Stable isotope profiles in whale shark (Rhincodon typus)suggest segregation and dissimilarities in the diet dependingon sex and size

Asunción Borrell & Alex Aguilar & Manel Gazo &

R. P. Kumarran & Luis Cardona

Received: 13 March 2010 /Accepted: 27 June 2011 /Published online: 7 July 2011# Springer Science+Business Media B.V. 2011

Abstract We investigated the sex- and size-relateddifferences in the diet of whale sharks from theArabian Sea (north-western Indian Ocean) usingcarbon and nitrogen stable-isotope analyses in whitemuscle. The samples were collected during thecommercial fishing season between April and May of2001 in Veraval (Gujarat, India). The overall isotopesignature was similar to that of the pelagic-neriticzooplanktivore Ilisha melastoma, which suggests thatboth species are feeding on similar prey. In whalesharks, a positive relationship was found between δ15Nand δ13C. This, together with a significant enrichmentof both heavy stable isotopes with total length indicatesthat the contribution to the diet of small fish and/orlarger zooplankton of higher trophic level increaseswith the movement from offshore areas to coastal areasas they grow. Gender differences in the isotopic ratioswere not statistically significant, but small sample sizecannot rule out completely the existence of somedegree of spatial or dietary segregation between sexes.

Keywords Habitat selection . Arabian Sea . IndianOcean .Whale shark . δ13C . δ15N

Introduction

The whale shark, Rhincodon typus, is the largest fishon Earth, with a maximum length of 20°m reported inTaiwan (Chen et al. 2002). It is found in all oceansfrom tropical to temperate latitudes (generally be-tween 30°N and 35°S), where it occupies bothoceanic and coastal waters, including the lagoons ofcoral atolls and reefs (Colman 1997). It migrates overthousands of kilometres (Eckert and Stewart 2001;Sleeman et al. 2010), and its movements are related tolocal productivity (Wilson et al. 2001; Stevens 2007;but see Sleeman et al. 2010). Its occurrence is oftenassociated with schools of pelagic fish that may feedon similar prey items (Compagno 1984).

In the 1990s, whale sharks from the Arabian Sea(north-western Indian Ocean) were caught annuallyfrom March to June in the Gujarat coasts. The numberof whale sharks caught during the 1988–1998 periodwas at least of 1823 units only in Verabal (Pravin2000). However, the catches declined in 2000,probably because of the excessive fishing pressure(Hanfee 2001). In 2001, the Government of Indiagranted protection from fishing to the whale sharkthrough the Wildlife (Protection) Act (Dutta 2001)and, in 2002, the Convention on Migratory Speciesincluded the species in Appendix II (IUCN 2003) thus

Environ Biol Fish (2011) 92:559–567DOI 10.1007/s10641-011-9879-y

A. Borrell (*) :A. Aguilar :M. Gazo : L. CardonaIrBio; Institut de Recerca de la Biodiversitat andDepartment of Animal Biology, Faculty of Biology,University of Barcelona,Avinguda Diagonal 645,08028 Barcelona, Spaine-mail: xonborrell@ub.edu

R. P. KumarranMullai Nagar, Anna Nagar,Chennai 600040, India

implementing restrictions on its international com-merce. However, poaching activities and illegal tradecontinued on small scale in the region (Riley et al.2009).

Despite the short but intense period in which thewhale shark was commercially exploited, the mainbiological characteristics, which could be investigatedfrom individuals captured by the fishery, remainunknown, particularly habitat use and diet composi-tion. The sparse available information indicates thatwhale sharks are probably born in either open oceanor deep waters and that juveniles move closer to theshore to form coastal aggregations that may be sex-biased, often in favour of males (Eckert and Stewart2001; Graham and Roberts 2007; Rowat et al. 2007;Rowat et al. 2008; Hobbs et al. 2009).

Whale sharks are generally considered suction-filter feeders that forage on dense aggregations ofzooplankton. However, other organisms such as algae,squids, tunas and other nektonic species of medium orlarge size have been identified as potential prey(Compagno 1984; Taylor 1994; Norman 1999; Wilsonand Newbound 2001; Duffy 2002; Stevens 2007).This information comes from the examination of thestomach contents of opportunistically studied strand-ed individuals (Compagno 1984; Last and Stevens1994; Colman 1997) and from observations ofindividuals from coastal aggregations while feeding(Clark and Nelson 1997; Heyman et al. 2001; Duffy2002; Nelson and Eckert 2007; Rowat et al. 2007).The sample size of stomach content studies isinvariably limited to a single individual. Moreover,although these analyses offer a high degree oftaxonomic precision, they provide only a snapshot intime of the consumers’ diet, and they are, therefore,limited in scope (Cortés 1999). In addition, stomachcontents may contain incidentally ingested items suchas plant material or mangrove seedpods (Beckley etal. 1997). On the other hand, the prey consumedwhile engaged at the coastal feeding aggregations isof unclear relevance because whale sharks are highlymobile and spend only a short time at any particularlocation (Eckert and Stewart 2001; Eckert et al. 2002;Wilson et al. 2006; Graham and Roberts 2007; Rowatet al. 2007; Sleeman et al. 2010).

The analysis of stable isotopes offers an alternativemethod for investigating diet compositions and hasproved reliable for establishing trophic levels in manyspecies including sharks (Rau et al. 1983; Fisk et al.

2002; Estrada et al. 2003; Domi et al. 2005; MacNeilet al. 2005; Estrada et al. 2006). This technique doesnot produce species-specific dietary information butprovides a measure of the assimilated prey over aperiod of time, depending on the tissue analysed, andcomplete isotope turnover is muscle shark may takemore than 2 years (Logan and Lutcavage 2010).

Animal tissues are typically more enriched in theheavy isotopes of both nitrogen (15N) and carbon(13C) than their food source; as a result, the values ofthese isotopes increase with trophic level (DeNiro andEpstein 1978; DeNiro and Epstein 1981; Minagawaand Wada 1984; Peterson and Fry 1987; Cabana andRasmussen 1996). This effect is more pronounced innitrogen (N) than in carbon (C), and, in the muscle oflarge sharks, it leads to increases of ~2.3‰ in δ15Nand of ~0.9‰ in δ13C above their prey items (Husseyet al. 2010).

Moreover, the primary producers of marine eco-systems show strong spatial gradients in isotopiccarbon ratios (Fry and Wainright 1991; Hemmingaand Mateo 1996; Rau et al. 2001) with valuestypically increasing from offshore to near-shoreecosystems and peaking in macrophyte dominatedecosystems (kelp and seagrass beds). This pattern ofvariation makes C isotopic ratios useful for investi-gating habitat use by predatory species (Burton andKoch 1999).

In this paper, we determined the stable isotopeprofiles of C and N in the muscle of 19 whale sharkscaught in Gujarat, India, to investigate potentialintrapopulation segregation in feeding habitats anddifferences in migratory patterns associated to the sizeand sex of the sharks.

Material and methods

Sampling

The samples used in this study were obtained fromthe whale shark fishing operations off the coast ofVeraval in Gujarat, India, (Fig. 1). During April andMay of 2001, we examined 19 whale sharks that hadbeen caught with hooks and lines and subsequentlytowed to the shore. We do not know the exactlocations where these sharks were captured but whalesharks captured in previous years were caught in areasshallower than 70 m (Pravin 2000). We determined

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the sex and the standard length (SL) before they werecut into pieces while afloat in the water. Once thebody parts were brought to shore, we collected forisotopic determination a sample of white muscle nextto the dorsal fin and below the collagen-like materialand the red muscle (Table 1). The total length (TL)was calculated using the equation TL=1.125*SL+0.0203, as described in Wintner (2000).

We also collected muscle from two teleost fish of wellknown feeding habits, captured from the same area. Theywere 1) the longarmmullet, Valamugil cunnesius (n=10;TL range 10.6–12.5 cm), which is a benthic, coastalfish that mainly feeds on decaying organic matter(Sommer et al. 1996), and 2) the Indian ilisha, Ilishamelastoma, (n=10; TL range 20–28 cm), a pelagiczooplanktivorous fish (Whitehead 1985; Carpenter et al.

Date of capture Standard length (m) Total length (m) Sex δ 15 N δ 13C C:N

11-apr-01 8.1 10.2 F 15.3 −15.1 3.2

11-apr-01 9.0 11.3 F 13.3 −16.4 4.1

12-apr-01 6.0 7.5 F 13.8 −16.2 3.1

12-apr-01 6.0 7.5 F 12.0 −16.3 3.0

17-apr-01 3.2 4.0 M 11.2 −17.9 2.8

30-apr-01 6.8 8.5 ? 14.7 −14.7 3.3

30-apr-01 4.9 6.2 ? 13.6 −16.2 3.7

01-may-01 6.3 7.9 M 15.2 −15.4 3.7

04-may-01 4.2 5.3 ? 15.2 −16.1 4.5

07-may-01 7.0 8.8 F 15.3 −15.7 3.8

08-may-01 15.0 18.8 F 15.1 −15.7 3.9

08-may-01 4.9 6.2 F 15.2 −16.8 3.3

08-may-01 5.9 7.4 M 15.5 −15.5 3.9

08-may-01 9.0 11.3 M 14.6 −15.1 3.6

08-may-01 ? ? M 15.5 −15.5 3.6

09-may-01 4.0 5.0 F 13.2 −16.1 3.3

09-may-01 5.4 6.8 F 14.6 −15.9 4.1

10-may-01 2.4 3.0 F 11.0 −17.0 3.1

11-may-01 6.3 7.9 F 12.0 −17.4 3.4

Table 1 Biological charac-teristics of the sampledwhale sharks and results ofthe stable isotope analysesconducted in their muscle

Fig. 1 Sampling location

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1997). As a consequence of their diet, these teleostswere expected to differ both in the δ15N (higher for theilisha than for the mullet) and in the δ13C (higher for themullet than for the ilisha) and were therefore consideredto provide a suitable baseline for interpreting the stableisotope values of whale sharks.

The collected samples were initially preserved incooking salt, a method considered suitable forpreserving fish muscle tissue (Arrington and Wine-miller 2002). Once in the laboratory, the sampleswere soaked for 30 h in distilled water and thenrinsed to remove the salt; subsequently, they werelong-term preserved at −25°C.

Stable isotope analysis

Prior to analysis, the muscle tissue (1 g) was dried for3 days at 70°C. After being ground, its lipids wereremoved by rinsing the ground tissue several timeswith a 2:1 chloroform: methanol mixture followingthe Folch method (Folch et al. 1957). Approximately1 mg of the powdered sample was weighed in tincapsules, automatically-loaded and combusted at1000°C to be analysed in a continuous flow isotoperatio mass spectrometer (Flash 1112 IRMS Delta CSeries EA Thermo Finnigan).

The results were presented according to the delta (δ)notation, where the relative variation of stable isotoperatios are expressed in parts-per-thousand from prede-fined standards. This variation is calculated as:

d ¼ RS RR=ð Þ � 1½ �»1000where RS is the ratio of the heavy isotope to the lightisotope of the sample, and RR is the ratio of the heavyisotope to the light isotope in the reference.

The Rstandard for13C and 15N are the Vienna Peedee

Belemnite (V-PDB) standard and atmospheric nitrogen(air), respectively. The international isotope secondarystandards of known 13C/12C ratios in relation toV-PDB, namely, polyethylene (IAEA CH7; δ13

C=−31.8‰), graphite (USGS24; δ13C=−16.1‰) andsucrose (IAEA-CH6; δ13C=−10.4‰), were used forthe calibration of δ13C at a precision of 0.2‰. Fornitrogen, the international isotope secondary standardsof known 15N/14N ratios in relation to air, namely,ammonium sulphate (IAEA N1; δ15N=+0.4‰ andIAEA N2; δ15N=+20.3‰) and potassium nitrate(IAEA NO3; δ15N=+4.7‰), were used for thecalibration of δ15N to a precision of 0.3‰. Atropine

(70.56%C, 4.84%N) was used as a standard forelemental composition of C and N. The experimentalprecision based on the standard deviation of replicatesof an atropine standard was 0.3‰ for both carbon andnitrogen.

The reference materials used for the analysis aredistributed by the International Atomic Energy Agency(IAEA). The analyses were carried out in the laborato-ries of the University of Barcelona.

Statistics

Prior to any statistical analysis, the data were testedfor normality with a Kolmogorov-Smirnov test ofgoodness of fit and for homogeneity of variances withLevene’s test. As all data sets were normallydistributed, the differences between groups wereinvestigated using the Student’s t-test. To investigatesex-related variation, we only used the subsample ofwhale sharks that measured more than 4.0 m (i.e.,three with known lengths of 7.4, 7.9, and 11.3 m andone with unknown length) and six females of similarsize (i.e., 7.5, 7.5, 7.9, 8.8, 10.2 and 11.3 m). A simpleleast squares linear regression was used to determine therelationships between δ15N and δ13C and betweenisotopic signatures and the total shark length afterlogarithmic transformation. For the latter analyses thelength values were converted to loge of length becausethe relationship of length with delta values fitted betteran exponential than a linear relationship.

All the statistical calculations were carried outusing the SPSS-15 statistical package.

Results

The main biological characteristics, the δ15N and δ13Cvalues and the C:N ratio of the muscle tissue of thesharks analysed are summarised in Table 1. The meanvalue of the C:N ratio was 3.5 (SD ±0.43), whichindicates that the lipid extraction process was effectivein the tissues analysed. The stable isotope ratios in themuscle tissue of whale shark ranged from −17.9to −14.7‰ for δ13C and from 11 to 15.5‰ for δ 15N(Table 1, Fig. 2); this implied a difference between theextreme values of 4.5‰ in δ15N and of 3.2‰ in δ13C.A statistically significant and positive correlation wasfound between the δ15N and δ13C values (R2=0.6; p<0.001; Fig. 2).

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As for the whale shark, the C:N ratio of the muscletissue of the Indian ilisha (3.06±0.05) and thelongarm mullet (3.14±0.1) were lower than 4 andhence the lipid extraction process was considered tobe effective. The Indian ilisha, a pelagic zooplankti-vore, had a mean δ15N of 14.64‰ (SD ±0.83) and amean δ13C of −15.23‰ (SD ±0.34), values thatoverlapped with the isotopic profiles of the majorityof whale sharks. As a consequence, the mean δ15N ofwhale sharks was statistically indistinguishable fromthat of the Indian ilisha (p>0.2). However, the meanδ13C was lower in the sharks than in the Indian ilisha(t=3.85, df=27, p<0.001; Fig. 2).

The longarm mullet, a coastal and benthic detri-tivore, showed a mean value of δ15N of 8.55‰ (SD ±0.56) and a mean value of δ13C of −13.27‰ (SD ±1.13). As expected, the longarm mullet was enrichedin 13C (t=5.27, df=10.6, p<0.001) but depleted in15N when compared with the Indian ilisha (t=19.2,df=18, p<0.001; Fig. 2) and both stable isotope ratioswere significantly different from those of the whalesharks (δ13C: t=7.61, df=27, p<0.001; δ15N: t=14.07, df=25, p<0.001; Fig. 2). The low variability inthe δ15N of the longarm mullet could be attributed toa low variability in the δ15N of its prey. On the otherhand, the broad range of the δ13C values in the

muscle of this species does not appear to be due to aninconsistent removal of lipids from the tissue becausethe C:N ratio was lower than 4 (see above) butprobably indicate that the analyzed individualsexploited heterogeneous sources of detritus (phyto-plankton, marine macrophytes and terrestrial detritus).

The isotopic signatures of whale sharks wereaffected by their body size, as regression analysesproved significant and positive both for δ13C andδ15N (δ15N: R2=0.30, df=16, p=0.022; δ13C: R2=0.32, df=16, p=0.019; Fig. 3).

Differences between the sexes were non-significantboth for δ13C (males: −15.4±0.2‰; females: −16.1±0.7‰; t=2.07, df=8, p=0.073) and for δ15N (males:15.2±0.4‰; females: 13.8±1.5‰; t=1.95, df=8, p=0.087), although in both cases the p values were onthe verge of significance (p<0.1).

Discussion

The total body length (TL) distribution of the whalesharks in this study (3–11.3 m; except for one largefemale of 18.8 m) falls within the range of variation

Fig. 3 Relationship between total length and δ15N and δ13C inthe muscle of whale shark (females: ♦; males: ●; unknown: ♦).Regression lines: δ15N=10.01+1.97*Ln (length); p<0.02 andδ15C=−18.30+1.11*Ln (length); p<0.02

Fig. 2 Relationship between δ13C and δ15N in the muscle ofwhale shark (females: ♦; males: ●; unknown: ♦; Indian Ilisha:Δ; longarm mullet: ○). Regression line fitted to shark values:δ15N=36.68+1.41* δ13C; R2=0.6; p<0.001

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previously observed both in India (n:164; range:3.15–14.5 m; modal length: 5.1–6.0 m) and world-wide (most specimens reported to measure 4–12 m)(Compagno 1984; Colman 1997; Pravin 2000; Duffy2002; Graham and Roberts 2007; Norman andStevens 2007). However, several authors havereported a maximum length of 20 m for this species(Compagno 2001; Chen et al. 2002). Sexual maturityis reached at a TL of about 9 m (Colman 1997;Norman and Stevens 2007), which means that themajority of the individuals in this study were stillimmature. This is consistent with the fact that inVeraval the fishery was conducted with relativelysmall boats which towed the catch to shore, andtherefore could not operate far away from the coast,and that immature whale sharks prevail in coastallocations worldwide (Eckert and Stewart 2001; Eckertet al. 2002; Meekan et al. 2006; Wilson et al. 2006;Graham and Roberts 2007; Rowat et al. 2007; Hobbset al. 2009). This indicates that this fishery exploitedan on-shore fishing ground, as previously suggestedby Hanfee (2001), although the exact locationremained unknown for the authors. However, in otherlocations such aggregations of immatures are mostlycomposed of males shorter than 9 m of TL, while oursample was dominated by females. This may indicatea female-biased sex ratio in the area, a featureinfrequently described in other localities (Eckert andStewart 2001; Hobbs et al. 2009).

The analysis of stable isotopes indicated that theoverall δ15N values for all the species in the currentstudy was high, possibly resulting from the influenceof the high levels of organic pollution that occur inthe coastal ecosystem of the Gujarat coast. Pollutionis particularly intense in the waters around Veraval,due to extensive raw urban and industrial sewage aswell as by runoff of agricultural products (Zingde2005). Thus, previous studies in similarly organicpolluted areas in the Adriatic Sea have shown ahigher than expected enrichment of the δ15N signal incoastal invertebrates such as Anemonia sulcata(Dolenec et al. 2005).

When isotopic values of whale sharks caught inVeraval are compared with those of other componentsof the food web, the results corroborate that, at leastin the Arabian Sea, the species has a primarilyzooplanktivorous diet. Thus, despite the wide spec-trum of prey that it is known to consume (Compagno1984; Taylor 1994; Colman 1997; Norman 1999;

Wilson and Newbound 2001; Duffy 2002) its stableisotope profile is close to that of the Indian ilisha.However, the positive relationship between δ15N andδ13C (explaining 60% of the variance) and therelationship between each isotope ratio and the totallength of sharks (explaining 30% of the variance) allsuggest that, as the size of the sharks increases, thecontribution to the diet of small fish and/or ofzooplankton of larger size and higher trophic levelincreases. The lack of highly developed filteringstructures on the gill-rakers in neonatal whale sharkscompared to adults (Garrick 1964) could explain thelow ability of small individuals to forage on largerprey. Indeed, studies on the intrapopulation variabilityof isotope signatures in fish frequently show apositive relationship between δ15N values and size, arelationship that is commonly attributed to ontogenet-ic or size-based changes in diet (Overman and Parrish2001).

Nevertheless, a reduction with age of the 15Ntissue-diet fractionation factor, as described in otherspecies (Focken 2001; Gaye-Siessegger et al. 2003;Trueman et al. 2005), may also contribute to theobserved increase of δ15N with total length. Indeed,this effect would explain why the slope of increasewith size of δ15N, and to a lesser degree of δ13C, wasmore abrupt in the group of smaller, growing fasterindividuals than in those of larger size and compar-atively lower growth rates. Whale shark neonatallength is approximately 50 cm±10 (Joung et al. 1996)and the growth rate is extremely high during the fewyears after birth (more than 100 cm per year) but,after reaching 3–4 m in length, it slows down byabout 75% (Chang et al. 1997; Hsu et al. 2000;Uchida et al. 2000; Nishida 2001). In this growingscenario, the sharp increase of muscle δ15N values insharks 3–4 m or longer may be partially caused bythis effect.

On the other hand, the increase of δ13C with totalsize is likely attributable to intraspecific differences inmovement patterns and heterogeneities in the locationwhere individuals forage. Thus, whale sharks smallerthan ca 4 m showed a δ13C of about −17‰, while inlarger individuals it increased to about −15‰, achange that indicates a transition from a pelagicoffshore life to a relatively more coastal habitat.

Despite the lack of knowledge of where and whenwhale sharks give birth or about the way how earlylife develops, the fact that all neonatal captures have

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occurred in, or very close to, deep waters seems toindicate that nursery areas are located offshore (Wolfson1983; Kukuyev 1995; Rowat et al. 2008). This appearsto be confirmed by the scarcity of records of whalesharks of <4 m in length, especially in the IndianOcean (Rowat et al. 2008), which would suggest thatneonates and very small whale sharks frequent deeperwaters than their larger conspecifics. Such preferencemay be due to natal philopatry, a behaviour describedin other species of sharks (Hueter et al. 2005) or,alternatively, to a protective behaviour of very youngwhale sharks, which would remain in deep waters toavoid predators (Rowat et al. 2008).

When comparing sharks of similar total length,differences between sexes in the stable isotope signalwas found to be marginally non-significant (p<0.1), aresult likely to be due to the low statistical power ofthe small-sized sample than to an actual homogeneityin signatures (Hoem, 2008). Thus, males appeared tohave higher values of δ13C and δ15N than females.Virtually nothing is known about abundance, segre-gation and migration patterns of the whale shark andwhether there are sex-related differences in these traits(Taylor 1996; Colman 1997; Heyman et al. 2001;Wilson and Newbound 2001; Duffi 2002; Stewart andWilson 2005; Rowat 2007), but coastal aggregationsare often observed to be sex-biased (Eckert andStewart 2001; Wilson et al. 2006; Graham andRoberts 2007; Rowat et al. 2007). In our case,regardless of isotope signal signatures, the higherproportion of females in the catch in a coastal fishery,as discussed above, would support the hypothesis ofspatial segregation between sexes.

Sex and size segregation have been observed inother shark species (Bres 1993; Sims 2005). Thus,tagged individuals and DNA studies have establishedthat male white sharks (Carcharodon carcharias) mayundertake transoceanic movements, whereas femalestend to remain in the coastal waters of the continentwhere they had been born (Pardini et al. 2001;Boustany et al. 2002); likewise, intermediate-sizefemales of scalloped hammerhead sharks (Sphyrnalewini) have been observed to form schools thatsegregate from males and feed more frequently onpelagic prey (Klimley 1987). Like other sharks, whalesharks may perform either short distance (Gunn et al.1999) or large-scale migrations (Eckert et al. 2002;Schmidt et al. 2009). These latter migrations may takeyears to complete, and their range seems to be

governed by the timing and location of productionpulses as well as by breeding behaviour, which differsbetween sexes (Colman 1997; Eckert and Stewart2001). Further research, potentially conductedthrough skin biopsies obtained from free-rangingindividuals, should be made to clarify whether thesedifferential patterns of movement result in actualsegregation between sexes in foraging habitat.

Acknowledgments We acknowledge the Fundació pelDesenvolupament Sostenible (FDS) for providing funds forthe sampling fieldwork, to the Serveis Cientifico Tècnics (SCT)of the University of Barcelona for analytical assistance, and tothe Vicerectorate of International Relations of the University ofBarcelona for travel funding during the elaboration andcorrection of the manuscript.

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