Possibility of using a Carbol fuchsine for the coloration of larvae of Lepidoptera

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 We were based on the method proposed by the N. A. Gura and A. V. Shipulin (unpublished) for Diaspididae (Hemiptera).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.

本研究探讨了使用石炭酸品红（Carbol fuchsine）对印度谷螟（Plodia interpunctella，鳞翅目（Lepidoptera）：螟蛾科（Pyralidae））幼虫进行染色的可行性。本次实验共使用87头印度谷螟幼虫，其中59头饲养时长超过50年，28头饲养时长为1年。本研究基于N. A. 古拉与A. V. 希普林针对盾蚧科（Diaspididae，半翅目（Hemiptera））提出的未发表方法展开。将幼虫从固定液中取出，置于装有石炭酸品红的试管中，设置不同的处理时长。使用搭载佳能EOS 6D相机的蔡司Stereo Discovery V12体视显微镜拍摄样本图像，光照参数固定不变。体视显微镜对焦于前胸的不同结构，并选取不同的刚毛用于后续分析。相机参数固定为ISO 400，快门速度1/25秒。使用相同的相机参数并关闭光源，拍摄暗场帧。基于上述图像开展刚毛对比度分析。首先挑选分析用刚毛清晰度最优的图像。使用开源软件Dcraw对原始图像进行暗场帧扣除处理，将其转换为线性单色16位TIFF文件。转换过程中以固定色温完成色彩通道转换，未进行去拜耳解码处理。将得到的图像导入开源软件ImageJ进行分析。随后在图像中选取两个区域：其一为刚毛旁的背景区域，其二为刚毛所在区域。借助ImageJ程序，以模数转换器计数（analog-to-digital converter counts，ADU）为单位，分别计算两个区域的平均亮度。进而按照公式K=|Is-Ib|/Ib计算目标结构的对比度，其中K代表对比度，Is与Ib分别为刚毛与背景的亮度，||表示取绝对值。K、Is、Ib的数值详见表1。不同刚毛及背景的亮度随石炭酸品红处理时长的变化关系如图1所示，数据通过指数衰减模型拟合至稳态值。由图2可知，经石炭酸品红染色5秒至1分钟的幼虫，其不同刚毛的对比度差异显著。这是由于背景始终保持明亮，而刚毛的染色极不均匀：部分刚毛清晰可见，其余则难以分辨。当染色时长达到2分钟及以上时，刚毛与背景的对比度始终大于0.1，所有刚毛均可清晰辨识。当染色时长超过10分钟时，所有样本的刚毛对比度均与对照组存在显著差异。当染色时长超过1小时，刚毛与背景的染色程度将趋于稳定，且在1小时至5小时范围内不再发生变化。当染色时长为2至20分钟时，刚毛对比度相对较高，且不存在未着色的刚毛。