Trypsinogen expression during the development of the exocrine pancreas in winter flounder (Pleuronectes americanus)

Histological, biochemical and molecular techniques were used to describe the functional development of the pancreas in winter flounder (Pleuronectes americanus) with specific reference to the expression of three trypsinogen genes. The pancreas was identified shortly following hatch, appearing as a compact structure situated dorsal and slightly posterior to the liver. As the larval fish approached metamorphosis, the pancreas became diffuse, spreading throughout the mesentery surrounding the stomach, the upper intestine and the pyloric caecae. Trypsin 2 expression was detected from 5 days post-hatch (dph). Two other related trypsinogen genes isolated from the pyloric caecae (Trypsin 1) and the intestine (Trypsin 3) showed contrasting results. Trypsin 1 showed very low levels of expression and only in late larval stages and metamorphosis. Trypsin 3 showed expression only after 20 dph. In order to determine tissue-specific expression of the three trypsinogen genes, the RNA from seven gastrointestinal-associated tissues was examined. Trypsin 1 and Trypsin 2 expression was most notably associated with the pyloric caecae, cardiac stomach, pyloric stomach and the rectum, although some variation in expression level between tissues was observed. Trypsin 3 expression had a narrower tissue distribution and was only associated with the pyloric caecae and the rectum. The tissue expression patterns observed here are likely due in part to the diffuse nature of the pancreas. Trypsin-like activity was evident from hatch and continued at significant levels through to at least 25 dph.     

The role of N and C termini in the antifreeze activity of winter flounder (Pleuronectes americanus) antifreeze proteins

Antifreeze proteins (AFPs) are found in many marine fish and have been classified into five biochemical classes: AFP types I-IV and the antifreeze glycoproteins. Type I AFPs are alpha-helical, partially amphipathic, Ala-rich polypeptides. The winter flounder (Pleuronectes americanus) produces two type I AFP subclasses, the liver-type AFPs (wflAFPs) and the skin-type AFPs (wfsAFPs), that are encoded by distinct gene families with different tissue-specific expression. wfsAFPs and wflAFPs share a high level of identity even though the wfsAFPs have approximately half the activity of the wflAFPs. Synthetic polypeptides based on two representative wflAFPs and wfsAFPs were generated to examine the role of the termini in antifreeze activity. Through systematic exchange of N and C termini between wflAFP-6 and wfsAFP-2, the termini were determined to be the major causative agents for the variation in activity levels between the two AFPs. Furthermore, the termini of wflAFP-6 possessed greater helix-stabilizing ability compared with their wfsAFP-2 counterparts. The observed 50% difference in activity between wflAFP-6 and wfsAFP-2 can be divided into approximately 20% for differences at each termini and approximately 10% for differences in the core. Furthermore, the N terminus was determined to be the most critical component for antifreeze activity.     

Developmental fate of the yolk protein lipovitellin in embryos and larvae of winter flounder, Pleuronectes americanus.

The developmental fate of the vitellogenin-derived yolk protein, lipovitellin (Lv), was investigated in winter flounder embryos and yolk-sac larvae. Since Lv is present as only one major polypeptide in ovulated winter flounder eggs, unlike the multiple yolk polypeptides found in the mature eggs of most teleosts, this system is presented as a simpler model of yolk protein structure and utilization during teleostean development. Winter flounder Lv is cleaved during embryogenesis from a 94 kD polypeptide at fertilization to 67 kD and 26 kD polypeptides at hatching. The rate of this proteolytic processing is slow during early embryonic development, but enters a more rapid phase between days 8 and 12 post-fertilization in embryos reared at 4-5 degrees C, and approaches 50% completion at day 10. Lv processing is essentially complete 3 days before hatching; nevertheless, major degradation of the Lv peptide by the developing winter flounder does not occur until after hatching. The Stokes radius of Lv changes only moderately following processing, from 4.50 nm in unfertilized eggs to 4.19 nm in late embryos and newly hatched larvae, whereas the processed Lv retains its heat stability relative to other yolk polypeptides. Nearly 50% of its lipid content, however, is released from the Lv particle during embryogenesis, concomitant with cleavage of the Lv 94 kD polypeptide. Lv processing may thus render a portion of the yolk protein-associated lipid more accessible to the developing embryo, whereas other yolk components are retained for later use by the winter flounder larva. Alternately, removal of lipid may lead to proteolytic vulnerability of the Lv polypeptide. In either case, only a portion of the lipid moiety of the Lv particle appears to play a significant nutritive role for the embryo, whereas its protein component is reserved for larval use. J. Exp. Zool. 284:686-695, 1999.     

Bifurcation of gill filaments in winter flounder (Pleuronectes americanus Walbaum) from Long Island Sound

Morphological and histological examinations of gills excised from adult winter flounder, Pleuronectes americanus Walbaum, collected at clean and contaminated areas of Long Island Sound were undertaken to assess possible biological consequences of contamination. On the basis of previous studies, three collection sites were chosen: Shoreham, New York, a relatively clean, unindustrialized area, and New Haven Harbor, Connecticut and Hempstead Harbor, New York, both industrialized and heavily populated.
Gill samples were taken monthly at all three sites for light and scanning electron microscopy (SEM) examination. Results from both techniques suggest a relationship between contamination of the sediments and the prevalence of bifurcated gill filaments, the condition being most severe at New Haven Harbor. The bifurcations were not consistently associated with any parasitological or pathological conditions when examined by light microscopy.
Gill samples were subsequently taken from juvenile winter flounder caught in New Haven Harbor to determine how early in the life cycle gill bifurcations develop. The data show that the majority of these anomalies begin in juvenile flounder rather than in embryos, larvae, or adults. Gill bifurcations were found in 27% of the 2-year-old flounder, compared to 12% of the 1-year-old fish and 15% of the adults from the same area.     

Cardiac function and critical swimming speed of the winter flounder (Pleuronectes americanus) at two temperatures

Using Transonic flow probes and a uniquely designed swimming flume, we directly measured cardiac parameters (Q, cardiac output; SV, stroke volume; and fH, heart rate) in winter flounder (Pleuronectes americanus) before and during critical swim speed (Ucrit) tests at 4 and 10 degrees C. Resting Q, SV and fH averaged 9.8 ml min(-1) kg(-1), 0.5 ml kg(-1) (1.0 ml g ventricle(-1)) and 21 beats min(-1) at 4 degrees C and 15.5 ml min(-1) kg(-1), 0.5 ml kg(-1) (0.95 ml g ventricle(-1)) and 34 beats min(-1) at 10 degrees C (Q10 values of 2.13, 0.91 and 2.35, for Q, SV and fH, respectively). Cardiac output, SV and fH increased by approx. 170%, 70% and 60% at both temperatures during the Ucrit test. However, cardiac parameters generally reached near maximal levels almost immediately upon swimming and remained at these levels until Ucrit (0.65 +/- 0.06 bl s(-1) at 4 degrees C and 0.73 +/ -0.07 bl s(-1) at 10 degrees C). This rapid rise in cardiac function to near maximal levels did not appear to be the result of stress alone, as Q only fell slightly when flounder were swum for 75 min at < 0.4 bl s(-1), speeds at which they appeared to swim comfortably. Our results suggest that both Q and Ucrit have been significantly overestimated in flatfishes, and that "lift-off"/slow swimming is energetically expensive. Furthermore, they show that maximum and resting stroke volume (per g of ventricle) are extremely high in the flounder as compared with other teleosts.     

Lymphocystis disease in the winter flounder, Pseudopleuronectes americanus.

Nodular lesions on the fins of winter flounder, Pseudopleuronemphocystis disease. Hypertrophied, encapsulated connective tissue cells contained cytoplasmic inclusions composed of icosahedral virus particles. The winter flounder is a new host for lymphocystis disease and is the second flatfish in the western North Atlantic to have the disease.     

Localization of vitellogenin-related protein in the developing oocytes of winter flounder (Pleuronectes americanus) by protein A-gold immunocytochemistry

Vitellogenin-related proteins were localized within the oocytes of a marine teleost, the winter flounder, using protein A-gold immunocytochemical electron microscopy. A homologous vitellogenin antisera was used along with 15-nm protein-A gold on thin sections of oocytes. Localization was significantly confined to organelles described as yolk spheres within the oocyte cytoplasm. This technique provides specific identification of yolk-containing organelles within the developing winter flounder oocyte.     

A critical period for nutritional control of early gametogenesis in female winter flounder, Pleuronectes americanus (Pisces: Teleostei)

When female winter flounder ( Pleuronectes americanus ) were subjected to periods of satiation feeding alternating with starvation it was found that the first part of their normal six-month feeding period could be associated with subsequent gametogenesis, whereas feeding in the later part of the normal feeding period was not necessary. Conversely if females were not fed during the first part of the normal feeding season they were likely to become non-reproductive, although high condition fish or a non-spawner could become gametogenic in spite of starvation at this time. Feeding restricted to the later part of the normal feeding season was therefore not generally associated with successful gametogenesis for the following year's spawn. When the first part of the feeding period was further subdivided with satiation feeding limited to one or more months within it, most fish became non-reproductive. The exceptions which became reproductive were females which had high post-spawn condition maintained in the month following the immediate spawning period for the individual fish. A nutritionally sensitive period for early gametogenesis in female winter flounder therefore appears to occur in the early part of the normal feeding season, close to the normal spawning period. The experimentally produced non-reproductive females were generally halted in a previtellogenic stage of development for the most advanced oocytes which is consistent with a nutritionally related inhibition of gametogenesis close to the previous spawning period.     

Seasonal changes in hepatocyte ultrastructure correlated with the cyclic synthesis of secretory proteins in the winter flounder (Pleuronectes americanus)

Liver tissue was sampled from flounder (Pleuronectes americanus) throughout the year with the intention of documenting changes in the ultrastructure coincident with the production and secretion of antifreeze proteins. In the winter, hepatocytes are dedicated to the production of these proteins and, in the female, also reproductive proteins. In both sexes, liver cells in the summer contain abundant lipid and glycogen stores. In the female, there is a conspicuous hepatocyte transformation from a fat-filled cell in the summer to one with well-developed rough endoplasmic reticulum in the winter. Large amounts of rough endoplasmic reticulum (11.2 mg/gm) were recovered after subcellular fractionation of female wintertime liver. The increased appearance of secretory organelles and the high number of nucleolar profiles observed in winter animals is consistent with the elevated demand for protein secretion and synthesis in both sexes. The fractional volumes occupied by lipid droplets and mitochondria were different when comparisons were made between sex and season. Females contained a greater volume of lipid than did males, and summer animals contained more lipid than those in winter.     

Epithelial cell proliferation in the intestine of the winter flounder,Pseudopleuronectes americanus

Summary To study epithelial cell proliferation in the North American flounder (Pseudopleuronectes americanus), fed and fasted fish received intravenous injections of ³H-thymidine and were killed 11/2 to 2 h later. Radioautographs of proximal, middle, and distal intestinal segments revealed proliferating epithelial cells at all levels of intestinal folds including the crest although labelled nuclei were most abundant in the epithelial cells on the lower half of folds and between folds. Mature appearing goblet cells with labelled nuclei were observed at all levels of the folds. The mean labelling index was greater in the epithelium of fed than fasted flounder. In fed flounder the mean labelling index was greatest in the proximal segment and least in the distal segment; no substantive differences in mean labelling indices were observed in the various segments of intestine from fasted fish. Electron microscopy revealed no major structural differences among epithelial cells along the base of folds compared to cells near the crest of folds. These findings indicate that 1) epithelial cell proliferation occurs at all levels of the folds of flounder intestine and is not compartmentalized to the base of the folds and interfold epithelium as reported in other teleosts, and 2) epithelial cell proliferation in the flounder intestine varies with feeding status.Supported be research grants AM 17537 and RR 05764 from the National Institutes of Health, Bethesda, Maryland and grant DEB7826821 AO1 from the National Science Foundation, Washington, D.C.The authors are grateful to Dr. Michael Field for stimulating discussions and suggestions and for providing facilities for collecting material from fish     

Individual variability in the epidermis of prespawning inshore winter flounder, Pseudopleuronectes americanus

Prespawning winter flounder, Pseudopleuronectes americanus Walbaum, manifest epidermal thickening as a secondary sexual characteristic, but with considerable individual variability. The correlation between epidermal thickness and fish size in post-spawned flounder is less apparent in prespawning flounders due to factors associated with sexual maturation. Thus, epidermal thickness in individual prespawning flounder displays significant correlation with gonadosomatic index and. in females, with the degree of final maturation of vitellogenic oocytes. However, both epidermal thickness and gonadosomatic index of individual flounders also display significant correlation with condition factor. It is proposed that body size and condition factor represent important primary physiological determinants influencing individual variability in seasonal epidermal thickness, and that factors associated with sexual maturation represent a secondary physiological determinant.     

Ion transport across the isolated intestinal mucosa of the winter flounder,Pseudopleuronectes americanus

The isolated intestinal mucosa of the flounder, Pseudopleuronectes americanus, when bathed in a 20 mM HCO3-Ringer's solution bubbled with 1% CO2 in O2, generated a serosa-negative PD and, when short-circuited, absorbed Cl at almost 3 times the rate of Na. Reducing HCO3 to 5 mM decreased the net Cl flux by more than 60%. The following results suggest that, despite the PD, Na and Cl transport processes are nonelectrically coupled: replacing all Na with choline abolished both the PD and net Cl flux; replacing all Cl with SO4 and mannitol abolished the PD and the net Na flux; and adding ouabain (to 0.5 mM) abolished the PD and the net Cl flux. Nearly all of the unidirectional serosa-to-mucosa Cl flux (JClsm) seemed to be paracellular since it varied with PD and Cl concentration in a manner consistent with simple diffusion. JClsm was only about one-fourth of JNasm, suggesting that the paracellular pathway is highly cation-selective. The data can be explained by the following model: (i) Na and Cl uptake across the brush border are coupled 1 : 1; Na is pumped into the lateral space and Cl follows passively, elevating the salt concentration there; (ii) the tight junction is permeable to Na but relatively impermeable to Cl; and (iii) resistance to Na diffusion is greater in the lateral space (considered in its entirety) than in the tight junction. If these assumptions are correct, the serosa-negative transmural PD is due mainly to a salt diffusion potential across the tight junction and, under short-circuit condition, most of the Na pumped into the lateral space diffuses back into the luminal solution, whereas most of the Cl enters the serosal solution. Morphological features of the epithelium support this interpretation: the cells are unusually long (60 micrometer); there is little distension of the apical 12 micrometer of the lateral space during active fluid absorption; and distension distal to this region is intermittently constricted by desmosomes.     

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder (Pleuronectes americanus): comparison to the effects of Saint John's Wort

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6beta- and 16beta-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder (Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6beta-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16beta-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8 x), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.     

Structural studies on the freezing-point-depressing protein of the winter flounder Pseudopleuronectes americanus

The freezing-point-depressing protein from the winter flounder, $\text{Pseudopleuronectes americanus}$ has been shown from circular dichroism measurements to possess a large proportion (～85%) of the α-helical conformation in aqueous solution (pH 8.0) at ?1°C. The helical content decreases as the temperature is raised. Viscosity data at ?1°C indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this freezing-point-depressing protein as well as its primary structure (reported elsewhere), are found to be different from its counterpart glycoproteins isolated from the Antarctic fish.     

Photoperiod and temperature effects on gametogenesis in winter flounder Pseudopleuronectes americanus

The winter flounder is an in‐shore flatfish living in shallow waters on the east coast of North America from Labrador to Georgia. In the St Lawrence estuary, the reproductive season is May and June. Our objective was to test the effects of winter‐spring photoperiod and temperature conditions on the timing of sexual maturation in both males and females. Groups (16 animals each) of winter flounder breeders were maintained from mid‐January to mid‐May under five different experimental conditions: (1) natural photoperiod and temperature conditions; (2) natural photoperiod, 6° C; (3) 15L : 9D, natural temperature conditions; (4) 15L : 9D, 6° C; (5) accelerated photoperiod increase from winter to spring conditions, 6° C. Natural photoperiod and temperature conditions correspond to a gradual increase in light period from 8L : 16D (January) to 15L : 9D (May) and in temperature from −1° C (January to April) to 6° C (May). GSI and condition factor did not differ among the treatments ( P  > 0·05). In males, milt production occurred simultaneously in the different treatments and histological examination did not indicate any significant effect of either photoperiod or temperature on testes development. In females, seven stages of oocyte development were observed. Both the number of oocytes at the cortical alveoli stage and number of atretic oocytes increased at 6° C (warm temperature conditions). Overall, neither photoperiod nor temperature modified the reproductive period. Warm winter‐spring temperature conditions, however, may decrease egg numbers and egg quality.     

Effect, distribution, and prevalence of Glugea stephani (Microspora) in winter flounder (Pleuronectes americanus) living near two pulp and paper mills in Newfoundland

A study was conducted to determine the effects, geographical distribution, and prevalence of a microsporan parasite, Glugea stephani, in winter flounder (Pleuronectes americanus) in Newfoundland. Fish were captured by SCUBA divers in several coastal areas, including 2 embayments where pulp and paper mill effluent was discharged, as well as a number of pristine sites. Fish health was assessed by comparing histological profiles, condition factors (K), organosomatic indices and blood values between infected and uninfected samples. Multifocal xenomas of G. stephani were observed in several organs of fish taken near contaminated sites, whereas infected samples captured at a pristine site harbored the cysts only in the wall of the digestive tract. Proliferative inflammation, granuloma formation, and focal necrosis were associated with the infection primarily in the liver and kidney. Condition factors and blood values were lower and ovarian development inhibited or delayed in infected flounder. The multifocal infection occurred only in flounder in 2 embayments in western Newfoundland where pulp and paper mill effluent was discharged. Prevalence varied seasonally, with a peak in autumn and a low in spring. It is likely that the multifocal infection was associated with immunodepression after exposure to the contaminant.     

The effect of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus

Synopsis The effects of constant and diurnally fluctuating levels of dissolved oxygen on the growth of young-of-the-year winter flounder,Pseudopleuronectes americanus, were examined under controlled laboratory conditions. Fish were exposed for either 10 or 11 weeks to constant levels of 6.7 (high) and 2.2 (low) mg l^–1, and a diurnal fluctuation, ranging from 2.5 to 6.4 mg 0₂l^–1. Growth rates, calculated for both standard length and weight, for fish exposed to low and diurnally fluctuating levels were significantly reduced (p < 0.001) as compared to those for fish exposed to the high level. Growth rates of fish exposed to the high level were over twice those of fish held under low oxygen conditions. Under fluctuating conditions, fish grew at intermediate rates. Following these exposures, all fish were subsequently held at 7.2 mg Oz l^–1 for five weeks. Growth rates increased over two and a half times for fish previously exposed to the low oxygen level and were significantly (p < 0.001) higher than for the other two groups.     

Epidermal characteristics of reproductive subsets in an inshore population of winter flounder, Pseudopleuronectes americanus

The winter flounder, Pseudopleuronectes americanus (Walbaum), population off Newfoundland can include a 'non-reproductive post-mature' subset at any time together with 'reproductive' and 'immature' subsets. Skin samples were taken from male and female specimens from each subset in the winter, after reproductive flounder have normally undergone a pre-winter phase of gonadal recrudescence, and also during the spring prespawning period and in the summer following spawning. Results for immature flounder demonstrate that fish size influences epidermal thickness in this species. The epidermis of reproductive flounders is thick, representing a secondary sexual characteristic, during gonadal recrudescence. A thick epidermis is most pronounced on the blind-side, particularly in prespawning males. Although the epidermis of non-reproductive post-mature fish was thinner than in prespawning males and females, it was thicker than in immature flounders. Also, the epidermal thickness of non-reproductive post-mature individuals can still display some seasonal change although gonadal recrudescence has not been sustained. There is also a relationship between condition factor and epidermal thickness which is manifested particularly clearly in immature and non-reproductive post-mature flounder.