Jasmonic

Acid-Induced

Changes

in

Brassica

oleracea

Affect

Oviposition

Preference

of

Two

Specialist

Herbivores

Jasmonic

acid

-LRB-

JA

-RRB-

is

a

key

hormone

involved

in

plant

defense

responses

The

effect

of

JA

treatment

of

cabbage

plants

on

their

acceptability

for

oviposition

by

two

species

of

cabbage

white

butterflies

Pieris

rapae

and

P.

brassicae

was

investigated

Both

butterfly

species

laid

fewer

eggs

on

leaves

of

JA-treated

plants

compared

to

control

plants

We

show

that

this

is

due

to

processes

in

the

plant

after

JA

treatment

rather

than

an

effect

of

JA

itself

The

oviposition

preference

for

control

plants

is

adaptive

as

development

time

from

larval

hatch

until

pupation

of

P.

rapae

caterpillars

was

longer

on

JA-treated

plants

Total

glucosinolate

content

in

leaf

surface

extracts

was

similar

for

control

and

treated

plants

however

two

of

the

five

glucosinolates

were

present

in

lower

amounts

in

leaf

surface

extracts

of

JA-treated

plants

When

the

butterflies

were

offered

a

choice

between

the

purified

glucosinolate

fraction

isolated

from

leaf

surface

extracts

of

JA-treated

plants

and

that

from

control

plants

they

did

not

discriminate

Changes

in

leaf

surface

glucosinolate

profile

therefore

do

not

seem

to

explain

the

change

in

oviposition

preference

of

the

butterflies

after

JA

treatment

suggesting

that

as

yet

unknown

infochemicals

are

involved

Introduction

Plants

can

be

attacked

by

many

herbivorous

insects

and

have

evolved

a

variety

of

defense

strategies

including

morphological

barriers

synthesis

of

toxic

or

repellent

secondary

metabolites

and

the

release

of

synomones

that

attract

natural

enemies

of

the

herbivores

These

defenses

can

be

constitutive

i.e.

expressed

independent

of

the

presence

of

an

attacker

or

inducible

in

which

case

defense

compounds

accumulate

in

response

to

attack

-LRB-

Karban

and

Baldwin

-RRB-

Herbivores

can

detect

induced

defensive

compounds

and

respond

by

avoiding

these

plants

which

signal

lower

suitability

as

a

host

plant

-LRB-

Landolt

De

Moraes

et

al.

Kessler

and

Baldwin

Meiners

et

al.

-RRB-

Induced

plant

defense

can

affect

herbivorous

insects

directly

through

the

production

of

toxic

compounds

or

indirectly

through

the

production

of

cues

that

indicate

intra

or

interspecific

competition

for

the

herbivores

-LRB-

Schoonhoven

et

al.

-RRB-

Moreover

induced

plant

defense

signals

can

reduce

the

enemy-free

space

for

the

herbivores

For

parasitoids

and

predators

induced

infochemicals

may

indicate

the

presence

of

their

host

or

prey

on

the

plant

-LRB-

Turlings

et

al.

Dicke

and

Vet

-RRB-

Phenotypic

changes

in

individual

plants

may

therefore

affect

insects

at

different

trophic

levels

and

thus

the

composition

of

the

insect

community

and

food

web

associated

with

the

plant

-LRB-

Price

et

al.

Van

Zandt

and

Agrawal

Takabayashi

et

al.

-RRB-

Already

in

the

century

Kirby

and

Spence

-LRB-

-RRB-

observed

that

Pieris

brassicae

females

preferred

to

lay

their

eggs

on

plants

devoid

of

eggs

Later

this

was

confirmed

under

more

controlled

conditions

by

Rothschild

and

Schoonhoven

-LRB-

-RRB-

for

both

P.

brassicae

and

Pieris

rapae

This

avoidance

of

infested

plants

is

caused

by

a

physiological

response

of

the

plant

to

oviposition

rather

than

by

compounds

excreted

by

the

butterflies

themselves

-LRB-

Blaakmeer

et

al.

-RRB-

The

butterflies

also

avoid

egg

deposition

on

leaves

with

feeding

larvae

-LRB-

Rothschild

and

Schoonhoven

-RRB-

It

was

postulated

that

butterflies

avoid

laying

eggs

on

herbivore-infested

plants

because

herbivore

attack

induces

defense

compounds

in

plants

that

can

influence

the

performance

of

their

offspring

and

to

reduce

the

risk

of

inter

or

intraspecific

competition

and

parasitism

-LRB-

Thompson

and

Pellmyr

Shiojiri

et

al.

-RRB-

Egg-induced

chemical

changes

in

Brassica

plants

are

also

known

to

arrest

Trichogramma

parasitoids

that

parasitize

Pieris

eggs

-LRB-

Fatouros

et

al.

-RRB-

Oviposition-site

selection

involves

an

important

behavioral

decision

in

the

life

cycle

of

an

herbivorous

insect

because

hatching

larvae

have

limited

dispersal

capacity

-LRB-

Renwick

and

Chew

-RRB-

P.

rapae

is

a

solitary

butterfly

that

lays

one

egg

at

a

time

whereas

P.

brassicae

is

gregarious

and

lays

batches

of

about

eggs

P.

rapae

appears

to

spread

the

risk

of

larval

mortality

laying

few

eggs

within

any

patch

This

has

the

advantage

of

being

able

to

exploit

isolated

plants

-LRB-

Davies

and

Gilbert

-RRB-

P.

brassicae

however

needs

patches

of

plants

because

one

large

egg

batch

will

require

more

than

one

plant

for

all

caterpillars

to

develop

into

adults

Oviposition-site

selection

is

performed

in

consecutive

phases

of

searching

and

contact

evaluation

P.

rapae

butterflies

use

visual

olfactory

and

tactile

cues

during

these

phases

-LRB-

Rothschild

and

Schoonhoven

Renwick

and

Radke

-RRB-

Acceptance

of

a

site

may

be

determined

by

the

balance

of

positive

and

negative

factors

-LRB-

Renwick

and

Radke

-RRB-

Renwick

and

Radke

-LRB-

-RRB-

suggest

that

olfaction

does

not

play

a

role

in

attraction

to

a

host

plant

but

may

be

involved

in

avoidance

of

non-host

plants

P.

rapae

and

P.

brassicae

are

crucifer

specialists

and

are

known

to

use

glucosinolates

toxic

secondary

metabolites

characteristic

for

Brassicaceae

as

oviposition

stimulants

Glucobrassicin

and

sinigrin

are

effective

oviposition

stimulants

for

P.

brassicae

and

P.

rapae

-LRB-

Renwick

et

al.

Van

Loon

et

al.

-RRB-

A

major

signal-transduction

pathway

involved

in

induced

plant

defense

is

the

octadecanoid

pathway

-LRB-

Arimura

et

al.

-RRB-

A

central

compound

in

the

pathway

is

jasmonic

acid

-LRB-

JA

-RRB-

which

has

an

important

role

in

direct

and

indirect

defense

in

many

plant

species

In

response

to

JA

or

methyl

jasmonate

-LRB-

MeJA

-RRB-

treatment

increased

concentrations

of

several

compounds

have

been

documented

in

a

range

of

plant

species

e.g.

proteinase

inhibitors

-LRB-

Moura

and

Ryan

-RRB-

polyphenol

oxidases

-LRB-

Thaler

et

al.

-RRB-

nicotine

-LRB-

Baldwin

et

al.

-RRB-

trypsin

inhibitors

-LRB-

Cipollini

and

Sipe

-RRB-

glucosinolates

-LRB-

Cipollini

and

Sipe

Van

Dam

et

al.

Mewis

et

al.

-RRB-

and

increased

volatile

emission

-LRB-

Boland

et

al.

Dicke

et

al.

Koch

et

al.

-RRB-

Herbivores

are

reported

to

be

affected

by

JA

treatment

of

plants

Several

studies

have

focused

on

the

larval

stage

of

the

herbivores

and

have

shown

reduced

relative

growth

rates

and

leaf

consumption

-LRB-

Van

Dam

et

al.

Gols

et

al.

Van

Dam

et

al.

-RRB-

In

field

experiments

spraying

of

JA

decreased

the

abundance

of

caterpillars

flea

beetles

aphids

and

thrips

-LRB-

Thaler

et

al.

-RRB-

Other

studies

addressed

the

influence

of

JA

application

to

plants

on

oviposition-site

selection

behavior

of

adult

herbivores

These

studies

showed

that

JA

application

can

result

in

induced

resistance

as

well

as

induced

susceptibility

-LRB-

Stanjek

et

al.

Kessler

and

Baldwin

Lu

et

al.

-RRB-

Here

we

studied

how

JA

application

affects

oviposition

of

two

specialist

herbivores

on

cabbage

Pieris

rapae

L.

and

P.

brassicae

L.

-LRB-

Lepidoptera

Pieridae

-RRB-

that

are

closely

related

yet

differ

drastically

in

the

amount

of

eggs

they

put

on

one

plant

Our

study

is

the

first

to

compare

closely

related

herbivores

with

a

different

oviposition

strategy

which

might

affect

the

consequences

of

JA-induced

responses

JA

is

known

to

mediate

the

induction

of

chemical

defense

responses

in

plants

to

feeding

damage

and

deposition

of

eggs

-LRB-

Dicke

and

Van

Poecke

Hilker

and

Meiners

Mumm

and

Hilker

-RRB-

By

using

JA

we

were

able

to

examine

the

effects

of

induced

defense

responses

in

cabbage

Moreover

JA

application

has

the

advantage

that

visually

detectable

damage

and

the

presence

of

herbivores

or

eggs

are

avoided

Finally

JA

allows

control

over

the

strength

of

induction

through

controlled

dosages

We

hypothesized

that

JA

treatment

would

inhibit

the

oviposition

of

the

butterflies

We

made

solvent

extracts

to

address

the

identity

of

the

active

plant

compounds

that

influenced

butterfly

behavior

Rather

than

testing

whole-leaf

extracts

however

we

extracted

the

glucosinolates

from

the

surface

of

both

control

and

JA-treated

plants

and

tested

the

oviposition

preference

of

the

butterflies

for

these

glucosinolate

fractions

on

a

neutral

substrate

Furthermore

we

included

a

control

experiment

to

exclude

a

potential

direct

effect

of

JA

on

oviposition

behavior

We

addressed

the

following

questions

-LRB-

-RRB-

does

JA

treatment

of

cabbage

plants

affect

host

plant

selection

of

the

two

Pieris

butterfly

species

-LRB-

-RRB-

are

there

differences

between

solitary

and

gregarious

butterflies

-LRB-

-RRB-

does

JA

treatment

affect

glucosinolate

levels

in

leaf

surface

extracts

and

-LRB-

-RRB-

do

changes

in

glucosinolate

levels

determine

the

changes

in

oviposition

preference

Methods

and

Materials

Plants

and

Insects

Brussels

sprouts

plants

Brassica

oleracea

var

gemmifera

L.

-LRB-

Brassicaceae

-RRB-

cultivar

Cyrus

were

grown

from

seed

in

a

greenhouse

in

plastic

pots

-LRB-

cm

-RRB-

at

°C

RH

and

a

dark

photoperiod

All

experiments

were

conducted

with

to

plants

Stock

colonies

of

the

large

cabbage

white

P.

brassicae

and

the

small

cabbage

white

P.

rapae

were

maintained

on

Brussels

sprouts

in

a

climatized

room

at

°C

RH

and

a

dark

photoperiod

Chemical

Analysis

Brussels

sprouts

plants

were

sprayed

with

mM

JA

or

control

solution

JA

-LRB-

-LRB-

-RRB-

jasmonic

acid

purity

Sigma-Aldrich

St

Louis

MO

USA

-RRB-

was

applied

to

the

surface

of

the

leaves

i.e.

plants

were

sprayed

with

a

JA

solution

with

Tween

until

run-off

or

just

with

Tween

for

the

control

The

next

day

glucosinolates

-LRB-

GLS

-RRB-

were

extracted

from

the

surface

of

the

intact

Brussels

sprouts

leaves

Each

sample

consisted

of

four

leaves

-LRB-

between

the

third

to

sixth

leaf

from

the

base

of

a

plant

-RRB-

that

were

cut

at

the

base

of

the

petiole

Directly

after

cutting

the

lamina

was

dipped

for

sec

in

ml

of

dichloromethane

and

after

a

interval

it

was

dipped

for

sec

in

ml

of

methanol

-LRB-

Städler

and

Roessingh

Van

Loon

et

al.

Griffiths

et

al.

-RRB-

The

methanol

was

evaporated

from

the

crude

methanol

dip-volume

with

a

rotary

evaporator

-LRB-

IKA-Werke

GmbH

Staufen

Germany

-RRB-

For

each

treatment

plants

were

sampled

The

extract

was

redissolved

in

methanol

desulphatased

on

a

DEAE-Sephadex

column

and

separated

on

a

reverse

phase

column

by

using

high

performance

liquid

chromatography

-LRB-

HPLC

-RRB-

as

described

in

Van

Dam

et

al.

-LRB-

-RRB-

Glucosinolate

detection

was

performed

with

a

photodiode

array

-LRB-

PDA

-RRB-

detector

-LRB-

nm

-RRB-

with

nm

as

the

integration

wavelength

Sinigrin

-LRB-

sinigrin

monohydrate

ACROS

NJ

USA

-RRB-

was

used

as

an

external

standard

We

used

the

correction

factors

at

nm

from

Buchner

-LRB-

-RRB-

and

the

EC

-LRB-

EC

-RRB-

to

calculate

the

concentrations

of

the

glucosinolates

Desulfoglucosinolate

peaks

were

identified

by

comparison

of

HPLC

retention

times

and

UV

spectra

with

standards

kindly

provided

by

M.

Reichelt

MPI

Chemical

Ecology

and

a

certified

rape

seed

standard

-LRB-

Community

Bureau

of

Reference

Brussels

code

-RRB-

The

surface

area

was

measured

directly

after

dipping

and

the

dry

mass

of

the

leaves

was

measured

after

drying

at

°C

for

hr

The

GLS

content

was

calculated

in

pmol

per

leaf

material

Herbivore

Oviposition

Preference

Test

Pieris

adults

emerged

from

pupae

in

a

large

oviposition

cage

-LRB-

cm

-RRB-

in

a

greenhouse

compartment

at

°C

and

RH

Apart

from

natural

daylight

cages

were

illuminated

by

sodium

vapor

lamps

-LRB-

type

SON-T

W

Philips

The

Netherlands

-RRB-

from

a.m.

until

p.m.

In

this

cage

they

were

provided

with

a

sucrose

solution

and

an

oviposition

substrate

depending

on

the

experiment

a

plant

or

an

artificial

leaf

made

of

green

cardboard

paper

sprayed

with

sinigrin

For

the

experiments

one

male

and

one

female

butterfly

were

introduced

per

oviposition

cage

-LRB-

cm

-RRB-

in

the

same

greenhouse

compartment

on

the

day

before

the

experiment

In

these

cages

the

butterflies

were

also

provided

with

sucrose

solution

At

a.m.

the

treated

leaves

or

papers

and

respective

controls

were

introduced

into

the

cages

and

the

butterflies

were

allowed

to

oviposit

until

the

beginning

of

the

afternoon

At

p.m.

the

leaves

were

removed

and

the

number

of

eggs

was

counted

The

experiments

were

carried

out

in

several

cages

per

day

and

d

per

treatment

with

new

pairs

of

butterflies

each

day

adding

up

to

a

total

of

independent

replicates

Surface

Application

of

JA

The

effect

of

JA-induced

changes

in

Brussels

sprouts

plants

on

butterfly

behavior

was

tested

in

oviposition

experiments

with

P.

brassicae

and

P.

rapae

Three

concentrations

of

JA

solution

and

mM

corresponding

to

approximately

and

μg

JA/g

fresh

weight

-LRB-

or

and

nmol

-RRB-

respectively

were

sprayed

on

the

plants

and

tested

against

a

control

-LRB-

plants

treated

with

Tween

-RRB-

The

next

morning

just

before

the

start

of

the

experiment

the

fourth

fifth

and

sixth

leaves

from

the

base

of

the

plants

were

cut

and

their

petioles

were

placed

directly

into

a

vial

with

tap

water

and

introduced

into

the

cages

with

butterflies

Systemic

Uptake

of

JA

For

P.

rapae

two

application

methods

were

used

to

assess

the

effect

of

JA-induced

changes

in

Brussels

sprouts

on

oviposition

preference

For

the

second

application

method

the

fourth

fifth

and

sixth

leaves

were

cut

from

untreated

plants

and

placed

in

a

aqueous

JA

solution

hr

before

the

start

of

the

experiment

Total

uptake

of

the

solution

was

on

average

ml

per

control

leaf

and

ml

for

JA-treated

leaves

-LRB-

corresponding

to

approximately

μg

JA/g

fresh

weight

or

nmol

assuming

homogeneous

distribution

over

the

leaf

tissue

after

uptake

-RRB-

Effect

of

Pure

JA

on

Oviposition

Preference

In

the

next

experiment

green

cardboard

paper

sprayed

with

an

oviposition

stimulant

was

used

to

test

the

effect

of

pure

JA

on

the

oviposition

behavior

of

P.

rapae

on

an

inert

substrate

Sinigrin

has

been

shown

to

be

a

suitable

oviposition

stimulant

for

Pieris

butterflies

-LRB-

Van

Loon

et

al.

-RRB-

and

was

therefore

used

to

stimulate

oviposition

on

the

artificial

substrate

in

this

experiment

The

paper

-LRB-

cm

-RRB-

was

treated

with

ml

of

a

sinigrin

solution

-LRB-

Janssen

Pharmaceutica

Tilburg

The

Netherlands

-RRB-

by

spraying

it

with

a

Desaga

chromatographic

sprayer

-LRB-

Heidelberg

Germany

-RRB-

Subsequently

after

drying

papers

were

sprayed

with

either

ml

of

a

JA

solution

or

water

-LRB-

control

substrates

-RRB-

just

before

the

test

-LRB-

μg

JA/carton

or

nmol

-RRB-

Bioassays

with

Purified

Glucosinolate

-LRB-

GLS

-RRB-

Fractions

GLS

were

extracted

from

the

leaf

surface

as

described

for

the

chemical

analysis

For

each

treatment

control

and

mM

JA

plants

were

used

for

the

extraction

of

which

four

to

five

leaves

per

plant

were

dipped

Subsequently

the

extracts

were

fractionated

following

the

protocol

of

Sørensen

-LRB-

Sørensen

-RRB-

The

GLS

fractions

were

stored

in

the

freezer

until

analysis

The

GLS

were

dissolved

in

methanol

to

make

two

concentrations

one

corresponding

to

the

amount

of

GLS

extracted

from

the

material

of

two

plants

in

ml

and

one

concentration

corresponding

to

the

amount

of

GLS

from

one

leaf

in

ml

Hereafter

we

will

express

these

concentrations

in

gram

leaf

equivalents

-LRB-

gle

-RRB-

One

gle

corresponds

to

the

amount

of

GLS

extracted

from

g

fresh

and

intact

leaf

With

an

average

weight

of

g

per

leaf

the

highest

concentration

corresponds

to

gle

and

the

lower

concentration

to

gle

With

a

sprayer

a

volume

of

ml

of

one

of

the

solutions

was

sprayed

on

green

paper

following

the

same

method

as

described

above

for

the

test

of

pure

JA

P.

rapae

butterflies

were

offered

a

two-choice

situation

with

one

paper

sprayed

with

GLS

extracted

from

control

plants

and

one

paper

with

GLS

from

JA-treated

plants

Performance

of

P.

rapae

Caterpillars

The

development

of

first

instar

caterpillars

to

pupae

was

observed

on

control

and

JA-treated

plants

Control

plants

were

sprayed

with

a

Tween

solution

and

JA-treated

plants

with

a

solution

of

mM

JA

with

Tween

Thirty

newly

hatched

P.

rapae

caterpillars

were

evenly

distributed

over

two

plants

per

treatment

hr

after

treatment

and

were

placed

in

cages

-LRB-

cm

-RRB-

in

a

greenhouse

compartment

at

°C

and

RH

The

plants

were

replaced

with

new

plants

twice

a

week

so

that

the

maximum

time

between

induction

and

larval

feeding

never

exceeded

d

The

number

of

days

until

pupation

and

pupal

weight

were

recorded

Statistical

Analyses

Each

individual

butterfly

female

was

subjected

to

a

two-choice

situation

in

which

most

individuals

oviposited

on

both

control

and

JA-treated

leaves

As

the

egg

load

differed

between

individuals

the

number

of

eggs

on

each

treatment

per

individual

was

treated

as

a

paired

sample

The

oviposition

data

for

P.

rapae

were

normally

distributed

therefore

they

were

analyzed

with

a

paired

t-test

The

oviposition

data

for

P.

brassicae

were

not

normally

distributed

and

therefore

analyzed

with

the

nonparametric

equivalent

of

the

paired

t-test

the

Wilcoxon

matched-pair

signed-ranks

test

The

data

on

the

developmental

time

of

the

caterpillars

in

the

performance

test

were

not

normally

distributed

and

were

analyzed

with

a

Mann

Whitney

U

test

for

differences

between

the

treatments

Pupal

weight

was

normally

distributed

and

analyzed

with

an

analysis

of

variance

-LRB-

ANOVA

-RRB-

Changes

in

GLS

content

were

analyzed

with

a

Mann

Whitney

U

test

Statistical

analyses

were

performed

with

SPSS

Results

Chemical

Analysis

Five

GLS

were

detected

in

B.

oleracea

leaf

surface

samples

glucoiberin

sinigrin

glucobrassicin

and

-LRB-

Table

-RRB-

No

significant

difference

was

detected

between

JA-treated

and

control

leaves

for

the

total

amount

of

GLS

per

The

amounts

of

glucobrassicin

the

most

abundant

glucosinolate

in

these

samples

and

sinigrin

did

not

significantly

differ

between

control

and

JA-treated

leaves

The

amounts

of

glucoiberin

and

collected

in

the

leaf

surface

extracts

were

significantly

lower

for

JA-treated

leaves

compared

to

control

leaves

-LRB-

Table

-RRB-

The

same

results

were

obtained

when

calculated

for

the

GLS

content

expressed

as

nmol

per

mg

dry

weight

-LRB-

not

shown

-RRB-

Table

content

in

surface

extracts

of

Brassica

oleracea

leaves

in

for

control

and

JA-treated

plantsCompoundControl

Treatment

Median

-LRB-

range

-RRB-

a

bJA

Treatment

Median

-LRB-

range

-RRB-

a

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Total

amount

of

-LRB-

-RRB-

-LRB-

-RRB-

aInterquartile

range

from

first

to

third

quartilebN

Herbivore

Oviposition

Preference

Surface

Application

of

JA

As

P.

brassicae

lays

its

eggs

in

batches

both

the

number

of

egg

batches

and

the

number

of

eggs

per

leaf

were

counted

For

the

mM

JA

treatment

the

number

of

batches

was

significantly

lower

on

JA-treated

leaves

than

on

control

leaves

-LRB-

N

Z

P

Wilcoxon

matched-pair

signed-ranks

test

-RRB-

and

the

total

number

of

eggs

was

significantly

lower

as

well

-LRB-

N

Z

P

Wilcoxon

matched-pair

signed-ranks

test

-RRB-

For

the

JA

treatment

the

result

was

similar

-LRB-

N

batches

Z

P

eggs

Z

P

Wilcoxon

matched-pair

signed-ranks

test

-RRB-

-LRB-

Fig.

-RRB-

Experiments

with

mM

JA

application

did

not

show

discrimination

by

the

butterflies

between

the

treated

and

control

leaves

-LRB-

results

not

shown

-RRB-

Also

P.

rapae

butterflies

significantly

preferred

to

oviposit

on

control

leaves

compared

to

JA-treated

leaves

-LRB-

Fig.

-RRB-

The

leaves

treated

with

the

two

highest

concentrations

of

JA

tested

and

mM

were

avoided

in

favor

of

the

control

leaves

-LRB-

paired

t-test

respectively

t

df

P

and

t

df

P

-RRB-

The

lowest

concentration

of

JA

tested

i.e.

mM

did

not

affect

the

distribution

of

eggs

over

the

leaves

-LRB-

t

df

P

paired

t-test

-RRB-

Fig.

brassicae

oviposition

on

control

and

JA-treated

plants

Two

concentrations

of

JA

were

tested

against

a

control

The

box

represents

the

interquartile

range

from

first

to

third

quartile

the

line

across

the

box

indicates

the

median

asterisks

indicate

statistical

differences

between

the

preference

for

control

and

JA-treated

plants

-LRB-

P

P

Wilcoxon

matched-pair

signed-ranks

test

-RRB-

a

Egg

batches

per

female

per

leaf

b

Eggs

per

female

per

leafFig

rapae

oviposition

preference

-LRB-

measured

as

the

number

of

eggs

per

female

per

leaf

-RRB-

between

control

and

JA-treated

B.

oleracea

Three

concentrations

of

JA

were

tested

against

a

control

in

replicated

experiments

for

each

concentration

Mean

numbers

of

eggs

per

female

SEM

are

given

asterisks

indicate

statistical

differences

between

the

preference

for

control

and

JA-treated

plants

-LRB-

n.s.

P

P

paired

t-test

-RRB-

Systemic

Uptake

of

JA

In

the

experiment

with

P.

rapae

that

employed

systemic

uptake

of

JA

through

the

petiole

the

same

result

was

obtained

the

number

of

eggs

on

the

JA-treated

leaves

-LRB-

-RRB-

was

lower

than

on

the

control

-LRB-

-RRB-

leaves

-LRB-

t

df

P

paired

t-test

-RRB-

Effect

of

Pure

JA

on

Oviposition

Preference

When

paper

treated

with

sinigrin

and

JA

was

compared

to

paper

with

only

sinigrin

there

was

no

difference

in

the

number

of

eggs

the

butterflies

deposited

on

the

two

substrates

-LRB-

t

df

P

paired

t-test

-RRB-

-LRB-

Fig.

-RRB-

These

results

show

that

the

observed

effect

of

the

JA

treatment

on

herbivore

oviposition

behavior

was

due

to

induced

changes

in

leaf

tissue

rather

than

to

a

direct

repellent

or

deterrent

effect

of

JA

itself

Fig.

of

P.

rapae

on

green

paper

sprayed

with

sinigrin

plus

JA

and

green

paper

with

sinigrin

only

Mean

number

of

eggs

per

female

per

paper

leaf

SEM

-LRB-

n.s.

P

paired

t-test

N

-RRB-

Bioassays

with

Purified

Glucosinolate

Fractions

The

butterflies

did

not

discriminate

between

the

two

GLS

fractions

-LRB-

Table

-RRB-

the

number

of

eggs

on

paper

with

the

GLS

from

control

plants

and

the

number

of

eggs

on

paper

with

GLS

from

JA-treated

plants

was

not

different

for

either

concentration

-LRB-

concentration

gle

Z

N

P

Wilcoxon

matched-pair

signed-ranks

test

gle

t

df

P

paired

t-test

-RRB-

-LRB-

Fig.

-RRB-

Table

-LRB-

GLS

-RRB-

content

in

fractions

from

leaf

surface

extracts

from

control

and

JA-treated

plants

in

Z

and

P-values

of

Mann

Whitney

U

testGlucosinolateGLS

from

control

plantGLS

from

JA-treated

plantGlucoiberin4.93.9Sinigrin3.15.14-HydroxyglucobrassicinNot

detectedNot

detectedGlucobrassicin16.081.04-MethoxyglucobrassicinNot

Fig.

of

P.

rapae

on

green

paper

sprayed

with

purified

GLS-fractions

from

leaf

surface

extracts

of

control

and

JA-treated

plants

Mean

number

of

eggs

per

female

per

paper

leaf

SEM

-LRB-

n.s.

P

paired

t-test

N

-RRB-

Performance

of

P.

rapae

Caterpillars

About

two-thirds

of

the

caterpillars

survived

until

pupation

and

a

similar

number

of

caterpillars

reached

the

pupal

stage

on

both

treatments

on

control

and

on

JA-treated

plants

-LRB-

Fig.

-RRB-

The

caterpillars

on

JA-treated

plants

pupated

on

average

after

d

while

the

caterpillars

on

the

control

plants

pupated

significantly

sooner

on

average

after

d

-LRB-

Mann

Whitney

U

Z

P

-RRB-

The

average

pupal

weight

on

the

control

plants

mg

was

similar

to

that

on

the

JA-treated

plants

mg

-LRB-

ANOVA

F

df

P

-RRB-

Fig.

time

of

P.

rapae

caterpillars

from

hatching

until

pupation

on

control

-LSB-

open

squares

-LRB-

-RRB-

-RSB-

and

JA-treated

-LSB-

closed

triangles

-LRB-

-RRB-

-RSB-

plants

Cumulative

number

of

pupae

per

treatment

Discussion

Our

data

show

that

JA

treatment

of

Brussels

sprouts

leaves

reduces

the

acceptance

of

leaves

for

oviposition

by

P.

rapae

and

P.

brassicae

in

a

similar

way

Treatment

of

leaves

with

or

mM

JA

reduced

the

proportion

of

eggs

the

butterflies

laid

on

these

leaves

A

concentration

of

mM

JA

did

not

change

oviposition

preference

The

former

concentrations

are

comparable

to

the

concentrations

of

JA

or

MeJA

that

were

applied

to

several

plant

species

in

other

studies

and

that

reduced

development

of

Spodoptera

exigua

Trichoplusia

ni

Manduca

sexta

thrips

and

aphids

-LRB-

Thaler

et

al.

Avdiushko

et

al.

Van

Dam

et

al.

Omer

et

al.

-RRB-

and

abundance

of

Manduca

quinquemaculata

S.

exigua

thrips

and

flea

beetles

in

the

field

-LRB-

Kessler

and

Baldwin

Thaler

et

al.

-RRB-

For

cabbage

plants

Lu

et

al.

-LRB-

-RRB-

found

inducible

resistance

in

a

susceptible

Brassica

species

-LRB-

Chinese

cabbage

Brassica

campestris

L.

-RRB-

and

induced

susceptibility

in

a

resistant

Brassica

species

-LRB-

common

cabbage

B.

oleracea

-RRB-

for

Plutella

xylostella

L.

To

exclude

that

JA

itself

caused

the

above

effect

we

tested

the

phytohormone

on

an

inert

substrate

and

studied

two

different

application

methods

to

the

leaf

material

We

considered

this

an

essential

control

that

is

lacking

in

other

studies

The

results

of

these

experiments

show

that

it

was

not

JA

itself

that

caused

the

difference

in

oviposition

preference

between

control

and

JA-treated

leaves

thus

providing

proof

that

processes

in

the

plant

induced

by

the

JA

treatment

changed

the

acceptability

of

the

leaves

It

has

also

been

reported

previously

that

development

of

cabbage

looper

or

tobacco

hornworm

larvae

is

not

affected

when

MeJA

is

added

to

an

artificial

diet

but

it

is

retarded

when

MeJA

was

applied

to

cabbage

or

tobacco

plants

-LRB-

Avdiushko

et

al.

-RRB-

In

leaf

surface

extracts

of

JA-treated

and

untreated

Brussels

sprouts

we

found

five

glucosinolates

After

JA

application

glucobrassicin

the

major

glucosinolate

in

the

B.

oleracea

cultivar

we

used

occurred

at

a

level

twice

as

high

as

in

control

plants

and

glucoiberin

and

concentrations

decreased

The

total

glucosinolate

content

did

not

change

significantly

However

most

other

studies

on

glucosinolate

content

in

Brassicaceous

plants

after

induction

by

JA

or

MeJA-treatment

or

insect

attack

have

reported

an

increase

in

glucosinolates

although

there

is

substantial

variation

among

different

species

or

even

genotypes

and

type

of

induction

-LRB-

Bodnaryk

Cipollini

and

Sipe

Mikkelsen

et

al.

Mewis

et

al.

-RRB-

Moreover

glucosinolates

may

not

be

evenly

distributed

throughout

the

leaf

We

measured

glucosinolate

content

in

a

surface

extract

after

hr

whereas

most

studies

have

measured

glucosinolate

content

in

whole

leaf

extracts

and

after

a

longer

induction

time

Recently

Reifenrath

et

al.

-LRB-

-RRB-

postulated

that

the

wax

layer

does

not

contain

glucosinolates

and

the

polar

glucosinolates

that

are

found

by

using

the

solvent

extraction

method

are

washed

from

the

inner

leaf

to

the

outside

through

the

stomata

Nevertheless

we

chose

a

surface

extraction

method

because

the

butterflies

retrieve

chemosensory

information

from

the

leaf

surface

as

they

do

not

damage

the

leaf

before

ovipositing

Surface

extracts

are

thus

likely

to

give

a

better

reflection

of

the

chemosensory

information

used

than

whole

leaf

extracts

Both

butterfly

species

distinguish

between

induced

and

non-induced

leaves

most

likely

based

on

chemical

differences

as

JA-induced

leaves

do

not

display

herbivore

presence

or

damage

The

different

levels

of

two

out

of

five

glucosinolates

in

the

surface

extracts

may

provide

a

chemosensory

basis

for

the

oviposition

preference

observed

although

the

isolated

GLS

from

the

two

treatments

yielded

no

differences

in

acceptance

of

the

paper

for

oviposition

While

the

isolated

GLS

on

paper

stimulated

oviposition

behavior

they

appear

not

to

be

the

main

cue

to

discriminate

between

the

JA-induced

and

non-induced

cabbage

plants

We

did

not

quantify

other

chemicals

stimulants

deterrents

or

precursors

that

might

mediate

preference

behavior

such

as

isothiocyanates

terpenoids

other

glycosides

or

amino

acids

-LRB-

Huang

et

al.

Renwick

and

Chew

Soldaat

et

al.

Agrawal

and

Kurashige

-RRB-

Both

P.

rapae

and

P.

brassicae

can

perceive

a

broad

range

of

chemicals

-LRB-

Van

Loon

et

al.

Hern

et

al.

-RRB-

Electroantennogram

responses

to

a

range

of

plant

volatiles

are

similar

for

both

species

-LRB-

Van

Loon

et

al.

-RRB-

although

host

plant

selection

by

Pieris

butterflies

appears

largely

based

on

contact

chemoreception

rather

than

olfaction

-LRB-

Renwick

and

Chew

-RRB-

Host

plant

selection

is

suggested

to

depend

on

a

balance

of

stimulants

and

deterrents

and

not

just

on

the

detection

of

presence

or

absence

of

particular

compounds

-LRB-

Huang

et

al.

Bruce

et

al.

-RRB-

Therefore

glucosinolates

in

combination

with

other

stimulants

or

deterrents

may

determine

the

acceptance

of

a

host

plant

by

the

butterflies

The

octadecanoid

pathway

in

which

JA

is

a

key

molecule

is

involved

in

induction

of

synomones

in

response

to

oviposition

and

to

herbivore

damage

-LRB-

Meiners

and

Hilker

Dicke

and

Van

Poecke

Hilker

and

Meiners

Mumm

and

Hilker

-RRB-

JA

treatment

of

plants

results

in

emission

of

synomones

that

attract

natural

enemies

like

predatory

mites

and

parasitoids

-LRB-

Dicke

et

al.

Hilker

and

Meiners

Van

Poecke

and

Dicke

Hilker

and

Meiners

-RRB-

This

attraction

results

in

a

higher

natural

enemy

density

around

damaged

plants

and

therefore

it

is

advantageous

for

the

herbivores

to

avoid

oviposition

on

induced

plants

Moreover

intact

plants

lack

competitors

either

intra

or

interspecific

Herbivores

may

use

induced

plant

cues

to

detect

the

presence

or

absence

of

other

herbivores

especially

because

plant

cues

are

although

less

reliable

often

easier

to

detect

than

cues

from

the

herbivores

themselves

-LRB-

Vet

and

Dicke

-RRB-

Furthermore

induced

plants

may

affect

herbivores

directly

by

influencing

the

performance

of

their

offspring

For

P.

rapae

the

development

time

differed

between

caterpillars

feeding

on

JA-induced

and

caterpillars

feeding

on

non-induced

plants

Development

of

the

caterpillars

to

pupae

took

longer

on

the

induced

plants

which

exposes

them

to

natural

enemies

for

a

longer

time

and

gives

them

a

disadvantage

in

the

competition

for

resources

with

other

herbivores

These

results

comply

with

those

of

Agrawal

and

Kurashige

-LRB-

-RRB-

who

showed

that

growth

of

P.

rapae

larvae

was

reduced

on

herbivore-induced

Brassicaceae

In

summary

-LRB-

-RRB-

JA

treatment

of

B.

oleracea

results

in

avoidance

of

host

plants

by

the

two

Pieris

butterflies

-LRB-

-RRB-

the

related

gregarious

and

solitary

butterfly

species

tested

responded

in

a

similar

fashion

to

JA-treated

plants

-LRB-

-RRB-

JA

treatment

reduced

the

contents

of

two

out

of

five

glucosinolates

in

leaf

surface

extracts

of

Brussels

sprouts

and

-LRB-

-RRB-

the

purified

GLS

fractions

could

not

explain

the

observed

avoidance

behavior

The

results

indicate

that

JA-induced

infochemicals

play

an

important

role

in

host

plant

selection

behavior

of

these

butterflies

however

the

phytochemicals

involved

still

have

to

be

elucidated

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

host plant selection
glucosinolates
pieris rapae
pieris brassicae
cabbage
performance
octadecanoid pathway


Risk of Fecal Diversion in Complicated Perianal Crohn 's Disease The purpose of the study was to determine the overall risk of a permanent stoma in patients with complicated perianal Crohn 's disease , and to identify risk factors predicting stoma carriage . A total of 102 consecutive patients presented with the first manifestation of complicated perianal Crohn 's disease in our outpatient department between 1992 and 1995 . Ninety-seven patients -LRB- 95 % -RRB- could be followed up at a median of 16 years after first diagnosis of Crohn 's disease . Patients were sent a standardized questionnaire and patient charts were reviewed with respect to the recurrence of perianal abscesses or fistulas and surgical treatment , including fecal diversion . Factors predictive of permanent stoma carriage were determined by univariate and multivariate analysis . Thirty of 97 patients -LRB- 31 % -RRB- with complicated perianal Crohn 's disease eventually required a permanent stoma . The median time from first diagnosis of Crohn 's disease to permanent fecal diversion was 8.5 years -LRB- range 0 -- 23 years -RRB- . Temporary fecal diversion became necessary in 51 of 97 patients -LRB- 53 % -RRB- , but could be successfully removed in 24 of 51 patients -LRB- 47 % -RRB- . Increased rates of permanent fecal diversion were observed in 54 % of patients with complex perianal fistulas and in 54 % of patients with rectovaginal fistulas , as well as in patients that had undergone subtotal colon resection -LRB- 60 % -RRB- , left-sided colon resection -LRB- 83 % -RRB- , or rectal resection -LRB- 92 % -RRB- . An increased risk for permanent stoma carriage was identified by multivariate analysis for complex perianal fistulas -LRB- odds ratio -LSB- OR -RSB- 5 ; 95 % confidence interval -LSB- CI -RSB- 2 -- 18 -RRB- , temporary fecal diversion -LRB- OR 8 ; 95 % CI 2 -- 35 -RRB- , fecal incontinence -LRB- OR 21 , 95 % CI 3 -- 165 -RRB- , or rectal resection -LRB- OR 30 ; 95 % CI 3 -- 179 -RRB- . Local drainage , setons , and temporary stoma for deep and complicated fistulas in Crohn 's disease , followed by a rectal advancement flap , may result in closing of the stoma in 47 % of the time . The risk of permanent fecal diversion was substantial in patients with complicated perianal Crohn 's disease , with patients requiring a colorectal resection or suffering from fecal incontinence carrying a particularly high risk for permanent fecal diversion . In contrast , patients with perianal Crohn 's disease who required surgery for small bowel disease or a segmental colon resection carried no risk of a permanent stoma . Introduction Crohn 's disease was initially described as a nonspecific inflammatory bowel disease , affecting mainly the terminal ileum and characterized by a subacute or chronic necrotizing and cicatrizing inflammation .1 Eventually , gastrointestinal Crohn 's disease became recognized as a full-thickness disease of the gastrointestinal wall that may affect the entire gastrointestinal tract , including the perianal region .2 -- 6 Perianal lesions are common in patients with Crohn 's disease .7 -- 13 Clinical manifestations vary from asymptomatic skin tags to severe , debilitating perianal destruction and sepsis . Asymptomatic perianal lesions require no treatment , but because they become painful and disabling , they may require surgical treatment . Surgical management needs to be conservative and should focus on the drainage of septic sites , preserving sphincter function and palliating symptoms .10 -- 13 Medical management has had some success in improving symptoms , but as yet , it has not been able to eliminate most perianal complaints permanently .14,15 Fecal diversion was successfully used to achieve remission in colonic Crohn 's disease . Moreover , it was utilized to allow severe perianal disease to settle , thereby avoiding proctectomy .16 -- 19 However , restoring the intestinal passage carries the risk of recurrent perianal disease activity , possibly resulting in a decreased quality of life compared to the situation with fecal diversion . We investigated the overall risk of a permanent stoma in patients with severe perianal Crohn 's disease and tried to identify risk factors predicting permanent stoma carriage . Methods Patients A total of 102 consecutive patients with Crohn 's disease presented with the first manifestation of a perianal fistula or a perianal abscess in our outpatient department between 1992 and 1995 . Patients were investigated in Trendelenburg 's position by perianal inspection , proctoscopy , rectoscopy , and rigid sigmoidoscopy . Endoanal ultrasound was performed in case of suspected perianal abscess formation , and MRI was conducted of the pelvic floor in case of complicated fistulizing disease or intrapelvic abscess formation . All patients were documented prospectively . Follow-up data of 97 patients -LRB- 95 % -RRB- were available by a standardized questionnaire mailed to the patients and by a standardized chart review . There were 50 female and 47 male patients -LRB- ratio 1.06:1 -RRB- with a median age of 23 years -LRB- range 8 -- 51 years -RRB- . Patients were evaluated with respect to the recurrence of perianal abscesses , fistulas , or surgical treatment of Crohn 's disease over the years . The median interval between the first diagnosis of Crohn 's disease and last follow-up was 16 years -LRB- range 8 -- 37 years -RRB- . Four patients had isolated small intestinal disease , 11 patients had isolated colonic disease , and 82 patients had small intestinal and colonic disease . The abscess location was categorized as subcutaneous , intersphincteric , deep perianal , ischiorectal , and above the pelvic floor . Abscess formations were categorized into simple -LRB- subcutaneous , intersphincteric , deep perianal , and ischiorectal , circular extension less than 90 ° , pelvic floor not involved -RRB- and complicated -LRB- circular extension more than 90 ° -LSB- horse shoe abscess -RSB- or pelvic floor involved -RRB- . Fistulas were classified according to Parks et al. 20 into subcutaneous , intersphincteric , extrasphincteric , transsphincteric , rectovaginal , and suprasphincteric , as described previously .21 We divided fistulas into simple fistulas -LRB- no more than two perianal openings -RRB- and complex fistulas -LRB- rectovaginal , three or more perianal openings -RRB- . A variety of factors , such as sex of the patient , perianal fistula , rectovaginal fistula , abscess formation , anal stricture , fecal incontinence , or abdominal surgery were evaluated with regard to their predictive character for permanent stoma carriage by univariate and multivariate analysis . Statistical Analysis Kaplan -- Meier analysis using a log-rank test was used for comparing risk rates over time . Factors that might influence permanent fecal diversion were analyzed using the chi-square test . Fisher 's exact test was used for univariate analysis and multiple logistic regression and Wald 's test for multivariate analysis . Subgroup analysis was performed for rectovaginal fistulas , as present in female patients only . Data are given as numbers of cases and percentages or median and interquartile ranges . A P < 0.05 was considered as significant . Results Overall Risk of Permanent Fecal Diversion Thirty of 97 patients -LRB- 31 % -RRB- with perianal Crohn 's disease eventually required a permanent stoma . Nineteen patients were female and 11 male -LRB- ratio 1.73:1 -RRB- . The median time from first diagnosis of Crohn 's disease to permanent fecal diversion was 8.5 years -LRB- range 0 -- 23 years ; Fig. 1 -RRB- . Increased rates of permanent fecal diversion were observed in 53 % of patients with previous temporary fecal diversion , in 54 % of patients with complex perianal fistulas , in 54 % of female patients with rectovaginal fistulas , and in 83 % of patients with fecal incontinence -LRB- Table 1 -RRB- . Patients who required subtotal colon resection -LRB- 60 % -RRB- , left-sided colon resection -LRB- 83 % -RRB- , or rectal resection -LRB- 93 % -RRB- needed a permanent stoma at high rates -LRB- Table 2 -RRB- . Figure 1Relative proportion of patients without permanent stoma in complicated perianal Crohn 's disease -LRB- n = 97 -RRB- during follow-up . Table 1Patients with First Manifestation of Complicated Perianal Crohn 's Disease -LRB- n = 97 -RRB- and the Rate of Fecal Diversion During Follow-up PatientsTemporary StomaPermanent StomaOverall9751 -LRB- 53 % -RRB- 30 -LRB- 31 % -RRB- Abscess formation7532 -LRB- 43 % -RRB- 21 -LRB- 28 % -RRB- Simple abscess formation33 -LRB- 44 % -RRB- 13 -LRB- 39 % -RRB- 7 -LRB- 21 % -RRB- Complex abscess formation42 -LRB- 56 % -RRB- 23 -LRB- 55 % -RRB- 15 -LRB- 36 % -RRB- Fistulas8851 -LRB- 58 % -RRB- 26 -LRB- 30 % -RRB- Simple perianal fistulas42 -LRB- 48 % -RRB- 17 -LRB- 40 % -RRB- 8 -LRB- 19 % -RRB- Complex perianal fistulas46 -LRB- 52 % -RRB- 34 -LRB- 74 % -RRB- 23 -LRB- 50 % -RRB- Rectovaginal fistulas26 -LRB- 54 % of ♀ -RRB- 18 -LRB- 69 % of 26 -RRB- 14 -LRB- 54 % of 26 -RRB- Overlap between abscess formation and presence of fistulas exists , and abscesses led to fecal diversion in combination with fistulas only.Table 2Abdominal Procedures and the Rate of Fecal Diversion During Follow-up in Patients with Complicated Perianal Crohn 's Disease -LRB- n = 97 -RRB- Patient NumberPermanent Stoma -LRB- % of Patients -RRB- Small Bowel ProceduresStricturplasty70Small bowel resection because of stenosis230Small bowel resection because of enteroenteric fistula70Anastomosis resection because of inflammatorystenosis300Stoma revision40Large bowel proceduresSegmental colon resection140Right-sided colon resection204 -LRB- 20 -RRB- Ileocecal resection6824 -LRB- 35 -RRB- Subtotal colon resection3521 -LRB- 60 -RRB- Left-sided colon resection65 -LRB- 83 -RRB- Rectal resection1312 -LRB- 92 -RRB- Patients that required small bowel resections carried no risk of fecal diversion , whereas patients with colon resections carried an increased risk of fecal diversion . Abscess Formation Seventy-five of 97 patients -LRB- 77 % -RRB- had at least one perianal abscess at first presentation or during follow-up . Surgical therapy for abscesses consisted of seton drainage , mushroom catheter drainage , or incision and drainage , as described previously .21 A permanent fecal diversion because of recurrent abscess formations , always in combination with fistula problems and perianal sepsis , became necessary in 21 of 75 cases -LRB- 28 % -RRB- . Simple abscess formations occurred in 33 patients , and 7 of those patients -LRB- 21 % -RRB- required permanent fecal diversion -LRB- Fig. 2 -RRB- . Complex abscess formations were present in 42 patients , with permanent fecal diversion being necessary in 14 cases -LRB- 33 % ; Table 1 -RRB- . Figure 2Relative proportion of patients without permanent stoma in complicated perianal Crohn 's disease with simple -LRB- n = 42 -RRB- and complex -LRB- n = 46 -RRB- fistulas during follow-up . The presence of a complex fistula significantly increased the risk of permanent fecal diversion -LRB- P < 0.001 -RRB- . Perianal and Rectovaginal Fistulas Perianal fistulas affected 88 of 97 patients -LRB- 91 % -RRB- , including 26 female patients with rectovaginal fistulas . Symptomatic fistulas with abscess formation were treated by simple drainage procedures and provided with a seton . Azathioprine therapy was tried if purulent secretion persisted . Infliximab was rarely used in recent years at the discretion of the referring gastroenterologist . Overall , permanent fecal diversion became necessary in 26 of 88 patients -LRB- 30 % -RRB- . Forty-two patients -LRB- 48 % -RRB- had simple fistulas , whereas 46 patients -LRB- 52 % -RRB- had complex fistulas . Of these , permanent fecal diversion was documented in 14 patients -LRB- 54 % -RRB- , whereas simple fistulas eventually required a permanent stoma in only 8 cases -LRB- 16 % -RRB- . Twenty-six of 48 female patients had rectovaginal fistulas , of which 14 -LRB- 54 % -RRB- eventually required permanent fecal diversion -LRB- Table 1 -RRB- . Thirty-four of 97 patients -LRB- 35 % -RRB- developed anal strictures , whereas fecal incontinence was documented in 12 patients -LRB- 12 % -RRB- during the follow-up . In 14 of 34 patients -LRB- 41 % -RRB- with anal stricture , a permanent fecal diversion became necessary , whereas 10 of 12 patients -LRB- 83 % -RRB- suffering from fecal incontinence required a permanent stoma . Abdominal Surgery Eighty-three of 97 patients with perianal Crohn 's disease -LRB- 86 % -RRB- underwent abdominal surgery at least once during follow-up because of Crohn 's disease activity , with a total of 227 abdominal procedures being performed . In 68 of 227 operations -LRB- 29 % -RRB- , intestinal stenosis required surgery . Segmental colon resection was performed 108 times -LRB- 46 % -RRB- , whereas subtotal colectomy for fulminant colitis was performed 35 times -LRB- 15 % -RRB- . One patient required surgery because of a fistula carcinoma . Patients with complicated perianal Crohn 's disease who required a small bowel resection carried no risk of permanent fecal diversion , whereas the majority of patients with left-sided colon resection , subtotal colon resection , or rectal resection needed permanent fecal diversion -LRB- Table 2 -RRB- . Patients that needed abdominal surgery repeatedly carried an increased risk for a permanent stoma -LRB- permanent stoma rate : three or more abdominal operations , n = 20 , 50 % ; less than two abdominal operations , n = 20 , 15 % ; P < 0.043 -RRB- . Patients with permanent fecal diversion had undergone previous abdominal surgery three times as often as those patients without -LRB- P < 0.05 ; Table 3 -RRB- . Eventually , 17 patients -LRB- 18 % -RRB- needed proctectomy , of which 13 were female patients with rectovaginal fistulas . In 14 patients with a permanent stoma , a rectal stump remained in place and was controlled at yearly intervals . Patients with complicated perianal Crohn 's disease and large bowel resection carried a significantly increased risk of permanent fecal diversion compared to patients with complicated perianal Crohn 's disease and small bowel resection -LRB- 48 vs 0 % , P < 0.001 -RRB- -LRB- Fig. 3 -RRB- . Table 3Abdominal Procedures per Patient and Percentage of Patients with Abdominal SurgeryAbdominal ProceduresMedian -LRB- 25 -- 75 % -RRB- Patients with Abdominal Surgery -LRB- % -RRB- P ValuePermanent fecal diversion3 -LRB- 2 -- 4 -RRB- 100P < 0.05 Without permanent fecal diversion1 -LRB- 1 -- 3 -RRB- Temporary fecal diversion3 -LRB- 2 -- 4.25 -RRB- 80P < 0.05 Without temporary fecal diversion1 -LRB- 0 -- 2.5 -RRB- Anal stricture2 -LRB- 1 -- 4 -RRB- 60n . s.Without anal stricture2 -LRB- 1 -- 3 -RRB- Incontinence2 -LRB- 2 -- 4.5 -RRB- 10n . s.Without incontinence2 -LRB- 1 -- 3 -RRB- Patients with Crohn 's disease and complicated perianal fistulas who required temporary or permanent stoma had more abdominal procedures than patients who did not need a stoma , whereas patients with anal stricture or fecal incontinence had no increased abdominal procedure rate -LRB- the number of abdominal procedures per patient is given as median with interquartile ranges -RRB- . Abdominal surgery and fecal diversion correlated on univariate analysis -LRB- P < 0.05 -RRB- . Figure 3Treatment algorithm for patients with complicated perianal Crohn 's disease . Temporary Fecal Diversion Temporary fecal diversion , done mostly by loop ileostomy , became necessary in 51 of 97 patients -LRB- 53 % -RRB- . Increased rates of temporary fecal diversion were observed in 55 % of patients with complex abscess formations , including those with severe perianal sepsis , in 77 % of patients with complex perianal fistulas , and in 69 % of female patients with rectovaginal fistulas . In 24 of 51 patients -LRB- 47 % -RRB- the temporary stoma could be removed after perianal disease had subsided . Fistulas were closed in these patients by rectal advancement flaps ,23 whereas cutting setons , fistulectomy , or infliximab infusions were not employed in these patients . Risk Factors Predictive of a Permanent Fecal Diversion Univariate Analysis Complex perianal fistulas -LRB- P < 0.04 -RRB- , fecal incontinence -LRB- P < 0.001 -RRB- , and rectovaginal fistulas in female patients -LRB- P < 0.001 -RRB- carried an increased risk for a permanent stoma . Patients with high rates of abdominal surgery had a significantly increased risk for fecal diversion , whereas the frequency of abdominal surgery was not influenced by the presence of anal stricture or fecal incontinence -LRB- Table 3 -RRB- . The need for subtotal colon resection -LRB- P < 0.001 -RRB- , rectal resection -LRB- P < 0.001 -RRB- , or temporary fecal diversion -LRB- P = 0.001 -RRB- also resulted in an increased risk of permanent fecal diversion . A variety of factors did not increase the risk of a permanent stoma , such as sex of the patient , anal stenosis , perianal abscesses , and abdominal surgery , excluding subtotal colon and rectal resection -LRB- Table 4 -RRB- . Table 4Risk Factors for Permanent Fecal Diversion by Univariate and Multivariate Analysis in Patients with Complicated Perianal Crohn 's DiseaseRisk FactorsUnivariate AnalysisMultivariate AnalysisPermanent Fecal Diversion -LRB- % -RRB- P ValueOR95 % CIP ValueRectal resection92P < 0.001305 -- 179P < 0.002 Fecal incontinence83P < 0.001213 -- 165P < 0.02 Subtotal colectomy60P < 0.001 Rectovaginal fistulas54P < 0.001 Temporary fecal diversion51p = 0.00182 -- 35P < 0.02 Complex perianal fistulas54P < 0.0452 -- 18P < 0.03 Multivariate Analysis According to multivariate analysis , complex perianal fistulas -LRB- P < 0.03 -RRB- , fecal incontinence -LRB- P < 0.02 -RRB- , temporary fecal diversion -LRB- P < 0.02 -RRB- , or rectal resection -LRB- P < 0.002 -RRB- still carried a significantly increased risk for permanent fecal diversion -LRB- Table 4 -RRB- . Discussion Perianal disease is a feature of Crohn 's disease that afflicts approximately one third of patients , but prevalence rates vary between 8 and 90 % .19 Patients with colonic involvement will have perianal disease in more than 50 % of cases , whereas patients with small bowel disease are affected in less than 20 % .24 Perianal Crohn 's disease can present as minor lesions , such as skin tags , skin excoriations , and fissures , which rarely need surgical treatment . In contrast , perianal fistulas often result in abscesses or perianal sepsis , requiring urgent surgery , and fistulas and abscesses are considered as suppurative complications of perianal Crohn 's disease .25 We conducted surgery as conservatively as possible because sphincter function should be preserved as much as possible .19,22,23 Throughout the study period , 77 % of patients developed abscess formations , which is considerably more than the 48 % rate reported previously with a shorter mean follow-up of 32 months .22 The incidence of perianal fistulas in Crohn 's disease is somewhat lower , affecting 10 -- 34 % of patients .26 Healing rates of 60 -- 70 % in patients with Crohn 's disease and perianal fistulas were reported in earlier series .21,26 -- 28 However , these series contained a large proportion of simple fistulas , whereas most of our patients had complex transsphincteric or rectovaginal fistulas and fistulas that did not heal through conservative treatment . Perianal fistulas that resulted in abscess formation were drained surgically , and in case of massive purulent secretion or perianal sepsis , oral antibiotics were used additionally . Setons were put into place to prevent recurrent abscess formation , a treatment that is well known as a possible means for controlling perianal sepsis and preventing recurrent abscess formation .19,21 -- 23,29 We did not use cutting setons nor fistulotomy for transsphincteric fistulas , as fecal incontinence may succeed .19,22,23 Infliximab was successfully shown to reduce fistula secretion , and outer fistula openings may heal .30 However , fistula tracks persist with varying degrees of residual inflammation , which may cause recurrent fistulas and perianal abscesses .31 Rectal advancement flaps can be used to close the inner opening of transsphincteric or rectovaginal fistulas when perianal disease has abated and the rectal mucosa is not inflamed . However , fistulas eventually recurred in about one third of patients after a mean follow-up of 19 months , and some patients developed new fistulas .23 Hyman32 reported an initial 79 % healing rate in a series of 14 patients with Crohn 's disease and perianal fistulas , but observed longer term success in only 50 % of patients . Our current approach is to use rectal advancement flaps for transsphincteric fistulas while delaying surgery if there is an acute flare of Crohn 's disease , in particular , if proctitis is present .23,33 Perianal fistulas in Crohn 's disease closed by a rectal advancement flap have a recurrence rate of 50 -- 60 % , but can be attempted repeatedly .23,34 If a second rectal advancement flap fails , the failure rate increases up to 75 % ,34 and a temporary stoma is suggested to the patient before a third attempt is made . By this approach , we were able to heal perianal fistulas in 24 out of 51 patients -LRB- 47 % -RRB- who required a temporary stoma . In a previous study , diversion was the most powerful factor influencing healing according to multiple regression analysis .23 The chances of a symptomatic fistula recurrence were increased without a stoma -LRB- 52 % vs 14 % , 21 -RRB- , and patients with Crohn 's colitis carried an increased risk of fistula recurrence .23 A particular problem is rectovaginal fistulas , which almost always open at the dentate line .19 They occur in 3 -- 10 % of patients with Crohn 's disease .19 In our series , 54 % of female patients had rectovaginal fistulas , indicating that a large patient proportion had complex perianal fistulas ; referral bias might have contributed to this high proportion of rectovaginal fistulas , as our outpatient clinic for Crohn 's disease is well known regionally . Rectovaginal fistulas carry a poor prognosis ,18 and a 70 % recurrence rate was observed 24 months after a rectal advancement flap was performed .23 In the experience of Keighley et al. 35 , 11 of 13 patients with rectovaginal fistulas required proctectomy with a permanent stoma . In our series , 8 out of 26 rectovaginal fistulas healed by a rectal advancement flap . Eighteen patients required a temporary stoma , of which four could be removed after a rectal advancement flap was tried again successfully -LRB- overall healing rate 12 of 26 , 46 % -RRB- , resulting in a permanent stoma rate of 14 of 26 patients -LRB- 54 % -RRB- . Healing rates of rectovaginal fistulas associated with Crohn 's disease vary widely , and low patient numbers are usually reported .18 In the Cleveland clinic , 16 of 37 rectovaginal fistulas -LRB- 43 % -RRB- healed using an endorectal advancement flap , but patients not having Crohn 's disease were included in the study .36 In low anovaginal fistulas , higher healing rates of up to 68 % were reported in patients with Crohn 's disease .37 Recurrent rectovaginal fistulas were shown to heal after repeated rectal advancement flap procedures were conducted at about the same rate even in Crohn 's disease , but surgery should be delayed for at least 3 months after a previous repair ,38 which is standard policy at our clinic . It is interesting to note that Halverson et al. 38 reported that the presence of a diverting stoma significantly increased the fistula recurrence rate -LRB- 67 vs 50 % -RRB- . After years of perianal or transvaginal pus secretion and recurrent abscess formations , patients are often tired of the suffering involved . Antibiotics , azathioprine , or 6-mercaptopurine may be used successfully in suppurative perianal Crohn 's disease .15,18,38,39 In our department , this is routinely tried if perianal disease does not settle after surgical drainage is provided . However , azathioprine or 6-mercaptopurine are not tolerated by all patients and may also fail to relieve perianal Crohn 's disease . In these cases , fecal diversion can be offered as a relatively minor procedure with low morbidity ,18 and might be performed laparoscopically in selected cases .40 However , overall healing rates of perianal disease are only around 40 % , with 87 % of those being diverted eventually retaining their stoma .18 We diverted 51 patients , of which 27 retained there stoma -LRB- 53 % -RRB- , which compares favorably , but indicates a considerable risk for a permanent stoma if diversion is tried , and being a significant risk factor according to multivariate analysis in our series . Perianal disease in combination with fecal incontinence might necessitate fecal diversion as well . Factors that may contribute to fecal incontinence in patients with Crohn 's disease are reduced stool consistency , sphincter injuries after abscess incisions , and keyhole deformities after laying open perianal fistulas . The latter was reported to result in fecal incontinence in 5 of 27 patients with perianal fistulas in Crohn 's disease ,41 which is why we did not use this technique or cutting setons in transsphincteric fistulas . We always incise perianal abscesses along a circular perianal line , meticulously preserving sphincter integrity . Nevertheless , fecal incontinence became prevalent in 12 % of our patients during follow-up and was a significant predictor of permanent fecal diversion by multivariate analysis . Rectal advancement flap procedures may influence continence ,22 and a 9 % rate of worsened continence was reported thereafter .33 In particular , the risk of fecal incontinence was increased after flap repairs in patients with previous surgical fistula repairs ,33 but only 2 out of 12 patients with fecal incontinence had a previous rectal advancement flap repair in our study . A high rate of abdominal procedures was associated with an increased risk for a temporary or a permanent stoma , possibly reflecting high intestinal disease activity of Crohn 's disease in these patients . All 30 patients that ended with a permanent stoma had frequently undergone abdominal procedures -LRB- median of three procedures -RRB- . Because of the suffering involved and unsuccessful previous attempts to heal perianal fistulas , about half of our patients eventually opted for temporary ileostomy , being brought into contact with a stoma nurse and stoma carriers with Crohn 's disease before , and 31 % of all patients remained with a permanent stoma . In general , the need for a stoma was reduced during recent decades in Crohn 's disease ,43 but the overall long-term risk for a stoma in patients with Crohn 's disease who require abdominal surgery was reported between 30 and 40 % .44,45 Fecal diversion remains an option to subside perianal disease activity , with an early response rate of about 70 -- 80 % .46 -- 48 Unfortunately , 75 % of those patients eventually experienced a relapse , and the restoration of intestinal continuity was achieved in 10 % only .48 In 130 patients with surgically treated perianal Crohn 's disease , a permanent stoma became necessary in 24 % , and this rate increased to 53 % in patients with Crohn 's colitis .45 In this light , considering that all of our patients had complicated perianal disease and almost all -LRB- 93 of 97 patients , 96 % -RRB- had colonic involvement , we judge a 31 % permanent stoma rate as a success in a subset of patients carrying a particularly high risk for a stoma . Local drainage , setons , and , if not successful , a temporary stoma after a rectal advancement flap , resulted in closure of the stoma in 47 % of the time , which is an improvement compared to previous studies where restoration of intestinal continuity was reported in only 10 -- 40 % of patients , with most studies providing the lower end percentages .44,46 -- 48 If perianal Crohn 's disease was the indication for creating a stoma , stoma closure was successful in 2 out of 15 patients only -LRB- 13 % -RRB- .47 Eventually , proctectomy became necessary in 18 % of patients , which is in the range others have reported .24,44,49,50 However , if the presence of colonic disease was considered separately , seven out of eight patients with complicated perianal Crohn 's disease needed proctectomy , again indicating that an 18 % proctectomy rate in our high-risk population is rather low . All of our patients who needed proctectomy had colonic disease , and 13 of 17 had a rectovaginal fistula . In a recent study investigating the quality of life in patients with Crohn 's proctocolitis , patients in remission had a health-related quality of life similar to controls . Patients with active disease had a reduced quality of life , and only the symptom index negatively predicted a reduced quality of life , whereas neither previous colonic surgery nor the presence of a stoma did .51 Accordingly , carrying a stoma does not necessarily mean a reduced quality of life , although a slight reduction in two out of eight domains -LRB- physical and emotional role -RRB- was detected .51 However , in patients with perianal disease , the cumulative abscess rate during 3 years of follow-up and the symptomatic fistula recurrence rate with a mean follow-up of 22 months were both greatly reduced with a stoma -LRB- 76 vs 13 % and 52 vs 14 % -RRB- , indicating reduced suffering and need for surgery .21,22 In summary , patients with complicated perianal Crohn 's disease , colonic involvement , and a high rate of abdominal procedures carried a significant risk for a permanent stoma . However , if the risk factors identified were taken into account , the rate of patients eventually requiring a permanent stoma seemed low and showed a decrease compared to previous studies , indicating that multiple treatment episodes and complex surgery , including temporary fecal diversion , might eventually heal at least some patients in this high-risk population , as was also observed by others .50

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

fecal diversion
perianal abscesses
fistulas