An effective method for accurate nymphal-stage delimitation of the cicada Hyalessa fuscata

Abstract

• A robust method is necessary for better demarcation among cicadas’ developmental stages. • Ln-transformation and PCA were applied to obtain an orthogonal linear transformed variable. • Best-suited model was determined by k-means clustering and simple linear regression analysis. • Five developmental stages was the best fit model for Hyalessa fuscata nymphs. • The developed framework can serve as an effective protocol for studies of cicadas’ life history. Subterranean nymphal development in cicadas presents challenges to researchers in accurately estimating the number of their developmental stages, although such information is crucial to understanding and predicting their population dynamics. While most studies have relied on head width as an attribute for life-stage determination to date, such character in cicadas can be highly variable and thus differentiation solely based on such morphology is prone to subjectivity in practice. Here, we propose a reliable method for instar estimation that is applicable to Hyalessa fuscata nymphs. We first obtained morphometrics of nymphs in all stages. Second, we computed logarithm-transformation and principal component analysis to extract a transformed variable that captures most of the variance of morphological characteristics. Third, k-means were computed to divide the dataset into distinct clusters assuming four-, five- and six life-stage scenarios for the best interferences of life stages. Finally, simple linear regression analysis was conducted to compare and select the best fit model. Our result shows that five nymphal stages best fit for H. fuscata nymphs. This method is expected to provide an easy-to-handle ecological tool for the study of life history of cicadas as well as other insects that have long life cycles and multiple developmental stages.