Circadian plasticity evolves via regulatory changes in a neuropeptide gene

Many organisms, including cosmopolitan drosophilids, display circadian plasticity, varying their activity with changing dawn/dusk intervals. How this behaviour evolves is unclear. Here, we compare Drosophila melanogaster with Drosophila sechellia, an equatorial, ecological specialist that experiences minimal photoperiod variation, to investigate the mechanistic basis of circadian plasticity evolution. D. sechellia has lost the ability to delay its evening activity peak time under long photoperiods. Screening circadian mutants in D. melanogaster/D. sechellia hybrids identifies a contribution of the neuropeptide Pigment-dispersing factor (Pdf) to this loss. Pdf exhibits species-specific, temporal expression, due in part to cis-regulatory divergence. RNAi and rescue experiments in D. melanogaster employing species-specific Pdf regulatory sequences demonstrate that modulating this neuropeptide’s expression impacts the degree of behavioural plasticity. The Pdf regulatory region exhibits sign..., Confocal images were obtained using the Zeiss microscopy ZEN 2.3 SP1 software. Drosophila Activity Monitor data were collected using the DAM System software, version 311X. All image analysis was performed in Fiji running v2.9.0 of ImageJ. RNA spots were detected using the RS-FISH macro, and Scholl analysis was performed using the neuroanatomy plug-in. Sequences were analyzed and visualised using SnapGene (www.snapgene.com) running MUSCLE (v3.8.1551), and Jalview (v2.11.2), and using the R package Phangorn (v2.11.2). Sequence alignments were perfomed using Samtools (v1.19.2), and tests of neutrality were performed using the DNA sequence polymorphism software (v6). Drosophila Activity Monitor data were analysed using Rethomics in R (v3.6.3). All R code used for data analysis are available at: github.com/mshahandeh/circ_plasticity. No statistical methods were used to predetermine sample size. For behavioural experiments, we aimed for a sample size of 25-30 individuals, as significant diffe..., , # Circadian plasticity evolves via regulatory changes in a neuropeptide gene

README

* /DAM: All files used for analysis of Drosophila activity monitoring (DAM) are contained within this folder.
* /DAM_metadata_files contains metadata files listing time, date, genotype, sex, status of flies recorded
* /DAM_monitor_files contains the raw data produced from the DAM system software
* /DATA: contains excel files with all data quantifications required for analyses, organized by Figure (details for each figure organized by panel below)
* /Images: contains all .czi confocal Z-stack images taken (specific quantifications can be found in the /Data folder for each image panel)
* /R: contains all R code required to reproduce the analyses

### Specific metadata, xcel, and image files for each figure, organized by panel, are provided below:

Figure1- data visualisation and analysis code in Rethomics.R; DAM metadata or excel data files listed below
c: Dmel+Dsec_all_1212LD.csv, metadata_14-10LD.c...

诸多生物（包括广布果蝇科类群）均表现出昼夜节律可塑性，其活动模式会随晨昏间隔的变化而发生改变。目前该行为的演化机制仍未明确。本研究以黑腹果蝇（Drosophila melanogaster）与塞氏果蝇（Drosophila sechellia）为研究对象，后者为仅经历极微光周期变化的热带专性生态物种，旨在探究昼夜节律可塑性演化的分子机制。
塞氏果蝇已丧失在长光周期条件下推迟晚间活动峰值的能力。通过对黑腹果蝇/塞氏果蝇杂交个体的节律突变体进行筛选，发现神经肽色素分散因子（Pigment-dispersing factor, Pdf）在该能力丢失过程中发挥了作用。
Pdf的表达具有物种特异性的时间模式，这部分源于顺式调控序列的分化。在黑腹果蝇中利用物种特异性Pdf调控序列开展的RNA干扰（RNAi）与拯救实验表明，调控该神经肽的表达水平会影响行为可塑性的程度。Pdf调控区域存在显著[原文截断]。

共聚焦图像采用蔡司显微镜ZEN 2.3 SP1软件采集。果蝇活动监测数据通过DAM系统软件v311X版本收集。所有图像分析均在运行ImageJ 2.9.0版本的Fiji软件中完成。RNA斑点通过RS-FISH宏命令进行识别，肖尔分析（Scholl analysis）采用神经解剖学插件完成。序列分析与可视化使用SnapGene（www.snapgene.com）搭配MUSCLE（v3.8.1551）、Jalview（v2.11.2），以及R包Phangorn（v2.11.2）实现。序列比对使用Samtools（v1.19.2）完成，中性检验采用DNA序列多态性分析软件v6进行。果蝇活动监测数据通过R语言中v3.6.3版本的Rethomics包进行分析。所有用于数据分析的R代码均可在以下网址获取：github.com/mshahandeh/circ_plasticity。
本研究未采用统计方法预先确定样本量。行为学实验的目标样本量为25-30只个体，以保证[原文截断]。

# 神经肽基因的调控变异驱动昼夜节律可塑性演化

## README
* /DAM：所有用于果蝇活动监测（Drosophila Activity Monitoring, DAM）数据分析的文件均收纳于此文件夹。
* /DAM_metadata_files：包含元数据文件，记录了果蝇的记录时间、日期、基因型、性别及实验状态
* /DAM_monitor_files：包含由DAM系统软件生成的原始数据
* /DATA：包含所有分析所需量化数据的Excel文件，按实验图表分组（各图表的详细信息将按子面板在下方列出）
* /Images：包含所有采集到的.czi格式共聚焦Z-stack图像（各图像子面板的具体量化数据可参见/Data文件夹）
* /R：包含所有可复现分析流程的R代码

### 各实验图表的专属元数据、Excel及图像文件将按子面板在下方提供：
图1：数据分析与可视化代码位于Rethomics.R；DAM元数据或Excel数据文件如下：
c: Dmel+Dsec_all_1212LD.csv、metadata_14-10LD.c...