Variation in blubber thickness and histology metrics across the body topography of a false killer whale (Pseudorca crassidens).

Blubber is a multifunctional tissue essential to the survival of cetaceans. Histological assessment of blubber may be useful in determining odontocete nutritional state but a greater understanding of specific variation across the body is needed. We report on morphological variation of the blubber according to girth axes and sampling planes in a sub-adult male, bycaught false killer whale (Pseudorca crassidens) using metrics of blubber thickness (BT), adipocyte area (AA), and adipocyte index (AI). 48 full depth blubber samples were taken along 6 girth axes at 5 equidistant sampling points on both sides of the body. At these sampling locations BT was recorded, and AA and AI were determined for three distinct blubber layers. Linear mixed effect models were used to assess variation of the blubber across layers and body topography. BT was somewhat non-uniform across the body but was generally thicker in the dorsal region and thinner laterally. AA was greater cranially and AI was greater caudally. The middle and inner layer blubber showed significant differences dorsoventrally with larger AA and smaller AI in the ventral region of the body. Variation of the blubber metrics across the body are indicative of variable functions of the blubber within an individual. Due to the variability observed, we expect that AI of the dynamic inner layer blubber is most informative of overall body condition and that biopsy samples of the outer and middle blubber may still be useful in determining the nutritional status of live false killer whales.

Novel cetacean morbillivirus in a rare Fraser's dolphin (Lagenodelphis hosei) stranding from Maui, Hawai'i.

Cetacean morbillivirus (CeMV) is a global threat to cetaceans. We report a novel morbillivirus from a Fraser's dolphin (Lagenodelphis hosei) that stranded in Maui, Hawaii in 2018 that is dissimilar to the beaked whale morbillivirus previously identified from Hawaii and to other CeMV strains. Histopathological findings included intranuclear inclusions in bile duct epithelium, lymphoid depletion, rare syncytial cells and non-suppurative meningitis. Cerebellum and lung tissue homogenates were inoculated onto Vero.DogSLAMtag cells for virus isolation and cytopathic effects were observed, resulting in the formation of multinucleated giant cells (i.e., syncytia). Transmission electron microscopy of infected cell cultures also revealed syncytial cells with intracytoplasmic and intranuclear inclusions of viral nucleocapsids, consistent with the ultrastructure of a morbillivirus. Samples of the cerebellum, lung, liver, spleen and lymph nodes were positive for morbillivirus using a reverse transcription-polymerase chain reaction. The resulting 559 bp L gene sequence had the highest nucleotide identity (77.3%) to porpoise morbillivirus from Northern Ireland and the Netherlands. The resulting 248 bp P gene had the highest nucleotide identity to porpoise morbillivirus in Northern Ireland and the Netherlands and to a stranded Guiana dolphin (Sotalia guianensis) in Brazil (66.9%). As Fraser's dolphins are a pelagic species that infrequently strand, a novel strain of CeMV may be circulating in the central Pacific that could have additional population impacts through transmission to other small island-associated cetacean species.

Rapid weight loss in free ranging pygmy killer whales (Feresa attenuata) and the implications for anthropogenic disturbance of odontocetes.

Understanding the impacts of foraging disruptions to odontocete body condition is fundamental to quantifying biological effects of human disturbance and environmental changes on cetacean populations. Here, reductions in body volume of free-ranging pygmy killer whales (Feresa attenuata) were calculated using repeated measurements of the same individuals obtained through Unoccupied Aerial System (UAS)-photogrammetry during a prolonged disruption in foraging activity arising from a 21-day stranding event. Stranded individuals were used to verify UAS-derived volume and length estimates through 3D-imaging, water displacement, and post-mortem measurements. We show that (a) UAS estimates of length were within 1.5% of actual body length and UAS volume estimates were within 10-13% of actual volume, (b) foraging disruption resulted in a daily decrease of 2% of total body mass/day, and (c) pygmy killer whales can lose up to 27% of their total body weight within 17 days. These findings highlight the use of UAS as a promising new method to remotely monitor changes in body condition and animal health, which can be used to determine the potential effects of anthropogenic disturbance and environmental change on free-ranging odontocetes.

Life history and social structure as drivers of persistent organic pollutant levels and stable isotopes in Hawaiian false killer whales (Pseudorca crassidens).

False killer whales are long-lived, slow to mature, apex predators, and therefore susceptible to bioaccumulation of persistent organic pollutants (POPs). Hawaiian waters are home to three distinct populations: pelagic; Northwestern Hawaiian Islands (NWHI) insular; and main Hawaiian Islands (MHI) insular. Following a precipitous decline over recent decades, the MHI population was listed as "endangered" under the Endangered Species Act in 2012. This study assesses the risk of POP exposure to these populations by examining pollutant concentrations and ratios from blubber samples (n = 56) related to life history characteristics and MHI social clusters. Samples were analyzed for PCBs, DDTs, PBDEs, and some organochlorine pesticides. Skin samples (n = 52) were analyzed for stable isotopes δ13C and δ15N to gain insight into MHI false killer whale foraging ecology. Pollutant levels were similar among populations, although MHI whales had a significantly higher mean ratio of DDTs/PCBs than NWHI whales. The ∑PCB concentrations of 28 MHI individuals (68%) sampled were equal to or greater than suggested thresholds for deleterious health effects in marine mammals. The highest POP values among our samples were found in four stranded MHI animals. Eight of 24 MHI adult females have not been documented to have given birth; whether they have yet to reproduce, are reproductive senescent, or are experiencing reproductive dysfunction related to high POP exposure is unknown. Juvenile/sub-adults had significantly higher concentrations of certain contaminants than those measured in adults, and may be at greater risk of negative health effects during development. Multivariate analyses, POP ratios, and stable isotope ratios indicate varying risk of POP exposure, foraging locations and potentially prey items among MHI social clusters. Our findings provide invaluable insight into the ongoing risk POPs pose to the MHI population's viability, as well as consideration of risk for the NWHI and pelagic stocks.

Perfluorinated Alkyl Acids in Hawaiian Cetaceans and Potential Biomarkers of Effect: Peroxisome Proliferator-Activated Receptor Alpha and Cytochrome P450 4A.

Perfluorinated alkyl acids (PFAAs) are persistent in marine biota and are toxic to many species, including marine mammals. We measured the concentrations of 15 PFAAs in liver and kidney samples of 16 species of stranded cetaceans from Hawai'i and other tropical North Pacific regions utilizing high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eleven PFAAs in liver and nine PFAAs in kidney were detected, including substantial perfluorooctanesulfonate (PFOS) and perfluoroundecanoic acid (PFUnA). Regression models indicated that phylogenetic family and age class significantly influenced concentrations of certain PFAAs. PFAAs can activate transcription factor peroxisome proliferator-activated receptor alpha (PPARα), which induces transcription of cytochrome P450 4A (CYP4A). Relative expression of PPARα and CYP4A mRNA was quantified using real-time PCR (qPCR) and CYP4A protein expression, using Western blot and then compared to PFAA concentrations in liver and kidney. Concentrations of four PFAA congeners, summation of perfluoroalkyl carboxylic acids (ΣPFCAs), and ΣPFAAs correlated significantly with PPARα mRNA expression and CYP4A protein expression in kidney, suggesting either may be biomarkers of PFAA exposure in cetaceans. This is the first study to quantify PFAAs in marine mammals from this region and the first observation of a direct relationship between PFAA exposure and PPARα and CYP4A expression in cetaceans.

BLOOD CHEMISTRY AND HEMATOLOGY VALUES IN HEALTHY AND REHABILITATED ROUGH-TOOTHED DOLPHINS ( STENO BREDANENSIS).

Rehabilitation efforts for live stranded marine mammals are guided by diagnostic measures of blood chemistry and hematology parameters obtained from each individual undergoing treatment. Despite the widespread use of blood parameters, reference values are not available in the literature from healthy rough-toothed dolphins ( Steno bredanensis) with which to infer the health status of an animal. We examined serum or plasma chemistry and hematology data from 17 rough-toothed dolphins either housed at Dolphin Quest French Polynesia or during their rehabilitation at the Dolphin and Whale Hospital in Sarasota, Florida, US between 1994 and 2005. Blood parameters were compared among healthy animals, rehabilitation animals that were eventually released, and rehabilitation animals that died. This study indicated significant differences in many blood parameters for the poorly known rough-toothed dolphin that are likely to vary between healthy and sick animals. These included aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, bicarbonate, and globulins, which were greater in sick dolphins, and alkaline phosphatase and total protein which were greater in healthy individuals. Total white blood cell counts were lower in healthy animals as were the absolute numbers of neutrophils, monocytes, and eosinophils. Analysis of first blood sample levels for glucose, sodium, and erythrocyte sedimentation rate may have value for triage and prognostic evaluation.

Initial characterization of novel beaked whale morbillivirus in Hawaiian cetaceans.

Cetacean morbillivirus (CeMV) is a causative factor in epizootics that have resulted in thousands of deaths throughout the Atlantic and Mediterranean since 1987, but less is known of its presence and significance in the Pacific. The first case of CeMV reported in Hawai'i was in a Longman's beaked whale that stranded in 2010. The initial CeMV sequence from this individual indicated the possibility of a novel strain. To address this, archived samples from cetaceans that stranded in Hawai'i between 1997 and 2014 were screened for CeMV. The beaked whale morbillivirus (BWMV) was detected in 15 individuals representing 12 different species (24% of Code 1 and 2 stranded cetaceans). The earliest detected case was a humpback whale that stranded in 1998. Sequence comparisons of a 2.2 kb sequence spanning the phosphoprotein (P) and nucleocapsid (N) genes strongly suggest that the BWMV represents a novel strain of CeMV present in Hawai'i and the Central Pacific. In contrast to recently reported isolates from Brazil and Australia that may represent a distinct clade, BWMV appears to be more closely related to known strains of CeMV (dolphin morbillivirus; porpoise morbillivirus; and pilot whale morbillivirus). Detection rates with repeat sampling of positive lymph nodes were between 2 and 61%, illustrating the extreme heterogeneity that can occur in affected tissues. Taken together, these results suggest that BWMV may be common and established in Hawaiian cetacean populations. BWMV will be important for understanding CeMV and health threats in the relatively understudied cetaceans of the Pacific.

Using cytochrome P4501A1 expression in liver and blubber to understand effects of persistent organic pollutant exposure in stranded Pacific Island cetaceans.

Elevated levels of persistent organic pollutants (POPs) have been reported in tropical Pacific Island cetaceans and their environment. In addition, recent health concerns in cetacean populations have warranted investigation into potential physiological effects from POP exposure for this region. Cytochrome P450 1A1 (CYP1A1) is a candidate for examining such effects. This well-studied biomarker of exposure and effect was examined in stranded cetacean liver using immunoblot (n = 39, 16 species) and blubber using immunohistochemistry (n = 23, 10 species). Paired tissue samples allowed for CYP1A1 comparisons not only between species but also within each individual animal to examine differences between tissue types. Liver CYP1A1 expression correlated positively and significantly with blubber concentrations of all POP categories (n = 39, p < 0.050) except octachlorostyrene and pentachlorobenzene (p > 0.100). Among Stenella species, liver CYP1A1 tissue expression was correlated negatively with the sum of all blubber layer endothelial cell CYP1A1 expression (n = 14, p = 0.049). Overall, elevated expression of liver CYP1A1 confirms its use as a biomarker of POP exposure to cetaceans stranded in the tropical Pacific basin.

Coinfection and vertical transmission of Brucella and Morbillivirus in a neonatal sperm whale (Physeter macrocephalus) in Hawaii, USA.

The viral genus Morbillivirus and the bacterial genus Brucella have emerged as important groups of pathogens that are known to affect cetacean health on a global scale, but neither pathogen has previously been reported from endangered sperm whales (Physeter macrocephalus). A female neonate sperm whale stranded alive and died near Laie on the island of Oahu, Hawaii, US, in May of 2011. Congestion of the cerebrum and enlarged lymph nodes were noted on the gross necropsy. Microscopic findings included lymphoid depletion, chronic meningitis, and pneumonia, suggesting an in utero infection. Cerebrum, lung, umbilicus, and select lymph nodes (tracheobronchial and mediastinal) were positive for Brucella by PCR. Brucella sp. was also cultured from the cerebrum and from mediastinal and tracheobronchial lymph nodes. Twelve different tissues were screened for Morbillivirus by reverse-transcriptase (RT)-PCR and select tissues by immunohistochemistry, but only the tracheobronchial lymph node and spleen were positive by RT-PCR. Pathologic findings observed were likely a result of Brucella, but Morbillivirus may have played a key role in immune suppression of the mother and calf. The in utero infection in this individual strongly supports vertical transmission of both pathogens.

Persistent organic pollutant concentrations in blubber of 16 species of cetaceans stranded in the Pacific Islands from 1997 through 2011.

Persistent organic pollutants (POPs) are toxic man-made chemicals that bioaccumulate and biomagnify in food webs, making them a ubiquitous threat to the marine environment. Although many studies have determined concentrations of POPs in top predators, no studies have quantified POPs in stranded cetaceans within the last 30 years around the Hawaiian Islands. A suite of POPs was measured in the blubber of 16 cetacean species that stranded in the tropical Pacific, including Hawai'i from 1997 to 2011. The sample set includes odontocetes (n=39) and mysticetes (n=3). Median (range) contaminant concentrations in ng/g lipid for the most representative species category (delphinids excluding killer whales [n=27]) are: 9650 (44.4-99,100) for ∑DDTs, 6240 (40.8-50,200) for ∑PCBs, 1380 (6.73-9520) for ∑chlordanes, 1230 (13.4-5510) for ∑toxaphenes, 269 (1.99-10,100) for ∑PBDEs, 280 (2.14-4190) for mirex, 176 (5.43-857) for HCB, 48.1 (<5.42-566) for ∑HCHs, 33.9 (<2.42-990) for ∑HBCDs, 1.65 (<0.435-11.7) for octachlorostyrene and 1.49 (<2.07-13.1) for pentachlorobenzene. ∑PCB concentrations in these Pacific Island cetaceans approach and sometimes exceed proposed toxic threshold values. Backward stepwise multiple regressions indicated the influence of life history parameters on contaminant concentrations when performed with three independent variables (species category, year of stranding, and sex/age class). No temporal trends were noted (p>0.063), but sex/age class influences were evident with adult males exhibiting greater contaminant loads than adult females and juveniles for ∑DDT, ∑PCBs, ∑CHLs, and mirex (p≤0.036). POP concentrations were lower in mysticetes than odontocetes for many compound classes (p≤0.003). p,p'-DDE/∑DDTs ratios were greater than 0.6 for all species except humpback whales, suggesting exposure to an old DDT source. These POP levels are high enough to warrant concern and continued monitoring.

Thyroid hormone concentrations in relation to age, sex, pregnancy, and perinatal loss in bottlenose dolphins (Tursiops truncatus).

This study evaluated circulating concentrations of thyroid hormones in relation to age, sex, pregnancy status, and perinatal loss in bottlenose dolphins (Tursiops truncatus) under human care. A total of 373 blood samples were collected from 60 individual dolphins housed at nine aquariums/oceanariums. Serum concentrations of total and free thyroxine (T4) and triiodothyronine (T3) were analyzed with commercial RIA kits validated for use with dolphins. While the effect of age was indicated by higher (P<0.0001) concentrations of total and free T4 and T3 in juveniles than adults, the effect of sex on thyroid hormones was inconclusive. The effect of pregnancy was indicated by higher (P<0.035) total and free T4 and T3 during early pregnancy compared to non-pregnancy. For both successful and unsuccessful pregnancy outcomes, maternal concentrations of thyroid hormones were highest during early, intermediate during mid, and lowest during late pregnancy (P<0.07 to P<0.0001). Compared to live and thriving births, concentrations of total and free T4 and total T3 were lower (P<0.08 to P<0.001) in dolphins with perinatal loss. Lower concentrations ranged from 10% to 14% during early, 11% to 18% during mid, and 23% to 37% during late pregnancy. In conclusion, the effects of age, reproductive status and stage of pregnancy on thyroid hormone concentrations are necessary factors to take into account when assessing thyroid gland function. Since perinatal loss may be associated with hypothyroidism in dolphins, analysis of serum T4 and T3 should be considered for those dolphins that have a history of pregnancy loss.

Relaxin and progesterone during pregnancy and the post-partum period in association with live and stillborn calves in bottlenose dolphins (Tursiops truncatus).

The objectives of this study were to validate a relaxin and progesterone RIA for use in bottlenose dolphins, and quantify and characterize both hormones in extracts of placental tissue and serum collected during pregnancy and the post-partum period, and compare the results between dolphins with live and stillborn calves. In Experiment 1, validation of a heterologous relaxin and progesterone RIA involved specific displacement of antibody-bound radiolabeled human relaxin or progesterone in response to increasing volumes of pooled pregnant dolphin serum and amounts of respective hormone standards added to a fixed volume of serum. The displacement curves were considered parallel and additive relative to respective standard curves. In Experiment 2, immunoreactive relaxin and progesterone were detected in placental extracts and, in corresponding serum samples, concentrations of both hormones were higher during the pre-partum than post-partum periods. Circulatory concentrations of progesterone decreased (P < 0.05) from relatively high concentrations during early and mid-pregnancy to intermediate concentrations by late pregnancy (month effect, P < 0.0001) in dolphins with live births, whereas, in dolphins with stillbirths, the decrease in progesterone began earlier (month-by-birth status interaction, P < 0.007); mean concentrations were lower at mid- (37%, P < 0.06) and late (25%) pregnancy. Temporally, relaxin increased (P < 0.05) progressively from relatively low concentrations during early pregnancy to high concentrations during late pregnancy (month effect, P < 0.0001) and was not different between birth statuses (birth status effect, P = 0.76; month-by-birth status interaction, P = 0.17). Even though the interaction did not reach significance, mean relaxin concentrations were 42%, 29%, and 34% lower at early, mid-, and late pregnancy, respectively, in dolphins with stillbirths than in those with live births. In conclusion, the pregnancy-specific increase in serum concentrations of relaxin and lower concentrations of both relaxin and progesterone in association with stillbirths suggest the potential for relaxin to be used diagnostically to determine pregnancy status, and one or both hormones to be used to assess placental function, and, perhaps, fetal well-being in bottlenose dolphins and other cetaceans.