Does substrate matter in the deep sea? A comparison of bone, wood, and carbonate rock colonizers.

Continental margins host methane seeps, animal falls and wood falls, with chemosynthetic communities that may share or exchange species. The goal of this study was to examine the existence and nature of linkages among chemosynthesis-based ecosystems by deploying organic fall mimics (bone and wood) alongside defaunated carbonate rocks within high and lesser levels of seepage activity for 7.4 years. We compared community composition, density, and trophic structure of invertebrates on these hard substrates at active methane seepage and transition (less seepage) sites at Mound 12 at ~1,000 m depth, a methane seep off the Pacific coast of Costa Rica. At transition sites, the community composition on wood and bone was characteristic of natural wood- and whale-fall community composition, which rely on decay of the organic substrates. However, at active sites, seepage activity modified the relationship between fauna and substrate, seepage activity had a stronger effect in defining and homogenizing these communities and they depend less on organic decay. In contrast to community structure, macrofaunal trophic niche overlap between substrates, based on standard ellipse areas, was greater at transition sites than at active sites, except between rock and wood. Our observations suggest that whale- and wood-fall substrates can function as stepping stones for seep fauna even at later successional stages, providing hard substrate for attachment and chemosynthetic food.

Methanotrophic bacterial symbionts fuel dense populations of deep-sea feather duster worms (Sabellida, Annelida) and extend the spatial influence of methane seepage.

Deep-sea cold seeps are dynamic sources of methane release and unique habitats supporting ocean biodiversity and productivity. Here, we describe newly discovered animal-bacterial symbioses fueled by methane, between two species of annelid (a serpulid Laminatubus and sabellid Bispira) and distinct aerobic methane-oxidizing bacteria belonging to the Methylococcales, localized to the host respiratory crown. Worm tissue δ13C of -44 to -58‰ are consistent with methane-fueled nutrition for both species, and shipboard stable isotope labeling experiments revealed active assimilation of 13C-labeled methane into animal biomass, which occurs via the engulfment of methanotrophic bacteria across the crown epidermal surface. These worms represent a new addition to the few animals known to intimately associate with methane-oxidizing bacteria and may further explain their enigmatic mass occurrence at 150-million year-old fossil seeps. High-resolution seafloor surveys document significant coverage by these symbioses, beyond typical obligate seep fauna. These findings uncover novel consumers of methane in the deep sea and, by expanding the known spatial extent of methane seeps, may have important implications for deep-sea conservation.

RNA sequencing by direct tagmentation of RNA/DNA hybrids.

Transcriptome profiling by RNA sequencing (RNA-seq) has been widely used to characterize cellular status, but it relies on second-strand complementary DNA (cDNA) synthesis to generate initial material for library preparation. Here we use bacterial transposase Tn5, which has been increasingly used in various high-throughput DNA analyses, to construct RNA-seq libraries without second-strand synthesis. We show that Tn5 transposome can randomly bind RNA/DNA heteroduplexes and add sequencing adapters onto RNA directly after reverse transcription. This method, Sequencing HEteRo RNA-DNA-hYbrid (SHERRY), is versatile and scalable. SHERRY accepts a wide range of starting materials, from bulk RNA to single cells. SHERRY offers a greatly simplified protocol and produces results with higher reproducibility and GC uniformity compared with prevailing RNA-seq methods.

Chemosymbiotic bivalves contribute to the nitrogen budget of seagrass ecosystems.

In many seagrass sediments, lucinid bivalves and their sulfur-oxidizing symbionts are thought to underpin key ecosystem functions, but little is known about their role in nutrient cycles, particularly nitrogen. We used natural stable isotopes, elemental analyses, and stable isotope probing to study the ecological stoichiometry of a lucinid symbiosis in spring and fall. Chemoautotrophy appeared to dominate in fall, when chemoautotrophic carbon fixation rates were up to one order of magnitude higher as compared with the spring, suggesting a flexible nutritional mutualism. In fall, an isotope pool dilution experiment revealed carbon limitation of the symbiosis and ammonium excretion rates up to tenfold higher compared with fluxes reported for nonsymbiotic marine bivalves. These results provide evidence that lucinid bivalves can contribute substantial amounts of ammonium to the ecosystem. Given the preference of seagrasses for this nitrogen source, lucinid bivalves' contribution may boost productivity of these important blue carbon ecosystems.

Atypical biological features of a new cold seep site on the Lofoten-Vesterålen continental margin (northern Norway).

A newly discovered cold seep from the Lofoten-Vesterålen margin (Norwegian Sea) is dominated by the chemosymbiotrophic siboglinid Oligobrachia haakonmosbiensis like other high latitude seeps, but additionally displays uncharacteristic features. Sulphidic bottom water likely prevents colonization by cnidarians and sponges, resulting in fewer taxa than deeper seeps in the region, representing a deviation from depth-related trends seen among seeps elsewhere. O. haakonmosbiensis was present among carbonate and barite crusts, constituting the first record of frenulates among hard substrates. The presence of both adults and egg cases indicate that Ambylraja hyperborea skates use the site as an egg case nursery ground. Due to sub-zero ambient temperatures (-0.7 °C), we hypothesize that small, seepage related heat anomalies aid egg incubation and prevent embryo mortality. We place our results within the context of high-latitude seeps and suggest they exert evolutionary pressure on benthic species, thereby selecting for elevated exploitation and occupancy of high-productivity habitats.

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing.

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tCO ) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tCO compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.

Astronomical and atmospheric impacts on deep-sea hydrothermal vent invertebrates.

Ocean tides and winter surface storms are among the main factors driving the dynamics and spatial structure of marine coastal species, but the understanding of their impact on deep-sea and hydrothermal vent communities is still limited. Multidisciplinary deep-sea observatories offer an essential tool to study behavioural rhythms and interactions between hydrothermal community dynamics and environmental fluctuations. Here, we investigated whether species associated with a Ridgeia piscesae tubeworm vent assemblage respond to local ocean dynamics. By tracking variations in vent macrofaunal abundance at different temporal scales, we provide the first evidence that tides and winter surface storms influence the distribution patterns of mobile and non-symbiotic hydrothermal species (i.e. pycnogonids Sericosura sp. and Polynoidae polychaetes) at more than 2 km depth. Local ocean dynamics affected the mixing between hydrothermal fluid inputs and surrounding seawater, modifying the environmental conditions in vent habitats. We suggest that hydrothermal species respond to these habitat modifications by adjusting their behaviour to ensure optimal living conditions. This behaviour may reflect a specific adaptation of vent species to their highly variable habitat.

Fluorescence Turn-On Sensing of DNA Duplex Formation by a Tricyclic Cytidine Analogue.

Most fluorescent nucleoside analogues are quenched when base stacked and some maintain their brightness, but there has been little progress toward developing nucleoside analogues that markedly increase their fluorescence upon duplex formation. Here, we report on the design and synthesis of a new tricyclic cytidine analogue, 8-diethylamino-tC (8-DEA-tC), that responds to DNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. These results, supported by DFT calculations, suggest a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.

Using Stable Isotopes to Infer the Impacts of Habitat Change on the Diets and Vertical Stratification of Frugivorous Bats in Madagascar.

Human-modified habitats are expanding rapidly; many tropical countries have highly fragmented and degraded forests. Preserving biodiversity in these areas involves protecting species-like frugivorous bats-that are important to forest regeneration. Fruit bats provide critical ecosystem services including seed dispersal, but studies of how their diets are affected by habitat change have often been rather localized. This study used stable isotope analyses (δ15N and δ13C measurement) to examine how two fruit bat species in Madagascar, Pteropus rufus (n = 138) and Eidolon dupreanum (n = 52) are impacted by habitat change across a large spatial scale. Limited data for Rousettus madagascariensis are also presented. Our results indicated that the three species had broadly overlapping diets. Differences in diet were nonetheless detectable between P. rufus and E. dupreanum, and these diets shifted when they co-occurred, suggesting resource partitioning across habitats and vertical strata within the canopy to avoid competition. Changes in diet were correlated with a decrease in forest cover, though at a larger spatial scale in P. rufus than in E. dupreanum. These results suggest fruit bat species exhibit differing responses to habitat change, highlight the threats fruit bats face from habitat change, and clarify the spatial scales at which conservation efforts could be implemented.

Carbon fixation by basalt-hosted microbial communities.

Oceanic crust is a massive potential habitat for microbial life on Earth, yet our understanding of this ecosystem is limited due to difficulty in access. In particular, measurements of rates of microbial activity are sparse. We used stable carbon isotope incubations of crustal samples, coupled with functional gene analyses, to examine the potential for carbon fixation on oceanic crust. Both seafloor-exposed and subseafloor basalts were recovered from different mid-ocean ridge and hot spot environments (i.e., the Juan de Fuca Ridge, the Mid-Atlantic Ridge, and the Loihi Seamount) and incubated with (13)C-labeled bicarbonate. Seafloor-exposed basalts revealed incorporation of (13)C-label into organic matter over time, though the degree of incorporation was heterogeneous. The incorporation of (13)C into biomass was inconclusive in subseafloor basalts. Translating these measurements into potential rates of carbon fixation indicated that 0.1-10 nmol C g(-1) rock d(-1) could be fixed by seafloor-exposed rocks. When scaled to the global production of oceanic crust, this suggests carbon fixation rates of 10(9)-10(12) g C year(-1), which matches earlier predictions based on thermodynamic calculations. Functional gene analyses indicate that the Calvin cycle is likely the dominant biochemical mechanism for carbon fixation in basalt-hosted biofilms, although the reductive acetyl-CoA pathway and reverse TCA cycle likely play some role in net carbon fixation. These results provide empirical evidence for autotrophy in oceanic crust, suggesting that basalt-hosted autotrophy could be a significant contributor of organic matter in this remote and vast environment.

Comparison of Archaeal and Bacterial Diversity in Methane Seep Carbonate Nodules and Host Sediments, Eel River Basin and Hydrate Ridge, USA.

Anaerobic oxidation of methane (AOM) impacts carbon cycling by acting as a methane sink and by sequestering inorganic carbon via AOM-induced carbonate precipitation. These precipitates commonly take the form of carbonate nodules that form within methane seep sediments. The timing and sequence of nodule formation within methane seep sediments are not well understood. Further, the microbial diversity associated with sediment-hosted nodules has not been well characterized and the degree to which nodules reflect the microbial assemblage in surrounding sediments is unknown. Here, we conducted a comparative study of microbial assemblages in methane-derived authigenic carbonate nodules and their host sediments using molecular, mineralogical, and geochemical methods. Analysis of 16S rRNA gene diversity from paired carbonate nodules and sediments revealed that both sample types contained methanotrophic archaea (ANME-1 and ANME-2) and syntrophic sulfate-reducing bacteria (Desulfobacteraceae and Desulfobulbaceae), as well as other microbial community members. The combination of geochemical and molecular data from Eel River Basin and Hydrate Ridge suggested that some nodules formed in situ and captured the local sediment-hosted microbial community, while other nodules may have been translocated or may represent a record of conditions prior to the contemporary environment. Taken together, this comparative analysis offers clues to the formation regimes and mechanisms of sediment-hosted carbonate nodules.

Thrombophilia testing has limited usefulness in clinical decision-making and should be used selectively.

Management of venous thromboembolism (VTE) includes evaluation for hypercoagulable state, especially if the VTE occurs in young patients, is recurrent, or is associated with a positive family history. These laboratory tests are costly, and surprisingly, there is little evidence showing that testing leads to improved clinical outcomes. Evidence based on observational prospective studies suggests that optimal duration of anticoagulation should be based on clinical risks resulting in VTE, such as transient, permanent, and idiopathic or unprovoked risks, and less on abnormal thrombophilia values. Thrombophilia screening is important in a subgroup of clinical scenarios, such as when there is clinical suspicion of antiphospholipid antibody syndrome, heparin resistance, or warfarin necrosis; with thrombosis occurring in unusual sites (such as mesenteric or cerebral deep venous thrombosis); and for pregnant women or those seeking pregnancy or considering estrogen-based agents. Thrombophilia screening is not likely to be helpful in most cases of first-time unprovoked VTE in the setting of transient risks, active malignant disease, deep venous thrombosis of upper extremity veins or from central lines, two or more VTEs, or arterial thrombosis with pre-existing atherosclerotic risk factors. The desire by both patient and physician for a scientific explanation of the clotting event may alone lead to testing, and if so, it should be with the understanding that an abnormal test result will likely not change management, and normal results do not accurately exclude a thrombophilic defect because there are likely factors yet to be discovered. Such false assumptions may lead to shorter durations of treatment than are optimal.

Using stable isotope compositions of animal tissues to infer trophic interactions in Gulf of Mexico lower slope seep communities.

We analyzed the tissue carbon, nitrogen, and sulfur stable isotope contents of macrofaunal communities associated with vestimentiferan tubeworms and bathymodiolin mussels from the Gulf of Mexico lower continental slope (970-2800 m). Shrimp in the genus Alvinocaris associated with vestimentiferans from shallow (530 m) and deep (1400-2800 m) sites were used to test the hypothesis that seep animals derive a greater proportion of their nutrition from seeps (i.e. a lower proportion from the surface) at greater depths. To account for spatial variability in the inorganic source pool, we used the differences between the mean tissue δ(13)C and δ(15)N of the shrimp in each collection and the mean δ (13)C and δ(15)N values of the vestimentiferans from the same collection, since vestimentiferans are functionally autotrophic and serve as a baseline for environmental isotopic variation. There was a significant negative relationship between this difference and depth for both δ(13)C and δ(15)N (p=0.02 and 0.007, respectively), which supports the hypothesis of higher dependence on seep nutrition with depth. The small polychaete worm Protomystides sp. was hypothesized to be a blood parasite of the vestimentiferan Escarpialaminata. There was a highly significant linear relationship between the δ(13)C values of Protomystides sp. and the E. laminata individuals to which they were attached across all collections (p < 0.001) and within a single collection (p = 0.01), although this relationship was not significant for δ(15)N and δ(34)S. We made several other qualitative inferences with respect to the feeding biology of the taxa occurring in these lower slope seeps, some of which have not been described prior to this study.

Identification of putative quantitative trait loci associated with a flavonoid related to resistance to cabbage seedpod weevil (Ceutorhynchus obstrictus) in canola derived from an intergeneric cross, Sinapis alba × Brassica napus.

KEY MESSAGE: Kaempferol 3- O -sinapoyl-sophoroside 7- O -glucoside was putatively identified as the major component of a characteristic HPLC peak previously correlated with the reduction of cabbage seedpod weevil larval infestation in a novel canola genotype. The cabbage seedpod weevil (Ceutorhynchus obstrictus [Marsham]) (CSPW) is a serious pest of brassicaceous oilseed crops such as canola in both Europe and more recently in North America. At present, the only control strategy against CSPW is the application of insecticides. As an alternative more environmentally-friendly control strategy, we developed novel canola germplasm resistant to weevil attack through introgression of Sinapis alba DNA into Brassica napus by making the wide cross followed by embryo rescue and backcrossing to the B. napus parent. We have previously characterized resistant canola lines by metabolic profiling and were able to correlate reduction of larval infestation to the presence of a characteristic HPLC peak. In this study, we have putatively identified the major component in the peak using mass spectrometry as kaempferol 3-O-sinapoyl-sophoroside 7-O-glucoside (KSSG). We have also identified quantitative trait loci (QTL) associated with this HPLC peak in a mapping population consisting of more than 200 individual doubled haploid (DH) lines derived from a cross between CSW428 (the resistant parent) and SC030686 (the susceptible parent). This QTL accounted for approximately 9.5 % of the phenotypic variation in KSSG content. The observation that only one QTL was identified as surpassing the LOD threshold of 3.0 suggests that both parents may possess the positive alleles for other QTL that have not been detected in our study. This finding also indicates a complex regulatory mechanism for KSSG levels and provides an appropriate explanation for the large transgressive segregation observed in the DH lines of the QTL mapping population.

Diversity in skeletal architecture influences biological heterogeneity and Symbiodinium habitat in corals.

Scleractinian corals vary in response to rapid shifts in the marine environment and changes in reef community structure post-disturbance reveal a clear relationship between coral performance and morphology. With exceptions, massive corals are thought to be more tolerant and branching corals more vulnerable to changing environmental conditions, notably thermal stress. The typical responses of massive and branching coral taxa, respectively, are well documented; however, the biological and functional characteristics that underpin this variation are not well understood. We address this gap by comparing multiple biological attributes that are correlated with skeletal architecture in two perforate (having porous skeletal matrices with intercalating tissues) and two imperforate coral species (Montipora aequituberculata, Porites lobata, Pocillopora damicornis, and Seriatopora hystrix) representing three morphotypes. Our results reveal inherent biological heterogeneity among corals and the potential for perforate skeletons to create complex, three-dimensional internal habitats that impact the dynamics of the symbiosis. Patterns of tissue thickness are correlated with the concentration of symbionts within narrow regions of tissue in imperforate corals versus broad distribution throughout the larger tissue area in perforate corals. Attributes of the perforate and environmentally tolerant P. lobata were notable, with tissues ∼5 times thicker than in the sensitive, imperforate species P. damicornis and S. hystrix. Additionally, P. lobata had the lowest baseline levels of superoxide and Symbiodinium that provisioned high levels of energy. Given our observations, we hypothesize that the complexity of the visually obscured internal environment has an impact on host-symbiont dynamics and ultimately on survival, warranting further scientific investigation.

Isothermal amplification method for next-generation sequencing.

We report an approach for generating immobilized monoclonal templates for next- generation sequencing applications. Our isothermal amplification method is based on a template walking mechanism using a pair of low-melting temperature (Tm) solid-surface homopolymer primers and a low-Tm solution phase primer. The method can generate more than one billion submicrometer-sized colonies in a single lane of a next-generation sequencing flowchip. An alternative paired-end sequencing method using interstrand DNA photo cross-linking to covalently link the complementary strands of the original templates to the solid surface is also demonstrated.

Arthropod food webs become increasingly lipid-limited at higher trophic levels.

Understanding why food chains are relatively short in length has been an area of research and debate for decades. We tested if progressive changes in the nutritional content of arthropods with trophic position limit the availability of a key nutrient, lipid, for carnivores. Arthropods at higher trophic levels had progressively less lipid and more protein in their bodies compared with arthropods at lower trophic levels. The nutrients present in arthropod bodies were directly related to the nutrients that predators extracted when feeding on those arthropods. As a consequence, nutrient assimilation shifted from lipid-biased to protein-biased as arthropods ascended trophic levels from herbivores to secondary carnivores. Successive changes in the nutritional consequences of predation may ultimately set an upper limit on the number of trophic levels in arthropod communities. Further work is needed to examine the influence of lipid and other nutrients on food web traits in a range of ecosystems.

Evidence for the role of endosymbionts in regional-scale habitat partitioning by hydrothermal vent symbioses.

Deep-sea hydrothermal vents are populated by dense communities of animals that form symbiotic associations with chemolithoautotrophic bacteria. To date, our understanding of which factors govern the distribution of host/symbiont associations (or holobionts) in nature is limited, although host physiology often is invoked. In general, the role that symbionts play in habitat utilization by vent holobionts has not been thoroughly addressed. Here we present evidence for symbiont-influenced, regional-scale niche partitioning among symbiotic gastropods (genus Alviniconcha) in the Lau Basin. We extensively surveyed Alviniconcha holobionts from four vent fields using quantitative molecular approaches, coupled to characterization of high-temperature and diffuse vent-fluid composition using gastight samplers and in situ electrochemical analyses, respectively. Phylogenetic analyses exposed cryptic host and symbiont diversity, revealing three distinct host types and three different symbiont phylotypes (one ε-proteobacteria and two γ-proteobacteria) that formed specific associations with one another. Strikingly, we observed that holobionts with ε-proteobacterial symbionts were dominant at the northern fields, whereas holobionts with γ-proteobacterial symbionts were dominant in the southern fields. This pattern of distribution corresponds to differences in the vent geochemistry that result from deep subsurface geological and geothermal processes. We posit that the symbionts, likely through differences in chemolithoautotrophic metabolism, influence niche utilization among these holobionts. The data presented here represent evidence linking symbiont type to habitat partitioning among the chemosynthetic symbioses at hydrothermal vents and illustrate the coupling between subsurface geothermal processes and niche availability.

Adaptive radiation in extremophilic Dorvilleidae (Annelida): diversification of a single colonizer or multiple independent lineages?

Metazoan inhabitants of extreme environments typically evolved from forms found in less extreme habitats. Understanding the prevalence with which animals move into and ultimately thrive in extreme environments is critical to elucidating how complex life adapts to extreme conditions. Methane seep sediments along the Oregon and California margins have low oxygen and very high hydrogen sulfide levels, rendering them inhospitable to many life forms. Nonetheless, several closely related lineages of dorvilleid annelids, including members of Ophryotrocha, Parougia, and Exallopus, thrive at these sites in association with bacterial mats and vesicomyid clam beds. These organisms are ideal for examining adaptive radiations in extreme environments. Did dorvilleid annelids invade these extreme environments once and then diversify? Alternatively, did multiple independent lineages adapt to seep conditions? To address these questions, we examined the evolutionary history of methane-seep dorvilleids using 16S and Cyt b genes in an ecological context. Our results indicate that dorvilleids invaded these extreme habitats at least four times, implying preadaptation to life at seeps. Additionally, we recovered considerably more dorvilleid diversity than is currently recognized. A total of 3 major clades (designated "Ophryotrocha," "Mixed Genera" and "Parougia") and 12 terminal lineages or species were encountered. Two of these lineages represented a known species, Parougia oregonensis, whereas the remaining 10 lineages were newly discovered species. Certain lineages exhibited affinity to geography, habitat, sediment depth, and/or diet, suggesting that dorvilleids at methane seeps radiated via specialization and resource partitioning.

Estimating niche width using stable isotopes in the face of habitat variability: a modelling case study in the marine environment.

Distributions of stable isotopes have been used to infer an organism's trophic niche width, the 'isotopic niche', and examine resource partitioning. Spatial variation in the isotopic composition of prey may however confound the interpretation of isotopic signatures especially when foragers exploit resources across numerous locations. In this study the isotopic compositions from marine assemblages are modelled to determine the role of variation in the signature of prey items and the effect of dietary breadth and foraging strategies on predator signatures. Outputs from the models reveal that isotopic niche widths can be greater for populations of dietary specialists rather than for generalists, which contravenes what is generally accepted in the literature. When a range of different mixing models are applied to determine if the conversion from δ to p-space can be used to improve model accuracy, predator signature variation is increased rather than model precision. Furthermore the mixing models applied failed to correctly identify dietary specialists and/or to accurately estimate diet contributions that may identify resource partitioning. The results presented illustrate the need to collect sufficiently large sample sizes, in excess of what is collected under most current studies, across the complete distribution of a species and its prey, before attempts to use stable isotopes to make inferences about niche width can be made.