Daniel Makrinos*, Amanda Marro, Blake Read, Timothy Bowden Daniel.Makrinos@gmail.com | The University of Maine School of

Food and Agriculture

VARIATIONS IN EXPRESSION OF ZEBRAFISH (DANIO RERIO) TOLL-LIKE RECEPTOR 9 IS

LINKED TO THE CIRDADIAN RHYTHM

Introduction Results

Aim

Materials and Methods

Conclusion

AcknowledgementsI would like to thank Amanda Marro and Blake Read for their persistence in

processing samples, Timothy Bowden for his constant support throughout this project, the University of Maine, and Fish Vet Group for funding the research.

• Determine if TLR9 gene expression in zebrafish and salmon oscillates on a 24 hour circadian rhythm?

• Zebrafish (N=81, 40 days post hatch, ~60 mg) were acclimated for 14 days to a 12:12 hr light cycle at 22ºC

• Atlantic salmon parr (N=81, ~6.0 g) were acclimated for 21 days to a 12:12 hr light cycle tank at 10.0ºC

• Zebrafish (N=27) and salmon (N=27) were sampled every 4 hours over a 32 hour period

• RNA was extracted using TRIzol, 1 μg of mRNA was used in RT-PCR to yield cDNA, and qPCR quantified the expression of zebrafish TLR9 relative to normalized expression of the reference genes β-actin and GAPDH

• Salmon gene expression was normalized using EF-1 and 18S reference genes

• Circadian rhythms are biological mechanisms that physiologically control gene expression over the course of 24 hours, which are induced by zeitgebers

• The time of day is linked to fluctuations in mouse innate immunity involving activation of antigen presenting cells through toll-like receptors (TLRs)

• TLRs are pattern recognition receptors (PRRs) which recognize pathogen associated molecular patterns (PAMPs) such as LPS and peptidoglycan

• TLR9 specifically functions to recognize unmethylated CpG sequences associated with bacterial and viral DNA sequences

• Silver et al. (2012).observed gene expression of TLR9, TLR7, and TLR4 in mice over 24 hrs and reported significant differences in TLR9 expression on a circadian rhythm

• Expression of TLR9 is not consistent throughout the 24 hours• Zebrafish and salmon TLR9 expression do not appear to follow the

same circadian rhythm• An up regulation of TLR9 in zebrafish and salmon occurs at 8 AM

and 4 PM

• The decrease in TLR9 expression between 8 AM and 4 PM is not seen at 24 hrs and beyond

• TLRs functioning on circadian rhythms could provide insight in teleost innate immunity

• Clock genes such per1b will be analyzed to confirm presence of 24 hour circadian rhythms

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Overall Zebrafish TLR9 Gene Expression

Time of Sampling

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8 AM 12 PM 4 PM 8 PM 12 AM 4 AM 8 AM 12 PM 4 PM

Literature Cited

Figure 1. (A) Gene expression of TLR9 over 32 hours in zebrafish (N=81). (B) Gene expression of TLR9 over 32 hours in Atlantic salmon (N=27). The standard error of measurement demonstrates the variability in gene expression of triplicate samples normalized to GAPDH and β-actin for zebrafish and elongation factor (EF-1) and 18S for salmon.

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Overall Salmon TLR9 Gene Expression

Time of Sampling

Mea

n Cq

Val

ue

8 AM 12 PM 4 PM 8 PM 12 AM 4 AM 8 AM 12 PM 4 PM

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B

Silver, A.C., Arjona, A., Walker, W.E. & Fikrig, E (2012) The Circadian Clock Controls Toll-like Receptor 9-Mediated Innate and Adaptive Immunity. Immunity, 36, 251-261.

Skjaeveland, I., Iliev, D., B., Zou, J., Jorgensen, T. & Jorgensen, J., B. (2008) A TLR9 homolog that is upregulated by IFN-y in Atlantic salmon (Salmo salar). Developmental and Comparative Immunology, 32, 603-607.