1. Development and Validation of
Reproductive Behavioral
Endpoints for Mummichog, an
Important Estuarine Model
Tanya Lama, Chelsea Blatchley & Thijs Bosker
tanya.lama@uconn.edu
UCONN Dept. of Natural Resources & the
Environment
2. Mimic hormone function, may accelerate or inhibit natural
hormone signaling pathways
EDCs that affect the hypothalamic pituitary gonadal axis
may affect reproductive capability and development of
offspring
◦ Intersex
◦ Altered mating behavior
◦ Reduced fecundity
Sources:
◦ Treated sewage effluent
◦ Agricultural run off
◦ Pulp-mill effluents
3. Behavioral stability provides the best opportunity for
survival and reproductive success
Measurable endpoint for sublethal toxicity
◦ Behavioral changes from toxicological exposure will alter an
organism’s response to environmental stimuli >>directly affect
fitness
4. Both freshwater and saltwater habitats receive inputs of
EDCs
◦ Current research has focused on freshwater habitats in EDC
exposure studies
Estuaries are valuable economically and ecologically
◦ Breeding and rearing grounds for fish and birds
◦ Receive high in-put use
5. Environmentally-relevant model to North America
◦ Native along the Atlantic coast
Sexually dimorphic
Hardy
Daily spawners
Euryhaline
Fish were captured with seine nets and minnow traps
◦ Barn Island Wildlife Management Area, Stonington CT
6. Develop behavior endpoints for
mummichog
◦ 1. Describe baseline behavior
◦ 2. Evaluate behavioral effects when
exposed to an androgenic EDC
7. 21-day Reproductive Bioassay
◦ Method used by USEPA
◦ Static daily renewal
◦ Video data collected after daily water changes and feedings
Model compound: 5α-Dihydrotestosterone (DHT)
◦ Potent & non-aromatizable
◦ Metabolite of testosterone
◦ Studies have shown measurable effects from DHT exposure
◦ 4 treatments: 0.00µg/L DHT, 0.05µg/L, 0.50µg/L and 5.00µg/L
Pre -
Exposure
Exposure
(Week 1)
Exposure
(Week 2)
Exposure
(Week 3)
8. Pre -
Exposure
Exposure
(Week 1)
Exposure
(Week 2)
Exposure
(Week 3)
Baseline
Behavior
Ethogram
Reproductive
Behavioral
Endpoints
Assessment of Behavioral Changes when
exposed to an androgenic EDC
9. Pre -
Exposure
Exposure
(Week 1)
Exposure
(Week 2)
Exposure
(Week 3)
• N = 5 tanks/treatment
• 20 total observations
Pre - Exposure
10 min Video
Segment
• Describe general behavior
• Behavioral endpoints which are measurable,
predictable and recognizable
Ethogram
10. Pinpoint endpoints for
reproductive behavior study
◦ Measurable, recognizable, high
frequency
◦ Ecological relevance to
reproductive success/ fecundity,
Ex: aggressive behavior, courtship
behavior
11. Behavior Description
Chasing/ Darting
Fish swims directly/aggressively toward another fish, causing it to increase its speed
and possibly change direction, and actively pursues the fish or returns to its original
position
Nipping
Fish clearly opens its mouth and bites other fish, often on the tail or ventral fins.
Often occurs during/immediately following a chase.
Spar
Fish slowly approaches another fish its fins flares and offers his/her body flank to the
other fish, making it appear larger. The opposing fish either moves away or
reciprocates, sometimes resulting in elaborate circling motion between the 2 fish.
Tail-Nose Nudging Male chasing the female rapidly, nudging her flanks with his snout.
Present Male presents his flank to a female of interest, showing off his yellow belly
Lead
Male attempts to escort a female to a spawning site by swimming alongside her, may
include tail-nose behavior
Approach
Female approaches male and makes contact with his flank, may result in male
encircling or tail-nose behavior
Escort Female follows a male side by side to a spawning site (accepting a lead)
14. Fig. 4. Adult mummichog (Fundulus heteroclitus) median behavioral counts by dosage of 5a-
dihydrotestosterone (DHT). Median number of chasing/darting, nipping and nudging behaviors observed in a
ten minute observation period after 21 day exposure to DHT. The same letters within each treatment reflect
results that are not significantly different from each other by Tukey’s multiple comparisons test.
a a
ab
b
ac
ab
b
c
a
a
a
b
0
1
2
3
4
5
6
7
8
Control
MedianBehavioralCount
Behavior
Median Behavior Count by Treatment, 21 days exposed
Control
0.05 µg/L DHT
0.50 µg/L DHT
5.00 µg/L DHT
Chasing/Darting Nipping Tail/Nose Nudging
15. Table 3. Kruskal-Wallis results for adult Mummichog (Fundulus heteroclitus) behavior after 21 days of
exposure to 5a-dihydrotestotsterone (DHT).
Behavior
Treatment
(DHT/L) N Median
Ave
Rank Z P Grouping1
Chasing/Darting
0.0µg 5 1 11.9 0.61
0.043
ab
0.05µg 5 0 5.9 2.01 a
0.50µg 5 0 8.3 0.96 ab
5.00µg 5 3 15.9 2.36 b
Nipping
0.0µg 5 4 12.6 0.92
0.007
ac
0.05µg 5 2 7.5 1.31 ab
0.50µg 5 1 5 2.40 b
5.00µg 5 6 16.9 2.79 c
Tail/Nose
Nudging
0.0µg 5 4 9.5 0.44
0.031
a
0.05µg 5 3 7.5 1.31 a
0.50µg 5 4 7.8 1.18 a
5.00µg 5 7 17.2 2.92 b
1
Grouping indicates significant differences based on individual Mann Whitney U-tests among treatments
16. 1. Described baseline behavior for adult mummichog
2. Evaluated behavioral effects when exposed to an
androgenic EDC (5a-DHT) in a 21-day reproductive
test
Future Work:
◦ Working on power analysis
◦ Refine adult behavioral endpoints – high variability, very time
consumptive video analysis, low sample size
17. Coe, T.S., Söffker, M.K., Filby, A.L., Hodgson, D., Tyler, C.R., 2010. Impacts of early life exposure to estrogen
on subsequent breeding behavior and reproductive success in zebrafish. Environ. Sci. Technol. 44, 6481-6487.
Colman, J.R., Baldwin, D., Johnson, L.L., Scholz, N.L., 2009. Effects of the synthetic estrogen, 17 alpha-
ethinylestradiol, on aggression and courtship behavior in male zebrafish (Danio rerio). Aquat. Toxicol. 91, 346-
354.
Weis, J.S., Weis, P., 1995. Swimming performance and predator avoidance by mummichog (Fundulus
heteroclitus) larvae after embryonic or larval exposure to methylmercury. Can. J. Fish. Aquat. Sci. 52, 2168-2173.
Brewer, S., Little, E., DeLonay, A., Beauvais, S., Jones, S., Ellersieck, M., 2001. Behavioral dysfunctions
correlate to altered physiology in rainbow trout (Oncorynchus mykiss) exposed to cholinesterase-inhibiting
chemicals. Arch. Environ. Contam. Toxicol. 40, 70-76.
Kane, A., Salierno, J., Brewer, S., 2005. Fish models in behavioral toxicology: Automated techniques, updates
and perspectives. Methods in Aquatic Toxicology (Chapter 32), Volume 2. 2, 559-590.
Mimic the functions of hormones with ability to accelerate or inhibit normal hormone signaling pathways
EDSs that target the hypothalamic-pituitary-gonadal axis
Impair reproductive capability
Affect offspring development
EDSs can target any of the endocrine glands in the body, but it is of particular concern when they target the hypothalamic pituitary gonadal axis because they can negatively impact reproductive capability and development of offspring.
Some examples of reported negative impacts of EDSs include intersex conditions reported to wild fish populations, altered mating behavior and reduced fecundity.
Treated sewage effluent – often pharmaceuticals do not sufficiently break down in the waste water treatment process as shown by this graph with levels of natural and synthetic steroid levels after primary and secondary treatment.
Behavior is a new and novel endpoint in aquatic ecotoxicology, as it is sensitive, nonlethal and environmentally relevant.
Behavioral stability provides the best opportunity for survival and reproductive success, allowing an organism to efficiently exploit resources and define suitable habitats (Little & Brewer, 2001)
Most behavioral endpoints used in toxicology have been developed for freshwater species including zebrafish, fathead and japanese medaka. Few behavioral endpoints for estuarine species have been developed.
Poor understanding of ecologically relevant behavior
Lack of test standards, variation in measured endpoints, small sample sizes (high time investment) (Vogl et al.,1999)
Lack of descriptive behavioral work in field or lab studies on many species (including mummichog)
Barn island was selected as a clean reference site because it is undeveloped and is managed for wildlife conservation
Androgenic EDCs may not be as omnipresent as estrogenic EDSs, but they are certainly present in the environment and a better understanding of their effects is needed. Like estrogens, androgens are vitally important to the development and functioning or reproductive organs.
We chose DHT because it will act as a model androgen. DHT is actually a naturally occurring metabolite of testosterone in mammals and it is responsible for differentiation of sex organs. It is not naturally occurring in fish so it will act as an androgen mimic.
We also chose this compound to work with because some studies with other species of fish have shown measureable results from the impacts of DHT.
Explain why we chose these concentrations:
We based the concentrations on similar levels to what we saw garnered effects in literature. It was important for us to see effects so we could compare behavioral endpoints.
Give detail about complete water changes every day followed by fresh dosing.
Explain why observations were scheduled after water changes and after feeding and during a normal 9-5 working day (reproducibility)
Impacts of DHT exposure on adult reproductive behavior
10 minute observations on days 7,14, 21 of exposure
n=5 tanks/treatment, high salinity group only
Use ethogram to assess frequency of courtship and aggressive behavior
Pinpoint 4 characteristic behaviors chosen from the ethogram for their ease of recognition, measurability and frequency
Observing and recording mummichog behavior over a 3 day period to obtain baseline activities and changes in behaviors over the course of the day
Tanks filmed for 10 minute segments
Total of 20 observations used to develop a behavioral ethogram of common reproductive behavior
Construction of an ethogram/inventory of general behaviors
To better understand and describe general and reproductive behavior in mummichog and to select potential behavioral endpoints which are measurable, predictable and recognizable
To evaluate the sensitivity of behavioral endpoints for adult mummichog in a short-term reproductive test to DHT.
**What is the connection between aggressive behavior and courtship behaviors
Three of these endpoints met our criteria: they were easily recognizable, measurable and exhibited frequently. All three behaviors entail a quick change in speed which is easily recognizable in the video footage. Futhermore, behavioral studies on zebrafish and fathead minnow exposed to an estrogenic EDC (EE2) have demonstrated decreased aggressive behavior, which led to a subsequent loss in dominance which was reflected in their reproductive success. I’m going to briefly show you what these behaviors look like.
Chasing/Darting: Fish swims directly/aggressively toward another fish, causing it to increase its speed and possibly change direction, and actively pursues the fish or returns to its original position
Nipping: Fish clearly opens its mouth and bites other fish, often on the tail or ventral fins. Often occurs during/immediately following a chase.
Tail-Nose Nudging: Male chasing the female rapidly, nudging her flanks with his snout
non parametric KW, if sig effect was found we conducted a MW U test
High variability endpoints
Future work: good addition to standardized test for esturine model using mummichog.
Aquatic Tox
Marine Pollution Bulletin
J of Env Monitoring
Check where most of the behavior papers have been published
Identify areas we need to work on
Make deadlines to get it submitted before Africa
