Ionotropic Glutamate Receptors Mediate Inducible Defense in the Water Flea Daphnia pulex

<div><p>Phenotypic plasticity is the ability held in many organisms to produce different phenotypes with a given genome in response to environmental stimuli, such as temperature, nutrition and various biological interactions. It seems likely that environmental signals induce a variety of mechanistic responses that influence ontogenetic processes. Inducible defenses, in which prey animals alter their morphology, behavior and/or other traits to help protect against direct or latent predation threats, are among the most striking examples of phenotypic plasticity. The freshwater microcrustacean <i>Daphnia pulex</i> forms tooth-like defensive structures, “neckteeth,” in response to chemical cues or signals, referred to as “kairomones,” in this case released from phantom midge larvae, a predator of <i>D</i>. <i>pulex</i>. To identify factors involved in the reception and/or transmission of a kairomone, we used microarray analysis to identify genes up-regulated following a short period of exposure to the midge kairomone. In addition to identifying differentially expressed genes of unknown function, we also found significant up-regulation of genes encoding ionotropic glutamate receptors, which are known to be involved in neurotransmission in many animal species. Specific antagonists of these receptors strongly inhibit the formation of neckteeth in <i>D</i>. <i>pulex</i>, although agonists did not induce neckteeth by themselves, indicating that ionotropic glutamate receptors are necessary but not sufficient for early steps of neckteeth formation in <i>D</i>. <i>pulex</i>. Moreover, using co-exposure of <i>D</i>. <i>pulex</i> to antagonists and juvenile hormone (JH), which physiologically mediates neckteeth formation, we found evidence suggesting that the inhibitory effect of antagonists is not due to direct inhibition of JH synthesis/secretion. Our findings not only provide a candidate molecule required for the inducible defense response in <i>D</i>. <i>pulex</i>, but also will contribute to the understanding of complex mechanisms underlying the recognition of environmental changes, which form the basis of phenotypic plasticity.</p></div

GO terms in the molecular function ontology significantly enriched among genes with differential responses to the kairomone (FDR < 0.1).

<p>GO terms in the molecular function ontology significantly enriched among genes with differential responses to the kairomone (FDR < 0.1).</p

Effect of ionotropic glutamate receptor agonists on the three different indicators of the defensive morph.

<p>The indicators are incidence of neckteeth (left), average number of neckteeth (center), and crest thickness normalized to body-length (right). The final concentration of all chemicals used in this experiment was 10 μM. Error bars indicate 95% confidence intervals. Different letters indicate significant differences (p < 0.01). N: number of specimens.</p

Up- or down-regulated genes following exposure to the kairomone.

<p>Up- or down-regulated genes following exposure to the kairomone.</p

Effect of ionotropic glutamate receptor antagonists on the three different indicators of the defensive morph.

<p>The indicators are incidence of neckteeth (left), average number of neckteeth (center), and crest thickness normalized to body-length (right). Error bars indicate 95% confidence intervals. Asterisks indicate significant differences (p < 0.01) as compared with <i>Daphnia</i> exposed to the same kairomone conditions without antagonists (-). N: number of specimens.</p

Effect of coexposure of methyl farnesoate (MF) and MK-801 (a) or NBQX (b) on the three different indicators of defensive morph.

<p>The indicators are incidence of neckteeth (left), average number of neckteeth (center), and crest thickness normalized to body-length (right). Kairomone (x1/16) was used for neckteeth induction. The final concentrations of chemicals used in this experiment were as follows: 10 μM of MK-801; 100 μM of NBQX; 100 μg/L (400 nM) of MF. Error bars indicate 95% confidence intervals. Different letters indicate significant differences (p < 0.01). N: number of specimens.</p

Metabolomics reveals target and off-target toxicities of a model organophosphate pesticide to roach (rutilus rutilus): implications for biomonitoring

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Whole-Organism Transcriptomic Analysis Provides Mechanistic Insight into the Acute Toxicity of Emamectin Benzoate in <i>Daphnia magna</i>

Emamectin benzoate (EMB) is an antisea lice chemical widely used in the aquaculture that may also unintentionally affect nontarget crustaceans in the environment. Although the adverse effects of this compound are well documented in various species, the full modes of action (MoAs) are still not well characterized. The current study was therefore conducted to characterize the MoAs of EMB and link perturbations of key toxicological pathways to adverse effects in the model freshwater crustacean <i>Daphnia magna</i>. Effects on molting and survival were determined after 48 h exposure to EMB, whereas global transcriptional changes and the ecdysone receptor (EcR) binding potency was determined to characterize the MoA. The results showed that the molting frequency and survival of <i>D. magna</i> decreased in a concentration-dependent manner, and the observed changes could not be attributed to direct interactions with the EcR. Major MoAs such as activation of glutamate-gated chloride channels and gamma-aminobutyric acid signaling, disruption of neuroendocrine regulation of molting, perturbation of energy homeostasis, suppression of DNA repair and induction of programmed cell death were observed by transcriptional analysis and successfully linked to the adverse effects. This study has demonstrated that acute exposure to intermediate and high pM levels of EMB may pose hazards to nontarget crustaceans in the aquatic environment

Metabolomics Reveals Target and Off-Target Toxicities of a Model Organophosphate Pesticide to Roach (Rutilus rutilus): Implications for Biomonitoring

The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a “red list” pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach (Rutilus rutilus) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish

Metabolomics Reveals Target and Off-Target Toxicities of a Model Organophosphate Pesticide to Roach (Rutilus rutilus): Implications for Biomonitoring

The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a “red list” pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach (Rutilus rutilus) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish