[MARMAM] New Publication: Whiskers provide time-series of toxic and essential trace elements, Se:Hg molar ratios, and stable isotope values of an apex Antarctic predator, the leopard seal

[Charapata, Patrick]

Dear MARMAM Community,

On behalf of my colleagues and myself, I am excited to share with you all our 
new research article: “Whiskers provide time-series of toxic and essential 
trace elements, Se:Hg molar ratios, and stable isotope values of an apex 
Antarctic predator, the leopard seal”, now available in Science of the Total 
Environment. The article can be obtained Open Access by following this specific 
link
 for the next 50 days.

Charapata, P., Clark, C. T., Miller, N., Kienle, S. S., Costa, D. P., Goebel, 
M. E., Gunn, H., Sperou, E. S., Kanatous, S. B., Crocker, D. E., Borras-Chavez, 
R., & Trumble, S. J. (2023). Whiskers provide time-series of toxic and 
essential trace elements, Se:Hg molar ratios, and stable isotope values of an 
apex Antarctic predator, the leopard seal. Science of The Total Environment, 
854, 158651. 
https://doi.org/10.1016/j.scitotenv.2022.158651

Abstract

In an era of rapid environmental change and increasing human presence, 
researchers need efficient tools for tracking contaminants to monitor the 
health of Antarctic flora and fauna. Here, we examined the utility of leopard 
seal whiskers as a biomonitoring tool that reconstructs time-series of 
significant ecological and physiological biomarkers. Leopard seals (Hydrurga 
leptonyx) are a sentinel species in the Western Antarctic Peninsula due to 
their apex predator status and top-down effects on several Antarctic species. 
However, there are few data on their contaminant loads. We analyzed leopard 
seal whiskers (n = 18 individuals, n = 981 segments) collected during 2018–2019 
field seasons to acquire longitudinal profiles of non-essential (Hg, Pb, and 
Cd) and essential (Se, Cu, and Zn) trace elements, stable isotope (ẟ15N and 
ẟ13C) values and to assess Hg risk with Se:Hg molar ratios. Whiskers provided 
between 46 and 286 cumulative days of growth with a mean ~ 125 days per whisker 
(n = 18). Adult whiskers showed variability in non-essential trace elements 
over time that could partly be explained by changes in diet. Whisker Hg levels 
were insufficient (<20 ppm) to consider most seals being at “high” risk for Hg 
toxicity. Nevertheless, maximum Hg concentrations observed in this study were 
greater than that of leopard seal hair measured two decades ago. However, 
variation in the Se:Hg molar ratios over time suggest that Se may detoxify Hg 
burden in leopard seals. Overall, we provide evidence that the analysis of 
leopard seal whiskers allows for the reconstruction of time-series ecological 
and physiological data and can be valuable for opportunistically monitoring the 
health of the leopard seal population and their Antarctic ecosystem during 
climate change.

Please email me 
(patrick_charapa...@baylor.edu) with any 
questions on the paper. Additionally, I will happily send over a PDF copy if 
anyone requests it! Thank you!!

Cheers,

Patrick Charapata, PhD – Baylor University
Postdoctoral Researcher  – NWFSC/NOAA






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[MARMAM] New Publication: Steroid hormones in Pacific walrus bones collected over three millennia indicate physiological responses to changes in estimated population size and the environment

[Charapata, Patrick]

Hello MARMAM Members,

On behalf of my colleagues and myself, I am excited to share with you all our 
new research article: “Steroid hormones in Pacific walrus bones collected over 
three millennia indicate physiological responses to changes in estimated 
population size and the environment”, now available in Conservation Physiology. 
The article is Open Access and can be found here.

Charapata P, Horstmann L, Misarti N (2021) Steroid hormones in Pacific walrus 
bones collected over three millennia indicate physiological responses to 
changes in estimated population size and the environment. ConservPhysiol 9(1): 
coaa135; doi:10.1093/conphys/coaa135.

Abstract

The Pacific walrus (Odobenus rosmarus divergens) is an iconic Arctic marine 
mammal and an important resource to many Alaska Natives. A decrease in sea ice 
habitat and unknown population numbers has led to concern of the long-term 
future health of the walrus population. There is currently no clear 
understanding of how walrus physiology might be affected by a changing Arctic 
ecosystem. In this study, steroid hormone concentrations (progesterone, 
testosterone, cortisol and estradiol) were analysed in walrus bones collected 
during archaeological [3585–200 calendar years before present (BP)], historical 
[1880–2006 common era (CE)] and modern (2014–2016 CE) time periods, 
representing ~ 3651 years, to track changes in reproductive activity and 
cortisol concentrations (biomarker of stress) over time. Our results show that 
modern walrus samples have similar cortisol concentrations (median = 43.97 ± 
standard deviation 904.38 ng/g lipid) to archaeological walruses (38.94 ± 
296.17 ng/g lipid, P = 0.75). Cortisol concentrations were weakly correlated 
with a 15-year average September Chukchi Sea ice cover (P = 0.002, 0.02, r2 = 
0.09, 0.04, for females and males, respectively), indicating a possible 
physiological resiliency to sea ice recession in the Arctic. All steroid 
hormones had significant negative correlations with mean walrus population 
estimates from 1960 to 2016 (P < 0.001). Progesterone in females and 
testosterone in males exhibited significant correlations with average September 
Chukchi Sea ice cover for years 1880–2016 (P < 0.001 for both, r2 = 0.34, 0.22, 
respectively). Modern walruses had significantly lower (P = < 0.001) 
reproductive hormone concentrations compared with historic walruses during 
times of rapid population increase, indicative of a population possibly at 
carrying capacity. This is the first study to apply bone as a tool to monitor 
long-term changes in hormones that may be associated with changes in walrus 
population size and sea ice cover.

Please email me 
(patrick_charapa...@baylor.edu) if you 
have any questions or are interested in the manuscript. If for any reason you 
do not have access to the article through the link, I would be happy to provide 
a PDF of the manuscript upon request.

Cheers,

Patrick Charapata
PhD Candidate, Baylor University
patrick_charapa...@baylor.edu

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[MARMAM] New Publication: Whiskers as a novel tissue for tracking reproductive and stress-related hormones in North Pacific otariid pinnipeds

[Charapata, Patrick]

Hello MARMAM Members,

On behalf of my colleagues and myself, I am pleased to share with you all our 
new research article: “Whiskers as a novel tissue for tracking reproductive and 
stress-related hormones in North Pacific otariid pinnipeds”, now available in 
Conservation Physiology. The article is Open Access and can be found 
here.

Keogh MJ, Charapata P, Fadely BS, Zeppelin T, Rea L, Waite JN, Burkanov V, 
Marshall C, Jones A, Sprowls C, Wooller MJ (2021) Whiskers as a novel tissue 
for tracking reproductive and stress-related hormones in North Pacific otariid 
pinnipeds. Conserv Physiol 9(10): coaa134; doi:10.1093/conphys/coaa134.

Abstract

Keratinized tissues, including whiskers, are ideal for acquiring a record of 
physiological parameters. Most tissues provide a snapshot of physiological 
status; however, whiskers may support longitudinal sampling for reproductive 
and stress-related hormones, if hormones are incorporated as whiskers grow and 
concentrations change with physiological state. Whiskers from female Steller 
sea lions (Eumetopias jubatus) and northern fur seals (Callorhinus ursinus) 
were serially sectioned and pulverized and steroid hormones were extracted. 
Standard methods were used to validate enzyme immunoassay kits for cortisol, 
progesterone, 17β-estradiol and testosterone. All hormones were measurable in 
whisker segments from both species with progesterone concentrations showing 
cyclical patterns, which appear to signify previous pregnancies or luteal 
phases. Yearly progesterone concentrations were greater in years a pup was 
produced compared with years when no pup was observed. Free-ranging female 
Steller sea lions had reproductive rates between 0 and 1.0 (0.53 ± 0.33, n = 
12) using a yearly progesterone concentration of 30 pg/mg or greater to 
classify a reproductive year as producing a pup and below 30 pg/mg as 
non-reproductive. Cortisol concentrations were greater near the root and 
rapidly declined, lacking any obvious patterns, throughout the rest of the 
whisker. Progesterone and testosterone concentrations were able to help 
determine sex of unknown individuals. Immunohistochemistry revealed that 
steroid hormones most likely do not leach out of whiskers based on the 
deposition patterns of progesterone and cortisol being present throughout the 
whisker length. Overall, measuring steroid hormones in whiskers can reveal 
individual reproductive histories over multiple years in sea lions and fur 
seals. Cyclical patterns of δ15N were useful for identifying periods of up to 
~10 years of growth within whiskers, and measuring both stable isotopes and 
hormones may be useful for differentiating periods of active gestation from 
diapause and potentially track multi-year reproductive histories of female 
otariids.

Please email lead and corresponding author (Mandy Keogh, 
mandyjke...@gmail.com) if you have any questions 
or are interested in the manuscript. If for any reason you do not have access 
to the article through the link, I would be happy to provide a PDF of the 
manuscript upon request 
(patrick_charapa...@baylor.edu).

Cheers,

Patrick Charapata
PhD Candidate, Baylor University
patrick_charapa...@baylor.edu



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[MARMAM] New Publication - Stress-related and reproductive hormones in hair from three north Pacific otariid species: Steller sea lions, California sea lions and northern fur seals

[Charapata, Patrick]

Hello MARMAM Members,

On behalf of my colleagues and myself, I am pleased to share with you all our 
new research article “Stress-related and reproductive hormones in hair from 
three north Pacific otariid species: Steller sea lions, California sea lions 
and northern fur seals”, now available in Conservation Physiology. The article 
is Open Access and can be found here.

Keogh MJ, Gastaldi A, Charapata P, Melin S, Fadely BS (2020) Stress-related 
and reproductive hormones in hair from three north Pacific otariid species: 
Steller sea lions, California sea lions and northern fur seals. ConservPhysiol 
00(00): coaa069; doi:10.1093/conphys/coaa069.

Abstract

Assessing the physiological impact of stressors in pinnipeds is logistically 
challenging, and many hormones are altered by capture and handling, limiting 
the utility of metabolically active tissues. Hair is increasingly being used to 
investigate stress- related and reproductive hormones in wildlife populations 
due to less-invasive collection methods, being metabolically inert once grown 
and containing multiple biomarkers of ecological interest. We validated enzyme 
immunoassays for measuring aldosterone, cortisol, corticosterone, and 
testosterone in lanugo (natal hair grown in utero) samples collected from 
Steller sea lions (Eumetopias jubatus), California sea lions (Zalophus 
californianus), and northern fur seals (Callorhinus ursinus). We applied 
laboratory validation methods including recovery of added mass, parallelism and 
dilution linearity. We found no effects due to differences in alcohol- versus 
detergent-based cleaning methods. Further, there were no significant 
differences in hormone concentrations in hair samples collected immediately 
after the molt and the subsequent samples collected over 1 year, indicating 
steroid hormones are stable once deposited into pinniped hair. We found no sex 
differences in any hormone concentrations, likely due to the lanugo being grown 
in utero and influenced by maternal hormone concentrations. For Steller sea 
lion and California sea lion pups, we found hormone concentrations 
significantly differed between rookeries, which warrants future research. Hair 
provides a novel tissue to explore the intrinsic or extrinsic drivers behind 
hormone measurements in otariids, which can be paired with multiple 
health-related metrics to further investigate possible drivers of physiological 
stress.

Please email lead and corresponding author (Mandy Keogh, 
mandyjke...@gmail.com) if you have any questions 
or are interested in the manuscript. If for any reason you do not have access 
to the article through the links, I would be happy to provide a PDF of the 
manuscript upon request 
(patrick_charapa...@baylor.edu).

Cheers,

Patrick Charapata
PhD Candidate, Baylor University
patrick_charapa...@baylor.edu

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Re: [MARMAM] New Publication - Reproductive and stress‐related hormones in whiskers from two North Pacific phocids: Harbor and ringed seals

[Charapata, Patrick]

Hello MARMAM Members,

On behalf of my colleagues and myself, I am pleased to share with you all our 
new research note “Reproductive and stress‐related hormones in whiskers from 
two North Pacific phocids: Harbor and ringed seals”, now available in early 
view at Marine Mammal Science. The article can be found here: 
https://bit.ly/38mVbJT.

Keogh MJ, Charapata P, Karpovich S, Jones A, Sprowls C, Marshall CD. 
Reproductive and stress-related hormones in whiskers from two North Pacific 
phocids: Harbor and ringed seals. Mar Mam Sci. 2020;1–12. 
https://doi.org/10./mms.12709

Summary

Several populations of North Pacific pinnipeds are currently listed as 
depleted under the Marine Mammal Protection Act, endangered under the 
Endangered Species Act, of the United States or with unknown status, 
highlighting the need for new methods to assess the reproductive rates of these 
populations. Most phocids are annual breeders with estrus and parturition 
occurring on terrestrial or ice platforms. In phocids, serum progesterone 
concentrations remain elevated during late gestation, supporting identification 
of pregnancy after implantation (Gardiner, Boyd, Follett, Racey, & Reijnders, 
1999; Gardiner, Boyd, Racey, Reijnders, & Thompson, 1996; Mellish & Iverson, 
2005; Reijnders 1990). However, current sampling methods based on blood and 
feces only provide a snapshot of reproductive status. Recently, methods were 
developed to measure cortisol in whiskers (Karpovich, Skinner, Kapronczai, 
Smith, & Janz, 2019), highlighting the potential to measure reproductive 
hormones in whiskers from free-ranging phocid seals. Unlike other tissues 
currently used for determining reproductive status, whiskers do not require 
special storage or handling, which can be challenging in remote field 
conditions. More importantly, the potential to use whiskers to measure 
reproductive hormones may alleviate problems associated with a single sample by 
capturing reproductive hormone concentrations sequentially along the length of 
the whisker, allowing for the examination of hormone con- centrations over the 
course of one year for phocids (Greaves, Hammill, & Eddington, 2004; Hirons, 
Shell, & St. Aubin, 2001; Lübcker, Condit, Beltran, Bruyn, & Bester, 2016; Zhao 
& Schell, 2004). Given the potential utility of measuring reproductive hormones 
in phocid whiskers, our objectives were to (1) validate enzyme immunoassays 
(EIA) to measure reproductive and stress-related steroid hormones in phocid 
whiskers, (2) compare the patterns of multiple steroid hormones along the 
length of whiskers to evaluate the retention of steroid hormones in phocid 
whiskers, (3) apply immunohistochemistry (IHC) methods to explore deposition of 
progesterone and cortisol along harbor seal whiskers, and (4) investigate the 
influence of age class (i.e., adult vs. subadult) and reproductive state on 
hormone concentrations in whiskers.

In this study, we report a novel method to measure reproductive hormones in 
phocid whiskers, providing another tool for future studies. We found hormones 
are deposited throughout the length of a phocid whisker, helping to validate 
the utility of whiskers as a reliable matrix for measuring reproductive 
hormones. We found differences in whisker progesterone concentrations between 
adults and subadults from two phocid species and between pregnant and 
nonpregnant adult harbor seals. There may be other reproductive steroid 
hormones, such as testosterone, that may also be useful for assessing age 
class. The timing of the whisker collection for both phocid species may 
influence detection of the rise in progesterone in the whisker likely 
associated with active gestation. Nevertheless, phocid whiskers contain 
hormones incorporated during the estimated years' worth of growth (Beltran et 
al., 2015, Hirons et al., 2001, Lübcker et al., 2016). Analysis of hormone 
concentrations from whiskers could be beneficial in accruing long-term 
physiological data from keratinized tissues in phocids. Further, whiskers in 
archived collections from museums, stranding networks, and gov- ernment 
agencies could serve as a reservoir of samples to perform retrospective studies 
on phocid reproductive and stress physiology, helping to understand how future 
environmental changes may impact phocid physiology.

Please email lead and corresponding author (Mandy Keogh, 
mandyjke...@gmail.com) if you have any questions 
or are interested in the manuscript. I would be happy to provide a PDF of the 
manuscript upon request 
(patrick_charapa...@baylor.edu).

Cheers,

Patrick Charapata
PhD Candidate, Baylor University
patrick_charapa...@baylor.edu


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[MARMAM] New publication on steroid hormones in ancient and modern walrus bone

[Charapata, Patrick]

Hi All,

We are pleased to announce the recent publication of our manuscript in Rapid 
Communications in Mass Spectrometry and it is available open access 
here.

Title: A novel method to measure steroid hormone concentrations in walrus bone 
from archaeological, historical, and modern time periods using liquid 
chromatography/tandem mass spectrometry

Authors: Patrick Charapata, Lara Horstmann, Amber Jannasch, Nicole Misarti

Abstract:

Rationale: A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method 
was validated and utilized to measure and analyze four steroid hormones related 
to stress and reproduction in individual samples from a novel tissue, Pacific 
walrus (Odobenus rosmarus divergens, herein walrus) bone. This method 
determines steroid hormone concentrations in the remote walrus population over 
millennia from archaeological (>200 BP), historical (200–20 BP), and modern 
(2014–2016) time periods.
Methods: Lipids were extracted from walrus bone collected from these periods 
using methanol before LC/MS/MS analysis. Isotopically labeled internal 
standards for each target hormone were added to every sample. Analytical and 
physiological validations were performed. Additionally, a tissue comparison was 
done among paired walrus bone, serum, and blubber samples. A rapid resolution 
liquid chromatography system coupled to a QqQ mass spectrometer was used to 
analyze all samples after derivatization for progesterone, testosterone, 
cortisol, and estradiol concentrations. Multiple reaction monitoring was used 
for MS analysis and data were acquired in positive electrospray ionization mode.
Results: Progesterone, testosterone, cortisol, and estradiol were linear along 
their respective standard calibration curves based on their R2 values (all > 
0.99). Accuracy ranged from 93–111% for all hormones. The recovery of 
extraction, recovery of hormones without matrix effect, was 92–101%. The 
overall process efficiency of our method for measuring hormones in walrus bone 
was 93–112%. Progesterone and testosterone concentrations were not affected by 
reproductive status among adult females and males, respectively. However, 
estradiol was different among pregnant and non‐pregnant adult females. Overall, 
steroid hormones reflect a long‐term reservoir in cortical bone. This method 
was also successfully applied to walrus bone as old as 3585 BP.
Conclusions: LC/MS/MS analysis of bone tissue (0.2–0.3 g) provides stress and 
reproductive data from elusive walruses that were alive thousands of years ago. 
Based on physiological validations, tissue comparison, and published 
literature, steroid hormone concentrations measured in walrus cortical bone 
could represent an accumulated average around a 10–20‐year time span. By 
investigating how stress and reproductive physiology may have changed over the 
past ~3000 years based on bone steroid hormone concentrations, this method will 
help answer how physiologically resilient walruses are to climate change in the 
Arctic.
--

Please contact me 
(patrick_charapa...@baylor.edu) with any 
questions regarding the paper.

Thank you for your time,

Patrick Charapata
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