Analysis of Vibrio dynamics in the Neuse River Estuary, NC using next generation sequencing amplicon data Kelsey Jesser, PhD Candidate UNC Chapel Hill, Institute of Marine Sciences May 16, 2017

Vibrio Vibrio ecology Diverse and naturally occurring genus Over 100 species, includes several human pathogens V. parahaemolyticus V. vulnificus Warm water Exist along a salinity gradient Response environmental parameters not well understood, vary across species and ecosystems Freshwater Brackish Water Coastal Water Pelagic Water V. anguillarum V. cholerae V. fluvialis V. navarrensis V. parahaemolyticus V. vulnificus V. logei V. alginolyticus Salinity Oysters aren’t prominent in the NRE, but they coinhabit estuarine ecosystems (shallow, eutrophic, lots of phytoplankton); Both oysters and Vibrio are prominent in estuaries Facultative fermentative- can use oxygen to undergo aerobic respiration, but can switch to fermentative metabolism if oxygen is not present Chemoorganotrophic- utilize chemical energy from breaking bonds in organic (not inorganic) molecules Graph shows seasonality of various clinically-relevant Vibrio in the Black Sea, off the coast of Georgia (Kovashil et al. 2015) Photos: Vibrio fisheri (flagella), Vp gram stain [figure adapted from Urakawa and Rivera 2006]

Vibrio detection Easily culturable Molecular methods (ex: QPCR) Selective and differential, but not effective Molecular methods (ex: QPCR) Thinking about NGS approaches 16S rDNA and hsp60 amplicon sequencing Want species-level resolution Applications for microbial ecology and monitoring Change out one figure for a DNA figure [1, 2]

Study site: the Neuse River Estuary (NRE) Estuary in eastern North Carolina Minimal tidal influence Flow dominated by river inflow and wind forcing Stratified Freshwater on top of saltwater Monitored biweekly by the ModMon monitoring program Shallow, bar-built estuary Minimal tidal influence, flow usually dominated by river inflow and wind forcing Stratified, fresh water on top of saltwater Eutrophic, anthropogenic as well as environmental gradients [3]

Sampling methods Samples collected biweekly May through early September, 2016 in coordination with Modmon Modmon stations 30, 70, and 120 Surface and bottom water Enhanced sampling after Tropical Storm Collin in early June, 2016 Large rainfall event 100 mL filtered onto 0.4 µm polycarbonate filters Genomic DNA extracted with MoBio Powersoil kit Extreme climatic events- tropical storms, hurricanes, drought We’re especially interested in storms as precipitation and wind-driven resuspension events 15 km, 28 km, and 42 km Collin [4]

Vibrio community hsp60 No sample No sample

Vibrio community 16S No sample No sample

Vibrio heatmap De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

The x-axis is highly correlated (Spearman’s rho>0. 5, p<0 The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a. NMDS Month De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

The x-axis is highly correlated (Spearman’s rho>0. 5, p<0 The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a. NMDS Storm sampling De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

The x-axis is highly correlated (Spearman’s rho>0. 5, p<0 The x-axis is highly correlated (Spearman’s rho>0.5, p<0.01) with salinity, DO, DOC, DIC, DON, TDN and chlorophyll-a. NMDS Site and depth De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

Network analysis correlation networks Positive Spearman rank correlations R2 > 0.3 De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

Network analysis correlation networks Negative Spearman rank correlations R2 > 0.3 De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

Conclusions The hsp60 universal target had much higher taxonomic resolution for Vibrio species than 16S The Vibrio communities in the NRE over the summer were very similar to one another Vibrio communities sampled in response to Tropical Storm Collin were very similar to others taken during June Network analyses can identify interactions between important pathogenic species (V. vulnificus and V. parahaemolyticus), total Vibrio, and environmental gradients Potential applications for monitoring/modeling Excellent tool for studying microbial ecology De novo otu picking– reads are clustered without any external reference. Clusters all reads based on sequence similarity

Acknowledgements Rachel Noble Noble lab Committee members Hans Paerl, Jill Stewart, Alecia Septer, and Adrian Marchetti Modmon monitoring project and the Paerl lab UNC ROI Kelsey Jesser PhD Candidate, Marine Sciences UNC Chapel Hill Institute of Marine Sciences kjesser@live.unc.edu 208-308-5653

References Other figures Centers for Disease Control and Prevention (2016). Foodborne Diseases Active Surveillance Network (FoodNet): FoodNet Surveillance Report for 2016 (Draft report). Urakawa, H., I.N.G. Rivera. (2006). Ch. 12. Aquatic environment. In: F.L. Thompson, B. Austin and J. Swings (eds). The Biology of the Vibrios. ASM Press. Washington, D.C. pg. 175-189. Hsieh, J.L., J.F. Fries, R.T. Noble (2008). Dynamics and predictive modelling of Vibrio spp. in the Neuse River Estuary, North Carolina, USA. Environmental Microbiology 10: 57-64. Other figures CHROMagar: http://www.uphs.upenn.edu/bugdrug/antibiotic_manual/gram4.htm; accessed 3/3/2016 3:52 p.m. DNA: http://media.gettyimages.com/photos/unaligned-dna-sequences-viewed-on-lcd-screen-picture- id157649758?s=170667a; accessed 5/15/2017 3:51 p.m. Neuse River color image: http://ims.unc.edu/home/research/; accessed 5/11/16 11:01 a.m. Tropical Storm Collin: https://www.nasa.gov/feature/goddard/2016/colin/; accessed 5/9/17 2:19 p.m.