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Assessment of the humane aspects of electric lancing of whales by measurement of current densities in the brain and heart of dead animals.

The potential physiological effects of the electric lance are assessed, as used in Japanese whaling operations. Current densities are measured in the brains and hearts of six whales to which a controlled current of 5 A is applied by two electrodes inserted at various sites in the carcasses. The whales vary in size from 1.8 m (22 kg) to 16 m (40 t). The minimum current density in the brain necessary to cause depolarisation of neurones is estimated to be 10 mA cm-2 and to cause ventricular fibrillation is estimated to be 0.5 mA cm-2. No current densities exceeding 4.8 mA cm-2 are recorded in the brain. Very few recordings of current density from the heart are above 0.5 mA cm-2, and they occur only when electrodes are in optimal positions. When electrodes are placed as in whaling operations, no whale over 3 m in length would receive current densities in the heart or brain sufficient to cause permanent dysfunction. It is concluded that electric lancing is ineffective as a secondary method of killing whales and that the current densities recorded could cause pain and suffering to an already distressed animal.

Animals↗

Molecular cloning and characterization of CD4 in an aquatic mammal, the white whale Delphinapterus leucas.

Given the importance of the cell surface recognition protein, CD4, in immune function, the cloning and characterization of CD4 at the molecular level from an odontocete cetacean, the white whale (Delphinapterus leucas), was carried out. Whale CD4 cDNA contains 2662 base pairs and translates into a protein containing 455 amino acids. Whale CD4 shares 64% and 51% identity with the human and mouse CD4 protein, respectively, and is organized in a similar manner. Unlike human and mouse, however, the cytoplasmic domain, which is highly conserved, contains amino acid substitutions unique to whale. Moreover, only one of the seven potential N-linked glycosylation sites present in whale is shared with human and mouse. Evolutionarily, the whale CD4 sequence is most similar to pig and structurally similar to dog and cat, in that all lack the cysteine pair in the V2 domain. These differences suggest that CD4 may have a different secondary structure in these species, which may affect binding of class II and subsequent T-cell activation, as well as binding of viral pathogens. Interestingly, as a group, species with these CD4 characteristics all have high constitutive expression of class II molecules on T lymphocytes, suggesting potential uniqueness in the interaction of CD4, class II molecules, and the immune response. Molecular characterization of CD4 in an aquatic mammal provides information on the CD4 molecule itself and may provide insight into adaptive evolutionary changes of the immune system.

Adaptation, Biological↗

Electron spin resonance spectrum of Tyr-151 free radical formed in reactions of sperm whale metmyoglobin with ethyl hydroperoxide and potassium irridate.

A five-line ESR spectrum was observed at room temperature in reactions of sperm whale metmyoglobin with ethyl hydroperoxide (EtOOH) at pH 9.5 and with potassium irridate at pH's 7.0 and 9.5. A spectrum with the same g value and hyperfine splitting constant appeared in a reaction of sperm whale apomyoglobin with potassium irridate and was assigned to a tyrosyl radical on the basis of optical spectrum data obtained under the same reaction conditions. It was concluded that this radical arose from Tyr-151 for the following reasons. (i) This ESR spectrum could not be observed in the reaction of horse heart metmyoglobin, which lacks Tyr-151. (ii) Sperm whale metmyoglobin no longer gave this spectrum when treated with tetranitromethane (TNM) under conditions in which approximately one tyrosine is lost in sperm whale metmyoglobin but none is lost in horse heart metmyoglobin. (iii) A complex ESR spectrum observed in the reaction of sperm whale metmyoglobin with EtOOH at neutral pH was found to be a mixture of this five-line spectrum and one arising from an unidentified free radical formed in the reaction of horse heart metmyoglobin with EtOOH. The TNM-treated sperm whale metmyoglobin gave the same ESR spectrum as that observed in the reaction of horse heart metmyoglobin with EtOOH.

Animals↗

Blue-green color and composition of Stejneger's beaked whale (Mesoplodon stejnegeri) milk.

Two hundred ml of milk were obtained from a lactating Stejneger's beaked whale stranded at Ninilchik, Alaska on 21 Oct, 1980. Total solids (41%) were similar to values reported for sperm and belukha whales, while fat (17%) was half as great and crude protein (17%) was 2-4 times greater than in milk of these species. Lactose was not detected. Calcium (0.22%) was greater than reported for pigmy sperm whales but less than for blue whales. Phosphorus (0.07%) was less than for any of the above species. Sodium and potassium concentrations were 0.13% and 0.11%, respectively. Values (microgram/g) for other elements analyzed (magnesium, 42; iron, 35; copper, 2.6; zinc, 1.5; manganese, 0.3; selenium, 0.36) have not been reported for whale milk. Based on SDS-gel electropherograms, this whale milk did not contain a whey protein corresponding to cattle milk alpha-lactalbumin. A blue-green pigment in the milk was identified as biliverdin.

Animals↗

Use of chemical tracers in assessing the diet and foraging regions of eastern North Pacific killer whales.

Top predators in the marine environment integrate chemical signals acquired from their prey that reflect both the species consumed and the regions from which the prey were taken. These chemical tracers-stable isotope ratios of carbon and nitrogen; persistent organic pollutant (POP) concentrations, patterns and ratios; and fatty acid profiles-were measured in blubber biopsy samples from North Pacific killer whales (Orcinus orca) (n=84) and were used to provide further insight into their diet, particularly for the offshore group, about which little dietary information is available. The offshore killer whales were shown to consume prey species that were distinctly different from those of sympatric resident and transient killer whales. In addition, it was confirmed that the offshores forage as far south as California. Thus, these results provide evidence that the offshores belong to a third killer whale ecotype. Resident killer whale populations showed a gradient in stable isotope profiles from west (central Aleutians) to east (Gulf of Alaska) that, in part, can be attributed to a shift from off-shelf to continental shelf-based prey. Finally, stable isotope ratio results, supported by field observations, showed that the diet in spring and summer of eastern Aleutian Island transient killer whales is apparently not composed exclusively of Steller sea lions.

Adipose Tissue↗

A novel method for in situ fixation of whale brains.

A new method of in situ formalin fixation was used on 38 brains from minke whales (Balaenoptera acutorostrata). The method was developed because traditional ways of fixing brains are poorly suited to the collection of whale brains. The whole brain was preserved uncut in its meninges and then excised undamaged from the skull at a later opportunity. There was no handling of the brain in the fresh state. Fixation was started within a couple of hours post mortem. All brains were subjected to gross and light microscopy examination. The results showed that both the gross and microscopic architecture of the brains were adequately preserved, with no massive gross or histological changes due to insufficient fixation apparent. The occurrence of fixation artifacts was low. Microscopic examination showed well-preserved cells and myelin in all parts of the brain. We report the mean fixed weight of the minke whale brain as 2741 g, which is the lowest among the baleen whales. The cerebellum constituted 22% of the total brain weight, which conforms to findings in other baleen whales. This in situ method can probably be used without any particular modifications in other whale species and also in large terrestrial mammals.

Animals↗

Levels and patterns of persistent organochlorines in minke whale (Balaenoptera acutorostrata) stocks from the North Atlantic and European Arctic.

Regional variation in PCBs and organochlorine (OC) pesticide concentrations was examined using the blubber of 155 minke whales (Balaenoptera acutorostrata) sampled in seven regions in the North Atlantic and European Arctic, including western and southeastern Greenland, the Norwegian Sea, the North Sea and the Barents Sea. The levels and relative proportions of OCs were also used to examine the boundaries for North Atlantic minke whale stocks previously defined by the International Whaling Commission (IWC). Concentrations of major OC groups (sigmaPCB, 89.1-22 800 ng/g lipid; sigmaDDT, 65.3-6280 ng/g lipid; sigmaCHL, 33.3-2110 ng/g lipid) generally increased from west to east, while HCH concentrations (sigmaHCH, < 1-497 ng/g lipid) showed the opposite trend. Statistical comparison between six regions using sex-adjusted least squared mean concentrations showed that minke whales from the Barents Sea had significantly higher concentrations of EPCBs than those from the Vestfjorden/Lofoten, the North Sea, and west Svalbard, as well as significantly higher sigmaDDT concentrations compared to west Greenland animals. The differences in concentrations suggest that west and southeast Greenland minkes may represent one group of whales, which are distinct from both the Jan Mayen minkes and those from other IWC defined stocks in northern European waters. Principal components analysis using proportions of 71 PCB congeners and 20 OC pesticides (of total OCs) did not reveal any major differences among groups although minkes from the North Sea were distinguished from those from Greenland waters by higher loadings of more highly chlorinated PCBs and recalcitrant OC pesticides. The general similarity in mean levels of sigmaPCBs, sigmaDDT and sigmaCHL, as well as mean principal components analysis scores, among minkes sampled at Jan Mayen, Svalbard, Vestfjorden/Lofoten, the North Sea and the Barents Sea suggests that the whales are quite mobile and may feed in multiple areas within the northeastern Atlantic.

Analysis of Variance↗

Comparison of the beluga whale (Delphinapterus leucas) expressed genes for 5-aminolevulinate synthase with those in other vertebrates.

The cDNA and inferred amino acid sequences were determined for beluga whale (Delphinapterus leucas) erythroid (E) and housekeeping (H) forms of 5-aminolevulinate synthase (ALS), and they were compared with known sequences for five other vertebrates with particular attention to regulatory features. The cDNAs for whale ALS-E and -H encode, respectively, proteins of 582 and 640 amino acids. Sequence alignments suggest that the whale ALS-H, like those for rat and chicken, has an N-terminal mitochondrial targeting sequence of 56 amino acids. There is a high degree of amino acid conservation between the beluga whale proteins and those of other vertebrates, including regulatory elements and functional residues that have been defined in other ALSs. Both whale proteins contain three heme regulatory motifs suggesting that mitochondrial uptake may be regulated by heme. The ALS-E mRNA contains an iron responsive element in its 5'-untranslated region indicating that its expression may be post-transcriptionally regulated by cellular iron. This extensive structural similarity and the presence of the same regulatory elements found in other ALSs indicate that regulation of ALS in beluga whale is similar to that in other vertebrates.

5' Untranslated Regions↗

Complete amino acid sequence of the major component myoglobin from the humpback whale, Megaptera novaeangliae.

The complete primary structure of the major component myoglobin from the humpback whale, Megaptera novaeangliae, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Over 80% of the amino acid sequence was established from the three peptides resulting from the cleavage of the acetimidated apomyoglobin at the three arginine residues with trypsin. The further digestion of the central cyanogen bromide peptide with trypsin and S. aureus strain V8 protease enabled the determination of the remainder of the covalent structure. This myoglobin differs from that of sperm whale, Physeter catodon, at 12 positions, and dwarf sperm whale, Kogia simus, at 14 positions, finback whale Balaenoptera physalus at 3 positions, minke whale, Balaenoptera acutorostrata at 2 positions, and California gray whal Eschrichtius gibbosus, at 1 position. All of the substitutions observed in this sequence fit readily into the three-dimensional structure of sperm whale myoglobin.

Amino Acid Sequence↗

Molecular evidence from retroposons that whales form a clade within even-toed ungulates.

The origin of whales and their transition from terrestrial life to a fully aquatic existence has been studied in depth. Palaeontological, morphological and molecular studies suggest that the order Cetacea (whales, dolphins and porpoises) is more closely related to the order Artiodactyla (even-toed ungulates, including cows, camels and pigs) than to other ungulate orders. The traditional view that the order Artiodactyla is monophyletic has been challenged by molecular analyses of variations in mitochondrial and nuclear DNA. We have characterized two families of short interspersed elements (SINEs) that were present exclusively in the genomes of whales, ruminants and hippopotamuses, but not in those of camels and pigs. We made an extensive survey of retropositional events that might have occurred during the divergence of whales and even-toed ungulates. We have characterized nine retropositional events of a SINE unit, each of which provides phylogenetic resolution of the relationships among whales, ruminants, hippopotamuses and pigs. Our data provide evidence that whales, ruminants and hippopotamuses form a monophyletic group.

Animals↗

A newly discovered species of living baleen whale.

In the late 1970s eight Balaenoptera specimens of unknown identity were caught in the lower latitudinal Indo-Pacific waters by Japanese research whaling vessels. The combination of the allozyme patterns and physical maturity of the eight specimens separated them from all acknowledged Balaenoptera species. In September 1998 we collected a medium-sized baleen whale carcass on a coastal island in the Sea of Japan. This specimen and the previously collected eight specimens resembled Balaenoptera physalus (fin whale) in external appearance but were much smaller. Comparison of external morphology, osteology and mitochondrial DNA data grouped the nine specimens as a single species but separated them from all known baleen whale species. Therefore, here we describe a new species of Balaenoptera, which is characterized by its unique cranial morphology, its small number of baleen plates, and by its distant molecular relationships with all of its congeners. Our analyses also separated Balaenoptera brydei (Bryde's whale) and Balaenoptera edeni (Eden's whale) into two distinct species, raising the number of known living Balaenoptera species to eight.

Animal Structures↗

Eocene evolution of whale hearing.

The origin of whales (order Cetacea) is one of the best-documented examples of macroevolutionary change in vertebrates. As the earliest whales became obligately marine, all of their organ systems adapted to the new environment. The fossil record indicates that this evolutionary transition took less than 15 million years, and that different organ systems followed different evolutionary trajectories. Here we document the evolutionary changes that took place in the sound transmission mechanism of the outer and middle ear in early whales. Sound transmission mechanisms change early on in whale evolution and pass through a stage (in pakicetids) in which hearing in both air and water is unsophisticated. This intermediate stage is soon abandoned and is replaced (in remingtonocetids and protocetids) by a sound transmission mechanism similar to that in modern toothed whales. The mechanism of these fossil whales lacks sophistication, and still retains some of the key elements that land mammals use to hear airborne sound.

Air↗

The antibody response to myoglobin is independent of the immunized species. Analysis in terms of replacements in the antigenic sites and in environmental residues of the cross-reactions of fifteen myoglobins with sperm-whale myoglobin antisera raised in different species.

The recent determination of the entire antigenic structure of sperm-whale myoglobin with rabbit and goat antisera has permitted the examination of whether the antigenic structure recognized by antibodies depends on the species in which the antisera are raised. Also, by knowledge of the antigenic structure, the molecular factors that determine and influence antigenicity can be better understood in terms of the effects of amino acid substitutions occurring in the antigenic sites and in the environmental residues of the sites. In the present work, the myoglobins from finback whale, killer whale, horse, chimpanzee, sheep, goat, bovine, echidna, viscacha, rabbit, dog, cape fox, mouse and chicken were examined for their ability to cross-react with antisera to sperm-whale myoglobin. By immunoadsorbent titration studies with radioiodinated antibodies, each of these myoglobins was able to bind antibodies to sperm-whale myoglobin raised in goat, rabbit, chicken, cat, pig and outbred mouse. It was found that the extent of cross-reaction of a given myoglobin was not dependent on the species in which the antisera were raised. This indicated that the antibody response to sperm-whale myoglobin (i.e. its antigenic structure) is independent of the species in which the antisera are raised and is not directed to regions of sequence differences between the injected myoglobin and the myoglobin of the immunized host. Indeed, in each antiserum from a given species examined, that antiserum reacted with the myoglobin of that species. The extent of this auto-reactivity for a given myoglobin was comparable with the general extent of cross-reactivity shown by that myoglobin with antisera raised in other species. The cross-reactivities and auto-reactivities (both of which are of similar extents for a given myoglobin) can be reasonably rationalized in terms of the effects of amino acid substitutions within the antigenic sites and within the residues close to these sites. These findings confirm that the antigenicity of the sites is inherent in their three-dimensional locations.

Amino Acid Sequence↗

Social structure in migrating humpback whales (Megaptera novaeangliae).

Although largely solitary, humpback whales exhibit a number of behaviours where individuals co-operate with one another, for example during bubble net feeding. Such cases could be due to reciprocal altruism brought on by exceptional circumstances, for example the presence of abundant shoaling fish. An alternative explanation is that these behaviours have evolved through kin selection. With little restriction to either communication or movement, diffuse groups of relatives could maintain some form of social organization without the need to travel in tight-nit units. To try to distinguish between these hypotheses, we took advantage of the fact that migrating humpback whales often swim together in small groups. If kin selection is important in humpback whale biology, these groups should be enriched for relatives. Consequently, we analysed biopsy samples from 57 groups of humpback whales migrating off Eastern Australia in 1992. A total of 142 whales were screened for eight microsatellite markers. Mitochondrial DNA sequences (371 bp) were also used to verify and assist kinship identification. Our data add support to the notion that mothers travel with their offspring for the first year of the calf's life. However, beyond the presence of mother-calf/yearling pairs, no obvious relatedness pattern was found among whales sampled either in the same pod or on the same day. Levels of relatedness did not vary between migratory phases (towards or away from the breeding ground), nor between the two sexes considered either overall or in the north or south migrations separately. These findings suggest that, if any social organization does exist, it is formed transiently when needed rather than being a constant feature of the population, and hence is more likely based on reciprocal altruism than kin selection.

Animal Migration↗

Limbs in whales and limblessness in other vertebrates: mechanisms of evolutionary and developmental transformation and loss.

We address the developmental and evolutionary mechanisms underlying fore- and hindlimb development and progressive hindlimb reduction and skeletal loss in whales and evaluate whether the genetic, developmental, and evolutionary mechanisms thought to be responsible for limb loss in snakes "explain" loss of the hindlimbs in whales. Limb loss and concurrent morphological and physiological changes associated with the transition from land to water are discussed within the context of the current whale phylogeny. Emphasis is placed on fore- and hindlimb development, how the forelimbs transformed into flippers, and how the hindlimbs regressed, leaving either no elements or vestigial skeletal elements. Hindlimbs likely began to regress only after the ancestors of whales entered the aquatic environment: Hindlimb function was co-opted by the undulatory vertical axial locomotion made possible by the newly evolved caudal flukes. Loss of the hindlimbs was associated with elongation of the body during the transition from land to water. Limblessness in most snakes is also associated with adoption of a new (burrowing) lifestyle and was driven by developmental changes associated with elongation of the body. Parallels between adaptation to burrowing or to the aquatic environment reflect structural and functional changes associated with the switch to axial locomotion. Because they are more fully studied and to determine whether hindlimb loss in lineages that are not closely related could result from similar genetically controlled developmental pathways, we discuss developmental (cellular and genetic) processes that may have driven limb loss in snakes and leg-less lizards and compare these processes to the loss of hindlimbs in whales. In neither group does ontogenetic or phylogenetic limb reduction result from failure to initiate limb development. In both groups limb loss results from arrested development at the limb bud stage, as a result of inability to maintain necessary inductive tissue interactions and enhanced cell death over that seen in limbed tetrapods. An evolutionary change in Hox gene expression--as occurs in snakes--or in Hox gene regulation--as occurs in some limbless mutants--is unlikely to have initiated loss of the hindlimbs in cetaceans. Selective pressures acting on a wide range of developmental processes and adult traits other than the limbs are likely to have driven the loss of hindlimbs in whales.

Animals↗

Production rates of volatile fatty acids in the minke whale (Balaenoptera acutorostrata) forestomach.

Minke whales (Balaenoptera acutorostrata) have developed a compartmentalized stomach system, which includes a non-glandular forestomach containing high concentrations of indigenous bacteria. The forestomach contents serve as microbial substrate, and samples were collected from five adult minke whales eating capelin (Mallotus villosus) and crustaceans (Thysanoessa sp.). Chemical analysis of the forestomach contents revealed that they consisted of crude protein (650 (SD 58) g/kg DM), lipid (330 (SD 77) g/kg DM) and water-soluble carbohydrates (53.3 (SD 7.3) g/kg DM). The contribution of energy from volatile fatty acids (VFA), produced by forestomach bacterial fermentation, to the total energy budget was estimated. The forestomach concentration of VFA ranged from 13.2 to 68.5 mmol/l, and the pH was 5.83 (SD 0.41). VFA pool size ranged from 72.8 to 638.1 mmol and represented from 0.169 to 2.107 kJ/kg live weight (W)0-75. Maximal recorded forestomach VFA production rate was 1694 mmol/h in one capelin-eating minke whale with 42.6 litres of forestomach fluid. Energy from VFA produced by forestomach fermentation represented 6-107 kJ/kg (W)0-75 per d, which accounts for only 0.9-16.9% of the average daily energy expenditure of minke whales. This study suggests that the bacterial fermentation in the minke whale forestomach varies, depending on the volume and the quality of substrate available, influencing fermentation rates and concentration of VFA. Due to the small relative size of the forestomach, the contribution of VFA to the daily energy requirement in minke whales would be of less importance than in ruminants even when assuming the same production rate of VFA as in a ruminant.

Animals↗

Nucleotide sequence of the D-loop region of the sperm whale (Physeter macrocephalus) mitochondrial genome.

We have amplified, by the polymerase chain reaction, and have sequenced the D-loop region of the mitochondrial DNA from the sperm whale (Physeter macrocephalus). The sperm whale D-loop was aligned with D-loop sequences from four other cetaceans (Commerson's dolphin, orca, fin whale, and minke whale) and an out-group (cow). This alignment showed the sperm whale sequence to be larger than that of other cetaceans. In addition, some sequence blocks were highly conserved among all six species, suggesting roles in the functioning of mitochondrial DNA. Other blocks that were previously reported to be well conserved among cetaceans showed little sequence conservation with the sperm whale D-loop, which argues against the functional importance of these sequence blocks in cetaceans.

Animals↗

Beaked whales echolocate on prey.

Beaked whales (Cetacea: Ziphiidea) of the genera Ziphius and Mesoplodon are so difficult to study that they are mostly known from strandings. How these elusive toothed whales use and react to sound is of concern because they mass strand during naval sonar exercises. A new non-invasive acoustic ording tag was attached to four beaked whales(two Mesoplodon densirostris and two Ziphius cavirostris) and recorded high-frequency clicks during deep dives. The tagged whales only clicked at depths below 200 m, down to a maximum depth of 1267 m. Both species produced a large number of short, directional, ultrasonic clicks with significant energy below 20 kHz. The tags recorded echoes from prey items; to our knowledge, a first for any animal echolocating in the wild. As far as we are aware, these echoes provide the first direct evidence on how free-ranging toothed whales use echolocation in foraging. The strength of these echoes suggests that the source level of Mesoplodon clicks is in the range of 200-220 dB re 1 microPa at 1 m. This paper presents conclusive data on the normal vocalizations of these beaked whale species, which may enable acoustic monitoring to mitigate exposure to sounds intense enough to harm them.

Animals↗