Axiophot 2

To visualize the overall morphology of head glands and facilitate their comparison, 3D-reconstructions of the head glands were conducted for 22 of the 26 investigated species. Even though for one Trachypus and three Philanthus species no complete series of sections were available (Table 1), the histological sections were sufficient to allow for the determination of gland characters. For 3D-reconstruction, continuous series of semithin sections of one individual per species were photographed using a digital microscope camera (Olympus DP20; Olympus, Japan) attached to a light microscope (Zeiss Axiophot 2; Carl Zeiss Microscopy GmbH, Jena, Germany). The digitalized sections were automatically aligned with regard to each other using the software package TrakEM2 [41 ] for the open source image processing software Fiji [42 (link)], and the alignment was subsequently corrected manually where necessary. The relevant structures within the head capsule were then marked as 3D-objects in TrakEM2 by manually outlining them in each picture of a series. Finally, 3D-reconstructions were calculated and visualized using Fiji’s 3D-viewer plug-in [43 ].

Immunofluorescence staining was performed on frozen graft tissue sections after 10-min fixation with ice-cold acetone and/or methanol (7 μm thickness). Sections were blocked for 1 hour at room temperature (RT) with 3% fetal bovine serum in phosphate buffered saline before incubation with primary antibodies against hC7 LH7.2 (Sigma-Aldrich) in a 1:500 dilution (Supplementary Table S1 online), desmoglein-1 (Fitzerald Industries, Acton, MA), involucrin (Sigma-Aldrich), keratin 10 (in-house), complex IV subunit II MTCO2 (Abcam, Cambridge, UK) overnight at 4 °C. Secondary antibody incubation with Alexa Fluor goat antimouse 488 (Invitrogen, Paisley, UK), goat antirabbit Cy3 (Life Technologies, Paisley, UK), and strep 488 was followed for 1 hour at RT. Sections were stained with 4'.6-diamidino-2-phenylindole (5 mg/ml) and mounted using a ProLong Gold antifade agent (Life Technologies). These were also stained by a hematoxylin and eosin histochemical technique. Staining was visualized and imaged using a Leica DMLB upright microscope (Leica Microsystems CMS, Wetzlar, Germany) and a Zeiss Axiophot 2 (Zeiss, Oberkochen, Germany) and processed using Image-Pro 6.2 (MediaCybernetics, Rockville, MD). Confocal imaging was carried out on a Zeiss LSM 510 Meta laser confocal microscope (Zeiss). Postprocessing was carried out using ImageJ.

For bright field image acquisition of DAB staining on DRG cryosections, we used a Leica DMI IL LED inverted microscope with a DMC2900 CCD camera (Leica Microsystems, Wetzlar, Germany). Images were acquired with the Leica Application Suite X software (Leica Microsystems, Wetzlar, Germany) at 20× magnification and were used for cell counting of CD11b⁺ and CD3⁺ cells in DRG sections. For fluorescence image acquisition, we used the fluorescence microscope Zeiss Imager M.2 with an Apotome 2 device for structured illumination, Colibri 7 LED as a light source, and an Axiocam 506 mono CCD camera (all Zeiss, Oberkochen, Germany), and the fluorescence microscope Zeiss Axiophot 2 (Zeiss, Oberkochen, Germany) equipped with a SPOT INSIGHT^TM 4.0 Mp Color Camera and the SPOT Advanced Software (both SPOT imaging, Sterling Heights, MI, USA). Images were acquired with the Zeiss ZEN Blue edition software at 20× magnification.

Wasps were cold anesthetized and decapitated. The heads were fixed using different fixing agents (Table 1). For large heads, both compound eyes were laterally cut off after fixation using sharp razor blades to facilitate the infiltration of the embedding medium. Heads were then thoroughly rinsed in 70 % or (in case of formalin-ethanol-acetic acid fixation) 80 % ethanol, dehydrated in a graded ethanol series and propylene oxide, and embedded in Epon 812 according the suppliers instructions (Polysciences Europe GmbH, Eppelheim, Germany). Sagittal semithin sections (4 μm) were cut with a microtome (Reichert Ultracut; Leica Microsystems AG, Wetzlar, Germany) equipped with a diamond knife and subsequently stained with toluidine blue [40 ]. The resulting series of histological sections were used for histological investigation of the head glands by light microscopy (Zeiss Axiophot 2; Carl Zeiss Microscopy GmbH, Jena, Germany) using bright field and differential interference contrast settings and for 3D-reconstructions of the head glands.

After demineralization, specimens were submitted to routine histological processing, embedding paraffin, and sectioned in 5 µm slices. Sections were stained with hematoxylin and eosin (HE) and observed in a light microscope Zeiss Axiophot 2 (Carl Zeiss, Oberköchen, Germany).

Heads of 0-24 hr old adult flies were detached and glued in an anterior up orientation to a microscope slide using a thin layer of clear nail polish. A drop of oil (Carl Zeiss Immersol, 518N) was placed on the sample and the eyes were examined under an upright light microscope (Zeiss Axiophot2). Shining a narrow bright beam of light from below the sample and optically neutralizing the cornea in an appropriate medium made the rhabdomeres visible (Franceschini and Kirschfeld, 1971a (link)).

Analysis was performed using a fluorescence microscope (Axiophot 2, Zeiss, Oberkochen, Germany) with appropriate filter sets. Each section, containing typically 2–5 labeled cell profiles, was examined using a 40× objective by focusing through the section thickness starting from the top surface. All profiles, except those visible at the top surfaces of sections, were counted (Simova et al., 2006 (link)). The application of this simple stereological principle prevented double counting of labeled cells and allowed precise evaluation of cell numbers.