Proc. NatL Acad. Sci. USAVol. 80, pp. 3382-3385, June 1983Evolution

Spider leg autotomy induced by prey venom injection: An adaptiveresponse to "pain"?*

(chemical defense/chemoreception/coevolution/pharmacology)

THOMAS EISNER AND SCOTT CAMAZINESection of Neurobiology and Behavior, Cornell University, Ithaca, New York 14853

Contributed by Thomas Eisner, February 22, 1983

ABSTRACT Field observations showed orb-weaving spiders(Argiope spp.) to undergo leg autotomy if they are stung in a legby venomous insect prey (Phymatafasciata}. The response occurswithin seconds, before the venom can take lethal action by spreadto the body of the spiders. Autotomy is induced also by honeybeevenom and wasp venom, as well as by several venom components(serotonin, histamine, phospholipase A2, melittin) known to be re-sponsible for the pain characteristically elicited by venom injec-tion in humans. The sensing mechanism by which spiders detectinjected harmful chemicals such as venoms therefore may be fun-damentally similar to the one in humans that is coupled with theperception of pain.

Bee stings hurt. So do wasp stings, scorpion stings, the bites ofcentipedes, and the venom injections of many other animals,including snakes. To inflict pain is not necessarily to the ad-vantage of an animal that uses its venom strictly for incapaci-tation of prey. In fact, it may be to its disadvantage because painmay induce increased struggling on the part of the prey. Butvenoms are also used' defensively, and it is in that context thatthey may derive their effectiveness largely, if not exclusively,from their pain-inducing qualities. It is principally becausevenoms are painful that they can function in defense.

Pain, in the sense of a consciously perceived experience, re-mains a subjective notion applicable to humans but untestablewith animals. But when defined operationally as a physiologicalphenomenon induced in an animal by stimuli that are painfulto us and resulting in a protective stimulus-avoidance responsein that animal, pain is amenable to testing with nonhuman sub-jects. We here report that' certain chemical venom componentsthat are painful to humans are also responsible for eliciting anatural self-preserving response in spiders: leg autotomy.

Leg autotomy had previously been noted to occur in spiderswhen a leg was pulled or injured. It occurs consistently at thelevel of the coxa-trochanter joint near the base' of the leg. Aspecial mechanism provides for minimization of bleeding at thesite of leg detachment, and spiders can withstand the loss ofseveral legs (1, 2). We found autotomy to occur also when a spi-der is stung in the leg while attempting to capture venomousinsect prey. Here we present evidence that, under such cir-cumstances, the response is adaptive to spiders because it pre-vents sytemic spread of the poisons and that it is triggered byaction of the venom itself rather than by the mechanics of cu-ticle perforation or fluid injection. Moreover, we show that cer-tain well-known pain-inducing compounds found in venoms arecapable of inducing the autotomy response.

EXPERIMENTAL PROCEDURES ANDOBSERVATIONS

Our study was prompted by field observations made whilestudying prey-capture behavior in orb-weaving spiders of thegenus Argiope. A small ambush bug, Phymatafasciata, had justbeen seen to fly into a web of Argiope aurantia, when the spi-der pounced upon it in typical fashion, in anticipation of wrap-ping it in silk and killing it. Suddenly the behavior came to ahalt. The phymatid had grasped the tip of one of the spider'slegs with its prehensile forelegs, and it was holding on whileseemingly attempting to puncture the leg with its proboscis.The spider remained motionless at first, but then abruptly au-totomized the seized appendage and retreated to the hub of theweb. The relinquished leg remained in the hold of the phy-matid (Fig. 1 D-F).

Tests with captured phymatids that were flipped individ-ually from vials into natural webs of Argiope (A. aurantia andA. trifasciata) showed autotomy to follow almost invariably whena spider's leg was grasped by a phymatid. But leg seizure oc-curred only rarely. Of 43 phymatids that were offered, 36 failedto gain a hold on the spider and were quickly spun in and killed.Six of the remaining seven phymatids seized the spider's legand induced autotomy. The seventh also seized a leg, but au-totomy did not follow. The encounter had an unexpected out-come. The spider seemed to attempt to pull away from the phy-matid as soon as it had wrapped the insect, but it was unableto do so and became stationary and, within seconds, entirelymotionless. For a full 20 min, during which the phymatid neverreleased its hold and the spider remained still, the pair was keptunder observation. When at the end of this period the spiderwas prodded, it failed to respond and was found to be dead (Fig.1G).

Phymatids can inflict a noticeable "sting" with their pro-boscis. They do this whenever given the opportunity (as whenthey are held between the fingers) to bring the tip 'of the pro-boscis to bear against the skin. The sting mechanism is essen-tially similar to what it is in the well-known relatives of phy-matids called assassin bugs (family Reduviidae). Four needle-sharp stylets, tightly apposed to form a slender bundle, effectthe puncture, which occurs with simultaneous injection of ven-omous saliva (3). Prey insects, including powerful species suchas bumblebees, are quickly immobilized by phymatid venom(Fig. 1 A and B). In humans, the sting induces an instantaneoussharply localized pain followed by a mild itch.

Laboratory observations showed that it is the sting, ratherthan the mere mechanical grasp of the spider's leg by the phy-matid, that induces the autotomy. Spiders (A. trifasciata) were

* This is report no. 74 of the series "Defense Mechanisms of Arthro-pods." Report no. 73 is Nowicki, S. & Eisner, T., Psyche, in press.

3382

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. NatL Acad. Sci. USA 80 (1983) 3383

FIG. 1. (A-C) Phymata fasciata as predator and prey (arrows point to dorsum of thorax of phymatid): (A) feeding on syrphid fly; (B) feedingon a hesperid butterfly which has been caught by its slender proboscis; (C) being eaten by an araneid spider. (D-F) Sequence of events in an attackofA. aurantia upon P. fasciata: (D) as spider inspects phymatid, one of its legs is caught by the latter; (E) stung in the leg, the spider autotomizesthat leg and retreats to the hub; (F) moments later the spider preens in its normal upside-down stance at the hub, while the detached leg remainsin the hold of the phymatid. (G) Comparable encounter, of fatal outcome to Argiope; the phymatid, already spun in, has grasped and stung a legof the spider, but the latter failed to autotomize and died as a result. The spider is here shown hanging limply by its seized appendage shortly afterdying. (H) Close-up view of phymatid stinging an Argiope leg (photograph of dead specimen, killed by immersion in liquid Freon at - 190'C whileit was stinging, and subsequently lyophilized while frozen). Bars: D, 10 mm; H, 1 mm.

fastened ventral side down on a flat surface with wax dropletsapplied to the venter of the "abdomen" (technically the spider'sopisthosoma) and to the tips of their outstretched legs. Indi-vidual phymatids attached by their backs to tethers were thenbrought into contact with an articular joint (the femur/patellaor patella/tibia joint) of a spider's leg. They reached out with

the forelegs, grasped the leg, and proceeded to probe the jointmembrane with the proboscis. Autotomy never occurred dur-ing such preliminary probings, but it followed quickly (within5 see in 10 of 10 cases) once the phymatid began pressing itsproboscis against a given site of the membrane, a moment thatis coincident with the onset of stinging. Stylet penetration at

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3384 Evolution: Eisner and Camazine

such moments could be seen through the translucent jointmembrane by observation with a stereomicroscope.

Further testing with immobilized spiders showed that meremechanical perforation of a leg was no substitute for the sting.A single puncture was made, at the femur/patella joint or at themidsection of the femur or tibia, in one leg of each of eightspiders (four A. aurantia and four A. trifasciata) by using a flame-sterilized steel pin. Autotomy followed in one case only. Therewas some bleeding from the wounds, but the spiders showedno long-range ill-effects.

That failure to autotomize following a phymatid sting maylead to lethal systemic envenomization in Argiope was alreadyapparent from the single field encounter of fatal outcome. Ar-giope are unable to withstand phymatid stings to the body. Eightspiders (three A. aurantia and five A. trifasciata) that were causedto be stung on the dorsal surface of the abdomen by tetheredphymatids all eventually died. Kept under observation in testarenas immediately after the sting, they showed locomotoryimpairment within seconds and total immobility and lack of re-sponsiveness within minutes.

Quantitative data on the autotomy-inducing capacity of ven-oms and venom components was obtained by injecting fixedvolumes (1.0 /.l) of test substance into individual legs of A. tri-fasciata. Injection was effected into the femur/patella or pa-tella/tibia joint of a leg with a glass micropipette operated bya micrometer-advanced microsyringe. The injection took a frac-tion of a second. The delay to autotomy was timed to the near-est second from the moment of injection, by using a foot-op-erated stopwatch. Up to four legs per spider were injected, eachnever more than once; at least 1 min transpired between con-secutive leg injections. All autotomies occurred within 19 sec(x ± SD, 4.5 + 3.7; n = 101 legs); failure to autotomize within30 sec was scored as no response (experience had shown thatautotomy is not usually delayed beyond that period).

Because hemipteran venoms are poorly characterized chem-ically and unavailable commercially, we used honeybee and wasp

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(Vespa maculata) venom for our tests, as well as known majorcomponents of hymenopteran venoms. The venom compo-nents were injected in physiological saline (4), at concentrationsapproximating those in natural honeybee or wasp venoms (5, 6);the venoms, available in lyophilized form, were brought to nat-ural concentrations by dilution with distilled water. Saline andhypertonic NaCl (7%) were injected as controls.

As is clear from the results (Fig. 2), the venoms themselvesas well as four of the venom components (serotonin, histamine,phospholipase A2, and melittin) were active, inducing autotomyin upward of 47% of cases. Other venom components were in-active (acetylcholine, bradykinin, hyaluronidase, adrenaline,and dopamine). The controls were inactive or minimally active.

DISCUSSIONThree of the four active components-serotonin, phospholi-pase A2, and histamine-are known to be pain-inducing in hu-mans (7-9). Whatever the neural basis of their detection in spi-ders-whether it be comparable to the pain-coupled venom-sensing mechanism in humans or not-it is clear that spidersare highly sensitive to these substances and that they responddefensively to them in a manner that prevents their systemicspread. All these compounds are of relatively widespread oc-currence in animal (and even plant) venoms (10-12), a reflec-tion perhaps of their broad potential effectiveness vis a vis ene-mies. Arthropods in particular may need to cope with vertebrateand arthropod predators alike, and it is interesting that sero-tonin, phospholipases, and histamine are present in many oftheir venoms (10). The fourth active component, the polypep-tide melittin, is also pain-inducing in humans but it is of re-stricted distribution and known from honeybee venom only (13).Of the inactive venom components tested, three-hyal-

uronidase, dopamine, and adrenaline-are reportedly pain-less, or at least not potently pain-inducing (8, 9), in humans. Al-though these compounds contribute to the overall pharma-

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FIG. 2. Incidence of leg autotomy in A. trifasciata in response to venom or venom component injected. Numbers above bars or in parenthesisafter substance give sample size (= number of legs injected). Percentages give concentration (wt/vol) in physiological saline; honeybee and waspvenom concentrations were 12% and 7.5%, respectively (weight lyophilized venom/volume distilled water).

Proc. Natl. Acad. Sci. USA 80 (1983)

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Proc. Natl. Acad. Sci. USA 80 (1983) 3385

cological effect of venoms and may even (as in the case of hy-aluronidase) (8) act to potentiate the activity of other venomcomponents, they are apparently not pain-inducing as such. Buttwo components, acetylcholine and bradykinin, which provedineffective vis a vis Argiope (at least at the concentrations tested),are definitely pain-inducing in humans (8, 9). Therefore, theremay be no absolute concordance in the chemical sensitivities ofthe venom-detecting systems of humans and Argiope. Giventhe phyletic gap between arachnids and vertebrates, this shouldcome as no surprise. What is remarkable is that there shouldeven be substantial overlap in the pharmacological spectrum ofwhat induces pain in us and what acts as if it were painful toArgiope.

The five inactive substances that failed to induce autotomyinvariably proved nonlethal to the spiders. But death did notalways follow in spiders that retained their leg after injectionof active samples. Although honeybee venom and phospholi-pase A2 were always lethal to nonautotomizing spiders (threeand six spiders respectively), histamine, serotonin, and melittinwere consistently not (eight, four, and five spiders). And, some-what surprisingly, neither was wasp venom (three spiders). Thechemosensory system that "forewarns" spiders against systemictoxins is thus not attuned strictly to molecules that are lethal.Our tests with phymatids offered no evidence that these in-

sects might secure their release from predators by defensiveuse of their venoms. They remained trapped in the Argiopewebs whether they induced leg autotomy or not, and all wereeventually eaten. But tests that we did with a jumping spider,Phidippus audax, a most likely natural enemy of phymatids,gave different results. Four individual Phymata were offeredto an equal number of female Phidippus, held in small arenasin which the spiders had been maintained for months on a dietof live insects. Three of the phymatids were promptly killed bythe spiders and eaten. The fourth was also pounced upon, butit grasped a leg of the spider, stung it, and caused the append-age to be autotomized. The spider backed away, leaving thephymatid unencumbered and potentially free to make an im-mediate escape.

Leg autotomy occurs also in arthropods other than spiders.If seized by a leg, katydids often relinquish the appendage, as

do many grasshoppers, crickets, and cockroaches. Tests that wedid with unidentified katydids showed them to autotomize read-ily in response to Phymata stings (n = 5 katydids), indicatingthat they too have the means for quick-sensing of potentiallyhazardous toxins. And so do spiders other than Argiope. Eachof seven unidentified spiders of three families (Salticidae,Thomisidae, and Agelenidae) autotomized in response to phy-matid stings. But one spider-the common house spiderAchaearanea tepidariorum (family Theridiidae)-proved ex-ceptional in that it never autotomized (n = 11 spiders) despiteits evident susceptibility to envenomization (7 of 11 died).Whether this spider lacks the appropriate venom-sensingmechanism or is physically unable to autotomize and whetherits vulnerability to venoms is correlated with a life style thatprovides for minimization of exposure to venoms remains un-known.

The initial field observations and laboratory tests were made at theHuyck Preserve, Rensselaerville, NY, in collaboration with Maria Eis-ner, who also helped with the photography. This study was supportedbyGrant AI-02908 from the National Institutes of Health and by a gen-erous stipend from Cornell University.

1. Parry, D. A. (1957) Q. J Microsc. Sci. 98, 331-340.2. Wood, F. D. (1926)J. Morphol Physiol 42, 143-195.3. Weber, H. (1930) Biologie der Hemipteren (Springer, Berlin).4. Griffiths, J. T. & Tauber, O. E. (1943) J. Gen. Physiol 26, 541-

558.5. O'Connor, R. & Peck, M. L. (1978) in Arthropod Venoms, ed.

Bettini, S. (Springer, Berlin), pp. 613-659.6. Edery, H., Ishay, J., Gitter, S. & Joshua, H. (1978) in Arthropod

Venoms, ed. Bettini, S. (Springer, Berlin), pp. 691-771.7. Schmidt, J. 0. (1982) Annu. Rev. Entomol 27, 339-368.8. Chahl, L. A. & Kirk, E. J. (1975) Pain 1, 3-49.9. Keele, C. A. & Armstrong, D. (1964) Substances Producing Pain

and Itch (Edward Arnold, London).10. Bettini, S. (1978) Arthropod Venoms (Springer, Berlin).11. Habermehl, G. (1976) Gift-Tiere und ihre Waffen (Springer, Ber-

lin)..12. Thurston, E. L. & Lersten, N. R. (1969) Bot. Rev. 35, 393-412.13. Habermann, E. (1971) in Venomous Animals and Their Venoms,

Venomous Invertebrates, eds. Bucherl, W. & Buckley, E. E. (Ac-ademic, New York), Vol. 3, pp. 61-93.

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