(Epi)genomic adaptation driven by fine geographical scale environmental heterogeneity after recent biological invasions.

Elucidating processes and mechanisms involved in rapid local adaptation to varied environments is a poorly understood but crucial component in management of invasive species. Recent studies have proposed that genetic and epigenetic variation could both contribute to ecological adaptation, yet it remains unclear on the interplay between these two components underpinning rapid adaptation in wild animal populations. To assess their respective contributions to local adaptation, we explored epigenomic and genomic responses to environmental heterogeneity in eight recently colonized ascidian (Ciona intestinalis) populations at a relatively fine geographical scale. Based on MethylRADseq data, we detected strong patterns of local environment-driven DNA methylation divergence among populations, significant epigenetic isolation by environment (IBE), and a large number of local environment-associated epigenetic loci. Meanwhile, multiple genetic analyses based on single nucleotide polymorphisms (SNPs) showed genomic footprints of divergent selection. In addition, for five genetically similar populations, we detected significant methylation divergence and local environment-driven methylation patterns, indicating the strong effects of local environments on epigenetic variation. From a functional perspective, a majority of functional genes, Gene Ontology (GO) terms, and biological pathways were largely specific to one of these two types of variation, suggesting partial independence between epigenetic and genetic adaptation. The methylation quantitative trait loci (mQTL) analysis showed that the genetic variation explained only 18.67% of methylation variation, further confirming the autonomous relationship between these two types of variation. Altogether, we highlight the complementary interplay of genetic and epigenetic variation involved in local adaptation, which may jointly promote populations' rapid adaptive capacity and successful invasions in different environments. The findings here provide valuable insights into interactions between invaders and local environments to allow invasive species to rapidly spread, thus contributing to better prediction of invasion success and development of management strategies.

Single Ultraviolet-C light treatment of early stage marine biofouling delays subsequent community development.

Past studies of Ultraviolet-C (UV-C) radiation as a marine antifoulant have focused on repeated doses. However, single or very low frequency exposures of UV-C could create more plausible applications for certain marine structures. The objective of the study reported here was to apply a single treatment of UV-C radiation to an early stage marine biofouling community to observe subsequent effects on biofouling development. Biofouling formed over a 2-week field immersion received UV-C treatments of 0 (control), 4, 20, or 120 min, and subsequent progression was then monitored weekly for 16 weeks. Analysis of acute effects and later macrofouling development suggested direct toxicity of UV-C illumination to invertebrate recruits caused reduction of subsequent biofouling (compared to controls) that persisted for up to 16 weeks following the longest UV-C treatment. Thus, UV-C treatments spaced by days or even weeks could be an option for some applications of UV-C radiation as an antifoulant.

Olfactory navigation in aquatic gastropods.

Gastropod diversity is substantial in marine and freshwater habitats, and many aquatic slugs and snails use olfactory cues to guide their navigation behaviour. Examples include finding prey or avoiding predators based on kairomones, or finding potential mates using pheromones. Here, I review the diversity of navigational behaviours studied across the major aquatic taxa of gastropods. I then synthesize evidence for the different theoretical navigation strategies the animals may use. It is likely that gastropods regularly use either chemotaxis or odour-gated rheotaxis (or both) during olfactory-based navigation. Finally, I collate the patchwork of research conducted on relevant proximate mechanisms that could produce navigation behaviours. Although the tractability of several gastropod species for neurophysiological experimentation has generated some valuable insight into how turning behaviour is triggered by contact chemoreception, there remain many substantial gaps in our understanding for how navigation relative to more distant odour sources is controlled in gastropods. These gaps include little information on the chemoreceptors and mechanoreceptors (for detecting flow) found in the peripheral nervous system and the central (or peripheral) processing circuits that integrate that sensory input. In contrast, past studies do provide information on motor neurons that control the effectors that produce crawling (both forward locomotion and turning). Thus, there is plenty of scope for further research on olfactory-based navigation, exploiting the tractability of gastropods for neuroethology to better understand how the nervous system processes chemosensory input to generate movement towards or away from distant odour sources.

Periodic ultraviolet-C illumination for marine sensor antifouling.

Ultraviolet light has intriguing potential as a marine antifoulant, targeting almost any species and applicable to almost any surface, while not accumulating in the environment. This study field-tested the effects of periodic ultraviolet-C illumination on marine macrofouling. Across four experiments, several UV illumination duty cycles were tested against controls with no illumination. Duty cycles between 1:2 (time with UV:total time per cycle) and 1:20 were all similarly effective, inhibiting almost all macrofouling at three different temperate Northeast Pacific and Northwest Atlantic sites. Susceptible taxa included barnacles, bryozoans, tunicates (colonial and solitary), and, to a slightly lesser extent, mussels. Duty cycles of 1:30 and 1:60 reduced but did not eliminate biofouling. Measurements of ultraviolet illumination on oceanographic sensors showed similar results. The results suggest further investigation of ultraviolet light as an antifoulant for marine sensors, including susceptibility of other taxa, optimizing illumination patterns, and exploring the potential for evolved resistance.

One rhinophore probably provides sufficient sensory input for odour-based navigation by the nudibranch mollusc Tritonia diomedea.

Tritonia diomedea (synonymous with Tritonia tetraquetra) navigates in turbulent odour plumes, crawling upstream towards prey and downstream to avoid predators. This is probably accomplished by odour-gated rheotaxis, but other possibilities have not been excluded. Our goal was to test whether T. diomedea uses odour-gated rheotaxis and to simultaneously determine which of the cephalic sensory organs (rhinophores and oral veil) are required for navigation. In a first experiment, slugs showed no coherent responses to streams of odour directed at single rhinophores. In a second experiment, navigation in prey and predator odour plumes was compared between animals with unilateral rhinophore lesions, denervated oral veils, or combined unilateral rhinophore lesions and denervated oral veils. In all treatments, animals navigated in a similar manner to that of control and sham-operated animals, indicating that a single rhinophore provides sufficient sensory input for navigation (assuming that a distributed flow measurement system would also be affected by the denervations). Amongst various potential navigational strategies, only odour-gated positive rheotaxis can produce the navigation tracks we observed in prey plumes while receiving input from a single sensor. Thus, we provide strong evidence that T. diomedea uses odour-gated rheotaxis in attractive odour plumes, with odours and flow detected by the rhinophores. In predator plumes, slugs turned downstream to varying degrees rather than orienting directly downstream for crawling, resulting in greater dispersion for negative rheotaxis in aversive plumes. These conclusions are the first explicit confirmation of odour-gated rheotaxis as a navigational strategy in gastropods and are also a foundation for exploring the neural circuits that mediate odour-gated rheotaxis.

Baseline monitoring of contaminant concentrations in American lobster (Homarus americanus) tissues from coastal Northumberland Strait, Nova Scotia, Canada.

A baseline survey was conducted in 2018 to characterize contaminants in American lobsters, Homarus americanus in the Northumberland Strait, Canada. Sampling included three age classes of lobsters at sites 4, 20, and 70 km from the Boat Harbour estuary, a historically contaminated site set to undergo remediation. Lobster tissues were measured for metal(loids), methylmercury, polycyclic aromatic hydrocarbons, and polychlorinated dibenzo-p-dioxins and polychlorinated dibenzo-p-furans. Contaminant concentrations were generally below the guidelines set by the Canadian Council of Ministers of the Environment and the Canadian Food Inspection Agency, except for arsenic which was elevated in all age classes from all sites (4.8-12.68 mg kg-1). Mercury and methylmercury (both ~0.04 mg kg-1) minimally exceeded one guideline in some age-classes and sites. There was also no consistent pattern of contaminant accumulation across either age classes or at particular sites. This study serves as a baseline for future monitoring following remediation of Boat Harbour.

Ecological Review of the Ciona Species Complex.

AbstractThe set of four closely related solitary ascidians Ciona spp. were once considered a single cosmopolitan species, Ciona intestinalis, but are now recognized as genetically and morphologically distinct species. The possibility of ecological differences between the species was not widely considered in studies preceding the schism of Ciona spp. Consequently, there may be an over-generalization of the ecology of Ciona spp., with potential implications for the broad range of studies targeting these species, encompassing the evolution, development, genomics, and invasion biology of Ciona spp. We completed a comprehensive review of the ecology of Ciona spp. to establish the similarities and differences between the widely distributed Ciona robusta and C. intestinalis (and what little is known of the two other species, Ciona sp. C and Ciona sp. D). When necessary, we used study locations and the species' geographic ranges to infer the species in each study in the review. As expected, ecological similarities are the norm between the two species, spanning both abiotic and biotic interactions. However, there are also important differences that have potential implications for other aspects of the biology of Ciona spp. For example, differences in temperature and salinity tolerances likely correspond with the disparities in the geographic distribution of the species. Asymmetries in topics studied in each species diminish our ability to fully compare several aspects of the ecology of Ciona spp. and are priority areas for future research. We anticipate that our clarification of common and unique aspects of each species' ecology will help to provide context for future research in many aspects of the biology of Ciona spp.

Effect of Animal Stocking Density and Habitat Enrichment on Survival and Vitality of Wild Green Shore Crabs, Carcinus maenas, Maintained in the Laboratory.

The wide geographic distribution, large size and ease of capture has led to decapod crustaceans being used extensively in laboratory experiments. Recently in the United Kingdom decapod crustaceans were listed as sentient beings, resulting in their inclusion in animal care protocols. Ironically, little is known about how captive conditions affect the survival and general condition of wild decapod crustaceans. We used the green shore crab, Carcinus maenas, to investigate the effects of stocking density and shelter on survival and vitality indices during a 6 month period in the laboratory. Neither stocking density nor the presence of shelter affected survival. Stocking density also had no effect on the vitality indices (limb loss, claw strength, BRIX, righting time, leg flare and retraction). The presence of shelter did affect the number of limbs lost and the leg retraction response, but had no effect on the other vitality indices. All vitality indices changed, and mortality increased over time, independent of treatment: this became most apparent after 8 to 11 weeks storage in the laboratory. This decline in condition may have been due to repeated handling of the crabs, rather than the stocking conditions. In support of this, untracked, non-handled (control) individuals sustained a 4% mortality rate compared with 67% mortality in experimental crabs during the 6 month period. Although simple experimental monitoring of crabs with biweekly vitality tests only produced transient short-term stress events, the repeated handling over time apparently led to a cumulative stress and a deterioration in animal health. Bringing wild crustaceans into the laboratory and holding them, even with modest experimental manipulation, may result in high mortality rates. Researchers and animal care committees need to be aware that wild captive invertebrates will respond very differently to laboratory-bred vertebrates, and plan experiments accordingly.

Analysis of rhodopsin G protein-coupled receptor orthologs reveals semiochemical peptides for parasite (Schistosoma mansoni) and host (Biomphalaria glabrata) interplay.

Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Identification of Schistosoma mansoni miracidia attractant candidates in infected Biomphalaria glabrata using behaviour-guided comparative proteomics.

Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.

Identification of Putative Neuropeptides That Alter the Behaviour of Schistosoma mansoni Cercariae.

Elucidating the infectivity of Schistosoma mansoni, one of the main etiological agents of human schistosomiasis, requires an improved understanding of the behavioural mechanisms of cercariae, the non-feeding mammalian infective stage. This study investigated the presence and effect of cercariae-derived putative neuropeptides on cercarial behaviour when applied externally. Cercariae were peptidomically analysed and 11 neuropeptide precursor proteins, all of which were specific to the Schistosoma genus and most of which highly expressed in the cercarial stage, were identified in cercariae for the first time. Protein-protein interaction analysis predicted the interaction of various neuropeptide precursors (e.g., Sm-npp-30, Sm-npp-33, Sm-npp-35) with cercarial structural proteins (e.g., myosin heavy chain and titin). In total, nine putative neuropeptides, selected based on their high hydrophobicity and small size (~1 kilodalton), were tested on cercariae (3 mg/mL) in acute exposure (1 min) and prolonged exposure (360 min) behavioural bioassays. The peptides AAYMDLPW-NH2, NRKIDQSFYSYY-NH2, FLLALPSP-OH, and NYLWDTRL-NH2 stimulated acute increases in cercarial spinning, stopping, and directional change during active states. However, only NRKIDQSFYSYY-NH2 caused the same behavioural changes at a lower concentration (0.1 mg/mL). After prolonged exposure, AAYMDLPW-NH2 and NYLWDTRL-NH2 caused increasing passive behaviour and NRKIDQSFYSYY-NH2 caused increasing body-first and head-pulling movements. These findings characterise behaviour-altering novel putative neuropeptides, which may inform future biocontrol innovations to prevent human schistosomiasis.

Rare-Earth Magnets Influence Movement Patterns of the Magnetically Sensitive Nudibranch Tritonia exsulans in Its Natural Habitat.

AbstractThe nudibranch Tritonia exsulans (previously Tritonia diomedea) is known to have behaviors and neurons that can be modified by perturbations of the Earth's magnetic field. There is no definitive evidence for how this magnetic sense is used in nature. Using an exploratory approach, we tested for possible effects of magnetic perturbations based on underwater video of crawling patterns in the slugs' natural habitat, with magnets of varying strength deployed on the substrate. For analysis, we used a paired comparison of tracks of animals between segments 25-50 cm distant from the magnets and segments of the same tracks 0-25 cm from the magnets, to determine whether any differences depended on the strength of the magnet. Most track measurements (length, displacement, velocity, and tortuosity) showed no such differences. However, effects were observed for the changes in track headings between successive points. These results showed that tracks had relatively higher heading variability when they moved closer to stronger magnets. We suggest that this supports a hypothesis that T. exsulans continuously uses a magnetic sense to help maintain straight-line navigation. Further specific testing of the hypothesis is now needed to verify this new possibility for how animals can benefit from a compass sense.

A technical review and guide to RNA fluorescence in situ hybridization.

RNA-fluorescence in situ hybridization (FISH) is a powerful tool to visualize target messenger RNA transcripts in cultured cells, tissue sections or whole-mount preparations. As the technique has been developed over time, an ever-increasing number of divergent protocols have been published. There is now a broad selection of options available to facilitate proper tissue preparation, hybridization, and post-hybridization background removal to achieve optimal results. Here we review the technical aspects of RNA-FISH, examining the most common methods associated with different sample types including cytological preparations and whole-mounts. We discuss the application of commonly used reagents for tissue preparation, hybridization, and post-hybridization washing and provide explanations of the functional roles for each reagent. We also discuss the available probe types and necessary controls to accurately visualize gene expression. Finally, we review the most recent advances in FISH technology that facilitate both highly multiplexed experiments and signal amplification for individual targets. Taken together, this information will guide the methods development process for investigators that seek to perform FISH in organisms that lack documented or optimized protocols.

1-Phenoxy-2-propanol is a useful anaesthetic for gastropods used in neurophysiology.

Anaesthesia is often used in neurophysiological, surgical, and neuroanatomical protocols. Several anaesthetics, including magnesium chloride, volatiles (halothane, etc.), and barbiturates, have been used in gastropod neurobiology. 1-Phenoxy-2-propanol (PP) is another anaesthetic option that has not yet been used extensively. We provide an analysis of the neural, muscular and behavioural effects of PP in gastropods. PP eliminates action potentials and reduces muscular contraction force in Hermissenda crassicornis, and eliminates behavioural activity in Tritonia diomedea. Our results show these effects are reversible, with complete action potential recovery, at least partial muscular recovery, and full behavioural recovery. Survival after surgery in T. diomedea was longer with PP than without anaesthetic, and PP also reduced contraction during tissue fixation in Lymnaea stagnalis. Moreover, PP can be bath applied, has low toxicity, and is biodegradable. Thus, PP is an effective anaesthetic in three species of gastropods, and useful in neurophysiological dissection, surgical, and fixation protocols.

Tissue-specific evaluation of suitable reference genes for RT-qPCR in the pond snail, Lymnaea stagnalis.

Reverse transcription quantitative PCR (RT-qPCR) is a robust technique for the quantification and comparison of gene expression. To obtain reliable results with this method, one or more reference genes must be employed to normalize expression measurements among treatments or tissue samples. Candidate reference genes must be validated to ensure that they are stable prior to use in qPCR experiments. The pond snail (Lymnaea stagnalis) is a common research organism, particularly in the areas of learning and memory, and is an emerging model for the study of biological asymmetry, biomineralization, and evolution and development. However, no systematic assessment of qPCR reference genes has been performed in this animal. Therefore, the aim of our research was to identify stable reference genes to normalize gene expression data from several commonly studied tissues in L. stagnalis as well as across the entire body. We evaluated a panel of seven reference genes across six different tissues in L. stagnalis with RT-qPCR. The genes included: elongation factor 1-alpha, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, beta-tubulin, ubiquitin, prenylated rab acceptor protein 1, and a voltage gated potassium channel. These genes exhibited a wide range of expression levels among tissues. The tissue-specific stability of each of the genes was consistent when measured by the standard stability assessment algorithms: geNorm, NormFinder, BestKeeper, and RefFinder. Our data indicate that the most stable reference genes vary among the tissues that we examined (central nervous system, tentacles, lips, penis, foot, mantle). Our results were generally congruent with those obtained from similar studies in other molluscs. Given that a minimum of two reference genes are recommended for data normalization, we provide suggestions for strong pairs of reference genes for single- and multi-tissue analyses of RT-qPCR data in L. stagnalis.

A Biomphalaria glabrata peptide that stimulates significant behaviour modifications in aquatic free-living Schistosoma mansoni miracidia.

The human disease schistosomiasis (or bilharzia) is caused by the helminth blood fluke parasite Schistosoma mansoni, which requires an intermediate host, the freshwater gastropod snail Biomphalaria glabrata (the most common intermediate host). The free-swimming parasite miracidia utilise an excellent chemosensory sense to detect and locate an appropriate host. This study investigated the biomolecules released by the snail that stimulate changes in the behaviour of the aquatic S. mansoni miracidia. To achieve this, we have performed an integrated analysis of the snail-conditioned water, through chromatography and bioassay-guided behaviour observations, followed by mass spectrometry. A single fraction containing multiple putative peptides could stimulate extreme swimming behaviour modifications (e.g. velocity, angular variation) similar to those observed in response to crude snail mucus. One peptide (P12;-R-DITSGLDPEVADD-KR-) could replicate the stimulation of miracidia behaviour changes. P12 is derived from a larger precursor protein with a signal peptide and multiple dibasic cleavage sites, which is synthesised in various tissues of the snail, including the central nervous system and foot. P12 consists of an alpha helix secondary structure as indicated by circular dichroism spectroscopy. This information will be helpful for the development of approaches to manipulate this parasites life cycle, and opens up new avenues for exploring other parasitic diseases which have an aquatic phase using methods detailed in this investigation.

GABA-, histamine-, and FMRFamide-immunoreactivity in the visual, vestibular and central nervous systems of Hermissenda crassicornis.

Hermissenda crassicornis is a model for studying the molecular and cellular basis for classical conditioning, based on its ability to associate light with vestibular stimulation. We used confocal microscopy to map histamine (HA), FMRF-amide, and γ-aminobutyric acid (GABA) immunoreactivity in the central nervous system (CNS), eyes, optic ganglia and statocysts of the nudibranchs. For HA immunoreactivity, we documented both consistently and variably labeled CNS structures across individuals. We also noted minor differences in GABA immunoreactivity in the CNS compared to previous work on Hermissenda. Contrary to expectations, we found no evidence for GABA inside the visual or vestibular systems. Instead, we found only FMRFamide- and HA immunoreactivity (FMRFamide: 4 optic ganglion cells, 4-5 hair cells; HA: 3 optic ganglion cells, 8 hair cells). Overall, our results can act as basis for comparisons of nervous systems across nudibranchs, and suggest further exploration of intraspecific plasticity versus evolutionary changes in gastropod nervous systems.

A simple automated system for appetitive conditioning of zebrafish in their home tanks.

We describe here an automated apparatus that permits rapid conditioning paradigms for zebrafish. Arduino microprocessors were used to control the delivery of auditory or visual stimuli to groups of adult or juvenile zebrafish in their home tanks in a conventional zebrafish facility. An automatic feeder dispensed precise amounts of food immediately after the conditioned stimuli, or at variable delays for controls. Responses were recorded using inexpensive cameras, with the video sequences analysed with ImageJ or Matlab. Fish showed significant conditioned responses in as few as 5 trials, learning that the conditioned stimulus was a predictor of food presentation at the water surface and at the end of the tank where the food was dispensed. Memories of these conditioned associations persisted for at least 2days after training when fish were tested either as groups or as individuals. Control fish, for which the auditory or visual stimuli were specifically unpaired with food, showed no comparable responses. This simple, low-cost, automated system permits scalable conditioning of zebrafish with minimal human intervention, greatly reducing both variability and labour-intensiveness. It will be useful for studies of the neural basis of learning and memory, and for high-throughput screening of compounds modifying those processes.

Field behavior of the nudibranch mollusc Tritonia diomedea.

The nudibranch mollusc Tritonia diomedea has been a useful model system for studies of how the brain controls behavior. However, no broad study of T. diomedea field behavior exists--an important deficit since laboratory behaviors may differ from what occurs in nature. Here we report analysis of time-lapse video of the slugs in their natural habitat to describe behaviors and their relationships to sensory cues. We found that movements relative to conspecifics, prey, and predators correlated with direction of water flow. These observations lead to three new navigational hypotheses: regardless of the actual heading to the target, T. diomedea crawls (1) upstream toward potential mates, (2) upstream toward food, and (3) downstream away from predators. We also describe both the behavior and its sensory context for feeding, escape swims, mating, and egg-laying, among other behaviors. Field behaviors were similar to published descriptions of laboratory behavior. However, the field observations add contextual detail, including preceding and subsequent behaviors and interactions with suites of habitat features not present in the laboratory. For example, the escape swim, previously studied as an isolated behavior in response to a single stimulus, appears to be affected by multiple sensory modalities and coordinated with several other behaviors. Our work will provide a basis for future neuroethological experimentation and also is the first step in the study of navigation in T. diomedea.

Orientation and navigation relative to water flow, prey, conspecifics, and predators by the nudibranch mollusc Tritonia diomedea.

Progress in understanding sensory and locomotory systems in Tritonia diomedea has created the potential for the neuroethological study of animal navigation in this species. Our goal is to describe the navigational behaviors to guide further work on how the nervous system integrates information from multiple senses to produce oriented locomotion. Observation of T. diomedea in its habitat has suggested that it uses water flow to navigate relative to prey, predators, and conspecifics. We test these hypotheses in the field by comparing slug orientation in time-lapse videos to flow direction in circumstances with and without prey, predators, or conspecifics upstream. T. diomedea oriented upstream both while crawling and after turning. This trend was strongest before feeding or mating; after feeding or mating, the slugs did not orient significantly to flow. Slugs turned downstream away from an upstream predator but did not react in control situations without an upstream predator. These data support the hypothesis that T. diomedea uses a combination of odors (or some other cue transported downstream) and water flow to navigate relative to prey, predators, and conspecifics. Understanding the context-dependent choice between upstream and downstream crawling in T. diomedea provides an opportunity for further work on the sensory integration underlying navigation behavior.