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RESEARCH ARTICLE
Individual Spawning Duration of Captive
Atlantic Bluefin Tuna (Thunnus thynnus)
Revealed by Mitochondrial DNA Analysis of
Eggs
Ana Gordoa
1
*, Nuria Sanz
2
, Jordi Viñas
2
1Department of Marine Ecology, Centro de Estudios Avanzados de Blanes, Spanish National Research
Council (CSIC), Blanes, Girona, Spain, 2Laboratori d'Ictiologia Genètica, Departament de Biologia, Facultat
de Ciències, Universitat de Girona, Girona, Spain
*gordoa@ceab.csic.es
Abstract
This study presents the first results on Atlantic bluefin tuna (Thunnus thynnus) individual
spawning duration and its short-term temporal behavior. The study was based on direct
measurements resulting from mtDNA analysis of the offspring of spawners held in transport
cages during the 2013 spawning monitoring survey in Balearic waters. The number of
females consisted of approximately 259 individuals with an average weight of 186 kg. The
survey began on May 22 and ended on July 3. Spawning started on May 30 and was
observed every night afterwards. The sampling of eggs for genetic monitoring was con-
ducted for 9 days interspersed from the beginning of spawning to the end of the survey. A
total of 946 eggs were analyzed and revealed 129 different haplotypes; 77 of these were
not previously detected in the Mediterranean. A total of 69 haplotypes were observed in
more than one spawning event and those with higher frequency lasted their maximum pos-
sible duration. The haplotypes present at the beginning of spawning were also identified at
the end of the sampling, indicating a minimum spawning duration of 34 days, and individual
annual fecundity was estimated at around 1290 eggs gr
-1
. These results differed from those
generally presumed until now and are indicative of a much higher fecundity. Females exhib-
ited a regular spawning schedule but with the capacity to shift the spawning hour during the
spawning season. These results were observed for the eastern population of Atlantic bluefin
tuna and before extrapolating to the western population, their validity should be proved.
Introduction
The Atlantic Bluefin tuna (Thunnus thynnus) is a highly migratory species, widely distributed
throughout the North Atlantic Ocean and the Mediterranean Sea. This species comprises two
different populations [1]: western and eastern Atlantic, which are managed separately by the
International Commission for the Conservation of Atlantic Tunas (ICCAT). These populations
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 1/13
OPEN ACCESS
Citation: Gordoa A, Sanz N, Viñas J (2015)
Individual Spawning Duration of Captive Atlantic
Bluefin Tuna (Thunnus thynnus) Revealed by
Mitochondrial DNA Analysis of Eggs. PLoS ONE 10
(8): e0136733. doi:10.1371/journal.pone.0136733
Editor: Hanping Wang, The Ohio State University,
UNITED STATES
Received: May 13, 2015
Accepted: August 8, 2015
Published: August 28, 2015
Copyright: © 2015 Gordoa et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: Sequences were
submitted to Genbank with accession numbers
KR337331-KR337459.
Funding: This study was funded by the Balfego Tuna
SL Group under a programme of scientific
cooperation with the Spanish National Research
Council and by theSpanish Ministry of the Economy
and Competitiveness (CTM2011-29525-C04-04). The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
differ greatly in size, the eastern is considered to be around 10 times the size of the western
[1,2]. The observed difference in population size may be increasing, particularly considering
the latest ICCAT stock assessments [3] where a clear recovery was only observed for the eastern
stock. The stocks share a wide range of feeding grounds [4], implying a wide spatial mixing
that makes management more difficult. In addition, the results from recent studies appear to
indicate that stock mixing might be higher than previously presumed [4,5], contributing to
increasing the observed uncertainty in the western stock assessment [3] or hindering its recov-
ery [6]. In spite of their spatial mixing, these populations are genetically distinct [7] as a result
of separate reproductive basins, Mediterranean (hereinafter, Med) and Gulf of Mexico (herein-
after, GOM) for the eastern and western stocks respectively [4,8].
The reproduction of Atlantic Bluefin Tuna (ABFT, here after) is characterized by serial
batch spawning and asynchronous oocyte development, distinctive of tunas [9]. The length of
the reproductive period of ABFT populations differs from other tunas by its shortness [9]. The
reproductive period varies between and within reproductive regions; from April to June in
GOM [4,10,11], and within the Mediterranean basin it varies in correlation with a progressive
east-to-west increase of the sea surface temperature throughout spring [12]. In the Western
Mediterranean ABFT spawning has been reported from June to July [13–19] and a month in
advance in the Levantine Sea [20].
The first studies on the reproductive biology of ABFT, traditionally approached by histolog-
ical analysis, were published eight decades ago [21], while the first estimation of ABFT relative
fecundity [17] was made four decades ago. The study of fecundity remains a key issue, as it is
an essential reproductive feature for stock assessment, allowing a better estimation of the stock
reproductive potential and of the intrinsic rate of population increase [22]. Although fecundity
is related to recruitment variation [23], recruitment is the net output of egg production (fecun-
dity) and offspring survival. Stock recruitment models are still widely used today despite their
limited or null explanatory capacity, and in the particular case of ABFT these models have
been proved highly unsatisfactory for both stocks [24]. However, they are still in use, but due
to their inefficiency different recruitment scenarios had to be adopted for both stocks and are
still used [3]. In 2009, and in order to provide scientific advice on the condition of ABFT, with
respect to the criteria applied for commercial exploited aquatic species under CITES, an alter-
native method of estimating ABFT productivity was developed [25]. This alternative, called the
potential rate of population increases, was first proposed by Jennings et al., [26] and expressed
by fecundity at the length at which 50% of the stock attains maturity (L50) and its correspond-
ing age (A50). The differences between ABFT stocks, in age at maturity and fecundity, resulted
in differences of their potential rate of population increase, which was found to be appreciably
higher for the eastern stock. However, fecundity comprised different specific terms, and when
used indistinctly might lead to flawed conclusions. The fecundity at L50 considered for ABFT
stocks [25] could be either the batch fecundity—total number of eggs spawned per batch [27]—
or total fecundity—number of advanced oocytes at any time in the ovary [28]—but neither of
these are indicative of annual fecundity. Thus, the apparent differences found between western
and eastern stocks in their potential rates of increase should be questioned until annual fecundi-
ties are estimated for each stock.
The annual fecundity is defined as the number of eggs released per female and year [28] and
should be estimated from the product of three parameters: the number of oocytes released per
spawning event (batch fecundity), the spawning frequency and the individual duration of the
spawning. The first two parameters for ABFT eastern stock are known and were estimated
from histological analysis; batch fecundity around 59 eggs g
-1
or 48 eggs g
-1
in the most recent
estimations, and with spawning periodicity of 1.2 days [29,30]. The above-mentioned spawn-
ing periods of ABFT, known for different reproductive areas, are inferred from the presence of
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 2/13
Competing Interests: This study has been partly
funded by a commercial company ("Balfego Tuna SL
Group") and this does not alter the authors’
adherence to all the PLOS ONE policies on sharing
data and materials.
spawners and larvae and from the results of histological analysis of gonad development. How-
ever, a key open issue is to ascertain the individual duration of spawning, the remaining
unknown parameter for estimating the annual fecundity. In recent years, major advances have
been made in tuna reproductive biology and also at an individual level, as a consequence of the
introduction of studies on cultured tuna and new technologies, such as electronic tagging and
genetics.
The genetic monitoring of captive spawning populations of tuna has been shown to be pos-
sible by analyzing the mitochondrial DNA (mtDNA) markers. Since the mtDNA control
region of the genus Thunnus is highly polymorphic [31,32], it displays enough variation to dis-
criminate among individuals [33]. These techniques have been successfully carried out in the
broodstock of different species of cultured tuna such as Pacific Bluefin tuna [33–36] or Yellow-
fin tuna [37], providing information about their spawning frequency and periodicity at individ-
ual level, key issues for identifying the more fertile females for culture. However, as ABFT
spawning was observed and defined quite recently [38], its culture is still in its early stages [39]
and the genetic tracking has not yet been studied for either offspring or broodstock. Thus, the
limited knowledge of ABFT individual spawning behavior comes from wild fish spawners
tracked by remote sensing and inferred from the duration of a particular swimming behavior;
first observed in GOM [11] and recently in the Mediterranean [40]. This inference is justified
because the particular swimming behavior coincides with the spawning hours observed for the
eastern population [41].
The direct observation of ABFT spawning in the natural system is a difficult task, and its
long-term monitoring was unfeasible until tuna transport cages were revealed to be effective
spawning observatories [38]. Their monitoring during four consecutive seasons provided rele-
vant information on spawning behavior [41] but the individual spawning temporal behavior is
still unresolved: neither the individual spawning extension nor the individual fidelity to spawn-
ing hours. The purpose of this investigation was to answer these questions by analyzing the var-
iability of the mtDNA CR sequence of eggs spawned by ABFT adults held and monitored in a
transport cage during the 2013 spawning season.
Materials and Methods
Each spawning season, since 2009, a group of ABFT spawners captive in the Balfegó fattening
facilities is transferred to a transport cage which is towed and transported to the Balearic ABFT
spawning region for research purposes (Fig 1). The transport of tuna groups under monitoring
required previous authorization from the Spanish Directorate-General for Fisheries. The study
was carried out offshore, where no specific permission is further required. The cage is moni-
tored from the middle of May to the beginning of July, covering most of the spawning season
in the western Mediterranean [41], when it is transported back to the farm. The objective of
this particular study, the mitochondrial DNA analysis of eggs, was included in the 2013 survey.
In 2013 the number of tuna transported was 563, with an average weight of 186 kg. The sur-
vey began on May 22
nd
and ended on July 3
rd
. Egg sampling followed the plankton protocol for
transport cages already tested successfully in earlier studies [38,41]. Samples were collected
using bongo nets fitted with 0.3 mm-mesh nets deployed behind the transport cages at a depth
of three metres. The towing speed of the transport vessels was constant at around 0.6 knots.
Sampling time (local time = UTC + 2 hours) was established within the spawning time interval
found for this species [41]. Three consecutive hours were sampled per night: 2:00–2:55 a.m.,
3:00–3:55 a.m. and 4:00–4:55. Upon retrieval of the gear, plankton samples were immediately
preserved in 5% buffered formalin. From each sample with eggs, a subsample of 2 ml of eggs
was separately preserved in absolute ethanol. Semi-quantitative measurements of the egg
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 3/13
volumes collected at each sampling station were estimated. The volume of eggs (in millilitres)
collected at each station was estimated after settling in 250-ml translucent jars. At several sta-
tions 250-ml jars were too small to hold all the eggs collected, so the spare volume of eggs was
measured before being returned to the sea or kept for hatching experiments.
The mitochondrial analysis of each spawning event was unaffordable and only eggs from a
determined subset of samples were analysed. The selection criteria of the samples were grounded
to reach the objectives set and based on the results of the daily spawning pattern (Fig 2). To
Fig 1. Map showing the sampling and farm locations. Primary location of the tuna transport cage during
the spawning monitoring (█) and the location of the fattening facilities from where the captive tuna was
transported every spawning season (▲).
doi:10.1371/journal.pone.0136733.g001
Fig 2. Temporal spawning pattern of ABFT monitored group. Volume (ml) of eggs collected per day and
time interval from Atlantic bluefin tuna caged group in 2013 spawning survey.
doi:10.1371/journal.pone.0136733.g002
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 4/13
achieve the first objective, the individual spawning duration, the first and the last observed
spawning events were selected and in between samples every 4/7 days were also analysed. To
determine if the spawning hour at the individual level was variable, three pair samples corre-
sponding to spawning events at different hours in the same day were selected. A total of 12
spawning events corresponding to 9 different days were analysed. The minimum number of eggs
analysed for each spawning event was 50 and doubled to 100 for events with higher spawning
intensity. The eggs were preserved in 96% absolute ethanol until analyzed.
To avoid cross-contamination between eggs in the same sampling tube, previous to DNA
extraction, all eggs were individualized and rinsed twice with 96% alcohol using 0.1 mm mesh.
The cleansed eggs were DNA extracted individually using DNA E.Z.N.A.Mollusc DNA Kit
(OMEGA Biotek, USA). Approximately 400 bp of the mtDNA control region was amplified
and subsequently sequenced as described in Viñas and Tudela [32]. The sequences obtained
were aligned, edited and checked for quality using the Geneious version 7.1.5 software (http://
www.geneious.com,[42]). Haplotype inference was carried out using DNAsp version 5.10 [43]
and haplotype diversity (h) was estimated in Arlequin version 3.5 [44]. Sequences were submit-
ted to Genbank with accession numbers KR337331-KR337459.
Results
A total of 946 eggs were analyzed. Of these, nine presented high sequence similarity with Thun-
nus alalunga and Thunnus orientalis, a consequence of the mtDNA introgression between
Thunnus species as has been reported previously [45], and so these sequences were removed
from the analysis. Then a total of 937 eggs were analyzed and revealed 129 haplotypes, and 53
of them were represented by one single egg. Haplotype diversity per sample (spawning event)
ranged from 0.697 to 0.971 with average haplotype diversity for all samples of 0.965 (Table 1).
It is important to point out the high percentage of haplotypes (60%) that have not previously
been detected in the Mediterranean, a total of 77, with some of them observed in more than 3
spawning events. As expected, some of the most common haplotypes in the Mediterranean
[45] were also present in our results (Table 2).
The number of total haplotypes per spawning event, indicative of the number of females
sampled in each spawning event, increased with the spawning intensity (Fig 3A) and a similar
pattern was observed with the number of unique haplotypes per sample (one single egg per
sample). Consequently, the relative frequency of unique haplotypes, which varies from 35% to
Table 1. Sampling and haplotipic details. Sampling dates and hours, number of analyzed eggs, number of haplotypes per sample, number of unique hap-
lotypes (present in one single egg) and haplotypic diversity.
Date Local time Analyzed eggs N° haplotypes N° unique Haplotypes Haplotypic diversity(SD)
30 May 3:00–4:00 66 11 5 0.798 (0.027)
1 Jun 2:00–3:00 60 8 4 0.697 (0.039)
1 Jun 3:00–4:00 66 6 0 0.801 (0.023)
6 Jun 3:00–4:00 109 39 18 0.946 (0.006)
14 Jun 3:00–4:00 107 30 16 0.933 (0.011)
18 Jun 3:00–4:00 113 41 17 0.967 (0.006)
23 Jun 3:00–4:00 58 18 8 0.909 (0.019)
23 Jun 4:00–5:00 49 14 5 0.901 (0.018)
27 Jun 3:00–4:00 51 20 10 0.922 (0.019)
27 Jun 4:00–5:00 109 31 16 0.939 (0.009)
30 Jun 4:00–5:00 56 23 13 0.930 (0.107)
3 Jul 4:00–5:00 93 42 21 0.971 (0.006)
doi:10.1371/journal.pone.0136733.t001
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 5/13
56% of the total, was independent of the spawning intensity (Fig 3B), excluding one single
event with total absence of unique haplotypes. Those frequencies are indicative of the represen-
tativeness of each sample to identify the active number of females at each spawning event: the
higher the percentage of unique haplotypes, the higher the sample’s representativeness.
The extent of the time interval at which each haplotype was observed could be considered as
a proxy of the spawning duration of each female if sampling is under optimal conditions. It
should be noted that the results of our estimations are highly limited by the length of the sam-
pled period, which constrains the maximum duration to 34 days. Additionally, the small sam-
ple size rules out the possibility of sampling the offspring of all female spawners, and there is
no likelihood of detecting all active females in each spawning event. The results showed that as
the frequency increased (number of events each haplotype was identified) the minimum values
of duration also increased, while the maximum duration was independent of the frequency
(Fig 4). The maximum possible duration, the 34 days of survey duration, was observed in sev-
eral haplotypes with sampling presence varying from 3 to 10. Individual haplotype duration is
also limited by the date each haplotype was first identified in our sampling: the later it was
identified, the shorter the estimations. However, considering the date of the first occurrence,
more than fifty percent of the haplotypes extended up to the last sampling day, so they exhibit
the potential maximum duration (Fig 4).
The annual individual fecundity was estimated on 1290 eggs gr
-1
resulting from the product
of batch fecundity and the number of batches during the individual spawning duration. The
number of batches were estimated by dividing the estimated individual spawning duration
(34 days) by the spawning interval, 1.2 days [29] and then multiplied by batch fecundity, 45,56
eggs gr
-1
[46].
Table 2. Time tracking of haplotypes with high frequency (>3 spawning events).
30 May
3–4
1 Jun
2–3
1 Jun
3–4
6 Jun
3–4
14 Jun
3–4
18 Jun
3–4
23 Jun
3–4
23 Jun
4–5
27 Jun
3–4
27 Jun
4–5
30 Jun
4–5
3 Jul
4–5
Hap_9
b
x xxxxx xxxx
Hap_1 xx xxxxx x x
Hap_32
b
x xxxx xxxx
Hap_4 x xxx xxxxx
Hap_31
b
xxxx xxxx
Hap_3
b
xx xxx x
Hap_50
b
xxxxx x
Hap_23
a
xxx xx
Hap_27 xx xxx
Hap_47
a
xx x x x
Hap_58 xxxx x
Hap_6 xx x x x
Hap_7 xxxxx
Hap_12 xx xx
Hap_14 xxx x
Hap_17 xx xx
Hap_2
a
xxx x
Hap_39 xx xx
Hap_57
a
xx xx
Hap_90 xxxx
a
haplotypes not detected in the Mediterranean population.
b
haplotypes most frequent (>3%) in the Mediterranean
doi:10.1371/journal.pone.0136733.t002
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 6/13
The results on the spawning hours shown in Fig 2 revealed that the spawning interval was
quite narrow and did not vary randomly between days. The spawning was tuned or synchro-
nized between 3:00–4:00 during the first 3 weeks of June, but minor spawning pulses were
observed from 2:00 to 3:00 during three days. Throughout the third week of June the spawning
intensity decreased and it took place during two consecutive hours, from 3:00 to 4:00 and from
4:00 to 5:00. The results over the last week showed an important upturn in spawning and the
spawning hour was highly synchronized and restricted from 4:00 to 5:00. At the individual
level, the results of mtDNA of eggs spawned at different hours showed the same pattern
(Table 2). At the beginning of spawning, the first two days, the same females spawned indis-
tinctly at 3:00–4:00 or at 2:00–3:00, then converging rapidly to the second hour until the third
week. In the third week the females shifted their spawning towards the third spawning hour,
4:00–5:00. It is notable that the day before they definitively shifted to the later hour some
females had two spawning pulses at hours, the old and the new one.
Discussion
This study presents the first results on ABFT individual spawning duration and temporal
behavior, based on direct measurements resulting from mtDNA analysis of the offspring of
Fig 3. Haplotypes descriptors. Total haplotypes (N° H) and unique haplotypes (N° UH) as a function of
spawning intensity (A) Frequency. (B) Proportion of.
doi:10.1371/journal.pone.0136733.g003
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 7/13
spawners held in transport cages. This study reveals once again the capability of tuna transport
cages as monitoring observatories and experimental platforms [38,41]. The results are dis-
cussed and contrasted with those previously inferred from indirect approaches [11,40]. How-
ever, the discussion of these results requires a preliminary overview concerning their
representativeness.
The size of the captive tuna was substantially higher than in studies of a similar nature
[33,36,37] and therefore also the potential number of released eggs. The number of captive
females was around 259, 46% of the captive group, according to the sex ratio reported for this
region, period and size of tuna [29,47]. In the Balearic waters, 84% of the female spawners’pop-
ulation contributes to the daily spawning [29], which in this study represent 217 females. The
potential numbers of eggs released daily per female result from the product of its individual
weight and the batch fecundity. The average weight of captive tuna was around 186 kg and the
batch fecundity for the ABFT eastern population is 45.56 eggs gr
-1
, in accordance with the latest
and most conservative figure [46]. Consequently, the daily egg production of each captive
female will be around 8.5 million eggs and the potential daily egg production of the whole cap-
tive group close to 2000 million. Thus, the fraction taken for genetic analysis represented an
infinitesimal fraction of the total number of eggs potentially produced at each spawning event.
These figures are essential to frame the inference of our results and are also essential in any
study of a similar nature. On the other hand, the high haplotype diversity of ABFT in the Medi-
terranean (above h= 0.992, [45,48]) and the low probability (p = 0.008) of sampling in the wild
population two individuals with the same haplotype [45,48] simplify the interpretation of the
results. Therefore, eggs with the same haplotype could be considered spawned by the same
female parent. This was also supported by the low levels of haplotype diversity in our samples,
result of sampling siblings and consequently significantly lower (p= 0.009) than the average
haplotype diversity found in the Mediterranean (above 0.991; [45,48,49]). In addition, there
Fig 4. Duration of haplotypes as a function their spawning event frequency; at the sides the
histograms of observed frequencies of spawning duration and spawning frequency.
doi:10.1371/journal.pone.0136733.g004
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 8/13
was a notably high number of new haplotypes found in this study, indicating that we are still in
the early stages of analysis of ABFT haplotypes.
A total of 129 different haplotypes were identified in this study and they are indicative of
the minimum number of active spawning females, c.a. 50% of the total captive females. Thus,
despite the limitations of the sample size, the proportion of identified females was substantial,
suggesting that most females could be active spawners. Furthermore, the proportion of females
identified from their offspring was similar to those reported for other tuna species in similar
studies [33,37]. However, at each spawning event the representativeness of the analyzed sample
was low; the maximum number of haplotypes identified in one spawning event (42) scarcely
represented the 16% of the total captive females, but this flaw did not prevent achieving the
purposes outlined in this study. The specific goal line was to probe deeper into the individual
reproductive parameters and temporal behavior, and particularly into the determination of
individual spawning duration and spawning hour. These specific objectives required collecting
eggs from the same females in different spawning events and this was achieved. The proportion
of females identified in more than one spawning event was 53%, and 20 females could be iden-
tified in more than 3 events. The results showed that all the females with high frequency of
occurrence (>3 spawning events) displayed similar temporal patterns, which made it possible
to attain the predefined objectives.
The results showed that most females displayed their maximum feasible duration: generally
they were present until the end of the sampling, but the individual durations were constrained
by the first date of appearance of each haplotype. It is also worth mentioning that out of the
total of the 20 females detected at the beginning of the spawning, May 30th–June 1st, 35% were
also detected on the very last day of sampling (July 3rd). These results indicate that the individ-
ual spawning duration can last over a minimum of 34 days. This estimation is limited by the
end of the sampling, before the end of spawning, so the individual spawning duration might be
longer, but also variable, because the lengthening of individual spawning can vary with age,
longer as they get older, as observed in different species of multiple-batch spawners [50–53].
Thus, we cannot rule out this possibility in ABFT, especially considering that larger tuna seem
to arrive and to leave the spawning grounds earlier and according to length frequency from
Spanish traps [54]. Nonetheless, the results clearly showed that the individual spawning period
is longer than the ones might be inferred from the swimming behavior of wild spawners. Dif-
ferences in the reproductive potential between wild and cultured tuna may exist despite the
similarity observed in their daily spawning behavior [41].
Electronic tags have been proved essential for studying ABFT spatial distribution and
behavior as a particular nighttime diving behavior, which was reported solely during the breed-
ing season and in the spawning grounds, and consequently linked to spawning behavior. This
particular behavior was first observed in the western population during the spawning season in
the GOM: the duration was first estimated in two weeks [55] and later around 17 ± 7d [11].
Recently, in 2012, a similar behavior has been observed in the Balearic spawning ground, with
an average duration of 23.9 ± 4.1 d [40] with maximum around 31 d, which is comparable to
the spawning duration observed in this study. Nevertheless, it is likely that both figures are
underestimating the real spawning duration because the electronic tags were released after the
onset of spawning, at least 10 days after the beginning of spawning was observed in 2012 [41],
and in the present study the sampling ended before the spawning ceased. The diving behavior
was associated with ABFT spawning because it was only observed in the spawning grounds,
and at least for the eastern stock additional studies corroborates the hypothesis: in the Balearic
spawning grounds, where the spawning hour interval has been clearly defined [41] and
comprises the hours on which the particular swimming was observed [40]. Thus, the western
and eastern differences in diving duration can be actual differences in their reproductive
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 9/13
parameters. Another difference between western and eastern diving behavior, according to the
data published [11,40], is the amplitude of the spawning hour interval; up to ten hours in the
GOM and a maximum of three hours in Balearic waters.
Although the studies on the reproductive behavior of the eastern population revealed that
the diving behavior [40] and the spawning [41] match in the hour interval, it should be empha-
sized that they differ in duration. While, according to published information [40], diving lasted
90 min on average, the spawning events take place in less than 15 min [41]. It has been hypoth-
esized that this diving may play an important thermoregulatory role during spawning and
associated with courtship behavior [40]. This is consistent with the courtship swimming behav-
ior observed in captive yellowfin tuna: the duration was similar (1 to 4 h) and was prior to
actual spawning, which took place in less than 90 sec. [56]. Thus, as courtship swimming
behavior it would take place before the spawning but as a thermoregulatory mechanisms
would be more necessary after the spawning. More research is needed to determine the func-
tionality of this swimming behavior as well as to explore if it is gender-related. In a recent com-
parative study of reproductive status between the two populations [46], the authors found
differences in batch fecundity, 62% higher in the eastern (45.56 eggs. g
-1
) than in the western
(28.14 eggs. g
-1
), but the difference could not prove statistically significant. Similarly, these
authors did not find differences in the spawning frequencies between populations. The differ-
ence in the reproductive parameters of the two populations are continuously under debate, and
in particular the age at maturity, an important parameter estimated around 3–4 y for the east-
ern [57,58] and 8–14 for the western [59,60]. However, in a recent study it has been suggested
that the western population matures at a much younger age, similar to that one considered for
the eastern [61].
The results on the spawning time of the captive group corroborate the hour interval (2:00–
5:00) found in previous studies [38,41]. At the individual level each female had the potential to
spawn at different hours but not haphazardly through the spawning period. Before the 23
rd
June, spawning was synchronized from 3:00 to 4:00 and after the 27
th
from 4:00 to 5:00, and
some females could be tracked on both periods. Of high interest is the transition period,
between the 23
rd
and 27
th
of June, when females could spawn at both hour intervals. This is
indicative that each female had the capability to spawn in two separated pulses, and they appar-
ently did so when they changed the spawning hour. The three-period spawning pattern was
previously observed, but the possibility that the same females were contributing to the spawn-
ing for so many weeks was disregarded [41]. Furthermore, the shift in spawning hour was used
to infer the individual spawning duration, c.a. 21 days, but this proved to be wrong according
to the results presented in this study.
This study, the first genetic monitoring of ABFT offspring, has provided the first approxi-
mation of individual spawning duration for ABFT eastern population, the missing parameter
for estimating the individual annual fecundity of this population, c.a. 1289 egg.g
-1
. There are
no bases for extrapolating the results presented here to the western stock: in fact the differences
in spawning-swimming duration found in other studies indicated the contrary. In addition to
molecular techniques, further studies in electronic tagging will help to determine individual
spawning duration for the western population and intra and inter variability of the two
populations.
Acknowledgments
The authors wish to thank Balfegó team for its continuous support and the crews of the trans-
port vessels “Estela Nova”and “Coyote”for their help with sample collection; to Alex Fonollosa
Spawning Duration of Atlantic Bluefin Tuna by mtDNA Analysis of Eggs
PLOS ONE | DOI:10.1371/journal.pone.0136733 August 28, 2015 10 / 13
and Marc Clos, Luis Peñarubia and Judith Oller for their excellent field and laboratory work;
and Paul Martin for revising the original English of the manuscript.
Author Contributions
Conceived and designed the experiments: AG. Performed the experiments: AG. Analyzed the
data: JV NS. Contributed reagents/materials/analysis tools: AG NS JV. Wrote the paper: AG
NS JV.
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