Phylogenetic community structure and stable isotope analysis of the parasitoid community associated with Eastern spruce budworm, Choristoneura fumiferana (Lepidoptera: Tortricidae)

Eastern spruce budworm, Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae), is a major pest of eastern North American forests. Outbreaks of spruce budworm occur every 30–40 years, causing high tree mortality. Researchers have established that higher proportions of hardwood trees within stands (higher hardwood content) may reduce the defoliation and mortality of balsam fir and spruces during spruce budworm outbreaks. One mechanism posited to explain these patterns is that hardwood trees positively impact the parasitoids of spruce budworm. Indeed, parasitism of spruce budworm by parasitoids has been found to be impacted by hardwood content. However, more research is needed to understand how hardwood content impacts the parasitoid community as a whole. In this study, we trialled the use of two analyses, phylogenetic community structure and stable isotope analysis, to examine how hardwood content influenced the parasitoid community associated with spruce budworm. We found that phylogenetic community structure differed between forest stands with different hardwood content. Furthermore, the trophic relationships between several parasitoids and caterpillars on balsam fir or hardwood trees changed within and between years. Our study highlights the potential of these two analyses for understanding how hardwood content influences the parasitoid community associated with spruce budworm.


INTRODUCTION
Every 30-40 years, Eastern spruce budworm, Choristoneura fumiferana Clemens (Lepidoptera: Tortricidae), has massive outbreaks in eastern North American forests (Royama et al., 2017). These outbreaks last about 5-15 years, severely defoliating balsam fir and spruce trees and causing high growth loss and tree mortality (Hennigar et al., 2008). Spruce budworm outbreaks have been known to damage millions of hectares of North American forests per outbreak and have large impacts on the forestry sector (Chang et al., 2012). Consequently, finding methods to reduce the severity of spruce budworm outbreaks is important to maximize forestry economic activity while minimizing losses of balsam fir and species of spruce.
Hardwood trees have long been thought to reduce the severity of spruce budworm outbreaks. Since the 1920s, the importance of tree diversity to spruce budworm control has been periodically brought up (Miller & Rusnock, 1993). More recently, researchers have evaluated how the proportion of hardwood trees within stands (hardwood content) impacts the growth, defoliation, and mortality of balsam fir and spruces. Research on balsam fir growth found that spruce budworm-caused growth reductions of balsam fir during the 1972-1992 outbreak was significantly mitigated by hardwood content (Campbell et al., 2008). Research on balsam fir defoliation found defoliation was lower in mixed forest stands containing hardwood trees compared to balsam fir dominated stands during spruce budworm outbreaks (Su et al., 1996;Zhang et al., 2018Zhang et al., , 2020. In contrast, Mac- Kinnon and MacLean (2003) found no effect of surrounding forest type on spruce budworm defoliation of balsam fir. Instead, Mac-Kinnon and MacLean (2003) found that spruce budworm defoliation of white spruce was reduced in stands surrounded by mixed wood forest. Finally, research on balsam fir mortality found mortality due to spruce budworm defoliation was greater in extensive conifer stands than fir stands surrounded by deciduous forest or on islands in the middle of a lake (Cappuccino et al., 1998). Researchers have also tested the effect of hardwood content on spruce budworm abundances and densities. Quayle et al. (2003) found that relative basal area of nonhost tree species had a significant negative effect on the abundance of spruce budworm and Eveleigh et al. (2007) found lower outbreak peak spruce budworm densities in heterogeneous plots compared to homogeneous plots. Overall, the evidence points to a complicated yet important impact of hardwood content on spruce budworm outbreaks.
One proposed mechanism behind hardwood content impacting spruce budworm outbreaks is that hardwood content affects the community of insects that parasitize and then kill spruce budworm caterpillars (parasitoids). Among the natural enemies of spruce budworm, parasitoids have arguably the strongest impact on spruce budworm mortality causing between 30% and 90% mortality depending on the surrounding forest composition and the point in the spruce budworm cycle (Cappuccino et al., 1998;Royama et al., 2017). Several researchers, examining how hardwood content impacts the parasitism of spruce budworm, have found that, depending on the parasitoid species, there was either no effect of tree composition or an increase in parasitism with higher diversity of trees (Kemp & Simmons, 1978;Quayle et al., 2003;Simmons et al., 1975).
However, these studies have examined parasitoid species individually.
An important further research direction is how hardwood content influences the parasitoid community as a whole. Currently, we know the parasitoid community responds strongly to spruce budworm density with increases in diversity cascading up parasitoid trophic levels (the bird feeder effect) (Eveleigh et al., 2007) and the parasitoid community responds largely indiscriminately to changing spruce budworm and other caterpillar abundances on balsam fir (Greyson-Gaito et al., 2021). Indeed in an initial survey, Eveleigh et al. (2007) did find increased diversity and abundance of primary parasitoids in plots with greater proportions of hardwood trees. Marrec et al. (2018) also found that variation in spruce budworm parasitoid community composition was mostly explained by surrounding forest structure. Eveleigh et al. (2007) and Marrec et al. (2018) research show that examining how hardwood content influences the parasitoid community as a whole is a useful endeavour.
Analysing phylogenetic community structure could be useful in examining how hardwood content impacts the parasitoid community associated with spruce budworm. Phylogenetic community structure is defined as the nonrandom patterns of evolutionary relatedness between species in a community (Kraft et al., 2007). These nonrandom patterns can be produced from the interaction of ecological processes, including habitat filtering and competitive exclusion, with the evolutionary history of species (i.e., how closely related are different species). With the assumption that closely related species have higher competition than distantly related species, the ecological processes can be inferred from the phylogenetic community structures found when sampling communities. Researchers test for three phylogenetic community structures: phylogenetic clustering, where communities are made up of closely related species; overdispersion, where communities are made up of distantly related species; and neither clustering nor overdispersion (Webb et al., 2002) (Figure 1a). F I G U R E 1 (a) Hypothetical phylogenies showing either clustered, overdispersed or neither phylogenetic community structures. In these hypothetical communities, the "sample" has 10 species from the 20 potential species in the species pool. Presence of a species is denoted by diamonds and black branches with absence denoted by grey branches. Note how closely related the "sampled" species are in the clustered structure compared to the overdispersed structure. (b) Conceptual diagram illustrating the stable isotopes (δ15N & δ13C) of basal resources, intermediate consumers, and a top consumer for two resource compartments (food chains). Note in this example, the top consumer is coupling the two resource compartments. The δ13C of this top consumer can even change depending on which resource compartment the top consumer is feeding on in time and space. related species competitively excluding each other or distantly related species converging on similar niches. Finding neither clustering nor overdispersion generally indicates distantly related species with convergent traits are competitively excluding each other (Webb et al., 2002). Overall, including the evolutionary history of species can illuminate fundamental processes behind the assembly of communities leading to key insights into how the community functions (Kembel & Hubbell, 2006;Ricklefs, 2006). Similarly, for the spruce budwormparasitoid system including the evolutionary history of the parasitoids can help us to identify how hardwood content might be influencing the parasitoid community associated with spruce budworm leading to insights into how to use the parasitoid community to reduce the severity of spruce budworm outbreaks.
Another analysis, stable isotope analysis, could similarly be useful for examining how hardwood content impacts the parasitoid community associated with spruce budworm. Stable isotope analysis aims to identify trophic relationships (Boecklen et al., 2011) and involves measuring the ratio of heavy to light isotopes of different chemical elements (often carbon and nitrogen). In fact, the ratio of heavy to light carbon isotopes in a consumer will be similar to that of the consumer's diet and the ratio of heavy to light nitrogen isotopes increases at each level of a trophic food chain ( Figure 1b). From this information, a food web of the different organisms measured can be elucidated (Boecklen et al., 2011). Furthermore, using carbon isotopes, researchers can examine whether the consumers feed on multiple resource compartments (food chains) within the food web, otherwise called coupling (McMeans et al., 2016). Importantly for this study, the ratio of heavy to light carbon isotopes differs between softwood and hardwood trees (Brooks et al., 1997;Risk et al., 2009). This difference is consistent even with environmental fluctuations and between different locations (Brooks et al., 1997). Thus, if we measure the stable isotopes of parasitoids, we can essentially measure the relative attack rates of parasitoids on caterpillars, either spruce budworm or other species, on the softwood and hardwood resource compartments. Hardwood content likely influences these relative attack rates. Thus, stable isotope analysis can be used to examine the extent of coupling by parasitoids of the softwood and hardwood resource compartments.
In this study, we illustrate how phylogenetic community structure and stable isotope analyses could be used to examine the impact of hardwood content on the parasitoid community associated with spruce budworm. We provide some preliminary findings from these analyses. Specifically, using Malaise caught parasitoids from years where spruce budworm were at low density and reared parasitoids from years where spruce budworm were at high density, we tested whether the phylogenetic community structure differed along a hardwood gradient. Second, using stable isotope analysis of Malaise caught parasitoids sampled immediately prior to and after a spruce budworm outbreak peak, we identified how trophic relationships between parasitoids and caterpillars on balsam firs and on hardwood trees changed within and between years. Our preliminary findings indicate that hardwood content does impact the parasitoid phylogenetic community structure, and the utilization of caterpillars on balsam fir or hardwood trees changes, depending on the parasitoid, within and between years.

Phylogenetic community structure along a hardwood gradient
Low density spruce budworm and once a month during July and August. We separated out individuals belonging to insect families that we knew contained species that attack spruce budworm. These families included, but were not limited to, Tachinidae, Sarcophagidae, Braconidae, and Ichneumonidae. We stored the collected parasitoids in 70% ethanol and in a refrigerator at 4 C, until they were barcoded. Note, we use the term spruce budworm-associated parasitoid community to acknowledge that although Malaise sampling will capture parasitoids attracted to the implanted spruce budworm, the Malaise sampling will also capture hyperparasitoids and other parasitoids that do not attack spruce budworm.
To examine phylogenetic community structure, we used DNA barcoding where a region of an organism's DNA is sequenced and compared to the same region in other organisms (Ratnasingham & Hebert, 2007). Tissue samples were taken using 1-6 legs and placed in 30 μl of 95% ethanol and stored at À20 C. Mitochondrial DNA from the cytochrome c oxidase I (COI) region (the standard animal DNA barcode locus) was amplified and sequenced at the Biodiversity Institute of Ontario (BIO; University of Guelph, Ontario). High resolution photographs were taken of wet specimens under a dissecting microscope using Leica Application Software V4.9. Sequences and photographs were uploaded to the Barcode of Life Data System (BOLD) (Ratnasingham & Hebert, 2007). We used Barcode Index Numbers (BINs), a DNA-based delineation of species based on patterns of intra and interspecies variations outlined by Ratnasingham F I G U R E 2 (a) Map of New Brunswick, Canada with the three plots from the 1980s when spruce budworm were at high density indicated. b) Map of the Acadia Research Forest. The nine plots used in the 2016 Malaise trapping of parasitoids when spruce budworm were at low densities are indicated by circles. Plots 1, 2, 3 are balsam fir dominated, plots 4, 5, 6 are mixed wood plots, and plots 7, 8, 9 are hardwood dominated. Two of the three plots from the 1980s when spruce budworm were at high density are indicated by squares (the third plot is indicated in the map of New Brunswick). and Hebert (2013), to identify species using the BOLD database. We constructed a single-representative maximum likelihood tree in MEGA6 based on the estimation of the best substitution models in MEGA6 (Nei & Kumar, 2000;Tamura et al., 2013).

Statistical analyses
To examine how hardwood content affected the phylogenetic community structure of spruce budworm-associated parasitoids, we calculated the mean nearest taxon distance (MNTD) using maximum likelihood trees between the three forest types for the Malaise caught parasitoids. Maximum likelihood trees used a general time-reversible model with discrete gamma distribution under the assumption that sites were evolutionarily invariable (Nei & Kumar, 2000;Tamura et al., 2013). The standard effect size of the MNTD was then calculated and phylogenetic clustering and dispersion assessed by performing 999 random permutations of hardwood content associations to simulate a distribution of MNTD for each community. The significance of the observed MNTD values for each community was examined with a two-tailed test of significance (p = 0.05) (function ses. mntd, R package Picante, version 1.7, (Kembel et al., 2010)).  parasitoids between the three plots. A subset of these parasitoid species were preserved at À20 C then DNA barcoded to explore how genetic estimates of isolation and species identification changed the estimates of food web connectance (connectance was reduced as the number of nodes increased) (Smith et al., 2011). However, Smith et al. (2011) did not report estimates of phylogenetic community structure for the parasitoids of these three plots, and so in this study, we add an examination of the phylogenetic community structure of parasitoids sampled in the 1980s when spruce budworm were at high density and compare with the phylogenetic clustering of parasitoids sampled along a hardwood gradient in 2016 when spruce budworm were at low density.

Statistical analyses
We calculated the mean nearest taxon distance (MNTD) and assessed phylogenetic clustering and dispersion (function ses.mntd, R package Picante, version 1.7, (Kembel et al., 2010)) of reared parasitoids collected from the three plots in Eveleigh et al. (2007).
Stable isotope analysis of parasitoid community trophic relationships Sampling All parasitoid sampling was performed in a single balsam fir dominated plot in ARF for the years of 1982, 1983, 1986, and 1987 (in this plot, spruce budworm peaked in 1985). Because this analysis used historical parasitoid sampling, the sampling was limited to the single plot and time points from the original study. This plot was 98% Abies balsamea, 1% Picea rubens Sarg., and 1% Acer rubrum L. by basal area (Lethiecq & Regniere, 1988). Parasitoids were collected using modified 1 m 3 Malaise traps (Nyrop & Simmons, 1982). Flying insects were collected daily, immediately stored in 70% ethanol, and frozen at À7 C until preparation for stable isotope analysis in 2017 (except insects collected in 1982 which were stored without ethanol but still in the freezer).
In 2017, as an initial attempt to understand how parasitoids with different life cycles utilize caterpillars, either spruce budworm or other species, on balsam fir and hardwood trees, we separated the 1980s Malaise caught parasitoids into three groups (see Table S1): Group 1, univoltine parasitoid species that attack one caterpillar species within a year and do not require an alternate caterpillar in which to overwinter (Elliott et al., 1987;O'Hara, 2005); Group 2, multivoltine parasitoid species that overwinter away from a host or where overwintering status was unknown; and Group 3, multivoltine parasitoid species that require an alternate caterpillar in which to overwinter (O'Hara, 2005;Thireau & Régnière, 1995). All parasitoid species are common parasitoids of spruce budworm. These parasitoid species are capable of attacking multiple caterpillar species but differ in the frequency and life cycles of attacking spruce budworm and other caterpillar species. The parasitoids had previously been identified using representative specimens provided by taxonomists from the Canadian National Collection of Insects, Arachnids, and Nematodes (CNC).
These three groups were then further split into three periods to capture the phenology of the parasitoid emergences from spruce budworm and other caterpillar species: May/June, July, and August/ September. When there were fewer than 50 total individuals in a group and sampling period, all individuals were used for stable isotope analysis. When there were more than 50 total individuals in a group and sampling period, we randomly selected 50 individuals and ensured the proportions of selected individuals of each species matched the proportions of total number of individuals for each species (within the group and sampling period). We removed legs and wings from all individuals, keeping the mass of legs and wings approximately constant between individuals and species. Legs and wings were combined for each group and sampling period and were dried at 60 C for at least 48 hours. We used legs and wings because many parasitoids as adults consume nonhost nutrient sources, and legs and wings have a slower turnover rate compared to other body parts (Benelli et al., 2017;Gratton & Forbes, 2006).
In stable isotope analysis, carbon and nitrogen stable isotopes are measured in samples from resources at the bottom of the food chain (basal resources) and from intermediate consumers of each resource compartment (food chain) (see Figure 1b). From these measurements, called baselines, researchers can deduce the trophic relationships of the focal organisms. In this study, balsam fir plus its inhabitant caterpillars and hardwood trees plus their inhabitant caterpillars were the two resource compartments. Thus, our baselines consisted of balsam fir and hardwood foliage, and caterpillars from these sampled foliage. In 2017 beginning on May 30th and ending on June 27th, once a week we sampled 1 m long, mid-canopy branch from 5 balsam fir trees in each of the nine plots used to study the phylogenetic community structure (one branch per tree, five trees per plot, 45 branches per week). Each week, we also sampled 1 m long branch from multiple hardwood tree species in each plot. These multiple hardwood species were the most abundant in each plot as found by the original plot ground truthing. On the 17th July and on the 4th August, we randomly sampled a single balsam fir branch from each plot, and we sampled branches from the same hardwood species as we sampled in June (a branch per species in each plot). We sampled foliage without any noticeable herbivory damage from all branches. This foliage was rinsed with distilled water and dried at 60 C for at least 48 h. We ground the foliage and ensured that the combination of different hardwood species in each plot's ground sample matched the proportions of hardwood trees found in each plot. This was repeated for June, July, and August. From the balsam fir branches and the hardwood branches, we collected all caterpillar individuals and separated them into caterpillars from balsam fir or hardwoods and by plot and by sampling period. The caterpillar samples were dried at 60 C for at least 48 h. All parasitoid, caterpillar, and foliage samples were analysed for carbon and nitrogen isotope ratios at the University of Windsor GLIER (Windsor, ON, Canada) laboratories.

Statistical analyses
Normal practice when using stable isotopes is to use mixing models, where both δ13C and δ15N are included to establish the trophic levels and percentage of diet from multiple resource pathways (Phillips et al., 2014). However, the δ13C of the parasitoid samples were enriched by 16% compared to the foliage and caterpillar baselines probably because the parasitoid samples were stored in ethanol and frozen for about 30 years whereas the foliage and caterpillars were sampled in 2017 (Jesus et al., 2015). Because mixing models are unable to account for this enrichment, we were not able to use mixing model analyses with both δ13C and δ15N. Instead, we used δ13C only by comparing δ13C between years, sampling periods, and groups because we knew that there were consistent differences in δ13C between hardwood and softwoods which were transferred to the caterpillars (Balsam fir and hardwood foliage Welch t-test: t = 2.813, df = 40.219, p = 0.00756. Balsam fir caterpillars and hardwood caterpillars Welch t-test: t = 3.161, df = 39.161, p = 0.00303). Note, from the three sampling periods above (May/June, July, August/ September), we simplified the periods into two sampling periods, May/June and July/August/September, by averaging the δ13C values of the July and August/September periods. These two sampling periods were chosen to coincide with when spruce budworm were larvae (approximately May/June) and when they were moths/eggs/L1 (approximately July/August/September). We ran a generalized least squares regression to test the effects of year, sampling period (May/June or July/August/September), parasitoid group, and all interactions on the δ13C of sampled parasitoid legs and wings (function gls, R package nlme, version 3.1-137, (Pinheiro et al., 2018)). We added a varIdent variance structure to account for the different variation in the residuals between the sampling periods. We fitted the full model using maximum likelihood estimation and then used backwards selection with log likelihood ratio tests to select the final fixed effects.
We refitted the final model using restricted maximum likelihood estimation to give unbiased maximum likelihood predictors (Zuur et al., 2009).

Phylogenetic community structure along a hardwood gradient
Low density spruce budworm  Figure 3b).

Stable isotope analysis of parasitoid community trophic relationships
The final model explaining δ13C included year, group, sampling period (May/June or July/August/September), and the interactions of year with group (year: group interaction, L = 13.230, p = 0.0013, df = 1, log likelihood ratio test, Figure 4) and group with sampling period (group: sampling period interaction, L = 28.900, p < 0.0001, df = 1, log likelihood ratio test, Figure 4, see Table 1 for ANOVA output of model). Group one parasitoids became slightly more negative by approximately 0.5% each year, and group one parasitoids caught when spruce budworm were absent had more negative δ13C values by 2.4% compared to group one parasitoids caught when in May/-June. δ13C values for group two parasitoids became less negative overtime by approximately 1.6% each year. Group three parasitoids showed a difference of 12.2% in δ13C between May/June and July/ August/September. In May/June, group three parasitoids had more negative δ13C values. In July/August/September, group three parasitoids had less negative δ13C values. In comparison to the difference in δ13C between May/June and July/August/September, δ13C for group three parasitoids changed little with no noticeable trend between years.
research. Using Malaise caught and reared parasitoids, the phylogenetic community structure of the parasitoid community was consistently clustered in balsam fir dominated plots when spruce budworm were at low and high density. From comparing the stable isotopes of parasitoids during a spruce budworm outbreak, we found that several parasitoids changed their attack rates between caterpillars, including spruce budworm, on balsam fir and caterpillars on hardwoods within and between years. Taken together, our study highlights the potential for these analyses to illuminate how hardwood content could impact the severity of spruce budworm outbreaks through affecting the parasitoid community.
The hardwood content of the stands did appear to impact the spruce budworm-associated parasitoid phylogenetic community structure. We found that the balsam fir dominated plots exhibited phylogenetic clustering in 2016 when spruce budworm were at low densities and in the 1980s when spruce budworm were high densities. Clustered phylogenies indicate that habitat filtering is a major factor determining the community composition (Webb et al., 2002). Because closely related parasitoid species are more likely to share host species or search within the same plant species than distantly related parasitoid species (Ives & Godfray, 2006), we speculate that the habitat filtering is likely due to the differences in caterpillar composition maintained by balsam fir dominated stands compared to stands with greater hardwood content (Summerville & Crist, 2008). Potentially, balsam fir dominated plots host a subset of caterpillar species thus filtering closely related parasitoid species. Similarly, Marrec et al. (2018) found environmental (habitat) filtering to be important in shaping spruce budworm parasitoid communities. One caveat to our habitat filtering pattern is that our sampling does not differentiate between primary parasitoids and hyperparasitoids. Because hyperparasitoids may be key in driving spruce budworm outbreaks (Nenzén et al., 2018), examining the differential impacts of hardwood content on primary parasitoids and hyperparasitoids is critical. Overall, our phylogenetic community structure analysis indicates that hardwood content likely impacts the spruce budworm-associated parasitoid community through influencing the caterpillar communities. Further research should extensively sample caterpillar communities on all tree types along a hardwood gradient as well as sample and differentiate between primary parasitoids and hyperparasitoids.
Our preliminary stable isotope analysis found that our three groups of parasitoids differed in how they utilized caterpillars on balsam fir and hardwood trees within and between years. The parasitoids that within a single year must attack caterpillars at the beginning of the summer, usually spruce budworm on softwoods, and then overwinter in other caterpillar species usually on hardwoods (group three) provide us with the clearest comparison of trophic relationships between balsam fir and hardwood. The δ13C of group three parasitoids sampled in May/June was more negative than the δ13C of group three parasitoids sampled in July/August/September. Our sampled hardwood foliage was similarly more negative in δ13C compared to our sampled balsam fir foliage (hardwood foliage = À30.222 δ13C, balsam fir foliage = À29.521 δ13C). This correspondence of the differences between group three in the two sampling periods and the differences in balsam fir and hardwood δ13C matches what we know of the life history of group three parasitoids because, in May/June, group three parasitoids emerge from other caterpillar species often on hardwood trees to attack caterpillars, usually spruce budworm, on balsam firs and other softwoods. Then in July (within the July/August/ September sampling period), group three parasitoids emerge from these caterpillars to attack other caterpillars often on hardwoods.
Therefore, we suggest any comparable changes in δ13C for the other groups should be due to the parasitoids changing their attack rates on caterpillars, including spruce budworm, on balsam fir and other caterpillar species on hardwoods.
The parasitoids that attack one caterpillar species within a year (group one) seemingly did not change their relative utilization of caterpillars, either spruce budworm or other species, on balsam fir and caterpillars on hardwoods within a year nor between years. Group one parasitoids not changing relative utilization within a year is unsurprising because these parasitoids are univoltine. Group one parasitoids not changing utilization between years as spruce budworm densities change is consistent with other studies that concluded that these parasitoids attack spruce budworm more than other caterpillar species (Cossentine et al., 2007;O'Hara, 2005). Furthermore, the populations of group one parasitoids are supported by other caterpillar species that feed on balsam fir as suggested by Apanteles fumiferanae Vier. (Hymenoptera: Braconidae) and Glypta fumiferanae Vier. (Hymenoptera: Ichneumonidae) attacking other caterpillar species on balsam fir (Greyson-Gaito et al., 2021). In contrast to group one parasitoids, the multivoltine parasitoid species that overwinter away from a host or where overwintering status was unknown (group two) exhibited greater change in δ13C between years, from more to less negative, suggesting that these parasitoids likely attacked caterpillars on hardwoods when spruce budworm had lower densities and then attacked spruce budworm (or other caterpillar species) on balsam fir when spruce budworm had higher densities.
Overall, there are indications that certain parasitoids may be coupling the softwood and hardwood resource compartments within and between years. However, increased resolution of this stable isotope analysis is required and we encourage future researchers to measure the stable isotopes of individual parasitoid, caterpillar, and tree species within a year and between years. We also recommend that researchers include understory plants as stable isotope baselines because parasitoids gain nutrients from nonhost sources including nectar from understory plants (Benelli et al., 2017) and caterpillars consume understory plants (Seifert et al., 2020).
Two techniques that would complement stable isotope analysis for examining how parasitoid utilize caterpillars on softwoods and hardwoods are fatty acid analysis and the quantitative polymerase chain reaction (qPCR) TaqMan assay. Fatty acid analysis has the same overall goal of stable isotope analysis because fatty acid compositions often differ between different resources and these differences get passed onto any consumers. Indeed, fatty acid compositions differ between softwoods and hardwoods more than δ13C (Mueller et al., 2012) and thus fatty acid analysis could be powerful to unpack the trophic relationships of the spruce budworm-associated parasitoids. The qPCR TaqMan assay can be used to identify individual species from bulk samples with high accuracy. In the spruce budworm system, a qPCR TaqMan assay has been created to determine whether and by what a spruce budworm larva has been parasitized (Nisole et al., 2020). So far this method is limited to 20 common natural enemies of spruce budworm as a compromise between time/costs and broad applicability. Yet, this assay has great potential to quickly identify a parasitoid species attacking spruce budworm sampled from the field. This assay similarly has great potential to be used to quantify the relative attack rates of parasitoids on spruce budworm and other caterpillar species. Thus, we suggest that DNA libraries of spruce budworm parasitoids be expanded to include representation from hardwood forest parasitoid communities. Overall, comprehensive sampling of parasitoids and caterpillars on softwoods and hardwoods throughout the spruce budworm cycle is required to evaluate the trophic relationships between parasitoids and the caterpillars on softwood and hardwood trees. Stable isotope analysis, fatty acid analysis, and qPCR would all be highly complementary techniques.
Hardwood trees in forest stands have long been thought to be important to reducing the severity of spruce budworm outbreaks. Key to reducing the severity of outbreaks could be hardwood trees impacting the abundances and composition of the parasitoids of spruce budworm. In this study, we have highlighted two useful analyses that we encourage spruce budworm researchers use to examine how hardwood content impacts the spruce budworm-associated parasitoid community: phylogenetic community structure analysis and stable isotope analysis. Our preliminary exploration using these analyses found that hardwood content influenced the phylogenetic structure of parasitoid communities and several parasitoids change their relative utilization of caterpillars on balsam fir and hardwoods within and between years. Taken together, we have shown some potential uses of the phylogenetic community structure and stable isotope analyses with some preliminary findings that point to the important influence of hardwood content on the spruce budworm-associated parasitoid community.