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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Sperm chemotaxis as revealed with live and synthetic eggs.

Fertilization is one of the least understood fundamental biological processes. How sperm search for and find an egg remains enigmatic. Sperm attraction to egg-derived chemical cues may be significant evolutionarily for maintaining species barriers and important ecologically for increasing gamete encounters. New tools are needed, however, to resolve the functional consequences of these dissolved signal molecules. Freshly spawned eggs from red abalone (Haliotis rufescens) naturally release l-tryptophan, which stimulates chemotactic responses by conspecific sperm. Here, microspheres were manufactured to the approximate size and the same shape as female gametes and formulated to emit controlled doses of chemoattractant, imitating natural l-tryptophan release rates. When experimentally tested for effectiveness, male gametes did not distinguish between chemically impregnated mimics and live eggs, demonstrating that l-tryptophan alone is both necessary and sufficient to promote chemotaxis, and confirming the identity of a native sperm attractant. The techniques that we describe can be used to create synthetic eggs for most animal and plant species, including humans. Egg mimics increase the capacity for experimental manipulation and enable realistic studies of sperm behavior even in the absence of female gametes.

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.

Chemical neuroecology and community dynamics.

Chemical neuroecology examines the relationships between chemosensory physiology, behavior, and population and community dynamics. A keystone species, for example, is one whose impact on communities is far greater than would be predicted from its relative abundance and biomass. Neurotoxins, then, could function in keystone roles. Rare within natural habitats, they 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 potent neurotoxins function as chemical defenses by binding to voltage-gated sodium channels on nerve and muscle cells. When borrowed by resistant consumer species, however, they are used in chemical defense against higher-order predators or as chemosensory excitants in mediating critical behavioral interactions. Through a combination of diverse physiological traits, TTX and STX exert profound impacts reverberating across multiple trophic levels and determining a wide range of community-wide attributes. Such traits ultimately render TTX and STX fully functional as keystone molecules, with vast ecological consequences for species assemblages and rates of material exchange.

Endogenous signaling pathways and chemical communication between sperm and egg.

Red abalone (Haliotis rufescens) sperm detect a waterborne chemical cue released by conspecific eggs, and change their swimming behavior to increase the likelihood of fertilization success. Previously, we isolated the natural sperm attractant by bioassay-guided fractionation and high-performance liquid chromatography, and chemically identified it as the free-amino acid l-tryptophan (l-Trp). In the present study, levels of this ecologically meaningful compound were quantified in various abalone tissues, and in freshly spawned eggs. Tryptophan was the least abundant of 19 dissolved free amino acids (DFAAs) in ovary, testis, foot muscle, gill, stomach and hemolymph. As a proportion of the DFAA pool, however, Trp concentrations were significantly elevated in eggs (three- to seven-times higher) relative to all other sampled tissues. Natural rates of Trp release from eggs also were measured and correlated with fertility. Fertilization success peaked during an initial 30 min period (post-spawn), but decreased to nil over the next 50 min. Closely paralleling these events, Trp accumulated in seawater around freshly spawned eggs for the first 45 min (post-spawn) before decaying rapidly from solution. Older eggs stopped releasing Trp approximately when they became infertile, revealing a critical link between gamete physiology and chemical signaling. This apparent negative feedback loop did not arise from tryptophan oxidation, uptake by bacteria in seawater, or a degrading enzyme released by eggs. As a metabolic precursor critical to development of the larval nervous system, Trp could be an honest indicator of egg fitness for prospective sperm suitors. Our results suggest that endogenous signaling pathways have been co-opted for external communication between gametes, as an adaptation to increase reproductive success by promoting sperm navigation towards fertile eggs.

Dynamic scaling in chemical ecology.

Natural rates of chemical production, release, and transport of fluid-borne molecules drive fundamental biological responses to these stimuli. The scaling of the field signaling environment to laboratory conditions recreates essential features of the dynamics and establishes ecological relevance. If appropriately scaled, laboratory simulations of physical regimes, coupled with natural rates of chemical cue/signal emission, facilitate interpretation of field results. From a meta-analysis of papers published in 11 journals over the last 22 years (1984-1986, 1994-1996, 2004-2006), complete dynamic scaling was rare in both field and laboratory studies. Studies in terrestrial systems often involved chemical determinations, but rarely simulated natural aerodynamics in laboratory wind tunnels. Research in aquatic (marine and freshwater) systems seldom scaled either the chemical or physical environments. Moreover, nearly all research, in all environments, focused on organism-level processes without incorporating the effects of individual-based behavior on populations, communities, and ecosystems. As a result, relationships between chemosensory-mediated behavior and ecological function largely remain unexplored. Outstanding exceptions serve as useful examples for guiding future research. Advanced conceptual frameworks and refined techniques offer exciting opportunities for identifying the ecological significance of chemical cues/signals in behavioral interactions and for incorporating individual effects at higher levels of biological organization.