ADVANCING MARINE                                              BIOTECHNOLOGY: USE OF OCS OIL PLATFORMS AS SUSTAINABLE SOURCES OF MARINE NATURAL PRODUCTS

Principal Investigators: Russell J. Schmitt, Jenifer Dugan, Scott Hodges,      Robert Jacobs, Mark Page, Leslie Wilson, and Steven Gaines

Summary of Research

The potential for sustainable harvest of marine organisms that contain valuable natural products, is an important question for the future management and use of ocean.  Many species of marine organisms potentially contain compounds that could become valuable natural products for a variety of applications including medicine, industry, and food.  However, harvest of these organisms in sufficient quantities for applied uses could potentially have significant ecological impacts, particularly for those species inhabiting natural reefs. 

The intertidal and subtidal portions of OCS offshore oil and gas production platforms provide habitats for marine organisms, many of which also occur on benthic and intertidal reefs.  In waters off the coast of California, platforms are known to provide habitat for a rich and productive marine community containing over 50 species of algae and invertebrates and reaching high biomass.  The feasibility of obtaining natural products having promising pharmaceutical applications or other uses from species that grow attached to submerged portions of offshore oil and gas production platforms is virtually untested.  Growth rates of some invertebrate species on platforms can be quite high, for example, mussels and goose barnacles on oil platforms in the region grow at rates equal to or higher that the highest reported anywhere else in the world.  Furthermore, many of the organisms inhabiting oil platforms are encrusting forms which rely upon antifouling compounds, rapid growth rates and alleopathic effects to compete for and maintain space on hard substrates and avoid predation (e.g., tunicates and sponges).  Such characteristics have been shown to be associated with compounds with potential pharmaceutical and other applications. 

The organisms growing on platforms are sacrificed periodically  when the platforms are cleaned for structural safety.  Using this untapped source of marine organisms for natural products research (and perhaps eventual harvesting) could eliminate virtually all of the direct ecological effects that accrue from harvesting on natural reefs.  For example, in the Southern California Bight, mussels for human consumption are harvested on a sustained basis from offshore oil platforms, raising the possibility that platform species with desired natural products also could be 'cultured' with minimum impacts on natural reefs.  Further, organisms that naturally dwell at very great depths occur within scuba diving depths on the offshore platforms in our region.  This unique situation provides us with remarkably easy access to a suite of species that otherwise would be difficult and expensive to examine, and raises the possibility that eventual mariculture of these species on OCS oil platforms also may be feasible. 

Overall, our objective is to investigate the feasibility of using OCS oil platforms for the development of marine biotechnology.  Specifically, we plan to explore the use of organisms growing on OCS oil platforms for the development of pharmacological compounds.  To realize these objectives we will integrate research across three disciplinary areas here at UCSB: 1, community and population ecology of reef organisms (Dr. Jenifer Dugan, Dr. Mark Page), 2, population genetics and natal sources (Dr. Scott Hodges, Dr. Steven Gaines), and 3, pharmacology with an emphasis on natural products identification and mechanisms of action (Dr. Robert Jacobs, Dr. Les Wilson).  We believe that the long-term success of using OCS oil platforms for biotechnology development will require basic information derived from each of these disciplinary areas.  For this proposal, the major objectives addressed in each of these areas are:

COMMUNITY AND POPULATION ECOLOGY.  To survey OCS oil platforms for organisms with potential applications to biotechnology, particularly those with pharmacological applications.  To determine the factors affecting the growth and distribution of these organisms on OCS oil platforms. 

POPULATION GENETICS AND NATAL SOURCES.  To generate molecular-genetic markers for the accurate identification of species with potential applications to biotechnology.  To determine the degree of genetic diversity of organisms growing  on OCS oil platforms relative to natural reefs.  Identify sources of populations for organisms growing on OCS oil platforms. 

PHARMACOLOGY.  To identify novel compounds, particularly eicosanoids and coumarins, affecting inflammation, wound healing or cell division from organisms growing on OCS oil platforms.  To investigate the mechanisms of action of these compounds for the potential development of new drug targets.  

ECOLOGY:

Background

Interest in marine natural products continues to grow worldwide. However, concern over the impact of the harvest of organisms that contain these products on the environment has arisen because large quantities of organisms are typically needed to extract a small amount of a natural product. Harvest of organisms from man-made structures, particularly oil and gas platforms, may alleviate impacts to natural reefs as many species of invertebrates grow on these artificial structures. Unfortunately, little information is available on the distribution and abundance and dynamics of invertebrates on oil platforms.  To examine the possibility of using OCS oil platforms as sustainable sources of, or as culturing sites for, invertebrates with important marine natural products, we have: 1) investigated spatial and temporal patterns in the distribution and abundance of invertebrates on selected offshore oil platforms in the Santa Barbara Channel, 2) explored whether the population dynamics (recruitment and growth) of common invertebrates vary among platforms (both spatially and temporally), and 3) examined the relationship between patterns of distribution and abundance and recruitment found at the platforms, and selected environmental factors (e.g., location, water temperature). Data collection on the Ecology component of this project is complete and manuscripts are in preparation. 

Study Sites 

We conducted our research at seven oil and gas platforms in the Santa Barbara Channel (Table 1, Fig. 1).  The platforms are arranged along the channel from the southeast to northwest in a region characterized by strong environmental and biogeographic gradients. 

Table 1.  Characteristics of study platforms.  Key to abbreviations:  Gi-Gina, Ga-Gail, Gil-Gilda, Gr-Grace, Hog-Hogan, Hou-Houchin, Hol-Holly.

Preliminary Results

Spatial variation in platform invertebrate communities

 
Across all platforms, the most widely distributed and abundant taxa, accounting for 83% of the total cover in our photoquadrats, were anemones (e.g., Corynactis californicus, Metridium sp.), tubiculous amphipods, (primarily Ericthonius sp.), hydroids (Plumaria, Agalophenia), and sponges (e.g., Haliclona spp., Halichondria panicea).  Other widespread taxa included mussels, (Mytilus californianus, M. edulis), barnacles (Megabalanus californicus, Balanus spp.), and tunicates (e.g., Styela montereyensis).  Exotic species were conspicuous on two platforms; the encrusting bryozoan, Watersipora cucullata, was observed only on Platform Gilda and the anemone, Diadumene sp. was recorded only on Platform Gail .  Filamentous red algae were the most widely distributed algal taxon.  However, the cover of algae was low (~5%) overall.

The structure of invertebrate communities varied greatly among platforms (P<0.001, F=13.729, df=120, 1082.43, MANOVA:  Fig. 2).  Anemones occurred in higher cover overall (up to 50 to 60%) than most other invertebrates, but the dominant species varied with location.  Corynactis californicnus was the dominant anemone on platforms at southeast end of the channel (e.g., Gina, 59 ± 18%); cover of this anemone tended to be lower on platforms to the northwest (e.g., 5 ± 2% at Holly).  An exception to this pattern occurred at Gail where mean cover of C. californicus was only 2 ± 1% and the most abundant anemone was the exotic species, Diadumene sp. (25%).  In contrast, mean cover of Metridium sp. was generally highest at the most northwest platforms  (Holly, 51 ± 13%) and lower on platforms to the southeast (Gina, 3 ± 2%).  An exception to this pattern was evident at Hogan where cover of Metridium was only 2 ± 1%. 

Tubiculous amphipods, hydroids, and mussels also generally occurred in higher cover on platforms with increasing distance along the channel from the southeast to the northwest (Fig. 2).  For example, tubiculous amphipods occurred at 15 to 20% cover on Hogan and Houchin, but <5% on Gail and Gilda.  Highest cover of mussels was recorded for Grace and Hogan (up to 25%) and lowest cover at Gilda (<3%).  In contrast, the cover of sponges was more variable, with highest cover at Gail (up to 35%) and the two most northerly platforms (Houchin, Holly).  The bryozoan, Watersipora cullcullata, occurred only on Gilda with mean cover of 41% (data not shown). 

Figure 2.  Comparison of the distribution and abundance of the anemones, Corynactis californicus and Metridium senile, sponges, tubiculous amphipods, mussels (Mytilus spp.), and hydroids among study platforms.

Community patterns

Discriminant Function Analysis (DFA) revealed that the communities of Gail and Gilda were clearly different from the other platforms, a pattern that can be attributed, in part, to the presence of introduced species on these platforms (Fig. 3a).   Canonical Discriminant Functions (CDF) 1 and 2 explained 80% of the variation in the data.  Cover of the anemone, Diadumene sp. was positively correlated (0.482) with CDF1, and an important source of the separation of Gail from the other platforms along the CDF1 axis.  The negative correlation of cover of the bryozoan, Watersipora cucullata, with CDF2 (-0.379) for Gilda contributed to the separation of this platform from the others (Fig. 3a).

To explore the effect that the exotic species might have on community patterns, we repeated the DFA, but excluded Diadumene sp. and Watersipora cucullata from the analysis (Fig. 3b).  The significant positive correlation of sponges (0.584) and negative correlation of Corynactis californicus (-0.614) with CDF1 contributed to the separation of all platforms except Gail along the CFD1 axis.  In contrast, the positive correlation of cover of Metridium sp (0.605) and negative correlation of hydroids (-0.428) with CDF2 contributed to the separation of Gail from the other platforms along the CDF2 axis.  Removal of W. cullculata from the analysis reduced variability in the Gilda data and community patterns at this platform tended to become more similar to those of the next closest platform (Gina).  In contrast, the structure of the invertebrate community at Gail remained distinct from the other platforms (Fig. 3b). 

Figure 3.  Results of Canonical Discriminant Function Analysis of invertebrate communities on the seven study platforms: a) all species, b) exotics species excluded.

Community patterns and environmental variables

To explore relationships between community patterns and environmental variables, we used the calculated values of CDF1 for each platform (calculated including and excluding Diadumene sp. and Watersipora cullculata), and the independent variables of location along the channel, water depth, proximity to shore, and platform size (Table 1) in stepwise multiple regression analysis.  Prior to this analysis, we tested for co-linearity among the independent variables.  There was a significant correlation between platform size and both water depth (P<0.001, r = 0.974) and proximity to shore (P = 0.049, r=0.758).  However, depth and proximity to shore were not significantly correlated (P>0.1).  Therefore, we excluded platform size from the analysis, but included water depth and distance from shore.  There was no relationship (P>0.1) between variation in CDF1 and any of the independent variables if the data from Gail were included in the analysis.   If the data from Gail were excluded from the analysis, variation in CDF1 was best explained by location along the channel (P=0.014:  Fig.4).

Figure 4.  Relationship between canonical discriminant function 1 and location of platforms along the Santa Barbara Channel.  R² value calculated excluding data from Platform Gail.  Taxa most positively or negatively correlated with CDF1 are also shown on the y-axis.

Measurements of water temperature during deployment of experimental modules

The prevailing gradient in oceanographic conditions in the Santa Barbara Channel is evident in satellite images showing the intrusion of warm water into the channel from the south and cold water from the west.

To examine variation in water temperature among platforms, which could help to explain variation in community patterns, a HOBO temperature logger was attached to one of the experimental modules at each platform.  Water temperature was recorded hourly, with the loggers retrieved and downloaded at the end of each experimental period.  To compare temperatures among sites we calculated the frequency of the number of hours at each water temperature (Fig. 5).

Overall, the water temperatures at the three northern platforms were similar to each other throughout the year.  Likewise, water temperatures of the four southern platforms were similar during all seasons, except in the summer.  During the summer, one site (Gina) experienced large daily fluctuations in water temperatures, resulting in a broad range of temperatures throughout this season (Fig. 5).  Such large temperature ranges were not recorded at the other southern sites.

Figure 4.  Water temperature frequencies by degree hours for Summer 2001 at a) southern and b) northern platforms.

Figure 5.  Distribution of water temperature, expressed as percent of total number of hours at a given water temperature, for the study platforms.

Recruitment Patterns and Oceanographic Factors

Recruitment data are useful for evaluating which platforms may provide sustainable sources of marine natural products.  There were significant spatial and temporal differences in patterns of recruitment of several taxa, although to varying degrees (Fig. 6).  For example, for some species, recruitment was higher at the southern platforms (barnacles; Balanus trigonus and B. regalis), while for others recruitment was higher at the northern platforms (hydroids; Plumularia sp.).  Further, for some species recruitment was spatially limited to just one (encrusting bryozoans; Watersipora cucullata) or two platforms (tunicates; Diplosoma literianum).  Likewise, temporal patterns of recruitment varied among taxa, with recruitment of some species occurring seasonally while for others it was more continuous (e.g., tunicates).

Figure 6.  Comparison of spatial and temporal patterns of recruitment of four taxa, the barnacles, Balanus trigonus, the hydroids, Plumularia sp., the encrusting bryozoans, Watersipora cucullata and the tunicates, Diplosoma listerianum, among study platforms.

We used the nonparametric Spearman’s Rho (Rs) to explore relationships between patterns of recruitment of selected invertebrates onto plates at the study platforms and location in the Santa Barbara Channel, platform depth, distance from shore and oceanographic factors (Table 2).  For the three barnacle species, there was a significant effect of location, with higher recruitment densities occurring at the southern compared to the northern platforms.  This pattern was consistent with predictions of oceanographic conditions bringing warm water masses and the longer-lived planktonic larvae of southern taxa into the channel.  Recruitment density was also associated with location along the channel for the hydroid, Plumularia sp.  For this species, recruitment was higher at the northern than at the southern platforms. 

In contrast, patterns of recruitment were variable for the majority of invertebrate taxa with short-lived larvae or direct development. 

For most of these species, there was no relationship between recruitment and location; many of these organisms recruited at only a few platforms (e.g., Diplosoma listerianum) or a single platform (Watersipora cucullata) where mature colonies occurred in high abundance.  This recruitment pattern is consistent with the short larval development time and limited dispersal of these species.  Although there was an association between recruitment and location for hydroids, their limited dispersal ability (crawl away larvae) suggests that oceanographic factors associated with water masses likely had little influence on transport of the hydroid larvae and subsequent recruitment.  Instead, biological interactions (predation, competition) or other factors may have influenced the recruitment patterns of this taxon. 

Table 2.  Relationships between recruitment of selected invertebrates and location in the channel. Rs = Spearman correlation coeffiencient. * < 0.05; ** < 0.01; *** < 0.001. nsv = no significant variation in recruitment.  nr=no recruitment

Species                                Summer 2001         Fall 2001       Winter 2002      Spring 2002

Barnacles

  Balanus trigonus                      -0.857**            -0.893***             0.607            0.509

  Megabalanus californicus        nsv                     -0.321                     nsv            -0.786*

  Balanus regalis                         nsv                       nr                          nsv             -0.821*

Encrusting bryozoans

  Watersipora cucullata              -0.204               -0.204                      nr               -0.204

  Other encrusting bryozoans        nsv                    -0.107                  -0.054              0.071

Branching bryozoans                                

  Crisia complex/

      Bugula neritina                    0.071                  0.036                  -0.286              0.029

Hydroids                                 

  Plumaria sp.                             0.901***            nsv                      0.056               0.089

Tunicates

  Diplosoma listerianum             0.445                  0.0490                0.0490             0.045

Our water temperature data provide support for the hypothesis that oceanographic factors influenced recruitment patterns in the summer for those species with longer-lived planktonic larvae, as a gradient in water temperature occurred along the Santa Barbara Channel during this season.  In particular, warm water intrusion was detected at the southern, but not at the northern platforms (Fig. 5).  There was a significant correlation between barnacle (B. trigonus) recruitment and water temperature in the summer, both with (p = 0.05, Spearman’s Rho) and without (p = 0.0003) the outlying data from Platform Gina (Fig. 7).  This suggests that larvae of warm water species could have been transported in these water masses during the summer.

Figure 7. The relationship between recruitment of Balanus trigonus and water temperature (mode) at each location.  Open circle = Platform Gina.

GENETICS:

We have focussed on determining the genetic variation among populations of Bugula neritina.  Primarily, we analyzing DNA sequence variation for the mtDNA segment we have PCR amplified. A preliminary analysis of  samples that are clearly closely related to the cryptic B. neritina species that harbors the bacterium that produces Bryostatin-1  appear in Figure 1. This analysis has two particularly interesting outcomes. First, it strongly supports that we have identified a new cryptic species of B. neritina. Second, it suggests that all of the samples from two OCS oil platforms are members of a single clade and thus that colonization of platforms may be a relatively rare event. We have also designed a new pair of primers for amplification from the bacterial symbiont in order to assess whether the new B. neritina clade also harbors a unique lineage of symbionts (one that may produce a unique Bryostatin compound). 

Currently, we are analyzing the mtDNA sequence data and will  begin conducting the bench-work to amplify and sequence DNA from the bacterial symbiont. Our goal is to determine if there is a unique genetic lineage of the bacterial symbiont in the new bryozoan lineage we have identified (Figure 1). 

Figure 1. Neighbor joining analysis of DNA sequences from the 1.4 Kb mtDNA region of Bugula neritina. Numbers above lines indicate bootstrap support (1000 replicates). The large box encompasses the lineage containing samples from platform Hugo (HG) and platform Houchin (HU). The smaller box encompasses the new lineage of B. neritina found from samples from the Channel Islands.

PHARMACOLOGY:

We are optimizing a number of organic extraction procedures in order to obtain a favorable yield of pure extract from Waterispora cucullata (WC). We confirmed that the activity was retained by testing the pure compounds in the Sea Urchin Embryo assay.

<>Fresh samples were extracted from Waterispora Cucullata (WC) and assayed on HeLa S3 cells for evaluation on cell proliferation and viability. 

Results

Inhibition of Cell Proliferation and Viability

Plots were made of % inhibition of cell proliferation (average of 2 samples) vs. drug concentration.  Percent % inhibition data are shown in figures 1 and 2.  IC50 values were approximated from plots and determined to be 12 μg/ml for WC1 and 16 μg/ml for WC2.  Cell viabilities were determined for each drug concentration by trypan blue dye exclusion and were found to be equal or less than controls in all cases (control viability 98.9%)

Mitotic Index, Spindle and Chromosome Abnormalities

The mitotic index was determined from DAPI-stained coverslips of untreated and drug treated samples.  (Table 1 below).  A 3 fold increase in mitotic index was seen in the two highest WC1 samples but the mitotic index of the WC2 sample was slightly below that of the control.  A high percentage of mitotic cells on the WC1 treated coverslips were noted to have abnormal chromosome distributions (see attached PowerPoint Slide for example).  The most common abnormal cell type was a mitotic cell with a distinguishable metaphase chromosome alignment but with 1 or more chromosomes stuck at the pole.  Several years ago Mary Ann Jordan developed a classification scheme for these where she grouped the abnormal cells as having either type I, type II or type III spindles.  (Jordan et al. 1992 J. Cell Science 102:401-16).  Type I spindles have elongated astral microtubules (we suspect most of these were supposed to be kinetochore microtubules but never made the right connections) and a metaphase chromosome distribution with one to 4 chromosomes at the pole.  Type II spindles have a shorter than normal interpolar distance (although still with long astral microtubules) and more than 4 chromosomes at the poles but still have recognizable metaphase accumulation of the majority of chromosomes at the equator.    Type III spindles form a ball of chromosomes as a result of a collapse of the spindle into a monoastral configuration in place of the normal bipolar orientation.  We have observed these types of cells as a result of treatments with drugs such as the Vinca alkaloids, podophyllotoxin, and taxol type compounds.   This morphology is observed when nanomolar concentrations of these drugs are used; concentrations that do not alter microtubule polymer mass.  From studies of interphase cells treated with these concentrations of drugs we know that microtubule dynamic instability is being suppressed by these drugs at these concentrations.

Most of the abnormal cells in WC1 treated cells fell into the category of type I and type II spindles.  The proportion of type I and type II mitotic spindles to total metaphase cells was determined and is reported in Table 1.  The abnormal mitotic cells were rare in control populations and were not observed in WC2-treated cells.  Also notable was the lack of increased numbers of multipolar spindles in WC1-treated cells (HeLa cells spontaneously develop 1-2% of these as a proportion of total mitotic cells but no increase in this number was seen in WC1 treated cells-data not shown).  Multipolar spindles are common in cells treated with compounds like taxol and epothalone B, but not with Vincas and other drugs that at higher concentrations cause microtubule depolymerization.   I think we can safely conclude that the crude extracts contain at least one compound that promotes microtubule stabilization in a manner similar to that of the Vinca alkaloids.

Table 1

Figure 1

Figure 2

A graduate student , Daniel Day, is working in the structure elucidation of the pure compound extracted from Waterispora Cucullata (WC). After studying a number of established protocols we were able to once again significantly increase the yield of pure compound extracted.  The methods and results are described in the following paragraphs and figures.

Figure 3.

Figure 4. 

Figure 5.

Figure 6.

Figure 7.

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