The effect and mechanism of variable particle size microplastics and levofloxacin on the neurotoxicity of Rana nigromaculata based on the microorganism-intestine-brain axis.

Microplastics (MPs) usually appear in the aquatic environment as complex pollutants in combination with other environmental pollutants, such as levofloxacin (LVFX). After a 45-day exposure to LVFX and MPs with different particle sizes at environmental levels, LVFX was neurotoxic to Rana nigromaculata tadpoles. The order of the effects of the exposure treatment on tadpole behavior was: LVFX-MP3>LVFX-MP1>LVFX-MP2 ≥ LVFX. Results of transcriptome analysis of tadpole brain tissue showed that LVFX in combination with 0.10 and 10.00 µm MP interferes with the nervous system through the cell adhesion molecules pathway. Interestingly, the order of effects of the co-exposure on oxidative stress in the intestine was inconsistent with that of tadpole behavior. We found that Paraacteroides might be a microplastic indicator species for the gut microbiota of aquatic organisms. The results of the targeted metabolism of neurotransmitters in the intestine suggest that in the LVFX-MP2 treatment, LVFX alleviated the intestinal microbiota disorder caused by 1.00 µm MP, by regulating intestinal microbiota participating in the TCA cycle VI and gluconeogenesis and tetrapyrrole biosynthesis I, while downregulating Met and Orn, and upregulating 5HIAA, thereby easing the neurotoxicity to tadpoles exposed to LVFX-MP2. This work is of great significance for the comprehensive assessment of the aquatic ecological risks of microplastics-antibiotic compound pollutants.

Expression and functional analysis of fam76b in zebrafish.

FAM76B is nuclear speckle-localized protein with a molecular weight of 39 kDa. The amino sequence of FAM76B protein is highly conserved among species, suggesting that FAM76B has important biological functions. However, the biological function of FAM76B is currently still unclear. To explore the biological function of FAM76B, we firstly used zebrafish as the experimental model to study the distribution and expression level of Fam76b. The results indicated that fam76b is highly expressed in hematopoiesis and immune systems of zebrafish by real-time quantitative PCR, in situ hybridization and Tg(fam76b: eGFP) transgenic zebrafish. Then, the fam76b gene was knocked out by CRISPR/Cas9 in zebrafish and fam76b rescue in fam76b-/- zebrafish was performed using the TOL2 transposable system. fam76b gene knockout zebrafish exhibit reduced thymus, excessive inflammatory response, and increased mortality. FAM76B was further found to be involved in regulating the development of hematopoiesis and immune system, and participate in the process of inflammatory response. Our findings in the study lay the groundwork for elucidating the function of the new molecule Fam76b and provide new insights into the development of zebrafish hematopoietic and immune system.