ANALYSIS OF THE MAIN IMMUNE GENE BIOMARKERS IN Ruditapes decussatus AFTER EXPERIMENTAL INFECTION BY Perkinsus olseni

Introduction

The grooved carpet shell clam (Ruditapes decussatus ) is a highly desirable species in  the European market.  Its population has been in a constant decline due to biotic and abiotic factors, namely, habitat degradation by anthropogenic actions in the case of abiotic factors but also due to competition with alien species and pathogens in the case of biotic factors. The introduction of the alien species Ruditapes philippinarum in the decade of 60´s in France and England constitutes one of the main problems due to habitat loss and competition against a faster growth clam

 , also, some introductions of the alien species from Asia in the 80´s brings the Apicomplexan parasite Perkinsus olseni to European waters

[2, 3]

 . This parasite not only provoke mortalities in the native clams but also it is able to impairs their reproduction by decreasing the quality of gametes and survival of the larvae

.

 Taking all  these details into account, our study is focused on the effect of different doses of the parasite in the ability of clams to survive and to fight against the disease. Also, some genes previously identify as potentially involved in the response to infection were studied in gill and hemocytes during the progression of the infection.

Material and methods

 A total of 540 R. decussatus clams (43.7 ± 2.9 mm length) were purchased from the fisheries association of San Bartolomé de Noia (Galicia, NW Spain) a known  spot for being P. olseni free bed. Clams were transferred to CIIMAR facilities and they were placed in 10 tanks (54 clams per tank) at 19ºC . After that, clams  from 9 tanks  were notched in the shell and injected  in the adductor muscle  with  100µL of two different doses of parasites (3 tanks with  5000 cells/clam – Low Infection (LI) ; 3 tanks with  500,000 cells/clam – High infection (HI)) and 3 tanks with marine filtered sea water (control), also one tank was untouched to check it out the effect of the injection into the clams (C-).

At 24 hours, 48 hours, 1, 2 and 4 weeks after injection, 5 clams per tank (15 per treatment) were sampled. Hemolymph was extracted by a 1mL insulin syringe, centrifuged at 2500 rpm, 10 min and 4ºC to separate hemocytes from plasma. Hemocytes were immediately stored at -80ºC. One piece of gill was collected and placed in 0.5 ml of RNA later (Invitrogen, US) and stored at -80ºC. Also, one hemigill was placed in 10 ml of RFTM medium (Casas & Villalba 2012) for P. olseni diagnosis and quantification and other was placed in ethanol 96% for DNA extraction and qPCR diagnosis of the parasite according to

.

Results

No differences in mortality was seen among the different treatments during the 3 months of trial (Fig.1). Infection trial was successful and a high correspondence parasite load was obtained by two diagnosis methods: RFTM and qPCR.

Regarding the gene expression results,  a high expression was seen in adiponectin in both tissues at all times and infection load, while most of the other genes seem to be down-regulated at first 48 hours to recover after that in gills while no differences were seen in hemocytes.

P. olseni was not able to provoke a high mortality in a small period of time in adult clams although  some studies reported high mortalities in juveniles

 . Regarding the experimental infection, all parasites migrate quickly from adductor muscle to gills and a high parasite load was seen 24 hours post-infection in HI clams suggesting an active infection and confirming  the effectiveness  of the  infection protocol. Also, no differences in mortality were recorded between  both control  tanks suggesting that notched and injection have not any harmful effect on clams.

Finally, gene expression study  shows that adiponectin seems to be the most relevant marker of infection for Perkinsus infection in clams.

Acknowledgements

 This research was supported by the project Tools4Breed – Challenge test and genetic markers for  Perkinsus  as a tool for Ruditapes decussatus´ selective breeding with reference FA_05_2017_025 financed by Fundo Azul and República Portuguesa.

Bibliography

1. Cordero, D., et al., Population genetics of the Manila clam (Ruditapes philippinarum) introduced in North America and Europe. Sci Rep, 2017. 7: p. 39745.

2. Vilas, R., Cao, A., Pardo, B. G., Fernández, S., Villalba, A., Martínez, P., Very low microsatellite polymorphism and large heterozygote deficits suggest founder effects and cryptic structure in the parasite Perkinsus olseni. Infection, Genetics and Evolution, 2011. 11: p. 904-911.

3. Azevedo, C., Fine structure of Perkinsus atlanticus n. sp. (Aplicomplexa, Perkinsea) parasite of the clam Ruditapes decussatus from Portugal. Journal of Parasitology, 1989. 75: p. 627-635.

4. Casas, S.M. and A. Villalba, Study of perkinsosis in the grooved carpet shell clam Ruditapes decussatus in Galicia (NW Spain). III. The effects of Perkinsus olseni infection on clam reproduction. Aquaculture, 2012. 356-357: p. 40-47.

5. Rios, R., et al., Development and validation of a specific real-time PCR assay for the detection of the parasite Perkinsus olseni. J Invertebr Pathol, 2020. 169: p. 107301.

6. Shimokawa, J., T. Yoshinaga, and K. Ogawa, Experimental evaluation of the pathogenicity of Perkinsus olseni in juvenile Manila clams Ruditapes philippinarum. J Invertebr Pathol, 2010. 105(3): p. 347-51.