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Applied and Environmental Microbiology, April 2000, p. 1685-1691, Vol. 66, No. 4
College of Marine Studies, University of
Delaware, Lewes, Delaware 19958
Received 16 September 1999/Accepted 10 January 2000
The Teredinidae (shipworms) are a morphologically diverse group of
marine wood-boring bivalves that are responsible each year for millions
of dollars of damage to wooden structures in estuarine and marine
habitats worldwide. They exist in a symbiosis with cellulolytic
nitrogen-fixing bacteria that provide the host with the necessary
enzymes for survival on a diet of wood cellulose. These symbiotic
bacteria reside in distinct structures lining the interlamellar
junctions of the gill. This study investigated the mode by which these
nutritionally essential bacterial symbionts are acquired in the
teredinid Bankia setacea. Through 16S ribosomal DNA (rDNA)
sequencing, the symbiont residing within the B. setacea gill was phylogenetically characterized and shown to be distinct from
previously described shipworm symbionts. In situ hybridization using
symbiont-specific 16S rRNA-directed probes bound to bacterial ribosome
targets located within the host gill coincident with the known location
of the gill symbionts. These specific probes were then used as primers
in a PCR-based assay which consistently detected bacterial rDNA in host
gill (symbiont containing), gonad tissue, and recently spawned eggs,
demonstrating the presence of symbiont cells in host ovary and
offspring. These results suggest that B. setacea ensures
successful inoculation of offspring through a vertical mode of symbiont
transmission and thereby enables a broad distribution of larval settlement.
Shipworms (family Teredinidae) are a
morphologically and physiologically diverse group of marine bivalve
mollusks notorious for their wood-boring lifestyle. Their ability to
bore into wood results in millions of dollars of damage annually to
wooden structures in both marine and estuarine systems worldwide. The
amazing diversity of this family is reflected in the wide range of
reproductive strategies, ranging from the brooding of young for the
entire 4-week larval development period to the broadcast spawning of gametes.
Like nearly all animals, shipworms are unable to metabolize wood
cellulose directly but rely on an obligate association with cellulolytic bacteria (22, 34, 35). Electron microscopy has
localized the symbionts to the Gland of Deshayes, a specialized region
of the host gill corresponding to the interlamellar junction of the
right and left demibranches (7, 25, 28, 32). Unlike symbioses of bivalves with chemoautotrophic bacteria, in the shipworm associations the symbionts can be cultured separately from the host.
Symbionts isolated from seven shipworm host species are currently
maintained in culture (this study and reference 35). In vitro gill isolates synthesize both cellulolytic and proteolytic enzymes for digestion, as well as nitrogenase for atmospheric nitrogen
fixation (14, 15, 16). It has been suggested that these
microbially derived enzymes provide the bivalve host with the ability
to thrive on a diet of wood alone.
A previous phylogenetic analysis of teredinid symbionts demonstrated
that four isolates from geographically distinct regions had identical
16S rRNA gene sequences, placing this consensus bacterium
(Teredinibacter turnerae) within the gamma subdivision of
the Proteobacteria (7). This study also
demonstrated that a 16S rRNA isolate-specific probe hybridized with the
resident bacteria in teredinid gill tissue (7). Linking the
physiological capabilities of these isolates to the symbiotic microbes
within the shipworm host further validated the notion that the
shipworm's nutritional success on a wood substrate is made possible by
a suite of microbially derived enzymes.
The success of the shipworm life strategy hinges on an obligate
symbiosis with cellulolytic microbes, but the mode by which this vital
association is established in each successive host generation has not
been described. The utility of nucleic acid probe technology for
studying symbiont transfer has been demonstrated in numerous marine
invertebrate systems (4, 5, 6, 17, 20). These studies rely
on the sensitivity of nucleic acid hybridization techniques to identify
the presence or absence of symbiont genes within the host gonad and
eggs, thus inferring a mode of symbiont transfer. Successful gene
amplification and in situ hybridization with symbiont 16S rRNA
gene-directed probes demonstrated the presence of symbionts in the
follicle cells surrounding the primary oocytes of the deep-sea
vesicomyid clams (5) and in the eggs and larvae of the
Protobranch bivalve Solemya reidi (4). These
experiments suggested that the chemoautotrophic endosymbiotic bacteria
resident in these bivalves are vertically transmitted. Using a similar approach, the endosymbionts from the hydrothermal vent tube worm Riftia pachyptila have never been detected in host
reproductive tissues or eggs, thus implying transmission of the
symbionts from the environment rather than through a direct transfer
from parent to offspring (6).
Here, we seek to ascertain the mode of symbiont transmission in
Bankia setacea (Tryon), a broadcast spawning teredinid
species indigenous to the Pacific Northwest coast of the United States. B. setacea causes extensive damage in the Pacific Northwest,
where forestry concerns depend on transportation and storage in marine systems (12). In light of data demonstrating that
geographically distinct and diverse species of shipworms harbor
symbionts that are genetically identical (7), an hypothesis
of horizontal (i.e., environmental) transmission was proposed for
B. setacea. However, phylogenetic characterization of the
B. setacea gill symbiont 16S rRNA and in situ hybridization
studies suggested that the symbiont of this species was unique.
Symbiont-specific 16S rRNA primers were designed and used in a
PCR-based detection strategy for symbiont genes within the host
reproductive tissue and freshly spawned eggs to elucidate the mode of
symbiont transmission in the shipworm B. setacea.
Specimens.
Adult B. setacea organisms were
collected from Yaquina Bay, Oreg. (44°N, 124°W), between September
1996 and October 1997. Animals were obtained through the deployment of
pine collection panels (31) that remained submerged for a
6-month period. Panels were retrieved from Yaquina Bay, wrapped in
moist paper towels, and transported to a quarantined recirculating
seawater system (10°C) at the University of Delaware College of
Marine Studies, Lewes. Adults were removed from the wood by hand, and
each was maintained separately in filtered (0.2-µm-pore-size filter)
sterile seawater. Gravid adults often spawned spontaneously upon
removal from the wood. After release, eggs were immediately collected, washed in sterile seawater, and prepared for nucleic acid extraction. Following a spawning event, the adults were dissected aseptically and
processed for either microscopic or molecular studies. Ovarial tissue
was always the first tissue to be dissected. To prevent contamination
of ovarial tissue with symbiont-containing gill tissue, ovaries were
sampled in the ventral region, separated from the dorsally located gill
lamellae. All samples were flushed multiple times with sterile seawater
to reduce external contaminants.
Symbiont isolation.
The control symbiont T. turnerae strain TBTC was isolated from the shipworm Teredo
bartschi, collected from Twin Cays, Belize, Central America
(17°N, 88°W), as described by the protocol of Waterbury et al.
(35).
Histological preparations.
Gill samples for electron
microscopy were fixed in 1% glutaraldehyde-1% paraformaldehyde in
phosphate buffer (pH 7.6) (60 min at room temperature) and postfixed (3 h) in 1% OsO4 in phosphate buffer. Samples were stained
overnight at room temperature in 1% aqueous uranyl acetate. Following
ethanol dehydration, specimens were embedded in araldite epoxy resin
and heat polymerized (60°C) for 3 days. Ultrathin sections were
stained with lead citrate and examined using a Zeiss CEM 902 transmission electron microscope.
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Bacterial Symbiont Transmission in the
Wood-Boring Shipworm Bankia setacea (Bivalvia:
Teredinidae)
and
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
Nucleic acid extraction. Bulk nucleic acids were extracted from aseptically dissected gill (symbiont containing), ovary, eggs, and non-symbiont-containing tissues (siphons) using an IsoQuick nucleic acid extraction kit (ORCA Research Inc., Bothell, Wash.) according to the manufacturer's instructions. The nucleic acid yield was quantified spectrophotometrically.
Gene sequencing.
To identify target sites for
symbiont-specific PCR primers and in situ hybridization probes, the 16S
ribosomal DNA (rDNA) of the B. setacea symbiont was fully
sequenced. Nucleic acids from host gill tissue were used as the
template in the PCR with eubacterial universal primers complementary to
the termini of the bacterial 16S rRNA gene (27f [AGA GTT TGA TCM TGG
CTC AG] and 1518r [5' AAG GAG GTG ATC CAN CCR CA 3'])
(13) according to the protocol described by Cary and
Giovannoni (5). The amplification product was cloned into
the plasmid vector pCR II and transformed into One Shot INV
F'
competent Escherichia coli cells according to the
instructions of the manufacturer (Invitrogen, San Diego, Calif.). The
entire 16S rRNA genes from four of the clones were amplified with the
same eubacterial universal primer set (27f-1518r) as described above.
For initial screening, a portion of the resulting amplification product
(7 µl) was digested with 2.5 U of the endonuclease HaeIII
(Promega, Madison, Wis.) at 37°C for 2 h and resolved on a 3%
agarose gel. Since the four clones displayed identical restriction patterns, a single clone was chosen for full sequencing.
Phylogenetic analysis. The 16S rDNA gene sequence of the putative B. setacea symbiont was aligned manually in the Genetic Data Environment (29) with a subset of eubacterial 16S rDNA gene sequences obtained from GenBank (2). The percent similarity between sequences was expressed as the number of different nucleotides divided by the total number of bases used in the analysis. Phylogenetic trees were constructed using the PHYLIP 3.5c ed. computer software package (11). To calculate evolutionary distances, DNADIST was used with a Kimura two-parameter model, a 2.0 transition/transversion ratio, and one category of substitution rates (19). Neighbor-joining trees were constructed based on evolutionary distance data using the program NEIGHBOR (27), and parsimony trees were constructed using the program DNAPARS. Branching confidence was assessed through 100 replicate resamplings of the data using the SEQBOOT general bootstrapping tool with global rearrangements. Reference 16S rDNA sequences used in these analyses were obtained from GenBank and include accession numbers M11224, M96399, M64338, M64339, M64340, Z31658, M99446, M99445, X56578, J01695, M32020, M11223, M26636, and M59298.
Oligonucleotide probe and primer design.
Alignments of the
B. setacea gill bacterium 16S rRNA sequence with those of
close relatives identified several regions of variability appropriate
for the development of probes specific to the teredinid symbionts and
an additional probe that would distinguish the B. setacea
gill symbiont strain from T. turnerae (Table
1). The 1243-1261 region (based on the
E. coli numbering system) provided the variability to design
a general shipworm symbiont-directed probe that would not discriminate
between the two symbiont strains (ter1243r). The shipworm symbiont
nucleotide sequences are identical in this probe region but differ from
that of their closest relative by at least four nucleotides. An
additional general shipworm symbiont primer (ter637f) was designed
based on a modification (shortened length and one base substitution) of
a published probe sequence (7). With two closely positioned mismatches between the two target molecules, the 613-632 region was
the most suitable target site for the B. setacea-specific probe with capabilities to distinguish between the B. setacea gill bacterium and T. turnerae (bs613r and
bs613f). Oligonucleotides were synthesized and high-pressure liquid
chromatography purified (Operon Technologies Inc., Alameda, Calif.) for
in situ hybridization (bs613r and ter1243r)- and PCR (bs613f, ter1243r,
and ter637f)-based assays.
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Clone library screening. To assess the possible diversity of bacteria within a single shipworm host, a second clone library of 16S amplicons from B. setacea gill DNA extract was constructed for restriction digest screening. Based on sequence data, a diagnostic region of the 16S gene (E. coli numbering 637 to 1243) was identified that would distinguish the symbiont T. turnerae from the B. setacea gill bacterium by a simple restriction analysis. Primers flanking this region (ter637f and ter1243r) were synthesized and used to amplify each of the 48 clones. Amplification reaction conditions were identical to those stated above but with an annealing temperature of 61°C. Amplification products were digested with the endonuclease HaeIII as described above. To verify that identical cut patterns reflected actual gene sequence identity, five clones were sequenced with the universal primer 907r (21).
In situ hybridizations. To determine the specificity of the teredinid symbiont probes (bs613r and ter1243r) in situ, hybridization reactions were conducted on sections of fixed gill tissue from the shipworm host. Oligonucleotide probes ter1243r, bs613r, eub338r, and 11f were labeled with DIG as described above. The eub338r eubacterial universal probe was used as a positive control to demonstrate the presence and accessibility of the bacterial target. A nonhomologous probe specific for blue mussel collagenous proteins, 11f (5' AAG CTT TCG CAA CGG AAG AC 3') (K. Coyne, unpublished data), functioned as a control for nonspecific binding. A no-probe control treatment was included in all experiments to ensure that hybrids were not an artifact of the reaction conditions.
The tissue sectioning and preparation for in situ hybridization were by the methacrylate embedding, acetone de-embedding technique of Warren et al. (33). Each hybridization experiment consisted of five individual treatments with either bs613r, ter1243r, eub338r, 11f, or a no-probe control. A total of 25 to 50 sections from three different embedded tissue samples were represented in each treatment. Prehybridizations were performed in hybridization buffer [2× SSC, 1× Denhardt's solution, 0.5 mg of sheared herring sperm DNA per ml, 5 mM EDTA, 0.1 mg of poly(A) per ml, and 0.1% Tween 20] for 60 min at 40°C. Following the prehybridization, the DIG-labeled probes were added to a final concentration of 1 ng/µl. Hybridization proceeded for 7 to 10 h at 40°C in a humid glass chamber. Posthybridization washes were conducted for 50 min each in 300 ml of wash 1 (2× SSC, 0.1% Tween 20) and 300 ml of wash 2 (0.2× SSC, 0.1% Tween 20). Detection of hybrids was accomplished using Enzyme-Labeled Fluorescence (ELF) (Molecular Probes, Eugene, Oreg.) by a protocol modified from that of the manufacturer. Following posthybridization washes, slides were washed twice (5 min each) in 1× wash buffer and then treated with blocking reagent (30 min) in a humid chamber. The alkaline phosphatase-conjugated anti-DIG antibody (1:500 in blocking reagent) was applied to sections and incubated (2 h) in a humid chamber. Following three washes (5 min each) in 1× wash buffer, sections were incubated (50 min) in freshly prepared substrate working solution. Sections were washed (5 min) in 1× wash buffer and postfixed (15 min; 2% formaldehyde in phosphate-buffered saline with 20 mg of bovine serum albumin per ml) for signal stabilization. After counterstaining (5 min) with DAPI (4',6'-diamidino-2-phenylindole) (1 µg/ml), slides were mounted in ELF mounting medium and visualized on a Zeiss LSM 410 inverted scanning confocal laser microscope equipped with a UV laser. The fluorescence produced by ELF was visualized using a 590-nm long-pass emission filter. DAPI fluorescence was visualized with a 480- to 520-nm band-pass emission filter and assigned a red pseudocolor.PCR detection of symbiont genes. The B. setacea gill bacterium-specific primer set (bs613f-ter1243r) was used in a series of PCR experiments to resolve the presence or absence of symbiont 16S rDNA in nucleic acid extracts from B. setacea gill, ovary, and recently spawned eggs. Symbiont-containing gill extract was included as a positive control, as was the plasmid containing the cloned 16S gene amplicon from B. setacea gill. Nucleic acid extracts were also amplified with primers specific for the termini of the eukaryotic small-subunit 18S rRNA gene (EukA, 5' AAC CTG GTT GAT CCT GCC AGT 3'; EukB, 5'GAT CCW TCT GCA GGT TCA CCT AC 3') (23) as a positive control. PCR mixtures consisted of 0.2 mM deoxynucleoside triphosphates (dATP, dCTP, dTTP, and dGTP) (Stratagene, La Jolla, Calif.), 2.0 µM MgCl2, 1× reaction buffer, 1 µM concentrations of each primer, 25 ng of purified DNA extract as the template, and 2.5 U of Taq DNA polymerase (Promega) in a final volume of 25 µl. For the bs613f-ter1243r primer set, cycling conditions consisted of 35 cycles each of denaturation at 94°C (1.5 min), annealing at 65°C (2 min), and extensions at 72°C (2 min), with a final extension time of 7 min. Cycling conditions for the 18S rRNA gene primer set were as described above, with the substitution of a 55°C annealing temperature. The authenticity of amplification products was verified by restriction fragment length polymorphism (RFLP) analysis with the restriction enzyme HaeIII (as described above) and by DNA sequencing.
Nucleotide sequence accession number. The full 16S rDNA sequence of the B. setacea gill symbiont has been deposited in GenBank under accession number AF102866.
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RESULTS AND DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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The goal of this study was to determine the mode by which bacterial symbionts are acquired by successive generations of the shipworm B. setacea. In a previous study, four shipworm species representing three different genera which were collected from varied ocean regions (California, North Carolina, Australia, and Massachusetts) were reported to harbor a phylogenetically common symbiont strain, T. turnerae (7). It was suggested by those authors that it was likely that most shipworm species would harbor the same symbiont. However, the teredinid B. setacea, a primary wood borer in the Pacific Northwest of the United States, was not included in these analyses. Our approach in this study was to systematically characterize the symbiont of B. setacea to develop molecularly based methodologies for the detection of the symbiont in the host reproductive tissues.
Clone library.
Bacterial 16S rRNA genes were successfully
amplified from B. setacea gill nucleic acid extract using
universal eubacterium-specific primers (13). The
amplification product was cloned, and a library consisting of 48 clones
was constructed. All of the clones in the library amplified with the
teredinid symbiont primer set (ter637f-ter1243r), producing a
predictable ~648-bp product. The digestion of the PCR products from
each of the clones with the restriction enzyme HaeIII
yielded identical cut patterns. The cut pattern diagnostic for T. turnerae was not represented by any of the clones in the library,
suggesting that T. turnerae was not present within B. setacea gill tissue (Fig. 1).
Furthermore, five of these clones were sequenced with the 907r primer,
yielding identical nucleotide sequences. The results of the clone
library screening suggested that the B. setacea gill was
colonized by a single microorganism that was distinct from T. turnerae.
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Detection and localization of the B. setacea symbiont. Oligodeoxynucleotide probes were designed based on comparative 16S rDNA sequence alignments of the B. setacea gill bacterium with T. turnerae and close relatives. The specificities of the 19-oligomer B. setacea gill bacterium probe (bs613r) and 20-oligomer teredinid symbiont-directed probe (ter1243r) were tested by solid-support dot blot hybridization. Experiments performed on the cloned 16S rRNA gene from the B. setacea gill bacterium and T. turnerae TBTC verified that bs613r bound exclusively to the target of interest and not to the T. turnerae target (data not shown). The experiments demonstrated that the positive control probe eub338r bound with both templates, confirming the accessibility of the DNAs for probing.
In situ hybridization experiments were essential to demonstrate that the newly designed B. setacea symbiont probe (bs613r) was specific for microbes residing within the host gill and not as external contaminants. Initial electron microscopic analysis of the gill tissue localized the bacterial symbionts to an intracellular location in host cells comprising distinct structures lining the interlamellar junction of the B. setacea gill (Fig. 3a). The eubacterial-universal control probe eub338r hybridized with bacteria within the gill (Fig. 3b). Both teredinid-directed probes (ter1243r and bs613r) hybridized in situ with the bacterial symbionts residing in the shipworm Gland of Deshayes (Fig. 3c and d). Both of the probes hybridized with target nucleic acids located in clusters (symbiont-containing vesicles, or bacteriocytes) along the gill lamellae but not with non-symbiont-containing tissue. As was expected, the nonhomologous probe control (11f) failed to form detectable hybrids (Fig. 3e). A no-probe control treatment demonstrated that the hybrid signal was not an artifact of the reaction conditions (data not shown). These experiments corroborate previous localization of teredinid symbionts within the bacteriocytes lining the gill interlamellar junctions of the other teredinid species (7).
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Evidence for vertical transmission. The results of the in situ hybridizations demonstrated that the B. setacea gill symbiont identified through cloning and 16S rDNA sequencing efforts resides within the B. setacea host gill. Because both the bs613r and ter1243r probes hybridized with the bacterial rRNA located within bacteriocytes in the host gill, it was concluded that these probes could be used as primers in the PCR to detect the symbiont target genes within the B. setacea gill and other tissues. A reverse complement of the bs613r probe (bs613f) was used in conjunction with the teredinid symbiont-specific probe (ter1243r) as a primer in the PCR to detect the presence of symbiont rDNA within host tissues. Both B. setacea and T. turnerae TBTC DNA templates were found to amplify with these primers. This cross-reactivity, however, was not a concern, since these two amplification products could be easily distinguished based on HaeIII RFLP analysis (Fig. 1). This was also the case with the closely related SCB11 bacterial strain. The published 16S rDNA gene sequence of SCB11 indicates the absence of an HaeIII recognition site at bp 846 (E. coli numbering), where a site exists in the 16S rDNA gene sequence of the gill bacterium in B. setacea. If the SCB11 template were to amplify with the B. setacea symbiont-directed primers, it could also be differentiated from the symbiotic template based on HaeIII digestion.
Conceivably, only a single bacterium would be necessary to initiate the symbiosis in a developing host larva. Detection at this level of resolution is not yet possible with conventional biochemical approaches. Nucleic acid amplification technologies based on the PCR provide the sensitivity and reproducibility necessary to identify the presence of extremely low copy numbers of a particular target sequence. This approach has been very successful in studies of symbiont transmission in other bivalve-bacterium associations (4, 5, 8, 9, 20). Bulk nucleic acids extracted and purified from gills, ovaries, eggs, and non-symbiont-containing siphon tissue were used as templates in PCR amplifications using the eukaryotic primer set (EukA-EukB) and the B. setacea symbiont primer set (bs613r-ter1243r). PCR amplification of the eukaryotic 18S ribosomal gene resulted in a band of the appropriate size (~1,800 bp) in all extracts, verifying the accessibility of the nucleic acid extracts for PCR amplification. The teredinid primer set clearly amplified its specific rDNA target (648 bp) in DNA extracts from gills, ovaries, and eggs. Amplification did not occur in the non-symbiont-containing siphon tissue which served as the negative control (Fig. 4). All positive amplifications were screened by RFLP and sequence analysis. PCR products from gill, ovary, and egg nucleic acid extracts, as well as the product amplified from the B. setacea 16S rRNA gene clone used in the original sequence analysis, all demonstrated identical restriction patterns. Direct sequencing of each purified PCR product further verified that the sequence of the symbiont-specific target amplified from the ovarial tissue and eggs was identical to the 16S rRNA gene sequence obtained from the symbiont-containing gill tissue. It was therefore concluded that the B. setacea gill symbiont was present in the ovary tissue and freshly spawned eggs, suggesting that this bacterium was transmitted vertically. While the presence of the DNA in the eggs and ovary as detected through PCR is highly suggestive of a vertical transmission strategy (4, 5, 8, 9, 20), verification that the bacteria are indeed viable and capable of initiating the symbiosis would require cultivation experiments which were beyond the scope of this investigation.
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Conclusions and implications. Although the present study indicates that the teredinid B. setacea harbors a unique symbiont that is transmitted vertically, previous work has shown that four different shipworm species representing different genera share a common symbiont strain (7). Clearly, the symbiotic relationships between teredinids and their bacterial counterparts are not as constrained as in other systems (8, 9, 20). This scenario is similar to another highly specific symbiosis between a single bacterial species (Vibrio fischeri) and several sepiolid squid host species (24). Given the great diversity of life history patterns, reproductive strategies, morphologies, and geographical distributions of species within the Teredinidae, varied strategies of symbiont acquisition among the species would not be surprising. Conceivably, teredinids may have the ability to acquire symbionts both vertically and from the environment. The diverse nature of the Teredinidae makes this family a model system for examining the mechanism of symbiont acquisition associated with clearly different life histories between closely related organisms.
The basis of this research is the hypothesis that the symbionts represent the weak link in the teredinid host life strategy and may be targeted in attempts to curb biodegradation of marine timber. Research is under way to characterize shipworm antifoulants derived from tropical wood species that demonstrate superior resistance to wood borers. Once characterized, these antifoulant compounds will be tested for their ability to inhibit the growth of the teredinid bacterial symbionts and thereby prevent teredinids from digesting the cellulose of treated wood.| |
ACKNOWLEDGMENTS |
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This research was supported by grants to S.C.C. from The Delaware Sea Grant Program (R/B 32) and the Smithsonian Institute Caribbean Coral Reef Ecosystem (CCRE) program.
We are indebted to K. Rützler and I. Feller for providing the unique opportunity to collect shipworm specimens in Belize. We thank R. Turner for encouragement and assistance early in this project. We thank K. Czymmek for technical assistance with confocal microscopy and R. Rhatigan for shipworm collection in Oregon. We also thank B. Campbell, K. Coyne, and A. Hacker for critically reviewing the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: College of Marine Studies, University of Delaware, 700 Pilottown Rd., Lewes, DE 19958. Phone: (302) 645-4078. Fax: (302) 645-4007. E-mail: caryc{at}udel.edu.
Present address: Department of Biological Sciences, University of
North Carolina-Wilmington, Wilmington, NC 28403.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results and Discussion
References
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