EP4085155A1 - Procédé de détection de la résistance au peroxyde d'hydrogène chez les crustacés - Google Patents

Procédé de détection de la résistance au peroxyde d'hydrogène chez les crustacés

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Publication number
EP4085155A1
EP4085155A1 EP20839316.5A EP20839316A EP4085155A1 EP 4085155 A1 EP4085155 A1 EP 4085155A1 EP 20839316 A EP20839316 A EP 20839316A EP 4085155 A1 EP4085155 A1 EP 4085155A1
Authority
EP
European Patent Office
Prior art keywords
seq
sea lice
protein
lice
group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20839316.5A
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German (de)
English (en)
Inventor
Tor Einar HORSBERG
Marit JØRGENSEN BAKKE
Celia AGUSTI-RIDAURA
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Norges Miljo Og Biovitenskapelige Universitet
Patogen As
Original Assignee
Norges Miljo Og Biovitenskapelige Universitet
Patogen As
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Application filed by Norges Miljo Og Biovitenskapelige Universitet, Patogen As filed Critical Norges Miljo Og Biovitenskapelige Universitet
Publication of EP4085155A1 publication Critical patent/EP4085155A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Definitions

  • TITLE Method for detecting hydrogen peroxide resistance in crustaceans
  • the present invention relates to a method for detecting whether or not crustaceans, in particular sea lice, such as Lepeophtheirus salmonis and Caligus rogercresseyi is resistant towards hydrogen peroxide (H2O2).
  • 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 H2O2.
  • the present invention furthermore relates to oligonucleotide sequences and kits useful in the 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.) are 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 €300 million in 2009 (Costello M. J. (2009), The global economic cost of sea lice to the salmonid farming industry. Journal of Fish Diseases. 32.
  • IPM integrated pest management
  • 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.
  • cytochrome P450 is involved in the detoxification of pyrethroids, as well as biomarkers in the mitochondrial DNA (Bakke et al., 2018,"DeItamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krpyer): Maternal inheritance and reduced apoptosis.” Scientific Reports, Vol. 8, 8450).
  • PCR based methods for detection of resistance genes have been published for azamethiphos (EP 3 033 433 Al), 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 Bl).
  • Interox ® Paramove ® 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 reinfection. 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 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.
  • the use of H 2 O 2 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).
  • Pesticide Resistance in 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.
  • detoxifying enzymes such as catalase, glutathione reductase, glutathione synthetase, superoxide dismutase, and glucose-6-phosphate dehydrogenase.
  • 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 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).
  • sea lice 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 H 2 O 2 is well established. Reduced sensitivity towards hydrogen peroxide (H 2 O 2 ) was first reported in Scotland (Treasurer et al.
  • 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.
  • NA yet unknown protein
  • the sea lice that may be analysed according to the present invention is one or more copepods, e.g. belonging to the family Caligidae.
  • the copepod is selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus, and Caligus rogercresseyi.
  • the copepod is selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei and Caligus elongates.
  • the copepod is Caligus rogercresseyi.
  • the copepod is Lepeophtheirus salmonis.
  • a method 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • G1p1_v2 aquaglyceroporin
  • ERP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • 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.
  • 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. 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.
  • said method 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 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 ID No. 10.
  • said method 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;
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10 is determined.
  • G1p1_v2 aquaglyceroporin
  • EBP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • nesprin-like and the protein of SEQ ID No. 10 is determined.
  • 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 (G1p1_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 ID 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.
  • the present invention provides a method for determination of H 2 O 2 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 (G1p1_v2) is downregulated compared with the expression level of aquaglyceroporin (G1p1_v2) in one or more H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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: (cl) 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 ID No. 5. SEQ ID No. 7, 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, SEQ ID No. 9, respectively; and
  • RNA expression level of said genes in the collected sea lice wherein said sea lice is resistant to H 2 O 2 if having reduced levels of expression of the genes encoding aquaglyceroporin (G1p1_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.
  • G1p1_v2 aquaglyceroporin
  • ERP29 endoplasmic reticulum resident protein 29
  • the present invention provides a method for determination of H 2 O 2 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;
  • a method 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 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • 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 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 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 ID No. 10.
  • said method 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.
  • 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.
  • 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 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.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 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 H 2 O 2 sensitive sea lice.
  • the present invention provides a method for determination of H 2 O 2 resistance in one or more sea lice, wherein step (c) of the fourth aspect of the present invention comprise the steps of:
  • (cl) 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.
  • the present invention provides a method for determination of H 2 O 2 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;
  • 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. 1, SEQ ID No. 3, SEQ ID No. 5.
  • 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.
  • 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 ID No. 3, SEQ ID No. 5.
  • sea lice is selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus and Caligus rogercresseyi
  • 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.
  • 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.
  • a DNA molecule 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. 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.
  • a DNA 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.
  • a DNA molecule 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.
  • Figure 1 show gene expression data (normalized counts) of catalase and the five genes significantly differentially expressed in the 2013, P0 and F2-H 2 O 2 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-P0 (white triangles), Ls V-P0 (grey triangles), Ls L2- H 2 O 2 -S (sensitive lice, white diamonds), Ls L2- H 2 O 2 -R (resistant lice, grey diamonds). Solid lines represent the mean in each group. Dark grey and black diamonds in the Ls L2-H H 2 O 2 -R group represent the same individual lice in both catalase and G1p1_v2 graphs.
  • Ls A-2013 white circles
  • Ls V-2013 grey circles
  • Ls V-P0 white triangles
  • Ls V-P0 grey triangles
  • Ls L2- H 2 O 2 -S sensitive lice, white diamond
  • Figure 2 illustrates the number of genes differentially expressed in the H 2 O 2 resistant lice (2013, P0 and F2- H 2 O 2 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.
  • 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 (G1p1_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 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 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • G1p1_v2 aquaglyceroporin
  • EBP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • said method may also involve the determination of the expression level of the gene encoding catalase.
  • the sea lice to be analysed is one or more adult female sea lice.
  • adult female sea lice selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus.
  • 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.
  • ERP29 endoplasmic reticulum resident protein 29
  • delta subunit 3 DNA polymerase
  • said method may also involve the determination of the expression level of the gene encoding catalase.
  • G1p1_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 aquaglyceroporins (G1p1_v1,
  • G1p1_v2, G1p2, G1p3_v1 and G1p3_v2) and two unorthodox aquaporins (Aqp12-like1 or “Aqp12L1” and Aqp12-like 2 or “Aqp12L2”).
  • Aquaporins are protein channels that facilitate transport of water, other small solutes such as H 2 O 2 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.
  • Parasite aquaporins Current developments in drug facilitation and resistance, Biochimica et biophysica acta. 1840: 1566-1573).
  • Faghiri and Skellv 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.
  • SmAQP putative aquaglyceroporin
  • the aquaglyceroporin LmAQPl transports Sblll 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 LmAQPl (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.
  • Catalase activity can also be regulated by reversible phosphorylation via kinase enzymes by increasing the affinity of the enzyme for H 2 O 2 (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 H 2 O 2 sensitive and resistant lice (data not shown).
  • 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 (ER
  • the sea lice is an adult female sea lice.
  • sea lice selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus,
  • the sea lice is Caligus rogercresseyi
  • the above method may optionally also involve the determination of the expression level of catalase in the analysed one or more sea lice.
  • 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 ( G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No.
  • step (c) involved determining the expression level of the genes encoding aquaglyceroporin (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No.
  • the method of the invention involves the determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2) and endoplasmic reticulum resident protein 29 (ERP29), and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1pI_v2) and DNA polymerase (delta subunit 3), and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1pl_v2) and nesprin-like, and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of aquaglyceroporin (G1ll_v2) and the protein of SEQ ID No. 10, and optionally also determination of the expression level of a gene encoding catalase.
  • 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.
  • ERP29 endoplasmic reticulum resident protein 29
  • delta subunit 3 DNA polymerase
  • 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.
  • ERP29 endoplasmic reticulum resident protein 29
  • nesprin-like a gene encoding catalase
  • 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.
  • 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.
  • 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 ID No. 10, and optionally also determination of the expression level of a gene encoding catalase.
  • 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.
  • a method 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 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 three 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.
  • G1p1_v2 aquaglyceroporin
  • ERP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • the sea lice is an adult female sea lice.
  • sea lice selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus.
  • the sea lice is Caligus rogercresseyi
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2), 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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), and nesprin-like, and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2), DNA polymerase (delta subunit 3), and nesprin-like, and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2), nesprin-like, and the protein of SEQ ID No. 10, and optionally also determination of the expression level of a gene encoding catalase.
  • 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.
  • 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.
  • ERP29 endoplasmic reticulum resident protein 29
  • delta subunit 3 DNA polymerase
  • SEQ ID No. 10 the protein of SEQ ID No. 10
  • the method of the invention involves determination of the expression level of the genes encoding 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.
  • ERP29 endoplasmic reticulum resident protein 29
  • nesprin-like the protein of SEQ ID No. 10
  • SEQ ID No. 10 the protein of SEQ ID No. 10
  • 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.
  • a method 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 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 four 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.
  • G1p1_v2 aquaglyceroporin
  • ERP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • the sea lice is an adult female sea lice.
  • sea lice selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus.
  • the sea lice is Caligus rogercresseyi
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_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.
  • 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), nesprin-like and the protein of SEQ ID No. 10; and optionally also determination of the expression level of a gene encoding catalase.
  • the method of the invention involves determination of the expression level of the genes encoding aquaglyceroporin (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), 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.
  • 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.
  • 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 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.
  • 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 Invitrogen® provided ThermoFisher Scientifc (https://www.thermofisher.com/no/en/home/life-science/gene- expression-analvsis-genotvping/quantigene-rna-assavs.html ' ).
  • Real-time PCR based methods 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.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • G1p1_v2 aquaglyceroporin
  • EBP29 endoplasmic reticulum resident protein 29
  • DNA polymerase delta subunit 3
  • nesprin-like the protein of SEQ ID No. 10.
  • 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.
  • 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.
  • sea lice selected from the group consisting of Lepeophtheirus salmonis, Caligus clemensei, Caligus elongatus.
  • the sea lice is Caligus rogercresseyi
  • louse or "sea lice” is to be understood to mean one or more copepod belonging to the family Caligidae.
  • sea lice or “sea louse” refer to the species Lepeophtheirus salmonis.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10, respectively.
  • 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 genomic material may be e.g. mRNA or DNA.
  • 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.
  • 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.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • G1p1_v2 aquaglyceroporin
  • EBP29 endoplasmic reticulum resident protein 29
  • 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.
  • 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 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.
  • the oligonucleotide probe according to the present invention comprises about 10 to 25 consecutive nucleotides.
  • LNA hybridization probes allow for the use of extremely short oligonucleotide probes (You Y.; Moreira B.G.; Behlke M.A. and Owczarzy R.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No.
  • 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.
  • the oligonucleotide primer according to the present invention comprises about 14 - 25 consecutive nucleotides, more preferably about 15 nucleotides.
  • 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).
  • PCR polymerase chain reaction
  • 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 oligonucleotide primer according to the present invention typically comprises the appropriate number of nucleotides allowing that said primer align with the target sequence to be analyzed.
  • Oligonucleotide probes and oligonucleotide primers may be manufactured according to methods well known to the skilled person.
  • oligonucleotides may be used determine the expression level of a gene encoding a protein 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.
  • oligonucleotides 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.
  • % 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.
  • BLAST Basic Local Alignment Search Tool
  • 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.
  • genomic material i.e. any part of the sea lice
  • any tissue i.e. any part of the sea lice
  • 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 preadult (chalimus stages 1-4), or the adult stage (adult male or adult female).
  • the sea lice are adult female.
  • 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 protocol of the manufacturer (http://lycofs01.lycoming.edu/ ⁇ gcat- seek/protocols/DNeasy_Blood_&_Tissue_Handbook.pdf ).
  • DNA extraction/isolation methods such as e.g. DNeasy DNA Tissue Kit according to the protocol of the manufacturer (http://lycofs01.lycoming.edu/ ⁇ gcat- seek/protocols/DNeasy_Blood_&_Tissue_Handbook.pdf ).
  • 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.
  • the detection reagents in a kit according to the present invention may furthermore include other components commonly included in such kits, e.g.
  • 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 method according to the present invention.
  • kits 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.
  • 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 (G1p1_v2), endoplasmic reticulum resident protein 29 (ERP29), DNA polymerase (delta subunit 3), nesprin-like and the protein of SEQ ID No. 10.
  • 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. Examples Salmon louse strains
  • Ls A sensitive to all anti-salmon lice chemicals used in Norway (tested by bioassays)
  • 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 H 2 O 2 treatment efficacy.
  • Example 1 Crossing experiment and bioassays
  • a batch crossing experiment was designed. The experiment was performed as described by Bakke et al. (2016) in 2015 (Bakke et al., (2016), “Deltamethrin resistance in the salmon louse, Lepeophtheirus salmonis (Krpyer): Maternal inheritance and reduced apoptosis”, Scientific Reports 8, Article number: 8450).
  • two Atlantic salmon one fish per aquarium
  • F2 parasites were allowed to develop to adults, and were selected for sensitivity towards hydrogen peroxide (Interox Paramove 50, H 2 O 2 50%, w/w, Solvay Chemicals, Belgium). The selection was performed in vitro using two-dose bioassays at Faculty of Veterinary 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 H 2 O 2 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 H 2 O 2 were included to check the general performance of the parasites.
  • Parasites affected/immobilized at the lowest H 2 O 2 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 or floating at the surface).
  • lice were fixed in RNAlater and kept at -80 °C following ⁇ 24h at room-temperature.
  • H 2 O 2 sensitive and resistant F2 adult females were used in the RNAseq analysis.
  • H 2 O 2 sensitive and resistant lice In order to obtain H 2 O 2 sensitive and resistant lice for the RNAseq study, F2 adult females were selected with two-dose H 2 O 2 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 H 2 O 2 doses. For adult females, there were no significant differences between family groups, indicating that inheritance of resistance was not gender-specific.
  • Family group 1 females from the sensitive Ls A strain were crossed with males from the FLCh-resistant Ls V strain in the P0 generation.
  • Family group 2 males from the sensitive Ls A strain were crossed with females from the FLCL-resistant Ls V strain in the P0 generation.
  • 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.
  • RNAseq RNAseq
  • RNA samples were 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 (Illumina, 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 bcl files were generated using RTA v2.4.11 and were later demultiplexed (using the sample specific index) and converted to fastq format using bcl2fastq v2.17.1.14.
  • RNAseq gene expression analysis (DESeq2) showed that the groups Ls V-2013 and Ls F2- H 2 O 2 -R each had more than 2000 genes differentially regulated compared to the corresponding, presumably sensitive groups, Ls A-2013 and Ls-F2- H 2 O 2 -S.
  • the Ls V-P0 lice had less than 150 genes differentially regulated compared to Ls A-P0 (Fig. 2).
  • the 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.
  • An R -script was developed to identify the shared genes across all the groups (Ls 2013, Ls P0 and Ls F2- H 2 O 2 ) or between two of the groups (Ls 2013 vs Ls P0, Ls 2013 vs Ls L2- H 2 O 2 and Ls P0 vs Ls L2- H 2 O 2 ).
  • qPCR Quantitative polymerase chain reaction
  • G1p1_v2 was significantly downregulated in the three groups of presumed H 2 0 2 -resistant parasites in the RNAseq study (Ls V-2013, Ls V-PO, Ls F2- H 2 O 2 -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-PO and Ls V-PO.
  • RNA extraction, DNase treatment and RNA cleaning were performed for every sample the same way as samples prepared for RNAseq.
  • First strand cDNA was produced from 1 pg of cleaned RNA using the qScriptTM cDNA synthesis (reverse transcriptase) kit (Quanta Biosciences, MD, USA).
  • the cDNA was cleaned with the DNA Clean & ConcentratorTM -5 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), following manufacturer’s protocol.
  • Each qPCR reaction was optimized for 11 m ⁇ total reaction volume, 150/150 or 300/300 nM primer concentration and 2 m ⁇ of template, corresponding to 0.2 pg 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.
  • the catalase gene was previously found differentially expressed in PGOi-sensitive and - resistant lice (Helgesen et al., 2017, supra ) and its expression level has recently been introduced as H 2 O 2 resistance marker in the salmon industry (Helgesen et al., 2017, supra, EP 3 164 502 Bl).
  • 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).
  • Ls V-P0 parasites which were not exposed to H 2 O 2 for two years, did not differ significantly from Ls A-P0.
  • the qPCR validation data showed a similar gene expression pattern compared to the DESeq2 analysis for 2013 and P0 RNAseq samples (Figs. 1 and 3).
  • Ls V-2013 had statistically significantly higher catalase expression than Ls A-2013 and Ls A-P0 (Fig. 3).
  • Ls V P0 had a statistically similar catalase expression as Ls A-2013 and Ls A-P0.
  • the sensitivity of the unexposed Ls V strain was tested after completion of the RNAseq study.
  • DNA- 2013 374 (331 _ 447) 464 (390 - 505) 0.318 0.044 polymerase P0 585 (495 - 658) 930 (812 - 1134) 0.669 0.024*
  • ERP29 2013 90 (77 - 102) 56 (40 - 74) -0.692 0.015*
  • KR005661.1 Aqp12L1, KR005665.1; Aqp12L2, KR005666.1; G1p2, KR005662.1; G1p3_v1, KR005663.1.
  • the induction of the catalase gene after H 2 O 2 exposure was demonstrated in a penaeid shrimp (Wang et al., 2012, supra).
  • the gene was significantly up-regulated at 2 h after injecting 0.1% H 2 O 2 in the shrimp body.
  • H 2 O 2 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.
  • the log2 fold change ranged from ⁇ 0.2
  • 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) and an aquaporin protein (G1p1_v2).
  • ERP29 plays an important role in the processing of secretory proteins within the endoplasmic reticulum.
  • Ls V-2013 and Ls V P0 had statistically significantly lower G1p1_v2 expression than Ls A-2013 and Ls A-P0 (Fig. 3).
  • G1p 2 was significantly down-regulated in two groups, Ls V-2013 and Ls F2-H 2 O 2 -R, but the expression of this gene was low.
  • G1p3_v1 was up -regulated in Ls V-2013.
  • the unorthodox aquaporins, Aqp12Ll and Aqp12L2 were statistically significantly down- regulated in Ls V-2013 and Ls F2-H 2 O 2 -R groups.
  • G1p1_v2 was down-regulated in all three groups of presumed H 2 O 2 resistant adult female lice, indicating a possible involvement in H 2 O 2 transport: The lower the number of
  • G1p1_v2 channels is, the less amount the exogenous H 2 O 2 would enter the louse body and cause toxic effects.
  • the downregulation of Aqp12Ll and Aqp12L2 in presumably resistant lice exposed to H 2 O 2 may also indicate a role of these proteins as H 2 O 2 channels. This goes especially for Aqp12L2, with a ⁇
  • G1ps Stavang et al. (2015), supra, also found an open pore configuration in the 3D modelling of Aqp12L2.
  • F2 lice is a louse population arisen from the mix of sensitive and resistant lice, with a wide range of H 2 O 2 sensitivities. This gene expression overlap suggests that H 2 O 2 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 ppm H 2 O 2 . Only the expression of catalase was able to clearly separate sensitive from resistant F2 lice.
  • the two F2-H 2 O 2 resistant lice with high G1p1_v2 reads are the ones with higher catalase expression, possibly suggesting a compensatory effect: high G1p1_v2 reads could mean that more exogenous H 2 O 2 would enter the louse body, needing the louse more catalase for breaking down the H 2 O 2 and survive the exposure (Fig. 1, dark grey and black triangles).
  • Table 6 Overview of sequence numbering
  • EBP29 endoplasmic reticulum resident protein 29
  • 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 ID No. 5.
  • SEQ ID No. 7 DNA sequence encoding nesprin-like identified in Lepeophtheirus salmonis
  • SEQ ID No. 8 Amino acid sequence of nesprin-like encoded by SEQ ID No.7
  • SEQ ID No. 9 DNA sequence encoding a hitherto unknown protein identified in Lepeophtheirus salmonis
  • SEQ ID No. 11 DNA sequence encoding catalase of L. salmonis
  • SEQ ID No. 14 DNA sequence encoding catalase of C. elongatus
  • 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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Abstract

La présente invention concerne un procédé permettant de détecter si des crustacés, en particulier les poux de mer, tel que Lepeophtheirus salmonis et Caligus rogercresseyi sont résistants au peroxyde d'hydrogène (H2O2). En particulier, le procédé de l'invention concerne la détection du niveau d'expression de gènes qui s'avèrent être régulés à la hausse ou régulés à la baisse dans les poux de mer résistants à H2O2. La présente invention concerne en outre des séquences oligonucléotidiques et des kits comprenant des séquences oligonucléotidiques utiles dans le procédé de la présente invention.
EP20839316.5A 2020-01-02 2020-12-22 Procédé de détection de la résistance au peroxyde d'hydrogène chez les crustacés Withdrawn EP4085155A1 (fr)

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