Mechanisms of Egg Yolk Formation and Implications on Early Life History of White Perch (Morone americana).

The three white perch (Morone americana) vitellogenins (VtgAa, VtgAb, VtgC) were quantified accurately and precisely in the liver, plasma, and ovary during pre-, early-, mid-, and post-vitellogenic oocyte growth using protein cleavage-isotope dilution mass spectrometry (PC-IDMS). Western blotting generally mirrored the PC-IDMS results. By PC-IDMS, VtgC was quantifiable in pre-vitellogenic ovary tissues and VtgAb was quantifiable in pre-vitellogenic liver tissues however, neither protein was detected by western blotting in these respective tissues at this time point. Immunohistochemistry indicated that VtgC was present within pre-vitellogenic oocytes and localized to lipid droplets within vitellogenic oocytes. Affinity purification coupled to tandem mass spectrometry using highly purified VtgC as a bait protein revealed a single specific interacting protein (Y-box binding protein 2a-like [Ybx2a-like]) that eluted with suramin buffer and confirmed that VtgC does not bind the ovary vitellogenin receptors (LR8 and Lrp13). Western blotting for LR8 and Lrp13 showed that both receptors were expressed during vitellogenesis with LR8 and Lrp13 expression highest in early- and mid-vitellogenesis, respectively. The VtgAa within the ovary peaked during post-vitellogenesis, while VtgAb peaked during early-vitellogenesis in both white perch and the closely related striped bass (M. saxatilis). The VtgC was steadily accumulated by oocytes beginning during pre-vitellogenesis and continued until post-vitellogenesis and its composition varies widely between striped bass and white perch. In striped bass, the VtgC accounted for 26% of the vitellogenin-derived egg yolk, however in the white perch it comprised only 4%. Striped bass larvae have an extended developmental window and these larvae have yolk stores that may enable them to survive in the absence of food for twice as long as white perch after hatch. Thus, the VtgC may play an integral role in providing nutrients to late stage fish larvae prior to the onset of exogenous feeding and its composition in the egg yolk may relate to different early life histories among this diverse group of animals.

Machine learning reveals sex-specific 17ß-estradiol-responsive expression patterns in white perch (Morone americana) plasma proteins.

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17ß-estradiol (E2 ) induction. Semiquantitative nanoLC-MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two-way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E2 -induced plasma samples using the protein expression data. E2 -induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain-containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E2 -responsive proteins in fishes and therefore may be useful indicators of estrogen induction.

Compartment proteomics analysis of white perch (Morone americana) ovary using support vector machines.

Compartment proteomics enable broad characterization of target tissues. We employed a simple fractionation method and filter-aided sample preparation (FASP) to characterize the cytosolic and membrane fractions of white perch ovary tissues by semiquantitative tandem mass spectrometry using label-free quantitation based on normalized spectral counts. FASP depletes both low-molecular-weight and high-molecular-weight substances that could interfere with protein digestion and subsequent peptide separation and detection. Membrane proteins are notoriously difficult to characterize due to their amphipathic nature and association with lipids. The simple fractionation we employed effectively revealed an abundance of proteins from mitochondria and other membrane-bounded organelles. We further demonstrate that support vector machines (SVMs) offer categorical classification of proteomics data superior to that of parametric statistical methods such as analysis of variance (ANOVA). Specifically, SVMs were able to perfectly (100% correct) classify samples as either membrane or cytosolic fraction during cross-validation based on the expression of 242 proteins with the highest ANOVA p-values (i.e., those that were not significant for enrichment in either fraction). The white perch ovary cytosolic and membrane proteomes and transcriptome presented in this study can support future investigations into oogenesis and early embryogenesis of white perch and other members of the genus Morone.

Masculinizing effect of background color and cortisol in a flatfish with environmental sex-determination.

Environmental sex-determination (ESD) is the phenomenon by which environmental factors regulate sex-determination, typically occurring during a critical period of early development. Southern flounder (Paralichthys lethostigma) exhibit temperature-dependent sex-determination that appears to be restricted to the presumed XX female genotype with the extremes of temperature, both high and low, skewing sex ratios toward males. In order to evaluate other environmental factors that may influence sex-determination, we investigated the influence of background color and cortisol on sex-determination in southern flounder. Experiments involving three sets of tanks, each painted a different color, were conducted at different temperatures using southern flounder of mixed XX-XY genotype. The studies involved rearing juvenile southern flounder in either black, gray, or blue tanks and sex-determination was assessed by gonadal histology. In both studies, blue tanks showed significant male-biased sex ratios (95 and 75% male) compared with black and gray tanks. The stress corticosteroid cortisol may mediate sex-determining processes associated with environmental variables. Cortisol from the whole body was measured throughout the second experiment and fishes in blue tanks had higher levels of cortisol during the period of sex-determination. These data suggest that background color can be a cue for ESD, with blue acting as a stressor during the period of sex-determination, and ultimately producing male-skewed populations. In a separate study using XX populations of southern flounder, cortisol was applied at 0, 100, or 300 mg/kg of gelatin-coated feed. Fish were fed intermittently prior to, and just through, the period of sex-determination. Levels of gonadal P450 aromatase (cyp19a1) and forkhead transcription factor L2 (FoxL2) messenger RNA (mRNA) were measured by qRT-PCR as markers for differentiation into females. Müllerian-inhibiting substance mRNA was used as a marker of males' gonadal development. Control fish showed female-biased sex ratios approaching 100%, whereas treatment with 100 mg/kg cortisol produced 28.57% females and treatment with 300 mg/kg cortisol produced only 13.33% females. These results suggest that cortisol is a critical mediator of sex-determination in southern flounder by promoting masculinization. This linkage between the endocrine stress axis and conserved sex-determination pathways may provide a mechanism for adaptive modification of sex ratio in a spatially and temporally variable environment.

Sex determination in flatfishes: Mechanisms and environmental influences.

Flounder of the genus Paralichthys exhibit a unique mode of sex determination where both low and high temperatures induce male-skewed sex ratios, while intermediate temperatures produce a 1:1 sex ratio. Male differentiation is thus easily induced in genetic females creating a combination of genetic (GSD) and environmental sex determination (ESD). Since male flounder become reproductively fit at substantially smaller body sizes than females, temperature or other environmental variables that elicit lower growth rates may also influence sex differentiation toward male development. This review covers our current knowledge of sex determination and differentiation in flatfishes including possible adaptive significance of ESD and involvement of factors such as aromatase (cyp19).

Temperature affects insulin-like growth factor I and growth of juvenile southern flounder, Paralichthys lethostigma.

Temperature profoundly influences growth of heterothermic vertebrates. However, few studies have investigated the effects of temperature on growth and insulin-like growth factor I (IGF-I) in fishes. The aim of this study was to examine effects of temperature on growth and establish whether IGF-I may mediate growth at different temperatures in southern flounder, Paralichthys lethostigma. In two experiments, juvenile flounder were reared at 23 and 28 degrees C and growth was monitored for either 117 or 197 days. Growth was similar across treatments in both experiments until fish reached approximately 100 mm total length. Body size then diverged with fish at 23 degrees C ultimately growing 65-83% larger than those at 28 degrees C. Muscle IGF-I mRNA, plasma IGF-I, and hepatosomatic index (HSI) were significantly higher in flounder at 23 degrees C, whereas hepatic IGF-I mRNA abundance did not differ with treatment. Muscle IGF-I mRNA was correlated with HSI, while plasma IGF-I was correlated with body size, hepatic IGF-I mRNA, and HSI. These results demonstrate a strong effect of temperature on flounder growth and show that temperature-induced variation in growth is associated with differences in systemic IGF-I and local (i.e., muscle) IGF-I mRNA levels. The results also support the use of plasma IGF-I and HSI as indicators of flounder growth status.

Aromatase cytochrome P450: cloning, intron variation, and ontogeny of gene expression in southern flounder (Paralichthys lethostigma).

Aromatase cytochrome P450 (P450arom) is the enzyme complex responsible for conversion of androgens to estrogens in vertebrates. Consequently, in some fishes its activity appears critical to ovarian differentiation. Southern flounder (Paralichthys lethostigma) is a commercially important flatfish in which females grow larger than males and sex determination is temperature sensitive. Through cloning of the P450arom gene in ovary and quantitative reverse transcription-polymerase chain reaction, we developed a biomarker for early female differentiation in southern flounder. The deduced amino acid sequence for southern flounder P450arom is similar to other teleosts. Comparison of P450arom intron sequences from fish of different populations revealed substantial inter-individual variation. Adult ovary and spleen exhibited high levels of P450arom mRNA, while P450arom mRNA was only weakly detected in testes. Brain, liver, intestine, kidney, gill, muscle, and heart showed little or no P450arom mRNA expression. Gonads of wild and hatchery-produced juvenile flounder of sizes spanning the period of sex differentiation initially exhibited low levels of P450arom mRNA followed by increases in some individuals and bifurcation into two clearly segregated groups (i.e., putative males and females) beginning at approximately 65 mm in total length. Gonadal histology confirmed predictions of sex based on P450arom expression in juvenile flounder, demonstrating that the patterns of P450arom expression observed relate to sex-specific differentiation. This research represents a unique approach to assessing sex differentiation in a natural population, and a powerful technique for better understanding mechanisms of flounder sex determination and rapidly defining conditions for controlling sex for aquaculture.