For marine species with complex life cycles, a critical question concerns the response of larval stages to warming. In species with feeding larvae, warming and food limitation may result in negative (additive or synergetic) effects on growth and survival. We experimentally evaluated the combined effects of temperature and food limitation on larval performance of the invasive crab Hemigrapsus sanguineus (local population of Helgoland, North Sea, German Bight). We then compared the performance of H. sanguineus with the native competitor (shore crab Carcinus maenas). Within the studied temperature range (15-24 °C) and under the conditions of food limitation (access to food for 6 hours a day), the primary driver of survival was temperature (low temperatures decreased survival especially at the more advanced stages). Food limitation decreased survival and growth at all temperatures but larvae metamorphosed to megalopa at all food limited conditions. The response of H. sanguineus differed from that of C. maenas where effects low temperatures have little impact on performance but high temperatures enhance the negative effects of food limitation. Hence, these two coexisting species appear to have different larval responses to warming and food limitation, with warming leading to a higher performance of the invasive relative to the native species.

Larval performance of the exotic Hemigrapsus sanguineus under increased temperature and food limitation

Ocean acidification and warming caused by increased anthropogenic carbon dioxide emissions poses a tremendous challenge to marine calcifiers such as intertidal bivalves. The approach to studying acclimation potential or adaptation to climate change are often conducted under artificial conditions where food is provided ad libitum and do not consider tidal emersion. These limitations present problems for extrapolating results to natural ecosystems. For instance, food provisioning and regular emersion can enhance or limit acclimation capacities of marine organisms. Here we investigate the physiological response of young Pacific oyster Crassostrea gigas exposed for 3 months to ambient and future (+3°C, -0.3 pH unit) climate scenarios under two food regimes (ad libitum vs restricted) and two tidal levels (subtidal vs intertidal, emersion twice daily) under laboratory conditions. Oysters were sampled for biometric and physiological rate measurements (respiration and food intake), biochemistry (energetic reserves and enzyme assay) and histology to evaluate their reproductive status. At the end of the experiment, oysters were further challenged with a viral disease to investigate the physiological cost of acclimation to these conditions and their immune response. Preliminary analyses suggest that food level was the most influential factor, followed by climate scenario and tidal level. We indeed found that growth, food intake, reproduction and disease resistance of oysters were negatively affected by food restriction. Future conditions enhanced oyster physiological rates and sexual maturation, particularly at high food levels. Finally, tidal level had no major effect on these variables, suggesting physiological compensatory mechanisms during emersion. Our study suggests that the oyster is robust to future climatic conditions, even when feeding was restricted. A detailed analysis of the results will be presented at the conference.

Physiological and immune response of oysters to climate change as a function of diet and tidal regime

Land plants cover their epidermis with a specialised protective layer called the cuticle, a feature which is crucial for surviving the perils of a terrestrial environment, including desiccation, UV damage and pathogen attack, dangers which may worsen with our escalating climate crisis. Several cereal staples such as barley and wheat show further epidermal specialisation with a distinctive, glaucous wax bloom on reproductive stage tissues, a feature associated with improved drought tolerance. Understanding the mechanisms underpinning wax bloom development may help breed more climate resilient varieties. To date, studies on barley eceriferum mutants have identified several enzymes involved in cuticular wax biosynthesis. However, little is known about the upstream regulation of this process. My research addresses this knowledge gap by exploiting genetic resources to identify a key regulatory player in barley cuticle development. Here, I will discuss how I used high density genotyping, comparative gene expression and wax compositional analyses to identify a gene essential for wax bloom formation and the expression of cuticular metabolic genes in barley.

Identification of a key regulator controlling cuticular wax in barley

Hydrogen peroxide (H2O2) is widely used as a signalling molecule. It is critical to measure H2O2 with a high spatial and temporal resolution to better understand its role as a messenger. The most frequent approach to detect H2O2 has been using chemical probes, which have the disadvantage of limited specificity and cannot report any dynamics or spatial information. Genetically encoded probes have made significant advances to overcome these limitations. Probes of the HyPer family consist of a circularly permuted yellow fluorescent protein (cpYFP) core and a sensing domain from a bacterial H2O2-activated transcription factor (OxyR). These, however, have a pronounced pH sensitivity. Other H2O2 sensors, such as roGFP2-Orp1, have shown to be pH-insensitive but lack the responsiveness to detect low nanomolar H2O2 changes, possibly due to the strong reducing effect of the glutathione redox potential (EGSH). A new ultrasensitive HyPer7 fuses the OxyR of N. meningitidis to an optimized cpYFP largely pH-independent. Here, we have generated and characterized stable Arabidopsis thaliana plants expressing a cytosolic version of HyPer7 and prove that the reduction of this sensor is mostly EGSH-independent and highly responsive to externally imposed oxidation via H2O2 feeding. Furthermore, HyPer7 could sense stress-induced H2O2 in live-cell plants exposed to herbicides, such as methyl viologen or the flagellin-induced apoplastic ROS burst, much better than the previously mentioned H2O2 probes. HyPer7 sensitivity also allowed us to see that the laser light used for excitation is sufficient to induce H2O2 release from chloroplasts in amounts only detected by this ultrasensitive sensor.

The latest HyPe(r) in plant H2O2 biosensing

We study the potential of a recent invader, the Asian brush-clawed crab (Hemigrapsus takanoi), to expand its distribution range further into the Baltic Sea. H. takanoi has been documented in the southwestern Baltic Sea since 2014. The ability to persist and further expand into the Baltic Sea will depend on their potential to sustain all stages of their complex life cycle, including pelagic larvae, under the Baltic Sea’s conditions. Range limits may be established by the tolerance to low salinity, which in addition may be affected by temperature. A key question is whether local populations at the distribution limit (within the Baltic) are adapting to low salinities and hence promote further expansion. We quantified the combined effects of salinity (10-32 PSU) and temperature (15-24°C) on four populations of H. takanoi (two from the Baltic and two from the North Sea). Our results show substantial differences in larval performance between the populations from the Baltic and North Sea. Larvae from the North Sea populations show higher survival and faster development compared with those from the Baltic Sea. No evidence of elevated tolerance towards low salinity were found in the larvae from the Baltic Sea. In addition, larvae from the population located at the range limit (Neustadt) show very low survival. We did not find evidence of adaptation to low salinity by any of the local populations of H. takanoi we studied. Thus, in order to the population to persist, larvae must either migrate to waters of increased salinity, and then return to the habitat of origin; or such populations are sustained by source-sink dynamics. 


Living at the frontier - salinity and temperature during larval development determine the potential for invasion of the Baltic Sea

Energy metabolism (encompassing energy assimilation, conversion and utilization) plays a central role in all life processes and serves as a link between the organismal physiology, behavior, and ecology. Metabolic rates define the physiological and life-history performance of an organism and have direct implications for Darwinian fitness. Anthropogenic and natural stressors affect energy balance because the stress-induced disruption of homeostasis must be corrected to ensure the organism's survival. Pollutants can negatively affect different aspects of energy metabolism of an organism interfering with energy assimilation and conversion or increasing energy costs for basal maintenance. This can diminish the energy fluxes available for other fitness functions and negatively affect the organism’s growth, reproduction and ability to deal with additional stressors or diseases. In this talk, I will discuss the role of bioenergetics in toxicity of legacy and emerging pollutants (including trace metals, nanoparticles and pharmaceuticals) and the pollutant interactions with other abiotic stressors such as temperature, salinity or oxygen deficiency. I will address the mechanisms underlying the pollutant-induced bioenergetics disturbances and discuss the use of bioenergetics-based approaches in linking the molecular and cellular stress responses to the whole-organism fitness in coastal environments exposed to multiple stressors.

Bioenergetic aspects of pollutant toxicity and their role in determining the tolerance limits to multiple stressors in aquatic organisms

The evolution of lignification in vascular plants conferred improved structural support, defence, and water transport, improving the success of plants on land. Despite lignification being a land plant specific expansion, lignin-like compounds have been identified in the cell walls of intertidal seaweeds. Here, I aim to characterise the function and structure of the ‘phycolignin’ fraction of the chlorophyte seaweed Ulva compressa. UV excitation of Ulva cell wall autofluorescence using confocal microscopy induced a blue-green emission profile comparable to that of lignin, which was significantly elevated under hyposaline stress (1.7X fold change to control, ***) and significantly reduced under hypersaline stress (0.5X fold change, ***), indicating a possible osmoprotective role. Cell walls were then isolated and fractionated into pectin, hemicellulose, and ⍺-cellulose to identify this fluorophore, with plant biomass also fractionated as a ‘lignin positive’ control. Several biochemical tests were performed to identify which polysaccharides lignin-like structures were associated with, followed by ATR-FTIR and NMR. ‘Phycolignin’ in Ulva appeared to be associated with cellulose and hemicellulose, with potential aromatic groups identified by ATR-FTIR and 1H NMR (solution-state). Positive identification of lignin within plant extracts showed that Ulva ‘phycolignin’ displayed fundamental structural differences to true lignin. However, the lack of available reference spectra for FTIR of chlorophyte phenolics, and the general insolubility of the Ulva cell wall in conventional NMR solvents limited data interpretation. Better structural characterisation of ‘phycolignin’ in Ulva will increase our understanding on how multicellularity, the metabolome, and stress responses evolved and diverge within the green lineage.

Characterisation of 'phycolignin' in the cell wall of chlorophyte seaweeds

Nonpollinating fig wasps (Hymenoptera: Chalcidoidea) are the most remarkable exploiters of the Ficus (Moraceae) and pollinating fig wasp (Hymenoptera: Agaonidae) mutualism. Both pollinating and nonpollinating fig wasps lay their eggs inside the inflorescences of the fig trees (Ficus), where their larvae grow. The nonpollinating fig wasps (NPFWs) negatively impact the pollinator population and fig seed production because, for each NPFW hatch in a fig, a pollinator or a seed has been terminated. Moreover, NPFWs can kill all the pollinating males in a fig and doom all the wasps in a single fig, as pollinating males dig the exit hole out of a fig. Up to now, only experimental studies based on the absence/presence of NPFWs have been done. To better quantify the impact of NPFWs, we conducted bagging experiments using a controlled number of fig wasps. Pollinating wasps were introduced in bags containing receptive figs and different numbers of NPFWs (from none to five). For this experiment, we used the pollinating wasp, Ceratosolen wui, and the NPFW species, Philotrypesis taida living on the fig tree Ficus benguetensis in Northern Taiwan. We found that higher numbers of NPFWs increased the fig abortion rate before maturation and decreased the number of emerging wasps and the proportion of pollinators. In conclusion, we suggest that selective abortion in the early stage of fig development is a density-dependent defense mechanism against NPFWs.

Fig abortion as a defense mechanism for Ficus trees and pollinating fig wasps

Evolution as a contemporary process that can affect changes from one generation to the next has only recently been considered in toxicology. While rapid evolution has recently been linked with the increase of invasive capacity of Invasive Alien Species (IAS), it is still not taken into consideration for Invasive Species Predictive Schemes and risk assessments. 
The Louisiana crayfish, Procambarus clarkii, has a strong adaptive capacity to a variety of aquatic environments, including highly contaminated water bodies, and different salinities. Here, we studied three invasive populations of P. clarkii that inhabit water bodies at different salinities and pollution levels in the South of France. Our results showed that physiological and molecular responses are population-specific when they are exposed to pollutants and salinity shock. Our results suggest that in P. clarkii, the capacity to inhabit polluted environments and the capacity to inhabit water bodies with different salinity levels are not related. While the capacity to face pollutants seems to share a common ancestor in these three populations, the capacity of dealing with salinity change seems to be a more recently acquired trial.

Physiological and molecular responses of P. clarkii to different levels of pollutants and salinity exposure: an Interpopulation study.

Reproductive success (and therefore selection) is ultimately related to how well an organism performs in the context of its environment. Performance reflects the interactive trade-off between physiological traits, behaviour, resource availability and mortality. Time-integrated field metabolic rate (FMR) captures the realised energetic cost of performing in a natural setting, accounting for multi-generational acclimation to the environmental and ecological context. In-situ measurements of FMR may be needed to fully appreciate the evolutionary significance of variation in metabolic traits. 

Here we draw on data compiled using a new emerging proxy capable of providing estimates of individual-level FMR together with experienced temperature and growth rate derived from the otoliths of marine fishes. We quantify the extent and nature of among- and within-species variation in FMR observed so far in marine fishes. Among other patterns we show that in most observed populations operating within their natural thermal range, realised FMR is relatively insensitive to temperature, with no clear relationship between individual-level variation in FMR and growth rate. However, temperature has a stronger effect on realised FMR during early juvenile life stages (including in the same individuals), implying that in fishes, thermal effects on selection may be stronger during early juvenile life history stages. 








Variation in field metabolic rates of wild marine fishes: thermal, ontogenetic, phylogenetic and macroecological drivers.

Fluid mechanics is an important tool for understanding the biology and ecology of marine life. A major challenge in studying how microscopic aquatic organisms function in their natural habitats is integrating the different scales at which critical physical and biological processes occur. How can we make large-scale field measurements and models that include the behaviors and physical features of real organisms? Conversely, how can we design small-scale experiments to measure those biological factors under hydrodynamic conditions that reflect what the organisms actually experience in the large- scale ocean? I will discuss examples of some of the approaches we have used to span different scales in our studies of how the interaction between the locomotion of microscopic organisms and the turbulent, wave-driven water flow around them determines how they move through the environment. We studied how the microscopic larvae of bottom-dwelling marine animals navigate to suitable habitats on the sea floor by combining field flow measurements in marine environments with flume studies, experiments in fluidic devices, experiments with dynamically-scaled physical models, and agent-based models of different locomotory strategies in measured turbulent flow fields. 

Locomoting in a turbulent environment: Ways to study microscale processes in a large-scale ocean

Thermal acclimation of some temperate fish species can stimulate changes in the physiological capacity of the heart. This is due to modifications to the active and passive properties of the myocardium. These modifications include changes in the Ca2+ sensitivity of the contractile element and to the amount and composition of myocardial collagen. Interestingly, this response is sex specific. Using cultured cardiac fibroblasts, from male and female trout, we have been working to determine how a change in physiological temperature leads to a change in deposited collagen. This work has integrated experiments examining the effect of biomechanical stretch, TGF-B, testosterone, angiotensin II, and microRNAs on the cell signalling pathways that regulate collagen deposition and turnover. We propose that increased biomechanical stimulation of the heart, caused by an increase in vascular resistance and blood viscosity, triggered by a decrease in physiological temperature, activates mitogen-activated protein kinase signalling pathways through stimulation of mechanosensitive membrane proteins and the release of TGF-B. The end result is changes in the expression of gene transcripts and cellular proteins that support an increase in collagen deposition including matrix metalloproteinases, and collagen monomers. It was also found that cellular pathways associated with the deposition of collagen are stimulated by physiologically relevant levels of testosterone, and that these can be reversed through the expression of microRNA-29b. These studies suggest that the phenotypic response of the heart to a change in environmental temperature is under complex regulatory control that is sex specific.  

Underline digital video library

Larval performance of the exotic Hemigrapsus sanguineus under increased temperature and food limitation

Physiological and immune response of oysters to climate change as a function of diet and tidal regime

Identification of a key regulator controlling cuticular wax in barley

The latest HyPe(r) in plant H2O2 biosensing

Living at the frontier - salinity and temperature during larval development determine the potential for invasion of the Baltic Sea

Bioenergetic aspects of pollutant toxicity and their role in determining the tolerance limits to multiple stressors in aquatic organisms

Characterisation of 'phycolignin' in the cell wall of chlorophyte seaweeds

Fig abortion as a defense mechanism for Ficus trees and pollinating fig wasps

Physiological and molecular responses of P. clarkii to different levels of pollutants and salinity exposure: an Interpopulation study.

Variation in field metabolic rates of wild marine fishes: thermal, ontogenetic, phylogenetic and macroecological drivers.

Locomoting in a turbulent environment: Ways to study microscale processes in a large-scale ocean

Mechanotransduction and the regulation of collagen deposition by trout cardiac fibroblasts during thermal acclimation

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