Supporting data for tiger beetle stable isotope analysis

Stable isotope ratios give insight into food web interactions, but interpretation can be clouded by dietary shifts and the associated timing of isotopic change, as well as the difference in isotope ratios between consumers and their diets at equilibrium. Typically, the 15N/14N ratio (δ15N) increases with each trophic transfer as 15N becomes enriched, whereas the 13C/12C ratio (δ13C) remains relatively constant with each trophic transfer but can be influenced by the lipid content of the study organism. This study reports the trophic discrimination factors and isotopic half-lives in tiger beetles (Coleoptera: Cicindelidae) collected near a large river in central Canada. Wild-caught tiger beetle larvae were reared in a laboratory setting, subjected to a diet switch experiment, and sampled over a period of 36 days. Quadratic plateau models were used to characterize the change in δ15N, δ13C, and the lipid-corrected carbon ratio (δ13Ccorr) over time, and trophic discrimination factors were calculated by subtracting the mean prey δ15N, δ13C, and δ13Ccorr from that of the tiger beetle asymptotic δ15N, δ13C, and δ13Ccorr values, respectively. The tiger beetle trophic discrimination factor for δ15N was 1.7 ±0.2‰ with a half-life of 11.4 days. For δ13C, the trophic discrimination factor and half-life were –0.6 ±0.2‰ and 3.9 days, respectively. After correcting for lipids (δ13Ccorr), the trophic discrimination factor was –0.2 ±0.2‰ with a half-life of 4.7 days. Our findings show that isotopic turnover of carbon and nitrogen in tiger beetles occurs relatively quickly and is comparable to rates reported for other insects. The trophic discrimination factors and turnover rates calculated in our study could be applied to future studies on wild tiger beetles.

Methods
This repository contains datasets and code used to analyze tiger beetle stable isotopes and body sizes. The main purpose of this study was to calculate the δ15N, δ13C, and δ13Ccorr TDFs and half-lives for tiger beetles. The δ15N TDF is important for calculating an organism's trophic level because N15 is enriched between trophic levels. The δ13C TDF, on the other hand is used to determine the underlying food source of an organism's energy pathway, i.e., C3 or C4 plants, because δ13C stays relatively constant between trophic levels. The δ13Ccorr TDF is more accurate than the δ13C TDF for food web studies because an increase in fat storage can drive δ13C's TDF negatively. TDFs vary between taxa, so having species- or genus-specific values is important for food web studies. Stable isotope half-lives give insight into an organism's metabolism. We used three species to represent tiger beetles (Cicindela duodecimguttata, C. hirticollis, and C. repanda). This was done by subjecting wild-caught tiger beetle larvae to a diet-switch experiment in the laboratory. They were fed a constant diet of Trichoplusia ni caterpillars, and larvae were sampled throughout the experiment. These larvae, along with samples of pupae, exuviae, adults, and T. ni, were sent for stable isotope analysis, where the resulting values were used for most calculations here. With this data, we also chose to compare the mean isotope values at equilibrium between life stages (and exuviae) to see if there were any changes due to pupation rather than diet. Additionally, we calculated the change in C/N over time in the larvae to observe the increase in fat storage. We allowed some tiger beetles to emerge as adults, which we took elytron measurements of and compared to wild-caught adult tiger beetles to estimate the health of the lab-raised adults. We concluded that 1.7‰ is an appropriate TDF for δ15N and 0‰ is appropriate for δ13C because, after correcting for lipids, it was not significantly different from 0. The isotopic half-lives were short, which is consistent with small, fast-growing organisms. However, these rates should be used with caution because they were calculated using the mean initial isotopic ratio, which in our case had wide standard deviations, meaning that our samples may not have been representative of the wild population. We only found at-equilibrium larvae and exuviae to be significantly different in mean δ15N and δ13Ccorr. The C/N ratio did increase linearly throughout the experiment in larvae, suggesting that they did grow fat stores. Lastly, the lab-raised adults were smaller on average compared to wild-caught adults, but this was insignificant.

稳定同位素比率可为食物网相互作用研究提供关键视角，但同位素数据的解译易受膳食转变、同位素变化的相关时序，以及消费者与其平衡态膳食间同位素比率差异的干扰。通常而言，随着15N的富集，每一次营养级传递都会使15N/14N比值（δ15N）升高；而13C/12C比值（δ13C）在各营养级传递中相对稳定，但会受研究生物的脂类含量影响。本研究针对采集自加拿大中部大型河流周边的虎甲（鞘翅目：虎甲科），测定了其营养判别因子（trophic discrimination factors, TDF）与同位素半衰期。研究人员将野外采集的虎甲幼虫置于实验室环境中饲养，开展饮食转换实验，并在36天的周期内进行多次采样。本研究采用二次平台模型刻画δ15N、δ13C与脂质校正碳比值（lipid-corrected carbon ratio, δ13Ccorr）随时间的变化规律，并分别通过虎甲的渐近δ15N、δ13C及δ13Ccorr值，减去对应猎物的平均δ15N、δ13C及δ13Ccorr值，计算得到营养判别因子。虎甲的δ15N营养判别因子为1.7±0.2‰，同位素半衰期为11.4天；δ13C的营养判别因子与半衰期分别为-0.6±0.2‰与3.9天；经脂质校正后（δ13Ccorr），其营养判别因子为-0.2±0.2‰，半衰期为4.7天。本研究结果表明，虎甲体内碳、氮的同位素周转速率相对较快，与已报道的其他昆虫周转速率相当。本研究测得的营养判别因子与周转速率可应用于未来的野生虎甲相关研究。

方法
本研究数据集与代码仓库包含用于分析虎甲稳定同位素与体型的相关数据及代码。本研究的核心目标为计算虎甲的δ15N、δ13C与δ13Ccorr营养判别因子及同位素半衰期。其中，δ15N营养判别因子是计算生物营养级的关键指标，因为15N在营养级间会发生富集。而δ13C营养判别因子则可用于确定生物能量通路的潜在食物来源，即C3或C4植物，因为δ13C在营养级间相对稳定。经脂质校正的δ13Ccorr营养判别因子在食物网研究中精度更高，因为脂类储存的增加会使δ13C的营养判别因子呈现负值偏移。不同类群的营养判别因子存在差异，因此获取物种或属水平的特异性数值对食物网研究至关重要。稳定同位素半衰期可反映生物的代谢特征。
本研究选取3种虎甲作为代表类群：12斑虎甲（Cicindela duodecimguttata）、毛颈虎甲（C. hirticollis）与曲纹虎甲（C. repanda）。实验通过将野外采集的虎甲幼虫置于实验室开展饮食转换实验完成：幼虫以恒定的粉纹夜蛾（Trichoplusia ni）幼虫为食，并在实验全程进行采样。采集的幼虫样本、蛹、蜕、成虫以及粉纹夜蛾样本均被送至实验室进行稳定同位素分析，所得数据用于本研究的绝大多数计算环节。
基于上述数据，本研究还对比了各生命阶段（及蜕）在平衡态下的平均同位素值，以探究是否存在因化蛹而非膳食变化导致的同位素差异。此外，本研究计算了幼虫体内C/N比值随时间的变化，以观测其脂类储存的增长情况。研究人员还将部分虎甲饲养至成虫羽化，对其鞘翅进行测量，并与野外采集的成虫对比，以评估实验室饲养成虫的健康状况。
本研究得出结论：δ15N的营养判别因子采用1.7‰较为合适，而δ13C的营养判别因子以0‰为宜，因为经脂质校正后，其数值与0并无显著差异。本研究测得的同位素半衰期较短，这与小型、快速生长的生物代谢特征相符。但使用该速率时需谨慎：本研究通过初始平均同位素比值计算半衰期，而本研究的初始样本存在较大的标准差，表明样本可能无法完全代表野生种群。本研究仅发现处于平衡态的幼虫与蜕在平均δ15N与δ13Ccorr值上存在显著差异。幼虫体内的C/N比值在实验期间呈线性增长，表明其确实积累了脂类储存。最后，实验室饲养的成虫平均体型小于野外采集的成虫，但该差异并不显著。