Retrospective analysis of the lifetime endocrine response of southern right whale calves to gull wounding and harassment: A baleen hormone approach.

Physiological measurements are informative in assessing the relative importance of stressors that potentially impact the health of wildlife. Kelp Gulls, Larus dominicanus (KG), resident to the region of Península Valdés, Argentina, have developed a unique behavior of landing on the backs of southern right whale adults and calves, Eubalaena australis (SRW), where they feed on their skin and blubber. This parasitic behavior results in large open wounds on the dorsal surface of the whale. Coincidently, the SRW population off the coast of Península Valdés has experienced elevated calf mortality. We quantified levels of glucocorticoids and thyroid hormone extracted from baleen of dead calves to evaluate, retrospectively, the endocrine response of whale calves to gull wounding and harassment. Baleen accumulates hormones as it grows, allowing evaluation of long-term trends in physiological condition. While glucocorticoids (GCs) are known to increase in response to stressors such as disturbance, the metabolic hormone triiodothyronine (T3) has been shown to decrease under sustained food deprivation but is largely unaffected by disturbance stress. We quantified lifetime patterns of GCs and T3 in baleen recovered at necropsy from 36 southern right whale calves with varying severity of wounding from KGs. GC levels in baleen correlated positively with the degree of wounding, while T3 levels remained stable irrespective of the severity of the wounding. Our results suggest no evidence of malnutrition in low vs. severely wounded whales. However, the positive correlation of GCs with wound severity indicates that heavily wounded calves are suffering high levels of physiological stress before they die. This suggests that KG wounding may have contributed to the high southern right whale calf mortality observed in the Península Valdés region of Argentina.

Domoic acid in a marine pelagic food web: Exposure of southern right whales Eubalaena australis to domoic acid on the Península Valdés calving ground, Argentina.

The gulfs that surround Península Valdés (PV), Golfo Nuevo and Golfo San José in Argentina, are important calving grounds for the southern right whale Eubalaena australis. However, high calf mortality events in recent years could be associated with phycotoxin exposure. The present study evaluated the transfer of domoic acid (DA) from Pseudo-nitzschia spp., potential producers of DA, to living and dead right whales via zooplanktonic vectors, while the whales are on their calving ground at PV. Phytoplankton and mesozooplankton (primary prey of the right whales at PV and potential grazers of Pseudo-nitzschia cells) were collected during the 2015 whale season and analyzed for species composition and abundance. DA was measured in plankton and fecal whale samples (collected during whale seasons 2013, 2014 and 2015) using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The genus Pseudo-nitzschia was present in both gulfs with abundances ranging from 4.4×102 and 4.56×105 cell l-1. Pseudo-nitzschia australis had the highest abundance with up to 4.56×105 cell l-1. DA in phytoplankton was generally low, with the exception of samples collected during a P. australis bloom. No clear correlation was found between DA in phytoplankton and mesozooplankton samples. The predominance of copepods in mesozooplankton samples indicates that they were the primary vector for the transfer of DA from Pseudo-nitzschia spp. to higher trophic levels. High levels of DA were detected in four whale fecal samples (ranging from 0.30 to 710µgg-1 dry weight of fecal sample or from 0.05 and 113.6µgg-1 wet weight assuming a mean water content of 84%). The maximum level of DA detected in fecal samples (710µg DA g-1 dry weight of fecal sample) is the highest reported in southern right whales to date. The current findings demonstrate for the first time that southern right whales, E. australis, are exposed to DA via copepods as vectors during their calving season in the gulfs of PV.

Effects of acidification, lipid removal and mathematical normalization on carbon and nitrogen stable isotope compositions in beaked whale (Ziphiidae) bone.

RATIONALE: The analysis of stable isotopes in tissues such as teeth and bones has been used to study long-term trophic ecology and habitat use in marine mammals. However, carbon isotope ratios (δ(13) C values) can be altered by the presence of (12) C-rich lipids and carbonates. Lipid extraction and acidification are common treatments used to remove these compounds. The impact of lipids and carbonates on carbon and nitrogen isotope ratios (δ(15) N values), however, varies among tissues and/or species, requiring taxon-specific protocols to be developed. METHODS: The effects of lipid extraction and acidification and their interaction on carbon and nitrogen isotope values were studied for beaked whale (Ziphiidae) bone samples. δ(13) C and δ(15) N values were determined in quadruplicate samples: control, lipid-extracted, acidified and lipid-extracted followed by acidification. Samples were analyzed by means of elemental analysis isotope ratio mass spectrometry. Furthermore, the efficiency of five mathematical models developed for estimating lipid-normalized δ(13) C values from untreated δ(13) C values was tested. RESULTS: Significant increases in δ(13) C values were observed after lipid extraction. No significant changes in δ(13) C values were found in acidified samples. An interaction between both treatments was demonstrated for δ(13) C but not for δ(15) N values. No change was observed in δ(15) N values for lipid-extracted and/or acidified samples. Although all tested models presented good predictive power to estimate lipid-free δ(13) C values, linear models performed best. CONCLUSIONS: Given the observed changes in δ(13) C values after lipid extraction, we recommend a priori lipid extraction or a posteriori lipid normalization, through simple linear models, for beaked whale bones. Furthermore, acidification seems to be an unnecessary step before stable isotope analysis, at least for bone samples of ziphiids. Copyright © 2015 John Wiley & Sons, Ltd.

Organochlorine pesticides and PCBs in Southern Right Whales (Eubalaena australis) breeding at Península Valdés, Argentina.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) were assessed in blubber from 35 dead Southern Right Whales (SRW - Eubalaena australis) stranded at Península Valdés, Argentina. The life cycle includes a feeding period in high productivity areas of the South West Atlantic and a reproductive period in coastal template waters of Argentina. Organochlorine pesticides showed higher concentrations (22.6±13.8 ng·g(-1)ww) than PCBs (7.5±10 ng·g(-1)ww). Among pesticides, HCHs, DDTs, endosulfans, dieldrin, chlordans, heptachlor epoxide, and trans-nonachlor were detected. p,p'-DDE and p,p'-DDT were present in 69% and 26% of samples, respectively. p,p'-DDT/p,p'-DDE ratio showed low values (<0.33) as a result of aged DDT inputs. However, the occurrence of only p,p'-DDT in some samples suggests a recent pesticide input. α-HCH/γ-HCH ratio (

Stable isotopes indicate population structuring in the southwest Atlantic population of right whales (Eubalaena australis).

From the early 17th century to the 1970s southern right whales, Eubalaena australis, were subject to intense exploitation along the Atlantic coast of South America. Catches along this coast recorded by whalers originally formed a continuum from Brazil to Tierra del Fuego. Nevertheless, the recovery of the population has apparently occurred fragmentarily, and with two main areas of concentration, one off southern Brazil (Santa Catarina) and another off central Argentina (Peninsula Valdés). This pattern suggests some level of heterogeneity amongst the population, which is apparently contradicted by records that traced individuals moving throughout the whole geographical extension covered by the species in the Southwest Atlantic. To test the hypothesis of the potential occurrence of discrete subpopulations exploiting specific habitats, we investigated N, C and O isotopic values in 125 bone samples obtained from whaling factories operating in the early 1970s in southern Brazil (n=72) and from contemporary and more recent strandings occurring in central Argentina (n=53). Results indicated significant differences between the two sampling areas, being δ13C and δ18O values significantly higher in samples from southern Brazil than in those from central Argentina. This variation was consistent with isotopic baselines from the two areas, indicating the occurrence of some level of structure in the Southwest Atlantic right whale population and equally that whales more likely feed in areas commonly thought to exclusively serve as nursing grounds. Results aim at reconsidering of the units currently used in the management of the southern right whale in the Southwest Atlantic Ocean. In the context of the current die-off affecting the species in Peninsula Valdés, these results also highlight the necessity to better understand movements of individuals and precisely identify their feeding areas.

Could feeding habit and migratory behaviour be the causes of different toxicological hazard to cetaceans of Gulf of California (Mexico)?

In this work, a suite of diagnostic biomarkers was applied to seven cetacean species to evaluate the role of the feeding habits and migratory behavior in the toxicological status of these species from the Gulf of California, Mexico. We investigate the interspecific differences in cytochrome P450 1A1 and 2B (CYP1A1 and CYP2B, respectively), aryl hydrocarbon receptor and E2F transcription factor 1 and the contaminants levels [organochlorine compounds, polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs)] in four odontocete species (common bottlenose dolphin, long-beaked common dolphin, sperm whale and killer whale) and three mysticete species (blue whale, fin whale, and Bryde's whale) using skin biopsy. Differences in contaminant levels and molecular biomarker responses between the odontocete and mysticete species have been pointed out. The canonical discriminant analysis on principal component analysis factors, performed to reveal clustering variables, shows that odontocete are characterised by the highest levels of lipophilic contaminants compared to the mysticete, with the highest levels of polychlorinated biphenyls, dichlorodiphenyltrichloroethanes and PBDEs detected in killer whale and the lowest levels in Bryde's whale. The biomarker data show interspecific differences amongst the seven species, revealing highest CYP1A and CYP2B protein levels in the mysticete fish-eating species (Bryde's whale). In conclusion, three main factors seem to regulate the biomarker responses in these species: (a) the inductive ability of persistent organic pollutants and PAHs; (b) the different evolutionary process of the two CYPs related to the different feeding habits of the species; (c) the migratory/resident behaviour of the mysticete species in this area.

Trace metal concentrations in southern right whale (Eubalaena australis) at Península Valdés, Argentina.

The presence of essential (Fe, Mn, Zn, Cu, Ni and Al) and non-essential trace metals (Cd and Pb) was tested in liver (N=26) and kidney (N=42) from dead Southern Right Whale (SRW--Eubalaena australis) calves found beached in Península Valdés, Argentina. Essential metals were detected in all samples, particularly in hepatic tissue; though Ni and Al were accumulated mainly in the kidney. Cd and Pb were not detected in any samples. Sex and length of calves did not influence metal levels found, nor did the geographic location of carcasses. Our findings for essential metals were similar to those reported for mysticetes in other parts of the world. Except for a previous report on one SRW calf, this is the first data on trace metals for this species in Patagonia. This information is vital for SRW management considering increasing human pressures impacting their feeding and breeding grounds.

Differential antioxidant protection in tissues from marine mammals with distinct diving capacities. Shallow/short vs. deep/long divers.

The diving response in marine mammals results in bradycardia and peripheral vasoconstriction, with blood flow redistributing preferentially to nervous and cardiac tissues. Therefore, some tissues are rendered ischemic during a dive; with the first breath after a dive, blood flow to all tissues is reestablished. In terrestrial mammals, reactive oxygen species (ROS) production increases in response to ischemia/reperfusion and oxidative damage can occur. The capacity of marine mammals to tolerate repeated ischemia/reperfusion cycles associated with diving appears to be due to an enhanced antioxidant system. However, it is not known if diving depth and/or duration elicit differences in tissue capacity to produce ROS and antioxidant defenses in marine mammals. The objective of this study was to analyze ROS production, antioxidant defenses and oxidative damage in marine mammal species that perform shallow/short vs. deep/long dives. We measured production of superoxide radical (O(2)(•-)), oxidative damage to lipids and proteins, activity of antioxidant enzymes, and glutathione levels in tissues from shallow/short divers (Tursiops truncatus) and deep/long divers (Kogia spp.). We found that differences between the diving capacity of dolphins and Kogia spp. are reflected in O(2)(•-) production and antioxidant levels. These differences suggest that shallow/short and deep/long divers have distinct mechanisms to successfully maintain redox balance.

Histological, immunohistochemical and pathological features of the pituitary gland of odontocete cetaceans from the Western gulf of Mexico.

Pituitary glands were recovered from dolphins and small whales found stranded along the Texas coast of the Gulf of Mexico over a 15-year period (1991-2006). One hundred animals of 14 species were found to be suitable for inclusion in this study. Of these, 72 were Atlantic bottlenose dolphins (Tursiops truncatus). Other species included were the melon-headed whale (Peponocephala electra), spinner dolphin (Stenella longirostris), striped dolphin (Stenella coeruleoalba), pantropical spotted dolphin (Stenella attenuata), pygmy sperm whale (Kogia breviceps), dwarf sperm whale (Kogia sima), Risso's dolphin (Grampus griseus), the short finned pilot whale (Globicephala macrorhyncha), false killer whale (Pseudorca crassidens), Fraser's dolphin (Lagenorhynchus hosei), rough-tooth dolphin (Steno bredanensis), Gervais's beaked whale (Mesoplodon europaeus) and an infant sperm whale (Physeter catodon). The pituitary weights in T. truncatus ranged from 0.69 g in a 109-cm long neonate to 3.44 g in a large (277 cm) male. More typical weights were in the range of 0.95-2.35 g (mean=1.65+/-0.70 g) The cetacean pituitary consisted of two distinct parts, the adenohypophysis and the neurohypophysis, which were separated by a thin fibrous membrane in all species examined, in contrast to terrestrial mammals in which the parts are apposed and joined through a pars intermedia. Cell types were identified with conventional stains and immunohistochemistry. Cells positive for adrenocorticotropic hormone, follicle stimulating hormone, growth hormone, melanocyte stimulating hormone, luteinizing hormone and prolactin were identified with appropriate antibodies. Lesions, which were few, included one pituicytoma of the pars nervosa and a squamous cyst in T. truncatus, and colloid cysts in several species. Nodular aggregates of single cell types were common, probably representing a physiological variant.

Origin of the anomalous circular dichroism spectra of many apomyoglobin mutants.

Several authors have reported that many sperm whale apomyoglobin mutants show anomalous circular dichroism spectra. These mutants have a low molar ellipticity compared to the wild-type protein but in several cases have the same stability of unfolding. A model in which native apomyoglobin is not folded in the same manner as that in other proteins and in which mutants show progressive reductions in their degree of folding has been suggested to explain this phenomenon. However, nuclear magnetic resonance of the native apomyoglobin conformation has shown that this state is folded and compact, raising the possibility that the anomalous circular dichroism spectra could have another explanation. We studied several mutants with anomalous circular dichroism spectra and found that these proteins were all contaminated with nucleic acid that contributed to the ultraviolet absorption and caused uncertainty in the determination of protein concentration. The resulting overestimation of the concentration of apomyoglobin explains the phenomenon of anomalous circular dichroism spectra. We describe a procedure to remove the contaminant nucleic acid which yields accurate protein concentration measurements and provides the normal circular dichroism spectra. Our findings support a well-structured native conformation for apomyoglobin and may also be of the interest to scientists working with the purification of recombinant proteins.

Trace metals in tissues of gray whale (Eschrichtius robustus) carcasses from the Northern Pacific Mexican Coast.

Samples of liver, lung, heart, muscle, and blubber tissue from the carcasses of juvenile gray whales (Eschrichtius robustus) found stranded on the coast off the Sea of Cortez, México were analyzed for a range of trace metals (Cu, Fe, Zn, Mn, Ni, Pb, and Cd). The highest concentrations of copper, iron, zinc, and manganese were found in liver; nickel and lead in heart, and cadmium in kidney. In all tissues analyzed, iron, zinc and copper were present in the highest concentrations; however, some whales also showed high levels of cadmium in the kidney which could be related to their diet. Elevated concentrations of copper were found only in the liver of one whale. In general, where low levels of iron were found in the liver, they were associated with poor nutrition. Lead, nickel, manganese and zinc levels in liver were within the normal range, indicating that these whales had not been exposed to high levels of these metals.