1. The Effects of Vegetation, Nutrition, and Sex Ratio on the Reproductive Cycle of Fundulus heteroclitus in the Laboratory College of Arts and Sciences, 11300 NE 2 nd Avenue, Miami Shores, FL 33161 The long term objective of this project is to monitor the reproductive cycle of Fundulus heteroclitus. In a previous study, we demonstrated that the fish can spawn successfully in a laboratory environment. This was done by feeding the fish an adequate amount of fish flake food (Tetramin ® Fish Flake) and brine shrimp (live and frozen) to provide enough caloric energy for gamete formation. At this time, we are further investigating the effects of vegetation, nutrition, and male to female sex ratio on the reproductive cycle. Fish were maintained at stable conditions [water temperature at 26 ± 2 °C; salinity (28-30 ppt); 14 hr light and 10 hr dark photoperiod]. Three sets of experimental variables were carried out in order to find the most optimal conditions to keep the fish reproductively active: a) tanks were set to several ratios of female to male [6:2, 4:4]; b) several screened trays (with or without vegetation) were placed within each tank to determine their inclination to spawn; c) fish were fed a variety of diets: enriched diet (live and frozen brine shrimp and flake food four times daily) vs. control diet (flake food once daily). The data collected will provide information on optimal laboratory conditions for Fundulus heteroclitus spawning success. In addition, this study will serve as a protocol for establishing a standard husbandry procedure for our future experiments. Supported by NIH-NIGMS MBRS RISE Grant R25 GM059244-09 and Department of Energy Grant No.-DE- FG02-06CH11438, Barry University The purpose of our project is to find the optimal laboratory conditions for Fundulus heteroclitus spawning success. In their natural habitat, F. heteroclitus typically have their reproductive behavior adapted to their tidal environment and spawn during spring tides among a selection of plants (Hsiao et al., 1994). This fish shows cyclic changes in ovarian follicle development following a semilunar periodicity (Taylor, 1984). Our main focus in this set of experiments was to examine the effect of nutrition, vegetation, and sex ratio on F. heteroclitus spawning cycle in our aquatic facility. In this study, the eggs were collected from each tank and recorded daily to get a collective sample of how suitable our aquarium setting is in comparison to their natural habitat and the previous laboratory setting of Hsiao (1994). ResultsAbstract Introduction Materials and Methods Fig. 5: Core Aquarium Facility Discussion References 1.Hsiao Shyh-Min, Greeley MS, and Wallace RA. 1994. Reproductive cycling in female Fundulus heteroclitus. Biol. Bull. 186:271-284. 2.Taylor MH. 1984. Lunar synchronization of fish reproduction. Trans. Am. Fish. Soc. 113:484-493. Fundulus heteroclitus were collected in St. Augustine, FL, and transported to our aquarium at Barry University (Fig. 5) in November 2009. Fish (8 per tank) were maintained in aerated 10 L tanks filled with saltwater (28-30 ppt) at 26 ± 2° C, under fluorescent photoperiods of 14 hr light and 10 hr dark. To catch the eggs during each successful spawning cycle, we designed containers [Pyrex trays (17x12 cm)] with a screen on top to allow the eggs to fall in (Fig.1). The egg production in each tank was monitored and removed from the container every day. Diet experiments: To examine the effect of nutrition on reproduction, control diet fish were feed only TetraMin ® flake food once every morning. The experimental group had an enriched diet with live and frozen brine shrimp in addition to the flake food four times a day. Effects of the diet on fish spawning (Fig. 2): The egg count within the first two cycles was similar between the two groups. By the third cycle (21 days after the beginning of the experiment), we saw a decrease in the number of eggs spawned by the control diet group (fed only once a day), while the enriched diet group maintained relatively high levels of egg production. The data collected indicate that the enriched diet contributed to an overall greater reproductive yield per female than the control diet, over time. Effects of vegetation on fish spawning (Fig. 3): We hypothesized that fish would prefer to lay eggs in places with vegetation to provide protection for the eggs. In contrast, more eggs were found in the trays without artificial vegetation throughout all the cycles examined. This indicates that artificial vegetation does not enhance the spawning activity of the fish. The fish seem to prefer spawning over trays without artificial vegetation. Effects of sex ratio on fish spawning (Fig. 4): The sex ratio of 4 female:4 male produced similar numbers of eggs per female as the 6 female:2 male ratio indicating that the presences of more males does not influence the production of eggs. Based on this study we can optimize the conditions for future reproductive experiments. In conclusion, the results show that an enriched diet 4 times a day, use of screened trays without vegetation and equal female to male sex ratio inside the tanks can maintain a good cyclic reproductive activity in F. heteroclitus kept in our core aquarium facility. Sex ratio experiments: To determine whether the number of females and males present in the tank will affect the spawning behavior, a female:male ratio of 6:2 and 4:4 were compared while maintaining the same number of fish (8) per tank. We hypothesized that the 4:4 ratio will produce more eggs per female. Vegetation experiments: To evaluate the behavioral tendencies of fish to either lay eggs in an area with vegetative coverage or in areas absent of vegetation, two screened trays were placed in each tank; one of the screened trays had artificial aquarium plants placed on top of the screen, and the other did not. Fig. 1: Screened Trays Alton Johnson, Alex Brehm, Blandine Victor, Stefanie Sveiven, Jodi-Ann Browning, Steven Johnson, Teresa Petrino, and Yu Wai P. Lin Email: Alton.Johnson@mymail.barry.edu