Field Verification of Predator Attraction to Minnow Alarm Substance

Fishes such as minnows in the superorder Ostariophysi possess specialized alarm substance cells (ASC) that contain an alarm cue. Alarm substance can only be released by damage to the epidermis; thus, the release of alarm substance is a reliable indicator of predation risk. When nearby minnows detect the cue, they adopt a range of antipredator behaviors that reduce their probability of predation. Predator–predator interactions afford prey an opportunity to escape and, thus, a fitness benefit that maintains alarm substance calls over evolutionary time. Here, we present data from a simple field experiment verifying that nearby predators are attracted to minnow alarm substance because it signals an opportunity to pirate a meal. Fishing lures were baited with sponge blocks scented with either (1) water (control for sponge odor and appearance), (2) skin extract from non-ostariophysan convict cichlids (superorder Acanthopterygii, Archocentrus Cichlasoma nigrofasciatus) to control for general injury-released cues from fish, or (3) skin extract from fathead minnows (superorder Ostariophysi, Pimephales promelas). Predator strike frequency on each sponge type was 1,1, and 7 for water, cichlid, and minnow cues, respectively. These data provide the first field test using fish predators of the predator-attraction hypothesis for the evolution of Ostariophysan alarm substance cells.     

Chemical labeling of northern pike (Esox lucius) by the alarm pheromone of fathead minnows (Pimephales promelas)

In previous experiments, chemical stimuli from northern pike (Esox lucius) elicited fright responses from pike-naive fathead minnows (Pimephales promelas) only if the pike had recently eaten conspecific minnows. We used a behavioral assay to determine if the fright response is the result of the incorporation of the minnow alarm pheromone into the chemical signature of the pike. Because the alarm substance cells (epidermal club cells) of fathead minnows are seasonally lost by males, we used chemical stimuli from pike that had eaten breeding male minnows as a control stimulus. In independent laboratory and field experiments, pike-naive minnows exhibited fright reactions (e.g., increased shelter use, avoidance) when exposed to water from tanks containing pike that had eaten nonbreeding fatheads (with alarm substance cells), but not to water from tanks containing pike that had eaten breeding male fatheads (without alarm substance cells). These data indicate that the fathead minnow alarm pheromone chemically labels northern pike as dangerous to pike-naive receivers.     

Conditioned Alarm Behavior in Fathead Minnows (Pimephales promelas) Resulting from Association of Chemical Alarm Pheromone with a Nonbiological Visual Stimulus

Fathead minnows (Pimephales promelas) adopt antipredator (alarm) behavior when they detect alarm pheromone released from an injured conspecific. This is an adaptive response since alarm pheromone is generally released only in the context of a predation event. Alarm reactions may also occur in response to chemical and visual stimuli that minnows learn to associate with release of alarm pheromone. Here, we tested if fathead minnows can learn to associate a nonbiological, visual stimulus with predation risk. Minnows were simultaneously exposed to red light and conspecific alarm pheromone, inducing an alarm reaction. When retested using red light alone, small shoals of minnows displayed an antipredator response: dashing movements and disorganized swimming followed by decreased height in the water column and increased shoal cohesion. This resulted from a single-trial exposure to the combined cues and demonstrates a robust ecological mechanism by which minnows learn to recognize indicators of predation risk that may vary in space and time. However, learning to associate risk with biologically irrelevant stimuli may be an ecological liability. How minnows discern between relevant and irrelevant stimuli in nature is not known.     

Alarm response by a plethodontid salamander (Desmognathus ochrophaeus): Conspecific and heterospecific “Schreckstoff”

The detection of chemical alarm cues plays an important role for predator avoidance in many taxonomic groups, but little is known about the presence of such chemical cues in adult or caudate amphibians. We investigated the response (i.e., aversion or nonaversion) to chemical cues from damaged salamander skin and mealworms (Tenebrio molitor) in the plethodontid salamander,Desmognathus ochrophaeus. Avoidance responses were demonstrated to skin extracts of both conspecific and heterospecific salamanders. However, salamanders (D. ochrophaeus) did not avoid heated conspecific skin, fresh conspecific viscera, fresh mealworm, or freshPlethodon richmondi skin extracts. These results indicate that chemical alarm cues are: (1) present in the skin ofDesmognathus salamanders, (2) not present in mealworm or the viscera ofDesmognathus salamanders, and (3) denatured or deactivated by heating. These results also suggest that an avoidance response to chemical cues from damaged conspecifics has adaptive value in predator avoidance in terrestrial as well as aquatic vertebrates.     

Contrasting Behavioral Responses by Detritivorous and Predatory Mayflies to Chemicals Released by Injured Conspecifics and Their Predators

Larvae of the mayfly Siphlonisca are predators of the detritivorous mayfly Siphlonurus in floodplain wetlands in Maine (USA). Both mayflies are natural prey of brook trout (Salvelinus fontinalis). We exposed larvae of Siphlonurus and Siphlonisca to chemicals from injured conspecifics and their predators. Significant decreases in movement activity by Siphlonurus were elicited by chemicals released from Siphlonisca, chemicals released from brook trout fed conspecifics, and by chemicals released from injured conspecifics. A significant decrease in movement activity by Siphlonisca was elicited by chemicals released from brook trout fed either conspecifics or Siphlonurus. Movement activity by either Siphlonurus or Siphlonisca was not significantly affected by chemicals released from trout feeding on brine shrimp (Artemia). Both Siphlonurus and Siphlonisca were able to detect chemicals that provided information about past feeding behaviour by brook trout. However, their response to the chemicals used in this study was context-specific. A reduction in movement activity, a behavior that presumably reduces the probability of being consumed by visual predators, occurred only when mayflies were exposed to chemicals released by brook trout feeding on conspecific (Siphlonurus) or confamilial (Siphlonisca) prey.     

Alarm substance of the marine mud snail,Nassarius obsoletus: Biological characterization and possible evolution

The gastropod snailNassarius obsoletus shows a dramatic self-burial response to the presence of crushed conspecifics. After it was shown that this burial alarm response could be reliably replicated in laboratory tests, a further characterization of the alarm substance was undertaken. Dilution experiments showed a very high response threshold resulting in a short effective radius of the substance in agreement with earlier field reports. Longevity experiments showed that the substance had lost some activity after 16 hr standing over marsh mud in sea water at room temperature; it became inactive after 24 hr. Superthreshold concentration in sea water was not necessary to keep the snails buried: Mud apparently provides an adsorption surface which can remain a stimulus source for previously unalarmed snails, and snails tend to remain buried after a short exposure to alarm substance, even when given a fresh environment. The substance is present in the snail's blood and tissues and is passively released. A potential natural predator capable of such release isCarcinus maenas, the green crab. Predator odor alone did not cause burial alarm responses. Preliminary chemical analysis indicates that the substance is water soluble, heat stable, and of high apparent molecular weight (over 100,000). A comparison with fish alarm substance and response is made in a discussion of the possible evolution of chemically triggered alarm responses. It is argued thatN. obsoletus may have developed an alarm response to an existing nonspecific substance rather than a true alarm pheromone.This paper is based in part upon a thesis submitted by D.S. to Boston University for the degree of Master of Arts.     

Chemical mediation of behavior in hermit crabs: Alarm and aggregation cues

Field studies with three common local species of hermit crabs,Clibanarius vittatus, Pagurus longicarpus, andPagurus pollicaris, showed that these crabs responded behaviorally to chemicals originating from crushed conspecifics. Hermit crabs are attracted specifically and in a manner similar to previously reported crab responses to odors from dead gastropods. Responses byC. vittatus to both kinds of odor are of three types: (1) aggregation/shell investigation responses (previously reported for odors from dead gastropods), characterized by increased locomotor activity, investigation of shells in the vicinity, and switching into empty shells; (2) alarm responses, in which crabs flee the area; and (3) withdrawal responses, in which crabs pull into their shells and do not come out. Studies withC. vittatus showed that the stimulatory chemicals originate from hemolymph, are less than 500 D, adsorb to octadecyl silica, and are recovered by elution with 20% methanol. Responses ofC. vittatus are dependent upon crab size, type of shell occupied, and shell fit. Chemicals originating from dead conspecifics provide a forum for shell acquisition by crabs in relatively small shells and alarm by crabs in relatively large shells.     

The role of olfaction in chemosensory-based predator recognition in the fathead minnow,Pimephales promelas

Solitary fathead minnows (Pimephales promelas) were rendered anosmic and exposed to chemical stimuli from a predatory northern pike (Esox lucius) to determine the role of olfaction in the minnow's ability to recognize predators on the basis of chemical stimuli. Anosmic fish did not respond to the pike stimuli with a typical fright reaction, while control fish, with intact olfactory receptors, did. These results demonstrate that the olfactory system is necessary for the ability of fathead minnows to recognize northern pike as a predator and that the gustatory and single-celled chemosensory systems are not sufficient for this recognition in the absence of olfactory input. Olfactory impairment was behaviorally confirmed by exposing minnows to alarm substance (Schreckstoff).     

Alarm response of the marine mud snail,Nassarius obsoletus: Specificity and behavioral priority

Nassarius obsoletus, N. vibex, andN. trivittatus were exposed to juices of conspecifics and taxonomically and ecologically related gastropod species. Conspecific juices caused the strongest alarm responses, while those from taxonomically related species (congeners) induced less pronounced responses. Juices from nonrelated species induced no alarm response with one notable exception, the strong alarm response ofN. trivittatus toUrosalpinx cinerea juices. Nonrelated gastropod species induced stronger feeding responses than more closely related species. The strongest feeding responses, however, were induced by juices from nongastropod mollusks. The apparent balance between feeding attraction to less related species and alarm response to more related species was further examined by manipulating the snails' diet. Starvation resulted in a great reduction of conspecific alarm responsiveness. Following the resumption of feeding, the original responsiveness was again observed. Although the alarm response is likely an antipredator device, conclusive evidence for its effectiveness in reducing predation is lacking. One of the predators ofNassarius obsoletus which may be responsible for maintenance of an alarm response is the green crab,Carcinus maenas.This paper is based in part upon a thesis submitted by D.S. to Boston University for the degree of Master of Arts.     

Shifts in Life History as a Response to Predation in Western Toads (Bufo boreas)

Larval western toads (Bufo boreas) are known to exhibit antipredator behavior in response to both chemical alarm cues released from injured conspecifics and chemical cues of predatory invertebrates. In this study, we tested whether long-term exposure to predator and alarm cues resulted in an adaptive shift in life history characteristics of the toads. We raised groups of tadpoles in the presence of: (1) predatory backswimmers (Notonecta spp.) that were fed toad tadpoles, (2) nonpredatory water boatman (Corixidae), and (3) chemical alarm cues of injured conspecifics. Tadpoles raised in the presence of both chemical alarm cues and cues of predators fed tadpoles metamorphosed in significantly shorter time than those raised in the presence of the nonpredator control. Reducing time taken to reach metamorphosis would reduce exposure to aquatic predators. There was no difference among treatments in the size at metamorphosis. Our results suggest that this shift in metamorphic characteristics may represent a facultative alteration in life history.     

Volatile Stimuli Related to Feeding Activity of Nonprey Caterpillars, Spodoptera exigua, Affect Olfactory Response of the Predatory Mite Phytoseiulus persimilis

The effect of volatiles related to feeding activity of nonprey caterpillars, Spodoptera exigua, on the olfactory response of the predatory mites Phytoseiulus persimilis was examined in a Y-tube olfactometer. At a low caterpillar density (20 caterpillars on 10 Lima bean leaves), the predators were significantly more attracted to volatiles from infested leaves on which the caterpillars and their products were present or from infested leaves from which the caterpillars and their products had been removed when compared to volatiles from uninfested leaves. The predators, however, significantly avoided odors from 20 caterpillars and their products (mainly feces) removed from bean leaves. In contrast, at a higher caterpillar density (100 caterpillars on 10 Lima bean leaves), the predators avoided volatiles from caterpillar-infested bean leaves. Volatiles from infested leaves from which the caterpillars and their products had been removed were not preferred over volatiles from uninfested leaves. Volatiles from feces collected from 100 caterpillars were strongly avoided by the predators, while the behavior of the predatory mites was not affected by volatiles from 100 caterpillars removed from a plant. The data show that carnivorous arthropods may avoid nonprofitable herbivores. This avoidance seems to result from an interference of volatiles from herbivore products with the attraction to herbivore-induced plant volatiles.     

Changes in Chemical Signature and Host Specificity from Larval Retrieval to Full Social Integration in the Myrmecophilous Butterfly Maculinea rebeli

The ant social parasite, Maculinea rebeli shows high levels of host specificity at a regional scale. While 68-88% of caterpillars in the field are adopted by nonhost Myrmica ants, 95-100% of the butterflies emerge from the natural host M. schencki the following year. While retrieval of preadoption caterpillars is specific to the genus Myrmica, it does not explain differential survival with different Myrmica species. We present survival data with host and nonhost Myrmica species suggesting that, with nonhosts (M. sabuleti and M. rubra), survival depends on the physiological state of the colony. We also compared the similarities of the epicuticular surface hydrocarbon signatures of caterpillars that were reared by host and nonhost Myrmica for 3 weeks with those from tending workers. Counterintuitively, the hydrocarbons of postadoption caterpillars were more similar (78%, 73%) to the ant colony profiles of the nonhost species than were caterpillars reared in colonies of M. schencki (42% similarity). However, caterpillars from M. schencki nests that were then isolated for 4 additional days showed unchanged chemical profiles, whereas the similarities of those from nonhost colonies fell to 52 and 56%, respectively. Six compounds, presumably newly synthesized, were detected on the isolated caterpillars that could not have been acquired from M. sabuleti and M. rubra (nor occurred on preadoption caterpillars), five of which were found on the natural host M. schencki. These new compounds may relate to the high rank the caterpillars attain within the hierarchy of M. schencki societies. The same compounds would identify the caterpillars as intruders in non-schencki colonies, where their synthesis appeared to be largely suppressed. The ability to synthesize or suppress additional compounds once adopted explains the pattern of mortalities found among fully integrated caterpillars in Myrmica colonies of different species and physiological states.     

Gustatory Responses of Ghost Crab Ocypode quadrata to Seawater Extracts and Chemical Fractions of Natural Stimuli

The efficacy of seawater-extracted fresh and decomposing blue crab (Callinectes sapidus) claw muscle homogenates as stimulants of feeding behavior by the ghost crab (Ocypode quadrata) was tested with cheliped flexion as a bioassay. Stimulatory components of extracts were heat-stable and <1 kDa. Fresh seawater extracts of muscle tissue homogenate elicited the most responses and decreased in efficacy with decomposition. Ultrafiltrates <1 kDa also became less stimulating with increasing decay of the homogenate. When ultrafiltrates were extracted with ethyl ether, the aqueous phase elicited the most responses. To some degree, active components were soluble in ether. Ion-exchange chromatography of the aqueous phase yielded eluates containing neutral and acidic compounds, which, following a peak in activity, became less stimulatory over time. In contrast, eluates containing amphoteric and basic compounds remained highly effective throughout bacterial degradation. However, their activity was significantly suppressed when they were mixed with neutral and acidic compounds isolated from the same samples. Mixture suppression may function as a mechanism ensuring the consumption of high-quality foods. The ability of O. quadrata to respond to both fresh and decomposing tissues contributes to this species' flexibility in foraging strategies and its success as an inhabitant of sandy beaches.     

Response of the Seven-spot Ladybird to an Aphid Alarm Pheromone and an Alarm Pheromone Inhibitor is Mediated by Paired Olfactory Cells

Electrophysiological responses of adult seven-spot ladybirds, Coccinella septempunctata, to (E)--farnesene, an aphid alarm pheromone, and (–)--caryophyllene, a plant-derived alarm pheromone inhibitor, were investigated by recording from single olfactory cells (neurons) on the antenna. Cells having high specificity for each of the two compounds were identified. Furthermore, these two cell types were frequently found in close proximity, with a larger amplitude consistently recorded for the cell responding specifically to (E)--farnesene. Preliminary behavioral studies in a two-way olfactometer showed that walking adults were significantly attracted to (E)--farnesene; this activity was inhibited with increasing proportions of (–)--caryophyllene. The possible ecological significance of colocation or pairing of olfactory cells for semiochemicals with different behavioral roles is discussed.     

Chemoreceptive responses of two sympatric water snakes to extracts of commonly ingested prey species

Newborn of two locally sympatric species of water snakes,Nerodia fasciata andN. erythrogaster, were exposed to skin extracts of frequently ingested prey. In southern Louisiana, fish account for 78% of the diet of adultNerodia fasciata and about 15% forNerodia erythrogaster. Anurans comprise most of the remainder of the diets of these water snakes. Responses of naive individuals were compared to the chemoreceptive response profiles of groups of snakes reared on limited diets of fish and/or frogs. All snakes were tested seven times over a nine-month period and ontogenetic changes were apparent. NewbornNerodia fasciata have a strong chemoreceptive response for fish extract that remains unchanged by early dietary restrictions for the first six months of life. After that time their responses are more variable and can be altered by recent feeding experience. In southern Louisiana, this species is the least specialized water snake, an observation supported by these laboratory results. While newbornN. erythrogaster did not respond preferentially to any of the prey extracts, a significant response rate for fish extract is established by two months of age that persists for several months regardless of dietary restrictions. A subsequent shift of the response to frog extract at eight months of age regardless of diet may reflect a maturational process. Field studies are in agreement with these results and support the notion of ontogenetic changes related to predator size and age. This is the first demonstration of an ontogenetic change in chemoreceptive response that is not related to dietary experience.     

Attraction of predatorily naive postlarval lobsters to extracts of metabolites of common prey:Mytilus edulis,Mya arenaria,Cancer irroratus,andAsterias vulgaris

Postlarval lobsters (4th–7th stage) exclusively fed frozen brine shrimp (Artemia saline) were assayed for food-search response to extracts and metabolites from four common prey: soft clams (Mya arenaria), blue mussels (Mytilus edulis), rock crabs (Cancer irroratus), and sea stars (Asterias vulgaris). Concentrations of soluble primary amines, protein, and ammonia in prey tissues and metabolites were determined. No significant responses were observed for any prey metabolites diluted to 1 and 10%, while onlyA. vulgaris evoked a significant response at full strength, suggesting that predatorily naive lobsters have yet to develop more pronounced chemosensory responses shown by field-collected lobsters. Removal of protein with retention of small-molecular-weight polar molecules did not appear to affect response to prey extracts. EC₅₀s, as micromoles per liter amines, computed from prey extract dose-response curves indicate differences per unit amine between prey extracts, withA. vulgaris extract more potent as an attractant than either bivalve extract.C. irroratus extract was equally attractive as the other three extracts. Ammonia levels excreted into seawater over 3 hr were similar for all prey species, while soluble primary amines and proteins were undetectable. Ammonia and protein per gram whole prey varied significantly between extracts of prey species, while primary amines were similar. Lobsters may be attracted preferentially to carrion species with higher concentrations of amines and/or higher potency of attractants per unit amine.     

Learned Recognition of Predation Risk by Enallagma Damselfly Larvae (Odonata, Zygoptera) on the Basis of Chemical Cues

We studied two populations of damselfly larvae (Enallagma boreale): one population cooccurred with a predatory fish (northern pike, Esox lucius); the other did not. Damselflies that cooccurred with pike adopted antipredator behavior (reduced activity) in response to chemical stimuli from injured conspecifics, and to chemical stimuli from pike, relative to a distilled water control. Damselflies from an area where pike do not occur responded only to chemical stimuli from injured conspecifics. In a second set of experiments, we conditioned pike-naive damselflies to recognize and respond to chemical stimuli from pike with antipredator behavior. Damselfly larvae that were previously unresponsive to pike stimuli learned to recognize pike stimuli after a single exposure to stimuli from pike and injured damselflies or pike and injured fathead minnows (Pimephales promelas). The response to injured fathead minnows was not a general response to injured fish because damselfly larvae did not respond to chemical stimuli from injured swordtails (Xiphophorus helleri), an allopatric fish. Taken together, these data suggest a flexible learning program that allows damselfly larvae to rapidly acquire the ability to recognize local predation risk based on chemical stimuli from predators, conspecifics, and heterospecific members of their prey guild.     

Response of the ichneumonid parasiteNemeritis canescens to Kairomones from the flour moth,Ephestia kuehniella

2-Acylcyclohexane-1,3-diones, a novel class of compounds in the larval mandibular glands ofEphestia(=Anagasta) kuehniella Zeller, act as kairomones. Their relative activities in eliciting oviposition responses from the larval parasiteNemeritis(=Venturia) canescens (Grav.) are reported.     

Chemical defense of a dorid nudibranch,Glossodoris quadricolor,from the red sea

The nudibranch,Glossodoris quadricolor (Doridacea) feeds on the red spongeLatrunculia magnifica, which grows in the reefs of the Gulf of Aqaba, Red Sea. The ichthyotoxic substance from the sponge, latrunculin B, was also indentified in the mucous secretion of the mollusk by TLC, indicating the use of this substance as defense allomone.     

Chemical communication in the primitive antAneuretus simoni: The role of the sternal and pygidial glands

Trail and alarm communication inAneuretus simoni are mediated by the secretions of the sternal and pygidial glands, respectively. The sternal gland is composed of a glandular epithelium and an associated reservoir located in the 7th sternum. This gland produces a relatively long-lived mass recruitment pheromone. The pygidial gland opens between the 6th and 7th tergites and produces a secretion that releases aggressive alarm. The evolution of glandular physiology in the Aneuretinae and Dolichoderinae are discussed in view of these findings.