Infection with an acanthocephalan helminth reduces anxiety-like behaviour in crustacean host

Organisms Once a month from October 2021 to February 2022, we collected P. tereticollis-infected and uninfected G. fossarum individuals in the river Chiron in Burgundy, France (47° 27' 25.7"N, 5° 18' 21.2"E). They were maintained in large tanks filled with a mix of oxygenated dechlorinated ultraviolet-treated water (conditioned water; CW) and water from their river, in a room at 16°C with a 12L:12D photoperiod. Rocks were available as substrate and refuge, and gammarids were fed with dry elm leaves and chironomid larvae. Water was partly renewed every week. Gammarids were acclimatized to the room conditions for at least three weeks before taking part in the experiment. Treatments: acute and chronic stress using a semi-automated electric shock device Gammarids were placed in plastic containers (150 x 105mm, height: 60mm) with 400ml of CW by batches of 15 to 20 individuals, separated by treatments and according to the presence or absence of cystacanths. The cystacanths and old acanthellae of P. tereticollis form orange spots visible through G. fossarum exoskeleton, which allows straightforward diagnosis in living hosts; younger acanthellae have a lighter colouration and their presence can be assessed after host dissection. They were provided a small stone and half a terracotta saucer as refuges, and an elm leaf as food. Exposure to ES was done using a semi-automatic ES device (ESD) made of a rectangular piece of 3D-printed polymer containing two electrodes connected to an electronic driver 30, that fitted inside the above-mentionned plastic containers. In the “low intensity” (LI) stress treatment, once a day for 6 days at varying hours, the “chronically stressed” (CS) gammarid groups were placed in the ESD that delivered an ES program consisting in ten 9V pulses of 2sec administered every minute for 10min. Water was daily renewed at the time of the ES treatment. “Chronic controls” (CC) were not placed in ESDs but their water was renewed at the same time. Chronic treatment ended the day before behavioural tests (see below). On the seventh day, the “acutely stressed” (AS) gammarids were placed individually in plastic containers holding 200ml of CW, inside an ESD. They were given 5min to acclimatize before the ES program started. “Acute controls” (AC) were in the same conditions, except that they were not placed in ESDs, and their refuge was moved to the centre of their container. When the ES program ended, we removed the ESDs and placed the refuges back to the top edge of each container. In the “high intensity” (HI) stress treatment, the same protocol was applied to another set of gammarids except that a higher number of pulses and/or voltage was used. The HI CS treatment consisted in twenty 9V pulses of 2sec administered every 30sec for 10min once a day for the three first days, and then ten 15V pulses of 2sec every minute for 10min once a day for the last three days. On the seventh day, the AS gammarids received the 10x15V ES program before the refuge use test. Finally, to differentiate between a truly chronic effect of the CS treatment and a persistent effect of the last ES session, we exposed uninfected gammarids to a 10x15V ES session and conducted refuge use tests 24 hs later; controls were kept in the same conditions, but they did not receive ES. Although we configured the ESD to administer 9V or 15V pulses, effective voltage inside the device was lower and peaked at around 4V and 9V, respectively (measured with Voltcraft Digital Multimeter VC830). Refuge use test Refuge use tests were conducted on a bench receiving light between 90 and 130 lux. The choice of dimmed light (around sunset/sunrise values) was motivated by the decreased photophobia of uninfected gammarids at low illuminance 27, thus allowing the detection of a possible increase in photophobia in stressed gammarids. Five minutes after the end of the AS ES program (acclimation time), we started recording gammarids’ position every thirty seconds for ten minutes (focal and time sampling). Each gammarid’s score, thereafter abbreviated “RUs” was calculated as the sum of observations during which the gammarid was inside the refuge (from 0 to 20). Refuge use tests were generally conducted in blocks of 12 gammarids in individual plastic containers, each of the 8 treatments being represented by at least one individual in each block (with a few exceptions). After each refuge use test, gammarids were sedated in a 0.6g/l MS222 solution until they stopped moving 30, weighted and dissected to count the number of cystacanth(s) and acanthella(e).

**生物样本采集与饲养**
2021年10月至2022年2月期间，我们每月一次在法国勃艮第地区希龙河（47°27'25.7"N，5°18'21.2"E）采集感染了P. tereticollis（Polymorphus tereticollis）的富氏钩虾（Gammarus fossarum, G. fossarum）以及未感染的同物种个体。将采集到的钩虾饲养于大型养殖缸中，缸内水体为充氧脱氯紫外处理水（conditioned water, CW）与原河水的混合液，饲养环境温度维持在16℃，光周期设置为12小时光照:12小时黑暗。养殖缸内放置石块作为底质与躲避场所，每日投喂干燥榆树叶与摇蚊幼虫。每周更换部分饲养用水。实验开始前，所有钩虾需在该饲养环境中至少驯化3周。

**实验处理：基于半自动电击装置的急性与慢性应激模型构建**
将钩虾按感染与否（以囊棘蚴（cystacanths）与成熟棘头体（acanthellae）的存在情况划分）及处理组别分批置于塑料养殖盒（150×105mm，高60mm）中，每批15~20只，每盒加入400mL脱氯紫外处理水（CW）。P. tereticollis的囊棘蚴与成熟棘头体可在富氏钩虾外骨骼下形成橙色斑点，可直接对活体宿主进行诊断；未成熟棘头体颜色较浅，需通过宿主解剖确认其存在。每盒内放置一块小石块与半片陶土浅碟作为躲避场所，同时投喂一片榆树叶作为食物。
电击暴露（electric shock, ES）通过半自动电击装置（electric shock device, ESD）完成，该装置为一块3D打印聚合物制成的矩形构件，内置两枚连接至电子驱动模块的电极，可嵌入上述塑料养殖盒内。
在“低强度应激（low intensity, LI）”处理组中，“慢性应激（chronic stressed, CS）”组钩虾于6天内每日不定时放置于ESD中，施加的电击程序为：每分钟发放1次2秒时长的9V脉冲，共发放10次，持续10分钟。每日于电击处理时更换饲养用水。“慢性对照组（chronic controls, CC）”不放置于ESD中，但于相同时间点更换饲养用水。慢性应激处理于行为学测试前一日结束。
实验第7天，“急性应激（acute stressed, AS）”组钩虾被单独置于盛有200mL CW的塑料盒中，放入ESD内。电击程序开始前，给予5分钟适应时间。“急性对照组（acute controls, AC）”处于相同环境，但不放入ESD，且其躲避场所被移至养殖盒中心。电击程序结束后，移除ESD，将躲避场所放回每个养殖盒的顶部边缘。
“高强度应激（high intensity, HI）”处理组采用相同实验流程，但使用更高的脉冲数量与/或电压。HI组的慢性应激处理为：前3天每日发放1次电击程序，即每30秒发放1次2秒时长的9V脉冲，共发放20次，持续10分钟；后3天每日发放1次电击程序，即每分钟发放1次2秒时长的15V脉冲，共发放10次，持续10分钟。实验第7天，HI组的急性应激处理组钩虾在躲避行为测试前接受10次15V脉冲的电击程序。
为区分慢性应激处理的真实长效效应与末次电击的持续效应，我们将未感染钩虾暴露于10次15V脉冲的电击程序，并于24小时后开展躲避行为测试；对照组保持相同环境，但不接受电击。尽管ESD被设置为输出9V或15V脉冲，但装置内的实际有效电压更低，峰值分别约为4V与9V（使用Voltcraft Digital Multimeter VC830型数字万用表测量）。

**躲避行为测试**
躲避行为测试在光照强度为90~130 lux的实验台面上开展。选择该弱光环境（近似日落/日出时的光照水平）的原因是：未感染钩虾在低照度下的畏光性会降低，从而便于检测应激处理后钩虾畏光性是否出现升高。
在急性应激电击程序结束5分钟（适应时间）后，开始以每30秒一次的频率记录钩虾的位置，持续10分钟（焦点扫描与时间采样法）。每只钩虾的得分记为“RUs”（即躲避次数得分，为缩写），计算方式为其处于躲避场所内的总观测次数（取值范围为0~20）。
躲避行为测试通常以12只钩虾为一批次开展，每只钩虾置于独立塑料盒中，每个批次需覆盖全部8种处理组（少数批次除外）。
每轮躲避行为测试结束后，将钩虾置于0.6g/L的MS222溶液中麻醉至停止活动，随后称重并解剖，计数其体内的囊棘蚴与棘头体数量。