Mitochondria-rich cells in gills and skin of an African lungfish, Protopterus annectens

We used scanning electron microscopy, the vital dye DASPEI and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills and skin (opercular, dorsal and ventral) of the lungfish Protopterus annectens in its free-swimming conditions and at the beginning of aestivation. In the free-swimming period, the gills were short and thick and the pavement cells were extremely large (30-40 microns). The mitochondria-rich cells, which were distributed in the secondary and primary epithelium, occurred as two morphologically different types, i.e. elongated and oval, similar to the alpha and beta chloride cells of fresh water teleosts. In the skin, only one type of mitochondria-rich cells was found, resembling the alpha chloride cells. All the mitochondria-rich cells distributed in the gills and skin were labelled with anti Ca(2+)-ATPase serum indicating the possible uptake of Ca2+ at freshwater chloride cell level. At the start of aestivation, the skin and gills were covered by a thick layer of mucus and the epithelium of the gills was reduced. The mitochondria-rich cells were almost completely covered by the pavement cells.     

Differential expression of aquaporin 3 in Triturus italicus from larval to adult epidermal conversion

By using immunohistochemical techniques applied to confocal microscopy, the presence of aquaporin 3 water channel in the epidermis of Triturus italicus (Amphibia, Urodela) has been shown. We analysed the expression of aquaporin 3 (AQP3) during the larval, pre-metamorphic and adult phases; we also showed the localization of the water-channel protein AQP3 in free-swimming conditions and during aestivation in parallel with histological analysis of the skin, focusing on the possible relationship between protein expression and terrestrial habitats. Our results indicate that aquaporin is produced as the epidermis modifies during the functional maturation phase starting at the climax. Moreover, our data suggest an increase in enzyme expression in aestivating newts emphasizing the putative functional importance of differential expression related to a distinct phase of the biological cycle.     

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during Pregnancy Induced Hypertension

Changes in [³⁵S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.Abbreviations dpm radioactive disintegrations per minute - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - SRP stress related protein     

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.     

Arousal and re-feeding rapidly restores digestive tract morphology following aestivation in green-striped burrowing frogs

During aestivation, the gut of the green-striped burrowing frog, Cyclorana alboguttata undergoes significant morphological down-regulation. Despite the potential impact such changes might have on the re-feeding efficiency of these animals following aestivation, they appear to be as efficient at digesting their first meals as active, non-aestivating animals. Such efficiency might come about by the rapid restoration of intestinal morphology with both arousal from aestivation and the initial stages of re-feeding. Consequently, this study sought to determine what morphological changes to the intestine accompany arousal and re-feeding following 3 months of aestivation. Arousal from aestivation alone had a marked impact on many morphological parameters, including small and large intestine masses, small intestinal length, LF heights, enterocyte cross-sectional area and microvilli height and density. In addition, the onset of re-feeding was correlated with an immediate reversal of many morphological parameters affected by 3 months of aestivation. Those parameters that had not returned to control levels within 36 h of feeding generally had returned to control values by the completion of digestion (i.e. defecation of the meal). Re-feeding was also associated with several changes in enterocyte morphology including the incorporation in intracytoplasmic lipid droplets and the return of enterocyte nuclear material to the 'active' euchromatin state. In conclusion, morphological changes to the gut of aestivating frogs which occur during aestivation are transitory and rapidly reversible with both arousal from aestivation and re-feeding. The proximate causes behind these transitions and their functional significance are discussed.     

The impact of prolonged fasting during aestivation on the structure of the small intestine in the green-striped burrowing frog, Cyclorana alboguttata

The effects of short‐term fasting and prolonged fasting during aestivation on the morphology of the proximal small intestine and associated organs were investigated in the green‐striped burrowing frog, Cyclorana alboguttata (Anura: Hylidae). Animals were fasted for 1 week while active or for 3–9 months during aestivation. Short‐duration fasting (1 week) had little effect on the morphology of the small intestine, whilst prolonged fasting during aestivation induced marked enteropathy including reductions in intestinal mass, length and diameter, longitudinal fold height and tunica muscularis thickness. Enterocyte morphology was also affected markedly by prolonged fasting: enterocyte cross‐sectional area and microvillous height were reduced during aestivation, intercellular spaces were visibly reduced and the prevalence of lymphocytes amongst enterocytes was increased. Mitochondria and nuclei were also affected by 9 months of aestivation with major disruptions to mitochondrial cristae and increased clumping of nuclear material and increased infolding of the nuclear envelope. The present study demonstrates that the intestine of an aestivating frog responds to prolonged food deprivation during aestivation by reducing in size, presumably to reduce the energy expenditure of the organ.     

Effect of aestivation on muscle characteristics and locomotor performance in the Green-striped burrowing frog, Cyclorana alboguttata

The Green-striped burrowing frog, Cyclorana alboguttata survives extended drought periods by burrowing underground and aestivating. These frogs remain immobile within cocoons of shed skin and mucus during aestivation and emerge from their burrows upon heavy rains to feed and reproduce. Extended periods of immobilisation in mammals typically result in muscle atrophy and a decrease in muscle performance. We examined the effect of aestivation and hence prolonged immobilisation, on skeletal muscle mass, in vitro muscle performance, and locomotor performance in C. alboguttata. Frogs were aestivated in soil for 3 months and were compared with control animals that remained active, were fed, and had a continual supply of water. Compared to the controls, the wet mass of the gastrocnemius, sartorius, gracilus major, semimembranosus, peroneus, extensor cruris, tibialis posticus and tibialis anticus longus of aestivators remained unchanged indicating no muscle atrophy. The in-vitro performance characteristics of the gastrocnemius muscle were maintained and burst swimming speed was unaffected, requiring no recovery from the extended period of immobilisation associated with aestivation. This preservation of muscle size, contractile condition and locomotor performance through aestivation enables C. alboguttata to compress their life history into unpredictable windows of opportunity, whenever heavy rains occur.     

Metabolic depression during aestivation does not involve remodelling of membrane fatty acids in two Australian frogs

Changes in membrane lipid composition (membrane remodelling) have been associated with metabolic depression in some aestivating snails but has not been studied in aestivating frogs. This study examined the membrane phospholipid composition of two Australian aestivating frog species Cyclorana alboguttata and Cyclorana australis. The results showed no major membrane remodelling of tissue in either frog species, or in mitochondria of C. alboguttata due to aestivation. Mitochondrial membrane remodelling was not investigated in C. australis. Where investigated in C. alboguttata, total protein and phospholipid content, and citrate synthase (CS) and cytochrome c oxidase (CCO) activities in tissues and mitochondria mostly did not change with aestivation in liver. In skeletal muscle, however, CS and CCO activities, mitochondrial and tissue phospholipids, and mitochondrial protein decreased with aestivation. These decreases in muscle indicate that skeletal muscle mitochondrial content may decrease during aestivation. Na⁺K⁺ATPase activity of both frog species showed no effect of aestivation. In C. alboguttata different fat diets had a major effect on both tissue and mitochondrial phospholipid composition indicating an ability to remodel membrane composition that is not utilised in aestivation. Therefore, changes in lipid composition associated with some aestivating snails do not occur during aestivation in these Australian frogs.     

Metabolic depression during aestivation in Cyclorana alboguttata

The green striped burrowing frog, Cyclorana alboguttata, spends, on average, nine to ten months of every year in aestivation. Recently, C. alboguttata has been the focus of much investigation regarding the physiological processes involved in aestivation, yet our understanding of this frog's capacity to metabolically depress remains limited. This study aimed to extend our current knowledge of metabolic depression during aestivation in C. alboguttata. C. alboguttata reduced whole animal metabolism by 82% within 5 weeks of aestivation. The effects of aestivation on mass specific in vitro tissue metabolic rate (VO₂) varied among individual organs, with muscle and liver slices showing significant reductions in metabolism; kidney VO₂ was elevated and there was no change in the VO₂ of small intestine tissue slices. Organ size was also affected by aestivation, with significant reductions in the mass of all tissues, except the gastrocnemius. These reductions in organ size, combined with changes in mass specific VO₂ of tissue slices, resulted in further energy savings to aestivating animals. This study shows that C. alboguttata is capable of selectively down- or up-regulating individual tissues, using both changes in metabolic rate and morphology. This strategy allows maximal energy savings during aestivation without compromising organ functionality and survival at arousal.     

The NOS/NO system in an example of extreme adaptation: The African lungfish

African dipnoi (lungfish) are aestivating fish and obligate air breathers that, throughout their complex life cycle, undergo remarkable morpho-functional organ readjustment from biochemical to morphological level. In the present review we summarize the changes of the NOS/NO (Nitric Oxide Synthase/Nitric Oxide) system occurring in lungs, gills, kidney, heart, and myotomal muscle of African lungfish of the genus Protopterus (P. dolloi and P. annectens), in relation to the switch from freshwater to aestivation, and vice-versa. In particular, the expression and localization patterns of NOS, and its protein partners Akt, Hsp-90 and HIF-1α, have been discussed, together with the apoptosis rate, evaluated by TUNEL technique.We hypothesize that all these molecular components are crucial in signalling transduction/integration networks induced by environmental challenges (temperature, dehydration, inactivity)experienced at the beginning, during, and at the end of the dry season.     

Carbohydrate and amino acid metabolism in fasting and aestivating African lungfish (Protopterus dolloi)

The potential importance of carbohydrates and amino acids as fuels during periods of fasting and aestivation in the African lungfish, Protopterus dolloi, were examined. No significant decreases in tissue glycogen levels were observed following 60 days of fasting or aestivation, suggesting lungfish may undergo 'glycogen sparing'. Yet glycogenolysis may be important during aestivation based on the differing responses of two flux-generating enzymes of the glycolytic pathway, hexokinase (HK) and pyruvate kinase (PK). PK is required for glycogen breakdown whereas HK is not. HK activity is significantly down-regulated in the heart and gill tissues during aestivation, while PK activity is sustained. The significant negative correlation between the activity of HK and glucose levels in the heart of aestivating lungfish suggests HK may be regulated by glucose concentrations. There was no indication of anaerobic glycolytic flux during aestivation as lactate did not accumulate in any of the tissues examined, and no significant induction of lactate dehydrogenase (LDH)activity was observed. The increase in glutamate dehydrogenase (GDH) and aspartate aminotransferase (Asp-AT) activities in the liver of aestivating P. dolloi suggests some energy may be obtained via increased aminoacid catabolism, leading to the generation of tricarboxylic acid (TCA) cycle intermediates. These findings indicate the importance of both carbohydrate and amino acid fuel stores during aestivation in aphylogenetically ancient, air-breathing fish.     

Metabolic depression and Na+/K+ gradients in the aestivating Australian goldfields frog,Neobatrachus wilsmorei

During aestivation the metabolic rate of the Australian goldfields frog Neobatrachus wilsmorei was reduced by 80% from its standard metabolic rate. The in vitro rate of oxygen consumption of isolated muscle and skin from aestivating frogs was up to 50% lower than that of the non-aestivating frogs. This in vitro rate of oxygen consumption was maintained for 6–12 h, indicating an intrinsic metabolic depression of tissues during aestivation. Frogs became dehydrated during aestivation. Muscle, skin and liver also became dehydrated during aestivation, but brain and kidney did not. Na⁺ and K⁺ contents and extracellular space measurement for muscle indicated that ion gradients were maintained across the muscle cell membrane during aestivation. Increases in plasma concentrations of Na⁺ and K⁺ were matched with similar increases in muscle intracellular ion concentrations. Extracellular space measurements were unsuccessful in the other tissues, but K⁺ content in all tissues (per dry weight) was maintained during aestivation, and the concentration of plasma K⁺ did not increase above that which can be accounted for by dehydration, indicating that K⁺ gradients were maintained.Abbreviations bm body mass - DPM disintegrations per minute - dw dry weight - MR metabolic rate - vO₂ rate of oxygen consumption - ww wet weight     

Hot and steady: Elevated temperatures do not enhance muscle disuse atrophy during prolonged aestivation in the ectotherm Cyclorana alboguttata

Animals that undergo prolonged dormancy experience minimal muscle disuse atrophy (MDA) compared to animals subjected to artificial immobilisation over shorter timeframes. An association between oxidative stress and MDA suggests that metabolic depression presumably affords dormant animals some protection against muscle disuse. Because aerobic metabolism is temperature sensitive, we proposed that MDA in dormant (aestivating) ectotherms would be enhanced at elevated temperatures. In the green‐striped burrowing frog, Cyclorana alboguttata, the thermal sensitivity of skeletal muscle metabolic rate is muscle‐specific. We proposed that the degree of atrophy experienced during aestivation would correlate with the thermal sensitivity of muscle metabolic rate such that muscles with a relatively high metabolic rate at high temperatures would experience more disuse atrophy. To test this hypothesis, we examined the effect of temperature and aestivation on the extent of MDA in two functionally different muscles: the M. gastrocnemius (jumping muscle) and M. iliofibularis (non‐jumping muscle), in C. alboguttata aestivating at 24 or 30°C for 6 months. We compared a range of morphological parameters from muscle cross‐sections stained with succinic dehydrogenase to show that muscle‐specific patterns of disuse atrophy were consistent with the relative rates of oxygen consumption of those muscle types. However, despite muscle‐specific differences in thermal sensitivity of metabolic rate, aestivation temperature did not influence the extent of atrophy in either muscle. Our results suggest that the muscles of frogs aestivating at high temperatures are defended against additional atrophy ensuring protection of muscle function during long periods of immobilisation. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

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.     

Physiology of drought tolerance and cold hardiness of the Mediterranean tiger moth Cymbalophora pudica during summer diapause

Prepupae of the arctiid moth Cymbalophora pudica spend spring and summer months in a summer diapause (aestivation), the duration of which is photoperiodically controlled. Cold hardiness, drought tolerance and some physiological and biochemical parameters were measured in aestivating prepupae. Large amounts of metabolic reserves, in the form of lipids and glycogen, accumulated prior to aestivation. Glycogen served as the main metabolic fuel for aestivating prepupae. Metabolic rate decreased rapidly after the onset of the inactive prepupal stage and remained low (5-15% of the level in active larva) during aestivation. A spontaneous increase of the respiration rate occurred before pupation. Neither low mol. wt sugars or alcohols (polyols) accumulated nor the haemolymph osmotic pressure changed during aestivation. Drought tolerance of aestivating prepupae was high (no decrease in survival after exposure to r.h.<10% at a temperature of 23 degrees C for a substantial part of diapause) owing to their extensive capacity to stabilize the relative body water content irrespective of the r.h. of surrounding air. Cold hardiness was low (>90% decrease in survival after exposure to -7 degrees C for 24h). Cold and drought acclimations did not lead to significant changes in the measured physiological and biochemical parameters but cold (not drought) acclimation caused a significant increase in cold hardiness. Neither drought tolerance nor the increase in cold hardiness after cold acclimation appear to be related to presence/accumulation of polyols in aestivating C. pudica prepupae.     

Facultative aestivation in a tropical freshwater turtle Chelodina rugosa

• 1.1. Chelodina rugosa dug from aestivation sites at the end of the dry season were immediately alert and well coordinated.
• 2.2. Compared with non-aestivating animals, aestivating turtles had 20% higher plasma osmotic pressure and 7% higher sodium. Coupled with a small, but significant weight gain upon return to the water, this suggested the occurrence of minor dehydration in aestivating animals.
• 3.3. Plasma lactate levels of aestivating animals were low, averaging 1.99 mmol/l, consistent with aerobic rather than anaerobic metabolism having sustained their long period under ground.
• 4.4. No evidence was seen of dramatic physiological specialization. Aestivation in this species is interpreted as a primarily behavioural adaptation, made possible by typically reptilian abilities to tolerate a wide range in plasma electrolytes and to survive long periods without feeding.

     

Sublethal Effect of Copper Toxicity Against Histopathological Changes in the Spiny Lobster, <Emphasis Type="Italic">Panulirus homarus</Emphasis> (Linnaeus, 1758)

The tissue damage induced by various organic pollutants in aquatic animals is well documented, but there is a dearth of information relating to the histological alterations induced by copper in the spiny lobster. In the present study, intermoult juveniles of the spiny lobster Panulirus homarus (average weight 150–200 g) were exposed to two sublethal concentrations of the copper (9.55 and 19.1 μg/l) for a period of 28 days. The muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of the lobsters were then dissected out and processed for light microscopic studies. Exposure to copper was found to result in several alterations in the histoarchitecture of the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of P. homarus. The alterations included disruption and congestion of muscle bundle in muscle tissue; blackened haemocytes; distended lumen and F cell; necrosis of the tubules of the hepatopancreas; disarrangement of circular muscle of the midgut; accumulation of haemocytes in the haemocoelic space; swelling and fusion of lamellae; abnormal gill tips; hyperplastic, necrotic, and blackened secondary gill lamellae of the gills; damaged neurosecretory cell and sensory and motor fibre; necrotic of the thoracic ganglion; dispersedly arranged muscle bands; clumped satellite cells and nucleus of the heart. The results obtained suggest that the muscle, hepatopancreas, midgut, gills, thoracic ganglion and heart of lobsters exposed to copper were structurally altered. Such alterations could affect vital physiological functions, such as absorption, storage and secretion of the hepatopancreas, digestion of gut and respiration, osmotic and ionic regulations of the gills, which in turn could ultimately affect the survival and growth of P. homarus. Thus, all possible remedial measures should be adopted to prevent the occurrence of copper contamination in the aquatic environment.     

Metabolic depression and sodium-potassium ATPase in the aestivating frog, Neobatrachus kunapalari

In aestivation the metabolic rate of the Australian desert frog Neobatrachus kunapalari was 50–67% lower than in the non-aestivating state. The rate of O₂ consumption of isolated muscle, skin and brain was measured in both metabolic states. The average rate of O₂ consumption of muscle was 30% lower and brain 50% lower in aestivating frogs, while the rate of O₂ consumption of skin was the same. The reduction in muscle could account for a large proportion of whole animal metabolic depression. To look for evidence of a reduction in energy demand in the tissues we measured the ouabain-sensitive fraction of tissue rate of O₂ consumption, which is considered to be the proportion of metabolism used for transmembrane Na⁺/K⁺ pumping. Ouabain inhibited the in vitro rate of O₂ consumption of skin by a average of 20% and of brain by an average of 30%. However, in muscle, ouabain stimulated in vitro O₂ consumption. Despite the 50% reduction in the in vitro rate of O₂ consumption of brain during aestivation, neither the ouabain-sensitive nor ouabain-insensitive fractions were found be statistically different, possibly because of the large individual variation in the degree of ouabain inhibition. A reduction in the level of ion pumping during aestivation was therefore not demonstrated in any tissue. Measurement of the level of the enzyme Na⁺K⁺-ATPase in skeletal muscle, ventricle, kidney and brain showed that there was no change in the amount of this enzyme in the aestivating frogs. Measurement of the levels of adenylates in muscle and liver showed that the adenylate energy charge was maintained in aestivation, but that there was a reduction in ATP in liver and a reduction in the level of total adenylates in both tissues, which could be an adaptation of the tissues to a lower energy turnover. Accepted: 22 July 1996     

Ultrastructural changes in the gill epithelium of the green crab Carcinus maenas in relation to the external salinity

Observation of semi-thin and ultrathin sections performed in the gills of green crabs (Carcinus maenas) kept in 100% and in dilute 30% sea water respectively reveals marked differences between the six anterior and the three posterior pairs of gills. The anterior gill lamellae are almost entirely lined by a thin pavement epithelium (0.9 to 3 mum thick) which does not undergo any noticeable change when crabs are acclimated from full to dilute sea water. This supports the view it is chiefly involved in the respiratory function. In addition to the pavement epithelium, the posterior gills exhibit small areas corresponding to a thick prismatic epithelium (10 mum) the ultrastructure of which is similar to that of most of the so-called 'salt transporting epithelia'. When submitted to reduced external salinity, this epithelium undergoes structural changes consisting of elaboration of an extensive apical plasma membrane infolding system, enlargement of the subcuticular compartment and close association of mitochondria with basolateral membrane infoldings. Pilaster cells exhibit ultrastructural features of either thin (respiratory) or thick (salt transporting) epithelial differentiation according to their localization within the gill. Their peculiar organization suggests they ensure, in addition, mechanical reinforcement of the gill lamellae against blood hydrostatic pressure. The fact that salt-transporting epithelium areas do not exceed, at most, 30% of the total lamellar surface is probably related to the weak osmoregulatory capabilities of the shore crab.