Chemical defense of an opilionid (Acanthopachylus aculeatus)
1 Department of Neurobiology and Behavior, Cornell University, Ithaca, NY
14853, USA
2 Facultad de Química, Universidad de la República,
Montevideo, Uruguay
* Author for correspondence (e-mail: te14{at}cornell.edu)
Accepted 5 January 2004
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Summary |
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Key words: opilionid, predation, exocrine gland, 1,4-benzoquinone, repellent, Formicidae, Lycosidae
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Introduction |
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We have recently had occasion to study live A. aculeatus in the laboratory. We were able to confirm the earlier claim that the secretion is quinonoid in nature, and in addition obtained detailed photographic documentation of the glandular discharge mechanism, and proof that the secretion has defensive potency. We also obtained evidence that the opilionid might produce additional, as yet unknown chemical factors that contribute to its unacceptability to spiders.
A. aculeatus belongs to the suborder Laniatores of the Opiliones.
Quinonoid secretions may be of common occurrence in this suborder, as one
might conclude from a number of chemical investigations (for references, see
Acosta et al., 1993). The
discharge mechanisms of the glands are not precisely the same in the different
quinone-producing Laniatores. However, the mechanisms have one feature in
common, namely that the quinones, upon discharge from the glands, are mixed
with a diluent, in the form of regurgitated enteric fluid. Such preparative
mixing of a glandular defensive product with gut fluid appears to be without
parallel in other animals.
The first Laniatores species in which the glandular discharge mechanism was
described is Vonones sayi (Eisner
et al., 1971). When this animal is disturbed, it promptly emits a
droplet of fluid from the mouth. The fluid does not remain in place, but seeps
almost instantly to the margins of the body along two linear clefts, formed by
the closely apposed bases (coxae) of the first and second legs. The fluid is
thus conveyed in about equal measure to each of the two gland openings, beside
which it accumulates, forming two distinct droplets. The animal then injects a
small amount of its glandular quinonoid paste into each droplet, and proceeds
to administer the mixture through brushings of its forelegs. We here present
photos of this discharge mechanism (Fig.
1), to provide a basis for visual comparison of the defensive
emission strategies of A. aculeatus and V. sayi.
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Other species of Laniatores that have been studied mix enteric fluid with
glandular quinone, as does V. sayi, but they do not all administer
the fluid with the forelegs. Rather, in some cases, they allow the fluid to
spread along two specialized channels on their flanks, with the result that
they become laterally coated with the fluid (for references, see
Acosta et al., 1993). As we
here demonstrate, A. aculeatus belongs to the latter category of
Laniatores.
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Materials and methods |
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Chemistry
Secretion was obtained by holding individual A. aculeatus by the
body, or by some of the legs, in forceps, and squeezing them gently until
droplets of the yellow fluid (mixture of enteric liquid and glandular
secretion) appeared at the edge of the carapace. The fluid was then picked up
in glass capillary tubing, weighed, and analyzed. Individuals were squeezed
repeatedly, until no further yellowish fluid appeared on their flanks.
Qualitative analysis was effected by GC-MS and NMR spectrometry. GC-MS data were obtained with a Hewlett-Packard (Palo Alto, CA, USA) 5890 gas chromatograph, coupled to a Hewlett-Packard 5971 mass selective detector [fused-silica capillary column coated with DB-5 stationary phase (25 mx0.25 mm; 0.25 µm film thickness)]. Oven temperature conditions were 60°C for 2 min, increased to 200°C at 5°C min1. Samples were injected with CH2Cl2 as solvent. NMR spectra (1H-NMR, 500 MHz) were obtained in d6-benzene, using a Varian Unity 500 spectrometer.
For quantitative determination of quinone content, fluid samples were mixed with 50 µl CH2Cl2, bearing 1,4-benzoquinone as internal standard, and analyzed by gas chromatography (same column and oven conditions as above) using a Hewlett Packard 5890 gas chromatograph equipped with a flame ionization detector. The relative proportion of the three benzoquinones in the fluid samples was calculated by GC peak comparisons.
The total amount of quinone per sample was calculated as the sum of the net quantities of the three quinones present in the mixture. The quantity of each quinone was calculated from the linear regression equation (r2=0.987) of a calibration curve constructed for 2,5-dimethyl 1,4-benzoquinone. All quinone amounts were therefore expressed as 2,5-dimethyl 1,4-benzoquinone equivalents.
Glands and discharge mechanism
Individual A. aculeatus were variously stimulated, by pinching
either the body or individual appendages with forceps, thereby causing them to
emit their defensive fluid, while the animals were under observation using a
Wild M400 Photomakroscope (Heerbrugg, Switzerland), which provided the
opportunity to record the events photographically.
For scanning electron microscopy, specimens were dehydrated in ethanol and gold-coated.
Irritancy of secretion
We made use of a bioassay that we had developed earlier for the assessment
of irritancy of test substances (Eisner,
1961). The assay is based on the observation that when a droplet
of an irritating chemical is placed on one side or the other of the fifth
abdominal tergite of a decapitated nymph of the cockroach Periplaneta
americana, the animal scratches the site with the hindleg of the side
stimulated. The time interval between application of the sample and scratching
provides a measure of the irritant effectiveness of the sample. Details of
this assay, which we have used for assessment of irritancy of a variety of
natural products, are given elsewhere
(Eisner et al., 1976
). Under
exceptional circumstances, when a sample is especially active, the cockroach
reacts to mere proximity of the test substance rather than only after contact
with it. The liquid effluent of A. aculeatus proved to be active on
near contact.
For our purposes we used last-instar P. americana nymphs, decapitated on the preceding day. Decapitation was effected by ligating the neck and severing the head just anterior to the ligation, thus preventing the animal from bleeding when decapitated.
Tests were effected by collecting a sample of fresh liquid effluent from an A. aculeatus, and bringing this liquid immediately to within close range of one side of the fifth abdominal tergite of the decapitated cockroach. The fluid sample was collected by pinching the body or a leg of the opilionid with forceps, so as to cause it to emit yellow effluent, and taking up the liquid from the animal's flanks, in a glass capillary tube (0.03 mm2 bore). The capillary tube, filled to the brim, was then promptly pointed from a distance of 46 mm at the tergal surface, and the delay to onset of scratching was timed to the nearest second with a foot-operated stopwatch.
Cockroaches were used only once, each with a sample from a separate opilionid. For control purposes, to check whether the oral effluent might itself contain irritant components, samples of liquid were taken up from the edges of the carapace, immediately after an A. aculeatus was stimulated, but before it injected secretion into the fluid. Such fluid, was presented at 46 mm from the tergal surface, in capillary tubes, as were the experimental samples bearing secretion.
Predation tests (ants)
The test with ants involved introducing individual A. aculeatus
into separate Petri dishes (9 cm diameter), each containing eight worker ants
(Formica exsectoides) collected outdoors (Ithaca, Tompkins County,
New York, USA) several hours beforehand. After 30 min exposure to the ants,
the opilionid was removed from the dish and transferred to a small humidified
chamber where it had access to water. After 12 h of such confinement, the
opilionid was subjected to stimulation (pinching of the body and appendages
with forceps), to check whether it still possessed secretory ability.
Predation tests (spiders)
Two series of tests were carried out, one to check on the deterrency of the
effluent of A. aculeatus, the other to determine the acceptability of
the opilionid itself. The tests were done with wolf spiders (Lycosa
ceratiola), captured weeks beforehand in the environs of Lake Placid,
Highland County, Florida, USA, and maintained in individual cages on a
substrate of sand and a diet of mealworms.
To test for the effectiveness of the defensive fluid, individual spiders were offered a nearly full-grown mealworm, such as they had been obtaining at biweekly intervals as a matter of routine, and then, once they had killed the mealworm and had commenced feeding on it, were stimulated by the addition to the surface of the mealworm, directly at the site where the spider had inserted the chelicers, of 2 µl of A. aculeatus effluent (oral fluid plus secretion). The fluid had been taken up in calibrated glass capillary tubes from an opilionid immediately before application of the liquid to the mealworm. The effect of the applied fluid on the spider was noted.
To test for the acceptability of the opilionid itself, a number of A. aculeatus were individually released in the spider cages, and the ensuing events were noted.
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Results |
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Glands and discharge mechanism
A. aculeatus respond consistently when disturbed. When merely
prodded or picked up gently by the body with forceps, they may not respond at
all. But if the body is squeezed, or appendages are pinched, they tend quickly
to activate their defense. Onset of the response is signaled by the
appearance, along the full length of the light-colored channels that demark
the lateral margins of the carapace, of clear enteric fluid (Figs
1C,
2C). Almost immediately
thereafter, the liquid in the channels takes on a yellow appearance, as the
animal injects some of its quinonoid secretion into the fluid
(Fig. 2D). Typically, the fluid
builds up at the posterior end of the channels, there to form two bulging
yellow drops (Fig. 1D). It is
also not uncommon for the oral effluent to be delivered in pulses onto the
channels (Fig. 2E), as
indicated by the discontinuous egress of fluid from between the bases of legs
1 and 2.
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Fig. 5 summarizes the sequence of events that accompany a secretory discharge in A. aculeatus. At the outset, enteric fluid (red) is emitted from he mouth, from where it spreads right and left along the clefts between the coxae of legs 1 and 2. The fluid then spreads upward between legs 1 and 2 onto the lateral channels, passing by the gland openings, and picking up secretion (blue) as it flows posteriorly. The mixed product then accumulates at the posterior end of the channels.
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The photographs illustrate additional details. Note, for instance, that the clefts between the coxae of legs 1 and 2, which convey fluid from the mouth to the gland opening, glisten as a consequence of their wetness following an oral discharge (Fig. 2A). Note also the two dorsal apophyses on the coxa of the second leg (denoted by asterisks in Fig. 4C, and seen also in Fig. 2B). It is possible that these apophyses help guide the oral fluid onto the carapace channels as the fluid is conveyed from the mouth. Fig. 2F,G illustrate a phenomenon repeatedly noted, namely the transference, in the course of normal leg movements, of ejected defensive fluid onto the femur of the hindlegs.
Fig. 3B shows the hindlegs
of a male of A. aculeatus, with its projecting spines. We noted
repeatedly, as have others (Capocasale and
Trezza, 1964), that A. aculeatus attempts to use its
hindlegs as pinching devices when seized by hand, by pushing the femora
together and exerting pressure with the spines. The spines are larger in the
males than in the females (Capocasale and
Trezza, 1964
), raising the possibility that the structures play
yet additional roles.
Irritancy of secretion
Eighteen effluent samples (regurgitant plus secretion) were tested, and in
16 cases the cockroaches responded within 1 s of presentation of the sample.
The two exceptional cockroaches responded within 2 and 4 s, respectively. Only
four samples of pure regurgitant were available for testing, but they proved,
without exception, to be inert (the cockroaches failed to scratch within 60 s
of presentation of the sample).
Predation test (ants)
Only four A. aculeatus were available for testing. The results
were similar with each. No sooner had the opilionid been introduced into the
Petri dish, than the ants attacked (Fig.
3C). They scurried over the body of the opilionid, causing the
latter to `freeze' and to adopt a stilted stance, with its legs straightened
out and the body lifted off the substrate. Events proceeded quickly, but it
was clear in each case that several ants succeeded in clamping their mandibles
onto legs of the opilionid. They did so only within the first minute after
introduction of the opilionid, and they did not long persist in their hold.
The opilionid, in each case, activated its defenses, as indicated by the
visible appearance of yellowish fluid along its flanks (the times, from test
onset to the appearance of the defensive fluid, were recorded as 25, 35, 35
and 50 s). Following such emission the opilionid appeared to be safe from
attack. The ants, as a group, responded instantly to the emission by scurrying
about quickly while showing distinct avoidance of the opilionid, and of the
dabs of the opilionid's effluent that had rubbed off on the dish floor. As
they dashed about, the ants paused frequently to preen, wiping antennae with
the forelegs (Fig. 3D), and
legs against legs.
Neither the ants nor the opilionid suffered ill-effects from the encounters. The ants, after removal of the opilionid from their midst, gradually returned to normal, resuming their ordinary ambulatory pace, and cutting back on the frequency of preening. 12 h following the encounter they seemed all to have recovered. The opilionids showed no signs of injury, even though they had been sprayed by the ants (ants that had seized the opilionid were observed in some cases to flex their abdomen forward beneath their body as they typically do when ejecting their formic acid-containing secretion) and when stimulated by pinching, 12 h after the encounter, produced yellow defensive effluent in what seemed to be normal quantity. The opilionids evidently had not expended their entire glandular reserves in the course of the ant assaults, or incurred a lasting deficit in enteric fluid. If indeed they had depleted their enteric reserves in the course of the assaults, they had evidently reacquired enough liquid by drinking to be able to produce normal amounts of diluent for their quinonoid emissions.
Predation tests (spiders)
Application of A. aculeatus effluent to the site where the spider
had inserted its chelicers into the mealworm had surprisingly little effect.
In five of 15 spiders thus tested, the spiders failed to respond altogether.
They continued to feed on the mealworm, without even momentarily dislodging
their chelicers. In eight of the remaining ten cases, the spiders did
extricate the fangs, but they did so only briefly, for no longer than it took
them to reinsert their fangs at a point adjacent to where the effluent had
been applied. In two cases the spiders did release their hold altogether, and
as they then backed away from the mealworm, proceeded to drag their mouthparts
in the sand. But they undertook such cleansing behavior only briefly, and
within 2 min in one case and 4 min in the other, returned to the mealworm to
finish the meal. In all 15 cases the spiders reduced the mealworms to small
packets of indigestible remains, as lycosids typically do with insect
prey.
The tests with actual A. aculeatus also gave consistent results. Not one of the 13 opilionids offered to individual spiders sustained injury. Six were pounced upon the moment they were introduced into a spider's cage, but the spider released them instantly, by relaxing their hold and enabling the opilionid to walk away. The other seven individuals were also spurned, but after mere palpation. They were not immediately noticed by the spider when released in the spider's cage, but when eventually detected were rejected after being touched briefly with the palps or legs (Fig. 3E,F). All 15 spiders killed and ate the mealworms they were offered as controls after the test.
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Discussion |
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1,4-benzoquinones are now known to be among the principal defensive
substances produced by arthropods, having been isolated from, among others,
millipedes, cockroaches, termites, earwigs, grasshoppers, hemipterans and
carabid, tenebrionid and staphylinid beetles
(Blum, 1981). Their presence in
opilionids appears to be restricted to species of the families Gonyleptidae
(to which A. aculeatus belongs) and Cosmetidae, both members of the
suborder Laniatores (Eisner et al.,
1978
; Roach et al.,
1980
; Holmberg,
1986
; Acosta et al.,
1993
). However, 1,4-benzoquinones are not the exclusive defensive
compounds of the Laniatores. Some Laniatores produce phenols
(Acosta et al., 1993
;
Eisner et al., 1977
;
Duffield et al., 1981
), and one
other, the Uruguayan Parampheres ronae, produces a mixture of vinyl
alkyl ketones (C. Rossini, unpublished observation). The members of the second
principal suborder of the opilionids, the Palpatores, produce an array of
volatile branched-chain alcohols, ketones and aldehydes
(Eisner et al., 1978
;
Ekpa et al., 1985
). The single
species of this suborder known to produce a quinonoid product, secretes
naphthoquinones rather than 1,4-benzoquinones
(Wiemer et al., 1978
).
The strategy of mixing glandular secretion with oral effluent appears to be restricted to the Laniatores. When Estable, Fieser and their associates did the original work on A. aculeatus, they had no evidence that the quinonoid mixture they isolated from this opilionid plays a defensive role. Our demonstration that quinone emission is in fact protective in this opilionid, at least against ants, provides a functional context for the pioneering chemical discovery by these investigators.
A. aculeatus is not alone within the Gonyleptidae in spreading its
defensive fluid along its flanks. The excellent description by Acosta et al.
(1993) indicates that the
gonyleptid Pachyloidellus goliath generates its protective effluent
by mixing secretory product with regurgitant, much as does A.
aculeatus, and that it also routes its effluent along carapace channels.
The animal conveys its regurgitant to the gland openings by way of intercoxal
clefts, as does A. aculeatus, and it has notches adjacent to the
gland openings by which the mixed fluid is guided onto the channels. One is
tempted to predict (in agreement with
Acosta et al., 1993
) that the
Gonyleptidae all mix and route their defensive fluid in this fashion, but the
mechanism could be subject to variation. Thus, while in the gonyleptid
Zygopachylus albomarginis, which secretes a mixture of
1,4-benzoquinones and a phenol, the discharge mechanism appears to be
identical to that in A. aculeatus and P. goliath
(Cokendolpher, 1987
), in
another gonyleptid, Goniosoma spelaeum, the mechanism is modified in
that the animal is capable both of spreading its effluent along carapace
channels and of ejecting its secretion forcibly as a spray. The latter species
is said to have a secondary gland opening that serves specifically for spray
ejection (Gnaspini and Cavalheiro,
1998
). Spray ejection has also been reported for an African
Laniatores, Larifugella natalensis
(Lawrence, 1938
).
Also subject to variation may be the gonyleptids' involvement of the legs
in defense. Both the incidental wetting of legs with effluent, that we noted
in A. aculeatus, and the attempts to inflict pinches with the hind
legs, occur also in some other gonyleptids
(Cokendolpher, 1987;
Gnaspini and Cavalheiro,
1998
). Also noted in other gonyleptids
(Cokendolpher, 1987
) is the
tendency to assume a rigid stance in response to disturbance, with legs
outstretched and body raised, such as A. aculeatus manifested when
attacked by ants.
Opilionids of the second major family within the Laniatores, the
Cosmetidae, appear not to have the carapace channels present in the
Gonyleptidae. Whether they all resort to leg dabbing to administer their
defensive effluent, as does V. sayi
(Eisner et al., 1971) remains
unknown, although it is clear that some species at least, including the
phenol-secreting Cynorta astora, do make use of the dabbing technique
(Eisner et al., 1977
).
The finding that the defensive effluent of A. aculeatus has
irritant potency comes as no surprise. 1,4-Benzoquinones had been shown to be
strongly effective in the Periplaneta scratch test, as might well be
expected, given the widespread occurrence of these compounds in defensive
secretions of arthropods. Interestingly, alkylated 1,4-benzoquinones, such as
are present in opilionid secretions, had been shown to be more effective as
irritants than unsubstituted 1,4-benzoquinone itself
(Peschke and Eisner, 1987),
explaining perhaps why the alkylated forms of these compounds should dominate
in arthropod defensive secretions (Blum,
1981
). The response to the A. aculeatus effluent in the
scratch test was essentially immediate (in 13 of 15 cases), even though the
samples were tested on near contact rather than actual contact, and the
quinones were tested in dilute rather than pure form. Given that the
regurgitant itself proved inactive in the tests, we assume the activity of the
mixed effluent to have been due exclusively to the quinones.
The tests with ants showed clearly that against these predators at least,
the chemical defense of A. aculeatus is strongly effective. While we
would obviously have liked to have had a greater number of the opilionids for
testing, the results were unambiguous. They were also expected.
1,4-Benzoquinones from the secretion of a number of insects, including
cockroaches, carabid beetles and tenebrionid beetles, had been shown to be
highly repellent to ants (Eisner,
1958a,b
;
Peschke and Eisner, 1987
).
Somewhat surprising were the findings with the wolf spiders. These predators seemed minimally inconvenienced by A. aculeatus effluent, even when the fluid was applied directly to their mouthparts. Based on these findings, we had expected the opilionid to be vulnerable to lycosid attack. However, the animals were consistently rejected by the spiders immediately on contact, before they were even prompted to emit effluent, from which we conclude that A. aculeatus contains additional chemical factors, repellent to spiders, but distinct from the quinones that convey protection against such other enemies as ants.
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Acknowledgments |
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