fads2, Δ4 Fads2, Elovl5, and Elovl4. We previously reported a direct interaction between the transcription factor Hnf4α and the promoter regions of Δ4 and Δ6Δ5 Fads2, which suggested that Hnf4α was involved in the transcriptional regulation of fads2 in rabbitfish. For functionally investigating it further, a full-length cDNA of 1736-bp-encoding rabbitfish Hnf4α with 454 amino acids was cloned, which was highly expressed in intestine, followed by liver and eyes. Similar to the expression characteristics of its target genes Δ4 and Δ6Δ5 fads2, levels of hnf4α mRNA in liver and eyes were higher in fish reared at low salinity than those reared in high salinity. After the rabbitfish primary hepatocytes were, respectively, incubated with alverine, benfluorex or BI6015, which were anticipated agonists or antagonist for Hnf4α, the mRNA level of Δ6Δ5 and Δ4 fads2 displayed a similar change tendency with that of hnf4α mRNA. Furthermore, when the mRNA level of hhf4α was knocked down using siRNA, the expression of Δ6Δ5 and Δ4 fads2 also decreased. Together, these data suggest that Hnf4α is involved in the transcriptional regulation of LC-PUFA biosynthesis, specifically, by targeting Δ4 and Δ6Δ5 fads2 in rabbitfish.

l-amino acid oxidase expression profile and biochemical responses of rabbitfish (Siganus oramin) after exposure to a high dose of Cryptocaryon irritans.

Cryptocaryon irritans is an important protozoan parasite which infects almost all kinds of marine teleosts, causing heavy economic losses. In our previous studies, we found that rabbitfish (Siganus oramin) displayed high resistance to C. irritans infection, and a novel protein, l-amino acid oxidase (LAAO), was identified from the serum that was lethal to C. irritans. In this study, the rabbitfish were firstly infected with a high dose of C. irritans, then the LAAO mRNA expression pattern and the activity of three enzymes [superoxide dismutase (SOD), Na+/K+-ATPase and Ca2+/Mg2+-ATPase] were measured in various tissues. The results indicated that, after infection, the feeding and swimming of rabbitfish was normal, and the infection intensity in the host was low. Tissue distribution analysis showed that LAAO mRNA was most pronounced in the head kidney and gill, with lower expression observed in the muscle. After infection with C. irritans, the LAAO mRNA was up-regulated early post infection (from 6 to 24 h) in both gill and spleen, but then returned to normal levels, implying that LAAO may play an important role in the host's early immune response. The SOD activity in the liver was significantly higher in the infection group than in the control group by 48 h post infection, while Na+/K+-ATPase and Ca2+/Mg2+-ATPase activities in the gill were decreased by 12 and 24 h after infection; no significant difference was detected at the other time points throughout the experiment. Together, these results suggest that biochemical responses of rabbitfish are relatively mild after infection with a high dose of parasite, and the LAAO may play an important role in the host's defense against C. irritans.

Dietary arachidonic acid affects immune function and fatty acid composition in cultured rabbitfish Siganus rivulatus.

The marbled spinefoot rabbitfish (Siganus rivulatus) is an economically valuable fish species that has potential for commercial production in aquaculture. To overcome challenges in its sustainable production, a formulated diet is required for imparting health and robustness. This study evaluates the effect of dietary supplementation with arachidonic acid (ARA; 20:4n-6) on growth, survival, immune function and fatty acid composition of red blood cells (RBCs) in rabbitfish. We conducted two feeding trials using juvenile fish (to evaluate growth and survival) and adults (to evaluate immune function and fatty acid incorporation). Fish were fed diets supplemented with three different levels of ARA (in % of total fatty acids): 0.6 (unsupplemented control), 2.6 (moderate) and 4.7 (high). The fish fed with moderate ARA levels exhibited improved (p < 0.05) growth over the control and the high ARA level groups. During an outbreak of Streptococcus iniae, fish fed with moderate ARA survived significantly (p < 0.05) better (89%) than the control and the high ARA groups (59% and 48%, respectively). Moderate ARA supplementation resulted in elevated lysozyme and complement levels in the plasma of rabbitfish. A significant increase in the total serum immunoglobulin levels was observed in both the medium and the high ARA level groups; however, a decrease in antiprotease activity was recorded in the supplemented groups as compared to the control. Fatty acid analysis in fish red blood cells revealed a significant (p < 0.05) increase in the proportion of ARA of total fatty acids in the groups fed with the medium and the high ARA level diets (9.5% and 11.2%, respectively, compared to 7.1% in the control). Concomitantly, there was a decrease in the proportion of eicosapentaenoic acid (EPA; 20:5n-3), dihomo-γ linolenic acid (DGLA; 20:3n-6) and several 18-carbon unsaturated fatty acids in these groups. In conclusion, ARA in rabbitfish feeds improved growth, survival as well as innate and acquired humoral immune functions. Thus ARA supplementation in the diet of this species could be a valuable step towards establishing the commercial culture of rabbitfish.

Goldspotted spinefoot Siganus punctatus (Siganidae) age-based reproductive life history and fisheries vulnerability.

Between February 2015 and 2016, samples of the Indo-Pacific goldspotted spinefoot Siganus punctatus were taken from local fish markets, feeding sites and nursery grounds on the main island of Pohnpei, Micronesia, to ascertain sexual pattern, reproductive seasonality, age, growth and mortality. Microscopic examinations of gonads identified two seasonal peaks in reproduction: February to May and September to December, with evidence of some spawning activity in most months. Ripe females were observed 4 days on either side of the new moon. Females first matured at c. 180 mm fork length (LF ) and 1 year of age, which coincides with their entry into the fishery. Ninety five per cent of individuals were less than 3 years and the oldest fish were 8 years. To examine the species vulnerability to fishing, a tag-and-recapture study was conducted over 4 months in 2015 in a locally managed marine area and at an unprotected site. Findings suggest high residency and high vulnerability to fishing at shallow-water feeding sites and restricted migration overall. The placement of marine protected areas within critical habitat appears to be an effective conservation strategy for this species, particularly when combined with gear and seasonal market restrictions during vulnerable life-history phases.

miR-17 is involved in the regulation of LC-PUFA biosynthesis in vertebrates: effects on liver expression of a fatty acyl desaturase in the marine teleost Siganus canaliculatus.

Biosynthesis in vertebrates of long-chain polyunsaturated fatty acids (LC-PUFA) such as arachidonic (ARA; 20:4n-6), eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids requires the catalysis by fatty acyl desaturases (Fads). A vertebrate Fad with Δ4 activity catalyzing the direct conversion of 22:5n-3 to DHA was discovered in the marine teleost rabbitfish Siganus canaliculatus. Recent studies in vertebrates have shown that miRNAs may participate in the regulation of lipid metabolism at post-transcription level. However, their roles in LC-PUFA biosynthesis were not known. In the present study, in silico analysis predicts that the rabbitfish Δ4 Fad may be a target of miR-17 and thus we cloned miR-17, which is located at the forepart of the miR-17-92 cluster. Dual luciferase reporter assays demonstrated that miR-17 targeted the 3'UTR of Δ4 Fad directly. Furthermore, the expression level of miR-17 displayed an inverse pattern with that of Δ4 Fad mRNA in gill, liver and eyes, and also the Δ4 Fad protein quantity in rabbitfish liver. Incubation of rabbitfish primary hepatocytes with linoleic acid (LA; 18:2n-6), α-linolenic acid (LNA; 18:3n-3), EPA or DHA showed differential effects on miR-17, Δ4 Fad and Δ6/Δ5 Fad expression. LNA promoted the expression of miR-17 and Δ6/Δ5 Fad, but suppressed the expression of Δ4 Fad. In contrast, LA and EPA decreased the expression of miR-17 and Δ6/Δ5 Fad, but had no effect on Δ4 Fad. However, all the above were down-regulated by DHA. These data indicate that miR-17 was involved in the regulation of LC-PUFA biosynthesis in rabbitfish liver by targeting Δ4 Fad.

De Novo Assembly, Characterization, and Comparative Transcriptome Analysis of Mature Male and Female Gonads of Rabbitfish (Siganus oramin) (Bloch & Schneider, 1801).

The rabbitfish, Siganus oramin, is a commercially important table fish in southeastern China. However, there have been few studies on its gonad development and reproduction regulation. Comparative transcriptome analysis was first performed on adult male and female gonads of S. oramin. In total, 47,070 unigenes were successfully assembled and 22,737 unigenes were successfully annotated. Through comparative transcriptome analysis of male and female gonads, a total of 6722 differentially expressed genes were successfully identified, with 3528 upregulated genes and 3154 downregulated genes in the testes. In addition, 39 differentially expressed reproduction-related genes were identified. Finally, quantitative real-time PCR was used to validate the expression levels of several differentially expressed genes. These results provide important data for further studying the function of reproduction-related genes and the molecular mechanism regulating gonad development and reproduction in S. oramin.

Toward Naturalistic Neuroscience of Navigation: Opportunities in Coral Reef Fish.

The ability to navigate in the world is crucial to many species. One of the most fundamental unresolved issues in understanding animal navigation is how the brain represents spatial information. Although navigation has been studied extensively in many taxa, the key efforts to determine the neural basis of navigation have focused on mammals, usually in lab experiments, where the allocated space is typically very small; e.g., up to one order of magnitude the size of the animal, is limited by artificial walls, and contains only a few objects. This type of setting is vastly different from the habitat of animals in the wild, which is open in many cases and is virtually limitless in size compared to its inhabitants. Thus, a fundamental open question in animal navigation is whether small-scale, spatially confined, and artificially crafted lab experiments indeed reveal how navigation is enacted in the real world. This question is difficult to study given the technical problems associated with in vivo electrophysiology in natural settings. Here, we argue that these difficulties can be overcome by implementing state of the art technology when studying the rivulated rabbitfish, Siganus rivulatus as the model animal. As a first step toward this goal, using acoustic tracking of the reef, we demonstrate that individual S. rivulatus have a defined home range of about 200 m in length, from which they seldom venture. They repeatedly visit the same areas and return to the same sleeping grounds, thus providing evidence for their ability to navigate in the reef environment. Using a clustering algorithm to analyze segments of daily trajectories, we found evidence of specific repeating patterns in behavior within the home range of individual fish. Thus, S. rivulatus appears to have the ability to carry out its daily routines and revisit places of interest by employing sophisticated means of navigation while exploring its surroundings. In the future, using novel technologies for wireless recording from single cells of fish brains, S. rivulatus can emerge as an ideal system to study the neural basis of navigation in natural settings and lead to "electrophysiology in the wild."

Is the Intestinal Bacterial Community in the Australian Rabbitfish Siganus fuscescens Influenced by Seaweed Supplementation or Geography?

We recently demonstrated that dietary supplementation with seaweed leads to dramatic improvements in immune responses in S. fuscescens, a candidate species for aquaculture development in Asia. Here, to assess whether the immunostimulatory effect was facilitated by changes to the gut microbiome, we investigated the effects of those same seaweed species and four commercial feed supplements currently used in aquaculture on the bacterial communities in the hindgut of the fish. Since we found no correlations between the relative abundance of any particular taxa and the fish enhanced innate immune responses, we hypothesised that S. fuscescens might have a core microbiome that is robust to dietary manipulation. Two recently published studies describing the bacteria within the hindgut of S. fuscescens provided an opportunity to test this hypothesis and to compare our samples to those from geographically distinct populations. We found that, although hindgut bacterial communities were clearly and significantly distinguishable between studies and populations, a substantial proportion (55 of 174 taxa) were consistently detected across all populations. Our data suggest that the importance of gut microbiota to animal health and the extent to which they can be influenced by dietary manipulations might be species-specific or related to an animals' trophic level.

Insulin activates LC-PUFA biosynthesis of hepatocytes by regulating the PI3K/Akt/mTOR/Srebp1 pathway in teleost Siganus canaliculatus.

Insulin is well known an important metabolic regulator in glucose and lipid metabolism. It has been proved to activate long-chain (≥ C20) polyunsaturated fatty acids (LC-PUFA) biosynthesis in mammals, but little is known about such a role in fish. To explore the effects and molecular mechanisms of insulin in fish LC-PUFA biosynthesis, we treated the rabbitfish S. canaliculatus hepatocyte line (SCHL) cells with 65 nM insulin for 12 h, and the results showed that the mRNA levels of genes encoding the key enzymes and transcription factor involved in rabbitfish LC-PUFA biosynthesis such as Δ6Δ5 fads2, elovl5 and srebp1, as well as those of PI3K pathway genes including pdk1, akt2 and mtor increased significantly. Moreover, SCHL cells treated with different PI3K/Akt pathway inhibitors (LY294002, Wortmannin, AKTi-1/2) alone or combined with insulin decreased the mRNA levels of PI3K/Akt/mTOR downstream signaling genes, including Δ6Δ5 fads2, Δ4 fads2, elovl5, elovl4 and srebp1. While PI3K/Akt agonists (740 Y-P, IGF-1, SC-79) had the opposite results. The results of fatty acid composition analysis of hepatocytes showed that insulin stimulation increased the Δ6Δ5 Fads2-dependent PUFA desaturation indexes, while Elovl5-dependent PUFA elongation indexes had upward trends, and consequently LC-PUFA contents increased. Taken together, these results indicated that insulin activated LC-PUFA biosynthesis probably through PI3K/Akt/mTOR/Srebp1 pathway in S. canaliculatus hepatocytes.

Evolutionary History and Taxonomic Reappraisal of Coral Reef Rabbitfishes (Siganidae): Patterns of Lineage Diversification and Speciation.

Rabbitfish (Siganidae) are coral reef fish that are distributed across diverse habitats that include estuaries, mangroves, reefs, and even seaweed mats. Given their ecological diversity and natural widespread distributions across the Indo-Pacific region, we were interested to investigate the evolutionary history of this group and patterns of divergence that have contributed to their present-day distributions. In the present study, samples were collected from the South China Sea to study taxonomic and phylogenetic relationships, and divergence times. We investigated the taxonomic relationships among modern rabbitfish species, reconstructed their molecular phylogeny, and estimated divergence times among selected lineages based on a fragment of the mtDNA cytochrome oxidase I (COI) and sequences of the nuclear rhodopsin retrogene (RHO). Our results indicate that modern rabbitfish likely originated in the Indo-West Pacific during the late Eocene [37.4 million years ago (mya)], following which they diverged into three major clades during the Pliocene/Pleistocene. Subsequent diversification and origins of the majority of siganids may likely be associated with episodes of paleo-oceanographic events, including greenhouse and glaciation events (Eocene-Miocene) as well as major plate tectonic events (Pliocene-Pleistocene). Some modern siganid species may naturally hybridize with congeneric species where their geographical ranges overlap. A comprehensive taxonomic analysis revealed that the phylogeny of Siganidae (cladogenesis of Clades I, II, and III) is characterized by divergence in several external morphological characters and morphometric parameters. Our study demonstrates that morphological characteristics, geographical heterogeneity, and environmental change have contributed to siganids' historical diversification.

Human health risk assessment of heavy metals via consumption of fish from Kao Bay.

Water pollution by heavy metals due to discharge from gold mining activity has threatened the aquatic environment and human health of the community around Kao Bay. This report review explores the level of mercury and arsenic in the fish and the health risk of fish consumption within the community around Kao Bay. Fish from 10 spots in the bay were analyzed for mercury and arsenic using Atomic Absorption Spectrophotometry. Community members around Kao Bay were interviewed for details of their fish consumption. Daily intake of metals and health risk level were also calculated. All of the fish caught contained mercury (mean of 0.2110 ug/g) and arsenic (mean of 0.422 ug/g). This heavy metal concentration exceeds the allowable level for food standard. The human health risk assessment showed that the fish caught from Kao Bay were not safe for human consumption (RQ>1). The hazard risk quotient based on cancer and non-cancer was more than one. As many as 49 of 52 people living around Kao Bay have a risk from mercury and arsenic exposure via fish consumption. The magnitude of HQ and ECR values for most fish indicates that it is not safe for consumption.

Hepatocyte nuclear factor 4α (Hnf4α) is involved in transcriptional regulation of Δ6/Δ5 fatty acyl desaturase (Fad) gene expression in marine teleost Siganus canaliculatus.

As the first marine teleost demonstrated to biosynthesize long-chain polyunsaturated fatty acids (LC-PUFAs) from C18 precursors such as linoleic acid (LOA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3), the rabbitfish (Siganus canaliculatus) contains the complete enzymatic system for LC-PUFA biosynthesis, including Δ6/Δ5 fatty acid desaturase (Fad), Δ4 Fad, and elongase 5 (Elovl5). Previously, our group demonstrated that hepatocyte nuclear factor 4α (Hnf4α) is a transcription factor (TF) for rabbitfish Δ4 fad and elovl5, and interacts with the core promoter of Δ6/Δ5 fad. To fully clarify the role of Hnf4α in the regulation of LC-PUFA biosynthesis, the present study aimed to explore the regulatory role of Hnf4α on Δ6/Δ5 fad gene expression. First, Hnf4α overexpression and agonist assays identified the Hnf4α response region in the Δ6/Δ5 fad core promoter as -456 bp to +51 bp. Bioinformatic analysis predicted four potential Hnf4α binding elements in the core promoter, which were confirmed by site-directed mutation and functional assays in a dual luciferase assay system. Moreover, the mRNA expression levels of hnf4α, Δ6/Δ5 fad, and Δ4 fad were significantly increased in the S. canaliculatus hepatocyte line (SCHL) cells after treatment with Hnf4α agonists (Alverine and Benfluorex) or its mRNA overexpression. By contrast, the expression levels of these three genes were markedly decreased after hnf4a small interfering RNA (siRNA) transfection. The results indicated that Hnf4α has a regulatory effect on rabbitfish Δ6/Δ5 fad gene transcription, identifying Hnf4α as a TF of Δ6/Δ5 fad in vertebrates for the first time.

Synergism of Dietary Co-Supplementation with Lutein and Bile Salts Improved the Growth Performance, Carotenoid Content, Antioxidant Capacity, Lipid Metabolism, and Lipase Activity of the Marbled Spinefoot Rabbitfish, Siganus rivulatus.

A 60-day feeding trial was conducted to assess the effects of dietary supplementation with bile salts (BS), lutein (LTN), and their combination on growth, survival, carotenoid content, and antioxidant status of rabbitfish (Siganus rivulatus) larvae. Fish were fed four isonitrogenous (34.39% protein) and isoenergetic (20.57 kJ/g) diets supplemented with BS (0.15 g kg-1), LTN (0.1 g kg-1), BS+LTN (0.15 and 0.1 g kg-1, respectively), and a non-supplemented control diet. The results revealed that fish fed BS+LTN had the highest significant specific growth rate (4.37% day-1), feed efficiency (46.55%), and survival (97.78%). Lutein supplementation improved whole-body protein content, meanwhile, fish fed a BS-supplemented diet had a higher lipid content. The carotenoid deposition was significantly increased with LTN and BS+LTN in skin, muscle, and whole body compared to the control and BS treatment. All dietary supplementation of BS and LTN showed significant improvement in total antioxidant capacity, catalase, and glutathione peroxidase activities. Additionally, LTN alone or BS+LTN significantly reduced malondialdehyde levels by 5.30 and 29.91%, respectively compared to the control. BS supplementation modulated aminopeptidases activities, triglycerides, cholesterol, and increased the activity of pancreatic lipase. Therefore, it could be inferred that dietary supplementation with LTN in combination with BS could improve the growth performance, carotenoid deposition, antioxidant status, lipid digestion, and metabolism of S. rivulatus.

The miR-15/16 Cluster Is Involved in the Regulation of Vertebrate LC-PUFA Biosynthesis by Targeting pparγ as Demonostrated in Rabbitfish Siganus canaliculatus.

Post-transcriptional regulatory mechanisms play important roles in the regulation of long-chain (≥ C20) polyunsaturated fatty acid (LC-PUFA) biosynthesis. Here, we address a potentially important role of the miR-15/16 cluster in the regulation of LC-PUFA biosynthesis in rabbitfish Siganus canaliculatus. In rabbitfish, miR-15 and miR-16 were both highly responsive to fatty acids affecting LC-PUFA biosynthesis and displayed a similar expression pattern in a range of rabbitfish tissues. A common potential binding site for miR-15 and miR-16 was predicted in the 3'UTR of peroxisome proliferator-activated receptor gamma (pparγ), an inhibitor of LC-PUFA biosynthesis in rabbitfish, and luciferase reporter assays revealed that pparγ was a potential target of miR-15/16 cluster. In vitro individual or co-overexpression of miR-15 and miR-16 in rabbitfish hepatocyte line (SCHL) inhibited both mRNA and protein levels of Pparγ, and increased the mRNA levels of Δ6Δ5 fads2, Δ4 fads2, and elovl5, key enzymes of LC-PUFA biosynthesis. Inhibition of pparγ was more pronounced with co-overexpression of miR-15 and miR-16 than with individual overexpression in SCHL. Knockdown of miR-15/16 cluster gave opposite results, and increased mRNA levels of LC-PUFA biosynthesis enzymes were observed after knockdown of pparγ. Furthermore, miR-15/16 cluster overexpression significantly increased the contents of 22:6n-3, 20:4n-6 and total LC-PUFA in SCHL with higher 18:4n-3/18:3n-3 and 22:6n-3/22:5n-3 ratio. These suggested that miR-15 and miR-16 as a miRNA cluster together enhanced LC-PUFA biosynthesis by targeting pparγ in rabbitfish. This is the first report of the participation of miR-15/16 cluster in LC-PUFA biosynthesis in vertebrates.

miR-146a is involved in the regulation of vertebrate LC-PUFA biosynthesis by targeting elovl5 as demonstrated in rabbitfish Siganus canaliculatus.

Rabbitfish Siganus canaliculatus is the first marine teleost demonstrated to have the ability to synthesize long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and thus provides us a unique model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. MicroRNAs (miRNAs) were shown to play important roles in the regulation of LC-PUFA biosynthesis in rabbitfish at posttranscriptional level in our previous studies. Here, we focused the roles of miR-146a in such regulation. The expression of miR-146a displayed an inverse pattern with that of elongase 5 (Elovl5), a key enzyme catalyzing the elongation of C18 (18:4n3 and 18:3n6) and C20 (20:5n3 and 20:4n6) PUFA in the LC-PUFA biosynthesis, in vivo in liver of rabbitfish reared under different salinities, as well as in vitro in S. canaliculatus hepatocyte line (SCHL) cells incubated with different fatty acids. Bioinformatics analysis predicted that miR-146a may target the 3'UTR of elovl5 directly, which was confirmed by the dual luciferase reporter assays in HEK 293T cells. Overexpression of miR-146a significantly downregulated the expression of elovl5 in SCHL cells, while knockdown of miR-146a showed an opposite effect. Moreover, up-regulation of miR-146a in SCHL cells significantly suppressed the elongation indexes 20:3n6/18:3n6, 20:4n3/18:4n3 and 22:5n3/20:5n3 associated with Elovl5 catalyzing activity, and consequently reduced the contents of LC-PUFA. These results indicate that miR-146a is involved in the regulation of LC-PUFA biosynthesis through inhibiting the mRNA expression and activity of Elovl5 in rabbitfish, which was for the first time to focus on the role of miR-146a in LC-PUFA biosynthesis in vertebrates and will provide a new insight into the regulatory mechanisms of LC-PUFA biosynthesis in teleosts.

Spatial distributions, feeding ecologies, and behavioral interactions of four rabbitfish species (Siganus unimaculatus, S. virgatus, S. corallinus, and S. puellus).

Clarifying the underlying mechanisms that enable closely related species to coexist in a particular environment is a fundamental aspect of ecology. Coral reefs support a high diversity of marine organisms, among which rabbitfishes (family Siganidae) are a major component The present study aimed to reveal the mechanism that allows rabbitfishes to coexist on coral reefs in Okinawa, Japan, by investigating the spatial distributions, feeding ecologies, and behavioral interactions of four species: Siganus unimaculatus, S. virgatus, S. corallinus, and S. puellus. All four species had a size-specific spatial distribution, whereby small individuals were found in sheltered areas that were covered by branching and bottlebrush Acropora spp. and large individuals were found in both sheltered and exposed rocky areas. However, no clear species-specific spatial distribution was observed. There was some variation in the food items taken, with S. unimaculatus primarily feeding on brown foliose algae, red foliose algae, and red styloid algae, and S. virgatus and S. puellus preferring brown foliose algae and sponges, respectively. However, S. corallinus did not show any clear differences in food preferences from S. virgatus or S. unimaculatus, mainly feeding on brown foliose algae and red styloid algae. The four species exhibited differences in foraging substrate use, which was probably related to differences in their body shape characteristics: S. unimaculatus has a slender body with a remarkably protruding snout and mainly used concave substrates for feeding, whereas S. virgatus has a deeper body with a low degree of snout protrusion and mainly used convex substrates. The other two species have a low degree of snout protrusion combined with a deeper body in the case of S. corallinus and a slender body in the case of S. puellus and used concave, flat, and convex substrates to an equal degree for feeding. Behavioral interactions were categorized into "agonistic behaviors" (attack and agonistic displays) and "no interactions." For all four species, a greater frequency of agonistic behaviors was observed when two conspecific pairs approached each other than when two heterospecific individuals encountered each other. Together, these results suggest that food item partitioning is one of the main factors enabling the coexistence of these four syntopic rabbitfish species, which is enhanced by species-specific differences in feeding substrates as a result of their different body shape and behavioral characteristics.