The overall goal of this procedure is to determine how the decomposition rate of organic matter is influenced by the body chemical composition of animal carcasses following stress from predation. This is accomplished by first creating experimental rearing conditions that subject prey to risk stress of predation under field conditions and risk stress-free control conditions. The second step is to collect the grasshoppers from the experimental treatments and after verification of their metabolic state using respiratory sacrifice them for body isotopic chemical content analysis in the lab and for priming decomposition processes in the field.
Next, the grasshopper carcasses are placed into enclosed plots and left to decompose for 40 days. At the same time, plants within mesocosms are labeled with carbon 13 carbon dioxide. Grass material is then harvested from these mesocosms around 10 days before the completion of the 40 day grasshopper carcass decomposition for air drying.
The dried grass is then placed into the enclosure at the 40 day time point. The final step is to monitor the decomposition rate of the grass litter in situ across 75 days by tracking both soil respiration and the respiration of carbon 13 carbon dioxide using a flow through chamber technique involving a piro cavity ring down spectroscope. Ultimately, these methods quantify how chemical changes in the body of herbivores stressed by predation can regulate the rate of decomposition by microbes in the soil pool.
This shows that even animals with low abundance can nonetheless have important multiplier effects. In ecosystems, Though, the method that we talk about today is applicable in this old field ecosystem. It's more broadly applicable to other grassland systems like African savannas or the Yellowstone National Park Prairie Grasslands, where you oftentimes get a lot of herbivore carcass inputs into the soil and the decomposition of that carcass can influence in an important way.
The the ecosystem nutrient cycling that you see in those, those locations, 0.5 meter circular closed mesocosms are used to prevent immigration or immigration of animal species. Each mes Cosm cage is constructed from 2.4 meter lengths of 1.5 meter high quarter inch mesh aluminum fence that is covered with 2.5 meter lengths of 1.75 meter high aluminum screening. Set the cage into the soil in the field by digging a 10 centimeter deep by four centimeter wide trench around the base of the cage.
Then sink the cage into the trench to a depth of 10 centimeters and tamp the so of the trench around the sun. Part staple a circular piece of window screening to the top of the meso cosm cage. The mesocosms should be arranged in a replicated paired experimental design in the field plot location should be selected to match the plant species identity and plant relative cover.
Use a sweep net to collect second in star grasshopper nymphs and stock them into the mesocosms at field densities. Then use a sweep net to capture individuals of a dominant sit and weight hunting, not web weaving spider predator species. After using fast drying cement to glue the spider colliery shut, stock the spiders at field densities to one meso cosm of each pair.
This will be the stress treatment mesocosms without spiders will be the stress-free treatment after grasshopper. Nymphs have been developed to the fourth and fifth instar stages. Collect all individuals from the cages and randomly assign individuals from each cage to one of three subsequent assay groups.
Group one validation of physiological stress. State group two, validation of shift in body elemental stoichiometry and group three microbial decomposition to measure microbial decomposition. Place replicated pairs of PVC collars in the field site.
Remove all vegetation within each collar via clipping at the soil surface. Additionally, establish a set of PVC collars across the field site to act as carbon 13 natural abundance controls to which neither grasshoppers nor grass litter are added. Then to one color in each pair, add one intact freeze-dried carcass of a grasshopper reared with predator risk as previously described, using field cages, be sure to record the biomass added to the other collar.
In each pair, add two intact freeze dried carcasses reared without predator risk. Again, recording the added biomass. Cover the PVC collars with the screen to prevent grasshopper removal by scavengers from the plots and let the grasshopper carcasses decomposed for 40 days.
During this time, label grass litter with carbon 13 by first sinking a square, 60 centimeter by 60 centimeter wooden frame with a rubber seal coated with silicone grease five centimeters into the ground. Then slide a clear plexiglass chamber with an inlet and outlet valve on top of the wooden frame so that the rubber seals the chamber erector base measurement stations central to all of the plots containing PVC colors. Vental attachment of the cavity ring down spectroscopy instrument one week after carbon 13 labeling as described in the text protocol, use a thermo delta plus isotope ratio mass spectrometer or similar to compare delta carbon 13 of the grass litter with natural abundance values collected from a random sample of identical grass species around 10 days before the end of the 40 day grass aper carcass decomposition harvest the labeled grass litter to be air dried after 40 days.
A 10 grams of air dried carbon, 13 labeled grass litter to each color that had previously been amended with grasshopper carcasses. Monitor the mineralization rate of the grass litter in situ across 75 days by first capping each color and tracking both total soil respiration and the respiration of carbon 13 carbon dioxide using a flow through chamber technique. Gas samples from each color are monitored in real time per eight minutes each using a piro cavity ring down, spectroscope cavity ring down spectroscopy enables simultaneous tracking of both total and delta carbon 13 of soil respiration.
Estimate the contribution of carbon 13 labeled grass litter to total soil respiration using isotope mixing equations. According to the instructions in the text protocol, this plot shows herbivore standard metabolic rate in relation to herbivore body mass. The data are divided into two classes according to experimental treatment, grasshoppers from mesocosms containing predators to induce stress and control mesocosms without predators and hence no induced stress due to body mass differences among individual grasshoppers and the fact that metabolic rate varies with body mass plots should present metabolic rates in relation to grasshopper body mass.
Parallel trends for the different treatments indicate that metabolic rate rises as a constant multiple of standard metabolic rate IE.There is no body mass by metabolic rate interaction for all stressed individuals, grasshopper body, carbon, and nitrogen elemental contents in risk and risk-free conditions are presented in this table. It is noteworthy that there is a very small 4%difference in body carbon to nitrogen ratios between treatments. Nevertheless, these small differences can translate into large differences in grass litter decomposition by the soil microbial pool.
These curves demonstrate cumulative CO2 release by the microbial pool while decomposing experimental grass litter inputs in PVC colors plotted values, a mean plus minus one standard error. The graft demonstrates that soils primed with stress. Grasshopper carcasses IE the predator situation results in 19%lower plant litter decomposition rates than control soils primed with stress-free grasshopper carcasses.
The method can help us answer key questions in ecosystem, ecology and bio geochemistry by allowing us to trace the carbon that's that's in plant leaves into different soil pools, and also allow us to track the fate of nutrients from from those pools back into the above ground plant parts.
