Changes in selected aspects of immune function in the leopard frog, Rana pipiens, associated with exposure to cold

The effect of exposure to low temperatures (5 °C) on lymphocyte proliferation, leukocyte populations, and serum complement levels was examined in the northern leopard frog, Rana pipiens. Proliferation of T lymphocytes in response to phytohemagglutinin stimulation was significantly decreased in frogs kept for 2, 3, and 5 months at 5 °C compared to that of animals kept at 22 °C. A significant increase in the average percentage of neutrophils and a decrease in the mean percentage of eosinophils was observed in the blood of frogs held for 5 months in the cold compared to animals held at 22 °C for the same length of time. Mean serum complement activity after 1 month at 5 °C was significantly reduced in comparison to animals held at 22 °C and was not detectable after 5 months in the cold. Recovery of complement levels at room temperature (22 °C) was also examined after cold exposure. Complement levels were significantly higher than controls (at 22 °C) in frogs returned to 22 °C for 7 and 14 days after 5 months in the cold. After frogs were held at 5 °C for 1 month, serum complement levels increased significantly within 2 days after returning to 22 °C and continued to rise 5 and 9 days after warming. Injections with Aeromonas hydrophila following a 5-week exposure to 5 °C failed to cause death or observable symptoms of disease in frogs that were returned to 22 °C. Accepted: 20 November 1996     

Pond canopy cover: a resource gradient for anuran larvae

1. The gradient in pond canopy cover strongly influences freshwater species distributions. This study tested the effects of canopy cover on the performance of two species of larval anurans, a canopy cover generalist (Rana sylvatica, the wood frog) and an open‐canopy specialist (R. pipiens, the leopard frog), and tested which factors co‐varying with canopy cover mediate these effects. 2. A field transplant experiment demonstrated that canopy cover had negative performance effects on both species. However, leopard frogs, which grow faster than wood frogs in open‐canopy ponds, were more strongly affected by closed‐canopy pond conditions. 3. Closed‐canopy ponds had lower temperature, dissolved oxygen (DO), and food nutritional quality as indicated by carbon‐to‐nitrogen ratio (C : N) analysis of field‐sampled food types, and of gut contents of transplanted larvae. 4. Laboratory experiments demonstrated that higher temperature and food quality but not DO substantially increased larval growth. However, only food quality increased growth rates of leopard frogs more than wood frogs. 5. The strong correlation of growth rates to gut content C : N in the field, and the similarity of growth curves as a function of resource quality in the field and laboratory, strongly suggest that resources are of primary importance in mediating intraspecific, and especially interspecific differences in performance across the canopy cover gradient.     

Osmotic and metabolic responses to dehydration and urea-loading in a dormant,terrestrially hibernating frog

Physiological responses to dehydration in amphibians are reasonably well documented, although little work has addressed this problem in hibernating animals. We investigated osmotic and metabolic responses to experimental manipulation of hydration state in the wood frog (Rana sylvatica), a terrestrial hibernator that encounters low environmental water potential during autumn and winter. In winter-conditioned frogs, plasma osmolality varied inversely with body water content (range 69–79%, fresh mass) primarily due to increases in sodium and chloride concentrations, as well as accumulation of glucose and urea. Decreased hydration was accompanied by a marked reduction in the resting rate of oxygen consumption, which was inversely correlated with plasma osmolality and urea concentration. In a separate experiment, resting rates of oxygen consumption in fully hydrated frogs receiving injections of saline or saline containing urea did not differ initially; however, upon dehydration, metabolic rates decreased sooner in the urea-loaded frogs than in control frogs. Our findings suggest an important role for urea, acting in concert with dehydration, in the metabolic regulation and energy conservation of hibernating R. sylvatica.     

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: In vivo and in vitro studies

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7 d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7 d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.     

Characterization of γ-glutamyltranspeptidase in the liver of the frog: 3. Response to freezing and thawing in the freeze-tolerant wood frog Rana sylvatica

The freeze tolerant wood frog Rana sylvatica was studied to determine the impact of the freezing and thawing of this frog on the activity of γ-glutamyltranspeptidase in the liver. On exposure to ?2·5°C, for 1, 12 and 24 h, frogs were found to be cool, covered with ice crystals and frozen, respectively. Thawing for 24 h at 4°C recovered the frogs completely. A 45 per cent decrease in the liver weight: body weight ratio was notable after 1 h at ?2·5°C, suggestive of an early hepatic capacitance response. A glycemic response to freezing was observed: blood glucose levels exhibited a 55 per cent decrease after 1 h at ?2·5°C on cooling; a 10·5-fold increase after 12 h at ?2·5°C on the initiation of freezing; and a 22-fold increase after 24 h at ?2·5°C in the fully frozen state. Blood glucose levels remained elevated four-fold in the thawed state. Plasma insulin levels were increased twofold in the frozen state and 1·8-fold in the thawed state, while plasma ketone levels were increased 1·8-fold in the frozen state and 1·5-fold in the thawed state. Plasma total T₃ levels were decreased by 22 per cent in the frozen state and normalized on thawing. In homogenates and plasma membranes isolated from the livers of Rana sylvatica, the activity of γ-glutamyltranspeptidase was found to be elevated at all stages of the freeze–thaw process. After 1, 12 and 24 h at ?2·5°C, activities were increased 2·5-, 2·3-, 2·4-fold respectively in the homogenates and 2·5-, 2·2-, 2·4-fold respectively in the plasma membranes. After thawing, activities were still increased 1·9-fold in both homogenates and plasma membranes. In homogenates prepared from the kidneys of Rana sylvatica, the activity of γ-glutamyltranspeptidase was increased 1·4-fold after 1 h at ?2·5°C after which it returned to normal. The role of thyroid hormone in producing the increase in γ-glutamyltranspeptidase in the liver of Rana sylvatica in response to freezing is discussed as is the significance of the enzyme increase in terms of hepatic cytoprotection and freeze tolerance.     

Regulation of pyruvate kinase in skeletal muscle of the freeze tolerant wood frog,Rana sylvatica

The wood frog (Rana sylvatica) can survive the winter in a frozen state, in which the frog’s tissues are also exposed to dehydration, ischemia, and anoxia. Critical to wood frog survival under these conditions is a global metabolic rate depression, the accumulation of glucose as a cryoprotectant, and a reliance on anaerobic glycolysis for energy production. Pyruvate kinase (PK) catalyzes the final reaction of aerobic glycolysis, generating pyruvate and ATP from phosphoenolpyruvate (PEP) and ADP. This study investigated the effect of each stress condition experienced by R. sylvatica during freezing, including dehydration and anoxia, on PK regulation. PK from muscle of frozen and dehydrated frogs exhibited a lower affinity for PEP (K_m = 0.098 ± 0.003 and K_m = 0.092 ± 0.008) than PK from control and anoxic conditions (K_m = 0.065 ± 0.003 and K_m = 0.073 ± 0.002). Immunoblotting showed greater serine phosphorylation on muscle PK from frozen and dehydrated frogs relative to control and anoxic states, suggesting a reversible phosphorylation regulatory mechanism for PK activity during freezing stress. Furthermore, PK from frozen animals exhibited greater stability under thermal and urea-induced denaturing conditions than PK from control animals. Phosphorylation of PK during freezing may contribute to mediating energy conservation and maintaining intracellular cryoprotectant levels, as well as increase enzyme stability during stress.     

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     

Canopy Closure,Competition, and the Endangered Dusky Gopher Frog

Abstract: A major challenge facing wildlife biologists is understanding why some species go extinct while others persist in the same habitat. To address this question, we investigated whether tree canopy closure over ponds affects growth and survival of rare and common tadpoles within ponds and mediates competitive interactions among species. We conducted 2 experiments to test whether canopy closure and competition may have contributed to the decline of the endangered dusky gopher frog (Rana sevosa), but allowed the persistence of the southern leopard frog (R. sphenocephala). We explored the response of both species to canopy closure in single-species and mixed- (1:1) species treatments of identical total tadpole density. An experiment using aquatic enclosures in temporary ponds showed that canopy closure reduced tadpole growth approximately 20% for both species. Survival of dusky gopher frog tadpoles was higher in mixed-species enclosures than in single-species enclosures. In a complementary experiment using artificial ponds, dusky gopher frogs had lower survival to metamorphosis, reduced size at metamorphosis, and produced a lower total biomass of metamorphosed juveniles in shaded ponds. Southern leopard frogs exhibited reduced body size at metamorphosis only when shaded. These studies suggest that pond canopy closure, not larval competition, may be contributing to the decline of the dusky gopher frog. The different responses to canopy closure suggest a potential mechanism for the loss of dusky gopher frogs and the persistence of southern leopard frogs. Removal of trees from historically open-canopy ponds may help facilitate the recovery of dusky gopher frogs and benefit similar species.     

Detoxification of environmental sulfide to sulfane sulfur in the intertidal sipunculid Phascolosoma arcuatum

The capability of Phascolosoma arcuatum to detoxify sulfide in anaerobic conditions was examined. Sulfane sulfur, which underwent cold cyanolysis, was the major excretory end product of sulfide detoxification during anoxia. Thiosulfate was not excreted into the external medium. Instead, it was absorbed by P. arcuatum and its absorption was stimulated by the presence of sodium sulfide (Na₂S) in the incubation medium. The effective formation and excretion of sulfane sulfur by P.␣arcuatum required the presence of both Na₂S and sodium thiosulfate (Na₂S₂O₃). Results obtained indicate that rhodanese might be involved in sulfide detoxification in this sipunculid. Rhodanese could act as a catalyst in the transfer of sulfur atoms from thiosulfate to HS⁻. The body wall and the introvert were the main sites of sulfide detoxification. However, it is unlikely that epibiotic bacteria associated with the outside surface of the worm were involved in the detoxification process. A time-course study on the contents of thiosulfate and sulfane sulfur in the body wall of P. arcuatum incubated anaerobically in the presence of Na₂S + Na₂S₂O₃ verified that thiosulfate absorbed was utilized to detoxify sulfide to sulfane sulfur. Accepted: 24 October 1996     

Crosstalk between cAMP and pheromone signalling pathways in Ustilago maydis

In the phytopathogenic basidiomycete Ustilago maydis mating and dikaryon formation are controlled by a pheromone/receptor system and the multiallelic b locus. Recently, a gene encoding a G protein α subunit, gpa3, was isolated and has subsequently been implicated in pheromone signal transduction. Mutants deleted for gpa3 are sterile and nonpathogenic, and exhibit a morphology that is similar to that of mutants with defects in the adenylate cyclase gene uac1. We have found that the sterility and mutant morphology of gpa3 deletion strains can be rescued by exogenous cAMP. In these mutants and in the corresponding wild-type strains, exogenous cAMP stimulates pheromone gene expression to a level comparable to that seen in the pheromone-stimulated state. In addition, we demonstrate that uac1 is epistatic to gpa3. We conclude that Gpa3 controls the cAMP signalling pathway in U.maydis and discuss how this pathway feeds into the pheromone response. Received: 4 May 1998 / Accepted: 24 July 1998     

High vapour pressure deficit exacerbates xylem cavitation and photoinhibition in shade-grown Piper auritum H.B. & K. during prolonged sunflecks

Water relations dynamics during simulated sunflecks at high (36°C) and medium (27°C) temperatures and high and low vapour pressure deficits beween leaf and air (VPD) were studied on shade-grown Piper auritum H.B. & K. plants, a pioneer tree, common in gaps and clearings of tropical rain forests. The leaves of P. auritum wilt rapidly when exposed to high light. Exposure to high VPD and high light caused substantial and rapid dehydration of leaves. Dehydration could be prevented under high humidity irrespective of temperature. Water stored in leaf cells served as initial source for transpiration upon high light exposure. This effect increased with increasing VPD and temperature. The pronounced decrease in leaf water content over time in high light caused a rapid decrease in leaf water potential (Ψ_l) and a concomitant increase in water potential gradient (ΔΨ/Δx) between trunk and leaf, yet the high leaf elasticity (small bulk elastic modulus, ε) allowed turgor maintenance under most conditions. Under high VPD and high temperature, stomata remained open and ΔΨ/Δx frequently exceeded 0.95 MPa · m⁻¹, the cavitation-inducing threshold (ΔΨ/Δx _cav) causing high rates of acoustic emissions from stems and leaf petioles and leading to concomitant losses in hydraulic conductance per leaf area (k _l). At medium temperature (high VPD), stomatal closure contained xylem embolism by keeping ΔΨ/Δx at or below this threshold. We argue that wilting substantially contributes to creating a sufficient driving force for water uptake from the soil, and reducing the VPD (through a decrease in radiation load and thus leaf temperature) to avoid excessive dehydration. Received: 3 March 1996 / Accepted: 10 November 1996     

Effects of hydration state on hormonal and renal responses during moderate exercise in the heat

The effects of hydromineral hormones and catecholamines on renal concentrating ability at different hydration states were examined in five male volunteers while they performed three trials. Each of these trials comprised a 60-min exercise bout on a treadmill (at 50% of maximal oxygen uptake) in a warm environment (dry bulb temperature, 35°C; relative humidity, 20–30%). In one session, subjects were euhydrated before exercise (C). In the two other sessions, after thermal dehydration (loss of 3% body mass) which markedly reduced plasma volume (PV) and increased plasma osmolality (osm_pl), the subjects exercised either not rehydrated (Dh) or rehydrated (Rh) by drinking 600 ml of mineral water before and 40 min after the onset of exercise. During exercise in the Dh compared to C state, plasma renin, aldosterone, arginine vasopressin (AVP), noradrenaline and adrenaline concentrations were increased (P < 0.05). A reduction in creatinine clearance and urine flow was also observed (P < 0.05) together with a decrease in urine osmolality, osmolar clearance and sodium excretion, while free water clearance increased (P < 0.05). However, compared to Dh, Rh partially restored PV and osm_pl and induced a marked reduction in the time courses of both the plasma AVP and catecholamine responses (P < 0.05). Values for renal water and electrolyte excretion were intermediate between those of Dh and C. Plasma atrial natriuretic peptide presented similar changes whatever the hydration state. These results demonstrate that during moderate exercise in the heat, renal concentrating ability is paradoxically reduced by prior dehydration in spite of high plasma AVP levels, and might be the result of marked activation of the sympatho-adrenal system. Rehydration, by reducing this activation, could partially restore the renal concentrating ability despite the lowered plasma AVP. Accepted: 23 April 1997     

Stress-induced antioxidant defense and protein chaperone response in the freeze-tolerant wood frog <Emphasis Type="Italic">Rana sylvatica</Emphasis>

Freeze tolerance is an adaptive response utilized by the wood frog Rana sylvatica to endure the sub-zero temperatures of winter. Survival of whole body freezing requires wood frogs to trigger cryoprotective mechanisms to deal with potential injuries associated with conversion of 65–70% of total body water into ice, including multiple consequences of ice formation such as cessation of blood flow and cell dehydration caused by water loss into ice masses. To understand how wood frogs defend against these stressors, we measured the expression of proteins known to be involved in the antioxidant defense and protein chaperone stress responses in brain and heart of wood frogs comparing freezing, anoxia, and dehydration stress. Our results showed that most stress proteins were regulated in a tissue- and stress-specific manner. Notably, protein levels of the cytosolic superoxide dismutase (SOD1) were upregulated by 1.37?±?0.11-fold in frozen brain, whereas the mitochondrial SOD2 isoform rose by 1.38?±?0.37-fold in the heart during freezing. Catalase protein levels were upregulated by 3.01?±?0.47-fold in the brain under anoxia stress, but remained unchanged in the heart. Similar context-specific regulatory patterns were also observed for the heat shock protein (Hsp) molecular chaperones. Hsp27 protein was down-regulated in the brain across the three stress conditions, whereas the mitochondrial Hsp60 was upregulated in anoxic brain by 1.73?±?0.38-fold and by 2.13?±?0.57-fold in the frozen heart. Overall, our study provides a snapshot of the regulatory expression of stress proteins in wood frogs under harsh environment conditions and shows that they are controlled in a tissue- and stress-specific manner.     

Novel tetranucleotide microsatellite DNA markers for the wood frog,Rana sylvatica

Fifteen tetranucleotide microsatellite loci were identified and characterized for wood frogs (Rana sylvatica) collected from three vernal pools in the southeastern US. These markers revealed a high degree of genetic diversity (nine to 34 alleles per locus), heterozygosity (30.6–92.3%) and allelic heterogeneity (69% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.40 and 0.55 and all F_ST values (0.02–0.05) were statistically significant. Genotypic assignment tests correctly classified 103 of 113 individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and metapopulation dynamics.     

Past and Present Risk: Exposure to Predator Chemical Cues Before and after Metamorphosis Influences Juvenile Wood Frog Behavior

Animals are exposed to different predators over their lifespan. This raises the question of whether exposure to predation risk in an early life stage affects the response to predators in subsequent life stages. In this study, we used wood frogs (Rana sylvatica) to test whether exposure to cues indicating predation risk from dragonfly larvae during the wood frog larval stage affected post‐metamorphic activity level and avoidance of garter snake chemical cues. Dragonfly larvae prey upon wood frogs only during the larval stage, whereas garter snakes prey upon wood frogs during both the larval stage and the post‐metamorphic stage. Exposure to predation risk from dragonflies during the larval stage caused post‐metamorphic wood frog juveniles to have greater terrestrial activity than juvenile wood frogs that were not exposed to larval‐stage predation risk from dragonflies. However, exposure to predation risk as larvae did not affect juvenile wood frog responses to chemical cues from garter snakes. Wood frogs exposed as larvae to predation risk from dragonfly larvae avoided garter snake chemical cues to the same extent as wood frog larvae not exposed to predation risk from dragonfly larvae. Our results demonstrate that while some general behaviors exhibit carry‐over effects from earlier life stages, behavioral responses to predators may remain independent of conditions experienced in earlier life stages.     

Regulation of cell cycle components during exposure to anoxia or dehydration stress in the wood frog, Rana sylvatica

The wood frog (Rana sylvatica) exhibits a well-developed natural anoxia and dehydration tolerance. The degree of stress tolerance depends on numerous biochemical adaptations, including stress-induced hypometabolism that helps to preserve long-term viability by reducing ATP demand. We hypothesized that the mechanisms involved in cell cycle control could act to aid in the establishment of the hypometabolic state required for stress survival. Selected proteins involved in the proliferation of cells were evaluated using immunoblotting in liver and skeletal muscle of wood frogs comparing controls with animals subjected to either 24-hr anoxia exposure under a nitrogen gas atmosphere or dehydration to 40% of total body water lost (all at 5°C). Levels of cyclins (type A, B, D, and E) decreased significantly under both stresses in liver and skeletal muscle. Similar reductions were seen for Cyclin-dependant kinases (Cdk) types 2, 4, and 6 in both liver and skeletal muscle; however, an increase in the relative amount of phosphorylated inactive p-Cdk (Thr14/Tyr15) was observed in liver under both stresses. Levels of positive regulators of Cdk activity (Cdc25 type A and C) were significantly reduced in both tissues under both stresses, whereas negative regulators of Cdk activity (p16(INK4a) and p27(KIP1) ) increased significantly in liver under both anoxia and dehydration stress (but not in muscle). This study provides the first report of differential regulation of cell cycle components in an anoxia and dehydration tolerant vertebrate, the wood frog, suggesting that cell cycle suppression is an active part of stress resistance and life extension in hypometabolic states.     

Sensory modulation and behavioral choice during feeding in the Australian frog, Cyclorana novaehollandiae

This study investigates how visual and tactile sensory information, as well as biomechanical effects due to differences in physical characteristics of the prey, influence feeding behavior in the frog Cyclorana novaehollandiae. Video motion analysis was used to quantify movement patterns produced when feeding on five prey types (termites, waxworms, crickets, mice and earthworms). Twelve kinematic variables differed significantly among prey types, and twelve variables were correlated with prey characteristics (including mass, length, height and velocity of movement). Results indicate that C.␣novaehollandiae uses a different strategy to capture each prey type. Visual assessment of prey characteristics appeared to be more important in modulating feeding behavior than tactile cues or biomechanical effects. We propose a hierarchical hypothesis of behavioral choice, in which decisions are based primarily on visual analysis of prey characteristics. In this model, the frogs first choose between jaw prehension and tongue prehension based on prey size. If they have chosen jaw prehension, they next choose between upward or downward head rotation based on length and height of the prey. If they have chosen tongue prehension, they next choose between behavior for fast and slow prey. Final decisions may be the result of behavioral fine tuning based on tactile feedback. Accepted: 5 August 1996