Chemical egg defense in Photuris firefly “femmes fatales

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  Female Photuris fireflies contain defensive chemicals of two types. They sequester steroidal pyrones (lucibufagins) from male fireflies of the genus Photinus that they eat, and themselves produce the defensive betaine N-methylquinolinium
  Chemoecology 9:177–185 (1999)0937–7409 / 99 / 040177-09 $1.50 + 0.20© Birkha¨user Verlag, Basel, 1999 Chemical egg defense in  Photuris  firefly ‘‘femmes fatales’’* Andre´s Gonza´lez 1 , James F. Hare 2 and Thomas Eisner 1 1 Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853, USA 2 Department of Zoology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada Summary.  Female  Photuris  fireflies contain defensivechemicals of two types. They sequester steroidal py-rones (lucibufagins) from male fireflies of the genus Photinus  that they eat, and themselves produce thedefensive betaine  N  -methylquinolinium 2-carboxylate.Chemical analyses of   Photuris  eggs showed that femalesthat fed on  Photinus  males endow their eggs with bothlucibufagin and the betaine, while those that did notfeed on  Photinus  lay eggs that contain betaine, butvirtually no lucibufagin.  Photuris  females collected inthe field during the  Photinus  flight season laid eggs thatinvariably contained betaine, but lucibufagin only attimes. Predation experiments showed that  Photuris  eggsare essentially unacceptable to larvae of a coccinellidbeetle ( Harmonia axyridis ) and an ant ( Leptothoraxlongispinosus ), but moderately acceptable to an earwig( Forficula auricularia ). When applied experimentally topalatable insect eggs, lucibufagin proved deterrent tothese three predators, while the betaine proved deter-rent to the ant and coccinellid larva only. Both types of defensive compound decreased egg predation in thefield. By endowing their eggs with both exogenous andendogenous chemicals,  Photuris  females are essentially‘‘maximizing their options’’ – when feeding on  Photi  - nus , their eggs are doubly protected, but they are notentirely defenseless when the females are unable toprocure lucibufagin. Key words.  Photuris  –   Photinus ignitus  – Coleoptera – Lampyridae – lucibufagin – steroidal pyrone – betaine – egg predation Introduction Fireflies (Coleoptera: Lampyridae) are known to bedistasteful to vertebrate and invertebrate predators(Lloyd 1973; Blum & Sannasi 1974; Sydow & Lloyd1975; Eisner  et al  . 1978, 1997). They are even lethal tocertain lizards and frogs (Knight  et al  . 1999).Fireflies of the genus  Photinus  ( P .  ignitus ,  P .  pyralis , P .  marginellus ) have been shown to contain mixtures of steroidal pyrones (lucibufagins † ), which are, at least inpart, responsible for their distastefulness and toxicity(Eisner  et al  . 1978, 1997; Meinwald  et al  . 1979; Goetz  etal  . 1979, 1981).Female fireflies of the genus  Photuris , appropriatelyknown as ‘‘femmes fatales’’, also contain lucibufagin(Fig. 1A), but their content is minimal unless they fedon  Photinus  (Eisner  et al  . 1997; Gonza´lez  et al  . 1999a).Specifically, female  Photuris  respond to the flash signalsof   Photinus  males by emitting light pulses imitative of those of   Photinus  females, thereby luring the  Photinus males to within graspable range. They then pounceupon the males and devour them, leaving only legs andbits of wings uneaten (Lloyd 1965). The  Photuris  fe-males sequester the defensive lucibufagin from the Photinus  prey, thereby obtaining protection against ene-mies such as spiders (Eisner  et al  . 1997). Fig. 1  Structures of the main components of the lucibufagin mixturein  Photuris  fireflies ( A ), and of the betaine N-methylquinolinium2-carboxylate ( B ) Correspondence to : T. Eisner, e-mail:* Paper 163 in the series ‘‘Defense Mechanisms of Arthropods’’.Paper 162 is Eisner T and Aneshansley DJ (1999) Spray aiming in thebombardier beetle: Photographic evidence. Proc Natl Acad Sci USA96:9705–9709.  † We refer to the compounds herein in the collective, as lucibufagin.  A. Gonza´lez, J. F. Hare and T. Eisner C HEMOECOLOGY 178 In addition to the lucibufagin acquired by the adultfemales,  Photuris  larvae and adults of both sexes con-tain the betaine N-methylquinolinium 2-carboxylate(herein referred to as the betaine; Fig. 1B) (Gonza´lez  etal  . 1999b), which appears to be biosynthesized endoge-nously (Gonza´lez  et al  . unpubl.).In this study, we investigate the defensive chemicalendowment of   Photuris  eggs, and how such endowmentaffects the eggs’ vulnerability to certain insect preda-tors. Specifically, we show that ( i  )  Photuris  females thatfed upon  Photinus  males endow their eggs with part of the lucibufagin they acquire; ( ii  )  Photuris  females alsoendow their eggs with the betaine, and they do sowhether they fed on  Photinus  or not; ( iii  ) field-collected Photuris  females and their eggs invariably contain thebetaine, but they may or may not contain lucibufagin;( i   )  Photuris  eggs are distasteful to some predators,irrespective of their lucibufagin content; and (  ) bothtypes of defensive chemical, the lucibufagin and thebetaine, show anti-insectan potency when applied ex-perimentally to palatable insect eggs, and contribute tothe chemical protection of   Photuris  eggs in the field. Materials and methods All fireflies were collected in the vicinity of Ithaca, Tompkins Co.,New York, USA. All  Photinus  were identified as  P .  ignitus . They werecollected in the field at night, and kept live individually in plasticvials, with access to water only.The  Photuris  were identified as  P .   ersicolor , but will be referredto as  Photuris  throughout this paper, since there is some question asto whether  P .   ersicolor  is in fact a complex of sibling species (Eisner et al  . 1997).The  Photuris  were either field-collected or laboratory-reared. Thefield-collected individuals (females only) were captured during the  P . ignitus  flight season, at the same sites as  P .  ignitus . These  Photuris females were assumed to have mated and were kept unfed in Petridishes (4.8 cm diameter), where they eventually laid eggs. Upon theirnatural death, the females were weighed and stored frozen for chem-ical analysis.The laboratory-reared  Photuris  (males and females) were raisedfrom field-collected larvae following procedures described elsewhere(Eisner  et al  . 1997; Gonza´lez  et al  . 1999a). The larvae were collectedin their late instars, on soil, at night in early fall, and kept in the darkat 10°C until May. They were then transferred to soil-filled chambersto induce pupation (24°C, 16L:8D photoperiod), thus ensuring thatadult emergence (June–mid July) coincided with the time of   P .  ignitus availability. Some  Photuris  larvae were frozen upon collection andstored for chemical analysis.Upon emergence, laboratory-raised  Photuris  adults were keptunfed (with access to water only) in individual Petri dishes (4.8 cmdiameter) under natural lighting conditions. The females were matedby enclosing them individually with single laboratory-reared  Photuris males, shortly before dusk, in humidified plastic cages (18 × 13 cmbase, 10 cm height). The pairs were checked every 5 min for 3 h.Mating typically took place within the first hour of enclosure, andlasted 7–15 min. After the partners spontaneously uncoupled, theywere returned to their individual dishes. Females that failed to matewere re-paired on subsequent evenings with a different male eachevening. Of the 164 females tested, 102 (62%) were observed to mate,and were used thereafter in the experimental feedings. (  A  )   Experimental feedings About half of the mated, laboratory-reared females (N = 53) wereoffered freshly collected  P .  ignitus  males. The  P .  ignitus  were intro-duced singly into the Petri dishes that housed the  Photuris  females.After a female consumed an entire male, usually overnight, a second P .  ignitus  male was introduced. After consuming the second prey, the Photuris  female was transferred to a new Petri dish where she laideggs. Control  Photuris  females (N = 49) were kept unfed, withoutaccess to  P .  ignitus  at any time. (  B   )   Egg collection Eggs were collected from  Photinus -fed and  Photinus -unfed labora-tory-raised females [herein called ( + ) and ( − )  Photuris  eggs, respec-tively], and from field-collected females. Females usually laid eggs inclusters, shortly before dying. Upon collection, the eggs were counted,weighed, and either stored frozen for chemical analysis, or kept onmoistened filter paper for egg predation experiments. A few femaleslaid a second egg cluster after the first had been collected. Thesesecond clusters were analyzed separately, and the values were aver-aged with those of the first cluster to obtain a single data point perfemale (first and second clusters of individual females showed similarvalues). (  C   )   Chemistry Egg clusters were homogenized in water / acetonitrile (19:1; 100   l),extracted while sonicated for 30 min, and centrifuged. The superna-tant was transferred to a 200   l vial insert, and subjected to quantita-tive HPLC analysis. Photuris  larvae, females and males were ground in water / acetoni-trile (14:1; 700   l), extracted overnight, centrifuged, and re-extractedwith 300   l water (3 h). The two extracts were combined, filteredthrough a 45   m membrane filter, and analyzed by HPLC.Quantitative HPLC analyses of lucibufagins and the betainewere performed with a Hewlett Packard 1090 Series II HPLC system,using a BDS Hypersil C-18 column (25 cm × 4.6 mm, 5   m; KeystoneScientific Inc.). The column was eluted with 5% acetonitrile in waterfor an initial 10 min, increasing the amount of acetonitrile to 30%over the next 30 min. Injection volume was 25   l, the flow was 1ml / min, and the analyses were monitored at   = 300 and 325 nm forquantification of the individual lucibufagins and the betaine, respec-tively. A pure sample of the lucibufagin 3- O -acetyl-5  ,11  -dihy-droxy-12-oxobufalin, isolated from  P .  ignitus  (Gonza´lez  et al  . 1999a),and a synthetic sample of the betaine (prepared according to Quast &Schmitt 1970), were used to construct the calibration curves for theHPLC analysis, using coumarin as internal standard. The curvescorrelated the net amounts of both the lucibufagin and betainestandards with the ratios of the chromatographic peak areas of eachcompound and that of the internal standard. The regression coeffi-cients for the calibration curves used in the analysis of   Photuris  eggswere 0.992 and 0.995 for the lucibufagin and the betaine standards,respectively. The corresponding coefficients used in the analysis of  Photuris  adults and larvae were 0.992 and 0.999. Since the lucibufaginin  Photuris  is present as a mixture of related compounds (Gonza´lez  etal  . 1999a), the total quantity of lucibufagin was calculated as the sumof individual lucibufagin components. (  D  )   Egg predation experiments We conducted field and laboratory egg predation experiments. In thelaboratory we used three insect predators: larvae of the coccinellidbeetle  Harmonia axyridis  (Coleoptera: Coccinellidae), colonies of theant  Leptothorax longispinosus  (Hymenoptera: Formicidae), and  Forfi - cula auricularia  earwigs (Dermaptera: Forficulidae). H  .  axyridis . This coccinellid is an exotic species that has becomeestablished in much of the eastern United States (Hoebeke & Wheeler1996). Both larva and adult are known to be voracious predators of small invertebrate prey (Hodek & Honek 1996). We used 3 rd and 4 th instar larvae from a laboratory colony established from adults col-lected in the vicinity of Ithaca, NY. The colony was kept in agreenhouse on alfalfa plants infested with the aphid  Acyrthosiphon pisum , which constituted the only food source for the beetle. Furtherdetails on the maintainance of the colony are given elsewhere (Rossini et al  ., 1999). Prior to the predation experiments the larvae werestarved for 48 h, during which they had access to water only.  Chemical defense in firefly eggs 179Vol. 9, 1999 Leptothorax longispinosus . This is a native ant species known toconsume insect prey in the laboratory (Hare & Eisner 1993). It occursthroughout eastern Canada and the northeastern United States(Creighton 1950). Although it is a polydomous ant (Alloway  et al  .1982), a group of workers and their queen(s) collected from a singlehickory nut were considered to be a colony. The ants were collectedat two different locations: Ithaca, NY, and Mississauga (Ontario,Canada). Unless otherwise indicated, we used colonies collected nearIthaca. The colonies were kept in plastic culture dishes (15 cmdiameter, 4 cm height) and fed weekly on mealworms, 10% honey-wa-ter, and a diet developed by Bhatkar and Whitcomb (1970). Oneweek before conducting the tests, the ants were deprived of food, andgiven water only. Colonies collected in Mississauga were maintainedin identical fashion, except that fruit flies were given instead of mealworms. F  .  auricularia . This earwig is also an introduced species, and it isnow widely distributed throughout southern Canada and the north-ern United States. It feeds on small prey, decaying animal matter, andplants (Crumb  et al  . 1941). The earwigs were collected near Ithaca,NY and in Sharon, VT. Some specimens were tested shortly aftercapture. These were kept unfed until testing, on sand in individualplastic cages (11 × 11 cm base, 4 cm height). Earwigs used in latertests were kept in the laboratory in communal plastic cages (18 × 13cm base, 10 cm height) and maintained on freshly cut-up mealworms(larvae of   Tenebrio molitor ). They were deprived of food for twoweeks prior to testing.1. Predation on ( + ) and ( − )  Photuris  eggs a .  Laboratory experiments .  H  .  axyridis  larvae were placed individu-ally in Petri dishes (4.8 cm diameter) lined with moistened filter paper.Each larva was offered a choice between ( + ) and ( − )  Photuris  eggs(5 eggs / category), and the number of uneaten eggs was scored at 2, 4,6 and 24 h. L .  longispinosus  colonies were offered single clusters of 9–12 Photuris  eggs [either ( + ) or ( − )]. Eggs were scored as consumedwhen they were either taken to the nest or eaten  in situ  by workers.Records of egg consumption were made at 10 min intervals duringthe first hour, and at 2, 24, 48, and 72 h. This experiment wasconducted at Brandon University, Manitoba (Canada), using antscollected in Mississauga and  Photuris  eggs from Ithaca.Freshly collected  F  .  auricularia  earwigs were each given a singleoffering of 5  Photuris  eggs [( + ) or ( − )] placed on wax papersquares. The experiment was conducted in Petri dishes (9.5 cmdiameter) lined with moistened filter paper, and checked at 1, 2, 4,and 24 h for missing eggs. b .  Field experiment . This experiment was conducted in late Julyon the outskirts of Ithaca, using ( + ) and ( − ) Photuris eggs freshlyobtained in the laboratory. Egg clusters (10–24 eggs / cluster) fromindividual females were placed at the center of soil-filled Petri dishes(4.8 cm diameter), and taken to the edge of a small pond, where Photuris  fireflies occur naturally. The Petri dishes were placed inscooped-out depressions in the soil, so that their soil contents wereflush with the ground. A plastic overhead shield protected the dishesfrom rain. The eggs were tested in pairs of ( + ) and ( − )  Photuris  eggclusters. The two dishes containing the clusters of a pair were spaced1 m apart, and their relative positions were switched every two days.The distance between neighboring pairs was at least 7 m. The numberof missing eggs was counted every 2 days until day 10, at which timethe  Photuris  larvae began hatching.2. Predation on lucibufagin- and betaine-treated food items( Utetheisa  eggs)We used eggs from a laboratory culture of an arctiid moth,  Utetheisaornatrix , as palatable food items. In nature,  U  .  ornatrix  eggs aredistasteful to predators by virtue of pyrrolizidine alkaloids of dietarysrcin they contain (Eisner & Meinwald 1995, and references therein).We maintain a colony of   U  .  ornatrix  on a pinto bean diet devoid of pyrrolizidine alkaloids (Miller  et al  . 1976), which provides us with asource of alkaloid-free eggs that have been shown to be acceptable toinsect predators (Dussourd  et al  . 1988; Hare & Eisner 1993). Toobtain such eggs, mated females were confined to containers linedwith wax paper, upon which they readily laid their egg clusters.The egg clusters (9–12 eggs / cluster) were treated by topicaladdition of a pure lucibufagin (2  -acetoxy-5  ,11  -dihydroxy 12-oxobufalin) that had been isolated from  Photuris  fireflies (Gonza´lez  etal  . 1999a), or by addition of the betaine (synthesized in accord withQuast & Schmitt 1970). The chemicals were administered in methanolsolution (2.0   g /  l) with a micropipette, at a dosage of 0.5   l / egg.Control eggs were treated by addition of methanol only (0.5   l / egg).While applying the compounds, it was noted that some solutioninevitably spilled from the eggs onto the underlying wax paper.Therefore, to estimate the actual amount of lucibufagin and betaineapplied to the eggs, we treated additional egg clusters and analyzedthem by HPLC. These analyses showed that the quantity of lucib-ufagin and betaine actually added to the eggs was in the range of 0.2–0.6   g / egg. a .  Laboratory experiments . These predation experiments wereconducted as were those with ( + ) and ( − )  Photuris  eggs.  H  .  axyridis larvae were offered a choice of two  U  .  ornatrix  egg clusters, onetreated with either lucibufagin or betaine, the other with solvent. Theexperiments were checked every 20 min for 3 h. Larvae that did notfeed after 3 h were not tallied.  L .  longispinosus  colonies were offeredsingle egg clusters treated with lucibufagin, the betaine, or solvent,and the number of eggs consumed or taken to the nest was scored at10 min intervals for the first hour, and then at 2 and 24 h.  F  . auricularia  earwigs were individually offered a choice of two eggclusters (one control, one treated with either lucibufagin or betaine),and egg predation was scored hourly for 5 h. b .  Field experiment . This test was conducted in August, immedi-ately following the field experiment with ( + ) and ( − )  Photuris  eggs,at the same field site. The eggs were of three kinds, betaine-treated,lucibufagin-treated, and controls. They were tested in groups of threeegg clusters (one cluster per treatment in every triad, 11–20 eggs / clus-ter). The three clusters forming a triad were placed, equidistant to oneanother, in a single sand-filled Petri dish (9.5 cm diameter). Neighbor-ing dishes were placed at distances of at least 7 m from one another.The number of missing eggs was scored at 2-day intervals for 10 days.Midway through the experiment all uneaten eggs were replaced byfreshly collected, equally-treated ones, since  U  .  ornatrix  eggs hatch atage of 5 days. (  E   )   Statistics All values are given as mean  SE. Data from all laboratory preda-tion experiments were expressed as proportion of eggs consumed, andwere subjected to the arc sine transformation for proportions(Snedecor & Cochran 1989). The transformed data were analyzed byrepeated-measures analyses of variance, with time taken as the re-peated-measures variable. Unless otherwise indicated, the Mann-Withney test was used in all two-sample comparisons. Otherstatistical procedures are specified where used. Results and conclusions (  A  )   Experimental feedings and   (  B   )   egg collection The laboratory-raised  Photuris  females eagerly as-saulted and devoured the two  P .  ignitus  males they wereoffered (only one of 53 females took a single  P .  ignitus ).Fed and control females did not differ as to lifespan(fed: 16  1; control: 17  1 days, P = 0.37) or bodymass at death (fed: 65  3; control: 63  3 mg, P = 0.33).Not all  Photuris  females laid eggs: only 25 (of 49)control and 34 (of 53)  P .  ignitus -fed laboratory-raisedfemales, and 13 (of 29) field-collected females, laid eggsin quantities sufficient for chemical analysis or preda-tion experiments. A comparison between fed and con-trol females showed no differences in the probabilitythat a female would lay eggs in the laboratory (G-test,  A. Gonza´lez, J. F. Hare and T. Eisner C HEMOECOLOGY 180 Fig. 2  Lucibufagin (solid bars) and betaine (open bars) in ( − ) and( + )  Photuris  eggs [( − ) eggs: N = 12 clusters; ( + ) eggs: N = 14clusters]. ( + ) Eggs contained more lucibufagin than ( − ) eggs (P  0.001), but the betaine endowment was equal (P = 0.94). Error barsrepresent standard errors Fig. 3 A  – Frequency distribution of lucibufagin content in eggs of field-collected  Photuris  females.  B  – Correlation between the lucib-ufagin content in field-collected  Photuris  females and their eggs(Spearman’s rank correlation test,   = 0.738, P  0.005; N = 13) P = 0.12), in the number of eggs laid (fed: 26  6;control: 33  7 eggs / female, P = 0.11), or in the meanegg mass (fed: 0.16  0.01; control: 0.19  0.02 mg,P = 0.14). (  C   )   Chemistry 1. ( + ) and ( − )  Photuris  eggsHPLC analyses of ( + )  Photuris  eggs from the experi-mental feedings showed that fed females endow theireggs with part of the total lucibufagin they sequesterfrom  P .  ignitus  (Fig. 2). Most ( − ) egg clusters werefree of lucibufagin, although some contained traceamounts of the chemicals (range 0.006–0.04   g / egg).This result was not unexpected, inasmuch as  Photuris females had been shown to contain some lucibufagineven if they had no access to  P .  ignitus  (Gonza´lez  et al  .1999a). Furthermore, analyses of ( + ) and ( − )  Pho - turis  eggs showed that the females allocate the betaineto their eggs, in amounts that do not depend onwhether they fed on  P .  ignitus  (Fig. 2).2. Field-collected females and their eggsThese females were collected as adults, and thereforehad the opportunity to feed on  P .  ignitus  under naturalconditions. Analysis by HPLC showed that both theeggs laid by these females and the bodies themselves of the females contained lucibufagin (N = 13 females, 1egg cluster / female), although the amounts were highlyvariable. Three females did not contain detectable levelsof lucibufagin, and the lucibufagin net amounts in theremaining females ranged from 2 to 63   g. Likewise,there was variability in the lucibufagin concentration inthe eggs of these females (the concentration in the eggscorrelated positively with that in the body of themother) (Fig. 3A and 3B). Five egg clusters containedlucibufagin at levels below 0.04   g / egg, which could beexpected for eggs from females that had no access to  P . ignitus  [( − ) eggs, previous section]. Another three clus-ters contained 0.40–0.60   g lucibufagin / egg, an amountcomparable to that in eggs of females that ate two  P . ignitus  males [( + ) eggs, previous section]. The remain-ing five egg clusters contained intermediate lucibufaginlevels (range 0.13–0.23   g / egg), such as one mightexpect from eggs laid by females that fed on a single  P . ignitus .Field-collected females and eggs also contained thebetaine. HPLC analyses showed that all females andegg clusters contained detectable levels of the com-pound. The females contained a betaine net amount of 55  11   g, and the eggs contained a betaine level of 0.45  0.05   g / egg, which is equal to the betaine con-tent of eggs from laboratory-reared females (previoussection) (P = 0.23).Interestingly, while there was a 5-fold enrichment inthe concentration of lucibufagin in the eggs when com-pared to the concentration in the females, no compara-ble enrichment was noted for the betaine. Thelucibufagin concentration in the eggs of the field-col-lected females that contained the chemicals was 1.0  0.3   g / mg, while the concentration in the femalesthemselves was 0.2  0.1   g / mg (Wilcoxon signed-ranktest, P  0.02, N = 10). The betaine concentration in theeggs was 1.8  0.3   g / mg, while that in females was1.3  0.3   g / mg (Wilcoxon signed-rank test, P  0.4,N = 13).  Chemical defense in firefly eggs 181Vol. 9, 1999 Fig. 4  Betaine net amounts ( A ) and concentration ( B ) in  Photuris larvae, males and females. Betaine concentration in  Photuris  eggs ( B )was included for comparison. Different letters above bars indicatesignificant differences in post-ANOVA Fisher’s pairwise comparisons(individual error rate 0.05). Error bars give standard errors (larvae,males and females: N = 10; eggs: N = 26 clusters) Fig. 5  Predation rates on ( − ) (open bars) and ( + ) (solid bars) Photuris  egg clusters in laboratory experiments with ( A )  L . longispinosus  ants [( − ) eggs: N = 7 clusters; ( + ) eggs: N = 6 clus-ters], and ( B )  F  .  auricularia  earwigs [( − ) eggs: N = 9 clusters; ( + )eggs: N = 8 clusters]. Error bars represent standard errors 3. Betaine in  Photuris  larvae, and adult males andfemalesIn addition to our analyses of the betaine contents of  Photuris  eggs, we determined by HPLC the betainecontents of   Photuris  larvae, and of adult males andfemales. The larvae were field-collected, and the femalesand males were laboratory-raised from field-collectedlarvae. Photuris  larvae contained about four times theamount of betaine of adults (Fig. 4). Although rela-tively little is known about the predation ecology of  Photuris  larvae (Lloyd 1973; Sivinski 1981), these re-sults suggest that the betaine may also serve as achemical defense for the larvae. The betaine net amountin females was higher than that in males, although therewas no gender difference in the concentration of thecompound (Fig. 4A and 4B). The concentration inlaboratory-raised females was equal to that in field-col-lected females (previous section) (P = 0.72). (  D  )   Egg predation experiments 1. Predation on ( + ) and ( − )  Photuris  eggs a .  Laboratory experiments . Both ( + ) and ( − )  Photuris eggs were decidedly unpalatable to  H  .  axyridis  larvae.No eggs from either category were eaten within 24 h(N = 5 larvae). The eggs were then removed, and fivealkaloid-free  U  .  ornatrix  eggs were offered in theirplace, to ensure that it was not for lack of hunger thatthe  H  .  axyridis  rejected the  Photuris  eggs. The  U  . ornatrix  eggs were all consumed within 2–24 h.Likewise, ( + ) and ( − )  Photuris  eggs were equallydistasteful to  L .  longispinosus  ants (P = 0.92) (Fig. 5A).No eggs were consumed or taken to the nests within 24h, and only few eggs from each category were con-sumed within 48 and 72 h. Previous studies had shownthat under comparable experimental conditions  L . longispinosus  consume palatable eggs within the firstfew hours after presentation (Hare & Eisner, 1993).Our third predator,  F  .  auricularia , consumed someeggs from both the ( + ) and ( − ) categories (Fig. 5B).Although ( + ) eggs were subjected to slightly lowerpredation rates, the difference was not significant (P = 0.27). On several occasions the earwigs were observedseizing an egg in their mandibles and dropping itpromptly, then engaging in mouth-dragging behavioragainst the paper substrate. This behavior was observedat least once in 9 of the 17 earwigs tested, and itsincidence was equal in tests with ( + ) (4 out of 8 trials)and ( − ) eggs (5 out of 9 trials). Interestingly, some
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