The possibility of using a Slide Dryer BIS-N 209-1 for the preparation of male genitalia of tribe Phycitini

We studied the possibility of using a Slide Dryer BIS-N 209-1 for the preparation of male genitalia of tribe Phycitini (Lepidoptera: Pyralidae). 31 males of Plodia interpunctella from a single sticky insert from pheromone trap were used. The male abdomens were separated and placed in a plastic Petri dish (of diameter 3.5 cm) with 2 ml of 10% KOH solution. The samples were prepared at a fixed temperature of 80 C and over various time intervals. Being prepared, the samples were placed in glycerol on a glass slide and covered with a coverslip. Images of the samples were made using Zeiss Axio Imager 2 (lighting settings were fixed) with Canon EOS 6D. The microscope was focused on various structures of the genitals. Two structures were selected for analysis: the cranial, the darkest part of the gnathos, and the lightest part of the tegumen. The samples were examined and imaged from the ventral side. The aedeagus was previously removed. The camera settings were fixed at ISO 100 and shutter speed of 1/200 s. A dark frame was shot with the same camera settings and the lighting was turned off. Then, the brightness of the light passing through the samples was analyzed based on these images. At first, we chose images in which the analyzed structures were as sharp as possible. The raw images were converted into linear monochrome 16-bit TIFF files with dark file subtraction using the open-source Dcraw software. The color channels were converted at a fixed color temperature, no debayering was performed. The resulting images were analyzed in the open-source ImageJ software. The images were downsampled by averaging the brightness values over the 4*4-pixel area (binning). Next, two areas of the image were selected: one on a background next to the sample, the other one on the structure under study. The most homogeneous areas of the image were selected for the analysis. The ImageJ program determined the average brightness for both areas in analog-to-digital converter counts (ADU). Further, the brightness of the structure under study was normalized to the background brightness, the obtained light transmittance of the structure was entered in Table 4. The dependences of the light transmittance of various structures on the time of treatment with KOH were plotted in Figure. The data were fitted with an exponential decay to the stationary value. The abdomens that stayed in the KOH for 5 minutes turned out to be completely unsuitable for research because the sclerotized structures were too rigid, and the soft tissues were not completely macerated. As a result, it was shown that the degree of transmittance of the genitals increases with time. This is best seen in the dark area of the gnathos. However, after 80 minutes in KOH, the genitals reach their maximum transmittance transparency.本研究探讨了使用玻片干燥器BIS-N 209-1（Slide Dryer BIS-N 209-1）制备螟蛾族（Phycitini，鳞翅目：螟蛾科）雄性生殖器的可行性。实验采用单个性诱捕器粘虫板上采集的31头印度谷螟（Plodia interpunctella）雄虫作为样本。将雄虫腹部剥离后，置于直径3.5 cm的塑料培养皿中，加入2 ml 10%氢氧化钾（KOH）溶液。样本在固定80℃的温度下，经不同时长的处理完成制备。制备完成后，将样本置于滴有甘油的载玻片上，加盖盖玻片。图像采集使用蔡司Axio Imager 2（Zeiss Axio Imager 2）显微镜搭配佳能EOS 6D（Canon EOS 6D）相机，光照参数固定；显微镜对焦于生殖器的不同结构，最终选取两处结构开展分析：颚突（gnathos）中颜色最深的颅侧部位，以及背兜（tegumen）中颜色最浅的部位。所有样本均从腹面进行观察与成像，且实验前已移除阳茎（aedeagus）。相机参数固定为感光度ISO 100、快门速度1/200秒。采用相同相机参数在关闭光照的条件下拍摄暗场帧，随后基于上述图像分析透过样本的光亮度。首先选取分析目标结构清晰度最优的原始图像，通过开源Dcraw软件执行暗帧减法，将原始图像转换为线性单色16位TIFF文件；转换过程中采用固定色温处理色彩通道，未进行去拜耳阵列处理。使用开源ImageJ软件对所得图像进行分析：通过对4×4像素区域的亮度值取平均进行降采样（分箱处理）。随后选取图像中的两处区域：一处为样本旁的背景区域，另一处为待测结构区域，均选择图像中亮度最均匀的区域进行分析。ImageJ软件将以模数转换器计数（ADU）为单位计算两处区域的平均亮度。将待测结构的亮度归一化至背景亮度，所得结构透光率数据录入表4。绘制不同结构的透光率随KOH处理时长变化的曲线，并对数据进行向稳态值的指数衰减拟合。经5分钟KOH处理的腹部样本完全不适用于研究，原因是骨化结构过硬，且软组织未完全消解。研究结果表明，生殖器的透光率随处理时长增加而升高，该现象在颚突的深色区域表现最为显著；但经KOH处理80分钟后，生殖器的透光率达到最大值。