Many animals protect themselves from predation with chemicals, both self-made or sequestered from their diet. The potential drivers of the diversity of these chemicals have been long studied, but our knowledge of these chemicals and their acquisition mode is heavily based on specialist herbivores that sequester their defenses. The wood tiger moth (Arctia plantaginis, Linnaeus, 1758) is a well-studied aposematic species, but the nature of its chemical defenses has not been fully described . Here, we report the presence of two methoxypyrazines, 2-sec- butyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine, in the moths’ defensive secretions. By raising larvae on an artificial diet, we confirm, for the first time, that their defensive compounds are produced de novo rather than sequestered from their diet. Pyrazines are known for their defensive function in invertebrates due to their distinctive odor, inducing aversion and facilitating predator learning. While their synthesis has been suspected, it has never previously been experimentally confirmed. Our results highlight the importance of considering de novo synthesis, in addition to sequestration, when studying the defensive capabilities of insects and other invertebrates. Key words: pyrazine, insect, chemical defense Many animals protect themselves from predation using a large vari- to as ‘neck fluid’) and one from the anus. Recent work by Rojas et al. ety of chemicals, both produced by themselves or sequestered from (2017) suggests that these two fluids are targeted toward different their diet. The potential drivers of this diversity have been subjected predator types; neck fluids are particularly effective against birds. to considerable research by evolutionary biologists. However, knowl- Neck fluids are secreted in response to pressure, and, because birds edge of these chemicals and their acquisition mode is heavily based typically attack the head of the moth (pers. obs.), they are likely to on species that rely on their sequestration (e.g., Optiz and Müller come into direct contact with the fluid. Previous research with wild- 2009). Wood tiger moths (Arctia plantaginis, formerly Parasemia caught birds has demonstrated the deterrent effect of this fluid even plantaginis: Rönkä et al. 2016) have been extensively studied due without wing color cues (Rojas et al. 2017). Furthermore, chem- to their wing color polymorphism and are experimental model ical assays found that the neck fluids contain 2-sec-butyl-3-meth- species for the study of predator–prey interactions . Despite this, oxypyrazine (SBMP), which is suggested to underlie the aversive the nature of their chemical defenses has yet to be fully described. reaction seen in bird predators. A. plantaginis is a capital breeder, Their larvae are polyphagous; in Finland alone, they are known to so adults do not feed. Thus, toxin sequestration from the diet could feed on plants from four different genera spanning four families: only take place during the larval stage. Given that the moth popula- Taraxacum, Plantago, Rumex, and Vaccinium (pers. obs.). Unlike tion used in this study was fed predominantly on dandelion, which is other tiger moths (subfamily Arctiinae: Lepidoptera: Erebidae), they not known to contain pyrazines (Schütz et al. 2006), this compound do not sequester pyrrolizidine alkaloids, nor do they appear to uti- is unlikely to have been sequestered from the moths’ diet. However, lize any dietary sources of other well-known defense compounds in populations are sometimes supplemented with lettuce, some culti- the group, such as cardenolides or lichen phenolics. Furthermore, vars of which do contain 3-alkyl-2-methoxypyrazines (Murray and previous studies have shown that A. plantaginis does not seem to Whitfield 1975); therefore, in order to confirm the de novo produc- sequester iridoid glycosides (IGs), although it is capable of feeding tion of methoxypyrazines in this species, the exact diet contents must on plants with high IG content (Lindstedt et al. 2010). be known. Here, we raised wood tiger moths from the same popula- Adult moths release two defensive fluids when threatened: one tion on an artificial diet to test the hypothesis that the methoxypyra- from the cervical gland at the base of the head (henceforth referred zines found in their chemical defenses are not sequestered from their © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact email@example.com Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/28/4939106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 2 diet. We used a solid-phase micro extraction (SPME) technique for detected using selected ion monitoring of ions 124, 138, and 151. sampling, and gas chromatography with mass selective detection Following the findings of Rojas et al. (2017), standards of SBMP (GC/MSD) to detect the presence/absence of methoxypyrazines in were measured in a scan mode and we then chose the targeted ions the neck fluids. based on the ion responses. The chromatograms and mass spectra were evaluated using Agilent Chemstation (v. G1701CA) software and the Wiley 8th edition mass spectral database. Methoxypyrazines Materials and Methods were identified using the ratio of these detected ions from the NIST webbook page (see Supp Material [online only] for electron impact Larvae were taken from six matings of unrelated adults from a labo- mass spectrum of both pyrazines detected) (Stein), as well as by com- ratory stock founded from moths collected in Southern and Central parison with standards of SBMP and 2-isobutyl-3-methoxypyrazine Finland in 2011. Upon hatching, families were split into two treat- (IBMP) (see Supp Material [online only]). To confirm that SBMP and ments: one fed on dandelion (Taraxacum sp.) and one on an arti- IBMP were not being produced in the artificial diet, approximately ficial diet. Dandelion was collected from the area surrounding the 400 mg of food was transferred into glass vials and allowed to sit at University of Jyväskylä, central Finland. Artificial diet was made on room temperature for 1 and 2 h. The headspace was then tested with site, under sterile conditions, and a 250-g batch contained the fol- the same methods described earlier. Additional tests using agar made lowing: 150-ml distilled water, 4.58-g agar, 32.1-g semolina, 8.58- with known concentrations of IBMP confirmed our ability to detect g yeast, 8.3-g wheat germ, and 1.75-g vanderzant vitamin mix. All pyrazines from the medium, if present. individuals were reared under laboratory conditions with an average temperature of 25°C during the day and 15–20°C at night. Upon emergence, all adult moths were stored in a climate cabinet at 4°C. Results Defensive neck fluids of adult moths were sampled between 0 and 10 d after eclosion. Before sampling, all moths were removed Individually analyzed neck fluids from A. plantaginis (both male and from the climate cabinet and sprayed with water. They were then female) from both treatments (N = 3 for each) were found to con- given 1 h to drink and become active. We stimulated fluid release tain IBMP and SBMP (Fig. 1, see Supp Material [online only] for ion by pinching the anterior end of the thorax, behind the head and the chromatograms). Both ranged in concentration from 0.1 to 1 ng/µl. neck, using tweezers. Individual secretions of three males from each SBMP was found in all pooled samples (11 from the artificial diet treatment were collected with 10-µl glass capillaries, and immedi- and 12 from the dandelion diet). IBMP was found in all but three ately transferred to glass vials containing 200-µl NaCl solution (3%). pooled samples: one from the artificial and two from the dandelion Secretions from a further 46 individuals (both male and female) were diet, although this absence may be due to poor sample quality in pooled in groups of two in 15 μl of autoclaved double-distilled H O. these cases. Neither pyrazine was detected from the headspace of Samples from stock individuals were included in the dandelion treat- the artificial diet. ment, as their rearing conditions were identical. Measurement of the pyrazines was done following the methods Discussion of Cai et al. (2007). Pyrazines were extracted from the headspace of fluid samples using SPME fibers (StableFlex 1-cm fibers with Our results confirm that, unlike other well-studied tiger moths, wood Divinylbenzene/Carboxen/Polydimethylsiloxane coating, Sigma- tiger moths can produce at least part of their defensive compounds, Aldrich, Darmstadt, Germany) for 30 min at 37°C. GC/MSD anal- methoxypyrazines, de novo instead of sequestering them from their yses were carried out on an Agilent 6890 series GC system equipped diet. This novel finding has important implications for our under - with a Zebron ZB-5HT Inferno (Phenomenex Inc., Torrance, CA) standing of chemically defended insects. While pyrazines are known column (length 30 m, 0.25 mm I.D. with a film thickness of 0.25 µm) to play an important role in insect defense (Guilford et al. 1987) connected to an Agilent 5973N MSD. Fibers were manually loaded and have been previously described in tiger moths (Rothschild et al. into the injector using a splitless injection mode, and the inlet tem- 1984), experimental tests of their acquisition mode have been lack- perature was set to 260°C. Helium was used as a carrier gas at a ing. Early work on pyrazines often failed to find clear dietary sources constant flow rate of 0.8 ml/min. The oven temperature was pro- of the compounds. Indeed, Rothschild et al. (1984) reported that they grammed as follows: 3 min at 60°C then ramped to 170°C at a rate sometimes detected pyrazines in butterflies whose host plants lacked of 7°C/min and from 170 to 260°C at a rate of 20°C/min and kept them, insinuating a role of de novo synthesis, as did Moore et al. (1990) at that temperature for an additional 5 min. Methoxypyrazines were who further noted that methoxypyrazines were found in the adults of Fig. 1. Results of GC-MS analysis monitoring ions 124, 138, and 151. Methoxypyrazines detected from (A) male-fed artificial diet, (B) male-fed dandelion, and (C) artificial diet (1 h). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/2/28/4939106 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 2 3 some species but not the larvae. This observation was, however, largely or even replace, sequestered defenses with those produced de novo can overlooked because lepidopteran larvae are known to frequently nib- influence the evolutionary trajectories of chemically defended species. ble chemically defended plants, other than their host plants, poten- tially using them as a ‘drug source’ for their own defense (Singer et al. Supplementary Data 2009). Thus, the focus on plant-derived defenses may have led us to underestimate the prevalence of de novo synthesis, and its evolutionary Supplementary data are available at Journal of Insect Science online. implications. Our results support those early findings by Rothschild and Moore, by confirming de novo pyrazine production in an arctiid species. Acknowledgments Pyrazines have long been known for their defensive function in Thanks to Anni Mäkelä and all the greenhouse staff for help with moth rear- insects and other invertebrates due to their distinctive odor, not only ing and to Nathan Morehouse for the artificial food recipe. The comments of inducing aversion (Rothschild et al. 1984, Guilford et al. 1987) but also several anonymous reviewers greatly improved this manuscript. The Center of facilitating predator learning (Rowe and Guilford 1996). Their presence Excellence in Biological Interactions provided funding. in the moths’ neck fluids may explain their antipredator function, as birds react similarly to SBMP within the concentration range reported References Cited from the fluids, even in the absence of color cues or previous experi- Ali, J. G., and A. A. Agrawal. 2012. 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Journal of Insect Science – Oxford University Press
Published: Mar 1, 2018
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