Biogenic methane in freshwater food webs

1. It has long been known that substantial amounts of methane are produced in anoxic lake sediments, and the components of the methane cycle in lakes have been well described. At oxic–anoxic interfaces, methane‐oxidising bacteria (MOB) convert methane to microbial biomass and can be highly productive. However, only recently has methane been recognised as a potentially important carbon and energy source for lake food webs, and some instances have also been reported of methane contribution to river food webs. Stable isotope analysis (SIA) has provided compelling evidence in this respect and has been supplemented by other lines of evidence. 2. In the benthic food webs of lakes, profundal chironomid larvae appear to be the main conduits for trophic transfer of biogenic methane via grazing on MOB. The mode of feeding of these larvae and the microhabitats they generate both promote larval ability to exploit MOB production. Support to chironomid larvae from methane is rather widespread, but its degree is highly variable; estimates suggest that in some lakes methane‐carbon might contribute more than 60% of chironomid carbon biomass. 3. Evidence of crustacean zooplankton in lakes deriving part of their carbon from methane is currently more limited. Reports from some lakes have indicated Daphnia with a substantial (>50%) contribution of methane‐carbon in their biomass. However, for this to happen, an oxic–anoxic interface where sufficient MOB production can occur needs to be within the range of vertical migrations by zooplankton, which may only rarely be the case. Hence, a significant methane subsidy of pelagic food webs in lakes is probably much less widespread than for benthic food webs. 4. There is also recent and currently very limited evidence that some stream benthos derives biomass carbon (reported values up to 30%) from methane. This can occur in stagnant backwater pools where conditions can be analogous to those in lake sediments. However, groundwater aquifers can also supply water supersaturated with methane to some rivers, providing a basis for a microbially‐mediated transfer of methane‐carbon to river benthos. 5. Evidence for significant transfer of methane‐derived carbon to higher trophic levels is still very limited. Within some lakes, those fish species that feed extensively on chironomid larvae can derive a substantial part (perhaps up to 20%) of their carbon biomass from methane. It is also likely that methane‐carbon produced in lakes or rivers is exported to riparian ecosystems when emerging chironomids or other insects are eaten by invertebrate or avian predators. 6. We argue that conceptual models of freshwater food webs, and especially those for lakes, need to be modified to enable incorporation of biogenic methane as a carbon and energy source. For some types of lakes, carbon and energy budgets certainly need to take account of the production and utilisation of biogenic methane, and the accumulating evidence indicates that this is a more widespread phenomenon that has generally been acknowledged hitherto.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Unravelling complexities in benthic food webs using a dual stable isotope (hydrogen and carbon) approach

1. Stable carbon isotope studies have been an essential component of research regarding the contribution of methane (CH₄)-derived carbon to freshwater food webs and results have suggested that benthic macroinvertebrates in billabongs, streams and lakes may be partially, and in some instances, significantly 'fuelled' by methanotrophic biomass. However, the singular use of carbon isotopes can lead to ambiguous interpretations concerning the origin of carbon, especially in systems where phototrophs are likely to be using carbon respired sources and hence show more ¹³C-depleted values.
2. These uncertainties can be further resolved by the inclusion of additional isotopic data. Stable hydrogen isotopes are being increasingly used in food web studies with a marked advantage that sources may be isotopically distinct by one or two orders of magnitude greater than stable carbon or nitrogen, the isotopes most commonly used to delineate trophic interactions. By using hydrogen as a second biogeochemical tracer we provide further supportive evidence for the assimilation of methanotrophic microbial biomass by chironomid larvae.
3. Moreover, the hydrogen and carbon isotope values we found in chironomid tissues appear to reflect the original substrate used during methanogenesis; either acetate fermentation or carbonate reduction. Use of the former tends to result in relatively heavy carbon and light hydrogen isotope values due to kinetic isotope effects, whereas use of the latter results in relatively lighter carbon and heavier hydrogen isotope values.
4. We provide preliminary evidence to suggest that hydrogen and carbon isotope values in macroinvertebrates may be used to distinguish between CH₄ formation pathways and help to explain inter-depth and inter-specific differences between co-existing chironomid species found in the same lake.     

Trophic relationships in a tropical stream food web assessed by stable isotope analysis

1. Stable isotope analysis, coupled with dietary data from the literature, was used to investigate trophic patterns of freshwater fauna in a tropical stream food web (Guadeloupe, French West Indies).
2. Primary producers (biofilm, algae and plant detritus of terrestrial origin) showed distinct δ¹³C signatures, which allowed for a powerful discrimination of carbon sources. Both autochthonous (¹³C-enriched signatures) and allochthonous (¹³C-depleted signatures) resources enter the food web. The migrating behaviour of fishes and shrimps between marine and freshwater during their life cycles can be followed by carbon isotopes. Here, shrimp δ¹³C signatures were shown to shift from −16‰ (for juveniles under marine influence) to −24.7‰ (for adults in freshwater habitats). For resident species, δ¹³C values partly reflected the species' habitat preferences along the river continuum : species living in river mouths were ¹³C-enriched in comparison with those collected upstream.
3. Nitrogen isotopic ratios were also discriminating and defined three main trophic guilds among consumers. The δ¹⁵N values of herbivores/detritivores were 5.0–8.4‰, omnivores 8.8–10.2‰ and carnivores 11–12.7‰.
4. Mixing model equations were employed to calculate the possible range of contribution made by respective food sources to the diet of each species. The results revealed the importance of omnivorous species and the dependence of riverine biota on terrestrial subsidies, such as leaf detritus and fruits. Finally, the abundance of shrimps and their feeding habits placed in relief their key role in tropical freshwater food webs. Isotopic analysis provides a useful tool for assessing animal feeding patterns.     

Comparison of the exploitation of methane-derived carbon by tubicolous and non-tubicolous chironomid larvae in a temperate eutrophic lake

Carbon and nitrogen stable isotope ratios (δ¹³C and δ¹⁵N) in three sympatric species of larval chironomids were analyzed in a temperate eutrophic shallow lake in Japan. Markedly lower δ¹³C values were reported in Chironomus plumosus (?51.2 ‰) and Tanypus sp. (?43.5 ‰) than those in photoautotrophic carbon sources [particulate organic matter (POM) and sediment]. There were positive correlations between δ¹³C and δ¹⁵N in the two chironomid species. These results indicated that they assimilated carbon derived from biogenic methane by exploiting methane-oxidizing bacteria (MOB). In contrast, Propsilocerus akamusi exhibited similar δ¹³C values to those of POM or sediment. A δ¹³C-based isotope mixing model was used to estimate the dietary contributions of MOB to each chironomid species. The mean contributions ranged from 11 to 15 % in C. plumosus, 13 to 19 % in Tanypus sp., but only up to 5 % in P. akamusi. In an aquarium, we observed that individuals of C. plumosus and Tanypus sp., which exhibited low δ¹³C values, built U-shaped larval tubes in the sediment, and an oxidized layer developed around these tubes. Propsilocerus akamusi did not exhibit this behavior. These results suggest that tube building may provide larval chironomids with greater access to methane-derived carbon through increased opportunities to feed on MOB.     

A trophic pathway from biogenic methane supports fish biomass in a temperate lake ecosystem

Although some primary consumers such as chironomid larvae are known to exploit methane‐derived carbon via microbial consortia within aquatic food webs, few studies have traced the onward transfer of such carbon to their predators. The ruffe Gymnocephalus cernuus is a widespread benthivorous fish which feeds predominantly on chironomid larvae and is well adapted for foraging at lower depths than other percids. Therefore, any transfer of methanogenic carbon to higher trophic levels might be particularly evident in ruffe. We sampled ruffe and chironomid larvae from the littoral, sub‐littoral and profundal areas of Jyväsjärvi, Finland, a lake which has previously been shown to contain chironomid larvae exhibiting the very low stable carbon isotope ratios indicative of methane exploitation. A combination of fish gut content examination and stable isotope analysis was used to determine trophic linkages between fish and their putative prey. Irrespective of the depth from which the ruffe were caught, their diet was dominated by chironomids and pupae although the proportions of taxa changed. Zooplankton made a negligible contribution to ruffe diet. A progressive decrease in δ¹³C and δ¹⁵N values with increasing water column depth was observed for both chironomid larvae and ruffe, but not for other species of benthivorous fish. Furthermore, ruffe feeding at greater depths were significantly larger than those feeding in the littoral, suggesting an ontogenetic shift in habitat use, rather than diet, as chironomids remained the predominant prey item. The outputs from isotope mixing models suggested that the incorporation of methane‐derived carbon to larval chironomid biomass through feeding on methanotrophic bacteria increased at greater depth, varying from 0% in the littoral to 28% in the profundal. Using these outputs and the proportions of littoral, sub‐littoral or profundal chironomids contributing to ruffe biomass, we estimated that 17% of ruffe biomass in this lake was ultimately derived from chemoautotrophic sources. Methanogenic carbon thus supports considerable production of higher trophic levels in lakes.     

Seasonal increase of methane in sediment decreases δ13C of larval chironomids in a eutrophic shallow lake

Recent studies have shown that larval chironomids assimilate ¹³C-depleted carbon derived from biogenic methane by feeding on methane-oxidizing bacteria (MOB). The dietary contribution of MOB is known to be maximized in the autumn overturn period or winter in eutrophic dimictic lakes due to the increase of MOB biomass following the supply of oxygenated water, but in polymictic lakes, such seasonal variability has not been revealed. We investigated the seasonal patterns of larval δ¹³C and methane concentrations in the sediment of a eutrophic polymictic lake, Izunuma, Japan. Larval δ¹³C decreased in late summer and autumn. Methane concentrations above a 6 cm depth peaked in late summer or autumn, while those in the 10–11- and 20–21-cm layers peaked in October. Negative correlations between methane concentrations in the 5–6/10–11-cm layers and larval δ¹³C were found. This suggests that an increase in the supply of methane stimulated the activity of MOB in a polymictic lake, where water above the lake bottom rarely became anoxic because of frequent overturn, thus increasing the dietary contribution of MOB to larval chironomids.     

Selective grazing of methanotrophs by protozoa in a rice field soil

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10⁴ MPN (g soil dry weight)⁻¹], and three out of four Methylocystis spp. yielded significantly fewer numbers [10²–10³ MPN (g soil dry weight)⁻¹]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.     

Methane emission from Texas rice paddy soils. 2. Seasonal contribution of rice biomass production to CH4 emission

Measurements focused on seasonal contribution of rice productivity to methane emission were made in three experiments conducted in Texas flooded paddy soils during 1994 and 1995 growing seasons. A total of five rice cultivars representing two distinct groups in methane emission were involved. Over a 10-week period after permanent flooding, total seasonal methane emission was positively correlated with rice above-ground biomass ( r ² = 0.845, n = 11). A very strong dependence of daily methane emission on above-ground vegetative biomass ( r ² = 0.887, n = 93) and on root biomass ( r ² = 0.816, n = 33) was also observed. Calculation from three developmental periods (vegetative, reproductive and ripening) of rice plant indicated that more than 75% of total seasonal methane was emitted during the last 5-week period in concert with reproductive and ripening stages, while rice biomass production during the same period amounted to ≈ 50% of the seasonal total. According to the correlation of cumulative methane emission with above-ground biomass increment between every two-week interval ( r ² = 0.490, n = 93, P = 0.000), the carbon released as methane is approximately equivalent to 3% and 4.5% of photosynthetically fixed carbon in the biomass for low and high emission cultivars, respectively. A further investigation showed that these fractions are related to plant growth and development. The carbon ratio of methane emitted to net photosynthetic production during vegetative, reproductive, and ripening periods averaged 0.9%, 3.6% and 7.9%, respectively, for low emission cultivars, and 2.0%, 5.0% and 8.3%, respectively, for high emission cultivars. Moreover, the ratio was strongly dependent on plant biomass, resulting in r 2 values from 0.775 to 0.907.     

Tracing the slow growth of anaerobic methane-oxidizing communities by 15N-labelling techniques

The anaerobic oxidation of methane (AOM) is an important methane sink in marine ecosystems mediated by still uncultured Archaea . We established an experimental system to grow AOM communities in different sediment samples. Approaches to show growth of the slow-growing anaerobic methanotrophs have been either via nucleic acids (quantitative PCR) or required long-term incubations. Previous long-term experiments with ¹³C-labelled methane led to an unspecific distribution of the ¹³C-label. Although quantitative PCR is a sensitive technique to detect small changes in community composition, it does not determine growth yield. Therefore, we tested an alternative method to detect a biomass increase of AOM microorganisms with ¹⁵N-labelled ammonium as N-source. After only 3 weeks, significant ¹⁵N-labelling became apparent in amino acids as major structural units of microbial proteins. This was especially evident in methane-containing incubations, showing the methane-dependent uptake of the ¹⁵N-labelled ammonium by microorganisms. Cell counts demonstrated a two- and fourfold increase at ambient or elevated methane concentrations. With denaturing gradient gel electrophoresis, over 6 months incubation no changes in community composition of sulphate-reducing bacteria and archaea were detected. These data indicate doubling times for AOM microorganisms between 2 and 3.4 months. In conclusion, the ¹⁵N-labelling approach proved to be a sensitive and fast way to show growth of extremely slow-growing microorganisms.     

Effects of water stress and nocturnal temperature on carbon allocation in the perennial grass, Leymus chinensis

Water deficit and high temperature often occur simultaneously, but their effects on plants are usually investigated separately. The aim of this study was to test how interactions between water stress and nocturnal warming affect carbon allocation in the perennial grass, Leymus chinensis . Plant biomass, dry mass allocation, ¹⁴C partitioning and carbon isotope composition (δ¹³C) were measured. Severe and extreme water stress during nocturnal warming decreased the allocation of dry mass and ¹⁴C partitioning below ground to the roots, but moderate water stress significantly increased the below-ground allocation of dry mass and ¹⁴C, especially at the lower night temperature. The δ¹³C values were more positive at day/night temperatures of 30/20°C than at 30/25°C, and greater in the roots than in the leaves. By plotting the δ¹³C values of the leaves against the δ¹³C values of the roots, the slopes of regressions were steeper at low than at high night temperature, also indicating that nocturnal warming reduces carbon allocation below ground to the roots. The results suggest that nocturnal warming may weaken acclimation during water stress in this species by regulating carbon allocation between source and sink organs.     

Methylmercury enters an aquatic food web through acidophilic microbial mats in Yellowstone National Park, Wyoming

Microbial mats are a visible and abundant life form inhabiting the extreme environments in Yellowstone National Park (YNP), WY, USA. Little is known of their role in food webs that exist in the Park's geothermal habitats. Eukaryotic green algae associated with a phototrophic green/purple Zygogonium microbial mat community that inhabits low-temperature regions of acidic (pH ∼ 3.0) thermal springs were found to serve as a food source for stratiomyid (Diptera: Stratiomyidae) larvae. Mercury in spring source water was taken up and concentrated by the mat biomass. Monomethylmercury compounds (MeHg⁺), while undetectable or near the detection limit (0.025 ng l⁻¹) in the source water of the springs, was present at concentrations of 4–7 ng g⁻¹ dry weight of mat biomass. Detection of MeHg⁺ in tracheal tissue of larvae grazing the mat suggests that MeHg⁺ enters this geothermal food web through the phototrophic microbial mat community. The concentration of MeHg⁺ was two to five times higher in larval tissue than mat biomass indicating MeHg⁺ biomagnification occurred between primary producer and primary consumer trophic levels. The Zygogonium mat community and stratiomyid larvae may also play a role in the transfer of MeHg⁺ to species in the food web whose range extends beyond a particular geothermal feature of YNP.     

Methane carbon supports aquatic food webs to the fish level

Large amounts of the greenhouse gas methane (CH(4)) are produced by anaerobic mineralization of organic matter in lakes. In spite of extensive freshwater CH(4) emissions, most of the CH(4) is typically oxidized by methane oxidizing bacteria (MOB) before it can reach the lake surface and be emitted to the atmosphere. In turn, it has been shown that the CH(4)-derived biomass of MOB can provide the energy and carbon for zooplankton and macroinvertebrates. In this study, we demonstrate the presence of specific fatty acids synthesized by MOB in fish tissues having low carbon stable isotope ratios. Fish species, zooplankton, macroinvertebrates and the water hyacinth Eichhornia crassipes were collected from a shallow lake in Brazil and analyzed for fatty acids (FA) and carbon stable isotope ratios (δ(13)C). The fatty acids 16∶1ω8c, 16∶1ω8t, 16∶1ω6c, 16∶1ω5t, 18∶1ω8c and 18∶1ω8t were used as signature for MOB. The δ(13)C ratios varied from -27.7‰ to -42.0‰ and the contribution of MOB FA ranged from 0.05% to 0.84% of total FA. Organisms with higher total content of MOB FAs presented lower δ(13)C values (i.e. they were more depleted in (13)C), while organisms with lower content of MOB signature FAs showed higher δ(13)C values. An UPGMA cluster analysis was carried out to distinguish grouping of organisms in relation to their MOB FA contents. This combination of stable isotope and fatty acid tracers provides new evidence that assimilation of methane-derived carbon can be an important carbon source for the whole aquatic food web, up to the fish level.     

Making eggs from nectar: the role of life history and dietary carbon turnover in butterfly reproductive resource allocation

The diets of many butterflies and moths change dramatically with development: from herbivory in the larvae to nectarivory in the adults. These diets are nutritionally distinct, and thus are likely to contribute differentially to egg manufacture. We examine the use of dietary resources in egg manufacture by four butterfly species with different patterns of oviposition and lifespan; three in the Nymphalidae ( Euphydryas chalcedona, Speyeria mormonia and Heliconius charitonia ), and one in the Pieridae ( Colias eurytheme ). Each species was fed two isotopically distinct adult diets based on sucrose, both of which differed from the larval hostplant in ¹³C content. Egg isotopic composition was analyzed to quantify the contribution of carbon from the larval and adult diets to egg manufacture. In all four species, egg ¹³C content increased to an asymptotic maximum with time, indicating that adult diet is an increasingly important source of egg carbon . The ¹³C increase closely resembled that of a nectar-feeding hawkmoth, and was well-described by a model of carbon flow proposed for that species. This similarity suggests that the turnover from larval to adult dietary support of egg manufacture is conserved among nectar-feeding Lepidoptera. Species varied widely in the maximum % egg carbon that derives from the adult diet, from 44% in E. chalcedona to nearly 80% in S. mormonia . These differences were related both to the extent of oocyte provisioning prior to adult emergence, and to egg composition. A species' lifetime use of larval vs adult resources in egg manufacture reflected both the carbon turnover of the eggs and the timing of oviposition. Thus, the extent to which dietary resources are important in egg manufacture in butterflies depends on development (egg provisioning in teneral adults), behavior (timing of oviposition) and nutritional physiology (nutrient synthesis and turnover).     

Contribution of fungal biomass to the growth of the shredder, Pycnopsyche gentilis (Trichoptera: Limnephilidae)

1.  1. It has been accepted that aquatic hyphomycetes colonising submerged leaves increase the nutritional value of leaf detritus and suggested that fungal biomass plays a greater role in the growth of shredders than leaf tissue itself. However, it is not clear what proportion of the nutritional needs of shredders is met by fungal biomass.
2.  We fed Pycnopsyche gentilis larvae with tulip poplar ( Liriodendron tulipifera ) leaf discs colonised by the aquatic hyphomycete, Anguillospora filiformis , which had been radiolabelled to quantify the contribution of fungal carbon to the growth of the shredder at different larval developmental stages. Instantaneous growth rates of larvae on this diet were also estimated.
3.  When provided with fungal-colonised leaves (14–16% fungal biomass), the third and the fifth instar larvae of P. gentilis grew at the rates of 0.061 and 0.034 day⁻¹, respectively, but on a diet of sterile leaves, both larval instars lost weight. The incorporation rates of fungal carbon were 31.6 μg C mg⁻¹ AFDM day⁻¹, accounting for 100% of the daily growth rate of the third instar larvae and 8.6 μg C mg⁻¹ AFDM day⁻¹, accounting for 50% of the daily growth rate of the fifth instar larvae.
4.  These results suggest that leaf material colonised by A. filiformis is a high quality food resource for P. gentilis larvae, and that fungal biomass can contribute significantly to the growth of these larvae. Differences in feeding behaviour and digestive physiology may explain the significantly greater assimilation of fungal biomass by the earlier instar than the final instar. To satisfy their nutritional needs the fifth instar larvae would have to assimilate detrital mass that may have been modified by fungal exoenzymes.     

13C-NMR studies on the influence of pH and nitrogen source on polyol pool formation in Aspergillus nidulans

Abstract The composition of the polyol pools in Aspergillus nidulans mycelium during active growth on sucrose depends strongly on pH. At pH 2.5, only mannitol is present. A comparison between nitrate- and ammonium-grown cultures shows stimulation of the arabitol content with nitrate a former nitrogen source. When starved mycelium is incubated either with natural-abundance or ¹³C-enriched glucose, label appears rapidly in mannitol and arabitol, regardless of the nitrogen source or the pH used.     

The underestimated importance of belowground carbon input for forest soil animal food webs

The present study investigated the relative importance of leaf and root carbon input for soil invertebrates. Experimental plots were established at the Swiss Canopy Crane (SCC) site where the forest canopy was enriched with ¹³C depleted CO₂ at a target CO₂ concentration of c . 540 p.p.m. We exchanged litter between labelled and unlabelled areas resulting in four treatments: (i) leaf litter and roots labelled, (ii) only leaf litter labelled, (iii) only roots labelled and (iv) unlabelled controls. In plots with only ¹³C-labelled roots most of the soil invertebrates studied were significantly depleted in ¹³C, e.g. earthworms, chilopods, gastropods, diplurans, collembolans, mites and isopods, indicating that these taxa predominantly obtain their carbon from belowground input. In plots with only ¹³C-labelled leaf litter only three taxa, including, e.g. juvenile Glomeris spp. (Diplopoda), were significantly depleted in ¹³C suggesting that the majority of soil invertebrates obtain its carbon from roots. This is in stark contrast to the view that decomposer food webs are based on litter input from aboveground.     

δ 13C of CO2 respired in the dark in relation to δ13C of leaf carbohydrates in Phaseolus vulgaris L. under progressive drought

The variations in δ ¹³C in both leaf carbohydrates (starch and sucrose) and CO₂ respired in the dark from the cotyledonary leaves of Phaseolus vulgaris L. were investigated during a progressive drought. As expected, sucrose and starch became heavier (enriched in ¹³C) with decreasing stomatal conductance and decreasing p _i/ p _a during the first half (15 d) of the dehydration cycle. Thereafter, when stomata remained closed and leaf net photosynthesis was near zero, the tendency was reversed: the carbohydrates became lighter (depleted in ¹³C). This may be explained by increased p _i/ p _a but other possible explanations are also discussed. Interestingly, the variations in δ ¹³C of CO₂ respired in the dark were correlated with those of sucrose for both well-watered and dehydrated plants. A linear relationship was obtained between δ ¹³C of CO₂ respired in the dark and sucrose, respired CO₂ always being enriched in ¹³C compared with sucrose by ≈ 6‰. The whole leaf organic matter was depleted in ¹³C compared with leaf carbohydrates by at least 1‰. These results suggest that: (i) a discrimination by ≈ 6‰ occurs during dark respiration processes releasing ¹³C-enriched CO₂; and that (ii) this leads to ¹³C depletion in the remaining leaf material.     

Benthic algae support zooplankton growth during winter in a clear-water lake

We used stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotopes to assess the importance of benthic algae for the zooplankton individual growth in winter in a shallow, clear subarctic lake. The δ¹³C values of calanoid ( Eudiaptomus graciloides ) and cyclopoid ( Cyclops scutifer ) zooplankton in autumn suggest a food resource of pelagic origin during the ice-free period. The zooplankton δ¹³C values were high in spring compared to autumn. E. graciloides did not grow over winter and the change in δ¹³C was attributed to a decrease in lipid content during the winter. In contrast, the increase in δ¹³C values of C. scutifer over the winter was explained by their growth on organic carbon generated by benthic algae. The δ¹⁵N of the C. scutifer food resource during winter was low compared to δ¹⁵N of the benthic community, suggesting that organic matter generated by benthic algae was mainly channelled to zooplankton via ¹⁵N-depleted heterotrophic bacteria. The results demonstrate that benthic algae can sustain zooplankton metabolic demands and growth during long winters, which, in turn, may promote zooplankton growth on pelagic resources during the summer. Such multi-chain omnivory challenges the view of zooplankton as mainly dependent on internal primary production and stresses the importance of benthic resources for the productivity of plankton food webs in shallow lakes.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.