Materials and methods
Sampling
Organisms were collected by epi- to bathypelagic trawling in canyons of
the Bay of Biscay continental slope (North-East Atlantic) during EVHOE
scientific cruises (“Evaluation Halieutique de l’Ouest de
l’Europe ”; https://doi.org/10.18142/8) that took place between 2002
and 2021. Trawls were conducted at night between 25 m to 2000 m depth at
25 stations (Figure 1). The trawl net was 192 m long with a headline of
76 m and a foot rope of 70 m. The average vertical mean mouth opening
was about 24 m and the horizontal opening of about 58 m. The mesh size
gradually decreased from a very large 8 m (stretched mesh) at the mouth
to 20 mm (stretched mesh) in the cod-end. To allow the capture of very
small specimens, the trawl was also equipped with a 7.5 m long sock with
a 12 mm mesh size. Each haul was made at a specifically chosen immersion
depth. Once the trawl reached the selected depth it was towed
horizontally (i.e., constant immersion depth) for 1 hour at 4 kn.
Datasets
Two different datasets were used to study ontogenetic changes in
deep-sea pelagic fish species from the Bay of Biscay. The trawling
(first) dataset included all data collected by trawls (i.e. number of
individuals per species per sampling depth and total body length of each
individual; n = 4165). To study the trophic aspects of ontogeny (see
methodology below), muscle sampling was performed on 12 species of the
trawling dataset to access the δ15N values of
individuals (n = 682). This constituted the isotopic (second) dataset.
The size measured for the individuals sampled for the isotopic dataset
was the standard length. The size distribution of the individuals
composing the species included in the isotopic dataset was
representative of the size distribution observed in the trawling dataset
(see appendix 1).
Nitrogen stable isotope
analysis
A total of 682 muscle samples belonging to 12 of the most abundant
species (seven migratory and five non-migratory (Loutrage et al., 2023)
were collected (table II). For each individual, the standard length (cm)
was measured on board and a small piece of muscle was collected and
frozen at -20°C. To have sufficient material for stable nitrogen isotope
analysis, the muscles of the smallest individuals were pooled. Within
each of these pools, the individuals were of equivalent size and were
sampled at the same depths. At the laboratory, muscle samples were
freeze-dried (72h). To reduce the samples to a fine powder, samples
containing a single individual were manually homogenised, while samples
containing a pool of individuals were homogenised using a ball mill
(MM400 Retsch®) with zirconium oxide-coated bowls and balls. A fraction
of this powder (0.50 ± 0.05mg dry mass) was weighed in tin cups.
Analyses were then performed with an isotope ratio mass spectrometer
(Delta V Advantage with a Conflo IV interface, Thermo Scientific)
coupled to an elemental analyser (Flash EA, 2000; Thermo Scientific).
Results are presented in the usual δ notation relative to the
deviation from an international standard (atmospheric nitrogen, forδ 15N values), in parts per thousand (‰). Based
on repeated measurements of USGS-61 and USGS-62 used as laboratory
internal standards, the experimental analytical precision was< 0.15‰.
The isotopic dataset included individuals sampled from different years
(i.e. between 2007 and 2021), which could have affected our data.
However, more than 90% of the muscle samples were collected between
2019 and 2021 and nearly 75% in 2021, which reduces the potential
inter-annual effect. Furthermore, if a significant temporal change inδ 15N values had occurred, we expect that a
common pattern would have been observed for the different species
considered, which was not the case. Thus, we hypothesize that changes in
size and depth have a much greater influence on theδ 15N values than does the temporal aspect.
Details on the sampling by year for each species are presented in the
appendix 2.
Relationships between size distribution and
depth
The different depth layers were defined as follows: the epipelagic zone
between 25 and 175m, the upper mesopelagic zone between 175 and 700m,
the lower mesopelagic zone between 700 and 1000m, and the bathypelagic
zone below 1000m. This division corresponds to the one used in the
literature (Sutton, 2013) and is congruent with the depth structuration
observed in the canyons of the Bay of Biscay (Loutrage et al., 2023). To
study the changes in size distribution with depth, the trawling dataset
was used. At both community and specific levels, a linear model was
performed, the sampling depth corresponding to a continuous explanatory
variable. Results were considered significant when the p-valuewas ≤0.05.
Relationships betweenδ 15N values and
size
The relationship between δ 15N values and
individual size was explored with the isotopic dataset at both community
and species levels. In the case of pooled samples for nitrogen isotope
analysis, the size data is the mean size of all pooled individuals. A
linear model was used in each case (species and community levels). For
non-significant relationships between fish size andδ 15N values (i.e. p-value ≥0.05) at the
species level, coefficients of variation were calculated to explore the
dispersion of values.
Variance partitioning
Variance partitioning was used to calculate the variance explained by
the different variables included in a model (Borcard et al., 1992;
Legendre and Legendre, 2012). This is done by developing a set of
partial models (in a multivariate or univariate framework) created using
a subset of predictor variables. Here, the objective was to test to what
extent the individual size and the sampling depth influence theδ 15N values at the specific level. Due to the
restricted depth range at which Aphanopus carbo and Stomias
boa were captured (≤ 100m), the variance partitioning was not performed
on these two species. The model results are composed of the proportion
of δ 15N values influenced by size and depth
separately, and a third fraction representing the shared fraction of
variation explained when both variables are included in the model. An
ANOVA-type permutation test was performed for each model to test the
significance of the influence of each variable (depth and size) on
δ15N values. Since the third fraction is deduced from
the sum of variances, it cannot be tested statistically. The R packagevegan was used to perform the tests (Oksanen et al., 2022). All
the graphics were performed with the ggplot2 R package and all
statistical analyses were performed in the R environment version 4.3
(Wickham et al., 2016; R Core Team, 2023).