[Histonet] Re: Mealworm larvae

From:"David A. Wright"

Hello Shirley & Histonet

I used to do a lot of microscopy with different insects. Here
are some general suggestions, mostly for the early part of
tissue isolation - if that's the challenge, but it would help
to know:
a) which species of mealworm? (Because they vary in size and
that would obviously affect the choice of method)
b) which tissue?

Mealworms might include the tiny flour beetles, Tribolium,
since they are used for genetic research, the midsized common
petstore mealworm, Tenebrio, and the giant Super Mealworm,
Zophobas (also petstores).

Insects are easy to take apart since the cuticle holds stuff
together after cutting, different organs are largely separate,
and there is no vasculature beyond a simple tubular "heart"
(along the back). There are white spidery tubes of air
(tracheae) everywhere but these can be ignored.  If you want
to you can dissect in a general Insect Ringer solution (e.g.
NaCl 9.1 g/L, KCl 0.52 g/L, CaCl2.2H2O, 1.2 g/L MgCl2.6H2O .8
g/L).  Cells will stay alive for quite a while.  Pin the
insect out on wax or Sylgard, using "Minuten" pins (from e.g.
Carolina Biological & used for mounting very tiny insects in
museum collections) in a drop of ringer.

Before you try cutting them up (or immobilizing below), a
simple way to narcotize them is to drown them in degassed
water until they stop wriggling (10-15'?).  Use a little vial
or bottle with a slight lip, sideways in a tray of water. 
Duck the larva under the lip and then if it bobs upward it
can't come to the surface.  Try to eliminate trapped bubbles
(I use a paintbrush).  Chilling is also effective.

For epidermis (technically hypodermis), simply make whole
mounts of the cuticle (if it is transparent enough) with the
attached cells - they form a monolayer and no sectioning is
necessary.  Chop out the part of interest with a single edge
razor blade on some soft plastic (polyethylene of "nalgene'
softness).  Rip this part away from the carcass with forceps
and immediately plunge into a vial of fixative.  I used fresh
Carnoy for haematoxylin staining.  You don't even need any
saline, though you might have to trim off odd bits of fat body
 and trachea after fixation (which is of 'tissue culture"
rapidity since it's a monolayer).

If you want to section (and infiltrate) I suggest you
immobilize the insect (e.g. with little strips of modelling
clay on a slab of the same on top of a bottle cap or petri lid
that you can manipulate easily).  Then inflate the body by
rapidly injecting your chosen fix.  They stretch a bit and the
cuticle usually stops them from bursting.  

You could use a regular hypodermic & fine needle in the larger
guys.  In smaller ones, draw out the tip of a Pasteur pipette
in a flame, break off the tip to a sharp point of suitable
diameter and use that to inject the fixative.  The
intersegmental cuticle (between the hard rings) is a good
place to impale.  You can also cut off a leg (tiny in larvae),
or even the head, and inject through the hole so made - just
make sure the needle is nearly the same diameter as the hole
(the taper on a drawn pipette makes this easier).

Once the body has fixed a bit the increased firmness makes it
easier to cut off the ends of the larvae (razor or fine
scissors) and process the middle bit, a hollow tube,
conventionally.  You can also slit the tube longitudinally
with fine scissors.

The outer layer of insect cuticle does not like being wax
embedded (it separates) - there was a recent brief thread
started by Anthony Gatt on how to improve things.  Resin is
easier, since that's ok with your final goal.

Hope this helps some
David A. Wright, PhD
Section of Neurosurgery
University of Chicago
Histonet Digest, Vol 42, Issue 44  Message: 14
Date: Thu, 31 May 2007 11:37:28 -0400
From: Shirley Powell 
Subject: [Histonet] Mealworm larvae
To: histonet@lists.utsouthwestern.edu

Hi Guys,

I need help with this one.  One of my investigators is
requesting histology
on mealworm larvae, which eventually turn into mealworm
beetles.  Anyone who
has worked with or has information on processing these lovely
creatures in the larvae stage, please share your processing
procedure with
me.  Paraffin or plastic.

Thanks in advance.
Shirley Powell

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