Trophic ecology of intertidal harpacticoid copepods, with emphasis on their interactions with bacteria

In the era of rapidly changing natural environments, there is an urgent need for understanding biodiversity-ecosystem functioning. Trophic interactions play a key role in structuring the ecosystem. In marine systems, the feeding ecology of the larger-sized species i.e. (top) predators of food webs, is fairly well documented, yet at the basal level of food webs, the complexity of grazer interactions is much higher and trophic linkages are potentially still missing. In this PhD thesis, the focus is on trophic interactions between harpacticoid copepods and bacteria. The meiofauna (organism size between 38 µm and 1 mm), and in particular harpacticoid copepods (the second most abundant group of the meiofauna, after nematodes), transfer microphytobenthos to higher trophic levels, predominantly larval and juvenile fish. Harpacticoids have highly complex feeding habits. They are able to consume a wide range of food sources, e.g. microalgae, cyanobacteria, flagellates, ciliates, mucoid substances, fungi and heterotrophic bacteria. They are therefore sometimes called ‘indiscriminate feeders’. However, laboratory feeding experiments and recently obtained in situ data, demonstrate their highly selective behaviour and their species-specific food preferences. Under experimental conditions, harpacticoids discriminated among substrates with poor- versus well-developed bacterial films although copepods are considered to be morphologically incapable of consuming small-sized particles such as bacteria. Based on their potential interaction with a broad range of food sources, the general importance of their herbivorous feeding strategy is being questioned and the recent development of dietary tracers such as stable isotopes and fatty acid analysis, now allows to investigate direct assimilation of bacterial matter or that of other food sources. Interest into the pathways of transfer of benthic bacterial matter upwards the food web, for instance with harpacticoids as intermediates, is due to the lack of insight into the general fate of the extensive benthic bacterial biomass and potential pathways of transfer to higher trophic levels are still unknown. A proportion of energy flows back to the microbial-detrital pool as copepod fecal pellets which are efficiently degraded and remineralized. Beside the upward fluxes of biomass and energy, the degradation and reminerilization of waste products resulting from grazing activity is crucial for the recycling of biomass and thus the efficiency of ecosystem functioning. We gained insight into food utilization by harpacticoids in a heterogeneous estuarine intertidal ecosystem, into species-specific feeding patterns and into the trophic importance of bacteria for harpacticoids. Feeding ecology was studied based on both field data of the harpacticoid species assemblages and on experimental laboratory data using individual species. The process of fecal pellet degradation was investigated with focus on the contribution of internal and external fecal pellet bacteria during early degradation and remineralisation of fecal pellets. In an attempt to gain information on the presence of internal bacteria and their position in the fecal pellet, Atomic Force Microscopy and Laser Scanning Confocal Microscopy (AFM-LSCM), a high-resolution imaging tool, was applied. It offers new prospects for studying microbial degradation of copepod fecal pellets. Harpacticoid assemblages for chapters 2 and 3 were studied at the Paulina intertidal area (Schelde estuary, The Netherlands) encompassing a gradient from tidal flat to salt marsh, from seasonal periods at five stations differing in sediment characteristics, tidal exposure, presence of vegetation, etc. To address the structural role of environmental factors, including abiotic sediment characteristics and food-related factor, for harpacticoid assemblages and disclose resource partitioning within harpacticoid assemblages, chapter 2 presents a field study into spatial-temporal heterogeneity in resource availability and in intertidal copepod assemblage structure (density, diversity and composition). An in-depth analysis of the most influential factors for species distribution added relevant information to the autoecology of intertidal harpacticoid species. Spatio-temporal harpacticoid assemblage variation was assigned to variables relating to total organic matter, microphytobenthic biofilms (characterised by pigments and their degradation products), differences in detrital origin and NH4+, suggesting a primary influence of food availability and quality. Nevertheless, harpacticoid assemblages of tidal flats were seemingly more structured by abiotic factors (granulometry and tidal exposure) and especially the harpacticoid assemblage of the sand flat (station H2, species Paraleptastacus spinicauda, Asellopsis intermedia) which was highly station-specific and constant over time. Assemblages of salt marsh stations are considerably similar in copepod family composition despite of differences in salt marsh granulometry, suggesting a primary influence of food availability and food quality. Variability in Microarthridion littorale abundances is related to microphytobenthos biomass. For Ectinosomatidae and Tachidius discipes, the limited number and low correlations for all biotic and abiotic factors indicates a generalistic occurrence. For some species, linkages between habitat characteristics and species distributions were little decisive (e.g. Platychelipus littoralis, Paronychocamptus nanus, Amphiascus sp. 1). Overall, high intercorrelation between a broad range of environmental factors hinders us to draw strong conclusions about the main regulating factors for harpacticoids distribution. Analysis of spatio-temporal variability in resource utilization by harpacticoid copepods, by means of copepod carbon isotopic profiles and fatty acid profiles (chapter 3), proved that the majority of intertidal harpacticoid species relied strongly on microphytobenthos (MPB) as a food source, with potentially fine-scaled selectivity among diatom species or other MPB components. In addition, harpacticoids spanned two trophic levels suggesting also an indirect pathway of MPB transfer to harpacticoids. Furthermore, intertidal harpacticoids showed dietary differences and species-specific spatio-temporal variability in food utilization with contributions of suspended particulate organic matter (Paronychocamptus nanus, Amphiascus sp. 1, Microartridion littorale), flagellates (M. littorale), and bacteria (Delavalia palustris) but not of Spartina detritus. Resource partitioning by harpacticoid assemblages occurred in all stations but was especially clear in the sand flat, comprising a diatom feeder (Asellopsis intermedia), a diatom feeder with temporal reliance on dinoflagellates (Tachidius discipes) and a bacterial feeder (Paraleptastacus spinicauda). In the muddy salt marsh, a trophic role of bacteria was found for Delavalia palustris and Cletodidae, the latter being unique by the utilization of a chemoautotrophic food source. In chapter 4, results from a microcosm feeding experiment using 13C-labelled bacteria, showed that bacterial feeding is linked to diatom grazing and that overall assimilation of bacterial carbon is low for all tested harpacticoid species. In contrast to the bacterivorous copepod Delavalia palustris, non-bacterivorous harpacticoid species (Nannopus palustris, Microarthridion littorale, Platychelipus littoralis) responded negatively on bacterial feeding, as deduced from their mortality and PUFA (polyunsaturated fatty acid) impoverishments. These findings suggest that bacterial biomass may complement feeding on MPB and that an exclusive bacterial diet does not meet copepod nutritional requirements. Delavalia palustris was able to biosynthesized PUFA from a bacterial diet but generally, bacteria represent a minor and low-quality food for these intertidal harpacticoid copepods. In a food-patch choice experiment with 13C-labelled bacterial biofilms (chapter 5), the ability of the harpacticoids Platychelipus littoralis and Delavalia palustris to select between bacterial species with potential different nutritional value (Gramella sp., Jannaschia sp. and Photobacterium sp.) was investigated. In line with chapter 4, bacteria carbon assimilation was low and significant bacterial fatty acid transfer was lacking. A low degree of selectivity was found (preference for Photobacterium sp.), and extracellular metabolites rather than biochemical content and bacterial densities are suggested to be the driver of selective feeding behaviour towards bacteria. Furthermore, the energetic cost of differential bacterivory resulted in a negative fatty acid balance for P. littoralis while D. palustris showed an improved fatty acid profile and thus a positive response to the low-quality bacterial food (similar as in chapter 4). As the ingested bacterial biomass is of limited use for the majority of harpacticoid species, the largest fraction of ingested bacteria returns to the microbial-detrital food web in the form of fecal pellets. Chapter 6 and 7 demonstrate that these ‘internal’ fecal pellet bacteria are viable cells with high densities and represent a diverse active community able to significantly participate in fecal pellet degradation and overall recycling of carbon for the grazer food web. In chapter 6, molecular (RNA-based PCR-DGGE) and metabolic profiling (Biolog Ecoplate assay) of freshly egested copepod fecal pellets proved the general presence of internal active bacteria with a broad metabolic potential in fecal pellets of different copepod species and with different fecal pellet content. The strong impact of the food source on the bacterial diversity of the fecal pellet, indicates the direct transfer of ingested food bacteria to the fecal pellets. Furthermore, the colonization of fecal pellets by bacteria from the surrounding water was relatively low. Consequently, internal bacteria diversity was not replaced by a new external bacterial assemblage. About half of the internal fecal pellet bacteria persisted after 60 h of fecal pellet degradation and, hence, internal fecal pellet bacteria deliver a significant contribution to fecal pellet recycling with Vibrio sp. as a potential important participant. These findings refute the hypothesis of high bacterial fp colonization, as observed for planktonic fecal pellets. AFM-LSCM (Atomic Force Microscopy - Laser Scanning Confocal Microscopy, chapter 7) imaging confirmed the presence of high densities of viable bacteria packed inside the fecal pellet. Furthermore, AFM—LSCM revealed the fibrillar network structure of the peritrophic membrane from a Paramphiascella fulvofasciata fecal pellet, similar to marine polysaccharides and α-chitin and allowed precise measurement of the membrane thickness (0.7-5.9 nm) and bacterial cell volumes (range 0.006-0.117 µm3, in liquid). This protocol enables high-resolution interrogation of biochemical structural changes and bacterial dynamics within the copepod fecal pellets and other heterogeneous particles such as marine snow under environmental conditions. AFM-LSCM generally allows studying bacterial cell size, cell shape and cell-cell interactions. Here it was applied (1) to visualize the ultra-structure of the peritrophic membrane and (2) to locate and quantify bacterial presence (cell size measurements) both inside and outside the fecal pellet. In conclusion, it is clear that the majority of intertidal harpacticoid species primarily relies on MPB, in particular diatoms, and their diet can include small contributions of other food sources such as suspended particulate organic matter, protozoa and bacteria. Despite indications that food availability and MPB shape harpacticoid assemblages on the spatio-temporal scale, the ’real’ importance of MPB, other food-related factors or physical habitat factors for structuring harpacticoid assemblages remains unclear. Furthermore, bacteria are a nutritional food source for some harpacticoid taxa but overall, transfer of bacterial biomass to harpacticoids seems rather limited and in the intertidal microbial food web, bacteria remain a sink. Although harpacticoids consume predominantly substrate-attached bacteria and often co-incidentally during grazing on a primary food source, there are indications that bacterivorous harpacticoids have special adaptations for consuming a poor-quality food source. Selective feeders Paraleptastacus spinicauda and Cletodidae proved, the latter consuming chemoautotrophic bacteria, proves harpacticoids ability to discriminate for bacteria and for bacterial groups but more fine-scale selectivity for bacterial species or for bacterial nutritional content, remains unclear. Although bacterial biomass is (passively) ingested by harpacticoids, the majority will be channeled back to the microbial-detrital food web in the form of fecal pellets. As a consequence of the relative higher contribution of ‘internal’ fecal pellet bacteria compared to external bacteria, the process of microbial degradation of benthic fecal pellets deviates from degradation of planktonic fecal pellets. This may imply that the functioning of the benthic microbial-detrital loop is not necessarily similar to the plantonic microbial-detrital loop

Copepods controlling bacterial communities on fecal pellets

The traditional view of the marine food web depicts bacteria and copepods (mainly planktonic species) as separate units, indirectly connected via nutrient cycling and trophic cascade processes. In contrast, several recent studies have demonstrated that zooplankton and bacteria directly interact, physically, e.g. bacteria attached to zooplankton bodies and biologically, e.g. zooplankton feeding supports bacterial growth through their excretions. Copepods produce large numbers of fecal pellets in the marine environment. Almost immediately after egestion, pellets host extensive bacterial communities. Low amounts of fecal material in sediment traps indicate most part of fecal pellet production is retained in the water column as a result of high microbial degradation rates and planktonic copepods reworking the fecal pellets. First observations on the re-use of feces by benthic copepods points out that these crustaceans profit in a yet unknown way from fecal pellet bacteria. Recently it was illustrated that the benthic species Paramphiascella fulvofasciata increases its fecal pellet production according to its food source. Presumably the bacteria associated with fecal pellets create a trophic upgrading of the fecal material. A detailed characterization of these bacteria is crucial to understand the trophic pathways in the lower marine food web. Culture-independent molecular techniques (e.g. DGGE) showed the specificity of these communities. Shifts in the bacterial communities are caused by age, original food source (e.g. diatoms) and producer of the fecal pellet. Moreover, an additional grazing experiment illustrated the importance of the freshness of the initial food source for grazing preferences but also for the bacterial communities on the fecal pellets. Food of low quality was compensated by more diverse bacterial communities that were available for additional grazing. These results illustrated the importance of fecal bacteria in the transformation of organic matter and energy transfer in marine sediments

Increased production of faecal pellets by the benthic harpacticoid Paramphiascella fulvofasciata: importance of the food source

The re-use of faecal pellets in the water column before sinking to the seafloor is known as an important pathway in marine food webs. Especially planktonic copepods seems to be actively use their faecal pellets. Since benthic copepods (order Harpacticoida) live in the vicinity of their pellets, it remains unclear how important these pellets are for their feeding ecology. In the present study a laboratory experiment was conducted to analyse the importance of faecal pellets for the feeding ecology of the harpacticoid Paramphiascella fulvofasciata and its grazing pressure on two diatom species (Seminavis robusta, Navicula phyllepta). By quantifying the amount and volume of the produced faecel pellets in different treatments, it was tested to what extent the food source and the lack of faecal pellets would influence the production of faecal pellets. We found that the grazing pressure of P. fulvofasciata depended on the diatom density since only a top-down effect could be found on the smaller Navicula cells during its initial exponential growth phase. The grazer had a negative effect on the diatom growth and controlled the cell density to about 4,000 cells/cm(2). In spite of the fact that the addition of faecal pellets did not show a significant positive effect on the assimilation of diatoms, the removal of faecal pellets strongly promoted the pellet production. Especially when grazing on Navicula the harpacticoid P. fulvofasciata produced significantly more and smaller faecal pellets when the pellets were removed. This outcome illustrates the need for faecal pellets of this harpacticoid copepod when grazing on the diatom Navicula. Apart from its selection for smaller diatom cells, it was suggested that the colonisation of heterotrophic bacteria enriched these pellets. This study is the first to indicate that trophic upgrading occurs on faecal pellets and not only on the initial autotrophic food sources per se

Bacterial communities on copepod faecal pellets: biodiversity, factors and functions

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Substrate-dependent bacterivory by intertidal benthic copepods

The trophic importance of bacteria to harpacticoid copepods in intertidal areas remains poorly understood, and so do the mechanisms of bacterial feeding. It is, for instance, unclear whether harpacticoids directly target bacterial cells or merely co-ingest them with substrates to which bacterial cells may be attached. Here, we investigate bacterial uptake and substrate requirement for four mud intertidal species (Microarthridion littorale, Platychelipus littoralis, Delavalia palustris and Nannopus palustris) by means of C-13-labeled bacteria and biomarker fatty acids (FA). Bacterial uptake strongly depended on grazing on a primary food source but bacterial ingestion rates were low, and no clear indication of copepods directly targeting bacteria was found. Delavalia was the only species that accumulated bacteria-derived FA and gained in polyunsaturated FA (PUFA) probably through bioconversion of bacteria-derived FA. In general, however, our results suggest that bacteria represent a relatively minor and low-quality food for intertidal harpacticoid copepods

Limited feeding on bacteria by two intertidal benthic copepod species as revealed by trophic biomarkers

Harpacticoids can discriminate between biofilms of different bacterial strains. We investigated whether assimilation of bacteria is selective and whether harpacticoids select the most nutritional bacteria. We specifically focused on the role of bacterial characteristics in copepod food selection. Trophic biomarkers (stable isotopes, fatty acids) were used to test selective assimilation of three bacteria by the harpacticoids Platychelipus littoralis and Delavalia palustris, all isolated from a salt marsh. The bacteria Gramella sp., Jannaschia sp. and Photobacterium sp. with contrasting ribosomal protein and fatty acid contents, were 13C-labelled and offered in a food-patch choice experiment with monospecific and combination (respectively single and two strains per microcosm) treatments. Low assimilation of bacterial carbon and lack of significant FA transfer proved that bacteria were a poor food source for the harpacticoids. Assimilation was copepod species-specific and bacteria strain-specific (preference for Photobacterium). However, only a low degree of selective feeding occurred; it can partly be explained by extracellular metabolites rather than by biochemical content and densities. Finally, the energetic cost of differential bacterivory resulted in a negative fatty acid balance for Platychelipus, while Delavalia showed an improved fatty acid profile and thus a positive response to the low-quality bacterial food

Structural and functional patterns of active bacterial communities during aging of harpacticoid copepod fecal pellets

Copepod fecal pellet (fp) dissolved organic matter is consumed by free-living bacteria, while particulate matter is degraded by bacteria packed inside the fp (‘internal’) or attached to the fp surface after colonization from the environment (external). This study analyzed the contribution of ‘internal’ and external fp bacteria to the active bacterial community associated with the fp from two copepod species, Paramphiascella fulvofasciata and Platychelipus littoralis, during 60 h of fp aging in seawater. Despite early colonization (within 20-40 h), fp enrichment by seawater bacteria as deduced from RNA-based DGGE after 60 h was limited. In contrast, ‘internal’ bacteria showed high phylotype richness. The majority of ‘internal’ bacterial phylotypes persisted on aged fp and together represented half of the active bacterial community. Food source strongly impacted ‘internal’ bacterial diversity, though the exact origin of fp ‘internal’ bacteria, as either undigested food-associated bacteria or as copepod gut bacteria, could not be unambiguously determined. ‘Internal’ bacteria of fresh fp showed a high functional diversity (based on Biolog assays) to which Vibrio sp. contributed significantly. In terms of bacterial diversity and functional potential, degradation of copepod fp by ‘internal’ bacteria is equally important as by bacteria which colonize fp from the outside