Environ Biol Fish (2012) 95:37–52 DOI 10.1007/s10641-011-9919-7
Carbon and nitrogen discrimination factors for elasmobranch soft tissues based on a long-term controlled feeding study
Sora Lee Kim & Dave R. Casper & Felipe Galván-Magaña & Ruth Ochoa-Díaz & Sandra Berenice Hernández-Aguilar & Paul L. Koch
Received: 18 November 2010 /Accepted: 17 August 2011 /Published online: 3 September 2011 # Springer Science+Business Media B.V. 2011
Abstract The foraging ecology of elasmobranchs because taxon-specific isotope discrimination factors (sharks, skates, and rays) is difficult to study because from a controlled experiment are unavailable. Trophic species have spatially and temporally diverse diets. discrimination factors for plasma, red blood cells, and Many diet and habitat preference studies for mammals, muscle were determined from an experiment with birds, and teleosts use stable isotope analysis, but leopard sharks (Triakis semifasciata)fedaconstant interpretations are limited for elasmobranch studies diet of squid over 1000 days. The δ13Cvaluesfor shark tissues at equilibrium with the squid diet did not vary significantly among individuals, but plasma and Electronic supplementary material The online version of this red blood cell δ15N values differed significantly among article (doi:10.1007/s10641-011-9919-7) contains supplementary individuals and sampling day. Individual variation of material, which is available to authorized users. δ15 : muscle N averages was observed and likely related S. L. Kim P. L. Koch to growth. Overall, carbon and nitrogen discrimination Department of Earth and Planetary Sciences, factors corresponded to previous studies featuring high- University of California, Santa Cruz, 1156 High St, protein diets and carnivorous taxa. The muscle-to-diet Santa Cruz, CA 95064, USA discrimination factors from the controlled feeding study were applied to blue sharks (Prionace glauca)and D. R. Casper smooth hammerhead sharks (Sphyrna zygaena)caught Long Marine Laboratory, University of California, Santa Cruz, offshore from Baja California, Mexico. This case study 100 Shaffer Rd., demonstrates the potential of stable isotope analysis to Santa Cruz, CA 95060, USA illuminate differences in foraging patterns between elasmobranch species. F. Galván-Magaña : R. Ochoa-Díaz : S. B. Hernández-Aguilar Centro Interdisciplinario de Ciencias Marinas-IPN, Keywords Diet . Stable isotope analysis . Blue shark . Av. Instituto Politécnico Nacional S/N, Po Box 572, La Paz, Smooth hammerhead shark . Baja California Mexico Baja California Sur, Mexico C.P. 23096
Present Address: S. L. Kim (*) Introduction Department of Geology and Geophysics, University of Wyoming, Elasmobranchs occupy a wide range of habitats as 1000 University Ave. #3006, Laramie, WY 82071, USA meso- and apex predators in marine ecosystems, but e-mail: [email protected] relatively little is known about individual species’ 38 Environ Biol Fish (2012) 95:37–52 abundance, diet, or movement (Dulvy et al. 2000, directly measured (Hussey et al. 2010). In this study, a 2008). Resolving aspects of foraging ecology within controlled feeding experiment was conducted and the populations or among species will help illuminate carbon and nitrogen isotope values of leopard shark trophic structure and food web dynamics, which is (Triakis semifasciata) tissues (plasma, red blood cell especially important in ecosystems affected by the [RBC], and muscle) and their diet were monitored for recent sharp decline in shark populations (Baum et al. 1000+ days. This extensive data set allowed us to 2003; Ward and Myers 2005; Myers et al. 2007; determine trophic discrimination factors and critically Ritchie and Johnson 2009). Elasmobranch dietary evaluate isotopic variation within tissues and among studies could benefit from stable isotope techniques, individual sharks. In addition, a case study is presented which are routinely used to study the ecology of comparing the results of stomach/gut content analysis mammals, birds, and teleosts (Kelly 2000; Vander with those of stable isotope analysis to assess the Zanden and Rasmussen 2001; Crawford et al. 2008; foraging ecology of blue (Prionace glauca)and Inger and Bearhop 2008; Newsome et al. 2010). smooth hammerhead (Sphyrna zygaena)sharkscaught However, the interpretation of elasmobranch ecology offshore from Baja California, Mexico. from stable isotope analysis is hindered by the lack of specific trophic discrimination factors (i.e., the isotopic Overview of stable isotope analysis difference between consumer tissues and diet; Fisk et al. 2002;Ostrometal.1993; Estrada et al. 2003;Domiet Carbon and nitrogen isotope values track nutrient- al. 2005), a biological parameter that should be flow through a food web and vary spatially at the base calculated from long-term controlled feeding experi- of the food web in response to differences in ments or well-characterized field studies (Martínez del productivity, upwelling, and other oceanographic Rio et al. 2009). factors (Saupe et al. 1989; Clementz and Koch Stable isotope analysis is a biogeochemical tool 2001; Post 2002; Barnes et al. 2009). Isotopes of that can supplement dietary and movement data from both of these elements are sorted (or fractionated) traditional methods (i.e., observation, stomach/gut during metabolic processes, such that consumer content analysis, and tagging), especially for wide- tissues become enriched in the rare heavier isotopes ranging and elusive taxa, such as elasmobranchs. (13Cand15N) relative to prey (Wada et al. 1991; Martínez With robust estimates of discrimination factors and del Rio et al. 2009). IsotopeÀÁÀÁ ratios will be discussed using h » tissue turnover rates, ecological studies can use stable δ values, where d X ¼ Rsample=Rstandard 1 1000 isotope ratios of carbon (13C/12C) and nitrogen and X is the element, h is the high mass number, (15N/14N) to help resolve a consumer’s diet composition R is the high mass-to-low mass isotope ratio, and and trophic level, as well as ontogenetic shifts in these Rstandard is V-PDB for carbon and AIR for nitrogen. variables. Furthermore, comparisons among species and Units are part per thousand (per mil, ‰). The populations through time can elucidate patterns of difference between prey and a consumer’stissueis trophic interaction and baseline changes (i.e., Estrada referred to as the trophic discrimination factor, with the h et al. 2003;Laymanetal.2007; Newsome et al. 2007). following notation and relationship: Δ Xtissue prey ¼ h h 1 Currently, there are no published discrimination d Xtissue d Xprey (Martínez del Rio et al. 2009). factors from a controlled feeding study on elasmo- A review of data for aquatic and terrestrial branchs. Although teleosts may share similar habitats invertebrates and vertebrates from laboratory and field and lifestyles to elasmobranchs, physiological differ- studies reported average Δ13CandΔ15Nvaluesof0.4‰ ences in osmoregulatory and buoyancy control may lead and 3.4‰, respectively (Post 2002). Physiological and to differences in trophic discrimination factors between dietary differences can affect discrimination factors, these taxa. In addition, Chondrichthyes, the clade which depend on the biochemical processes used to containing elasmobranchs, is the sister taxon to the Teleostomi, the clade containing teleosts and tetrapods 1 such as mammals and birds, which are the main subjects For ease of communication, we will refer to these as Δ Δ of controlled feeding studies. A previous study has discrimination factors and omit the subscripts on values. values are most often used because they are trivial to calculate published elasmobranch-specific discrimination factors, and accurate as long as the difference in δ values between diet but the isotopic values of prey were modeled rather than and tissue is small (< 10‰, Cerling and Harris 1999). Environ Biol Fish (2012) 95:37–52 39 metabolize and incorporate nutrients (Gannes et al. 1997; that suggests carbon and nitrogen discrimination Koch 2007;MartínezdelRioetal.2009). Nitrogen factors increase depending on dietary protein content fractionation occurs during deamination and transamina- (Hilderbrand et al. 1996; Oelbermann and Scheu tion of amino acids (Macko et al. 1986; Fogel et al. 2002; Pearson et al. 2003; Robbins et al. 2005; Florin 1997;Gannesetal.1998) and its magnitude is likely et al. 2011). Because most elasmobranchs are carni- regulated by the concentration of nitrogen lost in vores, carbon and nitrogen discrimination factors are excreted waste products (Koch 2007). The processes likely greater than previously reported average values. controlling dietary carbon isotope fractionation are less Elasmobranchs retain urea ((NH2)2CO) to maintain well understood, but in protein-rich tissues, are likely osmotic balance. The isotopic effects of this physiol- related to isotopic routing of dietary amino acids during ogy are not well understood. Urea isotope values may metabolism (Gannes et al. 1997, 1998; Martínez del Rio differ from those of body tissues, so high concen- et al. 2009). trations of urea in tissues may skew ecological Results from previous feeding studies illustrate that interpretations (Kim and Koch, in press). In addition, discrimination factors differ between diets in relation the urea cycle in elasmobranchs is different from that to protein quality and quantity (Focken 2001; Pearson in other taxa (reviewed by Ballantyne 1997 and et al. 2003; Robbins et al. 2005; Martínez del Rio et Hazon et al. 2007) and it is unknown if the additional al. 2009; Florin et al. 2011). Vertebrate carnivores are enzymatically-catalyzed step affects urea isotope rarely kept in captivity for feeding experiments, but values in elasmobranchs relative to other taxa. The published studies featuring carnivorous mammals and only urea-related δ13C values we found were for teleosts fed a natural carnivorous diet (in captivity or human and bovine urine, which were 3–5‰ and 0– well-known wild individuals) found Δ15N values for 3.5‰ greater than diet, respectively (Ivlev et al. 1996; blood (Kurle 2002), collagen (Fox-Dobbs et al 2007) Knobbe et al. 2006). Kim and Koch (in press) and muscle (Sweeting et al. 2007a) to be greater than reported average 1.3‰ depletion in δ15N values from the average of 3.4‰ reported by Post (2002)(Table1). elasmobranch muscle containing urea. Additionally, Additionally, there is a growing body of literature there may be biochemical adaptations specific to featuring omnivores (e.g., bears, spiders, birds, etc.) elasmobranch physiology that alter isotopic fraction-
15 Table 1 Discrimination factors calculated for controlled feeding mammals, wolf, and lynx) have greater Δ N tissue-diet values than experiments featuring carnivorous teleosts and mammals. Note subjects fed pellets or supplement additives, except sharks that that most subjects fed pure carnivorous diets (seals, carnivorous were not extracted of urea (Hussey et al. 2010)
13 15 Taxa Tissue Number of Δ Ctissue-diet ‰ Δ Ntissue-diet ‰ Diet type Reference consumers
Northern Fur Seal Plasmaa 2 0.6 5.2 C Kurle 2002 RBC 1.4 4.1 Harp, Ringed, and Harbor Blood 14 1.7 1.7 C+S Hobson et al. 1996 Seals Harp Seals Muscle 2 2.4 1.3 C+S Hobson et al. 1996 Rainbow Trout Muscle 2.5 3.3 P Pinnegar and Polunin 1999 Atlantic Salmon Muscle 25 2.1 2.3 P Trueman et al. 2005 Sharks Muscle 4 0.9 2.3 D Hussey et al. 2010 Carnivorous mammals Collagen 15 5–6 D Ambrose and DeNiro 1986 Wolf Collagen 5 0.7–1.3 3.4–3.9 D Bocherens and Drucker 2003 Lynx 4 0.4–1.2 4.0–4.2 Wolf Collagen 42 1.3 4.6 D Fox-Dobbs et al. 2007 Average of invertebrates 107 & 56 0.4 3.4 Post (2002) & vertebrates a Plasma sampled without clotting factors Abbreviations for diet type: carnivorous (C), supplemental additives (S), pellet meals containing wheat (P), and diet reconstructions (D) 40 Environ Biol Fish (2012) 95:37–52 ation patterns and ultimately require calculation of From February 2006 to November 2008, blood and elasmobranch-specific discrimination factors. muscle were sampled approximately every 21 days. Sharks were individually placed in a smaller tank Case study (1.2 m diameter, 0.2 m water depth) where an anesthetic, tricaine methanesulfonate (MS-222) was Blue and smooth hammerhead sharks are seasonally dissolved. Once the shark lost mobility, length and abundant and frequently caught in artisanal fisheries weight measurements and a 0.7 cc sample of blood targeting sharks in the northeast Pacific offshore from was taken (25-gauge needle into the caudal vein) and Baja California, Mexico (Cartamil et al. 2011). Blue sub-divided into a no-additive Vacutainer® and 2 sharks (Prionace glauca) are a cosmopolitan pelagic hematocrit tubes (capillary glass tubes filled with species with a wide distribution throughout the heparin). To prevent coagulation and collect well- Pacific. The species is thought to segregate by size separated plasma samples, the whole blood sample and sex with extensive migration throughout the north was immediately centrifuged and the plasma was Pacific and Atlantic (Nakano and Stevens 2008). transferred into a glass test tube. Muscle biopsies Recent stomach/gut content analysis revealed cepha- were taken above the lateral line between the first and lopods and red crab to be the primary prey of blue second dorsal fins with a histo-cut needle (14 gauge× sharks near our study location (Hernández-Aguilar 16 cm, Avid Medical Supplies). All samples were 2008). Stomach/gut content analysis suggests that stored in glass tubes and immediately frozen until hammerhead sharks (Sphyrna zygaena) also have a isotopic analysis. After sampling, hematocrit tubes cephalopod-dominated diet (Ochoa-Díaz 2009), but were centrifuged and packed cell volume (length of they mostly inhabit the continental shelf along the RBC in tube/length of whole blood in tube) was California and Baja California coast in the northeast measured to monitor the sharks’ health. Pacific (Casper et al. 2005). In this case study, estimates of dietary contributions from isotopic Case study sampling analysis are qualitatively and quantitatively compared to stomach/gut content analysis results. The blue and hammerhead sharks were caught off the coast of Baja California Sur, Mexico, in July 2004 and September 2005. Artisanal fishermen used long-line Methods techniques to capture individuals. All blue sharks (n=10; total length [TL] range=136–260 cm) and one Controlled feeding experiment sampling hammerhead (TL=142.5 cm) were caught offshore from Isla Magdalena (24º 46′N, 112º 15′W) and the remaining The leopard shark (Triakis semifasciata) is a coastal hammerheads (n=10; TL range=47–143 cm) were species found from Washington to Mazatlán, Mexico caught offshore from Las Barrancas (26º 0′N, 112º (including the Gulf of California; Carlisle and Starr 12′W). The average of three TL measurements were 2009; Farrer 2009) that is ideal for experimental taken and ~10 g of muscle tissue were sampled from study; individuals are relatively small (<2 m), locally below the first dorsal of each shark. Samples were kept abundant, and relatively easy to maintain in captivity. on ice for up to 5 days until a freezer was available to Leopard sharks were caught in otter trawls between store the samples. After transport to UCSC, samples August–December 2005 in San Francisco Bay, were frozen at −20°C until prepared for isotopic analysis. acclimated to captivity, and transported (1–2sharksata time) to Long Marine Lab at the University of California, Stable isotope analysis Santa Cruz (UCSC). Two sharks were maintained per polyethylene tank (2.3 m diameter, 1.2 m water depth) All shark tissues were freeze-dried and plasma and RBC with a continuous flow of filtered seawater from the samples were not chemically treated before isotopic Monterey Bay and fed squid (Loligo opalescens)from analysis. Small shark muscle samples (from the feeding Monterey Bay. Data for three control individuals study) were packed in Accelerated Solvent Extractor maintained on a constant diet from a larger diet- (ASE®) cells between glass fiber filters (GF/F), lipid switching experiment are presented here. extracted with 2 rinses of petroleum ether and urea Environ Biol Fish (2012) 95:37–52 41 extracted with 3 rinses of de-ionized water (9 ml Dobbs et al. 2007), the δ13Candδ15N values of solution at 50°C, 1500 Psi, and 60% volume for 5 min; cephalopod muscle and beaks have reported differ- Dobush et al. 1985; Bodin et al. 2009; Kim and Koch ences of 0.6–1.2‰ and 3.3–5‰,respectively(Hobson in press). Larger muscle samples were lipid extracted and Cherel 2006; Ruiz-Cooley et al. 2006). Whole with 2 rounds of petroleum ether (~10 ml) and 10 min squid and squid beaks were analyzed to determine a of sonication and then rinsed with 3 rounds of de- correction factor that converts isotopic data from squid ionized water (~10 ml) and 15 min of sonication to beaks to whole squid. remove urea. All muscle samples were dried in an oven Isotope values from plasma, RBC, and muscle set to 50°C or freeze-dried overnight. The isotopic collected after day 600 were used to calculate trophic effects of this lipid and urea extraction method are discrimination factors. Leopard shark muscle tissue detailed in Kim and Koch (in press). was assumed to be in equilibrium with the squid diet All squid fed to the leopard sharks were from a single based on the following: 1) calculation of 1σ from all shipment. Throughout the experiment, 1–3 squid were individuals over 3 consecutive sampling days <0.4‰ randomly analyzed from each box fed to the sharks to and 2) the estimate by MacNeil et al. (2006) of 95% insure the homogeneity of diet. Each complete individual muscle turnover in 422 days. Additionally, Logan and squid (n=43) was individually homogenized in a Lutcavage (2010) estimated 95% muscle turnover in blender, decalcified with 0.5 N hydrochloric acid 555 and 340 days for carbon and nitrogen, respec- overnight at 4°C, and lipid extracted with 3 rinses of tively, though this estimate is based on assumed petroleum ether. Beaks from another 24 squid were discrimination factors and a 46–55 day experiment. dissected, cleaned, and lipid extracted with 5 rinses of Finally, preliminary diet-switching results from leop- petroleum ether (10 mL). All squid samples were dried ard sharks indicate that plasma, RBC, and muscle will in an oven set to 50°C, and then further homogenized undergo a minimum of two half-lives in 600 days using a mortar and pestle before isotopic analysis. (Kim et al., unpubl. data). All the samples were weighed to 500 μg(±50)into The sampling of multiple individuals fed a constant 3×5 mm tin boats (Costech Supplies) and analyzed at the diet allowed the determination of individual mean Stable Isotope Laboratory at UCSC with an elemental values, estimation of uncertainty, and evaluation of analyzer coupled to a Thermo-Scientific Delta+XP significant differences for prey and shark tissues. For the continuous flow, isotope-ratio-monitoring mass spec- squid, means and standard deviations (SD) were based trometer (CF-IRMS). A gelatin of known C and N on 1–2 squid collected from each box (weight=2.3 kg) isotope composition was replicated within and between fed to the sharks starting on day 1 in captivity until day runs for corrections. Comparisons of this standard 1020, the last sampling day in this data set. Differences yielded standard deviations of <0.1‰ and <0.2‰ for betweenbeakandwholesquidδ13Candδ15Nvalues δ13Candδ15N values, respectively (n=178). All C:N were assessed with a Wilcoxon rank sum test, a ratios were also corrected to this gelatin standard and conservative comparison between two related sample calculated as the atomic ratio. populations that are not normally distributed. For shark plasma, RBC, and muscle, mean isotope values were Data analysis for controlled feeding study compared among tissues and isotope systems with a Kruskal-Wallis rank sum test, which is similar to the Most elasmobranchs ingest whole prey or large Wilcoxon test for data containing more than 2 groups. chunks of prey, so discrimination factors were based A one-way Analysis of Co-Variance (ANCOVA) on the analysis of whole squid samples. However, assessed if there was a non-random interaction between whole prey isotope values are often unavailable, individual and sampling day on plasma, RBC, and especially for large prey items, and isotopic values muscle isotope values. If there was no correlation for protein in hard tissues that are preserved in between individual and sampling day, then the signif- stomach/gut contents (i.e., chitin, keratin, or bone) icance for each model effect (individual and sampling are often substituted. Although previous studies day) was independently determined with an Analysis comparing muscle and collagen have reported similar of Variance (ANOVA) test. All statistical analysis of isotopic patterns (i.e., Sholto-Douglas et al. 1991; these data was performed in JMP® (version 7.0, SAS Hobson and Clark 1992a, b; Nardoto et al. 2006; Fox- Institute Inc. 2007). 42 Environ Biol Fish (2012) 95:37–52
A suite of Δ13CandΔ15N values for plasma, RBC, were included in the blue shark and hammerhead shark and muscle relative to whole squid were determined for mixing models, respectively, based on stomach/gut captive leopard sharks fed squid. These values were content results (Hernández-Aguilar 2008; Ochoa-Díaz based on the population mean values (all individuals 2009). Offshore cephalopods (Ommastrephes bartrami, from days 600–1020 and all squid); SD was propagated Berryteuthis anonychus, Octopoteuthis deletron, for each tissue. Histioteuthis dojleini, Taonius pavo; Gould et al. 1997) were included for blue sharks, because sub- Data analysis for case study adults are thought to migrate to the central Pacific and often caught as bycatch in cephalopod targeted Isotope values between blue and hammerhead sharks fisheries (Nakano and Stevens 2008). All cephalo- were compared using the Wilcoxon rank sum test and pod beak data was converted to represent whole ontogenetic relationships between δ15N values and TL cephalopods using the isotopic differences from the were evaluated with JMP. The diets of blue and squid in the controlled feeding study. The average hammerhead sharks were estimated from a Bayesian isotope values and SD for relevant prey categories stable isotope mixing model (MixSIR v. 1.0.4; Moore were entered into MixSIR (Appendix A). and Semmens 2008). Advantages of this model are 1) the incorporation of uncertainty in discrimination factors and prey sources and 2) the model results are posterior Results distributions of each source in a potential diet mix. The model parameter details are listed in Appendix A. Each Controlled feeding study potential dietary mix within the mixing model sums to 100% (Moore and Semmens 2008), but median values The plasma, RBC, and muscle of leopard sharks had of prey contribution distributions may not sum to significantly different mean δ13C and δ15N values precisely 100%. The isotope mixing model was (Kruskal-Wallis test HC=81.9, d.f.C=2, pC<0.0001; 13 15 performed with two sets of Δ CandΔ Nvalues HN=73.2, d.f.N=2, pN<0.0001; Fig. 1 and Table 2). (and SD): the values from this feeding study and those The C:N ratio (SD) of plasma, RBC, and muscle reported by Post (Δ13C=0.4‰ [SD=1.4] and Δ15N= equaled 1.3 (1.0), 2.2 (0.1), 2.9 (0.1), respectively. 3.4‰ [SD=1.0]; 2002). The values from Hussey et al. The ANCOVA and ANOVA tests for plasma, RBC, (2010) were not used because the isotopic value of diet and muscle isotope values demonstrated variability was not directly analyzed and urea was not removed attributed to individual and sampling day. The plasma from the muscle tissue before isotopic analysis. and RBC δ15N values co-varied for individual x
Potential prey species were identified in stomach/gut sampling day (pplasma=0.0007, pRBC=0.0148), but content studies of blue and hammerhead sharks caught in δ13C values were not significantly different for these similar locations to those sampled for isotopic analysis model effects (Table 3). Although the individual model (Hernández-Aguilar 2008;Ochoa-Díaz2009). Potential effect for RBC δ13C results had a low p-value (0.0547), prey isotope values were found in the literature and individual means only varied 0.1‰ throughout the unpublished data from samples caught in the same sampling period (Fig. 1b,Table2), which is within region as the sharks. The prey isotope data were analytical variability. There was no co-variance of categorized based on taxon and region. Jumbo cepha- individual and sampling day for δ13Candδ15Nvalues lopod (Dosidicus gigas; Ruiz-Cooley et al. 2006)and of muscle (Table 3). The ANOVA results for individual coastal cephalopod (caught on the continental shelf— and sampling day were not significant for muscle δ13C Ancistrocheirus lesueurii, Onychoteuthis banksii, values, but the individual effect for muscle δ15Nvalues Pholidoteuthis boschmai, Sthenoteuthis oualaniensis, had a low p-value (0.0515) and individual means Thysanoteuthis rhombus; Ochoa-Díaz 2009) categories differed (Tables 2 and 3). The growth rates for all three were used in mixing models for both blue and individuals were similar (0.0167, 0.0217 and hammerhead sharks. Red crabs (Pleuroncodes planipes; 0.0255 cm/day). However, total length measurements Peckham and Newsome, unpublished data) and sardines from sampling days indicated relatively steady growth (Sardinops sagax, Etrumeus teres; Gendron et al. 2001) for two individuals, but one individual (represented in Environ Biol Fish (2012) 95:37–52 43
A. 17 R Fig. 1 Carbon and nitrogen isotope values for (a) plasma, (b) RBC, and (c) muscle for 3 individual leopard sharks (each represented by a different symbol) from January 2006–October 16
‰ 2008. Only data collected after 600 days (shown beyond the
N, N, vertical dashed line) were analyzed to calculate trophic
15 15 discrimination factors δ
0 all graphs by “•” symbol) had a reduced growth rate and low hematocrit values (Supplemental Material A).
–15 Accurate calculation of isotopic discrimination factors
‰ requires a diet with a uniform and well known isotopic
C, C, composition and consumer tissues that are in equilibrium –16 13 13 15
δ with this diet. The mean δ Candδ N values for whole squid (n=43) and squid beaks (n=24) were significantly –17 400 600 800 1000 different (Table 2;Z =5.27, p =<0.0001; Z =−6.77, Days C C N pN<0.0001). The equations to convert beak data to 17 13 B. represent whole cephalopods are as follows: δ Cbeak- 13 15 15 0.6=δ Cwhole and δ Nbeak+4.7=δ Nwhole. Isotopic 16 means for each tissue were compared to isotopic means of whole squid and errors were propagated to determine N, ‰ N,
15 15 trophic discrimination factors and SD (Table 2). δ
0 Case study
Blue shark and hammerhead shark muscle δ13C and –15 15 ‰ δ N values were significantly different (Wilcoxon rank
C, C, sum test; ZC=−2.15, pC=0.029; ZN=0.0043, pN= 13 –16 13 15 δ 0.0039). The mean δ Candδ Nvalues(SD)for blue shark muscle (n=10) were −17.3‰ (0.6) and –17 ‰ 400 600 800 1000 17.8 (0.6), respectively and for hammerhead shark Days muscle (n=11) were −16.7‰ (0.4) and 18.9‰ (0.8), respectively. There were linear relationships be- C. 19 tween blue shark and hammerhead shark δ15N 15 18 values and TL (cm; δ Nblue=15.5+0.0127× TLblue 2 15 [r =0.75, d.f.=8, p=0.0012] and δ Nhammerhead= 17 2
N, ‰ N, 17.5+0.020× TLhammerhead [r =0.48, d.f.=9, p=
15 16 0.018]; Fig. 2). The smaller hammerhead sharks δ had a lower mean δ15Nvalue(by1.2‰)andawider 0 variation in δ15N values than the larger hammerhead sharks (>100 cm TL, Fig. 3b). There was a significant 15 –16 difference between δ N values of small and large ‰ individuals, but not for δ13C values (Wilcoxin Rank C, C,
13 –17 Sum test; ZC=0.0451, pC=0.034, ZN=0.91, pN=0.81). δ Dietary preferences, as dictated by stable isotope
–18 analysis, are qualitatively illustrated by adding 400 600 800 1000 muscle-to-whole prey discrimination factors from the Days feeding study to all potential prey data (Fig. 3) and quantitatively evaluated using MixSIR (Table 4). As expected, MixSIR determined different relative con- 44 Environ Biol Fish (2012) 95:37–52
Table 2 Plasma, RBC, and muscle isotopic averages across all compared to whole squid to estimate discrimination factorsa and individuals and sampling days from three leopard sharks fed a standard deviations. The statistical significance of individual and constant squid diet over 1000 days. Isotopic averages were sampling day effects are shown in Table 3
Tissue δ13C (SD), ‰ Δ13C (SD), ‰ δ15N (SD), ‰ Δ15N (SD), ‰
Individual Population Individual Population
Squid (whole) −18.5 (0.5) 13.3 (0.7) Squid (beak) −17.9 (0.3) 8.6 (0.4) Plasma −15.8 (0.4) −15.7 (0.4) 2.8 (0.6) 15.6 (0.4) 15.5 (0.3) 2.2 (0.7) −15.8 (0.4) 15.5 (0.3) −15.6 (0.3) 15.5 (0.2) RBC −16.1 (0.3) −16.2 (0.2) 2.3 (0.5) 15.5 (0.2) 15.7 (0.2) 2.4 (0.5) −16.1 (0.1) 15.7 (0.1) −16.2 (0.1) 15.8 (0.2) Muscle −16.8 (0.2) −16.8 (0.2) 1.7 (0.5) 17.0 (0.5) 17.0 (0.4) 3.7 (0.4) −16.8 (0.1) 16.9 (0.3) −16.7 (0.2) 17.3 (0.4) a h While Δ Xtissue-prey values are widely used in ecological studies (Martínez del Rio et al. 2009), a mathematicallyÀÁ more exact approach is to calculate isotopicÀÁ enrichment (PasseyÀÁ et al. 2005) with the following relationship: "tissue prey ¼ atissue prey 1 h h 1000, where atissue prey ¼ d Xtissue þ 1000 = d Xprey þ 1000 . These values are referred to as ε* values to indicate they are associated with fractionations not related to chemical equilibrium (Cerling and Harris 1999). In this study, all ε* values equal h Δ Xtissue-prey values except for plasma carbon, which equals 2.9‰. tributions of prey based on the discrimination factors red crab than stomach/gut content analysis (Table 4). used (Table 4). The δ15N values of blue shark muscle All hammerhead sharks had δ15N values between were positioned between those of coastal and offshore those of coastal cephalopods and sardines, similar to cephalopods. Red crabs and jumbo cephalopods were jumbo cephalopod (Fig. 3b). The mixing model with 13C-enriched relative to blue shark muscle (Fig. 3a). discrimination factors from this study and Post (2002) Mixing model results using different discrimination estimated greater median sardine contributions than factors determined smaller median contributions of stomach/gut content results (Table 4).
Table 3 Plasma, RBC, and muscle mean values and Tissue Response variable Model effects df F p-value statistical results of 13 ANCOVA and ANOVA tests Plasma δ C Individual × day 2, 32 0.775 0.469 for individual sharks and Individual 2, 34 1.022 0.371 sampling day (referred to as Day 1, 35 0.0068 0.935 “day”) during days 600– δ15 1020, when shark tissues N Individual × day 2, 32 9.208 0.0007* were equilibrated to the RBC δ13C Individual × day 2, 31 2.170 0.13 squid diet. Individual means Individual 2, 33 3.187 0.0547 are listed from the slowest Day 1, 35 0.190 0.666 to fastest growth rates and 15 significant effects are δ N Individual × day 2, 32 4.866 0.0148* indicated with an asterisk Muscle δ13C Individual × day 2, 31 0.701 0.504 α ( =0.05) Individual 2, 33 0.704 0.502 Day 1, 35 0.628 0.434 δ15N Individual × day 2, 31 0.425 0.658 Individual 2, 33 3.259 0.0515 Day 1, 35 0.638 0.430 Environ Biol Fish (2012) 95:37–52 45
Fig. 2 The ontogeny of Blue sharks blue sharks and hammer- Hammerhead sharks head sharks as described by > 100 cm muscle δ15N values. A 20 < 70 cm linear regression of each species total length by δ15N values yielded significant 19 results (see Results for 2
equations, r , and p values) N(‰)
15 18 δ
17
0 100 200 300 Total Length (cm)
Fig. 3 The qualitative die- A. tary analysis of isotope val- 24 ues from (a) blue sharks and Jumbo cephalopods Coastal cephalopods (b) smooth hammerhead Offshore cephalopods shark muscle collected in 22 Red crab Baja California Sur, Blue sharks Mexico. Potential prey items were selected based 20 on stomach/gut content analysis from sharks caught 18
in this region (Ochoa-Díaz ‰ N,
2009). Discrimination 15 δ factors from this study 16 added to the prey isotopic values. Quantitative stomach/gut content and 14 dietary mixing model results are presented in Table 4 –18 –17 –16 –15 –14 –13 δ13C,‰
B. 24 Jumbo cephalopods Coastal cephalopods Sardines 22 Hammerhead sharks Large (> 100 cm) Small (< 70 cm) 20 N, ‰ N,
15 18 δ
16
14
–18 –17 –16 –15 –14 –13 δ13C,‰ 46 Environ Biol Fish (2012) 95:37–52
Table 4 Dietary results for blue and hammerhead Prey Estimated prey contributions, Median % (SD) sharks from stomach/gut content and isotope mixing Stomach/gut content MixSIR, discrimination MixSIR, discrimination model analyses. The dis- factors from this study factors from Post (2002) crimination factors used in the two MixSIR analyses Blue sharks were from this study Jumbo cephalopods 1.2 1.5 (1.7) 28.4 (9.9) (Δ13C=1.7‰ and Δ15 Coastal cephalopods 53.3 52.8 (4.0) 21.1 (6.5) N=3.7‰) and Post (2002; Δ13C=0.4‰ and Δ15 Offshore cephalopods n/a 41.4 (4.4) 26.1 (5.8) N=3.4‰) Red crab 39.5 2.9 (2.9) 23.8 (11.9) Large hammerhead sharks Jumbo cephalopods 85 14.3 (4.8) 53.3 (8.6) Coastal cephalopods 11 44.3 (5.7) 35.0 (6.0) Sardines 1 41.4 (6.6) 11.2 (6.4)
Discussion rate slowed from days 400–800 (• in all figures and the first individual mean in Table 2; Supplemental Material Controlled feeding study A). The two healthy sharks in this experiment had an inverse relationship between growth rate and mean a. Variation among and within individuals muscle δ15N values, as expected from the results of Controlled feeding studies with vertebrates that are Trueman et al. (2005). However, the significance of this difficult to keep in captivity often sample deceased correlation must be tested with a larger sample size that specimens and perform dietary reconstructions to encompasses adult elasmobranchs, which have lower determine prey isotope values (Ambrose and DeNiro growth rates after maturity (Araya and Cubillos 2006). 1986; Hobson et al. 1996; Bocherens and Drucker Elasmobranches maintain high concentrations of urea 2003; Fox-Dobbs et al. 2007; Hussey et al. 2010)or and TMAO, which contain carbon and nitrogen that sacrifice specimens incrementally throughout their likely have different isotopic values from tissue. Previous experiment (Pinnegar and Polunin 1999; Trueman et urea concentration measurements for leopard shark al. 2005). However, these experimental designs may plasma revealed large variability (400–650 mM), likely lack accuracy because isotope values of prey are caused by variations between individuals, seasonal water estimated and individuals may vary. In this study, chemistry, or time since last feeding (Wood et al. 2005; sharks were kept in captivity and tissues were Kim and Koch in press). For all individuals and tissues, repeatedly sampled for isotopic analysis for 1000+ ANCOVA and ANOVA tests found no significant days. The individual mean δ13C values differed by differences among δ13C values, which indicates little 0.1‰ for all tissues, which is within instrument variation among individuals and sampling day and variability, but individual mean δ15N values differed suggests the urea and TMAO carbon input has little by 0.1‰, 0.3‰, and 0.4‰ for plasma, RBC, and effect. Although there was a low p-value for individual muscle, respectively. Despite the small number of effects on RBC δ13C values, this result is likely an subjects, the results of this study illustrate potential artifact of the one individual with health complications. individual variability when subjects are fed a constant The statistical results for δ15N values vary between diet and kept in a controlled environment. Two tissues; plasma and RBC results have significant differ- possible explanations for the variation in individual ences based on ANCOVA tests, but muscle results have mean δ15N values could be differences in either no significant differences based on ANCOVA or growth rate or urea retention. ANOVA tests. The different responses between these Variation among individual growth rates alters the rate tissues (plasma and RBC vs. muscle) could be related to or efficiency of nutrient assimilation and therefore, urea content. Currently, there are no methods to remove individual δ15Nvalues(Truemanetal.2005). All urea from blood, although there is a negative correlation captive sharks were juveniles with similar growth rates between urea concentration and plasma δ15Nvalues and squid consumption, but one individual’sgrowth (Kim and Koch, in press). Previous isotopic results from Environ Biol Fish (2012) 95:37–52 47 shark muscle indicate an increase in δ15Nvaluesby valves increase the surface area of elasmobranch guts 0.3–2.2‰ and decrease in δ15N variation with urea (despite their short length) to maximize nutrient removal techniques (Kim and Koch, in press). Once absorption. The specialized physiology of elasmo- urea removal techniques for blood are developed, the branchs maximizes nitrogen use efficiency and likely significant differences associated with individual and decreases the degree of expression of isotopic fraction- sampling day effects may be mitigated for plasma and ation associated with metabolic processes to values RBC samples. Future studies using shark plasma and more like those for omnivores than carnivores. δ15 RBC N values should consider the additional error c. Differences in isotope values between whole squid associated with urea during data interpretation. and squid beaks b. Discrimination factors The 13C-enrichment for samples analyzed in this For shark plasma, RBC, and muscle, the Δ13C study and by Hobson and Cherel (2006) and the 15 values are higher than average values but Δ15Nvalues consistent N-depletion in beaks relative to muscle or are similar to average values reported in Post (2002). whole squid across various cephalopod species suggest The quantity and quality of protein an organism that these isotopic differences may be an effect of consumes relative to its metabolic needs can generate different amino acid compositions between muscle and variation in Δ13C values (Martínez del Rio et al. beak protein (Hobson and Cherel 2006; Ruiz-Cooley et 2009). In studies of omnivorous taxa with variable al. 2006). Cephalopod beaks are composed of chitin and dietary protein, there is a positive linear correlation contain proteins with high concentrations of glycine and between protein content and Δ13C values (Hilderbrand histidine (Broomell et al. 2007). Previous studies 13 et al. 1996; Pearson et al. 2003). For all leopard shark analyzing δ C values of individual amino acids in tissues, the Δ13C values were higher than averages terrestrial invertebrates, terrestrial mammals, and marine 13 reported in Post (2002) and Sweeting et al. (2007b; birds found glycine to be C-enriched relative to bulk 13 Table 1). There are published studies of carnivorous δ C values (Hare et al. 1991; Fogel and Tuross 2003; taxa with low Δ13C values, which are likely an artifact O’Brien et al. 2005; Lorrain et al. 2009; studies did not 13 of high lipid concentration (Kurle 2002) and inaccurate report δ C values for histidine). The relationships 15 prey value estimates (Bocherens and Drucker 2003; between bulk tissue, glycine, and histidine δ Nvalues Hussey et al. 2010). Leopard sharks in this study were vary to a greater extent and are more difficult to assess fed squid, prey with high quality and quantity of because glycine and histidine are considered source protein, and the Δ13C value of plasma, RBC, and amino acids. These amino acids are not affected by 15 muscle was greater than previously reported averages trophic fractionation and instead, reflect δ Nvaluesof in Post (2002). primary producers in the food web (McClelland and 15 The Δ15N values we calculated for elasmobranchs Montoya 2002). Thus, the relative N-depletion of may resemble values for herbivores and omnivores glycine and/or histidine relative to a bulk tissue sample because elasmobranchs retain and recycle nitrogenous depends on the consumer’s trophic level. For example, 15 waste for osmotic balance. Transamination and deam- δ N values of glycine do not differ from bulk samples ination lead to 14N-enrichment in nitrogenous waste in zooplankton (histidine not reported; McClelland and 15 (urea, uric acid) relative to body tissues (Macko et al. Montoya 2002), but glycine and histidine are N- 1986; Fogel et al. 1997;Sponheimeretal.2003). depleted (~8–13‰) relative to bulk samples in tuna Under simple mass balance models, high proportional (Popp et al. 2007). fluxes of N lost as such waste (rather than as shed tissues or enzymes in fecal matter) lead to 15N- Case study enrichment of body tissues relative to diet (Gannes a. Ontogenetic shifts et al. 1997; Sponheimer et al. 2003;Koch2007). However, protein-limited species can decrease urinary The correlation between blue and hammerhead nitrogen loss, thereby reducing the nitrogen isotope shark δ15N values and size (Fig. 2) represent a difference between consumer tissues and diet (Gannes gradual increase in trophic level (e.g., consumption et al 1997;Sponheimeretal.2003). Elasmobranchs of larger cephalopods, Ruiz-Cooley et al. 2006)or also have a modified digestive physiology—intestinal foraging in habitats with a higher 15N baseline as 48 Environ Biol Fish (2012) 95:37–52 animals mature. For example, smaller blue sharks 2008;Cartamiletal.2011). Because blue sharks are caught near Baja California, Mexico could be recent offshore for approximately half the year, offshore immigrants from the 15N-depleted central Pacific near cephalopods likely compose a similar proportion of Hawaii and larger individuals could be sub-adults that their diet; however, remains are not present in stomach/ have been resident in the 15N-enriched coastal Pacific gut contents from individuals caught nearshore. The (Saino and Hattori 1987; Nakano and Stevens 2008). mixing model results using the discrimination factors The sampling of more individual blue sharks over a from this study support the extended time blue sharks greater size range will clarify potential migrational are not caught in Baja artisanal fisheries. segregation within the population. Hammerhead c. Isotope mixing model for hammerhead sharks sharks do not have large-scale movement patterns on- and offshore, but may migrate latitudinally to Stomach/gut content analysis of juvenile hammerhead continuously occupy temperate waters. The large sharks caught in the eastern Pacific offshore from Baja variation among young hammerhead sharks (TL< California Sur, Mexico, revealed a diet dominated by 70 cm and umbilical scars) likely reflects opportunistic jumbo cephalopod (85%; Ochoa-Díaz 2009). In contrast, foraging. Isotopic analysis of more hammerhead isotope mixing models with discrimination factors from sharks >100 cm will help characterize potential this study and Post (2002) suggest smaller contributions ontogenetic dietary shifts in this species. of jumbo cephalopods and larger contributions of sardines (Table 4). These differences are likely because b. Isotopic mixing model for blue sharks cephalopods have beaks made of chitin and are more The variation in blue shark isotope values likely resistant to digestion than sardine, as evidenced in reflects foraging in coastal and offshore areas that studies of penguins and grey seals (Wilson et al. 1985; have different baseline isotopic values. Stomach/gut Grellier and Hammond 2006). According to the content analysis results suggest that blue sharks have a discrimination factors from this study, coastal cephalopods diet dominated by red crabs and coastal cephalopods and sardine dominate hammerhead shark diet, whereas the (Hernández-Aguilar 2008). The isotope mixing models Post (2002) discrimination factors estimate a diet with discrimination factors calculated in this study and dominated by coastal and jumbo cephalopods. The Post (2002) determined a much smaller contribution analysis of more hammerhead sharks >100 cm would from red crab. The discrepancy between stomach/gut allow an isotope mixing model analysis of ontogenetic content and isotope mixing model results may occur dietary switches. Stable isotope analysis may improve because stomach/gut content results only provide a our understanding of smooth hammerhead shark diet snapshot of diet and cannot account for seasonal and habitat preferences, two biological characteristics variation. Although red crab may dominate blue shark that are not well known (Casper et al. 2005). diets in coastal Baja California, Mexico, in February and March (Hernández-Aguilar 2008), their annually integrated contribution to muscle growth may be Conclusion smaller. The isotope mixing model using discrimination factors from this study estimated a similar contribution of Previous studies suggest using trophic discrimination coastal cephalopods as the stomach/gut content analysis factors determined from studies featuring similar taxa, (Table 4). The isotope values for red crab, coastal diet, and tissue (Gannes et al. 1997, 1998; Martínez cephalopods, and jumbo cephalopods, the major prey del Rio et al. 2009). Although the Δ15N values from according to stomach/gut content analysis, did not fully Post (2002) and this study are similar, the difference characterize blue shark diet. Isotopic analysis of between average Δ13C values (0.4‰; Post 2002) and Atlantic blue sharks by Estrada et al. (2003)and elasmobranch muscle (1.8‰) may be enough to change MacNeiletal.(2005) demonstrated little seasonal diet some ecological interpretations. Furthermore, it is variation, but fisheries catch data suggests migratory important to consider the prey tissue analyzed and to movement of blue sharks in the north Pacific between apply appropriate discrimination factors. Because the coastal and offshore areas (Nakano and Stevens 2008). isotopic values of whole cephalopods and their beaks Blue sharks are frequently caught in Baja’s artisanal vary, the interpretation of diet and habitat preference is fisheries from February–June (Hernández-Aguilar affected by a conversion from beak to whole body or Environ Biol Fish (2012) 95:37–52 49 muscle, especially in ecosystems with subtle isotopic sharks throughout the duration of this study; the Marine variation. Isotopic analysis of blue and hammerhead Science Institute (Redwood City, California) that catches leopard sharks for educational demonstrations and donated sharks revealed ontogenetic dietary variation. A com- specimens for this project; S. Carleton, K. Fox-Dobbs, and two parison of results from stomach/gut content and isotopic anonymous reviewers for their constructive input on this analysis illustrated the potential overestimation of prey manuscript. The Institutional Animal Care and Use Committee with hard parts resistant to digestion with stomach/gut (IACUC) at UCSC approved sampling procedures according to the National Institutes of Health Policy. All squid fed to the content analysis (i.e., chitin and shells). sharks was donated from the Monterey Bay Aquarium. The infrastructure for the study was funded by NSF-OCE 0345943 Acknowledgements We thank J. Adams, A. Bennett, M. and largely executed by N. Moore (Long Marine Lab, UCSC). Gorey, L. Krol, S. Perry, S. Rumbolt, A. Sjostrom, A. Thell, and An IGPP Mini-Grant provided the funding for analytical C. Spencer for their assistance maintaining and sampling the analysis.
Appendix A
Table 5 Potential prey data for blue and hammerhead sharks used in MixSIR
Prey group Species Tissue Lipid # δ13C δ15N Location Citation extracted? (SD), ‰ (SD), ‰
Jumbo cephalopod Dosidicus gigas Muscle Y 8 −16.2 (0.3) 14.7 (0.4) southern Gulf of Ruiz-Cooley et al. 2006 CA/ 25° 12′ N, 110° 49′ W Coastal cephalopods Onychoteuthis Beak Y 1 −17.8 13.3 West coast of Baja Ochoa-Díaz 2009 banksii CA Sur/ 23°24′- Coastal cephalopods Thysanoteuthis Beak Y 1 −18.7 12.1 25°59′N,110°13′- rhombus 112°11′W Coastal cephalopods Ancistrocheirus Beak Y 1 −16.1 15.1 lesueurii Coastal cephalopods Onychoteuthis Beak Y 1 −17.8 12.5 banksii Coastal cephalopods Ancistrocheirus Beak Y 1 −18 12.7 lesueurii Coastal cephalopods Thysanoteuthis Beak Y 1 −18.1 11.3 rhombus Coastal cephalopods Pholidoteuthis Beak Y 1 −20.4 12 boschmai Average −18.1 (1.3) 12.7 (1.2) Offshore cephalopods Ommastrephes Muscle Y 44 −18.4 (0.2) 11.7 (0.4) N Central Pac/ Gould et al. 1997 bartrami 35-46° N, Offshore cephalopods Berryteuthis Muscle Y 5 −18.5 (0.4) 11.6 (0.9) 170°E-148°W anonychus, Octopoteuthis deletron, Histioteuthis dojleini, Taonius pavo Average −18.5 (0.4) 1.7 (1.9) Red crab Pleuroncodes Muscle N 8 −17.4 (0.8) 14.8 (1.3) West coast of Baja Peckham and Newsome, planipes CA Sur/ 23°24′- unpublish-ed data 25°59′N, 110°13′- 112°11′W Sardines Sardinops sagax Whole Y 4 −18.5 (0.6) 13.3 (1.2) southern Gulf of Gendron et al. 2001 Sardines Etrumeus teres Whole Y 4 −18.5 (0.6) 13.3 (1.2) CA /Bahia de la Paz Average −18.5 (0.8) 13.3 (1.7)
Non-informative priors were used to reduce data handling. MixSIR suggests a minimum of 1000 possible posterior distributions for robust results and therefore, blue shark and small smooth hammerhead shark analyses were set to 1×106 iterations and large smooth hammerhead shark analyses to 2×106 iterations. The maximum importance ratio of the MixSIR output was less than 0.001 for all analyses, as suggested by Moore and Semmens (2008). 50 Environ Biol Fish (2012) 95:37–52
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