Research on the larval age and DNA barcoding of Glyptotendipes tokunagai
Author of the article:LEI Qi** LIU Bang-Miao WEI Xiang-Cheng LIU Ze-Yuan DANG Hao YANG Tie-Zhu GUO Xu-Sheng WANG J
Author's Workplace：College of Fisheries, Xinyang Agriculture and Forestry University, Xinyang 464000, China
Key Words：Glyptotendipes tokunagai; larval instar division; morphometry; DNA barcoding; COⅠgene; genetic distance
Abstract：[Aim] To determine the optimal morphological indicators for identifying the instars of Glyptotendipes tokunagai larvae, and to explore DNA barcoding differences among different geographic populations. [Methods] Larvae were reared in the laboratory, and ten sclerotized morphological characteristics (head capsule length, length from tip of the median teeth of the mentum to the leading coronoid, head mentum width, mentum width, width of the median mentum tooth, antenna length, length of antennal basal segment, mandible length, ventromental plate length and ventromental plate width) were measured. Instar classification was based on the frequency distributions of these variables, the Brooks/Crosby index and regression analysis. The COⅠgene was extracted and sequenced, and MEGA 7.0 was used to analyze sequence composition, genetic distances and to construct a phylogenetic tree. [Results] The results confirm that G. tokunagai larvae have four instars. Among the measured characteristics, mentum width (Y4) had the best fit to the cubic regression model (R2 = 0.997 0). Both the Crosby index and coefficient of variation conformed to Dyar’s law, confirming mentum width as the optimal characteristic for instar identification. Three additional characteristics, namely antenna length (Y6), mandible length (Y8) and ventromental plate length (Y9), can serve as auxiliary indicators. The estimated durations for 1st to 4th instar larvae were approximately 2, 3, 6 and 8 days, respectively. COⅠsequences had a distinct AT bias (A+T content: 63.75%). The phylogenetic tree divided populations into two clades; one comprised of the Chinese population and another comprised of the Japanese and Korean populations. The average genetic distances between the Chinese population and the Japanese and Korean populations were 8.31% and 8.70%, respectively. The genetic distance between the Japanese and Korean populations was only 1.14%. [Conclusion] G. tokunagai larvae have four instars, and mentum width is the most reliable indicator for instar identification. The larval period is estimated to be 19 days. Cryptic species may exist among populations of this species in China, Japan and Korea.