Early and abrupt salinity reduction impacts European eel larval culture.

Reducing water salinity towards iso-osmotic conditions is a common practice applied in euryhaline fish farming to limit osmoregulation costs and enhance growth. In this respect, the present study investigated the timing of salinity reduction in an abrupt manner during European eel (Anguilla anguilla) larval culture by examining associated impacts on morphological and molecular levels. Larvae from 3 different parental combinations (families) were reared at constant 36 psu for 6 days (control) or subjected to a direct reduction to 18 psu on 1, 2, or 3 days post-hatch. Overall, salinity reduction enhanced growth and survival, resulting from more efficient energy resource utilization. In the control group, expression of growth-related igf2 remained constant, demonstrating a steady growth progression, while igf1 expression increased over time only for the salinity reduced treatments, potentially qualifying as a useful biomarker for growth performance. Even though each parental combination seems to have a different capacity to cope with salinity alterations, as observed by family-driven water-transport-related aquaporin (aqp1, aqp3) gene expression, it could be inferred that the abrupt salinity change is generally not stressful, based on non-upregulated heat shock proteins (hsp70, hsp90). However, the applied salinity reduction (irrespective of timing) induced the development of pericardial edema. As such, we conclude that despite the positive effect of salinity reduction on early growth and survival, the long-term benefit for eel larval culture lies in establishing a protocol for salinity reduction, at a precise developmental time point, without causing pericardial malformations.

Type of hormonal treatment administered to induce vitellogenesis in European eel influences biochemical composition of eggs and yolk-sac larvae.

Egg biochemical composition is among the main factors affecting offspring quality and survival during the yolk-sac stage, when larvae depend exclusively on yolk nutrients. These nutrients are primarily embedded in the developing oocytes during vitellogenesis. In aquaculture, assisted reproduction procedures may be applied enabling gamete production. For the European eel (Anguilla anguilla), reproductive treatment involves administration of pituitary extracts from carp (CPE) or salmon (SPE) to induce and sustain vitellogenesis. In the present study, we compared the influence of CPE and SPE treatments on offspring quality and composition as well as nutrient utilization during the yolk-sac stage. Thus, dry weight, proximal composition (total lipid, total protein), free amino acids, and fatty acids were assessed in eggs and larvae throughout the yolk-sac stage, where body and oil-droplet area were measured to estimate growth rate, oil-droplet utilization, and oil-droplet utilization efficiency. The results showed that CPE females spawned eggs with higher lipid and free amino acid contents. However, SPE females produced more buoyant eggs with higher fertilization rate as well as larger larvae with more energy reserves (estimated as oil-droplet area). Overall, general patterns of nutrient utilization were detected, such as the amount of total lipid and monounsaturated fatty acids decreasing from the egg stage and throughout the yolk-sac larval stage. On the contrary, essential fatty acids and free amino acids were retained. Notably, towards the end of the yolk-sac stage, the proximal composition and biometry of surviving larvae, from both treatments, were similar.

Temperature induced variation in gene expression of thyroid hormone receptors and deiodinases of European eel (Anguilla anguilla) larvae.

Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3'-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 °C) from hatch until first-feeding. We identified 2 subtypes of thr (thrα and thrß) with 2 isoforms each (thrαA, thrαB, thrßA, thrßB) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel.

Functional Characterisation of Eel Dopamine D2 Receptors and Involvement in the Direct Inhibition of Pituitary Gonadotrophins.

In various vertebrate species, dopamine (DA) exerts an inhibitory action on reproduction. In the European eel, DA plays a pivotal role in the inhibitory control of gonadotroph function and the blockade of puberty. In vivo studies have suggested that this effect is mediated by receptors pharmacologically related to the D2 family. In the European eel, two distinct D2 receptor (D2-R) paralogous genes have been identified (D2A-R and D2B-R) and both were shown to be expressed in the pituitary. We investigated the potential role of each paralogue in the control of gonadotroph function in this species. Eel recombinant D2A-R or D2B-R were expressed in HEK 293 cells, with a universal Gα subunit, and receptor activation was followed by inositol phosphate production. Recombinant D2-Rs exhibited a comparable affinity for DA, although they had differential affinities for mammalian D2-R agonists and antagonists, supporting subtle structure/activity differences. Furthermore, using eel pituitary cell primary cultures, the expression by gonadotroph cells of both native eel D2-R paralogues was examined by in situ hybridisation of D2A-R or D2B-R transcripts, coupled with immunofluorescence of luteinising hormone (LH)ß or follicle-stimulating (FSH)ß. LH and to a lesser extent, FSH cells expressed both D2-R transcripts but with a clear predominance of D2B-R. Notably, D2B-R transcripts were detected for the majority of LH cells. Accordingly, using these cultures, we showed that DA potently inhibited basal and testosterone-stimulated LHß expression and less potently basal and activin-stimulated FSHß expression. We also tested some D2-R antagonists, aiming to select the most adequate one to be used in innovative protocols for induction of eel sexual maturation. We identified eticlopride as the most potent inhibitor of DA action on basal and stimulated LH expression in vitro. Our data suggest a differential functionalisation of the duplicated receptor genes and demonstrate that mainly D2B-R is involved in the dopaminergic inhibitory control of eel gonadotroph function.

First Production of Larvae Using Cryopreserved Sperm: Effects of Preservation Temperature and Cryopreservation on European Eel Sperm Fertilization Capacity.

Sperm cryopreservation is a useful tool in captive fish reproduction management, that is to synchronize gamete production, especially in the case of species as the European eel, where the time of female spawning readiness is unpredictable. Several protocols to cryopreserve sperm of this species have been described, but until recently fertilization trials were not feasible. This study evaluated the effect of cold storage of diluted sperm prior to fertilizations and tested whether a previously defined protocol for European eel sperm cryopreservation can be successfully applied in fertilization trials to produce viable offspring. In our experiment, the sperm motility was evaluated after the extraction and the best samples were selected and pooled. Until stripping of eggs and fertilization, diluted sperm samples were maintained at either 4 or 20°C, or cryopreserved, following existing protocols. Fertilization of two egg batches was attempted. Diluted sperm caused a similar percentage of fertilized eggs and a similar number of embryos and larvae, independently of storage temperature (4 or 20°C). The cryopreserved sperm resulted in a lower percentage of fertilized eggs, but embryos developed and a few larvae ('cryolarvae') were obtained 55 h after fertilization in one of the two egg batches. This result evidences that the tested cryopreservation protocol is applicable for eel reproduction management, although improvements will be required to enhance fertilization success.

Evaluation of methods to determine sperm density for the European eel, Anguilla anguilla.

European eel, Anguilla anguilla, is a target species for future captive breeding, yet best methodology to estimate sperm density for application in in vitro fertilization is not established. Thus, our objectives were to evaluate methods to estimate European eel sperm density including spermatocrit, computer-assisted sperm analysis (CASA) and flow cytometry (FCM), using Neubauer Improved haemocytometer as benchmark. Initially, relationships between spermatocrit, haemocytometer counts and sperm motility were analysed, as well as the effect of sperm dilution on haemocytometer counts. Furthermore, accuracy and precision of spermatocrit, applying a range of G-forces, were tested and the best G-force used in method comparisons. We found no effect of dilution on haemocytometer sperm density estimates, whereas motility associated positively with haemocytometer counts, but not with spermatocrit. Results from all techniques, spermatocrit, CASA and FCM, showed significant positive correlations with haemocytometer counts. The best correlation between spermatocrit and haemocytometer counts was obtained at 6000 × g (r = 0.68). Of two CASA variants, one or three photographic fields (CASA-1 and CASA-2), CASA-2 showed a very high accuracy to haemocytometer counts (r = 0.93), but low precision (CV: CASA-2 = 28.4%). FCM was tested with and without microfluorospheres (FCM-1 and FCM-2), and relationships to haemocytometer counts were highly accurate (FCM-1: r = 0.94; FCM-2: r = 0.88) and precise (CV: FCM-1 = 2.5; FCM-2 = 2.7%). Overall, CASA-2 and FCM-1 feature reliable methods for quantification of European eel sperm, but FCM-1 has a clear advantage featuring highest precision and accuracy. Together, these results provide a useful basis for gamete management in fertilization protocols.

Changes in women's fear of success and fear of appearing incompetent in business.

The primary purpose of this research was to examine whether fear of success and of appearing incompetent among women have changed recently. Another purpose was to examine whether such fears differed among women who hold Traditional views and those who hold Progressive views about the roles of women in the workplace. The Fear of Success Scale, the Fear of Appearing Incompetent Scale, and the Attitude Toward Women Scale were completed by 61 male and 52 female graduating seniors. Significant differences were found between the groups for scores on the Attitude Toward Women Scale, but none between the sexes for scores on the Fear of Success Scale or the Fear of Appearing Incompetent Scale. Significant differences were found, however, on the latter two scales when women were separated into Traditional and Progressive groups.

The impact of perceptions and stereotypes on the managerial mobility of African Americans.

Although African Americans have made progress in reaching middle and upper management positions, a disproportionate number are stalled in lower levels of management where their remuneration often lags behind that of Whites in comparable positions. Further penetration into executive ranks may depend largely on the perceptions and attitudes of employers. Negative stereotypes held by top managers may have a deleterious effect on African American opportunities. Among a sample of White middle managers, Schein's (1973, 1975) descriptive index revealed a significant resemblance between ratings of Whites and ratings of managers (r' = .54), whereas the resemblance between ratings of Blacks and ratings of managers was nonsignificant (r' = .17). Managers were perceived to possess characteristics more commonly ascribed to Whites than to African Americans.

Perceptual differences in racial descriptions of Euro-American and Hispanic persons.

This study examined negative stereotypes held about Hispanic persons by 116 Euro-American graduating MBA students. Schein's 92-item Descriptive Questionnaire (1973) was rated by subjects as either positive (e.g., intelligent, persistent, ambitious, etc.) or negative (e.g., uncertain, passive, nervous, etc.). 53 items were characterized as positive while 18 were considered negative. When applied to Euro-American and Hispanic persons, zero and 8 negative items, respectively, could be used to describe these two groups in general. Conversely, while 26 positive items were used exclusively to describe Euro-American persons, only one could be applied exclusively to Hispanic persons. The potential effects of such perceptions on Hispanic persons job opportunities are discussed.