Symbiosis of disciplines: how can developmental biologists join conservationists in sustaining and restoring earth's biodiversity?

What can developmental biology contribute toward mitigating the consequences of anthropogenic assaults on the environment and climate change? In this Spotlight article, we advocate a developmental biology that takes seriously Lynn Margulis' claim that 'the environment is part of the body'. We believe this to be a pre-condition for developmental biology playing important roles in conservation and environmental restoration. We need to forge a developmental biology of the holobiont - the multi-genomic physiologically integrated organism that is also a functional biome. To this end, we highlight how developmental biology needs to explore more deeply the interactions between developing organisms, and their chemical, physical and biotic environments.

Bacterial lipopolysaccharide induces settlement and metamorphosis in a marine larva.

How larvae of the many phyla of marine invertebrates find places appropriate for settlement, metamorphosis, growth, and reproduction is an enduring question in marine science. Biofilm-induced metamorphosis has been observed in marine invertebrate larvae from nearly every major marine phylum. Despite the widespread nature of this phenomenon, the mechanism of induction remains poorly understood. The serpulid polychaete Hydroides elegans is a well established model for investigating bacteria-induced larval development. A broad range of biofilm bacterial species elicit larval metamorphosis in H. elegans via at least two mechanisms, including outer membrane vesicles (OMVs) and complexes of phage-tail bacteriocins. We investigated the interaction between larvae of H. elegans and the inductive bacterium Cellulophaga lytica, which produces an abundance of OMVs but not phage-tail bacteriocins. We asked whether the OMVs of C. lytica induce larval settlement due to cell membrane components or through delivery of specific cargo. Employing a biochemical structure­function approach with a strong ecological focus, the cells and OMVs produced by C. lytica were interrogated to determine the class of the inductive compounds. Here, we report that larvae of H. elegans are induced to metamorphose by lipopolysaccharide produced by C. lytica. The widespread prevalence of lipopolysaccharide and its associated taxonomic and structural variability suggest it may be a broadly employed cue for bacterially induced larval settlement of marine invertebrates.

Laser ablation of the apical sensory organ of Hydroides elegans (Polychaeta) does not inhibit detection of metamorphic cues.

Larvae of many marine invertebrates bear an anteriorly positioned apical sensory organ (ASO) presumed to be the receptor for settlement- and metamorphosis-inducing environmental cues, based on its structure, position and observed larval behavior. Larvae of the polychaete Hydroides elegans are induced to settle by bacterial biofilms, which they explore with their ASO and surrounding anteroventral surfaces. A micro-laser was utilized to destroy the ASO and other anterior ciliary structures in competent larvae of H. elegans. After ablation, larvae were challenged with bacterial biofilmed or clean surfaces and percentage metamorphosis was determined. Ablated larvae were also assessed for cellular damage by applying fluorescently tagged FMRF-amide antibodies and observing the larvae by laser-scanning confocal microscopy. While the laser pulses caused extensive damage to the ASO and surrounding cells, they did not inhibit metamorphosis. We conclude that the ASO is not a required receptor site for cues that induce metamorphosis.

High bacterial diversity in nearshore and oceanic biofilms and their influence on larval settlement by Hydroides elegans (Polychaeta).

Settlement of many benthic marine invertebrates is stimulated by bacterial biofilms, although it is not known if patterns of settlement reflect microbial communities that are specific to discrete habitats. Here, we characterized the taxonomic and functional gene diversity (16S rRNA gene amplicon and metagenomic sequencing analyses), as well as the specific bacterial abundances, in biofilms from diverse nearby and distant locations, both inshore and offshore, and tested them for their ability to induce settlement of the biofouling tubeworm Hydroides elegans, an inhabitant of bays and harbours around the world. We found that compositions of the bacterial biofilms were site specific, with the greatest differences between inshore and offshore sites. Further, biofilms were highly diverse in their taxonomic and functional compositions across inshore sites, while relatively low diversity was found at offshore sites. Hydroides elegans settled on all biofilms tested, with settlement strongly correlated with bacterial abundance. Bacterial density in biofilms was positively correlated with biofilm age. Our results suggest that the localized distribution of H. elegans is not determined by 'selection' to locations by specific bacteria, but it is more likely linked to the prevailing local ecology and oceanographic features that affect the development of dense biofilms and the occurrence of larvae.

Animals in a bacterial world, a new imperative for the life sciences.

In the last two decades, the widespread application of genetic and genomic approaches has revealed a bacterial world astonishing in its ubiquity and diversity. This review examines how a growing knowledge of the vast range of animal-bacterial interactions, whether in shared ecosystems or intimate symbioses, is fundamentally altering our understanding of animal biology. Specifically, we highlight recent technological and intellectual advances that have changed our thinking about five questions: how have bacteria facilitated the origin and evolution of animals; how do animals and bacteria affect each other's genomes; how does normal animal development depend on bacterial partners; how is homeostasis maintained between animals and their symbionts; and how can ecological approaches deepen our understanding of the multiple levels of animal-bacterial interaction. As answers to these fundamental questions emerge, all biologists will be challenged to broaden their appreciation of these interactions and to include investigations of the relationships between and among bacteria and their animal partners as we seek a better understanding of the natural world.

Complex embryos displaying bilaterian characters from Precambrian Doushantuo phosphate deposits, Weng'an, Guizhou, China.

Three-dimensionally preserved embryos from the Precambrian Ediacaran Doushantuo Formation, Weng'an, Guizhou, southern China, have attracted great attention as the oldest fossil evidence yet found for multicellular animal life on Earth. Many embryos are early cleavage embryos and most of them yield a limited phylogenetic signal. Here we report the discovery of two Doushantuo embryos that are three-dimensionally preserved and complex. Imaging techniques using propagation phase-contrast based synchrotron radiation microtomography (PPC-SR-microCT) reveal that the organization of cells demonstrates several bilaterian features, including the formation of anterior-posterior, dorso-ventral, and right-left polarities, and cell differentiation. Unexpectedly, our observations show a noticeable difference in organization patterns between the embryos, suggesting that they represent two distinct taxa. These embryos provide further evidence for the presence of bilaterian animals in the Doushantuo biota. Furthermore, these bilaterians had already diverged into distantly related groups at least 40 million years before the Cambrian radiation, indicating that the last common ancestor of the bilaterians lived much earlier than is usually thought.

Understanding Drivers of Variation and Predicting Variability Across Levels of Biological Organization.

Differences within a biological system are ubiquitous, creating variation in nature. Variation underlies all evolutionary processes and allows persistence and resilience in changing environments; thus, uncovering the drivers of variation is critical. The growing recognition that variation is central to biology presents a timely opportunity for determining unifying principles that drive variation across biological levels of organization. Currently, most studies that consider variation are focused at a single biological level and not integrated into a broader perspective. Here we explain what variation is and how it can be measured. We then discuss the importance of variation in natural systems, and briefly describe the biological research that has focused on variation. We outline some of the barriers and solutions to studying variation and its drivers in biological systems. Finally, we detail the challenges and opportunities that may arise when studying the drivers of variation due to the multi-level nature of biological systems. Examining the drivers of variation will lead to a reintegration of biology. It will further forge interdisciplinary collaborations and open opportunities for training diverse quantitative biologists. We anticipate that these insights will inspire new questions and new analytic tools to study the fundamental questions of what drives variation in biological systems and how variation has shaped life.

Developmental Symbiosis: A Sponge Larva Needs Symbiotic Bacteria to Succeed on the Benthos.

Settlement and metamorphosis of marine invertebrate larvae are processes of profound developmental, morphological, physiological and ecological change. That these processes in larvae of a marine sponge critically rely on products supplied by endosymbiotic bacteria signals the importance of developmental symbiosis among the most basal metazoans.

The tree snail on Rota Island, Northern Mariana Islands, long identified as Partula gibba (Partulidae), is a different species.

Tree snails in the family Partulidae are widespread across the tropical Pacific, with endemic species occurring on most high islands. Partulid species have faced catastrophic range reductions and extinctions due primarily to introduced predators. Consequently, most extant species are threatened with imminent extinction. The U.S. administered Mariana Islands, consisting of Guam in the South and the Commonwealth of the Northern Mariana Islands (CNMI) in the north, historically harbored six endemic partulid species, half of which are thought to be extinct. While conducting a phylogenetic assessment of Partula gibba, an extant tree-snail with a range spanning at least seven islands within the archipelago, it was discovered that what has been identified as P. gibba on the island of Rota is a misidentified cryptic species. Here we use molecular phylogenetics, shell morphometrics and reproductive anatomy to describe it as a new species, Partula lutaensis sp. nov.. Because the new species has suffered population declines and has a restricted range, consisting solely of the small island of Rota, we highlight the urgent need for conservation measures.

The natural sequence of events in larval settlement and metamorphosis of Hydroides elegans (Polychaeta; Serpulidae).

The broadly distributed serpulid worm Hydroides elegans has become a model organism for studies of marine biofouling, development and the processes of larval settlement and metamorphosis induced by surface microbial films. Contrasting descriptions of the initial events of these recruitment processes, whether settlement is induced by (1) natural multi-species biofilms, (2) biofilms composed of single bacterial species known to induce settlement, or (3) a bacterial extract stimulated the research described here. We found that settlement induced by natural biofilms or biofilms formed by the bacterium Pseudoalteromonas luteoviolacea is invariably initiated by attachment and secretion of an adherent and larva-enveloping primary tube, followed by loss of motile cilia and ciliated cells and morphogenesis. The bacterial extract containing complex tailocin arrays derived from an assemblage of phage genes incorporated into the bacterial genome appears to induce settlement events by destruction of larval cilia and ciliated cells, followed by attachment and primary-tube formation. Similar destruction occurred when precompetent larvae of H. elegans or larvae of a nudibranch gastropod were exposed to the extract, although neither of them metamorphosed. We argue that larvae that lose their cilia before attachment would be swept away from the sites that stimulated settlement by the turbulent flow characteristic of most marine habitats.

Formation and Function of the Primary Tube During Settlement and Metamorphosis of the Marine Polychaete Hydroides elegans (Haswell, 1883) (Serpulidae).

AbstractThe serpulid polychaete Hydroides elegans has emerged as a major model organism for studies of marine invertebrate settlement and metamorphosis and for processes involved in marine biofouling. Rapid secretion of an enveloping, membranous, organic primary tube provides settling larvae of H. elegans firm adhesion to a surface and a refuge within which to complete metamorphosis. While this tube is never calcified, it forms the template from which the calcified tube is produced at its anterior end. Examination of scanning and transmission electron micrographs of competent and settling larvae revealed that the tube is secreted from epidermal cells of the three primary segments, with material possibly transported through the larval cuticle via abundant microvilli. The tube is composed of complexly layered fibrous material that has an abundance of the amino acids that characterize the collagenous cuticle of other polychaetes, plus associated carbohydrates. The significance of the dependence on surface bacterial biofilms for stimulating settlement in this species is revealed as a complex interaction between primary tube material, as it is secreted, and the extracellular polymeric substances abundantly produced by biofilm-residing bacteria. This association appears to provide the settling larvae with an adhesion strength similar to that of bacteria in a biofilm and significantly less when larvae settle on a clean surface.

Evolutionary genomics of endangered Hawaiian tree snails (Achatinellidae: Achatinellinae) for conservation of adaptive capacity.

Phylogenomic studies can provide insights into speciation, adaptation, and extinction, while providing a roadmap for conservation. Hawaiian tree snails are a model system for an adaptive radiation facing an extinction crisis. In the last 5 years, nearly all populations of Hawaiian tree snails across the 30 remaining species in the subfamily Achatinellinae (Achatinellidae) have declined from hundreds or thousands in the wild down to undetectable levels. Nearly 100 species historically occurred across dramatic environmental gradients on five of the Hawaiian Islands, but habitat loss, overcollection, and predation by invasive species have decimated populations. As such, this system offers the opportunity to integrate efforts to conserve evolutionary potential into conservation planning for a rapidly declining subfamily. Here, we used genome-wide, restriction-site associated DNA sequencing (RADseq), along with mitochondrial genome reconstruction, to resolve evolutionary relationships to inform conservation efforts. Phylogenetic analysis of nearly 400k genome-wide SNPs from 59 populations and 25 species across six genera in the family Achatinellidae, was generally concordant with taxonomy, geography, and mtDNA with several notable exceptions; mtDNA was unable to resolve some deeper nodes (e.g., the monophyly of Achatinella), while SNP data did not resolve as many shallow nodes. Both phylogenetic and coalescent analysis revealed deep divergences between populations within Achatinella mustelina that were consistent with species-level differences. Given cryptic species-level divergence within populations that are geographically proximate, they are at higher risk of extirpation from invasive predators and climate change than previously assumed. This study clarifies evolutionary relationships within this model system for adaptive radiation, forming the basis for conservation strategies such as translocation, captive rearing, and hybridization trials to prevent the loss of capacity to adapt to rapidly changing environmental conditions.

Marine biofilms on submerged surfaces are a reservoir for Escherichia coli and Vibrio cholerae.

The enteric bacterium and potential human pathogen, Escherichia coli, is known to persist in tropical soils and coastal waters. Vibrio cholerae causes the disease cholera and inhabits marine environments including microbial films on submerged surfaces. The abundances of E. coli and V. cholerae were quantified in biofilm and water-column samples from three harbors in Honolulu, Hawai'i, which differ in their local and international ship traffic. E. coli and, in some cases V. cholerae, occurred in relatively high abundances in marine biofilms formed on abiotic surfaces, including the exterior hulls of ships. The community fingerprints of the biofilms and the water harboring these pathogens were further analyzed. The community compositions of biofilms from different locations were more similar to each other than to water-column communities from the same locations. These results suggest that biofilms are an overlooked reservoir and a source of dissemination for E. coli and V. cholerae.

Effects of initial surface wettability on biofilm formation and subsequent settlement of Hydroides elegans.

Hydroides elegans is a major fouling organism in tropical waters around the world, including Pearl Harbor, Hawaii. To determine the importance of initial surface characteristics on biofilm community composition and subsequent colonization by larvae of H. elegans, the settlement and recruitment of larvae to biofilmed surfaces with six different initial surface wettabilities were tested in Pearl Harbor. Biofilm community composition, as determined by a combined approach of denaturing gradient gel electrophoresis and fluorescence in situ hybridization, was similar across all surfaces, regardless of initial wettability, and all surfaces had distinct temporal shifts in community structure over a 10 day period. Larvae settled and recruited in higher numbers to surfaces with medium to low wettability in both May and August, and also to slides with high wettability in August. Pearl Harbor biofilm communities developed similarly on a range of surface wettabilities, and after 10 days in Pearl Harbor all surfaces were equally attractive to larvae of Hydroides elegans, regardless of initial surface properties.

Antifouling character of 'active' hybrid xerogel coatings with sequestered catalysts for the activation of hydrogen peroxide.

Halide-permeable xerogel films prepared from sols containing 50 mol% aminopropyltriethoxysilane (APTES)/50 mol% tetraethoxysilane (TEOS) or 10 mol% APTES/90 mol% TEOS and 0.015 M selenoxide or telluride catalyst in the sol gave reduced settlement of cypris larvae of the barnacle Balanus amphitrite and larvae of the tubeworm Hydroides elegans in the presence of artificial seawater (ASW) and hydrogen peroxide (5-100 microM) relative to glass controls. Settlement of Ulva zoospores was lower on both the 50 mol% APTES/50 mol% TEOS and 10 mol% APTES/90 mol% TEOS xerogel formulations in comparison with glass controls with or without the added catalyst. The 50 mol% APTES/50 mol%TEOS xerogel containing telluride catalyst gave reduced settlement of Ulva zoospores in the presence of 100 microM H(2)O(2) in ASW compared with the same coating without added peroxide. Scanning electron microscopy and XPS data suggest that exposure to H(2)O(2) does not lead to chemical or morphological changes on the xerogel surface.

Complete Genome Sequence of Thalassotalea euphylliae Strain H2.

A bacterial isolate of Thalassotalea euphylliae H2 was collected from the coral Montipora capitata. MinION long reads were employed for scaffolding and complemented with short-read MiSeq sequences to permit complete genome assembly. The genome is approximately 4.36 Mb long, with 3,669 protein-coding genes, 92 tRNAs, and 21 rRNAs.

Two intracellular and cell type-specific bacterial symbionts in the placozoan Trichoplax H2.

Placozoa is an enigmatic phylum of simple, microscopic, marine metazoans1,2. Although intracellular bacteria have been found in all members of this phylum, almost nothing is known about their identity, location and interactions with their host3-6. We used metagenomic and metatranscriptomic sequencing of single host individuals, plus metaproteomic and imaging analyses, to show that the placozoan Trichoplax sp. H2 lives in symbiosis with two intracellular bacteria. One symbiont forms an undescribed genus in the Midichloriaceae (Rickettsiales)7,8 and has a genomic repertoire similar to that of rickettsial parasites9,10, but does not seem to express key genes for energy parasitism. Correlative image analyses and three-dimensional electron tomography revealed that this symbiont resides in the rough endoplasmic reticulum of its host's internal fibre cells. The second symbiont belongs to the Margulisbacteria, a phylum without cultured representatives and not known to form intracellular associations11-13. This symbiont lives in the ventral epithelial cells of Trichoplax, probably metabolizes algal lipids digested by its host and has the capacity to supplement the placozoan's nutrition. Our study shows that one of the simplest animals has evolved highly specific and intimate associations with symbiotic, intracellular bacteria and highlights that symbioses can provide access to otherwise elusive microbial dark matter.

Analysis of nitric oxide-cyclic guanosine monophosphate signaling during metamorphosis of the nudibranch Phestilla sibogae Bergh (Gastropoda: Opisthobranchia).

The gas nitric oxide (NO), and in some cases its downstream second messenger, cyclic guanosine monophosphate (cGMP) function in different taxa to regulate the timing of life-history transitions. Increased taxonomic sampling is required to foster conclusions about the evolution and function of NO/cGMP signaling during life-history transitions. We report on the function and localization of NO and cGMP signaling during metamorphosis of the nudibranch Phestilla sibogae. Pharmacological manipulation of NO or cGMP production in larvae modulated responses to a natural settlement cue from the coral Porites compressa in a manner that suggest inhibitory function for NO/cGMP signaling. However, these treatments were not sufficient to induce metamorphosis in the absence of cue, a result unique to this animal. We show that induction of metamorphosis in response to the settlement cue is associated with a reduction in NO production. We documented the expression of putative NO synthase (NOS) and the production of cGMP during larval development and observed no larval cells in which NOS and cGMP were both detected. The production of cGMP in a bilaterally symmetrical group of cells fated to occupy the distal tip of rhinophores is correlated with competence to respond to the coral settlement cue. These results suggest that endogenous NO and cGMP are involved in modulating responses of P. sibogae to a natural settlement cue. We discuss these results with respect to habitat selection and larval ecology.

Microbial biofilms facilitate adhesion in biofouling invertebrates.

Much interest has focused on the role of microbial layers--biofilms--in stimulating attachment of invertebrates and algae to submerged marine surfaces. We investigated the influence of biofilms on the adhesion strength of settling invertebrates. Larvae of four species of biofouling invertebrate were allowed to attach to test surfaces that were either clean or coated with a natural biofilm. Measuring larval removal under precisely controlled flow forces, we found that biofilms significantly increased adhesion strength in the ascidian Phallusia nigra, the polychaete tubeworm Hydroides elegans, and the barnacle Balanus amphitrite at one or more developmental stages. Attachment strength in a fourth species, the bryozoan Bugula neritina, was neither facilitated nor inhibited by the presence of a biofilm. These results suggest that adhesive strength and perhaps composition may vary across different invertebrate taxa at various recruitment stages, and mark a new path of inquiry for biofouling research.

Brood Reduction, Not Poecilogony, in a Vermetid Gastropod with Two Developmental Outcomes within Egg Capsules.

A small vermetid gastropod broods capsules containing nurse eggs and embryos that develop into small veligers. A few of these veligers continue development and growth while nurse eggs and developmentally arrested sibling veligers disappear. Survivors hatch as crawling pediveligers and juveniles. None of the veligers, if removed from capsules, swim in a directed way or withdraw into their shells, indicating that even the developing veligers are unsuited for extracapsular life until they can crawl. The shells of arrested veligers decalcify while their siblings grow. Few of the developmentally arrested veligers that were isolated from siblings and fed algal cells resumed detectable growth. Nurse eggs rather than cannibalism provide most of the food, but full growth of developing veligers depends on limited sharing; arrest of some siblings is a necessary adjunct of the nurse-egg feeding. Here, two developmental outcomes for larvae produced by developmental arrest of some (often termed poecilogony) serves instead as a means of brood reduction. Brood reduction is often attributed to family conflicts resulting from genetic differences. Another hypothesis is that a mother who cannot accurately sort numbers of nurse eggs and developing eggs into capsules could rely on brood reduction to adjust food for her offspring. At the extreme, an entirely random packaging would produce a binomial distribution of embryos in capsules, a very uneven distribution of food per embryo, and some capsules with no embryos. Males have yet to be found in this species, but even if reproduction is asexual, selection could still favor brood reduction.