Keeping up with advances in qPCR pathogen detection: an example for QPX disease in hard clams.

With marine diseases on the rise and increased reliance on molecular tools for disease surveillance, validated pathogen detection capabilities are important for effective management, mitigation, and response to disease outbreaks. At the same time, in an era of continual evolution and advancement of molecular tools for pathogen detection, it is critical to regularly reassess previously established assays to incorporate improvements of common practices and procedures, such as the minimum information for publication of quantitative real-time PCR experiments (MIQE) guidelines. Here, we reassessed, re-optimized, and improved the quantitative PCR (qPCR) assay routinely used for Quahog Parasite Unknown (QPX) disease monitoring. We made 19 significant changes to the qPCR assay, including improvements to PCR amplification efficiency, DNA extraction efficiency, inhibition testing, incorporation of linearized standards for absolute quantification, an inter-plate calibration technique, and improved conversion from copy number to number of cells. These changes made the assay a more effective and efficient tool for disease monitoring and pathogen detection, with an improved linear relationship with histopathology compared to the previous version of the assay. To support the wide adoption of validated qPCR assays for marine pathogens, we provide a simple workflow that can be applied to the development of new assays, re-optimization of old or suboptimal assays, or assay validation after changes to the protocol and a MIQE-compliant checklist that should accompany any published qPCR diagnostic assay to increase experimental transparency and reproducibility amongst laboratories.

A Microcosm Experiment Reveals the Temperature-Sensitive Release of Mucochytrium quahogii (=QPX) from Hard Clams and Pallial Fluid as a Stable QPX Reservoir.

Mucochytrium quahogii, also known as QPX or Quahog Parasite Unknown, is the causative agent of QPX disease in the hard clam (Mercenaria mercenaria). Host-pathogen-environment interactions between M. quahogii, the hard clam, and temperature were explored in a microcosm experiment. Hard clams were housed in individual tanks with sterile seawater under two temperature regimes: low (13 °C) temperature, which is thought to be optimal for QPX disease development, and high (20 °C) temperature, which has been shown to promote "healing" of QPX-infected clams. Hard clam tissue, pallial fluid, seawater, and shell biofilms were collected and assayed for M. quahogii. The release of M. quahogii from naturally infected live hard clams into seawater was detected only in the low temperature treatment, suggesting that temperature influences the release of potentially infectious cells. M. quahogii was commonly found in hard clam pallial fluid, even after 9 weeks in the lab, suggesting pallial fluid is a stable reservoir of M. quahogii within its primary host and that M. quahogii is not a transient component of the hard clam microbiota. Overall, results support a host-specific relationship and that M. quahogii is a commensal member of the hard clam microbiota, supporting its classification as an opportunistic pathogen.

An apicomplexan parasite drives the collapse of the bay scallop population in New York.

The bay scallop, Argopecten irradians, represents a commercially, culturally and ecologically important species found along the United States' Atlantic and Gulf coasts. Since 2019, scallop populations in New York have been suffering large-scale summer mortalities resulting in 90-99% reduction in biomass of adult scallops. Preliminary investigations of these mortality events showed 100% prevalence of an apicomplexan parasite infecting kidney tissues. This study was designed to provide histological, ultrastructural and molecular characteristics of a non-described parasite, member of the newly established Marosporida clade (Apicomplexa) and provisionally named BSM (Bay Scallop Marosporida). Molecular diagnostics tools (quantitative PCR, in situ hybridization) were developed and used to monitor disease development. Results showed that BSM disrupts multiple scallop tissues including kidney, adductor muscle, gill, and gonad. Microscopy observations allowed the identification of both intracellular and extracellular stages of the parasite. Field surveys demonstrated a strong seasonal signature in disease prevalence and intensity, as severe cases and mortality increase as summer progresses. These results strongly suggest that BSM infection plays a major role in the collapse of bay scallop populations in New York. In this framework, BSM may synergistically interact with stressful environmental conditions to impair the host and lead to mortality.

Mucochytrium quahogii (=QPX) Is a Commensal, Opportunistic Pathogen of the Hard Clam (Mercenaria mercenaria): Evidence and Implications for QPX Disease Management.

Mucochytrium quahogii, commonly known as QPX (Quahog Parasite Unknown), is the causative agent of QPX disease in hard clams (Mercenaria mercenaria), but poor understanding of the relationship between host and pathogen has hindered effective management. To address this gap in knowledge, we conducted a two-year study quantifying the distribution and abundance of M. quahogii in hard clam tissue, pallial fluid, and the environment. M. quahogii was broadly distributed in clams and the environment, in areas with and without a known history of QPX disease. M. quahogii in clams was not strongly related to M. quahogii in the environment. M. quahogii was always present in either the tissue or pallial fluid of each clam, with an inverse relationship between the abundance in the two anatomical locations. This study suggests that the sediment-water interface and clam pallial fluid are environmental reservoirs of M. quahogii and that there is a host-specific relationship between M. quahogii and the hard clam, supporting its classification as a commensal, opportunistic pathogen. There appears to be minimal risk of spreading QPX disease to naïve clam populations because M. quahogii is already present and does not appear to be causing disease in hard clam populations in locations unfavorable for pathogenesis.

Erection of a New Genus and Species for the Pathogen of Hard Clams 'Quahog Parasite Unknown' (QPX): Mucochytrium quahogii gen. nov., sp. nov.

Quahog Parasite Unknown (QPX) is a facultative parasite of the hard clam, Mercenaria mercenaria. Although it has been observed in clams since the 1960's and cultivated since the 1990's, conflicting reports on important aspects of its biology have prevented its formal description. 18S rRNA gene sequences identify QPX as a thraustochytrid, but its production of copious mucus is atypical for this group. There are also conflicting reports about whether QPX shares common features of thraustochytrids, such as the production of an ectoplasmic net and biflagellate zoospores. This study reaffirms the previous descriptions of zoospore production by QPX in culture, in multiple strains from several geographic locations, and provides detail on how to maintain QPX cultures under conditions that promote the production of zoospores. Furthermore, we describe new aspects of the life cycle not previously observed. Finally, we erect Mucochytrium quahogii gen. nov., sp. nov. to accommodate this unusual thraustochytrid.