Identification of potential recruitment bottlenecks in larval stages of the giant fan mussel Pinna nobilis using specific quantitative PCR

Abstract

Pinna nobilis is an endangered species of fan mussel found along coastal Mediterranean waters requiring special attention for conservation. Populations are restricted in number, due to anthropogenic disturbances, disease, and in some areas, low rates of recruitment. To date, the difficulties associated with the identification of planktonic stages have prompted the use of benthic collectors as a proxy for quantifying larval supply, despite important information being lost regarding planktonic processes. We present evidence of spawning utilizing a qPCR assay developed for detecting genomic DNA of P. nobilis to enable specific identification of planktonic stages to augment knowledge of P. nobilis life history. In the Ebro Delta, Spain, it has been used to study what might be limiting their reproduction locally. We demonstrate the ability to differentiate DNA of P. nobilis from other bivalve mollusks and distinguish between fertilized and unfer-tilized eggs of P. nobilis, which may be a crucial point for understanding the low level of recruitment seen in this natural population. We also show evidence of larval presence during the expected spawning period, although abundance in positive samples were so low that they pose new questions about factors controlling the availability of planktonic stages of P. nobilis.

Breeding, planktonic and settlement factors shape recruitment patterns of one of the last remaining major population of Pinna nobilis within Spanish waters

“…20 to 26°C (Deudero et al, 2017). During the study, larval availability was not estimated in May-June 2016 since the qPCR technique was still under development (Andree et al, 2018), and therefore the presence of an earlier peak could have been overlooked.…”

“…Given the strong similarity in larval bivalve morphology, identification to species level is very challenging (Malchus & Sartori, 2013), and no studies have been conducted to assess natural variability in the availability of P. nobilis larvae. Yet, recent methodological approaches using q-PCR detection are now available to allow the estimation of larval abundances "per se" (Andree et al, 2018) and might provide a better understanding of early planktonic processes.…”

“…To this end we conducted: (1) long-term (>10 years) and mid-term (3 years, 2016 to 2018) assessment of recruitment events based on age determination from shells and on the abundance of juveniles (field densities) and settlers (artificial collectors), respectively. Further, we investigated the possible relationship between these data and long-term series of seawater temperature, salinity and phytoplankton availability; (2) evaluations of larval availability in the water column using qPCR analysis (Andree et al, 2018), and (3) histological examination of the gonads during the fertility period were also conducted in 2016. Both larval availability and early benthic stages were investigated in three areas of the bay with distinctive adult densities in order to establish a potential relationship between the number of breeding individuals and reproductive outputs.…”

“…Full methodological details of DNA extraction, qPCR amplification, calibration curves and assay optimization can be obtained from Andree et al (2018). Briefly, specific primers designed for the amplification of a small fragment (less than 200 bp) of the ribosomal DNA of P. nobilis were designed for assessing specific identity and abundance of target sequences in water samples, or for species identification among other bivalve larvae.…”

“…Briefly, specific primers designed for the amplification of a small fragment (less than 200 bp) of the ribosomal DNA of P. nobilis were designed for assessing specific identity and abundance of target sequences in water samples, or for species identification among other bivalve larvae. Due to the methodology of qPCR a higher abundance of specific target is inversely correlated to Ct values; that is, a low Ct value correlates to high abundance (Andree et al, 2018).…”