Stereomicroscope 475 110 9904

To analyse the frequency of embolus breakage and plugging in L. geometricus depending on the developmental stage of the female, we staged matings with late-subadult (N = 60) and adult females (N = 42) using virgin males (N = 102). After mating, males and females were fixed in 70% ethanol. The copulatory organs of 50 males mated to late-subadult females and 31 males mated to adult females were checked under a stereomicroscope (Carl Zeiss) to establish the occurrence of embolus loss. It was recorded whether a pedipalp lacked the tip or a larger part of the embolus. In all mated females (53 late-subadult and 37 adult), the genital area with the underlying spermathecae and copulatory ducts were dissected out with a micro-scissors and transferred into a vial containing 5% potassium hydroxide. Herein, the genitalia were macerated at 60 °C on a heating plate for two days. The cleared and transparent copulatory ducts and spermathecae were transferred into 70% ethanol and inspected for mating plugs under a stereomicroscope (Carl Zeiss stereomicroscope 475,110–9904). We recorded the occurrence, number and size of embolus pieces, as well as their position (spermathecal entrance, copulatory duct). For illustration, selected pedipalps and female genitalia were photographed using a customised Visionary Digital BK Plus imaging system as described above.

External genitalia of one virgin early-subadult female, one virgin and one mated late-subadult female and one virgin and one mated adult female were fixed in 70% ethanol and photographed using a customised Visionary Digital BK Plus imaging system (Dun, Inc., Palmyra, Virginia, USA). All image adjustments were carried out using either Adobe Photoshop CS6 and Illustrator CS4 (Adobe systems, Inc., San José, California, USA) or CorelDRAW 2017 and Corel PHOTO-PAINT 2017 (all Corel Corp., Ottawa, Ontario, Canada). Image stacking was processed using “Zerene Stacker” stacking software (Zerene Systems LLC, Richland, Washington, USA).
We inspected cuticles shed by virgin (N = 15) and mated (N = 15) subadult females during their final moult for a presence of spermathecae under a stereomicroscope (Carl Zeiss stereomicroscope 475,110–9904).