Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

The granular leucocytes of an active, mature female tuatara, Sphenodon punctatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and beta-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus. In the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electron-dense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles, lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, beta-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared 'altered' or degenerate and most of these contained microtubules. The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.     

Social network structure and parasite infection patterns in a territorial reptile, the tuatara (Sphenodon punctatus)

We investigated whether the parasite load of an individual could be predicted by its position in a social network. Specifically, we derived social networks in a solitary, territorial reptile (the tuatara, Sphenodon punctatus), with links based on the sharing of space, not necessarily synchronously, in overlapping territories. Tuatara are infected by ectoparasitic ticks (Amblyomma sphenodonti), mites (Neotrombicula spp.) and a blood parasite (Hepatozoon tuatarae) which is transmitted by the tick. We recorded the location of individual tuatara in two study plots twice daily during the mating season (March) in 2 years (2006 and 2007) on Stephens Island, New Zealand. We constructed weighted, directed networks to represent pathways for parasite transmission, where nodes represented individual tuatara and edges connecting the nodes represented the extent of territory overlap among each pair of individuals. We considered a network-based hypothesis which predicted that the in-strength of individuals (the sum of edge weights directed towards a node) in the derived network would be positively related to their parasite load. Alternatively, if the derived social network did not reflect actual parasite transmission, we predicted other factors such as host sex, size or territory size may better explain variation in parasite infection patterns. We found clear positive relationships between the in-strength of tuatara and their tick loads, and infection patterns with tick-borne blood parasites. In particular, the extent that individuals were connected to males in the network consistently predicted tick loads of tuatara. However, mite loads of tuatara were significantly related to host sex, body size and territory size, and showed little association with network measures. The results suggest that the pathway of transmission of parasites through a population will depend on the transmission mechanism of the parasite, but that social networks provide a powerful predictive tool for some parasites.     

Ultrastructural observations on the sporogony of Eimeria maxima

Pittilo R. M. and Ball S. J. 1985. Ultrastructural observations on the sporogony of Eimeria maxima. International Journal for Parasilology15: 617–620. Sporogony in Eimeria maxima was found to commence with a contraction of the oocyst cytoplasmic mass which was limited by a single membrane. By 9 h (at 29°C) dense bodies, surrounded by polysaccharide granules, were closely applied to the zygote limiting membrane. By 12 h nuclear division had occurred and by 15 h separate sporoblasts could be discerned. Sporozoite formation occurred from 21 to 30 h.     

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea)

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.     

Humoral immune responses of channel catfish (Ictalurus punctatus) fry and fingerlings exposed toEdwardsiella ictaluri

The ability of channel catfish to develop antibody responses to Edwardsiella ictaluri was evaluated. Fish were produced and reared under specific pathogen free (SPF) conditions. At the ages of 1, 2, 3 and 4 weeks and 2, 3, 4, 5, and 6 months post-hatch, a group of these naive fish were given a primary immersion exposure to E. ictaluri (mean doses of 6·4×10⁴ cfu ml⁻¹of tank water). Each group received a secondary immersion exposure 4 weeks after the primary exposure and were sampled 2 weeks after each exposure. Control groups were exposed to sterile culture medium. Specific antibody titres were first detected in fish exposed at 4 weeks post-hatch at an average weight of 85 mg. A secondary response was first demonstrated by fry that received a primary exposure at 4 weeks post-hatch and a secondary exposure at 8 weeks post-hatch. However, a true boosting effect was first demonstrated in fish that received their primary exposure at 2 months of age. Fish given a primary exposure before 4 weeks of age and a secondary exposure failed to produce a significant antibody response, even though they were the same age at secondary exposure as fish that produced strong antibody responses upon primary exposure. This phenomenon suggests that immunological tolerance was induced and indicates that channel catfish may not be capable of generating a humoral immune response before four weeks of age.     

The effect of thermal adaptation on the level of nitrogenous excretion of Channa punctatus (bloch)

The rates of ammonia-N and urea-N excretion have been measured in summer- winter-adapted Channa punctatus, a representative of an Indian freshwater air-breathing teleost. The season-specific difference in these rates have been analyzed with respect to thermal adaptation during the variation of seasonal temperature of summer and winter months. Due to cold adaptation (either in nature's laboratory or a man made laboratory) the rates of ammonia-N and urea-N excretion are decreased, suggesting a lesser mobilization-N excretion during warm adaptation (either summer acclimation or 32°C acclimation) are noticed, suggesting a greater mobilization of protein and ureogenesis.     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary Each ovarian follicle of Triops cancriformis is four-celled; these cells (one oocyte and three nurse cells) are interconnected by cytoplasmic bridges. In the course of differentiation, the nurse cells are early recognizable; they increase in size more than the oocyte and their nuclei contain many nucleoli. For the first time in Arthropoda, yolk globules are reported to be present in nurse cell cytoplasm; these globules arise from the smooth endoplasmic reticulum. The functional significance of the intercellular bridges and the trophic role of the nurse cells are discussed.The authors are grateful to Dr. Bruno Sabelli for his support and to Mr. Francesco Monte for his technical assistance     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

Cytochemical, immunocytochemical and ultrastructural observations on leukocytes and thrombocytes of fat snook (Centropomus parallelus)

The cytochemical, immunocytochemical and ultrastructural characteristics of leukocytes and thrombocytes in the peripheral blood of the fat snook (Centropomus paralellus) - a fish occurring in Brazil - were investigated. The cytochemical methods were performed to demonstrate four enzymatic reactions - o-toluidine-hydrogen peroxide, naphtol AS-MX phosphate, naphtol AS-BI phosphate and alpha-naphtil acetate to detect myeloperoxidase (MPO), alkaline phosphatase (ALP), acid phosphatase (ACP) and non-specific esterase (α-NAE), respectively - and two non-enzymatic ones - Periodic-Acid Schiff (PAS) and Sudan black B (SBB) to detect the occurrence of glycogen and phospholipids, respectively. Immunocytochemical method utilizing polyclonal rabbit antibody against mammal metalloproteinases (MMPs) 2 and 9 were done. Standard method for Electron Microscopy (EM) was applied for the ultrastructural study. The cytochemical reactions were positive in neutrophils for MPO, ACP, α-NAE, glycogen and phospholipids; in lymphocytes for ACP and α-NAE; in monocytes for ACP and α-NAE and in thrombocytes for ACP, α-NAE and glycogen. Only neutrophils were positive for MMPs 2 and 9, and none of the cells studied were positive for ALP. Ultrastructurally: 1) neutrophil showed a spherical shape with a spherical, indented or lobulated euchromatic nucleus, and cytoplasm containing granules of varied sizes and mitochondria of varied shapes and sizes. The nucleus/cytoplasm relation and the size of granules suggest neutrophil maturation in peripheral blood; 2) lymphocytes showed partially heterochromatic nucleus and minimal cytoplasm; 3) monocytes had long cytoplasmic projections, an indented nucleus, evident nucleolus and cytoplasm with granules of varied sizes and vacuoles; 4) thrombocytes were predominantly elliptical or roughly spherical in shape, had a partially heterochromatic nucleus and cytoplasm containing electron-dense granules, intricate canalicular system and vacuoles occasionally holding phagocytic material.     

Morphological,cytochemical, and biochemical observations on the blood of the tuatara,Sphenodon punctatus

Abstract

Morphological features of the circulating blood cells of an adult female tuatara are described and illustrated. Mitosis in circulating erythrocytes and the occurrence of an intraerythrocytic haemogregarine parasite are noted. The packed cell volume (haematocrit) was 35%. There were about 310 000 erythrocytes and 7200 leucocytes per mm³ of blood. The lymphocyte was the predominant leucocyte, followed by the eosinophil, monocyte, neutrophil, and basophil. The eosinophils and neutrophils reacted positively to acid phosphatase and peroxidase; the neutrophils were also esterase positive, and all three granulocytes were PAS positive. Haemoglobin, plasma protein, glucose, sodium, potassium, cholesterol, and triglyceride levels were determined. The primitive nature of the tuatara is reflected in the morphological. mensural, and biochemical data presented.     

Identification and characterization of the tumor suppressor p53 in channel catfish (Ictalurus punctatus)

Herein is presented the sequence of a catfish full-length p53 cDNA obtained from a cloned B cell line cDNA library. Southern blot analyses determined that a restriction fragment linked polymorphism (RFLP) existed with PstI among outbred catfish. Western blot analyses demonstrated that, when compared to PBLs, the catfish leukocyte lines express higher levels of p53 protein. Additionally, the results of Western blot analyses and in vitro translation experiments suggest that the catfish leukocyte lines may produce truncated forms of p53 due to internal initiation.     

Ultrastructural observations on Barroussia schneideri (apicomplexa,eucoccidiida) in the centipede Lithobius forficatus

The ultrastructure of the principal stages of Barroussia schneideri in the intestinal cells of the centipede, Lithobius forficatus, is described. The structure of the merozoites has much in common with that of other Eimeriidae: pellicle of an outer and two inner membranes, microtubules, mitochondria, micronemes, rhoptries, endoplasmic reticulum, and polysaccharide granules. The mature macrogamete has a large nucleus with a distinct nucleolus, wall-forming bodies of type 1 (WFB1), and presumably of type 2 (WFB2), and a large number of polysaccharide granules. The microgametocyte includes smaller polysaccharide granules closely associated with the cisternae of the endoplasmic reticulum. The microgametocyte surface is smooth in outline and has peripherally arranged nuclei. Microgametes have a curved nucleus, a mitochondrion, polysaccharide granules, and two flagella. One of the flagella is attached for some distance along the body.     

Ultrastructural and biochemical observations on interphase nuclei isolated from chicken erythrocytes.

Adult hen erythrocyte nuclei are isolated from cells or haemolysed in situ by acting on the plasma membrane with rotating knives or with non-ionic detergents. When the isolation medium contains magnesium ions (1 mM), sucrose (0-4 M) and Tris buffer (0.01 M, pH 7-5) called SMTOG (see text), the ultrastructure in thin sections through the condensed chromatin bodies, after staining with either uranyl-lead or phosphotungstic acid (PTA), is similar to that found in the intact cell. Hence it can be concluded that the 2 phases which comprise chromatin, the o- and e-phase, survive nuclear isolation. These are so called because the structural units in chromatin are arranged at the surface of the nucleus into one or more layers and give rise to oddly (o) and evenly (e) numbered bands. The 0-phase is also largely retained after extensive washing in 0-07 M NaC1 as shown by electron microscopy and biochemical measurements; only 6% of the total nuclear protein is removed, a value small compared with the fractional amount of the chromatin protein calculated to lie in the o-phase, about 70%. After extensive washing in saline-EDTA there are structural changes in chromatin, but biochemical data show that the molecules in the o-phase are also largely retained; loss of protein amounts to between 5 and 11%. These data suggest that the o-phase is a structural component of the chromatin bodies. They support the hypothesis that condensed chromatin is formed by folding superunit threads. These units consist of a central thread-like element about 17 nm diameter which stains preferentially with uranyl-lead and forms the e-phase, with an outer cylindrical shell forming the o-phase of total diameter about 28nm. The 5-10% proteins removed by salt washes are located exclusively in a particulate component, quite likely the chromatin. They have been examined by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. There are about 10 or more protein species, ranging in molecular weight from 21000 upwards. The groups of large granules previously found in the nuclear sap of intact erythrocytes are shown to be associated with an amorphous or finely fibrillar body.     

Developmental stages and molecular phylogeny of Hepatozoon tuatarae, a parasite infecting the New Zealand tuatara, Sphenodon punctatus and the tick, Amblyomma sphenodonti

The developmental stages of Hepatozoon tuatarae were elucidated in both the tuatara host, Sphenodon punctatus and the tick, Amblyomma sphenodonti. PCR amplicons from A. sphenodonti samples identified DNA matching H. tuatarae. Dissection of tick samples showed oogenesis and sporogony occurring in the haemocoel of A. sphenodonti with the average mature oocyst size being 236 × 228 μm. Partial sequence data of the parasite’s small subunit ribosomal gene, obtained by PCR, was used for phylogenetic comparison. Characterisation of the H. tuatarae lifecycle will help in conservation management of the tuatara.     

Multiple paternity and kinship in the gray fox (Urocyon cinereoargenteus)

Despite mounting evidence that extra-pair copulations (EPCs) are common in the Canidae, no studies have examined the most basal member of this family, the gray fox (Urocyon cinereoargenteus). In this study we explored the possibility that gray foxes may be socially, but not genetically, monogamous. Multiple paternity was confirmed in one litter and suspected in three others (n=7), thus 14.3–57.1% of all litters had more than one father. In this high-density population, multiple paternity may be one strategy to reduce inbreeding, although only one pair was significantly related to each other (r=0.36). Mother–daughter pairs were more common than father–son pairs. These results coupled with a previous study showing a female-biased sex ratio suggest male-biased dispersal and the potential for helper females.     

Ultrastructural and cytochemical observations on the granulocytes of the sturgeon, Acipenser brevirostrum (Chondrostei)

Granulocytes from cranial granulopoietic tissue were studied under the electron microscope, and cytochemistry carried out oncranial and peripheral blood granulocytes of two sturgeons, Acipenser brevirostrum . Ultrastructurally, eosinophils and basophils had homogeneous electron-dense granules similar to those of teleosts and some higher vertebrates. Neutrophils contained two granule types: small elongated fibrillar granules and large (<3.8μm long) usually homogeneous granules.
Neutrophil fibrillar granules were positive for alkaline phosphatase (ALP), acid phosphatase (ACP), α-naphthyl acetate esterase (ANAE), acetyl-l-tyrosine-α-naphthyl esterase (ATNE) and periodic acid Schiff (PAS) reaction. The large homogeneous granules were negative for all enzymes, and were only PAS positive. Eosinophils had granular, cyanide-, azide- and aminotriazole-resistant peroxidase (PO) and were ACP, ATNE, tosyl-l-lysine-α-naphthyl esterase (TLNE) and Luxol fast blue positive.
Ultrastructure and cytochemistry are discussed in relation to other vertebrates, and eosinophils identified as the main phagocytic leucocyte.