Rapid Assessment of Maturation Stage and Reproductive Mode in Centrolecytic Eggs of Stick Insects (Phasmatodea) Using DAPI Stain

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Abstract
A rapid three-step DAPI technique is proposed for detecting meiotic stages and sperm head evolution in yolky, fertilized stick insect eggs, which were difficult to analyze with other methods. Fixed eggs were freed from chorionic envelopes and stained directly in DAPI/PBS solution. After rinsing, eggs were singly squashed in a drop of mounting buffer and examined under a microscope with incident fluorescent illumination. The method was almost uniformly successful, and direct observation of nuclear structures, coupled with fluorometry, allowed easy recognition of bivalents, diads, pronuclei and their DNA content. The DAPI method proposed here appears particularly helpful for investigating unusual reproductive modes in eggs with large amounts of yolk.