Temporal changes in digestive enzyme activities in the gastrointestinal tract of European eel (Anguilla anguilla) (Linneo 1758) following feeding

Changes occurring after feeding in the digestive enzyme activities of European eel were investigated to provide some insights into the digestive physiology of this fish. Total and specific proteases, amylase and lipase activities were measured using standard biochemical assays over a 24 h cycle in fed eels, compared to starved ones, under the same rearing conditions. In the gastrointestinal tract of fed eels quantitative changes started 4 h after feeding and continued later on; conversely, in starved eels enzyme activities remained unchanged over time. In fed eels, total and specific protease activities showed an overall increasing trend in the intestine, while in the stomach they progressively decreased to values 22–50% lower than those measured at the pre-feeding time; this behaviour probably reflected the progression of digesta along the intestinal tract. The prolonged secretory response of European eel to food ingestion proved its extended activity in the digestive process.     

Burrowing behaviour of the European eel (Anguilla anguilla): Effects of life stage

The European eel (Anguilla anguilla) is a fascinating species, exhibiting a complex life cycle. The species is, however, listed as critically endangered on the IUCN Red List due to an amalgam of factors, including habitat loss. This study investigated the burrowing behaviour and substrate preference of glass, elver and yellow stages of A. anguilla. Preference was determined by introducing eels in aquaria with different substrates and evaluating the chosen substrate for burrowing. In addition, burrowing was recorded using a camera in all substrate types and analysed for kinematics. The experiments showed that all of these life stages sought refuge in the sediments with particle sizes ranging from sand to coarse gravel. Starting from a resting position, they shook their head horizontally in combination with rapid body undulations until half of their body was within the substrate. High-speed X-ray videography revealed that once partly in the sediment, eels used only horizontal head sweeps to penetrate further, without the use of their tail. Of the substrates tested, burrowing performance was highest in fine gravel (diameter 1–2 mm; lower burrowing duration, less body movements and/or lower frequency of movements), and all eels readily selected this substrate for burrowing. However, glass eels and elvers were able to use coarse gravel (diameter >8 mm) because their smaller size allowed manoeuvring through the spaces between the grains. Further, burrowing performance increased with body size: glass eels required more body undulations compared to yellow eels. Interestingly, the urge to hide within the sediment was highest for glass eels and elvers. Documentation of substrate preference and burrowing behaviour of A. anguilla provides new information about their potential habitat use. Considering that habitat alterations and deteriorations are partly responsible for the decline of the eel, this information can contribute to the development of more effective conservation measures.     

Isolation and characterization of expressed sequence tag-linked microsatellite loci for the European eel (Anguilla anguilla)

A European eel (Anguilla anguilla) expressed sequence tag database consisting of 795 contigs and 4008 singletons was screened for microsatellites sequences. Primers were designed to amplify 96 repeats, of which 86 gave good quality amplification products. Twenty-eight microsatellites were selected for further microsatellite genotyping. Only two loci were found to be monomorphic; out of the 26 polymorphic loci, number of alleles per locus ranged from two to 14, while the observed and expected heterozygosities ranged from 0.05 to 0.93, and from 0.05 to 0.95, respectively. All 28 primer sets tested revealed positive amplification in American eel (Anguilla rostrata).     

Vanadate inhibition of active Ca2+ transport across human red cell membranes

(1) Vanadate (pentavalent vanadium) inhibits with high affinity ($\text{K}{}_{0.5}\text{= 3 μ}\text{M}$) the ATP-dependent Ca²⁺ efflux in reconstituted ghosts from human red cells. (2) To inhibit Ca²⁺ efflux vanadate has to have access to the inner surface of the cell membrane. (3) The inhibitory effect of vanadate is potentiated by intracellular Mg²⁺ and by intracellular K⁺. (4) Ca²⁺ in the external medium antagonizes the inhibitory effect of vanadate.     

Comparative studies of the protein composition of red blood cell membranes from eight mammalian species

The polypeptide pattern of red blood cell (RBC) membranes from cow, sheep, horse, rabbit, guinea pig, rat, mouse, analyzed by polyacrylamide gel electrophoresis, was compared to human RBC counterpart. Some qualitative and quantitative differences were noted. Among the high molecular weight components the bands 2.1- 2.3 appeared slightly decreased in rabbit and rat and increased in sheep RBC membranes. Band 3 appeared to have a higher molecular weight in the cow, guinea pig and mouse RBCs, and a lower molecular weight in the sheep RBCs. Band 4.1 from the RBC membranes of cow, sheep, rabbit and guinea pig was splitted into two sub-bands, while band 4.2 overlapped with band 4.1 in horse and guinea pig RBC membranes. There are marked differences in the number and position of bands in the 4.5 region, while band 4.9 is present in higher amounts in horse, rabbit and guinea pig RBC membranes. Band 6 (glyceraldehyde 3-phosphate dehydrogenase) was undetectable in horse, rat and mouse RBC membranes and was decreased in sheep, rabbit and guinea pig. There are also major differences in the region of band 7 and below ("post-7"). Band 8 was undetectable in horse, cow and guinea pig, and was in higher amounts in rat. A band corresponding to a molecular weight of about 22 kD in the "post-8" region was present only in guinea pig RBC membranes.     

Occurrence of Phyllodistomum folium (Olfers) (Digenea) in the eel, Anguilla anguilla (Linnaeus), in a small river

The occurrence of Phyllodistomum folium in a population of eels was studied throughout one year. Abundance exhibited a summer peak. Gravid adults were found in all months, indicating that eels can serve as a definitive host for this parasite.     

Genetic systems of red cell blood groups in goats.

Evidence is presented for six genetic systems of goat red cell blood groups. The A system presently consists of one specificity, two alleles, two phenotypes (A1 and no-A1) and appears to be homologous to the A system of sheep. The B system, like its homologue in sheep, is very complex. Fourteen of 21 specificities detected in the present study, i.e. B2, B3, B4, B5, B7, B8, B9, B10, B11, B14, B15, B16, B17 and B20, belong to the B system which involves a large number of phenogroups (31 different B phenogroups identified in 26 sires). Because of their homology with sheep C and R systems, two other genetic systems of goat blood groups are named C and R respectively. Each of the two goat systems is presently a one blood group specificity, two phenotype (C12 and no-C12; R and no-R detectable on the red cells) two allele system. Two specificities, namely E6 and E18, belong to a genetic system called E in which four phenotypes are under the control of two alleles codominant and one recessive at a single locus. The F system involves but a single pair of alleles and two phenotypes (F19 and no-F19). Because of its low frequency in the goats tested, the X13 specificity remains unassigned.     

Comparison of the red blood cell Ca2+-ATPase in ghost membranes and after purification

We have compared properties of the red blood cell Ca²⁺-ATPase in two types of preparations: red cell membrane ghosts (enzyme in unfractionated membranes) and after purification (detergent-soluble enzyme). The Ca²⁺-ATPase activity was studied with respect to its requirement for: calmodulin, calcium, magnesium, monovalent cations, ionic strength, pH, and temperature. Sensitivity of the Ca²⁺-ATPase activity in the two preparations to anticalmodulin drugs and to engineered calmodulins with amino acid substitutions was determined. Finally, stoichiometry of the formation of phosphorylated enzyme intermediate (EP) and titrations of the ATP binding region with fluorescein 5-isothiocyanate (FITC) were characterized. For the first time a high phosphorylation level of 2.0–2.4 mmol EP/mg of purified enzyme is reported.The two enzyme preparations have been found to be very similar with respect to the dependency of all the regulating factors described here. These results complement findings reported from various laboratories on the similarity of other kinetic properties as well as the similarity of modulation of the Ca²⁺-ATPase activity by phospholipids and proteolysis in the membranous and purified enzyme. Thus, the purified detergent-soluble enzyme is very well suited for kinetic characterization of the red cell Ca²⁺-ATPase.     

Genetic variability is unrelated to growth and parasite infestation in natural populations of the European eel (Anguilla anguilla)

Positive correlations between individual genetic heterozygosity and fitness-related traits (HFCs) have been observed in organisms as diverse as plants, marine bivalves, fish or mammals. HFCs are not universal and the strength and stability of HFCs seem to be variable across species, populations and ages. We analysed the relationship between individual genetic variability and two different estimators of fitness in natural samples of European eel, growth rate (using back-calculated length-at-age 1, 2 and 3) and parasite infestation by the swimbladder nematode Anguillicola crassus . Despite using a large data set of 22 expressed sequence tags-derived microsatellite loci and a large sample size of 346 individuals, no heterozygote advantage was observed in terms of growth rate or parasite load. The lack of association was evidenced by (i) nonsignificant global HFCs, (ii) a Multivariate General Linear Model showing no effect of heterozygosity on fitness components, (iii) single-locus analysis showing a lower number of significant tests than the expected false discovery rate, (iv) sign tests showing only a significant departure from expectations at one component, and, (v) a random distribution of significant single-locus HFCs that was not consistent across fitness components or sampling sites. This contrasts with the positive association observed in farmed eels in a previous study using allozymes, which can be explained by the nature of the markers used, with the allozyme study including many loci involved in metabolic energy pathways, while the expressed sequence tags-linked microsatellites might be located in genes or in the proximity of genes uncoupled with metabolism/growth.     

Telemetric observations on the spawning migration of the eel (Anguilla anguilla) west of the European continental shelf

Synopsis In the Northern Bay of Biscay and west of the Iberian continental shelf five silver eels (Anguilla anguilla L.) have been tagged with ultrasonic transmitters and tracked 13 to 23 hours over a depth of 200 to 2500 m. Their mean direction from release to the final position of tracking was 288° and significantly closer to the direction of the Sargasso Sea (250° west) than silver eels tracked earlier in the North Sea (341°), possibly 260°. Four of the transmitters were equipped with pressure sensing devices capable of indicating depths of at least 400 m. Three eels tracked at night, during full moon, preferred mean depths of 125, 166 or 215 m. One eel chose a depth of 100 m during moonlight and 50 m after the setting of the moon. Major depth changes, usually occurring one per hour, ranged up to a maximum of 200 m at a maximum vertical speed of 0.6 m sec^–1; this is close to the eels' normal horizontal speed. At dawn all but one dived to a depth of 400 m or more. The eels generally swam below the thermocline and often crossed it.     

Cryopreservation of European eel (Anguilla anguilla) spermatozoa: effect of dilution ratio, foetal bovine serum supplementation, and cryoprotectants

The main aim of the present study was to investigate the effect of sperm freezing medium dilution ratio (1:1, 1:2, and 1:5 v/v), two cryoprotectants: dimethyl sulphoxide (Me(2)SO) and methanol (MeOH), and the addition of foetal bovine serum (FBS) on the cryopreservation of European eel sperm. The effect of these factors was evaluated comparing post-thawing viability with fluorescent staining (Hoechst bisbenzimide 33258) and the spermatozoa head morphometry, determined with computer-assisted morphology analysis (ASMA). The 1:5 (v/v) dilution ratio resulted in a lower viability in comparison with 1:1 and 1:2 (52.8+/-2.3% vs. 67.4+/-2.3% and 65.1+/-2.3%, respectively, p=0.0001), but without effects on the head morphology. Although the viability was not significantly different between Me(2)SO and MeOH (60.4+/-1.9 vs. 63.2+/-1.9%, respectively, p=0.305), a decrease of spermatozoa head area and perimeter was found when spermatozoa were frozen with methanol (6.19+/-0.01 vs. 6.36+/-0.01 microm(2) and 17.28+/-0.05 vs. 17.49+/-0.05 microm, for area and perimeter and MeOH and Me(2)SO, respectively, p=0.0001). Finally, a higher viability (75.1+/-1.7 vs. 48.5+/-1.7, with or without FBS, respectively, p=0.0001) and higher spermatozoa head size (6.40+/-0.01 vs. 6.15+/-0.01microm(2) and 17.88+/-0.05 vs. 16.89+/-0.05 microm, for area and perimeter, with or without FBS, respectively, p=0.0001) were found when cells were frozen-thawed in freezing media supplemented with FBS. Based on the above findings, dilution ratios lower than 1:5 (v/v) and the addition of serum improved the viability results after cryopreservation. Future studies are required in order to understand the spermatozoa membrane interchange mechanisms in response to the changes in spermatozoa head size caused by cryoprotectants and freezing media supplements.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions

Summary The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H⁺-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H⁺ on both sides of the red blood cell membrane.     

Association of HLA-DRB1 and DQB1 alleles with red blood cell alloimmunization in Chinese

Few systematic investigations have assessed the correlations between red blood cell (RBC) antibodies and human leukocyte antigen (HLA)-DRB1 alleles in the Chinese population. In this case-control study, we investigated whether specific HLA-DRB1 alleles were associated with RBC alloimmunization by calculating the odds ratios for the frequencies of HLA alleles associated with alloimmunization to different RBC antigens. Three hundred and eight patients harboring RBC alloantibodies were analyzed as the case group, and the frequencies of the HLA-DRB1and HLA-DQB1 alleles in control individuals were analyzed by collecting data from the China Marrow Donor Program (including more than 1.6 million healthy people). HLA alleles were genotyped by single specific primer-polymerase chain reaction. The development of anti-C was associated with DRB1*07, DQB1*06, and DQB1*08; anti-C,e was associated with DRB1*07 and DQB1*06; and anti-E and anti-M were associated with DQB1. Other associations were identified between anti-E and DRB1*09 and between anti-Le^a and DRB1*01. Thus, our findings confirmed that HLA-DRB1 and DQB1 restriction played an important role in the generation of RBC alloantibodies in Chinese individuals.     

Population biology of Proteocephalus macrocephalus (Creplin) in the European eel, Anguilla anguilla (Linnaeus), in two small rivers

Seasonal changes in populations of Proteocephalus macrocephalus (Creplin) were investigated for 17 months in the European eel, Anguilla anguilla (Linnaeus), in two rivers in Devon, SW England, but no clear seasonal patterns in prevalence and abundance were apparent. Population levels of the cestode are low in both localities, and it is suggested that natural population levels of P. macrocephalus may generally be low. However, growth and maturation of the cestode show marked seasonality with both occurring mainly in early summer.     

Development and characterization of a continuous cell line (EL) from the liver of European eel Anguilla anguilla

In the present study, a new hepatic tissue‐origin cell line from European eel Anguilla anguilla has been developed and characterized. This cell line designated EL has been maintained in Leibovitz L‐15 supplemented with 10% fetal bovine serum over 72 months, and subcultured more than 90 times. The EL cell line consisted predominantly of fibroblast‐like cells, which could survive over 100 days in vitro, and could grow at 15–32°C. The optimum temperature for growth was 27°C. The chromosome analysis revealed a modal diploid karyotype of 2n = 38. The origin of this cell line was confirmed by the 18S recombinant (r)RNA sequencing. The susceptibility test indicated significant cytopathic effects in the EL cells with regard to the Rana grylio virus and the Herpesvirus anguillae. The viral replication was confirmed by transmission electron microscopy and polymerase chain reaction analysis. Following poly (I:C) exposure, the expression levels of the immune‐related molecules interferon regulatory factor‐7 (irf7) and transforming growth factor‐β (TGF‐β) were downregulated in EL cells, whereas the expression levels of the rf3 and the cytochrome P450 (CYP450) were upregulated. All four genes were significantly upregulated following inflammation by lipopolysaccharide (LPS). These data suggested the application of EL cell line for viral identification, as well as for immunodiagnosis and pharmacological targeting.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Seasonal population dynamics of Paraquimpevia tenevvima (Linstow) (Nematoda) in the European eel Anguilla anguilla (Linnaeus) in three localities of southwest England

Seasonal population dynamics of Puraquzmperia tenerrima (Linstow) in the European eel Anguillu anguillu (Linnaeus) have been investigated in three localities in Devon, southwest England. The nematode is common and exhibits very similar seasonal patterns in changes of prevalence and abundance in the three localities. The population increases over winter through to late spring or early summer as the recruitment of the new generation occurs throughout this period. Reproduction of the nematode starts in spring. A sharp decrease of prevalence and abundance accompanies or follows reproduction, and infection levels are very low in autumn and early winter. The dispersion pattern of the nematode varies with the infection levels from over-dispersion in spring and early summer to almost random dispersion in autumn and winter. It is suggested that P. tenerrimu is a common specific parasite of eels, especially in small streams or rivers and that insect larvae or species of crustaceans might serve as intermediate hosts for the nematode.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.