A method for selective stimulation of leg chemoreceptors in whole crustaceans.

The integration of sensory information with adequate motor outputs is critical for animal survival. Here, we present an innovative technique based on a non-invasive closed-circuit device consisting of a perfusion/stimulation chamber chronically applied on a single leg of the crayfish Procambarus clarkii. Using this technique, we focally stimulated the leg inside the chamber and studied the leg-dependent sensory-motor integration involving other sensory appendages, such as antennules and maxillipeds, which remain unstimulated outside the chamber. Results show that the stimulation of a single leg with chemicals, such as disaccharides, is sufficient to trigger a complex search behaviour involving locomotion coupled with the reflex activation of antennules and maxillipeds. This technique can be easily adapted to other decapods and/or other sensory appendages. Thus, it has opened possibilities for studying sensory-motor integration evoked by leg stimulation in whole aquatic animals under natural conditions to complement, with a direct approach, current ablation or silencing techniques.

A Life Outside.

How do the morphologies of organisms affect their physical interactions with the environment and other organisms? My research in marine systems couples field studies of the physical habitats, life history strategies, and ecological interactions of organisms with laboratory analyses of their biomechanics. Here, I review how we pursued answers to three questions about marine organisms: (a) how benthic organisms withstand and utilize the water moving around them, (b) how the interaction between swimming and turbulent ambient water flow affects where small organisms go, and (c) how hairy appendages catch food and odors. I also discuss the importance of different types of mentors, the roadblocks for women in science when I started my career, the challenges and delights of interdisciplinary research, and my quest to understand how I see the world as a dyslexic.

Do Hydrothermal Shrimp Smell Vents?

Deep-sea species endemic to hydrothermal vents face the critical challenge of detecting active sites in a vast environment devoid of sunlight. This certainly requires specific sensory abilities, among which olfaction could be a relevant sensory modality, since chemical compounds in hydrothermal fluids or food odors could potentially serve as orientation cues. The temperature of the vent fluid might also be used for locating vent sites. The objective of this study is to observe the following key behaviors of olfaction in hydrothermal shrimp, which could provide an insight into their olfactory capacities: (1) grooming behavior; (2) attraction to environmental cues (food odors and fluid markers). We designed experiments at both deep-sea and atmospheric pressure to assess the behavior of the vent shrimp Rimicaris exoculata and Mirocaris fortunata, as well as of the coastal species Palaemon elegans and Palaemon serratus for comparison. Here, we show that hydrothermal shrimp groom their sensory appendages similarly to other crustaceans, but this does not clean the dense bacterial biofilm that covers the olfactory structures. These shrimp have previously been shown to possess functional sensory structures, and to detect the environmental olfactory signals tested, but we do not observe significant attraction behavior here. Only temperature, as a signature of vent fluids, clearly attracts vent shrimp and thus is confirmed to be a relevant signal for orientation in their environment.

Identification of putative ingestion-related olfactory receptor genes in the Chinese mitten crab (Eriocheir japonica sinensis).

BACKGROUND: Olfaction plays a central role in mating, spawning, obtaining food and escaping predators, which is essential for survival and reproduction of animals. The nature of the olfactory perception in crabs, which is a major group of crustaceans, has remained elusive. OBJECTIVE: This project aims to explore the molecular mechanism of olfaction in crabs and further improve our understanding of olfactory perception in crustaceans. METHODS: The olfactory receptors and ingestion-related gene expression in Eriocheir japonica sinensis were studied by transcriptomic techniques. The de novo assembly, annotation and functional evaluation were performed with bioinformatics tools. RESULTS: A series of chemosensory receptors associated with olfaction were identified including 33 EsIRs, 24 EsIGluRs, 58 EsVIGluRs, 1 EsOR and 1 EsGC-D. We found IRs were key odorant receptors demonstrating a specific species evolutionary trend in crustaceans. Furthermore, we identified ORs in E. j. sinensis and Litopenaeus vannamei. The incomplete EsOR and LvOR1 structures implied that ORs exist in crustaceans, and may have been degenerated or even lost in the olfactory evolutionary process. In addition, comparative transcriptome analysises demonstrated two possible olfactory transduction pathways of E. j. sinensis: the cGMP-mediated olfactory pathway related to vegetable odor molecules and the cAMP-mediated olfactory pathway related to meat odor molecules. The above results were consistent with its omnivorous ingestion of E. j. sinensis. CONCLUSIONS: Our study revealed the unique olfactory molecular mechanism of omnivorous crabs and provided valuable information for further functional research on the chemoreception mechanisms in crustaceans.

Environmentally relevant atrazine exposures cause DNA damage in cells of the lateral antennules of crayfish (Faxonius virilis).

The herbicide atrazine is heavily applied in agricultural areas in the Midwestern United States and can run-off and seep into surrounding aquatic habitats where concentrations can reach over 300 ppb. It is known that acute exposures to 80 ppb atrazine cause lasting deficiencies in the chemoreception of food and mate odors. Since atrazine impairs chemosensory responses, the goal of this study was to determine the effect of atrazine on cells, including olfactory sensory neurons, located in the lateral antennules of crayfish. In this experiment, we treated crayfish for 10 days with ecologically relevant concentrations of 0, 10, 40, 80, 100 and 300 ppb (µg L-1) of atrazine. Following treatments, the distal portion of the lateral antennules was cryosectioned. We used a TdT mediated dUTP nick-end labeling (TUNEL) assay to determine if any cells had DNA damage and may be thus undergoing apoptosis. We found that as atrazine concentrations increase above 10 ppb, the number of TUNEL-positive cells, visualized in the lateral antennules, significantly increases. Our data show that atrazine exposure causes DNA damage in cells of the lateral antennules, including olfactory sensory neurons, thus leading to impairments in chemosensory abilities. Because crayfish rely heavily on chemoreception for survival, changes in their ability to perceive odors following atrazine exposure may have detrimental effects on population size.

Expression of ionotropic receptors in terrestrial hermit crab's olfactory sensory neurons.

Coenobitidae are one out of at least five crustacean lineages which independently succeeded in the transition from water to land. This change in lifestyle required adaptation of the peripheral olfactory organs, the antennules, in order to sense chemical cues in the new terrestrial habitat. Hermit crab olfactory aesthetascs are arranged in a field on the distal segment of the antennular flagellum. Aesthetascs house approximately 300 dendrites with their cell bodies arranged in spindle-like complexes of ca. 150 cell bodies each. While the aesthetascs of aquatic crustaceans have been shown to be the place of odor uptake and previous studies identified ionotropic receptors (IRs) as the putative chemosensory receptors expressed in decapod antennules, the expression of IRs besides the IR co-receptors IR25a and IR93a in olfactory sensory neurons (OSNs) has not been documented yet. Our goal was to reveal the expression and distribution pattern of non-co-receptor IRs in OSNs of Coenobita clypeatus, a terrestrial hermit crab, with RNA in situ hybridization. We expanded our previously published RNAseq dataset, and revealed 22 novel IR candidates in the Coenobita antennules. We then used RNA probes directed against three different IRs to visualize their expression within the OSN cell body complexes. Furthermore we aimed to characterize ligand spectra of single aesthetascs by recording local field potentials and responses from individual dendrites. This also allowed comparison to functional data from insect OSNs expressing antennal IRs. We show that this orphan receptor subgroup with presumably non-olfactory function in insects is likely the basis of olfaction in terrestrial hermit crabs.

The hermit crab's nose-antennal transcriptomics.

In the course of evolution, crustaceans adapted to a large variety of habitats. Probably the most extreme habitat shift was the transition from water to land, which occurred independently in at least five crustacean lineages. This substantial change in life style required adaptations in sensory organs, as the medium conveying stimuli changed in both chemical and physical properties. One important sensory organ in crustaceans is the first pair of antennae, housing their sense of smell. Previous studies on the crustacean transition from water to land focused on morphological, behavioral, and physiological aspects but did not analyze gene expression. Our goal was to scrutinize the molecular makeup of the crustacean antennulae, comparing the terrestrial Coenobita clypeatus and the marine Pagurus bernhardus. We sequenced and analyzed the antennal transcriptomes of two hermit crab species. Comparison to previously published datasets of similar tissues revealed a comparable quality and GO annotation confirmed a highly similar set of expressed genes in both datasets. The chemosensory gene repertoire of both species displayed a similar set of ionotropic receptors (IRs), most of them belonging to the divergent IR subtype. No binding proteins, gustatory receptors (GRs) or insect-like olfactory receptors (ORs) were present. Additionally to their olfactory function, the antennules were equipped with a variety of pathogen defense mechanisms, producing relevant substances on site. The overall similarity of both transcriptomes is high and does not indicate a general shift in genetic makeup connected to the change in habitat. IRs seem to perform the task of olfactory detection in both hermit crab species studied.

Evolution of antennules of cytheroidean ostracods (Crustacea).

Phylogenetic analysis and fossil records indicate that antennules with whip-like setae are the most plesiomorphic state in cytheroidean ostracods and that antennulae with claw-like setae are derived from antennulae with whip-like setae. Character distributions on the 18S rDNA molecular phylogenetic tree suggest that two phenotypic features of cytheroidean antennules (W/L ratio and claw-like/whip-like setae) have morphological plasticity. These features have evolved as an ethological adaptation rather than due to phylogenetic constraints such as the evolution of shell outline in cytheroideans. However, the species of the Leptocytheridae-Trachyleberididae clade generally have stout, robust antennules, indicating a phylogenetic constraint. The character state of setae (claw/whip) in cytheroidean ostracods is reflected more through their ethology than the W/L ratio of the antennules. On the basis of the present analysis and the fossil record, diversification in the morphology of the antennules seems to have occurred during the early Mesozoic.