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Light intensity increases the susceptibility of Vallisneria natans to snail herbivory
Aquatic Botany 81 (2005) 265–275 www.elsevier.com/locate/aquabot
Light intensity increases the susceptibility of Vallisneria natans to snail herbivory Yongke Li, Dan Yu *, Xinwei Xu, Yonghong Xie Laboratory of Aquatic Plants, College of Life Sciences, Wuhan University, Wuhan 430072, PR China Received 15 October 2003; received in revised form 5 January 2005; accepted 7 January 2005
Abstract Palatability to snail herbivory (Radix swinhoei H. Adams) and C/N ratios were assessed for Vallisneria natans (Lour.) Hara, in three different experimental light regimes (midday fluxes respectively 280 mmol m2 s1, 15 mmol m2 s1, and a variable intensity between these two). Higher light intensity as well as prolonged photoperiods increased palatability and growth, and improved C/N ratio by decreasing N content. Snail growth was slightly increased but juvenile survivorship decreased under higher light. The results suggest that the availability of light may affects intraspecific variation in palatability of V. natans. # 2005 Elsevier B.V. All rights reserved. Keywords: CNB hypothesis; Light; Palatability; Resource availability; Snail; Submerged plant
1. Introduction It is well documented that invertebrate herbivory can influence relative abundance and diversity of macrophyte species in lake communities (Sheldon, 1987; Jacobsen and SandJensen, 1992; Gross et al., 2001; Pieczyn´ska, 2003). Some macrophyte tissue is assumed to be protected against herbivore attacks by repellent substances or a hard texture (Bro¨nmark, 1990; Underwood, 1991). However, numerous submerged plants lacking tougher surface and secondary compounds are reportedly grazed only little (Otto and Svensson, 1981; Lodge, 1991). Several hypotheses have been raised to explain interspecific and * Corresponding author. Tel.: +86 27 87686834; fax: +86 27 87882661. E-mail address: [email protected] (D. Yu). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.01.005
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intraspecific variation in plant palatability (Bryant et al., 1983; Coley et al., 1985; Yates and Peckol, 1993; Cronin and Hay, 1996; Bolser et al., 1998; Cebria´ n and Duarte, 1998; Dorn et al., 2001; Elger et al., 2002; Cronin and Lodge, 2003; Elger and Willby, 2003). Plant palatability has been explained by the resource availability theory (Coley et al., 1985), which hypothesizes that the strategy of plants coping with herbivory is related to availability of abiotic resources needed for plant growth (such as water, nutrients and light). Firstly, the theory predicts that levels of anti-herbivore defense should be high for plant species adapted to resource-limited habitats and with low vegetative turnover. Secondly, it argues that terrestrial plants often utilize carbon-based defenses (such as phenolics) rather than nitrogen-based ones, since nutrients are often more limiting than light. Thus, when carbon availability is high relative to nitrogen supply, an excess of carbon would be allocated to carbon-based defensive substances (Bryant et al., 1983; Vergeer and van der Velde, 1997). The theory has been confirmed in terrestrial plants (Dudt and Shure, 1994; Crone and Jones, 1999), but less so in freshwater ecosystems (but see Elger et al., 2002; Cronin and Lodge, 2003; Elger and Willby, 2003). Light is often a limiting and variable factor for submerged plants in lakes (Chambers and Kalff, 1985; Durate et al., 1987), and the same was observed in the Yangtze River Basin due to seasonal flooding (Yang et al., 2004). Based on this information, we chose light as an experimental factor to evaluate the influences of resource availability on intraspecific palatability of freshwater plants by using pulmonate snails. Pulmonate herbivory was found to be suitable to evaluate palatability (Elger et al., 2002). We asked the following questions: (1) Do snails have a preference for plants grown under specific conditions of light intensity? (2) If so, is this preference conforming to the resource availability hypothesis? (3) Is snail performance (growth, survivorship) on plants growing in different light intensity consistent with snail feeding preference?
2. Materials and methods 2.1. Plant and snail species Vallisneria natans (Lour.) Hara. is a perennial submerged plant forming rosettes of straplike leaves at the hooks of a creeping rhizome. Radix swinhoei (H. Adams) is a freshwater snail commonly found in flooded pools or shallow regions of Liangzi Lake (in Hubei province of China, 308160 N, 1148330 E). These snails are generalist plant-grazers and detritus-feeders, which may associate with living macrophytes (e.g. Spirodela polyrhiza, Trapa japonica, Potamogeton crispus, and V. natans), and decomposed plant material. 2.2. Experiment 1: Measuring plant growth Juvenile V. natans (3–4 leaves, 9–17 cm leaf length) germinated from seeds were transplanted to ten high-density polyethylene buckets (height = 27 cm, top diameter = 30 cm, had 16 L tap water and 6 cm sediment mixed with 3 g commercial compost) in a greenhouse. Three of these buckets always received ambient greenhouse daylight, which denoted by H-buckets (high light, averaged 280 and 85 mmol m2 s1 at 11:00 a.m. for
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sunny, and cloudy days, respectively). Three other buckets always received shading by black plastic net, which denoted by L-buckets (low light, averaged 15 and 5 mmol m2 s1, respectively). mmol m2 s1 was calculated from luxmeter by the equation: 1 K lx = 18 mmol m2 s1 (McCree, 1981). Three light observations were used per bucket. The remaining four were regularly swapped between the H and L condition once a week, which denoted by V-buckets (variable light). Of these four V-buckets, two firstly received light (HL) whilst the others firstly received shading (LH). Temperature was 18– 24 8C (heated by central heating when necessary); water temperature differences were not more than 2 8C among buckets. Water was renewed once a week. The experiment lasted from March 4 to May 28, 2003, and started 15 days after transplanting. Total leaf number (N), and length (H) and width (lwid) of the longest leaf were recorded for each plant (or ramet) at 6 days intervals (total four times). The relative initiating rate of new leaves (NGR) and relative growth rate (RGR) were calculated for each sampling interval according to the following formulas: NGR ¼ ln
ðNf =Ni Þ ; days
RGR ¼ ln
ðWf =Wi Þ days
where Nf and Ni is final and initial leaf number (dry mass) per bucket, Wf and Wi is final and initial dry mass per bucket, respectively. Dry mass estimates (Wf and Wi) were obtained nondestructively using allometric relationship, that is plant (ramet) drymass = TA/SLA/ LWR (Causton and Venus, 1981), where TA, SLA, and LWR are total leaf area per plant (ramet), specific leaf area and leaf weight ratio, respectively. SLA and LWR were assumed to be nearly constant during the short time interval. Therefore, RGR was determined by summary of TA per bucket. We did the regression of total leaf length (TL) with H N, which was fitted to a power line [TL = (H N)0.896 + 1.042, R2 = 0.92, 56 shoots], then estimated TA from multiplying TL by lwid. At harvest, plant leaves were oven dried at 70 8C, and ground into fine powder for chemical analysis. 2.3. Experiment 2: Assessing plant palatability Experimental snails were collected from Liangzi Lake, kept in tanks under dripping water and starved for 3 day before each trial. The V. natans leaves used in bioassay were freshly collected from experiment 1 after week 8. Equal amounts of leaf segments (central sections of mature leaves, 5–8 cm length, 0.4–0.6 cm width, 350 cm2 areas, being washed and no epiphytic coverage noticeable) were cut from each bucket and pooled together according to different light treatments. Two or three pairs of segments were placed in one plastic cup (400 ml, contained 8 cm depth of dechlorinated tap water) with a snail for 48 h. There were 26, 45, and 50 cups for H versus L, L versus V, and H versus V, respectively. The cups were covered by net and placed indoors in laboratory, with temperature of 22 4 8C and natural room light. The segment area images were scanned before and after feeding among plastic sheets or microscope slides. Then the pre- and post-feeding images were compared using Photoshop 7.0 software (Adobe Systems Inc.).The two tips of leaf segments were shaped into various kinds of markers (with shapes like ‘‘P’’, ‘‘L’’, ‘‘M’’, or ‘‘N’’ on both ends of a leaf strip) beforehand to identify the corresponding segments. Area was calculated by counting the
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green pixels occupied on the image (118.112 pixel/cm2 resolution was used). Daily feed consumption (DFC, cm2 g1 day1) was calculated in terms of area eaten per gram ash free dry weight of shell free snails (shell-free AFDW) in 1 day. Any cup containing a dead snail or leaf fraction indistinguishable from each other was rejected (<15%). Non-choice test of leaf consumption was carried out with five trials. Each trial had per treatment 16 feeding cups that holding single leaves with individual snails. One trial calculating on dry mass consumption paired the feeding cups with non-snail controls, following the procedure by Elger et al. (2002). The other four trials were based on the area consumption described above. 2.4. Experiment 3: Evaluating snail performance Twenty-four small buckets (height = 22 cm, top diameter = 20 cm, five plants per bucket) were divided into three equal groups to received H, V and L treatment separately. Supplemental light with metal halide bulbs and natural room light gave total irradiances averaged 125, and 20 mmol m2 s1 (observed three times) for H and L treatment, respectively. Room temperature was 25 4 8C. One month later, five juvenile snails with 0.3 g wet Wt. each, were introduced to six buckets (denoted by +S) of each group, the other two (denoted by S) served as plant controls, and additional two buckets with snails only were added to each group as snail controls. The buckets were covered by white nylon net to confine snails. The shell width, wet weight of individual snails, and snail survival rate were recorded at biweekly intervals (total three times). After 45 days, plants were harvested, divided into leaves, roots and other components, oven dried at 70 8C, and weighted, then ground for chemical analysis. Specific growth rate (SGR, %/day; Rosen et al., 2000) was calculated for the snail survivals in a bucket. 2.5. Leaf chemical analyses Leaf N content was determined by micro-Kjeldahl method and C content by wet oxidation method (Moore and Chapman, 1986). Three sub-samples were used to obtain a bucket mean. Ash content (%) was determined in 0.5 g of pooled leaf samples. Polyphenolic content was determined by Folin-Ciocalteau method (Choi et al., 2002), after leaves being dried (40 8C), ground, and extracted (with 50% acetone). 2.6. Statistical analyses Repeated-measures ANOVA was used to evaluate the light effect on the NGR and approximate RGR in experiment 1, with time intervals as a repeated measures variable. Snail feeding rates were compared using two-way ANOVAs (with light as a fixed effect and snail as a random effect) and separate paired t-tests (area eaten of each snail combined, Crone and Jones, 1999). The residuals of ANOVAs excluded among-snail variability, and were found to be normally-distributed when DFC values were log(x + 1) transformed. After log-transformation, results of paired t-tests were similar to those of ANOVAs. All statistical procedures were performed using SPSS software (SPSS Inc., version 11.5).
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Fig. 1. Relative initiating rate of new leaves (NGR) and approximate relative growth rate (RGR) of V. natans under different light conditions. Bars show 1S.E.
3. Results 3.1. Plant growth Of experiment 1, relative growth rate of V. natans in high light intensity was about twice as high as in the shade (Fig. 1). Repeated measures ANOVA of light effects on growth rates revealed significant or marginally significant results ( F 2,7 = 8.27, p = 0.014 for NGR; and F 2,7 = 4.21, p = 0.063 for RGR). The growth of plants was not significantly different between HL and LH treatments (t-test, p = 0.762, d.f. = 10 for NGR; and p = 0.332, d.f. = 10 for RGR). In experiment 3, the final production of dry mass averaged 1.10 0.11 (S.E., the same below), 0.49 0.07, and 0.27 0.05 g for H-, V-, and L-bucket, respectively; 0.73 0.16 and 0.58 0.10 g for +S and S bucket, respectively. Two way ANOVA testing light and snail effect showed only light effect was significant (explained 53% of total variation, p < 0.001) on dry mass production. Whereas snail effect (explained 8% of total variation, p = 0.073) and the interactive effect between snail and light (explained 2% of total variation, p = 0.595) was not significant. 3.2. Leaf palatability The DFC value ranged from 0.99 to 59.25 cm2 g1 day1 in the choice tests. Palatabilities estimated based on DFC ranked H > V > L (Table 1). Paired t-tests showed all the consumption differences were significantly differed (Table 1). In addition, we observed a strong snail light effect when using ANOVAs testing the palatability (the interactive effect explained 27% of total variation, p = 0.013 for H versus V, and 19.3%, 0.042 for V versus L, respectively). A possible distinction between palatabilities of HL-plants and LH-plants was assessed. The DFC difference between HL and LH was
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Table 1 Comparisons of palatability of V. natans based on DFC (cm2 g1 day1) obtained in dual choice tests Dual choice tests H vs. V Valid N Mean Minimum Maximum S.E.
V vs. L
43 20.98 0.55 59.25 2.56
Difference Paired-t P-value Palatability
43 13.75 0.48 47.64 2.20 7.23 2.15 0.038 H>V
H vs. L
41 26.58 0.99 51.86 2.20
41 7.36 0.59 34.48 1.41 19.22 5.90 <106 V>L
22 17.96 0.14 37.66 1.99
22 2.90 0.45 12.07 0.99 15.06 8.04 <107 H>L
Mean differences were tested by paired-t tests after log-transformation.
1.32 cm2 g1 day1 for pair of H versus V (t =0.67, d.f. = 84, p = 0.505), and 3.00 cm2 g1 day1 for pair of V versus L (t = 2.00, d.f. = 120, p = 0.048). In the non-choice tests, DFC on dry mass consumption ranged from 37.7 13.2 mg g1 day1 for H to 29.9 9.7 mg g1 day1 for L (Kruskal–Wallis Test, d.f. = 2, n = 55, H = 0.41, p > 0.05). DFC on leaf area consumption did not show significant differences among treatments either (Fig. 2). 3.3. Snail performance Survivorship was higher for snails with L-plants than that of snails with H-plants and Vplants at the middle interval of experiment 3 (Fig. 3, Chi-square test, x2 = 7.23, d.f. = 2, p = 0.027). While there was no significant difference for the controlled (without plants) buckets (x2 = 1.53, d.f. = 2, p = 0.466). Snail growth was not significantly enhanced in high light buckets (Table 2).
Fig. 2. Feeding rates (cm2 g1 day1) of V. natans to snails in non-choice tests under different light availability (ANOVA result: F3,123 = 1.945, P = 0.123). Bars show 1S.E.
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Fig. 3. The time course of snail survivorship with or without V. natans as influenced by different light availability (H, V and L). Bars show 1S.E. Table 2 Kruskal–Wallis test of SGR (%/day) of snails with H-, V-, and L-plants Date interval
17/4–29/4 29/4–15/5
Mean rank of SGR H
V
L
11.0 12.2
9.8 10.5
7.7 5.8
N
H
d.f.
P
18 18
1.2 4.5
2 2
0.548 0.103
3.4. Leaf chemistry The C/N ratio increased with the increasing light availability in experiments 1 and 3 (Tables 3 and 4). Leaf N content was more likely to be affected than C content, as indicate by only one set of significant differences occurring in C content. Two way ANOVAs of Table 3 Comparison of carbon C (mg/g DW, 1S.E.), nitrogen N (mg/g DW, 1S.E.), C/N ratios and ash contents (% of DW) of V. natans leaves sampled in experiment 1 Treatment
Sampling date
C content
N content ab
C/N ratio
Ash
H
April 17 May 24
320.0 9.2 328.9 3.4a
28.6 0.5 24.3 0.6a
11.2 13.5
14.2 14.9
LH
April 17 May 24
316.0 6.2 323.8 4.3ab
28.0 0.6a 28.0 0.4b
11.3 11.6
12.4 14.0
HL
April 17 May 24
317.2 2.7 329.6 3.1ab
30. 5 0.8b 30.6 0.6b
10.4 10.8
11.8 11.2
L
April 17 May 24
332.6 3.4 310.1 7.9b
31.3 1.0ab 28.5 1.0b
10.6 10.9
12.2 11.6
Ambient
April 17
290.2 1.8
23.5 0.6
12.4
14.1
Ambient leaves were field collected from Liangzi Lake. Identically lettered superscripts indicate homogeneous groups of treatments (ambient was excluded) in each time identified by Tukey’s HSD test.
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Table 4 Comparison of carbon C (mg/g DW, 1S.E.), nitrogen N (mg/g DW, 1S.E.), C/N ratios and ash contents (% of DW) of V. natans leaves harvested at the end of experiment 3 Treatment
C content
N content
C/N ratio
Ash
H+S HS V+S VS L+S LS
337.7 2.1 330.1 0.3 342.9 4.0 355.9 3.2 337.1 2.1 334.7 0.4
26.3 0.6a 28.3 1.1ab 30.1 0.3bc 31.6 0.7c 31.7 0.8c 31.4 0.4c
12.8 11.7 11.4 11.3 10.6 10.7
13.7 15.3 13.4 15.0 15.6 16.2
Treatment indicates different light levels (H, V and L) and different grazing pressures (+S and –S). Identically lettered superscripts indicate homogeneous groups identified by Tukey’s HSD test.
Fig. 4. Comparison of the phenolic contents (expressed as TAE, i.e. tannin acid equivalents) of V. natans under different light conditions. Also showing contents of all reducing compounds (PVPP, i.e. not adding polyvinylpolypyrrolidone) and those from non-phenolic origin (+PVPP). Bars show standard error of six sub-samples.
light and date in Table 3 showed that, both light ( F 3,12 = 6.16, p < 0.01) and date ( F 1,12 = 36.22, p < 0.001) had significant effects on N content, but not on C content. Two way ANOVAs of light and snail in Table 4 showed that N content ( p < 0.001) rather than C content ( p = 0.759) was affected by light. Furthermore, leaf N content was not affected by snail ( F 1,12 = 3.03, p = 0.107). High light intensity led to a significantly high content in leaf polyphenolics (Fig. 4).
4. Discussion 4.1. Snail preference Increase of light availability resulted in greater leaf consumption in choice tests, indicating that light condition has detectable effect on plant palatability. The feeding choices showed a rank of snail preference was high light plant > moderate-high light
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plant > low light plant. This rank is consistent with the results observed in some terrestrial plants (Karban and Thaler, 1999), which indicated light promoted plant defenses, but is contrary to Crone and Jones (1999). For submerged plants, Cronin and Lodge (2003) found no detectable difference between shaded and unshaded leaves of Potamogeton amplifolius. However, snail preference did not have an apparent pattern in non-choice test. The nonchoice test revealed non-significant results and even a departure from the expectation of the choice test. This may result from the inaccuracy substitute of preferences by consumption rates in non-choice test, or choice test may amplify the consequence of palatability differences. There is no notably interactive effect of light and the snail grazing on plant production in experiment 3, which implies that the grazing impact is not distinguished by treatment. 4.2. Role of resource availability Adaptive growth rates had been induced for V. natans, mostly due to the differences in light availability. Increased light exposure also resulted in higher polyphenolics accumulation and C/N ratio in leaves, which was consistent with the carbon–nitrogen balance (CNB) hypothesis (Bryant et al., 1983). It would indicate that the variation in palatability of V. natans is in accordance with the resource availability theory, if the snail preference differences detected in choice test were intrinsically related to growth rates. The leaf segments used were washed; hence the influence of epiphytes was little. However, the leaves of low-light and variable-light plants were observed to be narrower than those of high-light plants; thus the higher consumption of high-light plants could also be related to their wider leaves that may offer a more favorable surface to snails. Snail preferences in neither choice nor non-choice test were negatively related to leaf polyphenolics content, which is contrary to the CNB hypothesis. However, the polyphenolics content was quite low for V. natans (only about 10 mg/g DW); same was observed in most species of submersed plants (Smolders et al., 2000). Such a low concentration may not result in apparent responses of snail herbivores (Lodge, 1991; Bolser et al., 1998). Some studies even suggested that aquatic invertebrate herbivores have digestive strategies of overcoming the deleterious effects of polyphenolics (Grac¸a and Ba¨ rlocher, 1998; Gross et al., 2001). The average C (330 mg/g) and N (29 mg/g) contents measured for V. natans fit within the range of most freshwater macrophytes established by Duarte (1992). The N level was slightly decreased by high light (5–17% lower relative to shaded), which led to an increase of the C/N ratio. However, C/N ratio is not necessarily a repellent factors for snails (Otto and Svensson, 1981; Grantham et al., 1993), hence the snail preference did not relate to C/ N ratio, as suggested by this study. 4.3. Snail performance Snail growth rates were quite low and death rates were high, suggesting suboptimal growing conditions in the buckets (perhaps due to the use of tap water). Shading favored survivorship when snails were with plants; this is related to snail preference in neither choice nor non-choice tests. It is not ascertained whether the high mortality rate of snails
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Light intensity increases the susceptibility of Vallisneria natans to snail herbivory Yongke Li, Dan Yu *, Xinwei Xu, Yonghong Xie Laboratory of Aquatic Plants, College of Life Sciences, Wuhan University, Wuhan 430072, PR China Received 15 October 2003; received in revised form 5 January 2005; accepted 7 January 2005
Abstract Palatability to snail herbivory (Radix swinhoei H. Adams) and C/N ratios were assessed for Vallisneria natans (Lour.) Hara, in three different experimental light regimes (midday fluxes respectively 280 mmol m2 s1, 15 mmol m2 s1, and a variable intensity between these two). Higher light intensity as well as prolonged photoperiods increased palatability and growth, and improved C/N ratio by decreasing N content. Snail growth was slightly increased but juvenile survivorship decreased under higher light. The results suggest that the availability of light may affects intraspecific variation in palatability of V. natans. # 2005 Elsevier B.V. All rights reserved. Keywords: CNB hypothesis; Light; Palatability; Resource availability; Snail; Submerged plant
1. Introduction It is well documented that invertebrate herbivory can influence relative abundance and diversity of macrophyte species in lake communities (Sheldon, 1987; Jacobsen and SandJensen, 1992; Gross et al., 2001; Pieczyn´ska, 2003). Some macrophyte tissue is assumed to be protected against herbivore attacks by repellent substances or a hard texture (Bro¨nmark, 1990; Underwood, 1991). However, numerous submerged plants lacking tougher surface and secondary compounds are reportedly grazed only little (Otto and Svensson, 1981; Lodge, 1991). Several hypotheses have been raised to explain interspecific and * Corresponding author. Tel.: +86 27 87686834; fax: +86 27 87882661. E-mail address: [email protected] (D. Yu). 0304-3770/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquabot.2005.01.005
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intraspecific variation in plant palatability (Bryant et al., 1983; Coley et al., 1985; Yates and Peckol, 1993; Cronin and Hay, 1996; Bolser et al., 1998; Cebria´ n and Duarte, 1998; Dorn et al., 2001; Elger et al., 2002; Cronin and Lodge, 2003; Elger and Willby, 2003). Plant palatability has been explained by the resource availability theory (Coley et al., 1985), which hypothesizes that the strategy of plants coping with herbivory is related to availability of abiotic resources needed for plant growth (such as water, nutrients and light). Firstly, the theory predicts that levels of anti-herbivore defense should be high for plant species adapted to resource-limited habitats and with low vegetative turnover. Secondly, it argues that terrestrial plants often utilize carbon-based defenses (such as phenolics) rather than nitrogen-based ones, since nutrients are often more limiting than light. Thus, when carbon availability is high relative to nitrogen supply, an excess of carbon would be allocated to carbon-based defensive substances (Bryant et al., 1983; Vergeer and van der Velde, 1997). The theory has been confirmed in terrestrial plants (Dudt and Shure, 1994; Crone and Jones, 1999), but less so in freshwater ecosystems (but see Elger et al., 2002; Cronin and Lodge, 2003; Elger and Willby, 2003). Light is often a limiting and variable factor for submerged plants in lakes (Chambers and Kalff, 1985; Durate et al., 1987), and the same was observed in the Yangtze River Basin due to seasonal flooding (Yang et al., 2004). Based on this information, we chose light as an experimental factor to evaluate the influences of resource availability on intraspecific palatability of freshwater plants by using pulmonate snails. Pulmonate herbivory was found to be suitable to evaluate palatability (Elger et al., 2002). We asked the following questions: (1) Do snails have a preference for plants grown under specific conditions of light intensity? (2) If so, is this preference conforming to the resource availability hypothesis? (3) Is snail performance (growth, survivorship) on plants growing in different light intensity consistent with snail feeding preference?
2. Materials and methods 2.1. Plant and snail species Vallisneria natans (Lour.) Hara. is a perennial submerged plant forming rosettes of straplike leaves at the hooks of a creeping rhizome. Radix swinhoei (H. Adams) is a freshwater snail commonly found in flooded pools or shallow regions of Liangzi Lake (in Hubei province of China, 308160 N, 1148330 E). These snails are generalist plant-grazers and detritus-feeders, which may associate with living macrophytes (e.g. Spirodela polyrhiza, Trapa japonica, Potamogeton crispus, and V. natans), and decomposed plant material. 2.2. Experiment 1: Measuring plant growth Juvenile V. natans (3–4 leaves, 9–17 cm leaf length) germinated from seeds were transplanted to ten high-density polyethylene buckets (height = 27 cm, top diameter = 30 cm, had 16 L tap water and 6 cm sediment mixed with 3 g commercial compost) in a greenhouse. Three of these buckets always received ambient greenhouse daylight, which denoted by H-buckets (high light, averaged 280 and 85 mmol m2 s1 at 11:00 a.m. for
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sunny, and cloudy days, respectively). Three other buckets always received shading by black plastic net, which denoted by L-buckets (low light, averaged 15 and 5 mmol m2 s1, respectively). mmol m2 s1 was calculated from luxmeter by the equation: 1 K lx = 18 mmol m2 s1 (McCree, 1981). Three light observations were used per bucket. The remaining four were regularly swapped between the H and L condition once a week, which denoted by V-buckets (variable light). Of these four V-buckets, two firstly received light (HL) whilst the others firstly received shading (LH). Temperature was 18– 24 8C (heated by central heating when necessary); water temperature differences were not more than 2 8C among buckets. Water was renewed once a week. The experiment lasted from March 4 to May 28, 2003, and started 15 days after transplanting. Total leaf number (N), and length (H) and width (lwid) of the longest leaf were recorded for each plant (or ramet) at 6 days intervals (total four times). The relative initiating rate of new leaves (NGR) and relative growth rate (RGR) were calculated for each sampling interval according to the following formulas: NGR ¼ ln
ðNf =Ni Þ ; days
RGR ¼ ln
ðWf =Wi Þ days
where Nf and Ni is final and initial leaf number (dry mass) per bucket, Wf and Wi is final and initial dry mass per bucket, respectively. Dry mass estimates (Wf and Wi) were obtained nondestructively using allometric relationship, that is plant (ramet) drymass = TA/SLA/ LWR (Causton and Venus, 1981), where TA, SLA, and LWR are total leaf area per plant (ramet), specific leaf area and leaf weight ratio, respectively. SLA and LWR were assumed to be nearly constant during the short time interval. Therefore, RGR was determined by summary of TA per bucket. We did the regression of total leaf length (TL) with H N, which was fitted to a power line [TL = (H N)0.896 + 1.042, R2 = 0.92, 56 shoots], then estimated TA from multiplying TL by lwid. At harvest, plant leaves were oven dried at 70 8C, and ground into fine powder for chemical analysis. 2.3. Experiment 2: Assessing plant palatability Experimental snails were collected from Liangzi Lake, kept in tanks under dripping water and starved for 3 day before each trial. The V. natans leaves used in bioassay were freshly collected from experiment 1 after week 8. Equal amounts of leaf segments (central sections of mature leaves, 5–8 cm length, 0.4–0.6 cm width, 350 cm2 areas, being washed and no epiphytic coverage noticeable) were cut from each bucket and pooled together according to different light treatments. Two or three pairs of segments were placed in one plastic cup (400 ml, contained 8 cm depth of dechlorinated tap water) with a snail for 48 h. There were 26, 45, and 50 cups for H versus L, L versus V, and H versus V, respectively. The cups were covered by net and placed indoors in laboratory, with temperature of 22 4 8C and natural room light. The segment area images were scanned before and after feeding among plastic sheets or microscope slides. Then the pre- and post-feeding images were compared using Photoshop 7.0 software (Adobe Systems Inc.).The two tips of leaf segments were shaped into various kinds of markers (with shapes like ‘‘P’’, ‘‘L’’, ‘‘M’’, or ‘‘N’’ on both ends of a leaf strip) beforehand to identify the corresponding segments. Area was calculated by counting the
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green pixels occupied on the image (118.112 pixel/cm2 resolution was used). Daily feed consumption (DFC, cm2 g1 day1) was calculated in terms of area eaten per gram ash free dry weight of shell free snails (shell-free AFDW) in 1 day. Any cup containing a dead snail or leaf fraction indistinguishable from each other was rejected (<15%). Non-choice test of leaf consumption was carried out with five trials. Each trial had per treatment 16 feeding cups that holding single leaves with individual snails. One trial calculating on dry mass consumption paired the feeding cups with non-snail controls, following the procedure by Elger et al. (2002). The other four trials were based on the area consumption described above. 2.4. Experiment 3: Evaluating snail performance Twenty-four small buckets (height = 22 cm, top diameter = 20 cm, five plants per bucket) were divided into three equal groups to received H, V and L treatment separately. Supplemental light with metal halide bulbs and natural room light gave total irradiances averaged 125, and 20 mmol m2 s1 (observed three times) for H and L treatment, respectively. Room temperature was 25 4 8C. One month later, five juvenile snails with 0.3 g wet Wt. each, were introduced to six buckets (denoted by +S) of each group, the other two (denoted by S) served as plant controls, and additional two buckets with snails only were added to each group as snail controls. The buckets were covered by white nylon net to confine snails. The shell width, wet weight of individual snails, and snail survival rate were recorded at biweekly intervals (total three times). After 45 days, plants were harvested, divided into leaves, roots and other components, oven dried at 70 8C, and weighted, then ground for chemical analysis. Specific growth rate (SGR, %/day; Rosen et al., 2000) was calculated for the snail survivals in a bucket. 2.5. Leaf chemical analyses Leaf N content was determined by micro-Kjeldahl method and C content by wet oxidation method (Moore and Chapman, 1986). Three sub-samples were used to obtain a bucket mean. Ash content (%) was determined in 0.5 g of pooled leaf samples. Polyphenolic content was determined by Folin-Ciocalteau method (Choi et al., 2002), after leaves being dried (40 8C), ground, and extracted (with 50% acetone). 2.6. Statistical analyses Repeated-measures ANOVA was used to evaluate the light effect on the NGR and approximate RGR in experiment 1, with time intervals as a repeated measures variable. Snail feeding rates were compared using two-way ANOVAs (with light as a fixed effect and snail as a random effect) and separate paired t-tests (area eaten of each snail combined, Crone and Jones, 1999). The residuals of ANOVAs excluded among-snail variability, and were found to be normally-distributed when DFC values were log(x + 1) transformed. After log-transformation, results of paired t-tests were similar to those of ANOVAs. All statistical procedures were performed using SPSS software (SPSS Inc., version 11.5).
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Fig. 1. Relative initiating rate of new leaves (NGR) and approximate relative growth rate (RGR) of V. natans under different light conditions. Bars show 1S.E.
3. Results 3.1. Plant growth Of experiment 1, relative growth rate of V. natans in high light intensity was about twice as high as in the shade (Fig. 1). Repeated measures ANOVA of light effects on growth rates revealed significant or marginally significant results ( F 2,7 = 8.27, p = 0.014 for NGR; and F 2,7 = 4.21, p = 0.063 for RGR). The growth of plants was not significantly different between HL and LH treatments (t-test, p = 0.762, d.f. = 10 for NGR; and p = 0.332, d.f. = 10 for RGR). In experiment 3, the final production of dry mass averaged 1.10 0.11 (S.E., the same below), 0.49 0.07, and 0.27 0.05 g for H-, V-, and L-bucket, respectively; 0.73 0.16 and 0.58 0.10 g for +S and S bucket, respectively. Two way ANOVA testing light and snail effect showed only light effect was significant (explained 53% of total variation, p < 0.001) on dry mass production. Whereas snail effect (explained 8% of total variation, p = 0.073) and the interactive effect between snail and light (explained 2% of total variation, p = 0.595) was not significant. 3.2. Leaf palatability The DFC value ranged from 0.99 to 59.25 cm2 g1 day1 in the choice tests. Palatabilities estimated based on DFC ranked H > V > L (Table 1). Paired t-tests showed all the consumption differences were significantly differed (Table 1). In addition, we observed a strong snail light effect when using ANOVAs testing the palatability (the interactive effect explained 27% of total variation, p = 0.013 for H versus V, and 19.3%, 0.042 for V versus L, respectively). A possible distinction between palatabilities of HL-plants and LH-plants was assessed. The DFC difference between HL and LH was
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Table 1 Comparisons of palatability of V. natans based on DFC (cm2 g1 day1) obtained in dual choice tests Dual choice tests H vs. V Valid N Mean Minimum Maximum S.E.
V vs. L
43 20.98 0.55 59.25 2.56
Difference Paired-t P-value Palatability
43 13.75 0.48 47.64 2.20 7.23 2.15 0.038 H>V
H vs. L
41 26.58 0.99 51.86 2.20
41 7.36 0.59 34.48 1.41 19.22 5.90 <106 V>L
22 17.96 0.14 37.66 1.99
22 2.90 0.45 12.07 0.99 15.06 8.04 <107 H>L
Mean differences were tested by paired-t tests after log-transformation.
1.32 cm2 g1 day1 for pair of H versus V (t =0.67, d.f. = 84, p = 0.505), and 3.00 cm2 g1 day1 for pair of V versus L (t = 2.00, d.f. = 120, p = 0.048). In the non-choice tests, DFC on dry mass consumption ranged from 37.7 13.2 mg g1 day1 for H to 29.9 9.7 mg g1 day1 for L (Kruskal–Wallis Test, d.f. = 2, n = 55, H = 0.41, p > 0.05). DFC on leaf area consumption did not show significant differences among treatments either (Fig. 2). 3.3. Snail performance Survivorship was higher for snails with L-plants than that of snails with H-plants and Vplants at the middle interval of experiment 3 (Fig. 3, Chi-square test, x2 = 7.23, d.f. = 2, p = 0.027). While there was no significant difference for the controlled (without plants) buckets (x2 = 1.53, d.f. = 2, p = 0.466). Snail growth was not significantly enhanced in high light buckets (Table 2).
Fig. 2. Feeding rates (cm2 g1 day1) of V. natans to snails in non-choice tests under different light availability (ANOVA result: F3,123 = 1.945, P = 0.123). Bars show 1S.E.
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Fig. 3. The time course of snail survivorship with or without V. natans as influenced by different light availability (H, V and L). Bars show 1S.E. Table 2 Kruskal–Wallis test of SGR (%/day) of snails with H-, V-, and L-plants Date interval
17/4–29/4 29/4–15/5
Mean rank of SGR H
V
L
11.0 12.2
9.8 10.5
7.7 5.8
N
H
d.f.
P
18 18
1.2 4.5
2 2
0.548 0.103
3.4. Leaf chemistry The C/N ratio increased with the increasing light availability in experiments 1 and 3 (Tables 3 and 4). Leaf N content was more likely to be affected than C content, as indicate by only one set of significant differences occurring in C content. Two way ANOVAs of Table 3 Comparison of carbon C (mg/g DW, 1S.E.), nitrogen N (mg/g DW, 1S.E.), C/N ratios and ash contents (% of DW) of V. natans leaves sampled in experiment 1 Treatment
Sampling date
C content
N content ab
C/N ratio
Ash
H
April 17 May 24
320.0 9.2 328.9 3.4a
28.6 0.5 24.3 0.6a
11.2 13.5
14.2 14.9
LH
April 17 May 24
316.0 6.2 323.8 4.3ab
28.0 0.6a 28.0 0.4b
11.3 11.6
12.4 14.0
HL
April 17 May 24
317.2 2.7 329.6 3.1ab
30. 5 0.8b 30.6 0.6b
10.4 10.8
11.8 11.2
L
April 17 May 24
332.6 3.4 310.1 7.9b
31.3 1.0ab 28.5 1.0b
10.6 10.9
12.2 11.6
Ambient
April 17
290.2 1.8
23.5 0.6
12.4
14.1
Ambient leaves were field collected from Liangzi Lake. Identically lettered superscripts indicate homogeneous groups of treatments (ambient was excluded) in each time identified by Tukey’s HSD test.
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Table 4 Comparison of carbon C (mg/g DW, 1S.E.), nitrogen N (mg/g DW, 1S.E.), C/N ratios and ash contents (% of DW) of V. natans leaves harvested at the end of experiment 3 Treatment
C content
N content
C/N ratio
Ash
H+S HS V+S VS L+S LS
337.7 2.1 330.1 0.3 342.9 4.0 355.9 3.2 337.1 2.1 334.7 0.4
26.3 0.6a 28.3 1.1ab 30.1 0.3bc 31.6 0.7c 31.7 0.8c 31.4 0.4c
12.8 11.7 11.4 11.3 10.6 10.7
13.7 15.3 13.4 15.0 15.6 16.2
Treatment indicates different light levels (H, V and L) and different grazing pressures (+S and –S). Identically lettered superscripts indicate homogeneous groups identified by Tukey’s HSD test.
Fig. 4. Comparison of the phenolic contents (expressed as TAE, i.e. tannin acid equivalents) of V. natans under different light conditions. Also showing contents of all reducing compounds (PVPP, i.e. not adding polyvinylpolypyrrolidone) and those from non-phenolic origin (+PVPP). Bars show standard error of six sub-samples.
light and date in Table 3 showed that, both light ( F 3,12 = 6.16, p < 0.01) and date ( F 1,12 = 36.22, p < 0.001) had significant effects on N content, but not on C content. Two way ANOVAs of light and snail in Table 4 showed that N content ( p < 0.001) rather than C content ( p = 0.759) was affected by light. Furthermore, leaf N content was not affected by snail ( F 1,12 = 3.03, p = 0.107). High light intensity led to a significantly high content in leaf polyphenolics (Fig. 4).
4. Discussion 4.1. Snail preference Increase of light availability resulted in greater leaf consumption in choice tests, indicating that light condition has detectable effect on plant palatability. The feeding choices showed a rank of snail preference was high light plant > moderate-high light
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plant > low light plant. This rank is consistent with the results observed in some terrestrial plants (Karban and Thaler, 1999), which indicated light promoted plant defenses, but is contrary to Crone and Jones (1999). For submerged plants, Cronin and Lodge (2003) found no detectable difference between shaded and unshaded leaves of Potamogeton amplifolius. However, snail preference did not have an apparent pattern in non-choice test. The nonchoice test revealed non-significant results and even a departure from the expectation of the choice test. This may result from the inaccuracy substitute of preferences by consumption rates in non-choice test, or choice test may amplify the consequence of palatability differences. There is no notably interactive effect of light and the snail grazing on plant production in experiment 3, which implies that the grazing impact is not distinguished by treatment. 4.2. Role of resource availability Adaptive growth rates had been induced for V. natans, mostly due to the differences in light availability. Increased light exposure also resulted in higher polyphenolics accumulation and C/N ratio in leaves, which was consistent with the carbon–nitrogen balance (CNB) hypothesis (Bryant et al., 1983). It would indicate that the variation in palatability of V. natans is in accordance with the resource availability theory, if the snail preference differences detected in choice test were intrinsically related to growth rates. The leaf segments used were washed; hence the influence of epiphytes was little. However, the leaves of low-light and variable-light plants were observed to be narrower than those of high-light plants; thus the higher consumption of high-light plants could also be related to their wider leaves that may offer a more favorable surface to snails. Snail preferences in neither choice nor non-choice test were negatively related to leaf polyphenolics content, which is contrary to the CNB hypothesis. However, the polyphenolics content was quite low for V. natans (only about 10 mg/g DW); same was observed in most species of submersed plants (Smolders et al., 2000). Such a low concentration may not result in apparent responses of snail herbivores (Lodge, 1991; Bolser et al., 1998). Some studies even suggested that aquatic invertebrate herbivores have digestive strategies of overcoming the deleterious effects of polyphenolics (Grac¸a and Ba¨ rlocher, 1998; Gross et al., 2001). The average C (330 mg/g) and N (29 mg/g) contents measured for V. natans fit within the range of most freshwater macrophytes established by Duarte (1992). The N level was slightly decreased by high light (5–17% lower relative to shaded), which led to an increase of the C/N ratio. However, C/N ratio is not necessarily a repellent factors for snails (Otto and Svensson, 1981; Grantham et al., 1993), hence the snail preference did not relate to C/ N ratio, as suggested by this study. 4.3. Snail performance Snail growth rates were quite low and death rates were high, suggesting suboptimal growing conditions in the buckets (perhaps due to the use of tap water). Shading favored survivorship when snails were with plants; this is related to snail preference in neither choice nor non-choice tests. It is not ascertained whether the high mortality rate of snails
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with high light plants was caused by intrinsic characteristics of plants, simple physical or chemical variations of water, or amount and composition shifting of periphyton coverage. Some studies showed that snail densities were significantly reduced in shaded enclosures (Lamberti et al., 1989; Thomas and Daldorph, 1991), which due to scant of food. We only observed a slight decrease of snail survival rate and growth rate in shading buckets when plants were absent. Acknowledgements We thank Mr. Jan Vermaat and the two anonymous referees for providing constructive comments. We are grateful for the financial support received from the State Key Basic Research and Development Plan of China (Grant No. G2000046802), the Project of National Natural Science Foundation of China (Grant No. 30300046). References Bolser, R.C., Hay, M.E., Lindquist, N., Fenical, W., Wilso, D., 1998. Chemical defenses of freshwater macrophytes against crayfish herbivory. J. Chem. Ecol. 24, 1639–1658. Bro¨ nmark, C., 1990. How do herbivorous freshwater snails affect macrophyte? A comment. Ecology 71 (3), 1212–1215. Bryant, J.P., Chapin, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants in relation to vertebrate herbivory. Oikos 40, 357–368. Causton, D.R., Venus, J.C., 1981. The Biometry of Plant Growth. Edward Arnold Ltd., London, pp. 17–64. Cebria´ n, J., Duarte, C.M., 1998. Patterns in leaf herbivory on seagrasses. Aquat. Bot. 60, 67–82. Chambers, P.A., Kalff, J., 1985. Depth distribution and biomass of submersed aquatic macrophyte communities in relation to Secchi depth. Can. J. Fish. Aquat. Sci. 42, 701–709. Choi, C., Bareiss, C., Walenciak, O., Gross, E.M., 2002. Impact of polyphenols on growth of the aquatic herbivore Acentria ephemerella. J. Chem. Ecol. 28, 2245–2256. Coley, P.D., Bryant, J.P., Chapin, F.S., 1985. Resource availability and plant antiherbivore defense. Science 230, 895–899. Crone, E.E., Jones, C.G., 1999. The dynamics of carbon-nutrient balance: effects of cottonwood acclimation to short and long-term shade on beetle feeding preferences. J. Chem. Ecol. 25, 635–656. Cronin, G., Hay, M.E., 1996. Effects of light and nutrient availability on the growth, secondary chemistry, and resistance to herbivory of two brown seaweeds. Oikos 77, 93–106. Cronin, G., Lodge, D.M., 2003. Effects of light and nutrient availability on the growth, allocation, carbon/nitrogen balance, phenolic chemistry, and resistance to herbivory of two freshwater macrophytes. Oecologia 137, 32– 41. Dorn, N.J., Cronin, G., Lodge, D.M., 2001. Feeding preferences and performance of an aquatic lepidopteran on macrophytes: plant hosts as food and habitat. Oecologia 128, 406–415. Duarte, C.M., 1992. Nutrient concentration of aquatic plants: patterns across species. Limnol. Oceanogr. 37, 882– 889. Durate, C.M., Kalff, J., Peters, R.H., 1987. Patterns in biomass and cover of aquatic macrophytes in lakes. Can. J. Fish. Aquat. Sci. 43, 1900–1908. Dudt, J.F., Shure, D.J., 1994. The influence of light and nutrients on foliar phenolics and insect herbivory. Ecology 75, 86–98. Elger, A., Barrat-Segretain, M.H., Amoros, C., 2002. Plant palatability and disturbance level in aquatic habitats: an experimental approach using the snail Lymnaea stagnalis (L.). Freshwater Biol. 47, 937–940. Elger, A., Willby, N.J., 2003. Leaf dry matter content as an integrative expression of plant palatability: the case of freshwater macrophytes. Funct. Ecol. 17, 58–65.
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