Pharmacological modulation of fish-induced depth selection in D. magna: the role of cholinergic and GABAergic signalling

Animal behaviour is closely related to individual fitness, which allows animals to choose suitable mates or avoid predation. The central nervous system regulates many aspects of animal behaviour responses. Therefore, behavioural responses can be especially sensitive to compounds with a neurodevelopmental or neurofunctional mode of action. Phototactic behavioural changes against fish in the freshwater crustacean Daphnia magna have been the subject of many ecological investigations. The aim of this study was to identify which neurotransmitter systems modulate phototactic behaviour to fish kairomones. We used a positive phototactic D. magna clone (P132,85) that shows marked negative phototactism after exposure to fish kairomones. Treatments included up to 16 known agonists and antagonists of the serotonergic, cholinergic, dopaminergic, histaminergic, glutamatergic and GABAergic systems. It was hypothesized that many neurological signalling pathways may modulate D. magna phototactic behaviour to fish kairomones. A new custom-designed device with vertically oriented chambers was used, and changes in the preferred areas (bottom, middle, and upper areas) were analysed using groups of animals after 24 h of exposure to the selected substance(s). The results indicated that agonists of the muscarinic acetylcholine and GABAA receptors and their equi-effective mixture ameliorated the negative phototactic response to fish kairomones, whereas antagonists and their mixtures increased the negative phototactism to fish kairomones. Interestingly, inhibition of the muscarinic acetylcholine receptor abolished positive phototaxis, thus inducing the phototactic response to fish kairomones. Analysis of the profile of neurotransmitters and their related metabolites showed that the D. magna behavioural responses induced by fish depend on changes in the levels of acetylcholine, dopamine and GABA.

Acute Toxicity of Insecticide Thiamethoxam to Crayfish (Astacus leptodactylus): Alterations in Oxidative Stress Markers, ATPases and Cholinesterase

Thiamethoxam (Thmx) is a globally used neonicotinoid pesticide contaminated in freshwater ecosystems with residues detected in fishery products. Astacus leptodactylus is a popular freshwater crustacean that is cultivated and exported in many countries. In this study, we investigated the acute toxic effects of Thmx on A. leptodactylus using various biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, glutathione, superoxide dismutase, glutathione peroxidase, glutathione reductase, and adenosinetriphosphatases). The 96-h LC50 value of Thmx was calculated as 8.95 mg active ingredient L–1. As the dose of Thmx increased, oxidative stress was induced by the inhibition/activation of antioxidant enzymes, while the activities of acetylcholinesterase, carboxylesterase and adenosinetriphosphatases were inhibited. As a result, it can be said that Thmx has highly toxic effects on crayfish, therefore they are under threat in the areas where this pesticide is used.

Early presence of Bythotrephes cederströmii (Cladocera: Cercopagidae) in lake sediments in North America: evidence or artifact?

The spiny water flea (Bythotrephes cederströmii), a freshwater crustacean considered to be the world’s best-studied invasive zooplankter, was first recorded in North America in the Laurentian Great Lakes during the 1980s. Its arrival is widely considered to be the result of ocean-going cargo ships that translocated contaminated ballast water from Eurasia to the Great Lakes during the 1970–1980s. The subsequent first discovery of the species in inland lakes is consistent with the hypothesis that propagules dispersed initially from established Great Lakes populations. Here we present evidence of exoskeletal remains, including mandibles, tail spines, and resting eggs, in 210Pb-dated lake sediment cores, which suggests that B. cederströmii was already resident in four inland North American lakes (two in Minnesota, USA; two in Ontario, Canada) by at least the early 1900s. Densities of exoskeletal remains were low and relatively steady from first appearance until about 1990, after which time they increased in all cores. The earliest evidence that we found was a mandible at 33-cm depth (pre-1650) in the sediments of Three Mile Lake, Ontario, Canada. These unexpected findings challenge the current paradigm of B. cederströmii invasion, renew uncertainty about the timing and sequence of its colonization of North American lakes, and potentially question our ability to detect invasive species with traditional sampling methods. We attempted to eliminate errors in the dated stratigraphies of the exoskeletal remains that might have been introduced either methodologically (e.g., core-wall smearing) or naturally (e.g., bioturbation). Nonetheless, given the very low numbers of subfossils encountered, questions remain about the possible artifactual nature of our observations and therefore we regard our results as ‘preliminary findings’ at this time.

Thwarting predators? A three-dimensional perspective of morphological alterations in the freshwater crustacean Daphnia

Predation is a major selective agent, so that many taxa evolved phenotypically plastic defensive mechanisms. Among them are many species of the microcrustacean genus Daphnia, which respond to an increased predation risk by developing inducible morphological alterations. Some of these features are obvious and easily recognized, e.g., crests in D. longicephala, while others are rather hidden, such as the bulkier shape of D. magna induced by the presence of the tadpole shrimp Triops. In this study we investigated the extraordinary diversity of morphological adaptations in the presence of predators with different foraging strategies in six predator-prey systems. For the first time we were able to analyze the unexposed and predator-exposed morphs comprehensively using three-dimensional scanning and reconstruction. We show that morphological changes are manifold in appearance between species and predators, and go beyond what has been known from previous 2D analyses. This further demonstrates the enormous trait flexibility of Daphnia. Interestingly, we found that among this variety some species share morphological strategies to counter a predator, while others use a different strategy against the same predator. Based on these intra- and interspecific comparisons, we discuss the mechanisms by which the respective defense might operate. These data therefore contribute to a deeper understanding of the inducible defenses’ morphology as well as their diversified modes of operation in Daphnia, being a cornerstone for subsequent investigations, including the determination of costs associated with morphological change.

Brain volume increase and neuronal plasticity underly predator-induced morphological defense expression in Daphnia longicephala

Predator-induced phenotypic plasticity describes the ability of prey to respond to an increased predation risk by developing adaptive phenotypes. Upon the perception of chemical predator cues, the freshwater crustacean Daphnia longicephala develops defensive crests against its predator Notonecta spec. (Heteroptera). Chemical predator perception initiates a cascade of biological reactions that leads to the development of these morphological features. Neuronal signaling is a central component in this series, however how the nervous system perceives and integrates environmental signals is not well understood. As neuronal activity is often accompanied by functional and structural plasticity of the nervous system, we hypothesized that predator perception is associated with structural and functional changes of nervous tissues. We observe structural plasticity as a volume increase of the central brain, which is independent of the total number of brain cells. In addition, we find functional plasticity in form of an increased number of inhibitory post-synaptic sites during the initial stage of defense development. Our results indicate a structural rewiring of nerve-cell connections upon predator perception and provide important insights into how the nervous system of prey species interprets predator cues and develops cost–benefit optimized defenses.

Higher, Faster, Better: Maternal Effects Shorten Time Lags and Increase Morphological Defenses in Daphnia lumholtzi Offspring Generations

Prey species can respond to the presence of predators by inducing phenotypic plastic traits which form morphological, life history or behavioral defenses. These so-called inducible defenses have evolved within a cost-benefit framework. They are only formed when they are needed, and costs associated with defenses are saved when predators are not present. However, a disadvantage compared to permanent defenses are lag phases between predator perception and the full formation of defenses. This may be especially important when the predation risk persists for longer periods, e.g., outlasts one generation and challenges prey offspring. We hypothesized that transgenerational induced phenotypic plasticity reduces lag phases in situations where hazards threaten specimens over several generations. We tested this in three generations of the freshwater crustacean Daphnia lumholtzi using the three-spined stickleback Gasterosteus aculeatus as predator. In the presence of chemical cues from fish D. lumholtzi expresses elongated head and tail spines. In the F0 generation defenses are constraint by a comparatively long lag phase and are not developed prior to the 3rd instar. In the F1, and F2 of induced animals this lag phase is shortened and defenses are developed upon birth. We show that induction of TGP in the mothers takes place already during the juvenile stages and transfers to the offspring generation in forms of shortened time lags and enhanced trait expression. When progeny is additionally exposed to fish cues as embryos, the addition of maternal and embryonic effects further enhances the magnitude of defense expression. Our findings detail a distinguished strategy of transgenerational phenotypic plasticity which allows to shorten lag phases of trait changes in phenotypic plasticity.

Vacuolar H+-ATPase and Na+/K+-ATPase energize Na+ uptake mechanisms in the nuchal organ of the hyperregulating freshwater crustacean Daphnia magna.

The nuchal organ of the embryos and neonates of the cladoceran, Daphnia magna, has been shown to be a site of Na+ influx and H+, NH4+ and Cl− efflux. This study combines the scanning-ion selective electrode technique with application of inhibitors of specific transporters to assess the mechanisms of Na+ transport across the nuchal organ. Na+ influx across the nuchal organ was inhibited both by inhibitors of the Na+/K+-ATPase (ouabain, bufalin) and by inhibitors of the vacuolar H+-ATPase (bafilomycin, N-ethylmaleimde, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, KM91104, S-nitrosoglutathione). Na+ influx was unaffected by the epithelial Na+ channel blocker benzamil, but was sensitive to ethylisopropyl amiloride and elevated external ammonium concentrations, consistent with roles for Na+/H+ and Na+/NH4+ exchangers in the apical membrane but not Na+ channels. Transport across the basolateral membrane into the hemolymph is proposed to involve the Na+/K+-ATPase and a thiazide-sensitive Na+:Cl− cotransporter. Keywords: Daphnia magna, Na+/K+-ATPase, V-ATPase, Iionoregulation, Nnuchal organ