RESEARCH ARTICLE Defense through sensory inactivation: sea ... · determine whether sea hare ink applied to the antennules causes sensory inactivation. We monitored changes in the

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INTRODUCTIONAntipredator defenses assume many forms. Most of them operatethrough one or more senses of the predators. One possible defensivemechanism is sensory inactivation, in which the predators’ sensesare impaired by the would-be prey. However, experimental supportfor sensory inactivation as an antipredatory defense is largelylacking. For example, there is only anecdotal evidence for a ‘flash-bulb effect’ where animals deliver a bright light to temporarily‘blind’ predators (e.g. Morin, 1983; Morin, 1986; Mackie, 1995;Deheyn and Wilson, 2011) or for inactivation of predators’ chemicalsenses by inking cephalopods (e.g. Eibl-Eibesfeldt and Scheer, 1962;MacGinitie and MacGinitie, 1968; Fox, 1974; Kittredge et al., 1974;Prota et al., 1981; Moynihan and Rodaniche, 1982). One candidatefor sensory inactivation as an anti-predatory defense is the inksecretion of sea hares. Sea hares (Aplysia spp.) have many defenses,including crypsis and large size (Johnson and Willows, 1999), butchemical defenses are dominant (Derby, 2007; Derby and Aggio,2011). Passive chemical defenses are plentiful and effective, andinclude deterrent molecules in their skin and digestive gland (e.g.Carefoot, 1987; Johnson and Willows, 1999; Wägele andKlussmann-Kolb, 2005; Kamiya et al., 2006). Inking is an active,inducible chemical defense. Sea hares contain two active defensiveglands, the ink gland and the opaline gland, whose secretions arereleased into the mantle cavity and then pumped via a siphon towardsthe site of predatory attack. Ink is purple due to pigments derivedfrom the sea hare’s diet of red algae. Opaline is a whitish liquid

that polymerizes and becomes highly viscous upon contact withwater. Ink and opaline glands are under separate neural control bydifferent ganglia and can release independently or together (Carewand Kandel, 1977; Tritt and Byrne, 1980; Walters and Erickson,1986; Nolen and Johnson, 2001). Healthy animals do not releaseeither secretion without provocation, and significant mechanicalmanipulation such as being bitten or pinched is required.

Although no predator is known to make a regular meal of seahares, perhaps because they are so well defended, a number ofgeneralist predators from several different phyla, including spinylobsters, will take sea hares on occasion (reviewed by Johnson andWillows, 1999). Field studies in the marine reserves of CatalinaIsland, California, demonstrate that spiny lobsters Panulirusinterruptus prey on sea hares, probably because their highabundances there have led to depletion of their favorite foods,resulting in hunger-induced acceptance of less-preferred, chemicallydefended prey including sea hares (W. Wright, personalcommunication). Laboratory studies show that spiny lobsters willattack and in some cases consume sea hares (Kicklighter et al., 2005).

Sea hare ink has been shown in laboratory behavioral experimentsto play a significant protective role against predatory sea anemones,fish and crustaceans including spiny lobsters (Nolen et al., 1995;Kicklighter et al., 2005; Kicklighter and Derby, 2006; Nusnbaumand Derby, 2010b). Sea hare ink acts as a chemical defense throughmultiple molecules and mechanisms. One mechanism is as aphagomimetic decoy, by virtue of its high concentration of amino

SUMMARYAntipredator defenses are ubiquitous and diverse. Ink secretion of sea hares (Aplysia) is an antipredator defense acting throughthe chemical senses of predators by different mechanisms. The most common mechanism is ink acting as an unpalatablerepellent. Less common is ink secretion acting as a decoy (phagomimic) that misdirects predatorsʼ attacks. In this study, wetested another possible mechanism – sensory inactivation – in which ink inactivates the predatorʼs reception of food odorsassociated with would-be prey. We tested this hypothesis using spiny lobsters, Panulirus argus, as model predators. Ink secretionis composed of two glandular products, one being opaline, a viscous substance containing concentrations of hundreds ofmillimolar of total free amino acids. Opaline sticks to antennules, mouthparts and other chemosensory appendages of lobsters,physically blocking access of food odors to the predatorʼs chemosensors, or over-stimulating (short term) and adapting (longterm) the chemosensors. We tested the sensory inactivation hypotheses by treating the antennules with opaline and mimics of itsphysical and/or chemical properties. We compared the effects of these treatments on responses to a food odor for chemoreceptorneurons in isolated antennules, as a measure of effect on chemosensory input, and for antennular motor responses of intactlobsters, as a measure of effect on chemically driven motor behavior. Our results indicate that opaline reduces the output ofchemosensors by physically blocking reception of and response to food odors, and this has an impact on motor responses oflobsters. This is the first experimental demonstration of inactivation of peripheral sensors as an antipredatory defense.

Supplementary material available online at http://jeb.biologists.org/cgi/content/full/216/8/1364/DC1

Key words: chemical defense, chemical sense, chemoreception, neuroecology, predation.

Received 22 October 2012; Accepted 7 December 2012

The Journal of Experimental Biology 216, 1364-1372© 2013. Published by The Company of Biologists Ltddoi:10.1242/jeb.081828

RESEARCH ARTICLE

Defense through sensory inactivation: sea hare ink reduces sensory and motorresponses of spiny lobsters to food odors

Tiffany Love-Chezem, Juan F. Aggio and Charles D. Derby*Neuroscience Institute and Department of Biology, Georgia State University, Atlanta, GA 30303, USA

*Author for correspondence ([email protected])

THE JOURNAL OF EXPERIMENTAL BIOLOGY

1365Defense through sensory inactivation

acids stimulating chemoreceptors that mediate feeding in predatorycrustaceans (Kicklighter et al., 2005). A second mechanism is as arepellent, as demonstrated against predatory fish, spiny lobsters,crabs and sea anemones (Nolen et al., 1995; Johnson and Willows,1999; Kicklighter and Derby, 2006; Nusnbaum and Derby, 2010a).Several repellent molecules have been identified, includingaplysioviolin, phycoerythrobilin and compounds in the ‘escapin’pathway that include hydrogen peroxide, α-keto-ε-aminocaproicacid, Δ1-piperidine-2-carboxylic acid and others (Aggio and Derby,2008; Nusnbaum and Derby, 2010a; Nusnbaum and Derby, 2010b;Kamio et al., 2009; Kamio et al., 2010). A candidate mechanismthat has been proposed (Kicklighter et al., 2005; Derby, 2007) butnot experimentally tested is sensory inactivation, in which thesecretion decreases the activity of the predator’s sensors such thatthe predator cannot detect appetitive stimuli. Anecdotal evidencesuggesting that ink may disrupt the sensory systems of predators isthat attacks of spiny lobsters on sea hares often result with thelobsters’ sensory appendages being covered with the ink secretionand with the lobsters spending considerable time grooming theirappendages, especially their antennules (Kicklighter et al., 2005).

Sensory inactivation could be effected through either of twoactions of ink secretion. First, the viscous and sticky secretionmight cover the predators’ sensors and physically preventappetitive chemicals from reaching and stimulating itschemosensory neurons (i.e. masking). Second, the highlyconcentrated amino acids in the secretion might cause high-amplitude, long-lasting stimulation of chemoreceptor neurons, sothat they become unresponsive to the food stimulus either throughsaturation or adaptation.

We tested the sensory inactivation hypothesis and explored itsmechanisms using the antennular chemosensory pathway of spinylobsters (Fig.1). Antennular chemoreceptors of crustaceans,including spiny lobsters, play important roles in many chemosensorybehaviors. First, they are necessary to mediate many responses tointraspecific cues, including alarm cues in the hemolymph thatmediate avoidance of injured animals (Shabani et al., 2008) andsocial cues in the urine that mediate aggression (Shabani et al., 2009).More relevant to their role in predation, they are necessary to initiatesearching and orientation towards the source of a distant chemicalstimulus (Horner et al., 2004) and complex behaviors such aslearning and discrimination (Steullet et al., 2002). They may alsoplay a role in motivation to feed (J.F.A. and C.D.D., unpublishedobservations). The antennules of spiny lobsters have 10 types ofsetae comprising two major types and pathways (Cate and Derby,2001; reviewed by Caprio and Derby, 2008; Schmidt and Mellon,2011). The aesthetascs are unimodal chemosensilla located in adense tuft located on the ventral and distal half of the antennularlateral flagella. Environmental chemicals access the dendrites of thechemoreceptor neurons within the aesthetascs by passing throughthe porous cuticular walls of the setae (Derby et al., 2007). Theirchemoreceptor neurons project to the olfactory lobe in the brain andthus are part of the olfactory pathway. All the other types ofchemosensilla – of which there are 10 types in spiny lobsters – arebimodal, innervated by both chemosensory neurons andmechanosensory neurons. Collectively, these are called non-aesthetasc, or distributed, chemoreceptor neurons. Chemicalsstimulate their chemoreceptor neurons by entering each seta thougha distally located pore, and the axons of these neurons project tothe lateral antennular neuropil (Cate and Derby, 2001; Schmidt andDerby, 2005).

We performed two types of electrophysiological experiments todetermine whether sea hare ink applied to the antennules causes

sensory inactivation. We monitored changes in the sensitivity tofood-related chemicals for (1) chemoreceptor neurons in thedistributed (non-aesthetasc) antennular system and (2) antennularmotor neurons controlling food odor-activated movement of theantennules (Maynard, 1966; Daniel and Derby, 1988; Schmidt andAche, 1993; Schachtner et al., 2005). In both experiments, we usedopaline, several other treatments and controls to determine whethereffects were due to either of two effects. One possible effect is thephysical properties of ink, where the sticky secretion adheres to thesurface of the setae, thus impeding chemicals from moving throughthe setal pores and preventing them from reaching the setal lumenand therein the dendrites of the chemoreceptor neurons. The otherpossible effect is that chemical in the ink secretion – the millimolarconcentrations of amino acids and other chemostimulants – over-stimulate the chemoreceptor neurons such that they become adaptedand are unable to respond to subsequent chemical stimuli.

MATERIALS AND METHODSAnimals

Caribbean spiny lobsters, Panulirus argus (Latreille 1804), werecollected in the Florida Keys and shipped to Georgia StateUniversity. They were kept individually in 40l (50×25×30cm)aquaria containing artificial seawater (ASW) (Instant Ocean,Aquarium Systems, Mentor, OH, USA) until used in experiments.Sea hares Aplysia californica (Cooper 1863) were collected inCalifornia by commercial suppliers and shipped to Georgia StateUniversity. Upon arrival, they were cooled and anesthetized withan injection of 60ml of a 0.37moll–1 MgCl2 solution, after whichopaline glands were removed by dissection and stored at −80°Cuntil further processing.

Chemical stimuliChemical stimuli were delivered to the antennule before and aftertreatments to determine the effect of those treatments onchemosensory responses to appetitive chemical stimuli. Threechemical stimuli were tested: two concentrations of ‘shrimp juice’and a negative control (ASW). Shrimp juice was made by soaking1g of shredded shrimp in 1l of ASW for 1h, with occasional stirring.The large shrimp pieces were removed and the solution was filteredusing a 0.2μm membrane (Whatman, Kent, UK). Samples werealiquoted and stored at −20°C. Each test day, an aliquot of shrimpjuice was thawed and diluted in ASW to 1 and 10% of the initialconcentration.

TreatmentsFive treatments were applied to the medial or lateral flagella of theantennules to test their effects on responses to chemical stimuli.Treatments were applied to the sensory end of the antennules usinga small paintbrush. Approximately 0.5ml was used for eachapplication, which is a sufficient volume to cover the entirestimulation area. This application is meant to simulate the coverageof ink occurring during natural behavioral interactions between spinylobsters and inking sea hares. An example of ink secretion coveringthe antennules following an inking bout is shown in supplementarymaterial Movie1.

The five treatments were as follows:(1) Water-soluble fraction of opaline (opalineWSF). To obtain

opalineWSF, opaline glands were freeze-dried, crushed with a mortarand pestle, and extracted with 100% methanol. The methanolfraction, which contains amino acids and other major food attractantchemicals, was removed, leaving the pellet, which was stored at−80°C until prepared for testing by rehydrating with ASW to 100%


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concentration, thus forming opalineWSF. OpalineWSF is sticky andmimics the physical nature of opaline.

(2) Carboxymethylcellulose (CMC). When mixed at 0.3gl−1

ASW, CMC yielded a sticky substance that mimics the physicalnature of opaline. CMC lacks any of the amino acids or chemicalattractant components of opaline.

(3) A mixture of the five amino acids in highest concentrationin opaline (AAs): taurine (226.1mmoll−1), L-lysine (105.3mmoll−1),L-histidine (12.21μmol l−1), L-aspartic acid (6.867mmoll−1) and L-glutamic acid (2.793mmoll−1) (based on Derby et al., 2007). Thistreatment simulated the appetitive chemical stimuli in opaline forspiny lobsters (see Kicklighter et al., 2005).

(4) A combination of CMC and AAs (CMC+AAs), each asdescribed above. This treatment created a mixture that simulatesthe chemical and physical properties of opaline.

The Journal of Experimental Biology 216 (8)

(5) A negative control treatment of ASW (i.e. Instant Ocean).When released naturally, opaline (i.e. ‘raw’ opaline) is viscous but

with a fluid consistency. See supplementary material Movie1 for aview of the consistency of opaline and the ink secretion during naturalrelease by a sea hare. For our laboratory work on opaline, we collectedit by dissecting out the glands and freezing them at −80°C for storage.For each experiment, the frozen glands were allowed to reach roomtemperature and were then gently squeezed. The resulting opalinewas very clumpy, less consistent and less fluid than raw opaline. Thispreviously frozen opaline could not be evenly applied to the antennulein a way that is similar to raw opaline in natural encounters betweensea hares and lobsters, because it tended to lump together and couldnot be distributed along the antennule. We found that opalineWSF wasphysically much more like raw opaline, and thus we used it ratherthan the frozen opaline in our studies.

Aesthetasctuft

Antennule

Antenna

Abdomen

Legs Guard seta

Asymmetric seta

Companionseta

BA

Aesthetasc seta

200 µmC

Setuled seta

Hooded seta

Hooded seta Plumose seta

Mediumsimple setae

Medium simple setae

Short setuled seta

Plumose seta Hooded seta

Hooded seta

Plumose seta

D E

Telson

Medialflagellum

Lateral flagellum

60 µm

50 µm 50 µm

Fig.1. Chemical sensors on spiny lobsters.(A)Chemical sensors are present on most bodysurfaces of spiny lobsters, including appendagessuch as the first antennae (=antennules), each with amedial and a lateral flagellum, second antennae,legs and mouthparts, but also body regions includingthe cephalothorax, abdomen and telson. Thechemosensors are organized as sensilla, which arecuticular extensions of the body surface that areinnervated by the dendrites of chemosensoryneurons. Drawing from Lynn Milstead. (B)Scanningelectron micrograph of the tuft region of the lateralflagellum of the antennule, showing the types ofsetae: the unimodal (chemosensory) aesthetascsetae, representing the olfactory pathway; and thebimodal (chemosensory and mechanosensory)asymmetric, guard and companion setae,representing the non-olfactory or distributedchemosensory pathway of the antennules. Photocredit: Manfred Schmidt. (C–E) Scanning electronmicrographs of the medial flagellum of the antennule,which has an organization much like that of thelateral flagellum of the antennule outside of the tuft.Shown are the major setal types: hooded, simple,plumose and setuled setae. Modified from Cate andDerby (Cate and Derby, 2001).



In summary, the five treatments differ in two respects (Table1):some are sticky and therefore may physically prevent chemicalstimuli from reaching the receptors on the antennule (opalineWSF,CMC); some contain amino acids and by long-term application canlead to sensory adaptation and thus inactivation of chemoreceptorneuronal responses (AAs); some are both (opalineWSF, CMC+AAs);and some are neither (ASW).

Electrophysiological assay of antennular chemoreceptorneurons

The aim of this experiment was to determine whether responses ofchemoreceptor neurons of spiny lobsters to chemical stimulationchanged following application of opaline and other treatments tothe chemosensory organ, the antennule. We recorded spikingactivity from chemosensory neurons in response to a chemical foodstimulus, and monitored how responses changed in the varioustreatments.

Preparation and electrophysiological recordingsElectrophysiological recordings were made from chemoreceptorneurons in the medial or lateral flagellum of the antennule usingstandard techniques (Derby, 1995). The flagellum was dissected,placed in an olfactometer and supplied with oxygenated saline at~2mlmin–1 by cannulating its artery. The olfactometer had a U-shaped depression in which the antennular flagellum was situatedsuch that the distal portion of the flagellum (i.e. aesthetasc tuftregion) remained submerged at all times with seawater or treatment(see below). It was this distal portion that was chemically stimulated.Extracellular differential recordings were made using a glass suctionelectrode with silver chloride wire on nerves exposed at the proximalend of the antennule. Electrical activity was amplified and digitizedusing AxoScope (Molecular Devices, Sunnyvale, CA, USA).Recorded neural activity was in the form of action potentials and,following spike sorting (as described below), was resolvable asresponses of single receptor cells. This method is thought to recordselectively from non-aesthetasc (distributed) chemoreceptor neurons(e.g. Caprio and Derby, 2008; Schmidt and Mellon, 2011).

Stimulation and treatmentsThe antennule was stimulated with a 2s pulse of 1% shrimp juicevia an automated solution changer (Bio-Logic, Claix, France). Thestimulus or seawater flowed from the proximal to the distal end ofthe antennule at 2mlmin−1 and was removed via suction beforereaching the exposed nerves. Responses to the stimulation describedabove were recorded for the following conditions: before treatment,during treatment and after treatment. All three conditions wererepeated for as many stimuli as possible for each antennule. Becausea continuous flow of ASW would wash off the treatment, we avoidedthis using a protocol in which background flow was ofteninterrupted. In this protocol, one stimulation cycle consisted of thefollowing: (1) turn off background flow; (2) apply treatment andwait 1min; (3) stimulate with either ASW or shrimp juice and recordfor 20s; (4) turn on background flow to rinse off the chemical

stimulus (40s); (5–8) repeat steps 1 to 4 using the same treatmentin step 2 but the other stimulus in step 3. Steps 1–8 constitute onestimulus cycle, and each chemoreceptor cell was subjected to threecycles. In the first and last cycle, the treatment was ASW (beforeand after treatment conditions), and in the middle cycle it was thetreatment being tested (treatment condition). Steps 1–4 took ~2min.The persistence of each treatment was guaranteed by interruptingboth the background flow and suction from the moment of itsapplication until stimulus onset. In addition, in the case of the stickystimuli, it could be evaluated visually: they remain on the antennuleafter application and before chemical stimulation, and even duringthe 2s chemical stimulation. As described above, after each chemicalstimulation and subsequent rinse, we reapplied the treatment beforestimulating with a new chemical.

Data analysisData analysis was performed using Spike2 (Cambridge ElectronicDevices, Cambridge, England), to sort spikes based on waveform,thus allowing us to quantify responses of single chemoreceptorneurons. For each stimulation, the number of spikes in the 1s periodimmediately preceding stimulation was subtracted from the numberof spikes in the first 1s of the response, and the response to ASWwas subtracted. Thus, for each treatment, we obtained three suchnet responses: before treatment, during treatment and after treatment.The overall response was obtained by dividing the during treatmentresponse by the average of the before treatment and after treatmentresponses. This yielded ΔR, in which ΔR<1 indicates that thetreatment reduces the response, ΔR=1 indicates that the treatmenthas no effect and ΔR>1 indicates that the treatment enhances theresponse. ΔR values were compared statistically usingKruskal–Wallis tests and post hoc Dunn’s tests.

Electrophysiological assay of chemically stimulatedantennular motor neurons

The aim of this experiment was to determine whether a behavioralresponse to chemical stimulation of a chemosensory organ of spinylobsters changed following application of opaline or relatedtreatments to that organ. As a measure, we used motor neuronactivity associated with movement of the antennules in responseto its chemical stimulation with a food chemical stimulus, becauseantennular movement is a reliable component of chemical-stimulated food-seeking behavior in spiny lobsters (Zimmer-Faustet al., 1984; Zimmer-Faust, 1987; Daniel and Derby, 1988; Derbyet al., 2001).

Preparation and electrophysiological recordingsEach spiny lobster was placed in a restraining device dorsal sideup, blindfolded by placing aluminum foil caps over its eyes, andthen placed into a bath of aerated ASW, ensuring the gills werecovered. The lateral flagellum of one of the antennules was securedin an olfactometer, separated from the water bath, allowing forstimulation and application of the treatment as described above.Extracellular differential recordings were made from a pair of silver

Table1. Treatments and their physical and chemical properties

Treatment Physical masking (sticky) Chemical inactivation (contains amino acids)

Water-soluble fraction of opaline (opalineWSF) + –Carboxymethylcellulose (CMC) + –Amino acid mixture (AAs) – +Carboxymethylcellulose + amino acid mixture (CMC+AAs) + +Artificial seawater (ASW) – –


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wire electrodes, insulated with Teflon except for the tip. Oneelectrode of the pair was placed in the antennule joint where thelateral and medial flagellum bifurcate, near the motor neuronscontrolling movement of the antennular lateral flagellum; the otherelectrode of the pair was placed in the ASW bath. That we recordedthe activity of motor neurons and not mechanoreceptor neurons issupported by several observations. First, the amplitudes of the actionpotentials were generally very large, as expected for the large-diameter axons of motor neurons. Second, the responses lasted muchlonger than the duration of the stimulus (see Fig.3). Third, therewere instances of spontaneous bursts of action potentials in theabsence of mechanical stimulation (i.e. when the water flow wasturned off). Each lobster was given a 30min rest period afterimplanting electrodes before presentation of odor stimulus. Motorneuronal activity was recorded using the same amplifiers andsoftware as above.

Stimulation and treatmentsStimulation of the antennule was performed using the sameequipment exactly as described above, except that two shrimpconcentrations (1 and 10%) were tested for each animal. This wasdone to determine whether the treatment had a different effect ondifferent concentrations of shrimp. A negative control of ASW wasstill used. Each stimulus was presented twice in random order foreach animal and each day. Activity was recorded for both beforeand during treatments but not for after treatment due to the toleranceof spiny lobsters under testing conditions. Each spiny lobsterreceived only one treatment per day. The protocol used was thesame as for the chemoreceptor cells (see above) with the exceptionthat no after treatment condition was required because thepreparations were stable.

Data analysisData were amplified, digitized and imported into Spike2 as above.The analysis was also similar, but because we were often unable toidentify single units from the recordings, we used the followinganalysis: traces were rectified and smoothed (time constant=0.25s)and instead of counting the number of spikes, we calculated the areaunder the resulting curve. For each response, we identified the peakof the rectified and smoothed trace and calculated a 5s area under it(0.5s before the peak + 4.5s after the peak), and divided it by thearea under the equivalent length of pre-stimulus curve endingimmediately before stimulus onset. Because stimuli were appliedtwice, we then calculated an average for each one. In this analysis,ΔR for each stimulus was calculated as the response during treatmentdivided by the response before treatment, but the expected values arethe same as above. Data were statistically compared with a repeated-measures ANOVA. The different treatments were compared usingplanned comparisons and relevant stimulus–treatment combinationswith Bonferroni correction for multiple comparisons.

RESULTSOpaline affects sensory reception of food odors

Electrophysiological recordings from non-aesthetasc (distributed)chemoreceptor neurons in the antennules of spiny lobsters were usedto evaluate the effect of ink secretions on sensory reception of afood-related chemical stimulus, shrimp juice. Ink secretion of seahares is composed of two glandular secretions that are co-released:ink, a purple product of the ink gland; and opaline, a white productof the opaline gland (see supplementary material Fig.S1, Movie1).We focused on opaline because it is the more viscous of the twosecretions. Five treatments related to opaline were applied to the


antennules to evaluate whether sensory inactivation occurs and todetermine its mechanism (Table1).

Fig.2A,B shows examples of the effects of two treatments onresponses of distributed chemoreceptor neurons to 1% shrimp juice.Fig.2A shows that treatment with ASW, the negative control, didnot affect the response of one chemoreceptor neuron to stimulationwith the food odor. Fig.2B shows that treatment with opalineWSFreversibly reduces the intensity of the odor-evoked response inanother chemoreceptor neuron. Fig.2C shows composite data forall five treatments. Applying either opalineWSF, CMC or CMC+AAsto the antennule reversibly decreased the neural response to shrimpjuice when compared with ASW. Treatment with AAs caused adecrease that was statistically non-significant. Based on effects offractions and mimics of opaline, we conclude that the sensoryinactivation is principally due to the secretion physically coveringthe antennular chemosetae and thus blocking chemicals fromaccessing chemoreceptor neurons.

Opaline affects chemically evoked motor responsesElectrophysiological recordings from antennular motor neurons wereused to evaluate the effect of ink secretions on motor responses tochemical stimulation of a chemosensory organ of spiny lobsters.The activity of antennular motor neurons was used as an assaybecause antennular movement is a reliable feature of chemicallystimulated food-seeking behavior in spiny lobsters (Zimmer-Faustet al., 1984; Zimmer-Faust, 1987; Daniel and Derby, 1988; Derbyet al., 2001) and is a behavior that was reliably quantified in ourexperiments. We used the same five treatments as in thechemoreceptor neuron assay. Fig.3A,B shows two examples of theeffect of treatments on motor neuronal responses to stimulation with10% shrimp juice. Fig.3A shows that treatment with ASW did notaffect the motor neuronal responses of one animal tochemostimulation, but treatment with opalineWSF profoundlyreduced the intensity of the odor-evoked motor neuronal responsein a different animal (Fig.3B). Composite data for all five treatmentsare shown in Fig.3C. Treatment with opalineWSF, CMC orCMC+AAs significantly decreased the motor neuronal response toshrimp juice compared with treatment with ASW, but effect oftreatment with AAs was not different than treatment with ASW.Thus, the effect of the secretion on the chemically evoked motorresponse is due to it physically covering the antennular chemosetaeand blocking access of the chemicals to the chemoreceptor neurons.

DISCUSSIONSensory inactivation as an antipredatory chemical defense by

sea haresOpaline inactivates sensory responses to food odors

Our study examined whether ink secretion protects sea hares frompredators by inactivating the predators’ sensory systems. We usedelectrophysiological recordings from chemosensory neurons andmotor neurons in a major chemosensory organ, the antennules, ofpredatory spiny lobsters to evaluate the effect of ink secretions onresponses to food odors. We showed that covering the antennuleswith opaline, which is one of the two glandular secretions comprisingthe ink secretion of sea hares, significantly reduced the responsesof both types of neurons (Figs2, 3). This result supports theconclusion that opaline acts as a chemical defense because it causessensory inactivation. Our experimental treatment of the antennuleswith opaline, by applying 0.5ml over the cuticle surface using abrush, was meant to simulate the covering, matching what weobserved in behavioral interactions between spiny lobsters andinking sea hares. Supplementary material Movie1 presents one



example of the coating of the antennules with ink secretion thatoccurs during natural encounters between a spiny lobster and a seahare. The coating of antennules in natural encounters may be morepatchy across the antennular surface, but the coverage, where appliedon the antennule in our experiments, is, to our best approximation,similar to that occurring in natural encounters. Thus, even if naturalencounters lead to more patchy covering of the antennularchemosensors, our techniques should reveal at least qualitativelythe inactivating effect of opaline on responsiveness of thechemosensors.

What is the mechanism underlying sensory inactivation by seahare ink?

Two properties of opaline might contribute to sensory activation byopaline. First is its physical property: its stickiness. By covering thesetae containing the antennular chemoreceptor neurons, opalinemight prevent food odors and other soluble appetitive chemicalsfrom moving into the lumen of the setae, binding to the receptormolecules on the neuronal dendrites, and activating these cells. Asecond property is the chemicals constituting opaline. Opaline andink contain very high concentrations of small nitrogenouscompounds, such as amino acids and ammonium, which are knownto be highly stimulatory to chemoreceptor neurons of spiny lobstersand other crustaceans (reviewed by Caprio and Derby, 2008;Schmidt and Mellon, 2011). These stimulatory chemicals, especiallyembedded into the sticky matrix of opaline, might provideunnaturally long durations of stimulation of these receptor neurons,which would be followed by a sustained response adaptation (e.g.Gomez and Atema, 1996), both of which cause the neurons to berelatively unresponsive to food odors during their exposure toopaline. It is also possible that opaline or ink contains chemicalsthat suppress the responses of the receptor neurons, as these cellsare known to have such inhibitory transduction cascades (Ache andYoung, 2005). Our data provide strong support for the firsthypothesis – physical blocking. This is especially apparent from theobservation that carboxymethylcellulose prepared to a sticky

consistency similar to that of opaline had an inactivating effectsimilar to that of opaline (Figs2, 3). Because carboxymethylcellulosedoes not appear to be a chemical stimulant by itself (J.F.A. andC.D.D., personal observations) and it does not contain any otherchemicals, its effect appears to be due to its stickiness, probably byphysically blocking movement of chemicals through pores in theantennular sensilla (Fig.1) and into the sensillar lumen where thetransduction apparatus of the chemoreceptor neurons is located.

Carboxymethylcellulose spiked with a mixture of the major aminoacids in opaline at their natural concentrations was equally effectiveas carboxymethylcellulose alone in reducing responses to food odors(Figs2, 3), suggesting either that the inactivating effect of opalineis due either solely to its physical properties, or that the effect ofits physical properties is so dominant that any additional effect ofchemicals is inconsequential. Treatment with the amino acid mixtureby itself was not at all effective in reducing responses to food odors.This provides additional support for a physical-only effect. However,the absence of an effect of treatment with the amino acid mixturemight be partially due to technical issues. During treatment, weexposed the antennule to treatments for 1min without allowing anyseawater flow or rinsing over the antennule. In the ensuing test ofthe effect of treatment, we restarted the seawater flow and presentedthe food odor. Two factors in this protocol might have made it moredifficult to observe an effect of the amino acid mixture. First, becausethe amino acid mixture lacks the sticky consistency of opaline orcarboxymethylcellulose, it does not adhere to the antennular cuticle.Thus, although the apparatus kept the antennule bathed in the aminoacid mixture, the amino acids were quickly washed off when theflow was restarted. Taken together, our experiments provide strongsupport that the sensory inactivation is principally due to thesecretion physically covering the antennule and thus blockingchemicals from accessing chemosensory neurons. Our experimentsdo not provide evidence for the chemical properties of opaline, eitherexcitatory or suppressive, contributing to the inactivating effect, butexperimental design issues allow that there might be some chemicaleffect that we could not resolve.

* *

Shrimp odor Shrimp odor2 s2 s

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During

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3

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N=

A B C

Fig.2. The effect of components of ink secretion and their mimics on responses of antennular chemoreceptor neurons to a food odor in the spiny lobsterPanulirus argus. (A,B)Examples of responses to a 2s presentation (denoted by horizontal bar) of 1% shrimp juice for two chemoreceptor cells recorded fromdifferent preparations before (top), during (middle) and after (bottom) being treated with (A) artificial seawater (ASW) or (B) the water-soluble fraction of opaline(opalineWSF). While ASW had no effect on responses to shrimp odor, opalineWSF dramatically reduced the response intensity (i.e. number of action potentials).(C)Summary figure. There is a strong treatment effect (Kruskal–Wallis, P=0.008), and all treatments except the amino acid mixture (AAs) significantly decreasethe response to shrimp odor when compared with ASW treatment (*P<0.05, Dunnʼs). The data are depicted as median (horizontal line), interquartile range(boxes), and minimum and maximum values (whiskers). CMC, carboxymethylcellulose; N, the number of neurons tested in each treatment.


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What are the behavioral consequences of sensory inactivation?Our demonstration of sensory inactivation focused on antennularchemoreception. The antennular chemoreceptors play a specific rolein feeding behavior and its chemical activation. The fact that theantennules of spiny lobsters are many centimeters long and can beactively moved allows the animal to use them to sample the chemicalspace over a large three-dimensional area in front of the animal.This helps the antennular chemoreceptors in their major function,which is to initiate searching upon identifying appetitive chemicalsand to orient within an odor plume during tracking towards its source(Horner et al., 2004; Weissburg, 2011). Our demonstration thatsensory inactivation of the antennules impairs the ability of motorneurons to respond to food odors is consistent with an effect ofsensory inactivation on initiating searching and tracking duringsearches.

Typically, a sea hare is in the grasp of a spiny lobster before thesea hare inks. Our observations are that the ink sticks to all of thesensory appendages in the anterior end, including the antennules,mouthparts and anterior legs. We would expect an effect on theseother chemoreceptors similar to that we have demonstrated forantennular chemoreceptors. While all of the chemoreceptors in theanterior end of the animal will be affected, one might expect themouthpart and oesophageal chemoreceptors to be major functionaltargets of the defensive ink. Chemosensilla in the legs, mouthpartsand esophagus control other aspects of feeding behavior. Legchemoreceptors control local searching and grasping responses, anddelivery to the mouth. Once the material is in the lobster’s mouth,chemoreceptors on the mouthparts (mandibles, maxillae andmaxillipeds) mediate manipulation of the food and biting (Derbyet al., 2001; Garm et al., 2003; Garm et al., 2005). The decision toingest food is controlled by appetitive and deterrent receptors in themouthparts and esophagus (Garm et al., 2003; Garm et al., 2005;Aggio et al., 2012). Thus, if our demonstration of ink’s sensoryinactivation of antennules generalizes to chemosensors on othersensory appendages, which we expect that it does, then sea hare inkwill have effects on other aspects of feeding behavior.

Comparative biology of sensory inactivationSensory inactivation as an antipredatory defense

This is the first experimental demonstration of sensory inactivationas a chemical defense. While it has been proposed previously as a


mechanism of defense by cephalopods, in which ink over-stimulateschemoreceptors of predators so that those predators can no longersense chemicals released by prey (Eibl-Eibesfeldt and Scheer, 1962;MacGinitie and MacGinitie, 1968; Fox, 1974; Kittredge et al., 1974;Prota et al., 1981; Moynihan and Rodaniche, 1982), it has not beenexperimentally demonstrated. Sensory inactivation has been suggestedas functioning in the visual modality, but the evidence there too isanecdotal. For example, a ‘flash-bulb effect’ has been hypothesized,whereby animals deliver a bright light to temporarily ‘blind’ predators(Morin, 1983; Morin, 1986; Mackie, 1995; Deheyn and Wilson, 2011).In the acoustic channel, some moth species produce ultrasound, whichcauses jamming of echolocating bats (Tougaard et al., 1998; Corcoranet al., 2009). The mechanism underlying this jamming is notinactivation of the bats’ periphery auditory system, as bats can detecttheir ultrasonic echoes bouncing off moths. Rather, the moths’ultrasound interferes with the ability of the bats’ central nervoussystem to calculate correctly the distance of the moth (Corcoran etal., 2011). Other moth species use their ultrasound as an aposematicdefense, or as Batesian or Müllerian mimics, rather than for jamming(Hristov and Conner, 2005a; Hristov and Conner, 2005b; Conner andCorcoran, 2012). Our demonstration of the effect of sea hare ink onspiny lobsters is the first experimental demonstration of sensoryinactivation as a chemical defense.

Sensory inactivation as one of several antipredatory defensesInk of sea hares acts through multiple mechanisms of chemicaldefense against predators. These mechanisms includephagomimicry, in which the chemical acts as a decoy (Kicklighteret al., 2005); chemical deterrency, in which the chemical is arepellent (Kicklighter et al., 2005; Aggio and Derby, 2008; Kamioet al., 2009; Kamio et al., 2010; Nusnbaum and Derby, 2010a;Nusnbaum and Derby, 2010b; Nusnbaum et al., 2012); and, as shownin this paper, sensory inactivation. These various forms of chemicaldefense in sea hares prove to be effective anti-predatory chemicaldefenses against diverse species under various conditions, such ashunger state of the predator and environmental availability of certainspecies of algae and thus diet-derived acquisition of deterrentcompounds by the herbivorous sea hares (Derby, 2007; Derby andAggio, 2011).

Taxa throughout the animal kingdom have been shown to usesimilar antipredator defensive strategies. Decoys, in which the

** * ASW (8)

AAs (9) CMC (8)OpalineWSF (8)CMC+AAs (6)

ASW 1% Shrimp 10% Shrimp

Rel

ativ

e re

spon

se

1.5

1.0

0.5

02 s

Shrimp odor

Shrimp odor2 s

Before treatment After treatment

A

B

C

Fig.3. The effect of components of ink secretion and their mimics on the responses of antennular motor neurons to a food odor in the spiny lobsterPanulirus argus. (A,B)Examples of the responses to a 2s presentation (denoted by horizontal bar) of 10% shrimp odor recorded from two lobsters before(black) and after (red) applying (A) ASW or (B) opalineWSF. (C)Summary figure. A repeated-measures analysis shows strong treatment (P<0.001) andstimulus (P=0.01) effects and no interaction effect (P=0.43). Planned comparisons between each treatment showed that CMC (P=0.005), opalineWSF

(P=0.009) and CMC+AAs (P=0.047) differed significantly from ASW but AAs did not (P=0.216). *P<0.05, with Bonferroni correction. Values are means ±s.e.m., and the numbers in parentheses denote the number of lobsters tested in each condition.



defense is produced to distract the predator from the would-be prey,can also be seen in other sensory channels. For example, limbautonomy, in which an appendage is detached and distracts thepredator, is found in octopuses and lizards (Arnold, 1994; Batemanand Fleming, 2009). Decoys can also be visual, such as thebioluminescent ink clouds that are released in many marine species,including squid and ostracods, and act as misdirectional cues(Morin, 1983; Morin, 1986; Grober, 1990; Herring, 1990; Bush andRobison, 2007; Zoerner and Fischer, 2007; Haddock et al., 2010).Repellent or deterrent chemicals are commonly used to create startleor escape responses in predators; for example, the spray of skunks(Wood et al., 2002) and bombardier beetles (Eisner et al., 2006;Eisner et al., 2007). Startle responses can also be produced throughother sensory channels to repel or startle predators. Visual cuesmediating startle include the sudden flashing of eyespots on thewings of butterflies (Caro, 2005; Stevens, 2005; Langridge, 2009;Janzen et al., 2010) and bioluminescent flashes by planktonicanimals (Morin, 1983; Grober, 1990; Mackie, 1995). Auditory cuesused in defense include stridulation noises by wasps and beetlesagainst wolf spiders (Masters, 1979) and by spiny lobsters againstoctopuses or other predators (Bouwma and Herrnkind, 2009),which can be used as a startle defense, an aposematic signal or otherdefensive functions (Hoy, 1989; Sargent, 1990; Ruxton et al., 2004;Staaterman et al., 2010). In summary, the ubiquity of decoy andrepellent defenses indicates that sensory inactivation may beprevalent in antipredator defenses in diverse taxa, but at presentexperimental tests of this hypothesis are lacking. Further study ofsensory inactivation could provide crucial information onpredator–prey interactions in diverse groups of animals.

ACKNOWLEDGEMENTSWe thank the staff of the Keys Marine Laboratory for providing spiny lobsters. Wealso thank the entire Derby laboratory for their help and support.

AUTHOR CONTRIBUTIONSAll authors were involved in designing the experiments and writing the article, andT.L.-C. and J.F.A. executed the experiments.

COMPETING INTERESTSNo competing interests declared.

FUNDINGThis work was supported by National Science Foundation grants IOS-0614685and IOS-1036742 to C.D.D.

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RESEARCH ARTICLE Defense through sensory inactivation: sea ... · determine whether sea hare ink applied to the antennules causes sensory inactivation. We monitored changes in the