carried out for MSFD-D2 implementation. Different trials were conducted to identify
the optimal sample volume of seawater (1L, 2L, 3L) and filter’s typologies of 0.45 µm
pore size: mixed cellulose ester (MCE), Polyvinylidene difluorite (PVDF),
Polyethersulfone (PES); besides this, multiple markers (e.g. 18S, COI, rbcl) were tested
for detecting NIS taxa belonging to different trophic groups.
Results
Overall, eDNA metabarcoding analyses carried out on all samples collected allowed the
identification of 837 marine species validated according to WoRMS database
(marinespecies.org). Preliminary results obtained by using different molecular markers
on eDNA extracted from 2L of seawater filtered on MCE filters and from sediments,
showed the genetic signature of >500 taxa. The taxa were matched against the
Mediterranean NIS list (CIESM Atlas of Exotic Species in the Mediterranean
https://www.ciesm.org and Zenetos et al., 2022) and the Italian waters NIS list
(provided by ISPRA). Among the 18 NIS identified only 8 are already reported from
Italian waters, and only Pseudodiaptomus marinus Sato, 1913 and Styela plicata
(Lesueur, 1823) have been found between 2015-2020 with traditional monitoring in the
same area. According to our analysis, eDNA of 10 NIS that have never been identified
in the Italian seas, was recovered.
Discussion and conclusions
The high-throughput sequencing of eDNA provides a valid complementary tool supporting
traditional morpho-taxonomic analyses for investigating marine biodiversity (Pawlowski et al.,
2021). The advantage of eDNA metabarcoding for biodiversity analyses relies on its capacity to
detect on a relatively short-time scale a broad range of species, including small size organisms that
are difficult to identify by the classical taxonomic approach (Ruppert et al., 2019). However,
caution should be posed in using eDNA metabarcoding data for NIS surveillance given: 1) the
inability of used gene regions to reliably detect some taxa at species level, 2) the incompleteness of
reference databases (NCBI, BOLD) for marine organisms, 3) the transient nature of eDNA being
produced and degraded with a different extent in different marine environments, and 4) errors that
may occur in the reference data (Duarte et al., 2021). Results presented in this study allowed the
identification of 10 NIS that potentially can be recent new introductions or species undetected by
traditional monitoring analyses. These findings encourage the use of eDNA to identify the
presence of new NIS and/or harmful species, which can support the traditional monitoring and
mitigation strategies for avoiding the spreading of alien species.
Bibliography
DUARTE S., VIEIRA P., LAVRADOR A.S., COSTA F.O. (2021) - Status and prospects of
marine NIS detection and monitoring through (e)DNA metabarcoding. Science of The Total
Environment, 751, 141729.
PAWLOWSKI J. et al., (2022) - Environmental DNA metabarcoding for benthic monitoring: A review of
sediment sampling and DNA extraction methods. Science of The Total Environment, 818, 151783.
RUPPERT K.M., KLINE R.J., RAHMAN M.S. (2019) - Past, present, and future perspectives
of environmental DNA (eDNA) metabarcoding: A systematic review in methods,
monitoring, and applications of global eDNA. Glob. Ecol. Conserv, 17, e00547.
ZENETOS A., ALBANO P. G., LÓPEZ GARCIA E., STERN N., TSIAMIS K., GALANIDI M. (2022) -
Established non-indigenous species increased by 40% in 11 years in the Mediterranean Sea.
Mediterranean Marine Science, 23(1).
2nd Mediterranean Symposium on the Non-Indigenous Species (Genoa, Italy, 22-23 September 2022)
49
the optimal sample volume of seawater (1L, 2L, 3L) and filter’s typologies of 0.45 µm
pore size: mixed cellulose ester (MCE), Polyvinylidene difluorite (PVDF),
Polyethersulfone (PES); besides this, multiple markers (e.g. 18S, COI, rbcl) were tested
for detecting NIS taxa belonging to different trophic groups.
Results
Overall, eDNA metabarcoding analyses carried out on all samples collected allowed the
identification of 837 marine species validated according to WoRMS database
(marinespecies.org). Preliminary results obtained by using different molecular markers
on eDNA extracted from 2L of seawater filtered on MCE filters and from sediments,
showed the genetic signature of >500 taxa. The taxa were matched against the
Mediterranean NIS list (CIESM Atlas of Exotic Species in the Mediterranean
https://www.ciesm.org and Zenetos et al., 2022) and the Italian waters NIS list
(provided by ISPRA). Among the 18 NIS identified only 8 are already reported from
Italian waters, and only Pseudodiaptomus marinus Sato, 1913 and Styela plicata
(Lesueur, 1823) have been found between 2015-2020 with traditional monitoring in the
same area. According to our analysis, eDNA of 10 NIS that have never been identified
in the Italian seas, was recovered.
Discussion and conclusions
The high-throughput sequencing of eDNA provides a valid complementary tool supporting
traditional morpho-taxonomic analyses for investigating marine biodiversity (Pawlowski et al.,
2021). The advantage of eDNA metabarcoding for biodiversity analyses relies on its capacity to
detect on a relatively short-time scale a broad range of species, including small size organisms that
are difficult to identify by the classical taxonomic approach (Ruppert et al., 2019). However,
caution should be posed in using eDNA metabarcoding data for NIS surveillance given: 1) the
inability of used gene regions to reliably detect some taxa at species level, 2) the incompleteness of
reference databases (NCBI, BOLD) for marine organisms, 3) the transient nature of eDNA being
produced and degraded with a different extent in different marine environments, and 4) errors that
may occur in the reference data (Duarte et al., 2021). Results presented in this study allowed the
identification of 10 NIS that potentially can be recent new introductions or species undetected by
traditional monitoring analyses. These findings encourage the use of eDNA to identify the
presence of new NIS and/or harmful species, which can support the traditional monitoring and
mitigation strategies for avoiding the spreading of alien species.
Bibliography
DUARTE S., VIEIRA P., LAVRADOR A.S., COSTA F.O. (2021) - Status and prospects of
marine NIS detection and monitoring through (e)DNA metabarcoding. Science of The Total
Environment, 751, 141729.
PAWLOWSKI J. et al., (2022) - Environmental DNA metabarcoding for benthic monitoring: A review of
sediment sampling and DNA extraction methods. Science of The Total Environment, 818, 151783.
RUPPERT K.M., KLINE R.J., RAHMAN M.S. (2019) - Past, present, and future perspectives
of environmental DNA (eDNA) metabarcoding: A systematic review in methods,
monitoring, and applications of global eDNA. Glob. Ecol. Conserv, 17, e00547.
ZENETOS A., ALBANO P. G., LÓPEZ GARCIA E., STERN N., TSIAMIS K., GALANIDI M. (2022) -
Established non-indigenous species increased by 40% in 11 years in the Mediterranean Sea.
Mediterranean Marine Science, 23(1).
2nd Mediterranean Symposium on the Non-Indigenous Species (Genoa, Italy, 22-23 September 2022)
49