Bulletin of Entomological Research (2004) 94, 283–289 DOI: 10.1079/BER2004299

Effectiveness of the defence mechanism of the turnip sawfly, rosae (: ), against predation by lizards

L. Vlieger1, P.M. Brakefield1 and C. Müller1, 2 * 1Institute of Biology, Leiden University, PO Box 9516, NL-2300 RA Leiden, The Netherlands: 2Institut für Biowissenschaften, Universität Würzburg, Julius-von-Sachs-Platz 3, D-97082 Würzburg, Germany

Abstract

The turnip sawfly, Athalia rosae Linnaeus, is a pest on cruciferous crops. Larvae sequester secondary plant compounds, namely glucosinolates, in their haemolymph. When attacked, their integument is easily disrupted and a droplet of haemolymph is exuded (‘easy bleeding’). This has been shown to be an effective, chemical-based, defence against invertebrate predators. The efficiency of this proposed defence was tested against a vertebrate predator, using groups of the iguanid lizard Anolis carolinensis Voigt as a model predator. Caterpillars of Pieris rapae Linnaeus and Pieris brassicae Linnaeus served as control prey species that do not sequester glucosinolates. Lizards attacked far fewer sawfly larvae than pierid caterpillars. Several of the sawfly larvae were rejected after an initial attack, demonstrating unpalatability to the lizards, while the Pieris larvae were not rejected. However, P. rapae larvae topically treated with extracts of haemolymph of A. rosae had no deterrent effect on the lizards and no avoidance learning occurred over a period of two weeks. Adult sawflies do not easy bleed but have glucosinolates carried over from the larval stage. Lizards attacked them at a higher rate than larvae and they were never rejected. The results suggest that for the defensive effectiveness of the pest sawfly species against vertebrates the chemical cue is not necessarily sufficient. Movement and colour may be important additional factors triggering the behaviour of vertebrate predators.

Introduction Linnaeus (Hymenoptera: Tenthredinidae), against a vertebrate predator. The black larvae are very effective in Many herbivorous are specialized on plants with storing glucosinolates from various host plant species of the deterrent secondary compounds, and several of these , and concentrate them in their haemolymph specialists are able to sequester the plant compounds for more than ten-fold in comparison to their host plant (Müller their own defence against predators (e.g. Nishida & Fukami, et al., 2001). Athalia rosae can be destructive to cultivated 1990; Bowers, 1992; Trigo, 2000). Often, unpalatability is vegetables within the Brassicaceae (Benson, 1950). signalled by a conspicuous warning coloration, called Glucosinolates are characteristic secondary compounds aposematism (Guilford, 1990). This study focuses on the of Brassicaceae and form an effective defence system against defence of the herbivorous turnip sawfly, Athalia rosae generalist aquatic and terrestrial herbivores by reducing leaf palatability (Louda & Mole, 1991; Newman et al., 1992; Giamoustaris & Mithen, 1995). Glucosinolates and their * Author for correspondence hydrolysis products also show fungicidal, bactericidal, Fax: + 49 / 931 888 62 35 nematocidal and allelopathic properties (Chew, 1988; Fahey E-mail: [email protected] et al., 2001 and references therein). Several species, 284 L. Vlieger et al. however, are specialized on glucosinolate-containing plant Materials and methods species and even use these compounds as feeding and/or Lizard predators oviposition stimulants (Nielsen, 1988; Chew, 1995; Schoonhoven et al., 1998; Renwick, 2002), or for Eighteen specimens of A. carolinensis were obtained a sequestration (Weber et al., 1986; Müller et al., 2001; Aliabadi year before the experiments started. They were kept in six et al., 2002). Upon harassment, larvae of A. rosae exude cages (dimensions: 46 × 32 × 57.5 or 51 cm, length × breadth glucosinolate-containing haemolymph in a droplet through × height) in a climate cell (31/25°C day/night, 70% relative a readily rupturing integument. This phenomenon has been humidity, 12/12 h light/dark). Lizards were painted with described as ‘easy bleeding’, and occurs in larvae of several numbers to distinguish individuals. Every cage contained tenthredinid sawfly species (Heads & Lawton, 1985; Heads, one male and two female lizards, a substrate of bark chips 1986; Boevé & Schaffner, 2003). The haemolymph of A. rosae (premium reptibark, Zoomed Laboratories Inc., Canada), a has been shown to be deterrent against ants, Myrmica rubra maize plant, Zea mays L. (Poaceae), several bamboo perches, Linnaeus (Hymenoptera: Formicidae), even when diluted and a shallow Petri dish with water (diameter 90 mm), ten-fold or more (Schaffner et al., 1994; Müller et al., 2002). renewed daily. The cage was misted twice daily. Lizards Predatory wasps, Vespula vulgaris Linnaeus (Hymenoptera: were fed buffalo worms, Alphitobius laevigatus Fabricius Vespidae), rejected sawfly larvae after an initial attack and (Coleoptera: Tenebrionidae) dusted with calcium or vitamin also avoided palatable prey, such as caterpillars of Pieris supplement, sometimes complemented with live crickets, rapae Linnaeus (Lepidoptera: Pieridae), when these were Gryllus bimaculatus De Geer (Orthoptera: Gryllidae). treated with haemolymph of A. rosae (Müller & Brakefield, 2003). Thus, the defensive mechanism of the easy bleeding Prey species against invertebrates is clearly based on the chemistry. The black sawfly larvae were also avoided by chicks, Gallus Athalia rosae, P. rapae and P. brassicae were kept in culture gallus Linnaeus (Aves: Phasianidae), mainly due to warning using a stock of A. rosae established from a field collection in coloration (Ohara et al., 1993). The acceptance of A. rosae to Germany, and pierid eggs obtained from the University of another vertebrate was tested to gain more insight into the Wageningen. Insects were reared on S. alba plants at 21°C, defence mechanisms of the sawfly against this predator 70% relative humidity and 16/8 h light/dark. Larvae of group. Both larvae and adults were used since the orange comparable size were used (A. rosae fifth instar, Pieris spp. and black adults of A. rosae do not exhibit ‘easy bleeding’ but second to third instar). The two pierid species served as still contain the glucosinolates sequestered by the larvae in functional controls that do not show ‘easy bleeding’ and do their haemolymph albeit in a five-fold lower concentration not sequester glucosinolates, in contrast to A. rosae. (Müller et al., 2001). In this study, A. rosae were reared on white mustard, Sinapis Feeding assays with larvae of different prey species alba L. (Brassicaceae), from which they sequester the glucosinolate sinalbin (p-hydroxybenzylglucosinolate) (Müller Groups of lizards were offered one of the prey species et al., 2001). Caterpillars of Pieris brassicae Linnaeus daily in their cages for one week. The species of prey was (Lepidoptera: Pieridae) and P. rapae were reared on the same changed on a weekly basis for two consecutive periods of host plant species and used for comparison of the effectiveness three weeks each (table 1). Each group (1–3) received prey and mechanism of defence. According to Aplin et al. (1975), species in a different order. A group consisted of two cages pupae of P. brassicae contain glucosinolates and their and thus six lizards. Prey larvae were starved for 1 h prior to hydrolysis products, isothiocyanates, the latter also found in P. introduction into the cages to induce more mobility. They rapae. However, no glucosinolates could be found in larvae of were offered in a small Petri dish (diameter 25 mm) that was either species in a more recent study (Müller et al., 2003). While placed within a larger Petri dish (diameter 90 mm). The outer larvae of P. brassicae are conspicuously coloured (yellowish- dish was filled with water to prevent prey from escaping. green with black stripes), those of P. rapae are green. Five prey items were presented in each feeding trial. To investigate the palatability of A. rosae, larvae of this The lizard cages were visually isolated from one another. sawfly species as well as control caterpillars of P. rapae and P. Two cages (one group) were observed simultaneously for 1 h brassicae were offered for consecutive weeks to groups of the after introduction of prey from behind a one-way mirror. lizard Anolis carolinensis Voigt (Reptilia: Iguanidae), a Attacks and the fate of the larvae (consumption or rejection) generalist insectivore (Sexton, 1964). Lizards have been were noted. Rejected larvae were recovered at the end of the recorded as predators of sawflies (Benson, 1950) and observations and kept with food for 24 h to observe whether Lepidoptera (Sexton, 1964), and their role as selective agents they survived the attack. If lizards refused to feed during the on evolution of unpalatability discussed controversially experiment they were offered some larvae of A. laevigatus (Odendaal et al., 1987). Anolis carolinensis is a small, diurnal afterwards to test for their feeding activity. Statistical lizard from the south-eastern part of the USA and the analyses used the Chi square-test for homogeneity except Caribbean area (Roughgarden, 1995; Lyytinen et al., 2003). It with expected frequencies below five when the Fisher’s was captive reared in the laboratory, and used here as a exact test was performed. model vertebrate predator. To investigate whether the haemolymph of A. rosae larvae, after predator attack often Feeding assays with haemolymph-coated P. rapae larvae emitted as ‘easy bleeding’ is deterrent to lizards, palatable prey was treated with extracts of haemolymph and offered Sawfly larvae often show ‘easy bleeding’ when touched for a period of two weeks. Finally, to evaluate the defensive by predators. To test whether the sawfly haemolymph was effectiveness of different developmental stages of A. rosae deterrent, the lizards were offered P. rapae larvae coated with that do or do not evoke ‘easy bleeding’ adult turnip sawflies an extract of either A. rosae haemolymph (Athalia-treated) or were offered to lizards. P. rapae haemolymph (Pieris-treated). Two groups of lizards Defence mechanism of sawfly against lizards 285

Table 1. The number of larvae of Athalia rosae, Pieris rapae and P. brassicae offered, eaten and rejected by groups 1–3 of the lizard Anolis carolinensis. Group Larvae First period (weeks 1–3) Second period (weeks 4–6) Offered Attacked Eaten Rejected Offered Attacked Eaten Rejected Week 1 Week 4 1 P. rapae 40 19 19 0 25 14 14 0 2 P. brassicae 45 13 13 0 40 15 15 0 3 A. rosae 600 00401 01 Week 2 Week 5 1 A. rosae 50 2 1 1 50 7 6 1 2 P. rapae 25 19 19 0 10 7 7 0 3 P. brassicae 500 00500 00 Week 3 Week 6 1 P. brassicae 50 22 22 0 50 35 34 1 2 A. rosae 50 7 2 5 40 15 (1) 13 (1) 2 (0) 3 P. rapae – – – – 50 22 22 0

Group 3 was not offered any P. rapae larvae during the first period. Values in brackets are the results excluding data of one individual female lizard. were tested. Group A daily received Athalia-treated P. rapae number of attacks comparable to the other two groups. larvae in the first week and Pieris-treated P. rapae larvae in the During the third week no P. rapae could be offered to this second week, group B received the prey items in the reverse group due to shortage of larvae. Therefore, it was excluded order. Prior to the experiment, haemolymph had been from statistical analyses. collected subsequently from larvae reared on S. alba by The prey species were attacked in different frequencies piercing the integument with a needle without damaging the (table 1). The attack rate on P. rapae larvae was significantly gut. Haemolymph was collected in glass capillaries, higher than on P. brassicae (P < 0.05), and both pierid species transferred into vials (2.5 ml), and immediately frozen at were attacked significantly more than A. rosae (P < 0.001 for –80°C. Each larva was only milked once. After lyophilization, both) (fig. 1). As the lizard groups received the prey species haemolymph was suspended in ethanol, ground with a in different orders, order had no influence on the attack rate. spatula, vortexed for 60 s, centrifuged for 2 min, and the One female lizard from group 2 was responsible for most supernatant transferred to a new vial. Extraction was done of the consumptions and rejections of A. rosae during week 6, three times. Ethanol was evaporated under a low air stream. first rejecting (see below) two larvae in two days, then eating Equivalents of 6 µl fresh haemolymph were re-dissolved in 3 12 in the following three days. In attacks of all other trials, at µl ethanol, which is sufficient to cover a P. rapae larva and least two lizards were involved. corresponds to the amount that could typically be collected Prey offered during the second period were in most cases from a single A. rosae larva (fifth instar, similar size). Solvent attacked more than in the first period. This was significant was allowed to evaporate under a low air stream and freshly for group 1 attacking P. brassicae and for group 2 attacking A. treated larvae offered to the lizards. rosae (P < 0.01 in both cases). However, when excluding the For this experiment, 21 lizards in eight cages (containing data from the female lizard mentioned above, the number of one male and one or two females) were used, including attacks on A. rosae was not significantly different between some newly acquired individuals. All lizards were familiar P. rapae with as prey at that time. Observations were done as ab c described above. A group consisted of four cages with lizards. One cage from each group (A and B) was observed simultaneously in order to spread offering of the two 50 treatments equally over a day. 40

Feeding assays with adult A. rosae 30 Six lizards in two cages were offered adult A. rosae daily 20 for a period of two weeks. A Petri dish with lid (diameter Attacked larvae (%) 10 50 mm) containing six sawflies was introduced to each cage, the lid removed and the lizard behaviour observed for 30 0 min as described above. Athalia Pieris Pieris rosae rapae brassicae (190) (100) (185) Results Fig. 1. Percentages of larvae attacked (= eaten + rejected) by Attacks on larvae of different prey species Anolis carolinensis lizards belonging to groups 1 and 2. The number of each larval species offered is given in brackets below The three groups of lizards responded differently to the the columns. Bars with different lower case letters are offered prey (table 1). Group 3 did not attack any prey until significantly different at P < 0.001 (ab and ac) or P < 0.05 (bc) the fourth week, and only during the sixth week was the (Chi square-test for homogeneity). 286 L. Vlieger et al. the two periods for group 2. Compared to group 1, group 2 attacked a higher frequency of P. rapae and A. rosae larvae throughout the whole experimental period, but this was abab only significant for the attack on P. rapae during the second 50 period (P < 0.01). Compared to group 2, group 1 attacked 40 significantly more P. brassicae larvae during the second period (P < 0.01). 30 The tendency to attack prey more during the second period was similar for all groups. There was no overall 20 tendency for one group of lizards to attack prey more than

Consumed larvae (%) 10 the other group, independent of larval species. Therefore it was considered justified to pool the data for the two periods 0 and for the two groups. All lizards were, in general, hungry (37) (50) (17) (21) as they always accepted A. laevigatus larvae offered after Group 1 Group 2 trials in which no prey were eaten. Attacked prey could either be eaten or rejected. When Fig. 2. The percentages of haemolymph-treated Pieris rapae rejecting prey, lizards typically dropped the larva larvae consumed by the lizard Anolis carolinensis. Pieris rapae immediately after grasping it, without further mastication, were either treated with haemolymph of Athalia rosae (black and rubbed the sides of their beak against the substrate on bars) or P. rapae (white bars). The number of offered larvae per the spot (beak-wiping). Of the offered pierid larvae, only one treatment is given in brackets below the columns. The left column of a pair represents data from week 1, the right column P. brassicae was rejected. This larva was quite large (fourth from week 2. Bars with different lower case letters are instar) and after 6 min of trying to swallow it, the lizard spat significantly different at P < 0.01 (Chi square-test for it out. Although the number of sawfly larvae attacked was homogeneity). N = 100 were offered per week and per group. lower, a larger proportion of A. rosae was rejected. The two groups (1 and 2) of lizards did not differ significantly in the proportion of prey consumed and rejected per period for each of the three larvae species (Fisher’s exact Table 2. The number of Athalia rosae adults offered, attacked, test). Thus, the data are pooled for the groups. But, in eaten and rejected by Anolis carolinensis lizards of two cages. For contrast to the data on attacks, there was a significant comparison, the pooled data for A. rosae larvae are included. difference between periods. During the first period, A. rosae was rejected significantly more than either pierid species (P < Offered Attacked Eaten Rejected 0.001 for each, Fisher’s exact test; with no significant Adults week 1 48 27 27 0 difference between the pierids). During the second period week 2 48 31 31 0 there was no difference in the lizard rejection behaviour pooled 96 58 58 0 towards A. rosae and P. rapae even when data from the female Larvae 190 31 22 9 lizard noted above were excluded (Fisher’s exact test). In total, nine A. rosae larvae were rejected by six different lizards. Three larvae could not be found after the observational period so their fate remained unknown. Of the remaining larvae two died and four survived. pooled. In comparison to the larvae (feeding assay with larvae), the adults were attacked significantly more (P < 0.001), and eaten or rejected in significantly different Attacks on haemolymph-coated P. rapae proportions (P < 0.001; after Bonferroni correction) (table 2). Both A. rosae-treated and P. rapae-treated larvae were attacked by the lizards throughout the experimental period Discussion in comparable fashion. During both weeks group A consumed significantly more larvae than group B, Lizards attacked larvae of the pest species A. rosae irrespective of treatment (P < 0.01 for the first week, P < significantly less often than the pierid larvae (fig. 1). Several 0.001 for the second week) (fig. 2). None of the A. rosae- different prey traits could be responsible for the lizard treated larvae were rejected, although beak-wipe behaviour behaviour. Athalia rosae larvae move less than those of either was observed on three occasions near the end of the second Pieris species, and thus might present a less strong visual week following consumption. One of the P. rapae-treated stimulus to A. carolinensis. Lizards are known to be highly larvae was rejected. This larva had crawled out of the dish visual predators, depending on movement to detect prey and was picked up together with some wood chips. The (Burghardt, 1964; Curio, 1970; Goodman, 1971; Fleishman, lizard took it to the back wall of the cage, where it beak- 1986, 1992). The rate of attack was highest on P. rapae. wiped several times, thereafter dropping the larva which Different attack rates on the two pierid species had been survived the attack. observed before (Aplin et al., 1975) and could be due to the hairiness of the P. brassicae larvae. Several authors have described avoidance of hairy food by vertebrate predators, A. rosae adults such as birds (e.g. Whelan et al., 1989; Hilker & Köpf, 1994). Two-thirds of the adult sawflies were attacked and eaten Warning coloration might also have played a role; the dark within 5 min after introduction into the cages. No rejections colour of the A. rosae larvae could have been a more were observed (table 2). The data did not differ significantly aposematic signal than the yellow and black of P. brassicae, between the two weeks (Fisher’s exact test), and so were both being more warningly than the uniform green larvae of Defence mechanism of sawfly against lizards 287

P. rapae. However, the lizards had had no experience with larvae even in a manifold dilution (Schaffner et al., 1994; either prey before the experiments. At the beginning of the Müller et al., 2001) and wasps avoided P. rapae caterpillars experiments they immediately attacked only rarely sawfly (which are usually accepted as food) when these were larvae, making the aposematism hypothesis less likely. coated with sawfly haemolymph (Müller & Brakefield, Additionally, A. rosae larvae were rejected significantly 2003). However, in the latter experiment, fresh, not further more often than the pierid caterpillars. This rejection was processed haemolymph was applied. immediate; lizards dropped the larvae without further Adult A. rosae were attacked more rapidly by the lizards mastication and beak-wiped. Beak-wiping behaviour has and in higher numbers emphasizing the crucial importance been noted for A. carolinensis (Sexton, 1960, 1964; Stanger- of movement for prey detection by the lizards. Movement Hall et al., 2001; Sword, 2001) and other lizard species seems to overrule colour as well as chemistry. Thus the (Boyden, 1976) as a clear indicator of prey unpalatability. bright orange and black pattern of the adults, which could Thus, it appears that the sawfly larvae were also chemically be viewed as warning coloration, together with defended. Unpalatable prey can be recognized by lizards glucosinolates carried over from the larval feeding, was using taste buds on their tongue (Schwenk, 1995), a apparently not sufficiently deterrent (table 2). This contrast perception which can be reinforced by the prey colour, with the larvae could be due to the fact that adults do not leading to association between the two stimuli (Sexton, 1960, easy bleed, and perhaps the glucosinolate concentrations in 1964; Sexton et al., 1966; Shafir & Roughgarden, 1994; Sword, adults fall below the detection threshold of the lizards. It 1999, 2001; Sword et al., 2000; Stanger-Hall et al., 2001). was not observed whether the lizards would reject adult A. However, the rate of attacks on A. rosae increased during rosae after some (long) time lag from commencing to take the second period and fewer rejections occurred. Most them, but this seems unlikely since the consumption rate noteworthy was the single female lizard in group 2 that first remained unaltered and no rejections occurred over the rejected two A. rosae larvae but then ate twelve on the course of two weeks. subsequent three days without any sign of rejection In summary, these observations demonstrate that the behaviour. Thus, at least some individual lizards may be defence mechanism of the larvae of the pest species A. rosae able to overcome their initial taste aversion. known to be effective against invertebrate predators can also No comparable rejection to that of A. rosae was observed be effective against vertebrates. The larvae were frequently for the pierid larvae. The one P. brassicae larva that was rejected and some survived lizard attacks. However, while eventually rejected was only dropped after a 6 min struggle, against invertebrate predators the deterrent principle of the and was clearly too large to be swallowed whole. Our sawfly haemolymph overrules other cues, this is not the case finding of higher acceptance of both Pieris species than A. for lizards. The results suggest that only the combination of rosae could be correlated to the fact that the larvae of P. several strategies such as low mobility, warning coloration, brassicae and P. rapae do not sequester bitter-tasting easy bleeding, and distastefulness offers an effective glucosinolates (Müller et al., 2003) unlike the sawfly larvae protection against this vertebrate predator. (Müller et al., 2001). However, although A. rosae larvae were rejected, it was Acknowledgements not possible to demonstrate with the coating experiments that the active chemically deterrent principle of the sawfly Niels Würzer and Mariël Lavrijsen are acknowledged for haemolymph is sufficient on its own. Both A. rosae-treated assisting in the lizards’ maintenance, rearing plants and and P. rapae-treated P. rapae larvae were eaten, and other practical help; Bas Zwaan for help with statistical consumption rates tended to increase during the second analysis. The authors are also grateful to the University of week, regardless of treatment (fig. 2). Lizards might not have Wageningen for Pieris rapae eggs. This research was in part yet re-associated the appearance of P. rapae with supported by the European Community’s Improving unpalatability. The lizards had eaten P. rapae before in the Human Potential Programme under contract HPRN-CT- first experiment and found them palatable. Since the visual 1999–00054, INCHECO. system is of paramount importance in A. carolinensis (Burghardt, 1964; Fleishman, 1986), contradictory chemical References information might need more time to overrule established preferences. Indeed, near the end of the second week some Aliabadi, A., Renwick, J.A.A. & Whitman, D.W. (2002) lizards in group B started wiping their beaks after Sequestration of glucosinolates by Harlequin bug consuming A. rosae-treated pierid larvae but there was no Murcantia histrionica. Journal of Chemical Ecology 28, learning of avoidance. Any unpalatable compounds of A. 1749–1762. rosae haemolymph might also not have been extracted by the Aplin, R.T., D‘Arcy Ward, R. & Rothschild, M. (1975) procedure or were diluted too much in the solutions. 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