DOI: 10.5061/dryad.jh9w0vtmv
title: Supporting data for tiger beetle stable isotope analysis

description: 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.
description: 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
 (<em>Cicindela duodecimguttata</em>, <em>C.
 hirticollis</em>, and <em>C. repanda</em>). 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
 <em>Trichoplusia ni</em> caterpillars, and larvae were sampled
 throughout the experiment. These larvae, along with samples of pupae,
 exuviae, adults, and <em>T. ni</em>, 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.
description: # Supporting data for tiger beetle stable isotope analysis
 [https://doi.org/10.5061/dryad.jh9w0vtmv](https://doi.org/10.5061/dryad.jh9w0vtmv) ## Description of the data and file structure Data archive for: Establishing isotopic turnover rates and trophic discrimination factors in tiger beetle (Coleoptera: Cicindelidae) larvae through a diet switch experiment **Definitions** δ15N = [([15N/14N]sample/[15N/14N]standard) −1] × 1000. In our case, sample = our samples and standard = atmospheric N2. δ13C = [([13C/12C]sample/[13C/12C]standard) −1] × 1000. In our case sample = our samples and standard = Vienna Pee Dee Belemnite. δ13Ccorr = δ13C values that were mathematically corrected for lipids using the following formula: δ13C – (2.056 – 1.907 × ln(C/N)) (Logan et al., 2008). TDF = trophic discrimination factor, the isotopic difference between an organism and it's food source. Logan JM, Jardine TD, Miller TJ, Bunn SE, Cunjak RA, Lutcavage ME. 2008. Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods. *Journal of Animal Ecology* 77:838–846. DOI: 10.1111/j.1365-2656.2008.01394.x. ### Files and variables #### File: Cduodecimguttata\_Sizes.csv **Description:** A dataset containing elytra lengths (µm) and widths (µm) for lab-raised and wild-caught adult *Cicindela duodecimguttat*a. ##### Variables * Code: project specific code used to uniquely identify each tiger beetle and it's life stage. For codes beginning in "CrA", "Cr" = *Cicindela*, "A" = adult, and the number is the unique identifier. For codes beginning in "Cduodecimguttata_WSA", "Cduodeci" = species, and "WSA" = Water security Agency (who lent us the samples). The Water Security Agency had a collection of &gt;10 specimens of *C. duodecimguttata* so we used random.org to decide which specimens to measure between 1 and n with n = the collection size. The number associated with codes beginning in "Cduodeci_WSA" reflect these random numbers and thus denote the n^th specimen in the collection. * Category: Denotes whether the specimen was lab-raised or wild-caught as adults. * ElytraLenAvg: The mean length of each specimen's right and left elytron (µm). * ElytraWidAvg: The mean width of each specimen's right and left elytron (µm). #### File: TniSamples.csv **Description:** A dataset containing information on the tiger beetle food source, *Trichoplusia ni*, and their associated stable isotope values. The mean isotope values in this dataset are how we calculated the tiger beetle TDFs by subtracting the mean isotopic values of the *T. ni* from that of the plateau values. Additionally, the mean δ13C and δ13Ccorr values are used to create a dotted horizontal line in two of the quadratic plateau graphs to visualize this difference. ##### Variables * Date: The date that the sample was taken. * Code: Project specific code assigned to identify each individual sample. "tn" stands for *T. ni*, the first number is the batch number, and the second number is the n^th organism sampled within its batch. * Delta_13C: The organism's δ13C value in parts-per-mille. * Delta_15N: The organism's δ15N value in parts-per-mille. * Delta_13Ccorr: The organism's δ13Ccorr value in parts-per-mille. #### File: TigerBeetle\_Larvae.csv **Description:** A dataset containing information and stable isotope values for the tiger beetle larvae that were subjected to the diet switch experiment. ##### Variables * Day: The day in which the sample was taken relative to when the larva was caught from the wild. * Code: Project specific code used to uniquely identify each tiger beetle and it's life stage. "Cr" = *Cicindela*, "L" = larva, and the number is the unique identifier. * Delta_13C: The organism's δ13C value in parts-per-mille. * Delta_13Ccorr: The organism's δ13Ccorr value in parts-per-mille. * Delta_15N: The organism's δ15N value in parts-per-mille. * CN: The carbon to nitrogen ratio. #### File: Chirticollis\_Sizes.csv **Description:** A dataset containing mean elytron lengths (µm) and widths (µm) for individual lab-raised and wild-caught adult *Cicindela hirticollis*. ##### Variables * Code: Project specific code used to uniquely identify each tiger beetle and it's life stage. For codes beginning in "CrA", "Cr" = *Cicindela*, "A" = adult, and the number is the unique identifier. For codes beginning in "Chirticollis_WSA", "Chirticollis" = species, and "WSA" = Water security Agency (who lent us the samples). The Water Security Agency had a collection of &gt;10 specimens of *C. hirticollis* so we used random.org to decide which specimens to measure between 1 and n with n = the collection size. The number associated with codes beginning in "Chirticollis_WSA" reflect these random numbers and thus denote the n^th specimen in the collection. * Category: Denotes whether the specimen was lab-raised or wild-caught as adults. * ElytraLenAvg: The mean length of the specimen's right and left elytron (µm). * ElytraWidAvg: The mean width of the specimen's right and left elytron (µm). #### File: TigerBeetle\_LifeStages.csv **Description:** A dataset containing information on the at-equilibrium (i.e. post-diet switch) tiger beetle samples and their associated stable isotope values. ##### Variables * Day: The day in which the sample was taken relative to when the larva was caught from the wild. Null is used for exuviae because we do not have the exact date in which the larvae would have moulted. * Code: Project specific code used to uniquely identify each tiger beetle and it's life stage. "Cr" = *Cicindela*, "L" = larva, "E" = exuviae, "P" = pupa, "A" = adult, and the number is the unique identifier. * Category: Specifies the type of sample, i.e. what life stage the sample was or if the sample was exuviae. * Delta_13C: The organism's δ13C value in parts-per-mille. * Delta_13Ccorr: The organism's δ13Ccorr value in parts-per-mille. * Delta_15N: The organism's δ15N value in parts-per-mille. #### File: Crepanda\_Sizes.csv **Description:** A dataset containing elytra lengths (µm) and widths (µm) for lab-raised and wild-caught adult *Cicindela repanda*. ##### Variables * Code: Project specific code used to uniquely identify each tiger beetle and it's life stage. For codes beginning in "CrA", "Cr" = *Cicindela*, "A" = adult, and the number is the unique identifier. For codes beginning in "Crepanda_WSA", "Crepanda" = species, and "WSA" = Water security Agency (who lent us the samples). The Water Security Agency had a collection of &gt;10 specimens of *C. repanda* so we used random.org to decide which specimens to measure between 1 and n with n = the collection size. The number associated with codes beginning in "Crepanda_WSA" reflect these random numbers and thus denote the n^th specimen in the collection. * Category: Denotes whether the specimen was lab-raised or wild-caught as adults. * ElytraLenAvg: The mean length of the specimen's right and left elytron (µm). * ElytraWidAvg: The mean width of the specimen's right and left elytron (µm). ## Code/software All scripts were written in R version 4.3.1 (R Core Team) and the quadratic plateau models used the package rcompanion version 2.4.36 (Mangiafico, 2024). #### File: ElytronSizesStatistics.R **Description:** Used to calculate and compare mean elytron lengths and widths of lab-raised adults to wild-caught adults. **Inputs:** * Cduodecimguttata_Sizes.csv, Chirticollis_Sizes.csv, and Crepanda_Sizes.csv **Outputs:** * The means and standard deviations of elytron lengths (µm) and widths (µm) for lab-raised and wild-caught adult *C. duodecmguttata*, *C. hirticollis*, and *C. repanda*. * Welch's two-sample t-tests comparing mean elytron lengths and widths of lab-raised vs. wild-caught *C. hirticollis* and lab-raised vs. wild-caught *C. repanda*. #### File: LifeStageStatistics.R **Description:** A script to compare the mean isotope ratios of tiger beetle life stages + exuviae. We are aware that our data is not normal or homogeneous, even after transformations, but we decided to proceed regardless. **Inputs:** * TigerBeetle_LifeStages.csv **Outputs:** * Mean and SD of each stable isotope ratio (δ15N, δ13C, and δ13Ccorr) per life stage (larva, pupa, adult, exuviate). * ANOVA tables for each stable isotope ratio. * Tukey HSD tables for δ15N and δ13Ccorr. * Bar graphs comparing the mean isotope ratio for δ15N and δ13Ccorr with SD error bars. #### File: LinearRegression\_CN.R **Description:** A script to plot the C/N ratios of tiger beetle larvae over time. A linear regression line calculated and fit to the graph. The code was adapted from Mangiafico (2016). **Inputs:** * TigerBeetle_Larvae.csv **Outputs:** * Summary statistics of the linear model, most notably the R^2 and F-statistic. * A graph of the larval C/N ratios with a linear regression best fit line. #### File: QuadraticPlateauModel\_Delta13C.R **Description:** Fits a quadratic plateau model to larval tiger beetle δ13C values over time subjected to a diet-switch experiment. This is also where the half-life is calculated. Most code is taken directly from Mangiafico (2016). **Inputs:** * TigerBeetle_Larvae.csv * TniSamples.csv * Initial value for a (= intercept). * Initial value for b (= slope). * Initial value for clx (= critical x value i.e. x value of when the plateau is reached). **Outputs:** * Estimated best-fit value of a. * Estimated best-fit value of b. * Estimated best-fit value of clx. * Half-life of the stable isotope. * Statistics associated with the estimated parameters. * Pseaudo R^2 values (Mcfadden, Cox and Snell, and Nagelkerke). * The plateau value and the isotopic TDF. * A graph of the δ13C values of individual larva over time with the quadratic plateau best-fit line. #### File: QuadraticPlateauModel\_Delta13Ccorr.R **Description:** Fits a quadratic plateau model to larval tiger beetle δ13Ccorr values over time subjected to a diet-switch experiment. This is also where the half-life is calculated. Most code is taken directly from Mangiafico (2016). **Inputs:** * TigerBeetle_Larvae.csv * TniSamples.csv * Initial value for a (= intercept). * Initial value for b (= slope). * Initial value for clx (= critical x value i.e. x value of when the plateau is reached). Outputs: **Outputs:** * Estimated best-fit value of a. * Estimated best-fit value of b. * Estimated best-fit value of clx. * Half-life of the stable isotope. * Statistics associated with the estimated parameters. * Pseaudo R^2 values (Mcfadden, Cox and Snell, and Nagelkerke). * The plateau value and the isotopic TDF. * A graph of the δ13Ccorr values of individual larva over time with the quadratic plateau best-fit line. #### File: QuadraticPlateauModel\_Delta15N.R **Description:** Fits a quadratic plateau model to larval tiger beetle δ15N values over time subjected to a diet-switch experiment. This is also where the half-life is calculated. Most code is taken directly from Mangiafico (2016). **Inputs:** * TigerBeetle_Larvae.csv * TniSamples.csv * Initial value for a (= intercept). * Initial value for b (= slope). * Initial value for clx (= critical x value i.e. x value of when the plateau is reached). **Outputs:** * Estimated best-fit value of a. * Estimated best-fit value of b. * Estimated best-fit value of clx. * Half-life of the stable isotope. * Statistics associated with the estimated parameters. * Pseaudo R^2 values (Mcfadden, Cox and Snell, and Nagelkerke). * The plateau value and the isotopic TDF. * A graph of the δ15N values of individual larva over time with the quadratic plateau best-fit line. R Core Team. 2019. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Mangiafico SS. 2016. Summary and analysis of extension program evaluation in R, version 1.20.05, revised 2023. Rutgers Cooperative Extension, New Brunswick, NJ. Mangiafico, SS. 2024. rcompanion: Functions to support extension education program evaluation. version 2.4.36. Rutgers Cooperative Extension. New Brunswick, NJ. ## Access information Data was derived from the following sources: * All data is primary. Stable isotope data was processed by the National Hydrology Research Centre (Saskatoon, Saskatchewan, Canada).