Experimental infection of American winter flounder (Pseudopleuronectes americanus) with Glugea stephani (Microsporida)

Young-of-the-year Pseudopleuronectes americanus were captured in Sandy Hook Bay, New Jersey and maintained in aquaria at 16–19° C for 115 days in 1982 and 127 days in 1983. Half of them were experimentally exposed to Glugea stephani and the others were sham exposed. During the course of these experiments fish from both groups died as a result of G. stephani infection, demonstrating Glwgea -induced mortality in this species. Infections were found in both control and experimentally exposed fish. Mortality of Glwgea -infected fish in the exposed group was 63% while that in the controls was 28.5%.     

Seasonal prevalence of the microsporidan, Glugea stephani (Hagenmuller), in winter flounder, Pseudopleuronectes americanus (Walbaum), from the New York-New Jersey Lower Bay Complex

During a 32 month period 26 monthly collections of winter flounder were conducted from various locations in the New York-New Jersey Lower Bay Complex. A total of 3125 flounder were captured and examined for the presence of the microsporidan, Glugea stephani. Of the total number of fish collected, 260 (8.32%) were infected with the protozoan. At least one G. stephani infected flounder was captured and identified each month, which indicates that the infection is present on a year-round basis. The monthly infection prevalence ranged from 0.63 to 25%. Increased parasitism corresponded with elevated water temperatures. Fish size was not a statistically significant factor for infection.     

The immunomodulating effect of the microsporidan Glugea stephani on the humoral response and immunoglobulin levels in winter flounder, Pseudopleuronectes americanus

A quantitative study of serum immunoglobulin levels and haemagglutination titres has shown that an injection of the microsporidan Glugea stephani caused a decrease in the humoral response of the winter flounder. These examples demonstrate the effect of temperature on the decrease in total IgM levels and haemagglutination titres in winter flounder simultaneously injected with either G. stephani or horse red blood cells.
Transfer of serum from fish injected with spores 3 weeks earlier may also implicate soluble serum factors, such as prostaglandins or leukotrienes, causing the observed decrease in serum IgM levels.     

The occurrence of Glugea stephani (Hagenmuller, 1899) in American winter flounder, Pseudopleuronects americanus (Walbaum) from the New York-New Jersey lower bay complex

American winter flounder, Pseudopleuronectes americanus , Collected in the New York-New Jersey bay complex, contained cysts in the intestinal tract which were subsequently identified as the microsporidan Glugea stephani . These fish represent 13 collections taken at approximately monthly intervals over a one and a half year period. The winter flounder varied in size from 8 to 33 cm and consequently represented a fish population of diverse age. During this study, a total of 1840 apparently healthy fish were dissected and examined for the presence of Glugea cysts , with an overall parasite infection rate of 6.63%. There was no correlation between size of the fish and percentage of infection. This percentage represents the mean carrying rate of this parasite in the fish population, since these are the fish that have survived the initial infection. This report extends the reported host range to the New York-New Jersey area. As a further aid in the characterization of G. stephani , we have included ultrastructural details of the spore morphology.     

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.     

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.     

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.     

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.     

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.     

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.     

Ribosomal DNA sequences of Glugea anomala, G. stephani, G. americanus and Spraguea lophii (Microsporidia): phylogenetic reconstruction

The microsporidian species Glugea anomala, G. stephani, G. americanus and Spraguea lophii were compared by using sequence data derived from their small subunit rDNA genes which were amplified by polymerase chain reaction and directly sequenced. These sequence data and published data of G. atherinae were analyzed and were used to infer a phylogenetic tree. The 5 microsporidian fish parasites appeared to be closely related. The higher sequence similarities demonstrated among G. anomala, G. stephani and G. atherinae suggest that these 3 parasites are in fact only 1 species of Glugea. Moreover, the higher sequence similarities between S. lophii and G. americanus support the transfer of the latter Glugea species into the genus Spraguea.     

Thermoregulatory behavior and diel activity of yearling winter flounder,Pseudopleuronectes americanus (Walbaum)

Synopsis Sixteen yearling winter flounder, tested individually for 3-day periods in electronic shuttleboxes, voluntarily occupied an 8–27°C range of temperature, with a modal final thermal preferendum of 18.5°C (mean 18.7°C, median 19°C, midpoint 17.5°C, s.d. 1.9°C, S _k + 0.33). The locomotor activity pattern of the yearling fish in the laboratory was markedly nocturnal, with mean hourly nocturnal activity exceeding mean hourly diurnal activity by a factor of 3.4. Maximum activity occurred at 0300 EST, minimum activity at 1400 EST. While activity generally increased with temperature, a local activity minimum occurred at 18.7°C, coinciding with the 24-hour mean final thermal preferendum. Comparison of these laboratory data with previously published field data suggest that behavioral responses to temperature and light play significant roles in determining age- and size-specific differences in seasonal depth stratification and onshore-offshore distributions in this species.