Olfactory learning in Pieris brassicae butterflies is dependent on the intensity of a plant-derived oviposition cue

Butterflies, like many insects, use gustatory and olfactory cues innately to assess the suitability of an oviposition site and are able to associate colours and leaf shapes with an oviposition reward. Studies on other insects have demonstrated that the quality of the reward is a crucial factor in forming associative memory. We set out to investigate whether the large cabbage white Pieris brassicae (Linnaeus) has the ability to associate an oviposition experience with a neutral olfactory cue. In addition, we tested whether the strength of this association is dependent on the gustatory response to the glucosinolate sinigrin, which is a known oviposition stimulus for P. brassicae. Female butterflies were able to associate a neutral odour with an oviposition experience after a single oviposition experience, both in a greenhouse and in a semi-natural outdoor setting. Moreover, butterflies performed best when trained with concentrations of sinigrin that showed the strongest response by specif..., Butterflies were conditioned by spraying 1 ml sinigrin solution on a green paper disc (9.5 cm in diameter) as an unconditioned stimulus, coupled with 20 microliters of vanilla scent as a conditioned stimulus. Three different concentrations of sinigrin solution were used. 3 different conditioning treatments plus one group of non-conditioned naive animals --> four different treatments. Tests consisted of releasing one animal in a testing environment provided with four green paper discs, identical to the ones used during training, but without sinigrin. Two of these were scented with vanilla scent, while the other two were scented with 30% alcohol-water solution. Each test lasted 30 minutes. Parameters recorded for each test are listed and explained below. Please note that not all of the recorded parameters have been used for analysis. Electrophysiology Response of the trichoid taste sensilla to sinigrin was assessed with single sensillum recordings. Sinigrin was dissolved in 1 mM KCl, 4..., , # Olfactory learning in *Pieris brassicae* butterflies is dependent on the intensity of a plant-derived oviposition cue ## Folder structure and contents: **These files are related to the behavioural observations in both indoor and outdoor settings:** * GHdata.xlsx * OUTdata.xlsx * GH_Sinigrin_Behaviour.R * OUT_Sinigrin_Behaviour.R * Peftuloglu_et_al_Electronic_Supplementary_Material.pdf **These files are related to the electrophysiological taste recordings performed:** * pierislegtaste.csv * Pierislegtaste script.R **Pieris_ovi.mp4**: Mp4 video of oviposition instance by a female Pieris brassicae on green paper disc impregnated with sinigrin. **Peftuloglu_et_al_Electronic_Supplementary_Material.pdf**: This file contains a picture of the indoor set-up, as well as graphs showing the proportion of time spent on vanilla-scented vs. unscented discs for control butterflies as well as butterflies trained with different concentrations of sinigrin in the indoor trails (figure S1). The fil...与多数昆虫类似，大菜粉蝶（*Pieris brassicae*，Linnaeus）天生会借助味觉与嗅觉线索评估产卵位点的适宜性，且能够将颜色、叶片形状与产卵奖励建立关联。已有针对其他昆虫的研究证实，奖励的质量是形成关联记忆的关键因素。本研究旨在探究该物种能否将产卵经历与中性嗅觉线索建立关联。此外，我们还测试了该关联的强度是否依赖于对黑芥子苷（sinigrin）的味觉响应——黑芥子苷是已知的*P. brassicae*产卵刺激物。 无论是在温室还是半自然室外环境中，雌性大菜粉蝶仅需一次产卵经历，即可将中性气味与产卵经历建立关联。此外，当以能够引发最强味觉响应的黑芥子苷浓度进行训练时，菜粉蝶的表现最佳（原文此处未完整呈现：by specif...）。 本实验通过将1毫升黑芥子苷溶液喷涂于直径9.5厘米的绿色纸碟上作为非条件刺激，同时搭配20微升香草气味作为条件刺激，对菜粉蝶进行条件化训练。实验共设置3种不同浓度的黑芥子苷溶液，由此产生3种条件化处理组，外加1组未经过条件化的空白对照组，总计4种处理方式。 测试环节为：将单只菜粉蝶释放至测试环境中，环境内放置4张与训练阶段一致的绿色纸碟，但未涂抹黑芥子苷。其中2张纸碟喷洒香草气味，另外2张则喷洒30%乙醇水溶液作为对照。每项测试持续30分钟。本次测试记录的各项参数如下文所述并予以解释。请注意，并非所有记录参数均用于后续数据分析。 ## 电生理学实验 采用单感器记录技术评估毛形味觉感器对黑芥子苷的响应。黑芥子苷溶解于1 mM氯化钾（KCl）溶液中，[原文此处未完整呈现：4..., ,] # 菜粉蝶*Pieris brassicae*的嗅觉学习依赖于植物来源产卵刺激的强度 ## 文件夹结构与内容 ### 与室内外行为观测相关的文件： * GHdata.xlsx * OUTdata.xlsx * GH_Sinigrin_Behaviour.R * OUT_Sinigrin_Behaviour.R * Peftuloglu_et_al_Electronic_Supplementary_Material.pdf ### 与味觉电生理记录相关的文件： * pierislegtaste.csv * Pierislegtaste script.R **Pieris_ovi.mp4**：雌性菜粉蝶在涂有黑芥子苷的绿色纸碟上产卵的MP4视频。 **Peftuloglu_et_al_Electronic_Supplementary_Material.pdf**：该文件包含室内实验装置的实拍图，以及展示对照组与不同黑芥子苷浓度训练组菜粉蝶在室内测试中，停留于香草气味纸碟与无气味纸碟的时间占比的统计图（图S1）。[原文此处未完整呈现：The fil...]