Sz61 stereomicroscope

After mutant lines and RNAi knockdown progeny were scored for pigmentation, they were preserved in a 3:1 ethanol/glycerol solution and stored at 4°C until dissection for imaging. The fly cuticles were dissected from the abdomen and mounted to a glass slide using Permount and a glass cover slip. All photographs were taken with an Olympus DP25 microscope camera on an Olympus SZ61 stereo microscope.

Cells were grown on YPD plates for 2 days at 25 °C and the colonies were photographed with an Olympus SZ61 stereo microscope equipped with a CAM-E3CMOS6.3 camera using the ImageView software, and the size measurement performed was using the 3-point circle tool. For the collection of images of the cells, phase contrast microscopy was performed using a Zeiss Observer Z1 inverted microscope Ph3 stage with the Pln Apo 100X/1.4 objective by immobilizing cells from an overnight culture in a 1 mm layer of 1% agarose in PBS. Cells were photographed using the Hamamatsu ORCA-Flash4.0 LT + C11440 camera and the ZEN software. For cell measurement, the cells were photographed with an Olympus CX43 microscope equipped with a CAM-E3CMOS6.3 camera using the ImageView software, and the size measurement was performed with the 5-point ellipsis tool. All figures were prepared with Adobe Illustrator.

A Burg-Wächter PS 7215 digital caliper (Burg-Wächter, Wetter-Volmarstein, Germany) was used to determine the dimensions of the specimens. Olympus SZ61 stereo microscope (Olympus, Shinjuku, Japan) and KEYENCE VK-X210 3D scanning microscope (Keyence, Osaka, Japan) were used to determine the strand width and pore size of our inversely 3D printed ceramics. The voids within the ceramics were measured using Image-J software (Fiji version 1.52 h). The macroporosity of our samples was calculated from the measured values for strand width and pores using the following equation: $\mathrm{Macro} \mathrm{porosity} \left[\%\right]=\frac{\mathrm{pore} \mathrm{volume} \left[{\mathrm{mm}}^3\right]}{\mathrm{scaffold} \mathrm{volume} \left[{\mathrm{mm}}^3\right]}\times 100$

Strains were grown overnight in LB with agitation and ampicillin when required. A volume of 3.5 μL of culture was plated on Congo Red Agar (1% tryptone, 1% agar, 40 μL/mL of Congo Red Dye, 0.8 μL/mL of Coomassie Blue) and ampicillin when required. Plates were incubated at 30 °C or 37 °C for 72 h. Pictures of colonies were taken using an Olympus DP22 digital camera and its software with Olympus SZ61 stereomicroscope.

The prevalence of acanthocephalan parasites was assessed macroscopically and semi-quantitatively categorised as mild, moderate or severe [50 (link),51 (link)]. Parasites were collected in tap water and transferred to 70% ethanol after one hour. Other parasitic infections were noted on occasion, when grossly visible or when parasites were visible in the routine histologic examination. Acanthocephalans of a subset of 25 animals were identified further. Selected individuals were identified by PCR as a reference and the remaining specimens were identified based on morphological characteristics, using a stereomicroscope (Olympus SZ61 Stereo Microscope, Olympus, Tokyo, Japan) [52 ].

Dried dead bodies of both sexes were collected from the culture bottle. To digest their internal organs, we boiled the female specimens in 20% NaOH solution for two hours. Treatment in alkaline solutions dissolves soft tissues (including mesospermaleges and the mass of scar substances on the soft tissues) but leaves exoskeletons and scars (i.e., traces of copulation) on them [7] . The digested content was carefully removed by making a small hole in the intersegmental membrane between the metathoracic and first abdominal tergite using fine forceps. The exoskeleton samples were then carefully washed in water and observed from the ventral side under an SZ61 stereo microscope (Olympus, Tokyo, Japan). Digital images of the samples were obtained using a X-cam α CCD camera and Digi-Acquis software (Matrix Optics, Selangor, Malaysia). As a control for this observation, the exoskeleton of virgin females (n = 20) was observed by the same method.
Aberrant males with duplicated intromittent organs have been reported for C. lectularius (only two examples out of 3575 males examined; [13] ) and C. hemipterus ( [7] ; with unknown frequency). These males (D-males) were considered to be sterile [7] , [13] . In the current study, dead male bodies were also examined under the stereo microscope to check for the occurrence of D-males.