Over the past several years a disease similar to disseminated neoplasia (DN) previously described in other bivalves has been observed in the hard clam, Mercenaria mercenaria , in Wellfleet, Massachusetts. Work on DN in other bivalves has indicated that the disease is not only transmissible (Metzger et al. 2015) but that the neoplastic cell lineages tend to be specific for different bivalve species (Metzger et al. 2016). A retroelement (Steamer ) has been determined to show increased expression in diseased soft shell clams (Arriagada et al. 2014), but whether it is responsible for the disease is still unknown. To better define and understand this disease in hard clams, we set out to identify the origin of the neoplastic cells and to examine whether similar retroelements were present and showed increased expression in infected individuals.
To determine the origin of the neoplastic cells, w e acquired and compared the mitochondrial COI (mtCOI) gene sequences of hemocyte cells from normal and infected clams, and from tissues collected from a subset of normal and infected clams. This showed that haplotypes of the hemocyte cells from infected individuals were different than the corresponding tissue haplotypes and confirmed that the neoplastic cells originated from Mercenaria mercenaria . It was also noted that the mtCOI haplotypes of most transformed cells arose from previously identified haptophyte lineages that were likely uninfected, suggesting that more than one possible transformation event had occurred.
Transcriptome sequencing was accomplished on hemolymph from 5 infected hard clams and 3 uninfected hard clams collected from the adductor muscle sinus using sterile needles and preserved in RNALater at -80°C. The transcriptome was assembled using all 8 samples (529,795 assembled transcripts), and eukaryote BUSCO analysis indicated very good completeness (96.1% complete, 3.1% fragmented, 0.8% missing). Differential expression analysis indicated the up-regulation of 1,303 transcripts in the infected hemolymph, and 73% of these were annotated . Most of these transcripts were associated with cellular growth and cancer pathways, as expected for cells undergoing rapid replication. While t here were no retroelements similar to the Steamer element from soft shell clam, there were oncogene-related sequences, RNA-directed DNA polymerases and Jockey-like retroelements upregulated in the infected hard clam transcripts. I t is still not known whether this expression is a consequence or a cause of the transformation, but this data has revealed several potential targets for diagnostic methods.
Metzger, MJ , C Reinisch , J Sherry & SP Goff ( 2015). Cell 161: 255-263.
Metzger, MJ, A Villalba , JM Carballal , D Iglesias, J Sherry, C Reinisch , AF Muttray , SA Baldwin & SP Goff (2016). Nature 534: 705-709.
Arriagada , G, MJ Metzger, AF Muttray , J Sherry, C Reinisch , C Street, WI Lipkin, & SP Goff (2014). PNAS 111: 14175-14180.