Great tit predation on colour morphs of the wood tiger moth at different relative frequencies

Polymorphic warning signals in aposematic systems are enigmatic because predator learning should favor the most common form, creating positive frequency-dependent survival. However, many populations exhibit variation in warning signals. There are various selective mechanisms which can counter positive frequency-dependent selection and lead to temporal or spatial warning signal diversification. Examining these mechanisms and their effects requires first confirming whether the most common morphs are favored at both local and regional scales. Empirical examples of this are uncommon and often include potentially confounding factors such as a lack of knowledge of predator identity and behavior. We tested how bird behavior influences the survival of three coexisting morphs of the aposematic wood tiger moth Arctia plantaginis offered to a sympatric predator (great tits, Parus major) at different frequencies. We found that although positive frequency-dependent selection is present, its strength is affected by predator characteristics and varying prey profitability.  These results highlight the need to understand predator foraging in natural communities with variable prey defences, in order to better examine how behavioral interactions shape evolutionary outcomes.

Methods
Freeze-killed adult wood tiger (Arctia plantaginis) moths from a multi-year laboratory stock, were thawed and spread so that their hindwing color was visible. The forewing length of each moth was measured with calipers prior to the start of the experiment. Specimens were then laid unpinned on their ventral side, with the dorsal side visible on individual Petri dishes. Each experiment contained a total of 24 moths laid out in a 4x6 grid in one of two enclosed indoor aviaries. The floor of each aviary was covered in dark green sheeting (tarp) to approximately mimic a natural background. Four different frequency treatments were created as follows: Control – 8 white males, 8 yellow males, 8 orange/red females (hereby just called red); Red biased- 6 white males, 6 yellow males, 12 red females; White biased – 12 white males, 6 yellow males, 6 red females; and Yellow biased – 6 white males, 12 yellow males, 6 red females. Each bird was assigned to a single treatment. Moth position on the grid was randomized.

Wild Great tits (Parus major) were caught from baited traps at Konnevesi Research Station (Central Finland), where this experiment took place, in October 2015. Once trapped, all birds were measured, aged, sexed, housed individually in plywood cages (80x65x50 cm) with a daily light period of 11h:13h (light:dark), fed on sunflower seeds, peanuts and vitamin-enriched tallow, and provided with fresh water ad libitum. After the experiment, all birds were ringed for identification purposes before being released at the capture site. Wild birds were used with permission from the Central Finland Centre for Economic Development, Transport and Environment, licensed from the National Animal Experiment Board (ESAVI/9114/04.10.07/2014) and the Central Finland Regional Environment Centre (VARELY/294/2015), and used according to the ASAB guidelines for the treatment of animals in behavioral research and teaching.

Birds were trained in groups overnight to forage in the experimental room and take palatable food (peanuts and sunflower seeds) from the petri dishes laid out in the grid, before being returned to their home cages.  Trials were run the following day after training. Prior to the start of each trial the participating bird was food deprived for one to two hours to ensure they were motivated to forage. Twenty-four moths were then laid out on petri dishes in a 4x6 experimental grid (Figure 1) and a single bird was released. We observed each trial through a one-way mirror and recorded the timing, order, and outcome (i.e. eaten or rejected/dropped) of each attack, as well as other bird behaviors such as beak wiping or cleaning. Trials lasted until each bird had attacked at least twelve moths, or two hours had passed, whichever came first. All treatments included 10 trials and birds were only used for a single trial, for a total of 40 birds used in the entire experiment. Following their use in the experiment, birds were given at least six hours to feed in their home cages before being released.

警戒色（aposematic）系统中的多态警戒信号一直令人困惑：捕食者的学习偏好应倾向于最常见的信号形态，进而产生正频率依赖的生存优势，但许多种群的警戒信号仍存在变异。多种选择机制能够对抗正频率依赖选择（positive frequency-dependent selection），并引发警戒信号在时间或空间上的分化。要探究这些机制及其效应，首先需要验证最常见的形态在局域和区域尺度上是否均受到青睐。相关实证案例十分罕见，且往往包含潜在的混淆因素，例如对捕食者种类与行为缺乏了解。

本研究以同域捕食者大山雀（Parus major）为对象，测试其行为对三种共存的警戒色木虎蛾（Arctia plantaginis）形态存活率的影响，实验中各形态以不同频率设置。结果显示，尽管正频率依赖选择确实存在，但其强度受捕食者特征与猎物收益差异的调控。该结果凸显了在猎物防御存在变异的自然群落中，探究捕食者觅食行为的必要性，以便更深入地解析行为互作如何塑造进化结局。

## 方法

本研究使用的木虎蛾（Arctia plantaginis）成虫均来自多年实验室饲养种群，经冷冻致死保存后解冻，展开后翅以露出其色彩。实验开始前，使用卡尺测量每头蛾的前翅长度。随后将标本以腹面贴地、背面朝上的方式放置，无需固定，每个培养皿（Petri dish）放置一头蛾。实验在两间封闭室内鸟舍中的一间开展，每间鸟舍内以4×6的网格布局摆放共24头蛾。每间鸟舍的地面铺设深绿色防水布（tarp），以近似模拟自然背景。

共设置4种不同的频率处理组：对照组——8头白色雄蛾、8头黄色雄蛾、8头橙红色雌蛾（下文简称红色型）；红色偏倚组——6头白色雄蛾、6头黄色雄蛾、12头红色雌蛾；白色偏倚组——12头白色雄蛾、6头黄色雄蛾、6头红色雌蛾；黄色偏倚组——6头白色雄蛾、12头黄色雄蛾、6头红色雌蛾。每只大山雀仅被分配至一个处理组，蛾在网格中的位置均经过随机化处理。

野生大山雀（Parus major）于2015年10月在本实验开展地芬兰中部科涅韦西研究站（Konnevesi Research Station）通过诱饵陷阱捕获。捕获后，研究人员对所有个体进行体长测量、年龄鉴定与性别判定，并将其单独饲养于胶合板笼（80×65×50 cm）中，每日光照周期为11小时光照:13小时黑暗，饲喂向日葵籽、花生与维生素强化牛脂，并自由提供新鲜饮水。实验结束后，所有个体均佩戴脚环用于标识，随后放归捕获地点。

本研究使用野生鸟类已获得芬兰中部经济发展、交通与环境中心许可，实验许可由国家动物实验委员会（ESAVI/9114/04.10.07/2014）与芬兰中部区域环境中心（VARELY/294/2015）颁发，实验操作符合行为研究与教学中动物处理的ASAB指南。

实验前一晚，大山雀以群体形式接受训练，使其熟悉在实验室内觅食，并能从网格布局的培养皿中取食适口食物（花生与向日葵籽），训练结束后将其放回原饲养笼。训练次日开展正式试次。每一试次开始前，受试大山雀需禁食1至2小时，以确保其具备觅食动机。随后，以4×6的实验网格（图1）布局将24头蛾放置于培养皿中，放入单只受试大山雀。

研究人员通过单向镜观察每一试次，记录每一次攻击的时间、顺序与结果（即被取食或被拒绝/丢弃），以及大山雀的其他行为，例如喙部擦拭或清洁。试次持续至受试大山雀至少攻击12头蛾，或时长达到2小时，以先发生者为准。每个处理组均设置10个试次，每只大山雀仅参与一次试次，整个实验共使用40只个体。实验结束后，受试大山雀需在饲养笼中至少进食6小时后方可放归。