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A Comparison of Bacteria Cultured from Unionid Mussel Hemolymph

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A Comparison of Bacteria Cultured from Unionid Mussel Hemolymph A Comparison of Bacteria Cultured from Unionid Mussel Hemolymph between Stable Populations in the Upper Mississippi River Basin and Populations Affected by a Mortality Event in the Clinch River Authors: Eric Leis, Sara Erickson, Diane Waller, Jordan Richard, and Tony Goldberg Source: Freshwater Mollusk Biology and Conservation, 22(2) : 70-80 Published By: Freshwater Mollusk Conservation Society URL: https://doi.org/10.31931/fmbc.v22i2.2019.70–80 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles in the biological, ecological, and environmental sciences published by nonprofit societies, associations, museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-o-use. 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Downloaded From: https://bioone.org/journals/Freshwater-Mollusk-Biology-and-Conservation on 03 Jan 2020 Terms of Use: https://bioone.org/terms-of-use Freshwater Mollusk Biology and Conservation 22:70–80, 2019 Ó Freshwater Mollusk Conservation Society 2019 REGULAR ARTICLE A COMPARISON OF BACTERIA CULTURED FROM UNIONID MUSSEL HEMOLYMPH BETWEEN STABLE POPULATIONS IN THE UPPER MISSISSIPPI RIVER BASIN AND POPULATIONS AFFECTED BY A MORTALITY EVENT IN THE CLINCH RIVER Eric Leis1*, Sara Erickson1, Diane Waller2, Jordan Richard3, and Tony Goldberg4 1 La Crosse Fish Health Center, Midwest Fisheries Center, U.S. Fish and Wildlife Service, 555 Lester Ave, Onalaska, WI 54650 USA 2 Upper Midwest Environmental Sciences Center, U.S. Geological Survey, 2630 Fanta Reed Rd, La Crosse, WI 54603 USA 3 Southwestern Virginia Field Office, U.S Fish and Wildlife Service, Southwestern Virginia Field Office, 330 Cummings Street, Abingdon, VA 24210 USA 4 University of Wisconsin–Madison, Department of Pathobiological Sciences and Global Health Institute, 1656 Linden Drive, Madison, WI 53706 USA ABSTRACT The diagnosis of bacterial disease in freshwater unionid mussels has been hindered by a lack of baseline information regarding the microbial communities associated with these animals. In this study, we cultured and identified bacteria from the hemolymph of stable mussel populations from Wisconsin portions of the upper Mississippi River basin and compared the results to those from mussel populations experiencing a mortality event in the Clinch River in Virginia and Tennessee. Several bacterial genera were consistently identified across mussel species and locations, appearing to be part of the natural bacterial flora. One noteworthy bacterial species identified from the Clinch River was Yokenella regensburgei, which occurred in relatively high prevalence during the mortality event but was absent from samples acquired afterward. Its role in the mortality event, if any, is unknown but deserves further investigation. We suggest that future studies of freshwater mussel health incorporate hemolymph as a sample type due to its relative separation from the aquatic environment, its role in the circulatory system, and the fact that it can be collected nonlethally. KEY WORDS: freshwater mussel, Unionidae, microflora, bacteriology, hemolymph, disease INTRODUCTION emergence of microbiome research examining correlations Freshwater mussels are exposed to the microorganisms that between bacterial and archaeal communities and health and they filter and accumulate from the aquatic environment. resilience across a variety of animal species (e.g., gut biota of Bacteria are a food source, but also can be found in body humans and fish, livestock, etc.; see Ingerslev et al. 2014; tissues outside of the gut, including the hemolymph, in Ghanbari et al. 2015; Reese et al. 2018; Trinh et al. 2018). In apparently healthy animals (Starliper et al. 1998, 2008; mussels, bacterial diversity in fluids and tissues has been Antunes et al. 2010). In general, the characteristic bacterial associated with healthy, responsive animals (Starliper et al. flora of freshwater mussels is largely unknown, despite the 2008), whereas high bacterial loads have been associated with sick or moribund animals (see Sparks et al. 1990, Starliper et *Corresponding Author: [email protected] al. 2011). 70 Downloaded From: https://bioone.org/journals/Freshwater-Mollusk-Biology-and-Conservation on 03 Jan 2020 Terms of Use: https://bioone.org/terms-of-use BACTERIA CULTURED FROM FRESHWATER MUSSEL HEMOLYMPH 71 Historically, mass mortality events of freshwater mussels Virginia. Our primary objective was to determine the have occurred that suggest infectious causes (Neves 1987), but community composition of the culturable bacteria present in most cases, the causative agent has not been identified (see within these populations and the prevalence of specific taxa. review in Grizzle and Brunner 2009). While samples may be collected and analyzed from such events, the lack of data regarding the normal microbiota of healthy mussel populations METHODS has made it difficult to identify potential pathogens as well as We collected a variety of freshwater mussel species on June other potential commensal or mutualistic relationships (Star- 16, 2017, August 23, 2017, August 29, 2017, October 6, 2017, liper et al. 1998; Starliper 2008). The reports by Starliper, and October 26, 2017, from the Wisconsin stretches of the La focused on populations from the southeastern USA (Starliper Crosse River (43854052.5100 N, 9184034.9300 W), Chippewa River et al. 1998; Starliper and Morrison 2000; Starliper 2001, 2005, (44845038.8000 N, 91840044.8000 W), Lake Onalaska (a backwater 2008, 2011; Starliper et al. 2008, 2011), constitute much of the lake of Pool 7 of the UMR; 43853045.8500 N, 91816010.4100 W), knowledge base regarding the bacterial communities associ- Black River (43852018.6500 N, 91814042.3900 W), and Goose ated with freshwater mussels. To develop much-needed Island (Pool 8 of the UMR; 43844046.2000 N, 91813034.1700 W), specific diagnostic assays, we must better understand mus- respectively (Fig. 1). We obtained samples from Pheasantshell sel-microbe interactions and identify pathogens, tasks feasible (Actinonaias pectorosa) mussels from the Tennessee reaches only with bacteriology data from diverse unionid species (Wallen Bend, 3683502.6500 N, 8380049.9600 W; Kyle’s Ford, across broader geographic regions. 36833057.0500 N, 8382029.5700 W) of the Clinch River on Standardized diagnostic methods for freshwater fish typi- November 2, 2017, during an active mortality event (Richard cally utilize the kidney for the collection of bacteriological 2018) and postmortality event on February 1, 2018, again from samples (USFWS and AFS–FHS 2012) due to its function as a Kyle’s Ford as well as Sycamore Island (Virginia, filtration organ. However, similar methods are lacking for 36837016.3600 N, 8284906.2000 W) (Fig. 1). Mussels were hand- freshwater mussels, primarily because of limited attention to the collected and held in source water until a sufficient sample size diseases of these taxa (Grizzle and Brunner 2009). Typically, (~20–30) was acquired for each site. During the postmortality previous bacteriology studies of unionids have utilized lethal sampling event, we processed approximately half of the sampling to collect fluids and mixed tissue or whole body Pheasantshells in the field and, due to inclement weather, homogenate samples (Starliper et al. 1998, 2008, 2011; Starliper transported the others in source water to the laboratory, where and Morrison 2000; Starliper 2001, 2005), while others they were held (up to 72 h) before sampling. Following compared the microbiota between specific tissues (Sparks et collection, we used either a reverse pliers or a child’s nasal al. 1990; Chittick et al. 2001; Nichols et al. 2001; Antunes et al. speculum and stopper to open the shell slightly. The anterior 2010). Although bacteria were cultured from most tissue types, adductor mussel was cleaned using an individually wrapped interpretations are confounded by lack of organ specificity sterile rayon swab soaked in 70% isopropyl alcohol. We (mixed tissue or whole body homogenates) as well as the risk to withdrew approximately 100 lL of hemolymph from the sample integrity due to the closeness of the internal organs to anterior adductor muscle using a 1 mL insulin needle and the aquatic environment and the disinfection procedures used to syringe. The hemolymph sample was then sinuously streaked reduce contamination. Moreover, whole body and soft tissue onto a tryptic soy agar (TSA) plate using a sterile inoculation samples generally require sacrifice of the mussel, which should loop. TSA plates were incubated for 1 wk in a 218Cincubator. be avoided, especially for imperiled fauna. Bacterial colonies with unique macroscopic morphologies were A sample type that has received less attention in the sampled from each plate using a sterile bacteriology loop and assessment of freshwater mussel health is hemolymph (Sparks placed in sterile 2.0 mL microconical screw-cap collection et al. 1990; Starliper 2008; Antunes et al. 2010). This fluid, tubes.
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