Book lung development in the embryo,postembryo and first instar of the cobweb spider,Parasteatoda tepidariorum C. L Koch, 1841 (Araneomorphae,Theridiidae)

Light and electron microscopy were used to compare spider book lung development with earlier studies of the development of horseshoe crab book gills and scorpion book lungs. Histological studies at the beginning of the 20th century provided evidence that spider and scorpion book lungs begin with outgrowth of a few primary lamellae (respiratory furrows, saccules) from the posterior surface of opisthosomal limb buds, reminiscent of the formation of book gills in the horseshoe crab. In spider embryos, light micrographs herein also show small primary lamellae formed at the posterior surface of opisthosomal limb buds. Later, more prominent primary lamellae extend into each book lung sinus from the inner wall of the book lung operculum formed from the limb bud. It appears most primary lamellae continue developing and become part of later book lungs, but there is variation in the rate and sequence of development. Electron micrographs show the process of air channel formation from parallel rows of precursor cells: mode I (cord hollowing), release of secretory vesicles into the extracellular space and mode II (cell hollowing), alignment and fusion of intracellular vesicles. Cell death (cavitation) is much less common but occurs in some places. Results herein support the early 20th century hypotheses that 1) book lungs are derived from book gills and 2) book lungs are an early step in the evolution of spider tracheae.     

Myoanatomy and serotonergic nervous system of the ctenostome Hislopia malayensis: evolutionary trends in bodyplan patterning of ectoprocta

Background

Near the end of the nineteenth century the hypothesis was presented for the homology of book lungs in arachnids and book gills in the horseshoe crab. Early studies with the light microscope showed that book gill lamellae are formed by outgrowth and possibly some invagination (infolding) of hypodermis (epithelium) from the posterior surface of opisthosomal limb buds. Scorpion book lungs are formed near the bilateral sites of earlier limb buds. Hypodermal invaginations in the ventral opisthosoma result in spiracles and sac-like cavities (atria). In early histological sections of embryo book lungs, widening of the atrial entrance of some lamellae (air channels, air sacs, saccules) was interpreted as an indication of invagination as hypothesized for book gill lamellae. The hypodermal infolding was thought to produce the many rows of lamellar precursor cells anterior to the atrium. The ultrastructure of scorpion book lung development is compared herein with earlier investigations of book gill formation.

Results

In scorpion embryos, there is ingression (inward migration) of atrial hypodermal cells rather than invagination or infolding of the atrial hypodermal layer. The ingressing cells proliferate and align in rows anterior to the atrium. Their apical-basal polarity results in primordial air channels among double rows of cells. The cuticular walls of the air channels are produced by secretion from the apical surfaces of the aligned cells. Since the precursor cells are in rows, their secreted product is also in rows (i.e., primordial air channels, saccules). For each double row of cells, their opposed basal surfaces are gradually separated by a hemolymph channel of increasing width.

Conclusions

The results from this and earlier studies show there are differences and similarities in the formation of book lung and book gill lamellae. The homology hypothesis for these respiratory organs is thus supported or not supported depending on which developmental features are emphasized. For both organs, when the epithelial cells are in position, their apical-basal polarity results in alternate page-like channels of hemolymph and air or water with outward directed hemolymph saccules for book gills and inward directed air saccules for book lungs.     

Development of respiratory structures in embryos and first and second instars of the bark scorpion,Centruroides gracilis (Scorpiones: Buthidae)

The SEM was used to study the development of respiratory structures in successive stages in relation to the overall changes occurring in the scorpions. Book lung development is a slow process, starting with spiracles and a sac‐like atrium in the early embryo and continuing lamellar formation to 150 or more in the adult. In the embryo, the primordial epithelial cells become aligned in a planar pattern as they secrete granules of material that aggregate spontaneously to form the cuticular walls of the lamellae. A blade‐like structure is formed consisting of cells sandwiched within the two cuticle walls they secreted. These cells are in the primordial air channel. The adjacent hemolymph channel is nearly devoid of cells, but cross‐bridges develop and help stabilize the cuticle walls and maintain the width of the channel. The cells in the primordial air channel undergo cytolysis, leaving it open for air except for cuticular cross‐bridges. Development continues in the newborn (first instars); the air channels of some lamellae still contain cells and are not yet functional for gas exchange. The first instars are weak and relatively inactive. They climb up on the mother's dorsum until the first molt (about 8 days). With the cuticular walls of the lamellae in place, cells adhering to the wall in the hemolymph channel produce a thin, new tissue layer (epithelium) on the lamellar wall facing the hemolymph channel. This layer has many discontinuities as though it is slowly developing. Formation of the tissue layer and cytolysis of the cells in the air channels continue through the first molt in which little book lung cuticle is shed as exuvium. The air channels of the second instars (foraging nymphs) are now cell free and open for air passage except for the cross‐bridges. The tissue layer is still incomplete and continues to be formed. It may provide the hypodermal primordium for cuticle replacement in later molts, but development was not studied beyond the second instar except for comparison with book lungs in the adult. The blade‐like lamellae in the adult are larger and more numerous than in the second instar, but in the anterior book lung the shape of the cuticle wall and cross‐bridges and the widths of the air and hemolymph channels are about the same as in the second instar. The air channels in the posterior part of the lamellae have distinctive, vein‐like space‐holders. The similarity of the adult anterior lamellae with those in the second instar suggests retention of this part through the 4–5 molts to maturation, and/or cell processes like those in the embryo are repeated, but this needs to be examined in further studies of cell and cuticle changes before and during the molts. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

The morphology and vasculature of the lungs and gills of the soldier crab,Mictyris longicarpus

The five gill pairs of Mictyris longicarpus have the lowest weight specific area reported for any crab. The cuticle of the gill lamellae is lined with epithelial cells which have structural features characteristic of iontransporting cells. Pillar cells are regularly distributed in the epithelium and serve to maintain separation of the two faces of the lamellae. The central hemolymph space is divided into two sheets by a fenestrated septum of connective tissue cells. The dorsal portion of the marginal canal of each lamella receives hemolymph from the afferent branchial vessel and distributes it to the lamella while the ventral portion of the canal collects hemolymph and returns it to the efferent branchial vessel. The lung is formed from the inner lining of the branchiostegite and an outgrowth of this, the epibranchial membrane. Surface area is increased by invagination of the lining which forms branching, blind-ending pores, giving the lung a spongy appearance. The cuticle lining the lung is thin and the underlyng epithelial cells are extremely attenuated, giving a total hemolymph/gas distance of 90–475 nm. Venous hemolymph is directed close to the gas exchange surface by specialised connective tissue cells and by thin strands of connective tissue which run parallel to the cuticle. Air sacs are anchored in position by paired pillar cells filled with microtubules. Afferent hemolymph is supplied from the eye sinus, dorsal sinus, and ventral sinus. Afferent vessels interdigitate closely with efferent vessels just beneath the respiratory membrane. The two systems are connected by a “perpendicular system” which ramifies between the airways and emerges to form a sinus beneath the carapace and then flows back between the air sacs to the efferent vessels. The afferent side of the perpendicular system is the major site of gas exchange. Efferent vessels return via large pulmonary veins to the pericardial cavity. P_aO₂ levels were high (95.5 Torr), indicating highly efficient gas exchange.     

The morphology of the gills of the freshwater African crab Potamon niloticus (Crustacea: Brachyura: Potamonidae): A scanning and transmission electron microscopic study

The gills of the African freshwater crab Potamon niloticus -Ortmann have been investigated by scanning and transmission electron microscopy. Potamon has seven pairs of phyllobranchiate gills contained in the branchial chambers. From the central axis of the gills arise bilaterally situated thin flaps, the lamellae. The afferent branchial vessel (the epibranchial vessel) is located on the dorsal aspect of the gill arch and the efferent vessel (the hypobrancial vessel) on the ventral side. Between these two blood vessels, the blood percolates through the lamellar vascular channels where it is oxygenated. The lamellae consist of an epithelial cell layer covered by a thin cuticle which consists of tightly fused but distinct layers. The epithelial cells approach each other at regular intervals and fuse in the middle of the lamellar sinus delineating the vascular channels. Apical profuse membranous infoldings and numerous mitochondria characterize the epithelial cells, features typical of cells involved in active transport of macro- and micromolecules. In Potamon , however, there were no distinct gas exchange and osmoregulatory regions of the gills. On average, the cuticle was 0.78 μm thick while the epithelial cell was 6 μm. Cells that were morphologically similar to the renal glomerular podocytes of the vertebrates were observed in the efferent gill vessel of Potamon. These cells have been said to be phagocytic and may play an important defensive role in the crustaceans. Although basically the morphology of the gills of Potamon is similar to that of the other decapods, fine structural differences were evident as would be intuitively expected in a group of animals that has undergone such remarkable adaptive radiation.     

Organization of a phyllobranchiate gill from the green shore crab Carcinus maenas (Crustacea,Decapoda)

Summary The phyllobranchiate gills of the green shore crab Carcinus maenas have been examined histologically and ultrastructurally. Each gill lamella is bounded by a chitinous cuticle. The apical surface of the branchial epithelium contacts this cuticle, and a basal lamina segregates the epithelium from an intralamellar hemocoel. In animals acclimated to normal sea water, five epithelial cell types can be identified in the lamellae of the posterior gills: chief cells, striated cells, pillar cells, nephrocytes, and glycocytes. Chief cells are the predominant cells in the branchial epithelium. They are squamous or low cuboidal and likely play a role in respiration. Striated cells, which are probably involved in ionoregulation, are also squamous or low cuboidal. Basal folds of the striated cells contain mitochondria and interdigitate with the bodies and processes of adjacent cells. Pillar cells span the hemocoel to link the proximal and distal sides of a lamella. Nephrocytes are large, spherical cells with voluminous vacuoles. They are rimmed by foot processes or pedicels and frequently associate with the pillar cells. Glycocytes are pleomorphic cells packed with glycogen granules and multigranular rosettes. The glycocytes often mingle with the nephrocytes. Inclusion of the nephrocytes and glycocytes as members of the branchial epithelium is justified by their participation in intercellular junctions and their position internal to the epithelial basal lamina.     

A new specimen ofWeinbergina opitzi (Chelicerata: Xiphosura) from the Lower Devonian Hunsriick Slate, Germany

A new specimen of the synziphosurine arthropodWeinbergina opitzi is described from the Lower Devonian (Lower Emsian) Hunsrück Slate of Germany (Rhenish Slate Mountains). It is the smallest and only the fifth specimen of this taxon to be described and is preserved in ventral aspect with exceptional preservation of prosomal and opisthosomal appendages. This specimen confirms the presence of a seventh appendage, similar in morphology to the preceding prosomal appendages, associated with opisthosomal segment one. In addition, at least three opisthosomal plates fringed with teeth are confirmed. Correlation of prosomal appendage podomeres betweenWeinbergina and selected chelicerate taxa shows that appendage structure is most similar to eurypterid appendages III–IV and Araneae appendages III–VI. This is in contrast to modern horseshoe crabs which have fewer podomeres in appendages II–V due to an undifferentiated tibiotarsus.      

Diverse adaptations of an ancestral gill: a common evolutionary origin for wings,breathing organs,and spinnerets

Changing conditions of life impose new requirements on the morphology and physiology of an organism. One of these changes is the evolutionary transition from aquatic to terrestrial life, leading to adaptations in locomotion, breathing, reproduction, and mechanisms for food capture. We have shown previously that insects' wings most likely originated from one of the gills of ancestral aquatic arthropods during their transition to life on land. Here we investigate the fate of these ancestral gills during the evolution of another major arthropod group, the chelicerates. We examine the expression of two developmental genes, pdm/nubbin and apterous, that participate in the specification of insects' wings and are expressed in particular crustacean epipods/gills. In the horseshoe crab, a primitively aquatic chelicerate, pdm/nubbin is specifically expressed in opisthosomal appendages that give rise to respiratory organs called book gills. In spiders (terrestrial chelicerates), pdm/nubbin and apterous are expressed in successive segmental primordia that give rise to book lungs, lateral tubular tracheae, and spinnerets, novel structures that are used by spiders to breathe on land and to spin their webs. Combined with morphological and palaeontological evidence, these observations suggest that fundamentally different new organs (wings, air-breathing organs, and spinnerets) evolved from the same ancestral structure (gills) in parallel instances of terrestrialization.     

Ultrastructural and histochemical study on gills and skin of the Senegal sole, Solea senegalensis

This study was undertaken to identify the normal ultrastructural features of gills and skin of the Senegal sole, Solea senegalensis, for a comparative measure to morphological alterations caused by environmental stressors such as reduced water quality and diseases. In the Senegal sole skin, four morphologically distinct layers were identified: cuticle, epidermis, dermis and hypodermis. The epidermis was composed of stratified epithelium containing three cellular layers: the outermost or mucosa layer, the middle or fusiform layer and the stratum germinativum or the basal layer. In the mucosa, two mucous cell types were differentiated: type A cells containing several round vesicles of different electron density and type B cells containing mucosomes of uniform electron density. Senegal sole have five pairs of gill arches, each containing two rows of well‐developed and compactly organized primary filaments and secondary lamellae. Fingerprint‐like microridges were observed on the surface of epithelial cells. The branchial lamellae epithelium consisted of different cell types: pavement, mucous and chloride. Between the chloride cells and the larger pavement cells, accessory cells were observed. Complexes of tight junctions and desmosomes were frequently observed between adjacent chloride and epithelial cells. Neutral mucosubstances and/or glycoconjugates were observed in the epidermis, dermis and hypodermis of S. senegalensis skin. Proteins rich in different amino acids, such as arginine and cysteine, reacted negatively or weakly positive in the epidermis, dermis and hypodermis. In gills, some mucous cells responded weakly positive to periodic acid‐Schiff (PAS) reaction but were strongly stained with Alcian Blue at pH 0.5, 1 and 2.5. When Alcian Blue pH 2.5–PAS reaction was performed, most mucous cells were stained blue (carboxylated mucins) and some mucocytes stained purple, indicating a combination of neutral and acid mucins. Proteins rich in cysteine‐bound sulphydryl (‐SH‐) and cystine disulphide (‐S‐S‐) groups were strongly detected in branchial and epidermal mucous cells, whereas lysine, tyrosine and arginine containing proteins showed very weak staining in both epidermal and branchial mucous cells. Protein reactions were strongly positive in the pillar cells, except for those rich in tryptophan, whereas the branchial cartilaginous tissue did not show an important reaction. The performed lipid reactions were negative in goblet and chloride cells. It is concluded from this study that ultrastructural and cytohistochemical features of the Senegal sole skin and gills may serve as control structures in both natural and aquaculture systems to monitor or detect environmental stress responses at the histological level.     

Developmental changes in the embryo, pronymph, and first molt of the scorpion Centruroides vittatus (scorpiones: buthidae)

For the first time the scanning electron microscope was used to compare developmental changes in scorpion embryos and the first and second stadia. In the buthid species of this study, Centruroides vittatus, and all other scorpions, the newborn climb up on their mother's back and remain there without feeding for several days. At this location, they undergo their first molt and in a few days they disperse, fully capable of foraging in the terrestrial environment. The results here support earlier suggestions that the first stadium (pronymph) is a continuation and extension of embryological development. The first molt results in a nymph with exoskeletal features much like those in the adult. In the first molt the metasoma becomes relatively longer, and the sting (aculeus) becomes sharp and functional. The metasomal segments are modified for dorsal flexion and sting use. The embryos and the pronymphs have spiracles that open into an invagination near the posterior margin of flap-like abdominal plates in segments 4-7 of the ventral mesosoma. The second instars have spiracles that lead to book lungs farther anterior in sternites. Tubular legs with cylindrical segments in embryos and pronymphs become more sculptured and oval in the transverse plane. Each leg in the pronymph has a blunt, cup-shaped tip while distal claws (ungues, dactyl) are present in the second instar and subsequent stages. There are some sharp bristles and primordial sensilla in the pronymphs, but the second stadium has adult-like surface features: rows of knobs or granulations (carinae), serrations on the inner surfaces of cheliceral and pedipalpal claws, filtering hairs at the mouthparts, peg sensilla on the pectines, and mechano- and chemoreceptor sensilla on the body and appendages. Scorpion embryos and pronymphs have some structures like fossil scorpions thought to have been aquatic. There is a gradual development of features that appear to be terrestrial adaptations. Evidence is provided for the formation of the sternum from third and fourth leg coxal primordia and possibly from the first abdominal segment. This study is the first to provide evidence for a forward shift of the gonopore along with other structures in the anterior abdomen.     

Book lung development in juveniles and adults of the cobweb spider, Parasteatoda tepidariorum C. L. Koch, 1841 (Araneomorphae,Theridiidae)

Light and transmission electron microscopy were used to study the development of new book lung lamellae in juvenile and adult spiders (Parasteatoda tepidariorum). As hypothesized earlier in a study of embryos, mesenchyme cells dispersed throughout the opisthosoma (EMT) are a likely source of precursor epithelial cells (MET) for the new lamellae. The precursor cells in juveniles and adults continue many of the complex activities observed in embryos, e.g., migration, alignment, lumen formation, thinning, elongation, and secretion of the cuticle of air channel walls and trabeculae. The apicobasal polarity of precursor cells for new channels is apparently induced by the polarity pattern of precursor cells of channels produced earlier. Thus, new air and hemolymph channels extend and continue the alternating pattern of older channels. At sites more distant from the spiracle and atrium, new channels are usually produced by the mode II process (intracellular alignment and merging of vesicles). These air channels have bridging trabeculae and are quite stable in size throughout their length. At sites closer to the spiracle and atrium, new channels may be produced by mode I (coalescence of merocrine vesicle secretion). This raises the hypothesis that structural and functional differences in mode I and II channels and differing oxygen and fluid conditions with distance from the spiracle and atrium determine the mode of formation of new channels. Observations herein support an earlier hypothesis that there is some intercellular apical/apical and basal/basal affinity among the opposed surfaces of aligned precursor cells. This results in the alternating pattern of air channels at the apical and hemolymph channels at the basal cell surfaces.     

Gill morphometrics in relation to gas transfer and ram ventilation in high‐energy demand teleosts: Scombrids and billfishes

This comparative study of the gill morphometrics in scombrids (tunas, bonitos, and mackerels) and billfishes (marlins, swordfish) examines features of gill design related to high rates of gas transfer and the high‐pressure branchial flow associated with fast, continuous swimming. Tunas have the largest relative gill surface areas of any fish group, and although the gill areas of non‐tuna scombrids and billfishes are smaller than those of tunas, they are also disproportionally larger than those of most other teleosts. The morphometric features contributing to the large gill surface areas of these high‐energy demand teleosts include: 1) a relative increase in the number and length of gill filaments that have, 2) a high lamellar frequency (i.e., the number of lamellae per length of filament), and 3) lamellae that are long and low in profile (height), which allows a greater number of filaments to be tightly packed into the branchial cavity. Augmentation of gill area through these morphometric changes represents a departure from the general mechanism of area enhancement utilized by most teleosts, which lengthen filaments and increase the size of the lamellae. The gill design of scombrids and billfishes reflects the combined requirements for ram ventilation and elevated energetic demands. The high lamellar frequencies and long lamellae increase branchial resistance to water flow which slows and streamlines the ram ventilatory stream. In general, scombrid and billfish gill surface areas correlate with metabolic requirements and this character may serve to predict the energetic demands of fish species for which direct measurement is not possible. The branching of the gill filaments documented for the swordfish in this study appears to increase its gill surface area above that of other billfishes and may allow it to penetrate oxygen‐poor waters at depth. J. Morphol. 2010. © 2009 Wiley‐Liss, Inc.     

Characters in the book lungs of Scorpiones (Chelicerata,Arachnida) revealed by scanning electron microscopy

The fine structure of the book lungs in 29 species representing ten monophyletic taxa of the Scorpiones (Arachnida) was investigated using scanning electron microscopy (SEM). Scorpion lungs are not homogeneous across the group. Here we describe and score three sets of phylogenetically informative characters: (1) the surface ornament of the lung lamellae, (2) the distal margins of the lamellae and (3) the fine structure of the spiracle margin. Provisional results suggest that reticulation on the surface of the lung lamellae is characteristic of the Buthidae. By contrast, non-buthid scorpions maintain the air space between adjacent lamellae using projecting trabeculae. Typically they are simple struts, but the trabeculae are distally branched in all investigated Scorpionidae, plus at least one species belonging to the Liochelidae. Simple thorns on the lamellar margins probably represent the plesiomorphic condition, while more complex, branched, arcuate morphologies appear to be homoplastic, occurring sporadically in numerous scorpion sub-groups. The tightly packed, hexagonal pillars around the posterior margin of the spiracle support a close relationship between Scorpionidae and Liochelidae, to the exclusion of the Urodacidae.     

Die Vaskularisation der Kiemen von Myxine glutinosa L. (Cyclostomata)

The angioarchitecture of the gills in Myxine glutinosa L. was studied by scanning electron microscopy of vascular corrosion casts (methylmethacrylate). It was found that the afferent branchial artery may be connected to the sinus peribranchialis by a papilla. The sinus is connected to delicate vessels and sinuses, which are interposed between meridionally arranged radial arteries. These delicate vascular formations continue into the interior of the gill and form a plexus on both poles of the gill folds. Light and scanning electron microscopical studies on the vascular endothelium of the afferent vessels of the gill lamellae reveal rounded endothelial cells, which are characterized by a high content of granula, by long cellular processes and by bridge-formations towards neighbouring cells. Supporting columns within the vascular system of the lamellae were found to be set up by several spirally arranged cells. SEM observations reveal goblet cells and numerous superficial epithelial cells with various numbers of microvilli and microridges forming the epithelial surface of the gill folds.     

Effect of salinity on osmoregulatory patch epithelia in gills of the blue crab Callinectes sapidus

In euryhaline crabs, ion-transporting cells are clustered into osmoregulatory patches on the lamellae of the posterior gills. To examine changes in the branchial osmoregulatory patch in the blue crab Callinectes sapidus in response to change in salinity and to correlate these changes with other osmoregulatory responses, crabs were acclimated to a range of salinities between 10 and 35 ppt. When crabs that had been acclimated to 35 ppt were subsequently transferred to 10 ppt, both the size of the osmoregulatory patch on individual gill lamellae and the specific activity of Na+, K+-ATPase in whole-gill homogenates increased only after the first 24 h of exposure to dilute seawater. Enzyme activity and size of patch area increased gradually and reached their maxima (increasing by 200% and 60%, respectively) 6 days following transfer to 10 ppt seawater and then remained at these levels. Patch size at acclimation varied inversely with the salinity for seawater dilutions below 26 ppt (the isosmotic point of the crab), although it did not vary in salinities at or above 26 ppt. Thus, the size of the patch clearly is modulated with acclimation salinity, but it increases only in those salinities in which the crab hyperosmoregulates. An increase in the total RNA/DNA ratio in gill homogenates, the lack of mitotic figures in the lamellae, and the lack of incorporation of bromodeoxyuridine into nuclei of lamellar epithelial cells during acclimation to dilute seawater were interpreted as evidence that no cell proliferation had occurred and that increases in the size of the osmoregulatory patch occurred through differentiation of existing gas exchange cells or of undifferentiated epithelial cells into ion-transporting cells.     

Pathology of Hematodinium infections in snow crabs (Chionoecetes opilio) from Newfoundland, Canada

Bitter crab disease (BCD) of snow crabs, Chionoecetes opilio, is caused by a parasitic dinoflagellate, Hematodinium sp. The disease has shown an alarming increase in prevalence in the commercial fishery in eastern and northeastern areas of Newfoundland and Labrador since it was first recorded there in the early 1990s. We documented histopathological alterations to the tissues in snow crabs with heavy infections of Hematodinium sp. and during sporulation of the parasite. Pressure necrosis was evident in the spongy connective tissues of the hepatopancreas and the blood vessels in most organs. In heavy infections, little remained of the spongy connective tissues around the hepatopancreas. Damage to the gills varied; in some cases it was severe, particularly during sporulation, involving apparent thinning of the cuticle, loss of epithelial cells, and fusion of the membranous layers of adjacent gill lamellae. Affected lamellae exhibited varying degrees of distention with a loss of trabecular cells, hemocyte infiltrations, and swelling or "clubbing" along the distal margins. Large numbers of zoospores were located along the distal margins of affected lamellae suggesting that sporulation may cause a lysis or bursting of the thin lamellar cuticle, releasing spores. Pressure necrosis, due to the build up of high densities of parasites, was the primary histopathological alteration in most tissues. Hematodinium infections in the snow crab are chronic, long-term infections that end in host death, during sporulation of the parasite.     

Morphology and ultrastructure of the gills of terrestrial crabs (Crustacea,Gecarcinidae and Grapsidae): Adaptations for air-breathing

Summary The terrestrial crabsGeograpsus grayi, Geograpsus crinipes, Cardisoma hirtipes andGecarcoidea natalis have a reduced number of gills and show a reduced planar gill surface (SA) compared to aquatic species. Gill lamellae are stiffened and thickened (increasing blood/gas (BG) diffusion distances) and nodules maintain wide spacing between lamellae. Haemolymph is directed through the gill lamellae by rows of pillar cells and in the afferent region an intralamellar septum splits the haemolymph into two parallel networks. Gaps in the lines of pillar cells allow movement of haemolymph between adjacent channels. The afferent vessel distributes haemolymph to the lamella via a number of direct channels including the marginal canal and in large gills with the aid of a long, forked sinus which supplies the ventral and central regions of the lamellae. The marginal canal functions in both distribution and collection of haemolymph; the role varies with species. Potential flow-control sites were identified at the junctions between afferent and efferent areas and where the efferent channels enter the efferent branchial vessel. Each gill receives a branch from the sternal artery which supplies all the lamellae. Transport epithelia is the principal cell type in the gills of all species examined though its location varies between species, either being confined to certain gills or specific parts of the lamellae.The gill lamellae of air-breathing crabs are clearly modified to breathe air (stiffening and presence of nodules), though the overall contribution of the gills to gas exchange has been reduced (smaller SA and longer BG diffusion distances). The role of the gills in air-breathing crabs thus appears to have switched from one of an efficient aquatic gas-exchanger (thin with large surface area) and transport tissue, to one that is predominantly set up for ion-regulation.Abbreviations a afferent branchial vessel - ac afferent channels - art arteriole - ass artifactual subcuticular space - bl basal lamina - c cuticle - col collagen - ct connective tissue - e efferent branchial vessel - ec efferent channels - epi epithelium - f folds - g Glycogen - h haemolymph - hc haemocyte - is intralamellar septum - m marginal canal - mi mitochondria - mt microtubules - n nucleus - p pillar cell - s shaft of efferent vessel - sd septate desmosome     

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.