Abstract Nematostella vectensis, the starlet sea anemone, is a common marine invertebrate found in salt marsh estuaries along the East Coast. These animals are classified in Phylum Cnidaria, and have become a model system in the field of developmental biology. These animals reproduce both sexually and asexually, and have the ability to regenerate body parts lost to amputation. In this study, we explored the effects of chronic exposure to 17β-estradiol on the regeneration of N. vectensis. Amputated individuals were immersed for 96 hours in one of several different concentrations of estradiol (0 through 100 ng/l). We then collected body length, area, and weight data at 0, 48, and 96 hours post amputation (hpa) and ran one-way ANOVAs to test for significant differences in these variables across treatments. Our results showed that there were no significant differences between treatments at any time point. Typically, N.vectensis can fully regenerate a missing oral end in ~96 hpa, thus, we suspected that exposure to 17β-estradiol would hinder regenerative abilities, perhaps by causing undifferentiated cellular proliferation at the regenerating end. Future studies could explore the effects of 17β-estradiol on the Hedgehog signaling pathway because 17β-estradiol may promote tumor-like cell proliferation to a wound site, ultimately hindering regeneration. Background Nematostella vectensis •  Sea anemone - Phylum Cnidaria. Found in salt marsh mud. •  Model organism for developmental biology and environmental toxicology. •  Two body axes: Primary: oral – aboral. Secondary: perpendicular to primary. Estradiol •  Naturally occurring hormone •  Causes adverse effects in the environment at relevant concentrations. •  Enters marine environments via runoff polluted with feces, urine, pharmaceuticals, natural agricultural waste, and potential leeching from plastic •  In some systems, estradiol affects the downstream signaling of the Hedgehog (HH) signal transduction pathway, which plays a key role in regeneration. Aims •  Do measurement techniques affect N. vectensis vitality?

•  Does chronic exposure to estradiol affect regeneration of N.vectensis? •  Due to concentration? •  Mechanism?

•  Can cellular proliferation at the wound site be quantified via nuclear staining?

Methods:

•  Animals were collected from Sippewisset Marsh, Falmouth MA •  Maintained in aquaria at 25.2° C, 18ppt salinity, and fed Artemia or mussel tissue 5x/week.

Pilot Study: •  Pilot studies to test if weighing the affected animal vitality •  10 amputated, regenerating individuals were placed in vials with FSW •  Animals were measured once a day for 7 days •  10 non-amputated individuals were placed in vials with FSW •  Animals were measured once a day for 5 days •  One-Way ANOVAs test was then composed using Stats-Plus statistics software.

Regeneration Study:

Effects of Chronic Exposure to 17β-Estradiol on Regenerating Nematostella vectensis

Olivia D’Alleva and Justin S. McAlister Department of Biology, College of the Holy Cross

Results:

Conclusions: •  Estradiol did not negatively affect the regeneration rate of Nematostella vectensis under chronic exposure to concentrations up to 100ng/mL. •  Potential explanation could be concentration levels -- greater than 100ng/mL should be examined. •  Changes may be at the molecular level, thus unquantifiable by these morphological measurements. •  No evidence of tumor-like cellular-proliferation. •  Further assessments include a custom designed cellular staining protocol to explore cellular proliferation at the wound site under chronic concentration treatments.

Acknowledgements: We would like to thank Prof. Robert Bertin, Elizabeth Tobey, Erin Emmons, Asmani Adhav, Caroline Fleming, CJ Aprea and Margaret Mirabella for their work in the lab.

Figure 1: N. vectensis body plan. Figure 4: Laboratory set up. Yellow and blue are FSW and DMSO controls, green are estradiol treatments.

Figure 3: N.vectensis regeneation DMSO control vs. 100ng/mL treatment at 0,48 and 96 hpa

Figure 5: Weights of Nematostella vectensis FSW treatments to assess impact of weighing protocol over time.

Figure 6: Averages (+1SE) metrics of Nematostella vectensis under chronic exposure treatments for regenerating versus non-regenerating individuals. 6A weights of regenerating N.vectensis. One-way ANOVA comparison tests showed no significant differences between all treatments. 6B weights of un-amputated N.vectensis under treatment concentrations. Average area (6C) and length (6D) of regenerating N.vectensis. One-way ANOVAs found no significant difference across treatment for these metrics over the 96 hour period.

Figure 2: Estradiol chemical structure.

A B

C D

•  Morphological analysis of results. •  Weights of non-regenerating individuals are not significantly different across treatments. •  No significant difference in body weight, length, or area, of regenerating individuals. •  One-Way ANOVA test with proportioned ratios showed that tested concentrations did not affect

regeneration of the wound site.

Figure 7: Cellular staining protocol trials we developed to assess cellular proliferation at a molecular level. From left to right, 24hpa cellular staining with Hoechst stain emitted under 498 nm. White arrow shows regenerating oral end 24hpa.

Control 0hpa

Control 48hpa

Control 96hpa

100ng/mL 0hpa

100ng/mL 48hpa

100ng/mL 96hpa

-3

-2

-1

0

1

2

3

4

5

6

7

8

FSW DMSO 0.1ng/mL 1.0ng/mL 10.0 ng/mL 100.0 ng/mL

Wet

Wei

ght

(mg)

Individuals

Average Weights of Chronic Exposure 17β-estradiol on Regenerating N.vectensis

0

46

96

0

5

10

15

20

25

30

35

40

FSW DMSO 0.1 ng/mL 1.0 ng/mL 10.0 ng/mL 100.0 ng/mL

Wet

Wei

ght

(mg)

Treatment

Average Weights of Chronic Exposure 17β-estradiol on Un-Amputated N.vectensis

0

46

96

1

1.5

2

2.5

3

3.5

4

FSW DMSO 0.1 ng/mL 1.0 ng/mL 10.0 ng/mL 100.0 ng/mL

Leng

th (m

m)

Treatment

Average Length of Chronic Exposure 17β-estradiol on Regenerating N.vectensis

0

48

96

0

0.5

1

1.5

2

2.5

3

FSW DMSO 0.1 ng/mL 1.0 ng/mL 10.0 ng/mL 100.0 ng/mL

Are

a (m

m2 )

Treatments

Average Body-Area of Chronic Exposure 17β-estradiol on Regenerating N.vectensis

0

48

96

•  24 adult N. vectensis were amputated horizontally across the midline, under oral structures.

•  17β-Estradiol was dissolved in dimethyl sulfoxide (DMSO). •  Animals were exposed to one of six chronic exposure treatments: Filtered Sea

Water (FSW) control, DMSO control, 0.1, 1.0, 10, or 100 ng estrsdiol/mL. •  All treatments and controls were replicated four times for both amputated and

uninjured N. vectensis. •  Measures of weight, length and area were taken at 0, 48, and 96 hours post

amputation (hpa) •  Differences in weight, length, and area were tested using One-Way ANOVAs

using SPSS

y = -0.9893x + 9 R² = 0.89095

0

2

4

6

8

10

12

24 hrs 48 hrs 72 hrs 96 hrs 120 hrs 144 hrs 168 hrs

Wet

Wei

ght (

mg)

Time Passed Post Amputation (hrs)

Regenerating individual wet weight over 7 Days time

1

2

3

4

5

6

7

8

9

10

Linear (1)