The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration 1,2 . Plant litter comprises the majority of detritus 3 , and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores 4,5 . However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed 6 . A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass 7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism 6 . Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption 7 . Ultimately, prey stressed by predation risk increase their body C:N ratio 7,10 , making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production 6 . Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter 6,10,11 . We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants 9 .
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Rearing Grasshoppers Under Stress and Stress Free Conditions1. Use 0.5 m 2 circular, closed mesocosms to prevent emigration or immigration of animal species (Figure 1). Construct mesocosms using 2.4 m lengths of 1.5 m high ¼" mesh aluminum fence as a scaffolding. Cover the fencing with 2.5 m lengths 1.75 m high aluminum window screening folded over the top and bottom of the fencing and stapled together along fold. Join the fencing ends to form a closed circle and then staple the overlapping window screening together to create a seal. Set the mesocosm into the soil in the field by digging a 10 cm deep by 4 cm wide trench around the base of the mesocosom, sink the mesocosm into the trench and then tamp the sod of the trench around the sunken part of the mesocosm. Staple a circular piece of window screening to the top of...