Quit bugging me: Phorid fly parasitoids affect expression of an immune gene in foraging fire ant workers

Fire ant (S. invicta) colonies were collected and split into two sub-colonies. Containers were designed and created to serve as behavioral assay arenas. Behavioral assays were conducted in a climate-controlled laboratory, started between 13:30 and 14:20 pm, and performed over 48 hours. Each treatment colony was exposed to 10 decapitating flies (P. curvatus). Each experiment lasted 48 hours. Food was measured in grams (g) using a scale. Data was hand-collected and then transferred to Excel where basic statistics were performed (t-tests). Fire ant workers were collected during each experiment at hours: 0, 6, 24, 30, and 48 h. They were snap-frozen on dry ice and stored at -80°C prior to RNA extraction. We assayed relative expression of Sifor, OBP11, Hym, CYP4C1-like, abaecin, and Def-2, mRNA from pooled whole body fire ant workers/foragers relative to RPL18 (an endogenous control gene) using real-time qPCR techniques on a QuantStudioTM Pro System with PowerUpTM SYBRTM Green Master Mix and manufacturer’s protocols. Statistical analyses were conducted using paired t-tests of the DCt scores to compare differences in gene expression levels in fire ant workers at each sampling time point in the control (i.e., decapitating flies absent) and treatment (i.e., decapitating flies present) colonies.

本研究采集红火蚁（Solenopsis invicta，S. invicta）蚁群，并将其拆分为两个亚蚁群。设计并制作了专用容器作为行为测定试验箱。行为测定实验在控温实验室中开展，实验起始时间为13:30至14:20之间，持续时长48小时。每个处理组蚁群均暴露于10只弯角平腹蝇（又称斩首寄生蝇，Pseudacteon curvatus，P. curvatus）。每一轮实验均持续48小时。食物使用称重装置以克（g）为单位进行定量。实验数据采用手工采集记录，随后导入Excel软件完成基础统计分析（t检验）。在每轮实验的0、6、24、30和48小时节点，采集红火蚁工蚁样本。样本经干冰快速冷冻后，于-80℃条件下保存，以待后续RNA提取。我们采用实时荧光定量PCR（real-time qPCR）技术，以RPL18作为内参基因，基于QuantStudio™ Pro System、PowerUp™ SYBR™ Green Master Mix并按照厂商官方操作手册，对混合的红火蚁工蚁/觅食蚁整体组织的Sifor、OBP11、Hym、CYP4C1-like、abaecin及Def-2基因的mRNA相对表达量进行检测。统计分析采用配对t检验，基于ΔCt值比较各采样时间点下，对照组（即无斩首寄生蝇处理）与处理组（即有斩首寄生蝇处理）红火蚁工蚁的基因表达水平差异。