Early development and orientation of the acoustic funnel provides insight into the evolution of sound reception pathways in cetaceans.

Whales receive underwater sounds through a fundamentally different mechanism than their close terrestrial relatives. Instead of hearing through the ear canal, cetaceans hear through specialized fatty tissues leading to an evolutionarily novel feature: an acoustic funnel located anterior to the tympanic aperture. We traced the ontogenetic development of this feature in 56 fetal specimens from 10 different families of toothed (odontocete) and baleen (mysticete) whales, using X-ray computed tomography. We also charted ear ossification patterns through ontogeny to understand the impact of heterochronic developmental processes. We determined that the acoustic funnel arises from a prominent V-shaped structure established early in ontogeny, formed by the malleus and the goniale. In odontocetes, this V-formation develops into a cone-shaped funnel facing anteriorly, directly into intramandibular acoustic fats, which is likely functionally linked to the anterior orientation of sound reception in echolocation. In contrast, the acoustic funnel in balaenopterids rotates laterally, later in fetal development, consistent with a lateral sound reception pathway. Balaenids and several fossil mysticetes retain a somewhat anteriorly oriented acoustic funnel in the mature condition, indicating that a lateral sound reception pathway in balaenopterids may be a recent evolutionary innovation linked to specialized feeding modes, such as lunge-feeding.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes.

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60 approximately 81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Production of cloned sei whale (Balaenoptera borealis) embryos by interspecies somatic cell nuclear transfer using enucleated pig oocytes

In this study, we examined the feasibility of using subzonal cell injection with electrofusion for interspecies somatic cell nuclear transfer (iSCNT) to produce sei whale embryos and to improve their developmental capacity by investigating the effect of osmolarity and macromolecules in the culture medium on the in vitro developmental capacity. Hybrid embryos produced by the electrofusion of fetal whale fibroblasts with enucleated porcine oocytes were cultured in modified porcine zygote medium-3 to examine the effects of osmolarity and fetal serum on their in vitro developmental capacity. More than 66% of the whale somatic cells successfully fused with the porcine oocytes following electrofusion. A portion (60~81%) of the iSCNT whale embryos developed to the two- to four-cell stages, but no embryos were able to reach the blastocyst stage. This developmental arrest was not overcome by increasing the osmolarity of the medium to 360 mOsm or by the addition of fetal bovine or fetal whale serum. Our results demonstrate that sei whale-porcine hybrid embryos may be produced by SCNT using subzonal injection and electrofusion. The pig oocytes partly supported the remodeling and reprogramming of the sei whale somatic cell nuclei, but they were unable to support the development of iSCNT whale embryos to the blastocyst stage.

Comparative anatomy of the foramen ovale in the hearts of cetaceans.

The structure of the cardiac foramen ovale from 17 species representing six cetacean families, the Monodontidae, Phocoenidae, Delphinidae, Ziphiidae, Balaenidae and the Balaenopteridae, was studied using the scanning electron microscope. Eight white whale fetuses (Delphinapterus leucas) and a narwhal fetus (Monodon monoceros) represented the Monodontidae; one fetal and nine neonatal harbour porpoises (Phocoena phocoena) and a finless porpoise fetus (Neophocoena phocoenoides) represented the Phocoenidae; two white-beaked dolphin fetuses (Lagenorhynchus albirostris), four fetal and one neonatal Atlantic white-sided dolphins (Lagenorhynchus acutus), a Risso's dolphin fetus (Grampus griseus), two common bottle-nosed dolphin neonates (Tursiops truncatus), a female short-beaked common dolphin fetus (Delphinus delphis), four killer whale fetuses (Orcinus orca) and two long-finned pilot whale fetuses (Globicephala melas) represented the Delphinidae; two northern bottlenose whale fetuses (Hyperoodon ampullatus) represented the Ziphiidae; one bowhead whale fetus (Balaena mysticetus) represented the Balaenidae and five Common minke whale fetuses (Balaenoptera acutorostrata), one blue whale fetus (Balaenoptera musculus), nine fin whale fetuses (Balaenoptera physalus) and four humpback whale fetuses (Megaptera novaeangliae) represented the Balaenopteridae. The hearts of an additional two incompletely identified toothed and four baleen whale fetuses were also studied. In each species the fold of tissue derived from the cardiac septum primum and subtended by the foramen ovale had the appearance of a short tunnel or sleeve which was fenestrated at its distal end. In the toothed whales the tissue fold was tunnel-shaped with the interatrial septum as the floor whereas in baleen whales it was more sleeve-like. In toothed whales thin threads extended from the fold to insert into the interatrial septum whereas a network of threads covered the distal end of the sleeve in the baleen whales. Similar structures were present in the corresponding cardiac tissues of neonatal Hippopotamidae.

Relationship between the appearance of preantral follicles in the fetal ovary of Antarctic minke whales (Balaenoptera bonaerensis) and hormone concentrations in the fetal heart, umbilical cord and maternal blood.

The present study aimed to determine the relationship among changes in the number of preantral follicles and concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), androstenedione (A) and estradiol-17beta (E2) in the fetal heart, umbilical cord and maternal blood. Primordial follicles had already appeared in a 20 cm fetus and primary follicles were observed in a 50 cm fetus. In a 70 cm fetus, the number of primordial and primary follicles increased rapidly and secondary follicles were present. The concentrations of LH and FSH did not change between 20 cm and 160 cm in fetal length. When the fetal length became > 70 cm, serum levels in the fetus, umbilical cord and mothers, and E2 levels in umbilical cord increased synchronously (p < 0.05). These results showed increases in the number of preantral follicles in the Antarctic minke whale fetal ovary along with fetal growth during the early gestation period. These findings suggest that the change in preantral follicles was associated with changes in the concentration of steroids in early gestation periods. The changes in steroid concentrations in the fetal and umbilical cord blood and the increased number of preantral follicles were coincident at around 70 cm in fetal length, whereas the growth and differentiation of primordial and primary follicles appeared to be independent of FSH and LH.

Immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin during tooth development and degeneration in fetuses of minke whale, Balaenoptera acutorostrata.

The immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin were observed in the tooth buds of fetuses of minke whale, Balaenoptera acutorostrata. Distributions of extracellular matrices (ECMs) examined in this study except for tenascin were generally similar to those of terrestrial mammalian species during development of the tooth bud. Tenascin in the fetuses of minke whale showed characteristic distributions in the dental lamina and the enamel organ in the early tooth developmental stage. In the physiological degeneration stage of tooth bud development, immunoreactivity of the ECMs were very weakly and limitedly detected in the dental papilla and the surrounding mesenchyme. Immunoreactivity of tenascin and type I and III collagens were positively detected in the developing baleen plate germ which was associated with the degenerating tooth bud. These findings suggested that expressions of the ECMs were related to the formation of the tooth bud and baleen plate germ, and that the lack of the ECMs was related to the degeneration of the tooth bud in the fetal minke whale.

Ontogenesis of the sperm whale brain.

The development of the sperm whale brain (Physeter macrocephalus) was investigated in 12 embryos and early fetuses to obtain a better understanding of the morphological and physiological adaptations in this most exotic cetacean concerning locomotion, deep diving, and orientation. In male adult sperm whales, the average absolute brain mass and the relative size of the telencephalic hemisphere are the largest within the mammalia, whereas the ratio of the brain mass to the total body mass is one of the smallest. In the early sperm whale fetus, the rostral part of the olfactory system (olfactory nerves and bulbs) is lost, whereas the nervus terminalis seems to persist. Several components of the limbic system show signs of regression (hippocampus, fornix, mamillary body). In contrast, some components of the auditory system (trapezoid body, inferior colliculus) show marked enlargement in the early fetal period, thereby reflecting their dominant position in the adult. The cerebellum and pons grow slower than in most smaller toothed whales. The pyramidal tract develops poorly (reduction of the limbs), whereas marked growth of the striatum and inferior olive may be related to the animal's locomotion via trunk and tail. In the early fetal period, the trigeminal, vestibulocochlear, and facial nerves are the dominant cranial nerves (besides the vagus nerve). Whereas the number of axons in the vestibulocochlear nerve is high in adult, toothed whales and their diameters are considerable, the trigeminal nerve of the sperm whale may be the thickest of all cranial nerves and has the largest number of axons (innervation of the huge forehead region). A similar situation seems to exist for the facial nerve: It innervates the blowhole musculature that surrounds the very large spermaceti organ and melon (generation and emission of sonar clicks).

Morphology of the tracheobronchial tree and the route of the pulmonary artery in the fetal minke whale (Balaenoptera acutorostrata).

Molecular and statistical studies support the close phylogenetic relation between Cetacea and Artiodactyla. The presence of the tracheal bronchus has been pointed out as one of the common traits between only these groups. Nakakuki (1980) has investigated the mammalian tracheobronchial trees in 50 species based on his new nomenclature, and his study has consequently demonstrated the above-mentioned indication. Therefore, comparative anatomy of the tracheobronchial tree based on the nomenclature seems to be useful for investigations of the cetacean phylogeny. Three pairs of fetal lungs of the minke whale (Balaenoptera acutorostrata) were supplied from the Institute of Cetacean Research, Tokyo (1991) to observe their tracheobronchial trees and the ramification of the pulmonary artery. The fetal tracheobronchial tree consisted of one tracheal, four lateral, four dorsal and one medial secondary bronchi in the right lung, and five or six lateral and four or five dorsal secondary bronchi in the left, using the new nomenclature. The right pulmonary artery crossed the right axial bronchus from the ventral side to the dorsal over the third lateral bronchus. The tracheal bronchus in the fetal minke whale seems to belong to the type II of Nakakuki's nomenclature, and this type is seen in one species of the river dolphins. The other conspicuous traits of the fetal lungs are the defect of the second lateral broncus and the route of the pulmonary artery in the right side. This route is very different from that of many other mammals.

Observations on the external morphology and vasculature of a fetal heart of the bowhead whale, Balaena mysticetus.

BACKGROUND: Specialized demands within the aquatic environment for over some 60 million years have shaped unique morphological expressions in the whales, dolphins, and porpoises (Cetacea). Detailed consideration of these features, particularly in the great whales, has often been constrained by difficulties in securing adequate specimens for study. We had the opportunity to examine external heart morphology in a rarely obtained and prepared specimen from the bowhead whale, Balaena mysticetus. METHODS: The external morphology and in situ relations of a formalin-perfused heart were examined grossly in a near-term bowhead fetus. Latex injections assisted visualization of coronary vasculature. Magnetic resonance imaging was used to clarify heart positioning within the thoracic cavity in two younger (early and mid-gestational) intact fetuses. RESULTS: The heart was globular in form, with a blunt apex and wide base; it was laterally broad relative to height yet considerably compressed between nearly planar atrial (diaphragmatic) and auricular (sternocostal) surfaces. The heart constituted 0.01 of body mass in the near-term fetus. Within the thoracic cavity, the heart tilted forward on its long axis, placing the great basal vessels in the region of the thoracic inlet. The aorta extended forward from mid-base in parallel with the pulmonary trunk, arched sharply to the left, producing in succession the brachiocephalic trunk, left common carotid artery, and the left subclavian artery. Bifurcation of the brachiocephalic trunk yielded the right common carotid and right subclavian arteries. The distal portion of the aortic arch was linked to the pulmonary trunk via the ductus arteriosus. The aorta then swung caudally over the heart base, descending beneath the bodies of the thoracic vertebrae. The ascending aorta featured three bulbous sinuses immediately distal to the three semilunar cusps of the aortic valve. Originating along the distal boundaries of the left and right sinuses were the left and right coronary arteries. The arteries were similar in size and, because each sent contributions along their respective coronary and interventricular grooves, the heart can be described as bilateral relative to arterial supply. Anastomoses were common within and between the two arteries. Venous return from the heart was comprised of the great, middle, and right cardiac veins, all three converging in the coronary sinus. The right cardiac vein also included tributaries that emptied directly into the right atrium. CONCLUSIONS: External heart morphology in the fetal bowhead whale examined was distinguished by a laterally broad conformation with significant compression between its cranial and caudal surfaces. Aortic bulb configuration in combination with an expandable aortic arch may support blood service to the heart during diastole. Vascular service to the heart featured a complex vessel network with extensive intraarterial and intravenous anastomoses that enable many alternate blood perfusion pathways and may be adaptive to water-column-pressure fluctuations experienced by a large diving mammal.

Development and physiological degradation of tooth buds and development of rudiment of baleen plate in southern minke whale, Balaenoptera acutorostrata.

The development and degradation of temporary tooth buds and the development of rudiment of baleen plate were observed by gross-anatomical and histological examinations in twenty-four fetuses of the southern minke whale, Balaenoptera acutorostrata. The primary patterns of development of tooth buds were similar to those of deciduous tooth buds in the terrestrial species. Degradation of tooth buds was observed in the fetuses more than 615 mm body length (BL) and might proceed throughout the dental surface of the tooth buds. That degradation pattern was a little different from that of deciduous tooth buds in terrestrial species, which has a limited degradation area at the root of the tooth buds. In the fetuses with 135 and 153 mm BL, the upper jaw had a larger number of tooth buds than the lower jaw, although the number of buds varied in different individuals. Formation of rudiment of baleen plate was observed with degraded tooth buds in the fetus of 903 mm BL and it may be induced by the degradation of tooth buds.

On the origin of the so-called Meckelian ossicles in the nasal skull of odontocetes.

Although Cave (1987) accepts the theory that the Meckelian ossicles originate from the maxilloturbinals, evidence given in his study in fact supports the opinion of Klima and van Bree (1985) that the Meckelian ossicles arise from elements of the nasal floor, solum nasi, of the embryonic nasal capsule, in particular from the lamina transversalis anterior and the cartilago paraseptalis.

[Histologic studies of cartilaginous structures in the anterior region of the head of the sperm whale Physeter macrocephalus]. / Histologische Untersuchungen an Knorpelstrukturen im Vorderkopf des Pottwals Physeter macrocephalus.

Recently discovered cartilaginous structures in the forehead of the sperm whale (Behrmann and Klima 1985) were investigated histologically. The largest and most important of these structures is the nasal roof cartilage which can be derived from the tectum nasi, a part of the embryonic nasal capsule (Klima et al. 1986). In the investigated sperm whale fetuses, this structure consists of embryonic hyaline cartilage which is well suited for morphogenetic processes and fast growth. In the investigated adult sperm whale, the originally hyaline cartilage has been transformed into a special kind of elastic cartilage. The arrangement of cells, territories, and extracellular substance resembles hyaline cartilage. This component represents an adaptation to pressure load. The appearance and arrangement of elastic fibres resembles elastic cartilage. This component is an adaptation to distortion forces. Obviously, pressure and distortion are the strongest mechanical strains that the nasal roof cartilage is exposed. We see the function of this cartilage structure therein that, being a pressure-elastic skeletal support and following the left nasal meatus along its whole extension through the massive and soft forehead, it secures the only direct respiratory passage. Additionally, fibre bundles of transversely striated muscles are anchored in the perichondrium of the nasal roof cartilage. The function of this delicately interwoven muscle system is seen by us in the fine tuning of contraction and dilatation of the respiratory passage. Moreover, a possible function as a sound conducting cartilaginous structure serving the echolocation system is considered (c.f. Pilleri et al. 1983).

Early development of the olfactory and terminalis systems in baleen whales.

The development of the olfactory and terminalis systems was studied in tissue from eight embryonic and early fetal specimens belonging to three species of baleen whales. In contrast to toothed whales, baleen whales, particularly in these ontogenetic stages, are much less specialized in nasal organ morphology. The nasal cavity and peripheral olfactory system are well developed and do not show signs of reduction. However, as in toothed whales, there is no trace of a vomeronasal organ or nerve. The terminalis neuroblasts can already be distinguished from the olfactory material in the embryonic period, and they form compact masses medial and caudal to the developing olfactory bulb. As in most prenatal toothed whales, there are two large intrameningeal terminalis ganglia. These are connected with the telencephalic wall by central rootlets and with the septal mucosa by fiber bundles running through the level of the future cribriform plate. Clusters of terminalis neuroblasts also lie near the septal mucosa and along the peripheral terminalis fiber bundles. The functional implications of the olfactory and terminalis systems in whales are discussed.

Developmental changes of the fore- and hind-limbs in the fetuses of the southern minke whale, Balaenoptera acutorostrata.

15 fetuses of southern minke whale, Balaenoptera acutorostrata (from 8.2 mm C.R.L. to 38.9 mm C.R.L.), obtained from the Kyodo whale company in Japan (in whaling seasons from 1982 to 1986), were used for the present study. The fore-limb first appears in the 11.4 mm fetus, and the hind-limb in the 15.3 mm fetus. The fore-limb develops progressively during gestation, while the hind-limb disappears by the 38.9 mm fetus. The critical period of limb development in southern minke whale was shown to be at a stage between that in the small sized species; Common Porpoise, Phocoena communis and Striped Dolphin, Prodelphinus caeruleoalbus, and in large one; Humpback Whale, Megaptera nodusa.

Development of the external genitalia in fetuses of the southern minke whale, Balaenoptera acutorostrata.

The developmental changes of the anogenital distance and external genitalia were studied in 81 fetuses of the southern minke whale. It was difficult to sex the fetuses with less than 55.8 mm crown-rump length (CRL) even by histological means, but it was easy in the fetuses with more than 77.0 mm CRL and less than 110.0 mm CRL. The histologically determined male fetuses had a ratio of anogenital distance to CRL or body length of more than 5%, while females had a value of less than 4%. At later stages with a CRL of more than 113.0 mm, male fetuses had an umbilicus-directed genital tubercle, while in females a tail-directed tubercle was observed. The present study suggests that the stage with sexual dimorphism might be earlier in the southern minke whale than that previously reported.

Morphogenesis of the digestive tract in the fetuses of the southern minke whale, Balaenoptera acutorostrata.

Gross and histological observations on the digestive tract in 15 fetuses of the southern minke whale (Balaenoptera acutorostrata) with C.R.L. from 37.2 to 1,940mm revealed that the fetal stomach formation in this species resembled that of the ruminant, i.e., the compartment 1 arose from the stomach bud, but not from the esophagus to form the forestomach. The differentiated gastric glands, the parietal cells, were detected in fetuses with more than 213.0mm C.R.L. The circular folds resembling intestinal haustra like circular folds without tenia were detected on the cecum in fetuses with more than 650mm C.R.L. and less than 1,070mm C.R.L. The similar structure was seen in large intestine in fetuses with more than 650mm C.R.L.

Existence of vocal folds in the larynx of odontoceti (toothed whales).

Odontocetes (toothed whales) vocalize for communication and echolocation. The mechanisms of sound production, however, remain unclear. Their larynx has long been thought to lack vocal folds and, thus, was considered incapable of generating sounds. This study investigates internal anatomy of the odontocete larynx to: 1) describe the morphology of any folds found, 2) determine any structural homologies between these folds and the vocal folds of terrestrial mammals, and 3) assess their possible function in sound production. Larynges of 24 odontocetes representing ten genera (Delphinus, Stenella, Lagenorhynchus, Tursiops, Grampus, Delphinapterus, Globicephala, Kogia, Mesoplodon, and Phocoena) were studied post mortem. Nine specimens were cut midsagittally, and the remainder were dorsally opened to reveal internal anatomy. Results show that, contrary to established belief, vocal folds are consistently present. They are not isolated bands or "cords," but appear continuous with the internal laryngeal membrane. The attachments of these folds are the same as in terrestrial mammals, thus indicating homology with true mammalian vocal folds. These folds extend from the midline of the thyroid cartilage to the base of the arytenoid cartilages, sometimes to a discrete process. The vocal folds are elongated and oriented in a vertical plane, parallel to airflow direction. Vocal fold morphology varies, appearing as true bifurcated structures, a trifurcated fold, or a single midline fold. Laryngeal ventricles and vestibular folds are also consistently found lateral to the vocal folds. The vocal folds may divide the airstream within the larynx into three separate air currents. Fold vibrations may produce initial laryngeal sound used in echolocation or communication.

Trace elements intake in the Faroe Islands. I. Element levels in edible parts of pilot whales (Globicephalus meleanus).

We examined the distribution of copper, zinc, selenium, arsenic, cadmium and mercury (total and methyl mercury) in samples of muscle, liver, kidney and blubber from pilot whales (Globicephalus meleanus) caught off the Faroe Islands in 1977 and 1978. The very high total mercury level in the mature pilot whale exhibited differences among tissues and was highest in the liver. The total mercury concentration increased with body size. With increasing body size the ratio of methyl mercury to total mercury was relatively constant in muscle and kidney, but it decreased in liver. The concentrations of total mercury in the tissues of immature whales were much lower than those of mature whales. Selenium levels increased with body size. Significant correlation coefficients were found between the total mercury and selenium in liver and kidney. Selenium was present in the kidney in molar excess relative to mercury, whereas the opposite was the case in the muscle tissue. High cadmium contents were found in kidney and liver. In muscle and liver no significant correlations were found between cadmium and selenium, but a weak correlation between these elements was recorded in the kidney.