Pre-copulatory reproductive behaviours are preserved in Drosophila melanogaster infected with bacteria

The activation of the immune system upon infection exerts a huge energetic demand on an individual, likely decreasing available resources for other vital processes, like reproduction. The factors that determine the trade-off between defensive and reproductive traits remain poorly understood. Here, we exploit the experimental tractability of the fruit fly Drosophila melanogaster to systematically assess the impact of immune system activation on pre-copulatory reproductive behaviour. Contrary to expectations, we found that male flies undergoing an immune activation continue to display high levels of courtship and mating success. Similarly, immune-challenged female flies remain highly sexually receptive. By combining behavioural paradigms, a diverse panel of pathogens and genetic strategies to induce the fly immune system, we show that pre-copulatory reproductive behaviours are preserved in infected flies, despite the significant metabolic cost of infection.

Methods
Bacterial infection: The bacterial strains used in this study include Serratia marcescens (DB11), Staphylococcus aureus (SH1000), Listeria monocytogenes (EGD-e), Escherichia coli (DH5α), Pectinobacterium carotovorum carotovorum 15 (ECC15) and Micrococcus luteus (clinical isolate, gift from Prof William Wade, King's College London). The bacterial strains were cultured overnight (see extended methods) at and cultures were pelleted by centrifugation at 4500g for 2 minutes. The pellet was diluted in filter-sterilised PBS (phosphate buffered saline) to a defined concentration. 50nl of diluted bacterial solution was injected employing a nano-injector (MPP1-3 Pressure Injector, Applied Scientific Instrumentation) into the abdomen of anaesthetised flies.

Survival assay:Infected flies and controls were placed in groups of 10-15 in vials at 29°C. The number of live flies infected with pathogenic strains was counted at regular intervals until all the infected flies were dead. Flies injected with non-pathogenic strains were counted at regular intervals for 72 hours.

Behavioural assays and parameters: All behavioural experiments were done in between zeitgeber time (ZT) 01 and ZT10 at 25°C. Mating assays were carried out in courtship chambers (20mm in diameter, 5mm in height), which have built-in dividers that allow separation of the flies before the experiment. For single pair mating assay, flies were injected with bacteria or vehicle solution (PBS) and immediately placed in the courtship chamber with food. Before the behavioural measurement began, the uninfected flies of the opposite sex were introduced using a fly aspirator. The dividers were opened before the assay and behaviours were recorded for one hour. For mate choice assays, a focal fly was given a choice between an infected and a healthy (PBS) mate. The infected and healthy flies were marked with acrylic paint 48 hours before experimentation. After injection, both infected and PBS flies were transferred to a courtship chamber with food. The focal fly was aspirated into the chamber before behavioural experimentation and behaviours were recorded for one hour. Courtship index (in experiments with infected males and controls) was measured as the proportion of time the male spends courting from the beginning of courtship until 10 minutes or end of copulation. Mating success was measured as the percentage of flies that mated within one hour. Copulation latency (in experiments with infected females and controls) was measured as the time taken to copulate from the start of courtship. For competitive mating assays, the focal fly’s first mate choice was recorded (i.e., if the fly chose to mate with a healthy or infected mate).

Locomotion Assay: A single fly was aspirated into a circular chamber (2 cm in diameter, 0.5cm in height), and locomotion was recorded for 5 minutes at 60 frames per second using Blackfly S U3-13Y3M camera. Individual trajectories were computed using custom-written Bonsai software. From the trajectories, two parameters were computed: walking speed and total distance travelled. A fly was considered to be walking if the instantaneous speed was greater than 4mm/s. The parameter “walking speed” refers to the average instantaneous speed over 5 minutes. The parameter “total distance” refers to the total Euclidean distance travelled by the fly in 5 minutes.

感染触发的免疫系统激活会对宿主造成极高的能量需求，很可能削减繁殖等其他重要生命过程可利用的资源。目前，决定防御与繁殖性状间权衡关系的调控因子仍未被充分阐明。本研究利用黑腹果蝇（Drosophila melanogaster）的实验可操作性，系统评估免疫系统激活对交配前繁殖行为的影响。与预期相悖的是，经历免疫激活的雄性果蝇依然表现出高水平的求偶行为与交配成功率；同样，受免疫挑战的雌性果蝇仍保持较高的性接受度。本研究结合行为实验范式、多种病原菌菌株以及诱导果蝇免疫系统激活的遗传手段，证实即便感染会带来显著的代谢成本，受感染果蝇的交配前繁殖行为仍得以维持。

## 实验方法
### 细菌感染实验
本研究使用的细菌菌株包括粘质沙雷氏菌（Serratia marcescens，DB11）、金黄色葡萄球菌（Staphylococcus aureus，SH1000）、单核细胞增生李斯特菌（Listeria monocytogenes，EGD-e）、大肠杆菌（Escherichia coli，DH5α）、胡萝卜软腐果胶杆菌胡萝卜亚种（Pectinobacterium carotovorum carotovorum 15，ECC15）以及藤黄微球菌（Micrococcus luteus，临床分离株，由伦敦国王学院William Wade教授惠赠）。所有细菌菌株均过夜培养（详细方法见补充材料），随后以4500g离心2分钟收集菌体沉淀。将沉淀用滤过除菌的PBS（磷酸盐缓冲液）稀释至既定浓度。使用纳米注射器（MPP1-3型压力注射器，Applied Scientific Instrumentation公司）向麻醉果蝇的腹部注射50nl稀释后的菌液。

### 存活实验
将感染组与对照组果蝇以10-15只每管的密度置于29℃环境中培养。对于注射致病菌株的果蝇，定期统计存活数量直至全部感染果蝇死亡；对于注射非致病菌株的果蝇，则在72小时内定期统计存活数量。

### 行为实验与参数测定
所有行为实验均于25℃环境下的授时时间（zeitgeber time, ZT）01至ZT10区间内开展。交配实验在求偶箱（直径20mm，高度5mm）中进行，箱体内置分隔挡板，可在实验前将果蝇分隔开。
对于单对交配实验：将注射菌液或对照溶液（PBS）的果蝇立即移入带有食物的求偶箱；在行为测量开始前，使用果蝇吸捕器引入未感染的异性果蝇，实验前打开分隔挡板，记录1小时内的行为表现。
对于配偶选择实验：将目标果蝇置于感染与健康（PBS注射）配偶间进行选择。实验前48小时，使用丙烯酸颜料对感染组与健康组果蝇进行标记。注射完成后，将感染组与PBS组果蝇均转移至带有食物的求偶箱，实验开始前将目标果蝇吸入箱内，记录1小时内的行为表现。
求偶指数（针对雄性感染与对照实验）定义为：从求偶开始至10分钟或交配结束期间，雄性用于求偶的时间占总时长的比例。交配成功率定义为1小时内完成交配的果蝇占比。交配潜伏期（针对雌性感染与对照实验）定义为从求偶开始至完成交配所需的时间。对于竞争性交配实验，记录目标果蝇的首次配偶选择结果（即选择与健康果蝇还是感染果蝇交配）。

### 运动能力测定
将单只果蝇吸入圆形观测箱（直径2cm，高度0.5cm），使用Blackfly S U3-13Y3M相机以每秒60帧的速率记录其5分钟内的运动情况。使用自主编写的Bonsai软件计算单个果蝇的运动轨迹，进而提取两项参数：行走速度与总移动距离。当果蝇瞬时速度大于4mm/s时，判定为处于行走状态；“行走速度”指5分钟内的平均瞬时速度，“总移动距离”指果蝇在5分钟内移动的总欧氏距离。