Interaction between fish and freshwater mussels

Transcription

1 Interaction between fish and freshwater mussels Determination of functional hosts for the thick-shelled river mussel, U. Crassus. Mattias Larsson Degree project for Master of Science in Biology Animal Ecology, 45 hec, ht 2015 Department of Biological and Environmental Sciences University of Gothenburg Supervisors: Johan Höjesjö & Niklas Wengström Examiner: Lotta Kvarnemo

2 Abstract The primary aim of this study is to determine what species that acts as functional hosts for the thick-shelled river mussel, Unio crassus in its natural habitat by analysis of hatched juvenile mussels from fish infested in situ. Infested fish were collected from two streams, Virån and Svennevadsån, inhabited with recruiting populations of U. crassus. Both attached glochidia larvae obtained from sacrificed fish in laboratory and fully developed juvenile mussels from the hatchery were identified with molecular techniques. The hatchery successfully produced 26 juvenile mussels from five different species of fish. Juvenile thick-shelled river mussels were hatched only from Bleak, Alburnus alburnus, confirming it as functional host in Virån. Additionally Bleak, A. alburnus, Burbot, L. lota, Perch, P. fluviatilis, Trout, S. trutta and Bullhead, C. gobio are functional hosts for U. tumidus in Virån. Although no U. crassus were hatched from Svennevadsån, C. gobio is most likely a functional host in Svennvadsån where it also can be confirmed as a functional host for Pseudandonta complanata and U. tumidus. Our results show that the method of allowing infested fish to release its juvenile mussels in hatchery was a successful method. This managed to confirmed A. alburnus as a functional host for U. crassus in Virån.

3 Introduction Background The unionid mussels are spread over several continents; North America, Europe, Asia, Africa and the Indonesian Archipelago (Graf and Cummings, 2015). The decline of the unionid mussels is prominent all over its range (Master, 1990, Bauer & Wachtler, 2001, Bogan, 2008 and Christian & Harris, 2008) and primary causes are considered to be water pollution, dam construction, drainage, sedimentation and increased predation. All derive from human alteration of terrestrial and aquatic ecosystems, which affect both mussels and their hosts (Douda, 2010, Galbraith et al., 2010, & Hus et al., 2006). It is hypothesized that extinction of host-dependent species due to decline of their hosts represents the most comprehensive loss of biodiversity (Dunn et al., 2009). The extinction of one single species will in most cases affect several others and hostdependent species (e. g. dependent parasites) will face the risk of co-extinction as their hosts decline (Colwell et al., 2012). Mussels associated to the family Unioidae are one of many which are affected by this phenomenon. The thick-shelled river mussel, Unio crassus Distribution and statues The most threatened unionid mussel in Europe is the thick-shelled river mussel, Unio crassus. Due to IUCN s latest calculations the decline of U. crassus has reached 90% and is still decreasing which classifies it as Endangered on both the Swedish and the global red list (Lopes-Lima et al., 2014). The thick-shelled river mussel can be found throughout Europe with Spain, Italy and parts of the British islands as exceptions (Lopes-Lima et al., 2014). In Sweden U. crassus has an eastern distribution that stretches from the most southern parts up to a few miles north of Stockholm (von Proschwitz, 1999). The species is known from approximately 100 localities in Sweden but only of them have observed successful reproduction, which reflects one of the reasons to its decline in Sweden (Bergengren, 2008). Biology and reproductive cycle U. crassus lives particularly in running water but can be found in flowing parts of lakes. The mussel grows to about 4-7 cm and lives for years but even larger and older specimens do occur (von Proschwitz, 1999). U. crassus has as the other six native unionid mussels (eg. freshwater pearl mussel) in Sweden a complex reproduction with an obligate parasitic life stage. It starts with the release of sperm in the water from the males. Some of the sperm end up in the females through the influx water and fertilize the female eggs. After two to three weeks the eggs have developed into a larva called glochidium (Lang, 1998 & von Proschwitz, 1999). The female then releases up to glochidia into the water. What triggers the release of glochidia is not fully understood but chemical substances from the host fish have been suggested (Bauer & Wächtler, 2001). Glochidia have a short free-living period and are dependent on finding a host-fish for their survival (Mackie, 1984). Glochidia from U. crassus are equipped with hooks on its shell which they use to attach to their host, either on the gills, fins or body. Once encountered with a host, sensitive cilia on the glochidia trigger it to close its shell and attach with its hooks (Pekkarinen & Englund 1995). Once attached, the glochidia are

4 encysted within the epidermis of the host fish and surrounded with a hyaline layer (Wood 1974, Silva-Souza & Eiras 2002). During the attachment period, which last for approximately a month the glochidia convert its inner structure where muscles, nervous system, digestive apparatus and mantle are developed. Once the metamorphosis is complete the glochidia have gone from larva to juvenile mussel. The juvenile mussels then releases from its host and fall to the bottom where it start to dig itself down in the substrate. After two to three years the mussel emerges up from the gravel bed and starts its life as an adult mussel (von Proschwitz, 1999 & Bauer & Wächtler, 2001). In Germany the reproduction period occurs from April to July (Hochwald, 1997). Definitions For further reading it is of importance to distinguish the terms possible and functional host. In this report possible host is a host where glochidia can attach and be encysted but not necessarily develop into a juvenile mussel. While functional host is a host where successful attachment, encystment and metamorphosis to juvenile mussel can definitely be proved. Host fish European studies Investigations about U. crassus and its host-parasite-interaction have been performed in many different parts of Europe but due to the presence of sub-species (eg. U. crassus cytherea) and local variations of U. crassus, results and knowledge regarding compatible hosts can not necessarily be transferred from one stream to another (Taeubert et al., 2012 & Hochwald, 1997). Several studies in Germany suggest a number of species as hosts for U. crassus; Leuciscus cephalus, Phoxinus phoxinus, Scardinius erythrophtalmus, Leuciscus leuciscus, Gasterosteus aculeatus, Pungitius pungitius, Cottus gobio, Perca fluviatilis and Gymnocephalus cernuus (Nagel, 1990 & Hochwald, 1997). Swedish studies In the Swedish LIFE+ Nature project ( ) results from two investigated streams (Emån & Bräkneån) preliminary indicates that U. crassus uses a wide range of possible hosts. In the two streams glochidia from U. crassus were found on nine different species of fish; Alburnus alburnus, G. cernuus, P. phoxinus, Salmo trutta, Abramis vimba, P. fluviatilis, Sander lucioperca, Rutilus rutilus and Squalius cephalus. The results were obtained from DNA-analysis of attached glochidia and are not fully examined and published yet (Niklas Wengström, personal communication). In Virån, which is one of the examined streams in this study, investigations concerning suitable hosts have been performed before with analysis of attached glochidia from fish infested in situ. In this study R. rutilus, A, alburnus and Lota lota are considered to be possible hosts with A. alburnus as the most important one (Wengström, 2009). Although, C. gobio and P. phoxinus are believed to be the most suitable and functional hosts for U. crassus in Sweden, mostly because they dominate streams were U. crassus have successful reproduction (Lundberg et al., 2008) both C. gobio and P. phoxinus are resident and potential host species which generally stay in smaller areas of their stream

5 or lake throughout the year. Exceptionally C. gobio can migrate longer distances, usually downstream, if suitable habitats are already occupied (Frost, 1943 & Junker 2010). Incompatible host fish If a glochidium attach to an incompatible host it will be expelled after a few days. This can occur even on suitable hosts because of an immune response with antibody production against glochidial tissue (Meyers et al., 1980, O Connell & Neves, 1999). It is suggested that the extent of the immune response depends on both fish condition (Frost, 1943) and if the host has been infested before (Nagel, 1991). Thus, both condition and size can affect the intensity of infestation. Determination of host fish There are two methods that are commonly used to determine suitable hosts for fresh water mussels; either by collecting fish in the wild and identifying the encysted glochidia by molecular methods; or artificially infesting different species of fish and identify the juvenile mussel after completed metamorphosis (Khym & Layzer 2000). Because glochidia can be encysted without completing metamorphosis there is a risk of misjudgment in terms of functional hosts (Jansen et al., 2001). Using infestation in laboratory gives understanding of functional hosts under standardized conditions that will not occur in nature (Taeubert et al., 2012). Thus, both methods lack information needed to evaluate functional hosts in natural conditions. According to Blažek & Gelnar (2006) the only way to clarify the importance of host species in wild mussel populations is to identify glochidia from fish caught from field surveys. Identifying mussels and glochidia Identification of adult fresh water mussels is usually done looking at morphologic characters on the shell. Identification can also be done on glochidia by scanning electron microscopy (Pekkarinen & Englund 1995) but due to the variation of the characters within species there is a risk of misidentification (O Brien et al 2003). One alternative for both adult mussels and their glochidia to avoid misidentification is by using molecular techniques and analysis of the DNA (Kandl et al., 2001). One can choose to sequence all DNA or analyze parts of interest depending on the question and what is most time and cost efficient without affecting the result. One method to determine fresh water mussels is to analyze the length of the gene ITS-1, or internal transcribed spacer, which is a noncoding gene of the ribosomal DNA. ITS-1 is particularly effective when DNA yield is low (Mindell & Honeycutt 1990 & White et al., 1994). There are several methods to analyze the length of ITS-1. The method used in this study derives from the work done by Zieritz et al., 2012 and further developed at University of Skövde (Heléne Lindholm, personal communication). Conservation The main focus when trying to conserve and restore populations of U. crassus in Europe has been to protect areas and habitats where they appear and eliminate water pollutions without investigating whether or not suitable hosts are present (Engel & Wachtler, 1989). In addition to the restoration and conservation, knowledge regarding compatible hosts is vital when trying to rescue the unionid mussels (Williams et al., 1993, King et al., 1999).

6 Most studies and data related to host suitability for U. crassus originate from artificial infestations (Bednarczuk, 1986, Maaß, 1987, Hochwald, 1997 & Taeubert et al., 2012) which not necessarily reflect the situation under natural conditions. To my knowledge, no study has yet tested the metamorphosis success of U. crassus from fish infested in situ. To hatch juvenile mussels, which have completed successful metamorphosis, from fish infested under natural conditions would give a more reliable result. Aim and questions The primary aim of this study was to determine functional hosts for U. crassus in its natural habitat by analysis of hatched juvenile mussels from fish infested in situ. Hopefully the results will give useful information when trying to restore and protect U. crassus and give answers to following questions: 1. Which species of fish are functional hosts for U. crassus? 2. Are there any local differences between the two examined streams regarding functional hosts? 3. Do the mussels have any preferences in terms of fish size of their compatible hosts? Method and Material Investigated streams Virån (fig. 1) is located in the southeast of Sweden (RT , ) and has its well flow in Hultsfred and flows into the Baltic Sea, close to Figeholm. Virån within the county of Kalmar is protected as a Natura 2000 area. The protected parts measure app. 40 km (excluding lakes) and show big variations in surrounding landscape with elements of coniferous forest, deciduous forest, agriculture and partly populated areas. Virån contains many rare and typical species directly associated to water (eg. otter, Lutra lutra, kingfisher, Alcedo atthis). Nesting birds, rare algae and rich flora together with four species considered in the annexes II and IV of the EU Habitat Directive 92/43/EEC contributes to the protection of the area. One of them is U. crassus which is considered in the EU Habitat Directive. There have also been findings of Pseudanodonta complata (Musselportalen, 2015), which is redlisted on both the global and the Swedish redlist. In the conservation plan performed by county administration of Kalmar the status of U. crassus is, despite the decline, considered to be stable with regular reproduction of both mussels and host-fish in areas of favorable biotope. Thus, Virån is suitable for research regarding the interaction between host and mussel.

7 Svennevadsån (fig. 1) is located in the county of Örebro 35 km south of Örebro city (RT , ) and belongs to the Basin of Nyköpingsån, which eventually meet the Baltic Sea. The stream is protected as a Natura 2000 area mostly because of the presence of reproducing U. crassus, the northernmost known population in Sweden. As in Virån, Svennevadsån has populations of the redlisted P. complanata (Musselportalen 2015). The majority of Svennevadsån is slow flowing with mostly deciduous forests and bogs in the surrounding environment. In contrast to Virån, with its 20 species of fish, Svennevadsån has a scarce composition of fish. During electrofishing 2005 five species of fish were caught; C. gobio, Esox lucius, P. fluviatilis, R. rutilus, L. lota and G. cernuus. Svennevadsån is in many aspects similar to Virån apart from fish composition and geographic position, which makes Svennevadsån an interesting object of study. Fig. 1 Map with visited streams marked with red stars. Field survey Fish were collected by standardized electrofishing (Degerman, E. & Sers, B. 1999) from Svennevadsån in Örebro and Virån in Oskarshamn. In both streams four localities were fished in the beginning of June (table 1). All fish were measured for weight (g), length (mm) and infestation intensity (classified in four groups; 0=0 glochidia; 1<10 glochidia; 2= glochidia; 3>100, using a stereomicroscope). Only fish with an obvious infestation of glochidia were kept for further study. Approximately half of the infested fish were used in the hatchery and the other half were sacrificed for glochidial DNA analysis.

8 Table 1. The eight visited localities where fish was collected with associated coordinates (RT90). Stream Locality Date Coordinates (RT90) Svennevadån Lagmansbacka Svennevadån Restaureringslokal Svennevadån Nedan Skogasjön Svennevadån Mussellokal Virån Mussellokal ovan Bosjön Virån Ovan vägbro Virån Nedan E Virån Ovan Bosjön Hatchery The fish were brought in aerated tanks into the hatchery at SJölyckan, Göteborg, where two large containers measuring 200 x 200 cm and a height of 52 cm with a constant water level of 10 cm were used. In each large container aquariums measuring 38 x 48 cm with a height of 40 cm were set up on cement blocks just above the water level in the large tanks. A constant supply of fresh water from the adjacent Delsjön ran to each aquarium from a pipe above. The aquariums were equipped with a well centered at the bottom with a strainer with a mesh size of 2,5 mm. Water from each aquarium was transported from the well through a 40 mm wide pipe to a smaller filter container measuring 24 x 32 cm and a height of 15 cm standing next to each aquarium. The filter in the small containers had a mesh size of 25 μm, enough to catch eventual juvenile mussels. Because the small filter containers stood at the bottom of the large tanks they obtained the same constant water level of 10 cm as the large tanks. The water level in the aquariums was adjusted changing the height and angle of the pipes (fig. 2). Fig. 2 Drawning of the hatchery at Sjölyckan, Gothenburg.

9 Fish from Virån arrived and fish from Svennevadsån One species of fish from one stream was placed in each aquarium (eg. perch from Virån). Fish were fed every third day with either maggot or worms and all aquariums and filter containers were checked for eventual mussels. The filter was carefully rinsed and gathered material was put in a tin. Material at the bottom of aquarium was collected using a siphon and put in a second tin. The tins were filtered again to get rid of redundant water and the remaining material was put in a petri dish. The material was checked for juvenile mussels with a stereomicroscope. All found mussels were given a unique ID-number and information regarding species of host-fish, stream and date was noted for each mussel. The mussels were also categorized as dead or alive based on their activity and movement of the foot observed. Collected mussels were put in separately Eppendorf tubes in 95 % ethanol and kept in cooler at 5 C. The temperature could not be regulated in the hatchery, but followed the temperature in the adjacent Delsjön. After the experiment the remaining fish were sacrificed and put in 95 % ethanol for further study. Sacrificed infested fish from the two streams and the hatchery were checked more closely in laboratory environment with a stereomicroscope. All encysted glochidia were counted on gills and fins of each fish. Gills and fins with encysted glochidia were removed using pincette or scissor and put in tubes with 95 % ethanol. Each tube was marked with fish-id, date, stream, locality and number of glochidium. Calculations and statistics Infestation abundance (infested fish/total number of fish), infestation intensity (glochidia/infested fish) and mean intensity of infestation (average number of glochidia/infested fish) were calculated for each stream. Correlation between fish length and infestation intensity was tested with a Poission regression, scaled by deviance (11,752) as a compensation for overdispersion (Joacim Näslund, personal communication). C. gobio was used as model species in the correlation test. Difference of mean intensity of infestation between the two streams was tested with independent t-test (equal variances not assumed). DNA-analysis Briefly, glochidia attached to the prepared gills and fins were removed and put in Eppendorf tubes and DNA isolated (QuickExtract DNA extraction solution, Epicentre). The same procedure was performed with material from juvenile mussels collected from the hatchery at Sjölyckan. In each PCR reaction 2 μl of the glochidia-dna and a PCR mastermix (Failsafe PCR System, Epicentre) with added ITS-1 specific primers (Zieritz et al., 2012) was used. After completed PCR at university of Skövde all samples were sent to Karolinska Institutet where the length of the amplified ITS-1 gene product was determined and compared to the expected lengths for each species obtained from their Genbank entries.

10 Results From Svennevadsån 66 fish were caught, 25 were used in the hatchery, 26 were sacrificed for glochidial analysis and 15 released. From Virån 61 fishes were caught, 33 were used in the hatchery, 14 were sacrificed for glochidial analysis and 14 released (table 2). Table 2. Summary of collected fish from both Virån and Svennevadsån. Virån Svennevadsån Hatchery Killed Released Hatchery Killed Released C. gobio P. fluviatilis R. rutilus A. alburnus L. planeri G. cernuus S. trutta L. lota Total The hatchery produced 26 juvenile mussels from five different species of fish; four hatched from fish from Svennevadsån and 22 from Virån. The first juvenile mussel of U. crassus was found in the hatchery after 14 days and the last one after 27 days. There were no juvenile mussels found the last seven days of the experiment and the experiment ended after 38 days. The average water temperature in the facility was 17,25 C with 15 C in the start of the experiment and 19,1 C on the last day of the experiment. Svennevadsån Fish from Svennevadsån showed an infestation abundance of %. The fish that were sacrificed had a total number of 370 glochida attached to gills and fins. All were attached to C. gobio. 142 of them were used for further DNA-analysis and 103 gave readable results (table 3). 98 % were U. crassus 2 % were Unio tumidus. The two findings of U. tumidus were attached to fish infested also with U. crassus. In the hatchery four juvenile mussels were observed and collected. They all derived from C. gobio and two of them were categorized as dead and two as alive when found. All four of them were analyzed for identification and two gave readable result that showed to be P. complanata. Nine of the fish in hatchery from Svennvadsån had glochidia attached when the experiment ended. A total number of 200 glochidia were observed on the nine fish and 52 of them were analyzed for identification. Six of them gave no readable result. One showed to be U. crassus and was attached to C. gobio. The rest gave a result that could not be related to freshwater mussels.

11 Virån Fish from Virån showed an infestation abundance of approximately 70 %. The sacrificed fish had 4305 glochidia attached to fins and gills. Glochidia were found on Burbot, L. lota, Perch, P. fluviatilis and Roach, R. rutlius glochidia were attached to one single fish and were counted as glochidia despite uncertain identification during counting. 216 of the 4305 were used for further DNA-analysis and 190 gave readable result. In the hatchery 22 juvenile mussels were hatched from fish from Virån. All were analyzed and 19 gave readable result. Glochidia attached to L. lota were U. tumidus and U. crassus. All of the glochidia found on P. fluviatilis were U. tumidus. Four different glochida were found on R. rutilus; U. tumidus; Adonata cygnea or Pseudanodonta complanata; U. crassus and Unio pictorum (fig. 3). Fig. 3 Distribution of glochidia on fish from Virån (n=190). Hatched juvenile mussels from Virån derived from five different species of fish. Three juvenile mussels from L. lota where only one gave readable result from the DNA-analysis showed to be U. tumidus. 12 juvenile mussels from P. fluviatilis where 11 gave readable result were all U. tumidus. S. trutta and C. gobio each dropped one juvenile mussel were both showed to be U. tumidis. Eight mussels derived from A. alburnus were five gave readable result. One was U. tumidus and four were U. crassus (fig. 4).

12 Fig. 4 Hatched juvenile mussels from Virån with number of mussels on y-axis and species of fish on x-axis (n=25). Four of the fish in hatchery from Virån had glochidia attached when the experiment ended. A total number of 1218 glochidia were observed on the four killed fish and 26 of them were analyzed for identification (table 4) 1200 of them were attached to one of the fishes. Six gave no readable result, 15 were U. tumidus attached to L. lota, A. alburnus and C. gobio. The rest (four) were A. cygnea or P. complanata attached to L. lota and A. alburnus. Length and infestation intensity C. gobio showed a variation in infestation intensity (fig. 5). There was a significant positive correlation between length of C. gobio (n=24) and infestation intensity (X 2 =4.564, p=0.033).

13 Figur 5. Histogram over distribution of glochidia on C. gobio. Comparison There was no significant difference (t=1.186, p=0.258) of mean intensity of infestation between the two streams (fig. 6). Fig. 6 Comparison of mean intensity of infestation (y-axis) between Virån (n=11) and Svennevadsån (x-axis, n=24).

14 Discussion The primary aim of this study was to confirm functional host for juvenile freshwater mussels from fish infested in their natural habitat. At hatchery, 26 juvenile mussels were successfully hatched, four from Svennevadsån and 22 from Virån. Hatched juvenile mussels show that Bleak, A. alburnus can be confirmed as functional host to U. crassus in Virån. Wengström (2009) suggested Bleak, A. alburnus to be the most important host fish to U. crassus in Virån. My study can confirm that it is indeed a functional host in Virån. C. gobio is considered to be a generally suitable host fish to U. crassus in Sweden, mostly because it is present in many of the streams with populations of U. crassus (Lundberg et al., 2008). Surprisingly the hatchery failed to confirm C. gobio as a functional host for U. crassus in Svennevadsån despite the large amount of infested C. gobio. Nevertheless, C. gobio seems to be an important and, most likely, a functional host for U. crassus in Svennevadsån. Two of the localities fished during field survey have relatively high densities of U. crassus with detected recruitment. C. gobio was the only species caught in these localities, indicating that C. gobio in fact is a functional host. In addition, almost all of the sacrificed and examined C. gobio from those localities were infested with U. crassus. Result from the hatchery also show that Bleak, A. alburnus, Burbot, L. lota, Perch, P. fluviatilis, Trout, S. trutta and Bullhead, C. gobio are functional hosts for U. tumidus in Virån and Bullhead, C. gobio can be confirmed as functional host for U. tumidus and P. complanata in Svennevadsån. Analysis of attached glochidia provides answers only to possible hosts and statements of higher dignity should be done carefully. Attached and encysted glochidia will not necessarily develop into juvenile mussels. Still, the method of glochidial analysis is useful and gives clues to which fish might be functional host. It is also less time consuming and has fewer steps compared to the hatchery method. Analyzed glochidia in this study show that L. lota and R. rutilius are possible hosts for U crassus in Virån which is also suggested in the study from Wengström (2009). It contradicts the German studies where none of these species have been suggested as possible hosts (Nagel, 1990 & Hochwald, 1997). It can depend on the fact that most of the German studies have used a subspecies (U. crassus cytherea) in their studies. To determine whether or not L. lota and R. rutilus is functional hosts for U. crassus a method like the hatchery used in this study would be appropriate. Some of the glochidia from Virån gave results that indicated on either P. complanata or A. cygnea. A. cygnea has been observed close to the locality where fish infested with these glochida were collected (Musselportalen, 2015) which suggests that it is A. cygnea. The correlation between length and infestation intensity gave a significant result (p=0.033) using C. gobio as a model species to reflect this. From an anatomical view larger fish have larger areas where glochidia could attach, which is one explanation to the correlation. Most likely the distribution of glochidia can be due to many other factors

15 (eg. immune response, behavior) so the question to why some specimens have higher infestation intensity remains unsolved. According to Taeubert et al., (2014) fish infested with U. crassus held in a temperature of 17 C release their mussels after days. The average water temperature in the hatchery was 17,25 C suggesting that all U. crassus should have released after four weeks. To be on the safe side the experiment went on for 38 days and the remaining fish were killed and examined for attached glochidia. The experiment was closed down both because there were no findings of juvenile mussels several days in a row and because the fish started to die due to fungal infection. One of the fish in the hatchery had U. crassus still attached when the experiment ended suggesting that U. crassus can stay attached longer than what Taeubert et al., (2014) demonstrate. It is not known if glochidia attached for longer period than known to be normal will develop into a juvenile mussel. The differences in terms of fish composition in Svennevadsån and Virån are reflected in the results where Virån showed a larger number and different species of possible hosts. Virån and Svennvadsån both have recruiting populations of U. crassus and regardless of their differences in fish composition they show almost equal abundance of infestation, approximately % and no significant difference of mean intensity of infestation was observed. The abundance of infestation is calculated from the infestation intensity determined during field survey. Due to the difficulty to examine the infestation on Bullheads, C. gobio, as they have an extra skin flap on their operculum, the calculation of abundance cannot be done exactly without harming the fish. Thus, the abundance of infestation is an estimate. The hatchery was a successful method but had its flaws. There were mainly two big problems observed. The incoming water contained much detritus, which made it hard to detect juvenile mussels in the samples. Filtration of the incoming water would solve this problem. In some aquariums, especially the one with L. lota, the water was much cleaner and almost all detritus was completely gone. I suspect that the fish ate the detritus. It made samples much cleaner and observation for juvenile mussels easier but most likely some of the juvenile mussels also became food for the fish. A horizontal net the size of the aquarium a few centimeters above the bottom should prevent the unwanted eating. With an improved design the hatchery will most likely be useful as a method to determine host fish but also when hatching juvenile mussels with the purpose to boost weak populations with none or scarce recruitment. This study, among others with focus on unionid mussels, shows the importance of biodiversity and conservation and restoration of natural habitats. When trying to conserve rare and threatened species and environments the common species is equally important because of the interaction between them.

16 Conclusions This study has expanded the knowledge of the interaction between fish and freshwater mussels with several confirmed host fish for different species of fresh water mussels. For instance, A. alburnus can be confirmed as a functional host for U. crassus in Virån and C. gobio is most likely a functional host for U. crassus in Svennevadsån. Hatchery of juvenile mussels can be done with simple methods and is a suitable method in future conservation of unionid mussels. The study also demonstrates the importance of host fish surveys in each stream or population due to the observed differences of host fish preferences between populations. Acknowledgements Without some people this study could not have been completed. Big thanks to Niklas Wengström (external supervisor), Johan Höjesjö (internal supervisor), University of Skövde with Heléne Lindholm, Annie Jonsson and Mikael Ejdebäck (DNA-analysis), Sportfiskarna (lending of local and equipment) and my family.

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