Response of moulting genes and gut microbiome to nano-plastics and copper in juvenile horseshoe crab Tachypleus tridentatus

Nanoplastics (NPs) and heavy metals are typical marine pollutants, affecting the gut microbiota composition and molting rate of marine organisms. Currently, there is a lack of research on the toxicological effects of combined exposure to horseshoe crabs. In this study, we investigated the effects of NPs and copper on the expression of molt-related genes and gut microbiome in juvenile tri-spine horseshoe crabs Tachypleus tridentatus by exposing them to NPs (100 nm, 10⁴ particles L⁻¹) and/or Cu²⁺ (10 μgL⁻¹) in seawater for 21 days. Compared with the control group, the relative mRNA expression of ecdysone receptor (EcR), retinoid x receptor (RXR), calmodulin-A-like isoform X1 (CaM X1), and heat shock 70 kDa protein (Hsp70) were significantly increased under the combined stress of NPs and Cu²⁺. There were no significant differences in the diversity and abundance indices of the gut microbial population of horseshoe crabs between the NPs and/or Cu²⁺ groups and the control group. According to linear discriminant analysis, Oleobacillus was the most abundant microorganism in the NPs and Cu²⁺ stress groups. These results indicate that exposure to either NPs stress alone or combined NPs and Cu²⁺ stress can promote the expression levels of juvenile molting genes. NPs exposure has a greater impact on the gut microbial community structure of juvenile horseshoe crabs compared to Cu²⁺ exposure. This study is helpful for predicting the growth and development of horseshoe crabs under complex environmental pollution.