Humoral responses of congenital immunity in the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work presents analysis of changes of humoral protective factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The total protein concentration and the titers of hemagglutinins and hemolysins in starfish coelomic fluid, as well as the time of human hemoglobin elimination from circulation were estimated for 6–144 h of the experiment. The hemagglutinin titer was determined in hemagglutination reactions, the hemolysin titer—in hemolysis reaction. Time of human hemoglobin elimination from coelomic fluid was determined in a color enzymatic reaction. The starfish coelomic fluid was revealed to contain soluble factors that are able to interact with antigen— antibody complexes of mammals and have an opsonizing activity. It is established that injection of HE does not change the total protein concentration per 1 ml coelomic fluid, but affects dynamics of changes of the hemagglutinins titer. Time of hemoglobin elimination from circulation does not exceed 24 h. Humoral factors of coelomic fluid of the starfish Asterias rubens play an auxiliary role in congenital immunity reactions.Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 1, 2005, pp. 23–28.Original Russian Text Copyright © 2005 by Kudryavtsev, Dyachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Expression of the starfish complement component C3 gene homolog under the influence of bacterial lipopolysaccharide

A fragment of a homolog of the complement component C3 gene was cloned and sequenced from the starfish Asterias rubens. A phylogenetic analysis of the gene, termed ArC3-like, demonstrated its close similarity to the C3 gene homolog of deuterostome invertebrates. A high level of ArC3-like mRNA expression was observed in circulating cells (coelomocytes), a gut derivate (hepatopancreas), and the male gonad, but not in the stomach, female gonad, and rectal gland of A. rubens. ArC3-like gene expression was detected in all types of starfish coelomocytes, including lymphocyte-like cells and granular and nongranular amebocytes. Bacterial lipopolysaccharide (LPS) injected into the coelomic cavity of starfish increased the ArC3-like gene expression in coelomocytes and the hepatopancreas as compared to the control level (sterile sea water injection). The level of ArC3-like gene expression in coelomocytes in response to LPS reached its maximum 6 h after the injection and decreased to the baseline level 24 h after the injection. In the hepatopancreas, the level of ArC3-like gene expression reached its maximum 6–12 h after the LPS injection stimulation and remained high even 48 h after the injection. A long-term upregulation in response to LPS was demonstrated for the ArC3-like gene.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

Ultrastructure of coelomic epithelium and coelomocytes of the starfish Asterias rubens L. in norm and after wounding

Samples of coelomic epithelium (CE) and coelomocyte suspension of intact and wounded starfish Asterias rubens L. were studied by electron microscopy. The CE was shown to be composed of three types of cells: flagellar (approximately 60%), secretory (approximately 3%), and myoepithelial (approximately 37%); flagellar and secretory cells form the CE apical surface. Secretory cells are represented by two subtypes, i.e., granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. In 4–5% of cases, adjacent flagellar cells are separated by various sizes of intercellular gaps. These gaps seem to be lacunae left by the flagellar cells after their release into the coelomic cavity. The morphological pattern of the conversion of CE flagellar cells into coelomocytes was characterized. After a moderate wounding used in the present study, no significant structural alterations in the CE organization were revealed. In coelomocyte suspension, small rounded young coelomocytes (approximately 3%) and the larger mature coelomocytes (approximately 97%) were found. On the surface of one of the young coelomocytes, a flagellum was revealed. Surface of the mature coelomocytes forms processes of various size and structure; their cytoplasm contains lysosomes and phagocytic vacuoles of different size. After wounding, a coelomocyte activation was found that consisted of a sharp rise in the number and length of filopodia on their surface, as well as the formation of multicellular aggregates. The complex of ultrastructural data allows it to be suggested that the histogenesis of coelomocytes from CE flagellar cells is a process of cell transdifferentiation.     

Comparative analysis of behavior and proliferative activity in culture of cells of coelomic fluid and of cells of various tissues of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> L. Isolated from normal and injured animals

The sources of coelomocytes in Asteroidea are suggested to be the coelomic epithelium, the axial organ, or Tiedemann’s bodies. To study the problem of whether the cells are replenished at the expense of divisions, we analyzed the incorporation of bromodeoxyuridine (BrdU) in vivo into cells from different tissues of the sea star Asterias rubens L. To study differentiation in vitro, methods of isolating and cultivating cells from various tissues were elaborated and an analysis of the behavior and incorporation of BrdU in culture was performed. The reproduced BrdU incorporation was detected in coelomic epithelial cells. The behavior of coelomocytes and the coelomic epithelial cells in culture depended on the time after the injury of the animals in which the cells were isolated, whereas, for the axial organ and Tiedemann’s bodies, no differences were revealed. After 2 months of cultivation, the formation of BrdU-incorporating, colony-like cells with high nuclear-cytoplasmic ratios was characteristic of coelomic epithelial cells. Thus, the most prospective object for studying the processes of A. rubens cell differentiation in vitro seems to be the coelomic epithelium.     

The small cells of coelomic fluid and coelomic epithelium isolated from starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> and <Emphasis Type="Italic">Asterias amurensis</Emphasis> (Echinodermata: Asteroidea): Comparative analysis of cell morphology and proliferative activity in vivo and in vitro

Earlier we revealed the probable candidates for the role of Asteroidea stem cells in the starfish Asterias rubens L., small coelomic epithelial cells (SECs-1) with a high nuclear–cytoplasmic ratio that were able to proliferate in vivo and in vitro. To check the existence of a similar cell type in other members of Asteroidea, the small cells in suspensions of coelomic fluid (CF) and coelomic epithelium (CE) of A. amurensis were analyzed with respect to their morphology and proportion in the total cell pool. The morphology of proliferating cells and the proliferative activity of CF and CE cells in vivo and in vitro were studied. The small cells with parameters identical to those of A. rubens SECs-1, were found both in CF and CE of related species. The subpopulation of weakly attached CE cells, highly enriched with SECs-1, was detected. These cells were able to migrate from CE and to proliferate in vivo and in vitro. Additionally, large proliferating cells were described in both starfish. The dynamics of proliferative activity in primary cell cultures of these starfish had some distinctions. Moreover, for the first time, the formation of “crystals”, the potential centres of spiculogenesis, was observed in primary culture of A. amurensis CE cells. The data prove that SECs may fulfil the common functions in two members of Asteroidea.     

The functional activity of fractions of coelomocytes of the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> Linnaeus, 1758

As a result of the centrifugation of the circulating cells of the starfish Asterias rubens in a discontinuous density gradient of sodium diatrizoate, three cell fractions were separated. Small coelomocytes with a high nuclear–cytoplasmic ratio and the lack of granules prevailed in the upper fraction, coelomocytes with small granules evenly distributed in their cytoplasm dominated in the middle fraction, and large coelomocytes with a high content of large granules and vesicles in the perinuclear space were predominant in the bottom fraction. The coelomocytes of the separated fractions were tested for the production of reactive oxygen species, neutral red uptake, capture and internalization of the labeled bacteria Escherichia coli and Staphylococcus aureus, as well as for lytic activity and expression of the ArC3-like gene, which is a homologue of the C3 component of the mammalian complement cascade. Cells of the upper fraction manifested the most pronounced ability for the expression of the C3 gene homologue in response to stimulation with bacterial lipopolysaccharide. Coelomocytes of the middle fraction were distinguished by a pronounced ability to produce reactive oxygen species and phagocytosis, whereas the cells of the lower fraction had a high level of hemolytic activity and neutral red uptake.     

An autoradiographic investigation of the proliferative activity of pyloric caeca and gondas of Asterias rubens

The proliferative activity of the pyloric caeca of Asterias rubens was investigated. Autoradiographic experiments using intracoelomically injected (methyl-³H)-thymidine were performed throughout the year and incorporation into pyloric caeca and into gonads was studied. Tritiated thymidine was found to be incorporated mainly in the coelomic lining of both organs. Cell divisions in the coelomic lining may be necessary for the growth of these organs, for the production of coelomocytes or, in the case of the pyloric caeca, for growth of the digestive epithelium. Proliferative activity of the digestive epithelium of the pyloric caeca was only observed in the median duct. It is hypothesized that new cells, arising from mitosis, grow from the median duct to the side lobes and differentiate into storage cells, for example. The existence of a mitosis-inducing or mitosis-stimulating substance is discussed. In the ovaries follicle cells were found to incorporate (methyl-³H)-thymidine; in the testis, proliferation of the germinal epithelium occurred simultaneously in all spermatogenic columns. First, the spermatogonia and then later the spermatocytes became labeled. Absorption of substances from the coelomic fluid is discussed.     

Identité immunologique et métabolique entre le liquide coelomique,les coelomocytes et les ovocytes de Perinereis cultrifera (Annélide Polychète)

Immunological study of Perinereis cultrifera reveals the existence of an identical antigen in the coelomic fluid of females (oocyte diameter > 140 (μm), in oocytes, and in coelomocytes. This factor is not found in the body fluid of males or young females.

The elution patterns obtained after Sephadex chromatography shows a similar glycoprotein fraction (fraction I) in the coelomic fluid, in coelomocytes, and in soluble oocyte extracts. This fraction includes the main part of the antigenic components of the coelomic fluid.

Gas chromatography reveals that identical monosaccharides are present, albeit in varying proportions, in samples of fraction I obtained from the different sources.

The metabolic interrelationships of coelomocytes, coelomic fluid and oocytes is discussed. Glycoprotein synthesized by coelomocytes may be discharged into the coelomic fluid and contribute to the development of the cortical alveoli of the oocytes. No evidence of an involvement of this material in yolk synthesis has been obtained.     

The Influence of Salinity on Early Ontogeny of the Mussel Mytilus edulisand the Starfish Asterias rubensfrom the White Sea

The influence of increased and decreased salinity on early larvae of the mussel Mytilus edulisL. and the starfish Asterias rubensfrom the White Sea is studied. Fertilized egg cells, embryos, larvae, and yearlings of the two species were compared. Salinity tolerance changes with the stage of development of mussels and starfish. Morphological changes occurring in eggs and embryos exposed to extremely high and low salinities are reported.     

Induced in vitro phagocytosis of the Antarctic starfish Odontaster validus (Koehler 1906) at 0°C

Phagocytosis was studied in vitro using coelomic fluid of the Antarctic starfish Odontaster validus at 0°C. The number of coelomocytes present was determined and the phagocytic activity of the phagocytic amoebocytes (PA) was quantified with yeast during incubations of 1 and 2 h. The percentage of PA phagocytosing increased significantly from 42.29 ± 10.50% (SD) at 1 h to 52.57 ± 13.96% at 2 h. Numbers of yeast per PA also rose significantly from 2.27 to 2.45 cells per amoebocyte, indicating that phagocytic activity was maintained. In vitro phagocytosis of an Antarctic invertebrate at 0°C is shown for the first time, and the types of amoebocytes involved identified. Rates of phagocytosis were similar to, or higher than, reported data for temperate starfish, although this conclusion must be treated cautiously because of scarcity of data and differences in methods used. However, the data suggest that phagocytosis in O. validus is well adapted to low temperature. Accepted: 27 September 1999     

Evolutionary radiation of cytotoxic effects in bivalve and gastropod haemolymph

A comparative immunological survey of cytotoxic properties of whole and cell-free haemolymph (plasma) was performed on four species of marine molluscs. Diverse target cells were used: auto- and heterologous mollusc haemocytes, starfish Asterias rubens coelomocytes, trematode Himasthla elongata rediae, and human erythrocytes. While all the four mollusc species were found to contain cytotoxic factors in their plasma, functional features of these molecules in bivalve and gastropod species are essentially different. Cell-free haemolymph of two clams, Mya arenaria and Mytilus edulis, contains pre-synthetized cytolytic complexes capable of immediate and generalized activation, leading to destruction of a broad range of target cells. On the contrary, two gastropod species, Buccinum undatum and Littorina littorea, possess haemolytic factors which display high specificity of recognition of receptor molecules on the target cells and slow cytolytic reaction. Overall, molecular strategies of cytolytic cascade activation in Bivalvia and Gastropoda can be described as low- and highly selective, respectively.     

Behavioral response of the starfish asterias forbesh to a thermal gradient

ThirtyAsterias fbrbesii (Echinodermata: Asteroidea: Asteriidae) were tested as a group for 3 days in a long linear trough with a thermal gradient encompassing 5–45°C. The modal thermal preferendum of the starfish was between 20 and 25°C. The starfish avoided temperatures below 15°C or above 35°C.     

Über den Feinbau des hyponeuralen Nervensystems des Seesternes (Asterias rubens L.)

Zusammenfassung Langes Nerv, ein Teil des hyponeuralen Nervensystems der Asteroiden, wurde licht-, fluoreszenz- und elektronenmikroskopisch untersucht. Er zeigt die typische Gliederung in Kern- und Faserschicht. Die großen unipolaren Ganglienzellen reichen mit kolbenförmigen Ausläufern in das Cölom. Ihre basalen Fortsätze ziehen in die Faserschicht. Cölomepithelzellen liegen den Nervenzellen in unregelmäßigen Abständen als Deckzellen kappenartig auf oder sind als Stützzellen ausgebildet. Deck- und Stützzellen werden als Gliazellen aufgefaßt. Unterhalb der Kernschicht wurden licht- und elektronenmikroskopisch Zellen beobachtet, die für neurosekretorische Elemente gehalten werden. Der Feinbau der erwähnten Zelltypen wird beschrieben und diskutiert. Der histochemische Nachweis auf biogene Amine in Langes Nerv fiel negativ aus.

---

The fine structure of the hyponeural nerve system of the starfish (Asterias rubens L.)

Summary The nerve of Lange, part of the starfish hyponeural nervous system, was investigated with the light-, fluorescence- and electron microscopes in Asterias rubens. It consists of two layers, one containing perikarya, the other fibres. The big nerve cells are unipolar and possess club-shaped processes which extend into the coelom. At their basis, the axon merges into the fibrous layer. Irregularly spaced coelomic epithelial cells cover partly the ganglion cells or constitute supporting elements. Covering and supporting cells are interpreted to represent glia cells. Underneath the perikarya layer, cells have been found by both the light and electron microscopes which are considered to be neurosecretory elements. The fine structure of the various cell types is described and discussed. Histochemical tests for biogenic amines were negative.

Herrn Prof. Dr. G. Birukow zum 60. Geburtstag gewidmet.     

Über die Pylorusanhänge des Seesterns (Asterias rubens L.), insbesondere ihre Innervation

Zusammenfassung Die Pylorusanhänge (P. A.) von Asterias rubens L. wurden licht- und elektronenmikroskopisch untersucht. — Die Wandung der P. A. gliedert sich in eine hohe Epithelschicht, in deren basalen Abschnitten zahlreiche Nervenfasern verlaufen, eine bindegewebige Basallamelle und ein Coelomepithel, dessen Zellen eine lockere Lage von glatten Muskelzellen und Axonen bedecken.Die Zellen des Epithels der P. A. sind mit einem dichten, gleichmäßig ausgebildeten Bürstensaum ausgestattet, ferner mit je einer langen, mit einer Wimperwurzel verbundenen Cilie (9+2-Typus), die sich in die Lichtung der Caeca erhebt. Die Wimperwurzeln besitzen eine periodische Gliederung. Vereinzelte Epithelzellen tragen ein plumperes, unregelmäßig gestaltetes Geäst von Mikrovilli. Die bereits lichtmikroskopisch wahrnehmbare zarte Streifung der Zellapices beruht auf dem Vorhandensein von parallelisierten Mikrotubuli. Starke Vesikulation der Saumzellen und das Vorhandensein pinozytotischer Bläschen an der Basis der Mikrovilli ist als Ausdruck lebhafter resorptiver Tätigkeit des Epithels der P. A. anzusehen. Vor allem in den basalen Abschnitten der Epithelzellen liegen kugelige Lipideinschlüsse, in anderen Zellen Mukopolysaccharidgranula. An der Oberfläche der Epithelzellen werden Mukopolysaccharide ausgestoßen, teilweise in Form zusammengesinterter, unregelmäßig gestalteter Bildungen. Das morphologische Äquivalent der von anderen Autoren beschriebenen sog. Zymogenkörnchen konnte mit Sicherheit nicht ermittelt werden. Intraepitheliale Sinneszellen wurden nicht beobachtet.Die nackten Axone innerhalb des Epithels der P. A. lagern sich den Basalteilen der Saumzellen an, bilden jedoch mit ihnen keine typischen, d. h. durch Membranverdickungen ausgezeichneten Synapsen. Ihr Axoplasma enthält außer Neurotubuli Bläschen mit massendichtem Inhalt, die Granula aminerger oder peptiderger Nervenfasern ähneln. Möglicherweise sind bei Asterias Nervenfasern jeweils verschiedenen Inhalts ausgebildet, da Profile von Axonen mit kleineren (1000–1200 Å Durchmesser) und größeren Granula (1200–1600 Å) nachzuweisen sind. Das Vorkommen von Ergastoplasmastrukturen und Golgimembranen in den Axonen sowie die Abschnürung von Vesikeln mit massendichtem Inhalt vom Golgiapparat spricht für eine Entstehung von Elementargranula in der Peripherie der entsprechenden Neurone. Das Bild synaptischer Bläschen kann durch entleerte Elementargranula vorgetäuscht werden.Die glatten Muskelzellen unter dem Coelomepithel stehen mit Nervenendigungen, die dense-cored vesicles enthalten, in Berührung, doch sind Synapsen mit Membranverdickungen, wie sie bei den Vertebraten angetroffen werden, nicht ausgebildet.

---

Summary The pyloric ceca (p. c.) of the starfish, Asterias rubens L., are investigated light- and electronmicroscopically.The wall of the p. c. consists of 1. a high columnar epithelium the basal part of which is permeated by many axons, 2. a basal lamina, 3. a loose layer of smooth muscle cells intermingled with thin nerve fibres, and 4. the coelomic epithelium. Intraepithelial sensory elements are lacking in the epithelium.The epithelial cells of the p. c. are provided with a very well developed brush border built up by microvilli of equal length and diameter. In addition each cell bears a long cilium (9 + 2 pattern) connected with a periodically structured rootlet. Only single cells are characterized by a more or less irregular seam of plump ramified microvilli. The parallel orientation of microtubules in the apical parts of the cells causes the striation already to be observed under the light microscope.The intense vesiculation of the cytoplasm and the occurrence of many pinocytotic invaginations on the basis of the brush border is considered to be the equivalent of the high absorption activity of the epithelial cells.Spheroidal lipid inclusions, mucopolysaccharide granules and large masses of unknown nature are embedded in the cytoplasm of the epithelial cells. Irregularly shaped mucopolysaccharide substances are extruded into the lumen of the p. c. The occurrence of so-called zymogen granules has not been observed in our material.The intraepithelial axons are closely attached to the basal parts of the epithelial cells without forming typical synapses with membrane thickenings. Apart from neurotubules, the axoplasm of these tiny fibres contains dense-cored vesicles (diameter 1000–1200 Å, 1200–1600 Å) resembling the elementary granules of aminergic and peptidergic nerve fibres. The presence of ergastoplasmic structures and Golgi membranes within the axoplasm and the gemmation of dense-cored vesicles from the Golgi apparatus speaks in favour of a peripheral elaboration of elementary granules in the axons of the starfish.The smooth muscle cells covered by the coelomic epithelium of the p. c. are in contact with axonal terminals containing dense-cored and empty vesicles. Typical synaptic structures as described for vertebrates apparently do not exist in the starfish.

     

Über die Verbindung der Muskulatur mit dem Skelett bei dem Echinodermen Asterias rubens L.

Zusammenfassung Die Skelettmuskeln des Seesterns Asterias rubens L., der hier als Paradigma der Echinodermen untersucht wurde, inserieren mit Hilfe von Sehnenschlaufen, die außen um die Kalkbälkchen einer dünnen Oberflächenschicht der Sklerite herumgelegt sind. Die Sehnen gehen aus der Basalmembran hervor, welche die Muskelzellfortsätze umhüllt, und bestehen aus parallelisierten zarten Filamenten. Kollagene Fasern, von denen sie sich deutlich unterscheiden, dienen der Muskelinsertion ebensowenig wie die spärliche organische Matrix in der Hartsubstanz.

---

On the connection between skeletal muscles and skeleton of the echinoderm asterias rubens L.

Summary The skeletal muscles of the starfish Asterias rubens L. — as a paradigm of the echinodermes — are fixed by means of tendinous loops running around the calcareous beamlets of a thin superficial layer of the ossicles. The tendons emerge from a basement membrane which envelops the terminal processes of the muscle cells. They consist of thin parallel filaments. Collagenic fibers which can be clearly distinguished from these filaments, and the sparce organic matrix of the calcareous parts, do not participate in the muscle insertion.

---

---

Herrn Prof. Dr. phil. Dr. med. A. Kiesselbach zum 60. Geburtstag gewidmet.     

Functional morphology of vibratile urnae in the synaptid holothuroid Leptosynapta inhaerens (Echinodermata)

Summary Vibratile urnae of Leptosynapta inhaerens are organized in three longitudinal bands along the mesenteries. An individual of 10 cm length usually houses about 4500 urnae. These are minute (300 Gmm high), S-shaped and hollow peritoneal organs consisting of an intracoelomic projection of the body wall mesothelium supported by a thin connective tissue layer. The urnal cavity is strongly ciliated. Each urna harbours a clump of coelomocytes at the lower part of its aperture. The clump is attached to the urna through spot-like desmosomes occurring between its inner-most coelomocytes and apical urnal cells. Clumps and urnae form functional units. Urnal cilia produce steady water currents through urnal cavities and whirls along urnal bands. The particulate material conveyed by the coelomic fluid enters the urnal cavity and is either trapped by coelomocyte pseudopodia or agglutinated by a mucoid substance that covers the clump's outer surface. Depending on individuals, clearance of coelomic fluid occurs from 2 to 3 h after experimental injection of particulate material. The effectiveness of coelomic fluid clearance appears to be due to the particular organization and location of urnae, viz. in longitudinal bands along the mesenteries.     

Development of the starfish <Emphasis Type="Italic">Asterias amurensis</Emphasis> under laboratory conditions

Under laboratory conditions the development of the starfish Asterias amurensis Lütken from Vostok Bay (Sea of Japan) was studied at 14 and 17°C. At 14°C and a salinity of 31.6–32.6, ciliated coeloblastulae hatched from egg envelopes 19 h after fertilization. At this temperature the development proceeded slowly and stopped at the stage of bipinnaria. At 17°C and normal salinity of seawater, the development of A. amurensis was successful. The swimming blastula appeared in 14 h. It took 30.5 h for the embryos to reach the gastrula stage. The larvae began swimming in a horizontal position with the apical tip ahead. The dipleurula appeared at 60 h. These larvae began feeding. At 71 h after the beginning of development, the early bipinnaria has developed. In the larva, the edged ciliated band, the preoral plate, and the anal plate were already formed. At the age of 4.2 days, the larvae reached the stage of bipinnaria and the brachiolaria stage developed by 26–28 days after fertilization. The larvae had three identical brachiolar arms with attachment papillae on their tips and an attachment disk. In 37–44 days (at 17°C) the pelagic phase of A. amurensis development was completed by the attachment of larvae to the bottom plates and termination of metamorphosis. Most likely, the specificity to a substrate is not expressed in the brachiolaria of A. amurensis. They can settle on almost any hard substrate which is coated with a bacterial film. The newly settled juvenile starfish had five well-developed arms and moved using their ambulacral podia.Original Russian Text Copyright © 2005 by Biologiya Morya, Kashenko.     

Activity and immunodetection of lysozyme in earthworm Dendrobaena veneta (Annelida)

In the present study, lysozyme-like activity against Micrococcus luteus was detected in the coelomic fluid, the extract from coelomocytes, intestine and in the homogenates from cocoons of Dendrobaena veneta. Four hours after immunization with Escherichia coli, the lysozyme activity in the coelomic fluid increased about three times and in the extract of coelomocytes - four times, in comparison to the control. In three cases: of the coelomic fluid, the homogenates from cocoons and the extract from coelomocytes, the antibody against HEWL (hen egg white lysozyme) recognized only one protein with a molecular mass of about 14.4 kDa. In the coelomic fluid, apart from the protein with molecular mass of 14.4 kDa the antibody directed against human lysozyme recognized an additional protein of 22 kDa. Using the bioautography technique after electrophoretic resolution of native proteins in acidic polyacrylamide gels, two lytic zones of M. luteus were observed in the case of the coelomic fluid and three after the analysis of the extract of coelomocytes and the egg homogenates. The results indicated the existence of several forms of lysozyme with a different electric charge in the analyzed D. veneta samples. The highest lysozyme activity in the intestine of D. veneta was observed in the midgut. The antibody directed against human lysozyme indicated a strong positive signal in epidermal and midgut cells of earthworm.