Page 5 - Thunnus_2020

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Figure 2. Boxplot of δ C and δ N values from spines of T unnus thynnus. Top panels report the boxplots only
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for samples where it was possible to replicate the isotopic analyses on both extracted and non-extracted samples.
Bottom panels boxplot compares the isotopic values between Adults/Juveniles (A/J) and young of the year
(YOY) considering only non-extracted samples.

with growth. T e comparison between the dynamics of trophic shif in BFT and the one observed in T unnus
albacares evidenced strong dif erences mainly for smaller specimens. Weng et al. (2015) and Graham et al. (2007)
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reported a sigmoid relationship between δ N values and FL, thus smaller yellowf n tuna (FL range 20–40 cm)
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showed an almost constant δ N values up to FLs > 40 cm. For this species, the diet shif was very rapid and the
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δ N plateau was reached between 40 and 50 cm. T e δ N values of bluef n tuna YOY (in this study) were charac-
terized by a continuous increase up to 80 cm (FL). We believe that this dif erence between the two species could be
linked to dif erent factors such as: 1) the same prey in dif erent waters may exhibit dif erent stable isotopes values
due to dif erences in the nutrient sources that af ect the N values of food webs; 2) dif erences in physiology and
development (e.g. dif erences in the development of gape size); 3) dif erences in feeding strategies and/or prey
selectivity.
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On the other hand, the high variability found in δ N values of Juveniles/Adults bluef n tuna suggested that
for tuna with FL > 50 cm diet passes to be based mainly on cephalopods and f sh 21,30 . Furthermore, this variability
may be af ected by geographical location of prey and by dif erent prey assemblages. Indeed, during migration,
from spawning locations to feeding grounds, bluef n tuna probably fed as they encountered patches of prey but
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it will be also possible that groups of migratory bluef n tuna stop to feed at unidentif ed locations during seasonal
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migrations (e.g. ). T e high variability of δ N values in Juveniles/Adults specimens was also shown when the
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isotopic values, related to consecutive opaque and hyaline bands, were considered (Fig. 5).
When direct determination of δ N was obtained on powder micromilled growth layers of spine of adults
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bluef n tuna, the amount of material did not permit to determine elemental composition of nitrogen (and conse-
quently of C:N ratios), inhibiting also the evaluation of lipids ef ects in the growth layers.
The significant differences in δ C values, observed by comparing isotopic values from extracted and
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non-extracted collagen from spines of Juveniles/Adults tuna, is due to the presence of lipids and bioapatite that
can enrich or deplete these values, respectively. Despite sub-samples of spines were subject to acidif cation with
HCl before the isotopic analysis and no ef ect was recorded during this process, we assumed that the ef ect of
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bioapatite on δ C values could be negligible in our samples, in agreement to . On the contrary, the δ C values,
lower in non-extracted collagen samples, evidenced the presence of lipid-bond carbon. Lipids are generally 2–3‰
more depleted in C than in other organic molecules 35,36 . Moreover, also when direct determination of δ C val-
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ues was obtained on powder micromilled growth layers of spine of adults bluef n tuna, Tukey HSD post-hoc test
highlighted that δ C values in the core were signif cantly lower (p < 0.05) than the ones recorded in the growth
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layers. Consequently, we recommend to consider only samples that fall in the C:N range 2.6–3.5 to conf rm that
lipid extraction is not necessary. Since C:N ratio was not always determinable in untreated samples and, to our
knowledge, no standard protocol for δ C correction exists for T. thynnus, in this work we decided to use only the
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Scientific RepoRtS | (2020) 10:9899 | https://doi.org/10.1038/s41598-020-66566-w 5