Study site and sample collection

This study was conducted on the up-to-10 years practiced CR model in waterlogged rice paddy fields in Huai’an City, Jiangsu Province, China. The region has a humid, subtropical monsoon climate with a 1500 mm average annual rainfall. A peripheral trench (3.0 × 1.5 m) was excavated and crayfish larvae (weighing 5 ± 2 g) were stocked at a density of 1.5 × 10⁵ larvae·ha⁻¹, and the crayfish self-propagated inside the rice paddies.

Healthy and robust crayfish weighing approximately 15–25 g were collected from this ecosystem. They were acclimatized at ambient temperature (22 ± 1 °C) in air-pumped circulated artificial freshwater before the treatment. Five individuals were randomly captured and the intestine was sampled respectively (mark with S1–S5). The intestine was separated and its contents were collected using sterilized equipment, in which there was no contact with soil or other pollution sources. All samples were stored at − 80 °C immediately before DNA extraction.