Chemoecology 10:8187 (2000)09377409:00:00008107 $1.500.20 Birkhauser Verlag, Basel, 2000

Chemical defense of an earwig (Doru taeniatum)*Thomas Eisner1, Carmen Rossini2 and Maria Eisner1

1Department of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA2Facultad de Qumica, Universidad de la Republica, Montevideo, Uruguay CP 11624

Summary. The earwig Doru taeniatum (Dermaptera,Forficulidae) has a pair of defensive glands, opening onthe 4th abdominal tergite, from which it discharges aspray when disturbed. It aims the discharges by revolv-ing the abdomen, a maneuver that enables it simulta-neously to use its pincers in defense. The secretioncontains two quinones (methyl-1,4-benzoquinone and2,3-dimethyl-1,4-benzoquinone) present in the glands asa crystalline mass, together with pentadecane and a(presumably) aqueous phase. The gland openings areminute, with the result that virtually no quinone crys-tals are expelled with the spray. Only the two liquidphases are discharged, together with the ca. 1% quinonethey carry in solution. Such a solute-economizing dis-charge mechanism appears to be without parallelamong insect defensive glands.

Key words. Defensive glands discharge mechanism quinones hydrocarbon

Introduction

The insects of the order Dermaptera, the so-calledearwigs, are doubly protected against predation. Thepincers at the end of their abdomen provide for me-chanical protection, while the dischargeable glands thatmany of them have in the abdomen (Pawlowsky 1927;Vosseler 1890) provide for chemical protection.

Most thoroughly studied has been the Europeanearwig, Forficula auricularia (family Forficulidae). Thisearwig has two pairs of defensive glands, opening onthe posterior margins of the 3rd and 4th abdominaltergites, from which it discharges a secretion consistingof a mixture of methyl-1,4-benzoquinone and ethyl-1,4-benzoquinone (Schildknecht & Weiss 1960). It ejects thesecretion in the form of jets, which it is able to directwith some accuracy toward parts of the body subjectedto assault. It uses its glands and pincers in combination.When attacked, it first revolves the abdomen in such

fashion as to bring the pincers to bear upon the at-tacker. In so doing it automatically aims the glands, sothat if it then discharges it inevitably targets the bodysite under assault. Ants and frogs were shown to bedeterred by these defenses (Eisner 1960).

Recently we had occasion to study another speciesof earwig, Doru taeniatum (henceforth referred to asDoru) (Fig. 1A). We found this insect, also a member ofthe Forficulidae, to use its pincers and aim its dis-charges in much the same manner as F. auricularia,although we noted it to produce a different quinonoidmixture than the latter species and to have but twoinstead of four glands. More interesting, however, wasthe finding that Doru has a special glandular dischargemechanism by which it economizes on the amount ofquinone it expels when spraying. The mechanism issimple and seemingly without parallel among arthropo-dan defensive glands.

We here describe our findings. We succeeded incollecting only eighteen Doru. Our data are thereforenot nearly as extensive as we would have liked.

Materials and methods

The earwig

The Doru stemmed from Lake Placid, Highlands County, Florida,where they were collected at lights on the grounds of the ArchboldBiological Station. They were maintained in the laboratory in smallplastic containers and given freshly cut up mealworms (larvae ofTenebrio molitor) and water (soaked cotton wad).

Gland morphology

Dissections were done under saline solution. For light microscopy,preparations were examined fresh (mounted in glycerin) or afterconventional histological preparation (Bouins fixation; hematoxylinstaining).

For scanning electronmicroscopy, preparations were critical-point dried and gold-coated.

Spray emission

We knew from the odor of the secretion that it was quinonoid andthat we would therefore be able to use the same indicator paper fordepiction of the glandular discharges that we had used with otherquinone-spraying arthropods (Eisner 1958, 1960). That paper is pre-pared by soaking a sheet of filter paper in a freshly-made acidifiedsolution of potassium iodide and starch. The paper is then laid out on

Correspondence to : T. Eisner, e-mail: te14@cornell.edu* Paper no. 170 in the series Defense Mechanisms of

Arthropods. Paper 169 is Eisner T and Eisner M, Defensive use ofa fecal thatch by a beetle larva (Hemisphaerota cyanea). Proc NatlAcad Sci USA 97:26322636

T. Eisner, C. Rossini and M. Eisner CHEMOECOLOGY82

a sheet of glass and blotted off. It changes instantly to an intenseblue-black where contacted by benzoquinones.

To elicit the discharges, we used the same technique employedpreviously with F. auricularia (Eisner 1960). Individual Doru wereaffixed to a metal rod with a droplet of wax placed dorsally on thethorax, and the rod was adjusted in such manner as to cause theearwig to be positioned in a normal stance on the indicator paper.The earwig was then stimulated by pinching parts of its body withfine (watchmakers) forceps. The ensuing spray registered instantly asa pattern on the paper.

Chemistry

All chemicals were obtained from Sigma Chemical Co. (St. Louis,MO). Gas chromatographic analyses were done using a HewlettPackard (HP) 5890 instrument with a split:splitless injector and aflame ionization detector. Secretion extracts were introduced by splitinjection. The HP-ChemStation software program was used to ac-quire and integrate data. Low-resolution electronionization massspectra were obtained using a HP Gas Chromatograph coupled to aHP 5971 Mass Selective Detector. In both cases, the same columnwas used [25 m0.25 mm fused-silica capillary column coated withHP-5 (5% phenylmethylsilicone) stationary phase (0.25 mm filmthickness)], under the following oven temperature condition: 60C for4 min, increased to 260C at 10C:min.

Results

The glands

The two glands of Doru are situated immediately be-neath the 4th abdominal tergite and open on the poste-rior margin of that tergite (Fig. 1B, C). The cuticle oftergite 4 bulges outward conspicuously on each side toaccommodate the glands within (Fig. 1C).

The glands are capacious (Fig. 2B). A calculation ofgland volume, based on linear measurements from ascanning electronmicrograph (Fig. 2C), and certain ge-ometric assumptions (legend, Fig. 2C), yields a value of0.34 ml per replete gland. The gland opening is elongateand minute (Fig. 1B, 3A).

The glands are devoid of compressor muscles [ex-amination of a fresh glycerin mount of a glandular sacin polarized light failed to reveal presence of envelopingmuscle fibers (Fig. 1E), and so did surface examinationof a gland by scanning electronmicroscopy (Fig. 2A)].However, a muscle does occur in association with thegland opening. We assume this muscle, which insertsjust inside the gland opening on the narrowed, ordinar-ily collapsed exit duct of the gland, and originates onthe tergite wall, to serve as the opener muscle thatclears the path for glandular emission. The same musclehas been noted to occur in F. auricularia (Vosseler1890). Given the absence of enveloping muscles, weassume gland compression to be effected by a rise inhemocoelomic pressure, triggered perhaps by momen-tary telescoping of the abdominal segments. We envi-sion glandular ejections to occur when such a rise inblood pressure is coupled with contraction of theopener muscle.

The glands of Doru are integumental structures andas such are endowed with a thin cuticular lining thatsurvives KOH treatment (Fig. 2B). Microscopic exami-nation of whole mounts of the gland wall showedpresence of the large specialized cells that presumablysecrete the gland contents (Fig. 2DF). These cells are

Fig. 1 (A) Doru taeniatum (3.3 ). (B) Base of abdomen, in transmitted light, showing the defensive glands beneath tergum of 4th segment; notethe tiny white circles denoting the gland openings (preparation treated with KOH, consisting of cuticle alone) (18 ). (C) Dorsal view of base ofabdomen, showing gland openings at posterior margin of fourth abdominal tergite (30 ). (D) Inside view of freshly isolated 4th abdominaltergite, with glands attached; the glands are conspicuously filled with yellow crystalline quinone (12 ). (E) Whole mount of gland in glycerin,flattened out under a cover slip, photographed in partially polarized light. The yellow mass of quinone crystals is clearly discernable, as are someof the brown droplets of the oily phase (presumably pentadecane, bearing dissolved quinone). Most of the oil has been squeezed from the gland(some has drifted beyond the field of vision). The aqueous phase, also having been squeezed out, has become mixed with the mounting medium.The dark rectangle is a portion of the 4th tergite to which the gland is attached (25 ). (F) Same as preceding, in fully polarized light, showinga better resolution of the crystals of the quinonoid mass (30 )

Vol. 10, 2000 Chemical defense of an earwig (Doru taeniatum) 83

Fig. 2 (A) Surface view of gland (Bouins solution-fixed, critical point-dried); the nodular structures that beset the surface are the gland cells(90 ). (B) Isolated 4th abdominal tergite (KOH-treated and consisting of cuticle alone) with glands attached (one empty, one distended) (30 ).(C) Same distended gland as in B, indicating the two dimensions, radius (r0.3 mm) and height (h0.8 mm), used in the calculation ofapproximate gland volume. That volume was considered to be equal to two half spheres and a cylinder (4:3 p r3p r2 h0.34 ml). (D) Surfaceview of gland (Bouins solution, hematoxylin stain) showing large, intensely basophilic gland cells (360 ). (E, F) Two views of the same glandcell at different optical planes (940 ); the secretory vesicle is denoted by arrow in (E); at a plane closer to the cuticular lining of the gland, theduct that drains the vesicle is discernable (F). (G) Drainage duct of gland cell (2260 ); the duct has been isolated by KOH treatment. The insetshows site where a duct opens through the cuticular lining of the gland into the gland lumen (4275 )

Fig. 3 (A) Close-up view of gland opening(660 ). (B) The crystalline quinonoid masswithin a gland (partially polarized light) (78 );the inset shows the gland opening to scale (dimen-sions of opening taken from A). Bar in B10 mm

large enough to bulge out from the gland wall (Fig.2A). They are distributed singly. An actual count madeon one gland wall showed presence of 387 such cells.Each has a central vesicle (Fig. 2E, arrow), drained bya cuticular duct (Fig. 2F), an arrangement frequentlyfound in secretory cells of insectan defensive glands(Noirot & Quennedey 1974). The duct opens directly

into the gland lumen. It survives KOH treatment and isevidently cuticular (Fig. 2G).

Although chemical analysis was to show the secre-tion to consist of benzoquinones and a hydrocarbon,compounds that one might expect to be produced bydifferent cells, no second type of secretory cell wasfound to be associated with the Doru gland.

T. Eisner, C. Rossini and M. Eisner CHEMOECOLOGY84

Spray emission

As is clear from Fig. 4, Doru discharged its secretion asa coarse spray. It is also clear that Doru exercised somecontrol over the directionality of the emissions. Thus,stimulation of a front leg or of the head resulted inforwardly-directed discharges (Figs. 4B and D), whilestimulation of a prong of the pincers resulted in anejection accurately directed toward that prong (Fig.4C). As is evident from the broadness of the spraypatterns, the secretion was not always efficientlytargeted. In Figs. 4A and 4B, for instance, where thestimulus was to an individual leg, some spray wasmisdirected in each case to the side opposite of the leg,and in Fig. 4D, some spray was misdirected to the rear.

Doru aimed its ejections in the same manner as F.auricularia. As soon as a stimulus was inflicted, itflexed its abdomen and attempted to bite the offend-ing forceps with its pincers. The discharges never oc-curred prior to these abdominal adjustments, but theymay have occurred in some cases while the adjustmentswere taking place. Secretion ejected prematurely duringthe adjustments could account for the misdirected por-tion of discharges (the droplets to the rear of theearwig in Fig. 4D could have been ejected before theabdomen was fully coiled forward toward the head).Misdirection of part of the spray to an opposite side(as in Figs. 4A and B) could be a reflection of the factthat the two glands, as set in the abdomen, point

divergently and are therefore unable ever to dischargesynchronously in convergence upon a target. Theglands need not, of course, always discharge simulta-neously. Sharply focussed spray deliveries, when theyoccurred (Fig. 4D), could have been due to unilateralglandular emission.

Only 1 to 3 discharges could be elicited from eachof the 5 Doru that were stimulated in this fashion.

Chemistry

We had seven individual Doru available for analyticalwork. We divided these into two groups, which weretreated as follows:

a. (individuals 13). These were killed by freezing,then thawed out and dissected under saline solution.The glandular sacs were then excised intact, and ex-tracted with dichloromethane for analysis.

b. (individuals 47). These were first renderedlethargic by exposure to 4C, then held in forceps andsqueezed, so a to cause them to eject secretion. Theeffluent from each was taken up in a microcapillarytube, held over the gland openings as the earwig wasbeing stimulated. The animals were then killed by freez-ing and their glands dissected out. The weighed effluentand the glands were separately extracted with di-chloromethane for analysis.

Qualitatively the secretion was shown to consist ofthree volatile components, evidenced by three distinct

Fig. 4 Spray patterns, on indicator paper, of discharges elicited from individual Doru by stimulation of: the right midleg (A); the left foreleg (B);the right prong of pincers (C); and the head (D). The diagram of the earwig is included for positional and size reference purposes only; the animalsare not shown in the special stance they adopted (that is, with abdomen coiled and pincers brought toward the site stimulated) when theydischarged. The spray patterns were traced from photocopies made of the sheets of indicator paper on which the patterns were originally registered(1.2 )

Vol. 10, 2000 Chemical defense of an earwig (Doru taeniatum) 85

Fig. 5 A reconstructed ion chro-matogram of volatiles in a di-chloromethane extract of the defensivesecretion of Doru (compound abbrevia-tions as in Table 1)

peaks in the gas chromatograms (Fig. 5). These peakswere identified by GC:MS as corresponding to methyl-1,4-benzoquinone, 2,3-dimethyl-1,4-benzoquinone, andpentadecane. The characterizations were confirmed bycomparison with authentic samples.

The quantities in which the three components arestored in the glands are given in Table 1. For the 4earwigs in group (b) these quantities were calculated foreach compound by adding the amount present in theeffluent and the amount present in the glands aftereffluent emission.

It is clear from these values, that the compoundsare stored in substantial quantity in the glands. Indeed,upon dissection, the glands were noted to be ladenwith golden-yellow crystals that we took to be the

quinones (Fig. 1D). Examination of glycerin mounts offreshly dissected glands showed the secretion withinto be triphasic. The quinone crystals, which weredensely packed, made up the solid phase (Fig. 1E, F).Of the other two phases, both liquid, one was oilyand consisted mostly of large light-brown droplets(Fig. 1E), while the other, more plentiful and morefaintly colored, provided the carrier fluid in whichthe solid and oily phases were suspended. We presumethe carrier phase to be aqueous, and the oily phase tobe made up of pentadecane (indeed, earwig no. 6,whose effluent was noted to be free of oily droplets,proved upon analysis to have no pentadecane in theeffluent; see Table 2). We attribute the coloration ofthe two liquid-phases to quinones that they have

Body MassDoru Total per earwig (mg) % of Body Mass

DMQ 15C15C MQDMQno.:sex (mg) MQ

681 F 25.4 0.180.4145380.040.1513123933.92 F

3 M 30.0 46 36 15 0.26 0.054 F* 22.4 55 33 14 0.39 0.065 F* 0.050.2515603430.2

46.5 0.040.07168226 M*0.030.2411286237.07 M*

0.2790.12 0.0690.0232.298.0(X9SD) 1891147916 30917

Table 1 Total quinone andpentadecane content of theglands of 7 individual Doru.Asterisk denotes individuals ofgroup b for which totals werecalculated as the sum of theamount of chemical in effluentdischarged by glands, and theamount detected in glands af-ter emission of effluent (seeTable 2). MQmethyl-1,4-benzoquinone; DMQ2,3-dimethyl-1,4-benzoquinone;15Cpentadecane

Total in Effluent (mg)Doru % of EffluentEffluent

15C MQDMQ 15Cno.:sex (mg) MQ DMQ

0.110.484 F 0.37330 0.92 0.662.42 1.005 F 410 2.65 0.591.44

0.566 M 530 1.89 1.061.667 M 260 2.36 1.96 1.60 0.62

0.9290.543839116 1.9590.76 1.2890.56 0.4390.281.4691.03

Table 2 Volume and chemi-cal content of glandular fluid(effluent) discharged by the 4individual Doru of group b.Abbreviations as in Table 1

T. Eisner, C. Rossini and M. Eisner CHEMOECOLOGY86

taken up by partition from the solid phase. Penta-decane can be expected to have the greater affinity forquinones (Peschke & Eisner 1987), hence the darkercolor of the oily phase.

The sum total of quinones and pentadecane in thetwo glands ranged from 46151 mg (Table 1). Assuminga density of l mg:ml for these materials, this corre-sponds to 0.050.15 ml, or 722% of the summedcapacity of the two glands. The aqueous carrier phasecan therefore be expected to make up in the order of7893% of the gland contents.

The effluent itself of the glands, when taken up inmicrocapillary tubes, was noted to be virtually free ofquinone crystals, and to consist (by visual estimate) ofa few large droplets of the oily phase suspended withina much larger quantity of the aqueous phase.

It follows from the preceding that the dischargedsecretion should have a much lower quinone contentthan the mixture stored in the gland. Analysis bore thisout (Table 2). The amount of fluid that we collected aseffluent, which can be expected to provide a measure ofthe amount of secretion ordinarily voided by the earwigwhen spraying, ranged from 260530 mg, an equivalent(if one again assumes a density of 1 mg:ml) of 3878%of the combined capacity of the two glands. Yet thetotal amount of quinone in the effluent was only in therange of 1.64.3 mg or, on average, 0.92% of thesample. Doru evidently discharges a dilute solution ofquinones, such as one could envision occurring if itejects solvent only (in other words, quantities of thetwo liquid phases) and holds back on quinone crystalemission. We suggest that this is precisely what theearwig does, and that the reason the quinone crystalsare not expelled is that they are not readily mobilizedand flushed out during ejection. Physically the crystalsseemed to form a pasty aggregate. Individually theyseemed on the whole to be too large to fit through thetiny gland openings (Fig. 3A, B).

A simple calculation yields a figure for the volumeof secretion that the earwig would need to accommo-date if it stored its entire quinone supply at the concen-tration prevalent in the effluent: to be stored at aconcentration of 0.92%, 77 mg of quinone would require8.4 ml of solvent, the equivalent of 12 times the com-bined volume of the two glands.

An explanation is needed for why pentadecane wasalso present in minimal quantities in the effluent. Onepossibility is that the compound is not readily dislodgedfrom the gland lumen because of its physical affinity forthe crystalline quinone mass and the cuticular lining ofthe gland. We noted that when we compressed a freshlydissected gland mounted in glycerin under a coverslip,the aqueous phase would be squeezed out first, togetherwith only a fraction of the oily material.

Discussion

Although much of our argument about the operationof the Doru glands remains speculative, the basic fea-

tures of the mechanism seem established. The earwigstores a substantial quantity of the quinones in crys-talline form in the glands, and on ejection avoidsexpelling these crystals, discharging instead a dilutesolution of the quinones. The solvent, according toour argument, is the liquid mix in which the quinonesare suspended in the glands, a mix consisting of anoily phase (pentadecane) and an aqueous phase. Theadaptive advantage of such a mechanism is that theearwig can thereby avoid incurring a shortage ofquinones. For as long as it is able to replenish thesolvent fluids when these are lost as a consequence ofdefensive use of the glands, it is ready for action.It would clearly have been desirable to determine therate at which these fluids are secreted into the glands,just as it would have been interesting to establishwhen and at what rate the quinones are produced.One wonders whether the gland contents are lost atmolting, and if so, whether quinone production is ata peak immediately after molting. Unfortunately noneof our Doru were immatures.

While we carried out no tests with predators,there can be little doubt that Doru derives defensivebenefit from its glands. Ants must certainly be de-terred by the secretion, as they typically are by qui-none-bearing fluids (Blum 1981; Eisner 1958, 1960).At the concentration of about l% at which Doru ex-pels its quinones, these compounds (including methyl-1,4-benzoquinone itself) have been shown to bepotently irritating in a bioassay with cockroaches(Peschke & Eisner 1987).

Pentadecane may contribute to the defensive effec-tiveness of the secretion in several ways. Support forthe notion that the compound serves as solvent forthe quinones is provided by evidence that it effec-tively takes up quinones by partition (Peschke & Eis-ner 1987). But pentadecane can also promote spreadof the secretion over the integument of the enemy,and be deterrent in its own right. Indeed, penta-decane has been shown to be an effective surfactant,as well as a repellent to ants and a topical irritant tocockroaches (Peschke & Eisner 1987).

We know of no other arthropods with a defensiveglandular discharge mechanism comparable to that ofDoru. One mechanism that bears some similarity isthat of the opilionid Vonones sayi. This arachnidstores crystalline benzoquinones in two glands thatopen anterolaterally on the edge of the carapace.When disturbed, the animal mixes a small amount ofthese crystals with regurgitated enteric fluid, and theneffects dosaged delivery of the mixture by brushing iton the attacker with the tips of its forelegs. Ants areeffectively repelled. It was estimated that at any onetime a Vonones stores enough quinone in its glands tomix 55 such potions (Eisner et al. 1971).

A final point concerned the possibility that F.auricularia shared some of the adaptive featuresof the defensive apparatus of Doru and that thismight have escaped notice in previous studies. Wecollected a number of F. auricularia in Ithaca, NY

Vol. 10, 2000 Chemical defense of an earwig (Doru taeniatum) 87

and dissected these and milked them of secretion. Theglands were found to be replete with brown fluid, anddevoid of both yellow crystals and an oily phase. Thesecretion was found to consist of methyl-1,4-benzo-quinone and ethyl-1,4-benzoquinone, as previously re-ported (Schildknecht & Weiss 1960), and to lackhydrocarbon.

Acknowledgements

We acknowledge with thanks partial support of thisresearch by National Institutes of Health grantAI02908 (T. E.) and a fellowship from the Johnson &Johnson Corporation (C. R.). We are also very grate-ful to the personnel of the Archbold Biological Sta-tion, and especially Mark Deyrup, for hospitality andhelp during our stay at the Station.

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Received 14 February 2000; accepted 21 February 2000.

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