Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water?

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water? © The Author(s) 2021 Published version Hajisafarali, Mahsa; Aaltonen, Sari; Pulkkinen, Katja; Taskinen, Jouni Hajisafarali, M., Aaltonen, S., Pulkkinen, K., & Taskinen, J. (2022). Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water?. Hydrobiologia, 849(4), 1067-1081. https://doi.org/10.1007/s10750-021-04769-6 2022 PRIMARY RESEARCH PAPER Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water? Mahsa Hajisafarali .Sari Aaltonen .Katja Pulkkinen .Jouni Taskinen Received: 3 February 2021 / Revised: 19 November 2021 / Accepted: 22 November 2021 ÓThe Author(s) 2021 Abstract Global decline of freshwater mussels (Unionoida) is threatening biodiversity and the essential ecosystem services that mussels provide. As filterfeeding organisms, freshwater mussels remove phytoplankton and suspended particles from the water. By filtering bacteria, freshwater mussels also decrease pathogen loads in the water. The objective of this study was to evaluate whether the common freshwater bivalve Anodonta anatina (duck mussel) could remove the bacterial fish pathogen Flavobacterium columnare from the water. Mussels reduced bacteria in both of the two experiments performed, so that the bacterial concentration at the end of the 96-h monitoring in mussel treatments was only 0.3–0.5 times that of the controls. Surprisingly, mussels did not reduce algal cell concentration statistically significantly. Mussel behavior (shell openness, foot position, and movement) was not affected by the presence of bacteria or algae, except for biodeposition formation, which was greatest in algal-fed treatments, followed by bacterial-fed treatments and controls, respectively. The intestines of bacteria-incubated A. anatina harbored F. columnare, suggesting that mussels ingested the bacteria. Present results suggest that freshwater mussels may also have a potential to mitigate aquaculture pathogen problems, as well as play a role in water quality management. Keywords Biofiltration Columnaris disease Duck mussel Unionidae Bacteria Ecosystem services Introduction Freshwater mussels (Unionoida) are one of the most threatened animal group in the world (Lydeard et al., 2004; Lopes-Lima et al., 2017). In the recent decades, a number of freshwater mussel species have gone extinct (Haag & Williams, 2014). Mussel populations are declining due to pollution, habitat degradation and fragmentation, introduced species, and the loss of obligatory fish hosts (Bogan, 2008; Geist, 2010; Ferreira-Rodrı ´guez et al., 2019), reducing the biological diversity in lakes and rivers. Freshwater mussels carry out important ecosystem services via their filtration and burrowing activity, including nutrient recycling and the deposition of organic matter from the water column to the bottom in the form of feces and pseudofeces (e.g., Vaughn, 2018). The Handling editor: Eric R. Larson Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/ s10750-021-04769-6. M. Hajisafarali (&)S. Aaltonen  K. Pulkkinen J. Taskinen Department of Biological and Environmental Science, University of Jyva ¨skyla ¨, P.O. Box 35, 40014 Jyva ¨skyla ¨n, Finland e-mail: [email protected] 123 Hydrobiologia https://doi.org/10.1007/s10750-021-04769-6(0123456789().,-volV)(0123456789().,-volV) biodeposition of feces and pseudofeces (the noningested particles loosely wrapped in mucus and ejected without undergoing the digestive process; Berg et al., 1996) by freshwater mussels provides a nutrient-rich and easily assimilated food source for benthic microbes and invertebrates, which supports fish populations and links multiple trophic levels in aquatic ecosystems (Howard & Cuffey, 2006; Vaughn et al., 2008; Vaughn, 2018). Therefore, in addition to biodiversity reduction per se, the decline of freshwater mussels can have a marked effect on the ecosystem. Bivalves’ potential role in water quality management, their ‘‘bivalve environmental services’’, has already been recognized in marine systems (e.g., nutrient trapping connected with mussel production) (Nielsen et al., 2016; Clements & Comeau, 2019; Taylor et al., 2019; Kotta et al., 2020). Mussels’ effectiveness in nutrient and particle removal and in wastewater treatment has also been shown in freshwater systems (Strayer, 2014; Lummer et al., 2016; Mezzanotte et al., 2016; Hoellein et al., 2017; Kreeger et al., 2018). Thus, freshwater mussels can potentially be utilized in water quality management to reduce nutrient effluents and eutrophication (Kreeger et al., 2018). Depending on their ability to remove bacteria, freshwater mussels could also be used to reduce disease outbreaks. Several researchers have found that the dreissenid mussel Dreissena polymorpha (Pallas, 1771) can clear the pathogenic bacterium Escherichia coli (Migula, 1895) Castellani & Chalmers, 1919 from water in laboratory experiments (Silverman et al., 1995), in field conditions (Cotner et al., 1995), and from treated effluents of municipal waste water (Mezzanotte et al., 2016). Unionidae freshwater mussels are also capable of filtering bacteria (Jørgensen et al., 1984; Kryger & Riisga ˚rd, 1988; Nichols & Garling, 2002; Christian et al., 2004Vanderploeg et al., 2011b), including E. coli (Silverman et al., 1995; Ismail et al., 2015). However, the ability of freshwater mussels to eliminate bacterial fish pathogens has not been investigated. The aim of the present study was to investigate the capability of the duck mussel, Anodonta anatina (Linnaeus, 1758), to remove the bacterium Flavobacterium columnare Bernardet & Grimont 1989 from the water column in laboratory conditions. Anodonta anatina is a unionid mussel that commonly occurs in the lakes and rivers of Europe (Lopes-Lima et al., 2016). This mussel has been shown to filter Diplostomum pseudospathaceum Niewiadomska, 1984 larvae from water, thereby reducing the transmission of this trematode parasite from aquatic snails to its fish host (Gopko et al., 2017). More importantly, A. anatina has the potential to filter and ingest colonial and filamentous cyanobacteria (Bontes et al., 2007). Flavobacterium columnare is a freshwater bacterium and an opportunistic fish pathogen that causes columnaris disease (‘‘warm water disease’’) in fish. Flavobacterium columnare can infect a range of fish species throughout the world, such as salmon, trout, rainbow trout, channel catfish, carp, perch, pike, eel, and tilapia (Declercq et al., 2013). Columnaris disease is one of the most harmful diseases in channel catfish, Ictalurus punctatus (Rafinesque, 1818) farming in the USA, leading to approximately $30 million yearly losses (Wagner et al., 2006). It is also a very difficult problem in young salmonid culture, causing high mortality and the use of antibiotics (Pulkkinen et al., 2010). This pathogen can also be found in natural lakes and rivers (Kunttu et al., 2012) and has been isolated from tissues of the unionid mussel Villosa iris (I.Lea, 1829) (Clinch River, USA; Starliper et al., 2008). Our hypothesis was that A. anatina would be able to remove F. columnare from the water, manifested as a lower average bacterial concentration in the water with mussel treatment as compared to the control water without mussel. As a methodological control for the feeding of mussels, we established a treatment group fed with a commercial microalgae product commonly used for mussel feeding. As the bacterial suspension may affect the behavior of mussels, especially the filtration activity, monitoring of mussel behavior was included to the study. In addition, as the biodeposit formation can indicate filtration of matter from water, biodeposition formation was also monitored during the experiment. The present study is the first effort to evaluate the efficiency of freshwater mussels to remove the harmful bacterial fish pathogen from the water. Materials and methods Mussel collection and preparation In total, 30 A. anatina individuals (Table 1) were collected from Lake Koija ¨rvi, Finland (60.97°N, 123 Hydrobiologia 23.73°E) during September 2016. The mussels were transported to Konnevesi Research Station, University of Jyva ¨skyla ¨and were kept in a 163 L flow-through tank with sand on the bottom until the experiments began. The mussels were not fed, but they received food from incoming Lake Konnevesi water during the maintenance period. In January 2017, the mussels were transported to the laboratory of the Department of Biological and Environmental Science, University of Jyva ¨skyla ¨. The mussels were placed in a large bucket of well water, aerated with an aquarium air pump, and kept on a 12 h light–dark cycle at *17 ° C. In the laboratory during the pre-experiment maintenance, the mussels were fed ad libitum with a commercial phytoplankton product (Shellfish Diet 1800Ò, SFD, Reed Mariculture, USA) containing six marine microalgae: Isochrysis sp., Pavlova sp., Tetraselmis sp., Chaetoceros calcitrans (Paulsen) H.Takano, Thalassiosira weissflogii (Grunow) G.A.Fryxell & Hasle, and Thalassiosira pseudonana Hasle & Heimdal.The mussels were kept without food for 24 h before starting the experiments until they no longer released excrement. The water temperature at Konnevesi Research Station during the maintenance period was initially 17 °C in September; it gradually declined to 3 °C in December 2016, but it was artificially gradually increased to 17 °C in the last two weeks before the experiments, which were conducted at 17 °C. Preparation of bacterial suspension, Shieh medium and agar plates The F. columnare strain (B613) used in the experiments was isolated from a water sample taken from the shore of Lake Kynsivesi (62°2500 N, 26°1500 E) in 2014; it was then cultivated in a modified Shieh medium (Song et al. 1988) and stored at -80 °C with 10% glycerol and 10% fetal calf serum. In order to prepare the bacterial solution for the experiments, 20 ll from the frozen stock was added to 5 ml of modified Shieh medium and incubated with continuous stirring at 120 rpm for 24 h at room temperature. One ml of the overnight-grown bacterial suspension was further added to 9 ml of modified Shieh medium, with another incubation at room temperature overnight. The optical density (OD) of the bacterial suspension was measured with a spectrophotometer at 595 nm and converted to a bacterial concentration using a preestablished relationship between OD and colonyforming units (CFUs); this was done to allow the researchers to adjust the initial bacteria concentration levels during the experiments. Modified Shieh medium was prepared according to Song et al. (1988) to serve as a selective growth medium to isolate F. columnare. Agar plates were used to plate-count the viable bacterial concentrations; here, 10 g of agar powder were added per liter and supplemented with tobramycin in order to inhibit the growth of bacteria other than F. columnare (Decostere et al., 1997). Experiment 1 The first experiment to evaluate A. anatina’s ability to remove F. columnare from water began in February 2017 and included 5 treatments with 5 replicates each (replicate = 1 sediment/sand-free aquarium filled with 5 L of well water with or without a mussel individual; see below). A schematic presentation of the experimental design is given in the Supplementary Fig. 1. The aquaria were aerated and kept on a light–dark cycle of 12 h:12 h at *17 °C. The experimental treatments were as follows: Table 1 The number of mussels (Anodonta anatina), average length (mm, mean ±SD) and age (y, mean ±SD), sex (F= female, M= male), intensity of trematode infection (Rhipidocotyle fennica) (zero, light, moderate, heavy; see text for categorization), and occurrence of glochidia in mussels used in the 1st and 2nd experiment Number of mussels Length Age Sex Trematode Glochidia Mean Mean F M Unknown Zero Light Moderate Heavy Number 1st experiment 15 83 ±4 5.6 ±16 4 5 1 4 4 6 0 2nd experiment 9 87 ±4 6.1 ±11 4 4 2 2 2 3 0 123 Hydrobiologia (1) Bacteria and mussel: A known concentration (5 910 5 CFU ml -1 )ofF. columnare with an individual mussel to evaluate the potential of A. anatina to filter out F. columnare. (2) Bacteria control: A known concentration (5 910 5 CFU ml -1 )ofF. columnare without a mussel was used to study changes in the bacterial concentration of the water in the absence of the mussel. The null hypothesis was that there was no difference in bacterial concentration between treatments (1) and (2). (3) Algae and mussel: A known concentration of algae with a mussel; this served as a reference treatment to study A. anatina’s algal filtration. (4) Algae control: A known concentration of algae without a mussel was used to track changes in algae concentration in the water in the absence of the mussel. In treatments (3) and (4), 35 ml of Shellfish Diet 1800Òwas added to 1.1 L water; then, 100 ml of this suspension was added to each aquarium. The null hypothesis was that there was no difference in algae concentration between treatments (3) and (4). (5) Mussel control: A mussel without F. columnare and without algae was used to study mussel behavior without the presence of bacteria and algae. The null hypothesis was that there was no difference in behaviors between treatments (1), (3), and (5). Experiment 2 The second experiment began in March 2017 to verify the results of Experiment 1 in regard to bacterial filtration; it used a bacterial inoculation that was an order of magnitude lower than in Experiment 1 (see the Results section and Fig. 1). A schematic presentation of the experimental design is given in the Supplementary Fig. 2. The aquaria were aerated and kept on a light–dark cycle of 12 h:12 h at *17 °C. The experimental treatments were as follows: (1) Bacteria and mussel: A known concentration of F. columnare with an individual mussel to evaluate A. anatina’s potential to filter F. columnare; 9 replicate aquaria with a single mussel in each were used. (2) Bacteria control: A known concentration of F. columnare without a mussel was used to study changes in bacterial concentration in the absence of the mussel; 5 replicate aquaria were used. The null hypothesis was that there was no difference in bacterial concentration between treatments (1) and (2). Bacterial sampling from the water Water samples were taken from all the aquaria prior to starting each experiment to exclude the possibility of prior contamination by F. columnare. The concentration of bacteria was evaluated from 100 ll water samples from treatments with added bacteria (bacteria and mussel, bacteria control) at time points 0 h, 6 h, and 24 h; 1 sample per aquarium was evaluated. In order to decrease the observational error associated with reducing bacterial levels at time points 48 h and 96 h, the number of repeated water samples taken per aquarium was increased to three. In Experiment 2, 100 ll of water was taken at time points 0 h, 48 h, and 96 h; 2 repeated samples were taken per aquaria. A dilution series (10 -1 –10 -7 ) of each water sample was prepared and cultured on Shieh agar plates; colony formation was checked after 48 h. At the end of both experiments, treatments without bacteria were checked as described above (2 9100 ll water samples per aquarium) to exclude potential contamination from added bacteria during the experiment. Bacterial sampling from intestines and gonads To examine if mussels can ingest bacteria and become infected with F. columnare, bacterial samples were taken from intestine and gonad of the mussels from all treatments at the end of both experiments. To avoid bacterial contamination from the aquarium water, mussel shell surfaces were sprayed with 70% ethanol before dissection. After opening the valves, the mussel body was cut in 2 so that a cross-section through the gonads—and the intestine winding through the gonads—could be accessed. Then, the bacterial samples were collected with a sterile loop and cultured on Shieh agar plates supplemented with tobramycin. In Experiment 1, bacterial sampling was done first from the intestine and then from the gonads. In Experiment 2, the sampling order was reversed to minimize 123 Hydrobiologia possible contamination of the gonads from the intestine. Algal cell sampling To investigate A. anatina’s filtration capability, algal cell concentration was monitored from 3 replicate water samples per aquarium at different time points (0 h, 6 h, 24 h, 48 h) using a microscope counting chamber (Bu ¨rker, Marienfeld, Germany, depth = 0.1 mm, square width = 0.25 mm). After 24 h, algal cell concentration declined sharply in both treatments, and monitoring was discontinued. Mussel behavior In order to check the possible effect of the bacterial and algal diets on the mussels’ behavior (for example, a complete inactivation of mussels when incubated with bacteria), in both experiments, A. anatina behavior was recorded 3 times per day at 9:00, 12:00, and 15:00 h in a 2 min visual inspection for 99 h. The traits that were monitored included shell openness, foot position (extension or contraction), movement, and biodeposition (feces and pseudofeces) formation since the previous monitoring. Shell openness was scored as follows: fully closed (siphons completely invisible) = 0, slightly opened (siphons are still invisible) = 1, half open (siphons visible, partly protruding) = 2, fully open (siphons fully visible, fully protruding) = 3. Foot position was graded as follows: foot in = 0, foot partly out = 1, foot out = 2. Movement (position changes in the mussel) was monitored by comparing mussel position to a mark on the lid of each aquarium denoting the position at the previous inspection. It was scored as follows: movement = 1, no movement = 0. Biodeposition presence was classified into 2 categories: Fig. 1 Bacterial concentration (CFU ml -1 ) (A) in aquaria with mussels and without mussels at different time points (N = 5) (mean ±SD) in the 1st experiment, in the 2nd experiment (B), and algal cell concentration (cell ml -1 )(C) in aquaria with mussels and without mussels at different time points (N = 5) (mean ±SD) 123 Hydrobiologia without biodeposition = 0, and with biodeposition = 1. Trematode parasitism, sex, and reproduction of mussels Possible trematode parasitism (parasite species, intensity of infection) in the mussels was examined at the end of each experiment when the bacterial sampling from the intestine and gonads was performed; this was done because trematodes can have a marked influence on mussel physiology (Taskinen, 1998; Jokela et al., 2005). The gonads are the main site of trematode infection in A. anatina (Taskinen et al., 1997). Therefore, a 2–3 mm slice across the gonads was dissected after the bacterial sampling was conducted, pressed between large glass plates, and examined microscopically for trematode sporocysts using transmitted light. This was accompanied with the sex determination of the mussel and an estimation of the intensity of the infection (Taskinen et al., 1994; Taskinen & Valtonen, 1995). The intensity of the trematode infection was scored as follows: uninfected = 0, light infection (one sporocyst tubule or one group of sporocyst tubules) = 1, moderate infection from [1 group of sporocyst tubules to almost complete replacement of mussel gonad tissue by sporocysts = 2, and heavy infection (gonad tissue completely replaced by trematode sporocysts) = 3 (Taskinen et al., 1994). Statistical analyses The statistical analyses of bacterial and algal concentrations were performed using IBM SPSS software (version 24.0.0). Cell concentrations (the CFUs of F. columnare and the counts of algal cells) were used as response variables; aquaria were used as the statistical units. General Linear Model (GLM) repeated measures analyses were performed to compare differences in bacterial and algal concentration in time (the within-subject factor) between treatments with and without a mussel (the between-subject factor). The differences between different time points were further examined using within-subject contrast (‘‘simple’’). Levene’s test was used to assess the homogeneity of variance, and the Greenhouse–Geisser correction was applied to correct the number of degrees of freedom when the sphericity assumption was not valid. Differences in mussel behavior (shell openness, foot position, movement, biodeposition formation) at different time points between the treatments (mussel only, mussel ?bacteria, mussel ?algae) were analyzed using mixed effects logistic regression (GLMM) in R (version 4.0.2.) statistics software and Lme4 package (R Development Core Team, 2015). The interdependence of repeated observations from the same individual mussel during the experiment was accounted for by treating mussel identity as a random variable. To be able to fit the models using binomial (logit) distribution, all behavioral data were recoded as 0 or 1 before the analysis. Thus, shell openness was recategorized as 0 (valves fully closed) and 1 (valves slightly, half or fully open), and foot position as 0 (foot in) and 1 (foot partly or completely out). The differences in the intensity of trematode infection between treatment groups in Experiment 1 scored from zero (uninfected) to 3 (heavily infected) could not be tested with a Chi-square test because expected values were less than 5. Instead, we used Fishers exact test extended to 3 93 contingency table (McDonald , 2014). In addition, the difference in the mean intensity of the trematode infection was tested between the ‘‘bacteria and mussel’’ treatments in both experiments with a t-test. Differences between treatments were considered significant if p\0.05. Results Effect of mussel filtration on bacterial concentration (Experiment 1) At the beginning of Experiment 1, the mean concentration of bacteria was similar in both treatment groups with F. columnare (Fig. 1A). During the next 48 h, the bacterial concentration increased in both treatments, but the increase was more pronounced in the treatment without a mussel than in the treatment with a mussel. Between 48 and 96 h, the bacterial concentration decreased in both treatments (Fig. 1A). The bacterial concentration was lower in the treatment with a mussel than in the treatment without a mussel (Table 2, between-subject effects, F 1,8 = 16.31, p= 0.004). However, this difference became significant only after 96 h of the experiment, as indicated by the significant interaction between time and treatment and the contrasts between the levels of the within-subject 123 Hydrobiologia factor and treatment (time x treatment) (Table 2). Thus, it took 48 h from the beginning of the experiment before the removal of the bacteria by the mussels was apparent (Fig. 1A). Thereafter, the mussels reduced the bacteria so that the bacterial concentration in mussel treatment was 0.7–0.5 times that of the controls at 48 and 96 h, respectively (Fig. 1A). Thus, the mussels removed half the bacteria within 96 h as compared to the controls. Effect of Mussel Filtration on Bacterial Concentration (Experiment 2) In Experiment 2, a similar pattern in bacterial concentrations to those in Experiment 1 was observed. The bacterial concentrations were similar in the beginning of the experiment in the treatments with and without a mussel, respectively (Fig. 1B). At 48 h, the mean concentration of F. columnare had increased in both treatments, but with a mussel the increase was approximately half of that in the treatment without a mussel. Between 48 and 96 h, the bacterial Table 2 Results of GLM repeated measures ANOVA for the mean concentration of Flavobacterium columnare (CFU ml -1 ) in the two treatments (with and without mussel) at different time points in the 1st and 2nd experiment Source First experiment Second experiment df MS F pdf MS F p Between subjects Intercept 1 2.95E?12 654.47 \0.001 1 4.75E?11 180.30 \0.001 Treatment 1 7.36E?10 16.31 0.004 1 489E?10 18.55 0.001 Error 8 3.61E?10 12 2.63E?09 Within subjects Time 2.335 1.78E?12 61.63 \0.001 2 2.83E?11 61.17 \0.001 Time 9treatment 2.335 1.52E?11 a 5.26 0.012 2 2.74E?10 5.91 0.008 Error 18.676 2.88E?10 a 24 4.64E?09 Within-subject contrasts b Time Time 6 h vs time 0 h 1 8.26E?09 3.79 0.087 Time 24 h vs. time 0 h 1 1.55E?12 47.93 \0.001 Time 48 h vs. time 0 h 1 5.95E?12 139.97 \0.001 1 9.09E?11 107.46 \0.001 Time 96 h vs. time 0 h 1 2.11E?12 61.64 \0.001 1 4.22E?09 0.46 0.511 Time 9treatment Time 6 h vs time 0 h 1 6.76E?07 0.03 0.865 Time 24 h vs. time 0 h 1 1.37E?09 0.04 0.842 Time 48 h vs. time 0 h 1 1.72E?11 4.06 0.079 1 1.09E?11 12.92 0.004 Time 96 h vs. time 0 h 1 4.19E?11 12.21 0.008 1 3.36E?10 3.64 0.081 Error Time 6 h vs time 0 h 8 2.18 E?09 Time 24 h vs. time 0 h 8 3.23 E?10 Time 48 h vs. time 0 h 8 4.25 E?10 12 8.46 E?09 Time 96 h vs. time 0 h 8 3.43 E?10 12 9.22 E?09 Df = degrees of freedom, MS = mean squares a Greenhouse–Geisser corrected values b Within-subject contrast for the interaction between time (hour) and treatments. The contrast is ‘‘simple’’, which compares each time point to the first time point (time 0 h). Note that for the 2 nd experiment measurements were made at time points 0, 48 and 96 h 123 Hydrobiologia concentrations decreased in both treatments (Fig. 1B). The bacterial concentration was lower in the treatment with a mussel than in the treatment without a mussel (Table 2, between-subject effects, F 1,12 = 18.55, p= 0.001). Contrasts between the levels of the within-subject factor and treatment (time x treatment) indicated that this difference was significant after 48 h but not after 96 h of beginning the experiment (Table 2). Mussels reduced the bacterial concentration in mussel treatment to 0.5 and 0.3 times that of the controls at 48 and 96 h, respectively (Fig. 1B). Thus, the mussels removed two-thirds of the bacteria within 96 h as compared to the controls. Gonad and Intestine Analysis (Experiments 1 and 2) In Experiment 1, F. columnare was isolated both from the mussels’ intestines and gonads in the treatment with added bacteria, but not in the other treatment groups (i.e., the mussels with algae and the mussels kept in clean water). At the individual (mussel) aquarium level, among the 5 replicates of mussel ?bacterium treatment, 3 mussels had F. columnare in both the intestine and the gonads, 1mussel had F. columnare in the intestine but not in the gonads, and 1 tested negative (no bacterium in either the intestine or in the gonads). In Experiment 2, among the 9 replicates of mussel and bacteria, F. columnare was only found in the intestines of 8 individuals; it was not found in the gonads. Effect of Mussel Filtration on Algal Cell Concentration (Experiment 1) The mean concentration of algal cells (Shellfish Diet) decreased between the start of the experiment and 48 h in both treatments with and without a mussel (Fig. 1C, Table 3, within-subject effect [time], F 3,24 = 83.20, p= 0.001). However, the presence of A. anatina did not affect algal cell concentration (Table 3, F1,8 = 1.872, p= 0.208). Mussel Behavior Shell openness (Experiments 1 and 2) In both experiments, the mussels in all treatments had open or partly open valves at the beginning of the experiment. The mussels gradually closed their valves during the first 24 h but started opening their shells again toward the end of the experiment (Fig. 2A and E). In Experiment 1, the presence of bacteria did not affect the mussels’ shell openness as compared to the mussels with algal cells or those in clean water (GLMM, p [0.05, Table 4). Foot position (Experiments 1 and 2) Foot position scores revealed that the mussels kept their feet substantially contracted (Fig. 2B and F). In Experiment 1, the bacterial presence did not affect the foot position as compared to the mussels exposed to the algae or those kept in clean water (GLMM p[0.05, Table 4). Movement (Experiments 1 and 2) Anodonta anatina in all treatment groups in both experiments exhibited a continuous and coherent pattern in movement behavior throughout the experiment (Fig. 2C and G). The presence of bacteria had a slight effect in decreasing mussels’ movement activity (GLMM, the relative change in comparison to mussel only treatment 0.69 ±0.36, p = 0.053, Table 4), while the movement activity of mussels with algae was lower than mussels in clean water (GLMM, the relative change in comparison to mussel only treatment 0.75 ±0.36, p = 0.036, Table 4). Table 3 Results of GLM repeated measures ANOVA for the mean of algal cell concentration (cell ml -1 ) in the two treatments with and without mussel at different time points. Df = degrees of freedom, MS = mean squares Source df MS Fp Between subjects Intercept 1 3.68E?13 1681.82 \0.001 Treatment 1 4.10E?10 1.87 0.208 Error 8 2.19E?10 Within subjects Time 3 2.46E?12 83.16 \0.001 Time 9treatment 3 8.33E?10 2.82 0.061 Error 24 2.96E?10 123 Hydrobiologia subadult unionidae. Journal of Shellfish Research 21: 875–881. Nielsen, P., P. Cranford, M. Maar & J. Petersen, 2016. Magnitude, spatial scale and optimization of ecosystem services from a nutrient extraction mussel farm in the eutrophic Skive Fjord, Denmark. Aquaculture Environment Interactions 8: 311–329. Pulkkinen, K., L.-R. Suomalainen, A. F. Read, D. Ebert, P. Rintama ¨ki & E. T. Valtonen, 2010. Intensive fish farming and the evolution of pathogen virulence: the case of columnaris disease in Finland. Proceedings of the Royal Society B: Biological Sciences Royal Society 277: 593–600. Rubiolo, J. A., L. M. Botana, & P. Martinez, 2019. Insights into Mussel Microbiome. Microbial Communities in Aquaculture Ecosystems 95–120. R Development Core Team, 2015. R: A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna: Seiderer, L., C. Davis, F. Robb & R. Newell, 1984. Utilisation of bacteria as nitrogen resource by kelp-bed mussel Choromytilus meridionalis. Marine Ecology Progress Series 15: 109–116. Silverman, H., E. C. Achberger, J. W. Lynn & T. H. Dietz, 1995. Filtration and utilization of laboratory-cultured bacteria by Dreissena polymorpha,Corbicula fluminea, and Carunculina texasensis. The Biological Bulletin 189: 308–319. Silverman, H., S. J. Nichols, J. S. Cherry, E. Achberger, J. W. Lynn & T. H. Dietz, 1997. Clearance of laboratorycultured bacteria by freshwater bivalves: differences between lentic and lotic unionids. Canadian Journal of Zoology 75: 1857–1866. Starliper, C. E., R. J. Neves, S. D. Hanlon & P. Whittington, 2008. A survey of the indigenous microbiota (bacteria) in three species of mussels from the Clinch and Holston Rivers, Virginia. Journal of Shellfish Research 27: 1311–1317. Strayer, D. L., 2008. Freshwater mussel ecology: a multifactor approach to distribution and abundance, University of California Press, Berkeley: Strayer, D. L., 2014. Understanding how nutrient cycles and freshwater mussels (Unionoida) affect one another. Hydrobiologia 735: 277–292. Taskinen, J., 1998. Influence of trematode parasitism on the growth of a bivalve host in the field. International Journal for Parasitology 28: 599–602. Taskinen, J., T. Ma ¨kela ¨& E. T. Valtonen, 1997. Exploitation of Anodonta piscinalis (Bivalvia) by trematodes: parasite tactics and host longevity. Annales Zoologici Fennici 34: 37–46. Taskinen, J. & E. T. Valtonen, 1995. Age-, size-, and sexspecific infection of Anodonta piscinalis (Bivalvia: Unionidae) with Rhipidocotyle fennica (Digenea: Bucephalidae) and its influence on host reproduction. Canadian Journal of Zoology 73: 887–897. Taskinen, J., E. T. Valtonen & D. I. Gibson, 1991. Studies on bucephalid digeneans parasitising molluscs and fishes in Finland I. Ecological data and experimental studies. Systematic Parasitology 19: 81–94. Taskinen, J., E. T. Valtonen & T. Ma ¨kela ¨, 1994. Quantity of sporocysts and seasonality of two Rhipidocotyle species (Digenea: Bucephalidae) in Anodonta piscinalis (Mollusca: bivalvia). International Journal for Parasitology 24: 877–886. Taylor, D., C. Saurel, P. Nielsen & J. K. Petersen, 2019. Production characteristics and optimization of mitigation mussel culture. Frontiers in Marine Science Frontiers 6: 698. Tuttle-Raycraft, S. & J. Ackerman, 2018. Does size matter? Particle size vs. quality in bivalve suspension feeding. Freshwater Biology 63: 1560–1568. Vanderploeg, H., J. R. Liebig & T. F. Nalepa, 1995. From picoplankton to microplankton: temperature-driven filtration by the unionid bivalve Lampsilis radiata siliquoidea in Lake St. Clair. Canadian Journal of Fisheries and Aquatic Sciences 52: 63–74. Vaughn, C. C., 2018. Ecosystem services provided by freshwater mussels. Hydrobiologia 810: 15–27. Vaughn, C., S. Nichols & D. Spooner, 2008. Community and foodweb ecology of freshwater mussels. Journal of the North American Benthological Society 27: 409–423. Wagner, B. A., D. J. Wise, L. H. Khoo & J. S. Terhune, 2006. The Epidemiology of bacterial diseases in food-size channel catfish. Journal of Aquatic Animal Health 18: 263–272. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 123 Hydrobiologia