A simple method to disperse eggs from lepidopteran scalelike egg masses and to observe embryogenesis

Abstract

Various insect species lay tiny, thin‐ and soft‐shelled eggs in a connected “egg mass”. Especially in several lepidopteran species, the structure of such clustered eggs is covered with complicated scale‐like secretion, which has so far prevented analysis of individual embryos. However, few studies on methods to disperse egg clusters of such insects and to compare different methods have been carried out. To overcome these problems, we developed methods to separate egg masses into individual eggs, using two Tortricidae pests, Homona magnanima Diakonoff and Adoxophyes honmai Yasuda (Lepidoptera: Tortricidae). The eggs were successfully separated from each other using potassium hydroxide and sodium hypochlorite. Although the separated eggs no longer continued their embryogenesis, fixation with heptane–paraformaldehyde, permeabilization with heptane–methanol, and staining with several dyes enabled easy observation of embryogenesis. This protocol is expected to be applicable to other insect taxa and will facilitate further morphological and genetic studies in insects that lay egg masses.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima

“…To visualize DNA damage by performing TUNEL assays, mature embryos (108 hpo) and midguts of the dissected nearly hatched mature embryo (132 hpo) were fixed with 4% (wt/vol) paraformaldehyde phosphate-buffered saline with Tween 20 (PBST; 137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.68 KCl, 1.47 KH 2 PO 4 , and 0.05% Tween 20 [pH 7.4] as described in Arai et al [ 58 ]) solution for 15 min, washed twice with PBST for 5 min, incubated with 100 μL proteinase K solution (20 μg/mL) for 20 min at 25°C, and washed twice with PBST for 5 min. Positive controls were treated with 50 μL DNase solution (TaKaRa) for 20 min.…”

Distinct Effects of the Male-Killing BacteriaWolbachiaandSpiroplasmaand a Partiti-Like Virus in the Tea Pest Moth,Homona magnanima

“…To visualize DNA damage by performing TUNEL assays, mature embryos (108 hpo) and midguts of pharate larvae (132 hpo) were fixed with 4% (w/v) paraformaldehyde phosphate-buffered saline with Tween-20 (PBST; 137 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.68 KCl, 1.47 KH 2 PO 4 , 0.05% Tween-20, pH 7.4 as described in Arai et al (61) solution for 15 min, washed twice with PBST for 5 min, incubated with 100 μL proteinase K solution (20 μg/mL) for 20 min, and washed twice with PBST for 5 min. Positive controls were treated with 50 μL DNase solution (TaKaRa) for 20 min.…”