Light and Electron Microscopy of the Spore of Myxobolus heckelii n. sp. (Myxozoa), Parasite from the Brazilian Fish Centromochlus heckelii (Teleostei, Auchenipteridae)

ABSTRACT. A myxosporean parasitizing the gill filaments of the freshwater teleost fish Centromochlus heckelii collected in the Tocantins River (Lower Amazonian Region, Brazil) is described using light and electron microscopy. This parasite produces spherical to ellipsoidal cyst-like plasmodia up to 250 μm in diameter, with a thick wall strengthened by several stratified juxtaposed crossed collagen layers, whose thickness varies according to the number of the layers. Several compressed fibroblasts are observed among the collagen fibrils. Deposits of spherical dense material are scattered at the internal periphery of the cysts. Plasmodia and different developmental stages, including immature and mature spores, filled the central region of the cysts. The spore body is ellipsoidal in valvar view and biconvex in sutural view. It is formed by two equal-sized and symmetric valves measuring 12.7 μm long (12.2–13.1) ( n =50), 6.6 μm wide (6.3–6.9) ( n =25), and 4.0 μm (3.7–4.4) ( n =20) thick. A thin layer formed by fine and anastomosed microfibrils is observed at the spore surface. Two equal, elongated pyriform polar capsules measure 2.9 μm (2.7–3.3) × 1.7 μm (1.4–2.0) ( n =25), each containing four or five oblique polar filament coils. The binucleated sporoplasm contains numerous spherical sporoplasmosomes, glycogen particles, and a large vacuole with fine granular matrix. Based on the morphological and ultrastructural differences and specificity of the host, we describe this isolate as a new myxosporidian, Myxobolus heckelii n. sp. (Myxozoa, Myxosporea).     

Eimeria procyonis sp. n., an Isospora sp., and a Redescription of E. nuttalli Yakimoff and Matikaschwili, 1932 (Protozoa: Eimeriidae) from the American Raccoon (Procyon lotor)

SYNOPSIS. Thirty-two of 48 raccoons examined were infected with a previously undescribed species of Eimeria which is herein named E. procyonis. Of the 32 infected animals, 10 also harbored E. nuttalli and 1 had Isospora sp. oocysts.
The ellipsoid to ovoid oocysts of E. procyonis measured 23.4 × 18.0 (16–29 × 13–24) μm; its sporocysts measured 12.1 × 9.3 (11.5–15 × 7–10) μm, each containing a slightly flattened substiedal body. The sporocyst residuum consisted of numerous scattered granules each ∼1 μm in diameter. The oocyst wall was double-layered. The outer layer appeared rough and pitted, measuring 1.5 μm, except at the micropyle where it was 1 μm thick.
The oocysts of the Isospora sp. measured 16.8 × 13.7 (16–18.5 × 12.5–15.5) μm. The wall consisted of a single layer ∼0.5 μm thick. The sporocysts measured 11.2 × 9.1 (9.5–11.5 × 8–10) μm, and each contained 4 elongate sporozoites. The oocysts of E. nuttalli measured 17.5 × 13.6 (12-21 × 11-15) μm, with a smooth single-layered wall approximately 0.7 μm thick. The sporocysts measured 12.2 × 7.1 (9-13 × 5.5–11) μm. Each sporocyst had a thin, dark, Stieda body and the sporocyst residuum consisted of many fine granules.     

Eimeria tarentolae n. sp. from the Moorish Gecko, Tarentola mauritanica

ABSTRACT. A new species of Eimeria from the Moorish gecko, Tarentola mauritanica , of Minorca, Spain, is described. Oocysts of Eimeria tarentolae are ellipsoidal and measure 17.8 (17.6–18.7) μm by 13.5 (12.9–14.0) μm. Micropyle, oocyst residuum, and polar granule are absent. Each oocyst contains four spherical to slightly subspherical sporocysts 6.8 (6.4–7.0) μm in diameter. A sporocyst residuum is present, while a Stieda body is lacking. Sporulation is completed within 24–32 h at 21 ± 2°C.     

Eimeria crotalviridis sp. n. from Prairie Rattlesnakes, Crotalus viridis viridis, in New Mexico with Data on Excystation of Sporozoites and Ultrastructure of the Oocyst Wall

SYNOPSIS Oocysts of Eimeria crotalviridis sp. n. are described from prairie rattlesnakes, Crotalus viridis viridis in New Mexico on the basis of light and electron microscopy and in vitro excystation of sporozoites. Sporulated oocysts of E. crotalviridis are elliptical, 26.4 × 22.3 (23–29 × 20–24) μm with ovoid sporocysts 11.7 × 8.1 (11–13 × 7–9) μm. A micropyle, micropyle cap and polar bodies are absent, but oocyst and sporocyst residua and Stieda and substieda bodies are present. Excysted sporozoites are 12.4 × 2.8 (11–13 × 2–3) μm and have 1 large posterior refractile body and a nucleus with a prominent nucleolus. Ultrastructurally, the oocyst wall has 2 layers, a thick, electron-dense, highly sculptured outer layer composed of a fine granular matrix and a thin, granular, osmiophilic inner layer, separated from the outer layer by at least one unit membrane. These layers are 441 (353–510) and 21.6 (19–29) nm thick, respectively. Within 15 min after exposure to a trypsin-sodium taurocholate fluid, sporozoites of E. crotalviridis excysted from 5-month-old sporocysts.     

Water permeability of plant cuticular membranes: the effects of humidity and temperature on the permeability of non-isolated cuticles of onion bulb scales

Abstract. Water permeability of cuticular membranes (CM) from the inner bulb scales of Allium cepa has been investigated. CM have a thickness ranging from 0.6 to 1.3 μm. They are composed of a thin (120–200 nm) lamellated cuticle proper and a thicker (300–900 nm) cuticular layer. Permeability coefficients for diffusion of water across these thin membranes are very low (4 × lO⁻¹⁰ms⁻¹⁰). There was no difference in permeability of CM from successive scales of the same onion. Extraction of soluble cuticular lipids (SCL) with chloroform increased permeability by a factor of 1350 to 2050. Preliminary data indicate that only 1 μg cm⁻¹⁰ of SCL are removed by this treatment, hydrocarbons being the main (75%) consistuent. Permeability coefficients of cuticular transpiration were little affected by relative humidity, showing that transport is limited by a hydrophobic barrier that lacks dipoles. However, following extraction, permeability of the membranes depended strongly on humidity due to the presence of polar functional groups in the polymer matrix. Soluble cuticular lipids undergo a phase transition around 47°C. Temperatures higher than that irreversibly increased water permeability.     

Two ultrastructurally distinct types of transporting tissues,the branchiostegal and the gill epithelia,in an estuarine tanaid,Sinelobus stanfordi (Crustacea,Peracarida)

Summary On each lateral side of the cephalothorax segments, the adult Sinelobus stanfordi has a branchial chamber which contains an elongated bag-shaped gill and is covered by a thick branchiostegite. The ultrastructural study revealed that the inner surface of the branchiostegite is composed of a transporting-type epithelium which is morphologically distinct from the gill epithelium. Both epithelia are covered by extremely thin (about 80 nm) cuticle layers, suggesting high permeability to gases, ions, and water. In contrast, the outer surface of the branchiostegite consists of ordinary epithelium covered by a very thick cuticle layer in common with the body surface. The inner branchiostegal epithelium (4–10 m thick) has a shallow (about 1 m deep) apical infolding system of the cell membrane (AIS) and an extensive three-dimensional tubular network (about 120 nm in diameter) which is formed by the invagination of the basolateral cell membrane (TNB). The TNB is associated with slender mitochondria and occupies the majority of the cytoplasmic area of the epithelial cell. The gill epithelium, on the other hand, is about 10 m thick and characterized by an abundance of oval mitochondria, well-developed (4–5 m deep) AIS, and a smooth basal cell membrane lacking any infoldings. These morphological features indicate that not only the gill epithelia, but also the inner branchiostegal epithelia, are involved in the ion-transporting processes. The ultrastructural differences between these two kinds of epithelia also suggest their different roles in the osmoregulation of this animal, since it inhabits estuaries which are subject to extreme changes in salinity.     

Electron microscopical localization of chitin in the cuticle of Halicryptus spinulosus and Priapulus caudatus (Priapulida) using gold-labelled wheat germ agglutinin: phylogenetic implications for the evolution of the cuticle within the Nemathelminthes

 The ultrastructure of the cuticle of adult and larval Priapulus caudatus and Halicryptus spinulosus is investigated and new features of cuticle formation during moulting are described. For the localization of chitin by TEM wheat germ agglutinin coupled to colloidal gold was used as a marker. Proteinaceous layers of the cuticle are revealed by digestion with pronase. The cuticle of larval and adult specimens of both species consists of three main layers: the outer, very thin, electron-dense epicuticle, the electron-dense exocuticle and the fibrillar, electron-lucent endocuticle. Depending on the body region, the exocuticle comprises two or three sublayers. The endocuticle can be subdivided into two sublayers as well. In strengthened parts such as the teeth, the endocuticle becomes sclerotized and appears electron-dense. Only all endocuticular layers show an intense labelling with wheat germ agglutinin-gold conjugates in all investigated specimens. Additional weak labelling is observed in the exocuticle III layer of the larval lorica of P. caudatus. All other cuticular layers remain unlabelled. Chitinase dissolves the unsclerotized endocuticular layers almost completely, but also exocuticle II and partly the loricate exocuticle III. The epicuticle, the homogeneous exocuticle I and the sclerotized endocuticle are not affected by chitinase. The labelling is completely prevented in all layers after incubation with chitinase. Pronase dissolves all exocuticular layers, but not evenly. The presumably sclerotized regions of exocuticle I are not affected as well as the complete epicuticle and the endocuticle. All cuticular features of the Priapulida are compared with the cuticle of each high-ranked taxon within the Nemathelminthes with special regard to the occurrence of chitin. Based on this out-group comparison it can be concluded that: (1) a two-layered cuticle with a trilaminate epicuticle and a proteinaceous basal layer represents an autapomorphic feature of the Nemathelminthes, (2) the stem species of the Cycloneuralia have already evolved an additional basal chitinous layer, (3) such a three-layered cuticle is maintained as a plesiomophy in the ground pattern of the Scalidophora and (4) in the Nematoida, the chitinous basal layer is replaced by a collagenous one at least in the adults; the synthesis of chitin is restricted to early developmental phases or the pharyngeal cuticle. Accepted: 12 March 1998     

New Eimeriid Coccidia from the Canarian Lizard, Gallotia galloti Oudart, 1839

One species of Eimeria and one species of Isospora have been discovered in several specimens of the Canarian lizard, Gallotia galloti , collected on Tenerife, Canary Islands, and are described herein as new. The oocysts of Eimeria gallotiae n. sp. are elongate-ellipsoidal and measure 30.6 (29.1–32.6) by 16.0 (14.0–17.9) μm. Micropyle, oocyst residuum, and polar granule are absent. Oocysts each contain four ellipsoidal sporocysts measuring 14.6 (12.2–17.3) by 9.2 (8.2–11.2) μm. Sporocysts have sporocyst residuum but not a Stieda body. The majority of oocysts are fully sporulated when shed. Few oocysts are excreted unsporulated or in the beginning phase of sporulation. The oocysts of Isopora gallotiae n. sp. are spherical and 16.5 (15.3–17.6) μm in diameter. Micropyle, oocyst residuum, and polar granule are absent. Each oocyst has two lemon-shaped sporocysts, 11.5 (10.2–12.2) by 7.3 (6.6–8.2) μm, which take up almost the entire space of the oocyst. A sporocyst residuum is present as well as a knob-like Stieda body and a substieda body. Most oocysts are in the beginning phase of sporulation when excreted and have two spherical sporoblasts. Sporulation is completed within 24 to 28 h at 21 ± 2°C.     

Isospora viridanae N. Sp., an Intranuclear Coccidian Parasite from the Canarian Skink, Chalcides viridanus (Lacertilia: Scincidae)

A caryotropic species of coccidium, Isospora viridanae n. sp. (Apicomplexa: Eimeriidae). has been found as a parasite in the Canarian skink, Chalcides viridanus Gravenhorst, 1851, from Tenerife, Spain, and is described here as a new species. Fully sporulaled oocysts of Isospora viridanae are spherical and measure 21.6 (1 7.6–23.4) nm in diameter. Mtcropyle, oocyst residuum and polar granule are ali absent. Oocysts of this coccidian, with a smooth bilayered wall, contain 2 ovoid sporocysts 13.2 (11.7–14.0) by 9.5 8.2–10 5) μm. A sporocyst residuum is present as well as a Stieda body and a substieda body. Most oocysts are found to be at the beginning of sporulanon when excreted and show 2 spherical sporoblasts. Sporulation is completed within 24 to 48 h at 23 × 2°C Sporozoites are 13–14 μm long and are about 2.5–3 μ m wide. Endogenous stages of schizogony and gamogony develop in the nuclei of epithelial cells from the small intestine of the skink. Comparisons with other species of the genus found in lacertilian hosts indicate that it is a new species.     

The cocoon and hatchling of Nais variabilis (Naididae: Oligochaeta)

SUMMARY. 1. The cocoon of Nais variabilis is ovoid with an average length of 527.6±8.4 μm (95% CL) and an average breadth of 378.1±9.9 μm. The cocoon wall is about 7 μm thick and the average volume of the cocoon cavity is 0.035±0.002 mm³. Most cocoons contain one ovum; only single-ovum cocoons developed.
2. Cocoons hatch in about 80 days at room temperature (average 20°C. range 17–23°C). Hatchlings emerged posterior end first and were, on hatching, about one-third the adult live length of 8.5±10.3 mm.
3. It is estimated that adult Nais variabilis are unlikely to produce more than an average of ten cocoons each. Adults ceased asexual budding on becoming nature and died shortly after the completion of cocoon production. There was a brief co-existence of generations.     

Carbohydrate and lipid metabolism during the last larval moult of the tobacco hornworm, Manduca sexta

Abstract. At 25°C and with a light regime of 17 h light and 7h dark, the last larval moult of the tobacco hornworm, Manduca sexta , lasts approximately 32 h, during which profound changes of metabolism were observed. At the onset of the moult, which coincides with the cessation of feeding, the proportion of active fat body glycogen phosphorylase increased from 10 (-2h) to 25–30% (Oh). A biphasic pattern with peak activities of 45–50% after t – 12 h and again just prior to the shedding of the cuticle (32 h) was subsequently observed. Haemolymph trehalose concentration decreased significantly from c. 35 (Oh) to 20mM (8h), but then recovered to an intermediate level (30mM; 12h). After completion of the moult, the trehalose concentration was 35–40 mM. The haemolymph glucose level in feeding fourth instar larvae was 4–5 mM, but decreased sharply before the onset of the moult to c. 1 mM, followed by a slow 6-fold increase over the next 20h. Prior to the shedding of the cuticle, the glucose level dropped again dramatically. The haemolymph lipid level increased slowly from an initial level of 1.2–1.4mg/ml during the early part of the moult, reaching a maximum of 1.8mg/ml after /= 16 h. Afterwards, a decrease of c. 50% was observed until ecdysis occurred. Oxygen consumption per animal decreased steadily from 30–35 μl/min pre-moult by approximately 70% to c. 10 μl/min but started to increase about 5 h before the animals resumed feeding.     

Time Sequence of Early Events in Fertilization in the Medaka Egg

The time sequence of early events in fertilization was examined in eggs of the medaka Oryzias latipes . The mean time after insemination required for sperm attachment to the egg surface through the micropyle depended on sperm concentrations. It was 3 ± 1 sec with a range from 1 to 6 sec after insemination when concentration of spermatozoa was high (about 2 × 10⁸/ml at 23°–25°C). The mean time from sperm attachment until cessation of its movement on the egg surface was 4 ± 1 sec with a range from 1 to 9 sec. Small cortical alveoli at the animal pole region within 15 μm of the sperm attachment point began to undergo exocytosis 9 ± 0.3 sec (range 5–16 sec) after sperm attachment. The velocity at which the exocytosis wave propagated increased from the earliest initiation point of exocytosis up to the 100 μm area, and became constant at about 12 μm/sec from 100 μm to 500 μm from the sperm attachment point. The present results suggest that at the time of fertilization in the fish egg, exocytosis of small cortical alveoli in the area about 15 μm away from the sperm attachment point occurs simultaneously.     

Discovery of Stretch Receptor Organs in Nematodes–Structure, Arrangement and Functional Analysis

Filamentous organs, termed metanemes and interpreted as stretch receptor organs, have been discovered in nematodes of the order Enoplida. They have not been found in species of 11 other nematode orders. According to light microscopical observations, a metaneme consists of a 5–15 μm long scapulus, a 20–400 μm long frontal and a 30–170 μm long caudal cuticularized filament; the latter is lacking in some taxa. Metanemes are arranged serially in the lateral epidermal cords; they are parallel or at an angle of 10–30° to the longitudinal body line. The function of metanemes is likely to coordinate the serpentine movement and to control the actual body volume.     

An ultrastructural study of the cuticle in the marine annelid Heterodrilus (Tubificidae, Clitellata)

The ultrastructure of the cuticle in four species of the marine Heterodrilus (H. paucifascis, H. pentcheffi, H. flexuosus, H. minisetosus) is investigated with transmission electron microscopy. The noncellular cuticle consists of several parts; closest to the epidermis is a thick zone of collagen fibers embedded in a matrix. The matrix continues outside the fiber zone, forming a layered epicuticle. The external surface of the epicuticle is covered by evenly distributed, membrane-bound bodies, termed epicuticular projections. The epicuticular projections have their longitudinal axis perpendicular to the surface of the cuticle and are attached to the surface by either the surrounding membrane itself or by short pedestals. Microvilli, extensions from the epidermal cells, penetrate and sometimes pass completely through the cuticle. There is interspecific variation in the morphology of the cuticle. The four studied species differ in the arrangement of the collagen fibers, from irregularly distributed fibril bundles to orthogonally arranged fiber layers, as well as in the number and density of layers in the epicuticle. One of the studied species, H. paucifascis, shows intraspecific variation, which is associated with sample locality. The Bahamian specimens of H. paucifascis have four layers in the epicuticle, club-shaped epicuticular projections, and collagen fibers forming a less defined orthogonal grid, while the Belizean specimens have three layers in the epicuticle, epicuticular projections with a bulging part at midlevel, and a distinct orthogonal grid. Based on these findings the variation in the morphology of the cuticle appears to be dependent on both phylogenetic constraints, and functional and environmental factors.     

Spironucleus vortens N. Sp. from the Freshwater Angelfish Pterophyllum scalare: Morphology and Culture

ABSTRACT. A new diplomonad flagellate, Spironucleus vortens n. sp., is described from the intestinal lumen of the freshwater angelfish, ( Pterophyllum scalare ), bred in Florida. Live organisms are pyriform, and measure 12.5–20.5 μm long by 5.0–11.2 üm wide. Scanning electron microscopy shows that the trophozoite bears two compound lateral longitudinal ridges, each originating posterior to three emerging anterior flagella, and continuing posteriorly to the emergence of the posterior flagellum. Each ridge comprises a broad central part, surrounded by a peripheral ridge. At the opening of the flagellar pocket, the broader right peripheral ridge crosses to the other side of the body, and then back again. The posterior end of the body bears two papillae. Transmission electron microscopy shows that the compound lateral ridges are supported by microtubules, and bear microfibrillar structures in discrete longitudinal plaques. The serendipitous growth of S. vortens in a culture system with lip tumor tissue, facilitated axenic cultivation in a modified TYM medium (trypticase, yeast extract, maltose). The flagellate is now routinely maintained in an axenic TYI-S-33 medium (trypticase, yeast extract, iron serum), and is stabilized in the cryopreserved state. Spironucleus vortens is an aerotolerant anaerobe that can be cultured at 25° C, 28° C and 30° C.     

Ultrastructure and cytochemistry of the integument of Modiolicola gracilis,parasitic copepod in mussel gills (Mytilus galloprovincialis and Mytilus edulis)

Of mussels taken from the Ebro Delta River (E. Spain), 3% have a nonmodified copepod, Modiolicola gracilis, in the gill tissues. The cuticle of different segments of the body has an epicuticle with two layers, which show external microvilli-like projections. Weakly positive reactivity to the PTA technique has been detected in the external region. The procuticle has the helicoidal architecture of the chitinous tegument in arthropods, whereas the cuticle shows discontinuities in the regions of ducts in tegumental glands. The integument is comprised of three types of cells. Epidermal cells are flat with numerous mitochondria. Muscle cells show well-developed mitochondria with several longitudinally distributed cristae. A third and secretory cell shows a well-developed rough endoplasmic reticulum and Golgi complex in the basal zone. Its apical portion is full of secretory granules. Through the cuticle, these integumental glands open directly to the cuticular surface via a short duct coated by epicuticle. The composition and specializations of this complex cuticular architecture differ markedly from those shown by an endoparasitic copepod detected in the digestive gland of the mussel. It does not appear that the specializations detected in the cuticle of M. gracilis lead to any histopathological alteration in host tissues. © 1994 Wiley-Liss, Inc.     

Eimeria sinaitae n. sp. (Apicomplexa: Eimeriidae) from the Rock Agama (Agama sinaita) in Saudi Arabia

Eimeria sinaitae n. sp. is described from the gall bladder of Agama sinaita from Wasie, Saudi Arabia. Sporulated oocysts are elongate-ellipsoid 34.4 times 22.0 (29.0–40.0 times 17.4–24.5) μm. Oocyst wall is smooth, greenish yellow, 1.2 (1.0–1.4) μm thick, and two-layered. Micropyle, polar granule, and oocyst residuum are absent. Sporocysts are ellipsoid 11.4 times 7.6 (9.8–15.0 times 6.7–9.0) μm. Sporocyst residuum is present. The sporocysts lack a Stieda body. Sporozoites are crescent-shaped, blunt at one end and tapered at the other. Eimeria species from Agamidae are compared.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

Temperature- and salinity-dependent development of a Nordic strain of Ichthyophthirius multifiliis from rainbow trout

During the twentieth century evidence was presented which suggested the presence of various strains and races of the parasite Ichthyophthirius multifiliis Fouquet. However, ecological profiles of various parasite isolates from different climatic zones are sparse. Such stringent characterizations of parasite development at defined abiotic conditions could provide valuable criteria for the different races; profile comparison from various localities is one way to differentiate these strains. Baseline investigations were therefore performed on the associations between abiotic factors (temperature/salinity) and the development of theronts in tomocysts of I. multifiliis isolated from rainbow trout in a Danish trout farm. It was shown that tomocyst formation and theront development took place between 5 and 30°C. Development rates and sizes of theronts were clearly affected by temperature: theronts escaped tomocysts already after 16–27 h at 25°C and 30°C, whereas this process took 8–9 days at 5°C. Likewise, theront size decreased steadily from a maximum of 57.4 × 28.6 μm at 5°C to 28.6 × 20.0 μm at 30°C. This size variation was only partly associated with the number of theronts that appeared at different temperatures. The lowest number of theronts escaping from one tomocyst was indeed found at 5–7°C (mean 329–413). At 11.6, 17.0 and 21°C, the highest number of theronts appeared (mean 546–642). However, at 25 and 30°C, the number decreased (458 and 424, respectively). Additional studies on the salinity dependent development of the parasite (at 11.6°C) showed that salinities above 5 p.p.t. totally inhibited development. Even at 5 p.p.t. the developmental time significantly increased and the number of theronts produced from one tomocyst decreased.