Field experiment
To validate the panel of markers using actual samples, zooplankton
samples were collected on 21 October 2019 in waters around Howth Head
(see Appendix S3 for further details), where mussel beds are
known to occur and expected to be spawning at the time of sampling.
Samples were collected at five stations in five replicates per station
(n = 25) by means of vertical tows using a 100 μm mesh plankton net with
a diameter of 40 cm. Depth and vertical profiles of temperature and
salinity were recorded using a YSI Castaway CTD probe (SonTek). Upon
collection, samples were equally split into two 50 ml tubes and
preserved in Lugol’s iodine solution (for microscopical analysis), and
70% ethanol (for DNA analysis), respectively. Microscopic analysis was
carried out using a Cobra Micro Zoom MZ1000 stereo microscope, where
larvae belonging to the class Bivalvia and Malacostraca were counted,
and their relative abundance (larvae/m3) was
calculated by dividing the total number of larvae by the approximate
volume (m3) of seawater sampled (estimated by
multiplying the net opening area by the depth). The 25 ethanol samples
were extracted with the Power soil Pro kit (Qiagen), using a customized
protocol for plankton samples (see Appendix S4 for for a
detailed protocol). The HT-qPCR run was conducted using 96.96 IFCs
configuration and following relevant protocols as indicated above with
the inclusion of BSA (see also results section). For quantification the
conventional and modeled approach were used to calculate the total
copies detected by the HT-qPCR, the copies were averaged per site and
compared to the microscopic data. For statistical analysis, ANOVA with
Tukey tests were used to determine significant difference between sites
for both indirect and direct methods (microscopy and genetic analysis).