Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro

Accepted Manuscript Title: Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro Author: Ashley Booth Enmin Zou PII: DOI: Reference:

S0166-445X(16)30174-6 http://dx.doi.org/doi:10.1016/j.aquatox.2016.06.011 AQTOX 4418

To appear in:

Aquatic Toxicology

Received date: Revised date: Accepted date:

20-5-2016 12-6-2016 13-6-2016

Please cite this article as: Booth, Ashley, Zou, Enmin, Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab, Callinectes sapidus, in vitro.Aquatic Toxicology http://dx.doi.org/10.1016/j.aquatox.2016.06.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Impact of molt-disrupting BDE-47 on epidermal ecdysteroid signaling in the blue crab,

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Callinectes sapidus, in vitro

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Ashley Booth a and Enmin Zou a*

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a

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*

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Enmin Zou

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Department of Biological Sciences

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Nicholls State University

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Thibodaux, LA 70310

Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310, USA Corresponding author:

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USA

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Email address: [email protected] (E. Zou)

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Phone: 985-448-4711

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Highlights:

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1. A partial sequence of NAG cDNA from Callinectes sapidus epidermis was acquired.

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2. An epidermis-with-exoskeleton (EWE) tissue culture method was described. 3. The expression of epidermal NAG gene was inducible in vitro by 20-hydroxyecdysone. 4. BDE-47 upregulated NAG mRNA in the cultured epidermis. 5. The mechanism for disruption of crustacean molting by BDE-47 was discussed.

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ABSTRACT Polybrominated diphenyl ethers (PBDEs) are environmentally pervasive flame retardants

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that have been linked with endocrine disruption in a variety of organisms. BDE-47, one of the most

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prevalent congeners found in aquatic environments, has recently been shown to inhibit crustacean

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molting, but little is known about the specific mechanism through which molt-inhibition occurs.

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This study examined whether the inhibitory effect on molting arises from the disruption of hormone

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signaling in the epidermis using the blue crab, Callinectes sapidus, as the model crustacean. First,

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we partially sequenced cDNA of N-acetyl-β-glucosaminidase (NAG) from the epidermis, a terminal

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enzyme in the molting hormone-signaling cascades that is commonly used as the biomarker for

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ecdysteroid signaling. This partial cDNA sequence was then used to create primers for

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quantification of NAG gene expression. Then, a new tissue culture technique was developed and

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dubbed the epidermis-with-exoskeleton (EWE) method, wherein epidermal tissue, along with the

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overlying exoskeleton, is immersed in a medium of physiologically relevant osmolarity. Using this

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EWE tissue culture method, we assessed the inducibility of NAG mRNA by 20-hydroxyecdysone

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(20-HE) in vitro. Exposures to 1 µM 20-HE were found to induce NAG mRNA at a significantly

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higher level than the control. Using NAG expression as a biomarker for ecdysteroid signaling, the

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effects of BDE-47 were measured. BDE-47 alone at 100 nM and a combination of 1 µM BDE-47

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and 1 µM 20-HE were found to significantly increase NAG mRNA. A trend of increasing NAG

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gene expression in the binary BDE-47 exposure as compared to 1 µM BDE-47 and 1 µM 20-HE is

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suggestive of a synergistic effect of these two chemicals on ecdysteroid signaling in the cultured

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epidermis. Discussion on the mechanism for inhibition of crustacean molting by BDE-47 is

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presented.

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KEYWORDS: Molting, PBDE, Endocrine disruption, Tissue culture, N-Acetyl-β-glucosaminidase,

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Callinectes sapidus

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1. INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are commercially produced flame retardants that

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are ubiquitous in the environment, despite being banned in many developed countries.

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Commercially produced mixtures of PBDE congeners were historically used on textiles, furniture,

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electrical equipment and plastics, but the chemical nature of PBDEs is such that they do not

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covalently bind with the item to which they are applied and can easily leach into the environment

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(Rahman et al., 2001). Theoretically, 209 PBDE congeners exist, varying based on the number of

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bromines surrounding two phenyl rings linked by an ether bond. All 209 congeners are hydrophobic

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and many are known to bioaccumulate, particularly in adipose and liver tissues (de Wit, 2002). Of

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these 209 congeners, BDE-47 is amongst the most prevalent in biotic samples (Shaw et al., 2009;

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Wollenberger et al., 2005). Levels of BDE-47 have been measured at concentrations ranging from

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16 to 4110 ng/g lipid weight in a variety of crustacean species worldwide (Boon et al., 2002; La

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Guardia et al., 2007; Verslycke et al., 2005; Yu et al., 2009).

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Previous research in vertebrates has shown the ability of BDE-47 to cause hormonal

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alterations linked to developmental and neurobehavioral disorders (Costa et al., 2015; Darnerud et

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al., 2001). In zebrafish, sublethal doses of BDE-47 altered mRNA expression of several thyroid

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receptor, thyroid hormone, and hormone transport genes (Chan and Chan, 2012). Hallgren and

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Darnerud (2002) demonstrated decreased levels of thyroxin and increased thyroid sizes following

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exposure of rats to BDE-47, and numerous studies have established a relationship between exposure

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to BDE-47 and an increase in neural deficits in both rodents and humans (Costa et al., 2015;

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Eriksson et al., 2001; Herbstman et al., 2010; Ta et al., 2011). While the endocrine-disrupting

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effects of BDE-47 have been well investigated in vertebrates, little attention has been attracted to

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the possibility that this environmentally persistent chemical can also interfere with hormonally

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regulated processes in invertebrates. Davies and Zou (2012) examined the effects of several

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prevalent PBDEs on molting in the cladoceran, Daphnia magna, and identified BDE-47 as one of

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the congeners capable of delaying crustacean molting. However, the mechanisms for BDE-47’s

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inhibition of molting remain unknown.

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The molting process in decapod crustaceans is regulated by hormones from the X-organ-

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sinus gland (XOSG) complex and Y-organ. A molting cycle consists of five primary stages, denoted

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by the letters A, B, C, D, and E. During the post-molt (A, B) and intermolt (C) stages, the XOSG

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produces high levels of molt-inhibiting hormone (MIH) that prevents the occurrence of precocious

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ecdysis (stage E). The beginning of the pre-molt stage (D) is marked by a decrease in MIH and a

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responsive, increased production of ecdysteroids, or molting hormones (MH), in the Y-organ

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(Chang and Mykles, 2011; Lee et al., 1998). Ecdysteroids produced by the Y-organ are

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hydroxylated and proceed to interact with an ecdysteroid receptor (EcR), which dimerizes with the

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crustacean retinoid X receptor (RXR). This dimer then binds to the ecdysteroid response element,

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resulting in the expression of enzymes responsible for exoskeleton degradation (Zou, 2010, 2005).

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Terminal enzymes in the molting process, such as chitinolytic enzyme N-acetyl-β-glucosaminidase

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(NAG) (also known as chitobiase), act on the exoskeleton and allow apolysis and, ultimately,

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ecdysis to occur (Zou, 2005). Because of the inducibility of epidermal NAG activity in vivo (Zou

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and Fingerman, 1999) and epidermal NAG mRNA in vitro (Meng and Zou, 2009b) by 20-HE, the

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level of expression of NAG gene can be used as a biomarker to demonstrate the impact on molting

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hormone signaling.

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Xenobiotics may disrupt the molting process by interfering with ecdysteroid disposition or

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ecdysteroid signaling in the epidermis (Zou, 2005). Either of these modes of action could negatively

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affect molting. Gismondi and Thome (2014) recently evaluated the effects of BDEs-47 and -99 on

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the chitinolytic enzymes of Gammarus pulex. This study was the first to examine the

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bioaccumulation of PBDEs and their mechanism of interference on crustacean molting. The work of

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Gismondi and Thome (2014) provided insight into the general effects of PBDEs on chitinolytic

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enzymes, but the use of whole-body homogenate for enzymatic assays and the presence of

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chitinolytic enzymes in the hepatopancreas led to a failure to distinguish whether PBDEs targeted

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enzymatic activity in the epidermis alone. Therefore, clarification is needed to determine whether

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the inhibitory effect on molting involves the disruption of ecdysteroid signaling in the epidermis.

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This study seeks to evaluate the effects of BDE-47 on epidermal ecdysteroid signaling in vitro as

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reflected by epidermal NAG gene expression, thereby illustrating a mechanism for crustacean

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molting disruption.

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To achieve the above goal, we acquired a partial sequence of NAG cDNA from the

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epidermis of the blue crab, Callinectes sapidus, developed a novel epidermal tissue culture

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technique termed the epidermis-with-exoskeleton (EWE) method, verified the inducibility of NAG

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mRNA by an ecdysteroid in vitro, and examined the effect of BDE-47 on NAG gene expression in

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cultured epidermal tissues. The reason for using the blue crab as the model crustacean is two-fold.

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Firstly, this particular organism is widespread throughout the Gulf Coast region and requires

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relatively minor effort to obtain in the field and maintain in the laboratory. Secondly, Callinectes

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sapidus is large in size compared to other model crustaceans like Daphnia magna, and the fiddler

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crab, Uca pugilator. The large size of Callinectes sapidus allows for the collection of multiple

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epidermal tissue sections from a single organism for both control and treatment exposures, thus

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eliminating differences in individual sensitivity in an in vitro exposure experiment.

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2. MATERIALS AND METHODS

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2.1 NAG cDNA sequence acquisition

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Blue crabs were collected from Pointe aux Chenes Wildlife Management Area (Pointe-aux-

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Chenes, LA) and Bayou Lafourche (Thibodaux, LA) using hoop nets. Tissue samples were

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collected from individual crabs the same day as capture when possible or were maintained in

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aerated artificial seawater with a salinity of 10-15 ppt until the time of tissue sample collection.

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Epidermal tissues were collected from beneath the carapace of a Callinectes sapidus

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specimen. Epidermal tissues were homogenized and total RNA was extracted and purified using

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Qiagen’s Total RNA Purification, DNase Digestion, RNA Cleanup, and reverse transcription

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protocols. RNA concentration was calculated using the formula 44 µg/mL x Abs260nm x dilution

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factor of 10. Sample quality was evaluated based on the ration of absorbance at 260/280 nm. A

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sample with an absorbance ration of greater than or equal to 1.7 was considered of sufficient quality

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for further use. A pair of NAG gene primers (forward primer: 5’-

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CACTGGCACATCACCGACTCC-3’; reverse primer: 5’-GTAGGGGCTGCACCAGTTGTT-3’),

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designed on the basis of the conservative regions of crustacean NAG cDNA (Meng and Zou,

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2009a), were used to acquire a partial sequence of NAG cDNA in Callinectes sapidus. The PCR

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reaction was performed immediately following sample collection using the protocols of Qiagen’s

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Taq PCR Master Mix, and the resultant PCR product was resolved on a 2% agarose gel. After a

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PCR product of roughly 730 bp was obtained (Fig. 1), the resultant DNA band was excised from the

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2% agarose gel. The DNA sample was purified using Promega’s Wizard SV Gel and PCR Clean-

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Up System prior to submission for commercial sequencing by the ACGT Inc. (Wheeling, Illinois).

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Upon receiving the partial cDNA sequence from ACGT Inc., the provided forward sequence

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was input into NCBI’s Basic Local Alignment Search Tool (BLAST) to evaluate the similarity of

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the elucidated NAG cDNA sequence with previously identified NAG cDNA sequences in related

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organisms. The NCBI’s open reading frame finder (ORF) was then used to deduce the amino acid

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sequence produced by the target gene. This amino acid sequence was processed using BLAST to

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elucidate the similarity of amino acid sequences produced by the Callinectes sapidus NAG gene and

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that of phylogenetically similar organisms. Clustal W was then used to identify regions of similarity

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in NAG amino acid sequences between Callinectes sapidus and five other related organisms.

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2.2 Epidermis-with-exoskeleton (EWE) tissue culture method

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A tissue culture medium of HEPES, Grace’s insect medium, 10% Fetal Bovine Serum and

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100 unit/mL penicillin, 0.10 mg/mL streptomycin, and 0.2 mg/mL neomycin was mixed and stored

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at -20 °C. Sodium bicarbonate and sodium chloride were used to adjust tissue culture medium to a

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pH of 7 and an osmolarity of 666 mOsm/L (Osmette II, Precision Systems, Inc.), similar to the

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hemolymph osmolarity of blue crabs acclimated to 10-15 ppt seawater. Blue crabs were collected

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from Pointe-aux-Chenes or Cocodrie, LA and maintained in the laboratory for an acclimation

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period of at least 24 hours. Crabs were molt-staged and juveniles in D1 or D2 (Mangum, 1985) were

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selected for use in this portion of the experiment. The carapace was cleaned with 70% ethanol and

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separated from the body of the crab. All hepatopancreatic tissue was removed, and the EWE

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sections were excised, taking care to ensure that epidermal tissues remained attached to the

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carapace. The EWE sections were then randomly placed in individual wells of a 6-well cell culture

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plate along with 4 mL of tissue culture medium for in vitro testing. This tissue culture technique,

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which uses epidermal tissue and the overlying exoskeleton, is termed the epidermis-with-

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exoskeleton method.

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With the EWE method, epidermal cells are surrounded by an environment more closely

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resembling the natural setting than when detached, naked epidermal tissues are immersed in a

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culture medium. Following use of the EWE method, NAG mRNA expression was easily

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measurable using qPCR, validating the use of this technique as a method for exposing tissues prior

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to evaluation of gene expression. Additionally, the use of a large-sized crustacean, such as the blue

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crab, allows for the use of multiple EWE tissue sections from a single crab and allowed for the

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elimination of intra-specific variations.

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2.3 Induction of epidermal NAG mRNA by 20-HE

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Five EWE sections were excised from each of three juvenile crabs. For each specimen, one

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section served as a control, exposed to 0.1% v/v ethanol, and the remaining sections from the same

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specimen were exposed to physiologically relevant concentrations of 1 nM, 10 nM, 100 nM, and 1

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µM 20-HE (Lee et al., 1998), respectively. Tissues were allowed to remain in the tissue culture

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medium for one hour. After incubation, tissues were harvested and immediately frozen at -80˚ C

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until total RNA extraction and NAG mRNA quantification using real-time PCR (qPCR) could be

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performed.

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2.4 Quantification of NAG mRNA

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RNA samples were gathered by removing epidermal tissue from each EWE section of a

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Callinectes sapidus specimen. This tissue was homogenized, then purified using Qiagen’s Total

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RNA Purification, DNA Digestion, and RNA Cleanup protocols. RNA concentration was calculated

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as described above. Any remaining genomic DNA was eliminated by following Qiagen’s reverse

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transcription PCR protocols through the genomic DNA elimination reaction and subsequent

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incubation. Following this step, two RNA samples were prepared. One was prepared as prescribed

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per the reverse-transcription PCR protocol, and one without the addition of the Quantiscript Reverse

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Transcriptase (QRT) enzyme. Both samples were then processed according to the final steps of

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Qiagen’s reverse-transcription PCR protocol. Average total RNA used in reverse-transcription was

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3.8 ng/µL, with a total of 12 µL RNA used in each reverse-transcription sample. The resultant PCR

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products were then evaluated for expression of NAG gene using Qiagen’s qPCR protocol and

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Applied Biosystems’ StepOne Real-Time PCR system. NAG primers were designed from the

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previously elucidated NAG cDNA sequence using Primer Express 1.0 (Applied BioSystems, Inc.),

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and bracketed an amplicon of 80 bp from bp 588-667 on Callinectes sapidus NAG cDNA and

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amino acid 204-235 as visualized on a 5’3’ protein motif (Figs. 1 and 2) (forward primer, 5’-

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AGGCGTCGTCATTGCTGAAG-3’; reverse primer, 5’CTGGAGCACGCAGGATGAC-3’). The

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β-actin gene (Accession number: DQ084066.1, GenBank) was used as the internal control and

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primers for this gene bracketed an amplicon of 75 bp (forward primer, 5’-

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GCAGAGCAAGCGTGGTATCC-3’, reverse primer, 5’-TCCATGTCGTCCCAGTTGGT-3’).

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Amplification efficiencies for both the target gene and internal control were found to be around 2.0.

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Thermocycler conditions included denaturing at 50 ºC for 2 minutes then at 95 ºC for 15 minutes,

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followed by 40 cycles of amplification (94 ºC for 15 s, 53 ºC for 30 s, and 72 ºC for 30 s), and a

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melt curve stage of 95 ºC for 15 s, 60 ºC for 60 s, 95 ºC for 15 s, and 40 ºC for 15 s. Two qPCR

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replicates were performed for each tissue sample, using 2.5 µL template cDNA.

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The real-time PCR protocol was considered successful if the difference in CT value between

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positive QRT and negative QRT samples was greater than or equal to seven. For each crab, NAG

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expression levels were calculated for each treatment and expressed as relative abundance as

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compared to the control. This calculation was performed using the 2 -ΔΔCT method (Livak and

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Schmittgen, 2001). The concentration of 20-HE that caused the greatest NAG mRNA induction was

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used as the positive control for further experimentation in order to verify the responsiveness of

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cultured epidermal tissues to this molting hormone.

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2.5 Exposure to BDE-47

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BDE-47, purity 100%, was purchased from AccuStandard, Inc. (New Haven, CT). Five

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juvenile blue crabs were used for treatment with BDE-47. Tissues and tissue culture medium were

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prepared according to the EWE method. Five EWE sections were excised from each specimen. One

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section served as a negative control (exposed to 0.1% v/v ethanol) and another as a positive control

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(treated with 1 µM 20-HE). Two other sections were treated with either 100 nM or 1 µM PBDE,

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and the remaining section was exposed to a combination of 1 µM 20-HE and 1 µM PBDE. The

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treatment exposed to 1 µM 20-HE and 1 µM PBDE, or binary exposure, was designed to represent a

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more realistic response to PBDE exposure, as may occur in vivo. Tissues were harvested after one

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hour, immediately frozen at -80˚C until NAG mRNA could be quantified using qPCR as described

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above.

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2.6 Statistical analysis

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Levene’s test for homogeneity of variances was run and data was arctangent-transformed to

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account for unequal variance as needed. Then one-way ANOVA and LSD post hoc test were used

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to evaluate any differences between treatment and control tissues. A probability value less than 0.05

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was considered significant.

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3. RESULTS

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3.1 Partial sequence of NAG cDNA

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Sequencing of the reverse-transcription PCR product from the Callinectes sapidus epidermis

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revealed a 731 bp DNA sequence (Fig. 1) consistent with the size of NAG genes previously

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identified in Uca pugilator (Meng and Zou, 2009a). Using BLAST, this DNA sequence was found

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to have a 77% similarity with NAG cDNA in Uca pugilator (Meng and Zou, 2009a), 72% similarity

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with Portunus trituberculatus (GenBank accession no. KF914668.1), 70% similarity with

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Litopenaeus vannamei (GenBank accession no.FJ888482.1), 70% similarity with Fenneropenaeus

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chinensis (GenBank accession no. DQ280379.1), and 69% similarity with Cherax quadricarinatus

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(GenBank accession no. KP968829.1).

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The NAG amino acid sequence for Callinectes sapidus was deduced using ORF and

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evaluated by BLAST. The sequence in Callinectes sapidus was found to have 78% identity with the

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NAG amino acid sequence in Uca pugilator, 70% identity with Portunus trituberculatus, 69%

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identity with Cherax quadricarinatus, and 66% and 65% identity with Litopenaeus vannemei and

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Fenneropenaeus chinensis, respectively. The highest level of identity amongst all available

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sequenced NAG amino acids was with Uca pugilator. Clustal W (Thompson et al., 1994) revealed

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the alignment and conserved areas of NAG amino acid sequences in Callinectes sapidus and five

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other arthropods, Uca pugilator, Portunus trituberculatus, Litopenaeus vannamei, Fenneropenaeus

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chinensis, and Cherax quadricarinatus (Fig. 2).

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3.2 In vitro NAG mRNA induction by 20-HE

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The EWE sections from each of three juvenile crabs were exposed to varying levels of 20-

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HE to elicit NAG gene expression in vitro and create a dose-response relationship. Mean NAG

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mRNA induction was greatest in tissues treated with 1 µM 20-HE, and mean values for subsequent

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treatments decreased with decreasing 20-HE concentrations (Fig. 3). Treatments with 1 µM 20-HE

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were found to be significantly different from the control (p = 0.011). Therefore, 1 µM 20-HE was

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used as the positive control for subsequent tissue exposures.

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3.3 Effect of BDE-47 on NAG mRNA in vitro

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The EWE sections from each of five juvenile crabs were exposed to a negative control (0.1% v/v ethanol), two different concentrations of BDE-47 (100 nM and 1 µM), a binary exposure

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to BDE-47 and 20-HE, and a positive control (1 µM 20-HE). Mean NAG mRNA induction

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increased for all treatments, and exposures of 100 nM BDE-47 (p = 0.007, Fig. 4), binary exposure

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(p = 0.001), and the positive control (p = 0.036) were all significantly different from the negative

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control. Relative abundance of NAG mRNA exhibited a trend of greater expression for the binary

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exposure as compared to the positive control (p = 0.092). The binary exposure also showed a trend

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of increased NAG mRNA expression as compared to treatment with 1 µM BDE-47 (p = 0.061).

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4. DISCUSSION

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The partial cDNA sequence acquired from Callinectes sapidus epidermal tissue using

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reverse transcription, Taq PCR, and DNA gel electrophoresis was of a consistent size with the NAG

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gene previously identified in Uca pugilator (Meng and Zou, 2009a). Variations in similarity

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amongst NAG gene and NAG amino acid sequences can potentially be explained by the existence of

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paralogous NAG genes in these decapod crustaceans or the evolutionary divergence of these

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orthologs over time (Koonin, 2005). However, the high level of similarity between Callinectes

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sapidus epidermal NAG cDNA and that of phylogenetically similar organisms is evidence that the

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acquired PCR product was indeed a partial cDNA sequence of the NAG gene of Callinectes sapidus

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(Figs. 1 and 2).

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The lack of an ecdysteroid-responsive crustacean cell line has hampered the advancement of

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crustacean molting hormone endocrinology, as well as endocrine toxicology. Using an in vitro

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screen based on the ecdysteroid-responsive Drosophila melanogaster BII-cell line, Wollenberger et

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al. (2005) observed neither an agonistic nor an antagonistic activity of BDE-47. The negative result

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of this study on the effects of BDE-47 is a testimony to the importance of using a crustacean cell-

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based in vitro assay system. The EWE method, an efficient and effective technique for exposing

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epidermal tissues to exogenous substances while maintaining tissue integrity and viability, provides

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a means for future studies concerning molting hormone signaling in the epidermis.

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Following exposure of epidermal tissues to 20-HE, Callinectes sapidus tissues exhibited a

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dose-response relationship of NAG mRNA expression to 20-HE concentration. With increasing 20-

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HE concentrations, relative abundance of NAG mRNA also increased, and treatment with 1 µM 20-

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HE was significantly different than the control (p = 0.011) (Fig. 3). This dose-response relationship

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and the significant difference of exposures to 1 µM 20-HE indicate that NAG gene expression can

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be induced by the molting hormone 20-HE. The location of NAG as a terminal enzyme in the

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molting hormone signaling cascade and its verified inducibility by 20-HE validate the use of NAG

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gene expression as a biomarker for epidermal ecdysteroid signaling.

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Following exposure of epidermal tissues to BDE-47, NAG mRNA for the positive control

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was found to be significantly different from the negative control (p = 0.036, Fig. 4), suggesting that

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cultured epidermal tissues were responsive to 20-HE. It is important to note that all five EWE tissue

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samples for each experimental replicate were acquired from the same individual. Variations

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amongst relative abundance for individual crabs (Figs. 3 and 4) may have varied due to

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physiological differences, such as molt stage. However, all exposed individuals experienced an

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increase in NAG expression as compared to the negative control, demonstrating the ability of these

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organisms to effectively express NAG mRNA. The binary exposure, which mimics the hormonal

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milieu of specimens impacted by PBDEs, also showed a trend of increased NAG expression as

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compared with the positive control (p = 0.092) and as compared with exposures to the 1 µM BDE-

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47 treatment (p = 0.061). It appears that the presence of BDE-47 can enhance the 20-HE effect, and

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the amplitude of differences in expression between the binary exposure and the 1 µM 20-HE or 1

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µM BDE-47 treatments may indicate a synergistic interaction of these two chemicals on epidermal

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ecdysteroid signaling. BDE-47 at 100 nM was also found to significantly increase NAG mRNA.

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The difference in relative abundance between 100 nM and 1 µM BDE-47 exposures was not

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statistically significant (p = 0.334). Increased gene expression may result from direct interaction of

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BDE-47 with the EcR that leads to expression of chitinolytic enzymes. The increase in NAG gene

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expression following BDE-47 exposure could also stem from an increase in the number of receptors

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available, with which 20-HE can bind and thereby increase NAG gene expression (Lema et al.,

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2008; Zhang et al., 2012). Additionally, non-genomic mechanisms underlying BDE-47-induced

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upregulation of NAG gene expression in the epidermal cells cannot be excluded. The elucidation of

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how BDE-47 triggers the expression of NAG gene in the epidermis awaits future investigations.

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Davies and Zou (2012) examined the effects of several PBDE congeners on molting in

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Daphnia magna. Notably, they found that specimens exposed to 20 µg/L BDE-47 experienced

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greater than 50% mortality. Daphnids that survived during this exposure demonstrated delayed

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molting and a significantly decreased number of molts when compared to daphnids exposed to the

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control. This study demonstrated an inhibitory effect of BDE-47 on molting in Daphnia magna, but

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did not elucidate a mechanism by which this inhibition occurred. However, in view of the

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observation that high mortality rate was not always accompanied by inhibition of molting in

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Daphnia magna as is the case with BDE-100, Davies and Zou (2012) noted that BDE-47-induced

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molt inhibition, even mortality, may be tied to its endocrine toxicity. In contrast to the work of

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Davies and Zou (2012), Gismondi and Thome (2014), without examining the effect on molting

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frequency, documented both stimulating and inhibitory effects of BDE-47 on NAG activity

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measured from the whole body homogenate in the amphipod, Gammarus pulex. BDE-47 effects

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varied according to sex, length of exposure, and BDE-47 concentration. The discrepancy in findings

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between these two studies may cloud understanding of the effects of BDE-47 on crustacean

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molting. However, a careful examination may illuminate this apparent discrepancy in effects. NAG

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activity, assayed from the whole body homogenate, was significantly inhibited following a 4-day

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exposure of male amphipods to BDE-47 at 0.1 and 1 µg/L, while 0.1 µg/L, but not 1 µg/L, BDE-47

333

increased enzymatic activity in females (Gismondi and Thome 2014). Since the effect in males

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exhibits a clear concentration-response relationship, the inhibitory effect of BDE-47 on NAG

335

activity must be genuine, whereas stimulating effect on NAG activity in female could be accidental

336

because of the lack of a concentration-response relationship and the small margin of increase (1.2-

337

fold). Therefore, the inhibitory effect of BDE-47 on NAG activity could be considered consistent

338

with BDE-47-induced inhibition of molting in daphnids.

339

In the present study, BDE-47 showed a tendency of promoting molting hormone signaling

340

in the cultured epidermal tissues. Such a result is seemingly not in agreement with BDE-47’s

341

inhibitory effect on molting and NAG activity. Perhaps, BDE-47’s effect on NAG gene expression

342

is biphasic, i.e. initial stimulation followed by inhibition, which would account for the molt-

343

inhibitory effect of this PBDE congener. It is also likely that BDE-47 renders its molt-inhibitory

344

effect primarily through suppressing ecdysteroid titers in the hemolymph. Whether a longer

345

exposure time can result in an inhibitory effect on epidermal ecdysteroid signaling in vitro and

346

whether BDE-47 can impact ecdysteroid titers in the hemolymph in vivo need to be investigated.

347

The results from these future investigations would give a more definitive delineation of the

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mechanism for BDE-induced inhibition of crustacean molting.

349

5. CONCLUSIONS

350

BDE-47 is known to be capable of inhibiting crustacean molting. The goal of the present

351

investigation was to understand whether this inhibition involves disruption of molting hormone

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signaling in epidermal cells. To that end, we acquired a partial sequence of NAG cDNA from

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epidermal tissues of Callinectes sapidus, developed a novel epidermal tissue culture technique

354

called the EWE method, verified the inducibility of NAG mRNA by 20-HE to validate its use as a

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biomarker for ecdysteroid signaling, and examined the effect of BDE-47 on NAG mRNA in

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cultured epidermal tissues.

357

The validation of the use of NAG as a biomarker in this and other studies (Meng and Zou

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2009a, Gismondi and Thome 2014) and creation of the EWE method have far-reaching implications

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for future research on crustaceans. Identification and publication of a Callinectes sapidus partial

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NAG cDNA sequence will facilitate further investigation into the effects of xenobiotics on the

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endocrinology of these well-known crustaceans. The cost- and time-efficient EWE method will

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enable more effective assessment of the effects of xenobiotics on crustaceans, as this method can be

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extrapolated for use on epidermal tissues from other organisms within this group.

364

Using the EWE method to expose epidermal tissues to BDE-47, and qPCR to evaluate NAG

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gene expression, we found that exposure at 100 nM alone and to a combination of 1 µM BDE-47

366

and 1 µM 20-HE significantly upregulated NAG gene expression. Additionally, there appears to be

367

a synergistic interaction between BDE-47 and 20-HE. While this up-regulation of NAG mRNA

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expression appears to be in conflict with findings of previous studies that demonstrated inhibition of

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molting by BDE-47 (Davies and Zou 2012, Gismondi and Thome 2014), we propose that exposure

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to BDE-47 may initially upregulate NAG mRNA expression then subsequently suppress this

371

response, or that BDE-47 may interfere with ecdysteroid disposition. Possible mechanisms

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underpinning the upregulation of NAG mRNA in the epidermis and the inhibition of crustacean

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molting by BDE-47 are complex in nature and require further investigation to elucidate a clearer

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mechanism for molt disruption. However, it is clear that BDE-47 interferes with expression of NAG

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mRNA, thereby influencing the molting process in our crustacean model. The finding that BDE-47

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may adversely affect biological processes in Callinectes sapidus, similar results from other studies

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on crustaceans exposed to BDE-47, and the global ubiquity of this BDE congener support the need

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for further research and a closer investigation into the safety and continued use of these

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environmentally pervasive flame retardants.

380

Acknowledgements

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This study was sponsored by the National Oceanic and Atmospheric Administration through

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the Institute of Seafood Studies.

383

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Figure captions

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Figure 1. Gel electrophoresis results with DNA bands and partial NAG cDNA sequence from

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Callinectes sapidus epidermal tissue. Bolded letters indicate the 80 bp region amplified by NAG

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qPCR primers, highlighted areas indicate the location of forward and reverse primers.

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Figure 2. Multiple sequence alignment of deduced amino acid sequence of NAG mRNA from

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Callinectes sapidus as compared with NAG gene products in Portunus trituberculatus (GenBank

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accession no. AHJ81101.1), Cherax quadricarinatus (GenBank accession no. ALC79577.1),

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Litopenaeus vannamei (GenBank accession no. ACR23316.1), Fenneropenaeus chinensis

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(GenBank accession no. ABB86961.1), and Uca pugilator. Highlighted areas indicate highly

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conserved regions. Protein motif of 5’3’ open reading frame for acquired partial NAG cDNA and

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region amplified by NAG forward and reverse primers during qPCR.

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Figure 3. Relative abundance of NAG mRNA for each treatment with varying concentrations of 20-

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hydroxyecdysone. Epidermal tissues attached to exoskeleton were incubated in tissue culture

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medium and respective hormone treatment for one hour prior to analysis using qPCR. N = 3. Error

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bars indicate standard error.

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Figure 4. Effect of BDE-47 on NAG mRNA level in cultured epidermal tissues from Callinectes

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sapidus. Epidermal tissues attached to exoskeleton were incubated in tissue culture medium and

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respective treatment chemical for one hour prior to analysis using qPCR. N = 5. Error bars indicate

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standard error.

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489 490 491 492 493 494 495 496 497 498

Fig. 1

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500 501 502 503 504

Fig. 2

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Fig. 3

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510 511 512 513

Fig. 4