Risk of predation increases susceptibility to parasitism via trait-mediated indirect effects

The presence of natural enemies can cause organisms to change habitat use, foraging behaviour and/or resource allocation in response to a perceived risk; responses that may come at the cost of other fitness-related traits. Since most species encounter multiple natural enemies, defensive behaviours against one attacker may make the focal organism more vulnerable to attack by a different natural enemy. Anti-predator behaviours can lead to trait-mediated indirect effects such as an increased risk of attack by parasites, and vice versa. Few empirical studies have examined the response of a single focal species to the risk of attack by multiple species. Our experiments provided Drosophila nigrospiracula with opportunities to prioritise either anti-predator or anti-parasite behaviour at the cost of increased infection or predation, respectively. When exposed to parasites in the presence of predator cues, D. nigrospiracula experienced increased infection compared to flies without predator cues, but the presence/absence of parasite cues had no analogous effect on predation rates. We suggest that flies perceived parasitic infection to be a lesser threat and responded more strongly to predation risk at the cost of increased infection. In an ecological context, we shows how trait-mediated indirect effects could regulate community structure by increasing susceptibility to infection.

Methods
Flies, mites, and spiders: Drosophila nigrospiracula flies were cultured on Drosophila media (Formula 4–24 Instant Drosophila Medium; Carolina Biological Supply Company, Burlington, NC), instant mashed potato, and autoclaved necrotic tissue of Saguaro cacti (Carnegiea gigantea). Macrocheles subbadius mites were maintained in a mix of 2:1 mix of wheat bran to wood chips and co-cultured with free-living nematodes as a food source, hereafter referred to as ‘mite media’. Drosophila nigrospiracula and M. subbadius were both collected from the Sonoran Desert, Arizona, USA, in 2019 and maintained separately in continuous culture at the University of Alberta in climate controlled incubators (Percival Scientific, Perry, IA, USA) at 25 °C, 70 % relative humidity (RH) and a 12 h:12 h light:dark regime. Salticus scenicus spiders were collected by hand on the campus of the University of Alberta, Canada. Spiders were maintained in 100 mL specimen jars and provided with water and one D. nigrospiracula fly twice per week.

Experimental arenas: we custom designed a two-level arena system that could expose flies to parasites (M. subbadius) on the lower level or predators (S. scenicus) on the upper level (see schematic - Figure 1). Our design allowed us to test the effects of indirect contact (odour and visual cues) of one threat type while the focal species was simultaneously and directly exposed to the other threat type. The arena system was composed of two clear plastic Petri dishes (diameter 6 cm, height 1.5 cm) stacked one on top of the other. Mesh-covered holes in the floor of the upper dish and the roof of the lower dish allowed odour cues to flow between arenas. We placed a hole in the roof of the upper dish so that spiders and/or flies could be aspirated into this arena; this hole was plugged with adhesive tac (UHU Tac Adhesive Putty, UHU GmbH & Co. KG, Germany) during trials. The inner surfaces of the upper dish were abraded with course sandpaper so that the spiders could access all areas of the arena when hunting flies. This system allowed us to test our two alternate hypotheses using four separate scenarios. Our first hypothesis - flies prioritise anti-predator behavioural defenses over risk of infection - used scenarios 1) direct contact between flies and mites with indirect contact (visual and odour cues only) from spider and 2) direct contact between flies and mites, no spider cues. Our second hypothesis - flies prioritise anti-parasite behaviour at an increased risk of predation - used scenarios 3) direct contact between flies and spider with indirect contact with mites and 4) direct contact between flies and spider but no mite cues.

In Scenarios 1 and 2, approximately 15 g of mite media from our in-lab mite colonies was packed into the lower arena (Figure 1A-iv) and two intersecting trenches (0.5 mm wide, 0.5 mm deep) were made in the media with a short length of thin wood (Figure 1B-iv). These trenches replicated the small interstices or necrotic pockets of the cactus, where flies would normally encounter mites. The number of mites in the mite media varied among days, and therefore among trials, due to patchy aggregation of mites within colony containers. We assumed that any subsequent variability in infection would affect both treatments equally (see statistical analysis section). In both scenarios, five flies were aspirated into the trenches in the lower arena before attaching the upper arena. In Scenario 1, a spider was added to the upper arena, while in Scenario 2, no spider was added. In Scenarios 3 and 4, one fly and one spider were aspirated into the upper level; the fly was added first to avoid an immediate attack by the spider. In Scenario 3 mite media was packed into the lower level, but no trenches were made, while mite-free media was added to the lower level in Scenario 4, again with no trenches. Mite-free media contained wheat bran, wood chips and nematodes, but no mites, allowing us to control for any fly behavioural responses to the odour cues generated by the media and nematodes.

Experiment One: predator present and predator absent trials were conducted over 7 days in November and December 2023. Flies were directly exposed to mites with (n = 34) and without (n = 33) indirect exposure to spiders; arenas were then placed into a dark box inside the incubator for 1.5 hours. After the trial was complete, the flies were anaesthetised with CO2 and inspected under a dissecting microscope for mites. We calculated three response variables to quantify infection success (Rózsa et al. 2000): infection prevalence - the mean proportion of flies that were infected; infection intensity - the average number of mites among all infected flies, and infection abundance – the average number of mites per fly, including uninfected and infected flies.

Experiment Two: parasite present/absent trials were completed over four days in December 2023. Flies were directly exposed to spiders with (n = 32) and without (n = 32) indirect exposure to mites; trials lasted 30 minutes and were run in replicates of four: two with parasites present and two with parasites absent. Trials were limited to 30 minutes because after that time spiders began to spin webs that inhibited fly movement and confounded the behavioural responses of interest. After the spider and fly were loaded, an opaque plastic container (interior light intensity: 136 lux) was placed over top of the arena unit to limit visual disturbances that might affect spider or fly behaviour. The plastic container had a hole in the top that was covered with a lid, which was removed briefly at five-minute intervals to assess fly mortality. Spider and fly were observed until either the fly was killed or the trial ended, at which time we recorded mortality (yes/no) and time until death. Trials were conducted in-lab, where temperature and relative humidity were relatively consistent (18-19 °C and 22-24% RH).

Statistical Analysis

Experiment One: A generalised linear mixed effect model (glmm - glmer function in the package lme4 (Bates et al. 2014)) was used to analyse infection prevalence data. This model used infection prevalence as the response variable, treatment as the fixed effect, and trial number as a random effect to compensate for unknown variables in a given day. A general linear model (glm – glm function in the package stats) was used to test for differences of infection prevalence caused by variability in mite density among trials. This model used infection prevalence as the response variable and trial as fixed effect. Both models used a binomial error family and logit link function, and a dispersion parameter for our glmm was calculated using the dispersion_glmer function in the package blmeco (Korner-Nievergelt et al. 2019).

We used a Wilcoxon signed-rank non-parametric tests (wilcox.test function in the stats package) to analyse infection intensity and infection abundance data as neither met the assumptions of normality. Infection intensity or infection abundance were the response variables and treatment, predator present or predator absent, was the fixed effect. Due to variable mite density among trials, we used a Kruskal Wallace non-parametric test (kruskal.test function in the stats package) to look for any significant degree of variability in infection caused by trial number, and a pairwise Nemenyi test with a Tukey distribution (NemenyiTest function in the DescTools package (Signorell et al. 2024)) to find significant pairwise differences between trials.

Experiment Two: Fly survival was analysed using both a glmm (as described above) and a cox proportionate hazard model with random effects (coxme function in the package coxme (Therneau 2012)). The glmm analyzed end point survival only, with fixed effects of treatment (parasite present and parasite absent), media mass, spider mass, and trial date as a random effect. The proportional hazards model incorporated time of death of each treatment (parasite present and parasite absent), spider mass and media mass and again with the random effect of trial date. In this experiment, variable mite density was not considered to be an issue, as mite media provide only a parasite present signal. However, in the interest of completeness, we tested for any effect of trial date as a proxy for mite density using a glm with fly mortality as response variable and trial date as fixed effect, and a cox proportionate hazard model with time of death as response variable and trial date as fixed effect.

天敌（natural enemies）存在时，生物会为应对感知到的风险而改变栖息地利用、觅食行为及/或资源分配，这类响应往往会以其他与适合度相关的性状为代价。由于多数物种会遭遇多种天敌，针对一种攻击者的防御行为，可能会使得靶标生物（focal organism）更易受到其他天敌的攻击。反捕食行为可引发性状介导的间接效应（trait-mediated indirect effects），例如增加被寄生虫攻击的风险，反之亦然。目前鲜有实证研究探讨单个靶标物种对多种物种攻击风险的响应。本实验为暗斑果蝇（Drosophila nigrospiracula）提供了权衡选择的机会：优先采取反捕食行为则以增加感染风险为代价，优先采取抗寄生虫行为则以增加捕食风险为代价。当暴露于寄生虫且存在捕食者信号（predator cues）时，暗斑果蝇的感染率相较于无捕食者信号的果蝇更高；但寄生虫信号（parasite cues）的有无对捕食率并无类似影响。我们推测，果蝇将寄生虫感染视为威胁程度更低的风险，因此对捕食风险的响应更为强烈，代价则是感染风险上升。从生态学角度而言，本研究阐明了性状介导的间接效应如何通过增加感染易感性来调控群落结构。

实验方法

实验材料（果蝇、螨与蜘蛛）
暗斑果蝇（Drosophila nigrospiracula）饲养于即食果蝇培养基（Formula 4–24 Instant Drosophila Medium，Carolina Biological Supply Company，美国北卡罗来纳州伯灵顿市）、即食土豆泥以及高压灭菌的巨人柱（Carnegiea gigantea，萨瓜罗仙人掌）坏死组织中。亚双革螨（Macrocheles subbadius）饲养于麦麸与木屑按2:1比例混合的基质中，并以自由生活线虫作为食物来源，该基质后文统称为“螨培养基（mite media）”。暗斑果蝇与亚双革螨均于2019年采自美国亚利桑那州索诺兰沙漠，并在加拿大阿尔伯塔大学的控温培养箱（Percival Scientific，美国艾奥瓦州佩里市）中分别进行连续传代培养，培养条件为25 ℃、70%相对湿度（RH）以及12 h:12 h的光暗周期。斑马跳蛛（Salticus scenicus）于加拿大阿尔伯塔大学校园内徒手采集。蜘蛛饲养于100 mL标本瓶中，每周提供两次饮水及1只暗斑果蝇作为食物。

实验装置
本研究定制了双层实验装置，可使果蝇分别暴露于下层的寄生虫（亚双革螨Macrocheles subbadius）与上层的捕食者（斑马跳蛛Salticus scenicus）中（详见示意图——图1）。该装置可实现：当靶标生物同时直接暴露于一种威胁时，检测另一类威胁的间接接触（气味与视觉信号，odour and visual cues）所产生的效应。该装置由两个堆叠放置的透明塑料培养皿组成，直径6 cm，高度1.5 cm。上层培养皿底部与下层培养皿顶部均设有覆网小孔，可使气味信号在两层装置间流通。上层培养皿顶部设有小孔，用于将蜘蛛和/或果蝇吸入装置；实验过程中该小孔使用胶粘泥（UHU Tac Adhesive Putty，德国UHU股份公司两合公司）封堵。上层培养皿内壁用粗砂纸打磨，以便蜘蛛在捕食果蝇时可抵达装置内所有区域。借助该装置，我们可通过四组独立实验场景验证两个对立假说。第一个假说——果蝇优先采取反捕食行为防御而非应对感染风险——对应的实验场景为：1）果蝇与螨直接接触，同时接收蜘蛛的间接接触信号（仅视觉与气味信号）；2）果蝇与螨直接接触，无蜘蛛信号。第二个假说——果蝇优先采取抗寄生虫行为，代价为捕食风险上升——对应的实验场景为：3）果蝇与蜘蛛直接接触，同时接收螨的间接接触信号；4）果蝇与蜘蛛直接接触，无螨信号。

实验场景设置
在场景1与场景2中，取约15 g实验室螨种群的螨培养基装入下层装置（图1A-iv），并用细短木条在培养基中刻出两条交叉沟槽（宽0.5 mm，深0.5 mm，图1B-iv）。该沟槽模拟了巨人柱仙人掌的微小缝隙与坏死囊腔——果蝇通常在此处与螨接触。由于螨在种群容器内呈斑块状聚集，螨培养基中的螨数量会随饲养天数变化，进而导致不同实验批次间存在差异。我们假设后续感染率的任何差异对两组处理的影响是均等的（详见统计分析部分）。两组场景中，均在组装上层装置前，将5只果蝇吸入下层装置的沟槽内。场景1中向上层装置加入1只蜘蛛，场景2则不添加蜘蛛。在场景3与场景4中，将1只果蝇与1只蜘蛛吸入上层装置；为避免蜘蛛立即攻击果蝇，需先加入果蝇。场景3中，下层装置装入螨培养基，但不刻沟槽；场景4中下层装置装入无螨培养基，同样不刻沟槽。无螨培养基包含麦麸、木屑与线虫，但不含螨，可用于控制果蝇对培养基及线虫产生的气味信号的行为响应。

实验一
捕食者存在与缺失组实验于2023年11月至12月间开展，为期7天。将果蝇分别置于有（n=34）与无（n=33）蜘蛛间接信号的环境中，使其直接接触螨；随后将实验装置放入培养箱内的暗盒中静置1.5小时。实验结束后，用二氧化碳对果蝇进行麻醉，在体视显微镜下观察是否感染螨。我们计算了三个响应变量以量化感染成功率（Rózsa等，2000）：感染率（infection prevalence）——受感染果蝇的平均比例；感染强度（infection intensity）——所有受感染果蝇体内的平均螨数量；感染丰度（infection abundance）——每只果蝇的平均螨数量（包含未感染与已感染个体）。

实验二
寄生虫存在/缺失组实验于2023年12月间开展，为期4天。将果蝇分别置于有（n=32）与无（n=32）螨间接信号的环境中，使其直接接触蜘蛛；实验时长为30分钟，以4个重复为一组开展：2个重复设置寄生虫存在组，2个重复设置寄生虫缺失组。实验时长限定为30分钟，因为超过此时长后蜘蛛会开始结网，阻碍果蝇活动，干扰目标行为的观测。蜘蛛与果蝇被放入装置后，在装置上方覆盖一个不透明塑料容器（内部光照强度：136 lux），以减少可能影响蜘蛛或果蝇行为的视觉干扰。该塑料容器顶部设有带盖小孔，每5分钟短暂掀开盖子以评估果蝇存活情况。持续观察蜘蛛与果蝇，直至果蝇被杀死或实验结束，此时记录果蝇是否死亡（是/否）以及死亡所需时间。实验在实验室环境中开展，温度与相对湿度相对稳定（18~19 ℃，22%~24% RH）。

统计分析
实验一
使用广义线性混合模型（generalised linear mixed effect model，GLMM，lme4包中的glmer函数，Bates等，2014）分析感染率数据。该模型以感染率为响应变量，处理方式为固定效应，实验批次号为随机效应，以抵消单日实验中未知变量的影响。使用一般线性模型（general linear model，GLM，stats包中的glm函数）检验不同实验批次间螨密度差异导致的感染率差异。两个模型均采用二项误差族与logit连接函数；广义线性混合模型的离散参数通过blmeco包中的dispersion_glmer函数计算（Korner-Nievergelt等，2019）。由于感染强度与感染丰度数据不符合正态分布假设，我们使用Wilcoxon符号秩非参数检验（stats包中的wilcox.test函数）分析这两类数据。以感染强度或感染丰度为响应变量，处理方式（捕食者存在/缺失）为固定效应。由于不同实验批次间螨密度存在差异，我们使用Kruskal-Wallis非参数检验（stats包中的kruskal.test函数）检测实验批次号对感染率的显著影响，并使用基于Tukey分布的成对Nemenyi检验（DescTools包中的NemenyiTest函数，Signorell等，2024）分析批次间的显著成对差异。

实验二
果蝇存活情况同时采用广义线性混合模型（同前所述）与带随机效应的Cox比例风险模型（coxme包中的coxme函数，Therneau，2012）进行分析。广义线性混合模型仅分析终点存活情况，固定效应包括处理方式（寄生虫存在/缺失）、培养基质量、蜘蛛质量，实验日期为随机效应。Cox比例风险模型纳入了各处理组的死亡时间、蜘蛛质量与培养基质量，同样以实验日期为随机效应。本实验中，螨密度差异并非影响因素，因为螨培养基仅提供寄生虫存在的信号。但为确保分析的完整性，我们以实验日期作为螨密度的替代变量，分别采用以果蝇死亡率为响应变量、实验日期为固定效应的一般线性模型，以及以死亡时间为响应变量、实验日期为固定效应的Cox比例风险模型，检验实验日期的影响。