1 INTRODUCTION

Several factors, from host genetic background and sex to environmental conditions, contribute to host susceptibility to parasite infections (Carius et al., 2001; Debes et al., 2017; Nunn et al., 2009; Wilson et al., 2002). Many of these effects are at least partly mediated by differences in host immune function, which is the primary physiological defence against parasites (reviewed in Janeway et al., 2005) and an essential determinant of organismal fitness (reviewed in Seppälä, 2015). The immune system is costly to maintain and use because it requires a lot of energy (Moret & Schmid-Hempel, 2000; Sheldon & Verhulst, 1996). Therefore, parasite resistance typically depends on host nutritional state, which can have broad ecological and evolutionary consequences. For example, individuals in poor physiological condition can be more susceptible to infections (Knutie et al., 2017; Kolluru et al., 2006; Murray et al., 1998; Wiehn & Korpimäki, 1998). Thus, deteriorating living conditions may predispose populations to disease outbreaks (reviewed in Lloyd, 1995; Wakelin, 1989). Additionally, variation in host resource level can affect the expression of trade-offs related to parasite resistance (Brzęk & Konarzewski, 2007; McKean et al., 2008; Moret & Schmid-Hempel, 2000) and maintain genetic polymorphism in host defences through genotype-by-environment (G × E) interactions (Blanford et al., 2003; Mitchell et al., 2005; Seppälä et al., 2011).

Research on the condition dependence of host immune function is typically conducted in laboratory experiments that manipulate either the quantity or composition of available resources (Brunner et al., 2014; Brzęk & Konarzewski, 2007; Ponton et al., 2020; Siva-Jothy & Thompson, 2002; Slater & Keymer, 1986; Stahlschmidt et al., 2013). Such experiments are essential because they demonstrate the condition dependence of immune defence while controlling for possible confounding factors (e.g., individuals may differ in their foraging efficiency) and they prove causality. However, such experiments often compare extreme resource levels (e.g., ad libitum food supply vs. no food). In nature, the variation in host resource level is likely to be more subtle at any given time point, making it difficult to generalise the results of such simplified laboratory experiments to natural populations. Therefore, expanding the work on condition dependence of immune defence to consider natural variation in host physiological condition in field populations is a high priority.

Here, we examined the condition dependence of immune function in a generalist freshwater consumer, Lymnaea stagnalis (Gastropoda), using field-collected individuals. We quantified the activity of two immune parameters of snail haemolymph that represent different branches of the immune system, phenoloxidase (PO)-like (a component of oxidative defences) and antibacterial activity (Langeloh et al., 2017; Seppälä & Jokela, 2010; Seppälä & Leicht, 2013). These immune traits respond to immune challenges (Seppälä & Leicht, 2013), contribute to snail fitness (Langeloh et al., 2017) and show considerable within-population genetic variation (i.e., evolutionary potential; Leicht et al., 2017; Seppälä & Jokela, 2010; Seppälä & Langeloh, 2016). Furthermore, snail immune activity depends on environmental factors such as food availability (Seppälä & Jokela, 2010; Seppälä et al., 2021) and ambient temperature (Leicht et al., 2013, 2017; Salo et al., 2017, 2019; Seppälä et al., 2021). When the access of snails to food is removed experimentally under laboratory conditions, the PO-like activity is strongly decreased within a day, whereas the level of the antibacterial activity reduces a few days later (Seppälä & Jokela, 2010). These findings suggest that variation in resource availability in nature could contribute to disease outbreaks in snail populations. Furthermore, the dependence of the snail immune system on food availability shows within-population family-level variation (i.e., a G × E interaction determining immune activity; Seppälä & Jokela, 2010). This interaction suggests that variation in environmental conditions may promote the maintenance of genetic variation in snail defences.

To relate the variation in immune activity to the resource level of the snails, we measured several factors that reflect their condition based on the quantity and composition of resources consumed both recently (i.e., within the past few days; amount and stable isotope composition [¹⁵N:¹⁴N ratio denoted as δ¹⁵N and ¹³C:¹²C ratio denoted as δ¹³C] of produced faeces) and over a longer time period (i.e., weeks to months; stable isotope composition of tissues; Li et al., 2018). We found that under natural conditions (i.e., natural variation in snail resource level and use), the PO-like activity of the snails’ haemolymph was condition dependent. Snails that had recently consumed food with high δ¹⁵N values had a stronger defence. Considering the covariation between δ¹⁵N and C:N ratio of faeces, the result suggests that resource consumption from higher trophic levels, potentially including more animal protein, enhances the haemolymph PO-like activity. Additionally, snails with high δ¹³C values in tissues had high PO-like activity. Based on the covariation between δ¹³C and C:N ratio of tissues, this result suggests a negative relationship between the snails’ lipid reserves and the PO-like activity, which could arise from the energetic costs of immune activity.

中文翻译：

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稳定同位素揭示淡水蜗牛的条件依赖性免疫功能

1 简介

从宿主遗传背景和性别到环境条件，有几个因素会导致宿主对寄生虫感染的易感性（Carius 等人，  2001 年；Debes 等人，  2017 年；Nunn 等人，  2009 年；Wilson 等人，  2002 年）。许多这些影响至少部分是由宿主免疫功能的差异介导的，这是对寄生虫的主要生理防御（Janeway 等人，  2005 年综述）和有机体适应性的重要决定因素（Seppälä，2015 年综述）。免疫系统的维护和使用成本很高，因为它需要大量能量（Moret & Schmid-Hempel,  2000 ; Sheldon & Verhulst,  1996）。因此，寄生虫抗性通常取决于宿主的营养状态，这可能会产生广泛的生态和进化后果。例如，生理状况不佳的个体可能更容易受到感染（Knutie 等人，  2017 年；Kolluru 等人，  2006 年；Murray 等人，  1998 年；Wiehn 和 Korpimäki，  1998 年）。因此，不断恶化的生活条件可能使人群容易爆发疾病（Lloyd，  1995 年；Wakelin，  1989年综述）。此外，宿主资源水平的变化会影响与寄生虫抗性相关的权衡的表达（Brzęk & Konarzewski,  2007 ; McKean et al.,  2008; Moret & Schmid-Hempel,  2000 ) 并通过基因型与环境 (G × E) 相互作用维持宿主防御中的遗传多态性 (Blanford et al.,  2003 ; Mitchell et al.,  2005 ; Seppälä et al.,  2011 )。

对宿主免疫功能的条件依赖性的研究通常在实验室实验中进行，这些实验可以操纵可用资源的数量或组成（Brunner 等人，  2014 年；Brzęk & Konarzewski，  2007 年；Ponton 等人，  2020 年；Siva-Jothy & Thompson，  2002 年；Slater & Keymer，  1986 年；Stahlschmidt 等人，  2013 年）。这样的实验是必不可少的，因为它们证明了免疫防御的条件依赖性，同时控制了可能的混杂因素（例如，个体的觅食效率可能不同）并且它们证明了因果关系。然而，这样的实验经常比较极端的资源水平（例如，随意的食物供应与没有食物）。在自然界中，宿主资源水平的变化在任何给定时间点都可能更加微妙，因此很难将这种简化的实验室实验的结果推广到自然种群。因此，扩大免疫防御条件依赖性的工作以考虑田间种群中宿主生理条件的自然变化是当务之急。

在这里，我们使用现场采集的个体检查了普通淡水消费者Lymnaea stagnalis (Gastropoda)中免疫功能的条件依赖性。我们量化了蜗牛血淋巴的两个免疫参数的活性，它们代表免疫系统的不同分支，酚氧化酶 (PO) 样（氧化防御的一种成分）和抗菌活性（Langeloh 等人，  2017 年；Seppälä 和 Jokela，  2010 年； Seppälä 和 Leicht，  2013 年）。这些免疫特征对免疫挑战作出反应（Seppälä & Leicht,  2013），有助于蜗牛健康（Langeloh et al.,  2017）并显示出相当大的种群内遗传变异（即进化潜力；Leicht 等人， 2017 年；Seppälä 和 Jokela，  2010 年；Seppälä 和 Langeloh，  2016 年）。此外，蜗牛的免疫活性取决于环境因素，例如食物供应量 (Seppälä & Jokela,  2010 ; Seppälä et al.,  2021 ) 和环境温度 (Leicht et al.,  2013 , 2017 ;Salo et al.,  2017 , 2019 ; Seppälä等人，  2021 年）。当在实验室条件下通过实验去除蜗牛获取食物的途径时，PO 样活性会在一天内大幅降低，而抗菌活性水平会在几天后降低（Seppälä 和 Jokela，  2010）。这些发现表明，自然界资源可用性的变化可能导致蜗牛种群的疾病爆发。此外，蜗牛免疫系统对食物供应的依赖性显示出种群内家庭水平的变化（即，G × E 相互作用决定了免疫活性；Seppälä 和 Jokela，  2010 年）。这种相互作用表明，环境条件的变化可能会促进蜗牛防御中遗传变异的维持。

为了将免疫活性的变化与蜗牛的资源水平联系起来，我们根据最近消耗的资源的数量和组成测量了反映它们状况的几个因素（即在过去几天内；数量和稳定同位素组成 [ ¹⁵ N: ¹⁴ N 比率表示为 δ ¹⁵ N 和¹³ C: ¹² C 比率表示为 δ ¹³ C] 产生的粪便）和更长的时间段（即，数周至数月；组织的稳定同位素组成；Li 等人。 ,  2018）。我们发现，在自然条件下（即蜗牛资源水平和使用的自然变化），蜗牛血淋巴的 PO 样活性取决于条件。最近食用高 δ ¹⁵ N 值食物的蜗牛具有更强的防御能力。考虑到粪便的 δ ¹⁵ N 和 C:N 比率之间的协变，结果表明来自更高营养水平的资源消耗，可能包括更多的动物蛋白，增强了血淋巴 PO 样活性。此外，组织中具有高 δ ¹³ C 值的蜗牛具有高 PO 样活性。基于 δ ^{13之间的协变}组织的 C 和 C:N 比值，该结果表明蜗牛的脂质储备与 PO 样活性之间存在负相关关系，这可能源于免疫活动的能量消耗。