Karl B Andree
email: karl.andree@irta.es

Sergio Trigos

Nardo Vicente

Noelia Carrasco

Francesca Carella

Patricia Prado

Á P Carrasco

Prado

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

Keywords: Shellfish larvae, Western Mediterranean, Pinna, qPCR

Pinna nobilis is an endangered species of fan mussel found along coastal Mediterranean waters requiring special attention for conservation. Popula-tions 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 detect-ing genomic DNA of P. nobilis to enable specific identification of planktonic stages to augment knowl-edge 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.

Introduction

The endangered fan mussel Pinna nobilis L. is the largest and one of the most emblematic marine Mediterranean bivalves, reaching sizes of up to 120 cm, and 20 years of age [START_REF] Zavodnik | Synopsis on the fan shell Pinna nobilis L. in the eastern Adriatic Sea[END_REF][START_REF] Butler | Ecology of de pterioid bivalves Pinna bicolor Gmelin and P. nobilis L[END_REF]. Its endemic nature prompted an inclusion within the list of endangered Mediterranean species at the Barcelona convention (Protocol ASPIM Annex 2). The European Council Habitats Directive 92/43/EEC, on conservation of natural habitats and the wild fauna and flora, proclaims that P. nobilis is strictly protected under Annex IV EEC 1992, and all forms of deliberate capture or killing of fan mussels are prohibited by law [START_REF] Centoducati | Monitoring of the endangered Pinna nobilis Linne ´, 1758 in the Mar Grande of Taranto (Ionian Sea, Italy)[END_REF]. Yet, poaching activities continue (e.g., [START_REF] Siletic | Population study of the fan shell Pinna nobilis in Malo and Veliko Jezero of the Mljet National Park (Adriatic Sea)[END_REF], and an indirect threat is still imposed by a multiplicity of anthropogenic stressors such as fish-eries that use trawling, eutrophication of coastal embayments [START_REF] Marba | Growth and population dynamics of Posidonia oceanica on the Spanish Mediterranean coast: elucidating seagrass decline[END_REF], and emergent diseases. For instance, a significant large-scale mor-tality occurred during the summer of 2016 along the Spanish Mediterranean coast and the Balearic Islands, possibly associated with outbreaks of a haplosporidan parasite [START_REF] Darriba | First haplosporidan parasite reported infecting a member of the Superfamily Pinnoidea (Pinna nobilis) during a mortality event in Alicante (Spain, Western Mediterranean)[END_REF]. In certain areas such as the Mljet lakes (Croatia), and the Mar Grande Basin (Italy) P. nobilis populations have been reported to experience some increase (e.g., [START_REF] Siletic | Population study of the fan shell Pinna nobilis in Malo and Veliko Jezero of the Mljet National Park (Adriatic Sea)[END_REF][START_REF] Centoducati | Monitoring of the endangered Pinna nobilis Linne ´, 1758 in the Mar Grande of Taranto (Ionian Sea, Italy)[END_REF], although common abundances do not usually exceed one individual per 100 m 2 [START_REF] Guallart | Bases ecolo ´gicas preliminares para la conservacio ´n de las especies de intere ´s comunitario en Espan ˜a: Invertebrados[END_REF].

Recruitment rates of P. nobilis within benthic collectors are often used as a proxy for larval availability (e.g., [START_REF] Kersting | Preliminary data on Pinna nobilis larval recruitment using mesh collectors in Columbretes Islands Marine Reserve, NW Mediterranean[END_REF], but planktonic abundances per se have never been evaluated in any region. To distinguish bivalve species by larval morphology is not easily achieved as the veliger larval morphology reflects more ancestral states than adult morphology and therefore makes identification to species level of the plankton stages unlikely [START_REF] Malchus | Part N, Revised, Volume 1, Chapter 4: the early shell: ontogeny, features, and evolution[END_REF]). Yet, the abundance of planktonic stages of P. nobilis in open waters is not necessarily the same as in the benthos, and the pelagic-benthic coupling may not affect all species equally [START_REF] Ebert | Recruitment in echinoderms[END_REF]. For instance, it has been shown that large-scale environmental factors (e.g., currents, nutrients, temperature) were the most influential on larval availability, whereas benthic abundances were determined by the complexity of spatial distribution of preferential substrates for settlement, predation and migration occurring at smaller spatial scales [START_REF] Prado | Habitat and scale shape the demographic fate of the keystone sea urchin Paracentrotus lividus in mediterranean macrophyte communities[END_REF].

Alfacs Bay (Ebro Delta, Catalonia) has been reported to contain the second largest population of P. nobilis in the Mediterranean with an estimated number of over 90,000 individuals, with peak densities of up to 20 individuals per 100 m 2 , and mean densities of 1.61 m -2 [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF]. The population is characterized by an extremely superficial distribution of individuals growing over extensive meadows of the seagrass Cymodocea nodosa, at depths from 20 cm down to 130 cm [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF]. Although this is close to the shallow distribution limit established for the species (ca. 0.5-60 m; [START_REF] Butler | Ecology of de pterioid bivalves Pinna bicolor Gmelin and P. nobilis L[END_REF], it usually displays peak densities at depths from ca. 3-4 to 14-15 m (Garcı´a-March & [START_REF] Garcı ´a-March | Preliminary data on the distribution and density of Pinna nobilis and Pinna rudis in the Columbretes Islands Marine Reserve (Western Mediterranean, Spain)[END_REF][START_REF] Katsanevakis | Growth and mortality rates fan mussel Pinna nobilis in Lake Vouliagmeni (Korinthiakos Gulf, Greece): a generalized additive modelling approach[END_REF][START_REF] Coppa | The effect of hydrodynamics on shell orientation and population density of Pinna nobilis in the Gulf of Oristano (Sardinia, Italy)[END_REF]. Those animals inhabiting depths of less than 1 m are only rarely reported (see [START_REF] Zakhama-Sraieb | Density and population structure of Pinna nobilis (Mollusca, Bivalvia) in the Ghar El Melh lagoon (N-E Tunisia)[END_REF]. The origin of the Alfacs Bay population is uncertain and was apparently reduced to a few isolated individuals during the 1990s (J. Romero, pers. comm.), until a major recruitment event occurred. Nearby locations such as the Sierra de lIRTA, Columbretes Islands, or L'Ampolla de Mar may be possible sources for larval recruitment. Currently, the population displays a very homogeneous size structure (between 40 and 60 cm) and lacks individuals smaller than 20 cm. This suggests an important limitation in the availability of larvae and/or high mortality during the processes of benthic settlement and recruitment [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF]. The distribution of individuals in the bay is highly variable, and includes large unpopulated areas. This is presumably due to changes in habitat and/or the chemical characteristics of the water of the bay (e.g., agricultural runoff from extensive rice cultivation in the northern shore of the bay, and salt evaporation ponds along the inner part of the southern shore; [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF]. Therefore, developing an adequate tool for evaluating larval availability and fertilization success will aid in assessing the factors driving the demographics of the population within the bay (but may also be applicable to other regions). Such information could be very helpful for the implemen-tation of conservation programs that take into account how planktonic processes could be effected by local factors, such as agricultural runoff and/or the density of adults.

The use of quantitative polymerase chain reaction (qPCR) has proven to be useful for detection of low abundance and cryptic species in aquatic environments [START_REF] Ficetola | Species detection using environmental DNA from water samples[END_REF], or where morphological characteristics cannot be used for species discrimination [START_REF] Andree | Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea[END_REF]. More recently, qPCR has been shown to be an effective and sensitive tool for the detection and quantification of planktonic organisms (e.g., [START_REF] Vadopalas | Application of real-time PCR for simultaneous identification and quantification of larval abalone[END_REF][START_REF] Endo | Species-specific detection and quantification of common barnacle larvae from the Japanese coast using real time PCR[END_REF], whose identification through classical observational methods is extensive and arduous. More relevantly, qPCR has been used for the detection and quantification of larvae in other species of commercially important mollusks such as abalone [START_REF] Vadopalas | Application of real-time PCR for simultaneous identification and quantification of larval abalone[END_REF] and invasive alien species such as zebra and golden mussels [START_REF] Frischer | Specific amplification of the 18S rRNA gene as a method to detect zebra mussel (Dreissena polymorpha) larvae in plankton samples[END_REF][START_REF] Endo | Molecular based method for the detection and quantification of larvae of the golden mussel Limnoperna fortunei using real-time PCR[END_REF]. For these purposes planktonic abundances were estimated by plotting threshold cycle (C t ) values on the standard curve obtained from template DNA extracted from serial dilution of eggs/larvae (see for instance, [START_REF] Endo | Species-specific detection and quantification of common barnacle larvae from the Japanese coast using real time PCR[END_REF]. Therefore, one limitation to the devel-opment of such methods relies on the availability of planktonic material of the target species for construction of a proper calibration curve. Moreover, the interpretation of results requires some consideration be given to the chromosome endowment of the analyzed sample (fertilized or non-fertilized egg or larva), and the copy number of the genomic region being targeted by the assay.

A more direct need for assay development is a genetic marker that is specific and easily isolated. Ribosomal RNA genes occur in multiple copies within the genome, with many conserved regions interspersed with more variable portions, making them ideal for such use. Ribosomal DNA genes are one of the most common markers used for clarification of taxonomic assignment. There exist in genetic databases an ample variety of these sequences from many species whose genomes are otherwise completely unknown. The non-structural intergenic spacers have been shown to be especially useful for fine-scale phylogenetic assessment. The high rate of mutation during evolutionary time scales in ITS regions of the rDNA cistron, as compared to coding regions, means that there is a lower probability of encountering a similar sequence in closely related species to which species-specific primers can bind. Depending on the species under study this region may be useful as a genetic marker for population assignment [START_REF] Wren | Repeat polymorphisms within gene regions: phenotypic and evolutionary implications[END_REF].

In the particular case of P. nobilis, the apparent short period of larval planktonic life of between 5 and 10 days [START_REF] De Gaulejac | Ecologie de Pinna nobilis (L.) mollusque bivalve sur les co ˆtes de Corse. Essais de transplantation et expe ´riences en milieu contro ˆle ´[END_REF]) may help reduce variability in the number of larval cells among individuals collected at different time points. Among some commercial bivalve species studied more closely, a ''low …but uniform'' growth rate has been observed [START_REF] Robert | Bivalve hatchery technology: the current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France[END_REF], but only few data exist for P. nobilis. The fertilized oocytes are * 50 lm in size and in 5-10 days when they are ready to settle to the benthos they reach a diameter of * 100 lm [START_REF] Trigos | Estudio de la ecofisiologı ´a y ensayo de cultivo de la nacra Pinna nobilis Linnaeus[END_REF]. The modest increase in size suggests the number of cells should not vary considerably between different planktonic larvae. Hence, it was considered that quantitative PCR could be applied for the efficient detection and quantification of P. nobilis eggs and larvae. This leads to the following specific objectives: (1) to identify a species-specific marker for a qPCR assay; (2) to design primers for specific identification of P. nobilis, which utilize genomic regions that are well conserved at the species level, but with high interspecific variability to avoid false positives in screening environmental samples [START_REF] Andree | Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea[END_REF][START_REF] Dejean | Persistence of environmental DNA in freshwater ecosystems[END_REF][START_REF] Wilcox | Robust detection of rare species using environmental DNA: the importance of primer specificity[END_REF]; (3) to establish a useful calibration curve for fertilized and unfertilized eggs and larvae of P. nobilis that allow interpretation of C t values from field samples; and (4) to determine a field sampling method most effective for detecting and quantifying the presence of P. nobilis larvae or gametes. Meeting these objectives will provide the techniques for more expansive evaluations in other Mediterranean regions hosting P. nobilis.

Materials and methods

Sample collection

Tissue samples were collected from living P. nobilis in situ by pinching off a small piece of mantle using a 15-cm-long hemostat inserted between semi-opened valves, and preserving in 70% ethanol until later DNA extraction (Fig. 1). For the purpose of obtaining DNA sequence data, samples were initially collected from two sites: Alfacs Bay (n = 21) [40°35 0 40.59 00 N; 0°39 0 37.36 00 E] and to the south, outside of the bay, in the port of Las Casas de Alcanar (n =9) [40°33 0 09.32 00 N; 0°31 0 53.64 00 E] (Fig. 2).

Initially (from 06/01/16 to 07/01/16), only 500 ml water samples (n = 3 per site) were collected immediately adjacent to individual adult animals in situ and later filtered through a 10 lm nylon mesh. The mesh was rinsed with 400 ll lysis buffer to collect material for extraction and kept frozen until further processing. In June, when the first 500 ml samples were collected, they were also examined in the lab by light microscopy, and bivalve larvae identified and counted. However, the observation of single larvae in this volume was rare, therefore from 07/15/16 to 10/01/16, sampling was conducted every 15 days using a plankton net having an open aperture of 0.2 m diameter and 30 lm size mesh (sufficient for catching eggs and larvae). Samples were collected as before in 3 sites (n = 3 replicates per site) by walking ca. 30 m over the seagrass bed of Cymodocea nodosa, equivalent to a filtration volume of 3.77 m 3 . Samples were preserved in 70% ethanol and separated into different size fractions: 40 lm (egg sizes ranging from 40 to 70 lm and larval sizes from ca. 80-110; [START_REF] Trigos | Adult spawning and early larval development of the endangered bivalve Pinna nobilis[END_REF], 125 and 250 lm (to check for additional entangled material) using sieve nets. A preliminary observation of each fraction and sample was also conducted in order to determine the abundance of eggs and larvae and the feasibility of their separation. For eggs, given the large abundance and the absence of identification keys that could aid the distinction from other marine invertebrates, separation was considered unfeasible and not conducted. However, all bivalve larvae observed were collected. When the number was too large for accurate counting (hundreds to thousands of individuals), fractions were divided into smaller subsamples (1/2, 1/4, or 1/8 depending on the number), and later the total number of larvae in the sample calculated from Ct values obtained from these subsamples. Individual larvae were separated under the dissecting microscope (NIKON SMZ1500) using a zooplankton ''micro-spoon'' fabricated from a thin piece of platinum wire to which a loop of ca. 80 lm diameter was formed. All separated larvae from each replicate sample were kept in 1.5 ml vials with 30 ll of Qiagen AE buffer (to ease larvae release from the spoon) and stored at -80 °C until later DNA extraction (see below).

For the construction of calibration curves we used a limited supply of ethanol preserved D-veliger larvae (all of these ca. 85 lm in length), and fertilized and unfertilized egg samples of P. nobilis (Fig. 3) collected from an in vitro reproduction study performed previously in another institute [START_REF] Trigos | Adult spawning and early larval development of the endangered bivalve Pinna nobilis[END_REF] (Fig. 4). This same supply of eggs previously produced in the lab were also used for creating pseudofield samples to test the likelihood of collecting eggs from seawater and detecting the presence of P. nobilis DNA. This was done by collecting 10 eggs or 1 egg and placing them into different volumes of filtered seawater (15, 50, or 500 ml) that were filtered through a10lm nylon mesh for subsequent DNA extraction and testing by qPCR. Filters were rinsed with 400 ll of lysis buffer containing 40 ll proteinase K solution (Ref# 19131, Qiagen Valencia, CA, USA) to collect filtrate for DNA extraction. All of these experiments were performed with a somewhat low number of replicates and total N since the material used, eggs and larvae, was produced from experiments in P. nobilis captive spawning. Spawning for this species is still somewhat at the experimental stage, and not of a commercial nature as it is for cultured species such as Crassostrea gigas. This limited supply of eggs and larvae limited the number of experiments and the number of replicates per experiment.

DNA extraction, amplification, sequencing, and qPCR amplification Extraction of DNA utilized the Qiagen Blood and Tissue Kit (Ref# 69506, Qiagen, Valencia, CA, USA), and for all samples the A 260/280 ratios were examined Fig. 1 Non-lethal sample collection in situ from P. nobilis, using a hemostat by spectrophotometry to evaluate purity and concentration. Seawater samples were filtered using 10 lm mesh nylon filters and the filtrate treated the same as eggs and larvae samples using the same protocol.

DNA concentrations of all tissue samples were normalized to 100 ng/ll, based upon the values obtained by spectrophotometry, prior to use for PCR. For all other sample types (eggs, larvae, or seawater) The thermal cycler program used was 40 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, preceded by 5 min at 95°C, and followed by 10 min at 72°C. The resulting genomic fragment was approximately 1200 bp. Each PCR product was purified using the Qiagen PCR Purification Kit (Qiagen, Valencia, CA, USA), then sent to a private company for bidirectional sequencing using the same primers as those in the original amplification (Sistemas Genomicos, Valencia, Spain). The resulting sequence data were edited and trimmed using BioEdit ver 7.0.5.2 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT[END_REF] to remove terminal primer sequence artifacts and cleaned of any aberrant basecalling before aligning the forward and reverse reads to construct a consensus sequence representative of the specimens analyzed. The sequence was then subjected to analysis using BLAST to confirm the identity of the sequence obtained. Further, the BLAST results served to identify what species might have sequences already described in the GenBank database that are similar. The P. nobilis sequence was used for designing the species-specific primers that avoided the repeat regions to be applied in the qPCR assay. The primer pair Pin ITSF3 5 0 GTACCTGTGCCGAGTTCTCTC G 3 0 and Pin ITSR2 5 0 CGCCGTTGGATGAAACCG TACG 3 0 produced a 180 bp product, and this pair was chosen for use in assay optimization. The primer BLAST application (within GenBank NCBI) was used to test in silico the specificity of the primers and validate the absence of any other priming site within sequences currently in the NCBI database. After testing various annealing temperatures, the final qPCR program consisted of 45 cycles of 95°C for 20 s, 66°C for 30 s, preceded by 5 min at 95°C, and followed by a dissociation stage for determining the melt curve. All reactions contained 19 SYBR Green mix (Ref# 4364344, Thermo Fisher Scientific, USA) and 0.5 lM of each primer. Samples were analyzed in triplicate, and each plate (96 wells) contained negative and positive controls of genomic DNA from adult tissues to confirm validity of the reactions. For all positive results, comparison of melt curves of unknowns from all collection sites (3 subsites within Alfacs Bay, as well as Las Casas de Alcanar, and southeast of France) was made to melt curves of our known positive controls for confirmation of sequence identity. Calibration curves, specificity, and assay optimization ( To confirm the specificity of the assay, were tested using 50 ng of genomic DNA from the three most abundant commercially produced shellfish species: Crassostrea gigas, Cerastoderma edule, and Mytilus galloprovincialis. Further, the species-specific qPCR was tested on different numbers of eggs and larvae produced by captive spawning (as mentioned above) from individuals located in the Southeast of France and preserved in 70% ethanol [START_REF] Trigos | Adult spawning and early larval development of the endangered bivalve Pinna nobilis[END_REF]. Specific numbers of fertilized and unfertilized eggs, and larvae from these lab samples (n = 5, 10, 20 and 50; with two replicates of each), were used for the purpose of establishing a correlation between the C t values obtained and the number of individuals (fertilized/unfertilized eggs or larvae), i.e.,-a calibration curve. DNA extractions were performed as before with the Qiagen Blood and Tissue Kit, but with the additional step of bead-beating using a BeadBeater-8 (BioSpec Products, Bartlesville, OK, USA). Further, genomic DNA obtained from adult tissues was also tested for calibration and used to evaluate the overall efficiency of the assay. Additionally, a subset of DNA extracts was serially diluted (1:10, 1:20, 1:50, 1:100) to remove/dilute potential PCR inhibitors that may have originated in either the egg matrix or seawater as seen previously [START_REF] Andree | Quantitative PCR coupled with melt curve analysis for detection of selected Pseudo-nitzschia spp. (Bacillariophyceae) from the northwestern Mediterranean Sea[END_REF]. All DNA extracts were eluted in a final volume of 30 ll. Linear regression of the calibration curve established a slope that was used for the calculation of amplification efficiency (E%) following the equation: E%=10 -1/ slope) -1.

Results

Multiple repeat regions (short poly-A, poly-G, and poly-C stretches) were detected during sequence analysis of the intergenic transcribed spacer (ITS) rDNA sequences. These were problematic for sequencing and establishment of correct consensus sequences. Aside from the repeat regions, the remaining sequence fragments from all 30 isolates provided a clear consensus sequence. The two longest unambiguous sequences obtained have been uploaded to GenBank under accession numbers KX101234 and KX101235. Of the 447 bivalve mollusk ITS ribosomal DNA sequences currently in GenBank, results from BLAST analysis found only one species (other than P. nobilis), Perna perna that was a near match with a similarity index of 96.4%. No primer binding of the designed primers to the Perna perna sequence was evident in silico. Testing the different primer sets for P. nobilis demonstrated that each of the primer pairs tested had distinct melt curves (data not shown). The primer pair chosen amplified its respective target more robustly as compared to the other primer sets, as evidenced by the lower Ct value obtained when using the same quantity of DNA. Further, no amplification was seen when using genomic DNA from C. gigas, C. edule, or M. galloprovincialis, confirming in part, the specificity of the assay. Optimization of the assay using the DNA from adult tissues established an E% = 99.8% (R 2 = 0.997). This was similar for fertilized (97.6%; R 2 = 0.89) and unfertilized (99.8%; R 2 = 0.98) eggs, although the y intercepts were 50.33 and 54.23, respectively. The slope obtained when all egg samples were included indi-cated efficiency of 100.9% (Fig. 4).

Results of the calibration curve using larvae samples gave erratic results with no correspondence between extracted DNA and specific numbers of larvae per sample. We did obtain a standard curve using larvae, however the slope obtained was -3.73 indicating a E% = 85.4%. Another assay was conducted using seven replicates of only one larvae. The mean of the C t values from these was 36.9. Using the standard curve for diploid eggs, a C t of 36.9 is approximately 17,844 diploid cells. This value was used to correct for all the other calculations made from larvae isolated from field samples. Thus, the C t value obtained from one larva was used to normalize the quantity of P. nobilis larvae within plankton net field samples, giving a range from 0.2 to 32.4 (i.e., up to 8.6 larvaeÁm -3 ).

Field samples of 500 ml water collected in Alfacs Bay were all negative. Tests performed to establish possible losses of eggs and larvae during the filtration process were evaluated using pseudo-field samples (filtered seawater samples spiked with either ten eggs or one egg) and showed that samples with one egg in more than 50 ml were not consistently positive.

Further, although DNA was obtained from both field samples and preserved lab-cultured samples, there were some doubts about possible PCR inhibition since the expected amplification at C t values lower than 25 was not observed. Testing by qPCR of some samples serially diluted (1:10, 1:50, 1:100) was performed to eliminate potential inhibitors, but the expected reduction in Ct values (an indication of suppression of inhibition) was not observed. Yet, we continued using 1:10 dilutions in order to increase the quantity of sample available for experimentation.

Larvae from the plankton net samples collected manually using a micro-spoon under a dissecting microscope were used in a pool as DNA extraction material. From among these samples, several (15/62) were identified as containing P. nobilis (Table 1). The number of positives increased slightly from June-July to August-September. From the total number of positive samples, the mean C t value was 36.41.

Discussion

The present study constitutes the first approach conducted for quantifying the abundance of P. nobilis larvae in the water column, and proved to be effective in detecting P. nobilis DNA from larvae obtained from field samples. The assay did not amplify DNA of other mollusk and non-target bivalve species as confirmed by comparison to the melt curve profiles from known positive samples. Further, the few positives detected among the larvae collected from plankton net samples, (as well as the low number of ''hits'' seen by BLAST analysis) strongly suggest this assay has a high specificity. The assay performed as expected with P. nobilis DNA from three distinct populations (Alfacs Bay, Las Casas de Alcanar, and Southeast of France), supporting that it has broad applicability for similar quantitative assessments in other Mediterranean regions. We collected samples from all the local Pinna shellfish beds of which we were aware for evaluating the genetic diversity of the local populations. This was to ensure that the assay should be productive for this species with no ''cross reactivity'' with related shellfish. The conserved nature of the ribosomal DNA regions where the diagnostic primers bind provides support for this. Whether the sequence data obtained represent a consensus for all populations across the Mediterranean requires further sequence data, sampling, and utilization of this test for assessment. However, the results are suggestively robust considering some of the tested samples are from as far away as France. Additionally, [START_REF] Wesselmann | Genetic and oceanographic tools reveal high population connectivity and diversity in the endangered pen shell Pinna nobilis[END_REF] demonstrated using microsatellite markers that populations of P. nobilis from across the Mediterranean have low inter-population diversity, while genetically having high connectivity, which suggests this marker will be useful for areas outside the Ebro Delta and into the Eastern Mediterranean.

The documented ITS rDNA region used in this qPCR assay development was rich in homopolymer and repeat sequences (areas under less selective genetic pressure) that likely caused the difficulty in getting equivalent amplification from some primer sets initially tested despite their being designed to avoid these regions. Studies of genetic diversity may find this region useful since regions rich in repeat sequences, such as those found here, frequently expand or contract during the course of evolution [START_REF] Read | Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells[END_REF]; this being the basis for microsatellite analysis. Previous phylogenetic studies related to this genus have used more standard genetic markers such as cytochrome oxidase [START_REF] Sanna | Mitochondrial DNA reveals genetic structuring of Pinna nobilis across the Mediterranean Sea[END_REF][START_REF] Sanna | New mitochondrial and nuclear primers for the Mediterranean marine bivalve Pinna nobilis[END_REF], microsatellite DNA [START_REF] Gonzalez-Wanguemert | Highly polymorphic microsatellite markers for the Mediterranean endemic fan mussel Pinna nobilis[END_REF][START_REF] Wesselmann | Genetic and oceanographic tools reveal high population connectivity and diversity in the endangered pen shell Pinna nobilis[END_REF], or nuclear and mitochondrial ribosomal genes [START_REF] Lemer | First molecular phylogeny of the circumtropical bivalve family Pinnidae (Mollusca, Bivalvia): evidence for high levels of cryptic species diversity[END_REF]. In the case of the latter study, cryptic species were identified within the family Pinnidae. Hence, utilizing the ITS region of the rDNA cistron might reveal still more genetic diversity, adding new insights into variability among different populations.

Only few P. nobilis larvae were present during the expected summer spawning period (Kersting & Garcia-March, 2007). All samples displayed high Ct values (between 31.9 and 39.6 with mean value of 36.41), indicative of very low abundance of planktonic stages, yet still supporting the efficiency of the speciesspecific qPCR as a detection tool. The highest number of larvae visually observed among the sam-ples that tested positive by qPCR was 3237 collected from zone 3 of Alfacs Bay on Oct. 1. Given the Ct value obtained 37.49, it is possible that only one larvae among those could be considered as being P. nobilis.If the recruitment bottleneck resided with ecological processes occurring in the benthos, then a paucity of developmental stages would be expected in the substrata while there would be an abundance of planktonic stages in the water column. However, the low abundance of planktonic stages observed suggests some local processes are affecting the water column, rather than the benthos, and could be interfering with the production of viable larvae. Although during the study we chose three sites where population densities P. nobilis were significantly different [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF], the low Ct values observed prevented determination of a clear relationship between each zone and their larval abundance. Among potential causes of larval supply limitation at the broad spatial scale of the Alfacs Bay, endocrine disrupting organophosphates discharged from rice fields during the summer period might be causing alterations in the proper functional development of the reproductive system [START_REF] Terrado | Use of chemometric and geostatistical methods to evaluate pesticide pollution in the irrigation and drainage channels of the Ebro river delta during the rice-growing season[END_REF][START_REF] Frye | Endocrine disruptors: a review of some sources, effects, and mechanisms of action on behavior and endocrine systems[END_REF]. Several such com-pounds have been identified in environmental com-partments and wildlife of the Ebro Delta, and reproductive impacts on water fowl [START_REF] Man ˜osa | A review of the effects of agricultural and industrial contamination on the Ebro Delta Biota and wildlife[END_REF], as well as alterations of acetylcholinesterase (AChE) activity in Mytilus galloprovincialis [START_REF] Escartin | The use of cholinesterase and carboxylesterase activities from Mytilus galloprovincialis in pollution monitoring[END_REF] have been reported. In addition, lower salinity in Northern areas of the Bay due to persistent discharge of agricultural water also affects P. nobilis reproduction and ultimately may account for differences in adult abundances among the three zones [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF].

Due to funding limits, samples were collected only during the expected spawning season from July to October. While this is not a full year of sampling it should be regarded as significant considering that material was collected from three different sites within Alfacs Bay, and in triplicate subsites at each site. Those samples that tested positive were few, and simply a matter of the circumstances that point towards the very reason we performed this study. In all, only 15 samples were identified as containing P. nobilis DNA from mid-July to the beginning of October 2016. However, there were 30% more positive samples in September/October (n = 9) than in July/August (n = 6). In addition, the mean C t value of larval samples from Sept-Oct was ca. 2.7 times lower than in the July-Aug period, suggesting an increase in larval abundance, or in size of individual larvae. Yet, according to results using calibration curves, there were few P. nobilis in the water column (an average of 12.4 m -3 ), which indicates very low availability of larvae. Although the number of P. nobilis larvae has never been quantified in previous studies and it is impossible to establish common abundance ranges, the very low occurrence of juve-niles in the area [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF] suggests that they are abnormally low. This pattern is also confirmed by local recruitment data for 2016, which registered a total absence of individuals (Prado et al., in prep) whereas in other areas nearby such as the Columbretes Islands, located only 100 km south from Alfacs Bay, recruitment has been clearly demonstrated [START_REF] Kersting | Preliminary data on Pinna nobilis larval recruitment using mesh collectors in Columbretes Islands Marine Reserve, NW Mediterranean[END_REF]. In other species of bivalves such as the brown mussel Perna perna and the clam Ruditapes decussatus, larval peaks of hundreds to thousands of individuals per m -3 have been reported and result in high rates of recruitment [START_REF] Chı ´charo | A juvenile recruitment prediction model for Ruditapes decussatus (L.) (Bivalvia: Mollusca)[END_REF][START_REF] Porri | Spatio-temporal variability of larval abundance and settlement of Perna perna: differential delivery of mussels[END_REF]. The number of larvae shown in ''Subsample'' is the number of larvae in the extracted DNA samples tested. ''Total N'' is the number extrapolated from counting the subsample. All positives recorded had high Ct values between 31.9 and 39.6 (mean = 36.41). Larvae number are extrapolated from the calibration curve obtained from diploid eggs and the mean value of 7 replicates for amplification of one larvae: 36.9 = 17,844 diploid cells

Since the number of P. nobilis larvae all samples analyzed was small (mean C t value for all positives of 36.41), most of the observed larvae were likely from other locally abundant species such as oysters, mussels, clams, and/or cockles. For instance, in the case of the sample with the highest recorded abundance of larvae (15,272), collected on Sept. 15th, no P. nobilis DNA was detected. At present, the identification of morphometric characters requires extreme expertise and specialization in ontogenetic bivalve development (e.g., [START_REF] Malchus | Part N, Revised, Volume 1, Chapter 4: the early shell: ontogeny, features, and evolution[END_REF], so this information was not collected in this work. Yet, future multidisciplinary studies may also include this type of analysis prior to DNA extraction and qPCR.

In the use of 500 ml water samples, none tested positive. Although when using the plankton net protocol for sampling, the estimated volume of water filtered (ca. 3.77 m 3 ) was an increase of over 7000fold. Yet, negative results for seawater samples of 500 ml are coherent with low C t values and scarce P. nobilis-positive results from tests performed with collected larvae stages. This was surprising given that the bay harbors the second largest population of this species in the Mediterranean [START_REF] Prado | Habitat use by a large population of Pinna nobilis in shallow waters[END_REF]. A possible reason for the reduced number of positive results could be dilution of planktonic stages of the target species within the large dimensions of Alfacs Bay (ca. 49 Km 2 and average depth of 3.13 m). This dilution effect due to the large volume of the bay might be further enhanced by the continual renewal rate of the bay's water, even though tidal changes are low in the Mediterranean. This is in contrast to similar works in which water from ponds with little or no water renewal were analyzed by qPCR for the presence of frog (Rana catesbeiana) DNA [START_REF] Ficetola | Species detection using environmental DNA from water samples[END_REF] where sloughed epithelial cells contained in mucus from the frog's skin might accumulate in the absence of continual water renewal. Further, in this study when testing ''pseudo-field samples,'' only 15 and 50 ml volumes containing one egg of P. nobilis consistently tested as positive, whereas 500 ml volumes used were negative. However, all volumes were positive when ten eggs were added. This hints at some possible underestimation of DNA contents in field water samples of 500 ml; however, this volume was still preferred to maximize the potential of capturing multiple planktonic stages. Additional factors that may have contributed to the absence of positives from 500 ml water samples include low rate of adult mortality (a cause of release of necrotic tissue and cells from moribund animals), reduced spawning and larval abundance, or any combination of these factors.

Although the bay is also the site of intensive shellfish culture (C. gigas, C. edule, and Mytilus galloprovincialis) as well as artisanal harvest of other natural shellfish populations (Cerastoderma sp., carpet shell clam Ruditapes decussatus and the razor clam Solen marginatus), no false positives were detected, as would be evident from the melt curve profiles.

Calibration curves of fertilized and unfertilized eggs, and adult tissue (data not shown) showed very similar slopes but differed in their y-intercept, an indication of their cells being haploid or diploid. There was significant deviation among replicates that might have been due to artifacts such as free DNA in the aqueous fraction that was transferred with the eggs into the subsample tubes, and/or free DNA adherent to the surface of the eggs via DNA-binding cell surface receptors such as nucleolin [START_REF] Bennett | DNA binding to human leukocytes. Evidence for a receptormediated association, internalization, and degradation of DNA[END_REF][START_REF] Chen | Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA[END_REF]. Moreover, the eggs and larvae used were from experimental captive spawning in which all of the larvae produced eventually died. Therefore, it can be presumed that the larvae used may not have been in optimal condition, and if in a moribund state at the time of fixation, in which cells were undergoing apoptosis, the quality of the genetic material obtained may not have been consistent among animals within a sample, or cells within an individual animal. Additionally, ribosomal DNA copy number is likely to increase post-fertilization by regulatory mechanisms that implicate genes like fob1 or similar [START_REF] Johzuka | Replication fork block protein, Fob1, acts as an rDNA region specific recombinator in S. cerevisiae[END_REF], that would also contribute to variation between individual eggs and egg groups. Given that the absence or low rates of fertilization might be indicative of some type of infertility among adult individuals (e.g., [START_REF] Lewis | Infertility in male aquatic invertebrates: a review[END_REF], this ability to differentiate between fertilized and unfertilized eggs might be a useful tool in future studies aimed at establishing the causes of larval and recruitment failure.

To conclude, the development of analytical tools such as qPCR to assess the availability of planktonic stages of P. nobilis and the ability to differentiate between fertilized and non-fertilized eggs should help to address possible bottlenecks related to gamete viability, clarify concerns of agro-chemical impacts and species-specific sensitivity, and determine the influence of large-scale environmental factors on the larval recruitment of this species. Future research need to focus on using qPCR to monitor larval abundances in Alfacs Bay and in other Mediterranean areas, in order to address some of these questions to aid in understanding better how to improve management of wild stocks of this emblematic Mediterranean bivalve.

Fig. 2

 2 Fig. 2 Map indicating the location of each sampling zone within the study site (Z1 zone of high abundance, Z2 zone of intermediate abundance, Z3 zone of low abundance). Dotted lines indicate the irrigation canals used for managing water flows through the rice fields in the Ebro delta

Fig. 3

 3 Fig. 3 Exemplars of P. nobilis eggs (left) and larvae (right) produced in the laboratory using artificial gamete induction/fertilization

Fig. 4

 4 Fig. 4 Calibration curve demonstrating distinct results for fertilized and unfertilized eggs of P. nobilis. Squares = fertilized egg samples. Circles = unfertilized egg samples. Linear regression of results for fertilized or unfertilized eggs (solid

Table 1

 1 Summary of qPCR results from larvae collected between mid-July and September

	Date	Larval from plankton net samples		qPCR
		Site	Replicate	Subsample	Total N	±	Mean C t	# Larvae*
	07/15/2016	Zone 1	R1	174		-
			R2	129		-
			R3	141		-
		Zone 2	R1	0		-
			R2	3		?	38.39	0.4
			R3	154		-
		Zone 3	R1	14		?	38.04	0.5
			R2	11		?	36.19	1.6
			R3	102		-
	08/01/2016	Zone 1	R1	102		-
			R2	126		-
			R3	99		-
		Zone 2	R1	50		-
			R2	49		-
			R3	104		-
		Zone 3	R1	156		-
			R2	365		-
			R3	446	1279	-
	08/15/2016	Zone 1	R1	30		?	35.85	2.1
			R2	37		?	39.57	0.2
			R3	52		?	38.59	0.3
		Zone 2	R1	262	1368	-
			R2	141	566	-
			R3	116	233	-
		Zone 3	R1	750	2149	-
			R2	851	3143	-
			R3	472	1905	-
	09/01/2016	Zone 1	R1	22		?	36.11	1.7
			R2			-
			R3			-
		Zone 2	R1	33		-
			R2	29		?	37.32	0.8
			R3	56		?	31.86	32.4
		Zone 3	R1	30		-
			R2	302		-
			R3	39		?	37.76	0.6
	09/15/2016	Zone 1	R1	54		?	32.06	28.1
			R2	116		-
			R3	271	797	-
		Zone 2	R1	175		-
			R2	185		-
			R3	262		-
		Zone 3	R1	306	686	-
			R2	363	580	-
			R3	526	15,272	-

Acknowledgements

The authors wish to thank the Zoo Barcelona Foundation for an Antoni Jonch Grant 2015, which provided the economic support necessary to accomplish this work. Authors are very grateful to Dr. Miguel Alonso Garcı ´a-Amilivia for the construction of the zooplankton micro-spoon for the collection of P. nobilis eggs and larvae. Also, authors would like to thank Pep Cabanes, Lluis Jornet, and David Mateu for technical assistance during fieldwork sampling in Alfacs Bay.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


Sergio Trigos

Nardo Vicente

Patricia Prado

Francisco J Espinós

Adult spawning and early larval development of the endangered bivalve Pinna nobilis

Keywords: Pinna nobilis, Conservation, Larvae development, Rearing, Captivity

The development of aquaculture activities has posed an alternative solution for the preservation of some overexploited shellfish fisheries worldwide. In the same way, endemic Mediterranean bivalves such as Pinna nobilis, highly threatened by habitat loss and coastal pollution, could found in aquaculture a solution for pre-serving the continuity of the species. Given the endangered status of the species, the biological and ecological processes regulating natural populations have been well studied, but there are still important knowledge gaps preventing the development of viable artificial cultures. This study describes for the first time the larval de-velopment of P. nobilis (from fertilization until pediveliger larval stages) in captivity conditions. Moreover, different rearing tanks of 5, 16 and 80 L, larvae density from 1 to 600 larvae mL -1 , light conditions, food doses, were tested in order to establish the bases for the optimal rearing of the species and provide a source of in-dividuals for restoring field populations. Results showed that 16 L tanks with a concentration of 2 larvae mL -1 , constant temperature of 21 °C, 12/12 h photoperiod and fed with an "optimal" mixture of 25 cells per μL of Chaetoceros calcitrans + 33.3 cells per μL ofPavlova lutheri + 100 cells per μL of Isochrysis galbana" appear to be the best conditions to rear larvae of P. nobilis. Different captivity conditions such as lower or higher tank volume, larvae density, or food doses; light privation did not report better results for larval development.

Introduction

In the last decades the rearing of endangered bivalve species for both commercial and ecological purposes has received considerable attention [START_REF] Ellis | Nursery and grow-out techniques for giant clams (Bivalvia: Tridacnidae)[END_REF][START_REF] Knop | Riesenmuscheln: Arten und Pflege im Aquarium[END_REF]. In spite of the economic and scientific interest, the rearing of bivalve species has been proved to be rather difficult due to the great larval mortalities of the group [START_REF] Rumrill | Natural mortality of marine invertebrate larvae[END_REF]. Field studies show that larval mortality in natural conditions ranges from 25 to 80% during first days of life [START_REF] Talmage | The effects of elevated carbon dioxide concentrations on the metamorphosis, size, and survival of larval hard clams (Mercenaria mercenaria), bay scallops (Argopecten irradians), and eastern oysters (Crassostrea virginica)[END_REF][START_REF] Gazeau | Effect of ocean acidification on the early life stages of the blue mussel Mytilus edulis[END_REF]. Both natural and/or anthropogenic-driven mortality in bivalve larvae -i.e.., from egg to postlarval recruit-are often difficult to measure and are considered as a "constant" [START_REF] Philippart | Climate-related changes in recruitment of the bivalve Macoma balthica[END_REF]. Possible causes may include failure of fertilization, which is often related to sea acidification and may produce shape abnormal-ities in embryos or avoid development in over 50% of the larvae [START_REF] Kurihara | Effects of increased seawater pCO2 on early development of the oyster Crassostrea gigas[END_REF]; coastal development [START_REF] Gómez | The culture of true giant clam Tridacna gigas for conservation in the Philippines[END_REF][START_REF] Guest | Can giant clam (Tridacna squamosa) populations be restored on Singapore's heavily impacted coral reefs?[END_REF]; shortage of food resources [START_REF] Rico-Villa | Influence of phytoplankton diet mixtures on microalgae consumption, larval development and settlement of the Pacific oyster Crassostrea gigas (Thunberg)[END_REF], absence of a suitable substrate for benthic settlement [START_REF] Su | The effect of different substrates on pearl oyster Pinctada martensii (dunker) larvae settlement[END_REF]; combined effects of short planktonic durations and local patterns of marine circulation [START_REF] Shanks | Upwelling, downwelling, and cross-shelf transport of bivalve larvae: test of a hypothesis[END_REF]; and/or lethal environmental temperatures [START_REF] Philippart | Climate-related changes in recruitment of the bivalve Macoma balthica[END_REF]. Any of these scenarios usually leads to a reduction of recruitment rates and consequently, to the disappearance of populations.

Historically, mortality problems have been observed in the marine bivalve industry for decades (Samain and Mccombie, 2008;[START_REF] Barton | Impacts of coastal acidification on the Pacific northwest shellfish industry and adaptation strategies implemented in response[END_REF]. The main goal in hatchery production is to improve larval and post-larval survival by achieving larval growth and metamorphosis success. In this term, the proper formulation of larval diets has been considered as the most critical aspect in hatchery operations focusing on the type of microalgae used as feed [START_REF] Knuckey | Isolation of new nanoplanktonic diatom strains and their evaluation as diets for juvenile Pacific oysters (Crassostrea gigas)[END_REF][START_REF] Ponis | New microalgae for the Pacific oyster Crassostrea gigas larvae[END_REF][START_REF] Rico-Villa | Influence of phytoplankton diet mixtures on microalgae consumption, larval development and settlement of the Pacific oyster Crassostrea gigas (Thunberg)[END_REF][START_REF] Pettersen | Effects of different dietary microalgae on survival, growth, settlement and fatty acid composition of blue mussel (Mytilus galloprovincialis) larvae[END_REF][START_REF] Ragg | Optimising the delivery of the key dietary diatom Chaetoceros calcitrans to intensively cultured GreenshellTM mussel larvae, Perna canaliculus[END_REF][START_REF] Gui | Evaluation of the formulated diet MySpat for feeding hatchery-reared spat of the green-lipped mussel, Perna canaliculus (Gmelin, 1791)[END_REF] or even in artificial substitutes to phytoplankton

MARK

⁎ Corresponding author at: Innovation Network in Aquaculture Industries of the Valencian Community (RIIA-CV), Avda. Naranjos s/n. Edificio Colegio Mayor Galileo Galilei, Local 15, 46022, Valencia, Spain. E-mail address: strigos@riia.es (S. Trigos).

diet [START_REF] Coutteau | The use of algal substitutes and the requirement for live algae in the hatchery and nursery rearing of bivalve molluscs: an international survey[END_REF][START_REF] Knauer | A review of the nutritional requirements of bivalves and the development of alternative and artificial diets for bivalve aquaculture[END_REF][START_REF] Nevejan | Use of a formulated diet for mussel spat Mytilus galloprovincialis (Lamarck 1819) in a commercial hatchery[END_REF][START_REF] Gui | Evaluation of the formulated diet MySpat for feeding hatchery-reared spat of the green-lipped mussel, Perna canaliculus (Gmelin, 1791)[END_REF].

In other cases, recurrent mortality episodes are described as a result of virus or Vibrio-like bacteria infecting bivalve hatcheries and nurseries [START_REF] Renault | Herpes-like virus infections in hatchery-reared bivalve larvae in Europe: specific viral DNA detection by PCR[END_REF][START_REF] Dubert | Vibrio bivalvicida sp. nov. a novel larval pathogen for bivalve molluscs reared in a hatchery[END_REF][START_REF] Rojas | Genome sequence of Vibrio VPAP30, isolated from an episode of massive mortality of reared larvae of the scallop Argopecten purpuratus[END_REF] and reducing commercial production by approximately 40% as reported for French farming facilities of Pacific oyster (Crassostrea gigas) since 2008 [START_REF] Pernet | Associations between farming practices and disease mortality of Pacific oyster Crassostrea Gigas in a Mediterranean lagoon[END_REF].

From an ecological perspective, the rearing of endangered bivalves in captivity conditions may pose a potential solution for the rehabilitation of seriously damaged populations [START_REF] Ronquillo | Developmental stages and potential mariculture for coastal rehabilitation of endangered Pacific angelwing clam, Pholas orientalis[END_REF][START_REF] Vincie | Bivalvia: Unionidae), Reared in a Captive Environment[END_REF][START_REF] Thomas | Captive breeding of the endangered freshwater pearl mussel Margaritifera margaritifera[END_REF][START_REF] Theodorou | The recruitment of the endangered fan mussel (Pinna nobilis, Linnaeus 1758) on the ropes of a Mediterranean mussel long line farm[END_REF] and face the same early stages' bottlenecks than those reported for bivalve commercial farming [START_REF] Arnold | Application of Larval Release for Restocking and Stock Enhancement of Coastal Marine Bivalve Populations[END_REF]. One of these endangered species is the Mediterranean fan mussel, Pinna nobilis. The populations of this bivalve have significantly declined during the last decades (De Gaulejac and [START_REF] De Gaulejac | Ecologie de Pinna nobilis (L.) mollusque bivalve sur les coˆtes de Corse. Essais de transplantation et experiences en milieu contrôle´[END_REF][START_REF] Vicente | Statut de Pinna nobilis L. en Mediterranee (Mollusque Eulamellibranche)[END_REF][START_REF] Garcia-March | Aportaciones al conocimiento de la biología de Pinna nobilis Linneo. 1758 (Mollusca Bivalvia) en el litoral mediterráneo ibérico[END_REF][START_REF] Basso | The pen shell: a review of population status and recommended research priorities in the Mediterranean Sea[END_REF][START_REF] Rouanet | From youth to death of old age: the 50-year story of a Pinna nobilis fan mussel population at Port-Cros Island Port-Cros National Park, Provence, Mediterranean Sea[END_REF] as a result of different anthropogenic stressors such as coastal development, fishing pressure, and/or accidental harvesting by trawling and shell breakage by anchoring [START_REF] Katsanevakis | Population ecology of the endangered fan mussel Pinna nobilis in a marine lake[END_REF][START_REF] Acarli | Preliminary spat settlement of fan mussel Pinna nobilis Linnaeus. 1758 on a mesh bag collector in Karantina island (eastern Aegean Sea. Turkey)[END_REF][START_REF] Hendriks | Seagrass meadows modify drag forces on the Shell of the fan mussel Pinna nobilis[END_REF]. For these reasons, the European Union included it as an endangered species in the ANNEX IV of the Council Directive 92/43/EEC (EC Habitats Directive), and strictly forbids any kind of culling. Although these measures have helped certain recovery of populations in some Mediterranean regions [START_REF] Pérez-Vallazza | Cetacean diversity at the west coast of La Palma Island (Canary Islands)[END_REF][START_REF] Theodorou | Density and size structure of the endangered bivalve Pinna nobilis, (fan mussel) in the shallow water zone of Maliakos Gulf, Greece[END_REF], the species is still at risk by unknown factors such as those causing mass mortality event along the Spanish Mediterranean coast and the Balearic Islands during the summer of 2016 [START_REF] Darriba | First haplosporidan parasite reported infecting a member of the superfamily Pinnoidea (Pinna nobilis) during a mortality event in Alicante (Spain, western Mediterranean)[END_REF][START_REF] Vázquez-Luis | a mass mortality event in western Mediterranean Sea[END_REF]. Temperature effects due to climate change, coastal pollution, and pathogenic mechanisms are among potential explanatory factors, all of them mediated by human influence [START_REF] Rodrigues | Sensitivity of Mediterranean bivalve Mollusc aquaculture to climate change and ocean acidification: results from a producers' survey[END_REF]. In this scenario, the rearing of P. nobilis in captivity appears as an alter-native solution to obtain healthy stocks of recruits that could be re-introduced in suitable areas with declining population densities [START_REF] Trigos | Estudio de la ecofisiología y ensayo de cultivo de la nacra Pinna nobilis Linnaeus[END_REF]). Yet, the duration of the different larval stages from fertilized eggs, as well as the main factors influencing survival rates are still largely unknown, although they are proposed to follow similar patterns to other bivalves. Following this hypothesis, [START_REF] Vicente | La grande Nacre de Méditerranée. Parc national de Port-Cros[END_REF] described the larval stages of trochophore, veliger, and pediveliger followed by metamorphosis, and juvenile development of P nobilis. De Gaulejac (1989), observed larvae shells with electronic microscope and pointed the possibility of a time gap of 5 to 10 days between gametes expulsion and substrate settlement. [START_REF] Peharda | Modelling the recruitment effect in a small marine protected area: the example of saltwater lakes on the island of Mljet (Adriatic Sea)[END_REF] suggested that veliger fan mussel larvae could have a negative phototactism that allow vertical migration to deeper waters during the day to return to upper bathymetric ranges at night, as also indicated for other bivalves [START_REF] Gosling | Bivalve Molluscs: Biology. Ecology and Culture. Fishing News Books[END_REF], presumably to avoid UV light or predator activity [START_REF] Manuel | Veligers from different populations of sea scallop Placopecten magellanicus have different vertical migration patterns[END_REF]. However, there is still no available information on the behavioural responses of P. nobilis larvae to different environ-mental factors under captivity conditions that could provide a solid basis for designing and implementing a viable larval hatchery. In ad-dition, factors determining patterns of benthic recruitment are virtually unknown, although in other species is often linked to the availability of preferential substrates for settlement [START_REF] Prado | Habitat and scale shape the demographic fate of the keystone sea urchin Paracentrotus lividus in Mediterranean macrophyte communities[END_REF], which high-lights the importance of simulating optimal settlement conditions to rear P. nobilis in captivity. Other biological and physical aspects such as the possible correlation between adult size and numbers of oocytes expelled also require further investigation.

In this context, the main objective of this work was to describe, for the first time, the larval development of the ecologically important Mediterranean fan mussel (P. nobilis) with the aim of developing cultivation techniques and providing a practical guideline for problem identification during larval rearing in this species. More specifically we conducted experiments aimed (1) at different larval densities (2) with different tank volumes, (3) fed with three doses of microalgae mixture and (4) reared in light/dark conditions.

Materials and methods

Field sampling

A total of 40 individuals of P. nobilis were collected around the Embiez archipelago, South East of France from April 2012 to September 2014. The collected specimens were carefully transported to laboratory facilities within portable tanks and their total height (Ht) registered and labelled. Shells were brushed to remove epibiontic organisms, including other bivalves [START_REF] Rabaoui | Engineering effect of Pinna nobilis shells on benthic communities[END_REF] that could affect the fertilization process. All individuals were reintroduced to the field at the end of the experiments.

Gamete release and fertilization

Small groups of 4-6 individuals were placed in two 120 L tanks within controlled temperature facilities (20-21 °C) with filtered (1 μm) and treated (UV) seawater for a maximum of 24 h. A ≈ 10 °C gradient is necessary to induce a thermal shock causing gametes release in bi-valves [START_REF] Helm | Cultivo de bivalvos en criadero[END_REF] which takes place at various temperature thresholds, depending on the species [START_REF] Drent | Life history variation of a marine bivalve (Macoma balthica) in a changing world[END_REF]. Herein, in order to keep this temperature gradient two induction tanks were prepared; one tank was maintained at 15 °C while a second tank was heated to 25 °C thus respecting the conformability range of temperatures described for P. nobilis [START_REF] Trigos | Respiration rates of the fan mussel Pinna nobilis at different temperatures[END_REF].

Individuals were transferred to the induction tanks and individuals were translocated between both of them every 50 min for a maximum of 6 times. If no response was achieved, individuals were introduced in 2600 L tanks with constant aeration and water renovation, and the process repeated on the following days. When only males expelled gametes, small volumes of sperm were poured in the tanks on the following thermal shocks, with the aim to stimulate the females (if any) while the thermal shock was occurring. When male and female spawning occurred, individuals were introduced individually in 60 L tanks in order to avoid possible polyspermia. Both oocytes and spermatozoids were recovered from individual tanks using a sterilized 60 mL micropipette and then filtered through a 30 μm sieve in order to remove any possible fecal waste that could be attached (mainly to oo-cytes). Volumes of 4 mL of a homogenized sample of oocytes were counted (N = 3) in order to determine the possible correlation between adult size and numbers of oocytes expelled. After that, gametes were introduced together in a 15 L tank favouring the mixture, thus enhan-cing fertilization.

Larval cultures

Preliminary essays were conducted to determine the optimal rearing conditions according to survival time observed. To this aim and after fertilization, larvae were maintained in the same tank with constant aeration and temperature (21 °C) until they reached the trochophore stage. Then, they were transferred to different small rearing tanks of 5, 16 and 80 L (N = 3) in order to better control the larvae development and cultures were also adjusted at different larval concentrations of 1, 2 and 600 larvae mL -1 respectively, according to [START_REF] Helm | Cultivo de bivalvos en criadero[END_REF] with water renewal fluxes (600 mL•h -1 ) through a 35 μm strainer and constant aeration. In addition to exhaustive filtration of seawater circuits periodic, bacterial cultures were carried out to monitor the presence of pathogens within tanks. For each concentration and rearing tank, survival time was estimated daily by sampling (N = 1) of 4 mL and counting of living larvae.

Larvae diet was established using a mixture of three microalgae (Isochrysis galbana, Pavlova lutheri and Chaetoceros calcitrans), as suggested in the literature [START_REF] Pernet | Effect of varying dietary levels of ω6 polyunsaturated fatty acids during the early ontogeny of the sea scallop. Placopecten magellanicus[END_REF][START_REF] Milke | Growth of postlarval sea scallops, Placopecten magellanicus, on microalgal diets, with emphasis on the nutritional role of lipids and fatty acids[END_REF][START_REF] Milke | Comparison of early life history stages of the bay scallop, Argopecten Irradians: effects of microalgal diets on growth and biochemical composition[END_REF]. The final concentration mix of the three phytoplankton species was adjusted according to three different doses named as "low", "optimal" and "high" using the formula proposed by [START_REF] Helm | Cultivo de bivalvos en criadero[END_REF] for bivalves feeding in breeding facilities:

= ⋅ V L cell density needed V cell density available ( ) [μL] [μL]
dose tank where:

V dose = supplied dose in liters.

Cell density needed [μL] = cell concentration according to low, optimal or high dose.

V tank (L) = tank volume.

Cell density available [μL] = cell concentration in laboratory cultures.

For each concentration mix, the equation included the cell density suggested by the author:"15 Chaetoceros cells per μL + 25 Pavlova cells per μL + 50 cells per μL of Isochrysis" for the "low" dose, "25 Chaetoceros cells per μL + 33.3 Pavlova cells per μL + 100 cells per μL of Isochrysis" for the "optimal" dose, and "30 Chaetoceros cells per μL + 50 Pavlova cells per μL + 150 cells per μL of Isochrysis" for the "high" dose.

Effect of rearing conditions on larval development and settlement

Once previous essays allowed determination of the maximum survival time of larvae under the different parameters established, cultures were also used for testing the influence of light on larvae development due to negative phototactism, as observed for certain bivalve larvae [START_REF] Raby | Relationship between feeding and vertical distribution of bivalve larvae in stratified and mixed waters[END_REF]. To this end, six 16 L tanks were set with open circulation at densities of 2 larvae•mL -1 . Three of those tanks were kept at 12/12 h photoperiod whereas the other three tanks were covered with opaque plastic (darkness conditions). Microalgae doses were identically established as "low", "optimal" and "high" to elucidate if the use of any of them could affect directly the larvae growth. The latter was determined by registering larval length with a "Leica DM2500" microscope. A total of 60 fan mussel larvae were daily placed on a dig dish, and their length measured for average estimations ( ± SD). The process was carried out by triplicate using larvae coming from different spawns.

For all tanks, abiotic parameters were kept as stable as possible. Temperature: 19-21 °C; salinity: 32-37; pH: 7.7-8.4; and dissolved oxygen: 5.9-7.0 mg O 2 . Larvae concentrations were daily monitored (N = 1 per tank) to determine the conditions with lower mortality rates. Moreover, an artificial substrate made of 250 μm PVC mesh was deployed in all tanks in order to enhance larvae settlement. The substrate was autoclaved (120 °C. 30 min) to prevent the introduction of pathogens within rearing tanks.

Statistical analyses

All statistical analyses were conducted with SPSS® Statistics 21 program. The possible association between the size of broodstocks and the number of oocytes expelled was studied using the Pearson correlation factor.

The best survival rate was determined studying the effect of the tank volume (fixed factor, three levels) and the larvae density (fixed factor, three levels) and calculated by a two-way factorial ANOVA.

The effect of photoperiod (fixed factor, two levels), food dose (fixed factor, three levels) and rearing day on larvae survival, was investigated using a three-way factorial ANOVA followed by a post-hoc analysis (DHS-Tukey) to establish significant groupings. All data were tested for ANOVA assumptions of normality (Levene's test) and homogeneity of variances (Cochran's test).

Results

Gamete release and fertilization

A total of 31 individuals (47.10 ± 10.61 cm Ht), of the 40 sub-jected to thermal shock, released gametes for periods of 40 min and, in some instances for up to 3 h. Among individuals that responded posi-tively, 5 of them (16.1%) were strictly males and 14 (45.2%) expelled only oocytes. The remaining 12 specimens (38.7%) released almost simultaneously male and female gametes. The release of female gametes in P. nobilis was estimated in 1.9 10 6 oocytes L -1 (averaged for the 26 individuals releasing female gametes) with a mean Ht of 51.8 ± 9.98 cm (Fig. 1).

There was a significant a positive association between the size of individuals and the number of expelled oocytes (F = 0.765. p < 0.01) as reported for other bivalve species [START_REF] Helm | Cultivo de bivalvos en criadero[END_REF]. The smallest spawning female was 37.7 cm Ht, and 34.6 cm Ht in males. In those instances in which there was only male spawning, sperm was stored in a temperature controlled room at 4 °C and visual observations revealed that sperm remained alive for a maximum of 3 days.

Larval cultures

Viable oocytes were spherical with an average diameter of Ø=55 ± 1μm whereas spermatocytes hardly exceed 1 μm length. Embryonic development of P. nobilis started with the rapid fertilization of oocytes by surrounding spermatocytes at a temperature of 21 °C (Table 2). After 15-30 min the appearance of the first polar body and the formation of a perivitelline membrane confirmed successful ferti-lization (Fig. 2). The first zygote inclusions were observed after 40 min and gradually increase the number of blastomeres in a successive for-mation of inclusions until they attained a ciliated blastula stage 5 h later. Herein, the phase in which the motility of larvae begins (Fig. 3) and last for 24 h until the trocophore stage at an average size of 65 ± 5 μm. This was followed by a period of frenetic activity where larvae can reach speeds of 0.5 to 1 cm•s -1 and displayed a helical swimming pattern as observed in other bivalves [START_REF] Troost | Can bivalve veligers escape feeding currents of adult bivalves?[END_REF]. This speed was considerably reduced after the first 48 h when the larvae started to generate their own shell (Prodissoconch I) and become a D-larva or early veliger stage (85 ± 3 μm). Here, the appearance of a ciliary structure or "vellum" produced an incessant movement that generates a current of attraction that allows the capture of phyto-plankton cells. Later, the carbonate shell that protects the visceral cavity of the larvae and the food become more important, reducing cilia movement to an intermittent rotation. Progressively, the larvae secrete more calcium carbonate causing the thickening of shell layers and the development of the first growth rings (Prodissoconch II). Herein, the characteristic straight hinge that gives name to the "D" larva tends to bend thus reaching the umbonate phase. From this moment the larvae stopped swimming completely but few developed the foot that allows benthic settlement. (See Table 1.)

From the spawning to pediveliger stage the average growth of larvae was estimated at 8.57 μm•day -1 . However, at some point of the experiments (day 6 in 80 L tanks and day 7 in 16 L tanks) all growth was stopped and no more larval development was observed regardless of the rearing conditions and coinciding with the period when larvae stop swimming (red line) (Fig. 4). Thus, larvae remained alive for a maximum of 22 days and statistical analysis showed that there is a significant difference between survival of larvae and the parameters selected for the rearing activity occurring best survival results in 16 L tanks with an initial larvae density of 2 larvae•mL -1 (F = 13.542, p < 0.05).

Effect of rearing conditions on larval development and settlement

Results from ANOVA showed higher survival of larvae due to the presence of light in the tanks (F = 4.597, p < 0.05) and evidenced important differences according to the dose of food provided (F = 3.434, p < 0.05) while interaction between "Tank + Dose" was not significant (F = 2.910, p = 0.58). Post-hoc analysis showed that the "optimal" dose significantly improved (p < 0.05) larval survival compared to "low" and "high" doses where > 80% of larvae had died after 48 h and 96 h, respectively.

The relative daily mortality registered in tanks with photoperiod 12/12 showed mean values of 54.2% when the dose was "low", 18.3% when the dose was "optimal" and 34.4% when the dose was "high".In addition, the highest values of mortality were also recorded during mainly the first three days, during the trochophore and veliger phases (Fig. 5).

Pathologies observed

In most cases typical symptoms of bacterial infection (Fig. 6) are suspected to have caused the observed mortalities over 80% during the first 2-9 days. Herein, the loss of the larval "vellum" was observed during the veliger phase. Despite larvae continue alive, the absence of the ciliary structure prevents them to feed properly and after some days die. Another disease observed in dead larvae is easily identifiable by the continuous movement of the bacteria around and inside larvae shells referred as "swarming". In some cases, bacterial infection seems to affect larval motility, generating large clusters of larvae referred as "spotting" at the bottom of the tanks due to the secretion of mucous filaments.

Discussion

This study reports for the first time the successful spawning and larval rearing to pediveliger stage in captivity conditions of the endangered bivalve P. nobilis. This is also the first daily graphic documentation of the early development on this species.

Recent episodes of mass mortality occurred in southwestern Mediterranean coasts for P. nobilis accentuates the population regression registered in the last decades and consequently points captivity cultures as a potential solution to restore damaged populations. As described in [START_REF] Trigos | Estudio de la ecofisiología y ensayo de cultivo de la nacra Pinna nobilis Linnaeus[END_REF] the maintenance of large number of adults in captivity conditions for prolonged periods of time is necessary to obtain enough gametes of both sexes in a species in which the absence of external sexual dimorphism difficult the development of a hatchery protocol.

This study shows how 12 specimens (38.7%) from the total studied, released almost simultaneously male and female gametes, an event so far unknown in this species, which is described as a successive hermaphroditism, as a mechanism to prevent self-fertilization (De Gaulejac et al., 1995a,b). Interestingly, some expelled oocytes were still in a division process, suggesting that internal fertilization may occur as described for other bivalves such as Ostrea edulis [START_REF] Peteiro | Ciclo reproductivo de moluscos bivalvos[END_REF]. This observation coupled with the fact that oocytes present higher density than seawater and tended to sink after being released to the media could be indicate of internal fertilization as a common mechanism enhancing the survival of larvae, but further research is needed to confirm this hypothesis.

Spawned oocytes stayed at the tank bottom until the ciliated blastula stage (5 h post-spawning) when they became motile and swimming activity was observed. Life stages from late embryos to the veliger phase are considered as planktonic and potentially disperse by currents. Once the pediveliger stage was reached, larvae ceased to be planktonic and searched for a suitable substrate to start benthic development. Our study observed up to 22 days to achieve the pediveliger stage then to acquire the capability to get attached. In this context, the hypothesis proposed by De Gaulejac (1989) with a period between 5 and 10 days to settlement phase, contrast with our longer period which might be affected by captivity conditions. This could indicate that artificial conditions can be improved in order to obtain better results.

The massive mortality rates observed (up to 100% at day 4 and day 22, respectively in dark and light cultures), prevented observing more development phases for P. nobilis but as indicated by [START_REF] Hernández-Hernández | Distribución y abundancia de larvas de callo de hacha (Bivalvia: Pinnidae) en el sistema lagunar Corralero-Alotengo (Tesis de Licenciatura en Biología Marina)[END_REF] and [START_REF] Robles-Mungaray | Desarrollo de la biotecnología para la producción de semilla en laboratorio. diploide y triploide. de callo de hacha Atrina maura (Sowerby. 1835)[END_REF] for other species of Pinnidae, simultaneously to foot development, the progresses of the pediveliger phase (110 ± 10 μm size) is characterized by a gradual loss of the "vellum" that gives way to the formation of gills. Subsequently the process of metamorphosis begins with the secretion of new shell from the edge of the Prodissoconch II. Based on the type of growth of other specimens of the same family, the Prodissoconch II should continue its growth in a transverse direction to that previously recorded [START_REF] Robles-Mungaray | Desarrollo de la biotecnología para la producción de semilla en laboratorio. diploide y triploide. de callo de hacha Atrina maura (Sowerby. 1835)[END_REF]. The appearance of this new structure called dissoconch represents the turning point at which individuals reach the juvenile phase and acquire all adult characteristics such as the typical "pen" shape of the Pinnidae.

In our case, the mortality rates observed at this pediveliger phase were possibly associated to bacterial pathologies which are widespread in hatcheries of commercial species, as well as in regular experimental activities in the laboratory [START_REF] Andersen | Flow-through systems for culturing great scallop larvae[END_REF][START_REF] Prado | Warming and acidification-mediated resilience to bacterial infection determine mortality of early Ostrea edulis life stages[END_REF]. The infection of larval cultures may be caused by external or horizontal factors such as bacteria escaping the mechanisms of water filtration or other broodstock individuals [START_REF] Fontanez | Evidence for horizontal transmission from multilocus phylogeny of deep-sea mussel (Mytilidae) symbionts[END_REF]. The other type of transmission can be vertical when bacterial contamination occurs in gonads and intestinal tracts of broodstock and passes to offspring [START_REF] Beninger | Demonstration of nutrient pathway from the digestive system to oocytes in the gonad intestinal loop of the scallop Pecten maximus L[END_REF][START_REF] Prado | Vibriosin hatchery cultures of the razor clam. Solen marginatus (Pulteney)[END_REF]. These pathologies are thought to mainly affect larvae because they are much more susceptible to bacterial infections than adults [START_REF] Lambert | Specific inhibition of chemiluminescent activity by pathogenic Vibrios in Hemocytes of two marine bivalves: Pecten maximus and Crassostrea gigas[END_REF]. In particular, Vibrio species are regarded as central pathogens in larval bivalve cultures [START_REF] Gómez-León | Survival of eastern oyster Crassostrea Virginica from three lines following experimental challenge with bacterial pathogens[END_REF][START_REF] Elston | Re-emergence of Vibrio tubiashii in bivalve shellfish aquaculture: severity, environmental drivers, geographic extent and management[END_REF]Kesarcodi-Watson. 2009) with new species described in the last years [START_REF] Prado | Pathogenic bacteria isolated from disease outbreaks in shellfish hatcheries. First description of Vibrio neptunius as an oyster pathogen[END_REF][START_REF] Dubert | Vibrio bivalvicida sp. nov. a novel larval pathogen for bivalve molluscs reared in a hatchery[END_REF]. The most common problem arising from Vibrio action is the necrosis of soft tissues and ciliary structures [START_REF] Sugumar | Vibrio splendidus biovar II as the causative agent of bacillary necrosis of Japanese oyster Crassostrea gigas larvae[END_REF][START_REF] Neo | Spawning induction and larval development in the fluted giant clam. Tridacna squamosa (Bivalvia: Tridacnidae)[END_REF], thus preventing filtration and feeding mechanisms that cause the death of the larvae [START_REF] Dubert | Following the infection process of Vibriosis in Manila clam (Ruditapes philippinarum) larvae through GFP-tagged pathogenic Vibrio species[END_REF]. This disease is easily identifiable by the continuous movement of the bacteria around and inside larvae shells referred as "swarming" [START_REF] Beaz-Hidalgo | Diversity and pathogenicity of Vibrio species in cultured bivalve molluscs[END_REF]. The loss of the larval "vellum" during the veliger phase was a typical symptom of bacterial infection. Hence, Vibrio is suspected to have caused the observed mortalities over 80% during the first 2-9 days, depending on light conditions and food dose. A low dose of phytoplankton could weaken the larvae being consequently more susceptible to infection. By the contrary, the addition of high doses of the microalgae mixture could be responsible of an excess of non-profited food in the tanks, thus enhancing the proliferation of bacteria. The tanks exposed to the darkness registered mortalities higher than 80% on the second day and survival tended to improve with the "high" dose. Therefore, the photoperiod is presented as a limiting factor for the development of P. nobilis larvae and the high mortality observed could be explained by the absence of light which move the larvae away from natural conditions. This fact is supposed to stress considerably the larvae being consequently more susceptible to infection. According to our results, P. nobilis needs light to complete its larvae cycle in contrast with described by [START_REF] Peharda | Modelling the recruitment effect in a small marine protected area: the example of saltwater lakes on the island of Mljet (Adriatic Sea)[END_REF] who suggested that P. nobilis veliger larvae have a negative phototactism and migrates vertically to deeper waters during daylight and returns to superficial areas at night, as also indicated for other bivalves [START_REF] Gosling | Bivalve Molluscs: Biology. Ecology and Culture. Fishing News Books[END_REF].

The loss of vellum tissue also affected larval motility, generating large clusters of larvae referred as "spotting" at the bottom of the tanks due to the secretion of mucous filaments by the larval foot that may measure up to one meter in length [START_REF] Gérard | Criteres de compétence des larves à la métamorphose chez Pecten maximus[END_REF][START_REF] Bachelet | Adult larval and juvenile interactions in the suspension-feeding bivalve. Cerastoderma edule (L.): field observations and experiments[END_REF][START_REF] Rojas | Pathogenicity of a highly exopolysaccharide-producing Halomonas strain causing epizootics in larval cultures of the Chilean scallop Argopecten purpuratus (Lamarck. 1819)[END_REF]. Further studies need be conducted to determine whether these larvae clusters following the secretion of mucous filaments are a side effect of the veil loss or a mechanism to improve larval buoyancy and facilitate dispersal as proposed by [START_REF] Beninger | Demonstration of nutrient pathway from the digestive system to oocytes in the gonad intestinal loop of the scallop Pecten maximus L[END_REF] thus, discerning if it is a natural process or a negative consequence of bacterial activity.

In other Pinnidae such as Atrina maura, success in larvae rearing has not been achieved despite its commercial interest has prompted the study of aquaculture conditions for more than a decade. Coupled with pathologic problems, the larvae of A. maura appear to show a high hydrophobicity which causes the larvae adhesion to the water surface and consequently the death from desiccation and/or starvation [START_REF] Maeda-Martínez | Estado actual del cultivo y manejo de moluscos bivalvos y su proyección futura: factores que afectan su sustentabilidad en América Latina[END_REF]. According to [START_REF] González-Corona | Estudio de la fisiología reproductiva y gametogénesis del callo de hacha Atrina maura (Sowerby. 1835)[END_REF] and [START_REF] Robles-Mungaray | Desarrollo de la biotecnología para la producción de semilla en laboratorio. diploide y triploide. de callo de hacha Atrina maura (Sowerby. 1835)[END_REF] there are still some biological and technical aspects such as the adjustment of larval density or cleaning protocol, that need be optimized in order to reduce mortality and allow the sustainable commercial production of this bivalve. Therefore, there is a lack of empirical knowledge regarding the mechanisms that trigger disease transmission (Arechavala-Lopez et al., 2013), thus, the optimisation in the hatchery process is accordingly necessary and involves a better understanding of bivalve physiological requirements.

To conclude, this work presents the first detailed information on the biological cycle of Pinna nobilis and provides information concerning important variables determining larval mortality and settlement success, such as light conditions or food dose, establishing the bases for the rearing of the endangered fan mussel P. nobilis in captivity. Yet, the closure of its biological cycle in captivity appears to be rather difficult since large mortality rates are observed during first days of life (4 to 22 depending on light treatment). Given that our results are conclusive on the suitability of light conditions and "optimal" food doses, the experimental activity should be intended from a pathological approach, considering bacterial infection as one of the main bottlenecks in the rearing of P. nobilis larvae and preventing the development of this species at pediveliger stages.

Fig. 1 .

 1 Fig. 1. Correlation between broodstock size (Ht) and number of oocytes expelled per L. N = 26 adults.

Fig. 2 .

 2 Fig. 2. Early developmental stages of Pinna nobilis: (A) first polar body (00:15); (B) fertilized oocytes with double membrane (00:30); (C) first inclusion (00:40); (D) first complete division (01:00); (E) 3th and 4th division (03:00); (F) end of cell division phase (04:30). cp. polar body; ep. perivitelline membrane; ma. macromere; mi. micromere.

Fig. 3 .

 3 Fig. 3. Early larval stages in Pinna nobilis: (A) early trocophore (22:00); (B) late trocophore (30: 00); (C and D) isometric view of late veliger (72:00); (E and F) early umbonate (144:00); (G) pediveliger (168:00) (H) lateral view of attached larva. ci. cili; fa. Apical flagella; v. vellum; ma. posterior adductor muscle; gd. digestive glandule; u. umbo; p. foot; pI. Prodissoconch I; pII. Prodissoconch II.

Fig. 4 .

 4 Fig. 4. Larval growth at the different experimental volumes (5, 16, and 80 L). The red line indicates the moment when larvae stop swimming, concurring with the end of growth within 16 and 80 L tanks (arrows). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) Fig. 5. Daily evolution of larval survival according to photoperiod and phytoplankton dose.

Fig. 6 .

 6 Fig. 6. Common diseases observed in Pinna nobilis: (A) loss of vellum structure; (B) bacterial movement inside and around the larvae "swarming"; (C) larvae clusters attached by mucus "spotting".

Table 1

 1 Different established doses depending on volume and cell density.

		Low dose (L)	Optimal dose (L)	High dose (L)
	Tank 5 L
	Chaetoceros calcitrans	-	0.04	-
	Isochrysis galbana	-	0.14	-
	Pavlova lutheri	-	0.05	-
	Total (L)	-	0.23	-
	Tank 16 L
	Chaetoceros calcitrans	0.07	0.11	0.14
	Isochrysis galbana	0.23	0.46	0.69
	Pavlova lutheri	0.11	0.15	0.23
	Total (L)	0.41	0.72	1.05
	Tank 80 L
	Chaetoceros calcitrans	-	0.57	-
	Isochrysis galbana	-	2.29	-
	Pavlova lutheri	-	0.76	-
	Total (L)	-	3.62	-

Table 2

 2 Time of larvae development in Pinna nobilis at 21 °C. pH 8.50. O 2 6.5 mgO 2 •L -1 and salinity 38.0 psu.

	Stage	Cumulated time (h:min)	Size (μm)
	Spawning	0:00	50
	Sperm attachment	0:00	50
	1st polar body	0:15	50
	Double membrane	0:30	50
	1st inclusion	0:40	55
	Blastule	5:00	55
	Gastrule	8:00	55
	Early trocophore	22:00	65
	Late trocophore	30:00	70
	Early veliger	48:00	85
	Late veliger	72:00	90
	Early umbonade	144:00	100
	Pediveliger	168:00	110

Acknowledgements

The present study was financed by the Caisse d'Epargne Côte d'Azur. We are also grateful to the research crew of the Institut Océanographique Paul Ricard and the Catholic University of Valencia for their technical support and help collecting and maintaining fan mussels. Special thanks to the reviewers for their constructive and necessary suggestions.