Note: Descriptions are shown in the official language in which they were submitted.
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TITLE: Method for detecting hydrogen peroxide resistance in crustaceans
Field of the invention
The present invention relates to a method for detecting whether or not
crustaceans, in
particular sea lice, such as Lepeophtheirus salmonis and Caligus rogereresseyi
is resistant
towards hydrogen peroxide (f1/0/). In particular, the method of the invention
relates to the
detection of the expression level of genes found to be either upregulated or
downregulated
in sea lice resistant towards H202. The present invention furthermore relates
to
oligonucicotidc sequences and kits useful in thc method of the present
invention.
Background of the invention
Sea lice are naturally occurring marine ectoparasites that attach to the skin
and feed on the
mucus, blood and surface tissues of salmon and other species of fish. Sea lice
(Lepeophtheirus salmonis and Caligus spp.) arc the major pathogens affecting
global
salmon farming industry and have a significant impact in many areas. The
annual loss was
for the global salmon farming industry were estimated to Ã300 million in 2009
(Costello M.
J. (2009), The global economic cost of sea lice to the salmonid farming
industry. Journal of
Fish Diseases. 32. 115 118), and in 2013 it was estimated to USD 300 mill in
Chile alone
(Augusti C., Bravo S., Contreras G., Bakke M.J., Helgesen K.O., Winkler C.,
Silva M.T.,
Mendosa J., Horsberg T.E. (2016), Sensitivity assessment of (Vigils
rogereresseyi to anti-louse
chemicals in relation to treatment efficacy in Chilean salmonid farms.
Aquaculture 458, 195-
205).
Thus, aquaculture industry relays heavily on a few chemotherapeutants for sea
lice control.
Emerging resistance development to these drugs increase the necessity to
develop new
treatment methods (biological, prophylactic and drugs) and tools to avoid
increased loss
due to sea lice and to ensure a sustainable salmon farming industry in the
future. Control
measures have relied upon a limited number of chemothcrapeutants since the
1970s.
Parasite resistance and reduced efficacy have now been reported for the
majority of these
compounds (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 modeling)", Aquaculture 244, 19-27). A successful integrated louse-
management strategy requires free access to a range of effective, chemically
unrelated
active ingredients deployed according to current best practice. Over-reliance
on a limited
number of products will lead, inevitably to resistance, which is difficult to
counter.
Although various chemotherapeutants have been in use in the aquaculture
industry for
more than 35 years, it is only during the last 20 years that such use has been
part of some
kind of integrated pest management (IPM) system. Management practices include
coordinated salmon production within a defined area, use of single year class
of fish,
limited production period, fallowing, coordinated restocking, use of wrasse,
synchronized
treatments during the winter and targeting female lice to reduce the impact of
settlement
during the spring (Pike A., Wadsworth S. L., (2000), -Sea Lice: A review.
Advances in
Parasitology", Academic Press. 44. 232-337).
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Further tools are required to progress towards a true IPM-system common to
other forms of
food production. One key to succeeding with an IPM is to develop tools for the
management of resistance to the medicines in use (Brook K. (2009),
"Considerations in
developing an integrated pest management program for control of sea lice on
farmed salmon in
Pacific Canada-, Journal of Fish Diseases. 32, 59-73). Bioassays that require
comprehensive
numbers of parasites, equipment and labor were used to monitor sensitivity.
However,
these methods showed low sensitivity, and had limitations when comparing
resistance in
different regions, as well as predicting treatment efficacy. Resistance
mechanisms can be
identified in some cases, and molecular methods, with high precision, high -
throughput
potential and reduced total cost (Aaen et al., 2015, "Drug resistance in sea
lice: a threat to
salmonid aquaculture". Trends in Parasitology, Vol 31, No. 2). Resistance
towards
organophosphates has been linked to Phe362Tyr mutation in the AChE coding
gene. For
pyrethroids several factors are likely to be involved in resistance,
cytochrome P450 is
involved in the detoxification of pyrethroids, as well as biomarkers in the
mitochondrial
DNA (Bakke et al., 2018,"Deltamethrin resistance in the salmon louse.
Lepeophtheirus
salmonis (Kroyer): Maternal inheritance and reduced apoptosis." Scientific
Reports, Vol. 8,
8450). Using these genes, it was demonstrated that medicinal treatments drive
genetic
selection towards a more resistant salmon lice population within a very short
time span if
there is no influx of sensitive genotypes (Jensen et al., 2017, "A selection
study on
laboratory-designed population of salmon lice (Lepeophtherius salrnonis) using
organophosphatc and pyrcthroid pesticides", PLOS one12(5)e0178086).
PCR based methods for detection of resistance genes have been published for
azamethiphos
(EP 3 033 433 A 1 ), pyrethroids (EP 3 030 673 Al, EP 3 030 674 Al) as well as
the
catalase gene for hydrogen peroxide resistance (EP 3 164 502 B1).
Hydrogen peroxide has been demonstrated to be the least harmful to non-target
organisms, highly
effective against certain stages of louse development and most environmentally
responsible
(Burridge, L. 2013, -A review of potential environmental risks associated with
the use of
pesticides to treat Atlantic salmon against infestations of sea lice in
southwest New Brunswick,
Canada, DFO Can. Sci. Advis. Sec. Res. Doc. 2013/050. iv + 25 p). Interox
Paraniove 50 is a
commercially available hydrogen peroxide product for the treatment of sea
lice. It contains 50%
hydrogen peroxide.
Hydrogen peroxide is only efficacious on post-chalimus stages of sea lice; it
must be used in
conjunction with other pest management techniques to maximize treatment
benefits. There are
conflicting results regarding viability of sea lice post treatment as well as
the ability of lice re-
infection. Because there are no techniques currently developed to remove sea
lice from a tarp
treatment, timing of treatment to target post-chalimus stage lice is
essential. Recent studies of egg
viability and nauplii survival post-treatment do indicate that nauplii
survival reaches 0 within
days of hatch.
Hydrogen peroxide breaks down readily into water and is considered the anti-
louse treatment
with least environmental risk. Recent studies comparing the effects of Interox
Paramove 50,
Salmosan , and AlphaMax on American lobster, mysid shrimp and sand shrimp
showed that
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Interox Paramove 50 had the least impact on these non-target organisms.
Unlike other anti-
louse chemotherapeutants. hydrogen peroxide does not have a withdrawal period.
Hydrogen peroxide is believed to be available for use in all major salmon
farming countries. It
was a common louse treatment in the 1990's but was subsequently replaced by in-
feed louse
treatments and other bath treatments until a recent resurgence. In 2009, the
use of H202 in
aquaculture in Norway was 308 tons, in 2010 it was 3071 tons and in 2012 it
was 2538 tons,
further it was 8262 tons in 2013, 31577 tons in 2014, 43 246 tons in 2015,
26597 tons in 2016
and 9277 tons in 2017 (cf. Norwegian Institute of Public Health 2018
www.fhi.no).
Organisms naturally produce hydrogen peroxide as a by-product of oxidative
metabolism.
Consequently, nearly all living things (specifically, all obligate and
facultative aerobes)
possess enzymes known as catalase and peroxidases, which harmlessly and
catalytically
decompose low concentrations of hydrogen peroxide to water and oxygen. Two
theories
have been proposed to explain the therapeutic effects of hydrogen peroxide.
The first is
that bactericidal action is through the formation of hydroxyl radicals and its
effect on DNA
(Imlay J. A. (1987), "The mechanisms of toxicity of hydrogen peroxide", PhD
Thesis,
University of California, Berkeley). The second, proposed to explain toxicity
to protistans
and monogeneans, is the liberation of molecular oxygen as a result of catalase
action
(Schaperclaus et al., eds. (1979), Fishkrankheiten, 4th edn. Academic-Verlag.
Berlin).
Resistance of insects to pesticides develops through genetic selection of
individuals
(Soderlund D.M. & Bloomquist J.R. (1990), -Molecular mechanisms of insecticide
resistance. In: Pesticide Resistance in Arthropods" (ed. by R.T. Roush &
B.E.Tabashnik),
pp. 58-96. Chapman & Hall, London) and, in lice, this may be selection for
individuals
with cuticle that provides a barrier to penetration by hydrogen peroxide or
the presence of
detoxifying enzymes such as catalase, glutathione reductase, glutathione
synthetase,
superoxide dismutase, and glucose-6-phosphate dehydrogenase. An alternative
explanation
could be prior induction as reported for Aeromonas scampi-Jidda pre-exposed to
low
concentrations of hydrogen peroxide (Barnes et al., (1999a) "Superoxide
dismutase and
catalase in Photobacterium damselae suhsp. piscicida and their roles in
resistance to
reactive oxygen species", Microbiology 145, 483-494 & Barnes et al. (1999b),
"Peroxide-
inducible catalase in Aeromonas salmonicida subsp. salmonicida protects
against
exogenous hydrogen peroxide and killing by activated rainbow trout,
Oncorhynchus
mykiss. L. macrophages-, Microbial Pathogenesis 26, 149-158). These bacteria
had
catalase activity 20-fold higher when subsequently exposed to higher
concentrations than
in un-induced cultures.
Catalase is a common enzyme found in nearly all living organisms exposed to
oxygen. It
catalyzes the decomposition of hydrogen peroxide to water and oxygen
(Chelikani et
al.,January 2004, "Diversity of structures and properties among catalases",
Cell. Mol. Life
Sci. 61(2): 192-208). It is a very important enzyme in protecting the cell
from oxidative
damage by reactive oxygen species (ROS). Likewise, catalase has one of the
highest
turnover numbers of all enzymes; one catalase molecule can convert millions of
molecules
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of hydrogen peroxide to water and oxygen each second (Goodsell (2004-09-01),
''Catalase.
Molecule of the Month-, RCSB Protein Data Bank. Retrieved 2007-02-11).
The development of resistance by sea lice to medicines and its management is
one of the
main concerns in sea lice control, particularly when the range of medicines is
limited.
Resistance of sea lice to pesticides, organophosphates, pyrethroids and H202
is well established. Reduced sensitivity towards hydrogen peroxide (H202) was
first
reported in Scotland (Treasurer et al. (2000), "Resistance of sea lice,
Lepeophtheirus
salmonis (Kroyer), to hydrogen peroxide on farmed Atlantic salmon, Salmo salar
Aquaculture Research, 31, 855-860), and from Norway in 2013 (Helgesen et al.,
2015,
"First report of reduced sensitivity towards hydrogen peroxide found in the
salmon louse
Lepeophtheirus salmonis in Norway", Aquaculture Reports 1, 37-42). It has been
documented that increased catalase activity plays a role in H202 resistance in
salmon lice.
Increased catalase expression and subsequent enhanced catalase activity is a
mechanism for
H202 resistance in salmon lice, and molecular screening methods could be
developed on
this basis. (Helgesen et al.,2015 "First report of reduced sensitivity towards
hydrogen
peroxide found in the salmon louse Lepeophtheirus salmonis in Norway",
Aquaculture
Reports 1, 37-42). The sequence expressing the catalase gene and the methods
for using
this gene as a resistance marker for H202 resistance in sea lice is disclosed
in European
Patent application EP 3 164 502 Bl.
Data shows that the catalase gene is probably induced by the exposure of lice
to H202. It
has however been found that if I-1702 is not applied as sea lice treatment for
a period of
time, the catalase expressions could return to its normal expression values,
similar to the
sensitive louse levels (unpublished data). Thus, there is a need for improved
methods for
detection of H202 resistance in sea lice.
Efficient, sensitive, stable and reliable methods for diagnosing resistance
are crucial in
order to manage and control drug resistance. Early detection of reduced
sensitivity to a
chemical can enable effective countermeasures to be enforced at a time point
when these
have a greater probability of being effective. Therefore, accurate, stable and
speedy
identification of hydrogen peroxide (H202) resistant sea lice is crucial.
Detection of
hydrogen peroxide resistance prior to treatment, and the use of such analyses
after
treatment to evaluate treatment efficacy constitutes an important determinant
for the
integrated pest management (IPM) in the aquaculture industry.
Summary of invention
The present invention is based on the surprising finding that resistance
towards hydrogen
peroxide commonly used to combat sea lice infestation is linked to the
expression of a set
of genes that are either downregulated or upregulated in hydrogen peroxide
resistant sea
lice.
In particular, it has been found that the expression of a gene encoding an
aquaglyceroporin
(Glp1_v2) is downregulated in HA}, resistant adult female sea lice.
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Also, the endoplasmic reticulum resident protein 29 (ERP29) is downregulated
in 11202
resistant sea lice.
Furthermore, it has been found that the genes encoding a DNA polymerase (delta
subunit
5 3) is upregulated in sea lice resistant to H202
It has furthermore been found that expression of the gene encoding Nesprin-
like is
upregulated in H202 resistant sea lice.
It has furthermore been found that a gene encoding a yet unknown protein
(herein named
NA, SEQ ID No. 10) is upregulated in 11202 resistant sea lice.
The sea lice that may be analysed according to the present invention is one or
more
copepods, e.g. belonging to the family Caligidae.
According to one embodiment, the copepod is selected from the group consisting
of
Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus, and Caligus
rogercresseyi.
According to one embodiment, the copepod is selected from the group consisting
of
Lepeophtheirus salmonis, Caligus clemensei and Caligus elongates.
According to one embodiment, the copepod is Caligus rogercresseyi.
According to one embodiment the copepod is Lepeophtheirus salmonis.
According to a first aspect, a method is provided for the detection of
hydrogen peroxide
resistance in one or more adult female sea lice selected from the group
consisting of
Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus, and Caligus
rogercresseyi
comprising the steps of:
a) collecting one or more adult female sea lice from infested fish or water
samples;
b) isolating genomic material from the collected sea lice; and
c) determining the expression level of at least one of the genes encoding the
proteins selected from the group consisting of aquaglyceroporin (Glp1_v2),
endoplasmic reticulum resident protein 29 (ERY29), DNA polymer ase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10.
According to one embodiment of the first aspect, said method is provided for
the detection
of hydrogen peroxide resistance in one or more sea lice selected from the
group consisting
of Lepeophtheirus salmonis, Caligus clemensei and Caligus elongatus.
According to one embodiment of the first aspect, said method is provided for
the detection
of hydrogen peroxide resistance in one or more adult female sea lice wherein
the sea lice is
Lepeophtheirus salmonis.
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According to one embodiment of the first aspect, said method is provided for
the detection
of hydrogen peroxide resistance in one or more adult female sea lice wherein
the sea lice is
Caligus rogercresseyi.
According to one embodiment of the first aspect, said method is provided for
determining
the expression level of at least one of the genes encoding a protein having a
sequence
selected from the group consisting of:
SEQ ID No. 2 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 2;
SEQ ID No. 4 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 4;
SEQ ID No. 6 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ Ill No. 6;
SEQ ID No. 8 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 8;
SEQ ID No. 10 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 10.
According one embodiment of the first aspect, said method is provided for
determining the
expression level of at least one of the genes comprising a sequence selected
from the group
consisting of:
SEQ ID No. 1 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 1;
,SEQ ID No. 3 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 3;
SEQ ID No. 5 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 5;
SEQ ID No. 7 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 7;
SEQ ID No. 9 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 9.
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According to one embodiment of the first aspect, the method according to the
present
invention involves determination of the expression levels of at least two of
the genes
encoding the proteins selected from the group consisting of aquaglyceroporin
(Glpl _v2),
endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3),
nesprin-like and the protein of SEQ ID No. 10 is determined.
According to one embodiment of the first aspect, the method according to the
present
invention involves determination the expression levels of a gene encoding
catalase in
addition to one or more of the genes encoding the proteins selected from the
group
consisting of aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident
protein 29
(ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ
ID No.
10. Said catalase gene can be selected from the group consisting of SEQ Ill
No. 11, SEQ
ID No. 12, SEQ ID No. 13, and SEQ ID No. 14 or variants or fragments thereof
being at
least 70 % identical with SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ
ID No.
14, respectively.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H202 resistance in one or more sea lice, wherein the one
or more sea
lice is found to be resistant if the expression of the gene encoding
aquaglyceroporin
(Glp1_v2) is downregulated compared with the expression level of
aquaglyceroporin
(Glpl v2) in one or more 11702 sensitive sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H202 resistance in one or more sea lice, wherein the one
or more sea
lice is found to be resistant if the expression of the gene encoding
endoplasmic reticulum
resident protein 29 (ERP29) is downregulated compared with the expression
level of
endoplasmic reticulum resident protein 29 (ERP29) in one or more 11202
sensitive sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H202 resistance in one or more sea lice, wherein the one
or more sea
lice is found to be resistant if the expression of the gene encoding DNA
polymerase (delta
subunit 3) is upregulated compared with the expression level of DNA polymerase
(delta
subunit 3) in one or more H2O, sensitive sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H202 resistance in one or more sea lice, wherein the one
or more sea
lice is found to be resistant if the expression of the gene encoding nesprin-
like is
upregulated compared with the expression level of nesprin-like in one or more
H902
sensitive sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H2O, resistance in one or more sea lice, wherein the one
or more sea
lice is found to be resistant if the expression of the gene encoding and the
protein of SEQ
ID No. 10 is upregulated compared with the expression level of and the protein
of SEQ ID
No. 10 in one or more H902 sensitive sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H202 resistance in one or more adult female sea lice
according to the
first aspect, wherein step (c) of the present invention comprise the steps of:
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(c1) providing one or more isolated oligonucleotide sequence(s) comprising at
least 8
contiguous nucleotides of the sequence of SEQ ID No. 1, SEQ ID No. 3, SEQ TD
No. 5.
SEQ ID No. 7, SEQ ID No. 9 or a complementary oligonucicotide of SEQ ID No. 1,
SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7, SEQ ID No. 9, respectively; and
(c2) determining the expression level of at least one of the genes selected
from the
group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7,
and
SEQ ID No. 9, or variants or fragments thereof being at least 70 % identical
with the
entire length of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7, and
SEQ
Ill No. 9, respectively, by measuring the RNA expression level of said genes
in the
collected sea lice, wherein said sea lice is resistant to H202 if having
reduced levels of
expression of the genes encoding aquaglyceroporin (Glpl_v2) and/or endoplasmic
reticulum resident protein 29 (ERP29), and/or elevated levels of RNA-
expression of the
genes encoding DNA polymerase (delta subunit 3), and/or nesprin-like and/or
the
protein of SEQ ID No. 10 compared with one or more non-resistant sea lice.
According to one embodiment of the first aspect, the present invention
provides a method
for determination of H702 resistance in one or more female adult sea lice
according to the
first aspect of the present invention, comprising the steps of
(a) collecting one or more sea lice from infested fish or water samples;
(b) isolating DNA or RNA from the collected sea lice;
(c) providing a pair of PCR primers capable of hybridising under stringent
conditions
to at least one of the genes selected from the group consisting of SEQ ID No.
1, SEQ
ID No. 3, SEQ ID No. 5. SEQ ID No. 7, and SEQ ID No. 9, respectively;
(d) performing PCR on the isolated DNA or RNA using the pair of PCR primers of
(c);
(e) determining the expression level of one or more of said genes comparing
the level
of the expression of said genes in the collected sea lice with the level of
control
standard or a gene expression sample control.
The expression of the one or more genes in the collected sea lice or water
sample is
according to one aspect of the invention compared with the expression level of
said genes
in one or more non-resistant sea lice.
According to a second aspect, a method is provided for the detection of
hydrogen peroxide
resistance in one or more sea lice wherein the sea lice is of Lepeophtheirus
salmonis,
Caligus clemensei, Caligus elongatus and Caligus rogercresseyi comprising the
steps of:
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from any life stage of the collected sea lice;
and
c) determining the expression level of at least one of the genes encoding the
proteins selected from the group consisting of endoplasmic reticulum resident
protein 29 (ER P29), DNA polymerase (delta subunit 3), nesprin-like and the
protein of SEQ ID No. 10.
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According to one embodiment of the second aspect, said method is provided for
determining the expression level of at least one of the genes encoding a
protein having a
sequence selected from the group consisting of:
SEQ ID No. 4 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ Ill No. 4;
SEQ ID No. 6 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 6;
SEQ ID No. 8 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 8;
SEQ ID No. 10 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ Ill No. 10.
According one embodiment of the second aspect, said method is provided for
determining
the expression level of at least one of the genes comprising a sequence
selected from the
group consisting of:
SEQ ID No. 3 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 3;
SEQ ID No. 5 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 5;
SEQ ID No. 7 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 7;
SEQ ID No. 9 or variants or fragments thereof being at least 70 % identical,
such as 80 %
identical, such as 85% identical, such as 90% identical, such as 92%
identical, such as 95%
identical, such as 98% identical, such as 99% identical with SEQ ID No. 9.
According to one embodiment of the second aspect, the method according to the
present
invention involves determination of the expression levels of at least two of
the genes
encoding the proteins selected from the group consisting of endoplasmic
reticulum resident
protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the
protein of
SEQ ID No. 10 is determined.
According to one embodiment of the second aspect, the method according to the
present
invention involves determination the expression levels of a gene encoding
catalase in
addition to one or more of the genes encoding the proteins selected from the
group
consisting of endoplasmic reticulum resident protein 29 (ERP29), DNA
polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10. Said catalase gene
can be
selected from the group consisting of SEQ ID No. 11, SEQ ID No. 12, SEQ ID No.
13, and
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SEQ ID No. 14 or variants or fragments thereof being at least 70 % identical
with SEQ ID
No. 11, SEQ ID No. 12, SEQ ID No. 13, and SEQ ID No. 14, respectively.
According to one embodiment of the second aspect, the present invention
provides a
method for determination of H202 resistance in one or more sea lice, wherein
the one or
5 more sea lice is found to be resistant if the expression of the gene
encoding endoplasmic
reticulum resident protein 29 (ERP29) is downregulated compared with the
expression
level of endopl asmic reticulum resident protein 29 (ERP29) in one or more 1-
1202 sensitive
sea lice.
According to one embodiment of the second aspect, the present invention
provides a
10 method for determination of H202 resistance in one or more sea lice,
wherein the one or
more sea lice is found to be resistant if the expression of the gene encoding
DNA
polymerase (delta subunit 3) is upregulated compared with the expression level
of DNA
polymerase (delta subunit 3) in one or more H202 sensitive sea lice.
According to one embodiment of the second aspect, the present invention
provides a
method for determination of H202 resistance in one or more sea lice, wherein
the one or
more sea lice is found to be resistant if the expression of the gene encoding
nesprin-like is
upregulated compared with the expression level of nesprin-like in one or more
H202
sensitive sea lice.
According to one embodiment of the second aspect, the present invention
provides a
method for determination of H202 resistance in one or more sea lice, wherein
the one or
more sea lice is found to be resistant if the expression of the gene encoding
and the protein
of SEQ ID No. 10 is upregulated compared with the expression level of and the
protein of
SEQ ID No. 10 in one or more H202 sensitive sea lice.
According to one embodiment of the second aspect, the present invention
provides a
method for determination of H202 resistance in one or more sea lice, wherein
step (c) of the
fourth aspect of the present invention comprise the steps of:
(el) providing one or more isolated oligonucleotide sequence(s) comprising at
least 8
contiguous nucleotides of the sequence of SEQ ID No. 3, SEQ ID No. 5. SEQ ID
No. 7,
SEQ ID No. 9 or a complementary oligonucleotide of SEQ ID No. 3, SEQ ID No. 5.
SEQ ID No. 7, SEQ ID No. 9, respectively; and
(c2) determining the expression level of at least one of the genes selected
from the
group consisting of SEQ Ill No. 3, SEQ Ill No. 5. SEQ ID No. 7, and SEQ Ill
No. 9, or
variants or fragments thereof being at least 70 % identical with the entire
length of
SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7, and SEQ ID No. 9, respectively, by
measuring the RNA expression level of said genes in the collected sea lice,
wherein
said sea lice is resistant to H202 if having reduced levels of expression of
the genes
encoding endoplasmic reticulum resident protein 29 (ERP29), and/or elevated
levels of
RNA-expression of the genes encoding DNA polymerase (delta subunit 3), and/or
nesprin-like and/or the protein of SEQ ID No. 10 compared with one or more non-
resistant sea lice.
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According to one embodiment of the second aspect, the present invention
provides a
method for determination of H202 resistance in one or more sea lice according
to the
second aspect of the present invention, comprising the steps of
(a) collecting one or more sea lice from infested fish or water samples;
(b) isolating DNA or RNA from the collected sea lice;
(c) providing a pair of PCR primers capable of hybridizing under stringent
conditions
to at least one of the genes selected from the group consisting of SEQ ID No.
3,
SEQ ID No. 5. SEQ ID No. 7, and SEQ ID No. 9, respectively;
(d) performing PCR on the isolated DNA or RNA using the pair of PCR primers of
(c);
(e) determining the expression level of one or more of said genes comparing
the level
of the expression of said genes in the collected sea lice with the level of
control
standard or a gene expression sample control.
The expression of the one or more genes in the collected sea lice or water
sample is
according to one aspect of the invention compared with the expression level of
said genes
in one or more non-resistant sea lice.
In a third aspect the present invention furthermore provides the usc of one or
more
isolated oligonucleotide sequence(s) comprising at least 8 contiguous
nucleotides of the
sequence SEQ Ill No. 1, SEQ Ill No. 3, SEQ Ill No. 5. SEQ Ill No. 7, and SEQ
Ill No. 9,
or a complementary oligonucleotide of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.
5. SEQ
ID No. 7, and SEQ ID No. 9, respectively, for in vitro determination of
hydrogen peroxide
resistance in sea lice according to the method of the first aspect.
In one embodiment according to the third aspect, the one or more isolated
oligonucleotide
sequence is used for determination of hydrogen peroxide resistance in adult
female sea lice
selected from the group consisting of Lepeophtheirus salmonis, Caligus
clemensei, Caligus
elongatus.
According to a fourth aspect the present invention furthermore provides the
use of one or
more isolated oligonucleotide sequence(s) comprising at least 8 contiguous
nucleotides of
the sequence SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7, and SEQ ID No. 9, or a
complementary oligonucleotide of SEQ Ill No. 3, SEQ Ill No. 5. SEQ Ill No. 7,
and SEQ
ID No. 9, respectively, for in vitro determination of hydrogen peroxide
resistance in sea
lice according to the second aspect wherein the sea lice is selected from the
group
consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus
and Caligus
rogercresseyi,
According to yet another aspect, one or more mentioned isolated
oligonucleotide sequence
according to the third or fourth aspect may be used together with isolated
oligonucleotide
sequence for determining catalase expression wherein the isolated
oligonucleotide
sequence used in determining hydroxy peroxide resistance according to the
present use is
selected from the group consisting of SEQ ID No. 18, SEQ ID No. 19, SEQ ID NO.
20 and
SEQ ID No. 21.
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The present invention furthermore provides according to yet another aspect a
kit for
detection of hydrogen peroxide resistance in sea lice comprising one or more
isolated
oligonucleotide sequence(s) comprising at least 8 contiguous nucleotides of
the sequence
SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5. SEQ ID No. 7, and SEQ ID No. 9, or a
complementary oligonucleotide of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5. SEQ
ID
No. 7, and SEQ ID No. 9, respectively, and wherein the one or more isolated
oligonucleotide sequence is not SEQ ID No. 23.
The present invention provides according to yet a further aspect a DNA
molecule encoding
a protein comprising an amino acid sequence selected from the group consisting
of SEQ ID
No. 4, SEQ ID No. 6. SEQ ID No. 8, and SEQ ID No. 10.
According to yet another aspect, a DNA molecule is provided comprising a
sequence
selected from the group consisting of
SEQ ID No. 1 or variants thereof being at least 80 % identical, such as 85%
identical, such
as 90% identical, such as 92% identical, such as 95% identical, such as 98%
identical, such
as 99% identical with the entire length of SEQ ID No. 1,
SEQ ID No. 3 or variants thereof being at least 80% identical, such as 85%
identical, such
as 90% identical, such as 92% identical, such as 95% identical, such as 98%
identical, such
as 99% identical with the entire length of SEQ ID No. 3,
SEQ ID No. 5 or variants thereof being at least 80 % identical, such as 85%
identical, such
as 90% identical, such as 92% identical, such as 95% identical, such as 98%
identical, such
as 99% identical with the entire length of SEQ ID No. 1, SEQ ID No. 3, SEQ ID
No. 5
SEQ ID No. 7 or variants thereof being at least 80 % identical, such as 85%
identical, such
as 90% identical, such as 92% identical, such as 95% identical, such as 98%
identical, such
as 99% identical with the entire length of SEQ ID No. 1, SEQ ID No. 3, SEQ ID
No. 5.
SEQ ID No. 7, and
SEQ ID No. 9, or variants thereof being at least 80 % identical, such as 85%
identical, such
as 90% identical, such as 92% identical, such as 95% identical, such as 98%
identical, such
as 99% identical with the entire length of SEQ ID No. 9.
According to yet another aspect, a DNA molecule is provided comprising a
sequence
selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No.
5. SEQ ID
No. 7, and SEQ Ill No. 9.
According to yet another aspect, a DNA molecule is provided comprising a
sequence
selected from the group consisting of SEQ ID No. 3, SEQ ID No. 5. SEQ ID No.
7, and
SEQ ID No. 9.
The present invention and its various embodiments will be described in more
detail in the
following.
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13
Figures
Figure 1 show gene expression data (normalized counts) of catalase and the
five genes
significantly differentially expressed in the 2013, PO and F2-H202 groups (DNA-
polymerase delta subunit 3, Nesprin-like, NA, ERP29 and G1p1_v2): Ls A-2013
(white
circles), Ls V-2013 (grey circles), Ls A-PO (white triangles), Ls V-PO (grey
triangles), Ls
F2- H202-S (sensitive lice, white diamonds), Ls F2- H202-R (resistant lice,
grey diamonds).
Solid lines represent the mean in each group. Dark grey and black diamonds in
the Ls F241
H202-R group represent the same individual lice in both catalase and Glp1_v2
graphs.
Figure 2 illustrates the number of genes differentially expressed in the H202
resistant lice
(2013, PO and F2- H202 groups), separately for up- and down-regulated genes.
Numbers in
the circles represent the unique genes differentially expressed in each group.
Numbers in
the intersection of the circles represent the differentially expressed genes
shared between
two or three groups.
Figure 3. qPCR validation for catalase and Glp1_v2 genes in the RNAseq louse
groups: Ls
A-2013 (white circles), Ls V-2013 (grey circles), Ls A-PO (white triangles)
and Ls V-PO
(grey triangles) lice. Solid lines represent the mean in each group. Data
shown as fold
change (log2A-(AACt)) referred to Ls A 2013 and Ls A PO lice (calibrator
sample). Letters
above the data points (a,b and c) represent the statistically significant
differences between
the louse groups, groups sharing the same letter are not statistically
different (a = 0.05).
Detailed description of the invention
The present invention provides an in vitro method and means for determination
of
hydrogen peroxide resistance in crustaceans, in particular hydroxy peroxide
resistance in
sea lice. The method is based on the findings that determination of the
expression levels of
the genes encoding the proteins selected from the group consisting of
aquaglyceroporin
(Glp1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase
(delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10 with an unknown
function can
be used to reveal whether sea lice is sensitive towards hydroxy peroxide
treatment or not.
The present invention thus provides method for the detection of hydrogen
peroxide
resistance in one or more adult female sea lice selected from the group
consisting of
Lepeophtheirus sari-fortis, Caligus clernensei, Caligus elongatus, and Caligus
rogercresseyi
comprising the steps of:
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from adult female of the collected sea lice; and
c) determining the expression level of at least one of the genes encoding the
proteins selected from the group consisting of aquaglyceroporin (Glp1_v2),
endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10.
Optionally, in addition to the determination of the at least one mentioned
genes of step (c),
said method may also involve the determination of the expression level of the
gene
encoding catalase.
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According to one aspect, the sea lice to be analysed is one or more adult
female sea lice.
According to one aspect adult female sea lice selected from the group
consisting of
Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus.
In particular, it has been found that in hydroxy peroxide resistant adult
female sea lice the
expression of the gene encoding aquaglyceroporin (Glp1_v2) is significantly
downregulated compared with the expression levels of said genes in sea lice
being sensitive
to hydroxy peroxide.
The present invention further provides method for the detection of hydrogen
peroxide
resistance in one or more sea lice wherein the sea lice is selected from the
group consisting
of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus and Caligus
rogercresseyi comprising the steps of:
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from any life stage of the collected sea lice;
and
c) determining the expression level of at least one of the genes encoding the
proteins selected from the group consisting of endoplasmic reticulum resident
protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the
protein of SEQ ID No. 10.
Optionally, in addition to the determination of the at least one mentioned
genes of step (c),
said method may also involve the determination of the expression level of the
gene
encoding catalase.
Further, it has been found that in hydroxy peroxide resistant, the expression
of the gene
encoding cndoplasmic rcticulum resident protein 29 (ERP29)is significantly
downregulated
compared with the expression levels of said genes in sea lice being sensitive
to hydroxy
peroxide.
Furthermore, it has been found that expression of genes encoding DNA
polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10, respectively, in
resistant sea lice
is significantly upregulated compared with the expression level of said genes
in hydroxy
peroxide sensitive sea lice.
Glp1_v2 is one of the aquaglyceroporins identified by Stavang et al. (2015) in
L. salmonis
(Stavang et al, 2015, Phylogenomic and functional analyses of salmon lice
aquaporins
uncover the molecular diversity of the superfamily in Arthropoda. BMC Genomics
16:618.
Stavang et al. (2015) identified a total of seven aquaporins in the salmon
louse: two
classical aquaporins (Bib and Prip-like or "PripL"), three aquaglyccroporins
(Glp1_v1,
Glp1_v2, Glp2, Glp3_v1 and G1p3_v2) and two unorthodox aquaporins (Aqp12-like
1 or
"Aqp12L1" and Aqp12-like 2 or "Aqp12L2").
Aquaporins are protein channels that facilitate transport of water, other
small solutes such
as H202 and gasses (Bienert et al., 2007, Specific Aquaporins Facilitate the
Diffusion of
Hydrogen Peroxide across Membranes. The Journal of biological chemistry. 282.
1183-92;
Herrera and Garvin, 2011, Aquaporins as gas channels. Pflugers Arch - Eur J
Physiol 462:
623 ¨ 630; Miller et al., 2010, Aquaporin-3 mediates hydrogen peroxide uptake
to regulate
downstream intracellular signalling, Proceedings of the National Academy of
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Sciences, 107 (36) 15681-15686; Stavang et al., 2015, supra; Thiagarajah et
al., 2017,
Aquaporin-3-mediated colonic epithelial responses, Proceedings of the National
Academy
of Sciences, 114 (3) 568-573; Zwiazek et al., 2017, Significance of oxygen
transport
through aquaporins, Scientific Reports, 7:40411).
5 Stavang et al. (2015), supra, identified a total of seven aquaporins in
the salmon louse: two
classical aquaporins (Bib and Prip-like or -PripL"), three aquaglyceroporins
(Glpl vl,
Glpl _v2, Glp2, Glp3_v1 and Glp3_v2) and two unorthodox aquaporins (Aqpl 2-
likel or
"Aqp12L1" and Aqp12-like 2 or "Aqp12L2"). Glp3 v2 was found to be expressed
mostly
in Nauplius II stage.
10 It has been demonstrated that certain aquaglyceroporins and unorthodox
aquaporins are
able to transport H202 through cell membranes in several organisms (Miller et
al., 2010,
supra; Thiagarajah et al., 2017, supra). Glps have an open pore configuration
in L.
salmonis (Stavang et al., 2015, supra), also allowing bigger molecules than
water, like urea
and glycerol, to pass through the channel. Miller et al. (2010), supra, found
that one
15 aquaglyceroporin (AQP3) and one unorthodox aquaporin (AQP8) transported
11202 through
mammalian cell membranes. However, the classical aquaporin AQP1, did not
transport
H202.
Several authors have reported the role of aquaporins as drug transporters in
other parasites,
and the link between aquaporins and drug resistance (reviewed in Song et al.,
2014,
Parasite aquaporins: Current developments in drug facilitation and resistance,
Biochimica
et biophysica acta. 1840: 1566-1573). In Faghiri and Skelly (2009), The role
of tegumental
aquaporin from the human parasitic worm, Schistosoma mansoni, in
osmoregulation and
drug uptake, FASEB J. 23(8): 2780-2789, it was shown that the presence of a
putative
aquaglyceroporin (SmAQP) in the tegument of the parasitic worm Schistosoma
mansoni. It
was proven that SmAQP can transport water and an anti-parasitic compound
(potassium
antimonyl tartrate) across the parasite tegument. In addition, parasites with
reduced levels
of SmAQP exhibited a greater resistance to the anti-parasitic agent. In
Trypanosomatid
parasites, like Leishmania or Trypanosoma spp., certain aquaporins transport
trivalent
metalloids (SbIII and AsIII) through the parasite membranes (reviewed in
Mandal et al.,
2014, Trypanosomatid Aquaporins: Roles in Physiology and Drug Response.
Diseases).
The aquaglyceroporin LmAQP1 transports SbIII in Leishmania spp (Gourbal et
al., 2004,
Drug Uptake and Modulation of Drug Resistance in Leishmania by an
Aquaglyceroporin.
The Journal of biological chemistry. 279. 31010-7). Drug resistant parasites
showed down-
regulation of LmAQP1 (Marquis et al., 2005, Modulation in aquaglyceroporin
AQP1 gene
transcript levels in drug-resistant Leishmania, Mol Microbiol. Sep;57(6):1690-
9), and the
RNA levels correlated with the drug concentration. Resistance to melarsoprol
and
pentamidine is common among African trypanosomes (Baker et al., 2012,
Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in
African
trypanosomes, Proceedings of the National Academy of Sciences of the United
States of
America, 109(27), 10996-11001).
The authors found that the loss of function of an aquaglyceroporin, AQP2, was
linked to
drug resistance. Interestingly, a mitogen activated protein kinase 2 (MPK2)
stabilizes
LmAQP1 protein by phosphorylation in Leishmania major (Mandal et al., 2012),
and
dephosphorylation made LmAQP1 more susceptible to degradation. Altered AQP1
and
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16
MPK2 (by site-directed mutagenesis) reduced the drug uptake and drug
sensitivity.
Catalase activity can also be regulated by reversible phosphorylation via
kinase enzymes
by increasing the affinity of the enzyme for H202 (Dawson and Storey, 2016,
The
reversible phosphorylation of catalase from the freeze-tolerant North American
wood frog,
Rana sylvatica, Biochim Biophys Acta. 1860(3):476-85). These studies suggest
that drug
sensitivity can be linked to regulation of gene expression, but also to post-
translational
modifications of proteins. In our L. salmonis RNAseq data (cf. examples
below), we found
four putative mitogen activated protein kinases differentially expressed in
14202 sensitive
and resistant lice (data not shown).
Based on the finding of the inventors, determination of the expression level
of one of the
genes mentioned above each in particular is suitable to indicate whether one
or more sea
lice are resistant or sensitive towards hydrogen peroxide resistance. However,
without
being limited by theory, and based on the inventors findings regarding
determination of
hydroxy peroxide resistance based on catalase gene expression level in sea
lice, it is
according to one aspect provided a method for the detection of hydrogen
peroxide
resistance in one or more sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus clemensei. Caligus elongatus, and Caligus rogercresseyi
comprising the
steps of:
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from any life stage of the collected sea lice;
and
c) determining the expression level of at least two of the genes encoding the
proteins selected from the group consisting of aquaglyceroporin (G1p1_v2),
endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10.
According to one aspect the sea lice is an adult female sea lice.
According to one aspect the sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus clemensei, Caligus elongatus,
According to one aspect the sea lice is Caligus rogercresseyi
According to yet another aspect, the above method may optionally also involve
the
determination of the expression level of catalase in the analysed one or more
sea lice.
According to another aspect, step (c) involves determining the expression
level of at least
three, such as at least four of the genes encoding the proteins selected from
the group
consisting of aquaglyceroporin (Glpl_v2), endoplasmic reticulum resident
protein 29
(ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ
ID No.
10. According to yet another aspect, step (c) involved determining the
expression level of
the genes encoding aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident
protein 29
(ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ
ID No.
10.
According to yet another aspect, the method of the invention involves the
determination of
the expression level of the genes encoding aquaglyceroporin (Glp1_v2) and
endoplasmic
reticulum resident protein 29 (ERP29), and optionally also determination of
the expression
level of a gene encoding catalase.
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According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (GlpI v2) and DNA
polymerase
(delta subunit 3), and optionally also determination of the expression level
of a gene
encoding catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Glpl v2) and nesprin-
like. and
optionally also determination of the expression level of a gene encoding
catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of aquaglyceroporin (GlpI_v2) and the protein of SEQ ID No.
10, and
optionally also determination of the expression level of a gene encoding
catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29)
and DNA polymerase (delta subunit 3), and optionally also determination of the
expression
level of a gene encoding catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29)
and nesprin-like, and optionally also determination of the expression level of
a gene
encoding catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29)
and the protein of SEQ ID No. 10, and optionally also determination of the
expression level
of a gene encoding catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding DNA polymerase (delta subunit 3) and
nesprin-like,
and optionally also determination of the expression level of a gene encoding
catalase.
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding DNA polymerase (delta subunit 3) and
the protein
of SEQ Ill No. 10, and optionally also determination of the expression level
of a gene
encoding catalase.
According to another aspect, the method of the invention involves
determination of the
expression level of the genes encoding nesprin-like and the protein of SEQ ID
No. 10, and
optionally also determination of the expression level of a gene encoding
catalase.
According to yet another aspect, a method is provided for detection of
hydrogen peroxide
resistance in one or more sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus clemensei, Caligus elongatus, and Catigus rogercresseyi
comprising the
steps of
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from any life stage of the collected sea lice;
and
c) determining the expression level of at least three of the genes encoding
the
proteins selected from the group consisting of aquaglyceroporin (Glp1_v2),
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endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10.
According to one aspect the sea lice is an adult female sea lice.
According to one aspect the sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus clemensei, Caligus elongatus.
According to one aspect the sea lice is Caligus rogercresseyi
According to yet another aspect, the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Glpl v2), endoplasmic
reticulum
resident protein 29 (ERP29), DNA polymerase (delta subunit 3), and optionally
also
determination of the expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding aquaglyceroporin (Glpl_v2), en doplasmi
c reticulum
resident protein 29 (ERP29), and the protein of SEQ ID No. 10, and optionally
also
determination of the expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding aquaglyceroporin (Glp l_v2),
endoplasmic reticulum
resident protein 29 (ERP29), and nesprin-like, and optionally also
determination of the
expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding aquaglyceroporin (Glp1_v2), DNA
polymerase
(delta subunit 3), and nesprin-like, and optionally also determination of the
expression
level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding aquaglyceroporin (Glp1_v2), DNA
polymerase
(delta subunit 3), and the protein of SEQ ID No. 10, and optionally also
determination of
the expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding aquaglyceroporin (Glpl v2), nesprin-
like, and the
protein of SEQ ID No. 10, and optionally also determination of the expression
level of a
gene encoding catalasc.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29),
DNA polymerase (delta subunit 3), and nesprin-like, and optionally also
determination of
the expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29),
DNA polymerase (delta subunit 3), and the protein of SEQ ID No. 10; and
optionally also
determination of the expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29),
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nesprin-like, and the protein of SEQ ID No. 10; and optionally also
determination of the
expression level of a gene encoding catalase.
According to yet another, the method of the invention involves determination
of the
expression level of the genes encoding DNA polymerase (delta subunit 3),
nesprin-like and
the protein of SEQ ID No. 10; and optionally also determination of the
expression level of
a gene encoding catalase.
According to yet another aspect, a method is provided for detection of
hydrogen peroxide
resistance in one or more sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus elemensei, Caligus elongatus, and Caligus rogereresseyi
comprising the
steps of
a) collecting one or more sea lice from infested fish or water samples;
b) isolating genomic material from any life stage of the collected sea lice;
and
c) determining the expression level of at least four of the genes encoding the
proteins selected from the group consisting of aquaglyceroporin (Glp l_v2),
endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3), nesprin-like and the protein of SEQ ID No. 10.
According to one aspect the sea lice is an adult female sea lice.
According to one aspect the sea lice selected from the group consisting of
Lepeophtheirus
sahnonis, Caligus elemensei, Caligus elongatus.
According to one aspect the sea lice is Caligus rogereresseyi
According to one embodiment, the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Cilp l_v2),
endoplasmic reticulum
resident protein 29 (ERP29), DNA polymerase (delta subunit 3), and nesprin-
like; and
optionally also determination of the expression level of a gene encoding
catalase.
According to one embodiment , the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Glpl v2), endoplasmic
reticulum
resident protein 29 (ERP29), DNA polymerase (delta subunit 3), and the protein
of SEQ ID
No. 10; and optionally also determination of the expression level of a gene
encoding
catalase.
According to one embodiment , the method of the invention involves
determination of the
expression level of the genes cncoding aquaglyceroporin (Glp l_v2),
endoplasmic reticulum
resident protein 29 (ERP29), nesprin-like and the protein of SEQ ID No. 10;
and optionally
also determination of the expression level of a gene encoding catalase.
According to one embodiment, the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Glp1_v2), DNA
polymerase
(delta subunit 3), nesprin-like and the protein of SEQ ID No. 10; and
optionally also
determination of the expression level of a gene encoding catalase.
According toone embodiment , the method of the invention involves
determination of the
expression level of the genes encoding endoplasmic reticulum resident protein
29 (ERP29),
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DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No.
10; and
optionally also determination of the expression level of a gene encoding
catalase.
According toone embodiment , the method of the invention involves
determination of the
expression level of the genes encoding aquaglyceroporin (Glp1_v2), endoplasmic
reticulum
5 resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-
like and the
protein of SEQ Ill No. 10; and optionally also determination of the expression
level of a
gene encoding catalase.
Based on the teaching herein, i.e. that the expression level of genes encoding
the proteins
selected from the group consisting of aquaglyceroporin (Glp1_v2), endoplasmic
reticulum
10 resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-
like and the
protein of SEQ ID No. 10 are linked with hydrogen peroxide resistance in sea
lice, the
skilled person will acknowledge that various well known methods are available
for the
determination of the expression level of genes, and to determine whether the
expression of
a certain gene are downregulated or upregulated.
15 When referring to that the expression of a gen is "upregulated" it is to
be understood to
mean that the expression of the gene in question is increased compared with
the expression
level of said gene in a sea louse being sensitive towards hydrogen peroxide.
When referring to that the expression of a gen is "downregulated" it is to be
understood to
mean that the expression of the gene in question is decreased compared with
the expression
20 level of said gene in a sea louse being sensitive towards hydrogen
peroxide.
The expression level may be measured by quantifying the levels of the gene
product in
question and may thus be determined by e.g. quantifying the amount of protein
or
quantifying the amount of mRNA.
The skilled person is aware of a number of methods that may be used to
determine the
mRNA level of an organism, such as a sea louse. For example, the skilled
person would
know the major methods of quantitatively detect mRNA levels including
electrophoretic
methods (e.g. Nothern bloting), DNA microarray-based methods and quantitative
PCT
(Real-time PCR). Various protocols for determining expression levels are
available, cf. e.g.
the step by step guide to Northern blot analysis by ThermoFisher Scientific
(https://www.thermofisher.com/no/en/home/life-science/dna-rna-purification-
analysis/nucleic-acid-gel-electrophoresis/northern-blotting.html). Other
available analysis
is the QuantiGene RNA Assay for gene expression profiling of Invitrogen0
provided
ThermoFisher Scientifc (https://www.thermofisher.com/no/en/home/life-
science/gene-
expression-analysis-genotyping/quantigene-rna-assays.html).
Real-time PCR based methods (also called quantitative PCR or qPCR) allows
monitoring
DNA amplification during the PCR run in real time via fluorescent dyes that
yield
increasing fluorescent signal in direct proportion to the number of PCT
product molecules
(amplicons) that are generated. Real time PCT methods is an efficient and by
many
considered as the preferred method for detection and quantification of DNA or
RNA. An
overview of the Real Time PCR technology is found in Methods in Molecular
Biology,
"Quantitative Real-Time PCR, Methods and Protocols, edited by Roberto Bias
soni and
Alessandro Raso, Springer Science+13usiness Media, New York, 2014
(https://www.gene-
quantification.de/biassoni-raso-quantitative-real-time-per-ebook-2014.pdf).
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It is to be understood that the methods used in EP 3 164 502 B1 to quantify
the expression
level of catalase gene in sea lice by quantification of genomic material
isolated from a sea
lice suspected to be hydrogen peroxidase resistant may be used to quantify the
expression
level of the genes encoding the proteins selected from the group consisting of
aquaglyceroporin (Glpl_v2), endoplasmic reticulum resident protein 29 (ERP29),
DNA
polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
Although the experimental data linking hydrogen peroxide resistance with the
expression
level of genes encoding aquaglyceroporin (Glp1_v2), endoplasmic reticulum
resident
protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the
protein of
SEQ ID No. 10, respectively, were identified in the sea lice species
Lepeophtheirus
salinonis, the skilled person will acknowledge, based on the teaching herein,
that the
present method and may be used to determine hydrogen peroxide resistance in
copepods
belonging to the family Caligidae.
In particular, it is to be understood that the present method may be used to
determine
hydrogen peroxide resistance in copepods affecting farmed fish, such as e.g.
fish belonging
to the family Salmonidae.
According to one embodiment disclosed herein, the present method is applicable
for
detection of hydrogen peroxide resistance in copepod selected from the group
consisting of
Lepeophteirus salmonis, Caligus clemensei, Caligus elongatus, and Caligus
rogercresseyi.
According to one aspect the sea lice selected from the group consisting of
Lepeophtheirus
salmonis, Caligus clemensei, Caligus elongatus.
According to one aspect the sea lice is Caligus rogercresseyi
Throughout the disclosure, the term "sea louse" or "sea lice" is to be
understood to mean
one or more copepod belonging to the family Caligidae. In the experimental
data provided
in the present disclosure, "sea lice" or "sea louse" refer to the species
Lepeophtheirus
salmonis.
Based upon the information provided herein in respect of the linkage between
hydrogen
peroxidase resistance and the expression levels of the genes encoding the
proteins selected
from the group consisting of aquaglyceroporin (Glp1_v2), endoplasmic reticulum
resident
protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the
protein of
SEQ ID No. 10, the skilled person would, based on his common general knowledge
of the
well-known analysis and methods for determining expression levels be able to
provide the
means necessary to perform such analysis. For example, the skilled person
will, based on
the teaching herein, identify and construct applicable oligonucleotides, such
as probes or
primers, useful in the various methods mentioned above.
As used herein, an ''oligonucleotide sequence" or "nucleic acid sequence" is
to be
understood to mean an oligonucleotide sequence or a nucleic acid sequence
useful in
determining the expression level of genes encoding the proteins selected from
the group
consisting of aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident
protein 29
(ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ
ID No.
10, respectively.
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An ''oligonucleotide sequence" or "nucleic acid sequence" used to determine
the expression
level of one or more of the above listed genes is capable of hybridize to a
nucleic acid
sequence with a complementary sequence, such as e.g. genomic material,
extracted from
the one or more sea lice to be analyzed for hydrogen peroxide resistance. The
skilled
person will understand that the genomic material may be e.g. mRNA or DNA.
The skilled person is also aware of the fact that nucleic acid molecules may
be double
stranded or single-stranded, and that reference to a particular site of one
strand refers, as
well, to the corresponding site on a complementary strand. Thus, reference to
an adenine
(A), a thymine (T) (uridine (U)), a cytosine (C) or a guanine (G) at a
particular site on one
strand of a nucleic acid is also to be understood to define a thymine
(uridine), adenine,
guanine, or cytosine, respectively, at the corresponding site on a
complementary strand of
the nucleic acid molecule. Thus, reference may be made to either strand in
order to refer to
a particular position. The oligonucleotide probes and oligonucleotide primers
according to
the present invention may be designed to hybridize to either strand.
An "isolated nucleic acid" useful in the detection method of the present
invention, i.e. such
as primers and probes, as used herein is generally one that contains at least
8 nucleotides
and which is capable of hybridizing a nucleic acid with a complementary
sequence, and is
separated from most other nucleic acids present in the natural source of the
nucleic acid,
and is thus substantially free of other cellular material.
The present invention provides the use of oligonucleotide probes and
oligonucleotide
primers being useful in determining the expression level of any one of the
genes encoding
the proteins selected from the group consisting of aquaglyceroporin (Glp1_v2),
endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta
subunit 3),
nesprin-like and the protein of SEQ ID No. 10. The determination of the
expression level
of a gene is widely applied in both human and veterinary diagnosis, wherein
nucleic acids
from e.g. pathogens present in biological samples are isolated and hybridized
to one or
more hybridizing probes or primers are used in order to amplify a target
sequence.
One or more oligonucleotide probes may be constructed based on the teaching
herein and
used in hybridization-based detection methods where upon the binding of the
oligonucleotides to the target sequence enables determination of the level of
expression of
a gene present in the sea lice to be tested.
The skilled person will acknowledge that an oligonucleotide probe according to
the present
invention may be a fragment of DNA or RNA of variable length used herein in
order to
hybridize to the target sequence, e.g. single-stranded DNA or RNA. The
oligonucleotide
probe according to the present invention may furthermore be labeled with a
molecular
marker in order to easily visualize that hybridization have been achieved.
Molecular
markers commonly known to the skilled person may be used, e.g. a radiolabel,
and more
preferably, a luminescent molecule or a fluorescent molecule enabling the
visualisation of
the binding of the probe(s) to a target sequence.
An oligonucleotide probe according to the present invention is able to
hybridize to another
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nucleic acid molecule, such as the single strand of DNA or RNA originating
from one or
more sea lice to be analysed, under appropriate conditions of temperature and
solution
ionic strength, cf. e.g. Sambrook et al., Molecular Cloning: A laboratory
Manual (third
edition), 2001, CSHL Press, (ISBN 978-087969577-4). The condition of
temperature and
ionic strength determine what the skilled person will recognise as the
"stringency'' of the
hybridization. The suitable stringency for hybridisation of a probe to target
nucleic acids
depends on inter alia the length of the probe and the degree of
complementation, variables
well known to the skilled person. A oligonucleotide probe according to the
present
disclosure typically comprises a nucleotide sequence which under stringent
conditions
hybridize to at least 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50 (or any
other number in-
between) or more consecutive nucleotides in a target nucleic acid molecule,
e.g. single-
stranded DNA or RNA isolated from the sea lice to be analyzed according to the
present
invention. According to one embodiment, the oligonucleotide probe according to
the
present invention comprises about 10 to 25 consecutive nucleotides. New
technology like
specific Locked Nucleic Acid (LNA) hybridization probes allows for the use of
extremely
short oligonucleotide probes (You Y.; Moreira B.G.; Behlke M.A. and Owczarzy
R.
(2006). "Design of LNA probes that improve mismatch discrimination". Nucleic
Acids
Res. 34(8): e60. doi:10.1093/narigk1 1 75. PMC 1456327. PMTD 16670427)
According to
one embodiment, probes are used in the present method or according to the
present use,
which hybridize under stringent conditions to a gene encoding a protein
selected from the
group consisting of aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident
protein
29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of
SEQ ID
No. 10.
Also oligonucleotide primers may be used in methods according to the present
method for
determination of the expression level of a gene encoding a protein selected
from the group
consisting of aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident
protein 29
(ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ
ID No.
10, wherein the oligonucleotide primers are used for amplification of any
given region of
said genes. An oligonucleotide primer according to the present disclosure
typically
comprises a nucleotide sequence at least 8, 10, 12, 14, 16, 18, 20, 22, 25,
30, 40, 50 (or any
other number in-between) or more consecutive nucleotides. According to one
embodiment,
the oligonucicotide primer according to the present invention comprises about
14 - 25
consecutive nucleotides, more preferably about 15 nucleotides.
As used herein, the term "oligonucleotide primer'' is to be understood to
refer to a nucleic
acid sequence suitable for directing an activity to a region of a nucleic
acid, e.g. for
amplification of a target nucleic acid sequence by polymerase chain reaction
(PCR).
The skilled person will acknowledge that an oligonucleotide primer according
to the
present invention may be a fragment of DNA or RNA of variable length used
herein in
order to determine the expression level of the target sequence, e.g. single-
stranded DNA or
RNA, upon alignment of the oligonucleotide probe to complementary sequence(s)
of the
said target sequence to be analyzed. An oligonucleotide primer according to
the present
invention may furthermore be labeled with a molecular marker in order to
enable
visualization of the results obtained. Various molecular markers or labels are
available. An
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oligonucleotide primer according to the present invention typically comprises
the
appropriate number of nucleotides allowing that said primer align with the
target sequence
to he analyzed.
Oligonucleotide probes and oligonucleotide primers may be manufactured
according to
methods well known to the skilled person.
According to one aspect, oligonucleotides (primers or probes) may be used
determine the
expression level of a gene encoding a protein selected from the group
consisting of
aquaglyceroporin (Glp1_v2), endoplasmic reticulum resident protein 29 (ERP29),
DNA
polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
According to
yet another aspect, oligonucleotides (primers or probes) may be used to
determine the
expression level of nucleic acid molecule comprising a sequence selected from
the group
consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID NO. 5, SEQ ID No. 7 and SEQ
ID No.
9 or variants or fragments thereof having at least 70% identity with SEQ ID
No. 1, SEQ ID
No. 3, SEQ ID NO. 5, SEQ ID No. 7 and SEQ ID No. 9, respectively.
The term "% identity' is to be understood to refer to the percentage of
nucleotides that two
or more sequences or fragments thereof contains, that are the same. A
specified percentage
of nucleotides can be referred to as e.g. 70% identity, 75% identity, 80%
identity, 85%
identity, 90% identity, 95% identity, 99% identity or more (or any number in
between)
over a specified region when compared and aligned for maximum correspondence.
The skilled person will acknowledge that various means for comparing sequences
are
available. For example, one non-limiting example of a useful computer homology
or
identity program useful for determining the percent sequence identity between
sequences
includes the Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990,
J. of
Molec. Biol., 215:403-410, "The BLAST Algorithm; Altschul et al., 1997, Nuc.
Acids
Res. 25:3389-3402, Karlin and Altschul 1990, Proc. Nat'l Acad. Sci. USA,
87:2264-68 ;
1993, Proc. Nat'l Acad. Sci. USA 90:5873-77).
Isolation of genomic material of sea lice
The method according to the present invention may according to one embodiment
involve
the isolation of a biological sample from one or more sea lice and measuring
the level of
expression of the genes of interest in order to determine whether the sea lice
is hydrogen
peroxide resistant.
Various methods for obtaining genomic material well known to the skilled
person are
available. The skilled person will acknowledge that any tissue (i.e. any part
of the sea lice)
may be used in order to extract genomic material. Furthermore, the genomic
material to be
analyzed according to the present invention may be obtained from sea lice of
any life
stages, e.g. the free-swimming stages (nauplius stage I and II), the copepod
stage, the pre-
adult (chalimus stages 1-4), or the adult stage (adult male or adult female).
In one embodiment, the sea lice are adult female.
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According to one embodiment, tissue removed from sea lice to be tested is
maintained in
70% ethanol or other conservation liquid prior to further isolation of genomic
material.
DNA may be extracted from the obtained tissue using commonly available DNA
extraction/isolation methods, such as e.g. DNeasy DNA Tissue Kit according to
the
5 protocol of the manufacturer (http://lycofs01.1ycoming.edu/¨gcat-
scck/protocols/DNcasy_Blood_&_Tissue_Handbook.pdf ).
Hydrogen peroxidase resistance kits
Based on the teaching herein, the skilled person will acknowledge that the
identification of
10 the link between hydrogen peroxide resistance and expression levels of
genes encoding the
proteins selected from the group consisting of aquaglyceroporin (Glp1_v2),
endoplasmic
reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3),
nesprin-like and
the protein of SEQ ID No. 10, in sea lice, reagents applicable in
determination of the
expression level of said genes can be developed for the determination of
hydrogen
15 peroxidase resistance.
For example, according to the present invention, a kit may comprise
oligonucleotide
probe(s) or oligonucleotide primer(s) or primer sets, arrays/microarrays of
nucleic acid
molecules, and beads that contain one more oligonucleotide probe(s),
oligonucleotide
primer(s) or other detection reagents useful in the method of the present
invention. It is
20 furthermore to he understood that the detection reagents in a kit
according to the present
invention may furthermore include other components commonly included in such
kits, e.g.
such as various types of biochemical reagents (buffers. DNA polymerase,
ligase,
deoxynucleotide triphosphates for chain extension/amplification, etc.),
containers,
packages, substrates to which detection reagents are attached., etc. necessary
to carry the
25 method according to the present invention.
According to one embodiment of the present invention, a kit is provided which
comprises
the necessary reagents to carry out one or more assays in order to determine
the catalase
gene expression level according to the method of the present invention. A kit
according to
the present invention may preferably comprise one or more oligonucleotide
probes that
hybridize to a nucleic acid target molecule (i.e. genetic material) enabling
determination of
the catalase gene expression level in the material analyzed. Multiple pairs of
probes may be
included in the kit to simultaneously analyze for determination of catalase
gene expression
at the same time. The probes contained in the kit according to the present
invention may
according to one embodiment be immobilized on a carrier, such as e.g. an array
or a bead.
According to one embodiment, a kit according to the present invention
comprises
oligonucleotide primer(s) and optionally further reagents useful in methods
for the
determination of the expression level of one or more of the genes encoding the
proteins
selected from the group consisting of aquaglyceroporin (Glp 1 v2), endoplasmic
reticulum
resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like
and the
protein of SEQ ID No. 10. According to one embodiment, the kit according to
the present
invention comprises a forward primer and a reverse primer for amplifying a
region of one
or more of said genes. Said kit may furthermore optionally comprise further
reagents
(enzymes and nucleotide triphosphates) necessary for conducting PCR or real
time PCR.
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Examples
Salmon louse strains
Two well-characterized laboratory L. sahnonis strains were used in this study:
Ls A,
sensitive to all anti-salmon lice chemicals used in Norway (tested by
bioassays), and Ls V,
resistant to azamethiphos, deltamethrin, emamectin benzoate and hydrogen
peroxide (field
reports and bioassays). Ls A was a strain originally collected on a fish farm
in the Northern
part of Norway in 2011. Ls V was collected from a fish farm in Mid-Norway in
October
2013 with high anti-louse chemical treatment pressure and reported diminished
H202
treatment efficacy. A total of 14 anti-louse chemical treatments were
performed from
August 2012 to September 2013 in that farm: six H202 treatments (up until one
month
before the lice collection), six combined treatments with deltamethrin and
azamethiphos,
one treatment with diflubenzuron and one with emamectin benzoate. The Ls V-
2013
samples referred to in the current study were the original field samples of
this strain. Ls A
and Ls V strains were reared in continuous cultures at the research facilities
of
Solbergstrand (The Norwegian Institute for Water Research, NIVA, DrOak,
Norway), as
described by Hamre et al. (2009), in "Establishment and characterization of
salmon louse
(Lepeophtheirus salmonis (Kroyer 1837)) laboratory strains". Parasitol. Int.
58, 451-460
Example 1 Crossing experiment and bioassays
In order to obtain a lice sample from the same generation and a range of
hydroxy peroxide
sensitivities, a batch crossing experiment was designed. The experiment was
performed as
described by Bakke et al. (2018) in 2015 (Bakke et at., (2018), Deltamethrin
resistance in
the salmon louse, Lepeophtheirus salmonis (Kroyer): Maternal inheritance and
reduced
apoptosis", Scientific Reports 8, Article number: 8450).
In short, two Atlantic salmon (one fish per aquarium) were infested with
approximately 50
Ls A copepodites each and another two fish (one fish per aquarium) with the
same number
of Ls V copepodites to produce the parental generation (PO). All salmon lice
were collected
from all fish when the lice were in the pre-adult II stage, before the mating
occurred. Then
10 pre-adults II Ls A females and 10 pre-adult II Ls V males from the PO
generation were
put back on 2 individual fish (5 on each fish) kept in individual tanks to
produce the Fl
generation of the family group 1. Two other fish in separate tanks were
infested with the
same number of Ls A males and Ls V females to produce the Fl generation of
family group
2. All PO lice from both family groups were preserved in RNAlater (Sigma)
after removal
of the egg strings which were set aside to hatch. After ¨24h at room-
temperature. the
preserved samples were stored at ¨80 C. Four fish were infested with
copepodites from
the Fl generation, two fish with family group 1 and two fish with family group
2. The lice
developed to the adult stage, mated, and egg strings for the F2 generation
were collected.
Approximately 500 copepodites from each of the family groups 1 and 2 (F2) were
used for
infestation of eight Atlantic salmon for each family group, with the two-
family groups
separated in different tanks.
F2 parasites were allowed to develop to adults. and were selected for
sensitivity towards
hydrogen peroxide (Interox Paramove 50, H20250%, w/w, Solvay Chemicals,
Belgium).
The selection was performed in vitro using two-dose bioassays at Faculty of
Veterinary
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Medicine, NMBU (University of Life Sciences, Oslo, Norway) within 6 hours
after
sampling. All exposures were done in 1 L glass bottles held at 10-12 C with
constant
aeration. Three bioassays were performed where the females were exposed to 600
and 1800
ppm 11202 for 30 min (two bioassays with lice from the family group 1 and one
from family
group 2) and recording of the results immediately following the exposure time
(Helges en et
al., (2015), supra).
Control groups not exposed to H202 were included to check the general
performance of the
parasites. Parasites affected/immobilized at the lowest H202 concentrations
were
considered sensitive, whereas parasites that were not visibly affected at the
highest
concentrations were considered resistant. Lice were classified as affected
when they were
unable to attach to the container wall (lice could show weak swimming pattern,
being
partially or completely immobilized at the bottom of the container at floating
at the
surface). Immediately after exposure and the immobilization rate recorded,
lice were fixed
in RNAlater and kept at ¨80 C following ¨24h at room-temperature. H202
sensitive and
resistant F2 adult females were used in the RNAseq analysis.
In order to obtain H202 sensitive and resistant lice for the RNAseq study, F2
adult females
were selected with two-dose H202 bioassays. Lice affected at the lowest dose
were
considered sensitive and lice unaffected at the highest were considered
resistant. Table 1
shows the number of lice affected at the different 14202 doses. For adult
females, there were
no significant differences between family groups, indicating that inheritance
of resistance
was not gender-specific.
Table 1. Number of F2 adult female lice affected in two-dose H202 bioassays
(30 min
exposure). Results indicated as fractions (number of affected lice out of
total lice per dose)
and percentages (in brackets)
Crossing and bioassays
Family group 1 Family group 1 Family group
2
0 ppm (Control) 1/18 (6 %) 0/5 (0 %) 1/18 (6 %)
600 ppm 2/16 (13 %) 1/18 (6 %) 8/32 (25 %)
1800 ppm 13/15 (87 %) 12/18 (67 %) 16/25 (64 %)
Family group 1: females from the sensitive Ls A strain were crossed with males
from the
H202-resistant Ls V strain in the PO generation. Family group 2: males from
the sensitive
Ls A strain were crossed with females from the H202-resistant Ls V strain in
the PO
generation.
Example 2 Transcriptome analysis
In total, 36 adult female parasites were enrolled. From the original strains
(2013), four
laboratory reared Ls A and five field collected Ls V were used. From the PO
generation for
the batch crossing experiment (2015), three Ls A and four Ls V parasites were
included.
None of these parasites were selected for sensitivity towards any agent prior
to sampling.
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From the F2 generation after the crossing experiment, 20 parasites were
included. These
were selected for sensitivity towards H202, prior to sampling. Additional
details are given
in Table 2.
Table 2. Information about the 36 samples (all adult female lice) enrolled in
the RNAseq
study. N, sample size.
Group N Description
Laboratory strain, sensitive to all delousing chemicals. Collected in Northern
Norway
Ls A-2013 4
in 2011. Sixth generation.
Field strain, resistant to azamethiphos, deltamethrin, emamectin benzoate and
Ls V-2013 5
hydrogen peroxide_ Collected in Mid-Norway in 2013.
Laboratory strain. 12th generation of Ls A (sensitive). Not exposed to
delousing
Ls A-PO 3
chemicals during cultivation of any generation.
Laboratory strain. Sixth generation of Ls V (resistant). Not exposed to
delousing
Ls V-PO 4
chemicals during cultivation of any generation.
2-
Second generation after crossing of Ls A-PO and Ls V-PO, affected by 600 ppm
}1)07
Ls F
8 for 30 min (sensitive). Three lice from family
group 1 and five lice from family
H202-S
group 2.
Second generation after crossing of Ls A-PO and Ls V-PO, unaffected by 1800
ppin
Ls F2-
12 H202 for 30 min (resistant). Seven lice from family group 1 and five lice
from family
H202-R
group 2.
RNA extraction
Total RNA was extracted from the 36 individual adult females using a Trizol
protocol
combined with RNeasy Mini kit for animal tissues (Qiagen, CA, USA) (one
individual per
extraction). Lice tissues were disrupted and homogenized in 1 ml Trizol using
TissueLyser
MM 301 (Qiagen Retsch) and one stainless steel bead of 5 mm diameter (Qiagen).
After
mixing with 0.2 ml of chloroform and a centrifugation step, the aqueous phase
was
transferred to a new vial and mixed with one volume of 70% ethanol. Total RNA
was then
isolated with RNeasy spin columns following manufacturer's protocol. Genomic
DNA was
removed from the extracted RNA (10 lag) with Turbo DNA-free TM kit (TURBOTm
DNase
Treatment and Removal Reagents, Ambion, Life Technologies Thermo Fisher
Scientific,
USA). Subsequently, the RNA was cleaned and concentrated with RNA Clean &
ConcentratorTM-5 (Zymo Research). The RNA was quantified with ND-100
Spectrophotometer (Thermo Fisher Scientific, DE, USA) and the quality was
checked with
a 2100 Bioanalyzer instrument (Agilent Technologies) and the Agilent RNA 6000
Nano
kit.
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RNAseq
Total RNA samples wcrc used for library preparation and Illumina sequencing at
the
Norwegian Sequencing Centre (Oslo, Norway). Thirty-six RNA-seq libraries (one
per
individual lice), each with unique index barcodes, were prepared using the
TruSeq
Stranded total RNA library preparation Kit v2 (I1lumina. USA) by following
manufacturer's protocol including the polyA enrichment step. Libraries were
pooled
together and sequenced on NextSeq500 platform (Illumina, USA) using 150 bp
paired end
High output reagents. Raw bel files were generated using RTA v2.4.11 and were
later
demultiplexed (using the sample specific index) and converted to fastq format
using
bc12fastq v2.17.1.14.
RNAseq gene expression analysis (DESeq2) showed that the groups Ls V-2013 and
Ls F2-
H202-R each had more than 2000 genes differentially regulated compared to the
corresponding, presumably sensitive groups, Ls A-2013 and Ls-F2- H202-S. The
Ls V-PO
lice had less than 150 genes differentially regulated compared to Ls A-PO
(Fig. 2).
Example 3: Gene expression analysis
Demultiplexed raw reads were cleaned using Trimmomatic v0.33 (Bolger et al..
2014,
"Trimmomatic: a flexible trimmer for Illumina sequence data", Bioinformatics
30, 2114-
2120) to remove/trim low quality reads and sequencing adapters as well as
using BBMap
v34.56 (https://sourceforge.net/projects/bbmap/) to remove reads mapping to
PhiX genome
(Illumina spike-in). Cleaned fastq reads for each parasite were aligned to the
L. salmonis
transcriptome (coding sequences) using Hisat2 v2.1.0 (Kim et al., 2015,
"HISAT: a fast-
spliced aligner with low memory requirements", Nature Methods 12, 357-360.
https://www.nature.com/articles/nmeth.3317). The transcriptome file
(ftp://ftp.ensemblgenomes.org/pub/metazoakelease-
44/fasta/lepeophtheirus_salmonis)
contained the predicted transcriptome from genomic data. It was modified for
the
aquaporin genes by substituting the predicted cds sequences in the original
transcriptome
with experimentally determined cds sequences from Stavang et al. (2015),
supra.
Unmapped reads were filtered out using SAMtools version 1.4 (Li et al., 2009,
The
Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079).
Gene
annotation files in GTF format were generated for each parasite and then
mcrgcd using
Cufflinks version 2.2.1. (Trapnell et al., 2010, -Transcript assembly and
quantification by
RNA-Seq reveals unannotated transcripts and isoform switching during cell
differentiation". Nat. Biotechnol. 28, 511-515). Counts of fragments aligning
to each
transcript were calculated using FeatureCounts version 1.5.2. (Liao et al.,
2014,
"FeatureCounts: an efficient general-purpose program for assigning sequence
reads to
genomic features", Bioinformatics 30, 923-930). Analysis of the differential
expression
within each group (Ls A-2013 versus Ls V-2013; Ls A-PO versus Ls V-PO; Ls F2-
H202-S
versus Ls H,07-R) were done using DESN2 (Love et al., "Moderated estimation of
fold
change and dispersion for RNA-seq data with DESeq2-, Genome Biol. 15, 550)
(default
settings for the count normalization method). The significance level was set
to a = 0.05.
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Differentially expressed genes shared between presumed hydrogen peroxide
resistant lice
Thc DESeq2 analysis generated two lists for each group, one list of genes up-
regulated in
resistant lice and another list for genes down-regulated in resistant lice,
both compared to
sensitive lice within the same group. We searched for genes that were
differentially
5 expressed in the same direction in at least two of the three groups. An R
-script was
developed to identify the shared genes across all the groups (Ls 2013, Ls PO
and Ls F2-
1-1202) or between two of the groups (Ls 2013 vs Ls PO, Ls 2013 vs Ls F2- H202
and Ls PO
vs Ls F2- H202). The Uniprot database (www.uniprot.org), NCBI-Non-redundant
protein
sequences (nr) database (ncbi.nlm.nih.gov) and "ENSEMBL Metazoa (transcript)-
10 "protein information" section
(http://rnetazoa.ensembLorg/Lepeophtheirus_salmonis/Info/Index), were used to
annotate
the genes.
qPCR study
15 Quantitative polymerase chain reaction (qPCR) was used to validate the
RNAseq results
for two genes, catalase and Glpl_v2. An elevated expression of catalase has
already been
associated with resistance towards hydrogen peroxide (Helgesen et al. (2017),
"Increased
catalase activity ___________ A possible resistance mechanism in hydrogen
peroxide resistant salmon
lice (Lepeophtheirus salmonis)", Aquaculture, 468 (1), 135-140), and the
expression of
20 G1p1_v2 was significantly downregulated in the three groups of presumed
H20,-resistant
parasites in the RNAseq study (Ls V-2013, Ls V-PO, Ls F2- H202-R). The samples
enrolled
in the validation study were not exposed adult females from the RNAseq study:
Ls A-2013,
Ls V-2013, Ls A-P0 and Ls V-PO.
RNA extraction, DNase treatment and RNA cleaning were performed for every
sample the
25 same way as samples prepared for RNAseq. First strand cDNA was produced
from 1 iug of
cleaned RNA using the qScriptTM cDNA synthesis (reverse transcriptase) kit
(Quanta
Biosciences, MD, USA). The cDNA was cleaned with the DNA Clean &
ConcentratorTM5
kit (Zymo Research) and diluted 1:10 before used as PCR template for qPCR
using gene
specific primers and SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, CA,
USA),
30 following manufacturer's protocol. Each qPCR reaction was optimized for
11 1 total
reaction volume, 150/150 or 300/300 nM primer concentration and 2 In of
template,
corresponding to 0.2 lig cDNA/RNA. Reactions were run in duplicate or
triplicate and two
negative controls were added, a non-template control and a no reverse
transcriptase control.
The range of efficiencies for qPCR reactions were 96-98% for reference and
gene specific
primers. The qPCR was run on a Bio-Rad CFX96 real-time system (Bio-Rad, CA,
USA)
under the following conditions: 95 C for 30 sec followed by 40 cycles of
amplification at
95 C for 10 sec and 60 C for 50 sec. After qPCR, the homogeneity and
specificity of the
PCR products was confirmed by melting curve analysis, agarosc gel
electrophoresis and
Sanger sequencing. Relative gene expression was determined by the ACt method
(ACt =
Otarget Ctreference), where Cttarget is the Ct values for catalase or Glp1_v2,
and Ctreterence the
average of the elongation factor 1-alpha (EF) and prohibitin-2 (Proh2) genes.
(see table 3
below for primer details). Fold change in gene expression was calculated
according to the
2^-(AACt) method, using the average of the Ct values for Ls A-2013 and Ls A-PO
lice as
calibrator sample. Expression levels (ACt) of catalase and Glp1_v2 were
separately
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subjected to analysis of variance (ANOVA) with Tukey HSD post hoc test for
multiple
comparisons. Group (Ls A-2013+P0, Ls V-2013 and Ls V-PO) was used as the model
effect
and the significance level was set to p<0.05.
Table 3: Primers used for the qPCR validation experiment. Genes: Cat,
catalase; Glpl,
aquaglyceroporin type 1; EF, elongation factor 1-alpha; Proh2, prohibitin-2.
Gene Primer name Primer sequence Primer
Product
concentration
size (bp)
LsCAT6 F CCACAGAACAACTTGCCAAC
_ _ Cat 150/150 nM
157
Ls_CAT_6 R GCCATTTCGTCCATAAATGC
Ls_ Glp1_2 F TCGGCTCCAGGAATTGTTCT
Glp1 300/300 nM
200
Ls_ Glp1_2 R GGTCCTAAATCTCTCGCTGGG
Ls_gEF_2 F ATGGCACGGAGACAACATGT
EF 150/150 nM
206
Ls_gEF_2 R CGGGCACTGTTCCAATACCT
Ls gProhib2 2 F GCTCATCACACAGCGTCAAC
Proh2 300/300 nM
176
Ls_gProhib2_2 R CAGCTCTTTGGGCCTCTTGT
Gene expression
The catalase gene was previously found differentially expressed in fl202-
sensitive and -
resistant lice (Helgesen et al., 2017, supra) and its expression level has
recently been
introduced as H202 resistance marker in the salmon industry (Helgesen et al.,
2017, supra,
EP 3 164 502 B1). The present RNAseq study was performed in order to validate
the use of
the catalase gene expression as a resistance marker in adult females, as this
developmental
stage was not included in the previous study (Helgesen et al., 2017, supra).
There were
significantly higher numbers of catalase transcripts in resistant lice
selected by H202 (Ls
V-2013 and Ls F2- H202-R) than in sensitive lice (Table 5 and Fig. 1).
However, the
number of catalase transcripts in Ls V-PO parasites, which were not exposed to
H202 for
two years, did not differ significantly from Ls A-PO. The qPCR validation data
showed a
similar gene expression pattern compared to the DESeq2 analysis for 2013 and
PO RNAseq
samples (Figs. 1 and 3). Ls V-2013 had statistically significantly higher
catalase expression
than Ls A-2013 and Ls A-PO (Fig. 3). Ls V PO had a statistically similar
catalase
expression as Ls A-2013 and Ls A-PO. The sensitivity of the unexposed Ls V
strain was
tested after completion of the RNAseq study. The EC so value for pre-adult II
females of the
Ls V lab-strain was 1635 ppm, eight times higher than the Ls A strain (216
ppm) (Table 4),
suggesting that Ls V -PO lice were still resistant to H202 when enrolled in
the RNAseq
study. In addition, more than 70% of the Ls V-PO descendants (Ls F2- H202)
were
unaffected at 600 ppm H202, and more than 10% at 1800 ppm (Table 1). DESeq2
analysis
for Ls F2- H202-R showed that the catalase transcript had the lowest p(adj)
value among
the genes differentially expressed in that group of lice, and they had on
average close to
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three times higher numbers of catalase transcripts than their grandparents, Ls
V-PO (Table
5.
Table 4. Bioassay results for pre-adult 11 (males and females) and young adult
males
exposed to H202 for 30 min. N: total number of lice used in the bioassays (all
chemical
concentrations together). ppm: mg L-1. EC50: concentration immobilizing 50% of
the lice.
CI: confidence interval. Ls A: sensitive strain. Ls V: H202-resistant strain.
H202 exposure data:
Louse strain Year; water temperature; EC50
(ppm) (90% CI)
N; doses (ppm)
Ls A - Lab strain
Helgesen et al. 2015, supra * 2013; 10-12 C 216
(153 - 305)
Ls V
Helgesen et al. 2015, supra *
2013; 10-12 C 2127
(1253 - 3610)
2017; 10-11 C
Females: 1635 (734 -
Ls V- Lab strain 25 females and 22 males 3643)
M
0, 600, 1400, 2200, 3000,
ales: 1795 (1095 - 2943)
4200
*Data for males and females together. 95% CI.
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Table 5. Gene expression data of several genes differentially expressed in the
louse groups
Ls 2013, PO and F2-H202. The ENSEMBL L salmonis transcriptome was used in the
analysis, but the sequences of genes coding for aquaporins were replaced by
GenBank
entries (Stavang et al., 2015). log2FC = 1og2 fold change (up-regulation is
indicated as
positive values, down-regulation as negatives); p(adj) = p-value for
normalized counts (a =
0.05). Statistical significance is indicated in bold. Asterisk (*) indicates
no overlap in the
range of normalized counts between the groups.
Average normalized counts (range)
Lice
Gene Ls A / F2-H202-S Ls V / F2-H202-R log2FC p(adj)
group
Catalase 2013 3429 (2236 -
818 (675 - 1055) 2.066 <0.001*
6165)
PO 954 (696 - 1326) 706
(491 - 963) -0.433 0.784
F2-H202 2072 (1580 -
1161 (891- 1386) 0.836 <0.001*
2821)
DNA- 2013 374 (331 - 447) 464
(390 - 505) 0.318 0.044
polymerase PO 585 (495 - 658) 930
(812 - 1134) 0.669 0.024*
F2-H202 217 (144 - 344) 320
(165 - 548) 0.561 0.045
Nesprin- 2013 3864 (3522- 5297 (4644 -
0.455 <0.001
like 4290) 6116)
PO 5066 (4837 - 7036 (5803 -
0.474 0.034*
5304) 7547)
F2-H202 3271 (2887 - 4021 (3158 -
0.298 0.005
4498) 4403)
NA 2013 14(8 - 17) 33 (19 - 52)
1.186 0.018*
PO 21(11 -41) 94 (57 - 164)
2.162 0.026*
F2-H202 10 (4 - 20) 21(5 - 38)
1.034 0.015
ERP29 2013 90 (77 - 102) 56 (40 - 74) -
0.692 0.015*
PO 114 (96 - 128) 50 (45 - 55) -
1.203 <0.001*
F2-H202 110 (76- 140) 81 (44 - 118) -
0.443 0.019
G1p1_v2 2013 112 (74 - 164) 15 (10 - 26) -
2.894 <0.001*
PO 77 (64 - 86) 40 (35 - 44) -
0.934 0.025*
F2-ff202 197 (39 - 292) 88 (40 - 181) -
1.161 0.002
Aqp12L1 2013 158 (140 - 173) 99
(73 - 152) -0.679 0.013
PO 162 (144- 192) 148
(130- 181) -0.127 0.957
F2-H202 182 (130 - 219) 141
(104 - 185) -0.370 0.010
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Aqp12L2 2013 56 (42 ¨ 75) 15 (13 ¨
20) -1.905 <0.001*
PO 29 (11 ¨46) 24 (19 ¨
30) -0.289 0.960
F2-H202 98 (69 ¨ 124) 66(31 ¨103)
-0.571 0.012
G1p2 2013 20 (15 ¨ 31)
5 (0¨ 14) -1.980 0.045*
PO 17 (7 ¨ 26) 20 (16 ¨
30) 0.222 0.976
F2-H202 24 (6 ¨ 42) 11(1 ¨ 17)
-1.119 0.008
G1p3_v1 2013 149 (110 ¨ 182) 297 (183 ¨
365) 0,990 <0.001*
PO 222 (185 ¨ 253) 174 (120 ¨
253) -0.351 0.855
F2-11702 134 (91 ¨ 203) 121 (82 ¨
148) -0.160 0.378
ENSEMBL/GenBank gene names for the genes: Catalase, EMLSAT00000007315; DNA-
polymerase, EMLSAT00000002584; Ncsprin-likc, EMLSAT00000005972; NA (not
annotated), EMLSAT00000005947; ERP29, EMLSAT00000009549; Glpl v2,
KR005661.1; Aqp12L1, KR005665.1; Aqp12L2, KR005666.1; Glp2, KR005662.1:
Glp3_v1, KR005663.1.
These results may indicate that the catalase gene is induced by H202 exposure.
The
induction of the catalase gene after H202 exposure was demonstrated in a
penaeid shrimp
(Wang ct al., 2012, supra). Thc gcnc was significantly up-rcgulatcd at 2 h
aftcr injccting
0.1% H202 in the shrimp body. In addition, H202 resistance is hereditary, as
demonstrated
by Helgesen et al (2015, 2017, supra). The heritable factor may thus be the
ability to
quickly induce catalase expression. This also indicates that induction of the
catalase gene
can be problematic when using the expression of this gene as a 11202
resistance marker,
since resistant lice not exposed to H202 may have a normal expression of this
gene and
could erroneously be classified as sensitive. On the other hand, after a short
exposure to
WO?, sensitive and resistant lice seem to be readily separable by expression
of the catalase
gene.
New putative molecular markers
To identify further genes associated with H202 resistance, differentially
expressed genes
from the Ls 2013, Ls PO and Ls F2-11202 generations were compared. The
resistant adult
female lice that had been exposed to H202 (Ls V-2013 and Ls F2-H202-R) shared
790
differentially expressed genes (Fig. 2). This support the hypothesis that F202
exposure
could induce the expression of several genes, even within a timespan of 30
minutes. Only
five genes (three up-regulated and two down-regulated in resistant lice) were
differentially
expressed in all three groups (Ls V-2013, Ls V-PO and Ls F2-H202¨R) (Fig. 2).
irrespective of H202 exposure. Table 5 shows the gene expression and
annotation data for
these genes. The log2 fold change ranged from ¨10.21 to ¨131. The three genes
consistently
up-regulated in presumed resistant lice were a DNA polymerase (delta subunit
3), the
Nesprin-like protein and a not annotated protein (NA). Nesprin-like, also
known as enaptin
or synaptic nuclear envelope protein 1, is an actin-binding protein involved
in the
maintenance of nuclear organization and structural integrity. The two genes
downregulated
in resistant adult female lice were the endoplasmic reticulum resident protein
29 (ERP29)
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and an aquaporin protein (Glp1_v2). ERP29 plays an important role in the
processing of
secretory proteins within the endoplasmic reticulum.
All but Glpl_v1 and Glp3_v2 were detected in our RNAseq data. Table 5 shows
the gene
expression data for several aquaporins in our study. There were no
statistically significant
5 differences in the expression of Bill or PripL within any of the Ls 2013,
Ls PO or Ls F2-
H202 groups (data not shown). However, Glpl v2 was statistically significantly
down-
regulated in all presumed H2O, resistant groups (Ls V-2013, Ls V-PO and Ls F2-
14202-R).
The qPCR validation data showed a similar gene expression pattern compared to
the
DESeq2 analysis for 2013 and PO RNAseq samples (Figs. 1 and 3). Ls V-2013 and
Ls V PO
10 had statistically significantly lower Glpl v2 expression than Ls A-2013
and Ls A-PO (Fig.
3). Glp 2 was significantly down-regulated in two groups, Ls V-2013 and Ls F2-
H202-R,
but the expression of this gene was low. Glp3_v1 was up-regulated in Ls V-
2013. The
unorthodox aquaporins, Aqp12L1 and Aqp12L2, were statistically significantly
down-
regulated in Ls V-2013 and Ls F2-H202--R groups.
15 Glp1_v2 was down-regulated in all three groups of presumed H202
resistant adult female
lice, indicating a possible involvement in H202 transport: The lower the
number of
Glp1_v2 channels is, the less amount the exogenous H202 would enter the louse
body and
cause toxic effects. The downregulation of Aqp12L1 and Aqp12L2 in presumably
resistant
lice exposed to I-1202 may also indicate a role of these proteins as H202
channels. This goes
20 especially for Aqp12L2, with a ¨121log2 fold change and no overlap in
the normalized
count range in the Ls 2013 groups (Table 5). As in the case of Glps, Stavang
et al. (2015),
supra, also found an open pore configuration in the 3D modelling of Aqp12L2.
There was an overlap in the normalized count range for DNA-polymerase, Nesprin-
like,
NA, ERP29, Glp1_v2, Aqp12L1 and Aqp12L2 genes between sensitive and resistant
F2 -
25 H202 lice (Table 5), although the differences were statistically
significant. F2 lice is a
louse population arisen from the mix of sensitive and resistant lice, with a
wide range of
WO, sensitivities. This gene expression overlap suggests that H2O, resistance
in F2 lice
comes from a number of up- and down-regulated genes combined in slightly
different ways
depending on each individual louse, giving to all of them the capability to
survive 1800
30 ppm 1-1702. Only the expression of catalase was able to clearly separate
sensitive from
resistant F2 lice. As an example, the two F2-1-1202 resistant lice with high
Glp1_v2 reads,
are the ones with higher catalase expression, possibly suggesting a
compensatory effect:
high G1p1_v2 reads could mean that more exogenous H202 would enter the louse
body,
needing the louse more catalase for breaking down the H202 and survive the
exposure (Fig.
35 1, dark grey and black triangles).
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Table 6: Overview of sequence numbering
Sequence number Description
SEQ ID No. 1 DNA sequence encoding aquaglyceroporin (Glpl
v2) identified in
Lepeophtheirus salmonis
SEQ ID No. 2 Amino acid sequence of aquaglyceroporin
(Glp1_v2) encoded by
SEQ ID No. 1
SEQ ID No. 3 DNA sequence encoding endoplasmic reticulum
resident protein 29
(ERP29) identified in Lepeophtheirus salmonis
SEQ ID No. 4 Amino acid sequence of endoplasmic reticulum
resident protein 29
(ERP29) encoded by SEQ ID No. 3
SEQ ID No. 5 DNA sequence encoding DNA polymerase (delta
subunit 3) identified
in Lepeophtheirus salmonis
SEQ ID No. 6 Amino acid sequence of DNA polymerase (delta
subunit 3) encoded
by SEQ Ill No. 5.
SEQ ID No. 7 DNA sequence encoding nesprin-like identified
in Lepeophtheirus
salmonis
SEQ Ill No. 8 Amino acid sequence of nesprin-like encoded by
SEQ Ill No.7
SEQ ID No. 9 DNA sequence encoding a hitherto unknown
protein identified in
Lepeophtheirus salmonis
SEQ ID No. 10 Amino acid sequence of the protein encoded by
SEQ ID No. 9
SEQ ID No. 11 DNA sequence encoding catalase of L. salrnonis
SEQ ID No. 12 DNA sequence encoding catalase of C. clemensei
SEQ Ill No. 13 DNA sequence encoding catalase of C.
rogercresseyi
SEQ ID No. 14 DNA sequence encoding catalase of C. elongatus
SEQ ID No. 15 Primer used to detect expression level of
catalase (Ls_CAT_6 F)
SEQ ID No. 16 Primer used to detect expression level of
catalase (Ls_CAT_6 R)
SEQ ID No. 17 Primer used to detect expression level of
aquaglyceroporin type 1
(Ls_Glp1_2 F)
SEQ Ill No. 18 Primer used to detect expression level of
aquaglyceroporin type 1
(Ls_Glp1_2 R)
SEQ ID No. 19 Primer used to detect expression level of
elongation factor 1-alpha
(Ls_gEF_2 F)
SEQ ID No. 20 Primer used to detect expression level of
elongation factor 1-alpha
(Ls gEF 2 R)
SEQ ID No. 21 Primer used to detect expression level of
prohibitin-2 (Ls_gProhib2_2
F)
SEQ ID No. 22 Primer used to detect expression level of
prohihitin-2 (Ls_
gProhib2_2 R)
SEQ ID No. 23 A revers primer of 24 nucleotides comprising
the sequence
CATACAAGTATAGGAACTGGCTCA wherein the primer is used
for detection of a SNP in the zinc finger gene PLAGL1 from sheep
(SEQ ID 12 of CN108866160 A)
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