persistence in massachusetts of the veined white butterfly due to use of the invasive form of cuckoo...

ORIGINAL PAPER

Persistence in Massachusetts of the veined white butterflydue to use of the invasive form of cuckoo flower

M. V. Herlihy • R. G. Van Driesche •

D. L. Wagner

Received: 2 August 2013 / Accepted: 7 April 2014

� Springer International Publishing Switzerland 2014

Abstract The native pierid butterfly Pieris oleracea

underwent a large range reduction in New England in

the twentieth century, likely due to the introduction the

invasive butterfly Pieris rapae (Lep.: Pieridae) to

North America in 1860, and later the oligophagous

parasitoid Cotesia glomerata (Hymenoptera: Bracon-

idae) in 1884. Thought extirpated from the state by the

1970s, one large dense population of the butterfly was

found in the mid 1980s in a flood plain meadow along

the Housatonic River in Lenox, Berkshire Co., Mas-

sachusetts. We examined how this native pierid was

able to maintain a relatively dense local population by

feeding on a novel, invasive host plant, Cardamine

pratensis (cuckoo flower), in a meadow habitat despite

known parasitoid presence. We approached this ques-

tion in three ways. First, we deployed trap host plants

(cuckoo flower and collards) stocked host larvae (first

and second instars of either P. rapae or P. oleracea) at

the Lenox site and other locations to determine current

rates of C. glomerata attack, for comparison with

historical information. Second, we used olfactometer

experiments to determine if C. glomerata females

could detect the cuckoo flower volatiles released

during P. oleracea larval feeding. Third, we used field-

cage experiments to determine if the plant architecture

found in the flood plain meadow inhibited the ability

of C. glomerata females to locate and parasitize hosts.

Specifically, we asked if overtopping vegetation

prevented or reduced parasitism of P. oleracea larvae

feeding on the covered basal rosettes of C. pratensis,

which is the physical form of host plant for three of the

four butterfly generations at the site.

Keywords Biological control � Non-target

impact � Enemy-free space � Parasitoid

displacement � Pieris oleracea � Pieris rapae �Pieris napi � Cotesia glomerata � Cotesia

rubecula � Cardamine pratensis

Introduction

In the mid-to-late nineteenth century, the native

butterfly Pieris oleracea Harris (formerly Pieris napi

oleracea) began a decline in Massachusetts (Scudder

1889) that culminated in its apparent disappearance

from most of the state, with populations persisting in

parts of Berkshire County in western Massachusetts.

In 1986, a large population was rediscovered in

western Massachusetts (Lenox, Berkshire Co.) by

Roger Pease, an amateur Lepidopterist. It was later

determined that the larvae of this population fed

primarily on an introduced subspecies of cuckoo

M. V. Herlihy (&) � R. G. Van Driesche

Department of Environmental Conservation, University

of Massachusetts, Amherst, MA 01003, USA

e-mail: [email protected]

D. L. Wagner

Department of Ecology and Evolutionary Biology,

University of Connecticut, Storrs, CT 06269, USA

123

Biol Invasions

DOI 10.1007/s10530-014-0698-x

flower (Cardamine pratensis [L.] var. pratensis) in

wet meadows and along edges of rivers (Mike Nelson

unpubl. data). The introduced subspecies of cuckoo

flower is found throughout Massachusetts, while the

native form C. pratensis [L.] var. palustris is state

listed as threatened (USDA and CAPRP 2012). The

introduced plant is a biennial, flowers in spring, and

overwinters as a rosette. Foliage suitable for P.

oleracea larvae is present throughout the growing

season, allowing for four annual generations at Lenox.

Throughout the summer and fall, most of the available

cuckoo flower is overtopped by growth of other

floodplain plants. It is worth noting that although C.

pratensis is a novel host plant for P. oleracea in the

United States, it is a common host for all three sub-

species of P. napi in the United Kingdom (Asher et al.

2001).

Reasons for the original statewide decline of P.

oleracea in Massachusetts are uncertain. Evidence

supports the hypothesis that an introduced biological

control agent, Cotesia glomerata (L.) (Hymenoptera:

Braconidae), likely played an important role (Benson

et al. 2003), although other potentially contributing

factors have been suggested, including the possible

regional loss of a key forest host plant, Cardamine

diphylla [Michx.] A.W. Wood (two-leaf toothwort)

(Keeler et al. 2006). However, the continued abun-

dance of this plant throughout most western Massa-

chusetts did not prevent loss of P. oleracea in that

region (Benson et al. 2003). Historically, the decline

of P. oleracea began after the invasion of the pest

butterfly Pieris rapae (L.) (Lepidoptera: Pieridae) into

North America ca 1860 in Quebec and ca. 1869 in

Massachusetts (Scudder 1889). In response to this

invasion, in 1884 the European parasitoid C. glomer-

ata was introduced into the United States for biolog-

ical control of P. rapae (Clausen 1978), although the

same parasitoid may have co-invaded with P. rapae as

suggested by Scudder (1889). This parasitoid is not

specific to P. rapae, but rather readily attacks a range

of white butterflies (Pierinae) in several genera (Pieris,

Pontia, Tatachila, and Aporia) in different parts of the

world (Herrera Gonzalez 1982). C. glomerata estab-

lished and spread widely across North America and

became the dominant parasitoid of P. rapae in North

America (Van Driesche and Bellows 1988). In labo-

ratory tests, P. oleracea was a physiologically suitable

and preferred host (relative to P. rapae) for C.

glomerata (Van Driesche et al. 2003). Because of

the rarity of P. oleracea in Massachusetts, field attacks

of this host by C. glomerata were not directly

observed. However, attack on artificially deployed

trap host larvae of P. oleracea was observed in both

northern Vermont, where P. oleracea populations

persisted, and in western Massachusetts, where they

largely disappeared (Van Driesche et al. 2004). In

2001 in western Massachusetts, Van Driesche et al.

(2004) found that 100 % of 59 larvae recovered from

450 first or second instars of P. oleracea placed in a

meadow on hedge mustard, Sisymbrium officinale [L.]

Scop., were parasitized by C. glomerata during a

7-day exposure. Of particular relevance is that the

Shelburne, MA (N 42�320 by W 72�380) site used in

Van Driesche et al. (2004) is in the same region of

forested hills only 52 km NE from Lenox, Massachu-

setts, the site of our current study.

Parasitoid pressure from C. glomerata, however,

has not remained stable. Historically C. glomerata was

common and widespread in Massachusetts until

sometime between 2001 and 2007 (Van Driesche

et al. 2004; Van Driesche 2008) during which interval

another introduced parasitoid (Cotesia rubecula [Mar-

shall]) (Hymenoptera: Braconidae) outcompeted it for

dominance of P. rapae hosts (Van Driesche 2008), the

species that previously was the most common host of

C. glomerata in western Massachusetts (Van Driesche

1988). While several introductions of C. rubecula in

North America occurred between 1960 and 1992

(Puttler et al. 1970; Williamson 1971, 1972; Parker

and Pinnell 1972; McDonald and Kok 1992; Wold-

Burkness et al. 2005; Lee and Heimpel 2005), this

species’ presence in New England evidently resulted

from a 1988 release of wasps collected near Beijing,

China (Van Driesche and Nunn 2002). After release,

C. rubecula established and spread rapidly, and by

2007 it accounted for 99 % of the spring (Van

Driesche 2008) and (in 2009) 90 % of the fall

parasitism (Herlihy and Van Driesche 2013) of P.

rapae larvae collected from organic vegetable farms in

western Massachusetts. Because this species has a

narrower host range than C. glomerata (Brodeur et al.

1998; Van Driesche et al. 2003), it has rarely been

observed attacking species other than P. rapae in the

field (van Driesche et al. 2003, 2004). With this

background, we can construct a likely timeline for

both the presence of the P. oleracea population at

Lenox, MA, site (noted to occur there since at least

1986) and the region-wide displacement of C.

M. V. Herlihy et al.

123

glomerata by C. rubecula in western Massachusetts

(no earlier than 2001), leaving 1986–2001 as a period

when population increase of P. oleracea in Lenox on

cuckoo flower had to have occurred in the presence of

C. glomerata as a relatively abundant parasitoid,

which is a key point for understanding the history of

parasitoid-butterfly interactions at the study site.

The objectives of our current study were to describe

the current levels of parasitoid pressure from C.

glomerata at the Lenox site using trap–host experi-

ments and to determine what aspects of use of cuckoo

flower may have allowed for parasitoid escape by P.

oleracea historically (at least 1986–2001, likely

longer) when both C. glomerata and P. oleracea were

known to both be present at the site.

To estimate the current level of C. glomerata

pressure at the Lenox site, we conducted trap–host

exposures for 3 years (2008–10) in the wet meadow

and riverside areas where this P. oleracea population

still exists. Broadly, we found that at our study

location, as in the rest of western Massachusetts, C.

glomerata is now uncommon and there is currently

little-to-no parasitism of P. oleracea.

To meet our second objective and understand how

P. oleracea achieved population growth at the site pre-

2001, when C. glomerata was likely still common, we

used a combination of olfactometer and field-cage

experiments to determine what aspect of this Lenox

meadow habitat provided protection from parasitism.

We investigated three mechanisms that might have

created at least partially enemy-free space (Jeffries

and Lawton 1984): (1) that C. glomerata was unable to

detect or respond to volatiles from cuckoo flower

foliage infested by P. oleracea, plausible since

European cuckoo flower has a different glucosinolate

profile than either American cuckoo flower or brassica

crops like cabbage, collards, etc. (Agerbirk et al. 2010;

Agerbirk and Chew unpubl. data); (2) that the more

complex leaf architecture of the pinnate leaves of C.

pratensis may have lowered wasp foraging efficiency

as compared to the simpler leaves of crops such as

collards and cabbage (but not kale); or (3) that the

position of cuckoo flower rosettes, growing beneath

taller vegetation in summer and fall, may have reduced

the ability of C. glomerata to find infested cuckoo

flower under overtopping meadow plants (commonly

ca 0.6 m tall) as found by Sato and Ohsaki (1987) in

Japan, working with C. glomerata and the closely

related Pieris napi japonica Shirozu.

More broadly, there are at least two other hypotheses

about factors that might have affected population growth

of P. oleracea at the Lenox site that were not considered in

this study. First among these is the invasion of the region

by garlic mustard (Alliaria petiolata [M. Bieb.]) (Keeler

et al. 2006). While this species acts as a toxic egg trap for P.

oleracea (Keeler and Chew 2008), it is an abundant host to

which the butterfly may be adapting (Courant et al. 1994;

Keeler and Chew 2008; Chew et al. 2012). However, now

and in the critical past period (1986–2001) under study, the

overwhelming majority of garlic mustard plants were toxic

to most P. oleracea larvae. Garlic mustard is also found in

many areas in Massachusetts where P. oleracea popula-

tions did not persist. Therefore we discard this factor as

unimportant to the issue under study.

Second, the size of the resource patch formed by the

cuckoo flower population at the Lenox site is both large

and available for a very long period each year. This

allows P. oleracea butterflies to have four generations

per year at the site, which raises the butterfly’s effective

annual population rate of increase and helps it support

mortality from parasitism without a decrease in density.

We view this factor as likely important, but not one

readily tested experimentally. Consequently here we

focused on C. glomerata response to cuckoo flower

volatiles, the potential effect of leaf architecture, and the

effect of overtopping vegetation in reducing parasitism

and allowing for butterfly population growth.

Although much ecological research on exotic plants

has been focused on negative impacts on native species

through competition, exotic plants have other ecological

effects, including providing enemy free space to native

lepidopterans. In California, 82 butterflies (40 % of the

state’s species) have been shown to use exotic host

plants as food resources and to gain enemy free space

(Graves and Shapiro 2003; Shapiro 2002). We propose

that, despite parasitoid pressure due to C. glomerata and

range reduction of the host plant C. diphylla due to

invasion of garlic mustard, P. oleracea was able to

persist in Massachusetts by exploiting the exotic form of

C. pratensis as a host plant.

Materials and methods

Trap host experiments

Trap host studies were carried out in various sections

of the Lenox, Massachusetts site (Fig. 1) in the

Persistence in Massachusetts of the veined white butterfly

123

floodplain of the Housatonic River in 2008, 2009 and

2010 to detect parasitism by Cotesia using first and

second instar larvae of Pieris spp. butterflies. Similar

host exposures were also made in some additional

locations in 2009 and 2010 as positive controls of the

trap host technique. The floodplain sites were all on

property of the MA Department of Fish and Game and

were mowed yearly. Trap host larvae deployed in

early 2008 were placed in the field on naturally

occurring cuckoo flower plants. Starting with the third

experiment in 2008 and thereafter, trap host larvae

were placed, in the laboratory, on potted plants, which

were transported to field sites and placed out for

exposure to parasitoids. Exposures based on potted

plants were done either on previously dug field cuckoo

flower rosettes (a 100 cm2 area of plants), green-

house–grown collards (var. Georgia, 25 cm tall, 5–6

leaves), or field-dug broccoli transplants (25 cm tall,

5–6 leaves). After deployment in the field for the

desired number of days (3–4 in most cases, but up to

12 days in some experiments, see Table 1 for details),

remaining larvae were recovered and either reared on

host plants to the pupal stage (2008, some experiments

of 2009) or dissected (some experiments of 2009, all of

2010), either process being suitable for detection of

parasitism by Cotesia and both being procedures our

laboratory has carried out many other times. Details of

all nine trap host experiments (three in 2008, four in

2009, and two in 2010) are given in Table 1.

2008 experiments

In 2008, only one location (Lenox, MA, Fig. 1) was

used to experimentally expose larvae of P. oleracea to

measure field parasitism rates. The Lenox, Massachu-

setts site is located near the intersection of New Lenox

Rd. and the Housatonic River in Berkshire Co.

(42�230N; 73�140W). Two floodplain meadows at this

location were used to expose trap hosts in 2008 (A and

B, Fig. 1) in a series of two experiments (Table 1).

Cuckoo flower was abundant and in bloom in both

sites during the first experiments in 2008 in May. The

larvae used as trap hosts were all P. oleracea in 2008,

reared from eggs obtained from field-caught P.

oleracea adults from spring and summer flights at

the Lenox, Massachusetts site (under a MA Depart-

ment of Fish and Game, Natural Heritage Program

permit). In 2008, three separate trap–host exposures of

P. oleraceae were made (Exps. 1, 2, and 3), the first

two of which were done by placing larvae on field-

Fig. 1 Aerial photograph of the Lenox study site (42�230N; 73�140W), showing locations A, B, and C where trap host larvae were

exposed to detect Cotesia parasitism, as well as area D, an organic vegetable farm where trap host exposures were also made


123

grown cuckoo flower plants and the third with larvae

on potted collards (an artificial but suitable host plant,

highly attractive to Cotesia parasitoids). Details of

each trap host exposure are summarized in Table 1.

2009 experiments

In 2009, four experiments were run, spanning a longer

period (May 24–October 2), with work at three

Table 1 History of trap host exposures (Pieris spp.) to detect Cotesia parasitism at the Lenox, Massachusetts, site where P. oleracea

has increased, as well as several sites used as positive controls (in bold), 2008–2010

Year Exp # Dates Place Plant Host #L/Pl Total# trap

hosts/#pla% Par Rb

(par/n)

% Par Gc

(par/n)

2008 1 May 17–29 Lenox area B CK Fd (in situ) Pieris oleracea 1L 97/91 n/a 0 (0/17)

2 May 23–29 Lenox area B CK F (in situ) Pieris oleracea 5–8L 35/6 n/a 0 (0/19)

3 July 18–24 Lenox areas A&B collards (potted) Pieris oleracea 5L 40e/8 n/a n/a

2009 1a May 24–June 2 Lenox areas A&B collards (potted,) P. rapae 5L 84/17 12.5 (1/12) 0 (0/12)

1b May 31–June 3 Lenox area B CK F (potted) Pieris oleracea 5L 120/24 n/a 0 (0/83f)

1c May 31–June 3 N’hamptonfComm. Garden CK F (potted) Pieris oleracea 5L 80/16 n/a 0 (0/34)

2a July 14–17 Lenox areas A&B CK F (potted) Pieris oleracea 4L 41/10 n/a 0 (0/4)i

2b July 14–17 Lenox areas A&B CK F (potted) Pieris rapae 4L 48/12 0 (0/9) 0 (0/9)i

3 Aug 13–16 Lenox area C CK F (potted) Pieris oleracea 3–9L 147/30 n/a 0 (0/75)

4a Sept 24–Oct 2g Lenox area C Broccoli (potted) Pieris oleracea 5L/11 55 n/a 0 (0/12)

4b Sept 24–Oct 2g Lenox area C Broccoli (potted) Pieris rapae 5L/11 55 0 (0/12) 0 (0/12)

4c Sept 24–Oct 2g Hadley farm area Broccoli (potted) Pieris oleracea 5L/10 50 n/a 22 (5/23)

4d Sept 24–Oct 2g Hadley farm area Broccoli (potted) Pieris rapae 5L/10 50j 53 (10/19) 11 (2/19)

2010 1 June 23–28 URI North Kingston Collards (potted) Pieris rapae 8L 32/8 0 (0/13) 0 (0/13)

Collards (potted) Pieris oleracea 8L 32/8 n/a 0 (0/7)

CK-F (potted) Pieris rapae 8L 32/8 0 (0/9) 0 (0/9)

CK-F (potted) Pieris oleracea 8L 32/8 n/a 0 (15)

June 23–28 Lenox area C (meadow) Collards (potted) Pieris rapae 8L 32/4 0 (0/20) 0 (0/20)



CK-F (potted) Pieris oleracea 8L 32/4 n/a 0 (0/22)

June 23–28 Lenox area D (organic farm) Collards (potted) Pieris rapae 8L 32/4 0 (0/20) 0 (0/20)




2 July 24–July 28 Lenox area D (organic farm) Collards (potted) Pieris rapae 6L 60/10 46 (13/28) 0 (0/28



Lenox area C (meadow) Collards (potted) Pieris rapae 6L 60/10 0 (0/38) 0 (0/38)



a The number of trap host larvae exposed and the number of plants on which they were dispersed

b R = Cotesia rubecula. This is a specific parasitoid of Pieris rapae and so its recovery is expected only when P. rapae this the trap host

c G = Cotesia glomerata. This parasitoid could be recovered from either P. rapae or P. oleracea

d CK F = cuckoo flower

e All lost to flood

f One of the 83 recovered larvae was parasitized by an unidentified nematode

g Hard frost occurred during this experiment, affecting quality of trap host plants

h Northampton Community Garden and Hadley farm area were two distantly located sites used as positive controls to demonstrate ability of method to detect C.

glomerata parasitism in areas where C. glomerata was present

i Most larvae lost due to heavy damage of plants by snails

j Two additional larvae of the 19 recovered were parasitized by unidentified tachinids


123

locations: (1) New Lenox (areas A and B as in 2008

plus a new area, C); (2) the Northampton Community

Gardens, Northampton, Massachusetts (Prince Street,

42�180N; 72�390W); and (3) an organic vegetable farm

in Hadley, Massachusetts (42�200N; 72�360W). The

latter two locations were intended as positive controls

to demonstrate the efficacy of the trap host method.

Area C at the New Lenox site was a large wet meadow

(ca 2 ha) (Fig. 1) with a water table close to surface

such that small variations in the surface produced wet

or dry areas. Wet areas contained abundant cuckoo

flower, which by summer were overtopped with other

wetland plants. Butterflies were detected by us in this

meadow for the first time in 2009, but clearly were

present there earlier.

In 2009, in addition to P. oleracea, we also exposed

similar-sized larvae of P. rapae, a primary host of the

Cotesia parasitoids under study. For experiments, both

species of butterflies were collected in the wild (P.

oleracea from Lenox, MA and P. rapae from Hadley,

MA) and held over host plants outdoors for oviposition

(collards for P. rapae and cuckoo flower for P.

oleraceae). Leaf fragments with eggs were excised

and placed on moist filter paper in petri dishes to track

hatch. Hatching larvae were allowed several hours to

feed and then were held under chill (ca. 10 �C) until

deployed in field experiments. Details of the four

experiments are in Table 1.

2010 experiments

In 2010, the main experimental area was again area C

at the New Lenox site (the large wet meadow).

Experiments intended as positive controls were run in

two additional locations: (1) a cabbage plot on farm

land of the University of Rhode Island in N. Kingston,

Rhode Island (41�290N; 71�320W), selected because it

was the last site in the region known to us from

previous surveys where C. glomerata still dominated

and (2) River Valley Farm (area D in Fig. 1), an

organic vegetable farm close to our main research site

area C in New Lenox.

Pieris oleracea and P. rapae butterflies larvae used

in experiments in 2010 were reared from butterflies

sourced as in previous years and reared in a similar

manner. Collard plants used in experiments were

grown outdoors from Hart’s brand seed in 10 cm

square pots in garden soil, while cuckoo flower plants

were again dug from the New Lenox site. Plants

bearing trap host larvae were elevated ca 45 cm above

the soil on wooden stands (a pole topped with a plastic

container (20 cm w 9 20 cm l 9 8 cm h). The pole

was treated with Tanglefoot as a barrier to snails, a

pest discovered to be abundant at the site, and the

container in which the pot sat was filled with water to

sustain the plant.

Two experiments were run in 2010, each with four

treatments (two plants [collards and cuckoo flower]

crossed with larvae of two Pieris species (P. rapae and

P. oleracea). Exp. #1 (June 23–June 28) was run at

three locations (North Kingston RI and areas C and D

at Lenox) during the second flight of the P. oleracea

meadow population at the Lenox site (Fig. 2). At the

North Kingston Site, each treatment was replicated

eight times, with eight trap–host larvae per plant,

placing the 32 plants in stands as described above, in

an existing field of cabbage (free of insecticide

treatment). At the Lenox, MA site, 16 plants (four of

each treatment; each plant with eight larvae) were

placed in areas C (meadow) and D (River Valley farm)

(Fig. 1). In area C, plants were arranged in two

transects in the meadow and vegetation was cleared

from 60 cm circles around pots to increase visibility of

exposed larvae. At area D (the farm), plants were

arranged in a single row between a row of cultivated

kale plants and the forest edge, spacing pots about

3–4 m apart. Exp. #2 (July 24–28) was again run at

Lenox (in Areas C and D) during the third flight of the

meadow P. oleracea population (Fig. 2). The same

protocol was used as in Exp. 1 of 2010. Details of

protocol for both experiments are summarized in

Table 1.

Sources of plants and insects for olfactometer

and field-cage experiments

Cuckoo flower (C. pratensis) plants used in our

olfactometer experiments were dug at the study site

in Lenox, MA (42�2303700N; 73�1403300W) and placed

in 10 cm-dia pots prefilled with Pro-Mix BX mycorise

growing mix. Plants were kept in a greenhouse and

watered every 2 days until used for experimentation.

Collards (Brassica oleracea L. var. Blue max hybrid)

for experiments were purchased as 4-week-old plants

from Harvest Farm in Whately, MA. They were

transplanted into 10 cm-dia pots prefilled with Pro-

Mix BX mycorise growing mix. The plants were kept

in a greenhouse and watered every 2 days until used.


123

Cotesia glomerata wasps used in experiments were

obtained from our laboratory colony, originally started

with cocoons collected in Massachusetts and supple-

mented with material from Virginia and Illinois

(because it became impossible to find adequate

numbers in Massachusetts). C. glomerata cocoon

masses were held at about 3 �C until needed for

experiments. Cocoons were then put into cages with

honey and sugar solution (‘‘Instant Hummingbird

Nectar,’’ Perky Pet Woodstream Corp. Lititz, PA), at

20 �C, natural light, and 50–65 % R. H. and left for

adult emergence. Once adults had emerged, wasps

were continually supplied with honey and a sugar

solution, but were not exposed to host larvae or host

plants. Naıve female wasps, exposed to males for

several days for mating, were used when they were

3–5 days old. After use in experiments, females were

returned to the rearing colony, where they were placed

into a separate rearing cage and exposed to first and

second instars of P. rapae or P. oleracea on fresh

collard leaves for oviposition. Parasitized P. rapae and

P. napi larvae were reared on collard leaves to produce

C. glomerata cocoon masses.

Pieris oleracea larvae used in our experiments were

obtained from our laboratory colony, initiated in 2010

with adult female butterflies collected at the Lenox,

Massachusetts site and supplemented by additional

butterflies from the Lenox, Massachusetts population

over a 3 year period (2010–2012) (under permit from

the State Natural Heritage Program). Butterflies were

reared through several generations in the laboratory in

the course of the summer of 2012 for olfactometer and

overtopping vegetation cage experiments. Pupae were

stored at about 3 �C until needed and were then moved

to Bug dorm cages (61 cm 9 61 cm 9 61 cm) (Bio-

Quip Products, Rancho Dominguez, CA, USA), where

emerging adults were provided with sugar solution

(‘‘Instant Hummingbird Nectar,’’ Perky Pet Wood-

stream Corp. Lititz, PA) and potted cuckoo flower

plants dug from Lenox, Massachusetts. Eggs laid on

plants were allowed to hatch and larvae to develop on

the cuckoo flower plants until larvae were used.

Excess larvae were returned to the P. oleracea colony

where they were transferred from cuckoo flower to

collard leaves, both being highly suitable host plants.

Olfactometer construction

A standard four-arm olfactometer was used for

experiments. A central chamber (55 mm in diame-

ter 9 100 mm in height) was connected to four glass

arms (50 mm in diameter 9 100 mm long), which

were each connected to bait chambers (100 mm in

diameter 9 200 mm long) containing an odor source.

Wire screens were placed between the openings of

each of the odor source chambers and the arm leading

to the central chamber, allowing air flow, but

preventing wasps from entering the odor source

chamber. The odor source chambers were connected

to a pressure control valve (allowing for air flow rate

through each arm to be equalized), and air was drawn

through a deionized water bubbler to control the

humidity and an activated charcoal filter to remove

unwanted volatiles. The central chamber was linked to

Fig. 2 Observed counts of

P. oleracea butterflies in

flight at the Lenox,

Massachusetts, meadow site

during the 2010 season


123

a vacuum pump via 10 cm-dia plastic tubing. The

pump was set at 400 ml min-1, to pull air evenly

through all four arms, without affecting the wasps’

ability to fly through the arms. Light brown foam

boards (43 cm h) were placed on all four sides of the

olfactometer, which was placed on a laboratory table,

to prevent directional bias in lighting. Fluorescent

lights were positioned above the center of the olfac-

tometer to balance the light environment.

Experimental design for olfactometer tests

As a preliminary control to ensure that the physical set

up of the olfactometer was unbiased, tests were run

with four empty odor-source chambers in which a

single, 3- to 5-day-old, mated female wasp was

released into the central chamber, and whose position

was recorded after 30 min. Also, as a positive control

to ensure the wasps being tested were physiologically

prepared to respond to odors, in an additional exper-

iment, P. oleracea-infested collards (a host plant–

species combination known to be attractive to C.

glomerata) were tested against empty control arms,

with the two controls being placed 180� opposite each

other.

For our experiments, the test protocol was similar to

that of Karimzadeh et al. (2012). During each exper-

imental run, one naıve, 3–5-day-old, mated female C.

glomerata wasp was released into the central chamber.

Two arms (180� apart) contained the same bait (plant

or plant–host complex) and the other two were

unbaited controls, giving the wasp a choice between

either two plants (or two plant host complexes) and

two controls. We tested (1) uninfested collards vs

uninfested cuckoo flower and (2) P. oleracea-infested

collards versus P. oleracea-infested cuckoo flower.

For tests with uninfested plants, collard leaves were

cut at the end of the petiole nearest the leaf and

weighed; cuckoo flower leaves were then cut along the

petiole to match the weight of the collard leaves. This

was done because cuckoo flower leaves are pinnate

and thus easier than collards to downsize to a desired

weight without cutting the actual leaf blade (which

would increase the release of volatiles attractive to

parasitoids). Uninfested leaves were placed in test

chambers 10 min before each experiment. For exper-

imental runs with infested leaves, 10 second instar P.

oleracea were placed on each collard leaf, or group of

cuckoo flower leaves. All leaves were weighed and

foliar weight standardized between treatments. Foli-

age with larvae was placed in the odor source

chambers and larvae were allowed to feed for 1 h in

each experimental run to allow time for plants to

respond to feeding by releasing both constituent and

induced compounds. Tests ran until wasps made a

decision and entered an arm. If wasps were unrespon-

sive, a run was terminated after 30 min. Wasps

entering and remaining in an olfactometer arm beyond

a line 4 cm into the arm were counted as having made

a decision and those remaining in the central chamber

were considered unresponsive. Wasps that reversed

direction within an arm were counted as unresponsive.

The position of odor sources was rotated 90� after each

run (=the trial of one wasp) by disconnecting the odor

source chambers and reconnecting them to a new arm.

After each group of replicates run on a given day (ca

15–20 per day), the odor source chambers were

removed and washed in an odorless detergent and

rinsed with water and allowed to air-dry overnight. In

total, there was a minimum of 30 replicates per

treatment (each being the trial of one wasp).

Overtopping vegetation field cage experiment

To determine if overtopping by other vegetation

affected the rate of host detection by C. glomerata,

field experiments were run on the University of

Massachusetts Amherst campus in a meadow using

open-bottom, cube cages (0.6 m3) with black mesh

fabric (EQUINOX No-See-Um Netting Eastern

Mountain Sports) sides. In each run (one replicate)

of the experiment, we placed four such cages at the

field site. In total, there were eight replicates, run from

June to August 2012, each replicate on a different day.

Each cage was a single replicate of one of four

treatments being tested: (1) collards and clipped native

vegetation, (2) collards and unclipped vegetation, (3)

cuckoo flower and clipped native vegetation, and (4)

cuckoo flower and unclipped vegetation. Test plants of

each species were 10 cm tall. Clipped vegetation was

cut with scissors within 2–5 cm of the soil. Unclipped

vegetation was ca 35 cm tall. There were two test

plants per cage and five first instars of P. oleracea

were placed on each plant (=10 larvae per cage). Then

two 3 to 5-day-old C. glomerata females, which had

been given the opportunity to mate but had no previous

exposure to C. pratensis or B. oleracea volatiles, were

released into each cage. The larvae were exposed to


123

parasitoids for 24 h. The test was then terminated and

the larvae removed, refrigerated to arrest develop-

ment, and then dissected within 48 h to detect

parasitoid oviposition.

Statistical analyses

All statistical analyses were conducted using the

program R (version 2.13.1). Binomial exact tests were

used to compare female wasp choices in the olfac-

tometer. Cage experiment percent parasitism data

were arcsine transformed in order to meet the

assumption of normality and then analyzed with

ANOVA, followed by a Tukey HSD test.

Results

Trap host recoveries and parasitism, 2008–2010

Over the 3 years of field deployment of P. oleracea

larvae as trap hosts, 903 larvae were deployed (172,

363, and 368 in 2008, 2009, and 2010, respectively)

and 417 (46 %) were recovered after field exposure

(Table 1). Rates of recovery of trap host larvae

improved (21, 48, and 56 % in 2008, 2009, and

2010) as problems from flooding and herbivory by

snails were resolved by use of elevated stands

protected with Tanglefoot). Of the 417 P. oleracea

larvae recovered after field exposure, only 3 (0.7 %)

were parasitized by C. glomerata and none by C.

rubecula. The only detection at the Lenox site of C.

glomerata parasitism occurred at area D, an organic

vegetable farm (Fig 1) close to area C, the meadow

where the largest P. oleracea numbers occurred. P.

rapae butterflies occurred at all of the Lenox research

areas (A, B, C, D) but were most common at the farm

(area D).

Trap host exposures of P. rapae larvae at the Lenox

site were also made because we knew this was a highly

attractive host for both C. glomerata and C. rubecula

(and thus a good probe for both parasitoids). In total

435 P. rapae larvae were placed on trap host plants

(2008–2010) and 163 were recovered. Of these, none

were parasitized by C. glomerata, while 14 (9 %) were

attacked by C. rubecula. Again, all parasitism detected

for larvae of this butterfly was in area D, the organic

vegetable farm described above.

While our trap host method has been used previ-

ously on an extensive basis to detect Cotesia attack on

Pieris species (Benson et al. 2003; Van Driesche et al.

2004) as part of this study, we set out trap hosts in three

areas where we believed C. glomerata populations

might still remain despite its largely having been

suppressed in the region by competition from C.

rubecula (Van Driesche 2008; Herlihy and Van

Driesche 2013). Of these sites (Northampton, MA;

Hadley, MA; and N. Kingston, Rhode Island), Cotesia

parasitism was detected only at the Hadley site (a farm

field planted to organic cole crops). At that site, we

observed 22 % (5/23) parasitism by C. glomerata of P.

oleracea larvae. At the same site and dates, P. rapae

trap host larvae suffered 53 % (10/19) parasitism by C.

rubecula and 11 % by C. glomerata. As a positive

control, these data showed that the trap host system

functioned and was able to detect Cotesia parasitism.

It also demonstrated that P. oleracea is an attractive

host, at least on collards, for C. glomerata.

Response of C. glomerata to host and plant

volatiles

As a preliminary assessment to detect potential

directional bias in the olfactometer, wasp response

(n = 32) to four unbaited arms was assessed and no

significant difference among arms was detected

(X2 = 0.25, df = 3, P = 0.969) (Fig. 3). As a posi-

tive control to demonstrate that the test parasitoids

were capable of responding as expected to a known

high quality host/plant combination, female wasps

were given the choice of collard foliage infested with

P. oleracea larvae versus empty (unbaited) arms, and

in this test 23 of 25 parasitoids chose infested collards

over empty arms (P = 0.56 in two-tailed binomial

exact test with significance set at 0.05), a highly

significant difference.

In the first experiment, C. glomerata females were

offered uninfested collards vs uninfested cuckoo

flower plants and both were found to be equally

attractive; of 105 responsive females, 59 chose

infested collards and 46 chose infested cuckoo flower,

with no significant difference in a two-tailed binomial

exact test at the 0.05 level (P = 0. 24) (Fig. 3). In the

final test, C. glomerata females were offered a choice

of P. oleracea-infested cuckoo flower plants versus P.

oleracea-infested collards and of 58 responsive


123

females, 30 chose infested collards and 28 chose

infested cuckoo flower, a difference that was not

statistically significant in a two-tailed binomial exact

test at the 0.05 level (P = 1.0).

Overtopping vegetation field cage experiment

results

Overtopping vegetation had a significant effect on

rates of parasitism of P. oleracera by C. glomerata for

both cuckoo flower and collards (F = 12.8, df = 3,

P \ 0.001) (Table 2). P. oleracea larvae on both

collards and cuckoo flower with overtopping vegeta-

tion experienced significantly lower parasitism by C.

glomerata (18 of the 81 recovered larvae from these

two treatments were parasitized) than P. oleracea

larvae on collards and cuckoo flower plants without

overtopping vegetation (62 of the 85 recovered larvae

from these two treatments were parasitized). There

was no significant difference in parasitism by C.

glomerata between plants within a given vegetation–

height treatment (using Tukey HSD test) (Table 2).

Discussion

Exposure of both P. oleracea and P. rapae larvae as

trap hosts for three consecutive years (2008–2010)

showed that while both C. rubecula and C. glomerata

were present at Lenox, MA, there was no appreciable

parasitism of P. oleracea by C. glomerata (\1 %) and

none by C. rubecula. P. rapae larvae were attacked at

the Lenox site by C. rubecula at a 9 % (18/163) rate,

while no parasitism of this butterfly species by C.

glomerata was detected. These data show that cur-

rently there is virtually no parasitism pressure by

Cotesia affecting P. oleracea at the study site, in sharp

contrast to a study in 2001 at a nearby (52-km distant)

similar site in western MA, in which 100 % of P.

oleracea recovered larvae (59/59) deployed as trap

hosts in a meadow habitat on hedge mustard were

attacked by C. glomerata (Van Driesche et al. 2004).

These findings are consistent with evidence in other

surveys (Van Driesche 2008; Herlihy et al. 2012) that

C. glomerata populations have been strongly sup-

pressed in P. rapae by competition with C. rubecula

(introduced to Massachusetts from China in 1988 [Van

Driesche and Nunn 2002]). Low levels of C. glomer-

ata parasitism at the site currently, however, are

consistent with the view, as supported by Van

Driesche et al. (2004), that C. glomerata abundance

in Berkshire Co. (where Lenox, MA is located) was

much higher in the 1980s and 1990s before the region-

wide suppression of this species by C. rubecula. (Note,

however, that while trap hosts give an idea of the

relative rates of parasitism across treatments, they do

not directly measure natural rates of parasitism

because placement of trap hosts is unlikely to mimic

natural host spatial dispersion and density.) Given,

however, that the P. oleraceae population at the Lenox

site was almost certainly subjected to high levels of

Fig. 3 Choices made by female C. glomerata wasps in

olfactometer tests comparing (1) empty arms vs uninfested

collards, (2) P. oleracea-infested cuckoo flower or P. oleracea-

infested collards, (3) uninfested cuckoo flower or uninfested

collards

Table 2 Parasitism ± 95 % CI (n) of P. oleracea larvae by C.

glomerata in field-cage experiments (Amherst, Massachusetts,

June–August 2012) comparing the effects of the presence or

absence of overtopping vegetation in a 24-h exposure. Para-

sitism rate was detected via larval dissection within 48 h after

exposure

Plant type Percent parasitizeda

Collards

Clipped 77 ± 11 (52) a

Overtopping vegetation 23 ± 12 (44) b

Cuckoo flower

Clipped 68 ± 16 (34) a

Overtopping vegetation 22 ± 13 (37) b

a Statistical differences are denoted by different lowercase

letters


123

parasitism for several decades or more in the past, it is

of interest to know how the butterfly population could

have evaded parasitism or compensated for it when

feeding on the invasive cuckoo flower stands at the

study site.

It is known that C. glomerata can detect hosts from

long distances via olfactory signals from the plant–

host complex, using volatiles released by the plant

(Steinberg et al. 1993). C. glomerata can also detect

densities of hosts on a host plant and the age of the

plant, allowing it to find and exploit plants with the

highest number of host larvae on younger host plants

(Mattiacci and Dicke 1995; Geervliet et al. 1998). The

goal of one of our experiments was to determine

whether these olfactory signals from the plant–host

complex were being exploited by C. glomerata on C.

pratensis plants. Some plants seem to be exploited by

herbivorous insects because they offer some degree of

enemy-free space because their parasitoids do not

detect or recognize volatiles from such plants when

they are infested. For example, the Japanese subspe-

cies Pieris napi japonica is thought to use a nutrition-

ally inferior host plant (Arabis sp.) in order to avoid

parasitism by C. glomerata (Ohsaki and Sato 1990).

However, in our case, we found that C. glomerata did

not distinguish between volatiles from C. pratensis

and B. oleracea, either from uninfested plants or ones

infested by P. oleracea (Table 2), suggesting that in

the field, C. glomerata should be able detect volatiles

produced when P. oleracea larvae feed on C.

pratensis.

Other factors able to modify parasitism risk in the

field, apart from plant chemistry, are features affecting

plant apparency and foliar complexity (Sato and

Ohsaki 1987; Meiners and Obermaier 2004; Oberma-

ier et al. 2008). In our field cage experiment, we found

that in cages without overtopping vegetation, C.

glomerata wasps were able to easily locate, and

parasitize P. oleracea larvae on C. pratensis, with no

difference in attack rates from that for larvae on

collards (B. oleracea), showing that the more struc-

turally complex pinnate foliage of C. pratensis did not

reduce the ability of the wasp to locate and parasitize

larvae. However, we did find that overtopping vege-

tation greatly decreased parasitism of P. oleracea, on

both C. pratensis and B. oleracea, reducing parasitism

to about one-third of our controls without overtopping

vegetation–which suggests that overtopping vegeta-

tion provided protection from C. glomerata

parasitism. Whether this was from physical complex-

ity slowing or impeding wasp movement or from

effects of vegetation on odor-plume detection or wasp

movement toward odors is unknown.

In summary, the adoption of a novel host, i.e.,

cuckoo flower, by P. oleracea at our Lenox, Massa-

chusetts site evidently conferred some degree of

enemy-free space from C. glomerata by virtue of the

host plant rosettes being obscured most of the year by

taller vegetation, despite the odor signature of infested

cuckoo flower being highly attractive to the parasitoid.

Also, cuckoo flower was an extremely abundant

resource that was available for the whole growing

season, allowing four generations of the butterfly per

year. Multiple generations and an abundant host plant

likely enhanced the population growth rate and hence

density of the butterfly, which in turn may have

allowed the population to tolerate significant levels of

parasitism.

Acknowledgments The authors thank Richard Casagrande of

the University of Rhode Island, George Boettner and Stephen

Donahue of the University of Massachusetts Amherst, Patrick

DeFlorio of Yankee Glassblower, Jian Duan and Craig Oppel of

USDA ARS Newark, DE, Lisa Dachinger of River Valley Farm,

Lenox, Massachusetts, and Emmet Van Driesche. For help with

the rearing of insects and conduct of sentinel larval studies at

Lenox in 2008 and 2009, we thank Alex Meleg, Sabina Perkins,

April Rodd, and Ryan Wagner. This material is based upon work

supported by the National Institute of food and Agriculture, U.S.

Department of Agriculture, the Massachusetts Agricultural

Experiment Station and the Department of Plant, Soil and

Insect Sciences under Project number MAS00957.

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persistence in massachusetts of the veined white butterfly due to use of the invasive form of cuckoo...

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