Protocol
AUTHOR
This article is modified from Perez-Harguindeguy et al (2013). The “New handbook for standardised measurement of plant functional traits worldwide” is a product of and is hosted by Nucleo Diversus (with additional Spanish translation). For more on this and on its context as part of the entire trait handbook visit its primary site Nucleo DiverSus at http://www.nucleodiversus.org/?lang=en
Contributing Authors
Lucas Enrico, Hans Cornelissen
OVERVIEW
Despite the vast diversity and complexity of herbivores, plants, and their interactions, a small number of components of leaf quality affect preference by generalist herbivores in a predictable way. Leaf palatability (as indicated by preference by herbivores) can be seen as an integrator of several underlying leaf-quality traits. Additionally, palatability tends to be correlated with litter decomposability among species because both are limited by similar constraints (e.g. low nutrient contents, high concentration of lignin and secondary metabolites).
One method for quantifying leaf palatability, as indicated by model herbivore preference, is a cafeteria assay in which generalist herbivores are allowed to feed selectively on leaf samples cut out from fresh leaves of a whole range of species distributed in random positions on a feeding arena. These experiments can provide useful information about herbivore preference for a broad range of plant species.
UNITS, TERMS, DEFINITIONS
LA – leaf area (units: mm2 ; range of values: 1 – >206 )
SLA – specific leaf area : the one-sided area of a fresh leaf, divided by its oven-dry mass (units: mm2 mg-1 or m2 kg-1; range of values: <1-300)
PROCEDURE
What and how to collect
Leaves are selected and collected as indicated in Specific leaf area (SLA), preferably from at least 10 individuals per species. Be aware that all species must be collected within 2 days before the trial and stored in refrigerated and moist conditions. If different species grow in different seasons, at least two cafeteria trials should be carried out, making sure common species of contrasting quality are included in both for cross-calibration.
Storing and processing
All leaves are kept in sealed bags at 4-5∘C until processed. Once all leaves have been collected, 10 1-cm2samples (one from each individual) should be cut from each fresh leaf and randomly placed, securing with a thin needle, on a numbered grid cell on a polystyrene feeding arena (Fig. 1). The surface is covered by transparent plastic in the case of trials with snails. Large veins should be avoided unless leaves are too tiny. For narrow leaves, an equivalent area is reached by cutting an appropriate number of 10-mm lengths from the mid-leaf section and pinning these down together into a star shape. Tiny leaves may also need to be grouped like this (with minimal overlap) to make up to ~1 cm2In the case of highly succulent and aphyllous species, a thin 1-cm2 fragment of epidermis and adjacent mesophyll (relatively young photosynthetic tissue) is used as a leaf analogue. While preparing all leaf samples be sure to keep the cut leaves in a water-saturated environment (e.g. plastic bags containing moistened paper towel) to preserve turgidity. Including additional samples from a known material, such as lettuce for human consumption, popular with some of the herbivores used for these cafeterias (snails and slugs), can be useful to test animal behaviour. If the animals do not eat from known preferred material, either the animals or the feeding conditions are probably not right.
Measuring
Once all samples have been pinned onto the arena, several herbivores (~10 per 500 leaf samples) are placed in a random position and the arena is closed with a plastic net (Fig. 1). Snails (e.g. Helix spp.) require a cool, dark and humid environment (spraying, dark plastic cover) that will stimulate consumption. Grasshoppers need a dry and light, and crickets a dry and dark environment. The arena should be covered with netting to avoid any escapes. After herbivores have been placed, consumption is measured by direct observation after 4, 8 and 12 h, and subsequently every 12 h for 3 days. The %LA (% Leaf Area) consumed can be estimated by eye (with 2-10% accuracy per sample) on the basis of the original cut shape. Actual LA may also be measured accurately (see SLA) before and after the trial, provided the samples do not deteriorate during this procedure. To be sure that the model herbivores do not have previous experience with the plants included in the trials, the herbivores may be bred, or collected when young and raised in captivity, without exposure to any of the plants included in the cafeteria experiments. This and a pre-trial 48-h starvation period (promoting consumption during the trial) are important to avoid unbiased results.
FIG. 1 DIAGRAMMATIC REPRESENTATION OF A CAFETERIA (FOLLOWING GRIME ET AL. 1996; CORNELISSEN ET AL. 1999). IF THE MODEL HERBIVORES ARE SNAILS, (A) THE ARENA CAN BE CONSTRUCTED ON POLYSTYRENE AND (B) THE NUMBERED GRID SHOULD BE WRAPPED IN PLASTIC, WHICH IS A SUBSTRATE THAT SNAILS LIKE FOR CRAWLING ON. (C) EACH LEAF SAMPLE SHOULD BE PRICKED ON THE GRID, AVOIDING THE CONTACT WITH THE PLASTIC SO AS TO PREVENT ROTTING.
NOTES AND TROUBLESHOOTING TIPS
- Independent feeding trials.
It is recommended to assess leaf palatability in at least two independent feeding trials, using different model herbivores to cover a wider range of preferences by generalist herbivores. Snails are recommended for their generalist-feeding habits, but they consume few graminoid monocots; grasshoppers and crickets are better at discriminating between leaf qualities within graminoids.
Instead of selecting one recording time, several consumption measurements can be compared with analyse-first choice and successive choices. Values of LA consumption can be transformed into values of leaf biomass if SLA is included in the calculations (see SLA).
- Palatability v.accessibility
Experiments can be designed to evaluate palatability v. accessibility, following the same theoretical background on which palatability tests are based. For example, by offering whole shoots with and without spines to different animals (in this case, model herbivores should be bigger than snails or grasshoppers) and recording how much biomass is consumed per unit time.
LITERATURE REFERENCES
References on theory, significance and large datasets:
Coley PD (1987) Patrones en las defensas de las plantas: ¿por qué los herbívoros prefieren ciertas especies Revista de Biologia Tropical 35, 151-164.
Cornelissen JHC, Pérez-Harguindeguy N, Díaz S, Grime JP, Marzano B, Cabido M, Vendramini F, Cerabolini B (1999) Leaf structure and defence control litter decomposition rate across species and life forms in regional floras on two continents. New Phytologist 143, 191-200. doi:10.1046/j.1469-8137.1999.00430.x
Grime JP, Blythe GM, Thornton JD (1970) Food selection by the snail Cepaea nemoralis L. In -Animal populations in relation to their food resources’. (Ed. A Watson) pp. 73-99. Blackwell Scientific Publications: Oxford, UK.
Grime JP, Cornelissen JHC, Thompson K, Hodgson JG (1996) Evidence of a causal connection between anti-herbivore defense and the decomposition rate of leaves. Oikos 77, 489-494. doi:10.2307/3545938
Hartley SE, JonesCG(1997) Plant chemistry and herbivory, or why the world is green. In Plant ecology. (Ed. MJ Crawley) pp. 284-324. Blackwell Science: Oxford, UK.
Singer MC (2000) Reducing ambiguity in describing plant-insect interactions: -preference’, -acceptability’ and -electivity’. Ecology Letters 3, 159-162. doi:10.1046/j.1461-0248.2000.00136.x
Southwood TRE, Brown VK, Reader PM (1986) Leaf palatability, life expectancy and herbivore damage. Oecologia 70, 544-548. doi:10.1007/BF00379901
More on methods:
Pérez-Harguindeguy N, Díaz S, Vendramini F, Cornelissen JHC, Gurvich DE, Cabido M (2003) Leaf traits and herbivore selection in the field and in cafeteria experiments. Austral Ecology 28, 642-650. doi:10.1046/j.1442-9993.2003.01321.x