Harry V. Daniels

Gonadal differentiation and effects of temperature on sex determination in southern flounder (Paralichthys lethostigma)

Abstract Southern flounder ( Paralichthys lethostigma ) support valuable North American fisheries and show great promise for aquaculture. Because females grow faster and reach larger adult sizes than males, monosex culture of females is desirable for commercial operations. A detailed understanding of sexual development and its timing is critical to control sex and optimize culture. Structural and cellular sex-distinguishing markers were identified histologically, and then used to describe ovarian development in female and testicular development in male flounder. In presumptive ovaries of southern flounder, development of an ovarian cavity first occurs in fish ranging from 75 to 100 mm total length (TL). This is considerably delayed relative to that observed in the Japanese congener, Paralichthys olivaceus , where an ovarian cavity is seen in fish as small as 40 mm TL. The smallest southern flounder that possessed primary oocytes in the early perinucleolus stage was 115 mm TL. In presumptive testes, the formation of seminiferous tubules first occurs in fish of approximately 100 mm TL. Spermatogonia remained quiescent until most fish were over 100 mm TL. Overall, gonads from southern flounder greater than 120 mm TL commonly possess gonial cells undergoing meiosis, clearly differentiating sex. The effect of temperature on sex determination in southern flounder was addressed in a separate experiment. Juvenile southern flounder were grown at 18, 23, or 28°C for 245 days. High and low temperatures induced phenotypic sex reversal in juvenile southern flounder, producing a higher proportion of males (96% males at high temperature, P P P. olivaceus , but possibly shifted towards warmer temperatures. These findings indicate that sex differentiation in southern flounder is distinguishable in most fish by 100–120 mm TL and that sex determination is sensitive to temperature. This information is critical to the development of strategies to maximize the number of faster-growing females for commercial flounder culture.

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).

An evaluation of co-extruded poultry silage and culled jewel sweet potatoes as a feed ingredient for hybrid Tilapia (Oreochromis niloticus×O. mossambicus)

Abstract Two experiments were conducted with hybrid tilapia to evaluate a meal made by extrusion co-processing culled sweet potatoes and poultry mortality silage (ESPFP). In both experiments, dried, cull sweet potatoes and fermented whole turkey carcasses (60:40 ratio, w/w wet basis) were co-extruded, dried, and hammer-milled to make the ESPFP test ingredient. The resulting ESPFP meal was included at 0%, 11%, 22%, and 33% by weight in isocaloric, isonitrogenous pelleted diets. In an 87-day growth trial, no significant differences were found in the growth parameters or carcass (market) yields among the treatment groups. Tank water quality parameters were also unaffected by inclusion of the experimental ingredient. A consumer panel found no significant differences in the sensory indices (aroma, flavor, and texture) of the fillets from fish fed with the graded levels of ESPFP in the diet versus those from fish fed the control diet. The digestibility of the diets containing the ESPFP meal was evaluated in a second trial. The apparent dry matter (DM), gross energy (GE), and crude protein (CP) digestibility coefficients were reduced linearly with increasing levels of the ESPFP meal in the diets (P

Effect of dietary protein level on growth and blood parameters in summer flounder, Paralichthys dentatus.

Abstract Summer flounder, Paralichthys dentatus, were fed diets with various protein levels (36.6%, 40.0%, 47.7%, 51.9%, and 56.5%), and the effect of these levels on growth and blood parameters was measured. Fish fed the highest protein level gained significantly (P< 0.05) more weight than those fed lower levels of protein. There were no significant (P> 0.05) differences among mean feed conversion ratios (FCR), protein efficiency ratios (PER), net protein utilization (NPU), or mean percent total body moisture, protein, lipid, or ash content. Fish fed diets with 52 or 56% protein had red blood cell counts that were significantly (P< 0.05) higher than fish fed diets with lower protein levels. In addition, hematocrits were significantly (P< 0.001) correlated to dietary protein. There were no significant differences among mean total protein, triglyceride, glucose or phosphate. However, blood calcium levels varied with diet. Data indicate that summer flounder protein requirements are near those suggested for other flatfish.

Weaning Success of Southern Flounder Juveniles: Effect of Changeover Period and Diet Type on Growth and Survival

Abstract Two studies were conducted to determine the effects of changeover period duration and diet type (dry versus semimoist) on growth and survival of postmetamorphic southern flounder Paralichthys lethostigma during weaning from live feed onto formulated feeds. In the first study, feeding with live brine shrimp Artemia sp. was abruptly stopped after changeover periods of 5, 10, 15, and 20 d. A changeover period of 20 d resulted in significantly higher (P 0.05) from the other treatments or the control (live brine shrimp). In the second study, feeding with live brine shrimp was gradually reduced from 100% to 0% over a 12-d changeover period. Brine shrimp were replaced with either a dry feed or a semimoist feed. Survival of fish fed either the dry feed (71.1 ± 30.1%) or the semimoist feed (71.1 ± 15.4%) was not si...

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.

Effects of Temperature on Egg Hatch, Larval Growth and Metamorphosis for Hatchery-Cultured Southern Flounder, Paralichthys lethostigma

ABSTRACT Egg hatch, larval growth, and metamorphosis of southern flounder, Paralichthys lethostigma, were examined at 13, 17, 21, and 25°C in laboratory experiments. The experiments were separated into four developmental phases: (1) from fertilization until hatch; (2) from hatch until mouth opening; (3) from first feeding until the onset of metamorphosis; (4) from the onset until the completion of metamorphosis. Time to egg hatch were 109, 58, 39, 30 hours at 13, 17, 21, and 25°C, respectively. Size at hatch varied little among temperatures, while percent hatch was significantly higher at 17°C (P < 0.05). There was no significant difference in total length at mouth opening among temperatures (P > 0.05). However, those larvae raised at higher temperatures reached mouth opening earlier, as it took on average 7.4, 3.9, 2.8, and 2.2 days from hatch at 13, 17, 21, and 25°C, respectively. During phase three, the growth rate was optimal at 17°C, as growth rates were 0.046, 0.110, 0.106 and 0.096 mm/day at 13, 17, 21, and 25°C, respectively. Larvae in phase four completed metamorphosis sooner at higher temperatures than at lower temperatures. It took 26.4, 15.7, and 13.1 days to complete metamorphosis at 17, 21, and 25°C, respectively, while only 16% of those at 13 progressed to stage “G” within the 27 days that phase four encompassed. No difference in total length was found among treatments (P > 0.05) at the completion of metamorphosis, but fish in the 21 °C treatment had a significantly higher survival (P < 0.05). It can be concluded that southern flounder could be produced in the highest quantity and at the largest size by raising them at 17°C from fertilization until the onset of metamorphosis, and at 21 °C during metamorphosis.

A Practical Guide to the Morphological Development of Southern Flounder, Paralichthys lethostigma from Hatch Through Metamorphosis

Abstract Southern flounder (Paralichthys lethostigma) larvae were cultured at 17°C and observations were made to describe the morphological development from hatch until the completion of metamorphosis. A complete nine-part staging scheme was described with diagrams to show how larvae transform from a pelagic form with eyes on opposite sides of the body to a benthic form with both eyes on the left side of the body. The descriptions track the development of features that are easy to identify under low power microscopy. Behavioral patterns are included for each stage, particularly as they pertain to feeding. Such a staging scheme will help hatchery managers monitor development and confirm normal morphology and behavior.

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 17β-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 17β‐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.