MARINE MAMMAL SCIENCE, 24(1): 128–137 ( January 2008)C© 2007 by the Society for Marine MammalogyDOI: 10.1111/j.1748-7692.2007.00174.x

Ontogenetic diet changes in bottlenose dolphins (Tursiopstruncatus) reflected through stable isotopes

AMANDA KNOFF

Department of Environmental Sciences,University of Virginia,

Clark Hall, 291 McCormick Road, P. O. Box 400123,Charlottesville, Virginia 22904, U.S.A.

E-mail: aknoff@e2inc.com

ALETA HOHN

National Marine Fisheries Service,Southeast Fisheries Science Center,

National Oceanic and Atmospheric Administration Laboratory,101 Pivers Island Road,

Beaufort, North Carolina 28516, U.S.A.

STEPHEN MACKO

Department of Environmental Sciences,University of Virginia, Clark Hall, 291 McCormick Road, P. O. Box 400123,

Charlottesville, Virginia 22904, U.S.A.

ABSTRACT

The ability of stable isotope analysis to provide insight into ontogenetic dietarychanges was examined using bottlenose dolphin tooth and skin samples. Teethwere subsampled to compare tissue produced early in life (outer tooth) to thatproduced later in life (inner tooth). Outer tooth had significantly higher �15Nvalues than the corresponding inner sample from the same tooth (n = 60, P =0.0041), indicating that there was a temporal shift to a lower �15N diet. Therewere no significant �13C differences. Higher �15N values in young have previouslybeen attributed to the period of suckling. Analysis of skin tissue from strandedanimals of different developmental stages similarly indicated that the �15N valueswere significantly higher in young animals. Further comparisons indicated that theprimary influence for this difference was animals with lengths less than or equal tothe largest neonatal dolphin. This difference likely reflects an ontogenetic dietaryshift from a sole reliance on milk to a combination of milk and prey species duringthe first year of life.

Key words: bottlenose dolphin, Tursiops truncatus, diet change, stable isotope,carbon, nitrogen.

Several studies have found evidence that the tissues of young marine mammalshave �15N values higher than those of adults. This �15N difference has typically been

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KNOFF ET AL.: DIET CHANGE 129

attributed to the fact that while young are nursing, the milk they are consumingis isotopically representative of the lactating female’s tissues. As there is a well-established increase in �15N of approximately 3% per trophic level (Michener andSchell 1994), it has been hypothesized that the tissues of nursing young have higher�15N values because the young are essentially “consuming” the tissues of the adultfemale. This effect has been observed in northern fur seal muscle (Hobson et al. 1997)and bone collagen (Newsome et al. 2006), muscle from ringed seals (Holst et al. 2001),blood from polar bears (Polischuk et al. 2001) and Weddell seals (Burns et al. 1998),as well as baleen from eastern Arctic bowhead whales (Hobson and Schell 1998). Inaddition, Hobson and Sease (1998) found Steller sea lions (Eumetopias jubatus) withhigher �15N values and lower �13C values in the first annulus of tooth dentine relativeto dentine deposited after the first year. The lower �13C values were attributed tothe dietary influence of lipid-rich milk, as lipids tend to have lower �13C values thanother tissues (Tieszen and Boutton 1988). Similarly, Aurioles et al. (2006) found thatthe �13C values for nursing northern elephant seals (Mirounga angustirostris) graduallydeclined over time whereas �15N values increased. These statistically insignificantdifferences were attributed to changes in the lactating females’ feeding locations.In their study of eleven species, which included moose, caribou, black-tailed deer,coyotes, grizzly bears, domestic rabbits, rats, cows, sheep, pigs, and cats, Jenkins etal. (2001) found inconsistent �13C and �15N differences between lactating femalesand nursing offspring. Based on these results, Jenkins et al. concluded that futurestudies should not assume the presence of a reliable and consistent �13C and �15Ndifference between nursing offspring and adults. Rather, this difference should beassessed independently for different species, environments, and diets.

The present study reports �13C and �15N values in tooth and skin tissue frombottlenose dolphins (Tursiops truncatus) to examine this ontogenetic diet change si-multaneously within individuals (i.e., within a tooth) and within the population asa whole (i.e., by comparing skin samples from animals at different developmentalstages). As the present study used tissue from stranded dolphins, it is important tonote that some studies have found evidence that nutritional stress and disease canresult in increased �15N values (Hobson and Clark 1992, Hobson et al. 1993, Whiteand Armelagos 1997, Katzenberg and Lovell 1999, Oelbermann and Scheu 2002,Voigt and Matt 2004).

The use of multiple tissue types in isotope studies of feeding ecology providesintegrated signals of diet over multiple temporal scales. Tissues that are rapidlyreplaced, such as skin, reveal the diet of an animal in the relatively recent past(e.g., months), whereas tissues that do not have high turnover rates, such as teeth,reflect diet over longer spans of time (e.g., several years to a lifetime). Integrateddietary information can therefore be obtained by evaluation of the isotope valuesfrom several different tissue types or by sampling different areas of a tissue (e.g.,through a cross section of a tooth or along a feather, whisker, or baleen plate) thatare deposited at different times (Schell et al. 1989, Best and Schell 1996, Hobsonet al. 1996, Dieudonne 1998, Hobson and Schell 1998, Walker and Macko 1999,Hirons et al. 2001, Knoff et al. 2002). Teeth are a particularly informative tissue fortracking the diet of individuals over their lifetimes. Dolphin tooth tissue is mostlycomposed of dentin layers that fill in the open root as aging progresses (Rommel1990). Under normal conditions, growth layers in teeth do not resorb and thereforeprovide a permanent dietary record for the individual (Klevezel 1996, Hohn 2003).It is therefore possible to subsample within dentin layers to evaluate changes in dietwith age (Hobson and Sease 1998, Walker and Macko 1999, Walker et al. 1999).

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This procedure can also provide valuable information about long-term temporal dietchanges.

Walker and Macko (1999) used stable carbon and nitrogen analysis to analyze teethof eight species of marine mammals, including bottlenose dolphins. Their results in-dicated that the stable isotope composition of teeth could be reliably used to indicatedietary differences. In particular, bottlenose dolphin populations living offshore andnear-shore along the eastern coast of the United States were separated based on di-etary differences as reflected in stable isotope values. Although isotope differenceswere found within subsamples taken from a sperm whale tooth, no significant dif-ferences were found between subsamples from bottlenose dolphin teeth. In anotherstudy, Walker et al. (1999) revealed that it is possible to isotopically compare marinemammal teeth that have been archived for approximately 100 yr with teeth frommodern individuals to examine long-term temporal diet changes. Similarly, Abendand Smith (1995) used �13C and �15N analysis of teeth from long-finned pilot whalesto study geographic and temporal dietary variation.

The present study therefore sought to establish the use of isotope analysis ofbottlenose dolphin tooth subsections as a tool for the study of temporal diet change inthese animals. In addition, by comparing the skin �13C and �15N values of dolphinsfrom various developmental stages, population-wide temporal diet changes wereexamined.

METHODS

Teeth (n = 60) were selected from bottlenose dolphins >250 cm in length to ensurethat the animals were at or near asymptotic body length (Fernandez and Hohn 1998)and that sufficient tooth material was available to obtain two samples per tooth. Teethwere collected from dead dolphins that stranded (n = 49) between 1997 and 2000along the coasts of Virginia and North Carolina, and from dolphins temporarily heldand released alive (n = 11) during 1999 and 2000. Whole teeth were dried for 3–4days in a 60◦C oven. They were then cleaned of outer gum material with a carbidebur attached to a drill. Cleaned whole teeth were broken in half with a stainlesssteel rod and platform so that the drill could be used to remove internal pulp. Theinterior portion of each tooth was drilled out and the resulting powder was reservedto represent tooth formed during the later life of the animal. The remaining outerpart of the tooth was then crushed into a fine powder to represent tooth formedduring early life (i.e., the result was two samples per tooth; Fig. 1). Although theprecision of the subsampling method was limited by the small size of the teeth,teeth were approximately drilled along the layer of dentin that was deposited at 1yr of age (Fig. 1). Therefore, the outer tooth samples approximately represent toothmaterial deposited during the first year of life and inner samples represent toothdeposited from 1 yr of age to death. All powdered tooth samples were acidified with30% hydrochloric acid (HCl) to remove biogenic carbonates, which could alter theorganic �13C measurements. Samples of approximately 3–4 mg of residual acidifiedtooth were used for �13C and �15N analysis.

Skin samples from recently stranded animals (n = 238) were collected between1993 and 2003 along the coasts of Virginia and North Carolina. Samples were storedin freezers until they were prepared for isotope analysis. In this procedure, sampleswere first rinsed with deionized water to remove any external contamination, thenplaced in vials and dried in a 60◦C oven for 2–3 d. To remove lipids, dried samples

KNOFF ET AL.: DIET CHANGE 131

Figure 1. Photograph of a dolphin tooth cross-section. Throughout life, growth layers ofdentine fill the tooth. The black line indicates the layer of dentine deposited by 1 yr of age,which was the approximate division between “inner” and “outer” tooth samples in the presentstudy.

were placed in distilled dichloromethane (DCM), reflux extracted for 1 h, and filteredon glass-fiber filters to separate the skin from the DCM. Filtered skin samples werethen rinsed several times with clean DCM, dried in a 60◦C oven for an additional24 h, and weighed for analysis (typically 0.4–0.6 mg for a �13C and �15N analysis).

All samples (tooth and skin) were analyzed for their stable isotope compositions(�13C, �15N) using an elemental analyzer (EA) connected to a Micromass Optima

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Isotope Ratio Mass Spectrometer (IRMS; GV Instruments, Manchester, UK). Thegoal of preparation is the conversion of the organic samples into gases of suitablepurity that can then be analyzed by the mass spectrometer. The reproducibility ofisotope measurements varies depending on the specific technique, instruments, andtype of sample used, but it is typically around ±0.2% for the carbon and nitrogentechniques utilized in the present study (MacAvoy et al. 2000). Isotope ratios ofsamples (Rsam) are compared to the isotope ratio of a standard for that element (Rstd).R is the abundance ratio of the heavy isotope (N) to that of the light isotope of theelement (E) and differences in the ratios are expressed in “delta” (�) notation and arereported in per mil (‰):

�N E (‰) = (Rsam/Rstd − 1) × 1,000.

The standard for carbon is Pee Dee Belemnite (PDB), whereas the standard fornitrogen is atmospheric molecular nitrogen (N2). ANOVA with a Tukey post hocanalysis (SAS, version 8, SAS Institutue, Inc.) was used for all statistical comparisons;the maximum type-1 error rate was set at 0.05 for all tests.

RESULTS

Samples taken from the outer part of teeth had significantly higher �15N valuesthan the inner portions of the same teeth (P = 0.0041; Table 1); fifty-six of the sixtyteeth analyzed had higher �15N values in the outer tooth portion relative to the innerportion. Of the other four, two had no difference between outer and inner �15N andtwo had inner values 0.3 and 0.8 ‰ higher than outer values. Unlike the results ofHobson and Sease (1998), there was no significant difference in the �13C values ofthe inner and outer tooth portions (P = 0.9126).

For thirty-two of the sixty teeth sampled, corresponding skin samples were alsoavailable. Skin �13C was significantly more negative than the �13C value of toothmaterial from the same animal (P < 0.0001). On average, tooth �13C was 3.5‰ higherthan skin values (Table 2). The differences between skin �15N and corresponding toothwere not as consistent. The outer portion of tooth had significantly higher �15N thanskin (P = 0.0246), but there was no significant difference between the �15N of skinand inner tooth material.

Using predicted ranges of body length at different ages (Read et al. 1993), itwas possible to compare skin �13C and �15N values from stranded North Car-olina and Virginia dolphins of various body lengths to examine development-relatedpatterns of isotope values within the population. Although body length is not aspecific measure of age in bottlenose dolphins, it can provide a relative indicationof age.

Table 1. Mean (±SE) �13C and �15N values for inner and outer tooth subsamples.

Tooth sample n �13C �15N

Outer 60 −12.8 ± 0.1 17.6 ± 0.2Inner 60 −12.8 ± 0.1 16.8 ± 0.2

KNOFF ET AL.: DIET CHANGE 133

Table 2. Mean (±SE) �13C and �15N values of skin from dolphins categorized as“neonates,” “neonates” to 1 yr, 1–3 yr of age, and >3 yr of age. Body length ranges rep-resented in each of the age classes are in parentheses after the categories.

Age class n �13C �15N

“Neonates” (<132 cm) 43 −15.6 ± 0.1 19.3 ± 0.2“Neonates”–1 yr (132–183 cm) 32 −16.5 ± 0.2 17.7 ± 0.31–3 yr (184–211 cm) 45 −16.9 ± 0.2 17.3 ± 0.2>3 yr (>211 cm) 117 −16.6 ± 0.1 17.0 ± 0.1

Although the average age at weaning is 18–20 mo, lactation serves as the primarysource of nutrition for approximately 1 yr after birth (Wells and Scott 1999). Readet al. (1993) predicted an average length of 183-cm for 1-yr-old bottlenose dolphinsbased on modeled length-at-age curves. Therefore, the present study compared isotopevalues of animals less than or equal to 183 cm in length with those longer than 183cm (i.e., likely to be consuming prey as the primary source of nutrition). Bottlenosedolphins more dependent on milk (i.e., those less than or equal to 183 cm in length)had significantly higher �13C and �15N values than those more likely to be consumingprey (P < 0.0001 for both).

Following this finding, finer-scale length-class comparisons were conducted. Thelargest neonatal bottlenose dolphin found along the U.S. Atlantic coast was 132 cm(Mead and Potter 1990). In addition, Fernandez and Hohn (1998) found a similarmaximum neonatal length of 128 cm among animals from the Texas coast. In bothstudies, neonate was defined using morphological characteristics indicating that ananimal was within 24 h of birth. Using the length of 132 cm and less as actual orpossible neonates, a “neonates” category was defined that included specimens withinthis length range. Additional categories were then defined using predicted length-at-age information from growth curves (Read et al. 1993, Fernandez and Hohn 1998).Following “neonates” (132 cm) were animals from “neonates” to 1 yr of age (132–183 cm), animals from 1–2 yr of age (184–211 cm), and greater than 3 yr of age(greater than 211 cm) (Table 2). Within each of these developmental stages, isotopevalues of males and females were also compared, but no significant differences werefound between sexes.

There were significant differences in both �13C and �15N values between dolphinsin the “neonate” and “neonates” to 1 yr of age categories (Table 2). Overall, “neonates”(less than 132 cm body length) had higher �13C and �15N values than all otherage categories in pairwise comparisons (P < 0.0001 for all except P = 0.0009 for“neonates” and “neonates” to 1 yr �13C), whereas there were no other significantisotope differences between age classes (Fig. 2, 3).

DISCUSSION

As the outer part of the teeth was produced early in the life of the animal andthe inner part was produced later in life (Hohn 2003), the significant difference in�15N indicates that, overall, the diet of young animals is higher in �15N comparedto adult diet. As young bottlenose dolphins may nurse for 1–2 yr before they becomenutritionally self-sufficient (Wells and Scott 1999), the isotope compositions of the

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9

11

13

15

17

19

21

23

25

80 130 180 230 280

length (cm)

δ15N

(‰

)

“neonates” “neonates” to 1year

1 to 3years

> 3 years

Figure 2. �15N values and linear regression of skin from dolphins categorized by bodylength using a growth curve as “neonates,” “neonates” to 1 yr, 1–3 yr of age, and >3 yr of age.

outer tooth layers likely reflect this time period during which the young animal isconsuming a diet with a higher �15N value.

As outer tooth material is representative of diet during the early life of the animal,outer tooth would be expected to have a higher �15N value than skin formed by theanimal during adult life (lower �15N diet). This skin/tooth difference correspondsdirectly to the consistent difference in �15N observed between the inner and outer

-24

-22

-20

-18

-16

-14

-12

-10

80 130 180 230 280

length (cm)

δ13C

(‰

)

“neonates” “neonates” to 1year

1 to 3 years > 3 years

Figure 3. �13C values and linear regression of skin from dolphins categorized by bodylength using a growth curve as “neonates,” “neonates” to 1 yr, 1–3 yr of age, and >3 yr of age.

KNOFF ET AL.: DIET CHANGE 135

portions of teeth. It should be noted that this observation is based on the assumptionthat the �15N fractionation between diet and skin is similar to �15N fractionationbetween diet and tooth bulk organic matter. Although this assumption has not beendirectly tested by the present study, amino acid analyses indicate that the proteinsin the bulk organic matter of dolphin tooth are noncollagenous protein similar tomuscle and skin protein.

This tooth/skin result is consistent with the results obtained from the tooth sub-samples: Tissue produced while young animals are obtaining most of their nutritionfrom the lactating female through milk consumption have higher �15N values, thusindicating that the young are feeding at a higher trophic level. Similar to �15N,�13C values also increase with trophic level of the diet, and although this increase isnot as large as for �15N, an increase of approximately 1‰ is typically observed pertrophic level increase (Michener and Schell 1994). Therefore, the higher �13C valuesin dolphins 183 cm provides additional support for the assertion that young feedingon milk would occupy a higher trophic level than adults.

As the “neonate” category is representative of newborn dolphins and young calves,the significantly higher isotope values of these animals relative to all of the older ageclasses suggests that the isotope composition of tissues produced during likely periodsof exclusive milk consumption are higher than those produced during consumptionof adult diet or a combination of milk and adult diet. Although the differencesbetween the other age classes are not statistically significant, the �15N values arenegatively correlated with body length (Table 2). In early life stages, this trendwould indicate a decreasing contribution of milk to the diet, and continuation of thetrend beyond weaning would reflect additional ontogenetic diet shifts or metabolicchanges. Several studies have found no evidence of diet to tissue fractionation changeswith age for �13C or �15N (Minagawa and Wada 1984, Sutoh et al. 1987, Roth andHobson 2000, Hobson and Clark 1992), so ontogenetic diet changes may be a morelikely explanation for the negative correlation between �15N values and body lengthobserved in the present study.

The results of the present study suggest that comparison of tooth sections producedat different times can be used to document significant temporal dietary variation.With improved analytical techniques, more detailed subsampling of dolphin teethfor isotope analysis may become possible. With this, a more thorough understandingof temporal diet changes throughout life may be achieved.

ACKNOWLEDGMENTS

We are grateful for the help of the Cetacean and Sea Turtle Team at the NOAA Beaufortlaboratory, especially Annie Gorgone, Robin Baird, and Patti Haase, in the acquisition ofsamples used in this study. Financial assistance was provided by University of Virginia Gover-nor’s and Dean’s Fellowships and the Virginia Coast Reserve Long Term Ecological ResearchProject (National Science Foundation grants BSR-8702333-06 and DEB-9411974), and theNOAA/NMFS/Southeast Fisheries Science Center. The experiments performed in the currentstudy comply with the current laws of the United States.

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Received: 11 May 2006Accepted: 22 August 2007