Gregarines modulate insect responses to sublethal insecticide residues

Study organism and rearing A rearing of P. cochleariae was maintained for several generations at Bielefeld University under constant climatic conditions (20°C, 16 h: 8 h light:dark, 70% r.h.). Adult beetles were mixed randomly for mating and groups of 100-200 individuals kept in boxes (20 × 20 × 6.5 cm) covered with gauze lids. Every year the rearing population was replenished with individuals collected in the field (51°51′21″ N, 8°41′37″ E). Larvae and beetles were supplied with leaves of non-flowering 8-10-week old cabbage plants (Brassica rapa L. spp. pekinensis), which were cultivated in pots in a greenhouse (20˚C, 16 h: 8 h light:dark, 70% r.h.). For the experiment, only middle-aged leaves were offered, and larvae were provided with greenhouse-grown cabbage, while adults received cabbage bought from an organic store due to plant shortage. General experimental set-up and measurements of larval food consumption and life-history traits A full-factorial design was set up to test the influences of gregarine infection and sublethal insecticide exposure on larval consumption and life-history of P. cochleariae. Three-week-old adults (about 200 individuals) were provided with cabbage leaves and their offspring was subsequently used for the experiment. Female beetles bite little cavities in the leaf surface, lay individual eggs in these cavities and cover the eggs with secretion. Infection of larvae of P. cochleariae with gregarines occurs via the uptake of spores excreted by infected conspecifics. Faecal remains can also cover the eggs. Thus, after 24 h of oviposition time, eggs were carefully removed from the leaves and the secretion and potential faeces removed with a paintbrush and tap water. Eggs were placed on fresh cabbage leaves and randomly distributed over two rearing boxes, one assigned to the uninfected gregarine treatment (G-: N = 325), the other to the gregarine infection treatment (G+: N = 320). Hatching larvae (G-: N = 195, G+: N = 204) were supplied with the respective food sources for four days as described below (experimental infection of P. cochleariae larvae with gregarines) to ensure a G- or G+ treatment. At the fourth day after hatching, the larvae were divided into groups of 5-10 larvae in large Petri dishes (9 cm diameter) lined with filter paper and provided with cabbage leaf pieces (3 × 4 cm). From day five on, half of both G- and G+ larvae were assigned to one of the two insecticide treatment groups, either receiving no insecticide (I-) or receiving λ-cyhalothrin-treated leaf discs (I+) for 48 h (procedure see below), resulting in four treatment groups (G-I-: N = 50, G-I+: N = 113, G+I-: N = 60, G+I+: N = 130). Much higher numbers of I+ animals were set up to account for a lower survival of insecticide-treated individuals. From day seven after hatching, all individuals were provided with untreated cabbage leaves. To investigate the effects of the different treatments on consumption, the amount of leaf mass consumed within 24 h by each larva was measured. At day 9 after hatching, two larvae were randomly selected from each Petri dish. Each larva was weighed (micro balance, ME36S, Sartorius AG, Göttingen, Germany), placed in a small Petri dish (5.5 cm diameter) lined with moistened filter paper and offered a leaf disc (24 mm diameter) of known mass (balance, LA120S-OCE, Sartorius AG, Göttingen, Germany). After 24 hours, the larvae were weighed again and the remaining leaf discs were scanned (Samsung ProXpress SL-M3375FD, Schwalbach/Taunus, Germany; resolution: 600 dpi). The remaining leaf area was determined with ImageJ (v 1.52a) and used to calculate the consumed leaf mass [initial leaf mass x (initial leaf disc area – remaining leaf area)/initial leaf disc area]. Afterwards, these larvae remained in their individual Petri dishes and were used for counting of gregarines (see below). To test the effects of the different treatments on life-history traits, pupae were individually placed in small Petri dishes lined with filter paper and the time from larval hatching until adult eclosion was noted. The body mass of adult individuals was measured 24 hours after adult eclosion and the sex of the individuals determined. Adults were kept individually in small Petri dishes and supplied with cabbage leaves. Eight days after adult eclosion females were mated with males of the same treatment group, placing each male to one female for 24 hours. After mating, the pairs were separated again, and the number of eggs laid within the subsequent four days per female was counted. The hatching rate was determined by counting the number of hatched larvae relative to the number of eggs laid. Moreover, the survival of all individuals kept under the four treatment regimes was noted until day ten after adult eclosion. The experiment was ended when adults were 15 days old, while adults can live up to three months (Bogdanov-Katjkov 1923). Experimental infection of P. cochleariae larvae with gregarines To ensure larval infection with gregarines (G+ treatment), leaves covered with faeces were taken that had been placed in the boxes of the insect rearing stock for 24 hours. Random examinations of the rearing stock showed that all tested beetles were infected with gregarines and that infectious spores were present in their faeces. Larvae of the G- treatment were offered leaves that had been kept for 24 hours in a box without conspecifics and were damaged by regular cuttings to imitate feeding and thus provide leaves of comparable quality to those provided to the G+ group. Such leaves were offered to hatching larvae of the respective treatment groups and replaced every 2 days until larvae were 4 days old. Microscopy of larvae and adults (see below) confirmed that all dissected individuals of the G- treatment (N = 40 larvae, N = 46 adults) were not infected, while all individuals of the G+ treatment were successfully infected with gregarines (N = 40 larvae, N = 73 adults). Preparation of sublethal λ-cyhalothrin concentration and insecticide exposure treatment The pyrethroid λ-cyhalothrin was chosen as contact insecticide because it is widely used in agriculture, including fields with crops of Brassicaceae. A sublethal concentration was prepared from the insecticide LAMBDA WG, which contains 5% of the active toxin λ-cyhalothrin. The insecticide was dissolved in methanol (HPLC-grade, VWR International GmbH, Darmstadt, Germany), centrifuged and the supernatant used to prepare a 0.6 mg/L λ-cyhalothrin stock solution. This concentration was considered as sublethal, as less than 50% of the larvae died during the exposure period (following the definition by de França et al. 2017). In an agricultural system, this concentration is approximately less than half of the application recommended by the supplier and may thus occur in areas close to treated fields as residue. The experimental larvae were fed on day five and six after hatching with cabbage leaf discs (24 mm diameter) treated with 76.8 µl methanol (I-) or λ-cyhalothrin solution (I+). The surfaces of the leaf discs were evenly covered with the respective solutions using a pipette and leaf discs were kept under a fume hood until evaporation of the solvent (at least 20 min) before offering them to the larvae. Depending on the number of larvae, two to four leaf discs were offered per Petri dish to provide food ad libitum and after one day the leaf discs were exchanged by discs of the respective treatments for another day. Counting of gregarines Subgroups of both larvae and adults of P. cochleariae were examined to test whether the number of gregarines depends on the different treatments and developmental stage of the host. At day 12 after larval hatching, a subset of larvae of the different treatments (two larvae per Petri dish, used for consumption assay, N = 20 larvae per treatment group) was taken and individuals were frozen (-20 °C). Likewise, adult males and females of each treatment group (G-I-: N = 9 females, N = 12 males; G-I+: N = 12 females, N = 13 males; G+I-: N = 12 females, N = 15 males; G+I+: N = 26 females, N = 20 males) were randomly selected and frozen at day 15 after adult eclosion. Afterwards, individuals were thawed, the midgut dissected, spread on a microscope slide in 25 µl sodium phosphate buffer (0.1 M, pH = 7.2), and the number of gregarines (likely as trophozoites and gamonts) counted under a light microscope at 200 to 400 times magnification (ZEISS Axiophot, Carl Zeiss Microscopy GmbH, Jena, Germany) (ESM Fig. S2). Statistical analyses All statistical analyses were performed in R version 3.6.3 (R Core Team 2020) with RStudio version 1.2.5033 (RStudio Team 2019). Data were analysed using linear models (lm), generalised linear models (glm) (package MASS; Venables and Ripley 2002) and cox proportional hazards model (package survival; Therneau 2020). Model assumptions for lms and glms (normal distribution and homoscedasticity of residuals) and cox proportional hazard models (proportional hazard and influential observations) were checked using diagnostic plots. Stepwise backward model selection was applied to obtain the minimal adequate models. Based on F test or Chi-square test results (package MASS; Venables and Ripley 2002), non-significant (P-value > 0.05) interaction terms and/or predictors were excluded from the models. The effects of the predictors gregarine treatment and insecticide treatment as well as their interaction on development time (from larval hatching to adult eclosion) were tested using a glm (poisson distribution, identity link function). The influences of the predictors larval body mass, gregarine treatment and insecticide treatment and their interaction on the food consumption of larvae were analysed using a lm. The effects of the predictors gregarine treatment, insecticide treatment as well as their interaction on the adult body mass were analysed separately for males and females using lm. Glms were performed to investigate the effects of gregarine treatment, insecticide treatment and their interaction on the number of eggs laid by adult females (poisson distribution, log link function) and the hatching rate of larvae [binomial family (success/failure of hatching), logit link function]. To investigate the effects of insecticide treatment on the gregarine load in the larval stage a glm (poisson distribution, identity link function) was calculated. For the gregarine load in adults a glm (poisson distribution, identity link function) with insecticide treatment and sex as predictors was performed. The influences of the predictors gregarine treatment, insecticide treatment and their interaction on survival probability were tested using a cox model, followed by pairwise log rank post hoc tests. Survival data were plotted using Kaplan-Meyer curves (package survival; Therneau 2020). R Core Team (2020) R: a language and environment for statistical computing. R Foundation for statistical computing, Vienna, Austria. https://www.R-project.org/. R Studio Team (2019) RStudio: integrated development for R. RStudio, Inc., Boston, MA. http://www.rstudio.com/. Therneau T (2020) A package for survival analysis in R. R package version 3.2-7. https://CRAN.R-project.org/package=survival. Venables WN, Ripley BD (2002) Modern applied statistics with S, 4. edn. Springer, New York.研究生物与饲养：本研究的菱斑食植瓢虫（P. cochleariae）种群在比勒费尔德大学的恒定气候条件下（20℃，光周期16h:8h，相对湿度70%）多代饲养。成虫随机混群交配，以100~200头为一组置于覆盖纱网盖的饲养盒（20×20×6.5cm）中。每年均从野外（51°51′21″N，8°41′37″E）采集个体补充饲养种群。幼虫与成虫均饲喂8~10周龄未开花的白菜叶片（大白菜（Brassica rapa L. spp. pekinensis）），该白菜种植于温室（20℃，光周期16h:8h，相对湿度70%）盆钵中。实验仅使用中位叶片，幼虫饲喂温室种植的白菜，而成虫因植株短缺则采购自有机商店的白菜。 实验总体设计与幼虫取食量、生活史性状测定：本研究采用全因子设计（full-factorial design），以探究簇虫感染（gregarine infection）与亚致死杀虫剂暴露（sublethal insecticide exposure）对菱斑食植瓢虫幼虫取食行为与生活史的影响。选取3周龄成虫（约200头）饲喂白菜叶片，其后代用于本实验。雌虫会在叶片表面咬出小凹穴，将单个卵产于其中并以分泌物覆盖。菱斑食植瓢虫幼虫通过摄取被感染同种个体排出的孢子而感染簇虫。粪便残留物也可附着于卵上。因此，在产卵24h后，小心从叶片上取下卵，并用画笔和自来水清除卵上的分泌物与潜在粪便。将卵置于新鲜白菜叶片上，并随机分配至两个饲养盒：一组为非簇虫感染处理组（G-：N=325），另一组为簇虫感染处理组（G+：N=320）。孵化幼虫（G-：N=195，G+：N=204）按下述方法饲喂4天（见“菱斑食植瓢虫幼虫的簇虫实验感染”部分），以确保其分别处于G-或G+处理状态。孵化后第4天，将幼虫以每皿5~10头的密度置于铺有滤纸的大培养皿（直径9cm）中，饲喂白菜叶块（3×4cm）。自第5天起，将G-与G+组幼虫各随机分为两组，分别置于无杀虫剂处理组（I-）与氯氟氰菊酯（λ-cyhalothrin）处理组（I+），暴露48h（操作步骤见下文），最终得到4个处理组：G-I-（N=50）、G-I+（N=113）、G+I-（N=60）、G+I+（N=130）。I+组设置更多样本量以抵消杀虫剂处理个体较低的存活率。孵化后第7天起，所有个体均饲喂未处理的白菜叶片。 为探究不同处理对取食量的影响，测定每头幼虫24h内的取食叶片质量。孵化后第9天，从每个培养皿中随机选取2头幼虫。使用微量天平（ME36S，赛多利斯股份公司，哥廷根，德国）称量其体重，随后将其置于铺有湿润滤纸的小培养皿（直径5.5cm）中，饲喂已知质量的白菜叶碟（直径24mm）（使用LA120S-OCE天平称量，赛多利斯股份公司，哥廷根，德国）。24h后，再次称量幼虫体重，并扫描剩余叶碟（使用三星ProXpress SL-M3375FD扫描仪，陶努斯施瓦巴赫，德国；分辨率600dpi）。使用ImageJ（v1.52a）测定剩余叶面积，并通过公式[初始叶质量×(初始叶碟面积-剩余叶面积)/初始叶碟面积]计算取食叶片质量。此后，这些幼虫保留在各自的培养皿中，用于簇虫计数（见下文）。 为测试不同处理对生活史性状的影响，将蛹单独置于铺有滤纸的小培养皿中，记录从幼虫孵化至成虫羽化的时长。成虫羽化后24h称量其体重，并鉴定性别。将成虫单独饲养于小培养皿中，饲喂白菜叶片。成虫羽化后第8天，将同处理组的雄虫与雌虫配对，每头雄虫与1头雌虫共处24h以完成交配。交配后将雌雄分离，统计每头雌虫后续4d内的产卵量。孵化率通过孵化幼虫数与产卵总数的比值计算。此外，记录4个处理组所有个体直至成虫羽化后第10天的存活情况。实验在成虫15日龄时终止，而该物种成虫寿命可达3个月（Bogdanov-Katjkov 1923）。 菱斑食植瓢虫幼虫的簇虫实验感染：为确保幼虫被簇虫感染（G+处理组），采集饲养种群饲养盒中放置24h的带有粪便的叶片。随机抽检饲养种群发现，所有检测甲虫均感染簇虫，且其粪便中存在传染性孢子。G-处理组幼虫饲喂的叶片则置于无同种个体的饲养盒中24h，并通过常规切割造成损伤，以模拟取食行为，保证其叶片质量与G+组叶片相当。将此类叶片分别提供给对应处理组的孵化幼虫，每2天更换一次，直至幼虫4日龄。对幼虫与成虫的显微镜检测（见下文）证实：G-处理组所有解剖个体（幼虫N=40，成虫N=46）均未感染簇虫，而G+处理组所有个体均成功感染簇虫（幼虫N=40，成虫N=73）。 亚致死氯氟氰菊酯浓度配制与杀虫剂暴露处理：选用拟除虫菊酯类（pyrethroid）触杀杀虫剂氯氟氰菊酯（λ-cyhalothrin），因其广泛应用于农业生产，包括十字花科（Brassicaceae）作物田块。从活性成分含量为5%的农药制剂LAMBDA WG中配制亚致死浓度溶液。将原药溶于甲醇（HPLC级，VWR国际股份有限公司，达姆施塔特，德国），离心后取上清液制备0.6mg/L的氯氟氰菊酯储备液。该浓度被认定为亚致死浓度，因为暴露期间幼虫死亡率低于50%（遵循de França等2017年的定义）。在农业生产场景中，该浓度约为供应商推荐施用量的一半以下，因此可能作为残留存在于施药田块周边区域。实验幼虫在孵化后第5、6天饲喂经76.8μl甲醇（I-组）或氯氟氰菊酯溶液（I+组）处理的白菜叶碟（直径24mm）。使用移液枪将对应溶液均匀涂布于叶碟表面，将叶碟置于通风橱中直至溶剂完全挥发（至少20min）后再饲喂幼虫。根据幼虫数量，每个培养皿提供2~4片叶碟以保证充分取食，1天后更换为同处理组的新鲜叶碟，持续处理2天。 簇虫计数：选取部分幼虫与成虫样本，以探究簇虫数量是否受不同处理与宿主发育阶段影响。幼虫孵化后第12天，从各处理组中选取用于取食量测定的样本（每个培养皿2头幼虫，每处理组N=20），将其冷冻保存（-20℃）。同样，在成虫羽化后第15天，从各处理组中随机选取雌雄成虫（G-I-：雌虫N=9，雄虫N=12；G-I+：雌虫N=12，雄虫N=13；G+I-：雌虫N=12，雄虫N=15；G+I+：雌虫N=26，雄虫N=20）并冷冻保存。随后将样本解冻，解剖其中肠，置于载玻片上，滴加25μl磷酸钠缓冲液（0.1M，pH=7.2）平铺展开，在200~400倍光学显微镜（ZEISS Axiophot，卡尔蔡司显微股份公司，耶拿，德国）下计数簇虫（大概率为滋养体与配子体）（ESM图S2）。 统计分析：所有统计分析均在R版本3.6.3（R核心团队2020）与RStudio版本1.2.5033（RStudio团队2019）中完成。使用线性模型（linear models, lm）、广义线性模型（generalised linear models, glm）（MASS包；Venables与Ripley 2002）与Cox比例风险模型（survival包；Therneau 2020）分析数据。通过诊断图检验lm与glm的模型假设（残差正态分布与同方差性）以及Cox比例风险模型的假设（比例风险与异常观测值）。采用逐步向后选择法获取最小充分模型。基于F检验或卡方检验结果（MASS包；Venables与Ripley 2002），将无统计学意义（P>0.05）的交互项和/或预测变量从模型中剔除。使用广义线性模型（泊松分布，恒等连接函数）检验簇虫处理、杀虫剂处理及其交互作用对发育时长（从幼虫孵化至成虫羽化）的影响。使用线性模型分析幼虫取食量与幼虫体重、簇虫处理、杀虫剂处理及其交互作用的关系。分别针对雌雄个体，使用线性模型分析簇虫处理、杀虫剂处理及其交互作用对成虫体重的影响。使用广义线性模型探究簇虫处理、杀虫剂处理及其交互作用对雌虫产卵量的影响（泊松分布，对数连接函数）以及幼虫孵化率（二项分布族，logit连接函数）。为探究杀虫剂处理对幼虫期簇虫负荷的影响，使用广义线性模型（泊松分布，恒等连接函数）进行分析。针对成虫期簇虫负荷，使用广义线性模型（泊松分布，恒等连接函数），以杀虫剂处理与性别作为预测变量。使用Cox模型检验簇虫处理、杀虫剂处理及其交互作用对存活概率的影响，随后进行成对对数秩检验事后分析。存活数据使用Kaplan-Meier曲线（Kaplan-Meier curves）进行可视化（survival包；Therneau 2020）。 R核心团队（2020）R：统计计算语言与环境。R统计计算基金会，维也纳，奥地利。https://www.R-project.org/。 RStudio团队（2019）RStudio：R集成开发环境。RStudio股份公司，波士顿，马萨诸塞州。http://www.rstudio.com/。 Therneau T（2020）R语言生存分析包。R包版本3.2-7。https://CRAN.R-project.org/package=survival。 Venables WN, Ripley BD（2002）现代应用统计与S语言，第4版。施普林格，纽约。