Sea Lice Resistance to
Chemotherapeutants (SEARCH):
diagnosis, mechanisms, dynamics and
control
Resources
6 partners
Norwegian School of Veterinary Science
National University of Ireland Galway
Irish Marine Institute
Institute for Arable Crop Research,Rothamsted
Marine Harvest Norway
Marine Harvest Scotland
University of Prince Edward Island, in
collaboration with NRC Institute for Marine
Biosciences
Period: Jan. 1. 2001 - Jan.1. 2004
Canadian Team:
John F. Burka, Ph.D.
Larry Hammell, D.V.M., M.Sc.
Department of Health Management, Atlantic
Veterinary College, UPEI
Neil W. Ross, Ph.D.
Department of Anatomy & Physiology , Atlantic
Veterinary College, UPEI
Institute for Marine Biosciences, National Research
Council
Technical personnel, graduate students,
summer students
Funding and/or in-kind support
EU 5th Framework Programme
AquaNet
NSERC/NRC Collaborative Research
Programme (with Salmon Health Consortium and AVC
Inc.)
Schering-Plough Animal Health
Moore-Clark (Nutreco)
New Brunswick Salmon Growers Association
Objectives
To develop methods for monitoring the
development of resistance against
chemotherapeutants commonly used for
treatment of Lepeophtheirus salmonis in
Norway, Scotland, Ireland, and eastern
North America
Organophosphates
Pyrethroids
Avermectins
100
90
80
70
60
50
40
30
20
10
0
Org-P.
H2O2
Chit.synth.inh
.
Pyrethroids
19
95
19
96
19
97
19
98
19
99
%
Relative treatment intensity
year
Emamectin
Resistance mechanisms for
organophosphates and pyrethroids
I
Enhanced detoxification capacity
• Elevated levels of unspecific esterases
• Elevated levels of unspecific P450 oxidases
I
Mutations in genes coding for target proteins,
resulting in structural changes
OPs
Na+
Na+
Na+
Pyr.
Na+
Na+
Na+
Factors affecting sea lice treatments
with emamectin
Oral ingestion
Fish must eat medicated feed
Concentrations at the site of parasite location
Distribution into subcompartments
Affected by health of fish, hierarchies, etc.
Lipophilic:
Emamectin distributed into skin/hypodermic tissues and
mucus
Sea lice feed on mucus, epidermis, and blood
Time of parasite exposure
Emamectin requires about a week to achieve sufficient levels
in sea lice
Sea lice-dependent
Larval vs. adult stages
P-glycoproteins affect uptake
Antiparasitic agents and
P-glycoproteins (P-GPs)
Avermectins are P-GP substrates
P-GPs are responsible for protecting the blood
brain barrier of the host from avermectininduced CNS depression
P-GPs can be up-regulated in parasites
Resistance mechanisms for
avermectins
Changes in glutamate-gated chloride channels
contribute to avermectin resistance in
Haemonchus contortus and Drosophila
(Paement et al., 1999; Kane et al., 2000)
Does alteration in the glu-gated chloride channels
occur in sea lice and reduce hyperpolarization?
P-GPs are involved in the mechanism of
nematode resistance to avermectins (Xu et al.,
1998)
Does upregulation of P-glycoproteins prevent
avermectins from accumulating in sea lice?
Approaches to detect and verify
resistance
I
I
Efficacy data from full-scale treatments
Bioassays
• Determination of LC 50 and LC90 (EC50 and EC90 )
I
Biochemical methods (only when resistance mechanisms are
known)
• OP resistance: altered target site (modified AChE)
• Enhanced detoxification capacity
– Esterases, Oxidases, Etc.
I
Molecular methods
• Organophosphate resistance: altered AChE-gene(s)
• Pyrethroid resistance: altered sodium channel gene(s)
• Avermectin resistance: altered glu-gated chloride channel
gene(s) and/or upregulated P-glycoproteins
Project plans
Specific plans
To develop bioassays capable of detecting resistance
towards chemotherapeutants
To monitor the effectiveness of sea lice treatments
Characterise underlying mechanisms for resistance
Develop rapid laboratory methods for diagnosis of resistance
Develop genetic markers capable of distinguishing between sea lice
populations
Monitor patterns of gene flow between farms and regions
Monitor spacial and temporal distribution of sensitivity in sea lice
populations
To develop strategies for the control of resistance
Where are we
now?
Designation of
collection sites
Sea lice collections: Bay of Fundy
Collecting from 4 sea lice
populations
Saint John
Halifax
Bioassays
Bioassay for azamethiphos
Apparent high sensitivity of sea lice to
azamethiphos in all samples collected in NB
so far
Bioassay for emamectin
Modifications required – lice need to eat
emamectin
In progress
Epidemiology of lice infestations and
treatment efficacy
Larry Hammell and Jillian Westcott
Field Trial
Individually PIT-tag 2200
fish (2001 year class)
Obtain lengths, weights
and sea lice counts
Obtain samples of 30
randomly selected fish for
prevalence estimates of
selected diseases (ISA
and BKD)
Tissues obtained and
stored for subsequent
ISA testing
PIT Tag
Emamectin Field Trial
Estimate effectiveness of emamectin treatments
on sea lice counts and weight gains
Comparison of individual fish variables on sea
lice burdens, ISA and BKD prevalence
Comparison of emamectin uptake by different
sized fish following treatment
Survey of pesticide use in Atlantic Canada
salmon aquaculture
In progress
Emamectin receptor studies
Shara Cody and John Burka
To determine whether molecular heterogeneity of avermectin
receptors exists in sea lice populations
Are receptors altered in resistant sea lice?
Emamectin transport protein (P-glycoprotein)
studies
Nick Tribble and John Burka
To measure emamectin concentrations in sea lice and
differential expression of avermectin transport proteins
Do emamectin concentrations in sea lice correlate with
resistance?