View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Berner Fachhochschule: ARBOR J Chem Ecol (2011) 37:1110–1116 DOI 10.1007/s10886-011-0015-4 Ability of the Oriental Fruit Moth Grapholita molesta (Lepidoptera: Tortricidae) to Detoxify Juglone, the Main Secondary Metabolite of the Non-host Plant Walnut Rafal Piskorski & Simon Ineichen & Silvia Dorn Received: 29 June 2011 /Revised: 30 June 2011 /Accepted: 23 August 2011 /Published online: 8 September 2011 # Springer Science+Business Media, LLC 2011 Abstract Many plant species produce toxic secondary Introduction metabolites that limit attacks by herbivorous insects, and may thereby constrain insect expansion to new hosts. Host range expansion by herbivorous insects is in many Walnut is a host for the codling moth Cydia pomonella, cases limited by plant secondary metabolites. Therefore, which efficiently detoxifies the main walnut defensive adaptation to or tolerance of defensive chemicals by compound juglone (5-hydroxy-1,4-naphthoquinone). The herbivorous species may have important ecological and oriental fruit moth Grapholita molesta, which also belongs agricultural consequences (Louda et al., 1997). In to the tribe Grapholitini, does not feed on walnut. We tested Juglandaceae, the toxicity of juglone (5-hydroxy-1,4- the performance of G. molesta, a highly invasive species, naphthoquinone) and other naphthoquinones to numerous on artificial diets containing juglone at levels mimicking lepidopteran species is well known (Yu, 1987; Thiboldeaux those found in walnut over the growing season. Juglone-fed et al., 1994;Sunetal.,2007). However, a few insect G. molesta survived relatively well to adulthood, but larval species, such as the luna moth Actias luna (Lepidoptera: and adult body weights were reduced, and larval develop- Saturniidae) and the codling moth Cydia pomonella mental time was prolonged in a dose-dependent fashion. (Lepidoptera: Tortricidae), include Juglandaceae in their Chemical analysis of frass from larvae that had been fed a diet. The luna moth and codling moth efficiently metabo- juglone-containing diet suggests that G. molesta reduces lize naphthoquinones even at high concentrations juglone to non-toxic 1,4,5-trihydroxynaphthalene in its gut. (Lindroth, 1989; Piskorski and Dorn, 2011). The luna | downloaded: 5.3.2020 This unexpected tolerance of G. molesta to high levels of moth possesses high levels of quinone reductase activity, juglone may facilitate expansion of the host range beyond an enzyme responsible for naphthoquinone detoxification, the current rosacean fruit trees used by this invasive pest. with highest activity levels measured in midgut micro- somes (Yu, 1987; Lindroth, 1989). The codling moth Key Words Grapholita (=Cydia) molesta . Host range . reduces juglone in the gut and excretes non-toxic 1,4,5- Invasive species . Juglone . Naphthoquinone . trihydroxynaphthalene when it feeds on diets containing Trihydroxynaphthalene . Toxicity juglone (Piskorski and Dorn, 2011). The oriental fruit moth Grapholita (=Cydia) molesta R. Piskorski : S. Ineichen : S. Dorn (*) Busck (Lepidoptera: Tortricidae) belongs to the same tribe Institute of Agricultural Sciences, Applied Entomology, (Grapholitini) as the codling moth, and both species infest ETH Zurich, fruit trees of the Rosaceae family that are devoid of Schmelzbergstrasse 9/LFO, naphthoquinones. Grapholita molesta uses peach as its 8092 Zurich, Switzerland e-mail: [email protected] primary and apple as a secondary host (Rothschild and Vickers, 1991; Piñero and Dorn, 2009), whereas the codling Present Address: moth uses apple as its primary host (Phillips and Barnes, R. Piskorski 1975). The larvae of both species are internal fruit feeders https://doi.org/10.24451/arbor.9145 Innovative Environmental Services (IES) Ltd., Benkenstrasse 260, and are morphologically difficult to distinguish, so that 4108 Witterswil, Switzerland molecular tools may be required to discriminate between source: J Chem Ecol (2011) 37:1110–1116 1111 them (Chen and Dorn, 2009). Despite these similarities, Effect of Juglone on G. molesta Performance First-instar infestation by the highly invasive pest G. molesta has not larvae of G. molesta (N=30–34 per juglone concentration) been reported on Juglandaceae or other plants that contain were placed individually with a fine brush into Petri dishes naphthoquinones, in contrast to the codling moth, which with artificial diet containing juglone or with juglone-free thrives on walnut (Phillips and Barnes, 1975; Piskorski and diet (Piskorski and Dorn, 2011). Insects were allowed to Dorn, 2011). Juglone contents in walnut fluctuate over the develop under controlled conditions with a 16 : 8 hL:D season (Radix et al., 1998; Colaric et al., 2005; Solar et al., cycle at 26/24°C and 60% r.h. Corrugated cardboard strips 2006; Stampar et al., 2006), and survival rates of the were attached to the inner wall of each Petri dish lid after codling moth were high at the lower juglone level and 7 d of feeding to allow for pupation. moderate at the higher level reported for this plant species. Development was monitored daily from day 7 onwards, Concentrations of juglone that were double the maximum i.e., before the larvae were expected to terminate feeding juglone content in walnut were lethal to the codling moth prior to pupation. Larval developmental time was recorded larvae (Piskorski and Dorn, 2011), indicating that detoxifi- as the number of days from introduction of the larva onto cation is limited even in a herbivorous species adapted to the diet until its entry into the cardboard strip for pupation. walnut (Barnes, 1991) and its naphthoquinones. We Pupal developmental time was recorded as the period hypothesized that the oriental fruit moth may be unable to between larval entry into the cardboard strip and adult detoxify juglone, thus limiting its potential to use walnut as emergence, thus including any non-feeding prepupal stage a host. However, by using performance bioassays we the larva may have undergone (Notter-Hausmann and Dorn, showed that G. molesta can survive on a diet containing 2010; Piskorski and Dorn, 2011). Larval survival was juglone, and chemical analysis of larval frass suggests the assessed as the proportion of individuals that entered the ability of this species to reduce this compound in its cardboard strip in relation to those that had been introduced digestive tract. as first-instar larvae. Pupal survival was assessed as the proportion of emerged adults in relation to the number of individuals that had entered the strip (Notter-Hausmann and Methods and Materials Dorn, 2010; Piskorski and Dorn, 2011). To track sublethal effects, the weight of adults was Insects Larvae of the oriental fruit moth were collected recorded upon emergence (Velten et al., 2007; Piskorski and from a peach orchard in Trentino, Italy, in 2007 and reared Dorn, 2011); within 24 h of emergence the adults were in the laboratory under controlled conditions [16 : 8 hL:D killed by freezing, dried for 24 h in a 60°C oven, and cycle at 26/24°C and 60% relative humidity (r.h.)]. Upon weighed. To determine the larval weight gain, fresh weights emergence, the adults were transferred to a paper cylinder of larvae were recorded on the 9th day of feeding (Piskorski with moist cotton (Hern and Dorn, 1999). Neonate larvae and Dorn, 2011), i.e., when all larvae were still feeding and were transferred to Petri dishes and allowed to feed on an none had yet entered the non-feeding prepupal stage. artificial diet as specified below. Corrugated cardboard strips were offered for pupation. Unfed first-instars (24± Fate of Juglone To follow the fate of juglone in insects 12 h-old) were used for bioassays. feeding on the diet that contained juglone at 25 mg/g d.w. the following samples were analyzed: (1) frass, which was Artificial Diet The diet offered to larvae was a soya flour- collected only from larvae (N=8), which successfully based artificial diet devoid of any fruit material, used in entered the pupal stage; (2) larval regurgitant (N=12), mass rearing of the oriental fruit moth in our laboratory collected by gently touching the head with a micropipette (Torriani et al., 2010). Juglone was added at the end of diet and collecting the fluid; (3) larval hemolymph (up to 1 μl preparation; the diet was homogenized and distributed into from each larva; N=12), collected with a micropipette plastic Petri dishes of 55-mm diam to yield a 4-mm thick directly from each insect after cutting its cuticle from head layer (Piskorski and Dorn, 2011). Juglone (purity≥97%; to abdomen on the ventral side; (4) fed late-instars, i.e., Sigma-Aldrich, Buchs, Switzerland) was added to the diet larvae containing filled guts (N=12); (5) starved late-instars at concentrations of 5, 25, and 50 mg/g dry weight (d.w.), (N=6), which were placed for 17 h in Petri dishes, devoid with the first two concentrations corresponding to the of any food, on humid filter paper to empty their gut prior minimum and maximum of the seasonally fluctuating to analysis; (6) pupae (5–7 d-old; N=8); (7) newly emerged juglone content in walnut husks (Radix et al., 1998; adults (N=12) (Piskorski and Dorn, 2011). Samples (2)–(5) Stampar et al., 2006; Piskorski and Dorn, 2011). The diet were collected after a 9-d-feeding period. Samples (1), (2), was prepared directly prior to conducting the bioassay. and (7) (frass, regurgitant, and adults) were taken from Juglone was stable throughout the diet preparation and insects subjected to the performance trial, whereas samples bioassay (Piskorski and Dorn, 2011). (3)–(6) (body tissues) were prepared in a separate trial. 1112 J Chem Ecol (2011) 37:1110–1116 Insects were killed by freezing, freeze-dried, and ground analysis of variance (ANOVA) followed by Tukey’s post before extraction. Samples of each type were pooled. hoc test. The χ2-test was used to test for differences in the All samples were extracted twice with a CHCl3:MeOH number of surviving individuals between treatments; (2:1) mixture (5 ml, or 0.5 ml for regurgitant and survival on the control diet served as baseline value.