Introduction

1. Influence of Cold Temperature and Anoxia on Red-eared Slider Turtle Gut MicrobiotaCatheryn Wilson1,2, Jonathan Stecyk1, and Khrys Duddleston1 1Department of Biological Sciences, University of Alaska Anchorage; 2University of North Carolina at Pembroke Introduction Methods and Materials Intestinal microbiota share symbiotic relationships with a host. The host benefits include the processing of ingested nutrients [1],contributions to development of gut immune system [2], and assistance with maturation [3]. The microbe benefits via access to nutrients and to an anaerobic environment. However, when dysbiosis (disruption of symbiosis) occurs, it negatively impacts the host. Dysbiosis has been implicated in diseases like obesity and inflammatory bowel disease. Numerous host-gut microbe relationship studies use mammals but little work has been done utilizing reptiles. Using ectothermic vertebrates offer potential new insights into, and provides a different perspective on, host-gut microbe interactions. Due to physiological tolerance adaptations of cold temperatures and anoxic conditions, red-eared slider turtles are an intriguing reptilian model we specifically used for our research [4],[5]. Purpose: To observe the differences in gut microbial diversity in various treatment (exposure) groups • Experimental design: 23 turtles acclimatized to normoxia and anoxia at different temperatures • Cecum mucosal extraction: dissect turtle cecum, collect cecum mucosal material • Extraction of DNA: via MoBIOPowerMicrobiome RNA Isolation kit, omitting the DNase step • Concentration of DNA: using the NanoDrop 2000 Spectrophotometer • Roche 454 Pyrosequencing: of microbial 16S rRNA genes • (Research and Testing Laboratories, Inc., Lubbock,TX) Results Figure 1. Reduced alpha diversity in 5⁰C anoxia Fig.1.Turtles at 21⁰C normoxia exhibited the greatest phylogenetic diversity in their gut microbiotas. The turtles held for 14 days at 5⁰C under the anoxic condition had the lowest alpha diversity. Phylogenetic Diversity Species Richness Number of OTUs Sequences Per Sample Sequences Per Sample Sequences Per Sample Figure 2. Shift in beta diversity of exposure groups in response to treatments Figure 3. Microbial diversity and composition shift in 5⁰C anoxic exposure at the phyla (A) & family (B) levels p. Proteobacteria (1): f. Enterobacteriaceae p. Proteobacteria (1); f. Aeromonadaceae p. Fusobacteria (2); f. Fusobacteriaceae p. Firmicutes (3); f. Erysipelotrichaceae, Ruminococcaceae, etc. p. Bacteroidetes (4); f. Bacteroidaceae Fig.2. The turtles at 5⁰C normoxia seem to be intermediate between those communities at 21⁰C normoxia and 5⁰C anoxia. This would suggest an effect of temperature, followed by an effect of oxygen (lack of) on the mucosal microbiota. Objectives To discover interrelationships between turtle physiology and its gut microbial community. To identify members of the turtle gut microbiota that may influence, or be influenced by turtle physiological condition, thereby contributing to the extreme anoxia tolerance of the animal. 5⁰C Anoxic Fig. 3. Bacterial taxonomic profiles are compared for each sample treatment. In (A), 21N shows the diversity of organisms in turtle microbiota prior to exposure to low temperatures represented for the major phyla; notice that Fusobacteriain 5N and 5A are significantly lower in relative abundance compared to 21N. Similarly for (B) comparisons of the diversity in 21N, like Aeromonadaceae, has a trace relative abundance compared with 5N and the highest relative abundance found in 5A. 21⁰C Normoxic 5⁰C Normoxic Discussion References • At the phylum level, the bacteria we identified are typical of gut microbiotas of other animals. • From the phylum Proteobacteria, the families Aeromonadaceae (associated with freshwater habitats as well as most members utilizing glucose as an energy source [6]) and • Enterobacteriaceae(produce lactic acid from carbohydrates) are of interest. • Further study and data collection, however, are necessary to understand why the families: • Aeromonadaceae had the greatest relative abundance in 5A compared to 5N and 21N, and • Enterobacteriaceae had a much higher relative abundance in 5N compared to 5A and 21N. • It is known that lactic acid bacteria, such as certain members from the phyla Firmicutes and Proteobacteria, convert lactose and other sugars into lactic acid; this creates an extremely acidic environment that inhibits the growth of certain bacteria. From our data, this may explain why the relative abundance of Bacteroidaceae was lower at 5N than at 21N and 5A. [1] Savage.,1986. Annu. Rev. Nutr. 6:155-178. [2] Chow et al., 2010. Adv. Immunol. 107:243-274. [3] Bates et al., 2006. Dev. Biol. 297:374-386. [4] Bickler and Buck, 2007. Annu. Rev. Physiol. 69:145-170. [5] Jackson and Ultsch., 2010. J. Exp. Zool. 313A: 311-327. [6] Colwell et al.,1986. IJSB. 36(3):473-477. Acknowledgements This work was supported in part by the UAA Summer REU program (NSF DBI-1263415), the UAA-INNOVATE 2014 Award, and the Alaska INBBRE Award. Special thanks are extended to Dr. Duddleston and Dr. Stecyk, the Research and Testing Laboratories, Inc., Jasmine Hatton, Tim Stevenson, Chloe Cayabyab, and Sara Cain.