Isolation and characterization of novel microsatellite loci for the short‐beaked common dolphin (Delphinus delphis) and cross‐amplification in other cetacean species

Seven tetranucleotide and three dinucleotide microsatellite loci were isolated from the short‐beaked common dolphin (Delphinus delphis). Number of alleles and observed heterozygosity ranged from three to twelve (mean 8.4) and from 0.087 to 0.978 (mean 0.741), respectively. All loci were tested in four other odontocete species and showed polymorphisms in most locus–species combinations. This panel of nuclear markers will be useful for the investigation of population ecology and social interactions in common dolphins and other cetacean species.     

The karyotype of Franciscana dolphin (Pontoporia blainvillei)

Despite the recent increase in studies on franciscana dolphin (Pontoporia blainvillei) molecular biology, there has been no published karyotype information, as opportunities for sampling live individuals are very rare. In the present study, the diploid number of the species was established from corneal cell cultures of 2 newborn male franciscanas live stranded (2n = 44). From the comparison of the chromosomal number to the cetacean karyotype phylogeny, we suggest that the most parsimonious hypothesis is that the ancestral character state in the group is the diploid number of 42, with an extra chromosome pair appearing independently twice during cetacean evolution, once in the suborder Odontoceti and once in the suborder Mysticeti. This information on chromosomal number may be useful to future genetic mapping projects of the species.     

Diffusion tensor imaging of dolphin brains reveals direct auditory pathway to temporal lobe

The brains of odontocetes (toothed whales) look grossly different from their terrestrial relatives. Because of their adaptation to the aquatic environment and their reliance on echolocation, the odontocetes'' auditory system is both unique and crucial to their survival. Yet, scant data exist about the functional organization of the cetacean auditory system. A predominant hypothesis is that the primary auditory cortex lies in the suprasylvian gyrus along the vertex of the hemispheres, with this position induced by expansion of ‘associative′ regions in lateral and caudal directions. However, the precise location of the auditory cortex and its connections are still unknown. Here, we used a novel diffusion tensor imaging (DTI) sequence in archival post-mortem brains of a common dolphin (Delphinus delphis) and a pantropical dolphin (Stenella attenuata) to map their sensory and motor systems. Using thalamic parcellation based on traditionally defined regions for the primary visual (V1) and auditory cortex (A1), we found distinct regions of the thalamus connected to V1 and A1. But in addition to suprasylvian-A1, we report here, for the first time, the auditory cortex also exists in the temporal lobe, in a region near cetacean-A2 and possibly analogous to the primary auditory cortex in related terrestrial mammals (Artiodactyla). Using probabilistic tract tracing, we found a direct pathway from the inferior colliculus to the medial geniculate nucleus to the temporal lobe near the sylvian fissure. Our results demonstrate the feasibility of post-mortem DTI in archival specimens to answer basic questions in comparative neurobiology in a way that has not previously been possible and shows a link between the cetacean auditory system and those of terrestrial mammals. Given that fresh cetacean specimens are relatively rare, the ability to measure connectivity in archival specimens opens up a plethora of possibilities for investigating neuroanatomy in cetaceans and other species.     

Occurrence of the ectoparasite Isocyamus deltobranchium (Amphipoda: Cyamidae) on cetaceans from Atlantic waters

The cyamids collected from a True's beaked whale (Mesoplodon mirus), a common dolphin (Delphinus delphis), and a harbour porpoise (Phocoena phocoena) stranded in Galicia (NW Spain) were identified as Isocyamus deltobranchium, extending the range of this species to the Atlantic Ocean. The three cetacean species examined are new hosts for this parasite. The microscopic examination of female specimens reveals that certain important taxonomic characters can be present or absent depending on the maturation stage, an aspect which should be considered in future studies describing cyamid species.     

Characterization of the cardiac phospholipidome of small cetaceans provides adaptational insight and a foundation for indirect population health screening

The maintenance of a tightly regulated heart lipidome is a general hallmark of mammal health. However, in the case of cetaceans, heart tissue lipid composition remains obscure. This work represents a first in-depth state-of-the-art characterization of the lipid content of small cetacean hearts, namely those of Delphinus delphis, Phocoena phocoena, and Stenella coeruleoalba. Phospholipid classes identified were similar to those found in terrestrial mammals. Regarding fatty acid profiles, they revealed a much higher n-3 fatty acid content than that reported for terrestrial counterparts. Phospholipidome characterization allowed identification of 198 lipid species, including phospholipids, lysophospholipids, and sphingomyelins, generally characterized by a high content of species with esterified n-3 fatty acids. The cardiac lipid profile of the three cetacean species is similar in terms of lipid classes, fatty acids, and even molecular lipid species present. However, quantitative differences suggest a divergence of S. coeruleoalba from the other species in terms of fatty acid and phospholipid profile. The identified differences regarding terrestrial counterparts will contribute to better understanding the details of small cetacean cardiac physiology. Moreover, the characterization of cardiac phospholipidome may represent an opportunity to establish lipidomic-based practical tools to reliably and reproducibly assess small cetacean health population status by remote sampling.     

DOES ELASTIN CONTRIBUTE TO THE PERSISTENCE OF CORPORA ALBICANTIA IN THE OVARY OF THE COMMON DOLPHIN (DELPHINUS DELPHIS)

The corpora albicantia (CAs) from the ovaries of 39 common dolphins ( Delphinus delphis ) caught in driving fisheries were used, and histologically and immunohistochemically examined. The area of each corpus albicans (CA) and the proportion of that area occupied by elastin were measured using NIH-image software. In all CAs, elastoid material (EM) was apparent although EM area varied in each CA. CAs increased in number with dolphin age. Smaller CAs contained a higher proportion of EM. EM was completely digested by elastase, but not by collagenase. Furthermore, EM was immunostained with anti-α-elastin antibody. These results demonstrated that EM was elastin. The present study is the first to describe the presence of elastin in cetacean CAs. The higher proportion of elastin in small-sized CAs of common dolphins is suggested as the likely cause of the persistence of CA.     

Eight polymorphic microsatellite loci for bottlenose dolphin and other cetacean species

We developed eight polymorphic microsatellite loci that can be used to elucidate population structure and aid management of bottlenose dolphins, Tursiops truncatus, in the northwest Atlantic. These loci averaged nine alleles, with no evidence for null alleles. Their cross‐species utility was examined in Cephalorhynchus commersonii, Delphinus delphis, Delphinapterus leucas, Eschrictius robustus, Globicephala macrorhynchus, Lagenorhynchus obliquidens, Orcinus orca, Steno bredanensis and Stenella clymene. On average, 75% of the loci were polymorphic in these species (range = 50–88%). Our results indicate that these loci will be useful for elucidating population structure of bottlenose dolphin as well as of other cetacean species.     

委内瑞拉东南加勒比海岸七种海洋鲸类的潜在地理分布

本研究阐述了委内瑞拉沿海最常见的海洋鲸类的潜在地理分布情况。通过GIS分析,鲸类分布与地形和深度是相反的,以此可以获得鲸类潜在的分布图。已报告确认了16个物种(有402个目击报告),其中小布氏鲸(Balaenoptera edeni)、座头鲸(Megaptera novaeangliae)、海豚(Delphinus spp.)、圭亚那侏型豚(Sotalia guianensis)、花斑原海豚(Stenella frontalis)、长吻原海豚(Stenella longirostris)和宽吻海海豚(Tursiops truncatus)是最常见的。小布氏鲸和宽吻海海豚有可能分布于整个海岸,包括江河区域。座头鲸则可能季节性地集中分布在大陆架上的海岛沿岸和浅水区域。海豚(Delphinus spp.)有可能分布于高边坡区或沿海上升流区。花斑原海豚可能分布在东北区的西部,中部沿海以及与委内瑞拉海岸平行的岛屿周围。长吻原海豚则分布于浅海和远海区域。在一些高产的生态系统内新的物种可能正在形成,而这些潜在可能的分布图可以作为在高产生态系统内确立关键栖息地的标准,由此我们可以在委内瑞拉水域为鲸类建立新的保护区。     

Evolution of Functional Genes in Cetaceans Driven by Natural Selection on a Phylogenetic and Population Level

Cetaceans represent an evolutionary lineage marked by drastic morphological and physiological changes during their adaptation to an exclusively marine existence. In addition, several cetacean species exhibit geographical ranges that encompass different marine environments, with genetic breaks being sometimes consistent with environmental breaks. As such, genes that underwent adaptation during the land-sea transition can also be potential candidates for adaptation to different oceanic environments. In this study, we analysed 3 milk protein genes (β-casein, κ-casein, and α-lactalbumin) and 2 immunity related genes (MHC DQβ1 and γ-fibrinogen) for selection based on available phylogenetic datasets of both mammals and cetaceans, and used the results from this analysis to assess adaptation to different environments on a population level in the European common dolphin (Delphinus delphis). We found that evidence for positive selection could be detected in all genes in the phylogenetic analyses, with β-casein showing a further increase in selective pressure in the cetacean lineage. At the population level, both the immune system locus DQβ1 and β-casein genes showed patterns of variation consistent with divergent selection, and in each case the same populations showed differentiation. One of these populations was also differentiated at neutral markers, while the other was not. We discuss possible inference, and the potential for the further development of these ideas using genomic technologies.     

Phylogeography and alpha taxonomy of the common dolphin (Delphinus sp.)

The resolution of taxonomic classifications for delphinid cetaceans has been problematic, especially for species in the genera Delphinus, Tursiops and Stenella. The frequent lack of correspondence between morphological and genetic differentiation in these species raises questions about the mechanisms responsible for their evolution. In this study we focus on the genus Delphinus, and use molecular markers to address questions about speciation and the evolution of population structure. Delphinus species have a worldwide distribution and show a high degree of morphological variation. Two distinct morphotypes, long-beaked and short-beaked, have been considered different species named D. capensis and D. delphis, respectively. However, genetic differentiation between these two forms has only been demonstrated in the Pacific. We analysed samples from eight different geographical regions, including two morphologically defined long-beaked form populations, and compared these with the eastern North Pacific populations. We found high differentiation among the populations described as long-beaked instead of the expected monophyly, suggesting that these populations may have evolved from independent events converging on the same morphotype. We observed low genetic differentiation among the short-beaked populations across a large geographical scale. We interpret these phylogeographical patterns in the context of life history and population structure in related species.     

Ultrastructure of the dolphin adenohypophysis

Summary The ultrastructural appearances of the cell types are described in the pars distalis of common (Pacific) dolphins, Delphinus delphis. Tentative functions are assigned to these cells and discussed in relation to some of the known adaptations of these mammals to a marine environment.     

Composition of the milks of the bottlenose dolphin (Tursiops trucatus) and the Florida manatee (Trichechus manatus latirostris)

Milk samples from four individual bottlenose (Tursiops truncatus) and two Florida manatees (Trichechus manatus latirostris) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of alpha-lactalbumin, the regulatory protein of lactose synthase. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of lactose, as determined by several criteria. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of lactose together with two components that were tentatively identified as oligosaccharides. alpha-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.     

Characterization of alpha-fetoprotein in fetal striped dolphin (Stenella coeruleoalba): purification of protein product and molecular cloning of the corresponding transcript

Alpha-fetoprotein (AFP) is a fetal glycoprotein that is known as a biomarker for monitoring pregnancy in many mammalian species. However, characterization of AFP has not yet been undertaken in any cetacean species. Here, we purified AFP from the serum of fetal striped dolphin by chemical precipitation followed by a combination of immunoadsorbent column chromatography and gel filtration. The molecular masses of native and denatured dolphin AFP were estimated to be ～78,000 Da by gel filtration and ～68,000 Da by SDS-PAGE, respectively, representing typical masses reported for mammalian AFPs. In fetal serum, only the AFP band (～68,000 Da) appeared to be immunoreactive to an antiserum against purified dolphin AFP, indicating sufficient specificity for the development of an AFP immunoassay. Full-length cDNA encoding for the dolphin AFP was cloned from fetal liver and revealed an open reading frame comprising 610 amino acid residues, which included a putative signal peptide of 18 amino acid residues. This was followed by a sequence identical to the N-terminus of purified AFP. The deduced amino acid sequence of dolphin AFP showed more than 80% identity to those of other mammalian AFPs. To our knowledge, the present report represents the first identification and characterization of AFP from any cetacean species.     

First report of major histocompatibility complex class II loci from the Amazon pink river dolphin (genus Inia)

We report the first major histocompatibility complex (MHC) DQB1 sequences for the two species of pink river dolphins (Inia geoffrensis and Inia boliviensis) inhabiting the Amazon and Orinoco River basins. These sequences were found to be polymorphic within the Inia genus and showed shared homology with cetacean DQB-1 sequences, especially, those of the Monodontidae and Phocoenidae. On the other hand, these sequences were shown to be divergent from those described for other riverine dolphin species, such as Lipotes vexillifer, the Chinese river dolphin. Two main conclusions can be drawn from our results: 1) the Mhc DQB1 sequences seem to evolve more rapidly than other nuclear sequences in cetaceans, and 2) differential positive selective pressures acting on these genes cause concomitant divergent evolutionary histories that derive phylogenetic reconstructions that could be inconsistent with widely accepted intertaxa evolutionary relationships elucidated with other molecular markers subjected to a neutral dynamics.     

Modular organizations of novel cetacean papillomaviruses

The phylogenetic position of cetacean papillomaviruses (PVs: Omikron-PVs and Upsilon-PVs) varies depending on the region of the genome analysed. They cluster together with Alpha-PVs when analysing early genes and with Xi-PVs and Phi-PVs when analysing late genes. We cloned and sequenced the complete genomes of five novel PVs, sampled from genital and oesophageal lesions of free-ranging cetaceans: Delphinus delphis (DdPV1), Lagenorhynchus acutus (TtPV3 variant), and Phocoena phocoena (PphPV1, PphPV2, and PphPV3). Using Maximum Likelihood and Bayesian approaches, all cetacean PVs constituted a monophyletic group with Alpha-, Omega-, and Dyodelta-PVs as inferred from E1-E2 early genes analyses, thus matching the shared phenotype of mucosal tropism. However, cetacean PVs, with the exception of PphPV3, were the closest relatives of Xi-PVs and Phi-PVs in L2-L1 late genes analyses, isolated from cow and goat, thus reflecting the close relationship between Cetacea and Artiodactyla. Our results are compatible with a recombination between ancestral PVs infecting the Cetartiodactyla lineage. Our study supports a complex evolutionary scenario with multiple driving forces for PV diversification, possibly including recombination and also interspecies transmission.     

In vitro metabolism of polychlorinated biphenyl congeners by beluga whale (Delphinapterus leucas) and pilot whale (Globicephala melas) and relationship to cytochrome P450 expression

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.     

Bottlenose dolphin (Tursiops truncatus) depredation resulting in larynx strangulation with gill-net parts

Cetacean interactions with fishing gear are reported regularly and most frequently involve incidental capture. However, limited records exist related to depredation resulting in fishing gear ingestion. Here, we present cases of depredation resulting from ingestion of gill-net parts and larynx strangulation documented for the first time in a cetacean species. The bottlenose dolphin ( Tursiops truncatus ) is the only resident species in the Adriatic Sea, where small-scale commercial and private fisheries use gill nets throughout the year. A total of 120 dead-stranded bottlenose dolphins found along the Croatian coast of the Adriatic Sea were examined from 1990 to March 2008; 12 of them (10%) were affected by larynx strangulation with gill-net parts. The larynx of all affected animals showed at least one of the following pathological changes: edema, mucosal injury, and hypergranulation. In the majority of cases, it was a chronic condition. Larynx strangulations were found only in adult animals and appeared more often in animals with reduced ability to catch free-swimming prey.     

Ultrastructure of light cells in the dolphin thyroid

Summary The ultrastructural appearance of the light cells is described in the thyroid gland of the common (Pacific) dolphinDelphinus bairdi. The appearances of active light cells are discussed in relation to the production of thyrocalcitonin and the special osteological characteristics of marine mammals. The presence of nerve processes possibly associated with light cells is discussed.Also known as parafollicular cells (Nonidez, 1932) and C cells (Pearse, 1966).     

Body size and skeletal muscle myoglobin of cetaceans: adaptations for maximizing dive duration

Cetaceans exhibit an exceptionally wide range of body mass that influence both the capacities for oxygen storage and utilization; the balance of these factors is important for defining dive limits. Furthermore, myoglobin content is a key oxygen store in the muscle as it is many times higher in marine mammals than terrestrial mammals. Yet little consideration has been given to the effects of myoglobin content or body mass on cetacean dive capacity. To determine the importance of myoglobin content and body mass on cetacean diving performance, we measured myoglobin content of the longissimus dorsi for ten odontocete (toothed whales) and one mysticete (baleen whales) species ranging in body mass from 70 to 80000 kg. The results showed that myoglobin content in cetaceans ranged from 1.81 to 5.78 g (100 g wet muscle)(-1). Myoglobin content and body mass were both positively and significantly correlated to maximum dive duration in odontocetes; this differed from the relationship for mysticetes. Overall, the combined effects of body mass and myoglobin content accounts for 50% of the variation in cetacean diving performance. While independent analysis of the odontocetes showed that body mass and myoglobin content accounts for 83% of the variation in odontocete dive capacity.     

The composition of serval (Felis serval) milk during mid-lactation

Milk was obtained from three captive servals. The average nutrient content was 158.3+/-44.4 g protein; 152.6+/-62.3 g fat; and 68.7+/-31.4 g lactose per kg milk. The protein fraction respectively consisted of 117.7+/-44.8 g caseins per kg milk and of 40.6+/-6.7 g whey proteins per kg milk. Electrophoresis and identification of protein bands showed a similar migrating sequence of proteins as seen in cheetah and cat milk, with small differences in the beta-caseins. The lipid fraction contains 313.3+/-18.8 g saturated and 338.6+/-11.9 g mono unsaturated fatty acids per kg milk fat respectively. The high content of 292.4+/-24.9 g kg(-1) milk fat of polyunsaturated fatty acids is due to a high content in linolenic acid. No short chain fatty acids, but substantial levels of uneven carbon chain fatty acids were observed. In general, serval milk has a higher protein and fat content than that of the domestic cat and cheetah, and a lower content of unsaturated fatty acids than that of the domestic cat.