AquaFeed 0724 PR NO BLEED - Edited - 240827 - 082823

Rev Fish Biol Fisheries
https://doi.org/10.1007/s11160-024-09875-x
ORIGINAL RESEARCH
Octopus diet during the settlement period using DNA

metabarcoding
Oscar Escolar ·
Fernando Á. Fernández‑Álvarez ·
Roger Villanueva
Received: 18 July 2023 / Accepted: 10 July 2024

© The Author(s) 2024
Abstract The settlement phase is a challenging amphipods in importance. Niche breadth index
period for meropelagic octopus, as they adapt to their assigned to small recently settled octopus the role
new life in the sea bottom after a planktonic period. of a specialised predator, increasing the diversity of
Their ecology and trophic interactions with the sur- prey as they grow. Larger juveniles displayed a higher
rounding fauna in the wild are practically unknown. number of prey taxa suggesting a decreasing preda-
To understand their predatory role in the littoral tory specialisation with octopus size and an ontoge-
zone, the diet of recently settled Octopus vulgaris netic shift in trophic ecology from diet mainly based
from a Mediterranean sandy bottom was studied on amphipods to a richer diet. The considerable arm
through molecular methods. Amphipods were present elongation and the strong muscular arm crown devel-
in all the analysed stomach contents, with a total of opment after settlement probably allow the juvenile
20 amphipod taxa recorded as prey. Jassa slatteryi benthic octopus to develop ambush predation and
and Microprotopus maculatus were the most com- speculative hunting, increasing the range of poten-
monly found amphipod species. Hydrozoans, deca- tial benthic prey species along its adaptation to the
pod crustaceans, cephalopods and bivalves followed benthos.
Supplementary Information The online version

contains supplementary material available at https://doi.
org/10.1007/s11160-024-09875-x.
O. Escolar (*) · F. Á. Fernández‑Álvarez · R. Villanueva

Institut de Ciències del Mar (ICM-CSIC), Passeig Marítim
de la Barceloneta 37–49, 08003 Barcelona, Spain
e-mail: escolar@icm.csic.es
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Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Graphic abstract
Keywords Trophic ecology · Juvenile · Ontogeny · especially when considering advanced paralarval
Cephalopoda · Octopoda · eDNA stages (Roura et al. 2019). Besides, few studies had
succeeded in obtaining benthic individuals in the
lab. The settlement phase is virtually unknown in
Introduction the wild with the exception of a study on the popu-
lation dynamics of Octopus bimaculatus in Southern
The early life of many marine organisms occurs in California, where recently settled individuals were
the water column as planktonic larvae, when they collected in kelp holdfasts along the year (Ambrose
passively dispersed with the movements of the water 1988). In the lab, the settlement of meropelagic octo-
masses (McManus and Woodson 2012). At the end of puses starts when individuals begin to adhere to the
this developmental stage, their morphologies change tank surfaces with their suckers during increasing
towards their adult forms. Benthic octopods have two periods of time and search for shelters to hide (Vil-
development modes: (1) meropelagic, which are tem- lanueva 1995; Dan et al. 2021b; Roura et al. 2023a).
porarily pelagic, producing thousands of eggs with During the settlement period, octopus should adapt
planktonic hatchlings, and (2) holobenthic, that pro- their morphology to their new life at the sea bot-
duce a relative smaller number of eggs with direct tom, which involves quick learning to change their
benthic hatchlings with similar ecology and habitat behaviour, such as learning to avoid new predators
as their parents (Villanueva and Norman 2008). For and adapt to new foraging techniques. They also face
meropelagic species (Lincoln et al. 1998), their eco- important morphological changes, such as the strong
logical role is expected to suffer a drastic change, as positive allometric arm growth, the genesis of new
they adapt from pelagic to benthic environments dur- chromatophores, iridophores and leucophores, the
ing a life-challenging step. While there are a number development of skin sculptural components and a
of studies dealing with the planktonic stage of octo- horizontal pupillary response (Villanueva and Nor-
pus under laboratory conditions (e.g., Iglesias et al. man 2008; Dan et al. 2021b). They also loss several
2014; Dan et al. 2019, 2021a, b; Uriarte et al. 2010), characters present during their planktonic life, such
their study in natural environments is challenging, as the Kölliker organs (Villanueva et al. 2021), the
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lateral line system and the oral denticles of the beaks is composed of a wide range of prey that reflect its
(Boletzky 1971; Lenz 1997; Villanueva and Norman opportunistic and plastic predatory behaviour, mostly
2008). composed of crustaceans, molluscs and teleost fish
The common octopus Octopus vulgaris is a spe- (see among others, Guerra 1978, b; Sánchez and
cies with high commercial value that supports the Obarti 1993; Quetglas et al. 1998; Rosa et al. 2024).
most intense octopus fishery of the world (Sauer Despite the fact that feeding habits of subadult
et al. 2021). Octopus vulgaris is a meropelagic spe- and adult cephalopods in the wild are relatively well
cies (Lincoln et al. 1998) whose paralarvae develop known in several species (e.g., Nixon 1987; Ibáñez
in the plankton for several weeks, depending on the et al. 2021; Ribas et al. 2021), including metabarcod-
water temperature (Villanueva et al. 2016). Dur- ing DNA studies of stomach contents in subadult and
ing laboratory rearings at 21 °C, settlement was adult octopus (Ramos et al. 2023), the diet of early
observed from octopuses aged 47 days (Villanueva stages in the wild is far more mysterious. This is due
1995; Iglesias and Fuentes 2014) with a transitional to the small size of early ontogenetic stages and to the
period to a full benthic life of around 10 days (Vil- high degree of prey fragmentation. The use of DNA
lanueva and Norman 2008). Studies on the develop- metabarcoding techniques is a good method for over-
ment and feeding of O. vulgaris paralarvae in the come this problem, as it allows the identification of
wild show that the paralarvae are active predators of digested unrecognizable food (e.g., Olmos-Pérez
zooplankton, predating mainly on larvae of decapod et al. 2017; Fernández-Álvarez et al. 2018; García-
crustaceans, and also feeding on other groups such as Mayoral et al. 2022). Contrasting with its ecological
molluscs, ophiuroids, amphipods, cladocerans, cope- and ontogenetic importance, the diet of newly settled
pods, chaetognaths and cnidarians (Roura et al. 2012, octopus is still unknown for any octopod species. The
2016, 2017; Olmos-Pérez et al. 2017). After settle- objective of the present study is to fill this knowledge
ment, benthic juvenile and subadult O. vulgaris indi- gap, which is paramount for understanding the chal-
viduals were collected during summer in the Azores lenging transition from planktonic to benthic life in
Archipelago using PVC pipes with different diam- meropelagic octopus.
eters offered as shelter, collecting more individuals in
sandy areas in comparison with rocky bottoms (Pham
and Isidro 2010). Subadult and adult O. vulgaris diet
Fig. 1 Map of sample area in Vilanova i la Geltrú (Latitude: 41.2136611, Longitude: 1.736531) (A). Recently settled common octo-
pus Octopus vulgaris of 327 mg in weight and 11.02 mm dorsal mantle length photographed in laboratory after collection (B)
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Material and methods settled individuals. Moreover, different sampling

gears were utilised in order to lower sampling bias of
Sample collection and laboratory procedures specific gears. Mean weight of the selected individu-
als was 3.55 ± 6.44 g, ranging from 0.17 to 37.45 g.
A total of 166 individuals of O. vulgaris collected Individuals were not sexed.
between March 2020 and March 2021 through the After finishing this sampling protocol and dur-
artisanal fishing fleet of Vilanova i la Geltrú (NW ing a recent related study also on juvenile octopus,
Mediterranean, Fig. 1), during 52 sampling days, we have observed that 5% MgCl2 anaesthesia during
were used to extract the stomach contents. Stomach 30 min used to euthanize the individuals, significantly
contents of individuals collected during spring (47%), reduced the weight of the juveniles. As far as we
summer (14%), autumn (27%) and winter (10%) were know, this is a previously unrecorded effect of M
gCl2
examined. The sampling was carried out between in cephalopods. For this reason, the fresh, pre-anaes-
05:00 and 11:00 h, on the sandy bottom of an artisa- thesia octopus weight of the individuals used during
nal octopus fishery ground and between 5 and 25 m the present study was estimated later. We obtained 35
depth. Individuals were obtained using experimen- additional O.vulgaris individuals 0.7 ± 1 g ranging
tal juvenile octopus collectors (Escolar et al. 2021) from 0.2 to 4.4 g in weight, collected from the same
(JOC, n = 80), cuttlefish (Sepia officinalis) basket sampling area during December 2023. Animals were
traps (CT, n = 68) and octopus (O. vulgaris) traps weighted before and after 5% MgCl2 anaesthesia dur-
(OT, n = 10) and octopus pots (OP, n = 8). After col- ing 30 min, then frozen at -20 °C and thawed after
lection, individuals were killed on board with an a few days to obtain their thawed weight, following
overdose of anaesthesia (5% M gCl2) following wel- a similar protocol as the individuals used for stom-
fare recommendations and guidelines for cephalopods ach analysis. The loss of weight due to 30 min in 5%
(Fiorito et al. 2015), and then stored at – 20 °C until MgCl2 was 18 ± 5%, ranging from 8 to 34%. It was
lab processing. observed that weight loss was inversely related to the
In the laboratory, individuals were weighed after juvenile weight, being higher in smaller individuals
thawing using a microbalance. The stomach contents (Online resource 1). Therefore, the estimation of the
of a total of 166 individuals were extracted and visu- corresponding pre-anesthesia weight was carried out
ally assessed during dissections under a binocular ste- according to the different octopus size groups (see
reoscope Leica S8AP0 (Leica Microsystems, Wetzlar, Table 1). For the largest group of juveniles (size class
Germany). Individuals with scarce stomach content 5, see below), it was not possible to estimate their pre-
were excluded. For DNA analyses, 91 octopuses were anesthesia weight due to the lack of large individuals
selected. Selection of individuals was focused on to test the degree of weight loss. When comparing
the smaller size-class, looking for the more recently
Table 1 Octopus vulgaris size classes in grams, number of cies richness of prey species in stomach contents, Operational
individuals, range, mean and standard deviation of octopus Taxonomic Units and identified species
individuals weight, estimated preanaesthesia weight and spe-
Octopus weight (g) OTUs Identified taxa
Size Class n Mean ± SD Range Estimated preanaesthesia Mean S ± SD Range S Mean S ± SD Range S
weight Mean ± SD (Range)
1 32 0.3 ± 0.1 0.2–0.4 0.4 ± 0.1 (0.2–0.5) 9.4 ± 5.3 3–21 5.3 ± 2.5 2–10
2 15 0.7 ± 0.1 0.5–0.9 0.8 ± 0.2 (0.6–1.1) 12.9 ± 12.2 4–54 5.9 ± 3.6 1–15
3 18 1.7 ± 0.4 1.0–2.4 2 ± 0.5 (1.2–2.8) 11.4 ± 6.7 4–28 6.1 ± 3.2 2–13
4 14 4.3 ± 1.3 2.7–6.4 4.8 ± 1.4 (3–7.1) 13.7 ± 6.6 7–27 6.1 ± 2.8 3–12
5 12 17.7 ± 8.5 9.1–37.5 N/A 17.3 ± 9.0 4–38 8.7 ± 4.5 3–16
Total 91 3.6 ± 6.4 0.2–37.5 0.2–N/A 12.1 ± 8.0 3–54 6.1 ± 3.2 1–16
g grams; Mean S mean value of species richness; n number of individuals; N/A not available; OTUs Operational Taxonomic Units; S
species richness; SD standard deviation
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post-anaesthesia and thawed weight, no significant After thawing in the laboratory, mesofauna was sep-
differences were obtained (Student t-test, p = 0.96). arated and identified to the lowest taxonomic level
To prepare the samples for the subsequent molecu- using taxonomic keys (Zariquiey 1968; Campbell
lar analysis, special attention was paid to avoid ambi- 1984; Riedl 1986; Ortiz and Jimeno 2001; Handoo
ent and cross contamination. In short, stomach con- and Hunt 2016). Five pools with a total of 70 differ-
tents were extracted carefully with sterilised tools. ent potential prey species (Decapoda, Amphipoda,
The sterilisation process consisted in several clean- Isopoda, Leptostraca, Mysidacea, Gastropoda, Bival-
ing steps: (1) tools were cleaned with jets of bleach via and Cephalopoda) were used to test the effect of
at 10% diluted in milli-Q water, (2) then with jets the blocking primers. DNA was extracted from each
of milli-Q water, and (3) finally with jets of ethanol of the five pools following the same methodology as
at 96%. After that, each surface of every tool was for the stomach content samples (see below).
carefully treated with ultraviolet light for at least A mix of DNA from the five pools of potential
20–30 min under the fume hood. After sterilisation, prey was used to develop a potential prey DNA solu-
sampling tools were transported and kept carefully tion, which was mixed with different amounts of O.
inside a plastic box also sterilised with the same vulgaris DNA for constructing the mock communi-
methodology. The extraction of the stomach content ties. Two mock communities were designed with dif-
of the individuals was executed inside a sterilised ferent proportions of predator–prey DNA solution as
work area, new sterile latex gloves were used for follows: 50:50 and 90:10, respectively. Three recently
each individual and sterile lab material was individu- settled octopus stomach samples and two mock com-
ally used for each dissection. Stomach contents were munities were used to prepare four different treat-
preserved in 96% ethanol and stored at – 20 °C until ments per sample by alternating the presence/absence
DNA extractions. of blocking primer, and using fixed (55 °C) or vari-
able (60 and 55 °C) annealing temperature.
Blocking primer design and testing through mock
communities of prey and stomach dissections DNA metabarcoding
Co-amplification of predator DNA together with the DNA was isolated from the samples using two dif-
prey is the main concern in dietary DNA metabarcod- ferent extraction kits according to sample size: the
ing studies, known as self-contamination (Fernández- Quick-DNA Microprep Plus Kit (Zymo Research)
Álvarez et al. 2018). To avoid the amplification of was used for the smaller samples and the E.Z.N.A
host cephalopod DNA in the stomach content analy- Tissue DNA kit (Omega Biotek) was used for the
sis, a pilot analysis was carried out to test and design larger ones. Both kits were used following the manu-
a blocking primer. The blocking primer (cytochrome facturer’s instructions and the DNA was resuspended
c oxidase subunit 1 (COI)-intF_OctopusBP 5′ CGT in a final volume of 10 or 100 µL for the small and
TTA CCC RCC TCT TTC AAG AAA TTT AGC large samples, respectively. An extraction blank was
TC 3′) was produced following the methodology included in every DNA extraction round and treated
described by Vestheim and Jarman (2008). It was as a regular sample to check for cross-contamination.
designed with Geneious 10.2.6 (Biomatters Ltd), DNA was quantified using the Qubit High Sensitivity
based on its complementarity to O. vulgaris COI dsDNA Assay (Thermo Fisher Scientific).
sequences downloaded from GenBank. A C3 CPG In the pilot study, the addition of blocking prim-
spacer was added to the 3’ end of the blocking primer ers and a fixed annealing temperature provided better
to prevent elongation. results (Online Resource 2). Thus, these conditions
Mesofauna (size range 0.2–20 mm) from the sam- were applied to the polymerase chain reaction (PCR)
pled area was collected through juvenile octopus of the stomach content DNA extractions. A 313-base
collectors, cuttlefish traps and octopus traps. The pair (bp) fragment of the mitochondrial gene COI
different sampling gears were sampled using a high- was amplified using the primer pair COI-intF (5′
pressure seawater flux inside a 100 L plastic bucket GGW ACW GGW TGA ACW GTW TAY CCY CC
with a 200 μm plankton net surrounding the bucket. 3′) (Leray et al. 2013) and HCO2198 (5′ TAA ACT
The collected mesofauna was preserved at – 20 °C. TCA GGG TGA CCA AAA AAT CA 3′) (Folmer
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et al. 1994). PCRs were carried out in a final volume specific identifications, and between 90 and 97% for
of 12.5 µL, containing 1.25 µL of template DNA, identifications at the family level. When BLAST hits
0.5 µM of each primer, 10 µM of the blocking primer, were sequences identified as more than a single spe-
6.25 µL of Supreme NZYTaq 2 × Green Master Mix cies or belonged to different taxonomic groups, they
(NZYTech), and ultrapure distilled water up to 12.5 were assigned to the lowest taxonomic level shared
µL. The reaction mixture was incubated as follows: an among the original identifications. As PCR produces
initial denaturation step at 95 °C for 5 min, followed amplification bias that will skew abundance estima-
by 35 cycles of 95 °C for 30 s, 55 °C for 45 s, 72 °C tion (O’Rorke et al. 2012), the number of reads in the
for 45 s, and a final extension step at 72 °C for 7 min. results of the genetic analysis has not been taken into
Illumina sequencing indexes were attached to these account for downstream analyses and all inferences
amplicons at their 5′ ends in 5 cycles following the are based on presence/absence of prey OTUs.
same PCR conditions but with an annealing tempera-
ture of 60 °C. Data and statistical analyses
De novo chimaera detection was performed
using the UCHIME algorithm (Edgar et al. 2011) Octopus were arranged in five groups according to
implemented in VSEARCH (Rognes et al. 2016). their weight to assess possible size-related differences
Sequences were clustered with the SWARM algo- in feeding (Table 1). Trophic ecology data were ana-
rithm (Mahé et al. 2015) with a value of 13 follow- lysed through several indexes. The Emptiness Index
ing Bakker et al. (2019). The taxonomic identification (EMI = Es/Ts × 100) evaluates the number of empty
was performed by querying the sequences resulting stomachs (Es) in relation to the total number of ana-
from clustering against the COI gene database build lysed stomachs (Ts). The taxa groups of Chromista,
by Robeson et al. (2020) (updated on July 2020) Fungi, Protozoa, Plantae, Insecta and Arachnida were
based on the Barcode of Life Data System (BOLD) excluded as prey intentionally ingested/selected for
library reference database (Ratnasingham and Hebert downstream analyses. The relative importance of a
2007). To do so, the script feature-classifier classify- particular prey was studied through the frequency of
consensus-vsearch implemented in Qiime2 (Boku- occurrence index (FO = Si/Ts × 100), where Si is the
lich et al. 2018) and the VSEARCH algorithm with number of octopus stomachs in which the prey was
a sequence similarity threshold of 90% were used. registered. Samples whose rarefaction curves did not
Singletons (OTUs containing a single sequence in the tend towards saturation (n = 4), were excluded to sta-
whole dataset) were excluded from the OTU table. In tistical analyses and Cardona’s index estimation to
DNA metabarcoding studies, the mistagging phenom- avoid errors in the comparisons between groups. Car-
enon has been reported (Esling et al. 2015, Bartram dona’s index (B’ = (SFO-s/100 × R) was used to esti-
et al. 2016) in which a low percentage of the reads of mate the trophic niche breadth using the frequency
a sample can be assigned to another as the result of of occurrence data, where s represents the standard
the misassignment of the indices during library prep- deviation of occurrence frequencies and R is the num-
aration, sequencing, and/or demultiplexing steps. To ber of prey-species used by the whole guild (Cardona
account for this error, sequences with less of 0.01% 1991). This index assesses the degree of specialisa-
frequency in each sample were removed. Additional tion with values ranging from 0.01 (highly specialist)
filters were used based on the taxonomic assign- to 1.0 (complete generalist).
ment: the Operational Taxonomic Units (OTUs) Statistical analyses were carried out with the
assigned to Mammalia and Fungi were removed from software PERMANOVA+ for PRIMER 6 (Ander-
the OTU table, whereas the unidentified OTUs were son et al. 2008). To evaluate the data without tak-
kept for downstream analyses. Identification of those ing into account the number of reads as abundance,
unassigned OTUs was performed through BLAST the number of reads of each OTU corresponding to
(Altschul et al. 1990) at the NCBI portal. Identifica- a prey category per sample was transformed to cre-
tion was considered successful only if the following ate a presence/absence matrix (i.e., “0” was consid-
parameters were fulfilled: percent identity of 90%, ered as absence of a prey category, and any number
e-value of 1e-05, and a minimum hit coverage of 80%. of reads codified as “1” and considered as presence
The similarity threshold of 97% or higher was set for of that prey category). After the transformation of
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reads to a presence/absence matrix, data were stand- larger proportions of O. vulgaris reads: 1.27–1.35%
ardised by the total of samples, and then a resem- for the mock community 50:50 and 10.6–13.6% for
blance matrix using the Jaccard Index was created. the 90:10. For the recently settled octopus stomach
For comparison, semiquantitative data was stand- content samples, host reads represented 0.0–0.3%
ardised, square root transformed and a resemblance with blocking primers and 2.4–39.7% without block-
matrix using the Bray–Curtis similarity method was ing primers.
applied. Non-metric multidimensional scaling (MDS)
(Clarke and Warwick 2005) analyses with the super- DNA metabarcoding and prey profiles
imposition of Cluster were applied. The MDS were
represented by single samples, factors groups and by A total of 5,399,108 reads were obtained, resulting in
the average of size class and sampling gear groups a mean of 59,330.9 ± 30,561.1 (range 889–125,387)
combined (Online Resource 2). A permutational mul- reads per sample. From those, 4,304,850 reads and
tivariate analysis of variance (PERMANOVA) with a a mean of 47,306 ± 33,095 (range 5–120,799) reads
Type III sum of squares (unrestricted permutation of per sample were identified, and a total of 1,050,915
raw data = 999) was used to test for significant dietary and a mean of 11,548.5 ± 17,428.3 (range 0–72,721)
differences among size class, sampling gears and remained taxonomically unassigned. DNA metabar-
season. Additionally, a similarity percentage analy- coding revealed a total of 352 OTUs on O. vulgaris
sis (SIMPER) detected the intra-group percentage of stomach contents (Online Resource 4). Initially, with
mean diet dissimilarity for each size class, gear and the use of the BOLD database in Qiime2 a total of
season considering prey contribution of over 75%. 108 assigned and 260 unassigned taxa were classi-
fied. After subsequent BLAST searches at NCBI, data
treatment and joining the reads belonging to the same
Results taxa but over-splitted in several OTUs, the number of
identified OTUs increased to 139, while 213 remained
A total of 121 out 166 dissected O. vulgaris showed unassigned. Identified OTUs belonged to 18 phyla,
food remains in their stomach content through visual 57 orders and 95 families. Mean self-contamination
assessment, resulting in an emptiness index (EMI) was 2.2 ± 8.7% (0–62.9%) of the reads. The number
of 27%. A total of 91 octopuses were selected for of OTUs present per stomach content, including sev-
DNA metabarcoding analysis, ranging in weight from eral unassigned species, was 12.1 ± 8 (range: 3–54).
0.17 to 37.45 g. Particular effort was made to select The number of identified taxa present per stomach
the smaller size class (n = 32) individuals between content was 6.2 ± 3.2 (range: 1–16). Species richness
0.17 and 0.5 g, which are the more recently settled increased with the octopus size class (Table 1).
individuals. Among the identified taxa present in the stomach
contents, the group Amphipoda was the order with the
Blocking primer performance highest frequency of occurrence, which was present
in all the analysed stomach contents (FO = 100%).
No substantial effect was detected in read counts Other prey groups by FO rank were: Hydrozoa (46%),
using fixed or variable annealing temperature. There Decapoda (31%), Ochrophyta (22%), Bivalvia (22%),
were not significative difference on the number of Copepoda (13%), Gastropoda (12%), Sepiida (12%)
reads induced by the use or not of the blocking prim- and Chordata (11%). The rest of groups with FO > 2%
ers on octopus’ prey, both in the mock communities are represented in Fig. 2.
(Student t-test, p > 0.64) and the stomach contents The species with higher FO were the amphipods
(p > 0.53). The presence of host (O. vulgaris) reads Jassa slatteryi (90%), Microprotopus maculatus
was markedly higher in PCRs without the blocking (49%), Amphipoda sp. (35%), Gammarus crinicornis
primer (Online Resource 3). For the mock communi- (34%), Phtisica marina (25%) and Caprella scaura
ties in PCRs with blocking primers, O. vulgaris reads (23%). These six identified amphipods were six of the
represented 0% of the total amount of reads for the top seven OTUs with higher FO in the analysed stom-
mock community proportion 50:50, and 0.03% for ach contents. The third taxon with the highest FO
90:10. The PCRs without blocking primers showed (47%) was not identified with the BOLD and NCBI
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Fig. 2 Frequency of Occur-

rence (FO, %) and number
of identified OTUs (orange
line) in the stomach con-
tents of O. vulgaris (n = 91).
Groups with FO > 2% are
shown. Chromista, Fungi,
Protozoa, Plantae, Insecta,
and Arachnida taxa were
excluded. Recently settled
common octopus Octo-
pus vulgaris of 327 mg
in weight and 11.02 mm
dorsal mantle length
photographed in laboratory
after collection. Silhouette
images from https://www.
phylopic.org/
databases. The identity of this species remained unas- species: Sparus aurata (3%), Mullus barbatus (3%),
signed, as there were no DNA barcodes available. The Conger conger (2%), Spondyliosoma cantharus
next species with higher FO were from group Hydro- (1%) and Scyliorhinus canicula (1%).
zoa, Obelia dichotoma (22%), Bougainvillia muscus There were several unassigned OTUs with
(20%) and Obelia geniculata (14%). Three species important values of FO (range 1–47%) that might
had the same FO of 12%: the cephalopod Sepia offici- represent an important role in the diet of the benthic
nalis, the amphipod Leucothoe incisa and the clam transition, but its identification was not possible at
Mactra stultorum. Interestingly, M. stultorum showed present (Table 3).
a remarkable increment in its FO from 0% by the The amphipod Jassa slatteryi was the species
smaller octopus size class 1 (0.2–0.4 g) to 41.7% with with greater contribution for defining each group
the larger size class 5 (9.1–37.5 g) (Table 2). (size class, gear and season) as it represents from
Decapod crustaceans were the following group in 40 to 100% of the contribution of similarity in the
importance as prey of FO: Philocheras bispinosus SIMPER analysis. Table 4 summarises the signifi-
(10%), Athanas nitescens (9%), Liocarcinus vernalis cant differences among size class, collector gear
(9%) and Macropodia rostrata (5%). The species P. and season groups (P(Perm) < 0.05) using a PER-
bispinosus and A. nitescens were more frequent in the MANOVA pairwise tests analysis. The MDS analy-
stomach content of larger individuals from the size sis showed for the assessment of single samples a
classes 4 (2.7–6.4 g) and 5 (9.1–37.5 g) (Table 2). value of two-dimensions stress of 0.22 with pres-
Hydrozoans were more frequently found in the stom- ence/absence assessment, and 0.21 with the use
achs than decapods. However, the number of identi- of semiquantitative data. In the case of the factors
fied hydrozoans (n = 11) was lower than decapods size class and collector gear combined, the value
(n = 15). of two-dimensions stress was 0.19 with presence/
Octopus vulgaris juveniles also preyed on other absence assessment, and 0.18 with the use of semi-
cephalopods apart from Sepia officinalis, such as quantitative data treatment (Online Resource 2).
Sepiola affinis (4%), and exceptionally the small- Respective values of two-dimensions stress near to
spot octopus Callistoctopus macropus (1%). The 0.2 provided a poor representation that should not
class Bivalvia was represented mainly by Mactra be placed on the detail of the plot (Clarke and War-
stultorum (12%) and Chamelea gallina (5%). The wick 2001). Thus, differences in stress values are
presence of fish was low, with only five identified
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Table 2 Frequency of occurrence (FO) of identified species (with FO > 3%) recorded from stomach contents of 91 samples of O.
vulgaris in percentage (%), assorted by size class
Phylum Order Species FO per Size Class (%)
All (n = 91) 0.2–0.4 g 0.5–0.9 g 1.0–2.4 g 2.7–6.4 g 9.1–37.5 g
(n = 32) (n = 15) (n = 18) (n = 14) (n = 12)
Arthropoda Amphipoda Jassa slatteryi 90.1 93.8 86.7 94.4 78.6 91.7
Arthropoda Amphipoda Microprotopus maculatus 49.5 43.8 73.3 61.1 35.7 33.3
Arthropoda Amphipoda Amphipoda sp. 35.2 40.6 13.3 44.4 28.6 41.7
Arthropoda Amphipoda Gammarus crinicornis 34.1 28.1 60.0 38.9 21.4 25.0
Arthropoda Amphipoda Phtisica marina 25.3 15.6 33.3 27.8 21.4 41.7
Arthropoda Amphipoda Caprella scaura 23.1 25.0 13.3 27.8 14.3 33.3
Cnidaria Leptothecata Obelia dichotoma 22.0 21.9 6.7 33.3 14.3 33.3
Cnidaria Anthoathecata Bougainvillia muscus 19.8 15.6 6.7 22.2 35.7 25.0
Cnidaria Leptothecata Obelia geniculata 14.3 21.9 6.7 11.1 14.3 8.3
Mollusca Sepiida Sepia officinalis 12.1 6.3 13.3 11.1 28.6 8.3
Arthropoda Amphipoda Leucothoe incisa 12.1 12.5 20.0 0.0 21.4 8.3
Mollusca Venerida Mactra stultorum 12.1 0.0 6.7 11.1 21.4 41.7
Arthropoda Decapoda Philocheras bispinosus 9.9 12.5 0.0 0.0 7.1 33.3
Arthropoda Decapoda Athanas nitescens 8.8 6.3 0.0 0.0 14.3 33.3
Arthropoda Decapoda Liocarcinus vernalis 8.8 0.0 13.3 11.1 14.3 16.7
Cnidaria Leptothecata Campanularia hincksii 7.7 9.4 0.0 5.6 7.1 16.7
Cnidaria Leptothecata Clytia hemisphaerica 6.6 9.4 13.3 0.0 7.1 0.0
Arthropoda Amphipoda Atylus swammerdami 6.6 9.4 0.0 5.6 7.1 8.3
Mollusca Venerida Chamelea gallina 5.5 3.1 6.7 0.0 7.1 16.7
Arthropoda Decapoda Macropodia rostrata * 5.5 0.0 0.0 16.7 14.3 0.0
Arthropoda Amphipoda Gammarella fucicola 4.4 3.1 0.0 0.0 7.1 16.7
Arthropoda Calanoida Paracalanus quasimodo 4.4 9.4 0.0 0.0 0.0 8.3
Mollusca Sepiolida Sepiola affinis * 4.4 3.1 0.0 5.6 0.0 16.7
Cnidaria Anthoathecata Pennaria disticha 4.4 3.1 6.7 5.6 7.1 0.0
Mollusca Venerida Spisula subtruncata 3.3 0.0 0.0 0.0 14.3 8.3
Arthropoda Amphipoda Ericthonius punctatus 3.3 0.0 6.7 5.6 7.1 0.0
Arthropoda Decapoda Paguridae sp. * 3.3 0.0 0.0 11.1 7.1 0.0
Arthropoda Decapoda Xantho pilipes * 3.3 0.0 0.0 5.6 0.0 16.7
Arthropoda Decapoda Liocarcinus depurator 3.3 0.0 0.0 5.6 0.0 16.7
Arthropoda Amphipoda Corophium sp. 3.3 0.0 6.7 5.6 7.1 0.0
Arthropoda Sessilia Balanus sp. 3.3 0.0 13.3 0.0 7.1 0.0
Chordata Spariformes Sparus aurata 3.3 3.1 6.7 0.0 7.1 0.0
Chordata Perciformes Mullus barbatus 3.3 0.0 6.7 5.6 7.1 0.0
Prey taxa are ordered by total FO (%). Taxa groups of Chromista, Fungi, Protozoa, Plantae, Insecta, Arachnida were excluded. The
taxonomic criterion of WoRMS Editorial Board (2022) was applied. Asterisks designate species identified through BLAST at NCBI
N/A not available; SO superorder
minimal between presence/absence and semiquanti- The assessment of the trophic niche breadth of all
tative treatments. 87 samples through Cardona’s index showed a value
As shown in Table 5, the factors size class, gear B’ = 0.05, which is indicative of specialised preda-
and season showed a significant difference (p < 0.05) tion. A weak trend towards a more generalist diet
with separated PERMANOVA analysis designs. was observed when data were studied separately
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Table 3 Results of the SIMPER analysis showing the average similarity in percentage (%) and the percentage of contribution of
prey (% > 5%) to each octopus size class, sampling gear and season
Group Av. Cumulative (%) Prey (% contribution to similarity)
Similar-
ity (%)
Size class group 1 33 81 Jassa slatteryi (59), Amphipoda sp. (12), Microprotopus maculatus (10)
Size class group 2 37 92 Jassa slatteryi (40), Microprotopus maculatus (32), Gammarus crinicornis (20)
Size class group 3 33 85 Jassa slatteryi (53), Microprotopus maculatus (18), Amphipoda sp. (8), Gammarus
crinicornis (6)
Size class group 4 20 77 Jassa slatteryi (54), Microprotopus maculatus (11), Bougainvillia muscus (11)
Size class group 5 23 75 Jassa slatteryi (59), Mactra stultorum (10), Caprella Scaura (6)
JOC5 45 91 Jassa slatteryi (47), Amphipoda sp. (29), Caprella scaura (15)
JOC15 27 87 Jassa slatteryi (50), Obelia dichotoma (11), Microprotopus maculatus (10), Phti-
sica marina (8), Gammarus crinicornis (8)
OT 16 100 Jassa slatteryi (100)
CT 31 85 Jassa slatteryi (47), Microprotopus maculatus (28), Gammarus crinicornis (10)
OP 30 92 Jassa slatteryi (52), Gammarus crinicornis (12), Mactra stultorum (10), Micropro-
topus maculatus (6), Bougainvillia muscus (6), Leucothoe incisa (6)
Autumn 32 83 Jassa slatteryi (53), Caprella Scaura (16), Bougainvillia muscus (7), Amphipoda
sp (7)
Summer 41 94 Jassa slatteryi (55), Amphipoda sp. (39)
Spring 33 85 Jassa slatteryi (45), Microprotopus maculatus (30), Gammarus crinicornis (10)
Winter 31 82 Jassa slatteryi (49), Gammarus crinicornis (17), Leucothoe incisa (10), Bougainvil-
lia muscus (8)
Av. Similarity % average similarity (%); Cumulative % Sum of cumulative percentages of the prey species included in the table;
JOC5 juvenile octopus collector at 5 m depth; JOC15 juvenile octopus collector at 15 m depth; CT cuttlefish trap; OT octopus trap;
OP octopus pot
by octopus size classes: B’ size class groups 1 also visually assessed, the EMI values range from 45
(0.2–0.4 g), 2 (0.5–0.9 g) and 4 (2.7–6.4 g) had a to more than 70% (Sánchez and Obarti 1993; Diatta
slightly more specialised index (B’ = 0.05) than the et al. 2001; Rosa et al. 2004; Ajana et al. 2018). The
other size groups. The size class 3 (1.0–2.4 g) showed higher presence of stomach with food contents of the
a value of B’ = 0.06, while the largest size group 5 juveniles examined in comparison with the adults
(9.1–37.5 g) displayed the highest value of B’ = 0.07. from previous studies could be related with several
undetermined factors. However, this fact suggests
that juvenile octopus maintain a notable predatory
Discussion activity.
A total of 131 taxa belonging to 18 phyla, 57
Most studies on cephalopod trophism are focused orders and 95 families were molecularly identified.
on adults and subadults, usually showing empti- When Chromista, Fungi, Protozoa, Plantae, Insecta
ness indexes higher than 45% that could be affected and Arachnida were excluded as plausible primary
by variables such as temperature, season, sex and predation, the species richness (S) of potential diet
food availability, among others (see Ibáñez et al. in O. vulgaris juveniles reached S = 96 (Online
2021). Here, a lower emptiness index value of 27% resource 6). This value represents a higher num-
was found during the present study, indicating that ber of taxa compared with previous studies from
roughly only one quarter of the stomach contents vis- the same species based on DNA metabarcoding of
ually examined in recently settled O. vulgaris did not paralarval diet, obtaining 54 (Olmos-Pérez et al.
have food remains. In subadult and adult O. vulgaris 2017) and 87 prey species (Roura et al. 2023b).
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Table 4 Results of the PERMANOVA pair-wise tests for differences amongst octopus size class, sampling gears and seasons
Source Similarity % t P (perm) Perms Cumulative % Prey (% contribution to dissimilarity)
Size class group 1 & 2 20.3 1.49 0.007 999 31 Microprotopus maculatus (9), Gammarus crinicornis
(9), Amphipoda sp. (8)
Size class group 2 & 4 16.2 1.24 0.038 999 26 Microprotopus maculatus (8), Gammarus crinicornis
(8)
Size class group 2 & 5 14.4 1.51 0.001 997 23 Microprotopus maculatus (11), Gammarus crini-
cornis (7)
JOC15 & JOC5 16.8 2.02 0.001 997 42 Amphipoda sp. (10), Caprella scaura (8), Micropro-
topus maculatus (6), Obelia dichotoma (6), Gam-
marus crinicornis (6), Phtisica marina (6)
JOC5 & OT 14.3 1.50 0.024 784 39 Amphipoda sp. (10), Caprella scaura (7), Bou-
gainvillia muscus (6), Gammarella fucicola (6),
Echinocardium cordatum (6)
JOC5 & CT 16.8 2.62 0.001 997 35 Amphipoda sp. (11), Microprotopus maculatus (9),
Caprella scaura (8), Gammarus crinicornis (7)
JOC5 & OP 19.2 1.65 0.002 996 32 Amphipoda sp. (8), Caprella scaura (7), Mactra
stultorum (7), Gammarus crinicornis (7)
Autumn & Summer 20.6 1.55 0.005 999 21 Amphipoda sp. (10), Caprella scaura (9), Bougain-
villia muscus (6), Microprotopus maculatus (6)
Autumn & Spring 16.33 2.03 0.001 997 25 Microprotopus maculatus (9), Caprella scaura (7),
Gammarus crinicornis (6), Amphipoda sp. (6)
Summer & Spring 16.77 2.10 0.001 997 25 Amphipoda sp. (12), Microprotopus maculatus (10),
Gammarus crinicornis (7)
Summer & Winter 16.7 1.62 0.003 995 11 Amphipoda sp. (8), Gammarus crinicornis (8),
Leucothoe incisa (7)
Only pair-wise tests that are significant were listed. Resemblance: Bray Curtis similarity. Sums of squares Type III. Unrestricted per-
mutation of raw data (999). Prey categories contribution to average dissimilarity percentages higher than 5% (SIMPER)
Cumulative % Sum of cumulative percentages of the prey species included in the table; CT cuttlefish trap; JOC5 juvenile octopus
collector at 5 m depth; JOC15 juvenile octopus collector at 15 m depth; OT octopus trap; OP octopus pot; Perms Unique permuta-
tions; P(Perm) permutation P-value; Similarity % percentage of similarity; t t-value
Table 5 Frequency of PERMANOVA analysis through the sum of squared Type III, with unrestricted permutations
Source df SS MS Pseudo-F P(Perm) Perms
Size class 4 18,856 4714 1.4205 0.012 996

Residuals 82 2.72E + 05 3318.6
Gear 4 32,479 8119.8 2.5757 0.001 998
Residuals 82 2.58E + 09 3152.5
Season 3 28,377 9458.9 2.9896 0.001 998
Residuals 83 2.62E + 05 3164
df degrees of freedom; MS mean squares; Perms permutations; P(Perm) permutation P-value; Pseudo-F Pseudo-F ratio; SS sums of
squares
Furthermore, results from visual analyses of sub- directly comparable. Prey species richness by
adult and adult individuals obtained lower richness octopus juveniles ranged from 1 to 16 prey with a
values of S = 35 (Guerra 1978, b) and S = 23 (Rosa mean of 6 prey. Mean prey S increased with size
et al. 2004). However, it must be pointed out that class (Table 1). The high number of identified prey
molecular and visual assessments of prey are not
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suggests that recently settled and early benthic octo- found in the diet of O. vulgaris and Alloteuthis media
pus could predate on a wide spectrum of animals. planktonic paralarvae diet study by Olmos-Pérez et al.
(2017). Also, Obelia dichotoma was found in the diet
Amphipods as main prey for O. vulgaris at settlement of O. vulgaris paralarvae by Roura et al. (2023b).
and other secondary prey groups Surprisingly, present data suggest that hydrozoans
constitute also an important feeding resource for
The most important prey of O. vulgaris juveniles was recently settled octopus.
a Mediterranean invasive amphipod species, Jassa Eggs of the European cuttlefish Sepia officinalis
slatteryi, present in 90% of the samples. It is an epi- are normally found in the fishing gears, which are a
benthic species of intertidal vegetated habitats, living hard substrate where mature females lay eggs (Bar-
at depths lower than 30 m (Jeong et al. 2006). Conlan rett et al. 2022). As cuttlefish egg capsule protect the
et al. (2021) pointed out that some species of Jassa had embryo and its predation is usually limited by the
been transported by human vectors since at least the mechanical power of the predator capture tools (Mar-
nineteenth century, which explains their wide distribu- tins et al. 2018), it is likely octopus predated on hatch-
tion across the world. Amphipods are primary consum- lings instead. Furthermore, analysing differences by
ers, eating detritus and phytoplankton, and are in turn octopus size classes, the bivalves were preyed pref-
preyed by higher trophic level consumers, such as fish erably by larger juveniles. Thereby, M. stultorum
and large crustaceans (Jeong et al. 2006; Dauvin 2024). increased its occurrence in the stomach contents of
Jassa species has been used as successful prey for par- larger octopus and C. gallina and S. subtruncata were
alarval rearings of the octopus Robsonella fontaniana also more consumed for members of the largest octo-
(González et al. 2011), O. vulgaris (Tur et al. 2020; Varó pus size class 5 (9.1–37.5 g) (Table 2). Octopus can
et al. 2022), hatchlings of the cuttlefish Sepia officinalis consume bivalves through two possible methods: (1)
(Baeza-Rojano et al. 2010), and other marine gammarid drilling a hole on the shell then injecting cephalotoxin
amphipods showed to be suitable prey for Octopus maya or (2) by pulling the shells using the strength of their
hatchlings (Baeza-Rojano et al. 2013). The present study suckers and arms (Fiorito and Gherardi 1999). The
on the diet of wild juvenile octopus confirms amphipods consumption of clams (Ruditapes phillipinarum) by
as a food resource to develop laboratory rearing experi- Octopus sinensis juveniles under laboratory condi-
ments on early stages of octopus. Different technolo- tions increased gradually with octopus size and were
gies for amphipod production have been published (see not drilled (Dan et al. 2021b). The fact that bivalves
between others, Fernández-González et al. 2018; Jimé- were more abundant in stomach contents of the larg-
nez-Prada et al. 2021; Vargas-Abúndez et al. 2021; Sha- est size class of O. vulgaris juveniles confirms the
hin et al. 2023) and probably can be adapted for octopus. observations of Dan et al. (2021b), and are probably
The amphipod species Caprella scaura, the sixth spe- related with the rapid development of the arm crown
cies with more FO, is also considered a Mediterranean in larger juveniles reaching enough arm strength to
invasive species, first recorded in Roses Bay in 2005, pull bivalve shells apart.
169 km north to the sampled area (Martínez & Adarraga Decapods were the most consumed prey by O. vul-
2008). The predation of juvenile octopus on invasive garis paralarvae in the water column (Olmos-Pérez
species is of interest, opening the role of recently set- et al. 2017; Roura et al. 2023b). In juveniles, this prey
tled octopus as a native controller on two Mediterranean group increased their presence in the stomach contents
invasive species. This fact furthers into feeding plasticity of larger juveniles, as for P. bispinosus and A. nites-
of octopus (e.g., Fernández-Álvarez et al. 2020), which cens, mostly predated by the largest juveniles from size
are able to adapt to the use of new feeding resources class 5 (9.1–37.5 g). Furthermore, other Decapoda with
available in the area. an important FO were not consumed by the group of
The class Hydrozoa is the second group in terms the recently settled size class 1 (0.2–0.4 g) (Table 2).
of FO. It cannot be ascertained if the consumption of These differences in FO among size class groups could
hydrozoans occurred over polyp or medusa stages. be explained by the strong morphological development
This would be an important difference, as the polyp along the settlement period. Morphological adaptations
stage is sessile and probably easier to catch than the as arm elongation would further enhance new hunting
swimming medusa stage. Obelia geniculata was also techniques, such as ambush predation and speculative
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Fig. 3 Schematic representation of prey diversity (black silou- pod crustaceans and bivalves (present study). Subadult and
ettes) of Octopus vulgaris along its life cycle. During plank- adult stages prey mainly on crabs, molluscs and teleost fishes
tonic stage most frequently detected prey are decapod zoea, (Guerra 1978, b; Sánchez and Obarti 1993; Quetglas et al.
followed by calanoid copepods, brittle stars and cladocerans 1998; Rosa et al. 2024), while brooding females stop feeding
(Olmos-Pérez et al. 2017; Roura et al. 2023a, b). After set- after spawning. Silhouette images from https://www.phylo
tlement, during juvenile stage amphipods are the prey with pic.org/. Central image of adult octopus by Ricardo Roberto
higher frequency of occurrence, followed by hydrozoans, deca- Fernández
hunting on the seafloor, predatory behaviours well molecular dietary studies (e.g., Olmos-Pérez et al.
known in subadults and adults (Hanlon and Messenger 2017; Fernández-Álvarez et al. 2018; García-Mayoral
2018) that may need a learning or adapting period after et al. 2022; Roura et al. 2023b). It is remarkable that
settlement in the new benthic environment (Fig. 3). Fish the values of self-contamination obtained in this study
were represented by four species, with a maximum FO of before the use of blocking primers (Online Resource
3%. Surprisingly, it included the small-spotted catshark 2) were lower than the values observed in previous
Scyliorhinus canicula. This shark inhabits normally studies. Importantly, we were not able to identify any
deeper waters and its size at hatching is far larger than O. amplifying bias induced by the blocking primers on
vulgaris juveniles. Probably, this prey item might actually octopus prey, both in the mock communities and the
represent carrion consumption, likely coming from trawl stomach contents. While careful dissections are able
fishery discards or artisanal fishery bait, which is used to lower self-contamination values (e.g., Fernández-
in the same fishery ground and/or secondary predation Álvarez et al. 2018), our results indicate that the use
mediated by amphipods. Subadult and adult octopuses of blocking primers are especially useful to obtain
are known to be scavengers, as they are attracted by the better dietary assessments, especially for tiny animals
dead bait inside traps (Sauer et al. 2021) or feeded by fro- difficult to dissect as paralarvae. However, blocking
zen prey and/or artificial food in the lab (Villanueva et al. primers should be species-specific and therefore mul-
2017), suggesting that scavenger behaviour of juvenile tispecies projects would need to use adequate species-
octopuses in the wild also can be expected. specific primers for each predator, or assume possi-
ble amplification bias if a single blocking primer is
Benefits of the use of blocking primers designed for the whole set of predators.
The use of blocking primers achieved an exception- Limitations of the study

ally low value of self-contamination (2.2 ± 8.7%),
which contrast with the comparatively higher values As DNA metabarcoding involves a PCR, one of the
(70–95%) commonly observed in other cephalopod main biases that occurs is the primers not hybridising
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with the whole spectrum of actual prey. Besides, they under laboratory conditions intensifies after settle-
also might create amplification bias among the DNA ment in aquaria, and cannibalism has been reported in
of different species within the sample, as the primers wild populations of octopus (Ibáñez and Keyl 2010).
might be more efficient amplifying the DNA of some Thus, the existence of a cannibalistic component
species than others. Additionally, the high precision in the diet of O. vulgaris juveniles was not assessed
of recognition of DNA metabarcoding evokes the here. Molecular methods can be used for cannibal-
possibility that some identified taxa could have origi- ism assessment, as it was studied in lionfish through
nated from secondary predation (Ibáñez et al. 2021, microsatellites (Dahl et al. 2018).
as “secondary digestion”), which means that the
taxon was actually predated by the prey instead from Recently settled octopus as a specialist predator
a direct predation event from the predator. Moreo-
ver, other organisms living in association with the During settlement, the young octopus probably lives
prey, such as epiphytes, symbionts or parasites, can a period of learning-dependent foraging and discov-
also be sequenced. It is important to note that Clarke ering and tasting new prey species. The movements
et al. (2020) showed that secondary digestion could and permanence in a specific area could be selected
be detected in fish diet samples, but the relative read based on the quantity or quality of the available prey.
abundance of these items would rarely be greater than According to Ibáñez et al. (2021), many studies con-
1%. Importantly, Nigmatullin et al. (2009) interpreted fuse the terms opportunistic vs. generalist predator
the presence of secondary digestion in 43.3% of the and selective vs. specialist predator. These authors
stomach content analysis from the neon flying squid also noted that although most cephalopods exhibit a
Ommastrephes bartramii using morphological assess- generalised diet and wide trophic niche, their hunting
ment methods. Fernández-Álvarez et al. (2018) iden- strategy is selective and specialised at the individual
tified 42% of reads of secondary digested prokaryotic level. During the present study, the value of Cardona’s
algae and chloroplasts in subadult and adult flying index was low (B’ = 0.05). However, B’ increased
squids, while this component was minimal in par- slightly from 0.05 to 0.07 along octopus growth.
alarvae. Moreover, it is expected that digestion time These results show that the diet of the recently settled
would be longer in species with hard structures. Thus, and early benthic O. vulgaris is typical of a specialist
crustaceans should have longer digestion times than predator, strongly focused in amphipods, and that this
hydrozoans, for instance. This fact could provoke specialised dietary habits blur with the increase in
differences between the frequency of occurrence size. However, a mean of six identified prey or twelve
appeared with the analysis and their real contribution OTUs and a range between 1 and 16 identified spe-
to the diet. The 91 analysed stomach contents were cies or between 3 and 54 OTUs were found inside the
all from juveniles collected in a shallow (5–25 m stomachs, suggesting that the juveniles feed on a wide
depth) Mediterranean sandy bottom, during daytime, diversity of prey.
and mostly collected during spring and autumn. This Anderson et al. (2008) obtained values very simi-
environment and sampling characteristics obviously lar for adult Octopus americanus (identified as O.
conditioned the results. It is possible that octopus vulgaris) in the southern Caribbean with B’ of 0.08
juveniles settling in rocky beds might prey on dif- analysing the middens of 38 octopus dens. A similar
ferent organisms, as the benthic communities would relationship was obtained by Alejo-Plata et al. (2018)
definitely differ. Future studies taking into account for Octopus hubbsorum from NE Pacific, obtain-
different environments and seasons will be necessary ing B’ of 0.03 for small octopuses (40–80 mm ML)
to understand the feeding dynamics of recently settled and B’ of 0.09 for larger octopuses (> 80 mm ML).
octopus. A slightly high index (B’ = 0.24) was observed for
In DNA metabarcoding studies, DNA for the host Octopus rubescens found in beer bottles on a sand/
species is commonly regarded as self-contamination, muddy habitat in the NE Pacific (Anderson et al.
but it can also be explained by cannibalism. The use 1999) and a B’ of 0.28 was found for O. cyanea in
of blocking primers for O. vulgaris prevents the pos- Hawaii (Mather, unpublished data, cited in Anderson
sibility to assess cannibalism. According to Dan et al. et al. 2008).
(2021b), the incidence of cannibalism in O. sinensis
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Some studies considered the planktonic stage of O. Acknowledgements Special thanks to Eduardo Escolar,
vulgaris paralarvae as a generalist predator, and oth- owner and captain of the artisanal fishing boat Nova Miriam.
Thanks to him for all his work and marine knowledge shared
ers, as a specialist during their first days of life in the along all the sampling days carried out to get the successful
pelagic realm based on the prey species detected and realization of this project. Thanks to Ainhoa Bernal (Instituto
their relative abundances in the zooplankton (Roura Español de Oceanografía, CSIC) for her support with statisti-
et al. 2012, 2023b; Nande et al. 2017; Olmos-Pérez cal analyses. DNA metabarcoding analyses were carried out by
AllGenetics & Biology SL (www.allgenetics.eu). Funding was
et al. 2017). Nevertheless, several studies defined provided by the Spanish Ministry of Science, Innovation and
octopus as generalist feeders and Hanlon & Mes- Universities (OCTOSET, Ref. RTI2018-097908-B-I00; ECO-
senger (2018) assigned the term ‘opportunistic feed- PHYN, Ref. PID2021-126824NB-C32; MCIU/AEI/FEDER,
ers’. However, there were some individuals that EU), and the Spanish government through the “Severo Ochoa
Center of Excellence” accreditation (CEX2019-000928-S).
were specialising on certain taxa or even single spe- F.Á.F.-Á. was supported by a JdC-I Postdoctoral Fellowship
cies (Anderson et al. 2008). The results found in our Grant (Ref. IJC2020-043170-I) awarded by MCIN/AEI /https://
study on a sandy bottom area showed recently settled doi.org/10.13039/501100011033 and by a Beatriu de Pinós
octopus as specialist predators mainly focused on fellowship from Secretaria d´Universitats i Recerca del Depar-
tament de Recerca i Universitats of the Generalitat de Catalu-
amphipods. nya (Ref. BP 2021 00035). We thank the Associate Editor and
anonymous reviewers for their helpful comments.
Conclusions Author contributions RV defined the initial project and

all authors contributed to the study conception and design.
Sampling was performed by OE. Morphological data collec-
Amphipods are the prey with higher frequency of tion and analyses were performed by OE. Data and statistical
occurrence in the stomach contents of the recently analyses were performed by OE. Molecular data curation by
settled and early benthic individuals of O. vulgaris OE and FÁF-Á. Funding acquisition by RV. The first draft of
the manuscript was written by OE and all authors commented
in a littoral sandy bottom of the Mediterranean Sea. on subsequent versions of the manuscript. All authors read and
A total of 20 amphipod taxa were present in the ana- approved the final manuscript.
lysed stomach contents and this group seems to play a
key role as a feeding resource during the octopus set- Funding Open Access funding provided thanks to the
tlement period in the studied area. Jassa slatteryi and CRUE-CSIC agreement with Springer Nature.
Microprotopus maculatus are the amphipod species Data availability Genetic data underlying this article are
with higher occurrence, present in 90% and 50% of available in the GenBank Nucleotide Database at https://www.
the stomach contents, respectively. As J. slatteryi (1st ncbi.nlm.nih.gov/genbank/ and can be accessed with the Gen-
FO) and C. scaura (6th FO) are invasive species, it is Bank accession numbers OR131649–OR131744. The FastQ
files can be accessed within the GenBank Nucleotide Database
possible that recently settled octopus are controllers with the BioProject accession number PRJNA984187. Addi-
of these non-native species. Hydrozoans, decapods, tional material is available in Online Resources.
S. officinalis and M. stultorum were also important
prey. Niche breadth index assigned to recently settled Declarations
octopus the role of a specialised predator, increasing Conflict of interest The authors declare no conflict of inter-
their prey diversity along the juvenile benthic growth. est.
The analysed larger juvenile size class has a higher
diversity of prey, suggesting a decreasing predatory Open Access This article is licensed under a Creative Com-
specialisation. Importantly, the presence of bivalves mons Attribution 4.0 International License, which permits
use, sharing, adaptation, distribution and reproduction in any
and decapods increased with the weight of the juve- medium or format, as long as you give appropriate credit to the
nile octopuses and suggests that the strong muscular original author(s) and the source, provide a link to the Crea-
development of the arm and suckers after settlement tive Commons licence, and indicate if changes were made. The
provides the juveniles with the skills to increase their images or other third party material in this article are included
in the article’s Creative Commons licence, unless indicated
trophic niche breadth along its adaptation to the ben- otherwise in a credit line to the material. If material is not
thos, where they will remain for the rest of their lives. included in the article’s Creative Commons licence and your
Less than one third of the stomach contents examined intended use is not permitted by statutory regulation or exceeds
were empty, suggesting that juvenile octopus main- the permitted use, you will need to obtain permission directly
tain a notable predatory activity.
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Rev Fish Biol Fisheries

Octopus diet during the settlement period using DNA

Received: 18 July 2023 / Accepted: 10 July 2024

Supplementary Information The online version

O. Escolar (*) · F. Á. Fernández‑Álvarez · R. Villanueva

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Material and methods settled individuals. Moreover, different sampling

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Fig. 2 Frequency of Occur-

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Size class 4 18,856 4714 1.4205 0.012 996

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The use of blocking primers achieved an exception- Limitations of the study

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Conclusions Author contributions RV defined the initial project and

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of octopus skin. Front Mar Sci 8:645738. https://​doi.​org/​

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