Drug resistance in sea lice: A threat to salmonid aquaculture

Abstract

Sea lice are copepod ectoparasites with vast reproductive potential and affect a wide variety of fish species. The number of parasites causing morbidity is proportional to fish size. Natural low host density restricts massive parasite dispersal. However, expanded salmon farming has shifted the conditions in favor of the parasite. Salmon farms are often situated near wild salmonid migrating routes, with smolts being particularly vulnerable to sea lice infestation. In order to protect both farmed and wild salmonids passing or residing in the proximity of the farms, several measures are taken. Medicinal treatment of farmed fish has been the most predictable and efficacious, leading to extensive use of the available compounds. This has resulted in drug-resistant parasites occurring on farmed and possibly wild salmonids.
Copyright © 2014 Elsevier Ltd. All rights reserved.

Full text

Drug
resistance
in
sea
lice:
a
threat
to
salmonid
aquaculture
Stian
Mørch
Aaen,
Kari
Olli
Helgesen,
Marit
Jørgensen
Bakke,
Kiranpreet
Kaur,
and
Tor
Einar
Horsberg
Norwegian
University
of
Life
Sciences,
School
of
Veterinary
Science,
Sea
Lice
Research
Centre,
Oslo,
Norway
Sea
lice
are
copepod
ectoparasites
with
vast
reproduc-
tive
potential
and
affect
a
wide
variety
of
fish
species.
The
number
of
parasites
causing
morbidity
is
propor-
tional
to
fish
size.
Natural
low
host
density
restricts
massive
parasite
dispersal.
However,
expanded
salmon
farming
has
shifted
the
conditions
in
favor
of
the
parasite.
Salmon
farms
are
often
situated
near
wild
salmonid
migrating
routes,
with
smolts
being
particu-
larly
vulnerable
to
sea
lice
infestation.
In
order
to
protect
both
farmed
and
wild
salmonids
passing
or
residing
in
the
proximity
of
the
farms,
several
measures
are
taken.
Medicinal
treatment
of
farmed
fish
has
been
the
most
predictable
and
efficacious,
leading
to
extensive
use
of
the
available
compounds.
This
has
resulted
in
drug-
resistant
parasites
occurring
on
farmed
and
possibly
wild
salmonids.
Massive
technological
progress
above
and
below
sea
level
Terrestrial
food
production
represents
the
main
protein
source
in
the
industrialized
world.
Due
to
a
rapidly
grow-
ing
human
population,
and
thus
a
deficiency
of
traditional
protein
resources,
alternative
ways
to
provide
for
the
increase
in
nutrient
demand
are
being
sought.
In
2006,
the
world
aquaculture
production
of
fish
contributed
to
47%
of
the
world’s
food
fish
supply,
mainly
carps
and
other
cyprinids
(59%)
but
also
salmonids.
Approximately
7%
of
the
world’s
fish
production
in
aquaculture
comes
from
salmonid
farming
[1].
Fish
farming
in
sea
water
possesses
vast
potential
for
protein
processing
[2],
with
the
most
heavily
industrialized
production
being
farming
of
Atlantic
salmon.
Developing
from
small-scale
production
to
a
mas-
sive
industry
within
40
years,
the
optimal
techniques
for
fish
farming
are
probably
yet
to
be
identified.
Several
pathogens
are
compromising
salmon
production,
most
of
which
are
being
addressed
through
vaccines
and
other
precautions.
However,
caligid
copepods
have
proved
to
be
a
major
constraint
to
biological
sustainability.
In
2002,
Denholm
et
al.
[3]
reviewed
the
development
of
sea
lice
resistance
towards
available
compounds.
Intensive
research
has
been
conducted
with
several
sea
lice
species
since
then,
catapulting
the
knowledge
about
parasite–
fish
interactions
and
molecular
mechanisms
to
the
next
level.
A
state
of
constant
increase
Commercial
farming
of
salmonids
was
initiated
in
mid-
Norway
in
the
late
1960s
(see
Store
Norske
leksikon
Review
Glossary
Acetylcholine
(ACh):
a
neurotransmitter
present
in
cholinergic
synapses.
Acetylcholine
esterase
(AChE):
an
enzyme
involved
in
inactivation
of
acetylcholine.
Azamethiphos
(AZA):
an
organophosphate
targeting
acetylcholine
esterase,
used
as
a
delousing
compound
applied
as
bath
treatment,
and
has
been
used
since
1994.
Benzoyl
ureas:
a
class
of
chemical
compounds
that
inhibit
molting
in
several
parasite
or
pest
species.
These
compounds
inhibit
the
synthesis
of
chitin.
Cypermethrin
(CYPER):
a
compound
within
the
class
of
pyrethroids;
it
is
derived
from
the
organic
compound
pyrethrin.
Deltamethrin
(DELTA):
a
compound
from
the
group
of
pyrethroids.
It
is
structurally
similar
to
cypermethrin.
Diflubenzuron
(DIFLU):
a
compound
from
the
group
of
benzoyl
ureas.
Emamectin
benzoate
(EMB):
a
chemical
compound
within
the
class
of
avermectins.
This
compound
acts
mainly
as
activator
of
chloride
channels
in
membranes.
Fitness
cost:
the
loss
of
a
feature
or
a
favorable
metabolic
pathway
through
changes
in
genetic
material
to
avoid
the
effects
of
xenobiotics.
Hydrogen
peroxide
(H
2
O
2
):
a
disinfectant
with
insecticidal
and
ovicidal
properties.
g-Aminobutyric
acid
(GABA):
a
neurotransmitter
present
in
both
vertebrates
and
invertebrates.
Glutamate
(GLU):
a
proteinogenic
amino
acid
and
neurotransmitter.
Glutathione
S-transferase
(GST):
a
class
of
isoenzymes
with
a
range
of
functions,
including
metabolism
of
xenobiotics.
Integrated
pest
management
(IPM):
a
theory
for
extirpation
of
pest
organisms
through
a
combination
of
complex
measures.
Mixed
function
oxidases
(CYP):
metabolic
enzymes
of
the
cytochrome
P450
class.
Organophosphates
(OPs):
a
class
of
chemical
compounds
that
have
been
utilized
as
insecticides
in
agriculture
for
more
than
50
years.
These
compounds
inhibit
acetylcholine
esterase,
leading
to
paralysis.
P-glycoprotein
(P-gp):
a
protein
of
the
cell
membrane
responsible
for
the
efflux
of
a
range
of
substances
from
the
cell.
P-gp
is
also
known
as
multidrug
resistance
protein
1.
Pyrethroids
(PYRs):
a
class
of
compounds
that
includes
the
substances
cypermethrin
and
deltamethrin.
Refugia:
parasites
not
treated
with
medicinal
compounds
to
let
them
spread
their
genetic
material
to
the
population
of
reduced
sensitivity.
In
theory,
refugia
will
slow
down
resistance
development.
Teflubenzuron
(TEFLU):
a
compound
from
the
group
of
benzoyl
urea
compounds.
Therapeutic
index:
a
mathematic
factor
describing
the
medicinal
dose
toxic
to
salmon/dose
used
to
remove
sea
lice
off
fish.
Voltage-dependent
sodium
channels
(VDSC):
channels
specific
to
sodium
ions,
opened
by
depolarisation
of
the
membrane.
Xenobiotics:
foreign
chemical
substances
present
in
living
organisms.
1471-4922/
ß
2014
Elsevier
Ltd.
All
rights
reserved.
http://dx.doi.org/10.1016/j.pt.2014.12.006
Corresponding
authors:
Aaen,
S.M.
(stian.morch.aaen@nmbu.no);
Horsberg,
T.E.
(tor.e.horsberg@nmbu.no).
Keywords:
salmon
lice;
resistance;
bioassays;
molecular
methods;
salmon
aquaculture.
72
Trends
in
Parasitology,
February
2015,
Vol.
31,
No.
2

(https://snl.no/fiskeoppdrett))
with
the
seawater
cultiva-
tion
phase
conducted
in
floating
net
pens,
allowing
for
the
exchange
of
water
and
its
contents
with
the
environ-
ment.
Fish
farms
are
considered
a
major
factor
for
spreading
sea
lice
to
wild
salmonids
in
Europe
and
North
America
[4].
When
the
critical
parasite
abundance
level
is
exceeded,
the
host
could
eventually
suffer
from
osmotic
stress
and
secondary
infections,
leading
to
mortalities
[5].
The
interplay
of
farmed
salmonids,
parasites,
and
wild
salmonids
is,
however,
a
complex
matter.
Species-depen-
dent
ability
to
reject
the
parasite,
interaction
between
the
parasite
and
the
host
immune
system,
self-imposed
delous-
ing
in
rivers,
and
fish
size
are
all
important
factors
deter-
mining
the
outcome
for
the
host
(reviewed
by
Torrisen
et
al.
[6]).
Thus,
sea
lice
have
varying
impacts
on
wild
salmonid
populations
[6,7].
Sea
lice
levels
in
salmon
farms
are
under
comprehensive
surveillance
throughout
the
sea
water
production
period.
Medicinal
treatments
have
historically
been
the
most
pre-
dictable
measures
to
prevent
the
occurrence
of
high
sea
lice
abundance.
The
possibility
of
infecting
vulnerable
wild
salmonids
residing
in
the
proximity
of
fish
farms
is
a
major
concern
behind
the
monitoring
of
sea
lice
levels.
Altogeth-
er,
sea
lice
parasites
constitute
a
serious
threat
to
sustain-
able
salmon
farming
in
the
main
producing
countries,
Canada,
Chile,
the
Faroe
Islands,
Ireland,
Norway,
and
Scotland,
as
the
total
production
of
Atlantic
salmon
has
almost
doubled
from
1.1
billion
tons
in
2002
to
2.1
billion
tons
in
2012
(FAO
Cultured
Aquatic
Species
Information
Programme
Salmo
salar
(http://www.fao.org/fishery/
culturedspecies/Salmo_salar/en)).
Spillover
from
medici-
nal
treatments
to
remove
sea
lice
is
potentially
harmful
to
other
organisms
such
as
lobsters
and
shrimps
[8].
Ex-
tensive
use
of
medicines
has
resulted
in
an
inevitable
drift
towards
resistance.
This
imposes
a
huge
threat
for
fish
health
and
welfare,
the
environment,
the
economy
in
salmonid
production,
and
for
seafood
production
in
gener-
al.
In
summary,
these
parasites
represent
a
massive
eco-
nomical
and
biological
obstacle
to
fish
farming
companies
in
all
salmon-producing
countries.
Life
cycle
and
current
combat
strategies
In
Europe
and
Canada,
the
most
frequently
occurring
sea
louse
is
Lepeophtheirus
salmonis
(Krøyer),
whereas
Cali-
gus
rogercresseyi
(Boxshall
and
Bravo)
is
its
counterpart
in
the
Southern
Hemisphere.
A
third
species,
Caligus
elon-
gatus
(Nordmann),
is
to
some
degree
prevalent
in
Europe
and
Canada,
affecting
a
variety
of
salmonid
and
non-
salmonid
species.
C.
elongatus
is
only
sporadically
subject
to
research
because
of
its
low
prevalence
and
limited
influence
on
fish
morbidity.
All
three
copepods
are
crusta-
ceans,
and
their
life
cycle
consists
of
eight
stages;
three
of
these
stages
are
planktonic
whereby
the
third
stage
(cope-
podid)
infects
the
fish,
and
five
of
these
stages
are
parasitic
instars.
Regarding
L.
salmonis,
two
instars
(chalimus
I
and
II)
attach
to
the
fish
by
a
protein
filament,
whereas
the
following
three
(pre-adult
I,
II,
and
adult)
are
able
to
move
on
the
host
while
grazing
on
mucus
and
blood.
The
Caligus
species
also
comprise
five
parasitic
instars:
the
first
four
(chalimus
I–IV)
attach
to
the
host
by
a
protein
filament,
with
the
latter
molting
to
the
adult
stage
[9].
The
developmental
time
from
the
extrusion
of
egg
strings
to
adults
is
temperature
dependent.
For
L.
salmonis,
it
is
approximately
40
days
for
males
and
50
days
for
females
at
108C,
but
shorter
for
C.
rogercresseyi
(reviewed
by
Costello
[10]).
Apart
from
chemical
intervention
through
bath
treat-
ments
and
medicated
feed,
several
non-chemical
methods
are
utilized
to
remove
or
reduce
the
number
of
parasites
attaching
to
farmed
fish.
Fallowing,
synchronized
treat-
ments
within
geographic
zones,
cleaner
fish,
delousing
laser,
and
plankton
shielding
skirts
are
already
in
use,
with
others
such
as
snorkel
cages
and
enclosed
cages
on
the
verge
of
commercial
introduction.
Despite
the
use
of
alter-
natives
to
chemical
treatment,
extensive
use
of
medicinal
compounds
combined
with
limited
access
to
effective
chem-
ical
compounds
has
led
to
widespread
resistance
towards
the
most
applied
medicinal
products.
The
evolving
reduced
sensitivity
in
sea
lice
is
a
good
example
of
microevolution,
and
shows
how
humans
can
influence
nature
considerably
in
a
relatively
short
time
frame.
Addressing
a
prevailing
parasite
in
fish
farming,
this
text
will
review
current
knowledge
of
resistance
against
chemi-
cal
treatment
agents
in
L.
salmonis
and
C.
rogercresseyi.
The
emergence
of
more
accessible
molecular
methods
has
already
contributed
to
a
rapid
increase
in
knowledge
regarding
this
issue.
General
definitions
of
resistance
The
World
Health
Organization
expert
committee
in
1957
defined
resistance
to
insecticides
as
the
‘development
of
an
ability,
in
a
strain
of
insects,
to
tolerate
doses
of
toxicants
which
would
prove
lethal
to
the
majority
of
individuals
in
a
normal
population
of
the
same
species’
[11].
The
degree
of
reduced
susceptibility
needed
to
survive
an
antiparasitic
treatment
differs
between
agri-
cultural
pests
and
parasites
attached
to
a
vertebrate
host.
Successful
treatments
of
agricultural
pests
are
mainly
dependent
on
the
dose,
whereas
for
parasitic
pests,
the
maximum
applicable
amount
is
determined
by
the
host’s
ability
to
withstand
the
toxicity
as
well
as
the
parasite’s
susceptibility
to
the
compound.
For
several
anti-sea
lice
medicines
the
therapeutic
index
(see
Glossa-
ry)
is
small;
thus,
only
minor
reductions
in
sea
lice
sus-
ceptibility
may
present
as
clinical
resistance
in
the
field
[12–14].
Resistance
development
is
driven
by
the
survival
and
reproduction
of
the
less
sensitive
individuals
[15].
The
expression
‘‘resistant
sea
lice’’
is
mainly
applied
for
populations
showing
a
high
degree
of
reduced
sensitivity
in
bioassays.
A
therapeutic
failure
in
the
field
could
be
caused
by
a
reduced
sensitivity
to
the
applied
medicinal
com-
pound,
but
also
by
suboptimal
treatment
regimens,
such
as
insufficient
drug
dispersal,
poor
medicine
management,
or
badly
adjusted
feeding
procedures.
Several
genetic
resistance
mechanisms
have
been
iden-
tified
in
arthropods.
These
include
point
mutations
in
the
chemical’s
target
gene
rendering
the
protein
insensitive
to
the
chemical,
upregulation
of
genes
for
detoxifying
metabo-
lism
of
the
chemical
by
the
parasite,
and
upregulation
of
efflux
pumps
in
the
parasite’s
intestine
leading
to
decreased
uptake
of
chemicals
given
to
the
fish
as
medicated
feed.
Similarly,
changes
in
genes
coding
for
cuticle
thickness
or
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
73

other
protective
mechanisms
that
decrease
the
penetration
of
the
chemical
into
the
parasite
can
be
a
resistance
mecha-
nism
[16].
Substances
from
five
groups
of
medicinal
compounds
are
used
to
combat
sea
lice:
avermectins,
benzoyl
ureas,
disin-
fectants,
organophosphates
(OPs),
and
pyrethroids
(PYRs).
Their
use
in
different
countries,
modes
of
action,
and
resistance
mechanisms
are
summarized
in
Tables
1
and
2.
Selection
of
highly
resistant
individuals
may
be
facilitated
by
the
use
of
off-label
medicines,
when
inade-
quate
treatment
regimens
are
upheld,
or
when
drugs
with
long
half-lives
are
utilized
[17].
It
may
be
argued
that
national
imposed
treatment
thresholds
are
an
important
factor
for
resistance
selection.
By
intending
to
reduce
the
Table
1.
A
systemic
approach
to
medicinal
compounds,
their
mode
of
action,
and
resistance
mechanisms
in
relation
to
sea
lice
treatment
a
Substance
Mode(s)
of
action
Resistance
mechanisms
Resistance
in
sea
lice
Refs
Emamectin
benzoate.
Oral
treatment
Activation
of
glutamate-
and
GABA-gated
chloride
channels,
thereby
reducing
the
cell’s
excitability.
Mechanisms
probably
not
fully
elucidated
Resistance
in
nematodes
and
arthropods.
Overexpression
of
P-gps,
CYPs,
GST,
carboxyl
esterases.
Changes
in
glutamate-
and
histidine-gated
ion
channels
Widespread.
Mechanisms
unclear.
Overexpression
of
metabolic
enzymes,
P-gps,
and
downregulation
of
GABA-gated
chloride
channels
and
neuronal
AChE
receptors
[55–62]
Benzoyl
ureas,
diflubenzuron
and
teflubenzuron.
Oral
treatment
Inhibition
of
chitin
synthesis,
rendering
the
parasites
unable
to
detach
from
their
exuviae
during
molting
Resistance
in
arthropods.
Overexpression
of
CYPs.
Mutation
in
the
chitin
synthase
gene
Not
reported
[63–65]
Hydrogen
peroxide.
Bath
treatment
Not
fully
elucidated.
Etching
action.
Gas
bubbles
in
the
body
rendering
the
parasites
unable
to
hold
on
to
a
surface.
Also
used
against
amoebic
gill
disease
Resistance
in
bacteria
and
fungi.
Overexpression
of
metabolic
enzymes
such
as
catalase,
glutathione
peroxidase,
GST
in
mammalian
cells,
bacteria,
and
fungi
Reported
from
Norway
and
Scotland.
Mechanism
not
elucidated
[66–72]
Azamethiphos.
Bath
treatment
Inhibition
of
AChEs
in
cholinergic
synapses,
leading
to
excitation
and
subsequently
paralysis
Resistance
in
arthropods.
Mutations
in
genes
coding
for
AChE.
Overexpression
of
metabolic
enzymes.
Described
already
in
the
1960s
Reported
from
the
North
Atlantic
Ocean.
Mutation
in
one
of
the
genes
coding
for
AChE
[3,73,74]
Pyrethroids,
deltamethrin
and
cypermethrin.
Bath
treatment
Disruption
of
neuronal
signals
through
interference
with
VDSCs,
leading
to
excitation
and
subsequently
paralysis
Resistance
in
arthropods.
Also
reported
in
non-target
aquatic
species.
Overexpression
of
metabolic
enzymes
such
as
CYPs,
GST,
superoxide
dismutase,
mutations
in
the
VDSC
gene,
and
reduced
cuticle
penetration
Widespread.
Metabolic
resistance
most
likely
[75–84]
a
Abbreviations:
AChE,
acetylcholine
esterase;
CYPs,
mixed
function
oxidases;
GABA,
g-aminobutyric
acid;
GST,
glutathione
S-transferase;
P-gps,
P-glycoproteins;
VDSC,
voltage-dependent
sodium
channel.
Table
2.
A
historical
presentation
of
medicinal
compounds;
where
and
when
they
have
been
used
to
treat
fish
for
sea
lice
infestations
Compound
Canada
Chile
Faroe
Islands
a
Ireland
Norway
Scotland
Refs
Organophosphates
Between
mid-
1990s
to
early
2000.
Now
sporadically,
emergency
drug
release
Metriphonate
1981–1985.
Dichlorvos
1985–
2000.
AZA
since
2013
Since
1997
Yes
Metriphonate
1974–1996.
Dichlorvos
1986–1997.
AZA
1994–
1999
and
since
2008
b
Dichlorvos
1979–1997.
AZA
since
1994
[6,43,85–88]
Pyrethroids
Emergency
drug
release
2009–2010
Since
2007
Since
1997
DELTA
since
2006
c
Since
1994
CYPER
since
1997,
DELTA
since
2008
c
[35,85,88,89]
Emamectin
benzoate
Since
1999
Since
1999.
Only
available
medicine
between
2000
and
2007
Since
1999
Yes
Since
1998
Since
2000
[33,86,87,90,91]
Benzoyl
ureas
No
Since
2009
Since
1997
Yes
Since
1996
b
Since
2007
[43,86,88,90]
Hydrogen
peroxide
Sporadically,
emergency
drug
release
Since
2007
Since
2011
No
marketing
authorization.
Sporadic
use
1993–1997.
Reintroduced
2009
b
1993–1999.
Reintroduced
in
2009
[12,43,87,88,92,93]
a
Data
provided
by
Mørkøre,
B.,
Food
and
Veterinary
Authority,
Faroe
Islands,
personal
communication.
b
Data
from
the
Norwegian
Institute
of
Public
Health
(http://www.fhi.no/artikler/?id=109416).
c
Data
provided
by
Martinsen,
B.,
Pharmaq
Norway,
personal
communication.
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
74

number
of
infective
copepodids,
the
treatment
threshold
for
L.
salmonis
is
in
some
areas
in
the
region
of
0.5–1
parasite
per
fish.
This
can
result
in
numerous
treatments
during
the
production
cycle,
thereby
inducing
a
highly
effective
process
of
selecting
resistant
individuals.
Resistance
to
more
than
one
xenobiotic
occurring
in
individuals
or
a
population
is
called
cross
resistance
or
side
resistance
when
the
underly-
ing
mechanism
is
identical.
When
several
mechanisms
are
simultaneously
carried
by
a
parasite,
this
is
called
multiple
resistance.
There
is,
however,
some
confusion
regarding
these
expressions,
as
there
is
no
current
standardized
definition
[18].
The
fitness
cost
of
resistance
mechanisms
in
sea
lice
are
to
some
degree
described.
Espedal
et
al.
[19]
found
no
fitness
cost
in
generation
F4
in
a
laboratory-reared
lice
strain
resistant
to
emamectin
benzoate
(EMB).
Bravo
et
al.
[20]
found
no
increase
in
sensitivity
of
C.
rogercresseyi
after
11
passages
of
an
EMB-resistant
strain
in
the
laboratory.
This
applies
also
for
OPs
and
PYRs.
In
our
laboratory,
OP-
and
PYR-resistant
strains
of
L.
salmonis
have
been
reared
for
more
than
ten
generations
without
loss
of
the
resistance
trait
(Aaen
et
al.,
unpublished).
Even
though
resistant
traits
against
the
most
commonly
used
medicinal
products
in
salmon
lice
may
be
associated
with
some
fitness
costs,
these
do
not
seem
to
be
substantial
for
any
genotype.
Refugia:
isolated
populations
Occurrence
of
refugia
is
important
in
resistance
develop-
ment,
as
untreated
parasites
may
possess
wild-type
genes,
and
thus
slow
down
the
drift
towards
highly
resistant
populations.
In
the
present
salmonid
farming
areas,
refu-
gia
will
exist
either
on
wild
hosts
or
on
hosts
in
parts
of
farms
not
treated.
The
latter
is
not
commonly
seen
in
fish
farming
compared
to
agriculture.
This
leaves
wild
salmo-
nid
fish
as
identified
refugial
hosts
for
L.
salmonis
in
Europe,
whereas
in
Western
Canada,
the
three-spine
stick-
leback
is
also
reported
to
carry
the
parasite
[21].
In
Chile,
the
most
common
native
host
for
C.
rogercresseyi
is
thought
to
be
Eleginops
maclovinus,
supplying
refugia
year-round,
probably
participating
to
slow
down
the
development
of
resistance
[22].
Resistance
development:
geographical
overview
Information
about
the
severity
of
reduced
sensitivity
in
sea
lice
populations
from
salmon-producing
countries
is
scarce.
Only
Norway
has
implemented
a
nationwide
surveillance
program
for
resistance
monitoring
based
on
bioassay
data.
In
other
countries,
there
are
some
organized
efforts.
Some
of
the
generated
data
are
published
through
various
chan-
nels,
whereas
some
are
kept
within
the
industry
and
research
groups.
Norway.
A
number
of
incidents
of
non-effective
treatments
with
EMB
and
PYRs
have
been
reported
since
2008.
Since
2009,
reports
of
treatment
failures
with
azamethiphos
(AZA)
exist.
The
reports
validate
the
significant
increase
in
the
relative
use
of
delousing
agents
in
Norway
[23].
How-
ever,
no
official
monitoring
to
determine
the
extent
of
the
problem
was
in
place
before
the
surveillance
program
initiated
by
the
Norwegian
Food
Safety
Authority
was
conducted
in
2013.
The
basis
for
the
survey
was
a
simplified
bioassay
method
[24],
demonstrating
that
re-
duced
sensitivity
towards
AZA,
deltamethrin
(DELTA),
and
EMB
was
present
in
all
counties
except
Finnmark,
the
northernmost
county
in
Norway
[25].
The
survey
also
revealed
that
the
areas
with
the
most
severe
problems
were
located
in
the
Hordaland
county
in
the
south
west,
the
Nord-Trøndelag
county
in
mid-Norway,
and
the
northern
part
of
the
adjacent
county
Nordland.
In
these
areas,
resistance
towards
all
agents
in
the
program
(AZA,
DEL-
TA,
and
EMB)
was
widespread,
whereas
in
many
cases
the
same
population
displayed
resistance
traits
towards
all
the
above-mentioned
compounds
simultaneously,
a
scenario
of
multiple
resistance.
In
the
other
areas,
the
situation
was
more
subtle,
with
variations
from
susceptible
parasites
to
resistance
towards
one
or
several
agents.
Resistance
to-
wards
hydrogen
peroxide
(H
2
O
2
),
a
compound
also
used
against
amoebic
gill
disease
[26],
has
also
been
reported
from
mid-Norway
[72].
For
the
chitin
synthesis
inhibitors
diflubenzuron
(DIFLU)
and
teflubenzuron
(TEFLU),
no
evidence
for
resistance
is
reported,
although
some
reduced
treatment
efficacy
was
seen
in
2011
and
2012
[25].
Scotland.
Failing
efficacy
of
EMB
has
been
reported
since
2008.
Heumann
et
al.
[27]
reported
a
sevenfold
reduced
sensitivity
towards
EMB
in
a
salmon
lice
population
isolat-
ed
from
the
west
coast
in
2008.
In
an
epidemiological
study,
Lees
et
al.
[28]
demonstrated
a
gradual
decline
in
efficacy
of
this
compound
since
2004.
However,
no
recent
sensitivity
data
have
been
published
for
AZA,
PYRs,
or
H
2
O
2
.
Ireland.
There
are
no
recently
published
reports
of
sensi-
tivity
issues
in
sea
lice
from
this
country.
Jackson
[29]
indicated
that
reduced
treatment
efficacy,
herein
possibly
reduced
sensitivity,
could
be
a
contributing
factor
to
an
observed
rise
in
the
average
number
of
parasites
per
fish
in
the
period
2005–2007.
Canada.
Several
publications
describe
surveillance
pro-
jects
for
sea
lice
sensitivity
towards
chemotherapeutants.
In
a
survey
conducted
on
the
west
coast
of
Canada
in
2010–
2012,
only
1
out
of
22
bioassays
clearly
indicated
development
of
tolerance
towards
EMB,
the
only
applied
treatment
in
the
region
[30].
Analysis
of
treatment
efficacy
data
also
indicated
that
reduced
sensitivity
towards
EMB
did
not
appear
to
be
a
problem
in
British
Columbia.
On
the
east
coast,
the
scenario
is
different,
emphasized
by
the
fact
that
Pacific
and
Atlantic
L.
salmonis
are
different
species
[31].
Furthermore,
the
presence
of
alternate
hosts
is
vast
on
the
Pacific
side,
and
almost
negligible
on
the
Atlantic
side.
Reduced
efficacy
of
EMB
became a
concern
in
the Bay
of
Fundy
in
2008
[32].
How-
ever,
a
statistical
analysis
of
EMB
treatment
efficacy
data
between
2004
and
2008
revealed
that
the
efficacy
of
this
compound
declined
gradually
during
these
years
[33,34].
In-
formation
about
other
treatments
is
scarce.
DELTA
was
used
in
the
region
in
2009
and
2010.
Analysis
of
treatment
efficacy
data
demonstrated
consistently
suboptimal
efficacy
on
adult
female
parasites
(<
70%
clearance)
and
bioassays
revealed
sensitivity
levels
elevating
tenfold
between
parasite
popula-
tions
[35].
PYR
had
not
been
used
in
the
region
prior
to
the
survey.
The
results
are
in
retrospect
explained
by
methodo-
logical
errors
[36].
In
Canada,
AZA,
TEFLU,
and
H
2
O
2
have
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
75

also
been
used
to
a
limited
extent
over
the
last
5
years
[32],
but
there
are
no
reports
of
reduced
sensitivity.
Chile.
Reduced
sensitivity
has
been
reported
towards
EMB
and
DELTA
in
the
sea
lice
species
causing
the
most
problems
in
Chilean
salmon
aquaculture,
C.
rogercresseyi.
Bravo
et
al.
[37]
reported
that
treatment
failures
with
EMB
had
occurred
since
2006,
and
bioassays
demonstrated
sensitivity
levels
that
corresponded
with
the
resistant
L.
salmonis.
Helgesen
et
al.
[23]
found
that
in
2012,
the
sensitivity
of
C.
rogercres-
seyi
had
decreased
by
a
factor
of
5–10
since
the
introduction
of
DELTA
as
a
delousing
agent
in
Chile
in
2008.
Information
about
the
sensitivity
status
for
the
other
available
treat-
ments,
AZA,
DIFLU,
and
H
2
O
2
,
is
unavailable.
In
conclusion,
resistance
towards
available
treatments
is
rapidly
emerging
in
all
major
salmon-producing
countries.
This
is
supported
by
the
findings
from
Besnier
et
al.
[38].
Using
a
novel
diagnostic
tool,
the
SNP-chip,
a
genome
sequence
connected
to
EMB
resistance
found
in
sea
lice
from
all
countries
examined
[Canada,
the
Faroe
Islands,
Ireland,
Norway,
and
the
UK
(Shetland)]
was
reported.
Although
there
is
limited
information
about
the
scale
of
the
problem
in
Scotland
and
Ireland,
there
is
reason
to
believe,
based
on
the
reports
noted
above,
that
resistance
causes
serious
problems
in
all
salmonid
farming
regions
except
Western
Canada.
Monitoring
the
sensitivity
level
of
sea
lice
Traditional
resistance
detection:
bioassays
Bioassays
(biological
assays)
are
experiments
in
which
a
living
organism,
tissues,
or
cells
are
used
as
a
test
subject
[39].
Regarding
sea
lice,
the
expression
is
commonly
used
for
quantitative
response
bioassays
using
a
binary
model
for
recording
response,
that
is,
dead
versus
alive,
and
one
explanatory
variable,
the
differing
quantities
of
a
treat-
ment
chemical.
To
assess
sensitivity
levels
in
L.
salmonis,
bioassays
for
PYR,
EMB,
and
AZA
have
been
developed
[24,40–42],
and
for
EMB
and
DELTA
for
C.
rogercresseyi
[24,37].
These
original
bioassay
protocols
are
relatively
comprehensive
with
regard
to
the
number
of
parasites,
equipment,
and
labor
needed
to
perform
the
assays.
Sub-
sequently
simpler
bioassay
protocols
have
therefore
been
developed
[24,44].
Several
biases
are
connected
to
sensi-
tivity
testing
by
bioassays,
such
as
equipment
setup
and
the
personnel
performing
the
tests.
The
final
outcome
is
eventually
connected
to
sea
lice
behavior,
as
dead
versus
alive
is
measured
by
parasite
mobility.
This
contributes
to
low
test
sensitivity.
Bioassays
therefore
have
limitations
when
comparing
resistance
levels
between
different
regions,
and
also
as
a
tool
to
predict
treatment
efficacies
as
shown
for
EMB
[42].
Although
bioassays
are
valuable
tools
in
resistance
detection
when
the
resistance
mecha-
nisms
are
unknown,
the
need
for
more
accurate
and
sim-
pler
methods
has
led
to
extensive
research
to
identify
biomarkers
and
to
develop
molecular
test
methods.
Novel
methods:
molecular
sequencing
Specific
resistance
mechanisms
can
in
some
cases
be
iden-
tified
through
phenotypic
traits
displayed
by
the
resistant
organism
(Figure
1).
Molecular
methods
can
be
developed
for
known
mechanisms.
They
do
not
require
that
the
para-
sites
are
alive
when
enrolled
and
can
be
set
up
as
automated
assays
with
high
precision,
high-throughput
potential,
and
a
reduced
total
of
costs.
Candidate
gene
screening
is,
however,
labor
intensive
and
may
require
substantial
work
before
a
suitable
biomarker
candidate
has
been
detected.
A
set
of
molecular
assays
for
resistance
mechanisms
in
L.
salmonis
has
either
just
become
available
or
are
on
the
verge
of
being
so.
Figure
2
provides
an
overview
of
appro-
aches
to
identify
suitable
molecular
markers
for
resistance.
Dispersal
and
mixing
of
sea
lice
genes
in
the
ocean
The
geographic
location
of
fish
farms,
even
down
to
each
net
pen,
has
a
major
impact
on
the
sea
lice
abundance
in
an
area.
As
dispersal
of
infective
larvae
is
largely
dependent
on
water
currents,
mapping
of
the
latter
phenomena
has
been
performed
thoroughly
in
relation
to
sea
lice
dispersal
and
reproduction
[21,45,46].
Salmonids
are
anadromous
fish,
and
several
of
the
species
are
potential
hosts
for
sea
lice.
Farmed
individuals
are
hatched
in
freshwater
hatcheries,
whereas
wild
indi-
viduals
are
hatched
in
fresh
water,
mainly
in
rivers,
and
live
there
for
up
to
a
few
years
before
migrating
to
the
oceans
to
graze.
Those
who
avoid
predators
migrate
back
to
the
exact
same
river
to
spawn.
Outgoing
smolts
may
pass
close
to
several
fish
farms
on
their
way
to
pelagic
or
coastal
waters,
and
are
thereby
susceptible
to
infestation
by
sea
lice
copepodids.
These
parasites
are
likely
to
be
reprodu-
cing
when
the
fish
arrives
in
the
open
sea.
Thus,
the
sea
lice
reservoir
has
historically
been
pelagic
waters,
at
least
for
the
Atlantic
parasites.
The
migrating
routes
for
Atlantic
salmon
are
yet
to
be
fully
discovered.
The
less
host
specific
C.
rogercresseyi
are
thought
to
have
their
reservoir
on
other
fish,
mainly
the
Chilean
rock
cod
[22].
In
summary,
the
host
conditions
for
sea
lice
differ
significantly
between
locations,
and
are
thus
not
really
comparable.
Where
do
the
sea
lice
come
from?
As
reviewed
by
Todd
[47]
and
Costello
[10]
in
2006,
no
distinction
of
genetic
material
could
be
made
between
sea
lice
on
wild,
living
salmonids
and
sea
lice
parasitizing
farmed
fish.
In
Western
Canada,
returning
salmon
are
considered
the
major
reservoir
for
new
infestations
[48].
On
the
other
hand,
two
reports
from
2008
and
2009
pointed
at
farmed
fish
being
responsible
for
the
majority
of
sea
lice
larvae
in
a
Scottish
sea
loch
[49,50].
Furthermore,
as
reviewed
by
Costello
in
2009
[4],
sea
lice
originating
from
fish
farms
may
have
a
negative
effect
on
wild
salmon
and
sea
trout
populations.
However,
as
reviewed
by
Torrisen
et
al.
[6]
and
Jackson
et
al.
[29],
sea
lice
shed
from
farmed
fish
had
little
impact
on
the
survival
of
wild
salmonids,
even
though
the
infection
pressure
close
to
fish
farms
can
be
elevated
several
orders
of
magnitude
[51].
All
of
this
indicates,
as
claimed
by
Brooks
in
2005
[21],
that
the
infection
pressure
on
both
farmed
and
wild
fish
is
site-
specific,
and
that
the
larval
dispersal
varies
considerably
with
local
geographic
and
hydrographic
factors.
In
general,
local
conditions
determine
the
origin
of
the
sea
lice.
Current
evidence
supports
the
theory
that
several
At-
lantic
sea
lice
populations
are
sharing
genetic
material
coding
for
resistance,
in
this
case
resistance
towards
the
compound
EMB
[38].
Following
this
argument,
sea
lice
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
76

insensitive
to
all
the
current
remedies
will
most
likely
be
prevailing
in
the
Atlantic
Ocean
in
the
near
future.
This
scenario
would
be
regardless
of
the
sea
lice
origin,
either
from
farms
self-infecting
or
from
wild-salmonid
coloniza-
tion.
In
Pacific
Canada
and
Chile,
the
situation
is
some-
what
different,
as
the
number
of
wild
hosts
in
Pacific
Canada
is
vast
compared
to
Atlantic
farming
sites.
In
Chile,
the
host
reservoir
is
thought
to
consist
of
several
species
as
C.
rogercresseyi
is
not
very
host
specific.
Thus,
the
better
prognosis
applies
for
the
Western
Canadian
sea
lice
population,
since
resistance
is
less
common
there
than
in
northern
Europe
and
Chile.
Integrated
pest
management
for
sea
lice
So
far,
every
country
has
its
own
guidelines
for
sea
lice
regulation.
As
salmon
migration
routes
are
not
clearly
identified
to
date,
a
mutual,
international
set
of
sea
lice
legislation
would
be
beneficial.
The
European
countries
could
then
be
considered
as
one
area.
Dispersal
of
resistant
parasites
from
Norway
to
adjacent
countries
would
possi-
bly
negatively
affect
the
industry.
The
Eastern
and
West-
ern
coast
of
North
America
would
then
be
another
two
zones,
with
Chile,
isolated
geographically
and
carrying
its
own
species
of
sea
lice,
should
be
considered
the
fourth
zone.
Integrated
pest
management
(IPM)
principles
have
been
important
in
salmon
farming
ever
since
Mordue
and
Pike
introduced
the
concept
in
2002
[52].
IPM
encom-
passes
several
precaution
measures
to
combat
parasites.
The
Food
and
Agriculture
Organization
of
the
United
Nations
states:
‘The
careful
consideration
of
all
available
pest
control
techniques
and
subsequent
integration
of
Resistant individual: Mutated
ion channel gene with low
affinity to modulator drugs
Sensive individual: High
affinity to drugs acng on ion
channels
Ion channel
modulator
Ion channel
Resistant individual:
Mutated enzyme gene
Chin
synthase
inhibitor
Chin synthase
Sensive individual:
Wild type enzyme gene
Chin
Substrate
Substrate
Substrate
Substrate
Mutated chin synthase
Sensive individual: Wild type
enzyme catabolizing
neurotransmier
ACh
Acetate
Choline
AChE
Resistant individual: Mutated
gene coding for enzyme
catabolizing neurotransmier
ACh
Mutated
AChE
(A)
(B)
(C)
AChE
inhibitor
Ion channel
TRENDS in Parasitology
Figure
1.
Frequent
use
of
medicinal
compounds
will
select
individuals
who
possess
features
favoring
medicine
avoidance
for
the
next
generation.
Such
features,
which
are
subject
to
molecular
studies,
are
obtained
by
mutations
from
the
wild
type.
In
example
(A)
the
mutated
enzyme
will
not
be
targeted
by
the
xenobiotic,
and
thus
provide
for
effective
neuronal
transmission.
(B)
A
mutated
gene
coding
for
ion
channels
responsible
for
neuronal
signaling
make
the
xenobiotic
incapable
of
binding
to
its
receptor,
continuing
the
neuronal
impulse
in
the
organism.
(C)
Xenobiotics
interfering
with
specific
metabolic
processes
in
target
cells
are
ineffective
in
individuals
possessing
mutated
enzymes,
where
physiological
processes
may
continue.
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
77

appropriate
measures
that
discourage
the
development
of
pest
populations
and
keep
pesticides
and
other
interven-
tions
to
levels
that
are
economically
justified
and
reduce
or
minimize
risks
to
human
health
and
the
environment.
IPM
emphasizes
the
growth
of
a
healthy
crop
with
the
least
possible
disruption
to
agro-ecosystems
and
encourages
natural
pest
control
mechanisms’
(http://www.fao.org/
agriculture/crops/thematic-sitemap/theme/pests/ipm/en/).
Brooks
et
al.
[53]
presented
a
similar
concept
in
2009,
retrospectively
reflecting
considerable
progress
regarding
measures
taken
by
farmers
and
authorities
5
years
later.
In
a
broader
10-year
perspective,
IPM
should
include
more
pathogens
in
the
same
program.
For
salmonids,
this
would
include
amoebic
gill
disease,
virus-borne
diseases
such
as
pancreatic
disease
and
infectious
salmon
anemia,
which
all
have
substantial
impact
on
the
fish
and
cause
economic
challenges
for
the
industry
[26]
[Store
Norske
leksikon
(https://snl.no/fiskeoppdrett)].
Future
perspectives
The
ongoing
whole
genome
sequencing
and
annotation
of
L.
salmonis
and
potentially
also
of
C.
rogercresseyi
opens
numerous
avenues
to
develop
new
diagnostic
methods,
essential
to
combat
resistance
effectively.
There
are,
how-
ever,
challenges
connected
to
such
studies.
Amidines,
such
as
amitraz,
have
been
used
against
ticks
for
more
than
30
years.
Still,
the
drug’s
mode
of
action
is
relatively
unknown
[54].
Similarly,
the
mechanisms
connected
to
the
pharmacology
of
EMB
in
sea
lice
are
still
subject
to
speculation
[55].
This
provides
indications
that
investigat-
ing
medicinal
compounds,
their
effects
on
sea
lice,
and
possible
resistance
mechanisms,
are
potentially
substan-
tial
tasks
to
deal
with.
Mutations
causing
resistance
may
be
appearing
in
nuclear
DNA
as
well
as
in
ribosomal
or
mitochondrial
DNA,
of
which
the
latter
in
particular
may
represent
hard
fought
discoveries.
Until
novel
medicinal
compounds
become
available,
salmonids
infested
with
sea
lice
will
to
some
degree
have
to
be
treated
with
compounds
the
parasites
have
developed
some
tolerance
against.
This
will
enhance
the
need
for
accurate
and
reliable
diagnostic
tests,
such
as
qPCR.
In
this
respect,
molecular
studies
of
all
instars
will
be
of
crucial
importance,
in
order
to
gain
knowledge
about
rem-
edies
and
their
specific
effect
on
defined
life
cycle
stages.
In
particular,
the
expression
of
various
proteins
in
the
cha-
limus
stage
is
less
known,
representing
a
potential
for
more
effective
extirpation
of
sea
lice.
Sea
lice
management
is
subject
to
intense
research
and
development
in
several
countries.
Currently,
large
resources
are
targeting
a
vaccine
against
the
parasites
in
question,
anti-attachment
diets
are
in
development,
offshore
farms
are
in
the
planning,
whereas
cleaner
fish
and
laser
treatments
are
already
in
use.
Regarding
medic-
inal
compounds,
additional
substances
with
effects
on
one
Molecular methods as tools for screening the sensivity status of
sea lice populaons
Gene mapping:
Finding resistance-
associated
genec loci
Screening for
candidate genes
Differenal
expression
studies: Finding
genes th
at
express
differently in
resistant versus
sensive
individual
s
Proteomic
studies:
Tran
scriptomics:
Epigenec
modificaons:
Finding heritable
changes in gene
acvity or
expression,
independent of
changes in gene
sequ
ence
Mod
elin
g
simulaons:
Differenal expression
studies: Finding genes
that express differently
during treatme
nt
Screening genes
based on biological
fun
cons
Finding resistance-
associated
protein
modificaons
Imitate internal
processes to predict
effects of different
acons in silico, e.g.,
effect of different
environmental condions
or assessment of
treatment strategies on
populaon dynamics
TRENDS in Parasitology
Figure
2.
Flow
chart
visualizing
areas
of
possible
approaches
in
molecular
biology-based
research
in
resistance.
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
78

or
more
parasitic
stage
could
be
introduced
if
discovered.
Medicinal
products
ought
to
be
the
last
preventive
tool
in
line,
instead
of
the
first,
which
they
traditionally
have
been.
Altogether,
these
novel
non-medicinal
methods
may
have
the
potential
to
prolong
the
effect
of
the
current
medicinal
compounds.
Acknowledgments
This
work
was
financed
by
the
Sea
Lice
Research
Centre
platform,
NFR
203514/030,
and
the
PrevenT
project
NFR
199778/S40.
References
1
FAO
Fisheries
and
Aquaculture
Department,
Rome
(2009)
The
State
of
World
Fisheries
and
Aquaculture
2008.
Bi-annual
Report,
Food
and
Agriculture
Organization
of
the
United
Nations
2
Naylor,
R.L.
et
al.
(2009)
Feeding
aquaculture
in
an
era
of
finite
resources.
Proc.
Natl.
Acad.
Sci.
U.S.A.
106,
15103–15110
3
Denholm,
I.
et
al.
(2002)
Analysis
and
management
of
resistance
to
chemotherapeutants
in
salmon
lice,
Lepeophtheirus
salmonis
(Copepoda:
Caligidae).
Pest.
Manag.
Sci.
58,
528–536
4
Costello,
M.J.
(2014)
How
sea
lice
from
salmon
farms
may
cause
wild
salmonid
declines
in
Europe
and
North
America
and
be
a
great
threat
to
fishes
elsewhere.
Proc.
Biol.
Sci.
http://dx.doi.org/10.1098/
rspb.2009.0771
5
Finstad,
B.
et
al.
(2000)
Laboratory
and
field
investigations
of
salmon
lice
Lepeophtheirus
salmonis
infestation
on
Atlantic
salmon
(Salmo
salar
L.)
post-smolts.
Aquac.
Res.
31,
795–803
6
Torrisen,
O.
et
al.
(2013)
Salmon
lice
–
impact
on
wild
salmonids
and
salmon
aquaculture.
J.
Fish
Dis.
36,
171–194
7
Skilbrei,
O.T.
et
al.
(2013)
Impact
of
early
salmon
louse,
Lepeophtheirus
salmonis,
infestation
and
differences
in
survival
and
marine
growth
of
sea
ranched
Atlantic
salmon,
Salmo
salar
L.,
smolts
in
1997-2009.
J.
Fish.
Dis.
36,
249–260
8
Fairchild,
W.L.
et
al.
(2010)
Acute
and
chronic
toxicity
of
two
formulations
of
the
pyrethroids
pesticide
deltamethrin
to
an
amphipod,
sand
shrimp
and
lobster
larvae.
Can.
Tech.
Rep.
Fish
Aquat.
Sci.
2876,
1–34
9
Hamre,
L.
et
al.
(2013)
The
salmon
louse
Lepeophtheirus
salmonis
(Copepoda:
Caligidae)
life
cycle
has
only
two
chalimus
stages.
PLoS
ONE
8,
e73539
10
Costello,
M.J.
(2006)
Ecology
of
sea
lice
parasitic
on
farmed
and
wild
fish.
Trends
Parasitol.
22,
475–483
11
WHO
(1957)
WHO
Technical
Report
Series
No.
125,
1957.
(Insecticides:
Seventh
Report
of
the
Expert
Committee),
WHO
12
Johnson,
S.C.
et
al.
(1993)
Laboratory
investigations
on
the
efficacy
of
hydrogen-peroxide
against
the
salmon
louse
Lepeophtheirus
salmonis
and
its
toxicological
and
histopathological
effects
on
Atlantic
salmon
Salmo
salar
and
Chinook
salmon
Oncorhynchus
tshawytscha.
Dis.
Aq.
Org.
17,
197–204
13
Richterova,
Z.
and
Svobodova,
Z.
(2012)
Pyrethroids
influence
on
fish.
Slov.
Vet.
Res.
49,
63–72
14
Roy,
W.J.
et
al.
(2000)
Tolerance
of
Atlantic
salmon,
Salmo
salar
L.,
and
rainbow
trout,
Oncorhynchus
mykiss
(Walbaum),
to
emamectin
benzoate,
a
new
orally
administered
treatment
for
sea
lice.
Aquaculture
184,
19–29
15
Kunz,
S.E.
and
Kemp,
D.H.
(1994)
Insecticides
and
acaricides
–
resistance
and
environmental
impact.
Rev.
Sci.
Tech.
13,
1249–1286
16
Brattsten,
L.B.
et
al.
(1986)
Insecticide
resistance
–
challenge
to
pest-
management
and
basic
research.
Science
231,
1255–1260
17
Alonso-Diaz,
M.A.
et
al.
(2013)
Amblyomma
cajennense
(Acari:
Ixodidae)
tick
populations
susceptible
or
resistant
to
acaricides
in
the
Mexican
tropics.
Vet.
Parasitol.
197,
326–331
18
Magiorakos,
A.P.
et
al.
(2011)
Multidrug-resistant,
extensively
drug-
resistant
and
pandrug
resistant
bacteria:
an
international
expert
proposal
for
interim
standard
definitions
for
acquired
resistance.
Clin.
Microbiol.
Infect.
18,
268–281
19
Espedal,
P.G.
et
al.
(2013)
Emamectin
benzoate
resistance
and
fitness
in
laboratory
reared
salmon
lice
(Lepeophtheirus
salmonis).
Aquaculture
416–417,
111–118
20
Bravo,
S.
et
al.
(2010)
Sensitivity
assessment
in
the
progeny
of
Caligus
rogercresseyi
to
emamectin
benzoate.
Bull.
Eur.
Assoc.
Fish
Pathol.
30,
99–105
21
Brooks,
K.
(2005)
The
effects
of
water
temperature,
salinity,
and
currents
on
the
survival
and
distribution
of
the
infective
copepodid
stage
of
sea
lice
(Lepeophtheirus
salmonis)
originating
on
Atlantic
salmon
farms
in
the
Broughton
Archipelago
of
British
Columbia,
Canada.
Rev.
Fish.
Sci.
13,
177–204
22
Gonzalez,
M.T.
(2012)
Fecundity
of
the
sea
louse
Caligus
rogercresseyi
on
its
native
host
Eleginops
maclovinus
captured
near
salmon
farms
in
southern
Chile.
Aquac.
Res.
43,
853–860
23
Helgesen,
K.O.
et
al.
(2014)
Deltamethrin
resistance
in
the
sea
louse
Caligus
rogercresseyi
(Boxhall
and
Bravo)
in
Chile:
bioassay
results
and
usage
data
for
antiparasitic
agents
with
reference
to
Norwegian
conditions.
J.
Fish.
Dis.
37,
877–890
24
Helgesen,
K.O.
and
Horsberg,
T.E.
(2013)
Single
dose
field
bioassay
for
sensitivity
testing
in
sea
lice,
Lepeophtheirus
salmonis:
development
of
a
rapid
diagnostic
tool.
J.
Fish
Dis.
36,
261–272
25
Grøntvedt,
R.N.
et
al.
(2014)
The
Surveillance
Program
for
Resistance
to
Chemotherapeutants
in
L.
salmonis
in
Norway
2013.
Annual
Report
Oslo,
Norwegian
Veterinary
Institute
26
Adams,
M.B.
et
al.
(2012)
Preliminary
success
using
hydrogen
peroxide
to
treat
Atlantic
salmon,
Salmo
salar
L.,
affected
with
experimentally
induced
amoebic
gill
disease
(AGD).
J.
Fish
Dis.
35,
839–848
27
Heumann,
J.
et
al.
(2012)
Molecular
cloning
and
characterization
of
a
novel
P-glycoprotein
in
the
salmon
louse
Lepeophtheirus
salmonis.
Comp.
Biochem.
Physiol.
C
198–205
28
Lees,
F.
et
al.
(2008)
The
efficacy
of
emamectin
benzoate
against
infestations
of
Lepeophtheirus
salmonis
on
farmed
Atlantic
salmon
(Salmo
salar
L.)
in
Scotland
between
2002
and
2006.
PLoS
ONE
3,
e1549
29
Jackson,
D.
(2011)
Ireland:
The
development
of
sea
lice
management
methods.
In
Salmon
Lice:
an
Integrated
Approach
to
Understanding
Parasite
Abundance
and
Distribution
(Jones,
S.
and
Beamish,
R.,
eds),
pp.
177–203,
Wiley-Blackwell
30
Saksida,
S.M.
et
al.
(2013)
Use
of
Atlantic
salmon,
Salmo
salar
L.,
farm
treatment
data
and
bioassays
to
assess
for
resistance
of
sea
lice,
Lepeophtheirus
salmonis,
to
emamectin
benzoate
(SLICE1)
in
British
Columbia,
Canada.
J.
Fish
Dis.
36,
515–520
31
Skern
Mauritzen,
R.
et
al.
(2014)
Pacific
and
Atlantic
Lepeophtheirus
salmonis
(Krøyer,
1838)
are
allopatric
subspecies:
Lepeophtheirus
salmonis
salmonis
and.
L.
salmonis
oncorhynchi
subspecies
novo.
BMC
Genet.
15,
32
32
Chang,
B.D.
et
al.
(2011)
Sea
louse
abundance
on
farmed
salmon
in
the
southwestern
New
Brunswick
area
of
the
Bay
of
Fundy.
In
Salmon
Lice:
an
Integrated
Approach
to
Understanding
Parasite
Abundance
and
Distribution
(Jones,
S.
and
Beamish,
R.,
eds),
pp.
83–115,
Wiley-
Blackwell
33
Jones,
P.G.
et
al.
(2012)
Effectiveness
of
emamectin
benzoate
for
treatment
of
Lepeophthe irus
salmonis
on
farmed
Atlantic
salmon
Salmo
salar
in
the
Bay
of
Fundy.
Can.
Dis.
Aquat.
Organ.
102,
53–64
34
Jones,
P.G.
et
al.
(2013)
Detection
of
emamectin
benzoate
tolerance
emergence
in
different
life
stages
of
sea
lice,
Lepeophtheirus
salmonis,
on
farmed
Atlantic
salmon,
Salmo
salar
L.
J.
Fish
Dis.
36,
209–220
35
Whyte,
S.K.
et
al.
(2014)
Assessment
of
sea
lice
(Lepeophtheirus
salmonis)
management
in
New
Brunswick,
Canada
using
deltamethrin
(AlphaMax1)
through
clinical
field
treatment
and
laboratory
bioassay
responses.
Aquaculture
422–423,
54–62
36
Beattie,
M.
(2009)
Industry
Update
on
Pesticide
Usage.
In
Integrated
Pest
Management
Sea
Lice
Treatments
Application
Requirements
and
Systemic
Use
Workshop.
Workshop
report
St.
Andrews
NB.
New
Brunswick
Salmon
Growers
Association
37
Bravo,
S.
et
al.
(2008)
Sensitivity
assessment
of
Caligus
rogercresseyi
to
emamectin
benzoate
in
Chile.
Aquaculture
282,
7–12
38
Besnier,
F.
et
al.
(2014)
Human-induced
evolution
caught
in
action:
SNP-array
reveals
rapid
amphi-atlantic
spread
of
pesticide
resistance
in
the
salmon
ecotoparasite
Lepeophtheirus
salmonis.
BMC
Genomics
15,
937
39
Robertson,
J.L.
et
al.,
eds
(2007)
Bioassays
in
Arthropods
(2nd
edn),
CMC
Press
40
Sevatdal,
S.
et
al.
(2005)
Monitoring
of
the
sensitivity
of
sea
lice
(Lepeophtheirus
salmonis)
to
pyrethroids
in
Norway,
Ireland
and
Scotland
using
bioassays
and
probit
modelling.
Aquaculture
244,
19–27
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
79

41
Sevatdal,
S.
and
Horsberg,
T.E.
(2003)
Determination
of
reduced
sensitivity
in
sea
lice
(Lepeophtheirus
salmonis)
against
the
pyrethroid
deltamethrin
using
bioassays
and
probit
modelling.
Aquaculture
218,
21–31
42
Westcott,
J.D.
et
al.
(2008)
Optimization
and
field
use
of
a
bioassay
to
monitor
sea
lice
Lepeophtheirus
salmonis
sensitivity
to
emamectin
benzoate.
Dis.
Aquat.
Organ.
79,
119–131
43
Burridge,
L.E.
et
al.
(2014)
The
acute
lethality
of
three
anti-sea
lice
formulations:
Alphamax1,
Salmosan1,
and
Interox1Paramove
TM
50
to
lobster
and
shrimp.
Aquaculture
420–421,
180–186
44
Whyte,
S.K.
et
al.
(2013)
A
fixed-dose
approach
to
conducting
emamectin
benzoate
tolerance
assessments
on
field-collected
sea
lice,
Lepeophtheirus
salmonis.
J.
Fish
Dis.
36,
283–292
45
Amundrud,
T.L.
and
Murray,
A.G.
(2009)
Modelling
sea
lice
dispersion
under
varying
environmental
forcing
in
a
Scottish
sea
loch.
J.
Fish
Dis.
32,
27–44
46
Stormoen,
M.
et
al.
(2012)
Modelling
salmon
lice,
Lepeophtheirus
salmonis,
reproduction
on
farmed
Atlantic
salmon,
Salmo
salar
L.
J.
Fish
Dis.
36,
25–33
47
Todd,
C.D.
(2006)
The
copepod
parasite
(Lepeophtheirus
salmonis
(Krøyer),
Caligus
elongatus
Nordmann)
interactions
between
wild
and
farmed
Atlantic
salmon
(Salmo
salar
L.)
and
wild
sea
trout
(Salmo
trutta
L.):
a
mini
review.
J.
Plank.
Res.
29,
161–171
48
Gottesfeld,
A.S.
et
al.
(2009)
Sea
lice,
Lepeophtheirus
salmonis,
transfer
between
wild
sympatric
adult
and
juvenile
salmon
on
the
north
coast
of
British
Columbia,
Canada.
J.
Fish.
Dis.
32,
45–57
49
Penston,
M.J.
et
al.
(2008)
Spatial
and
temporal
distribution
of
Lepeophtheirus
salmonis
(Krøyer)
larvae
in
a
sea
loch
containing
Atlantic
salmon,
Salmo
salar
L.,
farms
on
the
north-west
coast
of
Scotland.
J.
Fish
Dis.
31,
361–371
50
Penston,
M.J.
and
Davies,
I.M.
(2009)
An
assessment
of
salmon
farms
and
wild
salmonids
as
sources
of
Lepeophtheirus
salmonis
(Krøyer)
copepodids
in
the
water
column
in
Loch
Torridon,
Scotland.
J.
Fish
Dis.
32,
75–88
51
Krkos
ˇ
ek,
M.
et
al.
(2005)
Transmission
dynamics
of
parasitic
sea
lice
from
farm
to
wild
salmon.
Proc.
R.
Soc.
B
272,
689–696
52
Mordue,
J.
and
Pike,
A.
(2002)
Salmon
farming:
towards
an
integrated
pest
management
strategy
for
sea
lice.
Pest.
Manag.
Sci.
58,
513–514
53
Brooks,
K.M.
(2009)
Considerations
in
developing
an
integrated
pest
management
programme
for
control
of
sea
lice
on
farmed
salmon
in
Pacific
Canada.
J.
Fish
Dis.
32,
59–73
54
Guerrero,
G.
et
al.
(2012)
Acaricide
resistance
mechanisms
in
Rhipicephalus
(Boophilus)
microplus.
Braz.
J.
Vet.
Parasitol.
21,
1–6
55
Sutherland,
B.
et
al.
(2014)
Transcriptomic
responses
to
emamectin
benzoate
in
Pacific
and
Atlantic
Canada
salmon
lice
Lepeophtheirus
salmonis
with
differing
levels
of
drug
resistance.
Ev.
App.
http://
dx.doi.org/10.111/eva.12237
56
Geary,
T.G.
and
Morena,
Y.
(2012)
Macrocyclic
lactone
anthelmintics:
Spectrum
of
activity
and
mechanism
of
action.
Curr.
Pharm.
Biotech.
13,
866–872
57
He,
L.
et
al.
(2009)
Genetic
analysis
of
abamectin
resistance
in
Tetranychus
cinnabarinus.
Pestic.
Biochem.
Physiol.
95,
147–151
58
Ghosh,
R.
(2012)
Natural
variation
in
a
chloride
channel
subunit
confers
avermectins
resistance
in
C.
elegans.
Science
335,
573
59
Wolstenholme,
A.J.
and
Kaplan,
R.M.
(2012)
Resistance
to
macrocyclic
lactones.
Curr.
Pharm.
Biotech.
13,
873–887
60
Kwon,
D.H.
et
al.
(2010)
A
point
mutation
in
a
glutamate-gated
chloride
channel
confers
abamectin
resistance
in
the
two-spotted
spider
mite,
Tetranychus
urticae
Koch.
Insect
Mol.
Biol.
19,
583–591
61
Igboeli,
O.O.
et
al.
(2012)
Role
of
P-glycoprotein
in
emamectin
benzoate
(SLICE1)
resistance
in
sea
lice,
Lepeophtheirus
salmonis.
Aquaculture
344–349,
40–47
62
Carmichael,
S.N.
et
al.
(2013)
Salmon
lice
(Lepeophtheirus
salmonis)
showing
varying
emamactin
benzoate
susceptibilities
differ
in
neuronal
acetylcholine
receptor
and
GABA-gated
chloride
channel
mRNA
expression.
BMC
Genet.
14,
408
63
Merzendorfer,
H.
(2013)
Chitin
synthesis
inhibitors:
old
molecules
and
new
developments.
Insect
Sci.
20,
121–138
64
Pimprikar,
G.D.
and
Georghiou,
G.P.
(1979)
Mechanisms
of
resistance
to
diflubenzuron
in
the
house
fly
Musca
domestica
(L).
Pest.
Biochem.
Physiol.
12,
10–22
65
Van
Leeuwen,
T.
et
al.
(2012)
Population
bulk
segregant
mapping
uncovers
resistance
mutations
and
the
mode
of
action
of
a
chitin
synthesis
inhibitor
in
arthropods.
Proc.
Natl.
Acad.
Sci.
U.S.A.
109,
4407–4412
66
Bruno,
D.W.
and
Raynard,
R.S.
(1994)
Studies
on
the
use
of
hydrogen
peroxide
as
a
method
for
the
control
of
sea
lice
on
Atlantic
salmon.
Aquacult.
Int.
2,
10–18
67
Thomassen,
J.M.
(1993)
A
new
method
for
control
of
salmon
lice.
In
Fish
Farming
Technology
(Reinertsen,
H.
et
al.,
eds),
pp.
233–236,
A.A.
Balkema
68
Fiander,
H.
and
Schneider,
H.
(2000)
Dietary
ortho
phenols
that
induce
glutathione
S-transferase
and
increase
the
resistance
of
cells
to
hydrogen
peroxide
are
potential
cancer
chemopreventives
that
act
by
two
mechanisms:
the
alleviation
of
oxidative
stress
and
the
detoxification
of
mutagenic
xenobiotics.
Cancer
Lett.
156,
117–124
69
Spitz,
D.R.
et
al.
(1992)
Mechanisms
of
cellular
resistance
to
hydrogen-
peroxide,
hyperoxia
and
4-hydroxy-2-nonenal
toxicity
–
the
significance
of
increased
catalase
activity
in
H
2
O
2
-resistant
fibroblasts.
Arch.
Biochem.
Biophys.
292,
221–227
70
Nakamura,
K.
et
al.
(2012)
Microbial
resistance
in
relation
to
catalase
activity
to
oxidative
stress
induced
by
photolysis
of
hydrogen
peroxide.
Microbiol.
Immunol.
56,
48–55
71
Uhlich,
G.A.
(2009)
KatP
contributes
to
OxyR-regulated
hydrogen
peroxide
resistance
in
Escherichia
coli
serotype
O157:H7.
Microbiol.
Sgm
155,
3589–3598
72
Helgesen,
K.O.
et
al.
(2014)
First
report
of
reduced
hydrogen
peroxide
sensitivity
in
the
salmon
louse
Lepeophtheirus
salmonis
in
Norway.
Aquacult.
Rep.
In
press
73
Smissaert,
H.R.
(1964)
Cholinesterase
inhibition
in
spider
mites
susceptible
and
resistant
to
organophosphate.
Science
143,
129–131
74
Kaur,
K.
et
al.
(2015)
Identification
of
the
mechanism
behind
resistance
against
the
organophosphate
azamethiphos
in
salmon
lice
(Lepeophtheirus
salmonis).
PLoS
ONE
In
press
75
Weston,
D.P.
et
al.
(2013)
Multiple
origins
of
pyrethroid
insecticide
resistance
across
the
species
complex
of
a
nontarget
aquatic
crustacean,
Hyalella
azteca.
Proc.
Natl.
Acad.
Sci.
U.S.A.
110,
16532–
16537
76
Mu¨
ller,
P.
et
al.
(2007)
Transcription
profiling
of
a
recently
colonized
pyrethroids
resistant
Anopheles
gambiae
strain
from
Ghana.
BMC
Genet.
8,
36
77
Ranson,
H.
et
al.
(2011)
Pyrethroid
resistance
in
African
anopheline
mosquitoes:
what
are
the
implications
for
malaria
control?
Trends
Parasitol.
27,
91–98
78
Vontas,
J.G.
et
al.
(2001)
Glutathione
S-transferases
as
antioxidant
defence
agents
confer
pyrethroids
resistance
in
Nilaparvata
lugens.
Biochem.
J.
357,
65–72
79
Xu,
L.
et
al.
(2013)
Overexpression
of
multiple
detoxification
genes
in
deltamethrin
resistant
Laodelphax
striatellus
(Hemiptera:
Delphacidae)
in
China.
PLoS
ONE
8,
e79443
80
Bloomquist,
J.
(1996)
Ion
channels
as
targets
for
insecticides.
Annu.
Rev.
Entomol.
41,
163–190
81
Davies,
T.G.E.
et
al.
(2007)
DDT,
pyrethrins,
pyrethroids
and
insect
sodium
channels.
IUBMB
Life
59,
151–162
82
Rinkevich,
F.D.
et
al.
(2013)
Diversity
and
convergence
of
sodium
channel
mutations
involved
in
resistance
to
pyrethroids.
Pest.
Biochem.
Physiol.
106,
93–100
83
Koganemaru,
R.
et
al.
(2013)
Robust
cuticular
penetration
resistance
in
the
common
bed
bug
(Cimex
lectularius
L.)
correlates
with
increased
steady–state
transcript
levels
of
CPR-type
cuticle
protein
genes.
Pest.
Biochem.
Physiol.
106,
190–197
84
Sevatdal,
S.
et
al.
(2005)
Monooxygenase
mediated
pyrethroid
detoxification
in
sea
lice
(Lepeophtheirus
salmonis).
Pest.
Manag.
Sci.
61,
772–778
85
Bravo,
S.
et
al.
(2014)
Efficacy
of
deltamethrin
in
the
control
of
Caligus
rogercresseyi
(Boxshall
and
Bravo)
using
bath
treatment.
Aquaculture
432,
175–180
86
O’Donohoe,
P.
et
al.
(2012)
National
Survey
of
Sea
Lice
(Lepeophtheirus
salmonis
Krøyer
and
Caligus
elongatus
Nordmann)
on
Fish
Farms
in
Ireland
–
2011.
Irish
Fisheries
Bulletin
No
40,
Marine
Institute
87
Grave,
K.
and
Horsberg,
T.E.
(2000)
Terapianbefaling:
Behandling
mot
lakselus
i
oppdrettsanlegg
(2000:02),
Norwegian
Medicines
Control
Authority
88
Grave,
K.
et
al.
(2004)
Consumption
of
drugs
for
sea
lice
infestations
in
Norwegian
fish
farms:
methods
for
assessment
of
treatment
patterns
and
treatment
rate.
Dis.
Aquat.
Organ.
60,
123–131
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
80

89
Corner,
A.R.
et
al.
(2006)
A
Review
of
the
Sea
Lice
Bath
Treatment
Dispersion
Model
Used
for
Discharge
Consenting
in
Scotland.
A
Report
to
the
Scottish
Aquaculture
Research
Forum,
University
of
Sterling
90
Marin,
S.L.
et
al.
(2014)
Effects
of
Caligus
rogercresseyi
(Boxshall
&
Bravo
2000)
chalimus
stage
condition
(dead,
moribund,
live)
on
the
estimates
of
Cypermethrin
BETAMAX1
efficacy.
Aquac.
Res.
http://
dx.doi.org/10.1111/are.12460
91
Roth,
M.
(2000)
The
availability
and
use
of
chemotherapeutic
sea
lice
control
products.
Contrib.
Zool.
69,
1–2
92
Bravo,
S.
et
al.
(2010)
Effectiveness
of
hydrogen
peroxide
in
the
control
of
Caligus
rogercresseyi
in
Chile
and
implications
for
sea
louse
management.
Aquaculture
303,
22–27
93
Lees,
F.
et
al.
(2009)
Strategic
Sea
Lice
Control
Implementation
and
Impact
Within
Scottish
Tripartite
Wo-rking
Group
Area
Management
Agreements.
Report,
University
of
Strathclyde,
Glasgow
Review
Trends
in
Parasitology
February
2015,
Vol.
31,
No.
2
81