# Evidence for Use ofAlliaria petiolata in North America by the European Cabbage White Butterfly,Pieris rapae

Authors: Sam L.  Davis; Don Cipollini
Journal: Psyche
 (2016)

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## Abstract

Pieris rapae L., an invasive crop pest, may have recently begun usingAlliaria petiolata Bieb. (Cavara & Grande), a European invasive biennial. We investigated howP. rapae uses forest habitats for nectar and oviposition and examined larval performance onA. petiolata in the field and laboratory. Being known primarily to occupy open habitats, we found thatP. rapae regularly uses forest edge habitats, most surveyedA. petiolataplants hadP. rapae damage, andP. rapae successfully used both stages ofA. petiolata for larval development.


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## Body

## 1. Introduction

Although some of the 50,000 alien species introduced into the United States have economic value, organisms unintentionally introduced to novel habitats have been estimated to cost the United States almost $120 billion in agricultural and economic damages each year [1]. Invasive species also cause untold damages to natural habitats through changing nutrient cycles, altering resource competition, and affecting the physical landscape structure around them, including nutrient cycling [2]. Where rare species live, invasion by novel plants or animals can cause vulnerable species to become endangered or extinct [3].Pieris rapaeL. (small cabbage white; Lepidoptera: Pieridae) is a multivoltine European butterfly accidentally introduced to Quebec, Canada, in 1860. A specialist on glucosinolate-containing Brassicaceae host plants, it soon became a destructive crop pest in North America, moving south and west as far as Kentucky in just 12 years [4]. Being now ubiquitous and abundant across the United States and Canada, it is known as a butterfly of open meadows, crop plantings, and sunny areas where its cultivated and wild hosts are typically found [5, 6].Its primary hosts in its native range includeArmoracia rusticana,Brassicaspp.,Cardaminespp.,Crambe maritima,Sisymbrium officinale, andTropaeolum majus, among others, most of which are high light requiring plants [7]. In North America, it benefits from habitat fragmentation and disturbance favoring growth of its weedy hosts, such asBarbarea vulgaris, introducedBrassica species, andLepidium species, many of which are also nonnative [8–10]. As a common pest on commercial brassicaceous crops,P. rapae is highly visible as an adult and more cryptic in its larval stage and has been controlled in the past through application of DDT and Bt, along with introductions ofCotesia glomerataandC. rubecula parasitoid wasps [6, 11, 12].Although it regularly uses crop plants in North America,P. rapae may also use the invasive European biennialAlliaria petiolataBieb. (Cavara & Grande), in part due to the plant’s increasing abundance in the understory and close relationship with other host plants in the Brassicaceae. Unlike most other potential hosts,A. petiolata is unique in its shade-tolerance and occupancy of forest edges and understories. This invasive mustard allelopathically affects mycorrhizal forest plants as well as competes directly with neighboring plants for resources [13, 14]. Anecdotal observations suggest that this plant is much more abundant in North America than in Europe, and its presence may drawP. rapae into forests more often [15].There are not many herbivores that useA. petiolataas a food source in North America. Although Yates and Murphy [16] identified three arthropod herbivores present onA. petiolatain Ontario, Canada (Ceutorhynchus erysimiFabr.,Plutella xylostellaL., andPhilaenus spumarius), they did not observeP. rapaeconsumingA. petiolata, and no herbivore eats enough to control its spread or abundance. Even mollusks avoid consumingA. petiolata, instead preferring more palatable native plants [17, 18]. This suggests thatA. petiolatais generally well defended from most North American herbivores, and the damage that it does accrue rarely reduces plant fitness. However,P. rapaemay be able to use the European plant as a host in North America, especially since there is some evidence of it being used as a host in Europe [7]. At present, only rare, anecdotal observations exist of the use of forested habitats byP. rapae in North America [19–21].To investigate howP. rapaeis using forested habitats and the host plant,A. petiolata, in North America, we directly observedP. rapaeoviposition and nectaring behavior in forested habitats shared withP. virginiensis,a native congener. We also investigated howP. rapaeusesA. petiolatain forest edge habitats. Finally, we compared the performance ofP. rapaelarvae and adults fedA. petiolatato that observed on its more typical hosts,Brassica junceaandB. oleracea.


## 2. Methods

### 2.1. Direct Observations ofP. rapae in Forest Habitats

Observations ofP. rapaeoccurred from April to June in 2011, 2012, and 2013 at three sites known to be occupied byP. virginiensis: a private site in Morrow Co. (MCO), OH, Wooster Memorial Park (WMP) in Wooster, OH, and Allegany State Park in Salamanca (ASP), NY. Basic visual observations were recorded using field notebooks and photography. Sites were surveyed in tandem with surveys forP. virginiensis, a related native butterfly. More details about observation protocols and site histories can be found in Davis and Cipollini 2014a, b publications [21, 22].More detailed behavioral observations were made at WMP. Twenty-fivePieris rapaeindividuals were monitored between 1100 and 1600 on Apr. 15 and 18, 2012, at least 300 meters away from the nearest edge or agricultural habitat. Behaviors of individual butterflies were recorded in ten-second intervals until the butterfly left the area and included flying, gathering nectar, oviposition, and resting. Although ten seconds is longer than the time required forP. rapaeto oviposit, we gathered enough observations to capture the rate of oviposition through time.We identified all plants that the butterflies interacted with during oviposition and nectar gathering using the Newcomb [23] guide to wildflowers. Butterflies were identified asP. rapaeand not as the nativeP. virginiensis by distinct, dark spots on the dorsal wing surfaces and yellow scales on the ventral wing surfaces. In contrast,P. virginiensisis white with occasional wing-vein shading and light spots on the wings [4].We also observed herbivory byP. rapaecaterpillars at WMP during the same observation periods. Although the first instarPieriscaterpillars are difficult to identify to species, olderP. rapaecaterpillars develop a broken yellow line along the dorsal surface and yellow spots around the spiracles; these characters are missing in nativeP. virginiensiscaterpillars [4].


### 2.2. Herbivory byP. rapae on A. petiolata in Edge Habitats

We examined how frequentlyP. rapaeusesA. petiolataas a larval host plant in wooded habitats by measuring end-of-year herbivory on first-yearA. petiolataplants in maple-beech-oak forests surrounding Dayton, OH. All herbivory experiments that follow were performed in late fall, when anyP. rapaein the area would be in diapause as pupae. This ensured that we recorded a maximum amount of damage on individual plants.In 2011, we surveyed approximately 9000 m2 of a recreational trail in Beavercreek, OH, between Grange Hall Road and N. Fairfield Road (BCT, western corner: 39.734756N, 84.082472W; eastern corner: 39.724096N, 84.060070W). This trail has grass and unmanaged shrubs on the southern side and a strip of second-growth forest (20–60 m forest perpendicular to the trail) on the northern side. In 2013, we returned to resurvey BCT and also surveyed two other sites: Narrows Reserve in Beavercreek, OH (NAR, located at 39.691313N, 84.030293W), and Fairborn Community Park in Fairborn, OH (FCP, located at 39.789345N, 84.009446W). Approximately 3000 m2 and 2400 m2 were surveyed at NAR and FCP, respectively. All three sites had parking lots, recreation trails, and forest areas. We walked the perimeter of each study area and systematically examined every rosetteA. petiolatawithin 3 m of the open area. In patches with more than 10 rosettes clumped together, we randomly chose 10 plants to sample. We surveyed 99 plants at BCT in 2011. In 2013, we surveyed 136 plants at BCT, 53 plants at FCP, and 81 plants at NAR.Plants with at least one leaf larger than 5 cm in diameter (mostA. petiolata individuals) were surveyed for chewing damage from caterpillars (asymmetrical, smooth holes away from the leaf edge) on fully expanded leaves. Our observations ofP. rapaedamage are indirect only because the surveys were performed afterP. rapaecaterpillars had pupated for the winter. Damage was attributed primarily toP. rapaecaterpillars for several reasons. First, caterpillar damage is distinct from other causes of damage and disease, including deer herbivory, slug herbivory, and flea beetle damage (SLD and DC, personal observations). Second, we have observedP. rapae caterpillars feeding onA. petiolata throughout the year at these locations, andP. rapae is the only caterpillar that we have ever observed feeding in this area, despite reports ofPlutella xylostellaas another lepidopteran herbivore onA. petiolata [16]. Finally, several other researchers have confirmed these observations ofP. rapae feeding onA. petiolata in both Ohio and Massachusetts (John Stireman and Frances Chew, personal communications). Although some leaf tearing and disease were noted (especially the presence of a powdery mildew fungus [24]), these observations were excluded from analysis of herbivory. Each leaf on a chosen plant was scored for leaf area loss by caterpillars from 0 to 5 (undamaged, 1–20%, 21–40%, 41–60%, 61–80%, and 81–100% leaf loss). The damage rating was converted to percent leaf loss by weighing each leaf score as follows: 0 (0), 1 (0.1), 2 (0.3), 3 (0.5), 4 (0.7), and 5 (0.9). The leaf scores for each plant were averaged into a final plant score. We used the indices 1–5 because precise measurements of leaf damage in the field were not possible.


### 2.3. Pieris rapae Larval Performance Assay

In order to determine the suitability ofA. petiolataas a larval host, we examinedP. rapaelarval performance on both rosette and floweringA. petiolata(Wright State Forest, Dayton, OH) and on two commercial brassicaceous crops,B. junceaandB. oleracea(Meijer, Inc.).Pieris rapaeeggs (Carolina Biological Supply) were raised on eitherBrassica oleracea L. “green cabbage” (Meijer, Inc.) or floweringA. petiolata and allowed to emerge as adult butterflies. We used second generation butterflies because field grown rosetteA. petiolata(used below) was too small to be useful when the shipment of eggs arrived. Between ten and fifteen adults were placed in 75 L aquaria with artificial nectar (20% sucrose : water solution on delicate task wipes until moist) and allowed to oviposit on floweringA. petiolata. Eggs laid by the adult butterflies were used in the following larval performance experiment. We distributed eighty neonates evenly among the four treatments below (n = 20 per treatment).After hatching, second generation neonates were placed on either field-collected (June 2014) rosetteAlliaria petiolata, floweringA. petiolata, commercially purchased, nonorganicB. oleracea(green cabbage, Meijer, Inc.), orB. juncea (southern giant curled mustard, Meijer, Inc.) leaves in moist filter-paper lined Petri dishes and kept in a 16 : 8 L : D incubator at room 25 deg. C. Caterpillars were kept individually to mimic the solitary nature ofP. rapaecaterpillars in the wild. Commercial plants were rinsed with distilled water before use. We choseB. oleraceaandB. junceato represent commercial hosts available toP. rapaein the wild. Caterpillar habitats were cleaned daily and stocked with an overabundance of host plant material to prevent starvation or time without eating. After one week of monitoring daily for survival, we took daily measurements of caterpillar mass, until they neared pupation. Pupae were weighed and placed in 75 L aquaria according to their larval host plant, with artificial nectar and an oviposition substrate (rosetteA. petiolata). After eclosion, butterflies were allowed to mate and oviposit freely. When all butterflies died, the number of eggs and the number of females were counted to calculate the mean number of eggs laid per female, an indirect measure of fitness.


### 2.4. Statistical Analysis

All statistical analyses were performed inR [25]. We separated our field herbivory data into two sets: data from BCT alone and data from 2013 alone. These data were separated because only one site, BCT, was sampled for two years. For both datasets, we used a binomial model with a logit link function followed by Tukey’s HSD test (multcomp package) to examine how the number of leaves on a plant covaried with location (2013 data, predictor) or year (BCT data, predictor) to affect the presence or absence of damaged leaves (response variable) [26]. We also examined the same datasets (2013 and BCT data) for differences in the percent leaf loss score. We removed all zeroes because we were only interested in damaged plants, log-transformed the percent leaf loss scores to meet normality assumptions, and then evaluated the data using a generalized linear model followed by Tukey’s HSD post hoc testing when appropriate. Plots were constructed with the gplotspackage [27].For the larval performance experiments, we used the Kaplan-Meier estimator for survival data (survival package) and one-way ANOVA to compare pupal mass and relative growth rate across host plants [28]. Relative growth rate (RGR) was calculated as larval mass increase (from day 7 until pupation) divided by the initial larval mass times the number of days of recorded growth. Chi-square testing followed by chi-square tests with Bonferroni correction was used to evaluate differences between the number of eggs laid per treatment.


## 2.1. Direct Observations ofP. rapae in Forest Habitats

Observations ofP. rapaeoccurred from April to June in 2011, 2012, and 2013 at three sites known to be occupied byP. virginiensis: a private site in Morrow Co. (MCO), OH, Wooster Memorial Park (WMP) in Wooster, OH, and Allegany State Park in Salamanca (ASP), NY. Basic visual observations were recorded using field notebooks and photography. Sites were surveyed in tandem with surveys forP. virginiensis, a related native butterfly. More details about observation protocols and site histories can be found in Davis and Cipollini 2014a, b publications [21, 22].More detailed behavioral observations were made at WMP. Twenty-fivePieris rapaeindividuals were monitored between 1100 and 1600 on Apr. 15 and 18, 2012, at least 300 meters away from the nearest edge or agricultural habitat. Behaviors of individual butterflies were recorded in ten-second intervals until the butterfly left the area and included flying, gathering nectar, oviposition, and resting. Although ten seconds is longer than the time required forP. rapaeto oviposit, we gathered enough observations to capture the rate of oviposition through time.We identified all plants that the butterflies interacted with during oviposition and nectar gathering using the Newcomb [23] guide to wildflowers. Butterflies were identified asP. rapaeand not as the nativeP. virginiensis by distinct, dark spots on the dorsal wing surfaces and yellow scales on the ventral wing surfaces. In contrast,P. virginiensisis white with occasional wing-vein shading and light spots on the wings [4].We also observed herbivory byP. rapaecaterpillars at WMP during the same observation periods. Although the first instarPieriscaterpillars are difficult to identify to species, olderP. rapaecaterpillars develop a broken yellow line along the dorsal surface and yellow spots around the spiracles; these characters are missing in nativeP. virginiensiscaterpillars [4].


## 2.2. Herbivory byP. rapae on A. petiolata in Edge Habitats

We examined how frequentlyP. rapaeusesA. petiolataas a larval host plant in wooded habitats by measuring end-of-year herbivory on first-yearA. petiolataplants in maple-beech-oak forests surrounding Dayton, OH. All herbivory experiments that follow were performed in late fall, when anyP. rapaein the area would be in diapause as pupae. This ensured that we recorded a maximum amount of damage on individual plants.In 2011, we surveyed approximately 9000 m2 of a recreational trail in Beavercreek, OH, between Grange Hall Road and N. Fairfield Road (BCT, western corner: 39.734756N, 84.082472W; eastern corner: 39.724096N, 84.060070W). This trail has grass and unmanaged shrubs on the southern side and a strip of second-growth forest (20–60 m forest perpendicular to the trail) on the northern side. In 2013, we returned to resurvey BCT and also surveyed two other sites: Narrows Reserve in Beavercreek, OH (NAR, located at 39.691313N, 84.030293W), and Fairborn Community Park in Fairborn, OH (FCP, located at 39.789345N, 84.009446W). Approximately 3000 m2 and 2400 m2 were surveyed at NAR and FCP, respectively. All three sites had parking lots, recreation trails, and forest areas. We walked the perimeter of each study area and systematically examined every rosetteA. petiolatawithin 3 m of the open area. In patches with more than 10 rosettes clumped together, we randomly chose 10 plants to sample. We surveyed 99 plants at BCT in 2011. In 2013, we surveyed 136 plants at BCT, 53 plants at FCP, and 81 plants at NAR.Plants with at least one leaf larger than 5 cm in diameter (mostA. petiolata individuals) were surveyed for chewing damage from caterpillars (asymmetrical, smooth holes away from the leaf edge) on fully expanded leaves. Our observations ofP. rapaedamage are indirect only because the surveys were performed afterP. rapaecaterpillars had pupated for the winter. Damage was attributed primarily toP. rapaecaterpillars for several reasons. First, caterpillar damage is distinct from other causes of damage and disease, including deer herbivory, slug herbivory, and flea beetle damage (SLD and DC, personal observations). Second, we have observedP. rapae caterpillars feeding onA. petiolata throughout the year at these locations, andP. rapae is the only caterpillar that we have ever observed feeding in this area, despite reports ofPlutella xylostellaas another lepidopteran herbivore onA. petiolata [16]. Finally, several other researchers have confirmed these observations ofP. rapae feeding onA. petiolata in both Ohio and Massachusetts (John Stireman and Frances Chew, personal communications). Although some leaf tearing and disease were noted (especially the presence of a powdery mildew fungus [24]), these observations were excluded from analysis of herbivory. Each leaf on a chosen plant was scored for leaf area loss by caterpillars from 0 to 5 (undamaged, 1–20%, 21–40%, 41–60%, 61–80%, and 81–100% leaf loss). The damage rating was converted to percent leaf loss by weighing each leaf score as follows: 0 (0), 1 (0.1), 2 (0.3), 3 (0.5), 4 (0.7), and 5 (0.9). The leaf scores for each plant were averaged into a final plant score. We used the indices 1–5 because precise measurements of leaf damage in the field were not possible.


## 2.3. Pieris rapae Larval Performance Assay

In order to determine the suitability ofA. petiolataas a larval host, we examinedP. rapaelarval performance on both rosette and floweringA. petiolata(Wright State Forest, Dayton, OH) and on two commercial brassicaceous crops,B. junceaandB. oleracea(Meijer, Inc.).Pieris rapaeeggs (Carolina Biological Supply) were raised on eitherBrassica oleracea L. “green cabbage” (Meijer, Inc.) or floweringA. petiolata and allowed to emerge as adult butterflies. We used second generation butterflies because field grown rosetteA. petiolata(used below) was too small to be useful when the shipment of eggs arrived. Between ten and fifteen adults were placed in 75 L aquaria with artificial nectar (20% sucrose : water solution on delicate task wipes until moist) and allowed to oviposit on floweringA. petiolata. Eggs laid by the adult butterflies were used in the following larval performance experiment. We distributed eighty neonates evenly among the four treatments below (n = 20 per treatment).After hatching, second generation neonates were placed on either field-collected (June 2014) rosetteAlliaria petiolata, floweringA. petiolata, commercially purchased, nonorganicB. oleracea(green cabbage, Meijer, Inc.), orB. juncea (southern giant curled mustard, Meijer, Inc.) leaves in moist filter-paper lined Petri dishes and kept in a 16 : 8 L : D incubator at room 25 deg. C. Caterpillars were kept individually to mimic the solitary nature ofP. rapaecaterpillars in the wild. Commercial plants were rinsed with distilled water before use. We choseB. oleraceaandB. junceato represent commercial hosts available toP. rapaein the wild. Caterpillar habitats were cleaned daily and stocked with an overabundance of host plant material to prevent starvation or time without eating. After one week of monitoring daily for survival, we took daily measurements of caterpillar mass, until they neared pupation. Pupae were weighed and placed in 75 L aquaria according to their larval host plant, with artificial nectar and an oviposition substrate (rosetteA. petiolata). After eclosion, butterflies were allowed to mate and oviposit freely. When all butterflies died, the number of eggs and the number of females were counted to calculate the mean number of eggs laid per female, an indirect measure of fitness.


## 2.4. Statistical Analysis

All statistical analyses were performed inR [25]. We separated our field herbivory data into two sets: data from BCT alone and data from 2013 alone. These data were separated because only one site, BCT, was sampled for two years. For both datasets, we used a binomial model with a logit link function followed by Tukey’s HSD test (multcomp package) to examine how the number of leaves on a plant covaried with location (2013 data, predictor) or year (BCT data, predictor) to affect the presence or absence of damaged leaves (response variable) [26]. We also examined the same datasets (2013 and BCT data) for differences in the percent leaf loss score. We removed all zeroes because we were only interested in damaged plants, log-transformed the percent leaf loss scores to meet normality assumptions, and then evaluated the data using a generalized linear model followed by Tukey’s HSD post hoc testing when appropriate. Plots were constructed with the gplotspackage [27].For the larval performance experiments, we used the Kaplan-Meier estimator for survival data (survival package) and one-way ANOVA to compare pupal mass and relative growth rate across host plants [28]. Relative growth rate (RGR) was calculated as larval mass increase (from day 7 until pupation) divided by the initial larval mass times the number of days of recorded growth. Chi-square testing followed by chi-square tests with Bonferroni correction was used to evaluate differences between the number of eggs laid per treatment.


## 3. Results

### 3.1. Direct Observations ofP. rapae in Forest Habitats

At MCO and ASP, we regularly observedP. rapaeflying in heavily wooded areas but did not observe any nectar gathering or oviposition behavior. We found an unidentified first instar caterpillar in 2012 at MCO onA. petiolata that could have been eitherP. rapaeorP. virginiensis. At WMP, we observedP. rapaeadults gathering nectar in the understory from several plant species, includingClaytonia, Phlox, andViolaspecies, as well as fromA. petiolataitself. We also observed 3 femaleP. rapaeovipositing onCardamine diphyllaand photographed an olderP. rapaecaterpillar feeding onA. petiolata (Figure 1). Additionally,P. rapae caterpillars have been observed feeding onA. petiolataoutside of the Wright State University greenhouse (2012–1015), onA. petiolata in the Wright State University woods, and in residential and park areas in Fairborn and Beavercreek, OH (SLD, personal observations).Figure 1
MatureP. rapae caterpillar consuming rosetteA. petiolata in Wooster, OH. Photo taken on May 11, 2012, by SLD.


### 3.2. Herbivory byP. rapae on A. petiolata in Edge Habitats

Although overall percent leaf loss was low, 78.8% of plants were damaged by caterpillars in 2013, indicating thatP. rapaecommonly usesA. petiolatain North America. In 2013, both the number of leaves (z = 3.475, P<0.01) and the location (P<0.01) influenced the probability of plants being attacked. BCT was significantly different from NAR (z=2.614, P<0.05) and FCP (z=3.631, P<0.01), but the latter two were not significantly different from each other (z=-2.217, P>0.05). Across years at BCT, only the number of leaves was a significant factor in the model (z=3.622, P<0.01), indicating no difference in plant damage between years. Evaluating the percent leaf loss score revealed similar results, with BCT being significantly different from both NAR (z=2.387, P<0.05) and FCP (z=3.697, P<0.01), but NAR and FCP were not significantly different from each other, and the number of leaves per plant was not correlated with the percent leaf loss score. The model evaluating percent leaf loss score as influenced by date and number of leaves for the BCT site alone was not significant. Figure 2 shows the mean percent leaf loss score for both sites and years.Figure 2
Herbivory (percent leaf loss) onA. petiolata varied between sites and habitats in 2013. Gray bar represents data from 2011; black bars represent data from 2013.


### 3.3. Pieris rapae Larval Performance

Although there was a trend towards lower survival ofP. rapaecaterpillars feeding on floweringA. petiolata, we found no significant differences in survival ofP. rapaecaterpillars on the four hosts that we tested (χ2=7.4, df = 3, and P=0.0596, Figure 3). Pupal mass did not vary between treatments (F=2.213, df = 3, and P=0.109); however, there were differences in time to pupation (F=7.897, df = 3, and P<0.01). Caterpillars reared onB. junceapupated significantly earlier than those raised on rosetteA. petiolata(P<0.05, Tukey’s HSD) and on commercialB. oleracea(P<0.01). Relative growth rates also differed between treatments (F=4.428, df = 3, and P<0.01) because caterpillars on leaves ofB. juncea grew significantly faster than those on floweringA. petiolata (P<0.01, Tukey’s HSD). To summarize,P. rapaecaterpillars reared onB. junceagrew faster and pupated earlier with no significant loss of pupal mass, whereas caterpillars reared on floweringA. petiolatatook longer and grew slower than those onB. juncea. Means and standard error of each treatment are found in Table 1.Table 1
Mean percent survival, days to pupation, pupal weight, and relative growth rate with standard error ofP. rapae (both sexes) between four host plants (n=16 per treatment). Different letters indicate significant differences (P<0.05).
Treatment
Survival (%)
Days To pupation (d)
Pupal mass (g)
Relative growth rate (g g−1 d−1)
A. petiolatarosette
56.25
15.33 ± 0.85
0.128 ± 0.003
0.277 ± 0.046ab
A. petiolataflowering
20.00
15.33 ± 1.45
0.112 ± 0.008
0.182 ± 0.031b
B. oleracea “cabbage”
68.75
16.63 ± 0.28
0.134 ± 0.004
0.294 ± 0.019ab
B. juncea “mustard”
56.25
12.78 ± 0.46
0.132 ± 0.006
0.380 ± 0.044aFigure 3
Kaplan-Meier survival estimates ofP. rapaecaterpillars fed commercial cabbage (solid black), rosetteA. petiolata(dash grey), floweringA. petiolata(solid grey), or commercial mustard greens (dash black).Eclosed butterflies from the larval performance experiment were allowed to freely mate and lay eggs on rosetteA. petiolata. Butterflies raised onB. juncealaid 89.5 eggs per female (n=4 females, 3 males), those raised onB. oleracea laid 176.6 eggs per female (n=3 females, 3 males), those raised on rosetteA. petiolatalaid 119.5 eggs per female (n=2 females, 4 males), and the lone female raised on floweringA. petiolata laid 147 eggs (n=1 female, 1 male). A chi-square test for proportions revealed significant differences from the mean of 133 eggs per female (χ2=31.3284, df = 3, and P<0.01). Post hoc testing showed that females laid significantly fewer eggs when raised onB. junceathan any of the other groups, and females raised onB. oleracealaid significantly more eggs than eitherB. junceaor rosetteA. petiolataraised butterflies.


## 3.1. Direct Observations ofP. rapae in Forest Habitats

At MCO and ASP, we regularly observedP. rapaeflying in heavily wooded areas but did not observe any nectar gathering or oviposition behavior. We found an unidentified first instar caterpillar in 2012 at MCO onA. petiolata that could have been eitherP. rapaeorP. virginiensis. At WMP, we observedP. rapaeadults gathering nectar in the understory from several plant species, includingClaytonia, Phlox, andViolaspecies, as well as fromA. petiolataitself. We also observed 3 femaleP. rapaeovipositing onCardamine diphyllaand photographed an olderP. rapaecaterpillar feeding onA. petiolata (Figure 1). Additionally,P. rapae caterpillars have been observed feeding onA. petiolataoutside of the Wright State University greenhouse (2012–1015), onA. petiolata in the Wright State University woods, and in residential and park areas in Fairborn and Beavercreek, OH (SLD, personal observations).Figure 1
MatureP. rapae caterpillar consuming rosetteA. petiolata in Wooster, OH. Photo taken on May 11, 2012, by SLD.


## 3.2. Herbivory byP. rapae on A. petiolata in Edge Habitats

Although overall percent leaf loss was low, 78.8% of plants were damaged by caterpillars in 2013, indicating thatP. rapaecommonly usesA. petiolatain North America. In 2013, both the number of leaves (z = 3.475, P<0.01) and the location (P<0.01) influenced the probability of plants being attacked. BCT was significantly different from NAR (z=2.614, P<0.05) and FCP (z=3.631, P<0.01), but the latter two were not significantly different from each other (z=-2.217, P>0.05). Across years at BCT, only the number of leaves was a significant factor in the model (z=3.622, P<0.01), indicating no difference in plant damage between years. Evaluating the percent leaf loss score revealed similar results, with BCT being significantly different from both NAR (z=2.387, P<0.05) and FCP (z=3.697, P<0.01), but NAR and FCP were not significantly different from each other, and the number of leaves per plant was not correlated with the percent leaf loss score. The model evaluating percent leaf loss score as influenced by date and number of leaves for the BCT site alone was not significant. Figure 2 shows the mean percent leaf loss score for both sites and years.Figure 2
Herbivory (percent leaf loss) onA. petiolata varied between sites and habitats in 2013. Gray bar represents data from 2011; black bars represent data from 2013.


## 3.3. Pieris rapae Larval Performance

Although there was a trend towards lower survival ofP. rapaecaterpillars feeding on floweringA. petiolata, we found no significant differences in survival ofP. rapaecaterpillars on the four hosts that we tested (χ2=7.4, df = 3, and P=0.0596, Figure 3). Pupal mass did not vary between treatments (F=2.213, df = 3, and P=0.109); however, there were differences in time to pupation (F=7.897, df = 3, and P<0.01). Caterpillars reared onB. junceapupated significantly earlier than those raised on rosetteA. petiolata(P<0.05, Tukey’s HSD) and on commercialB. oleracea(P<0.01). Relative growth rates also differed between treatments (F=4.428, df = 3, and P<0.01) because caterpillars on leaves ofB. juncea grew significantly faster than those on floweringA. petiolata (P<0.01, Tukey’s HSD). To summarize,P. rapaecaterpillars reared onB. junceagrew faster and pupated earlier with no significant loss of pupal mass, whereas caterpillars reared on floweringA. petiolatatook longer and grew slower than those onB. juncea. Means and standard error of each treatment are found in Table 1.Table 1
Mean percent survival, days to pupation, pupal weight, and relative growth rate with standard error ofP. rapae (both sexes) between four host plants (n=16 per treatment). Different letters indicate significant differences (P<0.05).
Treatment
Survival (%)
Days To pupation (d)
Pupal mass (g)
Relative growth rate (g g−1 d−1)
A. petiolatarosette
56.25
15.33 ± 0.85
0.128 ± 0.003
0.277 ± 0.046ab
A. petiolataflowering
20.00
15.33 ± 1.45
0.112 ± 0.008
0.182 ± 0.031b
B. oleracea “cabbage”
68.75
16.63 ± 0.28
0.134 ± 0.004
0.294 ± 0.019ab
B. juncea “mustard”
56.25
12.78 ± 0.46
0.132 ± 0.006
0.380 ± 0.044aFigure 3
Kaplan-Meier survival estimates ofP. rapaecaterpillars fed commercial cabbage (solid black), rosetteA. petiolata(dash grey), floweringA. petiolata(solid grey), or commercial mustard greens (dash black).Eclosed butterflies from the larval performance experiment were allowed to freely mate and lay eggs on rosetteA. petiolata. Butterflies raised onB. juncealaid 89.5 eggs per female (n=4 females, 3 males), those raised onB. oleracea laid 176.6 eggs per female (n=3 females, 3 males), those raised on rosetteA. petiolatalaid 119.5 eggs per female (n=2 females, 4 males), and the lone female raised on floweringA. petiolata laid 147 eggs (n=1 female, 1 male). A chi-square test for proportions revealed significant differences from the mean of 133 eggs per female (χ2=31.3284, df = 3, and P<0.01). Post hoc testing showed that females laid significantly fewer eggs when raised onB. junceathan any of the other groups, and females raised onB. oleracealaid significantly more eggs than eitherB. junceaor rosetteA. petiolataraised butterflies.


## 4. Discussion

We looked for evidence of the nonnative butterfly,Pieris rapae, usingA. petiolatain both forest and edge habitats in North America, and also examined larval performance. Previous authors have observed occasional forest use byP. rapae[19–21], but we demonstrate thatP. rapaefrequents forested habitats, using both native and nonnative nectar and host plants. We also confirmed thatP. rapaesuccessfully usesA. petiolataas well as its more typical brassicaceous hosts in North America. In forests shared withP. virginiensis,P. rapaeuses the same nectar and oviposition resources asP. virginiensis, with one exception:P. rapaecan successfully useA. petiolataas a larval host, but the native congener cannot [22, 29, 30].One possible implication regarding the use of forested habitats byP. rapae is direct competition for oviposition sites (and, therefore, larval food resources) by nativePierisspecies. IfP. rapaeprefers to oviposit on the primary native host ofP. virginiensis,Cardamine diphylla, the caterpillars may occasionally compete for food. This competition could be limiting near pupation when native ephemeral plant hosts are in decline [31]. However, habitat sharing may benefitP. virginiensisifP. virginiensispractices egg avoidance like other congeners. IfP. rapaeprefers ovipositing onA. petiolata instead of on the nativeC. diphylla,P. virginiensismay not lay its eggs on already occupiedA. petiolata leaves.The presence ofP. rapaein forests may negatively affectadult native pierids only if nectar is a limiting resource. Nectar resources drive Lepidopteran habitat selection and fuel successful egg maturation and oviposition [32–34]. In some cases, Lepidoptera compete directly for nectar resources, attempting to dislodge other butterflies occupying desirable flowers [35]. The initial invasion ofP. rapaemay have caused a severe decline in the abundance of another native butterfly,P. oleracea, before the invasion ofA. petiolata [4], though more recent authors disagree [20]. Further work needs to be done to determine if nectar availability would be limiting for native pierids persisting in forest habitats, and whether competition for nectar withP. rapae is important.In North America,A. petiolatais an ideal host forP. rapae, providing nectar each spring, as well as plant material year round (rosettes persist through winter before flowering in the spring) for larval development. AlthoughP. rapae may reduce the fitness ofA. petiolata through folivore, any fitness reduction will not be substantial. Evans and Landis [36] found that the minor foliar damage recorded in field observations ofA. petiolata actually increased fecundity ofA. petiolata, much like grazing can positively affect grasses. Further work needs to examine howP. rapaeandA. petiolataaffect each other’s fitness and abundance.In addition to its use as a larval host, the nectar resources offered byA. petiolatamay draw moreP. rapaeto agricultural fields near forested areas and edges occupied byA. petiolata. Zhao et al. [37] found thatP. rapaewere more abundant in broccoli interplanted with nectar-producing plants than in broccoli monocultures. Future experiments should include an examination ofP. rapaepopulations in fields with and without nearby woodlands invaded byA. petiolata.There may be an increase in apparent competition for enemy-free space whenP. rapaeuse forest resources in habitats already occupied by nativePierisspecies. Benson et al. [6] found no evidence thatCotesia glomerataL. orC. rubeculawould attackP. virginiensissentinel caterpillars near meadows; however, lab work demonstrates no preferences by these wasps for differentPierisspp. caterpillars as potential hosts. Despite being not currently a problem,Cotesiamay be a problem for future generations ofP. virginiensis, P. oleracea, and other native pierid butterflies if they begin to followP. rapaeinto nearby forests.Finally,P. rapaemay interfere with volunteer-driven conservation efforts for the nativePierisspecies. There are many organizations that trackP. virginiensispopulations over time, but some volunteers estimate unusually high densities ofP. virginiensis (C. Lehn, unpublished data). Some of these observations may be ofP. rapaeutilizing forest habitat for its nectar and oviposition resources. Differentiating between these Pierids at a distance, by sight or behavior, is difficult [20, 31]. Volunteers may be overestimating population sizes by misidentifyingP. rapaeas nativePierisspp. and consequently missing instances where populations are in decline or extinct.In conclusion,P. rapaeis present in North American forest habitats with and without cooccurring native pierid species, and its use ofA. petiolata appears to facilitate its occupancy.Pieris rapaemay be simultaneously escaping pressure from competition and parasitism, as well as increasing herbivorous pressure on the exotic mustardA. petiolata. WhereP. rapaeoverlaps with native Pierids, there are opportunities for competition. However, more work needs to be done to investigate both the cause ofP. rapaehabitat expansion and the ecological implications of moving into forested habitat.



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*Source: 9671506-2016-02-21.xml*