Nonlinear laser scanning microscopy of oral multispecies-biofilms: Fixative induced fluorescence as a fast and economical in vitro screening method

In this letter we report a fast and easy method which could be used for initial screening of multispecies-biofilm development on putative new dental implant materials. Most staining methods require numerous washing steps that can result in detachment of loosely bound biofilms and therefore falsify the results. Thus, we used glutaraldehyde fixation, which induces autofluorescence through bacterial membrane protein cross-linking and concurrently stabilizes the biofilm structure. We analyzed the biofilms with nonlinear laser scanning microscopy and were able to (I) evaluate the multispecies-biofilm growth and (II) distinguish between bacterial species based on different two-photon autofluorescence intensities. © 2016 by De Gruyter

CO2 induced seawater acidification impacts sea urchin larval development I: Elevated metabolic rates decrease scope for growth and induce developmental delay

Anthropogenic CO(2) emissions are acidifying the world's oceans. A growing body of evidence is showing that ocean acidification impacts growth and developmental rates of marine invertebrates. Here we test the impact of elevated seawater pCO(2) (129Pa, 1271 atm) on early development, larval metabolic and feeding rates in a marine model organism, the sea urchin Strongylocentrotus purpuratus. Growth and development was assessed by measuring total body length, body rod length, postoral rod length and posterolateral rod length. Comparing these parameters between treatments suggests that larvae suffer from a developmental delay (by ca. 8%) rather than from the previously postulated reductions in size at comparable developmental stages. Further, we found maximum increases in respiration rates of +100% under elevated pCO(2), while body length corrected feeding rates did not differ between larvae from both treatments. Calculating scope for growth illustrates that larvae raised under high pCO(2) spent an average of 39 to 45% of the available energy for somatic growth, while control larvae could allocate between 78 and 80% of the available energy into growth processes. Our results highlight the importance of defining a standard frame of reference when comparing a given parameter between treatments, as observed differences can be easily due to comparison of different larval ages with their specific set of biological characters

Energy metabolism and regeneration impaired by seawater acidification in the infaunal brittlestar, Amphiura filiformis

Seawater acidification due to anthropogenic release of CO2 as well as the potential leakage of pure CO2 from sub-seabed carbon capture storage sites (CCS) may impose a serious threat to marine organisms. Although infaunal organisms can be expected to be particularly impacted by decreases in seawater pH, due to naturally acidified conditions in benthic habitats, information regarding physiological and behavioral responses is still scarce. Determination of pO2 and pCO2 gradients within the burrows of the brittlestar Amphiura filiformis during environmental hypercapnia demonstrated that besides hypoxic conditions, increases of environmental pCO2 are additive to the already high pCO2 (up to 0.08 kPa) within the burrows. In response to up to 4 weeks exposure to pH 7.3 (0.3 kPa pCO2) and pH 7.0 (0.6 kPa pCO2), metabolic rates of A.filiformis were significantly reduced in pH 7.0 treatments accompanied by increased ammonium excretion rates. Gene expression analyses demonstrated significant reductions of acid-base (NBCe and AQP9) and metabolic (G6PDH, LDH) genes. Determination of extracellular acid-base status indicated an uncompensated acidosis in CO2 treated animals, which could explain depressed metabolic rates. Metabolic depression is associated with a retraction of filter feeding arms into sediment burrows. Regeneration of lost arm tissues following traumatic amputation is associated with significant increases in metabolic rate, and hypercapnic conditions (pH 7.0, 0.6 KPa) dramatically reduce the metabolic scope for regeneration reflected in 80% reductions in regeneration rate. Thus, the present work demonstrates that elevated seawater pCO2 significantly affects the environment and the physiology of infaunal organisms like A. filiformis

Infrared neurostimulation in ex-vivo rat sciatic nerve using 1470 nm wavelength.

OBJECTIVE: To design and implement a setup for ex-vivo optical stimulation for exploring the effect of several key parameters (optical power and pulse duration), activation features (threshold, spatial selectivity) and recovery characteristics (repeated stimuli) in peripheral nerves. APPROACH: A nerve chamber allowing ex-vivo electrical and optical stimulation was designed and built. A 1470 nm light source was chosen to stimulate the nerve. A photodiode module was implemented for synchronization of the electrical and optical channels. MAIN RESULTS: Compound Neural Action Potentials (CNAPs) were successfully generated with infrared light pulses of 200-2000 µs duration and power in the range of 3-10 W. These parameters determine a radiant exposure for stimulation in the range 1.59-4.78 J/cm2. Recruitment curves were obtained by increasing durations at a constant power level. Neural activation threshold is reached at a mean radiant exposure of 3.16 ± 0.68 J/cm2 and mean pulse energy of 3.79 ± 0.72 mJ. Repetition rates of 2-10 Hz have been explored. In 8 out of 10 sciatic nerves, repeated light stimuli induced a sensitisation effect in that the CNAP amplitude progressively grows, representing an increasing number of recruited fibres. In 2 out of 10 sciatic nerves, CNAPs were composed of a succession of peaks corresponding to different conduction velocities. SIGNIFICANCE: The reported sensitisation effect could shed light on the mechanism underlying Infrared NeuroStimulation (INS). Our results suggest that, in sharp contrast with electrical stimuli, optical pulses could recruit slow fibres early on. This more physiological order of recruitment opens the perspective for specific neuromodulation of fibre population who remained poorly accessible until now. Short high-power light pulses at wavelengths below 1.5 µm offer interesting perspectives for neurostimulation

Analysing vagus nerve spontaneous activity using finite element modelling

Objective. Finite element modelling has been widely used to understand the effect of stimulation on the nerve fibres. Yet the literature on analysis of spontaneous nerve activity is much scarcer. In this study, we introduce a method based on a finite element model, to analyse spontaneous nerve activity with a typical bipolar electrode recording setup, enabling the identification of spontaneously active fibres. We applied our method to the vagus nerve, which plays a key role in refractory epilepsy. Approach. We developed a 3D model including dynamic action potential propagation, based on the vagus nerve geometry. The impact of key recording parameters – inter-electrode distance and temperature – and uncontrolled parameters – fibre size and position in the nerve – on the ability to discriminate active fibres were quantified. A specific algorithm was implemented to detect and classify action potentials from recordings and tested on six rats in vivo vagus nerve recordings. Main results. Fibre diameters can be discriminated if they are below 3 µm and 7 µm, respectively for inter-electrode distances of 2 mm and 4 mm. The impact of the position of the fibre inside the nerve on fibre diameter discrimination, is limited. The range of active fibres identified by modelling in the vagus nerve of rats is in agreement with ranges found at histology. Significance. The nerve fibre diameter, directly proportional to the action potential propagation velocity, is related to a specific physiological function. Estimating the source fibre diameter is thus essential to interpret neural recordings. Among many possible applications, the present method was developed in the context of a project to improve vagus nerve stimulation therapy for epilepsy

Long-term and trans-life-cycle effects of exposure to ocean acidification in the green sea urchin Strongylocentrotus droebachiensis

Anthropogenic CO2 emissions are acidifying the world’s oceans. A growing body of evidence demonstrates that ocean acidification can impact survival, growth, development and physiology of marine invertebrates. Here, we tested the impact of long-term (up to 16 months) and trans-life-cycle (adult, embryo/larvae and juvenile) exposure to elevated pCO2 (1,200 μatm, compared to control 400 μatm) on the green sea urchin Strongylocentrotus droebachiensis. Female fecundity was decreased 4.5-fold when acclimated to elevated pCO2 for 4 months during reproductive conditioning, while no difference was observed in females acclimated for 16 months. Moreover, adult pre-exposure for 4 months to elevated pCO2 had a direct negative impact on subsequent larval settlement success. Five to nine times fewer offspring reached the juvenile stage in cultures using gametes collected from adults previously acclimated to high pCO2 for 4 months. However, no difference in larval survival was observed when adults were pre-exposed for 16 months to elevated pCO2. pCO2 had no direct negative impact on juvenile survival except when both larvae and juveniles were raised in elevated pCO2. These negative effects on settlement success and juvenile survival can be attributed to carry-over effects from adults to larvae and from larvae to juveniles. Our results support the contention that adult sea urchins can acclimate to moderately elevated pCO2 in a matter of a few months and that carry-over effects can exacerbate the negative impact of ocean acidification on larvae and juveniles

Fine sediment reduces vertical migrations of Gammarus pulex (Crustacea: Amphipoda) in response to surface water loss

Surface and subsurface sediments in river ecosystems are recognized as refuges that may promote invertebrate survival during disturbances such as floods and streambed drying. Refuge use is spatiotemporally variable, with environmental factors including substrate composition, in particular the proportion of fine sediment (FS), affecting the ability of organisms to move through interstitial spaces. We conducted a laboratory experiment to examine the effects of FS on the movement of Gammarus pulex Linnaeus (Crustacea: Amphipoda) into subsurface sediments in response to surface water loss. We hypothesized that increasing volumes of FS would impede and ultimately prevent individuals from migrating into the sediments. To test this hypothesis, the proportion of FS (1–2 mm diameter) present within an open gravel matrix (4–16 mm diameter) was varied from 10 to 20% by volume in 2.5% increments. Under control conditions (0% FS), 93% of individuals moved into subsurface sediments as the water level was reduced. The proportion of individuals moving into the subsurface decreased to 74% at 10% FS, and at 20% FS no individuals entered the sediments, supporting our hypothesis. These results demonstrate the importance of reducing FS inputs into river ecosystems and restoring FS-clogged riverbeds, to promote refuge use during increasingly common instream disturbances

Effects of oil and global environmental drivers on two keystone marine invertebrates

Ocean warming (OW) and acidification (OA) are key features of global change and are predicted to have negative consequences for marine species and ecosystems. At a smaller scale increasing oil and gas activities at northern high latitudes could lead to greater risk of petroleum pollution, potentially exacerbating the effects of such global stressors. However, knowledge of combined effects is limited. This study employed a scenario-based, collapsed design to investigate the impact of one local acute stressor (North Sea crude oil) and two chronic global drivers (pH for OA and temperature for OW), alone or in combination on aspects of the biology of larval stages of two key invertebrates: the northern shrimp (Pandalus borealis) and the green sea urchin (Strongylocentrotus droebachiensis). Both local and global drivers had negative effects on survival, development and growth of the larval stages. These effects were species- and stage-dependent. No statistical interactions were observed between local and global drivers and the combined effects of the two drivers were approximately equal to the sum of their separate effects. This study highlights the importance of adjusting regulation associated with oil spill prevention to maximize the resilience of marine organisms to predicted future global conditions

Direct observations of the effect of fine sediment deposition on the vertical movement of Gammarus pulex (Amphipoda: Gammaridae) during substratum drying

Benthic macroinvertebrates inhabit the streambed sediments of temporary streams during drying events. Fine sediment (< 2 mm in diameter) deposition and clogging of interstitial pathways reduces the connectivity between benthic and subsurface habitats, potentially inhibiting macroinvertebrate vertical movements. Direct observations within subsurface sediments are, however, inherently difficult. As a result, confirmation of macroinvertebrate vertical movement, and the effect of fine sediment, is limited. We used laboratory mesocosms containing transparent gravel sized particles (10–15 mm) to facilitate the direct observation and tracking of vertical movements by Gammarus pulex in response to water level reduction and sedimentation. Seven sediment treatments comprised two fine sediment fractions (small: 0.125–0.5 mm, coarse sand: 0.5–1 mm) deposited onto the surface of the substrate, and a control treatment where no fine sediment was applied. We found that G. pulex moved into the subsurface gravel sediments in response to drying, but their ability to remain submerged during water level reduction was impeded by fine sediment deposition. In particular deposition of the coarser sand fraction clogged the sediment surface, limiting vertical movements. Our results highlight the potential effect of sedimentation on G. pulex resistance to drying events in streams