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The microphytobenthos (MPB) – Hydrobia ulvae trophic interaction is one of the main channels of material transfer to higher trophic levels in intertidal mudflats. A new non-invasive approach to evaluate the grazing activity of H. ulvae on microphytobenthos is proposed. The effects of season and period (combination of tide and day/night) on ingestion rates of H. ulvae (using 14Clabeled MPB) and egested pheopigments a (using HPLC pigment analysis) were also investigated. H. ulvae ingestion rate was found to vary significantly over season and period, being higher in summer and during diurnal low tide periods. This is possibly related to higher growth rates of H. ulvaein summer, as well as to an increase in surface MPB biomass during diurnal low tides. A highly significant relationship was found between ingested chl a and egested pheophorbide a, allowing the estimation of ingestion rate from the amount of egested pheophorbide a on H. ulvae faecal pellets. This new non-invasive methodology may allow the improvement of long-term studies of consumption rates and the evaluation of grazing of H. ulvae on MPB. We thank I. Macário, P. Pochelon and T. Salvaterra for assistance in field work; M. Ruivo for assistance in HPLC and A. Luísa Santos for assistance in the Radioisotope Laboratory; A. Almeida for hosting the radioisotope work in the Radioisotope Laboratory at the Department of Biology, University of Aveiro; and 3 anonymous reviewers for critical comments on the manuscript. H.C. was supported by FCT – Fundação para a Ciência e Tecnologia (SFRH/BD/23720/ 2005). This work is part of the research project Benthic link – trophic links regulated by tidal and daily rhythms: benthic microflora and fauna in estuaries’, funded by FCT (POCI/BIABDE/61977/2004). Both grants were allocated by FCT under the Support Community Framework III, Operational Programme Science, Technology and Innovation. The methods used in this study comply with Portuguese legislation on animal experimentation. published

International audience; Intertidal soft sediment microphytobenthic biofilms are often dominated by diatoms, which are able to regulate their photosynthesis by physiological processes (e.g. down-regulation through the xanthophyll cycle, referred to as non-photochemical quenching, NPQ) and behavioural processes (e.g. vertical cell movement in the sediment–biofilm matrix). This study investigated these 2 processes over a 6 h emersion period using chemical inhibitors under 2 light treatments (ambient and constant light at 300 µmol m–2 s–1). Latrunculin A (Lat A) was used to inhibit cell movement and dithiothreitol (DTT) to inhibit NPQ. HPLC analysis for chlorophyll a and spectral analysis (Normalised Difference Vegetation Index) indicated that Lat A significantly inhibited cell movement. Photosynthetic activity was measured using variable chlorophyll fluorescence and radiolabelled carbon uptake and showed that the non-migratory, Lat A-treated biofilms were severely inhibited as a result of the high accumulated light dose (significantly reduced maximum relative electron transport rate, rETRmax, and light utilisation coefficient, α, compared to the migratory DTT and control-treated biofilms). No significant patterns were observed for 14C data, although a decrease in uptake rate was observed over the measurement period. NPQ was investigated using HPLC analysis of xanthophyll pigments (diatoxanthin and the percentage de-epoxidation of diadinoxanthin), chlorophyll fluorescence (change in maximum fluorescence yield) and the 2nd order spectral derivative index (diatoxanthin index). Patterns between methods varied, but overall data indicated greater NPQ induction in the non-migratory Lat A treatment and little or no NPQ induction in the DTT and control treatments. Overall, the data resulted in 2 main conclusions: (1) the primary response to accumulated light dose was vertical movement, which when inhibited resulted in severe down-regulation/photoinhibition; (2) diatoms down-regulated their photosynthetic activity in response to accumulated light dose (e.g. over an emersion period) using a combination of vertical migration and physiological mechanisms that may contribute to diel and/or tidal patterns in productivity.

The effect of high (ambient) and low (shaded to 50 % of ambient) light on microphytobenthic biofilm primary production, vertical migration and allocation of photoassimilated carbon into extracellular carbohydrates was investigated over the low tide emersion period at Sarilhos Pequenos on the Tagus estuary, Portugal, in July 2000. Carbon uptake ( 14 C measurements), electron transport (pulse-amplitude modulated [PAM]-fluorescence) and carbon allocation into extracellular carbohydrate fractions were measured. There was a decrease in the maximum rate of primary production (P max ) ( 14 C) over the emersion period (16 decreasing to 5 mg C m -2 h -1 at midday and the end of emersion, respectively) that was independent of the incident light and the light history to which the biofilms had been exposed. Primary production ( 14 C) did not correlate with measurements of in situ relative electron transport rate (rel.ETR), due in part to the light-induced migration of the cells away from the sediment surface during periods of high irradiance (photosynthetic photon flux density [PPFD] > 1200 mol m -2 s -1 ). Downward migration coincided with the light level at which P max ( 14 C) was reached, 750 mol m -2 s -1 , and with a point of inflexion on the rel.ETR versus PPFD curve. Above this light level rel.ETR was over-estimated due to migration of the cells into a lower light field in the sediment. Shading significantly increased the extracellular polymeric substance (EPS) production rate (from 0.4 to 1.0 mg C m -2 h -1 in ambient and shaded light, respectively) and percentage allocation of photoassimilates into EPS (from 4 ± 1.0 to 18 ± 3.5 % of total production in ambient and shaded light, respectively). In contrast, percentage allocation of photoassimilates to low molecular weight extracellular carbohydrate was higher in the non-shaded treatments (35 ± 4.5 % compared with 13 ± 3.8% in ambient and shaded treatments, respectively), probably due to photosynthetic 'overflow'. In non-shaded biofilms there was little or no increase in the concentration of all extracellular carbohydrate fractions until the last hour before tidal immersion. Results indicate that diel patterns in microphytobenthic primary production are not predictable solely from light data.