Development of Two Diagnostic Tools to Detect the Putative Agent of Bacterial Gill Disease of Razor Clams (Siliqua patula) and Estimates of Prevalence and Load in Select Pacific Northwest Populations

Razor clams (Siliqua patula) are a dominant shellfish on coastal beaches of the NE Pacific providing food for numerous marine and terrestrial taxa. Additionally, they serve as a valuable fishery in Washington state, with the recreational fishery valued at USD $22 million per year. In the past ten years, poor survival of adult clams at Kalaloch Beach on the north-central Washington coast has resulted in more than eight full or partial closures of the fishery. Factor(s) driving this variability remain undetermined; however, it is hypothesized that mortality from bacterial gill disease, also called nuclear inclusion X (NIX) disease may be contributing. NIX – a gamma proteobacterium – infects the nucleus of razor clam gill epithelial cells and is associated with hypertrophied nuclei. Histological examination is often used as a diagnostic tool for NIX. The goal of this study was to develop and validate quantitative PCR (qPCR) and a chromogenic in situ hybridization (CISH) assay to detect NIX using DNA-specific probes. A newly developed qPCR test was then applied to determine NIX prevalence in razor clams from three coastal Washington populations, and two Alaskan populations sampled in spring and summer 2019. The in situ hybridization sections were cross-referenced against paired histological sections stained with hematoxylin and eosin. This comparison confirmed that the putative intranuclear bacterium observed by histological examination was the NIX agent. Quantitative PCR agreed with CISH and indicated 100% prevalence of the bacterium in all three Washington populations with a significantly higher gene copy number in the Kalaloch population. Consistent with prior histological observations, razor clams sampled from Cook Inlet in Alaska had 0% prevalence of NIX. Once validated, these tools will be used to assess spatial and temporal patterns of NIX and evaluate the role of NIX in changing razor clam demography.