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					                                                                          The Reproductive Cycle and Embryo Development of Fundulus
                                                                                         heteroclitus in the Laboratory
                                                                                                   College of Arts and Sciences, 11300 NE                                                                2nd        Avenue, Miami Shores, FL 33161
                                 Jodi-Ann Browning, Alton Johnson, Blandine Victor, Myrline Sterling, Teresa Petrino, Ph.D, and Yu Wai P. Lin, Ph.D
                                                                                                                                                             Fig. 2: Egg collection in (a) a screened Petri dish or
                                      Abstract                                                                   Fig. 1: Core aquarium facility
                                                                                                                                                             (b) Pyrex container                                                            Fig. 5: Developmental stages of Fundulus heteroclitus according to
                                                                                                                                                                                                                                                             Armstrong and Child (1965).
                                                                                                                                                              (a)                                  (b)
The objective of this project is to monitor the reproductive activity of the first batch of fish                                                                                                                                             1-Cell Stage                     2-Cell Stage                    4-Cell Stage
(Fundulus heteroclitus) brought into the aquarium in January 2009. 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                                                                                                                                                                     1 mm         1 mm                                              1 mm
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                                                                                                                                                              16-Cell Stage
results indicate that our aquarium environment appears to be suitable to support the                         Fig. 3: Graphs showing the spawning cycle of each tank over a 26 day period. Broken                                                                                  Growth & organodifferentiation; stage 29
reproductive activity of this species.                                                                       lines on each graph represents days in which eggs were not collected.
Supported by NIH-NIGMS MBRS RISE grant R25 GM059244 and Department of
                                                                                                                                                                     Tank C8
Energy Grant No.-DE-FG02-06CH11438                                                                                           Tank C7
                                                                                                                                                    100                                      100
                                                                                                                                                                                                         Tank C9
                                                                                                            E 100                                 E

                                                                                                            g                                     g 80
                                                                                                                  80                                                                          80
                                                                                                            C                                     C                                      g
                                                                                                            o                                     o 60                                   g
                                                                                                                  60                                                                          60
                                                                                                            u                                     u
                                                                                                                                                  t 40
                                                                                                                                                                                                                                                 1 mm                                        1 mm
                                                                                                                                                        20                               t
                                                                                                                  20                                                                          20
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                                0                                  0                                     0

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                                                                                                                       Tank C12
                                                                                                                                                                                                                                      Growth & organodifferentiation; stage 35      Growth & organodifferentiation; stage 35
                                                                                                                                                                    Tank C11
their exterior appearances (Armstrong and Child, 1965). The male’s dorsal turns into                         100
                                                                                                                          Tank C10                 100                                       100

transparent black with white radiant spots and the ventral area turns into a pale yellow.               E
                                                                                                        g                                              80                                     80

The female’s ventral has a pearly white to grayish appearance.
                                                                                                        g        80                               E                                      E
                                                                                                                                                  g                                      g
                                                                                                        C                                         g 60                                   g
                                                                                                        o        60                                                                           60
                                                                                                        u                                         C                                      C

During the fall season, the female’s ovarian eggs range in size from 0.16 mm to 0.4 mm.
                                                                                                        n                                         o                                      o
                                                                                                                 40                                 40                                        40
                                                                                                        t                                         u                                      u
                                                                                                                                                  n                                      n

After the month of April the egg’s size progressively increases to a maximum size of 1.3                         20
                                                                                                                                                                                                                                                                                                       1 mm
mm before ovulation (Armstrong and Child, 1965). In their natural habitat, F. heteroclitus                        0                                     0                                      0                                                   1 mm
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                                      Tank C13                                   Tank C14
                                                                                                                                                                                                     Tank C15
fish only spawns on a new moon; it usually means that during a new moon the female fish                      100                                       100

may release a larger amount of eggs, in comparison to the rest of the spawning period.                  E        80


                                                                                                                                                  g                                      g
                                                                                                        g                                                                                     60
                                                                                                                 60                               g 60
In this study, the eggs were collected from each tank and recorded daily to get a collective            C
sample of how suitable our aquarium setting is in comparison to their natural habitat and               n
                                                                                                        t        20
                                                                                                                                                  n 20
                                                                                                                                                                                         t    20

the previous laboratory setting of Hsiao (1994). In addition to collecting data on the                            0

                                                                                                                                                         0                                     0
spawning cycle, we observed and photographed all of the successive stages of the
development of F. heteroclitus.                                                                                                                                                                                                      Fish were housed for one month (Fig. 1) before the starting collection date (Feb. 25th).
                                                                                                                                                                                                                                     Data for each tank (Fig. 3) were examined separately and grouped together according to
                                                                                                                                                                                                                                     the dates the eggs were spawned (Fig. 4).
                         Material and Methods
                                                                                                                            Tank C6
                                                                                                        250                                                               Fig. 4: Spawning cycle of all tanks
                                                                                                                                                                                                                                     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
Eighty-two Fundulus heteroclitus were collected in St. Augustine, FL, and transported
                                                                                                    g   150
                                                                                                                                                                                                                                     collect all the eggs produced; subsequently, they were replaced with a bigger Pyrex

from the Whitney Laboratory to our aquarium at Barry University (Fig. 1) in January
                                                                                                    u   100
                                                                                                                                                  200                                                                                container [Fig. 2 (b)] . Also, this indicates that the collection is just a sample of the
2009. In our facility, the fish were sorted randomly and placed in 10L tanks in System C
                                                                                                                                                                                                                                     spawning activity as we did not intend to collect every spawned egg.

(C6-C15); Tank C6 [male:female ratio (6:5)], Tank C7 (4:4), C8 (7:2), C9 (2:4), C10                                                          g    150                                                                    Tank C#6

(2:2), C11 (3:7), C12 (5:4), C13 (3:2), C14 (5:5), and C15 (3:7). To catch the eggs during
                                                                                                             0                               C
                                                                                                                                                                                                                         Tank C#7
                                                                                                                                                                                                                         Tank C#8    In addition to egg production, we observed that the fertilized eggs were able to continue
each successful spawning cycle, we designed two containers [Pyrex tray (17x12 cm) or
                                                                                                                                             n     100
                                                                                                                                                                                                                         Tank C#9
                                                                                                                                                                                                                         Tank C#10
                                                                                                                                                                                                                                     development to hatching stage and beyond (Fig. 5).
                                                                                                                                             t                                                                           Tank C#11
Petri dish (100x15cm)], with a screen on top, and held together with elastic bands and                                                                                                                                   Tank C#12

cable ties respectively, to allow the eggs to fall in. Aquarium plants were placed either on                                                           50
                                                                                                                                                                                                                         Tank C#13
                                                                                                                                                                                                                         Tank C#14
                                                                                                                                                                                                                                     In conclusion, our project needs to continue for several more months in order to truly
top or around the container to encourage spawning (Fig. 2).                                                                                                                                                              Tank C#15   identify if there is a spawning cyclic with F. heteroclitus within our new aquarium facility.
                                                                                                                                                         0                                                                           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
All tanks were aerated and filled with saltwater at 26 ± 2°C and 28-30ppt. The lighting in                                                                                                                                           embryonic development is an indication that the aquarium conditions are adequate to
the aquarium is fluorescent with photoperiods of 14hr light and 10hr dark. Fish were fed                                                                                                                                             support the spawning of good quality eggs and an abundant amount of normal developing
3-4 times daily with TetraMin flake food, in addition to brine shrimp twice per week. The                                                                                                                                            embryo that are hatching to be healthy fry.
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
                                                                                                                                                              References                                                                                         Acknowledgements
photographs were taken of developmental cell stages. The unfertilized eggs were removed                 1. Armstrong PB, Child JS. 1965. Stages in the Normal Development of Fundulus heteroclitus. Marine
and the Petri dishes were left in the laboratory at room temperature. Once the eggs had                    Bio Lab 128:144-168.                                                                                                              Supported by NIH-NIGMS MBRS RISE grant R25 GM059244 and Department
hatched, the baby fish (fry) were fed with brine shrimp while they were still in the Petri              2. Hsiao Shyh-Min, Greeley MS, and Wallace RA. 1994. Reproductive Cycling in Female Fundulus                                         of Energy Grant No.-DE-FG02-06CH11438, Barry University
dishes. After 2 to 4 days, the fry were transported to our nursery in the aquarium.                        heteroclitus. Biol. Bull. 186:271-284.
                                                                                                        3. Taylor MH. 1984. Lunar synchronization of fish reproduction. Trans. Am. Fish. Soc. 113:484-493.

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