Díaz-Díaz LM , Rosario-Meléndez N , Rodríguez-Villafañe A , Figueroa-Vega YY , Pérez-Villafañe OA , Colón-Cruz AM , Rodríguez-Sánchez PI , Cuevas-Cruz JM , Malavez-Cajigas SJ , Maldonado-Chaar SM , García-Arrarás JE .

20270.001.000.XXXX.220.206350070017.00 Puerto Rico Science, Technology and Research Trust, 5R25GM061151-19 Research Training Initiative for Student Enhancement, HDR-1906130 Puerto Rico Louis Stokes Alliance For Minority Participation - Bridge to the Doctorate Program, 5R25NS080687 Neuroscience Research Opportunities to Increase Diversity (NeuroID)/NIH

	Figure 1. Rudiment growth after antibiotics exposure for 10-dpe. Sections of regenerating intestines from animals subjected to various antibiotic treatments including untreated controls (SW), and penicillin/streptomycin (PS) alone as well as kanamycin (KPS), vancomycin (VPS), and erythromycin (E4PS and E20PS) based cocktails-treated animals (A). Scale bar represents 100 μm. Percentage of the rudiments’ area in comparison with the PS group (B). Blastema size was measured using the program ImageJ. Bars show the mean of at least six (6) animals, ±SD graphed using GraphPad PRISM. Asterisk shows the result of t-test comparisons between SW and E20PS (* p < 0.05).
	Figure 2. Muscle dedifferentiation in antibiotic treated regenerating guts. Muscle dedifferentiation was detected by the presence of SLSs and muscle fibers, labeled with Phalloidin-TRITC (green), and nuclei, labeled with DAPI (magenta) in regenerating guts of animals treated with PS, KPS, VPS, E4PS, and E20PS (A). Scale bar represents 100 μm. Dedifferentiation grade was determined using the algorithm shown Scheme S1B (B). Bars represents the mean of at least five (5) animals, ±SD. Asterisks show t-test comparisons between SW and experimental groups * p < 0.05, ** p < 0.01. Results were graphed using GraphPad PRISM.
	Figure 3. ECM remodeling after antibiotic treatments. Collagen presence was determined using the antibody E6D9G3. Non-treated animals (SW), animals treated with PS and KPS, VPS, E4PS, and E20PS were labeled with anti-collagen (yellow) and DAPI staining (magenta) (A). Scale bar represents 100 μm. Remodeling grade was determined using the algorithm shown in Scheme S1A (B). Bars represent the mean of at least six (6) animals, ±SD. Asterisks show t-test comparisons between SW and experimental groups ** p < 0.01. Results were graphed using GraphPad PRISM.
	Figure 4. Antibiotic treatment effects on cellular proliferation. BrdU+ cells and DAPI labeled nuclei in the rudiment. Non-treated animals (SW), animals treated with PS and KPS, E20PS, E4PS, and VPS were labeled with anti-BrdU (green) and DAPI staining (red) (A). The light blue arrowheads point the coelomic epithelium and the dashed line delimits the rudiment area where cells were counted. Scale bar represents 100 μm. Cell division rate was calculated in coelomic epithelium and connective tissue by dividing the number of proliferative cells (BrdU+ cells) per total cells (DAPI+ nuclei) (B). Bars represent mean values of at least three (3) animals and SEM are displayed in the error bars. Asterisk shows t-test comparisons between the connective tissue of SW and experimental groups * p < 0.05. Results were graphed using GraphPad PRISM.
	Figure 5. Ex vivo effect of antibiotics on the enzymatic activity of holothurians muscle explants. Results from ex vivo toxicity assay on sea cucumbers’ longitudinal muscle explants after incubation with antibiotics treatments for 72 h in culture, and 1 h in MTT (A). Dose effects after 72 h in culture with PS (B). The dose effects of kanamycin (K), vancomycin (V), or erythromycin (E) alone on holothurians muscle explants (C,E,G). On the right, are represented the dose effect explants in PS (100 μg/mL)-based cocktails, additionally supplemented with K, V, and E, respectively (D,F,H). Results are shown as the rate of metabolic activity normalized to the tissue density (MTT OD/interpolated relative protein concentration) in comparison to non-treated explants. All tissues were plated in triplicates (3 wells for each condition) per plate. The values are the average of at least eight (8) plate culture replicates, including the SEM. Blue arrows show doses tested in vivo. Asterisks show t-test comparisons between non-treated explants and experimental groups * p < 0.05, ** p < 0.01. a All PS-based treatment samples also contain PS for a FC = 100 mg/mL.
	Figure 6. In vitro bacterial growth in antibiotic-selective media for minimum inhibitory concentration (MIC) determination. Plates with selective media incubated with holothurian gut detritus bacteria (A–C). A negative sign “-’’ was ascribed for no growth (A), “+” if at least one colony was seen (B), “++” if a full confluence lawn was seen (C). MIC was determined from plates with no colony formation (no bacteria growth), results from at least 4 replicates per dose were graphed (D).
	Figure 7. Summary of the effects of antibiotics. This scheme gathers the findings in this article on the effect of 100 µg/mL penicillin/streptomycin (PS) and PS-based cocktails including: 100 µg/mL kanamycin (KPS), 5 µg/mL vancomycin (VPS), and erythromycin 4 µg/mL (E4PS) and 20 µg/mL (E20PS) in the intestinal regeneration of sea cucumbers. Drawings were made accordingly the average results of each group to present the proliferating cells (blue dots), SLS (green ovals), muscle fibers (green lines) and collagen (yellow) localization in the regenerating rudiments and adjacent mesentery of 10-dpe animals treated with antibiotics. Briefly, administration of PS and KPS for 10-dpe only have an effect on cellular dedifferentiation while VPS, E4PS, and E20PS alter both cell dedifferentiation and ECM remodeling. Exposure to VPS also altered the cellular proliferation rate in the connective tissue of the regenerating gut, and higher doses of erythromycin (E20PS) perturbed rudiments’ growth.

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