The Reproductive Cycle and Embryo Development of Fundulus heteroclitus in the Laboratory Abstract Introduction Material and Methods Results Discussion ReferencesAcknowledgements Jodi-Ann Browning, Alton Johnson, Blandine Victor, Myrline Sterling, Teresa Petrino, Ph.D, and Yu Wai P. Lin, Ph.D College of Arts and Sciences, NE 2 nd Avenue, Miami Shores, FL The objective of this project is to monitor the reproductive activity of the first batch of fish (Fundulus heteroclitus) brought into the aquarium in January The aim is also to use these data as the baseline for future experiments on endocrine disruption. The reproductive activity of F. heteroclitus follows a semi-lunar pattern in their natural habitat; they spawn heavily during the full moon and new moon. Under the current conditions in the laboratory (water temperature at 26 ± 2°C; salinity 28ppt-30ppt; 14hr light and 10 hr dark photoperiod; fed on average 3 to 4 times each day), 10 tanks containing a total of 79 fish (42 males, 37 females) were monitored and their eggs removed from the tanks after each successful spawning cycle. A screened tray was placed within each tank to collect the eggs. Once the eggs were collected each day, the development of the embryos was closely observed and the successive developmental stages were photographed. With the current set up, we were able to collect fertilized eggs, which developed to a normal hatching stage. Although more data are needed to determine the periodicity of the spawning cycle, these results indicate that our aquarium environment appears to be suitable to support the reproductive activity of this species. Supported by NIH-NIGMS MBRS RISE grant R25 GM and Department of Energy Grant No.-DE-FG02-06CH11438 Supported by NIH-NIGMS MBRS RISE grant R25 GM and Department of Energy Grant No.-DE-FG02-06CH11438, Barry University The purpose of our project is to observe and document the reproductive cycle and the embryonic developmental stages of Fundulus heteroclitus as they were introduced to our new Aquarium facility at Barry University. The average length of an adult F. heteroclitus ranges from 5-15cm. During the breeding season, both sexes show intense changes in their exterior appearances (Armstrong and Child, 1965). The males dorsal turns into transparent black with white radiant spots and the ventral area turns into a pale yellow. The females ventral has a pearly white to grayish appearance. During the fall season, the females ovarian eggs range in size from 0.16 mm to 0.4 mm. After the month of April the eggs size progressively increases to a maximum size of 1.3 mm before ovulation (Armstrong and Child, 1965). 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). The fish will normally release their eggs when a new moon occurs (Taylor, 1984). This does not mean that the fish only spawns on a new moon; it usually means that during a new moon the female fish may release a larger amount of eggs, in comparison to the rest of the spawning period. 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). In addition to collecting data on the spawning cycle, we observed and photographed all of the successive stages of the development of F. heteroclitus. 1.Armstrong PB, Child JS Stages in the Normal Development of Fundulus heteroclitus. Marine Bio Lab 128: Hsiao Shyh-Min, Greeley MS, and Wallace RA Reproductive Cycling in Female Fundulus heteroclitus. Biol. Bull. 186: Taylor MH Lunar synchronization of fish reproduction. Trans. Am. Fish. Soc. 113: Fish were housed for one month (Fig. 1) before the starting collection date (Feb. 25 th ). Data for each tank (Fig. 3) were examined separately and grouped together according to the dates the eggs were spawned (Fig. 4). During the course of our study, we noticed there were many eggs on the filter and in the sump of our aquatic system indicating that the Petri dishes [Fig. 2 (a)] were too small to collect all the eggs produced; subsequently, they were replaced with a bigger Pyrex container [Fig. 2 (b)]. Also, this indicates that the collection is just a sample of the spawning activity as we did not intend to collect every spawned egg. In addition to egg production, we observed that the fertilized eggs were able to continue development to hatching stage and beyond (Fig. 5). In conclusion, our project needs to continue for several more months in order to truly identify if there is a spawning cyclic with F. heteroclitus within our new aquarium facility. From these preliminary results, we cannot yet determine the cyclic reproductive activity as previously demonstrated for this species. However, the fact that we have seen normal embryonic development is an indication that the aquarium conditions are adequate to support the spawning of good quality eggs and an abundant amount of normal developing embryo that are hatching to be healthy fry. Eighty-two Fundulus heteroclitus were collected in St. Augustine, FL, and transported from the Whitney Laboratory to our aquarium at Barry University (Fig. 1) in January In our facility, the fish were sorted randomly and placed in 10L tanks in System C (C6-C15); Tank C6 [male:female ratio (6:5)], Tank C7 (4:4), C8 (7:2), C9 (2:4), C10 (2:2), C11 (3:7), C12 (5:4), C13 (3:2), C14 (5:5), and C15 (3:7). To catch the eggs during each successful spawning cycle, we designed two containers [Pyrex tray (17x12 cm) or Petri dish (100x15cm)], with a screen on top, and held together with elastic bands and cable ties respectively, to allow the eggs to fall in. Aquarium plants were placed either on top or around the container to encourage spawning (Fig. 2). All tanks were aerated and filled with saltwater at 26 ± 2°C and 28-30ppt. The lighting in the aquarium is fluorescent with photoperiods of 14hr light and 10hr dark. Fish were fed 3-4 times daily with TetraMin flake food, in addition to brine shrimp twice per week. The egg production in each tank was monitored and removed from the container everyday.. After the eggs were collected, they were placed in labeled Petri dishes containing pre- mixed saltwater from our system. The eggs were rinsed to remove any flake food debris by replacing the water several times. They were examined under a microscope and photographs were taken of developmental cell stages. The unfertilized eggs were removed and the Petri dishes were left in the laboratory at room temperature. Once the eggs had hatched, the baby fish (fry) were fed with brine shrimp while they were still in the Petri dishes. After 2 to 4 days, the fry were transported to our nursery in the aquarium. Fig. 3: Graphs showing the spawning cycle of each tank over a 26 day period. Broken lines on each graph represents days in which eggs were not collected. Fig. 5: Developmental stages of Fundulus heteroclitus according to Armstrong and Child (1965). 1-Cell Stage4-Cell Stage 16-Cell Stage 2-Cell Stage Growth & organodifferentiation; stage 29 Growth & organodifferentiation; stage 35 Fig. 2: Egg collection in (a) a screened Petri dish or (b) Pyrex container (a) (b) Fig. 1: Core aquarium facility Fig. 4: Spawning cycle of all tanks 1 mm