Electron microscopic analysis of the fibroblast differentiation of amebocytes in the common snail, Lymnaea stagnalis L. (Mollusca, Gastropoda) in a lesion focus

The amoebocytes and fibroblasts of intact connective tissues of the common pond snail foot, as well as the amoebocytes of the burn wound were studied by means of electron microscope. The found transitional forms from the amoebocytes to the fibroblasts allowed to conclude that there were cells--precursors of fibroblasts among the amoebocytes migrating in the wound. A tentative scheme of histogenetic series of amoebocytes is given.     

The occurrence of inflammatory granulomas in the ctenidial marsupium of Corbicula fluminea (mollusca: Bivalvia): A consequence of larval incubation

A number of tumor-like cysts in the ctenidia of the freshwater bivalve Corbicula fluminea are described. The cysts result from the encapsulation of incubated larvae within the female inner demibranchs which are modified to form a marsupium. Corbicula fluminea is a protandric consecutive hermaphrodite, and larval incubation commences with sex reversal and the release of the first eggs. For some reason a few of the larvae are not released, and their retention and subsequent death results in the mobilization of the innate cellular defensive mechanism of the parent. This process involves, initially, an invasion of epithelioid cells and amoebocytes (granulocytes), resulting in hyperplasia. This is followed by structural changes to the epithelium bordering the interlamellar spaces (within which the larvae are incubated) to form ultimately the cyst wall. The epithelium is surrounded externally by layers of fibrocytes which eventually form a thick capsule. The autolyzed larval tissues are themselves invaded by fibrocytes, epithelioid cells, and amoebocytes and, in one specimen, had formed a three-layered capsule within the surrounding capsule. The amoebocytes probably reabsorb the larval cellular debris. This unusual example of a molluscan cellular defensive mechanism may assist in the diagnosis and separation of hyperplastic injury responses from neoplasmic conditions in invertebrates.     

Long-term in vitro generation of amoebocytes from the Indian horseshoe crab Tachypleus gigas (Müller)

Amoebocyte is the single type of cell circulating in the horseshoe crab hemolymph, which plays a major role in the defense system of the animal. Granules present in these cells are sensitive to nanogram quantities of bacterial endotoxins, which form the basis of the Limulus amoebocyte lysate (LAL) test. Normally, amoebocytes for the production of the LAL are collected by cardiac puncture; hence, development of the in vitro culture system for amoebocytes will reduce the variability of the lysate and help to conserve the 400 million-yr-old living fossil. In the present investigation we have attempted organ culture of gill flaps that have been shown to be the source of amoebocytes. The gill flaps were cultured at 28 degrees C on a rocker platform in a modified L-15 medium supplemented with 10% v/v horseshoe crab serum. This led to the release of amoebocytes outside the gill flaps for a period of 6-8 wk with a more or less steady number of amoebocytes during the weekly harvest. No significant difference was seen in the yield of amoebocytes from male and female horseshoe crabs. Confocal laser microscopy studies revealed significant difference in the size of amoebocytes released in vitro as compared with those obtained in vivo. Thus, we have optimized the culture conditions for the long-term generation of amoebocytes in vitro from the Indian horseshoe crab Tachypleus gigas by reducing the incidence of contamination, simulating in vivo conditions for the organ culture of gill flaps, and improvising the nutritional status using the modified L-15 medium, providing the desired osmolarity and pH.     

Quantitative aspects of carotenoid pigment circadian variations in the crayfish Astacus leptodactylus

The carotenoid pigment concentrations of whole animal, epidermis and hepatopancreas of the crayfish Astacus leptodactylus were studied every three hours during a period of 24 h. The extract of whole animal exhibits a total pigment concentration which remains noticeably stable. Circadian variations were observed in extracts from the hepatopancreas and epidermis; the maxima observed for the epidermis as well as the minima recorded for the hepatopancreas are related to the alternating light and dark periods. The symmetry of such variations suggests a transfer of pigment between the two tissues.     

The tubular endoplasmic reticulum in the amoebocytes of the shell-regenerating snail,Helix pomatia L.

Summary The tubular endoplasmic reticulum has been studied in the amoebocytes which are present in the connective tissue of the hepatopancreas of the snail, Helix pomatia. The reticulum is similar to that previously described within the glandular cells of the hepatopancreas. Two distinct components are recognizable in the reticulum—central main tubules approximately 100 m in diameter and connecting tubules about 20 m in width. The profile of this tubular network in cross-sections appears as a very regular, apparently crystalline array. The tubules are intimately associated with dense granular material, dense bodies and with mitochondria. The possible function of the tubular endoplasmic reticulum is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council, which are gratefully acknowledged. I am indebted to Miss G. Drugge for her technical assistance.     

Ultrastructural localization of highly variable 185/333 immune response proteins in the coelomocytes of the sea urchin, Heliocidaris erythrogramma

The 185/333 proteins of sea urchins represent a family of highly variable immune response molecules with unknown functions. In this study, we show that 185/333 proteins are expressed by three cell types: amoebocytes, colourless spherule cells and gut-associated amoebocytes. A sub-population of amoebocytes express 185/333 proteins on the membranes of vesicles emanating from the trans-Golgi and which later fuse with the plasma membranes of the cells. The previously uncharacterized gut-associated amoebocytes also show a high level of 185/333 protein expression on their internal vesicles and plasma membranes. Colourless spherule cells contain 185/333 proteins within large spherules (specialized intracellular vesicles). In the presence of bacteria and yeast, the ultrastucture of colourless spherule cells changes and 185/333 proteins disappear. In contrast, 185/333 proteins were not found in the phagosomes of coelomocytes. The 185/333-positive gut amoebocytes were often associated with anuclear bodies, which appeared to incorporate material of microbial origin that was surrounded by 185/333 proteins. The association between 185/333 proteins on gut amoebocytes and anuclear bodies suggests that these proteins may be involved in the phagocytosis of microbes in the gut epithelium.     

The Cellular Basis for Colony Form in Podocoryne carnea

During the past 10 years considerable data have appeared indicatingthat the growth of hydroids is not, as was earlier believed,similar to the meristematic growth of plants, but rather thatthe sites of cell proliferation are removed in space from thesites of utilization, that cells migrate individually, activelyas amoebocytes through the epidermis or pawively as epitheliocytescarried along in the hydroplasm, to the sites of utilization,and that considerable migration across the mesoglea occurs. A model of hydroid morphogenesis and morphostasis based on thisnew information provides a comprehensive picture, albeit onefilled in with numerous assumptions, of the manner in whichcolony form is produced as a consequence of cell proclivities.     

Repugnatorial glands with associated striated muscle and sensory cells inMelibe leonina (Mollusca,Nudibranchia)

Summary The nudibranch molluscMelibe leonina is repellant to some seastars and fish, but not to crab predators. Distinctive subepidermal glands, which are particularly abundant within the cerata and dorsal wall of the oral hood, can be easily seen within the translucent body wall of this slug. The glands can be individually discharged by touching the overlying epidermis and the collected secretion is repugnant to seasters. Ultrastructural analysis reveals that each repugnatorial gland is composed of two types of secretory cell encased by cross-striated muscle. Storage of secretory product within a single, large, intracellular vacuole and cytological features of the muscle investment are consistent with a rapid, all-or-none secretion event, as observed in live tissue. The secretory cells empty into a short duct that opens by a pore. The pore is surrounded by ciliated sensory cells that probably act as mechanoreceptors triggering gland discharge. I argue that one of the two secretory cells releases defensive chemical, whereas the second may produce a protein-rich mucin.     

Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio)

The first part of this article is a review of the current status of knowledge of the fish skin, with particular attention to its development. In the second part we present original results obtained in zebrafish (Danio rerio), with particular emphasis on the deposition and organisation of the dermal collagenous stroma. Using a series of zebrafish specimens aged between 15 hours postfertilization (hpf) and 4.5 years old, we have combined Transmission Electron Microscopy (TEM) observations and in situ hybridisation using type I collagen a2 chain (Col1a2) probe. Collagen fibrils, with a diameter of 22 nm, appear first in an acellular subepidermal space at 24 hpf, are first all oriented in the same direction, and form the primary dermal stroma. Subsequently, three events occur. (1) From 5-7 days pf (dpf) onwards the collagen fibrils self-organise into several lamellae arranged in a plywood-like structure, starting in the upper layers and progressing throughout the entire thickness of the dermis. (2) At 20-26 dpf, fibroblasts of unknown origin progressively invade the acellular collagenous stroma, some of them accumulating below the epidermis. (3) Concomitant with the invasion of fibroblasts, the collagen fibrils increase progressively in diameter to reach 160 nm towards the end of the fish life. In situ hybridisation experiments reveal that, between 24 and 48 hpf, the collagen matrix is produced by the epidermis only. From 72 hpf to 20-26 dpf, both the basal epidermal cells and the dermal cells bordering the deep region of the dermis are involved in the production of collagen. When the fibroblasts invade the plywood-like structure, the epidermal cells progressively cease to synthesise collagen, which from this point is produced only by the fibroblasts. This suggests that the fibroblasts secrete a still unidentified signalling molecule that downregulates collagen production by the epidermis.     

Ceratal autotomy and regeneration in the aeolid nudibranch Phidiana crassicornis and the role of predators

Abstract. Ceratal autotomy by the aeolid nudibranch Phidinna crassicornis is common in the field and was induced in the laboratory by mechanical and predatory stimuli. The ceras detaches from the body wall along an autotomy plane located at its basal constriction. Cerata released copious amounts of mucus during autotomy and exhibited a prolonged writhing response that continued for several hours after detachment. Regeneration of cerata autotomized in the field and in the laboratory was documented. Four days after autotomy, regenerating cerata appeared as small protuberances. By day 24 the regenerates acquired their mature structural organisation and vivid colour. The cerata subsequently increased in length and diameter and were indistin‐guishable from surrounding cerata by 41 to 43 days after autotomy. Regeneration rates of cerata induced to autotomize in the laboratory and regeneration of cerata autotomized in the field were similar, averaging 0.08 and 0.067 mdday, respectively. The sequence of morphological events involved with regeneration following experimental and natural induction of autotomy was identical. The kelp crab Pugettia productn induced autotomy by holding cerata with its chelae. This crab also fed on autotomized cerata and consumed locomotory and ceratal mucus. Ceratal autotomy may be an important mechanism of escape from this predatory crustacean. Other potential predators including hermit crabs and tidepool sculpins did not elicit defensive behaviour in P. crussicornis. Nematocysts were present in the enidosacs and their role in defense was investigated. Fired nematocysts were observed in podia of the asteroid Crossaster papposus following ceratal contact but were not seen in the podia of Pycnopodia helianthoides in a similar trial. For P. crassicornis, the cnidosacs may function primarily as a storage device for safe sequestering of nematoeysts that could pose a threat to the digestive system. They did not play a major defensive role against the predators tested, but may be important in the field against other predators.     

Antioxidant Enzymatic Activity of Coelomocytes of the Far East Sea Cucumber Eupentacta fraudatrix

The basal activity of superoxide dismutase (SOD), glutathione reductase (GR), and catalase in a pool of coelomocytes as well as in the fraction of amoebocytes and the mixed fraction of amoebocytes and morula-like cells of the sea cucumber Eupentacta fraudatrix is studied. For SOD and catalase, pH optima are in the range of values of pH optimum for tissues of mammals, the pH optimum for GR is shifted to a more acidic region in comparison with the latter enzymes. Temperature optima for all studied enzymes are higher than the usual temperature values of the sea cucumber habitation. A pronounced temperature dependence of all three enzymes is revealed. In coelomocytes, the activities of SOD and catalase, but not of GR, are lower than in the fraction of amoebocytes, but higher than in the mixed fraction of amoebocytes and morula-like cells. The rate of production of active forms of oxygen (AFO) is three times higher in amoebocytes than in the fraction relatively enriched in morula-like cells. Apparently, the main part of the SOD and catalase activities, as well as AFO production in coelomocytes is located in amoebocytes, which confirms the existence of cytophagic function in the latter cells as well as argues in favor of functional differentiation between individual types of coelomocytes.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

Structure of the Digestive Tube in the Holothurian Eupentacta fraudatrix (Holothuroidea: Dendrochirota)

In the holothurian Eupentacta fraudatrix,the gut wall exhibits trilaminar organization. It consists of an inner digestive epithelium, a middle layer of connective tissue, and an outer mesothelium (coelomic epithelium). The pharynx, esophagus, and stomach are lined with a cuticular epithelium composed of T-shaped cells. The lining epithelium of the intestine and cloaca lacks a cuticle and consists of columnar vesicular enterocytes. Mucocytes are also encountered in the digestive epithelium. The connective tissue layer is composed of a ground substance, which houses collagen fibers, amoebocytes, morula cells, and fibroblasts. The gut mesothelium is a pseudostratified epithelium, which is dominated by peritoneal and myoepithelial cells and also includes the perikarya and processes of the neurons of the hyponeural plexus and vacuolated cells.     

Amoebocytes facilitate efficient carbon and nitrogen assimilation in the Cassiopea-Symbiodiniaceae symbiosis

The upside-down jellyfish Cassiopea engages in symbiosis with photosynthetic microalgae that facilitate uptake and recycling of inorganic nutrients. By contrast to most other symbiotic cnidarians, algal endosymbionts in Cassiopea are not restricted to the gastroderm but are found in amoebocyte cells within the mesoglea. While symbiont-bearing amoebocytes are highly abundant, their role in nutrient uptake and cycling in Cassiopea remains unknown. By combining isotopic labelling experiments with correlated scanning electron microscopy, and Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging, we quantified the anabolic assimilation of inorganic carbon and nitrogen at the subcellular level in juvenile Cassiopea medusae bell tissue. Amoebocytes were clustered near the sub-umbrella epidermis and facilitated efficient assimilation of inorganic nutrients. Photosynthetically fixed carbon was efficiently translocated between endosymbionts, amoebocytes and host epidermis at rates similar to or exceeding those observed in corals. The Cassiopea holobionts efficiently assimilated ammonium, while no nitrate assimilation was detected, possibly reflecting adaptation to highly dynamic environmental conditions of their natural habitat. The motile amoebocytes allow Cassiopea medusae to distribute their endosymbiont population to optimize access to light and nutrients, and transport nutrition between tissue areas. Amoebocytes thus play a vital role for the assimilation and translocation of nutrients in Cassiopea, providing an interesting new model for studies of metabolic interactions in photosymbiotic marine organisms.     

Recognition of foreignness by blood cells of the freshwater snail Lymnaea stagnalis, with special reference to the role and structure of the cell coat

The structure and function of the cell coat of the blood cells (amoebocytes) of the freshwater snail Lymnaea stagnalis were studied with ultrahistochemical tests, including concanavalin A (Con A) labeling, and with in vitro phagocytosis experiments. The cell coat is intensely stained by ruthenium red and tannic acid. The cells possess binding sites for Con A. Proteolytic enzymes destroy the receptors for Con A and totally inhibit the phagocytic activity of amoebocytes. Incubation experiments with proteases, carbohydrases, and inhibition sugars revealed that (1) the Con A binding sites are anchored in the plasma membrane by proteins, and (2) glucose, fructose, mannose, and to a lesser extent N-acetylglucosamine and N-acetylgalactosamine, inhibit the binding of Con A to amoebocytes, suggesting that these carbohydrates might form part of these binding sites.     

Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes

Microscope techniques, light microscope (LM), transmission electron microscope (TEM), scanning electron microscope (SEM) were employed to describe and classify coelomocytes of the oligochaete Dendrobaena veneta. Three main cell types were distinguished in the coelomic fluid: eleocytes, amoebocytes and granulocytes. Eleocytes are large, oval cells containing characteristic granules called chloragosomes. Amoebocytes are most numerous coelomocytes and have been divided into two types (I and II). Both amoebocytes of the types I and II often form aggregations of a few to about a dozen cells. Granulocytes are oval cells with spherical nuclei and cytoplasm containing polymorphic, electron dense granules. Contrary to the amoebocytes, the granulocytes do not form aggregations. Morphology and ultrastructure of coelomocytes are presented on micrographs: similarities and differences are compared to coelomocytes of related species.     

Aeolid nudibranchs (Gastropoda: Opisthobranchia) of the family Glaucidae from New Zealand waters

Two new species of aeolid, a new genus being created for one of them, are described along with another two species, one Phidiana militaris (Alder & Hancock) (= Learchis militaris ) a new record for New Zealand, the other, Glaucus atlanticus , found only once before. Jason mirabilis gen. et sp. nov. is distinguished by a ventral tongue-like process with a ridged chitinous covering which replaces the radula (still present but vestigial) and by the greatly subdivided rhinophoral lamellae. Babaina caprinsulensis sp. nov. differs from its Californian relative in having papillate rhinophores. The New Zealand specimens of Phidiana militaris differ slightly in colouration from those described for elsewhere in the Indo-Pacific.
The Glaucidae is enlarged to include the Babainidae, Facelinidae and Favorinidae, the main familial characteristics being the simple oral glands, sharp radular tooth and pugnacious behaviour. Possible evolutionary trends within the family based on the arrangement of the cerata and penial structure are traced out. The Babaininae, pleuroproctic with a notal ridge and irregular disposition of the cerata, is considered the most primitive subfamily and the Hervellinae, with cerata in single rows, the most advanced. The many facelinid genera previously recognized are reduced to two, Antionetta Schmekel and Phidiana Gray.     

The origin of the endothelial cells: an evo-devo approach for the invertebrate/vertebrate transition of the circulatory system

Circulatory systems of vertebrate and invertebrate metazoans are very different. Large vessels of invertebrates are constituted of spaces and lacunae located between the basement membranes of endodermal and mesodermal epithelia, and they lack an endothelial lining. Myoepithelial differentation of the coelomic cells covering hemal spaces is a frequent event, and myoepithelial cells often form microvessels in some large invertebrates. There is no phylogenetic theory about the origin of the endothelial cells in vertebrates. We herein propose that endothelial cells originated from a type of specialized blood cells, called amoebocytes, that adhere to the vascular basement membrane. The transition between amoebocytes and endothelium involved the acquisition of an epithelial phenotype. We suggest that immunological cooperation was the earliest function of these protoendothelial cells. Furthermore, their ability to transiently recover the migratory, invasive phenotype of amoebocytes (i.e., the angiogenic phenotype) allowed for vascular growth from the original visceral areas to the well-developed somatic areas of vertebrates (especially the tail, head, and neural tube). We also hypothesize that pericytes and smooth muscle cells derived from myoepithelial cells detached from the coelomic lining. As the origin of blood cells in invertebrates is probably coelomic, our hypothesis relates the origin of all the elements of the circulatory system with the coelomic wall. We have collected from the literature a number of comparative and developmental data supporting our hypothesis, for example the localization of the vascular endothelial growth factor receptor-2 ortholog in hemocytes of Drosophila or the fact that circulating progenitors can differentiate into endothelial cells even in adult vertebrates.     

Coelomic Cells in the Connective Tissue Spaces of the Clitellar Epithelium of Lumbricus friendi Cognetti, 1904 (Oligochaeta): an Ultrastructural Study

The ultrastructure of the connective tissue spaces in the clitellar epithelium has been studied in the earthworm Lumbricus friendi. Four morphological types of coelomic cells are described: amoebocytes, mucocyte-like cells, pigment cells and crystal-containing cells. The amoebocytes are characterized by the presence of spherical to oval electron-dense granules, phagocytic vacuoles and numerous microtubules located in the Golgi areas. The mucocyte-like cells show the features of the mucocytes reported in enchytraeid worms (globular inclusions with filamentous and homogeneous, moderately electron-dense material, as well as a filopodous process). The pigment cells contain typical spindle-shaped osmiophilic granules, microtubules (not reported before) and glycogen particles. The crystal-containing cells show inclusions which are polygonal in section with a striated substructure (periodicity of about 4.5 nm). Apart from the mucocyte-like cells, the coelomocytes showed cytoplasmic processes attached to the basement membrane of the spaces. The possible functions of these cells are discussed and a common peritoneal origin is postulated on the base of their morphological and cytological features.