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Evidence for intraspecific endocrine disruption of Geukensia demissa
(Atlantic ribbed mussel) in an urban watershed
Zachery M. Halem, Dustin J. Ross, Rachel L. Cox
⁎
The Lisman Research Laboratories at Riverdale Country School, Bronx, NY, USA
abstractarticle info
Article history:
Received 15 October 2013
Received in revised form 28 April 2014
Accepted 30 April 2014
Available online 9 May 2014
Keywords:
COI gene
Dissolved oxygen
DNA barcoding
Endocrine disruption
Mussel
Populations undergo physiologica l adaptations in response to environm ental stressors. Our 5-year bio-
monitoring study of the Bronx River Estuary demonstrates comparatively low dissolved oxygen concentrations
in this urbanized watershed. Additionally, our current results establish altered hormonal levels, resulting from
endocrine disruption, in Geukensia demissa (Atlantic ribbed mussel) from the Bronx River Estuary. No studies
have yet investigated a correlation between low dissolved oxygen and endocrine disruption in field-collected
bivalves. Testosterone, estradiol, and progesterone levels were collected from male and female mussels in the
oxygen depleted Bronx River and well-oxygenated Greenwich Cove. Bronx Riv er mussels exhibited higher
testosterone levels and lower estradiol levels than Greenwich Cove mussels. The resulting abnormal hormonal
ratio seems to indicate that environmental conditions in the Bronx River facilitate an allosteric inhibition of the
cytochrome P450 aromatase enzyme, which aids conversion of testosterone to estradiol. Low progesterone levels
suggest that Bronx River mussels are experiencing a delay in sexual maturation, and morphometric data show a
stalling of shell and tissue growth. To confirm that the mussels collected from both sites are the same species, the
universal mitochondrial cytochrome c oxidase subunit I gene was analyzed, through DNA barcoding. Minimal
sequential heterogeneity confirmed the mussels are the same species. Such findings suggest intraspecific
divergence in various endocrine processes, resulting from environmentally induced stress.
© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
1. Introduction
Endocrine disruption is hazardous to the vitality of any marine pop-
ulation. Numerous recent studies identify mechanisms and effects of
endocrine disruption in invertebrates. Vertebrate steroid hormones
(testosterone, estradiol, and progesterone) that are synthesized from
cholesterol are found in mollusks, and evidence shows that such sex ste-
roids can serve as ideal biomarkers of endocrine disruption (Gust et al.,
2010). While the specific function of steroid hormones in mollusks' en-
docrine system is still speculative, reports emphasize steroids' influence
on gender differentiation, gametogenesis, gonadal maturation, fertiliza-
tion an d embryonic development, and reproduction (Mori, 1969;
Reis-Henriques and Coimbra, 1990; Matsumoto et al., 1997; Wang and
Croll, 2006; Ketata et al., 2008). Specifically, testosterone and estradiol
concentrations in the gonads vary during different stages in the repro-
duction process, largel y affecting gender determination and gamete
growth (Matsumoto et al., 1997; Gauthier-Clerc et al., 2006). Progester-
one also influences sex specific processes, such as gametogenesis and
gonadal development, and has been s hown to potentially impact
spawn ing in both sexes (Reis-Henriques and Coimbra, 1990; Wang
and Croll, 2006).
Endocrine disrupting compounds are frequently found in surface
water contamination, emanating from the sewage depositing of indus-
trial fa cilities (Gomes and Lester, 2003; Gültekin and Ince, 2007a,
2007b). Specific analysis of wastewater content through a hepatocyte
assay has shown industrially impacted water to possess estrogenic
activity (Islinger et al., 1999; Gagné and Blaise, 2000). Studies have
demonstrated that natural estrogens, such as 17β-estradiol, estriol and
estrone, ubiquitous in certain sewage effluents can induce a feminiza-
tion of fish (Björkblom et al., 2007). Certain heavy metals and chemicals
stimulate endocrine disruption in mollusks. Tributyltin (TBT) acts as a
neuro toxin and increases APGWamide, a neurotransmitter peptide,
which re leases the neurohormone Penis Morphogenic Factor (PMF)
(Oberdörster and McClellan-Green, 2000; Oberdörster et al., 2005;
Ketata et al., 2008). PMF generates male characteristi cs in both male
and female mollusks. However, no study has yet explored low dissolved
oxygen levels as an inducer of endocrine disruption in mollusks, or any
invertebrate.
Dissolved oxygen is vital for development, reproduction, and life in
nearly all aquatic organisms. The concentration of dissolved oxygen in
any given body of water is dependent on a multitude of factors, includ-
ing atmospheric reaeration, biochemical oxygen demand (BOD), the
Comparative Biochemistry and Physiology, Part A 175 (2014) 1–6
⁎ Corresponding author at: Riverdale Country School, 5250 Fieldston Road, Bronx, NY
10538, USA. Tel.: +1 718 549 8810x730.
E-mail address: rcox@riverdale.edu (R.L. Cox).
http://dx.doi.org/10.1016/j.cbpa.2014.04.016
1095-6433/© 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Contents lists available at ScienceDirect
Comparative Biochemistry and Physiology, Part A
journal homepage: www.elsevier.com/locate/cbpa
organismal rate of cellular respiration, and the rate of photosynthesis
(Dobbins, 1965; Edwards and Owens, 1965; Bennett and Rathburn,
1972; Cox, 2003). Low dissolved oxygen results as a consequence of
processes that consume oxygen at a greater rate than processes that
produce oxygen (Rowe, 2001). In addition to natural regulators of
dissolved oxygen, eutrophication can artificially reduce water's oxygen
content through runoff or sewage depositing. When dissolved oxygen
levels fall bel ow 2–3 mg/L, the water is identifi ed as hypoxic, and
most organisms will struggle for survival (Diaz, 2001). Water that is
borderline hypoxic remains detrimental to organisms' physiological
processes (Diaz, 2001).
Urbanization and human industrial activity have a severe impact on
water quality and the health of estuarine ecosystems (Limburg et al.,
2005; Astaraie-Imani et al., 2012; Chin et al., 2013). Since industrializa-
tion, the Bronx River estuary, the only river to run through the city of
New York, remains a site of substantial sewage contamination (Wang
and Pant, 2010). The construction of the Bronx River Parkway in 1909
was the first major disruption to the surrounding natural features.
This paralleling parkway reduced forest cover and contributed to poor
bank stabilization as urbanization grew dense (Rachlin et al, 2007).
The United States Environmental Protection Agency (EPA) labels “path-
ogens” as a major concern in the Bronx River, and speculates that CSOs
(combined sewer overflow) are the likely source of such affliction
(Crimmens, T., 2002. Bronx River Restoration: Report and Assessment).
The New York City sewage system is designed to overflow into nearby
rivers when waste is overly profuse. To date, the EPA has documented
31 “causes of impairment” in the Bronx River estuary, including patho-
genic contamination and oxygen depletion. The EPA has yet to indicate a
single “cause of impairment” for wildlife in the Greenwich Cove Estuary.
Despite various conservation efforts, our studies suggest that organ-
isms in the Bronx River are stressed, likely from persisting adverse envi-
ronmental conditions. Our research shows that the well-documented
stress responding protein, heat shock protein 70 (HSP70) displays
elevated baseline expression in Bronx River versus Greenwich Co ve
Geukensia demissa mussels as well as Spartina alterniflora marsh grass
(Hans et al., 2013; Magun et al., 2013). In addition, our work indicates
that Bronx River mussels serve as a valuable in situ model to study the
potential synergistic effect of multiple active stress responders includ-
ing acetylcholinesterase and heat sh ock pr oteins 70 and 90 (Magun
et al., 2013). Most significantly, our laboratory continues to document
lower dissolved oxygen levels in the Bronx Ri ver when compared to
Greenwich Cove (Shah et al., 2012).
To establish validity for our in situ study of comparative hormone
levels, we genetically confirmed the presence of Geukensia demissa
species in both sites by analyzing nucleotide divergence in cytochrome
c oxidase subunit I (COI) gene of the mitochondrial DNA, through DNA
barcoding. Since the 1990s, several systems have emerged for the iden-
tification of species (Sugita et al., 1998; Vincent et al., 2000; Floyd et al.,
2002). DNA barcoding emerged as a promising approach to make spe-
cies identification more quantitative for insects, and morphologically
challenging marine invertebrates, such as mollusks (Undheim et al.,
2010; Keskin and Atar, 2011). Confirmation that the populations from
Bronx River and Greenwich Cove are of the same species enables us to
infer that divergences in endocrine ratios are responses to the environ-
ment and not due to interspecific differences.
The current study demonstrates a correlation between comparative-
ly low dissolved oxygen levels in the Bronx River and endocrine
disruption in mussels from th at site. Further studies are required in
order to determine the mechanism responsible for this alteration. One
possibility is that low dissolved oxygen c oncentrations inhibit the
cytochrome P450 aromatase mediated conversion of testosterone to
estra diol, leading to a higher testosterone/es tradiol ratio in Bronx
River Geukensia demissa (Matthiessen and Gibbs, 1998; Alzieu, 2000;
Morcillo and Porte, 2000).
Another possibility is that energy limitations mediated by low dis-
solved oxygen and other factors such as food limitation delay growth
rate and maturation which could lead to altered levels of testosterone,
estradiol, and progesterone (Franz, 1996; Petes et al., 2007).
2. Materials and methods
2.1. Water analyses
Dissolved oxygen measurements were obtained using the Winkler
titration method. Oxygen was fixed at each site using manganous
sulfate, alkaline potassium iodide azide, and sulfamic acid. Sodium thio-
sulfate was used to titrate the water sample with starch indicator to a
clear endpoint. pH measurements were collected using a Flinn Scientific
pH Meter (AP8673).
2.2. Sample collection and dissection
Geukensia demissa specimens were collected at low tide from Harding
Lagoon in the Bronx River (40° 48′ 35.563″ N, 73° 51′ 40.893″ W), and
Todd's Point in Greenwich Cove (41° 0′ 31.296″ N, 73° 34′ 18.042″ W).
Our collections were performed during the months of June and July
when Geukensia demissa ranging in size from 25 to 36 mm (width) are
uniformly undergoing gametogenesis (Franz, 1996). Mussels were
transported to the laboratory in buckets with water collected from the
site. All samples, maintained in native water, were incubated at 4 °C
and allowed 24 h to equilibrate. The length, width, and total weight of
each mussel were recorded before dissection of tissues. Upon dissection,
shell weight and combined gill weight were also recorded. Both gills and
mantles were extracted from each organism, and stored at − 20 °C.
2.3. Sex determination
Two tests were used to determine the sex of the mussels. Upon
dissection, the color of the mussels' mantle was noted. In Geukensia
demissa, a brown pigment signi
fies a female, and a yellowish cre am
color is characteristic of males (Puglisi, 2008). For confirmation, the
mussel's gender was determined by the protocol of Jabbar and Davies
(1987) with slight mo dificati on. One mantle f rom each mussel was
placed in a Petri dish with 1 mL of 0.75% (w/v) thiobarbituric acid.
Males developed a yellow pigment, while females developed a pink
pigment. A 100% correlation was found between the results of the two
gender assays.
2.4. COI gene analysis
Genetic material was isolated from a 1 by 1 mm mantle sample from
12 organisms from each site (24 total) using the QIAGEN DNeasy Blood
and Tissue Kit (cat# 6904) and material was stored at − 20 °C. PCR was
used to amplify the COI gene using Geukensia demissa specific primers:
5′-CCGCGAATTAATAATTTCAGATTT-3′/5′-ACCAAAAAATCAAAATAAAT
GCAT-3′. Primers were designed using GenBank Sequence
FJ693154
and synth esized by Sigma Genesis Inc. in a 5 μM concentration.
Twenty-five microliters of PCR reactions, composed of 2 μL template
DNA and 11.5 μL of each primer in GE illustra PuReTaq Ready-To-Go
PCR Beads [cat# 27-9559-01], were subsequently prepared. Reactions
were run for 50 cycles with 30 s denaturation step at 94 °C, 45 s anneal-
ing step at 54 °C, and 45 s extending step at 72 °C using Techne Genius
Therm o Cycler. PCR products from the 24 individua ls produced
amplicons of approximately 440 bp as visualized on a 2% agarose gel
using pBR322/BstNI molecular weight standard from New England
Biolabs [cat #N2021L]. All products, along with Geukensia demissa cus-
tom primers, were sent to Genewiz I nc. for sequencing. Bronx River
and Greenwich Cove sequences were aligned by Nucleotide BLAS T,
and percent similarity was ascertained using the CLUSTAL W alignment
tool (Thompson et al., 1994; Ni et al., 2012).
2 Z.M. Halem et al. / Comparative Biochemistry and Physiology, Part A 175 (2014) 1–6
2.5. Testosterone, estradiol, and progesterone enzyme immunoassay (EIA)
Per each assay, one gill from each mussel was homogenized in
1.2 mL ice-cold 50:50 water:methanol. Homogenates were extracted
three times with 5 mL of high purity diethyl ether for the testosterone
assay, or 5 mL of dichloromethane for the estradiol and progesterone
assay. At room temperature, organic extracts were eva porated to
dryness. EIA buffer (500 μL; Cayman Chemical, Ann Arbor, MI, USA)
was used to reconstitute each sample.
Steroid hormones were quantified using commercial testosterone, es-
tradiol, and progestero ne enzyme immunoassay kits (Cayman Chemical,
cat#582701, 582251, 582601). The assay is based on the competitive
immunoreactions between the free hormone and steroid linked acetyl-
cholinesterase (AChE) conjugate for a finite amount of antiserum. EIAs
were performed in 96-well plate format, with each plate being read by
a Fisher Scientific* Multiskan* FC Microplate Reader (ca t# 14-387-360).
Standards, provided in each kit, were diluted to form standard curves
and assayed in duplicate on each plate. Testosterone standard concentra-
tions range from 500 to 3.9 pg/mL, estradiol standard concentrations
range from 6.6 to 4000 pg/mL, and progesterone standard concentrations
range from 7.8 to 1000 pg/mL. Two blank, two non-specific binding, three
maximum binding, and one total activity well(s) were also run on each
plate. Samples were run in triplicate at three dilutions: 1:1, 1:2, and
1:10. The intra- and inter-assay coefficients of variation (CVs) were less
than 10%. Cross reactivity of the testosterone antiserum with various ste-
roids is as follows: testosterone 100%, 5α-d ihydrot estoste rone 27.4%, 5β-
dihydrotestosterone 18.9%, methyltestosterone 4.7%, androstenedione
3.7%, 11-keto testosterone 2.2%, and lessthan1%forallothersteroidstest-
ed (Cayman Chemicals). Cross reactivity of the estradiol antiserum with
various steroids is as follows: estradiol 100%, estradiol-3-sulfate 14.5%,
estradiol-3-glucuronide 14%, estrone 12%, estradiol-17 glucuronide 10%,
and less than 0.5% for all other steroids tested (Cayman Chemicals).
Cross reactivity of the progesterone antiserum with various steroids is
as follows: progesterone 100%, 17β-estradiol 7.2%, 5β-Pregnan-3α-ol-
20-one 6.7%, pregnenolone 2.5%, and less than 1% for all other steroids
tested (Cayman Chemicals).
2.6. Statistical analyses
P-values for water data, morphometric data, and steroid hormone
concentrations were determined through a t-test. Any p-value less
than or equal to 0.05 is considered statistically significant.
3. Results
3.1. Water analyses
Bronx River water has consistently possessed lower dissolved
oxygen concentrations than Greenwic h Cove over our 5-year study.
In the past 2 years, our show data has shown decreasing diss olved
oxygen concentrations in the Bronx River as a trend. Difference be-
tween the temperature and pH of the water at the two sites was mini-
mal (Table 1).
3.2. Morphometric data
Bronx River mussels were consistently of smaller size (length/width)
and lower weight than Greenwich Cove mussels over our 5-year study.
Average combined gill weight was also significantly lower in Bronx
River mussels.
3.3. COI gene analysis
Among Bronx River mussels, CLUSTAL W alignment revealed negli-
gible heterogeneity (0–1%) in COI sequence. CLUSTAL W alignment
also showed insignificant (0– 1%) divergence in COI sequence within
the Greenwich Cove population. When compared across sites, however,
alignment disclosed a consistent 2% sequential heterogeneity. Organism
gender appeared to have no impact on COI gene sequence.
3.4. Tissue steroid levels
Steroid levels were determined in gill tissue. The same mussel test
cohort was used for the testosterone and estradiol assays, but different
individuals were used for progesterone assay. Testosterone concentra-
tions were significantly higher (p b 0.05) in Bronx River mussels rela-
tive to Greenwich Cove mussels (2.1 fold in males, 3.6 fold in females)
(Fig. 1A). In both sites, male mussels contained higher levels of testos-
terone than female mussels. The Bronx River mussels also possessed
Table 1
Average levels of dissolved oxygen levels (mg/L), water temperature (°C), and pH, over a
5-year survey of the Bronx River and Greenwich Cove.
Site Dissolved oxygen (mg/L) Temperature (°C) pH
Bronx River 3.9 ± 0.2 22.35 ± 0.24 7.29 ± 0.02
Greenwich Cove 10.0 ± 0.7 25.32 ± 0.94 7.97 ± 0.07
All water collections (3 each year) occurred in June and July, around low tide. Data are
presented as mean ± SEM (n = 15). p b 0.0001 for dissolved oxygen concentrations.
Male
Female
Male Female
Male Female
0
50
100
150
200
Bronx River Greenwich Cove
pg/ml testosterone
A
0
20
40
60
80
100
0
20
40
60
80
100
120
Bronx River Greenwich Cove
pg/ml estradiol
B
Bronx River Greenwich Cove
pg/ml progesterone
C
Fig. 1. Gill steroid hormone concentrations (pg/mL) for (A) testosterone, (B) estradi-
ol, and (C) progesterone. Male and female distinctions ar e presented. Data are shown
as mean ± SEM (n =16,8males,8females).p = 0.00885 for testosterone, p =
0.04175 for estradiol, and p = 0.0001 for progesterone.
3Z.M. Halem et al. / Comparative Biochemistry and Physiology, Part A 175 (2014) 1–6
lower estradiol concentrations than Greenwich Cove mussels (Fig. 1B).
Female mussels had higher tiss ue concentrations of estradiol than
male mussels at the Bronx River (57.6 ± 1.6 pg/mL versus 14.1 ±
0.8 pg/mL) and Greenwich Cove (86.4 ± 3.1 pg/mL versus 25.9 ±
2.5 pg/mL). Thus, the testosterone/estradiol ratio (combining concen-
trations in male and female mussels) is significantly higher in Bronx
River mussels (3.60) than Greenwich Cove mussels (0.93). Progesterone
levels were not influenced by gender, but varied between the two sites.
The concentration was higher in gill tissue of Greenwich Cove mussels
than Bronx River mussels (6.6- and 4.3-fold for male and female,
respectively) (Fig. 1C).
4. Discussion
Results of our comparative steroid a nalysis suggest that mussels
living in an urbanized and environmentally impacted watershed are
experiencing endocrine disruption, indicated by the relative concentra-
tions of all three endogenous steroids (testo sterone, estradiol, and
progesterone). The Bronx River mussels have significantly higher tes-
tosterone levels (2.1 fold in males, 3.6 fold in females) and significantly
lower estradiol concentrations (1.8 fold in males, 1.5 fold in females)
(Fig. 1A). The average ratio of testosterone/estradiol (pg/mL) is 3.60 in
Bronx River mussels, juxtaposed to the normal 0.93 in Greenwich
Cove mussels.
Our findings concur with Friesen et al. (2012) and other recent stud-
ies that correlate low dissolved oxygen with endocrine disruption in fish
species (Wu et al., 2003; Landry et al., 2007). The pathway for such an
alteration was identified as a hypoxia mediated inhibit ion of cyto-
chrome P450 aromatase activity (Alzieu, 2000; Matthiessen and Gibbs,
1998; Morcillo and Porte, 2000; Shang et al., 2006). This mechanism
may explain our observed alterations of the testosterone/estradiol
ratio in mussels from the oxygen depleted Bronx River. Such an inhibi-
tion could lead to a general masculini zation of both male and female
mussels, though more research is required.
Evidence also shows that low dissolved oxygen can impact growth
and development, RNA/DNA ratio, gonad and embryo formation,
fitness, and survival rates in fish species (Zhou et al., 2001; Gercken
et al., 2006; Hassell et al., 2008; Kolding et al., 2008; Wang et al.,
2008). These findings correlate well with our 5-year morphometric
studies demonstrating that Geukensia demissa from the hypoxic Bronx
River site have a consistently lower body mass index than ribbed
mussels from Greenwich Cove, CT.
In addition, studies show that heavy metals, specifically tributyltin
(TBT) inhibit cytochrome P450 aromatase, thus blocking the conversion
of testosterone to estradiol, and affecting the testosterone/estradiol ratio
(Matthiessen and Gibbs, 1998; Alzieu, 2000; Morcillo and Porte, 2000).
Other substances known to induce endocrine disruption and the
conversion of testosterone to estradiol in marine inv ertebrates include:
herbicides (diquat dibromide, atrazine, simazine, diuron), metals (cadmi-
um, selenium, zinc, mercury, lead), PCBs, alkylphenols (nonylphenol,
pentylphenol), and insecticides (DDT, ednrin, toxaphene, piperonyl
butoxide, methoprene) (Depledge and Billinghurst , 1999).
Endocrine disruption is also indicated by the significantly low level
of progesterone in the gill tissue of Bronx River mussels averaging
18.6 ± 1.3 pg/mL, as compared to an average of 98.6 ± 1.9 pg/mL in
Greenwich Cove mussels. Conditions in the Bronx River could potential-
ly alter energy demands of the mussels, forcing them to distribute the
limited available oxygen towards aerobic metabolic processes, rather
than the synthesis of progesterone. Our conclusion that progesterone
levels are not gender mediated supports the hypothesis of Reis-
Henriques and Coimbra (1990), which indicates that male and female
mussels require a similar pattern and amount of progesterone through-
out their reproductive cycle. We expect the progesterone deficiency to
have other physiological effects. During the maturation of oocytes in
M. edulis, an increase in ly sosomal enzyme activity is usually noted
(Peek and Gabott, 1990). In correlation, rising progesterone levels in
M. edulis have been shown to result in the destabilization of lysosomes
(Moore et al., 1978). Thus, studies suggest progesterone as a biomarker
of sexual maturation, in regard to oocyte progression (Siah et al., 2003).
Moreover, st udies show that progesterone levels peak during active
gametogenesis, when the male's gonads are maturing and females are
in the spawning stage (Reis-Henriques and Coimbra, 1990; Siah et al.,
2002). Due to a lack of progesterone, the Bronx River mussels may be
undergoing a deceleration of the rate of sexual maturation and oocyte
development. In addition, progesterone has shown to act as a gonad
messenger for neurohor mones (Siah et al., 2002). The Bronx River
mussels may be experiencing a de lay in germ cell proliferation, and
our results lead us to hypothesize that the lack of progesterone has
affected the production of neurohormones. To confirm this hypothesis,
further studies will be required to examine neurohormone-receptor
responses to oxygen depletion.
COI gene analysis revealed only slight heterogeneity for organisms
from the same site (0–1% Bronx, 0–1% Greenwich), thus indicating
that mussel populations at both sites contain only one species. The 2%
COI sequen ce divergence when comparing mussels across sites indi-
cates that mussels at both sites ar e of the same sp ecies (Geukensia
demissa). This supports the claims of Hebert et al. (2003) that for two
populations, intraspeci fic COI sequence divergence is approximately
2% (Avise, 2000; Cognato, 200 6). The consistent 2% heterogeneity in
COI sequence across sites indicates adaptive pressure, indicating the
possibility of allopatric speciation in the future.
As mentioned above, morphometric analysis demonstrates a
delayed shell growth and tissue growth in Bronx River mussels
(Table 2). The growth rate of mussels tends to vary by season, higher
in spring and summer, and lower in winter (Page and Hubbard, 1987;
Garen et al., 2004). In addition, shell and tissue growth are generally af-
filiated with the reproductive cycle of the given population (Bayne and
Worrall, 1980; Handå et al., 2011). However, in mussels, shell growth is
not directly correlated to tissue growth (Hilbish, 1986; Kautsky, 1982;
Rodhouse et al., 1984). Environmental stress and food limitations are
known to impede growth processes. The 31 causes of impairment (as
documented by the EPA) or the limitation of dissolved oxygen in the
Bronx River may require that mussels conserve energy for vital survival
processes, which would delay shell and tissue growth. A recent study
transplanted Mytilus galloprovincialis and Perna c analiculus to high-
Table 2
Average body mass, condition index (CI) total ((wet weight / total weight) × 100), CI shell ((wet weight / shell weight) × 100), and combined gill weight in Geukensia demissa mussels
taken over a 5-year span from the Bronx River and Greenwich Cove (Shah et al., 2012).
Bronx River Greenwich Cove
2009 2010 2011 2012 2013 2009 2010 2011 2012 2013
Length/Width (mm) 66.6/28.4 60.0/24.0 62.0/27.3 ND 72.8/30.9 77.0/31.1 85.0/31.0 81.1/32.6 ND 86.2/40.2
Average body mass (g) 17.5 18.16 20.39 30.1 37.80 21.8 35.88 36.03 39.7 46.76
Conditionindextotal 44414838424144514358
Condition index shell 84 72 89 63 79 74 80 106 76 89
Average combined gill mass (g) 0.87 1.08 0.80 ND 0.97 1.33 1.30 1.13 ND 1.21
ND = no data available.
p = 0.0099 for length, and p = 0.0150 for average combined gill mass.
4 Z.M. Halem et al. / Comparative Biochemistry and Physiology, Part A 175 (2014) 1–6
stress and low-stress elevation edges of an intertidal mussel bed to com-
pare growth and reproduction (Petes et al., 2007). Mussels living in the
high-stress edge exhibited reduced growth rate and a smaller tissue
mass (Petes et al., 2007). Additional studies demonstrate endogenous
steroid levels as regulators of growth. In the fresh water mussel, Elliptio
complanata (Gagné et al., 2001) demonstrated that high estrogen levels
promote total and soft tissue weights. The lack of estradiol in Bronx
River mussels may have stunted tissue growth, accounting for the re-
duced average total body weight and average combined gill weight
(Table 2).
5. Conclusion
Overall, this study is the first to demonstrate a correlation between
low dissolved oxygen emanating from an urbanized site and disruption
in endogenous steroid levels, physiological development, and sexual
maturation in Geukensia demissa.
Acknowledgments
We wish to acknowledge the Marjot Foundation and Riverdale
Country School for generous support of our research. We thank Eli
Sands and all of the researchers at the Lisman Research Laboratories
for their invaluable assistance. We wish to express additional gratitude
towards Kelley Nicholson-Flynn for editorial assistance and Kevin Bailey
for assistance with statistical analyses.
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