Biology Reference
In-Depth Information
Molecular analyses of gut DNA require that a series of steps be conducted
to validate the tests. These steps include sampling each potential prey species
found in the appropriate environment; sequencing appropriate genes from each
prey (choosing the appropriate number of each species to sample to obtain an
accurate estimate of the variability within each prey species); designing PCR
primers that are species-specific; amplification of a diagnostic, but relatively
small, fragment because the DNA is degraded in the gut to a greater or lesser
degree; and feeding each prey species to hungry predators and conducting PCR
analyses to determine the time it takes after feeding at different temperatures
for the PCR signal to be undetectable.
Variables that can affect the decay rate of the DNA in the predator's gut
include the number of prey consumed, the size of the prey (which affects the
quantity of the DNA present initially), the number of different prey species
consumed at nearly the same time, the preservation methods for the preda-
tors, the DNA extraction methods, and the temperature at which the predators
were held. It can be difficult to determine whether the predators fed on live
prey or on dead prey (
Foltan et al. 2005
) or whether a generalist predator fed
on another predator that had fed on a particular prey species (secondary preda-
tion) (
Sheppard et al. 2005
). Sensitivity tests for the PCR should be conducted
using prey DNA mixed with predator DNA in serial dilutions in order to deter-
mine how sensitive the tests are, and both positive and negative controls should
always be used to reveal problems with the reaction or with contamination,
respectively. Furthermore, the primers should be tested on predator DNA to con-
firm there is no cross amplification.
King et al. (2008)
reviewed the methods used in gut analyses of predators and
recommended specific practices to improve procedures. Primer design and test-
ing and assay optimization are described, and the need for negative controls
was emphasized. Multiplex PCR can sometimes be used to screen for different
prey species simultaneously, and quantitative PCR can potentially quantify pre-
dation rates (
Weber and Lundgren 2009
). However, if hundreds of predators
need to be studied for predation on many prey species, the cost of such stud-
ies is high, and the evaluations become tedious and lengthy.
King et al. (2008)
speculated that microarrays or pyrosequencing of DNA in predator guts might
be developed in the future for these assays.
Methods also are needed to ensure that PCR analyses of gut contents are not
reporting DNA contamination on the external insect (
Greenstone et al. 2012
).
Treatment with 2.5% commercial bleach reliably eliminated external DNA con-
tamination but 80% ethanol (EtOH) did not. The bleach treatment did not
affect the ability to detect the true prey species in the gut by the PCR.
