Temporal effects of heat waves on sex ratios and gene expression in a freshwater turtle. Anthony Breitenbach PhD Candidate Illinois State University
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1 Temporal effects of heat waves on sex ratios and gene expression in a freshwater turtle Anthony Breitenbach PhD Candidate Illinois State University
2 Climate Change and Heat Waves
3 IPCC 2014
4 Predicted changes in heat wave frequency (with respect to data from ) Fischer and Schär 2010
5 Predicted changes in heat wave amplitude (with respect to data from ) Fischer and Schär 2010
6 Reptiles with TSD Chelydra serpentina Chelonia mydas Chrysemys picta Trachemys scripta Sphenodon punctatus Alligator mississippiensis Agama agama
7 Crews et al. 1994
8 Modified from Lance and Valenzuela 2004
9 Male-producing temperatures (MPTs) 29 C Trachemys scripta Female-producing temperatures (FPTs) Cool temps. (26 C) Warm temps. (31 C) Modified from Lance and Valenzuela 2004
10 SEX HAS NOT BEEN DETERMINED SEX IS BEING DETERMINED SEX HAS BEEN DETERMINED Ovarian development Female Bipotential gonads Testicular development Male Oviposition Thermosensitive Period (TSP) (middle third) Hatching
11 Temperature affects TSP length and timing Matsumoto and Crews 2012
12 Male producing temperature Female producing temperature Dmrt1 (transcription factor) Sox9 (transcription factor) Rspo1 (growth factor) Foxl2 (transcription factor) Aromatase (estrogen synthase) SF1 (nuclear receptor/ mrna splicing factor) AMH P450 scc, P450 17ɑ, 3βHSD Testicular development Ovarian development cholesterol testosterone dihydrotestosterone Modified from Matsumoto and Crews 2012
13 Male producing temperature Female producing temperature Dmrt1 (transcription factor) Sox9 (transcription factor) Rspo1 (growth factor) Foxl2 (transcription factor) Aromatase (estrogen synthase) SF1 (nuclear receptor/ mrna splicing factor) AMH P450 scc, P450 17ɑ, 3βHSD Testicular development Ovarian development cholesterol testosterone dihydrotestosterone Modified from Matsumoto and Crews 2012
14 Matsumoto and Crews 2012
15 Male-producing temperature cue Testicular development dihydrotestosterone
16 Female-producing temperature cue Ovarian development
17 Steroid hormones Crews et al. 1994; Matsumoto and Crews 2012
18 Field versus Laboratory
19 Constant versus Fluctuating Daily/seasonal fluctuations Field Lab Paitz et al. 2010
20 Average temperatures are poor predictors of sex ratios Trachemys scripta Chrysemys picta 28.5 C 28.5 ± 3 C Les et al. 2007
21 Heat waves affect sex ratios Carter et al. 2018
22 Current Understanding 1978
23 Natural Fluctuations Chrysemys picta: Janzen 1994
24 Biased Sex Ratios Mrosovsky and Provancha 1989 Wibbels et al Mrosovsky and Provancha 1992 Marcovaldi et al Hanson et al Godley et al Öz et al Caretta caretta (Loggerhead Sea Turtle) Female biased nests, juvenile and sub-adult 89-99% females
25 Potential Responses Behavioral Plasticity Nesting phenology* Chrysemys picta nesting 27 days earlier on average over 13 year period (Schwanz and Janzen 2008) Spatial changes in nesting Lacks plasticity (Refsnider and Janzen 2012; Refsnider 2013) Evolve a sensitivity to a different pivotal temperature Migrate to cooler climate to balance sex ratios Depends on water body distributions
26 Overarching Hypothesis Climate change, in the form of heat waves, will influence the physiological and endocrinological underpinnings of TSD in turtles. 1) How does TSD operate in the field under natural conditions? 2) How will climate change affect sex ratios in reptiles with TSD?
27 Model Species- Trachemys scripta
28 Hypothesis 1 Heat wave timing will influence the physiological and endocrinological underpinnings of TSD. Prediction 1: A heat wave that occurs within the TSP will produce more female-biased sex ratios. Prediction 2: A heat wave applied during the TSP will trigger relatively higher levels of aromatase expression and relatively lower levels of Dmrt1 expression. a. Using fluctuating incubation temperatures, when does the TSP occur? b. How does aromatase and Dmrt1 expression respond to a simulated heat wave during the predicted TSP?
29 Hypothesis 1- Methods Male producing condition: 25 ± 3 C Baseline Female producing condition: 29.5 ± 3 C Heat wave 15-day heat wave varied temporally Eggs: gravid female (oxytocin)/nest Group eggs to avoid clutch/box/incubator effects 20 eggs per treatment (sex ratio) 15 extra eggs in control and (qpcr) Illinois Climate Network 2015; Carter et al. 2017
30 Hypothesis 1- Methods (continued) Macroscopic gonad examination after euthasol injection Matsumoto et al. 2012
31 Hypothesis 1- qpcr Sample embryonic tissues on days 38 and 43 in the control and day groups Gapdh as housekeeping gene Primer Name Sequence Reference Forward Gapdh GGC TTT CCG TGT TCC AAC TC Ge et al Reverse Gapdh GAC AAC CTG GTC CTC CGT GTA TC Ge et al Forward Aromatase CGA CAT GGA CTT TGC ATC ACA Ramsey et al Reverse Aromatase GAA CCA TCA TCT CCA ACA CAC ACT GGT TC Ramsey et al Forward Dmrt1 CAA CTA CTC CCA ATA CCA GAT GGC Shoemaker et al Reverse Dmrt1 GGC TTC GCA GGC TGT TTT TC Shoemaker et al. 2007
32 Sex ratio results: Heatwave treatment timing: χ 2 = , df = 4, p < 0.001
33 Middle third? Treatment Avg. Middle Third % Female Stat. Differences Control % Heat wave % a Heat wave % b Heat wave % b Heat wave % b Heat wave % a
34 Relative Aromatase expression (2^ dct) Aromatase df F value p value Treatment p = ** Day p < *** Treatment*Day p = ** Day Control Heatwave Treatment
35 Relative Dmrt1 expression (2^ dct) Dmrt1 df F value p value Treatment p < *** Day p < *** Treatment*Day p =.0193 * Day Control Heatwave Treatment
36 Control Day ± 3 C shifted to 29.5 ± 3 C Day Day Day Day Predicted TSP Hatch Day
37 Control Day ± 3 C shifted to 29.5 ± 3 C Day Day Day Day Hatch Actual TSP Day
38 Hypothesis 1 Conclusions TSP is likely around days using these temperature parameters Middle three heat waves have similar potency to produce females Heat waves induce aromatase expression MPTs induce Dmrt1 expression Treatment Avg. Middle Third % Female Stat. Differences Control % Heat wave % a Heat wave % b Heat wave % b Heat wave % b Heat wave % a
39 Next Steps: Natural Sex Ratios Multi-year T. scripta field incubations Eggs: gravid female (oxytocin)/nest Individual nests dug for each clutch in nesting area ibutton data loggers Macroscopic gonad examination a. What sex ratios are being produced in nature?
40 Next Steps: Hypothesis 2 Heat wave continuity will influence the physiological and endocrinological underpinnings of TSD in turtles. Prediction 1: With hot days held constant, more continuous heat waves will produce more female-biased sex ratios. a. How does heat wave continuity affect sex determination? Paitz et al. 2009
41 Hypothesis 2- Methods Heat Wave Length (Days) Proportion Females 8 24% 11 74% 14 94% 17 89% 20 90% 23 94% 26 90% % % % Carter et al. unpublished
42 Next Steps: Hypothesis 3 Aromatase and Dmrt1 expression will respond differentially to heat waves of varying lengths. Prediction 1: Longer heat waves will produce higher levels of aromatase expression. Prediction 2: Heat waves will down-regulate Dmrt1 expression. a. How does aromatase expression respond to a heat waves of varying lengths? b. How does Dmrt1 expression respond to a heat waves of varying lengths? Heat Wave Length (Days) 8 24% 11 74% 14 94% 17 89% 20 90% 23 94% 26 90% % % % Proportion Females Carter et al. unpublished
43 Hypothesis 3- Methods Sample on days 30, 33, & 36 Gapdh as housekeeping gene
44
45 Acknowledgements PhD Committee: Dr. Rachel Bowden Dr. Scott Sakaluk Dr. Ben Sadd Dr. Steven Juliano Dr. Ryan Paitz Community Outreach: Jill Wallace Jennifer Trimble Dr. Craig Gatto Funding: Dr. Rachel Bowden Marc Ashford Rosario Marroquin-Flores Dr. Ryan Paitz Dr. Amanda Wilson-Carter
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