Michal B&#237;l&#253; and Ond&#345;ej P. Simon were supported by grants from the Czech University of Life Science [Internal Grant Agency of Faculty of Environmental Sciences, CULS Prague (42110 1312 3175 (20164236))]. Support for Karel Douda came from the Czech Science Foundation (13-05872S). Data on the bioindication and present occurrence of pearl mussels were collected during the implementation of the Czech Action Plan for Freshwater Pearl Mussels managed by the Nature Conservation Agency of the Czech Republic, which is funded by the government of the Czech Republic and is available at

1. Mesh Cage
Note: See Figure 1.
<ol>
	<li>Prepare material
	<ol>
		<li>Prepare the material for the in-laboratory part of the experiment: ~1 - 2 L of river water per mesh cage, mesh cages (1 main plastic body, 2 plastic covers, 2 sheets of special technical sieves with 340 &#181;m pores, 4 bolts and 4 nuts per cage), pliers, a spanner, Pasteur pipettes, a strainer, a digital camera, a trinocular dissecting zoom stereo microscope, a calibration ...

<ol>
	<li>Goldberg, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mussel+watch-a+first+step+in+global+marine+monitoring.">The mussel watch-a first step in global marine monitoring.</a> Marine Pollution Bulletin. 6 (7), 111-114 (1975).</li><li>Vaughn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecosystem+services+provided+by+freshwater+mussels.">Ecosystem services provided by freshwater mussels.</a> Hydrobiologia. , (2017).</li><li>Lopes-Lima, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+status+of+freshwater+mussels+in+Europe:+state+of+the+art+and+future+challenges.">Conservation status of freshwater mussels in Europe: state of the art and future challenges.</a> Biological Reviews. 92 (1), 572-607 (2017).</li><li>Strayer, D. L., Malcom, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+recruitment+failure+in+freshwater+mussel+populations+in+southeastern+New+York.">Causes of recruitment failure in freshwater mussel populations in southeastern New York.</a> Ecological Applications. 22 (6), 1780-1790 (2012).</li><li>Douda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+nitrate+nitrogen+pollution+on+Central+European+unionid+bivalves+revealed+by+distributional+data+and+acute+toxicity+testing.">Effects of nitrate nitrogen pollution on Central European unionid bivalves revealed by distributional data and acute toxicity testing.</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 20 (2), 189-197 (2010).</li><li>Modesto, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+and+mussels:+importance+of+fish+for+freshwater+mussel+conservation.">Fish and mussels: importance of fish for freshwater mussel conservation.</a> Fish and Fisheries. , (2017).</li><li>Bauer, G., W&#228;chtler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ecology and evolution of the freshwater mussels Unionoida. 145, 1-394 (2001).</li><li>Neves, R. J., Widlak, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habitat+ecology+of+juvenile+fresh-water+mussels+(Bivalvia,+Unionidae)+in+a+headwater+stream+in+Virginia.">Habitat ecology of juvenile fresh-water mussels (Bivalvia, Unionidae) in a headwater stream in Virginia.</a> American Malacological Bulletin. 5, 1-7 (1987).</li><li>&#352;vanyga, J., Simon, O. P., Min&#225;rikov&#225;, T., Spisar, O., B&#237;l&#253;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Z&#225;chrann&#253; program pro perlorodku &#345;&#237;&#269;n&#237; v &#268;R (Action plan for the endangered freshwater pearl mussel in the Czech Republic). , (2013).</li><li>Schmidt, C., Vandr&#233;, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ten+years+of+experience+in+the+rearing+of+young+freshwater+pearl+mussels+(Margaritifera+margaritifera).">Ten years of experience in the rearing of young freshwater pearl mussels (Margaritifera margaritifera).</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 20 (7), 735-747 (2010).</li><li>Gum, B., Lange, M., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+reflection+on+the+success+of+rearing+and+culturing+juvenile+freshwater+mussels+with+a+focus+on+the+endangered+freshwater+pearl+mussel+(Margaritifera+margaritifera+L.).">A critical reflection on the success of rearing and culturing juvenile freshwater mussels with a focus on the endangered freshwater pearl mussel (Margaritifera margaritifera L.).</a> Aquatic Conservation: Marine and Freshwater Ecosystems. 21 (7), 743-751 (2011).</li><li>Denic, M., Taeubert, J. E., Lange, M., Thielen, F., Scheder, C., Gumpinger, C., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+stock+origin+and+environmental+conditions+on+the+survival+and+growth+of+juvenile+freshwater+pearl+mussels+(Margaritifera+margaritifera)+in+a+cross-exposure+experiment.">Influence of stock origin and environmental conditions on the survival and growth of juvenile freshwater pearl mussels (Margaritifera margaritifera) in a cross-exposure experiment.</a> Limnologica. 50, 67-74 (2015).</li><li>&#268;ern&#225;, M., Simon, O. P., B&#237;l&#253;, M., Douda, K., Dort, B., Galov&#225;, M., Volfov&#225;, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Within-river+variation+in+growth+and+survival+of+juvenile+freshwater+pearl+mussels+assessed+by+in+situ+exposure+methods.">Within-river variation in growth and survival of juvenile freshwater pearl mussels assessed by in situ exposure methods.</a> Hydrobiologia. , (2017).</li><li>Denic, M., Taeubert, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+relationships+between+the+larvae+of+two+freshwater+mussels+and+their+fish+hosts.">Trophic relationships between the larvae of two freshwater mussels and their fish hosts.</a> Invertebrate Biology. 134 (2), 129-135 (2015).</li><li>Douda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host-dependent+vitality+of+juvenile+freshwater+mussels:+implications+for+breeding+programs+and+host+evaluation.">Host-dependent vitality of juvenile freshwater mussels: implications for breeding programs and host evaluation.</a> Aquaculture. 445, 5-10 (2015).</li><li>Buddensiek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+juvenile+freshwater+pearl+mussels+Margaritifera+margaritifera+L.+in+cages:+a+contribution+to+conservation+programmes+and+the+knowledge+of+habitat+requirements.">The culture of juvenile freshwater pearl mussels Margaritifera margaritifera L. in cages: a contribution to conservation programmes and the knowledge of habitat requirements.</a> Biological Conservation. 74 (1), 33-40 (1995).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+semi-natural+breeding+program+of+freshwater+pearl+mussel+in+the+Czech+Republic.">Experience of semi-natural breeding program of freshwater pearl mussel in the Czech Republic.</a> Die Flussperlmuschel in Europa: Bestandssituation und Schutzma&#223;nahmen. , 69-75 (2001).</li><li>Barnhart, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Buckets+of+muckets:+a+compact+system+for+rearing+juvenile+freshwater+mussels.">Buckets of muckets: a compact system for rearing juvenile freshwater mussels.</a> Aquaculture. 254 (1), 227-233 (2006).</li><li>Simon, O. P., Van&#237;&#269;kov&#225;, I., B&#237;l&#253;, M., Douda, K., Patzenhauerov&#225;, H., Hru&#353;ka, J., Pelt&#225;nov&#225;, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+status+of+freshwater+pearl+mussel+in+the+Czech+Republic:+several+successfully+rejuvenated+populations+but+the+absence+of+natural+reproduction.">The status of freshwater pearl mussel in the Czech Republic: several successfully rejuvenated populations but the absence of natural reproduction.</a> Limnologica. 50, 11-20 (2015).</li><li>. A language and environment for statistical computing Available from: <a target="_blank" href="https://www.r-project.org/">https://www.r-project.org/</a> (2013)</li><li>Hastie, L. C., Yang, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Conservation of the freshwater pearl mussel I: captive breeding techniques. 2, (2003).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nahrungsanspr&#252;che+der+Flu&#223;perlmuschel+und+deren+halbnat&#252;rliche+Aufzucht+in+der+Tschechischen+Republik.">Nahrungsanspr&#252;che der Flu&#223;perlmuschel und deren halbnat&#252;rliche Aufzucht in der Tschechischen Republik.</a> Heldia. 4 (6), 69-79 (1999).</li><li>Scheder, C., Lerchegger, B., Jung, M., Csar, D., Gumpinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+experience+in+the+rearing+of+freshwater+pearl+mussels+(Margaritifera+margaritifera):+advantages+of+a+work-saving+infection+approach,+survival,+and+growth+of+early+life+stages.">Practical experience in the rearing of freshwater pearl mussels (Margaritifera margaritifera): advantages of a work-saving infection approach, survival, and growth of early life stages.</a> Hydrobiologia. 735 (1), 203-212 (2014).</li><li>Braun, A., Auerswald, K., Geist, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drivers+and+spatio-temporal+extent+of+hyporheic+patch+variation:+implications+for+sampling.">Drivers and spatio-temporal extent of hyporheic patch variation: implications for sampling.</a> PLoS ONE. 7 (7), e42046 (2012).</li><li>Franken, R. J. M., Storey, R. G., Williams, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological,+chemical+and+physical+characteristics+of+downwelling+and+upwelling+zones+in+the+hyporheic+zone+of+a+north-temperate+stream.">Biological, chemical and physical characteristics of downwelling and upwelling zones in the hyporheic zone of a north-temperate stream.</a> Hydrobiologia. , 183-195 (2001).</li><li>Roley, S. S., Tank, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pore+water+physicochemical+constraints+on+the+endangered+clubshell+mussel+(Pleurobema+clava).">Pore water physicochemical constraints on the endangered clubshell mussel (Pleurobema clava).</a> Canadian Journal of Fisheries and Aquatic Sciences. 73 (12), 1712-1722 (2016).</li><li>Larson, J. H., Eckert, N. L., Bartsch, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+variability+in+shell+and+soft+tissue+growth+of+the+freshwater+mussel+Lampsilis+siliquoidea.">Intrinsic variability in shell and soft tissue growth of the freshwater mussel Lampsilis siliquoidea.</a> PLoS ONE. 9 (11), e112252 (2014).</li><li>Lavictoire, L., Moorkens, E., Ramsey, A. D., Sinclair, W., Sweeting, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+substrate+size+and+cleaning+regime+on+growth+and+survival+of+captive-bred+juvenile+freshwater+pearl+mussels,+Margaritifera+(Linnaeus,+1758).">Effects of substrate size and cleaning regime on growth and survival of captive-bred juvenile freshwater pearl mussels, Margaritifera (Linnaeus, 1758).</a> Hydrobiologia. 766 (1), 89-102 (2015).</li><li>Hru&#353;ka, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience+of+semi-natural+breeding+programme+of+freshwater+pearl+mussel+in+the+Czech+Republic.">Experience of semi-natural breeding programme of freshwater pearl mussel in the Czech Republic.</a> Die Flussperlmuschel in Europa: Bestandssituation und Schutzmassnahmen. , 69-75 (2000).</li><li>Bayne, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+components+of+growth+differences+between+individual+oysters+(Crassostrea+gigas)+and+a+comparison+with+Saccostrea+commercialis.">Physiological components of growth differences between individual oysters (Crassostrea gigas) and a comparison with Saccostrea commercialis.</a> Physiological and Biochemical Zoology. 72 (6), 705-713 (1999).</li><li>Tamayo, D., Azpeitia, K., Markaide, P., Navarro, E., Ibarrola, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+regime+modulates+physiological+processes+underlying+size+differentiation+in+juvenile+intertidal+mussels+Mytilus+galloprovincialis.">Food regime modulates physiological processes underlying size differentiation in juvenile intertidal mussels Mytilus galloprovincialis.</a> Marine Biology. 163 (6), (2016).</li></ol>

Exposure time:
Even one-month exposed mesh cages show a visible growth reflecting differences between localities (Figure 3), so they are very usable for the quick and easy detection of a locality characterization. Nevertheless, the relevance of the results depends on the short-term state of the conditions, which can oscillate. In particular, short rainfall events can play a role. In contrast, unpredictable episodic pollution may not always be recorded. In locality V ...

The exposure of experimental organisms in situ with the subsequent evaluation of their condition is one possible way to get information about the environmental quality and (especially) the site suitability for a species. Within animals, such a bioindication is applicable primarily for small invertebrates which are able to live in a limited bounded space. Young stages of bivalves (Bivalvia) are one such suitable organism group1.
Bivalves of the family Unionidae are a very important component of aquatic ecosystems2. However, these species are often critically endangered, especially in streams and rivers. Some of them are characterized as &#39;umbrella species&#39; whose conservation is closely related to the conservation of the whole stream biotope and which require a comprehensive approach3. These animals have a life cycle associated with many environment components, from water chemistry4,5 to changes in the populations of fish which serve as mussel larvae hosts6. Because mussel juveniles often represent a critical phase of the mussel life cycle, the site suitability for their development at this stage is crucial for a successful species population development in a locality.
The freshwater pearl mussel (FWPM, Margaritifera margaritifera; Unionida, Bivalvia) is a critically endangered bivalve occurring in oligotrophic European streams. Their numbers have fallen drastically during the 20th century across the occurrence area. It seems that the current decline in species reproduction in the majority of the central European populations is primarily caused by very low to zero survival of juveniles during the first few years of their life. It is assumed that juvenile FWPMs live for many years in the shallow hyporheic zone7, of which the conditions and their variability are still not well described. Moreover, until their second year of life, the juveniles only have a dimension of up to about 1 mm, so they are very difficult to find in large volumes of sediment under natural conditions8. Therefore, experiments with captive juveniles are necessary for the study of their ecology.
Within the Czech Action Plan for Freshwater Pearl Mussel9, there are thousands of juveniles rising every year from a semi-natural breeding program. Nevertheless, there is a question of which localities and habitats are suitable for successful population support by these juveniles or for eventual species reintroduction. In situ bioindications present a way of finding the answer.
Despite the fact that inconsistent survival rates of juvenile mussels in exposure cages were observed in some earlier works that questioned the suitability of juvenile mussels as bioindicators10, several recent studies have confirmed the applicability of juvenile exposure methods for water quality testing11,12,13. Additionally, it has been demonstrated that several factors need to be considered when interpreting the results of these particular studies, such as the stock origin14 and the persisting effects of larval conditions15.
The question arises of how to install experimental juveniles in tested localities and how to most effectively evaluate their condition. The first rigorous application of in situ exposure methods with juvenile FWPMs was published by Buddensiek16. Juvenile FWPM individuals were kept in sheet cages, exposed in the free-flowing water of streams, and their survival and growth were quantified after several weeks of exposure. The approach was originally developed as a semi-artificial breeding method, but the author also highlighted its applicability for the assessment of habitat requirements and water quality. Although the FWPM juvenile survival is naturally very low on a scale of months/years and only a very small number of animals will survive, the survival rate can be a good marker of the environmental effect on a scale of several weeks16. Over years of research, exposure methods were developed further to hold experimental juvenile mussel in-stream habitats and to evaluate their growth and survival rates; these include sandy boxes17, mussel silos based on an upwelling principle18, and various other exposure cages (summarized by Gum and colleagues)11. Because juveniles occur naturally in shallow hyporheic zone7, the application of experimental devices within the stream bottom is very desirable.
In our article, we describe the use of two exposure devices for FWPMs: i) modified Buddensiek sheet cages (&#34;mesh cages&#34;) also enabling bioindication testing in hyporheal conditions; and ii) Hru&#353;ka sandy boxes (&#34;sandy cages&#34;). The protocol describes the application of both methods in open water and hyporheic conditions (i.e., four variants of exposure are described). The methods were gradually modified and expanded over more than 15 years of application within the Czech Action Plan for Freshwater Pearl Mussel9 and verified by a set of experiments.

<table>
	<tbody>
		<tr>
			<td>biological material maintenance and care</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freshwater pearl mussel juveniles</td>
			<td>any</td>
			<td>NA</td>
			<td>from a FWPM breeding programme</td>
		</tr>
		<tr>
			<td>plastic boxes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>thermobox</td>
			<td>MERCI</td>
			<td>212,070,600,030</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>field thermobox (ca25 l)</td>
			<td>any</td>
			<td>NA</td>
			<td>cold box (insulated box) commonly used for food transport</td>
		</tr>
		<tr>
			<td>river water</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic Pasteur pipettes with balloon bulb (droppers)</td>
			<td>any</td>
			<td>NA</td>
			<td>hole diameter 1 mm</td>
		</tr>
		<tr>
			<td>hydrogen peroxide</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic container (ca 50 l) for river water</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic tea strainer</td>
			<td>any</td>
			<td>NA</td>
			<td>commonly used in kitchen</td>
		</tr>
		<tr>
			<td>mesh cages construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>main plastic bodies</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plactic covers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 340 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 20 T</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 100 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 67 M</td>
			<td></td>
		</tr>
		<tr>
			<td>rubber hose (diameter&#160; 5.5 mm)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel bolts</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel nuts</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>spanner</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>steel spikes</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>pliers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>beakers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic dishes (ca. 25x15x3-5cm)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>squirt bottle</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>field protocols</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>stationery</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic container</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>string</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>hammer</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>sandy cages construction and use</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sieve 1 mm</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>sieve 2 mm</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>special technical sieves 340 &#181;m</td>
			<td>Silk &#38;Progress</td>
			<td>UHELON 20 T</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic boxes with tight-fitting lid</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>hot melt adhesive</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic box (ca 250 x 150 x 100 cm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>big plastic box (ca 25 l)</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>flat stone</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>net</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>river sand</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>round containers</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>magnifying glasses</td>
			<td>Carson</td>
			<td>Carson CP 60</td>
			<td>There ar many possibilities. This is one example only</td>
		</tr>
		<tr>
			<td>cages installation and maintenance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>field temperature dataloggers</td>
			<td>ONSET</td>
			<td>UA-001-64</td>
			<td>http://www.onsetcomp.com/products/data-loggers/ua-001-64</td>
		</tr>
		<tr>
			<td>spade</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>toothbrush</td>
			<td>any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>experiment evaluation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>trinocular dissecting zoom stereo microscope</td>
			<td>Bresser optic</td>
			<td>ICD 10x-160x</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>digital camera/ electronic eyepiece</td>
			<td>Bresser optic</td>
			<td>MikroCamLab 5M</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>Calibration&#160; gird</td>
			<td>Am Scope</td>
			<td>SKU: MR100</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>external power source with two movable light guides</td>
			<td>Arsenal</td>
			<td>K1309010150021</td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>Image software</td>
			<td>ImageJ software</td>
			<td></td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
		<tr>
			<td>table processor</td>
			<td>MS excel</td>
			<td></td>
			<td>There are many possibilities. This is one example only.</td>
		</tr>
	</tbody>
</table>

bioindication testing of stream environment suitability for young freshwater pearl mussels using in situ exposure methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

The four bioindication methods (open water sandy cages, within-bed sandy cages, open water mesh cages, and within-bed mesh cages) were applied to investigate the environment condition suitability for FWPMs in the upper Vltava River Basin (Bohemian Forest, Czech Republic). This river represents one FWPM residual locality within central Europe19. Here, we present a specially selected set of results illustrating the most important aspects of the four methods. Further ...

Watch this Scientific Journal Video about Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods at JoVE.com

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

This method can help answer key questions in the river ecosystem preservation field such as indications of stages favorable for young mussels that augment as well, as prediction of this which I propagate. The main advantage of this technique is that we can test juvenile growth and survival in hyporheic conditions which represent the natural juvenile environment. Demonstrating the procedures will be my colleagues Simona Nemcikova, Jan Svanyga, and Vojtech Barak.When assembling the cages, always insert one plastic cover first, then one sheet of the plastic sieve and finally the main body on top. Put the cage into the plastic dish containing river water at the FWPM storage temperature. Ensure that the chambers are half full.Next, load the FWPMs into a petri dish. Using a squirt bottle and strainer, sift through the juveniles to clear the detritus. Then, pipet one individual from the dish and transfer it to a dish under the microscope to check it's fitness.Select only FWPM of good fitness. Take two photographs of each laid length wise at about 80 times magnification with the goal of measuring maximum shell length. Proceed to load all the appropriate chambers of the cage with the selected and documented FWPM.Then, put the plastic sieve on the cage followed by the plastic cover and secure all the parts with the nuts. To set up a mesh cage for a hyporheic zone installation, pass one of the hose ends through one of the chambers and fix it in this position. Then, take the anti-clogging mesh and bind it on the bottom end of the hose.Temporarily store the loaded cages in river water in the thermal box. For a sandy cage preparation, collect river sand and sift it through a two millimeter sieve. Then sift it through a one millimeter sieve and keep the particles between one and two millimeters.Place an empty cage in the plastic box and then load the bottom third of the cage with sand. Next, add river water until the water is 10 millimeters above the sand level. Load the sandy cage into a 25 liter water tank and let it equilibrate to the water temperature for 12 hours before loading them with FWPM.Transport the FWPM juveniles to the site in a field thermal box. At the field site, first immerse the thermal box in the river and remove the cage from the field thermal box. Now, start planning the cage positions.Choose conditions typical for FWPMs such as at the edge of the main water flow. Do not place the cage in direct water flow, nor in standing water. To install mesh cages in open water use a pair of the steel spikes.Fix the cage to the river bottom on it's side and level with the river bottom. Angle 45 degrees to the river flow downstream facing the center of the river. The lower horizontal edge should be 10 to 15 centimeters above the river bottom.To install a sandy cage in open water fasten it to a flat stone using a net and place the cage on the river bottom. Angle the larger end against the flow at a 45 degree angle. To install either cage type in the hyporheic zone dig them into the river bottom perpendicularly to the flow of water so that the upper horizontal edge of the cage is parallel to the river bottom surface and the chambers the testing depth in the river bottom.For mesh cages in the hyporheic zone pull out the rubber hose from under the bottom surface for water sampling during the experiment. After the planned exposure period, pull the cages out of the water and clear them of fine sediment, as well as of drifted material. Then, load them into a field thermal box filled with river water.Once back at the lab, evaluate the FWPM by photo documenting them as before. The described methods were used to investigate the environmental suitability of FWPMs in the upper Vltava River Basin in the Czech Republic from 2014-2015. A longitudinal river profile was represented by mainstream localities at sites A through E.Tributaries of different pollution stages were tested at sites R and V.These localities were tested using sandy cages and mesh cages in free flowing water.In addition, a gravel hyporheic zone was tested by within bed sandy cages in localities B, C, and D.The localities could be clearly distinguished based on the growth rate in the open water mesh cages despite the high within cage variability even in different growth favorable periods. In the more growth favorable exposure in 2015, the growth rate increased downstream using the mesh cages. Importantly, the survival rate was equivalently high in both seasons.By contrast, the growth rate of open water sandy cages in 2014 varied by location. The location with the greatest growth, C, was also the location of greatest growth in 2015. While growth overall was about the same in both years, mortality was higher in 2014.The within bed mesh cages showed a significant effect of the substrate composition on mortality. Oxygen saturated stony bottoms had a survival rate close to 100%and poorly oxygenated sand had high mortality. After watching this video, you should have a good understanding of how to install experimental mussel juveniles in the riverside to test local conditions for day

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Research

JoVE Journal

Environment

7.2K vues.  Czech University of Life Sciences Prague. This method can help answer key questions in the river ecosystem preservation field such as indications of stages favorable for young mussels that augment as well, as prediction of this which I propagate. The main advantage of this technique is that we can test juvenile growth and survival in hyporheic conditions which represent the natural juvenile environment. Demonstrating the procedures will be my colleagues Simona Nemcikova, Jan Svanyga, and Vojtech Barak.When assembling the cages...