Bacterial Consumption of Humic and Non-Humic Low and High Molecular Weight DOM and the Effect of Solar Irradiation on the Turnover of Labile DOM in the Southern Ocean

The decomposition of dissolved organic matter (DOM) in pelagic ecosystems is mediated primarily by heterotrophic bacteria, but transformation by short-wave solar radiation may play an important role in surface waters, in particular when humic substances constitute a substantial fraction of the DOM pool. Most of the studies examining bacterial decomposition and photochemical transformation of DOM stem from limnetic and coastal marine systems and much less information is available from oceanic environments. To examine the bacterial decomposition of humic and non-humic DOM in the Southern Ocean we carried out microcosm experiments in which we measured bacterial growth on isolated fractions of humic and non-humic DOM of the size classes <3 kDa and >3 kDa. Experiments carried out at the Polar Front showed a preferential bacterial growth on non-humic DOM and in particular on the size fraction <3 kDa. Bacterial growth, measured as bacterial biomass production, on non-humic DOM accounted for 74% to 88% of the total growth on all four DOM fractions. In experiments in the Antarctic circumpolar current and the coastal current under pack ice, bacterial growth was 6× lower than at the Polar Front, and humic and non-humic DOM was consumed to equal amounts. The size fraction <3 kDa was always preferred. Experiments examining the effect of solar radiation on the release of dissolved amino acids (DAA) and carbohydrates (DCHO) and their subsequent bacterial utilization showed a stimulating effect on glucose uptake and the release of DAA at the Polar Front but an inhibition in the eastern Weddell Sea. Ultraviolet-B was the most effective component of the solar radiation spectrum tested. Effects of UV-B on glucose uptake and release of DAA were positively correlated with concentrations of humic-bound DAA. The data imply that at low concentrations, e.g., <100 nM (amino acid equivalent), UV-irradiation reduces, whereas at concentrations >100 nM UV-irradiation stimulates glucose uptake and release of DAA as compared to dark conditions.     

Use of electrospray ionization (ESI) mass spectrometry to investigate complex dissolved organic matter (DOM) and its potential applications in phytoplankton research

Organic nutrients are one of many factors considered to be important in the growth and proliferation of phytoplankton including many species that cause harmful algal blooms (HABs). Several studies have investigated the effects of known organic compounds on phytoplankton growth, however, the role of natural dissolved organic matter (DOM) in phytoplankton nutrition remains understudied at the compound level. This lack of research is due in part to analytical limitations for the characterization of DOM compounds. Electrospray ionization (ESI) mass spectrometry (MS) provides an unprecedented level of chemical information on thousands of organic compounds that comprise the bulk DOM pool. In this paper we provide a brief overview of some of the benefits and caveats of using ESI to investigate DOM in natural freshwater and marine systems and show an example of ESI-MS DOM characterization for a natural bloom of the raphidophyte Chattonella cf. verruculosa.     

Competition for dissolved glucose between meiobenthos and sediment microbes

Meiobenthos, small invertebrates inhabiting the surface layers of marine sediments, can absorb dissolved organic matter (DOM). Experiments were performed to test if meiobenthos can compete with sediment microbes for uptake of small amounts of [¹⁴C]glucose. Meiofaunal glucose uptake rates were measured by themselves and in the presence of sediment microbes. Glucose uptake by meiofauna was not inhibited by the presence of bacteria, nor did it appear that bacterial uptake was inhibited by meiofauna. Thus, there was no direct or interference competition. Uptake rates by 1 cm³ of sediment (bacteria) were four orders of magnitude greater than those of individual meiofauna, but on a biomass specific basis, meiofaunal uptake was in the same range if not higher than that of sediment bacteria. Thus, the potential for indirect or resource competition exists. Since bacterial biomass dominated the system studied, uptake of glucose was dominated by bacteria. The results support the hypothesis that in natural sediments, where the biomass of bacteria is higher than that of meiofauna, heterotrophic uptake is primarily a microbial process. However, resource competition between meiofauna and bacteria for DOM in sediments probably exists where bacterial biomass is low relative to meiofaunal biomass.     

Bacterial--Invertebrate Interactions in Uptake of Dissolved Organic Matter

SYNOPSIS. AS compared to integumentary uptake systems of soft-bodiedmarine invertebrates, bacterial systems, in terms of transportconstants, are much better adapted to the low concentrationsof dissolved organic nutrients encountered in coastal and offshorewaters. Bacteria respond to the presence of suitable dissolvedorganic substrates with induction, uptake and multiplication,maintaining the concentrations of dissolved organic matter (DOM)permanently low. At realistic in situ concentrations, epidermaluptake by pelagic and epibenthic animals proceeds at such lowrates that scarcely a substantial proportion of their metabolicneeds is provided by absorption. In marine sediments, wherethe life processes of bacteria and animals are closely interrelated,the macrofauna is sheltered by shells, firm tubes and burrows,which are irrigated by means of overlying water of the watercolumn. Hence, interstitial water with its sometimes higherconcentrations of DOM is scarcely available to sediment-dwellinglarger metazoans. The meiofauna mainly inhabits the few millimetersof the upper sediment layers and the thin halos surroundingirrigated macrofaunal burrows, where sufficient oxygen is available.Unless the magnitude of horizontal water movement, the amountsof diffusional nutrient supply and the percentages, by whichnutrient concentrations are reduced by meiofaunal uptake, areknown, estimations of nutritional benefits from uptake of DOMby meiofauna cannot be made. For all infaunal taxa, bacteriaappear to represent a major food supply.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

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 hosts larval stage, which is exceptionally vulnerable to predators.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Extrapolating non-target risk of Bt crops from laboratory to field

The tiered approach to assessing ecological risk of insect-resistant transgenic crops assumes that lower tier laboratory studies, which expose surrogate non-target organisms to high doses of insecticidal proteins, can detect harmful effects that might be manifested in the field. To test this assumption, we performed meta-analyses comparing results for non-target invertebrates exposed to Bacillus thuringiensis (Bt) Cry proteins in laboratory studies with results derived from independent field studies examining effects on the abundance of non-target invertebrates. For Lepidopteran-active Cry proteins, laboratory studies correctly predicted the reduced field abundance of non-target Lepidoptera. However, laboratory studies incorporating tri-trophic interactions of Bt plants, herbivores and parasitoids were better correlated with the decreased field abundance of parasitoids than were direct-exposure assays. For predators, laboratory tri-trophic studies predicted reduced abundances that were not realized in field studies and thus overestimated ecological risk. Exposure to Coleopteran-active Cry proteins did not significantly reduce the laboratory survival or field abundance of any functional group examined. Our findings support the assumption that laboratory studies of transgenic insecticidal crops show effects that are either consistent with, or more conservative than, those found in field studies, with the important caveat that laboratory studies should explore all ecologically relevant routes of exposure.     

Consequences of Parasitism to Marine Invertebrates: Host Evolution?

Parasitism among aquatic invertebrates is common, if not ubiquitous,and can be pathological to hosts. However, host evolution inresponse to parasitism has received little attention, particularlyfor marine invertebrates. Drawing on the rich literature demonstratingprey adaptations to predators, I develop analogous predictionsfor the ways in which host life histories may be molded by theirparasites. Such adaptations are expected when the effects ofparasites are severe and when the probability of infection ishigh. Predicted life history changes include the evolution ofsemelparity, reduced age at first reproduction and reduced sizeat first reproduction. Using Recent and fossil populations oftwo bivalves species in the genusTransennella, I show that theincidence of trematode parasites may explain a trend of reducedsize through time and contribute to the maintenance of sexualdimorphism for size.     

Nutrient-limited growth rates: quantitative benefits of stress responses and some aspects of regulation

Young sunflower plants (Helianthus annuus L.) under stress oflow nitrate or phosphate availability exhibited increases inroot: shoot ratio and in kinetic parameters for uptake. Theyshowed no significant changes in photosynthetic utilizationof either nutrient. Increases in root: shoot ratio were achievedby early and persistent suppression of shoot growth, but notroot growth. Affinity for phosphate uptake, 1/K_m(P), increasedwith phosphate stress, as did affinity for nitrate uptake, 1/K_m(N),with nitrate stress. Maximal uptake rate, V_max, for phosphateuptake increased with phosphorus stress; V_max for nitrate didnot increase with nitrogen stress. Phosphate V_max was relatedstrongly to root nutrient status. Decreases in V_max with plantage were not well explained by changes in age structure of roots.Estimated benefits of acclimatory changes in root: shoot ratioand uptake kinetics ranged up to 2-fold increases in relativegrowth rate, RGR. The relation of RGR to uptake physiology followedpredictions of functional balance moderately well, with somesystematic deviations. Analyses of RGR using growth models implyno significant growth benefit from regulating Vmax, specifically,not from down-regulating it at high nutrient availability. Quantitativebenefits of increases in root: shoot ratio and uptake parametersare predicted to be quite small under common conditions whereinnutrient concentrations are significantly depleted by uptake.The root: shoot response is estimated to confer the smallestbenefit under non-depleting conditions and the largest benefitunder depleting conditions. Even then, the absolute benefitis predicted to be small, possibly excepting the case of heterogeneoussoils. Depleting and non-depleting conditions are addressedwith very different experimental techniques. We note that atheoretical framework is lacking that spans both these cases,other than purely numerical formulations that are not readilyinterpreted. Key words: Nutrient stress, nutrient uptake, nutrient use efficiency, relative growth rate, Helianthus annuus     

Exploring the regulatory role of nitric oxide (NO) and the NO-p38MAPK/cGMP pathway in larval settlement of the bryozoan Bugula neritina

The bryozoan Bugula neritina is a cosmopolitan marine fouling species that causes major fouling problems in sub-tropical waters. Settlement of B. neritina larvae can be triggered without an obvious external cue. Here, the negative regulatory role of nitric oxide (NO) during larval settlement of B. neritina was demonstrated to be mediated by cyclic guanosine monophosphate (cGMP). Although the regulatory role of the NO-p38 MAPK signaling axis in larval settlement was not evident, inhibition of nitric oxide synthase (NOS) led to the deactivation of p38 MAPK. Exclusive localization of NO and NO signaling components in sensory-related organs of the larvae is consistent with its signal transduction function in metamorphosis. Overall, this study provides new insights into the regulatory roles of the NO-p38MAPK/cGMP pathway in B. neritina settlement.     

Integrating development and environment to model reproductive performance in natural populations of an intertidal gastropod

Functional challenges can differ among life-history stages,yet performance at one stage may be linked to the outcome ofperformance at others. For example, adult performance, in termsof the location or timing of reproduction in response to environmentalsignals, can set conditions that affect the performance of developmentalstages. In marine invertebrates, however, early performancehas been studied primarily in the laboratory. I outline an integrativeapproach to the study of field reproductive performance in amarine gastropod that undergoes development in intertidal habitats.Embryos within gelatinous masses experience high variabilityin development temperature and frequent exposure to thermalstress. In laboratory experiments, developmental performancewas measured as a function of maximum temperature (T_max) experiencedduring fluctuations that mimicked field tidal profiles. Performancecurves showed declines that coincided with temperature thresholdsfor heat shock protein (Hsp) expression, a signal of cellularstress. Application of laboratory results to field records ofT_max predicted large variation in the survival of embryos depositedon different days. Timing of field reproduction was non-randomwith respect to T_max, suggesting that adults could help to bufferembryos from environmental stress. Embryo survival, however,was not predicted to benefit from the non-random pattern ofadult reproduction. Adults may be constrained to respond toinformation that only weakly predicts conditions that embryoswill experience. Studies that incorporate linkages between lifecycle stages in the field may better reveal how performancecapacities and constraints at one stage can influence performanceand selection at others.     

DNA-stable isotope probing (DNA-SIP) identifies marine sponge-associated bacteria actively utilizing dissolved organic matter (DOM)

Sponges possess exceptionally diverse associated microbial communities and play a major role in (re)cycling of dissolved organic matter (DOM) in marine ecosystems. Linking sponge-associated community structure with DOM utilization is essential to understand host–microbe interactions in the uptake, processing, and exchange of resources. We coupled, for the first time, DNA-stable isotope probing (DNA-SIP) with 16S rRNA amplicon sequencing in a sponge holobiont to identify which symbiotic bacterial taxa are metabolically active in DOM uptake. Parallel incubation experiments with the sponge Plakortis angulospiculatus were amended with equimolar quantities of unlabelled (¹²C) and labelled (¹³C) DOM. Seven bacterial amplicon sequence variants (ASVs), belonging to the phyla PAUC34f, Proteobacteria, Poribacteria, Nitrospirae, and Chloroflexi, were identified as the first active consumers of DOM. Our results support the predictions that PAUC34f, Poribacteria, and Chloroflexi are capable of organic matter degradation through heterotrophic carbon metabolism, while Nitrospirae may have a potential mixotrophic metabolism. We present a new analytical application of DNA-SIP to detect substrate incorporation into a marine holobiont with a complex associated bacterial community and provide new experimental evidence that links the identity of diverse sponge-associated bacteria to the consumption of DOM.     

Aerobic and anaerobic metabolism of the freshwater oligochaete Tubifex sp.

The freshwater oligochaete Tubifex shows several mechanisms of metabolic adaptations, enabling the worms to occupy saprobial habitats of extremely variable oxygen content. Under normoxic conditions the metabolism of the worms is mainly aerobic with a respiratory ratio of 0.7. Under hypoxic conditions, metabolism of energy sources via aerobic and anaerobic pathways is observed. During complete anoxia acetate and propionate are the main products of glycogen degradation and they are excreted in constant rates into the water. A retransfer of the worms to aerobic conditions enables them to regain aerobic metabolic state within about 60 min.In two Tubifex habitats, which we have characterized, concentrations of dissolved organic material (DOM) were low in the surface water, but high in the interstitial water from sediments. The short-chain fatty acids acetate and propionate reached concentrations up to 1 mmole/liter. Employing radioisotope techniques, we demonstrated that Tubifex can achieve an integumentary uptake of acetate and propionate from artificial tap water at naturally occurring concentrations of 5 to 1000 M. Levels of uptake (600 to 800 nmoles/g wwt.hr) and transport characteristics are very similar to those of marine invertebrates associated with detritus rich sediments. The uptake is susceptible to inhibition by structurally analogous compounds and to metabolic inhibition. Furthermore, DOM uptake in Tubifex is susceptible to inhibition by oxygen depletion, ouabain and Na⁺-depletion. The results may suggest that a carrier system for DOM transport exists in the integument of the worms. The uptake system is highly specific for aliphatic C2 and C3 carboxylic acids. The absorbed volatile fatty acids are rapidly metabolized. Only 15 min after absorption, a considerable amount of radioactivity is present in the glycogen storage of the animals. Depending on the substrate concentration assumed to be available for uptake, up to 40 per cent of the oxidative requirement of the worms may be attained by using dissolved organic material from the interstitial water of their habitat.Supported by the Deutsche Forschungsgemeinschaft (Ho 631/9-9).     

Experimentally induced foraging mode shift by sympatric charrs in a Japanese mountain stream

Foraging mode shifts may allow animals flexibility to adaptto a variety of ecological conditions. Theory holds that ectothermssuch as fishes should shift from ambush to active search modeswhen prey density declines, to maintain a minimum encounterrate. Salvelinus malma (Dolly Varden) sympatric withS. leucomaenis(white-spottedchirr) in a northern Japan mountain stream were observed toshift from ambushing drifting invertebrates at fixed focal pointsto picking benthic invertebrates from the substrate along circuitousroutes throughout pools, as drift declined throughout summer.Experimentally reducing drift in two pools during early summerwhen all charr were drift foragers caused subordinate malmato make marked shifts from drift to benthic foraging within1 h, whereas nearly all leucomaenis remained drift foragers.Some charr of both species responded to reduced drift by emigratingfrom pools. Combined results of three replicate experimentsrevealed that malma shifted to benthic foraging when their rateof drift forays was reduced below a specific threshold, therebyupholding predictions of the theory. Adaptive foraging modeshirts may promote coexistence of these congeneric charrs inreaches of Japanese mountain streams     

Photooxidation of wetland and riverine dissolved organic matter: altered copper complexation and organic composition

In natural waters, the uptake of transition metals such as copper (Cu) by aquatic biota depends on the activity of the free cupric ion ({Cu²⁺}) rather than on total Cu concentration. Thus, an important ecological function of dissolved organic matter (DOM) in aquatic ecosystems is Cu–DOM complexation, which greatly decreases the {Cu²⁺}. However, Cu bioavailability is greatly modified by source and environmental history of DOM because DOM affinity for Cu varies by orders of magnitude among DOM sources; moreover, DOM is photochemically unstable. During 72-h irradiation experiments at intensities approximating sunlight with DOM from a palustrine wetland and a third-order river, we investigated photooxidative effects on DOM complexation of Cu as well as spectral and chemical changes in DOM that might explain altered Cu complexation. Irradiation decreased Cu complexation by riverine DOM, but unexpectedly increased Cu complexation by wetland DOM, resulting in 150% greater {Cu²⁺} in riverine DOM at the same dissolved organic carbon concentrations. The specific ultraviolet absorption (SUVa) and humic substances tracked photochemical changes in the conditional stability constants of Cu–DOM complexes, suggesting that the aromaticity of DOM influences its affinity for Cu. Carbonyl concentration in ¹³C nuclear magnetic resonance spectra (¹³C-NMR) covaried directly with Cu binding-site densities in DOM. However, no aspect of Cu–DOM complexation consistently covaried with fluorophores (i.e., the fluorescence index) or low molecular weight organic acids. Our results suggest that global increases in UV radiation will affect Cu–DOM complexation and subsequent Cu toxicity depending on light regime as well as DOM source. Handling editor: K. Martens     

Evidence for the Biosynthesis of Bryostatins by the Bacterial Symbiont “Candidatus Endobugula sertula” of the Bryozoan Bugula neritina

The marine bryozoan, Bugula neritina, is the source of the bryostatins, a family of macrocyclic lactones with anticancer activity. Bryostatins have long been suspected to be bacterial products. B. neritina harbors the uncultivated gamma proteobacterial symbiont “Candidatus Endobugula sertula.” In this work several lines of evidence are presented that show that the symbiont is the most likely source of bryostatins. Bryostatins are complex polyketides similar to bacterial secondary metabolites synthesized by modular type I polyketide synthases (PKS-I). PKS-I gene fragments were cloned from DNA extracted from the B. neritina-“E. sertula” association, and then primers specific to one of these clones, KSa, were shown to amplify the KSa gene specifically and universally from total B. neritina DNA. In addition, a KSa RNA probe was shown to bind specifically to the symbiotic bacteria located in the pallial sinus of the larvae of B. neritina and not to B. neritina cells or to other bacteria. Finally, B. neritina colonies grown in the laboratory were treated with antibiotics to reduce the numbers of bacterial symbionts. Decreased symbiont levels resulted in the reduction of the KSa signal as well as the bryostatin content. These data provide evidence that the symbiont E. sertula has the genetic potential to make bryostatins and is necessary in full complement for the host bryozoan to produce normal levels of bryostatins. This study demonstrates that it may be possible to clone bryostatin genes from B. neritina directly and use these to produce bryostatins in heterologous host bacteria.     

Overview of the chemical ecology of benthic marine invertebrates along the western Antarctic peninsula

Thirteen years ago in a review that appeared in the American Zoologist, we presented the first survey of the chemical and ecological bioactivity of Antarctic shallow-water marine invertebrates. In essence, we reported that despite theoretical predictions to the contrary the incidence of chemical defenses among sessile and sluggish Antarctic marine invertebrates was widespread. Since that time we and others have significantly expanded upon the base of knowledge of Antarctic marine invertebrates' chemical ecology, both from the perspective of examining marine invertebrates in new, distinct geographic provinces, as well as broadening the evaluation of the ecological significance of secondary metabolites. Importantly, many of these studies have been framed within established theoretical constructs, particularly the Optimal Defense Theory. In the present article, we review the current knowledge of chemical ecology of benthic marine invertebrates comprising communities along the Western Antarctic Peninsula (WAP), a region of Antarctica that is both physically and biologically distinct from the rest of the continent. Our overview indicates that, similar to other regions of Antarctica, anti-predator chemical defenses are widespread among species occurring along the WAP. In some groups, such as the sponges, the incidence of chemical defenses against predation is comparable to, or even slightly higher than, that found in tropical marine systems. While there is substantial knowledge of the chemical defenses of benthic marine invertebrates against predators, much less is known about chemical anti-foulants. The sole survey conducted to date suggests that secondary metabolites in benthic sponges are likely to be important in the prevention of fouling by benthic diatoms, yet generally lack activity against marine bacteria. Our understanding of the sensory ecology of Antarctic benthic marine invertebrates, despite its great potential, remains in its infancy. For example, along the WAP, community-level non-consumptive effects occur when amphipods chemically sense fish predators and respond by seeking refuge in chemically-defended macroalgae. Such interactions may be important in releasing amphipods from predation pressure and facilitating their unusually high abundances along the WAP. Moreover, recent studies on the sensory biology of the Antarctic keystone sea star Odontaster validus indicate that chemotactile-mediated interactions between conspecifics and other sympatric predatory sea stars may have significant ramifications in structuring community dynamics. Finally, from a global environmental perspective, understanding how chemical ecology structures marine benthic communities along the WAP must increasingly be viewed in the context of the dramatic impacts of rapid climatic change now occurring in this biogeographic region.     

Adaptations by Invertebrate Larvae for Nutrient Acquisition from Seawater

Unlike life on land, animals that live in seawater are surroundedby a medium that contains organic nutrients in dilute solution.Larval forms of soft-bodied marine invertebrates are adaptedto take advantage of the fact that most of the organic carbonin their environment is in solution as dissolved organic material(DOM). New evidence for the importance of DOM to metazoans ispresented by showing that larval forms can increase in biomass,even in the absence of paniculate foods. Such increases occurredonly in those species capable of transporting DOM. The physiologicalbasis for using DOM as an energy source is dependent upon anincreased transport capacity for DOM as growth proceeds. Usingbivalve larvae, mass coefficients and exponents were determinedfor (i) alanine transport rates and (ii) metabolic rates. Thesecoefficients were not statistically different when determinedover the life span of a larva. Thus, as growth proceeds, theselarvae increase their ability to obtain a potential supply ofmetabolic fuel (DOM) in direct proportion to the increase intheir metabolic demand. The percent of this increased transportcapacity that larvae could actually utilize in nature will dependupon the substrate concentrations in their environment. Currentviews on what these concentrations are in seawater may be alteredas more attention is given to the fine scale distributions oforganic chemicals in the ocean. After DOM has been transportedby the animal, its metabolic fate can now be rigorously studiedusing bacteria-free larvae. Measurements of amino acid synthesisin larvae cultured under axenic conditions suggest that a muchgreater plasticity may exist in the biochemical requirementsof larvae for dietary amino acids.