Ontogenetic diet changes in bottlenose dolphins - Wiley Online Library

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

Ontogenetic diet changes in bottlenose dolphins (Tursiops truncatus) 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: [email protected]

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 dietary changes was examined using bottlenose dolphin tooth and skin samples. Teeth were subsampled to compare tissue produced early in life (outer tooth) to that produced later in life (inner tooth). Outer tooth had significantly higher 15 N values than the corresponding inner sample from the same tooth (n = 60, P = 0.0041), indicating that there was a temporal shift to a lower 15 N diet. There were no significant 13 C differences. Higher 15 N values in young have previously been attributed to the period of suckling. Analysis of skin tissue from stranded animals of different developmental stages similarly indicated that the 15 N values were significantly higher in young animals. Further comparisons indicated that the primary influence for this difference was animals with lengths less than or equal to the largest neonatal dolphin. This difference likely reflects an ontogenetic dietary shift from a sole reliance on milk to a combination of milk and prey species during the 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 mammals have 15 N values higher than those of adults. This 15 N difference has typically been 128

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attributed to the fact that while young are nursing, the milk they are consuming is isotopically representative of the lactating female’s tissues. As there is a wellestablished increase in 15 N of approximately 3% per trophic level (Michener and Schell 1994), it has been hypothesized that the tissues of nursing young have higher 15 N values because the young are essentially “consuming” the tissues of the adult female. 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). In addition, Hobson and Sease (1998) found Steller sea lions (Eumetopias jubatus) with higher 15 N values and lower 13 C values in the first annulus of tooth dentine relative to dentine deposited after the first year. The lower 13 C values were attributed to the dietary influence of lipid-rich milk, as lipids tend to have lower 13 C values than other tissues (Tieszen and Boutton 1988). Similarly, Aurioles et al. (2006) found that the 13 C values for nursing northern elephant seals (Mirounga angustirostris) gradually declined over time whereas 15 N values increased. These statistically insignificant differences 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 et al. (2001) found inconsistent 13 C and 15 N differences between lactating females and nursing offspring. Based on these results, Jenkins et al. concluded that future studies should not assume the presence of a reliable and consistent 13 C and 15 N difference between nursing offspring and adults. Rather, this difference should be assessed independently for different species, environments, and diets. The present study reports 13 C and 15 N values in tooth and skin tissue from bottlenose dolphins (Tursiops truncatus) to examine this ontogenetic diet change simultaneously within individuals (i.e., within a tooth) and within the population as a whole (i.e., by comparing skin samples from animals at different developmental stages). As the present study used tissue from stranded dolphins, it is important to note that some studies have found evidence that nutritional stress and disease can result in increased 15 N values (Hobson and Clark 1992, Hobson et al. 1993, White and 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 provides integrated signals of diet over multiple temporal scales. Tissues that are rapidly replaced, 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). Integrated dietary information can therefore be obtained by evaluation of the isotope values from 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) that are deposited at different times (Schell et al. 1989, Best and Schell 1996, Hobson et 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 for tracking the diet of individuals over their lifetimes. Dolphin tooth tissue is mostly composed of dentin layers that fill in the open root as aging progresses (Rommel 1990). Under normal conditions, growth layers in teeth do not resorb and therefore provide a permanent dietary record for the individual (Klevezel 1996, Hohn 2003). It is therefore possible to subsample within dentin layers to evaluate changes in diet with 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 diet changes. Walker and Macko (1999) used stable carbon and nitrogen analysis to analyze teeth of eight species of marine mammals, including bottlenose dolphins. Their results indicated that the stable isotope composition of teeth could be reliably used to indicate dietary differences. In particular, bottlenose dolphin populations living offshore and near-shore along the eastern coast of the United States were separated based on dietary differences as reflected in stable isotope values. Although isotope differences were found within subsamples taken from a sperm whale tooth, no significant differences were found between subsamples from bottlenose dolphin teeth. In another study, Walker et al. (1999) revealed that it is possible to isotopically compare marine mammal teeth that have been archived for approximately 100 yr with teeth from modern individuals to examine long-term temporal diet changes. Similarly, Abend and Smith (1995) used 13 C and 15 N analysis of teeth from long-finned pilot whales to study geographic and temporal dietary variation. The present study therefore sought to establish the use of isotope analysis of bottlenose dolphin tooth subsections as a tool for the study of temporal diet change in these animals. In addition, by comparing the skin 13 C and 15 N values of dolphins from various developmental stages, population-wide temporal diet changes were examined.

METHODS Teeth (n = 60) were selected from bottlenose dolphins >250 cm in length to ensure that 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. Teeth were collected from dead dolphins that stranded (n = 49) between 1997 and 2000 along the coasts of Virginia and North Carolina, and from dolphins temporarily held and released alive (n = 11) during 1999 and 2000. Whole teeth were dried for 3–4 days in a 60◦ C oven. They were then cleaned of outer gum material with a carbide bur attached to a drill. Cleaned whole teeth were broken in half with a stainless steel rod and platform so that the drill could be used to remove internal pulp. The interior portion of each tooth was drilled out and the resulting powder was reserved to represent tooth formed during the later life of the animal. The remaining outer part of the tooth was then crushed into a fine powder to represent tooth formed during early life (i.e., the result was two samples per tooth; Fig. 1). Although the precision 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 1 yr of age (Fig. 1). Therefore, the outer tooth samples approximately represent tooth material deposited during the first year of life and inner samples represent tooth deposited from 1 yr of age to death. All powdered tooth samples were acidified with 30% hydrochloric acid (HCl) to remove biogenic carbonates, which could alter the organic 13 C measurements. Samples of approximately 3–4 mg of residual acidified tooth were used for 13 C and 15 N analysis. Skin samples from recently stranded animals (n = 238) were collected between 1993 and 2003 along the coasts of Virginia and North Carolina. Samples were stored in freezers until they were prepared for isotope analysis. In this procedure, samples were first rinsed with deionized water to remove any external contamination, then placed in vials and dried in a 60◦ C oven for 2–3 d. To remove lipids, dried samples


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Figure 1. Photograph of a dolphin tooth cross-section. Throughout life, growth layers of dentine 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 present study.

were placed in distilled dichloromethane (DCM), reflux extracted for 1 h, and filtered on glass-fiber filters to separate the skin from the DCM. Filtered skin samples were then rinsed several times with clean DCM, dried in a 60◦ C oven for an additional 24 h, and weighed for analysis (typically 0.4–0.6 mg for a 13 C and 15 N analysis). All samples (tooth and skin) were analyzed for their stable isotope compositions (13 C, 15 N) using an elemental analyzer (EA) connected to a Micromass Optima

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Isotope Ratio Mass Spectrometer (IRMS; GV Instruments, Manchester, UK). The goal of preparation is the conversion of the organic samples into gases of suitable purity that can then be analyzed by the mass spectrometer. The reproducibility of isotope measurements varies depending on the specific technique, instruments, and type of sample used, but it is typically around ±0.2% for the carbon and nitrogen techniques utilized in the present study (MacAvoy et al. 2000). Isotope ratios of samples (R sam ) are compared to the isotope ratio of a standard for that element (R std ). R is the abundance ratio of the heavy isotope (N) to that of the light isotope of the element (E) and differences in the ratios are expressed in “delta” () notation and are reported in per mil (‰): N E (‰) = (Rsam /Rstd − 1) × 1,000. The standard for carbon is Pee Dee Belemnite (PDB), whereas the standard for nitrogen is atmospheric molecular nitrogen (N 2 ). ANOVA with a Tukey post hoc analysis (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 15 N values than the inner portions of the same teeth (P = 0.0041; Table 1); fifty-six of the sixty teeth analyzed had higher 15 N values in the outer tooth portion relative to the inner portion. Of the other four, two had no difference between outer and inner 15 N and two had inner values 0.3 and 0.8 ‰ higher than outer values. Unlike the results of Hobson and Sease (1998), there was no significant difference in the 13 C values of the inner and outer tooth portions (P = 0.9126). For thirty-two of the sixty teeth sampled, corresponding skin samples were also available. Skin 13 C was significantly more negative than the 13 C value of tooth material from the same animal (P < 0.0001). On average, tooth 13 C was 3.5‰ higher than skin values (Table 2). The differences between skin 15 N and corresponding tooth were not as consistent. The outer portion of tooth had significantly higher 15 N than skin (P = 0.0246), but there was no significant difference between the 15 N of skin and inner tooth material. Using predicted ranges of body length at different ages (Read et al. 1993), it was possible to compare skin 13 C and 15 N values from stranded North Carolina and Virginia dolphins of various body lengths to examine development-related patterns of isotope values within the population. Although body length is not a specific measure of age in bottlenose dolphins, it can provide a relative indication of age. Table 1.

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

Tooth sample

n

13 C

15 N

Outer Inner

60 60

−12.8 ± 0.1 −12.8 ± 0.1

17.6 ± 0.2 16.8 ± 0.2

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Table 2. Mean (±SE) 13 C and 15 N 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 represented in each of the age classes are in parentheses after the categories. Age class “Neonates” (3 yr (>211 cm)

n

13 C

15 N

43 32 45 117

−15.6 ± 0.1 −16.5 ± 0.2 −16.9 ± 0.2 −16.6 ± 0.1

19.3 ± 0.2 17.7 ± 0.3 17.3 ± 0.2 17.0 ± 0.1

Although the average age at weaning is 18–20 mo, lactation serves as the primary source of nutrition for approximately 1 yr after birth (Wells and Scott 1999). Read et al. (1993) predicted an average length of 183-cm for 1-yr-old bottlenose dolphins based on modeled length-at-age curves. Therefore, the present study compared isotope values of animals less than or equal to 183 cm in length with those longer than 183 cm (i.e., likely to be consuming prey as the primary source of nutrition). Bottlenose dolphins more dependent on milk (i.e., those less than or equal to 183 cm in length) had significantly higher 13 C and 15 N values than those more likely to be consuming prey (P < 0.0001 for both). Following this finding, finer-scale length-class comparisons were conducted. The largest 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 similar maximum neonatal length of 128 cm among animals from the Texas coast. In both studies, neonate was defined using morphological characteristics indicating that an animal was within 24 h of birth. Using the length of 132 cm and less as actual or possible neonates, a “neonates” category was defined that included specimens within this length range. Additional categories were then defined using predicted length-atage 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, isotope values of males and females were also compared, but no significant differences were found between sexes. There were significant differences in both 13 C and 15 N values between dolphins in the “neonate” and “neonates” to 1 yr of age categories (Table 2). Overall, “neonates” (less than 132 cm body length) had higher 13 C and 15 N values than all other age categories in pairwise comparisons (P < 0.0001 for all except P = 0.0009 for “neonates” and “neonates” to 1 yr 13 C), whereas there were no other significant isotope differences between age classes (Fig. 2, 3).

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

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25 “neonates” to 1 year

“neonates”

23

1 to 3 years

> 3 years

21

17

15

δ N (‰)

19

15 13 11 9 80

130

180

230

280

length (cm)

Figure 2. 15 N values and linear regression of skin from dolphins categorized by body length 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 is consuming a diet with a higher 15 N 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 15 N value than skin formed by the animal during adult life (lower 15 N diet). This skin/tooth difference corresponds directly to the consistent difference in 15 N observed between the inner and outer -10 “neonates” to 1 year

“neonates” -12

1 to 3 years

> 3 years

13

δ C (‰)

-14

-16

-18

-20

-22

-24 80

130

180

230

280

length (cm)

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


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portions of teeth. It should be noted that this observation is based on the assumption that the 15 N fractionation between diet and skin is similar to 15 N fractionation between diet and tooth bulk organic matter. Although this assumption has not been directly tested by the present study, amino acid analyses indicate that the proteins in the bulk organic matter of dolphin tooth are noncollagenous protein similar to muscle and skin protein. This tooth/skin result is consistent with the results obtained from the tooth subsamples: Tissue produced while young animals are obtaining most of their nutrition from the lactating female through milk consumption have higher 15 N values, thus indicating that the young are feeding at a higher trophic level. Similar to 15 N, 13 C values also increase with trophic level of the diet, and although this increase is not as large as for 15 N, an increase of approximately 1‰ is typically observed per trophic level increase (Michener and Schell 1994). Therefore, the higher 13 C values in dolphins 183 cm provides additional support for the assertion that young feeding on 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 age classes suggests that the isotope composition of tissues produced during likely periods of exclusive milk consumption are higher than those produced during consumption of adult diet or a combination of milk and adult diet. Although the differences between the other age classes are not statistically significant, the 15 N values are negatively correlated with body length (Table 2). In early life stages, this trend would indicate a decreasing contribution of milk to the diet, and continuation of the trend beyond weaning would reflect additional ontogenetic diet shifts or metabolic changes. Several studies have found no evidence of diet to tissue fractionation changes with age for 13 C or 15 N (Minagawa and Wada 1984, Sutoh et al. 1987, Roth and Hobson 2000, Hobson and Clark 1992), so ontogenetic diet changes may be a more likely explanation for the negative correlation between 15 N values and body length observed in the present study. The results of the present study suggest that comparison of tooth sections produced at different times can be used to document significant temporal dietary variation. With improved analytical techniques, more detailed subsampling of dolphin teeth for isotope analysis may become possible. With this, a more thorough understanding of 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 Beaufort laboratory, especially Annie Gorgone, Robin Baird, and Patti Haase, in the acquisition of samples used in this study. Financial assistance was provided by University of Virginia Governor’s and Dean’s Fellowships and the Virginia Coast Reserve Long Term Ecological Research Project (National Science Foundation grants BSR-8702333-06 and DEB-9411974), and the NOAA/NMFS/Southeast Fisheries Science Center. The experiments performed in the current study comply with the current laws of the United States.

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