Consequences of the colonisation of leaves by fungi and oomycetes for leaf consumption by a gammarid shredder

Consequences of the colonisation of leaves by fungi and oomycetes for leaf consumption by a gammarid shredder

CHRISTINE ABMANN', KARSTEN RINKE,·l, JAN NECHWATAL t AND ERIC VON ELERTt 'Limnological Institute, University of Konstanz, Germany

tDepartment of Biology, Phytopathology, University of Konstanz, Germany tZoological Institute, University of Koln, Germany

SUMMARY

1. Leaf litter breakdown by shredders in the field is affected by leaf toughness, nutritional value and the presence of secondary compounds such as polyphenols. However,

experiments involving the use of single fungal strains have not supported the assumption that leaf parameters determine food selection by shredders perhaps because of a failure to test for high consumption prior to isolation of fungal strains, overrepresentation of hyphomycetes or the potential effects of accompanying bacteria. In this study, we used bacteria-free, actively growing fungi and oomycetes isolated from conditioned leaf litter for which a shredder had already shown high consumption rates.

2. Black alder (Alnus glutinosa) leaf litter was exposed to the littoral zone of Lake Constance in autumn, and subsamples were analysed for leaf parameters and consumption by Gammarus roeselii under standard conditions at regular intervals. On dates with a high consumption rate of the exposed leaves, 14 single strains of fungi and oomycetes were isolated, freed of bacteria and grown on autoclaved leaves.

3. Six of eight measured leaf parameters of exposed leaves were significantly correlated with Gammarus consumption rates, with high colinearity among leaf parameters hampering the identification of causal relations between leaf parameters and feeding activity.

4. When single strains of fungi and oomycetes were grown on autoclaved leaf litter, toughness of colonised leaves was always lower than in the control and the content of protein, Nand P were increased. There were pronounced strain-specific effects on leaf parameters. Consumption rates also differed significantly, with nine of fourteen isolates consumed at higher rates than controls and none proving to be a deterrent. Protein and polyphenol content were significantly correlated with consumption rates. Oomycete- colonised leaves were consumed at similar rates but were of lower food quality than fungi- colonised leaves.

5. We argue that direct strain-specific attractant or repellent effects of fungi and oomycetes on consumption by G. roeselii are not important. However, we found indirect strain- specific role operating via effects on leaf parameters.

Keywords: food preference, fungi, Gammarus, leaf litter, oomycete

Correspondence: Eric von Elert, Cologne Biocenter, Zillpicher Strasse 47b, University of Cologne, 50674 K61n, Germany.

E-mail: Evelert@uru-koeln.de

1 Present address: Helmholtz Centre for Environmental Research (UFZ), Magdeburg, Germany.

Introduction

Leaves that have entered freshwaters lose soluble inorganic and organic substances during 'leaching' and are colonised by microorganisms, the two pro- cesses referred to as 'conditioning' (Abelho, 2001).

839 http://dx.doi.org/10.1111/j.1365-2427.2010.02530.x

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Secondary compounds such as polyphenols are lost during conditioning (Barlocher & Gra~a, 2005), whereas nitrogen, protein and phosphorus content increases (Barlocher, 1985; Suberkropp, 1992; Gra~a,

Maltby & Calow, 1993a), leaf toughness is reduced

(Gra~a & Zimmer, 2005) and leaves become more palatable to shredders. In the field, feeding and abundance of shredders have been shown to be correlated with increased conditioning of leaves and with fungal biomass on leaf litter (Chergui & Pattee, 1993; Gra~a et ai., 1993a; Gra~a, Maltby & Calow, 1994b; Robinson, Gessner & Ward, 1998), raising the question of what the proximate cues are for shredder food preference.

The microbial community associated with decom- posing plant material is dominated by fungi, consti- tuting up to 16% of total detritus mass in freshwaters and regarded as the main microbial decomposers of leaf litter (Abelho, 2001; Gessner et al., 2007). Hence, the association of shredders with leaf litter colonised by fungi may be attributable to (i) leaf traits that have been affected by leaching; (ii) the fungal biomass itself; or (iii) effects of the fungi on leaf traits. While field observations indicate that leaf parameters deter- mine shredder food selection, laboratory experiments have failed to support this correlative evidence. For example, no leaf parameter was correlated with food preference of Gammarus pulex in experiments with different fungal strains growing on autoclaved leaf discs (Gra~a et ai., 1993a; Gra~a, Maltby & Calow, 1994a). Similarly, Gammarus tigrinus consumption of six hyphomycetes growing on autoclaved leaf discs was not correlated with leaf protein, ph~nols, lipids or ergosterol (Rong, Sridhar & Barlocher, 1995). Such results have led to the conclusion that observed differences in preference cannot be explained by strain-specific effects on leaf parameters but may be related to differences in the synthesis of micronutri- ents by fungi or the production of feeding stimulants or distasteful compounds (Arsuffi & Suberkropp, 1989).

Proper assessment of the role of fungi in preferen- tial feeding by Gammarus spp. requires the investiga- tion into microbes growing on conditioned leaf litter that indeed is preferentially fed on. However, this aspect of leaf litter consumption was not included in earlier studies, where fungi were isolated from con- ditioned leaves without prior assessment of their preference by shredders. Most information on the

effects of aquatic fungi has involved hyphomycetes (a group comprising the asexual stages of ascomycetes and basidiomycetes), because of the ease of their isolation (via spores) rather than their prevalence on conditioned leaf litter, and constituting a possible bias in our understanding. When exploring the role of single strains of fungi, sterile leaf litter is usually used as a carbon source, but in most cases, without ensuring the isolates are free of bacteria, which may have confounded interpretation.

In an attempt to overcome these shortcomings, we investigated leaf conditioning in an exposure exper- iment in the field and the consequences for consump- tion by Gammarus roeselii, an important shredder in the littoral zone of lakes (Mortl, 2003; Baumgartner, Mortl & Rothhaupt, 2008). Leaching and colonisation were allowed to act simultaneously. Bacteria-free strains of fungi and oomycetes were isolated from conditioned leaves, and single isolates were grown on leaves that had been extensively leached by autoclav- ing. Leaf parameters and consumption by G. roeselii were determined from autoclaved leaves inoculated with single strains, allowing us to quantify (i) the relationship between isolates and leaf parameters;

(ii) the relationship between leaf parameters and

shredder consumption; and (iii) the relationship between shredder consumption and isolates. We expected strain-specific effects on G. roeselii prefer- ence, but we anticipated that using bacteria-free strains would allow the identification of leaf param- eters that explain these effects. We further expected to shed light on the functional role of oomycetes in the interaction between conditioned leaf litter and G. roese/ii.

Methods Gammarids

Specimens of G. roeselii, a common shredder in Lake Constance (Mortl, 2003), were collected with a dip net (mesh size 200 11m) in the littoral zone near the Limnological Institute of the University of Konstanz.

Body length was measured according to Gergs &

Rothhaupt (2008) using a stereomicroscope (Zeiss Stemi 2000-C; Carl-Zeiss, Jena, Germany) connected to a digital imaging system. Adults (of both sexes, body lengths 7-12 mm) were starved for 1 day prior to the experiments. The animals were reared, and the

experiments were run in a climate chamber at a constant temperature (15 °C) with a photoperiod of twelve hours.

Leaf litter

Freshly fallen black alder leaves (Alnus glutinosa [L.) GAERTNER) were collected from the ground in autumn 2003 and used for the exposure experiment.

For the single isolate experiment, black alder leaves were collected by means of a nylon net mounted above the ground in autumn 2005. All collected leaves were air-dried and stored at room temperature in the dark until needed for the experiments.

Exposure experiment

To determine how different rates of fungal colonisa- tion influence shredder consumption, we established three different treatments of leaf conditioning in the exposure experiment (as described in ABmann et a/., 2010): (i) autoclaving; (ii) exposure to the littoral; and (iii) incubation in running tap water. From an earlier study (ABmann et al., 2010), we knew that exposure to tap water produces slower fungal colonisation than in lake water. Therefore, in our statistical analysis, the results from tap water exposure constitute an addi- tional treatment that provides contrasting dynamics of leaf parameters and shredder consumption rates in comparison with lake-water exposure.

Starting in October 2005, black alder leaves were exposed to the littoral of upper Lake Constance (47°41.5'N; 9°12.2'E) in cages excluding shredders at 0.4 m water depth with contact to the sediment. The cages were made of polyethylene tubes (0 = 125 mm, length 31 mm) covered on both sides by gauze with a mesh size of 30 /lm. Each cage contained eight pre- soaked alder leaves (c. 2 g dry wt each). In parallel, 5-1 containers each holding about 50 leaves were exposed to tap water at a flow rate of 4300 L day-', simulating the continuous water exchange in the littoral. During the experiment (which lasted for more than 6 weeks), leaves from the littoral and the tap water treatments were harvested weekly on days 8, IS, 22, 29, 36 and 43.

In the autoclaved treatment, pre-soaked leaves were autoclaved (30 min, 121°C) before each experiment, providing leaf material that was physically softened and leached but not chemically modified through microbial colonisers.

Food assays with leaves from the exposure experiment Leaf discs (0 14 mm) were cut from different leaves of each of the three treatments in the exposure experiment using a cork borer near the leaf edge (to avoid larger leaf veins). The wet weight of the leaf discs was measured (Mettler AE 240, Mettler-Toledo, Giessen, Germany) four times to obtain a variation of less than

±0.1 mg. Prior to weighing, the discs were dipped in deionised water and dabbed with a paper towel to reduce weight fluctuations. Although dry weight is usually measured in feeding assays (e.g. Gra~a et al., 1993a, 1994a; Rong et al., 1995), we used wet weight to avoid affecting microbial colonisation.

Single discs from each of the three treatments were simultaneously offered to a single G. roeselii in a four- chambered polyethylene container (108 x 108 x 40 mm). Every chamber contained a shelter (a stone of c. 4 g). A single leaf disc from each of the three different treatments was placed in each of three chambers, the container was filled with 250 mL filtered (30 ILm) lake water, and a single G. roeselii was added. The feeding assays were stopped after 48 h or at a threshold of a third of a leaf disc remaining in any of the three discs offered (visual inspection). For each feeding assay, the absolute consumption rate for each leaf was calculated as the difference between initial and resid- ual wet weights divided by the duration of the experiment, expressed in mg leaf consumed per indi- vidual per hour. The total consumption rate (mg ind.-¹ h-¹⁾ was calculated by summing the abso- lute consumption rates for each of the three discs. The assays with conditioned leaf material were replicated using different individuals of G. roese/ii (n = 14--19).

Isolation and identification of fungi and oornycetes Fungi' and oomycetes were isolated from the littoral exposure treatment on days 22 and 36, by which time we noted a high consumption of the littoral-exposed leaves (Fig. 1a), suggesting that the microbial leaf community was palatable to the gammarids. The leaves from which fungi and oomycetes were to be isolated were harvested on day 21 and (as according to ABmann et al., 2010) stored in petri dishes (15

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Exposure time (days) Exposure time (days)

Fig. 1 Results from the exposure experiment. The three treatments consisted of black alder leaves exposed to the littoral zone of Lake Constance (filled circles), exposed to tap water (open triangles) or autoclaved (open circles). (a) Absolute consumption rates (mean value ± SE, /"I = 14-19) of the three differently treated food items offered simultaneously to Gammarus roese/ii. (b) Mean leaf tough- ness ± SE (from 11 = 5 leaves). Mean values of two analyses for each sampling date and treatment are depicted for all other leaf parameters. The coefficients of variance (CV) were: (c) polyphenoI3.5%; (d) protein 3.4%; (e) phosphorus 3.4%; (f) nitrogen 3.2%; (g) carbon 0.9%; and (h) ergosterol content 5.2%.

completed. Pieces of littoral-conditioned leaf litter (c.

2.5 x 2.5 mm) were aseptically cut with a scalpel and transferred to petri dishes containing water agar (2%

agar) containing antibiotics (90 mg L -1 ampicillin, 150 mg L -1 streptomycin sulphate). The leaves pro- vided the carbon source; no other carbon sources were added. The petri dishes were incubated at 20°C with a 12-h photoperiod for 3 days. Actively growing single hyphae extending over the leaf pieces onto the agar were selected and transferred onto malt extract agar (MEA; 1.5% malt extract, 2% agar).

Fungal and oomycete cultures were purified from bacteria according to the method of Abdelzaher, Ichitani & Elnaghy (1994): fungal and oomycete hyphae grew vertically through MEA containing antibiotics, and bacteria-free hyphae were scraped from the surface of the MEA. New MEA petri dishes were inoculated with these hyphae, establishing the stock cultures. The isolates were examined with a stereomicroscope and preliminarily grouped accord- ing to their macroscopic appearance.

Mycelium from pure cultures of the thirteen isolates of fungi and oomycetes was used for taxonomical classification. DNA extraction, amplification of the internal transcribed spacer (ITS) regions 1 and 2 including the 5.8S gene of the ribosomal RNA (rDNA) genes, restriction fragment length polymorphism (RFLP) analyses and sequence analyses were carried out as described in ABmann et al. (2010). Mycelium from each of the fungal and oomycete isolates was scraped off the MEA and homogenised using a pestle in 50 /lL sterile water in micro-centrifuge tubes. Che- lex 100 resin (10%; Bio-Rad, Munich, Germany) was added and incubated for 40 min at 65°C and for 5 min at 90 °C (Wirsel, 2002). The homogenate was centri- fuged (2300 g, 15 min), and the supernatant containing the DNA was stored at -20°C. ITS regions 1 and 2 including the 5.8S gene of the ribosomal RNA genes (rDNA) were amplified using the primer pair IT- SlIITS4, as described in White et al. (1990) and Gardes

& Bruns (1993). PCR products were separated on 1.5%

agarose gels (70 x 80 mm; 1 x T AE buffer; 45 min, 85 V, 400 rnA), and bands were visualised with ethi- dium bromide. When multiple or weak bands appeared on the gels, the DNA was extracted using a DNeasy Plant Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol and subse- quently amplified. Amplified DNA was digested with restriction endonucleases MspI and AluI (Fermentas,

St. Leon-Rot, Germany) according to the manufac- turer's instructions to identify groups of isolates with identical RFLP banding patterns on 3% agarose gels (70 x 80 mm; 1 x T AE buffer; 106 min, 70 V,400 rnA).

PCR products of isolates showing unique RFLP pat- terns were sequenced using the aforementioned for- ward and reverse primers by Eurofins MWG Operon (Ebersberg, Germany). BLAST was used to identify the closest related species in GenBank. Fungal and oomyc- ete sequences obtained from GenBank were aligned using BIOEDIT, version 7.0.5.3 (http://www.rnbio.

ncsu.edu/BioEdit/bioedit.html). RFLP analyses and comparison of sequence data were used for the identification of the isolates. The four different sequences obtained during this study have been submitted to GenBarik® (see Table 1 for accession numbers). Because of the unexpectedly high abun- dance of identical oomycete isolates, we selected two independent isolates with identical RFLP patterns and sequences (36e and 36c; Table 1).

Single isolate experiment

A total of 14 different strains of fungi and oomycetes were used. Nine of these isolates were obtained from leaves exposed to the littoral zone of Lake Constance in July/August 2005 in the study of ABmann et al. (2010).

To increase the number of identified isolates in this study, we repeated the procedure of isolating strains in OctoberlNovember 2005 during our exposure exper- iment. This second isolation of fungiloomycete strains resulted in another five strains. Autoclaved leaves were inoculated with each of the 14 strains.

The inoculation with fungi and oomycetes and the incubation of the leaves were conducted as described in ABmann et al. (2010). Leaf litter was soaked in tap water and then autoclaved (30 min, 121°C) because earlier studies have suggested that autoclaved leaves constitute an appropriate model system for leached It:!aves (ABmann, von Elert & Gergs, 2009; ABmann et al., 2010).

Single leaves were placed on a cellulose filter (0 = 70 mm) saturated with a mineral solution (0.01 g MgS04'7 H20, 0.01 g CaCl2·2 H20, 0.01 g KN03, 0.01 g K2HP04 and 0.5 g 2-[N-morpho- lino) ethanesulfonic acid per litre, pH 6.0; Duarte et al.

2006) under sterile conditions in a petri dish (0 = 90 mm). Each leaf was inoculated with the mycelium of an isolate (agar plug placed in the centre

844

Table 1 Fungi and oomycetes isolated from leaf litter conditioned in lake water in summert (ABmann et al., 2010) and autumn 2005 (exposure experiment)'. Isolates were identified by BLAST analysis of amplified sequences of the internal transcribed spacer regions 1 and 2 (ITS1, ITS2) and the 5.8S rONA; similarity of the sequences refers to pair-wise alignments with the closest match

GenBank

Phylum or accession

class Species number

Ascomycota EpicocculII sp. PV Wi 22e' EU740394 Ascomycota EpicoCClIIIl sp. PV Wi 36a' EU740397 Ascomycota Cylindrocarpon sp. PV Wi 22k' EU740396 Ascomycota Cylindroclndielln pnrva t EU637905 Ascomycota Cylindrocnrpon sp. 94-2057t EU637906 Ascomycota Cylindrocnrpon sp. 4/97-1 t EU637900 Ascomycota Fusnriw/I sporotrichioides t EU637901 Ascomycota MicrodochiulIl sp. PV S02t EU637902 Ascomycota Ascomycete sp. PV So8t EU669082 Oomycetes Pythilllrl litorale^t EU637904 Oomycetes Pythiul1l sp. IN I_bt EU637903 Oomycetes Pythium sp. PV S07t EU669081 Oomycetes PythiulrI sp. PV Wi 36c' FJ882625 Oomycetes Pythiwn sp. PV Wi 36e' EU740398

of the leaf). Petri dishes were incubated at 20°C with a 12-h photoperiod. When a fungus or oomycete had fully colonised the surface of the petri dish or had grown through the matrix of the leaf (tips of hyphae grew out of or over the cellulose filter, signifying the 'fully conditioned phase'; Barlocher, 1985; determined visually), the leaves were used in the preference assays. Autoclaved leaves (control) and the colonised leaves .were offered in food assay experiments. Leaf discs were cut from leaves of the two different treatments (i.e. colonised or autoclaved) as described earlier (food assay with leaves from the exposure experiment). One disc from the conditioned leaves and one from the control leaves were offered together to a single G. roeselii in the four-chambered polyeth- ylene container as described earlier. In a control assay, two autoclaved leaf discs were offered to G. roeselii.

Absolute consumption rates were calculated as described earlier. We replicated the food assays for each isolate and the control with at least nine individuals (n = 9-12).

Leaf parameters

Samples of the littoral-exposed and tap water-condi- tioned leaves were sampled weekly. Leaves from the same treatment and the same sampling day were pooled, freeze-dried and then homogenised with

Best BLAST hit; accession no.; similarity (%)

Epicocculll nigrulrI (LINK); A Y787697; 100.0

EpicocculIl nigrul1l isolate H2Fl (LINK); EU529998; 100.0 Cylindrocarpon sp. EXP0565F (WOLLENWEBER); DQ914670;

98.9

Cylindroclndielln pnrvn ASICP1(ANOERSON); DQ779786; 100 Cylil'ldrocarpol'l sp. 94-2057 (WOLLENWEBER); A Y295305;

100

Cylilldrocnrpon sp. 4/97-1 (WOLLENWEBER); AJ279490; 100 FusnriulIl sporotrichioides var. lrIinor BBA 62425

(SHERBAKOFF); AF414973; 100

Microdochiultl sp. 4/97-103 (SPRAGUE); AJ279489; 99.6 Leaf litter ascomycete its261(GILBERT); AF502786; 96.8 PythiulrI litorale P.03 (NECHWATAL); OQl44637; 100 PythiulIl sp. IN-lb (NECHWATAL); DQ230904; 100 Pythium sp. IN-12 (NECHWATAL); OQ237932; 90.3 Pythium litorale (NECHWATAL); EU637904; 96.3 PythiulrI litorale (NECHWATAL); EU637904; 96.3

mortar and pestle and stored at -80°C. Samples from autoclaved leaves and from leaves inoculated with a single fungus or oomycete were treated alike. All measurements of leaf litter samples were taken in duplicate to reduce the chance of systematic errors.

Particulate organic carbon and nitrogen content was determined with an NCS-2500 analyzer (Carlo Erba Instruments, Thermo Fisher Scientific, Dreieich, Germany), and particulate phosphorus was deter- mined according to ABmann et al. (2010). Protein content was measured according to Baerlocher (2005), and total polyphenol content was determined photo- metrically as described by Barlocher & Gra~a (2005).

We measured the ergosterol content of the littoral- exposed leaves to monitor fungal development (Gessner et al., 2007). Ergosterol was extracted in alkaline methanol at 80°C, followed by a C_I8 solid- phase extraction (Sep-Pak® VactC18 6cc; Waters, Eschborn, Germany) according to Gessner (2005).

The extract was quantified using HPLC (LiChro- spher® 100 RP-18 column, 5 ^11m, 250 x 4 mm; Merck, Darmstadt, Germany) as described by Gessner (2005). Note that oomycetes do not contain ergosterol (Weete

& Gandhi, 1996). A penetrometer (Pabst et al., 2008)

was used to determine leaf toughness by puncturing five points (littoral exposure) or ten points (single isolate experiment) at the edge of five leaves from each treatment.

Statistical analyses

Statistical analysis of the absolute consumption rates in the exposure experiment with littoral-exposed and tap water-exposed leaf litter was conducted using an ANOV Aliinear model approach. First, we averaged the measured consumption rates and leaf parameters in each treatment (littoral-exposed, tap-exposed and autoc1aved) and on each sampling day. Since all the single leaf discs from each treatment were offered together, the absolute consumption on one leaf disc depended not only on the preference of the gamma rid for that leaf disc but also on its preference for the other discs. The autoc1aved leaves served as a control, because the quality of this food source remained constant over the whole experiment. Based on the average consumption of the control leaves, we then adjusted the consumption rates on littoral-exposed and tap water-exposed leaves (Le. the mean con- sumption rate of autoc1aved leaves was subtracted from the consumption rates of littoral-exposed and tap water-exposed leaves). We made the same adjustment based on the control treatment for all leaf parameters (leaf toughness, protein content, etc.).

Finally, we calculated ANOV Asliinear models be- tween the adjusted consumption rate as a dependent variable and the adjusted leaf parameters as indepen- dent variables.

The absolute consumption rates in the single isolate experiment were also examined by ANO- vA/linear models. We calculated the average con- sumption rates and leaf parameters for each isolate and for the control. Since we wanted to find out which isolates were significantly preferred over the control treatment, we then subtracted the average consumption rate in the control treatment (0.065 mg wet weight x h-¹ x ind.-¹) from the consumption rates of the isolates. This means that a consumption rate of zero in the transformed data of a treatment corresponds to the consumption rate in the control treatment. Finally, we calculated an ANOV Aliinear model with an intercept of zero, with the consump- tion rate as a dependent variable and the isolate as the independent variable. Since the controls by definition had a mean of zero, we removed them from the analysis. All statistical analyses were performed using R (http://www.r-project.org/), and the significance level of the analyses was P

=

0.05.

Table 2 Total consumption rate of Gnmlllnrlls roeselii for littoral-exposed, tap water-conditioned and autoc1aved leaf litter from the different feeding assays of the exposure experiment

Exposure Number of Total consumption day replicates (n) rate (mg ind.-¹ h-¹⁾ ± 2 SE

1 19 0.23 ± 0.07

8 14 0.15 ±

om

15 19 0.21 ± 0.03

22 19 0.30 ± 0.07

29 18 0.29 ± 0.04

36 18 0.30 ± 0.07

43 18 0.43 ± 0.05

Results

Relationship between shredder consumption and leaf parameters in the exposure experiment

The feeding assays with G. roeselii were stopped at a threshold of a third of leaf disc remaining (for any of the three discs offered) to ensure that Gammarus requirements in terms of food quantity were always met. Nevertheless, total consumption showed an increase with sampling date (Table 2), suggesting that this reflects an increase in food quality over time.

In all three treatments, the absolute consumption rate for black alder leaf litter (littoral-exposed, tap water-conditioned and autoc1aved) changed over time. While at the beginning, almost exclusively autoc1aved leaves were consumed, and at a very high rate, littoral-exposed leaves were increasingly pre- ferred later (Fig la). From day 22 onwards, the consumption rate of littoral-exposed leaves was about 0.2 mg ind.-¹ h-¹. Leaves incubated in tap water were hardly eaten in the first few weeks, but consumption of these leaves increased steadily over the course of the experiment. Nevertheless, consumption of tap water-conditioned leaves always remained below 0.15 mg ind.-¹ h-¹. On days 8 and 15, we noted similar consumption rates on littoral-exposed and autoc1aved leaves, but afterwards, the consumption rate of autoc1aved leaves remained at a very low level (below 0.05 mg ind.-¹ h-¹).

The ergosterol content of littoral-exposed leaves increased during exposure, indicating colonisation by fungi on the leaves starting between day 15 and 22 and reaching a maximum of 35 /lg g-l dwt on day 36 (Fig. 1h). The other leaf parameters (littoral-exposed leaves) also changed considerably during the

Table 3 Pearson product-moment correlation coefficients (Pearson's r) between experiment day and leaf parameters in the exposure experiment. Since the development of the leaf parameters over time was qualitatively similar in the littoral-exposed and tap water experiments, results from the two treatments were merged. Bold numbers and asterisks indicate significant differences: *P < 0.05,

**P < 0.01, ***P < 0.001

Variable Leaf toughness Polyphenol Protein

Day -0.758** -0.932*** -0.010

Leaf toughness 0.647* -0.044

Polyphenol 0.060

Protein P N C N:C

experiment (Fig. Ib-g): while leaf toughness as well as polyphenol and nitrogen content decreased strongly, protein and phosphorus content showed only minor changes.

There was considerable co linearity between the leaf parameters in the littoral- and tap water--exposed leaves (i.e. the different leaf parameters were correlated with each other and developed similarly over time). We quantitatively explored these correlations by calculat- ing a matrix of Pearson product-moment correlation coefficients between all leaf parameters (including N : C and P : C ratios) and exposure time. Fourteen of 36 combinations were significant (Table 3). In fact, many parameter combinations showed significant cor- relations because of their similar development over exposure time. For example, the strong decrease in leaf toughness was accompanied by a corresponding decrease in polyphenol content.

The statistical analysis of the absolute consumption rate of the littoral-exposed leaves (exposure experi- ment) as a dependent variable and leaf parameters as independent variables showed that six of eight parameters were significantly correlated with con- sumption (Table 4), with polyphenol content and leaf toughness explaining a relatively high degree of variability (r > 0.8; Table 4, Exposure) of absolute leaf

P N C N:C P:C

0.547* -0.167 -0.286 0.091 0.422

-0.773** -0.041 0.358 -0.428 -0.563*

-0.425 0.342 0.334 0.081 -0.408

-0.401 0.348 0.714** -0.270 -0.655*

-0.034 -0.665* 0.645* 0.875***

0.637* 0.623* -0.407 -0.205 -0.938***

0.442

consumption. In addition, correlations of protein and P content and the P : C ratio with absolute consump- tion were highly significant (r > 0.7; Table 4, Expo- sure). However, since most of these parameters were simultaneously correlated with each other as indi- cated above, we could not distinguish potential causal relationships.

Relationship between isolates and leaf parameters

In the single isolate experiment, the leaf toughness of fungal- and oomycete-colonised leaves was always lower than in the control (Fig. 2b). In the case of protein and N content, the fungal- and oomycete-colonised leaves had higher values than the control (autoclaved leaves, Fig. 2c,d), and in most cases, their P content was also higher (Fig. 2e). However, the different fungal and oomycete isolates had very different effects on the measured parameters, indicating strain-specific effects. For example, leaf colonisation by Fusarium sporotrichioides made the leaves softer (low leaf tough- ness) and increased the content of protein, Nand P compared to the control (Fig. 2); whereas Pythium sp.

IN

I-b increased nitrogen to similar levels, it had considerably smaller effects on leaf toughness, protein and phosphorus (Fig. 2). Oomycetes affected some leaf

Table 4 Effects of adjusted leaf parameters (see Methods) on adjusted consumption rates in the exposure experiment (exposure to the littoral) and in the single isolate experiment (leaves colonised with single fungus or oomycete). Pearson product-moment correlation coefficients (r) and P-values are given (P). Bold letters indicate significant P-values

Experiment Leaf toughness Polyphenol Protein P N C N:C P:C

Exposure (11 = 13-18) -0.806 -0.811 -0.754 0.753 0.279 -0.485 0.576 0.759

P 3.810-' 1.310-' 0.002 0.002 0.335 0.079 0.031 0.002

Single isolates (11 = ^{8-15) -0.478 -0.678 -0.658 0.031 -0.012 0.358 -0.077} 0.008

P 0.084 0.008 0.010 0.917 0.969 0.209 0.793 0.979

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~ 'C 200

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parameters differently than fungi. For example, leaves inoculated with oomycetes had, on average, higher leaf toughness and higher polyphenol content but reduced phosphorus content (Table 5).

Fig. 2 Consumption by Gnmmnrtls roeselii and leaf parameters of black alder leaves autoc1aved (control, hatched) or colonised with different isolates of fungi or oomycetes. Isolates from the summer experiment (grey) and from the autumn experiment (white) are depicted. (a) Absolute consumption by G. roeselii (mean value ± SE, n = 8--15). (b) Mean leaf toughness ± SE (from n = 10 leaves). Mean values of two analyses for each sampling date and treatment are depicted for all other leaf parameters. The coefficients of variance (CV) were: (c) protein 4.5%; (d) nitrogen 0.8%; and (e) phosphorus 5.7%. Asterisks in the top panel indicate significantly different consumption rates between control and the respective strain. Fus., FusariulII sporo- trichioides; Micro., MicradochiulrI sp. PV 502; Pyth. a, Pythium sp.

IN 1-b; Pyth. b, Pythiuln litornle; Cylin., Cylindrocladiel/a parvn;

Pyth. c, Pythium sp. PV 507; Asc., Ascomycete sp. PV 508; Cyl.

a, Cylindrocnrpon sp. 94-2057; Cyl. b, Cylindrocarpon sp. 4/97-1;

Epi. a, Epicoccum sp. PV Wi 22e; Cyl. c, Cylindrocnrpon sp. PV Wi 22k; Epi. b, Epicoccllln sp. PV Wi 36a; Pyth. d, PytiliulrI sp. PV Wi 36c; Pyth. e, Pythium sp. PV Wi 36e; Cont., control.

Table 5 Statistical analysis of leaf parameters and Gnmmarus consumption rate of oomycetes and fungi by ANOVA. Negative values for difference between means indicate lower values for oomycetes. Significant differences are given in bold

Difference between

Variable Unit means F P

Consumption mg Ind-1 h-1 -0.064 F1•129 = 3.13 0.076 rate

Leaf N 0.435 F_1•135 = 14.0 2.710-' toughness

Polyphenol Ilg (mg DW)-1 7.284 F1•26 = 11.3 2.410-³ Protein Ilg (mg DW)-1 7.493 F1•26 = 0.61 0.44 P Ilg (mg DW)-1 -0.121 F1•26 = 15.5 2.710-⁵ N Ilg (mg DW)-1 -2.026 F1•26 = 2.96 0.097 C Ilg (mg DW)-1 1.847 F1•26 = 0.35 0.56

N:C -4.210-³ F1•26 = 3.89 0.059

P:C -2.410-' F1•26 = 16.3 4.210-'

Relationship between shredder consumption and isolates

Absolute consumption varied between leaves inocu- lated with different fungal and oomycete isolates compared to the control (autociaved leaves). Nine of fourteen isolates (six ascomycetes and three oomyce- tes) were significantly preferred by G. roeseIii over the control (Fig. 2a and Table 6), and no strain had a repellent effect. Different fungal and oomycete iso- lates had very different effects on the absolute consumption by G. roeselii, which indicates that not fungal colonisation in general leads to increased consumption, but rather that strain-specific effects determine consumption by G. roese/ii. This study is

848

Table 6 Results of the statistical analysis of absolute consumption rates of Gammarus roeselii for fungal-and oomycete-colonised leaves (single isolate experiment), by an A NovA/linear model. Negative estimates for the regression coefficient indicate lower consump- tion rates than in the control treatment ^(I'I = 15); positive values indicate higher consumption rates. ANOV A of the model indicated high significance (FI4.117 = 11.53, P < 0.001). Differences from the control values significantly different from zero are given in bold

Number of Difference

replicates from

Leaf conditioning n Abbreviation control 5E I-value P-value

Fusarium sporotrichioides 10 Fus.

Microdochium sp. PV 502 10 Micro.

Pythiwn sp. IN I-b 9 Pyth. a

Pythiwn litorale 10 Pyth. b

Cylindrocladie/la parvn 9 Cylin.

Pylhium sp. PV 507 10 Pyth. c

Ascomycete sp. PV 508 9 Asc.

Cylindrocarpon sp. 94-2057 10 Cyl. a

Cylindrocarpon sp. 4/97-1 9 Cyl. b

Epicoccum sp. PV Wi 22e 8 Epi. a

Cylindrocarpon sp. PV Wi 22k 10 Cyl. c

Epicoccum sp. PV Wi 36a 8 Epi. b

Pylhiu!n sp. PV Wi 36c 10 Pyth. d

Pythiuln sp. PV Wi 36e 9 Pyth. e

the first report in which oomycete isolates were found to positively affect Gammal'us consumption (Table 6).

However, the shredder's consumption rate was not systematically different between fungi and oomycetes (Table 5).

Relationship between shredder consumption and leaf parameters in the single isolate experiment

In the case of Cylindrocladiella parva, relatively minor increases in protein were associated with increased consumption (Fig. 2a), whereas F. sporotrichioides sub- stantially reduced leaf toughness and increased the content of protein, Nand P, but leaf colonisation by this fungus had no effect on G. roeselii consumption (Fig. 2a). These effects are in contrast to the wide- spread view that fungal colonisation generally in- creases leaf palatability to shredding invertebrates

(Gra~a, 2001).

The statistical analysis of the single isolate experi- ment revealed that polyphenol and protein content of the leaves explain a large proportion of the variability observed in consumption (1' > 0.65; Table 4, single isolates). Six of eight parameters were significantly correlated with absolute consumption in the exposure experiment, whereas in the absence of leaching (single isolate experiment), only polyphenol and protein content were significantly correlated with absolute consumption, suggesting that only a limited set of leaf

-0.04205 0.05583 -0.753 0.45286

0.15625 0.05583 2.799 0.00600

0.09836 0.05885 1.671 0.09732

0.08125 0.05583 1.455 0.14826

0.38803 0.05885 6.593 1.3010-⁹

0.15465 0.05583 2.770 6.5210-³

0.25414 0.05885 4.318 3.3110-⁵

0.27435 0.05583 4.914 2.9310-⁶

0.32336 0.05885 5.495 2.3210-⁷

0.06012 0.06242 0.963 0.33742

0.15705 0.05583 2.813 5.7610-³

0.11175 0.06242 1.790 0.07599

0.14375 0.05583 2.575 1.110-¹

0.13525 0.05885 2.298 2.310-¹

parameters are affected by fungal colonisation of leached leaves.

Discussion

Leaf toughness and polyphenol content were impor- tant leaf parameters in the exposure experiment, providing support for the contention of Gra~a (2001) that leaf toughness and content of secondary com- pounds are principle factors determining shredder preference. However, although leaf toughness may represent an important parameter in the field, it was not significant in our single isolate experiment, a fact that is not surprising as autoc1aved leaves were used.

The single strain experiments showed that the effects of fungi and oomycetes on polyphenol and protein content were major determinants of feeding prefer- ence. It is important to note that this does not necessarily imply that leaf toughness is of minor releyance for shredder preference in the field. It merely indicates that microbial colonisers affect shredder activity in the late stage of leaf conditioning by changing the content of polyphenol and protein.

Dynamics of consumption and leaf parameters during exposure

In the exposure experiment, the preference by G. roes- elii shifted from autoc1aved leaf litter to littoral-

exposed leaves with increasing incubation time. As has already been shown for shredder organisms (Kaushik & Hynes, 1971), consumption by G. roeselii increased with increasing conditioning time of leaf litter. The negative relation of consumption with polyphenols is in accordance with the observations of several authors (Rosset, Barlocher & Oertli, 1982;

Pennings et ai., 2000; Abelho, 2001) who found them to be repellents of invertebrate grazers.

In general, the leaf litter content of nitrogen and protein increase during decomposition, leading to an enhanced preference by shredders (Barlocher, 1985;

Suberkropp, 1992; Abelho, 2001). An increase in protein content from 4 to 5.8% dry wt has been shown for maple leaves exposed in bags with 3 mm mesh size after 4 weeks (Barlocher & Kendrick, 1974).

When alder leaves were exposed in bags of 9 mm mesh size, protein content increased from 13 to 22%

dry wt in 6 weeks (Gessner, 1991). In another 6-week experiment (Canhoto & Gra~a, 1996) using bags with a mesh size of 0.5 mm, an increase in nitrogen content from 2.6 to 3.3% dry wt was observed. In our exposure experiment, however, both protein and N content of the littoral-exposed leaves decreased over the first 2 weeks of exposure. We assume that proteins and N-containing compounds such as amino acids were leached out and that simultaneously growing microbes (fungi and oomycetes) could not compen- sate for this loss. Only after 3 weeks, did the protein and nitrogen levels begin to increase slightly, indicat- ing that microbial growth became more important than loss processes. The decline in polyphenols in this study provides no evidence that leaching rates were substantially higher than in the aforementioned cases, indicating that either exposure to the littoral of a lake or the substantially smaller mesh size used here (30 {1m) has led to a significantly later growth of microbes, so that leaching of proteins and N-contain- ing compounds rather than biomass increase in microbes was the dominant process during the early weeks of exposure.

Effects of single strains on leaf parameters and consumption

To assess the effects of single strains of fungi and oomycetes, we used autoclaving to experimentally decouple leaching from colonisation in the single isolate experiment. Such a decoupling might occur in

the field for leaves with high levels of tannins and thick cuticles, such as in Eucalyptus (Canhoto & Graca, 1999), or with high lignin concentrations that have been associated with slower microbial colonisation rates (Schindler & Gessner, 2009). Autoclaving re- duces leaf toughness and dissolves soluble organic and inorganic substances out of the leaf matrix. Note that leaf toughness of the autoclaved leaves in the single isolate experiment was of approximately the same range as that of the littoral-exposed leaves in the last stage (days 29-43) of the exposure experiment.

Hence, it has been suggested that autoclaved leaves constitute an appropriate model system for leached leaves (AlSmann et ai., 2009, 2010).

Compared to the initial parameters of the auto- claved leaves, all leaf parameters were affected by the colonisation by single isolates. However, the magni- tude of the effects was strongly strain-specific, in agreement with Gra~a et ai. (1993a) and Rong et al.

(1995).

The majority of the single isolates were preferred over control leaves, and none were rejected. Although this corroborates the findings of Gra~a et ai., 1993a, 1994a), it is in contrast to Rong et al. (1995), who reported that a third of the tested hyphomycetes were not fed upon. In agreement with Gra~a et ai. (1994b) and Rong et ai. (1995), consumption rate varied considerably between strains, because of specific effects on leaf parameters.

Polyphenols can be actively degraded by fungi (Bhat, Singh & Sharma, 1998), and the statistical analysis of our single isolate experiment revealed polyphenol and protein levels to be of particular importance in determining consumption rate of G. roeselii, indicating that fungi and oomycetes may indirectly determine consumption by altering the leaf litter content of protein and polyphenols. The inter- pretation of the consumption rates in the single isolate experiments deserves a more detailed analysis regard- ing the effects of protein content. We noted a strain- specific increase in protein content for all isolates, confirming Barlocher & Kendrick (1973) and Rong et al. (1995), and with only one exception, the isolated strains lead to higher consumption rates of the leaf discs. However, on a quantitative basis, we found a negative relationship between consumption rate and protein content. We conclude that colonisation of leaf discs is associated with rising protein content and a higher consumption rate by shredders. However, the

protein content is not the ultimate cause of the inten- sified consumption, because the strains achieving the highest protein content were not those most strongly consumed.

The importance of polyphenols and proteins strongly indicates that the observed strain-specific effects on consumption by G. roeselii are attributable to strain-specific effects on these two leaf parameters.

In earlier studies with single strains of fungi, leaf parameters were not correlated with the preference of Gammarus spp. (Gra~a et aI., 1993a, 1994a; Rong et aI., 1995), which led to the assumption that feeding stimulants or distasteful compounds explained the strain-specific effects on consumption by shredders (Arsuffi & Suberkropp, 1989). In our study, however, the strong correlation between preference and poly- phenol and protein content does not point to the involvement of attractants or repellents, supporting the conclusion of ABmann & von Elert (2009).

Role of oomycetes in leaf conditioning

Although the importance of fungi as microbial decomposers of leaves is widely acknowledged, the role of oomycetes in leaf conditioning is not well understood, a fact that is partly attributable to the absence of an easily accessible parameter for the determination of abundance. The five strains of oomycetes that we isolated belong to Pythium

(PRINGSHEIM), a genus that is well represented in freshwater habitats (Dix & Webster, 1995) and has been regularly found on conditioned leaf litter in streams and lakes (Chamier, Dixon & Archer, 1984;

Dix & Webster, 1995; Nechwatal & Mendgen, 2006;

Wielgoss et al., 2009).

This study is the first report in which oomycete isolates were found to positively affect Gammarus consumption rates. This positive effect was not different from that of fungi, but colonisation by oomycetes resulted in higher values for leaf toughness and polyphenol content and lower P content of the autoclaved leaves than colonisation by fungi. This strongly suggests that colonisation by oomycetes results in a lower food quality for shredders than colonisation by fungi. Because of the presence of oomycetes in the early stages of conditioning (Wiel- goss et aI., 2009), our findings indicate that oomycetes may have a greater impact on early leaf litter decom- position in freshwaters and on the coupling of this

process to higher trophic levels than hitherto as- sumed.

Acknowledgments

We thank two anonymous reviewers who contributed substantially to the improvement in the study. We are grateful to M.O. Gessner for substantial help in establishing the ergosterol analysis, C. Gebauer and P. Merkel for their assistance with leaf litter analyses, C. Geiss for her assistance with the food choice assays and M. Wolf for help in building experimental components. F. Bartlett helped with English editing.

This study was supported by a grant to E. v. E. from the German Research Foundation (DFG) within the Collaborative Research Centre SFB 454 Littoral Zone of Lake Constance.

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