Island fly culture
Island flies used for the scans were obtained from cultures maintained at the Waite Insectary, School of Agriculture Food and Wine, University of Adelaide, Urrbrae, South Australia, at 27 °C; RH 70–80% and L:D 13:11 (natural light supplemented by fluorescent lighting). Flies for the culture were sourced from Waikerie and Mypolonga in South Australia’s Riverland, male and female flies were trapped in McPhail traps containing Putrescine (FFP) and ammonium acetate (FFA) lures (Suterra LLC, Bend, OR, USA). Adult flies were provided with a diet of hydrolysed yeast and water ad libitum supplemented with Enterobacter spp isolated from the gut of wild D. pornia collected from Loxton in 2011 [15]. Fresh oranges were poked with the help of a needle and placed in adult cages for oviposition and larval development.
Preliminary dissections
Prior to CT and SEM scans a series of 100 dissections, 50 male and 50 female, of adult D. pornia were conducted with the help of a Nikon SMZ25 stereomicroscope. The age of male and females flies dissected in this manner ranged from 24 h to 3 weeks post eclosion to observe any developmental changes that may have affected outcomes.
Preparation of island flies for CT scans
Methods for dehydration of the flies were modified from Alba-Trecedor [20]. Our method differs from his mainly in the fixing and dehydration process. In our case, when the samples were fixed with 4% Paraformaldehyde in PBS + 4% Sucrose at a pH of 7.2 prior to dehydration as described in his method, the resulting images appeared unclear. This may have occurred due to fluids trapped during the fixing process. Therefore we did not fix the sample. Instead we directly dehydrated the sample in a graded series of ethanol. Dehydrating of the sample at 70% which was the method used by Alba-Trecedor also resulted unclear images and tissue damage. In order to avoid this we dehydrated the specimen using a graded series of ethanol at 30, 50, 70, 80, 90 and 100% which showed better results. Similarly, staining the sample with 1% Iodine for more than 6 h showed better contrast of the softer tissues in the CT scans than staining them with 1% Iodine for 3 h only.
For this study, 5 day-old male flies were selected from the culture and euthanized at − 18 °C for 3 min. Euthanized flies were dehydrated in a graded series of ethanol starting at 30, 50, 70, 80, 90 and 100% for 30 min each. The dehydrated flies were then stained with 1% iodine in 100% ethanol for over 6 h. The stained flies were then critically dehydrated in hexamethyldisilazane (HMDS) for 2 h with one fresh change of HMDS in between. They were left to dry overnight under a fume hood. One fly per scan was selected and then mounted over the top of the axis of a micro-CT scanner (SkyScan 1072, Bruker microCT, Belgium) by sticking it with Araldite® glue (Selleys®, Padstow, New South Wales, Australia) and left to dry for 20–30 min.
Micro-CT scanning in SkyScan 1072
The micro-CT scan and analysis of the head of D. pornia was performed at Adelaide Microscopy (Medical School North, Frome Road, The University of Adelaide, SA, Australia) with a Skycan 1072 (Bruker microCT, Kontich, Belgium). The specifications used were; beam energy set at 23 kV, current set at 120 μA, cross-section pixel size set to of 3.67 μ; exposure set at 3.4 s and rotation set to 180° with images captured every 0.225°.
Post-scan image processing
Scanned images that were saved in tagged image file format (TIFF) were reconstructed with the help of the NRecon software (Bruker microCT, Kontich, Belgium). The region of interest (ROI) was identified and the dynamic range selected, following which the image was adjusted for misalignment compensation and in this instance fine tuning/ beam hardening was performed. The image data were then converted into bitmap (BMP) format. The new reconstructed image data were then used to segment, recolour and visualise the alimentary tract of the fly in 3D with the help of the Avizo® Fire 8.1 software (ThermoFisher Scientific Hillsboro, Oregon, USA). A volume rendering module was attached to the data file and the alpha scale adjusted in order to reveal a semi-transparent scanned image of the fly. This was followed by the creation of a label field. An interactive segmentation was performed for the various structures and organs of the scanned 3D image. After the segmentation was complete the new surface generated was visualised with the help of the surface-view and volume rendering modules. Images were captured and saves as TIFF files.
Dissection of D. pornia to study the alimentary tract
Seven 24 h-old adult males were removed from the culture and provided with dyed sucrose solution (Queen Rainbow Food Colours, Australia) for 12 h. One adult was selected and dissected with the help of a Nikon SMZ25 stereomicroscope. The dye helped locate the alimentary tract during dissection. Images of the dissected areas were captured with the help of the attached camera.
Scanning electron microscopy studies of the oesophageal diverticulum of D. pornia
For the SEM studies, 8 adult male D. pornia collected from an orchard at Waikerie, SA, were selected. The flies were caught on the leaves and immediately chilled to transportation and processing. The oesophageal diverticuli were removed and fixed in in 4% Paraformaldehyde in PBS, 4% sucrose at a pH of 7.2 for 30 min. 2–3 washes were made in PBS + 4% sucrose for 5 min each. The washed oesophageal diverticuli were then post-fixed in 2% OsO4 in PBS for 1 h. They were then dehydrated in a graded series of ethanol at 70, 90 and 100% with 2 changes per concentration of ethanol at a 15 min interval, with an additional third change made for the last concentration of 100% ethanol. This was followed by critical point dehydration in a Bal-Tec CPD 030 Critical Point Dryer. The dried oesophageal diverticuli were then mounted on SEM stubs and coated with platinum. They were observed under a Philips XL20 Scanning electron microscope set at beam energy of 10 keV the next day. Images were captured with the help of the CCD camera and were saved in TIFF format.