Host fish species of the thick-shelled river mussel (Unio crassus) in Swedish rivers Technical report

Host fish species of the thick-shelled river mussel (Unio crassus) in Swedish rivers Technical report LIFE10 NAT/SE/000046 ( The thick-shelled river mussel brings back life to rivers ) 1

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UnioCrassusforLIFE (LIFE10 NAT/SE/000046) Host fish species of the thick-shelled river mussel Unio crassus in seven Swedish river systems LIFE+ nature project The thick-shelled river mussel brings back life to rivers www.ucforlife.se Authors: Martin Österling, Valentina Zülsdorff & Lea D. Schneider Karlstad University Department of Environmental and Life Sciences - Biology 651 88 Karlstad Sweden E-mail: martin.osterling@kau.se 3

Abstract As part of the LIFE+ project The thick-shelled river mussel brings life back to rivers (LIFE10 NAT/SE/000046), the host fish use and suitability was investigated for the endangered thick-shelled river mussel (Unio crassus) in seven Swedish rivers systems. Fishing was conducted twice a year (2012-2015) during the reproduction season of U. crassus. and 1260 fish individuals were analyzed for encystment of the mussels glochidia larvae on the fish gills. Over 3000 larvae were picked from the fish gills, and determined to the species level using DNA-analyses. Forty percent of the analyzed glochidia larvae were U. crassus mussel larvae. Alburnus alburnus, Cottus gobio, Phoxinus phoxinus were generally considered the most suitable hosts for U. crassus. There was however a high variability in host fish use among river systems. The most suitable host fish species was P. phoxinus and S. trutta in Bråån; A. alburnus and P. phoxinus in Bräkneån; A. alburnus and P. phoxinus in Emån, A. alburnus, Lota lota, C. gobio and Gymnocephalus cernua in Kilaån; G. cernua, A. alburnus and Perca fluviatilis in Storån; A. alburnus and G. cernua in Svärtaån; and P. phoxinus and C. gobio in Tommarpsån. The results have implications for conservation incentives for U. crassus, namely 1) habitat restoration measures can be adapted to the ecological requirements of U. crassus and its local host fish species, 2) fish inventories can be directed towards known host fish species 3) mussel propagation and re-introduction projects gain from the knowledge of host suitability and availability in rivers. Latin names Unio crassus = thick-shelled river mussel Alburnus alburnus = bleak, Abramis brama = common bream, Anguilla anguilla = European eel, Barbatula barbatula = stone loach, Blicca bjoerkna = silver bream, Cottus gobio = bullhead, Esox lucius = pike, Gasterosteus aculeatus = three-spined stickleback, Gymnocephalus cernua = ruffe, Lota lota= burbot, Perca fluviatilis = perch, Phoxinus phoxinus = minnow, Salmo trutta = brown trout, Scardinius erythrophthalmus = common rudd, Squalius cephalus = european chub, Rutilus rutilus = roach, Tinca tinca = tench, Vimba vimba = vimba bream 4

Introduction The thick-shelled river mussel (Unio crassus) used to be abundant in European rivers (BANK ET AL, 2006). Over the last century U. crassus populations have however declined all over its distribution and is classified as protected and endangered in Europe (IUCN Red List; LYDEARD ET AL., 2004), including Sweden (Anonymous, 2015), while it is critically endangered in Germany and Austria (REISCHÜTZ & REISCHÜTZ, 2007, BINOT HAFKE ET AL. 2011). The decline of U. crassus is partly linked to habitat alteration and degradation, as well as predation by the invasive muskrat (Ondatra zibethicus). One proposed local major threat is a lack of host fish (TAEUBERT ET AL. 2012; STOECKL ET AL, 2014). In Sweden, there is almost no knowledge of the host fish suitability. U. crassus has an obligatory one month parasitic larval life stage on the gills of suitable host fish. For that reason, it is important to adapt restoration measures that match the biology of the mussel and its host fish species. The present LIFE+ project The thick-shelled river mussel brings back life to rivers has, as one of its main focuses, to improve the ecological status of the thick-shelled river mussel in Sweden. Restoration measures, such as river re-meandering, bypasschannels and substrate restoration, are meant to improve the habitat conditions and status of U. crassus. In order to design and monitor these restoration measures, it is of outmost importance to study host fish suitability over the mussel s distribution area. Several fish species have been suggested to be suitable hosts for U. crassus in Europe (LOPES- LIMA ET AL. 2016). However, as for large parts of the mussel s distribution, the knowledge of host fish use by U. crassus in Sweden is mainly anecdotal. In the present LIFE+ project, we therefore performed the first large scale inventory on U. crassus host fishes over its Swedish distribution. The aim was to investigate the suitability, composition and importance of host fish for U. crassus in the LIFE+ project rivers. In order to do so, we studied the natural infestation rates on wild fish and performed host fish suitability tests in the laboratory. The aim of this study was to investigate and compare the suitability of fish species to U. crassus in seven rivers all over the mussel s Swedish distribution. Specifically, we aimed to answer the following questions: a. Which fish species are hosts for U. crassus? b. Is there a difference in the natural encystment rates of U. crassus larvae among fish species? c. Does the host fish use differ among rivers? d. Are wild fish with larval encystment also releasing living juvenile mussels? e. How efficient is the different fishing methods? 5

Methods In order to evaluate the host suitability of U. crassus, we investigated natural encystment rates on wild fish. We also collected metamorphosed juveniles from wild fish at the laboratory facility that was built within The thick shelled river mussel brings back life to rivers project at Hemmestorps Mölla. Juvenile Encystment Our investigations included seven river systems, located in the Counties Skåne (Bråån and Tommarpsån Rivers), Blekinge (Bräkneån River), Jönköping (Emån River), Östergötland (Storån River) and Södermanland (Kilaån and Svärtaån Rivers). Fishing was conducted during the reproduction season of U. crassus between May and July during the years 2012-2015. To maximise the catch of fish naturally infested with U. crassus, we fished at locations with the most dense mussel beds in each river. Aiming for a collection of all fish species present at the mussel sites in each river, we used a variety of fishing methods, such as electrofishing, stream nets and fyke nets. Electrofishing (600 V LUGAB, L-600) was performed during the day at sites that could be waded. Stream nets and fyke nets were placed out overnight, both at shallow and deep river sites. To gather information on glochidia encystment rates of naturally infested wild fish, a proportion of the captured fish was preserved in ethanol (95%). Each fish individual was measured for length (± 1 mm), weight (± 1 g) and marked with an individual fin cut before it was stored in 95% ethanol for further gill examination (Figure 1) Figure 1. Marking of P. phoxinus with an individual fin cut. The seven unionoid mussel species that occur in Sweden often co-exist (Table 1). At sites with several mussel species, fish individuals are known to be encysted with a variety of different mussel species larvae. However, it is difficult to distinguish the different mussel species at the larval stage by the eye, and thus DNA-analyses were used for species identification. Prior to DNA analysis, fish gills from every fish species caught were dissected, whereupon the number of mussel larvae attached to the fish gills was counted using microscope binoculars (Figure 2). To determine which fish species U. crassus can use as hosts, a subsample of the glochidia was collected and identified to mussel species using ITS rdna-analyses (nuclear ribosomal internal transcribed spacer region) at the Museum of Natural History, Stockholm (KÄLLERSJÖ ET AL. 2005). 6

Table 1 Presence of mussel species in the project rivers and locations of fishing sites. * Does not occur at the fishing site, but occurs in a side-stream or a nearby lake, 1 several mussels found/ good populations, 2 only few or single mussels found. Stream Mussel species present GPS-coordinates of fishing sites (RT90/NorthEast) Bråån 1U. crassus, 2 U. pictorum, 2 U. tumidus, 1 A. anatina, A. cygnea* 6187823, 1363317 6187942, 1364505 Bräkneån 1M. margaritifera, 1 U. crassus, 2 A. anatina 6229596, 1455989 Emån 1M. margaritifera, 1 U. pictorum, 1U. tumidus, 1 U. crassus, 1 A. anatina, 1 A. cygnea, 1 P. complanata Kilaån 1U. pictorum, 1 U. tumidus, 1 U. crassus 1 A. anatina, 2 A. cygnea, 1P. complanata Storån 2U. crassus, *U. tumidus, 1 A. anatina, 2 A. cygnea, 2 P. complanata Svärtaån 2U. pictorum, 1 U. tumidus, 1U. crassus, 1 A. anatina, *A. cygnea 6364761, 1483237 6513471, 1544037 6513795, 1545474 6513509, 1549673 6446065, 1524876 6446143, 1524715 6445930, 1524428 6522559, 1574425 6520282, 1573972 6518081, 1573955 Tommarpsån 1U. crassus, 1 A. anatina, 2 A. cygnea 6159159, 1394977 6159236, 1395094 6159543, 1395513 Figure 2. Fish gill with encysted glochidia, ranging from 200 to 230 µm (left). Juvenile mussels two weeks after falling off the host fish (right). Juvenile mussel release In order to study juvenile excystment from naturally infested fish, 142 fish individuals (A. alburnus, n = 29; B. bjoerkna, n = 17; C. gobio, n = 30; G. cernua, n = 3; L. lota, n = 22; P. fluviatilis, n = 13; R. rutilus, n = 26; T. tinca, n = 2) were caught in Kilaån and Svärtaån in June 2015. The fish were transported to the lab facility at Hemmerstorps Mölla. At the laboratory, the fish were placed in specially constructed hatchery tanks (40L or 80L) constructed after EYBE AND THIELEN (2010) and adjusted to our needs. The fish were kept separated according to fish species and river 7

origin, except for three G. cernua individuals from Kilaån and Svärtaån, which were held together in one tank. Juvenile mussels that were released from the fish gills ended up in a collection net from which they were transferred to Petri Dishes (Figure 3). A day/night light cycle was installed in the laboratory. Water temperature was measured constantly using data-loggers (Onset, Hobo pendant temp logger UA-002-64), and was around 15 C during the experimental period. The hatchery tanks were cleaned once a week as was the water exchanged. Fish were fed with frozen chironomids, gammarus or fish (E. lucius) every third day. The juvenile collection nets were checked daily for juvenile release. All living juveniles were counted and stored in 95% ethanol in Eppendorf tubes. The juveniles were then identified to mussel species using DNA methods as described above. In order to evaluate the host suitability, the number of mussel larvae per fish individual and per gram fish weight was calculated for each fish species. Figure 3. Mussel hatchery tanks in the aquarium laboratory at the Hemmestorps mölla. To avoid the fish from jumping out of the tanks, the tanks were covered with nets. Water was constantly pumped through an external filter creating water flow between the fish tank and a storing tank placed at a lower level (the floor). When the juvenile mussels released from the host fish, they followed the water current to the collection nets in the storing tank. The nets were controlled every day whereupon the juveniles were transferred to Petri Dishes for further examination. Results Fishing methods Electrofishing was the most efficient method in shallow river stretches. Electrofishing was also an efficient method for relatively stationary fish species/life stages such as young-of-the-year S. trutta, C. gobio and P. phoxinus, but was also efficient for more ephemeral species such as A. alburnus. 8

Fyke nets were generally less efficient compared to electrofishing, although fish survival was high. The fishing efficiency was lowest for P. phoxinus, G. aculeatus, B. barbatula and E. lucius. The fishing efficiency was highest for A. alburnus, L. lota, R. rutilus, P. fluviatilis, A. bjoerkna, T. tinca and S. erythrophthalmus. Stream nets was an efficient method for A. alburnus, and partly also for G. cernua and A. bjoerkna (Table 2). Table 2. Number of fish caught and preserved during 2012-2015 for the different fishing methods (E: Electro-fishing; S: Stream net; F : Fyke nets). Fish caught and released are not indicated in this table. Fish species Fishing method E S S+F F A. alburnus 111 148 38 111 A. bjoerkna 35 15 1 77 A. brama 1 A. vimba 1 B. barbatula 16 C. gobio 83 E. lucius 1 G. cernua 7 29 15 54 L. lota 34 P. fluviatilis 62 26 20 118 P. phoxinus 110 1 R. rutilus 27 22 10 119 S. erythrophthalmus 1 S. trutta 41 S. cephalius 2 3 T. tinca 1 Total 528 240 84 487 Glochidia encystment In total, 15 404 glochidia larvae were found on 14 out of 17 fish species (1260 fish individuals in total). Of these, 3310 glochidia were picked from the fish gills and were subsequently DNAanalyzed. Since there were no results for some glochidia, a total number of 2838 individual glochidia larvae could be identified to species. A total number of 1143 glochidia (40% of all glochidia) were thick-shelled river mussels. No glochidia were found on A. brama, L. idus and S. erythrophthalmus. Encysted thick-shelled river mussel larvae were found on the following fish species: A. alburnus, B. bjoerkna, B. barbatula, C. gobio, G. cernua, L. lota, P. fluviatilis, P. phoxinus, R. rutilus, S. trutta, S. cephalus and T. tinca, V. vimba (Table 3). In Bråån, P. phoxinus, S. trutta and B. barbatula were infested with U. crassus glochidia. All glochidia found on those fish were U. crassus. In Bräkneån, P. phoxinus carried most U. crassus glochidia per fish individual, which was more than two times higher than A. alburnus, and up to four times higher than S. trutta. However, S. trutta carried most U. crassus glochidia per gram fish. Furthermore, U. crassus glochidia were also found on G. cernua, and had almost as high infestation 9

rates as A. alburnus, and on P. fluviatilis and V. vimba. R. rutilus was not infested with glochidia larvae at all. In Emån, U. crassus was identified on five fish species, namely A. alburnus, P. fluviatilis, P. phoxinus, R. rutilus and S. cephalius. Only 6 % of the mussel species attached to S. cephalius were U. crassus. In contrast, all glochidia on P. phoxinus were U. crassus, while 55 % of the glochidia attached to A. alburnus was U. crassus. Ten different fish species were caught in Kilaån. No glochidia were found on S. trutta or on S. erythrophthalmus. A. alburnus, C. gobio, G. cernua, S. cephalus, L. lota, P. fluviatilis and R. rutilus carried mussel larvae. S. cephalus had the highest U. crassus infestation, albeit only one fish individual was investigated. A. alburnus, C. gobio and L. lota were more important hosts for U. crassus in terms of fish catch and high encystment rates. In Svärtaån, G. cernua had the highest U. crassus infestation rates. However, only 8 % of the glochidia was U. crassus, while 92% belonged to other mussel species. U. crassus infestation rates were lower on A. alburnus, but higher than on B. bjoerkna and P. fluviatlis. T. tinca had similar infestation rates as A. alburnus, but only one fish individual was caught. No U. crassus glochidia were found on B. barbatula, L. idus, or R. rutilus from Svärtaån. In Storån A. alburnus, A. bjoerkna, G. cernua, P. fluviatilis and R. rutilus carried U. crassus glochidia. In Tommarpsån, U. crassus glochidia was identified on C. gobio and P. phoxinus (Table 3). Juvenile hatching Glochidia encystment rates of fish preserved in the field (n = 106) were about two to five fold higher than juvenile excystment rates, with the largest difference in L. lota and P. fluviatilis. Generally, U. crassus glochidia were found on 58% of the preserved fish, with highest infestation rates per fish individual on L. lota, followed by C. gobio from Kilaån. However, the weight related infestation on C. gobio which was about two fold higher than on A. alburnus (Table 4). In total, 146 fish individuals were caught in Svärtaån and Kilaån in 2015, and were placed in the hatchery tanks in the laboratory facility. Three hundred and fifty-one mussel juvenile mussels were collected from a total of 142 fish individuals. Fourteen percent of all juvenile mussels collected were identified as U. crassus, and were released from A. alburnus, followed by L. lota, C. gobio, and R. rutilus (Table 4). Unio tumidus, Unio pictorum and Pseudanodonta complanata juveniles were also found on the fish from these rivers. 10

Table 3. Encystment rates of U. crassus on fish from the project rivers. The number of fish individuals is the total number that was caught. The numbers of glochidia on fish is an estimation of the total number of U. crassus glochidia that was found on the fish in relation to other mussel species on the fish gills. Stream Fish species Examined fish Glochidia on fish Glochidia DNA analysed UC glochidia UC glochidia UC on fish [NR gram.fish-1] ± sd [%] [NR] [NR] [%] [NR fish-1] ± sd Bråån B. barbatula 32 4 100 0.13 ± 0.34 0.07 ±0.19 100 P. phoxinus 94 2541 100 27.03 ± 4.93 11.10 ±11.44 100 S. trutta 40 409 100 10.23 ± 8.89 0.64 ±1.36 100 Bräkneån A. alburnus 94 537 95 7.42 ± 9.39 0.73 ±0.89 99 G. cernua 11 40 100 3.44 ± 2.93 0.29 ±0.43 97 P. fluviatilis 3 2 100 1.00 ± NA 0.01 ± NA 100 P. phoxinus 20 405 100 17.37 ±12.47 6.75 ±5.06 95 R. rutilus 2 0 NA NA NA NA S. trutta 24 36 94 4.70 ±5.35 14.27 ±19.36 89 V.vimba 1 3 100 2.00 ± NA 0.01 ± 100 Emån A. alburnus 2 13 100 3.00 ±2.83 0.24 ±0.27 55 P. fluviatilis 9 592 99 1.51 ±2.48 0.03 ±0.06 5 P. phoxinus 22 47 100 3.21 ±3.21 7.08 ±7.10 100 R. rutilus 33 935 98 0.39 ±1.50 0.01 ±0.04 3 S.cephalus 2 289 100 14.95 ± NA 0.39 ± NA 6 Kilaån A. alburnus 42 106 74 2.86 ±2.59 0.31 ±0.26 96 C. gobio 41 144 79 3.40 ±5.91 0.83 ±1.63 76 E. lucius 1 1 100 0.00 ± NA 0.00 ± NA NA G. cernua 4 119 100 3.93 ±5.97 0.59 ±0.97 9 L. lota 34 257 66 5.88 ±6.81 0.41 ±0.35 90 P. fluviatilis 12 77 99 1.60 ±1.82 0.01 ±0.01 11 R. rutilus 71 49 84 1.47 ±4.47 0.08 ±0.19 56 S. cephalus 1 20 100 15.00 ± NA 0.02 ± NA 100 S. erythrophthalmus 1 0 NA NA NA NA S. trutta 5 0 NA NA NA NA Storån A. alburnus 39 92 87 2.37 ±2.71 0.18 ±0.19 94 A. brama 1 0 NA NA NA NA B. bjoerkna 36 65 100 0.86 ±0.69 0.03 ±0.02 10 G. cernua 30 543 54 4.62 ±11.61 0.53 ±1.08 45 P. fluviatilis 41 385 82 3.06 ±2.96 0.13 ±0.17 38 R. rutilus 11 3 100 0.67 ±0.58 0.04 ±0.05 100 Svärtaån A. alburnus 80 242 83 3.53 ±4.78 0.35 ±0.48 76 B. barbatula 5 1 0 NA NA NA B. bjoerkna 49 38 74 0.33 ±0.71 0.02 ±0.05 16 G. cernua 49 1842 40 6.25 ±17.51 0.45 ±1.55 8 L. idus 1 0 NA NA NA NA P. fluviatilis 77 4317 72 0.74 ±2.26 0.04 ±0.12 3 R. rutilus 12 7 57 0.00 ±0.00 0.00 ±0.00 0 T. tinca 1 112 100 3.73 ± NA 0.00 ± NA 4 Tommarpsån C. gobio 102 442 51 7.59 ±6.70 4.85 ±5.77 100 P. phoxinus 90 630 54 10.60 ±9.36 10.59 ±7.16 100 S. trutta 35 59 0 NA NA NA TOTAL 1260 15404 68 6.29 ±12.71 2.31 ±5.75 42 11

Species fish ind [NR] fish with UC [%] glochidia [NR fish -1 ] glochidia [NR fish -1 ] from fish ind. [NR] all juveniles [NR] UC juveniles [%] UC juveniles Table 4. Average encystment and juvenile excystment rates (± SD) of fish from Kilaån and Svärtaån in 2015. NA indicates no data available. Stream Fish Unio crassus on fish Juvenile mussels collected [NR fish -1] UC juveniles [NR gram.fish -1 ] Kilaån A. alburnus 1 100 2.0 ± NA 0.15 ± NA 10 11 23.9 1.10 ± 0.14 0.09 ± 0.02 C. gobio 20 70 4.5 ± 7.6 1.14 ± 2.08 30 59 15.2 0.23 ± 0.12 0.03 ± 0.03 L. lota 12 42 5.6 ± 7.2 0.66 ± 0.46 22 8 17.4 0.39 ± 0.18 0.26 ± 0.23 R. rutilus 9 22 0.7 ± 0.18 0.18 ± 0.26 12 7 15.2 0.53 ± 0.46 0.02 ± 0.02 S. trutta 1 0 NA NA NA NA NA NA NA Svärtaån A. alburnus 30 60 3.3 ± 4.3 0.33 ± 0.49 19 20 28.3 0.57 ± 0.48 0.04 ± 0.03 A. bjoerkna 10 0 NA NA 17 0 0.0 0.00 ± 0.00 0.00 ± NA L. idus 1 0 NA NA NA NA NA NA NA P. fluviatilis 20 100 0.3 ± 1.3 0.01 ± 0.06 13 190 0.0 0.00 ± 0.00 0.00 ± 0.00 R. rutilus 2 50 0.0 ± NA 0.00 ± NA 14 2 0.0 0.00 ± 0.00 0.00 ± 0.00 T. tinca NA 0 NA NA 2 0 0.0 0.00 ± NA 0.00 ± NA Svärteån & Kilaån G. cernua NA 0 NA NA 3 17 0.0 0.00 ± NA 0.00 ± NA TOTAL 106 58 2.6 ± 5.1 0.43 ± 1.10 142 314 0.31 ± 0.39 0.06 ± 0.11 Discussion In this first large scale investigation of host fish suitability of the thick-shelled river mussel in Sweden, several suitable host fish species were identified. A. alburnus, P. phoxinus and C. gobio were the most suitable host fish overall as they carried the highest number of glochidia, released functional juveniles and occurred at relatively high densities. G. cernua occurred in four rivers and was identified to be a suitable host, although the few individuals that were tested for juvenile release did not release any U. crassus juveniles. The high encystment on S. trutta seems to be more locally important, due to its suitability in only two rivers. L. lota was a functional host and released juveniles, but with a varying importance due to their large difference in abundance among sites and rivers. The function of R. rutilus is probably also relatively varying, and locally the large abundance of this species may be of large importance as a host for U. crassus, although the U. crassus infestation rates are relatively low. P. fluviatilis appeared to be a less suitable host, even if large individuals can carry many glochidia. Likewise, A. brama, B. bjoerkna, T. tinca seemed to be relatively unsuitable as hosts. The comparison between encystment rates and juvenile hatching rates revealed that investigations of encystment rates are a generally good method to estimate hosts fish functionality. It may thus be enough to investigate the parasitic stage on the fish, and not the juvenile hatching. In fact, if the photo method (Österling, 2011) is used, there is a possibility to investigate encystment without sacrificing fish, even if mussel species are difficult to distinguish at the glochidial stage. Furthermore, in earlier experiments, not shown in the present report, we found that few 12

glochidia that were encysted on host fish three days after the infestation event died during the parasitic stage on fish. This strengthens the view that when glochidia are found on a fish, that fish species is a potential functional host, such as P. phoxinus and C. gobio tested in our studies. There was a large variation in the number of host fish species that were present in the rivers, which may affect growth and survival of the mussel populations. For example, migratory behaviour differs among fish species and can affect dispersal of the mussels, which may counteract reductions in genetic variation in small populations. Migratory fish species may also be of importance at anthropogenic disturbance, since migrating species that carry glochidia can spread mussels between sites and rivers. Thus, mussel populations may differ in their sensitivity, depending on the host fish composition. A river with many host fish species may be more tolerant to disturbances than a river with only one or a few host species. Host fish composition can thus be a measure of resilience, where rivers with many fish species may have higher resilience than rivers with single host species. In Bråån, P. phoxinus was a dominant host fish occurring in high densities, while S. trutta was a less suitable host. P. phoxinus occurred at the mussel distribution sites in Bråån over the reproduction season of U. crassus. The mussels in Bråån thus seem to have hosts present that can sustain the mussel population with juvenile mussels. Besides, S. trutta is a migratory species that also move within rivers. Thus, the mussel population can also be spread over large areas. Bräkneån probably had at least six host fish species. The high numbers of A. alburnus that were caught in Bräkneån probably mean that this species is a key host fish. It was noticed that spawning A. alburnus appeared in large schools during the reproduction season of U. crassus, why movement of mussels is high within the river when the fish leave their spawning grounds. The composition of the fish community showed that there were hosts from at least four fish families, and a large variation of the fish species ecological niches and behavioral types. Bräkneån thus seems to be a river with a high resilience for the glochidial stage on host fish. In Emån, there were at least five host fish species, and there may actually be more functional host species in Emån, since it is a large river with many fish species. However, the fish species that we found were probably the most important hosts for U. crassus, since they were present at the mussel sites. Like Bräkneån, Emån is likely a river with a relatively high resilience, given the large numbers of host fish species. Kilaån also had large schools of A. alburnus. The relatively stationary C. gobio was also found in high numbers, which probably secured the addition of juvenile mussels at the sites where adult mussels occur. Kilaån also had large numbers of L. lota at one out of three river stretches investigated. L. lota can, however, be considered as an important host in this river. Generally, host fish were sufficiently abundant in terms of densities and fish species. Storån was one of two rivers that had many G. cernua with high encystment rates of U. crassus. Here, also A. alburnus was a suitable host, which shows that A. alburnus seem to be an important host fish in general, as it was found in five of the seven rivers. While P. fluviatilis was also 13

identified as a functional host, the cyprinids B. bjoerka and R. rutilus were found suitable, although with much lower encystment rates. Svärtaån was the only river where T. tinca was caught and was found to have glochidia on its gills. However, only one individual was caught and preserved, which is why further investigations are needed to evaluate its function as a host. A. alburnus, B. bjoerka, G. cernua and P. fluviatilis were also important host fish species in Svärtaån, which underline the variety of possible hosts for U. crassus in this river. Lastly, Tommarpsån had three functional host fish species. Even if few host fish species were identified in this stream, C. gobio and P. phoxinus were highly functional species occurring at high densities. Together with S. trutta, there is still a variety of host fish composition that can sustain the mussel populations with juvenile recruitment. Our investigations showed that there was a large difference in host fish composition among the rivers. Likewise, the suitability and importance of fish species differed among rivers. Thus, it can be stated that the resilience, in terms of the number of host fish species and the ecological differences among host fish species, differs. It is thus essential to study the suitability and composition of host fish species in individual rivers when restoration measures are planned and implemented. It is also of importance to perform tests of the host species function before mussel re-introductions are carried out, since mussels from different rivers may differ in their compatibility to local host fish species. Lastly, to be able to re-introduce mussels at sites where functional host fish species occur, host fish distribution should also be investigated. References Anonymous, 2015. SLU. Artdatabanken. Rödlistade arter i Sverige 2015. 1-211. Binot-Hafke, M., Balzer, S., Becker, N., Gruttke, H., Haupt, H., Hofbauer, N., Ludwig, G., Matzke-Hajek, G. & Strauch, M. (2011). Red List of Endangered Aanimals, Plants and Fungi Germany, Invertebrates (Part 1) (Volume 3). Bundesamt für Naturschutz, Bonn-Bad Godesberg. Eybe, T. and Thielen, F. 2010. Restauration des populations de moules perlières en Ardennes. Technical Report: Action A1 /D1 /F3 Mussel Rearing Station. Källersjo, M. et al. 2005. Evaluation of ITS rdna as a complement to mitochondrial gene sequences for phylogenetic studies in freshwater mussels: an example using Unionoidae from north-western Europe. - Zool Scripta 34: 415 424. Lopes-Lima et al. 2015. Conservation status of freshwater mussels in Europe: state of the art and future challenges. Biological Reviews. doi: 10.1111/brv.12244 Lydeard, C., Cowie, RH., Ponder, WF., Bogan, AE., Bouchet, P., Clark, SA., Cummings, KS., Frest, TJ., Gargominy, O., Herbert, DG., et al. 2004. The global decline of nonmarine molluscs. Bioscience 54: 321 329. 14

Österling, E.M. (2011) Test and application of a non-destructive photo-method investigating the parasitic stage of the threatened mussel Margaritifera margaritifera on its host fish Salmo trutta. Biological Conservation, 144, 2984-2990. Reishütz, A.; Reischütz, L.P.. 2007, Austria red list of molluscs. In Red List of Endangered Animals of Austria (part 2; eds. K.P.Zulka), pp. 363-433. Böhlau Verlag, Wien. Stoeckl,K., Taeubert, J-E., Geist, J. 2014. Fish species composition and host fish density in streams of the thick-shelled river mussel (Unio crassus)- implications for conservation, Aquatic Conservation: Marine and Freshwater Ecosystems, 25, 2, 276 Taeubert, J-E., Gum, B. Geist, J. 2012. Host-specificity of the endangered thick-shelled river mussel (Unio crassus, Philipsson 1788) and implications for conservation. Aquatic Conservation: Marine and Freshwater Ecosystems, 22: 36-46. 15

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