MORPHOLOGICAL RESPONSES OF DAPHNIA PULEX TO CHAOBORUS AMERICANUS KAIROMONE IN THE PRESENCE AND ABSENCE OF METALS

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1 Environmental Toxicology and Chemistry, Vol., No. 5, pp , SETAC Printed in the USA /04 $ MORPHOLOGICAL RESPONSES OF DAPHNIA PULEX TO CHAOBORUS AMERICANUS KAIROMONE IN THE PRESENCE AND ABSENCE OF METALS KIM HUNTER and GREG PYLE* Department of Biology, Nipissing University, 100 College Drive, North Bay, Ontario P1B 8L7, Canada (Received July 00; Accepted October 00) Abstract Daphnia pulex neonates develop neck teeth in the presence of predatory kairomone from Chaoborus americanus that are fed D. pulex. These neck teeth reduce the susceptibility of the neonates to predation. Evidence suggests that aqueous metals interfere with chemical communication in fish. The objective of our study was to determine if Cu or Ni at environmentally relevant concentrations affects predatory kairomone response in D. pulex. To test this possibility, D. pulex were placed in increasing waterborne concentrations of Cu or Ni in the presence or absence of predatory kairomone. Both Cu and Ni reduced neck tooth induction in D. pulex neonates in the presence of predatory kairomone. Copper had a significant nonlinear effect on neck tooth length consistent with a hormetic response, where neck tooth length was highest at 5 g/l Cu, but not significantly different than 0 g/l Cu at higher Cu concentrations. A Ni concentration of 00 g/l caused D. pulex to become hypersensitive to Chaoborus regardless of Chaoborus diet, leading to increased neck tooth number but decreased neck tooth length. Neither Ni nor Cu produced any significant effects on body length or brood size. These results suggest that metal inhibition of neck tooth induction probably occurs along the signal transduction pathway. Impairment of chemosensory response to predatory chemical cues may have widespread ecological consequences in aquatic systems contaminated by metals. Keywords Chemical communication Predatory kairomone Daphnia pulex Chaoborus americanus Morphological antipredator defense INTRODUCTION Many animal species, both vertebrates and invertebrates, develop behavioral and morphological defenses in the presence of predators to increase their chance of survival against predation [1]. One example of such a relationship is that between the water flea, Daphnia pulex (prey), and phantom midge larvae, Chaoborus spp. (predator). In the presence of phantom midge larvae, D. pulex develop neck teeth in the first through third instar of development (body length 1. mm) [ 8]. The induction of an antipredator morphological defense in D. pulex is thought to be mediated by the presence of a predatory kairomone emitted by Chaoborus feeding on D. pulex. The kairomone is thought to be a low-molecular weight, nonolefinic hydroxy-carboxylic acid [8] that induces morphological changes in D. pulex. Third- and fourth-instar Chaoborus larvae are gape-limited predators that are unable to ingest large Daphnia [9,10]. Therefore, these larvae select small- to medium-sized daphnids ( mm body length) as prey [11 1]. Neck teeth enlarge D. pulex, which reduces the predator s handling efficiency and decreases the prey s susceptibility to predation []. Induction of neck teeth helps maintain viable populations of D. pulex in ecosystems because it prevents local extinction by Chaoborus. However, the induction of neck teeth may be inhibited if the prey does not perceive the kairomone. Beyers and Farmer [14] demonstrated that olfactory ability in Colorado pikeminnow (Ptychocheilus lucius) declined in the presence of Cu at concentrations g/l. Hansen et al. [15] studied the olfactory epithelial structure of chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (O. mykiss) * To whom correspondence may be addressed (gregp@nipissingu.ca). and found that olfactory receptors in these two species were significantly damaged by exposure to Cu at 5 g/l. Moreover, chinook salmon were unable to avoid potentially lethal concentrations of Cu after being acclimated to only g Cu/L [1]. These studies show that metals can affect chemoreception in fish, and suggest the possibility the same metals could interfere with invertebrate chemical communication systems, such as that which operates between D. pulex and Chaoborus. Contaminant-inhibited chemical communication systems between D. pulex and Chaoborus could result in poor neck tooth development in D. pulex, leading to increased predation by Chaoborus. Zooplankton (i.e., D. pulex) population declines could lead to a reduction of available food for Chaoborus, and an increase in abundance of phytoplankton which are typically grazed by D. pulex. This sort of bottom-up ecosystem disturbance could potentially contribute to eutrophication. Both D. pulex and Chaoborus occupy important ecological niches in the food web. Therefore, conditions or events disturbing D. pulex or Chaoborus populations could generate an ecological cascade leading to an unpredictable outcome [17]. The purpose of this study was to examine the effects of Cu and Ni at environmentally relevant concentrations on Chaoborus-induced neck tooth development and growth in D. pulex. Because previous studies have demonstrated a failure to detect and respond to chemical stimuli by fish in the presence of metals, we hypothesized that dissolved metals could impair the ability of D. pulex to respond to Chaoborus kairomone, and therefore fail to produce neck teeth as a defense against potential predation. MATERIALS AND METHODS Culture methods Daphnia pulex were obtained from a laboratory culture at Natural Resources Canada in Ottawa (ON, Canada) and Chao- 111

2 11 Environ. Toxicol. Chem., 004 K. Hunter and G. Pyle borus were collected from a fishless pond in North Bay (ON, Canada). Daphnia pulex and Chaoborus were cultured in the laboratory for the duration of the experiment. Daphnia pulex were reared at C under 1:8-h light:dark in dechlorinated North Bay tap water (mean standard deviation; ph 7.7, dissolved organic carbon mg/l, hardness mg/l as CaCO, alkalinity mg/l as CaCO, turbidity farazin turbidity units, Cu. 0. g/ L, Ni g/l) [18] and fed Chlorella emersonii ( ml/week; cell density,. 10 cells/ml). Chaoborus were held in densities of approximately 10 organisms per liter of dechlorinated tap water at 8 C in constant dark, and were fed either D. pulex or brine shrimp (Artemia salina; Inve Group, Grantsville, UT, USA) nauplii ad libitum. preparation Three experimental stimuli were established to test neck tooth induction in D. pulex neonates in the presence or absence of metals. The three stimuli were dechlorinated water ([DW] negative control, no kairomone), dechlorinated water conditioned by Chaoborus fed D. pulex (K[ ]; experimental treatment, predatory kairomone), or dechlorinated water conditioned by Chaoborus fed brine shrimp (K[ ]; positive control, no kairomone). All stimuli were stored under the same conditions at 8 C. A 50% water change was conducted in each stimulus container once per week. Chaoborus fed D. pulex and Chaoborus fed brine shrimp were placed on these feeding regimes more than one month before the experiments started. Each replicate metal concentration that was added to these stimuli was prepared from a stock solution. Stock solutions of Cu (CuCl.H O, Anachemia Canada, Montreal, QC, Canada) and Ni (NiCl.H O, Anachemia Canada) were prepared from chloride salts dissolved in double deionized water. Exposure solutions were prepared by diluting the appropriate stock solution with dechlorinated municipal tap water to yield double the appropriate concentration. These solutions were then diluted in half by a stimulus solution in order to achieve the final desired concentration. Nominal Ni concentrations tested were 0, 40, 10, and 00 g/l and Cu concentrations were 0, 5, 15, and 5 g/l. These concentrations tested reflect measured metal concentrations in lakes around Sudbury, Ontario, and were selected to provide maximum ecological relevance. Table 1. Summary of two-way analysis of variance testing the effect of Cu or Ni on Daphnia pulex neonate neck tooth number, neck tooth length, body length, and brood size after exposure to one of three stimuli: K( ), K( ), and dechlorinated water Source Neck tooth number stimulus Neck tooth length (mm) stimulus Body length (mm) stimulus Brood size stimulus Exposure to Cu df F ratio p Exposure to Ni df F ratio p Experimental setup and design Effects of Cu and Ni on D. pulex neonate responses to predatory kairomone were tested in separate experiments. Each experiment examined four metal concentrations and three experimental stimuli; K( ), K( ), and DW. Each combination of metal and stimulus was replicated four times; therefore, each experiment had a 4 4 design. Experiments were conducted in -ml glass test tubes. A single gravid D. pulex female was randomly selected from the stock culture and placed into a test tube with 40 l of culture water. Then 90 l of either K( ), K( ), or DW was added to each test tube. solutions were then added as 1 ml aliquots in order to yield the appropriate metal concentration and a final volume of ml. One drop of C. emersonii culture (. 10 cells/ml) was added daily as food. Experimental replicates were arranged randomly to minimize gradient effects associated with position in the environmental chamber. Experimental conditions matched D. pulex culture conditions. Every 4 h, all experimental solutions and metal concentrations were replaced to avoid bacterial degradation of the predatory kairomone and another drop of C. emersonii culture was added as food. Data collection Daphnia pulex were acclimated to experimental conditions for 48 h. Each test tube containing a single gravid female was examined every 4 h to collect neonates. If neonates were present, they were removed and examined, leaving the adult D. pulex in the original test tube. The neonates were counted to assess 4 h neonate production (i.e., brood size), and examined under a microscope to measure body length (excluding caudal spine), neck tooth length, and to count the number of neck teeth. Neck tooth and body length were measured using an ocular micrometer. In the event of the death of an adult D. pulex, the adult was replaced and acclimated for 48 h before its neonates were examined. Each metal exposure was conducted for up to d. Data analysis Data were analyzed using JMP Ver 5.0 [19]. A two-way analysis of variance was used to examine main effects of metal (Cu or Ni), stimulus (K[ ], K[ ], or DW), and metal stimulus interaction on D. pulex neonate neck tooth number, length, and body length. Main effects were further analyzed by oneway analysis of variance, followed by a Tukey Kramer multiple range test or least-squares regression. RESULTS Effects of Cu Neither Cu nor stimulus had a direct effect on D. pulex neck tooth number (p 0.08, Table 1). However, there was a significant Cu stimulus interaction (p 0.049). At 0 g/ L Cu, D. pulex exposed to K( ) had at least twice as many neck teeth as those exposed to either K( ) ordw(f,8 11.8, p 0.004; Fig. 1A). At higher Cu concentrations, neck

3 inhibition of antipredator defenses in Daphnia pulex Environ. Toxicol. Chem., Fig.. Effect of Cu on D. pulex neck tooth length. Data are represented as means standard error of the mean (n 1). * indicates significant difference from 0 g/l Cu. The quadratic equation of the line, its coefficient of determination, and significance are indicated on the graph. Fig. 1. Effect of Cu (A) and Ni (B) ondaphnia pulex neonate neck tooth number in the presence of dechlorinated municipal North Bay tap water (DW), water conditioned by Chaborus fed D. pulex [K( )], or water conditioned by Chaoborus fed Artemia salina [K( )]. Data are represented as means standard error of the mean (n 4). Mean differences were considered significant when p * indicates significant difference from DW or K( ). Bars sharing horizontal lines are not significantly different from one another. tooth number did not vary among K( ), K( ), or DW (p 0.05). Copper concentration had a marginally significant effect on neck tooth length (Table 1; p 0.055). Daphnia pulex exposed to 5 g/l Cu had significantly longer neck teeth than those exposed to 0 g/l Cu (F,44.87, p 0.047). However, at higher Cu concentrations, this effect disappeared (p 0.05). Neck tooth length demonstrated a significant nonlinear relationship with Cu concentration that reflected longer neck teeth at low Cu concentrations relative to controls, but not at higher concentrations (r 0.1, p 0.019; Fig. ). There was no significant interaction between Cu concentration and stimulus (p 0.4; Table 1). Neither Cu (p 0.180) nor stimulus (p 0.519) had a significant effect on D. pulex body length (Table 1). There was also no significant interaction between Cu and stimulus on body length (p 0.50). Similarly, neither Cu (p 0.914) nor stimulus (p 0.55) significantly affected brood size, and there was no significant interaction between the two (p 0.4; Table 1). Effects of Ni Both Ni (p 0.049) and stimulus (p ) had a significant effect on neck tooth number (Table 1). Daphnia pulex exposed to 40 g/l Ni had 49% fewer neck teeth than those exposed to 00 g/l Ni. However, the effect was not significant when analyzed independently of the whole model (F, , p 0.15). Daphnia pulex exposed to K( ) produced significantly more neck teeth than those exposed to either K( ) ordw(f, , p 0.001). The interaction between Ni and stimulus was only marginally significant (p 0.08). In 0 g/l Ni, D. pulex exposed to K( ) had approximately twice as many neck teeth as those exposed to either K( ) ordw(f,9 5.5, p 0.0; Fig. 1B). At 40 (F,9.85, p 0.0) and 10 g/l Ni (F,9.91, p 0.10) there was no significant difference in neck tooth number among D. pulex exposed to K( ), K( ), or DW. However, at 00 g/l Ni, D. pulex exposed to K( ) had significantly more neck teeth relative to those exposed to DW (F,9 4.0, p 0.051). Neck tooth length was significantly affected by stimulus (p 0.01; Table 1). Daphnia pulex exposed to K( ) had 18% longer neck teeth than those exposed to K( ) (F,45.5, p 0.0); however, there was no difference in neck tooth length between those exposed to DW and those exposed to K( ) ork( ) (Fig. ). As with Cu, neither Ni (p 0.841) nor stimulus (p 0.54) had a significant effect on D. pulex body length (Table 1). There was also no significant interaction between Ni and stimulus on body length (p 0.815). Similarly, brood size was not affected by Ni (p 0.17) or stimulus (p 0.49), and there was no significant interaction between Ni and stimulus (p 0.857) affecting brood size (Table 1). DISCUSSION The results of this study indicate that the chemical communication system between Chaoborus and D. pulex was impaired by environmentally relevant concentrations of Cu and Ni. Neck tooth induction was inhibited in D. pulex exposed to K( ) and Cu, although neck tooth length increased at low concentrations of Cu (5 g/l), but not by higher concentrations (Fig. ). Although Cu had a clear inhibitory effect on neck tooth induction, the effect of Ni was less clear (Fig. 1). Neck tooth induction was inhibited in D. pulex exposed to K( ) and Ni. However, at 00 g/l Ni, D. pulex exposed to K( )

4 114 Environ. Toxicol. Chem., 004 K. Hunter and G. Pyle Fig.. Effect of stimulus on neck tooth length in D. pulex during exposure to Ni. Data are shown as means standard error of the mean (n 1). Bars sharing the same letter are not significantly different from one another (p 0.05). DW dechlorinated municipal North Bay tap water; K( ) water conditioned by Chaoborus fed D. pulex; K( ) water conditioned by Chaoborus fed Artemia salina. showed significant neck tooth induction relative to those exposed to DW at the same Ni concentration. These results are somewhat counterintuitive because, in general, increasing waterborne contaminant concentrations should yield increasing effects following a typical dose-response curve. Nonetheless, these results have considerable environmental relevance as many water bodies around industrial regions are contaminated at these low concentrations. concentrations in most industrially contaminated environments may not be high enough to cause acute toxicity to D. pulex; however, this study suggests that typical metal contaminated aquatic ecosystems could have metal concentrations that could affect chemical communication between Chaoborus and D. pulex. Hannah, Whitson, and Ramsey Lakes, located in the industrial region of Sudbury (ON, Canada) have Cu concentrations of.7, 17.8, and 11. g/l [0], and Ni concentrations of 181, 155, and 5 g/l, respectively (G. Pyle et al., Nipissing University, North Bay, ON, USA, unpublished data). These values are all below the reported 48-h LC50 (lethal concentration required to kill 50% of the test organisms during a 48-h exposure) values for daphnids. Daphnia magna was reported to have a Cu 48-h LC50 value of 54 g/l [1] and Daphnia pulicaria was reported to have a Ni 48-h LC50 of 97 g/l []. Although water quality (e.g., hardness, ph, alkalinity, and dissolved organic matter) can moderate the acute toxicity of metals, observed concentrations of metals in the industrially polluted water bodies around Sudbury are probably not acutely toxic to daphnid species. However, results reported here indicate that metal concentrations measured in Sudbury-area lakes may be great enough to inhibit chemical communication between zooplankton and their predators. Several possible reasons can be offered for why Cu and Ni seem to impair the D. pulex response to predatory kairomone, including: Cu or Ni may interfere with some process along the signal transduction pathway from kairomone reception to neck tooth production, the presence of elevated concentrations of Cu or Ni may alter the chemistry of the kairomone itself, or Cu or Ni may out-compete kairomone molecules at kairomone receptor sites. The molecular mechanism that underlies chemoreception in invertebrates has been investigated only in Caenorhabditis elegans and Drosophila melanogaster and is not well understood in other invertebrate species []. Although there are probably some differences between invertebrate and vertebrate chemosensory mechanisms, there is a high degree of conservation among phyla [4]. Signal transduction is initiated upon kairomone binding to a G-protein-coupled chemoreceptor, probably in the antennae []. The kairomone-bound receptor activates a G-protein, which in turn activates adenylyl cyclase to produce cyclic adenosine monophosphate from adenosine triphosphate. The cyclic adenosine monophosphate then binds to a cyclic nucleotide-gated channel, causing an influx of small cations (i.e., Na,Ca ), and cell depolarization. Depolarization to threshold ( 0 mv) yields an action potential which conducts the signal to higher neurological processing centers. The downstream mechanism to induce neck tooth development after the signal has been received by higher processing centers is unknown. Two lines of evidence suggest that the mechanism interfering with the D. pulex response to K( ) in the presence of Cu or Ni may be in the signal transduction pathway. The observation that Cu inhibits the number of neck teeth produced at all concentrations tested, but increases neck tooth length at 5 g/l suggests that Cu may impair the signal transduction pathway leading to neck tooth production, but stimulates the pathway leading to neck tooth length. The apparent stimulatory effect of low Cu concentrations on neck tooth length is consistent with the phenomenon of hormesis (i.e., a stimulatory response upon exposure to a toxicant) [5]. A Cu concentration of 5 g/l may have been sufficient to disrupt homeostasis, leading to an over-compensatory effect along the signal transduction pathway and a corresponding increase in neck tooth length []. The second line of evidence relates to the observation that at 00 g/l Ni, D. pulex produced significantly more neck teeth when exposed to K( ) than DW (Fig. 1). However, an increase in the number of neck teeth produced in D. pulex exposed to K( ) was offset by their shorter length (Fig. ). This finding suggests that exposure to Ni may render D. pulex more sensitive to the presence of Chaoborus in general, regardless of its diet, leading to more but shorter neck teeth. This sort of metal-induced hypersensitive chemosensory response at high metal concentration has been observed in fish olfactory and gustatory systems [7]. Kairomone-induced neck tooth production in D. pulex previously was shown to be reduced under low ph conditions relative to higher ph, suggesting that H bound to the kairomone molecule may alter kairomone chemistry [8]. It is possible that dissolved metal ions may bind to the kairomone molecule causing a change in its chemistry, reducing its efficacy for inducing neck tooth production in D. pulex. However, this explanation seems unsatisfactory for explaining increased neck tooth length at 5 g/l Cu, or an increased sensitivity to Chaoborus at 00 g/l Ni given the nonmonotonic nature of the responses under these conditions. Similarly, Cu or Ni may simply out-compete kairomone molecules for chemosensory binding sites. However, this also seems unlikely for the same reason. At present, the underlying mechanism of metal impairment of chemosensory function in this predatorprey system remains unknown and warrants further investigation. Sherwood et al. [8] found that yellow perch were unable to perform normal ontogenetic diet shifts (i.e., zooplankton to macroinvertebrates to fish) in metal-contaminated lakes, resulting in stunted growth of yellow perch in those lakes. The

5 inhibition of antipredator defenses in Daphnia pulex Environ. Toxicol. Chem., absence of healthy macroinvertebrate populations is thought to be responsible for an energetic bottleneck leading to reduced growth in yellow perch [8]. Because metal concentrations in contaminated systems are rarely high enough to be acutely toxic to macroinvertebrates, it is unlikely that acute toxicity alone can account for the absence of macroinvertebrates. Results from the current study provide a possible explanation for the decline of macroinvertebrates in metal contaminated aquatic ecosystems, especially those having metal concentrations below acute toxicity thresholds. If neck tooth development is inhibited in metal-contaminated ecosystems, D. pulex may become more susceptible to predation by Chaoborus or other gape-limited predators. Increased predation could lead to significant reductions in D. pulex populations. Insect larvae that feed on D. pulex, such as Chaoborus, could lose an important food resource, which in turn could lead to reductions in Chaoborus populations. Moreover, this study only examined a single predator-prey system that demonstrated metal inhibition of chemical communication. This raises an important question about the extent to which industrial metal pollution may be affecting chemical communication among a great variety of aquatic organisms, and how this might affect their ecology. The presence of potentially toxic metals might not affect aquatic organisms through direct toxicity, but still impede trophic interactions through chemosensory impairment and reduced energy flow at the ecosystem level. Exposure to environmentally relevant concentrations of Cu or Ni caused a marked reduction in neck tooth induction relative to controls (Fig. 1A), but no corresponding change in body or brood size. It is thought that neck teeth serve to increase the size of neonatal D. pulex as an antipredation strategy against gape-limited predators such as Chaoborus. The increased size of neonatal D. pulex conferred by the presence of neck teeth increases the probability of escape by decreasing handling efficiency by Chaoborus [9]. Lüning [0] found that in the presence of Chaoborus flavicans kairomone, D. pulex were 0% larger relative to controls. This is different than our results because body size was not significantly different between kairomone-exposed D. pulex and controls in any experimental treatment. Lüning [0] also found that female D. pulex exposed to Chaoborus flavicans kairomone produced fewer but larger offspring than controls. These discrepancies may be related to differences in experimental conditions, or to genetic differences between D. pulex clones used in each study. Only one source of D. pulex was used in this study. Consequently, results do not necessarily account for genetic variability because D. pulex reproduce parthenogenetically [1]. Spitze [5] found significant variability among different genotypes of D. pulex with respect to body length and the expression of neck teeth, with some clones having a larger body length in the presence of Chaoborus compared to control, and expressing variability in the number of neck teeth induced among individuals of the same clone. In conclusion, this study is among the first to demonstrate that low, environmentally relevant concentrations of Cu and Ni impair the ability of D. pulex to induce antipredator morphological defenses in the presence of predatory kairomone from Chaoborus. We speculate that both Cu and Ni interfere with some process along the signal transduction pathway leading to an increase in neck tooth length in very low concentrations of Cu, and an increased sensitivity to Chaoborus (regardless of diet) in the presence of Ni. 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