Name: laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers
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We thank Silke Classen from the Research Institute for Ecosystem Analysis and Assessment (gaiac), RWTH Aachen for her assistance in the establishment of the group-specific primer sets used in this protocol. We also thank Peter Martin and Laura Schynawa from the University of Kiel for the cooperation in the water mites&#39; experiments. We also thank the technical lab assistants for keeping the lab running smoothly and preparing the register of companies and catalogue numbers. This work was supported by the German Research Foundation (DFG; project GE 2219/3-1).

1. Collect&#160;Macroinvertebrates from the Field and Prepare the Samples for Genetic Gut Content Analyses.
<ol>
	<li>Collect macroinvertebrates at each site, sort out individuals of consumer species of interest into single tubes, and directly freeze them in liquid nitrogen or dry ice to prevent gut clearance and degradation of DNA. 
	NOTE: Avoid storing samples in alcohol, because some macroinvertebrates tend to regurgitate before the alcohol kills them.</li>
	<li>Only use the gastro-intestinal tract of m...

<ol>
	<li>Carreon-Martinez, L., Heath, 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=Revolution+in+food+web+analysis+and+trophic+ecology:+diet+analysis+by+DNA+and+stable+isotope+analysis.">Revolution in food web analysis and trophic ecology: diet analysis by DNA and stable isotope analysis.</a> Mol Ecol. 9 (1), 25-27 (2010).</li><li>Hardy, C. M., Krull, E. S., Hartley, D. M., Oliver, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+source+accounting+for+fish+using+combined+DNA+and+stable+isotope+analyses+in+a+regulated+lowland+river+weir+pool.">Carbon source accounting for fish using combined DNA and stable isotope analyses in a regulated lowland river weir pool.</a> Mol Ecol. 19 (1), 197-212 (2010).</li><li>Baxter, C. V., Fausch, K. D., Saunders, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tangled+webs:+reciprocal+flows+of+invertebrate+prey+link+streams+and+riparian+zones.">Tangled webs: reciprocal flows of invertebrate prey link streams and riparian zones.</a> Freshwater Biol. 50 (2), 201-220 (2005).</li><li>Baxter, C. V., Fausch, K. D., Murakami, M., Chapman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fish+invasion+restructures+stream+and+forest+food+webs+by+interrupting+reciprocal+prey+subsidies.">Fish invasion restructures stream and forest food webs by interrupting reciprocal prey subsidies.</a> Ecol. 85 (10), 2656-2663 (2004).</li><li>Traugott, M., Kamenova, S., Ruess, L., Seeber, J., Plantegenest, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirically+Characterising+Trophic+Networks:+What+Emerging+DNA-Based+Methods,+Stable+Isotope+and+Fatty+Acid+Analyses+Can+Offer.">Empirically Characterising Trophic Networks: What Emerging DNA-Based Methods, Stable Isotope and Fatty Acid Analyses Can Offer.</a> Adv. Ecol. Res. Vol 49: Ecological Networks in an Agricultural World. 49, 177-224 (2013).</li><li>Diaz Arredondo, M. A., Guzman del Proo, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+habits+of+the+spiny+lobster+(Panulirus+interruptus+Randall,+1840)+in+Bahia+Tortugas,+Baja+California+Sur.">Feeding habits of the spiny lobster (Panulirus interruptus Randall, 1840) in Bahia Tortugas, Baja California Sur.</a> Cienc Mar. 21 (4), 439-462 (1995).</li><li>Letourneur, Y., Galzin, R., Harmelin-Vivien, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+in+the+diet+of+the+damselfish+Stegastes+nigricans+(Lacep&#232;de)+on+a+R&#233;union+fringing+reef.">Temporal variations in the diet of the damselfish Stegastes nigricans (Lacep&#232;de) on a R&#233;union fringing reef.</a> J Exp Mar Biol Ecol. 217 (1), 1-18 (1997).</li><li>Akin, S., Winemiller, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seasonal+variation+in+food+web+composition+and+structure+in+a+temperate+tidal+estuary.">Seasonal variation in food web composition and structure in a temperate tidal estuary.</a> Estuar Coast. 29 (4), 552-567 (2006).</li><li>Redd, K. S., Jarman, S. N., Frusher, S. D., Johnson, C. 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+molecular+approach+to+identify+prey+of+the+southern+rock+lobster.">A molecular approach to identify prey of the southern rock lobster.</a> Bull Entomol Res. 98 (3), 233-238 (2008).</li><li>Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dreissena+polymorpha+in+Lake+Constance:+An+example+of+a+keystone+engineer?.">Dreissena polymorpha in Lake Constance: An example of a keystone engineer?.</a> Universit&#228;t Konstanz. , (2009).</li><li>Sheppard, S. K., Harwood, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+molecular+ecology:+tracking+trophic+links+through+predator-prey+food-webs.">Advances in molecular ecology: tracking trophic links through predator-prey food-webs.</a> Funct Ecol. 19 (5), 751-762 (2005).</li><li>Jarman, S. N., Ward, R. D., Elliott, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligonucleotide+primers+for+PCR+amplification+of+coelomate+introns.">Oligonucleotide primers for PCR amplification of coelomate introns.</a> Mar Biotechnol (NY). 4 (4), 347-355 (2002).</li><li>Verma, S. K., Singh, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+universal+primers+establish+identity+of+an+enormous+number+of+animal+species+for+forensic+application.">Novel universal primers establish identity of an enormous number of animal species for forensic application.</a> Mol Ecol Notes. 3 (1), 28-31 (2003).</li><li>Deagle, B. E., 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=Molecular+scatology+as+a+tool+to+study+diet:+analysis+of+prey+DNA+in+scats+from+captive+Steller+sea+lions.">Molecular scatology as a tool to study diet: analysis of prey DNA in scats from captive Steller sea lions.</a> Mol Ecol. 14 (6), 1831-1842 (2005).</li><li>Jarman, S. N., Deagle, B. E., Gales, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group-specific+polymerase+chain+reaction+for+DNA-based+analysis+of+species+diversity+and+identity+in+dietary+samples.">Group-specific polymerase chain reaction for DNA-based analysis of species diversity and identity in dietary samples.</a> Mol Ecol. 13 (5), 1313-1322 (2004).</li><li>Jarman, S. N., Redd, K. S., Gales, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group-specific+primers+for+amplifying+DNA+sequences+that+identify+Amphipoda,+Cephalopoda,+Echinodermata,+Gastropoda,+Isopoda,+Ostracoda+and+Thoracica.">Group-specific primers for amplifying DNA sequences that identify Amphipoda, Cephalopoda, Echinodermata, Gastropoda, Isopoda, Ostracoda and Thoracica.</a> Mol Ecol Notes. 6 (1), 268-271 (2006).</li><li>Hayashi, K., Yandell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+sensitive+is+PCR-SSCP?.">How sensitive is PCR-SSCP?.</a> Hum Mutat. 2 (5), 338-346 (1993).</li><li>Glava&#269;, D., Dean, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+single-strand+conformation+polymorphism+(SSCP)+technique+for+detection+of+point+mutations.">Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations.</a> Hum Mutat. 2 (5), 404-414 (1993).</li><li>M&#252;lhardt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Der Experimentator Molekularbiologie / Genomics. , (2013).</li><li>Lee, S. V., Bahaman, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discriminatory+Power+of+Agarose+Gel+Electrophoresis+in+DNA+Fragments+Analysis.">Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis.</a> Gel Electrophoresis - Principles and Basics. , (2012).</li><li>Wang, X. W., 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=A+new+electrophoresis+technique+to+separate+microsatellite+alleles.">A new electrophoresis technique to separate microsatellite alleles.</a> Afr J Biotechnol. 8 (11), 2432-2436 (2009).</li><li>Gemmer, I., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+first+twelve+microsatellite+loci+for+the+amphipod+Gammarus+roeselii+(Crustacea:+Amphipoda).">Characterization of the first twelve microsatellite loci for the amphipod Gammarus roeselii (Crustacea: Amphipoda).</a> Conserv Genet Resour. 5 (4), 955-957 (2013).</li><li>Olafsson, K., Hjorleifsdottir, S., Pampoulie, C., Hreggvidsson, G. O., Gudjonsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+set+of+multiplex+assays+(SalPrint15)+for+efficient+analysis+of+15+microsatellite+loci+of+contemporary+samples+of+the+Atlantic+salmon+(Salmo+salar).">Novel set of multiplex assays (SalPrint15) for efficient analysis of 15 microsatellite loci of contemporary samples of the Atlantic salmon (Salmo salar).</a> Mol Ecol Resour. 10 (3), 533-537 (2010).</li><li>Baric, S., Hollrigl, A., Fureder, L., Petutschnig, J., Dalla Via, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+analysis+of+genetic+variability+in+Carinthian+populations+of+the+white-clawed+crayfish+Austropotamobius+pallipes.">First analysis of genetic variability in Carinthian populations of the white-clawed crayfish Austropotamobius pallipes.</a> Bull. Fr. Peche Piscicult. (380-381), 977-990 (2006).</li><li>Koester, M., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+intraguild+predation+of+Dikerogammarus+villosus+(Sowinsky,+1894)+at+an+invasion+front+in+the+Untere+Lorze,+Switzerland.">No evidence for intraguild predation of Dikerogammarus villosus (Sowinsky, 1894) at an invasion front in the Untere Lorze, Switzerland.</a> Aquat Invasions. 9 (4), 489-497 (2014).</li><li>Sonnenberg, R., Nolte, A. W., Tautz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+LSU+rDNA+D1-D2+sequences+for+their+use+in+species+identification.">An evaluation of LSU rDNA D1-D2 sequences for their use in species identification.</a> Front Zool. 4 (6), 6 (2007).</li><li>Koester, M., Bayer, B., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+Dikerogammarus+villosus+(Crustacea,+Gammaridae)+a+'killer+shrimp'+in+the+River+Rhine+system?.">Is Dikerogammarus villosus (Crustacea, Gammaridae) a 'killer shrimp' in the River Rhine system?.</a> Hydrobiologia. 768 (1), 299-313 (2016).</li><li>Koester, M., Cla&#223;en, S., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+group-specific+PCR+primers+for+the+identification+of+freshwater+macroinvertebrates.">Establishment of group-specific PCR primers for the identification of freshwater macroinvertebrates.</a> Conserv Genet Resour. 5 (4), 1091-1093 (2013).</li><li>Martin, P., Koester, M., Schynawa, L., Gergs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+detection+of+prey+DNA+in+Hygrobates+fluviatilis+(Hydrachnidia,+Acari):+a+new+approach+for+determining+predator-prey+relationships+in+water+mites.">First detection of prey DNA in Hygrobates fluviatilis (Hydrachnidia, Acari): a new approach for determining predator-prey relationships in water mites.</a> Exp Appl Acarol. 67 (3), 373-380 (2015).</li><li>Deagle, B. E., 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=Studying+Seabird+Diet+through+Genetic+Analysis+of+Faeces:+A+Case+Study+on+Macaroni+Penguins+(Eudyptes+chrysolophus).">Studying Seabird Diet through Genetic Analysis of Faeces: A Case Study on Macaroni Penguins (Eudyptes chrysolophus).</a> PLoS One. 2 (9), e831 (2007).</li><li>Sint, D., Niederklapfer, B., Kaufmann, R., Traugott, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group-Specific+Multiplex+PCR+Detection+Systems+for+the+Identification+of+Flying+Insect+Prey.">Group-Specific Multiplex PCR Detection Systems for the Identification of Flying Insect Prey.</a> PLoS One. 9 (12), e115501 (2014).</li><li>Zarzoso-Lacoste, D., Corse, E., Vidal, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+PCR+detection+of+prey+in+molecular+diet+studies:+importance+of+group-specific+primer+set+selection+and+extraction+protocol+performances.">Improving PCR detection of prey in molecular diet studies: importance of group-specific primer set selection and extraction protocol performances.</a> Mol Ecol Resour. 13 (1), 117-127 (2013).</li><li>Vestheim, H., Jarman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blocking+primers+to+enhance+PCR+amplification+of+rare+sequences+in+mixed+samples+-+a+case+study+on+prey+DNA+in+Antarctic+krill+stomachs.">Blocking primers to enhance PCR amplification of rare sequences in mixed samples - a case study on prey DNA in Antarctic krill stomachs.</a> Front Zool. 5 (1), 12 (2008).</li><li>Harper, G. L., 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=Rapid+screening+of+invertebrate+predators+for+multiple+prey+DNA+targets.">Rapid screening of invertebrate predators for multiple prey DNA targets.</a> Mol Ecol. 14 (3), 819-827 (2005).</li></ol>

Here, we present an easy and cost-efficient method for PCR-based determination of predator-prey interactions from field samples via group-specific primers for rDNA regions, which can be combined with other non-molecular analyses of the same specimens. However, there are several critical steps within the protocol. Firstly, assuring the specificity of the primers used is essential. Secondly, it is crucial to avoid contamination and loss of gut content material during the preparation of the gastro-intestinal tract and the s...

Predator-prey interactions, which constitute the majority of trophic interactions and food web dynamics, are key aspects to characterize the fluxes of matter and energy throughout food webs within and between ecosystems, which is one of the major goals of ecology1. The determination of the source and flow of carbon and nutrients is furthering the understanding of ecological connectivity between ecosystems2. However, ecosystems, such as rivers and their catchments, are not only linked by fluxes of organic matter and nutrients but also by the movements of organisms3. Thus, habitat alterations interrupting the flow of resources that link those systems can strongly alter the food webs of both ecosystems, not only directly but also indirectly by changing the respective composition of predator-prey communities. For instance, changes of food webs have been shown to be linked to the movements of single predator species (e.g., rainbow trout)4. Such changes potentially threaten biodiversity and the functioning of aquatic ecosystems. Therefore, analyzing predator-prey interactions in the field is essential to determine the impact of human-induced environmental changes, such as water management practices, on the native biodiversity of aquatic ecosystems.
Since tracking trophic linkages is difficult in complex systems, several approaches have been established that enable the assessment of feeding interactions in the field5. Traditionally, investigations of feeding interactions in the field are based on visual identification of prey remains in dissected guts and require an extensive knowledge about morphologic prey diversity. Visual gut content analyses have provided insights in resource use of several groups of consumers (e.g., seasonal variation in diet of lobster6 and fish7,8 or feeding preferences of copepods). However, the physical process of digestion makes visual gut content analyses difficult and usually misses soft bodied prey-organisms9. For species feeding by liquid ingestion or for some invertebrate consumers that intensively comminute their food before ingestion, like amphipods, visual identification of prey species in gut contents is impossible10. Due to these limitations, molecular analysis provides a promising alternative.
Molecular analyses have now become a common tool allowing rapid and precise prey detection in gut contents. The range of such techniques is diverse: strategies based on monoclonal antibodies or polymerase chain reactions (PCR) are often used, because of their high specificity and sensitivity11. The development of new monoclonal antibodies is time- and cost-intensive, therefore, the application of other molecular techniques is more useful when antibodies do not already exist11. Another common approach is the amplification of regions of deoxyribonucleic acid (DNA), like ribosomal ribonucleic acid (RNA) genes present in most species, using universal primer sets12,13. When using this technique, it is often not possible to identify the whole range of prey organisms within mixed sources of DNA14. An effective approach to avoid such a drawback is to use group-specific primer sets for genetic gut content analyses. Designed to amplify only DNA regions of particular target groups and exclude all other species15,16, group-specific primers enable identification of prey organisms on the taxonomic level of the specified groups without time- and cost-intensive secondary analyses. However, like all gut content analysis, such analyses provide only a snapshot of feeding behavior. Therefore, combining molecular gut content analyses with analyses of time-integrating natural tracers (e.g., stable isotopes) is considered beneficial1,2.
Here, we describe a detailed method for PCR-based investigations of predator-prey interactions using group-specific primer sets for nuclear ribosomal DNA (rDNA) regions to be combined with stable isotope analyses of the same specimen. We describe the detection of the DNA of single prey groups via agarose gel electrophoresis. Additionally, we present an opportunity for further downstream analyses of PCR products of such group-specific primers applicable whenever a higher taxonomic resolution than the primers&#39; specificity is required. Because single stranded DNA (ssDNA) fragments form tertiary conformations that are determined by their primary sequence17, small variations in fragments amplified by such group-specific primers lead to conformational changes. Such changes can be detected by single strand conformation polymorphism (SSCP) analyses with polyacrylamide gels17,18, enabling a more precise identification of prey organisms (down to the species level).
While agarose gel electrophoresis is a common and inexpensive tool to visualize DNA fragments and determine their approximate length19, the resolution depends on the amount of DNA and the staining dye used20. Usually, the visualization is straight forward when working with pure DNA samples, but potentially low amounts of prey DNA in the gut contents of consumers can complicate the scoring of agarose gel electrophoresis results. Still, this detection method is feasible to screen a low number of consumer specimens from the field for one or a few prey groups, but complication in the scoring makes the screening of a high number of samples for multiple prey taxa extremely time intensive and thus impracticable. A more sensitive detection method is the automated analysis of fragments via capillary electrophoresis, which additionally allows the determination of the exact length of fragments21. Several microsatellite based studies have shown that by using different fluorescent dyes as labels, it is possible to detect and determine different fragments of comparable length by automated fragment analysis22,23,24. Therefore, we also present a detailed protocol for parallel detection of DNA from multiple prey groups using PCR with labeled group-specific primer sets and detection via automated fragment analysis with an automated sequencer. Additionally, we present results from a case study demonstrating that the detection of prey DNA via automated fragment analysis is an approach which also enables a relative quantification of ingested prey.

<table>
	<tbody>
		<tr>
			<td>liquid nitrogen</td>
			<td>Any</td>
			<td>NA</td>
			<td>For transport of field samples of macroinvertebrates to the laboratory.</td>
		</tr>
		<tr>
			<td>1.5 ml reaction tubes - Safelock</td>
			<td>Any</td>
			<td>NA</td>
			<td>For collection of consumer specimens in the field and subsequent conservation in liquid nitrogen reaction tubes do not necessarily need to be PCR clean. To avoid exploding of the tubes, the lid of the tubes should be punctured with a needle.</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Any</td>
			<td>NA</td>
			<td>Make sure magnification is suitable to prepare the gastro-intestinaltract of the size of consumer of interest.</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Any</td>
			<td>NA</td>
			<td>To dissect the gastro-intestinal tract of consumer specimens under the stereomikroscope.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps No. 7</td>
			<td>Bioform</td>
			<td>B40d</td>
			<td>This curved stainless steel forceps can sometimes be very helful to hold specimen fixed inbetween the curved part without puncturing the gastro-intestinal tract.</td>
		</tr>
		<tr>
			<td>Stainless steel foceps: Dumont forceps Electronic No. 5</td>
			<td>Bioform</td>
			<td>B40b</td>
			<td>This fine stainless steel forceps is ideally suited for the preparation of the gastro-intestinal tract of consumer specimens.</td>
		</tr>
		<tr>
			<td>DNA-ExitusPlus</td>
			<td>AppliChem</td>
			<td>A7409,1000RF</td>
			<td>Decontamination solution for the removal of DNA and RNA contaminations</td>
		</tr>
		<tr>
			<td>fuzz-free paper towel</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab scale</td>
			<td>Any</td>
			<td>NA</td>
			<td>Precision needs to be at least 0.01 g.</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 2.0 ml</td>
			<td>VWR</td>
			<td>211-2165</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Safe-Lock Tubes 1.5 ml</td>
			<td>VWR</td>
			<td>211-2164</td>
			<td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td>
		</tr>
		<tr>
			<td>Sodium chloride &#8805;99.5%, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>3957.3</td>
			<td>For salt extraction buffer (SEB) and 5M NaCl-solution required for extraction of DNA.</td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)-aminomethane Tris</td>
			<td>carlroth</td>
			<td>4855.2</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &#956;L)</td>
			<td>carlroth</td>
			<td>8043.1</td>
			<td>For salt extraction buffer (SEB) and TBE buffer</td>
		</tr>
		<tr>
			<td>Boric acid &#8805;99.8 %, p.a., ACS, ISO</td>
			<td>carlroth</td>
			<td>6943.2</td>
			<td>For TBE buffer. Not needed if commercialy available TBE buffer is obtained.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>carlroth</td>
			<td>4360.1</td>
			<td>For extraction of DNA</td>
		</tr>
		<tr>
			<td>Proteinase K lyophil.</td>
			<td>carlroth</td>
			<td>7528.1</td>
			<td>Prepare a working solution containing 10 mg/ml.</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Quiagen</td>
			<td>85300</td>
			<td>Bead mill for homogenization of the samples.</td>
		</tr>
		<tr>
			<td>Stainless Steel Beads, 5 mm</td>
			<td>Quiagen</td>
			<td>69989</td>
			<td>For homogenization of samples.</td>
		</tr>
		<tr>
			<td>Heating Thermoshaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge: himac CT 15 RE</td>
			<td>Hitachi</td>
			<td>NA</td>
			<td>Refrigerated centrifuge used for centrifugation during the extraction of DNA (Protocol 1).</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>carlroth</td>
			<td>6752.5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.8 %, p.a.</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Molecular biology grade (ddH2O)</td>
			<td>AppliChem</td>
			<td>A7398,1000</td>
			<td>nuclease-free (stream sterilized and DEPC treated) water used for re-suspension of DNA and whenever refered to ddH2O within the manuscript</td>
		</tr>
		<tr>
			<td>Unmodified Olegonucleotides</td>
			<td>Eurofins MWG Operon</td>
			<td>NA</td>
			<td>Primers unlabeled</td>
		</tr>
		<tr>
			<td>Modified olegonucleotides</td>
			<td>Metabion International AG</td>
			<td>NA</td>
			<td>Primers labelled</td>
		</tr>
		<tr>
			<td>peqGOLD Taq all inclusive</td>
			<td>VWR Peqlab</td>
			<td>01-1000</td>
			<td>Taq all inclusive for PCR reaction mixtures (reaction buffer Y and S, dNTPs and MgCl2)</td>
		</tr>
		<tr>
			<td>Primus 96 advanced gradient thermocycler or peqStar96x Universal Gradient</td>
			<td>Peqlab</td>
			<td>NA</td>
			<td>Primer sets were originally established on a Primus 96 advanced gradient thermocycler. As this cycler was quite old, we than checked if our primer sets are efficient and specific under the same conditions using the peqSta96x (which also could simulate the older model). However, tests showed that our primers work well and are still specific (see Koester et al. 2013) without using the simulation of the old machine.</td>
		</tr>
		<tr>
			<td>Standard 96 Well PCR Plates</td>
			<td>VWR Peqlab</td>
			<td>732-2879</td>
			<td>Used for PCR reactions.</td>
		</tr>
		<tr>
			<td>Low Profile Tube Stripes 0.15 ml with Flat Cap Stripes</td>
			<td>VWR Peqlab</td>
			<td>732-3223</td>
			<td>Cap stripes used to close the PCR plates.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Peqlab</td>
			<td>35-1020</td>
			<td>Used for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Microwave</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical flask</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring cylinder</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal electrophoresis unit and respective combs with teeth</td>
			<td>Any</td>
			<td>NA</td>
			<td>For agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply PS3002</td>
			<td>Life Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide solution 1 %</td>
			<td>carlroth</td>
			<td>2218.1</td>
			<td>Used to prepare staining bath for agarose gels.</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>carlroth</td>
			<td>6749.1</td>
			<td>Needded for the denaturation buffer for SSCP analyses.</td>
		</tr>
		<tr>
			<td>Bromphenol blue</td>
			<td>AppliChem</td>
			<td>A3640.0010</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>carlroth</td>
			<td>4621.1</td>
			<td>Needed for the loading dye for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>100 bp-DNA-Ladder extended</td>
			<td>carlroth</td>
			<td>T835.1</td>
			<td>As size standard for agarose gel electrophoresis.</td>
		</tr>
		<tr>
			<td>UV-Documentation system</td>
			<td>Any</td>
			<td>NA</td>
			<td>To visualize gel electrophoresis results.</td>
		</tr>
		<tr>
			<td>Rotiphorese NF-Acrylamide/Bis-solution 40 % (29:1)</td>
			<td>carlroth</td>
			<td>A121.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Ammonium peroxydisulphate</td>
			<td>carlroth</td>
			<td>9592.2</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>foldback clips (32 mm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>square-shaped plastic box (21 &#215; 6.5 &#215; 5 cm)</td>
			<td>Any</td>
			<td>NA</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>carlroth</td>
			<td>23.67.1</td>
			<td>For the preparation of polyacrylamid gels.</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Any</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>RC 10 Digital chiller</td>
			<td>VWR</td>
			<td>462-0138</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Vertical electrophoresis unit P10DS (OWL Separation Systems)</td>
			<td>VWR</td>
			<td>700-2361</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Notched glass plate</td>
			<td>VWR</td>
			<td>730-0261</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Blank glass plate</td>
			<td>VWR</td>
			<td>730-0260</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Spacers</td>
			<td>VWR</td>
			<td>730-0262</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Comb with 25 teeth</td>
			<td>VWR</td>
			<td>730-0294</td>
			<td>For SSCP analyses using polyacrylamide gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Shaking platform</td>
			<td>Any</td>
			<td>NA</td>
			<td>For constant shaking of the staining bath.</td>
		</tr>
		<tr>
			<td>GelStar Nucleic Acid Stain (Lonza Rockland, Inc.)</td>
			<td>Biozym</td>
			<td>850535 (50535)</td>
			<td>For staining of polyacrylamide gels.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 400bp</td>
			<td>AB Sciex</td>
			<td>608098</td>
			<td>For automated fragment analyses. Size stanard for Batch B.</td>
		</tr>
		<tr>
			<td>GenomeLab DNA Size Standard Kit 600bp</td>
			<td>AB Sciex</td>
			<td>608095</td>
			<td>For automated fragment analyses. Size stanard for Batch A.</td>
		</tr>
		<tr>
			<td>Sample Loading Solution (SLS)</td>
			<td>AB Sciex</td>
			<td>608082</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzing plate - 96 Well Polypropylene PCR Microplate</td>
			<td>Costar</td>
			<td>6551</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Plate centrifuge, PerfectSpin P</td>
			<td>VWR Peqlab</td>
			<td>NA</td>
			<td>mini plate centrifuge used to spin down PCR products.</td>
		</tr>
		<tr>
			<td>Buffer plate - 96 Well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate</td>
			<td>Corning</td>
			<td>9017</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GenomeLab Seperation buffer</td>
			<td>AB Sciex</td>
			<td>608012</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Separation Gel - LPAI</td>
			<td>AB Sciex</td>
			<td>608010</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>Sequenzer - CEQ8000 Genetic Analysis System</td>
			<td>Beckman Coulter</td>
			<td>NA</td>
			<td>For automated fragment analyses.</td>
		</tr>
		<tr>
			<td>GeneMarker V1.95</td>
			<td>Softgenetics</td>
			<td>NA</td>
			<td>To analyze results of automated fragment analyses.</td>
		</tr>
	</tbody>
</table>

laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

We successfully used steps described in this protocol for diet analyses within different studies. In this section, we present examples describing the applicability of the different sections of the protocol.
As an example, to demonstrate the effectiveness of group-specific primer sets established by the authors28 and the potential of further downstream analyses of PCR products by SSCP analyses, a feeding e...

Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

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