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Article

The Lethal and Sublethal Effects of Aerial Applications of Bacillus thuringiensis subsp. kurstaki on the Spruce Budworm and Its Parasitism

by
Christian Hébert
1,*,
Jean-Michel Béland
1,
Alain Dupont
2 and
Richard Berthiaume
2
1
Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, P.O. Box 10380, Stn. Sainte-Foy, Québec, QC G1V 4C7, Canada
2
Société de Protection des Forêts Contre les Insectes et Maladies (SOPFIM), 1780 Rue Semple, Québec, QC G1N 4B8, Canada
*
Author to whom correspondence should be addressed.
Forests 2025, 16(11), 1666; https://doi.org/10.3390/f16111666 (registering DOI)
Submission received: 9 September 2025 / Revised: 23 October 2025 / Accepted: 28 October 2025 / Published: 31 October 2025
(This article belongs to the Special Issue Integrated Pest Management and Control in Forestry)
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Abstract

The bioinsecticide Bacillus thuringiensis subsp. kurstaki (Btk) is applied over large areas to reduce defoliation caused by the spruce budworm, an insect which affects millions of hectares of coniferous forests every 30 to 40 years in eastern North America. The aim of our study was to determine whether, in addition to its direct lethal effects, aerial spraying of Btk had sublethal effects on spruce budworm populations and their parasitism. Four sites were sprayed with Btk and compared to four control sites in two regions, one where the outbreak had started three years earlier and the other where it had been going on for 10 years. Insects were collected to compare budworm pupal mass and parasitism at different stages (L5, L6 and pupae). Budworm pupae were significantly lighter in Btk-treated sites than in controls, and in the older population than in the younger one. However, pupae collected from Btk-treated sites had the same mass in both regions, suggesting a minimum pupal mass threshold, which may affect population dynamics. Larval parasitism was low, but pupal parasitism was high and strongly influenced by an interaction between Btk and region, with a higher parasitism observed in Btk-treated sites of the younger population than in those of the older population. A significant interaction was observed between Btk treatment and region on the proportion of larvae that failed to complete development, which was particularly high in Btk-treated sites of the older population. Our study confirms the effectiveness of Btk in controlling spruce budworm populations directly but also indirectly through sublethal effects on budworm development, capacity to complete development, pupal size and parasitism. To maximize control efficacy, the timing of Btk applications could vary according to the age of populations during the budworm outbreak cycle.

1. Introduction

Spruce budworm (SBW), Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae), is the most damaging insect defoliator of coniferous forests in eastern North America [1,2]. Outbreaks have occurred periodically over the past few centuries, with an average interval of 35 years [3,4]. In Quebec, at the peak of the last outbreak in 1975, defoliation affected over 35 million hectares [5]. Nearly half a billion cubic meters of wood were lost during this outbreak [6]. This represents twice the amount of wood harvested by industry during this period. Since the 1950s, insecticides have been used over huge areas against this insect to protect tree foliage, reduce tree mortality and limit economic losses.
The tree species most severely affected by spruce budworm is balsam fir, Abies balsamea (L.) Mill., followed by white spruce, Picea glauca (Moench) Voss, red spruce, P. rubens Sarg., and black spruce, P. mariana (Mill.) B.S.P. [7,8]. Larvae feed on needles of current-year shoots, but in extremely high populations they can also feed on previous-year foliage [8,9]. Spruce budworm defoliation reduces tree growth and leads to the death of balsam fir after 4–5 successive years of severe defoliation [2,10,11,12,13] or 6–7 years for white spruce [14].
Public pressure against aerial spraying of chemical insecticides over large areas of forest led the Quebec government to adopt the bioinsecticide Bacillus thuringiensis subsp. kurstaki de Barjac and Lemille (hereinafter Btk) in 1987 for reducing spruce budworm defoliation [15]. Btk commercial products are now widely used around the world to control forest-defoliating Lepidoptera and reduce defoliation and tree mortality [16,17,18,19,20]. This is an environmental improvement, but we still do not know the full extent of Btk’s effectiveness on insect pests and its impact on other organisms in forest ecosystems. For instance, in addition to its lethal effects, Btk has shown sublethal effects on several insect pests in laboratory experiments, including the spruce budworm [17,18,21,22,23]. Similarly, sublethal effects of Btk have been reported on non-target dipteran Drosophila melanogaster Meigen in laboratory studies [24]. The sublethal effects of pesticides are usually overlooked, the lethal effects receiving all the attention [25]. In our study, sublethal effects are defined as physiological or biological effects on spruce budworm individuals that survive aerial applications of Btk, presumably due to ingestion of a sublethal dose of the biopesticide (definition inspired by [22]). The most common sublethal effects observed are delays in larval developmental and pupal mass [17,18,21,22,23].
In spruce budworm, larvae fed on artificial diet containing Btk, and which survived, developed more slowly and produced lighter pupae than those fed on a diet without Btk [17,23,26]. A feeding inhibition period begins immediately after larvae are fed a diet containing Btk [27]. Budworm larvae can compensate these sublethal effects by going through additional instars, thereby producing pupae of a similar mass to those of larvae that had not been fed on a Btk-based diet [22]. These laboratory observations need to be validated by field data, as pesticide sublethal effects affect various insect physiological parameters, including reproductive success [25] and therefore have the potential to influence insect pest population dynamics and resulting damage.
There is also much to learn about the interactions between Btk and natural enemies. The use of Btk against the diamondback moth, Plutella xylostella L., reduced parasitism by the braconid Cotesia vestalis Haliday by 5%–10%, which was considered a minor impact compared with other insecticide groups [28,29]. Similarly, among six biopesticides tested, Btk proved to be safest for the braconid Bracon nigricans Szépligeti, a parasitoid of Tuta absoluta (Meyrick), an invasive moth attacking tomatoes in Europe [30]. However, the braconid Habrobracon hebetor Say, a parasitoid of the cotton bollworm, Helicoverpa armigera (Hübner), showed a longer larval and pupal development, and a lower fecundity when exposed to hosts fed on diets containing sublethal doses of Btk [31]. The use of transgenic plants incorporating the Btk gene in commercial crops adds a further layer of complexity in herbivore–natural enemy interactions. For instance, in Btk cotton flowers, significantly greater predation was observed on larvae of the bollworm, Helicoverpa zeae Boddie, by the lady beetle Hippodamia convergens Guérin-Méneville at higher sublethal Btk doses [32]. Bollworm growth and development are then slowed by feeding on a Btk diet, which favors the predator as it benefits from a better predator–prey size ratio and a longer period of bollworm availability.
Btk is a broad-spectrum biological insecticide that can reduce populations of all lepidopteran larvae at the time of application [33,34]. Most spruce budworm parasitoids are multivoltine and use alternate lepidopteran hosts to complete their life cycles [35,36,37]. By decreasing the availability of alternate hosts [35], Btk applications could indirectly affect spruce budworm parasitism. Parasitoids play an important role in spruce budworm population dynamics. They are particularly efficient in reducing populations during the declining phase of outbreaks and between outbreaks, when they are instrumental in maintaining budworm populations at very low densities [36,38,39]. Despite the importance of parasitism in spruce budworm population dynamics and the widespread use of Btk for managing outbreaks, little is known about the effects of Btk applications on parasitism. The only study addressing this issue [40] has used molecular analyses developed by [41] to determine whether budworm larvae were parasitized or not. Based on 213 larvae collected in low budworm populations, they found no significant effect of Btk sprays on overall parasitism [40].
The objectives of our study were to determine whether operational aerial applications of Btk in forests have (1) sublethal effects on spruce budworm fitness and (2) parasitism rates.

2. Materials and Methods

2.1. Site Selection and Experimental Design

Four large blocks of mature forests were selected, two in each of the Côte-Nord and Bas-Saint-Laurent regions in the province of Quebec, Canada, where spruce budworm outbreaks began in 2006 and 2013, respectively (Figure 1). The selected balsam fir forest blocks had identical eco-forest classifications (Table 1), similar altitudes and similar sizes (from 50 to 243 ha). Both had been lightly defoliated in 2016 (Figure 1), the year preceding the study, but the outbreak was older in Côte-Nord than in Bas-Saint-Laurent, having started in 2006 and 2013, respectively. All spruce budworm overwintering populations reached densities justifying a double application of Btk (more than 40 L2/45 cm branch tips). The Société de protection des forêts contre les insectes et maladies (hereinafter SOPFIM) sprayed Btk operationally in one block of each region, the other one being used as control. Bioprotec HP™, a Btk strain HD-1 commercial formulation at nominal potency of 20 billion international units per litre (BIU/L) (AEF Global Inc., Levis, QC, Canada) was used in the experiment. The aircraft used, a C-GMTG Air tractor 504TM equipped with six MicronairTM atomizers (Micronair Sprayers Ltd., Bromyard, UK), was calibrated before spraying. It flew at 161 km/h, with 80 m spray widths. Spraying was performed when it was not raining, and wind speed did not exceed 16 km/h. The nozzles were calibrated to deliver 1.5 L/ha or 30 BIU/ha. To achieve the entire annual spray program (e.g., 250,600 ha in 2025), Btk applications usually begin when spruce budworm reaches the peak of the third larval instar as there is no difference in treatment efficacy for applications timed at the peak of 3rd or 4th instar [42]. Each treated block received two applications of Btk: the first, on 12 and 15 June in the Côte-Nord and Bas-Saint-Laurent regions, respectively, and the second on 23 and 26 June, 11 days later in each region.
Four sampling sites were set up in each forest block, each along a different flight line in the Btk-treated blocks and thus spaced by at least 80 m. Fuentealba et al. [43] used this approach to reduce variability in a field experiment testing the effectiveness of various Btk treatments. Replicating Btk spray experiments in several forest blocks increase variability unrelated to the Btk treatment itself. Variability in forest composition, tree density and topography are greater between forest blocks than within each one, and this influences local microclimate, which affects insect development. Moreover, spraying several forest blocks requires complex logistic operations and treatments can rarely be applied to all blocks on the same day, by the same aircraft and the same pilot, thus increasing variability unrelated to the Btk treatment itself. Using flight lines to replicate Btk treatments in a small number of blocks reduces variability unrelated to the treatment itself in field experiments. Experimental conditions are better controlled in such design as a 250 ha area can be treated in less than an hour. Favourable weather conditions are required to apply Btk, and this only occurs for a few hours per day.

2.2. Spruce Budworm Sampling and Defoliation Estimation

Overwintering spruce budworm populations (L2 larvae) were estimated by collecting one 75 cm branch from the mid-crown of three balsam firs, using a pole pruner, in each forest block during fall 2016. Overwintering larvae were extracted from the branches using the method described by [44]. Spruce budworm seasonal development was monitored in each forest block twice weekly in 2017 from L2 to moth emergence (15 May–27 July). One 45 cm branch was collected from the upper part of the mid-crown of 5 to 25 balsam firs, using a pole pruner equipped with a basket. As our goal was to collect at least 100 insects in each forest block, the number of trees sampled varied because trees in Btk-treated blocks had lower larval densities. Branches were carefully examined in laboratory to extract larvae, determine instar of each individual and count all budworm living larvae and pupae, as well as empty pupae to assess moth emergence. Defoliation was estimated by trained SOPFIM technicians to avoid bias, using the Fettes [45] grid, on 30 current-year shoots from each of 25 branches (1/tree) when the spruce budworm had reached the pupal stage and completed defoliation.

2.3. Spruce Budworm Pupal Mass and Parasitism

Additional balsam fir branches were collected at the peak of the 5th and 6th larval instars, as well as at the peak of pupae in the four sites of each treatment and region, for a total of 16 sites. We targeted the late-instar larvae and pupae because most spruce budworm parasitoids emerge from these stages, and all parasitoid species attacking budworm after Btk application emerge from these stages [46]. One 45 cm branch was collected from each of 5 to 25 balsam firs, in order to collect 75 insects in each site at the peak of 5th and 6th larval instars and at the peak of pupae. These branches were rapidly shipped to the laboratory where the 75 budworms were extracted. Therefore, for estimating pupal mass, each site estimate was obtained from 75 pupae, split between sexes.
Spruce budworm larvae were individually reared in 1 oz Solo cups and fed on fresh current-year balsam fir shoots, renewed twice weekly, to complete their development at 21 °C, 16L:8D and 40%–60% RH. At the third collection, collected pupae were sexed and weighed using an electronic scale (Mettler AE163; Fisher Scientific, Waltham, MA, USA) before being placed individually in Solo cups. Budworms were observed twice weekly to monitor parasitoid or moth emergence. Dead larvae and pupae from which adults had not emerged (two months after collection) were dissected to check for parasitism. Parasitoids were pinned and identified at the species level whenever possible, using general keys [46] and specialized ones for Chalcidoidea [47], Ichneumonidae [48] and Tachinidae [49] parasitoids attacking the spruce budworm. We also used specimens of the René-Martineau Insectarium at Laurentian Forestry Centre, which contains many specimens authenticated by specialists. Vouchers of each parasitoid species found in our study were deposited in the collection of the René-Martineau Insectarium.

2.4. Statistical Analysis

We used ANOVAs followed by Sidak’s all pairwise comparisons (post hoc analysis) to compare budworm larval densities (pre and post Btk sprays) and defoliation between treatments and region. The anova function from the stats package and the emmeans function (using estimated marginal means) from the emmeans package were used for the pairwise comparisons. We used the t.test function from the stats package to compare the average development stage of the spruce budworm of control and Btk-treated sites of each region.
Our sample unit was the site (n = 4 per treatment in each region, for a total of 16 sites) to compare budworm pupal mass and parasitism between treatments and regions, as Btk treatments were applied at this scale. For estimating pupal mass, each site estimate was obtained from 75 pupae, split between sexes. To determine whether Btk sprays caused sublethal effects on the spruce budworm, we compared the pupal mass of both sexes in Btk-treated sites with those in control sites of both regions, using the lm and anova functions of the stats package (normal distribution and homogeneity of variance were verified; t-values based method for confidence intervals) [50] and the emmeans function of the emmeans package (pairwise comparisons with Tukey method for p values). The lm model and the emmeans values were used for mean mass comparison per group combination [51]. The interaction between fixed-effects factors (Btk treatment, region and sex) was tested and integrated. The effect size parameters (η2) were calculated, to obtain the percent of the total variance associated with each variable, using the eta_squared function from the effectsize package.
We compared parasitism in Btk-treated sites with those in control sites, for both regions (n = 4 estimates for each Btk treatment/region), in separate models for each budworm collection (peaks of L5, L6 and pupa). Parasitism was calculated for all species combined (including unidentified parasitoids) and for each species separately. We cumulated budworm larvae or pupae collected from each site and reared at each collection to calculate the percentage of apparent parasitism, according to [52], using the following equation:
% apparent parasitism = (Nb SBW parasitized/Nb SBW reared) × 100
This is the most conservative estimate of parasitism (it cannot be lower for a dataset). Since a high proportion of reared larvae did not complete development (mainly from Côte-Nord), we wanted to provide a more complete picture of the impact of Btk treatments on budworm mortality in populations of different ages in the two regions. Thus, we also calculated the percentages of development failure and corrected parasitism, according to the definition of [53]-, using the following equations:
% development failure = (Nb SBW died from unknown causes/Nb SBW reared) × 100
% corrected parasitism = (Nb SBW parasitized/(Nb SBW reared − Nb SBW died from unknown causes)) × 100
Corrected parasitism considers that budworms that died of unknown causes were not available to parasitoids. In fact, even if dead hosts were dissected, parasitoid eggs and young larvae may have been difficult to detect. In addition, females of many parasitoid wasps feed on the body fluids of their host to mature their eggs [54] or when their egg load is low [55]. This host feeding causes more host mortality than parasitism itself for the polyphagous ichneumonid parasitoid Itoplectis conquisitor (Say) as it punctures spruce budworm pupae five to six times for each egg deposited [54]. On the spongy moth, Lymantria dispar (L.), pupae were killed much more frequently by ichneumonid wasp stinging than by successful parasitism; I. conquisitor sting more than 200 times more pupae than they produce offsprings [56].
We used the glm function from the stats package (logistic distribution; profile likelihood method for confidence intervals) and the emmeans function for pairwise comparisons (Tukey method for p values) to obtain mean probabilities per group combination. Interaction terms between fixed-effects factors (Btk treatment and region) were always tested and included. Figures were produced using the ggplot function from the ggplot2 package [57]. Statistical analyses were performed using R version 4.5.1 [50].

3. Results

3.1. Effectiveness of Btk Sprays in Reducing Spruce Budworm Populations and Defoliation

Pre-treatment spruce budworm larval populations showed no significant difference, but post-treatment populations were markedly different, being lower in Btk-treated blocks than in controls (Table 2). Spruce budworm larval populations were reduced by 79.7% and 92.2% in Btk-treated blocks compared with 56.5% and 61.7% in controls, in the Bas-Saint-Laurent and Côte-Nord regions, respectively. Compared to control sites, this represents additional larval mortality of 23.2% and 30.5% in Bas-Saint-Laurent and Côte-Nord, respectively. Annual defoliation was total (100%) or almost total (92.8%) in control blocks of the Côte-Nord and Bas-Saint-Laurent regions, respectively, and significantly higher than in Btk-treated blocks of Côte-Nord where defoliation was 56.1% while it was 76.7% in Bas-Saint-Laurent (not significantly different in this region); this represents additional foliage protection of 43.9% and 16.1% in each region (Table 2).

3.2. Sublethal Effects of Btk Sprays on the Spruce Budworm

The first application was synchronized with the peak of the 4th instar in Côte-Nord while the application occurred at an average development stage of 4.36 in Bas-Saint-Laurent (Figure 2). Differences in the average development stage (μ) over 0.68 (t = −3.65) between Btk-treated blocks and controls were significantly different (p < 0.001) from the mean 0.46 (C.I. 0.34–0.58). In Côte-Nord, the difference was highly significant between Btk-treated and control blocks as soon as four days after the first spray and remained highly significant until the end of insect development. This first application had an immediate effect on the Côte-Nord region, where the spruce budworm average development stage even regressed, while in Bas-Saint-Laurent, development continued to progress, but a little more slowly than in the control (Figure 2). In the Bas-Saint-Laurent region, the difference in average developmental stage between Btk-treated and control blocks was significant only 10 days after the second Btk treatment and remained moderately significant until the end of the insect development. In fact, spruce budworm larvae were still in the 4th instar on Côte-Nord when the second Btk treatment was applied, whereas in Bas-Saint-Laurent, the insect already passed the peak of the 5th instar (average development stage of 5.49). Overall, Btk treatments delayed spruce budworm development in both regions, but more strongly in Côte-Nord where the first treatment was more efficient as evidenced by the lower defoliation recorded and the stronger impact on budworm development (Table 2).
Pupae of both sexes were heavier in control sites of Bas-Saint-Laurent (females: 75 ± 1 mg, males: 62 ± 1 mg) than in Côte-Nord (females: 60 ± 1 mg, males: 44 ± 1 mg), but even if they were lighter in Btk-treated sites, pupal mass was nearly the same in both regions (females: 46 ± 1 mg in both regions, males: 33 ± 1 mg and 34 ± 1 mg) (Figure 3). A significant interaction was found between Btk treatment and region (Table 3) (η2 = 0.05, small effect). Budworm pupal mass was lower in Btk-treated sites than in control ones (η2 = 0.27, large effect), regardless of sex (η2 = 0.13, medium effect), the reduction being greater in Bas-Saint-Laurent than in Côte-Nord (η2 = 0.03, small effect) (Figure 3). Finally, female pupae were significantly heavier than males, regardless of region or treatment.

3.3. Spruce Budworm Parasitism and Development Failure

A total of 3 313 spruce budworms were reared in this study, leading to the recovery of 426 parasitoids, among which 406 (>95%) were identified at the species level. Overall, 12 parasitoid species were identified among four families (Table 4). Apparent parasitism of 5th instar larvae was low (1%–4%) and not influenced by Btk treatments and region (Table 4; Figure 4). Glypta fumiferanae (Viereck), which attacks small larvae (L1 and L2 instar), was the most common parasitoid in spruce budworm larvae collected at the 5th instar and was present in both regions and treatments (Table 4). Apparent parasitism of 6th instar larvae was also low (<5%) except in Côte-Nord sites in which Btk treatments were applied, where parasitism was significantly higher than in controls (Table 4; Figure 4a). As many larvae died of unknown causes on Côte-Nord, corrected parasitism was much higher, with 26% in Btk-treated sites and 16% in controls of Côte-Nord, than in Bas-Saint-Laurent where it remained low at 6% and 3%, respectively, in controls and Btk-treated sites (Figure 4b). Parasitism by Smidtia fumiferanae (Tothill), the most common tachinid parasitoid of the spruce budworm, was significantly lower in Btk-treated sites than in controls (Côte-Nord: 1% vs. 3.04%; Bas-Saint-Laurent: 0.33% vs. 1.71%) (Dev = 6.3925, df = 1, p = 0.0115).
The highest incidence of parasitism measured on spruce budworm in our study was at the pupal stage, with 29% overall parasitism. The ichneumonid Dirophanes maculicornis (Stephens) was the most prevalent species with 21.4% parasitism, followed by Apechthis ontario (Cresson) with 4.4%. Both Btk treatment and region had significant effects on spruce budworm pupal parasitism, with a significant interaction between the two factors (Table 4; Figure 4a). In fact, spruce budworm pupal parasitism was higher in the Btk-treated sites of the Bas-Saint-Laurent region (55.3 ± 3.3%) than in control sites of this region (21.3 ± 1.4%) or in both control and treated sites of the Côte-Nord region (Figure 4a). The difference was much higher when using corrected parasitism with 73% in Btk-treated sites vs. 23% in controls of the Bas-Saint-Laurent region (Figure 4b). Parasitism by D. maculicornis and A. ontario were both significantly affected by Btk treatment and region, with a significant interaction between factors for D. maculicornis (Dev = 11.5319, df = 1, p = 0.0007) but not for A. ontario (Btk treatment: Dev = 33.427, df = 1, p < 0.0001; Region: Dev = 77.380, df = 1, p < 0.0001). Two ichneumonid parasitoids of spruce budworm pupae, A. ontario and Itoplectis conquisitor (Say), the Pteromalid Mesopolobus tortricis (Brues), as well as two tachinid parasitoids of late-instar larvae, Eumea caesar (Aldrich) and Phryxe pecosensis (Townsend), were not found in pupae of the Côte-Nord region (Table 4).
Many reared spruce budworms died before the emergence of a moth or a parasitoid. Both Btk treatment and region significantly affected the number of 5th instar larvae that failed to complete development, with an interaction between factors, the impact of Btk being greater in Côte-Nord where it reached 84.6 ± 2.6% compared to 36 ± 2.8% in controls of this region (Figure 5; Table 5). The number of 6th instar larvae that failed to complete development was also influenced significantly by an interaction between region and Btk treatment (Figure 5; Table 5). Development failure was greater in the Btk-treated sites (45.7 ± 2.9%) than in controls (16.7 ± 2.2%) of the Bas-Saint-Laurent region while it was highly prevalent in both controls and Btk-treated sites in Côte-Nord (57.6 ± 4.5% and 71.3 ± 2.6% for Btk-treated sites and controls, respectively). Finally, Btk treatment and region also influenced the number of pupae that failed to complete development, but without interaction between the two factors, Btk increased the number of failed completions similarly in both regions, Côte-Nord being more affected than Bas-Saint-Laurent (Figure 5; Table 5). For all budworm stages studied, the number of budworms that failed to complete development was always higher in the Côte-Nord region than in Bas-Saint-Laurent (Figure 5).

4. Discussion

Our results clearly demonstrate the effectiveness of aerial applications of Btk in controlling spruce budworm populations, reducing defoliation, and improving forest protection. Aerial applications were particularly effective in reducing defoliation in the old budworm population of the Côte-Nord region where it protected more than 40% more foliage than in controls. In Côte-Nord, the first Btk treatment was applied at the peak of the 4th larval instar, which is in the optimal budworm developmental window to maximize efficacy [42]. It had a significant impact on larval development as shown by the regression of the budworm average development stage, resulting from larval feeding cessation for at least 3 days after Btk consumption [58]. In Bas-Saint-Laurent, Btk was sprayed slightly later, when the budworm average development stage was at 4.36 (i.e., beginning of 5th larval instar) and there was no significant effect on budworm development. The 6.2 mm of rain that fell the day after Btk was sprayed may have reduced the effectiveness of the treatment. In addition to the reduced impact on budworm development, the difference in efficacy of Btk sprays between the two regions was reflected in differences in defoliation. Budworm population health might also be involved, Btk being more efficient in the old population of Côte-Nord.
Our results also revealed significant sublethal effects of Btk on budworm population fitness. Regardless of sex, larvae that fed sublethal doses of Btk produced smaller pupae. In general, pupal mass is positively correlated to female fecundity in Lepidoptera [59], including the spruce budworm [60] and therefore, Btk may influence population dynamics and the trajectory of spruce budworm outbreaks. Female pupal mass was reduced by 39% in Btk-treated sites in Bas-Saint-Laurent, compared with 23% in Côte-Nord, showing that the reduction in budworm fecundity is much greater in a young population than in an older one, underlining the importance of controlling an outbreak as early as possible. This is particularly relevant to consider for the early intervention strategy tested since 2014 in New Brunswick, Canada [61]. Similarly, pupae from the old population (Côte-Nord) were lighter than those from the young population (Bas-Saint-Laurent), most likely due to poor food quality and the precarious health of an old population. After two years of severe defoliation, the fiber content of balsam fir foliage increases, and the larval development of the spruce budworm lengthens [62]. At the time of sampling on Côte-Nord, we were already in the 10th year of a very severe outbreak and foliage biomass on trees was already strongly reduced. Food quality was undoubtedly of poor quality, as evidenced by the lighter pupae and the high level of development failure at all stages in control sites of Côte-Nord compared to Bas-Saint-Laurent where the population was much younger. In addition, it is interesting to highlight that pupae of each sex collected in Btk-treated sites had the same mass in both regions, even if they were much heavier in controls of the young population of Bas-Saint-Laurent, suggesting that the minimum mass for spruce budworm pupae could be around 43 mg for females and 36 mg for males.
The Btk treatment and region had no effect on parasitism of 5th instar larvae, but a significant interaction between these factors was observed on the inability to complete development. In fact, more larvae failed to complete development in Btk-treated sites. The difference was greatest on Côte-Nord, where over 70% of larvae died from other causes than parasitism in Btk-treated sites, suggesting a fragile health of larvae in this old population. Insects affected by sublethal diseases and/or poor food quality are probably more vulnerable to the addition of another sublethal stress (Btk), which results in a higher percentage of larvae that failed to complete development in the old population. The failure to complete development was also very high for L6 in Côte-Nord and was even higher in the controls than in the Btk-treated sites, indicating that the health of this population was poor and probably affected by pathogens and poor food quality. The incidence of pathogens, such as the microsporidian Nosema fumiferanae, increases in spruce budworm populations as the outbreak progresses [63,64,65]. In the Bas-Saint-Laurent region, the pattern observed in L5 was also observed in L6 but with a stronger effect of Btk on the inability to complete development.
A significant interaction was detected between Btk treatment and region for parasitism of the 6th instar larvae. Some species attacking late-instar larvae seem to respond in opposite ways to Btk treatments. For instance, parasitism by the tachinid Smidtia fumiferanae was significantly higher in control sites of both regions while parasitism by the braconid Meteorus trachynotus showed a reverse pattern. Parasitism by the tachinids Compsilura concinnata (Meigen) and Blepharipa pratensis (Robineau-Desvoidy) was also reduced after Btk treatment against the spongy moth, Lymantria dispar (L.) [66]. Tachinid flies are strongly stimulated by host movement, as their host selection behavior involves vision [67,68]. However, budworm larvae activity decreases after Btk treatment, as surviving larvae had consumed sublethal doses that led them into a feeding inhibition period. As a result, larvae affected by Btk treatment are probably less perceptible to tachinid parasitoids. Moreover, the window of availability of 6th instar budworm larvae, the preferred stage of S. fumiferanae [69], might not be well synchronized with its flight activity in Btk-treated sites, as it delays budworm larval development. Adult females of S. fumiferanae emerge when budworm larvae are at the 2nd to 5th instars, but the maturation of their eggs is well synchronized with the appearance of 6th instar larvae of the spruce budworm [70]. On the other hand, M. trachynotus can attack and develop in all larval instars of the spruce budworm, but it is more efficient in attacking small (L3 to L5) than large L6 larvae [69]. This parasitoid overwinters in an alternate host, Choristoneura rosaceana (Harris) [37], and its adults emerge when the spruce budworm is mainly at the 6th larval instar. As Btk slows down larval development of the budworm, it extends the window of availability of this host to attacks by M. trachynotus. In the spongy moth, such a positive effect of delayed development due to Btk sprays (1-wk temporal lag) has been reported to favor another braconid parasitoid, Cotesia melanoscela (Ratzeburg), going from 1% and 4.4% in untreated blocks to 13.3% and 32.3% in Btk-treated blocks [66,71]. Moreover, in one of these studies [66], only half of the commonly used Btk dose was applied without compromising defoliation reduction targets (8.2% in Btk-treated blocks vs. 40.9% in untreated blocks), suggesting that it could be possible to reduce treatment costs while improving parasitoid efficacy.
Pupal parasitism was particularly high in our study compared with previous studies [72,73,74]. It was significantly affected by an interaction between Btk and region, with much higher parasitism being observed in Btk-treated stands in the Bas-Saint-Laurent region than in those of the Côte-Nord region. This was partly due to the complete absence of A. ontario in Côte-Nord, this species parasitizing over 15% of pupae in Btk-treated sites of Bas-Saint-Laurent compared to 2.4% in control stands. The absence of this species in Côte-Nord may result from the unavailability of alternate hosts essential for A. ontario to complete its life cycle [75]. The Côte-Nord region belongs to the boreal forest, where plant diversity is lower than in the Bas-Saint-Laurent region, which belongs to the mixed forest [76]. Therefore, alternate hosts should be more abundant and diverse in Bas-Saint-Laurent than in Côte-Nord. However, the most prevalent species of the study, D. maculicornis, followed the same pattern as A. ontario in Bas-Saint-Laurent with 31.7% parasitism in Btk-treated sites compared with 18.3% in controls. However, it was nearly the same in both treatments in Côte-Nord (16% in Btk-treated sites vs. 19.7% in controls). These regional differences in the response of D. maculicornis parasitism to Btk treatments might result from complex interactions among the number of available hosts, their health status and the synchrony between adult parasitoids and pupal hosts.
In Bas-Saint-Laurent, Btk was applied later in the budworm’s larval development, and the first treatment had little effect on budworm development. The second Btk treatment was applied very late, when budworm larvae were already at the 5th and even 6th instar. Late application of Btk may have a lesser effect on larval development and allow better synchrony between adult parasitoids and budworm pupae. However, unknown effects on pupae could also explain why pupal parasitism was so high in the Bas-Saint-Laurent region. This will need further studies, which could hopefully elucidate the mechanism behind this surprising result. Spraying Btk later in budworm development might be a good strategy to maximize reductions in pest populations, even if it means tolerating slightly more defoliation. This could be the best approach in the early stages of a spruce budworm outbreak when the objective is to reduce pest populations to avoid outbreak [61].

5. Conclusions

Our study confirms the effectiveness of Btk in controlling spruce budworm populations and their damage directly, but also indirectly through sublethal effects on budworm development, pupal size and parasitism. Forest insect control programs aim to reduce pest populations and the damage they cause by using the maximum authorized dose of insecticide, without considering mortality caused by natural enemies [66]. In fact, with a biopesticide like Btk, we hope above all to avoid disrupting the natural control of pest populations. Even if these programs successfully reduce pest populations to acceptable levels, they may use more insecticide than necessary, thus resulting in high costs and mortality of non-target organisms, including some natural enemies. Our study suggests that Btk is a flexible tool that can be used in various ways depending on the status of populations during the epidemic cycle. At the beginning of an outbreak, when populations are healthy and defoliation is not yet an issue, Btk could be applied later in the development of the budworm (L5) to maximize sublethal effects on both pupal size and pupal parasitism, increasing impact of Btk on the population dynamics of the spruce budworm. This might be particularly relevant for the early intervention strategy recently deployed against the spruce budworm in New Brunswick [19]. Later in the outbreak cycle, when population health decreases, Btk sprays properly timed with the peak of the 4th larval instar are highly efficient for protecting foliage.

Author Contributions

Conceptualization, C.H. and A.D.; methodology, C.H. and A.D.; formal analysis, J.-M.B.; investigation, C.H. and R.B.; resources, A.D. and C.H.; data curation, J.-M.B. and C.H.; writing—original draft preparation, C.H.; writing—review and editing, C.H., J.-M.B., R.B. and A.D.; supervision, C.H., A.D. and R.B.; project administration, C.H., A.D. and R.B.; funding acquisition, A.D., C.H. and R.B. All authors have read and agreed to the published version of the manuscript.

Funding

The study was funded by SOPFIM, the Pest Risk Management program of the Canadian Forest Service and by a NSERC discovery grant (RGPIN/436015-2013) to Dr. Christian Hébert.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We thank SOPFIM field technicians for collecting the branches needed to estimate budworm density, development, parasitism and defoliation. We would also like to thank Jessica Girona, then employed by the Canadian Forest Service of Natural Resources Canada (NRCan-CFS), for extracting the budworms from the branches, rearing them, and weighing the pupae. She also pinned and labeled the parasitoids that are kept at the René-Martineau Insectarium at NRCan-CFS’s Laurentian Forestry Centre. Parasitoids were identified by M Georges Pelletier, then biologist in insect identification at NRCan-CFS Laurentian Forestry Centre.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Location of forest blocks studied in Quebec in 2017, based on defoliation caused by the spruce budworm since the beginning of the outbreak. The dots represent the control blocks, while the triangles represent forest blocks sprayed twice with Btk.
Figure 1. Location of forest blocks studied in Quebec in 2017, based on defoliation caused by the spruce budworm since the beginning of the outbreak. The dots represent the control blocks, while the triangles represent forest blocks sprayed twice with Btk.
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Forests 16 01666 g002
Figure 2. Seasonal trends in spruce budworm average development stage in blocks treated or not treated with Btk in 2017, in two regions of Quebec. The large black arrows indicate the first and second Btk applications in each region. Grey and black vertical bars and asterisks represent significant differences in budworm development between the Btk-treated and control blocks (grey: p < 0.001; black: p < 0.0001).
Figure 2. Seasonal trends in spruce budworm average development stage in blocks treated or not treated with Btk in 2017, in two regions of Quebec. The large black arrows indicate the first and second Btk applications in each region. Grey and black vertical bars and asterisks represent significant differences in budworm development between the Btk-treated and control blocks (grey: p < 0.001; black: p < 0.0001).
Forests 16 01666 g002
Forests 16 01666 g003
Figure 3. Pupal mass (EMMS ± SE) of male and female spruce budworms in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons (p < 0.05) share the same letters.
Figure 3. Pupal mass (EMMS ± SE) of male and female spruce budworms in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons (p < 0.05) share the same letters.
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Forests 16 01666 g004
Figure 4. Apparent parasitism (a) and corrected parasitism (EMMS ± SE) (b) of spruce budworm larvae (L5 and L6) and pupae in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons share the same letters.
Figure 4. Apparent parasitism (a) and corrected parasitism (EMMS ± SE) (b) of spruce budworm larvae (L5 and L6) and pupae in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons share the same letters.
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Forests 16 01666 g005
Figure 5. Inability of late-instar larvae (L5 and L6) and pupae of the spruce budworm to complete development (EMMS ± SE) in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons share the same letters.
Figure 5. Inability of late-instar larvae (L5 and L6) and pupae of the spruce budworm to complete development (EMMS ± SE) in balsam fir forests of two regions in Quebec, treated or untreated with Btk in 2017. Estimated marginal means that are not significantly different following Tukey’s pairwise comparisons share the same letters.
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Table 1. Characteristics of each forest block studied based on the designations on ecoforest maps.
Table 1. Characteristics of each forest block studied based on the designations on ecoforest maps.
RegionTreatmentEcoforest map designationAltitude
Composition aDensity/height bAge(m)
Bas-Saint-LaurentBtkSBSBB350–70467
ControlSBSBB350–70388
Cote-NordBtkSBSBB370333
ControlSBSBB370220
a Refers to the dominant and 1st companion tree species. SBSB, for “Sapin Baumier-Sapin Baumier” (in French), which means pure Balsam fir stands. b Tree density and height classes. “B” refers to a 60%–80% tree cover and “3” to 12–17 m high trees.
Table 2. Spruce budworm population and balsam fir defoliation estimated marginal means (EMMs ± SE) to assess efficacy of Btk spray treatments applied in two regions in Quebec. In each column, values followed by the same letters do not differ significantly at p < 0.05.
Table 2. Spruce budworm population and balsam fir defoliation estimated marginal means (EMMs ± SE) to assess efficacy of Btk spray treatments applied in two regions in Quebec. In each column, values followed by the same letters do not differ significantly at p < 0.05.
RegionTreatmentLarvae/Branch 1Mortality 2Defoliation 3
Pre-treat.Post-treat.(%)(%)
Bas-Saint-LaurentBtk21.7 ± 4 a4.4 ± 1 b79.776.7 ± 9 ab
Control33.4 ± 4 a14.0 ± 3 a56.592.8 ± 9 a
Côte-NordBtk23.2 ± 7 a1.8 ± 1 b92.256.1 ± 9 b
Control42.8 ± 13 a16.4 ± 4 a61.7100.0 ± 9 a
1 estimated in spring 2017. 2 larval mortality estimated in 2017 using the following equation: ((Pre-treat − Post-treat)/Pre-treat) × 100. 3 estimated in summer 2017.
Table 3. Summary of analysis of variance comparing spruce budworm pupal mass as a function of sex, region (Bas-Saint-Laurent and Côte-Nord) and Btk spray treatment.
Table 3. Summary of analysis of variance comparing spruce budworm pupal mass as a function of sex, region (Bas-Saint-Laurent and Côte-Nord) and Btk spray treatment.
TreatmentsDfFp > F
Btk application 1613.77<0.001
Region167.98<0.001
Sex1286.57<0.001
Btk application*Region1112.47<0.001
Btk application*Sex10.370.543
Region*Sex10.430.509
Btk application*Region*Sex11.430.233
Table 4. Parasitoids emerged from 5th or 6th instar larvae or from pupae of the Spruce Budworm, collected in balsam fir stands treated or not with aerial applications of Btk, in the Bas-Saint-Laurent and Côte-Nord regions in Quebec, Canada.
Table 4. Parasitoids emerged from 5th or 6th instar larvae or from pupae of the Spruce Budworm, collected in balsam fir stands treated or not with aerial applications of Btk, in the Bas-Saint-Laurent and Côte-Nord regions in Quebec, Canada.
Bas-Saint-LaurentCôte-Nord
StageOrderFamilySpeciesBtk SpraysControlBtk SpraysControl
L5HymenopteraBraconidaeApanteles fumiferanae 2
Meteorus trachynotus 1
IchneumonidaeEnytus montanus1
Glypta fumiferanae9828
DipteraTachinidaeEumea caesar 1
Number of SBW—L5 reared300300195300
L6HymenopteraBraconidaeMeteorus trachynotus314
Undetermined 1
IchneumonidaeDirophanes maculicornis 2 *
Glypta fumiferanae 112
Undetermined 12
DipteraTachinidaeEumea caesar 41
Phryxe pecosensis 3
Smidtia fumiferanae1519
Undetermined 1 21
Number of SBW—L6 reared300300118300
PupaHymenopteraIchneumonidaeApechthis ontario467
Dirophanes maculicornis95554859
Itoplectis conquisitor5
Undetermined4 1
PteromalidaeMesopolobus tortricis2
Mesopolobus verditer6 1
DipteraTachinidaeEumea caesar 1
Phryxe pecosensis 1
Smidtia fumiferanae5 32
Undetermined 3 22
Number of SBW pupae reared 300300300300
* Probably attacked as prepupae.
Table 5. Influence of Btk sprays and study region (Bas-Saint-Laurent and Côte-Nord) on spruce budworm parasitism or developmental failure after collections at 5th or 6th larval or pupal stage.
Table 5. Influence of Btk sprays and study region (Bas-Saint-Laurent and Côte-Nord) on spruce budworm parasitism or developmental failure after collections at 5th or 6th larval or pupal stage.
SBW StageFactorsProbability Metric
Parasitism Developmental Failure
DfDev.Pr > Chi DfDev.Pr > Chi
L5Btk treatment 10.800.371 168.27<0.001
Region10.390.530 1155.60<0.001
Btk treatment*Region12.810.094 128.89<0.001
L6Btk treatment 10.070.785 12.520.112
Region16.070.014 1150.94<0.001
Btk treatment*Region19.650.002 147.21<0.001
PupaBtk treatment 136.10<0.001 157.91<0.001
Region153.09<0.001 17.10<0.001
Btk treatment*Region138.28<0.001 11.750.186
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Hébert, C.; Béland, J.-M.; Dupont, A.; Berthiaume, R. The Lethal and Sublethal Effects of Aerial Applications of Bacillus thuringiensis subsp. kurstaki on the Spruce Budworm and Its Parasitism. Forests 2025, 16, 1666. https://doi.org/10.3390/f16111666

AMA Style

Hébert C, Béland J-M, Dupont A, Berthiaume R. The Lethal and Sublethal Effects of Aerial Applications of Bacillus thuringiensis subsp. kurstaki on the Spruce Budworm and Its Parasitism. Forests. 2025; 16(11):1666. https://doi.org/10.3390/f16111666

Chicago/Turabian Style

Hébert, Christian, Jean-Michel Béland, Alain Dupont, and Richard Berthiaume. 2025. "The Lethal and Sublethal Effects of Aerial Applications of Bacillus thuringiensis subsp. kurstaki on the Spruce Budworm and Its Parasitism" Forests 16, no. 11: 1666. https://doi.org/10.3390/f16111666

APA Style

Hébert, C., Béland, J.-M., Dupont, A., & Berthiaume, R. (2025). The Lethal and Sublethal Effects of Aerial Applications of Bacillus thuringiensis subsp. kurstaki on the Spruce Budworm and Its Parasitism. Forests, 16(11), 1666. https://doi.org/10.3390/f16111666

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