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Tobacco Hornworms


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Antibacterial response (hemolymph)

Changes In Hemolymph During the Antibacterial Response of M. sexta

 

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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March 1999