Chrysomelidae: Cassidinae) As Chemically Mediated Defence Against Predators

Faecal shield of the tortoise beetle Plagiometriona aff. ﬂavescens (Chrysomelidae: Cassidinae) as chemically mediated defence against predators

Flavia´ Nogueira-de-Sa´ and Jose´ Roberto Trigo1

Laboratorio´ de Ecologia Qu´ımica, Departamento de Zoologia, Instituto de Biologia, CP 6109, Universidade Estadual de Campinas, 13083-970, Campinas, SP, Brazil (Accepted 16 August 2004)

Abstract: Larvae of Plagiometriona aff. flavescens carry a structure on their back made of faeces and exuviae, called faecal shield, which may protect larvae against natural enemies. Previous investigations suggested that the nature of such protection was chemical. To test if chemicals found in the faecal shield of Plagiometriona aff. flavescens provided defence for larvae, experiments in the field and in the laboratory (using the ant Camponotus crassus,andchicksGallus gallus as model predators) were undertaken. Both field and laboratory experiments showed that live larvae with faecal shields, as well as baits treated with faecal shield extracts, were rejected by predators, confirming the chemical nature of this kind of defence.

Key Words: Aureliana fasciculata, chemical defence, larval defence, natural enemies, predation, Solanaceae

INTRODUCTION also cases in which faecal shields can serve as attractive cues for generalist predators and parasitoids (Muller¨ & Predators and parasitoids represent an important source Hilker 1999, Schaffner & Muller¨ 2001). of mortality for phytophagous insects (Cornell et al. 1998, Some authors have suggested that shields provide Denno et al. 1990, Gratton & Denno 2003, Hawkins et al. physicalprotectiontolarvae(Eisner&Eisner2000,Muller¨ 1997, Keese 1997, Oksanen & Ericson 1987). Natural 2002, Muller¨ & Hilker 2003, Root & Messina 1983). How- enemies of Chrysomelidae (leaf beetles) belong to many ever, others have demonstrated that in different chryso- different groups of organisms or taxa, varying from melids, including another species of the genus Plagio- intracellular parasites such as fungi and bacteria to large metriona, this kind of physical protection does not occur predators like birds and lizards (see review in Nogueira- (Morton & Vencl 1998, Nogueira-de-Sa´ & Trigo 2002, de-Sa´ & Vasconcellos-Neto 2003). Selective pressures Vencl & Morton 1998, Vencl et al. 1999). In fact, shield imposed on these insects by natural enemies probably res- effectiveness was demonstrated to be due to chemical ulted in a diversity of defensive attributes including phys- components of host plant origin (Gomez´ et al. 1999, ical, behavioural and chemical adaptations (Olmstead Morton & Vencl 1998, Muller¨ & Hilker 2003, Vencl et al. 1996, Pasteels et al. 1988, Vencl et al. 1999). Chrysomelid 1999). Therefore, the objective of this study was to verify beetles frequently use enteric products and exuviae as if chemical compounds found in the shields of Plagiometri- protection barriers, especially for their eggs and larvae ona aff. flavescens were able to deter predators from attack- (Olmstead 1994). Larvae of Chrysomelidae belonging to ing larvae, under both field and laboratory conditions. different subfamilies can carry masses of moulted skins and faeces, forming a shield-like structure (Eisner & Eisner 2000, Eisner et al. 1967). Various functions such as METHODS camouflage, protection against radiation and natural enemies, have been attributed to these structures (Muller¨ & Field bioassay – test for the chemical defence by faecal Hilker 2003 and references therein), although there are shield of Plagiometriona aff. flavescens

This experiment was conducted at Parque Estadual 1Corresponding author. Email: [email protected] Intervales, in Sao˜ Paulo State, south-eastern Brazil 190 FLAVIA´ NOGUEIRA DE SA´ AND JOSE´ ROBERTO TRIGO

(24◦12–24◦25S; 48◦03–48◦30W), where Plagiomet- on the larvae by natural enemies (see Nogueira- riona aff. flavescens (Boheman 1855) and its host plant de-Sa´ & Trigo 2002 for more details). Aureliana fasciculata (Vellozo) Sendt. (Solanaceae) occur (2) Experimental: baits were treated topically with shield (see Nogueira-de-Sa´ & Trigo 2002 for more details on the extract of Plagiometriona aff. flavescens, and placed on research area). Voucher specimens were deposited at the unprotected plants. entomologicalcollectionoftheDepartmentofBiodiversity (3) Solvent control: baits were treated topically with and Evolutionary Taxonomy, University of Wroclaw, MeOH (the solvent used to dilute the extract), and Poland (CAS 153/86/1/2004, CAS 153/86/2/2004). placed on protected plants as described in (1). Our goal was to verify the importance of the chemicals (4) Solvent control: baits were treated topically with present in the faecal shield in the protection of Plagiometri- MeOH, and placed on unprotected plants. ona aff. flavescens without the influence of the physical (5) Untreated control: natural baits without the appli- barrier provided by the shield. We used frozen third- cation of any substance, and placed on protected instar larvae of the noctuid moth Spodoptera frugiperda as plants as described in (1). palatable baits, to which chemicals from the shield were (6) Untreated control: natural baits without the appli- topically applied with a 10-µl syringe. The shields were cation of any substance, and placed on unprotected removed with fine forceps from larvae of all five instars of plants. Plagiometriona aff. flavescens and maintained at − 70 ◦C until chemical extraction (maceration in a mixture of CH2Cl2:EtOH:H20 1:1:1, using 10 × volume:weight, in The experiment was carried out with 30 host plant order to extract the majority of compounds). The mixture individuals submitted to the treatments described above was sonicated for 2 min, vortexed at medium speed during six experimental periods of 2 d each. Preliminary for 2 min and then left at 4 ◦C until the aqueous and experiments showed that the period of 2 d was enough to organic layers were completely separated. This extraction expose larvae to predators. The treatment to be applied procedure was repeated once. The aqueous phase was to each of the 30 experimental plants was randomly freeze-dried and the organic phase was evaporated under selected in the beginning of each experimental period. vacuum at 25 ◦C. Although this procedure may eliminate The same host plant individual was submitted to the volatile compounds present in the organic phase, same treatment only once. Therefore, at the end of the six preliminary extractions of shields by other methods (e.g. experimental periods, each host plant had been submitted SPME) and further GC-MS analysis did not show volatile to all treatments once, resulting in 30 replicates for each compounds. Both phases were diluted and recombined in treatment. The experiments were carried out during the MeOH. We calculated the amount of extract to be applied summer, when the populations of the cassidines and on Spodoptera frugiperda larvae following the ratio faecal predators of herbivorous insects were large (Nogueira- shield fresh weight/Plagiometriona aff. flavescens larval de-Sa´ & Vasconcellos-Neto 2003). At the end of each body fresh weight, which was 1.033 (n = 18). As the experimental period, we recorded if baits had disappeared ratio was close to 1, extracts obtained from portions of or if they were intact. For analysis, baits that had shields with the same weight of the Spodoptera frugiperda disappeared were scored as one and intact baits were larva were applied to the frozen bait (diluted in 30 µl scored as zero. Baits that had been partially attacked MeOH). (recorded as bite marks) but not completely consumed We used potted Aureliana fasciculata plants grown in the were discarded from analysis. Cochran’s Q test was used greenhouse in order to minimize cues due to herbivory to compare the proportions of disappeared and intact (which might attract natural enemies) and to standardize baits among treatments with a post hoc test for multiple host size to a 1 m height. Host plants were transferred comparisons (Zar 1999, p. 268). to the field and placed along a trail at 10-m intervals. Soon after the application of shield extract or solvent to the baits, these were glued on host plant leaves with a Laboratory bioassays drop of paper glue (C & C CapitaniTM). A single bait was glued, on the abaxial surface of a randomly chosen leaf of Carpenter ants, Camponotus crassus (Formicidae: each plant. Because experimental plants were of similar Myrmicinae) were used as an invertebrate predator size, the baits were glued at similar heights as well. The model, and chicks, Gallus gallus (Galliformes: treatments were as follows: Phasianidae), as a vertebrate model. Two kinds of biossays were carried out, with the capturing frequencies by these predators as variable. In one bioassay, we (1) Experimental: baits were treated topically with shield used live 3rd or 4th instar larvae with and without extract of Plagiometriona aff. flavescens, and placed on shields, removed experimentally with fine forceps. In a plants protected by cages. This prevented the attack second bioassay we used individual baits (frozen larvae Faecal shield defence in tortoise beetle 191 of Spodoptera frugiperda) submitted to one of the three the colony. This procedure was applied to check if the chemical treatments described below. experimental larva or bait was not consumed because the ants rejected it or if the colony was no longer hungry. (1) Experimental: baits were treated topically with faecal When the ants consumed the second control within 24 h, shield extract of Plagiometiona aff. flavescens (see the behavioural category ‘not captured’ was confirmed. above). When the second control was not consumed, that trial (2) Solvent control: baits were treated topically with was rejected from further analysis. MeOH. In both larva and bait bioassays frequencies of captured (3) Untreated control: baits without chemical treatment. and not captured organisms were compared using a G test forcontingencytable,followedbysubdividedcontingency table as a post hoc test when needed (Zar 1999, p. 504). Camponotus crassus bioassay Gallus gallus bioassay We collected Camponotus crassus colonies at Estaçao˜ Experimental de Mogi Guaçu (Fazenda Campininha), One-day-old chicks were obtained from a commercial in Sao˜ Paulo State, south-eastern Brazil (22◦18S, hatchery and taken to the laboratory, where they were 47◦10W). In the laboratory, the colonies were kept in kept together for 1 wk. The animals were maintained a plastic container (20 × 20 × 5 cm) which walls were under25 ◦C,naturalphotophaseandfedwithcommercial treated with FluonTM to prevent the ants from escaping. corn-based food and water ad libitum.After7d,the We placed a test tube (20 cm length, 2 cm diameter, birds were placed singly in experimental cages 2 cm height) with damp cotton in the bottom covered (30 × 30 × 40 cm) and were visually isolated from each with red plastic film, in the container where the ants other. On their 9th, 10th and 11th day of life, chicks nested. The nest container was linked to a foraging arena were deprived of food for 2 h, and then trained to (anotherplasticcontainerof16cmdiameter,5cmheight) become familiar with the palatable larvae of mealworm, where food was supplied and where the experiments were Tenebrio molitor (Coleoptera: Tenebrionidae), offered in carried out. The colonies were fed daily with a honey- Petri dishes. They were given 2 min to accept or reject the water solution; and a frozen 5th or 6th instar larvae of mealworm. There were one or two training sessions every Spodoptera frugiperda was supplied weekly. We conducted day.Anindividualbirdthatnevermanagedtofind/eatthe the bioassays in the foraging arena to avoid defensive larvae was not used in the experiment. behaviours of the ants that might occur due to proximity On the day following the third training day we to the nesting chamber. Colonies of Camponotus crassus conducted the double quantification bioassay. Chicks were kept under 14/10 h, LD photoperiod, at 24 ◦C. were deprived of food for 2 h, and then offered the A double quantification bioassay was carried out as first control larva: a mealworm to check if the chick follows. Two days before the beginning of every bioassay was hungry. When the first control was eaten, one a larva of Spodoptera frugiperda was offered to the colony as experimental larva or bait was offered, depending on the described above, as the first control. This indicated if the bioassay (see above), and the bird response in relation to colony was hungry and the ability to feed on the tested the offered organism was recorded as: (1) captured, when prey. When the larva was not consumed, the colony was the larva or bait was consumed, and (2) not captured, considered unable to attack prey and was not used in when the larva or bait was pecked and released. When the experiments. When the first control was consumed, the chick did not try to prey upon or attack the larva we started the bioassay 48 h after that was offered, by or bait, the result for that individual was not considered offering an individual larva or bait, depending on the (as in Chai 1990). In the response category (2), after bioassay, as mentioned above. The treatment was defined offering the experimental larvae or bait, we offered to randomly. Larvae or baits were placed on a fresh leaf of the chick another mealworm (second control). When the Aureliana fasciculata with the petiole dipped into a vial bird ignored or rejected the second control, that trial with water to prevent wilting. This system was kept in the was rejected for further analysis. Each individual chick centre of the foraging arena. One day later, the behaviour was never used in more than one trial. The frequency of the ants towards the test organisms was recorded. of captured and non-captured larvae or baits in each The recorded behavioural categories were: (1) captured, bioassay were compared as in the ant experiments. when the experimental larva or bait disappeared from host plant leaf after 24 h, or (2) not captured, when ants left the experimental larva or bait intact after 24 h. In RESULTS this case, we offered a second Spodoptera frugiperda control larva, which was placed on an Aureliana fasciculata leaf In the field bioassay, the disappearance of Spodoptera as well, after removing the experimental larva or bait of frugiperda was significantly different among the 192 FLAVIA´ NOGUEIRA DE SA´ AND JOSE´ ROBERTO TRIGO

than upon solvent controls (91.7%, n = 24) or untreated controls (88.2%, n = 17) (G = 14.3, df = 2, P < 0.001). Some chicks that pecked and released live larvae with shields and baits treated with shield extract tried to clean their beaks by pressing them onto the cage ﬂoor.

DISCUSSION

Earlyinourstudies,wedemonstratedthatthefaecalshield of Plagiometriona aff. flavescens larvae has a role in defence in the field (Nogueira-de-Sa´ & Trigo 2002). According to the results presented in the previous paper, shields are important in protecting larvae, and comparisons of larvae mortality without shields or with natural shields substituted by an artificial non-noxious shield showed that such protection was not physical, suggesting that the protection must be due to chemicals in this structure. In thiswork we have tested and confirmed thissuggestionfor Figure 1. Disappearance of baits submitted to three different treatments Plagiometriona aff. flavescens larvae in field and laboratory to test for chemical protection of shield of Plagiometriona aff. flavescens in the field. Different letters mean significant difference at the 5% level. conditions. Sample size = 30 for each treatment. Results of field bioassays testing predation upon baits treated with shield extracts agree with the chemical protection suggested by Nogueira-de-Sa´ & Trigo (2002). treatments (Q = 50.6, df = 5, P < 0.01; Figure 1). On Other authors also suggested that chemicals from the protected plants, disappearance was always lower faecal shield, which may be derived from the host compared with unprotected ones, but there was no plant, were responsible for the protection of cassidine significant difference among treatments. On unprotected larvae against predators (Evans et al. 2000, Gomez´ et al. plants the disappearance of baits treated with shield 1999, Morton & Vencl 1998, Muller¨ & Hilker 2003, extract was significantly lower than solvent controls and Vencl et al. 1999). However, in those studies no field untreated controls. bioassays testing the chemical nature of protection in In the laboratory bioassays using ants, capturing leaf beetles against natural predators were conducted frequencies of live larvae with shields (6.67%, n = 30) or on their host plants. When undertaking this experiment, without shields (13.8%, n = 29) did not differ significantly arthropods were collected on the vegetation along the (GYates corr = 0.23, df = 1, P = 0.63). However, the studied trail using a sweep net; most potential predators frequency of predation upon baits treated with shield werespiders,andtherewereafewantsandheteropterans. extract was significantly lower (40%, n = 20) than upon On three occasions we observed the spiders Misumenops solvent controls (90%, n = 20) or untreated controls sp. (Thomisidae), Achaearanea tesselata and Theridion (86.7%, n = 15) (G = 14.6, df = 2, P < 0.001). Observing calcynatum (Theridiidae) preying upon cassidine larva ant behaviour in relation to the offered prey, it was (Nogueira-de-Sa´ 2004). verified that ants tried to drag the prey away at first Ant bioassays showed that the capturing percentage of contact with the larvae or the baits. However, when live larvae was lower than that of baits treated with shield larvaewithshieldsorbaitstreatedwithshieldextractwere extracts. We verified that Plagiometriona aff. flavescens offered, some ants groomed their antennae and cleaned larvae have another defensive mechanism against contaminated mouthparts by rubbing them against the Camponotus crassus ants, namely chemical camouflage, substrate. This behaviour is described in the literature as a in which larvae matched the pattern of cuticular typical reaction to chemical repellents (Eisner et al. 1967, hydrocarbons of its host plant (Nogueira-de-Sa´ 2004). Morton & Vencl 1998). This kind of defence strategy was first verified for larvae In the laboratory bioassays using chicks, the frequency of the ithomiine butterfly Mechanitis polymnia that feed ofpredationuponlivelarvaewithshieldswassignificantly on Solanum species (Portugal 2001). Because chemical lower than larvae without them (57.1% n = 21 and cues are so important for ants, such alternative defensive 89.5% n = 19, respectively) (GYates corr = 3.92, df = 1, mechanisms could be the main factor responsible for the P = 0.048). Nevertheless, pecked and released larvae higher survivorship of live larvae – even without shields – alwaysdied.Thefrequencyofpredationuponbaitstreated when compared with baits treated with chemicals from with shield extract was significantly lower (50%, n = 28) shields in the ant bioassays. Another explanation is Faecal shield defence in tortoise beetle 193 the lack of defensive behaviours in baits. Chrysomelidae is part of the Ph.D. thesis of FNS in the Curso de Pos-´ larvae rely on some behavioural characteristics described Graduaçao˜ em Ecologia, UNICAMP. as responses to natural enemies (see Jolivet & Verma 2002 and references therein). Specifically, moving the body, fortifying defensive compounds and waving the shield LITERATURE CITED after a disturbance to cover stimulated areas of the body were observed for larvae of different Chrysomelidae BEGOSSI, A. & BENSON, W. W. 1988. Host plants and defense mecha- species (Blum 1994, Olmstead 1996). nisms in Oedionychina (Alticinae). Pp. 57–71 in Jolivet, P. H., Although chicks preyed upon larvae with shields less Petitpierre, E. & Hsiao, T. (eds.). Biology of Chrysomelidae. Kluwer frequently than larvae without it, those prey always died Academic Publishers, Dordrecht. after being pecked, independent of the presence of the BLUM, M. S. 1994. Antipredator devices in larvae of the Chrysomelidae: shield. 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