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Figure 1. Study area with sampling locations (Map generated by using the QGis 2.8 - https://qgis.org/).

T e present study proposes a method able to estimate stable isotope values in metabolically non-active tissues
(with a life history memory), verifying if it could be used also when the amount of sample material is not enough
to perform the traditional collagen extraction procedure, (such as in very small f sh species, in archaeological
remains or in hard tissues of species stored in museums).
T e isotopic analysis was here applied on the f rst dorsal spine of both adults and young of the year (YOY)
bluef n tuna specimens, caught in the central Mediterranean Sea, to address important questions on the ecology
and feeding behaviour of this species during its growth. Spine sections of adult tuna, of en chosen for growth
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studies (e.g. ), were also considered to combine isotopic results with growth layers.
T e bluef n tuna, T unnus thynnus, is a highly migratory and long-living f sh at the top of the pelagic food
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web and plays a key role in the stability of marine food webs by exerting top-down control on its prey . It feeds
on a broad spectrum of prey items, such as small pelagic f sh, cephalopods and shrimps 22–25 ; furthermore, in
some Mediterranean and Atlantic feeding grounds, the component of mesopelagic f sh is dominant in its diet 26,27 .
However, the diet composition of bluef n tuna varies in relation to its growth, seasons and migratory patterns,
making it dif cult to evaluate spatial and temporal ef ects. T is latter aspect is further complicated during the
f rst months of life, when T. thynnus specimens have a rapid growth rate leading to changes in its trophic status.
Moreover, the Reg. CE 302/2009 prohibits f shing tuna specimens of size smaller than 115 cm (specimen weight
<30 kg), making the trophic position in the early life stage dif cult to be determined.
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In the present study, stable nitrogen and carbon isotopes (δ N and δ C) values and elemental nitrogen (%N)
and carbon (%C) composition were obtained from: (1) extracted and non-extracted collagen samples obtained
from the same spines of bluef n tuna adults, in order to evaluate the possible dif erences in the isotopic val-
ues between the two approaches; (2) whole spine of both YOY and adults tuna (without collagen extraction),
to highlight dif erences in feeding behavior between the two macro-size classes (YOY and Juveniles/Adults);
(3) untreated micromilled powder obtained from individual growth layers of spine section, to provide a more
detailed trophic ecology in each layer representing a dif erent period of animal’s stage.
Results
A total of 51 T. thynnus specimens were collected in two sampling sites (Fig. 1): 29 Juveniles/Adults (with fork
length FL > 50 cm) and 22 young of the year (YOY with FL < 50 cm) tuna. Ages of the specimens ranged between
the f rst year of life to f f een years (Table 1).
Paired Wilcoxon test, carried out on δ N and δ C values obtained on extracted and non-extracted collagen
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samples of 28 Juveniles/Adults tunas (Fig. 2), showed non-signif cant dif erences for δ N values, while a signif -
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cant dif erence was observed for δ C (W = 0, p-value = 7.451e-09). Mean δ C values of non-extracted collagen
was 4.8‰ lower than the same extracted collagen samples (Fig. 2).
Looking at dif erences between YOY and Juveniles/Adults (only for non-extracted collagen samples), signif -
cant dif erences were found in terms of δ N (W = 55, p-value = 5.384e-07), while non-signif cant dif erence was
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evidenced for δ C values. In particular, δ N values were higher in Juveniles/Adults specimens than in YOY, with
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absolute dif erences in median values of 1.4‰ (see Fig. 2). Finally, when δ N and δ C values were compared
among core, non-opaque and opaque bands in spines section, non-signif cant dif erences were found in terms of
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δ N, while signif cant dif erences were observed for δ C. In particular, a Tukey HSD post-hoc test highlighted
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Scientific RepoRtS | (2020) 10:9899 | https://doi.org/10.1038/s41598-020-66566-w 2