Changes In Hemolymph During M. sexta
Development
Protocol: Hemolymph Collection
Introduction
Hemolymph is collected from M. sexta larvae by
clipping one of the fleshy prolegs on the abdomen. Hemolymph
is collected from pupae by clipping the proboscis. In both
cases, the insect is chilled on ice prior to collection.
Materials
- scissors; microsurgery scissors work best but lab
scissors are also sufficient
- microfuge tubes (1.5 ml) containing
phenylthiocarbamide (also called phenylthiourea;
available from Sigma)
- microfuge tubes (1.5 ml)
- ice buckets
- 70% ethanol in a narrow 50 ml beaker
- microfuge
- water blanks for balancing in microfuge
- scotch tape
Hazards
Phenylthiocarbamide is very harmful when
swallowed, absorbed through the skin or splashed in the
eyes. It may be fatal if swallowed or absorbed through the
skin. Gloves, protective glasses, and lab coats should be
worn when working with this compound.
Method
Larval Hemolymph Collection:
1. Obtain your larva(e). It is critical that you
do not become confused about the identities of the
caterpillars if you are bleeding more than one (as in an
experiment where you are comparing bacteria-treated and
untreated specimens!) Rinse them briefly under running water
to remove any food or fecal particles. Identify the third
proleg. Bury each of the insects in ice. If the insect gets
too cold it will become flaccid and will not bleed well. If
this happens, just warm the insect at room temperature
briefly.
2. Set up the following: scissors, 70% ethanol, lab
tissues, 1.5 ml microfuge tubes each containing
phenylthiourea (PTU) crystals. The phenylthiourea will
inhibit the action of phenyloxidase in the insect hemolymph
and prevent the hemolymph from turning black (melanizing).
Label the tubes clearly. Cover the label with a small piece
of scotch tape to protect it. Put the microfuge tubes on
ice. Get the scissors from the faculty or TA. Sterilize the
scissors by dipping them in the ethanol and laying them on a
blanket of lab tissues. Cover the scissors' tips with the
lab tissues to keep them sterilized.
3. Read the following steps very carefully before
proceeding. You will want to work quickly but carefully, and
everything needs to be ready before you begin.
a. Once one of the caterpillars has stopped moving (about
5-10 min.), dip it in ethanol to cover the third proleg. Dry
the caterpillar on a blanket of lab tissues.
b. Working over the microfuge tube, bend the caterpillar
gently in the vicinity of the third proleg. Use the
scissors to clip the third proleg (do not cut it off). Rest
the proleg against the rim of the microfuge tube so that the
hemolymph flows into the tube. The hemolymph will be bluish
in color. You may have to use a gentle milking action to
encourage the hemolymph to flow. Do not squeeze the insect.
Avoid collecting white tissue (fat body) and immediately
stop if a yellowish tissue protrudes from the wound. This is
the gut and contains many proteolytic enzymes. Close the
tube and mix the contents by gently flicking the microfuge
tube with your index finger. Keep the tube on ice. Return
the animal to its container.
c. Place the animal(s)s in the freezer for disposal.
4. Microfuge the hemolymph for 2 min. Place the tubes in
the microfuge with the tabs facing out; this will cause your
pellet to form under the tab and you will know where to look
for it. This centrifugation step gives cell-free hemolymph;
the hemocytes (blood cells) and any undissolved PTU crystals
will be pelleted.
5. While the tubes are spinning, label new microfuge
tubes. After the centrifugation, transfer most of the blue
supernatant to the appropriately labeled new tube. Be
careful not to mix-up the samples. Also be careful to leave
the pellet undisturbed. Keep the new tubes on ice; discard
the tubes containing the pellets. Hemolymph should be stored
in the freezer.
Pupal Hemolymph Collection:
1. Obtain the insects and bury each in ice for 3 to 10
minutes. The proboscis is the curved extension that comes
off the head and ends on the abdomen.
2. Set up the following on your lab bench: scissors, 1.5
ml microfuge tubes each containing phenylthiourea (PTU)
crystals. The phenylthiourea will inhibit the action of
phenyloxidase in the insect hemolymph and prevent the
hemolymph from turning black (melanizing). Label the tubes
clearly and cover the label with a small piece of scotch
tape to protect it. Put the microfuge tubes on ice.
Sterilize the scissors by dipping them in the ethanol and
laying them on a blanket of lab tissues. Cover the scissors'
tips with the lab tissues to keep them sterilized.
3. Clip the proboscis half a centimeter or so from the
head. Collect hemolymph into the microfuge tube. You may
have to squeeze gently on the abdomen to get hemolymph to
flow from the wound. The hemolymph will be a yellow-green
color. Dispose of the pupa(e) by freezing.
4. Microfuge the hemolymph sample for pin for 2 min.
Place the tubes in the microfuge with the tabs facing out.
This will cause your pellet to form under the tab and you
will know where to look for it. This centrifugation step
gives cell free hemolymph; the hemocytes (blood cells) and
any undissolved PTU crystals will be pelleted.
5. While the tubes are spinning, label new microfuge
tubes. After the centrifugation, transfer most of the
supernatant to the appropriately labeled new tube. Be
careful not to mix-up the samples. Also be careful to leave
the pellet undisturbed.
6. Hemolymph samples should be stored in the freezer.
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