DOI: 10.5061/dryad.8kprr4xvx
title: Seasonal patterns of resource use within natural populations of burying beetles

description: For organisms in temperate environments, seasonal variation in resource
 availability and weather conditions exert fluctuating selection pressures
 on survival and fitness, resulting in diverse adaptive responses. By
 manipulating resource availability on a local spatial scale, we studied
 seasonal patterns of resource use within natural populations of burying
 beetles (Nicrophorus vespilloides) in a Norfolk woodland. Burying beetles
 are necrophagous insects that breed on vertebrate carcasses. They are
 active in Europe between April and October, after which they burrow into
 the soil and overwinter. Using breeding and chemical analyses, we compared
 the fecundity and physiological state of beetles that differed in their
 seasonal resource use. We found seasonal variation in carrion use by wild
 burying beetles and correlated differences in their reproductive success
 and cuticular hydrocarbon profiles. Our results provide novel insights
 into the seasonal correlates of behaviour, physiology, and life history in
 burying beetles.
description: <strong>PART A: EXPERIMENTAL PROTOCOLS AND
 METHODS</strong> <strong>Question 1 or
 3: Is there seasonal variation in the trapping frequency of
 burying beetles on chick and mice carrion between June and
 August?</strong> Study area
 and trapping methods We sampled the burying
 beetle population at Thetford Forest (52°20'39.5" N
 0°32'14.9" W), Norfolk, UK from May to October 2017 at 10
 different trap locations (See Methods table 1 below), under permit from
 Forestry Commission England. We used carrion-baited beetle traps (Japanese
 Beetle Trap Kit from Scotts Co., not treated with any pheromones),
 suspended in vegetation 1-2m above ground. The bottom half of the trap was
 filled with Miracle-Gro compost, and a small dead vertebrate was placed on
 the top as bait. The contents of the trap were collected at intervals and
 brought back to the lab for processing. The trap was then refilled and
 rebaited. After processing in the lab, no beetles were released back into
 the field. Beetles were sampled using a paired-trap
 arrangement, in which we placed two beetle traps- one baited with a dead
 domestic chick and the other baited with a dead mouse- near each other at
 each trap location and recorded the number of beetles found in each trap.
 The traps within each experimental pair were placed 1-2 m apart. Pairs of
 traps were placed 200- 400 m apart from each other. With this design,
 beetles were given a simultaneous choice between a dead mouse and a dead
 chick. Each time we rebaited a trap with carrion, we rebaited it with the
 alternate carrion type. Therefore, if a mouse carcass had been placed in
 the trap previously, it was replaced by a chick carcass on the next
 sampling trip to ensure that the trap location itself did not bias beetle
 catch. The mice and chick carcasses used were matched in weight (30-40
 g). Processing field-caught
 beetles At the lab, we used carbon dioxide to
 immobilise each beetle and brush off any mites stuck to it. We recorded
 the pronotum width and sex of each <em>N. vespilloides</em>
 beetle we trapped. We compared beetles collected at two different time
 points during the burying beetle season: the first set was collected in
 June 2017 after 10 days of trapping between 4 June and 14 June, and the
 second set was collected in August 2017 after 15 days of trapping between
 4 August and 19 August. The 10 trapping locations (Methods Table 1) were
 the same across both sampling periods.
 <strong>Question 2 or 3: Does reproductive success
 vary with carrion substrate and/or
 season?</strong> <em>Measuring
 reproductive performance</em> After collecting
 beetles from the traps, measuring and identifying them, we put each
 <em>N. vespilloides</em> individual into its own personal
 small plastic box (12 cm × 8 cm × 2 cm) and fed it 1 g of beef mince. The
 beetles were stored alone in their boxes for 7-10 days to ensure that any
 newly eclosed individuals had had sufficient time to become sexually
 mature before we measured their reproductive performance.
 For breeding, we placed a pair of beetles (one male and one
 female) in a larger plastic breeding (17 cm x 12 cm x 6 cm) box
 half-filled with Miracle-Gro compost and provided with either a chick or
 mouse carcass. Each member of the pair had been trapped on the same type
 of carrion and we bred them on the same carrion they were trapped upon.
  This method was used twice, once for beetles collected in June and once
 for those collected in August, yielding four treatments in all.
 The mass of the carcass provided for reproduction was recorded
 and kept consistent within each treatment. We then placed the breeding box
 inside a cupboard so that it was shielded from light to mimic the low
 light conditions typically experienced by beetles as they breed below
 ground. Eight days after pairing the beetles (i.e., the point at which the
 larvae had completed development and were starting to disperse away from
 the remains of the carcass), we counted and weighed the surviving larvae
 from each pair. We used the following measures to
 record reproductive success in our experiments: Brood failure: We
 recorded the total number of broods that failed to produce any larvae: 0
 denoted broods that failed, 1 denoted those that had at least one
 surviving larva at 8 days post dispersal. Brood size:
  The total number of dispersing larvae 8 days post-breeding.
 Average larval mass: Total mass of the brood at dispersal (g)
 divided by the brood size. Larval density: Brood size
 divided by the mass of the carrion used for breeding (g).
 Carcass use efficiency: (total brood mass (g) divided by
 original carrion mass (g)) x 100% In June,
 53 pairs of beetles trapped on mice (MM) and 24 pairs of beetles trapped
 on chicks (CC) successfully produced broods with at least one larva. There
 were 4 failed broods (3 on mice carcasses and 1 on a chick carcass). In
 August, 16 pairs of beetles trapped on mice (MM) and 25 pairs of beetles
 trapped on chicks (CC) produced broods with at least one larva. There were
 7 failed broods (2 on mice carcasses and 5 on chick carcasses). The failed
 broods were excluded from analyses of reproductive success.
 <strong>Question 3 or 3: Do beetles that are
 attracted to different types of carrion also differ predictably and
 seasonally in their CHCs? </strong> For
 this experiment, we sampled a total of 63 females; 32 were trapped on
 chicks and 31 were trapped on mice. 40 females were collected on 23 May
 2017 (“early” season- 20 on chicks and 20 on mice). 6 females were
 collected on 14 June 2017 (“mid” season- 3 on chicks and 3 on mice). 17
 females were collected on 4 September 2017 (“late season”- 9 on chicks and
 8 on mice). After removing the mites from the body of the beetles, we
 isolated up to two female beetles from each trap individually in a glass
 vial for 15-20 min before storing them in a fresh vial at -80 °C. Later,
 we processed the beetles for CHC extraction by allowing them to thaw at
 room temperature for 30 min. We then soaked them in 4 ml of solvent (99%
 hexane, HPLC grade) for 20 mins. We transferred the extract obtained to a
 clean vial and allowed it to evaporate completely in a fume hood under
 nitrogen gas. At this stage, the sealed vials were shipped to Prof.
 Patrizia d'Ettorre’s lab at Université Sorbonne Paris Nord for
 analysis and characterisation. <em>CHC analysis
 and characterisation</em> We resuspended the
 extract in 400 µl of pentane (HPLC grade) and added an internal standard
 (C18, Octadecane at 16ng/µL) to each extract. The internal standard was
 used to determine the absolute amount of cuticular compounds present in
 each sample. We then analysed 2µl of the extracts using GC-MS (Agilent
 Technologies 7890A gas-chromatograph coupled to a 5975C Mass Spectrometer
 equipped with a HP5MS GC column (30 m × 0.25 mm × 0.25 μm) and operated at
 70 eV in the electron impact ionization mode). The carrier gas used was
 helium at 1 ml/min. The column oven was programmed as follows: an initial
 hold of 1 min at 70°C, then increased to 200°C at 35°C/min, to 320°C at
 4°C/min (held for 20 min). We identified cuticular
 hydrocarbons based on their retention times (compared to standards) and
 fragmentation patterns. We manually integrated the chromatograms and
 converted the peak areas of the total hydrocarbon fraction using the MSD
 ChemStation software by Agilent Technologies, Inc.
 <strong>PART B: DATA VISUALISATION AND STATISTICAL
 ANALYSES</strong> <em>Field and
 reproductive success data</em> We carried out all
 statistical analyses to test our predictions using R (RStudio version
 1.3.959) with generalised linear models (GLM) and generalised linear mixed
 models (GLMM) using the lme4, glmmsr, and MASS packages.
 Analysis-of-variance tables for model objects were calculated using the
 ‘car’ package. Post-hoc comparisons using Tukey’s HSD test were carried
 out using the package ‘lsmeans’. The asymptotic test for the equality of
 coefficients of variation (CV) was carried out using the ‘cvequality’
 package (Feltz & Miller 1996).
 <strong>Question 1 or 3: Is there seasonal
 variation in the trapping frequency of burying beetles on chick and mice
 carrion between June and August?</strong>
 We calculated the average number of beetles per day by dividing
 the total number of <em>N. vespilloides</em> beetles found in
 a trap by the number of days the traps had been left out. We focussed on
 the two different time points for which we also measured reproductive
 outcome, namely June and August 2017, using a GLMM that included carrion
 type and sampling month as fixed effects, and trap ID and sampling date
 (to account for any differences in sampling effort) as random factors with
 a Poisson error structure. The total number of <em>N.
 vespilloides</em> beetles found in a trap on the sampling day was
 used as the response variable. <strong>Question 2
 or 3: Does reproductive success vary with carrion substrate
 and/or season?</strong> We examined the
 effect of month-trapped, carcass type used for breeding and their
 interaction on the following measures of reproductive success:
 brood
 success versus failure, using a multivariate logistic regression model
 with a binomial error term  the number of dispersing
 larvae, using a GLM with a Poisson error term 
 average larval mass using a linear model 
 larval density using a linear model carcass
 uses efficiency using a linear model When
 arriving at a minimal model using GLMs and GLMMs to explain our results,
 we removed non-significant terms and interactions using stepwise
 elimination. When presenting the results from post-hoc analyses, we list
 all the terms that were tested, and their statistics at the last point
 when they were retained in the model.
 <strong>Question 3 or 3: Do beetles that are
 attracted to different types of carrion also differ predictably and
 seasonally in their CHCs? </strong> To
 analyse the chemical profile of both sets of beetles, we selected the 17
 most regularly occurring GC-MS peaks (Methods Table 2). These represented
 the hydrocarbons we had identified and integrated using the MSD
 ChemStation software. We carried out the principal
 component analysis, hierarchical clustering, and visualisation of the data
 using <em>ggplot2</em>,<em> dplyr, pvclust, FactoMineR,
 </em>and<em> factoextra</em> packages in R (RStudio
 version 1.3.959). We log-normalised the peak areas
 within each sample using the following formula (Aitchison 1982):
 Z<sub>ij</sub>
 = ln [Y<sub>ij</sub>/g(Y<sub>j</sub>)]
 where Z<sub>ij </sub>is the transformed area of
 peak<em> i </em>for beetle <em>j</em>;
 Y<sub>ij</sub> is the area of peak <em>i</em> for
 beetle <em>j</em>; and g(Y<sub>j</sub>) is the
 geometric mean of the areas of all peaks for beetle
 <em>j</em>. We used the standardised area
 values of the 17 peaks for hierarchical cluster analysis with Ward’s
 classification method to classify our samples. The significance of each
 node in the cluster was determined by multiscale bootstrap clustering with
 10,000 iterations using the ‘pvclust’ package in R (Suzuki et al. 2019).
 We set the confidence level for the p-value threshold to 95% and ensured
 that only the most significant clusters (with a p-value lower than 0.05)
 were highlighted. To examine how the CHC compounds
 found in our samples contribute to the discrimination of these samples, we
 performed a principal component analysis (PCA) on the log-normalised peak
 areas. We implemented the PCA using singular value decomposition (Hartman
 et al. 2023) for better numerical accuracy. Plotting standard deviations
 of principal components (PCs) and proportion of variances against all 17
 PCs, we used the elbow method to determine the optimal number of PCs that
 explained the maximum amount of variance in our data (Jolliffe 2002). 
 Based on the visual inspection of the elbow plots, we retained the first
 10 principal components,  which capture most of the variance in the data
 (96.3%). We then visualised the data using a heatmap depicting the
 loadings of the first 10 PCs. To check the correlation between the first
 two Principal Components and the original variables, we calculated the
 squared cosine value (cos2) for each variable by squaring the cosine of
 the angle between the vector with the variable's coordinates and the
 origin of the graph. Cluster validation of our data
 indicated one outlier (Sample M13E). We confirmed this visually by using a
 2-dimensional scatterplot before removing the outlier. We then repeated
 our PCA and clustering analysis without this data point.
 <strong>PART C: APPENDIX</strong>
 <strong>Methods Table 1: Geographical coordinates of
 trapping locations at Thetford Forest</strong> 
 
 <strong>Trap location number</strong>
 
 <strong>Latitude</strong> 
 <strong>Longitude</strong>
 1
 52.3443120’N 
 0.5374620’W 
 2 
 52.3428090’N 
 0.5382080’W 
 3 
 52.3446849’N 
 0.5406787’W 
 4 
 52.3435720’N 
 0.5398280’W 
 5 
 52.3443100’N 
 0.5397050’W 
 6 
 52.3451601’N 
 0.5406294’W 
 7 
 52.3434220’N 
 0.5414500’W 
 8 
 52.3449940’N 
 0.5430960’W 
 9 
 52.3440520’N 
 0.5441710’W 
 10 
 52.3471700’N 
 0.5435390’W 
 <strong>Methods Table 2:
 </strong>Identification of the 17 most regularly occurring peaks in
 the cuticular hydrocarbon profile of <em>N. vespilloides.
 </em>Diagnostic ions are provided. 
 
 <strong> </strong> 
 <strong>Retention time (min)</strong>
 
 <strong>Compound</strong> 
 <strong>Diagnostic EI ions
 (m/z)</strong> 
 Internal standard 
 7.47 C18 (IS)
 (Octadecane) 254
 1
 10.59 
 C21 (Heneicosane) 
 296 
 2 11.95
 C22 (Docosane) 
 310 
 3 
 13.46 C23
 (Tricosane) 324
 4
 14.57 
 3MeC23 (3-methyltricosane) 
 57, 309, 281, 323 
 5 
 16.25 C25:1
 (Pentacosene) 350
 6
 16.34 
 C25:1 (Pentacosene) 
 350 
 7 16.66
 C25 (Pentacosane) 
 352 
 8 
 17.45 5MeC25
 (5-methylpentacosane) 85, 309,
 281, 351 
 9 17.85
 3MeC25 (3-methylpentacosane)
 57, 337, 309, 351 
 10 
 18.42 
 3,9diMeC25 (3,9dimethylpentacosane) 
 57, 155, 252, 351, 365 
 11 
 19.57 C27:1
 (Heptacosene) 378
 12
 19.66 
 C27:1 (Heptacosene) 
 378 
 13 19.92
 C27 (Heptacosane) 
 380 
 14 
 21.13 3MeC27
 (3-methylheptacosane) 57, 365,
 337, 379 
 15 21.69
 3,9 diMeC27
 (3,9-dimethylheptacosane) 57,
 155, 281, 379, 393 
 16 22.81
 C29:1 (Nonacosene) 
 406 
 17 
 23.17 C29
 (Nonacosane) 408
description: # Seasonal patterns of resource use within natural populations of burying
 beetles
 [https://doi.org/10.5061/dryad.8kprr4xvx](https://doi.org/10.5061/dryad.8kprr4xvx) # Description of the data and file structure The data are structured in the same way as the manuscript- with separate data files for each question addressed: **Q1. Is there seasonal variation in the trapping frequency of burying beetles on chick and mice carrion between June and August?** Data file name: *traps.csv* *traps.csv* has the following columns (in bold). An italicised description has been added below each column heading. The term "beetle(s)" here refers to an individual(s) of the species *Nicrophorus vespilloides*. | trip number | day | month | trap setup on | beetles collected on | number of days collected for | trap | carcass | females | males | total | per day | | :---------------------------------------------------------------------------------------------------------------------------- | :------------------------------------------------------------------------------------------------------------------------------------------------------------------ | :---------------------------------------------------------- | :------------------------------------------------------------------------------------------- | :------------------------------------------------------------------------------------------ | :---------------------------------------------------------------------------------------------- | :---------------------------------------------------------------------------- | :----------------------------------------------------------- | :--------------------------------------------- | :------------------------------------------ | :----------------------------------------------------------------------------------------- | :--------------------------------------------------------------------------------------------------------------------------------- | | *The collection trip in June marked the 4th trip of the 2017 season and the collection trip in August 2017 was the 10th trip* | *Indicates the number of days since the trapping experiment was started in Thetford (22nd day for the June collection & 88th day for the August collection trip)*  | *Month of collection ("Jun" for June and "Aug" for August)* | *Date of baiting the trap with a chick or mouse carcass for that particular collection trip* | *Date of collecting beetles from the trap previously baited with a chick or mouse carcass*  | *Sampling effort or the total number of days the trap was left in the field to collect beetles* | *The trapping location (#1 to #10). For locational coordinates see "Methods"* | *Carcass type the trap was baited with ("Chick" or "Mouse")* | *Number of female beetles baited in the trap* | *Number of male beetles baited in the trap* | *Total number of beetles baited in the trap (Sum of male and female beetles in each trap)* | *Total number of beetles baited in the trap divided by the total number of days the trap was left in the field to collect beetles* | **Q2. Does reproductive success vary with carrion substrate and/or season?** Two data files, one for each "sub-question" addressed. *2.1 Testing the relationship between sampling month and reproductive performance in wild Thetford beetles* Data file name: *fitness.csv* *fitness.csv* has the following columns (in bold). An italicised description has been added below each column heading. The term "beetle(s)" here refers to an individual(s) of the species *Nicrophorus vespilloides*. | collected | carcass | cwt | female | male | broodsize | broodmass | avlwt | efficiency | density | | :--------------------------------------------------------------------- | :---------------------------------------------------------------------------------------------------------------------- | :---------------------------------------------------------------------------- | :------------------------------------------------------- | :----------------------------------------------------- | :--------------------------------------------------------------------------------------- | :--------------------------------------------------- | :-------------------------------------------------------------------------- | :------------------------------------------------------------------------------- | :------------------------------------------------------------------------------- | | *The month of beetle collection ("jun" for June and "aug" for August)* | *The carcass type the beetle was baited on and subsequently bred on ("m" for mouse carcass and "c" for chick carcass)* | *Mass of the carcass (in grams) provided to the pair of beetles for breeding* | *Identity marker of the female collected from the field* | *Identity marker of the male collected from the field* | *The total number of dispersing larvae at 8 days post setting up of breeding experiment* | \*Total mass of the brood at dispersal (in grams) \* | *Total mass of the brood at dispersal (in grams) divided by the brood size* | *Total brood mass (in grams) divided by original carrion mass (in grams) x 100%* | *Brood size divided by the mass of the carrion used for breeding (unit: /grams)* | *2.2 Testing the relationship between carcass size and reproductive performance in Thetford-derived beetles* Data file name: *consolidated.csv* *consolidated.csv* has the following columns (in bold). An italicised description has been added below each column heading. The term "beetle(s)" here refers to an individual(s) of the species *Nicrophorus vespilloides*. | carcass type | csize | bred on | dispersed on | carcass wt | larval number | total mass | average larval mass | efficiency | larval density | | :----------------------------------------------------------------------------------------------------- | :---------------------------------------------------------------------------------------------------------------------------------------------------------------- | :----------------------------------------------- | :---------------------------------------------------------------------------------------------------------------------- | :---------------------------------------------------------------------------- | :--------------------------------------------------------------------------------------- | :--------------------------------------------------- | :-------------------------------------------------------------------------- | :------------------------------------------------------------------------------- | :------------------------------------------------------------------------------- | | *The carcass type the beetles were breed on ("mice" for mouse carcass and "chicks" for chick carcass)* | *The carcass size the beetles were breed on \["large" for large carcass with mass 20.64±1.20 S.D (g) and "small" for small carcass with mass 8.23 ±0.70 S.D (g)]* | *The date of setting up the breeding experiment* | *The date of dispersal/larval counts of the breeding experiment. This was. always 8 days after the date of setting up.* | *Mass of the carcass (in grams) provided to the pair of beetles for breeding* | *The total number of dispersing larvae at 8 days post setting up of breeding experiment* | \*Total mass of the brood at dispersal (in grams) \* | *Total mass of the brood at dispersal (in grams) divided by the brood size* | *Total brood mass (in grams) divided by original carrion mass (in grams) x 100%* | *Brood size divided by the mass of the carrion used for breeding (unit: /grams)* | **Q3. Do beetles that are attracted to different types of carrion also differ predictably and seasonally in their CHCs?** Data file name: *chcvespilloides.csv* The first data column in chcvespilloides.csv represents the identities of beetles sampled during the CHC experiment. In the sample name, the first letter indicates the carrion type the individual was trapped on (C- chick, M- mouse). The following number is the sample ID/ beetle number, and the last letter indicates the trapping season (E- early, M- mid, L- late). The first row of *chcvespilloides.csv* indicates the 17 most regularly occurring GC-MS peaks in the cuticular hydrocarbon profiles of the beetles we sampled. More information on these 17 compounds can be found in Methods Table 2.  The matrix is, therefore, composed of peak area values (see "Question 3" in the Methods section for more details on how these are calculated) of each compound across the entire sample set of 62 beetle samples. ## Code/Software All data visualisation and analyses were done using R (RStudio version 1.3.959). Please refer to the "Methods" section for a detailed description. The questions addressed in our analyses have been demarcated using annotations in the "Seasonality MS combined.R" R script.