Chronic stress from fishing gear entanglement is recorded in baleen from a bowhead whale (<i>Balaena mysticetus</i>)

Abstract

Arctic ecosystems are experiencing rapid environmental changes (e.g., reduction in sea-ice extent, rising temperatures) coupled with the effects of increasing human activity associated with resource extraction, vessel traffic, and commercial fishing (Pompa et al. 2011, Doney et al. 2012, Moore et al. 2012). As a result, anthropogenic impacts on Arctic wildlife such as bowhead whales (Balaena mysticetus) are expected to escalate (Reeves et al. 2012). Assessing stress responses in marine wildlife by quantifying adrenal steroid hormones (i.e., glucocorticoids; cortisol, corticosterone, and/or their metabolites) has been a valuable tool for evaluating the physiological impacts of anthropogenic disturbance (Ayres et al. 2012; Rolland et al. 2012, 2017; Atkinson et al. 2015; Kellar et al. 2015), and for monitoring the impacts of ecological changes (Romero and Wikelski 2001, Bechshøft et al. 2012). Glucocorticoid hormones (GCs) have been analyzed in several sample types in marine mammals, including blood (i.e., serum, plasma), blubber, feces, hair, saliva, exhaled respiratory vapor (“blow”), cerumen (i.e., earwax, ear plug), and baleen (e.g., Hogg et al. 2005, 2009; Amaral 2010; Rolland et al. 2012, 2017; St. Aubin et al. 2013; Trumble et al. 2013; Hunt et al. 2014; Kellar et al. 2015; Champagne et al. 2016; Robeck et al. 2017; Burgess et al. 2018). The temporal signature of the physiological response manifested as concentrations of GCs in these different types of biological samples varies from real-time (blood) or near real-time (blow), to hours or days (blubber, feces) or months to years (cerumen, hair, and baleen). Experimental design and data interpretation should consider the temporal dynamics of the hormones accumulated or sequestered in different sample types. Thus, concentrations of stress hormones in blood and blow reflect acute stress responses, while those measured in feces, blubber, baleen, cerumen, and hair indicate chronic or repeated hypothalamic-pituitary-adrenal (HPA) axis activity (Dickens and Romero 2013, Dantzer et al. 2014, Rolland and Moore 2018). In this study, we analyzed GC hormones in baleen from a western Arctic bowhead whale that endured a severe, prolonged entanglement in fishing gear to investigate whether a chronic stress response from the entanglement could be detected. Analysis of steroid hormones in baleen is uniquely valuable in that a retrospective longitudinal record of hormone concentrations over many years can be documented on a single plate (depending on the species and length of the plate) with at least monthly resolution (Hunt et al. 2014). Baleen is a keratinous, integumentary tissue similar in structure to hair, nails, and feathers. These tissues grow continuously throughout life, and steroid hormones have been quantified in all of these matrices using immunoassay techniques (e.g., Davenport et al. 2006, Bortolotti et al. 2008, Bechshøft et al. 2011, Hunt et al. 2014, Izawa et al. 2015). Researchers have hypothesized that hormones are incorporated into these integumentary tissues by passive diffusion from capillaries feeding the highly vascularized follicles from which they grow (Meyer and Novak 2012). Thus, the hormone content of a baleen sample from a given location along the plate reflects an integrative measure of the circulating levels of that hormone during the period of tissue growth. Studies have shown that hormone concentrations in keratinized tissues increase during prolonged or repeated elevation of circulating levels, but not in response to single, acute hormone elevations (Endo et al. 2017, Tallo-Parra et al. 2017). Therefore, GC concentrations in these integumentary tissues are being explored as a method for assessing a record of chronic stress responses (Meyer and Novak 2012, Mack and Fokidis 2017). In bowhead whales, we previously validated immunoassays for progesterone and cortisol (i.e., immunoreactive hormones and/or their metabolites) in baleen, and found that levels of both hormones varied according to sex, maturity, and pregnancy status (Hunt et al. 2014). In the current study, we validated an immunoassay system in bowhead baleen for an additional adrenal cortical hormone, corticosterone. Increased GCs in baleen in response to both anthropogenic and natural stressors have been reported recently in right whales (Eubalaena sp.). Lysiak et al. (2018) found elevated corticosterone in a baleen plate from a North Atlantic right whale (E. glacialis) with a chronic fishing gear entanglement that was eventually fatal. Fernández Ajó et al. (2018) attributed postnatal increases in baleen GCs to chronic stress from repeated wounding by kelp gulls (Larus dominicanus) in two southern right whale calves (E. australis). Variation in baleen GCs associated with energetically demanding life history stages such as pregnancy and lactation has been reported in E. glacialis (Hunt et al. 2017a). Additional steroid and thyroid hormones have also been extracted and quantified in baleen from mysticete whales (Hunt et al. 2017b), potentially expanding insights into reproductive physiology, metabolic activity, and nutritional status. Here, we analyzed baleen samples for both GC hormones (i.e., immunoreactive cortisol and corticosterone and/or their metabolites) along the length of a baleen plate from an 11.1 m juvenile male bowhead whale that was severely entangled in fishing rope when it died. For comparison, we present data on the same adrenal hormones measured in baleen samples from seven apparently healthy, male bowhead whales as a nonentangled reference group. The objectives of this study were to (1) determine whether we could detect an increase in GCs in baleen that might reflect sustained activation of the HPA axis related to the entanglement; (2) investigate the approximate timeframe of the entanglement event based on the location on the plate of the initial elevation of GCs above baseline concentrations for the whale, and estimated baleen growth rates; (3) assess the relative variation in cortisol and corticosterone in baleen in response to chronic stress from entanglement; (4) compare baleen GC concentrations in the entangled whale to those of nonentangled male bowheads; and (5) evaluate GC concentrations in a second sample type, feces, collected shortly after death during a time when the whale was known to be severely entangled. The entangled bowhead whale (17B6) was hunted for subsistence on 5 May 2017 by Inupiaq hunters from Utqiaġvik (Barrow), Alaska, under a quota approved by the International Whaling Commission. The captain and crew described the whale as lethargic (“resting at the surface”) as they approached it by boat, and they noted a rope trailing from the whale. They assumed it was a whale that may have been struck earlier, but lost. The hunters killed the whale instantly with a penthrite projectile that struck the cervical vertebrae. The whale was towed and hoisted (or “landed”) onto shorefast sea ice, where a severe, multicontact rope entanglement became apparent; the hunters estimated that 100–200 m of 19 mm diameter rope was attached to the whale. The rope was tightly anchored to the baleen rack, wrapped once around the left pectoral flipper, and wrapped tightly six times around the peduncle, penetrating ∼10 cm through the epidermis into the underlying blubber tissue (Fig. 1). (Note that the rope was briefly used as a towline, but not to hoist the whale onto the ice, nevertheless some penetration into subsurface tissues may have partially been a handling artifact.) The rope was cut off the whale by the hunters during harvest, and fraying of the rope ends was consistent with cut ends that had not been heat sealed or whipped (Fig. 2). It could not be determined from the available evidence whether the fishing gear was in active use when the whale became entangled. The rope removed from the whale measured 84.02 m in length and weighed 17.27 kg, although not all of the rope was retrieved or available for examination, so these are minimum measurements. There appeared to be two different types of rope including a white rope that was probably nylon and a blue flecked rope that was likely a copolymer fishing line (Fig. 2). Growth of algae and some marine worms on the rope suggested either prolonged fishing gear deployment or chronic entanglement. The gear was not marked; therefore, the specific fishery of origin was unknown, although rope characteristics (type, diameter and length) were consistent with Bering Sea fixed-gear pot fisheries (e.g., crab and cod). Pathological findings included an open traverse laceration (∼35 cm long) in the posterior body of the tongue. This lesion extended into the underlying muscle and the wound bed appeared somewhat necrotic (“pus” was noted). Histopathological results for the glossal rope injury were characterized by myofiber degeneration (multifocal, mild with stromal edema), and mucosal epithelial hyperplasia (diffuse, moderate with stromal fibrosis and lymphoplasmacytic glossitis). About 50% of the white chin patch was discolored yellow likely due to colonization by diatoms or algae. Incidental findings included a nuclear cataract (left eye) that obscured about 50% of the field of vision (RS, unpublished data). The right eye was not collected. Body condition appeared poor to hunters and observers based on a thin body shape, especially in the posterior region (see Fig. 1A). This observation was supported by the umbilical girth measurement (594 cm), which was the smallest measured for bowheads of this body length (11.0–11.2 m length, n = 9, umbilical girth (mean ± SD) = 676.9 ± 71.5 cm). Although the axillary girth (730 cm) was typical for a whale of this size there was some doubt about the accuracy of this measurement, and Miller et al. (2012) found that the umbilical area (i.e., 60% of the body length from the snout) showed the largest variation in width with body condition changes in closely related Eubalaena sp. Additionally, while blubber thickness (19 cm ventral, 23 cm dorsal) was considered normal for a whale in this age-class, the hypodermis was thin (1–2 cm; North Slope Borough, unpublished data), and George et al. (2015) showed that blubber thickness scaled with body length, not body condition (or presumably lipid percentage) and Rosa (2006) noted large variation in the percent lipid in bowhead blubber. While it is possible that the whale was able to feed while entangled, foraging efficiency would have been compromised by the damage to the tongue and baleen. Further, van der Hoop et al. (2016) showed that severely entangled right whales (Eubalaena glacialis) experience greatly increased energetic expenditures from the drag created by carrying fishing gear, which would have accelerated the loss of body condition in this whale. Gross examination results including extensive scar tissue formation associated with the rope embedded around the peduncle, algal growth and marine worms on the fishing lines, and the poor body condition of the whale were all evidence of a chronic entanglement of unknown duration. The opinion of the hunters and attending biologists was that the entangled whale was severely compromised physically, there was a low probability that the rope would have been shed, and had it not been harvested, the whale would not have survived. A single 227 cm long baleen plate was extracted from the rostrum, gum tissue (mammaq) was manually scraped off the base (closest to the maxilla), and the plate was stored at ambient temperatures in Utqiaġvik until shipment to the New England Aquarium Laboratory (Boston, MA). Prior to sampling, the plate was of using A was to the to sample along the plate (Fig. of baleen were using a tool with a baleen was collected by thickness through the plate cm the growth the of the plate (Fig. The and baleen were with to were to the of data on the entanglement event from baleen and to baseline levels of GCs to the of the entanglement using samples of the of the baleen. Baleen in whales, an in bowheads et al. 2008, George et al. a method such as to a specific for the on the an estimated baleen growth was used for this study which is likely especially for the samples the whale was and the baleen growth was was the for the samples during the we based the on an estimated of baleen growth for bowhead whales et al. this whale was to based on body length et al. et al. at the plate samples were collected cm for the and cm for the of the plate to cm of the plate was thin to baleen for on the estimated baleen growth for the cm of the plate of data at the These were used to the timeframe of a physiological response by the whale in response to the entanglement. A of samples were from plate for the of through For ± of baleen was weighed into mm and extracted with of were for for to the baleen from the of was into a and under and heat The sample was in of and briefly to and the hormone into a were in a to a from baleen and the was into a and stored at until hormone were measured using and immunoassay for the for bowhead baleen samples were previously by (see Hunt et al. 2014). and were for the corticosterone to that the system could detect and measure corticosterone in bowhead whale baleen was on sample of both baleen growth cm from the plate and growth cm from the These two were to that hormones in baleen similar of prolonged to in the Baleen were to were as relative hormone was used to that the of the of the was not different from the (i.e., accuracy was by a with an of baleen at were as expected and and were The corticosterone and accuracy for bowhead baleen For the both the and showed but corticosterone. The of the was not different from the for = = or = = growth baleen, the system could detect the hormone of in both growth and baleen that the to an immunoreactive in the sample with an that is similar to the results showed that the of the expected was with an = This the of that there were the baleen with measurement of immunoreactive corticosterone in this on baleen samples were the for the cortisol where was for use of the included with the samples were analyzed in low and hormone were in and and were on all to the data of variation were for samples were and for cortisol = and and for corticosterone = Hunt et al. the of for both hormone results are as of immunoreactive hormone of baleen concentrations for immunoreactive cortisol and corticosterone in baleen from whale were from the hormone data using an that all data the until this Lysiak et al. 2018). were considered elevated they were above this baseline were also (mean ± SD) for the data The the two GCs in baleen from was analyzed using and data to data based on and were using For all was considered Additionally, a sample was collected from the of at and stored until hormone In to the longitudinal hormone record in baleen, this sample an integrative measure of concentrations of circulating GCs to based on estimated time for Rolland et al. The sample was and GC were extracted with using similar to those for North Atlantic right whales (Hunt et al. The sample was in the for and GC concentrations were measured using a corticosterone which was previously validated for bowhead whale unpublished data). are as of immunoreactive hormone of The of corticosterone in this whale at was to concentrations for bowhead whales of this and determined in unpublished data). Baleen from the reference of male bowheads = were collected during subsistence in Utqiaġvik and Alaska, and This included juvenile and two and based on body length et al. et al. This reference GC data was from baleen at cm along the plate of using the same method described above for baleen samples from were to for to either or on the available sample A of samples were analyzed from the reference with whales and baleen Baleen cortisol and corticosterone concentrations in these samples were using the same described for whale The baseline using the described and cortisol and corticosterone concentrations were determined for the and two to compare with baleen GC concentrations in whale Glucocorticoid concentrations along the length of baleen plate from to for cortisol and from to for corticosterone. and corticosterone were similar = n = of both GCs were with for a the entanglement and corticosterone was present at levels cortisol during this (Fig. hormone concentrations SD) during this period were ± for cortisol and ± for corticosterone, and these were also the baseline concentrations there were 1). The baseline cortisol and corticosterone concentrations in were the SD) in the male = ± corticosterone = ± and ± ± 1). Baleen cortisol concentrations in the at cm from the plate base with a increased was elevated least for and increased to the about to death (Fig. to in the sample of baleen highly elevated at due to the severely of the whale. The cortisol was over the baseline in this whale, a adrenal stress response was in baleen. cortisol levels in were to the nonentangled male and the found in these reference In baleen corticosterone showed a response to the entanglement to was elevated several months cortisol and above the baseline throughout the baleen samples (Fig. The corticosterone was about this baseline It was the for the reference of ± and ± and was by the in two and two concentrations of both GCs in the baleen samples from the two hormone concentrations in the male reference group. This increase in GC levels was likely related to associated with the of was a juvenile whale with the increased baleen GCs in this whale were not related to reproductive maturity, for and for cortisol, greatly both this baseline and the reference baseline GCs in were which is over reference levels for male bowhead whales (mean ± ± n = unpublished data), the stress response present in this whale in the days et al. In blood a consistent with chronic stress in white blood low Atkinson et al. 2015). of this study the that chronic adrenal activation by a fishing rope entanglement was in the baleen from this bowhead whale. elevated baleen GCs were also reported in a North Atlantic right whale that was entangled in fishing gear et al. 2018). Studies in have also documented chronic adrenal activation in hair and keratinized tissues et al. 2011, et al. 2013, Mack and Fokidis 2017). using to HPA axis activation in and calves showed that acute in circulating GCs were not in that elevated hair GCs likely from activation of the HPA axis (Endo et al. 2017, Tallo-Parra et al. 2017). These from evidence that the elevation of GCs over months found in baleen from an adrenal response to the chronic entanglement by this whale. In increases in GCs at an stress response in this whale using a second sample The corticosterone was consistent with the of GC concentrations reported for entangled North Atlantic right whales et al. 2017). in this right whale have been and from months to years (mean of in highly elevated stress poor body and a prolonged to death et al. et al. 2013; Rolland et al. 2017). in fishing gear is the anthropogenic to large whales et al. 2011, et al. 2012, et al. Although not a for the of about 50% of bowhead whales in the western Arctic showed evidence of fishing gear entanglement based the of et al. 2017). fishing has with to an increased of fishing gear for bowhead whales and marine the of baleen growth in and several months in the responses of the two the when became entangled be but the hormone data that it and of This approximate timeframe is consistent with the of bowhead whales from feeding in the and to the Bering Sea in where there is extensive commercial fishing Analysis of in with hormone in could the therefore, the location of this entanglement et al. 2018). cortisol and corticosterone were both in baleen from and baseline corticosterone concentrations were over baseline This is consistent with of GCs in baleen, in which corticosterone concentrations were to cortisol in baleen (Hunt et al. Lysiak et al. 2018). Although the specific for of circulating GCs into baleen have not been the different of the two both and of the two GCs in the baleen For the for corticosterone is that of cortisol for the in and is related to the lipid of the and the to with and Therefore, baseline of corticosterone baleen may be increased relative to cortisol due to or that the baleen In the effects of of GCs in baleen are unknown this as is the possible of hormone as reported for GCs measured in hair follicles et al. 2012). Although both GCs baseline in for months presumably due to the entanglement in the current study, the response by cortisol and was to corticosterone. is to be the of circulating GC in and Atkinson et al. et al. and the results of the current study indicate a for cortisol in the adrenal response to this chronic entanglement. GCs from a North Atlantic right whale had concentrations of cortisol to corticosterone, that a cortisol response was also in this highly right whale et al. 2017). Lysiak et al. (2018) found that baleen corticosterone concentrations were and cortisol levels were the baseline during a chronic entanglement in an North Atlantic right whale. Hunt et al. (2018) suggested that both GCs were increased during the approximate when a North Atlantic right whale a mild entanglement along with the of white In the study, baleen corticosterone was at concentrations cortisol, although in both GCs was for the data and elevation in GCs was in the current The in baleen GC responses to entanglement in these may be they are based on from single whales, species in adrenal responses, variation in the biological responses of these to of or temporal in the GC responses in these different Additional on a of whales of and is to determine the relative of both GCs to different types of both natural (e.g., and anthropogenic to a reference of baleen GC levels, and to the biological and temporal of increased baleen hormone on both and marine species has suggested that the two GCs may therefore, of acute and chronic stress should of both hormones et al. 2012, et al. et al. In of adrenal hormones extracted from baleen is a tool for monitoring the physiological effects of chronic in bowheads and and of GCs along the length of a baleen plate can a longitudinal of adrenal responses potentially over a or which can with on entanglement the coupled with the location of This can also be to assess chronic stress responses to environmental and anthropogenic baleen whales they are through the same physiological of the integrative of adrenal stress hormones measured in baleen, a by the of and of this to investigate the of and to investigate of stress responses based on In baleen hormone as an for monitoring of stress to assess bowhead whale responses to the environmental changes and increased human activity in a Arctic greatly the for this study from the North Slope of biologists and are to the Whaling and the and from Utqiaġvik (Barrow), and St. Alaska, for to samples from these their and this would not have been also to for with sample and and and for are to Burgess for a of this Lysiak for baleen from the reference of whales, and for with and of large whale This study was by the Studies tissues collected for this study under to the North Slope of The have of to