Intraspecific variation in host susceptibility to individual parasite species is common, yet how these effects scale to mediate the structure of diverse parasite communities in nature is not as well understood. To address this knowledge gap, we tested how host genetic identity affects parasite communities on restored reefs seeded with juvenile oysters from different sources – a regional commercial hatchery or one of two wild progenitor lines. We assessed the prevalence and intensity of three micro- and two macro-parasite species for four years following restoration. Despite the spatial proximity of restored reefs, oyster source identity strongly predicted parasite community prevalence across all years, with sources varying in their relative susceptibility to different parasites. Oyster seed source also predicted reef-level parasite intensities across space and through time. Our results highlight that host intraspecific variation can shape parasite community structure in natural systems, and reinforce the importance of considering source identity and diversity in restoration design.

Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Supplemental Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Funding

Oyster Reef Restoration Experiment
In May 2017, we created nine oyster reefs in Quonochontaug Pond, Rhode Island (RI) (41°20'22.6"N, 71°43'41.2"W), with three separate reefs each located within three distinct regions (West, Northeast, East). Each ~22 square meter (m²) reef (0.5-0.8 m height) was constructed from a base layer of steam-shucked clam shell topped with clean, recycled oyster shell, and then seeded with remote-set spat on shell (see Davenport et al. 2022 for a detailed description of reef construction). A goal of this study was to compare the performance of different oyster sources and the consequent structure of associated parasite communities, so one replicate reef per oyster source was constructed in each of the three regions of Quonochontaug Pond (hereafter, blocks). The oyster seed sources included one line from a regional commercial hatchery, as well as two wild progenitor lines spawned from broodstock collected from nearby existing wild populations in Green Hill Pond, RI and Narrow River, RI. All oyster lines were spawned at local hatcheries, transferred to Roger Williams University Shellfish Hatchery and set on oyster shell in June 2016, and then stored in cages on an oyster lease in Quonochontaug Pond until reef construction in May 2017.

Reef Monitoring and Oyster Collection
In the fall of each year for four years post-restoration (2017-2020), we monitored oyster density and size distribution by non-destructively sampling six haphazard 0.25 m² quadrats per reef and recording the number of live and dead oysters, as well as shell height of a subsample of up to 50 live and 30 dead oysters (following methods of Griffin et al. 2012 (see Supplemental Files) and guidance of Baggett et al. 2015). Coincident with each fall monitoring event, we harvested ~35 haphazardly-selected live oysters from each reef for analysis of parasite communities; samples were transported to the Northeastern University Marine Science Center on ice and then stored at -80 degrees Celsius prior to processing.

Parasite Prevalence and Intensity
Crassostrea virginica is commonly infected by a variety of micro- and macro-parasites simultaneously. We assessed the prevalence (proportion of sampled oysters infected per oyster reef) and intensity (parasite concentration per infected host) of five common parasite species (microparasites: Perkinsus marinus (Dermo disease), Haplosporidium nelsoni (MSX disease), and Haplosporidium costale (SSO disease); macroparasites: Cliona spp. and Polydoraspp.).

To assess microparasite prevalence and intensity, DNA was extracted from up to 32 oysters per reef and then amplified using both a polymerase chain reaction (PCR) assay modified from Stokes & Burreson (2001) SSO protocol, and a multiplex quantitative polymerase chain reaction (qPCR) assay modified from De Faveri et al. (2009) Dermo protocol and Wilbur et al. (2012) MSX protocol. DNA was extracted using 20-40 milligrams (wet weight) of gill and mantle tissue with the Omega Bio-Tek E-Z 96® Tissue DNA Kit.

We used a modified version of the De Faveri et al. (2009) Dermo protocol and the Wilbur et al. (2012) MSX protocol to analyze the samples on a Bio-Rad CFX96TM Real-Time System with Bio-Rad CFX Manager Software (version 3.1). Each reaction consisted of 1 microliter (μl) template DNA, 3 μl water, 5 μl TaqMan Multiplex Master Mix, and 0.5 μl of each 20X primer/probe master mix, which contained 18 μM of each P. marinus primer and 5 μM of TaqManTM QSY probe (De Faveri et al. 2009) for Dermo, and 18 μM of each H. nelsoni primer and 5 μM of TaqManTM MGB probe (Wilbur et al. 2012) for MSX. qPCR cycling conditions included initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. We used gBlocks® (gene fragments containing the target regions from P. marinus and H. nelsoni; Integrated DNA Technologies) to develop standard curves, and extracted DNA from cultures of P. marinus and H. nelsoni with known cell quantities to use as positive controls. All standards, samples, and positive and negative controls were run in duplicate; if samples differed by >1 Cq, they were rerun to confirm presence/absence and/or parasite concentration.

We used a modified version of the Stokes & Burreson (2001) SSO protocol to assess H. costale prevalence on a Bio-Rad CT100 thermal cycler. PCR cycling conditions included initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30 sec, 59°C for 30 sec, and 72°C for 60 sec, and final extension at 72°C for 5 min. PCR products were visualized on a 1% agarose gel and photographed for analysis of band intensity using ImageJ, following the methods of DeLong & Hanley (2013). For the SSO assay, we used gBlocks® containing the target region from H. costale as positive controls and as standards for estimating parasite concentration based on quantification of band intensity using ImageJ (Abràmoff et al. 2004).

To assess the prevalence and intensity of two common macroparasites, boring sponge (Cliona spp.) and mud blister worm (Polydora spp.), we photographed the inside (mud blisters) and/or outside (sponge holes) of both top and bottom valves for all samples with holes characteristic of boring sponges and blisters characteristic of mud blister worms to quantify the proportion of affected shell area (i.e., [(total infected area/total shell area)*100]) using ImageJ (Abràmoff et al. 2004), following the methods of Hanley et al. (2019).

Data Processing
For oyster density, we calculated the mean number of live oysters per reef as the average density across the six replicate quadrats sampled. For oyster size, we calculated the mean shell height of up to 50 live oysters per quadrat and then took the average across the six replicates per reef. For each parasite, we calculated reef-level prevalence as [number of infected oysters] / [number of oysters sampled], and reef-level intensity as the mean parasite concentration per infected host (i.e., uninfected hosts were not included).

BCO-DMO Processing
- replaced "NA" with "nd" (no data value);
- renamed fields to comply with BCO-DMO naming conventions.

Coverage: Coastal New England

NSF Award Abstract:
Disease outbreaks in the ocean are increasing, causing losses of ecologically important marine species, but the factors contributing to these outbreaks are not well understood. This 5-year CAREER project will study disease prevalence and intensity in two marine foundation species - the seagrass Zostera marina and the Eastern oyster Crassostrea virginica. More specifically, host-disease relationships will be explored to understand how genetic diversity and population density of the host species impacts disease transmission and risk. This work will pair large-scale experimental restorations and smaller-scale field experiments to examine disease-host relationships across multiple spatial scales. Comparisons of patterns and mechanisms across the two coastal systems will provide an important first step towards identifying generalities in the diversity-density-disease relationship. To enhance the broader impacts and utility of this work, the experiments will be conducted in collaboration with restoration practitioners and guided by knowledge ascertained from key stakeholder groups. The project will support the development of an early career female researcher and multiple graduate and undergraduate students. Students will be trained in state-of-the-art molecular techniques to quantify oyster and seagrass parasites. Key findings from the surveys and experimental work will be incorporated into undergraduate courses focused on Conservation Biology, Marine Biology, and Disease Ecology. Finally, students in these courses will help develop social-ecological surveys and mutual learning games to stimulate knowledge transfer with stakeholders through a series of workshops.

The relationship between host genetic diversity and disease dynamics is complex. In some cases, known as a dilution effect, diversity reduces disease transmission and risk. However, the opposite relationship, known as the amplification effect, can also occur when diversity increases the risk of infection. Even if diversity directly reduces disease risk, simultaneous positive effects of diversity on host density could lead to amplification by increasing disease transmission between infected and uninfected individuals. Large-scale field restorations of seagrasses (Zostera marina) and oysters (Crassostrea virginica) will be utilized to test the effects of host genetic diversity on host population density and disease prevalence/intensity. Additional field experiments independently manipulating host genetic diversity and density will examine the mechanisms leading to dilution or amplification. Conducting similar manipulations in two marine foundation species - one a clonal plant and the other a non-clonal animal - will help identify commonalities in the diversity-density-disease relationship. Further, collaborations among project scientists, students, and stakeholders will enhance interdisciplinary training and help facilitate the exchange of information to improve management and restoration efforts. As part of these efforts, targeted surveys will be used to document the perceptions and attitudes of managers and restoration practitioners regarding genetic diversity and its role in ecological resilience and restoration.