Linda C. Weiss

More than one genotype: how common is intracolonial genetic variability in scleractinian corals?

In recent years, a few colonial marine invertebrates have shown intracolonial genetic variability, a previously unreported phenomenon. Intracolonial genetic variability describes the occurrence of more than a single genotype within an individual colony. This variability can be traced back to two underlying processes: chimerism and mosaicism. Chimerism is the fusion of two or more individuals, whereas mosaicism mostly derives from somatic cell mutations. Until now, it remained unclear to what degree the ecologically important group of hermatypic (reef building) corals might be affected. We investigate the occurrence of intracolonial genetic variability in five scleractinian corals: Acropora florida, Acropora hyacinthus, Acropora sarmentosa, Pocillopora species complex and Porites australiensis. The main focus was to test different genera for the phenomenon via microsatellite markers and to distinguish which underlying process caused the genetic heterogeneity. Our results show that intracolonial genetic variability was common (between 46.6% for A. sarmentosa and 23.8% for P. species complex) in all tested corals. The main process was mosaicism (69 cases of 222 tested colonies), but at least one chimera existed in every species. This suggests that intracolonial genetic variability is widespread in scleractinian corals and could challenge the view of a coral colony as an individual and therefore a unit of selection. However, it might also hold potential for colony survival under rapidly changing environmental conditions.

Chaoborus and Gasterosteus Anti-Predator Responses in Daphnia pulex Are Mediated by Independent Cholinergic and Gabaergic Neuronal Signals

Many prey species evolved inducible defense strategies that protect effectively against predation threats. Especially the crustacean Daphnia emerged as a model system for studying the ecology and evolution of inducible defenses. Daphnia pulex e.g. shows different phenotypic adaptations against vertebrate and invertebrate predators. In response to the invertebrate phantom midge larvae Chaoborus (Diptera) D. pulex develops defensive morphological defenses (neckteeth). Cues originating from predatory fish result in life history changes in which resources are allocated from somatic growth to reproduction. While there are hints that responses against Chaoborus cues are transmitted involving cholinergic neuronal pathways, nothing is known about the neurophysiology underlying the transmission of fish related cues. We investigated the neurophysiological basis underlying the activation of inducible defenses in D. pulex using induction assays with the invertebrate predator Chaoborus and the three-spined stickleback Gasterosteus aculeatus. Predator-specific cues were combined with neuro-effective substances that stimulated or inhibited the cholinergic and gabaergic nervous system. We show that cholinergic-dependent pathways are involved in the perception and transmission of Chaoborus cues, while GABA was not involved. Thus, the cholinergic nervous system independently mediates the development of morphological defenses in response to Chaoborus cues. In contrast, only the inhibitory effect of GABA significantly influence fish-induced life history changes, while the application of cholinergic stimulants had no effect in combination with fish related cues. Our results show that cholinergic stimulation mediates signal transmission of Chaoborus cues leading to morphological defenses. Fish cues, which are responsible for predator-specific life history adaptations involve gabaergic control. Our study shows that both pathways are independent and thus potentially allow for adjustment of responses to variable predation regimes.

Morphology of the Daphnia nervous system: A comparative study on Daphnia pulex, Daphnia lumholtzi, and Daphnia longicephala

The freshwater crustacean Daphnia is well known for its phenotypic plasticity, in which environmental cues are perceived by the nervous system and transformed into phenotypic adaptations beneficial under current conditions. Critical knowledge regarding the distribution and localization of neuronal antigens or neurotransmitters and differentially expressed proteins is sparse. Here, we applied immunohistochemical and histological‐staining methods in combination with confocal laser scanning microscopy on whole mount preparations in Daphnia pulex, Daphnia longicephala, and Daphnialumholtzi. We document the nervous system, elements of the sensory system, and cell clusters with secretory characteristics in the Daphnia head. This is the first report on the nervous system of the species D.longicephala and D.lumholtzi. The methods established in this investigation will help to foster research on specific neuronal mechanisms in this rapidly advancing model system of life science research. J. Morphol., 2012.

Predator-induced defences in Daphnia longicephala: location of kairomone receptors and timeline of sensitive phases to trait formation

ABSTRACT The freshwater crustacean Daphnia adapts to changing predation risks by forming inducible defences. These are only formed when they are advantageous, saving associated costs when the defence is superfluous. However, in order to be effective, the time lag between the onset of predation and the defence formation has to be short. Daphnia longicephala develop huge protective crests upon exposure to chemical cues (kairomones) from its predator the heteropteran backswimmer Notonecta glauca. To analyse time lags, we determined kairomone-sensitive stages and the developmental time frames of inducible defences. Moreover, we looked at additive effects that could result from the summation of prolonged kairomone exposure. Kairomones are perceived by chemoreceptors and integrated by the nervous system, which alters the developmental program leading to defence formation. The underlying neuronal and developmental pathways are not thoroughly described and surprisingly, the location of the kairomone receptors is undetermined. We show that D. longicephala start to sense predator cues at the onset of the second juvenile instar, defences develop with a time lag of one instar and prolonged kairomone exposure does not impact the magnitude of the defence. By establishing a method to reversibly impair chemosensors, we show the first antennae as the location of kairomone-detecting chemoreceptors. This study provides fundamental information on kairomone perception, kairomone-sensitive stages, developmental time frames and lag times of inducible defences in D. longicephala that will greatly contribute to the further understanding of the neuronal and developmental mechanisms of predator-induced defences in Daphnia. Highlighted Article: Chemoreceptors for kairomone perception are localized on the first antennae of Daphnia longicephala and their role in inducible defences contributes greatly to our understanding of the neuronal and developmental mechanisms of predator-induced defences.

Density-dependent adjustment of inducible defenses

Predation is a major factor driving evolution, and organisms have evolved adaptations increasing their survival chances. However, most defenses incur trade-offs between benefits and costs. Many organisms save costs by employing inducible defenses as responses to fluctuating predation risk. The level of defense often increases with predator densities. However, individual predation risk should not only depend on predator density but also on the density of conspecifics. If the predator has a saturating functional response one would predict a negative correlation between prey density and individual predation risk and hence defense expression. Here, we tested this hypothesis using six model systems, covering a taxonomic range from protozoa to rotifers and crustaceans. In all six systems, we found that the level of defense expression increased with predator density but decreased with prey density. In one of our systems, i.e. in Daphnia, we further show that the response to prey density is triggered by a chemical cue released by conspecifics and congeners. Our results indicate that organisms adjust the degree of defense to the acute predation risk, rather than merely to predators’ densities. Our study suggests that density-dependent defense expression reflects accurate predation-risk assessment and is a general principle in many inducible-defense systems.

Transcriptional profiling of predator-induced phenotypic plasticity in Daphnia pulex

BackgroundPredator-induced defences are a prominent example of phenotypic plasticity found from single-celled organisms to vertebrates. The water flea Daphnia pulex is a very convenient ecological genomic model for studying predator-induced defences as it exhibits substantial morphological changes under predation risk. Most importantly, however, genetically identical clones can be transcriptionally profiled under both control and predation risk conditions and be compared due to the availability of the sequenced reference genome. Earlier gene expression analyses of candidate genes as well as a tiled genomic microarray expression experiment have provided insights into some genes involved in predator-induced phenotypic plasticity. Here we performed the first RNA-Seq analysis to identify genes that were differentially expressed in defended vs. undefended D. pulex specimens in order to explore the genetic mechanisms underlying predator-induced defences at a qualitatively novel level.ResultsWe report 230 differentially expressed genes (158 up- and 72 down-regulated) identified in at least two of three different assembly approaches. Several of the differentially regulated genes belong to families of paralogous genes. The most prominent classes amongst the up-regulated genes include cuticle genes, zinc-metalloproteinases and vitellogenin genes. Furthermore, several genes from this group code for proteins recruited in chromatin-reorganization or regulation of the cell cycle (cyclins). Down-regulated gene classes include C-type lectins, proteins involved in lipogenesis, and other families, some of which encode proteins with no known molecular function.ConclusionsThe RNA-Seq transcriptome data presented in this study provide important insights into gene regulatory patterns underlying predator-induced defences. In particular, we characterized different effector genes and gene families found to be regulated in Daphnia in response to the presence of an invertebrate predator. These effector genes are mostly in agreement with expectations based on observed phenotypic changes including morphological alterations, i.e., expression of proteins involved in formation of protective structures and in cuticle strengthening, as well as proteins required for resource re-allocation. Our findings identify key genetic pathways associated with anti-predator defences.

Dopamine is a key regulator in the signalling pathway underlying predator-induced defences in Daphnia

The waterflea Daphnia is a model to investigate the genetic basis of phenotypic plasticity resulting from one differentially expressed genome. Daphnia develops adaptive phenotypes (e.g. morphological defences) thwarting predators, based on chemical predator cue perception. To understand the genomic basis of phenotypic plasticity, the description of the precedent cellular and neuronal mechanisms is fundamental. However, key regulators remain unknown. All neuronal and endocrine stimulants were able to modulate but not induce defences, indicating a pathway of interlinked steps. A candidate able to link neuronal with endocrine responses is the multi-functional amine dopamine. We here tested its involvement in trait formation in Daphnia pulex and Daphnia longicephala using an induction assay composed of predator cues combined with dopaminergic and cholinergic stimulants. The mere application of both stimulants was sufficient to induce morphological defences. We determined dopamine localization in cells found in close association with the defensive trait. These cells serve as centres controlling divergent morphologies. As a mitogen and sclerotization agent, we anticipate that dopamine is involved in proliferation and structural formation of morphological defences. Furthermore, dopamine pathways appear to be interconnected with endocrine pathways, and control juvenile hormone and ecdysone levels. In conclusion, dopamine is suggested as a key regulator of phenotypic plasticity.

Push or Pull? The light-weight architecture of the Daphnia pulex carapace is adapted to withstand tension, not compression.

Daphnia (Crustacea, Cladocera) are well known for their ability to form morphological adaptations to defend against predators. In addition to spines and helmets, the carapace itself is a protective structure encapsulating the main body, but not the head. It is formed by a double layer of the integument interconnected by small pillars and hemolymphatic space in between. A second function of the carapace is respiration, which is performed through its proximal integument. The interconnecting pillars were previously described as providing higher mechanical stability against compressive forces. Following this hypothesis, we analyzed the carapace structure of D. pulex using histochemistry in combination with light and electron microscopy. We found the distal integument of the carapace to be significantly thicker than the proximal. The pillars appear fibrous with slim waists and broad, sometimes branched bases where they meet the integument layers. The fibrous structure and the slim‐waisted shape of the pillars indicate a high capacity for withstanding tensile rather than compressive forces. In conclusion they are more ligaments than pillars. Therefore, we measured the hemolymphatic gauge pressure in D. longicephala and indeed found the hemocoel to have a pressure above ambient. Our results offer a new mechanistic explanation of the high rigidity of the daphniid carapace, which is probably the result of a light‐weight construction consisting of two integuments bound together by ligaments and inflated by a hydrostatic hyper‐pressure in the hemocoel. J. Morphol. 277:1320–1328, 2016.

Daphnia longicephala neuropeptides: Morphological description of crustacean cardioactive peptide (CCAP) and periviscerokinins in the Ctenodaphnia central nervous system

The publication of the Daphnia genome has driven research in this ecologically relevant model organism in many directions. However, information on this organisms physiology and the relevant controlling factors is limited. In this regard, especially neuropeptides are important biochemical regulators that control a variety of cellular processes, which in combination influence physiological conditions and allow the adaptation of the internal physiological state to external conditions. Thus, neuropeptides are prime in understanding an organisms physiology. We here aimed to detect and describe the distribution of evolutionary conserved neuropeptides including the crustacean cardioactive peptide (CCAP) and peptides of the family periviscerokinins (PVKs) in the central nervous system and the periphery of the Daphnia longicephala head region. We were able to identify a large pair of CCAP immunoreactive cells within central nervous system. In addition, in the periphery we found CCAP immunoreactive cells in the epidermis of the head with processes indicating cuticular secretion. Furthermore, we were able to identify and describe a complex neuronal circuit of PVK neuropeptides in the central nervous system. The data obtained in this study will provide important background information for future investigations aiming to unravel the cellular, neuronal and physiological pathways in a highly adaptive organism such as Daphnia.

Biomechanical properties of predator-induced body armour in the freshwater crustacean Daphnia

The freshwater crustacean Daphnia is known for its ability to develop inducible morphological defences that thwart predators. These defences are developed only in the presence of predators and are realized as morphological shape alterations e.g. ‘neckteeth’ in D. pulex and ‘crests’ in D. longicephala. Both are discussed to hamper capture, handling or consumption by interfering with the predator’s prey capture devices. Additionally, D. pulex and some other daphniids were found to armour-up and develop structural alterations resulting in increased carapace stiffness. We used scanning transmission electron microscopy (STEM) and confocal laser scanning microscopy (CLSM) to identify predator-induced structural and shape alterations. We found species specific structural changes accompanying the known shape alterations. The cuticle becomes highly laminated (i.e. an increased number of layers) in both species during predator exposure. Using nano- and micro-indentation as well as finite element analysis (FEA) we determined both: the structure’s and shape’s contribution to the carapace’s mechanical resistance. From our results we conclude that only structural alterations are responsible for increased carapace stiffness, whereas shape alterations appear to pose handling difficulties during prey capture. Therefore, these defences act independently at different stages during predation.