OCS Study 

MMS 2009-059 

Coastal Marine Institute 

Evaluation of Oil and Gas Platforms 

on the Louisiana Continental Shelf 

for Organisms with Biotechnology Potential 

U.S. Department of the Interior

Minerals Management Service 

Gulf of Mexico OCS Region 

Cooperative Agreement

Coastal Marine Institute 

Louisiana State University 
U.S. Department of the Interior Cooperative Agreement Minerals Management Service Coastal Marine Institute 

Gulf of Mexico OCS Region Louisiana State University 

OCS Study 

 MMS 2009-059 

Coastal Marine Institute 

Evaluation of Oil and Gas Platforms 

on the Louisiana Continental Shelf 

for Organisms with Biotechnology Potential 

Editor 

L. Rouse 

November 2009 

Prepared under MMS Cooperative Agreement 

1435-01-99-CA-30951-17809 

by 

Louisiana State University 

Coastal Marine Institute 

Baton Rouge, Louisiana 70803 

Published by 
iii

DISCLAIMER 

This report was prepared under contract between the Minerals Management Service (MMS) and 

Louisiana State University. This report has been technically reviewed by the MMS, and it has 

been approved for publication. Approval does not signify that the contents necessarily reflect the 

view and policies of the MMS, nor does mention of trade names or commercial products 

constitute endorsement or recommendation for use. It is, however, exempt from review and 

compliance with MMS editorial standards. 

REPORT AVAILABILITY 

This report is available only in compact disc format from the Minerals Management Service, 

Gulf of Mexico OCS Region, at a charge of $15.00, by referencing OCS Study MMS 2009-059. 

The report may be downloaded from the MMS website through the Environmental Studies 

Program Information System (ESPIS). This report can also be obtained from the National 

Technical Information Service. The addresses are below. Copies can also be inspected at 

selected Federal Depository Libraries. 

 Minerals Management Service U.S. Department of Commerce 

 Gulf of Mexico OCS Region National Technical Information Service 

 Public Information Office (MS 5034) 5285 Port Royal Road 

 1201 Elmwood Park Boulevard Springfield, Virginia 22161 

 New Orleans, Louisiana 70123-2394 Phone: (703) 605-6040 

Telephone requests may be placed at Fax: (703) 605-6900 

 (504) 736-2519, 1-800-200-GULF, or Email: bookstore@ntis.gov

Rush Orders: 1-800-553-6847 

 Fax: (504) 736-2620 

CITATION 

Rouse, L., ed. 2009. Evaluation of oil and gas platforms on the Louisiana continental shelf for 

organisms with biotechnology potential. U.S. Dept. of the Interior, Minerals Management 

Service, Gulf of Mexico OCS Region, New Orleans, LA. OCS Study MMS 2009-059. xi + 

53 pp. 

ABOUT THE COVER 

Day and night images of the semisubmersible petroleum platform “Nakika” located in the Gulf 

of Mexico at Mississippi Canyon Block 474. Images by David L. Nieland, Coastal Fisheries 

Institute, Louisiana State University, Baton Rouge, Louisiana. 
v 

PROJECT OVERVIEW 

This project was developed in response to MMS's recognition that offshore oil and gas 

platforms may serve as a harvestable source of organisms with pharmaceutical or other 

commercial applications. The first concern of this study, therefore, was, to identify the 

organisms that make up the biofouling or epifaunal communities on platforms. The second 

objective of this study was to preliminarily determine the potential of any of these organisms to 

be source of pharmaceuticals or any other commercially important products. Finally, the 

concentration and distribution of potentially important species on the structures and the 

variability of that distribution with platform location, and the density of organisms at various 

depths on the platform and seasonal variation of occurrence was considered. 

Some of the groups of organisms common to the Gulf of Mexico platforms that show 

potential for biotechnology applications were included in this study. These include bacteria, 

particularly members of the Class Actinobacteria, that have yielded numerous bioactive 

compounds valued as a source of pharmaceuticals and enzymes; marine algae that provide a 

range of natural products from agar to pharmaceuticals; benthic foraminiferans, especially 

agglutinated wall species; byssate molluscs that have potential in the production of bioadhesives 

for many uses from transdermal drug delivery systems to surgical adhesives; and bryozoans, 

particularly Bugula neritina, the only source of bryostatin, a drug used in cancer treatment. 

Two collecting trips were made from Port Fourchon, Louisiana, on the M/V Spree. On the 

first cruise, June 9, 2001, Tropical Storm Allison prevented sampling except at one of the five 

chosen platforms, ST 23. On the second cruise, May 4-5, 2002, four platforms were sampled and 

one platform could not be sampled due to dangerous diving conditions. 

At each sampling site, divers outlined a 25 cm x 25 cm area, using a metal template. A diver 

suctioned loose surface material from this area with a 60 cc syringe. All encrusting organisms 

were then scraped and preserved by methods appropriate to the taxa studied. The following is a 

summary of the findings from each group of organisms. 

BACTERIA

The study demonstrated that the biomatrix associated with gas and oil platforms contains 

large numbers of bacterial species many of which are novel and belong to taxonomic groups 

known to be of biotechnological importance. 

The construction of total genome libraries from the biomatrix of oil and gas platforms and 

their expression will no doubt provide novel bioactivities from both the micro and macro 

components of these complex systems. This study established that the biomatrix encrusted on oil 

and gas platforms represents a potential starting material for such studies. 

ALGAE

The total number of algal taxa collected during this study was 24. The best represented 

group was Rhodophyta (approximately 50% of the taxa collected). Seven new taxa were added 

to the list of macroalgae identified from the platforms and one new report of Antithamnionella 

breviramosa from the Gulf of Mexico. Approximately 50% of the taxa collected are known to 

have biotechnological potential. 
vi 

FORAMINIFERA

A rich assemblage of benthic Foraminifera was recognized in the scrapings and the syringe 

samples. The species included both grazers and attached forms. The assemblage density was 

highly variable. 

The most conspicuous aspect of the foraminiferal distribution was the abundance of attached 

taxa; some sessile species that are rare or absent in soft substrates live in relative profusion on 

the platforms. In addition, the foraminiferal community includes many vagrant foraminiferal 

species. Agglutinated foraminiferal species hold promise as a source of bioadhesives for 

biotechnological and biomedical applications, because they can secrete and then harden adhesive 

organic compounds in an aqueous medium although no species with known biotechnical 

potential were found. 

BRYOZOA

To date the only marine compound to enter phase II clinical trials is bryostatin 1. Bryostatin 

1 combats the growth of cultured cancer cells and has shown some promise aiding patients 

suffering from non-Hodgkin's lymphoma and lymphocytic leukemia. Bryostatin 1 was initially 

isolated from the bryozoan Bugula neritina, but in California that named species turned out to 

be composed of several cryptic species, only one of which produces bryostatin I. The compound 

occurs in an endosymbiotic bacteria present within B. neritina. 

B. neritina as currently recognized in US waters comprises three cryptic species: the deep 

water Pacific form, a Shallow/Southern form present both in shallower Pacific waters and along 

the Atlantic coast south of Cape Hatteras, and a third form present in the Atlantic north of the 

Cape Hatteras region. This study found that neither the species collected from the Gulf of 

Mexico platforms nor those found in the Atlantic produce the commercially important bryostatin 

I. 

MOLLUSCS

The platforms sampled hosted a low diversity molluscan assemblage dominated by byssate 

bivalves. Twenty-seven bivalve and gastropod species were identified, with Isognomon bicolor 

and Barbatia candida making up about 90% of the total assemblage. Changes in relative 

abundance of taxa among platforms were in part caused by mollusks collected in this study with 

the greatest biotechnology potential include the byssate bivalves Isognomon and Barbatia that 

produce bioadhesives. The byssus is a bundle of proteinaceous threads secreted by some 

bivalves that attaches to a substrate by an adhesive plaque. These adhesives are of 

biotechnological interest because they provide strong, durable adhesion to wet surfaces. 

Taxonomic and genetic heterogeneity of marine organisms must be anticipated prior to 

surveys for biotechnologically useful molecules and studies of ecological processes. This study 

examined the genetic variability of the bivalve community associated with northern GOM oil 

platforms in order to (1) determine if there were cryptic species and (2) examine the genetic 

variation within and among platforms. Data collected indicated that all of the individuals were 

Barbatia candida. 
vii 

TABLE OF CONTENTS 

Page 

PROJECT OVERVIEW ................................................................................................................. v 

FIGURES....................................................................................................................................... ix 

TABLES ........................................................................................................................................ xi 

I. PROJECT OVERVIEW ........................................................................................................ 1 

II. GULF OF MEXICO PLATFORM STUDY SITES AND FIELD COLLECTION 

METHODS ............................................................................................................................ 3 

III. BACTERIA............................................................................................................................ 5 

Introduction ........................................................................................................................... 5 

Methods................................................................................................................................. 5 

Sites Sampled ..................................................................................................................5 

Total Counts and Isolation ..............................................................................................5 

Selection of Isolates.........................................................................................................6 

Identification Using ARDRA and 16S rRNA Gene Sequencing ....................................6 

Establishment of a Culture Collection.............................................................................6 

Results ................................................................................................................................... 6 

Bacterial Counts..............................................................................................................6 

Diversity of Organisms Recovered .................................................................................7 

Products of This Study ....................................................................................................8 

 Novel Isolates............................................................................................................8 

 Novel Strain Collection.............................................................................................9 

Conclusions ........................................................................................................................... 9 

References ........................................................................................................................... 10 

IV. MACROALGAE ................................................................................................................. 11 

Introduction ......................................................................................................................... 11 

Methods............................................................................................................................... 11 

Results ................................................................................................................................. 12 

Conclusions......................................................................................................................... 15 

References ........................................................................................................................... 15 

V. FORAMINIFERA................................................................................................................ 17 

Introduction ......................................................................................................................... 17 

Methods............................................................................................................................... 17 

Laboratory Methods ......................................................................................................17 

Results ................................................................................................................................. 18 

Assemblage Density......................................................................................................18 

Foraminiferal Diversity and Dominance.......................................................................19 

Attached Species ...........................................................................................................19 
viii 

Discussion .....................................................................................................................20 

Biotechnology Potential ................................................................................................26 

Conclusions ......................................................................................................................... 26 

Literature Cited.................................................................................................................... 26 

VI. BRYOZOANS ..................................................................................................................... 29 

Introduction ......................................................................................................................... 29 

Methods............................................................................................................................... 30 

Population Sampling .....................................................................................................30 

Genetic Variation within B. neritina .............................................................................31 

Genetic Variation of Bacteria Associated with B. neritina ...........................................31 

Results ................................................................................................................................. 31 

Cryptic Species within Bugula neritina ........................................................................31 

Discussion ........................................................................................................................... 32 

Conclusions ......................................................................................................................... 33 

Acknowledgments............................................................................................................... 34 

References ........................................................................................................................... 34 

VII. MOLLUSCS ........................................................................................................................ 37 

Introduction ......................................................................................................................... 37 

Previous Work...............................................................................................................37 

Methods............................................................................................................................... 38 

Sample Preparation........................................................................................................38 

Results ................................................................................................................................. 38 

Conclusions ......................................................................................................................... 40 

Potential Significance of Platform Molluscs to Biotechnology ....................................40 

Literature Cited.................................................................................................................... 41 

VIII. GENETIC VARIATION OF MOLLUSCS......................................................................... 43 

Introduction ......................................................................................................................... 43 

Methods............................................................................................................................... 43 

Collection of Samples ...................................................................................................43 

Genetic Methods............................................................................................................44 

Data Analysis ................................................................................................................44 

Results ................................................................................................................................. 45 

Discussion ........................................................................................................................... 47 

Acknowledgments............................................................................................................... 47 

Literature Cited.................................................................................................................... 47 

IX. CONCLUSIONS.................................................................................................................. 51 

Bacteria................................................................................................................................ 51 

Macroalgae .......................................................................................................................... 51 

Foraminifera ........................................................................................................................ 52 

Bryozoa ............................................................................................................................... 52 

Molluscs .............................................................................................................................. 52 
ix 

FIGURES 

Page 

Figure II-1. Platform locations. .................................................................................................. 3 

Figure III-1. Variation in cfu/g biomatrix with depth and culture media for Platforms GI 

82A and GI 42C...................................................................................................... 7 

Figure III-2. Diverse colony morphologies observed in this isolation study. ............................. 8 

Figure IV-1. Vertical distribution of algae ................................................................................ 14 

Figure V-1. Estimated foraminiferal density (number/m2

) on platform surface. ..................... 18 

Figure V-2. Attached agglutinated species; a, b, d, digital photomicrographs, others 

scanning electron micrographs. a, Ammobaculites sp. cf. A. agglutinans

(GI95A-W10A); b, Glaphyrammina americana (ST67H-W20A); c, 

Placopsilina sp. (GI95A-W10A); d, e, Sagenina divaricans (d: GI95A-W10A, 

e: GI42C-E20A); f, Textularia sp. (ST67H-E10B); g, Trochammina

sp.(GI95A-W10A). ............................................................................................... 21 

Figure V-3. Attached calcareous species; a, c, d, f, digital photomicrographs, others 

scanning electron micrographs. a (including inset), Acervulina sp. (GI42CE20A); b-e, Calcituba polymorpha (sensu Arnold; GI82A-W0A); f, g, 

Cibicides lobatulus (f: GI42C-E20A, g: GI95A-W20A)...................................... 22 

Figure V-4. Attached calcareous species (contd.); e, digital photomicrograph, others 

scanning electron micrographs. a, Cornuspira involvens (GI95A-W10A); b, 

Hanzawaia concentrica (ST67H bottom); c, Neoconorbina sp. (GI95AW20B); d, Melonis sp. (GI95A-W10A); e-g, Nodobacularia bonairensis (e, f: 

GI82A-W20A, g: GI95A-W10A)......................................................................... 23 

Figure V-5. Attached calcareous species (contd.); a-c, digital photomicrographs, others 

scanning electron micrographs. a, Nubeculinita sp. cf. N. inhaerens (ST67HW20A); b, c, Parrina bradyi (b, detached specimen) (GI42C-E20A); d, 

Patellina corrugata (ST67H-E10B); e, f, Planorbulina mediterranensis (e: 

GI82A-W10B, f: GI82A-W0A)............................................................................ 24 

Figure V-6. Attached calcareous species (contd.); a, e, digital photomicrographs, others 

scanning electron micrographs. a, Rectocibicides sp. cf. R. miocenicus

(ST67H-W20A); b, c, Rosalina globularis (GI95A-W0A); d, Spirillina 

vivipara (GI95A-W10A); e, Rosalina sp. (GI42C-E20A).................................... 25 

Figure VII-1 Relative abundance of molluscan species from each platform and for all 

platforms combined (labeled total) ....................................................................... 39 

Figure VII-2. Relative abundance of molluscan species at each sampling level and for all 

sampling levels combined (all depths).................................................................. 40 

Figure VIII-1. Number of species collected and species diversity............................................... 45 

Figure VIII-2. Comparison of mitochondrial COX I DNA sequence data with previously 

published data on Arcidae (Marko & Moran, 2002; Marko, 2002)...................... 46 
xi 

TABLES 

Page 

Table II-1. Platform locations and water depths....................................................................... 3 

Table II-2. Sample replicates collected..................................................................................... 4 

Table III-1. Number of strains collected on each platform sampled.......................................... 9 

Table IV-1. Algal Taxa Collected ............................................................................................ 13 

Table V-1. Foraminiferal density data from five samples. ..................................................... 19 

Table VIII-1. Numbers of Barbatia candida sampled from five oil platforms for RFLP 

analysis.................................................................................................................. 45 

Table VIII-2. Results of AMOVA analysis showing genetic variation of Barbatia candida

within and among five oil platforms in the northern Gulf of Mexico. ................. 47 
1 

I. PROJECT OVERVIEW 

This project was developed in response to MMS's recognition that offshore oil and gas 

platforms may serve as a harvestable source of organisms with pharmaceutical or other 

commercial applications. The project also considered recommendations from the 1998 National 

Ocean Conference Report (Turning to the Sea: America’s Ocean Future, 1999; available at 

http://www.publicaffairs.noaa.gov/oceansreport/) to (1) increase support for sustainable 

harvesting and testing of marine compounds by both government agencies and commercial 

pharmaceutical companies as possible treatments for AIDS, inflammatory or infectious diseases, 

and cancers; and (2) support research on the environmental effects of extracting marine 

organisms for biotechnology purposes. 

Over 3000 oil and gas platforms in the Gulf of Mexico provide hard substrate for the 

attachment and growth of large concentrations of fouling organisms in areas otherwise devoid of 

hard substrate. These platforms, consisting of vertical and horizontal steel members, could form 

the substrate basis for harvesting organisms with potential for biotechnology applications. 

The first objective of this study was, therefore, to identify the organisms that make up the 

biofouling or epifaunal communities on platforms. The second objective of this study was to 

preliminarily determine the potential of any of these organisms to be a source of pharmaceuticals 

or any other commercially important products. Finally, the concentration and distribution of 

potentially important species on the structures and the variability of that distribution with 

platform location, density of organisms at various depths on the platform and seasonal variation 

of occurrence was considered. Knowledge of spatial and temporal variation will be necessary in 

determining the cost effectiveness of harvesting any potentially important organisms. 

Alternately, the knowledge of the combination of factors necessary to maximize biomass 

production of individual species may lead to the design of artificial substrates used exclusively to 

farm commercially important organisms. 

Some of the groups of organisms common to the Gulf of Mexico oil and gas platforms that 

show potential for biotechnology applications were included in this study. These include 

bacteria, particularly members of the class Actinobacteria, that have yielded numerous bioactive 

compounds valued as a source of pharmaceuticals and enzymes; marine algae that provide a 

range of natural products from agar to pharmaceuticals; benthic foraminiferans, especially 

agglutinated wall species; byssate molluscs that have potential in the production of bioadhesives 

for many uses from transdermal drug delivery systems to surgical adhesives; and bryozoans, 

particularly Bugula neritina, a source of bryostatin in some areas, a drug used in cancer 

treatment. 

For this project, six teams of scientists were assembled from the Coastal Marine Institute at 

Louisiana State University in collaboration with University of Louisiana in Lafayette to address 

research objectives relative to the chosen taxa. The Project Director was Dr. Lawrence Rouse, 

and the co-principal investigators were Drs. Fred Rainey (bacteria), Russell Chapman (algae), 

Barun San Gupta (benthic foraminiferans), Michael Hellberg (bryozoans), Laurie Anderson 

(molluscs) and David Foltz (genetic analyses of molluscs). Reports of these investigators (and 

co-investigators) resulting from the relevant studies of their respective taxonomic groups are 

presented in full with the exception of the platform locations and field collection methods of the 

samples shared among all investigators and described below. 
3 

II. GULF OF MEXICO PLATFORM STUDY SITES AND FIELD 

COLLECTION METHODS 

Two collecting trips were made from Port Fourchon, Louisiana, on M/V Spree. On the first 

cruise, June 9, 2001, Tropical Storm Allison prevented sampling, except at one of the five 

chosen platforms, ST 23. On the second cruise, May 4-5, 2002, one platform could not be 

sampled due to dangerous diving conditions. The latitude/longitude locations of the platforms 

sampled are given in Table II-1 and their relative locations are shown in Figure II-1. 

Table II-1 

 

Platform Locations and Water Depths 

 Platform Location Depth (m) Collection Date

ST 23 (Chevron South Timbalier Block 

23) 29o 01.40' N, 90o 10.17' W 15.8 June 9, 2001 

ST 67H (ExxonMobil South Timbalier 

Block 67H) 28o 47.935' N, 90o 24.889' W 19.2 May 5, 2002 

GI 42C (Vastar Grand Isle Block 42C) 28o 59.947' N, 89o 56.264' W 30.5 May 4, 2002 

GI 82A (El Paso Grand Isle Block 82A) 28o 43.301' N, 89o 57.916' W 51.8 May 5, 2002 

GI 95A (Vastar Grand Isle Block 95A) 28o 30.962' N, 90o 07.366' W 61 May 5, 2002 

Figure II-1. Platform locations.
4 

Scrapings from an easterly (E) and westerly (W) leg were taken from each platform, using 

two teams of three divers. Ideally, three levels were to be sampled: near surface (0 m), 10 m, and 

20 m. Three (25 cm x 25 cm) areas (A, B, and C) were to be scraped at each sampling level on 

each leg. This protocol could not be strictly followed due to rough seas. No surface samples 

were collected at ST 23, and only one surface sample was collected at GI 42C. One surface 

sample per leg was sampled at the other three platforms. Only five samples were taken at the 10 

m level at ST 23 and GI 42C because of diving gear problems. One replicate from 10 m on the 

west leg of ST 42C was collected but lost before reaching the lab. A summary of the samples 

collected from each platform leg is given in Table II-2. At sites where bottom water depth was 

less than 20 m, samples labeled “20 m” were actually taken 1 m above the bottom. 

Table II-2

 

Sample Replicates Collected 

Platform Leg 0 m 10 m 20 m 

 

ST 23 E 0 3 3 

 W 0 2 3 

ST 67H E 0 3 3 

 W 1 3 3 

ST 42C E 1 3* 3 

 W 0 2 3 

GI 83A E 1 3 3 

 W 1 3 3 

GI 95A E 1 3 3 

 W 1 3 3 

* Sample Lost in Transit. 

At each sampling site, divers outlined a 25 cm X 25 cm area, using a metal template, and the 

labeled quadrat was photographed. The diver then suctioned loose surface material off this area 

with a 60 cc syringe. All incrusting organisms were then chiseled off into a large, prelabeled, 

resealable, plastic bag. At two of the shallower platforms (ST 23 and ST 67), divers collected 

bottom samples within 1 m of the platform leg, using a 1-l Histoplex jar to scoop up sediment. 

At these stations, bottom sediment samples were also collected away from the platform, 

deploying a petite ponar grab from the boat. 

Once samples were brought aboard, bacteria subsamples were immediately taken from the 

scrapings. Each bag of scrapings was then emptied into a plastic tub, an index card with the 

sample name was added, and any specimens of algae or the bryozoan, Bugula, were removed. 

The remaining contents of the tub were returned to the plastic bag, using seawater to rinse down 

the sides. The sample was double-bagged, if necessary, and frozen. Syringe samples were placed 

in an ice water bath. Bottom sediment samples were kept in the 1-liter collection jars and 

preserved in ethanol (unstained). 

These samples were used for analyses of the bacteria, algae, foraminiferans, bryozoans and 

molluscs in the studies that follow. Additional sampling locations from off the east coast from 

Florida to Massachusetts for comparative genetic analysis of the bryozoan Bugula are addressed 

in Section VI. 
5 

III. BACTERIA 

Survey of the Numbers and Diversity of Bacteria Associated with the 

Biomatrix of Oil and Gas Platforms on the Louisiana Continental Shelf 

Fred A. Rainey 

Department of Biological Sciences 

Louisiana State University 

INTRODUCTION 

Here we report the data obtained on the numbers and diversity of members of the domain 

Bacteria associated with the biomatrix on oil and gas platforms off the Louisiana coast. This 

study was part of a multi-taxa investigation to look at the biodiversity associated with gas and oil 

platforms with the overall aim of using these organisms for biotechnological applications. The 

specific aims of the project were to (1) determine the numbers and diversity of the bacteria 

associated with the biomatrix on the oil and gas platforms, (2) determine the location of these 

organisms with respect to depth above or below the water line, and (3) determine if any of the 

bacteria associated with the biomatrix have potential for biotechnological applications. There is 

no previous work described in the literature regarding the bacterial populations associated with 

the encrusted biomatrix on oil and gas platforms. 

METHODS

Sites Sampled 

In 2001 the biomatrix on a single platform ST 23 was sampled. The following year in May 

2002 five platforms were sampled as described in Section II. Samples taken from the biomatrix 

encrusting the platform legs were very heterogeneous both in structure and macro-organism 

composition. 

Total Counts and Isolation 

Ten grams of biomatrix was added to 90 ml of sterile artificial seawater (Instant Ocean) at 4 

°C and homogenized using a stainless steel blender for 5 mins. The resulting slurry was serially 

diluted in sterile artificial seawater before plating on solid media. Two approaches were taken for 

the analysis of the bacterial populations. One was the approach of determination of the total 

heterotrophic bacteria that could be sultured from the biomatrix samples and the other was using 

specific media to target the isolation of specific groups of organisms known to have 

biotechnological potential. Total counts were determined on Marine Agar (Difco) and one-tenth 

strength Marine Agar (prepared in full strength artifical seawater). For specific isolations Starch 

Casein Agar (10g soluble starch, 1g casein, 0.5g K2HPO4, 20g Agar, 1L Instant Ocean) and 

YME Agar (4g yeast extract, 10g malt extract, 4g dextrose, 20g Agar, 1L Instant Ocean) were 

used. To these isolation media cyclohexamide (100ug/ml) and rifampicin (5ug/ml) were added. 

These filter-sterilized antibiotics were added after the agar was autoclaved. 
6 

Selection of Isolates 

Since numerous isolates have been recovered from isolation studies of marine environment 

samples a selection strategy was used to attempt to isolate and establish a collection of diverse 

organisms for further study. Organisms for molecular identification and inclusion in the culture 

collection were selected on the basis of colony morphological characteristics including colony 

size, colony color and texture of the colony material. 

Identification Using ARDRA and 16S rRNA Gene Sequencing 

Genomic DNA was isolated from the strains and the 16S rRNA gene amplified using PCR. 

The ARDRA technique was used to group the isolates and reduce duplication prior to 

sequencing. The amplification and sequencing was done as described by Rainey et al. (1996). 

The 16S rRNA gene sequences were compared with all available sequences in the GenBank 

database using the BLAST tool (Altschul, et al., 1990). 

Establishment of a Culture Collection 

A culture collection was established in which each of the isolates selected for identification 

was purified and preserved in liquid media containing 15% (v/v) glycerol at -80 °C. The strains 

have been preserved in triplicate. 

RESULTS

Bacterial Counts 

Total colony forming units (cfu) per gram of biomatrix were determined for each of the five 

platforms studied. Counts were determined for samples taken at 0, 10 and 20 meters and at one 

meter above the surface for sites in less than 20m of water (ST23 and ST67H). Samples were 

dilution plated on Marine Agar or 1/10 strength Marine Agar. The 1/10 strength Marine Agar 

contains a lower amount of carbon and energy sources (sugars and yeast extract) and was used 

based on previous observations in attempts to recover novel organisms from low nutrient 

environments. The total colony forming units per gram of biomatrix for all five platforms 

sampled ranged from 5.8 x 104

 to 9.0 x 106. The lowest numbers of cfus were found for platform 

GI82A-W at a depth of 20m (plated on Marine Agar) and the highest counts were found for 

platform GI42CW at a depth of 10m (plated on 1/10 strength Marine Agar). Figure III-1 shows 

a graphical representation of the cfu/g of biomatrix for the platforms GI-82Aand GI-42C. The E 

and W designations indicate the east and west supports of the platform. From the comparison of 

these two data sets we can see there are significant differences in the numbers of colony forming 

units recovered from the different platforms, the different locations (east and west support 

structures) and the depth sampled. In the case of samples from the west support structure of 

platform GI-82A it can be seen on both MA and 1/10 MA the cfu/g of biomatrix are significantly 

lower than that found on the east support structures of the same platform at a depth of 20m. 

Interestingly at 10m the reverse is the case with the higher numbers of cfu/g of biomatrix being 

recovered from the west support structures. 

These differences between depths and east/west location are a common trend in the data sets 

obtained. The numbers of cfu/g of biomatrix recovered will be directly related to the macro 

composition of the samples from which the bacteria are isolated. Samples containing higher 
7 

numbers of macro scale organisms will contain higher numbers of culturable bacteria when 

compared to samples containing low numbers of living macro scale organisms. 

Diversity of Organisms Recovered 

The biomatrix of the platforms studied harbor an extensive diversity of bacterial species. This 

is not unexpected considering the large diversity of bacteria present in the majority of marine 

environments. The biomatrix does provide a location for the accumulation of diversity that in the 

case of the open ocean would be much more dilute and require extensive concentration to 

recover a similar level of diversity. The use of 16S rRNA gene sequencing allows us to realize 

this diversity and put it into a taxonomic context. When looking at the diversity in terms of 

similarity to other species or strains it should also be considered that strains of the same species 

can produce very different secondary metabolites and bioactive products. 

Figure III-1. Variation in cfu/g biomatrix with depth and culture media for Platforms GI 82A 

and GI 42C. 

From the serial dilution plate counts on Marine Agar and 1/10 strength Marine Agar bacteria 

from three phyla of the domain Bacteria are recovered. The phyla represented by these isolates 

are the Firmicutes, Proteobacteria and Actinobacteria. The isolates for further study were 

selected based on the diversity of their colony morphologies when grown on agar plates. The 

majority of the isolates, 69% of them fall within the phylum Proteobacteria and represent species 

of genera which are typically found in the marine environment eg. Vibrio, Photobacterium, 

Pseudoalteromonas, Microbulbifer, Shewanella and Ruegeria. Some 29% of the isolates from 

serial dilution plating are representatives of species of genera in the Firmicutes phyla. These 

isolates are mostly Bacillus species with a small number of Plannococcus species. The remaining 

2% of the isolates are actinobacteria of the genera Streptomyces and Gordonia. Figure III-2
8 

provides an example of the diverse types of colony morphologies observed in this isolation 

study. 

Figure III-2. Diverse colony morphologies observed in this 

isolation study. 

Using the more selective approach of additional culture media (as described in methods 

section) that contained the antibiotics cyclohexamide and rifampicin the Gram negative members 

of the Proteobacteria were eliminated and large numbers of Gram positives especially 

actinobacteria that were only represented at low levels on Marine Agar cultivation could be 

enriched. It is these actinobacteria that have traditionally been a great source of secondary 

metabolites and bioactive compounds. Using this approach we cultivated a large number (156) of 

actinobacteria including members of the genera Streptomyces, Micromonospora, and Agromyces. 

Species of the genera Streptomyces and Micromonospora are well known for their production of 

bioactive molecules including antibiotics. Based on their 16S rRNA gene sequence data these 

isolates represent new species of these genera and therefore have the potential to produce novel 

compounds. 

Products of This Study 

Novel Isolates 

Although the isolates for the most part represent species of already described genera the 

majority of them have 16S rRNA gene sequence similarities of less than 98% to other described 

species. 16S rRNA gene sequence similarity values below 98% point to the novelty of these 

isolates at the species level and in some cases at the genus level. The large number of 

actinobacteria that can be recovered from the biomatrix on these platforms is encouraging both in 

the fact that these organisms are part of the renewable biomatrix but in addition these 

actinobacterial components contain novel and as yet undescribed species. The large number of 

Bacillus species could be by common view written off as insignificant with respect to 

biotechnological applications. However, in collaboration with a colleague at University of 
9 

Louisiana Monroe (ULM) we have found similar Bacillus strains from sea sponges that act as 

biocatalysts producing compounds that have anti-tumor properties (Elsayed et al., 2007). These 

novel isolates represent a resource that is currently being studied both at LSU and at ULM for 

their biotechnological applications. In 2003 as a result of the data obtained in this project the 

Louisiana BOR (through the Governor’s Biotechnology Initiative) funded a permanent faculty 

line and a technical line in the area of natural products science at LSU. 

Novel Strain Collection 

A collection of some 400 strains (purified and preserved) has been established at LSU. The 

number of strains from each of the five platforms sampled is shown in Table III-1. Some of 

these strains have been provided to researchers at LSU and ULM for further study. A database of 

the strains, source and their growth conditions has been established. 

Table III-1

 

Number of Strains Collected on Each Platform Sampled 

Platform Strains Included in the Collection 

GI 82A 81 

GI 95A 31 

GI 42C 78 

ST 23 101 

ST 67H 109 

CONCLUSIONS 

The study demonstrated that the biomatrix associated with oil and gas platforms contains 

large numbers of bacterial species many of which are novel and belong to taxonomic groups 

known to be of biotechnological importance. There are a number of factors that would seem to 

influence the bacterial load and diversity of the biomatrix recovered from a given location. The 

composition and density of the macroorganisms would seem to influence the numbers and 

diversity of the bacterial species recovered by conventional isolation techniques. The isolation 

approach used also influences the types and diversity of the strains recovered. The use of general 

culture media such as Marine Agar provides both proteobacterial and Gram positive bacteria 

with the majority being Proteobacteria which are known to be dominant in marine environments. 

Using a selective approach can result in the isolation of more Gram positives and actinobacterial 

species which in general are considered to have greater potential for biotechnological 

applications. This study clearly shows that the biomatrix on the structures of oil and gas 

platforms is the habitat of novel bacterial species that represent a biotechnological resource 

waiting to be exploited. Isolation and natural product screening still has its place in 

biotechnology but the application of whole genome studies to such environments is the way 

forward. The construction of total genome libraries from the biomatrix of oil and gas platforms 

and their expression will no doubt provide novel bioactivities from both the micro and macro 

components of these complex systems. Here we have established that the biomatrix encrusted on 

oil and gas platforms represents a potential starting material for such studies. 
10 

REFERENCES 

Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment 

search tool. J. Mol. Biol. 215:403-410. 

El Sayed K.A., S. Laphookhieo, H.N. Baraka, M. Yousaf, A. Hebert, D. Bagaley, F.A. Rainey, 

A. Muralidharan, S. Thomas, and G.V. Shah. 2008. Biocatalytic and semisynthetic 

optimization of the anti-invasive tobacco (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-

diol. Bioorganic & Medicinal chemistry 16(6):2886-2893. 

Rainey, F. A., N. Ward-Rainey, R. M. Kroppenstedt, and E. Stackebrandt. 1996. The genus 

Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete 

lineage: proposal of Nocardiopsaceae fam. nov. International Journal of Systematic 

Bacteriology 46:1088-1092. 
11 

IV. MACROALGAE 

Biodiversity and Potential Biotechnological Uses of Marine Macroalgae from the 

Offshore Oil and Gas Platforms in the Gulf of Mexico 

Juan M. López-Bautista1

 and Suzanne Fredericq 

 University of Louisiana at Lafayette 

Russell L. Chapman2

 and Debra A. Waters 

Louisiana State University 

INTRODUCTION

The more than 3000 standing oil and gas platforms in the northwestern Gulf of Mexico 

(GOM) provide habitat for a significant community of marine organisms. As part of a larger 

study to determine the potential for organisms inhabiting oil rigs to provide a harvestable source 

of organisms with biotechnology potential, this study was designed to determine species 

diversity, relative abundance, and vertical, horizontal, and seasonal distribution of macroalgae. 

METHODS

The May 2002 cruise visited four platforms where algae were collected GI 42C, GI 82A, 

GI 95A, ST 23, and ST 67H (Figure II-1).These sites were selected to follow a horizontal 

gradient that allowed comparison of near-shore to outer-shelf assemblages. 

At each site, divers sampled the east-west legs of the platform as described in Section I.

After sample bags were carried on board, the algal component was selected with forceps or 

spatulas and transferred to labeled bags. The thalli were sorted as follows: 

• 48 sample bags fixed in 90% ethanol for algal identification, curation on semipermanent microscope slides, and DNA extraction and molecular study; 

• 14 sample bags of algae were kept alive in a cool chamber in seawater for 

further culture studies in the laboratory at University of Louisiana at Lafayette 

(ULL). 

Cultures were maintained at 18o

C with a light/dark regime of 16/8 hrs in a Percival Scientific 

benchtop plant growth chamber Model E-30B in seawater enriched with Alga/Gro (Carolina 

Biological Supply Co.). A collection of isolates in a culture chamber was kept in seawater 

medium for further detailed studies. DNA studies for ongoing molecular systematic analysis 

were done on some samples using the following methods: 

For the DNA extraction, a small portion of the algal sample was ground in liquid nitrogen 

with a mortar and pestle. DNeasy Plant Mini Kit from QIAGEN was used following the 

manufacturer’s protocol. For gene amplification, 2:l of the resulting extraction was used as 

template for a 50:l PCR consisting of 10:l 5M betaine, 6:l 10x PCR buffer (Perkin Elmer Corp.), 

 1

 Currently at the University of Alabama, Tuscaloosa, Alabama. 2

 Currently at Scripps Institutions of Oceanography, University of San Diego, California. 
12 

6:l 25 mM MgCl2 solution, 8:l of 500 mM dNTP stock, 2:l each of the appropriate primers at 10 

mM, and 0.3-0.5:l Amplitaq® DNA Polymerase. Amplification conditions for rbcL consisted of 

4 minutes at 96°C for denaturation, followed by 30 cycles of 60 seconds at 94°C, 60 seconds at 

either 45°C or 42°C, and 90 seconds at 72°C, with a final 10 minute extension cycle at 72°C, and 

soak cycle at 4°C. The amplification reactions were performed on a PE GeneAmp PCR system 

9700 or 2400. For automated gene sequencing, amplification products were cleaned of excess 

primer, enzyme, and dNTPs by PEG precipitation (Hillis et al., 1996). 

The sequences were determined over both strands using an ABI Prism 3100 Genetic 

Analyzer (PE Applied Biosystems, Foster City, CA) with the ABI Prism BigDye Terminator 

Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA, USA). Reaction mixtures 

comprised 4 :l Terminator Ready reaction mix with 4 :l 2.5X buffer or 8:l Terminator Ready 

reaction mix, 1-2 :l template, 3.2 pmol primer, and deionized water q.s. up to a total volume of 

20 :l. The cycle sequencing reactions were performed on a PE GeneAmp PCR system 9700 or 

2400 for 25 cycles (96°C for 10 seconds, rapid thermal ramp to 50°C, 50°C for 5 sec., rapid 

thermal ramp to 60°C, 60°C for 4 min, rapid thermal ramp to 4°C). Resulting products were then 

purified using Centri-Sep spin columns (Princeton Separations P/N CS-901) following the 

manufacturer’s instructions. 

RESULTS 

A total of 24 taxa have been identified from the samples. Table IV-1 is a list of algal taxa 

found. The number of taxa by division/class were as follows: 

• Chlorophyta: 39 

• Rhodophyta: 42 

• Phaeophyceae: 19 

• Cyanophyta: 3 

The macroalgae collected from the platforms are known to occur in the northwestern GOM 

with the exception of one red algal species, Antithamnionella breviramosa. It is reported here for 

the first time from the GOM. It was previously known from Pacific Mexico, eastern Australia, 

the Solomon Islands, North Carolina, and Brazil. Six other species, although known to occur in 

the Gulf of Mexico, were reported for the first time from the oil and gas platforms in Louisiana. 

They included Ulva fasciata, Chaetomorpha aerea, Gelidium pusillus, Jania capillacea, 

Callithamniella tingitana, Centrocerus clavulatum. All but one belong to genera with 

biotechnological potential. 
13 

Table IV-1 

 

Algal Taxa Collected 

Chlorophyta *Bryopsis pennata Lamouroux 

*Chaetomorpha aerea (Dillwyn) Kützing 

*Cladophora sp. 

*Enteromorpha spp. 

Entocladia viridis Reinke 

*Ulva fasciata Delile 

Ulvella lens P. Crouan & H. Crouan

Rhodophyta Acrochaetium hypneae Børgesen 

Acrochaetium microscopicum (Nageli ex Kützing) Nägeli +

Antithamnionella breviramosa (Dawson) Wollaston in Womersley & Bailey+ 

Callithamniella tingitana (Schousboe ex Bornet) Feldman-Mazoyer 

*Centroceras clavulatum (C. Agardh in Kunth) Montagne in Durieu de 

Maisonneuve 

*Ceramium flaccidum (Kützing) Ardisson 

Erythrotrichia carnea (Dillwyn) J. Agardh 

*Gelidium pusillum (Stachouse) Le Jolis 

Herposiphonia secunda (C. Agardh) Ambronn f. tenella (C. Agardh) Wynne 

*Jania capillacea Harvey 

Kylinia crassipes (Børgesen) Kylin 

Sahlingia subintegra (Rosenvinge) Kornmann

Phaeophyceae Kuetzingiella elaschistaeformis (Heydrich) Balakkrishnan & Kinkar 

Sphacelaria rigidula Kützing

Cyanobacteria Gloeocapsa sp. 

Oscillatoria acuminata Gomont 

Spirulina sp. +

First report from the Gulf of Mexico. 

*Taxa have been reported as being of biotechnological potential. 

Approximately half of the macroalgae collected from the platforms are known to have 

biotechnological potential. Some of their properties that are or may be of potential use are given 

below. 

• Agar Producing: Gelidium spp. (Cooper and Johnstone, 1944) 

• Antibacterial: Cladophora spp., Gelidium spp. (Trono, 1999), Centroceras 

clavulatum (Caccamese et al., 1981) 

• Antifungal: Bryopsis spp. (Tringali, 1997), Cladophora spp. (Trono, 1999; 

Ballesteros et al., 1992), Centroceras clavulatum, (Caccamese et al., 1981), 

Gelidium pusillum, Jania spp. (Ballesteros et al., 1992) 

• Antiherpetic: Chaetomorpha spp., Cladophora spp., Eneromorpha spp., 

Centroceras clavulatum (Hayashi et al., 1996), Ulva fasciata (Santos et al., 

1999) 

• Anti-inflammatory: Gelidium spp. (Baker, 1984) 
14 

• Anti-influenza: Ceramium spp., Gelidium spp. (Serkedjieva et al., 2000) 

• Antitumoral: Bryopsis spp. (Tringali, 1997), Enteromorpha spp. (HagashiOkai et al., 1999) 

• Antiviral: Bryopsis spp, Ulva fasciata (Tringali, 1997), Cladophora spp., 

Gelidium spp. (Trono, 1999), Enteromorpha spp. (Kim et al., 1997), 

Centroceras clavulatum (Caccamese et al., 1981) 

• Diuretic: Enteromorpha spp. (Naqvi et al., 1980). Additionally, the agar 

produced from red algae is widely known to have antirheumatic properties 

and alginates from brown algae (Phaeophyceae) have been studied for spinal 

chord, and nerve regeneration (Novikov et al.; 2002), bone repair (FelicioFernandex and Laranjeira, 2000) and angiogenesis (Zemani et al., 2005). 

Spirulina (Cyanophyta) is used as a food additive (Miranda et al, 1998). 

Figure IV-1 shows the vertical distribution of the algae commonly collected at the platforms. 

Figure IV-1. Vertical distribution of algae. 

A zonation of algal communities in the intertidal area was visible at all stations and consisted 

of barnacles and small blades of green algae, mainly Ulva fasciata and several species of 
15 

Enteromorpha. From low intertidal to shallow intertidal, common algae included Chaetomorpha 

aerea, Cladophora sp., Kuetzingiella elachistaeformis, Gelidium pusillum and epiphytes, 

Antithamnionella breviramosa, Herposiphonia secunda f. tenella and Centroceras clavulatum. 

From 10 m below mean low water, the most common species were Oscillatoria acuminata, 

Jania capillacea and Bryopsis pennta. 

In addition to the vertical distribution of the algae on the platforms, the biodiversity and 

abundance of macroalgae was found to increase from nearshore to offshore and decrease from 

intertidal to depth. 

CONCLUSIONS

The total number of taxa collected during this study was 24. The best represented group was 

Rhodophyta (approximately 50% of the taxa collected). There was a vertical distribution of taxa 

found on the platforms themselves, and there was also a trend identified for biodiversity and 

abundance of macroalgae to increase from nearshore to offshore and decrease from intertidal to 

depth. 

In all, seven new taxa were added to the list of macroalgae identified from the platforms and 

one new report of Antithamnionella breviramosa from the Gulf of Mexico. Approximately 50% 

of the taxa collected are known to have biotechnological potential and approximately 20% of the 

taxa collected are found only on oil and gas platforms. 

The macroalgal taxa that occur on the platforms in the Gulf of Mexico are still not well 

known. There is a need for further work on this group including comparisons of offshore and 

nearshore macroalgae and seasonal diversity as well as determining the importance of these 

macroalgae to herbivores. With further elucidation of the taxa that occur in the Gulf, their 

potential use in biotechnology can be more definitively assessed. 

REFERENCES

Baker, J.T. 1984. Seaweeds in pharmaceutical studies and applications. Hydrobiologia 

116/117:29-40. 

Ballesteros, E., D. Martin, and M.J. Uriz. 1992. Biological activity of extracts from some 

Mediterranean macrophytes. Botanica Marina 35:481-485. 

Caccamese, S., R. Azzolina, G. Furnari, M. Cormaci, and S. Grasso. 1980. Antimicrobial and 

antiviral activities of extracts from Mediterranean algae. Botanica Marina 23:285-288. 

Cooper, N.C., G.R. Johnstone. 1944. The seasonal production of agar in Gelidium 

carthilagineum, a perennial red alga. American Journal of Botany 31(10):638-640. 

Felicio-Fernandez, G., and Laranjeira, M.C.M. 2000. Calcium phoshophate biomaterials from 

marine algae. Hydrothermal synthesis and characterization. Quinn. Nova 23(4):441-446. 

Hayashi, K., J. Hamada, and T. Hayashi. 1996. A screening strategy for selection of anti-HSV -

1 and anti-HIV extracts from algae. Phytotherapy Research 10(3):233-237. 
16 

Haqashi-Okai, K, S. Otani, and Y. Okai. 1999. Potent suppressive effect of a Japanese edible 

seaweed, Enteromorpha prolifera (Sujiaopnori) on initiation and promotion phases of 

chemically induced mouse skin tumonigenesis. Cancer Letters 140(1):21-25. 

Hillis, D.M., B.K. Mable, A. Lason, S.K. Davis, and E.A. Zimmer. 1996. Nucleic Acids IV: 

sequencing and cloning. pp. 321-381. In Hillis, D.M., C. Moritz, and B.K. Mable, eds. 

Molecular Systematics. Sinauer Associates, Inc. Sunderland MA. 655 pp. 

Kim, J.H., J.B. Hudson, A.M. Huang, K. Bannister, H. Jin, T.J. Choi, G.H.N. Towers, Y.K. 

Hong, and R.E. DeWreede. 1997. Biological activity of seaweed extracts from British 

Columbia, Canada and Korea. I. Antiviral activity. Canadian Journal of Botany. 75(10): 

1656-1660. 

Miranda, M.S., R.J. Cintra, S. B. M. Barros, and J. Mancini-Filho. 1998. Antioxidant activity of 

the microalga Spirulina maxima. Brazilian Journal of Medical and Biological Research. 

31(8):1075-1079. 

Naqvi, S.W.A., S.Y. Kamat, L. Fernandes, and C.V.G Redsdy. 1980. Screening of some marine 

plants from the Indian coast for biological activity. Botanica Marina 24:1-55. 

Novikov, L.N., L. N. Novikova, A. Mosahebi, M. Weiberg, G. Terenghi, and J. Kellerth. 2002. 

A novel biodegradable implant for neuronal rescue and regeneration after spinal chord injury. 

Biomaterials: 23(16):3369-3376. 

Serkedjieva, J., M. Konaklieva, S. Dimitrova-Konaklieva, V. Ivanova, K Stefanov, and S. Popov. 

2000. Antiinfluenza virus effect of extracts from marine algae and invertebrates. Z. 

Naturforsch 55c:87- 93. 

Santos, M.G.M., M. H. C. Lagrota, M.M.F. Miranda, Y. Yoneshigue-Valentin, and M.D.A. 

Wigg. 1999. A screening for the antiviral effect of extracts from Brazilian marine algae 

against a cyclovir resistant herpes simplex virus type 1. Bot Mar. 42:iii-v. 

Tringali, C. 1997. Bioactive Metabolites from Marine Algae: Recent Results. Current Organic 

Chemistry 1:375-395. 

Trono, G.C. 1999. Diversity of the seaweed flora of the Philippines and its utilization. 

Hydrobiologia 398-399:1-6. 

Zemani F., D. Benisvy, I. Galy-Faroux, A. Lokajczyk, S. Colliec-Jouault, G. Uzan, A.M. 

Fischer, and C. Boisson-Vidal. 2005. Low-molecular-weight fucoidan enhances the 

proangiogenic phenotype of endothelial progenitor cells. Biochemical Pharmacology 

70(8):1167-1175. 
17 

V. FORAMINIFERA 

Evaluation of Oil and Gas Platforms on the Louisiana Continental Shelf for 

Organisms with Biotechnology Potential 

Barun K. Sen Gupta 

Department of Geology and Geophysics 

Louisiana State University 

and 

Lorene E. Smith 

Museum of Natural Science 

Louisiana State University 

INTRODUCTION

Benthic foraminiferal communities of the northwestern Gulf of Mexico continental shelf 

have been the subject of numerous studies, mainly with regard to broad patterns of geographic 

and water-depth distributions, but also in the context of ecological questions such as the effects 

of seasonal hypoxia on populations. There is, however, no published account of foraminiferal 

species living on oil and gas platforms in the Gulf. With this background, the focus of the 

present investigation was the taxonomic diversity and dominance trends of such species. 

Particular attention was given to species with agglutinated wall structure, because of their 

potential as a source of bioadhesives. 

METHODS

Laboratory Methods 

In the laboratory, samples of scrapings were thawed, washed over a series of sieves (63 μm, 

500 μm, 1 mm, and 2 mm), and preserved in Rose-Bengal-stained ethanol. One replicate from 

each depth and platform was selected for analysis. After a minimum of 48 hours, the two smaller 

size fractions (63–500 μm and 500 μm –1 mm) were rinsed, oven dried, and examined using a 

dissecting stereomicroscope. All benthic foraminifers, loose and attached, were removed. If the 

foraminifers were especially numerous, the fraction was subsampled using a microsplitter. For 

five selected samples, the larger size fractions were also picked. Selected species were illustrated 

by digital photomicrography and scanning electron micrography. 
18 

RESULTS

Assemblage Density 

The degree of platform leg fouling varied from site to site. A rich assemblage of benthic 

Foraminifera was recognized in the scrapings and the syringe samples; the species included both 

grazers and attached forms. The assemblage density was highly variable. The estimated density 

for the fine (63 μm – 1 mm) fraction in 19 samples varied between 2,112 and 107,488/m2

. The 

estimated density for both fine and coarse fractions in five samples varied between 15,728/m2 

and 143,920/m2 (Figure V-1). The gray bars for 14 samples represent only the fine (63 μm – 1 

mm) fractions; for five samples, the estimates for the coarse fraction (>1 mm) are black bars. 

Figure V-1. Estimated foraminiferal density (number/m2) on platform surface. 

The proportion of living (i.e., Rose-Bengal-stained individuals) to total foraminiferal counts 

in these five samples varied between 12% and 70% (Table V-1). Given the small number of 

samples with living foraminiferal data, generalizations are hard to make, but we note that the 

relative abundance of living Foraminifera was lowest at the base of the shallowest platforms. 

These populations were possibly affected by hypoxia that is known to prevail in the area in 

spring and summer (Rabalais et al., 2002). 

0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000

GI95A-W-20-A 

GI95A-W-10-A 

GI95A-W-0-

GI95A-E-20-C

GI95A-E-10-C

GI82A-W-20-A 

GI82A-W-10-A 

GI82A-W-0-

GI82A-E-20-B

GI42C-W-20-B 

GI42C-W-10-B 

ST67-W-20-A 

ST23-W-20-A 

ST23-W-10-B

ST23-E-10-A

ST67-W-0- 

GI42C-E-10-A

GI42C-E-20-A

GI42C-E-0-
19 

Table V-1 

Foraminiferal Density Data from Five Samples 

Fine and Coarse Fractions 

Sample No. of Forams Picked Forams / m2 % Living Living Forams / m2

ST 23 -W-20-A 3,321 110,208 11.9 13,120 

ST 67-W-20-A 924 21,440 29.3 6,288 

GI 42C-E-20-A 6,023 143,920 45.8 65,936 

 983 15,728 70.3 11,056 

G I95A-W-10-A 4,636 116,048 45.2 52,416 

Foraminiferal Diversity and Dominance 

Other than allogromiids (species with organic-walled shells), all major groups (agglutinated, 

porcelaneous, and calcareous shells) were represented in the samples. An abundance of juveniles 

and extraordinary morphological variations in attached species make species-level taxonomy 

particularly difficult, but we recorded over 20 species in most samples; the highest number was 

51 in sample GI95A-W10A. Free-moving species (grazers) were dominated by Ammonia 

parkinsoniana, Astrononion gallowayi, Bolivina spp., and Eubuliminella morgani. Attached 

species were dominated by Cibicides spp. (especially C. lobatulus), Cornuspira involvens, 

Patellina corrugata, Planorbulina spp., and Trochammina sp. 

Attached Species 

The most striking difference between the foraminiferal assemblage of the platforms and that 

of seafloor sediment from the area is in the abundance and diversity of attached species. These 

species are listed below: 

(1) Agglutinated group: Ammobaculites sp. cf. A. agglutinans (Figure V-2a), 

Glaphyrammina americana (Figure V-2b), Placopsilina sp (Figure V-2c), 

Sagenina divaricans (Figure V-2d, e), Textularia sp. (Figure V-2f), 

Trochammina sp. (Figure V-2g). 

(2) Calcareous group: Acervulina sp. (Figure V-3a), Calcituba polymorpha (of 

the type described by Arnold (1967); Figure V-3b-e), Cibicides lobatulus

(Figure V-3f, g), Cornuspira involvens (Figure V-4a), Dyocibicides sp., 

Hanzawaia concentrica (Figure V-4b), Melonis sp. (Figure V-4d), 

Neoconorbina sp. (Figure V-4c), Nodobacularia bonairensis (Figure V-4eg), Nubeculinita sp. cf. N. inhaerens (Figure V-5a), Parrina bradyi (Figure

V-5b, c), Patellina corrugata (Figure V-5d), Planorbulina mediterranensis

(Figure V-5e, f), Rectocibicides sp. cf. R. miocenicus (Figure V-6a), 

Rosalina globularis (Figure V-6b,c), Rosalina sp. (Figure V-6e), Spirillina 

vivipara (Figure V-7d), Webbinela rugosa, unidentified miliolids. 

Most attached Foraminifera adhere to living or dead barnacles (e.g., Figures V-2c,g, V-3d,e, 

V-4c, f, V-5a, V-6b, c,d) and bivalves (e.g., Figure V-2b, V-4a,e, V-6a) that cover the 
20 

platforms. Other foulers that provide attachment surfaces to the Foraminifera include sponges, 

tunicates, erect bryozoans (Figure V-3g), encrusting bryozoans (Figures V-3b,c), hydroids 

(Figures V-3a,f), algae (Figure V-5c), calcareous and chaetopterid polychaete tubes (Figures 

V-5e, V-6e, respectively), unidentified shell fragments (Figure V-2a,d-f, V-4b,g, V-5d,f), and 

motile organisms such as crabs. 

Within the succession of fouling, Foraminifera were found both on top and beneath 

invertebrate settlers. Firmly cemented foraminifers (e.g. Sagenina divaricans, Placopsilina sp., 

Nubeculariidae, some tube-like foraminifers) were typically present on more exposed, harder 

substrates. Planorbulina spp. were usually found firmly attached to hard substrates such as 

barnacle and mollusk shells, coralline algae, and calcareous polychaete tubes, but they were 

occasionally found on sponges. At one station, Melonis sp. was attached to a bivalve (probably 

arcid) byssus (Figure V-4d). At sites where barnacles were covered with sponges, tunicates, and 

octocorals, loosely attached Spirillina, Cornuspira, and miliolids were more common. Rosalina 

globularis was loosely attached to both calcareous and non-calcareous substrates. This species 

was also found in pits on shell surfaces. For some foraminiferal species (e.g., Planorbulina 

mediterranensis, Acervulina sp., Cibicides spp., Rosalina globularis), the shell morphology is 

modified by the shape of the attachment surface; misshapen shells and aberrant chamber 

arrangements are common. 

Discussion 

The most conspicuous aspect of the foraminiferal distribution is the abundance of attached 

taxa; some sessile species that are rare or absent in soft substrates live in relative profusion on 

the platforms. In addition, the foraminiferal community includes many vagrant foraminiferal 

species that have been recruited from soft-bottom natural substrates. Many sessile species of 

Foraminifera secrete an organic film for attachment (e.g., Poag, 1982), and are easily detached 

from the substrate by mechanical forces. Our sample coverage may be inadequate for a proper 

census of these species. The distribution of errant polychaetes (Gallaway and Lewbel, 1982) 

suggests that depressions and crevices on the surface of the artificial reefs (i.e., on or between the 

surfaces of live or dead, shelled epifauna) would be particularly suitable microhabitats for 

Foraminifera, providing both food and shelter. Judging by the high diversity of motile species on 

natural reefs (e.g., Rose and Lidz, 1977; Hallock, 1999; Sen Gupta, 1999), it is likely that further 

work would reveal the presence of a large suite of motile species on GOM platforms, feeding on 

bacteria, microscopic algae (including diatoms), and particulate organic debris (Goldstein, 1999). 

We postulate that foraminiferal colonization of platform legs starts with (1) migration of 

individuals from the surrounding seafloor, (2) settlement of benthic zygotes following the union 

of free-swimming gametes (or settlement of benthic gamonts produced by meiosis), (3) transport 

by motile invertebrates (e.g., mollusks, arthropods), or (4) accidental settlement of living 

individuals after the resuspension of physically disturbed seafloor sediment (Brunner and 

Biscaye, 1997). A relevant question is the distance of migration between the natural habitat 

(provenance) of a species and the platform (artificial reef). Published data from the Flower 

Garden Banks, the nearest living coral reef (Poag and Tresslar, 1981), show that many species 

present there are also present on the platforms, but these are all widespread species of the Gulf of 
21 

Figure V-2. Attached agglutinated species; a, b, d, digital photomicrographs, others scanning 

electron micrographs. a, Ammobaculites sp. cf. A. agglutinans (GI 95A-W10A); b, 

Glaphyrammina americana (ST 67H-W20A); c, Placopsilina sp. (GI 95A-W10A); d, e, 

Sagenina divaricans (d: GI 95A-W10A, e: GI 42C-E20A); f, Textularia sp. (ST 67HE10B); g, Trochammina sp.(GI 95A-W10A). 
22 

Figure V-3. Attached calcareous species; a, c, d, f, digital photomicrographs, others scanning 

electron micrographs. a (including inset), Acervulina sp. (GI 42C-E20A); b-e, Calcituba 

polymorpha (sensu Arnold; GI82A-W0A); f, g, Cibicides lobatulus (f: GI 42C-E20A, g: GI 

95A-W20A). 
23 

Figure V-4. Attached calcareous species (contd.); e, digital photomicrograph, others scanning 

electron micrographs. a, Cornuspira involvens (GI 95A-W10A); b, Hanzawaia 

concentrica (ST 67H bottom); c, Neoconorbina sp. (GI 95A-W20B); d, Melonis sp. (GI 

95A-W10A); e-g, Nodobacularia bonairensis (e, f: GI 82A-W20A, g: GI 95A-W10A). 
24 

Figure V-5. Attached calcareous species (contd.); a-c, digital photomicrographs, others 

scanning electron micrographs. a, Nubeculinita sp. cf. N. inhaerens (ST 67H-W20A); 

b, c, Parrina bradyi (b, detached specimen) (GI 42C-E20A); d, Patellina corrugata

(ST 67H-E10B); e, f, Planorbulina mediterranensis (e: GI 82A-W10B, f: GI 82A-W0A). 
25 

Figure V-6. Attached calcareous species (contd.); a, e, digital photomicrographs, others scanning 

electron micrographs. a, Rectocibicides sp. cf. R. miocenicus (ST 67H-W20A); b, c, 

Rosalina globularis (GI 95A-W0A); d, Spirillina vivipara (GI 95A-W10A); e, Rosalina sp. 

(GI 42C-E20A). 
26 

Mexico. Our samples do not contain Amphistegina, the most typical reefal foraminifer of the 

Caribbean biogeographic province (including the Flower Garden Banks), but whether this genus 

can occur as a "displaced" taxon on oil and gas platforms (i.e., at "some distance from their usual 

home range"), as is the case with several reef-type invertebrates (Lewbel et al., 1987), is an open 

question. 

Biotechnology Potential 

Agglutinated foraminiferal species hold promise as a source of bioadhesives for 

biotechnological and biomedical applications, because they can secrete and then harden adhesive 

organic compounds in an aqueous medium (Bowser et al., 1992). So far, only an Antarctic 

species, Astrammina rara, chosen on the basis of its relatively large size (average ~3 mm 

diameter) and shallow-water habitat (depth ~25 m, which facilitates harvesting) has been studied 

for this purpose (e.g., Bowser et al., 1992; Bowser and Bernhard, 1993). The species uses tensile 

cables of a bioadhesive to bind coarse sand-sized particles (>300 μm) and construct a shell. The 

chemistry of this bioadhesive has not been fully investigated, but SEM studies indicate the 

presence of at least two proteinaceous components (fiber and resin). In this context, a task of the 

MMS/CMI biotic survey of oil and gas platforms was a search for agglutinated foraminiferal 

species. Both attached species (listed earlier) and free-moving agglutinated species (e.g., 

Bigenerina irregularis, Spiroplectammina sp.) were found on the platforms, but not high 

densities of relatively large specimens (>1 mm) that could be exploited to extract bioadhesives. 

CONCLUSIONS

1. The foraminiferal community of Gulf of Mexico platforms includes a great 

variety of species, of both agglutinated and calcareous wall structures. 

2. Many attached species of platforms are rare or absent in adjacent seafloor 

sediment. 

3. No species with biotechnology potential could be identified. Platform related 

foraminiferans with potential as a source for bioadhesives remains but was not 

further investigated. 

LITERATURE CITED

Arnold, Z.M. 1967. Biological observations on the foraminifer Calcituba polymorpha Roboz. 

Archiv für Protistenkunde 110:280–304.

Bowser, S.S., and J.M. Bernhard. 1993. Structure, bioadhesive distribution and elastic 

properties of the agglutinated test of Astrammina rara (Protozoa: Foraminiferida). Journal of 

Eukaryotic Microbiology 40:121–131. 

Bowser, S.S., J.M. Bernhard, and S.P. Alexander. 1992. Test morphogenesis and bioadhesives 

in a giant Antarctic foraminifer. Antarctic Journal of the United States 26:165–167. 

Brunner, C.A., and P.E. Biscaye. 1997. Storm-driven transport of foraminifers from the shelf to 

the upper slope, southern Middle Atlantic Bight. Continental Shelf Research 17:491–508. 
27 

Gallaway, B.J., and G.S. Lewbel. 1982. The ecology of petroleum platforms in the 

northwestern Gulf of Mexico: A community profile. U.S. Fish and Wildlife Service, Office 

of Biological Services, Washington, D.C. FWS/OBS-82/27. Bureau of Land Management, 

Gulf of Mexico OCS Regional Office, Open-File Report 82-03, xiv + 92 pp. 

Goldstein, S.T. 1999. Foraminifera: A biological overview. In: B.K. Sen Gupta (ed.). Modern 

Foraminifera. Kluwer, Dordrecht, pp. 37–55. 

Hallock, P. 1999. Symbiont-bearing Foraminifera. In: B.K. Sen Gupta (ed.). Modern 

Foraminifera. Kluwer, Dordrecht, pp. 123–139. 

Lewbel, G.S., R.L. Howard, and B.J. Gallaway. 1987. Zonation of dominant fouling organisms 

on northern Gulf of Mexico platforms. Marine Environmental Research 21:199–224. 

Poag, C.W. 1982. Environmental implications of test-to-substrate attachment among some 

modern sublittoral foraminifera. Geological Society of America Bulletin 93:252–268. 

Poag, C.W., and R.C. Tresslar. 1981. Living foraminifers of West Flower Garden Bank, 

northernmost coral reef in the Gulf of Mexico. Micropaleontology 27:31–70. 

Rabalais, N.N., R.E. Turner, and W.J. Wiseman. 2002. Hypoxia in the Gulf of Mexico, a.k.a. 

“The Dead Zone.” Annual Review of Ecology and Systematics 33:235–263. 

Rose, P.R., and B. Lidz. 1977. Diagnostic foraminiferal assemblages of shallow-water modern 

environments: South Florida and the Bahamas. Sedimenta VI, Univ. Miami, 55 pp. 

Sen Gupta, B.K. 1999. Foraminifera in marginal marine environments. In: B.K. Sen Gupta 

(ed.). Modern Foraminifera. Dordrecht, The Netherlands: Kluwer Academic Publishers. 

Pp. 141–159. 
29 

VI. BRYOZOANS 

Geographical Variation in 

Chemical Defenses in the Byozoan Bugula neritina: 

Cryptic Species, Cryptic Endosymbionts 

Tamara M. McGovern3 and Michael F. Hellberg 

Department of Biological Sciences 

Louisiana State University 

INTRODUCTION 

Natural marine products may eventually provide many useful medicines, however to date the 

only marine compound to enter phase II clinical trials is bryostatin 1. Bryostatin 1 was initially 

isolated from the bryozoan Bugula neritina, along with other members of a pyran ring family of 

compounds now termed bryostatins. Bryostatin 1 combats the growth of cultured cancer cells 

(Hornung et al., 1992) and has shown some promise aiding patients suffering from nonHodgkin's lymphoma and lymphocytic leukemia (Varterasian et al., 2000) by promoting 

lymphocyte survival during therapy via action on the protein kinase C signal transduction 

pathway (see references in Davidson et al., 2001). 

Bryostatin 1 has yet to be synthesized in the lab. Isolating gram quantities of bryostatin 1 

requires over 10,000 gallons for wet bryozoans as starting material (Schaufelberger et al., 1991), 

the equivalent of perhaps millions of colonies. Aside from the direct impact that collecting such 

quantities of B. neritina from natural populations, the collateral damage done to other benthic 

species could be very great. Large sources of B. neritina that could be harvested from artificial 

substrate thus might serve a medical need while sparing natural communities from damage. 

Bugula neritina is a common member of temperate fouling communities worldwide. Along 

the Atlantic and Gulf coasts of North America, B. neritina is common on natural and artificial 

hard substrates (Osman 1977). Significantly, B. neritina has been reported as a dominant 

member of the fouling communities on the legs of shelf oil and gas platforms in the Gulf of 

Mexico (Gallaway et al., 1981). Whether these platforms could serve as a source of bryostatin 1, 

however, remains unknown because recent studies reveal that individuals identified 

morphologically as B. neritina actually belong to at least two cryptic species, only one of which 

harbors the symbiont that produces bryostatin 1 (Davidson and Haygood 1999). Furthermore, if 

bioactive compounds like bryostatins are produced to defend against predators, then populations 

might be expected to vary latitudinally in chemical defenses (Harvell et al., 1993) in association 

with geographic changes in predation rates (Vermeij, 1978, Bertness et al., 1981, Lankford et al., 

2001). Along the Atlantic coast of North America, the Cape Hatteras region separates southern 

waters, where predation pressure is high, from comparatively low predation to the north 

(Vermeij,1978; Bertness et al., 1981; Lankford et al., 2001). Chemical defenses in several marine 

animals are known to vary to either side of Cape Hatteras (Stachowicz & Hay, 2000; Sotka & 

Hay, 2002). 

 3

 Currently at Clemson University, Clemson, South Carolina. 
30 

Bugula neritina is likewise geographically variable in its palatability to predators. Larvae 

(Lindquist & Hay, 1996) and adults (Stachowicz & Hay, 1999) of B. neritina from North 

Carolina are chemically defended and unpalatable to fish and crab predators, respectively. In 

contrast, the larvae of morphologically indistinguishable bryozoans from Delaware, also 

identified as B. neritina, are palatable to the same fish that avoid larvae from North Carolina 

(Lopanik et al., 2004). The larvae of B. neritina are large, conspicuous and nutrient rich (Wendt, 

2000). They are released during the day (Lindquist & Hay, 1996), swim slowly (Wendt 2000), 

and spend little time in the plankton (Keough & Chernoff 1987; Wendt 2000). Lindquist & Hay 

(1996) argued that such larvae may face especially strong selective pressure for defensive 

adaptations because of their association with the benthos where predation is great. Unlike the 

crustaceans mentioned above, larvae, particularly large nonfeeding larvae of taxa such as B. 

neritina, tend to defend themselves chemically via the internal production of secondary 

compounds (Lindquist et al., 1992; Lindquist & Hay, 1996). Bryozoans have proven rich sources 

of novel organic compounds, several with potential pharmaceutical values (Pettit, 1991), as have 

many other sessile marine invertebrates (including sponges, gorgonians and tunicates; Faulkner, 

2002). Interestingly, these compounds often appear to be produced not by the animal itself, but 

rather by bacterial endosymbionts (Lee et al., 2001). The larvae of B. neritina harbor such 

bacterial symbionts (Woollacott 1981; Haygood & Davidson 1997). 

Davidson et al. (2001) have confirmed previous suspicions (Anthoni et al., 1990) that 

bryostatins are produced by an endosymbiotic γ-proteobacteria, which they have identified 

genetically as Endobugula sertula (Davidson & Haygood, 1999). The array of bryostatins 

produced by B. neritina varies among populations (Pettit, 1991). Davidson & Haygood (1999) 

surveyed genetic variation within B .neritina along its Pacific range in California. They found 

two cryptic species of B. neritina that harbored different strains of E. sertula. The different 

strains of symbionts produced different bryostatins. Davidson & Haygood (1999) suggested (but 

did not demonstrate) that the suite of bryostatins produced by each endosymbiont may determine 

the predators to which its animal host is susceptible. If so, or if endosymbionts produce defensive 

compounds other than bryostatins, then bryozoan hosts and their bacterial endosymbionts may 

co-evolve in response to geographical variation in predation pressure. 

In this study, we genetically examined whether the Bugula neritina growing on shelf oil and 

gas platforms in the Gulf of Mexico might serve as a source of the putative chemotheraputic 

agent bryostatin 1. This question was addressed within a larger study (McGovern & Hellberg 

2003) in which we examined genetic differentiation among populations of both B. neritina and 

its bacterial associates from its Atlantic range between the Gulf of Mexico and southern New 

England. Specifically, we asked (i) whether populations of B. neritina from north and south of 

Cape Hatteras, which differ in their palatability, belong to a single genetically mixed species or 

constitute genetically distinct cryptic species, and (ii) whether differently defended bryozoan 

populations harbor genetically distinct endosymbionts. 

METHODS

Population Sampling 

We sampled 9–11 colonies of Bugula neritina from five populations south of Cape Hatteras 

(‘Southern’ populations: South Terrebonne platform ST67H, LA (28°48′ N, 90°25′ W); Turkey 

Point, FL (29°54′ N84°29′ W); Cedar Key, FL (29°08′ N, 83°02′ W); Mosquito Lagoon, FL 

(28°45′ N, 80°45′ W); Beaufort, NC (32°25′ N,80°40′ W)), including the population from 
31 

Beaufort, North Carolina that was shown to be unpalatable to fish predators (Lopanik et al., 

2004). (.We also sampled 10 individuals from each of two ‘Northern’ populations (Indian River, 

DE (38°35′ N, 75°17′ W); Waterford,CT (41°20′ N, 72°08′ W)), including the Delaware site 

from which palatable animals were collected (Lopanik et al., 2004). In addition, we sampled two 

or three colonies of B. stolonifera from the same Delaware site where B. neritina was collected 

and from Woods Hole, Massachusetts (Marine Biological Laboratory Supply Center, (41°31′ N, 

72°08′ W)), and two colonies of B. turrita from Woods Hole. 

Genetic Variation within B. neritina 

We used the polymerase chain reaction (PCR) to amplify a portion of cytochrome oxidase c 

subunit I (COI) with primers LCO1490 and HCO2198 (Folmer et al., 1994). 624 base pairs (bp) 

of the resulting COI sequence were aligned by eye; there were no indels or stop codons. COI 

sequences of both deep- and shallow-water forms of B. neritina (Davidson & Haygood,1999; 

GenBank accession numbers AF061422, AF061424, AF061425, AF061432) were included in 

the alignment. Bugula stolonifera and B. turrita were used as outgroups. Phylogenetic trees 

were constructed using the neighbor joining method in PAUP* (Swofford, 2001) with Kimura 

two-parameter distances. Maximum parsimony trees were generated using both equal and 8:1 

weightings of transversions and transitions. We used Modeltest (Posada & Crandall, 1998) to 

determine most appropriate model for maximum likelihood analysis. Bayesian posterior 

probabilities were then generated using MrBayes (Huelsenbeck & Ronquist, 2001). 

Genetic Variation of Bacteria Associated with B. neritina 

To identify the bacterial associates of the different bryozoans, we amplified and sequenced > 

1,000 bp of the bacterial gene encoding the small subunit ribosomal RNA (SSU rRNA). SSU 

amplification was a two-step process that first amplified using general bacterial primers, then 

targeted the Endobugula symbionts using specific primers (Haygood & Davidson, 1997, Table 1 

in McGovern & Hellberg 2003). We obtained sequence from a minimum of three clones per 

individual bryozoan colony using the amplification primers, cloning vector primers and a 

combination of internal sequencing primers. 

In the SSU alignment, we included all of the sequences we obtained, as well as the E. sertula

sequences obtained by Haygood & Davidson (1997; GenBank accession numbers AF006606–

AF006608) and the four most similar bacterial sequences identified using blast searches: 

Oceanopirillum maris (AB006763, Satomi et al., 1998), O. multiglobuliferum (AB006764; 

Satomi et al., 1998) and two uncultured γ - proteobacteria (AB015541; Li et al., 1999 and 

AF228694). The two most distant of the sequences obtained from our BLAST search (the two 

Oceanopirillum spp.) were used as outgroups. Equally weighted unrooted neighbor-joining and 

parsimony trees were generated using PAUP*. We also generated a maximum likelihood tree 

using a model and settings determined by Modeltest. 

RESULTS

Cryptic Species within Bugula neritina 

COI sequences from Bugula neritina sampled from the Gulf of Mexico and from the Atlantic 

coast south of Cape Hatteras were identical (Figs 2A, 3A in McGovern & Hellberg 2003). These 

sequences were also identical to those of Davidson & Haygood (1999) from Beaufort, NC over 

the 618 bp where they aligned, thus falling within their Shallow clade of Californian B. neritina.
32 

There was a deep divergence (11.5%), however, between these Southern/Shallow form 

sequences and those obtained from B. neritina populations in Delaware and Connecticut. This 

Northern Atlantic form of B. neritina differs not only from the Shallow/Southern form, but also 

from two other species of Bugula found north of Cape Hatteras, B. stolonifera (AY173427) and 

B. turrita (AY173428), and from the Pacific deep water form of B. neritina identified by 

Davidson & Haygood (1999). Qualitatively similar trees were obtained using the parsimony 

with both transition:transversion ratios and using neighbor-joining methods. These trees placed 

the Southern/Shallow and deep forms as sister taxa, with the Northern clade falling basal within 

a monophyletic B. neritina. Maximum likelihood trees differed topologically, placing the 

Northern form as the sister to the deep-water clade. 

For the bacterial SSU rRNA sequences, all tree building methods produced trees that were 

topologically similar with regard to relationships among variants of Endobugula sertula and 

between this symbiont and other γ- proteobacteria (Figs 2B, 3B in McGovern & Hellberg 2003). 

All bacterial sequences (AY173429–AY173431) obtained from the Southern populations of B. 

neritina are identical. They differ by only 2 bp (0.2%) and 4–5 bp (0.5%) from SSU rRNA 

sequences reported for Haygood & Davidson’s (1997) shallow- and deep-water E. sertula, 

respectively. In contrast, the bacterial sequences obtained from Bugula found north of Cape 

Hatteras (AY173432–AY173454) differ by from 5% to 11% from the Endobugula sequences 

from the Southern populations and by 0% to 10% from each other. The bacterial sequences from 

the Northern Bugula show no apparent correlation with either geography or host phylogeny; 

bacteria from the three species and the six collecting sites (Woods Hole, Connecticut and 

Delaware) are interspersed. Whereas all Southern bryozoan colonies were associated with 

identical bacterial sequences, only two sequences associated with the Northern B. neritina and 

the other Bugula species were found more than once. 

DISCUSSION 

The reciprocal monophyly and high level of genetic divergence between the Southern and 

Northern populations of Bugula neritina indicate that this nominal species is most probably a 

complex of cryptic species along the east coast of the United States, just as Davidson & Haygood 

(1999) found for B. neritina living along the west coast. We suggest that B. neritina as currently 

recognized in US waters comprises three cryptic species: the deep water Pacific form delineated 

by Davidson & Haygood (1999), a Shallow/Southern form present both in warmer waters along 

the Pacific coast and along the Atlantic coast south of Cape Hatteras including the Gulf of 

Mexico, and a third form present in the Atlantic north of the Cape Hatteras region (our 

‘Northern’ form), identified here for the first time (Figure 2A in McGovern & Hellberg, 2003). 

Thus, no Atlantic Bugula species can serve as a source of bryostatin 1. 

The identification of two allopatric cryptic species means that differences in larval 

palatability between Northern and Southern B. neritina (Lopanik et al., 2004) do not result from 

intraspecific responses to geographical variation in predation pressure, as suggested for other 

species (Sotka et al., 1999; Stachowicz & Hay, 2000; Sotka & Hay, 2002.). Instead, our results 

indicate that the geographical differences in larval (and perhaps adult) palatability within B. 

neritina result from the presence of two cryptic and allopatric species, each with different 

defensive capabilities. Other instances of apparent intraspecific variation in morphology and 

behavior have likewise been found to be the result of grouping together morphologically similar 

cryptic species (Knowlton et al., 1992; Rowan et al., 1997; reviewed in Shaw, 2001). We cannot 

eliminate the possibility that past adaptation to differing predation pressures initiated the 
33 

differentiation of the two cryptic species, but at present the evidence suggests that these are fully 

isolated species, and current local adaptation need not be invoked. 

The Shallow/Southern species of B. neritina has a widespread distribution despite the low 

dispersal potential of larvae (Keough & Chernoff, 1987; Wendt, 2000), probably reflecting both 

anthropogenic transport (Carlton & Geller, 1993) and this species’ success as a fouling organism. 

The absence of intraspecific variation seen here is consistent with the possibility that both the 

Southern/Shallow and Northern Bugula species may have been introduced to the ranges studied 

here. In the face of this demonstrated colonization ability, the failure of the Southern/Shallow 

species to establish populations north of the Cape Hatteras area, and the failure of the Northern 

species to establish populations further south, suggests that ongoing ecological forces may act to 

restrict range expansion of both cryptic species . These factors may include different abiotic 

tolerances for the two bryozoan species, perhaps for temperature (Davidson & Haygood, 1999). 

However, differences between the two species in their associations with bacteria may provide a 

better-supported explanation. 

Our results demonstrate that the Northern and Southern species of B. neritina differ in their 

associations with γ- proteobacteria. The Southern B. neritina populations appear to have an 

endosymbiotic relationship with a single species of γ-proteobacteria: Endobugula sertula. In 

contrast, in the Northern populations there was no evidence of a close (or indeed any) symbiotic 

relationship between B. neritina and E. sertula or any other γ-proteobacteria. 

The suite of bryostatins produced by E. sertula may determine the predators to which its 

bryozoan host is susceptible (Davidson & Haygood, 1999; Davidson et al., 2001). Because the 

Northern form of B. neritina lacks E. sertula, their larvae should also lack both bryostatins and 

any defensive capabilities these compounds confer, capabilities that should be present in larvae 

of the Southern form. Consistent with this reasoning, the larvae of the Northern form of B. 

neritina are palatable to fish predators whereas the Southern form is unpalatable (Lopanik et al., 

2004). Bryostatins were not specifically isolated and tested for their deterrent properties (N. 

Lopanik, personal communication), but even if the bryostatins are not the source of deterrence, 

the correlation of deterrence with the presence of E. sertula suggests a role for the Bugula–

Endobugula symbiosis in the response to predators. 

CONCLUSIONS

Genetic markers have allowed us to investigate the source of geographical variation in 

predator resistance within Atlantic populations of the bryozoan B. neritina (Lopanik et al., 2004). 

Using mitochondrial COI sequences, we found that this geographical variation in chemical 

defense does not result from the local adaptation of a single widespread species to different 

predation regimes. Rather, the populations surveyed by Lopanik et al. (2004) belong to two 

genetically differentiated cryptic species: one northern, one southern. Using bacterial small 

ribosomal subunit sequences, we found that the southern bryozoan species is engaged in a 

symbiotic relationship with a bacterium (Endobugula sertula) known to produce compounds 

(bryostatins) that may serve in the host’s chemical defense. The northern species showed no 

evidence of such an association. The Gulf of Mexico species also does not produce Bryostatin 1. 

The ability to identify genetically both cryptic species of Bugula neritina and their bacterial 

associates has allowed us to reject a hypothesis of intraspecific geographical variation in 

chemical defenses. Instead, the pattern of chemical defenses, and possibly the geographical 

range of the undefended species, may be determined by species-specific patterns of 

endosymbiosis. 
34 

ACKNOWLEDGMENTS 

Specimens were graciously provided by Greg Boland, MMS, Kevin Krajick, Nicole Lopanik, 

Frank Maturo of the University of Florida Marine Laboratory, Melanie Mead, Barbara Shoplock, 

Jay Stachowicz, Cheryl Swanson, Jeff Terwin, Linda Walters and the Woods Hole 

Oceanographic Institute. 

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Marinospirillum gen. nov., with descriptions of Marinospirillum megaterium sp. nov., 

isolated from kusaya gravy, and transfer of Oceanospirillum minutulum to Marinospirillum 

minutulum comb. nov. Int. J. Syst. Bact. 48:341–1348. 
36 

Schaufelberger, D.E., M.P. Koleck, J.A. Beutler, A.M. Vatakis, A.B. Alvarado, P. Andrews, 

L.V. Marzo, G.M. Muschik, J. Roach, J.T. Ross, W.B. Lebherz, M.P. Reeves, R.M. 

Eberwein, L.L. Rodgers, and A.J. Shaw. 2001. Biogeographic patterns and cryptic 

speciation in bryophytes. J. Biogeography 28:253-261. 

Sotka, E.E., and M.E. Hay. 2002. Geographic variation among herbivore populations in 

tolerance for a chemically-rich seaweed. Ecology 83:2721-2735. 

Sotka, E.E., M.E. Hay, and J.D. Thomas. 1999. Host-plant specialization by a non-herbivorous 

amphipod: advantages for the amphipod and costs for the seaweed. Oecologia 118:471-482. 

Stachowicz, J.J., and M.E. Hay. 2000. Geographic variation in camouflage specialization by a 

decorator crab. American Naturalist 156:59-71. 

Swofford, D.L. 2001. Phylogenetic analysis using parsimony. Version 4. Sunderland, MA: 

Sinauer Associates. 

Varterasian, M.L., R.M. Mohammad, M.S. Shurafa, K. Hulburd, P.A. Pemberton, D.H. 

Rodriguez, V. Spadoni, D.S. Eilender, A. Murgo, N. Wall, M. Dan, and A.M. Al-Katib. 

2000. Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's 

lymphoma and chronic lymphocytic leukemia. Clin. Cancer Res. 6:825-828. 

Vermeij, G.J. 1978. Biogeography and Adaptation: Patterns of Marine Life. Cambridge, MA: 

Harvard University Press. 

Wendt, D.E. 2000. Energetics of larval swimming and metamorphosis in four species of Bugula

(Bryozoa). Biological Bulletin 198:356-356. 

Woolacott, R.M. 1981. Association of bacteria with bryozoan larvae. Mar. Biol. 65: 155-158. 
37 

VII. MOLLUSCS 

Mollsca from Oil and Gas Platforms on the Louisiana Continental Shelf: 

A Biotechnology Survey 

Laurie C. Anderson 

Dept. of Geology & Geophysics 

Louisiana State University 

INTRODUCTION

Previous Work 

Gallaway and Lewbel (1982) considered offshore platforms to represent a new biological 

habitat for the northwestern Gulf of Mexico, and a habitat characterized by distinct faunal 

successions. They recognized three faunal groupings by water depth: coastal (1-30 m), offshore 

(30-60 m), and blue water (>60 m) (Gallaway et al., 1981; Gallaway and Lewbel, 1982). 

Changes in faunal composition are apparently influenced by onshore-offshore trends in turbidity; 

seasonal variation of temperature, salinity, dissolved oxygen, and primary productivity; and 

exposure to Caribbean water masses (Gallaway et al., 1981; Gallaway and Lewbel, 1982). 

Coastal platforms in the western Gulf of Mexico are dominated by barnacles on the shallower 

portions of platforms, although bivalves can be common on deeper portions of these platforms 

(at 10 m) (Gallaway et al., 1981; Gallaway and Lewbel, 1982). Bivalves previously reported 

from Louisiana coastal platforms include several oyster species (Crassostrea virginica, Ostrea 

equestris, Lopha frons, and Hyotissa thomasi (10 m) and Chama macrophylla (1-30 m). In 

Texas, Anadara transversa (3-12 m), Chama macrophylla, Diplothura smithyi (21.5 m), and 

Isognomon bicolor (to 12 m) are reported (Fotheringham, 1981; Gallaway and Lewbel, 1982). 

Anadara transversa is, however, an infaunal byssate bivalve, and it is possible that this reported 

occurrence is a misidentification of another (epifaunal) arcid species. 

Bivalves make up the largest portion of the biomass on offshore platforms in the western 

Gulf of Mexico (Gallaway et al., 1981; Gallaway and Lewbel, 1982). Bivalves reported include 

Isognomon (dominant at 1 m), Hyotissa thomasi (dominant from 10-20 m), Chama macrophylla

(1-20 m), and Arca imbricata (1-10 m). Blue water platforms have a lower biomass than those 

in shallow water, and bivalves are abundant at greater depths on these platforms (Gallaway and 

Lewbel, 1982). 

Bysssate bivalves were included in this study for the biotechnological potential of the 

bioadhesive used by the byssus.. Byssal bioadhesives provide the mollusc a strong and durable 

adhesion to underwater surfaces. These non-toxic, biodegradable and non-polluting adhesive 

proteins have many potential uses including medical and dental adhesives and fillers (Rzepecki 

and Waite, 1995) 
38 

METHODS

Sample Preparation 

Samples of molluscs were frozen in the field. Each sample was thawed in the lab and 

preserved in 95% ethanol. Large pieces (ca. > 4 mm) were stained with Rose Bengal and 

retained in ethanol. Finer sediment and biota were screened using 63 u, 0.5 mm, 1 mm, and 2 

mm sieves, stained with Rose Bengal and preserved in ethanol. Mollusks were identified from 

the > 2 mm fraction of samples. To compare sampling levels, differences in the number of 

replicates per sampling level had to be taken into account. However, because the “A” replicate 

of most sampling levels had a larger volume of biotic material, one replicate per level was not 

chosen at random. Instead, abundance data per level was averaged across replicates. 

RESULTS

The platforms sampled hosted a low diversity molluscan assemblage dominated by byssate 

bivalves (Figures VII-1 and VII-2). Twenty-seven bivalve and gastropod species were 

identified, with Isognomon bicolor and Barbatia candida making up about 90% of the total 

assemblage. Changes in relative abundance of taxa among platforms (Figure VII-1) were in 

part caused by uneven sampling at the 0-m level. No 0-m samples were collected at ST23 and 

only one sample was collected at ST67H and GI42C (Table II-2). These platforms had a lower 

relative abundance of I. bicolor, a species that is most abundant at very shallow depths (Figure 2, 

and Galloway and Lewbel, 1982). The high dominance of I. bicolor at GI82A and GI95A is a 

result of the large numbers of I. bicolor in comparison to numbers of other taxa found in the 

single replicates taken at 0-m at these platforms. 
39 

Figure VII-1. Relative abundance of molluscan species from each platform and for all 

platforms combined (labeled total) 

A similar pattern of change in relative abundance with sample depth can be detected for all 

platforms (Figure VII-2). Isognomon bicolor dominated (nearly 100%) of 0-m samples. This 

species also dominated 10-m samples (> 60%), although Barbatia candida was also common 

(nearly 20%) at this level. Lithophaga spp. were rare in 0-m samples, but the abundance of these 

boring bivalves increased with depth, and they were especially common at platform ST 23. 

Chama macrophylla was the fourth most abundant species identified, and although individuals 

were not numerically abundant, the valves of this species from both live and dead individuals 

formed an important substrate for other members of the encrusting community. 
40 

Figure VII-2. Relative abundance of molluscan species at each sampling level and for all 

sampling levels combined (all depths). 

Bivalve composition was similar to that previously reported from oil and gas platforms on 

the Louisiana and east Texas continental shelf (e.g., Fotheringham, 1981; Gallaway and Lewbel, 

1982), where Isognomon was most common from 0-12 m; bivalves of the Family Arcidae 

(arcids) were common from 3-12 m; and Chama macerophylla was reported between 1 and 20 

m. However, previous reports indicate that oysters were common several meters below the water 

surface on near shore platforms (Gallaway and Lwebel, 1982). In the platforms examined for 

this study, three species of oysters were identified (Ostrea equestris, Crassostrea virginica, and 

Lopha frons) but none were numerically abundant. 

The bivalve assemblages identified in this study resembled assemblages found on shorelines 

characterized by hard substrates in Texas and Mexico (Britton and Morton, 1989). Communities 

found on naturally hard shorelines in Texas and Mexico contain I. bicolor in areas exposed to 

wave energy and Barbatia candida in sheltered areas. Isognomon bicolor is also common in the 

midlittoral zone of jetties and other artificial shoreline structures. 

CONCLUSIONS

Potential Significance of Platform Molluscs to Biotechnology 

The mollusks collected in this study with the greatest biotechnology potential include the 

byssate bivalves Isognomon and Barbatia that produce bioadhesives. The byssus is a bundle of 

proteinaceous threads secreted by some bivalves that attaches to a substrate by an adhesive 

plaque (Rzepecki and Waite, 1991; Burzio et al., 1997). These adhesives are of biotechnological 
41 

interest because they provide strong, durable adhesion to wet surfaces (Rzepeckiand Waite 1991, 

1995). In addition, some of the proteins in the adhesive can chelate metal ions (Martell, 1982; 

Deming, 1999). The most widely studied byssal protein is mussel adhesive protein (MAP) from 

Mytilus edulis. This compound is used as an attachment factor for cells and tissues in culture 

(Waite, 1991; Deming, 1999); as an immobilization agent for antigens, antibiotics, and enzymes 

(Burzio et al., 1997); and as an anticorrosive coating for metals and metal sequestering reagent 

(Burzio et al., 1997; Rzepecki and Waite, 1995). Additional potential uses are as medical and 

dental adhesives and fillers; microencapsulating agents; as sizing agents for textiles; and waterresistant inks (Rzepecki and Waite, 1995; Burzio et al., 1997). 

Byssal composition is highly variable among taxa (Waite, 1983). Therefore, an examination 

of byssate bivalves other than M. edulis and including those taxa (I. bicolor and B. candida)

found in abundance in this study on Louisiana platforms may lead to the extraction of a new 

compound with similar or new applications. 

LITERATURE CITED 

Britton, J.C., and Morton, B. 1989. Shore Ecology of the Gulf of Mexico. The University of 

Texas Press, Austin, TX. 387 pp. 

Burzio, L., Burzio, V., Silva, T., Burzio, L. and Pardo, J. 1997. Environmental bioadhesion: 

themes and applications. Current Opinion in Biotechnology, 8: 309-312. Deming, T. (1999). 

Mussel byssus and biomolecular materials. Current Opinion in Chemical Biology 3:100-105. 

Fotheringham, N. 1981. Observations on the effects of oil field structures on their 

bioticenvironment: platform fouling community. In: Middleditch, B.S., ed. Environmental 

effects of offshore oil production. The Buccaneer Gas and Oil Field Study. Marine Science 

14:179-208. 

Gallaway, B. and G. Lewbel. 1982. The ecology of petroleum platforms in the northwestern 

Gulf of Mexico: a community profile. U.S. Fish and Wildlife Service, Office of Biological 

Services, Washington, D.C. FWS/OBS-82-27. Bureau of Land Management, Gulf of Mexico 

OCS Regional Office, Open-File Report, 82-03, 92 pp. 

Gallaway, B.J., M.F. Johnson, L.R. Martin, F.J. Margraf, G.S. Lewbel, R.L. Howard, and G.S. 

Bowland. 1981. The artificial reef studies. Vol 2. In: Bedinger, C.A., and L.Z. Kirby, eds. 

Ecological investigations of petroleum production platforms in the central Gulf of Mexico. 

SWRI Project 01-5245. U.S. Dept. of the Interior, Bureau of Land Management, New 

Orleans, LA. 199 pp. 

Martell, A.E. 1982. Stability constants of metal-ion complexes. Suppl. No. 1, Part II, Special 

Publication no. 25. London: The Chemical Society. 

Rzepecki, L., and J. Waite. 1991. DOPA proteins: versatile varnishes and adhesives from 

marine fauna. Bioorganic Marine Chemistry, 4: 119-148. 

Rzepecki, L. and Waite, J. 1995. Wrestling the muscle from mussel beards: research and 

applications. Molecular Marine Biology and Biotechnology 4:313-322. 
42 

Waite, J. 1983. Quinone-tanned scleroproteins. In: Hochachka, P.W., ed. The Mollusca, Vol.1, 

Metabolic Biochemistry and Molecular Biomechanics. New York: Academic Press. Pp. 

467-504 

Waite, J. 1991. Mussel beards: a coming of age. Chemistry and Industry, 17: 607. 
43 

VIII. GENETIC VARIATION OF MOLLUSCS 

Genetic variation of Barbatia candida (Arcidae; Bivalvia) Populations 

From Oil Platforms in the Gulf of Mexico 

Christopher S. Gregg and David W. Foltz 

Department of Biological Sciences, 

Louisiana State University 

INTRODUCTION 

Offshore oil and gas platforms represent a unique, man-made hard substrate for fouling 

organisms in the northern Gulf of Mexico (GOM). Oil and gas platforms in the northern GOM 

extend from coastal (shore to > 27 m depth), to offshore (27 to 64 m), to blue water (> 64 m) 

placing them in different water masses that are subject to different hydrodynamic regimes 

(Gallaway et al., 1981). Consequently, the larvae that colonize the platforms may originate from 

different sources and dispersal among platforms may be limited. Therefore, the fouling 

organisms inhabiting platforms in different zones may be cryptic species or exhibit population 

structure due to a lack of gene flow. Molecular analyses of genetic structure within some 

nominal species representing diverse marine taxa have revealed heterogeneity suggestive of 

interspecific differences (Knowlton, 1993; 2000). To date, there have been few studies 

investigating the taxonomic diversity of the fouling organisms inhabiting oil and gas platforms 

(e.g., Howard et al., 1980; Gallaway et al., 1981; Gallaway and Lewbel, 1982) and all have relied 

on morphological identifications. Prior to the present group of studies, none have explored the 

genetic variability of these organisms in order to confirm their taxonomic status or to evaluate 

their population structure; however, there have been two studies that examined the genetic 

diversity of harpacticoid copepods inhabiting the sediments around GOM oil platforms (Street 

and Montagna, 1996; Gregg et al., 2006). 

Taxonomic and genetic heterogeneity of marine organisms must be anticipated prior to 

surveys for biotechnologically useful molecules and studies of ecological processes. The 

purpose of the present study was to examine the genetic variability of the bivalve community 

associated with northern GOM platforms in order to (1) determine if there are cryptic species and 

(2) examine the genetic variation within and among platforms. To do this, we examined the 

variability in the mitochondrial cytochrome c oxidase subunit I (COX I) gene. Mitochondrial 

genes have been used extensively in studies of bivalves to discriminate cryptic species (e.g., 

O’Foighil et al., 1996; Peek et al., 1997) and to determine population structure (e.g., Reeb and 

Avise, 1990). 

METHODS 

Collection of Samples 

Samples of attached bivalve mollusks were collected as described in Section II and stored in 

70% ethanol until they were processed for genetic analysis. Processing consisted of dissecting 

the soft tissues from each individual and storing the tissues in TE buffer (10 mM Tris pH 8.0; 0.1 
44 

mM EDTA) at -20°C. The shells were retained as vouchers for further morphological 

examination. 

Genetic Methods 

A small (≈ 2 mm2

) piece of soft tissue was removed from each bivalve. DNA was extracted 

from this tissue using the CTAB protocol described by Winnepenninckx et al. (1993). A 720-bp 

portion of the COX I gene was amplified by the polymerase chain reaction (PCR) using the 

universal primers of Folmer et al. (1994). The PCR was carried out by adding 1 μl of the DNA 

extract to 5 μl GeneReleaser (BioVentures, Inc.) in a 0.5-μl tube, then incubating the mixture in a 

thermal cycler using the profile recommended by the manufacturer. After incubation, a PCR 

master mix was added to each tube. Reactions were done at either a 25 or 50 μl final volume, 

and the final concentrations of the reagents were as follows: 1X Promega Reaction Buffer A; 2.5 

mM MgCl2; 0.2 mM total dNTPs; 1 μM of both forward and reverse primers; 2 units Promega 

Taq polymerase. The PCR was done in a Perkin-Elmer DNA Thermal cycler under the 

following conditions: 1 cycle of 95°C for 3 min; 40 cycles of 95°C for 30 sec, 47°C for 45 sec, 

and 74°C for 1 min; 74°C for 7 min. Three μl of each PCR product was electrophoresed into a 

2% agarose gel, stained with ethidium bromide, and viewed by UV transillumination. 

The genetic variability of the COX I gene was determined by cutting with restriction 

enzymes and DNA sequencing. After the PCR, a portion of the amplicon was digested with the 

restriction enzyme MseI (recognition sequence AATT; New England Biolabs) according to the 

manufacturer’s protocol. The restriction fragments were then electrophoresed on 2% agarose 

gels, stained with ethidium bromide, and viewed by UV transillumination. DNA sequences were 

then obtained from randomly chosen individuals from within each restriction profile. The PCR 

products were purified using Exo-SAP IT (USB) following the manufacturer’s protocols, then 

sequenced using the ABI-Prism Big-Dye cycle sequencing chemistry (Applied Biosystems). The 

sequencing reactions were scaled down to 10-μl reaction volumes, as described by RochaOlivares et al. (2001), and run on an ABI-377 Gene Analyzer (Applied Biosystems). 

Data Analysis 

The raw DNA sequence data were proofread and assembled into contigs using Sequencher 

4.0 (Gene Codes). Previously published sequence data from bivalves in the family Arcidae 

(Marko 2002; Marko and Moran 2002) were downloaded from the GenBank database 

(http://www.ncbi.nlm.nih.gov/). The processed sequence data were then aligned using ClustalX 

(Thompson et al., 1997). Phylogenetic analysis was performed in MEGA 2.0 (Kumar et al., 

2001) using the Neighbor-joining method (Saitou and Nei 1987) for tree construction with the 

Kimuratwo-parameter model (Kimura 1980) of DNA evolution. The reliability of the 

phylogenetic tree was determined by the nonparametric bootstrap test (Felsenstein 1985). 

Frequencies of the different COX I restriction profiles within and among platforms were used to 

determine the population structure by an Analysis of Molecular Variance (AMOVA, Excoffier et 

al., 1992) using Arlequin 2.0 (Schneider et al., 2000). There were two levels in the hierarchical 

design of the AMOVA including variability among platforms and the variability within 

platforms. 
45 

RESULTS 

Species abundance and diversity are shown in Figure VIII-1 for each of the five sampled 

platforms. Further details are provided by Anderson (‘Mollusca from Oil and Gas Platforms on 

the Louisiana Continental Shelf: A Biotechnology Survey’) in Section VII. RFLP analysis of 

the COX-I gene was done for 195 Barbatia candida (Table VIII-1). 

Figure VIII-1. Number of species collected and species diversity. 

Table VIII-1 

 

Numbers of Barbatia candida Sampled from Five Platforms for RFLP Analysis 

RFLP Haplotype 

Platform 

Depth 

(m) A B C D Total 

GI 42 C 31 17 37 1 1 56 

GI 82 A 52 2 4 0 0 6 

ST 67 H 19 6 14 2 1 23 

ST 23 15 8 20 3 0 31 

GI 95 A 61 19 60 0 0 79 

Total 52 135 6 2 195 

Twenty DNA sequences from six species within the family Arcidae were downloaded from 

the GenBank database for comparison with 21 DNA sequences collected in the present study. 

These species included Barbatia gradata, B. domingensis, B. reeveana, B. candida, Arca 

mutabilis, and A. imbricata. Each species formed a separate clade with high (96100%) bootstrap 

support (Figure VIII-2). The 21 DNA sequences from the present study all grouped within the 

same clade as previously-published B. candida sequences, suggesting that there are no cryptic 

species among the arcid molluscs from the platforms examined. The average uncorrected 

distance among sequences from the present study was 0.006 with a maximum of 0.0075. 

Maximum p-distance = 1.7% (3.4%); overall mean p-distance = 0.6% (0.8%). Genetic distance is 

consistent with intraspecific variation, i.e., no cryptic species. 

Restriction fragment profiles were analyzed to determine the level of genetic variation 

present and to find evidence of cryptic species. The restriction fragment length analysis revealed 
46 

the presence of four haplotypes at the three platforms, which will be referred to as A - D. DNA 

sequences have been obtained from individuals consisting of haplotypes A and B (A - Bc 9, 10, 

19, 28; B – Bc 13, 32, 35; see Figure VIII-2) confirming that they are located within the B. 

candida clade and are likely the same species; however, no sequence data have yet been 

collected to confirm the phylogenetic status of haplotypes C and D. For the following analysis, 

we assumed that these 2 haplotypes also fell within the B. candida lineage. No significant 

population structure was detected using AMOVA (FST = 0.014; p>0.05) with essentially all the 

variation found among individuals within platforms compared to between platforms (Table 

VIII-2). The negative estimate of FST is likely a result of the true value being close to zero. 

Taxonomic units labeled Bc # (in Table VIII-2) are from the present study. Barbatia candida

sequences are monophyletic. 

Figure VIII-2. Comparison of mitochondrial COX I DNA sequence data with 

previously published data on Arcidae (Marko & Moran, 2002; 

Marko, 2002). 
47 

Table VIII-2

 

Results of AMOVA Analysis Showing Genetic Variation of Barbatia candida within 

and among Five Platforms In The Northern Gulf of Mexico. 

Source of Variation Variance Component % of Variation 

Among Platforms -0.0058 -1.45 

Within Platforms 0.2456 101.45 

DISCUSSION 

The genetic analysis of the Arcidae collected from five platforms in the northern Gulf of 

Mexico has shown no evidence of cryptic species or population structure. The RFLP and 

sequence data collected both indicate that all of the individuals are Barbatia candida. The 

Arcidae are known to show significant intraspecific morphological variation among populations 

in different habitats suggesting the potential for cryptic species (Marko and Jackson, 2001). 

Collecting data for more populations from differing habitats (i.e., coastal, offshore, and blue 

water) may reveal greater taxonomic heterogeneity within this group. The RFLP analysis 

suggested that there was no structuring of the arcid populations inhabiting the five sampled 

platforms. Bivalves in the family Arcidae disperse by means of planktotrophic larvae that may 

remain in the plankton for up to one to two weeks (Chanley and Andrews, 1971). Given the 

closeness of the platforms, for which samples have been studied thus far, it is not surprising that 

gene flow among them is sufficient to homogenize the populations. Nonetheless, genetic 

discontinuities have been observed among geographically close bivalve populations when 

hydrographic barriers exist (e.g., Reeb and Avise, 1990). Analysis of samples from platforms 

located in differing hydrographic regimes may reveal restricted gene flow among populations. 

ACKNOWLEDGMENTS

We thank Lorene Smith for processing samples on board ship, Nannette Crochet for 

assistance with DNA sequencing and Jennifer Kiger, Collin Richie and Kinda Seaton for 

assistance in the laboratory. 

LITERATURE CITED 

Chanley, P., and J.D. Andrews. 1971. Aids for the identification of bivalve larvae of Virginia. 

Malacologia 11(1):45-120. 

Excoffier, L., P.E. Smouse, and J.M. Quattro. 1992. Analysis of molecular variance inferred 

from metric distances among DNA haplotypes: application to human mitochondrial DNA 

restriction data. Genetics 131:479-491. 

Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. 

Evolution 39:783-791. 

Folmer, O., M. Black, R. Hoeh, R. Lutz, and R. Vrijenhoek. 1994. DNA primers for 

amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan 

invertebrates. Mol. Mar. Biol. Biotech. 3(5):294-299. 
48 

Gallaway, B.J., and G.S. Lewbel. 1982. The ecology of petroleum platforms in the 

northwestern Gulf of Mexico: a community profile. U.S. Fish and Wildlife Service, Office 

of Biological Services, Washington D.C. FWS/OBS-82/27. Bureau of Land Management, 

Gulf of Mexico OCS Regional Office, Open File Report 82-03. xiv + 92 pp. 

Gallaway, B.J., M.F. Johnson, L.R. Martin, F.J. Margraf, G.S. Lewbel, R.L. Howard, and G.S. 

Bowland. 1981. The artificial reef studies. Vol 2. In: Bedinger, C.A., and L.Z. Kirby, eds. 

Ecological investigations of petroleum production platforms in the central Gulf of Mexico. 

SWRI Project 01-5245. Bureau of Land Management, New Orleans OCS, LA. 199 pp. 

Gregg, C. S., D. W. Foltz, and J. W. Fleeger. 2006. Chapter 13: Genetic diversity of 

harpacticoid copepods. In: Continental Shelf Associates, Inc. Effects of Oil and Gas 

Exploration and Development at Selected Continental Slope Sites in the Gulf of Mexico. 

Volume II: Technical Report. U.S. Dept. of the Interior, Minerals Management Service, Gulf 

of Mexico OCS Region, New Orleans, LA. OCS Study MMS 2006-045. Pp. 441-457. 

Howard, R.L., G.S. Boland, B.J. Gallaway, and G.D. Dennis. 1980. Effects of gas and oil field 

structures and effluents on fouling community production and function. Vol V. In: Jackson, 

W.B., and E.P. Wilkens (eds). Environmental assessment of Buccaneer gas and oil fields in 

the northwestern Gulf of Mexico, 1978–1979. NOAA Technical Memorandum NMFSSEFC-39, 60p. 

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions 

through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120. 

Knowlton, N. 1993. Sibling species in the sea. Annu. Rev. Ecol. Syst. 24:189-216. 

Knowlton, N. 2000. Molecular genetic analysis of species boundaries in the sea. Hydrobiologia 

420:73-90. 

Kumar, S., K. Tamura, I.B. Jakobsen, and M. Nei. 2001. MEGA2: Molecular Evolutionary 

Genetics Analysis software, Arizona State University, Tempe, AZ. 

Marko, P.B. 2002. Fossil calibration of molecular clocks and the divergence times of geminate 

pairs separated by the Isthmus of Panama. Mol. Biol. Evol. 19(11):2005-2021. 

Marko, P.B., and J.B.C. Jackson. 2001. Patterns of morphological diversity among and within 

arcid bivalve species pairs separated by the Isthmus of Panama. J. Paleontol. 75:590-606. 

Marko, P.B., and A.L. Moran. 2002. Correlated evolutionary divergence of egg size and a 

mitochondrial protein across the Isthmus of Panama. Evolution 56(6):1303-1309. 

O’Foighil, D., T.J. Hilbish, and R.M. Showman. 1996. Mitochondrial gene variation in 

Mercenaria clam sibling species reveals a relict secondary contact zone in the western Gulf 

of Mexico. Mar. Biol. 126(4):675-683. 
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Peek, A.S., R.G. Gustafson, R.A. Lutz, and R.C. Vrijenhoek. 1997. Evolutionary relationships 

of deep-sea hydrothermal vent and cold-water seep clams (Bivalvia: Vesicomyidae): results 

from mitochondrial cytochrome oxidase subunit I. Mar. Biol. 130(2):151-161. 

Reeb, C.A., and J.C. Avise. 1990. A genetic discontinuity in a continuously distributed species: 

mitochondrial DNA in the American oyster, Crassostrea virginica. Genetics 124:397-406. 

Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing 

phylogenetic trees. Mol. Biol. Evol. 4:406-425. 

Schneider, S., D. Roessli, and L. Excoffier. 2000. Arlequin ver. 2.000: a software for population 

genetics data analysis. Genetics and Biometry Laboratory, University of Geneva, 

Switzerland. 

Street, G.T., and P.A. Montagna. 1996. Loss of genetic diversity in Harpacticoida near offshore 

platforms. Mar. Biol. 126:271-282. 

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51 

IX. CONCLUSIONS 

The Gulf of Mexico has over 3,000 oil and gas platforms that provide hard substrate for the 

attachment of a large and diverse assemblage of epifaunal or fouling organisms. Many of these 

organisms have the potential to provide compounds useful in industrial and medical applications. 

Bioprospecting for new compounds requires preliminary elucidation and clarification of the 

taxonomy of the organisms present and, in some instances, their chemical ecology. The 

macrofaunal assemblages unique to the oil and gas platforms of the Gulf are not well known and, 

in this study, the taxonomy and relative abundance with depth and distance offshore of major 

taxa of epifaunal organisms were addressed. This study involved investigation of various aspects 

of five major taxa : 

 Bacteria 

 Macroalgae

 Foraminifera 

 Bryozoa *

Molluscs 

*The study of the Bryozoa centered on a single genus, Bugula, known to be the source of 

an important chemical, bryostatin, used in cancer teatment. 

Conclusions relative to the study of these taxa are as follows. 

BACTERIA

The study demonstrated that large numbers of bacterial species, many of which are novel and 

belong to taxonomic groups known to be of biotechnological importance, were present in the 

fauna. There are a number of factors that would seem to influence the bacterial load and diversity 

of the biomatrix recovered from a given location. 

Examination of the bacterial species present in the biomatrix revealed that this is a habitat of 

novel bacterial species that represents a biotechnological and scientific resource waiting to be 

exploited. Isolation and natural product screening still has its place in biotechnology but the 

application of whole genome studies to such environments is the future path. The construction of 

total genome libraries from the biomatrix of oil and gas platforms and their expression will no 

doubt provide novel bioactivities from both the micro and macro components of these complex 

systems. This study established that the biomatrix encrusting oil and gas platforms represents a 

baseline of species and a potential starting point for material for such studies. 

MACROALGAE

The total number of macroalgal taxa collected during this study was 24. The best represented 

group was Rhodophyta (approximately 50% of the taxa). There was a vertical distribution of 

taxa found on the platforms themselves, and there was also a trend identified for biodiversity and 

abundance of macroalgae to increase from nearshore to offshore and decrease from intertidal to 

depth. 

In all, seven new taxa were added to the list of macroalgae identified from the platforms and 

there was one new report of Antithamnionella breviramosa from the Gulf of Mexico. 
52 

Approximately 50% of the taxa collected are known to have biotechnological potential and 

approximately 20% of the taxa collected are found only on oil and gas platforms. 

FORAMINIFERA

Agglutinated foraminiferal species hold promise as a source of bioadhesives for 

biotechnological and biomedical applications because they can secrete and then harden adhesive 

organic compounds in an aqueous medium. The foraminiferal community of Gulf of Mexico oil 

and gas platforms includes a great variety of species, of both agglutinated and calcareous wall 

structures. No species with biotechnology potential, however, could be identified in this 

particular study although further study of the foraminferal fauna may reveal biotechnological 

potential. Taxonomical work with this group will be necessary prior to identifying their potential 

contribution to any biotechnological potential. 

BRYOZOA

This study found that B. neritina as currently recognized in US waters comprises three 

cryptic species: the deep water Pacific form, a Shallow/Southern form present both in shallower 

Pacific waters and along the Atlantic coast south of Cape Hatteras, and a third form present in 

the Atlantic north of the Cape Hatteras region. The species collected from the Gulf of Mexico 

platforms does not produce the commercially important bryostatin I although its cytochrome 

oxidase subunit sequences were identical to those collected from the Atlantic south of Cape 

Hatteras. 

MOLLUSCS

Bivalve composition was similar to that previously reported from platforms on the Louisiana 

and east Texas continental shelf where Isognomon was most common found from 0-12 m. 

Bivalves of the Family Arcidae (arcids) were common from 3-12 m; and Chama macerophylla 

was reported between 1 and 20 m. 

The bivalve assemblages identified in this study resembled assemblages found on shorelines 

characterized by hard substrates in Texas and Mexico. The byssus are of biotechnological 

interest because they provide strong, durable adhesion to wet surfaces. In addition, some of the 

proteins in the adhesive can chelate metal ions. 

The most widely studied byssal protein is mussel adhesive protein (MAP) from Mytilus 

edulis. This compound is used as an attachment factor for cells and tissues in culture; as an 

immobilization agent for antigens, antibiotics, and enzymes; and as an anticorrosive coating for 

metals and metal sequestering reagent. Additional potential uses are as medical and dental 

adhesives and fillers; microencapsulating agents; sizing agents for textiles; and water-resistant 

inks. 

Byssal composition is highly variable among taxa. Therefore, an examination of byssate 

bivalves other than M. edulis and including those taxa (I. bicolor and B. candida) found in 

abundance in this study on Louisiana platforms may lead to the extraction of a new compound 

with similar or new applications. 

The genetic analysis of the Arcidae collected from five platforms in the northern Gulf of 

Mexico has shown no evidence of cryptic species or population structure. The RFLP and 

sequence data collected both indicate that all of the individuals are Barbatia candida. The 

Arcidae are known to show significant intraspecific morphological variation among populations 
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in different habitats suggesting the potential for cryptic species. Collections from more 

populations from differing habitats (i.e., coastal, offshore, and blue water) in the Gulf of Mexico 

may reveal greater taxonomic heterogeneity within this group. Analysis of samples from 

platforms located in differing hydrographic regimes and more spatially isolated may reveal 

restricted gene flow among populations. 

This study provides a foundation for future analyses of epifauna that may be of 

biotechnological potential. Organisms may be initially screened for use in medical and industrial 

applications with little knowledge of their ecology. There is, however, a need for much more 

information about the taxonomy, seasonality, and ecology of these organisms before any 

questions can be resolved regarding ways to farm and harvest them economically if they are 

determined to have medical or industrial applications. 
The Department of the Interior Mission

As the Nation's principal conservation agency, the Department of the Interior has responsibility 

for most of our nationally owned public lands and natural resources. This includes fostering 

sound use of our land and water resources; protecting our fish, wildlife, and biological diversity; 

preserving the environmental and cultural values of our national parks and historical places; 

and providing for the enjoyment of life through outdoor recreation. The Department assesses 

our energy and mineral resources and works to ensure that their development is in the best 

interests of all our people by encouraging stewardship and citizen participation in their care. 

The Department also has a major responsibility for American Indian reservation communities 

and for people who live in island territories under U.S. administration. 

The Minerals Management Service Mission 

As a bureau of the Department of the Interior, the Minerals Management Service's (MMS) 

primary responsibilities are to manage the mineral resources located on the Nation's Outer 

Continental Shelf (OCS), collect revenue from the Federal OCS and onshore Federal and Indian 

lands, and distribute those revenues. 

Moreover, in working to meet its responsibilities, the Offshore Minerals Management Program 

administers the OCS competitive leasing program and oversees the safe and environmentally 

sound exploration and production of our Nation's offshore natural gas, oil and other mineral 

resources. The MMS Minerals Revenue Management meets its responsibilities by ensuring the 

efficient, timely and accurate collection and disbursement of revenue from mineral leasing and 

production due to Indian tribes and allottees, States and the U.S. Treasury. 

The MMS strives to fulfill its responsibilities through the general guiding principles of: (1) being 

responsive to the public's concerns and interests by maintaining a dialogue with all potentially 

affected parties and (2) carrying out its programs with an emphasis on working to enhance the 

quality of life for all Americans by lending MMS assistance and expertise to economic 

development and environmental protection.