4. Research report (Follow the guideline on the next page)

Title: Effects of azithromycin on river periphyton: Structure, meta-transcriptional
process and ecological functions
Jiayi Liang1, Hisato Iwata2, Yulu Tian1, Jiahua Guo1, *1 Shaanxi Key Laboratory of Earth Surface System and Environmental Carrying Capacity, Collegeof Urban and Environmental Sciences, Northwest University, Xi’an, 710127, China
2Center for Marine Environmental Studies, Ehime University, Bunkyo-cho 2-5, Matsuyama,

Ehime prefecture, 790-8577, Japan

1. Purposes
The aims of this study are: 1) to evaluate the inhibition effects of azithromycin on thephotosynthesis and carbon metabolism processes in periphyton; 2) to investigate the effectsof azithromycin on the periphytic composition using 16S & 18S metabarcoding; 3) toelucidate the molecular mechanism underlying the structural and functional changes using
meta-transcriptomic analysis.
2. Methods
2.1 Periphyton culturing and testing
Pebbles that were gathered from pristine natural rivers (the Qinling reach of Taiyi River in
Xi'an), China (109.0192◦ N, 34.0517◦E)) were selected as culture substrates. These pebbles wereplaced in a 2L beaker, immersed with BG11 medium and cultured in a light incubator at thefollowing conditions for 21 days: 2800 lux light intensity, a 14 h/10 h light/dark circle, and acontrolled temperature of 22 ± 2 °C. The culture medium was freshly prepared at an interval of
5 days. After 21 days of incubation, a sterile toothbrush was used to harvest the periphyton fromthe stones.
A total of twenty-one 250-mL Erlenmeyer flasks, which contain 0.1 grams (g) wet weight (ww)of periphyton and 150 mL of BG11 medium, respectively, were initially prepared and placed in theculture chamber. After 7 days of inoculation, the medium was replaced by a range of AZI testsolutions, comprising 150 mL of 5 μg/L, 50 μg/L, 200 μg/L, 500 μg/L, 5,000 μg/L, and a control
group (0 μg/L), which were all prepared in BG11 medium. A 50 μg/L treatment group without
periphyton was prepared for the evaluation of potential abiotic degradation. All groups were intriplicates and the incubation lasted for 14 days, as the periphyton start decaying after 16 days inour earlier growth curve measurement. After that, 50 mL of periphyton samples from each groupwere subjected to filtration via a 0.22 μm membrane (Jintang Company, Tianjin, China), flash
freeze with liquid nitrogen and kept in a -80 °C refrigerator before DNA metabarcoding andmetatranscriptomic analysis.

2.2 Periphyton structure analysis
The taxonomic composition of the periphytic microbial community in response to AZI

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exposure was revealed by DNA metabarcoding technology. Briefly, the DNA extraction wasconducted for the filtered periphyton on day 14, and 16s & 18s rRNA sequencing were analyzed
using the Illumina MiSeq platform of Personalbio Technology Co, Ltd (Nanjing, China).
2.3 ChlorophyII a fluorescence and carbon metabolism
The chlorophyll a fluorescence parameter, reflecting the activity and energy conversion ofphotosystem II (PSII) in autotrophic organisms of periphyton, was measured on day 14 using theAquaPen AP110-P (Photon Systems Instrument, Czech Republic). To evaluate the carbon
utilization capacity for the heterotrophic organisms in periphyton, a Biolog EcoPlate™ (Biolog Inc.,Hayward, CA, USA) test that contains 31 different sources of carbon, each with three replicates,
was performed following the manufacturer’s protocol.

2.4 The meta-transcriptomic analysis
The total RNA was extracted and the purity and concentration were quantified by NanoDrop
NC 2000 (Thermo Scientific, Wilmington, Delaware, US), followed by next-generation sequencing(NGS) using the Illumina HiSeq X Ten (Nanjing Personal Gene Technology Co, LTD, China).
Detailed information for metatranscriptomic sequencing could be found in the published paper.
The "DESeq" package was used to screen the differentially expressed genes (DEGs) in thetreatment group based on the following criteria: adj p value < 0.05 and |log2 Fold Change (FC)|> 1. The enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway wasthen analyzed to reveal the primary biological functions. The Pathview package of R software wasused to visualize the metabolic pathways.

3. Results
3.1 Effects of AZI on periphytic photosynthesis
Exposure to AZI significantly inhibited PSII reaction centers involved in plant photosynthesis,
especially the overall maximum photochemical quantum yield (Fv/Fm) of PS II that was decreased
in a concentration dependent manner (Fig. 1). In particular, exposure to AZI at 5,000 μg/L resulted
in a 63.64% decrease of Fv/Fm compared to the control group. In the 50 to 5,000 μg/L of AZI
treatment, the ABS/RC, ETo/RC, TRo/RC, and DIo/RC levels involved in energy flux of PS II werepromoted by AZI exposure compared with the control group, with the increased values of 63.0086.16%, 16.09-29.35%, 19.55-36.55%, and 79.83-94.56%, respectively.
a4 / 6bFig.1 Effect of azithromycin on photosynthesis of periphyton. (a) OJIP parameter results of AZIexposure; (b) The photosynthetic process at the molecular and physiological levels.
3.2 Effect of AZI on periphyton carbon source utilization
In the present study, the PCA analysis indicated that the periphytic capacity to utilize different
carbon sources in medium (50 μg/L) and high (5,000 μg/L) treatments departed from controls (Fig.
2). Particularly, the metabolism of carbohydrates and phenolic acids were increased 46.70% and39.54% in 5,000 μg/L AZI treatment, respectively, where no evident alteration was detected forother four carbon sources (Fig. 2). A positive correlation between the periphytic bacterial
community and five carbon sources, in addition to polymers, was further detected (Fig. 2).
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Fig. 2 Carbon source utilization capacity and correlation between periphytic composition andfunction analyses. (a) the utilization of six carbon sources in control and treatment groups; (b)PCA plots of AWCD; (c) Correlation between composition and Chl a fluorescence parameters; (d)Correlation between composition and carbon sources (*p < 0.05; **p < 0.01).
A total of 14 compounds and 46 pathways related to the carbon source utilization weresignificantly enriched, where energy and carbohydrate metabolism were the dominant pathways.
These processes were in relation to the energy and carbon source supply for microbial activities,
which were pivotal for the growth and metabolism of microorganisms. In the case of energy
metabolism, the increase in the utilization of phenolic acids at AZI 5,000 μg/L group was mainly
caused by the increased 4-Hydroxybenzoic acid, which was governed by the related metabolic
pathways, including ubiquinone and other terpenoid-quinone biosynthesis (ko00030), benzoate
degradation (ko00362), aminobenzoate degradation (ko00627) and folate biosynthesis(ko00790), in which all the DEGs involved were up-regulated. In the matter of carbohydrates, theelevated utilization of D-Galactonic acid -lactone, D-Cellobiose, i-erythritol, alpha-D-Glucose-1phosphate, D-xylose, and N-Acetyl-D-glucosamine contributed to the enhanced carbohydrate
consumption in 500 μg/L and 5,000 μg/L AZI treatment groups. The metabolism of thesecarbohydrates was primarily concerning the starch and sucrose metabolism (ko00500), ABC
transporters (ko02010), galactose metabolism (ko00052), glycolysis/gluconeogenesis (ko00010),
amino sugar and nucleotide sugar metabolism (ko00520), phosphotransferase system (ko02060),
etc. where most of the DEGs were up-regulated.
Due to the copyright of publisher, other detailed results of the altered periphytic composition,
KEGG secondary metabolic pathway composition, and the proposed transformation pathway ofAZI can be found in the published paper (Liang et al., 2024, Water Res. 251: 121140, 2024)

4. Future challenges
Our next plan is to investigate the plasmid mediated conjugation transfer of ARGs inperiphyton under azithromycin pressure.

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