Biology Reference
In-Depth Information
Other preservation methods include acetone, 2-propanol, diethyl ether,
and ethyl acetate, which allow insects to be stored for
≈
6 months (
Fukatsu
1999
).
Fukatsu (1999)
recommended the use of acetone, which preserved the
DNA of insects (as well as the DNA of microbial organisms within the insects)
for
>
2 years at room temperature. Detecting microbial symbionts within
stored insects can offer a challenge because symbionts may be low in titer;
PCR-inhibiting substances may be present in the insect gut.
Zaspel and Hoy
(2008)
compared the titer of
Wolbachia
in specimens of
Ephestia kuehniella
stored in 95% EtOH or 100% acetone and held at room temperature or at
−
80 °C for up to 101 weeks using high-fidelity and standard PCR protocols and
real-time quantitative PCR methods. The least-effective storage method was
the 95% EtOH and room-temperature protocol, but all protocols resulted in
the detection of
Wolbachia
over the 2-year period.
Critical-point drying or other drying techniques are used to preserve many
small insects in museums (
Austin and Dillon 1997
). Such dried insects sometimes
can be used for molecular studies, especially if the target DNA is short and abun-
dant (such as mitochondrial and ribosomal DNA). Specimens that were killed
in 100% EtOH, stored at 5 °C, and then dried yielded good quality DNA upon
extraction (
Austin and Dillon 1997
). Amplification of long segments of single-
copy genes from insects that have been poorly preserved is likely to be difficult.
8.3.8 Preparing DNA Samples
Template DNA used in the PCR generally should be free of proteases that could
degrade the DNA polymerase. It should be free of nucleases that could degrade
DNA, and free of DNA-binding proteins or high levels of heat-precipitable pro-
teins that would inhibit amplification. Ideally, 10
5
to 10
6
template DNA mole-
cules are available, although successful PCRs have been achieved with only a few
DNA molecules.
Relatively crude DNA preparations
sometimes
can be used for the PCR. For
example, when large numbers of individual insects must be processed, it is pos-
sible to do PCR on undissected larval or adult insects without prior isolation of
the DNA (
Grevelding et al. 1996
). Apparently, the repeated denaturation steps
at high temperature are sufficient to lyse cells so that sufficient template DNA
is available, especially when the target DNA is present in high copy number in
each cell (such as mitochondrial genes or ribosomal RNA genes). The advantage
of using a crude lysate is that it reduces the time and costs to prepare the sam-
ple, which is important when hundreds or thousands of specimens must be eval-
uated. Such crude preparations do not allow the DNA to be stored.
