A Balancing Act: The Immune System Supports Neurodegeneration and Neurogenesis.

Decapod crustaceans, like mammals, retain the ability to make new neurons throughout life. In mammals, immune cells are closely associated with stem cells that generate adult-born neurons. In crayfish, evidence suggests that immune cells (hemocytes) originating in the immune system travel to neurogenic regions and transform into neural progenitor cells. This nontraditional immune activity takes place continuously under normal physiological conditions, but little is known under pathological conditions (neurodegeneration). In this study, the immune system and its relationship with neurogenesis were investigated during neurodegeneration (unilateral antennular ablation) in adult crayfish. Our experiments show that after ablation (1) Proliferating cells decrease in neurogenic areas of the adult crayfish brain; (2) The immune response, but not neurogenesis, is ablation-side dependent; (3) Inducible nitric oxide synthase (iNOS) plays a crucial role in the neurogenic niche containing neural progenitors during the immune response; (4) Brain areas targeted by antennular projections respond acutely (15 min) to the lesion, increasing the number of local immune cells; (5) Immune cells are recruited to the area surrounding the ipsilateral neurogenic niche; and (6) The vasculature in the niche responds acutely by dilation and possibly also neovascularization. We conclude that immune cells are important in both neurodegeneration and neurogenesis by contributing in physiological conditions to the maintenance of the number of neural precursor cells in the neurogenic niche (neurogenesis), and in pathological conditions (neurodegeneration) by coordinating NO release and vascular responses associated with the neurogenic niche. Our data suggest that neural damage and recovery participate in a balance between these competing immune cell roles.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

FEATURES ULTRASTRUCTURAL ORGANIZAITION OF SENSORY NEURONS IN THE ABDOMINAL MUSCLES RECEPTOR ORGANS CRAYFISH ASTACUS LEPTODACTYLUS.

Despite the fact that abdominal muscle receptor organ crayfish is one of the most thoroughly studied objects in neurobiology, the ultrastructural mechanisms that make up its two different operating systems are disclosed insufficiently as the focus of these papers was paid to the area of representation of dendrites of sensory neurons in the receptor muscles. Detailed comparative analysis of the fine structure of the soma sensory cells was not conducted. In the present study, we have investigated the ultrastructure of soma of slow- and fast-adapting sensory neurons of abdominal muscle receptor organ Astacus leptodactylus with special attention to the differences between the two systems. Differences in the fine structure of the main cell organelles have been identified and quantified. We have discovered a relatively high concentration of mitochondria in sensitive cells of slow type receptor as well as a large total area of tanks of the endoplasmic reticulum cells in neurons of faster system. We have assumed that the differences revealed in the fine structure of organelles in soma of sensory neurons between the two types of receptors might be partially responsible for the speed of adaptation and for sensitivity thresholds observed in the physiological experiments.

Ultrastructure of egg activation and cortical reaction in the noble crayfish Astacus astacus.

Morphological changes in the gamete of female noble crayfish at four developmental stages were studied. Mature ovarian oocytes are enclosed by the first envelope that consists of two layers. Numerous pores are visible on the surface of the outer layer of this envelope and the inner layer contains bottlebrush-shaped objects. Three types of vesicles, including some that are highly and moderately electron-dense and some multi-layered, occur in the cortex of the oocyte. In freshly ovulated eggs, the pores in the outer layer are closed, and the bottlebrush-shaped objects in the inner layer are replaced by a spongiform electron-lucent matrix containing scattered electron-dense vesicles. One hour post-spawning, the first envelope, especially its inner layer, is condensed. The highly and moderately dense vesicles discharge their contents into the perivitelline space, where they combine and form a second envelope around the egg. Twenty-four hours post-ovulation, a second envelope is visible in the perivitelline space and the outer part of egg cortex. The highly and moderately electron-dense vesicles continue to release and combine contents to further construction of the second envelope after 24h post-spawning. The egg attachment stalk is derived from the first envelope. The fertilization coat in the noble crayfish consists of first and second envelopes that are produced by the ovary and the cortical reaction, respectively.

Adult neurogenesis: ultrastructure of a neurogenic niche and neurovascular relationships.

The first-generation precursors producing adult-born neurons in the crayfish (Procambarus clarkii) brain reside in a specialized niche located on the ventral surface of the brain. In the present work, we have explored the organization and ultrastructure of this neurogenic niche, using light-level, confocal and electron microscopic approaches. Our goals were to define characteristics of the niche microenvironment, examine the morphological relationships between the niche and the vasculature and observe specializations at the boundary between the vascular cavity located centrally in the niche. Our results show that the niche is almost fully encapsulated by blood vessels, and that cells in the vasculature come into contact with the niche. This analysis also characterizes the ultrastructure of the cell types in the niche. The Type I niche cells are by far the most numerous, and are the only cell type present superficially in the most ventral cell layers of the niche. More dorsally, Type I cells are intermingled with Types II, III and IV cells, which are observed far less frequently. Type I cells have microvilli on their apical cell surfaces facing the vascular cavity, as well as junctional complexes between adjacent cells, suggesting a role in regulating transport from the blood into the niche cells. These studies demonstrate a close relationship between the neurogenic niche and vascular system in P. clarkii. Furthermore, the specializations of niche cells contacting the vascular cavity are also typical of the interface between the blood/cerebrospinal fluid (CSF)-brain barriers of vertebrates, including cells of the subventricular zone (SVZ) producing new olfactory interneurons in mammals. These data indicate that tissues involved in producing adult-born neurons in the crayfish brain use strategies that may reflect fundamental mechanisms preserved in an evolutionarily broad range of species, as proposed previously. The studies described here extend our understanding of neurovascular relationships in the brain of P. clarkii by characterizing the organization and ultrastructure of the neurogenic niche and associated vascular tissues.

Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus.

In this study, we explored the pathogenic mechanism of white spot syndrome virus (WSSV) in crayfish, Cherax quadricarinatus, by investigating activities of enzymes related to innate immune function during infection. After 6-12 h of exposure to WSSV, the activities of four enzymes, phenoloxidase (PO), peroxidase (POD), superoxide dismutase (SOD) and lysozyme (LSZ), increased in the gills of C. quadricarinatus but then sharply decreased during longer infection times. Except for PO, the activities of other enzymes in the WSSV-infected crayfish (Group II) were significantly lower than those of the controls at 72 h post-exposure (P < 0.01). Interestingly, the enzyme activities in the group treated with polysaccharides before challenge with WSSV (Group III) were higher than those in Group II. This phenomenon demonstrated that the polysaccharides could improve the immuno-enzyme activities and enhance the organism's antiviral defenses. Morphological examination by transmission electron microscopy revealed abundant WSSV particles and significant damage in the gills of infected crayfish. WSSV infection caused parts of the gill epithelium and microvilli to be reduced in number and size or damaged; meanwhile, the mitochondria morphology changed, with parts of the cristae diminished leaving large vacuoles. Moreover, electron dense deposits appeared and heterochromatinized nuclei could be seen in blood cells with ruptured nuclear membranes and outflow of nucleoplasm. The findings of this study furthers our understanding of the biochemical alterations induced by viral infections, including changes in the antioxidant status, oxidative stress and lysozyme activity, which could help to advance strategies for control of WSSV in crayfish.

[Glio-neuronal and glio-glial syncytial cytoplasmic connections in peripheral nerve trunks of the crayfish Astacus leptodactylus].

The paper considers various aspects of glial sheaths of neuritis in the crayfish peripheral nerve trunks and roots. There are revealed dotted glio-neurite tight junctions and a varicose deformation of the intercellular glio-neurite cleft. Rupture of membranes in the area of contact leads to formation of the glio-neurite pore (less than 10 nm) that is enlarged and forms wide (up to 240 nm) syncytial perforations. At the edge of perforation, either remnants of tight junctions are present or damaged membranes that fuse and are rounding. The lumen of perforations always contains residual membranous bodies in the form of vesicles. Their deviation from the median line can indicate a mutual translocation of substances of the glio- and neuroplasm. In the adjacent layers of the multilayer glial sheath there is noted a similar phenomenon of formation of the glio-glial syncytial connection terminating by fusion of neighbor glial layers, which is terminated by fusion of neighbor glial layers into the single lamina. The process begins from the varicose deformation of interglial clefts, which appears as a result of massive formation of dotted and expanded tight membranous contacts. As a result of transformation of ellipsoid varicose deformations into the spherical ones, syncytial pores (less than 10 nm) between them are formed, which are enlarged and break the paired gliolemmas into fragments. As a result, the adjacent glial layers are united. Since this process in intact animals occurs on the background of undamaged nerve structures, a suggestion is put forward about its reversibility and the functional nature.

Response properties of crayfish antennules to hydrodynamic stimuli: functional differences in the lateral and medial flagella.

Antennules have been reported to influence localization of distant food odors, sex discrimination, and agonistic and social behaviors of decapod crustaceans. Although olfaction by the antennules is largely recognized, information on the sensitivity of antennules to hydrodynamic stimuli has been scant. In red swamp crayfish Procambarus clarkii antennules, mechanosensory setae outnumber the chemosensory setae. We studied the mechanosensitivity of crayfish antennules by recording neural activities from isolated antennules in response to sinusoidal dipole stimuli. Both the lateral and the medial flagellum of the antennules responded to hydrodynamic stimuli, although the medial flagellum showed more sensitivity at frequencies higher than 60 Hz. The most dominant setae present on the stimulated site were the simple setal type. Although both lateral and medial flagella are capable of detecting chemical and hydrodynamic cues, results from neural responses, morphological observations and antennular behavior observations indicate that the lateral flagellum of P. clarkii functions as an olfactory organ whereas the medial flagellum complements as a hydrodynamic receptor. It appears that in crayfish antennular sensory processing, crayfish simultaneously use chemical and hydrodynamic information. We have compared our data with the threshold of fish lateral line to the same stimuli and we discuss probable similarities in response properties.

Ultrastructure of neuroglial contacts in crayfish stretch receptor.

In order to explore neuroglial relationships in a simple nervous system, we have studied the ultrastructure of the crayfish stretch receptor, which consists of only two mechanoreceptor neurons enwrapped by glial cells. The glial envelope comprises 10-30 glial layers separated by collagen sheets. The intercellular space between the neuronal and glial membranes is generally less than 10-15 nm in width. This facilitates diffusion between neurons and glia but restricts neuron communication with the environment. Microtubule bundles passing from the dendrites to the axon through the neuron body limit vesicular transport between the perikaryon and the neuronal membrane. Numerous invaginations into the neuron cytoplasm strengthen glia binding to the neuron and shorten the diffusion pathway between them. Double-membrane vesicles containing fragments of glial, but not neuronal cytoplasm, represent the captured tips of invaginations. Specific triads, viz., "flat submembrane cisterns - vesicles - mitochondria", are presumably involved in the formation of the invaginations and double-membrane vesicles and in neuroglial exchange. The tubular lattice in the glial cytoplasm might transfer ions and metabolites between the glial layers. The integrity of the neuronal and glial membranes is impaired in some places. However, free neuroglial passage might be prevented or limited by the dense diffuse material accumulated in these regions. Thus, neuroglial exchange with cellular components might be mediated by transmembrane diffusion, especially in the invaginations and submembrane cisterns, by the formation of double-walled vesicles in which large glial masses are captured and by transfer through tubular lattices.

Habitat-related divergence among tailfan sensory systems in reptantian Decapod crustaceans.

Squat lobsters (Galatheidae) and mole sand crabs (Hippidae) differ in posture and locomotion from each other and from crayfish, their surrogate ancestor for neurobehavioral features. Galatheids resemble crayfish more closely in general behavior and niche, but are intermediate between crayfish and hippids with respect to morphology and neuromusculature. The tailfan is inverted under the abdomen in both, due to the flexed abdominal posture, but its morphology has diverged considerably. Nothing is known about adaptations of the tailfan exteroceptors to the new sensory world of either group. We used SEM, vital staining, and extracellular electrophysiological techniques to survey the sensory structures on the telsons of the galatheid Munida quadrispina and the hippid Emerita analoga for comparison with published data on the homologous mechanosensory system in crayfish. Both telsons bear plumose, peg, and non-annulate (natatory or guard) setae. In addition, M. quadrispina has singly-innervated smooth setae and E. analoga a previously undescribed type of small seta the outer face of which is covered by transversely-oriented, thin setules that are much broader than they are long and angled outward toward the seta's distal end, overlapping loosely. The 'stack-of-scales' appearance of its distal portion viewed from the side engendered the name: scaly seta. Some shared features with other small setae that are chemo- and mechanoreceptive suggest scaly setae might be bimodal sensilla. The telson of M. quadrispina is very flexible. Plumose setae on its dorsal surface are arranged into hemi-circlets and most, if not all, appear not to be innervated. They may contribute to adjacent smooth setae's mechanosensitivity via mechanical coupling through adjacent cuticle, as occurs between feathered and smooth setae on crayfish antennae. Sensory nerve recordings show many afferents to have low thresholds to mechanical disturbance, suggesting they are hydrodynamic receptors. The telson of E. analoga is rigid, and all dorsal setae are relegated to the margins. Patches of scaly setae on the anterior lateral dorsal telson are strategically located to sense the substrate when the crabs are in sand. Scaly and peg setae are arrayed along shallow grooves, one along each side, that are flanked laterally by a fringe of plumose and pappose setae. Substantial deflection from resting position of the latter was required to reliably elicit afferent activity, suggesting most function as touch receptors. The different, non-random distributions of tailfan setae match these animals' divergent sensory worlds and might have engendered species-specific alterations in their central sensory systems.

Cellular structures involved in the transport processes and neuroglial interactions in the crayfish stretch receptor.

In order to explore neuroglial relationships in a simple nervous system, the ultrastructure of crayfish stretch receptor, which consists of only two sensory neurons enveloped by satellite glial cells, was studied. Neuronal Golgi complex was oriented such that its output trans-Golgi network usually faced the bundles of microtubules within the neuronal cytoplasm and very rarely to the outer membrane. Therefore, it participates mainly in the processing of proteins transported along microtubules to distal neuron parts rather than those transported to glial cells. Structural triads of submembrane cisterns-vesicles-mitochondria were involved in formation of glial protrusions into the neuronal cytoplasm. The double-wall vesicles within the neuron body were the captured parts of such glial protrusions. Glial protrusions and double-wall vesicles facilitated the neuroglial transport and large-scale delivery of the glial material into the neuron. The neuroglial transport could also be performed by diffusion across the intercellular space. These data indicate the significant neuroglial exchange with cellular components.

A scanning electron microscopic study on the appendage morphology of Astacus leptodactylus (Eschscholtz, 1823) and Pacifastacus leniusculus (Daña, 1852) (Crustácea:Decapoda: Astacoidea)

This study compares the morphology of rostrum, pereipods 1,2,4 and mouthparts of juvenile Astacus leptodactylus with those oí Pacifastacus leniusculus. Differences in morphology were observed, in particular with regard to the mouthparts e.g. including setal armature and number of teeth on the mandible. In general, the shape of the rostra in the two species is similar in that both taper to a point with a pair of sharp spines distally. Laterally the rostrum of A. leptodactylus is bordered by a regular row of setae, which is not so well defined in P leniusculus. The observations also showed that in addition to an increase in size, changes in morphology in the feeding apparatus between the developmental stages of the two species were present. It was concluded that both species have similar rostra, but different setal patterns and there are differences between the two species in the armature of mouthparts as development progresses. Therefore, important differences in the morphology of mouthparts between P. leniusculus and A. leptodactylus and in the different stages of the species might cause a difference in the feeding behavior and food choice of the species.

Este estudio compara la morfología del rostro, pereiópodos 1,2,4 y piezas bucales de los Astacus leptodactylus jóvenes con los de Pacifastacus leniusculus. Se observaron las diferencias en la morfología, en particular, con respecto a las piezas bucales, por ejemplo incluyendo la armadura setal y el número de dientes en la mandíbula. En general, la forma del rostro en las dos especies es similar, tanto cónicas, como en punta, con un par de espinas distalmente. Lateralmente al rostro, A. leptodactylus está bordeada por un fila de setas, que no está tan bien definida en P leniusculus. Las observaciones también muestran que, además de un aumento en el tamaño, estaban presentes cambios en la morfología en el aparato masticatorio, entre las etapas de desarrollo de las dos especies. Se llegó a la conclusión que ambas especies tienen rostros similares, pero diferentes patrones setales y hay diferencias entre las dos especies en la armadura de piezas bucales como evolución del desarrollo. Por lo tanto, importantes diferencias en la morfología de piezas bucales entre P leniusculus y A. leptodactylus y en las distintas etapas de la especie podrían causar una diferencia en la conducta de alimentación y opciones de alimentación de la especie.

[Synthesis of nucleic acids and localization of atrial natriuretic peptide in the crayfish haemocytes].

Three types of cells circulate in haemolymph of the crayfish Astacus astacus: agranular haemocytes (HCs I), small-granule haemocytes (HCs II) and large-granule haemocytes (HCs III). Their proliferation, differentiation and function remain poorly understood. By means of light and electron microscopic autoradiography using [3H]-thymidine, we have revealed that only HCs I are capable of DNA synthesis and mitosis whereas HCs II and HCs III are replicatively inactive. To determine whether the HCs I are proliferating progenitor cells for the granular HCs, we have analyzed autographs of HC population in 1, 2, 7 and 21 days after a single [3H]-thymidine administration. Contrary to the expectation, we have failed to find labeled HCs II and HCs III. These findings raise doubts on the capacity of the HCs I to differentiate into two other types of HCs. By autoradiography using 3H-uridine, it has been detected that intensity of the RNA synthesis was the greatest in HCs I and less by a factor of two and four in HCs II and HCs III, respectively. Additionally, by EM immunocytochemistry, ANP-like immunoreactivity was revealed in the large granules of the HCs III. We assume that availability of ANP in secretory granules extends the possible functions of the crayfish HCs and suggests their participation in regulation of water-salt balance and immune responses.

[Endocellular regulation of oxygen consumption in the neurone of isolated crawfish stretch receptor].

Morphological correlations of functional regulation of oxygen consumption have been investigated in single of isolated crustacean stretch receptor neuron. The increase in oxygen consumption is promoted by: 1) redistribution of mitochondria and increase in cytochrome oxidase (CO) activity in mitochondria near to the plasmatic membrane; 2) coordination of mitochondria aggregation rhythms with pO2 rhythms in external environment of a cell; 3) reduction of the area with high CO and mitochondria activity, and reduction of the way of oxygen diffusion; 4) increase in CO activity gradient from periphery to the center of the neuron body; 5) carry of oxygen by water current under hydration of the neuron body, and cytoplasm dilution under transition of a part of gel in sol; 6) cyclic changes in the neuron body and hillock sizes ratio determining carry of oxygen by water current into the neuron body, oxygen absorption by mitochondria in the neuron body, and transition of the water released from oxygen from the neuron body into hillock and further into the external environment.

[A phototube connecting digital cameras with light optic microscopes].

We present here a phototube model making possible connection of a digital camera with light optic microscopes in order to obtain images of microobjects and for their further computer treatment. The advantage of this model is simplicity of its manufacturing and small required expenses as well as an increase in information density for microobject studies. This phototube has been covered by a patent for a useful model N2 48228 registered in the Public Register of RF on September 27, 2005.

Desarrollo embrionario del camarón de río Cryphiops caementarius (Decapoda: Palaemonidae) en condiciones de laboratorio / Embryonic development of the freshwater shrimp Cryphiops caementarius (Decapoda: Palaemonidae) under laboratory conditions

We describe seven stages for the embryonic development of Cryphiops caementarius (Molina, 1782) using both light and scanning electron microscopy. Thirty ovigerous females were captured in Limarí River, Chile, and maintained separately in three 35 liter tanks with temperatures of 15, 22 and 28 ºC. At the same time, some embryos were separated from the females and placed in a 100 ml beaker for in vitro culture at 15 ºC. Duration of development at 15, 22, and 28 ºC was of 36-39, 30-32 and 25-28 days, respectively, with no differences observed between in vitro and in vivo cultures. Seven development stages were observed: (I) homogeneously distributed vitellum; the embryo cleavage leads to the morulae stage; (II) first embryonic stage; antennule, antenna and ocular globe are present; (III) primordium lengthens, and the outlines of the pereiopods can be observed; ocular pigmentation appears as a curved line; (IV) ocular pigmentation in hemispherical form; the antennae develop, and the first red pigmentation appears near the eye; heartbeat is visible; (V) the pereiopods touch the base of the antennae; the ocular globe is more evident, in a oval form; (VI) eyes are faceted, and both antenna and third abdominal segment with chromatophores; ocular pigmentation as spherical form; (VII) ocular globe with spherical eye pigmentation and facets on the surface; star-shaped chromatophores on the appendages. Egg volume increased from 0.082 mm3 at the beginning of the development to 0.125 mm3 close to hatching, representing a rise of 65.6 %. The diameter of the embryo and eye pigmentation may be used as growth parameters.

Se describen siete estados del desarrollo embrionario de Cryphiops caementarius Molina, 1782), tanto a microscopía de luz, como electrónica de barrido, y se establece el tiempo de desarrollo en embriones cultivados a tres temperaturas. Además se realizó un cultivo in vitro a 15 ºC en vasos precipitados de 100 ml. Hembras ovígeras capturadas en el río Limarí, Chile se ubicaron en estanques de 35 litros a temperaturas de 15, 22 y 28ºC. La duración del desarrollo a 15, 22 y 28 ºC fue de 36-39, 30-32 y 25-28 días, respectivamente. El cultivo in vitro a 15 ºC no presentó diferencias con los cultivos in vivo. Se establecieron siete estados de desarrollo. (I) vitelo distribuido homogéneamente, durante este estado comienza dividirse llegando a estado de mórula; (II) aparece el primordio embrionario con los esbozos de la anténula, antena y mandíbula; (III) se observan los esbozos de los pereiópodos y abdomen; pigmentaciónocular en forma de línea curva; (IV) pigmentación ocular en forma de una semiesfera, aparece pigmentación roja en las antenas, cerca del ojo; se observa latido cardíaco; (V) los pereiópodos se extienden hasta la base de las antenas; la pigmentación ocular es oscura y con forma ovalada; (VI) pigmentación ocular esférica y con algunas facetas en la superficie; cromatóforos en la antena y tercer segmento abdominal; (VII) la pigmentación ocular tiene forma esférica y aparecen cromatóforosestrellados en los pereiópodos. El embrión aumenta su volumen en 65.6 %, desde 0.082 mm3 en el estado I hasta 0.125 mm3 en del estado VII. El diámetro del embrión y la pigmentación del ojo pueden ser utilizados como parámetros de crecimiento en esta especie.

A novel Spiroplasma pathogen causing systemic infection in the crayfish Procambarus clarkii (Crustacea: Decapod), in China.

A novel disease of crayfish Procambarus clarkii appeared in the summer of 2004 in freshwater aquaculture in Jiangsu province of China. Light and transmission electron microscopy (TEM), molecular biological methods and in vitro culture were used to identify the pathogen. The agent was unique in having a helical morphology and rotary motility as observed by phase-contrast light microscopy and was found in haemolymph, muscles, nerves and connective tissues by smear method and TEM. Ultra-thin sections under TEM revealed the wall-free membrane of the microbe. The agent could pass through membrane filters with pores 220 nm in diameter and was cultivated in vitro in M1D medium. 16S rDNA of the crayfish pathogen was amplified by PCR using primers specific for Spiroplasma-specific 16S rDNA. The resultant 271bp PCR product showed 99% identity with Spiroplasma mirum 16S rDNA, having a close relationship with the spiroplasma from the Chinese mitten crab Eriocheir sinensis. This is the second time a spiroplasma has been found in a freshwater crustacean. The 271bp PCR product was also amplified from the bottom mud in the ponds associated with the disease. The PCR molecular method is an effective way to detect spiroplasma in freshwater environment. The results from this study are significant in expanding the host range of spiroplasma and freshwater ecology.

Activity-dependent change in morphology of the glial tubular lattice of the crayfish medial giant nerve fiber.

An evaluation of electron micrographs of stimulated nerve fibers used to investigate the effect of action potential generation on the structure-function relationship between axons and its associated glial cells revealed that what was at first thought to be stimulation-induced damage to the glia was, in fact, limited to volume expansion and disaggregation of the glial tubular lattice. All other structures appeared well preserved and otherwise normal. Using a 4-point subjective scale for evaluation by two investigators, 50-Hz stimulation for 2 min was observed to cause a volume expansion and disaggregation of the tubular lattice. Quantitatively, the internal diameter of the stimulated tubular lattice increased 65% above the unstimulated control (50.96 +/- 2.09 nm and 30.81 +/- 0.87 nm, respectively, P < or = 0.001). Stimulation had its greatest effect on tubular lattice volume and organization in the adaxonal glial layer and a decreasing effect as distance from the giant axon increased. These effects are reversible since the tubular lattice diameter and degree of disaggregation preserved 10 min after the cessation of stimulation were not found to be different from their unstimulated paired controls. Axons injected with TEA, a voltage-gated potassium channel blocker, prevented stimulation-induced volume expansion and disaggregation of tubular lattice structure. These results are consistent with an active uptake of K+ with obligated water or, alternatively, hyperosmotic K+ uptake and a fixation-induced increase in water permeation. Either mechanism of K+ uptake would result in tubular lattice volume expansion and disaggregation and suggests that the tubular lattice serves a larger role than a simple trans-glial diffusion pathway.