Potentialities of iodine-enhanced micro-CT imaging in the morphological study of adult Aedes (Stegomyia) aegypti (Linnaeus, 1762) mosquitoes

Abstract

X-ray micro-computed tomography (CT) produces three-dimensional images of samples on a micrometer scale. This technique has several advantages, such as its nondestructive character and low measurement time, compared with other techniques. However, when applied to biological samples of soft tissue, the low attenuation and low effective contrast between structures pose difficulties in creating appropriate images for morphological studies. Diffusible iodine-based contrast-enhanced CT (DICE-CT), which uses iodine solutions to enhance contrast, is a viable alternative for addressing the aforementioned challenges. Given the variety of biological samples, an appropriate methodology must be adapted depending on the dimensions and morphological characteristics of the investigated object. A specimen that has not been morphologically studied by micro-CT and is of high sanitary importance is the adult Aedes aegypti mosquito. This study investigated the stage of iodine staining in the treatment of the A. aegypti mosquito to determine the most suitable staining time for the morphological study of this mosquito in adulthood. After determining the appropriate staining time, we discuss the potential of applying DICE-CT and methodology to mosquito studies. Seven A. aegypti females were treated using fixation steps with Bouin's solution, dehydration in a graded ethanol series, staining with iodine solution (1%), and washing in absolute ethanol. Only the staining step was different between samples. Each mosquito spent a varying amount of time (6–72 h) in the iodine solution (1%). For comparison, one of the mosquitoes was not stained. After treatment, the samples were scanned using the Bruker SkyScan 1172 micro-CT scanner. The reconstructed volumes and histograms were compared to determine the most suitable time. In addition, a quantitative analysis was performed based on a comparison of the attenuation profiles of the mosquito brains. Thereafter, the most suitable treatment process was selected, and two other samples were scanned after applying the selected process. Although fewer than 18 h was insufficient for an effective increase in attenuation and effective contrast, surpassing 24 h proved unnecessary and resulted in saturating the gray tones visualized through the histograms, leading to information loss. Therefore, a time of approximately 24 h was the most suitable staining time for studying adult A. aegypti. It was possible to isolate the organs of the digestive and reproductive systems of the mosquito stained for 24 h. Thus, micro-CT was confirmed to be an excellent technique in studies of individual structures of adult A. aegypti mosquitoes.