中华绒螯蟹蜕壳过程中肌肉的组织学、超微结构及主要蛋白质含量的变化

为探讨中华绒螯蟹(Eriocheir sinensis)蜕壳前后肌肉组织的形态特征变化, 采用石蜡切片、电镜及生物化学方法, 研究了中华绒螯蟹蜕皮过程中步行足和腹部肌肉的组织学、超微结构及主要蛋白质含量的变化。结果显示: 相对于蜕皮间期, 步行足在蜕皮前后组织学形态特征无明显变化; 超微结构在蜕皮前无明显变化, 蜕皮后可见肌原纤维纵裂及肌小节横裂现象, 表明蜕皮后外骨骼硬化的过程伴随着肌肉的生长。相对于蜕皮间期, 腹部肌肉在蜕皮前后组织学特征变化明显: 蜕皮前肌束间隙增大, 蜕皮后肌束内肌纤维间隙增大。电子显微镜观察显示, 蜕皮前肌原纤维在内部降解, 出现空洞, 肌原纤维边缘降解, 导致肌原纤维间隙增大; 蜕皮后肌原纤维重新组装、重建, 恢复到间期正常形态。生物化学研究发现, 蜕皮前后步行足和腹部肌肉中肌原纤维蛋白和可溶性蛋白含量的变化同其结构特征的变化相一致。以上研究结果表明, 中华绒螯蟹肌肉组织的结构特征同蜕皮周期密切相关。     

A morphological study on gills of the brown shrimp, Penaeus aztecus

The gills of Penaeus aztecus were examined by light and electron microscopy. They are dendrobranchiate, consisting of a central axis with biserially arranged branches that subdivide into bifurcating filaments. A septum divides the lumina of these structures into afferent and efferent channels. Hemolymph from the sternal sinus flows through the afferent channels into the filaments where it is directed into the efferent channels and finally to the pericardial cavity. In addition to these channels, numerous blood vessels permeate the gill. The cuticle covering the gill overlies a thin epithelium which is separated from hemolymph by a basal lamina. The epithelium, which is active in cuticle secretion, has a series of pillar processes that form subcuticular lacunae. The apical membranes of epithelial cells become folded in shrimp exposed to hypo- and hyperosmotic salinities. Granular cells that contain elaborate Golgi apparati and several types of granules are present throughout the gill. Nephrocytes resembling glomerular podocytes line the efferent channels. A large nerve traverses the septum in the axis.     

Proteasomal activities in the claw muscle tissue of European lobster, Homarus gammarus, during larval development

Decapod crustaceans grow discontinuously and gain size through complex molt processes. The molt comprises the loss of the old cuticle and, moreover, substantial reduction and re-organization of muscles and connective tissues. In adult lobsters, the muscle tissue of the massive claws undergoes significant atrophy of 40-75% before ecdysis. The degradation of this tissue is facilitated by calcium-dependent proteases and by the proteasome, an intra-cellular proteolytic multi-enzyme complex. In contrast to the adults, the involvement of the proteasome during the larval development is yet not validated. Therefore, we developed micro-methods to measure the 20S and the 26S proteasomal activities within mg- and sub-mg-quantities of the larval claw tissue of the European lobster, Homarus gammarus. Within the three larval stages (Z1-3) we distinguished between sub-stages of freshly molted/hatched (post-molt), inter-molt, and ready to molt (pre-molt) larvae. Juveniles were analyzed in the post-molt and in the inter-molt stage. The trypsin-like, the chymotrypsin-like, and the peptidyl-glutamyl peptide hydrolase activity (PGPH) of the 20S proteasome increased distinctly from freshly hatched larvae to pre-molt Z1. During the Z2 stage, the activities were highest in the post-molt animals, decreased in the inter-molt animals and increased again in the pre-molt animals. A similar but less distinct trend was evident in the Z3 stages. In the juveniles, the proteasomal activities decreased toward the lowest values. A similar pattern was present for the chymotrypsin-like activity of the 26S proteasome. The results show that the proteasome plays a significant role during the larval development of lobsters. This is not only reflected by the elevated activities, but also by the continuous change of the trypsin/chymotrypsin-ratio which may indicate a shift in the subunit composition of the proteasome and, thus, a biochemical adjustment to better cope with elevated protein turnover rates during larval development.     

Release of lysozyme from hemolymph cells of Mercenaria mercenaria during phagocytosis

Activity of the lysosomal enzyme, lysozyme, has been quantitatively determined in the serum and cells of the hemolymph of Mercenaria mercenaria which had been exposed to known quantities of Bacillus megaterium and also in the serum and cells of hemolymph which had not been exposed to bacteria. The results indicate that the level of enzyme activity is greater in serum of hemolymph that had been exposed to B. megaterium and concurrently, there is an equivalent decrease in the level of activity in the cells. This evidence indicates that the amount of lysozyme released from cells into serum is enhanced during phagocytosis of the bacteria.It has also been demonstrated that the release of lysozyme from cells occurs during the process of phagocytosis and is not a delayed phenomenon.Enzyme release by secondary phagosomes is reflected morphologically by what is commonly referred to as degranulation. This process does not involve the rupture of the plasma membrane of the hemolymph cells since biochemical studies have revealed that there is no release of the cytoplasmic enzyme, lactate dehydrogenase.     

Lipase activity in the serum and hemolymph cells of the soft-shelled clam, Mya arenaria, during phagocytosis.

Quantitative determinations of lipase activity in the sera and hemolymph cell homogenates of Mya arenaria that had been challenged with heat-killed Bacillus megaterium and those of control clams have revealed that there is an increase in both fractions of the hemolymph during phagocytosis.The occurrence of lipase in serum and cells suggests that suitable substrates could be degraded by hydrolysis extracellularly as well as within phagocytes.     

Experimentally induced elevation of aminopeptidase activity in hemolymph cells of the American oyster,Crassostrea virginica

The levels of aminopeptidase activity in the serum and hemolymph cells of the American oyster, Crassostrea virginica, have been determined to be 2.89 ± 2.25 and 0.62 ± 0.39 Sigma units/ml, respectively. Low levels of aminopeptidase activity have been demonstrated cytochemically within cytoplasmic granules, i.e., secondary phagosomes, of circulating hemolymph cells. Exposure of whole hemolymph to heat-killed Bacillus megaterium or to sterile sea water results in a significant increase in cellular aminopeptidase activity, with the level of activity being the highest in cells that had been exposed to bacteria. The level of aminopeptidase activity in serum is unaltered in similarly challenged whole hemolymph. It is concluded that aminopeptidase synthesized in cells during phagocytosis or as a result of stimulation by exposure to sea water is not released into the serum but is retained intracellularly. Our studies suggest that it is the intracellular and not the serum aminopeptidases whith are of primary importance in the degradation of B. megaterium.     

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.     

Adenylate energy charge, arginine phosphate and ATPase activity in juvenile Homarus americanus during the molt cycle

Neither gill nor hepatopancreas exhibited significant differences in Na+, K+-ATPase activity with molt stage. Hepatopancreatic residual ATPase activity was significantly higher (F = 6.273) in post-molt animals; while gill residual ATPase activity exhibited no significant differences. Muscle AEC did not change with molt stage, but levels of ATP (F = 8.050) and ADP (F = 4.130) were significantly higher in premolt (D3 pleopod stage 5.0-5.5) animals; while levels of arginine phosphate (F = 6.981) were significantly higher in post-molt animals. Arginine phosphate/ATP and ATP/ADP ratios were highest in post-molt animals, but were not statistically significant. Although not significant, changes in Na+, K+-ATPase activity and AEC did suggest alterations in: enzyme activity that correlate with known osmotic compensations occurring during the water uptake and hardening/mineralization processes; and energy metabolism which occur during the molt cycle, respectively.     

The chiton gill: Ultrastructure in Chiton olivaceus (Mollusca,Polyplacophora)

Gills of Chiton olivaceus, a primitive mollusc, are relatively simple in their structure and ultrastructure but are well adapted to a life in the intertidal zone. In contrast to some other molluscs, there is no differentiation of the gill epithelium into functional regions other than respiratory ones. Ciliation of the epithelium in certain areas may optimize water flow from the outer to the inner part of the mantle cavity. The hemolymph sinuses are oriented so that hemolymph flows in the opposite direction. Interstitial cells link epithelial cells with nerve endings. Muscle cells as well as the collagenous matrix in the connective tissue differ within the main gill axis and the lateral lamellae. The life cycle of immunoactive cells within the connective tissue and the hemolymph is described.     

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.     

Na,K-ATPase activity and epithelial interfaces in gills of the freshwater shrimp Macrobrachium amazonicum (Decapoda,Palaemonidae)

Diadromous freshwater shrimps are exposed to brackish water both as an obligatory part of their larval life cycle and during adult reproductive migration; their well-developed osmoregulatory ability is crucial to survival in such habitats. This study examines gill microsomal Na,K-ATPase (K-phosphatase activity) kinetics and protein profiles in the freshwater shrimp Macrobrachium amazonicum when in fresh water and after 10-days of acclimation to brackish water (21‰ salinity), as well as potential routes of Na⁺ uptake across the gill epithelium in fresh water. On acclimation, K-phosphatase activity decreases 2.5-fold, Na,K-ATPase α-subunit expression declines, total protein expression pattern is markedly altered, and enzyme activity becomes redistributed into different density membrane fractions, possibly reflecting altered vesicle trafficking between the plasma membrane and intracellular compartments. Ultrastructural analysis reveals an intimately coupled pillar cell-septal cell architecture and shows that the cell membrane interfaces between the external medium and the hemolymph are greatly augmented by apical pillar cell evaginations and septal cell invaginations, respectively. These findings are discussed regarding the putative movement of Na⁺ across the pillar cell interfaces and into the hemolymph via the septal cells, powered by the Na,K-ATPase located in their invaginations.     

Respiratory mechanisms and metabolic adaptations of an intertidal crab,Chasmagnathus granulata (Dana, 1851)

• 1.1. The ventilatory mechanism, gill area, sites of oxygen uptake, oxygen consumption and activity of a crab from south Brazil, Chasmagnathus granulata, were investigated.
• 2.2. The oxygen uptake seems to be restricted to the gill lamellae.
• 3.3. The gill area varies with the wet body weight, being relatively higher in smaller animals. There is not a significative reduction of the gill area in relation to species of the infralittoral zone.
• 4.4. C. granulata presents a mechanism for recirculating the water of its branchial chamber when exposed to atmospheric air.
• 5.5. The oxygen consumption and activity are reduced when the animals are exposed to atmospheric air. The reduction in the oxygen consumption may be related to the poorly adapted respiratory system, while the decrease in activity may be a mechanism for saving energy during this hypoxic period.

     

Oyster-MSX interactions: Alterations in hemolymph enzyme activities in Crassostrea virginica during the course of Minchinia nelsoni disease development

Hemolymph enzyme activities were investigated during the course of Minchinia nelsoni (MSX) disease development in Crassostrea virginica. Aspartate aminotransferase, alanine aminotransferase, and phosphohexose isomerase activities increase significantly during the gill lesion stage of MSX disease. Enzyme activities are not significantly elevated during the general infection stage of MSX disease. The alteration of hemolymph enzyme activity is discussed with respect to host metabolism and possible humoral defense mechanisms.     

Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata

The structure of the epithelia of the branchial chamber organs (gills, branchiostegites, epipodites) and the localization of the Na(+),K(+)-ATPase were investigated in two caridean shrimps, the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. The general organization of the phyllobranchiate gills, branchiostegites and epipodites is similar in P. adspersus and in R. exoculata. The gill filaments are formed by a single axial epithelium made of H-shaped cells with thin lateral expansions and a basal lamina limiting hemolymph lacunae. In P. adspersus, numerous ionocytes are present in the epipodites and in the inner-side of the branchiostegites; immunofluorescence reveals their high content in Na(+),K(+)-ATPase. In R. exoculata, typical ionocytes displaying a strong Na(+),K(+)-ATPase specific fluorescence are observed in the epipodites only. While the epipodites and the branchiostegites appear as the main site of osmoregulation in P. adspersus, only the epipodites might be involved in ion exchanges in R. exoculata. In both species, the gill filaments are mainly devoted to respiration.     

Biomineralisation markers during a phase of active growth in Pinctada margaritifera

To search for the biochemical parameters involved in calcium and carbonate transport during crystal formation and biomineralisation in nacreous molluscs, the carbonic anhydrase activity, the levels of calciotropic hormones in hemolymph and in tissues and the circulating concentration of calcium were measured in pearl oysters (Pinctada margaritifera) during a phase of active growth. Activity of carbonic anhydrase in gill tissue increased linearly with age of the animals, while no age variation in activity was noted for the mantle. The circulating level of total calcium increased during the growth of the animals. Calciotropic hormones were radioimmunoassayed in gill, mantle and hemolymph. Only a calcitonin-gene related peptide (CGRP) could be detected and its concentration decreased as a function of growth, both in hemolymph and mantle. No variation in CGRP concentration with age was observed in gill tissue. Our data demonstrate that carbonic anhydrase and a molecule biologically and immunologically related to CGRP are involved during growth of the animals. In addition, this study shows the presence of three main calcium compartments, gill, hemolymph and mantle, involved in the biomineralisation process.