Fasting in the ureotelic Lake Magadi tilapia, Alcolapia grahami, does not reduce its high metabolic demand, increasing its vulnerability to siltation events.

Lake Magadi, Kenya, is one of the most extreme aquatic environments on Earth (pH~10, anoxic to hyperoxic, high temperatures). Recently, increased water demand and siltation have threatened the viable hot springs near the margins of the lake where Alcolapia grahami, the only fish surviving in the lake, live. These Lake Magadi tilapia largely depend on nitrogen-rich cyanobacteria for food and are 100% ureotelic. Their exceptionally high aerobic metabolic rate, together with their emaciated appearance, suggests that they are energy-limited. Therefore, we hypothesized that during food deprivation, Magadi tilapia would economize their energy expenditure and reduce metabolic rate, aerobic performance and urea-N excretion. Surprisingly, during a 5-day fasting period, routine metabolic rates increased and swimming performance (critical swimming speed) was not affected. Urea-N excretion remained stable despite the lack of their N-rich food source. Their nitrogen use switched to endogenous sources as liver and muscle protein levels decreased after a 5-day fast, indicating proteolysis. Additionally, fish relied on carbohydrates with lowered muscle glycogen levels, but there were no signs indicating use of lipid stores. Gene expression of gill and gut urea transporters were transiently reduced as were gill rhesus glycoprotein Rhbg and Rhcg-2. The reduction in gill glutamine synthetase expression concomitant with the reduction in Rh glycoprotein gene expression indicates reduced nitrogen/ammonia metabolism, most likely decreased protein synthesis. Additionally, fish showed reduced plasma total CO2, osmolality and Na+ (but not Cl-) levels, possibly related to reduced drinking rates and metabolic acidosis. Our work shows that Lake Magadi tilapia have the capacity to survive short periods of starvation which could occur when siltation linked to flash floods covers their main food source, but their seemingly hardwired high metabolic rates would compromise long-term survival.

Air breathing and aquatic gas exchange during hypoxia in armoured catfish.

Air breathing in fish is commonly believed to have arisen as an adaptation to aquatic hypoxia. The effectiveness of air breathing for tissue O2 supply depends on the ability to avoid O2 loss as oxygenated blood from the air-breathing organ passes through the gills. Here, we evaluated whether the armoured catfish (Hypostomus aff. pyreneusi)-a facultative air breather-can avoid branchial O2 loss while air breathing in aquatic hypoxia, and we measured various other respiratory and metabolic traits important for O2 supply and utilization. Fish were instrumented with opercular catheters to measure the O2 tension (PO2) of expired water, and air breathing and aquatic respiration were measured during progressive stepwise hypoxia in the water. Armoured catfish exhibited relatively low rates of O2 consumption and gill ventilation, and gill ventilation increased in hypoxia due primarily to increases in ventilatory stroke volume. Armoured catfish began air breathing at a water PO2 of 2.5 kPa, and both air-breathing frequency and hypoxia tolerance (as reflected by PO2 at loss of equilibrium, LOE) was greater in individuals with a larger body mass. Branchial O2 loss, as reflected by higher PO2 in expired than in inspired water, was observed in a minority (4/11) of individuals as water PO2 approached that at LOE. Armoured catfish also exhibited a gill morphology characterized by short filaments bearing short fused lamellae, large interlamellar cell masses, low surface area, and a thick epithelium that increased water-to-blood diffusion distance. Armoured catfish had a relatively low blood-O2 binding affinity when sampled in normoxia (P50 of 3.1 kPa at pH 7.4), but were able to rapidly increase binding affinity during progressive hypoxia exposure (to a P50 of 1.8 kPa). Armoured catfish also had low activities of several metabolic enzymes in white muscle, liver, and brain. Therefore, low rates of metabolism and gill ventilation, and a reduction in branchial gas-exchange capacity, may help minimize branchial O2 loss in armoured catfish while air breathing in aquatic hypoxia.

Gill paracellular permeability and the osmorespiratory compromise during exercise in the hypoxia-tolerant Amazonian oscar (Astronotus ocellatus).

In the traditional osmorespiratory compromise, fish increase their effective gill permeability to O2 during exercise or hypoxia, and in consequence suffer unfavorable ionic and osmotic fluxes. However oscars, which live in the frequently hypoxic ion-poor waters of the Amazon, actually decrease ionic fluxes across the gills during acute hypoxia without changing gill paracellular permeability, and exhibit rapid paving over of the mitochondrial-rich cells (MRCs). But what happens during prolonged exercise? Gill paracellular permeability, ionic fluxes, and gill morphology were examined in juvenile oscars at rest and during aerobic swimming. Initial validation tests with urinary catheterized fish quantified drinking, glomerular filtration, and urinary flow rates, and confirmed that measurements of gill paracellular permeability as [(3)H]PEG-4000 clearances were the same in efflux and influx directions, but far lower than previously measured in comparably sized trout. Although the oscars achieved a very similar proportional increase (90%) in oxygen consumption (MO2) to trout during steady-state swimming at 1.2 body lengths sec(-1), there was no increase in gill paracellular permeability, in contrast to trout. However, oscars did exhibit increased unidirectional Na(+) efflux and net K(+) rates during exercise, but no change in drinking rate. There were no changes in MRC numbers or exposure, or other alterations in gill morphology during exercise. A substantial interlamellar cell mass (ILCM) that covered the lamellae to a depth of 30% was unchanged by 4 h of swimming activity. We conclude that a low branchial paracellular permeability which can be dissociated from changes in O2 flux, as well as the presence of the ILCM, may be adaptive in limiting ionoregulatory costs for a species endemic to ion-poor, frequently hypoxic waters.

Gill remodeling in three freshwater teleosts in response to high environmental ammonia.

The present study aimed to determine whether gill macro- and microstructure show compensatory responses in three freshwater fish differing in their sensitivity to high environmental ammonia (HEA). The highly ammonia-sensitive salmonid Oncorhynchus mykiss (rainbow trout), the less ammonia-sensitive cyprinid Cyprinus carpio (common carp) and the highly ammonia-resistant cyprinid Carassius auratus (goldfish) were used as test species and were exposed for 0 h (control), 3h, 12h, 24h, 48 h, 84 h and 180 h to 1mM ammonia (as NH4HCO3; pH 7.9). In cyprinids, dramatic alterations were initiated quickly evident by thickening of filaments and lamellae, retraction of lamellae, enlargement of interlamellar cell mass (ILCM), and increase in the water-blood diffusion distance; while in trout, these modifications were absent or developed very slowly. These reorganizations may attempt to reduce the surface area presumably protecting against the water borne ammonia; and were more pronounced in goldfish marked by momentous enlargement of ILCM volume and the presence of rudimental and almost fused lamellae. Extensive mucus production in the gills of goldfish and carp and to a limited extent in trout may have been part of general stress response and/or may have played a protective role. While goldfish and carp showed shrinkage of apical crypts of mitochondrion rich cells (MRCs), probably aiding to regulate ion status, trout showed enlarged apical crypts of MRCs. All species displayed changes in the pattern of the microridges on the surface of pavement cells (PVCs). Overall, the present results connote that the goldfish with its minimal respiratory surface area and a large population of the MRCs with small apical crypts located on the edge of ILCM is better prepared for survival in ammonia polluted water compared to carp which maintain larger lamellae and especially the trout that did not show gill remodeling.

Interspecific differences in hypoxia-induced gill remodeling in carp.

The gills of many fish, but in particular those of crucian carp (Carassius carassius) and goldfish (Carassius auratus), are capable of extensive remodeling in response to changes in oxygen (O2), temperature, and exercise. In this study, we investigated the interspecific variation in hypoxia-induced gill modeling and hypoxia tolerance in 10 closely related groups of cyprinids (nine species, with two strains of Cyprinus carpio). There was significant variation in hypoxia tolerance, measured as the O2 tension (P(O2)) at which fish lost equilibrium (LOEcrit), among the 10 groups of carp. In normoxia, there was a significant, phylogenetically independent relationship between mass-specific gill surface area and LOEcrit, with the more hypoxia-tolerant carp having smaller gills than their less hypoxia-tolerant relatives. All groups of carp, except the Chinese bream (Megalobrama pellegrini), increased mass-specific gill surface area in response to 48 h of exposure to hypoxia (0.7 kPa) through reductions in the interlamellar cell mass (ILCM) volume. The magnitude of the hypoxia-induced reduction in the ILCM was negatively correlated with LOEcrit (and thus positively correlated with hypoxia tolerance), independent of phylogeny. The hypoxia-induced changes in gill morphology resulted in reduced variation in mass-specific gill surface area among species and eliminated the relationship between LOEcrit and mass-specific gill surface area. While behavioral responses to hypoxia differed among the carp groups, there were no significant relationships between hypoxia tolerance and the Po2 at which aquatic surface respiration (ASR) was initiated or the total number of ASR events observed during progressive hypoxia. Our results are the first to show that the extent of gill remodeling in cyprinids is associated with hypoxia tolerance in a phylogenetically independent fashion.

Interactions between hypoxia tolerance and food deprivation in Amazonian oscars, Astronotus ocellatus.

Oscars are often subjected to a combination of low levels of oxygen and fasting during nest-guarding on Amazonian floodplains. We questioned whether this anorexia would aggravate the osmo-respiratory compromise. We compared fed and fasted oscars (10-14 days) in both normoxia and hypoxia (10-20 Torr, 4 h). Routine oxygen consumption rates (O2) were increased by 75% in fasted fish, reflecting behavioural differences, whereas fasting improved hypoxia resistance and critical oxygen tensions (Pcrit) lowered from 54 Torr in fed fish to 34 Torr when fasting. In fed fish, hypoxia reduced liver lipid stores by approximately 50% and total liver energy content by 30%. Fasted fish had a 50% lower hepatosomatic index, resulting in lower total liver protein, glycogen and lipid energy stores under normoxia. Compared with hypoxic fed fish, hypoxic fasted fish only showed reduced liver protein levels and even gained glycogen (+50%) on a per gram basis. This confirms the hypothesis that hypoxia-tolerant fish protect their glycogen stores as much as possible as a safeguard for more prolonged hypoxic events. In general, fasted fish showed lower hydroxyacylCoA dehydrogenase activities compared with fed fish, although this effect was only significant in hypoxic fasted fish. Energy stores and activities of enzymes related to energy metabolism in muscle or gills were not affected. Branchial Na(+) uptake rates were more than two times lower in fed fish, whereas Na(+) efflux was similar. Fed and fasted fish quickly reduced Na(+) uptake and efflux during hypoxia, with fasting fish responding more rapidly. Ammonia excretion and K(+) efflux were reduced under hypoxia, indicating decreased transcellular permeability. Fasted fish had more mitochondria-rich cells (MRC), with larger crypts, indicating the increased importance of the branchial uptake route when feeding is limited. Gill MRC density and surface area were greatly reduced under hypoxia, possibly to reduce ion uptake and efflux rates. Density of mucous cells of normoxic fasted fish was approximately fourfold of that in fed fish. Overall, a 10-14 day fasting period had no negative effects on hypoxia tolerance in oscars, as fasted fish were able to respond more quickly to lower oxygen levels, and reduced branchial permeability effectively.

Ultrastructure of the spermatozoon of the diphyllobothriidean cestode Cephalochlamys namaquensis (Cohn, 1906).

This contribution provides the first ultrastructural and cytochemical data on the mature spermatozoon of a diphyllobothriidean cestode belonging to the family Cephalochlamydidae. The mature spermatozoon of Cephalochlamys namaquensis (Cohn, 1906), a parasite of the African clawed frog, Xenopus laevis (Daudin, 1802), from southern California, USA, has been examined using transmission electron microscopy and cytochemical staining with periodic acid-thiosemicarbazide-silver proteinate for glycogen. The male gamete is a filiform cell tapered at both extremities. Its moderately electron-dense cytoplasm possesses two parallel axonemes of unequal lengths with a 9 + "1" trepaxonematan pattern, a nucleus, parallel cortical microtubules, four electron-dense plaques/attachment zones, and electron-dense granules of glycogen. The crested body is absent. The anterior extremity of the cell exhibits a centriole surrounded by a semiarc of four parallel cortical microtubules. The number of cortical microtubules reaches its maximum (up to 37) at the beginning of the anucleated two-axoneme region II of the spermatozoon. In contrast to other diphyllobothriideans, a small membranous element appears in the anucleated region II. In addition, the nucleus is surrounded by a few cortical microtubules in region V. The distal extremity of the mature spermatozoon exhibits only one nucleus. Variations of spermatozoa ultrastructural characters within diphyllobothriideans as well as other Eucestoda are discussed.

Physiological, molecular, and cellular mechanisms of impaired seawater tolerance following exposure of Atlantic salmon, Salmo salar, smolts to acid and aluminum.

We examined the physiological, molecular, and cellular mechanisms of impaired ion regulation in Atlantic salmon, Salmo salar, smolts following acute acid and aluminum (Al) exposure. Smolts were exposed to: control (pH 6.5, 3.4 micrpg l(-1) Al), acid and low Al (LAl: pH 5.4, 11 microg l(-1) Al), acid and moderate Al (MAl: pH 5.3, 42 microg l(-1) Al), and acid and high Al (HAl: pH 5.4, 56 microg l(-1) Al) for two and six days. At each time-point, smolts were sampled directly from freshwater treatment tanks and after a 24h seawater challenge. Exposure to acid/MAl and acid/HAl led to accumulation of gill Al, substantial alterations in gill morphology, reduced gill Na(+)/K(+)-ATPase (NKA) activity, and impaired ion regulation in both freshwater and seawater. Exposure to acid/MAl for six days also led to a decrease in gill mRNA expression of the apical Cl(-) channel (cystic fibrosis transmembrane conductance regulator I), increased apoptosis upon seawater exposure, an increase in the surface expression of mitochondria-rich cells (MRCs) within the filament epithelium of the gill, but reduced abundance of gill NKA-positive MRCs. By contrast, smolts exposed to acid and the lowest Al concentration exhibited minor gill Al accumulation, slight morphological modifications in the gill, and impaired seawater tolerance in the absence of a detectable effect on freshwater ion regulation. These impacts were accompanied by decreased cell proliferation, a slight increase in the surface expression of MRCs within the filament epithelium, but no impact on gill apoptosis or total MRC abundance was observed. However, MRCs in the gills of smolts exposed to acid/LAl exhibited morphological alterations including decreased size, staining intensity, and shape factor. We demonstrate that the seawater tolerance of Atlantic salmon smolts is extremely sensitive to acute exposure to acid and low levels of Al, and that the mechanisms underlying this depend on the time-course and severity of Al exposure. We propose that when smolts are exposed to acid and moderate to high Al concentrations, impaired seawater tolerance results from extensive gill Al accumulation, damage to the epithelium, reduced MRC and transport protein abundance, and a synergistic stimulation of apoptosis in the gill upon seawater exposure. When smolts are exposed to acid and low levels of Al, loss of seawater tolerance appears to be independent of these mechanisms and may result instead from a shift in the phenotype of MRCs present in the gill epithelium.

The ionoregulatory responses to hypoxia in the freshwater rainbow trout Oncorhynchus mykiss.

We utilized the rainbow trout, a hypoxia-intolerant freshwater teleost, to examine ionoregulatory changes at the gills during hypoxia. Progressive mild hypoxia led first to a significant elevation (by 21%) in J(Na)(influx) (measured with 22Na), but at 4-h hypoxia when PCO2 reached approximately 110 mmHg, there was a 79% depression in J(Na)(influx). Influx remained depressed during the first hour of normoxic recovery but was restored back to control rates thereafter; there were no significant changes in J(Na)(efflux) or J(Na)(net). A more prolonged (8 h) and severe hypoxic (approximately 80 mmHg) exposure induced a triphasic response whereby J(Na)(influx) was significantly elevated during the first hour, as during mild hypoxia, but returned to control rates during the subsequent 3 h. Thereafter, rates started to gradually increase and remained significantly elevated by about 38% through to 8 h of hypoxia. A similar triphasic trend was observed with J(Na)(efflux) but with larger changes than in J(Na)(influx), such that negative Na+ balance occurred during the hypoxic exposure. Net K+ loss rates to the water approximately doubled. There were no significant alterations in ammonia excretion rates in either of the hypoxia regimes. Branchial Na+/K+-ATPase activity did not change during 4 h at PO2 approximately 80 mmHg or return to normoxia; H+-ATPase activity also did not change during hypoxia but was significantly depressed by approximately 75% after 6 h of normoxic recovery. Scanning electron microscopy revealed that within 1 h of exposure to PO2 approximately 80 mmHg, exposed mitochondria-rich cell (MRC) numbers increased by 30%, while individual MRC exposed surface area and total MRC surface area both increased by three- to fourfold. MRC numbers had decreased below control levels by 4 h of hypoxia, but surface exposure remained elevated by approximately twofold, a response that persisted through 6 h of normoxic recovery. Environmental hypoxia induces complex changes in gill ionoregulatory function in this hypoxia-intolerant species that are very different from those recently reported in the hypoxia-tolerant Amazonian oscar.

Regulation of gill transcellular permeability and renal function during acute hypoxia in the Amazonian oscar (Astronotus ocellatus): new angles to the osmorespiratory compromise.

Earlier studies demonstrated that oscars, endemic to ion-poor Amazonian waters, are extremely hypoxia tolerant, and exhibit a marked reduction in active unidirectional Na(+) uptake rate (measured directly) but unchanged net Na(+) balance during acute exposure to low P(O(2)), indicating a comparable reduction in whole body Na(+) efflux rate. However, branchial O(2) transfer factor does not fall. The present study focused on the nature of the efflux reduction in the face of maintained gill O(2) permeability. Direct measurements of (22)Na appearance in the water from bladder-catheterized fish confirmed a rapid 55% fall in unidirectional Na(+) efflux rate across the gills upon acute exposure to hypoxia (P(O(2))=10-20 torr; 1 torr=133.3 Pa), which was quickly reversed upon return to normoxia. An exchange diffusion mechanism for Na(+) is not present, so the reduction in efflux was not directly linked to the reduction in Na(+) influx. A quickly developing bradycardia occurred during hypoxia. Transepithelial potential, which was sensitive to water [Ca(2+)], became markedly less negative during hypoxia and was restored upon return to normoxia. Ammonia excretion, net K(+) loss rates, and (3)H(2)O exchange rates (diffusive water efflux rates) across the gills fell by 55-75% during hypoxia, with recovery during normoxia. Osmotic permeability to water also declined, but the fall (30%) was less than that in diffusive water permeability (70%). In total, these observations indicate a reduction in gill transcellular permeability during hypoxia, a conclusion supported by unchanged branchial efflux rates of the paracellular marker [(3)H]PEG-4000 during hypoxia and normoxic recovery. At the kidney, glomerular filtration rate, urine flow rate, and tubular Na(+) reabsorption rate fell in parallel by 70% during hypoxia, facilitating additional reductions in costs and in urinary Na(+), K(+) and ammonia excretion rates. Scanning electron microscopy of the gill epithelium revealed no remodelling at a macro-level, but pronounced changes in surface morphology. Under normoxia, mitochondria-rich cells were exposed only through small apical crypts, and these decreased in number by 47% and in individual area by 65% during 3 h hypoxia. We suggest that a rapid closure of transcellular channels, perhaps effected by pavement cell coverage of the crypts, allows conservation of ions and reduction of ionoregulatory costs without compromise of O(2) exchange capacity during acute hypoxia, a response very different from the traditional osmorespiratory compromise.

The effect of hypoxia on gill morphology and ionoregulatory status in the Lake Qinghai scaleless carp, Gymnocypris przewalskii.

Goldfish and crucian carp at low temperature exhibit plasticity in gill morphology during exposure to hypoxia to enhance gas exchange. Hypoxia-induced changes in gill morphology and cellular ultrastructure of the high altitude scaleless carp from Lake Qinghai, China, were investigated to determine whether this is a general characteristic of cold water carp species. Fish were exposed to acute hypoxia (0.3 mg O2 l(-1)) for 24 h followed by 12 h recovery in normoxic water (6 mg O2 l(-1) at 3200 m altitude), with no mortality. Dramatic alterations in gill structure were initiated within 8 h of hypoxia and almost complete by 24 h, and included a gradual reduction of filament epithelial thickness (>50%), elongation of respiratory lamellae, expansion of lamellar respiratory surface area (>60%) and reduction in epithelial water-blood diffusion distance (<50%). An increase in caspase 3 activity in gills occurred following 24 h exposure to hypoxia, indicating possible involvement of apoptosis in gill remodeling. Extensive gill mucous production during hypoxia may have been part of a general stress response or may have played a role in ion exchange and water balance. The large increase in lamellar surface area and reduction in diffusion distance presumably enhances gas transfer during hypoxia (especially in the presence of increased mucous production) but comes with an ionoregulatory cost, as indicated by a 10 and 15% reduction in plasma [Na+] and [Cl-], respectively, within 12-24 h of hypoxia. Within 12 h of hypoxia exposure, ;wavy-convex'-mitochondria rich cells (MRCs) with large apical crypts and numerous branched microvilli were transformed into small ;shallow-basin' cells with a flattened surface. As the apical membrane of MRCs is the site for active ion uptake from the water, a reduction in apical crypt surface area may have contributed to the progressive reduction in plasma [Na+] and [Cl-] observed during hypoxia. The changes in the macro- and ultra-structure of fish gills, and plasma [Na+] and [Cl-] during hypoxia were reversible, showing partial recovery by 12 h following return to normoxia. Although the large morphological changes in the gill observed in the scaleless carp support the hypothesis that gill remodeling during hypoxia is a general characteristic of cold water carp species, the reduced magnitude of the response in scaleless carp relative to goldfish and crucian carp may be a reflection of their more active lifestyle or because they reside in a moderately hypoxic environment at altitude.

Physiological, biochemical and morphological indicators of osmoregulatory stress in 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum) exposed to hypersaline water.

The salinity tolerance of the 'California' Mozambique tilapia (Oreochromis mossambicus x O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l(-1)) tilapia hybrids were exposed to salinities from 35-95 g l(-1), using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na+], [Cl-], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical (Na+, K(+)-ATPase) and morphological (number of mature, accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l(-1) to 65 g l(-1) with little or no change in osmoregulatory status; however, in fish exposed to 75-95 g l(-1) salinity, plasma osmolality, [Na+], [Cl-], Na+, K(+)-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l(-1), prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, 'California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l(-1) indicates they may be unable to withstand extreme salinities for extended periods of time.