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

Published: 12 December 2020| Version 1 | DOI: 10.17632/dkt4yy3f7s.1
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Description

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.

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