Nicholas M. Kellar

Overcoming the challenges of studying conservation physiology in large whales: a review of available methods

A description and comparison of the four major methods available for studying conservation physiology of large whales, namely analysis of faecal, respiratory vapour, and skin/blubber biopsy samples, and photographs.

Validation of Blubber Progesterone Concentrations for Pregnancy Determination in Three Dolphin Species and a Porpoise

Recent studies have validated the use of biopsies as a minimally invasive way to identify pregnant females in several species of wild cetaceans: Balaenaptera acutorostrata , Delphinus delphis , Lissodelphis borealis , and Lagenorhynchus obliquidens . These studies found that progesterone (P4) concentrations quantified from blubber attached to biopsy samples is diagnostic of pregnancy. Here we examine a broader group of cetacean species in efforts to investigate how progesterone levels vary between species with respect to pregnancy status. We compared P4 concentrations in blubber collected from fishery bycatch and beach-stranded specimens for 40 females of known reproductive condition from Delphinus capensis (n = 18), Stenella attenuata (n = 8), S . longirostris (n = 6), and Phocoenoides dalli (n = 8). The P4 concentrations were different (t = -7.1, p = 1.79E-08) between pregnant and non-pregnant animals in all species, with the mean blubber P4 concentration for pregnant animals 164 times higher than that of non-pregnant animals. There was no overlap in concentration levels between sexually immature or non-pregnant sexually mature animals and pregnant animals. No significant differences (F = 0.354, p = 0.559) were found between mature non-pregnant and immature D . capensis and P dalli , suggesting P4 level is not indicative of maturity state in female delphinoids. P4 concentrations in relation to reproductive state were remarkably similar across species. All samples were analyzed with two different enzyme immunoassay kits to gauge assay sensitivity to measure progesterone in small samples, such as biopsies. With the technique now validated for these cetacean species, blubber P4 is a reliable diagnostic of pregnancies across multiple species, and thus expands the utility of this method to study reproduction in free-ranging cetaceans using biopsies.

Blubber Cortisol: A Potential Tool for Assessing Stress Response in Free-Ranging Dolphins without Effects due to Sampling

When paired with dart biopsying, quantifying cortisol in blubber tissue may provide an index of relative stress levels (i.e., activation of the hypothalamus-pituitary-adrenal axis) in free-ranging cetacean populations while minimizing the effects of the act of sampling. To validate this approach, cortisol was extracted from blubber samples collected from beach-stranded and bycaught short-beaked common dolphins using a modified blubber steroid isolation technique and measured via commercially available enzyme immunoassays. The measurements exhibited appropriate quality characteristics when analyzed via a bootstraped stepwise parallelism analysis (observed/expected = 1.03, 95%CI: 99.6 – 1.08) and showed no evidence of matrix interference with increasing sample size across typical biopsy tissue masses (75–150mg; r2 = 0.012, p = 0.78, slope = 0.022ngcortisol deviation/ultissue extract added). The relationships between blubber cortisol and eight potential cofactors namely, 1) fatality type (e.g., stranded or bycaught), 2) specimen condition (state of decomposition), 3) total body length, 4) sex, 5) sexual maturity state, 6) pregnancy status, 7) lactation state, and 8) adrenal mass, were assessed using a Bayesian generalized linear model averaging technique. Fatality type was the only factor correlated with blubber cortisol, and the magnitude of the effect size was substantial: beach-stranded individuals had on average 6.1-fold higher cortisol levels than those of bycaught individuals. Because of the difference in conditions surrounding these two fatality types, we interpret this relationship as evidence that blubber cortisol is indicative of stress response. We found no evidence of seasonal variation or a relationship between cortisol and the remaining cofactors.

Heavy with child? Pregnancy status and stable isotope ratios as determined from biopsies of humpback whales.

Recent advances in analytical techniques have allowed cetacean pregnancy status to be diagnosed using reproductive hormones from biopsy samples. We tested the efficacy of blubber progesterone assays for diagnosing pregnancy in humpback whales, calculated pregnancy rates and examined the relationship between pregnancy and stable isotope ratios for these whales.

From Progesterone in Biopsies to Estimates of Pregnancy Rates: Large Scale Reproductive Patterns of Two Sympatric Species of Common Dolphin, Delphinus spp. off California, USA and Baja, Mexico

Abstract Blubber progesterone levels were measured in biopsy samples and used to predict the pregnancy status of 507 female common dolphins (204 long-beaked common dolphins, Delphinus capensis, and 303 short-beaked common dolphins, D. delphis). Samples were collected in the coastal waters of the eastern North Pacific between central California, USA and the southern end of Baja California, Mexico. The percentage of females pregnant was similar between the two species: 22.1% (n  =  45) of D. capensis and 28.1% (n  =  85) of D. delphis. For both species we found strong geographic patterns in pregnancy, suggesting that some areas were more conducive for pregnant females. A sizable drop in percent pregnant from early (38.8%, n  =  133) to late (25.3%, n  =  91) autumn was found in D. delphis but not in D. capensis. The potential for sample selectivity was examined via biopsies collected either from a large research ship or from a small, rigid-hull inflatable boat (RHIB) launched from the larger ship. An analysis of “Tandem Biopsy Sampling”, replicate biopsy effort on the same schools from each vessel/platform, yielded little evidence that disproportionately more pregnant female common dolphins were biopsied from one platform versus the other. This result plus an analysis of pregnancy status relative to the duration of biopsy operations failed to uncover strong evidence of unaccounted sampling bias with respect to pregnancy state. In total, these results demonstrate the utility of blubber progesterone concentrations to assess pregnancy status in free-ranging cetaceans and they highlight potential factors associated with population-level variation in dolphin pregnancy rates.

Seasonal Variation in the Skin Transcriptome of Common Bottlenose Dolphins (Tursiops truncatus) from the Northern Gulf of Mexico

As long-lived predators that integrate exposures across multiple trophic levels, cetaceans are recognized as sentinels for the health of marine ecosystems. Their utility as sentinels requires the establishment of baseline health parameters. Because cetaceans are protected, measurements obtained with minimal disruption to free ranging animals are highly desirable. In this study we investigated the utility of skin gene expression profiling to monitor health and contaminant exposure in common bottlenose dolphins (Tursiops truncatus). Remote integument biopsies were collected in the northern Gulf of Mexico prior to the Deepwater Horizon oil spill (May 2010) and during summer and winter for two years following oil contamination (2010-2011). A bottlenose dolphin microarray was used to characterize the skin transcriptomes of 94 individuals from three populations: Barataria Bay, Louisiana, Chandeleur Sound, Louisiana, and Mississippi Sound, Mississippi/Alabama. Skin transcriptomes did not differ significantly between populations. In contrast, season had a profound effect on gene expression, with nearly one-third of all genes on the array differing in expression between winter and the warmer seasons (moderated T-test; p<0.01, fold-change≥1.5). Persistent organic pollutants (POPs) in blubber changed concurrently, reaching >two-fold higher concentrations in summer compared to winter, due to a seasonal decrease in blubber thickness and loss of stored lipid. However, global gene expression did not correlate strongly with seasonally changing contaminant concentrations, most likely because the refractory, lipid-stored metabolites are not substrates for phase I or II xenobiotic detoxification pathways. Rather, processes related to cell proliferation, motility, and differentiation dominated the differences in expression in winter and the warmer seasons. More subtle differences were seen between spring and summer (1.5% of genes differentially expressed). However, two presumed oil-exposed animals from spring presented gene expression profiles more similar to the summer animals (presumed exposed) than to other spring animals. Seasonal effects have not previously been considered in studies assessing gene expression in cetaceans, but clearly must be taken into account when applying transcriptomic analyses to investigate their contaminant exposure or health status.

High pregnancy rates in humpback whales (Megaptera novaeangliae) around the Western Antarctic Peninsula, evidence of a rapidly growing population

Antarctic humpback whales are recovering from near extirpation from commercial whaling. To understand the dynamics of this recovery and establish a baseline to monitor impacts of a rapidly changing environment, we investigated sex ratios and pregnancy rates of females within the Western Antarctic Peninsula (WAP) feeding population. DNA profiling of 577 tissue samples (2010–2016) identified 239 males and 268 females. Blubber progesterone levels indicated 63.5% of the females biopsied were pregnant. This proportion varied significantly across years, from 36% in 2010 to 86% in 2014. A comparison of samples collected in summer versus fall showed significant increases in the proportion of females present (50% to 59%) and pregnant (59% to 72%), consistent with demographic variation in migratory timing. We also found evidence of annual reproduction among females; 54.5% of females accompanied by a calf were pregnant. These high pregnancy rates are consistent with a population recovering from past exploitation, but appear inconsistent with recent estimates of WAP humpback population growth. Thus, our results will help to better understand population growth potential and set a current baseline from which to determine the impact of climate change and variability on fecundity and reproductive rates.

Comprehensive Screening Links Halogenated Organic Compounds with Testosterone Levels in Male Delphinus delphis from the Southern California Bight

While environmental pollutants have been associated with changes in endocrine health in cetaceans, efforts to link contaminant exposure with hormones have largely been limited to a list of known, targeted contaminants, overlooking minimally characterized or unknown compounds of emerging concern. To address this gap, we analyzed a suite of potential endocrine disrupting halogenated organic compounds (HOCs) in blubber from 16 male short-beaked common dolphins ( Delphinus delphis) with known maturity status collected from fishery bycatch in the Southern California Bight. We employed a suspect screening mass spectrometry-based method to investigate a wide range of HOCs that were previously observed in cetaceans from the same region. Potential endocrine effects were assessed through the measurement of blubber testosterone. We detected 167 HOCs, including 81 with known anthropogenic sources, 49 of unknown origin, and 37 with known natural sources. The sum of 11 anthropogenic and 4 unknown HOC classes were negatively correlated with blubber testosterone. Evidence suggests that elevated anthropogenic HOC load contributes to impaired testosterone production in mature male D. delphis. The application of this integrative analytical approach to cetacean contaminant analysis allows for inference of the biological consequences of accumulation of HOCs and prioritization of compounds for future environmental toxicology research.

Initial validation of blubber cortisol and progesterone as indicators of stress response and maturity in an otariid; the California sea lion (Zalophus californianus)

Chronic stress can have detrimental effects on an individuals health and reproductive success. The use of cortisol quantification as an indicator of stress in free-ranging cetaceans and phocids is increasing but no studies have applied this technique on blubber in otariids. We measured cortisol concentrations in blubber samples obtained from California sea lions, Zalophus californianus, stranded in San Diego County and those incidentally killed in the California drift gillnet fishery. We also measured progesterone concentrations to assess female reproductive status and, in males, as a potential secondary measure of adrenal steroid production. Blubber cortisol and progesterone values were compared across demographic groups (sex and maturity), season, and proportion blubber lipid extracted. Stranded animals (247.3±70.767SEng/gblubber) had significantly higher cortisol concentrations compared to fishery bycaught (8.1±2.108SEng/gblubber) animals. These findings are likely driven by inherent differences in the cause of death and associated nutritional state coupled with the mean duration of expiration for these two groups of animals (i.e., the duration from an animals initial perception of the threat-to-self until death). The duration of transition from healthy state to death in stranded animals is on the order of many hours to weeks while in fishery bycaught animals, this transition occurs much more rapidly (i.e., seconds to tens of minutes). The presumed longer duration of the mortality event in stranded animals gives sufficient time for elevated cortisol to diffuse into the blubber. No significant differences between demographic groups, or season were found. However, blubber cortisol declined inversely with proportion blubber lipid extracted, suggesting utility in assessing long-term nutritional status. Blubber progesterone was significantly higher in mature females than immature females (153.8±54.546SEng/gblubber and 9.7±3.60SEng/gblubber respectively), containing on average 15 times more progesterone, irrespective of pregnancy state. Additionally, a significant relationship between mean cortisol and progesterone was found in males with >35% blubber lipid (p<0.0001). This study is an initial step in validating blubber cortisol and progesterone concentrations as a potential marker of stress response and reproductive state, respectively, in otariids. Especially when paired with dart biopsying, this approach could represent a relatively rapid way to assess baseline stress, nutritional status and reproductive states in otariids while minimizing the effects of sampling.

Natural Variation in Stress Hormones, Comparisons Across Matrices, and Impacts Resulting from Induced Stress in the Bottlenose Dolphin

Knowledge regarding stress hormones and how they vary in response to seasonality, gender, age, and reproductive status for any marine mammal is limited. Furthermore, stress hormones may be measured in more than one matrix (e.g., feces, blood, blubber), but the relationships between levels of a given hormone across these matrices are unknown, further complicating the interpretations of hormones measured in samples collected from wild animals. A study is underway to address these issues in a population of bottlenose dolphins trained for voluntary participation in sample collections from different matrices and across season and time of day.