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

## TL;DR

This study develops two fast molecular tests to detect a destructive pest, Hyblaea puera, in mangroves and teak forests, improving pest control and ecosystem protection.

## Contribution

The paper introduces two novel molecular assays (SS-PCR and LAMP) for rapid and accurate detection of Hyblaea puera.

## Key findings

- The mitochondrial COI gene was identified as the optimal marker for H. puera detection.
- Both SS-PCR and LAMP assays showed high specificity and sensitivity across all developmental stages and populations.
- LAMP is more sensitive (8.3 fg/µL DNA) and suitable for field use, while SS-PCR is better for lab-based large-scale testing.

## Abstract

The frequent outbreaks of Hyblaea puera represent a growing threat to mangrove ecosystems in China. Accurate identification of its immature stages (eggs, larvae, and pupae), however, remains challenging due to the inherent constraints of conventional morphological approaches. To address this, we aimed to develop a rapid and reliable molecular detection technique to facilitate timely and effective pest monitoring and management. After evaluating mitochondrial protein-coding genes, the mitochondrial cytochrome c oxidase I (COI) gene was selected as an optimal molecular marker. Based on this target marker, we established two specific detection assays: species-specific PCR (SS-PCR) and loop-mediated isothermal amplification (LAMP). Both methods exhibited high specificity and successfully distinguished H. puera from sympatric non-target species. These novel molecular tools enable forest managers to allocate pest control resources and equipment in a targeted manner, avoiding the waste of blanket treatments and improving the cost-effectiveness of the prevention and control of H. puera, thereby supporting the protection of economically and ecologically important forests and mangrove ecosystems from this invasive pest.

The teak defoliator, Hyblaea puera, 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 Avicennia marina, severely threatening local mangrove ecosystems. However, accurate morphological identification of H. puera 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 H. puera and related species were analyzed. Sliding window analysis was conducted to estimate nucleotide diversity (Pi), leading to the selection of the cytochrome c oxidase subunit I (COI) gene as the optimal target. Species-specific primers were designed based on the H. puera COI 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 H. puera 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 H. puera. These methods contribute to the early detection and effective management of H. puera populations, thereby safeguarding mangrove ecosystems.