Combinatorial metabolomic and transcriptomic analysis of muscle growth in hybrid striped bass (female white bass Morone chrysops x male striped bass M. saxatilis).

BACKGROUND: Understanding growth regulatory pathways is important in aquaculture, fisheries, and vertebrate physiology generally. Machine learning pattern recognition and sensitivity analysis were employed to examine metabolomic small molecule profiles and transcriptomic gene expression data generated from liver and white skeletal muscle of hybrid striped bass (white bass Morone chrysops x striped bass M. saxatilis) representative of the top and bottom 10 % by body size of a production cohort. RESULTS: Larger fish (good-growth) had significantly greater weight, total length, hepatosomatic index, and specific growth rate compared to smaller fish (poor-growth) and also had significantly more muscle fibers of smaller diameter (≤ 20 µm diameter), indicating active hyperplasia. Differences in metabolomic pathways included enhanced energetics (glycolysis, citric acid cycle) and amino acid metabolism in good-growth fish, and enhanced stress, muscle inflammation (cortisol, eicosanoids) and dysfunctional liver cholesterol metabolism in poor-growth fish. The majority of gene transcripts identified as differentially expressed between groups were down-regulated in good-growth fish. Several molecules associated with important growth-regulatory pathways were up-regulated in muscle of fish that grew poorly: growth factors including agt and agtr2 (angiotensins), nicotinic acid (which stimulates growth hormone production), gadd45b, rgl1, zfp36, cebpb, and hmgb1; insulin-like growth factor signaling (igfbp1 and igf1); cytokine signaling (socs3, cxcr4); cell signaling (rgs13, rundc3a), and differentiation (rhou, mmp17, cd22, msi1); mitochondrial uncoupling proteins (ucp3, ucp2); and regulators of lipid metabolism (apoa1, ldlr). Growth factors pttg1, egfr, myc, notch1, and sirt1 were notably up-regulated in muscle of good-growing fish. CONCLUSION: A combinatorial pathway analysis using metabolomic and transcriptomic data collectively suggested promotion of cell signaling, proliferation, and differentiation in muscle of good-growth fish, whereas muscle inflammation and apoptosis was observed in poor-growth fish, along with elevated cortisol (an anti-inflammatory hormone), perhaps related to muscle wasting, hypertrophy, and inferior growth. These findings provide important biomarkers and mechanisms by which growth is regulated in fishes and other vertebrates as well.

Monitoring post-spawning movement, habitat use, and survival of adult anadromous rainbow smelt using acoustic telemetry in a New Hampshire estuary.

Anadromous rainbow smelt (Osmerus mordax, [Mitchill 1814]) are found along the northeast Atlantic coastline of North America, with their range now limited to north of Cape Cod, Massachusetts, USA. Although their anadromous life cycles are described broadly, gaps remain regarding how adult rainbow smelt use estuaries post-spawning, including movement behaviors, habitats used, and specific timing of emigration to coastal waters. In spring 2021, we used acoustic telemetry to characterize movements during and after the spawning season of rainbow smelt captured in tributaries to Great Bay, New Hampshire, USA, a large estuarine system near the southern edge of their range. Forty-four adult rainbow smelt (n = 35 male, n = 9 female) were tagged with Innovasea V5 180-kHz transmitters and an array of 22,180 kHz VR2W receivers were deployed throughout Great Bay to detect movements of tagged fish from March to October 2021. Rainbow smelt were detected 14,186 times on acoustic telemetry receivers, with 41 (93%) of the tagged individuals being detected at least once post-tagging. Individuals were detected moving between tributaries, revealing that rainbow smelt can use multiple rivers during the spawning season (March-April). Mark-recapture Cormack-Jolly-Seber models estimated 83% (95% confidence interval 66%-92%) of rainbow smelt survived to the mainstem Piscataqua River, and a minimum of 50% (22 of 44) reached the seaward-most receivers and were presumed to have survived emigration. Most individuals that survived remained in the estuary for multiple weeks (average = 19.47 ± 1.99 standard error days), displaying extended use of estuarine environments. Downstream movements occurred more frequently during ebb tides and upstream movements with flood tides, possibly a mechanism to reduce energy expenditures. Fish emigrated from the estuary by mid-May to the coastal Gulf of Maine. Our results underscore that rainbow smelt need access to a variety of habitats, including multiple tributaries and high-quality estuarine habitat, to complete their life cycle.

The effects of stress, cortisol administration and cortisol inhibition on black sea bass (Centropristis striata) sex differentiation.

Sex differentiation in many lower vertebrates (e.g. reptiles, amphibians, and fishes) can be influenced by environmental factors experienced during sensitive developmental periods. Environmental stressors, acting through cortisol, masculinize some teleost fishes during development by limiting gonadal cytochrome P450 aromatase (cyp19a1a), the enzyme that irreversibly converts testosterone to 17ß-estradiol. In this study, we examined the influence of cortisol, cortisol inhibitors and a repeated, acute stressor (net-chasing) on sex differentiation in black sea bass (BSB; Centropristis striata), a protogynous hermaphroditic teleost. Wild-caught, sexually-undifferentiated, BSB juveniles (~90 mm) were collected from Rhode Island waters, raised in recirculating systems and fed diets supplemented with cortisol, a cortisol receptor antagonist (mifepristone), a cortisol synthesis inhibitor (metyrapone), or net-chased twice a week for two min until gonads were differentiated (77-89 days). Long term cortisol administration partially masculinized all female fish, but repeated net-chasing did not alter sex differentiation relative to the control group. Blocking cortisol receptor binding delayed sex differentiation in some individuals, but overall led to increased masculinization compared to control fish. The proportion of treatment fish that developed as males suggests a functionally, diandric protogynous reproductive strategy in this species. We also identified a glucocorticoid response element in the gonadal aromatase (cyp19a1a) promoter, indicating a possible relationship between cortisol and cyp19a1a gene expression.

Initiation of sex change and gonadal gene expression in black sea bass (Centropristis striata) exposed to exemestane, an aromatase inhibitor.

Many teleost fishes exhibit sequential hermaphroditism, where male or female gonads develop first and later undergo sex change. Model sex change species are characterized by social hierarchies and coloration changes, which enable experimental manipulations to better understand these processes. However, other species such as the protogynous black sea bass (Centropristis striata) do not exhibit these characteristics and instead receive research attention due to their importance in fisheries or aquaculture. Black sea bass social structure is unknown, which makes sex change sampling difficult, and few molecular resources are available. The purpose of the present study was to induce sex change using exemestane, an aromatase inhibitor, and assess gonadal gene expression using sex markers (amh, zpc2) and genes involved in steroidogenesis (cyp19a1a, cyp11b), estrogen signaling (esr1, esr2b), and apoptosis or atresia (aen, casp9, fabp11, parg, pdcd4, rif1). Overall, dietary exemestane treatment was effective, and most exposed females exhibited early histological signs of sex change and significantly higher rates of ovarian atresia relative to control females. Genes associated with atresia did not reflect this, however, as expression patterns in sex changing gonads were overall similar to those of ovaries, likely due to a whole ovary dilution effect of the RNA. Still, small but insignificant expression decreases during early sex change were detected for ovary-related genes (aen, casp9, fabp11, zpc2) and anti-apoptotic factors (parg, rif1). Exemestane treatment did not impact spermatogenesis or testicular gene expression, but testes were generally characterized by elevated steroidogenic enzyme and estrogen receptor mRNAs. Further research will be needed to understand these processes in black sea bass, using isolated ovarian follicles and multiple stages of sex change.

The effects of temperature on ovarian aromatase (cyp19a1a) expression and sex differentiation in summer flounder (Paralichthys dentatus).

Female summer flounder grow considerably faster and larger than males, and a tremendous increase in performance can therefore be realized through production of monosex female populations. Rearing temperature has been shown to affect sex differentiation in other teleost species by influencing expression of genes encoding transcription factors or enzymes involved in endocrine function. Cyp19a1a is a well-studied gene that had been shown to play a role in ovarian development, and exhibits sexually dimorphic expression in other species. In the present study, summer flounder (37 days post-hatch; DPH) were raised at 13, 16 or 19 °C. Fish from all three treatments were sampled throughout development and analyzed in qPCR to determine cyp19a1a gene expression levels. Sex ratios of additional fish grown to ≥150 mm at each temperature treatment were determined. Low female production was achieved overall (26.9, 17.6 and 0% at 13, 16 and 19 °C, respectively). Cyp19a1a expression was significantly lower at 52 DPH (~15 mm total length) at the male-producing temperature (19 °C) and increased to similar levels as other treatments at 66 DPH. Expression levels later in juvenile development (66-191 DPH) largely decreased with fish size. The period of sex differentiation in summer flounder remains unknown, but cyp19a1a expression patterns suggest that it may occur earlier in development than that of congenerics. Further research is necessary to understand the sex-determining mechanisms in this species before sexually dimorphic growth can be used to achieve economic advantages in commercial production.

Brain aromatase (cyp19a1b) and gonadotropin releasing hormone (gnrh2 and gnrh3) expression during reproductive development and sex change in black sea bass (Centropristis striata).

Teleost fish exhibit diverse reproductive strategies, and some species are capable of changing sex. The influence of many endocrine factors, such as gonadal steroids and neuropeptides, has been studied in relation to sex change, but comparatively less research has focused on gene expression changes within the brain in temperate grouper species with non-haremic social structures. The purpose of the present study was to investigate gonadotropin releasing hormone (GnRH) and brain aromatase (cyp19a1b) gene expression patterns during reproductive development and sex change in protogynous (female to male) black sea bass (Centropristis striata). Partial cDNA fragments for cyp19a1b and eef1a (a reference gene) were identified, and included with known gnrh2 and gnrh3 sequences in real time quantitative PCR. Elevated cyp19a1b expression was evident in the olfactory bulbs, telencephalon, optic tectum, and hypothalamus/midbrain region during vitellogenic growth, which may indicate changes in the brain related to neurogenesis or sexual behavior. In contrast, gnrh2 and gnrh3 expression levels were largely similar among gonadal states, and all three genes exhibited stable expression during sex change. Although sex change in black sea bass is not associated with dramatic changes in GnRH or cyp19a1b gene expression among brain regions, these genes may mediate processes at other levels, such as within individual hypothalamic nuclei, or through changes in neuron size, that warrant further research.

Identification and expression of GnRH2 and GnRH3 in the black sea bass (Centropristis striata), a hermaphroditic teleost.

We cloned two cDNAs for two gonadotropin-releasing hormones, GnRH2 (chicken GnRH-II) and GnRH3 (salmon GnRH), respectively, from the black sea bass (Centropristis striata). Black sea bass are protogynous hermaphroditic teleosts that change from females to males between 2 and 5 years of age. Similar to other GnRH precursors, the precursors of black sea bass GnRH2 and GnRH23 consisted of a signal peptide, decapeptide, a downstream processing site, and a GnRH-associated peptide. Our analyses failed to identify GnRH1. GnRH3 precursor transcript was more widely distributed in a variety of tissues compared with GnRH2. Further examination of GnRH expression and gonadal histology was done in black sea bass from three different size groups: small (11.4-44.1 g), medium (179.4-352.2 g) and large (393.8-607.3 g). Interestingly, GnRH3 expression occurred only in the pituitaries of males in the small and medium groups compared with expression of GnRH2. Future functional studies of the sea bass GnRHs will be valuable in elucidating the potential underlying neuroendocrine mechanisms of black sea bass reproduction and may ultimately contribute to management advances in this commercially important fish.

Identification of ovarian gene expression patterns during vitellogenesis in Atlantic cod (Gadus morhua).

Follicular maturational competence and ovulatory competence in teleost fish refer to the ability of the ovarian follicle to undergo final oocyte maturation and ovulation, respectively, in response to gonadotropin stimulation and other external cues. Some gene products related to competence acquisition are likely synthesized during vitellogenic growth, as these follicles gain in vivo responsiveness to exogenous gonadotropin stimulation and can be induced to undergo maturation and ovulation. In Atlantic cod (Gadus morhua), gonadotropin responsiveness has been shown to be oocyte size-dependent, and only ovaries containing late-stage vitellogenic follicles can be induced to ovulate. The purpose of the present study was to compare gene expression patterns between mid (unresponsive) and late (responsive) vitellogenic ovaries to identify genes involved in gonadotropin responsiveness and the acquisition of maturational and ovulatory competencies. Representational difference analysis was conducted in two reciprocal comparisons using intact ovarian fragments and follicle wall-enriched tissues, and genes of interest were used in real time quantitative PCR to confirm differential expression. Few differences were detected in intact ovarian fragments, but type IV ice-structuring protein and gephyrin were upregulated later in development and may be involved in lipid and sulfur metabolism, respectively. Candidate gene assays for luteinizing hormone receptor and aromatase also exhibited significant upregulation during vitellogenesis. Many genes were differentially expressed in follicle wall-enriched tissues, including endocrine maturational regulators and smooth muscle genes. Overall, maturational and ovulatory competencies during vitellogenesis in Atlantic cod are associated with up- and downregulation of many genes involved in lipid metabolism, endocrine regulation, and ovulatory preparation.

Characterizing ovarian gene expression during oocyte growth in Atlantic cod (Gadus morhua).

Vertebrate oocytes undergo dramatic changes during development as they accumulate many RNA transcripts, glycoproteins, and yolk proteins, necessary to ensure proper fertilization and embryogenesis. Oogenesis in teleosts often requires several years for completion, but very little is known about the early developmental stages. Recently, two-stage gene expression comparisons were made during oocyte growth in coho salmon (Oncorhynchus kisutch) and Atlantic cod (Gadus morhua), but more broad-scale, comprehensive assessments have not been conducted. The objectives of the present study were to characterize the gene expression patterns throughout oocyte growth in cod and compare them to changes previously identified in coho salmon. A quantitative PCR survey was conducted using 50 genes at six ovarian stages, ranging from the onset of primary growth (oocyte differentiation) to late vitellogenesis. Most expression patterns could be grouped into three major clusters, consisting of oocyte-derived (cluster 1) and likely follicle cell (clusters 2 and 3) genes. Oocyte genes were elevated during primary growth, while many follicle cell transcripts were abundant during oocyte differentiation and vitellogenesis. Few expression changes identified in coho salmon were evident in cod, which is likely due to differences in reproductive strategies. These results demonstrate that dynamic changes in gene expression occur during oocyte growth in teleost fish.