Otolith geochemistry reflects life histories of Pacific bluefin tuna.

Understanding biological and environmental factors that influence movement behaviors and population connectivity of highly migratory fishes is essential for cooperative international management and conservation of exploited populations, like bluefin tuna. Pacific bluefin tuna Thunnus orientalis (PBT) spawn in the western Pacific Ocean and then juveniles disperse to foraging grounds across the North Pacific. Several techniques have been used to characterize the distribution and movement of PBT, but few methods can provide complete records across ontogeny from larvae to adult in individual fish. Here, otolith biominerals of large PBT collected from the western, eastern, and south Pacific Ocean, were analyzed for a suite of trace elements across calcified/proteinaceous growth zones to investigate patterns across ontogeny. Three element:Ca ratios, Li:Ca, Mg:Ca, and Mn:Ca displayed enrichment in the otolith core, then decreased to low stable levels after age 1-2 years. Thermal and metabolic physiologies, common diets, or ambient water chemistry likely influenced otolith crystallization, protein content, and elemental incorporation in early life. Although similar patterns were also exhibited for otolith Sr:Ca, Ba:Ca and Zn:Ca in the first year, variability in these elements differed significantly after age-2 and in the otolith edges by capture region, suggesting ocean-specific environmental factors or growth-related physiologies affected otolith mineralization across ontogeny.

Introduction

Characterizing the complete life histories of highly migratory species is challenging due to their long-distance movements through remote habitats and difficulties in observing them. Knowledge of migration routes and population connectivity is important for effective (spatially explicit) management and conservation [1], especially for species that undertake transoceanic movements and cross international boundaries, where they are susceptible to fishery exploitation by multiple nations [2, 3]. A prime example is the Pacific bluefin tuna (Thunnus orientalis). Pacific bluefin tuna (PBT) exhibit a suite of adaptations enabling fast swimming speeds, and expansive migrations that connect disparate oceanic ecosystems across hemispheres.

Methods such as electronic and conventional tagging and analysis of catch data have enhanced our knowledge of life-stage specific behaviors of PBT [4, 5]. Documented spawning of PBT occurs only in the western Pacific Ocean (WPO) [4] around the Philippines and East China Sea in April-June and in the Sea of Japan in July-August [6, 7]. PBT spawning in the East China Sea are 8+ years old, while those spawning in the Sea of Japan are typically age 3–6 years [7, 8]. Larval PBT spawned in the East China Sea and near the Philippines are then transported northward by the Kuroshio Current to utilize coastal areas as nurseries along the southern coast of Japan [9, 10] and in the Sea of Japan [11]. Fish spawned in the Sea of Japan may remain in local waters seeking preferred temperatures ranging from 23 to 26°C [11]. Juvenile PBT (age 0–2) then seek favorable thermal habitats with abundant prey in the vicinity of the Kuroshio Current [12]. Some one to two year old PBT undergo transoceanic migrations to the eastern Pacific Ocean (EPO), with migration journeys ranging from 1.2 to 5.5 months and departure timings dependent on nursery foraging areas [6, 12]. PBT remain in the EPO for several years (ranging from 3 to 9) before returning to the WPO [13].

In addition to the EPO, historical catch records indicate some portion of the population also migrates across the equator into tropical south Pacific waters. For example, large PBT have been caught in long line and recreational fisheries of New Zealand [14, 15]. While previous tagging studies provide useful information on movements of PBT to the EPO and the SPO, these techniques are limited to fish behavior post-tagging, and do not provide birth-to-capture life histories. In contrast, natural tags such as the molecular and elemental compositions of fish tissues and hard parts, can offer unique opportunities to reconstruct more complete life histories.

Natural tags have been applied to investigate migration dynamics in PBT. Some studies examine the isotopic composition of muscle tissue including nitrogen stable isotopes [16, 17] and Fukushima-derived radionuclides [18]. As a metabolically active tissue, isotopes in muscle provide a time-window that is equal to the turnover rate [19]. For PBT, this time window is approximately a year, which limits the scale of question that can be addressed.

In comparison, calcified structures such as fish otoliths (ear stones) form continuously by radial growth and are metabolically inert once formed, thus otolith core-to-edge chemical compositions span entire life histories of specimens. Otoliths grow by accretion of calcium carbonate crystals on a protein matrix, within endolymph fluid that is influenced by blood chemistry [20-22]. Daily physiological processes can affect aspects of blood chemistry (e.g. pH, bicarbonate, protein content) [20, 23]. Environmental conditions such as temperature, salinity, and dissolved oxygen [24] can also influence blood chemistry, and consequently some elements incorporated within the calcium carbonate structure are useful environmental proxies. There is high uncertainty on the relative influence of intrinsic and extrinsic processes that affect element incorporation into fish otolith and responses are species specific [24]. Few studies have yet examined the influence of endothermic physiology on crystal formation biochemistry in pelagic fish species, due to the difficulty of conducting controlled experiments [25]. Bluefin tuna are regional endotherms that can elevate the temperature of their eyes and brain [26], therefore thermal physiology may influence otolith biomineral element incorporation and confound interpretation of fish movement.

To the extent that PBT move through geochemically distinct water masses and physiological influences on biomineralization can be accounted for, PBT otolith geochemical records could allow for reconstruction of migratory movements and identification of migratory contingents. Previous studies utilized otolith chemistry to characterize signatures of young-of-the-year PBT collected on spawning grounds [27, 28], to determine natal origins of PBT that migrated to the EPO [29], to document discrete profiles of juveniles collected in the EPO [30], and assess the timing of juvenile emigration from the WPO [31]. However, studies documenting continuous life histories for large PBF otoliths collected over wide areas of the north and south Pacific are needed to better understand influences of physiology and water mass environmental conditions on otolith chemical time series.

Here, otoliths of large PBT collected from the WPO, EPO and SPO were analyzed using laser-ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to document chemical chronologies from juvenile to adult life in sequentially deposited growth bands. By comparing complete elemental profiles of PBT sourced from different ocean basins, this study explores if 1) certain elements reflect ocean basin-scale migratory movements or regional residence and 2) how physiology, the environment and in situ water chemistries influence the element patterns across ontogeny in PBT otoliths.

Materials and methods

Otolith collection

PBT otoliths (n = 25) were collected from fishery-dependent sources in three regions of the Pacific Ocean: the eastern Pacific Ocean (EPO, n = 10), the western Pacific Ocean (WPO, n = 10) and south Pacific Ocean (SPO, n = 5) (Fig 1, Table 1).

Fig 1

Geographical map of opportunistic collected PBT otoliths samples from the eastern Pacific (EPO), western Pacific (WPO) and south Pacific (SPO); different colored ellipses represent approximate collection regions.

Table 1

Pacific bluefin tuna (PBT) metadata including date, ocean region of collection region, local region of collection; sex, gilled and gutted (GG) weight and fork length (FL) reported where available.

Estimated ages were derived from length-age relationships reported in [32]. Laser distance measured as in Fig 2B.

PBT_ID	Date PBT collected	Ocean PBT collected	Local region PBT collected	Sex	FL (cm)	Estimated age (yr)	GG weight (kg)	Laser distance (μm)
EP01	9/20/17	East Pacific	San Deigo, Califronia USA	M	197	9	156	3086
EP02	7/29/17	East Pacific	San Deigo, Califronia USA		182	7+	167	3165
EP03	7/20/17	East Pacific	San Deigo, Califronia USA		179	7		3258
EP04	8/8/16	East Pacific	San Deigo, Califronia USA		180	7		2818
EP05	8/8/16	East Pacific	San Deigo, Califronia USA		180	7		3310
EP06	10/30/17	East Pacific	San Deigo, Califronia USA		178	7	103	3284
EP07	7/29/16	East Pacific	San Deigo, Califronia USA	F	181	7+		2929
EP08	7/19/17	East Pacific	San Deigo, Califronia USA		173	6+		3127
EP09	7/7/17	East Pacific	San Deigo, Califronia USA		173	6+		2957
EP10	8/20/16	East Pacific	San Deigo, Califronia USA		174	7		2726
WP01	5/12/17	West Pacific	Nansei Islands	F	225	13	219	3342
WP02	5/5/17	West Pacific	Nansei Islands	F	222	12	205	3355
WP03	5/8/17	West Pacific	Nansei Islands	F	210	10	167	3345
WP04	5/7/17	West Pacific	Nansei Islands	F	208	10	160	2903
WP05	5/4/17	West Pacific	Nansei Islands	F	206	10		3083
WP06	5/11/17	West Pacific	Nansei Islands	M	222	12	214	3425
WP07	5/10/17	West Pacific	Nansei Islands	M	199	9	156	3145
WP08	5/12/17	West Pacific	Nansei Islands	F	223	13	241	3748
WP09	5/12/17	West Pacific	Nansei Islands		203	9	154	3230
WP10	5/10/17	West Pacific	Nansei Islands	F	212	10+	156	3439
NZ01	8/22/07	South Pacific	New Zealand	F	245	22	180	4187
NZ02	5/6/13	South Pacific	New Zealand	F	168	6	100	3212
SP01	3/31/18	South Pacific	Cook Islands	M	265	26+		4052
SP02	8/15/17	South Pacific	Cook Islands	M	228	14	255	3599
SP03	8/23/18	South Pacific	Cook Islands	F	253	26+		4135

Fig 2

Schematic of Pacific bluefin tuna otolith preparation for elemental analysis.

Otoliths of PBT were sectioned in the transverse plane (a, red shaded box) and the ~1 mm section was polished to expose the core; a laser scan (b, red dashed line) starting from the early life core region was scanned across the otolith growth bands encompassing the complete life history, with the laser path turning at the inflection point; the final 100 μm of data (small yellow box) was averaged to represent the recent otolith material deposited in the region of collection.

As some samples were collected opportunistically, it was not always possible to obtain full metadata for each tuna, as sometimes only the fish head was available. Region of collection (EPO, WPO, SPO) was used to compare elemental patterns. Where possible, the fork length (FL cm), weight (kg), sex and capture location were recorded. For EPO samples where only heads were available, the operculum length (OL cm) was used to estimate FL using the equation: FL = OL*3.802–13.794; r2 = 0.94 [33]. Following collection, dried, tissue-free otoliths were stored in labeled plastic vials. Whole sagittal otoliths were embedded in a clear epoxy resin EpoFix (Stuers) so that the distal lobe could be used to identify the location of the core. The resin was spiked with 30 ppm indium (115In) during mixing to serve as an internal elemental marker of the epoxy. Embedded otoliths were sectioned using a low-speed diamond blade saw to obtain a ~1 mm thick central section from the transverse plane (perpendicular to the longest otolith axis, Fig 2).

Central otolith sections were mounted on a petrographic slide using thermoplastic (Crystalbond™) adhesive, with the distal lobe facing up, then surface polished using 600–1200 grit silicone-carbide paper (Buehler) and ultrapure water until the distal lobe became transparent and the sulcus groove formed a sharp narrow ‘V’, indicating the core was reached (Fig 2). Final polished sections were remounted on new petrographic slides, such that each slide contained several closely spaced otoliths arranged in a random sequence. The slides were scanned at high-resolution to assist with placement of laser transects.

Elemental measurements and data analysis

Elemental concentrations were measured using a New Wave 193 nm laser coupled to an 7500ce Agilent inductively coupled plasma mass spectrometer at the University of Texas at Austin. All samples and standards were loaded into a large format cell with fast washout times (< 1 s). All laser scans began in the otolith core and moved outwards along the longest growth axis (Fig 2). Test scans revealed optimal ion counts using gas flows of 850 mL min-1 for Ar and 800 mL min-1 for He. Otolith sections and standards were pre-ablated to remove any surface contamination using a 75μm spot, 50μm s-1 scan rate, 20 Hz repetition rate and 40% power. Data acquisition parameters were 35% power, 20 Hz with a 50 μm spot moving at 5μm s-1. Laser fluence during analysis averaged 2.12 ± 0.02 J/cm2. The quadrupole time-resolved method measured 13 masses using integration times of 10 ms (24Mg, 43-44Ca, 88Sr, 115In), 20 ms (25Mg, 55Mn), and 50 ms (7Li, 59Co, 63Cu, 66Zn, 137-138Ba). Time-resolved intensities were converted to concentration (ppm) equivalents using Iolite software (Univ. Melbourne, [34]), with 43Ca as the internal standard and a Ca index value of 38.3 weight %. Baselines were determined from 30-s gas blank intervals measured while the laser was off, and all masses were scanned by the quadrupole. USGS MACS-3 was used as the primary reference standard and accuracy and precision were proxied from replicates of NIST 612 analyzed as an unknown. NIST 612 analyte recoveries were typically within 2% of GeoREM preferred values (http://georem.mpch-mainz.gwdg.de).

Concentration data (ppm) were converted to molar ratios to facilitate comparisons with previous studies. To remove high frequency noise, time-series were smoothed by sequential application of 7-point moving median and 7-point moving average filters. In order to remove edge effects associated with intersection of the laser with epoxy resin, otolith edges were defined at crossover points where 43Ca and 115In counts-per-second (CPS) were < 200,000 and > 1000 CPS, respectively. Using these criteria, there were some instances when Mg:Ca and Li:Ca increased shortly before the edge, which was likely due to thermoplastic Crystalbond™ cement penetrating the intersection of the otolith-epoxy edge. One otolith (NZ02) clearly had translucent Crystalbond™ covering the surface that was confirmed after analysis, which affects the Mg:Ca values. Therefore, Mg:Ca was not plotted for this specimen (see S1 Fig). Approximate core-to-edge distances of mean annuli distances along the laser path (see Fig 2 in [35]) were measured in Image J and superimposed on elemental time-series plots to discern how elemental patterns generally relate to age. The mean annuli distances were 1,223 μm in year one, 1,577 μm in year two, 2,054 μm for year 3+, as measured from the otolith core and along the laser path.

To document regional spatial variation in elemental signatures at times of capture, the molar element:Ca ratios at the edge of the otoliths were compared among EPO, WPO and SPO collection regions. For each otolith, the final 100 μm of the otolith edge was averaged to represent the recent otolith material accreted in the region of collection, which represents different time frames for individual fish. The otolith edge data was then inspected for outliers using a Grubbs test, with identified outliers removed from calculated averages and subsequent statistical analysis. Normality was assessed using a Kologorov-Smirnov test and only Mg:Ca was not normal distributed, and thus a log transform was used to meet normality assumption for Mg:Ca. A Brown-Forsythe test confirmed that the standard deviations were not significantly different among regions and thus the parametric ANOVA was appropriate, using PRISM 8 statistical program. To test if elemental differences were present among each region, a one-way analysis of variance (ANOVA) test was performed for followed by a Holm-Sidak multiple comparison test. For element:Ca ratios that were significantly different among regions, quadratic discriminant function analysis (QDFA) was used to determine whether PBT from each region could be chemically identified using a jackknifed classification matrix using SYSTAT 13. Due to low sample size from the SPO (N = 5), the number of elements used in the QDFA had to be limited to three. The first and second canonical scores were then plotted to visualize separations among regions.

Results

Study otoliths were collected from 25 PBT: 10 from EPO, 10 from WPO and 5 from SPO (Fig 1, Table 1). EPO and WPO collections were from 2016 and 2017, whereas SPO collection spanned from 2007 to 2018. Estimated specimen ages (based on length from Shimose et al. 2009) ranged from 6 to 26 year with most young fish coming from the EPO and one age 6 specimen from the SPO (Table 1).

Mean element:Ca profiles

Element:Ca profiles are remarkably similar among PBT otoliths in the general trend over the transect (Fig 3). Element:Ca data are grouped by region, but it is possible that PBT migrated across different regions throughout life. Li:Ca, Mg:Ca and Mn:Ca are elevated in core regions, then decrease to low values following the first (estimated) annuli position. Some specimens with peak Li:Ca and Mg:Ca near the otolith edge were removed as outliers (as discussed below). Highest Mn:Ca values occur in cores, with EPO slightly higher than WPO and SPO, but all specimens show a secondary peak between 800 to 1000 μm that rapidly decreases to near detection limits after ~age two (past 1500 μm). Note that all fish are assumed in the WPO spawning ground during the period that corresponds to the core. In contrast to Mn:Ca patterns, Zn:Ca, Sr:Ca and Ba:Ca are low in cores then show increasing trends after the second annuli that are differentiable by collection region (Fig 3). Zn:Ca gradually increase from core regions and plateau up to ages 1 to 3+, but increase again after 3000 μm. Sr:Ca show a small increase in the core that levels out through age-0 then further increases to 1800 μm at approximately age-3, before further increasing by region; continual increasing values characterize EPO and SPO, whereas lower stable values differentiate WPO. Ba:Ca diverges among capture regions after approximate age-3, with sharp, moderate, and much more gradual increases distinguishing the EPO, SPO, and WPO, respectively. In contrast to the other element:Ca ratios, otolith Ba:Ca patterns are highly oscillatory with high variability among individuals (S1 Fig), possibly reflecting differential movement among ocean regions prior to capture. Compared to EPO and SPO, oscillatory otolith Ba:Ca profiles have much lower amplitudes for WPO.

Fig 3

Mean element:Ca profiles for otoliths of Pacific bluefin tuna.

(see Table 1 for fish collection details) collected from the eastern Pacific (EPO = blue, N = 10), western Pacific (WPO = red, N = 10), and south Pacific (SPO = green, N = 5) Ocean; solid lines represent mean values and shading depicts ± standard deviation across laser distance from core (0 μm) to edge; dashed vertical lines denote approximate annuli distances derived from [35].

Mean otolith edge patterns

The Grubbs test revealed 3 outliers (Li:Ca = 7.53 for EP05; Mg:Ca = 0.563 for NZ02, Mn:Ca = 1.66 for EP05) that were removed before the 1-way ANOVA test. Li:Ca, Mg:Ca, and Mn:Ca are not significantly different among regions, while Zn:Ca, Sr:Ca, and Ba:Ca are significantly different (Table 2; Fig 4). Holm-Sidak’s multiple comparison tests indicate that otolith edge Zn:Ca was significantly higher in the SPO compared to the EPO (p = 0.02) and WPO (p = 0.04), but is not different between EPO and WPO (p = 0.52). Otolith edge Sr:Ca is significantly lower in the WPO versus EPO (p = 0.01) and SPO (p<0.001), but not different between the EPO and SPO (p = 0.07). The EPO has the highest otolith edge Ba:Ca, with significantly lower values in the WPO (p = 0.002), but EPO and SPO are not significantly different (p = 0.19) (Fig 4).

Table 2

One-way ANOVA results based on mean otolith edge (outer 100 μm) element:Ca values for Pacific bluefin tuna for exploring regional differences in otolith chemistry among the eastern Pacific (EPO), western Pacific (WPO) and south Pacific (SPO).

Only Mg:Ca was log transformed to meet normality assumption.

Element	Factor	SS	DF	MS	F	P value
Li:Ca	Region	1.38	2	0.6902	1.32	0.2883
	Residual	10.98	21	0.5228
	Total	12.36	23
log(Mg:Ca)	Region	0.3879	2	0.194	3.335	0.0552
	Residual	1.221	21	0.05816
	Total	1.609	23
Mn:Ca	Region	0.2734	2	0.1367	1.119	0.3452
	Residual	2.564	21	0.1221
	Total	2.838	23
Zn:Ca	Region	408.6	2	204.3	4.642	0.0208
	Residual	968.2	22	44.01
	Total	1377	24
Sr:Ca	Region	1.333	2	0.6664	10.73	0.0006
	Residual	1.366	22	0.0621
	Total	2.699	24
Ba:Ca	Region	176	2	87.99	8.085	0.0023
	Residual	239.4	22	10.88
	Total	415.4	24

Fig 4

Boxplots of otolith edge values (mean of final 100 μm) for each region.

EPO = blue, WPO = red, SPO = green. One-way ANOVA significant differences (see Table 1) among regions were further tested with a Holm-Sidak multiple comparison test with lower case letters indicating regions that are significantly different; regions that were not significantly different share the same lower case letter.

QDFA based on only Zn:Ca, Sr:Ca and Ba:Ca resulted in overall jackknifed classification success of 72% (Table 3). Classification success varies by each region, with 70% accuracy in the EPO with 1 misclassification occurring in the SPO and 2 in the WPO. The lowest classification success of 40% occurred in the SPO, with 3 misclassifications attributed to the EPO. WPO has the highest classification success of 90%, with only 1 misclassification in the EPO (Table 3). The regional classification differences were clear in the plot of the first and second discriminant scores (Fig 5). Two SPO fish clearly grouped together and were separated from the EPO and WPO, while 3 SPO fish more closely grouped to the EPO (Fig 5).

Table 3

Jackknifed classification success to the three regions (EPO, WPO, SPO) derived from quadratic discriminant function analysis based on otolith edge element:Ca values of Pacific bluefin tuna.

Known region of capture	Predicted region of capture			% correct classification
	EPO	SPO	WPO
EPO	7	1	2	70
SPO	3	2	0	40
WPO	1	0	9	90
Total	11	3	11	72

Fig 5

Quadratic discriminant function analysis canonical score plot.

EPO = blue circle, WPO = red square, SPO = green triangle. The overall classification accuracy using Pacific bluefin tuna otolith edge Zn:Ca, Sr:Ca and Ba:Ca was 72%.

Discussion

Reconstructing the life histories of migratory fishes requires tools that record endogenous and exogenous events experienced throughout ontogeny. This is the first study to investigate otoliths of PBT collected from the SPO, a poorly understood migratory path where samples have rarely been collected or investigated. The element:Ca profiles of PBT otoliths collected from three regions (WPO, EPO, SPO) are similar during early life from age-0 to age-1 for all element:Ca ratios examined. This consistency suggests either 1) similar water mass occupancy during early life, 2) a strong physiological control on element incorporation in early life independent of ambient water concentration [23, 36], or 3) a combination of both. This result is expected given that all fish originate from the WPO, however it is not possible to differentiate between the influence of regional water chemistry and physiological control. Wells et al. [29] found statistically significant differences in otolith core Mn:Ca, Mg:Ca, Sr:Ca and Zn:Ca between YOY fish from the East China Sea and Sea of Japan, suggesting at least these elements are influenced by regional conditions that vary interannually.

As juveniles, after the estimated age of 3, trends of three element:Ca profiles (Zn:Ca, Sr:Ca, and Ba:Ca) diverge by region of collection suggesting that environmental factors that vary in time and space (e.g., temperature and salinity) influence incorporation of those elements [24]. Thus, otolith element:Ca patterns characterized in this study align well with our current understanding of PBT life history, that includes a common spawning ground in the WPO with juveniles either remaining resident in the WPO or migrating to the EPO [4, 16, 17, 37] or larger fish moving into the SPO [14, 15].

Otolith geochemical records provide useful life history information for pelagic tuna species including natal origin determination [29, 38, 39], stock structure identification [38, 40, 41] and migrations with life history transects [30, 42–45]. Ontogenetic otolith chemistry patterns revealed by LA-ICP-MS core-to-edge transects in this study are similar to those derived from LA-ICP-MS discrete spot analyses [30] on PBT, as well as probe-based otolith studies of other migratory tuna species including south Pacific albacore Thunnus alalunga [42], skipjack tuna Katsuwanus pelamus [43] and southern bluefin tuna Thunnus maccoyii [44, 45]. Similarities in elemental profiles include higher Li and Mn in early life and general increases in Sr and Ba in older ages. Employing probe-based analysis to quantify continuous elemental patterns across sequential calcified and proteinaceous growth bands provides a chemical calendar revealing the life histories of highly migratory fish.

Identifying drivers of elemental variations during life requires understanding the relative influence of physiological and environmental processes on element incorporation and dissolved ion transfer from water and food, across gill and gut membranes, transport through blood-endolymph interfaces, and ultimately otolith biomineralization [24, 46]. Analytical limitations must also be recognized when interpreting laser scan profiles in biominerals. Optimizing laser setting (power (J/cm2), repetition rate (Hz), scan rate (um s-1) and ICP MS parameters (i.e. carrier gas (He, Ar) flow rate, cone distance, etc.) and using matrix-matched replicated standards is important for comparing studies conducted in different labs. Although laser spot diameters are standardized, crystal growth and accretion rates decrease with age [47], thus the laser integrates different time windows based on the age and growth rate of the fish. The geographic otolith edge comparison here integrated different time frames in the PBT since they differed in age, but early life otolith core comparisons should reflect similar growth and biomineral accretion rates in larval/juvenile PBT, regardless of collection location.

In PBT otolith cores, Li:Ca, Mg:Ca and Mn:Ca were enriched, then gradually decreased to low stable values after age 1. Physiological regulation of these elements have been shown in teleost fish [24, 48]. Thus, the enrichment of otolith Li:Ca, Mg:Ca and Mn:Ca, at early life may reflect periods of rapid juvenile growth rate, higher protein accumulation versus aragonite crystallization, and different metabolic rates before PBT exhibit endothermy. PBT undergo a metamorphosis from larvae to juveniles at 20–35 days post hatch, with rapid increases in protein synthesis and somatic growth that correspond with drastic increases of protein-DNA and RNA-DNA ratios [49]. This period of rapid somatic growth would result in increased otolith accretion rates [50]. The peak of Mn:Ca within the core, but following the primordium within age-0, may correspond with the larvae-to-juvenile transition to rapid growth, where otolith morphology and growth axes shift. The fact that element concentrations change at this inflection point, where the aragonite growth axes also change direction, is consistent with ontogenetic changes in mineral growth or crystal formation as a control influencing elemental uptake. After the first 1000 μm (~age 1), core-enriched elements (Li:Ca, Mg:Ca and Mn:Ca) decrease and remain low throughout ontogeny.

Results from other studies suggest that Mn:Ca and Mg:Ca are also influenced by environmental conditions. These ratios were most useful for multivariate assignment of YOY PBT collected from two separate spawning regions using both solution- [27, 51] and LA-ICP-MS-based analysis of core regions [29]. In addition, Mn:Ca and Mg:Ca within the first 500 μm of core otolith differed inter-annually, which may reflect interannual environmental variability experienced by PBT spawned in different years (e.g., ambient water temperature and chemistry). PBT metabolic rates, foraging behavior, and prey availability within the two known spawning grounds, may also affect differential incorporation of Mn [52] and Mg [36] during early life. All of these factors can vary from year to year, so annual juvenile otolith chemistry baselines from each spawning region are essential for inferring natal origins of age-classed matched unknowns [29].

Our finding that Zn:Ca, Sr:Ca, and Ba:Ca have similar values in otolith cores but differ significantly in the last 100 μm of otolith edges among the three capture areas supports that these element:Ca ratios may proxy ocean basin-specific differences. The outer 100 μm of otolith growth likely represent ≥ 33 days in larger individuals with slower growth rates based on direct validation studies of southern bluefin tuna (Thunnus maccoyii) with estimated mean otolith accretion rates of 3 μm day-1 [53]. For such time intervals it is reasonable that the specimens largely or completely resided within the capture regions, and thus associated element:Ca values were also obtained within capture regions.

Other studies have shown that otolith chemistry can distinguish among widely separated collection regions in the Pacific [40, 43]. For example, Arai et al. [43] found (EPMA-based) Sr profiles effective for distinguishing skipjack tuna migratory behavior between temperate and tropical waters, including a fish tagged in the WPO that migrated to the SPO and was recaptured [43]. For skipjack, Sr concentrations were lower in the cooler (19-22°C) temperate waters (Japan coast) and higher in warmer (29-30°C) tropical waters near the equator. Our finding that lowest PBT otolith edge Sr concentrations occur in specimens collected from the WPO, is consistent with the skipjack tuna patterns of Arai et al. [43], but we cannot confirm the specific temperature experiences of WPO fish were lower compared to EPO or SPO fish.

Based on differences in (EPMA-based) Na, Ca, Sr, S, K, and Cl profiles for PBT otolith, Proctor et al. [44] suggested that ontogenetic variability was greater than any environmental variability, and thus otolith chemistry would not be useful for delineating geographic stock structure due to the homogeneity of ocean chemistry for these conservative elements. However, injection of sea-cage PBT with SrCl2 in the same study resulted in a strong spike in otolith concentrations, suggesting transfer of dissolved Sr2+ from blood to otolith, but it is unknown how that experiment reflects natural conditions [20, 23, 54]. Sr2+ substitution for Ca2+ during calcium carbonate biomineralization is well documented and widely attributed to similar ionic radii [53, 55, 56]. That Sr2+ is only found in the non-protein salt fraction of otoliths and not under physiological regulation [22], supports otolith Sr:Ca as an environmental proxy of water temperature and salinity [57], but may be complicated by biochemical, biological and physiological interactions, including sexual maturation [20, 23, 54] and the development of increased endothermy with body size [58].

We find highest Ba concentrations in otoliths collected in the EPO, a well characterized upwelling region [59, 60]. Cold, nutrient rich upwelled water typically exhibits higher dissolved Ba concentrations than surface waters [61, 62]. Barium has been shown to proxy upwelling conditions in diverse calcified biominerals, including coral skeleton [63, 64], shark vertebrae [65] and fish otoliths [30, 40, 66]. Tagging studies demonstrate that PBT experience cold water temperatures when they occasionally dive below the thermocline, likely to forage on deep-water prey [67, 68]. EPO PBT mainly reside at the surface the water column [69], and move latitudinally over areas of coastal upwelling with high primary productivity [5]. The oscillations of otolith Ba:Ca detected after age 3 in our study, could follow movement into deeper, cooler water, when PBT forage on deep water prey and increased dissolved Ba is taken up through the gills or intestine. The width of oscillating Ba:Ca peaks are approximately seasonal (~300 μm) in transects of EPO and SPO otoliths. Ba:Ca amplitudes fluctuate between 10–20 μmol mol-1 in these specimens, compared to amplitudes < 10 μmol mol-1 in WPO specimens (see S1 Fig). These regional Ba:Ca patterns could reflect larger-scale seasonal movements to upwelling areas, as consistent with archival tagging data [5].

Highest otolith edge Zn concentrations correspond to SPO PBT, the region where the largest and oldest (3 PBT estimated 20+ years) fish were collected. Because Zn in seawater is commonly bound to organic complexes, dissolved Zn2+ is not readily available for uptake compared to Sr2+ and Ba2+ [54, 70]. Previous studies indicate that Zn2+ is under strong physiological control in fish, serving as co-factors in many enzymes and proteins [22, 54, 71]. Elevated Zn:Ca in otolith core regions, followed by decreasing levels with age, is a common ontogenetic pattern reported in other species [24]. Our data demonstrate an opposite pattern with Zn:Ca increasing with ontogeny and highest otolith edge Zn:Ca occurring in the oldest PBT. Increasing otolith Zn:Ca with age in PBT could indicate a physiological control, such as sexual maturity [54, 72] or reduced otolith accretion rate with age.

We found an overall average discrimination classification success of 72% among capture regions using Sr:Ca, Ba:Ca and Zn:Ca in the outer 100 μm of otolith edges. The limitation of using a constant otolith edge distance (100 μm) is that this distance will represent different time frames, equating to potentially years in the oldest PBT (7–20+ years in this study) as otolith increment growth slows down significantly with age in bluefin [73]. This outer-edge discrimination approach also assumes that the fish have been in capture regions long enough for local signatures to have been incorporated. All WPO PBT were collected in same month and year (May 2017), had similar estimated ages (range 9–13 y, mean ± standard deviation = 11±1.5 y) and exhibited the highest classification accuracy of 90% with only one misclassification. Thus, temporal variability of both collection location (all samples in one month) and otolith accretion rate was minimized for these specimens. The next highest classification accuracy was for EPO PBT at 70%, with two fish misclassified from WPO and one fish mistaken from SPO. The youngest PBT were also collected from the EPO, with a mean estimated age 7.2±0.7 years and collection dates within a year (July 2016 to Oct 2017). The lowest classification accuracy (40%) was obtained for SPO adults, likely due to wide ranging collection dates from 2007 to 2018, differences in fish size and age (6 to 20 y), and more disparate collection regions, including New Zealand and the Cook Islands separated by over 3,000 km in tropical waters, compared to EPO and WPO in temperate waters. The influence of interannual variability in oceanic conditions (i.e. temperature shifts for La Nina versus El Nino) on otolith biomineralization is a recognized factor degrading classification success using otolith geochemical signatures [74].

Conclusion

This study examined if elemental time series of PBT otoliths reflect ocean basin-scale migratory movements or regional residence. Elemental transects of PBT otoliths provide ontogenetic records of physiological and environmental histories, although it is often difficult to discern between the two. Similarities of Li:Ca, Mg:Ca, and Mn:Ca profiles for juvenile PBT 1–2 years old (to 1500 μm distance from core) among all three geographically distinct capture regions are potentially related to similar thermal physiology, rapid growth and otolith accretion rates and a common region of origin within the WPO. After 2–3 years, Sr:Ca, Ba:Ca, and Zn:Ca begin to diverge by region of collection, likely reflecting spatial and temporal oceanographic variability experienced when PBT undertake broadscale migrations, or physiological influences associated with changes in foraging and/or breeding behavior. Without controlled laboratory experiments, which are very difficult for large bodied and fast-moving tunas, the relative influences water chemistry, ambient temperature, diet and metabolic physiology on otolith elemental uptake will be premised on descriptive studies. Additional research on captive reared PBT or otoliths of tagged and recaptured individuals will expand knowledge on elemental uptake in otolith biominerals in PBT. Future otolith geochemical studies involving a greater number of older (larger) specimens should further advance understanding PBT life history and migratory behaviors. Refining the otolith chemistry approach to characterize behavior including migratory and resident contingents, can help mangers better understand stock dynamics and improve stock assessment models for highly migratory species.

(XLSX)

Click here for additional data file.

Elemental profiles of individual PBT otoliths collected from the eastern Pacific (EPO), western Pacific (WPO) and south Pacific (SPO); different colored lines represent individual fish from each region.

See Table 1 for details on collection. Asterisk (*) indicates visible crystal bond on surface of NZ02 that was removed from Fig 1.

(PDF)

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3 Aug 2022

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22 Sep 2022

Mohan Response to reviewers comments

Review Comments to the Author

This study presents an original research article on otolith trace element profile analysis of Pacific bluefin tuna (Thunnus orientalis), as an approach to provide records of their life history. Authors apply for the first time this technique in Pacific bluefin tuna collected across the species adult distributional range; covering distant areas from the Pacific Ocean (i.e., west, east, and south) and discuss on how different elements provide ontogenetic records of physiological and environmental histories.

Overall, the article is presented intelligibly and is easy to follow. Both experiments and statistics are performed to a high technical standard and are described in sufficient detail, so that other scientist can follow this study. Authors also state that data is available under request without restrictions. Results are rigorously reported and support the conclusions of the article. The motivation behind the work is clearly described, with straight and sound discussion of the observed results and in the limitations of the performed study.

I do believe that this research meets all criteria for publication in PLOS ONE journal. However, there are some minor issues that could be improved in my point of view before its publication as a high-quality manuscript:

My biggest concern is with the readability of the figures and tables. I don't know if it is a problem with the system, or with the quality of the figures themselves, but they should be thoroughly revised as they are difficult to read as I see them. Please check them for quality standards and size format.

Response: The figure files were uploaded following the specific requirements of PLOS PONE for PRISM:

Prism: Export your graph with the following settings: File format: TIFF; Resolution: 300; Color Mode: RGB; Size Make Width: 7.5 in; Enable Compression

I think the discussion lacks a little bit more, the microchemistry part is very well detailed and contextualized, but it seems to me a good opportunity to discuss a little bit more about migratory behaviours, contingents and discuss some implications of these findings for the Pacific bluefin tuna management and fisheries as well as and future research priorities. Besides, the introduction of the discussion needs to be refined a bit by looking at the connection between the ideas to be presented later.

Response: Thanks we have added lines 530 in conclusion: “Refining the otolith chemistry approach to characterize behavior including migratory and resident contingents, can help mangers better understand stock dynamics and improve stock assessment models for highly migratory species.”

If authors can address these minor issues, I do believe that this manuscript will be a great addition to the PLOS ONE articles collection and to the scientific community working with otolith or tuna management related studies. Bellow I have provided specific comments and/or questions to the authors that should be quite straightforward to address. I hope authors and editor can find these suggestions useful, and my congratulations for this nice piece of work, this is good science that needs to be shared!

Response: Thanks!

Introduction

Line 85: I suggest using “core-to-edge” as it is more common in the literature and then in MS you are referring to that part as “edge”.

Response: Done

Lines 83-96: I would add also a small sentence saying that there is also substantial uncertainty on how intrinsic and extrinsic processes affect elemental incorporation into the otolith, and also that responses can be species specific (e.g. Hüssy et al., 2020; Sturrock et al., 2015).

Response: Added on line 92 ” There is high uncertainty on the relative influence of intrinsic and extrinsic processes that affect element incorporation into fish otolith and responses are species specific (23). “

Line 93: You can cite Farwell et al. 2001 at the end of this sentence if you want, which says “The open ocean, pelagic environment of most scombrid species has made it challenging to conduct field research. Their high metabolic rates and specialized swimming result in significant space requirements if held in captivity. The major challenge is that tunas must swim continuously, which makes collecting and captive care difficult”.

Farwell, C. J. (2001). 10. Tunas in captivity. Fish physiology, 19, 391-412.

Response: Done

Line 99: And differentiate among contingents or fish with different life histories?

Response: Added line 102 ” …identification of migratory contingents.”

Line 108-109: I suggest replacing “LA ICP MS” by “LA-ICP-MS”

Response: Done

Line 109: From hatch time to adult?

Response: We prefer using juvenile to adult life. Hatch time would infer the laser could resolve larval stages, but the laser spot of 50 microns integrates on larger ‘weekly’ time scales and thus using juvenile stage is more appropriate.

Line 110: I feel that “comparing complete chemical profiles” would be more accurate than “comparing complete life histories”

Response: Changed to “By comparing complete elemental profiles of PBT sourced from different ocean basins, this study explores…”

Line 114: I suggest deleting “biominerals” at the end of the sentence.

Response: Done

Material and Methods

Lines 118-120: I think it will be nice to indicate here how many otoliths did you get from each region, and not later in Results section.

Response: Added samples sizes to each region as suggested.

Line 120: I feel that a map would be a great added value here, where you can show the location of the 3 regions you are considering (instead of figure S1), and the location of the spawning areas. I think this will be a plus for the manuscript and will help the non PBT familiarized readers to locate the regions you mention throughout the manuscript (I needed to check in google maps to get the whole picture in my mind).

Response: New Figure 1 added from supplement to manuscript.

Table 1: I think that table 1 needs from some restructuration for the paper, for example, I am not sure if both PBT_ID and Otolith ID are relevant for the reader (perhaps choose only one). Same I am not sure if the fishing methods adds something here? Like this you could use the space you have left over to incorporate a “Region” column where you indicate if samples belonged to WPO, EPO or SPO a with the Location only, readers can be confused (as it was my case because I am not familiarized with the Pacific). Please add to the legend that size is fork length (FL). Also check that are some measures of FL and GG that are in bold.

Response: Thank you for the suggestions. Only one PBT ID is now presented. The fishing method was removed, and Ocean region of collection column added. The bold font was also removed, and legend indicated the FL=fork length

Lines 129-130: “It was also impossible to know with certainty the migratory histories of sampled fish…” sure, but this is what you want to inspect no? I would suggest deleting this sentence and said only that fish were grouped by sampling region for comparisons.

Response: Deleted and revised as suggested: lines 143: “Region of collection (EPO, WPO, SPO) was used to compare elemental patterns.”

Figure 1b: Perhaps it is worthy to write core, inflexion point and edge in the figure to help the readers locate the otolith position afterwards? Not sure…

Response: Thanks for suggestion. Added additional labels to new Figure 2b.

Line 151. Crystalbond “adhesive” or “glue” instead of “cement”?

Response: Changed “cement” to “adhesive” as suggested

Line 165: Perhaps “Otolith sections and standards…”

Response: Changed as suggested.

Line 173: Perhaps you can cite Sturgeon et al., 2005 at the end of this sentence.

Sturgeon, R. E., Willie, S. N., Yang, L., Greenberg, R., Spatz, R. O., Chen, Z., ... & Thorrold, S. (2005). Certification of a fish otolith reference material in support of quality assurance for trace element analysis. Journal of Analytical Atomic Spectrometry, 20(10), 1067-1071.

Response: Since we did not use a certified otolith standard, we did not include this citation. We used a calcium carbonate standard MACS-3 as the primary reference standard, not a certified otolith standard.

Line 190: Are these mean annual annuli? If so please state it in the text

Response: Yes, mean annuli distances are stated in the text line 215: “The mean annuli distances were 1,223 µm in year one, 1,577 µm in year two, 2,054 µm for year 3+.”

Line 190: But this image only shows annual rings up to 3 years, how did you do for your older individuals?

Response: We only calculated mean annuli distance for the first 3 years, when the annuli are widely spaced. Older ages and annuli distances were not estimated because data was not available. In Pacific bluefin tuna, the annuli become tightly spaced together at older ages, thus mean distances would not be accurate or comparable among individuals.

Line 197: I know you mention later in the discussion, which is really nice, but I think it would also be worthy to mention here that time frame represented in each individual is different.

Response: Added line 221: “For each otolith, the final 100 µm of the otolith edge was averaged to represent the recent otolith material accreted in the region of collection, which represents different time frames for individual fish.”

Line 200-201: Was your data normally distributed and present homogeneity of variances so that it justifies the use of one way ANOVA? If so please state, if not, consider using another non-parametric test. I see that you mention latter that a Brown- Forsythe test was performed and confirmed that variances of the populations from which the samples are drawn are equal, but what about normal distribution?

Response: Thanks for suggesting testing the normality assumption. Only one element was not normal and thus was log transformed and then meet the normality assumption. Added lines 223: “Normality was assessed using a Kologorov-Smirnov test and only Mg was not normal distributed, and thus a log transform was used to meet normality assumption for Mg.”

Results

Lines 212-216: I am not sure whether this should go here or in M&M, I feel that it does not fit in results, and it fits better in M&M sampling description.

Response: Coauthors felt this was a result, so we have detailed the otolith collection sample sizes in both the M&M and results

Line 219: “...but it is possible that PBT…” ?

Response: Yes, thank you. “that” was added to text.

Line 236: Perhaps add “ratios” after “element:Ca”

Response: Done

Figure 2: “solid line represent mean values and shading depicts ± standard deviation across laser distance” I cannot see this in the figure I get. Please check just in case. “dashed vertical lines denote approximate annuli distances for the first 3 years of life…”, I think it will also be beneficial to add where the inflection point will be represented in the transect, as then you mention this in you discussion.

Response: New Figure 3 (previously Figure 2) been updated in full resolution to see details. It is likely the poor resolution of the original figure obscured shading of the standard deviation. We are unable to add the inflection point, since it differs between each individual fish otolith and this figure represents mean concentrations grouping fish by region.

Figure 3: I personally found confusing the fact that horizontal barckets join regions that are not statistically significant, when usually it is the other way around, I would just mark between which there are significant differences.

Response: Removed brackets that connected non-significant differences. Used lower case letter above groups that did display significant multiple comparison test, with non-significant groups sharing the same letter in new Figure 4 (previously Fig 3).

Table 2: This table need a little bit of restructuring and editing. Please also indicate in the legend that significant P values are highlighted in bold in the table. Also add that inspected regional differences are between WPO, EPO and SPO.

Response: Table 2 has been updated to reflect the log(MgCa) results and the legend has been updated: “One-way ANOVA results based on mean otolith edge (outer 100 �  m) element:Ca values for Pacific bluefin tuna for exploring regional differences in otolith chemistry among the Eastern Pacific (EPO), Western Pacific (WPO) and South Pacific (SPO). Only Mg:Ca was log transformed to meet normality assumption.”

Lines 282-283: I suggest expanding here and also to mention that although classification differences are more or less clear for WPO and EPO there is also some overlap. Also, that SPO is disperse, and not clear grouping can be observed. So, there are 2 SPO with similar values among them, 1 more “EPO like” and 2 more “WPO like”, as you can see in the classification of table 3.

Response: Added line 330: “Two SPO fish clearly grouped together and were separated from the EPO and WPO, while 3 SPO fish more closely grouped to the EPO (Fig 5).

Table 3: I recommend replacing “origin” by “capture” here. Perhaps you can highlight in bold the correct classifications of the table.

Response: Thank you. New table 3 updated.

Discussion

Lines 296-297: I feel that this sentence is a bit weak as it is, and you should contextualise it more.

Response: Added lines 351: “This is the first study to investigate otoliths of PBT collected from the SPO, a poorly understood migratory path where samples have rarely been collected or investigated.”

Lines 295-307: I think that some cohesion in the first paragraph of the discussion is missing.

Response: The first paragraph of discussion is providing a summary of overall results and linking to recent related work of Wells et al. 2020.

Line 303: All of your fish are originated from the WPO no? not only the juveniles

Response: Removed “juveniles”

Line 318: I suggest replacing “core-to-rim” by “core-to-edge”

Response: Done

Lines 318-322: Are all of these in the Pacific? Can you specify more on what similarities did you found? (e.g., enrichment/depletion for early life signatures etc.

Response: Added line 379: “Similarities in elemental profiles include higher Li and Mn in early life and general increases in Sr and Ba in older ages.”

Lines 322-324: And also continuous transects no?

Response: Yes, probe-based analysis refers to continuous transects.

Lines 325-333: I think you could also discuss here on the importance of the placement of the transect, as some elements do not precipitate equally in the otolith growth axis (you can check Artetxe-Arrate et al. 2021)

Artetxe-Arrate, I., Fraile, I., Clear, N., Darnaude, A. M., Dettman, D. L., Pécheyran, C., ... & Murua, H. (2021). Discrimination of yellowfin tuna Thunnus albacares between nursery areas in the Indian Ocean using otolith chemistry. Marine Ecology Progress Series, 673, 165-181.

Response: While we agree this is important, we placed the laser path on the same otolith growth axis for each individual. We would only be able to assess spatial variable with a 2-dimension elemental map, which is not available for these samples.

Line 365: What individual variability do you see in you fish for early life period of these elements as you are accounting for fish that were born at different years? Do you see some fish with signatures more similar to the East China Sea and other to Sea of Japan reported in Wells et al. 2020? Maybe you can discuss a bit more here

Response: Thank you for the comment. Unfortunately, we are unable to assess this as baseline YOY samples from the birth years of these fish are not available. Matched YOY baselines would be needed to address this, since interannual variability of juvenile baselines can affect interpretation of the natal chemical signatures.

Line 382: Are lower temperatures expected for the WPO than for EPO and SPO? If so please state it

Response: Thank you for the comment. We are not able to assess the temperature since we cannot define exact locations of the ocean that fish experienced, since they are highly migratory but also regionally endothermic. Added: lines 442 ”Our finding that lowest PBT otolith edge Sr concentrations occur in specimens collected from the WPO, is consistent with the skipjack tuna patterns of Arai et al. (2005), but we cannot confirm the specific temperature experiences of WPO fish were lower compared to EPO or SPO.”

Line 402-404: But are these occasional dives long enough to be recorded in the chemical signature? I don’t think so…

Response: Good point. However, if dives below the thermocline are consistent over several weeks occurring daily, then the integrated otolith signature might reflect that accumulative diving behaviour.

Lines 419-424: We also found that otolith borders were enriched in Zn in comparison with the rest of the otolith for few different tuna species, perhaps because of Crystalbond inclusion, or because the otolith crystal composition of the border. I don’t know if it is worthy to also consider this in small sentence here?

Response: Thank you for this information. Since we did not see a similar Zn enrichment near the edge, we prefer to not discuss here.

I do miss a little discussion on contingents and migratory behaviour, and also to recap the importance of this knowledge for management (following your intro)

Response: Added lines 530: “Refining the otolith chemistry approach to characterize behavior including migratory and resident contingents, can help mangers better understand stock dynamics and improve stock assessment models for highly migratory species.”

Conclusion

Perhaps recap your objectives and how you answer them with this study

Response: Added sentence line 513: “This study examined if elemental time series of PBT reflect ocean basin-scale migratory movements or regional residence.”

Lines 448-449: Perhaps you can add “although it is often difficult to discern between the two”

Response: Added lines 515: “Elemental transects of PBT otoliths provide ontogenetic records of physiological and environmental histories, although it is often difficult to discern between the two”

Lines 461: And in other fish in general, as these type studies are mostly lacking for any species.

Response: True, but this is especially true for large pelagic species that cannot be kept in captivity, such as the PBT studies here.

Best luck.

Reviewer #2: Mohan et al. used otolith geochemical transects (core-to-edge) to reconstruct life histories of adult Pacific Bluefin tuna (PBT). They analyzed trace elements in otoliths of adult PBT obtained from the western, eastern, and southern Pacific Ocean to investigate patterns across ontogeny. Results show that some elements (Li:Ca, Mg:Ca, Mn:Ca) were higher during the first 1-2 years and then decreased independently of fish capture location, while other elements (Sr:Ca, Ba:Ca, Zn:Ca) showed a similar pattern in the first 1-2 years but then showed varying patterns likely related to different migration pathways to the capture location. Overall, the manuscript is well written and showcases the utility of otolith geochemical signatures to investigate entire life histories (birth to capture). I have only a few comments that I would like the authors to address before the manuscript can be accepted for publication in Plos One.

The authors only describe the otolith elemental transect data and do not perform any formal statistical testing. It could be worthwhile to consider using a time-series clustering approach analyzing the first 1-2 years and the remaining years to help support the statement that all PBT spawning and nursery areas are located in the EPO and that after ~2 years PBT migrate to different areas.

Response: Thanks for the suggestion. Unfortunately, an additional time-series clustering approach is beyond the scope of this study. We feel the current graphs and statistical approach of comparing otolith edge regions sufficiently support the conclusion of the study.

Using 100 um from the edge as the capture location signature can be very misleading as you discuss in the Discussion section. Why didn´t you consider using individual age-related distance to obtain a edge signature that corresponds roughly to the same time frame in each individual?

Response: Since the ages were only estimated based on size and not based on individual annuli counts of these tuna otoliths, our approach of standardized edge distance is the best we can do. We clearly discuss the limitations and caveats of our approach.

Table 1 legend: Change to “Pacific bluefin tuna (PBT) metadata including collection date, region and method;…”

Response: Done

Table1: Why some of the number are in bold?

Response: Corrected this error.

Line 144: “Otoliths of PBT were sectioned…”

Response: Thank you for catching this. Corrected.

Line 192-193: Please clarify this sentence. As I understand it, you are referring to the distance from the core to age 1, age 2 etc, but can be interpreted as distance between annuli.

Response: As stated, this is estimated annuli distance based on the laser path direction, not distance between annuli. Added: “The mean annuli distances were 1,223 µm in year one, 1,577 µm in year two, 2,054 µm for year 3+, as measured from the otolith core and along the laser path.”

Line 357: YOY PBT

Response: Thank you. Corrected.

Figures 2-4: Need to be substantially improved for publication.

Response: Yes, agreed. Higher resolution images will be uploaded with revised figures.

27 Sep 2022

Otolith geochemistry reflects life histories of Pacific bluefin tuna

PONE-D-22-17432R1

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4 Oct 2022

PONE-D-22-17432R1

Otolith geochemistry reflects life histories of Pacific bluefin tuna

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