Histology. Insects were dissected in sterile PBS to extract full-length gastrointestinal tracts (guts). Full-length guts were linearized by removing connective tracheoles and allowed to relax in a droplet of PBS for photography. Images documenting the longitudinal extent of guts adjacent to a millimeter scale were acquired. Molten autoclaved 3% agarose was pipetted into a wax mold and dissected gut tissues were immersed into the agar while maintained in a linear orientation. Additional molten agar
was pipetted over the surface of the gut to encase the tissues. Agar-embedded guts were inserted into 10 mL vials containing Carnoy’s solution (100% ethanol, chloroform, and acetic acid were mixed in at a 6:3:1 ratio) and incubated at 25 ˚C with gentle agitation for 24 hours (Koga, Tsuchida, & Fukatsu, 2009). Fixative was removed and replaced with 70% EtOH and tissues were stored at 4 ˚C. Fixed tissues were trimmed of excess agar and dehydrated and embedded in wax in an automatic tissue processor using the following solvent exchange series: 1hr in 70% EtOH, 1hr in 80% EtOH, 1hr in 90% EtOH, 1hr in 95% EtOH, 1hr in 100% EtOH 1, 1hr in 100% EtOH 2, 1 hr in 50:50 EtOH/Xylene, 1hr in 100% Xylene, 4 hrs in 100% Xylene, 20 min in 30% periplast dissolved in xylene, 20 min in 100% periplast @ 60 ˚C, 20 min in 100% periplast @ 60 ˚C. To obtain suitable transverse tissue sections, custom molds were fashioned from 10ml syringes and mounted onto wooden blocks for clamping to a microtome. Agar embedded guts were pierced through the agar with a fine insect mounting needle at an approximately 45 degree angle and lowered into the block/mold assemblies, so that the gut was centered in the cylinder of the syringe bore. The mold was topped with molten wax with a heated syringe and large bore needle (16 ga). After the wax has hardened, the mold was loosened and the block was trimmed. Ten micron sections were prepared from the anal cavity to proventriculus and affixed to positively-charged slides. Each slide had 4-8 sections from the same gut sample and region, with about 20-30 slides per gut being prepared. Sections were fixed on slides using a hot plate at 55 ˚C for 10 min and dried for 1 hour on a slide dryer before at least 24h of drying at room temperature. Slides were dewaxed and rehydrated through the following xylene, ethanol, and water series: 10 min in 100% xylene, 10 min in 100% xylene 5 min in 50:50 xylene/ethanol, 1 min in 100% ethanol, 1 min in 100% ethanol, 1 min in 90% ethanol, 1 min in 80% ethanol, 1 min in 70% ethanol, 1 min in 50% ethanol, and 2 min in distilled H2O. Rehydrated slides were stained according to Heidenhain’s Azan Trichrome method (Schmid, 1989), with a 7.5 min soak in 50 ˚C azocarmine g in 1% acetic acid, water rinse, 30 sec in 0.1% aniline in EtOH, 2 min in acidified EtOH (0.1% HCL), 2 hrs in 5% phosphotungstic acid, and 3 hrs in 2% Orange G/0.5% Analine Blue in 7.5% aqueous acetic acid. Slides were rinsed in distilled H2O for approximately 10 seconds, until blue dye dissipated from the agar portion of the section and then slides were dehydrated: 10 sec in 100% ethanol, 10 sec in 100% ethanol, 10 sec in 50:50 xylene/ethanol, 10 sec in 100% xylene, 10 sec in 100% xylene. Slides were mounted and coverslipped with Cytoseal 60. Tissue Imaging and Measurement. Images of tissues were acquired on an Olympus bright field microscope with an Omax camera at 100x total magnification for crosssectional area and perimeter measurements and at 400x total magnification for muscle measurements. Five cross-sections were imaged per gut region per individual for a total
of 20 measurements per treatment per gut region for cross-sectional area and perimeter measurements. For muscle measurements, the same five cross sections per individual were imaged at higher magnification, capturing 4 micrographs per section, one from each quadrant, for a total of 80 images per treatment per gut region. Measurements were taken in ImageJ using a custom macro to threshold images, create masks, and record measurements in a semi-automated fashion. Pixel measures were converted to μm and μm2 units. Photos containing digestate and bacterial biomass directly adjacent to the gut lumen required manual demarcation of the luminal boundary to ensure
accurate thresholding. Images were converted to masks of total gut cross-sectional area and luminal area and the difference of these constituted the epithelial cross-sectional area (Figure 1). Perimeter measurements were obtained from the luminal mask and the total cross-sectional area mask (Figure 1). For muscle measurements, grid overlays were projected over micrographs to direct measurements in a random fashion to avoid measurement bias, as muscle thickness within a sample was often heterogeneous, and two measurements were taken per image. DAPI Imaging. Representative slides from three different insects were selected from both the hindgut and midgut regions and were dewaxed as previously described under histology methods. Rehydrated tissues were flooded with 600 nM aqueous 4',6- Diamidine-2'-phenylindole dihydrochloride (DAPI), allowed to rest for 15 minutes in the dark, and then were rinsed in 3 changes of PBS. Rinsed slides were coverslipped with
Dako Faramount antifade mountant, and immediately imaged. Hindgut slides were imaged at 400x at the luminal boundary to resolve both host and microbial biomass and both hindgut and midgut slides were imaged at 1000x within the lumen to resolve individual microbial cells. Multiple micrographs from different focal planes were merged using the utility Combine ZP to create composite micrographs.