Transmission electron microscopy (TEM)
Twenty-four larvae were left to develop in felled spruce longs up to
their 2nd or 3rd instar. They were then removed from the logs, surface
sterilized by rinsing in distilled water and 40% ethanol, which also
killed them gently. The larvae were then dissected under a binocular
magnifier in a drop of sterile buffer (10x PBS). The digestive tract of
each individual was then separated into 3 subsamples (foregut, midgut
and hindgut) to facilitate orientation in the sample and fixed by
following protocol. For TEM analysis, pieces of the digestive tract were
fixed for 24 h in a solution of 2.5% glutaraldehyde in 0,1 M cacodylate
buffer (pH 7.2) and postfixed in 2% OsO4 in the same cacodylate buffer.
Fixed samples were dehydrated through a standard ascending ethanol and
acetone series and embedded in Araldite - Poly/Bed® 812 resin mixture.
Thin sections were cut on an ultramicrotome (Reichert-Jung Ultracut E)
and stained using uranyl acetate and lead citrate following the Hayat
(2000). Sections were examined and photographed using JEOL JEM-1011
(JEOL Ltd., Japan) transmission electron microscope. Fine structure
measurements were performed using a Veleta camera and iTEM 5.1 software
(both Olympus Soft Imaging Solution GmbH, Germany). Photos were made
under accelerating voltage from 100 to 300 keV.