Introduction
The cockle shellfish beds of the Ría de Arousa (Galicia, NW Spain) were devastated in 2012 by the protozoan parasite Marteilia cochillia responsible for marteiliosis. The annual outbreak of this parasite threatens production and the ecosystem services provided by cockles In 2018, Villalba et al. (2021) suggested the presence of naturally resilient stocks, when compared their survival to naive cockles (never in contact with the parasite) from a northern estuary (Ría la Noia).
Generating preventive measures against parasitic infection is complex due to the lack of knowledge of the parasite’s biology and the difficulty of fighting against a parasite in a natural open environment. Therefore, developing resilient strains against M. cochillia through breeding programmes is an appealing approach, previously applied in other molluscs to control the impact of the parasite and restore cockle beds.
This study aimed to identify candidate genetic markers, single nucleotide polymorphisms (SNPs), associated with resilience to marteliosis for developing and validating a cost-effective genomic tool to assist in the restoration of cockle beds and preventing economic losses due to parasite spreading to other areas. For this, a common garden experiment involving naive and long-affected cockle stocks was carried out in Ría de Arousa in 2021 using families founded at hatchery.
Materials and Methods
To identify markers associated with Martelia resilience we followed two complementary approaches: (i) population genomics (PGA) using 77 samples, 39 collected in 2018 from a long term affected shellfish bed in the Arousa estuary, Lombos de Ulla, before and after the annual outbreak of marteiliosis, and 38 naive from 2012 before the first report of marteliosis in the area. Samples were genotyped using a 2b-RAD panel of 9.154 SNPs 2 mapped on the cockle’s genome and used to identify outlier loci . (ii) transcriptomic (TA), where cockles sorted by infection degree were compared to identify SNPs associated with 767 differentially expressed genes (DEG) 3 according to the infection status . SNPs from both approaches were filtered according to missing data, heterozygosity (He) and technical criteria, to select the most consistent SNPs to be genotyped using a MassARRAY platform 4.
For the common garden experiment, mature breeders (400 individuals) from naive and long-affected shellfish beds were used to produce families at hatchery, After a pre-growing stage in a raft, 300 cockles from each stock were settled in two shellfish beds in Ría de Arousa under marteiliosis pressure. Prevalence and mortality were recorded through histopathology. Individuals were histologically classified according to the level of infection and genotyped with the SNPs panel selected (183 naïve, and 197 long affected).
Results and Discussion
From the TA, a total of 121 DEG-SNPs showed significant differentiation depending on the cockle’s infection degree and another 110 outlier SNPs from the PGA were selected and mapped in the cockle’s genome. Finally, a panel of 45 SNPs including both approaches were combined in two multiplexes to be genotyped in a MassARRAY platform. Results from the common garden experiment showed that the naive stock was fully depleted after four months in both shellfish beds, while the affected stock barely showed marteiliosis.. Among the 45 selected SNPs, 28 showed significant divergence between naive and long affected beds (p value <0.0012; Bonferroni correction), despite no significant differentiation was detected with neutral markers, suggesting they were involved in directional selection after eight generations of marteiliosis pressure (long-term selection). Furthermore, 11 SNPs showed significative differentiation in the short period affecting the naive stock (short-term selection), most of them shared with those from the long-term comparison. Several SNPs were located within relevant immune genes pertaining to families such as proteasome, ubiquitine and glutation S-transferase.
Conclusions
Common cockle became resilient to marteiliosis after eight generations of selection. The genomic approach followed to identify SNP markers associated with resilience proved to be useful and a total of 28 SNPs are candidates to be applied in marker assisted selection programs to manage common cockle shellfish beds. Some of these markers are within genes involved in critical immune functions. A SNP panel can be applied to recover common cockle shellfish beds in Galicia.
Funding information
This study was possible in part due to funds granted by the European Union through the project COCKLES (EAPA_458/2016) and the Marine Science Programme for Galicia funded by the Resilience and Recovery Funds.
References
1. Villalba, A. et al. Increased resistance against marteiliosis in the cockle Cerastoderma edule population of the inner area of the Ría de Arousa (Galicia NW Spain) through natural selection. in European Association of Fish Pathologists 20th - International Conference on Diseases of Fish and Shellfish pp.146 (2021).
2. Vera, M. et al. Genomic survey of edible cockle (Cerastoderma edule) in the Northeast Atlantic: a baseline for sustainable management of its wild resources. Evol. Appl. 15, 262–285 (2022).
3. Pardo, B. G. et al. Transcriptome characterization of the common cockle (Cerastoderma edule) after exposure to a Marteilia cochillia outbreak. bioRixiv 10.18.512677 (2022) doi:10.1101/2022.10.18.512677.
4. Pampín, M. et al. Genetic markers associated with divergent selection against the parasite Marteilia cochillia in common cockle (Cerastoderma edule ) using transcriptomics and population genomics data. Front. Mar. Sci. 10, 1057206. (2023).