Sea hares use novel antipredatory chemical defenses

Numerous studies have demonstrated that chemical defenses protect prey from predation and have often assumed that these defenses function by repelling predators. Surprisingly, few have investigated the mechanisms whereby predators are affected by these defenses. Here, we examine mechanisms of chemical defense of sea hares (Aplysia californica), which, when attacked by spiny lobsters (Panulirus interruptus), release defensive secretions from ink and opaline glands. We show that ink-opaline facilitates the escape of sea hares by acting through a combination of novel and conventional mechanisms. Ink-opaline contains millimolar quantities of amino acids that stimulate chemoreceptor neurons in the spiny lobster's nervous system. Ink stimulates appetitive and ingestive behavior, opaline can elicit appetitive behavior but can also inhibit ingestion and evoke escape responses, and both stimulate grooming. These results suggest that these secretions function by "phagomimicry," in which ink-opaline stimulates the feeding pathway to deceive spiny lobsters into attending to a false food stimulus, and by sensory disruption, in which the sticky and potent secretions cause high-amplitude, long-lasting chemo-mechanosensory stimulation. In addition, opaline contains a chemical deterrent that opposes appetitive effects. Thus, chemical defenses may act in more complex manners than palatability assays of prey chemistry may suggest.     

Multiple components in ink of the sea hare Aplysia californica are aversive to the sea anemone Anthopleura sola

Sea hares of the genus Aplysia rely on an array of behavioral and chemical defenses, including the release of ink and opaline, to protect themselves from predation. While many studies have demonstrated that ink and opaline are repellent to predators, very little is known about which components of these secretions are active against predators. Ink was previously shown to facilitate the escape of Aplysia from predatory anemones (Anthopleura) by eliciting tentacle retraction and/or shriveling, and gastrovascular eversion, but the metabolites mediating this interaction were not identified. We investigated the metabolites in Aplysia californica secretions that were aversive to the anemone Anthopleura sola, as demonstrated by tentacle shriveling and/or retraction. We found that ink elicited tentacle shriveling and/or retraction, while opaline elicited a feeding response. The active components in ink do not appear to be diet-dependent, as ink was aversive regardless of diet (natural seaweed diet vs. Gracilaria ferox). Furthermore, metabolites extracted from G. ferox were not aversive, suggesting that the aversive components are produced by the sea hares. We then examined escapin, a protein in ink with antimicrobial properties. Escapin quickly forms reaction products when mixed with the amino acids l-lysine and l-arginine, which would occur when ink and opaline are released into the sea hare mantle cavity. Neither escapin alone nor escapin mixed with its amino acid substrate l-lysine elicited aversive behaviors either immediately before or 2 min before applying to the tentacles. In addition, escapin mixed with opaline and applied to tentacles after 2 min did not elicit a significant aversive response. Using bioassay-guided fractionation, we attempted to isolate the components in A. californica ink that are aversive to A. sola. We determined that multiple components in ink, including both lipophilic and hydrophilic constituents, elicited aversive responses. We hypothesize that these components may facilitate A. californica's escape from A. sola by eliciting tentacle shriveling and/or retraction, which lead to anemones dropping ensnared sea hares.     

Acidity enhances the effectiveness of active chemical defensive secretions of sea hares, <Emphasis Type="Italic">Aplysia californica</Emphasis>, against spiny lobsters, <Emphasis Type="Italic">Panulirus interruptus</Emphasis>

Sea hares such as Aplysia californica, gastropod molluscs lacking a protective shell, can release a purple cloud of chemicals when vigorously attacked by predators. This active chemical defense is composed of two glandular secretions, ink and opaline, both of which contain an array of compounds. This secretion defends sea hares against predators such as California spiny lobsters Panulirus interruptus via multiple mechanisms, one of which is phagomimicry, in which secretions containing feeding chemicals attract and distract predators toward the secretion and away from the sea hare. We show here that ink and opaline are highly acidic, both having a pH of ∼5. We examined if the acidity of ink and opaline affects their phagomimetic properties. We tested behavioral and electrophysiological responses of chemoreceptor neurons in the olfactory and gustatory organs of P. interruptus, to ink and opaline of A. californica within their natural range of pH values, from ∼5 to 8. Both behavioral and electrophysiological responses to ink and opaline were enhanced at low pH, and low pH alone accounted for most of this effect. Our data suggest that acidity enhances the phagomimetic chemical defense of sea hares.     

Descending antinociceptive mechanisms in the brainstem: Their role in the animal's defensive system

The identification of specialized mechanisms in the mammalian brainstem that function to inhibit the rostral transmission of nociceptive (pain-related) information in the spinal cord led to an explosion of research into the neuroanatomical and neurochemical substrates of these antinociceptive systems. As outlined in the present paper, most attention was directed at those mechanisms in the periaqueductal grey (PAG) and rostral ventromedial medulla (RVM). However, comparatively little attention has been paid to the functional role of these mechanisms in animal behaviour. The purpose of the present paper is to review research into the behavioural significance of those antinociceptive mechanisms in the PAG and RVM. It is concluded that these mechanisms function as part of the animal's fear or defensive system, serving to make a threatened animal insensitive to noxious stimulation and thereby allowing that animal to engage in defensive responses instead of recuperative activities. Further, it is argued that the organization of these antinociceptive circuits reflects the animal's increasing capacity for early detection of danger. Specifically, nociception itself is held to signify the presence of immediate threat, and consequently, nociceptive input directly activates antinociceptive circuits at either the spinal level (during intense noxious stimulation) or RVM (following exposure to moderate noxious stimuli). In contrast, events that are themselves innocuous but which signal threat (either learned or innate danger signals) activate fear and defensive systems in the amygdala and PAG which engage the descending antinociceptive projections in the RVM.     

Dynamic Processing of Nociception in Cortical Network in Conscious Rats: A Laser-evoked Field Potential Study

(1) Field potential study in conscious rats provides a convenient and effective animal model for pain mechanism and pharmacological research. However, the spatial-temporal character of nociception processing in cortex revealed by field potential technique in conscious rats remains unclear. (2) In the present study, multi-channel field potentials evoked by noxious laser stimulation applied to the hind paw of conscious rats were recorded through 12 chronically implanted skull electrodes. Independent component analysis (ICA) was used to remove possible artifacts and to extract the specific nociception-related component. (3) Two fast sharp responses and one slow blunt response were evoked by noxious laser stimulation. Systemic morphine (5 mg/kg, i.p.) preferentially attenuated the amplitude of the slow blunt response while had no significant effect on the first two sharp responses. ICA revealed that those responses came from activities of contralateral anterior parietal area, medial frontal area and posterior parietal area. A movement artifact was also detected in this study. Partial directed coherence (PDC) analysis showed that there were changes of information flows from medial frontal and posterior parietal area to anterior parietal area after noxious laser stimulation. (4) Characterization of the spatio-temporal responses to noxious laser stimulation may be a valuable model for the study of pain mechanisms and for the assessment of analgesia.     

Respiratory pumping in Aplysia fasciata as part of an integrated defensive response to increases and decreases in seawater concentration

Much of the neural circuitry controlling respiratory pumping in Aplysia has been well characterized, but the function of this movement is incompletely understood. To gain insight into possible functions of respiratory pumping, responses were examined for a 40 min exposure to two stimuli that modulate the movement: 1) increase and 2) decrease in seawater concentration. Thresholds were present for both stimuli to affect respiratory pumping. Above threshold, there were graded increases in the number of pumps elicited. There were decrements in respiratory pump frequency as a function of time exposed to the stimulus. Increased respiratory pumping did not contribute to volume regulation in response to exposure to altered seawaters, but was associated with increased defensive responses, such as escape locomotion (swimming) and inking. In addition, head shock, a well-established noxious stimulus, elicited temporal patterns of respiratory pumping similar to those elicited by altered seawaters. The data indicate that in our experimental conditions, respiratory pumping is elicited as part of an integrated defensive response to noxious seawaters.     

Modeling panic disorder in rodents

Panic disorder (PD) is a subtype of anxiety disorder in which the core phenomenon is the spontaneous occurrence of panic attacks. Although studies with laboratory animals have been instrumental for the understanding of its neurobiology and treatment, few review articles have focused on the validity of the currently used animal models for studying this psychopathology. Therefore, the aim of the present paper is to discuss the strengths and limits of these models in terms of face, construct and predictive validity. Based on the hypothesis that panic attacks are related to defensive responses elicited by proximal threat, most animal models measure the escape responses induced by specific stimuli. Some apply electrical or chemical stimulation to brain regions proposed to modulate fear and panic responses, such as the dorsal periaqueductal grey or the medial hypothalamus. Other models focus on the behavioural consequences caused by the exposure of rodents to ultrasound or natural predators. Finally, the elevated T-maze associates a one-way escape response from an open arm with panic attacks. Despite some limitations, animal models are essential for a better understanding of the neurobiology and pharmacology of PD and for discovering more effective treatments.     

Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions

Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay (RIA) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends an axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in "split-foot" preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.     

Involvement of the lateral habenula in the regulation of generalized anxiety- and panic-related defensive responses in rats

Recently obtained evidence points to the involvement of the lateral habenular nuclei (LHb) in the mediation of coping defensive responses to threatening/stressful stimuli. Nevertheless, the role of this brain area in the regulation of defensive responses that have been associated with specific subtypes of anxiety disorders recognized in clinical settings is presently unknown. To address this question, we investigated the effects of either electrolytic lesions or chemical stimulation of the LHb on the defensive behaviors generated in rats by the elevated T-maze. This experimental model allows the measurement, in a same rat, of two defensive behaviors, inhibitory avoidance and escape, that have been related in terms of psychopathology to generalized anxiety and panic disorders, respectively. Bilateral electrolytic lesions of the LHb (1 mA, 10 s) impaired inhibitory avoidance acquisition and facilitated escape performance. On the other hand, chemical stimulation of the LHb by bilateral microinjection of kainic acid (30-60 pmol/0.2 microL) had the opposite effect, i.e., facilitated inhibitory avoidance and impaired escape. The present results indicate that the LHb exerts an opposed regulatory control on generalized anxiety- and panic-related defensive responses in rats.     

大鼠扣带回前部对外侧缰核单位放电的抑制作用

电刺激扣带回前部,对75％的外侧缰核痛兴奋神经元(pain-excitative neuron of lateral habenular nucleus,LHPE)和75％的痛抑制神经元(pain-inhibitive neuron of lateral habenular nucleus,LHPI)的自发放电均产生抑制作用,并取消躯体和内脏伤害性刺激对外侧缰核(lateral habenular nucleus,LHN)单位放电的影响。扣带回内微量注射吗啡可以抑制LHPE的自发放电,并取消伤害性刺激对LHPE的增频效应。注射纳洛酮则使LHPE的自发放电增多,加强伤害性刺激对LHPE的增频作用,并可拮抗电针对LHPE伤害性刺激反应的抑制作用。     

Defensive strategies in planktonic coelenterates

Some planktonic coelenterates respond to potentially harmful stimulation by protective involution, others by escape behaviour. Examples of protective involution are seen in the ‘crumpling’ behaviour of various hydrome‐dusae (Sarsia, Euphysa) and of siphonophores such as Hippopodius. Involution may be accompanied by striking visual displays e.g. light emission in Euphysa, light emission and blanching in Hippopodius. These displays probably serve to startle or blind interlopers. In Hippopodius, light emission in the dark would have the same effect as blanching in the light, an example of behavioural self‐mimicry.

Animals employing escape locomotion include the ctenophore Euplokamis, the siphonophore Nanomia and the rhopalonematid medusa Aglantha. All of these forms have evolved giant axons that facilitate escape by reducing response time. The central nervous circuitry underlying locomotion in Aglantha is reviewed.

In a few cases (e.g. Aglantha and possibly Nanomia), the responses described can be seen as defensive against predators, but in the majority of cases, the responses probably serve primarily to reduce the risk of damage due to accidental contact with other organisms.     

Machaeridian locomotion

The discovery that machaeridians (class Machaeridia Withers, 1926) are annelids allows their mode of locomotion to be interpreted in the context of the body plan of this phylum. The Plumulitidae were errant epibenthic forms, moving with parapodia. The body of Turrilepadidae and Lepidocoleidae, however, was enclosed largely within the mineralized plates that make up the skeleton. Articulated specimens indicate that these machaeridians were able to burrow like other annelids using peristaltic locomotion. A lepidocoleid specimen indicates that multiple waves of shortened and contracted regions moved over the body. This is in contrast to the mode of locomotion in earthworms and most polychaetes, but similar to peristaltic progression in Polyphysia (Scalibregmidae). Either the rugose sculpture (turrilepadids) and/or the margins of the overlapping shell plates functioned as a burrowing sculpture, allowing forward movement but preventing backwards slipping. A trace from the Devonian Hunsrück Slate associated with a lepidocoleid indicates that considerable flexing of the skeleton was possible, but this is an escape trace and does not represent normal locomotion. Features of the skeleton of machaeridians are convergent on those of molluscs where the shells likewise function in protection and burrowing.     

Escape manoeuvres of schooling Clupea harengus

The escape behaviour of schooling herring startled by an artificial sound stimulus was observed by means of high speed video filming. Response latencies showed two distinct peaks, at 30 ms and c . 100 ms. Escape responses belonging to the two latency groups showed different turning rates during the first stage of the response, and showed different escape trajectories. We suggest that long latency escapes may be responses to startled neighbours or simply weak responses to the sound stimulus. In addition, the different contraction rates during the C-bend formation seen in the two latency groups may imply differences in the neuronal commands. The escape responses of herring were directed away from the stimulus more often than towards it (88% of the total). These away responses were more common in long latency responses, suggesting that the latter enable herring to be more accurate in discerning the direction of the threat. Startled fish contracting their body towards the stimulus (performing a towards response) appear to correct their escape course, since their escape trajectory distribution is non-uniformty distributed around 360° and directed away from the stimulus. We hypothesize that when herring are schooling, the ability of each fish to correct its trajectory following turns towards the stimulus is enhanced.     

Responses of bulbar neurons to stimulation of locomotor and inhibitory sites in the cat brainstem

Bulbar locomotor and inhibitory sites were located in the pons of mesencephalic decerebellate cats. Rhythmic stimulation of locomotor sites through microelectrodes at the rate of 60 Hz elicited stepping movements in the forelimbs which were halted when the inhibitory sites were rhythmically stimulated. Neuronal response was elicited by single or paired stimulation of locomotor sites at the rate of 1.5 Hz or by applying a series of 2–4 stimuli spaced 2 msec apart to the inhibitory site. Medial neurons generated synaptic responses (postsynaptic potentials or action potentials) to stimulation of the inhibitory site twice as frequently as when the locomotor site was stimulated. Responses in lateral neurons, however, occurred twice as frequently to stimulation of the locomotor site, while IPSP were only observed half as often as EPSP in neurons of both groups. In neurons excited by stimulation of the locomotor site, stimulation of the inhibitory site did not normally produce IPSP. Possible mechanisms underlying the halt of locomotion occurring in response to stimulation of the inhibitory site are discussed.Information Transmission Institute, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 18, No. 4, pp. 525–533, July–August, 1986.     

Responses of the sea catfish Ariopsis felis to chemical defenses from the sea hare Aplysia californica

Ink secretion of sea hares (Aplysia spp.), which is a mixture of co-released ink from the ink gland and opaline from the opaline gland, protects sea hares from predatory invertebrates through diverse mechanisms. These include both aversive or deterrent compounds and also high concentrations of amino acids that stimulate the predators' chemical senses and divert the attack through phagomimicry or sensory disruption. The aim of the present study was to examine if sea hares also defend themselves from predatory vertebrates by interacting with their chemical senses. We used sea catfish, Ariopsis felis, in behavioral and electrophysiological experiments. Behavioral tests on sea catfish show that ink is aversive: when ink is added to palatable food items (noodles with food flavoring), the noodles are no longer eaten, and when ink is added to noodles without food flavoring, the noodles are avoided more than unflavored noodles. Behavioral tests also show that opaline and the amino acid components of either opaline or ink are appetitive. Electrophysiological recordings of chemosensory neuronal activity in the olfactory epithelium and maxillary barbels show that the olfactory and gustatory systems of sea catfish are highly stimulated by ink and opaline, and that the amino acid components of ink and opaline significantly contribute to these responses. Compounds generated by the activity of escapin, an L-amino acid oxidase in the secretion, are moderately stimulatory to both olfactory and gustatory systems. Taken together, our results support the idea that sea hares are chemically defended from predatory sea catfish largely through unpalatable chemical deterrents in ink, but possibly also through amino acids stimulating olfactory and gustatory systems and thus functioning through phagomimicry or sensory disruption.     

Transfer of habituation in Aplysia: contribution of heterosynaptic pathways in habituation of the gill-withdrawal reflex

Habituation of the Aplysia gill-withdrawal reflex (and siphon-withdrawal reflex) has been attributed to low-frequency homosynaptic depression at central sensory-motor synapses. The recent demonstration that transfer of habituation between stimulation sites occurs in this model system has prompted the hypothesis that heterosynaptic inhibitory pathways also play a role in the mediation of habituation behavior. To test this hypothesis, the sites and mechanisms of neural plasticity which underlie transfer of habituation in Aplysia were examined. Transfer of habituation is a reduction in the reflex evoked at one stimulation site (siphon) due to repeated presentation of a stimulus to a second site (gill). Centrally mediated transfer of habituation, measured in a preparation lacking the siphon-gill peripheral nervous system (PNS), was associated with a reduced excitatory response in central motor neurons. Repeated tactile stimulation of the gill did not attenuate the gill response evoked by electrical stimulation of the branchial nerve nor the mechanoreceptor response recorded in LE sensory neurons. In contrast, repeated stimulation of siphon or gill at a site which was "off" the sensory field of a specific mechanoreceptor led to a diminution in synaptic transmission between that sensory neuron and its followers (motor neurons and inter-neurons). These data demonstrate that centrally mediated transfer of habituation results from heterosynaptic modulation of synaptic transmission at the sensory-motor (and sensory-interneuron) synapses. Therefore, habituation behavior in Aplysia is mediated through the conjoint action of homosynaptic and heterosynaptic inhibitory processes.     

Chemical heterogeneity of soldier defensive secretions in the desert subterranean termite, Amitermes wheeleri

Soldier defensive secretions were analyzed by GC/MS in eight spatially separated groups of Amitermes wheeleri collected in Arizona and California. Eleven sesquiterpenoids, four of known structure, were isolated. Quantitative and qualitative differences among the groups of termites were extensive; composition of the defensive secretions among colonies varied from one to six components. Intraspecific differences between pairs of sympatric colonies at six sites were much less pronounced. The variability found in A. wheeleri suggests that soldier defensive secretions in the genus Amiterines are not reliable markers for interspecific systematic comparisons.     

Sharks modulate their escape behavior in response to predator size,speed and approach orientation

Escape responses are often critical for surviving predator–prey interactions. Nevertheless, little is known about how predator size, speed and approach orientation impact escape performance, especially in larger prey that are primarily viewed as predators. We used realistic shark models to examine how altering predatory behavior and morphology (size, speed and approach orientation) influences escape behavior and performance in Squalus acanthias, a shark that is preyed upon by apex marine predators. Predator models induced C-start escape responses, and increasing the size and speed of the models triggered a more intense response (increased escape turning rate and acceleration). In addition, increased predator size resulted in greater responsiveness from the sharks. Among the responses, predator approach orientation had the most significant impact on escapes, such that the head-on approach, as compared to the tail-on approach, induced greater reaction distances and increased escape turning rate, speed and acceleration. Thus, the anterior binocular vision in sharks renders them less effective at detecting predators approaching from behind. However, it appears that sharks compensate by performing high-intensity escapes, likely induced by the lateral line system, or by a sudden visual flash of the predator entering their field of view. Our study reveals key aspects of escape behavior in sharks, highlighting the modulation of performance in response to predator approach.     

Effect of Dorsal and Ventral Hippocampal Lesions on Contextual Fear Conditioning and Unconditioned Defensive Behavior Induced by Electrical Stimulation of the Dorsal Periaqueductal Gray

The dorsal (DH) and ventral (VH) subregions of the hippocampus are involved in contextual fear conditioning. However, it is still unknown whether these two brain areas also play a role in defensive behavior induced by electrical stimulation of the dorsal periaqueductal gray (dPAG). In the present study, rats were implanted with electrodes into the dPAG to determine freezing and escape response thresholds after sham or bilateral electrolytic lesions of the DH or VH. The duration of freezing behavior that outlasted electrical stimulation of the dPAG was also measured. The next day, these animals were subjected to contextual fear conditioning using footshock as an unconditioned stimulus. Electrolytic lesions of the DH and VH impaired contextual fear conditioning. Only VH lesions disrupted conditioned freezing immediately after footshock and increased the thresholds of aversive freezing and escape responses to dPAG electrical stimulation. Neither DH nor VH lesions disrupted post-dPAG stimulation freezing. These results indicate that the VH but not DH plays an important role in aversively defensive behavior induced by dPAG electrical stimulation. Interpretations of these findings should be made with caution because of the fact that a non-fiber-sparing lesion method was employed.