We have had great
success extracting and amplifying DNA from fleas preserved
in 70-100% ETOH. If stored in the freezer at – 80 °C,
the specimens stay extractable for ca. 5 years after collection.
DNA is extracted from fleas without homogenization of tissues
by separating the thorax from the abdomen. To extract DNA
we usually use the DNeasy® Tissue Kit (Quiagen). However,
a regular SDS/Phenol/isoamyl alcohol: chloroform protocol
also works.
To mount the specimen it is removed from the extraction buffer
and stored in 70% Ethanol. This technique provides us with
an excellent source of DNA without destroying the structures
used for species identification. The incubation in extraction
buffer helps clear the specimen prior to KOH treatment, and
these specimens are almost always in better condition (key
features are easier to see) than specimens which have simply
gone through a KOH treatment without the DNA extraction. After
the 10% KOH treatment the specimen is transferred through
a series of ethanol concentration for dehydration, reaching
a final concentration of 96.7%. The specimen is then transferred
to oil-of-wintergreen and xylene and finally mounted on a
slide. We prefer Canada balsam as mounting medium. The flea
is mounted in a laterolateral fashion, with the dorsum being
located towards the lower margin of the slide and the head
pointing to right. In the end we have a complete voucher on
a slide with which every DNA extraction can be associated.
The vouchers are stored in the Entomological Collection of
the Monte L. Bean Life Science Museum.
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Traubella
grundmanni cleared and mounted using standard techniques
(without DNA extraction).
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Traubella
grundmanni
cleared and mounted after DNA extraction.This was an
ETOH preserved specimen. Notice how well the aedeagus
can be seen and that all the spines and setae are intact.
The extracted DNA is high quality and has been sequenced
for multiple loci in this taxon.
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Megabothris
abantis,
cleared and mounted using standard techniques ( without
DNA extraction).
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Megabothris
turbidis,
cleared and mounted after DNA extraction. This was an
ETOH preserved specimen. Notice how well the aedeagus
can be seen and that all the spines and setae are intact.
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