Open Archive Toulouse Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in: http://oatao.univ-toulouse.fr/ Eprints ID: 8935 To link to this article: DOI: 10.1007/s10750-011-0920-0 URL: http://dx.doi.org/10.1007/s10750-011-0920-0 To cite this version: Majdi, Nabil and Tackx, Michèle and Traunspurger, Walter and Buffan-Dubau, Evelyne Feeding of biofilm-dwelling nematodes examined using HPLC-analysis of gut pigment content. (2012) Hydrobiologia, vol. 680 (n° 1). pp. 219-232. ISSN 0018-8158 Any correspondence concerning this service should be sent to the repository administrator: [email protected] DOI 10.1007/s10750-011-0920-0 Feeding of biofilm-dwelling nematodes examined using HPLC-analysis of gut pigment contents Nabil Majdi • Miche`le Tackx • Walter Traunspurger • Evelyne Buffan-Dubau Abstract The natural feeding behaviour of the C. viridis fed opportunistically (non-selectively) on nematodes Chromadorina bioculata (Schultze in MPB within the biofilm. Only diatom biomarker Carus 1857) and Chromadorina viridis (Linstow pigments were found in nematode guts suggesting that 1876) was studied in situ, within epilithic biofilms of they could preferentially fed on diatoms among MPB the Garonne River (France). Based on their feeding- groups. However, the non-detection of biomarker type characteristics and population dynamics, it was pigments for other microphyte groups could be also hypothesised that these species feed selectively on linked to HPLC detection limits. It was estimated that microphytobenthos (MPB) within the biofilm, and that Chromadorina nematodes daily ingested on average among MPB groups, diatoms are preferred. High- 0.03–0.67% of the MPB standing stock. This grazing performance liquid chromatography (HPLC) was used covered only a small part of their energetic require- for separation, identification and quantification of ments, suggesting that besides MPB they probably pigments both in nematode guts and in the biofilm. also fed on other biofilm food sources. Some consid- This is the first time that nematode gut pigment erations on the applicability of the HPLC gut pigment contents were examined under natural conditions. analysis technique for the examination of nematode Diatoms dominated the MPB which also comprised feeding are also presented. cyanobacteria and green microalgae. The comparison between chlorophyll a content in nematode guts Keywords Selectivity Á Grazing Á Diatoms Á versus in the biofilm showed that C. bioculata and Periphyton Á Meiofauna Á Chromadorina Introduction N. Majdi (&) Á M. Tackx Á E. Buffan-Dubau EcoLab (Laboratoire Ecologie Fonctionnelle et Meiofauna is extremely species rich and abundant in Environnement), INP, UPS, Universite´ de Toulouse, freshwater benthos, contributing substantially to sec- 118 route de Narbonne, 31062 Toulouse, France ondary production, acting as food web intermediates e-mail: [email protected] and informing general ecological theories such as the N. Majdi Á M. Tackx Á E. Buffan-Dubau metabolic theory of ecology (Schmid-Araya & Sch- EcoLab, CNRS, 31062 Toulouse, France mid, 2000; Schmid-Araya et al., 2002; Bergtold & Traunspurger, 2005; Stead et al., 2005; Reiss et al., W. Traunspurger Department of Animal Ecology, University of Bielefeld, 2010; Reiss & Schmid-Araya, 2010). Free-living Morgenbreede 45, 33615 Bielefeld, Germany nematodes are among the most important contributors to meiofauna (Traunspurger, 2002). Nematodes feed technique was applied with some meiobenthic groups: on a variety of microorganisms including microphytes harpacticoid copepods (Buffan-Dubau et al., 1996; (Moens & Vincx, 1997; Ho¨ckelmann et al., 2004), Buffan-Dubau & Carman, 2000) and chironomids protozoans (Hamels et al., 2001), fungi (Ruess et al., (Goldfinch & Carman, 2000) in muddy salt marshes, 2002) and bacteria (Traunspurger et al., 1997) and but not with nematodes (Moens et al., 2006). Although probably also on organic matter through enzyme- both selective and non-selective feeding strategies sharing interactions with bacteria (Riemann & were observed for free-living marine bacterial feeding Helmke, 2002). or predaceous nematodes under laboratory conditions, In freshwater epilithic biofilms, microphytes, pro- nematode selectivity on microphytobenthos (MPB) in tozoans, fungi and bacteria are embedded in close situ and in freshwater habitats is poorly documented connection within a three-dimensional mucous matrix (Moens & Vincx, 1997; Moens et al., 2006). of self-produced exo-polymeric substances (EPS; In order to determine ingestion rates from gut Flemming & Wingender, 2010). These biofilms offer pigment contents, these have to be reported to gut a shelter and a rich variety of potential food items for passage times (GPT). However, information on nem- nematodes (Ho¨ckelmann et al., 2004; Peters & Traun- atode GPT and their dependence on environmental spurger, 2005). In return, nematode activity might factors remain scarce and mainly restricted to bacte- influence key biofilm processes such as detachment, rial-feeding nematodes (Moens et al., 1999, 2006). oxygen turnover and secondary metabolites release Thus, a careful approach is needed for determining (Sabater et al., 2003; Gaudes et al., 2006; Mathieu ingestion rates from measurements of gut pigment et al., 2007). Biofilm biomass dynamics can, to a contents by using literature GPT. Nonetheless, given considerable extent, be modelled as a function of our generally limited knowledge about the grazing hydrodynamics and self-detachment (e.g. Bouleˆtreau rates of freshwater nematodes (Borchardt & Bott, et al., 2006). However, functional field studies assess- 1995), even such estimations represent, at present, a ing nematode feeding habits within these biofilms are significant advancement in the evaluation of their lacking (Moens & Vincx, 1997), hampering an appro- grazing pressure on MPB. priate assessment of their trophic role within the mat In a recent study conducted in the Garonne River, and their potential feeding impact on biofilm biomass. Majdi et al. (2011) found a coupling pattern between This lack of in situ data is mostly due to the difficulty of epilithic diatom biomass and the density of the two measuring nematode feeding in such complex habitats: dominant biofilm-dwelling nematode species: Chrom- not only are epilithic biofilms composed of a complex adorina bioculata (Schultze in Carus 1857) and organic matrix containing a variety of potential food Chromadorina viridis (Linstow 1876). According to sources for nematodes, but the mucous nature of the their buccal morphology, both these species were biofilm itself poses practical experimental problems. classified as epistrate-feeders after Traunspurger The quantification of the chlorophyll a-equivalent (1997), and hence are expected to feed predominantly (Chl a-eq, i.e. Chl a ? phaeopigments) contained in guts on microphytes (Traunspurger, 2000). In marine allows to obtain in situ data on the grazing activity of environments, a diatom-feeding behaviour is well- post-mortem isolated taxa of animals. To date, this documented for Chromadoridae (i.e. the family technique is routinely used with e.g. planktonic cope- including Chromadorina spp. nematodes), which pods: the quantitative measurement of their gut Chl a-eq puncture or crack diatom frustules to suck inner content with regards to Chl a concentration in the cellular contents (Tietjen & Lee, 1977; Jensen, 1982; surrounding habitat has allowed to investigate their Romeyn & Bouwman, 1983; Moens & Vincx, 1997). selective grazing on phytoplankton, with a disproportion Examining the digestive physiology of Chromadorina between gut Chl a-eq content and Chl a concentration germanica Bu¨tschli 1874, Deutsch (1978) also sug- indicating a selective grazing (e.g. Price, 1988; Gasparini gested that it must have a fairly narrow diet primarily et al., 1999; Irigoien et al., 2000; Tackx et al., 2003). composed of diatoms. As stated above, river epilithic Gut pigment analyses using high-performance biofilms offer a vast variety of potential food items to liquid chromatography (HPLC) can inform on feeding the nematode community. Within this offer, MPB selectivity among various microphytic taxa by iden- seem a likely preferred food source considering the tifying and quantifying their biomarker pigments. This above mentioned knowledge on the feeding behaviour of the dominant nematode species (Chromadorina estimate the relative contribution of the different spp.). It can also be expected that epilithic diatoms are MPB groups to total MPB biomass in terms of selected among the other microphyte groups available chlorophyll a (Chl a) using CHEMTAX version 1.95 in the biofilm. software (Mackey et al., 1996). These procedures are In this context, this study aims: (1) to test the detailed in Majdi et al. (2011). hypothesis that biofilm-dwelling C. bioculata and For nematode gut pigment analysis, four more C. viridis nematodes feed selectively on biofilm cobbles were collected on each sampling occasion. MPB under natural conditions and that diatoms are The biofilm covering cobbles was collected in the field preferred among microphyte groups, (2) to estimate by scraping-off the upper cobble surface with a scalpel their grazing pressure on MPB biomass. and immediately immerged into liquid N2. This instant freezing minimises nematode gut content egestion (Moens et al., 1999). Frozen biofilm samples were