Costs

and

benefits

of

induced

resistance

in

a

clonal

plant

network

Plant

defense

theory

suggests

that

inducible

resistance

has

evolved

to

reduce

the

costs

of

constitutive

defense

expression

To

assess

the

functional

and

potentially

adaptive

value

of

induced

resistance

it

is

necessary

to

quantify

the

costs

and

benefits

associated

with

this

plastic

response

The

ecological

and

evolutionary

viability

of

induced

defenses

ultimately

depends

on

the

long-term

balance

between

advantageous

and

disadvantageous

consequences

of

defense

induction

Stoloniferous

plants

can

use

their

inter-ramet

connections

to

share

resources

and

signals

and

to

systemically

activate

defense

expression

after

local

herbivory

This

network-specific

early-warning

system

may

confer

clonal

plants

with

potentially

high

benefits

However

systemic

defense

induction

can

also

be

costly

if

local

herbivory

is

not

followed

by

a

subsequent

attack

on

connected

ramets

We

found

significant

costs

and

benefits

of

systemic

induced

resistance

by

comparing

growth

and

performance

of

induced

and

control

plants

of

the

stoloniferous

herb

Trifolium

repens

in

the

presence

and

absence

of

herbivores

Introduction

Plants

can

allocate

a

limited

pool

of

resources

to

the

three

main

functions

of

growth

reproduction

and

defense

suggesting

that

increased

investments

in

one

function

may

compromise

the

others

Empirical

studies

have

shown

that

constitutive

defense

can

be

costly

-LRB-

e.g.

tannins

Sagers

and

Coley

glandular

trichomes

Elle

et

al.

-RRB-

Plant

defense

theory

postulates

that

inducible

defense

mechanisms

have

evolved

to

reduce

these

costs

by

optimizing

the

temporal

match

between

resource

investment

into

defense

and

herbivory

threats

-LRB-

Herms

and

Mattson

Agrawal

et

al.

-RRB-

In

the

last

decades

numerous

studies

have

attempted

to

find

costs

of

induced

defense

-LRB-

reviewed

in

Bergelson

and

Purlington

and

Strauss

et

al.

-RRB-

but

evidence

for

costs

of

inducible

plant

defense

remains

scarce

and

inconclusive

More

recently

empirical

evidence

has

emerged

supporting

the

allocation

cost

theory

with

the

help

of

improved

experimental

designs

genetic

engineering

and

biochemical

methodology

-LRB-

Baldwin

Van

Dam

and

Baldwin

Heil

and

Baldwin

-RRB-

Inducible

resistance

is

a

form

of

phenotypic

plasticity

as

it

allows

plants

to

express

an

adequate

phenotype

in

response

to

temporally

and

spatially

variable

herbivore

damage

Herbivore-induced

changes

in

the

phenotypes

of

plants

often

relate

to

trait

alterations

which

reduce

the

palatability

and

digestibility

of

consumed

tissue

by

producing

toxic

metabolites

and/or

by

up-regulating

the

production

of

a

variety

of

defensive

compounds

The

ecological

viability

of

induced

resistance

as

an

efficient

defense

strategy

depends

on

the

balance

of

costs

and

benefits

associated

with

plastic

defense

induction

Assessing

the

benefits

of

induced

defense

in

conjunction

with

possible

costs

is

a

prerequisite

for

estimating

the

advantages

and

disadvantages

of

plastic

defense

induction

as

a

potentially

adaptive

form

of

phenotypic

plasticity

-LRB-

Dudley

and

Schmitt

Schmitt

et

al.

-RRB-

and

hence

for

understanding

potential

selection

pressures

leading

to

the

evolution

of

induced

plant

defenses

-LRB-

Agrawal

-RRB-

Costs

of

defense

have

been

traditionally

measured

in

terms

of

decreased

plant

fitness

Allocation

costs

refer

to

a

direct

fitness

decrease

as

a

consequence

of

resource-mediated

trade-offs

between

defense

investment

and

other

plant

functions

Recent

empirical

and

conceptual

work

has

provided

convincing

arguments

for

the

notion

that

defense

induction

can

also

affect

fitness

in

an

indirect

manner

via

a

multitude

of

potentially

complex

ecological

interactions

-LRB-

Van

Dam

and

Baldwin

Heil

and

Baldwin

Strauss

et

al.

-RRB-

These

costs

are

commonly

referred

to

as

ecological

costs

Allocation

theory

suggests

that

costs

of

plastic

defense

induction

should

be

more

apparent

in

low-resource

environments

than

under

optimal

growth

conditions

-LRB-

Herms

and

Mattson

Bergelson

Bergelson

and

Purlington

but

see

van

Dam

and

Baldwin

-RRB-

as

the

diversion

of

resources

to

defense

can

not

easily

be

compensated

for

by

enhanced

resource

acquisition

In

addition

experiments

to

detect

costs

of

defense

conducted

under

quasi-optimal

conditions

are

unlikely

to

reflect

realistic

situations

and

therefore

tend

to

underestimate

plasticity

costs

To

overcome

this

problem

several

studies

have

used

competitive

and/or

low-resource

environments

to

quantify

costs

of

induced

defense

-LRB-

Siemens

et

al.

and

studies

quoted

therein

-RRB-

Additionally

previous

studies

have

shown

that

controlling

the

genetic

background

of

plants

can

substantially

enhance

the

chances

to

detect

costs

by

removing

confounding

effects

due

to

genetic

variation

in

the

induced

response

-LRB-

Bergelson

and

Purlington

Strauss

et

al.

-RRB-

Stoloniferous

plants

consist

of

multiple

genetically

identical

individuals

-LRB-

ramets

-RRB-

that

are

interconnected

by

aboveground

horizontal

stems

-LRB-

stolons

-RRB-

Resource

transport

within

clonal

plant

networks

has

been

extensively

described

in

the

literature

-LRB-

Pitelka

and

Ashmun

Marshall

Alpert

Alpert

and

Stuefer

-RRB-

Nevertheless

the

importance

of

stolon

connections

for

the

transport

of

defense

agents

is

a

novel

aspect

-LRB-

Stuefer

et

al.

-RRB-

that

has

only

recently

been

demonstrated

-LRB-

Gómez

and

Stuefer

-RRB-

Ramets

of

the

stoloniferous

herb

Trifolium

repens

are

able

to

systemically

induce

other

ramets

after

local

herbivore

damage

On

the

one

hand

this

form

of

physiological

integration

may

confer

clonal

plant

networks

with

considerable

benefits

by

allowing

for

a

fast

specific

and

efficient

early-warning

system

among

interconnected

ramets

On

the

other

hand

the

potentially

large

spatial

scale

of

clonal

plant

networks

may

also

lead

to

substantial

costs

if

network

members

become

induced

without

being

threatened

by

herbivores

-LRB-

Gómez

and

Stuefer

-RRB-

These

costs

are

due

to

a

potential

mismatch

in

the

spatio-temporal

scale

of

plastic

defense

expression

and

the

dynamics

and

patterns

of

herbivore

attacks

To

assess

the

potentially

adaptive

nature

of

plastic

responses

it

is

necessary

to

demonstrate

that

the

phenotype

induced

in

each

relevant

environment

confers

higher

fitness

in

that

environment

relative

to

alternative

phenotypes

-LRB-

Schmitt

et

al.

-RRB-

This

is

analogous

to

stating

that

the

induced

phenotype

should

incur

costs

in

herbivore-free

environments

while

defense

induction

should

lead

to

benefits

in

herbivore-exposed

environments

To

quantify

costs

and

benefits

we

measured

traits

related

to

plant

fitness

and

performance

of

induced

and

uninduced

T.

repens

plants

in

the

absence

and

presence

of

herbivores

Growing

induced

and

uninduced

plants

in

the

absence

of

herbivores

allows

for

a

quantification

of

possible

costs

of

induced

resistance

simulating

localized

damage

-LRB-

e.g.

by

small

herbivores

with

a

low

mobility

-RRB-

and

the

activation

of

defense

in

ramets

beyond

the

feeding

range

of

the

herbivore

Benefits

of

induction

however

can

only

be

assessed

in

the

presence

of

herbivores

after

an

initial

attack

thereby

simulating

a

scenario

with

mobile

herbivores

showing

active

foraging

behavior

beyond

the

first

place

of

attack

In

this

study

we

tested

the

following

specific

hypotheses

In

the

absence

of

herbivores

systemically

induced

ramets

of

clonal

plants

perform

worse

than

uninduced

ramets

of

the

same

genotype

This

is

due

to

costs

of

defense

induction

when

defense

is

not

needed

In

the

presence

of

herbivores

induced

ramets

of

clonal

plants

perform

better

than

uninduced

plants

due

to

an

enhanced

protection

through

induced

defense

To

test

these

hypotheses

we

grew

induced

and

uninduced

-LRB-

control

-RRB-

plants

of

the

stoloniferous

herb

T.

repens

together

to

expose

them

to

mutual

competitive

interactions

resembling

sub-optimal

growing

conditions

in

a

sward

To

quantify

costs

and

benefits

of

induced

resistance

we

grew

plants

in

herbivore-free

and

herbivore-exposed

environments

respectively

Materials

and

methods

Study

organisms

Five

genotypes

of

the

stoloniferous

herb

T.

repens

L.

were

vegetatively

propagated

in

a

greenhouse

at

a

mean

temperature

of

°C

°C

-LRB-

day/night

-RRB-

and

at

a

h

-LRB-

light/dark

-RRB-

photoperiod

The

genotypes

originated

from

natural

riverine

grassland

populations

situated

along

the

river

Waal

The

Netherlands

They

had

been

collected

years

prior

to

the

start

of

this

experiment

and

were

grown

under

common

garden

conditions

eliminating

possible

maternal

and

environmental

carry-over

effects

The

beet

armyworm

-LRB-

Spodoptera

exigua

Hübner

-RRB-

used

in

this

study

is

a

generalist

caterpillar

with

a

broad

host

range

The

caterpillar

colony

was

maintained

at

a

constant

temperature

of

°C

and

h

-LRB-

light/dark

-RRB-

photoperiod

The

larvae

were

reared

on

an

artificial

diet

described

in

Biere

et

al.

-LRB-

-RRB-

Pre-growth

of

plant

material

We

started

the

experiment

with

cuttings

of

each

of

the

five

genotypes

The

cuttings

were

planted

in

pairs

in

plastic

trays

-LRB-

cm

cm

cm

-RRB-

using

sterilized

clay

grains

as

a

substrate

-LRB-

Seramis

Masterfoods

Germany

-RRB-

Each

tray

was

fertilized

weekly

with

ml

full-strength

Hoagland

solution

before

the

start

of

the

experiment

At

the

beginning

of

the

experiment

all

cuttings

consisted

of

a

main

stolon

with

at

least

eight

fully

developed

ramets

If

present

side

branches

were

removed

immediately

before

starting

the

experiment

Experimental

design

The

experimental

set-up

-LRB-

Fig.

-RRB-

to

measure

costs

and

benefits

of

systemic

induced

resistance

-LRB-

SIR

-RRB-

consisted

of

four

peripheral

trays

placed

around

a

central

tray

which

we

will

refer

to

as

the

competition

tray

All

trays

were

of

similar

dimensions

-LRB-

cm

cm

cm

-RRB-

Each

of

the

peripheral

trays

contained

two

cuttings

with

at

least

eight

ramets

each

The

cuttings

in

two

of

those

trays

received

a

treatment

to

induce

defense

during

the

entire

duration

of

the

experiment

-LRB-

for

details

see

below

-RRB-

while

the

cuttings

in

the

other

two

trays

remained

uninduced

-LRB-

control

-RRB-

Trays

receiving

the

same

treatment

were

placed

diagonally

opposite

each

other

The

competition

tray

was

placed

inside

a

metal

frame

-LRB-

cm

cm

cm

-RRB-

covered

by

mosquito

netting

-LRB-

mesh

gauge

-RRB-

with

four

small

openings

on

both

longitudinal

sides

The

two

youngest

ramets

of

each

cutting

were

inserted

through

the

mesh

openings

and

allowed

to

grow

-LRB-

proliferate

and

root

-RRB-

in

the

competition

tray

for

days

We

used

five

T.

repens

genotypes

each

of

which

was

replicated

times

to

measure

costs

and

times

to

measure

benefits

of

defense

induction

All

induced

and

control

plants

grown

together

in

the

same

experimental

set-up

-LRB-

as

described

above

-RRB-

belonged

to

the

same

genotype

The

experimental

systems

were

randomly

distributed

on

greenhouse

benches

Fig.

representation

of

the

experimental

set-up

to

measure

costs

and

benefits

of

systemic

induced

resistance

-LRB-

SIR

-RRB-

in

a

clonal

plant

network

Control

-LRB-

white

-RRB-

and

defense-induced

-LRB-

gray

-RRB-

plants

grew

from

four

peripheral

trays

into

a

common

central

competition

tray

The

circles

represent

petri

dishes

used

for

a

continued

controlled

herbivore

attack

-LRB-

defense

induction

treatment

-RRB-

To

measure

costs

of

SIR

plants

grew

together

in

the

absence

of

herbivores

in

the

competition

tray

-LRB-

upper

drawing

-RRB-

To

measure

benefits

ten

caterpillars

-LRB-

wavy

black

lines

-RRB-

were

added

to

the

competition

tray

-LRB-

lower

drawing

-RRB-

See

Materials

and

methods

for

more

details

Systemic

induction

of

resistance

Systemic

induction

of

resistance

was

achieved

through

a

controlled

herbivore

attack

One

S.exigua

larva

was

confined

with

two

leaves

in

one

petri

dish

mounted

on

the

plants

-LRB-

Gómez

and

Stuefer

-RRB-

The

corresponding

ramets

of

uninduced

control

plants

were

similarly

enclosed

in

modified

petri

dishes

but

without

adding

any

larvae

The

controlled

herbivore

attack

was

maintained

throughout

the

course

of

the

experiment

starting

on

the

ramet

on

the

eighth

position

-LRB-

counting

from

the

tip

of

the

stolon

-RRB-

from

each

cutting

When

the

two

ramets

inside

the

petri

dish

had

lost

at

least

of

leaf

tissue

the

petri

dish

was

moved

forward

on

the

stolon

and

the

adjacent

younger

ramet

was

inserted

into

the

petri

dish

Whenever

the

induction

treatment

was

moved

forward

on

the

induced

cuttings

a

comparable

leaf

area

was

removed

with

scissors

from

one

ramet

of

each

cutting

in

the

control

trays

This

was

done

to

compensate

for

the

leaf

area

loss

due

to

caterpillar

feeding

in

the

induced

plants

Cutting

the

leaves

with

scissors

does

not

induce

resistance

in

T.

repens

-LRB-

S.

Gómez

unpublished

data

-RRB-

The

induction

treatment

started

day

after

the

cuttings

were

placed

into

the

competition

tray

If

the

caterpillar

inside

the

petri

dish

died

it

was

replaced

by

a

new

one

to

maintain

defense

induction

In

order

to

enhance

plant

interactions

induced

and

control

plants

were

grown

together

in

the

competition

tray

Since

all

plants

growing

together

belonged

to

the

same

genotype

induction

effects

can

not

be

confounded

with

genetic

differences

in

plant

traits

including

competitive

ability

between

induced

and

control

plants

All

measurements

described

below

were

performed

on

ramets

growing

in

the

competition

trays

Costs

of

SIR

Costs

of

defense

induction

were

measured

as

a

reduction

in

plant

performance

Costs

can

be

measured

after

initial

herbivore

damage

-LRB-

and

consequent

defense

induction

-RRB-

in

the

absence

of

subsequent

herbivore

attacks

To

quantify

costs

of

defense

induction

we

measured

the

following

traits

which

are

known

to

be

closely

related

to

plant

performance

and

fitness

total

biomass

production

relative

biomass

allocation

to

leaves

petioles

stolons

and

roots

number

and

length

of

the

main

and

side

stolons

and

number

of

ramets

on

the

main

and

side

stolons

We

also

measured

the

petiole

length

petiole

dry

mass

leaf

area

leaf

dry

mass

of

the

fourth

and

fifth

youngest

ramets

of

each

cutting

Benefits

of

SIR

To

quantify

benefits

of

SIR

we

exposed

the

plants

in

the

competition

tray

to

a

second

controlled

herbivory

attack

-LRB-

referred

to

as

herbivory

treatment

-RRB-

We

released

five

fourth

instar

caterpillars

on

day

in

the

competition

tray

and

then

added

two

and

three

more

on

day

and

respectively

to

achieve

substantial

levels

of

herbivore

damage

The

plants

were

harvested

days

after

the

start

of

the

experiment

We

quantified

benefits

of

induced

resistance

by

scoring

herbivory

damage

in

the

induced

and

in

the

control

plants

At

the

time

of

harvesting

each

ramet

on

the

main

stolon

was

classified

according

to

the

leaf

area

consumed

We

visually

estimated

the

damage

and

assigned

each

ramet

a

damage

category

ranging

from

to

The

values

corresponded

to

the

following

amounts

of

damage

no

damage

and

of

leaf

area

consumed

We

also

recorded

the

position

of

the

damaged

ramet

on

the

stolon

to

investigate

possible

intra-clonal

variation

in

the

damage

pattern

according

to

ramet

age

In

addition

to

the

degree

of

damage

we

measured

the

dry

mass

of

leaves

petioles

stolons

and

roots

in

induced

and

control

plants

Herbivore

preference

test

One

day

before

releasing

the

caterpillars

-LRB-

herbivory

treatment

-RRB-

we

performed

two

dual

choice

tests

per

competition

tray

to

check

whether

plants

assigned

to

the

defense-induction

treatment

were

systemically

induced

For

each

competition

tray

we

cut

off

two

control

and

two

induced

ramets

of

a

similar

developmental

stage

-LRB-

third-youngest

fully

expanded

leaf

-RRB-

Each

control

ramet

was

paired

with

an

induced

one

and

placed

together

on

a

moist

filter

paper

in

a

petri

dish

to

perform

a

dual

choice

test

A

fourth

instar

S.

exigua

caterpillar

was

placed

in

the

middle

and

allowed

to

feed

until

more

than

of

one

of

the

leaves

was

consumed

or

for

h.

By

means

of

visual

estimates

the

leaf

with

the

largest

area

consumed

was

recorded

for

each

choice

test

In

of

the

cases

more

of

the

control

leaf

was

consumed

-LRB-

sign

test

M

P

n

-RRB-

than

the

induced

one

confirming

that

plants

in

the

competition

trays

that

had

received

local

herbivore

damage

-LRB-

defense

induction

treatment

-RRB-

were

induced

before

the

herbivory

treatment

started

Statistical

analysis

Central

competition

trays

were

considered

the

units

of

replication

in

all

statistical

analyses

To

avoid

pseudo-replication

and

a

consequent

inflation

of

df

-LRB-

Hurlbert

-RRB-

all

traits

measured

on

plants

-LRB-

cuttings

-RRB-

in

the

competition

trays

were

pooled

per

treatment

-LRB-

by

averaging

the

four

control

cuttings

and

the

four

defense-induced

cuttings

respectively

-RRB-

prior

to

data

analysis

Consequently

our

experiment

had

replicates

for

measuring

costs

and

replicates

for

assessing

benefits

Competing

plants

can

not

be

considered

independent

from

each

other

as

by

definition

they

change

each

other

's

environment

growth

and

development

To

take

this

dependence

into

account

we

used

a

repeated

measures

design

to

analyze

differences

between

competing

plants

that

belonged

to

different

treatment

groups

Repeated

measures

analysis

explicitly

considers

intrinsic

relationships

between

treatment

groups

-LRB-

Potvin

et

al.

-RRB-

Costs

of

SIR

Repeated

measures

ANOVA

was

performed

to

test

for

costs

of

defense

induction

in

number

and

length

of

the

main

and

side

stolons

and

number

of

ramets

on

the

main

and

side

stolons

relative

biomass

allocation

to

roots

stolons

petioles

and

leaves

and

petiole

length

petiole

dry

mass

leaf

area

leaf

dry

mass

of

the

fourth

and

fifth

youngest

ramets

Defense

induction

-LRB-

induced

vs.

control

-RRB-

was

considered

a

within-subjects

effect

and

genotype

was

treated

as

a

between-subjects

effect

Absolute

dry

masses

of

roots

stolons

petioles

and

leaves

were

analyzed

using

two-way

repeated

measures

ANOVA

-LRB-

within-subjects

effect

defense

induction

between-subjects

effects

plant

genotype

and

herbivory

-RRB-

Benefits

of

SIR

The

amount

of

damage

in

the

herbivory

treatment

was

assessed

with

doubly

repeated

measures

ANOVA

using

ramet

age

and

defense

induction

as

repeated

factors

and

genotype

as

main

effect

The

analysis

included

a

profile

analysis

-LRB-

SAS

procedure

GLM

profile

statement

-RRB-

to

test

for

differences

in

the

degree

of

damage

between

adjacent

ramets

on

the

stolons

To

correct

for

differences

in

the

developmental

stage

of

different

cuttings

we

used

only

the

six

youngest

ramets

of

each

cutting

in

the

damage

analysis

All

analyses

were

conducted

with

SAS

-LRB-

SAS

Institute

Cary

N.C.

-RRB-

Results

Costs

of

SIR

Total

dry

mass

did

not

differ

between

control

and

induced

plants

-LRB-

Table

-RRB-

However

defense

induction

caused

a

significant

reduction

in

petiole

dry

mass

Additionally

defense

induction

resulted

in

a

shift

in

biomass

allocation

to

the

different

plant

parts

Relative

biomass

allocation

to

leaves

increased

significantly

after

defense

induction

-LRB-

Table

P

-RRB-

The

percentage

of

biomass

allocated

to

roots

stolons

and

petioles

did

not

significantly

differ

between

control

and

induced

plants

-LRB-

Table

-RRB-

Table

measures

ANOVA

for

effects

of

genotype

herbivory

and

defense

induction

on

roots

stolons

petioles

leaves

and

total

dry

massSourcedfRootStolonPetiolesLeavesTotalMSFMSFMSFMSFMSFBetween-subject

effectsGenotype

-LRB-

Gen

-RRB-

Herbivory

-LRB-

Herb

-RRB-

Gen

Herb449.30.361240.141050.312920.291,1710.17Error301368993421,0146,714Within-subject

effectsInduction

-LRB-

Ind

-RRB-

Ind

Ind

Ind

Gen

P

P

Table

of

systemic

induced

resistance

-LRB-

SIR

-RRB-

Repeated

measures

ANOVA

for

effects

of

genotype

and

defense

induction

on

relative

dry

mass

allocation

to

roots

stolons

petioles

and

leaves

on

plants

without

an

herbivory

treatment

in

the

competition

traySourcedfRootsStolonsPetiolesLeavesMSFMSFMSFMSFBetween-subjects

effectsGenotype

-LRB-

Gen

-RRB-

effectsInduction

-LRB-

Ind

-RRB-

Ind

P

P

P

Table

-LRB-

SE

-RRB-

absolute

and

relative

dry

mass

allocated

to

roots

stolons

petioles

and

leaves

of

uninduced

and

induced

plants

in

the

absence

of

a

subsequent

herbivory

treatment

-LRB-

Costs

-RRB-

and

in

the

presence

of

a

subsequent

herbivory

treatment

-LRB-

Benefits

-RRB-

in

the

competition

trayRoot

-LRB-

mg

-RRB-

Stolons

-LRB-

mg

-RRB-

Petioles

-LRB-

mg

-RRB-

Leaves

-LRB-

mg

-RRB-

Total

-LRB-

mg

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

number

of

ramets

produced

on

the

main

stolon

was

lower

in

induced

as

compared

to

control

plants

-LRB-

Table

induction

effect

P

-RRB-

The

number

and

length

of

side

stolons

and

the

number

of

ramets

formed

on

them

did

not

change

after

defense

induction

Table

Costs

of

SIR

Repeated

measures

ANOVA

for

effects

of

genotype

and

defense

induction

on

plant

fitness

and

performance-related

traits

in

the

absence

of

herbivoresSourcedfRamet

no

main

stolonLength

main

stolonRamet

no

side

stolonsLength

side

stolonsSide

stolons

numberFourth

ramet

petiole

lengthFourth

ramet

areaMSFMSFMSFMSFMSFMSFMSFBetween-subjects

effectsGenotype

-LRB-

Gen

-RRB-

Error150.694.3110.718.91.231.200.55Within-subject

effectsInduction

-LRB-

Ind

-RRB-

7.572.451.250.250.780.230.150.314.385.22

Ind

.171.193.921.271.830.374.101.220.120.240.350.420.090.21

P

P

P

The

fourth

and

fifth

youngest

ramets

on

the

main

stolon

produced

petioles

shorter

in

the

induced

plants

-LRB-

Table

fourth

ramet

P

fifth

ramet

P

-RRB-

Leaf

area

leaf

dry

mass

and

petiole

dry

mass

measured

on

those

ramets

were

not

significantly

affected

by

the

induction

treatment

Benefits

of

SIR

Defense

induction

had

a

very

strong

effect

on

the

amount

of

damage

inflicted

by

S.

exigua

larvae

on

the

plants

-LRB-

Table

induction

effect

P

Fig.

-RRB-

The

number

of

ramets

that

were

partially

or

fully

consumed

during

the

herbivore

attack

was

consistently

higher

in

control

than

in

induced

plants

Most

of

the

damaged

ramets

lost

only

a

small

part

of

their

leaf

area

-LRB-

-RRB-

This

was

consistent

for

both

control

and

induced

plants

-LRB-

Fig.

-RRB-

In

induced

plants

up

to

of

the

ramets

on

the

main

stolon

were

not

damaged

whereas

in

control

plants

only

of

ramets

on

the

main

stolon

were

undamaged

Table

of

SIR

Doubly

repeated

measures

ANOVA

for

effects

of

genotype

defense

induction

and

ramet

age

on

leaf

area

loss

due

to

herbivorySourcedfMSFBetween-subjects

effectsGenotype

-LRB-

Gen

-RRB-

effectsInduction

-LRB-

Ind

-RRB-

Ind

Error

-LRB-

induction

-RRB-

Age

Error

-LRB-

age

-RRB-

Ind

Age

Error

-LRB-

Ind

Age

-RRB-

P

P

Fig.

damage

-LRB-

SE

-RRB-

inflicted

on

ramets

of

the

main

stolon

-LRB-

the

ramet

being

the

youngest

and

the

being

the

oldest

-RRB-

of

control

and

induced

plants

in

the

competition

tray

after

carrying

out

a

controlled

herbivore

attack

-LRB-

herbivory

treatment

-RRB-

Damage

categories

no

damage

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

The

asterisks

above

the

bars

indicate

the

statistical

significance

of

the

result

of

a

profile

analysis

-LRB-

SAS

procedure

GLM

profile

statement

-RRB-

to

test

for

differences

in

the

degree

of

damage

between

ramets

of

successive

age

classes

The

amount

of

damage

was

significantly

higher

for

control

than

for

induced

plants

in

all

age

classes

P

ns

not

significant

The

herbivory

treatment

significantly

reduced

the

biomass

of

leaves

and

petioles

-LRB-

Table

P

and

P

respectively

Table

-RRB-

in

both

induced

and

uninduced

plants

In

the

presence

of

herbivores

induced

and

uninduced

plants

had

a

comparable

total

biomass

However

induced

plants

showed

a

larger

percentage

of

biomass

in

their

leaves

-LRB-

Table

repeated

measures

ANOVA

F

P

-RRB-

suggesting

that

the

induced

plants

benefitted

from

increased

relative

biomass

in

those

organs

under

attack

Ramet

age

regardless

of

the

induction

state

had

a

very

strong

effect

on

herbivore

preference

-LRB-

Table

age

effect

P

-RRB-

Younger

ramets

especially

the

first

and

second

youngest

ones

were

heavily

preferred

over

older

ones

-LRB-

profile

analysis

Fig.

-RRB-

The

first

ramet

exhibited

particularly

severe

damage

in

both

induced

and

control

plants

-LRB-

average

leaf

area

consumed

Fig.

-RRB-

Defense

induction

had

a

significant

effect

on

leaf

area

loss

due

to

herbivory

in

all

ramet

age

classes

-LRB-

Fig.

-RRB-

The

degree

to

which

systemic

defense

induction

reduced

herbivory

damage

was

similar

for

ramets

of

all

age

classes

-LRB-

Table

no

age

induction

effect

-RRB-

There

was

a

marginally

significant

genotype

effect

on

the

feeding

of

the

caterpillars

-LRB-

Table

genotype

effect

P

-RRB-

Discussion

Our

study

provides

empirical

evidence

of

significant

costs

and

benefits

of

SIR

in

a

clonal

plant

network

In

agreement

with

our

hypotheses

induced

and

control

plants

showed

clear

differences

in

performance

and

fitness-related

traits

when

grown

in

the

absence

and

presence

of

herbivores

In

environments

without

herbivores

induced

plants

produced

fewer

ramets

shorter

petioles

and

exhibited

a

shift

in

biomass

allocation

patterns

In

environments

with

herbivores

control

plants

suffered

consistently

higher

degrees

of

leaf

damage

than

induced

plants

Even

though

defense

induction

resulted

in

changes

in

plant

growth

and

significantly

affected

the

amount

of

damage

caused

by

the

herbivores

total

plant

biomass

did

not

respond

as

expected

under

the

adaptive

plasticity

hypothesis

-LRB-

Dudley

and

Schmitt

Schmitt

et

al.

-RRB-

as

we

could

not

find

a

significant

induction

herbivory

interaction

effect

However

we

propose

that

the

differences

observed

in

our

study

-LRB-

e.g.

reduced

ramet

production

rates

and

shorter

petioles

in

the

cost

experiment

decreased

amount

of

leaf

damage

in

the

benefits

experiment

-RRB-

are

likely

to

translate

into

substantial

differences

in

plant

productivity

and

hence

biomass

in

the

longer

term

Costs

of

SIR

Biomass

production

and

allocation

Total

plant

biomass

production

did

not

change

as

a

consequence

of

defense

induction

implying

that

defense

induction

did

not

incur

direct

and

immediate

productivity

costs

After

induction

however

biomass

allocation

shifted

significantly

towards

the

leaves

We

suggest

that

this

allocation

shift

may

enable

plants

to

better

cope

with

current

and

future

herbivory

by

reducing

resource

allocation

to

those

organs

that

are

not

currently

impacted

by

herbivore

damage

While

potentially

beneficial

in

the

short

term

this

response

might

result

in

longer

term

indirect

costs

due

to

reduced

performance

under

certain

environmental

conditions

such

as

drought

root

herbivory

and

severe

root

competition

A

similar

shift

in

the

biomass

allocation

pattern

was

observed

in

Lepidium

virginicum

plants

after

defense

induction

Induced

plants

grown

at

a

high

density

showed

a

reduction

in

root

biomass

and

an

increase

in

aboveground

biomass

-LRB-

Agrawal

-RRB-

In

agreement

with

our

findings

total

biomass

production

was

not

significantly

altered

by

defense

induction

in

that

study

A

reduction

in

belowground

biomass

was

also

reported

for

induced

wild

parsnip

plants

In

this

case

however

the

aboveground

biomass

did

not

change

significantly

after

defense

induction

-LRB-

Zangerl

et

al.

-RRB-

Further

studies

are

necessary

to

assess

the

generality

functional

significance

-LRB-

including

costs

and

benefits

-RRB-

and

mechanistic

basis

of

changes

in

root

shoot

allocation

in

response

to

induced

resistance

to

herbivory

Reduction

in

developmental

growth

rate

Defense

induction

negatively

affected

plant

fitness

by

reducing

the

number

of

ramets

produced

This

delayed

developmental

growth

rate

was

expressed

as

a

reduction

in

the

number

of

ramets

on

the

main

stolon

produced

during

the

experiment

-LRB-

ramets

on

the

control

and

on

the

induced

plants

-RRB-

In

the

shorter

run

-LRB-

i.e.

time

span

of

this

experiment

-RRB-

this

effect

is

unlikely

to

translate

into

biomass

differences

In

the

longer

run

however

subtle

changes

in

the

developmental

growth

rate

are

known

to

result

in

very

major

divergences

in

performance

structure

and

clonal

fitness

of

stoloniferous

plants

-LRB-

Birch

and

Hutchings

Birch

and

Hutchings

Huber

and

Stuefer

Stuefer

and

Huber

-RRB-

Reduction

in

petiole

length

Defense

induction

had

significant

negative

effects

on

petiole

lengths

This

effect

can

have

severe

performance

and

fitness

consequences

for

a

stoloniferous

plant

like

T.

repens

which

often

grows

in

dense

herbaceous

canopies

and

which

relies

on

petiole

elongation

for

shade

avoidance

-LRB-

Huber

-RRB-

Petiole

length

largely

determines

the

ability

of

stoloniferous

plants

to

place

their

leaves

higher

up

in

the

canopy

-LRB-

Huber

and

Wiggerman

Weijschedé

et

al.

-RRB-

Even

a

small

reduction

in

petiole

length

could

have

serious

performance

costs

since

differences

in

the

relative

position

of

leaves

in

herbaceous

canopies

are

likely

to

be

amplified

by

asymmetric

competition

for

light

-LRB-

Weiner

Pierik

et

al.

-RRB-

Defense

induction

may

also

cause

physiological

trade-offs

which

impede

the

simultaneous

expression

of

plasticity

to

herbivores

and

to

shading

by

competitors

-LRB-

Cipollini

-RRB-

A

decrease

in

petiole

length

as

a

result

of

defense

induction

can

hence

compromise

the

competitive

ability

of

plants

and

result

in

an

enhanced

risk

of

induced

plants

being

over-shaded

by

neighbors

A

recent

study

by

Kurashige

and

Agrawal

-LRB-

-RRB-

supports

this

notion

by

showing

that

Chenopodium

album

plants

which

had

previously

been

damaged

by

herbivores

were

able

to

elongate

stems

to

a

similar

proportional

degree

as

undamaged

plants

when

grown

in

competition

for

light

However

the

damaged

plants

were

smaller

due

to

the

expression

of

induced

resistance

thereby

incurring

potential

opportunity

costs

due

to

asymmetric

competition

Benefits

of

SIR

Reduced

damage

Our

results

provide

direct

evidence

for

short-term

benefits

of

having

an

early-warning

system

in

clonal

plant

networks

In

the

presence

of

herbivores

induced

plants

suffered

considerably

less

damage

than

control

plants

As

many

as

fewer

ramets

were

attacked

in

induced

plants

as

compared

to

controls

Localized

damage

-LRB-

defense-induction

treatment

-RRB-

resulted

in

a

greater

degree

of

protection

against

herbivores

for

ramets

further

along

that

main

stolon

and

its

side

branches

The

reduced

damage

did

not

translate

into

a

significant

effect

of

defense

induction

on

biomass

production

due

to

the

fact

that

the

youngest

usually

not

fully

developed

leaves

were

heavily

preferred

by

the

herbivores

The

biomass

loss

due

to

young

leaf

consumption

is

very

likely

to

strongly

underestimate

the

negative

effects

of

herbivory

and

defense

induction

on

future

plant

growth

and

performance

Coleman

and

Leonard

-LRB-

-RRB-

demonstrated

how

leaf

area

consumption

and

its

consequences

for

plant

performance

can

be

severely

underestimated

if

the

developmental

stage

of

leaves

is

not

taken

into

account

They

showed

that

a

certain

amount

of

damage

inflicted

on

young

expanding

Nicotiana

tabaccum

leaves

is

more

detrimental

than

the

same

amount

received

by

mature

fully

developed

leaves

As

leaf

tissue

expanded

the

area

of

the

holes

increased

almost

fourfold

and

the

final

area

of

the

leaf

decreased

by

approximately

In

addition

they

observed

a

decrease

in

the

number

and

mass

of

fruits

on

the

plants

that

received

the

damage

to

expanding

young

tissues

Therefore

an

initially

small

amount

of

damage

inflicted

on

young

developing

leaves

may

have

dramatic

consequences

for

plant

performance

and

fitness

over

time

Similarly

the

differences

found

in

our

experiment

can

be

expected

to

result

in

considerable

performance

differences

between

induced

and

uninduced

plants

as

increased

damage

and

loss

of

young

leaves

in

uninduced

plants

will

compromise

plant

productivity

by

reducing

the

number

of

future

source

ramets

Our

results

show

that

ramet

age

largely

determines

herbivore

damage

The

first

and

second

ramets

were

heavily

attacked

as

compared

to

the

rest

This

damage

although

still

large

was

significantly

reduced

in

induced

plants

The

reduction

in

leaf

area

loss

in

induced

young

ramets

likely

increases

their

chance

of

survival

and

establishment

Young

ramets

in

clonal

plants

constitute

the

most

valuable

tissue

since

they

represent

the

future

reproductive

potential

of

the

plant

-LRB-

Huber

and

During

-RRB-

and

their

protection

is

critical

since

they

are

responsible

for

a

high

proportion

of

the

future

biomass

production

-LRB-

Beinhart

-RRB-

We

present

evidence

supporting

the

hypothesis

that

an

early-warning

system

after

herbivory

in

a

clonal

plant

network

grants

vulnerable

young

offspring

ramets

with

parental

support

-LRB-

Stuefer

et

al.

-RRB-

that

non-clonal

plants

are

unable

to

confer

their

offspring

at

the

moment

of

the

attack

-LRB-

but

see

Agrawal

et

al.

-RRB-

Our

study

provides

evidence

for

significant

costs

and

benefits

of

systemic

defense

induction

in

T.

repens

The

experimental

approach

used

in

this

study

however

does

not

allow

for

balancing

costs

and

benefits

in

terms

of

plant

fitness

and

overall

plant

performance

because

both

positive

and

negative

effects

of

induction

reported

here

although

likely

to

have

significant

longer-term

effects

on

productivity

and

ultimately

on

fitness

did

not

have

an

effect

on

biomass

at

the

short

time

scale

during

which

the

experiment

took

place

While

our

results

indicate

clear

advantages

and

disadvantages

of

network

induction

in

the

subsequent

presence

and

absence

of

herbivores

respectively

an

accurate

and

reliable

quantification

of

the

cost

benefit

ratio

should

make

use

of

long-term

experiments

In

conclusion

the

present

study

shows

that

in

the

short

term

the

activation

of

early-warning

responses

in

clonal

plant

networks

has

both

costs

and

benefits

In

the

absence

of

herbivores

the

performance

of

the

induced

phenotype

was

compromised

as

compared

to

the

uninduced

phenotype

in

terms

of

potential

competitive

ability

In

the

presence

of

herbivores

the

induced

phenotype

was

favored

by

suffering

considerably

less

herbivore

damage

suggesting

potential

advantages

for

the

phenotype

correctly

matching

its

environment

Whether

this

represents

an

adaptive

value

of

the

induced

responses

remains

to

be

demonstrated

in

longer-term

studies

where

the

initial

small

changes

observed

in

our

study

can

be

measured

directly

in

terms

of

fitness

The

long-term

balance

of

costs

and

benefits

of

induced

resistance

in

clonal

plant

networks

is

likely

to

be

strongly

context

dependent

and

a

function

of

the

match

between

spatio-temporal

aspects

of

systemic

defense

expression

and

the

feeding

behavior

of

herbivores

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

adaptive plasticity hypothesis
physiological integration
plant defense
trifolium repens


Recurrence of nephrotic syndrome/focal segmental glomerulosclerosis following renal transplantation in children The incidence of recurrence of nephrotic syndrome/focal segmental glomerulosclerosis -LRB- NS/FSGS -RRB- is variable -LRB- ~ 30 % -RRB- The incidence of recurrence is less in African-Americans than in whites and Hispanics Graft survival rates are decreased in recipients with FSGS , especially if remission of the NS is not achieved in those with recurrence Although controversial , the use of living donor -LRB- LD -RRB- transplants are not contraindicated ; however , obligatory heterozygote parental grafts with a podocin mutation should be used with caution Optimal treatment to induce a remission post-transplant has not been delineated Pre-transplant and/or prophylactic post-transplant pre-operative plasmapheresis -LRB- PP -RRB- for high-risk patients -- especially those with recurrence in a previous graft -- may be promising An international multicenter controlled study is required to delineate the optimal approach to prevent and/or treat the recurrence of NS/FSGS Introduction Focal segmental glomerulosclerosis -LRB- FSGS -RRB- is a histologic -LRB- pathologic -RRB- diagnosis that encompasses numerous specific clinical entities with potential varied etiologies . Steroid-resistant nephrotic syndrome -LRB- SRNS -RRB- is a specific clinical phenomenon that may vary histologically from minimal change nephrotic syndrome -LRB- MCNS -RRB- to FSGS , but it also may be associated with other specific histological diagnoses . The most frequently acquired disease resulting in end-stage renal disease -LRB- ESRD -RRB- in children is FSGS , accounting for 8.3 % of the chronic renal insufficiency -LRB- CRI -RRB- patients , 14.3 % of the dialysis patients , and 11.4 % of the transplant recipients included in the North American Pediatric Renal Transplant Cooperative Study -LRB- NAPRTCS -RRB- registry -LSB- 1 -RSB- . In 1972 , Hoyer et al. -LSB- 2 -RSB- from Minnesota described the recurrence of NS in 3/4 pediatric renal allograft recipients whose original disease was FSGS . At post-mortem examination , the lesion of FSGS was present in the grafts of two recipients -LSB- 2 -RSB- . An editorial accompanying the publication of this article in Lancet stated that `` renal transplantation may be contraindicated -LRB- in patients with FSGS -RRB- leaving dialysis as probably the only means of prolonging life '' -LSB- 3 -RSB- . The purpose of this teaching review article will be to address the factors that influence the incidence of recurrence of NS/FSGS in renal allografts , establish the known risk factors for recurrence , and delineate the current preventive and therapeutic approaches to the recurrence of NS/FSGS in pediatric renal allograft recipients . What is the incidence of recurrence of post-transplant NS/FSGS ? The general consensus is that 30 -- 40 % of recipients whose original disease was NS/FSGS will clinically manifest recurrence -LSB- 4 -RSB- . In most instances , recurrence is clinically apparent in the immediate post-transplant period , frequently with substantive proteinuria in the initial 24 hours following transplantation . However , the onset of clinical recurrence has been reported to occur at more than one year following transplantation . The delay in initial clinical manifestations may be coincident to the occurrence of acute tubular necrosis -LRB- ATN -RRB- , which occurs more frequently in live donor -LRB- LD -RRB- and deceased donor -LRB- DD -RRB- recipients with FSGS -LSB- 5 -RSB- . It has been proposed that the `` immediate fulminate recurrence of NS/FSGS post-transplant likely predisposes such grafts to ATN and explains the increased incidence of ATN in this patient population -LSB- 5 -RSB- . What is the incidence of subsequent recurrence in a patient who manifested clinical recurrence in a previous graft ? Re-transplantation of a recipient with recurrence of NS/FSGS in a previous graft has been associated with a ~ 80 % recurrence rate -LSB- 4 -RSB- . Recently , a 100 % recurrence rate has been reported in ten recipients of a second -LRB- 6 -RRB- , third -LRB- 3 -RRB- , and fourth -LRB- 1 -RRB- transplant -LSB- 6 -RSB- . Therefore , proceeding with a subsequent transplant in a patient who manifested previous recurrence has been discouraged . Recently , pre-operative plasmapheresis -LRB- PP -RRB- was successful in preventing clinical recurrence in 3/6 patients whose initial graft was lost in association with clinical recurrence -LSB- 7 -RSB- . What is the incidence of recurrence in recipients with mutations of genes that encode for podocin ? The association of mutations impacting on the structure of the foot processes in the region of the slit diaphragm of the glomerular basement membrane in patients with sporadic and familial NS has potentially delineated the pathophysiology of the NS in such patients -LSB- 8 -RSB- . Initial publications indicated that the incidence of recurrence of NS/FSGS in patients with podocin mutations was similar to that reported for all patients with NS/FSGS -LSB- 9 -RSB- . However , a recent analysis of 73 patients with either homozygous/compound heterozygous -LRB- 65 -RRB- or heterozygous -LRB- 8 -RRB- mutations revealed a 7.7 % incidence in the former and a 62.5 % incidence in the latter recipients -LSB- 10 -RSB- . If these data are confirmed , it would seem advantageous to test all potential recipients with ESRD secondary to NS/FSGS for such mutations in order to delineate an optimal transplant strategy . Do patients with the glomerular tip lesion who receive a renal allograft manifest recurrence of the NS ? Howie et al. -LSB- 11 -RSB- reported that 12 of 14 recipients whose native kidney manifested the glomerular tip lesion had clinical recurrence of the NS post-transplant . In all 12 recipients , the glomerular tip lesion was the only histologic abnormality on graft biopsy . These data support the concept that the glomerular tip lesion may be the earliest histologic abnormality in the NS/FSGS spectrum of disease . Precise incidence and outcome data await subsequent reports . Does race impact on the recurrence of NS/FSGS ? Although the disease process appears more virulent in African-American -LRB- AA -RRB- children with NS/FSGS -LSB- 12 -RSB- , it is interesting that the incidence of recurrence following transplantation in this ethnic population is decreased . At least three studies -LSB- 5 , 13 , 14 -RSB- have shown that the incidence of recurrence in the AA recipient was < 50 % of that in the Caucasian recipient . Hispanic recipients had an incidence similar to that of Caucasians . The precise reason for this disparity in incidence has not been delineated . Has the incidence of recurrence changed with the introduction of new immunosuppressive regimens ? Since the initial publication in 1972 of Hoyer et al. -LSB- 2 -RSB- , there has been a significant change in the available immunosuppressive agents and regimens to both prevent and treat rejections . Despite these regimens , which have substantially reduced the incidence of acute rejection episodes and markedly improved short-term allograft survival rates , the incidence of recurrent NS/FSGS has remained unaffected -LSB- 14 -RSB- . Bartosh et al. -LSB- 14 -RSB- reviewed the NAPRTCS database for the period 1998 -- 2003 and found the incidence of recurrence was 31 % , which was similar to the incidence in the initial NAPRTCS report for the period 1987 -- 1990 -LSB- 4 -RSB- . What are the risk factors for the recurrence of NS/FSGS ? Three factors identified in the 1970s to be associated as risk factors for recurrence were : -LRB- 1 -RRB- the rapid progression to ESRD in a period of < 3 years ; -LRB- 2 -RRB- the presence of mesangial hypercellularity on biopsy ; and -LRB- 3 -RRB- the age at onset of the clinical NS of > 6 years . More recently , the presence of a circulating permeability factor -LSB- 15 -RSB- , the use of induction therapy in the immunosuppressive regimen -LSB- 16 -RSB- , and native kidney nephrectomy either prior to or at the time of transplantation -LSB- 17 -RSB- have been reported to be associated with an increased risk of recurrence . Does the presence of an identified circulating factor predispose to the recurrence of NS/FSGS ? Savin et al. -LSB- 15 -RSB- showed that the presence of a permeability factor -LRB- Palb -RRB- with an activity of ≥ 0.50 was associated with a recurrence rate of 86 % -LRB- 6/7 -RRB- , whereas an activity of < 0.50 was associated with a recurrence rate of 17 % -LRB- 4/23 -RRB- . Plasmapheresis was associated with both a reduction in the Palb activity and in urinary protein excretion . Is induction therapy a risk factor for the recurrence of NS/FSGS ? Raafat et al. -LSB- 16 -RSB- noted a recurrence rate of NS/FSGS of 43 % between 1968 and 1997 . However , it was only 11 % -LRB- 1/9 -RRB- in those who did not receive induction , whereas it was 53 % -LRB- 15/28 -RRB- in those receiving some form of antilymphocyte serum -LRB- ALS -RRB- . Conversely , Gagnodoux -LSB- 17 -RSB- reported an incidence of recurrence of 31 % prior to 1985 when no induction therapy with antilymphocyte globulin/antithymocyte globulin -LRB- ALG/ATG -RRB- was used compared to 30 % following the introduction of induction therapy . Both Sheth et al. -LSB- 18 -RSB- and Hubsch et al. -LSB- 19 -RSB- noted a significant increase in the incidence of recurrence -LRB- 17.6 % vs 37.5 % and 38 % vs 83 % , respectively -RRB- with the use of anti-IL2 receptor antibody as induction therapy . In the most recent analysis of NAPRTCS for the period 1998 -- 2003 , Bartosh et al. -LSB- 14 -RSB- noted an overall incidence of recurrence of 31 % -LRB- 51/165 -RRB- , which was similar to no induction -LRB- 30 % -RRB- or the use of polyclonal antibody -LRB- 27 % -RRB- or monoclonal antibody -LRB- 33 % -RRB- . Therefore , advocating the restriction of induction therapy to reduce the incidence of recurrence awaits the performance of a multicenter prospective randomized controlled study . Is native nephrectomy a risk factor for the recurrence of NS/FSGS ? Analysis of the recent NAPRTCS database -LSB- 14 -RSB- indicated a significant -LRB- p < 0.001 -RRB- difference in the incidence of recurrence in recipients who had undergone prior native kidney nephrectomy -LRB- 50 % -RRB- compared to those whose native kidneys were not removed prior to or at the time of transplantation -LRB- 22 % -RRB- . A possible explanation for this phenomenon was that the native kidneys provided a `` sponge '' for any circulating putative antibody and that native nephrectomy facilitated a sufficient level of circulation of such antibodies which were deposited in the graft precipitating recurrence . Other reports -LSB- 20 -RSB- have come to the same conclusion . Does the recurrence of NS/FSGS impact on the allograft survival rate ? Baum et al. -LSB- 21 -RSB- in 2002 and Bartosh et al. -LSB- 14 -RSB- in 2004 reviewed the NAPRTCS database , which indicated inferior long-term graft survival rates in both recipients of LD and DD grafts whose original disease was FSGS . Analysis of the UNOS database demonstrated a similar disadvantageous graft survival rate for recipients whose original disease was FSGS compared to recipients with other primary renal diseases -LSB- 22 -RSB- . A consensus is that 50 % of the grafts with the recurrence of NS/FSGS are lost , presumably as a result of the recurrent pathologic process . However , data from Schachter and Harmon -LSB- 23 -RSB- noted that long-term survival rates significantly -LRB- p = 0.002 -RRB- improved in those recipients with recurrence who achieved remission with some therapeutic intervention compared to those who failed to go into remission and had persistent NS . Does age at the time of transplantation impact on graft survival in recipients with FSGS ? Adolescent recipients -LRB- 13 -- 17 years of age -RRB- with FSGS have a significantly poorer graft survival rate with both LR and DD grafts compared to younger recipients -LRB- 0 -- 12 years of age -RRB- -LSB- 21 -RSB- . In actuality , the 0 -- 12 year-old recipient DD graft survival rate was similar in the FSGS and no FSGS groups . The specific reason for this phenomenon is not known . However , Scientific Registry of Transplant Recipients -LRB- SRTR -RRB- data have consistently demonstrated that adolescent recipients have substantially poorer long-term graft survival rates , which approach that of recipients > 65 years of age at transplantation -LSB- 24 -RSB- . Should an LD graft be utilized in children with ESRD secondary to FSGS ? At least five reports from 1989 to 1999 -LSB- 4 , 25 -- 28 -RSB- concluded that recipients with FSGS had a greater risk of allograft failure from an LD graft than from a DD graft . A precise reason for this phenomenon was not delineated . In 2001 , Baum et al. -LSB- 5 -RSB- reviewed the NAPRTCS database and showed that the 5-year LD graft survival rate was 69 % for recipients with FSGS compared to 82 % with no FSGS -LRB- p < 0.001 -RRB- , whereas it was 60 % and 67 % , respectively , in the DD groups . The authors concluded that the long-term advantage of LD grafts was lost in recipients with FSGS . Conversely , Huang et al. -LSB- 22 -RSB- proffered that , although the LD advantage was not observed in AA pediatric recipients , the authors recommend that LD transplantation be considered for all racial groups with FSGS . With this potential excellent outcome with newer preventative and therapeutic regimens for recurrence to be detailed subsequently , the previous concerns regarding the use of an LD graft may not be appropriate . However , the recurrence of NS/FSGS in two recipients with a homozygous podocin mutation who received an LD graft from their obligatory heterozygote mother indicates that the obligatory heterozygote parental donor may not be ideal -LSB- 29 , 30 -RSB- . Similarly , the development of FSGS and ESRD in three donors -LSB- 28 , 31 -RSB- at some point following donation also indicates that appropriate informed consent should be obtained prior to utilizing an LD graft in recipients with FSGS . What are the therapeutic options available to produce a remission following the recurrence of NS in recipients with FSGS ? In 1988 , Laufer et al. -LSB- 32 -RSB- reported the remission of NS in two pediatric recipients following nine sessions of PP at 3 and 6 months post-transplant . Both recipients manifested prolonged ATN . This led to the general use of PP in patients with recurrence . However , no randomized control trial was ever performed and the rate of remission has been variable . Ingulli et al. -LSB- 33 -RSB- in 1990 reported the success in inducing a remission with high-dose cyclosporine in three pediatric recipients . The rationale for the increased dosage was that hyperlipidemia bound the cyclosporine and higher doses were required to achieve adequate bioavailability . The precise dose and length of treatment with the `` high-dose '' regimen were never delineated in a controlled trial . However , this report led to the general approach of using a higher maintenance dosage of cyclosporine once a remission was obtained . Recently , Raafat et al. -LSB- 34 -RSB- reported a recurrence rate of 67 % -LRB- 16/24 -RRB- in recipients with FSGS between 1991 and 2003 . The oral dose of cyclosporine was increased to 24 mg/kg/day until either remission was achieved or renal toxicity was evident . Complete remission was achieved in 69 % -LRB- 11/16 -RRB- within 14 days to 19 months -LRB- 10/11 within 3 months -RRB- and 12 % -LRB- 2/16 -RRB- had a partial remission . Concomitant PP was administered to 7/16 recipients with recurrence . Cochat et al. -LSB- 35 -RSB- in 1993 introduced a regimen of PP and cyclophosphamide in three patients who remitted within 12 -- 24 days . This regimen has been adopted by some centers , especially for patients who failed to remit with PP alone . However , again , no controlled trial has been performed . Plasma protein absorption was introduced by Dantal et al. -LSB- 36 -RSB- in 1994 for recipients with an identified circulating factor . Although proteineuria was reduced in the eight patients treated , it was permanent in only one patient following discontinuation of the procedure . Subsequent reports utilizing this technique in pediatric recipients are limited . Recently , Salomon et al. -LSB- 37 -RSB- reported an 82 % -LRB- 14/17 -RRB- remission rate utilizing IV Cyclosporine at 3 mg/kg/day to reach a cyclosporine level of 250 -- 350 mg/dl . These data add to the potential utility of high-dose cyclosporine in inducing a remission following the recurrence of NS/FSGS . With the use of angiotensin-converting enzyme/angiotensin receptor blocker -LRB- ACE/ARB -RRB- therapy to reduce proteinuria in patients with various forms of renal disease , it would seem logical to extend the use of these agents to recipients with the recurrence of NS/FSGS . However , there has been no controlled study of the use of these drugs either alone or as adjunctive to other therapeutic regimens . Can pre-operative PP reduce the risk of recurrence of NS/FSGS ? Ohta et al. -LSB- 38 -RSB- in 2001 reported the recurrence of NS/FSGS in 9/21 grafts . Pre-operative PP was performed in 15 and recurrence developed in five , whereas 4/6 without pre-operative PP developed recurrence . The authors concluded that pre-operative PP was effective in preventing the recurrence of NS/FSGS . At the International Pediatric Transplant Association -LRB- IPTA -RRB- Congress in August 2005 , Rainthavorn et al. -LSB- 39 -RSB- from the UCLA reported a recurrence rate of 100 % in three recipients of DD grafts who received one session of pre-operative PP , whereas it was 57 % -LRB- 4/7 -RRB- in recipients of LD grafts who received < 5 pre-operative PP and 0 % in three recipients who received > 5 pre-transplant PP sessions . The authors concluded that > 5 pre-operative PP sessions prevented the recurrence of NS/FSGS . Kawamura et al. -LSB- 40 -RSB- , at the same IPTA congress , reported a recurrence rate of 15.4 % -LRB- 2/13 -RRB- in LD recipients who received 1 -- 2 pre-operative PP sessions , as well as native kidney nephrectomy . Recurrence was evident at 6 days and 8 months post-transplant , respectively , and remitted with post-transplant PP . A recent publication involving primarily adult recipients -LRB- 9/10 were adult -RRB- utilized a combination of pre - and post-operative PP in high-risk patients -- six with recurrence in a prior graft and four with rapid progression to ESRD -LRB- < 3 years -RRB- -LSB- 7 -RSB- . The LD recipients received eight courses of PP over a two-week period -- one week prior to and one week following transplantation . The DD recipients initiated PP within 24 hours of transplantation and continued for two weeks post-transplant . There was no recurrence in the four recipients with rapid progression to ESRD and 3/6 recipients with a recurrence in a prior graft had no recurrence . Certainly , the utilization of pre-operative and prophylactic post-operative PP appears promising in high-risk patients . Controlled multicenter trials are warranted to delineate the optimal preventative and therapeutic approaches . Conclusions The incidence of recurrence of nephrotic syndrome/focal segmental glomerulosclerosis -LRB- NS/FSGS -RRB- has remained at ~ 30 % for the past four decades , despite the introduction of newer and more potent immunosuppressive regimens . Multiple risk factors have been identified to predispose or precipitate clinical recurrence . However , no prospective multicenter studies have validated the relationships . Mutations of the gene encoding for podocin may identify a population of patients with NS/FSGS who are at a lower risk for clinical recurrence . Because of the potential for an increased incidence of recurrence and/or graft loss , living donor -LRB- LD -RRB- grafts should be used with constraint . Graft survival rates in recipients with NS/FSGS are decreased . However , if a remission can be induced , the outlook for long-term graft survival is substantially improved . Various therapeutic regimens , including pre-operative plasmapheresis -LRB- PP -RRB- , high-dose cyclosporine , IV cyclosporine , and adjunctive cyclophosphamide , have proven to be effective . However , there have been no multicenter controlled studies to delineate the optimal therapeutic approach . Recent use of pre-operative and/or post-operative prophylactic PP to prevent the recurrence of NS/FSGS is exciting . Controlled trials are required to validate efficacy and delineate a precise preventative regimen . Questions -LRB- Answers appear following the references -RRB- The incidence of recurrence of FSGS is : Comparable in whites and African-AmericansLess in whites than HispanicsLess with re-transplants than with primary graftsNot influenced by pre-transplant plasmapheresisBetween 30 % and 40 % The use of a deceased donor is mandatory in a patient with FSGS when : A living donor graft is not availableThe initial graft was lost as a result of recurrenceThe parental donor is not an obligatory podocin heterozygoteThe potential live-donor is a haplotype mismatchThe potential live-donor is a B-antigen mismatchGraft survival following the recurrence of NS in a patient with FSGS is dependent upon : A reduction in the magnitude of proteinuria to 3.0 g/kg/dayThe lack of histologic evidence of FSGS during the initial post-transplant monthInducing a complete remission of the NSThe immediate use of post-transplant plasmapheresisThe use of a one haplotype-matched live-related donorThe optimal approach to prevent the recurrence of NS in patients with FSGS : Includes cyclophosphamide and plasmapheresisIncludes one week of pre-transplant plasmapheresisIncludes post-operative tacrolimus and plasmapheresisIncludes post-operative sirolimus and plasmapheresisHas not been delineatedThe recurrence of NS in patients with congenital nephrotic syndrome : Has not been reported with the `` Finnish '' typeIs > 80 % in recipients with diffuse mesangial sclerosisIs uniform with Fin-major/Fin-minor genotypeIs associated with the development of antinephrin antibodiesIs associated with anti-podocin antibodiesWhich of the following does not impact on the recurrence of NS in patients with FSGS : RaceNative kidney nephrectomyRecurrence in a prior graftPodocin mutationNone of the above

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

recurrent focal segmental glomerulosclerosis (fsgs)