Investigating reconstructed inflows and pathogen infection patterns between low-relief and high-relief subtropical oyster reefs

Abstract

Flood and drought events cause significant freshwater inflow fluctuations in estuaries, potentially leading to physiological stress and altered abundances of pathogens such as Vibrio vulnificus and Perkinsus marinus in oysters. To assess the effects of freshwater pulses to oyster reefs in subtropical estuaries in Texas, this study accomplished two goals: 1) reconstructed a reef-specific history of freshwater pulses through shell stable isotope analysis, 2) quantified the abundance of V. vulnificus and P. marinus through culture-dependent and culture-independent microbiology analyses. Oysters from a low-relief and high-relief reef experienced similar fluctuations in shell isotopes, indicating similar ranges of past environmental conditions. V. vulnificus and P. marinus were detected throughout the study but the abundance of these microorganisms was not correlated with environmental parameters or one another. Importantly, the P. marinus infection intensity was always lower at the high-relief reef, which suggests that high-relief reefs may experience lower infection frequencies.

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., 2012; Pollack et al., 2012). Oysters are subject to colonization by V. vulnificus which is a naturally occurring marine bacterium and one of the deadliest human pathogens (Strom & Paranjpye, 2000). 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, 2013). 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 10-15^oC 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, 1952). 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, 2013) followed by species confirmation of selected isolates by polymerase chain reaction (PCR) using primers targeting the species-specific vvhA gene (Kaysner & DePaola, 2004). 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 (2020). Environmental and pathogen data are available in full online at Turner (2020). Additional results are discussed in Walther et al. (2019).

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.