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.     

Immunofluorescent detection of erythrocyte sialoglycoprotein antigens on murine erythroid cells

A sialoglycoprotein fraction isolated from murine (DBA/2) erythrocytic ghosts (see companion article, Sarris and Palade, 1982, J. Cell. Biol. 93:583-590) was used to raise antibodies in rabbits. By immune-IgG (serum)-[125I] protein A overlays, the antibodies were found to react positively with the four sialoglycoprotein monomers (gp-2.1, gp-2.2, gp- 3.1, and gp-3.2) of the original fraction, with the sialoglycoproteins detected in erythrocytic ghosts (gp-2.1 and gp-3.1), with a diffuse component (probably a macroglycolipid) trailing around gp-3.1 in SDS polyacrylamide gel electrophoretograms of solubilized ghosts, and with a minor sialoglycoprotein hidden under this trail. IgG's isolated from immune and nonimmune rabbit sera were conjugated to tetramethylrhodamine isothiocyanate and used to survey, by fluorescence microscopy, the distribution of the cognate antigens on the three different erythroid lines known to succeed each other during the life span of the mouse. In the peripheral blood of the adult, the antibodies recognized only mature erythrocytes; they did not crossreact with either platelets, monocytes, or different types of granulocytes. In the spleen of adult anemic mice, the antibodies reacted weakly with proerythroblasts and strongly with all types of erythroblasts. In enucleating erythroblasts, antigens were preferentially segregated on the cell membrane of the nascent reticulocyte. In the 10-day-old embryo, antigens were already present on the primitive nucleated erythrocytes (produced by the blood islets of the yolk sack), and in the 14-d fetus they were found on all hepatic erythroblasts and derived non-nucleated erythrocytes. A positive immunoreaction was also obtained on Friend erythroleukemic cells, before or after induction by dimethyl sulfoxide. Nonimmune serum, or nonimmune IgGs gave negative reactions in all cases. The antibodies were species-specific: they did not crossreact with either human or rat erythrocytes.     

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.     

Morphological and biochemical characterization of mitochondria in Torpedo red blood cells

A study is presented on the morphology and respiratory functions of mitochondria from Torpedo marmorata red blood cells. In vivo staining of red blood cells and transmission electron microscopy showed the existence of a considerable number of vital and orthodox mitochondria which decreased from young erythroblasts to mature erythrocytes from 60-50 to 30-20 per cell. In erythrocytes mitochondria exhibited a canonical, functional respiratory chain. The content and activity of cytochromes in erythrocytes were, however, significantly lower as compared to mammalian tissues.     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.     

Observations on granulocyte peroxidase in New Zealand freshwater eels, Anguilla species

Peroxidase activity in the granulocytes of eels was investigated using o-tolidine, paraphenylene-diamine-pyrocatechol, and 4-chloro-l-naphthol as substrates, and cyanide, azide and aminotriazole as inhibitors. Most circulating neutrophils of Anguilla australis Richardson, 1848 and A. dieffenbachii Gray. 1842 showed no peroxidase activity at pH 7.6 and pH 9.0, but a few neutrophils, thought to be mature, were positive. Another granulocyte in the anterior kidney, spleen, parasitized gill tissue and, rarely, the blood contained a cyanide-resistant, azide-inhibited, peroxidase and was tentatively identified as the eosinophil. Neutrophils of A. anguilla (L.) showed granular peroxidase activity which was inhibited by cyanide. The eosinophil was not observed.
Absence of peroxidase from most circulating neutrophils in A. anguilla and A. dieffenbachii , and its pattern in the neutrophil precursors and mature neutrophils of A. anguilla , may be due to two morphologically indistinguishable granule types. Primary peroxidase-negative granules occur in precursors and immature neutrophils and secondary peroxidase-positive granules in mature neutrophils of all three eels. Circulating neutrophils in New Zealand eels seldom mature and are theretore peroxidase-negative, whereas A. anguilla neutrophils are mature and are usually peroxidase-positive.
Impairment of microbicidal activity in neutrophils lacking peroxidase is discussed.     

An enhanced humoral immune response against the swimbladder nematode, Anguillicola crassus, in the Japanese eel, Anguilla japonica, compared with the European eel, A. anguilla.

The humoral immune response in the two eel species, Anguilla japonica and Anguilla anguilla against two fractions of antigens in Anguillicola crassus were studied. Within species, both eel species showed significantly elevated titres compared with controls when immunized with antigens from Anguillicola crassus. In interspecific comparison, Anguilla japonica showed significantly elevated titres in comparison with Anguilla anguilla. Immunization of Anguilla anguilla caused a significantly decrease in the plasma levels of protein in comparison with control fish and all groups of Anguilla japonica. In contrast, Anguilla japonica showed significantly lower plasma levels of Ig in all groups compared with Anguilla anguilla. The different susceptibilities to Anguillicola crassus between the natural host, Anguilla japonica, and the naive, Anguilla anguilla, is partly due to differences in the ability of the two eel species to mount a humoral immune response.     

Effects of aconitine and veratrine on the isolated perfused heart of the common eel (Anguilla anguilla L.)

The effects of two alkaloids: aconitine and veratrine have been investigated in isolated perfused heart of the common eel (Anguilla anguilla L.). Low concentrations (less than 10(-6)M) of aconitine induced a decrease of the heart rate where high concentrations (greater than 10(-6)M) produced a tachycardia and finally led to a depolarization of cardiac cells. Veratrine induced a decrease of the heart rate depending on the concentration. A cardiac arrest was observed with high concentrations (greater than 10(-4)M). Removal of aconitine or veratrine from perfusion medium did not reverse the effects of high concentrations. The tachycardia and arrhythmias were triggered or enhanced.     

Histochemical Demonstration of the Inhibitory Effect of Nuvan® and Neguvon® on Cholinesterase Activity in Pseudodactylogyrus anguillae (Monogenea)

The nervous system of Pseudodactylogyrus anguillae, a gill parasite from European eel (Anguilla anguilla) is demonstrated histochemically using the acetylthiocholine iodide method. It was shown that the staining of the nervous system was reduced or eliminated due to the inhibitory effect of Nuvan® and Neguvon®.     

Effects of anaesthesia and surgery on levels of adrenaline and noradrenaline in blood plasma of the eel (Anguilla anguilla L.)

1. The effects of surgery and anaesthesia on adrenaline and noradrenaline plasma levels were investigated in the eel (Anguilla anguilla L.). 2. Effect of surgery: highest values were obtained when putting back the fish in water. Three hours after surgery, adrenaline and noradrenaline plasma levels were always significantly higher than those obtained 24 and 48 hr after surgery. 3. Effect of anaesthesia: anaesthesia only had no effect on adrenaline and noradrenaline plasma levels. 4. It was concluded that the trauma of surgery was mainly responsible for the elevation of CA plasma levels in the eel. A minimum post-operative period of 24 hr should be allowed before any blood sampling for estimation of resting CA plasma levels. Resting adrenaline and noradrenaline plasma levels, 48 hr after surgery, were respectively 1.31 +/- 0.38 and 3.37 +/- 0.41 pmol/ml.     

Nucleolar changes in maturing avian erythroblasts and erythrocytes

Maturing erythroblasts and erythrocytes were studied in chickens and adult hens to provide more information on the presence and frequency of various nucleolar types in these cells. Nucleoli were present at all stages of erythroblastic and erythrocytic development except in the case of a few reticulocytes and the mature erythrocytes. The number of nucleoli per cell (expressed as the nucleolar coefficient) reached a maximum at the stage of the polychromatic erythroblast. Early erythroblasts were characterized by the presence of compact nucleoli or nucleoli with nucleolonemata. Rings shaped nucleoli and micronucleoli increased in number with further maturation. Cells of the final erythroblast stage (orthochromatic erythroblasts) contained mostly micronucleoli, and micronucleoli alone were present in reticulocytes and mature erythrocytes.     

Malarial Parasites of the "Jesu Cristo" Lizard Basiliscus basiliscus (Iguanidae) in Panama

SYNOPSIS. The iguanid lizard Basiliscus basiliscus in Panama is parasitized by Plasmodium basilisci and P. achiotense sp. nov. P. basilisci in this host is characterized by schizonts containing 4–14 merozoites, with schizonts parasitizing proerythrocytes containing more merozoites than those in erythrocytes. Asexual parasites lack cytoplasmic projections, while mature gametocytes are round or oval with regular margins.
P. achiotense is characterized by the combination of prominently pigmented, large schizonts containing 36–56 merozoites and oval or round gametocytes which are about 1/3 larger than those of P. basilisci.
EE-schizonts of P. basilisci were observed commonly in thrombocytes and occasionally in lymphocytes, and appeared early in experimental infections induced by blood inoculation.     

Erythropoietin control of terminal erythroid differentiation: maintenance of cell viability, production of hemoglobin, and development of the erythrocyte membrane

Using the spleen cells of mice infected with the anemia-inducing strain of Friend leukemia virus, an in vitro model system of erythropoiesis has been developed in which a homogeneous population of murine proerythroblasts terminally differentiates in response to erythropoietin (EP). The biochemical events involved in EP's capacity to maintain viability, induce hemoglobin production, and promote the development of the specialized erythrocyte membrane were studied during the 48-72 hour period required for proerythroblasts to differentiate into reticulocytes. The results show that EP increases glucose uptake and the syntheses of RNA and protein in the first few hours after exposure of the erythroblasts to the hormone. A coordinated production of heme, alpha and beta globin occurs later and peaks at about 48 hours. This peak corresponds to the time at which the majority of cells are undergoing enucleation and becoming reticulocytes. The syntheses of the erythrocyte membrane and membrane skeletal proteins are not coordinated, and multiple patterns of synthesis are found with respect to the time of EP exposure. A number of proteins are lost from the membrane fraction while the characteristic proteins of the mature erythrocyte become prominent in the membrane fraction of erythroid cells as they develop from reticulocytes into erythrocytes.     

Cytamoeba bacterifera Labbé, 1894. I. Morphology and Host Incidence of the Parasite in California.

SVXOPSIS. Parasites belonging to the genus Cytamoeba have been found in amphibians from central and northern California. The infected animals and their numbers were (URODELA) Aneides flavipunctatus (2), A. lugubris (29), Batrachoseps a. atttnuatus (81). Dicamptodon ensatus (2), and (SALIENTIA) Rana b. boyli (1). The rates of infection varied between 0.6% and 29.3%, and the number of parasites per infected cell ranged from one to seven.
Preparations stained with haematoxylin and Romanowsky dyes indicated several types of parasite: some were homogeneous, others possessed several dark staining granules and some possessed large, dark staining central bodies or peripheral dark staining rims. Parasites were seen in the erythrocytes, the plasma and, occasionally, in endothelial cells and monocytes. They were found in the peripheral and heart blood and in the blood of the liver, spleen, kidneys and lungs. Cell division was common in erythrocytes, and yielded daughter cells of identical size. In some cases, chains of 3 to 5 individuals were seen and, rarely, three bodies, in a cloverleaf configuration, were produced as the result of a single division. Following acid hydrolysis, methylene blue-stained parasites showed a peripheral ring of granules, and, in some, a few stained centrally located bodies.     

Changes in the skin-scale complex with sexual maturation in the European eel, Anguilla anguilla (L.)

Structural features of the epidermis, dermis and scales were examined in adult female Anguilla anguilla of varying sexual maturity. Advanced sexual stages were obtained by hormone injection. The dermis increased in thickness with both size and sexual development. Epidermal thickness was unrelated to size or maturity but mucous cell densities decreased in more mature animals. Hormone treated eels showed a loss of mucous cells accompanied by degeneration of the epidermis. Scale areas increased from 50% in sexually immature adults to 145% in sexually maturing eels. Skin calcium values ranged from 0.27 to 0.83 mg cm⁻² and did not show any relation to either scale or sexual maturity. The possible relevance of structural changes to migration is discussed.     

Saurian Malarial Parasites in Eastern Panama

SYNOPSIS Plasmodium gonatodi sp. nov. is described from Gonatodes albogularis fuscus of eastern Panama. It is characterized by elongate gametocytes and polymorphic schizonts containing 12-46 nuclei when apparently mature. Both proerythrocytes and erythrocytes are commonly parasitized, host cells are hypertrophied and distorted, and their nuclei are displaced. Prematuration sexual stages may be irregularly shaped and larger than mature gametocytes.
Plasmodium diploglossi Aragão and Neiva , 1909 is reported from Mabuya mabouya in eastern Panama, and Plasmodium morulum sp. nov. is described from this host. P. morulum usually parasitizes immature erythrocytes, and is characterized by lenticular or oval to round gametocytes, and schizonts with 14-40 nuclei usually arranged in a globular mass. Host cells are slightly hypertrophied and distorted, and their nuclei are usually displaced. Inoculation of infected blood into clean hosts produces numerous schizonts in white cells as well as in the erythrocyte series.
Pigment in both P. gonatodi and P. morulum , if present, consists of a few minute dark dots which do not meet the polarized light test for hemozoin.     

Measurements on ground or sectioned otoliths: possibilities of bias

Back-calculation usually requires measurements of growth marks revealed on otoliths by specific preparations. The standardization of the grinding (or sectioning) plane is necessary, but difficult, especially along the antero-posterior axis. In order to show the importance of the grinding plane, tetracycline labelling of eel otoliths ( Anguilla anguilla L.) has been used. This marking has a calcio-traumatic effect on otoliths, which can be revealed with staining techniques. Unless the grinding plane is incorrect, the tetracycline labelling and the staining of the ground surface are then superposed.     

PRIMITIVE ERYTHROPOIESIS IN EARLY CHICK EMBRYOGENESIS : I. Cell Cycle Kinetics and the Control of Cell Division

The primitive line of embryonic chick blood cells develop as a relatively homogeneous cohort of cells. Using an analysis based on the continuous uptake of thymidine-³H, we have established the generation time, G1, S, and G2 for progressively more mature generations of these immature erythroblasts. The data indicate that after the initiation of hemoglobin synthesis, the average cell will yield six generations of hemoglobin producing erythroblasts. The older generations of erythroblasts exhibit a longer generation time, G1, S, and G2 than the earlier generations of erythroblasts. Other methods of analysis corroborated these findings. One of these methods, an estimate of total erythrocyte productivity from the primitive stem cells (hematocytoblasts), led to the conclusion that the erythroblast cell lineage might be initiated as early as the sixth or seventh division following fertilization. In addition, primitive erythroblasts characterized by one set of cell cycle parameters, when grown in serum associated with erythroblasts of different parameters, showed no alteration in mitotic behavior. These results suggest the presence of programmed cell division not immediately cued by extracellular influence.     

Nucleosome repeat lengths in the definitive erythroid series of the adult chicken

Morris [1] has suggested that the difference in nucleosome repeat length between chicken liver (200 base pairs) and mature chicken erythrocytes (212 base pairs) may be due to the presence of histone H5 which is found in chicken erythroid cells but not in other tissues. Levels of H5 increase during erythroid maturation in the adult chicken. To determine what influence H5 might have on repeat length, erythroid populations at various stages of maturation were isolated, and repeat lengths and levels of H5 were determined. Bone marrow cells from anemic chickens were cultured in vitro to permit non-cycling erythroblasts to mature and thus increase in density. Less dense cycling basophilic erythroblasts were then isolated by buoyant density centrifugation. This population has a repeat length of 205 base pairs and an H5 content roughly two-thirds that of mature erythrocytes, which have a repeat length of 212 base pairs. A population intermediate in maturation, consisting of cells of the anemic peripheral blood, has a repeat length of 218 base pairs, and the predominant cell type in this population has an H5 content greater than that of mature erythrocytes. Therefore, changes in histone H5 content are reflected by the nucleosome repeat length during erythroid maturation.     

Haemogregarina nototheniae n. sp., from the blood of Antarctic nototheniids

A new species of haemogregarina, Haemogregarina nototheniae, is described from the Southern ocean teleosts Notothenia neglecta and Notothenia rossii. Stages identified as macro- and microschizogony and gametogony are described in mononuclear leukocytes from fish caught during the austral summer. The mature gametocyte is the most commonly found stage: it is exoerythrocytic, but carries the host erythrocyte nucleus attached to its external surface near one end. The gametocyte has a central nucleus and 2–16 subterminal eosinophilic granules, but no polar cap. During microschizogony the schizont nucleus undergoes repeated division without cytoplasmic division to give 32 nuclear masses, all of which appear to be in metaphase. Cytoplasmic division yields free merozoites identifiable by the coarse chromatin of the nuclear area. During macroschizogony the intraleukocytic parasite swells to a subspherical mass with a median band of fine heterochromatin granules. The cytoplasm later divides, forming three merozoites. There appear to be two routes by which merozoites proceed to become gametocytes: in winter small merozoites are seen in mature erythrocytes; but in summer, in erythroblasts. The invertebrate definitive host and the means of transmission are unknown, but the parasite is provisionally assigned to the genus Haemogregarina.