NORTH PACIFIC RESEARCH BOARD PROJECT FINAL REPORT
Comparison of stable carbon and nitrogen isotope ratios in muscle and epidermis of
subsistence-harvested bowhead, beluga and gray whales
NPRB Project 635 Final Report
Larissa-A. Dehn 1,2, Erich H. Follmann 1
1
Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska 99775
Phone: (907) 459-7288, e-mail: lara.dehn@alaska.gov
Phone: (907) 474-7338, e-mail: ffehf@uaf.edu
2
Alaska Department of Fish and Game, Division of Commercial Fisheries, 1300 College
Road, Fairbanks, Alaska 99701-1599
October 2007
ABSTRACT
Collection of minimally invasive biopsy samples (i.e., epidermis) has become
increasingly important to establish normal reference ranges of stable isotopes and their biological
variability. These baseline data enhance the understanding of feeding ecology, habitat use and
potential food limitation in apparently healthy, free-ranging cetacean populations. Muscle and
epidermis samples were collected from subsistence-hunted bowhead (Balaena mysticetus) and
beluga whales (Delphinapterus leucas) from northern Alaska, and from subsistence-harvested
Russian gray whales (Eschrichtius robustus). Samples were also obtained from gray whales
stranded along the California coast during an unusual mortality event in 1999 and 2000. Stable
isotopes of nitrogen and carbon are good indicators of trophic position and benthic/pelagic
feeding, respectively in both muscle and epidermis of these Arctic whales. However, epidermis
tends to be enriched in 15N over muscle, while carbon-13 is more depleted in epidermis. Lipidextraction did not alter nitrogen isotope-ratios in either muscle or epidermis, but significantly
affected 13C in epidermis. Nitrogen-15 is enriched in muscle, but not epidermis of stranded
whales as compared to subsistence-harvested gray whales, indicating protein catabolism and
nutritional stress in stranded whales. Similarly, 13C in epidermis of whales harvested for
subsistence use was lower than in stranded whales, suggesting depleted lipid stores and/or food
limitation in stranded animals. Epidermal carbon and nitrogen isotope signatures were similar in
both present-day bowhead whales and in an ancient sample from the Northern Bering Sea region.
Though only a single specimen, this suggests that feeding ecology of bowhead whales has
remained stable for a millennium. However, biological variables, i.e., length and sex, are
unknown for the ancient sample and 15N is negatively correlated to length in epidermis of
present-day whales.
KEY WORDS
Feeding ecology, Food limitation, Stable isotopes, Bowhead whale, Beluga whale, Gray whale
CITATION
Dehn, L.-A., and Follmann, E.H. 2007. Comparison of stable carbon and nitrogen isotope
ratios in muscle and epidermis of subsistence-harvested bowhead, beluga and gray whales. North
Pacific Research Board Final Report 635, 24 p.
ii
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... ii
KEY WORDS ................................................................................................................................ ii
CITATION ..................................................................................................................................... ii
LIST OF TABLES........................................................................................................................ iii
LIST OF FIGURES...................................................................................................................... iii
INTRODUCTION ......................................................................................................................... 1
METHODS..................................................................................................................................... 4
Field Sampling ............................................................................................................................ 4
Stable Isotopes ............................................................................................................................ 4
Statistical Analysis ...................................................................................................................... 6
RESULTS ....................................................................................................................................... 6
Bowhead Whales......................................................................................................................... 8
Gray Whales................................................................................................................................ 9
Beluga Whales ............................................................................................................................ 9
Muscle versus Epidermis ............................................................................................................ 9
Lipid Extraction ........................................................................................................................ 10
DISCUSSION............................................................................................................................... 10
CONCLUSIONS .......................................................................................................................... 14
PUBLICATIONS......................................................................................................................... 14
OUTREACH ................................................................................................................................ 14
ACKNOWLEDGEMENTS ........................................................................................................ 15
REFERENCES ............................................................................................................................ 17
LIST OF TABLES
Table 1: Samples collected from subsistence-harvested whales in Alaskan and Russian
communities and from gray whales stranded along the California coast. .......................... 4
Table 2: Mean stable carbon and nitrogen isotope values ± standard deviation (SD), median,
range, and sample size (n) in muscle and skin of bowhead, beluga and gray whales
harvested in Alaska and Russia and gray whales stranded along the California coast (LE –
Lipid Extracted).................................................................................................................. 7
LIST OF FIGURES
Figure 1: Sample collection sites in Alaskan and Russian communities. ....................................... 3
iii
Figure 2: Sample of ancient bowhead whale muktuk recovered from an ice cellar in Gambell,
Alaska. Photo courtesy of C. George (North Slope Borough, Department of Wildlife
Management). ..................................................................................................................... 5
Figure 3: Patch of fetal epidermis and “new”, smooth skin at the rostrum of a juvenile (ingutuk)
bowhead whale harvested in fall, 2006. ............................................................................. 8
Figure 4: Length [cm] as a proxy for age versus 15N in bowhead whales. The red line and bar
illustrate the mean nitrogen isotope ratio of the ancient epidermis sample ± 1SD (n=4).
LOESS non-parametric smoothing was employed to visualize trends for epidermis
(dotted blue line) and muscle (dotted green line). ............................................................ 13
Figure 5: Flyer for community presentation in Barrow, Alaska as part of the Barrow Arctic
Science Consortium (BASC) Schoolyard Project. ........................................................... 16
iv
INTRODUCTION
Feeding ecology is a fundamental aspect in the understanding, management and
conservation of free-ranging marine mammals. Nutritional status of marine mammals is a factor
that can limit reproductive output and thus population growth. However, foraging of large
cetaceans can be difficult to assess when direct observation is not possible, and the use of
chemical feeding ecology (i.e., stable isotope and fatty acid analysis) has become increasingly
important (Schell et al., 1989; Hooker et al., 2001; Hoekstra et al., 2002; Krahn et al., 2004;
Caraveo-Patiño and Soto, 2005; Dehn et al., 2006a). Stable isotopes of carbon and nitrogen have
been established as powerful tools in animal ecology. They occur naturally, and nitrogen isotope
ratios of prey are reflected in tissues of the consumer, with slight enrichment occurring at each
trophic step (Kelly, 2000). Stable carbon isotopes are generally used to provide information on
spatial habitat use and carbon sources rather than trophic relationships as they enrich in consumer
tissues only to a minor degree (Schell et al., 1989; France, 1995; Burton and Koch, 1999).
Stable isotope ratios for marine mammals are typically reported in muscle as it represents
a tissue with low to medium metabolic turnover and thus provides dietary information over the
period of about one month (Tieszen et al., 1983). However, obtaining muscle samples via biopsy
from free-ranging Arctic cetaceans is particularly challenging as their blubber thickness can
exceed 30 cm (Lowry, 1993). Sampling of muscle tissue from healthy marine mammals is
therefore only possible during Native subsistence harvests. On the other hand, skin samples of
large whales can be obtained via biopsy dart and crossbow or biopsy pole and are minimally
invasive (Fossi and Marsili, 1997; Krahn et al., 2004). In addition, biopsy sampling presents the
advantage of re-sampling the same individual over its lifetime, allowing assessment of
chronological and seasonal dietary variation. However, it is often difficult to interpret stable
isotope ratios in epidermis compared to the growing database of published results for muscle due
to unpredictable or poorly understood turnover rates and tissue- and species-specific isotopic
fractionation (Gannes et al., 1997; Adams and Sterner, 2000; Bearhop et al., 2002; McCutchan et
al., 2003).
Bowhead whales (Balaena mysticetus) are large mysticetes and occupy the seasonally
ice-covered Arctic Ocean year round. The Bering-Chukchi-Beaufort seas stock of bowheads is
listed as endangered but is recovering at an estimated rate of 3.4 % annually, while sustaining a
controlled subsistence harvest (George et al., 2004). They migrate yearly from the Bering Sea in
winter to the Beaufort Sea in summer (Moore and Reeves, 1993) and subsistence whalers have
taken bowhead whales along their migratory path for centuries. Though bowhead feeding has
been observed in both the Beaufort and the Bering–Chukchi seas, the importance of either region
1
as feeding ground is still debated (Hoekstra et al., 2002; Lowry et al., 2004; Lee et al., 2005).
Despite these uncertainties, the diet of bowheads is well described. Bowheads are adapted to
filter-feed on even low-density patches of zooplankton (George et al., 1999). Though a wide
variety of prey species have been identified from bowhead stomach contents, their main prey
remains relatively stable and consists of copepods and euphausids (Lowry et al., 2004). The
stable and well-described diet of bowhead whales makes them the ideal choice to determine
isotopic enrichment factors in different tissues.
Five stocks of beluga whales (Delphinapterus leucas) are currently recognized in
Alaskan waters (O’Corry-Crowe et al., 1997). Belugas are odontocetes and consume a wide
variety of fish. It was suggested that beluga whales compete with piscivorous spotted seals
(Phoca largha) for prey (Seaman et al., 1982), though stable nitrogen isotope ratios illustrate that
belugas occupy a lower trophic level than spotted seals (Dehn et al., 2006b). This indicates that
belugas do not consume fish exclusively and 90-100% of stomachs analyzed by Seaman et al.
(1982) contained invertebrates (e.g., octopus, shrimp and polychaetes). The study of belugas
compared to mysticete whales offers the opportunity to investigate stable isotope ratios and
isotope fractionation in tissues of whales that rely on a wide variety of prey species from different
trophic levels.
Gray whales (Eschrichtius robustus) are primitive baleen whales and unique in their
reliance on benthic invertebrate prey (Rice and Wolman, 1971). The eastern Pacific stock of gray
whales migrates annually from their feeding grounds in the Bering and Chukchi seas in summer
to their calving grounds in Baja California and the Gulf of California in winter (Rice and
Wolman, 1971). Gray whale feeding ecology is relatively well documented, though the
importance of the Gulf of California as feeding area remains uncertain (Oliver et al., 1983;
Caraveo-Patiño and Soto, 2005). Benthic gammaridean amphipods (e.g., Ampelisca spp.) are
most commonly identified from stomach contents (Rice and Wolman, 1971; Zimushko and
Ivashin, 1980; Bogoslovskaya et al., 1981).
Hobson et al. (1993) found evidence that nutritional stress will lead to enrichment of
nitrogen-15 in tissues due to catabolism of body proteins that are already enriched relative to the
diet. Marine mammals are well known for their capability to fast for long periods of time, e.g.
during migration and rearing of young. However, fasting adapted species mobilize fat reserves
and produce ketone bodies, but will utilize body protein sparingly (Castellini and Rea, 1992).
Significant body protein mobilization occurs during phase III fasting or starvation and fasting
adapted marine mammals will avoid reaching phase III (Castellini and Rea, 1992). During 1999
and 2000 the number of gray whales involved in fatal strandings along the North Pacific coast
2
increased from an average of about 50 animals per year to 283 and 368 whales, respectively (Le
Boeuf et al., 2000; Gulland et al., 2005). Whales appeared emaciated and the blubber was low in
lipid content, thus pointing to nutritional stress as a likely cause in this mortality event (Moore et
al., 2001; Gulland et al., 2005). Reasons for this starvation event remain unknown, although
carrying capacity and environmental changes, such as El Niño, have been discussed and could
have led to diminished food sources and forced whales to utilize less nutritious prey (Le Boeuf et
al., 2000; Moore et al., 2001; Moore et al., 2003; Gulland et al., 2005).
The objectives of this study are 5-fold and aim i) to compare stable carbon and nitrogen isotope
ratios in muscle and epidermis of apparently healthy bowhead, belugas and gray whales taken
during Native subsistence harvests and make inferences on their general feeding ecology, ii) to
assess variability of carbon and nitrogen isotope ratios with regard to sex and age, iii) to
investigate the effect of lipid extraction on isotope ratios, iv) to compare stable carbon and
nitrogen isotope ratios of present-day bowhead whales to an ancient sample, and v) to evaluate
the nutritional stress hypothesis by comparing stable carbon and nitrogen isotope ratios of
subsistence-harvested gray whales to whales stranded along the California coast.
Figure 1: Sample collection sites in Alaskan and Russian communities.
3
METHODS
Field Sampling
Whale samples (epidermis and muscle) were obtained during Native subsistence harvests
in the Alaskan and Russian Arctic (Figure 1). Bowhead whale tissues were collected between
1997 and 2006 during either spring or fall migration. Further, the North Slope Borough,
Department of Wildlife Management in Barrow, Alaska made a sample of ancient bowhead
whale skin available to this study (Figure 2). The sample was obtained from an ancient ice-cellar
in Gambell, Alaska and was carbon dated to be 1050±70 years old. Beluga whale tissues were
sampled from 1996 to 1999, and gray whale samples were collected in 2001. In addition,
epidermis and muscle was obtained from gray whales stranded in 1999 and 2000 along the
California coast during an unusual mortality event. Whales were either freshly dead or only
slightly decomposed at the time of specimen collection. Samples were frozen, shipped to the
University of Alaska Fairbanks (UAF) and stored at -20ºC until analysis. Basic morphometrics
were recorded, e.g., standard body length (rostrum to fluke notch), blubber thickness and sex.
Standard body length was used as a proxy for age in cetaceans (Rice and Wolman, 1971; George
et al., 1999). Sampling locations and sample sizes are summarized in Table 1.
Table 1: Samples collected from subsistence-harvested whales in Alaskan and Russian
communities and from gray whales stranded along the California coast.
Species
Bowhead whale
Beluga whale
Gray whale
Sampling Location
Barrow
Kaktovik
Wainwright
Savoonga
Pt. Lay
Wainwright
Pt. Hope
Barrow
Kaktovik
Little Diomede
Lorino / Lavrentiya
California Coast - Stranded
Epidermis Muscle
n
116
115
11
17
2
6
32
32
2
2
9
5
4
1
1
3
25
17
18
11
n: sample size
Stable Isotopes
Approximately 5 g of tissue was sub-sampled in 7 ml scintillation vials, freeze dried for a
minimum of 48 hours and ground into a fine powder. A sub-sample of homogenized epidermis
4
was analyzed for the effect of lipid-removal following the procedure established by Pinnegar and
Polunin (1999) using a 10:5:4 methanol:chloroform:water mixture. Lipid-extracted samples were
frozen and returned to the freeze-dryer for 8 hours. Lipid extraction had no significant effect on
stable carbon and nitrogen ratios in muscle of Arctic cetaceans (Hoekstra et al., 2002). For each
sample, 0.2–0.4 mg of tissue was weighed into a 4.75 x 4 mm tin capsule, which was folded into
a cube. Stable carbon and nitrogen isotope ratios were determined at UAF following the
procedure described in Dehn et al. (2006a) using a Finnigan MAT DeltaPlusXL Isotope Ratio
Mass Spectrometer (IRMS) directly coupled to a Costech Elemental Analyzer (ESC 4010).
Enrichment of a particular isotope is reported using the following notation and equation:
R‰ = ((Rsample / Rstandard) - 1) x 1000,
where the differential notation (R) represents the relative difference between isotopic ratios of
the sample and standard gases (i.e., 13C/12C, 15N/14N). External instrument reproducibility for
both carbon and nitrogen isotope analysis was ±0.2‰. Stable isotope ratios in muscle of
subsistence-harvested cetaceans have been reported previously by Dehn et al. (2006a).
Figure 2: Sample of ancient bowhead whale muktuk recovered from an ice cellar in Gambell,
Alaska. Photo courtesy of C. George (North Slope Borough, Department of Wildlife
Management).
5
Statistical Analysis
Standard statistical tests were used to analyze and compare variables in the data set. All
variables were ranked prior to analysis to adjust for violations of normality and homogeneity
assumptions. Analysis of Variance (ANOVA) followed by Tukey’s multiple comparison test was
applied to compare variable means among cetacean species, between tissues (epidermis and
muscle), between lipid-extracted and non-extracted tissues, and between stranded and
subsistence-harvested gray whales. Spearman rank correlation was calculated within each species
to determine correlations of stable carbon and nitrogen isotope ratios with length (as a proxy for
age). All statistical analyses have been performed with the SAS software package (Version 9.1)
with =0.05. LOESS smoothing followed by nonlinear regression analysis was utilized on nonranked raw data to estimate suitable functions between two variables. Graphing and nonlinear
regression analyses were conducted using Sigma-Plot (Version 10). Results are reported as mean
± standard deviation (SD) unless otherwise noted. In addition, the sample median is reported as it
is robust to outliers.
RESULTS
Stable carbon and nitrogen isotope ratios were significantly different in both muscle and
epidermis for the three subsistence-harvested cetacean species analyzed (P=<0.0001 for both 13C
and 15N in muscle and P=<0.0001 for both 13C and 15N in epidermis). 15N was highest in
belugas (16.7 ± 0.6‰ and 16.8 ± 0.5‰ for muscle and skin, respectively), followed by bowheads
(13.3 ± 0.8‰ in muscle and 13.6 ± 0.9‰ in skin) then gray whales (12.0 ± 0.9‰ and 13.2 ±
1.1‰ for muscle and skin, respectively). Carbon isotope values were more enriched in gray
whales (-17.3 ± 1.0‰ for muscle and -17.6 ± 1.3‰ for skin) than in bowheads (-20.6 ± 0.9‰ for
muscle and -21.0 ± 0.7‰ for skin) and belugas were intermediate (-18.4 ± 0.6‰ and -19.3 ±
0.6‰ for muscle and skin respectively). Means, standard deviations, medians and ranges of stable
carbon and nitrogen isotope ratios of subsistence harvested whales and stranded gray whales are
given in Table 2.
6
Table 2: Mean stable carbon and nitrogen isotope values ± standard deviation (SD), median,
range, and sample size (n) in muscle and skin of bowhead, beluga and gray whales harvested in
Alaska and Russia and gray whales stranded along the California coast (LE – Lipid Extracted).
Bowhead Whale
Muscle
Mean ± SD
Median
Range
1047 ± 259
942
399 to 1770
n = 133
15N
Non-LE
13.29 ± 0.76
13.16
11.80 to 15.56
n = 134
Skin
Mean ± SD
Median
Range
1036 ± 282
941
273 to 1800
n = 130
13.63 ± 0.94
13.41
11.34 to 15.96
n = 133
-20.97 ± 0.73
-20.97
-22.81 to -19.10
n = 133
13.81 ± 0.84
13.69
11.93 to 16.53
n = 99
-20.49 ± 0.68
-20.42
-22.50 to -18.70
n = 99
Ancient
Muktuk
Mean ± SD
Median
Range
-
12.71 ± 0.33
12.64
12.43 to 13.12
n=4
-20.82 ± 0.10
-20.84
-20.92 to -20.68
n=4
-
-
13C
Non-LE
-17.32 ± 1.03
-17.05
-20.00 to -15.96
n = 17
15N
LE
-
13C
LE
-
Length [cm]
13C
Non-LE
-20.63 ± 0.85
-20.65
-25.06 to -18.84
n = 134
15N
LE
13.33 ± 0.60
13.27
12.22 to 14.71
n = 53
13C
LE
-20.27 ± 0.47
-20.31
-21.10 to -18.94
n = 53
Gray Whale - Subsistence
Muscle
Mean ± SD
Median
Range
1053 ± 196
990
810 to 1460
n = 15
15N
Non-LE
12.03 ± 0.86
11.87
11.12 to 14.62
n = 17
Skin
Mean ± SD
Median
Range
999 ± 192
967
800 to 1460
n = 23
13.21 ± 1.13
13.3
11.55 to 15.21
n = 25
-17.62 ± 1.32
-17.00
-21.38 to -15.90
n = 25
13.34 ± 0.93
13.28
11.45 to 15.17
n = 18
-16.72 ± 0.78
-16.81
-18.46 to -15.60
n = 18
13C
Non-LE
-16.58 ± 0.89
-16.56
-18.43 to -15.19
n = 11
15N
LE
-
13C
LE
-
Length [cm]
Gray Whale - Stranded
Muscle
Mean ± SD
Median
Range
844 ± 312
823
434 to 1341
n = 11
15N
Non-LE
12.93 ± 1.01
12.86
11.06 to 14.82
n = 11
Skin
Mean ± SD
Median
Range
1084 ± 275
1190
398 to 1341
n = 16
13.51 ± 0.80
13.76
11.57 to 14.62
n = 18
-16.52 ± 1.71
-16.73
-20.53 to -11.14
n = 18
13.59 ± 1.02
13.33
11.72 to 15.03
n = 14
-16.06 ± 0.92
16.17
-17.30 to -13.99
n = 14
Length [cm]
13C
Non-LE
-18.41 ± 0.62
-18.32
-20.75 to -17.21
n = 49
15N
LE
16.70 ± 0.27
16.64
16.22 to 17.21
n = 12
13C
LE
-18.26 ± 0.21
-18.39
-18.75 to -17.96
n = 12
-19.30 ± 0.55
-19.18
-21.34 to -18.53
n = 42
16.75 ± 0.74
16.74
15.05 to 17.92
n = 36
-18.67 ± 0.57
-18.59
-20.61 to -17.89
n = 36
Length [cm]
Beluga Whale
Muscle
Mean ± SD
Median
Range
368 ± 51
388
240-440
n = 45
15N
Non-LE
16.74 ± 0.56
16.72
15.48 to 18.34
n = 49
Skin
Mean ± SD
Median
Range
369 ± 58
390
206 to 440
n = 40
16.81 ± 0.53
16.88
15.56 to 17.84
n = 42
7
Bowhead Whales
Epidermal carbon and nitrogen isotope signatures were similar in both present-day
bowhead whales and the ancient sample (P=0.07 and P=0.84 for 15N and 13C, respectively).
Total body length (as a substitute for age) was significantly negatively correlated with 15N in
muscle (P=<0.0001), but not with 13C (P=0.95). Similarly, epidermal 15N was negatively
correlated to length (P=<0.0001), though 13C showed no correlation (P=0.13). Both stable
carbon and nitrogen isotope ratios did not differ between sexes in either muscle or skin (P=0.62
and P=0.86 for 15N and 13C in muscle, respectively and P=0.44 and P=0.06 for 15N and 13C
in epidermis, respectively).
Sampling of two juvenile bowheads offered the opportunity to obtain both newly
developed, smooth skin as well as fetal epidermis from the same animal (Figure 3). The presence
of both spongy, fetal skin and smooth neonatal epidermis has been described by Reeb et al.
(2005) in southern right whales (Eubalaena australis). The first whale was a 780 cm long
ingutuk, a morphological variant described as a short juvenile with wide axillary circumference
(Braham et al., 1980). 15N and 13C in “new” skin was 15.48‰ and -20.42‰, respectively, and
was 14.12‰ and -19.57‰, respectively in fetal skin. The second whale was a 630 cm calf and
15N in “new” skin measured 12.43‰, while the fetal epidermis was 14.80‰. Carbon isotope
values were -21.85‰ in “new” skin and -20.18‰ in fetal epidermis.
Fetal Epidermis
Baleen
“New” Smooth Epidermis
Figure 3: Patch of fetal epidermis and “new”, smooth skin at the rostrum of a juvenile (ingutuk)
bowhead whale harvested in fall, 2006.
8
Gray Whales
Nitrogen isotope ratios in muscle of gray whales harvested for subsistence use in
Chukotka were significantly lower than in stranded gray whales (P=0.008). This finding was not
repeated for 15N in epidermis (P=0.20). Carbon isotope ratios were not different between
stranded and subsistence harvested whales in muscle (P=0.06), but epidermal carbon-13 was
significantly enriched in stranded gray whales (P=0.01). In lipid extracted skin, 13C was similar
between subsistence-harvested and stranded gray whales (P=0.06). Lipid extraction had no other
significant effect on isotope ratios. Stable carbon and nitrogen isotope ratios were not different
between males and females of both subsistence-harvested and stranded gray whales in either
tissue. Total body length (as a proxy for age) was not correlated to 15N and 13C in muscle of
subsistence-harvested gray whales (P=0.09 and P=0.84 for 15N and 13C, respectively), but was
significantly correlated in epidermis (P=0.0002 for 15N and P=0.002 for 13C). Total body length
of stranded gray whales was not correlated with any of the variables in either epidermis or muscle
(P=0.29 and P=0.57 for 15N in muscle and skin, respectively and P=0.49 for 13C in muscle and
P=0.46 for 13C in skin).
Beluga Whales
O'Corry-Crowe et al. (1997) showed that belugas migrating past Point Hope, Alaska are
part of the Eastern Beaufort stock and mDNA markers of these whales are distinctly different
from belugas harvested in Point Lay, Alaska (Eastern Chukchi stock). In addition, Adams et al.
(1993) indicated that subsistence hunters from Diomede, Barrow and Kaktovik also harvest
belugas from the Eastern Beaufort stock. 13C and 15N were not significantly different in muscle
of Eastern Beaufort and Eastern Chukchi beluga whales (P=0.90 for both 13C and 15N). Only
few skin samples were available from Eastern Beaufort beluga whales. For further analysis,
samples from both stocks were pooled to increase samples size and statistical power. No sex
differences were found for both 15N (P=0.96 and P=0.64 for muscle and skin, respectively) and
13C (P=0.54 for muscle and P=0.77 for skin). Total body length, as an indicator for age, was not
correlated to 15N (P=0.86 and P=0.27 for muscle and epidermis, respectively) or 13C (P=0.18
for muscle and P=0.34 for epidermis).
Muscle versus Epidermis
A comparison of stable isotope ratios between tissues showed that nitrogen-15 was
significantly enriched in bowhead whale skin compared to muscle (P=0.0003) and carbon-13 was
9
significantly depleted in skin compared to muscle (P=0.0001). When tissues were lipid extracted
nitrogen-15 in skin was still enriched over muscle (P=0.0004), but the difference in 13C was no
longer significant (P=0.07). For beluga whales, 15N did not differ between muscle and epidermis
in both non-lipid-extracted (P=0.22) and extracted scenarios (P=0.71). As described for bowhead
whales, carbon-13 was significantly depleted in epidermis (P=<0.0001), but this difference
disappeared with lipid-extraction (P=0.08). Nitrogen-15 was significantly enriched in skin of
subsistence-harvested and stranded gray whales compared to muscle (P=0.0004 and P=0.04 for
subsistence and stranded whales, respectively), while 13C was not different between tissues
(P=0.50 for subsistence and P=0.85 for stranded whales).
Lipid Extraction
Lipid-extraction had no significant effect on 15N in epidermis (P=0.09, 0.76, and 0.81
for bowhead, beluga, and gray whales, respectively) of subsistence-harvested Arctic cetaceans
and stranded gray whales (P=0.92). Similarly, lipid extraction did not significantly alter 15N in
muscle (P=0.51 for bowhead and P=0.75 for beluga whales). Additional muscle tissue of
stranded and subsistence-harvested gray whales was not available for lipid-extraction procedures.
In contrast, carbon-13 was significantly enriched in lipid-extracted skin of all subsistenceharvested whales (P=<0.0001 for bowheads and belugas and P=0.045 for gray whales). However,
13C was not different in epidermis of extracted and non-lipid-extracted stranded gray whales
(P=0.12). In muscle, carbon-13 was depleted only in bowhead whales (P=0.0005), while lipidextraction did not affect 13C in belugas (P=0.90).
DISCUSSION
Stable nitrogen isotope ratios in muscle and epidermis are indicative of trophic position
in Arctic cetaceans. Beluga whales occupy a higher trophic level (based on 15N) than both
baleen whale species, in accordance with their piscivorous diet (Seaman et al., 1982). The diet of
bowhead whales is well described and their main prey consists of copepods and euphausids
(Lowry et al., 2004), while gray whales are unique in their reliance on ampeliscid amphipods
(Bogoslovskaya et al., 1981). Carbon isotope signatures of prey consumed by bowheads and gray
whales are isotopically distinct (Dehn et al., 2006a) and these differences are reflected in both
muscle and epidermis of bowheads and gray whales. However, this study showed that epidermis
is significantly enriched over muscle in nitrogen-15 in the species analyzed, with the exception of
beluga whales. The amino acids arginine and glutamine appear to preferentially incorporate 15N,
10
while some essential amino acids like phenylalanine and lysine are isotopically lighter (Patterson
et al., 1993). Arginine is generally described as a major component in keratinized structures
(Wilkerson, 1934; Block and Bolling, 1939) therefore explaining differences between epidermis
and muscle tissue. The lack of nitrogen-15 enrichment in beluga skin compared to muscle may be
associated with their highly variable diet (Seaman et al., 1982). For example, high protein diets
can induce catabolism and therefore influence amino acid abundance and whole body metabolism
(Zhao et al., 2006). Alternatively, beluga whales molt annually (St. Aubin et al., 1990) rather than
the continuous sloughing of epidermis typically described for mysticetes (Baum et al., 2001;
Reeb et al., 2005) that could lead to differences in protein metabolism and balance in beluga skin
compared to the baleen whales in this study.
Carbon-13 in epidermis was depleted over muscle in bowhead and beluga whales.
Selective fractionation of carbon isotopes leads to depletion of carbon-13 in body fat compared to
other tissues (DeNiro and Epstein, 1977). Lipids are an integral part of whale epidermis, and a
variety of different functional uses for these fat globules have been discussed, such as
permeability barrier, keratinization, thermogenesis, glycerol (anti-freeze) generation, and energy
utilization (Stormberg, 1985; Menon et al., 1986; Pfeiffer and Jones, 1993). The effect of lipid
removal on stable isotope ratios remains unclear. While Sotiropoulos et al. (2004) reported only
minimal shifts in 13C and 15N values of lipid-extracted fish muscle compared to non-extracted
tissue, the effect of lipid extraction on total body homogenate of juvenile fish was significant.
Sotiropoulos et al. (2004) suggested that the removal of structural fats also removes amino acids
attached to the lipids and thus induces nitrogen-15 enrichment. In this study, lipid-extraction did
not alter nitrogen and carbon isotope ratios in whale muscle and epidermis. As expected, lipidextracted skin was significantly enriched in carbon-13 over non-lipid-extracted epidermis. In
contrast, Abend and Smith (1997) showed that muscle and skin of long-finned pilot whales
(Globicephala melas) differed significantly in carbon and nitrogen isotope ratios, though sample
sizes were small and lipids were not removed prior to analysis. Todd et al. (1997) investigated
13C in muscle and epidermis of humpback whales (Megaptera novaeangliae) and found no
significant differences (lipids were extracted). Similarly, lipid extraction had little effect on 13C
in muscle in this study, implying lower lipid content associated with muscle as compared to
epidermis. In addition, carbon isotope ratios in lipid extracted epidermis are similar to 13C in
muscle (P=0.08 for belugas and P=0.07 for bowheads), therefore lipids should be extracted from
skin biopsies to be comparable to carbon isotope ratios published for muscle tissue. In general,
caution is warranted when assessing and interpreting feeding ecology of whales based on nitrogen
11
isotope ratios in biopsy/skin samples compared to values generated for muscle, where species
differences exist and different, tissue-specific enrichment factors may have to be applied.
A significant negative correlation exists for 15N and length/age in bowhead whales, with
fetuses and juveniles being more enriched in nitrogen-15 compared to adults (Figure 4). This
relationship was not found for beluga tissues or gray whale muscle, likely due to sample size
limitations. Several studies have reported enrichment of nitrogen-15 in marine mammal fetuses,
calves and pups (Hobson et al., 1997; Das et al., 2003; Dehn et al., 2006a). This is consistent with
increased nitrogen demands in growing juveniles as suggested by Roth and Hobson (2000), but
could also be associated with the relative low protein content of mysticete milk (Oftedal, 1993)
and consequently higher reliance on body-own nitrogen reserves. Similarly, the sample of fetal
epidermis from the bowhead whale calf was enriched in nitrogen-15 over both muscle and
smooth, neonatal skin, suggesting mobilization and incorporation of maternal nitrogen and
associated trophic enrichment. In contrast, neonatal epidermis sampled from the ingutuk was
enriched in nitrogen-15 over both muscle and fetal epidermis. Ingutuks are assumed to be freshly
weaned yearlings that have been described as benthic foragers due to their short baleen (Hazard
and Lowry, 1984). These young whales likely have high metabolic demands and tissue turnover
as well as limited access to protein via milk, leading to nitrogen-15 enrichment. Trophic shift and
enrichment factors are therefore dependent on nitrogen contents of food/prey consumed and
nitrogen demands of the animal. Carbon isotope ratios were not correlated with length in any
cetacean species analyzed, though other studies reported nursing juveniles showing depleted
carbon-13 signatures, likely due to transfer of carbon from maternal lipid-stores (Jenkins et al.,
2001; Dehn et al., 2007).
Epidermal carbon and nitrogen isotope signatures were similar in both present-day
bowhead whales and the ancient sample. Although only a single specimen, this suggests that
feeding ecology of bowhead whales has remained stable for a millennium. However, biological
variables, i.e., length and sex, are unknown for the ancient sample. Assuming that the ancient
sample was taken from an adult whale (>10m), carbon and nitrogen isotope signatures are similar
in both present-day bowhead whales and the ancient sample (Figure 4).
12
Figure 4: Length [cm] as a proxy for age versus 15N in bowhead whales. The red line and bar
illustrate the mean nitrogen isotope ratio of the ancient epidermis sample ± 1SD (n=4). LOESS
non-parametric smoothing was employed to visualize trends for epidermis (dotted blue line) and
muscle (dotted green line).
Muscle and epidermis were analyzed in this study from gray whales along the California
coast in 1999 and 2000. Muscle of stranded gray whales was significantly enriched in nitrogen-15
compared to subsistence harvested gray whales from Chukotka. Gray whales stranded during this
mortality event were described as emaciated, in over all poor condition and had low blubber lipid
content (Gulland et al., 2005). Body protein catabolism will lead to 15N enrichment (Hobson et
al., 1993), thus muscle isotope ratios of stranded whales in this study support the nutritional stress
hypothesis. Interestingly, nitrogen enrichment was not displayed in epidermis of stranded whales.
Zhang et al. (1998) described that, in contrast to muscle, a protein mass balance is maintained in
rabbit epidermis on either food-restricted, high fat or high protein diets by continuous proteolysis
and re-utilization of amino acids. It is therefore plausible that structural proteins in skin are
maintained even during starvation and body protein catabolism. This study also noted that
13
C
was significantly depleted in epidermis of subsistence-harvested gray whales compared to
stranded animals, but this effect was not noted if lipids were extracted from epidermis. This
indicates that epidermal fat droplets were not present in stranded whales, providing another line
of evidence that these whales were nutritionally stressed. Alternatively, Menon et al. (1986)
discussed that epidermal lipid globules are associated with thermogenesis and anti-freeze
13
generation as mentioned above. Therefore, gray whales migrating to warmer waters may not need
these droplets and, although unlikely, any association with the stranding events may be
coincidental.
CONCLUSIONS
In conclusion, epidermis samples are an adequate replacement for muscle tissue in
comparative feeding ecology studies using stable isotopes. However, different species and tissue
specific enrichment factors as well as turnover rates apply and care should be taken with the
interpretation. While stable nitrogen isotopes in muscle can provide insights into food limitation
and protein catabolism, isotope ratios in epidermis are unaffected. However, carbon isotope ratios
in whale skin are indicative of lipid stores and absence of a lipid signature can point to starvation.
In general, lipids should be removed from epidermis samples to avoid skewing carbon isotope
ratios, unless food limitation is suspected. Nitrogen isotope ratios are not affected by lipid
removal using chloroform and methanol.
PUBLICATIONS
Dehn, L.-A., Follmann, E.H., Zelensky, G., Rosa, C., George, C. In prep. Comparison of
stable carbon and nitrogen isotope ratios in muscle and epidermis of subsistence-harvested Arctic
cetaceans with reference to ancient whale skin and stranded gray whales. Marine Mammal
Science.
OUTREACH
Recognizing the importance of communicating the results of scientific investigations to
the public, we planned to present our findings in a community-based forum in the village of
Barrow, Alaska. The subsistence hunters who were responsible for providing us with bowhead
and beluga whale tissues have been very supportive of the various research investigations that
have been conducted on the North Slope for many years. We, therefore, felt obligated to share
with them the important results of this study. It was our intent to travel to Barrow in October to
present our findings, but a series of conflicting activities on the part of Barrow residents
prevented this from occurring. Therefore, the presentation was postponed until November 17. It
was coordinated through the Barrow Arctic Science Consortium (BASC) which takes
responsibility for hosting visiting scientists and scheduling community events such as ours
through the National Science Foundation Schoolyard Project.
14
On November 16 we flew to Barrow and met with Dr. Cheryl Rosa of the North Slope
Borough Department of Wildlife Management. We discussed our project with her and discussed
the possibility of follow-up studies on marine mammals using Barrow and other North Slope
villages as a source of samples. We were provided housing and meals by the BASC and they
advertised (Figure 5) and set up the meeting room for our presentation on Saturday afternoon.
Because of some conflicting activities in town our presentation was attended by only about 25
individuals that included scientists, health professionals, and other local residents. The
PowerPoint presentation “Muktuk Tells a Tale about Whales” went very well and a considerable
number of questions from the audience were fielded. All-in-all we were quite satisfied with the
gathering despite the attendance being lower than we anticipated.
In addition to the community presentation above, we plan to attend and present the results
of this investigation at the Alaska Marine Science Symposium in Anchorage in January 2008 (an
abstract has been submitted). As well, a manuscript is in preparation for submission to a peerreviewed journal (Marine Mammal Science) in 2008. In January 2007, preliminary results of this
study have been presented at the Alaska Marine Science Symposium in Anchorage (Dehn, L.-A.,
Follmann, E. H., Rosa, C., George, C. 2007. Comparison of stable isotope ratios in muscle and
epidermis of bowhead whales with reference to ancient whale skin).
ACKNOWLEDGEMENTS
We thank the subsistence hunters and whaling captains in Alaskan and Russian
communities who generously allowed the sampling of their whales. We greatly appreciate the
assistance of the North Slope Borough, Department of Wildlife Management with sampling,
logistics and access to the ancient whale sample. In particular, we thank Cheryl Rosa, Craig
George, Taqulik Hepa, Gennady Zelensky, Gay Sheffield, Victoria Woshner, Norma
Haubenstock, Tim Howe and many others for assistance in the field and with sample analysis.
We gratefully acknowledge the support of the Marine Mammal Center, in particular Frances
Gulland for providing samples of stranded gray whales. We thank Teri Rowles (Marine Mammal
Health and Stranding Response Program) and Aleria Jensen (Alaska Stranding Network) for their
help and support with permitting and sample transfers. Funding for this project was provided by
the North Pacific Research Board.
15
Figure 5: Flyer for community presentation in Barrow, Alaska as part of the Barrow Arctic Science
Consortium (BASC) Schoolyard Project
16
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