Euparal

All material was collected by the author in northern Iran. The specimens were collected by hand and preserved in 96% ethanol. The isopods were dissected and body parts were mounted in micropreparations using Euparal (Carl Roth, Karlsruhe). Drawings were made using a drawing tube fitted on a Nikon Y-IDT compound microscope. Micrographs were taken using a Hitachi S-2460N SEM.
Some material of the present study was deposited in the

Zoological Museum, University of Tehran

(ZUTC) and

Iranian Research Institute of Plant Protection

(IRIPP), and the others were kept in the

personal collection of the author

(PCGMK). A map with sampling localities for Hemilepistoidesmesserianus in Iran along with the type locality is presented (Fig. 1).

For pigmentation analyses, adult females between 3 and 5 days old were stored in ethanol 70% during ten days. Abdominal cuticles were cut just beyond the dorsal midline and dehydrated in ethanol 100% during 5 minutes. After dehydration, cuticles were mounted in Euparal (Roth).
For nEGFP analyses, pupal and adult abdomens were dissected in PBS, fixed 20 minutes in 3.7% paraformaldehyde in PBS, washed twice 10 minutes in PBS and mounted in Mowiol. As developmental time is sensitive to temperature, morphological markers (wing colour, location of meconium) were used to compare pupae grown at 18°C and 29°C at a similar stage of development [56 (link)].

The right wing of each specimen was removed and mounted under a cover slip (15 × 15 mm) with Euparal (Carl Roth, Karlsruhe, Germany). Pictures of each wing were taken under 20× magnification with a stereomicroscope (Leica M205 C, Leica Microsystems, Wetzlar, Germany). Fiji^{28 (link)} as bioscience package of ImageJ^{29 (link)} was used to digitize 18 landmarks (Figure S1). The landmark selection was in accordance with other studies analysing mosquito wing morphometry^{18 (link)–20 (link),30 (link)}. The wing pictures were divided among two observers (authors LE and FGS) and digitalised in random order to minimize a memory biased landmark collection between the mosquito specimens of the same species. One month later, the measurement was repeated for three specimens per species by four observers (authors LE, LJ, RL and FGS) to assess the degree of observer error and repeatability in landmark collection^{31 (link)}.

After fixation in Carnoy’s solution (60% ethanol, 30% chloroform and 10% glacial acetic acid), anthers were washed in 95% ethanol and incubated with 1 N HCl at 60°C for 10 minutes. Next, the anthers were washed with water and incubated in Schiff’s reagent for 1 h at room temperature in the dark, followed by rinsing in bisulfite solution (5 ml of 10% potassium metabisulfite, 5 ml of 1 N HCL, 90 ml of distilled water) in 3 washes of 5 min each and thorough rinsing with water. Then, smears were prepared on 2.5% gelatin-coated slides and frozen on dry ice. The slides were finally dehydrated in a 70%, 96% and 100% alcohol series for 2 min each, followed by mounting with Euparal (CARL ROTH GmbH, Karlsruhe, Germany) under coverslips.

Adult males, three to five days old, were used for analysis. Dehydrated wings were mounted in Euparal (Roth; Karlsruhe, Germany). Wing area was measured using Image J software (oval selection for total wing size in two measurements that were sampled). Cell number was determined by counting the individual trichoma on the wing blade in three defined 10.000 μm² squares localized in the L4/L5 field next to the posterior cross vein. Subsequently, the total cell number for the determined wing area was calculated, as was cell size. Pictures were assembled using Corel Draw and Corel Photo Paint. Flies and wings were photographed with an ES120 camera (Optronics; Goleta CA, USA) using Pixera Viewfinder software, version 2.0.
To investigate developmental timing, offspring of six parallel inter se crosses with the genotype w¹¹¹⁸; cycG^HR7/+ was counted at days 9 to 20. Since the cycG^HR7 mutant carries a mini-white⁺ gene [24 (link)], the homozygous cycG^HR7 and the heterozygous cycG^HR7/+ flies could be distinguished by red and orange eye color, respectively, from the white-eyed control siblings.

Adult flies of the respective genotype were collected and etherized. Pictures were taken with an ES120 camera (Optronics, Goleta, CA, USA) mounted to a Wild M5 stereomicroscope (Leica, Wetzlar, Germany). Dehydrated wings of female flies were embedded in Euparal (Carl Roth, Karlsruhe, Germany) and documented with an ES120 camera (Optronics, Goleta, CA, USA) coupled to a Zeiss Axiophot (Carl Zeiss, Jena, Germany). Pictures were recorded using Pixera viewfinder software version 2.0. Alternatively, uncoated etherized flies were pictured with a table top scanning electron microscope NeoScope (JCM-5000 SEM; Nikon, Tokyo, Japan) using proprietary software. The wing or eye area was determined using the freehand tool of Image J (open source). Only the SEM pictures of female flies were used for the quantification of micro- and macrochaetae, respectively. The numbers of bristles were determined as described before [57 ,58 (link),59 (link),60 (link)]. Statistical analysis was conducted by ANOVA two-tailed test for multiple comparisons using Tukey–Kramer’s or Dunnett’s approach, as indicated: *** p ≤ 0.001 highly significant; ** p ≤ 0.01 very significant; * p ≤ 0.05 significant; not significant (n.s.) p > 0.05. The figures were assembled using Corel Photo Paint, Corel Draw and BoxPlotR software.

Cells in M phase within the posterior compartment were counted based on PH3 signals. The posterior compartment was determined by GFP labelling from en-Gal4-GFP [46 (link)]. Cell number was related to total size of the respective wing discs using ImageJ. p53R-GFP expression in salivary glands was examined by measuring signal intensity of 12 nuclei from seven different glands each (n = 84 nuclei), using the mean intensity of Pzg signals as internal standard. Wings from female flies were dehydrated in ethanol, mounted in Euparal (Roth, Karlsruhe, Germany) and documented with an ES120 camera (Optronics, Goleta CA, USA) connected to a Zeiss Axiophot (Carl Zeiss AG, Jena, Germany) using Pixera Viewfinder software, version 2.0. Female flies were etherized before taking pictures from the heads with an ES120 camera coupled to a Leica Wild M3C Stereomicroscope (Leica, Wetzlar, Germany). Size of female eyes (UASp-lacZ/+; ey-Gal4/+ and ey-Gal4/+; UAS-mei-41/+) or wings (UASp-lacZ/+; en-Gal4-GFP/+ and en-Gal4-GFP/+; UAS-mei-41/+) was measured using Image J. Statistical analysis was conducted by ANOVA using a two-tailed Tukey-Kramer or Dunnett’s test for multiple comparisons. ***p < 0.001 highly significant; **p < 0.01 very significant; *p < 0.05 significant; not significant (ns) p > 0.05. Box plots were compiled using the online plotting tool BoxPlotR (http://shiny.chemgrid.org/boxplotr/).