环境浓度多西环素对斑马鱼焦虑行为、认知记忆能力的影响与肠道菌群变化的关联

ENVIRONMENTAL DOXYCYCLINE ON ANXIOUS BEHAVIOR, COGNITIVEMEMORY ABILITY AND INTESTINAL MICROFLORA OF ZEBRAFISH

为探讨环境浓度多西环素长期暴露对斑马鱼焦虑行为、学习记忆、认知灵活性及肠道菌群的影响,采用不同环境浓度多西环素(0、0.01、0.1和1μg/L)对斑马鱼成鱼进行水体暴露染毒21d后,采用新缸实验、Y迷宫、明暗实验和斑马鱼行为学高通量监测系统方法检测环境浓度多西环素对斑马鱼行为学和肠道菌群的影响。结果表明:在新缸实验中,与对照组相比, 0.01、0.1和1μg/L浓度组斑马鱼平均游泳速度显著降低;随着多西环素浓度升高,斑马鱼最大游泳速度和总游动距离与对照组相比呈现下降趋势,其中1μg/L浓度组同对照组相比差异显著;在底部区域停留时间上,与对照组相比, 0.01、0.1和1μg/L组均有升高, 0.01μg/L差异显著。在Y迷宫实验中, 0.01μg/L浓度组斑马鱼在探索臂停留时间与对照组相比显著增加, 0.1和1μg/L浓度组显著降低;0.01μg/L浓度组斑马鱼在探索臂的转弯角度与对照组相比增加,而0.1和1μg/L浓度组显著降低。在明暗实验中,斑马鱼在光区停留时间随着多西环素浓度增加具有上升的趋势。在斑马鱼行为学高通量监测系统实验中,斑马鱼总游动距离随着多西环素浓度的增加呈现下降趋势,其中1μg/L浓度组同对照组相比差异显著。基于16S rRNA测序技术对肠道菌群进行分析, 12份肠道样本共获得1454814条原始有效序列,筛选后获得1314404条优质序列供后续分析。同时利用DADA2方法降噪,以100%相似度聚类,发现多西环素各浓度组减少了斑马鱼肠道菌群的丰度,博斯氏菌属、副球菌属、不动杆菌等菌属丰度降低,棒状杆菌属和漫游球菌等菌属丰度升高。香农指数、辛普森指数和chao1指数染毒组同对照组相比显著降低,此外,主坐标分析中多西环素组和对照组之间的群落不同。综上,环境浓度多西环素长时间暴露能够引起斑马鱼行为改变,产生焦虑样行为, 0.01μg/L浓度组能够提高斑马鱼短期学习记忆能力和认知灵活性;多西环素长期暴露引起斑马鱼肠道菌群丰度和多样性改变。

The use of antibiotic can influence gut microbiota and impair memory function either positively or negatively.In order to investigate the effects of long-term exposure to doxycycline at environmental concentration on anxiety behavior,learning and memory,cognitive flexibility and intestinal flora,we took zebrafish as the animal model,and used doxycycline at different environmental concentrations(0,0.01,0.1 and 1μg/L)to expose adult zebrafish to water for 21 days.Behavioral responses and intestinal flora alterations were assessed using the new tank test,Y-maze,light and dark test and zebrafish behavior high-throughput monitoring system.The results showed as follows:in the new tank experiment,the average swimming speed of zebrafish in 0.01,0.1 and 1μg/L concentration groups decreased significantly compared with the control group.Additionally,higher doxycycline concentrations correlated with reduced maximum swimming speed and total swimming distance,with a pronounced difference observed in the 1μg/L group.Maze experiments indicated a significant increase in the residence time of zebrafish in the exploration arm at the 0.01μg/L concentration,contrasting with decreased times in the 0.1 and 1μg/L groups.The turning angle exhibited a similar pattern,increasing in the 0.01μg/L group but decreasing in the higher concentration groups.In the light and dark test,zebrafish demonstrated an increased residence time in the light zone with rising doxycycline concentration.Zebrafish behavioral high-throughput monitoring system experiments further confirmed a significant decrease in total swimming distance with increasing doxycycline concentration,particularly notable in the 1μg/L group.In terms of intestinal flora analysis,based on 16S rRNA sequencing technology,1454814 original valid sequences were obtained from 12 intestinal samples,and 1314404 high-quality sequences were obtained after screening for subsequent analysis.At the same time,DADA2 method was used to reduce noise and cluster with 100%similarity.It was found that the abundance of intestinal flora of zebrafish in each concentration group decreased,and the abundance of bacteria genera such as Bosteria,Paracococcus and Acinetobacter decreased,while genera like Corynebacterium and Romatococcus increased.The Shannon index,Simpson index and Chao1 index were significantly lower in the exposed group,indicating decreased diversity.In conclusion,long-term exposure to doxycycline in environmental concentration induced behavioral changes in zebrafish,resulting in anxiety-like behavior.The 0.01μg/L concentration improved short-term learning and memory ability and cognitive flexibility.Furthermore,doxycycline exposure led to changes in the abundance and diversity of zebrafish intestinal flora.