Freshwater Mussel Viromes Increase Rapidly in Diversity and Abundance When Hosts Are Released from Captivity into the Wild

Abstract

Freshwater mussels (order: Unionida) are highly imperiled globally and are increasingly the focus of captive propagation efforts to protect and restore wild populations. The Upper Tennessee River Basin (UTRB) in Virginia is a freshwater biodiversity hotspot hosting at least 45 of North America's ~300 species of freshwater mussels, including 21 threatened and endangered species listed under the U.S. Endangered Species Act. Recent studies have documented that viruses and other microbes have contributed to freshwater mussel population declines in the UTRB. We conducted a multi-year longitudinal study of captive-reared hatchery mussels released to restoration sites throughout the UTRB to evaluate their viromes and compare them to captive hatchery environments. We documented 681 viruses from 27 families. The hatchery mussels had significantly less viruses than those deployed to wild sites, with only 20 viruses unique to the hatchery mussels. After the hatchery mussels were released into the wild, their number of viruses initially spiked and then increased steadily over time, with 451 viruses in total unique to the mussels in the wild. We found Clinch densovirus 1 (CDNV-1), a virus previously associated with mass mortality events in the Clinch River, in all samples, but the wild site mussels consistently had significantly higher CDNV-1 levels than those held in the hatchery. Our data document substantial differences between the viruses in the mussels in the hatchery and wild environments and rapid virome shifts after the mussels are released to the wild sites. These findings indicate that mussel release programs might benefit from acclimatization periods or other measures to mitigate the potential negative effects of rapid exposure to infectious agents found in natural environments.

Keywords: aquaculture; conservation; die-off; endangered species; freshwater mussel; mass mortality event; restoration; unionid; virome; viruses.

Figure 1

Map of sampling sites and the Aquatic Wildlife Conservation Center (AWCC) mussel hatchery used in the 2020–2021 mussel silo study. Abbreviations used in text are CP (Clinchport, Clinch River, mile 213.2); SYC (Sycamore Island, Clinch River, mile 206.9); POW (Powell River, mile 120.2), BEN (Clinch River, mile 278.1), NFH (North Fork Holston River, mile 97.2), and MFH site (Middle Fork Holston River, mile 8.3).

Figure 2

KRONA diagram showing taxonomy of the 681 viruses observed in juvenile freshwater mussels deployed in sites from the Upper Tennessee River Basin. Groups representing <0.4% of all viruses are collapsed and shown with cross-hatch shading.

Figure 3

Boxplots showing average number of viruses by sample site (Above) and cumulative viral intensity (Below). Boxes depict median and first and third quartiles for each group. Upper whiskers represent the smaller of either the maximum observed value of 1.5× the 3rd quartile. Lower whiskers represent the larger of either the minimum observed value or 1.5× the 1st quartile subtracted from the value of the 1st quartile. Letters and colors depict results from Tukey HSD analysis. Boxes with the same letters and colors are not statistically different (i.e., adjusted p-value ≥ 0.05).

Figure 4

Non-metric multidimensional scaling (NMDS) plot showing virome results for all sequencing pools included in the Upper Tennessee River Basin mussel silo study. For both years, there was a clear separation of viromes in the mussels held at the hatchery (hollow inverted triangles; Aquatic Wildlife Conservation Center, AWCC) from those deployed to river sites (all other shapes). River viromes clustered largely by sample site. All three sample sites within the Clinch River (SYC, CP, and BEN) largely overlapped, while sites from the Middle Fork Holston River (MFH), North Fork Holston River (NFH), and Powell River (POW) did not overlap with samples from any other rivers.

Figure 5

Changes in freshwater mussel virome composition at two Clinch River sites over 4 sampling intervals in 2020. Top row: number of viruses. Second row: virome turnover plots showing total turnover rate (light blue line), disappearance rate (orange line), and appearance rate (green line). Third row: mean rank shifts representing the degree of virus abundance reordering between time points. Fourth row: rate of virome community change. Note that the top plot includes five values, including the baseline value from the outset of the study, while all other plots contain four values, representing observed changes from the previous interval.

Figure 6

Changes in freshwater mussel virome composition at 4 river sites (Clinch River sites BEN and SYC, Middle Fork Holston River (MFH), North Fork Holston River (NFH)), and the Aquatic Wildlife Conservation Center (AWCC) hatchery over 3 sampling intervals in 2021. Top row: number of viruses. Second row: virome turnover plots showing total turnover rate (light blue line), disappearance rate (orange line), and appearance rate (green line). Third row: mean rank shifts representing the degree of virus abundance reordering between time points. Fourth row: rate of virome community change. Note that the top plot includes four values, including the baseline value from the outset of the study, while all other plots contain three values, representing observed changes from the previous interval.