Possibility of using a Carbol fuchsine for the coloration of larvae of Lepidoptera
Description
We studied the possibility of using a Carbol fuchsine for the coloration of larvae of Plodia interpunctella (Lepidoptera: Pyralidae). 87 larvae of Plodia interpunctella were used (59 were kept for more than 50 years, 28 were kept for one year). We were based on the method proposed by the N. A. Gura and A. V. Shipulin (unpublished) for Diaspididae (Hemiptera). Larvae were extracted from the fixing liquid and placed them in a test tube with Carbol fuchsine for various time intervals. Images of the samples were made using Zeiss Stereo Discovery V12 microscope (lighting settings were fixed) with Canon EOS 6D camera. The stereomicroscope was focused on various structures of the prothorax. Different setae were selected for analysis. The camera settings were fixed at ISO 400 and shutter speed of 1/25 s. A dark frame was shot with the same camera settings and the lighting turned off. Then, the contrast of setae was analyzed based on these images. At first, we chose images in which the analyzed setae 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. Then two areas of the image were selected: one with a background next to the seta, the other one on the seta. We determined the average brightness for both areas in analog-to-digital converter counts (ADU) using the ImageJ program. Further, the contrast of the structure under study was calculated as K=|Is-Ib|/Ib, where K stands for contrast, Is and Ib is seta and background brightness and || means absolute value. K, Is, Ib values are presented in Table 1. The dependences of the brightness of various setae and of background on the time of treatment with Carbol fuchsine were plotted in Figure 1. The data were fitted with an exponential decay to the stationary value. As can be seen from Figure 2, the contrast of different setae in larvae stained with Carbol fuchsine for the time period from 5 seconds to 1 minute is very different. This is due to the fact that the background remains light, and the setae are colored extremely unevenly, that is, some setae are clearly visible, while others remain poorly distinguishable. Starting from the staining time of 2 minutes, the contrast of the setae and the background is consistently greater than 0.1, and all the setae are clearly visible. With a staining time of more than 10 minutes, the setae contrast of all samples differ from the control ones. The exposure time of more than one hour leads to the fact that the degree of staining of the setae and background becomes as stable as possible and does not change from one hour to five hours. When exposed to Carbol fuchsine for 2 to 20 minutes, the contrast of the setae is relatively high, and there are no unpainted setae anymore.