1. Hypothesis Effects of environmental temperature fluctuations on development and hatching success in the California grunion Fernando Vargas, Andres Carrillo, and Kathryn Dickson Department of Biological Science, California State University, Fullerton Methods Conclusion Acknowledgements I would like to thank the directors of the Southern California Ecosystem Research Program, Dr. Bill Hoese and Dr. Darren Sandquist. Funding from the National Science Foundation (DBI-1041203), from the Faculty-Student Research Creative Activities Grant, and a Southern California Academy of Science Research Grant. I would also like to thank everyone who has helped me with my project: Helena A., Doug B., John B., Andres C., Ernesto C., Lauren D., Faye C., Laura E., Justin G., Mauricio G., Brandon H., Katie H., Sean H., Chris K., Annie M., Kyle N., Rick N., Eric P., Cheryl S., Jacqulyne S., Lauren S., Dylan T., Tracie T., and Sean V. References 1. Walker, B. W. (1952). A guide to the grunion. Cal. Fish and Game Bull. 38, 409-420. 2. Treybig, T. Degrassi, A. Casillas, E. Hessom, E. Ramos, E. (2009). Environmental factors that affect delayed hatching on development and swimming speed in the California grunion, Leuresthes tenuis. Dimensions: The Journal of Undergraduate Research in Natural Sciences and Mathematics CSUF, 11, 36-42. 3. Smyder, E. A. and Martin, K. L. M. (2002). Temperature effects on egg survival and hatching during the extended incubation period of California grunion, Leuresthes tenuis. Copeia 2002, 313-320 4. Moffatt, N. M. and Thomson, D. A. (1978). Tidal influence on the evolution of egg size in the grunions, Leuresthes Atherinidae. Environ. Biol. Fish. 3, 267-274. The California grunion, Leuresthes tenuis, spawns on sandy beaches along the coast of California and Northern Baja California during the months of May through August (Fig 1, 2). Spawning cycles are consistent with the lunar cycle, occurring one to three days after new and full moons (1). Sand temperature fluctuates up to 11 o C at Cabrillo Beach in San Pedro, California, where grunion are known to spawn (2). Embryos incubated at 20 o C had an 80% chance of survival, whereas embryos incubated at 15 o C had a 3% chance of survival 9-13 days post-hatching (3). The sand temperatures at which grunion embryos incubate in the sand in southern California beaches are not known. Figure 1 California grunion (length ~150mm). Photo by Helena Ayafar Introduction Temperature sensors monitored sand temperature at Cabrillo Beach in San Pedro, CA, throughout the spawning season (Fig 4, 5). Gametes of multiple males and multiple females was collected in one container, where fertilization occurs, and split into nine containers. Three batches of fertilized eggs was placed for incubation either at variable temperatures recorded from the temperature sensors (experimental), at constant 20 o C or constant average temperature 16 o C(control). Minimum of 30 viable eggs were agitated to hatch Hatching success was quantified as follows: Hatching %= (Number of Embryos Hatched/ Total Number of Viable Embryos). A sample of the hatched larvae (n=5) was measured for notochord length, and yolk sac surface area from each hatching time by using Image J software (Fig 6, 7). Figure 8. Mean (+ SD) sand temperature ( o C) recorded from March 27 to May 29 [64 days], using five iButtons placed at depths where grunion eggs were found (10 cm) at Cabrillo Beach, Los Angeles, CA in 2011. The mean height (m above mean low-low water) of five iButtons and predicted tidal heights (http://tbone.biol.sc.edu/tide/) are plotted on the right axis. Figure 3 Fertilized grunion embryo, four days post- fertilization (2mm in diameter). Figure 4 Temperature housing protects sensor and keeps sensor stable Figures 6 Measurement of notochord length of grunion larvae (length ~7.5mm). Photo by Helena Aryafar. Figure 7 Measure of yolk sac surface area. Photo by Helena Aryafar. I hypothesize that incubation of eggs at fluctuating temperature will affect development by 1) decreasing hatching success, 2) decreasing notochord length at hatching, and 3) slowing the rate at which yolk is used, when compared with embryos incubated at a constant, 16.3 o C and optimal temperature of 20 o C. Figure 2 Distribution of California grunion (4) Figure 5 Placement of housing in the sand Temperature ( o C) Temperature sensors Results Tidal Height (m) Yolk Area rate of decrease was not significantly different in any treatment Figure 11 Mean (± SD) yolk area (mm 2 ) of grunion at hatching under different temperature treatments. The rate of decrease was not significantly different in any treatment (p>.05). Hatching success for grunion eggs incubated under fluctuating and constant 16.3 o C was significantly lower than 20 o C throughout experiment. Grunion eggs incubated under fluctuating temperatures did not affect rate of development when compared to constant temperature incubation. Knowing how grunion respond to their environment can help us protect this species from beach visitors at Cabrillo Beach. Side experiment of grunion embryos that get washed out before competent in sand can still develop and hatch in the ocean. Future work: Repeat hatching experiment but with sand temperature collected during the warmer months of July to investigate if hatching percentage increases Notochord length was not significantly different in any treatment Days post- fertilization (dpf) Figure 10 Mean (+ SD) notochord lengths (mm) of grunion larvae at hatching, after they were incubated under different temperature treatments. Notochord was not significantly different in any treatment. Figure 9 Mean (+ SD) hatching success (percent of viable eggs that hatched) after different incubation periods of grunion embryos incubated under different temperature treatments. Hatching success between constant 16 o C and variable temperature did not significantly differ 64 days (11.7 to 27.9 o C) Percent Hatching Notochord Length (mm) Yolk area (mm 2 ) Temperatures replicated in the lab for variable treatment