Introduction
The purpose of the project was to assess how variation in reconstructed freshwater inflow affects the abundance of pathogens in oysters from low-relief and high-relief reefs in subtropical estuaries in Texas. Punctuated flood events, such as those during Hurricane Harvey, bring large amounts of freshwater to oyster reefs which can have profound implications for oyster physiological stress and loading of harmful bacteria and parasites (Froelich et al., Reference Froelich, Williams, Noble and Oliver2012; Pollack et al., Reference Pollack, Cleveland, Palmer, Reisinger and Montagna2012). Oysters are subject to colonization by V. vulnificus which is a naturally occurring marine bacterium and one of the deadliest human pathogens (Strom & Paranjpye, Reference Strom and Paranjpye2000). Oysters are also subject to infection by the Dermo-causing parasite P. marinus which can cause significant mortality of oysters. Assessing links between inflows and V. vulnificus and P. marinus requires time series of salinity variations at the reef scale, which are often unavailable from instrumental monitoring. Fortunately, oyster shells themselves naturally record inflow fluctuations via stable isotope ratios of oxygen and carbon that are incorporated into shell growth increments (Walther & Rowley, Reference Walther and Rowley2013). Pairing this individual record of inflow stress with assessments of V. vulnificus and P. marinus abundance is a potential way to identify the effects of inflow variation on oyster health.
Objective
This study had two primary goals: 1) reconstruct a reef-specific history of inflow fluctuations through shell stable isotope analysis, and 2) quantify the abundance of V. vulnificus and P. marinus through culture-dependent and culture-independent microbiology analyses. This work was carried out in two locations with different reef types in Texas: the high-relief Shellbank Reef in Copano Bay (Latitude N 28°6’43’’, Longitude W 97°9’6’’) and a low-relief reef in St. Charles Bay (Latitude N 28°08’22’, Longitude W 96°58’11’). The study aimed to compare inflow histories and pathogen infection patterns between low-relief and high-relief reefs to assess if pathogen susceptibility differs among these reef types.
Methods
A total of 40 oysters (Crassostrea virginica) were collected by dredge from two reefs on five dates (04/08/2019, 04/29/2019, 05/29/2019, 07/09/2019, and 07/29/2019, 4 oysters collected per site each date). Vibrio analyses were not performed on samples collected on 05/29/2019 because of a temporary lack of available facilities, however Vibrio were analyzed from the remaining four dates. Environmental parameters (temperature, salinity, pH, and dissolved oxygen) were measured at 0.5 meters depth with a handheld YSI 556 sonde (YSI Inc., Yellow Springs, OH). Oysters were stored in a cooler between 10-15oC and returned to the lab where they were rinsed/cleaned with filter-sterilized seawater and shucked with flame-sterilized tools. Temperature variation during transit was monitored using a Kestrel Drop D1 (Kestrel Meters, Boothwyn, PA) and oysters were processed within four hours of collection. To quantify P. marinus abundance, a 5 mm2 section of mantle tissue was incubated for two weeks at room temperature in fluid thioglycollate media and the infection intensity was assigned a numerical value ranging from 0 (no infection) to 5 (heavy infection) (Ray, Reference Ray1952). Remaining tissue was used to quantify V. vulnificus abundance in a two-step process: selective culture of presumptive V. vulnifucus isolates using V. vulnificus X-gal (VVX) agar (Griffitt & Grimes, Reference Griffitt and Grimes2013) followed by species confirmation of selected isolates by polymerase chain reaction (PCR) using primers targeting the species-specific vvhA gene (Kaysner & DePaola, Reference Kaysner and DePaola2004). Six oyster shells (three from each reef), collected 5/29/2019, were processed to extract 5-14 subsamples of carbonate (0.3 mg powder each) from sectioned oyster shells for δ18O and δ13C isotope analyses following methods available online at http://dx.doi.org/10.17504/protocols.io.bbd7ii9n. Additional methods are provided in the Supplementary Information.
Results
The environmental parameters (temperature, salinity, pH, and dissolved oxygen) varied minimally over the study (Figure 1). The abundance of presumptive V. vulnificus varied moderately over the study, ranging from not detectable to 9,066 colony forming units (CFU) mL-1 (Figure 2). A species-specific PCR test determined that fewer than half of the isolates (54/120 or 45%) possessed the species-specific vvhA gene target. The infection intensity of P. marinus also varied minimally over the study, ranging from 0 (not detectable) to 1.67 (light infection; Figure 3). There were no obvious differences in isotope patterns between Shellbank Reef and St. Charles Bay. One-way repeated measures analyses of variance did not identify statistically significant differences in values of either δ18O (F(13,5)=1.05, p=0.41) or δ13C (F(13,5)=1.0, p=0.44) among shells (Figure 4). Oyster isotope data are available in full online at Walther and Hinson (Reference Walther and Hinson2020). Environmental and pathogen data are available in full online at Turner (Reference Turner2020). Additional results are discussed in Walther et al. (Reference Walther, Turner, Bachand, O’Donnell and Hinson2019).
Figure 1. The variation in temperature (oC), salinity (ppt), pH, and dissolved oxygen (% saturation) recorded over the three month study period in (A) low-relief St. Charles Bay and (B) high-relief Shellbank Reef.
Figure 2. The variation in the log-transformed abundance of presumptive V. vulnificus (CFU mL-1 oyster homogenate) over the three month study period in high-relief Shellbank Reef and low-relief St. Charles Bay. Columns show the average abundance (N = 4 oysters per site) and error bars show the standard error.
Figure 3. Variation in the infection intensity of P. marinus over the three month study period in high-relief Shellbank Reef and low-relief St. Charles Bay. Columns show the average infection intensity (N = 4 oysters per site) and error bars show the standard error.
Figure 4. Boxplots depicting variations in (A) δ18O and (B) δ13C values within individual shells from low-relief St. Charles Bay and high-relief Shellbank Reef.
Discussion
This work did not find major or consistently identifiable differences in either environmentally-dependent isotope reconstructions or pathogen prevalence. This result may have been due to the relatively similar environmental conditions found at both reef sites. Fewer than half of the isolates tested were confirmed as V. vulnificus, indicating that the media was semi-selective rather than selective (i.e., it permitted the growth of non-target species) at the incubation temperature of 30°C. Alternative and more accurate methods such as colony blot hybridization and real-time PCR are available but the cost and labor of these methods was prohibitive in this study.
Conclusions
This study attempted to assess how two distinct reefs responded to freshwater intrusion. The shell isotopes indicated that both reefs experienced similar inflow fluctuations over the lifetimes of the oysters. Thus, this study at least afforded a comparison of a low-relief reef versus a high-relief reef that experienced similar environmental conditions. The abundance of V. vulnificus and the infection intensity of P. marinus served as proxies for monitoring changes in oyster microbiota. The abundance of V. vulnificus was not significantly different between reefs but the Dermo infection intensity was often lower in the high-relief reef. This decreased infection intensity may relate to the reef height as high-relief reefs may experience less sedimentation and stress.
Acknowledgements
The authors thank Robert Duke and Jay Tarkington from the Texas A&M University-Corpus Christi (TAMU-CC) Center for Coastal Studies for their assistance with field sampling. Shell stable isotope analyses were conducted at the University of California Davis Stable Isotope Facility by Richard Doucette. Rosario Martinez at the Coastal Bend Bays and Estuaries Program provided assistance with project management and oversight. The authors also thank Dr. Jennifer Beseres Pollack and her Research Associate Natasha Breaux (TAMU-CC Harte Research Institute) for conducting the Dermo analysis. The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guides on the care and use of laboratory animals. Oysters were collected with permission under Texas Parks and Wildlife Department Scientific Research Permit No. SPR-0117-20.
Author Contributions
BDW and JWT conceived and designed the study. PTB, CAO, and AH conducted field sampling. PTB and CAO conducted molecular analyses. AH processed shells for analyses. BDW, PTB, AH, CAO and JWT analyzed data and wrote the article.
Funding Information
This work was funded by the Texas Commission on Environmental Quality and the Coastal Bend Bays and Estuaries Program (CBBEP Contract #1920).
Data Availability Statement
Shell isotope data are available online at: https://doi.org/10.6084/m9.figshare.11608347 Environmental and pathogen data are available online at: https://doi.org/10.6084/m9.figshare.11733426.v1
Conflict of Interest
BDW, PTB, AH, CAO, and JWT declare none.
Supplementary Materials
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/exp.2020.28.
Open access
Comments
Comments to the Author: The authors collected four oysters each from two sites over five days. Quantity of V vulnificus, and P marinus was measured, along with environmental parameters. The analysis seems incomplete, as it is unclear if the environmental parameters had any correction with vibrio abundance. Furthermore, it is unclear if the vibrio abundance data was presented as is, or after correcting for the PCR confirmation. Both of these can be completed without extra field or lab work. No explanation is provided why only four of the five samples have vibrio data. Finally, 6 oysters is often the minimum for performing this type of work, but four is still acceptable. L86: Forgot to replace X and Y with values. Figure 2: Does this figure consider the PCR data confirming VV?