Trophic inferences of blue shark (
Prionace glauca
) in the MexicanPaci
c from stable isotope analysis in teeth
Carlos J. Polo-Silva
1
, Felipe Galván-Magaña
2
*
and Antonio Delgado-Huertas
3
1
Posgrado en Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Apdo. Postal 70-305 CiudadUniversitaria, 04510 México, D.F., México
2
Centro Interdisciplinario de Ciencias Marinas, Ave. IPN s/n, P.O. Box 592, 23096 La Paz, Baja California Sur, México
3
Instituto Andaluz de Ciencias de la Tierra (CSIC-UGR), Avda. de las Palmeras 4, 18100 Armilla, Granada, Spain
RATIONALE:
Isotopic analysis of biogenic tissues such as teeth of elasmobranchs has been well recognized as animportant method to interpret present and past environmental conditions. However, few studies on shark teethhave focused their attention on making trophic inferences or reconstruction of diet.
METHODS:
We analyzed the carbon (
d
13
C) and nitrogen (
d
15
N) isotope composition of the tooth crown and root from blue sharks
Prionace glauca
caught in southern Baja California using a continuous-
ow system by means of an elementalanalyzer combined with a Delta Plus XL mass spectrometer, to describe their feeding ecology, considering sex andmaturity stage.
RESULTS:
Signi
cant differences in
d
13
C values for tooth root were found between immature and mature males, withdepleted values of
13
C in immature individuals. No statistical differences were found between maturity stages in femalesfor both the C and N isotopes in any part of the tooth, which suggests that females were consuming the same prey in thesame area. In addition, we observed signi
cant differences in
d
15
N values between the tooth crown and root.
CONCLUSIONS:
Isotopic analysis in the tooth crown (dentin) and root may represent a new tool to describe the feedingecology of different species of elasmobranchs, showing dietary change over a short timescale. Copyright © 2012 JohnWiley & Sons, Ltd.
The use of stable isotopes of nitrogen (
d
15
N values) andcarbon (
d
13
C values) to address ecological questions hasincreased, becoming one of the most widely used methodol-ogies in animal ecology.
[1
–
3]
This biogeochemical methodallows quantitative analysis of dietary composition andforaging patterns on spatial and temporal scales, providinginsight on the food webs dynamics and the energy
ow inaquatic ecosystems.
[4,5]
The application of stable isotope analysis (SIA) by massspectrometry in ecological research is based on a constantincrease in the heavier isotopic values (
15
N and
13
C) withprogression up the food chain. Ratios of nitrogen isotopes(
15
N/
14
N) are used to infer relative trophic level, becausetissues tend to become enriched on
15
N at each trophictransfer.
[6,7]
In contrast, carbon isotopic composition (
13
C/
12
C)iscommonlyusedtoinferbasalcarbonsources,ortodifferenti-ate between nearshore and offshore nutrient sources.
[3,8,9]
Shark dietary studies have been improved with the use of SIA, which is routinely used to study the ecology of mammals, birds, and teleosts.
[3,10,11]
Generally, thetissues used fortrophicstudies in sharks using isotopic analyses are liver, muscleand vertebrae, which provide information about the foodassimilated by individuals over the past months, 1
–
2 yearsprior to death and each year of the life, respectively.
[1,12
–
16]
Insharks, accretionary tissues that contain growth layers suchas vertebrae can record a chronology of ecological informationover the lifetime of a single individual.
[15,16]
In some sharkspecies, such as the white shark (
Carcharodon carcharias
) andthe silky shark (
Carcharhinus falciformis
), a single growth layerinvertebraerepresentsoneyearofgrowthandthiscanbeusedto accurately determine the age of individuals.
[16,17]
Teeth have been used to study paleo-environmentalconditions, because of their relative abundance in marinesediments through time. Shark teeth have good crystallinityand physical hardness which allow them to stay almostintact with little to no wear once they are lost. The isotopiccompositions of the
uorapatite of the enameloid are used tointerpret both present and past environmental conditions.
[18,19]
However, few isotopic studies in sharks have used such tissuestodescribetrophichabits.Isotopic analysesinteeth ofothertaxasuch as marine mammals have proven to be a useful tool thathelps to establish trophic relationships.
[19
–
22]
Shark teeth arecomposed of two types of calci
ed tissue: the crown covered by an enameloid matrix in the outer part and the osteodentineoccurring in the inside and tooth root.
[18,23]
Teeth occur inmultiple rows and are continuously replaced during the life of the shark. Tooth replacement rates (from birth until exfoliation)for sharks are only known for some species, varying from 8to12 days for lemon shark (
Negraprion brevirostris
) and leopardshark (
Triakis semifasciata
) and from about 32 to 35 days per*
Correspondence to:
F. Galván-Magaña, Centro Interdiscipli-nario de Ciencias Marinas. Ave. IPN s/n, P.O. Box 592, 23096La Paz, Baja California Sur, México.E-mail: galvan.felipe@gmail.com
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom.
2012
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Research Article
Received: 17 March 2012 Revised: 12 May 2012 Accepted: 13 May 2012 Published online in Wiley Online Library
Rapid Commun. Mass Spectrom.
2012
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, 1631
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1638(wileyonlinelibrary.com) DOI: 10.1002/rcm.6275
1 6 3 1
row of teeth for silky sharks (
Carcharhinus falciformis
). Thereplacement rates of mature sharks could be slower, takingmonths to occur rather than days.
[24
–
26]
The blue shark (
Prionace glauca
) is the most abundant andwidespread elasmobranch and one of the most prominentlarge predatory and nektonic species of the marine world.
[27]
This species is seasonally abundant and frequently caughtaround the world. Off the western coast of Baja California,Mexico, this species numerically dominates artisanal shark-
sheries.
[28,29]
The segregation model of this species (by sexand maturity stage) throughout the north Paci
c exhibits a broad spatial distribution.
[27]
In the north Paci
c matingoccurs in early summer between 20.0 and 30.0
N, and sexsegregation takes place only among juveniles. This modelsuggested that the distribution of the parturition areaextended around the Subarctic Convergence zone (SAC), between 35.0 and 45.0
N. The nursery areas of juvenilefemales and males would be located north and south of theSAC, respectively, while adult males and females should bedistributed from the breeding areas towards the equator. Thissegregation is associated with colder and richer waters, aswell as prey distribution.
[27,30]
Stomach content analysissuggests that blue sharks feed on cephalopods, red crab andteleosts along the Baja California Peninsula, with a higherdominance of squid over time.
[31,32]
The objectives of the present study were to examine theisotopic relationships between sex, maturity stage and timeacross two tissues. Based on previous feeding studies of thisspecies
[31,32]
in this area and published isotopic values of itspotential prey
[33]
and unpublished data from samples caughtinthesameregionasthesharks,weexpectedthat:(1)bluesharkwould have isotopic differences, by sex and maturity stages, because they have different foraging areas and feed of differentprey, and (2) a comparison of the isotopic values of root andcrown of tooth would re
ect different timelines. We predictedthat root collagen would re
ect a quicker timeline than crowncollagen, because previous studies have shown that duringthe tooth formation of shark the root is formed to the end.
[24,26]
EXPERIMENTAL
Sample collection
All blue shark samples were obtained from two artisanal
shing camps (Punta Belcher: 24
15
’
N; 112
05
’
W; and PuntaLobos: 23
25
’
N; 110
15
’
W) located in Baja California Peninsuladuring January, July and December of 2009. The total length(cm) was measured, and the sex and maturity stage (matureor immature) were recorded for each shark using the criteriaproposedbyPratt
[34]
:maturemaleshaveacompletelycalci
edclasper with 360
rotation,anopenriphiodonand thepresenceof semen; mature females showed copulation marks, a cloacalentrance of at least 3 cm in diameter and presence of vitellogenic follicles and embryos in uteri. Individuals thatdid not meet the above characteristics were consideredimmature sharks.
Sample preparation
The teeth were taken from the
rst row of the upper jaw. Eachtooth was cleaned of tissue debris and other external materialand washed with distilled water. Collagen, which is depositedcontinuously in the tooth, was sampled from both the crownand the root. Shark teeth consist largely of
uorapatite withlesser amounts of hydroxyapatite, which is a primarycomponent of dentin and contains about 25 to 30% organicmatter.
[19]
Each part of the tooth (crown and root) wasground to a
ne powder using a low-speed cutting drill with bits ranging in size from 800 to 1000
m
m. To isolate collagen,each powdered sample was demineralized in 1 mL of 4 Mhydrochloric acid (HCl), which was then evaporated at 50
Cfor 12 h.
[23]
Each sub-sample was then rinsed with distilledwater for neutralization and freeze-dried. Approximately1.0 mg of collagen from each part of tooth was weighed intotin cups for isotopic analysis.
Stable isotope analysis
All the samples were combusted at 1020
C and their carbon(
13
C/
12
C) and nitrogen (
15
N/
14
N) isotope ratios weredetermined using a continuous-
ow system involving a1500 NC elemental analyzer (Carlo Elba, Milan, Italy)combined with a Delta Plus XL isotope ratio mass spec-trometer (ThermoQuest, Bremen, Germany) at the StableIsotope Laboratory of the Estación Experimental del Zaidin(CSIC, Granada, Spain). The results are expressed instandard delta notation (
d
), de
ned as parts per thousand,as follows:
d
h
X
¼
R
sample
=
R
standard
1
1000
;
where X is the element,
h
is the mass of the heavy isotopeand R
sample
and R
standard
are the heavy to light isotope ratios(i.e.
13
C/
12
C and
15
N/
14
N) of the sample and standard,respectively.
[7]
The standards used were V-PDB (Vienna-PeeDee Belemnite) and atmospheric nitrogen (AIR) for C and N,respectively. The method precision was
0.1
%
for both Cand N stable isotopes. The weight-percent C:N ratios of eachsample were also measured, and these were in the expectedrange (2.7
–
3.3) for pure protein.
[35]
Statistical analyses
All samples (crown and root) were separated according tosex and maturity stage. The normality and homogeneity of variance were determined by using the Shapiro-Wilk andBarlett test, respectively. If the data met these assumptions,a Student
’
s t-test for signi
cant differences in the mean of
d
13
C and
d
15
N values was used. If the data were not normallydistributed a non-parametric Mann-Whitney U-test was used.To test whether a relationship existed between total length(LT) of shark and stable isotope values, simple linear regres-sions were
tted to shark LTand
d
15
N and
d
13
C data for bothtissues. Statistical analyses were conducted using Statistical8.0 (StatSoft Inc., Tulsa, OK, USA) software with a criterionfor signi
cance of
p
<
0.05.The results are expressed asmean
standard deviation (SD).Potential prey isotope values recorded in previous stomachcontent studies of blue shark
[31,32]
were taken from publishedand unpublished data from samples caught in the sameregion as the sharks (Table 1) with the goal of comparingthem qualitatively with the isotopic values recorded in both tissues of blue shark and to try to give more supportto our inferences. As the rates of dental replacement forC. J. Polo-Silva, F. Galván-Magaña and A. Delgado-Huertaswileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom.
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1 6 3 2
the blue shark are unknown, a replacement rate of 2 weeks or1
–
2 months was assumed based on studies in other species of carcharinid sharks such as silky shark and lemon shark.
[24,26]
RESULTS
Stable isotopic values by sex and maturity
Atotalof42teethwereanalyzed(22femalesand20males).Theisotopic comparisons (
d
13
C and
d
15
N values) show no signi
-cantdifferencesbetweensexesinanypartofthetooth(Table2).However, the
d
15
N values in the root and crown were slightlyhigher in females (16.9
%
; 16.0
%
) than in males (16.3
%
;15.0
%
)(Table2).Comparingthemeanisotopicvaluesbetweenmaturity stages for each sex showed no signi
cant differences between immature females (IF) and mature females (MF) forroot or crown in
d
13
C and
d
15
N values (Table 2), while thecomparison between immature males (IM) and mature males(MM) showed signi
cant differences in
d
13
C values for root(Mann-Whitney U-test,
U
=6,
p
<
0.01) and
d
15
N values forcrown (Student
’
s t-test,
t
=2.63,
p
=0.01) (Table 2). The isotopevalues for prey samples were corrected for trophic discrimi-nation of 1.7 and 3.7 for
d
13
C and
d
15
N values, respectively,estimated by Kim
et al
.,
[14]
to allow a direct comparison between isotopic data of consumer and their potential foodsources. The
d
15
N and
d
13
C values of both tissues for malesand females of each maturity stage were positioned betweenthree species of cephalopods
Dosidicus gigas
,
Ancistrocheiruslesueuri
,
Onychoteuthisbanksii
andredcrab
Pleuroncodesplanipes
.The
shes
Sardinops caeruleus
and
Scomber japonicus
were
5
N-enriched relative to blue shark teeth (Figs. 1 and 2).No linear relationships were found between LT and the
d
15
N or
d
13
C values in the crown tooth and between LT andthe
d
15
N values in the root tooth. However, the
d
13
C values inthe root tooth were positively correlated with LT (R
2
=0.18,
p
<
0.05), where values increase (less depleted) from 200 cmLT (Fig. 3).
Comparison between two parts of the tooth (root andcrown)
The comparison between the two parts of the tooth showed asigni
cant difference in
d
15
N values for immature females(Student
’
s t-test
t
=2.26;
p
=0.03) and immature males (Stu-dent
’
s t-test
t
=4.84;
p
<
0.001); with root values on average1.6
%
higher than crown values (Fig. 4). However, for maturefemales (Student
’
s t-test
t
=0.45;
p
=0.65) and mature males(Mann-Whitney U-test
U
=33;
p
=0.19), no signi
cant differ-ences were found.
DISCUSSION
The blue shark is a species that feeds on cephalopods,
shesand crustaceans, changing its feeding preferences in differentregions of the world.
[31,32,36]
However, offthe western coast of Baja California, this species feeds mainly on cephalopods andred crab.
[30,31]
Contrary to information reported in otherstudies based on stomach content analyses which indicatethat
P. glauca
have food segregation by sex (males feed mainlyon cephalopods and females on red crab),
[31]
our isotopiccomparison on both tissues (root and crown) between thesexes suggests that females and males are feeding on preywith similar isotopic values (Fig. 1). These observed differ-ences probably occur because each technique has differenttemporal resolution (depending on the number of samplesand the period covered). For example, the number of stomachcontents analyzed in previous studies was 300, with samplescollected between February and June of 2001 and 2006,making it possible to record dietary changes (speci
cally thetype of prey). On the other hand, the dental collagen of elasmobranch teeth has a rapid turnover rate, which providesinformation about the food assimilated by an individual overthe last week or 1
–
2 months prior to death.
[24
–
26]
Althoughconceptually stomach content analyses have been criticized by its
’
snapshot
’
that cannot account for seasonal variation,in this case the information provided by isotopic analyses inthe tooth was unable to detect the differences in feeding between sexes previously registered in stomach content.The isotopic comparison with the potential prey of sharksshowed that both sexes share some prey. It is also known thatthe red crab (
P. planipes
) is one of the prey consumed byfemalesoftheblueshark(Fig.1),corroboratingwhathasbeenfound in previous studies of stomach contents.
[31,32]
In this study we found signi
cant differences in the
d
13
Cvalues of the tooth root for mature and immature males,wherematureindividualshadhigher
d
13
Cvaluesthanimmatureindividuals. Although no differences were found in the toothcrown for this isotope, the mature individuals still maintainhigher
d
13
C values (Fig. 2). The
d
13
C values track productivity,with higher values found in productive nearshore areas, such
Table 1.
Isotopic compositions of potential blue shark prey from southern Baja California compiled from published andunpublished data used in Figs. 1 and 2ID Species prey
d
13
C
SD(
%
)
d
15
N
SD(
%
) TissueLipidextracted N Reference1
Dosidicus gigas
16.7
1.0 13.5
0.8 Muscle Y 2 Ochoa-Díaz
[33]
2
Octopoteuthis sicula
18.9 14.8 Beak Y 1 Ochoa-Díaz
[33]
3
Ancistrocheirus lesueurii
17.5
0.3 13.6
1.0 Muscle Y 6 Polo-Silva, unpublished data4
Onychoteuthis banksii
17.8 12.9 Muscle Y 1 Polo-Silva, unpublished data5
Pleuroncodes planipes
17.4
0.8 14.8
1.3 Muscle Y 8 Peckham and Newsome,unpublished data6
Sardinops caeruleus
17.3 17.8 Muscle Y 1 Ochoa-Díaz
[33]
7
Scomber japonicus
16.9 17.3 Muscle Y 1 Ochoa-Díaz
[33]
Trophic inferences of blue shark from SIA in teethwileyonlinelibrary.com/journal/rcm
Copyright © 2012 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom.
2012
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, 1631
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