Blood morphology of the European eel (Anguilla anguilla). IV. Thrombocytes and their developmental stages]

By the aid of histological special stainings, cytochemical proofs, phase contrast and electron microscopical methods the thrombocytes (spindle cells) of the European ell (Anguilla anguilla) and their stages of development were described as an independent series of cells. After an artificial blood loss thrombocytoblasts, prothrombocytes and mature thrombocytes could distinctly be demonstrates. Besides the kidney the spleen was found to be the main place of the thrombopoiesis. Phase contrast investigations provided evidence for the relatively strong locomotion of these cells. By means of cytochemical proofs a further differentiation of the thrombocytes from other white blood cells can be made. Electron microscopical investigations of the thrombocytes yielded a great similarity to the thrombocytes of the amphibians. The endoplasmatic ring and particular inclusions could be shown to be characteritstic features of the fine structure.     

Blood morphology of the European eel (Anguilla anguilla). II. Studies on granulopoiesis]

It was found that the granulocytes for Anguilla anguilla mainly form in the kidney. The stem cell of granulocytes is the large hemocytoblast. Sequences of development were established for the heterophil, cosinophil and basophil granulocytes. Myeloblasts, promyelocytes, metamyelocytes, mature red-shaped and segmented forms were described as stages of development of the heterophil granulocytes. In the case of the eosinophil and basophil granulocytes the myeloblastic and myelocytic stages could be demonstrated as well as the mature granulocyte. By the aid of special granular staining, phase contrast observations, supravital and cytochemical investigations the granulocytes could be described. The cytochemical proof for the granulocytes delivered a distribution pattern of the lipids, carbohydrates, RNA (RNS) and enzymes: unspecific esterase, alkaline and acid phosphatases, oxydase and peroxydase. By means of heart puncture a larger loss of blood was caused and the hematopoiesis stimulated. The following blood letting gave indications of how rapidly the granulopoiesis develops and how much time maturing takes. After a larger loss of blood the new-growth of the granulocytes will be completed nine days later.     

Studies on the morphology of blood of the European eel (Anguilla agnuilla). III. Studies on monocytic and lymphoid cells]

Investigations concerning the monocytic and lymphocytic cell series were carried out on the peripheral blood and dabbings of the kidney, the spleen and intestines of healthy eels (anguilla anguilla), and eels taken ill with vibrio anguillarum. By means of histological, cytochemical, phaseoptical, and electronmicroscopical examinations independent development lines of the monocytes and lymphocytes were established. The place of origination for the monocytes was found to be the kidney, for the lymphocytes the spleen and the intestines additionally. The evidence of the cytochemical assay of enzymes for the differentiation of these blood cells and their cellular systems is primarily dependent on the distribution of the activity of the unspecific esterase, the naphthol-AS-D-chloroacetate-esterase, the peroxidase and the alkalic phosphatase. Macrophages were - on the basis of enzymocytochemical results - seen as derivatives of the monocytes. Plasma cells and lymphoid cells are transformation forms of the lymphocytes. Our results emphasize the significance especially of cytochemistry for the hematological diagnostics of fish diseases.     

Stocking density at early developmental stages affects growth and sex ratio in the European eel (Anguilla anguilla)

To investigate the effect of stocking density on growth and sex ratio in European eel, four constant density conditions were tested during the transition from the glass to the elver stage for 90 days (Period 1). The test conditions combined the weight of fish per unit surface or volume (surface density or volume density) resulting in four experimental groups: low surface density (0.5 kg/m(2)) and low volume density (5 kg/m(3)) (group S(0.5)V(5)); low surface density (0.5 kg/m(2)) and high volume density (10 kg/m(3)) (group S(0.5)V(10)); high surface density (2 kg/m(2)) and low volume density (5 kg/m(3)) (group S(2)V(5)); and high surface density (2 kg/m(2)) and high volume density (10 Kg/m(3)) (group S(2)V(10)). Subsequently, fish from the S(0.5)V(5), S(2)V(5), and S(2)V(10) groups were transferred to low density conditions (0.1-0.4 kg/m(2) or 0.1-0.3 kg/m(3)) for another 21 months (630 days; Period 2). After Period 1, fish maintained at high surface density, regardless of the volume density, showed higher standard growth rates (SGRs) and RNA/DNA ratio in muscle than those cultured at low surface density. The percentage of mortality was similar in three of the groups (34.2%-41.8%), but not in the S(2)V(10) group (83.3%). At the end of Period 2, most fish (about 95%) exhibited fully differentiated gonads, but different sex ratios were observed in each group. Thus, the S(2)V(5) group showed a higher proportion of females (36.1%) and a lower proportion of males (56.8%) than the S(0.5)V(5) group (11.4% and 72.5%, respectively), while all survivor fish from the S(2)V(10) group developed into females. The gonadosomatic index and SGR were higher in females than in males. These results suggest that glass eels maintained at high surface density during the first months of growth tend to develop into females. The data also indicate that growth and sex ratio are linked processes during eel development, with growth seeming to be sex dependant rather than being influenced by the density conditions in which glass eels are maintained.     

Iron-rich particles in European eel (Anguilla anguilla L.)

Magnetic material in the European eel (Anguilla anguilla L.) was investigated by a combination of magnetic susceptibility measurements, energy dispersive X-ray fluorescence analysis and transmission electron microscopy. It was shown that the magnetic material is associated with iron. The main part of the iron is present in the form of iron-rich particles with irregular shapes about 100-3000 A large. The structures of magnetite (Fe3O4), hematite (alpha-Fe2O3) and alpha-iron (bcc structure) were identified. The particles are composed of more than one of these phases with magnetite being a minority phase when present. The iron-rich particles found in the eel are different from the materials reported for bacteria or bees.     

Development of the swimbladder in the European eel (Anguilla anguilla)

The swimbladder of the adult eel, Anguilla anguilla, with its bipolar countercurrent system, the rete mirabile, is a widely used model for swimbladder function, but very little is known about the development of this swimbladder. Our histological studies on the developing swimbladder revealed that during metamorphosis the swimbladder becomes present as a dorsal outgrowth of the esophagus. It is filled with surfactant, and gas was not detected in the swimbladder. In the young glass-eel, the epithelial (gas gland) cells of the swimbladder are columnar, but do not yet have the typical basolateral labyrinth established in adult animals. Few blood vessels are found in the swimbladder tissue, and the submucosa is present as a thick layer of connective tissue, giving a large diffusion distance between blood vessel and swimbladder lumen. Within the next 2 or 3 months of development, gas gland cells develop their typical basolateral labyrinth, and the thickness of the submucosa is significantly reduced, resulting in a short diffusion distance between blood vessels and the swimbladder lumen. The first filling of the swimbladder with gas is observed while the gas gland cells are still in a poorly differentiated status and it appears unlikely that these cells can accomplish their typical role in gas deposition. The presence of small gas bubbles in the swimbladder as well as in the ductus pneumaticus at the time of initial swimbladder inflation suggests that the swimbladder is filled by air gulping or possibly by taking up gas bubbles from the water.     

Olfactory sensitivity to amino acids in the juvenile stages of the European eel Anguilla anguilla (L.)

Scanning electron micrograph observations of the olfactory mucosa from both unpigmented glass eel(GE)andpigmentedelvers(EL)of the European eel, Anguilla anguilla (L.), revealed the presence of various cell types; amongst these, the ciliated and microvillous ones are likely to possess a chcmosensory function. Recording of underwater electro-olfactograms (EOGs) showed that various amino acids (glycine, L-alanine, L-valine, L-leucine, L-asparagine, L-glutamine and L-methionine) are effective stimulants for the olfactory mucosa. Dose response curves of stimulus concentrations v. EOG amplitudesfit regression linesat both GE and EL stages. Leucine was more stimulatory at the GE than at the EL stage. The stimulatory effect of the other six amino acids tested was similar at both developmental stages. The possible role of olfactory sensitivity in animal behaviour at different developmental stages is discussed.     

Replication banding in the chromosomes of the European eel (Anguilla anguilla)

The chromosomes of the European eel Anguilla anguilla have been analyzed with a replication banding technique from lymphocyte cultures treated with 5-BrdU. This technique allows us to identify with high resolution the individual chromosome pairs and to differentiate classes of chromatin by the order of replication. The replication banding obtained on the chromosomes of European eel can be related with the structural bands described in this species.     

Persistence of herpesvirus of eel Herpesvirus anguillae in farmed European eel Anguilla anguilla.

Herpesvirus of eel Herpesvirus anguillae (HVA) was isolated repeatedly from farmed eel of an outwardly healthy stock, but virus isolation was much greater in an experimental group of fish that were injected with dexamethasone. The results suggest that HVA can establish a latent infection in eel. Previous exposure of these eels to HVA virus was shown by detection of HVA-specific antibodies. These eels did not show clinical signs after a secondary infection with HVA. Tracing of seropositive eel stocks, which had previous contact with HVA, and of HVA carrier fish can be useful to control disease outbreaks due to HVA infection.     

Cloning and Expression of Guanylin from the European eel (Anguilla anguilla)

Extracts of intestinal epithelia from the European eel (Anguilla anguilla) stimulated cGMP production in the T84 human colon carcinoma cell line which suggested the presence of a guanylin-like peptide in this teleost fish. Degenerate oligonucleotide primers were subsequently used in RT-PCR resulting in the amplification, cloning, and sequencing of two cDNAs which represent possible 5' spliceoforms of an eel homologue of the mammalian peptide, guanylin. Northern blotting indicated that the main site of expression of the eel peptide is in the intestine with much lower signals also detected in the kidney. Intestinal expression of guanylin mRNA is up-regulated in both nonmigratory "yellow" and the more sexually mature, migratory "silver" eels following acclimation to the seawater environment. These results suggest that this peptide signalling system may play a role in osmoregulation in euryhaline teleost fish during migration between the marine and freshwater environments.     

Temperature acclimation and oxygen binding properties of blood of the European eel, Anguilla anguilla

Temperature acclimation of the European eel, Anguilla anguilla, resulted in red cell GTP/Hb molar ratios of 1.20, 1.77 and 0.80 at 2, 17 and 29 degrees C, respectively. A small increase in blood oxygen capacity was present in 29 degrees C acclimated eels. The CO2 Bohr effect and the shape of the oxygen binding curve (n-Hill) were invariant with both temperature and GTP/Hb. The significant differences in the GTP/Hb ratio corresponded with a strong enhancement of the temperature effect on blood oxygen affinity between 2 and 17 degrees C and a similarly strong compensation between 17 and 29 degrees C. Predicted in vivo P50 values were 3.0, 13.8 and 17.6 mmHg at 2 degrees C, 17 and 29 degrees C, respectively. The adaptational value of these findings are discussed in relation to standard metabolic rates at the various temperatures. A tentative hypothesis is proposed that the present study confirms and expands earlier work and supports the contention that adjustments in blood oxygen affinity of thermally acclimated teleosts serve to provide them with an unloading O2 tension for diffusion closely matching the standard oxygen requirements at the various temperatures.     

Induced oogenesis of the European eel (Anguilla anguilla L.) in freshwater condition

European eel is a catadromous fish species, which means that after living in freshwater premature individuals adapt to sea water, and migrate to the Sargasso Sea for spawning. Although male eel can be sexually matured even in freshwater, to date, it was believed that female eel can be matured only in seawater. Here we show that the process of sexual maturation may be induced in freshwater by treating female eels with carp pituitary (GSI = 9.87 ± 1.55%). It is thus proposed that seawater condition is not an obligatory environment for stimulating gametogenesis and for artificial maturation of the European eel in neither gender.     

Muscle fibre differentiation and vascularisation in the juvenile European eel (Anguilla anguilla L.)

Summary The development of the pituitary gland and its blood vessels is described in rat embryos (gestational day 12 through day 21) injected with India-ink via the umbilical vessels. The vascularization of all components of the pituitary gland develops from the surface network covering the prosencephalic vesicle. However, vascular connections exist between the prospective median eminence and the anterior pituitary gland in the earliest stages examined (day 12) but are not augmented by vessels from the stomatodeal roof until day 13. Primary portal veins appear initially on day 13, the vascularization of the pars distalis is visible on day 15. The Mantelplexus covering the floor of the diencephalon is discernible on day 16. Large-caliber portal veins appear immediately before birth, but otherwise there is no significant change in the vascular pattern during the last five days of gestation. The pars intermedia and the median eminence-pituitary stalk region remain avascular throughout embryonic life.     

Evidence for isolation by time in the European eel (Anguilla anguilla L.)

Life history traits of highly vagile marine species, such as adult reproductive success and larval dispersal, are strongly determined by oceanographic and climatic forces. Nevertheless, marine organisms may show restricted dispersal in time and space. Patterns of isolation by distance (IBD) have been repeatedly observed in marine species. If spawning time is a function of geographical location, temporal and spatial isolation, can easily be confounded or misinterpreted. In this study, we aimed at discriminating between various forces shaping the genetic composition of recruiting juveniles of the European eel (Anguilla anguilla L.). By controlling for geographical variation, we assessed temporal variation and tested for possible isolation by time (IBT) between recruitment waves within and between years. Using 12 polymorphic allozyme and six variable microsatellite loci, we show that genetic differentiation was low (F(ST) = 0.01-0.002) and significant between temporal samples. Regression analysis between genetic and temporal distance, was consistent with a subtle interannual pattern of IBT. Our data suggest that the population dynamics of the European eel may be governed by a double pattern of temporal variance in genetic composition: (i) a broad-scale IBT of spawning cohorts, possibly as a consequence of the large migration loop in anguillids and strong variance in annual adult reproductive contribution; and (ii) a smaller-scale variance in reproductive success (genetic patchiness) within cohorts among seasonally separated spawning groups, most likely originating from fluctuating oceanic and climatic forces. The consistency of both mechanisms remains to be verified with fine-scale analyses of both spawning/migrating aged adults and their offspring to confirm the stochastic/deterministic nature of the IBT pattern in eel.     

Studies on the chemical nature of feeding stimulants for the juvenile European eel, Anguilla anguilla (L.)

A study has been made of the chemical nature of feeding stimulants for juvenile European eels, Anguilla anguilla . Mixtures of L-amino acids were stimulatory, while neither the corresponding. D-amino acids nor the non-amino acid components were effective. Synergistic effects were observed both between L-amino acids and between L-amino acids and non-amino acid constituents. The results are discussed in the context of results obtained from electrophysiological and behavioural studies with other species.     

Structure and colour of the integument of the European eel Anguilla anguilla (L.)

The structure of the integument of 26 wild and nine artificially matured female Anguilla anguilla , and 30 sexually immature eels maintained in controlled lighting, was investigated. Melanophore densities varied in a way unrelated to maturity while xanthophores were progressively lost from lower levels in the skin. Reflecting layers made up of the purines, guanine and hypoxanthine were associated with chromatophore layers. Purine concentrations (as a percentage of total skin dry weight) decreased with maturity but a reorganization of the reflecting elements occurred. Colour changes associated with maturation were due to the redistribution of purines. Spectral and angular reflectance measurements indicate that the integument has camouflage value and colour changes can be related to the change in habitat occurring with migration. The usefulness of colour as an index of sexual maturity is discussed.     

Effects of serotonin on the cardio-circulatory system of the European eel (Anguilla anguilla) in vivo

The effects of serotonin on continuously recorded cardiac parameters (heart rate, cardiac output, cardiac stroke volume), ventral and dorsal aortic blood pressures, branchial and systemic vascular resistances were investigated in the European eel in vivo. Intravenous administration of serotonin (30 g · kg^–1) caused a marked bradycardia (45%) and a simultaneous decrease in cardiac output (50%), ventral (35%) and dorsal (50%) aortic blood pressures. Branchial resistance was markedly increased (60%) and systemic resistance decreased (30%). Cardiac stroke volume remained unchanged. The effects of serotonin on cardiac mained unchanged. The effects of serotonin on cardiac parameters were suppressed either by methysergide or a bilateral section of the cardiac vagus. Bradycardia could then be regarded as the consequence of a vagal mechanism triggered by serotonin action on central methysergide-sensitive serotonergic receptors. No inotropic effect of serotonin was observed. This lack of myocardiac contractility modification is discussed. The serotonin-mediated branchial vasoconstriction was attenuated by vagotomy, whereas the residual increase in branchial resistance (40%) was suppressed by methysergide. The serotonin-mediated branchial vasoconstriction could be the consequence of both a passive mechanism (compliance) caused by the decrease in cardiac output and an active mechanism involving methysergide-sensitive serotonergic receptors of the branchial vasculature. A possible involvement of this vasomotor effect in gill oxygen uptake is discussed. The serotonin-induced systemic vasodilation was insensitive either to cardiac vagotomy or to 5-HT_1/2, 5-HT₃ and 5-HT₄ receptor antagonists, suggesting the involvement of a local mechanism which remains to be assessed.Abbreviations CSV cardiac stroke volume - DAP dorsal aortic pressure - HR heart rate - QC cardiac output - VAP ventral aortic pressure - VR _b branchial vascular resistance - VR _s systemic vascular resistance - VR _t total vascular resistance - 5-HT 5-Hydroxytryptamine serotonin - RBI Research Biochemical Incorporated, metoclopramide HCl