Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys
CITATION SEARCH THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00103214/00001
 Material Information
Title: Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at Nearshore Sites in the Florida Keys
Series Title: PLoS One
Physical Description: Archival
Language: English
Creator: Spade, Daniel J.
Griffitt, Robert J.
Liu, Li
Kroll, Kevin J.
Feswick, April
Barber, David S.
Denslow, Nancy D.
Publisher: Public Library of Science
Publication Date: 2010
 Subjects
Genre:
Spatial Coverage:
 Notes
Funding: Publication of this article was funded in part by the University of Florida Open-Access publishing Fund. In addition, requestors receiving funding through the UFOAP project are expected to submit a post-review, final draft of the article to UF's institutional repository, IR@UF, (www.uflib.ufl.edu/ufir) at the time of funding. The Institutional Repository at the University of Florida (IR@UF) is the digital archive for the intellectual output of the University of Florida community, with research, news, outreach and educational materials
 Record Information
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: 10.1371/journal.pone.0012737
System ID: UF00103214:00001

Full Text






Queen Conch (Strombus gigas) Testis Regresses during

the Reproductive Season at Nearshore Sites in the

Florida Keys

Daniel J. Spade', Robert J. Griffitt'", Li Liu Nancy J. Brown-Peterson', Kevin J. Krolll, April Feswick',
Robert A. Glazev, David S. Barber', Nancy D. Denslow'*
1 Department of Physiological Sciences and Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida, United States of America,
21Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, Florida, United States of America, 3 Department of Coastal Sciences, University of
Southern Mississippi, Ocean Springs, Mississippi, United States of America, 4Fish and Wildlife Research Institute, Florida Fish and Wildlife Conservation Commission,
Marathon, Florida, United States of America


Abstract

Background: Queen conch (Strombus gigas) reproduction is inhibited in nearshore areas of the Florida Keys, relative to the
offshore environment where conchs reproduce successfully. Nearshore reproductive failure is possibly a result of exposure
to environmental factors, including heavy metals, which are likely to accumulate close to shore. Metals such as Cu and Zn
are detrimental to reproduction in many mollusks.

Methodology/Principal Findings: Histology shows gonadal atrophy in nearshore conchs as compared to reproductively
healthy offshore conchs. In order to determine molecular mechanisms leading to tissue changes and reproductive failure, a
microarray was developed. A normalized cDNA library for queen conch was constructed and sequenced using the 454 Life
Sciences GS-FLX pyrosequencer, producing 27,723 assembled contigs and 7,740 annotated transcript sequences. The
resulting sequences were used to design the microarray. Microarray analysis of conch testis indicated differential regulation
of 255 genes (p<0.01) in nearshore conch, relative to offshore. Changes in expression for three of four transcripts of interest
were confirmed using real-time reverse transcription polymerase chain reaction. Gene Ontology enrichment analysis
indicated changes in biological processes: respiratory chain (GO:0015992), spermatogenesis (GO:0007283), small GTPase-
mediated signal transduction (GO:0007264), and others. Inductively coupled plasma-mass spectrometry analysis indicated
that Zn and possibly Cu were elevated in some nearshore conch tissues.

Conclusions/Significance: Congruence between testis histology and microarray data suggests that nearshore conch testes
regress during the reproductive season, while offshore conch testes develop normally. Possible mechanisms underlying the
testis regression observed in queen conch in the nearshore Florida Keys include a disruption of small GTPase (Ras)-mediated
signaling in testis development. Additionally, elevated tissue levels of Cu (34.77 ng/mg in testis) and Zn (831.85 ng/mg in
digestive gland, 83.96 ng/mg in testis) nearshore are similar to reported levels resulting in reproductive inhibition in other
gastropods, indicating that these metals possibly contribute to NS conch reproductive failure.

Citation: Spade DJ, Griffitt RJ, Liu L, Brown-Peterson NJ, Kroll K), et al. (2010) Queen Conch (Strombus gigas) Testis Regresses during the Reproductive Season at
Nearshore Sites in the Florida Keys. PLoS ONE 5(9): el2737. doi:10.1371/journal.pone.0012737
Editor: Laszlo Orban, Temasek Life Sciences Laboratory, Singapore
Received April 28, 2010; Accepted August 2, 2010; Published September 15, 2010
Copyright: @ 2010 Spade et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This project was funded by the following sources: The United States Environmental Protection Agency (www.epa.gov; #X7974799-03), Florida Fish and
Wildlife Conservation Commission (www.myfwc.com; #F2410 and #NGO6-106) and the University of Florida College of Veterinary Medicine (www.vetmed.ufl.
edu). Publication of this article was funded in part by the University of Florida Open-Access Publishing Fund. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
E-mail: ndenslow@ufl.edu
a Current address: Department of Coastal Sciences, University of Southern Mississippi, Ocean Springs, Mississippi, United States of America


Introduction

Queen conch (Strombus gigas) is a species of significant ecological
and economic importance -la.n1.....~~ its range. For example, the
estimated economic value of the annual conch fishery in the
Bahamas is approximately $4.457 million, representing 9,800
seasonal jobs [1]. The queen conch is also a large benthic
invertebrate associated with coral reef ecosystems, and therefore
could serve as an indicator species for toxic effects contributing to
the decline of the Florida coral reef ecosystem. As a result of the


a,' PLoS ONE |www.plosone.org


queen conch population decline in Florida, a complete morato-
rium on the Florida conch fishery was declared in 1986 [2,3]. The
queen conch was listed under the Convention on International
Trade in Endangered Species' (CITES) Appendix II in 1992 [4r].
However, recovery of adult conchs in spawning aggregations
within the Florida Keys has been modest. In 2001, the number of
adult conchs in offshore (OS) spawning aggregations was estimated
at 27,000, up from a lowest observed estimate of 5,750 in 1992,
according to transect data collected by the Florida Fish and
Wildlife Conservation Commission [3]. It is believed that little or


September 2010 | Volurne 5 | Issue 9 | el2737


PLoS one


OPEN ACCESS Freelyavilable online







Queen Conch Testis Regression


no reproduction occurs in nearshore (NS) aggregations, and that
this might contribute to the slow recovery of the population [2,3].
A study of conch reproduction found that NS conchs failed to
develop adequate gonad tissue for reproduction, but that
translocation of NS conchs to the OS environment resulted in
development of normal gonad tissue and reproductive activity
within three months [2]. However, the causes of reproductive
failure of NS conchs remain unknown.
Human impacts on coastal marine ecosystems are ever-increasing,
and threats include inputs of nutrients, organic contaminants, and
metals, as well as changes in temperature, decreasing ocean pH, and
deoxygenation [5]. While many of these factors can theoretically
affect reproduction, one plausible cause for reproductive failure in a
NS marine gastropod is heavy metal exposure. A number of
gastropod studies have related heavy metal exposure, in particular
exposure to Cu [6-8] and Zn [I. "* 111 to reduced fecundity _
reproductive output usually measured in terms of egg laying. Despite
the link between exposure to Cu and Zn and decreased reproductive
output in gastropods, past studies consider mostly female-mediated
effects at the individual level. In the Florida Keys, both male and
female reproductive development is inhibited NS [2,3]. Given that
heavy metals are known to inhibit gastropod egg laying, and that
general and point sources for metal contamination exist close to
shore in the Florida Keys [11-13], our general hypothesis is that
heavy metals are likely to contribute to the reproductive failure
observed NS. For the present study, our specific hypotheses were:

1. that testis transcriptional data would identify candidate gene
expression pathways affected by NS environmental stressors, and

81,52' W


2. that tissue concentrations of heavy metals in NS conchs would
exceed those of OS conchs.

For this study we developed and used a microarray to identify
gene expression differences between the testes of NS and OS
conchs. Gene expression data was anchored in 1.: .....,~11...1.._ to
provide a more complete understanding of the dysfunction in testis
development in NS conchs. Additionally, we used inductively
coupled plasma-mass spectrometry to quantify nine metals,
including Cu and Zn, in conch tissues and to determine whether
their concentrations correlate with histological and gene expres-
sion evidence of NS reproductive dysfunction.

Methods

Field collections
For this study, all conchs were collected in the Florida
Keys (Figure 1). Tissue samples used for queen conch cDNA
library construction were collected from Sombrero Reef on 9 June
2004 and from East Sister's Rock and Eastern Sambo on 15
March 2005. Tissue samples used for the comparison of
reproductive (OS) versus non-reproductive (NS) queen conchs by
histology, real-time reverse-transcription polymerase chamn reac-
tion (real-time RT-P'I 14 and metals analysis were collected from
Pelican Shoal (OS) and Tingler Island (NS) on 15 February 2007
and from Eastern Sambo (OS) and East Sisters' Rock (NS) on 7
June-9 June 2007. Microarray analysis was conducted using 15
February 2007 samples from Pelican Shoal and Tingler Island.
Sampling was conducted by free diving or SCUBA diving. Only


80o51'W


Figure 1. Nearshore (NS) and offshore (OS) queen conch sampling sites in the Florida Keys. Microarray construction: ESR, East Sister's
Rock (NS); ES, Eastern Sambo (OS); SR, Sombrero Reef (OS). Microarray and real-time reverse-transcription polymerase chain-reaction experiments:
PS, Pelican Shoal (OS); TI, Tingler Island (NS). Validation of 185 rRNA for real-time RT-PCR: PS; TI; ES; ESR; DS, Delta Shoal (OS). Inductively coupled
plasma-mass spectrometry and histology: PS; TI; ESR, ES.
doi:1 0.1 371/journal.pone.001 2737.g001


SPLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | el2737







Queen Conch Testis Regression


adult queen conchs, identified by a fully flared lip [4], were
collected.
In all cases, adult male conchs were collected live and tran-
sported immediately to the Florida Fish and Wildlife Conservation
Commission's Fish and Wildlife Research Institute (FWRI)
laboratory in Marathon, FL. Conchs were then euthanized and
tissues were harvested. For molecular assays and determination of
tissue metal burdens, gonad, digestive gland, neural ganglia, blood,
and foot muscle samples were frozen immediately in liquid
nitrogen. Frozen tissue samples were maintained at -80'C until
further analysis. For histology, a piece of testis tissue approxi-
mately 1 cm3 in size from the middle of each conch's testis was
placed in an individually labeled cassette and fixed in 10% neutral
buffered formalin for a minimum of 7 days and retained for
further processing.

Histopathology
Histological examinations of the testes of experimental conchs
were conducted as described in Delgado et al. [2]. Briefly, fixed
tissue samples were rinsed in running tap water overnight,
dehydrated in a graded series of ethanols, cleared, embedded in
Paraplast (Leica Microsystems, Wetzlar, Germany), sectioned at
4 Ipm and stained with hematoxylin and eosin following standard
histological techniques.
Testicular development was assessed histologically to determine
reproductive capability of conchs. Conchs were classified into
reproductive phases based on a maturity scale presented in Delgado
et al. [2] (Table 1). In order to quantify the amount of testicular
tissue present, a series of photomicrographs were taken to enable
visualization of all gonadal tissue, resulting in 6-20 photographs for
each specimen. Three photographs were randomly selected for each
specimen, and the area of each testicular lobule in these
photographs was determined using ImageJ software. A sperm scale
based on the presence of developing sperm in the testis
(spermatogonia = 1, spermatocytes = 2, spermatids = 3, sperma-
tozoa = 4, vas deferens with spermatozoa = 5) was recorded for
each lobule. A spermatogenic index (SI) was calculated following
1!o 11....1..1... similar to that reported in Kofoed et al. [14]l; the area
of each lobule (mm ) was multiplied by the sperm scale for that
lobule. A total SI was determined for each specimen by summing
the SI for each lobule in the three photographic views.




ITable 1. Reproductive phases used to describe testicular
development in queen conch.


Construction, sequencing, and annotation of a
normalized CDNA library
cDNA library construction was carried out following the
method of Garcia-Reyero et al. [15]. Briefly, total RNA was
isolated from 12 individual conch tissue samples (neural ganglia,
gonad, and digestive gland from each of four conchs, one female
and one male from NS and one female and one male from OS)
using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the
manufacturer's protocol. RNA quality and integrity were assessed
using the NanoDrop ND-1000 spectrophotometer (NanoDrop
Technologies, Wilmington, DE, USA) and Agilent 2100 Bioana-
lyzer (Agilent Technologies, Santa Clara, CA, USA), respectively.
200 ng of each RNA sample was pooled and used as the template
for the cDNA library construction and normalization using the
SMART (Clontech/Takara Bio USA, Madison, WI, USA) and
Trimmer .1. I.... .. Joint Stock Company, Moscow, Russia) kits,
respectively. The normalized queen conch cDNA library was
sequenced using a 454 GS-FLX pyrosequencer (Roche/454 Life
Sciences, Branford, CT, USA) [16], at the University of Florida
Interdisciplinary Center for Biotechnology Research (ICBR).
Sequencing was conducted as described in Garcia-Reyero et al.
[15]. Sequence assembly and annotation were performed as
described in Farmerie et al. [17] using Newbler v1.1.03.24 and
Paracel TranscriptAssembler. Minimum confidence for a match
was set at e<1 x10-4. Each sequence and its top 100 hits from
BLAST search were retained in ICBR's BlastQuest database [17]
and annotated with Gene Ontology (GO) information and with
human and zebrafish (Danio rerio) homologues by referring to the
NCBI Gene database. A queen conch oligonucleotide microarray
was designed in an 8 x15 k format consisting of 15,208 60-mer
user probes and 536 internal control probes using the eArray
service from Agilent. The microarray was submitted as a platform
dataset to NCBI's Gene Expression Omnibus (GEO) database
(GPL8934r).

Extraction of RNA from experimental samples
For microarray and cloning procedures, four biological replicate
conch RNA samples were isolated from testis of male conchs
collected in February, 2007, at Pelican Shoal (OS) and Tingler
Island (NS) (Figure 1). One array from each group was determined
to be of insufficient quality during the quality control process, and
so the final sample size for microarrays was three individuals from
each location, OS and NS. The OS individuals are considered to
be the reference group, as they exhibit successful sexual
reproduction, while NS conchs do not. Total RNA was extracted
using the RNA STAT-60 reagent (Tel-Test, Friendswood, TX,
USA), reconstituted in RNAsecure (Ambion), and DNase-treated
with Turbo DNA-free (Ambion). In all cases, manufacturers'
protocols were followed. RNA samples used for real-time RT-
PCR assays were extracted using a more rigorous procedure, in
which STAT-60 extraction of RNA was followed by gradient
centrifugation -la..n _1. a 1.4 mL layer of 5.7 M CsC1, 0.5 M
EDTA in the Beckman Optima-TLX ultracentrifuge and
TLAl20.2 rotor. RNA cleanup was performed using the Qiagen
RNEasy Mini Kit. RNA quantity was assessed on the NanoDrop
ND-1000 and quality was assessed using the Agilent 2100
Bioanalyzer. We have observed that un-degraded conch total
RNA samples show no 28S rRNA band; therefore, no RIN can
be calculated to quantify RNA integrity for these samples. This
is likely due to the "hidden break" in invertebrate 28S rRNA
that has been described by Ishikawa [18] and that is present
in gastropods including Haliotis rufescens [19]. Despite the impossi-
bility of calculating RIN, only high-quality samples for which


Reproductive Phase Description


Early Developing
Developing

Spawning Capable

Regressing

Atretic

Regenerating
No Tissue


Only spermatogonia and spermatocytes present
All stages of spermatogenesis present;
no spermatozoa in vas deferens
All stages of spermatogenesis present,
spermatozoa present in vas deferens
Empty lobules, resorption of spermatozoa.
Some spermatogenesis occurring
Lobules degenerating, resorption of spermatozoa'
no active spermatogenesis
Only spermatogonia present
No spermatogenesis occurring and no spermatogonia
present; this is an abnormal condition in adult males.


IModified from Delgado et al. [2].
Sdoi:1 0.1 371 /journal.pone.001 2737.t001


a, : PLoS ONE | www.plosone.org


September 2010 | Volurne 5 | Issue 9 | el2737







Queen Conch Testis Regression


Bioanalyzer profiles appeared to be un-degraded were used for
further analysis.


Microarray processing
RNA samples were labeled with Cy3-CTP and hybridized to
the microarray following the Agilent Protocol titled One Color
Microarray-Based Gene Expression Analysis v5.5 (publication
no. G4140-90040). Specific activity of Cy3 label was at least
10.04 pmol/pLL in each sample, and averaged 13.14 pmol/LL.
Microarray scanning and feature extraction was performed at
ICBR using an Agilent G2505B Microarray Scanner and Agilent
Feature Extraction Software v9.5. All microarray data here
reported are MIAME compliant; raw and normalized microarray
data have been submitted to the GEO database (GSE17 -.,
according to MIAME standards [20].


Cloning of 5. gigas 185 ribosomal RNA
3 pLg of each conch RNA sample was reverse transcribed to
produce cDNA using Invitrogen SuperScript II Reverse Tran-
scriptase and random primers, per the manufacturer's protocol.
18S rRNA was cloned using primers designed in the program
Primer [21] based on alignment of 18S rRNA from the gastropod
Bursa rana (X94269.1) and the bivalve ~Nucula sulcata (AF207642.1)
(Table 2). 18S rRNA primers were used in a PCR reaction with
Invitrogen Taq polymerase, according to the manufacturer's
protocol. PCR products were cloned in the pGEM-T Easy vector
. ....- \1.11.. I. St. Louis, MO, USA) and Invitrogen One-shot
Topl0 chemically competent E. coli cells, per the manufacturer's
protocols. The sequence of the cloned 18S rRNA fragment was
confirmed by Sanger sequencing at ICBR (GUl98749).

Real-Time RT-PCR
Copper transporter 10 (C 1.` thiolester-containing protein II
(TeplI), Similar to Glutathione S-transferase (GST), and Start
domain-containing protein 7 (Stard7) were evaluated by real-time
RT-PCR. Primers for transcripts of interest (Table 2) were
developed from 454-derived cDNA library sequences using
Primer. All primer sets were verified using the same cloning
and sequencing methods as in the section ( .. of S. gigas 18S
ribosomal RNA, above. The 638 bp clone of 18S rRNA was used to


Table 2. Primers for 18S rRNA cloning and for real-time RT-PCR.



Sequence Purpose Direction Primer Sequence (5'-3')


design a second set of primers that was more optimal for real-time
RT-PCR. For all other clones, cloning primers were also used for
real-time RT-PCR. Plasmids containing each cloned sequence
were used to create a standard curve ... I .. .~ of eight points in a
serial dilution from 1x102 -la..n_~1. 1xtu' copies/reaction. Real-
time RT-PCR was performed as a two step process. In step one, 2
Cpg DNase-treated RNA was reverse transcribed using Invitrogen's
SuperScript II reverse transcriptase and random primers. In step
two, real-time PCR reactions were run using SYBR Green
Supermix (Bio-Rad, Hercules, CA, USA), per the manufacturer's
protocol, on the Bio-Rad iCycler real-time PCR thermal cycler,
using a two-step protocol with an initial denaturation at 95'C,
followed by 40 cycles of denaturation at 95'C and annealing and
extension at 58'C, during which real-time quantification was
enabled. Following amplification, a dissociation curve was run
beginning at 55'C and increasing to 95'C at 0.5'C intervals every
10s. Standards and experimental samples were run in duplicate,
along with two negative controls for each gene: a "no reverse
transcriptase (-RT)" control, in which DNAse-treated RNA
samples were pooled and water was used in place of reverse
transcriptase during the reverse transcription reaction, and a "no
template control (NTC)," in which water was used in place of
template cDNA during the real-time PCR reaction.
The use of 18S rRNA as a reference gene for RT-PCR was
validated by measuring 18S rRNA by the method described above
for 23 conch testis samples, collected in February, 2007, June,
2007, and March, 2009 (Figure 1). Initial quantity for each sample
was calculated as 18S rRNA copy number/ng total RNA. Data
were analyzed inJMP v8 using a two-way ANOVA on the factors
"collection (date)" and "location (OS vs. NS)." This analysis
showed nearly identical mean 18S levels between OS and NS,
with no statistically significant difference according to ANOVA
(Table Sl). While no reference gene is perfect [22,23], 18S rRNA
appears to be the best internal reference for this experiment. 18S
rRNA expression does vary across some sites and collection times,
but is remarkably consistent for the samples here presented,
collected in February, 2007. Moreover, at least one commonly
used reference gene, p-actin, was differentially regulated between
NS and OS in the microarray study (Table S2, ProbeName
UF_Sgi_AF_101275), making it a poor candidate for our internal
reference in real-time RT-PCR.


Amplicon Size (bp)
636


Accession Number


18S rRNA


18S rRNA


Ctr c


Tepll


Cloning


Forward


GTT TCC CAT CCT ACG CTT CC
AGA CAA ATC GCT CCA CCA AC
TCG GTC TTA TTT TGC TGG TTT
ATC GCT AGT TGG CAT CGT TT
ACA AGG GCG GAA GAA GAA GT
GGC TTT CAG TAC CCA AAC GA
GTC ACG GCT GAC TCC TTC TC
TAA AGA ACA CGC CGA TCT CC
TAT GGC AAG ACC AAC ATG GA
ATT CGC GTA AAA GCC AAA GA
GCG CTG TTG CTG AAC ATA AA
CTT CTT GCA CAC CAT CTC GTT


GVl98749


GVl98749


Reverse
Real-time Forward
Reverse
Real-time Forward
Reverse
Real-time Forward
Reverse
Real-time Forward
Reverse
Real-time Forward
Reverse


UF_Sgi_AF_100593


UF_Sgi_AF_105314


UF_Sgi_AF_101461


UF_Sgi_AF_103703


|"Accession Number" refers to the accession for the sequence in NCBI (18S rRNA) or the UF ProbeName for the corresponding microarray probe. Transcripts: Ctric,
copper transporter Ic; Tepll, thiolester containing protein II; GST, Similar to Glutathione S-transferase; Stard7, StAR-related lipid transfer (START) domain containing 7.
doi:1 0.1 371 /journal.pone.001 2737.t002


*,: PLoS ONE | www.plosone.org 4 September 2010 | Volurne 5 |Issue 9 | el2737




































































Table 3. Summary of testicular development observed in queen conchs collected from offshore (OS) and nearshore (NS) sites in
the Florida Keys in February and June 2007.


Gonadal Development OS February (n= 4) NS February (n= 4) OS June (n= 2) NS June (n= 5)

Early Developing 0 0 0 20
Developing 25 50 0 20
Spawning Capable 75 50 100 20
Regenerating 0 0 0 20
No gonadal tissue 0 0 0 20
SI 21.29'2.57a 3.74+0.72b 26.03+4.51a 0.60+0.47C

Data for reproductive phases presented as percentage. Data for spermatogenic index (SI) presented as mean + SE. Superscripts indicate differences in SI (nested
ANOVA, p<0.05).
doi:1 0.1 371 /journal.pone.001 2737.t003


*,' PLoS ONE |www.plosone.org 5 September 2010 |Volurne 5 |Issue 9 |el2737


Queen Conch Testis Regression


Metal analysis by inductively coupled plasma-mass
spectrometry (ICP-MS)
ICP-MS was used to determine levels of 58Ni, 65Cu, 66Zn, 88Sr,
l07Ag, 11 Cd, "8Sn, 202Hg, and 23U in blood, digestive gland,
foot, neural ganglia, and testis for male conchs collected in
February and June 2007 field collections (n= 2-8, varying by
sample type). Weighed tissue samples (approximately 50-200 mg)
were acid digested to completion in 0.5 mL 67-70% optima grade
HNO3 at 140' C for 2 hours. This was followed by addition of
0.5 mL 30% ultrapure HzOz and further digested at 110'C until
almost dry. The sample was quantitatively diluted to 5 mL using
ultrapure water for a final concentration of 2% HN03 and filtered
-la.._1. a 0.22 pLm nylon syringe filter. The reconstituted samples
were analyzed for total metal content using an XSeries 2 ICP-MS
(Thermo Electron Corporation, Winsford, Cheshire, UK) with
"5In as an internal standard. Samples were quantified against a
seven point standard curve with standard concentrations 1, 5, 10,
50, 100, 500, and 1000 ppb each analyte. The lower limit of
detectability for this assay was set at 0.5 ppb analyte in the
digested sample.

Statistical Analysis
Histological data were analyzed usingJMP y8 (SAS, Cary, NC,
USA). Differences in SI were analyzed using ANOVA with month
nested within location. A student's t-test based on least square
means of the nested ANOVA was calculated inJMP for all month
and location combinations. SI was tested for homogeneity of
variance (Levene's test) and normality of distribution (Kolmo-
gorov-Smirnov test), and values were logo transformed if
assumptions were violated. Raw microarray data were imported
into JMP Genomics v3.1 and analyzed as follows: Non-uniform
spots were :1 .~~ I and removed from the dataset. Next, 590 rows
not containing at least two data points for each treatment group
(OS and NS) were deleted, leaving 15,154 rows in the analysis,
Array data were then median-centered prior to performing
one-way ANOVA on the factor location (OS/NS) to identify
differentially regulated transcripts (p<0.01 or p<0.05 for further
analyses, FDR = 5%). Hierarchical clustering analysis of significant
transcripts (p<0.01) was performed using the program Cluster
[24,] and visualized in the programJava TreeView [25]. Data were
median-centered by gene and clustering was based on centered
correlation and complete linkage. Real-time RT-PCR data were
analyzed using the Kruskall-Wallis non-parametric test calcula-
tor available at http:/ /elegans.wsmed .edu/leon/ stats /ute st.html.
ICP-MS data were imported into JMP v7 and analyzed for
difference of means using two-way ANOVA, with the two factors


being tissue and location; this analysis was followed by the Post hoc
Tukey-Kramer HSD test for multiple comparisons (p<0.05). For
non-parametric correlation analysis, Spearman's p was calculated
in JMP.

Gene Ontology and Pathway Analysis
For microarray data, functional enrichment analysis of Gene
Ontology terms was performed by Fisher's exact test using the
FatiGO tool within the Babelomics suite [26]. All terms with a
nominal p-value of p<0.05 (no Post hoc correction) were considered
to be enriched. Finally, Pathway Studio 7 (Ariadne Genomics,
Rockville, MD, USA), operating on the ResNet 7.0 mammalian
database updated with zebrafish annotation, was used to identify
all shortest paths between genes falling under _,.,,1
enriched terms and cellular processes, in order to illustrate
important connections within these biological processes, based
on human and zebrafish (Danio rerio) homologs.

Results

Histopathology
Testis tissue from eight conchs (four OS, four NS) in February
2007 and seven conchs in June 2007 (two OS, five NS) was
analyzed by 1.: I.. .,ll...l... (Table 3). All OS conchs from both
months had normal, healthy testicular tissue present in >75% of
each histological section (Figure 2A). In contrast, the amount of
testicular tissue present in NS conchs was lower than OS conchs in
both February and June (Figure 2B, C). Differences in gonadal
development based on reproductive phase were not marked
in February between NS and OS conchs. All conchs from both
locations had testicular tissue undergoing active spermatogenesis
in February, Jul..... _1. a higher percentage of OS conchs (75%)
were Spawning Capable compared to NS conchs (50%; Table 3).
However, there was a significant difference in the SI between OS
and NS conchs in February (tl = 2.606, p =II I s: with OS
conchs having a SI value nearly 7 times greater than the NS value
(Table 3).
In contrast, by June all OS conchs were Spawning Capable
while only 60% of NS conchs exhibited active spermatogenesis,
with only one NS individual Spawning Capable (Table 3). The SI
was nearly 45 times higher in OS conchs compared to NS conchs
in June (tll = 6.05, p<0.001; Table 3). Overall, in both February
and June the SI was ;:_..:0. ,,.11 higher for OS conchs when
compared to NS conchs. There was no difference in SI between
months for OS conch (tll = 0.245, p = 0.81), suggesting OS conchs
were reproductively active from February-1.,...._1~June. Interest-
ingly, for NS conchs, the SI was ;:_..:0. ,~,11 higher in February



































than in June (t,, = -4.49, p = 0.0009; Table 3) suggesting that the
NS conchs were unable to maintain spermatogenic tissue during
the reproductive season.


Normalized CDNA library sequencing and assembly
Sequencing produced a total of 64,794,458 bases across 286,933
reads (average read 1. ,, _11. = 225.8 bases) (Table 4,). Sequences
were submitted to the NCBI Sequence Read Archive (SRA)
(experiment SRX017250). These sequences were assembled into
28,010 contigs, of which 7,740 matched to NCBI NR or NT
database with e<1 x10-4. Of 7,740 annotated sequences, at least
one GO term was assigned to 3,971 (51.3 ].. I..... human
homologues to 2,688 (34.7 ].. I. ... and zebrafish homologues to
2,681 (34.6 percent). The microarray was designed with probes
corresponding to all 7,740 annotated sequences and to 7,468
additional un-annotated sequences for a total of 15,208 user-
defined elements.


Microarray analysis of testicular transcription
255 .1:FC i. ,,.:,11 ... _,1,i~... probes (58 up and 197 down in NS
with respect to OS conchs) were identified by ANOVA (n = 3,
p<0.01, FDR= 5%) (Figure 3, Table S2). At a less stringent
p-value, 1147 sl~iff i. I:.11i -1. _..1.1..1 probes (341 up and 806
down) were identified (p<0.05, FDR= 5%) (Table S2). Based
on differentially-regulated probes, all OS and NS individuals
clustered separately from one another, indicating that the identi-
fied set of transcripts show a clear difference between these two
presumably outbred groups of wild conchs (Figure 3). Differen-
tially regulated genes were predominantly down-regulated in this
experiment; at a cutoff of p<0.01, the proportion of differentially


Table 4. Information on conch transcriptome assembly.


Queen Conch Testis Regression


C
r ..,.



-L
;L" : .;L
I:
e T:

. .
j- .
;L

-~'.,' .=-i;. -'*-

,_ *


Figure 2. Histological sections of testis tissue from queen conchs captured in the Florida Keys in 2007. A. Testicular tissue from an
offshore (OS), Spawning Capable male in February. All OS conchs captured in June had a similar appearance. B. Testicular tissue from a nearshore
(NS), Developing male in February. C. Testicular tissue from a NS male in June showing little spermatogenic tissue or spermatogenesis. ST-
spermatogenic tissue; VD-vas deferens.
doi:1 0.1 371/journal.pone.001 2737.g002


regulated transcripts that are down-regulated was 77.4 percent.
The two most up-regulated probes with annotation (p<0.01) were
Similar to Glutathione S-transferase (GST, 15.24-fold up-regulat-
ed NS) and Collagen 1, Alpha 1 (COLIA1, 10.26-fold up-
regulated NS). The two most down-regulated probes with
annotation (p<0.01) were RIKEN CDNA F730014IO5 Gene, a
mouse genome sequence (13.83-fold down-regulated NS), and
Dolichyl-phosphate Mannosyltransferase Polypeptide 2 Regulato-
ry Subunit (Dpm2, 4.92-fold down-regulted NS).
Functional enrichment analysis based on GO terms for biological
process identified 11 ;:_.,;0. ,,,.1 enriched terms in the differen-
tially-regulated gene list (Table 5). The most ;:_..:0. ,,.11 enriched
term was "proton transport," under which all but one of seven
differentially-regulated genes was down-regulated. Another notable
term was "small GTPase-mediated signal transduction." "Sper-
I!* *l- --. ... ; was the twelfth term on the list (p = 0.052). Pathway
analysis (Pathway Studio) further illustrated the results of the
enrichment analysis (Figure 4): most affected transcripts were down-
regulated (blue color), many of these transcripts are found in the
mitochondria, and there were a large number of associations with
the cell processes "respiratory chain," "cell proliferation," and
1.. "!' ...- "~ ') among others.

Real-time RT-PCR
Efficiencies of the real-time RT-PCR assays here reported
ranged from 92.5 percent to 108.6 percent (Table 6), and their
correlation coefficients ranged from 0.988 to 0.999. The difference
between threshold cycles of the last experimental sample to
amplify and the first negative control well to amplify in any
reaction was at least 6.49 cycles and 9.83 cycles for -RT and NTC
controls, respectively. For each assay, the dissociation curve
indicated that a single amplicon was produced. By real-time RT-
PCR (n= 4), two of the four genes, Stard7 and TepII, were
.;.,.1differentially regulated (p =0.029 and 0.014, respec-
tively); the direction of regulation was the same as determined by
microarray. The fold-change was similar to that determined by
microarray for Stard7(1.87 by real-time RT-PCR compared to
2.31 by microarray), but smaller for TeplI (5.66 by real-time RT-
PCR compared to 29.66 by microarray). For GST, the fold-
change was smaller, but the direction of regulation (4.71-fold up-
regulated NS) was similar to that determined by microarray
(15.24-fold up-regulated NS). This difference in real-time RT-
PCR was not significant according to the Kruskall-Wallis test
(p =0.100). For one gene, Ctrlc, the direction of regulation
determined by real-time RT-PCR was opposite that determined


Average
Run Bases Reads Length Alias


Titration
Production-1
Production-2
Production-3
Total


1,286,141
37,762,116
15,882,529
9,863,672
64,794,458


5,354
161,610
67,779
52,190
286,933


240.22
233.66
234.33
189.00
225.82


ERXZlSM
ES8A9FR
ETMYUMG
EU6CYYIF
S. gigas 454


"Alias" refers to the run alias listed in the SRA database.
doi:1 0.1 371 /journal.pone.001 2737.t004


a, : PLoS ONE | www.plosone.org


September 2010 | Volume 5 | Issue 9 | el2737







Queen Conch Testis Regression


Figure 3. Hierarchical clustering of significantly differentially regulated genes in conch testis. Red color represents expression of a gene
at a level greater than the row (gene) average, and blue color represents expression lower than the row average. The map shows a clear distinction
between nearshore (NS) and offshore (OS) testis samples based on the 256 differentially-regulated transcripts. Approximately one-fourth of the
regulated transcripts are up-regulated in NS relative to OS; the majority are down-regulated.
doi:1 0.1 371/journal.pone.001 2737.g003


by microarray, 1l...n_1. the difference was essentially zero (1.03-
fold down NS by RT-PCR compared to 1.74-fold up NS by
microarray). This change was not significant by Kruskall-Wallis
(p = 0.443). Therefore, RT-PCR results were similar to micro-
array, -l...n_1. each transcript's fold change and statistical
significance was reduced when measured by RT-PCR, compared
to microarray.


Tissue metal burdens
ICP-MS results for all nine analytes are given in Table S3
(sample size varies: n=2-8/group, specifically enumerated in
Table S3). 66Zn was present at a ;:_..:0. ,~,11 higher level in the
digestive gland of NS conchs (831.85 ng/mg) than OS conch
digestive gland (84.53 ng/mg), or any other tissue at either site
(Figure 5A). In addition, Jul...n ,_1. not statistically significant,
the concentration of Zn in the NS testis (83.96 ng/mg) was
approximately 15-fold higher than in the OS testis (5.43 ng/mg)
(Figure 5A). 65Cu, conversely, was not ;_ ..:0. ,~,11 higher in any
of the NS tissue means compared to the corresponding OS means.


However, there was a non-significant (p =0.65), approximately
five-fold difference between 65Cu levels in NS (34.77 ng/mg) and
OS (6.60 ng/mg) gonad (Figure 5B, Table S3). In the tissue term
of the two-way ANOVA, concentrations of 58Ni, 66Zn, '' Cd, and
238U were ;:_..:0. ,~,. higher in digestive gland than any other
tissue. "8Sn, despite being detected only at very low concentra-
tions in these samples, was found at its highest concentrations in
the neural ganglia. 65Cu levels were highest in the blood, which in
molluscs contains a copper-based hemocyanin pigment [27].


Correlations among microarray, histology, and metal data
Correlation analysis was based on testis histological conditions
(n = ; metal concentrations in testis and digestive gland (n = 3
and expression levels of ,1:11. ...:.11 -1. _,1.1l..1 transcripts under
the GO biological processes spermatogenesis and small GTPase-
mediated signal transduction as determined by microarray (n = 3)
(Table 7). SI was ;:_.,;0. ,,,.1 inversely correlated with digestive
gland Zn (p = -0.655), and inversely correlated with digestive gland
Cu (p= -(l .ll*, 1...,, i.I this was not statistically significant


Table 5. Functional enrichment analysis based on Gene Ontology (GO) biological process terms.


Biological Process

proton transport
membrane fusion

virus induced gene silencing

receptor clustering
aromatic compound metabolic process

seryl-tRNA aminoacylation
cilium biogenesis
small GTPase mediated signal transduction

prostaglandin biosynthetic process
protein kinase C activation
neuron differentiation

spermatogenesis


GO Term ID

GO:001 5992
GO:0006944
GO:0009616
GO:0043113
GO:0006725
GO:0006434
GO:0042384
GO:0007264
GO:0001516
GO:0007205
GO:0030182
GO:0007283


% of DR

2.90
0.83
0.83
0.83
1.24
0.83
0.83
2.90
0.83
0.83
0.83
1.66


% of other

0.75
0.00
0.00
0.00
0.13
0.03
0.03
1.07
0.07
0.07
0.10
0.52


p-value

0.005
0.005
0.005
0.005
0.011
0.015
0.015
0.023
0.029
0.029
0.045
0.052


|"%/ of DR" refers to the percent of differentially regulated transcripts falling under the term; "%/ of other" refers to the percent of all other transcripts with GO annotation
that fall under the term. P-value is the raw (nominal) p-value from Fisher's exact test.
doi:1 0.1 371 /journal.pone.001 2737.t005



*, : PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | el2737







Queen Conch Testis Regression


CRP







sleep
nOCFOSIS





cell proliferation





.- .,~~~ DGKZ nerbs itssecretion









vs lel




spermatogenesis ~







Figure ~ 1 4.Ptwy nlssof diferental result ensfrm ohtstismiraaysuyPthySudo(idnGemc)wa









sprmtoyt hmoog lpi dsaurse; GK, ypthtialLO5785 similr todayglycero inseioa;D l2dy inax em ,






Fintermediate hai nayi 2; DNJ1,DnaJ (Hsp40)l related, ufml Bne membe 13;c GeSTM4 glutoar thione S-tranfeae mudi 4;ian MOV10,cs sidey-8g
(Mol ofney aleue i vioruest 10); NAPA Nehylma hmlemi e sensitive fusiong proteinattachmentl pnrotein alha PilGDSl procstaglanin D2e tsythas. e,
hematorpoiestic uPICK1, hypothetialprotei(nS LOC9103 PPME1, zgc:56239 (pwnrogltein ph sphts me thlseaes 1);1 zg:28 APSME4,hypothtiofca ol ik LO 1538
(Tpro teaom (prosome, m+tacrpopain), acivtor subnditl 4); RAB1A, RB1tA membeier RASC1 oncgen faiy;RABL4, RAB mebr nsoftg RAS oncogne famly-
cmle 4 A1, RAP1A, moyembier ofTG RAS P onognte, famly SARSseryltRNA ytets mitochondrial;F opex uui SLM2, (slowmo homolog 2T (rsopthia)e
(rasimilrtong kiodiser) VAP (VAMP(eile-assocniated; memran sch13protein)-assoae proein A, 33 ktia. Organ) ells1, cokise from top genertier
semitochndrionen oplasmic restiuume) Go gi Z com plexnucaleus. 86 (iilrt icllyeo iae it) N Idyen xnm



( PLoSe ONEuei | wwrplsonr 80 SeA -tymliid estv uin rti tahetprtembe 2010a |GS Vrolume 5 | Isse |2 el2737































(p = 0.110). Digestive gland Zn was also _,;0. ,,,. ~1 and inversely
correlated with four of the 11 transcripts included in the analysis;
gonad Zn was correlated with two of the 11. SI was ;:_..:0. ,,.11
correlated with six of the 11 genes in the analysis.

Discussion

The testis histological data ._ .11.. I. .1 in this study show a strong
difference between NS and OS conchs in terms of development
-la..n_1..... the reproductive season (Table 3, Figure 2). Queen
conchs from NS sites had dramatically less spermatogenic tissue in
relation to OS conchs in both February and June. Additionally,
spermatogenesis was somewhat reduced in February and markedly
reduced in June in NS conchs, during the peak of the conch
reproductive season. The SI values give a clear picture of the
reduction in spermatogenic capability of NS conchs during both
February and June, and highlight the decrease in spermatogenic
capability of NS conchs between February and June. Ala... _ 1. all
NS conchs were undergoing active spermatogenesis in February,
there is evidence of developmental delay in February, as a lower
percentage of NS conchs were Spawning Capable compared to
OS conchs, as well as evidence of significant regression from
February toJune. The histological data fromJune collections show
that NS conchs are unable to maintain reproductive capability


A


Queen Conch Testis Regression


Table 6. Comparison of microarray and real-time RT-PCR results.


Microarray

Gene Fold Change


Real-Time RT-PCR

Fold Change
1.03
5.66
4.71
1.89


Direction

up
up
up
down


Direction

down
up
up
down


Efficiency
108.2%
92.5%
95.1%
100.7%


GST 15.24
Stard7 2.32


Real-time RT-PCR values were normalized to 18S rRNA (18S rRNA efficiency = 108.6%). "Direction" of regulation is given for nearshore samples, with respect to offshore.
Transcripts: Ctric, copper transporter Ic; Tepll, thiolester containing protein II; GST, Similar to Glutathione S-transferase; Stard7, StAR-related lipid transfer (START)
domain containing 7.
doi:1 0.1 371 /journal.pone.001 2737.t006


-la.. _1,...ni the spawning season. While all OS conchs collected in
June had high SI values and were Spawning Capable, only 20% of
the NS conchs were Spawning Capable, and all had very low SI
values. The current study only presents this histological data as a
physiological anchor for gene expression at two sites in the Florida
Keys. While we acknowledge that site-specific effects may play a
large role in testis development, these observations mirror results
from conchs collected at similar NS and OS areas of the Florida
Keys in 1999 [2], suggesting that NS conchs show a persistent,
long-term reduction in reproductive capability. Moreover, the
histology here reported showed a more dramatic reduction than
that reported for 1999.
These results complement the results of our microarray
and ICP-MS experiments. NS conch testis transcription differed
from OS in the GO biological processes proton transport
(GO:001 ."***: spermatogenesis (GO 111111-_ small GTPase-
mediated signal transduction (GO IIIIII- _1. and others (Table 5,
Figure 4). This supports specific hypothesis (1), and also suggests
that inhibition of small GTPase (Ras)-mediated signaling in NS
testis contributes to NS reproductive failure. ICP-MS analysis
indicated that Cu and Zn were elevated in some NS conch tissues,
providing preliminary support for specific hypothesis (2), and
creating the hypothesis that Cu and Zn may be a causative factor
in reproductive failure of NS conchs in the Florida Keys. It is


100


80









20


Blood DG Foot NG Testis


Blood DG Foot NG Testis


Organ Organ

Figure 5. Tissue distribution of Zn (A) and Cu (B) in offshore (OS) and nearshore (NS) conchs. Letters indicate significant difference in 2-
way ANOVA, with the two factors tissue and location, followed by Tukey-Kramer HSD (p<0.05). Note different y-axis for Cu and Zn. Break in Zn data
(A) omits 150-800 ng/mg. DG = digestive gland; NG = neural ganglia.
doi:1 0.1 371/journal.pone.001 2737.g005


*, : PLoS ONE | www.plosone.org


September 2010 | Volume 5 | Issue 9 | el2737


800 -





40 .


mn Cl r








Queen Conch Testis Regression


VE




hc E


c ar








I=E~







O~~~~ 0 00 o










o I~~ ~ I I d I I I









o a m I I.o





i;Eo




P~~~~~oS~~I ON | ww~lsn~r 0Spe br210|Vlre5|Ise9|e23







Queen Conch Testis Regression


important to note that site-specific differences in metal concen-
trations and gene expression surely exist. Future studies will
incorporate metal and gene expression data from additional sites
to determine whether differences in these parameters are as
consistent as the histological differences observed -la..n _1...n~I the
NS and OS Florida Keys.

Conch testis gene expression
The gene expression analysis in the conch testis reveals,
logically, that spermatogenesis-associated transcripts are down-
regulated NS. Correspondingly, mitochondrial transcripts are
,a.,.1down-regulated in NS testes. The effects on proton
transport identified by the GO enrichment analysis could be either
a cause or a result of the observed reduction in spermatogenesis in
NS testes, given the important role of mitochondria in sperma-
tozoa and in sperm maturation [28-30]. Our finding is likely the
result of the reduction in mature spermatozoa, and consequent
numeric reduction in mitochondria, in NS testes as opposed to
OS.
Under the Biological Process GO:0007283, spermatogenesis, we
identified differentially regulated transcripts with major roles in
spermatogenesis in species ranging from Drosophila to humans,
including degenerative spermatocyte homolog 1 (DEGS1) [31];
Similar to Kiser il.!..i. ..1...... to slowmo) [32]; proteasome
activator subunit 4 (PSME4/PA200) [33]; DnaJ related, subfamily
B, member 13 (DNAJBl3) [34,35], which is also related to the
TSARG genes in rats [36] and mice [37]; and nuclear auto-
antigenic sperm protein (histone-binding) (NASP) [38]. These
genes, important for the process of spermatogenesis in a wide
range of species, appear to be conserved in queen conch, and were
all down-regulated NS in the present study.
A surprising result of the GO enrichment analysis was the
enrichment of the term "small GTPase-mediated signal transduc-
tion."' Most of the genes under this term are related to Ras-GTPases,
proto-oncogenes involved in mammalian tumor formation and
developmental disorders [39]. Seven genes that fall under this GO
term were differentially regulated in our experiment, including
related Ras viral oncogene homolog i10 Ras related protein lb
(Raplb); RABlA member ofPas oncogene family; T-cell lymphoma
invasion and metastasis 1 (TIAM1); RAB member of ras oncogene
family 4-like (RABL4r); ADP ribosylation factor-like 1 (ARL1); and
4R79.2, a hypothetical GTP-binding protein identified in Caenor-
habditis elegans. All of these genes are down-regulated with the
exception of TIAM 1 (Table S2). Ras function has been described in
invertebrates including ascidians, for which Ras signaling is involved
in embryonic tissue development [40], and Drosophila, for which
Rapl is involved in cell adhesion and polarity during epidermal
1.. 11. factor receptor-mediated tissue ._1.. 11. [41l]. Ras genes are
also known to be involved in vertebrate and invertebrate testis
development. The Ras-cyclin D2 pathway is involved in mouse
spermatogonial stem cell development in citro [4,2] MAPK and Rap-
GEF ;: ..,1;:,. 1.,11. .are also involved in testis development and
renewal in Drosophila [4,3]. Therefore, Ras-GTPase signaling may
play a major role in conch testis tissue ._1.. 11. and differentiation.
Histological SI was correlated with six of the 11 differentially
regulated transcripts involved in spermatogenesis or small GTPase-
mediated signaling (Table 7). This suggests that transcription of these
genes is indicative of the overall maturation of the testis tissue in
queen conchs, and that perturbation of normal transcription of these
genes is detrimental to spermatogenesis.
Transcripts evaluated by real-time RT-PCR were selected
based on their differential regulation between NS and OS,
according to the microarray study (Table S2) and their varied and
interesting biological functions. GO biological processes of these


a,' PLoS ONE |www.plosone.org


gene products include: Ctrlc, copper transmembrane transport;
TepII, antibacterial humoral response; GST, __1~1.. .11,..... meta-
bolic process; Stard7, no biological process (but related to
steroidogenic acute regulatory (StAR) protein). The results of
our real-time RT-PCR assays were largely successful in validating
the changes observed in the microarray study. TepII, GST, and
Stard7 were confirmed by real-time RT-PCR, -1..... 1. the GST
result was not statistically significant. Ctrlc, however, was
essentially unchanged between NS and OS samples in real-time
RT-PCR, with a 1.03-fold change in the direction opposite that
determined by microarray. The difference in results between
platforms is possibly due in part to the small sample size (n = 4)
used for both assays; increased sample size would lend power to
the analyses. Unfortunately, permitting regulations limit sample
size for a protected species such as S. gigas. It is also possible that
for Ctrlc our probe was designed to a region with homology to
other proteins or isoforms in the SLC31 family of copper
transporters, causing the lack of consistency between microarray
and real-time RT-PCR. C I. ... _. in TepII, GST, and Stard7 may
indicate that stressors affecting NS conchs cause changes in
immune response, xenobiotic metabolism/redox balance, and
steroidogenesis, respectively. However, these are single gene
changes, and so should be interpreted carefully.

POtential YOle Of metalS BS a reproductive stressor
Our ICP-MS data indicated that Cu and Zn, two known
reproductive toxicants in gastropods, were elevated in some NS
conch tissues. Our study also included other analytes with known
toxic effects, including Ni, Ag, Cd, Sn, Hg, and U. Sr was included
due to its role in shell-building; it is known to be physiologically
beneficial in gastropods at low doses, but toxic at high levels [44d].
However, few differences were observed for the latter seven
analytes. The effects of Cu and Zn on gastropod reproductive
output have been well-documented, Jul...,, _1. most examples
relate to females. In laboratory exposures, Cu has resulted in
reduced fecundity in Helix aspersa [6], reduced egg-laying and a
dose-dependent reduction in hatching in Pomacea palludosa [7], and,
as copper oxychloride, reduced oocyte number in the ovotestis of
Helix aspersa [8]. Zn exposures, likewise have impacted reproduc-
tion in numerous studies, resulting in reduced fecundity and
population ._1 .. 11. rate in Valvata piscinalis [10], reduced fecundity
in Helix aspersa [6], and, as an emuent containing Zn, Cd, and Fe,
mortality and reduced egg laying in Lymnaea palustris [9].
General and point sources of heavy metals in south Florida
include storm water runoff, roadway contaminants, septic system
leachate, and boats, which may be responsible for high levels of
Hg, Pb, Zn, and Cu in waterways [12]. Elevated Cu, Zn, Cr, Hg,
Pb, and Ni levels have been identified in Biscayne Bay, adjacent to
the city of Miami, as well as at the outflow of canals [l l].
Additionally, heavy metals including Cu and Zn have been
detected in sediments and seagrass beds, both habitats occupied by
conchs, as well as in surface waters at multiple sites -la.n1.....~~
south Florida, with Cu sometimes exceeding guidelines for aquatic
life and sediment quality [13]. Taken -.._.11.. I this information
suggests that potential sources of Cu and Zn contamination exist in
the Florida Keys and are likely to be primarily on land or close to
shore, further n .I ... , _II~ 11.. plausibility of these metals interfering
with NS testis development.
In the present study, Zn was elevated in the digestive gland, and
possibly in the gonad, of NS conchs (Figure 5A). Coupled with the
knowledge that Zn causes reduced fecundity in other gastropod
species [I.'Il this finding suggests that Zn may contribute to the
reproductive failure of NS conchs. The observed NS digestive
gland mean concentration of 831.85 ng Zn/mg tissue is similar to


September 2010 | Volurne 5 | Issue 9 | el2737







Queen Conch Testis Regression


the body burden observed (approx. 200-500 Iag Zn/g tissue) in
emuent treatments resulting in mortality and reduced fecundity in
Lymnaea palustris [9]. While available data in the literature focus on
female-mediated reproductive inhibition measured as reduced
fecundity, studies of fecundity may miss mechanistic effects in both
males and females. Further, while the gonad is the apparent site of
action for any potential toxicant, accumulation of Zn in the
digestive gland in the present study is also likely to be a significant
finding. The digestive gland is adjacent to the gonad and is
believed to be a site of metal accumulation and detoxification in
gastropods [45-48]. While a recent study indicates that Zn
concentrations in the testis of the Japanese ( e. I. japonica track
the progression of spermatogenesis [4,9], it is important to note
that an excess of Zn from external sources could still have a
deleterious effect, as is possible in the present study. The
relationship between Zn and spermatogenesis is likely complex,
and should be the subject of further study.
No significant differences in Cu concentrations within any tissue
were found between NS and OS. The mean concentration of
34.77 ng Cu/mg tissue observed in NS conch testis in this study is
still only a fraction of the toxic levels accumulated in studies by
Rogevich et al. [7] (396.60 ng Cu/mg tissue) and Snyman et al.
[8] (260.47 ng Cu/mg tissue), but is approximately five times the
OS mean of 6.60 ng Cu/mg tissue. Further, the aforementioned
studies measured whole body Cu rather than tissue-specific
accumulation. Blood levels of Cu in our study (40.18 ng Cu/mg
tissue NS, 58.90 ng Cu/mg tissue OS) were the highest of any
tissue, and it would be difficult to separate the Cu contribution of
hemocyanin in a tissue to the amount actually bound up in cells. In
other words, blood Cu bound in hemocyanin might obscure
differences between tissues. Therefore, Cu might still be a factor in
NS reproductive failure, and future studies will attempt to test this
possibility. It should also be noted that many environmental
factors could be considered stressors in a complex environmental
mixture, and as with all real-world situations, multiple stressors are
likely involved at our NS sites. The inverse correlations between
Cu and Zn concentrations in the digestive gland and SI (Table 7)
provide support for the argument that accumulation of metals,
including Zn and possibly Cu, in the conch digestive gland affects
development of the conch testis. These hypotheses will be
examined in future studies.

High-throughput sequencing for gastropod
transcriptomics
The approximately 60,000 extant gastropods make up the
largest class within the 100,000-member phylum Mollusca, the
second-largest animal phylum [50]. However, very little work has
been done in the area of gastropod genomics. A PubMed search
for gastropodd microarray" on 16 July 2010 yielded only 14
results, one of which was non-germane. Two of the remaining 13
papers discussed toxicogenomics as a tool for understanding
endocrine disruption in invertebrates [51,52]. The remaining 11
papers applied to only five genera of gastropods: Helix [53],
Lymnaea [54], Haliotis [55], Aplysza [56], and Biomphalanza [57:' 1 or
to schistosomes that use both humans and gastropods as hosts [59-
63]. A fielded search for . II ...I..1 ... ~~~ !]" on GEO yielded
only 13 results, consisting of the two submissions here reported, mn
addition to two platforms (GPL3635 and GPL3636) and two gene
expression datasets (GSE4628 and GSE18783) for Aplysia
californica, one platform (GPL7421) and one gene expression
dataset (GSE13039) for Haliotis asinina, and two platforms
(GPL9129 and GPL9483) and two gene expression datasets
(GSE16596, GSE18705, and GSE22037) for Biomphalaria glabrata.
The use of 1.; _1..11.,...._1. .... sequencing allowed us to make a


a,' PLoS ONE |www.plosone.org


significant contribution to this growing field. Still, aside from
several heavily studied genera, one of which (Biomphalaria) has
direct importance for human health, the entire realm of gastropod
genomics remains to be developed.

Conclusions
This study has provided new information regarding the
reproductive failure of NS conchs in the Florida Keys. The major
findings of this study include the following: first, that failure of NS
conchs to reproduce is coupled with a reduction in NS testis
development, as previously reported [2], and premature regression
of NS testis. Second, the microarray results indicate that reduced
testis tissue in NS male conchs is concurrent with a decrease in the
expression of many genes related to spermatogenesis and
mitochondrial function. Transcription of small GTPase-related
signaling genes is clearly affected, and this may contribute to the
lack of testis tissue development, but this requires further study.
Finally, this study supports the hypothesis that heavy metals may
contribute to the reproductive failure of NS conchs. Zn and
possibly Cu are elevated in the NS conch digestive gland, and Zn
may be elevated in the testis. Given that Zn and Cu are known to
reduce gastropod fecundity, the possibility that these same metals
may also inhibit gametogenesis in both males and females merits
further consideration.
Note that this study characterized effects of the NS environment
on reproductive tissue of male conchs. While many gastropod
reproduction studies rely on egg-laying (i.e. female-mediated
effects) as the measure of average reproductive success [6-10], the
phenomenon observed in the NS Florida Keys affects both males
and females [2,3]. Conchs rely on mate-pairing and copulation
[4], rather than broadcast spawning or other mating strategies that
would require fewer reproductive males. Logically, this lack of
male reproductive maturity could have a significant impact on the
conch population. Future studies will aim to assess transcriptional
effects on the ovaries of affected NS females, in addition to males.
,\l11...n _1. the testicular regression in NS conchs appears to be a
persistent problem in the Florida Keys, it is apparently reversible
at the level of the individual, as many NS conchs transplanted to
OS areas become Spawning Capable [2]. This suggests that
transcriptional effects, which can immediately and transiently
respond to environmental factors, can play an important role in
,,..1. I I ....l., ll.. disparity in conch reproduction from NS to OS,
as well as identifying responsible factors. Therefore, the combina-
tion of microarray studies with more traditional approaches will
yield useful information for managers as they work to facilitate the
recovery of NS queen conch populations in the Florida Keys.

Supporting Information
Table SI Validation of 18S rRNA as a reference gene for real-
time RT-PCR. "Tukey" denotes whether interaction term
tissue*OS/NS is ;:_..:0. ,,.11 different by ANOVA (only if
p<0.05) followed by Tukey-Kramer HSD for multiple compar-
isons. Withmn each analyte, values not connected by the same letter
are differentfferen. *NS samples for 06/2007 were
contaminated with digestive gland. Microarray and real-time
RT-PCR reported mn the present study was conducted with 02/
2007 samples.
Found at: doi:10.1371/journal.pone.0012737.s001 (0.03 MB


Table S2 List of all differentially regulated probes from the
microarray experiment (p<0.05, FDR= 5%). No gene title is
given for probes with insufficient annotation. "Diff of treatment =
(NS)-(OS)" gives the 1.._~ _*.ls ... I; I ....i fold change with respect to


September 2010 | Volurne 5 | Issue 9 | el2737








Queen Conch Testis Regression


NS (OS as control). P-value as determined by one-way ANOVA
(FDR = 5%).
Found at: doi: 10.1371/journal.pone.0012737.s002 (0.15 MB
XLS)

Table S3 ICP-MS analysis. "Tukey" denotes whether interac-
tion term tissue*OS/NS is ;:_..:0. ,,.11 different by ANOVA (only
if p<0.05) followed by Tukey-Kramer HSD for multiple
comparisons. Within each analyte, values not connected by the
same letter are ;:_..:0. ,,.11 different.
Found at: doi: 10.1371/journal.pone.0012737.s003 (0.11 MB
DOC)




References

1. Chakalall B, Crispoldi A, Garibaldi L, Lupin H, Mateo J, et al. (2007) World
markets and industry of selected commercially-exploited aquatic species:
Caribbean queen conch (Strombus gigas). Food and Agriculture Organization of
the United Nations. http:/ /www~.fao.org/docrep/ 006/Y5 26 1E/y5261e07.htm.
2. Delgado GA, Bartels CT, Glazer RA, Brown-Peterson NJ, McCarthy KJ (2004)
Translocation as a strategy to rehabilitate the queen conch (Strombus gigas)
population in the Florida Keys. Fish Bull 102: 278-288.
3. Glazer RA, Delgado GA (2003) Towards a holistic strategy to managing
Florida's queen conch (Strombus gigas) population. In: Aldana-Aranda D, ed. El
caracol Strombus gigas: conocimiento integral para su manejo sustenable en el
Caribe. YficatanMe~xico: CYTED. Program Iberoamerican de Ciencia y
Tecnologia para Desarolllo. pp 73-80.
4. Davis M (2000) Queen conch (Strombus gigas) culture techniques for research,
stock enhancement and growout markets. In: Fingerman M, Nagabhushanam R,
eds. Recent Advances in Marine Biotechnology, Volume 4 Aquaculture,
Part A Seaweeds and Invertebrates. EnfieldNH: Science Publishers, Inc. pp
27-59
5. Doney SC (2010) The growing human footprint on coastal and open-ocean
biogeochemistry. Science 328: 1512-1516.
6. Laskowski R, Hopkin SP (1996) Effect of Zn, Cu, Pb, and Cd on fitness in snails
(Helix aspersa). Ecotoxicol Environ Saf 34: 59-69.
7. Rogevich EC, Hoang TC, Rand GM (2009) Effects of sublethal chronic copper
exposure on the growth and reproductive success of the Florida apple snail
(Pomacea paludosa). Arch Environ Contam Toxicol 56: 450-458.
8. Snyman RG, Reinecke AJ, Reinecke SA (2004) Changes in oocyte numbers in
the ovotestis of Helix aspersa, after experimental exposure to the fungicide copper
oxychloride. Bull Environ Contam Toxicol 73: 398-403.
9. Coeurdassier M, de Vaufleury A, Crini N, Scheifler R, Badot PM (2005)
Assessment of whole effluent toxicity on aquatic snails: bioaccumulation of Cr,
Zn, and Fe, and individual effects in bioassays. Environ Toxicol Chem 24:
198-204.
10. Ducrot V, Pery AR, Mons R, Queau H, Charles S, et al. (2007) Dynamic energy
budget as a basis to model population-level effects of zinc-spiked sediments in the
gastropod Valuata piscinalis. Environ Toxicol Chem 26: 1774-1783.
11. Carnahan EA, Hoare AA, Hallock P, Lidz BH, Reich CD (2008) Distribution of
heavy metals and foraminiferal assemblages in sediments of Biscayne Bay,
Florida, USA. J Coastal Res 24: 159-169.
12. Kruczynski WL (1999) Water quality concerns in the Florida Keys: Sources,
effects, and solutions. Florida Keys National marine Sanctuary Water Quality
Protection Program. 75 p.
13. Lewis MA, Dantin DD, Chancy CA, Abel KC, Lewis CG (2007) Florida
seagrass habitat evaluation: a comparative survey for chemical quality. Environ
Pollut 146: 206-218.
14. Kofoed TMN, Tomkiew~icz J, Pederson JS. Histological study of hormonally
induced spermatogenesis in European eel (Anguilla anguilla). In: Wyanski DS,
Brown-Peterson NJ, eds. 4th Workshop on Gonadal Histology of Fishes; 2010; El
Puerto de Santa Maria, Spain. http://hdl.handle.net/10261/24937, 83-86.
15. Garcia-Reyero N, Griffitt RJ, Liu L, Kroll KJ, Farmerie WG, et al. (2008)
Construction of a robust microarray from a non-model species (largemouth bass)
using pyrosequencing technology. J Fish Biol 72: 2354-2376.
16. Droege M, Hill B (2008) The Genome Sequencer FLX System-longer reads,
more applications, straight forward bioinformatics and more complete data sets.
J Biotechnol 136: 3-10.
17. Farmerie WG, Hammer J, Liu L, Sahni A, Schneider M (2005) Biological
workflow with Blastauest. Data Knowl Eng 53: 75-97.
18. Ishikawa H (1977) Evolution of ribosomal RNA. Comp Biochem Physiol B 58:
1-7.
19. GroppeJC, Morse DE (1993) Isolation of full-length RNA templates for reverse
transcription from tissues rich in RNase and proteoglycans. Anal Biochem 210:
337-343.
20. Brazma A, Hingamp P, QuackenbushJ, Sherlock G, Spellman P, et al. (2001)
Minimum information about a microarray experiment (MIAME)-toward
standards for microarray data. Nat Genet 29: 365-371.



*, : PLoS ONE | www.plosone.org


Acknowledgments

The authors would like to acknowledge Dejerianne Ostrow, William G.
Farmerie, and Christopher Martymiuk from UF, who aided in sequencmng
and microarray experiments, as well as Gabriel Delgado, from FWRI, who
has contributed to sampling efforts and discussions throughout this project.
We thank the Louisiana State University Veterinary Pathology laboratory
for histological processing.


Author Contributions

Conceived and designed the experiments: DJS RJG KK RAG DB ND.
Performed the experiments: DJS RJG NJBP KK AF RAG. Analyzed the
data: DJS RJG LL NJBP AF. Contributed reagents/materials/analysis
tools: RAG DB ND. Wrote the paper: DJS RJG NJBP AF RAG DB ND.




21. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for
biologist programmers. Methods Mol Biol 132: 365-386.
22. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse
transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169-193.
23. Bustin SA (2002) Quantification of mRNA using real-time reverse transcription
PCR (RT-PCR): trends and problems. J Mol Endocrinol 29: 23-39.
24. Eisen MB, Spellman PT, Brown PO, Botstein D (1998) Cluster analysis and
display of genome-wide expression patterns. Proc Natl Acad Sci U SA 95:
14863-14868.
25. Saldanha AJ (2004) Java Treeview-extensible visualization of microarray data.
Bioinformatics 20: 3246-3248.
26. Al-Shahrour F, Minguez P, Vaquerizas JM, Conde L, Dopazo J (2005)
BABELOMICS: a suite of web tools for functional annotation and analysis of
groups of genes in high-throughput experiments. Nucleic Acids Res 33:
W460-464.
27. van Holde KE, Miller KI (1995) Hemocyanins. Adv Protein Chem 47: 1-81.
28. Pena FJ, Rodriguez Martinez H, Tapia JA, Ortega Ferrusola C, Gonzalez
Fernandez L, et al. (2009) Mitochondria in mammalian sperm physiology and
pathology: a review. Reprod Domest Anim 44: 345-349.
29. Erkan M, Sousa M (2002) Fine structural study of the spermatogenic cycle in
M~tar rudis and Chamelea gallina (Mollusca, Bivalvia, Veneridae). Tissue Cell 34:
262-272.
30. Yaffe MP (1997) Mitochondrial morphogenesis: fusion factor for fly fertility.
Curr Biol 7: R782-783.
31. Ternes P, Franke S, Zahringer U, Sperling P, Heinz E (2002) Identification and
characterization of a sphingolipid delta 4-desaturase family. J Biol Chem 277:
25512-25518.
32. Reeve S, Carhan A, Dee CT, Moffat KG (2007) Slowmo is required for
Drosophila germline proliferation. Genesis 45: 66-75.
33. Khor B, Bredemeyer AL, Huang CY, Turnbull IR, Evans R, et al. (2006)
Proteasome activator PA200 is required for normal spermatogenesis. Mol Cell
Biol 26: 2999-3007.
34. GuanJ, Kinoshita M, Yuan L (2009) Spatiotemporal association of DNAJBl3
with the annulus during mouse sperm flagellum development. BMC Dev Biol 9:

35. GuanJ, Yuan L (2008) A heat-shock protein 40, DNAJBl3, is an axoneme-
associated component in mouse spermatozoa. Mol Reprod Dev 75: 1379-1386.
36. Yang HM, Liu G, Nie ZY, Nie DS, Deng Y, et al. (2005) Molecular cloning of a
novel rat gene Tsargl, a member of the DnaJ/HSP40 protein family. DNA Seq
16: 166-172.
37. Li L, Liu G, Fu JJ, Li LY, Tan XJ, et al. (2009) Molecular cloning and
characterization of a novel transcript variant of Mtsargl gene. Mol Biol Rep 36:
1023-1032.
38. Alekseev OM, Widgren EE, Richardson RT, O'Rand MG (2005) Association of
NASP with HSP90 in mouse spermatogenic cells: stimulation of ATPase activity
and transport of linker histones into nuclei. J Biol Chem 280: 2904-2911.
39. Karnoub AE, Weinberg RA (2008) Ras oncogenes: split personalities. Nat Rev
Mol Cell Biol 9: 517-531.
40. Nishida H (2002) Patterning the marginal zone of early ascidian embryos:
localized maternal mRNA and inductive interactions. Bioessays 24: 613-624.
41. O'Keefe DD, Gonzalez-Nino E, Burnett M, Dylla L, Lambeth SM, et al. (2009)
Rapl maintains adhesion between cells to affect Egfr signaling and planar cell
polarity in Drosophila. Dev Biol 333: 143-160.
42. Lee J, Kanatsu-Shinohara M, Morimoto H, Kazuki Y, Takashima S, et al.
(2009) Genetic reconstruction of mouse spermatogonial stem cell self-renewal in
vitro by Ras-cyclin D2 activation. Cell Stem Cell 5: 76-86.
43. Singh SR, Hou SX (2008) Immunohistological techniques for studying the
Drosophila male germline stem cell. Methods Mol Biol 450: 45-59.
44. Buchardt B, Fritz P (1978) Strontium uptake in shell aragonite from the
freshwater gastropod Limnaea stagnalis. Science 199: 291-292.
45. Gros O, Frenkiel L, Aranda DA (2009) Structural analysis of the digestive gland
of the queen conch Strombus gigas Linnaeus, 1758 and its intracellular parasites.
J Mollusc Stud 75: 59-68.


September 2010 | Volurne 5 | Issue 9 | el2737








Queen Conch Testis Regression


46. Desouky MM (2006) Tissue distribution and subcellular localization of trace
metals in the pond snail Lymnaea stagnalis with special reference to the role of
lysosomal granules in metal sequestration. Aquat Toxicol 77: 143-152.
47. Gimbert F, Vijver MG, Coeurdassier M, Scheifler R, Peijnenburg WJ, et al.
(2008) How subcellular partitioning can help to understand heavy metal
accumulation and elimination kinetics in snails. Environ Toxicol Chem 27:
1284-1292-
48. Nott JA, Nicolaidou A (1990) Transfer of Metal Detoxification Along Marine
Food-Chains. Journal of the Marine Biological Association of the United
Kingdom 70: 905-912-
49. Yamaguchi S, Miura C, Kikuchi K, Celino FT, Agusa T, et al. (2009) Zinc is an
essential trace element for spermatogenesis. Proc Natl Acad Sci U SA 106:

50. Rup ert E Fox RS, Barnes RD (2004) Invertebrate Zoology: A Functional





52. Iguchi T, Watanabe H, Katsu Y (2006) Application of ecotoxicogenomics for
studying endocrine disruption in vertebrates and invertebrates. Environ Health
Perspect ll4(Suppl 1): 101-105.
53. Guiller A, Bellido A, Coutelle A, Madec L (2006) Spatial genetic pattern in the
land mollusc Helix aspersa inferred from a 'centre-based clustering' procedure.
Genet Res 88: 27-44.
54. Azami S, Wagatsuma A, Sadamoto H, Hatakeyama D, Usami T, et al. (2006)
Altered gene activity correlated with long-term memory formation of
conditioned taste aversion in Lymnaea. J Neurosci Res 84: 1610-1620.
55. van Rensburg MJ, Coyne VE (2009) The role of electron transport in the
defence response of the South African abalone, Haliotis midae. Fish Shellfish
Immunol26: 171-176.


































































*, : PLoS ONE | www.plosone.org


56. Lee YS, Choi SL, Kim TH, Lee JA, Kim HK, et al. (2008) Transcriptome
analysis and identification of regulators for long-term plasticity in Aplysia kurodai.
Proc Natl Acad Sci U SA 105: 18602-18607.
57. Adema CM, Hanington PC, Lun CM, Rosenberg GH, Aragon AD, et al. (2009)
Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda,
Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma
paraensei (Digenea, Platyhelminthes). Mol Immunol 47: 849-860.
58. Lockyer AE, Spinks J, Kane RA, Hoffmann KF, Fitzpatrick JM, et al. (2008)
Biomphalan'a glabrata transcriptome: cDNA microarray profiling identifies
resistant- and susceptible-specific gene expression in haemocytes from snail
strains exposed to Schistosoma mansoni. BMC Genomics 9: 634.
59. Gobert GN, Moertel L, Brindley PJ, McManus DP (2009) Developmental gene

eprle sion profiles of the human pathogen Schistosoma japonicum. BMC Genomics


60 s osom smanoni: Mcro ray anal siso n ex resiron lidue by hosts

61. Gobert GN, McInnes R, Moertel L, Nelson C, Jones MK, et al. (2006)
Transcriptomics tool for the human Schistosoma blood flukes using microarray
gene expression profiling. Exp Parasitol 114: 160-172.
62. Fitzpatrick JM, Protasio AV, McArdle AJ, Williams GA, Johnston DA, et al.
(2008) Use of genomic DNA as an indirect reference for identifying gender-
associated transcripts in morphologically identical, but chromosomally distinct,
Schistosoma mansoni cercariae. PLoS Negl Trop Dis 2: e323.
63. Dillon GP, Feltwell T, SkeltonJ, Coulson PS, Wilson RA, et al. (2008) Altered
patterns of gene expression underlying the enhanced immunogenicity of
radiation-attenuated schistosomes. PLoS Negl Trop Dis 2: e240.


September 2010 | Volurne 5 | Issue 9 | el2737




University of Florida Home Page
© 2004 - 2011 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Powered by SobekCM