Development and Application of Two Rapid Molecular Detection Assays for Hyblaea puera Cramer (Lepidoptera: Hyblaeoidea), a Major Pest of Mangroves and Teak

The teak defoliator, <i>Hyblaea puera</i>, native to South Asia and Southeast Asia (e.g., India, Laos, and Myanmar), has recently caused frequent outbreaks in mangrove forests across Guangdong, Guangxi, and other regions of China. Its larvae feed extensively on the leaves of <i>Avicennia marina</i>, severely threatening local mangrove ecosystems. However, accurate morphological identification of <i>H. puera</i> across its eggs, larvae, and pupae remains challenging. Therefore, the development of rapid molecular detection methods is essential for effective pest identification and monitoring, thereby supporting timely management interventions. In this study, mitochondrial protein-coding genes (PCGs) were analyzed from <i>H. puera</i> and related species were analyzed. Sliding window analysis was conducted to estimate nucleotide diversity (<i>Pi</i>), leading to the selection of the cytochrome c oxidase subunit I (<i>COI</i>) gene as the optimal target. Species-specific primers were designed based on the <i>H. puera COI</i> sequence, and two molecular detection assays-SS-PCR and LAMP-were developed. Both assays exhibited high specificity, stability, and sensitivity, successfully amplifying target fragments from <i>H. puera</i> across all tested geographic populations and different developmental stages. The limit of detection of the SS-PCR method was 83 fg/µL DNA, while that of the LAMP method reached 8.3 fg/µL DNA. The newly developed assays offer reliable and robust tools: the SS-PCR method is suitable for precise, large-scale detection in laboratory settings, whereas the LAMP assay is preferable for rapid, field-based detection of <i>H. puera</i>. These methods contribute to the early detection and effective management of <i>H. puera</i> populations, thereby safeguarding mangrove ecosystems.