Can the impacts of cold-water pollution on fish be mitigated by thermal plasticity?

Increasingly, cold-water pollution (CWP) is being recognised as a significant threat to aquatic communities downstream of large, bottom-release dams. Cold water releases typically occur during summer when storage dams release unseasonably cold and anoxic hypolimnetic waters, which can decrease the temperature of downstream waters by up to 16°C. Depending on the release duration, these hypothermic conditions can persist for many months. The capacity of ectothermic species to tolerate or rapidly adjust to acute temperature changes may determine the nature and magnitude of the impact of CWP on affected species. This study assessed the impacts of an acute reduction in water temperature on the physiological function and locomotor performance of juvenile silver perch (Bidyanus bidyanus) and examined their capacity to thermally compensate for the depressive effects of low temperatures via phenotypic plasticity. Locomotor performance (Ucrit and Usprint) and energetic costs (routine and maximum metabolic rate) were measured at multiple points over a 10-week period following an abrupt 10°C drop in water temperature. We also measured the thermal sensitivity of metabolic enzymes from muscle samples taken from fish following the exposure period. Cold exposure had significant depressive effects on physiological traits, resulting in decreases in performance between 10% and 55%. Although there was partial acclimation of Ucrit (~35% increase in performance) and complete compensation of metabolic rate, this occurred late in the exposure period, meaning silver perch were unable to rapidly compensate for the depressive effects of thermal pollution. The results of this study have substantial implications for the management of cold water releases from large-scale dams and the conservation of native freshwater fish species, as this form of thermal pollution can act as a barrier to fish movement, cause reduced recruitment, ecological community shifts and disruptions to timing and success of reproduction.

Measuring Ucrit and endurance: equipment choice influences estimates of fish swimming performance.

This study compared the critical swimming speed (Ucrit ) and endurance performance of three Australian freshwater fish species in different swim-test apparatus. Estimates of Ucrit measured in a large recirculating flume were greater for all species compared with estimates from a smaller model of the same recirculating flume. Large differences were also observed for estimates of endurance swimming performance between these recirculating flumes and a free-surface swim tunnel. Differences in estimates of performance may be attributable to variation in flow conditions within different types of swim chambers. Variation in estimates of swimming performance between different types of flumes complicates the application of laboratory-based measures to the design of fish passage infrastructure.

Osmoregulation by juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, in hypo- and hyper-saline waters.

While there is a considerable body of work describing osmoregulation by elasmobranchs in brackish and saltwater, far fewer studies have investigated osmoregulation in hypersaline waters. We examined osmo- and ionoregulatory function and plasticity in juvenile brown-banded bamboo sharks, Chiloscyllium punctatum, exposed to three experimental salinities (25, 34 and 40‰) for two weeks. C. punctatum inhabits sheltered coastal areas and bays which can naturally become hypersaline as a consequence of evaporation of water but can also become hyposaline during flood events. We hypothesised that C. punctatum would demonstrate a phenotypically plastic osmoregulatory physiology. Plasma osmolality, urea, Na(+) and Cl(-) levels increased significantly with increasing environmental salinity. Rectal gland and branchial sodium-potassium ATPase (NKA) activities were unaffected by salinity. Using immunohistochemistry and Western Blotting we found evidence for the presence of the key ion-regulatory proteins vacuolar H(+)-ATPase (VHA), pendrin (Cl(-)/HCO3(-) co-transporter) and the Na(+)-H(+) exchanger isoform 3 (NHE3) in discrete cells within the branchial epithelia. These results indicate that C. punctatum is a partially euryhaline elasmobranch able to maintain osmo- and ionoregulatory function between environmental salinities of 25‰ and 40‰. As suggested for other elasmobranchs, the gills of C. punctatum likely play a limited role in maintaining Na(+) homeostasis over the salinity range studied, but may play an important role in acid-base balance.

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.

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.

Effect of incubation temperature on muscle growth of barramundi Lates calcarifer at hatch and post-exogenous feeding.

Muscle morphology was investigated in newly hatched barramundi Lates calcarifer larvae incubated at set temperatures (26, 29 and 31 degrees C) prior to hatching. Three days after hatching (the start of exogenous feeding), larvae from the 26 and 31 degrees C treatments were each divided into two groups and reared at that temperature or transferred over the period of several hours to 29 degrees C (control temperature). Incubation temperature significantly affected muscle cellularity in the developing embryo, with larvae incubated at 26 degrees C (mean +/-s.e. 223.3 +/- 7.9) having on average 14.4% more inner muscle fibres than those incubated at 31 degrees C (195.2 +/- 8.8) and 4.8% more than those incubated at 29 degrees C (213.5 +/- 4.7). Conversely, inner muscle fibre cross-sectional area significantly increased at the warm incubation temperature in L. calcarifer, so that the total cross-sectional muscle area was not different between treatment groups. The total cross-sectional area of superficial muscle fibres and the proportion of superficial to total fibre cross-sectional area in just hatched L. calcarifer were also affected by incubation temperature, with incubation at the cool temperature (26 degrees C) increasing both the total cross-sectional area and proportion of superficial muscle fibres. By 9 days post-hatch, the aforementioned differences were no longer significant. Similarly, there was no difference in total superficial fibre cross-sectional area between any treatment groups of L. calcarifer, whereas incubation temperature still significantly affected the proportion of superficial to total muscle fibre cross-sectional area. Larvae hatched and grown at 31 degrees C had a significantly reduced percentage of superficial muscle cross-sectional area (mean +/-s.e. 5.11 +/- 0.66%) compared with those incubated and grown at 29 degrees C (8.04 +/- 0.77%) and 26 degrees C (9.32 +/- 0.56%) and those incubated at 26 degrees C and transferred to 29 degrees C (7.52 +/- 0.53%), and incubated at 31 degrees C and transferred to 29 degrees C (6.28 +/- 0.69%). These results indicate that changes in muscle cellularity induced by raising or lowering the incubation temperature of L. calcarifer display varying degrees of persistence over developmental time. The significance of these findings to the culture of L. calcarifer is discussed.

Substantial changes in the genetic basis of tadpole morphology of Rana lessonae in the presence of predators.

Predator-induced morphological plasticity is a model system for investigating phenotypic plasticity in an ecological context. We investigated the genetic basis of the predator-induced plasticity in Rana lessonae by determining the pattern of genetic covariation of three morphological traits that were found to be induced in a predatory environment. Body size decreased and tail dimensions increased when reared in the presence of preying dragonfly larvae. Genetic variance in body size increased by almost an order of magnitude in the predator environment, and the first genetic principal component was found to be highly significantly different between the two environments. The across environment genetic correlation for body size was significantly below 1 indicating that different genes contributed to this trait in the two environments. Body size may therefore be able to respond to selection independently in the two environments to some extent.

Predator-induced phenotypic plasticity in tadpoles: extension or innovation?

Phenotypic plasticity, the ability of a trait to change as a function of the environment, is central to many ideas in evolutionary biology. A special case of phenotypic plasticity observed in many organisms is mediated by their natural predators. Here, we used a predator-prey system of dragonfly larvae and tadpoles to determine if predator-mediated phenotypic plasticity provides a novel way of surviving in the presence of predators (an innovation) or if it represents a simple extension of the way noninduced tadpoles survive predation. Tadpoles of Limnodynastes peronii were raised in the presence and absence of predation, which then entered a survival experiment. Induced morphological traits, primarily tail height and tail muscle height, were found to be under selection, indicating that predator-mediated phenotypic plasticity may be adaptive. Although predator-induced animals survived better, the multivariate linear selection gradients were similar between the two tadpole groups, suggesting that predator-mediated phenotypic plasticity is an extension of existing survival strategies. In addition, nonlinear selection gradients indicated a cost of predator-induced plasticity that may limit the ability of phenotypic plasticity to enhance survival in the presence of predators.

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.

Stenotherms at sub-zero temperatures: thermal dependence of swimming performance in Antarctic fish.

We examined the burst swimming performance of two Antarctic fishes, Trematomus bernacchii and T. centronotus, at five temperatures between -1 degrees C and 10 degrees C. As Antarctic fishes are considered one of the most cold specialised and stenothermal of all ectotherms, we predicted they would possess a narrow thermal performance breadth for burst swimming and a correlative decrease in performance at high temperatures. Burst swimming was assessed by videotaping swimming sequences with a 50-Hz video camera and analysing the sequences frame-by-frame to determine maximum velocity, the distance moved throughout the initial 200 ms, and the time taken to reach maximum velocity. In contrast to our prediction, we found both species possessed a wide thermal performance breadth for burst swimming. Although maximum swimming velocity for both T. bernacchii and T. centronotus was significantly highest at 6 degrees C, maximum velocity at all other test temperatures was less than 20% lower. Thus, it appears that specialisation to a highly stable and cold environment is not necessarily associated with a narrow thermal performance breadth for burst swimming in Antarctic fish. We also examined the ability of the Antarctic fish Pagothenia borchgrevinki to acclimate their burst-swimming performance to different temperatures. We exposed P. borchgrevinki to either -1 degrees C or 4 degrees C for 4 weeks and tested their burst-swimming performance at four temperatures between -1 degrees C and 10 degrees C. Burst-swimming performance of Pagothenia borchgrevinki was unaffected by exposure to either -1 degrees C or 4 degrees C for 4 weeks. Maximum swimming velocity of both acclimation groups was thermally independent over the total temperature range of 1 degrees C to 10 degrees C. Therefore, the loss of any capacity to restructure the phenotype and an inability to thermally acclimate swimming performance appears to be associated with inhabiting a highly stable thermal environment.

The calibre of the foramen of Panizza in Crocodylus porosus is variable and under adrenergic control.

The foramen of Panizza is located within the outflow tract of the crocodilian heart, between the left and right aortas. It has been suggested that the foramen of Panizza has a variable calibre, which could explain the profound changes in the distribution of flows and pressure profiles recorded in the right and left aortas. We investigated this possibility using a modified in-situ perfused heart preparation in combination with isolated strip preparations from the outflow tract. In the perfused heart preparation, bolus injections of adrenaline increased the resistance in the foramen of Panizza, indicating a decrease in its diameter. Isolated strip preparations from the outflow tract showed a concentration-dependent increase in tension in response to adrenaline, while vasoactive intestinal polypeptide caused a relaxation in adrenaline pre-contracted strip preparations. We propose that an increase in the diameter of the foramen of Panizza may be important during pulmonary to systemic shunts to allow blood to flow from the left to right aorta (reverse foramen flow) in order to supply the carotid and coronary arteries. During non-shunting conditions, a constricted foramen may prevent excess flow from the right to left aorta during diastole.

Nitric oxide, a potent vasodilator of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus.

The effects of five neuropeptides (CGRP, SOM, SP, NPY, VIP), L-NAME (nitric oxide synthase inhibitor), and adrenaline on the contractile tone of the aortic anastomosis in the estuarine crocodile, Crocodylus porosus, were investigated. None of the neuropeptides, which had previously been found to be present in the aortic anastomosis, had any direct effect on the tension developed by ring preparations. L-NAME itself significantly increased the basal tone of the vascular ring preparations, suggesting a tonic release of nitric oxide in the preparation. Adrenaline produced concentration-dependent vasoconstrictions that were counteracted by profound reflex vasodilatations that were susceptible to blockade by L-NAME. Immunohistochemistry revealed the presence of nitric oxide synthase and tyrosine hydroxylase-containing (indicating the presence of a adrenergic innervation) nerve fibres in the adventitia and adventitio-medial border of the aortic anastomosis. These data demonstrate opposing actions of adrenaline and nitric oxide on the vascular smooth muscle in the anastomosis of the C. porosus. The morphology of the anastomosis, with the extremely thick muscular vessel wall, suggests a sphincter-like function for this vessel that could be controlled mainly by adrenergic and nitrergic mechanisms.

Control of heart rate during thermoregulation in the heliothermic lizard Pogona barbata: importance of cholinergic and adrenergic mechanisms.

During thermoregulation in the bearded dragon Pogona barbata, heart rate when heating is significantly faster than when cooling at any given body temperature (heart rate hysteresis), resulting in faster rates of heating than cooling. However, the mechanisms that control heart rate during heating and cooling are unknown. The aim of this study was to test the hypothesis that changes in cholinergic and adrenergic tone on the heart are responsible for the heart rate hysteresis during heating and cooling in P. barbata. Heating and cooling trials were conducted before and after the administration of atropine, a muscarinic antagonist, and sotalol, a beta-adrenergic antagonist. Cholinergic and beta-adrenergic blockade did not abolish the heart rate hysteresis, as the heart rate during heating was significantly faster than during cooling in all cases. Adrenergic tone was extremely high (92.3 %) at the commencement of heating, and decreased to 30.7 % at the end of the cooling period. Moreover, in four lizards there was an instantaneous drop in heart rate (up to 15 beats min(-1)) as the heat source was switched off, and this drop in heart rate coincided with either a drop in beta-adrenergic tone or an increase in cholinergic tone. Rates of heating were significantly faster during the cholinergic blockade, and least with a combined cholinergic and beta-adrenergic blockade. The results showed that cholinergic and beta-adrenergic systems are not the only control mechanisms acting on the heart during heating and cooling, but they do have a significant effect on heart rate and on rates of heating and cooling.

Inability of adult Limnodynastes peronii (Amphibia: Anura) to thermally acclimate locomotor performance.

Despite several studies on adult amphibians, only larvae of the striped marsh frog (Limnodynastes peronii) have been reported to possess the ability to compensate for the effects of cool temperature on locomotor performance by thermal acclimation. In this study, we investigated whether this thermal acclimatory ability is shared by adult L. peronii. We exposed adult L. peronii to either 18 or 30 degrees C for 8 weeks and tested their swimming and jumping performance at six temperatures between 8 and 35 degrees C. Acute changes in temperature affected both maximum swimming and jumping performance, however there was no difference between the two treatment groups in locomotor performance between 8 and 30 degrees C. Maximum swimming velocity of both groups increased from 0.62+/-0.02 at 8 degrees C to 1.02+/-0.03 m s(-1) at 30 degrees C, while maximum jump distance increased from approximately 20 to >60 cm over the same temperature range. Although adult L. peronii acclimated to 18 degrees C failed to produce a locomotor response at 35 degrees C, this most likely reflected a change in thermal tolerance limits with acclimation rather than modifications in the locomotor system. As all adult amphibians studied to date are incapable of thermally acclimating locomotor performance, including adults of L. peronii, this acclimatory capacity appears to be absent from the adult stage of development.

Morphology of the cloaca in the estuarine crocodile, Crocodylus porosus, and its plastic response to salinity.

The cloacal complex of Crocodylus porosus is composed of three chambers (proctodaeum, urodaeum, and coprodaeum) separated by tight, muscular sphincters. The proctodaeum is proximal to the cloacal vent and houses the genitalia. The urodaeum is the largest chamber, is capable of storing large quantities of urine, and is lined with an epithelium with the capacity for transepithelial water and ion exchange. The coprodaeum, the most orad cloacal chamber, is a small, only marginally expandable chamber that has an epithelium composed almost entirely of mucus-secreting cells. The coprodaeum and lower intestine are reported to be the site(s) for urine modification in birds and bladderless lizards. A radiographic trace of urine storage in C. porosus kept for 2 months under hyperosmotic conditions showed no signs of retrograde movement of urine into the coprodaeum or rectum. Instead, urine was stored in the urodaeum of C. porosus. Examination of the mucosal surface of the urodaeum by SEM showed a plastic response to environmental salinity, with a possible increase in surface area in animals kept in hyperosmotic water compared with animals from fresh water. We propose the urodaeum as the primary site for postrenal modification of urine in C. porosus.

Allometric scaling relationships of jumping performance in the striped marsh frog Limnodynastes peronii.

We constructed a force platform to investigate the scaling relationships of the detailed dynamics of jumping performance in striped marsh frogs (Limnodynastes peronii). Data were used to test between two alternative models that describe the scaling of anuran jumping performance; Hill's model, which predicts mass- independence of jump distance, and Marsh's model, which predicts that jump distance increases as M(0.2), where M is body mass. From the force platform, scaling relationships were calculated for maximum jumping force (F(max)), acceleration, take-off velocity (U(max)), mass-specific jumping power (P(max)), total jumping distance (D(J)) and total contact time for 75 L. peronii weighing between 2.9 and 38. 4 g. F(max) was positively correlated with body mass and was described by the equation F(max)=0.16M(0.61), while P(max) decreased significantly with body mass and was described by the equation P(max)=347M(-)(0.46). Both D(J) and U(max) were mass-independent over the post-metamorph size range, and thus more closely resembled Hill's model for the scaling of locomotion. We also examined the scaling relationships of jumping performance in metamorph L. peronii by recording the maximum jump distance of 39 animals weighing between 0.19 and 0.58 g. In contrast to the post-metamorphic L. peronii, D(J) and U(max) were highly dependent on body mass in metamorphs and were described by the equations D(J)=38M(0.53) and U(max)=1.82M(0.23), respectively. Neither model for the scaling of anuran jumping performance resembled data from metamorph L. peronii. Although the hindlimbs of post-metamorphic L. peronii scaled geometrically (body mass exponent approximately 0.33), the hindlimbs of metamorphs showed greater proportional increases with body mass (mass exponents of 0.41-0.42).

Effect of ontogenetic increases in body size on burst swimming performance in tadpoles of the striped marsh frog, Limnodynastes peronii.

The effect of ontogenetic increases in total length on burst swimming performance was investigated in tadpoles of the striped marsh frog (Limnodynastes peronii) over the total-length range of 1. 5-4 cm and Gosner developmental stages 25-38. The burst swimming performance of tadpoles at 10 degrees and 24 degrees C was determined by videotaping startle responses with a high-speed video camera at 200 Hz and analysing the sequences frame by frame. Maximum swimming velocity (Umax) and acceleration (Amax) increased with total length (L) at a rate that was proportionally greater than the increase in total length (i.e., positive allometry; exponents >1) and was described by the allometric equations Umax=0.061L1.34 and Amax=1.15L1.11 at 10 degrees C and Umax=0.114L1.34 and Amax=1.54L1. 11 at 24 degrees C. Stride length increased with a total-length exponent of approximately 1 but was unaffected by temperature. Tail-beat frequency was not affected by total length and increased from 7.8+/-0.2 Hz at 10 degrees C to 21.7+/-0.7 Hz at 24 degrees C. Developmental stage did not significantly influence the relationship between total length and Umax or Amax. Furthermore, temperature and the associated changes in water viscosity did not affect the relationship between total length and burst swimming performance. At their Umax, Reynolds numbers ranged from approximately 1,500 in the smaller tadpoles up to 50,000 for the larger animals at 24 degrees C. We suggest the positive allometry of Umax in larval L. peronii was due in part to the increases in tail width (TW) with total length (TW=-1.36L1.66), possibly reflecting the increasing importance of burst swimming performance to survival during larval development.

Thermal acclimation of locomotor performance in tadpoles of the frog Limnodynastes peronii.

Previous analyses of thermal acclimation of locomotor performance in amphibians have only examined the adult life history stage and indicate that the locomotor system is unable to undergo acclimatory changes to temperature. In this study, we examined the ability of tadpoles of the striped marsh frog (Limnodynastes peronii) to acclimate their locomotor system by exposing them to either 10 degrees C or 24 degrees C for 6 weeks and testing their burst swimming performance at 10, 24, and 34 degrees C. At the test temperature of 10 degrees C, maximum velocity (Umax) of the 10 degrees C-acclimated tadpoles was 47% greater and maximum acceleration (Amax) 53% greater than the 24 degrees C-acclimated animals. At 24 degrees C, Umax was 16% greater in the 10 degrees C-acclimation group, while there was no significant difference in Amax or the time taken to reach Umax (T-Umax). At 34 degrees C, there was no difference between the acclimation groups in either Umax or Amax, however T-Umax was 36% faster in the 24 degrees C-acclimation group. This is the first study to report an amphibian (larva or adult) possessing the capacity to compensate for cool temperatures by thermal acclimation of locomotor performance. To determine whether acclimation period affected the magnitude of the acclimatory response, we also acclimated tadpoles of L. peronii to 10 degrees C for 8 months and compared their swimming performance with tadpoles acclimated to 10 degrees C for 6 weeks. At the test temperatures of 24 degrees C and 34 degrees C, Umax and Amax were significantly slower in the tadpoles acclimated to 10 degrees C for 8 months. At 10 degrees C, T-Umax was 40% faster in the 8-month group, while there were no differences in either Umax or Amax. Although locomotor performance was enhanced at 10 degrees C by a longer acclimation period, this was at the expense of performance at higher temperatures.

Hypoxia stimulates cerebral blood flow in the estuarine crocodile (Crocodylus porosus).

The effect of N2 respiration on cerebral blood flow (CBF) velocity on the dorsal surface of cerebellum was examined in the estuarine crocodile, Crocodylus porosus, using epi-illumination microscopy. Twelve minutes of N2 respiration resulted in a 126% increase in CBF velocity. N2 respiration had no effect on blood pressure, indicating an underlying cerebral vasodilation. In addition, heart rate increased significantly. Systemic injections of aminophylline and the NO synthase (NOS) inhibitor nitro-L-arginine (L-NA) did not affect the hypoxia induced increase in CBF. We conclude that C. porosus responds to hypoxia with adenosine and nitric oxide (NO) independent cerebral vasodilation, and that this is likely to be a mechanism protecting the brain from energy deficiency during prolonged dives.