Constitutive differences in immune gene expression and energy storage phenotypes co-vary with winter environment in wood frogs

Many terrestrial ectotherms have gone to great evolutionary lengths to adapt to long cold winters; some have even evolved the ability to tolerate the freezing of most of the water in the body. Now, however, high-elevation, and high-latitude winters are experiencing an accelerated period of warming. Specialized winter adaptations that promoted fitness in a seasonally frozen environment may soon be superfluous or even maladaptive. We ask whether winter adaptations include changes in immune functions, and whether changing winter conditions could exert disparate effects on populations of a wide-ranging terrestrial ectotherm, the wood frog (Lithobates sylvaticus). By rearing wood frogs from ancestral winter environments that vary in length and temperature in a common garden, and reciprocally crossing post-metamorphic frogs into unfrozen and frozen artificial winter conditions in the lab, we were able to decompose transcriptomic differences in ventral skin gene expression into those that were environmentally induced (responsive to temperature), genetically determined, and those that varied as an interaction between genotype and environment. We found that frogs from harsh ancestral winter environments upregulated immune processes, including cellular immunity, inflammatory processes, and adaptive immune processes, as compared to frogs from mild ancestral winter environments. Further, we saw that expression of several genes varied in an interaction between genotype and artificial winter environment, a pattern that was recapitulated at the level of hepatosomatic index (the proportion of body mass comprising liver). We suggest that just as winter climates likely served as the selective force resulting in remarkable winter adaptations such as freeze tolerance, they also induced constitutive changes in immune gene expression. We tested the hypothesis that immune gene expression would vary differentially based on whether expression is promoted by glycolysis or lipid metabolism, and with respect to an interaction between artificial winter treatment temperature and origin winter environments (‘harsh’ or ‘mild’). We performed a common garden and reciprocal exposure experiment by rearing wood frogs from egg masses in a laboratory. The egg masses were sourced from across the eastern US. Seventeen weeks post-metamorphosis, we sampled frogs for a "pre winter" timepoint, or exposed them to -2° or +2° C. Then we allowed them to overwinter for 9 days, euthanized them, and collected a ventral skin sample for tag-seq analysis of gene expression.

许多陆生变温动物在演化历程中费尽心力以适应漫长寒冷的冬季，其中部分类群甚至演化出耐受体内大部分体液结冰的能力。然而如今，高海拔与高纬度地区的冬季正经历加速升温。曾在季节性冰冻环境中提升个体适合度的特化冬季适应性性状，很快可能变得多余甚至产生适应不良效应。
本研究旨在探究两个核心问题：其一，冬季适应性是否包含免疫功能的改变；其二，冬季环境的变化是否会对广布陆生变温动物——林蛙（Lithobates sylvaticus）的不同种群产生迥异影响。
本研究借助同质园实验（common garden experiment）饲养来自冬季时长与温度存在差异的祖先环境的林蛙，并将变态后的林蛙交叉分配至未冰冻与人工冰冻的实验室模拟冬季环境中，从而将腹侧皮肤基因表达的转录组差异拆解为三类：环境诱导（对温度响应）的差异、遗传决定的差异，以及基因型与环境互作导致的差异。
研究发现，相较于来自温和冬季祖先环境的林蛙，来自严酷冬季祖先环境的林蛙会上调免疫相关通路，包括细胞免疫、炎症反应与适应性免疫通路。此外，本研究观察到多个基因的表达量会随基因型与人工冬季环境的互作发生变化，这一模式在肝体指数（hepatosomatic index，肝脏占体质量的比例）层面得到了重现。
本研究认为，正如冬季气候曾作为选择压力塑造了冰冻耐受等卓越的冬季适应性性状，其同样会诱导免疫基因表达的组成性改变。本研究验证了如下假说：免疫基因的表达差异会因调控其表达的代谢途径（糖酵解或脂质代谢）不同而存在区别，同时也会随人工冬季处理温度与原生冬季环境（"严酷"或"温和"）之间的互作发生变化。
本研究通过实验室饲养卵块来源的林蛙，开展了同质园与交叉暴露实验：实验所用林蛙卵块采自美国东部多地。在变态后第17周，研究人员对部分林蛙进行"冬季前"时间点采样，其余个体则分别暴露于-2℃与+2℃环境中；随后将所有个体越冬饲养9天，处死后采集腹侧皮肤样本用于标签测序（tag-seq）的基因表达分析。