DOI: 10.5061/dryad.tmpg4f57m
title: Impact of light intensity on sugar maple leaf physical traits and consequences for caterpillar preference and performance

description: This study investigates the impact of light intensity on the physical
 defenses of sugar maple (Acer saccharum) saplings and how these defenses
 affect interactions with insect herbivores. Conducted at the Kenauk Nature
 Reserve in Quebec, the study involved 12 sites representing both shaded
 and sunlit environments. Leaf traits, including thickness, specific leaf
 area (SLA), water content, and toughness, were measured from 216 leaves
 collected in 2021 and 40 in 2022. To explore herbivore interactions, two
 Erebid caterpillars including the invasive spongy moth (Lymantria dispar)
 and the native white-marked tussock moth (Orgyia leucostigma) were used in
 controlled bioassays that assessed caterpillar feeding preference and
 performance. The results revealed that sunlit leaves were
 significantly thicker and had lower SLA compared to shaded leaves across
 both years. While water content was lower in sunlit leaves in 2021, it
 showed no significant difference in 2022. Leaf toughness, measured only in
 2022, did not significantly differ between sunlit and shaded leaves.
 Caterpillar feeding preference tests indicated that white-marked tussock
 moth caterpillars consumed more sunlit leaves, showing a significant
 preference for them. However, spongy moth caterpillars did not exhibit a
 significant preference. In terms of performance, white-marked tussock moth
 caterpillars had a higher survival rate and larger pupal mass on sunlit
 saplings in 2021, but no significant differences were observed in
 laboratory feeding initiation assays with first-instar caterpillars in
 2022. These findings suggest that a tree's investment in sun
 leaves to maximise photosynthesis is not necessarily followed by increased
 defense against herbivory. This research contributes to our understanding
 of how environmental factors like light can shape plant defense strategies
 and impact insect herbivore dynamics in temperate forests.
description: Materials and Methods Study site and Plant
 species This study was conducted at the Kenauk Nature
 property, an old-growth natural forest spanning two regions from the
 Outaouais River valley to the Laurentian Mountains in southwestern Quebec,
 Canada (45°42'N; 74°53'W) in 2021 and 2022. In the first year,
 we selected 12 sites in this maple/bitternut hickory domain of temperate
 deciduous forest (Quebec Ministry of Forests, 2023) with each site over 10
 meters apart. Tree species composition was relatively uniform across all
 sites and site selection was made based on the similarity in tree size
 (1–2 m height) and ease of access to the branches. Each
 site comprised one healthy sugar maple sapling in the shade under almost
 full canopy cover and one healthy sugar maple sapling in the sun with less
 canopy cover. Temperature, humidity, and light intensity were measured 10
 times at 4-day intervals at different times of the day from late May to
 early July, and 3 more times in August at 10-day intervals during the
 bioassays in 2021. Canopy openness was measured once at each site in late
 August of the same year. We ensured the average value of light intensity
 and canopy openness showed meaningful differences between shaded and
 sunlit trees per site, but not between sites. For
 visual clarity and to present all experiments and their respective timings
 in one place, an infographic has been included as Figure 1.
 Measurements of leaf physical traits In
 2021, to evaluate the effect of light intensity on leaf defensive traits,
 leaf traits were measured three times. Once right before installing cages
 for a spongy moth performance test in late May, once in early June before
 conducting a spongy moth preference test in the lab, and last time before
 the performance bioassay and preference test with tussock moth in late
 June. Considering the completion of leaf growth, which leads to expected
 stability in leaf traits once leaves are mature, no further measurements
 were taken after this date. On each sampling date, 3 undamaged
 average-sized leaves per sapling were collected per site (In total 216
 leaves = (3 leaves* 2 saplings* 12 sites) * 3 sampling dates). Leaves were
 weighed and their thickness was measured using a digital caliper when they
 were fresh. Subsequently, the leaves were pressed, labeled, and dried in
 an oven for 72 hours. Dried leaves were then weighed again, and all leaves
 were scanned to measure the leaf area using ImageJ software. All efforts
 were made to ensure that dried leaves remained intact and free from any
 folding or breaking to maintain measurement accuracy. This allowed us to
 determine water content and specific leaf area (SLA) for all sampled
 leaves. In early June of 2022, we used 10 of the 12 sites from the
 previous year to collect 20 pairs of leaves (two sunlit and two shaded
 leaves per site) for leaf trait measurements. This year we added leaf
 toughness measurement using a digital penetrometer burrowed from Janice
 Cooke's laboratory at the University of Alberta, Canada.
 Insect defoliators To explore potential
 differences in leaf palatability and susceptibility to insect herbivores
 between sugar maple saplings in shade and sun, we focused on insect leaf
 cutters, which are primarily lepidopteran caterpillars in sugar maple
 forests (Thomas et al., 2010). As a representative of these lepidopterans,
 we selected two common leaf-eating caterpillars, both generalists from the
 Erebidae family which are also feasible to rear in the lab. One
 early-season species, spongy moth caterpillar (<em>Lymantria
 dispar</em>) is an invasive outbreaking pest (Boukouvala et al.,
 2022). The other late-season species named white marked tussock moth
 (<em>Orgyia leucostigma</em>) is native and non-outbreaking
 (Hebert et al., 2017). For the spongy moth, egg masses were collected from
 Angrignon Park in Montreal, QC, CA in late January 2021 and tussock moth
 egg masses were obtained in diapause from Insect Production and Quarantine
 Laboratories (IPQL) in Ontario, CA.  Both species were allowed to hatch
 and were reared in our lab before being placed in the field. All were fed
 on an artificial Bell diet until they finished the first instar and then
 were fed on non-experimental red oak (<em>Quercus rubra)</em>
 and Norway maple (<em>Acer platanoides</em>) leaves prior to
 the experiment. Caterpillars’ feeding
 Preference <em>Laboratory study:</em> We
 conducted a preference test using two caterpillar species in their later
 instars, on sugar maple sapling leaves from shaded and sunny branches in
 2021. The preference experiment was performed on June 17 with 4th instar
 spongy moth caterpillars (early season caterpillar) and on August 12th
 with 4th instar tussock moth caterpillars (late season caterpillar). From
 all previously mentioned 12 sites, 25 undamaged sunlit sugar maple leaves
 and 25 shaded sugar maple leaves were randomly harvested and each stored
 in a 2.0 ml microcentrifuge tube with a hole punched into the lid and
 filled with water. Leaf petioles were fixed and sealed in tubes using
 parafilm to avoid water loss. Subsequently, leaves were sorted by size,
 scanned, and their area measured using Image J. One sunlit and one shaded
 sugar maple leaf of similar size were placed on opposite ends of each of
 the 25 plastic containers (19cm L*13cm W), with a similar leaf area in
 each container. A 4th instar caterpillar was then introduced to each
 container, positioned equidistantly between the two maple leaves to allow
 free choice. After 48 hours, the caterpillars were removed. Then we
 measured the consumed leaf area by rescanning the leaves and calculating
 the area that was gone which was used as the measure for host preference
 (Fortin et al., 1997; DiTommaso & Losey, 2003; Foss &
 Rieske, 2003; Morrow et al., 2022). This whole process was repeated later
 in the season using white-marked tussock moth caterpillars.
 Caterpillars' performance bioassay
 <em>Field study: </em>To assess the performance of
 the caterpillars on sugar maple leaves, we installed one sleeve cage on a
 sunlit sapling and one on a shaded sugar maple sapling in all 12 sites in
 2021. Then, 10 caterpillars in their late 3rd instar were placed in each
 cage (10 caterpillars*12 sites = 120 caterpillars per treatment) and left
 there for approximately 3 weeks. The cages were checked regularly to
 collect pupae and count the remaining caterpillars until they all pupated
 or died. All pupae were weighed to compare the performance of caterpillars
 on shaded and sun branches. This performance test was conducted twice,
 once in the early season (cages and caterpillars set on May 30 and
 collected on June 30), with spongy moth and once in the late season with
 white marked tussock moth (cages and caterpillars set on July 30 and
 collected on August 31), to coordinate with their natural life
 cycles. <em>Laboratory study: </em>In 2022
 we conducted an experiment with early instar tussock moth larvae to
 determine if they would be more affected by the differences detected in
 leaf physical defenses between the two maple saplings considering that
 mortality in lepidopteran first instar larvae is reported to be highest
 compared to the later stages (Zalucki et al., 2002). We were not able to
 get spongy moth larvae in 2022. The same protocol as the previous year was
 followed for obtaining tussock moth egg masses and for collecting and
 storing sugar maple leaves from sunlit and shaded trees in early July in
 Kenauk. Egg masses were hatched and reared on Bell diet for a few days
 till their late first instar stage of life. For this bioassay, a total of
 52 caterpillars at their first instar larval stage were moved to the
 plastic containers (19cm L*13cm W) and were supplied with fresh sunlit
 maple leaves (26 containers * 2 first instar caterpillars per container),
 and the same number of caterpillars were fed on shaded sugar maple leaves.
 Due to our focus on the evaluation of the ability to establish feeding of
 young 1st instar leaf cutter insect herbivores, caterpillars were observed
 after 48, and their survival rate was recorded at this time point
 (Murakami & Wada, 1997b; Zalucki et al., 2001; DiTommaso &
 Losey, 2003). Statistical analysis
 All statistical analyses were conducted using R and RStudio
 (version 2024.04.1+748). Visualizations were generated using the ggplot2
 package in R (Wickham, 2016). <em>Leaf
 traits:</em> To examine differences in leaf traits such as specific
 leaf area, thickness, toughness, and water content between sunlit and
 shaded saplings, the average value measured for three leaves per sapling
 was used. Having normally distributed count data, we employed a linear
 mixed model with sunny and shaded treatments as fixed factors and site as
 a random factor for 2021 and 2022 datasets in separate models using the
 lme4 package (Bates et al., 2015). All model assumptions were eventually
 evaluated by using the DHARMa package (Hartig, 2022).
 <em>Preference test:</em> To assess the effect of
 light intensity on third instar larval preference after 48 hours, the
 experiment was designed with each caterpillar fed in a separate container,
 each containing one shaded and one sunlit sugar maple leaf, with the
 consumed leaf area as the response variable. Since the data from each
 light condition represent paired observations from the same caterpillars,
 a paired t-test was employed. This statistical test accounts for the
 natural variability between individual caterpillars and provides a precise
 evaluation of the effect of light intensity on leaf area consumption by
 comparing the consumption of the two leaf types by the same individual.
 Test assumptions, including normality and equal variance, were evaluated
 using the Shapiro-Wilk test and Levene's test for each caterpillar
 species separately. <em>Performance
 bioassays</em>: In the 2021 cage experiment, the proportion of
 surviving caterpillars per cage was calculated as the survival rate. This
 percentage data with a beta distribution correction was fit in a
 specialized package of betareg model (Cribari-Neto & Zeileis,
 2010). Since the DHARMa package does not work for models with beta
 distribution, we used a Q-Q residual plot for the evaluation of our model
 assumptions. Pupal mass data, as a normally distributed continuous
 response variable with cages having possible effect as a random factor,
 was analyzed using a linear mixed-effects model (LMM) with lmer from the
 lme4 package.  In 2022, having 2 caterpillars per container made the
 survival data a better fit as a binomial distribution to be analyzed with
 a glm with a binomial family. Due to a high incidence
 of spongy moth mortality caused by Nucleopolyhedrovirus (NPV), a common
 viral disease affecting insect larvae, only 4 individuals survived. As a
 result, their performance data was excluded from the analysis.
 Finally, the correlations between caterpillars’
 preference-performance measures and leaf traits, relationships between
 leaf trait variables, as well as differences between sun and shaded
 saplings, were analysed using separate Principal component analysis (PCA)
 for each experiment. For 2021 performance data, the average of three leaf
 trait measurements on May 20, June 10, and June 24 along with survival
 rate and pupal mass were used for PCA. For the preference experiments, we
 used early May leaf trait data for spongy moth and late June leaf trait
 data for tussock moth PCA analysis. Since the number of average leaf
 traits measured was less than the umber of leaves used for the preference
 test, missing data for PCA were handled using multiple imputations (van
 Ginkel, 2023).  Performance from 2022 data was not included in any PCA due
 to its lack of significant difference between treatments.
description: # Impact of light intensity on sugar maple leaf physical traits and
 consequences for caterpillar preference and performance
 [https://doi.org/10.5061/dryad.tmpg4f57m](https://doi.org/10.5061/dryad.tmpg4f57m) ## Description of the data and file structure ### Files and variables #### File: Per.leaf.traits.22.csv **Description:** Leaf traits measured before doing performance bioassay using 1st instar white marked tussock moth in early June of 2022 ##### Variables * site: each site includes two treatments (one sunlit sapling and one shaded sapling) used to collect leaves * leaf.type: These are our treatments including leaves collected from sunlit saplings versus leaves collected from shaded saplings * toughness (N/mm): Leaf toughness was measured for all collected leaf samples * thickness (mm): Leaf thickness was measured for all collected leaf samples * water.content (%): Leaf water content was measured for all collected leaf samples * leaf.area (cm^2): Leaf area was measured for all collected leaf samples * SLA (cm^2/g): Specific leaf area was measured for all collected leaf samples #### File: Per.tussock.22.csv **Description:** This data provides information regarding the non-choice performance test done in the lab using 1st instar white marked tussock moth caterpillars in 2024 ##### Variables * container.number: we used 26 containers per treatment (26 had sunlit leaves and 26 had shaded leaves) with two caterpillars inside per container * leaf.type: These are our treatments inducing: leaves collected from sunlit saplings versus leaves collected from shaded saplings * survived.48: The caterpillars' status was checked after 48 hours and recorded for two caterpillars per container * survived.96: The caterpillars' status was checked after 96 hours and recorded for two caterpillars per container #### File: Tussock.spongy.pereference.21.csv **Description:** This data includes information regarding the choice preference test done in the lab in 2021 using 4th instar spongy moth in June and later using 4th instar white marked tussock moth in August. ##### Variables * leaf.type: These are our treatments including leaves collected from sunlit saplings versus leaves collected from shaded saplings * container.number: 25 containers were used each time with one caterpillar inside and two types of leaves (two treatments) at the two ends of the container * Species: The experiment was done using the 4th instar spongy moth in June and later repeated using the 4th instar white marked tussock moth in August. * prop.eaten (%): Our response variable was the proportion of leaves eaten per container * consumed.area (cm^2): Our response variable was the amount of leaf area eaten per container #### File: Tussock.spongy.performance.csv **Description:**  ##### Variables * cage.number: We had two cages per site and cage. The number shows the site number actually * cage.type: These are our treatments including cages installed on sunlit saplings versus cages installed on shaded saplings * Av.temp (°C): the temperature was monitored and recorded through the field performance experiment and the average amount of all measurements is presented here   * Av.hum (%): Humidity was monitored and recorded through the field performance experiment and the average amount of all measurements is presented here  * Av.light (lux): Light intensity was monitored and recorded through the field performance experiment and the average amount of all measurements is presented here  * Av.canopy.openess (%): Canopy openness was monitored and recorded once through the field performance experiment and the average amount of all measurements is presented here  * survived (%): 10 caterpillars were entered per cage at the beginning of the experiment and their status was recorded per two weeks until they pupated. The number of survived caterpillars per cage was recorded at the end which is shown here  * dead (%): 10 caterpillars were entered per cage at the beginning of the experiment and their status was recorded per two weeks until they pupated. The number of dead caterpillars per cage was recorded at the end which is shown here  * Pupal.mass (gr): The average amount of pupal mass per cage for survived caterpillars was calculated at the end * n.male: number of males survived caterpillars to pupation  * n.female: number of females survived caterpillars to pupation  * Av.thickness (mm): Leaf thickness was measured three times (once before installing the cages, once during the experiment, and once after removing them), and the average of measurements was recorded here  * Av.water.content (%):  Leaf water content was measured three times (once before installing the cages, once during the experiment,t and once after removing them), and the average of measurements was recorded here  * Av.SLA (cm^2/g): Specific leaf area was measured three times (once before installing the cages, once during the experiment, and once after removing them), and the average of measurements was recorded here * Av.leaf.area (cm^2): Leaf area was measured three times (once before installing the cages, once during the experiment,t and once after removing them), and the average of measurements was recorded here ## Code/software See dataset Methods.