This study developed the brook stickleback as a novel bioindicator species for assessing exposure to endocrine disrupting compounds in freshwater environments in Canada. Sex-specific genes were differentially expressed between male and female brook stickleback. Exposure to model androgens and estrogens induced the expected responses, with androgens inducing spiggin expression in females and estrogens inducing vitellogenin expression in males and increasing liver size. Transcript levels proved to be more sensitive biomarkers of hormone exposure than somatic indices. The brook stickleback showed potential for use in both laboratory and field studies of endocrine disrupting compound exposure.

1. Created by Peter Downing – Educational Media Access and Production © 2011
The assessment of EDCs in aquatic environments in Canada is limited due to a lack of
quantifiable and reliable biomarkers to assess (anti-)androgens in currently used fish
model species. The three-spined stickleback has been used in coastal Canada and in
the UK to assess the effects of (anti-)androgens using a unique, quantifiable biomarker
called spiggin1,2. However, the three-spined stickleback habitat is limited to coastal
environments. On the other hand, the brook stickleback possess all of the same
attributes as the three-spined stickleback including the production of spiggin and they
are ubiquitous in freshwaters throughout interior Canada. Therefore, they hold
potentialasabioindicatorspeciesforEDCinfreshwaterenvironmentsinCanada.
1.KatsiadakiI.etal.2012.AquaticToxicology122-123:75-85
2.WartmanC.A.etal.2009.AquaticToxicology92:131-139
.
INTRODUCTION
1. Develop a bioassay to measure androgenic and estrogenic exposure in
brook stickleback.
2. Determine the sensitivity and responsiveness of bioassays and biomarkers
during in vivo exposure to a model androgen and estrogen.
OBJECTIVES
MATERIALS and METHODS RESULTS and CONCLUSIONS
ACKNOWLEDGEMENTS
Assistance from M. Gallant, S. Hanson, K. Steeves, J. Marshall, A. Wang, T. Gebreyohannes, J. Wang, M. Kautzman. Graduate stipend
to B. Muldoon provided by the U of S New Faculty Graduate Student Support Program and an NSERC CGS-M.
Developing the brook stickleback (Culaea inconstans) as a novel bioindicator of
exposure to androgenic and estrogenic compounds in aquatic environments
2. Collection of wild
brook stickleback
Cranberry Flats, SK
Spring/Summer/Fall
2013-14
Mature brook stickleback:
8 females + 8 males sacrificed
Kidney and liver collected
3. Differential expression of sex-specific genes
1. Primer design and optimization
Assess relative transcript levels of
vitellogenin and spiggin in males versus
females using qPCR
4. Hormone Exposures
Treatments: 17α-methyltestosterone (MT)
17α-ethinylestradiol (EE2)
Exposure: 21 days (sampled on 7 + 21 days)
Concentration: 0, 1, 10 & 100 ng/L
Endpoints: somatic indices, spiggin/vitellogenin
mRNA transcript levels , histology (not shown)
RESULTS
Breda Muldoon1 and Natacha S. Hogan1,2
1Toxicology Center, University of Saskatchewan, Saskatoon, SK, Canada, 2Animal and Poultry Science, University of Saskatchewan, Saskatoon, SK, Canada
1. Sex Differences in Basal Gene Expression
2. MT Exposure – Spiggin Expression
(A) Vitellogenin (B) Spiggin
3. EE2 Trial Results – Somatic Indices
3. EE2 Trial Results – Vitellogenin Expression
(A) 7-day (B) 21-day
• We have established a method to measure transcript levels of vitellogenin
and spiggin and these transcripts are differentially expressed in females
and males.
• MT exposure had no effect on somatic indices but did induce spiggin
expression after both 7 and 21 days in females.
• EE2 exposure resulted in increased HSI in females after 7 days and in both
males and females after 21 days as well as induced vitellogenin expression
after 7 and 21 days in males.
• Transcript levels are a more sensitive indicator of hormone exposure
compared to somatic indices as they respond earlier.
• Brook stickleback hold excellent potential as a bioindicator species for
exposure to EDCs in both lab and field studies.
3-spine characterized sequence
Degenerate primers
Sequence Vtg and Spg
Design real-time primers
Optimize (melt curve & standard curve)
Figure 1: Fold change in basal (A) vitellogenin and (B) spiggin transcripts in male and
female brook stickleback. Asterisk (*) indicates significant differences between the sexes.
Figure 2: Induction of spiggin transcript levels in females exposed to MT for (A) 7 and (B) 21
days. Data are expressed as fold change relative to control (0 ng/L). Asterisk (*) indicates
significant difference from control. Basal spiggin in males is shown for comparison.
Figure 3: Hepatosomatic index (HSI) for males and females following (A) 7 and (B) 21 days of
exposure to EE2. Asterisk (*) indicates significant difference from control (0 ng/L).
Figure 4: Induction of vitellogenin transcript levels in males exposed to EE2 for (A) 7 and (B) 21
days. Data are expressed as fold change relative to control (0 ng/L). Asterisk (*) indicates
significant difference from control. Basal vitellogenin in females is shown for comparison.
Scan this QR code with
your smartphone to
view a 3 minute
presentation of this
poster
What is spiggin?
• Glycoprotein produced in the kidney and
secretedintheurinarybladder.
• Produced in response to increases in circulating
endogenous androgens (solely in the males) to
buildneststohouseeggsandfry.
• Production can be induced in females with
exposuretoexogenousandrogens.
(B) 21-day(A) 7-day (A) 7-day (B) 21-day
Sticklebacknest