Not just the sum of its parts: geographic variation and non-additive effects of pyrazines in the chemical defence of an aposematic moth

Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within-and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-methoxypyrazine) are volatiles that deter bird attacks. We expected the variation to reflect populations’ predation pressures and early-life conditions. To understand the role of the methoxypyrazines, we experimentally manipulated synthetic SBMP and IBMP and tested the birds’ reactions. We found a considerable variation in methoxypyrazine amounts and composition, both from wild-caught and laboratory-raised male moths. In agreement with the “cost of defence” hypothesis, the moths raised in the laboratory had a higher amount of pyrazines. We found that SBMP is more effective at higher concentrations and that IBMP is more effective only in combination with SBMP and at lower concentrations. Our results fit findings from the wild: the amount of SBMP was higher in the populations with higher predation pressure. Altogether, this suggests that, regarding pyrazine concentration, more is not always better, and highlights the importance of testing the efficacy of chemical defence and its components with relevant predators, rather than relying only on results from chemical analyses.

1987, Guilford et al. 1987). Pyrazine has a distinctive smell and a disgusting taste that has 72 4 been shown to help predators learn the association between a warning signal and a secondary 73 defence (Rowe & Guilford, 1996). 74 Here we investigated whether different populations of the wood tiger moth, Arctia  86 We tested whether the efficacy of chemical defences can be extrapolated from their 87 composition, using European and Caucasian wood tiger moth populations both wild-caught 88 and raised in the laboratory. The wood tiger moth, Arctia plantaginis, is a chemically 89 defended, warningly-coloured species. The major components of this moth's defences, the 90 methoxypyrazines SBMP (2-sec-butyl-3-methoxypyrazine) and IBMP (2-isobutyl-3-91 methoxypyrazine), are synthesised de novo and secreted as reflex blood in response to attacks 92 by avian predators. First, we investigated within-and between-population variation in the 93 amount and composition of methoxypyrazines from wood tiger moths collected in Estonia, 94 Finland, Scotland and Georgia. We expected variation in chemical defences to reflect  are also known to be monomorphic; for example, in Northern Scotland and in Estonia, the 120 6 hindwings of adult males are either yellow or white, respectively (Hegna et al., 2015).  and misted with water daily. The only exception to this was the Georgian population, whose 144 diet in the laboratory, besides Taraxacum sp., was supplemented with Plantago sp. and 145 Rumex sp.. Tubs were cleaned daily as needed and uneaten food was replaced. Upon 146 pupation, individuals were kept individually in vials at 25°C until eclosion.

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In all cases, the protocol for sampling the thoracic fluids was the same and followed the   blend of SBMP and IBMP was also made such that each dilution (0.05, 0.1, 0.5, and 1 ng/µl) 194 was the total additive concentration of the two pyrazines combined. These dilutions were then 195 refrigerated at ~4°C for no more than one month before use in the experiment. 196 We used blue tits (Cyanistes caeruleus) as a model predator to test their response to the pure

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A total of 79 blue tits were used to measure bird responses to pure pyrazines. Each bird was 223 used in the experiment only once and was assigned a single treatment. Birds were first trained 224 to eat water-soaked oats before use in the assay. Each assay consisted of five trials. In the 225 first trial, birds were offered water-soaked oats to ensure they were motivated to feed and, in 226 the last trial, birds were again offered water-soaked oats to discard satiation. During trials 2,3 227 and 4 each bird was presented with 3 oats per trial on a small white dish. Each oat was 228 covered with 8µl of either water (as a control treatment) or one of the pure pyrazine 229 treatments. Therefore, only trials 2, 3, and 4 are used in the analysis. In each trial we recorded 230 hesitation time (measured as time in seconds from seeing the oat to pecking/eating the first 231 oat), the proportion of the oats eaten (to the nearest 10%), beak cleaning (a disgust behaviour 232 measured as the number of bouts where the bird wiped its beak against a surface such as the 233 perch), drinking (the number of times the bird drank water, which is a behaviour that can 234 increase in response to distasteful food), and trial duration (from the time the oats were seen 235 by the bird until they were consumedor max 300 seconds if some of the oats remained). All 236 trials were video recorded using a hole at the top of the experimental enclosure.    After hatching, larvae were fed with lettuce and Taraxacum sps., Georgian larvae, as 262 mentioned above, were also fed with Plantago sp. and Rumex sp. When the adults emerged 263 from pupae, they were given water and stored at 4 degrees to slow their metabolic rate. wiped its beak against a surface -e.g. the perch); the drinking (number of times the bird 292 drinks water, as a response to the distaste of the food); the trial duration (from the time the 293 oats were seen by the bird until they were consumedor max 300 seconds if the oat was not 294 eaten). All trials were video recorded.

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Predator assay statistical analysis 296 The statistical analyses were conducted using the software R v. 4.1.2 (R Development Core    Fig 1).

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The variance between laboratory and wild wood tiger moth was equal in the amount of

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Birds drank more water in later trials (estimate ± SE = 0.34 ± 0.07, z = 4.60, p < 0.001). In 381 addition, birds drank more water in response to the SBMP+IBMP 0.5 ng/µl concentration 382 compared to the control (estimate ± SE = 3.06 ± 1.41, z = 2.17, p = 0.030, Figure S3). There 383 was also a trend for birds to drink more water in response to the IBMP 0.05 ng/µl 384 concentration compared to the control (estimate ± SE = 2.46 ± 1.42, z = 1.74, p = 0.083, 385 Figure S3), but no concentrations of SBMP differed from the control (Table S10, Figure S3).  Figure 5B). We found no 402 significant difference in the beak wiping behaviour between birds exposed to oats soaked in 403 either fluid from laboratory and wild Finnish and Georgian wood tiger moths, and those 404 exposed to water-soaked oats (p>0.05, see Figure S4, Table S13, supplementary material).

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The thoracic defence from laboratory-raised Georgian wood tiger moths elicited longer 410 hesitation times in the predators' response than the defence fluid from Georgian wild moths, 411 and both Finnish laboratory and wild moths (p<0.05, see Table S15; supplementary material).

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The percentage of oat eaten per unit of time (minute), differs between Georgian laboratory 413 moths and Finnish and Georgian wild moths (p<0.05, see Table S16; supplementary 414 material). The water drinking behaviour does not differ between Finnish and Georgian 415 population (p>0.05, see Table S17; supplementary material).

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Insects present an extreme diversification in their antipredator defence strategies. To 434 understand how these defences evolve, and why they vary, it is necessary not only to study 435 the chemical composition of the defences but also to test them on relevant wild predators. In

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Our analysis of wild blue tit responses to pure pyrazines suggests that SBMP alone was a 476 more effective defence than IBMP: birds ate a smaller proportion of oats soaked with SBMP 477 and there was a trend for birds to hesitate longer to approach SBMP oats compared to the 478 control. In contrast, IBMP was a weak defence on its own, although there was a trend for 479 IBMP to cause birds to drink more water, which suggests that birds may find IBMP more 480 aversive after tasting it. Despite having no effect on bird hesitation to approach, the 50/50 481 blend of IBMP+SBMP influenced the greatest number of bird behaviours: reducing the 482 proportion eaten, increasing drinking behaviour, and there was a trend to increase beak wipes. PAs confers better defences to the moths (Winters et al., 2021). Because the laboratory-raised 548 moths in the current study did not sequester PAs from their diet, it is possible that they 549 invested more in the production of pyrazines. Smell is one of the first cues that birds perceive 550 when approaching prey, so this may also explain why laboratory moths seem to allocate more 551 resources to the production of pyrazine.

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Finally, it should be noted that wood tiger moths are capital breeders, meaning that adults do 553 not eat, and all resources must be acquired at the larval stage. For that reason, it is unknown 554 how effectively moths can recover their chemical defences after they have released them. 555 Moths can certainly produce defensive fluids multiple times over their lifespan, but the Overall, our results suggest that measuring the absolute amounts of chemical defences does 568 not give the full picture of their efficacy: it is also necessary to test chemical defences on 569 relevant predators. The early environment drives variation in methoxypyrazine chemical 570 defences, even though the defences are produced de novo. In addition, chemical variation 571 appears to correlate with previously measured predation pressure, suggesting that natural 572 selection may also drive investment in chemical defences in this species. Clearly, the study of 573 chemical defences may be complicated by non-additive interactions between the chemical 574 components of the defence, and caution must be used when extrapolating from chemical 575 measurements to predator responses.