Marine Biology Research

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/smar20

On Caretta caretta’s shell: first spatial analysis of

micro- and macro-epibionts on the Mediterranean
loggerhead sea turtle carapace

Monica Francesca Blasi, Alice Rotini, Tiziano Bacci, Monica Targusi, Giusy
Bonanno Ferraro, Luca Vecchioni, Rosa Alduina & Luciana Migliore

To cite this article: Monica Francesca Blasi, Alice Rotini, Tiziano Bacci, Monica Targusi, Giusy
Bonanno Ferraro, Luca Vecchioni, Rosa Alduina & Luciana Migliore (2021) On Caretta�caretta’s
shell: first spatial analysis of micro- and macro-epibionts on the Mediterranean loggerhead sea
turtle carapace, Marine Biology Research, 17:7-8, 762-774, DOI: 10.1080/17451000.2021.2016840

To link to this article: https://doi.org/10.1080/17451000.2021.2016840

Published online: 12 Jan 2022.

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 MARINE BIOLOGY RESEARCH
2021, VOL. 17, NOS. 7–8, 762–774
https://doi.org/10.1080/17451000.2021.2016840

On Caretta caretta’s shell: first spatial analysis of micro- and macro-epibionts

on the Mediterranean loggerhead sea turtle carapace
Monica Francesca Blasia, Alice Rotinib, Tiziano Baccib, Monica Targusib, Giusy Bonanno Ferraroa,
Luca Vecchioni c, Rosa Alduinac and Luciana Migliored
a
Filicudi Wildlife Conservation, Location Stimpagnato Filicudi Lipari (Me), Italy; bItalian National Institute for Environmental Protection and
Research (ISPRA), Rome, Italy; cDepartment of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University
of Palermo, Palermo, Italy; dDepartment of Biology, Tor Vergata Rome University, Rome, Italy

ABSTRACT ARTICLE HISTORY

The loggerhead sea turtle, Caretta caretta (Linnaeus, 1758), is the most common sea turtle Received 10 May 2021
species in the Mediterranean Sea, where it can experience severe anthropogenic impacts. Accepted 3 December 2021
Although C. caretta is known to host more than 200 epibiotic taxa (crustaceans, algae and Published online 12 January
cyanobacteria), no reports have included a detailed evaluation of the microbial community 2022
of its carapace scutes. Thus, this study aimed to determine the diversity and composition of SUBJECT EDITOR
the visible and invisible communities on the carapace scutes of wild loggerhead turtles from Dr. Carolin Löscher
the Aeolian Archipelago (Southern Tyrrhenian Sea, Italy) by using a combined approach of
morphological/spatial examination and molecular analyses. Altogether, our results displayed KEYWORDS
a higher abundance of crustaceans, macroalgae and Proteobacteria on the posterior Caretta caretta;
carapace scutes, while Firmicutes were more abundant on the anterior scutes. For the first cyanobacteria; firmicutes;
time, this study showed the complexity of the microbial (invisible) and visible epibionts of proteobacteria;
Kilonelliaceae; biofilm
the loggerhead sea turtles from the Mediterranean Sea and suggests the importance of
including evaluation of the microbial components when studying epibiont communities.

Introduction (Frick et al. 2002) and it may change according to the

host life stage (e.g. neritic vs. pelagic) or the sex (i.e.
Epibiosis is the association between two organisms in
nesting females contain more complex communities
which the epibiont grows on the surface of the host
due to their sedentary lifestyle during the nesting
organism (basibiont). The relative fitness of the host
period) (Frick and Pfaller 2013). The critical stages of
may depend on the ability of epibionts to cause or
epibiont succession in sea turtles have been poorly
buffer biotic and abiotic stress (Wahl 2008). Epibionts
understood, but undoubtedly it begins with microbial
range from vertebrates to invertebrates, from micro-
colonizers. It was reported that microbial biofilm for-
scopic algae to bacteria (Frick et al. 2002; Lee et al.
mation on artificial surfaces is initiated by the attach-
2008; Frick and Pfaller 2013), and can establish
ment of specific pioneer free-living bacteria, mainly
several ecological interactions with the basibionts
γ-Proteobacteria (Lee et al. 2008), present in the sea-
(commensalism, mutualism or parasitism) (Dang and
water (Dang and Lovell 2000, 2016). In addition, chemi-
Lovell 2016).
cals produced by bacteria can induce the settlement of
The loggerhead sea turtle (Caretta caretta, Linnaeus,
some macroinvertebrate larvae (Siddik and Satheesh
1758) is the most abundant marine turtle species in the
2019; Hadfield et al. 2021). Various factors may
Mediterranean Sea and is considered a vulnerable
disrupt epibiotic communities; for instance, migration
species globally, and yet one of the least concern in
to less favourable habitats might cause the less toler-
the Mediterranean in the IUCN Red List of Threatened
ant epibionts to die and slough off. Moreover, commu-
Species. Its carapace provides a suitable substrate for
nity succession may be disturbed when host turtles
more than 200 epibiotic taxa, that can be obligate
‘groom’ themselves by actively rubbing against sub-
commensals or parasites. The species composition of
merged structures (Frick and Mcfall 2007).
epibiotic communities largely depends on the geo-
Colonization of carapace scutes in the loggerhead
graphic region or habitat in which the turtle is found
turtle involves the settlement of both prokaryotic

CONTACT Rosa Alduina valeria.alduina@unipa.it Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF),
University of Palermo, 90128 Palermo, Italy
Supplemental data for this article can be accessed https://doi.org/10.1080/17451000.2021.2016840.
© 2022 Informa UK Limited, trading as Taylor & Francis Group

Published online 12 Jan 2022


and eukaryotic epibionts, such as algae (Kitsos et al.
2005; Pfaller et al. 2008; Kaleli et al. 2020) and crus-
taceans (Tanabe et al. 2017; Crespo-Picazo et al.
2017; Domenech et al. 2017; Zardus 2021), all of
which, in turn, attract small fishes predating epibionts,
and becoming potential food for loggerhead turtles
(Blasi and Mattei 2017; Blasi et al. 2018). Because of
its longevity, vagrant lifestyle and diverse foraging/
reproductive habits, the loggerhead turtle is an excel-
lent bio-indicator of marine pollution (Caracappa et al.
2016; Blasi et al. 2018; Matiddi et al. 2019; Sciortino
et al. 2021) as well as a carrier of both pathogenic
and environmental bacteria (Pace et al. 2018, 2019;
Arizza et al. 2019; Alduina et al. 2020; Sucato et al.
2021). Additionally, the hosted epibionts may
become putative indicators of water quality and
migratory patterns.
In the present study, combined morphological and
molecular analyses were applied to characterize the
composition and spatial distribution of epibiotic com-
munities in carapace scutes of wild-caught loggerhead
turtles from the Aeolian Archipelago, an important
feeding area located in the Southern Tyrrhenian Sea
(Italy). This study represents the first detailed investi-
gation of the bacterial communities in scutes of a sea
turtle species.
Methods
Study area and sampling
Dedicated boat surveys were performed from May to
June 2016 along a study area covering 400 km2 of
the coastal area around Filicudi and Alicudi, two
small islands located in the Aeolian Archipelago
(Southern Tyrrhenian Sea, Sicily, Italy; 38°35′ N, 14°
34′ E). Ten loggerhead sea turtles were captured by
hand in their feeding habitat and immediately released
into the sea after taking high-resolution pictures of the
carapace and collecting biological samples (Blasi et al.
2020; Zardus 2021). Sampling was carried out follow-
ing the regulations of the International Council for Lab-
oratory Animal Science Ethical Guidelines and was
authorized by the Italian Ministry of Environment
(PROT. No. 0001735, 02-02-2010; renewal: PROT No.
0006876, 25-01-2013). For each turtle (identified by
the letters A–L; see Table SI in Supplemental Data)
the curved carapace length (CCL notch-tip), curved car-
apace width (CCW) and weight were measured, and
the health status was evaluated according to official
Italian guidelines (Figure S1).
Macroinvertebrates and algae were identified using
samples collected from the posterior carapace scutes
MARINE BIOLOGY RESEARCH 763
of each of the 10 turtles by a small scalpel (Figure
1a). Sampling was performed from most of the pos-
terior scutes since algae and invertebrates were only
visible there. Biological samples were then preserved
in sterile polyethylene containers with 4% formalin
and stored at 4°C for morphological analysis.
Samples for the analysis of microbial communities
were collected with a sterile scalpel from some pos-
terior and anterior carapace scutes as indicated in
Figure 1a of three sea turtles (#A, #B and #D; see
Table SI in Supplementary Data) and stored at 4°C in
polyethylene tubes containing 200 mM Tris-HCl pH 8,
10 mM EDTA, and 0.24% Triton X-100 (Blasi et al.
2016) for further analysis, to preserve the genetic
material until the DNA extraction.
Analysis of macroinvertebrates and algae
Mapping of algal coverage on the whole carapace of
loggerhead turtles was accomplished from photo-
graphic images taken during the sampling phase.
The surface area they occupied on each carapace
scute of each individual turtle was estimated, and
used to calculate the coverage (%) of the whole cara-
pace of each individual turtle and the mean coverage
(%) for each scute. Samples for algae were fixed in
4% formalin in seawater, each sample was then
sieved through a 1 mm mesh to collect the crustaceans
clinging to algae. For algal morphological identifi-
cation, semi-permanent slides were prepared using a
25% Karo Syrup solution (ACH Foods, Memphis, TN,
USA) with a Leica Wild M5A dissecting microscope
and an optical microscope Aristoplan (Leica) equipped
by a Leica DFC 500 camera. Both seaweed and Cyano-
bacteria were classified to the lowest possible taxo-
nomic level according to dichotomous keys (Maggs
and Hommersand 1993; Komárek 1999; Kadivar and
Stapleton 2003; Norris 2014). Invertebrates were
counted and identified to the lowest possible taxo-
nomic level (i.e. species) (Schultz 1969; Naylor 1972;
Ruffo 1982, 1989, 1993; Giaccone et al. 2003) under a
Zeiss stereo Discovery.V20 optical microscope
equipped with a Zeiss Axiocam ERc5S. Values calcu-
lated by applying the Bray–Curtis index of similarity
to macroinvertebrate communities from each turtle
were subjected to non-metric Multi-Dimensional
Scaling (nMDS) using Kruskal’s stress formula 1, to
establish affinities among hosts. In addition, hierarchi-
cal clustering (group average) and similarity profile
(SIMPROF) permutation tests (n = 999 randomizations)
were performed to measure statistical significance (P <
0.05) and identify genuine clustering of samples which
are a priori unstructured (Riggio 1996). Statistical
764 M. F. BLASI ET AL.

Figure 1. Caretta caretta carapace scutes. (a) Samples were collected from some anterior (V2 and V3, indicated by light grey) and
some posterior carapace scutes (V5, C4, C5, S, M8, M9, M10, M11 indicated by dark grey). (b) Intensity map of the mean coverage
(as %) of algae for each scute of the 10 specimens investigated.

analyses were performed using PRIMER 6 software demultiplexed and the quality and chimera filtering
(Riggio 1996). were performed. Operational Taxonomic Units (OTUs)
were defined by clustering at 97% similarity with
usearch61. A representative sequence from each OTU
Bacterial identification cluster was chosen and taxonomy was assigned
using the SINA classifier on the latest ‘SILVA’ dataset
The epibiotic bacterial community colonizing two
available (Schmalenberger et al. 2001) (https://www.
areas of the carapace scutes of three sea turtles (speci-
arb-silva.de/ngs/, accessed on 11 September 2020).
mens #A, B and D, showing similar CCLs) was charac-
The complete set of raw sequences obtained in this
terized by Next Generation Sequencing (NGS –
study was deposited in GenBank under the study
Illumina platform). From each sea turtle, one sample
accession BioProject ID no. PRJNA681485.
was taken from an anterior carapace scute and
Rarefaction analysis was carried out plotting the
another from a posterior scute (Figure 1a). DNA extrac-
number of observed OTUs against the total number of
tion of scraped scute material was carried out using the
filtered reads per sample. PRIMER 6+PERMANOVA (soft-
Power Soil®DNA isolation kit (Mo Bio, Carlsbad, CA,
ware package from Plymouth Marine Laboratory, UK)
USA) according to the manufacturer’s instructions.
was used to determine the beta diversity among
The V4–V5 region of the bacterial 16S rRNA gene was
samples using the Bray–Curtis similarity matrix. Alpha
amplified using universal primers COM1 (5′ -CAG-
diversity, Abundance-based Coverage Estimator (ACE),
CAGCCGCGGTAATAC-3′ ) and COM2 (5′ -
′
Chao1, Shannon–Wiener diversity, H’, Simpson index,
CCGTCAATTCCTTTGAGTTT-3 ) (Clarke and Gorley
1-D, and evenness, e, were estimated to determine the
2006). DNA amplification was performed by PCR,
microbial richness and diversity. Good’s coverage was
with an initial denaturation step of 94°C for 3 min, fol-
estimated to evaluate the completeness of sampling.
lowed by 30 cycles of 94°C for 1 min; 50°C for 1 min
OTU Tables along with metadata were uploaded to
and 72°C for 1 min, with a final extension step of 72°
METAGENassist website (http://www.metagenassist.
C for 4 min. PCR amplicons were purified using Gel/
ca/METAGENassist/) (Pruesse et al. 2012). The analysis
PCR DNA fragments Extraction Kit (Mo Bio) and sent
was performed using the filtered data as per default
to MR DNA (www.mrdnalab.com, Shallowater, TX,
settings and samples were normalized using total
USA) for Illumina paired-end sequencing.
sum (sample vs sample) and Pareto scaling (taxon vs
Regarding the sequencing data processing for bac-
taxon; mean-centred and divided by the square root
teria, the sequence data were processed using Quanti-
of the standard deviation of each variable). The pro-
tative Insights Into Microbial Ecology (QIIME, v. 1.1.9).
cessed data were used to deduce taxonomic-to-phe-
Within QIIME, mapping files were validated and reads
notypic mapping of metabolism.
were depleted of barcodes. The sequences were

Results
Analysis of algae and macroinvertebrates
Per cent coverage of algae and invertebrate epibionts
measured across all the 10 loggerhead sea turtles was
on average low (11.6%) and unevenly distributed
among the carapace scutes. The highest total coverage
value (20.1%) was found on specimen #L. The mean
per cent coverage of algae for each carapace scute
was visualized as an intensity map (Figure 1b), which
highlighted the differences among the scutes: lowest
coverage was recorded on the anterior carapace
scutes, while the highest values were recorded on
the posterior vertebral, costal and marginal scutes.
The highest mean coverage (57.8%) was documented
on the supracaudal scutes.
The most abundant algal colonizer of the carapace,
observed in all turtles was Polysiphonia sensu lato Gre-
ville, a species of Rhodophyta. Within the same analy-
sis, three species of cyanobacteria were also identified
by microscopy on a single turtle (#A; see Table S1 in
Supplemental Data): Scytonematopsis crustacean
Figure 2. (a) Overall per cent abundance of the crustacean species identified in this study; (b): Percentage abundance of the crus-
tacean species identified for each of the 10 loggerhead turtles (#A–L).
MARINE BIOLOGY RESEARCH 765
Thuretex Bornet & Flahault, Leibleinia gracilis (Raben-
horstex Gomont) Anagnostidis and Lyngbya sordida
Gomont. A total of 1120 macroinvertebrates were
found, all crustaceans belonging to six species. The
number of epiphytic crustacean species remained
quite constant among the sea turtle specimens (3–5),
although the abundance of each species per host
was highly variable, ranging from 7 to 379 individuals.
The great majority were amphipods (1002 individuals;
3 species), followed by cirripeds (60; 1 species), tanaids
(57; 1 species) and isopods (1; 1 species). The most
abundant epibionts were the free-living amphipod
Caprella andreae (Mayer, 1890) (n = 585 individuals;
52.6%) and Protohyale grimaldii (Chevreux, 1891) (n =
416; 37.3%), which were recorded on all C. caretta
specimens (Figure 2). Less abundant was the cirriped
Lepas (Anatifa) hillii (Leach, 1818) (n = 60; 4.4%),
though it occurred on most C. caretta specimens
(Figure 2). The tanaid Hexapleomera robusta (Moore,
1894) (n = 57; 5.4%) was found less frequently, in 5
out of the 10 specimens (Figure 2). Lastly, both the
isopod Idotea metallica (Bosc, 1802) and the amphipod
766 M. F. BLASI ET AL.

Isaea montagui (H. Milne Edwards, 1830) were found
on one specimen in very low abundance (n = 1; 0.1%;
Figure 2). Multivariate analysis (nMDS, Figure 3) discri-
minated the loggerhead sea turtles based on their
crustacean assemblages, highlighting a cluster (speci-
mens #B,E,F,G,L), characterized by a more structured
assemblage, and a cluster (specimens #A,C,D,H,I)
characterized by low numbers of species and
specimens.
Bacterial analysis
The epibiotic bacterial community colonizing two
areas of the carapace scutes of three sea turtles (speci-
mens #A, B and D, showing similar CCLs,) was studied
by Next Generation Sequencing (NGS). The raw output
of sequencing resulted in a total of 438,018 bacterial
sequences, their processing with the QIIME software
resulted in a total of 432,258 sequences, assigned to
216 OTUs. Diversity indexes were low in samples col-
lected from the anterior scutes of turtle #A and the
posterior scutes of turtle #B (Table SII), highlighting
heterogeneity in both α diversity and the Shannon–
Wiener index. In addition, the bacterial community
was not evenly distributed within and among
samples, as shown by the evenness values (Table SII),
although the estimation of rarefaction curves indicated
that a good level of diversity was sampled (Figure S2).
The taxonomic composition of the microbial com-
munities found on the carapace, at phylum, order,
class and family levels, is reported in Figure 4. The
dominant phyla were Firmicutes (55.8%) and Proteo-
bacteria (43.5%), while Actinobacteria, Bacteroidetes,
Thermi, Cyanobacteria and Acidobacteriota accounted
for less than 1% of the identified bacteria. Bacillales
was the prevalent order (55.8%), mainly represented
by the family Staphylococcaceae (55.7%). Proteobac-
teria were mainly represented in the sea turtle
samples by the Alpha class (41.5%) while the most rep-
resented abundant family was Kiloniellaceae (41.3%),
encompassing a single genus Kiloniella.
Comparison of the microbial colonizers of anterior
vs posterior carapace scutes showed that anterior
ones were enriched in Firmicutes (Staphylococcaceae),
while Proteobacteria (Kilonelliaceae) were found to be
dominant in the posterior ones (Figure 5). In addition,
anterior scutes contained a larger microbial diversity,
200 OTUs, over the posterior ones, containing only
84 OTUs. Altogether 132 OTUs were uniquely found
in the anterior carapace scutes and 16 OTUs in the pos-
terior ones, respectively, whilst 68 OTUs were common
between the two groups (Figure 6). On the anterior
scutes in two out of the three specimens analysed
(#B and D), metagenomic analysis highlighted the
presence of Cyanobacteria, Pseudanabaenaceae and
Rivulariaceae.

Figure 3. Bray–Curtis similarity measures for nMDS ordination plot of crustacean assemblages. Clusters defined by SIMPROF split
the crustacean assemblages into two groups (green circle). Mean coverage of algae (as %) of the posterior vertebral, costal and
marginal plates for each loggerhead turtle was superimposed by bubble values in the ordination plot. In turtle #A algal coverage
data are not available. Spearman rank correlations between variables and the ordination are plotted as vectors (blue lines).
 MARINE BIOLOGY RESEARCH 767

Figure 4. Relative abundance (as %) of bacterial communities from the carapace scutes of the loggerhead sea turtles at different
taxonomic levels, phylum (a), class (b), order (c) and family (d).

METAGENassist analysis (Pruesse et al. 2012) Discussion

showed diverse metabolic pathways between the
In this study, the micro- and macro-components of the
anterior carapace scutes, in which ammonia oxidizers,
epibiotic community (prokaryotes and eukaryotes)
dehalogenating bacteria, nitrite reducers and nitrogen
from carapace scutes of wild-caught Mediterranean
fixing bacteria are equally present, and the posterior
loggerhead turtles were characterized. Algal coverage
scutes, in which bacteria characterized by unknown
(as percentage) on whole carapaces was low in all
metabolisms were detected (Figure 7).
768 M. F. BLASI ET AL.

Figure 5. Relative abundance (as %) of bacterial communities from the anterior (indicated by A) and posterior (indicated by P)
carapace scutes of the loggerhead sea turtles at different taxonomic levels, phylum (a), class (b), order (c) and family (d).

specimens, but variable among the scutes. Mean cov-
erage was highest on the supracaudal scute,
suggesting that epibiotic communities tend to
accumulate along the vertebral scutes and across the
posterior third of the carapace, as previously reported
(Frick and Pfaller 2013). This may be due to higher
current flow on the anterior scutes during swimming,
which may hamper the colonization rate. Microscopy
analysis identified Polysiphonia sensu lato as the most
abundant algal colonizer of the carapace in all
turtles; this species has been reported worldwide,
from subtropical to Arctic and Antarctic waters, as
both an epiphyte on other algae or seagrasses and
epizoic on sea turtles (Hollenberg 1968, 1969, 1971;

Hollenberg and Norris 1977; Arndt et al. 2012). More-
over, for the first time three species of cyanobacteria
were reported as epibionts of the Mediterranean log-
gerhead turtles. Cyanobacteria were previously
reported on sea turtles living in the Pacific Ocean
(Womersley 1979) and Atlantic Ocean (Cribb 1969)
and the Indian Ocean. Scytonematopsis crustacea, Lei-
bleinia gracilis and Lyngbya sordida are ubiquitous
marine species, well known marine epiphytic and epi-
lithic species, although they have never been reported
as loggerhead turtle epibionts in the Mediterranean
Sea. Crustaceans were confirmed as important coloni-
zers of the sea turtle carapace. The most abundant
crustacean epibionts (Caprella andreae and Protohyale
grimaldii), recorded in all the investigated sea turtles,
are commonly found as epibionts of C. caretta and
are considered as chelonophilic facultative commen-
sals (Abel Sentíes et al. 1999; Kitsos et al. 2005;
Sezgin et al. 2009; Zakhama-Sraieb et al. 2010; Casale
et al. 2012; Domènech et al. 2015; Crespo-Picazo
et al. 2017). Similarly, the cirriped Lepas (Anatifa) hillii,
encountered on most turtle specimens is a cosmopoli-
tan and very abundant sea turtle epibiont in the
Western and Central Mediterranean Sea, classified as
a facultative commensal of sea turtles (Zakhama-
Sraieb et al. 2010; Frick and Pfaller 2013; Cabezas
et al. 2013). The rarely recorded tanaid Hexapleomera
robusta, also shows a strong association with sea
turtles (Badillo et al. 2007; Sezgin et al. 2009); the
species has been considered as an obligate epibiont
of sea turtles or other marine motile organisms (Frick
and Pfaller 2013). The isopod Idotea metallica and the
amphipod Isaea montagui were found at very low
abundance; both have previously been reported as
facultative epibionts of C. caretta (Zakhama-Sraieb
et al. 2010; Blazewicz-Paszkowycz et al. 2012). The log-
gerhead carapace represents a microhabitat with
different features and trophic opportunities where
the settlement of some epibiont species may be
favoured by the presence of specific environmental
conditions. However, all epibiont species show rapid
turnover on turtles, with relatively short lives, rapid
growth and high juvenile mortality. It is worth noting
that acorn barnacles were not detected in this study,
although they are known as common obligate com-
mensals of the loggerheads in several Mediterranean
areas (Domenech et al. 2017). However, this could be
due to the specific migratory routes, foraging areas
and life stage of the specimens sampled in this
study. It was already reported (Abel Sentíes et al.
1999) that the composition of barnacles may vary
among loggerhead turtles frequenting not only
different geographic regions but also different habitats
MARINE BIOLOGY RESEARCH 769
(pelagic vs. benthic habitats) in the Mediterranean Sea
and a higher occurrence of balanomorph barnacles is
reported in areas on the continental shelf (Abel
Sentíes et al. 1999; Targusi et al. 2017). Since the
sampled turtles were mainly juvenile turtles (with a <
70 cm CCL), which are known to feed on pelagic prey
in open waters, it is not excluded that the colonization
of balanomorph barnacles on their carapace might be
a rare event. On the other hand, the higher occurrence
of Lepas spp. could be ascribed to an opportunistic
feeding behaviour of turtles. Indeed, it was reported
that in the study area loggerhead turtles can feed on
prey, such as algae, fish larvae, pelagic gooseneck bar-
nacles and Lepas spp., which have grown on Fishing
Aggregating Devices (FADs) and floating debris (Blasi
and Mattei 2017; Zardus 2021).
Multivariate analysis established the affinities
among crustacean assemblages of the 10 investigated
loggerhead sea turtles and the relationship between
the crustacean assemblage and the algal coverage of
the scutes in each sea turtle. More structured assem-
blages, characterized by many dozens of specimens,
were associated with higher values of algal coverage.
The rhodophyte Polysiphonia may form a secondary
substrate on the turtle carapace, allowing many
species to attach themselves to the algae, thus favour-
ing the presence of a diverse epiphyte community
(Gramentz 1988; Frick et al. 2000; Casale et al. 2004).
As an example, three specialist species, C. andreae,
P. grimaldi and H. robusta, are frequently found associ-
ated with Polysiphonia (Cabezas et al. 2013).
The study of the microbial communities showed as
dominant the phyla Firmicutes and Proteobacteria. Fir-
micutes and Proteobacteria, together with Bacteroi-
detes, are the main colonizers of gut and cloaca of
loggerhead and green turtles (Fuller et al. 2010;
Abdelrhman et al. 2016; Pace et al. 2018). Firmicutes
are structurally resistant Gram-positive bacteria due
to their cell wall and can thrive in organic enriched
environmental conditions; among them, Staphylococ-
caceae was the prevalent family (Order Bacillales)
which includes ubiquitous salt- and acid-tolerant
microorganisms. Gram-negative Proteobacteria,
mainly represented by the Alpha class, are an extra-
ordinarily diverse and ancient group of bacteria,
acting as either parasites or beneficial symbionts. Kilo-
niellaceae was the most highly represented family,
comprising a single genus Kiloniella. These are meso-
philic chemoheterotrophic aerobic bacteria, typically
marine, with the potential for denitrification. The
species Kiloniella laminariae has been isolated from
the marine macroalgae, Saccharina latissima (former
Laminaria saccharina), from the Baltic Sea and it is
770 M. F. BLASI ET AL.

Figure 6. Venn diagram showing the number of OTUs present on anterior (A) and posterior (P) scutes.

known to have antifungal activity. The genus has also
been found associated with animals, as Kiloniella majae
M56.1T (with a spider crab), Kiloniella spongiae
MEBiC09566T (with a sponge) and Kiloniella litopenai
P1-1 (with a white shrimp), all inhabiting different
marine habitats (Biagi et al. 2019). Recently, the
family of Kiloniellaceae was found to be abundant in
a sea turtle nest of Campania, South Italy (Vecchioni
et al., unpublished data). This last result suggests that
bacterial profiles could provide valuable biogeo-
graphic data to localize the origin of the specimens;
in the future, the study of carapace colonizing bacteria
on large samples of turtles collected from different
areas could possibly be useful in establishing their
migratory patterns.
The differences in microbial diversity and compo-
sition between anterior vs posterior carapace scutes
(with highest diversity and dominance of Firmicutes

Figure 7. Putative types of bacterial metabolism present in anterior (A) and posterior scutes (P).

in the anterior scutes and dominance of Proteobacteria
in the posterior ones) may be due to varying environ-
mental conditions at these locations. Gram-positive
bacteria, such as the Staphylococcaceae (Firmicutes),
may thrive in the most stressed and relatively dry
environment of the anterior scutes, which are often
directly exposed to air and sun. Furthermore, hydro-
dynamics at the anterior scutes may act by inhibiting
macroalgae settlement or growth.
The presence of Cyanobacteria, Pseudanabaena-
ceae and Rivulariaceae, mainly on the anterior scutes,
probably indicates a less competitive environment
for Cyanobacteria in the absence of macroalgae. Con-
versely, at the most colonized and predominantly sub-
merged posterior scutes, the Gram-negative
Kiloniellaceae dominate, probably thriving in associ-
ation with macroalgae. Algae and bacteria can
exchange numerous metabolites (Kouzuma and Wata-
nabe 2015; Wiese et al. 2020), including active antifoul-
ing molecules, potentially accounting for the limited
number of bacterial species in these scutes (De Rosa
et al. 2001; Krohn et al. 2005; Rao et al. 2007; Goecke
et al. 2010; Lever et al. 2019). Furthermore, algae
could also control bacterial colonization by interfering
with their quorum-sensing system, which regulates
genes for biofilm formation and surface adhesion
(Maggio et al. 2020).
The detection of Cyanobacteria by metagenomic
NGS analysis emphasizes how this approach can give
deeper insight into the microbial composition, if com-
pared with the evaluation of Cyanobacteria by light
microscopy, and calls for further investigations in this
field by using this high throughput methodology.
Standardized protocols for the study of whole cara-
pace associated microbiomes of sea turtles should be
adopted, as exemplified with seagrass (Jha et al.
2013), with particular attention to sampling pro-
cedures and data analysis.
Consistent with the varying distributions of bacteria
on the carapace, diverse metabolic pathways corre-
sponding to environment and lifestyle have been high-
lighted by the METAGENassist analysis (Figure 7). This
suggests some relationships among the epiphytic bac-
terial community of loggerhead sea turtles and the
other colonizers of the carapace: the epiphytic bac-
terial community can modulate and/or be modulated
by the algae, as reported for brown algae (Conte
et al. 2021). It must be emphasized that microalgae
were not examined in the present study, neither by
light microscopy nor by NGS, hence their presence
and role cannot be discussed.
To the best of our knowledge, this is the first study
aimed at characterizing the microbial community
MARINE BIOLOGY RESEARCH 771
associated with the sea turtle carapace by NGS, so far
only Cyanobacteria being studied by light microscopy.
Conclusion
This pioneering study, based on a limited number of
samples, reports for the first time the microbial com-
position of the carapace of wild loggerhead sea
turtles, highlighting the importance of including
the microbial component in such investigations. It
also provides an outline for setting up standardized
protocols to understanding of the complexity of
interactions between sea turtles and their epibionts.
Our results showed the rhodophyte Polysiphonia,
crustaceans and Proteobacteria were mainly on the
posterior carapace of the analysed specimens,
while microbial diversity was higher in the anterior
scutes. Further investigation is necessary to establish
the nature of association among epibionts–host and
to test the hypothesis that algae affect the coloniza-
tion or the growth of other microbial epibionts in
the posterior scutes. In fact, the reduced bacterial
colonization on the backs of the sea turtles could
also be partly attributed to the actions of detritivory
by crustaceans. Eventually, studies on a large scale
could be useful to evaluate if specific microbial
signatures could be used to establish migratory
patterns.
Our results highlight the importance of understand-
ing the physical and biological interactions of sea
turtles with epibiotic communities to establish rules
for the management of different coastal seascapes
and the need for knowledge of local processes for
the conservation of marine biodiversity.
Acknowledgements
We thank Dr Chiara Bruno for her support during turtle hos-
pitalization and sampling at Filicudi Wildlife Conservation
First Aid. The authors wish to thank Loretta Lattanzi and
Paolo Tomassetti for crustacean identification, Chiara Conte
and Nicoletta Perini for bioinformatics analyses. We wish to
thank Filicudi Wildlife Conservation for Logistic and in-kind
support.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
772 M. F. BLASI ET AL.
Availability of data and material
Regarding bacterial sequence data, the complete set of
raw sequences obtained in this study was deposited in
GenBank under the study accession BioProject ID no.
PRJNA681485.
Author contributions
Conceptualization and Methodology: Monica Blasi,
Luciana Migliore; Formal analysis and investigation:
Alice Rotini, Tiziano Bacci, Monica Targusi, Giusy
Bonanno Ferraro, Luca Vecchioni; Writing – original
draft preparation: Rosa Alduina, Alice Rotini, Tiziano
Bacci, Monica Targusi, Giusy Bonanno Ferraro; Writing –
review and editing: All authors; Resources: Monica
Blasi, Luciana Migliore; Supervision: Monica Blasi,
Luciana Migliore, Rosa Alduina. All authors read and
approved the final manuscript.
Ethics approval:
Sampling was carried out following the regulations of
the International Council for Laboratory Animal
Science Ethical Guidelines and was authorized by the
Italian Ministry of Environment (PROT. N° 0001735,
02-02-2010; renewal: PROT N° 0006876, 25-01-2013).
Consent to participate: not applicable.
Consent for publication: not applicable.
ORCID
Luca Vecchioni http://orcid.org/0000-0003-4325-9728
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