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stigmine and picrotoxin and suppressed by atropine. It was proposed that these chemicals were acting on neurosecretory cells that release the hormones ...
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Progress toward Understanding the Neurophysiological Basis of Predator-Induced Morphology in Daphnia pulex Michael J. Barry* Max Planck Institute for Limnology, Plo¨n, Germany Accepted 12/21/01

ABSTRACT Previous studies have demonstrated that certain pesticides, including carbaryl and endosulfan, can modulate the expression of predator-induced morphology in Daphnia. These pesticides affect the transmission of nervous impulses in vertebrates and invertebrates. The aim of this study was to determine the role of two neurotransmitter systems, excitatory cholinergic transmission and inhibitory g-aminobutyric acid (GABA)-mediated transmission, in the regulation of inducible defenses of Daphnia. The effects of chemicals with four different modes of action on the expression of Chaoborus-induced neckteeth in Daphnia pulex were measured. These chemicals included chemicals that could enhance transmission at cholinergic synapses (physostigmine, nicotine), inhibit cholinergic transmission (atropine), stimulate or enhance the effects of GABA (diazepam, muscimol, cis-4-aminocrotonic acid), or antagonise the action of GABA (picrotoxin, bicuculline, SR95531). The development of Chaoborus-induced neckteeth in D. pulex was enhanced by physostigmine and picrotoxin and suppressed by atropine. It was proposed that these chemicals were acting on neurosecretory cells that release the hormones necessary to induce neckteeth development. The results also indicate mechanisms through which anthropogenic pollutants could influence the expression of inducible defenses, leading to inappropriate expression in environments with low predator intensity or to suppression in environments with high risks of predation.

Introduction Predator-induced defenses have been described in a wide range of marine and freshwater taxa (reviewed in Tollrian and Harvell 1999). These induced morphological structures are usually de* Present address: School of Biological Sciences, Monash University, P.O. Box 18, Clayton, Victoria 3800, Australia; e-mail: [email protected]. Physiological and Biochemical Zoology 75(2):179–186. 2002. 䉷 2002 by The University of Chicago. All rights reserved. 1522-2152/2002/7502-01102$15.00

ployed when the prey species detects a chemical odour (kairomone) signaling the presence of predators in the proximate environment. The induced structures normally protect the prey species or reduce the efficiency of predation. The induction of neckteeth in Daphnia pulex by kairomones released by predatory larvae of the phantom midge, Chaoborus spp., is a paradigmatic example of this response (Krueger and Dodson 1981). However, despite abundant literature on ecological aspects of predator induction (reviewed in Tollrian and Dodson 1999), there have been relatively few insights into the physiological mechanisms governing their expression. Because the morphological structures lack innervation, Jacobs (1980) proposed that induction was induced by target-specific hormones released into the haemolymph, while Beaton and Hebert (1997), found large polynucleated cells at the base of the inducible structures and higher rates of cell division in the surrounding tissue. Hanazato (1991b, 1992) demonstrated that several insecticides could induce the development of a helmet in Daphnia ambigua that was morphologically indistinguishable from that induced by Chaoborus kairomones. Apparently, the insecticides were stimulating the physiological mechanisms responsible for helmet induction. The inducing pesticides included carbaryl, fenobucarb, temephos, diazinon, fenthrothion, and fenthion. There was no effect, however, with the herbicides thiobencarb and oxadiazon or the fungicide iprofenos (Hanazato 1992). In a further study, Hanazato and Dodson (1993) found that carbaryl could also induce helmets or neckteeth in D. pulex, Daphnia galeata mendotae, Daphnia retrocurva, and Daphnia lumholtzi, indicating that the pesticide was affecting some aspect of internal physiology common to all the species. Carbaryl and the other insecticides that Hanazato (1991b, 1992) found to be effective inducers of helmet development belong to the carbamate and organophosphate classes, and all act by inhibiting the action of acetylcholine esterase, an enzyme that degrades the neurotransmitter acetylcholine. They therefore increase the transmission of nerve impulses at synapses that use acetylcholine. I (Barry 1998) reported that the organochlorine pesticide, endosulfan, could induce and increase crest development in Daphnia longicephala at concentrations two orders of magnitude below levels that caused acute toxicity. Endosulfan inhibits the binding of g-aminobutyric acid (GABA) to its receptor (Lawrence and Casida 1984; Bloomquist and Soderlund 1985). Thus, it was proposed that GABA may be an inhibitory neurotransmitter, down-regulating the expression of inducible defenses in Daphnia. These findings sug-

180 M. J. Barry gested that crest development in Daphnia is regulated by the release of target-specific hormones from neuroendocrine glands for which acetylcholine is the main stimulatory neurotransmitter and GABA is the main inhibitory transmitter. The ability of certain pesticides to stimulate the development of inducible morphological structures indicates a potentially significant, but hitherto unrecognised, form of endocrine disruption (Hanazato 1999; Barry and Stoopman 2000). Endocrine disruption is defined as the deleterious modulation of hormone-regulated processes by an external agent. The effects of man-made chemicals on the expression of inducible defenses in Daphnia is of broad ecological concern for at least two reasons. First, they are an important member of many aquatic ecosystems. Second, Daphnia are a paradigmatic example of adaptive phenotypic plasticity. They may share common underlying neurological regulatory mechanisms with many other species; thus, effects on daphnids may indicate potential effects on a diverse range of taxa. From a different perspective, the effects of certain classes of pesticides on inducible morphology may provide clues that help to elucidate the regulatory processes underlying crest development. Such understanding is important for the eventual integration of ecological, genetic, and physiological data into a cogent model of daphnid biology. This, in turn, will aid in understanding the ecology and evolution of inducible defenses in general. The aim of this study was to test the model that the expression of predator-induced morphological defenses in Daphnia is regulated by excitatory cholinergic and inhibitory GABAergic neurotransmitters (Barry 1998). To achieve this aim, developing embryos of D. pulex were exposed to a range of drugs, with specific pharmacological actions, in conjunction with different concentrations of Chaoborus kairomone. The number of neckteeth expressed in the second neonatal instar was then measured. Two hypotheses were tested: (1) the expression of neckteeth would be increased by drugs that increased neural transmission at cholinergic synapses (cholinergic agonists) and reduced by drugs that blocked transmission (cholinergic antagonists) and (2) the expression of neckteeth would be decreased by drugs that mimic the neurotransmitter GABA or agonise the GABA receptor and increased by drugs that inhibit the binding of GABA to its receptor (GABA antagonist). Methods A single clone of Daphnia pulex, from the culture collection of L. J. Weider, at the Max Planck Institute for Limnology, was used. This clone was selected because preliminary experiments demonstrated that it could develop up to four large neckteeth and a neck pedestal and was most sensitive to Chaoborus kairomone during embryogenesis. Daphnia were cultured and experiments performed in a synthetic pond water (SPW) because previous studies have shown that the kairomone is sensitive to

degradation by bacteria present in natural lake water (Tollrian 1993). SPW was made by mixing 0.5 g L⫺1 of synthetic marine salts (Reef Crystals, Aquarium Industries) with Milli-Q water, adjusting the pH to 7.6 with 1 M NaOH and bubbling with 0.45-mm filtered air for 24 h. Daphnia were fed chemostatcultured Scenedesmus obliquus at a concentration equivalent to 1 mg C L⫺1 and maintained in 1-L autoclaved glass jars at a density of five daphnids per litre. Cultures were maintained at 20⬚ Ⳳ 1⬚C and illuminated by a bank of fluorescent lights with a 16L : 8D photoperiod. The daphnids were transferred to fresh media on alternate days. Experiments were performed using a modification of the embryological induction assay developed by Tollrian and von Elert (1994). They commenced by isolating between five and 10 third- to sixth-brood female daphnids holding black-eyed embryos (about 10–12 h before parturition). The embryos were carefully dissected from the mothers in a glass petri dish containing SPW. The embryos from several mothers were pooled in each experiment. They were then rinsed by serial transfer, through two more petri dishes containing fresh media, using a Pasteur pipette. A single embryo was then placed in each well of a 24-well culture plate that had previously been filled with 2 mL of culture media per well containing the appropriate concentrations of the test drug and Chaoborus kairomone. Each treatment combination was replicated 10 times. The total number of embryos collected varied depending on the number of treatments in the experiment being performed. Stock solutions of each drug were prepared by dissolving the appropriate amount of chemical in either Milli-Q water (atropine, nicotine hydrogen tartarate salt, bicuculline, SR95531, picrotoxin), 0.05 M HCl (muscimol), analytical grade ethanol (physostigmine, diazepam), or 52.5% ethanol/water (cis-4aminocrotonic acid; CACA). These stocks were frozen at ⫺20⬚C and thawed immediately before use. Drugs with four classes of activity were tested: (1) drugs that stimulated cholinergic transmission: physostigmine (PHY) and nicotine (NIC); (2) inhibitors of cholinergic transmission: atropine (ATR); (3) drugs that mimic GABA or modulate its activity: muscimol (MUS), diazepam (DIA), and CACA, a folded and conformationally restricted GABA analogue that at low concentrations is specific for mammalian GABAc receptors and a general GABA receptor agonist at high concentrations (Qian and Dowling 1996); (4) GABA antagonists, picrotoxin (PTX), bicuculline (BIC), and SR95531 (also known as gabazine). Preliminary experiments were performed with all chemicals to determine concentrations that caused significant toxicity, where toxicity is defined as mortality or failure to moult into the second instar. Unless otherwise stated, the maximum concentrations used were at least 50% below the threshold concentrations for toxicity. Test solutions were prepared by serial dilution of the stock solution with Milli-Q water, then the diluted stocks were mixed with SPW in glass vials to make a total volume of 20 mL. A kairomone extract from Chaoborus flavicans (Tollrian and von

Regulation of Neckteeth Induction in Daphnia 181 Elert 1994) was added to each vial at one of four concentrations: 0, 0.25, 0.5, or 1.5 mL mL⫺1. The extract had been stored in Eppendorf tubes at ⫺80⬚C for over 24 mo before the commencement of the study, but preliminary tests showed no loss of activity. The kairomone was thawed immediately before use. The range of concentrations selected was designed to induce the full range of neck morphologies expressed by this clone of D. pulex. Scenedesmus obliquus was added to each vial at a concentration of 0.5 mg C L⫺1. Tollrian and von Elert (1994) recommended culturing the embryos without food to reduce the possibility of bacterial degradation of kairomone; however, preliminary tests indicated that the presence of food increased the survival rate of embryos in the presence of some drugs but did not affect the number of neckteeth expressed. In each experiment, daphnid embryos were exposed to between four and six concentrations of a specific drug and one of four concentrations of kairomone. Thus, there were up to 24 treatments in a single experiment with each treatment replicated 10 times. Full details of the test concentrations are provided in Table 1. The culture plates were stored in the dark for 48 h at 20⬚ Ⳳ 1⬚C, then checked under a microscope for neonates that had moulted into their second instar. Animals in the second instar were easy to identify because the first-instar moult was visible in the culture well. Second-instar daphnids were removed for measurement, and the plates were returned to the incubation chamber for a further 24 h. Daphnids that had not moulted within 72 h of the commencement of the experiment were discarded. Daphnids were measured under a stereo-binocular microscope at a magnification of #40, followed by closer examination at #400. In a separate experiment, to illustrate the effect of high concentrations of chemicals on the induction of neckteeth, D. pulex embryos were exposed to a wide range of concentrations of

PTX in the presence of 1.5 mL kairomone mL⫺1, and the neck score was determined in the second neonatal instar. A modification of two previous systems was used for scoring neckteeth (Parejko and Dodson 1990; Tollrian 1993) and took into account the fact that induction in this clone involved the development both of neckteeth and of a dorsal keel or neck hump. Three neck shapes were recognised as representing stages from noninduced through maximal induction: normal, neckkeel formation, and neck keel with pedestal (see Fig. 1 of Tollrian 1993 for photographs of each neck type). Neck shape was scored as follows: normal p 0; neck-keel formation p 0.5; and neck keel with pedestal p 1. A scoring system was also used for the number and size of neckteeth. A large, fully developed necktooth was scored as 1; a necktooth that was !50% in height of a large necktooth, but still clearly visible, was scored as 0.5. A necktooth that was only visible under the highest power magnification (#400) was scored as 0.1. The maximum number of neckteeth observed in this study was four. A total measure of induction, called “neck score,” was then calculated as the sum of neck shape plus neckteeth scores. The effects of the kairomone and each drug on the expression of neckteeth was analysed using two-way ANOVA. Tukey’s test was used as a post hoc test, when the ANOVA indicated that factor was significant. Heteroscedastic data were log transformed before analysis. A probability of P ≤ 0.05 was selected as evidence of significance. Results are presented as means Ⳳ 1 SE. Results In all experiments, the number of neckteeth produced was positively correlated with kairomone concentration, but the number and size of neckteeth produced was also affected by

Table 1: Effects of nine drugs on induction of neckteeth in Daphnia pulex ANOVA Drug

Concentrations (mM)

Drug

Kairomone

Interaction

Physostigmine Nicotene Atropine Picrotoxin SR9553 Bicuculline Diazepam Muscimol CACAa

.001, .005, .01, .05, .10 .001, .01, .1, 1.0 1, 5, 10, 50 .05, .1, .5, 5 .001, .01, .10 .001, .01, .10 .005, .01, .05, .1, .5, 1.0 .01, .1, 1.0, 10, 50 .001, .01, .10, 1.0, 10

* * *** *** NS NS NS NS *

*** *** *** *** *** *** *** ** ***

*** NS NS *** NS NS NS NS NS

Note. ANOVA indicates the effects of the main factors (drug and kairomone concentration and interaction) on induction of neckteeth. a Cis-4-aminocrotonic acid. * P ! 0.05. ** P ! 0.01. *** P ! 0.001.

182 M. J. Barry the test chemicals. In the absence of the Chaoborus kairomone, none of the drugs induced morphological change (i.e., neckteeth scores almost always equal to zero), indicating that they were not mimicking the action of the kairomone but modulating a physiological response. Because there was no response in the absence of kairomone, these data were excluded from statistical analysis. The cholinesterase inhibitor, PHY, significantly increased the neck score at concentrations ≤0.001 mM but only at 0.25 mL kairomone mL⫺1 (P ! 0.05; Fig. 1A). In this experiment, there was no significant difference in neck score between 0.5 mg kairomone mL⫺1 and 1.5 mg kairomone mL⫺1, and PHY did not increase neck scores above these levels. There was also an effect of the prototype nicotinic agonist, NIC, on neck score; however,

the effect was minor (P ! 0.05). A Tukey’s test was unable to determine at what concentration this occurred, but mean neck scores increased from 2.24 Ⳳ 0.19 for controls to 2.99 Ⳳ 0.19 at 0.1 mM but decreased at 1.0 mM NIC (Table 2). The nonspecific muscarinic antagonist, ATR, reduced the number of neckteeth at a concentration of 5 mM (P ! 0.05; Fig. 1B). The effect of ATR on the expression of neckteeth was similar at all concentrations of kairomone. At 50 mM, ATR completely blocked the development of neckteeth at all but 1.5 mL kairomone mL⫺1. There was no effect of the GABA modulator, DIA, on neck score (P 1 0.05). Similarly, there was no effect of the GABA agonist, MUS, up to 50 mM (P 1 0.05). The GABAc agonist, CACA, induced a small increase in neck scores at intermediate

Figure 1. Effects of three drugs and three kairomone concentrations on the expression of neckteeth (neck score Ⳳ 1 SE) in second-instar Daphnia pulex. A, Physostigmine (PHY); B, atropine (ATR); C, picrotoxin (PTX). White bars p 0.25 mL mL kairomone mL⫺1; grey bars p 0.5 mL mL kairomone mL⫺1; black bars p 1.5 mL mL kairomone mL⫺1; asterisk p P ! 0.05.

Regulation of Neckteeth Induction in Daphnia 183 Table 2: Effects of nicotine (NIC) and cis-aminocrotonic acid (CACA) on expression of neckteeth in Daphnia pulex Kairomone (mL mL⫺1) Drug/concentrations (mM) NIC: 0A .001A .01A .1A 1.0A CACA: 0AB .001A .01B .1AB 1.0AB 10AB

.25

.5

1.5

1.76 1.41 2.37 2.72 1.12

(.35) (.37) (.35) (.33) (.37)

2.00 2.39 2.26 2.57 1.96

(.33) (.33) (.33) (.33) (.31)

2.94 3.06 3.11 3.69 3.30

(.33) (.35) (.33) (.37) (.31)

2.69 2.22 3.11 2.60 2.36 2.46

(.40) (.38) (.36) (.36) (.43) (.38)

3.00 2.71 3.80 2.75 3.34 3.37

(.33) (.36) (.34) (.40) (.38) (.40)

3.62 3.44 4.15 3.41 3.20 3.72

(.40) (.38) (.36) (.38) (.30) (.38)

Note. The effects of NIC, CACA, and three concentrations of Chaoborus kairomone on the expression of neckteeth (neck score) in second-instar Daphnia pulex. P ! 0.05 in ANOVA. Common alphabetic superscripts indicate no significant difference in Tukey’s test (P ! 0.05).

concentrations. Thus, mean scores (averaged over the three kairomone concentrations) increased from 3.10 Ⳳ 0.22 for controls to 3.69 Ⳳ 0.20 at 0.01 mM and decreased to 3.18 Ⳳ 0.20 at 10 mM (P ! 0.05). The increase in neck score was most noticeable at 0.25 and 0.5 mL kairomone mL⫺1 (Table 2). PTX, a GABA antagonist, significantly increased the neck score at concentrations ≥0.05 mM PTX in the presence of 0.25 and 0.5 mL kairomone mL⫺1 but had no effect at 1.5 mL kairomone mL⫺1 (P ! 0.05; Fig. 1C). There was no significant effect of the synthetic GABA antagonist SR95531 or BIC on neck scores (P 1 0.05). When D. pulex embryos were exposed to increasing concentrations of PTX in the presence of sufficient kairomone to cause maximal induction of neckteeth, there was no effect of the drug until a concentration of 5,000 mM was reached. At this point, there was significant inhibition of neckteeth, which resulted in a lower neck score (Fig. 2). Discussion The adaptive expression of inducible morphology involves a multistep pathway that is poorly understood. The first step is the discrimination of the predator kairomone from a chemical milieu that contains thousands of different types of molecules. Translation of information indicating presence of predators in the proximate environment into the morphogenic activity needed for development of neckteeth requires transmission of the information via the nervous system, hormonal system, or a combination of both. Based on the work of Jacobs (1980),

we can postulate that neural signals from the brain innervate neurosecretory cells that release hormones into the haemolymph. These hormones would ultimately bind to receptors on the polynucleated cells at the base of the inducible structures (Beaton and Hebert 1997) and these, in turn, would produce the morphogens necessary to stimulate mitosis. Neurosecretory cells, classically defined as neurones specialised for the synthesis, storage, and secretion of hormones, are quantitatively more important and play more varied roles in crustaceans than their counterparts in vertebrates. Sterba (1957) identified two groups of neurosecretory cells in the brains of Daphnia pulex and Daphnia magna, one of which is situated ventrally within the protocerebrum and consists of four large cells. A second group is symmetrical and consists of distinct cells near the midline of the brain. Zahid et al. (1980) identified four types of neurosecretory cells in the brain of Simocephalus vetulus. On the basis of the experiments of Hanazato (1991b, 1992), it was predicted that cholinergic agonists and cholinesterase inhibitors would stimulate neckteeth formation and that cholinergic antagonists would inhibit it. The cholinergic inhibitor, PHY, synergistically interacted with low concentrations of Chaoborus kairomone to induce maximal expression of neckteeth in D. pulex. However, it did not induce the growth of neckteeth in the absence of kairomone, nor would it induce larger neck scores than caused by high concentrations of kairomone alone. These findings suggest that it was stimulating the release of the neurohormone responsible neckteeth development. PHY carbamoylates the active serine residue of the cholinesterase molecule, greatly slowing the acyl-enzyme hydrolysis reaction compared with the acetylated enzyme (O’Brien 1969). In contrast to PHY, NIC, the prototypic nicotinic agonist, showed only very weak induction of neckteeth relative to controls. These results may indicate that the receptors that regulate release of the neurohormones are muscarinic in nature or that the generalised toxicity of NIC inhibited any inductive response. Previous studies have shown that the embryos of D. pulex are particularly vulnerable to inhibition at toxic concentrations of chemicals (Barry 2000). The cholinergic antagonist, ATR, competitively inhibits the binding of acetylcholine to its receptor. ATR inhibited the development of neckteeth at all concentrations of kairomone in a dose-dependent manner. In vertebrates, ATR is specifically a muscarinic antagonist; however, in invertebrates, this classification may not hold. GABA is generally accepted to be an inhibitory transmitter in the peripheral nervous system of invertebrates (Robinson and Olsen 1988). In this study, PTX interacted with low concentrations of kairomone to induce maximal neckteeth expression in a manner similar to PHY. PTX is thought to block the Cl⫺ ionophore either directly or by binding to a closely located site (Barker et al. 1983). PTX has also been reported to block GABA effects in lobster and crayfish muscles (Takeuchi and Takeuchi 1969), and it decreased the GABA-activated re-

184 M. J. Barry

Figure 2. The effect of increasing concentrations of picrotoxin (PTX) on the expression of neckteeth (neck score) in second-instar Daphnia pulex in the presence of 1.5 mL kairomone mL⫺1. Asterisk p P ! 0.05.

sponse by 70% in embryonic lobster neurones (Jackel et al. 1994). In contrast, there was no effect of BIC or the synthetic GABAa antagonist SR95531 on the induction of neckteeth, except at near toxic concentrations where they reduced the neck score. Jackel et al. (1994) also found no effects of BIC or SR95531 on the GABA receptor of embryonic and adult lobster neurones in cell culture. BIC is generally not an effective antagonist in invertebrates (Osborne 1996). It was predicted that drugs that mimicked or stimulated the effects of GABA would cause a reduction in the number of neckteeth. Neither the GABAa agonist, MUS, or the GABA modulator, DIA, significantly inhibited neck scores, except at concentrations that were close to toxic. However, this effect was observed with all chemicals, no matter what their mode of action. This result was demonstrated in the experiment in which embryos were exposed to high concentrations of PTX. It was therefore not possible to determine whether they were directly affecting the GABA receptor; however, the results suggest that the daphnid GABA receptor is conformationally different from the vertebrate one. In contrast, Jackel et al. (1994) found, in lobster neurones, that GABA-evoked currents could be mimicked by MUS and CACA, but there was no effect of DIA. There was, however, a small inductive effect of CACA, the mammalian GABAc agonist, at intermediate concentrations. Further experiments were performed in an attempt to identify a possible narrow range active concentration for CACA, but results were similar in all cases. It is possible that CACA only weakly binds to the GABA receptor in daphnids or at a site distant to the ionophore and thus actually inhibits the binding of GABA rather than mimicking it. The above results highlight one of the complexities of this

study, where drugs that are normally used with isolated cell preparations were used to measure whole-organism responses. All the chemicals used were neurotoxins and, at high concentrations, can cause generalised toxicity. This toxicity may inhibit vital processes such as feeding, respiration, and movement. Thus, the reduction in the expression of neckteeth at high concentrations of most drugs may have been due to a reduction in nutrition and inhibition of growth, rather than the direct inhibition of stimulatory neurohormones. Previous studies have demonstrated that morphological induction can be inhibited by poor nutrition (Hanazato 1991a; Barry 1995). Ethanol was used as a carrier for some drugs (PHY, DIA, CACA) and may have interacted with the effects of some chemicals. However, a direct inhibitory effect of ethanol is unlikely because no inhibition was seen with PHY at the highest concentrations used. In contrast, the reduction in neck scores caused by ATR showed quite a different pattern to that of the other drugs. There was a gradual decrease in induction, approaching complete inhibition at the highest ATR concentrations, suggesting a more specific response. It is now well established that chemically induced defenses are found in many aquatic taxa (Larsson and Dodson 1993; Tollrian and Harvell 1999). Chemical communication between planktonic species was recently characterised as a low-energy, information-rich network that parallels the flow of materials through ecosystems (Ringelberg 1997). Despite a growing awareness of the breadth of taxa that have evolved systems of chemically induced defenses and their importance to population- and ecosystem-level processes, insights into the physiological basis of regulation remain limited. An understanding of the neurological mechanisms that regulate expression of in-

Regulation of Neckteeth Induction in Daphnia 185 ducible morphology is necessary before the underlying molecular processes can be elucidated. These, in turn, may provide insights into the genetics and evolution of defense mechanisms. This study showed that the development of Chaoborusinduced neckteeth in D. pulex could be modulated by cholinergic and GABAergic drugs, and it proposed that these chemicals were acting on neurosecretory cells that released the hormones necessary to induce neckteeth development. The results also indicate mechanisms through which anthropogenic pollutants could influence the expression of inducible defenses, leading to inappropriate expression in environments with low predator intensity or to suppression in environments with high risks of predation. Over the past 2 decades, scientists have become increasingly concerned about the presence of manmade chemicals in the environment that could disrupt endogenous endocrine processes in humans and wildlife (Colborn et al. 1996). Previous studies have reported that several widely used pesticides, including carbaryl and endosulfan, can effect the expression of inducible defenses in Daphnia (Hanazato 1991b; Barry 1998). The effects of these chemicals fit the definition of endocrine-disrupting chemicals. This study demonstrates a likely mode of action and indicates that other chemicals that can modulate neurosecretion may also affect induction. It was also found that all the chemicals that were tested could inhibit the development of neckteeth at near-toxic concentrations. This supports previous work that showed that endosulfan (Barry 2000) and cadmium (Barry and Stoopman 2000) could inhibit inducible defenses at sublethal concentrations. Acknowledgments I thank W. Lampert and the Max Planck Society for supporting the project; R. Tollrian for providing the kairomone; and P. Wright and K. Way for helpful discussion. I also thank D. Lemcke for technical support. Literature Cited Barker J.L., R.N. McBurney, and D.A. Mathers. 1983. Convulsant-induced depression of amino acid responses in cultured mouse spinal neurones studied under voltage clamp. Br J Pharmacol 80:619–629. Barry M.J. 1995. The role of nutrition in regulation of predatorinduced crests of Daphnia carinata. Freshwater Biol 34: 229–239. ———. 1998. Endosulfan-enhanced crest induction in Daphnia longicephala: evidence for cholinergic innervation of kairomone receptors. J Plankton Res 20:1219–1231. ———. 2000. Effects of endosulfan on Chaoborus-induced lifehistory shifts and morphological defenses in Daphnia pulex. J Plankton Res 22:1705–1718.

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