Marine and terrestrial herbivores display convergent chemical ecology despite 400 million years of independent evolution.

Chemical cues regulate key ecological interactions in marine and terrestrial ecosystems. They are particularly important in terrestrial plant-herbivore interactions, where they mediate both herbivore foraging and plant defense. Although well described for terrestrial interactions, the identity and ecological importance of herbivore foraging cues in marine ecosystems remain unknown. Here we show that the specialist gastropod Elysia tuca hunts its seaweed prey, Halimeda incrassata, by tracking 4-hydroxybenzoic acid to find vegetative prey and the defensive metabolite halimedatetraacetate to find reproductive prey. Foraging cues were predicted to be polar compounds but instead were nonpolar secondary metabolites similar to those used by specialist terrestrial insects. Tracking halimedatetraacetate enables Elysia to increase in abundance by 12- to 18-fold on reproductive Halimeda, despite reproduction in Halimeda being rare and lasting for only ∼36 h. Elysia swarm to reproductive Halimeda where they consume the alga's gametes, which are resource rich but are chemically defended from most consumers. Elysia sequester functional chloroplasts and halimedatetraacetate from Halimeda to become photosynthetic and chemically defended. Feeding by Elysia suppresses the growth of vegetative Halimeda by ∼50%. Halimeda responds by dropping branches occupied by Elysia, apparently to prevent fungal infection associated with Elysia feeding. Elysia is remarkably similar to some terrestrial insects, not only in its hunting strategy, but also its feeding method, defense tactics, and effects on prey behavior and performance. Such striking parallels indicate that specialist herbivores in marine and terrestrial systems can evolve convergent ecological strategies despite 400 million years of independent evolution in vastly different habitats.

Conservation of progesterone hormone function in invertebrate reproduction.

Steroids play fundamental roles regulating mammalian reproduction and development. Although sex steroids and their receptors are well characterized in vertebrates and several arthropod invertebrates, little is known about the hormones and receptors regulating reproduction in other invertebrate species. Evolutionary insights into ancient endocrine pathways can be gained by elucidating the hormones and receptors functioning in invertebrate reproduction. Using a combination of genomic analyses, receptor imaging, ligand identification, target elucidation, and exploration of function through receptor knockdown, we now show that comparable progesterone chemoreception exists in the invertebrate monogonont rotifer Brachionus manjavacas, suggesting an ancient origin of the signal transduction systems commonly associated with the development and integration of sexual behavior in mammals.

Bromophycoic acids: bioactive natural products from a Fijian red alga Callophycus sp.

Bioassay-guided fractionation of extracts from a Fijian red alga in the genus Callophycus resulted in the isolation of five new compounds of the diterpene-benzoate class. Bromophycoic acids A-E (1-5) were characterized by NMR and mass spectroscopic analyses and represent two novel carbon skeletons, one with an unusual proposed biosynthesis. These compounds display a range of activities against human tumor cell lines, malarial parasites, and bacterial pathogens including low micromolar suppression of MRSA and VREF.

Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed.

Organism surfaces represent signaling sites for attraction of allies and defense against enemies. However, our understanding of these signals has been impeded by methodological limitations that have precluded direct fine-scale evaluation of compounds on native surfaces. Here, we asked whether natural products from the red macroalga Callophycus serratus act in surface-mediated defense against pathogenic microbes. Bromophycolides and callophycoic acids from algal extracts inhibited growth of Lindra thalassiae, a marine fungal pathogen, and represent the largest group of algal antifungal chemical defenses reported to date. Desorption electrospray ionization mass spectrometry (DESI-MS) imaging revealed that surface-associated bromophycolides were found exclusively in association with distinct surface patches at concentrations sufficient for fungal inhibition; DESI-MS also indicated the presence of bromophycolides within internal algal tissue. This is among the first examples of natural product imaging on biological surfaces, suggesting the importance of secondary metabolites in localized ecological interactions, and illustrating the potential of DESI-MS in understanding chemically-mediated biological processes.

Ecological leads for natural product discovery: Novel sesquiterpene hydroquinones from the red macroalga Peyssonnelia sp.

Pharmacologically-motivated marine natural product investigations have yielded a large variety of structurally unique compounds with interesting biomedical properties, but the natural roles of these molecules often remain unknown. While secondary metabolites may function as antimicrobial chemical defenses, few studies have examined this hypothesis. In the present investigation, chromatographic fractions from 69 collections of Fijian red macroalgae representing at least 43 species were evaluated for growth inhibition of three microbial pathogens and saprophytes of marine macrophytes. At least one microbe was suppressed by fraction(s) of all evaluated algae, suggesting that antimicrobial defenses are common among tropical seaweeds. From these leads, peyssonoic acids A-B (1-2), novel sesquiterpene hydroquinones, were isolated from the crustose red alga Peyssonnelia sp. At ecologically realistic concentrations, both compounds inhibited growth of Pseudoalteromonas bacteriolytica, a bacterial pathogen of marine algae, and Lindra thalassiae, a fungal pathogen of marine algae, and exhibited modest antineoplastic activity against ovarian cancer cells. The peyssonoic acids included one novel carbon skeleton and illustrated the utility of ecological studies in natural product discovery.

Genetic determinants of mate recognition in Brachionus manjavacas (Rotifera).

BACKGROUND: Mate choice is of central importance to most animals, influencing population structure, speciation, and ultimately the survival of a species. Mating behavior of male brachionid rotifers is triggered by the product of a chemosensory gene, a glycoprotein on the body surface of females called the mate recognition pheromone. The mate recognition pheromone has been biochemically characterized, but little was known about the gene(s). We describe the isolation and characterization of the mate recognition pheromone gene through protein purification, N-terminal amino acid sequence determination, identification of the mate recognition pheromone gene from a cDNA library, sequencing, and RNAi knockdown to confirm the functional role of the mate recognition pheromone gene in rotifer mating. RESULTS: A 29 kD protein capable of eliciting rotifer male circling was isolated by high-performance liquid chromatography. Two transcript types containing the N-terminal sequence were identified in a cDNA library; further characterization by screening a genomic library and by polymerase chain reaction revealed two genes belonging to each type. Each gene begins with a signal peptide region followed by nearly perfect repeats of an 87 to 92 codon motif with no codons between repeats and the final motif prematurely terminated by the stop codon. The two Type A genes contain four and seven repeats and the two Type B genes contain three and five repeats, respectively. Only the Type B gene with three repeats encodes a peptide with a molecular weight of 29 kD. Each repeat of the Type B gene products contains three asparagines as potential sites for N-glycosylation; there are no asparagines in the Type A genes. RNAi with Type A double-stranded RNA did not result in less circling than in the phosphate-buffered saline control, but transfection with Type B double-stranded RNA significantly reduced male circling by 17%. The very low divergence between repeat units, even at synonymous positions, suggests that the repeats are kept nearly identical through a process of concerted evolution. Information-rich molecules like surface glycoproteins are well adapted for chemical communication and aquatic animals may have evolved signaling systems based on these compounds, whereas insects use cuticular hydrocarbons. CONCLUSION: Owing to its critical role in mating, the mate recognition pheromone gene will be a useful molecular marker for exploring the mechanisms and rates of selection and the evolution of reproductive isolation and speciation using rotifers as a model system. The phylogenetic variation in the mate recognition pheromone gene can now be studied in conjunction with the large amount of ecological and population genetic data being gathered for the Brachionus plicatilis species complex to understand better the evolutionary drivers of cryptic speciation.

Long distance dispersal and vertical gene flow in the Caribbean brooding coral Porites astreoides.

To date, most assessments of coral connectivity have emphasized long-distance horizontal dispersal of propagules from one shallow reef to another. The extent of vertical connectivity, however, remains largely understudied. Here, we used newly-developed and existing DNA microsatellite loci for the brooding coral Porites astreoides to assess patterns of horizontal and vertical connectivity in 590 colonies collected from three depth zones (≤10 m, 15-20 m and ≥25 m) at sites in Florida, Bermuda and the U.S. Virgin Islands (USVI). We also tested whether maternal transmission of algal symbionts (Symbiodinium spp.) might limit effective vertical connectivity. Overall, shallow P. astreoides exhibited high gene flow between Florida and USVI, but limited gene flow between these locations and Bermuda. In contrast, there was significant genetic differentiation by depth in Florida (Upper Keys, Lower Keys and Dry Tortugas), but not in Bermuda or USVI, despite strong patterns of depth zonation in algal symbionts at two of these locations. Together, these findings suggest that P. astreoides is effective at dispersing both horizontally and vertically despite its brooding reproductive mode and maternal transmission of algal symbionts. In addition, these findings might help explain the ecological success reported for P. astreoides in the Caribbean in recent decades.

Gene expression of corals in response to macroalgal competitors.

As corals decline and macroalgae proliferate on coral reefs, coral-macroalgal competition becomes more frequent and ecologically important. Whether corals are damaged by these interactions depends on susceptibility of the coral and traits of macroalgal competitors. Investigating changes in gene expression of corals and their intracellular symbiotic algae, Symbiodinium, in response to contact with different macroalgae provides insight into the biological processes and cellular pathways affected by competition with macroalgae. We evaluated the gene expression profiles of coral and Symbiodinium genes from two confamilial corals, Acropora millepora and Montipora digitata, after 6 h and 48 h of contact with four common macroalgae that differ in their allelopathic potency to corals. Contacts with macroalgae affected different biological pathways in the more susceptible (A. millepora) versus the more resistant (M. digitata) coral. Genes of coral hosts and of their associated Symbiodinium also responded in species-specific and time-specific ways to each macroalga. Changes in number and expression intensity of affected genes were greater after 6 h compared to 48 h of contact and were greater following contact with Chlorodesmis fastigiata and Amphiroa crassa than following contact with Galaxaura filamentosa or Turbinaria conoides. We documented a divergence in transcriptional responses between two confamilial corals and their associated Symbiodinium, as well as a diversity of dynamic responses within each coral species with respect to the species of macroalgal competitor and the duration of exposure to that competitor. These responses included early initiation of immune processes by Montipora, which is more resistant to damage after long-term macroalgal contact. Activation of the immune response by corals that better resist algal competition is consistent with the hypothesis that some macroalgal effects on corals may be mediated by microbial pathogens.

Synergistic toxicity of Macondo crude oil and dispersant Corexit 9500A(®) to the Brachionus plicatilis species complex (Rotifera).

Using the marine rotifer Brachionus plicatilis acute toxicity tests, we estimated the toxicity of Corexit 9500A(®), propylene glycol, and Macondo oil. Ratios of 1:10, 1:50 and 1:130 for Corexit 9500A(®):Macondo oil mixture represent: maximum exposure concentrations, recommended ratios for deploying Corexit (1:10-1:50), 1:130 the actual dispersant:oil ratio used in the Deep Water Horizon spill. Corexit 9500A(®) and oil are similar in their toxicity. However, when Corexit 9500A(®) and oil are mixed, toxicity to B. manjavacas increases up to 52-fold. Extrapolating these results to the oil released by the Macondo well, suggests underestimation of increased toxicity from Corexit application. We found small differences in sensitivity among species of the B. plicatilis species complex, likely reflecting phylogenetic similarity. Just 2.6% of the water-accommodated fraction of oil inhibited rotifer cyst hatching by 50%, an ecologically significant result because rotifer cyst in sediments are critical resources for the recolonization of populations each Spring.

Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera).

RNA interference (RNAi) is a powerful technique for functional genomics, yet no studies have reported its successful application to zooplankton. Many zooplankton, particularly microscopic metazoans of phylum Rotifera, have unique life history traits for which genetic investigation has been limited. In this paper, we report the development of RNAi methods for rotifers, with the exogenous introduction of double-stranded RNA (dsRNA) through the use of a lipofection reagent. Transfection with dsRNA for heat shock protein 90, the membrane-associated progesterone receptor, and mitogen-activated protein kinase significantly increased the proportion of non-reproductive females. Additionally, a fluorescence-based lectin binding assay confirmed the significant suppression of four of six glycosylation enzymes that were targeted with dsRNA. Suppression of mRNA transcripts was confirmed with quantitative PCR. Development of RNAi for rotifers promises to enhance the ability for assessing genetic regulation of features critical to their life history and represents a key step toward functional genomics research in zooplankton.