Mzapo microscope

Morphological details were examined with a Leica MZAPO microscope. Color illustrations were taken with a KEYENCE VHX-5000. The specimens were identified as Paraleuctra species by the following features: The Paraleuctra species is small, generally no more than 10 mm, and brown or dark brown. At rest, the wings roll into a tube to the abdomen. The subanal probe and epiproct of the male are specialized (Figure 1C–E,H–J). The tergum 10 of the male terminalia is divided into two parts (Figure 2A,D,G,J). Cerci sclerotize and form a dentate process (Figure 1A,B,F,G). The sternum 8 of the female forms an obvious subgenital plate; the cercus is simple with no change (Figure 3B,C). The terminology used for description follows Yang et al. (2015) [20 ].

Specimens were collected by hand and preserved in 75% ethanol. Morphological details were examined with a Leica MZAPO microscope. Colour illustrations were taken with a KEYENCE VHX-5000 digital dissection microscope. All specimens used in this study are deposited in the Insect Collection of Yangzhou University (ICYZU), Jiangsu Province, China. All the type materials are deposited ICYZU. The morphological terminology follows that of Sivec et al. (2008) .

For colorimetric in situ hybridization, embryos were gradually dehydrated in 100% MeOH post in situ hybridization and gradually cleared into 100% glycerol for imaging. Images were taken on a Leica MZ APO microscope using the Lumenera Infinity 3 camera at a magnification of 50X together with the Infinity Analyze software. Images were cropped and post-processed in a consistent way in Adobe Photoshop 2023 for color balance, brightness, and contrast. For HCR-FISH, embryos were cleared for 1 h in Optiprep (Stem Cell Technology). For dorsal imaging of the developing head, embryos were mounted dorsally on 0.5%-1% agarose blocks made with a customized inverted mold including a ~250 μm lane accommodating the head, and ~400 μm accommodating the yolk. Mounted embryos were carefully placed on a 35 mm MatTek glass bottom dish. For lateral imaging of the developing head, embryos were mounted laterally on a glass slide in Optiprep and positioned as flat as possible using layers of double-sided tape. Images were taken on a Nikon AT-AT inverted confocal microscope, using a 4X magnification for both lateral imaging of the developing head (st20 embryos) and dorsal imaging of the developing head (st21-st23.5 embryos). Images were analyzed and post processed for brightness and contrast in Fiji-ImageJ and Adobe Photoshop 2023, then cropped and assembled into montages in Adobe Illustrator 2023.