Host Fish Species of Freshwater Mussels in Seven Swedish River Systems

Host fish species of freshwater mussels in seven Swedish river systems

Technical report

LIFE10 NAT/SE/000046 (“The thick-shelled river mussel brings back life to rivers”)

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: [email protected]

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, Carassius carassius = crucian carp, Cobitis taenia = spined loach, Cottus gobio = bullhead, Cottus poecilopus = siberian bullhead, Esox lucius = pike, Gasterosteus aculeatus = three-spined stickleback, Gymnocephalus cernua = ruffe, Lota lota= burbot, Oncorhynchus mykiss = rainbow trout, Perca fluviatilis = perch, Phoxinus phoxinus = minnow, Pseudorasbora parva = stone moroko, Pungitius pungitius = nine-spined stickleback, Salmo salar = Atlantic salmon, Salmo trutta = brown trout, Scardinius erythrophthalmus = common rudd, Squalius cephalius = european chub, Silurus glanis = Wels catfish, Rutilus rutilus = roach, Tinca tinca = tench, Vimba vimba = vimba brea

Introduction

Unionoid freshwater bivalves can be a dominant part of aquatic ecosystems (NEGUS, 1966), and in Sweden they can reach sizes up to 20cm (Anodonta cygnea). In fact, freshwater mussels provide several ecosystem services to human welfare (SCBD 2010). They have for example high capacity of water purification, pearl production and positive effects on biodiversity (HAAG, 2012). However, they are declining all over their distribution and are one of the most threatened organism groups (LYDEARD 2004).

In Europe, there are sixteen species of unionoid freshwater mussels, and seven species occur in Sweden. The most well-known mussels in Sweden are the freshwater pearl mussel (Margaritifera margaritifera) and the thick-shelled river mussel ( Unio crassus), which are both categorized as endangered on the IUCN and the Swedish Red list of species (2015). All unionoid mussels have a compulsory larval parasitic stage on fish. Thus, healthy mussel populations need functional host fish populations. Conservation measures should therefore include mussels and their associated host fish species.

The knowledge host fishes in Sweden are scarce, and due to the bad status of several of the species, there is an urgent need to study the host fish suitability. Also, in order to design and monitor restoration measures it is important to study host fish suitability and distribution area of freshwater mussels. This LIFE+ nature project "The thick-shelled river mussel brings back life to rivers" gathers information of host fish suitability of the thick-shelled river mussel.

Freshwater mussels can be categorized as specialists or generalists. Margaritifera margaritifera is host-specific, meaning that a specific host fish species is needed to complete their life cycle (TAEUBERT ET AL2010). Other species, such as Unio spp. have a broader range of host fish species (LOPES-LIMA 2016). However, the information is still scarce, and many fish species have not been tested for suitability to the mussels.

In Sweden, host fish suitability to Unio spp. is poorly known. In the present LIFE+ nature project, we therefore investigated patterns of host fish suitability in Sweden. The main focus was on Unio crassus. However, since U. pictorum, U. tumidus and Pseudoanodonta complanata have overlapping reproduction seasons with U. crassus, we studied natural encystment rates on wild fish for all these four species. The combination of information about the interaction these freshwater mussels and their host-fish species give valuable information prior to restoration measures.

The aim of this study was to investigate the host suitability of different fish species to U. crassus, U. pictorum, U. tumidus and Pseudodoanodonta complanata. We specifically asked the following questions:

a. Which fish species are hosts for these mussel species? b. What are the natural encystment rates of host fish for these mussel species? c. Does the host fish use vary between river systems? d. Are wild fish functional hosts in terms of juvenile metamorphosis?

Methods

The host fish mapping included two parts. In the first part, we investigated the natural encystment rates on wild fish from seven streams in the LIFE+ project. In the first part, we performed a lab-based host fish suitability test, where juvenile mussels were hatched from wild fish.

Encystment on fish

The investigation was carried out during the years 2012-2015 in the rivers Bråån, Bräkneån, Emån, Kilaån, Storån, Svartaån and Tommarpsån. Two fishing events were carried out every year, one in late May and one in late June. In order to maximise the probability to catch functional host fish species, the fishing sites with the highest mussel density in each stream were chosen (Table 1).

Table 1. Presence of mussel species in sampled streams and location of fishing sites. (*) Not in the stream directly, but occurs in a side-stream or the lake fed, 1healthy mussel population present, 2only few or single mussels found

Stream Mussel species present GPS-coordinates of fishing sites (RT90/NorthEast) Bråån 1U. crassus, 2U. pictorum, 2U. tumidus, 6187823, 1363317 1A. anatina, A. cygnea* 6187942, 1364505 Bräkneån 1M. margaritifera, 1U. crassus, 6229596, 1455989 2A. anatina Emån 1M. margaritifera, 1U. pictorum, 6364761, 1483237 1U. tumidus, 1U. crassus, 1A. anatina, 1A. cygnea, 1P. complanata Kilaån 1U. pictorum, 1U. tumidus, 1U. crassus 1A. 6513471, 1544037 anatina, 2A. cygnea, 6513795, 1545474 1P. complanata 6513509, 1549673 Storån 2U. crassus, *U. tumidus, 1A. anatina, 2A. 6446065, 1524876 cygnea, 2P. complanata 6446143, 1524715 6445930, 1524428 Svärtaån 2U. pictorum, 1U. tumidus, 6522559, 1574425 1U. crassus, 1A. anatina, *A. cygnea 6520282, 1573972 6518081, 1573955 Tommarpsån 1U. crassus, 1A. anatina, 2A. cygnea 6159159, 1394977 6159236, 1395094 6159543, 1395513

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 traps. Electrofishing (600 V LUGAB, L-600) was performed during the day at sites that could be waded. Stream nets and traps were placed out overnight, both at shallow and deep river sites.

To gather information of the encystment rates on naturally infested wild fish a proportion of the captured fish was sacrificed. Each fish was measured: length (mm), weight (g) and marked with an individual fin cut before it was stored in 95% ethanol until further analysis.

In order to determine the mussel species, fish gills were dissected and the number of mussel larvae on the gill takers and arches was counted using microscope binoculars (Figure 2, left). A proportion of the glochidia from the fish gills was collected and stored in ethanol (95%) in eppendorf-tubes. To determine which fish species' the mussels 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 (KÄLLERSJO ET AL. 2005).

Figure 1. Fish gills with encysted glochidia ranging from 200 to 300µm (left). Juvenile mussels after falling off the host fish (right).

Juvenile 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 was placed in specially constructed hatchery tanks (40L or 80L) (EYBE AND THIELEN, 2010). The fish were kept separated according to fish species and river origin, except for three G. cernua individuals from Kilaån and Svartaån, which were held together in one tank. Juvenile mussels that were released from the fish gills ended up in a collection net.

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 experiment period. The hatchery tanks were cleaned and the water was exchanged once a week. Fish were fed with frozen chironomids, gammarus or fish every third day. The juvenile collection nets were checked daily for juvenile release (Figure 2, right). All living juveniles were counted and stored in 95% ethanol in Eppendorf tubes. The juveniles were then analyzed to species using DNA methods as described above. In order to evaluate the host

7 suitability, the number of mussel larvae per fish individual and per gram fish weight was calculated for each fish species.

Results

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. trntta, C. gobio and P. phoxinus, but was also efficient for more ephemeral species such as A. alburnus. Fyke nets were generally less efficient compared to electrofishing, although fish survival was high. The fishing efficiency was lowest for P. phoxinus, G. acueatus, B. barbatula and E. lucius. The fishing efficiency was highest for A. albumus, L. lota, R rutilus, P. fluviatilis, B. bjoerkna, T. tinca and S. erythrophthalamus. Stream nets was an efficient method for A. alburnus, and partly also for G. cernua and B. bjoerkna (Table 2).

Table 2 Number of fish caught during 2012-2015 for the different fishing methods. (E: electrofishing; S: stream nets; F: fyke nets). Fish species Fishing method E S S+F F A. alburnus 111 148 38 111 B. 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

Encystment on fish

In total, 15 404 glochidia larvae were found on 1260 fish individuals. Of these, 3310 glochidia were picked from the fish gills and were subsequently DNA-analyzed. Since there were no results for some glochidia, a total number of 2838 individual glochidia larvae could be identified to species.

Anodonta spp. were just found in low numbers, and only on G. cernua and P. fluviatilis. This was due to the fact that they spawn earlier than the other mussel species, and were thus in the very end of their reproduction season in our investigation.

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 encystment rates as A. alburnus, and on P. fluviatilis and V. vimha. R. rutilus was not infested with glochidia larvae at all. No U. tumidus larvae were found, while A. alburnus, G. cernua, P. phoxinus and S. trutta were found to have encysted larvae of U. pictorum on their gills. P. complanata was not found on any fish in Bråån.

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. Mussel larvae from U. tumidus were found on P. fluviatilis, S. cephalius and R rutilus while U. pictorum larvae were found on A. alburnus, P. fluviatilis, R ruti/us and S. cephalius. P. complanata was only found on R. rutilus.

Ten different fish species were caught in Kilaån. No glochidia were found on S. trutta or on S. erythrophthalamus. A. alhurnus, C. gohio, G. cernua, S. cephalius, L lota, P. fluviatilis and R rutilus carried mussel larvae. S. cephalius had the highest U. crassus encystment, albeit only one fish individual was investigated. A. alburnus, C. gohio and L Iota were more important and had high encystment rates of U. crassus. The same patterns were found for U. tumidus, with the exceptions of S. cephalius and where no mussel larvae were found. U. pictorum larvae were only found on R rutilus. Lastly, C. gohio, G. cernua and L lota were all found to have larvae of P. complanata on their gills.

In Storån A. alhurnus, B. hjoerkna, G. cernua, P. fluviatilis and R rutilus carried U. crassus glochidia. For U. tumidus and U. pictorum the same patterns were found, with the exception of R rutilus, which had no larvae of U. tumidus or U. pictornm on their gills. B. hjoerkna, G. cernua had larvae from P. complanata on their gills.

In Svärtaån, G. cemua had the highest U. crassus encystment rates. U. crassus encystment rates were lower on A. alhurnus, but higher than on B. hjoerkna and P. fluviatilis. T. tinca had similar encystment rates as A. alhurnus, but only one fish individual was caught. No U. crassus glochidia were found on B. harbatu/a, L id11s, or R rutilus from Svärtaån. In contrast to the other rivers, U. tumidus larvae were found on the same fish species as U. crassus, but also on R rutilus and T. tinca. U. pictorum was not found on the fish gills at all in Svärtaån. Lastly, G. cernua, P. fluviatilis and R rutilus carried larvae of P. complanata on their gills.

In Tommarpsån U. crassus glochidia was identified on C. gobio and P. phoxinus. However no U. tumidus, U. pictorum or P. complanata was found on any fish in Tommarpsån (Table 3).

Table 3. Encystment rates of the mussels 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 glochidia that was found on the fish.

glochidia on fish for every mussel species [NR gram.fish-1] ± sd

fish fish species examined fish gill fish on glochidia analysed DNA glochidia stream [NR] [NR] [%] AA AC PC UC UT UP

B. barbatula 32 4 75 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.15 ± 0.26 0.00 ± 0.00 0.00 ± 0.00 P. phoxinus 94 2541 43 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 11.08 ± 12.49 0.00 ± 0.00 0.00 ± 0.00 Bråån S. trutta 40 409 94 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.12 ± 1.57 0.00 ± 0.00 0.00 ± 0.00 A. alburnus 94 537 95 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.73 ± 0.89 0.00 ± 0.00 0.01 ± 0.04

G. cernua 11 40 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.29 ± 0.43 0.00 ± 0.00 0.01 ± 0.02

P. fluviatilis 3 2 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 P. phoxinus 20 405 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 6.75 ± 5.06 0.00 ± 0.00 0.39 ± 0.98

R. rutilus 2 0 NA NA NA NA NA NA NA Bräkneån S. trutta 24 36 94 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 14.27 ± 19.36 0.00 ± 0.00 1.84 ± 3.13 V.vimba 1 3 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.01 ± NA 0.00 ± NA 0.00 ± NA A. alburnus 2 13 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.24 ± 0.27 0.00 ± 0.00 0.13 ± 0.18

P. fluviatilis 9 592 99 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.03 ± 0.06 0.18 ± 0.23 1.22 ± 2.09 P. phoxinus 22 47 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.08 ± 7.10 0.00 ± 0.00 0.00 ± 0.00 Emån R. rutilus 33 935 98 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.08 0.01 ± 0.04 0.00 ± 0.01 0.84 ± 1.10 S.cephalus 2 289 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.39 ± 0.00 0.92 ± 0.00 5.63 ± 0.00 A. alburnus 42 106 74 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.31 ± 0.26 0.01 ± 0.04 0.00 ± 0.00 C. gobio 41 144 79 0.00 ± 0.00 0.00 ± 0.00 0.06 ± 0.32 0.83 ± 1.63 0.13 ± 0.21 0.00 ± 0.00 E. lucius 1 1 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA G. cernua 4 119 100 0.00 ± 0.00 0.00 ± 0.00 3.72 ± 2.28 0.59 ± 0.97 0.19 ± 0.33 0.00 ± 0.00 L. lota 34 257 66 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.03 0.41 ± 0.35 0.03 ± 0.09 0.00 ± 0.02 P. fluviatilis 12 77 99 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.11 0.11 ± 0.23 0.00 ± 0.01 Kilaån R. rutilus 71 49 84 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.08 ± 0.19 0.00 ± 0.01 0.02 ± 0.04 S. cephalus 1 20 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.02 ± NA 0.00 ± NA 0.00 ± NA S. erythrophthalmus 1 0 NA NA NA NA NA NA NA S. trutta 5 0 NA NA NA NA NA NA NA A. alburnus 39 92 87 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.18 ± 0.19 0.01 ± 0.03 0.00 ± 0.01

A. brama 1 0 NA NA NA NA NA NA NA

B. bjoerkna 36 65 100 0.00 ± 0.00 0.00 ± 0.00 0.38 ± 1.02 0.03 ± 0.02 0.03 ± 0.09 0.01 ± 0.02

G. cernua 30 543 54 0.11 ± 0.46 0.22 ± 0.60 1.31 ± 2.24 0.53 ± 1.08 0.05 ± 0.12 0.01 ± 0.05 Storån P. fluviatilis 41 385 82 0.00 ± 0.00 0.01 ± 0.05 0.00 ± 0.01 0.13 ± 0.17 0.14 ± 0.18 0.01 ± 0.07 R. rutilus 11 3 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.04 ± 0.05 0.00 ± 0.00 0.00 ± 0.00 A. alburnus 80 242 83 0.00 ± 0.00 0.00 ± 0.02 0.00 ± 0.00 0.35 ± 0.48 0.09 ± 0.16 0.00 ± 0.01 B. barbatula 5 1 0 NA NA NA NA NA NA

B. bjoerkna 49 38 74 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.02 ± 0.05 0.13 ± 0.21 0.00 ± 0.00 G. cernua 49 1842 40 0.00 ± 0.00 0.00 ± 0.00 1.14 ± 2.03 0.45 ± 1.55 1.01 ± 1.74 0.00 ± 0.00 L. idus 1 0 NA NA NA NA NA NA NA Svärtaån P. fluviatilis 77 4317 72 0.00 ± 0.02 0.05 ± 0.21 0.24 ± 0.81 0.04 ± 0.12 4.13 ± 4.93 0.02 ± 0.09 R. rutilus 12 7 57 0.00 ± 0.00 0.00 ± 0.00 0.01 ± 0.01 0.00 ± 0.00 0.06 ± 0.10 0.00 ± 0.00 T. tinca 1 112 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.10 ± NA 0.00 ± NA

- C. gobio 102 442 51 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 4.85 ± 5.77 0.00 ± 0.00 0.00 ± 0.00 P. phoxinus 90 630 54 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 10.59 ± 7.16 0.00 ± 0.00 0.00 ± 0.00

arpsån S. trutta 35 59 0 NA NA NA NA NA NA Tomm

Grand Total 1260 15404 68 0.00 ± 0.08 0.01 ± 0.13 0.13 ± 0.72 2.31 ± 5.75 0.45 ± 1.95 0.11 ± 0.62

The results showed that U. crassus was the only mussel on several fish species, and also had the highest mean encystment rates generally. Unio pictorum and Unio tumidus were also found on many fish species, although in lower proportions than U. crassus. P. complanata, which occurs in low numbers in streams and rivers, had the lowest proportions of encystment, even if they in fact the highest encystment proportions in some rivers.

Table 4. Proportional encystment of the fish caught in the rivers investigated by different mussel species. AA: Anodonta anatina; AC: Anodonta cygnea, PC: Pseudanodonta complanata; UC: Unio crassus, UT: Unio tumidus, UP: Unio pictorum. % % % % % % stream fish species AA AC PC UC UT UP Bråån B. barbatula 0 0 0 100 0 0 P. phoxinus 0 0 0 100 0 0

S. trutta 0 0 0 100 0 0

Bräkneån A. alburnus 0 0 0 99 0 1 G. cernua 0 0 0 97 0 3 P. fluviatilis 0 0 0 100 0 0 P. phoxinus 0 0 0 95 0 5 S. trutta 0 0 0 89 0 11 V.vimba 0 0 0 100 0 0 Emån A. alburnus 0 0 0 55 0 45 P. fluviatilis 0 0 0 5 23 73

P. phoxinus 0 0 0 100 0 0

R. rutilus 0 0 1 3 1 96

S.cephalus 0 0 0 6 13 81

Kilaån A. alburnus 0 0 0 96 4 0 C. gobio 0 0 4 76 20 0 G. cernua 0 0 89 9 3 0 L. lota 0 0 1 90 8 1 P. fluviatilis 0 0 1 11 83 4 R. rutilus 0 0 0 56 11 33 S. cephalus 0 0 0 100 0 0 Storån A. alburnus 0 0 0 94 4 2 B. bjoerkna 0 0 80 10 8 2

G. cernua 2 6 40 45 5 2

P. fluviatilis 1 4 4 38 49 4

R. rutilus 0 0 0 100 0 0

Svärtaån A. alburnus 0 1 0 76 22 1 B. bjoerkna 0 0 0 16 84 0 G. cernua 0 0 68 8 24 0 P. fluviatilis 0 2 7 3 87 1 R. rutilus 0 0 50 0 50 0 T. tinca 0 0 0 4 96 0 Tommarpsån C. gobio 0 0 0 100 0 0 P. phoxinus 0 0 0 100 0 0 Mean prop 0 1 13 42 30 14

Juvenile release

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. Iota and P. fluviatilis. Generally, U. crassus glochidia were found on 58% of the preserved fish, with highest encystment rates per fish individual on L. lota, followed by C. gobio from Kilaån. However, the weight related encystment on C. gobio which was about two fold higher than on A. alburnus. There was a high encystment rate of U. tumidus on P. fluviatilis. U. tumidus was also found on C. gobio, R. rutilus and A. alburnus. P. complanata, was found on C. gobio and P. fluviatilis (Figure 3).

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. iota, C. gobio, and R. rutilus. The juvenile hatching rates of U. tumidus, U. pictorum and P. complanata generally followed the encystment rates, with the exception of P. complanata, which was found to have higher juvenile hatching rates compared to the encystment rates from C. gobio. In contrast to the encystment investigation where no P. complanata was also found on G. cernua, several juvenile mussels were hatched from P. complanata. (Figure 4).

4 UP 3 UT UC glochidia per gram fish 2 PC 1

L. L. lota

C. gobio C.

R. rutilus R. rutilus

A. alburnus A. alburnus P. fluviatilis Kilaån Svärtaån

Figure 3. Glochidia encystment rates of fish caught in Kilaån and Svärtaån in the year 2015. Values represent average glochidia encystment per gram fish. UP: Unio pictorum; UT: Unio tumidus-, UC: Unio crassus; PC: Pseudanodonta complanata.

0,6

0,5

0,4

0,3 UP

0,2 UT

juveniles per fish gram UC

0,1 PC

L. L. lota

C. gobioC.

R.rutilus R. rutilus

G. cernua

A. bjoerkna

A. alburnus A. alburnus P. fluviatilis Kilaån Svärteån Svärteån & Kilaån

Figure 4. Successfully metamorphosed juvenile mussels collected from fish caught in the rivers Kilaån and Svärtaån in the year 2015. Values represent average excystment rates per gram fish. UP: Unio pictorum; UT: Unio tumidus, UC: Unio crassus, PC: Pseudanodonta complanata.

Discussion

Even though freshwater mussels constitute an important part of freshwater ecosystems, little quantitative information of host fish suitability exists (LOPES-LIMA ET AL 2016). However, in the project "The thick-shelled river mussel brings life back to rivers" we documented several suitable host fish species unionoid freshwater mussels.

In this first large scale investigation of host fish suitability of unionoid mussels in Sweden, several suitable host fish species were identified. For U. crassus, A. alburnus, P. phoxinus and C. gobio were the most suitable host fish and carried the highest number of glochidia, released functional juveniles and occurred at relatively high densities. A. alburnus was also found on U. tumidus and U. pictorum, but n on on P. complanata, while U. tumidus but not U. pictorum was found on C. gobio, and the opposite patterns was found for P. phoxinus. G. cernua occurred in four rivers and was found to be a suitable h ost. Interestingly, G. cernua seems to be a key host species for P. complanata, since the encystment and juvenile hatching rates of P. complanata were high.

The high encystment on S. trutta seems to be more locally important, due to its suitability in few rivers. L Iota 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. P. fluviatilis appeared to be a not so suitable host for U. crassus, but seems to be an important host, especially for U. tumidus. In addition, large P. fluviatilis individuals can carry many glochidia, which mean that they may contribute to juvenile recruitment also of U. crassus. This shows that a large number of host fish species is a prerequisite to support mussel communities. 13

The comparison between encystment and juvenile hatching rates revealed that investigations of encystment rates are a good method to estimate hosts fish function, and it may actually be enough to investigate the parasitic stage. Furthermore, in earlier experiments, not shown in the present report, we showed that few glochidia that were encysted on host fish three days after the encystment 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 functional host.

The variation in the number of host fish species was large among rivers, which may affect the growth and survival of mussel populations. Migratory behaviour differs among fish species, can affect dispersal of the mussels, and may counteract reductions in genetic variation. Migratory fish species can also be of importance at anthropogenic disturbance, since migrating species that carries 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.

U. crassus was the only mussel species found in Bråån. Here, 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 Braan 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.

Bräknean probably had at least six host fish species of U. crassus. The high numbers of A . alburnus that were caught in Braknean probably mean that this species is a key host for U. crassus. It was noticed that A. alburnus appeared in large schools during the reproduction season, why movement of mussels is high within the river. However, A . alburnus did not seem to be a dominating host for U. tumidus or U. pictorum. 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 is at least five host fish species, and there may in fact 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, since they were present at the mussel sites. The different fish species that we found in Emån complement each other as hosts for the mussels. For example, P. fluviatilis was a good host for U. tumidus and U. pictorum, but appeared not to be equally important for U. crassus, which was instead more dependent on P. phoxinus. Like Bräkneån, Emån is likely a river with a relatively high resilience, given the large number of host fish species.

Kilaån also had large schools of A . alburnus, and seems to be important for the movement of U. crassus, while U. tumidus and U. pictorum again had low encystment rates on A. alburnus. The relatively stationary C. gobio was also found in large numbers, which probably secured the addition of juvenile mussels at the sites where adult mussels occur. Kilaån also had large numbers of L. lota, which can be considered as an important host in this river, at least for U. crassus. There was also a good number of fish species, and thus a variation in hosts that can sustain the mussel populations.

Storån was one of two rivers that had many G. cemua with high encystment rates, especially of P. complanata. Again, A. alburnus was a suitable host for U. crassus, which shows that A. alburnus. P. fluviatilis was also a functional host, especially for U. tumidus.

Svärtaån was the only river where T. tinca was caught and was found to have glochidia of U. tumidus on their gills. Again, different mussel species seemed to be especially dependent on different fish species. This shows that single or few host fish species are often needed for each mussel species, even if each mussel species has a number of functional host species that they can use.

In Tommarpsån, U. crassus, which was the only mussel species that was found here, had three functional host fish species. Even if so few host fish species were found, C. gobio and P. phoxinus were highly functional species. Together with S. trutta, there is still a variety in host fish composition that can sustain the mussel populations with recruiting juvenile mussels.

In summary, there was a large difference in host fish composition among the rivers. Likewise, the suitability and importance of host fish species differed among mussel species and rivers. Thus, it seems that the resilience, in terms of the number of host fish species and ecological differences among host fish species, differ. It is thus a need to know the suitability and composition of host fish species in single rivers when restoration measures are planned and implemented. It is also of importance to perform tests of host species function before mussel re-introductions, 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

Haag, W. R. 2012. North American Freshwater Mussels: Natural History, Ecology, and Conservation. Cambridge University Press, Cambridge. Killersjo, M. et al. 2005. Evaluation of ITS rDNA as a complement to mitochondrial gene sequences for phylogenetic studies in freshwater mussels: an example using Unionidae from north-western Europe. - Zool Scripta 34: 415-424. Lopes-Lima, M., et.al. 2016. 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, 1J., Gargominy, 0., Herbert, DG., et al. 2004. The global decline of nonmarine molluscs. Bioscience 54: 321-329. Negus, C.L. 1966. A quantitative study of growth and production of unionid mussels in the river Thames at Reading. Journal of Animal Ecology 35, 513-532. Secretariat of the Convention on Biological Diversity, 2010. Ecosystem Goods and Services in Development Planning:A Good Practice Guide. Montreal, 80 + iv pages. Taeubert,JE., Denic, M., Gum, B., Lange, M., Geist,J. 2010. Suitability of different salmonid strains as hosts for the endangered freshwater pearl mussel (MargaritifCra margaritifera L.) Aquat. Conserv.-Mar. Freshw. Ecosyst. 20:728-734. 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.

Appendix 1.

glochidia on fish for every mussel species [NR fish-1] ± sd

stream fish species examined fish gill fish on glochidia analysed DNA glochidia [NR] [NR] [%] AA AC PC UC UT UP

B. barbatula 32 4 75 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.13 ± 0.34 0.00 ± 0.00 0.00 ± 0.00 P. phoxinus 94 2541 43 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 27.03 ± 34.93 0.00 ± 0.00 0.00 ± 0.00 Bråån S. trutta 40 409 94 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 10.23 ± 28.89 0.00 ± 0.00 0.00 ± 0.00 A. alburnus 94 537 95 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.42 ± 9.39 0.00 ± 0.00 0.05 ± 0.38 G. cernua 11 40 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.44 ± 2.93 0.00 ± 0.00 0.10 ± 0.32 P. fluviatilis 3 2 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 P. phoxinus 20 405 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 17.37 ± 12.47 0.00 ± 0.00 1.07 ± 2.71

Bräkneån R. rutilus 2 0 NA NA NA NA NA NA NA S. trutta 24 36 94 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 4.70 ± 5.53 0.00 ± 0.00 0.52 ± 0.88 V.vimba 1 3 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 2.00 ± NA 0.00 ± NA 0.00 ± NA A. alburnus 2 13 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.00 ± 2.83 0.00 ± 0.00 2.50 ± 3.54

P. fluviatilis 9 592 99 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.51 ± 2.48 12.87 ± 11.61 55.12 ± 67.82 P. phoxinus 22 47 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 3.21 ± 3.21 0.00 ± 0.00 0.00 ± 0.00 Emån R. rutilus 33 935 98 0.00 ± 0.00 0.00 ± 0.00 0.52 ± 2.76 0.39 ± 1.50 0.05 ± 0.28 27.77 ± 31.52 S.cephalus 2 289 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 14.95 ± 0.00 34.88 ± 0.00 214.26 ± 0.00 A. alburnus 42 106 74 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 2.86 ± 2.59 0.09 ± 0.43 0.00 ± 0.00 C. gobio 41 144 79 0.00 ± 0.00 0.00 ± 0.00 0.12 ± 0.60 3.40 ± 5.91 0.67 ± 0.83 0.00 ± 0.00 E. lucius 1 1 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA 0.00 ± NA G. cernua 4 119 100 0.00 ± 0.00 0.00 ± 0.00 31.76 ± 8.53 3.93 ± 5.97 1.20 ± 2.08 0.00 ± 0.00

L. lota 34 257 66 0.00 ± 0.00 0.00 ± 0.00 0.28 ± 1.32 5.88 ± 6.81 0.82 ± 2.47 0.05 ± 0.21

P. fluviatilis 12 77 99 0.00 ± 0.00 0.00 ± 0.00 0.20 ± 0.45 1.60 ± 1.82 11.60 ± 21.74 0.60 ± 0.89 Kilaån R. rutilus 71 49 84 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1.47 ± 4.47 0.08 ± 0.29 0.25 ± 0.62 S. cephalus 1 20 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 15.00 ± NA 0.00 ± NA 0.00 ± NA S. erythrop- 1 0 NA NA NA NA NA NA NA hthalmus S. trutta 5 0 NA NA NA NA NA NA NA A. alburnus 39 92 87 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 2.37 ± 2.71 0.09 ± 0.30 0.04 ± 0.21

A. brama 1 0 NA NA NA NA NA NA NA

B. bjoerkna 36 65 100 0.00 ± 0.00 0.00 ± 0.00 6.71 ± 17.76 0.86 ± 0.69 0.71 ± 1.50 0.14 ± 0.38

Storån G. cernua 30 543 54 0.65 ± 2.67 1.39 ± 3.74 7.67 ± 12.04 4.62 ± 11.61 0.43 ± 1.21 0.08 ± 0.34 P. fluviatilis 41 385 82 0.04 ± 0.21 0.55 ± 2.01 0.30 ± 1.27 3.06 ± 2.96 4.29 ± 6.43 0.32 ± 1.53 R. rutilus 11 3 100 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.67 ± 0.58 0.00 ± 0.00 0.00 ± 0.00 A. alburnus 80 242 83 0.00 ± 0.00 0.03 ± 0.17 0.00 ± 0.00 3.53 ± 4.78 1.02 ± 1.73 0.03 ± 0.17 B. barbatula 5 1 0 NA NA NA NA NA NA

B. bjoerkna 49 38 74 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.33 ± 0.71 1.89 ± 3.14 0.00 ± 0.00

G. cernua 49 1842 40 0.00 ± 0.00 0.00 ± 0.00 15.80 ± 31.60 6.25 ± 17.51 16.37 ± 31.50 0.00 ± 0.00

L. idus 1 0 NA NA NA NA NA NA NA Svärtaån P. fluviatilis 77 4317 72 0.51 ± 3.64 2.21 ± 7.01 6.81 ± 19.98 0.74 ± 2.26 44.41 ± 42.37 0.31 ± 1.33 R. rutilus 12 7 57 0.00 ± 0.00 0.00 ± 0.00 0.67 ± 1.15 0.00 ± 0.00 0.33 ± 0.58 0.00 ± 0.00 T. tinca 1 112 100 0.00 ± NA 0.00 ± NA 0.00 ± NA 3.73 ± NA 89.60 ± NA 0.00 ± NA

C. gobio 102 442 51 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 7.59 ± 6.70 0.00 ± 0.00 0.00 ± 0.00

- P. phoxinus 90 630 54 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 10.60 ± 9.36 0.00 ± 0.00 0.00 ± 0.00

arpsån Tomm S. trutta 35 59 0 NA NA NA NA NA NA

Grand Total 1260 15404 68 0.07 ± 1.21 0.29 ± 2.38 1.71 ± 9.62 6.29 ± 12.71 5.66 ± 19.70 2.68 ± 16.08