Urbanization-driven climate change increases invertebrate lipid demand, relative to protein—a response to dehydration

1. Climatic change alters not only animal energy balance, but also water balance, but this latter topic has received less attention. Water can be obtained through consumption of moist food and metabolism of dry food. The breakdown of carbohydrates, lipids, and proteins can produce metabolic water. Metabolism of lipids produces large amounts of water, whereas excretion of nitrogenous waste related to protein metabolism requires water losses.

2. Here we tested the hypothesis that climatic shifts associated with urbanization influences animal lipid demand relative to protein, due to shifts in water balance.

3. We placed artificial diets high in lipid or protein, and either with or without supplemented water, at 16 pairs of sites along an urbanization gradient in Toledo, OH, USA.

4. Lipid consumption, relative to protein, increased with urbanization and mean temperature, but water supplementation reduced the magnitude of this association. Ants were ~50% of the observed consumers.

5. These results suggest that shifts in nutritional demand with climatic change are partially predictable from physiological first principles related to water balance and nutrient metabolism. Because ants and other arthropods play key roles in many food webs and ecosystems, increased demand for lipids with urbanization or climate change could have major consequences for ecosystem services (e.g. urban waste removal, seed predation). Overall, our results suggest that warming related to urbanization increases animal demand for lipids, in part to maintain water balance, and this could have important implications for both animal health and ecosystem services.

Methods
Study Sites

We selected 16 pairs of sites along an urbanization gradient (Figure 1, Table S1) within Toledo, OH and surrounding areas, examining the effects of impervious surface at local scales (50 m radius buffer) nested within a coarser scale (500 m radius buffer). Pairs were distributed throughout the region, no more than 15 km from the city center and no less than 3 km from each other. We selected each site pair by considering Toledo’s landscape features (e.g. impervious surface), accessibility of sites, and obtained permissions.

Diets and Consumption Measurement

Two artificial diets varying in lipid and protein were used in this study. The high-lipid diet was composed of 1:1:5 Protein: Carbohydrate: Lipid (P: C: L) and the high-protein diet had a 5:1:1 P: C: L ratio (Table S2). The protein components were composed of three different foods, because each food item did not offer an even and complete suite of amino acids. The diet’s final amino acid profile was validated by Lebensmittel Consulting Co, Fostoria, OH. Diets were deposited into clean metal bottlecaps and dried at 50°C in a drying oven (100L Gravity Oven, model 51030520, Fisher Scientific, Hampton, NH). Bottlecaps were then attached to small petri dishes (Figure S1) with a non-toxic glue dot (0.5” removable dot, Glue Dots Intl., Germantown, WI). The bottlecaps were used for simplicity, while the small petri dish captured particles of food displaced from the bottlecap (modified from Clissold et al. 2014). Food-filled bottlecaps were placed inside small labeled plastic bags and then weighed to 0.01 mg (Micro Balance, model XPE56, Mettler Toledo, Columbus, OH). When diets were collected from the field, they were placed into the same bags as before. Diets were then dried inside their open plastic bags, dirt and frass was removed, then they were weighed a final time within their bags, with differences in mass indicating consumption.

Consumption Trials

Within each site, we selected six trees that were no less than 10 m apart and placed one set of consumption measurement materials at each tree (Figure S1). Each consumption measurement setup was comprised of a high-lipid and high-protein diet, a wet or dry water pillow (small pouches filled with a polymer that absorbs water; Cricket water pillows, Zilla, Franklin, WI), and a cage to exclude mammals. The addition of a water source allowed us to isolate the effects of water balance from metabolic rate on consumption. The wet and dry pillows were assigned to measurement locations in a stratified order at each site. Cages were made of hardware wire laced together with green floral wire and were fixed to the ground with landscape staples. To prevent rain or UV radiation from altering the pillows and diet, we placed a covering on the cages, made of a large petri dish sprayed with translucent UV protectant (Model 1305 Gallery Series, Krylon Products Group, Cleveland, OH). The lid could also be removed to make observations with minimal disturbance to the arthropods inside.

We measured consumption three times at each site, from June – August 2016, shuffling the order in which sites were visited (pairs of sites were initially randomly assigned to one of four groups and within each group the order of site visits was reversed during the second trial).  Each measurement took place over three days: cages were placed on the first day, sites were visited during the morning and at night on the second day, to make observations of consumption, and cages were removed on the third day. Cages were not placed during a storm, but cages were visited on the second and third day, regardless of weather conditions.

Other Measurements 

We measured temperature and humidity for each survey using three data loggers (Thermochron iButton, model DS 1923, Maxim Inc., San Jose, CA), placed within cages, spread evenly within a site. Each iButton was attached to a Styrofoam covering protecting it from solar radiation while allowing exchange with the atmosphere. With each visitation, we measured soil moisture (SM 150 soil moisture sensor, Dynamax, Houston, TX) and canopy cover (Mobile application software, HabitApp v. 1.1, Scrufster) three times, within 0.5 m of each cage. Canopy cover measures via HabitApp were verified to be comparable to a densiometer before use. We also identified invertebrates located in our experimental setups during each visit via photographs. Most of these photographs were taken by the same person (J. Becker) and this person performed all identifications from photographs. Some invertebrates moved too quickly to be photographed. These individuals were noted, but not included in analyses, due to the lack of identification. This could have resulted in under-recording highly-mobile taxa.

1. 气候变化不仅会改变动物的能量平衡，还会影响其水平衡，但后者受到的研究关注相对较少。动物可通过取食湿润食物和代谢干燥食物获取水分：碳水化合物、脂质与蛋白质的分解均可产生代谢水（metabolic water）；脂质代谢可生成大量水分，而蛋白质代谢产生的含氮废物（nitrogenous waste）排泄过程则会造成水分流失。

2. 本研究验证了如下假说：与城市化相关的气候变化会通过改变动物的水平衡，进而影响其脂质相对于蛋白质的需求。

3. 我们在美国俄亥俄州托莱多市沿城市化梯度设置了16组样地，分别放置高脂质、高蛋白质两种人工饲料，且每组饲料均设置补水与不补水两个处理。

4. 相较于蛋白质，动物的脂质取食率随城市化程度与平均温度升高而上升，但补水可削弱这一关联的强度。实验中观察到的取食者中，蚂蚁占比约50%。

5. 研究结果表明，气候变化下动物的营养需求变化可部分通过与水平衡及养分代谢相关的生理学基本原理进行预测。由于蚂蚁与其他节肢动物（arthropods）在诸多食物网与生态系统中发挥关键作用，城市化或气候变化引发的脂质需求上升可能会对生态系统服务（ecosystem services）产生重大影响，例如城市垃圾清理、种子捕食等。综上，本研究结果显示，与城市化相关的气候变暖会提升动物对脂质的需求，这一过程部分是为了维持体内水平衡，这对动物健康与生态系统服务均具有重要意义。

## 研究方法

### 研究样地
我们在俄亥俄州托莱多市及其周边区域沿城市化梯度选取了16组样地（图1、附表S1），考察嵌套于粗尺度（500米半径缓冲区）下的局域尺度（50米半径缓冲区）不透水面（impervious surface）的影响。样地对分布于整个区域，距市中心不超过15千米，且样地对之间间距不小于3千米。样地对的选取综合考虑了托莱多的景观特征（如不透水面）、样地可达性以及场地许可情况。

### 饲料与取食量测定
本研究使用两种脂质与蛋白质比例不同的人工饲料：高脂质饲料的蛋白:碳水:脂质（P:C:L）比例为1:1:5，高蛋白质饲料的P:C:L比例为5:1:1（附表S2）。由于单一食物无法提供均衡完整的氨基酸组合，蛋白质组分由三种不同食物混合而成。饲料最终的氨基酸谱由俄亥俄州福斯托里亚市的Lebensmittel咨询公司完成验证。将饲料置于洁净的金属瓶盖中，于50℃的干燥烘箱（100L重力对流烘箱，型号51030520，Fisher Scientific，新罕布什尔州汉普顿市）中烘干。随后使用无毒胶点（0.5英寸可移除胶点，Glue Dots Intl.，威斯康星州日耳曼敦市）将金属瓶盖固定于小型培养皿中（附图S1，修改自Clissold等人2014年的方法），此举既可简化操作，又可通过培养皿收集从金属瓶盖脱落的食物碎屑。将装有饲料的金属瓶盖装入标注好的小型塑料袋中，以精度0.01毫克的微量天平（Micro Balance，型号XPE56，Mettler Toledo，俄亥俄州哥伦布市）称重。野外回收饲料后，将其放回原塑料袋中，烘干后去除污垢与虫粪，再次称重，通过前后质量差计算取食量。

### 取食实验
在每个样地中选取6棵间距不小于10米的树木，在每棵树上放置一套取食量测定装置（附图S1）。每套装置包含高脂质与高蛋白质饲料各一份、补水或不补水的水枕（填充吸水聚合物的小型袋状物；蟋蟀用水枕，Zilla，威斯康星州富兰克林市），以及用于排除哺乳动物的笼具。设置水源可帮助我们分离水平衡与代谢速率对取食行为的影响。每个样地的补水与不补水处理按分层随机原则分配至各测定点位。笼具由五金网用绿色花艺铁丝绑扎而成，并用景观固定钉固定于地面。为避免雨水与紫外线辐射影响饲料与水枕，我们在笼具上方加盖经半透明紫外线防护剂喷涂的大型培养皿（型号1305 Gallery Series，Krylon Products Group，俄亥俄州克利夫兰市），该盖子可移除，以便在尽量不干扰笼内节肢动物的前提下进行观察。

我们于2016年6月至8月间在每个样地开展三次取食实验，调整样地访问顺序：初始时将样地对随机分为四组，第二次实验时每组内的样地访问顺序反转。每次实验持续三天：第一天放置笼具，第二天在上午与夜间前往样地观察取食情况，第三天移除笼具。若遇暴雨则不放置笼具，但无论天气如何，均需在第二、第三天对笼具进行回访。

### 其他测定指标
每次调查时，我们在笼具内放置三台数据记录仪（Thermochron iButton，型号DS 1923，Maxim Inc.，加利福尼亚州圣何塞市），均匀分布于样地内，以测定温度与湿度。每台iButton均包裹有聚苯乙烯泡沫罩，既可避免太阳辐射干扰，又可保证与大气的气体交换。每次回访时，我们在每个笼具周边0.5米范围内三次测定土壤含水率（SM 150土壤湿度传感器，Dynamax，得克萨斯州休斯顿市）与冠层盖度（使用移动应用程序HabitApp v.1.1，Scrufster开发）。实验前已验证，通过HabitApp获取的冠层盖度数据与使用郁闭度计的测定结果具有可比性。此外，我们在每次回访时通过照片记录实验装置内的无脊椎动物，绝大多数照片由J. Becker拍摄并完成物种鉴定。部分移动过快无法拍摄的无脊椎动物仅作记录，未纳入数据分析，这可能导致高移动性类群的记录存在遗漏。