Effect of Gut Microbiota on the Enteric Nervous System of the Sea Cucumber

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1 Effect of Gut Microbiota on the Enteric Nervous System of the Sea Cucumber Sonya J. Malavez Cajigas, Julio M. Cuevas Cruz, Paola I. Rodríguez Sánchez, Lymarie Díaz- Díaz, Omar Delannoy- Bruno, José E. García Arrarás1 University of Puerto Rico, Río Piedras Campus Department of Biology

2 Microbiota Symbiosis (mutualis?c) All organisms have mutualis?c rela?ons. Mutualis(c

3 Gut Microbiota Crucial in essen?al physiological processes involved in homeostasis for different organisms. Also related to immune system and health in general. Responds to changes or altera?ons.

4 Seung Chul Shin et al. Scheperjans, Filip, et al.

5 Holothuria glaberrima

6

7 Holothuria glaberrima

8 Intestinal Regeneration First week Second week Third week

9 Nervous System Regeneration

10 Nervous System Regeneration

11 Objective Determine the effect gut microbiota on the regenera?on of the intes?nal tract of the sea cucumber Holothuria glaberrima. An?bio?c usage Measuring blastema size and regenerated intes?ne length Immunohistochemical analysis: RN1

12 Methodology 1 Collection and preparation 2 KCl.35 M injection 3 Antibiotic treatment

13 Methodology Antibiotic Treatment Control Group (Ar(ficial Seawater) Penicillin/ Streptomycin (100μg/mL) and Kanamycin (100μg/mL) Penicillin/ Streptomycin (100μg/mL) and Erythromycin (20μg/mL) Penicillin/ Streptomycin (100μg/mL) and Neomycin (100μg/mL) Penicillin/ Streptomycin (100μg/mL) 1L Water 1L Water 10mL Pen/Strep 1mL Kanamycin 1L Water 10mL Pen/Strep 1mL Erythromycin 1L Water 10mL Pen/Strep 10mL Neomycin 1L Water 10mL Pen/ Strep 4 animals per group Total number of animals: 20

14 Methodology An(bio(c treatment: 10 days post eviscera(on

15 Methodology 6 Immuno histochemistry/ Tissue measurement 5 Tissue sec?oning 4 SacriTice 10 dpe

16 Methodology 7 Fluorecence 8 microscopy Data analysis

17 Measurement of Intestine Length

18 Results: Measurement of Intestine Length 25.0 Effect of An(bio(cs on Lumen Forma(on Mean of Regenerated Lumen (%) Small intes?ne Large intes?ne Ar?ficial Water Kanamycin Erythromycin Treatment

19 Measurement of Blastema Size

20 Control vs. Kanamycin Control Kanamycin

21 Control vs. Erythromycin Control Erythromycin

22 Results: Measurement of Blastema Size Effects of An(bio(cs on the Area of Blastema Control Kanamycin Erythromycin

23 Comparison with Previous Experiments 140 Effect of the An(bio(cs in Rudiment Size (%) Percent Control Group Kanamicina

24 Comparison with Previous Experiments 140 Effect of the An(bio(cs in Rudiment Size (%) Percent Control Group Erythromycin

25 Observations As observed Kanamycin has no effect on lumen forma?on; however, there was an apparent effect on animals treated with Erythromycin. As seen in previous results, an?bio?cs have a nega?ve effect on rudiment size. Animals treated with Kanamycin showed a significant reduc?on in the size of the blastema. Recently, we have observed a smaller rudiment in animals treated with Kanamycin or Erythromycin.

26 Immunohistochemical analysis

27 Control

28 Immunohistochemistry Control Kanamycin

29 Immunohistochemistry Control Erythromycin

30 Observations of RN1 Marker on Previous Experiments Less fibers between 5 and 7 dpe and by 10 dpe no nerve fibers or few in rudiment. By 14 dpe re- innerva?on in the rudiment started.

31 Observations Animals treated with Kanamycin and Erythromycin showed more nervous fibers in the blastema. Animals in the control group has more neuron- like cells.

32 Conclusions Our results suggest that the intes?nal microbiota could be involved in the regenera?ve process. It appears that an?bio?cs delay both processes: enteric nervous system and?ssue regenera?on.

33 Ques(ons