Macroconsumer–Resource Interactions

Chapter 19

MacroconsumereResource Interactions Ashley H. Moerke1, Carl R. Ruetz, III 2, Troy N. Simon3 and Catherine M. Pringle3 1

School of Biological Sciences, Lake Superior State University; 2Annis Water Resources Institute, Grand Valley State University; 3Institute of Ecology, University of Georgia

19.1 INTRODUCTION Macroconsumers can play an important role in the structure and function of stream ecosystems. Most studies on macroconsumereresource interactions in streams have focused on fish, decapod crustaceans, and amphibians as macroconsumers, whereas semiaquatic avian and mammalian predators have received less attention (e.g., Benstead et al., 2001; Steinmetz et al., 2003; Harvey and Nakamoto, 2013; Wolff et al., 2015). Similarly, manipulative experiments examining consumereresource interactions in streams have emphasized top-down effects, which do not minimize the growing consensus of the importance of both top-down and bottom-up effects on the structure and function of food webs (Gruner et al., 2008). Although macroconsumers have been shown repeatedly to regulate prey and resource biomass (e.g., Power, 1990; Taylor et al., 2006), the strength of top-down effects by macroconsumers is not consistent among streams (Kurle and Cardinale, 2011), ranging from strong (Power, 1990; Pringle et al., 1999) to weak (Allan, 1982; Ruetz et al., 2004; Benstead et al., 2009; Ho and Dudgeon, 2016). The variability in the strength of top-down control by macroconsumers in stream food webs can be explained in part as being context-dependent and influenced by many factors (Kurle and Cardinale, 2011; Garcia et al., 2015). For example, seasonal flooding (Power et al., 2008; Winemiller et al., 2014), stream bedload (Schofield et al., 2004), macroconsumer abundance (Pringle et al., 1999; Greathouse et al., 2006; Connelly et al., 2008), macroconsumer community composition (Simon, 2015), and inputs of terrestrial arthropods to streams (Nakano et al., 1999; Baxter et al., 2005) have been shown to determine the strength of top-down control by macroconsumers in streams. Nevertheless, the true complexity of relationships that results from adding or removing macroconsumers to entire streams may become apparent only over large spatiotemporal scales (Estes et al., 2011), which often are logistically unfeasible, and therefore smaller-scale experiments are useful to provide important insights (Greathouse et al., 2006; Connelly et al., 2008). The effects of macroconsumers in stream food webs can occur via trophic and nontrophic pathways (Kéfi et al., 2012). Trophic interactions include detritivory, herbivory, predation, and omnivory. These top-down effects can be direct and indirect. Direct effects occur when the macroconsumer has a direct impact (often via consumption) on a resource, whereas indirect effects are mediated via another species or resource. A classic example of an indirect effect is a trophic cascade (Estes et al., 2011), where predation can indirectly affect basal resource biomass (e.g., benthic algal biomass) or ecosystem function (e.g., leaf breakdown rate) through chains in the stream food web (Holomuzki et al., 2010). With respect to predation, the top-down effects of macroconsumers can be density-mediated via direct consumption, or trait-mediated by stimulating defensive strategies such as changes in prey behavior or morphology (Preisser et al., 2005; Holomuzki et al., 2010). Thus, trait-mediated interactions are an example of a nontrophic interaction. Other nontrophic interactions include ecosystem engineeringdthe process by which a species modifies physical habitatdand bioturbation, which is a special case of ecosystem engineering in the form of a physical perturbation to streambed substrates (Moore, 2006; Tiegs et al., 2011). The role of macroconsumers can be strong in stream food webs across detritus-based and algal-based pathways. In detritus-based food webs, stream macroconsumers can affect organic matter dynamics. Many examples of such strong interactions involve decapod crustacean macroconsumers increasing the leaf breakdown rates (e.g., Mancinelli et al., 2013). The top-down effects of decapod crustacean macroconsumers are often caused by the direct shredding of leaf litter

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(Usio, 2000; March et al., 2001; Schofield et al., 2001), although many decapod crustacean macroconsumers are omnivorous, and therefore their effects on smaller invertebrates also can be manifested via predation and bioturbation (Rosemond et al., 1998; Usio and Townsend, 2004). Omnivorous and detritivorous decapods also have been found to decrease the quantity but increase the quality of deposited organic material and attached algae (Pringle et al., 1999; Greathouse et al., 2006). In at least one instance (a Pacific Island stream in Micronesia), however, decapods were found to have no direct top-down effects on leaf breakdown via shredding or indirect effects on smaller invertebrates (Benstead et al., 2009). Fish can also affect organic matter dynamics in streams via several different pathways. As one example, a growing body of evidence indicates that fish predation on shredding invertebrates can decrease leaf breakdown rates, causing a detrital-based trophic cascade (Konishi et al., 2001; Ruetz et al., 2002; Greig and McIntosh, 2006; Woodward et al., 2008; Simon, 2015). In contrast, the removal of detrital-feeding fish (typically in tropical streams) decreases downstream transport of particulate organic carbon because of decreased bioturbation, consumption, and egestion (Taylor et al., 2006). Pacific salmon migrations may have a similar influence on organic matter dynamics in temperate streams due to redd construction (Moore et al., 2007). In algae-based food webs, macroconsumers can affect invertebrate prey abundance and benthic algal biomass. Direct grazing by crayfish (Creed, 1994; Evans-White et al., 2001), fish (Power et al., 1985; Kohler et al., 2011; Capps and Flecker, 2015), and tadpoles (Lamberti et al., 1992; Ranvestel et al., 2004) can reduce benthic algal biomass and alter algal stoichiometry. Conversely, stream macroconsumers can indirectly affect benthic algal biomass via a trophic cascade by density-mediated and trait-mediated effects on grazing invertebrates. For instance, fish predation on the grazing invertebrates can increase benthic algal biomass in three-trophic-level food chains (Nakano et al., 1999; Garcia et al., 2015), and salamanders may have similar top-down effects in fishless streams (Keitzer and Goforth, 2013). Alternatively, in four-trophic-level chains, fish predation on smaller predators can release grazing invertebrates from predation pressure, thereby decreasing the biomass of benthic algae over some years (Power, 1990; Power et al., 2008). Finally, stream macroconsumers can have strong nontrophic effects on the algae-based food web. Spawning migrations of Pacific salmon can have positive and negative effects via nutrient additions (due to large numbers of spawning fish) and bioturbation (due to nest construction), respectively, on both invertebrates and benthic algal biomass (Collins et al., 2011; Janetski et al., 2009, 2014), and similar effects likely apply to other migrating macroconsumers beyond Pacific salmon (Flecker et al., 2010; Childress and McIntyre, 2015). In this chapter, we highlight three common methods used to test for these important top-down effects of macroconsumers in stream food webs via manipulative field experiments. These approaches illustrate the role of macroconsumers in shaping the prey communities and resource quality and availability. The most basic approach is to use mesh exclosures and open-cage controls (e.g., Ruetz et al., 2002) to test how stream invertebrate density, benthic algal biomass, and/or leaf breakdown rates respond to the absence of macroconsumers. Next, a more advanced approach to address the same ecological question (mentioned above) is to implement electric exclosures, which have the advantage over mesh exclosures in that electric exclosures will have a smaller effect on the physical environment than mesh exclosures (Pringle and Blake, 1994) and are resistant to high-discharge events (e.g., Pringle and Hamazaki, 1997). Finally, we describe an optional cage experiment, which allows for the direct manipulation of macroconsumer density in mesh cages (e.g., Stenroth and Nystrom, 2003; Ruetz et al., 2004; Keitzer and Goforth, 2013). Regardless of the approach implemented, the spatial and temporal scales of the experiment should be carefully considered. Stream macroconsumer manipulations can be carried out at the patch (e.g., Ruetz et al., 2002) or reach scale (Ranvestal et al., 2004; Taylor et al., 2006), and the density, distribution, and mobility of the macroconsumer and prey should be considered when planning experiments (see also Chapter 18). In field experiments, prey emigration may be increased for highly mobile taxa due to lack of refugia in small cages, and immigration can be restricted in small-mesh cages; both of which, along with behavioral responses of prey in the presence of predators, may alter the perceived effects of predation on stream prey (Cooper et al., 1990; Englund and Olsson, 1996; Dahl and Greenberg, 1999; Englund et al., 2001). Thus, when implementing exclosure studies and interpreting results, it is important to consider the size, density, and behavior of the macroconsumer and prey being studied in relation to the mesh size of cages or energy output of electric exclosures, as well as the spatial scale of the manipulation.

19.2 GENERAL DESIGN 19.2.1 Site Selection and Timing of Experiment Small- to moderate-sized (stream orders 2e5) wadeable streams that are hydrologically stable (i.e., groundwater-fed) are preferable for the methods described in this chapter. If hydrologically stable sites are not available, then consider timing the experiment to avoid high discharge events that may destroy experimental arenas used to manipulate macroconsumer

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FIGURE 19.1 Diagram of experimental design of field experiments for (A) Basic and Advanced Method, and (B) Optional Method, where grouped replicate treatment and control experimental arenas are randomly distributed across habitat units (i.e., blocks) within a stream reach. Control arenas may be open cages (Basic Method ), electric exclosures without electricity (Advanced Method ), or cage exclosures (Optional Method ). Treatment arenas may be cage exclosures (Basic Method ), electric exclosures (Advanced Method ), or cage enclosures (Optional Method ). The location of treatment and control arenas within a block also should be determined randomly.

density. Alternatively, if hydrological events are of interest (Pringle and Hamazaki, 1997) or unavoidable, then electric exclosures can be used since they are less prone to being washed out during high-flow conditions. When possible, select a stream where the distribution and density of the dominant macroconsumer is known and is high relative to other consumers, which will help to identify underlying mechanisms if a top-down effect is detected (this is especially important in exclosure experiments). In the study stream, select a reach containing several habitat units (riffles, runs, or pools; see Chapter 2) with similar physical characteristics including substrate, riparian shading, and depth. In this design, each habitat unit is considered a statistical block, which will contain each treatment combination to account for variation in habitat and predator-prey distribution across habitat units (Fig. 19.1). Thus, environmental conditions within each statistical block should be more homogenous than across blocks.

19.2.2 Field Experiments 19.2.2.1 Macroconsumer Exclosure Cages Exclosures can be made from simple and inexpensive materials to exclude macroconsumers and quantify macroconsumer effects on primary consumers and/or basal resources. Mesh exclosures, alone or paired with comparative field studies, have been used to demonstrate the importance of macroconsumers (native and non-native) on stream ecosystem structure and function, including top-down effects where macroconsumers reduced the standing stock of benthic organic matter and algal biomass (Capps and Flecker, 2015), reduced invertebrate densities (Garcia et al., 2015), caused detrital-based trophic cascades (Ruetz et al., 2002), and increased leaf-processing rates (Usio, 2000). One set of cages (exclosure and paired open control) is deployed in each stream section (e.g., multiple riffles throughout a stream reach) and sampling of all cages is conducted over time or once at the end of the experiment. The open-cage control is designed to contain two mesh sides, with the downstream side open (Fig. 19.1). This approach assumes that (1) any flow and sediment alteration is similar between exclosures and open controls, (2) macroconsumers can and will colonize open controls similar to natural habitat, and (3) prey movement and colonization is the same between exclosure and open-cage controls (i.e., the mesh of exclosures does not impede prey movement relative to open-cage controls). However, consideration also should be given to potential cage effects that result from altered flow, invertebrate drift, and increased sedimentation (see Walde and Davies, 1984; Cooper et al., 1990; Zimmerman and Vondracek, 2006), and possibly to altered macroconsumer behavior; each of these may confound interpretation of results. Samples of natural substrate outside of exclosure or open-cage controls also can be taken to evaluate cage effects (e.g., Tiegs et al., 2011).

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Although exclosures are relatively simple to construct and deploy, they often collect debris and may require frequent (e.g., at least daily) maintenance to minimize flow alteration and sediment accumulation.

19.2.2.2 Electric Exclosures to Manipulate Consumers An experimental technique using electricity was developed to meet some of the challenges of working in dynamic stream environments that are characterized by unidirectional flow and discharge fluctuations (Pringle and Blake, 1994; Pringle and Hamazaki, 1997, 1998). This electric exclosure technique allows manipulation of stream macroconsumers under realistic hydrodynamic conditions. The technique minimizes the reduction of stream velocity and increased sedimentation that can occur in caging experiments (discussed above). Electric exclosures are also preferable in streams with abundant floating debris, which can clog the mesh of cages and require frequent maintenance. Electrified plots (small squares or hoops placed over replicated areas of the stream bottom) are wired to solar- or battery-powered (6e12 V) fence chargers (mounted on the stream bank) that emit continuous electric pulses to repel macroconsumers. This technique has been used to examine the effects of fishes (Pringle and Hamazaki, 1998; Marshall et al., 2012), crayfish (Bobeldyk and Lamberti, 2010), shrimps (Pringle and Blake, 1994; Greathouse et al., 2006), and larval anurans/tadpoles (Ranvestel et al., 2004; Connelly et al., 2008) on algal and macroinvertebrate communities. Pringle and Hamazaki (1998) used electric exclosures to manipulate the presence of diurnal fishes and nocturnal shrimps by turning the electricity on and off during the daytime and nighttime hours, depending on the desired treatment. Their findings showed that fishes and shrimps separately exerted strong direct trophic effects, which resulted in cumulative effects on benthic algal biomass and community composition in addition to affecting benthic invertebrate communities. Whereas most field-based, experimental studies of stream grazing have been conducted during or were designed to simulate baseflow conditions (see Feminella and Hawkins, 1995), data on how trophic forces interact with abiotic disturbance in streams, such as high discharge, are lacking because severe physical conditions impose logistical constraints on in-situ experiments (e.g., floods can destroy cages). An advantage of the electric exclosure technique is that it allows for examination of top-down trophic effects of macroconsumers under natural hydrologic conditions including discharge fluctuations. For example, effects on algae can be assessed in a relatively natural depositional environment subject to natural background erosion and sloughing. Pringle and Hamazaki (1997) found that fishes played a key role in maintaining the stability of benthic algal assemblages during high-discharge events (160-fold increases in discharge over the base flow). This technique also has allowed evaluation of increases in stream bedload on top-down effects of macroconsumers in both algae- and detritus-based streams (Schofield et al., 2004). Results indicate that smalldyet environmentally realisticd increases in bedload affect benthic communities, primarily by altering fish effects. Electric exclosures have been used effectively to assess top-down effects of macroconsumers on rates of detrital processing, by measuring rates of leaf pack decomposition in the presence and absence of fishes (Rosemond et al., 1998), crayfishes (Schofield et al., 2001), shrimps (March et al., 2001), and crabs (Marshall et al., 2012). Whereas all of these studies have manipulated the presence and absence of macroconsumers, it is also possible to exclude smaller organisms such as aquatic insects using electricity. Biota are affected by electric fields in proportion to their body size (i.e., large organisms are affected more strongly than small organisms). The amount of energy produced by electric exclosures can be manipulated by changing the distance between electrodes or by choosing fence chargers with different energy (voltage or joules) outputs. Brown et al. (2000) used high-powered electric fence chargers to inhibit the grazing of mayflies (Ephemeroptera) in an Australian stream, as aquatic insect exclusion requires more powerful (higher voltage) chargers and/ or shorter distances between electrodes. In an experiment employing two intensities of electrical current, Moulton et al. (2004) excluded both shrimps and mayflies (high-intensity electric treatment) or only shrimps (low-intensity electric treatment) from benthic areas in a coastal neotropical Brazilian stream. The use of high- and low-intensity electric exclosures may be especially useful in isolating the role of omnivorous macroconsumers (e.g., shrimp, crabs, and crayfish) where trophic roles often overlap with smaller macroinvertebrates.

19.2.2.3 Optional MethoddEnclosure/Exclosure Density Manipulations Similar to the mesh exclosure method (see above), cages can be constructed from simple and inexpensive materials, but then stocked as enclosures to maintain known densities of macroconsumers (see also Chapter 18). This approach has been used commonly to evaluate the top-down effects of fish macroconsumers on invertebrates (e.g., Englund, 2005; Meissner and Muotka, 2006; Power et al., 2008; Woodward et al., 2008), salamander predation (Keitzer and Goforth, 2013), and grazing fish (Flecker and Taylor, 2004). An advantage of this approach relative to exclosure experiments is that a particular species of macroconsumers is manipulated, which makes drawing inferences regarding the cause of the effect more straightforward.

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However, this also can be a disadvantage, because few streams have a single species of macroconsumer, meaning the results of the experiment may not be easily transferable to streams with diverse assemblages of macroconsumers. Typically, enclosures are stocked with macroconsumer densities that are within the normal range of a system (e.g., Ruetz et al., 2004), represent a gradient of macroconsumer densities (e.g., Flecker and Taylor, 2004), or evaluate potential densities of an invasive macroconsumer (e.g., Stenroth and Nystrom, 2003). For a straightforward density manipulation, we recommend installing three cagesdhigh density, low density, and control (no macroconsumers)din each habitat unit (statistical block) (Fig. 19.1). Note that the control in this experimental design excludes macroconsumers. The design of these cages should be the same as the mesh exclosures discussed above. Cages should be installed randomly and in locations that minimize differences in flow and depth among all treatments within a block, and potential cage effects should be considered when interpreting results (see Macroconsumer Exclosure Cages above). The three general approaches that we have outlined for manipulating macroconsumer density are not exhaustive and mainly focus on the patch scale. When designing experiments to manipulate macroconsumers, an important factor to consider is the size of the experimental arenas (e.g., cages, electric exclosures), which may influence whether the results of predation experiments are consumption- or behavior-controlled (Englund et al., 2001; Englund, 2005). Small-scale enclosure/exclosure experiments may not be good predictors of the top-down effects of macroconsumers at the streamwide scale (Meissner and Muotka, 2006). If the questions of interest extend beyond a patch scale, then approaches addressing a reach or stream scale should be considered. For example, Taylor et al. (2006) conducted a split-stream removal experiment where a plastic divider was installed down the center of the stream and macroconsumers were removed from one side to measure the effects on ecosystem processes. A similar experimental approach was conducted in pools that were split longitudinally to manipulate macroconsumer density (e.g., Power et al., 1985). Macroconsumer removals conducted at the habitat scale (e.g., removal of fish from a pool) provide another approach that can work well in streams where macroconsumers are highly sedentary or block nets can be maintained for the duration of the experiment (e.g., Ho and Dudgeon, 2016).

19.2.2.4 Laboratory Analyses In the three methods described above, benthic algae, invertebrates, and/or leaf litter breakdown can be evaluated as the response variable(s). Benthic algae often is collected when evaluating the impact of a grazing macroconsumer, and samples can be analyzed for biomass and chlorophyll a content at the end of the experiment (see Chapter 12), although many other variables also can be measured if desired. Invertebrates are commonly sampled when examining the effects of a predatory macroconsumer, and invertebrate samples can be analyzed for density, community structure, and growth rates (see Chapter 15). If the focus is on a shredding macroconsumer, leaf litter decomposition is commonly assessed by measuring breakdown rates via leaf pack mass loss (see Chapter 27). Response variables also can be expanded to include multiple trophic levels to detect algae-based or detritus-based trophic cascades.

19.3 SPECIFIC METHODS 19.3.1 Basic Method: Macroconsumer Exclosure Cages 19.3.1.1 Initial Field Work 1. Visit your study stream and decide at what spatial scale (e.g., patch or stream reach) you wish to evaluate macroconsumer effects. Once this is decided, identify appropriate habitat units (riffles, runs, pools) throughout the study area in which you can deploy exclosures. Consider water depth, which will affect the height needed for exclosures to extend above the water level. You should consider that one arena (i.e., exclosure or open-cage control) is an experimental unit, which must be replicated for statistical analyses. At a minimum, plan for at least three exclosures and three open-cage controls for comparison (n ¼ 3 replicates). If stream habitat is patchy or heterogeneous, consider increasing the number of replicates to at least five. If pilot data or other information are available for expectations regarding the effect size (i.e., difference between exclosures and controls) and error variance for the proposed experiment, then power analysis is an excellent tool for planning the number of replicates in your experiment (Lenth, 2001). If you are unsure, it is better to err on the side of too many replicates than too few (increasing statistical power of the test), which also can be an advantage if any arenas are destroyed (e.g., by vandalism or floods) during an experiment. Any sample taken from one of the arenas (i.e., experimental unit) at a particular time contributes only one replicate; additional samples from that same arena constitute “subsamples” or pseudoreplication (Hurlbert, 1984). Samples taken over time from the same experimental arena are not independent observations (i.e., true replicates) but rather are “repeated measures” (Littell et al., 2000;

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Zar, 2010). Place replicates (typically a paired exclosure and open-cage control) at random within the habitat unit of interest, dispersed over the study reach. Measure other environmental covariables that might influence the outcome of the experiment, such as canopy cover, water velocity, and water depth. Analyze the results using an appropriate statistical test (see Data Analysis below).

19.3.1.2 Exclosure Construction and Installation 1. Construct five macroconsumer exclosures and five open-cage controls; this number can be increased or decreased depending on your experimental design (three at a minimum). Each exclosure is constructed in the stream from four pieces of rebar and plastic fence, poultry wire, or hardware cloth (Fig. 19.2). The material used for the exclosure will depend on the question and the mesh size needed to exclude the macroconsumer(s) of interest, while still allowing emigration and immigration of other invertebrate consumers. Avoid using cloth material that is less sturdy and easily degrades in the stream, possibly resulting in gaps and holes in exclosures. 2. At each habitat unit (i.e., block), pound four pieces of rebar into the substrate for one exclosure and one control cage, while attempting to match water velocities, canopy cover, and depth (if possible) within blocks. It is best to place an exclosure and open-cage control either side-by-side (if the stream is wide enough) or staggered (if one is placed upstream of the other) to minimize flow influences on each other. The rebar should be positioned to form a quadrat (e.g., 1 m  1 m square), and oriented so that one rebar is upstream and one is downstream to deflect flow as much as possible to minimize debris from accumulating on mesh. That is, one “point” of the quadrat should be positioned furthest upstream thereby allowing some debris to be swept by the angled sides of the cage. Larger or smaller arenas may be warranted depending on the size, density, and spatial distribution of macroconsumers, along with the patchiness of the habitat. Larger arenas may be required for larger, lower density, and/or more patchily distributed macroconsumers. 3. For exclosures, use plastic cable ties to attach the mesh to the rebar, creating a square cage that has mesh on all four sides to prevent macroconsumers from moving into and out of the arena, while not restricting movement of invertebrates (Fig. 19.2). Make sure that mesh is extended into the substrate and well above the water to avoid the exclosure being breached by the macroconsumers you are trying to exclude or becoming inundated during high flows. If macroconsumers can burrow, make sure to extend the mesh well into the substrate and/or add a bottom to the exclosure and bury the bottom mesh. For the open-cage control, add mesh to only the two upstream sides, leaving the two downstream sides open. This design will control for cage effects due to changes in the physical conditions (e.g., water velocity) but still allow access by macroconsumers. Optional: Pound 4 additional rebar pieces into the substrate to outline a cage-free arena to evaluate cage effects. Appropriate comparisons to evaluate cage effects may include testing

FIGURE 19.2 An example of a paired exclosure (bottom) and open-cage control (top) in a Michigan, USA, stream. Cages (1 m  1 m) were constructed of plastic fencing (25-mm mesh) and attached to rebar using plastic cable ties. Unglazed tiles were placed in each arena to evaluate changes in benthic algal biomass. Streamflow is from left to right.

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for differences in physical habitat, such as water velocity or sedimentation (Hendrick et al., 2013), among all treatment levels, and comparisons of invertebrate drift and benthic density between open-cage controls and cage-free arenas. 4. Measure canopy cover, water velocity, and water depth in the center of each arena. Water velocity can be measured using a flow meter (see Chapter 3), while canopy cover can be estimated by taking multiple directional measurements using a spherical densiometer at each plot (see Chapter 7).

19.3.1.3 Sampling Exclosures 1. At the end of the experiment (z14e90 days; length will vary depending on the consumer and resource of interest), sample the consumer/resource of interest (e.g., benthic algae, benthic invertebrates, and/or leaf litter) in each exclosure and open-cage control. If sampling multiple variables, consider whether larger or extra replicate exclosures are needed to accommodate all sampling. Each arena represents one replicate, so it is only necessary to collect one sample from each. However, if samplers are small or variability is high within an arena, then consider collecting multiple subsamples from each arena and pooling the subsamples (so that you have a single value for each arena) prior to analysis. 2. For benthic algae, collect one to three natural substrates, which will be subsamples of each arena, at the end of the experiment. Artificial substrates (e.g., unglazed clay tiles) can be used in place of natural substrates, but thought should be given to how well artificial substrates mimic natural substrates (Lamberti and Resh, 1985; Aloi, 1990; Lane et al., 2003). Place substrates in a labeled bag and store on ice in a cooler until the samples can be returned to the laboratory, pooled for each arena, and processed for chlorophyll a and algal biomass (see Chapter 12). Note: If using artificial substrates, they should be incubated in the stream for at least 28 days before sampling benthic algae and invertebrates (Lamberti and Resh, 1985) unless the focus on the experiment is how macroconsumers affect species colonization, which is different than studying macroconsumereresource interactions. 3. For benthic invertebrates, use a quantitative sampler, such as a Hess or Surber sampler (see Chapter 15), to collect one sample per arena. An alternative approach (that is not mutually exclusive) is to collect benthic invertebrates from the same substrates in which benthic algae are sampled (i.e., “whole-rock” sampling; see Wrona et al., 1986). Regardless of which approach is implemented, transfer the invertebrate sample into a bottle or bag, place a label internally and externally containing date, stream name, block number (i.e., habitat unit), arena number, and exclosure/control. Preserve invertebrate samples in 70% ethanol, and sort and identify invertebrates to the lowest taxonomic level possible in the laboratory. At a minimum, identify taxa to phylum or order using the key provided in Appendix 15.1. However, identification to the lowest practical taxonomic level is best (e.g., genus level is better than family-level identification). Count and record the number of individuals in each taxon. See Chapter 15 for details on laboratory sorting, identification, and enumeration of invertebrates. 4. For leaf litter breakdown, first determine the sampling period (1e3 months depending on the expected leaf breakdown rate) and sampling interval (weekly or biweekly). Prepare enough leaf packs for retrieval at multiple intervals over this time, considering what size leaf pack is most appropriate (e.g., Ruetz et al., 2006). Construct 1e3 preweighed leaf packs per experimental arena (i.e., exclosure and open cages) and per retrieval date (see Chapter 27 for detailed methods). At the specified sampling interval or retrieval schedule (e.g., weekly), collect 1e3 leaf packs from each arena and place them in a Ziploc bag with internal and external labels. Place all samples on ice and return them to the laboratory for processing. In the laboratory, clean the leaf packs of debris and invertebrates, dry them for 24 h at 50 C, and then weigh each leaf pack and record dry mass. Leaf breakdown rates can then be calculated using an exponential decay model (see Chapter 27). The invertebrates collected on leaf packs also can serve as an additional response variable when testing the top-down effects of macroconsumers (e.g., Ruetz et al., 2002, 2006).

19.3.2 Advanced Method: Macroconsumer Manipulation Using Electric Exclosures 19.3.2.1 Electric Exclosure Construction 1. Construct 10 metal frames (each 0.125 m2; five control and five treatment) consisting of two concentric rectangles of copper wire, connected by plastic cable ties (outer rectangle 0.25 m  0.50 m; inner, 0.08 m  0.30 cm). Each rectangle is constructed of one length of either 1.55-m or 0.81-m long 8-gauge noninsulated solid copper wire for outer or inner rectangles, respectively. Each copper rectangle should be bent into the above dimensions, leaving approximately 0.05 m of wire overlapping and connected using a copper split bolt for 8-gauge wire. The outer and inner rectangles of copper wire are connected with plastic cable ties (Fig. 19.3). All 10 frames should be constructed in the same manner. The only difference between the five control and five exclusion frames is that exclusions are connected to solar- or battery-powered fence chargers (electrified treatments), whereas controls are not. The resulting frame should resemble the one shown in Fig. 19.3.

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FIGURE 19.3 Electric exclosures deployed in the field. (A) Schematic diagram of one electric exclosure showing basic wiring design (solar-powered fence charger is preferable when sunlight is available). (B) Block of two frames (one electric and one control) installed in a low-order mountain stream in northern Trinidad. One electrified frame is connected to a solar-powered fence charger (not visible in photo but placed higher on the streambank in direct sunlight), while the control frame is not electrified. (C) Close-up of one electric exclosure frame installed on the stream bottom and containing both unglazed ceramic tiles and leaf packs (Simon, 2015).

19.3.2.2 Initial Field Work 1. In this study, the main effect is macroconsumer presence or absence, and the response variables are benthic algal biomass (e.g., chlorophyll a, AFDM; see Chapter 12) and algal assemblage composition (see Chapter 11). However, benthic insects (see Chapter 15) or leaf decomposition (see Chapter 27) could be included as additional or alternate response variables (see Rosemond et al., 1998; March et al., 2001; Simon, 2015 for modifications of leaf-breakdown experiments using electric exclosures). 2. Before running an experiment, the effectiveness of fence chargers to exclude macroconsumers must be directly tested in the study stream, since the strength of the electric charge of a given model of fence charger varies with water chemistry (i.e., conductivity) and size of the macroconsumer; thus, the electric pulses emitted by the charger may fail to repel macroconsumers. A 6-V fence charger successfully excluded all fishes in study streams in lowland Costa Rica (Pringle and Hamazaki, 1997), but it may not be effective in other streams with lower conductivity. While 6-V chargers completely excluded fishes in southern Appalachian streams of North Carolina and lowland streams of Costa Rica, 12-V chargers were most effective in lower conductivity waters in Panama (Ranvestel et al., 2004; Connelly et al., 2008) and Trinidad (Marshall et al., 2012; Simon, 2015). 3. The experimental design involves establishing five experimental blocks within a representative 100-m stream reach. Each block includes one electric exclosure and one adjacent control treatment frame, with locations of blocks occurring within standardized stream conditions (e.g., similar water velocity, depth, canopy cover) depending on the type of algal/ grazer assemblage being examined and the macroconsumer being manipulated. For example, in a study examining the effects of grazing tadpoles on algae and benthic insects, Connelly et al. (2008) established blocks in both runs and pools because different species of grazing tadpoles were abundant in each habitat type. 4. The duration of the experiment should be at least 28 days to allow establishment of algal assemblages on artificial substrates. At least six unglazed clay tiles should be placed within each treatment frame, with one tile retrieved from each frame every 5 days during the experiment. As previously discussed, consider preincubating the tiles in the stream prior to starting the experiment to allow algal colonization.

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19.3.2.3 Installation of Experiment 1. After placement of one control and one electric exclosure treatment frame within similar physical conditions in each of the five blocks (described above), each of the frames should be flush with the stream bottom and anchored in place using devices appropriate to the bottom substratum. For example, Simon (2015) affixed frames to metal spikes driven into the gravel bottom of a Trinidadian headwater stream, whereas Pringle et al. (1999) affixed frames onto flat bedrock surfaces using underwater epoxy in Puerto Rican streams. Each electric exclusion replicate should be connected to a fence charger by attaching appropriate lengths of insulated 14-gauge copper wire from the inner rectangle to the power source and from the outer rectangle to the ground. Attach insulated wire to the frames using the split-bolt connecter found on each copper wire frame. Frames should be placed to minimize any influence of macroconsumer exclusion treatments on adjacent control treatments (0.5-m gaps between adjacent treatments usually will suffice). 2. Tether six unglazed ceramic tiles (7.5  15 cm) with cable ties and binder clips to the solid copper wire within each experimental frame. 3. Monitor the experiment on a daily basis to: (1) ensure that fence chargers continuously emit pulses of electricity and to replace batteries when necessary (it may be necessary to change batteries every 3e5 days to maintain a strong, consistent electric charge), and (2) to remove any debris (leaves, twigs, etc.) that may have become caught on insulated cables or copper metal frames; such debris can alter water velocity and light, resulting in a “cage” effect.

19.3.2.4 Experimental Sampling 1. Retrieve one tile from each frame every 5 days for analyses of benthic algal biomass and assemblage composition through time (see Chapters 11 and 12). Tile retrieval will consist of cutting cable ties and removing the tile from the water within a fine-mesh hand net to prevent loss of invertebrates. Care should be taken not to disturb any sediment that may have accrued on tiles. Tiles and the contents of the hand net should be placed immediately into Ziplock bags and transported to the laboratory on ice in a cooler. 2. In the laboratory, the top surface of tiles should be scraped with a razor blade and scrubbed thoroughly with a toothbrush to remove algae. Once invertebrates are removed (and preserved in 70% ethanol for later identification), the homogenate of sediment, benthic algae, and other fine particulates should be subsampled for chlorophyll a, AFDM, and algal species composition (and algal biovolume, if desired; see Chapter 11). Other algal variables also can be measured, as desired, including stable isotopes (see Chapter 23) and elemental composition (see Chapter 36). Analyze the results using an appropriate statistical test (see Data Analysis below). 3. To identify those macroconsumers that visit the control treatment frames (but are repulsed by the electrified plots), establish a schedule of systematic observations. For example, to identify fishes and large crustaceans (e.g., shrimp or crabs) that were foraging on control tiles in an electric exclosure experiment in a Trinidadian stream, Simon (2015) recorded the number of each macroconsumer present in each treatment replicate frame using visual point counts, taken once a minute over a 15-min period (n ¼ 15 point counts). Observations were conducted on several dates during both day and night (under red light) to yield density estimates of each macroconsumer species calculated as an average over the 15-point counts in the 0.125-m2 treatment frames.

19.3.3 Optional Method: Macroconsumer Density Manipulation 19.3.3.1 Initial Field Work 1. As was discussed for the exclosure experiments (see above), identify appropriate habitat units (riffles, runs, pools) throughout a study reach in which to deploy enclosures. Measure water depth to determine if cages will need to be submerged and observe whether avian or small mammal predation may be high. If so, enclosures should be constructed with mesh on the top, which should be placed on all arenas to avoid potential differences in shading. 2. Each habitat unit should contain three experimental cages (i.e., treatment levels): high-density enclosure, low-density enclosure, and control cage (exclosure). Considerations for replication are the same as for the exclosure experimentsd at least 3 replicates (3 replicates  3 treatment levels ¼ 9 experimental arenas), but preferably 5 replicates (15 experimental arenas) distributed across blocks. Any sample taken from an arena at a particular time contributes only one replicate; additional samples from that same cage constitute “subsamples.” Measure other environmental covariables that might influence the outcome of the experiment, such as canopy cover, water velocity, and water depth. Analyze the results using an appropriate statistical test (see Data Analysis below).

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19.3.3.2 Enclosure Construction and Installation 1. The Optional Method is an extension of the Basic Method (see Exclosure construction above for details) with the following modifications. 2. Once cages are constructed, collect macroconsumers from the same water body and add them to cages in densities representing the high and low ranges of natural densities at that site. If in doubt, err on the side of selecting a high density that is borderline too high so that detecting a top-down effect of macroconsumers is more likely. The low density can be used to mimic a density that is more reasonable for the study stream. Backpack electrofishing (Chapter 16) can be used to collect many macroconsumers, including fishes, amphibians, and decapods (see also Chapter 18). The control cages should not have any macroconsumers, and if needed, macroconsumers may need to be removed from all cages at the onset of the experiment. Macroconsumers that are stocked in cages should be given a unique mark (or uniquely tagged) so that the macroconsumer density manipulation can be confirmed at the conclusion of an experiment. Marking also provides an opportunity to measure individual growth rates (i.e., change in mass) of macroconsumers during an experiment, which provides information on the wellbeing of macroconsumers during an experiment (e.g., a negative growth rate suggests that macroconsumers were food-limited or stressed during the experiment). Cages will need to be checked often throughout the experiment to clean the mesh (so that debris does not disrupt flow) and to verify (if possible without being too invasive) that macroconsumers are in cages. Any macroconsumers that die during an experiment should be replaced. 3. To characterize variation in environmental conditions among cages, measure canopy cover, water velocity, and water depth in the center of each as described above.

19.3.3.3 Sampling Enclosures 1. Sampling the cages is similar to sampling the mesh exclosures (see Mesh Exclosure Cages above for details). At the end of the experiment (z14e90 days, depending on the resource of interest), sample the consumer resource of interest (e.g., benthic algae, benthic invertebrates, and/or leaf litter breakdown rates) in each cage. If sampling multiple variables, then consider whether larger or extra replicate cages are needed to accommodate all sampling. Each cage represents one replicate for that treatment level, so it is only necessary to collect one sample from each. However, when samplers are small, or variability is high, consider collecting multiple samples and then pooling the samples for each cage prior to analysis.

19.3.4 Data Analysis All three experiments in this chapterdBasic Method, Advanced Method, and Optional Methoddpropose a complete randomized block design, meaning that each treatment level is randomly applied to each habitat unit, which is the blocking variable (Montgomery, 1991). This design accounts for variation in the response variable across the habitat units (blocks). For the Basic Method, response variables in the exclosure versus control can be tested using a paired t-test, which examines the difference between paired treatment and control experimental arenas. This approach is preferred over an independent t-test because it statistically removes variation in the response variable that is associated with differences in the habitat unit (blocks). In the Advanced Method, response variables are measured in two experimental arenas (treatment and control) multiple times during the study, and therefore a repeated-measures ANOVA should be used to account for the lack of independence between sampling events (Littell et al., 2000; Zar, 2010). Finally, for comparing three treatment levels in the Optional Method, a randomized block ANOVA should be used followed by pairwise comparisons (e.g., Tukey’s test, or see Day and Quinn (1989) for other approaches) among the three treatment levels (high density, low density, control) when a significant macroconsumer effect is detected by ANOVA. Similar to the paired t-test, the block accounts for variation in habitat units and can increase the power to detect differences among treatments. Optional Statistical Analyses: If there is interest in evaluating the importance of habitat variables (e.g., water velocity, canopy cover) on the response variables, then an analysis of covariance (ANCOVA) can be used to include the habitat variable(s) as a covariate. Additionally, to test for a cage effect in the Basic Method and Optional Method, an environmental variable (e.g., water velocity or sedimentation inside cage) can be used as the response variable in the recommended statistical analysis. For all analyses discussed above, if the assumptions of parametric statisticsdincluding that model errors meet normality and the treatments have equal variance (Montgomery, 1991)dare not met with transformations, then nonparametric statistics should be considered (Zar, 2010).

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19.4 QUESTIONS 1. Did macroconsumers have significant effects on the response variable (e.g., benthic algal biomass, invertebrate density, leaf litter breakdown rates)? If the macroconsumer exclusion resulted in significant accrual of the resource studied (e.g., high algal biomass, high invertebrate density, or lower leaf breakdown rates when compared to the control), what does this indicate about the overall importance of macroconsumers in the stream(s)? 2. Can the observed effect(s) of macroconsumers be extrapolated across the entire stream? Why or why not? Would you expect this same result during all seasons or in other streams within or outside the watershed/region? In temperate streams, very few field experiments have been executed during the winter months. How might the strength of top-down effects of macroconsumers differ between summer and winter? 3. If the effects of macroconsumers on invertebrate density were tested, consider how to determine whether macroconsumer effects were due to direct consumption (density-mediated) versus altered dispersal of invertebrates (traitmediated)? If you measured the response of invertebrates associated with leaf packs, then how might their response differ if you used leaves from a tree species with different leaf geometry or leaf packs of different size? 4. How do you predict your experimental results would change if larger arenas were used to manipulate macroconsumer density? What underlying mechanisms support your predictions? 5. Was your response variable affected by canopy cover, depth, or water velocity within or among your treatments? What can you conclude about the relative importance of light, velocity, and macroconsumers on the consumer resource studied? 6. Were the exclosures (cages or electric exclosures) effective at eliminating some or all macroconsumers? How might the characteristics of the cage (e.g., mesh size) or electric exclosures (e.g., electrical output) influence your results? 7. If the enclosure/exclosure approach was used, what was the relationship between macroconsumer density and benthic algal biomass, invertebrate density, and/or leaf litter breakdown rates? If macroconsumer growth was measured, how did this vary with macroconsumer density, and what can you conclude about the importance of intraspecific interactions among macroconsumers? What else might explain these patterns?

19.5 MATERIALS AND SUPPLIES Cage Exclosure Materials Plastic fencing, hardware cloth, or poultry wire with desired mesh size Large cable ties Rebar (4 bars per plot) Steel sledgehammer Electric Exclosure Materials 8-gauge noninsulated solid copper wire for inner and outer electrodes and frame of exclosures (10  1.55 m lengths for outer and 10  0.81 m lengths for inner) Copper split bolt for 8-gauge wire for each electrode frame (20) Copper wire stranded (14-gauge, insulated) Large cable ties (50) Either five 1-J or five 3-J output 12-V fence chargers (e.g., Speedrite by Tru-Test, http://www.speedrite.com/). Select output of fence charger based on initial field trials that assess response of macroconsumers to electric charge in your study stream. Unglazed clay tiles (60 tiles, each 7.5  15 cm) Medium-sized binder clips Rebar spikes to attach electrodes to stream bottom Field and Laboratory Materials (see Chapters 12, 15, and 27 for materials to collect benthic algae, macroinvertebrates, or leaf litter, respectively) 70% ethanol Paper labels Whirl-pak bags, Ziploc bags, or Nalgene bottles Field Equipment Meter stick Current velocity meter (optional) Spherical densiometer (optional)

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Backpack electrofishing unit, seine, or minnow traps (optional) Laboratory Equipment (see Chapters 12, 15, and 27 for all materials to analyze benthic algae, invertebrates, or leaf litter, respectively) Dissecting microscope Drying and ashing ovens Electronic balance (þ0.1 mg) Filtration apparatus Spectrophotometer

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