Enhancing seafood traceability: tracking the origin of seabass and seabream from the tuscan coast area by the analysis of the gill bacterial communities

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Me iggi e al. Animal Mic obiome (2024) 6:13
h ps://doi.o g/10.1186/s42523-024-00300-z Animal Mic obiome
†Niccolò Me iggi and Alessand o Russo con ibu ed equally o his
wo k.
*Co espondence:
Duccio Ca alie i
duccio.ca[email p o ec ed]
Full lis o au ho in o ma ion is a ailable a he end o he a icle
Abs ac
Backg ound The sea ood consump ion and ade ha e inc eased o e he yea s, and along i s expec ed expansion
pose majo challenges o he sea ood indus y and go e nmen ins i u ions. In pa icula , he global ade in ish
p oduc s and he consequen consump ion a e linked o eliable au hen ica ion, necessa y o gua an ee law ul ade
and heal hy consump ion. Al e a ions o e o s in his p ocess can lead o comme cial aud and/o heal h h ea s.
Consequen ly, he de elopmen o new in es iga i e ools became c ucial in ensu ing unwan ed scena ios. He e
we used NGS echniques h ough a ge ed me agenomics app oach on he V3-V4 egion o he 16S RNA genes
o cha ac e ize he gill bac e ial communi ies in wild-caugh seab eam (Spa us au a a) and seabass (Dicen a chus
lab ax) wi hin di e en ishe ies a eas o he “Cos a degli E uschi’’ a ea in he Tuscan coas . Ou challenge in ol ed he
possibili y o disc imina ing be ween he mic obio a o bo h ish species collec ed om h ee di e en ishing si es
e y close o each o he (all wi hin 100 km) in impo an a eas om a comme cial and ou is poin o iew.
Resul s Ou esul s showed a signi ican di e ence in he assembly o gill bac e ial communi ies in e ms o di e si y
(alpha and be a di e si y) o bo h seabass and seab eam in acco dance wi h he h ee ishing a eas. These di e ences
we e ep esen ed by a unique si e - ela ed bac e ial signa u e, mo e e iden in seab eam compa ed o he seabass.
Acco dingly, he co e membe ship o seab eam specimens wi hin he h ee di e en si es was minimal compa ed o
he seabass which showed a g ea e numbe o sequence a ian s sha ed among he di e en ishing si es. The e o e,
he LRT analysis highligh ed he possibili y o ob aining speci ic ish bac e ial signa u es associa ed wi h each si e;
i is no ewo hy ha speci ic axa showed a unique associa ion wi h he ishing si e ega dless o he ish species.
This s udy demons a es he e ec i eness o a ge -me agenomic sequencing o gills in disc imina ing bac e ial
signa u es o specimens collec ed om ishing a eas loca ed a a limi ed dis ance o each o he .
Enhancing sea ood aceabili y: acking he
o igin o seabass and seab eam om he
uscan coas a ea by he analysis o he gill
bac e ial communi ies
NiccolòMe iggi1†, Alessand oRusso2†, SoniaRenzi2, Benede aCe asuolo2, Ma aNe ini2, Albe oUgolini3,
MassimilianoMa asi2 and DuccioCa alie i2*
Page 2 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
Backg ound
Globaliza ion has enabled access o a wide a ie y o ood
esou ces a ound he wo ld, howe e i has imposed
new challenges o he con ol o ood supply chains and
ood secu i y. These condi ions a e o g ea es in e es
when we deal wi h sea ood, whose consump ion co e s
nea ly one i h o wo ld animal p o ein in ake. Indeed,
he consump ion a e o aqua ic oods wo ldwide has
doubled in he las 50 yea s [1], highligh ing he impac
on he economy and public heal h ha a possible p ob-
lem in he ade chain could cause. The pe ishabili y o
sea ood equi es a highe accu acy in ood sa e y con ol
p ocedu es and i s impo ance in he global ood ade
impose he de elopmen o new me hodologies o ensu e
e o s o aud in he global supply chain [2]. T aceabil-
i y plays a c ucial ole in suppo ing he sus ainabili y o
ishe ies by p o iding anspa ency, accoun abili y, and
he abili y o moni o and manage ishing ac i i ies e ec-
i ely [3]. T aceabili y suppo s sus ainabili y in ishe ies
in many ways: i) i helps o p e en Illegal, Un epo ed,
and Un egula ed (IUU) ishe y which deple es na u al
ish s ocks (EC No 1005/2008 o 29 Sep embe 2008); (ii)
imp o e consume con idence and ma ke access, includ-
ing p oduc ion such as wild-caugh o a med [4]; (iii)
suppo s ce i ica ions p og ammes ha demons a e
commi men o sus ainable p ac ices and gain access o
niche ma ke s ha p io i ize en i onmen ally esponsible
p oduc s (EU No 664/2014 o 18 Decembe 2013), (i )
aceabili y allows o be e managemen o he supply
chain, educing he likelihood o ish spoilage and was e
[5]. Finally, aceabili y echnologies will p o ide aluable
in o ma ion o manage s and policymake s o assess
he heal h o sea ood s ocks and de e mine sus ainable
ca ch le els [6]. The c i e ia o he co ec acking o
sea ood p oduc s a e egula ed by Council Regula ion
(EC) No. 1379/2013 and ega d he common p ocedu es
o he ma ke s in ishe y and aquacul u e p oduc s. The
g ea a en ion exe ed by he con ol au ho i ies is no
de e mined only by he isk o sea ood con amina ion o
w ong p ese a ion bu is also aimed a gua an eeing he
igh s o consume s and p oduce s who aim o eques
ce i ica ions o o igin.
Consequen ly, one o he main challenges in he sea-
ood indus y ega ds he possibili y o acking he p od-
uc ’s o igin. The egula o y in as uc u e is no su icien
o gua an ee aceabili y wi hou sui able echnological
and analy ical suppo . In his di ec ion, he de elopmen
and adop ion o new me hodological app oaches o he
iden i ica ion o ishes om di e en a eas can ep esen
a cen al esou ce in aceabili y managemen .
Mic obial communi ies analysis o indi iduals om
di e en habi a s demons a ed he o ensic alue o
his ype o app oach o ob ain dis inc i e signa u es
ep esen a i e o speci ic en i onmen s. Ac ually, an
inc easingly consis en numbe o di e en app oaches
we e explo ed o ace sea ood o igins [10, 11], hese
app oaches include DNA me aba coding app oaches,
s able iso ope analysis o machine lea ning app oaches
based on sequencing da a [2, 12–14]. Nex Gene a ion
Sequencing (NGS) echniques ep esen an ex emely
in e es ing ool as hey a e able o p o ide a comp ehen-
si e cha ac e iza ion o he mic obial communi ies o
he a ea o o igin. The p og essi e educ ion o he cos
o hese me hodologies has made he NGS app oaches
a ool ha can be easily used by egula o y au ho i ies,
con ol o ganisms and companies.
I we conside he app oaches based on he analysis o
bac e ial DNA sequences, a wide numbe o hese usu-
ally include PCR-DGGE-based analysis [15], howe e
he NGS app oaches along wi h he possibili y o ob ain
high- h oughpu and mo e accu a e in o ma ion ega d-
ing mic obial signa u e [16] has p og essi ely eplaced
he PCR-DGGE, which i was commonly used in a wide
numbe o case s udies [16, 17]. NGS p o ides, con-
e sely o PCR-DGGE, highe esolu ion leading o he
mic obial species iden i ica ions [18], de ec ion o a wide
ange o mic oo ganisms o gene ic a ia ions [19], las
bu no leas , he da a gene a ed by NGS can be eas-
ily s o ed o u u e e e ence and used o compa isons
agains po en ial audulen p oduc s. Acco ding o his
e idence, he exploi a ion o mic obial me agenomics
ools o aceabili y has been p oposed o assess he o i-
gin o ood in o he scena ios [16, 20].
Ta ge ed-me agenomic app oaches can also o e
solu ions in ensu ing heal h sa e y and sus ainable con-
sump ion. In ac , hese echniques allow o ollow he
a ia ions in he mic obial communi ies as indica o s
o de e io a ion, de ined as speci ic spoilage-associa ed
o ganisms (SSOs) [7, 8]. This allows o imp o e he
managemen o ish esou ces, acili a ing he con ol o
de e io a ion p ocesses wi hin he ish supply chain. The
possibili y o de e mining SSOs, combined wi h he pos-
sibili y o disc imina ing he o igin o he ish bo h a a
local and highe spa ial le els [9] can be use ul o gua -
an ee no only he o igin bu also sa e y and sus ain-
able sea ood consump ion. In ac , al hough Eu opean
Conclusions This s udy p o ides new in o ma ion ela ing he s uc u e o he gill mic obio a o seabass and
seab eam in a ishing a ea wi h a c ucial comme cial and ou is in e es , namely “Cos a degli E uschi”. This s udy
demons a ed ha mic obiome-based app oaches can ep esen an impo an ool o alida ing he sea ood o igins
wi h a cen al applica i e pe spec i e in he sea ood aceabili y sys em.
Page 3 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
legisla ion equi es he a ixing o labels o ce i ying he
a ea o o igin acco ding o he The Food and Ag icul u e
O ganiza ion (FAO) acking sys em (see ma e ials and
me hods sec ion o de ails on FAO e e ences), i is di -
icul o es ima e he eshness o he ish ca ch which, in
ac , depends on conse a ion pa ame e s no necessa -
ily ela ed o he local o o eign o igin o ish caugh [9].
Thus, de e mining he mic obial composi ion associa ed
wi h a gi en specimen, wi h possible SSOs iden i ica-
ion, can inc ease he ood sa e y, p o iding gua an ees
o sus ainable consump ion, allowing he consume o be
awa e o he o igin and heal h s a us o he ish p oduc ,
e en a a local le el.
The scien i ic li e a u e is mainly ep esen ed by s ud-
ies on he ish in es inal mic obio a [21], al hough he
gills ep esen an ideal issue o s udying ish aceabil-
i y gi en he high deg ee o a iabili y o he esiden
mic obial communi ies ollowing en i onmen al a ia-
ions [22]. Few s udies highligh he cen al ole o gills
in assessing he en i onmen al mic obial signa u e (use-
ul o aceabili y): The gill mic obio a, hen, could be a
ep esen a i e me ic o in e p e ing he en i onmen-
al mic obial signa u e as highligh ed in a ecen s udy
on seab eam which showed ha gill mic obio a is mo e
closely e lec ing he en i onmen han o he o gans
(gu , skin, ille ) [23]. The e o e, he gills a e ecei ing
inc easing a en ion, as hey may be sampled wi hou
des oying o comp omising he saleable pa s o he ish
(i.e. by swabbing o biopsy) [24, 25]. In his con ex , we
ound se e al s udies ocused on he mic obio a o mol-
luscs [14, 26], while hose ha analyzed ish conce ned
a med specimens [16]. We belie e ha he challenge o
hese echniques ep esen s he unde s anding o hei
applicabili y on on-boa d ozen ish caugh in open-sea
a eas, ec ea ing a s ep o con ol o he a ea o o igin
wi hin he ish supply chain.
He e, we analyzed he gill mic obio a o wo ish spe-
cies o cen al in e es in he global ish ade, i.e. Dicen-
a chus lab ax (he ea e seabass) and Spa us au a a
(he ea e seab eam), caugh in he Medi e anean Sea
om h ee di e en a eas wi hin he a ea o Tuscan
coas named “Cos a degli E uschi”. We pe o med 16S
RNA gene sequencing (V3-V4 egion) by using a ge ed-
me agenomic app oach. We desc ibe o he i s ime
he gill mic obial p o ile o he seabass and seab eam
specimens om his pa icula ishing a ea, in es iga ing
in e speci ic and in aspeci ic bac e ial a ia ions o wild
specimens caugh a a e y limi ed geog aphical dis ance.
Ou da a sugges he possibili y o disc imina e be ween
wild ish species acco ding o hei sampling a ea despi e
limi ed geog aphical esolu ion.
Resul s
Di e en ishing si e de e mine di e en gills bac e ial
di e si y assemblage
A e DADA2 analysis pipeline and subsequen qual-
i y il e ing a o al o 4’162’462 eads (median: 72’787.5)
clus e ed in 8’736 di e en bac e ial amplicon sequence
a ian s (ASVs) we e de ec ed. Seabass and seab eam
da ase s we e cha ac e ized by a signi ican ly di e -
en numbe o ASVs and ela ed ex apola ed di e si y
(In e se Simpson index) highligh ing signi ican di e -
ences in he s uc u e o he gill mic obio a be ween he
wo ish species (Fig. S1c-d). To exclude a me hodologi-
cal bias he a e ac ion cu es and Good’s co e age
es ima ion we e calcula ed. Ra e ac ion cu es eached
he pla eau condi ion demons a ing he e ec i eness
o sequencing in desc ibing he bac e ial di e si y o
each sample (Fig. S1a) and his was co obo a ed by he
Good’s co e age es ima o which anged om 99.99 o
100% (Fig. S1b). All he ollowing analyses we e ca ied
ou in o de o highligh a ia ions in he gills bac e ial
s uc u e o he wo ish species, be ween he h ee di -
e en coas al si es wi hin he a ea “Cos a degli E uschi”
in acco dance wi h he expe imen al design in Fig.1.
The mul idimensional analysis (PCoA based on B ay-
Cu is dis ance) showed a sha p sepa a ion be ween
samples acco ding o bo h ish species and collec ion
si es a iables (Fig.2a). The ish species ep esen s he
main a iable in he assembly o gills bac e ial di e si y
in acco dance wi h he explained a iance esul s om
adonis pe mano a (R2 in Fig.2a). The ish species and
ishing si e a iables a e e ec i e in desc ibing a ound
he 28% and 23% o he a iance explained, espec i ely
(R2 in Fig.2a). To clea ly unde s and he ac ual di e si y
be ween di e en collec ion si es, we di ided he da ase
in acco dance o he wo di e en ish species, hen we
calcula ed di e si y me ics and pe o med mul i a ia e
analysis as desc ibed abo e.
Mul i a ia e analysis (PCoA based on B ay-Cu is dis-
ance) p oduced a clea sepa a ion among g oups o
almos all ishing si es in he wo ish species da ase s
(Fig.21b-c). The o e all e ec o si e a iable, assessed
by adonis pe mano a, sugges ed a highe impac o di -
e en ishing si es in d i ing gills di e si y o seab eam
compa ed o seabass (R2 in Fig.2b-c). To es he signi i-
can di e ences be ween he indi idual si es, he pai -
wise adonis pe mano a analysis was pe o med o bo h
seabass and seab eam da ase s. The analysis showed sig-
ni ican di e ences in di e si y be ween samples om
di e en ishing si es o bo h ish species da ase s, wi h
he excep ion o he compa ison be ween samples col-
lec ed in Ce si e compa ed o Li si e in seabass da ase
(Fig.2d and Table S2).
Di e ences in alpha di e si y me ics among di e en
ishing si es o bo h ish species we e also es ima ed.
Page 4 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
Bac e ial di e si y (alpha di e si y) was a he uni o m in
seabass caugh in Ce si e and Li si e while whole di e si y
measu es epo ed signi ican ly highe alues in CdP si e
(Fig.2e). No signi ican di e ences in he o al numbe
o bac e ial ASVs we e obse ed in seabass caugh om
he h ee di e en si es (Obse ed in Fig.2e). The alpha
di e si y me ics in seab eam da ase showed a mo e he -
e ogeneous pa e n, epo ing a signi ican ly lowe num-
be o bac e ial ASVs in ish collec ed in Ce si e compa ed
o he o he s, whe eas ish collec ed in Li si e epo ed
signi ican ly highe di e si y measu e a es compa ed o
he o he s (Fig.2e). The e o e, he di e si y me ics (be a
and alpha) ep esen ed a unc ional disc imina ion c i e-
ion o map he gills bac e ial signa u es o di e en ish
species om di e en ishing si es.
The gill bac e ial signa u e swi ch acco ding o di e en
ishing si e
In o de o inspec he co e membe ship in he wo ish
species we pe o med an ASV-le el co e mic obiome
analysis ega dless o he ishing si e (Fig.3). The assem-
bly o gills bac e ial communi ies was s ongly dependen
on he di e en ishing si es and was highligh ed by he
o e all limi ed numbe o co e membe s in bo h ish spe-
cies da ase s (Fig.3a).
The ASV_1 (Esche ichia-Shigella) ep esen ed he
widely sha ed a ian in he co e gills mic obio a o sea-
bass p esen in all ishing si es while he o he co e a i-
an s we e la gely associa ed wi h he Psych obac e genus
(Seabass in Fig.3b). The seab eam showed a highe a i-
abili y in he gills mic obio a acco ding o each ishing
si es and his was e idenced by he educed co e mic o-
bio a, composed by h ee a ian s only, among hese he
mos widely ep esen ed was he ASV_2 (2013A k19i)
(Seab eam in Fig.3b).
We also es ima e occupancy-abundance me ics o
each sample si e in bo h ish species o be e desc ibe
he ish species co e membe ship. The analysis did no
explo e he di ec con ibu ion o be a di e si y bu
cha ac e ized he mos ep esen a i e ASVs o each si e
Fig. 1 Geog aphical dis ibu ion o ishing a eas in which he popula ions o he wo ish species D. lab ax and S. au a a a e caugh . The map shows he
sampling si es (colo code) acco ding o he name o he ela ed ishing po . The enla gemen s o he pa s o he coas a e shown on he le o he
map and e e o he coo dina es epo ed in he ma e ials and me hods sec ion. Map o Tuscan coas is gene a ed using QGIS e sion 3.30 (h ps://qgis.
o g) and edi ed using open sou ce g aphics edi o Inkscape 1.1.2 (h ps://inkscape.o g/) highligh ing he speci ic ishing a eas acco ding o he legend
men ioned in he igu e
Page 5 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
wi hin each ish species conside ing hei occupancy-
abundance a io (Fig. S2 and Addi ional ile 1: Table S3).
To deepen he axonomic a iabili y associa ed wi h each
si e we highligh ed he ASVs wi h occupancy = 1, i.e.
p esen in 100% o he samples associa ed wi h he si e
(The op 10 ASVs wi h ela i e axonomic assignmen
we e ep esen ed in Fig. S3). The analysis showed ha
some axa a e ep esen a i e o he gills co e membe ship
o he ish species hus de ec able in all si es wi hin he
ish species da ase , his condi ion was e iden o he
genus Psych obac e (Addi ional ile 1: Table S3). How-
e e , we obse ed ha speci ic ASVs ep esen he co e
ea u es (Occupancy = 1) o one speci ic si e only wi hin
speci ic ish species da ase . Conside ing only some an-
dom examples, ASV_85 (Halobacillus), ASV_114 (Cya-
nobium PCC- 6307), ASV_283 (Au an imonas) and
Fig. 2 Bac e ial dis ibu ion among si es in seabass and seab eam. a Mul idimensional scaling analysis (PCoA) based on B ay-Cu is dis ance acco ding
o he main wo a iables, ish species (shape pa e n) and si e (colo pa e n). R-squa ed alues and signi icance a e adonis pe mano a analysis es ed
on species and si e a iables a e epo ed inside he panel. b, c PCoA based on B ay-Cu is dis ance pe o med a e spli ing he da ase acco ding o he
ish species, i.e. seab eam (b) and seabass (c). PCoA epo ed sample dis ibu ion acco ding o he di e en si es (colo pa e n) while R-squa ed alues
and signi icance om adonis pe mano a analysis a e epo ed on op o he panels. d Hea maps display s a is ical signi icance and R-squa ed alues
om pai wise adonis pe mano a o each compa ison be ween g oups, om lowe alue (blue scale) o highe alue ( ed scale). Signi ican pai s a e
epo ed using as e isks (ns; no signi ica i e, *; P < 0.05, **; P < 0.01). e, Boxplo s epo ed he alpha di e si y me ics among di e en si es in seabass
(e) and seab eam ( ) da ase s. Pai wise compa isons among alpha di e si y measu es we e calcula ed by using he Wilcoxon es (Benjamini-Hochbe g
adjus men ). Signi ican pai s a e epo ed using as e isks (*; P < 0.05, **; P < 0.01, ***; P < 0.001, ****; P < 0.0001). The ishing si es a e epo ed acco ding o
he ollowing legend, CdP: Cas iglione della Pescaia, Ce: Cecina, Li: Li o no

Page 6 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
ASV_299 (Fic ibacillus) ep esen ed he co e membe s o
CdP si e in seab eam da ase only, while ASV_33 (Ca no-
bac e ium) and ASV_38 (Lysinibacillus) ep esen ed he
co e membe s o Ce in seabass da ase only.
These esul s co obo a e he p esence o a axonomic
si e- ela ed a iabili y in bo h seabass and seab eam
da ase s hus, highligh ing he p esence o speci ic co e
membe ship indica i e o di e en ishing si es.
In o de o inspec bac e ial ASVs dis ibu ion in di -
e en si es, he log-likelihood a io es (LRT o DESeq2)
on bo h ish species da ase was pe o med (Addi ional
ile 2: Table S4). We de ec a o al o 24 and 247 di e -
en ASVs o seabass and seab eam da ase s, espec i ely
(Addi ional ile 2: Table S4). The ASVs mean abun-
dance we e calcula ed oge he wi h ASVs p e alence
(see ma e ials and me hods o de ails) and ep esen ed
wi hin e na y space acco ding o he h ee ishing si es
(Fig.2a-b), highligh ing he ASVs signi ican ly selec ed
by LRT analysis (Fig.4). The dis ibu ion o ASVs signi i-
can ly in luenced by ishing si es (see colo ed scheme in
Fig.4 and clus e ing analysis in Fig. S4 and Addi ional ile
3: Fig. S5) based on hei abundance showed a axonomic
a iabili y consis en wi h di e en ishing si es.
This analysis sugges ed he p esence o he associa ion
among ishing si es and speci ic axonomic ea u es. Con-
side ing he seabass da ase , a o al o 10 di e en ASVs
assigned o he Psych obac e genus we e signi ican ly
associa ed wi h he CdP si e while he Ca nobac e ium
and Mic ococcaceae phylo ypes we e associa ed wi h he
Ce si e whe eas Ch yseobac e ium, Candida us Mic o-
h ix and Pa acoccus gene a wi h he Li si e (Fig.4a). The
same condi ion was depic ed in he seab eam da ase ,
wi h he p esence o a la ge numbe o gene a exclusi ely
associa ed wi h a ishing si e, among hese some an-
dom examples include, he gene a Ah ensia and Alkali-
bac e ium signi ican ly associa ed wi h he CdP si e, he
gene a Alcani o ax and Cycloclas icus signi ican ly asso-
cia ed wi h he Ce si e and he Cnexibac e and Glu a-
micibac e gene a we e signi ican ly associa ed wi h he
Li si e (Fig. 4b). In e es ingly, si e-speci ic associa ions
ega dless o he di e en ish species we e also isible,
o example, Mic ococcaceae amily was signi ican ly
Fig. 3 Bac e ial ASV-le el co e mic obio a. a Line cha shows he end o co e ASVs wi h hei p e alence (colo g adien ) ac oss he ela i e abundance
h esholds. b Hea map epo s he co e ASVs and ela ed axonomic assignmen wi h hei p e alence (colo g adien ) a di e en de ec ion h esholds
(Rela i e abundance) in each ish species da ase . The y-axis epo s he p e alence a e o he co e ASVs while he x-axis epo s he di e en de ec ion
h esholds ( ela i e abundance) ange
Page 7 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
associa ed wi h he Ce si e in bo h ish species, as well as
Mic o h ix genus signi ican ly associa ed wi h he Li si e
in bo h ish species (Fig.4a-b).
Clus e ing analysis based on s -scaled abundance
highligh ed he p esence o a si e- a iable gill mic obio a,
mo e e iden in seab eam da ase compa ed o he sea-
bass da ase , con i ming he e idence desc ibed in he
analyzes abo e and highligh ing he e ec i eness o he
use o a ge ed-me agenomic app oach o de ec si e-
dependen bac e ial signa u e in di e en ish species.
Discussion
The globaliza ion o he sea ood ade and he lack o
s anda ds o in o ma ion exchange wi hin he supply
chain ha e made acking o sea ood e y challenging
[27]. A anspa en and aceable sea ood supply chain
is necessa y o p omo e high-end a med sea ood and o
suppo a sea- o- o k scena io. The NGS echniques, in
conjunc ion wi h o he aceabili y echniques, will be -
e link all he ac o s in ol ed wi hin he supply chain
such as a me s, egula o s, policymake s, scien is s and
consume s. The applica ion o he animal mic obiome
in he aceabili y con ex , o example by using a ge ed
and un a ge ed me agenomic app oaches, will in oduce
a powe ul and eplicable ool o pu sue designa ion o
o igins allowing he usage o gua an ee adema k such
as P o ec ed Designa ion o O igin (PDO), P o ec ed
Geog aphical Indica ion (PGI), o au hen ica ion and
ood aud de ec ion.
He e, we es he applica ion o a widely used ech-
nique, easy o use o a company o ins i u ion o he
ac ually es ained cos and execu ion ime, on wo e y
Fig. 4 bac e ial p o iles swi ch acco ding o di e en ishing si es. Te na y plo s epo he ASVs mean ela i e abundances dis ibu ion wi h he loga i hm
o p e alence (size scale). The colo scheme ep esen s ASVs signi ican ly associa ed wi h di e en ishing si es acco ding o LRT analysis in seabass (a) and
seab eam (b) da ase s. Fo each signi ican ASVs he deepes axonomic assignmen is also epo ed. The b acke s oge he wi h he axonomic desc ip-
ion highligh he numbe o ASVs de ec ed o he same axonomic assignmen . The ishing si es a e epo ed acco ding o he ollowing abb e ia ion,
CdP: Cas iglione della Pescaia, Ce: Cecina, Li: Li o no
Page 8 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
impo an ish species o hei ele ance in ish ade in
a challenging con ex ep esen ed by ishing a eas highly
close o each o he , pe o ming he a ge ed me age-
nomic analysis on gills issue because hey a e desc ibed
o o e a comp ehensi e signa u e o he di e si y and
composi ion o ish mic obio a [25].
Ou main indings we e esumed by he ollowing
obse a ions, (i) The bac e ial di e si y (alpha and be a
di e si y) was signi ican ly a ec ed by bo h ishing spe-
cies and ishing si es. The ish species was he main
d i e in shaping he gills mic obio a, howe e speci ic
a ia ions acco ding o di e en ishing a eas we e also
obse ed. (ii) Si e-dependen changes in bac e ial di e -
si y we e no equally e iden in bo h ish species, in ac ,
seab eam specimens showed a clea pola iza ion o he
gills mic obio a acco ding o he h ee di e en ish-
ing si es while seabass did no show clea di e ences
be ween Ce s. Li si es. This da a sugges s ha he appli-
ca ion o hese echniques could be a ec ed by he ish
species conside ed, which implies a di e en ine- uning
o a ge ed me aba coding app oaches. (iii) The LRT
(DESeq2) analysis p oduced eliable axonomic p o iles
which, combined wi h clus e ing analysis, p o ide si e-
speci ic clus e s based on ASVs abundance dis ibu ion.
In de ail, all he obse a ions men ioned abo e we e
suppo ed by he ollowing speci ic esul s. The compo-
si ion o he gill bac e ial communi ies exhibi ed a si e-
dependen a iabili y and i is cons ained by a ela i ely
educed numbe o co e bac e ial membe s associa ed
wi h each ish species da ase s. Ou analysis shows ha
he co e is s ongly dependen on he ish species unde
examina ion: seab eam specimen p esen s a educed
co e in e ms o ASVs ( his condi ion was d i en by
high si e-speci ic di e si y), while he seabass specimen
showed a b oade co e mic obio a (a g ea e numbe
o ASVs sha ed by all samples in he sea bass da ase ).
The g ea e numbe o co e membe ships in he sea-
bass specimens explained he educed alues o a iance
explained in he be a di e si y me ics obse ed when
di e en ishing si es we e compa ed and he absence
o signi ican di e ence in he compa ison o Ce s. Li
si es. The genus Psych obac e ep esen ed one o he
mos sha ed axa wi hin bo h ish da ase s. The osmo-
ole an Psych obac e spp., iden i ied as a co e genus
in he seab eam in ou s udy, was also ound by Que o
and collabo a o s (2023) and assigned o he co e mem-
be s in sea- a med seab eam in he Medi e anean a ea
[23].In Pi edda and collabo a o s (2023), Psych obac e
was desc ibed as one o he mos ep esen a i e axa o
Scombe scomb us om FAO a eas 27.4, 27.7 and 27.8.
Fu he mo e, Psych obac e was iden i ied as SSOs in
he e alua ion o una spoilage, highligh ing i s possible
ole as a spoilage ma ke [28]. Taxa such as Ca nobac-
e ium and Pseudomonas, iden i ied in ou da ase can
be conside ed as SSOs o he assessmen o ish esh-
ness, mo eo e abundances o Esche ichia-Shigella and
S aphylococcus can be moni o ed o ensu e he ish
heal hiness. These aspec s highligh he plas ici y o his
sequencing app oach in di e en con ex s, all ela ed o
ish aceabili y.
Al hough a common axonomic signa u e is expec ed
in each ish species mic obio a, i was also in e es ing o
no e he high numbe o axa exclusi ely associa ed wi h
each ishing si e, especially o he seab eam da ase (e.g.
Ah ensia, Alkalibac e ium, Alcani o ax, Cycloclas icus,
Cnexibac e o Glu amicibac e ). These speci ic axa
could ep esen he componen o he gill mic obio a o
conside o u u e me aba coding-based analyses, de el-
oping an e en mo e p edic i e ish aceabili y sys em.
In e es ingly, a small numbe o signi ican ly selec ed
axa (see LRT) we e iden i ied in he same ishing si e in
bo h ish species, indica ing ha he gill mic obio a may
e lec he mic obial pa e n o he geog aphical a ea o
o igin. Simila e idence was ecen ly obse ed also in
he mussels My ilus gallop o incialis, which showed
si e-speci ic axonomic pa e ns [14, 26], co obo a -
ing he hypo hesis o i s possible applica ion as a ool o
aceabili y.
In he u u e, he numbe o axonomic ma ke s could
be inc eased by ex ending he coho o ished specimens
and expanding he mapped a ea. By doing ha , speci ic
da abases could be c ea ed o each a ea in acco dance o
speci ic ish species. This idea was sugges ed by Liu 2020
[29] in he s udy o so -shell clams mic obio a, highligh -
ing how he esea ch o solu ions in aceabili y con ex
based on he s udy o mic obial communi ies ga he s
g owing in e es in di e en sea ood b anches o s udy.
Finally, his s udy p o ides new da a on he mic obio a
o he gills o seabass and seab eam in a coas al a ea o
in e es o he local ish supply chain. This wo k u he
highligh s he impo ance o using a ge ed me agenomic
app oaches o acking ish species wi hin he ish supply
chain, p e en ing aud, e o s and ensu ing he co ec
ade and sa e y consump ion. This wo k demons a es
he possibili y o use gill mic obiome o di e en ia e he
o igin o ish cap u e a a small geog aphical scale. The
p oposal o bioma ke s analysis based on he gill mic o-
bio a can be in eg a ed wi hin he exis ing me hods o
con ol o he ish supply chain o egula o y agencies
guidelines in acco dance wi h he FAO acking sys em
and he Council Regula ion (EC) No. 1379/2013. Fu he -
mo e, he esul s p esen ed in his s udy can play a un-
damen al ole in p omo ing he use o hese me hods o
inc ease he ishing ecological sus ainabili y o in de in-
ing sa e s o age me hods, adding alue and encou ag-
ing he b oade concep o he consump ion o local ish
p oduc s.
Page 9 o 11Me iggi e al. Animal Mic obiome (2024) 6:13
Conclusion
This s udy p o ides an insigh o mic obial communi ies
o gill associa ed wild-caugh specimens o wo di e en
impo an comme cial species (D. lab ax and S. au a a),
demons a ing he possibili y o disc imina e be ween
he ish species and he sampling si e by analyzing he
espec i e mic obial signa u es. In addi ion, gills can be
easily collec ed om esh ish igh a e ishing and he
p ocedu e does no al e he appea ance and o ganolep-
ic quali ies o he p oduc . We he e o e sugges ha ou
p o ocol could be implemen ed by sea ood ma ke s ake-
holde s, such as egula o y agencies o companies. As
al eady sugges ed by del Rio-La in [26] and Liu [29], we
also would like o emphasize he necessi y o an upda ed
da abase o sea ood-associa ed mic obio a, o imp o e
he possibili y o implemen ing his me hod and subse-
quen echnical pe o mance and yield by esea ch ins i-
u ions and agencies.
Me hods
Fish collec ing and gills sampling
Fish specimens we e caugh by a p o essional ishing
company (San Leopoldo Piccola soc. coop. a .l La go
Mon e osa, 42–58,100 G osse o) in h ee di e en
sampling si es wi hin one nau ical mile (abou 1.8km)
om he coas , in he immedia e p oximi y o h ee
po s loca ed a he ollowing coo dina es: Li o no,
43.553634889212105, 10.301929423228817; Cecina,
43.303228810603976, 10.486780667967448; Cas iglione
della Pescaia, 42.76334583850374, 10.883166525614593
(Fig.1) belonging o he a ea o Tuscany coas named
“Cos adegli E uschi”, belonging o FAO ishing a ea
37.1.3, o GFCM Geog aphical Suba ea 9, FAO (2022)
[30]. The seabass specimens we e collec ed du ing la e
au umn/win e 2022–2023 whe eas seab eamwas col-
lec ed du ing la e summe /au umn 2022 in acco dance
wi h he seasonali y o each ish species. F ozen sam-
ples p o ided by he ishing company we e deli e ed o
he Depa men o Biology o he Uni e si y o Flo ence
(I aly) and s o ed a -20°C un il subsequen downs eam
p ocedu es. A e comple ely hawing he ish, a biopsy o
he igh gills o abou 2 cm2 was pe o med, including
bo h ca ilage and lamella issue acco ding o me hod-
ological p ocedu es men ioned in Clin on 2021 [25]. Fo
each sampling si e, en ish specimens o each ish spe-
cies we e p ocessed, o a o al o 60 di e en indi iduals.
All sampling p ocedu es we e ca ied ou in s e ile con-
di ions unde biological hood and all ins umen s ha e
been p e iously s e ilized wi h e hanol 70% o a oid any
con amina ion [31].
DNA ex ac ion, lib a y p epa a ion and a ge ed
me agenomic sequencing
Genomic DNA was ex ac ed om gills issue by DNeasy
Powe soil Ki (Qiagen, Hiledn, Ge many), acco ding o
manu ac u e ins uc ions. The genomic DNA was isu-
alized o quali y and in eg i y on s ained aga ose gel and
quan i ied using Qubi 4 Fluo ome e (The mo Fishe
Scien i ic,Wal ham, MA USA) 1x dsDNA High Sensi i -
i y ki . Fo each sample, PCR ampli ica ion o 16S V3-V4
hype a iable egions was pe o med, using p ime s 341
(5′-CCTACGGGNGGCWGCAG‐3)′ and 805 (5′‐GAC-
TACNVGGGTWTCTAATCC‐3′) [32]. Lib a ies we e
p epa ed acco ding o Illumina P o ocol 16 S Me age-
nomic Sequencing Lib a y P epa a ion (Pa # 15,044,223
Re . B; URL: h ps://www.illumina.com/con en /dam/
illumina-suppo /documen s/documen a ion/chem-
is y_documen a ion/16s/16s-me agenomic-lib a y-
p ep-guide-15044223-b.pd ). Pai ed end 2 × 300bp we e
pe o med on Illumina MiSeq Pla o m (Illumina Inc)
using MiSeq Reagen Ki 3 (600 cycle).
Amplicon sequence a ian s p oduc ion and s a is ical
analysis
The p ime pai sequences we e emo ed by using cu -
adap e sion 3.5 [33]. The amplicon sequence a ian s
(ASVs) in e ence was assessed by DADA2 pipeline e -
sion 1.26 [34]. Low quali y sequences we e emo ed
by using “ il e AndT im” wi h a maximum numbe o
expec ed e o h esholds o 2 o o wa d and e e se
ead pai s. The e o a e es ima ion was pe o med
using he “lea nE o s” and denoising was assessed by
he “dada” unc ion wi h de aul pa ame e s. Denoised
sequences we e me ged by using he “me gePai s” unc-
ion and chime ic sequences we e emo ed using he
“ emo eBime aDeno o” unc ion. The axonomic clas-
si ica ion was in e ed by using DECIPHER package e -
sion 2.26 [35]. ASVs ha we e no assigned o bac e ia
(unknown) o assigned o chlo oplas s and mi ochond ia
sequences we e emo ed o p ope ly pe o m he down-
s eam s a is ical analyses.
S a is ical analyzes we e pe o med in he R en i on-
men e sion 4.2.2 [36]. Be a di e si y was explo ed by
using he “ egan” package e sion 2.6.4 [37]. Ra e ac-
ion cu es we e calcula ed and depic ed by using he
“gg a e” unc ion o “ anacapa” package e sion 0.1 [38].
Co e mic obiome was assessed by using he “mic obi-
ome” package e sion 1.20 [39]. A e emo al o single
coun s and ans o ma ion in o ela i e abundances, he
dis ibu ion o samples was isualized by P incipal Coo -
dina e Analysis (PCoA) using he “cmdscale” unc ion o
he “s a ” package based on B ay-Cu is dis ance index.
Pe mu a ional mul i a ia e analysis o a iance using
dis ance ma ices (adonis pe mano a) was pe o med
o inspec di e ences be ween sample g oups using he