Tag: baleen

Optimizing hormone extraction protocols for whale baleen

By Alejandro Fernández Ajó, Postdoc, OSU Department of Fisheries, Wildlife, and Conservation Science, Geospatial Ecology of Marine Megafauna Lab

Large whale conservation is challenged by our limited understanding of the impacts of natural and anthropogenic disturbances on the whale´s health and its population level consequences. To better mitigate human-wildlife conflicts, we need to improve our ability to predict multi-scale responses of whales to disturbances, describe and identify disease dynamics, and understand the reproductive biology of whales (Madliger, 2020; McCormick and Romero, 2017). Conservation physiology and conservation endocrinology can provide tools to illuminate the underlying physiological mechanisms whales use to cope with changing environments and different stressors, thus filling information gaps to guide management and conservation actions.

In brief, conservation physiology is a multidisciplinary field wherein a broad suite of tools and concepts are used to understand how organisms and ecosystems respond to both environmental and anthropogenic change and stressors (Madliger et al., 2020). Conservation endocrinology is a subdiscipline within conservation physiology, which relies on endocrine measurements (hormone quantifications). However, monitoring the physiology of free ranging animals in wild populations presents many technical challenges and it is particularly difficult when studying whales. Traditionally, conservation endocrinology relied on laboratory analyses of plasma samples (derived from blood). Yet implementing this techniques for monitoring the physiology of mysticetes (baleen whales) is currently impossible, as there are no feasible, non- (or minimally) invasive, methods to obtain a blood sample from living large whales (Hunt et al., 2013).

Therefore, we are interested in the development and further validation of alternative sample types from whales to obtain endocrine data. During my Ph.D. dissertation I worked to develop and ground truth the endocrine analyses of whale baleen as a novel sample type that can be used for retrospective assessments of the whale´s physiology. Baleen, the filter-feeding apparatus of the mysticete whales (Figure 1), consists of long fringed plates of stratified, keratinized tissue that grow continuously and slowly downward from the whale´s upper jaw (Hunt et al., 2014). Baleen plates are readily accessible at necropsy and routinely collected from carcasses of stranded whales.

Figure 1: Southern Right Whale skim feeding at the surface. Baleen plates hanging downwards are visible in the image. Photo: Alejandro Fernández Ajó / Instituto de Conservación de Ballenas de Argentina.

Like hair, nails, feathers, spines, or horns of other animals, baleen is a keratinized tissue that can store steroid and thyroid hormones in detectable and relevant concentrations to provide an integrated measure of hormonal plasma levels over the period that the structure was growing. Thus, baleen contains a progressive time-series that captures months and often years of an individual’s endocrine history with sufficient temporal resolution to determine seasonal endocrine patterns allowing to explore questions that have historically been difficult to address in large whales, including pregnancy and inter-calving interval, age of sexual maturation, timing and duration of seasonal reproductive cycles, adrenal physiology, and metabolic rate. Additionally, their robust and stable keratin matrix allows baleen samples to be stored for years to decades, enabling the analysis and comparison of endocrine patterns from past and modern populations. Therefore, keratinized sample matrices are valuable tools to investigate reproductive and stress physiology in whales and other vertebrates.

However, due to its novelty, the extraction and analysis of hormones from baleen and other keratinized tissues requires both biological and analytical validations to ensure the method fulfills the requirements for its intended use. Baleen hormone analyses has already passed several essential assay validations, including parallelism and accuracy of immunoassays (Hunt et al., 2017b), and numerous biological validations, such as the study of animals with known physiological status (i.e., pregnancy, and known stress events such as entanglement in fishing gear or presence of lesions) to assess the degree to which the endocrine data reflect the physiology of the individual (Fernández Ajó et al., 2020, 2018; Hunt et al., 2018, 2017a; Lysiak et al., 2018; Palme, 2019). Yet, other questions essential for technical validation remain unknown, including choice and volume of extraction solvent, the effect of solvent-to-sample ratio (solvent:sample) on extraction yield, and the amount of sample (e.g., mg) needed for analysis to obtain reliable hormonal data.

In our recent contribution, Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation, we aimed to tackle two of these important questions: “1) what is the minimum sample mass of baleen powder required to reliably quantify hormone content of baleen samples analyzed using commercially available enzyme immune assays (EIAs); and 2) what is the optimal ratio of solvent volume to sample mass for steroids extracted from baleen, i.e., the ratio that yields the maximum amount of hormone with high accuracy and low variability between replicates.”

We performed the extraction with methanol and tested a variety of sample masses with the objective to provide methodological guidance regarding optimizing sample mass and solvent volume for steroid hormone extraction from powdered baleen. Our results suggest that the optimal sample mass for methanol extraction of steroid hormones from baleen samples is 20 mg, and that larger sample masses did not produce either better yield or less variation in the apparent hormone per g of baleen sample (Figure 2). In addition, when the extraction was performed keeping the volume of solvent proportional to the sample mass (namely, a solvent:sample ratio of 80:1), masses as small as 10 mg yielded reliable hormone measurement (Figure 2).

Figure 2. Extraction experiment results for variable (solid circles) or constant (open circles) solvent:sample ratio (mean progesterone concentration ± standard error of the mean). Extractions for 5, 10, 20, 50, and 75 mg of baleen powder were performed in triplicate for each sample mass. Letters indicate significant differences between groups after Tukey’s post hoc test. The right-side panel presents a plot for 100 mg of baleen powder extracted with 8 mL of solvent performed in triplicate; error bars are too small to show in the figure. Source: Fernández Ajó et al., 2021.

Our results indicate how baleen hormone analytic techniques can be more widely employed on small sample masses from rare specimens (i.e., less sample than is currently employed, which is typically 75 mg or 100 mg in most studies to date), such as from natural history museums and stranding archives. Thus, we demonstrate that greater use of this valuable technique to reconstruct the endocrine and physiological history of individual whales over time can be achieved with reduced sample size (so reduced damage to the sample). I hope these findings encourage researchers to apply these methods more broadly to analyze historical archives of baleen plates that can date back to the era of commercial whaling, and modern archives of baleen collected from stranded animals to help continue further developing techniques that can make headway in gaining conservation-relevant physiological knowledge of this particularly challenging taxon.

Bibliography:

Fernández Ajó, A., Hunt, K.E., Dillon, D., Uhart, M., Sironi, M., Rowntree, V., Loren Buck, C., 2021. Optimizing hormone extraction protocols for whale baleen: Tackling questions of solvent:sample ratio and variation. Gen. Comp. Endocrinol. 113828. https://doi.org/10.1016/j.ygcen.2021.113828

Fernández Ajó, A.A., Hunt, K.E., Giese, A.C., Sironi, M., Uhart, M., Rowntree, V.J., Marón, C.F., Dillon, D., DiMartino, M., Buck, C.L., 2020. Retrospective analysis of the lifetime endocrine response of southern right whale calves to gull wounding and harassment: A baleen hormone approach. Gen. Comp. Endocrinol. 296, 113536. https://doi.org/10.1016/j.ygcen.2020.113536

Fernández Ajó, A.A., Hunt, K.E., Uhart, M., Rowntree, V., Sironi, M., Marón, C.F., Di Martino, M., Buck, C.L., 2018. Lifetime glucocorticoid profiles in baleen of right whale calves: potential relationships to chronic stress of repeated wounding by Kelp Gulls. Conserv. Physiol. 6, 1–12. https://doi.org/10.1093/conphys/coy045

Hunt, K.E., Lysiak, N.S., Moore, M., Rolland, R.M., 2017a. Multi-year longitudinal profiles of cortisol and corticosterone recovered from baleen of North Atlantic right whales (Eubalaena glacialis). Gen. Comp. Endocrinol. 254, 50–59. https://doi.org/10.1016/j.ygcen.2017.09.009

Hunt, K.E., Lysiak, N.S., Robbins, J., Moore, M.J., Seton, R.E., Torres, L., Loren Buck, C., Buck, C.L., 2017b. Multiple steroid and thyroid hormones detected in baleen from eight whale species. Conserv. Physiol. 5. https://doi.org/10.1093/conphys/cox061

Hunt, K.E., Lysiak, N.S.J., Matthews, C.J.D., Lowe, C., Fernández Ajó, A., Dillon, D., Willing, C., Heide-Jørgensen, M.P., Ferguson, S.H., Moore, M.J., Buck, C.L., 2018. Multi-year patterns in testosterone, cortisol and corticosterone in baleen from adult males of three whale species. Conserv. Physiol. 6, 1–16. https://doi.org/10.1093/conphys/coy049

Hunt, K.E., Moore, M.J., Rolland, R.M., Kellar, N.M., Hall, A.J., Kershaw, J., Raverty, S.A., Davis, C.E., Yeates, L.C., Fauquier, D.A., Rowles, T.K., Kraus, S.D., 2013. Overcoming the challenges of studying conservation physiology in large whales: a review of available methods. Conserv. Physiol. 1, cot006–cot006. https://doi.org/10.1093/conphys/cot006

Hunt, K.E., Stimmelmayr, R., George, C., Hanns, C., Suydam, R., Brower, H., Rolland, R.M., 2014. Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus). Conserv. Physiol. 2, cou030–cou030. https://doi.org/10.1093/conphys/cou030

Lysiak, N.S.J., Trumble, S.J., Knowlton, A.R., Moore, M.J., 2018. Characterizing the Duration and Severity of Fishing Gear Entanglement on a North Atlantic Right Whale (Eubalaena glacialis) Using Stable Isotopes, Steroid and Thyroid Hormones in Baleen. Front. Mar. Sci. 5, 1–13. https://doi.org/10.3389/fmars.2018.00168

Madliger, C. L., Franklin, C. E., Love, O. P., & Cooke, S.J. (Ed.), 2020. Conservation Physiology: Applications for Wildlife Conservation and Management., 1st ed. Oxford University Press. https://doi.org/10.1093/oso/ 978019883610.001.0001

McCormick, S.D., Romero, L.M., 2017. Conservation Endocrinology. Bioscience 67, 429–442. https://doi.org/10.1093/biosci/bix026

Palme, R., 2019. Non-invasive measurement of glucocorticoids: Advances and problems. Physiol. Behav. 199, 229–243. https://doi.org/10.1016/j.physbeh.2018.11.021

Applying novel methods in conservation physiology to understand cases of large whale mortalities

By Alejandro Fernánez Ajó, PhD student at NAU and GEMM Lab research technician

Although commercial whaling is currently banned and several whale populations show evidence of recovery, today´s whales are exposed to a variety of other human stressors (e.g., entanglement in fishing gear, vessel strikes, shipping noise, climate change, etc.; reviewed in Hunt et al., 2017a). The recovery and conservation of large whale populations is particularly important to the oceanic environment due to their key ecological role and unique biological traits, including their large body size, long lifespan and sizable home ranges (Magera et al., 2013; Atkinson et al., 2015; Thomas and Reeves, 2015). Thus, scientists must develop novel tools to overcome the challenges of studying whale physiology in order to distinguish the relative importance of the different impacts and guide conservation actions accordingly (Ayres et al., 2012; Hunt et al., 2013).

To this end, baleen hormone analysis represents a powerful tool for retrospective assessment of patterns in whale physiology (Hunt et al., 2014, 2016, 2017a, 2017b, 2018; Lysiak et. al., 2018; Fernández Ajó et al., 2018; Rolland et al., 2019). Moreover, hormonal panels, which include multiple hormones, are helping to better clarify and distinguish between the physiological effects of different sources of anthropogenic and environmental stressors (Ayres et al., 2012; Wasser et al., 2017; Lysiak et al., 2018; Romero et al., 2015).

What is Baleen? Baleen is a stratified epithelial tissue consisting of long, fringed plates that grow downward from the upper jaw, which collectively form the whale´s filter-feeding apparatus (Figure 1). This tissue accumulates hormones as it grows. Hormones are deposited in a linear fashion with time so that a single plate of baleen allows retrospective assessment and evaluation of a whales’ physiological condition, and in calves baleen provides a record of the entire lifespan including part of their gestation. Baleen samples are also readily accessible and routinely collected during necropsy along with other samples and relevant information.

Figure 1: Top: A baleen plate from a southern right whale calf (Source: Fernández Ajó et al. 2018). Bottom: A southern right whale with mouth open exposing its baleen (photo credit: Stephen Johnson).

Why are the Southern Right Whales calves (SRW) dying in Patagonia?

I am a Fulbright Ph.D. student in the Buck Laboratory  at Northern Arizona University since Fall 2017, a researcher with the Whale Conservation Institute of Argentina (Instituto de Conservación de Ballenas) and Field Technician for the GEMM Lab over the summer. I focus my research on the application and development of novel methods in conservation physiology to improve our understanding of how physiological parameters are affected by human pressures that impact large whales and marine mammals. I am especially interested in understanding the underlaying causes of large whale mortalities with the aim of preventing their occurrence when possible. In particular, for my Ph.D. dissertation, I am studying a die-off case of Southern Right Whale (SRW) calves, Eubalaena australis, off Peninsula Valdés (PV) in Patagonia-Argentina (Figure 2).

Prior to 2000, annual calf mortality at PV was considered normal and tracked the population growth rate (Rowntree et al., 2013). However, between 2007 and 2013, 558 whales died, including 513 newborn calves (Sironi et al., 2018). Average total whale deaths per year increased tenfold, from 8.2 in 1993-2002 to 80 in 2007-2013. These mortality levels have never before been observed for the species or any other population of whales (Thomas et al., 2013, Sironi et al., 2018).

Figure 2: Study area, the red dots along the shoreline indicate the location where the whales were found stranded at Península Valdés in 2018 (Source: The Right Whale Program Research Report 2018, Sironi and Rowntree, 2018)

Among several hypotheses proposed to explain these elevated calf mortalities, harassment by Kelp Gulls, Larus dominicanus, on young calves stands out as a plausible cause and is a unique problem only seen at the PV calving ground. Kelp gull parasitism on SRWs near PV was first observed in the 1970’s (Thomas, 1988). Gulls primarily harass mother-calf pairs, and this parasitic behavior includes pecking on the backs of the whales and creating open wounds to feed on their skin and blubber. The current intensity of gull harassment has been identified as a significant environmental stressor to whales and potential contributor to calf deaths (Marón et al., 2015b; Fernández Ajó et al., 2018).

Figure 3: The significant preference for calves as a target of gull attacks highlights the impact of this parasitic behavior on this age class. The situation continues to be worrisome and serious for the health and well-being of newborn calves at Península Valdés. Left: A Kelp Gull landing on whale´s back to feed on her skin and blubber (Photo credit: Lisandro Crespo). Right: A calf with multiple lesions on its back produced by repeated gull attacks (Photo credit: ICB).

Quantifying gull inflicted wounds

Photographs of gull wounds on whales taken during necropsies and were quantified and assigned to one of seven objectively defined size categories (Fig. 4): extra-small (XS), small (S), medium (M), large (L), extra-large (XL), double XL (XXL) and triple XL (XXXL). The size and number of lesions on each whale were compared to baleen hormones to determine the effect of the of the attacks on the whales health.

Figure 4. Kelp gull lesion scoring. Source: Maron et al. 2015).

How baleen hormones are applied

Impact factors such as injuries, predation avoidance, storms, and starvation promote an increase in the secretion of the glucocorticoids (GCs) cortisol and corticosterone (stress hormones), which then induce a variety of physiological and behavioral responses that help animals cope with the stressor. Prolonged exposure to chronic stress, however, may exceed the animal’s ability to cope with such stimuli and, therefore, adversely affects its body condition, its health, and even its survival. Triiodothyronine (T3), is the most biologically active form of the thyroid hormones and helps regulate metabolism. Sustained food deprivation causes a decrease in T3 concentrations, slowing metabolism to conserve energy stores. Combining GCs and T3 hormone measures allowed us to investigate and distinguish the relative impacts of nutritional and other sources of stressors.

Combining these novel methods produced unique results about whale physiology. With my research, we are finding that the GCs concentrations measured in calves´ baleen positively correlate with the intensity of gull wounding (Figure 4, 1 and 2), while calf’s baleen thyroid hormone concentrations are relative stable across time and do not correlate with intensity of gull wounding (Figure 4 – 3). Taken together these findings indicate that SRW calves exposed to Kelp gull parasitism and harassment experience high levels of physiological stress that compromise their health and survival. Ultimately these results will inform government officials and managers to direct conservation actions aimed to reduce the negative interaction between Kelp gulls and Southern Right Whales in Patagonia.

Figure 4: Physiological stress correlates with number of gull lesions (1 and 2). According to the best-fit linear model, immunoreactive baleen corticosterone (B) and cortisol (F) concentrations increased with wound severity (i.e. number of gull lesions). However, nutritional status indexed by baleen immunoreactive triiodothyronine (T3) concentrations does not correlate with the number of gull lesions (3). (Fernández Ajó et al. 2019, manuscript under revision)

Baleen hormones as a conservation tool

Baleen hormones represent a powerful tool for retrospective assessments of longitudinal trends in whale physiology by helping discriminate the underlying mechanisms by which different stressors may affect a whale’s health and physiology. Moreover, while most sample types used for studying whale physiology provide single time-point measures of current circulating hormone levels (e.g., skin or respiratory vapor), or information from previous few hours or days (e.g., urine and feces), baleen tissue provides a unique opportunity for longitudinal analyses of hormone patterns. These retrospective analyses can be conducted for both stranded or archived specimens, and can be conducted jointly with other biological markers (e.g., stable isotopes and biotoxins) to describe migration patterns and exposure to pollutants. Further research efforts on baleen hormones should focus on completing biological validations to better understand the hormone measurements in baleen and its correlation with measurements from alternative sample matrices (i.e., feces, skin, blubber, and respiratory vapors).

References:

Atkinson, S., Crocker, D., Houser, D., Mashburn, K., 2015. Stress physiology in marine mammals: how well do they fit the terrestrial model? J. Comp. Physiol. B. 185, 463–486. https://doi.org/10.1007/s00360-015-0901-0.

Ayres, K.L., Booth, R.K., Hempelmann, J.A., Koski, K.L., Emmons, C.K., Baird, R.W., Balcomb-Bartok, K., Hanson, M.B., Ford, M.J., Wasser, S.K., 2012. Distinguishing the impacts of inadequate prey and vessel traffic on an endangered killer whale (Orcinus orca) population. PLoS ONE. 7, e36842. https://doi.org/10.1371/journal.pone.0036842.

Fernández Ajó, A.A., Hunt, K., Uhart, M., Rowntree, V., Sironi, M., Marón, C.F., Di Martino, M., Buck, L., 2018. Lifetime glucocorticoid profiles in baleen of right whale calves: potential relationships to chronic stress of repeated wounding by Kelp Gull. Conserv. Physiol. 6, coy045. https://doi.org/10.1093/conphys/coy045.

Hunt, K., Lysiak, N., Moore, M., Rolland, R.M., 2017a. Multi-year longitudinal profiles of cortisol and corticosterone recovered from baleen of North Atlantic right whales (Eubalaena glacialis). Gen. Comp. Endocrinol. 254: 50–59. https://doi.org/10.1016/j.ygcen.2017.09.009.

Hunt, K.E., Hunt, K.E., Lysiak, N.S., Matthews, C.J.D., Lowe, C., Fernández-Ajo, A., Dillon, D., Willing, C., Heide-Jørgensen, M.P., Ferguson, S.H., Moore, M.J., Buck, C.L., 2018. Multi-year patterns in testosterone, cortisol and corticosterone in baleen from adult males of three whale species. Conserv. Physiol. 6, coy049. https://doi.org/10.1093/conphys/coy049.

Hunt, K.E., Hunt, K.E., Lysiak, N.S., Moore, M.J., Rolland R.M., 2016. Longitudinal progesterone profiles in baleen from female North Atlantic right whales (Eubalaena glacialis) match known calving history. Conserv. Physiol. 4, cow014. https://doi.org/10.1093/conphys/cow014.

Hunt, K.E., Lysiak, N.S., Moore, M.J., Seton, R.E., Torres, L., Buck, C.L., 2017b. Multiple steroid and thyroid hormones detected in baleen from eight whale species. Conserv. Physiol. 5, cox061. https://doi.org/10.1093/conphys/cox061.

Hunt, K.E., Moore, M.J., Rolland, R.M., Kellar, N.M., Hall, A.J., Kershaw, J., Raverty, S.A., Davis, C.E., Yeates, L.C., Fauquier, D.A., Rowles, T.K., Kraus, S.D., 2013. Overcoming the challenges of studying conservation physiology in large whales: a review of available methods. Conserv. Physiol. 1: cot006. https://doi.org/10.1093/conphys/cot006.

Hunt, K.E., Stimmelmayr, R., George, C., Hanns, C., Suydam, R., Brower, H., Rolland, R.M., 2014. Baleen hormones: a novel tool for retrospective assessment of stress and reproduction in bowhead whales (Balaena mysticetus). Conserv. Physiol. 2, cou030. doi: https://doi.org/10.1093/conphys/cou030.

Lysiak, N., Trumble, S., Knowlton, A., Moore, M., 2018. Characterizing the duration and severity of fishing gear entanglement on a North Atlantic right whale (Eubalaena glacialis) using stable isotopes, steroid and thyroid hormones in baleen. Front. Mar. Sci. 5: 168. https://doi.org/10.3389/fmars.2018.00168.

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