Material and Methods
Insect collectionnext section
Adults of P. kuandianensis were collected at the Quanshan Forest Park (41°06’N, 125°02’E, elev. 650–800 m), Kuandian County, Liaoning Province in northeastern China from early July to August in 2015 and 2016.
Male and female adults were reared in three gauze cages (40 cm × 40 cm × 60 cm) in the laboratory under natural conditions following Zhong and Hua (2013). Absorbent cotton containing water was replaced each day in a Petri dish. Twigs with leaves were placed in the cages to simulate the habitat of the adults, and were replaced every three days. Each cage was supplied with five drops of honey per day to prevent low nutritional status (Jiang et al., 2014).
Mating behavior observation
Ten males and ten females were reared in each cage to investigate their mating behaviors. These adults were observed 24 h a day during 6–10 July 2015 to check the circadian rhythms of mating activity. Then adults were observed every 30 min from daybreak to noon to record the mating behaviors, including mating positions and copulatory processes. Photographs were taken with a Nikon D90 digital camera.
Freeze-fixation of copulating pairs
Pairs in copula were frozen through carbon dioxide aerosol spray compressed in hydraulic cans, and were immediately fixed in Carnoy’s solution at room temperature for 24–48 h to stabilize the interactions of their genital structures and preserved in 75% ethanol.
Light microscopy and scanning electron microscopy
Fifty adults (30 males and 20 females) fixed in Carnoy’s solution were dissected under a Nikon SMZ1500 microscope. Photographs were taken with a QImaging Retiga-2000R digital camera attached on the microscope and were stacked with the Syncroscopy Auto-Montage software.
For scanning electron microscopy (SEM), the dissected genital structures were cleaned ultrasonically for 90 s, and then dehydrated in a graded ethanol series, freeze-dried for 3 h, coated with gold in a sputter coater, and examined in a Hitachi S-3400N scanning electron microscope (Hitachi, Tokyo, Japan) at 5 kV.