Marine Teleost Research Articles

Dietary astaxanthin differentially affected the lipid accumulation in the liver and muscle of the marine teleost, tiger pufferTakifugu rubripes

Dietary astaxanthin differentially affected the lipid accumulation in the liver and muscle of the marine teleost, tiger puffer<i>Takifugu rubripes</i>

Patterns of Fish Reproduction at the Interface between Air and Water.

Although fishes by nature are aquatic, many species reproduce in such a way that their embryos are exposed to air either occasionally or constantly during incubation. We examine the ecological context and review specific examples of reproduction by fishes at the air-water interface, including fishes that do and do not breathe air. Four modes of reproduction at the air-water interface are described across 18 teleost orders, from fresh water, estuaries, and sea water. Mode 1, the most common type of reproduction by fishes at the air-water interface, includes 21 families of mostly marine teleosts that spawn in water onto a substrate surface, on vegetation, or into hollow objects such as shells that will later be continuously or occasionally exposed to air. Although the eggs are emerged into air, many of these species do not emerge into air as adults, and only about half of them breathe air. Mode 2 involves six families of freshwater fishes setting up and guarding a nest and guarding on the water surface, either with bubbles or in vegetation. Most of these species breathe air. In Mode 3, annual killifishes in at least two families in seasonally dry habitats bury eggs in mud in temporary pools, then die before the next generation emerges. These species neither guard nests nor breathe air. Mudskippers (Gobiidae) breathe air and use Mode 4, excavating burrows in a soft substrate and then storing air in a subterranean chamber. In a variation of Mode 4, eggs are placed on bubbles within a nesting burrow by swamp eels (Synbranchidae). No fishes from basal taxa are known to place their embryos where they will be exposed to air, although most of these species breathe air as adults. The widespread but still rare, diverse forms of fish reproduction at the air-water interface across a broad taxonomic spectrum suggest repeated independent evolutionary events and strong selection pressure for adult fishes to protect their embryos from hypoxic waters, aquatic predators, pathogens, and UV radiation. Air-breathing by adult fishes appears to be de-coupled from air exposure of developing embryos or aerial emersion of adults during spawning.

Combined effects of ocean acidification and crude oil pollution on tissue damage and lipid metabolism in embryo-larval development of marine medaka (Oryzias melastigma).

Ocean acidification (OA) and crude oil pollution have been highlighted as some of the most pervasive anthropogenic influences on the ocean.In marine teleosts, early life-history stages are particularly vulnerable to disturbance by CO2-driven acidification as they lack pH-mediated intracellular regulation. Embryos exposed to trace levels of crude oil constituents dissolved in water exhibit a common syndrome of developmental abnormalities. So far, little is known about the combined effects of OA and crude oil on the early life history of marine fish. Eggs and larvae of the marine medaka (Oryzias melastigma) were treated with CO2 (1080μatm atmospheric CO2), the water-soluble fraction (WSF) of crude oil (500μg/L) and a CO2 (1080μatm atmospheric CO2)/WSF (500μg/L) mixture within 4h after oviposition. Isolated and combined OA/WSF had no detectable effect on embryonic duration, egg survival rate and size at hatching. Histopathological anomalies of tissue and lipid metabolic disorder were significant when CO2 or WSF was given alone at 30days of age. Combination of CO2 and WSF enhanced their toxicity compared to their separate administration. Since the early life-history stage of marine fish is thought to be impacted more heavily by increasing CO2 partial pressure (pCO2) levels and crude oil pollution, OA and crude oil pollution have the potential to act as an additional source of natural mortality.

Osmoregulation in the Plotosidae Catfish: Role of the Salt Secreting Dendritic Organ.

Unlike other marine teleosts, the Plotosidae catfishes reportedly have an extra-branchial salt secreting dendritic organ (DO). Salinity acclimation [brackishwater (BW) 3aaa, seawater (SWcontrol) 34aaa, and hypersaline water (HSW) 60aaa] for 14 days was used to investigate the osmoregulatory abilities of Plotosus lineatus through measurements of blood chemistry, muscle water content (MWC), Na+/K+-ATPase (NKA) specific activity and ion transporter expression in gills, DO, kidney and intestine. Ion transporter expression was determined using immunoblotting, immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). HSW elevated mortality, plasma osmolality and ions, and hematocrit, and decreased MWC indicating an osmoregulatory challenge. NKA specific activity and protein levels were significantly higher in DO compared to gill, kidney and intestine at all salinities. NKA specific activity increased in kidney and posterior intestine with HSW but only kidney showed correspondingly higher NKA α-subunit protein levels. Since DO mass was greater in HSW, the total amount of DO NKA activity expressed per gram fish was greater indicating higher overall capacity. Gill NKA and V-ATPase protein levels were greater with HSW acclimation but this was not reflected in NKA activity, mRNA or ionocyte abundance. BW acclimation resulted in lower NKA activity in gill, kidney and DO. Cl- levels were better regulated and the resulting strong ion ratio in BW suggests a metabolic acidosis. Elevated DO heat shock protein 70 levels in HSW fish indicate a cellular stress. Strong NKA and NKCC1 (Na+:K+:2Cl- cotransporter1) co-localization was observed in DO parenchymal cells, which was rare in gill ionocytes. NKCC1 immunoblot expression was only detected in DO, which was highest at HSW. Cystic fibrosis transmembrane regulator Cl- channel (CFTR) localize apically to DO NKA immunoreactive cells. Taken together, the demonstration of high NKA activity in DO coexpressed with NKCC1 and CFTR indicates the presence of the conserved secondary active Cl- secretion mechanism found in other ion transporting epithelia suggesting a convergent evolution with other vertebrate salt secreting organs. However, the significant osmoregulatory challenge of HSW indicates that the DO may be of limited use under more extreme salinity conditions in contrast to the gill based ionoregulatory strategy of marine teleosts.

Population specific sperm production in European flounder Platichthys flesus: Adaptation to salinity at spawning.

Marine teleosts inhabiting the brackish Baltic Sea have adapted to the less saline water with activation of spermatozoa at low salinity hypo-osmotic conditions but with shorter longevity and lower swimming speed that affect the fertilization capacity. Aiming to elucidate if the fertilization capacity may be maintained by increasing the number of spermatozoa produced, testis size for the euryhaline flounder Platichthys flesus with external fertilization was assessed along a salinity gradient; with spawning at a salinity of c. 7, 10-18 and 30-35. Fulton's condition factor K = 0.881 ± 0.085 (mean ± S.D.), 0.833 ± 0.096 and 0.851 ± 0.086, for fish spawning at salinities of c. 7, 10-18 and 30-35, respectively, with no difference between areas, i.e. analysed fish were in similar nutritional condition. A general linear model, with testes dry mass as the dependent variable and somatic mass as covariate resulted in a significant difference between areas-populations with larger testes for P. flesus spawning at a salinity of c. 7 but no difference between fish spawning at a salinity of 10-18 and 30-35. The result suggests that adaptation by increasing the number of spermatozoa produced may be a key mechanism for marine teleosts spawning in areas with low salinities to sustain the fertilization capacity as shown here for the euryhaline P. flesus.

Eyes Wide Shut: the impact of dim-light vision on neural investment in marine teleosts.

Understanding how organismal design evolves in response to environmental challenges is a central goal of evolutionary biology. In particular, assessing the extent to which environmental requirements drive general design features among distantly related groups is a major research question. The visual system is a critical sensory apparatus that evolves in response to changing light regimes. In vertebrates, the optic tectum is the primary visual processing centre of the brain and yet it is unclear how or whether this structure evolves while lineages adapt to changes in photic environment. On one hand, dim-light adaptation is associated with larger eyes and enhanced light-gathering power that could require larger information processing capacity. On the other hand, dim-light vision may evolve to maximize light sensitivity at the cost of acuity and colour sensitivity, which could require less processing power. Here, we use X-ray microtomography and phylogenetic comparative methods to examine the relationships between diel activity pattern, optic morphology, trophic guild and investment in the optic tectum across the largest radiation of vertebrates-teleost fishes. We find that despite driving the evolution of larger eyes, enhancement of the capacity for dim-light vision generally is accompanied by a decrease in investment in the optic tectum. These findings underscore the importance of considering diel activity patterns in comparative studies and demonstrate how vision plays a role in brain evolution, illuminating common design principles of the vertebrate visual system.

Characterization of TLR5 and TLR9 from silver pomfret (Pampus argenteus) and expression profiling in response to bacterial components

Toll like receptor (TLR) 5 and 9 are important members of the TLR family that play key roles in innate immunity in all vertebrates. In this study, paTLR5 and paTLR9 were identified in silver pomfret (Pampus argenteus), a marine teleost of great economic value. Open reading frames (ORFs) of paTLR5 and paTLR9 are 2646 and 3225 bp, encoding polypeptides of 881 and 1074 amino acids, respectively. Sequence analysis revealed several conserved characteristic features, including signal peptides, leucine-rich repeat (LRR) motifs, and a Toll/interleukin-I receptor (TIR) domain. Sequence, phylogenetic and synteny analysis revealed high sequence identity with counterparts in other teleosts, confirming their correct nomenclature and conservation during evolution. Quantitative real-time PCR revealed that the that both TLRs were ubiquitously expressed in all investigated tissues, most abundantly in liver, kidney, spleen, intestine and gill, but lower in muscle and skin. In vitro immunostimulation experiments revealed that Aeromonas hydrophila lipopolysaccharide (LPS) and Vibrio anguillarum flagellin induced higher levels of paTLR9 and paTLR5 mRNA expression in isolated fish intestinal epithelial cells (FIECs) than Lactobacillus plantarum lipoteichoic acid (LTA), but all increased the secretion of IL-6 and TNF-α and induced cell apoptosis and necrosis. Together, these results indicate that paTLR5 and paTLR9 may function in the response to bacterial pathogens. Our findings enhance our understanding of the function of TLRs in the innate immune system of silver pomfret and other teleosts.

Male-specific Dmrt1 is a candidate sex determination gene in spotted scat (Scatophagus argus)

The Scatophagus argus is a cultured marine teleost fish in which females grow faster and larger than males, therefore cloning the master sex-determination (SD) gene will be helpful for its sex control in aquaculture. A well-conserved Dmrt1 (doublesex- and mab-3-related transcription factor-1) gene, encoding zinc finger-like DNA binding motif (DM-domain) plays a critical role in sex determination and differentiation in vertebrates. Here, two pairs of sex-specific primers, Dmrt3-Marker-F/R and Dmrt1-Marker-F/R were developed from Dmrt3 and Dmrt1 transcripts in S. argus respectively. The single-nucleotide polymorphisms (SNP) at position −151 and 166 bp on the DNA amplified by Dmrt3-Marker-F/R are all homozygous CC GG genotype in females and heterozygous CG GA genotype in the males. The Dmrt1-Marker-F/R could only amplify a 3.2 kb fragment in males. Both Markers were confirmed in 246 adult S. argus (113 males and 133 females) from five populations in China. These results indicate that the sexes of S. argus are controlled by sex chromosomes (XX females, XY males). The nucleotide at position 166 bp of the DNA amplified by Dmrt3-Marker-F/R is found to be a donor splice site mutation (c.400 + 1G > A) in Dmrt3 which generates a truncated isoform. The truncated Dmrt3 is linked to male sex and thereafter named as Dmrt3△-Y, and its allele is the normal Dmrt3 on the X chromosome. The DNA amplified by Dmrt1-Marker-F/R is a fragment between exon 2 and exon 3 of a normal Dmrt1 implying that Dmrt1 is male-specific and located on the Y chromosome. A truncated Dmrt1 (Dmrt1b) was found to exist in both males and females. The Dmrt1b is with high similarity with Dmrt1 indicating Dmrt1b is the allele of Dmrt1 located on the X chromosome. RT-PCR revealed that Dmrt1 is highly expressed in the testes, while it is not expressed in the ovaries. Dmrt3△-Y is only expressed weakly in some of the testes, while it is not expressed in the ovaries. The testicular specifically expressed Dmrt1 is the candidate SD gene on the Y chromosome in S. argus.

The cellular localization and redistribution of multiple aquaporin paralogs in the spermatic duct epithelium of a maturing marine teleost.

Aquaporin-mediated fluid transport in the mammalian efferent duct and epididymis is believed to play a role in sperm maturation and concentration. In fish, such as the marine teleost gilthead seabream (Sparus aurata), the control of fluid homeostasis in the spermatic duct seems also to be crucial for male fertility, but no information exists on the expression and distribution of aquaporins. In this study, reverse transcriptase-polymerase chain reaction and immunoblotting analyses, employing available and newly raised paralog-specific antibodies for seabream aquaporins, indicate that up to nine functional aquaporins, Aqp0a, -1aa, -1ab, -3a, -4a, -7, -8bb, -9b and -10b, are expressed in the spermatic duct. Immunolocalization of the channels in the resting spermatic duct reveals that Aqp0a, -1aa, -4a, -7 and -10b are expressed in the monolayered luminal epithelium, Aqp8b and -9b in smooth muscle fibers, and Aqp1ab and -3a in different interstitial lamina cells. In the epithelial cells, Aqp0a and -1aa are localized in the short apical microvilli, and Aqp4a and -10b show apical and basolateral staining, whereas Aqp7 is solely detected in vesicular compartments. Upon spermiation, an elongation of the epithelial cells sterocilia, as well as the folding of the epithelium, is observed. At this stage, single- and double-immunostaining, using two aquaporin paralogs or the Na+ /K+ -ATPase membrane marker, indicate that Aqp1ab, -3a, -7, -8bb and -9b staining remains unchanged, whereas in epithelial cells Aqp1aa translation is supressed, Aqp4a internalizes, and Aqp0a and -10b accumulate in the apical, lateral and basal plasma membrane. These findings uncover a cell type- and region-specific distribution of multiple aquaporins in the piscine spermatic duct, which shares conserved features of the mammalian system. The data therefore suggest that aquaporins may play different roles in the regulation of fluid homeostasis and sperm maturation in the male reproductive tract of fish.

Characteristics of the fads2 gene promoter in marine teleost Epinephelus coioides and role of Sp1-binding site in determining promoter activity

Δ6 fatty acyl desaturase (Fads2) is a rate-limiting enzyme in long-chain polyunsaturated fatty acids (LC-PUFA) biosynthesis. Comparative analysis of gene promoters of Fads2 between salmonids and carnivorous marine fish suggested that the lack of binding site for stimulatory protein 1 (Sp1) was responsible for the low expression of fads2 gene of carnivorous marine species. To confirm this speculation, the fads2 candidate promoter (2646 bp) was cloned from carnivorous marine teleost Epinephelus coioides, and 330 bp core regulatory region was identified. Several binding sites for transcriptional factors such as nuclear factor 1, nuclear factor Y, sterol regulatory element and hepatocyte nuclear factor 4γ were identified, while that for Sp1 was shown to be absent in the promoter by both bioinformatic analysis and site-directed mutation. Moreover, after the Sp1-binding site from the fads2 promoter of herbivorous Siganus canaliculatus, the first marine teleost demonstrated to have LC-PUFA biosynthetic ability, was inserted into the corresponding region of E. coioides fads2 promoter, activity was significantly increased. The results provided direct data for the importance of the Sp1-binding site in determining fads2 promoter activity, and indicated that its lack may be a reason for low expression of fads2 and poor LC-PUFA biosynthetic ability in E. coioides.

Comparison of the organic matrix found in intestinal CaCO3 precipitates produced by several marine teleost species

Marine bony fish poses the unique ability to hydrate from imbibed seawater. They accomplish this, in part, by the precipitation of inorganic carbonate mineral in their intestine, which lowers luminal osmotic pressure and allows for water uptake. It has recently been described that in the Gulf toadfish (Opsanus beta) this Ca(Mg)CO3 precipitation occurs under the regulation of an organic matrix. To date no investigations have aimed to determine if this phenomenon applies more generally to marine fish. Here, intestinally derived precipitates were collected from gray snapper (Lutjanus griseus), white grunt (Haemulon plumieri), European flounder (Platichthys flesus), as well as Gulf toadfish, and their matrices were extracted. The ability of these matrices to regulate CaCO3 production was determined using an in vitro calcification assay, which revealed that the matrix derived from each of the tested species increased precipitation at low concentrations, while inhibiting it at higher concentrations in full agreement with the earlier studies on toadfish. Matrix extracted from European flounder precipitates was then analyzed by mass spectrometry, leading to the identification of over 50 unique proteins. When the identities of these proteins were compared to previous investigation of toadfish precipitate matrix, nearly 35% were found to overlap between the flounder and toadfish analyses, suggesting conserved mechanisms of precipitation control. The effects of using different sodium hypochlorite (NaOCl) solutions during precipitate purification on the resulting organic matrix are also discussed.

Influence of supraphysiological cortisol manipulation on predator avoidance behaviors and physiological responses to a predation threat in a wild marine teleost fish.

The stress axis in teleost fish attempts to maintain internal homeostasis in the face of allostatic loading. However, stress axis induction has been associated with a higher predation rate in fish. To date, the physiological and behavioral factors associated with this outcome are poorly understood. The purpose of the present study was to investigate the impact of experimental cortisol elevation on anti-predator behavior and physiological responses to predator presence. We hypothesized that semi-chronic cortisol elevation would increase susceptibility to predation by increasing stress-induced risk-taking behaviors. To test this hypothesis, schoolmaster snapper were given cocoa butter implants without cortisol (sham) or with cortisol (50 mg/kg body weight) and tethered to cover. Fish were exposed to either a lemon shark or control conditions for 15-min. Space use and activity were recorded throughout and fish were terminally sampled for blood. Cortisol implantation, relative to shams, resulted in higher blood glucose and plasma cortisol concentrations with a lower plasma lactate concentration. Shark exposure, relative to controls, elicited higher blood glucose and lactate concentrations but had no effect on plasma cortisol concentration. No interactions were detected between shark exposure and cortisol treatment for any physiological trait. Behavioral metrics, including shelter use and activity, were unaffected by either cortisol implantation or shark exposure. Physiological responses to cortisol implantation likely resulted from enhanced gluconeogenic activity, whereas alterations under predator exposure may have been the product of catecholamine mobilization. Further work should address context-specific influences of stress in mediating behavioral responses to predation.

Toxicological effects of irregularly shaped and spherical microplastics in a marine teleost, the sheepshead minnow (Cyprinodon variegatus)

The increasing global contamination of plastics in marine environments is raising public concerns about the potential hazards of microplastics to environmental and human health. Microplastics formed by the breakdown of larger plastics are typically irregular in shape. The objective of this study was to compare the effects of spherical or irregular shapes of microplastics on changes in organ distribution, swimming behaviors, gene expression, and enzyme activities in sheepshead minnow (Cyprinodon variegatus). Both types of microplastics accumulated in the digestive system, causing intestinal distention. However, when compared to spherical microplastics, irregular microplastics decreased swimming behavior (i.e., total distance travelled and maximum velocity) of sheepshead minnow. Both microplastics generated cellular reactive oxygen species (ROS), while ROS-related molecular changes (i.e., transcriptional and enzymatic characteristics) differed. This study provides toxicological insights into the impacts of environmentally relevant (fragmented) microplastics on fish and improves our understanding of the environmental effects of microplastics in the ecosystem.

Performance and precision of double digestion RAD (ddRAD) genotyping in large multiplexed datasets of marine fish species

The development of Genotyping-By-Sequencing (GBS) technologies enables cost-effective analysis of large numbers of Single Nucleotide Polymorphisms (SNPs), especially in “non-model” species. Nevertheless, as such technologies enter a mature phase, biases and errors inherent to GBS are becoming evident. Here, we evaluated the performance of double digest Restriction enzyme Associated DNA (ddRAD) sequencing in SNP genotyping studies including high number of samples. Datasets of sequence data were generated from three marine teleost species (>5500 samples, >2.5 × 1012 bases in total), using a standardized protocol. A common bioinformatics pipeline based on STACKS was established, with and without the use of a reference genome. We performed analyses throughout the production and analysis of ddRAD data in order to explore (i) the loss of information due to heterogeneous raw read number across samples; (ii) the discrepancy between expected and observed tag length and coverage; (iii) the performances of reference based vs. de novo approaches; (iv) the sources of potential genotyping errors of the library preparation/bioinformatics protocol, by comparing technical replicates. Our results showed use of a reference genome and a posteriori genotype correction improved genotyping precision. Individual read coverage was a key variable for reproducibility; variance in sequencing depth between loci in the same individual was also identified as an important factor and found to correlate to tag length. A comparison of downstream analysis carried out with ddRAD vs single SNP allele specific assay genotypes provided information about the levels of genotyping imprecision that can have a significant impact on allele frequency estimations and population assignment. The results and insights presented here will help to select and improve approaches to the analysis of large datasets based on RAD-like methodologies.

Inorganic arsenic causes apoptosis cell death and immunotoxicity on European sea bass (Dicentrarchus labrax)

Inorganic arsenic (As) is one of the most toxic pollutants in the water. We have studied their effects on the marine teleost European sea bass (Dicentrarchus labrax) at 2 and 10 days of 5 μM of As2O3 (sub-lethal doses) waterborne exposure. Arsenic accumulates in liver and gill tissues. The expression profile of five genes (bax, blc2, casp3, casp8 and casp9) involved in apoptosis cell death confirmed apoptotic effects in liver, slight changes in gill and no effects in skin according with the histopathology findings. Total IgM level and peroxidase activities were increased at 2 and 10 days, respectively. The bactericidal activity was decreased at 2 days after As exposure. A general decrease of cellular immune activities with significant differences in the case of respiratory burst activity was observed after 2 and 10 days of exposure. This work describes for the first time the effects of As exposure on European sea bass.

Skin and scale regeneration after mechanical damage in a teleost

Skin wound healing has been widely studied in mammalian models but the information on teleost cutaneous healing is sparse and frequently considered in the context of viral or bacterial infections or parasitic infestations in aquaculture. In the present study a detailed time course (0 h, 6 h, 1, 2, 3 and 4 days) coupled to morphology and gene expression analysis revealed rapid regeneration of skin without scarring in a marine teleost after a superficial wound caused by the loss of a large area of scales. The integrity of the integument, as assessed by quantification of extracellular matrix (ECM) gene transcripts (fn1a, colIα1, colVα2, colXα1, ogn1, ogn2, crtac1a, cyr61, pcna, krt2 and mmp9) was restored within 2 days. Epithelial-mesenchyme interactions assessed by expression of edar and shh were associated with epidermal closure, the re-establishment of the basement membrane and also scale eruption. Histological observations suggested tissue re-epithelialization was independent of inflammation and that transcripts representing the humoral and cellular elements of the immune response (mpo, cyba and csf1r, cd48 and cd200) were modulated in the early stages of sea bream (Sparus aurata) skin repair after injury. Overall, the results indicate that after superficial skin damage tissue reconstitution started immediately with re-epithelialization, followed by ECM deposition and finally tissue maturation, indicating that in the skin regenerative process, reconstitution of the physical barrier was the priority over other integument functions, including immune surveillance.

Changes of visceral properties and digestive enzymes in the herbivorous marine teleost Siganus canaliculatus fed on different diets

The rabbitfish Siganus canaliculatus is one of the few cultured herbivorous marine teleosts. To better understand the digestive physiology of this fish and provide data for designing formulated feed using macroalgae as an ingredient, the changes of visceral properties and digestive enzyme activities were investigated after the juveniles were fed on different types of food including raw fish (RF), formulated diet (FD) or macroalgae Enteromorpha prolifra (EP) and Gracilaria lemaneiformis (GL) for eight weeks. The results showed that the hepatosomatic and viscerosomatic indices in the RF and FD groups, as well as the relative intestine length (RIL) in the EP and GL groups, were significantly higher than those in other groups. Additionally, differences in the histological structure of the liver and anterior intestine were also observed among different dietary groups. The hepatic nuclei were displaced to the periphery by lipid inclusions in fish fed RF. The highest levels of mucosal folds were found in the anterior intestines of fish fed macroalgae. Digestive enzyme activity profiles showed obvious fluctuations in the first three weeks, and then leveled off in the following weeks. The levels of protease, lipase and α-amylase in the alimentary tract showed changes related to the levels of dietary protein, lipid and carbohydrate, respectively. Although macroalgae significantly inhibited the activity of protease in the stomach, it increased RIL and the number of mucosal folds in the anterior intestine so as to compensate for the influences on protease activities in the stomach. This study suggests that the digestive tract of rabbitfish can well adapt to different diets, and needs about three weeks to physiologically acclimatize to the nutritional status, thus implying that rabbitfish are somewhat omnivorous.

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.

Cloning and characterization of ∆6/∆5 fatty acyl desaturase (Fad) gene promoter in the marine teleost Siganus canaliculatus

The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding ∆6/∆5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C18 PUFA to LC-PUFA. First, 2044 bp promoter sequence was cloned by genome walking, and the sequence from −456 bp to +51 bp was determined as core promoter by progressive deletion mutation. Moreover, binding sites of transcription factors (TF) such as CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), stimulatory protein 1 (Sp1), nuclear factor Y (NF-Y), activated protein 1 (AP1), sterol regulatory element (SRE), hepatocyte nuclear factor 4α (HNF4α) and peroxisome proliferator activated receptor γ (PPARγ) were identified in the core promoter by site-directed mutation and functional assays. Moreover, NF-1 and HNF4α were confirmed to interact with the core promoter region by gel shift assay and mass spectrometry. This is the first report of the promoter structure of a ∆6/∆5 Fad in a marine teleost, and a novel discovery of NF-1 and HNF4α binding to the ∆6/∆5 Fad promoter.

The role of maternal age and context-dependent maternal effects in the offspring provisioning of a long-lived marine teleost.

Despite evidence of maternal age effects in a number of teleost species, there have been challenges to the assertion that maternal age intrinsically influences offspring quality. From an evolutionary perspective, maternal age effects result in young females paradoxically investing in less fit offspring despite a greater potential fitness benefit that might be gained by allocating this energy to individual somatic growth. Although a narrow range of conditions could lead to a maternal fitness benefit via the production of lower quality offspring, evolutionary theorists suggest these conditions are seldom met and that the reported maternal age effects are more likely products of the environmental context. Our goal was to determine if maternal effects operated on offspring provisioning in a long-lived rockfish (genus Sebastes), and to evaluate any such effects as an intrinsic function of maternal age or a context-dependent effect of the offspring release environment. We found that offspring provisioning is a function of both maternal age and the timing of offspring release; older females exhibit increased provisioning over younger females throughout the spawning season despite a decrease in provisioning across all maternal ages as the season progresses. These findings suggest a role for both maternal age effects and a potential context-dependent maternal effect in population productivity, carrying important implications when modelling population persistence and resilience.