Flower

vs.

Leaf

Feeding

by

Pieris

brassicae

Glucosinolate-Rich

Flower

Tissues

are

Preferred

and

Sustain

Higher

Growth

Rate

Interactions

between

butterflies

and

caterpillars

in

the

genus

Pieris

and

plants

in

the

family

Brassicaceae

are

among

the

best

explored

in

the

field

of

insect

plant

biology

However

we

report

here

for

the

first

time

that

Pieris

brassicae

commonly

assumed

to

be

a

typical

folivore

actually

prefers

to

feed

on

flowers

of

three

Brassica

nigra

genotypes

rather

than

on

their

leaves

First

and

second-instar

caterpillars

were

observed

to

feed

primarily

on

leaves

whereas

late

second

and

early

third

instars

migrated

via

the

small

leaves

of

the

flower

branches

to

the

flower

buds

and

flowers

Once

flower

feeding

began

no

further

leaf

feeding

was

observed

We

investigated

growth

rates

of

caterpillars

having

access

exclusively

to

either

leaves

of

flowering

plants

or

flowers

In

addition

we

analyzed

glucosinolate

concentrations

in

leaves

and

flowers

Late-second

and

early-third-instar

P.

brassicae

caterpillars

moved

upward

into

the

inflorescences

of

B.

nigra

and

fed

on

buds

and

flowers

until

the

end

of

the

final

-LRB-

fifth

-RRB-

instar

after

which

they

entered

into

the

wandering

stage

leaving

the

plant

in

search

of

a

pupation

site

Flower

feeding

sustained

a

significantly

higher

growth

rate

than

leaf

feeding

Flowers

contained

levels

of

glucosinolates

up

to

five

times

higher

than

those

of

leaves

Five

glucosinolates

were

identified

the

aliphatic

sinigrin

the

aromatic

phenyethylglucosinolate

and

three

indole

glucosinolates

glucobrassicin

and

Tissue

type

and

genotype

were

the

most

important

factors

affecting

levels

of

identified

glucosinolates

Sinigrin

was

by

far

the

most

abundant

compound

in

all

three

genotypes

Sinigrin

and

phenylethylglucosinolate

were

present

at

significantly

higher

levels

in

flowers

than

in

leaves

In

response

to

caterpillar

feeding

sinigrin

levels

in

both

leaves

and

flowers

were

significantly

higher

than

in

undamaged

plants

whereas

leaf

levels

were

lower

Our

results

show

that

feeding

on

flower

tissues

containing

higher

concentrations

of

glucosinolates

provides

P.

brassicae

with

a

nutritional

benefit

in

terms

of

higher

growth

rate

This

preference

appears

to

be

in

contrast

to

published

negative

effects

of

volatile

glucosinolate

breakdown

products

on

the

closely

related

Pieris

rapae

Introduction

Most

plants

are

not

simply

homogeneous

resources

to

herbivorous

insects

but

produce

discrete

organs

with

different

functions

In

addition

to

the

vegetative

leaf

tissues

plants

produce

reproductive

organs

-LRB-

flowers

seeds

-RRB-

that

may

exhibit

significant

differences

in

levels

of

both

nutrients

and

defensive

secondary

metabolites

Specialized

guilds

of

herbivores

are

known

to

exploit

specific

plant

structures

-LRB-

Schoonhoven

et

al.

-RRB-

Flowers

and

seeds

may

be

attacked

by

insects

that

rarely

feed

on

leaf

tissues

and

vice

versa

Other

herbivores

exhibit

more

plasticity

in

food

selection

and

will

readily

feed

on

all

available

parts

of

the

plant

Even

the

latter

herbivore

types

display

some

preference

for

a

specific

plant

structure

presumably

as

a

means

of

obtaining

optimal

nutrient

intake

limiting

competition

or

obtaining

enemy-free

space

The

cabbage

white

butterflies

Pieris

brassicae

L.

Pieris

rapae

L.

and

Pieris

napi

L.

are

specialized

on

the

plant

family

Brassicaceae

Interactions

between

these

butterflies

and

their

host

plants

have

been

explored

in

detail

and

they

have

acquired

the

status

of

a

model

system

in

the

field

of

insect

plant

biology

-LRB-

Feltwell

Chew

and

Renwick

Renwick

-RRB-

Glucosinolates

characteristic

of

the

Brassicaceae

play

a

crucial

role

as

chemical

mediators

of

these

interactions

Adult

females

of

P.

brassicae

and

P.

rapae

as

well

as

their

larvae

exploit

glucosinolates

as

token

stimuli

during

selection

of

host

plants

for

oviposition

and

feeding

-LRB-

Renwick

et

al.

van

Loon

et

al.

Moyes

et

al.

Schoonhoven

and

van

Loon

-RRB-

The

majority

of

Pieris

Brassica

interaction

studies

has

focused

on

cultivated

forms

of

Brassica

oleracea

L.

which

are

biennials

Other

wild

crucifers

such

as

the

annual

black

mustard

Brassica

nigra

L.

-LRB-

Koch

-RRB-

are

reported

to

be

host

plants

for

P.

brassicae

in

Europe

as

well

-LRB-

Harvey

et

al.

-RRB-

We

studied

within-plant

feeding

site

location

in

detail

on

three

genotypes

of

B.

nigra

We

included

the

analysis

of

leaf

and

flower

glucosinolates

to

assess

whether

a

chemical

basis

for

selection

of

either

plant

organ

in

terms

of

quality

or

quantity

of

these

compounds

could

be

determined

In

addition

we

investigated

whether

growth

rates

differed

between

caterpillars

feeding

on

either

leaves

or

flowers

to

assess

whether

the

observed

preference

behavior

would

confer

a

nutritional

benefit

Methods

and

Materials

Plants

Seeds

of

B.

nigra

var

abyssinica

A.

Braun

from

three

early

flowering

accessions

were

obtained

from

the

Centre

for

Genetic

Resources

-LRB-

CGN

Wageningen

The

Netherlands

-RRB-

The

accessions

were

A

-LRB-

feral

population

collected

in

from

the

Peloponesus

Greece

-RRB-

B

Junius

-LRB-

advanced

cultivar

collected

in

in

Germany

-RRB-

and

C

Giebra

-LRB-

advanced

cultivar

donated

in

by

the

Botanical

Garden

of

the

Justus

Liebig

University

Giessen

Germany

-RRB-

Sowing

was

done

on

April

in

a

greenhouse

at

°C

and

RH

Two

weeks

later

plants

were

individually

transplanted

into

pots

and

transferred

to

another

greenhouse

compartment

on

April

This

compartment

was

kept

at

°C

RH

natural

light

supplemented

by

additional

illumination

by

SON-T

lamps

-LRB-

Philips

Eindhoven

The

Netherlands

-RRB-

for

hr

per

day

Flower

bud

formation

and

flower

opening

were

recorded

daily

to

determine

developmental

stage

-LRB-

Harper

and

Berkenkamp

-RRB-

Insects

Pieris

brassicae

caterpillars

were

obtained

from

a

laboratory

strain

established

in

and

reared

on

Brussels

sprouts

-LRB-

B.

oleracea

var

gemmifera

cv

Cyrus

-RRB-

plants

grown

in

a

climatized

greenhouse

under

the

same

conditions

as

given

above

for

B.

nigra

plants

The

colony

was

maintained

in

a

climatized

room

at

°C

RH

and

a

photoperiod

of

Caterpillar

Position

on

the

Plant

On

the

first

day

of

an

experiment

newly

hatched

P.

brassicae

larvae

were

collected

from

the

laboratory

culture

and

taken

to

the

greenhouse

Three

caterpillars

were

placed

on

the

first

true

leaf

below

the

lowest

flowering

branch

of

a

plant

in

growth

stage

-LRB-

Harper

and

Berkenkamp

-RRB-

Total

numbers

of

plants

used

from

each

accession

were

A

B

and

C

Caterpillar

position

on

the

plants

was

scored

each

morning

in

four

categories

on

a

true

leaf

on

a

small

leaf

in

the

inflorescence

on

a

flower

and

on

the

stem

Caterpillar

instar

stage

was

recorded

daily

until

the

fifth

instar

was

reached

In

the

late

fifth

instar

caterpillars

that

had

entered

the

wandering

phase

left

the

plants

Caterpillar

Growth

on

Leaves

and

Flowers

Three

neonate

caterpillars

were

inoculated

on

each

of

plants

of

accession

A

on

a

mature

leaf

just

below

the

inflorescence

The

inoculation

took

place

when

plants

had

just

started

to

flower

-LRB-

growth

stage

-RRB-

Just

after

caterpillars

had

molted

to

the

third

instar

they

were

transferred

to

the

lowest

branches

of

the

inflorescence

on

half

of

the

plants

We

applied

a

specially

constructed

water

barrier

to

prevent

caterpillars

from

migrating

from

leaves

to

the

inflorescence

or

vice

versa

while

not

influencing

the

microclimate

around

the

plant

-LRB-

Fig.

-RRB-

Fresh

body

weights

of

larvae

were

determined

to

the

nearest

milligram

on

a

Mettler

electronic

balance

at

three

time

points

separated

by

intervals

Fig.

drawing

-LRB-

top

and

lateral

views

-RRB-

and

dimensions

of

water

barrier

applied

around

the

stem

of

B.

nigra

plants

to

prevent

movement

of

caterpillars

from

leaves

to

flowers

and

vice

versa

A

circular

water

container

was

manufactured

of

plastic

The

middle

circle

consisted

of

a

central

platform

preventing

drowning

of

caterpillars

feeding

on

the

inflorescence

in

the

rare

events

of

falling

down

or

downward

migration

In

the

center

of

the

platform

a

hole

allowed

the

main

stem

to

grow

through

The

platform

was

put

in

place

around

the

main

stem

just

below

the

branch

carrying

the

first

flower

buds

The

circular

opening

between

the

central

hole

and

the

stem

was

blocked

with

soft

foam

-LRB-

not

drawn

-RRB-

Preparation

of

Plant

Samples

for

Glucosinolate

Analysis

Ten

seedlings

of

each

of

the

three

accessions

were

individually

transplanted

to

1 1/2

l

pots

Plants

were

maintained

at

°C

RH

during

the

photophase

-LRB-

Philips

SON-T

Agro

sodium

lamps

-RRB-

and

scotophase.At

day

after

sowing

leaf

and

flower

samples

were

taken

from

all

plants

of

each

accession

After

sampling

five

plants

of

each

accession

were

designated

untreated

-LRB-

controls

-RRB-

Three

groups

of

newly

hatched

P.

brassicae

caterpillars

were

placed

on

three

leaves

of

the

five

treated

plants

As

soon

as

the

caterpillars

had

migrated

to

the

flowers

which

took

place

late

in

the

second

or

early

in

the

third

instar

-LRB-

days

-RRB-

leaves

damaged

by

the

caterpillars

were

sampled

Leaves

of

a

similar

age

were

sampled

from

the

control

plants

and

intact

flowers

were

sampled

from

both

control

and

treated

plants.The

flower

and

leaf

samples

were

kept

in

paper

envelopes

and

stored

in

a

°C

freezer

before

and

after

freeze-drying

Samples

were

freeze-dried

in

the

envelopes

for

d

-LRB-

Labconco

Freedry

system

-RRB-

After

freeze-drying

the

envelopes

were

sealed

in

plastic

and

sent

to

the

laboratory

of

in

Denmark

for

chemical

analysis

Determination

of

Glucosinolate

Content

Freeze-dried

samples

were

weighed

accurately

into

centrifuge

tubes

-LRB-

excluding

the

petiole

part

of

leaves

-RRB-

For

samples

below

mg

the

entire

sample

-LSB-

mg

dry

weight

-LRB-

DW

-RRB-

-RSB-

was

used

to

avoid

errors

associated

with

subsampling

but

from

a

few

samples

above

mg

a

representative

subsample

was

taken

Each

sample

was

extracted

in

boiling

aq

methanol

with

benzylglucosinolate

as

internal

standard

The

combined

extracts

were

adjusted

to

ml

of

which

an

amount

was

applied

to

an

anion

exchange

column

-LRB-

Agerbirk

et

al.

-RRB-

To

keep

within

the

linear

range

of

the

additional

sample

preparation

steps

the

amount

of

crude

extract

applied

depended

on

the

weight

of

the

sample

extracted

as

follows

-LRB-

DW

extracted/volume

applied

-RRB-

ml

ml

and

ml

Glucosinolates

were

converted

to

desulfoglucosinolates

-LRB-

Agerbirk

et

al.

-RRB-

which

were

eluted

and

quantified

by

high-performance

liquid

chromatography

-LRB-

HPLC

-RRB-

-LRB-

Agerbirk

et

al.

-RRB-

and

identified

by

comparison

of

retention

times

and

diode

array

UV

spectra

with

those

of

authentic

standards

-LRB-

Agerbirk

et

al.

-RRB-

A

number

of

unidentified

trace

peaks

with

areas

typically

less

than

of

the

desulfosinigrin

area

were

ignored

Generally

accepted

relative

response

factors

were

used

for

calculation

of

glucosinolate

levels

based

on

the

HPLC

peak

areas

-LRB-

at

nm

band

width

nm

-RRB-

of

the

desulfoderivatives

-LRB-

Wathelet

et

al.

-RRB-

The

recoveries

of

internal

standard

in

the

three

extraction

series

were

-LSB-

mean

-LRB-

SD

-RRB-

-RSB-

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

as

compared

to

HPLC

peak

areas

obtained

after

application

of

the

internal

standard

only

to

DEAE

columns

in

parallel

control

experiments

Preliminary

experiments

without

the

addition

of

internal

standard

-LRB-

comprising

both

leaf

and

flower

samples

from

plants

both

with

and

without

caterpillars

feeding

-RRB-

confirmed

the

absence

of

benzylglucosinolate

as

an

endogenous

glucosinolate

in

the

material

in

accordance

with

previous

reports

-LRB-

Feeny

and

Rosenberry

Sang

et

al.

-RRB-

allowing

the

use

of

this

glucosinolate

as

internal

standard

The

identities

of

all

desulfoglucosinolate

peaks

were

confirmed

by

liquid

chromatograph

mass

spectrometry

-LRB-

LC

MS

-RRB-

of

representative

samples

with

an

Agilent

Series

LC

-LRB-

Agilent

Technologies

Waldbronn

Germany

-RRB-

coupled

to

a

Bruker

Esquire

ion

trap

mass

spectrometer

-LRB-

Bruker

Daltonics

Bremen

Germany

-RRB-

An

XTerra

MS

column

-LRB-

Waters

Milford

MA

USA

μM

mm

-RRB-

was

used

at

a

flow

rate

of

ml

min

The

mobile

phases

were

as

follows

A

-LRB-

v/v

-RRB-

HCOOH

and

μM

NaCl

B

-LRB-

v/v

-RRB-

HCOOH

and

-LRB-

v/v

-RRB-

MeCN

The

gradient

program

was

to

min

isocratic

-LRB-

v/v

-RRB-

B

to

min

linear

gradient

to

B

to

min

linear

gradient

to

-LRB-

v/v

-RRB-

B

to

min

linear

gradient

to

-LRB-

v/v

-RRB-

B

to

min

isocratic

B

The

mass

spectrometer

was

run

in

electrospray

mode

observing

positive

ions

Mass

spectral

data

were

treated

with

the

native

DataAnalysis

software.The

extraction

procedure

was

somewhat

simplified

compared

to

the

standard

protocol

-LRB-

Agerbirk

et

al.

originally

optimized

for

seed

extraction

-RRB-

as

variable

sample

weights

were

accepted

and

mechanical

homogenization

during

extraction

was

avoided

These

simplifications

were

justified

by

results

of

initial

control

experiments

A

number

of

both

leaf

and

flower

samples

of

varying

weights

-LRB-

up

to

mg

-RRB-

were

first

subjected

to

the

extraction

described

above

Then

the

residue

was

further

extracted

for

another

three

times

but

this

time

with

mechanical

homogenization

In

all

cases

low

relative

amounts

of

glucosinolates

-LRB-

compared

to

the

first

extract

-RRB-

were

detected

in

the

extract

obtained

with

mechanical

homogenization

-LRB-

with

a

tendency

for

increasing

percentage

with

increasing

sample

weight

-RRB-

Proportional

amounts

of

internal

standard

were

similarly

recovered

in

all

the

extracts

obtained

with

mechanical

homogenization

showing

that

the

internal

standard

had

already

been

completely

mixed

with

endogenous

analytes

during

the

extraction

without

homogenization

Hence

it

was

concluded

that

the

ratio

of

internal

standard

to

endogenous

glucosinolates

extracted

without

mechanical

homogenization

was

representative

of

their

initial

concentrations

Statistical

Analyses

Proportion

of

days

caterpillars

spent

on

different

positions

on

the

plant

was

treated

as

binomial

data

Distributions

over

the

four

positions

were

compared

by

using

the

Kolmogorov

Smirnoff

test

-LRB-

GenStat

release

Anonymous

-RRB-

Caterpillar

weights

were

analyzed

by

analysis

of

variance

The

amount

-LRB-

μmol/g

DW

-RRB-

of

each

glucosinolate

detected

in

the

leaf

and

flower

samples

were

analyzed

with

a

generalized

linear

model

after

a

logarithmic

transformation

The

effect

of

time

-LRB-

before

and

during

caterpillar

feeding

-RRB-

treatment

-LRB-

undamaged

and

damaged

by

caterpillars

-RRB-

plant

tissue

-LRB-

leaf

and

flower

-RRB-

and

genotype

-LRB-

accessions

-RRB-

and

all

possible

two

and

three-way

interactions

were

investigated

-LRB-

GenStat

release

-RRB-

Results

Position

on

the

Plant

First

and

second

instars

fed

primarily

on

leaves

whereas

third

instars

migrated

via

the

small

leaves

of

the

flower

branches

to

the

flower

buds

and

flowers

Once

flower

feeding

began

no

further

leaf

feeding

was

observed

-LRB-

Fig.

-RRB-

The

distribution

of

caterpillars

over

the

four

positions

was

statistically

similar

for

the

three

genotypes

-LRB-

Kolmogorov

Smirnoff

test

P

-RRB-

Fig.

distribution

of

caterpillar

position

on

B.

nigra

plants

observed

daily

over

a

period

of

d.

On

day

neonates

were

introduced

on

a

true

leaf

just

below

the

inflorescence

Four

organ

positions

were

distinguished

on

a

true

leaf

on

a

small

leaf

in

the

inflorescence

on

a

flower

or

on

a

stem

in

the

inflorescence

Percentages

are

averaged

based

on

three

caterpillars

per

plant

for

eight

seven

and

nine

plants

of

accessions

A

B

and

C

respectivelyCaterpillars

fed

in

discrete

meals

two

to

three

times

each

hour

interspersed

with

phases

without

feeding

On

a

leaf

after

termination

of

a

meal

they

moved

away

from

the

freshly

damaged

wound

after

which

they

rested

most

of

the

time

elsewhere

on

the

leaf

until

they

moved

back

to

the

feeding

site

By

contrast

in

the

inflorescence

during

a

single

meal

caterpillars

moved

from

flower

to

flower

along

flowering

branches

A

fifth-instar

caterpillar

consumed

on

average

buds

and

flowers

When

they

were

not

feeding

P.

brassicae

larvae

rested

on

stems

immediately

adjacent

to

the

flowers

Caterpillar

Growth

Caterpillars

feeding

on

flowers

of

accession

A

since

molting

to

the

third

instar

had

significantly

higher

fresh

body

weights

on

each

of

the

three

measuring

points

-LRB-

and

early

and

and

late

Fig.

-RRB-

than

those

that

were

confined

to

leaves

-LRB-

P

generalized

linear

model

-RRB-

Fig.

body

mass

of

P.

brassicae

on

either

flowers

-LRB-

circles

-RRB-

or

leaves

-LRB-

squares

-RRB-

of

B.

nigra

in

mid-fourth

mid-fifth

and

late-fifth

instar

developing

on

B.

nigra

accession

A.

Mean

and

SEM

are

plotted

for

caterpillars

Glucosinolate

Levels

Five

glucosinolates

were

identified

the

aliphatic

allylglucosinolate

-LRB-

sinigrin

-RRB-

which

occurred

in

large

amounts

and

low

levels

of

four

aromatic

glucosinolates

including

and

the

three

indole

glucosinolates

-LRB-

glucobrassicin

-RRB-

and

Despite

the

minor

levels

of

the

identified

aromatic

glucosinolates

they

were

quantified

and

included

in

the

statistical

analyses

because

of

their

well

established

chemosensory

influences

on

Pieris

species

-LRB-

Schoonhoven

and

van

Loon

-RRB-

No

other

significant

peaks

were

detected

was

either

below

the

detection

limit

in

leaves

-LRB-

accession

C

-RRB-

or

present

at

very

low

levels

-LRB-

range

μmol

g

DW

-RRB-

We

carefully

distinguished

the

peak

with

the

exact

retention

time

of

-LRB-

desulfo

-RRB-

from

an

unidentified

trace

peak

that

was

more

intense

in

leaf

chromatograms

-LRB-

estimated

levels

μmol/g

DW

in

leaves

μmol/g

DW

in

flowers

-RRB-

that

had

a

retention

time

min

longer

than

that

of

-LRB-

desulfo

-RRB-

and

that

did

not

coelute

with

the

authentic

standard

of

after

spiking.Genotype

and

tissue

type

were

the

most

important

factors

affecting

the

levels

of

identified

glucosinolates

and

these

factors

showed

a

significant

interaction

-LRB-

Table

-RRB-

No

significant

interactions

were

found

between

treatment

and

tissue

-LRB-

P

for

all

glucosinolates

-RRB-

Glucosinolate

concentrations

have

been

graphically

depicted

for

accession

A

in

Fig.

Sinigrin

was

by

far

the

most

abundant

compound

in

all

three

genotypes

with

levels

at

least

times

higher

-LRB-

range

-RRB-

in

leaves

and

at

least

times

higher

-LRB-

range

-RRB-

in

flowers

of

any

of

the

three

genotypes

and

treatments

than

the

second

most

abundant

compound

Sinigrin

and

phenylethylglucosinolate

were

present

at

significantly

higher

levels

in

flowers

than

in

leaves

Sinigrin

levels

were

ca

fivefold

higher

in

flowers

than

in

leaves

In

response

to

caterpillar

feeding

sinigrin

levels

in

leaves

were

higher

than

in

undamaged

plants

-LRB-

P

Table

-RRB-

In

flowers

of

accession

A

-LRB-

Fig.

-RRB-

there

was

a

similar

tendency

for

induction

of

sinigrin

by

caterpillar

feeding

but

such

induction

was

not

seen

with

flowers

from

the

other

two

genotypes

-LRB-

results

not

shown

-RRB-

Levels

of

other

glucosinolates

showed

moderate

variations

that

were

not

affected

by

the

experimental

variables

-LRB-

Table

Fig.

and

results

not

shown

-RRB-

There

was

no

systematic

induction

of

any

indole

glucosinolate

or

of

total

indole

glucosinolates

in

response

to

caterpillar

feeding

-LRB-

results

not

shown

-RRB-

Table

from

generalized

linear

model

analysis

on

log-transformed

data

of

glucosinolate

concentrations

in

two

tissue

types

-LRB-

leaves

and

flowers

-RRB-

for

three

genotypes

of

B.

nsTissue

ns

nsTreatment

nsns

nsGenotype

nsTissue

genotype

ns

nsTreatment

means

presence

or

absence

of

third-instar

caterpillars

feeding

on

flowers

during

d.

Effect

of

the

factor

time

refers

to

changes

over

d

in

intact

plantsPE

phenyethylglucosinolate

GB

glucobrassicin

P

P

P

Fig.

of

five

glucosinolates

-LRB-

mean

SEM

-RRB-

in

leaves

and

flowers

of

plants

of

accession

A.

For

both

leaves

and

flowers

the

initial

concentration

just

before

treatment

the

concentration

in

leaves

or

flowers

of

plants

damaged

by

feeding

caterpillars

during

d

and

the

concentration

determined

in

leaves

or

flowers

of

intact

plants

d

after

caterpillars

were

introduced

on

the

treated

plants

Discussion

Our

data

show

that

P.

brassicae

third

fourth

and

fifth

instars

preferentially

feed

on

B.

nigra

flowers

Preference

was

absolute

although

leaf

material

was

available

in

surplus

caterpillars

fed

exclusively

on

flowers

Historically

P.

brassicae

has

been

commonly

considered

a

folivore

-LRB-

Feltwell

-RRB-

with

its

impact

on

plants

examined

primarily

from

this

perspective

To

the

best

of

our

knowledge

this

is

the

first

detailed

study

showing

that

P.

brassicae

larvae

preferentially

feed

on

the

buds

and

flowers

of

its

host

plant

The

status

of

P.

brassicae

as

an

agricultural

pest

has

invariably

resulted

in

a

focus

on

cultivated

biennial

forms

of

B.

oleracea

in

studies

of

its

host

plant

relationships

We

also

observed

flower

and

silique

feeding

under

field

conditions

-LRB-

Smallegange

et

al.

unpublished

results

-RRB-

The

impact

of

folivores

on

plants

has

both

direct

and

indirect

effects

Directly

these

herbivores

reduce

the

leaf

area

available

for

photosynthesis

thereby

decreasing

the

plants

ability

to

garner

resources

for

defense

and

reproduction

-LRB-

Marquis

Mothershead

and

Marquis

Schoonhoven

et

al.

-RRB-

Indirectly

leaf

herbivory

can

delay

flowering

-LRB-

Meyer

and

Root

Strauss

et

al.

-RRB-

or

can

alter

developing

flowers

both

physically

-LRB-

Strauss

et

al.

Lehtilä

and

Strauss

Mothershead

and

Marquis

-RRB-

and

chemically

-LRB-

Lohman

et

al.

Karban

and

Baldwin

-RRB-

This

may

cause

a

reduction

in

plant

attractiveness

to

pollinators

or

in

the

efficiency

of

pollen

delivery

resulting

in

decreased

pollination

and

subsequently

reduction

of

seed

set

and

overall

fitness

of

the

plant

Florivory

feeding

on

flowers

has

received

less

attention

than

folivory

or

feeding

on

other

vegetative

plant

parts

despite

its

common

occurrence

and

potentially

high

impact

on

plant

fitness

-LRB-

recently

reviewed

by

McCall

and

Irwin

-RRB-

Feeding

on

flowers

causes

damage

to

structures

essential

for

plant

reproductive

output

-LRB-

Juenger

and

Bergelson

Adler

et

al.

-RRB-

Indirectly

petal

damage

leads

to

a

reduction

in

pollinator

visitation

that

not

only

reduces

reproductive

success

but

also

affects

male

fitness

-LRB-

via

pollen

removal

-RRB-

of

the

plant

as

well

-LRB-

Karban

and

Strauss

Krupnick

and

Weis

Adler

et

al.

-RRB-

However

the

compensatory

abilities

of

plants

can

partially

overcome

the

impact

of

floral

herbivory

-LRB-

Hendrix

Krupnick

and

Weis

-RRB-

Removal

of

reproductive

organs

as

a

preferred

mode

of

feeding

has

evolutionary

consequences

as

this

type

of

attack

is

likely

to

exert

a

stronger

selection

pressure

than

leaf

feeding

especially

in

annual

species

like

B.

nigra

Although

many

plant

species

accumulate

higher

concentrations

of

defensive

secondary

metabolites

in

reproductive

organs

than

in

vegetative

organs

-LRB-

Schoonhoven

et

al.

-RRB-

in

B.

nigra

these

higher

levels

are

not

sufficient

to

deter

the

specialist

feeder

P.

brassicae

A

putative

barrier

to

florivory

by

generalist

herbivores

lies

in

the

higher

quantities

of

secondary

plant

compounds

that

are

typically

found

in

these

organs

relative

to

the

levels

reported

for

leaves

-LRB-

Rask

et

al.

Fahey

et

al.

Brown

et

al.

Strauss

et

al.

this

study

-RRB-

The

location

of

high

concentrations

of

defensive

secondary

plant

compounds

including

glucosinolates

in

reproductive

structures

is

consistent

with

the

optimal

distribution

of

chemical

defenses

predicted

by

plant

defense

theory

-LRB-

Zangerl

and

Bazzaz

van

Dam

et

al.

Wallace

and

Eigenbrode

Strauss

et

al.

-RRB-

Myrosinases

β-thioglucosidase

enzymes

that

convert

glucosinolates

into

products

such

as

isothiocyanates

and

nitriles

which

are

volatile

in

the

case

of

e.g.

sinigrin

and

occur

in

different

forms

in

flowers

and

leaves

-LRB-

Rask

et

al.

-RRB-

By

using

headspace

techniques

isothiocyanates

and

nitriles

have

been

reported

from

mechanically

macerated

bud

samples

of

several

Brassica

species

-LRB-

Tollsten

and

Bergström

-RRB-

Phenylacetonitrile

was

detected

in

headspace

samples

of

intact

B.

rapa

L.

flowers

-LRB-

Omura

et

al.

-RRB-

Benzylcyanide

was

released

in

significantly

higher

amounts

from

Brassica

napus

L.

in

midflowering

than

in

bud

stage

-LRB-

Jönsson

et

al.

-RRB-

High

levels

of

isothiocyanates

are

potentially

toxic

to

both

specialist

and

generalist

herbivorous

insects

-LRB-

e.g.

Li

et

al.

Agrawal

and

Kurashige

-RRB-

while

even

low

levels

may

be

detected

by

specialized

sensory

receptors

and

assist

specialized

herbivores

in

host

plant

finding

-LRB-

Renwick

et

al.

-RRB-

The

growth

rate

we

observed

for

caterpillars

feeding

on

flowers

was

remarkable

The

feeding

site

preference

of

caterpillars

presents

a

case

of

within-plant

selective

foraging

the

adaptive

value

of

which

might

be

the

ingestion

of

higher

nutritional

content

in

flowers

known

to

act

as

nutrient

sinks

compared

to

leaves

Nutrient

acquisition

during

the

larval

stage

can

affect

pupal

survival

longevity

and

fecundity

of

Lepidoptera

-LRB-

Feltwell

Van

der

Meijden

et

al.

Metspalu

et

al.

-RRB-

For

example

female

pupae

of

Tyria

jacobaeae

were

significantly

larger

when

caterpillars

were

reared

on

leaves

and

inflorescences

of

Senecio

jacobaea

plants

than

when

reared

on

leaves

only

This

is

advantageous

because

large

pupae

have

a

higher

eclosion

success

and

it

may

explain

why

T.

jacobaeae

females

prefer

flowering

plants

for

oviposition

-LRB-

Vrieling

and

de

Boer

-RRB-

Faster

growth

has

often

been

interpreted

as

a

mortality-reducing

factor

It

is

assumed

that

by

reducing

the

amount

of

time

that

larvae

are

early

instars

when

they

are

most

vulnerable

to

attack

by

natural

enemies

they

will

experience

higher

survival

-LRB-

the

slow-growth

high-mortality

hypothesis

sensu

Clancy

and

Price

Benrey

and

Denno

-RRB-

This

has

been

experimentally

confirmed

under

field

conditions

for

the

interaction

between

P.

rapae

feeding

on

B.

oleracea

and

its

predators

and

parasitoids

such

as

Cladophora

glomerata

-LRB-

Loader

and

Damman

Benrey

and

Denno

-RRB-

However

for

other

tritrophic

interactions

faster

growth

was

associated

with

higher

mortality

due

to

natural

enemies

-LRB-

e.g.

Clancy

and

Price

-RRB-

Biere

et

al.

-LRB-

-RRB-

found

that

larvae

of

the

moth

Hadena

bicruris

exhibit

a

strong

preference

for

seeds

of

their

food

plant

Silena

latifolia

over

leaf

tissues

Herbivore

development

proceeded

more

rapidly

on

seeds

and

the

larvae

were

less

susceptible

to

parasitism

from

their

main

endoparasitoid

Microplitis

tristis

Herbivorous

insects

specialized

on

glucosinolate-containing

plants

typically

avoid

the

formation

of

toxic

isothiocyanates

by

employing

specialized

detoxifying

mechanisms

In

the

case

of

P.

brassicae

this

is

accomplished

by

a

nitrile

specifier

protein

-LRB-

NSP

-RRB-

in

the

gut

that

changes

the

products

of

the

myrosinase-catalysed

hydrolysis

of

glucosinolates

from

isothiocyanates

to

relatively

harmless

nitriles

-LRB-

Wittstock

et

al.

-RRB-

which

may

be

further

metabolized

before

excretion

depending

on

side

chain

structure

-LRB-

Agerbirk

et

al.

-RRB-

Shortly

before

this

mechanism

was

reported

it

was

discovered

that

allyl

isothiocyanate

the

volatile

hydrolysis

product

of

sinigrin

is

toxic

to

the

specialist

feeder

P.

rapae

when

feeding

on

an

artificial

diet

-LRB-

Agrawal

and

Kurashige

-RRB-

Assuming

a

DW

in

the

artificial

diet

as

used

by

Agrawal

and

Kurashige

-LRB-

-RRB-

the

maximum

concentration

to

which

P.

rapae

caterpillars

were

exposed

in

their

study

was

μmol/g

DW

This

dose

caused

significant

negative

effects

on

growth

rate

and

inhibited

development

In

contrast

sinigrin

levels

in

B.

nigra

flowers

were

more

than

times

higher

-LRB-

Fig.

-RRB-

and

flowers

sustained

higher

growth

rates

of

P.

brassicae

than

leaves

which

had

average

levels

of

sinigrin

two

times

higher

than

the

corresponding

amount

of

allylisothiocyanate

as

applied

in

the

artificial

diet

Assuming

that

isothiocyanates

were

produced

in

significant

amounts

during

ingestion

of

glucosinolate-containing

foliage

by

P.

rapae

an

involvement

of

the

glucosinolate

myrosinase

system

in

defense

against

this

specialist

species

was

suggested

Brassica

nigra

plants

that

had

been

induced

by

previous

P.

rapae

feeding

damage

had

a

increase

in

sinigrin

concentration

but

also

higher

trichome

density

-LRB-

Traw

and

Dawson

-RRB-

Our

data

confirm

the

reported

induction

of

sinigrin

in

leaves

upon

feeding

but

also

demonstrate

that

the

magnitude

of

the

induction

is

minor

relative

to

the

difference

between

leaves

and

flowers

With

the

elucidation

of

the

NSP-based

mechanism

for

detoxification

of

the

glucosinolate

myrosinase

system

the

suggested

involvement

of

the

glucosinolate

myrosinase

system

and

defense

to

specialists

now

needs

to

be

re-evaluated

Formation

of

isothiocyanates

is

to

be

expected

only

if

the

myrosinase

activity

in

the

plant

is

so

high

that

available

NSP

can

not

completely

direct

the

reaction

towards

nitriles

combined

with

glucosinolate

levels

sufficiently

high

to

produce

toxic

levels

of

isothiocyanates

The

selective

foraging

on

flowers

by

P.

brassicae

documented

here

demonstrates

that

the

caterpillars

are

able

to

detoxify

even

high

levels

of

sinigrin

such

as

those

present

in

B.

nigra

flower

tissue

Not

only

high

glucosinolate

levels

but

also

high

myrosinase

activity

would

be

needed

to

exert

toxic

effects

on

Pieris

caterpillars

as

has

been

experimentally

demonstrated

for

another

glucosinolate-adapted

herbivorous

insect

Plutella

xylostella

-LRB-

Li

et

al.

-RRB-

Glucosinolates

occur

in

cells

of

all

organs

of

Brassicaceous

plants

whereas

myrosinases

can

be

found

in

scattered

glucosinolate-free

cells

It

remains

to

be

examined

whether

flower

buds

of

different

age

flowers

and

siliques

of

B.

nigra

have

cells

that

contain

myrosinases

In

the

related

B.

napus

L.

expression

of

the

coding

for

particular

myrosinase

isoforms

has

been

demonstrated

to

occur

in

all

organs

investigated

including

petals

-LRB-

Falk

et

al.

-RRB-

although

myrosinase

was

absent

in

very

young

flower

buds

in

this

species

-LRB-

Andréasson

et

al.

-RRB-

Myrosinase

gene

promoters

fused

to

beta-glucuronidase

as

a

reporter

showed

expression

of

myrosinase

in

idioblast

myrosin

cells

in

immature

and

mature

seeds

and

phloem

myrosin

cells

of

B.

napus

-LRB-

Thangstad

et

al.

-RRB-

Data

on

headspace

composition

of

inflorescences

actually

fed

upon

by

Pieris

spp.

are

not

available

Follow-up

studies

on

the

types

and

quantities

of

volatile

breakdown

products

emitted

by

herbivore-damaged

flowers

are

required

to

quantify

myrosinase

activity

levels

in

flowers

If

these

levels

are

low

the

better

performance

associated

with

flower

feeding

might

be

reconciled

with

a

role

of

the

glucosinolate

myrosinase

system

in

defense

against

the

specialist

P.

brassicae

However

a

likely

and

simple

explanation

of

the

better

performance

associated

with

flower-feeding

may

be

that

the

caterpillars

are

able

to

efficiently

detoxify

the

glucosinolate

myrosinase

defense

system

in

both

organs

and

that

flowers

major

nutrient

sinks

on

a

plant

are

more

nutritious

than

leaves

The

fact

that

early

instars

did

not

migrate

to

flowers

may

be

an

adaptation

to

factors

other

than

nutrient

content

leaves

are

the

site

of

oviposition

are

present

earlier

in

the

season

than

flowers

and

may

provide

better

refuge

from

natural

enemies

for

small

instars

We

have

never

observed

P.

brassicae

or

P.

rapae

ovipositing

on

flowers

or

flowering

stems

Neither

P.

rapae

nor

P.

brassicae

sequester

glucosinolates

for

their

own

defense

against

natural

enemies

-LRB-

Müller

et

al.

-RRB-

in

contrast

to

what

had

been

published

earlier

by

Aplin

et

al.

-LRB-

-RRB-

The

intensely

green

clear

regurgitant

these

caterpillars

instantly

and

typically

produce

when

attacked

may

contain

highly

concentrated

glucosinolate

hydrolysis

products

-LRB-

nitriles

-RRB-

stored

in

the

foregut

Presence

of

concentrated

glucosinolate

hydrolysis

products

may

explain

the

obvious

repellent

effect

of

regurgitant

to

natural

enemies

such

as

Cotesia

wasps

and

predators

-LRB-

Brodeur

et

al.

-RRB-

Consequently

caterpillars

may

prefer

flower

tissues

that

contain

high

levels

of

these

compounds

thus

boosting

their

defense

against

both

specialist

and

generalist

natural

enemies

-LRB-

Soler

et

al.

-RRB-

Behavioral

experiments

with

parasitoids

and

predators

known

to

act

as

common

natural

enemies

of

P.

brassicae

-LRB-

Feltwell

-RRB-

are

required

to

test

this

hypothesis

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

herbivory
lepidoptera
myrosinase
isothiocyanates
brassica nigra