Neural correlates of swimming behavior in Melibe leonina

The nudibranch Melibe leonina swims by rhythmically bending from side to side at a frequency of 1 cycle every 2-4 s. The objective of this study was to locate putative swim motoneurons (pSMNs) that drive these lateral flexions and determine if swimming in this species is produced by a swim central pattern generator (sCPG). In the first set of experiments, intracellular recordings were obtained from pSMNs in semi-intact, swimming animals. About 10-14 pSMNs were identified on the dorsal surface of each pedal ganglion and 4-7 on the ventral side. In general, the pSMNs in a given pedal ganglion fired synchronously and caused the animal to flex in that direction, whereas the pSMNs in the opposite pedal ganglion fired in anti-phase. When swimming stopped, so did rhythmic pSMN bursting; when swimming commenced, pSMNs resumed bursting. In the second series of experiments, intracellular recordings were obtained from pSMNs in isolated brains that spontaneously expressed the swim motor program. The pattern of activity recorded from pSMNs in isolated brains was very similar to the bursting pattern obtained from the same pSMNs in semi-intact animals, indicating that the sCPG can produce the swim rhythm in the absence of sensory feedback. Exposing the brain to light or cutting the pedal-pedal connectives inhibited fictive swimming in the isolated brain. The pSMNs do not appear to participate in the sCPG. Rather, they received rhythmic excitatory and inhibitory synaptic input from interneurons that probably comprise the sCPG circuit.     

Different functions for homologous serotonergic interneurons and serotonin in species-specific rhythmic behaviours

Closely related species can exhibit different behaviours despite homologous neural substrates. The nudibranch molluscs Tritonia diomedea and Melibe leonina swim differently, yet their nervous systems contain homologous serotonergic neurons. In Tritonia, the dorsal swim interneurons (DSIs) are members of the swim central pattern generator (CPG) and their neurotransmitter serotonin is both necessary and sufficient to elicit a swim motor pattern. Here it is shown that the DSI homologues in Melibe, the cerebral serotonergic posterior-A neurons (CeSP-As), are extrinsic to the swim CPG, and that neither the CeSP-As nor their neurotransmitter serotonin is necessary for swim motor pattern initiation, which occurred when the CeSP-As were inactive. Furthermore, the serotonin antagonist methysergide blocked the effects of both the serotonin and CeSP-As but did not prevent the production of a swim motor pattern. However, the CeSP-As and serotonin could influence the Melibe swim circuit; depolarization of a cerebral serotonergic posterior-A was sufficient to initiate a swim motor pattern and hyperpolarization of a CeSP-A temporarily halted an ongoing swim motor pattern. Serotonin itself was sufficient to initiate a swim motor pattern or make an ongoing swim motor pattern more regular. Thus, evolution of species-specific behaviour involved alterations in the functions of identified homologous neurons and their neurotransmitter.     

Neuroethology of Melibe leonina Swimming Behavior

The nudibranch Melibe leonina swims by rhythmically flexingits body from side to side at a frequency of 1 cycle every 2–5sec. Melibe swim spontaneously, when they are dislodged fromthe substrate, or when they come in contact with predatory seastars,such as Pycnopodia helianthoides. Intracellular recordings obtainedfrom semi-intact swimming Melibe reveal a population of 15 swimmotoneurons (SMNs) in each pedal ganglion. In general, SMNsin one pedal ganglion fire out-of-phase with SMNs in the oppositepedal ganglion, resulting in rhythmic side-to-side bending movements.In isolated brains, recordings from SMNs yield similar results,indicating the existence of a swim central pattern generator(CPG). There is no evidence for synaptic interactions betweenSMNs and either inhibiting or exciting SMNs has no impact onthe swim pattern. The SMNs are driven by a CPG consisting of4 interneurons; 2 in the cerebropleural ganglia and 1 in eachpedal ganglion. Appropriate bursting activity in the swim interneuronsis necessary for swimming to occur. Either hyperpolarizationor depolarization of any of the 4 CPG interneurons disruptsthe normal swim pattern. Swimming behavior, and the fictiveswim motor program expressed by the isolated brain, are inhibitedby light and nitric oxide donors. NADPH-diaphorase stainingand nitric oxide synthase (NOS) immunocytochemistry of Melibebrains suggests the source of nitric oxide might be a pair ofbilaterally symmetrical cells located in the cerebropleuralganglia.     

Serotonin influences locomotion in the nudibranch mollusc Melibe leonina

Serotonin (5-HT) influences locomotion in many animals, from flatworms to mammals. This study examined the effects of 5-HT on locomotion in the nudibranch mollusc Melibe leonina (Gould, 1852). M. leonina exhibits two modes of locomotion, crawling and swimming. Animals were bath-immersed in a range of concentrations of 5-HT or injected with various 5-HT solutions into the hemolymph and then monitored for locomotor activity. In contrast to other gastropods studied, M. leonina showed no significant effect of 5-HT on the distance crawled or the speed of crawling. However, the highest concentration (10(-3) mol l(-1) for bath immersion and 10(-5) mol l(-1) for injection) significantly increased the time spent swimming and the swimming speed. The 5-HT receptor antagonist methysergide inhibited the influence of 5-HT on the overall amount of swimming but not on swimming speed. These results suggest that 5-HT influences locomotion at the behavioral level in M. leonina. In conjunction with previous studies on the neural basis of locomotion in M. leonina, these results also suggest that this species is an excellent model system for investigating the 5-HT modulation of locomotion.     

Modulation of swimming in Tritonia: excitatory and inhibitory effects of serotonin

1. In the mollusc Tritonia escape swimming is produced by a network of central pattern generator (CPG) neurons. The purpose of this study was to determine which neurotransmitters might be involved in the swim system.
2. Injection of serotonin (5HT) into whole animals elicited swimming followed by a long-lasting inhibition of swimming. In isolated brain preparations, bath-applied 5HT elicited a swim pattern at short latency and also caused a long-lasting inhibition of the swim pattern. The activation of swimming by 5HT was associated with a tonic depolarization of cerebral cell 2 (C2) and the dorsal swim interneurons (DSI) which form part of the swim CPG network.
3. In isolated brain preparations, bath applied glycine, histamine, proctolin, and FMFRamide had no effect on the swim motor pattern elicited by electrical stimulation of a peripheral nerve. Aspartate, carbacol, dopamine, glutamate, octopamine, pilocarpine, and small cardioactive peptide-B (SCP_B) inhibited the activation of swimming by nerve stimulation.
4. The 5HT antagonists cyproheptidine, tryptamine, and 7-methyltryptamine had no effect on swimming, but methysergide and fenfluramine inhibited swimming to both normal sensory stimuli and exogenously applied 5HT.
5. Staining with a polyclonal antibody indicated that one class of CPG neurons, the dorsal swim interneurons (DSI), was immunoreactive for 5HT.
6. Taken together, the data suggest that pattern generator interneurons, particularly the DSIs, use 5HT as a neurotransmitter.

     

Initiation of swimming activity by trigger neurons in the leech subesophageal ganglion

The aim of this study was to identify neurons in the subesophageal ganglion of the medicinal leech which initiate swimming activity and to determine their output connections. We found two bilaterally symmetrical pairs of interneurons, Tr1 and Tr2, located in the first division of the subesophageal ganglion which initiate swimming activity in the isolated nervous system when depolarized with brief (1-3 s) current pulses. Tr1 and Tr2 are considered trigger neurons because elicited swimming episodes outlast the stimulus duration, and because the length of elicited swim episodes is nearly independent of the intensity with which Tr1 and Tr2 are stimulated. Tr1 and Tr2 have similar morphologies. The neurites of both cells cross contralaterally in the subesophageal ganglion, project posteriorly, and exit the subesophageal ganglion in the contralateral connective. The axons of Tr1 and Tr2 extend as far posterior as segmental ganglion 18 of the ventral nerve cord. Tr1 provides direct excitatory drive to three groups of segmental neurons which are capable of initiating swimming: swim-initiating interneurons (cells 204 and 205), serotonin-containing interneurons (cells 61 and 21), and the serotonergic Retzius cells. In addition, all Retzius cells in the subesophageal ganglion are excited directly by Tr1. These three groups of neurons are excited even if Tr1 stimulation is subthreshold for swim initiation. In contrast to Tr1, Tr2 stimulation evokes transient inhibition in swim-initiating and serotonin-containing interneurons, and has little immediate effect on Retzius cells. In addition, Tr2 indirectly inhibits several oscillator neurons, including cells 208, 33, and 60. When Tr1 is stimulated during a swimming episode the swim period decreases for several cycles, while stimulation of Tr2 during swimming episodes reliably resets the ongoing swimming rhythm. Our findings indicate that Tr1 and Tr2 are trigger neurons which initiate swimming activity by different pathways. These neurons also have functional interactions with the swim oscillator network since either Tr1 or Tr2 stimulation during swimming can modulate the ongoing swimming rhythm.     

Evidence that the Central Pattern Generator for Swimming in Tritonia Arose from a Non-Rhythmic Neuromodulatory Arousal System: Implications for the Evolution of Specialized Behavior

Comparisons of the nervous systems of closely related invertebratespecies show that identified neurons tend to be highly conservedeven though the behaviors in which they participate vary. Allopisthobranch molluscs examined have a similar set of serotonin-immunoreactiveneurons located medially in the cerebral ganglion. In a smallnumber of species, these neurons have been physiologically andmorphologically identified. In the nudibranch, Tritonia diomedea,three of the neurons (the dorsal swim interneurons, DSIs) havebeen shown to be members of the central pattern generator (CPG)underlying dorsal/ventral swimming. The DSIs act as intrinsicneuromodulators, altering cellular and synaptic properties withinthe swim CPG circuit. Putative homologues of the DSIs have beenidentified in a number of other opisthobranchs. In the notaspid,Pleurobranchaea californica, the apparent DSI homologues (As1–3)play a similar role in the escape swim and they also have widespreadactions on other systems such as feeding and ciliary locomotion.In the gymnosomatid, Clione limacina, the presumed homologousneurons (Cr-SP) are not part of the swimming pattern generator,which is located in the pedal ganglia, but act as extrinsicmodulators, responding to noxious stimuli and increasing thefrequency of the swim motor program. Putative homologous neuronsare also present in non-swimming species such as the anaspid,Aplysia californica, where at least one of the cerebral serotonergicneurons, CC3 (CB-1), evokes neuromodulatory actions in responseto noxious stimuli. Thus, the CPG circuit in Tritonia appearsto have evolved from the interconnections of neurons that arecommon to other opisthobranchs where they participate in arousalto noxious stimuli but are not rhythmically active.     

Intracellular stimulation of sensory cells elicits swimming activity in the medicinal leech

Intracellular stimulation of each of three different types of mechanoreceptors, the T, P and N cells, evokes swimming behavior in leech preparations. Stimulation of an individual N cell or P cell evoked swimming in 75% and 53% respectively, of the preparations tested. Stimulation of an individual T cell was ineffective in eliciting swimming; however, simultaneous stimulation of two T cells evoked swimming in 59% of our preparations. Stimulation of mechanosensory neurons elicited swimming activity for a limited number of trials; i.e. the response habituated. The number of swim episodes evoked before habituation to criterion did not differ significantly for the different types of mechanoreceptors. The duration of swim episodes declined significantly over the course of N cell stimulation. The tendency for swim length to decline with repeated stimulation was present as well for swim episodes elicited by P or T cell stimulation. Swim initiation recovered spontaneously following habituation resulting from T cell stimulation. Spontaneous recovery following N cell stimulation was not demonstrated. However, N cell stimulation evoked swimming again after DP nerve shock or to a limited extent, after cell 204 stimulation. Spontaneous recovery of swim initiation to P cell stimulation was not investigated. A previous study detailed habituation of swimming activity to mechanical stimulation of the body wall (Debski and Friesen 1985). Only the T cells are activated significantly by this stimulus. Stimulation of sensory receptors other than mechanoreceptors was not effective in eliciting swimming in our preparation. We conclude that T cells mediate swim initiation elicited by stroking of the body wall and that the cessation of swimming to this stimulus is not due to sensory adaptation.     

Swimming ability in three Costa Rican dry forest rodents

We investigated the swimming abilities of three Costa Rican dry forest rodents (Coues' rice rat. Oryzomys couesi, hispid cotton rat, Sigmodon hispidus, and spiny pocket mouse, Liomys salvini) associated with a large marsh, Laguna Palo Verde, using 90 s swim trials in a plastic container. Swimming ability was evaluated by observing the use of limbs and tail in the water, inclination to the surface, and diving and floating behavior. Rice rats could float, swim and dive, suggesting that they can exploit surface and underwater resources. Cotton rats swam at the water's surface, but were less skilled swimmers than rice rats. Spiny pocket mice tired quickly and had difficulty staying at the water's surface. Results suggest that differential swimming ability is related to the distribution of the three sympatric species within the marsh and adjacent forest habitats.     

Effects of site of tactile stimulation on the escape swimming responses of hatchling Xenopus laevis embryos

Hatchling Xenopus laevis embryos usually swim when the skin is touched with a fine hair. Less common are small, local V-flexions and more general C-flexions. Simple flexions or the initial flexion at the start of swimming occur predominantly on the opposite side to the stimulus to direct the animal away from the stimulus. Strokes to the midline lead to random sidedness of responses.
The reliability of the sidedness of flexions and the first flexions of swimming decreases the more rostrally the stimuli are given. The range of directions of swimming paths are larger with more rostral stimuli so responses to head stimuli are unpredictable in direction.
In animals immobilized in α-bungarotoxin, strokes to the skin produce electrically recorded motor output which corresponds to: V-flexions, C-flexions and swimming. Fictive activity generally starts on the side opposite to the stimulus. The fictive responses suggest that the three basic behaviour patterns observed can be generated entirely within the central nervous system without any sensory feedback.
We discuss possible mechanisms for the generation of 'protean' responses to head stimulation which are unpredictable in direction.     

Patterns of EMG activity of rat plantaris muscle during swimming and other locomotor activities

The purpose of the study was to examine the patterns of electromyographic (EMG) activity of the rat plantaris during loaded swimming in comparison with other locomotor activities. Five female Sprague-Dawley rats were implanted with chronic bipolar electrodes in the plantaris muscle of the left hindlimb under pentobarbital anesthesia. Characteristics of EMG bursts recorded while the conscious rat was performing treadmill walking (0.24 m/s) were stable and reproducible 10-14 days postsurgery. Following this stabilization period, records of EMG activity were obtained during walking, loaded swimming (6.5 g attached to tail), and several other locomotor tasks. Compared to walking, EMG bursts during loaded swimming were significantly higher (67%) in maximum amplitude, one-third as long in duration, and occurred at a greater rate (4.4 vs. 1.7 bursts/s, P less than 0.05). Swimming bursts were of higher amplitudes than those of all other activities examined and reached 65% of the EMG amplitude recorded following stimulation of the sciatic nerve with supramaximal voltage. The addition of a mass to the animal's tail during swimming did not increase the EMG burst amplitudes but resulted in a higher frequency of bursts. Compared with treadmill walking, loaded swimming elicited burst of high variability in amplitude. Swimming in the rat involves rapid, extensive activation of plantaris, thus providing an exercise model to study the adaptability of the neuromuscular system to prolonged activity of this type.     

Differential metabolic capacity of mice selected for magnitude of swim stress-induced analgesia.

Maximum oxygen consumption (Vo(2)) elicited by swimming in 20 degrees C water or by exposure to -2.5 degrees C in helium-oxygen (Helox) atmosphere is higher in mice selected for low (LA) than for high (HA) stress-induced analgesia (SIA) produced by swimming. However, this line difference is greater with respect to swim- than to cold-elicited Vo(2). To study the relationship between the analgesic and thermogenic mechanisms, we acclimated HA and LA mice to 5 degrees C or to daily swimming at 20 or 32 degrees C. Next, the acclimated mice were exposed to a Helox test at -2.5 degrees C and to a swim test at 20 degrees C to compare Vo(2) and hypothermia (DeltaT). Cold acclimation raised Vo(2) and decreased DeltaT. These effects were similar in both lines in the Helox test but were smaller in the HA than in the LA line in the swim test. HA and LA mice acclimated to 20 or 32 degrees C swims increased Vo(2) and decreased DeltaT elicited by swimming, but only HA mice acclimated to 20 degrees C swims increased Vo(2) and decreased DeltaT in the Helox test. We conclude that the between-line difference in swim Vo(2) results from a stronger modulation of thermogenic capacities of HA mice by a swim stress-related mechanism, resulting in SIA. We suggest that the predisposition to SIA observed in laboratory as well as wild animals may significantly affect both the results of laboratory measurements of Vo(2) and the interpretation of its intra- and interspecific variation.     

Cardiovascular and sympathoadrenal responses to stress in swim-trained rats

Chronic exposure to swim stress (i.e., training) is associated with functional adaptations of the cardiovascular system. On the other hand, repeated exposure to tail shock, an emotional stress, often results in deleterious changes in resting blood pressure and myocardial pathology. We hypothesized that the pathological adaptation following chronic exposure to tail shock was associated with a larger acute physiological response compared with swim stress. Therefore, acute responses to swim and shock stress were compared. A second concern of this study examined the extent to which adaptation to swim training influences responses to predictable tail shock stress. The cardiovascular and sympathoadrenal responses to swim stress, using 1% body wt attached to the tail, were compared with predictable tail shock (0.2-0.4 mA intensity, 1-s duration, 1/min) in two groups of Long-Evans male rats. In the first, 11 rats were studied following 5-7 wk of swim training, consisting of daily 1-h sessions of swimming with 2% body wt attached to their tails. They were compared with an age-matched nontrained (NT) group (n = 8). During swimming, the trained animals showed significantly lower heart rate (387 +/- 10 vs. 449 +/- 18 beats/min) and significantly lower lactate (0.9 +/- 0.09 vs. 2.0 +/- 0.24 mmol/l), epinephrine (332 +/- 57 vs. 739 pg/ml), and corticosterone (32 +/- 10 vs. 62 +/- 9 micrograms/dl) responses. Systolic and diastolic blood pressures were elevated in swim stress by the same degree in trained (167/110 mmHg) and NT (177/116 mmHg) rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Factors influencing swimming in bay scallops, Argopectenirradians (Lamarck, 1819)

Factors influencing the probability, distance, and direction of swimming in bay scallops (Argopectenirradions Lamarck, 1819) were studied through a series of experimental releases in the field and in a 3-m tank. The probability of a scallop swimming was significantly influenced by the type of substratum on which it was released (sand vs. grassbed), by contact with two natural gastropod predators (Murex, Fasciolaria), and by the amount of rest allowed after a previous swim. The horizontal distance traveled by a swimming scallop was significantly influenced by artificial weight of a magnitude equivalent to a normal load of shell-encrusting organisms, by the amount of rest allowed after a previous swim, by the height attained in the water column, and by the scallop's size. The direction of scallop swimming was significantly influenced by the location along the mantle edge where a predator was contacted, and by factors probably related to the asymmetrical water flow pattern through the mantle cavity. Swimming in bay scallops apparently serves to maintain position in grassbeds and to avoid predators.     

Central generation of swimming activity in the hydrozoan jellyfish Aequorea aequorea

Swimming in Aequorea is controlled by a network of electrically coupled neurons (swim motorneurons) located in the inner nerve ring. The network is made up of the largest neurons in the ring, up to 22 microns in diameter. Intracellular recordings from swim motorneurons reveal slow membrane potential oscillations and a superimposed barrage of synaptic "noise." The synaptic noise, but not the slow oscillations, is eliminated in seawater containing an elevated Mg++ concentration. The swim motorneurons produce a rapid burst of two to eight action potentials preceding each contraction of the subumbrella. Spontaneous bursting persists in high-Mg++ seawater. Injected ramp currents indicated a "bursty" character of the swim motorneurons as suprathreshold depolarizations produced repetitive bursting with an increasing burst frequency with increased depolarization. Hyperpolarizing currents locally blocked spiking in swim motorneurons. Intercellular coupling was demonstrated with Lucifer Yellow injection and dual electrode recordings. In dye fills, only the large neurons of the inner nerve ring were dye-coupled. Two pieces of evidence suggest that swim motorneurons activate the overlying epithelial cells via chemical synapses. First, direct synaptic connections have been noted in ultrastructural examination of the inner nerve ring region. Second, dual recordings from a swim motorneuron and an epithelial cell reveal a 1:1 correspondence between neuron spikes and epithelial synaptic potentials. The synaptic potentials occur with a latency as short as 3 ms which is constant in any one recording session. The results suggest that the swim motorneuron network of Aequorea not only performs a motorneuron function, but also serves as the pattern generator for swimming activity.     

The influence of light on locomotion in the gastropod melibe leonina

In this study, we investigated the effects of light on both the locomotion of intact animals and the swim motor program expressed by isolated brains in the gastropod Melibe leonina. Spontaneous locomotion (crawling and swimming) was examined during a period of natural lighting (L:D) to establish normal behavior, and then under two different light regimes: constant darkness (D:D) and constant light (L:L). In L:D, there was significantly more locomotor activity at night than during the day and this pattern continued in D:D. However, in L:L, activity was substantially reduced at all times. Using isolated brain preparations, we further demonstrated that the swim motor program was rapidly inhibited by light, and that this inhibition was mediated by the eyes. These results indicate that M. leonina displays a nocturnal activity pattern, and that light has a strong inhibitory effect on locomotion in the intact animal and on the swim motor program expressed by the isolated brain.     

The recovery of locomotory activity following exhaustive exercise in juvenile rainbow trout (Oncorhynchus mykiss)

This study investigated the recovery of locomotory activity in exhausted juvenile rainbow trout (Oncorhynchus mykiss, approximately 6-10-cm fork length) in response to two conditions: (1) direct transfer to a range of velocities (0-15 cm s(-1)) in a swim flume (forced swimming) and (2) direct transfer to a pool downstream of a swim channel where a choice of velocities was presented: 2-3 cm s(-1) in the lower half of the pool, a range of velocities from 7 to 40 cm s(-1) in the upper half the pool near the channel entrance, and a velocity of 57 cm s(-1) in a swim channel emptying into the pool (volitional swimming). Exhausted trout showed a pronounced delay in the recovery of normal locomotory activity. With forced swimming, the time required to resume swimming was inversely proportional to water velocity. At 15 cm s(-1), almost all exhausted fish recovered immediately, whereas it took about 1 h for recovery at a current of 5 cm s(-1). In contrast, nonexhausted fish responded to imposed velocity with immediate rheotactic responses (orientation and station holding) at all test velocities. In voluntary swim trials, exhausted trout showed a marked preference for holding station in current in the downstream pool (approximately 11 cm s(-1)) but took, on average, 2 h longer than nonexhausted fish to make transits in the swim channel. Moreover, their ground speed in the swim channel was significantly slower. We conclude that swimming performance is impaired for at least 6 h by exhaustive exercise. Maladaptive behaviors during this time include a preference for current near the surface over cover and a reduced capacity for burst activity, both of which would translate into greater predation risk and reduced ability to forage.     

The effects of temperature and swimming speed on instantaneous fuel use and nitrogenous waste excretion of the Nile tilapia.

The effects of acclimation temperature (30 degrees, 20 degrees, and 15 degrees C) and swimming speed on the aerobic fuel use of the Nile tilapia (Oreochromis niloticus; 8-10 g, 8-9-cm fork length) were investigated using a respirometric approach. As acclimation temperature was decreased from 30 degrees C to 15 degrees C, resting oxygen consumption (Mo2) and carbon dioxide excretion (Mco2) decreased approximately twofold, while nitrogenous waste excretion (ammonia-N plus urea-N) decreased approximately fourfold. Instantaneous aerobic fuel usage was calculated from respiratory gas exchange. At 30 degrees C, resting Mo2 was fueled by 42% lipids, 27% carbohydrates, and 31% protein. At 15 degrees C, lipid use decreased to 21%, carbohydrate use increased greatly to 63%, and protein use decreased to 16%. These patterns at 30 degrees C and 15 degrees C in tilapia paralleled fuel use previously reported in rainbow trout acclimated to 15 degrees C and 5 degrees C, respectively. Temperature also had a pronounced effect on critical swimming speed (UCrit). Tilapia acclimated to 30 degrees C had a UCrit of 5.63+/-0. 06 body lengths/s (BL/s), while, at 20 degrees C, UCrit was significantly lower at 4.21+/-0.14 BL/s. Tilapia acclimated to 15 degrees C were unable or unwilling to swim. As tilapia swam at greater speeds, Mo2 increased exponentially; Mo2min and Mo2max were 5.8+/-0.6 and 21.2+/-1.5 micromol O2/g/h, respectively. Nitrogenous waste excretion increased to a lesser extent with swimming speed. At 30 degrees C, instantaneous protein use while swimming at 15 cm/s ( approximately 1.7 BL/s) was 23%, and at UCrit (5.6 BL/s), protein use dropped slightly to 17%. During a 48-h swim at 25 cm/s (2.7 BL/s, approximately 50% UCrit), Mo2 and urea excretion remained unchanged, while ammonia excretion more than doubled by 24 h and remained elevated 24 h later. These results revealed a shift to greater reliance on protein as an aerobic fuel during prolonged swimming.