Abstract |
Pinna nobilis, also known as the noble pen shell, is an endemic fan mussel, the largest bivalve
mollusk of the Mediterranean Sea, reaching up to 120 cm length, and it is found in a range of
depths between 0.5 and 60 m in soft substrate and seagrass meadows. During the last years, the
pen shell populations have decreased drastically, beginning from Spain, and spreading to the rest
of the Mediterranean basin, including Greece. The mass mortality event (MME) has been
attributed, among other causes, to a parasitic disease caused by the novel protozoan
Haplosporidium pinnae, which has been identified with histology and molecular techniques in
the tissues of sick individuals. The magnitude of the phenomenon led to the change of the fan
mussel’s status to Critically Endangered by the IUCN in December 2019. In this study, a PCR was
conducted to identify the presence of the parasite in 16 P. nobilis individuals from Amvrakikos
Gulf and in 26 benthic metazoans from locations where P. nobilis was known to exist, to assess
the new population’s health condition and to investigate the existence of intermediate hosts for
H. pinnae. In addition to that, PCR for Mycobacterium was conducted in 38 P. nobilis individuals.
Lastly, a metabarcoding analysis for the 18S rRNA and 16S rRNA genes was conducted to
investigate the presence of H. pinnae, Mycobacterium and Vibrio spp., but also to compare the
eukaryotic and prokaryotic microbiome of a presumably healthy population and that of affected
(sick) populations. None of the benthic organisms, except for the pen shell, didn’t give a positive
signal for H. pinnae. The results of the PCR and the metabarcoding analysis confirmed that the
pen shells from Amvrakikos Gulf were healthy (H. pinnae negative), but the other populations
were sick and positive to H. pinnae, Mycobacterium and Vibrio spp. The majority of the ASVs of
Mycobacterium spp. were identified as species of the M. simiae complex and the most abundant
ASVs of Vibrio spp. belonged to the Splendidus clade, while V. mediterranei was identified in few
copies in only two locations. LEfSe analysis pointed out 143 biomarker genera, which differed,
statistically significantly, in abundance between healthy and sick individuals. The most abundant
genera of healthy pen shells included Rubritalea, Novosphingobium, Bradyrhizobium, while in
sick pen shells the genera Mycobacterium, Haplosporidium και Vibrio stood out, which are well
known pathogens, but also other, potentially opportunistic pathogens, like Endozoicomonas,
Psychrilyobacter, Acinetobacter, Pseudoalteromonas and Legionella. To conclude, this study’s
results confirm the role of H. pinnae as the initial disease factor, which causes a disruption of the
microbiome and the subsequent increase of opportunistic pathogens, which eventually lead to
the pen shell mass mortality.
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