Bruno Jesus

Monitoring Migration and Measuring Biomass in Benthic Biofilms: The Effects of Dark/far-red Adaptation and Vertical Migration on Fluorescence Measurements.

Pulse modulated fluorescence has increasingly been used as an ecological tool to examine changes in the vertical distribution of microphytobenthic cells within the upper layers of estuarine sediments (most often using the minimum fluorescence yield Fo) as well as to indicate the health of the community (using the maximum PS II quantum efficiency Fv/Fm). However, the practicalities of in situ measurements, often dictates that short dark adaptation periods must be used (∼15 min). The use of far-red light as an alternative to dark adaptation was investigated in natural migratory microphytobenthic biofilms and artificial non-migratory biofilms. Prolonged periods of darkness (∼24 h) were not adequate to achieve ‘true’ measurements of Fo and Fv/Fm, which require complete oxidation of QA and full reversal of non-photochemical quenching (NPQ). In some instances, stable values were only achieved using far-red light. Prolonged exposure to dark/far-red light led to a downwards migration of cells in natural assemblages, as seen by a reduction in both Fo and the maximum fluorescence yield (Fm). In non-migratory biofilms, Fm increased in the dark and far-red treatments, indicating a reversal of NPQ, whereas Fo decreased in far-red light but increased in the dark. It is suggested that far-red light and darkness differentially affected the balance between NPQ reversal and QA oxidation that lead to the measured Fo yield. The use of far-red light as an alternative to dark adaptation is discussed and the implications of short (e.g., 15 min) dark adaptation times used in situ are discussed with reference to the vertical migration of cells within sediment biofilms.

Telomerase reverse transcriptase synergizes with calorie restriction to increase health span and extend mouse longevity

Caloric restriction (CR), a reduction of food intake while avoiding malnutrition, can delay the onset of cancer and age-related diseases in several species, including mice. In addition, depending of the genetic background, CR can also increase or decrease mouse longevity. This has highlighted the importance of identifying the molecular pathways that interplay with CR in modulating longevity. Significant lifespan extension in mice has been recently achieved through over-expression of the catalytic subunit of mouse telomerase (mTERT) in a cancer protective background. Given the CR cancer-protective effects in rodents, we set to address here whether CR impacts on telomere length and synergizes with mTERT to extend mouse longevity. CR significantly decreased tumor incidence in TERT transgenic (TgTERT) mice and extended their lifespan compared to wild-type (WT) controls under the same diet, indicating a synergy between TgTERT and CR in increasing mouse longevity. In addition, longitudinal telomere length measurements in peripheral blood leukocytes from individual mice showed that CR resulted in maintenance and/or elongation telomeres in a percentage of WT mice, a situation that mimics telomere dynamics in TgTERT cohorts. These results demonstrate that CR attenuates telomere erosion associated to aging and that synergizes with TERT over-expression in increasing “health span” and extending mouse longevity.

The application of variable chlorophyll fluorescence to microphytobenthic biofilms

Community assemblages of diatoms, green algae and cyanobacteria comprise the microphytobenthos (MPB), which inhabit benthic sediment ecosystems (Admiraal 1984; Underwood and Kromkamp 1999; Consalvey et al. 2004). Particular attention has been paid to the analysis of intertidal soft sediment systems, e.g. cohesive mudflat and sandy substrata typical of estuarine habitats. Variable chlorophyll fluorescence has been applied to these systems since the 1990s, in an attempt to investigate the primary productivity and photophysiology of the integrated biofilms, when viewed as a “black box system”, and also at the species level (Sections 5, 6 and 7). These transient (i.e. temporary) biofilms are not confined to such soft sediment habitats however, and more recently application of fluorescence methodologies has been applied to biofilms inhabiting rocky shores and stromatolite systems (Kromkamp et al. 2007; Perkins et al. 2007). However the large majority of published work has centred upon benthic soft-sediment biofilms, due to their important ecosystem functions of carbon flow and sediment stability (Underwood and Kromkamp 1999). In the former their high magnitude of productivity fuels carbon flow through invertebrate and bacterial food webs to support important trophic levels of anthropogenically exploited taxa, including coastal fish and shell fisheries and coastal avifauna. In the case of sediment stability, biogenic exopolymers, usually referred to as extracellular polymeric substances (EPS), produced by the MPB in part to facilitate mobility, may contribute significantly to sediment stability, hence increasing the sediment resistance to hydrodynamic stresses and thus resistance to coastal erosion (e.g. Underwood and Kromkamp 1999 and citations there-in). Finally, the photosynthetic production of oxygen can be regarded as an important ecosystem function.

Pheophorbide and pheophytin a-like pigments as useful markers for intertidal microphytobenthos grazing by Hydrobia ulvae

Pigment degradation by Hydrobia ulvae, feeding on an intertidal mudflat dominated by diatoms, was studied in the laboratory. HPLC analysis identified significantly higher amounts of pheopigments in grazed sediment and a decrease in the concentrations of major chlorophylls (a and c1+c2) and carotenoides (fucoxanthin, diadinoxanthin, and β-carotene). Grazing by H. ulvae leads to a stronger pheophorbide a accumulation relative to pheophytin a. Dominant chlorophyll a degradation products were the less polar pheophorbide a4 and pheophytin a2. These pigments were identified in very high concentrations in H. ulvae faecal pellets. Although a major disadvantage of the HPLC method is the uncertainty regarding the extent and rate of pigment conversion into colourless products, these data strongly suggest the possible use of pheophorbide a4 and pheophytin a2 as useful markers for intertidal microphytobenthos grazing by H. ulvae.

A metabolic signature predicts biological age in mice

Our understanding of the mechanisms by which aging is produced is still very limited. Here, we have determined the sera metabolite profile of 117 wild‐type mice of different genetic backgrounds ranging from 8 to 129 weeks of age. This has allowed us to define a robust metabolomic signature and a derived metabolomic score that reliably/accurately predicts the age of wild‐type mice. In the case of telomerase‐deficient mice, which have a shortened lifespan, their metabolomic score predicts older ages than expected. Conversely, in the case of mice that overexpress telomerase, their metabolic score corresponded to younger ages than expected. Importantly, telomerase reactivation late in life by using a TERT‐based gene therapy recently described by us significantly reverted the metabolic profile of old mice to that of younger mice, further confirming an anti‐aging role for telomerase. Thus, the metabolomic signature associated with natural mouse aging accurately predicts aging produced by telomere shortening, suggesting that natural mouse aging is in part produced by presence of short telomeres. These results indicate that the metabolomic signature is associated with the biological age rather than with the chronological age. This constitutes one of the first aging‐associated metabolomic signatures in a mammalian organism.

Photoacclimation in microphytobenthos and the role of xanthophyll pigments

Estuarine microphytobenthos are frequently exposed to excessively high irradiances. Photoinhibition in microalgae is prevented by various photophysiological responses. We describe here the role of the xanthophyll pigments in photoacclimation. The pigment composition of the microphytobenthos was studied in three European estuaries (Barrow, Ireland; Eden, UK; Tagus, Portugal). Using HPLC-analyses, microscale changes in biomass and pigment composition were monitored over short (hourly) and long (seasonal) time scales. In the Barrow estuary, the biomass of microphytobenthos (measured as chlorophyll a) increased significantly in the top 400–500 µm of the sediment surface within 1 h of emersion; simultaneously, the xanthophyll pool size (diadinoxanthin plus diatoxanthin, dd + dt) almost doubled. A more gradual conversion of dd into dt was observed, with the dt:dd ratio increasing from <0.1 at the start of emersion to >0.3 after 3 h emersion. Similar trends in the dt:dd ratio were observed in the surface sediments of the Eden and the Tagus estuaries. Higher ratios were recorded in the Tagus estuary, which may be explained by higher incident irradiance. In addition, seasonal studies carried out in the Eden and Tagus estuaries showed that the xanthophyll pool size increased by 10% in the summer months. The pool size was highest in the Tagus estuary. Concurrently, high values for the de-epoxidation state were recorded, with values for dt/(dt + dd) > 0.35 recorded in the summer. At the Eden, the ratio never exceeded 0.3. The de-epoxidation state was higher in winter than in summer, which was ascribed to the low winter temperatures. During a vertical migration study, a negative relationship between chlorophyll a and the de-epoxidation state was observed. It is suggested that this relationship originates from ‘micro-migration’ within the biofilm. Migration within the euphotic zone may provide an alternative means for cells to escape photodamage. In this paper, we propose that both xanthophyll cycling and ‘micro’-migration play an important role in photoacclimation and it appears that these processes operate in parallel to regulate the photosynthetic response.

Structure and diversity of intertidal benthic diatom assemblages in contrasting shores: a case study from the Tagus estuary1

The structure of intertidal benthic diatoms assemblages in the Tagus estuary was investigated during a 2‐year survey, carried out in six stations with different sediment texture. Nonparametric multivariate analyses were used to characterize spatial and temporal patterns of the assemblages and to link them to the measured environmental variables. In addition, diversity and other features related to community physiognomy, such as size‐class or life‐form distributions, were used to describe the diatom assemblages. A total of 183 diatom taxa were identified during cell counts and their biovolume was determined. Differences between stations (analysis of similarity (ANOSIM), R = 0.932) were more evident than temporal patterns (R = 0.308) and mud content alone was the environmental variable most correlated to the biotic data (BEST, ρ = 0.863). Mudflat stations were typically colonized by low diversity diatom assemblages (H′ ~ 1.9), mainly composed of medium‐sized motile epipelic species (250–1,000 μm3), that showed species‐specific seasonal blooms (e.g., Navicula gregaria Donkin). Sandy stations had more complex and diverse diatom assemblages (H′ ~ 3.2). They were mostly composed by a large set of minute epipsammic species (<250 μm3) that, generally, did not show temporal patterns. The structure of intertidal diatom assemblages was largely defined by the interplay between epipelon and epipsammon, and its diversity was explained within the framework of the Intermediate Disturbance Hypothesis. However, the spatial distribution of epipelic and epipsammic life‐forms showed that the definition of both functional groups should not be over‐simplified.

Tools providing new insight into coastal anoxygenic purple bacterial mats: review and perspectives

Coastal photosynthetic microbial mats are highly structured microbial communities that populate a variety of shallow environments such as estuaries, sheltered sandy beaches, intertidal flats, salt marshes and hypersaline salterns. In soft sediments, most of these microbial mats are formed of vertically stratified, multicolored cohesive thin layers, of several functional groups of microorganisms, such as cyanobacteria, colorless sulfur bacteria, purple sulfur bacteria and sulfate-reducing bacteria, distributed along vertical microgradients of oxygen, sulfide and light. These microbial communities are highly productive and significant contributors to carbon, nitrogen and sulfur cycles and to sediment stability in shallow-water habitats. Many examples of these communities have been cited in the past, but comparatively few microbial mats have been presented for which mass developments of anoxygenic purple bacteria have been observed. Yet, application of molecular approaches has provided fresh insight into the ecology, diversity and evolution of microbial mats. In situ measurements using electrochemical and optical microprobes led to detailed characterization of their physical and chemical environment, whereas reflectance measurements revealed the spatial and temporal heterogeneity of microbial mat surfaces. We hereby report the main discoveries due to introduction of these powerful techniques and we point out the potential insight to be gained from the study of anoxygenic purple bacterial mats.

DETECTION OF DIATOM XANTHOPHYLL CYCLE USING SPECTRAL REFLECTANCE1

Analysis of reflectance spectra was used to monitor the conversion of diadinoxanthin (DD) into diatoxanthin (DT) in two benthic diatom species, Amphora coffeaeformis (C. Agardh) Kütz. and Cylindrotheca closterium (Ehrenb.) J. C. Lewin et Reiman, cultured at high light (HL, 400 μmol · m−2 · s−1 PAR) and low light (LL, 25 μmol · m−2 · s−1 PAR). Cultures were exposed to saturating light for 32 min. HL cultures of both species showed higher (DT + DD) content, whereas LL cultures exhibited higher chl a and fucoxanthin content. DD to DT conversion, measured by HPLC, occurred mainly in the first 2 min (LL) or 5 min (HL) after exposure to saturating light. Nonphotochemical quenching (NPQ), measured by PAM fluorescence, showed the same pattern as DT/(DD + DT), resulting in a linear relationship between these parameters. Addition of dithiothreitol (DTT) blocked the conversion of DD into DT and significantly reduced NPQ induction. Reflectance spectra showed no obvious change after light exposure. However, second derivative spectra (δδ) showed a shift in reflectance from 487 to 508 nm, which was not present for DTT‐treated samples. Changes in δδ487 were strongly correlated with changes in DD (r = 0.76), while changes in δδ508 were strongly correlated with changes in DT (r = 0.94). The best index to estimate DD to DT conversion was δδ508/δδ630 (r = 0.87). This index was very sensitive to minute changes that occurred immediately after exposure to light and was species insensitive. Good relationships were observed between indices for xanthophyll cycle activation (DD to DT conversion and NPQ induction) and the second derivative spectra. With further in situ validation, this index may prove to be highly useful for investigation into aquatic global photoregulation mechanisms in diatom‐dominated samples.

Field spectroscopy of estuarine intertidal habitats

The recent introduction of portable, low‐cost hyperspectral radiometers for measuring the reflectance of marine intertidal habitats has considerable promise, first as a source of reference spectra for airborne and satellite remote sensing, and second as a survey technique in its own right. This paper reports on the results of an intercalibration exercise in which two designs of commercially available instruments were used to measure reflectance spectra across a diverse, intertidal site. The substrates sampled included seagrasses, macroalgae, and sediments which varied in wetness and grain size. Spectra were compared statistically using several measures of similarity, and the results showed a high degree of correlation between the measured reflectance spectra. There were no significant differences between instruments from different manufacturers in the shape of reflectance spectra, but the signal‐to‐noise ratio varied considerably between individual instruments. Spectra measured by operators with lesser experience in field spectrometry tended to be more variable than those of more experienced operators, indicating that training and adherence to set protocols are important when implementing this method in the field situation. Spatial plots derived from field spectra showed essentially the same trends in surface features as plots created using traditional sampling and remote sensing methods. Furthermore, the use of field spectrometry as a stand‐alone, low‐cost method for rapidly mapping the distribution of major habitat types was demonstrated by the results of a survey of a large intertidal sandbank.