Niels Lindquist

Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges.

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the worlds oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.

Constraints on chemically-mediated coevolution: multiple functions for seaweed secondary metabolites.

Diterpene alcohols produced by the brown seaweed Dictyota menstrualis deter feeding by numerous species of abundant herbivores. Here we show that these same compounds also may prevent fouling organisms from colonizing the surface of this alga. In the field, Dictyota menstrualis plants were less frequently and less heavily fouled than any of the other common seaweed species investigated. In laboratory assays, larvae of the common fouling bryozoan Bugula neritina failed to settle on Dictyota even though they contacted its surface as often as they contacted the surface of a preferred host alga. Rejection occurred only after direct contact with the algas surface. Rejection of Dictyota was not mediated by water-borne chemical cues or by surface wettability (a physical property of the surface that can affect fouling). The lipid-soluble extract from surface rubbings of Dictyota inhibited larval settlement when placed on other surfaces and contained the di- terpene alcohols pachydictyol A and dictyol E. Larvae exposed to these compounds ex- perienced mortality, abnormal development, or reduced rates of development. Although the potential for chemically mediated coevolution between plants and her- bivores has been the focus of scores of previous investigations, such coevolution will depend on selection altering the chemical defenses of the plant following the evolution of resistance by herbivores. Such a reciprocal response will be constrained if compounds play multiple roles that are ecologically important. Dictyota produces secondary metabolites that are broadly defensive against a wide variety of consumers and fouling organisms. Although certain consumers may evolve resistance to these metabolites, it is unclear that feeding by these consumers will result in reciprocal responses from the plant. We suggest that coevolved interactions may be uncommon, and that many interactions that appear to be coevolved may result from fortuitous and opportunistic preadaptations.

Palatability and chemical defense of marine invertebrate larvae

Risk of larval mortality is a critical component of models and debates concerning the ecology and evolution of the differing reproductive characteristics exhibited by marine invertebrates. In these discussions, predation often is assumed to be a major source of larval mortality. Despite limited empirical support, most marine larvae are thought to be palatable and broadly susceptible to generalist predators. Previous studies of larval—planktivore interactions have focused primarily on larvae that typically feed, grow, and develop for weeks to months in the plankton. Such planktotrophic species commonly produce large numbers of small larvae that disperse over vast distances. In contrast, the nonfeeding lecithotrophic larvae from sessile invertebrates that brood are often large and conspicuous, lack morphological defenses, and have limited dispersal distances because they typically are competent to settle minutes to hours after spawning. Interactions between lecithotrophic larvae and consumers are not well studied. This has limited the ability of previous authors to test broad generalities about marine larvae. We show that brooded larvae of Caribbean sponges (11 species) and gorgonians (three species) as well as brooded larvae of temperate hydroids (two species) and a bryozoan are unpalatable to co—occurring fishes. In contrast, brooded larvae of temperate ascidians (three species), a temperate sponge, and Caribbean hard corals (three species) are readily consumed by fishes, as are larvae from four of six species of synchronous broadcast—spawning gorgonians from the Florida Keys. Frequencies of survivorship for larvae attacked and rejected by fishes were high and statistically indistinguishable from frequencies for unattacked control larvae. Frequency of metamorphosis (when it occurred) of rejected larvae never differed significantly from that of unattacked control larvae. Assays testing for larval vs. adult chemical defenses for five species with distasteful larvae showed that larvae of all five species were chemically distasteful to fishes, whereas only three of five adult extracts deterred fish feeding. A comparison of larval palatability among chemically rich taxa showed that brooded larvae were significantly more likely to be unpalatable (86% of the species tested) than larvae of broadcasters (33%), and that palatable larvae were rarely released during the day (23%) while unpalatable larvae usually were (89%). Additionally, the frequency of bright coloration was high (60%) for unpalatable larvae and low (0%) for palatable larvae, suggesting that unpalatable larvae often may be aposematically colored. Results of this broad survey cast doubt on the widely accepted notion that virtually all marine larvae are suitable prey for most generalized planktivores. Among species that do not chemically or physically protect larvae against fishes, selection appears to favor the release of larvae at night, or the production of smaller more numerous offspring that grow and develop at sea as a way of escaping consumer—rich benthic habitats. Because distasteful larvae are not similarly constrained, distasteful species should exhibit reproductive and larval characteristics selected more by the fitness—related consequences of larval development mode and dispersal distance than by the necessity of avoiding benthic predators. Production of large larvae and retention of offspring in parental habitats that have proved to be suitable for growth and reproduction have both been proposed as advantageous, but these advantages often were assumed to be offset by losses due to increased larval apparency to fishes. This assumed trade—off is not mandatory because larvae can be defended chemically. Distasteful larvae tend to be conspicuous, localized dispersers that can co—occur with benthic fishes, and yet not be consumed.

Molecular microbial diversity survey of sponge reproductive stages and mechanistic insights into vertical transmission of microbial symbionts.

ABSTRACT Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission.

Potent cytotoxins produced by a microbial symbiont protect host larvae from predation

Larvae of the sessile marine invertebrate Bugula neritina (Bryozoa) are protected by an effective chemical defense. From the larvae, we isolated three bryostatin-class macrocyclic polyketides, including the novel bryostatin 20, that deterred feeding by a common planktivorous fish that co-occurs with B. neritina. A unique bacterial symbiont of B. neritina, Endobugula sertula, was hypothesized as the putative source of the bryostatins. We show that: (1) bryostatins are concentrated in B. neritina larvae and protect them against predation by fish; (2) the adults are not defended by bryostatins; and (3) E. sertula produces bryostatins. This study represents the first example from the marine environment of a microbial symbiont producing an anti-predator defense for its host and, in this case, specifically for the host’s larval stage, which is exceptionally vulnerable to predators.

Do associated microbial abundances impact marine demosponge pumping rates and tissue densities

The evolution of marine demosponges has led to two basic life strategies: one involving close associations with large and diverse communities of microorganisms, termed high microbial abundance (HMA) species, and one that is essentially devoid of associated microorganisms, termed low microbial abundance (LMA) species. This dichotomy has previously been suggested to correlate with morphological differences, with HMA species having a denser mesohyl and a more complex aquiferous systems composed of longer and narrower water canals that should necessitate slower seawater filtration rates. We measured mesohyl density for a variety of HMA and LMA sponges in the Florida Keys, and seawater pumping rates for a select group of these sponges using an in situ dye technique. HMA sponges were substantially denser than LMA species, and had per unit volume pumping rates 52–94% slower than the LMA sponges. These density and pumping rate differences suggest that evolutionary differences between HMA and LMA species may have resulted in profound morphological and physiological differences between the two groups. The LMA sponge body plan moves large quantities of water through their porous tissues allowing them to rapidly acquire the small particulate organic matter (POM) that supplies the majority of their nutritional needs. In contrast, the HMA sponge body plan is suited to host large and tightly packed communities of microorganisms and has an aquiferous system that increases contact time between seawater and the sponge/microbial consortium that feeds on POM, dissolved organic matter and the raw inorganic materials for chemolithotrophic sponge symbionts. The two evolutionary patterns represent different, but equally successful patterns and illustrate how associated microorganisms can potentially have substantial effects on host evolution.

DEFENSE OF ASCIDIANS AND THEIR CONSPICUOUS LARVAE: ADULT VS. LARVAL CHEMICAL DEFENSES'

Previous investigations, focused primarily on vertebrates, have noted substantial losses of eggs and embryos to predators and questioned why selection has not more commonly resulted in the evolution of chemically defended eggs or embryos. Hypotheses regarding the apparent rarity of such defenses have emphasized the potential incompatibility of actively developing tissues and toxic metabolites. Alternatively, this apparent pattern could be an artifact of our greater knowledge of vertebrates, which in general show few tendencies for synthesizing defensive metabolites in either juvenile or adult stages. In this study, we investigated adult and larval chemical defenses of a group of benthic marine invertebrates, the ascidians, in which the adults are often chemically rich, and we contrast our findings with what is known about chemical defenses of eggs and embryos from terrestrial and aquatic organisms. Our findings suggest that there is no fundamental incompatibility of rapidly developing juvenile tissues and bioactive metabolites, and that chemically defended eggs and larval stages may be common among some taxonomic groups. Ascidians are benthic invertebrates that often lack apparent physical defenses against predation, yet are common on coral reefs where predation by fishes is intense. In contrast to most co—occurring invertebrates, many ascidians also release large, conspicuous larvae during daylight hours when exposure to fish predation would be highest. Thus selection by predators might favor the evolution of distasteful larvae. In situ observations indicate that many conspicuous ascidian larvae are distasteful to potential consumers. We investigated the ability of secondary metabolites produced by taxonomically diverse ascidians from geographically distant locales to deter predation on both adults and larvae. Larvae from the Caribbean ascidian Trididemnum solidum were distasteful to reef fishes, and when organic extracts of individual larvae were transferred onto eyes of freeze—dried krill (a good larval mimic in terms of size and color), these eyes were rejected by fishes while control eyes (solvent only) were readily eaten. Larvae of the Indo—Pacific ascidian Sigillina cf. signifera were also distasteful to coral—reef fishes and contained the unpalatable bipyrrole alkaloid tambjamine C. When added to artificial foods at or below their natural mean concentrations and offered to consumers in field and laboratory feeding assays, the secondary metabolites produced by Trididemnum solidum (Caribbean Sea), Sigillina cf. signifera (Indo—Pacific), and Polyandrocarpa sp. (Gulf of California) significantly deterred feeding by co—occurring fishes and invertebrates. Secondary metabolites produced by Trididemnum cf. cyanophorum from the Caribbean Sea, Lissoclinum patella from the Indo—Pacific, and Aplidium californicum from the temperate Pacific, and the small stellate spicules common to many tropical didemnid ascidians did not significantly affect fish feeding. High—pressure liquid chromatography (HPLC) analyses of six didemnin cyclic peptides in individual colonies of Trididemnum solidum from one patch reef at Little San Salvador, Bahamas found large inter—colony differences in their concentrations. The mean concentration of didemnin B was more than double the concentration needed to significantly deter fish feeding in our field assays, and feeding tests with nordidemnin B showed that it deterred fish feeding across the entire range of natural concentrations. HPLC analysis of the extract from a combined collection of T. solidum larvae found adequate concentrations of didemnin B and nordidemnin B to account for their rejection by foraging fishes. We demonstrate that taxonomically diverse ascidians from habitats characterized by intense predation pressure produce secondary metabolites that significantly reduce predation on both adults and larvae, and suggest that this defensive chemistry may be crucial in allowing the release of large, well—provisioned larvae during daylight periods when larvae have the greatest probability of using photic cues to select physically appropriate settlement sites. Production of defensive secondary metabolites appears widespread among certain groups of ascidians, some of which are also known to concentrate acid and heavy metals as additional defensive strategies.

Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.

Vertical Transmission of a Phylogenetically Complex Microbial Consortium in the Viviparous Sponge Ircinia felix

ABSTRACT Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed “IF clusters,” that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.

CRAYFISH FEEDING PREFERENCES FOR FRESHWATER MACROPHYTES: THE INFLUENCE OF PLANT STRUCTURE AND CHEMISTRY

Abstract The omnivorous crayfish Procambarus clarkii fed selectively on several species of macrophytes, preferring delicate fresh plants that had filamentous or finely-branched architectures. When the macrophytes were dried, powdered, and reconstituted into an alginate gel (thus eliminating among-species differences in physical characteristics), crayfish preferences were altered; previously tough plants that were high in nitrogen and protein were preferred over previously delicate plants that were low in nitrogen and protein. Even though plant structure influences feeding decision of crayfish, the structurally identical macrophyte gels were fed upon differently, demonstrating that nonstructural traits are important feeding determinants. However, plant tissue constituents such as nitrogen, protein, phenolics, lignin, cellulose, or ash were not significantly correlated with feeding preferences. Two high-nitrogen plants that were avoided by crayfish as fresh and as reconstituted tissue (Nuphar luteum macrophyllum and Alternanthera philoxeroides) possessed extracts that reduced crayfish feeding in laboratory assays, demonstrating that macrophyte metabolites can deter some herbivores. As is often observed with large generalist herbivores and omnivores in terrestrial and marine systems, the freshwater crayfish made feeding decisions based upon multiple plant cues (structure, nutrition, chemical defenses).