Selective Feeding of Gammarus pseudolimnaeus on Fresh and Senescent Leaves of Watercress, Nasturtium officinale

David S. Ginsberg

University of Michigan Biological Station

General Ecology 1998

Claudia Jolls and Eric Hellquist

Watercress (Nasturtium officinale) is a freshwater macrophyte that possesses the glucosinolate-myrosinase chemical defense system. This metabolic pathway produces 3-phenyl proponitrile and 2-phenylethyl isothiocyanate, deterrent chemicals upon leaf tissue damage. This chemical defense is proposed to account for the apparent lack of consumption of living watercress leaves by generalist feeders in aquatic environments. Senescent leaves have lower levels of these toxic chemicals, but are also less nutritious than fresh leaves. This experiment tested the feeding preferences of Gammarus pseudolimnaeus, a freshwater amphipod, provided with fresh green and senescent yellowed watercress leaves. Amphipods were collected from Carp Creek, Michigan, starved for 12-h, and given a choice between green and yellow leaf disks for a 26-h period. The experiment (n = 24) was performed in a dark, 10^o C environmental chamber. The areas of leaf disk eaten were measured and statistically analyzed with a chi squared analysis and student’s t-tests. Yellow leaves were consumed to a much greater extent than green leaves (mean area yellow consumed = 88.96 mm², mean area green consumed = 1.08 mm², p < 0.05). The differences in leaf consumption are most likely attributed to the presence activity of the glucosinolate pathway in the green leaves.

The topic of herbivory is extremely broad and crosses the traditional divide between botany and zoology (Crawley 1983). Due to the wide variety of herbivorous interactions affecting populations and communities, the interdisciplinary nature of herbivory studies is appropriate. However, not all areas of herbivory have been studied intensely, especially the complicated freshwater systems. Since living macrophytes are rarely consumed, these plants were once thought to be functionally negligible in aquatic food systems (Shelford 1918, Hutchinson 1981). More recent studies have shown the importance of macrophyes in freshwater food webs (e.g. Lodge 1991). Low herbivory (Newman et al. 1990) and the presence of pungent compounds (Kerfoot 1988) have led us to reconsider the possibility that chemical interactions influence the dynamic relationships between herbivores and freshwater macrophytes.

The presence of secondary chemicals produced by plants for defense would explain the lack of extensive plant damage despite evidence of aquatic herbivore attack. The chemical systems implicated by most researchers to defend macrophytes have been increasingly investigated (e.g. Ostrofsky and Zettler 1986, Newman et al. 1990; 1992, Clark 1995, Cronin and Hay 1996). Ostrofsky and Zettler (1986) have found alkaloids, a widely distributed class of toxic compounds, in all fifteen of the aquatic macrophyte species they analyzed. Most of the species had a collection of toxins; one species had a total of nine different chemicals. This diversity is necessary to protect against the many generalist consumers that inhabit freshwater systems, including caddisflies, snails and amphipods (Newman et al. 1992). These creatures eat other animals, leaf litter, algae and cellulose, foraging and browsing throughout the entire habitat. The general defense system in freshwater plants protects against these different species, as opposed to terrestrial plants which are most frequently attacked by specialist herbivores and posses specific defenses.

Glucosinolates are another class of toxic compounds, found primarily in the mustard family, Cruciferae (Louda and Mole 1991). Watercress (Nasturtium officinale R. Br., or Ropippa nasturtium-aquaticum (L.) Hayek) uses the glucosinolate-myrosinase system (Newman et al. 1992). Myrosinase is released upon damage to watercress tissue, converting 2-phenylethyl glucosinolate into 3-phenyl proponitrile and 2-phenylethyl isothiocyanate. These chemicals give watercress its hot taste and are repellant and sometimes toxic to many generalist herbivores. Glucosinolate itself may also be toxic in large quantities with extended consumption (Bones and Rossiter 1996, Vageeshbaru and Chopra 1997). The glucosinolate is present in high quantities in fresh, living watercress leaves but at much lower levels in senescent yellow leaves (Newman et al. 1996). The chemical pathway degrades along with the health of the leaf.

Our experiment was designed to test the consumption of watercress by the generalist amphipod Gammarus pseudolimnaeus Bousfield (Arthropoda: Crustacea: Amphipoda). These two organisms frequently co-occur in alkaline spring streams, with the amphipod commonly living among the roots of watercress and adjacent moss species. We gave amphipods a choice between green or yellowed leaves of watercress. Analysis by Newman et al. (1990) showed that green leaves have a lower carbon nitrogen ratio than yellowed leaves (7.27 versus 12.7), making them more nutritious. This difference in nutrition would presumably make them more attractive to consumers than senescent leaves. However, the glucosinolate-myrosinase defense system should prevent the amphipods from consuming the green leaves. The lack of glucosinolate in yellow leaves would explain a preference for this lower quality food.

The experiment was performed using amphipods and watercress collected from Carp Creek, Cheboygan County, MI (45^o33’N; 84^o41’W: T36N, R3W, S4), on June 30, 1998, and conducted in a dark Percival growth chamber set at 10° C. Collected amphipods were divided into three size classes (less than 7 mm in length, between 7 and 10 mm, and over 10 mm in length). The smallest amphipods were not used. Two medium amphipods and one large amphipod were grouped together in order to keep a constant total length (about 27 mm) of amphipod in each sample. Sample amphipods were placed in petri dishes containing 30 ml of filtered stream water from Carp Creek, then starved for 12-h to increase their appetites and to equalize their consumption rates.

Watercress leaves were blotted out on paper towel, then cut into 201 mm² disks with a cork borer. The disks were soaked in filtered stream water for the duration of the starvation period to remove toxins released by damaged tissue. At the end of the 12-h starvation, one fresh green disk and one senescent yellowed disk were placed in each petri dish. The dishes were partially covered to prevent contamination or escape of the amphipods, but to avoid suffocation. Twenty four samples were constructed and left in the growth chamber for a 26-h period of consumption, after which the leaves were removed and measured. All amphipods were collected and returned to Carp Creek after completion of the experiment.

Partially consumed leaf disks were compared to outlines of whole leaf disks on graph paper, so that the missing leaf area could be counted. Statistical analyses were done with SYSTAT 5.2 (Wilkinson 1992). Chi squared analysis was used to check for differences between the distributions of total leaf area eaten. Paired students’ t-tests were used because of the dependant nature of the data collected (two leaf disks grazed by the same amphipods). The t-tests were performed on the means of consumption and the means of the arcsine transformed data of the proportion of leaf disks eaten (arcsine Ö x ).

Chi square analysis of the 24 samples showed that the total area of green leaf consumed was significantly different from the total area of yellow leaf consumed (X² = 2058, df = 1, p £ 0.05; Table 1). Mean tissue consumption was significantly different between green and yellow leaves (t = -7.301, df = 23, p < 0.001; Table 1), as was the mean proportion of yellow and green disks eaten (t = -9.062, df = 23, p < 0.001; Table 1). In all cases, yellow leaf consumption was greater than green leaf consumption (mean consumed » 90:1 ; Figure 1).

Gammarus pseudolimnaeus consumed different amounts of green and yellow leaves in our experiment. The condition of the watercress leaves made a difference, consistent with all previous studies (Newman et al. 1990; 1992; 1996, Clark 1995, Hamilton 1995). Chemical analysis (Newman et al. 1990) has shown that both carbon nitrogen ratios and glucosinolate content change with the health of watercress leaves. Glucosinolate content in particular can be related to leaf consumption. Amphipods in our study consumed a greater amount of yellow leaf than green leaf on average. Newman et al. (1990) noticed the same preference, and discovered greater glucosinolate concentrations in green leaves (8.85 mg glucosinolate / g leaf, versus 1.24 mg/g for yellowed leaves). A negative correlation could be found between glucosinolate levels in leaves and consumption by herbivores in choice tests. As is the case in previous studies, we found that a greater proportion of yellowed leaves was consumed than that of green leaves. However, Newman et al. (1990) report more fresh green leaf consumption that was exhibited in our study. The curious difference in total leaf areas consumed, two full orders of magnitude, had definitely not been demonstrated before, and would probably be reduced with greater amphipod acclimation time and additional repetitions. Newman et al. used laboratory grown watercress, and this might account for different levels of chemicals than found in the wild.

Despite the higher nutritional value of green leaves, the Gammarus pseudolimnaeus in our experiment consistently preferred senescent yellow leaves. In addition, most of the green leaves were partially consumed by the amphipods, in small amounts (mean = 1.08 ± 0.7 mm²). This evidence hints at the existence and effectiveness of the glucosinolate-myrosinase chemical defense system in Nasturtium officinale. The amphipods were probably repelled by toxins released from damage to the green leaf. This assumption is in accordance with Newman’s research (1992, 1996), in which amphipods readily consumed fresh green leaves whose myrosinase had been denatured by heat. While preference for green leaves with myrosinase removed by freezing and leeching in water has been documented (Clark 1995, Hamilton 1995), G. pseudolimnaeus demonstrated biomass loss and reduced survival when fed a constant diet of heat denatured green leaves (Newman et al. 1996). This could possibly be an artifact of the different denaturing methods, but it appears that amphipods could prefer senescent leaves over time, despite lower nitrogen ratios. Clearly a consistent multifactored study is necessary, as are extended experiments to test the full life span effects of a limited diet on the nutrition and metabolism of amphipods and other generalist feeders.

The glucosinolate-myrosinase system stands as an effective chemical defense for watercress. Its toxin is general enough to repel all generalist feeders in streams, judging by the consistent lack of damage. In addition, it protects the healthy living material of the plant while allowing useless senescent tissue to be consumed by herbivores and detritivores. The benefits of detritivory to both plant and generalist consumer will likely be documented in the near future. Our study has shown that the system works at its most basic level, that of herbivory defense.

Table 1. Mean areas of fresh green and senescent yellow leaf disk consumed by Gammarus pseudolimnaeus (mean ± 2 SE, n = 24 for all cases).