Nitrogen oxides elicit antipredator responses in juvenile channel catfish,but not in convict cichlids or rainbow trout: conservation of the ostariophysan alarm pheromone

Recent studies with cyprinid and characin (superorder Ostariophysi) fishes suggest that purine-N-oxides function as chemical alarm cues (alarm pheromones) and that the nitrogen oxide functional group acts as the chief molecular trigger. To further test the hypothesis that the nitrogen-oxide functional group is evolutionarily conserved as an active component of the Ostariophysan alarm pheromone system, we exposed juvenile channel catfish (Ictalurus punctatus, Siluriformes) to conspecific skin extract, hypoxanthine-3-N-oxide (the putative alarm pheromone) and a suite of structurally and functionally similar compounds. Conspecific skin extract and hypoxanthine-3-N-oxide elicited significant increases in species typical antipredator behaviors. A structurally dissimilar compound possessing a nitrogen oxide functional group (pyridine-N-oxide) elicited a significant, but less intense alarm response. Compounds lacking a nitrogen oxide functional group were not significantly different from control stimuli. In addition, two non-Ostariophysan species known to possess chemical alarm cues (convict cichlids, Acrchocentrus nigrofasciatus, Cichlidae, Acanthopterygii and rainbow trout, Oncorhynchus mykiss, Salmonidae, Protacanthopterygii) did not show any increase in antipredator behavior in response to hypoxanthine-3-N-oxide. These data demonstrate the conservation of chemical alarm cues within at least three orders of the superorder Ostariophysi.     

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.     

Chemical Ecology of Astigmatid Mites LXXXVII. S-(+)-Isopiperitenone: Re-identification of the Alarm Pheromone as the Female Sex Pheromone in Tyrophagus similis (Acari: Acaridae)

Behavioral analysis revealed that S-(+)-isopiperitenone [(S)-3-methyl-6-isopropenyl-2-cyclohexen-1-one], previously identified as an alarm pheromone, is also the female sex pheromone of Tyrophagus similis (Astigmata: Acaridae), showing maximum male attraction at a dose of 0.1 female equivalent. Although the antipode, R-(−)-isopiperitenone, was not detectable in the mite extract, this synthetic optical isomer (80% e.e.) also induced activity at a dose of 100 ng, a response indicative of S-(+)-isopiperitenone being the active compound. The average content was determined to be 38.5 ng per female and 19.8 ng per male. This is the first example of an astigmatid mite species possessing a compound that functions as an alarm as well as a sex pheromone.     

Responses of Mud Snails and Periwinkles to Environmental Odors and Disaccharide Mimics of Fish Odor

Estuarine snails, periwinkles (Littoraria irorata), and mud snails (Ilyanassa obsoleta) were tested for behavioral responses to aqueous extracts of tissue macerates, odors of living intact organisms, and to disaccharides derived from heparin. Extracts included salt-marsh cordgrass (Spartina alterniflora), pink shrimp (Penaeus duorarum), crushed periwinkles, and crushed mud snails. Odors included live periwinkles, mud snails, stone crab (Menippe mercenaria), striped hermit crab (Clibanarius vittatus), tulip snail (Fasciolaria hunteria), and mummichog (Fundulus heteroclitus). Responses were determined upon contact by snails of a ring of solution in the bottom of an otherwise dry bowl and by presenting snails in seawater with 25 l of solution on a cotton swab. In each test, the response of 30 individuals was determined. Behaviors were recorded as fed, alarm, and no response. The strongest alarm response (>80% of all snails tested) in both snails was elicited by crushed mud snails. The strongest feeding response was to shrimp and periwinkle extract for mud snails (>70%), and salt-marsh cordgrass extract attracted periwinkles. High percentages of mud snails and periwinkles fled in response to the odors of intact snail predators, stone crabs, tulip snails, and mummichogs. Mud snails and periwinkles did not flee in response to nonpredator odors, including each other's odor, as well as that of hermit crabs, shrimp, and marsh cordgrass. Heparin disaccharides were tested because other studies reported that biological activity of predatory fish odor is due to similar disaccharides originating from fish mucus. Mud snail responses to disaccharides indicate that they respond to the same kinds of molecules as crustacean larvae and that the modified amine on the disaccharide is essential for activity. The Q-tip assay is appropriate for bioassay-directed purification of alarm signals originating in fish body odor.     

The Mandibular Gland Secretions of the Leaf-Cutting Ants Atta sexdens sexdens and Atta opaciceps Exhibit Intercaste and Intercolony Variations

The mandibular gland secretions of worker castes from wild colonies of the leaf-cutting ants Atta sexdens sexdens and Atta opaciceps were analyzed quantitatively by gas chromatography–mass spectrometry. The secretions comprised a complex mixture of volatile, mainly oxygenated compounds, and their profiles exhibited considerable qualitative and quantitative variations among species and castes. The known alarm pheromone 4-methyl-3-heptanone was common to both species. The elevated relative proportions of this ketone found in the secretions of gardeners and generalists suggest that such castes are primarily responsible for the production and release of the alarm pheromone. Quantitative variations (but no qualitative differences) in the profiles of secretions of soldiers from different colonies of A. sexdens sexdens were detected, supporting the view that intraspecific colony recognition is mediated through mandibular gland secretions. Subsequent laboratory assays showed that, among the compounds identified by GC–MS, 4-methyl-3-heptanone elicited a strong alarm response in workers of A. sexdens sexdens and A. opaciceps.     

Are chemical alarm cues conserved within salmonid fishes?

A wide diversity of fishes possess chemical alarm signalling systems. However, it is not known whether the specific chemicals that act as alarm signals are conserved within most taxonomic groups. In this study we tested whether cross-species responses to chemical alarm signals occurred within salmonid fishes. In separate laboratory experiments, we exposed brook charr (Salvelinus fontinalis), brown trout (Salmo trutta), and rainbow trout (Oncorhynchus mykiss) to chemical alarm signals from each of the three salmonid species and from swordtails (Xiphophorus helleri). In each case, the test species responded with appropriate antipredator behavior to all three salmonids alarm cues, but did not react to swordtail cues. These data suggest that chemical alarm cues are partially conserved within the Family Salmonidae. For each species tested, the intensity of the response was stronger to conspecific alarm cues, than to heterospecific alarm cues, indicating that salmonids could distinguish between chemical cues of conspecifics versus heterospecifics. These results suggest that the chemical(s) that act as the alarm cues may be: 1) identical and that there may be other chemical(s) that allow the test fish to distinguish between conspecifics and heterospecifics, or 2) that the cues that act as signals are not identical, but are similar enough to be recognized.     

Chemistryvis-à-vis maternalism in lace bugs (Heteroptera: Tingidae): Alarm pheromones and exudate defense inCorythucha andGargaphia species

The hawthorn lace bug,Corythucha cydoniae, and the eggplant lace bug,Gargaphia solani, possess alarm pheromones that are produced in dorsal abdominal glands (DAGs). WhenG. solani nymphs are grasped, they emit secretion from both DAGs; the posterior DAG secretion alone elicits alarm, but the anterior DAG secretion may hasten the response. InC. cydoniae, the response is due to a synergism between the anterior and posterior DAG secretions, and nymphs are apparently unable to voluntarily release their DAG secretions; both DAGs must be ruptured for the pheromone to escape. The alarm pheromones are interspecifically active in patterns matching the intraspecific activities. Compounds identified from tingid DAG secretions that are involved in the alarm messages are: (E)2-hexenal, (E)-4-oxo-2-hexenal, acetaldehyde, geraniol, and linalool. A new natural product of unknown function (designated nerolidol aldehyde) was identified from the anterior DAG secretions of both species.     

Alarm Response of Arius felis to Chemical Stimuli from Injured Conspecifics

The hardhead catfish (Arius felis) showed increased activity when subjected to water containing chemical cues of injured conspecifics. This response was similar to the visually stimulated response to a model of a predatory fish. Arius felis also responded to chemical cues of injured sailfin mollies (Poecilia latipinna) with increased activity, but this was less than in response to conspecific chemical cues. A histological examination of Arius felis epidermis revealed that the alarm substance cells of marine catfish are similar to those of freshwater catfish. Responses to odors of injured individuals is common among ostariophysans, which are mostly freshwater fishes. This is the first demonstration of an alarm reaction in a marine ostariophysan.     

Mandibular Gland Secretion of <Emphasis Type="Italic">Melipona beecheii</Emphasis>: Chemistry and Behavior

The mandibular gland secretion of Melipona beecheii contains a rich mixture of terpenoid and oxygenated compounds and unsaturated and saturated hydrocarbons. However, it differs markedly from the 11 other Brazilian species examined in previous studies, both by the absence of 2-heptanol and the presence of rose oxides. The most abundant compound was geranyl hexanoate, whereas the most volatile compounds were cis- and trans-rose oxide and geraniol. The complete blend and five individual components found in the gland secretion were tested by electroantennography (EAG) and behavioral assays. The complete mandibular gland extract and geraniol elicited the strongest EAG responses, whereas these and farnesyl acetate induced the strongest attack response from workers. The role of the rose oxides remains to be elucidated, as they do not appear to play a major role as an alarm pheromone of this species.     

Isolation of a Pyrazine Alarm Pheromone Component from the Fire Ant, <Emphasis Type="Italic">Solenopsis invicta</Emphasis>

Alarm pheromones in social insects are an essential part of a complex of pheromone interactions that contribute to the maintenance of colony integrity and sociality. The alarm pheromones of ants were among the first examples of animal pheromones identified, primarily because of the large amount of chemical produced and the distinctive responses of ants to the pheromone. However, the alarm pheromone of the fire ant, Solenopsis invicta, eluded identification for over four decades. We identified 2-ethyl-3,6-dimethylpyrazine as an alarm pheromone component of S. invicta. Worker fire ants detect the pyrazine alarm pheromone at 30 pg/ml, which is comparable to alarm pheromone sensitivities reported for other ant species. The source of this alarm pheromone are the mandibular glands, which, in fire ants, are not well developed and contain only about 300 pg of the compound, much less than the microgram quantities of alarm pheromones reported for several other ant species. Female and male sexuals and workers produce the pyrazine, which suggests that it may be involved in fire ant mating flight initiation, as well as the typical worker alarm response. This is the first report of 2-ethyl-3,6-dimethylpyrazine from a Solenopsis species and the first example of this alkaloid functioning as an alarm pheromone.     

Behavioral response of solitary fathead minnows,Pimephales promelas,to alarm substance

Single fathead minnows,Pimephales promelas, were exposed to a range of concentrations of conspecific skin extract. Their responses were observed qualitatively and quantified by a computer linked to an activity meter. The response of fathead minnows to skin extract is complex, involving at least three separate types of behavior. The minnows responded over a 1000-fold range of extract concentrations with combinations of dashing, freezing, slowing, and exploring. The latency of the response increased at the lowest extract concentrations, suggesting summation of sensory cell responses. At low stimulus concentrations, a period of exploratory behavior sometimes preceded the more typical alarm responses. The active space generated by the alarm substance in 1 cm² of minnow skin may exceed 58,000 liters.     

Alarm response to venom by social waspsPolistes exclamans andP. fuscatus (Hymenoptera: Vespidae)

The venoms ofPolistes exclamans andP. fuscatus elicit alarm behavior and attract attacking wasps. The response is not species specific, for both hetero- and conspecific venoms elicit similar responses in both species. A test in a wind tunnel provided no support for the hypothesis that alarmed wasps release an alarm pheromone on the nest.     

Alarm responses in the crayfishOrconectes virilis andOrconectes propinquus

Individuals of two species of crayfish (Orconectes virilis andO. propinquus) were tested in the laboratory for responses to chemicals released from physically damaged conspecifics. Individuals ofO. propinquus did not show an alarm response to crushed conspecifics. Individuals ofO. virilis responded to a water-borne substance released from crushed conspecifics by assuming an intermediate posture and ceasing movement. Similar alarm responses were shown by individuals ofO. virilis to crushed congeneric individuals (O. propinquus), and these responses were not eliminated by either freeze-thawing the crayfish used to prepare the signal or by treating freshly crushed crayfish with the enzyme trypsin. Individuals ofO. virilis showed strong feeding responses to solutions prepared from frozen fish flesh but showed a mixture of alarm and feeding responses to freshly killed fish. These results indicate that the alarm substance used byO. virilis is widespread.     

Allelochemics produced by the hydrophyteMyriophyllum spicatum affecting mosquitoes and midges

An extract of the hydrophyteMyriophyllum spicatum L. was found to be toxic to first- and fourth-instar larvae of the mosquitoes,Culex quinquefasciatus Say,Culex tarsalis Coquillett,Culiseta incidens (Thomson),Aedes aegypti L., and chironomid midges in the laboratory. When first-stage larvae were exposed to the extract, the lowest concentration (6.4 mg extract/100 ml H₂O) produced 86,60, and 48% mortality inC. incidens, C. quinquefasciatus, andA. aegypti, respectively. Higher concentrations caused 100% mortality in both first and fourth instars (63.75 mg/100 ml H₂O or more). In general, the biocidal activity of the extract was found to be similar when first- and fourth-stage larvae were exposed. Some mortality in the successive pupal and adult stages was observed when fourth-stage larvae were exposed to the extract, but mortality occurred only in the various larval instars when first instars were exposed. Among the chironomids,Tanytarsus spp. was more tolerant to the extract thanChironomus spp. In addition to the biocidal activity against immature mosquitoes, the extract was found to show a unique activity acting as an attractant to both sexes of adultC. quinquefasciatus andA. aegypti. The response elicited inA. aegypti appeared to be somewhat higher than that inC. quinquefasciatus. In general, the percentage of males responding to water treated with the extract was higher than that of the females. The extract at higher concentrations seemed to show some repellency at the outset to both species, but induced positive responses later on.     

Alarm pheromone of pentatomid bug,Erthesina fullo Thunberg (Hemiptera: Pentatomidae)

In the pentatomid bug,Erthesina fullo Thunberg, the odor of male metathoracic scent gland elicits an alarm response, making the male individuals of the same species alert and disperse; the alarm response of males is more obvious than that of females. Chemical composition of the glandular secretion was identified by gas chromatography and mass spectrometry data in comparison with authentic compounds. No sexual dimorphism exists in the glandular composition in this species. A total of 9 compounds [(E)-2-hexenal, (E)-4-keto-2-hexenal, (E)-2-hexenyl acetate,n-undecane,n-dodecane, (E)-2-decenal,n-tridecane, (E)-2-decenyl acetate, andn-pentadecane] are identified, among whichn-tridecane and (E)-4-keto-2-hexenal comprised nearly 70% of the total secretion in both females and males.     

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.     

Risk-sensitive habitat use by brook stickleback (Culaea inconstans) in areas associated with minnow alarm pheromone

Brook stickleback (Culaea inconstans) share habitat and predators with cyprinid species, and they exploit the alarm pheromone of fathead minnows (Pimephales promelas) to avoid areas of high predation risk. In this field experiment, we measured the retention and duration of area avoidance by brook stickleback from areas marked with alarm pheromone of fathead minnows. Area avoidance was greatest during the first 2 hr after the source of the alarm pheromone was removed (P<0.05), but after 4 hr, area use was not significantly different from premarking levels. This study shows that brook stickleback: (1) use the alarm pheromone of fathead minnows to avoid high risk areas, (2) continue to avoid locations associated with predation risk after the source of the pheromone has gone, and (3) avoid risky areas temporarily, and resume use of risky areas after 2–4 hr. This behavioral response by stickleback to minnow alarm pheromone could serve to minimize risk of predation.     

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.     

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.     

Intra- and interspecific avoidance of areas marked with skin extract from brook sticklebacks (Culaea inconstans) in a natural habitat

The detection of a chemical alarm pheromone may allow receivers to avoid areas where a predator has captured the prey's conspecifics. We marked minnow traps with either brook stickleback (Culaea inconstans) skin extract or a control of distilled water and tested whether sticklebacks avoided the skin extract marked traps in a natural habitat. Significantly more sticklebacks were captured in traps marked with control water, thereby demonstrating avoidance of conspecific skin extract. The stickleback captured in traps marked with conspecific extract were significantly smaller than those captured in traps marked with control water, implicating ontogenetic factors (i.e., experience or physiological development) in the development of the response. We also captured significantly fewer finescale dace (Chrosomus neogaeus) and fathead minnows (Pimephales promelas) in traps marked with skin extract. These data suggest that dace and minnows may benefit by avoiding areas where predators have recently captured sticklebacks.