Forward shift of feeding buzz components of dolphins and belugas during associative learning reveals a likely connection to reward expectation, pleasure and brain dopamine activation.

For many years, we heard sounds associated with reward from dolphins and belugas. We named these pulsed sounds victory squeals (VS), as they remind us of a child's squeal of delight. Here we put these sounds in context with natural and learned behavior. Like bats, echolocating cetaceans produce feeding buzzes as they approach and catch prey. Unlike bats, cetaceans continue their feeding buzzes after prey capture and the after portion is what we call the VS. Prior to training (or conditioning), the VS comes after the fish reward; with repeated trials it moves to before the reward. During training, we use a whistle or other sound to signal a correct response by the animal. This sound signal, named a secondary reinforcer (SR), leads to the primary reinforcer, fish. Trainers usually name their whistle or other SR a bridge, as it bridges the time gap between the correct response and reward delivery. During learning, the SR becomes associated with reward and the VS comes after the SR rather than after the fish. By following the SR, the VS confirms that the animal expects a reward. Results of early brain stimulation work suggest to us that SR stimulates brain dopamine release, which leads to the VS. Although there are no direct studies of dopamine release in cetaceans, we found that the timing of our VS is consistent with a response after dopamine release. We compared trained vocal responses to auditory stimuli with VS responses to SR sounds. Auditory stimuli that did not signal reward resulted in faster responses by a mean of 151 ms for dolphins and 250 ms for belugas. In laboratory animals, there is a 100 to 200 ms delay for dopamine release. VS delay in our animals is similar and consistent with vocalization after dopamine release. Our novel observation suggests that the dopamine reward system is active in cetacean brains.

Vocal reporting of echolocation targets: dolphins often report before click trains end.

Bottlenose dolphins (Tursiops truncatus) wore opaque suction cups over their eyes while stationing behind an acoustically opaque door. This put the dolphins in a known position and orientation. When the door opened, the dolphin clicked to detect targets. Trainers specified that Dolphin S emit a whistle if the target was a 7.5 cm water filled sphere, or a pulse burst if the target was a rock. S remained quiet if there was no target. Dolphin B whistled for the sphere. She remained quiet for rock and for no target. Thus, S had to choose between three different responses, whistle, pulse burst, or remain quiet. B had to choose between two different responses, whistle or remain quiet. S gave correct vocal responses averaging 114 ms after her last echolocation click (range 182 ms before and 219 ms after the last click). Average response for B was 21 ms before her last echolocation click (range 250 ms before and 95 ms after the last click in the train). More often than not, B began her whistle response before her echolocation train ended. The findings suggest separate neural pathways for generation of response vocalizations as opposed to echolocation clicks.

Simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (Tursiops truncatus) utilizing flow cytometry.

Phagocytic and respiratory burst activity was simultaneously measured by flow cytometry in polymorphonuclear leukocytes (PMN) and monocytes in whole blood from bottlenose dolphins (Tursiops truncatus). Blood was collected from 16 adult dolphins, 12 males (6-34 years of age) and 4 females (11-30 years) and subsequently incubated with a bacteria-to-leukocyte ratio of 25:1 and 10 µl of 500 µM 2',7'-dichlorofluorescein diacetate for 70 min at 37°C. PMN (44.5 ± 3.2%) and monocytes (33.5 ± 3.0%) were positive for propidium iodide-labeled Staphylococcus aureus, indicating phagocytosis. Respiratory burst activity after 70 min as measured by the mean fluorescence intensity (MFI) was 68.0 ± 14.4 in PMN and 47.0 ± 10.3 in monocytes. There were no significant differences in MFI or percentage of phagocytizing PMN (p > 0.094) or monocytes (p > 0.275) after storage at 4°C for 24h when compared to activity measured in fresh blood. Nor was there an effect of storage on respiratory burst activity (MFI or percentage) in PMN (p > 0.420) or monocytes (p > 0.301). This assay may be particularly useful to assess the ability of dolphins to effectively combat bacterial pathogen challenges with minimal amounts of blood.

Cetacean brain evolution: Dwarf sperm whale (Kogia sima) and common dolphin (Delphinus delphis) - An investigation with high-resolution 3D MRI.

This study compares a whole brain of the dwarf sperm whale (Kogia sima) with that of a common dolphin (Delphinus delphis) using high-resolution magnetic resonance imaging (MRI). The Kogia brain was scanned with a Siemens Trio Magnetic Resonance scanner in the three main planes. As in the common dolphin and other marine odontocetes, the brain of the dwarf sperm whale is large, with the telencephalic hemispheres remarkably dominating the brain stem. The neocortex is voluminous and the cortical grey matter thin but expansive and densely convoluted. The corpus callosum is thin and the anterior commissure hard to detect whereas the posterior commissure is well-developed. There is consistency as to the lack of telencephalic structures (olfactory bulb and peduncle, olfactory ventricular recess) and neither an occipital lobe of the telencephalic hemisphere nor the posterior horn of the lateral ventricle are present. A pineal organ could not be detected in Kogia. Both species show a tiny hippocampus and thin fornix and the mammillary body is very small whereas other structures of the limbic system are well-developed. The brain stem is thick and underlies a large cerebellum, both of which, however, are smaller in Kogia. The vestibular system is markedly reduced with the exception of the lateral (Deiters') nucleus. The visual system, although well-developed in both species, is exceeded by the impressive absolute and relative size of the auditory system. The brainstem and cerebellum comprise a series of structures (elliptic nucleus, medial accessory inferior olive, paraflocculus and posterior interpositus nucleus) showing characteristic odontocete dimensions and size correlations. All these structures seem to serve the auditory system with respect to echolocation, communication, and navigation.

Morphology and evolutionary biology of the dolphin (Delphinus sp.) brain--MR imaging and conventional histology.

Whole brains of the common dolphin (Delphinus delphis) were studied using magnetic resonance imaging (MRI) in parallel with conventional histology. One formalin-fixed brain was documented with a Siemens Trio Magnetic Resonance scanner and compared to three other brains which were embedded in celloidin, sectioned in the three main planes and stained for cells and fibers. The brain of the common dolphin is large, with the telencephalic hemispheres dominating the brain stem. The neocortex is voluminous and the cortical grey matter thin but extremely extended and densely convoluted. There is no olfactory ventricular recess due to the lack of an anterior olfactory system (olfactory bulb and peduncle). No occipital lobe of the telencephalic hemisphere and no posterior horn of the lateral ventricle are present. A pineal organ could not be detected. The brain stem is thick and underlies a very large cerebellum. The hippocampus and mammillary body are small and the fornix is thin; in contrast, the amygdaloid complex is large and the cortex of the limbic lobe is extended. The visual system is well developed but exceeded by the robust auditory system; for example, the inferior colliculus is several times larger than the superior colliculus. Other impressive structures in the brainstem are the peculiar elliptic nucleus, inferior olive, and in the cerebellum the huge paraflocculus and the very large posterior interpositus nucleus. There is good correspondence between MR scans and histological sections. Most of the brain characteristics can be interpreted as morphological correlates to the successful expansion of this species in the marine environment, which was characterized by the development of a powerful sonar system for localization, communication, and acousticomotor navigation.

Neuron numbers in sensory cortices of five delphinids compared to a physeterid, the pygmy sperm whale.

With its large mass and enormous gyrification, the neocortex of whales and dolphins has always been a challenge to neurobiologists. Here we analyse the relationship between neuron number per cortical unit in three different sensory areas and brain mass in six different toothed whale species, five delphinids and one physeterid. Cortex samples, including primary cortical areas of the auditory, visual, and somatosensory systems were taken from both hemispheres of brains fixed in 10% buffered formalin. The samples were embedded in paraffin, sectioned at 25 microm thickness and stained with cresyl violet. Because cortical thickness varies among toothed whale species, cell counts were done in cortical units measuring 150mum in width, 25 microm in thickness, and extending from the pial surface to the white matter. By arranging the delphinid brains according to their total mass, 834-6052 g, we found decreasing neuron numbers in the investigated areas with increasing brain mass. The pigmy sperm whale (Kogia breviceps), a physeterid with an adult brain weight of 1000 g had a distinctly lower neuron number per cortical unit. As had been expected, an increase in adult brain weight in delphinid cetaceans (family Delphinidae) is not correlated with an increase in neuron number per cortical unit.

Asymmetry and symmetry in brain waves from dolphin left and right hemispheres: some observations after anesthesia, during quiescent hanging behavior, and during visual obstruction.

Studies of sleep in cetaceans (whales, dolphins, and porpoises), substantiated by electrophysiological data, are rare with the great majority of observations having been made by one group from Russia. This group employed hard-wired recording with low-noise cables for their EEG observations, whereas our report describes behavioral and EEG observations of dolphin sleep using telemetry. Marked asymmetry of the EEG was observed during behavioral sleep posture. At different times synchronized slow waves appeared in both left and right brain hemispheres concurrently with lower voltage, faster, desynchronized EEG activity in the opposite hemisphere. On the other hand, during one brief period of sleep behavior, sleep-like EEG activity appeared on leads from both hemispheres. When the animal was exposed to a loud sound, it woke with lower voltage, faster, relatively symmetrical, desynchronized EEG activity appearing from both hemispheres. Additionally, the EEG appeared relatively desynchronized and symmetrical between the two hemispheres when the animal was awake during recovery from pentothal-halothane anesthesia as well as during waking periods when one or both of the animal's eyes were covered by an opaque rubber suction cup.

Description of a poorly differentiated carcinoma within the brainstem of a white whale (Delphinapterus leucas) from magnetic resonance images and histological analysis.

In this study we used magnetic resonance imaging (MRI) to investigate neuroanatomical structure in the brain of a white whale (Delphinapterus leucas) that died from a large tumor within the brainstem. This specimen was also compared with a normal white whale brain using MRI. MRI scans of the white whale specimen show how the tumor deformed surrounding brain structure. Histopathological analysis indicated a poorly differentiated carcinoma of uncertain origin. These analyses demonstrate the usefulness of supplementing histological analyses of pathology with studies of gross morphology facilitated by MRI.

Opportunities for using Navy marine mammals to explore associations between organochlorine contaminants and unfavorable effects on reproduction.

The Department of Defense (DoD) has a unique marine mammal program maintained by the US Navy that includes the largest force of bottlenose dolphins, Tursiops truncatus, worldwide. In recent years, this population of cetaceans that lives in netted open water enclosures in San Diego Bay has been monitored for levels of organochlorine (OC) contaminants in blubber, blood and milk. Data generated from these studies have afforded insight into the fate and possible effects of OC contaminants in marine mammals. We now report preliminary findings on the effects of maternal OC exposure on pregnancy outcome. Blubber OC levels were compared between females whose calves survived beyond 6 months and females whose calves were stillborn or died within 12 days of birth. The mean concentration of SigmaDDT was more than 3 times as high among dolphins whose calves died as that among dolphins whose calves survived beyond 6 months (P = 0.002). Mean SigmaPCB was more than 2.5 times higher in females whose calves did not survive (P= 0.076). This population is a logical sentinel for the assessment of environmentally mediated disease. Biological tissues and fluids can be sampled on a regular basis from the dolphins for accumulation of tissue residues, facilitated by conditioned husbandry behaviors. These trained behaviors help preclude possible alterations in health measures resulting from capture stress. Animals' diets can be monitored for contaminant levels. With these data, the expertise and facilities available at the Navy laboratory and in collaboration with other experts in the field, controlled studies can be designed to monitor and assess dietary exposure, measurable immune and neurologic responses and assess reproductive and transgenerational effects of contaminants. Biomarkers can be developed to relate the health of individual animals relative to contaminant exposures. Such investigations of natural exposure and response scenarios are a logical adjunct to traditional laboratory toxicity studies.

Anatomy and three-dimensional reconstructions of the brain of the white whale (Delphinapterus leucas) from magnetic resonance images.

Magnetic resonance imaging offers a means of observing the internal structure of the brain where traditional procedures of embedding, sectioning, staining, mounting, and microscopic examination of thousands of sections are not practical. Furthermore, internal structures can be analyzed in their precise quantitative spatial interrelationships, which is difficult to accomplish after the spatial distortions often accompanying histological processing. For these reasons, magnetic resonance imaging makes specimens that were traditionally difficult to analyze, more accessible. In the present study, images of the brain of a white whale (Beluga) Delphinapterus leucas were scanned in the coronal plane at 119 antero-posterior levels. From these scans, a computer-generated three-dimensional model was constructed using the programs VoxelView and VoxelMath (Vital Images, Inc.). This model, wherein details of internal and external morphology are represented in three-dimensional space, was then resectioned in orthogonal planes to produce corresponding series of "virtual" sections in the horizontal and sagittal planes. Sections in all three planes display the sizes and positions of such structures as the corpus callosum, internal capsule, cerebral peduncles, cerebral ventricles, certain thalamic nuclear groups, caudate nucleus, ventral striatum, pontine nuclei, cerebellar cortex and white matter, and all cerebral cortical sulci and gyri.

Hearing and whistling in the deep sea: depth influences whistle spectra but does not attenuate hearing by white whales (Delphinapterus leucas) (Odontoceti, Cetacea).

Hearing is attenuated in the aerial ear of humans and other land mammals tested in pressure chambers as a result of middle ear impedance changes that result from increased air density. We tested the hypothesis, based on recent middle ear models, that increasing the density of middle ear air at depth might attenuate whale hearing. Two white whales Delphinapterus leucas made dives to a platform at a depth of 5, 100, 200 or 300 m in the Pacific Ocean. During dives to station on the platform for up to 12 min, the whales whistled in response to 500 ms tones projected at random intervals to assess their hearing threshold at each depth. Analysis of response whistle spectra, whistle latency in response to tones and hearing thresholds showed that the increased hydrostatic pressure at depth changed each whale's whistle response at depth, but did not attenuate hearing overall. The finding that whale hearing is not attenuated at depth suggests that sound is conducted through the head tissues of the whale to the ear without requiring the usual ear drum/ossicular chain amplification of the aerial middle ear. These first ever hearing tests in the open ocean demonstrate that zones of audibility for human-made sounds are just as great throughout the depths to which these whales dive, or at least down to 300 m.

Auditory and behavioral responses of bottlenose dolphins (Tursiops truncatus) and a beluga whale (Delphinapterus leucas) to impulsive sounds resembling distant signatures of underwater explosions.

A behavioral response paradigm was used to measure masked underwater hearing thresholds in two bottlenose dolphins and one beluga whale before and after exposure to impulsive underwater sounds with waveforms resembling distant signatures of underwater explosions. An array of piezoelectric transducers was used to generate impulsive sounds with waveforms approximating those predicted from 5 or 500 kg HBX-1 charges at ranges from 1.5 to 55.6 km. At the conclusion of the study, no temporary shifts in masked-hearing thresholds (MTTSs), defined as a 6-dB or larger increase in threshold over pre-exposure levels, had been observed at the highest impulse level generated (500 kg at 1.7 km, peak pressure 70 kPa); however, disruptions of the animals' trained behaviors began to occur at exposures corresponding to 5 kg at 9.3 km and 5 kg at 1.5 km for the dolphins and 500 kg at 1.9 km for the beluga whale. These data are the first direct information regarding the effects of distant underwater explosion signatures on the hearing abilities of odontocetes.

Temporary shift in masked hearing thresholds of bottlenose dolphins, Tursiops truncatus, and white whales, Delphinapterus leucas, after exposure to intense tones.

A behavioral response paradigm was used to measure masked underwater hearing thresholds in five bottlenose dolphins and two white whales before and immediately after exposure to intense 1-s tones at 0.4, 3, 10, 20, and 75 kHz. The resulting levels of fatiguing stimuli necessary to induce 6 dB or larger masked temporary threshold shifts (MTTSs) were generally between 192 and 201 dB re: 1 microPa. The exceptions occurred at 75 kHz, where one dolphin exhibited an MTTS after exposure at 182 dB re: 1 microPa and the other dolphin did not show any shift after exposure to maximum levels of 193 dB re: 1 microPa, and at 0.4 kHz, where no subjects exhibited shifts at levels up to 193 dB re: 1 microPa. The shifts occurred most often at frequencies above the fatiguing stimulus. Dolphins began to exhibit altered behavior at levels of 178-193 dB re: 1 microPa and above; white whales displayed altered behavior at 180-196 dB re: 1 microPa and above. At the conclusion of the study all thresholds were at baseline values. These data confirm that cetaceans are susceptible to temporary threshold shifts (TTS) and that small levels of TTS may be fully recovered.

Source levels and estimated yellowfin tuna (Thunnus albacares) detection ranges for dolphin jaw pops, breaches, and tail slaps.

Tuna fishers in the eastern Pacific Ocean often exploit an association between a few genus of dolphin (Stenella and Delphinus) and yellowfin tuna (Thunnus albacares) to locate and capture the tuna. Identification of a mechanism which facilitates the tuna/dolphin bond may provide a means of exploiting the bond and capturing tuna without catching dolphin. To investigate if tuna may be attracted to low-frequency sounds produced by dolphins, source levels of bottlenose dolphin (Tursiops truncatus) jaw pops, breaches, and tail slaps were experimentally measured and used to estimate the maximum range at which yellowfin could detect similar sounds produced by pelagic species. The effective acoustic stimulus to the tuna was defined as the maximum one-third-octave level between 200 and 800 Hz, the frequency range where T. albacares is most sensitive. Spherical spreading was assumed to predict transmission loss with range. Breaches and jaw pops produced maximum one-third-octave source levels between 200 and 800 Hz of 153 (+/-4) and 163 (+/-2) dB re: 1 microPa-m, respectively, which resulted in estimated detection ranges of 340-840 and 660-1040 m, respectively. Tail slaps had lower source levels [max. 141 (+/-3) dB re: 1 microPa-m] and a maximum detection range of approximately 90-180 m.

Relative volume of the cerebellum in dolphins and comparison with anthropoid primates.

According to the 'developmental constraint hypothesis' of comparative mammalian neuroanatomy, brain growth follows predictable allometric trends. Therefore, brain structures should scale to the entire brain in the same way across mammals. Evidence for a departure from this pattern for cerebellum volume has recently been reported among the anthropoid primates. One of the mammalian groups that has been neglected in tests of the 'developmental constraint hypothesis' is the cetaceans (dolphins, whales, and porpoises). Because many cetaceans possess relative brain sizes in the range of primates comparative tests of the 'developmental constraint hypothesis' across these two groups could help to delineate the parameters of this hypothesis. In this paper, we compare relative cerebellum volumes in two cetacean species, the bottlenose dolphin (Tursiops truncatus) and the common dolphin (Delphinus delphis), with published data from anthropoid primates. We found that relative cerebellum size is significantly greater in the two dolphin species than in any of the primates, including humans. These results suggest that there is possibly expansion of brain structures independent of strictly allometric processes.

Effects of in vitro hemolysis on serum biochemistry values of the bottlenose dolphin (Tursiops truncatus).

The effects of in vitro hemolysis on 23 biochemical analytes were assessed in sera from 14 clinically healthy Atlantic bottlenose dolphins (Tursiops truncatus). Each serum sample was divided into three portions for analysis: 1) nonhemolyzed control; 2) moderate hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 150 mg/dl Hb; and 3) severe hemolysis, simulated by adding hemolyzed serum to a final concentration of approximately 500 mg/dl Hb. Moderate hemolysis resulted in statistically significant increases in the mean values of iron, lactate dehydrogenase, potassium, and uric acid and a decrease in creatinine (P < 0.001). Severe hemolysis resulted in statistically significant changes in the mean values of the above analytes in addition to the following increases: alanine aminotransferase, calcium, and serum globulins (P < 0.001) and albumin and total protein (P < 0.01). Total bilirubin and gamma glutamyl transferase levels were lower in the severely hemolyzed sample (P < 0.001). Differences in mean values for alkaline phosphatase between nonhemolyzed and hemolyzed serum were not significant but did show a downward trend in the hemolyzed sera. The presence and severity of hemolysis must be considered in the interpretation of the serum chemistry values.

Brucella-induced abortions and infection in bottlenose dolphins (Tursiops truncatus).

Two bottlenose dolphins (Tursiops truncatus) aborted fetuses that died as a result of Brucella infection. Brucella placentitis occurred in both cases. Infected placenta and vaginal/uterine fluids may transmit Brucella species to other cetaceans. In a third case, an identical organism was cultured from lung necropsy tissue of an adult female T. truncatus. Microbiology, specific polymerase chain reaction, and pulsed-field gel electrophoresis results supported the designation of an additional genomic group(s), Brucella delphini, for isolates adapted to T. truncatus. Current serologic diagnostic tests reliable for known Brucella species are unreliable in detecting dolphin brucellosis. Our findings, together with previous reports, suggest that dolphin brucellosis is a naturally occurring disease that can adversely impact reproduction in cetaceans. The zoonotic significance of cetacean brucellosis is unknown, although the disease has not been reported in people who have frequent contact with dolphins. Further studies on the zoonotic aspects, distribution, prevalence, virulence, and impact of this disease in cetaceans and other marine mammal species are needed.