Objective The purpose of this study was to investigate the species and abundance of natural food for Chinese soft-shelled turtles (Pelodiscus sinensis) cultured in paddy fields under different feeding levels, explore the feeding ecology of P. sinensis in the rice-turtle co-culture mode, and provide a basis for optimizing the feeding strategy of comprehensive planting and breeding in rice fields and revealing the formation process and mechanism of ecological benefits.

Method The field feeding experiment was conducted with three feeding levels of 0.7%, 1.4% and 2.1%(w) for studying rice-turtle co-culture. Morphological analysis of stomach contents and environmental DNA analysis were conducted to determine the feeding habit of the natural food and commercial feed for P. sinensis in different treatment groups. The relative importance index of different food in treatments was also analyzed.

Result The feeding rate of P. sinensis increased with the increasing of feeding level. In morphological analysis of stomach contents, 8 species of fish, 2 species of shrimp, 2 species of molluscs, 9 species of insects and rotifers were identified. The species with high occurrence rate included Abbottina rivularis, Pseudorasbora parva, Macrobrachium nipponense, Cipangopaludina chinensis, Polyarthra sp., Chironomidae larvae and Limnodrilus sp.. The occurrence rate and relative importance index proportion (IRIP) of commercial feed significantly increased with the increase of feeding level, while the IRIP of natural food decreased. The IRIP of commercial feed was 91.97% when the feeding level was 2.1%, and the added IRIP of multiple natural food was only 8.03%. In environmental DNA analysis, there were 16 species of fish with DNA relative abundance higher than 0.5%, and among them, Abbottina rivularis, Pseudorasbora parva, Rhodeus sinensis, Hemiculter tchangi, Misgurnus anguillicaudatus, Paramisgurnus dabryanus, Carassius auratus and Hemisalanx brachyrostralis were also identified in morphology analysis of stomach contents. A total of 36 species of aquatic vertebrates were identified, and the species with DNA relative abundance higher than 1% in stomach content samples mainly included Pseudochaeta sp. Janzen14, Palaemom modestus, Cipangopaludina chinensis, Chironomidaelarvae, Limnodrilus sp., rotifer, etc.

Conclusion The natural food of P. sinensis included small fish, molluscs, arthropods and rotifers, etc. Higher feeding level of commercial feed results in lowerfeeding rate and IRIPof natural food, whichwould reduce the ecological benefits of co-culture.