Individual female differences in chemoattractant production change the scale of sea urchin gamete interactions.

Sperm selection by females is an important process influencing fertilization and, particularly in broadcast-spawning organisms, often occurs before sperm reach the egg. Waterborne sperm chemoattractants are one mechanism by which eggs selectively influence conspecific sperm behavior, but it remains an open question whether the eggs from different females produce different amounts of sperm chemoattractant, and how that might influence sperm behavior. Here, we quantify the differences in attractant production between females of the sea urchin species Lytechinus pictus and use computational models and microfluidic sperm chemotaxis assays to determine how differences in chemoattractant production between females affects their ability to attract sperm. Our study demonstrates that there is significant individual female variation in egg chemoattractant production, and that this variation changes the scope and strength of sperm attraction. These results provide evidence for the importance of individual female variability in differential sperm attraction and fertilization success.

Keystone predation and molecules of keystone significance.

Keystone species structure ecological communities and are major determinants of biodiversity. A synthesis of research on keystone species is nonetheless missing a critical component - the sensory mechanisms for behavioral interactions that determine population- and community-wide attributes. Here, we establish the chemosensory basis for keystone predation by sea stars (Pisaster ochraceus) on mussels. This consumer-resource interaction is prototypic of top-down driven trophic cascades. Each mussel species (Mytilus californianus and M. galloprovincialis) secretes a glycoprotein orthologue (29.6 and 28.1 kDa, respectively) that acts, singularly, to evoke the sea star predatory response. The orthologues (named "KEYSTONEin") are localized in the epidermis, extrapallial fluid, and organic shell coating (periostracum) of live, intact mussels. Thus, KEYSTONEin contacts chemosensory receptors on tube feet as sea stars crawl over rocky surfaces in search of prey. The complete nucleotide sequences reveal that KEYSTONEin shares 87% (M. californianus) or 98% (M. galloprovincialis) homology with a calcium-binding protein in the shell matrix of a closely related congener, M. edulis. All three molecules cluster tightly within the Complement Component 1 Domain Containing (C1qDC) protein family; each exhibits a large globular domain, low complexity region(s), coiled coil, and at least four of five histidine-aspartic acid tandem motifs. Collective results support the hypothesis that KEYSTONEin evolved ancestrally in immunological, and later, in biomineralization roles. More recently, the substance has become exploited by sea stars as a contact cue for prey recognition. As the first identified compound to evoke keystone predation, KEYSTONEin provides valuable sensory information, promotes biodiversity, and shapes community structure and function. Without this molecule, there would be no predation by sea stars on mussels.

A multifunctional chemical cue drives opposing demographic processes and structures ecological communities.

Foundation species provide critical resources to ecological community members and are key determinants of biodiversity. The barnacle Balanus glandula is one such species and dominates space among the higher reaches of wave-swept shores (Northeastern Pacific Ocean). This animal produces a cuticular glycoprotein (named "MULTIFUNCin") of 199.6 kDa, and following secretion, a 390 kDa homodimer in native form. From field and lab experiments, we found that MULTIFUNCin significantly induces habitat selection by conspecific larvae, while simultaneously acting as a potent feeding stimulant to a major barnacle predator (whelk, Acanthinucella spirata). Promoting immigration via settlement on the one hand, and death via predation on the other, MULTIFUNCin drives opposing demographic processes toward structuring predator and prey populations. As shown here, a single compound is not restricted to a lone species interaction or sole ecological function. Complex biotic interactions therefore can be shaped by simple chemosensory systems and depend on the multifunctional properties of select bioactive proteins.

Sperm chemotaxis promotes individual fertilization success in sea urchins.

Reproductive success fundamentally shapes an organism's ecology and evolution, and gamete traits mediate fertilization, which is a critical juncture in reproduction. Individual male fertilization success is dependent on the ability of sperm from one male to outcompete the sperm of other males when searching for a conspecific egg. Sperm chemotaxis, the ability of sperm to navigate towards eggs using chemical signals, has been studied for over a century, but such studies have long assumed that this phenomenon improves individual male fitness without explicit evidence to support this claim. Here, we assessed fertilization changes in the presence of a chemoattractant-digesting peptidase and used a microfluidic device coupled with a fertilization assay to determine the effect of sperm chemotaxis on individual male fertilization success in the sea urchin Lytechinus pictus We show that removing chemoattractant from the gametic environment decreases fertilization success. We further found that individual male differences in chemotaxis to a well-defined gradient of attractant correlate with individual male differences in fertilization success. These results demonstrate that sperm chemotaxis is an important contributor to individual reproductive success.

Sperm chemotaxis, fluid shear, and the evolution of sexual reproduction.

Chemical communication is fundamental to sexual reproduction, but how sperm search for and find an egg remains enigmatic. For red abalone (Haliotis rufescens), a large marine snail, the relationship between chemical signaling and fluid motion largely determines fertilization success. Egg-derived attractant plumes are dynamic, changing their size and shape in response to unique combinations of physical and chemical environmental features. Attractant plumes that promote sexual reproduction, however, are limited to a precise set of hydrodynamic conditions. Performance-maximizing shears are those that most closely match flows in native spawning habitats. Under conditions in which reproductive success is chronically limited by sperm availability, gametes are under selection for mechanisms that increase sperm-egg encounter. Here, chemoattraction is found to provide a cheap evolutionary alternative for enhancing egg target size without enlarging cytoplasmic and/or cell volume. Because egg signaling and sperm response may be tuned to meet specific fluid-dynamic constraints, shear could act as a critical selective pressure that drives gamete evolution and determines fitness.

Chemosensory Ca2+ dynamics correlate with diverse behavioral phenotypes in human sperm.

In the female reproductive tract, mammalian sperm undergo a regulated sequence of prefusion changes that "prime" sperm for fertilization. Among the least understood of these complex processes are the molecular mechanisms that underlie sperm guidance by environmental chemical cues. A "hard-wired" Ca(2+) signaling strategy that orchestrates specific motility patterns according to given functional requirements is an emerging concept for regulation of sperm swimming behavior. The molecular players involved, the spatiotemporal characteristics of such motility-associated Ca(2+) dynamics, and the relation between a distinct Ca(2+) signaling pattern and a behavioral sperm phenotype, however, remain largely unclear. Here, we report the functional characterization of two human sperm chemoreceptors. Using complementary molecular, physiological, and behavioral approaches, we comparatively describe sperm Ca(2+) responses to specific agonists of these novel receptors and bourgeonal, a known sperm chemoattractant. We further show that individual receptor activation induces specific Ca(2+) signaling patterns with unique spatiotemporal dynamics. These distinct Ca(2+) dynamics are correlated to a set of stimulus-specific stereotyped behavioral responses that could play vital roles during various stages of prefusion sperm-egg chemical communication.

Flagellar kinematics reveals the role of environment in shaping sperm motility.

Swimming spermatozoa from diverse organisms often have very similar morphologies, yet different motilities as a result of differences in the flagellar waveforms used for propulsion. The origin of these differences has remained largely unknown. Using high-speed video microscopy and mathematical analysis of flagellar shape dynamics, we quantitatively compare sperm flagellar waveforms from marine invertebrates to humans by means of a novel phylokinematic tree. This new approach revealed that genetically dissimilar sperm can exhibit strikingly similar flagellar waveforms and identifies two dominant flagellar waveforms among the deuterostomes studied here, corresponding to internal and external fertilizers. The phylokinematic tree shows marked discordance from the phylogenetic tree, indicating that physical properties of the fluid environment, more than genetic relatedness, act as an important selective pressure in shaping the evolution of sperm motility. More broadly, this work provides a physical axis to complement morphological and genetic studies to understand evolutionary relationships.

Dispersal pathways, seed rains, and the dynamics of larval behavior.

Dispersing propagules (larvae, seeds, and spores) establish and maintain populations, which serve as templates for subsequent species interactions. Connectivity among demes derives, in large part, from connectivity between consecutive steps, release, transport, and settlement, in dispersal pathways. Concurrent measurements of individuals in each step are a necessary precursor to identifying governing mechanisms. Here we directly and definitively resolved the roles of physics and behavior in mediating dispersal pathways of an estuarine parasite between its intermediate hosts. Planktonic cercariae of Himasthla rhigedana, a parasitic flatworm, are functionally similar to lecithotrophic larvae of many free-living marine invertebrates. The combination of parasite life cycle characteristics and the relatively simple tidal flows in their habitat renders this system an effective model for dispersal studies. Simultaneous field measurements of larval release, transport, settlement, and the flow regime, together with mechanistic experiments, led to empirical understanding of host colonization. All dispersal steps were highly and significantly correlated over time and in space. This tight coupling resulted, unequivocally, from a suite of larval behaviors. Cercariae emerged from first intermediate host snails only during daytime flood tides, enhancing larval retention in the marsh. Daylight triggered downward swimming, and within seconds, cercariae overpowered turbulent mixing, landing in benthic habitat of second intermediate host snails and crabs. Larvae settled (encysted) on external regions of snails/crabs that, presumably, were most vulnerable to ingestion by definitive host shorebirds. In total, cercarial behaviors greatly foreshortened dispersal distances, magnified local parasite prevalence, and increased the likelihood of large-scale transmission by definitive hosts. Cracking open the black box of dispersal thus revealed mechanisms, connectivity, and ecological consequences of the larval stage.

Neuroecology, chemical defense, and the keystone species concept.

Neuroecology unifies principles from diverse disciplines, scaling from biophysical properties of nerve and muscle cells to community-wide impacts of trophic interactions. Here, these principles are used as a common fabric, woven from threads of chemosensory physiology, behavior, and population and community ecology. The "keystone species" concept, for example, is seminal in ecological theory. It defines a species whose impacts on communities are far greater than would be predicted from its relative abundance and biomass. Similarly, neurotoxins could function in keystone roles. They are rare within natural habitats but exert strong effects on species interactions at multiple trophic levels. Effects of two guanidine alkaloids, tetrodotoxin (TTX) and saxitoxin (STX), coalesce neurobiological and ecological perspectives. These molecules compose some of the most potent natural poisons ever described, and they are introduced into communities by one, or only a few, host species. Functioning as voltage-gated sodium channel blockers for nerve and muscle cells, TTX and STX serve in chemical defense. When borrowed by resistant consumer species, however, they are used either in chemical defense against higher order predators or for chemical communication as chemosensory excitants. Cascading effects of the compounds profoundly impact community-wide attributes, including species compositions and rates of material exchange. Thus, a diverse array of physiological traits, expressed differentially across many species, renders TTX and STX fully functional as keystone molecules, with vast ecological consequences at multiple trophic levels.

Noxious newts and their natural enemies: Experimental effects of tetrodotoxin exposure on trematode parasites and aquatic macroinvertebrates.

The dermal glands of many amphibian species secrete toxins or other noxious substances as a defense strategy against natural enemies. Newts in particular possess the potent neurotoxin tetrodotoxin (TTX), for which the highest concentrations are found in species within the genus Taricha. Adult Taricha are hypothesized to use TTX as a chemical defense against vertebrate predators such as garter snakes (Thamnophis spp.). However, less is known about how TTX functions to defend aquatic-developing newt larvae against natural enemies, including trematode parasites and aquatic macroinvertebrates. Here we experimentally investigated the effects of exogenous TTX exposure on survivorship of the infectious stages (cercariae) of five species of trematode parasites that infect larval amphibians. Specifically, we used dose-response curves to test the sensitivity of trematode cercariae to progressively increasing concentrations of TTX (0.0 [control], 0.63, 3.13, 6.26, 31.32, and 62.64 nmol L-1) and how this differed among parasite species. We further compared these results to the effects of TTX exposure (0 and 1000 nmolL-1) over 24 h on seven macroinvertebrate taxa commonly found in aquatic habitats with newt larvae. TTX significantly reduced the survivorship of trematode cercariae for all species, but the magnitude of such effects varied among species. Ribeiroia ondatrae - which causes mortality and limb malformations in amphibians - was the least sensitive to TTX, whereas the kidney-encysting Echinostoma trivolvis was the most sensitive. Among the macroinvertebrate taxa, only mayflies (Ephemeroptera) showed a significant increase in mortality following exogenous TTX exposure, despite the use of a concentration 16x higher than the maximum used for trematodes. Our results suggest that maternal investment of TTX into larval newts may provide protection against certain trematode infections and highlight the importance of future work assessing the effects of newt toxicity on both parasite infection success and the palatability of larval newts to invertebrate predators.

Odorant receptors and olfactory-like signaling mechanisms in mammalian sperm.

Since their discovery in 1991, members of the odorant receptor (OR) family have been found in various ectopic tissues, including testis and sperm. It took, however, more than a decade for the first mammalian testicular ORs to be functionally characterized and implicated in a reproductively relevant scenario. Activation of hOR17-4 and mOR23 in human and mouse sperm, respectively, mediates distinct flagellar motion patterns and chemotactic behavior in various bioassays. For hOR17-4, receptor function and downstream signal transduction events are shown to be subject to pharmacological manipulation. Further insight into the basic principles that govern sperm OR operation as well as into the molecular logic that underlies OR-mediated signaling could set the stage for pioneering future applications in procreation and/or contraception.

Chemical Ecology of Wave-Swept Shores: the Primacy of Contact Cues in Predation by Whelks.

Wave-swept shores are valuable for developing and testing key ecological principles. A synthesis of research is nonetheless missing a critical component: the chemosensory basis for behavioral interactions that determine population- and community-wide attributes. Chemical signaling environments on wave-swept shores, given their intense, turbulent mixing and complex topographies, would be difficult or impossible to simulate in a laboratory setting. For this reason, appropriately scaled field studies are needed to advance understanding of chemical stimuli and their biotic effects. Here, we performed a field investigation to establish the relative roles of dissolved and contact cues in predation by whelks (Acanthinucella spirata) on barnacles (Balanus glandula), their preferred prey. Experiments tested responses of whelks to seawater drawn above dense prey patches (10,240-12,180 barnacles m-2) and also over adjacent sand flats (no prey present). There was no evidence of waterborne stimuli associated with prey, even when sea states were nearly tranquil. Field trials also tested faux prey, which were constructed from cleaned barnacle shells and flavored gels. Prospective contact cues were presented to whelks at concentrations typical of epidermal tissue and cuticle in live, intact barnacles. These compounds were highly effective inducers of attack behavior and feeding. Selective enzyme degradations showed that the bioactive material was proteinaceous. Moreover, whelks did not distinguish faux barnacles with a single, purified glycoprotein (named "MULTIFUNCin") from live counterparts. Combined field results thus demonstrate the importance of contact cues, and indicate little, if any, effect of waterborne cues on predation by whelks under native conditions. Our findings underscore the need for appropriately scaled field experiments, and highlight surface chemistry as a critical factor that drives trophic interactions on rocky, wave-swept shores.

MULTIFUNCin: A Multifunctional Protein Cue Induces Habitat Selection by, and Predation on, Barnacles.

Foundation species provide critical resources to ecological community members and are major determinants of biodiversity. The barnacle Balanus glandula is one such species and dominates space among the higher reaches on wave-swept shores. Here, we show that B. glandula produces a 199.6-kDa glycoprotein (named "MULTIFUNCin"), and following secretion, a 390-kDa homodimer in its native state. MULTIFUNCin expression is localized in the epidermis, cuticle, and new shell material. Consequently, this molecule can specify upon contact the immediate presence of a live barnacle. Shared, conserved domains place MULTIFUNCin in the α2-macroglobulin (A2M) subgroup of the thioester-containing protein family. Although previously undescribed, MULTIFUNCin shares 78% nucleotide sequence homology with a settlement-inducing pheromone (SIP) of the barnacle, Amphibalanus amphitrite Based on this and further evidence, we propose that the two proteins are orthologues and evolved ancestrally in structural and immunological roles. More recently, they became exploited as chemical cues for con- and heterospecific organisms, alike. MULTIFUNCin and SIP both induce habitat selection (settlement) by conspecific barnacle larvae. In addition, MULTIFUNCin acts as a potent feeding stimulant to major barnacle predators (sea stars and several whelk species). Promoting immigration via settlement on the one hand, and death via predation on the other, MULTIFUNCin simultaneously mediates opposing demographic processes toward structuring both predator and prey populations. As a multifunctional protein cue, MULTIFUNCin provides valuable sensory information, conveys different messages to different species, and drives complex biotic interactions.

Developmental dimorphism: consequences for larval behavior and dispersal potential in a marine gastropod.

Specific effects of alternative developmental programs on swimming and settlement behavior for marine larvae have not been identified experimentally. A major impediment to this research has been the rarity of species with variable development. Here, we compared traits related to movement and habitat selection for different ontogenetic stages of long-lived, feeding larvae (planktotrophic) and short-lived, nonfeeding larvae (lecithotrophic) of the herbivorous gastropod Alderia modesta. Newly hatched planktotrophic larvae swam in meandering paths with equal rates of upward and downward movement. As planktotrophic larvae developed towards competence (physiological ability to metamorphose), their swimming paths became straighter, faster, and increasingly directed towards the bottom, traits shared by newly hatched lecithotrophic larvae. Despite differing in developmental history, competent planktotrophic (32-d-old) and lecithotrophic larvae (competent upon hatching) exhibited qualitatively similar swimming behaviors and substrate specificity. However, lecithotrophic larvae moved downward at twice the speed of competent planktotrophic larvae, potentially producing a 5-fold higher rate of contact with the bottom in natural flows. Competent larvae swam downwards rather than passively sinking, even though sinking rates were faster than swimming speeds; active swimming may allow larvae to keep the velum extended, permitting rapid response to chemical settlement cues and promoting successful habitat colonization. Differences between larvae of the two development modes may reflect fine-tuning by selection of traits important for dispersal and settlement into patchy adult habitats.

Patterns and processes of larval emergence in an estuarine parasite system.

Trematode parasites in intertidal estuaries experience constantly varying conditions, with the presence or absence of water potentially limiting larval transport between hosts. Given the short life spans (< or =24 h) of cercariae, emergence timing should be optimized to enhance the probability of successful transmission. In the present study, field measurements and laboratory experiments identified processes that regulate the emergence of cercariae from their first intermediate snail hosts in an intertidal marsh. Larvae emerged over species-specific temperature ranges, exclusively during daylight hours, and only when snails were submerged. The three factors operate over different temporal scales: temperature monthly, light diurnally (24-h period), and water depth tidally (12-h period). Each stimulus creates a necessary condition for the next, forming a hierarchy of environmental cues. Emergence as the tide floods would favor transport within the estuary, and light may trigger direct (downward or upward) swimming toward host habitats. Abbreviated dispersal would retain asexually reproduced cercariae within the marsh, and local mixing would diversify the gene pool of larvae encysting on subsequent hosts. In contrast to the timing of cercarial release, emergence duration was under endogenous control. Duration of emergence decreased from sunrise to sunset, perhaps in response to the diminishing lighted interval as the day progresses. Circadian rhythms that control cercarial emergence of freshwater species (including schistosomes) are often set by the activity patterns of subsequent hosts. In this estuary, however, the synchronizing agent is the tides. Together, exogenous and endogenous factors control emergence of trematode cercariae, mitigating the vagaries of an intertidal environment.

The physics of broadcast spawning in benthic invertebrates.

Most benthic invertebrates broadcast their gametes into the sea, whereupon successful fertilization relies on the complex interaction between the physics of the surrounding fluid flow and the biological properties and behavior of eggs and sperm. We present a holistic overview of the impact of instantaneous flow processes on fertilization across a range of scales. At large scales, transport and stirring by the flow control the distribution of gametes. Although mean dilution of gametes by turbulence is deleterious to fertilization, a variety of instantaneous flow phenomena can aggregate gametes before dilution occurs. We argue that these instantaneous flow processes are key to fertilization efficiency. At small scales, sperm motility and taxis enhance contact rates between sperm and chemoattractant-releasing eggs. We argue that sperm motility is a biological adaptation that replaces molecular diffusion in conventional mixing processes and enables gametes to bridge the gap that remains after aggregation by the flow.

Quantifying tetrodotoxin levels in the California newt using a non-destructive sampling method.

Toxic or noxious substances often serve as a means of chemical defense for numerous taxa. However, such compounds may also facilitate ecological or evolutionary processes. The neurotoxin, tetrodotoxin (TTX), which is found in newts of the genus Taricha, acts as a selection pressure upon predatory garter snakes, is a chemical cue to conspecific larvae, which elicits antipredator behavior, and may also affect macroinvertebrate foraging behavior. To understand selection patterns and how potential variation might affect ecological and evolutionary processes, it is necessary to quantify TTX levels within individuals and populations. To do so has often required that animals be destructively sampled or removed from breeding habitats and brought into the laboratory. Here we demonstrate a non-destructive method of sampling adult Taricha that obviates the need to capture and collect individuals. We also show that embryos from oviposited California newt (Taricha torosa) egg masses can be individually sampled and TTX quantified from embryos. We employed three different extraction techniques to isolate TTX. Using a custom fabricated high performance liquid chromatography (HPLC) system we quantified recovery of TTX. We found that a newly developed micro-extraction technique significantly improved recovery compared to previously used methods. Results also indicate our improvements to the HPLC method have high repeatability and increased sensitivity, with a detection limit of 48 pg (0.15 pmol) TTX. The quantified amounts of TTX in adult newts suggest fine geographic variation in toxin levels between sampling localities isolated by as little as 3 km.

Community ecology and the evolution of molecules of keystone significance.

Molecules of keystone significance are vital in structuring ecological communities. Select bioactive compounds can cause disproportionately large effects by connecting such seemingly disparate processes as microbial loop dynamics and apex predation. Here, we develop a general theory and propose mechanisms that could lead to the evolution of keystone molecules. Introduced into a respective community by one, or only a few, autotrophic or microbial species, these compounds often originate as chemical defenses. When co-opted by resistant consumer species, however, they are used either in chemical defense against higher-order predators or as chemosensory cues that elicit courtship and mating, alarm, and predatory search. Requisite to these multifunctional properties, biosynthetic capacity evolves along with mechanisms for resistance and/or toxin storage in primary producers. Subsequently, consumers acquire resistances or tolerances, and the toxins are transferred through food webs via trophic interactions. In consumers, mechanisms eventually evolve for recognizing toxins as feeding cues and, ultimately, as signals or pheromones in chemical communication within or between species. One, or a few, active compounds can thus mediate a vast array of physiological traits, expressed differentially across many species in a given community. Through convergent evolution, molecules of keystone significance provide critical information to phylogenetically diverse species, initiate major trophic cascades, and structure communities within terrestrial, freshwater, coastal-ocean and open-ocean habitats.

Neuroecology and the need for broader synthesis.

Neuroecology combines physiological and ecological principles toward understanding behavioral mechanisms and their roles in establishing patterns of organismal abundances and species distributions. This amalgamation of research approaches incorporates the strengths of neuroethology to determine the cellular basis of behavior. It, however, treads where neuroethology does not by establishing critical linkages between neural processes and the population- and community-level consequences of individual behavior. Neuroecology also promotes understanding of nervous systems within a strong environmental context by encouraging use of keystone and foundation species as critical "ecological models" for studies of electrically excitable cells. Previous investigations of environmental stress, metabolism, and energy relations have proven the value of a combined cellular biochemical and biophysical approach toward predicting natural patterns of organismal abundances and species distributions. Borrowing from this approach, neuroecology would coalesce neuroscience with population and community ecology to establish how individual behavior functions, and how such behavior acts to determine higher-order biological processes.