Several marine mammals have evolved an echolocating system called biosonar. In particular, odontocetes echolocate by producing clicking sounds and receiving and interpreting the resulting echo. By means of this system, toothed whales and dolphins can identify objects and animals out of their sighting range, thus obtaining information on their size, number, shape, texture, and speed (Kuc, 1996; Hoffmann et al., 1998; Au, 2002).
The organs devoted to the production and propagation of the sound are the “monkey lips”/dorsal bursa (MLDB complex) and the melon, respectively. The echoed sounds are received and conducted through the lower jaw to the middle ear, inner ear, and then to hearing centers in the brain by means of the auditory nerve. The brain receives the sound waves in the form of nerve impulses, which relay the messages of sound and enable the dolphin to interpret the sound's meaning (Au, 2002). Age-related structural and compositional differences were reported for the melon of several odontocete species, thus suggesting that echolocation undergoes a maturation process through aging (Koopman et al., 2003; Scano et al., 2005).
Whereas the role of the melon in transmitting the sounds produced in the nasal sac to the water is clear, the contribution of the lower jaw in echolocation has been only supposed. The lower jaw construct of the dolphin consists of two mandibular bones (fused in one mandible), and the teeth. The mandible is hollow and thin, with a particular flared area, known as the “pan bone,” existing at the posterolateral third of the jaw (Wartzok and Ketten, 1999). The lower jawbone cavity is filled with fat bodies, rich in isovaleric acid. Externally, the pan bone region of each side of the lower jaw is covered with a fat deposit; it apparently continues with the lipids of the melon and of the channel inside the jaw, and extends toward the throat and from the posterior of the jaw to make contact with the tympano-periotic complex (Norris, 1968; Varanasi and Malins, 1970, 1971; Morris, 1986). This fat acts as a low density sound channel and conducts sounds from the flared portion of the lower jaw directly to the middle ear (Berta and Sumich, 1999).
Several studies report that the molecular composition of the mandibular fat is similar to that of the melon, but differs from blubber fat, and it is instrumental in sound transmission (Litchfield and Greenberg, 1974; Litchfield et al., 1975). It is worth noting that several studies reported that metabolism in this tissue is nominal (Cranford et al., 1996; Koopman et al., 2003; Houser et al., 2004). Some authors (Ames et al., 2002; Scano et al., 2005) report that this acoustic tissue contains few dietary lipids but high concentrations of endogenously synthesized branched iso-acids, among which isovalerate (iso5:0) is the most abundant molecular component. The presence of wax esters was also detected.
In a previous study (Scano et al., 2005), the melon of the striped dolphin (Stenella coeruleoalba), a species about which scanty information is available, was investigated by morphological and high-resolution 13C nuclear magnetic resonance (NMR) methods, the latter being the most suitable and nondestructive technique to analyze the lipid components in a complex mixture of compounds (Jie and Mustafa, 1997; Halliday et al., 1998). We characterized structure and composition at the histological and molecular level of two different regions (referred to as basal and apical) of the melon of both adult and juvenile individuals. The aim of the present work is to extend our above-mentioned study, by the same techniques, to the investigation of the molecular and histological structure of the fat bodies covering the pan bone of lower jaw of the striped dolphin. The analyses of samples belonging to adult and juvenile individuals were performed with the aim of seeking age-related differences.
iso5:0 = isovaleric acid; NMR = Nuclear Magnetic Resonance; TAG = triacylglycerols; WE = wax esters; mol% = molar; MUFA = mono unsaturated fatty acids; PUFA = poly unsaturated fatty acids; 2-MBA = 2-Methylbutyric acid; IBA = Isobutyric acid.
MATERIALS AND METHODS
We examined samples of extramandibular fat of the lower posterior jaw (Fig. 1) belonging to five individuals of Stenella coeruleoalba, here designated as adult 1 (age, 12–13 yr; sex, M), adult 2 (age, 15–17 yr; sex, F), adult 3 (age, 30–32 yr; sex, M), juvenile 1 (age, 3–5 months; sex, M), and juvenile 2 (age, 6–8 months; sex, F). The age of the dolphins was determined by counting dentine layers in sections of the teeth (Ridgway et al., 1975).
All tissue samples were dissected from dolphins found stranded along the shore of Sardinia (Italy) and the Mediterranean coasts of France in 2003 and 2004. Samples were from animals of approximately 2–12 hr postmortem time, and the data described in this study were obtained only from fresh or good-quality specimens. The heads were removed from the rest of the body, transferred to the Department of Experimental Biology, and completely dissected at the Department of Cytomorphology. After the complete excision of the extramandibular fat bodies, multiple fat samples were obtained from all different regions. Each sample was divided in two parts: the former was reduced in 1 × 5 cm logs, introduced in NMR tubes and immediately analyzed; the latter, cut in specimens of 2 × 1 cm for the morphological examination, was fixed in 10% buffered formalin for 24 hr and processed for paraffin embedding.
Because of the peculiar structure and composition of the tissues examined, we encountered difficulties in obtaining cryostat sections, because the tissue samples were not hard enough for cutting at −35°C. Thus, the study was carried out on formalin-fixed and paraffin-embedded microtome sections. Nevertheless, the histological treatment adopted for the paraffin-embedding technique, which consisted of dehydration in ethyl alcohol and diaphanation in xylene before inclusion, caused the solubilization of the lipids present in the tissues. Finally, the sections (6–7 μm) were stained with hematoxylin–eosin. Adjacent sections were used for the histochemical Masson's trichromic staining modified by Goldner for the elastic fibers, connective tissue, and collagen.
The histological features were evaluated by light microscopy using a Zeiss microscope (AxioPhot2, Carl Zeiss Vision GmbH, Hallbergmoos, Germany); images were acquired and processed by graphic card Matrox Meteor PCI Frame Grabber (Matrox Electronics Systems Ltd., Dorval, Canada) and by Zeiss AxioVision Software Rel. 3.0 (Carl Zeiss Vision GmbH), respectively.
Intact portions of tissue were examined in nonrotating 10-mm NMR tubes, without solvent, at 25°C. The 13C spectra were run on a Varian VXR-300 spectrometer at operating frequencies of 75.4 MHz. The 13C spectra were acquired using the NOE-suppressed, inverse-gated, proton decoupled technique (Waltz-16), with 1 sec of acquisition time, using a sweep width of 13 KHz. One thousand scans were collected using a 90-degree excitation pulse and a delay of 30 sec to allow for complete relaxation of each 13C signal.
Morphological analysis of the external fat of the posterolateral jaw showed that the lipid component is organized in an adipose tissue, with voluminous rounded cells (Fig. 2). In both adults and juveniles, this adipose tissue is morphologically similar to univacuolar adipose tissue composed of closely related cells, with the nucleus localized marginally between the plasma membrane and a big cytoplasmic lipid drop, and little extracellular matrix. Structural differences related to age were noticed: while in the juveniles a muscular component was observed, with bundles of fibers inserted in the adipose mass (Fig. 2d), not muscle fibers were found in the corresponding tissue of adults (Fig. 2a–c). The adipose tissue of adults presented instead large and irregularly shaped cavities (Fig. 2a–c). These cavities were generally empty or sometimes contained few blood cells. The cavities were bordered by a thin wall of flat cells, lying above a consistent layer of connective tissue, and were structurally organized as veins (Fig. 2a–c).
The 13C NMR spectra of intact tissues of the fat bodies of the lower posterior jaw of adults and juveniles of Stenella (Fig. 3) showed patterns of signals characteristic of lipid components, whereas no signal was observed for the molecular components of the muscular tissues. This result can be ascribed to the fact that the molecular components occurring in the muscles present so slow molecular motion that the related NMR signals are broadened beyond detection. The assignment of the signals, reported in Table 1, was obtained according to data reported in a previous study of the melon of Stenella and references cited therein (Scano et al., 2005).
Table 1. Peak assignments for 13C nuclear magnetic resonance spectrum of intact tissue of jaw fat bodies of a juvenile of Stenella
In all of the examined samples, triacylglycerols (TAG) and wax esters (WE), composed of linear, isovaleric, and longer isobranched fatty acids, were observed. In particular, the presence of 2-methylbutyric and isobutyric acids was detected in the NMR spectrum of the calves of Stenella. In the latter samples, the NMR spectrum showed signals at 182.68 and 20.03 ppm attributed to the muscle metabolite lactate. Furthermore, the peak at 54.66 ppm can be attributed to the -N+(CH3)3- functional group of phosphatidylcholine and/or to the -CH2- group of creatine/phosphocreatine.
The quantitative analysis was performed measuring the integrated areas of the 13C NMR signals; the data (mol %) are reported in Table 2. As far as the major lipid components (TAG and WE) are concerned, different results were found comparing the data concerning the jaw fat bodies of the adult individuals. In all of the investigated samples the concentration of the isovalerate compound was calculated as more than 50% of the total fatty acids. Smaller quantities of linear fatty acids and a lower degree of unsaturation were estimated in the adults as compared with the juveniles samples. Moreover, no topographical variations in the lipid composition of the extramandibular fat bodies of adults and juvenile dolphins was found.
Table 2. Percentage content (mol%) obtained by 13C nuclear magnetic resonance quantitative analysis of jaw fat of adults and juveniles of Stenella
Note. WE, wax esters; TAG, triacylglycerols.
Calculated from peaks 6 and 7.
The MUFA (monounsaturated fatty acids) and the PUFA (polyunsaturated fatty acids) contents were measured from the ratio of half area of peak 4 and 5, respectively, and area of the carboxyl signal which was normalized as 100, since peak 4 comprises signals from the PUFA, the PUFA content was then subtracted from the MUFA content.
The contents of isovaleric, isobranched and linear fatty acids, and alcohols were measured from areas of peaks 10, 12, 27, respectively.
Presents only as fatty acid.
The chain length of fatty acids, except of iso5:0, was calculated as the ratio between the sum of the areas of all the olefinic, methanetriyl methylene, and methyl functional groups (with the exclusion of the glycerol signals) and the acyl carbon atoms.
Au (1997) defines echolocation in dolphins as “the process whereby sounds are emitted and returning echoes are analyzed to detect and recognize objects underwater.” By echolocation, the presence of objects, their size, structure, material composition, and shape can be determined. It is widely accepted that the lower jaw acts as a specialized receiver that picks up and conducts the echoes bouncing back from the environments to the auditory bullae (Au, 1993). The lower jaw of the dolphin is hollow with a particularly thin area, known as the “pan,” located in the posterolateral region of the jaw.
The mandibular canals are filled with fatty deposits, rich in isovaleric acid, which extend from the posterior jaw to make contact with the tympano-periotic complex (Varanasi and Malins, 1970, 1971). This fat is an excellent sound conductor as it presents a low impedance with respect to water (Au, 1993). Sound energy traveling through water causes vibrations in the region of the lower jaw due to the thinness of the bone (Hughes, 1999). Once within the jaw, the sound waves can be transmitted to the bulla and subsequently toward the cochlea by the acoustic fat surrounding this area.
In Tursiops truncatus after the intravenous administration of 99mTc-bicisate followed by scanning with single photon emission computed tomography, Houser and colleagues (2004) detected a significant uptake of the ligand in the posterior region of the lower jaw indicative of perfusion to the fat bodies of this tissue. They suggest that such blood flow functions as a thermoregulatory control of lipid density in the lower jaw, as the sound velocity is inversely related to the temperature of acoustic lipids. This finding agrees with our finding, in Stenella coeruleoalba, of wide and enlarged veins that spread throughout the extramandibular fat. These vessels may regulate the blood flow in response to changing water temperature and stabilize thermal gradients within the lipids of the jaw by varying heat availability to this region. Houser et al. (2004) also reported a substantial 99mTc-bicisate uptake in the melon, although in our previous study (Scano et al., 2005), we did not find any hints of blood vessels in melon of Stenella, similar to those detected in extramandibular fat. This finding is probably due to the fact that we studied a sample obtained from the central part of melon, whereas Houser and coauthors revealed the greatest uptake in its frontal portion.
It is widely accepted that the echolocating organs involved in sound propagation and reception of sounds undergo a maturation process through aging (Gardner and Varanasi, 2003). In a previous study (Scano et al., 2005), we reported morphological and molecular differences between the melon of the adult and that of the juvenile of Stenella that may reflect different sound transmission and conduction properties related to the melon maturation stage. Koopman et al. (2003) reported that the patterns of accumulation of iso5:0 in different tissues (blubber, melon, and mandibular fats) of the harbor porpoises is age-related; isovalerate concentrations in melon increased from 12% to 35% in the first year of life, reaching a steady state by the age of 1 or 2 years. In the present study in Stenella, the level of iso5:0 (mol%) as calculated from the integrated areas of the 13C NMR spectrum of the fat bodies externally covering the jaw in the juvenile is comparable to those of the adult counterparts: it cannot be, therefore, regarded as an index of maturity of the organ. On the other hand, a much lower degree of unsaturation together with higher amounts of isobranched fatty acids were detected in the adults; all of these compounds are known to slow down sound transmission (Morris, 1975).
Age-related structural differences were also noticed in the extramandibular fat: a muscular component, with bundles of fibers inserted in the adipose mass, was observed in the juvenile, but not in the adults. This finding is confirmed by the presence in the 13C NMR quantitative spectrum of the samples of the juvenile of signals attributed to muscle metabolite lactate.
The morphological analysis of the extramandibular fat showed that, in both adults and juveniles, it is organized as univacuolar adipose tissue, constituted of closely related cells, with the nucleus localized marginally between the plasma membrane and a big cytoplasmic lipid drop, and little extracellular matrix; an analogous histological organization was previously reported in the adipose tissue of the melon of Stenella coeruleoalba (Scano et al., 2005). Furthermore, we observed that the adipose tissue of the juvenile lower jaw is composed of smaller cells than those of the adult counterparts. This finding was confirmed by the presence in the 13C NMR quantitative spectrum of the lower jaw fat bodies of the juvenile of signals attributable to phospholipids, that compose the cellular membranes. These results confirm the hypothesis of Gardner and Varanasi (2003) on Phocoena phocoena and Tursiops truncatus that the acoustic system is not fully developed at birth and that its biochemical structure changes throughout development.
In summary, the innovative integration of NMR and morphological techniques led us to demonstrate that the fat bodies of the posterolateral region of the jaw of Stenella coeruleoalba, as in the case of the melon, is morphologically organized as peculiar adipose tissue, the molecular components of which are different from those of blubber. Taking into consideration the structural differences between adult and juvenile, we can conclude that the molecular components and the histological organization may indicate a maturation of the organ with age that probably reflect different sound reception properties.
We thank the GECEM (Groupe d'Etude des Cétacés de Méditerranée) and the CSC (Centro Studi Cetacei, Italy) organizations for contributing and assisting with sample collection, and Mrs. Nicoletta Zinnarosu and Mr. Massimo Annis for their expert technical assistance.