Bryozoan Bugula Neritina Research Articles

Complex life cycles and offspring provisioning in marine invertebrates

Offspring size can have pervasive effects throughout an organism's life history. Mothers can make either a few large or many small offspring, and the balance between these extremes is determined by the relationship between offspring size and performance. This relationship in turn is thought to be determined by the offspring's environment. Recently, it has become clear that events in one life-history stage can strongly affect performance in another. Given these strong carryover effects, we asked whether events in the larval phase can change the relationship between offspring size and performance in the adult phase. We manipulated the length of the larval period in the bryozoan Bugula neritina and then examined the relationship between offspring size and various parameters of adult performance under field conditions. We found that despite the adult stage being outplanted into identical conditions, different offspring sizes were predicted to be optimal, depending on the experience of those adults as larvae. This work highlights the fact that the strong phenotypic links between life-history stages may result in optimal offspring size being highly unpredictable for organisms with complex life cycles.

Using latent effects to determine the ecological importance of dissolved organic matter to marine invertebrates

The uptake and utilization of dissolved organic matter (DOM) by marine invertebrates is a field that has received significant attention over the past 100 years. Although it is well established that DOM is taken up by marine invertebrates, the extent to which it contributes to an animal's survival, growth, and reproduction (that is, the ecological benefits) remains largely unknown. Previous work seeking to demonstrate the putative ecological benefits of DOM uptake have examined them within a single life stage of an animal. Moreover, most of the benefits are demonstrated through indirect approaches by examining (1) mass balance, or (2) making comparisons of oxyenthalpic conversions of transport rates to metabolic rate as judged by oxygen consumption. We suggest that directly examining delayed metamorphosis or the latent effects associated with nutritional stress of larvae is a better model for investigating the ecological importance of DOM to marine invertebrates. We also provide direct evidence that availability of DOM enhances survival and growth of the bryozoan Bugula neritina. That DOM offsets latent effects in B. neritina suggests that the underlying mechanisms are at least in part energetic.

Facilitation and inhibition of larval attachment of the bryozoan Bugula neritina in association with mono-species and multi-species biofilms

In this study, we investigated the effect of mono-species and multi-species biofilms on larval attachment of the bryozoan Bugula neritina. The effect of biofilms was examined through a double-dish choice bioassay in which larvae were given the choice of attaching either to a clean surface of a container or to surfaces covered with biofilms. Larvae attached in response to mono-species biofilms of 5 out of 7 bacterial isolates from a subtidal region, but they avoided surfaces covered by biofilms of 7 out of 8 isolates obtained from an intertidal region. In the follow-up choice experiments with multi-species biofilms developed for 2 days, 7 days, 14 days, 28 days and 30 days, larvae preferentially attached to filmed surfaces over the unfilmed surfaces. When biofilms from 2 different tidal regions (intertidal and subtidal) were offered as choices in the double-dish bioassay, larvae in all cases attached on the subtidal biofilms. Two-day-old subtidal biofilms with low densities of bacteria induced significantly higher ( p < 0.05) attachment than did 30- day-old intertidal biofilms, which had high bacterial density. Terminal Restriction Fragment Polymorphism (T-RFLP) analysis revealed that the bacterial communities were substantially different in the subtidal and intertidal regions during all periods of the experiment. Attachment of B. neritina on subtidal biofilms did not depend on the bacterial density but rather was negatively correlated with diatom density, thickness of the exopolysaccharide layer and biofilm age. Our results suggest that the larvae of B. neritina can discriminate between biofilmed and clean surfaces and between biofilms developed under different tidal zones.

Isolation of Two Polyketide Synthase Gene Fragments from the Uncultured Microbial Symbiont of the Marine Bryozoan Bugula neritina

Isolation of Two Polyketide Synthase Gene Fragments from the Uncultured Microbial Symbiont of the Marine Bryozoan Bugula neritina

Differential tolerance to metals among populations of the introduced bryozoan Bugula neritina

Resistance to heavy metals is a potentially important trait for introduced marine organisms, facilitating their successful invasion into disturbed natural communities. We conducted laboratory and field experiments to examine differential resistance to copper (Cu) between two source populations of the introduced bryozoan Bugula neritina, originating from a polluted (Port Kembla Harbour, NSW, Australia) and an unpolluted (Botany Bay, NSW, Australia) environment. A laboratory toxicity test was conducted to test the relative resistance of B. neritina recruits from the two sources, by measuring the attachment success, survival and growth of individuals exposed to a range of Cu concentrations (0, 25, 50 and 100 μg l−1 Cu). Upon completion, reciprocal transplantation of the colonies to the original polluted and unpolluted locations was carried out to assess ongoing survival and growth of colonies in the field. B. neritina colonies originating from the polluted Port Kembla Harbour had increased resistance to Cu relative to populations from an unpolluted part of Botany Bay. There appeared to be a cost associated with increased metal tolerance. In the laboratory, Botany Bay recruits displayed significantly higher growth in control treatments and significantly poorer growth at 100 μg l−1 Cu with respect to Port Kembla Harbour individuals, which showed unusually uniform and low growth irrespective of Cu concentration. No difference in attachment success or post-metamorphic survival was observed between populations. Field transplantation showed copper resistance in Port Kembla Harbour colonies constituted an advantage in polluted but not benign environments. The findings of this study provide evidence of the benefits to invasive species of pollution tolerance and suggest that human disturbance can facilitate the establishment and spread of invasive species in marine systems.

Suppression of Settlement of Larvae of Fouling Organism by Water-Soluble Substances from Epibiotic Bacteria

Twenty-nine bacterial strains were isolated from the surface of the green alga Ulva reticulata, the soft coral Dendronephthya sp., and the sponge Haliclona sp. The bacterial species Vibrio alginolyticus, Vibrio sp. 4, an unidentified α-Proteobacterium, Vibrio sp. 7, Pseudoalteromonas sp. 2, and Pseudoalteromonas sp. 4 were found to suppress the larval settlement of the polychaete Hydroides elegans (Haswell, 1883) and the bryozoan Bugula neritina (Linnaeus, 1758). Aqueous extracts of five bacteria (all those named above except Pseudoalteromonas sp. 2) prevented larval settlement. Bacteria V. alginolyticus, Vibrio sp. 4, and an unidentified α-Proteobacterium were first discovered to produce high-molecular substances (>100 kDa) preventing larval settlement. Their activity was inhibited by amylase treatment, while trypsin and papain did not influence their activity. The data obtained proved that bacteria from the surface of the number of marine organisms excrete water-soluble sacchariferous compounds preventing larval settlement.

Identifying bryostatins and potential precursors from the bryozoan Bugula neritina

The bryozoan species Bugula neritina contains the anticancer agent bryostatin. Bryostatin has been extracted from these sessile marine invertebrates since the late 1960s from the Gulf of California, Gulf of Mexico, as well as various locations on the eastern and western rims of the Pacific Ocean. In this work we are focusing on animals harvested in the Gulf of Mexico near Alligator Point (Florida). Using Inductively Coupled Plasma-Mass Spectrometry (ICP–MS) we measure the concentration of 70 elements in B. neritina, a sea squirt, and the sediment from the point of harvesting. This data has helped us generate an extraction process for marine natural products. Combining UV/VIS absorbance measurements with Matrix Assisted Laser Desorption Ionization–Time of Flight-Mass Spectrometer (MALDI–TOF-MS), we demonstrated that the specific form of bryostatin extracted is a function of the solvent. A 9.4 T Fourier Transform-Ion Cyclotron Resonance (FT-ICR) mass spectrometer, whose sensitivity, mass accuracy, and resolving power allowed the exact empirical formulas of potential precursors of bryostatin to be identified, was employed. Finally we examine extracts of 14 marine species of the Gulf of Mexico, from the sand trout (Cynoscion arenarius) to chicken liver sponge (Chrondrilla nucula), all recently collected, which had shown some medicinal activity thirty years ago in a National Cancer Institute study. By the MALDI–TOF-MS, we were able to identify mass spectral features that correspond to different variations of the basic bryostatin structure, which raises the question if the bryozoans are the original source of bryostatin.

Responses to damage in an arborescent bryozoan: Effects of injury location

A common consequence of the predation or physical disturbance of sessile modular animals is the removal of part of the colony. This damage is often sub-lethal, and leaves the remainder of the colony to repair and regenerate lost tissue. Damage may, however, have lasting effects on the life history characteristics of a colony. We examined the effects of damage on growth and reproduction in the arborescent bryozoan Bugula neritina (Linnaeus), with a particular focus on the effects of the location of that damage. We predicted that a damaged colony would initially allocate more energy to damage repair and subsequent growth, increasing in size to decrease the risk of mortality, at the expense of its short-term reproductive potential. A short-term experiment assessed the immediate regenerative capacities of colonies, while a longer-term experiment measured overall colony responses to damage at different colony locations. In this experiment, zooids were removed in three colony regions; all on one colony branch, spread evenly across all growing tips, and a third, intermediate pattern. A third experiment tested the effects of predator-induced damage versus damage alone, by measuring the recovery of colonies subjected to either a predation event by the nudibranch Polycera hedgpethi, or to an imitation predation event, where an equivalent section of the colony was removed manually. Regeneration was rapid, with budding resuming a few days after damage occurred. Removing around 20% of the colony did not consistently alter colony growth rates in any experiment, but the onset of reproduction was delayed by 7 to 20 days, and subsequent colony fecundity reduced by as much as 70% in one damage treatment. The removal of the same amount of tissue by nudibranch predators, or manually removed in a treatment that mimicked this predation, had no detrimental effect on the onset of reproduction or the eventual reproductive output. Nudibranchs removed tissue by consuming whole colony branches, and the experimental treatment that removed one branch also had the least effect of three damage treatments. These results suggest the severity of long-term effects depends on the location of the injury, and that B. neritina does not modify its growth or reproductive patterns to minimise these effects.

Geographical variation in offspring size effects across generations

Offspring size is thought to strongly affect offspring fitness and many studies have shown strong offspring size/fitness relationships in marine and terrestrial organisms. This relationship is strongly mitigated by local environmental conditions and the optimal offspring size that mothers should produce will vary among different environments. It is assumed that offspring size will consistently affect the same traits among populations but this assumption has not been tested. Here I use a common garden experiment to examine the effects of offspring size on subsequent performance for the marine bryozoan Bugula neritina using larvae from two very different populations. The local conditions at one population (Williamstown) favour early reproduction whereas the other population (Pt. Wilson) favours early growth. Despite being placed in the same habitat, the effects of parental larval size were extremely variable and crossed generations. For larvae from Williamstown, parental larval size positively affected initial colony growth and larval size in the next generation. For larvae from the other population, parental larval size positively affected colony fecundity and negatively affected larval size in the next generation. Traditionally, exogenous factors have been viewed as the sole source of variation in offspring size/fitness relationship but these results show that endogenous factors (maternal source population) can also cause variation in this crucial relationship. It appears offspring size effects can be highly variable among populations and organisms can adapt to local conditions without changing the size of their offspring.

The effect of bacterial and diatom biofilms on the settlement of the bryozoan Bugula neritina

Biofilms of marine bacteria and diatoms and their combinations were examined in laboratory choice assays to determine their effects on the attachment and successful metamorphosis of the larvae of the bryozoan Bugula neritina (Linnéus). The larval settlement in response to unfilmed surfaces, a natural biofilm (NBF) and adsorbed cells of three strains of bacteria, five strains of pennate diatoms and combinations of the two at different densities. Bacterial and diatom strains showed different effects on the larval settlement of B. neritina. Bacterial monospecific strains of an unidentified α- Proteobacterium and Vibrio sp. mediated the same percentage of settlement as a filtered seawater control. Biofilms of Pseudoalteromonas sp. caused significantly lower larval settlement. Larval settlement of B. neritina was negatively correlated with increasing densities of Pseudoalteromonas sp. The highest percentages of settlement were mediated by the biofilms of the diatom species Achnanthes sp., Amphora cofeaeformis, Amphora tenerrima, Nitzschia constricta and a 5-day-old natural biofilm, while the lowest settlement was found on a N. frustulum film. A three-way analysis of variance demonstrated that the density of bacteria and the presence of particular species of diatoms and bacteria in combined biofilms, significantly affected the settlement of B. neritina larvae. High settlement of larvae (50–90%) at all treatments indicated that B. neritina larvae are much more indiscriminate settlers than previously expected. Hence, using this species as a monitoring organism to trace ecologically relevant subtle changes of settlement cues in the natural environment should be carefully re-examined.

When the going gets rough: effect of maternal size manipulation on larval quality

Variation in larval size has been shown to be an important factor for the post-metamorphic performance of marine invertebrates but, despite its importance, few sources of this variation have been identified. For a range of taxa, offspring size is positively correlated with maternal size but the reasons for this correlation remain unclear. We halved the size of colonies in the bryozoan Bugula neritina 1 wk prior to reproduction (but during embryogenesis) to determine if larval size is a fixed or plastic trait. We manipulated colonies in such a way that the ratio of feeding zooids to reproductive zooids was constant between treatment and control colonies. We found that manipulating colony size strongly affects larval size; halved colonies produced larvae that were similar to13% smaller than those produced by intact colonies. We entered these data into a simple model based on previous work to estimate the likely post-metamorphic consequences of this reduction in larval size. The model predicted that larvae that came from manipulated colonies would suffer similar to300% higher post-metamorphic mortality and similar to50% lower fecundity as adults. Colonies that are faced with a stress appear to be trading off current offspring fitness to maximize their own long-term fitness and this may explain previous observations of compensatory growth in colonial organisms. This study demonstrates that larval size is a surprisingly dynamic trait and strong links exist between the maternal phenotype and the fitness of the offspring. The performance of settling larvae may be determined not only by their larval experience but also by the experience of their mothers.

Waterborne polar macromolecules act as algal antifoulants in the seaweed Ulva reticulata

Repetitive field observations in Hong Kong waters revealed that the green macroalga Ulva reticulata was conspicuously free of epibiotic macroorganisms. This phenomenon was indica- tive of some sort of natural antifouling control in a macroalga that has been typified as undefended thus far. In previous studies, we identified the alga itself and a thallus-associated epibiotic Vibrio sp. as sources of waterborne, macromolecular substances with an inhibitory effect on the larval attach- ment of the polychaete Hydroides elegans. Employing a less artifactual water collection technique under field conditions in this study, we demonstrated that naturally obtained algal exudates evoked a similar inhibitory effect as those obtained artificially under laboratory conditions. In order to inter- pret the chemical defense system of U. reticulata with respect to the production and nature of bioac- tive metabolites, the allelopathic activity of secondary metabolites obtained from Vibrio sp. and the macroalga was bioassayed and biochemically analyzed in parallel. In addition to H. elegans, bio- assays were performed with larvae of the bryozoan Bugula neritina. Different enzyme probes revealed chemical and structural features of the factor inhibitory to larval attachment in both sam- ples, and pointed to large polysaccharide units and sulfate esters as structural parts of the bioactive moiety. Specific susceptibilities of the algal and bacterial samples to proteolytic enzymes indicated that (a) different macromolecular constituents were causative of the inhibitory effect, and (b) that the bioactive moiety in the alga-derived sample comprised a structural connection between carbo- hydrate(s) and proteinaceous molecular domains. The results indicated an antifouling concept in U. reticulata that was presumably driven by at least 2 different waterborne macromolecules, and revealed that highly water soluble macromolecules may play an essential chemical ecological role in a marine system. This exemplifies a distinct concept in comparison to other prominent examples of algal antifoulants, which exclusively comprise comparatively small and non-polar algal metabolites.

Effect of amino acids on larval metamorphosis of the polychaete Hydroides elegans

The inductive compounds that are cues for for the settlement and metamorphosis of polychaete Hydroides elegans larvae, isolated from adult homogenate of the same species and leachate of the bryozoan Bugula neritina, are known to be a mixture of amino acids. Here we inves- tigated the toxicity of 20 amino acids on competent larvae. Nine amino acids were found to be toxic at certain concentrations. Subsequently, 12 out of 20 amino acids were assayed for their effect on lar- val metamorphosis of H. elegans. The effects of amino acids were decoupled from possible inductive effects of the bacteria in the bioassay system. The results showed that the amino acid isoleucine had no inductive effect on larval metamorphosis. Two acidic and 9 aliphatic amino acids directly induced larval metamorphosis. The effects of these amino acids were concentration-dependent. Among them, asparagine was the most active amino acid, and induced a high percentage of larval settlement at 1.0 × 10 -5 M, but became toxic at concentrations higher than 1.0 × 10 -4 M. The exact mechanisms by which amino acids directly induce larval metamorphosis are still unclear, although we assume that they act by binding to specific receptors and triggering the signal transduction pathway entirely or partially.

OFFSPRING SIZE AFFECTS THE POST-METAMORPHIC PERFORMANCE OF A COLONIAL MARINE INVERTEBRATE

The positive relationship between offspring size and offspring fitness is a fundamental assumption of life-history theory, but it has received relatively little attention in the marine environment. This is surprising given that substantial intraspecific variation in offspring size is common in marine organisms and there are clear links between larval experience and adult performance. The metamorphosis of most marine invertebrates does not represent a newbeginning, and larval experiences can have effects that carry over to juvenile survival and growth. We show that larval size can have equally important carryover effects in a colonial marine invertebrate. In the bryozoan Bugula neritina, the size of the non-feeding larvae has a prolonged effect on colony performance after metamorphosis. Colonies that came from larger larvae survived better, grew faster, and reproduced sooner or produced more embryos than colonies that came from smaller larvae. These effects crossed generations, with colonies from larger larvae themselves producing larger larvae. These effects were found in two populations (in Australia and in the United States) in contrasting habitats.

Drugs from the Sea - Opportunities and Obstacles

The supply problem with regard to drug development and sustainable production lies in the limited amounts of biomass of most marine invertebrates available from wild stocks. Thus, most pharmacologically active marine natural products can only be isolated in minute yields. Total synthesis of pharmacologically active natural products has been successfully established but is in many cases economically not feasible due to the complexity of the molecular structures and the low yields. To solve the pressing supply issue in marine drug discovery, other strategies appear to be more promising. One of these is mariculture which has successfully been established with the bryozoan Bugula neritina (the source of the bryostatins) and the tunicate Ecteinascidia turbinata (the source of ET-743). Another strategy involves partial synthesis from precursors which are biotechnologically available. An example is ET-743 that can be partially synthesized from safracin B which is a metabolite of Pseudomonas fluorescens. There have been many examples of striking structural similarities between natural products obtained from marine invertebrates and those of microbial origin which suggests that microorganisms living in their invertebrate hosts could be the actual producers of these secondary metabolites. With regard to sustainable biotechnological production of pharmacologically important metabolites from marine invertebrates and their “endosymbionts”, a more advanced strategy is to focus on cloning and expression of the respective key biosynthetic gene clusters. This molecular biological approach will open up new avenues for biotechnological production of drugs or drug candidates from the sea.

Aquaculture of three phyla of marine invertebrates to yield bioactive metabolites: process developments and economics

Large-scale, renewable supplies of chemical constituents derived from marine invertebrates have limited development of potential new natural product drugs. This paper describes the development of two in-sea aquaculture systems designed and engineered for production of large quantities of biomass for two species of marine invertebrates desired for their natural product chemical constituents. The two invertebrates and their products were: (1) the cosmopolitan, arborescent bryozoan Bugula neritina (Phylum Bryozoa) for its anticancer chemical constituent bryostatin 1; and (2) Ecteinascidia turbinate (Phylum Tunicata) the source of anticancer ecteinascidin 743. For the third invertebrate Phylum Porifera, and its representative sponge Acanthella cavernosa (desired for its anti-parasitic and anti-infective kalihinols) in-sea systems were not developed in favor of controlled environment tank aquaculture systems. For the bryozoan and tunicate, projected economics for commercial-scale in-sea production proved cost effective. This was in contrast to the controlled environment sponge culture tank system, which did not prove to be economical due to inherent slow growth and low natural product yields of the sponge in culture. A non-destructive method for “milking” natural product chemicals from sponges was tested and is described.

Drugs from the seas - current status and microbiological implications.

The oceans are the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds (especially the tunicate metabolite ET-743) show pronounced pharmacological activities and are interesting candidates for new drugs primarily in the area of cancer treatment. Other compounds are currently being developed as an analgesic (ziconotide from the mollusc Conus magus) or to treat inflammation. Numerous natural products from marine invertebrates show striking structural similarities to known metabolites of microbial origin, suggesting that microorganisms (bacteria, microalgae) are at least involved in their biosynthesis or are in fact the true sources of these respective metabolites. This assumption is corroborated by several studies on natural products from sponges that proved these compounds to be localized in symbiotic bacteria or cyanobacteria. Recently, molecular methods have successfully been applied to study the microbial diversity in marine sponges and to gain evidence for an involvement of bacteria in the biosynthesis of the bryostatins in the bryozoan Bugula neritina.

Relationships between environmental variables and the abundance of Peracarid fauna in Algeciras Bay (Southern Iberian peninsula)

The distribution and abundance of peracaridean fauna associated with the bryozoan Bugula neritina have been analysed according to their possible relationships with environmental stress on the coastline of Algeciras Bay. The spatial environmental variability along this bay has been established based on the analysis of five abiotic variables (hydrodynamism, siltation, suspended solids, and organic matter content) at six stations over a 12-month period. The temporal variability of these abiotic variables was not taken into account due to the method used in collecting the samples. The abundance and beta diversity of the peracarid fauna were also calculated monthly. Hydrodynamism was the only abiotic variable significantly correlated with beta diversity, with a linear regression model between the two variables (r = 0.81). The distributional pattern of the peracarids (based on the abundance matrix) was correlated with the environmental variables by matching samples' similarities using the Spearman rank correlation. This analysis showed that the combination of variables that best explains the patterns of distribution is hydrodynamism and siltation (ρs = 0.665). Of the species considered the amphipods Ischyrocerus inexpectatus, Jassa marmorata and Podocerus variegatus were negatively correlated with hydrodynamism and positively with temperature. The caprellidea Phtisica marina was also negatively correlated with hydrodynamism. On the other hand, the amphipods Cressa mediterranea and Stenothoe dollfusi preferred environments with high hydrological forces and low sedimentation rate. Due to the particular environmental conditions where they live and their requirements, Gammaropsis maculata, Corophium acutum, J. marmorata, I. inexpectatus, and P. variegatus were categorised as good indicator species in port environments. Others such as Aora spinicornis, Microjassa cumbrensis, and C. mediterranea were categorised as species well adapted to high hydrological forces.

SEASONALITY AND SPATIAL DISTRIBUTION OF PERACARIDS ASSOCIATED WITH THE BRYOZOAN BUGULA NERITINA IN ALGECIRAS BAY, SPAIN

Abstract The fouling community at Algeciras Bay exhibits the general features of other warm-temperate fouling communities in which the bryozoan Bugula neritina is a major competitor for space during summer. This bryozoan was sampled in order to study spatio-temporal changes of the peracaridan fauna of Algeciras Bay (Strait of Gibraltar). The results of this work point out the relevance of studying the life cycle patterns of both the living substratum and its associated fauna prior to analyzing the possible classification or ordination of the stations. There was a good correlation between the height of B. neritina and temperature and between the colony dry mass and the number of peracaridan individuals. Numerical analyses allowed the identification of two groups of stations based on the distribution and abundance of the peracaridan faunas in Algeciras Bay, the internal and the external stations. The external stations had higher diversity, regarding both evenness and species richness, than in the internal s...

Localisation of biogenic amines in larvae of Bugula neritina (Bryozoa: Cheilostomatida) and their effects on settlement

Distributions of serotonin and catecholamines in larvae of the marine bryozoan Bugula neritina (Bryozoa: Cheilostomatida) were investigated using immunohistochemistry with anti-serotonin antiserum and glyoxylic acid–induced fluorescence histochemistry. Anti-serotonin immunoreactive substances and glyoxylic acid–induced fluorescent substances had similar distributions in the equatorial neuromuscular ring, the neural plexus, the paired axial neuromuscular cords, and tracts connecting the neural plexus to ciliated cells bordering the pyriform organ. The effects of dopamine, noradrenaline, adrenaline, tyramine, octopamine, synephrine and serotonin, at 10−4, 10−5 and 10−6 M, on settlement were analysed. In filtered seawater, 98% of larvae settled in 3 h, but only 11%, 3% and 6% total settlement was observed after 8 h in 10−4 M dopamine, 10−4 M serotonin and 10−5 M serotonin, respectively. Total settlement was 70% in 10−4 M noradrenaline, 80% in 10−4 M adrenaline and 60% in 10−4 M tyramine. Less than 60% settlement was observed in 10−4 and 10−5 M octopamine and synephrine. Serotonin's inhibitory effect on settlement was mimicked by a range of serotonin receptor agonists and antagonists, among which 5-carboxamidotryptamine was the most potent.