Environ Biol Fish (2018) 101:1599–1612
https://doi.org/10.1007/s10641-018-0806-3
Feeding habits of the whale shark (Rhincodon typus) inferred
by fatty acid profiles in the northern Mexican Caribbean
Natali Cárdenas-Palomo & Elsa Noreña-Barroso &
Jorge Herrera-Silveira & Felipe Galván-Magaña &
Ana Hacohen-Domené
Received: 29 January 2018 / Accepted: 20 August 2018 / Published online: 1 September 2018
# Springer Nature B.V. 2018
Abstract Whale shark (Rhincodon typus, Smith, 1828)
is an endangered species with anthropogenic pressures
due to increasing demand of encounter tourism activi-
ties. Research efforts to identify management and con-
servation strategies for this species are needed. The
Northern Mexican Caribbean is one of the most impor-
tant feeding aggregation sites of whale sharks world-
wide. In this study, Mexican Caribbean whale shark
feeding habits are assessed by means of fatty acid (FA)
signature analysis, a biochemical non-destructive tech-
nique widely applied in trophic ecology studies. Sub-
dermal tissue biopsies of 68 whale sharks and samples
of their potential prey (zooplankton) were collected
during 2010 and 2011 in two areas with high R. typus
abundance. Zooplankton samples (n = 17) were divided
in two categories: mixed zooplankton (several groups of
zooplankton) and fish eggs (> 95% of sample compo-
nents were fish eggs). FA profiles of whale shark tissue
N. Cárdenas-Palomo : J. Herrera-Silveira
Centro de Investigación y Estudios Avanzados del IPN,
97310 Mérida, Yucatán, Mexico
N. Cárdenas-Palomo
Pronatura Península de Yucatán, AC, 97205 Mérida, Yucatán,
Mexico
E. Noreña-Barroso (*)
Facultad de Química, Universidad Nacional Autónoma de
México, 97356 Sisal, Yucatán, Mexico
e-mail: enorena@unam.mx
F. Galván-Magaña : A. Hacohen-Domené
Centro Interdisciplinario de Ciencias Marinas, Instituto
Politécnico Nacional, 23094 La Paz, BCS, Mexico
sampled between years showed significant variability;
while there was no intraspecific differences in FA sig-
nature related to sex, size and location. FA profiles of
whale sharks and their potential prey were dominated by
saturated fatty acids (SFA). R. typus FA signature was
significantly different from that of mixed zooplankton;
on the other hand, whale shark and fish egg FA profiles
formed groups with overlapping values and registered
high levels of oleic acid. PUFA average ω3/ ω6 ratio on
whale shark FA profiles for both years was below 1.
Arachidonic acid (ARA) percentage was higher in
whale shark biopsies (13.2% in 2010, 6.8% in 2011)
compared to values observed in fish eggs (2.0%) and
mixed zooplankton (1.4%). Similarity between FA pro-
files of whale sharks and fish eggs, low levels of bacte-
rial FA found in R. typus biopsies, as well as whale shark
feeding behavior observations in the study area, suggest
that R. typus is feeding mainly on surface zooplankton in
Mexican Caribbean; however, elevated ARA percent-
ages in whale shark samples may indicate that this
species has complementary feeding sources, such as
demersal zooplankton, which has been reported in other
aggregation sites. Results obtained contribute to the
knowledge of the whale shark trophic ecology in the
area, but are inconclusive. Further studies are recom-
mended to evaluate whale shark FA profiles from dif-
ferent tissues (muscle or blood); also, broader informa-
tion is needed about zooplankton FA signature in the
study area.
Keywords Fatty acid analysis . Feeding ecology . Whale
shark . Chondrichthyans . Elasmobranchs
1600
Introduction
Whale shark Rhincodon typus is known to be the
world’s largest living fish. Whale sharks receive great
attention worldwide because there is a growing demand
for highly lucrative encounter tourism involving this
species (Ziegler et al. 2012; Araujo et al. 2017). Unfor-
tunately, count data and modelled population estimates
show an overall global decline of R. typus population of
about 50% over the last 75 years, so it is considered as
an endangered (A2bd + 4bd) species by the IUCN Red
List of Threatened Species (Pierce and Norman 2016).
This scenario establishes the need to increase research
efforts in order to identify management strategies to
minimize anthropogenic impacts upon this species
(Graham 2007; Quiros 2007; Catlin and Jones 2010).
Although R. typus has a wide distribution around the
planet (Compagno 2001; Stevens 2007); the places
where it aggregates temporarily, probably to feed, are
few (Colman 1997; Compagno 2001; Rowat and
Brooks 2012). These feeding aggregations are common-
ly close to the coast and usually correspond to high
productivity areas, where plankton is abundant (Rowat
and Brooks 2012). Whale sharks are one of the three
existing filter-feeding shark species, catalogued as an
opportunistic (Colman 1997) and highly migratory spe-
cies (Eckert and Stewart 2001; Rowat and Gore 2007;
Stevens 2007; Hueter et al. 2013), whose movements
are mainly influenced by food availability (Taylor and
Pearce 1999; Heyman et al. 2001; Wilson et al. 2001;
Duffy 2002; Rowat and Brooks 2012; Robinson et al.
2013; Rowat et al. 2013; Cárdenas-Palomo et al. 2015;
Pacheco-Polanco et al. 2015). Information about whale
shark feeding habits can improve ecological understand-
ing of the underlying drivers of its movements and
support strategies for sustainable management of the
whale shark aggregation areas.
Whale sharks, as most of the elasmobranchs, have a
complex ecology and behavior (Compagno 1990;
Martin 2007), i.e. spend most of their time in oceanic
areas (Stevens 2007; Wilson et al. 2001; Rowat and
Brooks 2012; Hueter et al. 2013), in some aggregations
they feed mainly during night (Taylor 2007) and can
perform deep dives (Wilson et al. 2006; Rowat and Gore
2007; Brunnschweiler and Sims 2011; Tyminski et al.
2013). In the last decades, several studies have been
conducted on the trophic ecology of whale sharks. Most
of these studies are based on direct observation (Clark
and Nelson 1997; Gunn et al. 1999; Nelson and Eckert
Environ Biol Fish (2018) 101:1599–1612
2007; Taylor 2007; Motta et al. 2010; Cárdenas-Palomo
et al. 2010, 2015; Robinson et al. 2013), stomach con-
tents (Silas and Rajagopalan 1963; Chen et al. 1997;
Rohner et al. 2013), fecal samples (Jarman and Wilson
2004; Meekan et al. 2009) and recently, using biochem-
ical techniques which have proven their efficacy for
inferring food habits such as stable isotope and fatty
acid (FA) analysis (Hacohen 2007; Borrell et al. 2011;
Couturier et al. 2013a; Rohner et al. 2013; Marcus et al.
2016). The stomach content analysis, despite its high
taxonomic specificity, poses the disadvantage of
sacrificing the organism and only represents information
of prey recently consumed; in addition, it can lead to
underestimation of ingested organisms due to the differ-
ent stages of digestion they show at the sampling mo-
ment (Tripp 2010). Applying biochemical techniques in
trophic ecology studies has several advantages, like
requiring only a small amount of tissue from the organ-
ism (<5 g; Budge et al. 2006; Couturier et al. 2013a),
which can be extracted from free animals and without
significative damage; moreover, it has been established
that these analyses can provide information from the diet
consumed during a longer-term period (Budge et al.
2006; Iverson 2009).
In recent decades, the use of FA signature analysis to
study the diet of marine species, including elasmo-
branchs, has increased (Budge et al. 2006; McMeans
et al. 2012; Couturier et al. 2013a, b; Rohner et al. 2013;
Pethybridge et al. 2014; Marcus et al. 2016). These
studies are based on the idea that FA composition of
the prey has a direct influence on FA signature of high
trophic level marine animals, since some FA are gener-
ally assimilated intact, especially long chain polyunsat-
urated FA (Iverson et al. 2002, 2004; Bergé and
Barnathan 2005; DeLorenzo-Costa et al. 2006; Parrish
et al. 2009; Pethybridge et al. 2014; Meyer et al. 2017).
In the northern Mexican Caribbean, there is a whale
shark aggregation site that is recognized as one of the
most important worldwide (Hueter et al. 2013; Norman
et al. 2017). Most studies about whale shark trophic
ecology in this area are based on either observation of
whale sharks feeding and plankton tows (Motta et al.
2010; De la Parra Venegas et al. 2011; Cárdenas-Palomo
et al. 2015). FA analysis offers an alternative method for
investigating diet composition and a useful tool to con-
firm if potential preys are effectively assimilated by
predator.
Until now, there are only three recently published
studies about whale shark trophic ecology using FA
Environ Biol Fish (2018) 101:1599–1612
profile analysis (Couturier et al. 2013a; Rohner et al.
2013; Marcus et al. 2016). Rohner et al. (2013),
interpreting the information obtained from the FA pro-
filing of sharks in Mozambique and South Africa, sug-
gest the whale shark trophic spectrum could be wider
than expected and might include even bathypelagic and
mesopelagic food sources, as well as deep-water fish
and demersal zooplankton. Marcus et al. (2016) also
suggest that whale sharks’ food sources in Australia
might include deep-water organisms. There are no pub-
lished studies on whale shark trophic ecology using FA
profiling for the Mexican Caribbean whale shark aggre-
gation site. The principal aim of this research was to
describe the whale shark feeding habits, comparing FA
profiles obtained from whale sharks tissue and zoo-
plankton samples (potential prey) sampled during
2010 and 2011 on the whale shark aggregation located
at North of the Mexican Caribbean. Sources of variation
such as location, year, sex and age class of whale sharks
were also analyzed.
Materials and methods
Study area
The study area includes coastal and offshore areas to the
north of Mexican Caribbean. There are two locations
with high whale shark abundance: a protected area
known as Whale Shark Biosphere Reserve (WSBR)
and another known as BAfuera^, located southeast from
the Contoy Island (Fig. 1).
Zooplankton composition from each of the whale
shark’s locations (WSBR and Afuera) showed evident
differences. On the Whale Shark Biosphere Reserve,
zooplankton was dominated by copepods, other groups
like sergestidae, chaetognaths, appendicularians and
decapods were also present (Cárdenas-Palomo et al.
2010; Motta et al. 2010). Whereas in Afuera, zooplank-
ton is strongly dominated by fish eggs (De la Parra
Venegas et al. 2011; Cárdenas-Palomo et al. 2015).
Field sampling
Whale shark sub-dermal tissue samples were collected
between May to September 2010 and 2011 at the Mex-
ican Caribbean. Sub-dermal tissue was used because it is
the largest proportion of tissue obtained in non-invasive
biopsy sampling procedures on whale sharks. When the
1601
whale shark biopsy was collected, we registered posi-
tion (using a Garmin GS70-global positioning system
receiver), date and time.
Whale shark sub-dermal tissue (approximately two
cm deep) was taken close to the dorsal fin base, using a
hand spear with a modified tip. Once extracted, the
biopsies were kept at low temperatures until their arrival
to the laboratory where they were stored at −40 °C
temperature until analysis preparation. Total length of
whale sharks sampled was estimated comparing their
length with the size of the boat; also, sex of the organ-
isms was registered when possible. Whale shark length
was used to discriminate between young and adult indi-
viduals, since sharks >9 m total length, are considered
adult or sexually mature organisms (Taylor 1994;
Colman 1997). Gender was determined by swimming
underneath the whale shark to observe the presence or
absence of claspers.
Zooplankton samples were collected at each site
where a whale shark biopsy was obtained (Fig. 1) using
a 30-cm diameter, 300 μm mesh net towed horizontally
from a boat, following the recommendations of Coop-
erative Investigations of the Caribbean and Adjacent
Regions (UNESCO 1979). Each of the samples was
kept at low temperatures until their arrival to the labo-
ratory, where they were stored at −40 °C temperature.
Due to the evident differences in zooplankton composi-
tion collected in the locations, the zooplankton samples
were divided into zooplankton mix (collected inside
WSBR) and fish eggs (collected in Afuera).
Field survey was carried out under permits and ex-
emptions from the Mexican Federal Government Agen-
cy CONANP and the BDirección General de Vida
Silvestre^ SGPA/DGVS (04114/10, 05035/11).
Fatty acid analyses
FA profile analyses on whale shark and zooplank-
ton samples were made in the Chemical Laboratory
of National Autonomous University of Mexico at
Sisal, Yucatán. Prior to the analysis, samples were
freeze-dried and ground in a mortar to obtain a fine
powder. Lipids were extracted based on Folch ex-
traction procedure (Folch et al. 1957), using
dichloromethane:methanol (2:1, v/v). Lipid extracts
were saponified with 20% KOH:Methanol (w/v)
and free fatty acids were recovered in hexane from
the acidified saponifiable fraction (pH = 1–2). Fatty
acid methyl esters (FAMEs) were formed by
1602
Fig. 1 Study area showing the
main aggregation sites of whale
shark: Whale Shark Biosphere
Reserve (WSBR) and the Afuera
zone. Also, the map showing the
locations where biopsies and
zooplankton samples were col-
lected in 2010 and 2011
esterification with 10% BF3 in methanol (Fluka
15,716) for 60 min at 80 °C (Metcalfe et al.
1966). FAMEs were analyzed by capillary gas
chromatography in a Perkin Elmer Clarus 500 gas
chromatograph equipped with a Zebron ZB-WAX
capillary column (Phenomenex, 7FD-G007–08;
20 m of length, 0.18 mm i.d. and 0.18 μm of film
thickness) and a flame ionization detector (FID).
Hydrogen was used as carrier gas with a flow rate
of 40 mL min−1. Injector and detector temperatures
were 280 and 250 °C, respectively. Column tem-
peratures were programmed from 40 to 200 °C at a
rate of 20 °C min−1 and from 200 to 250 °C at 2.5.
Individual FAME were identified by comparing
retention times with reference standard containing
37 individual fatty acids (Supelco FAME Mix,
47,885-U). All FA were converted from chromato-
gram peak area to percentage of total area.
Statistical analyses
Fatty acids were categorized as saturated (SFA), mono-
unsaturated (MUFA) and polyunsaturated (PUFA), and
each FA expressed as a percentage of the total FA area.
Data are shown as mean ± standard error. The FA pro-
files of whale sharks included 31 fatty acids (Table 1),
but only those FA which were detected in both sampling
years and which represented more than 1% of the total
area, were considered for the statistical analyses.
Environ Biol Fish (2018) 101:1599–1612
PERMANOVA analysis (permutational multivariate
analysis of variance, based on 9999 permutations) was
applied to explore factorial differences (year, location,
sex and age class) within whale shark FA profiles and
within zooplankton samples. Non-parametric multi-di-
mensional scaling (MDS) was used to visualize possible
relationships of grouping between samples of whale
shark collected in different years (2010 and 2011). Hi-
erarchical cluster analysis based on group averages was
applied to the MDS plot to show clustering of similar
groups. No pre-treatment was applied to signature FA
data prior to computing a resemblance matrix based on
Bray-Curtis index.
One factor analysis of similarities (ANOSIM) was
carried out to explore the difference among FA profiles
of predator (different groups of whale shark were con-
sidered, A to G, according to the results obtained from
the cluster mentioned in the previous paragraph) and
prey (zooplankton mix and fish eggs). ANOSIM is a
non-parametric permutation procedure that generates a
statistic R, an absolute distance measure between
groups. Due the small sample size of some of the
groups, interpretation of the results focused on the
ANOSIM-R values rather than on significance level;
so R statistic values were used to evaluate at what level
the groups differed, with ANOSIM-R > 0.75 indicating
a clear and evident separation between groups, values
between 0.25 and 0.75 indicating separate groups but
with some overlapped data and < 0.25 as barely separat-
ed groups (Clarke and Gorley 2006; Rohner et al. 2013).
Environ Biol Fish (2018) 101:1599–1612 1603

Table 1 Fatty acid (FA) composition (% of total FA, mean ± SE) of whale shark biopsies and zooplankton collected in the northern Mexican
Caribbean (Mexico) during 2010 and 2011

Fatty acid Whale shark 2010 (n = 18) Whale shark 2011 (n = 50) Fish eggs (n = 12) Zooplankton Mix (n = 5)

10:0 0.0 ± 0.0 0.4 ± 0.4 0.3 ± 0.1 0.0 ± 0.0

11:0 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.2 0.1 ± 0.0
a
12:0 1.0 ± 0.1 0.4 ± 0.2 1.0 ± 0.4 0.5 ± 0.2
13:0 0.8 ± 0.1 0.0 ± 0.0 0.8 ± 0.3 0.6 ± 0.2
14:0a 3.3 ± 0.2 2.1 ± 0.3 8.9 ± 1.3 11.6 ± 2.7
15:0a 1.2 ± 0.1 0.9 ± 0.1 1.5 ± 0.1 1.5 ± 0.3
16:0a 21.6 ± 0.8 27.7 ± 1.1 28.3 ± 2.5 32 ± 2.3
a
17:0 1.7 ± 0.1 1.9 ± 0.1 1.6 ± 0.1 1.7 ± 0.3
18:0a 22.3 ± 1.6 30.1 ± 1.0 11.6 ± 2.2 10.6 ± 0.9
20:0 0.3 ± 0.0 0.3 ± 0.1 0.4 ± 0.1 0.5 ± 0.1
21:0 0.3 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
22:0 0.3 ± 0.0 0.5 ± 0.1 0.6 ± 0.3 0.3 ± 0.1
23:0 0.2 ± 0.0 0.3 ± 0.0 0.3 ± 0.2 0.1 ± 0.0
24:0 0.2 ± 0.0 0.4 ± 0.0 0.3 ± 0.1 0.4 ± 0.2
Total SFA 53.1 ± 2.1 65.3 ± 2.8 56.2 ± 2.2 59.9 ± 2.5
14:1 0.3 ± 0.0 0.1 ± 0.0 0.1 ± 0.1 0.0 ± 0.0
15:1 0.0 ± 0.0 0.0 ± 0.0 1.9 ± 1.0 0.7 ± 0.3
a
16:1 2.7 ± 0.2 1.9 ± 0.2 8.5 ± 1.7 8.7 ± 1.1
17:1a 2.4 ± 0.3 1.9 ± 0.2 0.6 ± 0.1 0.4 ± 0.1
18:1ω9c/ta 21.6 ± 0.7 18.0 ± 1.0 13.9 ± 2.7 6.2 ± 0.7
20:1ω9 0.5 ± 0.1 0.3 ± 0.0 0.7 ± 0.1 0.7 ± 0.3
22:1ω9 0.7 ± 0.1 0.8 ± 0.1 1.3 ± 0.6 0.3 ± 0.1
Total MUFA 28.1 ± 3.0 23.1 ± 2.5 27.0 ± 1.9 16.9 ± 1.3
18:2ω6ca 1.5 ± 0.1 0.8 ± 0.1 2.4 ± 0.9 2.1 ± 0.1
18:3ω6 0.1 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0
18:3ω3 0.4 ± 0.1 0.5 ± 0.2 1.6 ± 0.8 1.2 ± 0.2
20:2ω6 0.2 ± 0.0 0.2 ± 0.0 1.0 ± 0.4 0.6 ± 0.2
20:3ω6 0.2 ± 0.0 0.3 ± 0.2 1.1 ± 0.2 1.5 ± 0.3
20:3ω3 0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.1 0.3 ± 0.1
20:4ω6 (ARA)a 13.2 ± 0.9 6.8 ± 1.0 2.0 ± 0.8 1.4 ± 0.9
20:5ω3 (EPA)a 1.0 ± 0.1 0.7 ± 0.1 2.2 ± 0.4 5.8 ± 1.7
22:2ω6 0.3 ± 0.1 0.5 ± 0.1 0.3 ± 0.2 0.2 ± 0.1
a
22:6ω3 (DHA) 2.0 ± 0.1 1.9 ± 0.2 5.7 ± 1.5 10.2 ± 3.7
Total PUFA 18.8 ± 1.5 11.7 ± 6.3 16.9 ± 0.5 23.2 ± 1.1
ω3/ω6 0.22 0.36 1.5 3.1

Fish eggs correspond to zooplankton samples collected in the Afuera zone and mixed zooplankton were collected inside of Natural Protected
Area (WSBR)
SFA saturated FA, MUFA monounsaturated FA, PUFA polyunsaturated FA, EPA eicosapentaenoic acid, DHA docosahexaenoic acid, ARA
Arachidonic acid, c cis-configured MUFA, t trans-configured MUFA
a
denotes the FAs that were included in statistical analyses

Similarity Percentage Analysis (SIMPER) was carried dissimilarity between both groups, but do it consistently
out to identify the discriminating fatty acids between among all samples on both groups). Additionally, a
those groups (FA that not only contribute to the MDS (Multidimensional scaling) analysis was
1604
performed to visualize the grouping or separation be-
tween FA profiles of predator and prey (whale shark,
zooplankton mix and fish eggs). Analyses and plots
were done using the PRIMER6 software, version 6.1.2
(Plymouth Routines in Marine Environmental Research
programs, Clarke and Gorley 2006).
Results
Whale shark samples
A total of 68 whale shark biopsies and 17 zooplankton
samples (potential prey) were collected. From the total
whale shark biopsies, 29 corresponded to male organ-
isms, 13 to female and 26 samples the gender could not
be determined. In Afuera, 35 biopsies were collected,
while on the WSBR a total of 33 were obtained. Most of
the sampled organisms were young (39); only 14 adult
samples were collected and the size of 15 organisms
could not be determined.
FA profiles collected from whale sharks on different
years showed significant differences (pseudo-F = 11.7,
p < 0.001). There were no differences found between
whale sharks’ FA profiles from different sex (pseudo-
F = 0.47, p = 0.72) or age class (pseudo-F = 0.58, p =
0.63), nor between samples collected on different loca-
tions (pseudo-F = 2.8, p = 0.53).
In general whale shark fatty acids contained high
proportions of saturated fatty acids (16:0 and 18:0).
Between years, whale sharks from 2011 had higher
proportions of both of these fatty acids. Conversely,
whale sharks from 2010 had higher proportions of
18:1n-9 and 20:4n-6 (Table 1). Major FA on whale
shark tissue on both years was stearic acid (18:0) follow-
ed by palmitic acid (16:0). SIMPER analysis indicated
that higher percentages of 18:0 and 16:0 in 2010 sam-
ples contributed to the separation between sampling
groups; same as the higher ARA percentage (more than
double) detected in 2010. PUFA average ω3/ ω6 ratio
on whale shark FA profiles for both years was below 1.
Cluster analysis identified seven different groups of
whale shark samples based on their FA profiles (Fig. 2),
where one group corresponds exclusively to samples
collected in 2010 (Group A), Group B is comprised by
samples collected on both years and the five other
groups are comprised only by samples collected in
2011 (Groups C to G; Table 2). Most of the samples
were clustered in Groups B and C. The fatty acids that
Environ Biol Fish (2018) 101:1599–1612
characterize the sample profiles of each group and con-
tribute to the separation of them are described in Table 2.
SIMPER analysis indicated that the two most similar
whale shark groups were Groups B and C (Average
dissimilarity = 16.9). In contrast, the highest dissimilar-
ities occurred between Groups A and D (Average dis-
similarity = 40.3).
Potential prey samples
As mentioned before, a total of 17 zooplankton samples
were collected for this study, five were collected in 2010
(two in WSBR and three in Afuera) and twelve samples
were obtained in 2011 (three in WSBR and nine in
Afuera). FA profiles of zooplankton samples from dif-
ferent years did not show significant differences (pseu-
do-F = 1.53, p = 0.19).
Considering the FA profile of fish eggs and mixed
zooplankton, the major group was saturated fatty acids
(SFA) with 56.2 and 59.9%, respectively. Monounsatu-
rated fatty acids (MUFA) were the second most abun-
dant group for fish eggs with 27.0%; meanwhile for
zooplankton mix samples polyunsaturated fatty acids
(PUFA) had the second highest percentage (23.2%;
Table 1). EPA (5.8%) and DHA (10.2%) percentages
for mixed zooplankton samples were higher than those
for fish egg samples (2.2 and 5.7%, respectively).
PUFA’s average ratio ω3/ ω6 of fish eggs FA profile
was1.5, and for mixed zooplankton 3.1 (Table 1).
Palmitic acid (16:0) was the most abundant FA for
mixed zooplankton samples and fish eggs, followed by
oleic (18:1ω9c/t) in fish eggs and meristic acid (14:0) in
mixed zooplankton (Table 1).
Whale shark and potential prey FA profiles comparisons
Whale shark FA composition was significantly different
to zooplankton mix and fish eggs. Figure 3 shows the
grouping or separation between FA profiles of predator
and prey (whale shark, zooplankton mix and fish eggs).
ANOSIM-R values indicate separation with overlap-
ping values (ANOSIM-R = 0.69) between whale shark
FA profiles and fish egg FA profiles. On the other hand,
ANOSIM-R values indicate a clear difference between
signature FA profiles of whale sharks and mixed zoo-
plankton (ANOSIM-R = 0.91), where FA 18:0,
18:1ω9c/t, 16:0 and ARA were the main source for this
separation. FA profiles of Group G were the most sim-
ilar to FA profiles of mixed zooplankton (average
Environ Biol Fish (2018) 101:1599–1612 1605

Fig. 2 Multi-dimensional scaling

ordination of whale shark clusters
for 2010 and 2011

dissimilarity = 36.2); Groups A and C were the most
similar to fish eggs (average dissimilarity = 35.13); the
Group F was the most dissimilar to the FA of their
potential prey: fish egg (average dissimilarity = 46.8)
and mixed zooplankton (average dissimilarity = 51.8).
Stearic and oleic acid on whale shark samples,
showed higher levels compared to mixed zooplankton
and fish eggs; in fact, these two FA are the main sources
of separation between profiles of whale sharks and its
potential prey profiles. PUFA showed higher levels in
zooplankton samples than in whale sharks; ARA per-
centage was higher in whale shark biopsies (13.2% in
2010, 6.8% in 2011) compared to shown on fish eggs
(2.0%) and mixed zooplankton (1.4%; Table 1). Despite
not showing statistically significant dissimilarities be-
tween fish egg and mixed zooplankton FA profiles, it is
worth mentioning PUFA percentages on mixed zoo-
plankton were higher than those on fish eggs. Mixed
zooplankton obtained significantly higher levels of
DHA and EPA than fish eggs (Table 1).

Table 2 Groups identified in the cluster analysis

Year Sex Age class Aggregation site Characteristics of fatty acid profiles

Group A 2010 (n = 3) 3 males 3 juveniles 3 WSBR Much lower 18:0 than the rest of Groups; higher levels of
ARA, EPA and DHA than the rest of Groups (except
Group B)
Group B 2010 (n = 15) 19 males; 15 juveniles; 14 WSBR; Higher levels of ARA, EPA and DHA than the rest of Groups
2011 (n = 24) 10 females; 12 adults; 25 Afuera (except Group A)
10 n/a 12 n/a
Group C 2011 (n = 12) 5 males; 9 juveniles; 5 WSBR; Lower ARA and EPA than the rest of groups; moderate levels
1 female; 2 adults, 7 Afuera of 16:0 and 18:1ω9
6 n/a 1 n/a
Group D 2011 (n = 1) 1 male 1 juvenile 1 Afuera Much higher level of 12:0 than the rest of Groups; ARA and
EPA were not detected
Group E 2011 (n = 1) 1 n/a 1 juvenile 1 WSBR Much higher level of DHA than the rest of Groups; higher
level of 16:0; ARA and EPA were not detected
Group F 2011 (n = 1) 1 n/a 1 n/a 1 Afuera Slightly higher 18:0 and lower 18:1ω9 than the rest of
Groups; moderate levels of ARA and EPA
Group G 2011 (n = 11) 1 male; 10 WSBR; Slightly lower 17:1 and 18:1ω9 than the rest of Groups;
1 female; 1 Afuera lower levels of ARA and EPA
9 n/a

Fatty acid indicators and collection and biological information are included
WSBR Whale shark Biosphere Reserve, EPA eicosapentaenoic acid, DHA docosahexaenoic acid, ARA arachidonic acid
1606
Fig. 3 Multi-dimensional scaling
of mean fatty acid profiles (% of
total FA) of whale shark and their
potential prey (zooplankton mix
and fish eggs) collected in the
northern Mexican Caribbean
during 2010 and 2011
Discussion
FA profiles and feeding habits inferences
Whale shark aggregation in the northern Mexican Ca-
ribbean has been monitored steadily since 2005. During
these years, it has been confirmed that R. typus uses the
area as a feeding site taking advantage of zooplankton
abundance due to two events: Yucatan’s upwelling in-
tensification and the presence of dense fish egg masses
(De la Parra Venegas et al. 2011; Hueter et al. 2013;
Cárdenas-Palomo et al. 2015). In this research, FA anal-
ysis was applied to confirm if the zooplankton groups
(potential prey) that are abundant in this site were as-
similated by the whale shark, but results obtained were
inconclusive on their own.
In whale shark profiles of samples collected, saturat-
ed FA had the greater contribution. This result is con-
sistent with whale shark FA signatures of samples col-
lected in Mozambique, South Africa (Couturier et al.
2013a; Rohner et al. 2013) and Australia during 2013
(Marcus et al. 2016); nevertheless, Marcus et al. (2016)
report PUFAS as the major FA group on whale shark
samples collected during 2014, mainly due to elevated
percentages of ARA (16.4%).
For our study, the ratio ω3/ω6 was 0.22 (less than
one), because of PUFAω6 family dominance, with
ARA standing out as the highest percentage of FA-
PUFA. Whale sharks did not present a similar FA profile
compared to other planktivorous species where FA ω3
prevail, featuring EPA and DHA (Sargent and Falk-
Petersen 1988; Dalsgaard et al. 2003; Couturier et al.
Environ Biol Fish (2018) 101:1599–1612
2013b). Recent studies have suggested that the trophic
ecology of the whale shark could be more complex than
expected (Rohner et al. 2013; Marcus et al. 2016). Just
as in this paper, Couturier et al. (2013a) and Rohner
et al. (2013) report whale sharks’ FA profiles dominated
by PUFA ω-6 (and ω3/ω6 ratio lower than 1), also due
to the high amounts of arachidonic acid (20:4ω6,
ARA). Meyer et al. (2017) indicate that variation of
key essential FA such as ARA could be greatly affected
by the tissue from which FA profiles are derived, as well
as interpretation of the shark’s diet.
ARA levels detected for mixed zooplankton and fish
eggs (less than 3% of the total FA area each), were not
similar to ARA detected in whale shark samples. Phy-
toplankton and some macroalgae species such as
rhodophytes, can synthetize PUFA and report high
levels of ARA on their FA profiles (Johns et al. 1979;
Dunstan et al. 1994). Nevertheless, it has been sug-
gested that whale shark phytoplankton ingestion is inci-
dental, since their digestive apparatus is adapted to
larger sized prey (Motta et al. 2010; Rowat and
Brooks 2012).
ARA percentage obtained from this study in whale
shark samples, was relatively low compared to the other
FA profile studies reported for R. typus (Couturier et al.
2013a; Rohner et al. 2013; Marcus et al. 2016). On these
studies, it is also suggested that elevated ARA percent-
ages might be due to organisms feeding near the bottom
and being able to ingest sediments containing organisms
which carry high ARA levels or feeding demersal zoo-
plankton which could potentially contain high levels of
this FA-PUFA. Even, Rohner et al. (2013) report
Environ Biol Fish (2018) 101:1599–1612
similarities between FA profiles of whale sharks and
mysidacea, bathyal shrimp, cumacea and copepods
from more than 200 m depth, supporting the idea that
R. typus has an important feeding source on deep water
species (Rohner et al. 2013).
Whale sharks in Mexican Caribbean could be
feeding on demersal zooplankton as a complementa-
ry source of food, but it is important indicate that data
suggest that their main feeding source is the near-
surface zooplankton. Zooplankton FA profiles, spe-
cifically those of fish eggs, were similar to the FA
signature of whale shark samples. On the other hand,
Tyminski et al. (2013) report whale shark vertical
movement patterns from data recovered from 28 sat-
ellite tags attached on organisms in the Mexican
Caribbean. This study showed most organisms tend
to be near the surface most of the day during summer
(no more than 10 m depth), which matches the re-
ports for the species in other aggregation sites (Eckert
and Stewart 2001; Brunnschweiler et al. 2009); then,
after the sunset, they combine it with short shallow
dives. Deeper and longer dives, were registered
mainly during the rest of the year in oceanic water,
when organisms are no longer in the Mexican Carib-
bean aggregation site. These results combined with
the feeding behavioral evidence observed, support
the theory that, in the Mexican Caribbean, whale
sharks feed mainly near the surface and during the
day. It also is important to mention that the aggrega-
tion area of whale shark in the Mexican Caribbean is
shallow (< 50 m deep).
Comparing among FA profiles from prey and whale
sharks, fish egg FA profiles were more similar to those
of whale sharks. Except for the similarities between
saturated fatty acid abundance (since this suppose main-
ly new synthesis), the link between both profiles seems
to be the elevated amounts of oleic acid detected. In
relation to PUFAS, similarities between FA profiles
from whale sharks and potential prey can be identified
by the relatively high levels of linoleic acid (C:18:2n6c)
and DHA (C22:6n3). DHA has been identified as a
biomarker to trophic studies (Budge et al. 2006) and
linoleic acid has been suggested as a precursor of ARA
(Dalsgaard et al. 2003).
As mentioned previously, oleic acid (18:1n9c/t), was
particularly high on whale shark sub-dermal tissue.
Oleic acid generally rises with depth, since this FA have
been detected in higher levels in plankton or fish from
deep waters (Lewis 1967). These arguments served to
1607
Rohner et al. (2013) to suggest that whale sharks feed, at
least partly, from organisms which belong to waters
deeper than 200 m in Mozambique and South Africa.
In the Mexican Caribbean, high oleic acid in whale
shark tissue could be better explained by sharks feeding
on fish egg, which oleic acid percentages were relatively
high. Oleic acid has been identified as one of the main
FA present in fish egg and larvae (Castello 2013). High
levels of oleic acid on plankton organisms have been
registered in upwelling areas in Chile as well (Escribano
and Perez 2010). Other indicator that back up the sur-
face feeding theory, is that the fatty acids which indicate
bacterial activity (C15:0 and C17:0; Bergé and
Barnathan 2005) were scarcely represented on the whale
shark profiles (Table 1).
On the other hand, it is possible to compare between
zooplankton nutritional value generated by upwelling
(mixed zooplankton) and that of fish eggs using total FA
composition and/or polyunsaturated FA (PUFAs) con-
tent such as EPA and DHA. Under this premise, it was
observed that upwelling zooplankton (mixed zooplank-
ton) constitutes a more valuable food for whale sharks
than fish egg from a nutritional view, since it had a
higher PUFA proportion. This result matches the reports
by De la Parra Venegas et al. (2011) when comparing
the energetic value from mixed zooplankton and fish
eggs collected on the same study area. It is important to
indicate that even when mixed zooplankton seems to
have a higher nutrimental value, registered values of fish
eggs biomass were much more elevated than those of
mixed zooplankton (Cárdenas-Palomo et al. 2015).
Since whale sharks are opportunistic organisms, it has
been reported that larger groups prefer to aggregate in
spawning areas rather than areas where mixed zooplank-
ton patches are located.
Except for the year in which the samples were col-
lected, the sources of potential variation between FA
profiles from whale shark tissue (location, sex and size
class) did not indicate significant differences. The rea-
son could be that in the Mexican Caribbean it is com-
mon to find groups of different sex and age sharing the
same areas, unlike other whale shark aggregation sites
where sexual and size segregation has been observed
(Borrell et al. 2011; Ketchum et al. 2013). Likewise, it
has been proven using photo identification that organ-
isms in the Mexican Caribbean move between locations
(WSBR and Afuera) during the months when the spe-
cies is present (Ramírez-Macías et al. 2012; Hueter et al.
2013; Tyminski et al. 2015; McKinney et al. 2017).
1608
Limitations in the use of FA profile analysis
Similarity analysis indicated FA whale shark and poten-
tial prey profiles were separated groups, although we
would have been expected that these profiles were sim-
ilar, since there is feeding behavioral evidence in the
study area. This is possibly due to the constraints of the
technique itself and the lack of essential data to interpret
FA analysis correctly.
For elasmobranchs, many metabolic issues that
might influence the results are unknown, such as
the degree in which an elasmobranch modifies FA
from dietary before storing them (McMeans et al.
2012; Pethybridge et al. 2014). Additionally, it has
been reported that different elasmobranch species
might store different FA amounts and types depend-
ing on the tissue. It has been recorded that elasmo-
branch muscle tissue tend to reserve large quantities
of PUFA, in such a way that this tissue may be
adequate for elasmobranch trophic ecology studies
(Pethybridge et al. 2014). McMeans et al. (2012)
compared FA muscle, liver and blood (plasma) of
Greenland shark. They concluded that the best tissue
for studying shark diet is the plasma.
However, in this research we used subdermal tissue
because is the largest proportion of tissue obtained in
non-invasive biopsy sampling. Meyer et al. (2017) men-
tion that the sub-dermal tissue serves as a key structural
component, with a slower metabolic turnover rate than
muscle (assessed in relation to divergent isotopic
signatures by del Rio et al. 2009), but it can be reflecting
diets incorporated across different times. Couturier et al.
(2013b) compared FA profiles from whale sharks
(dermic tissue) and Manta alfredi (muscle tissue)
reporting a similarity between both profiles, which sug-
gests dermic tissue might be adequate to obtain dietary
information.
As mentioned before, whale shark is a highly
migratory species (Eckert and Stewart 2001;
Graham and Roberts 2007; Rowat et al. 2007;
Hueter et al. 2013) taking advantage of areas abun-
dant in zooplankton, sometimes moving great dis-
tances between plankton patches. Mexican Caribbe-
an organisms might be reflecting food consumed in
other aggregation sites. The connectivity among
known whale shark aggregation sites within Western
Central Atlantic Ocean has been reported on previ-
ous studies (Graham and Roberts 2007; Gifford
et al. 2007; McKinney et al. 2017). It would be
Environ Biol Fish (2018) 101:1599–1612
suggested to include samples collected from differ-
ent known aggregation sites of Caribbean in future
studies.
The knowledge about where whale sharks come
from or migrate to after they disperse is limited.
Hueter et al. (2013) characterize the horizontal move-
ments of whale sharks that come to the Mexican
Caribbean by conventional visual tags, photo-
identification and satellite tags. They report that in-
dividual sharks remained in the area for an estimated
mean duration of 24–33 days with a maximum resi-
dency up to about 6 months. Marcus et al. (2016)
mentioned that the FA profile technique would indi-
cate a diet from weeks or even months before, due to
the fibrous nature of R. typus subcutaneous tissue and
having low vascularity (therefore low metabolic ac-
tivity). Even Rohner et al. (2013), suggest that FA
profiles collected from whale sharks in Mozambique
might be reflecting preys assimilated by the organism
in oceanic waters, where they might be feeding on
deep organisms, with higher ARA levels. For future
studies, it would be recommendable collecting whale
shark tissue with slower metabolic turnover rate, such
as blood. The research group at Georgia Aquarium,
U.S., has developed a method to extract blood from
live sharks in captivity (Dove et al. 2013), which has
been tested on free organisms in the Caribbean. The
analysis of this sort of tissue might give useful infor-
mation to make inferences about trophic ecology of
the species when reflecting the recently assimilated
food.
Another limitation in the use of FA signature to
infer feeding habits of whale sharks in our study area,
is the lack of studies about FA profiles of tropical
zooplankton species which can be useful to compare
and identify possible prey of whale sharks. Research
about zooplankton FA profiles are mainly focused on
species distributed alongside the poles (Dalsgaard
et al. 2003). For future whale shark trophic ecology
studies applying FA analysis, it is recommended to
use vertical tow nets to collect samples along the
water column, being able to analyze demersal zoo-
plankton (or other potential prey) from the study
area; moreover, for comparative purposes, it would
be necessary to explore available information of oth-
er zooplanktivorous species whose knowledge of
their trophic spectrum is broader. It is also suggested
to increase the sample size, since this may rise the
analytic technique accuracy.
Environ Biol Fish (2018) 101:1599–1612
In conclusion, results of this study support the theory
that whale sharks are feeding mainly on surface zoo-
plankton in the Mexican Caribbean, although the possi-
bility that other sources of feeding may play additional
roles is not ruled out. The fact that results are not
consistent in relation to the whale shark feeding habits
in the different aggregation sites in the world, makes
evident the foraging plasticity of the species and their
ability to adapt to different conditions (e.g. heterogene-
ity of their habitat or diverse distribution of their prey
item). Even, when results obtained have not been con-
clusive on their own, they contribute to the knowledge
of the whale shark trophic ecology in one of the most
relevant sites for the species worldwide. In the western
central Atlantic Ocean, the whale shark population has
been estimated of 2167 sharks, and in our study area
1115 individuals have been identified (McKinney et al.
2017). The area attracts thousands of tourists per year
and researchers from different countries around the
world (Ziegler et al. 2015). The need for cooperation
between the different groups that research the species in
the area has become evident, to make more efficient
efforts and get information generated timely to be used
in the design and implementation of management and
conservation strategies of the whale shark.
Acknowledgements We would like to thank Santiago Capella
for his assistance with the design of this study and laboratory
techniques. Thanks to the personnel of the Chemistry Unit at Sisal
(Faculty of Chemistry, UNAM), and the Primary Production Lab-
oratory at CINVESTAV Merida for their help with analyses of
samples. We also thank the CONANP and PRONATURA Penin-
sula de Yucatan for logistical support; people who contributed to
field-work including tourist service providers and volunteers.
FGM thanks to Instituto Politecnico Nacional for fellowships
(EDI, COFAA). This research was funded by Consejo Nacional
de Ciencia y Tecnologia (FOMIX-Yucatán 10889) and the project
MEX-US (University of California-CONACYT): BDetermination
of movement, habitat use, filtration mechanics and diet/food pref-
erence of manta rays off the Yucatan peninsula^.
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