Screening and identification of resistant markers in crustacean

专利摘要:
A method for the detection of resistance of pests to pyrethroid chemotherapeutics, in particular to a method of determining single polymorphisms associated with pyrethroid resistance in the mitochondrial DNA of arthropods, in particular crustaceans, in particular copepods, and the use of such SNP´s in a screening method for determining whether such pests are sensitive to, or resistant to, pyrethroids.
公开号:DK201600138A1
申请号:DK201600138
申请日:2016-03-03
公开日:2016-03-21
发明作者:Frank Nilsen；Per Gunnar Espedal
申请人:Bergen Teknologioverføring As；Patogen Analyse As；
IPC主号:

专利说明:

Screening and identification of resistant markers in crustacean Field of the invention
The present invention relates to a method for the detection of resistance of pests to pyrethroid chemotherapeutics, in particular to a method of determining single nucleotide polymorphisms associated with pyrethroid resistance in arthropods, in particular crustaceans, in particular copepods, and the use of such SNP's in a screening method for determining whether such pests are sensitive to, or resistant to, pyrethroids.
Background of the invention
Currently, pyrethroids are the most used insecticides against arthropod plagues in agriculture and livestock as well as in the control of vectors of veterinary and human health importance.
Examples of such pests are species of crustacean, and in particular in the class copepod known as sea lice. Infestations of the parasitic copepods Lepeophtheirus salmonis and Caligus spp., commonly referred to as sea lice, represent a major challenge to commercial salmon aquaculture. Control measures have been reliant upon the use of a limited number of chemotherapeutants, in particular pyrethroid compounds, since the 1970’s, and reduced efficacy has now been reported for the majority of these compounds. Reduced sensitivity and potential resistance to currently available medicines are constant threats to maintaining control of sea lice populations in Atlantic salmon farms. Today, resistance against most of the available treatment options is widespread in almost every region where salmonids are cultured in sea water. Double- or triple resistance, in which sea lice is resistant to more than one type of chemotherapeutant, have been verified.
Drug resistance in sea lice has been detected by various types of bioassays as a significant increase in EC50/LC50. Although the bioassays can detect any type of resistance to a given drug, such methods are not very accurate or sensitive.
The genomes of all organisms undergo spontaneous mutations during their continuing evolution, forming variant forms of progenitor genetic sequences. A mutation may results in an evolutionary advantage or disadvantage relative to a progenitor form or may be neutral. A variant that result in an evolutionary advantage may eventually be incorporated in many members of the species and may thus effectively become the progenitor form. Furthermore, often various variant forms survive and coexist in a species population. The coexistence of multiple forms of a genetic sequence gives rise to genetic polymorphism, including single-nucleotide polymorphisms (SNPs).
A single-nucleotide polymorphism (SNP) is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes in an organism. For example, two DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide, commonly referred to as two alleles. Almost all common SNPs have only two alleles. The genomic distribution of SNPs is not homogenous; SNPs usually occur in non-coding regions more frequently than in coding regions or, in general, where natural selection is acting and fixating the allele of the SNP that constitutes the most favorable genetic adaptation (Adapted from Wikipedia).
Of particular relevance is the fact that occasionally a SNP may occur in a pest organism that provides that organism with resistance to known pesticides.
Up to date, however, no SNPs specifically associated with insecticide resistance in sea lice have been reported. In fact, no such SNPs have been identified for any crustacean species (Arthropods Pesticide Resistance Database, it is known in the art that in some species of arthropods, specific mutations in the gene coding for the voltage gated sodium channel have been reported to result in an altered function of this channel, resulting in a decreased ability for pyrethroids to interfere with its function (Ziotkin E, (1999). The insect voltage-gated sodium channel as target of insecticides. Anna Rev EntomoL 44:429-55).
Previously, non-mendelian inheritance of resistance by spider mites towards a hydrazine carbazate derivate have been linked to mutations in mitochondria! DNA of the spider mite (Van Leeuwen, T., Vanhoime, B., Van Potteiberge, S., Van Nieuwenhuyse, P., Nauen, R,s Tirry, L, & Denholm, I. (2010). Mitochondrial heteropiasmy and the evolution of insecticide resistance: non-Mendeiian inheritance in action. Proceedings of the National Academy of Sciences of the
United States of America, 105(18), 5980—5), Several factors are drastically different however between the observations regarding spider-mites, and inherited resistance towards pyrethroids in crustaceans, in particular copepods such as seal Sice. First of all, the spider-mite and sea lice are distantly related. Also, as the present inventors have observed, SNPs in non-coding regions of the mitochondrion is important for the regulation of resistance towards pyrethroids, while in the spider-mite it was highly conserved coding-regions that were observed to be of importance. No previous study has ever described non-coding regions of the mitochondrial DMA to be of importance to pesticide resistance in any species.
Genes encoded by mitochondrial DNA (mtDNA) exist in large numbers per cell but can be selected very rapidly as a result of unequal partitioning of mtDNA between germ cells during embryogenesis. Each mitochondrion is estimated to contain 2-10 mtDNA copies (Wiesner RJ, Ruegg JC, Morano I (1992). "Counting target molecules by exponential polymerase chain reaction, copy number of mitochondrial DNA in rat tissues". Biochim Biophys Acta. 183 (2): 553-559. doi: 10.1016/0008-291X02)90517-0. PMID 1550583.), and each ceil contains several mitochondria. However, empirical studies of this “bottlenecking" effect are rare because of the apparent scarcity of heteroplasmic individuals possessing more than one mtDNA haplotype. Previously, Van Leeuwen ei al, (2008)) (Van Leeuwen, T., Vanholme, B., Van Pottelberge, S., Van Nieuwenhuyse, P., Nauen, R., Tirry, L, & Denholm, I. (2008). Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-Mendelian inheritance in action. Proceedings of the National Academy of Sciences of the United States of America, 105(16), 5980-5) have reported an example of insecticide resistance in an arthropod pest (Tatranychus urticae) being controlled by coding-regions of the mtDNA and on its inheritance in a heteroplasmic mite strain. The present invention is believed to represent the first example of insecticide resistance in an arthropod pest, in particular in a crustacean pest, and more particularly in a copepod pest such as sea lice (L saimonis) being controlled by non-coding regions of the mtDNA and on its inheritance in a heteroplasmic lice strain. Also, this is the first report of resistance towards pyrethroids being controlled by mitochondrial genes in an arthropod.
in spider mite, resistance to the insecticide bifenazate is highly correlated with remarkable mutations in cytochrome b, a mitochondriaiiy encoded protein in the respiratory pathway (Van Leeuwen, T., Vanholme, B., Van Pottelberge, S., Van Nieuwenhuyse, P., Nauen, R., Tirry, L., & Denholm, I. (2008). Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-Mendelian inheritance in action. Proceedings of the National Academy of Sciences of the United States of America, 105(16), 5980-5). Four sites in the Q0 site that are absolutely conserved across fungi, protozoa, plants, and animals are mutated in resistant mite strains. The same conserved sites are also present in the L. saimonis rntDNA, but these sites are not mutated in any strains of the L. saimonis mtDNA, and the resistance traits are not linked to these sites as they are in the spider mite.
Partially resistant strains, consisting of heteroplasrnic individuals, transmit their resistant and susceptible hapiotypes to progeny in highly variable ratios consistent with a sampling bottleneck. Insecticide selection on heteroplasrnic individuals favors those carrying resistant hapiotypes at a frequency of 60% or more. This combination of factors enables very rapid evolution and accounts for mutations being fixed in most field-collected resistant strains (Van Leeuwen, T., Vanholme, B., Van Pottelberge, S., Van Nieuwenhuyse, P., Nauen, R., Tirry, L, & Denholm, I. (2008). Mitochondrial heteroplasmy and the evolution of insecticide resistance: non-Mendelian inheritance in action. Proceedings of the National Academy of Sciences of the United States of America, 105(16), 5980-5).
Of particular note, reciprocal crosses between susceptible and resistant se lice showed resistance to be inherited only maternally; offspring of resistant females were strongly resistant, but those of susceptible females remained fully susceptible. Such complete uniparental Inheritance is unprecedented for insecticide resistance and suggested mitochondrial control as one possible explanation. The fact that non-coding regions of the mtDNA controls resistance is highly surprising.
Mitochondria contain few genes as compared to ceil nuclei, but they exhibit some intriguing evolutionary characteristics. Their inheritance is essentially non-Mendelian in that recombination through meiosis is lacking and transmission to offspring is predominantly uniparental. In contrast to nuclear DNA, In which there are only two copies of each gene per cell, the mitochondrial DNA (mtDNA) copy number in somatic ceils is generally in the range of 103-—104 copies per ceil (Legros F , Malka F , Frachon P , Lombes A , Roja M (2004) Organization and dynamics of human mitochondrial DNA. J Cell Sci 117:2653-2662). Higher mutation rates in animal mtDNA together with limited DNA repair mechanisms render these genes susceptible to rapid evolution through random drift or natural selection. As a consequence, mtDNA is highly polymorphic and is widely used for studies in systematics and population genetics. However, most genetic variation within a species exists between individuals, and the occurrence of more than one mtDNA sequence variant in a single individual (heteroplasmy) is relatively rare (Jenuth JP , Peterson AC . Fu K . Shoubridge EA (1996) Random genetic drift in the female germ line explains the rapid segregation of mammalian mitochondrial DNA. Nat Genet 14:146-151.). No previous study has uncovered resistance traits connected to mutations in non-coding regions of the mitochondrion, but we strongly believe that this, as in the L. saimonis, is an important mechanism for the development of resistance in arthropods.
Another mechanism which has been suspected to contribute in the resistance towards pyrethroids is enhanced detoxification capability by the parasite. From other arthropods, increased activities of monooxydases and unspecific esterases have been identified as mechanisms in question. However, esterases seem to play an insignificant role in sea lice, as the parasite has a very low background activity of these enzymes. On the other hand, unspecific monooxidases seem to play a significant role, indicated by a correlation between enzyme activity and reduced sensitivity.
The mitochondrial genes COI, A6, Cytb and 16sRNA of Lepeophtheirus saimonis has shown to display high levels of polymorphisms (Tjensvoll et al., 2006, Diseases of Aquatic Organisms, vol, 68, pp. 251 - 259), resulting in a significant number of haplotypes. However, the prior art is silent as to whether the large number of polymorphic sites have had any effects on the characteristics of the sea lice.
Pyrethroids are one type of chemotherapeutics used for delousing of commercial salmon aquaculture sites infested with sea lice. In Norway, the synthetic deltamethrin (AlphaMax™) and cis-cypermethrin (BetaMax™) are the most widely used pyrethroids. In Scotland and Ireland, cypermethrin (ExisTM) has been commonly used. The pyrethroids affect the sodium channels thus inhibiting the transmittal of nerve impulses in the synapses. Failure of pyrethroid treatment has been reported in Norway (deltamethrin and cis-cypermethrin) since turn of the century (Sevatdal and Horsberg, 2000, Norsk Fiskeoppdrett, vol. 12, pp. 34-35), and reduced sensitivity towards pyrethroids was documented in 2003 (Sevatdal og Horsberg, 2003, Aquaqulture, vol. 218, pp. 21-31).
Efficient and sensitive 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 and speedy identification of pyrethroid resistant pests is crucial. Detection of resistance prior to treatment, and the use of such analyses after treatment to evaluate treatment efficacy constitutes an important determinant for an integrated pest management (IPM) system.
Summary of invention
The present invention is based on the surprising finding that resistance towards pyrethroid chemotherapeutics commonly used to combat arthropod pests, in particular the copepod pest sea lice, is linked to mutations in the mitochondrion genome. More particularly, the present invention is based on the identification of novel single-nucleotide polymorphisms (SNPs) in the mitochondrion genome of the sea lice (Lepeophtheirus salmonis) shown to be involved in the resistance towards pyrethroid-based chemotherapy. Specifically, the present inventors have identified two pyrethroid resistance-associated SNPs located in the mitochondrial Col gene (T8605C, A9035G) and three pyrethroid resistance-associated SNP in the mitochondrial Cyp b gene (C13957T, A14017G and C14065T).
It is further noted that the identification of the SNPs involved in pyrethroid resistance in the mitochondrial genome proved to be challenging due to unexpected characteristics such as numerous repeats etc. of the non coding parts of the mitochondrial genom (D-loop).
The present invention thus provides an in vitro method for determination of pyrethroid resistance in arthropod pests, in particular crustaceans, in particular copepods, comprising the steps of detecting one or more single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial genome of the organism to be analyzed.
According to one aspect, the pest is a member of a species having ZZ-ZO-type sex determination.
According to another aspect of the invention, the single nucleotide polymorphism (SNP) associated with pyrethroid resistance is present in the coding region of a gene coding for Cytochrom B.
According to another aspect of the invention, the single nucleotide polymorphism (SNP) associated with pyrethroid resistance is present in the coding region of a gene coding for Col.
According to another aspect of the invention, single nucleotide polymorphism (SNP) associated with pyrethroid resistance is present in a regulatory non-coding part of the mitochondria.
According to another aspect, the invention provides for isolated DNA sequences of mitochondrial DNA comprising a single nucleotide polymorphism (SNP) associated with pyrethroid resistance.
According to another aspect, the invention provides a method of identifying single nucleotide polymorphisms (SNP) associated with pyrethroid resistance in an arthropod pest, in particular a crustacean, in particular a copepod, comprising the steps of a) collecting a sample of the organism from its natural environment; b) isolating mitochondrial genomic material from the collected organism c) identifying the nucleotide at the polymorphic site of the isolated genomic material, d) comparing the nucleotide polymorphic site of the isolated mitochondrial genomic material with a standard mitochondrial nucleic acid sequence from a member of the species known to be susceptible to pyrethroids, wherein the collected organism is determined to be resistant to pyrethroids if the nucleotide at the polymorphic site differs from the nucleotide at the polymorphic site of the standard.
According to another aspect, the invention provides a method for determining whether a population of arthropod pests, in particular crustacean pests, in particular copepod pests, is resistant towards pyrethroids, comprising the steps of a) collecting a sample of the organism from its natural environment; b) isolating mitochondrial genomic material from the collected organism c) identifying the nucleotide at a polymorphic site in the mitochondrial DNA, the polymorphic site being the site of a single nucleotide polymorphism (SNP) known to be related to resistance towards pyrethroids, b) comparing the nucleotide polymorphic site of the isolated mitochondrial genomic material with a standard mitochondrial nucleic acid sequence from a member of the species known to be susceptible to pyrethroids, or in the alternative comparing the nucleotide polymorphic site of the isolated mitochondrial genomic material with a standard mitochondrial nucleic acid sequence from a member of the species known to be resistant to pyrethroids, wherein the collected organism is determined to be resistant to pyrethroids if the nucleotide at the polymorphic site differs from the nucleotide at the polymorphic site of the susceptible standard, or is the same as the resistant standard, respectively.
According to another aspect, the invention provides a method for identifying a single nucleotide polymorphism indicative of pyrethroid resistance in an arthropod pest, comprising the steps of: a) determining whether the species to which the organism belongs exhibits incidents of pyrethroid resistance, b) determining whether said pyrethroid resistance is inherited within the species from the female members to their progeny, c) in the event said pyrethroid resistance is inherited within the species from the female members to their progeny, then isolating mitochondrial DNA from a member of the species known to be resistant to pyrethroids and comparing said isolated DNA with the mitochondrial DNA of members of the species known to be susceptible to pyrethroids, d) identifying one or more single nucleotide polymorphisms in the mitochondrial DNA of the resistant member of the species, e) performing a control to determine whether said single nucleotide polymorphisms are indicative of pyrethroid resistance.
The present invention is in particular based upon the discovery of novel single nucleotide polymorphisms in the mitochondrial DNA of sea lice indicative of resistance to pyrethroids. These particular aspects of the invention, and supporting examples thereof, are describes as follows:
The present invention furthermore provides for novel isolated sequences comprising SNPs involved in the resistance towards pyrethroid-based chemotherapy, and their use in determination of pyrethroid resistance in crustaceans. In particular, said method is useful for detection of pyrethroid resistance in copepods, such as sea lice (Lepeophtheirus salmonis).
According to one example, the present inventors have identified single nucleotide polymorphisms (SNP) in the mitochondrial genome of a sea lice (Lepeophtheirus salmonis), wherein said sea lice is resistant to pyrethroids if at least one of the following nucleotides: i. C in position 8605; ii. G in position 9035; iii. T in position 13957; iv. G in position 14017; v. T in position 14065; or the complementary oligonucleotide thereof, is present in the mitochondrial genome sequence of the one or more sea lice to be analyzed, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
According to yet another aspect, a method is provided, comprising the steps of: a) collecting a sea lice individual from an infested population; b) isolating mitochondrial genomic material from the collected organism; c) determining the nucleotide polymorphic site at the positions 8605, 9035 and 14065 of the isolated mitochondrial DNA compared with of the mitochondrial nucleic acid sequence SEQ ID No. 1, wherein said sea lice is resistant to pyrethroids if at least one of the nucleotides, or a complementary oligonucleotide thereof, is present in position 8605, 9035, 13957, 14017, and 14065, as indicated above respectively.
According to one embodiment of the above method, said step c) is performed using a primer selected from the group consisting of SEQ ID No. 6-15.
A method according to yet another embodiment, said step c) is performed using at least one probe selected from the group consisting of SEQ ID No. 16-20.
According to yet another embodiment, said step c) comprises nucleic acid amplification, e.g. using polymerase chain reaction.
According to yet another embodiment, said step c) is performed by contacting mitochondrial DNA sequence of the sea lice to be analyzed with a detection reagent, and determining which nucleotide is present in position 8605, 9035, 13957, 14017 and 14065.
According to yet another embodiment, said detection reagent is an oligonucleotide probe.
According to yet another embodiment, said step c) is performed using SNP specific probe hybridization, SNP specific primer extension, SPN specific amplification, sequencing, 5’ nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis.
According to another embodiment of the present method of the invention, the pyrethroid is selected from the group consisting of deltamethrin, and cis-cypermethrin.
According to another embodiment, the present invention provides an isolated oligonucleotide sequence comprising small nucleotide polymorphism (SNP) associated with pyrethroid resistance in sea lice, wherein said isolated oligonucleotide sequence comprises nucleotides that distinguishes sea lice which are resistant to pyrethroids from non-resistance to pyrethroids, and wherein the SNP are present in the mitochondrial DNA.
According to one embodiment, the oligonucleotide of the present invention comprises a single-nucleotide polymorphism (SNP) associated with pyrethroid resistance in sea lice, wherein said isolated oligonucleotide sequence comprises nucleotides that distinguishes sea lice which are resistant to pyrethroids from non-resistant sea lice, and which is identical or has at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID No. 4and 5 or a fragment thereof, and complementary sequences of SEQ ID No. 4 and 5 and fragments thereof, provided that at least one SNP selected from the group of SNPs consisting of T8605C (U8605C in case of RNA), A9035G, C13957T (U13957T in case of RNA), A14017G, and C14065T (C14065U in case of RNA) is present, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
According to one embodiment, an oligonucleotide probe or oligonucleotide primer is provided comprising an oligonucleotide sequence being homologous to a fragment of an isolated oligonucleotide sequence according to SEQ ID 16, said probe or primer being specific for a mitochondrial DNA sequence of sea lice associated with pyrethroid resistance comprising at least one SNPs selected from the group consisting of T8605C and A9035G of SEQ ID No. 3, and C13957T, A14017G, and C14065T of SEQ ID 4, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
According to yet another embodiment, the probe or primer according to the present invention comprising at least 8 contiguous nucleotides of SEQ ID No. 1, including at least one of the nucleotide C, G, T, G, T or a complementary oligonucleotide thereof in the position corresponding to position 8605, 9035, 13957, 14017 and 14065, respectively, of SEQ ID 1.
According to one embodiment, a probe is provided, wherein the sequence of said probe is selected from the group consisting of SEQ ID No. 16, SEQ ID No. 17, SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, and sequences having at least 80% sequence identity therewith.
According to one embodiment, a primer is provided, wherein the sequence is selected from the group consisting of SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13 and SEQ ID No. 14, and SEQ ID No. 15, and sequences having at least 80% sequence identity therewith.
Furthermore, the present invention provides a kit for detection of pyrethroid resistance in crustaceans, such as copepods, e.g. in sea lice (Lepeophteirus salmonis), comprising a probe or primers according to the present invention.
Finally, the present invention provides an isolated oligonucleotide sequence comprising at least 8 contiguous nucleotides of the sequence selected from the group consisting of SEQ ID No. 4 and SEQ ID No. 5, and wherein said sequence comprises at least one of the nucleotide C, G, T, G, T or U or a complementary oligonucleotide thereof in the position corresponding to position 8605, 9035 and 14065, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
Figures
Figure 1 illustrates the hybridization experiment showing that pyrethroid resistance characteristics are transmitted from female sea lice to their progenies.
Figure 2 illustrates the experiment flow of the hybridization experiment. Two field-collected strains of lice were initially used to create two hybrid strains: /.sHybridG with Z-sGulen as its maternal strain, and /.sHybridV with /.sVikna as its maternal strain.
Figure 3 show percent active lice in deltamethrin bioassays 1 and 2. In bioassay 1 the concentrations 0.5 and 20 ppb were not included, and in bioassay 2 /.sGulen F7 was not tested for 20 ppb.
Figure 4 shows the organization of mitochondrial DNA of L. salmonis.
Figur 5 shows an alignment of mitochondria sequences from L. salmonis. The upper sequence is the reference sequence of the L. salmonis mtDNA deposited by Tjensvoll et al (2005) with NCBI accession number NC_007215 (hiip.//www.ncDi.niiri.nn':.gov/nuccoi'e/NC 007215.1 ), corresponding to SEQ ID No. 1. The start point in this sequence is in the D-loop (i.e. the non-coding region of the mtDNA). The sequence named LsGulen2 is isolated from a pyrethroid sensitive strain and is depicted in SEQ ID No. 2 and 3. The sequence named LsViknal is isolated from a strain resistant to pyrethroids, and is depicted in SEQ ID No. 4 and 5.
The present invention and its various embodiments will be described in more detail in the following.
Detailed description of the invention
The present invention provides an in vitro method for determination of pyrethroid resistance in arthropods, in particular crustaceans, including copepods, such as Lepeophtheirus salmonis and novel SNPs, based on the surprising findings that mutations linked with resistance against pyrethroid in sea lice was found in mitochondrial DNA.
Although pyrethroid resistance linked SNPs presented in the experimental data was identified in the sea lice species Lepeophtheirus salmonis, the skilled person will acknowledge, based on the teaching herein, that the present method and the present oligonucleotides may be used to determine pyrethroid resistance is crustaceans, in particular copepods, in particular copepods belonging to the family Caligidae. According to one embodiment, the present method and present oligonucleotides are useful for detection of pyrethroid resistance in copepod selected from the group consisting of Lepeophteirus salmonis, Caligus elongatus, and Caligus rogercresseyi.
Throughout the application, the term “sea lice” is to be understood to mean any copepod belonging to the family Caligidae. However, whenever the term “sea lice” is used in connection with the experimental data in the present application, sea lice refers to the specie Lepeophtheirus salmonis.
By hybridization of adult male sea lice shown to be sensitive to pyrethroids with preadult female sea lice shown to be resistant towards pyrethroids, the present inventors where able to show that the resistant female sea lice transmitted their pyrethroid resistant characteristics to their progenies (cf. figure 1, example 1). Resistant adult male sea lice did not transfer their pyrethroid resistant characteristics when crossed with sensitive females. The mechanism of action could therefore be linked to the mitochondrion of the sea louse. Based on further extensive sequence analysis of mitochondrial genomes of pyrethroid resistant sea lice, and comparison with the mitochondrial genomic sequence of a known reference strain (GenBank acc. No. NC_007215 (Tjensvoll et al., 2005, Gene, 353 (2), 218-230), SEQ ID No. 1) and mitochondrial sequences of a pyrethroid sensitive sea lice strain (SEQ ID No. 2 and 3), the present inventors have identified five single nucleotide polymorphism sites that are clearly linked to pyrethroid resistance (figure 5) in sea lice.
More particularly, sequences from 180 individual L. salmonis obtained from 6 different locations (see Tjensvoll et al 2006, supra) from Col and cyt b were compared to sequences from pyrethroid sensitive and resistant lice and the newly identified SNPs characteristic for resistant strains are only present in the resistant populations. Two of the SNPs uniquely associated with resistance to pyrethroids are found in the Col gene, more specifically in position 8605 and position 9035 (T8605C, A9035G). Three SNPs found to be uniquely associated with resistance to pyrethroids is found in the cyt b gene, more specifically in position 13957 (C13957T), 14017 (A14017G), and 14065 (C14065T).
The start point of the mitochondrial DNA sequence of SEQ ID No. 1 is in the D-loop (i.e. the non-coding region of the mtDNA). The numbering of the positions of the identified SNPs are based of the mitochondrial sequence of Tjensvoll et al, 2005, supra, deposited with the GenBank Acc. No. NC_007215 as depicted in SEQ ID No. 1. It is to be understood that whenever referring to the positions of the SNPs identified according to the present invention, the numbering is throughout the present description made according to the numbering of the reference strain sequence (GenBank Acc. No. NC_007215) if not otherwise stated.
Based on the identification of the five SNPs responsible for pyrethroid resistance in sea lice, a method for determination of pyrethroid resistance in crustaceans, such as copepods, in particular in sea lice, e.g. Lepeophtheirus salmonis PRLS have been provided. More particular, a method for detection of PRLS is provided comprising the steps of detecting single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial DNA of a sea lice to be analyzed, wherein said sea lice is resistant to pyrethroids if at least one of the following nucleotides: i. C in position 8605; ii. G in position 9035; iii. T in position 13957; iv. G in position 14017; v. T in position 14065; or the complementary oligonucleotide thereof, is present in the mitochondrial DNA sequence of the sea lice, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
In addition to the qualitative determination of the SNPs involved in pyrethroid resistance according to the present invention, the present invention also provides for a method for gradation of pest population being susceptible of developing resistance, i.e. taking into account the composition of haplotypes that are determined within a pest population (see also example 3 for practical details).
According to the present invention, “single-nucleotide polymorphisms (SNP)” is to be understood to refer to a nucleotide sequence variation occurring when a singe nucleotide, A, T (U), C or G in the genome, or other shared sequences (e.g. RNA) or fragments thereof, differs between members of a biological species, such as between variants of Lepeophtheirus salmonis. SNPs may fall within coding sequences of genes, or non-coding regions of genes, or in the regions between the genes in a genome. The five SNPs identified according to the present invention fall within the genes encoding the cytochrome oxidase subunit I (Col) and cytochrome B (Cytb), respectively.
Furthermore, as used herein, an “oligonucleotide sequence” or “nucleic acid sequence” is generally an oligonucleotide sequence or a nucleic acid sequence containing a SNP described herein, or one that hybridizes to such molecule such as a nucleic acid sequence with a complementary sequence.
The skilled person is well 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, when defining a SNP position, 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 SNP position. The oligonucleotide probes and oligonucleotide primers according to the present invention may be designed to hybridize to either strand, and SNP detection methods disclosed herein may thus also in general target either strand.
An “isolated nucleic acid” as used herein is generally one that contains at least one of the SNPs described herein or one that hybridizes to such molecule, e.g. 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.
Oligonucleotide probes and oligonucleotide primers
The present invention provides oligonucleotide probes and oligonucleotide primers that may be used for detection of the presence of the SNPs according to the present invention in mitochondrial DNA of a sea lice to be tested, and thus for determination of pyrethroid resistance. The detection of nucleic acids present in a biological sample 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 detection of the presence of at least one of the SNPs described herein if present in the sample 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 detect an SNP in a target sequence, e.g. single-stranded mitochondrial DNA or RNA, upon hybridization of the oligonucleotide probe to complementary sequence(s) of the said target sequence to be analyzed. The oligonucleotide probe according to the present invention may furthermore be labeled with a molecular marker in order to easily visualize that hybridization, and thus detection of the SNPs disclosed herein, 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.
A oligonucleotide probe according to the present invention is able to hybridize to another nucleic acid molecule, such as the single strand of mitochondrial DNA or RNA originating from a 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 invention typically comprises a nucleotide sequence which under stringent conditions hybridize to at least 8, 10, 12, 16, 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 mitochondrial 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 13 to 25 consecutive nucleotides. It is to be understood that the oligonucleotide probe according one embodiment comprise one of the SNPs described herein or the complement thereof. 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:10.10Q3/nar/gkM75. PMC 1456327. PMiP 16670427.) According to one embodiment, probes are provided which are selected from the group consisting of SEQ ID No. 16-20.
The present invention furthermore provides oligonucleotide primers useful for amplification of any given region of a nucleotide sequence, in particular a region containing one of the SNPs described herein. An oligonucleotide primer according to the present invention typically comprises a nucleotide sequence at least 8, 10, 12, 16, 20, 22, 25, 30, 40, 50 (or any other number in-between) or more consecutive nucleotides. According to one embodiment, the oligonucleotide primer according to the present invention comprises about 18-25 consecutive nucleotides, more preferably about 20 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). According to one embodiment of the present invention, “oligonucleotide primer pairs” is provided suitable for amplification of a region of mitochondrial genome material comprising the SNPs according to the present invention.
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 detect an SNP in a target sequence, e.g. single-stranded mitochondrial 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, dependent on the SNP detection method used.
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. It is to be understood that the oligonucleotide primer according to the present invention according to one embodiment comprises the SNP described herein or the complement thereof. According to one embodiment, the probes useful in order to determine pyrethroid resistant sea lice is selected from the group consisting of SEQ ID No. 16, SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, and SEQ ID No.20.
According to one embodiment of the present invention, primer pairs are provided selected from the group consisting of SEQ ID No. 6 - SEQ ID No. 15 (see also table 4 below).
Oligonucleotide probes and oligonucleotide primers according to the present invention may be synthesized according to methods well known to the skilled person.
The present invention furthermore relates to isolated nucleic acid sequences and variants or fragments thereof having at least 70% identity with the nucleic acid sequences depicted in SEQ ID NO. 4, or SEQ ID No. 5, or fragments thereof. 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, 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 homology 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).
SNP identification methods
Upon the identification of the SNPs associated with pyrethroid resistance in a pest according to the present invention, the skilled person will acknowledge that various methods commonly used in order to detect polymorphisms within a population of pests may be used. Both methods based on genome sequencing, hybridization and enzyme based methods are applicable for determining whether a pest is pyrethroid resistant in accordance with the present inventions.
Various enzyme based methods are available for the skilled person, of which a number of polymerase chain reaction (PCR) based methods are available.
For example, Perkin Elmer Life Sciences provides SNP detection kit that may be used in order to determine whether a sea louse is pyrethroid resistant (AcycloPrime™-FP SNP Detection). In said method, a thermostable polymerase is used which extends an oligonucleotide primer according to the present invention by one base, then ending the oligonucleotide primer one nucleotide immediately upstream of the relevant SNP position by the incorporation of fluorescent dye-labeled terminators. The identity of the base added is then determined by the increase fluorescence polarization of its linked dye
Oligonucleotide primers according to the present invention useful in such a method would thus be constructed in order to facilitate the extension of the primer by one base in the position selected from the group 8605, 9035, 13957, 14017, or 14064, relative to SEQ ID No. 1 in the case where the pest is sea lice.
Another enzyme based method that may be used is restriction fragment length polymorphism (RLFP), utilizing that various, highly specific endonuclease upon digestion of the target sample, i.e. mitochondrial genome, results in different fragments that may be separated by gel electrophoresis.
Yet another enzyme based method that may be utilized in accordance with the present invention is the flap endocuclease (FEN) method. In said method, a structure-specific endonuclease is used to cleave a three-dimensional complex formed by hybridization with the target DNA, e.g. mitochondrial genomic material from sea lice, and where annealing with a target sequence comprising the SNP of interest triggers cleavage by the endonuclease (Oliver, 2005, Mutat. Res., vol. 573 (1-2), pp. 103-110).
Yet another method applicable in respect of the present invention is based on the use of TaqMan® Assays (Invitrogen). In said assay the oligonucleotide primers used in order to detect an SNP is labeled in both the 5’- and the 3’ end, i.e. with a fluorophore at the 5’end of the oligonucleotide primer, and a quencher at the 3’-end of the oligonucleotide primer. Upon annealing of the oligonucleotide primer with a target sequence, the Taq polymerase will extend the oligonucleotide primer and form a nascent strand, followed by degradation of the oligonucleotide primer being annealed to the target, said degradation eventually resulting in the release of the fluorophore and provide a cleavage close to the quencher. The fluorescence signal produced is proportional to the fluorophore released. Various fluorophore labels may be used, such as e.g. 6-carboxyfluorescein, tetrafluorofluorescein. As quenchers, tetramethylrhodamine or dihydrocyclopyrroloindol may be used.
Several hybridization methods for detection of SNPs are available to the skilled person, and which may be utilized in accordance with the method of the present invention. For example, the SNPs according to the present invention may be detected utilizing molecular beacon technology. According to this aspect of the present invention, oligonucleotide primers may be synthesized comprising complementary regions at each end allowing the formation of a hairpin loop, and wherein a fluorophore is attached at one end of the oligonucleotide primer, and a quenching agent is attached to the other end, and wherein fluorescence signal is produced upon binding to a DNA target of interest, i.e. mitochondrial genomic material isolated from the sea louse to be analyzed.
Yet another method applicable in respect of the present invention is based on DNA or RNA sequencing, which is the process of determining the precise order of nucleotides within a molecule. It includes any method or technology that is used to determine the order of the four bases (adenine, guanine, cytosine, and thymine) in a strand of DNA. The skilled person is well known with the various commonly known DNA and RNA sequencing methods that may be used according to the present invention, such as e.g. shotgun sequencing or bridge PCR sequencing.
Isolation of sea lice genomic material
The method according to the present invention may according to one embodiment involve the isolation of a biological sample from a sea lice and testing for the presence of a SNP associated with PRLS in the mitochondrial genome. The skilled person will acknowledge that the SNPs identified according to the present invention may be detected by analyzing mitochondrial DNA as well as mitochondrial RNA, dependent upon the detection method used.
In order to determine whether a sea louse is pyrethroid resistant in accordance with the present invention, mitochondrial genomic material may be isolated. 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 mitochondrial genomic material. Furthermore, the mitochondrial 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). 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 protocol of the manufacturer (http://ivcofsQ1.iycominq.edu/~gcat-seek/protocols/DNeasy Blood & Tissue Handbook.pdf).
SNP detection Kits
Based on the teaching herein, the skilled person will acknowledge that, based on the identified SNPs and associated sequence information disclosed herein, detection reagents can be developed and used to determine any SNP described herein individually or in combination, and that such detection reagents can be readily incorporated into kits used for SNP detection known in the art. The term “kit” as used herein in the context of SNP detection reagents are intended to cover e.g. combinations of multiple SNP detection reagents, or one or more SNP detection reagents, such as oligonucleotide probe(s) and oligonucleotide primer(s) or primer sets, arrays/microarrays of nucleic acid molecules, and beads that contain one ore more oligonucleotide probe(s), oligonucleotide primer(s) or other detection reagents useful in the method of the present invention. It is furthermore to be understood that the SNP 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 SNP detection reagents are attached., etc. necessary to carry the 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 detect the SNP disclosed herein 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. mitochondrial genetic material) enabling detection of each target SNP position if present in the material analyzed. Multiple pairs of probes may be included in the kit to simultaneously analyze for the presence of the SNP disclosed herein 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, the oligonucleotide probes are suitable for the detection of the SNP T8605C. According to another embodiment, the oligonucleotide probes are suitable for the detection of the SNP A9035G.
According to yet another embodiment, the oligonucleotide probes is suitable for the detection of the SNP C13957T. According to yet another embodiment, the oligonucleotide probes is suitable for the detection of the SNP A14017G. According to yet another embodiment, the oligonucleotide probes is suitable for the detection of the SNP C14065T. According to yet another embodiment, the kit according to the present invention comprises oligonucleotide probes suitable for detection of all the SNPs described herein.
According to one embodiment, a kit according to the present invention comprises oligonucleotide primer(s) and optionally further SNP detection reagents useful in SNP detection methods utilizing oligonucleotide primers or primer pair(s).
According to one embodiment, the kit according to the present invention comprises a forward primer and a reverse primer for amplifying a region containing a SNP selected from SNP selected from the group of SNPs consisting of T8605C, (U8605C in case of RNA), A9035G, C13957T (or U13957T in case of RNA), A14017G, or C14065T (C14065U in case of RNA). Said kit may furthermore optionally comprise further SNP detection reagents (enzymes and nucleotide triphosphates) necessary for conducting PCR or real time PCR. According to one embodiment, the primer pairs are suitable for the detection of the SNP T8605C. According to another embodiment, the primer pairs are suitable for the detection of the SNP A9035G. According to yet another embodiment, the primer pairs is suitable for the detection of the SNP C13957T. According to yet another embodiment, the primer pairs is suitable for the detection of the SNP A14017G. According to yet another embodiment, the primer pairs is suitable for the detection of the SNP C14065T. According to yet another embodiment, the kit according to the present invention comprises primer pairs suitable for detection of all the SNPs described herein.
Table 1 Sequences related to the invention
According to one embodiment of the present invention, a method is provided for detection of the pyrethroids deltamethrin (CAS No. 52918-63-5) and cis-cypermethrin (CAS No. 52315-07-8). It is well known that resistance against one type of pyrethroid in general provides the possibility of resistance also against all type of pyrethroids; see e.g. Sevatdal S, Fallang A, Ingebrigtsen K, Horsberg TE. 2005. Monooxygenase mediated pyrethroid detoxification in sea lice (Lepeophtheirus salmonis). Pest Manag Sci. 2005 Aug;61 (8):772-8, Du Y, Nomura Y, Satar G, Hu Z, Nauen R, He SY, Zhorov BS, Dong K. 2013. Molecular evidence for dual pyrethroid-receptor sites on a mosquito sodium channel. Proc Natl Acad Sci USA. 2013 Jul 2. [Epub ahead of print], and Zhu F, Gujar H, Gordon JR, Haynes KF, Potter MF, Palli SR. 2013. Bed bugs evolved unique adaptive strategy to resist pyrethroid insecticides. Sci Rep. 2013;3:1456. doi: 10.1038/srep01456.
The skilled person will thus acknowledge, based on the teaching of the present invention, that the method according to the invention may be used to determine resistance towards various types of pyrethroids in crustaceans, such as e.g. allethrin, bifenthrin, cyfluthrin, cyphenothrin, esfenvalerat, etofenoprox, fenpropathrin, fenvalerate, flumethrin, flucythrinate, imiprothrin, lamda-cyhalothrin, metofluthrin, permethrin, prallethrin, resmethrin, silafluofen, sumithrin, fluvalinate, teflutrhin, and tetramethrin.
Examples
Example 1: Hybridization of sensitive and pyrethroid resistant sea lice
Crossing experiment
In order to investigate the inheritance and mechanism of pyrethroid resistance in L. salmonis, two strains (sensitive and resistant) and their reciprocal hybrids were used for a two-generation breeding experiment complimented with bioassays to measure tolerance (Fig. 2). The pyrethroid sensitive strain (hereon also referred to as Z-sGulen) was collected in June 2006 from newly slaughtered rainbow trout from Gulen in western Norway. The pyrethroid resistant strain (hereon also referred to as Z-sVikna) was collected in February 2009 from a salmon farm in Vikna, North Trøndelag, Norway. The resistant strain was collected on the basis of suspected reduced sensitivity to pyrethroids due to the fact that there had been reported a treatment failure on the farm from which it was collected. This was subsequently confirmed in bioassays with /.sGulen as sensitive control.
Prior to the initiation of the present study, /.sGulen and /.sVikna had been cultivated in laboratory for 13 and 4 generations respectively under standard rearing conditions at the Institute of Marine Research (IMR), Norway (Hamre et al., 2009, Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains. Parasitology International, 58, 451 -460).
In order to hybridise the lice strains for the inheritance experiment, the parental strains were first synchronised and multiplied up in numbers by simultaneously infecting 20 Atlantic salmon with copepods. In total, 1000 copepods per strain were added. At 32 days post infection (dpi), all salmon were anaesthetised and approximately half of the lice were carefully removed using tweezers. At this stage, lice were predominantly preadult 2 females and adult males, with a few percent adult females and preadult 2 males. The lice removed from the salmon were sexed, and then used to create the reciprocal hybrid strains. This was achieved by combining 4-5 females from the resistant strain and 4-5 males from the sensitive strain (/.sHybridV), and 4-5 females from the sensitive strain and 4-5 males from the resistant strain (/.sHybridG). Only preadult 2 females were used. These combinations were each placed into 2 replicate tanks in order to make two hybrid strains (4 groups and 8 tanks at this stage). At 67-68 dpi, all lice were removed from the four strains. Egg strings were removed from adult females and incubated in containers containing a single strain each. The number of adult lice, and harvested egg strings varied, but all were incubated (Table 2).
Table 2. Number of egg strings collected from each strain. Egg strings from replicate tanks were pooled.
Most egg strings were part of a pair.
Strain Generation Egg strings (n)
LsGulen F14 133
LsVikna F5 106
LsHybridG P 96
LsHybridV P 60
The subsequent generation (F2, Fig. 2) were both produced using the general procedure described above, although the hybrid strains were only crossed back to themselves to create multiple generation-hybrids. Collection of egg strings, which marked the establishment of a new generation, was done at 55 and 56 dpi respectively. Salmon that hosted the previous generation were re-used for reinfection with the next generation, but hosted only the same experimental strain.
Bioassay test
Each strain, including both types of hybrids obtained in example 1 were quantified in their tolerance of deltamethrin at sampling points 1 and 2 representing the F1 and F2 generations for the hybrids (Fig. 1 and 2). Bioassays were performed according to standard guidelines (SEARCH Consortium, 2006, Sea lice resistance to chemotherapeutants: A handbook in resistance management. 2 ed.), with minor modifications. Only adult male lice were used for the bioassay, which after sampling (at 55 or 56 dpi) had been incubated overnight in running seawater (ca.
10 °C). The 30 min exposure to the prepared deltamethrin concentration was also carried out at ca. 10 °C, and higher concentrations than those described in the given protocol were included (Table S3). After exposure to deltamethrin, the lice were kept in running seawater (24 h at ca. 10 °C) until evaluation of the bioassays. At evaluation, lice were characterised as active, or inactive (moribund and dead lice pooled). ECso values were calculated using probit analysis (PoloPlus 2.0;
LeOra Software, CA, USA). The results of two deltamethrin bioassay sets are shown in table 3a and 3b below.
Table 3a. Deltamethrin bioassays performed on sampling set 1
Lice: LsHybridG
Generation: F1
Fish tank: 01
Dose (ppb) Active (n) Inactive (n) % Inactive 5 17 5 (Γ 0.1 16 0 0 0.3 32 9 21 1 0 35 100 3 0 28 100 6 0 18 100 12 0 27 100
Lice: LsHybridV
Generation: F1
Fish tank: 02
Dose (ppb) Active (n) Inactive (n) % Inactive 0 16 0 (Γ 0.1 17 0 0 0.3 44 0 0 1 56 3 5 3 33 5 13 6 17 15 12 15 7 31
Lice: LsGulen
Generation: F15
Fish tank: 04
Dose (ppb) Active (n) Inactive (n) % Inactive 0 9 0 (Γ 0.1 40 0 0 0.3 29 27 48 1 0 37 100 3 0 20 100 6 0 14 100 12 0 9 100
Lice: LsVikna
Generation: F6
Fish tank: 05
Dose (ppb) Active (n) Inactive (n) % Inactive 5 12 5 (Γ 0.1 16 0 0 0.3 9 0 0 1 21 3 12 3 29 11 27 6 18 10 35 12 7 5 41
Lice: LsHybridV
Generation: F1
Fish tank: 06
Dose (ppb) Active (n) Inactive (n) % Inactive 0 26 0 (Γ 0.1 17 0 0 0.3 45 0 0 1 39 3 7 3 36 1 2 6 19 2 9 12 19 9 32
Lice: LsHybridG
Generation: F1
Fish tank: 07
Dose (ppb) Active (n) Inactive (n) % Inactive 5 14 5 Γ 0.1 18 4 18 0.3 21 10 32 1 0 38 100 3 0 46 100 6 0 21 100 12 0 20 100
Lice: LsVikna
Generation: F6
Fish tank: 09
Dose (ppb) Active (n) Inactive (n) % Inactive 0 16 0 (Γ 0.1 21 1 4 0.3 15 0 0 1 26 3 10 3 17 4 19 6 23 7 23 12 5 2 28
Lice: LsGulen
Generation: F15
Fish tank: 10
Dose (ppb) Active (n) Inactive (n) % Inactive 0 13 0 (Γ 0.1 26 0 0 0.3 10 10 50 1 0 41 100 3 0 9 100 6 0 17 100 12 0 22 100
Table 3b. Deltamethrin bioassays performed on sampling set 2 .
Lice: LsGulen
Generation: F16
Fish tank: 03
Dose (ppb) Active (n) Inactive (n) % Inactive 0 19 0 (Γ 0.1 16 1 5 0.3 28 26 48 0.5 7 43 86 1 0 18 100 3 0 23 100 6 0 7 100
Lice: LsGulen
Generation: F16
Fish tank: 04
Dose (ppb) Active (n) Inactive (n) % Inactive 5 24 5 Γ 0.1 23 2 8 0.3 16 20 55 0.5 1 20 95 1 0 16 100 3 0 15 100 6 0 6 100 12 0 3 100
Lice: LsHybridV
Generation: F2
Fish tank: 06
Dose (ppb) Active (n) Inactive (n) % Inactive 0 19 0 (Γ 0.1 13 0 0 0.3 14 0 0 0.5 15 0 0 1 32 0 0 3 25 5 16 6 22 1 4 12 11 3 21 20 6 4 40
Lice: LsHybridG
Generation: F2
Fish tank: 07
Dose (ppb) Active (n) Inactive (n) % Inactive 0 22 0 (Γ 0.1 17 0 0 0.3 14 14 50 0.5 1 6 85 1 1 10 90 3 0 18 100 6 0 32 100 12 0 27 100 20 0 32 100
Lice: LsVikna
Generation: F7
Fish tank: 08
Dose (ppb) Active (n) Inactive (n) % Inactive 0 35 0 (Γ 0.1 9 0 0 0.3 27 0 0 0.5 6 0 0 1 12 0 0 3 14 16 6 22 5 18 12 6 0 0 20 17 11 39
Lice: LsHybridV
Generation: F2
Fish tank: 09
Dose (ppb) Active (n) Inactive (n) % Inactive 0 16 0 (Γ 0.1 12 2 14 0.3 11 0 0 0.5 28 1 3 1 17 15 3 17 0 0 6 25 6 19 12 11 18 20 14 17 54
Lice:
LsHybridG
Generation: F2
Fish tank: 10
Dose (ppb) Active (n) Inactive (n) % Inactive 0 10 0 (Γ 0.1 19 1 5 0.3 25 19 43 0.5 6 16 72 1 0 10 100 3 0 23 100 6 0 19 100 12 0 25 100 20 0 10 100
The four experimental strains were successfully cultured through the two generations and exposed to two separate bioassays. At the control and lowest concentration (0.1 ppb), no differences in lice activity was observed between the four strains. This was the case for both sampling 1 and 2. However, at concentrations between 0.3 and 1 ppb, clear differences were observed between the strains (Fig. 2). Above 1 ppb, only lice from the resistant strain and its maternal hybrid strain survived the bioassay, while the sensitive strain and its associated maternal hybrid strain displayed 100% mortality. No differences between the hybrid strain and its maternal founder strain were observed for either strain, demonstrating that the resistance followed a maternal pattern of inheritance.
Exemple 2: Identification of SNPs associated with pyrethroid resistance
The presented outcome of crossing resistant and sensitive lice (example 1) showed that the mitochondria genome encodes the resistance. The L. salmonis mtDNA was characterized by Tjensvoll et al (2005) and encodes 13 proteins, two rRNAs and 22 tRNA genes.
The first approach to identify genetic differences between the LsSensitive (LsGulen) and Ls Resistent (LsVikna) was by sequencing a 1000 bp fragment from cytB from 6 L. salmonis specimens (3 sensitive and 3 resistant). The sequencing revealed 3 unique SNPs in the cytB from the LsResistent strain. We sequenced the cytB from 10 more individual from the LsResistent strain, which revealed that all except 1 individual had the same unique SNP (C14065T) in the cytB. This single atypical LsVikna individual was sampled from the control group during the bioassay (i.e. LsVikna incubated without deltamethrin). Tjensvoll et al (2006) sequenced cytB, A6. col and 16S from 180 L. salmonis specimens from six locations in the North Atlantic that were collected in 2000 or 2002. We aligned our cytB (from LsSensitive and LsResistent) sequences to these 180 cytB sequences from Tjensvoll et al (2006). None of the 180 cytB sequences from historic samples had the C14065T SNP. To further validate if the C14065T SNP is unique in resistant lice we obtained samples from 9 different lice strains, including several field strains with reduced sensitivity towards deltamethrin or cis-cypermethrin.
The presented outcome of crossing resistant and sensitive lice showed that the mitochondria genome encodes the resistance. Since L. salmonis is ZZ (male)/Z0 (female) the maternally encoded resistance properties must be in the mtDNA. The L. salmonis mtDNA was characterized by Tjensvoll et al (2005) and encodes 13 proteins, two rRNAs and 22 tRNA genes.
Genetic variation within the mtDNA
Tjensvoll et al (2006), supra, identified 158 haplotypes for cytb and 164 haplotypes for COI. In 19 L. salmonis individuals with know deltamethrin resistance only 2 haplotypes for COI were identified. The cytB was sequenced from 29 individuals with known deltamethrin resistance and only 1 haplotype was identified. Among 19 sensitive specimens 9 different haplotypes for cytB was present (Table 4).
Table 4. Number of haplotypes identified for each of CytB and Col in sensitive sea lice and resistant sea lice.
Example 3: Testing for PRLS
Based on the identification of SNPs responsible for pyrethroid resistance in L salmonis, we developed several sensitive Real-Time PCR (TaqMan) 5'-nuclease assays for single nucleotide polymorphism (SNP) detection (Real-Time PCR SNP-assay) (Table 1), and successfully applied these to differentiate between pyrethroid resistant and sensitive sea lice. Fluorogenic PCR probes, chemically modified with a minor groove binding agent to increase duplex stability, were used in single and multiplex probe closed tube formats. The probes were tested in a commercially available thermocycling fluorimeter (Applied Biosystems 7500 Real-Time PCR System) at PatoGen Analyse AS laboratory in Ålesund. All assays were used qualitatively to determine the relative amount of the SNP- in individual samples, and samples from different life stages of sea lice were use. Comparison of results obtained using sea lice samples with different Real-Time PCR SNP-assays detecting different SNPs specific for pyrethroid resistant sea lice showed no discrepancies from results obtained by genome sequencing. The reported SNPs and the relevant Real-Time PCR SNP-assays are ideal for the differentiating between pyrethroid resistant and sensitive sea lice in the aquaculture industry.
Sampling of sea lice
Sea lice samples were collected from sea lice with known sensitivity-status to pyrethroids, cultivated in The Sea Lice Research Centre laboratories at the University of Bergen. The sea lice had been tested with respect to sensitivity to pyrethroids by bioassays as previously described in example 1 above. Also, the genome sequences for the relevant genes were known from previous genomic sequencing performed as described in example 1 above.
In addition, sea lice were sampled from fish farms on the west coast of Norway.
Sea lice were collected using forceps, and approximately 10-50 lice per site were conserved in 70% ethanol and kept at 4°C. Samples were sent refrigerated to PatoGen by express mail carrier.
Nucleic acid purification
In PatoGens laboratory, RNA and/or DNA were extracted from samples by methods well known to the skilled person. In short, tissue samples were transferred to Micro Collection Tubes and lysed and homogenized using QIAzol Lysis Reagent, steel beads and vigorous shaking using a TissueLyser system, followed by nucleic acid extraction using an RNAeasy kit (Qiagen) or DNAeasy kit (Qiagen), all according to the manufacturer’s instructions, and by methods well known to the skilled person. Chloroform were added to the samples and shaken vigorously. After resting and centrifuging, the relevant liquid phase were collected for further extraction of either RNA or DNA by vacuum technology using a Qiagen robot system, all according to the manufacturer’s instructions, and by methods well known to the skilled person. Finally, nucleic acids were eluted in 25 ml of elution buffer and used for PCR by methods well known to the skilled person.
SNP-detection by Real-Time PCR
The primers and probes listed in table 5 are TaqMan® MGB Probe SNP Genotyping Assays using TaqMan® 5' nuclease assay chemistry for amplifying and detecting specific SNP alleles in purified genomic DNA or RNA. In this study, primers and probes SNP-14017, SNP-14065, & SNP-9035 listed in table 1 were ordered from Life Technologies Corporation. The primers and probes listed in table 1 for SNP-8605 and SNP-13957 serves as examples of assays for these SNPs, but were not included in this study. One-step amplification (45 cycles) was performed on an Applied Biosystems 7500 Real-Time PCR System performed at PatoGen Analyse AS laboratory in Ålesund, all according to the manufacturer’s instructions, and by methods well known to the skilled person. All assays were tested on both RNA and DNA, and used qualitatively to determine the relative amount of the relevant SNPs in individual samples.
Table 5: Primers and probes
Table 5: Primers and probes. Listing of primers and probes for each SNP identified. Probes are both listed with IUPAC nucleotide codes for SNPs, and with the alternative nucleotides for each position as indicated. IUPAC codes used are in accordance with Nomenclature for Incompletely Specified Bases in Nucleic Acid Sequences, of which the following are relevant here: Y = C or T, R = A or G (Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1984) http: // www. c h e m. q m u i. a c. u k/ i u b m b/ rn i s c/ n a s e q. h ί m I).
Results
The results from Real-Time PCR-assays are interpreted by looking at the deviation in Ct-values between the probes detecting the two variants of the SNP. For the Real-Time PCR-assay towards SNP-14017 performed using RNA or DNA, sensitive lice has a deviation lower than -2, and resistant sea lice has a deviation higher than -2. For the Real-Time PCR-assays towards SNP-14065 & SNP-9035 performed using RNA or DNA, sensitive lice has a deviation lower than zero, and resistant sea lice has a deviation higher than zero. In addition, there is a quantitative aspect where the highly resistant strains have a higher deviation value than moderately resistant sea lice. Most likely, genetically resistant strains that have not been exposed to pyrethroids for some time has a lower deviation value than genetically resistant strains that been repeatedly and newly exposed to pyrethroids.
The results from the Real-Time PCR SNP-analyses using assays directed towards SNP-14065 & SNP-9035 (according to Table 5) using RNA or DNA from sea lice samples with known sensitivity to pyrethroids showed good correlation with results obtained by genome sequencing, and correlated with the known resistance status in sea lice populations (Table 6 & 7). The Real-Time PCR SNP-assay directed towards SNP-14017 (according to Table 5) gave the same results using DNA or RNA, and correlated with the results from SNP-14065 & SNP-9035 for all but one sea lice sample (Table 6).
Also, analyses performed on a higher number of sampled field strains showed good correlation with the known resistance status and as shown in Table 8. Here, analyses were performed using SNP-14065, and RNA as template.
All tested Real-Time PCR-SNP-assays show very promising results, and we believe that these SNPs can be used separately and/or in combination with other SNPs to serve as a tool in the practical differentiation between pyrethroid- sensitive and resistant sea lice.
Thus the reported SNPs and the relevant Real-Time PCR SNP-assays represent an ideal tool for differentiating between pyrethroid resistant and sensitive sea lice in the aquaculture industry. Also, the prevalence of sensitive versus resistant sea lice in a population, and the deviation value for individual lice or average deviation values for populations, can be used to predict the best possible outcome of a treatment using pyrethroids in the population. Also, the technique can become an important tool for optimizing sea lice treatments using pyrethroids, and to monitor the resistant status of populations of sea lice before treatment.
Table 6. Conclusions regarding resistance status showed good correlations between different assays directed towards different SNPs, and regardless of whether RNA or DNA was used as a template. The results from the Real-Time PCR SNP-analyses using assays directed towards SNP-14065 & SNP-9035 (according to Table 5) using RNA or DNA from sea lice samples with known sensitivity to pyrethroids showed full correlation. The Real-Time PCR SNP-assay directed towards SNP-14017 (according to Table 5) gave correlating results using DNA or RNA, and correlated with the results from SNP-14065 & SNP-9035 for all but one sea lice sample (sample 17).
Table 7. SNP-analyses correlate well with known resistance status. SNP-analyses using SNP14065 correlates with known resistance status in sea lice (/.. salmonis) from the sea lice lab at the University of Bergen (based on bioassays and field observations), and with genotyping performed by the University of Bergen. Bioassays, genotyping and SNP-analyses are all based on different lice from the same populations, and thus the results cannot be correlated on individual sea lice level. Also, since the number of samples is very low, the slight differences with respect to prevalence of resistant genotype observed between sequencing and Real-Time PCR SNP-14065-analysis must be attributed to the low number of samples tested, and this is especially the case for lice population D. Still, the conclusions with respect to population resistance level correlates well. Also, populations A, B and C represent surviving sea lice from the same original population, but A, B and C are surviving lice that has been exposed to different concentrations of pyrethroid in a bioassay. The results show that pyrethroid treatment selects for more resistant sea lice, and this is reflected in both the prevalence of genetically resistant lice, and in the total average deviation in Ct-values between the probes detecting the two variants of the SNP.
Table 8. SNP-analyses correlate well with known resistance status in field strains. Field strains of sea lice (/.. salmonis) were characterized with respect to sensitivity to pyrethroids at the University of Bergen sea lice lab, and with the exception of strain P, all strains have been cultivated for one or more generations in the lab (up to 34 generations) before sampling. The conclusions from Real-Time PCR analyses towards SNP-14065 correlates very well with the known resistance characteristics from bioassays and field observations.
FUTURE EXAMPLES
In addition to the specific examples above related to L. salmonis, and in order to confirm the applicability of the observations for L. salmonis to other arthropods, in particular crustaceans, in particular copepods, the inventors plan to execute the following experimental protocol:
Crossing experiment Caligus elongatus
In order to investigate the inheritance and mechanism of pyrethroid resistance in Caligus elongatus, two strains (sensitive and resistant) and their reciprocal hybrids will be used for a two-generation breeding experiment complimented with bioassays to measure tolerance. A pyrethroid sensitive strain and a pyrethroid resistant strain will be selected on the basis of results from bioassays.
In order to hybridise the lice strains for the inheritance experiment, the parental strains will first be synchronised and multiplied up in numbers by simultaneously infecting 20 Atlantic salmon with copepodids. At the stage when lice are predominantly preadult 2 females and adult males, the lice will removed from the salmon, sexed, and then used to create the reciprocal hybrid strains. This will be achieved by combining 4-5 females from the resistant strain and 4-5 males from the sensitive strain, and 4-5 females from the sensitive strain and 4-5 males from the resistant strain. Only preadult 2 females will be used. These combinations will each be placed into 2 replicate tanks in order to make two hybrid strains (4 groups and 8 tanks at this stage). When egg strings develop in these hybrid strains, all lice will be removed from the four strains. Egg strings will be removed from adult females and incubated in containers containing a single strain each.
The subsequent generation (F2) will be produced using the general procedure described above, although the hybrid strains will only be crossed back to themselves to create multiple generation-hybrids. Collection of egg strings will mark the establishment of a new generation. Salmon that hosted the previous generation will be re-used for reinfection with the next generation, but will only host the same experimental strain.
Bioassay test
Each strain, including both types of hybrids obtained in this example will be quantified in their tolerance of deltamethrin at sampling points 1 and 2 representing the F1 and F2 generations for the hybrids. Bioassays will be performed according to standard guidelines (SEARCH Consortium, 2006, Sea lice resistance to chemotherapeutants: A handbook in resistance management. 2 ed.), with minor modifications. Only adult male lice will be used for the bioassay, which after sampling will be incubated overnight in running seawater (ca. 10 °C). The 30 min exposure to the prepared deltamethrin concentration will also be carried out at ca. 10 °C, and higher concentrations than those described in the given protocol will be included. After exposure to deltamethrin, the lice will be kept in running seawater (24 h at ca. 10 °C) until evaluation of the bioassays. At evaluation, lice will be characterised as active, or inactive (moribund and dead lice pooled). ECso values will be calculated using probit analysis (PoloPlus 2.0; LeOra Software, CA, USA).
The expected results from this experiment is that only lice from the resistant strain and its maternal hybrid strain will show resistance towards the pyrethroid treatment, and thus survive the bioassay. The sensitive strain and its associated maternal hybrid strain will displayed close to 100% mortality. No differences between the hybrid strain and its maternal founder strain will be observed for either strain, demonstrating that the resistance follow a maternal pattern of inheritance.
The presented outcome of crossing resistant and sensitive lice will show that the mitochondria genome encodes the resistance in this species. We will then amplify, sequence, and compare complete mitochondrial genomes of susceptible and resistant strains of Caligus elongatus strains to identify the specific SNPs associated to this resistance, but the above described experiment will provide enough information to conclude that the mitochondria is involved in the regulation of resistance towards pyrethroids in Caligus elongatus as described for the L. salmonis.
Crossing experiment Caligus rogercresseyi
In order to investigate the inheritance and mechanism of pyrethroid resistance in Caligus rogercresseyi, two strains (sensitive and resistant) and their reciprocal hybrids will be used for a two-generation breeding experiment complimented with bioassays to measure tolerance. A pyrethroid sensitive strain and a pyrethroid resistant strain will be selected on the basis of results from bioassays.
In order to hybridise the lice strains for the inheritance experiment, the parental strains will first be synchronised and multiplied up in numbers by simultaneously infecting 20 Atlantic salmon with copepodids. At the stage when lice are predominantly preadult 2 females and adult males, the lice will removed from the salmon, sexed, and then used to create the reciprocal hybrid strains. This will be achieved by combining 4-5 females from the resistant strain and 4-5 males from the sensitive strain, and 4-5 females from the sensitive strain and 4-5 males from the resistant strain. Only preadult 2 females will be used. These combinations will each be placed into 2 replicate tanks in order to make two hybrid strains (4 groups and 8 tanks at this stage). When egg strings develop in these hybrid strains, all lice will be removed from the four strains. Egg strings will be removed from adult females and incubated in containers containing a single strain each.
The subsequent generation (F2) will be produced using the general procedure described above, although the hybrid strains will only be crossed back to themselves to create multiple generation-hybrids. Collection of egg strings will mark the establishment of a new generation. Salmon that hosted the previous generation will be re-used for reinfection with the next generation, but will only host the same experimental strain.
Bioassay test
Each strain, including both types of hybrids obtained in this example will be quantified in their tolerance of deltamethrin at sampling points 1 and 2 representing the F1 and F2 generations for the hybrids. Bioassays will be performed according to standard guidelines (SEARCH Consortium, 2006, Sea lice resistance to chemotherapeutants: A handbook in resistance management. 2 ed.), with minor modifications. Only adult male lice will be used for the bioassay, which after sampling will be incubated overnight in running seawater (ca. 10 °C). The 30 min exposure to the prepared deltamethrin concentration will also be carried out at ca. 10 °C, and higher concentrations than those described in the given protocol will be included. After exposure to deltamethrin, the lice will be kept in running seawater (24 h at ca. 10 °C) until evaluation of the bioassays. At evaluation, lice will be characterised as active, or inactive (moribund and dead lice pooled). ECso values will be calculated using probit analysis (PoloPlus 2.0; LeOra Software, CA, USA).
The expected results from this experiment is that only lice from the resistant strain and its maternal hybrid strain will show resistance towards the pyrethroid treatment, and thus survive the bioassay. The sensitive strain and its associated maternal hybrid strain will displayed close to 100% mortality. No differences between the hybrid strain and its maternal founder strain will be observed for either strain, demonstrating that the resistance follow a maternal pattern of inheritance.
The presented outcome of crossing resistant and sensitive lice will show that the mitochondria genome encodes the resistance in this species. We will then amplify, sequence, and compare complete mitochondrial genomes of susceptible and resistant strains of Caligus rogercresseyi strains to identify the specific SNPs associated to this resistance, but the above described experiment will provide enough information to conclude that the mitochondria is involved in the regulation of resistance towards pyrethroids in Caligus rogercresseyi as described for the L. saimonis.
Other experiments may be performed on other arthropods, in particular crustaceans, in particular copepods following similar experimental protocols as described herein, with expected similar results.
SEQUENCE LI STI NG <11 0> Pat o Gen Analyse AS
<120> Screening and identification of resistant markers in crustaceans <130> P23482PC00 <160> 20 <170> Pat ent I n version 3.5 <210> 1 <211> 15445
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 1 agccccct at ttaggtgcag 11 aaat aaat at at 11 at 11 aaat t aact a t gt acct at a 60 t gagt cgccc tagaatcaat aaagt t aagg caat gggagc at agt 11 aat t at agt gt t g 120 agaaaagggg aggattatac ctttatacgg acgtggccac gggcattcaa tggcaggttc 180 t agacat at a 11 gaggaggg ggggggggag at t aggt aat aaat 11 at 11 aaat 11 at t a 240 tat getcgca aagttgaatt aggggtaat a gggagtatac ggettaacta tagtgtctag 300 ggtaaatcag tattagaagc taegtctccc aaagetat aa gttctggaca actagettct 360 acccgaagaa ggt at aaagt caagt t cct t t aat aaagaa tattatgaaa at aat acaag 420 ttcgaaagga t agaggact a agtaagattt aggeatttat aaget t aget aat accaggt 480 ttaggttcta tttaataaaa gatttggaaa gtcaaggagg tgattactta tagagtaagg 540 ttatcttctc cttcaaagta actttttccc tagggagtag ccaggtcage ettteagget 600 tataaaaaga ttagaataag cagggaaaat ctttgagtta aatttaaggt ttagaactaa 660 agaggactat tatttttttt tttttttttt tgageatgtc atgaaaagtc taggagagag 720 getgaegage aat ct at eet tet gageagt tgtgtgaatt ct ct ggget t agggagt aat 780 t aaat get 11 t at 11 aaat c aatctgcgaa gggggcaat a aggt at gt t a 11 aat aaat a 840 aggtttggag ct 11 aaat ct aaagt t gggg at at gt ggac t gat t aaccc agaat aat at 900 at at at atgc agtgctcaaa ågettgttgt caaaagaaag ccaggggcgg gegeagaget 960 gagaegeagt ccacgtgcta aaaaat t act at gggggt ag caaat at caa t aacaccgaa 1020 gacaaaatat ggaaagctgc cgaaaagaaa aaagttaagg gaattgaagt taattgaata 1080 agattttctc t agaat 11 cc 11 gcgaagca act gaaaat a at 111 at at t aaat agt at c 1140 ccgagaatta aggcaatega gattggaagt aggaaaagga ggaataaatt gagtaaagtt 1200 at agt agaga gaatctcggc tataagattt atagtggggg gccctccagc at t gagt aag 1260 caagct aaga at cagat 111 t act aaegt a gget gggt ag t aat t at gcc ct t at 11 aaa 1320 at aatgtgac gagtcccaga ggacaagtaa aat ataccag eggegagaaa tatcaaagag 1380 gaagt aaat c egt gagccac aaagt t aat t at gget ct ag aagt t eet gt ggaagagaga 1440 gagaaaactg tageagaage tatagacatg tgacctaegg aggagtaagc aat aataacc 1500 11 cat at eta ct t gagt t ae gcaaagcat t at agccaagg ct gccacaac t aeggagaaa 1560 gagat t at t a eet t ggt aaa aaat eet aaa aaaaaagagt 11 at at ggac aaat egaat a 1620 agcccatacc cccccaactt aagcaaaact cctgccagga ttatagaccc cataagaggg 1680 gcctctacgt gagetaaggg aagtcataaa tgtagggaaa agatgggcaa etttacaaga 1740 aat get at aa aagt cat t aa aagaat aat a ggggaagggg aggat gat t g ggegat aaaa 1800 aaaaaagtga aataggagag atgggggcca gcaatgataa caaaaaggag gggcaaagag 1860 cct at t aaag t at at at aat t at t at 111 g 11 agcaaaaa 11 egt t ct gg ct gat agcct 1920 caacccaaaa tgattaaaaa aatagggata aggtttåget cgaaagcaat ataaaaaaaa 1980 aggatattag agaeagagaa agtcaaaact gaaaaaaaaa aaagtaagtt egagtagaaa 2040 gaggt t aaag ccgct at 11 c gat agaaat c aacaaaagaa t agt cat aga aagaat aggg 2100 getctaacct egtccaagaa aaaagaagag gagaggttaa aggagatgtc ataaatggga 2160 atcagggtta taaaagaaaa aattaaagga aaagagataa gaaaattggg taatgaacta 2220 11 aaaagct g t aaaaaaagc 111 aagt aaa aaggaggat a t aaggaaaaa aat aagt 1 1 1 2280 aaact agaag tt aat teat a at aagt t aat tttgagaaga ggtctagggg ct agggt get 2340 aattggactt acaagaagca gattttttat tttttggagg gctgtcgaac t aaat act at 2400 cct gt 11 at t gt gat gt t gt ggttagaggg ggagaaaggc t ccgagt t ga ggggaat aaa 2460 at act t ct t a at t caagggt 11 aggt ccct at t aat agt c t gaggagt ct 111 at t at at 2520 aggat 1111 a at aat agt ag ggggt ct 111 aaagt tagga gt gt 11 cct t tccatttatg 2580 aagt ct agat ttagcagtaa ggagaagatg ggattgctgg ct gt at 11 aa tgagtttaca 2640 aaaagt cct a cccct acacc t agt agt aaa t gt aagagaa ggaagt aggg 1111 gagggg 2700 t gt aact ct a ct t aat 11 gg t agt t aggt g ct t agggagg cttcataaaa gt at at t aaa 2760 ggaat t at t a 1111 at t ct t cact at 11 aa cat aagat t a 11 gt t get at t gaggaget c 2820 geaet ct at c ct agt 11111 1111 eet t at 11 at aggaga 11 at t aat t c ct t gt t gggg 2880 gattttaaaa aactccgagg t agact acct geageaagt a catctaagaa gagggggatt 2940 at t åget 111 ct gt eget ga gagggt t ccc t eet agt ct g gggt 1111 gt acaagt at gg 3000 get at t caga ct 111 agt ca get t aaat t a t ggagt gt t a t act gt t gt a gggt t at act 3060 eet cagt ct c 111 at at 111 at at 11 at 11 aagagt gt 11 11 aeaget ga aat t aat egg 3120 aagaegaaea tttatgccaa ggatgagggg gaaagggggc tatctacttg ccccccaagc 3180 at 11 ct aat g at t aggggaa gat t gattet at at åget 11 t aaaagact a 1111 gaaaat 3240 agt caat gga gtttcttcaa åget at 11 gt ct aaegt aac t cgccaccct ttccatttag 3300 11gat gagt c teettggcct ctattaaggt ct111getgg agggt11gtg get11aggcc 3360 tattaggett atttttaaac agaagaagga gcctaatttt tacttccctg atttttttag 3420 ccct aat t at aat ccagt ga t ggt t agat g 111 eggt t ga gggeaet t at caagggct t c 3480 at t caaaagt egt t caaat t ggget aagat gagggat aat ct t at 11 at t agat cagaag 3540 1111 gt t ct t ct 11 agt 111 ttttggggat at tt teat ag cagat t agt a ccaagagt ag 3600 aaattggagg aggggagtgg ccccctttag gggttaeagg cctcagagct tgagaaatcc 3660 ctttactcaa tacaatcatt ct gt t gagat cgggggtgag agt cact tga agacatcaca 3720 gat t agt aat aggt aaacat caaaat geat ct 11 aagat t act gat tact gt at t act ag 3780 gaggt t at 11 t act t gt ct g cagggagt ag agt act t cga agcaagat t c agt at t agag 3840 at agagt gt a cggt t caact 11 ct t ct t ac t aacaggat t ccat ggt 11 a cacgt get ag 3900 t aggggccac 111111 aggg gt ct gt t act t ccgaat agt gt ct gggeat 111 ggt aaca 3960 gccaccattt tgggtttgaa gegget gegt ggtattgaca ctttgtagat gttgtttgac 4020 t at 1111 at t 111 gggggt t t act get t ag t aaaaaaagg ct gt t at t ag agt agt 11 at 4080 caaaatatta act11gggag 11aaagaaaa ct11agttct ctgataggtt agt aat11at 4140 acaaaatatt ågettgtcaa gccaaaaaaa aggaacteet tttagcctgg gaaacttaag 4200 11 aat aaaac t at cagt ct t caaaact gac aat aaaccct 111 åget t cc gaat t act at 4260 aact t at aaa gagtattaca 11 gaaget gt aaaggagget t agcct agt a at aat 11 1 1 1 4320 tattcttat g ggtgttaggt ctaaget11a ctat11eett attaat cat a 11actaggtt 4380 attctttaag acgaaggggg gaggegaatt tagataagtt aagacctttt gagtgtggat 4440 tcaatcctaa tgeaggteat egaaagat at tttetttaea tttcttccta gtctctttgg 4500 tgtttttagt cttegattta gagttggtaa ttcttttccc ctttctaagt gtaggaggag 4560 tggggaggag ggtagaaaga tacttaaggt gtgttttatt tttgtttgta ttaggattag 4620 gcct 111 at a egagt ggt t a at aaaaagt t t agagt gat g t gat t aaat g t aaaat acaa 4680 at 1111 aat t t aaat 11 ccg ggggt t at gg cttgttttga gaaggt 111 g agt act gggg 4740 ggggt 111 aa t at t eet 11 c åget 111 agg agt 11 aaagg aatcctcaat act agt at t g 4800 agagcaaaac taagaatttc aaatttggaa ctagaagtca gagggttggg tgaettttae 4860 t ct at at cct t caggggagt tgtgataata at t aggggt t cagt gt at ct ct act ccct t 4920 aggt at at ag aaact gaaaa gt at 111 aat cgatttatga ct ct cgt t ag gt t gt 11 at t 4980 111 agaat gc t agt gt t aat 11 acagaggg gatgtggtac aat t aat t ct agggt gggat 5040 ggcttaggtc ttaggtctta 111 act agt t t gt t act at a at aat aggaa 11 ct t ct aat 5100 gcaggggcac taacccttat actaaaccgt ttaggggatg tgggtttgtt tttaagaatt 5160 t acgt t at ag taaattccag gcat t caat t aacaggt t ca t aagcat ct c t agggt aat a 5220 ggaaggctat tattatttgt agcttttact aaaagggctc aattgccctt tagggcttgg 5280 11 acccgcag caat agct gc t cct act cct gt gt ct t ct c t agt ccat t c ct caact 11 g 5340 gt t act gcag gagt ct acgt at t aat t cga 1111 at gaca ggct aat 11 c t gt gt t at ga 5400 attaggttaa 11gtaggagt ctctactaga agattagcga ct11agt age aatagctgaa 5460 egegaeat aa aaaaaat t gt agct 11 ct ca act 11 aagac atttaggaat t at aat aagt 5520 at tet gagat t aggt t gagt egaaet agcc tttaggeatt taattttcca tgcttttttt 5580 aaagegt t at t at 11 ct agt agt ggggt at tgaatccaca get cct gegg 11 at caagac 5640 ctattaaaaa ttaatttgtt gagtaggeaa gagccagtaa tcagaagtct tggeggagtt 5700 t ct at aat aa gt ct 11 gt gg act t ccct at 11 aact gggt 111 at agaaa agact t at 1 1 5760 atagaageaa gtgtgeagtt taggagaagt cttcttggat taggttttat tttaagaagt 5820 t gt gt aggt t cagt aat gt a t agaat t egg atgtttctaa t agt t aat aa agt gggaagt 5880 gtctcaaggc ttttaaattg aggagggtct tttaaatttt accagttaag tataagetta 5940 11at acttct 111ccattat aggaggtaga 1111ggagaa acttctgagt tacttccggg 6000 agagt 111 at at gt ct 111 g agaagt aaaa at agt gat t a 11 aggt t aat cacat gcagg 6060 gggttget ag gagettatct taggaataca ggggtaaaaa aeaggt1111 gagtaaacta 6120 gggt acct aa gagacat aag aatccggctc cccaacatcc tcagaaaatc ettgataage 6180 aaaagagt aa aat t agat aa 11 ct 111 ct t agcagccat t acaat aaat a ct aeagagt a 6240 gaaggaggaa aat at 11 at t aaat agaggt aact t aaggt attetgaaet aat ct t agt a 6300 at at t ct 111 111 at gt gat at t at at gt g ct cagggggt aat t gt t eet caagcagaag 6360 tagt at attg aaacataatt 1111gtctat gtgteggtag tattggtgtt agggtattaa 6420 aat t at at 11 111 gt aaaga ggt aaget aa aaaagct gat agact cat aa t ct at ct at a 6480 ggatcccttc tct11aaagc 11aaaagagg aaat11ctaa 111ct11at g gettaggtcg 6540 ågettttage taataaaaag ggaattgeaa actccctatt cctcacaggg ttågettgtt 6600 t aggt aat 11 at at aaaat g cccaat t gt g gagt gggt aa ageat ct t gc ett aaacat a 6660 tgcctacttg atctcagt11 agtttccaag at getteet c cccagtaat a gaagagt11a 6720 1111 at t cca tgatgtgatt at agt 11 gt t t aggcct t at ct t agt at cc gt agggggt a 6780 tattaggggc agtgt11gta gaggggcctt atgtaggagg 111aat egae ggagagtgac 6840 t agagt gaat 11 gaaeaget 11 acct ggt t t agt act at t t ggggt åget at accct ct t 6900 t at ct 11 at t at at at at ae gatgaaataa caaattttga tttgggggta aaagt aaat g 6960 ggt at cagt g at at t ggagc t aegagat ae eet t ageaga gggcct t gt g ttagattgtt 7020 ttatggtccc cagaagggat acaaagttag gacaaatgcg cttacttgaa agagatgtgg 7080 ct 11 aact 11 acct at t aaa agaat get gc agt t gat agt gt ct t caaag gat gt at t ae 7140 at gettggge ggtccctage 11aggggtta aaatagaege tgtgccagga egaattaat a 7200 ccctatcaat at at agat 11 eggeegggat tgtattttgg gcaatgttca gaaatttgtg 7260 gggeaeat ca t aggt 11 at g eet at 111 ae t agagat agt aagggt t gac gattttgeta 7320 ggt gat t aaa gt tagt gaga gaagaat aag cat aaagt gc at t gt t at t a aagt gt t gt a 7380 ggt 1111 aat cat caggat t eet gt act t g t aaat gt t gc gt 11 at t acc ct act agaac 7440 gaaaaat t at t ggt t act ct caagcacgt a aaggacccaa t aaggt gt ct 11111 aggga 7500 ttcttcagcc 111 ct ccgat get at t aaat t at 11 gt aaa agaagt agge aggt t cagat 7560 11 gt aaat t a eagagtetae t ggat 11 ct c ccgt agt ggg t ct aagggt g geaet act aa 7620 t ct gaaat gt ct at ccct t a gagat aaggt 11 ggat ct t g aagaat 11 ct tgacttggac 7680 t gt t ageggg gat aagagt a agagtttacc eet t at 11 ct tagagggtga aggt ct aat t 7740 gt gt gt at t c acatattgga ggagt gegag gagt t get ca agt aat ct cc t aegaagt ag 7800 11111act at eet agt at11 tct11aat aa gattaagagg aagettaact 11agtgggga 7860 gt at act at t t agggagt at 1111 at gt 11 ae at at t aag t eet 11 at t a gt gt acct at 7920 gattcctaac aggtctageg gagaccaacc gaactccctt tgatttctct gagggggaaa 7980 gagagt tagt gt ccggat t c aataeagagt atgggggcag ggggt 11 get 11 aat 1111 a 8040 t ageaeagt a t get t egat c 11 at t gt t aa gaagtttatt t agat t at 11 11 act t aaaa 8100 gaagaggact 111 gt 111 at gt aat t agaa ggt t aggagg gttcttgtga gt gt gaaggc 8160 gggt t act t a ccct cggcac cggt at gact gat t aat aag at t at gt t ga aaat ct ct 1 1 8220 t accaagaat t ct act cggg ggagggggaa t aat 111 aat aat aat aaga at 11 aat t gt 8280 caact at aaa aaagt gat t a tgatcttgca accataaaga tattgggact ct at acct ac 8340 t aagt ggat t 11 gat ct gga 11 agt gggt t t agct at aag agtcatcatc cgtcttgagc 8400 t gt ct cagcc gggagcat at 11 aggggat t cccaggt 11 a t aat gt t at t gt aact get c 8460 at gcct t cat t at aat ct 11 11 cat agt aa t acct gt at t aat t gggggt 111 ggaaat t 8520 gat t agt tee 111 aat act g ggggcacct g acat agct 11 cccccgct t a aacaat at aa 8580 gattttggtt 111 aat accc tctttgagtt t at t get t at aagggcat t a gt agaaagt g 8640 gt gcaggaac t gggt gaaca gt at accccc ccct gt ct t c gggagt 1111 cact ccgggg 8700 cttcagtaga ttttgegatt ttttccctcc acttagctgg agtgtcttct ttattagggg 8760 ctgtaaactt tattagaact attacaaatt taeggtgett gggactttta gtggggcaaa 8820 t gccaat at t cccct gat cc gttttaatca ct get gt get 111 act ct t g t ct 11 accag 8880 t gt t ageggg agcaattact at act t ct t a ctgatcgaaa 111 aaat aca act 111111 g 8940 accctagagg tggaggggac cccattttat accagcattt attttgattt ttcgggcacc 9000 ct gaagt 11 a t at t ct aat t cttccagggt 11 ggat t aat ct ct cagat t at t acccaag 9060 aaact t gt aa agatgagget tttggttcgc 11 ggt at aat ttatgcaata gtagcaattg 9120 gagt at t ggg at 11 at t gt t t gaget cat c acat gt 11 ac agt t gggt t a gact cagaca 9180 ctcgagcgta 1111acggca gccactat ag taattget at ccctaeaggg attaaagtat 9240 11 agt t gat t agggact 11 a tatggaagta agat t at 11 a t accccct ca at 11 at t ggg 9300 tggtagggtt tctgtttctt tttaeagtgg gaggattaac aggtattgta ttågetaact 9360 ctgccttgga tatgaetttg catgacactt act at gt agt tgcccacttt cactatgttc 9420 11agaatagg ageagttttt gccctaat ag egggattcac tcactgat ae ccccttttga 9480 caggt11aag aatgaactta act ataagga aagegeagtt tat eet aat a 111attggtg 9540 t aaat 11 aac gt 11111 eet at gt act t cc t agggt t age aggt at acct eggeggt at a 9600 gggat t accc agaettetat tatttgtgga at caggt age t agt 11 eggt t ct t acct aa 9660 ct 11 at 111 c t gt ggt agt a tttgtaggga t aat tt gaga gt ct at agt t aggt t aegae 9720 cagt at t age ggt at t t gat aggggaagat ccat egagt t caaacat aeg t gt eet ccgt 9780 at aaccat t c 11 at aact cc t gcccccgac t gået t tage at aaat aaat t aat 11 aaaa 9840 aaatacctaa aattagttat 111 eet 11 cg tact aataag taagtaaaag ccctgagat a 9900 gaaaccaat c t gået t aegt egat ct t aac t caaat cat g t aagt aegaa aagt egaaea 9960 gacttaaaat aggacct t ct geageaet at at t act t aat t caacat ega ggt cacgaat 10020 atcttttaaa taagagetct aaaagtatta egegetgtta tccctaaggt acctatgtgt 10080 agcaaaat aa ttggttcaga at aact aagg 111 ct 11 at t at at t aagt g ggctatccca 10140 gccaaacatg gt aaaggt ct t agggt ct t a t egt ct t egg agagtatatc agcgtttgta 10200 ct gat åget a agat t caaga gat aagaat a aacatgatta accaat get a get t cat aca 10260 ggtttacaat t aat aaact a at t at t gcgc t act 11 aaag ccat aaaaaa t cat t cat t g 10320 ggcagat aca t cagacacag t ct t act at g t ct 11 gt t aa acagt 11 gga gaatctcaag 10380 11 at 11 agt t t at at 11 aat 11 aaaat t aa t aaaagt t at 11 aagt aagg aaat aat aaa 10440 agt at at 11 a ct agt gt aaa caagttataa ttctcagaat t aact accca aataataaga 10500 gaagct t aat aaat aaat t a ggtcaataat aat 11 at t at aacagagggg act at t at t c 10560 ct gaaat at t 111 agact ag 11 get cact a at gt at act c ageat aaget aaat acat t a 10620 1111 aaaat t cat t ct aaaa t gt 111 at 11 ct 11 gggaac cagat 111 aa aaagt accat 10680 at at egt gt t catccttgtc 11 at åget t a gt 111 ct acc t agget t aga gagaggat ag 10740 at at 1111 aa t cgggaaagt ttttattgat act aat t gt t t acccat gat gcaaaaggt a 10800 agagt t ct t a t act act 11 g aaggat t act aat aggggca gt t egeat aa ct at eet at c 10860 aagatggccc ctat acttcg ggcaccctat at111aaagg gggtggccca tgtctageae 10920 11 aaaget ag tgeaetttet ctgccacttt aaaaat aat t t at t aaat aa aagat t aagg 10980 at aat ct aeg at aaget t ct cccat agaga agt aaageat aaageagat t aagt t act t a 11040 11 gagaat aa t agcct at at aaaat t ct aa 111 acat gea at gaaat t gg act egeagt a 11100 t at ggat 11 a ct aaat aaaa aaagaagt ag agat t t t t aa gttctgaata agaaagt gcc 11160 ageat t egeg gt t at act 11 t agaaaaat a ggcatttaaa tctcaaaaat taacatttca 11220 t gaggt caaa 111 aaaaaat gt t agt aaaa 111 gat gaaa at agt ccagt gaat eggat t 11280 agt ccccgag t aat aaagat aagt 11111 a gt agt act ga at agaaaact aaact caaat 11340 ggcagaat t a t ct ct caaat t agggaaacc tgtattaaca gaat 11 act c 111 agat 111 11400 aat 111111 a aagt at ggct gt ct t act 11 at t cagaaat t gt ct t agt c aagt aat at t 11460 II at at aat t aat at 11 at a ggttgcatta aat t at aaaa t agagt at 11 aat agt aagc 11520 t aaaaaacgc tgaatatttt agt aat t agt tcaaattgtc cgtcactctc t taggagat a 11580 agt cgt agca aagt aat agt tatggaaata get at t aaat 11 ct 111 gaa gaaatttttt 11640 11aaattaat agttat11cg agagttgett gtact at aat tcttgggetg atteet agt c 11700 ctttcatgtt gt gt 11 at t a at t gt agt at t aact agagt t agat gt ggg at agt aagga 11760 111111gtag ccagtggtta ågetaegett tagt aattgt attcttgggg ggtat aat ag 11820 t at t gt 11 ae 11 aeget t ca agaat aagag t at cagat aa attgacaaaa agaaggagat 11880 gaaaaagact atttgtattt ctgattatgg ggttaacttc aagggccccc agaggattta 11940 aagt 11 at at aggaagact a t at t caaat a at gggggggt t at act 11 ca at ct t aacct 12000 III at 11 act eet gt gcct a 111 agagt gg t aaaaat agt agaagt gaga aaaggggct t 12060 tgatttetta gggt t aacat aat 11 aat 11 gcatttaaag ct t gccgt 11 ggct aacct t 12120 act tt tagag tgtaatcacg 11 at aat 111 at t at aaaaa t åget t aaag ct t aaaagt a 12180 t at 111 aagg t gt 11 ageae at aggat 111 gagt eet aag gagt t aagaa at t at ct aaa 12240 at aat t at 11 ggget aaagc at ct agt at t t acaaaat ae t cat 111 agt t aaact acca 12300 aat aaagt t a gcct aat aag t aaaaaaagt ggagttggat agtteagtag act 11 gaaeg 12360 gattgaagag at aagggcag caagccct at acat get t ca eagaet at aa ct gt cacaaa 12420 taaaaatctg at agttgagt aggttat act gaagaccgaa aaaaaaatgc aaataggaat 12480 tatggtagct gtaagagctt ctaataaaag tagcctgata agcaagtgcc ctttattaaa 12540 taaaaatcac ctagaggtga ataaaggagg gagagctaac aagtataaac tgetaaggtg 12600 aggt aaaaaa aaaat gagt a caagagttgt t aaaat agt t ctcatagagt at aaaaacaa 12660 t agct gt t ag ct t aaaagat aat ct aat aa agat gt t aaa 11111 act 11 aattatggaa 12720 ggt eet ct 11 t aat cccct a at t at 11 aag gtcttgcatt agaegt 11 ca ct gt t aat ga 12780 aaagaagagt 11 ct ct aaat atteagttag t at aagaat t aeaet t aat t tccaattaag 12840 agatttaaaa gt t aaact ga ct at t aat ca t agt aaat 11 t at at 11 cac 11 acaat gaa 12900 aaggtgccaa tggetgatta attcttat cg agcteet at c aattaagtag 111aataaaa 12960 accttaaatt gtaaat11aa aattagat t t tctcttaatt aaagccttag ggagtact aa 13020 t gaat t ct 11 at 111 ccaga t t t gat get a aeggat t gac aaact gat ae 11 acct t ct t 13080 t agcaagagg 111 at t act a tttccaaggt at t gat tage gggt aat aca ttaggctcca 13140 t gt gaagagg ggt 1111 agg get at t cat t ccgatgtaaa aat t at at 11 agt gaagggg 13200 cagggt 11 at get t at gaga 11 at 1111 aa gggt t ct aag aatgaatttt ct aggget 1 1 13260 11 ccgt acat 1111 act agg agt aggeaea tctgtgtgag at t aggt at a ggget gcct t 13320 tat gattagg cagtcaaata gtttgttgag tataeaggag agagcaaagg 111gcccacc 13380 t agt t ccaga gggggct eet agaggt tt aa ttcctctttt ggttttaatt gaaagaatta 13440 gaaggt t aat ccgacct ct a accct aaggg 11 egget t at agct aat at a at t gcaggcc 13500 acct act act aat 111 act t ggt agt aat g ct agagt ggg gt cccct ct t agaattctat 13560 t aact at gt t aggat t at t a 11 ct t agt cc t act agagt t aggggt gagt ctaattcaga 13620 gat acgt 111 t at at t at t a agct ct 11 at at gt t aggga gact ct t cac t aat gaat 1 1 13680 t gt t act gt c ctagtttcga cct aggct t a ggttttaacc agcaaaat aa tctaattatt 13740 t at gt 11 gt g t aat gcgggc agt t at t acg cctgtcaaat gact t caaac t aat aaaagt 13800 11 agct caca atccgtctga gt aaggt aag gacagt cct a ct ct gaagt a t aagacagt a 13860 aaaacttggc ccaggaaaat atagtttccc tctacaggtt ttgetccaat ccaggttaaa 13920 agaaaaaaaa tcccgataaa agctcaaaac ataageattt tcgaagggtt aaatgtagea 13980 tgtaeggegg ctcttttatc ccccagcaaa aatggaaaaa aaaacaaaat tcttacagac 14040 aaagetaaag ccactacccc ccccaactta tttgggatag accgcaaaat ageataagea 14100 aacaaaaaat accattcagg ttgaatgtga gcaggagtta ctaaaggatt agctggaata 14160 aagttttcag ggt ct get aa taatcaggga gagt aagaaa ct aaaget cc catgagacaa 14220 aaaataagac cgaagccaaa ggcgtcttta aaggtatagt aagggtcaaa aggaatetta 14280 t aat agt 11 c ct ggaacccc caat ggat 11 gaggaccct g 11 gaat gaag aaaaat t gt a 14340 tgagetataa ctccaactgt ggacaaaaga ggaatgaggt aatgaagage aaagaatege 14400 ettaaagtag ctttgtccac eetaaacccc ccccaaagtc aaattacaat ttcgtcccca 14460 at accaggga tgacagaaaa t agat 11 gt a at aaccgt ag ctcctcagaa agatatctgg 14520 ccccagggga gcacatagee caaaaaagct ctggetatga ttaaaattaa aatcgtagag 14580 cctacaactc aaacaggagt aagcttatac gaagaatagt aaaggccccg acccgtatga 14640 at gt at aaac acat aaaaaa t aacct cacg gt gt t get at gaat åget cg aat t cct cac 14700 ccat aat t aa cat cccgcat gat at ggt et aeggat aaaa aeget gt aga aat et et et t 14760 gaat aat gea t agccaaaaa aatteeggte ataatctggg t aat aagaca t agcccaagt 14820 aaagacccaa agttccaccc cgacgaaata ttaactgggg tgggaagtgt tagaagagga 14880 11 at agat ag aaagagt 111 at aagaat t c at aaat ggag ctttctggca gaaaaagt gc 14940 gt t aat 111 a gaaat t aagt at aagagat t et t gt aagct acaacat act act t aat agt 15000 at 11 at t aaa et cat aggag 11 aacaaat a gtcttgatcc at aagat 11 a ctgtgtgcaa 15060 aegget agat t at t agt 11 a gt at at t aac t agaacaaaa t agt t agt t a at at 11 at t a 15120 aaagt11aac caagttaagt ggaggagget tetgtagt aa ccgcggagtt tggt11aat g 15180 agaact et et t aaaget aac egaaaet agg tt gat et tea aaat at 1111 t aagat t ggt 15240 II aagct at g cacaagttgc t aat at 11 at taaaagattt aaat aagt ag aaagt t taga 15300 III aat act a agaagaat t a et agagaat a aagccagacc aaat at 11 at 11 aaagat 1 1 15360 agat aagt ag et aegget ag gt ggt gggga caaggt geat aatcaataca agt gagt t gg 15420 gggggggggg tatggggggg gatta 15445 <210> 2 <211> 1056
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 2 ctgtctcagc cgggggcat a 111 aggggat t cccaggt 11 at aat gt t at t gt aact get 60 catgccttca 11 at aat et t tt teat agt a at acct gt at t aat t ggggg gt 11 ggaaat 120 t gat tagt te et 11 aat act gggggcacct gacat åget t t ccct eget t aaacaat at a 180 agattttggt 1111 aat acc et et 11 gagt 11 at t ggt t a t aagggeat t agt agaaagt 240 ggtgcaggaa ctgggtgaac agt atacccc cccctgtctt cgggagtttt tcactccggg 300 getteagtag at 111 gegat 11111 ccct c cact t åget g gagt gt et t c 111 at t aggg 360 get gt aaact 11 at t agaac tattacaaat 11 aeggt get t gggact 111 agt ggggcaa 420 at gccaat at tcccctgatc egt 111 aat c act get gt gc 1111 act et t gt et 11 acca 480 gt gt t agegg gagcaat t ae t at act t et t act gat egaa at 11 aaat ae aact 111111 540 gaccct agag gtggagggga tcccatttta taccagcatt tattttgatt tttcgggcac 600 eet gaagt 11 at at t et aat tcttccaggg 111 ggat t aa teteteagat t at t acccaa 660 gaaact t gt a aagat gagge 1111 ggt t cg et t ggt at aa 111 at gcaat agt agcaat t 720 ggagt at t gg gat 11 at t gt 11 gaget cat cacatgttta cagt t gggt t agact eagae 780 actegagegt at111aegge agccactata gtaattget a tccctacagg gattaaagta 840 111 agt t gat t agggcct 11 at at ggaagt aagat t at t t at accccct c aat 11 at t gg 900 gt ggt agggt 11 et gt 11 et ttttacagtg gggggat t aa caggt at t gt at t åget aac 960 t et gcct t gg at at gået 11 geat gaeaet t act at gt ag 11 gcccact t t cact at gt t 1020 cttagaatag gageagtttt tcgacctagg ettagg 1056 <210> 3 <211> 996
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 3 1111 aaccag caaaat aat c t aat t at 11 a t gt 11 gt gt a at gcgggcag 11 at t acgcc 60 t gt caaat ga ct t caaact a at aaaagt 11 åget cacaat ccgt ct gagt aaggt aagga 120 cagccctact ctgaagtata agaeagtaaa aacttggccc aggaaaatat agtttccctc 180 t aeaggt 111 get ccaat cc aggt t aaaag aaaaaaaat c ccgat aaaag ct caaaacat 240 aageattttc gaggggttaa atgtageagg taeggeggtt cttttatccc ccagcaaaaa 300 tggaaaaaaa aacaaaattc ttacagacaa ågetaaagcc actacccccc ccaacttatt 360 t gggat agac cgcaaaat ag cat aagcaaa caaaaaat ae cattcaggtt gaatgtgagc 420 aggagttact aaaggattag ctggaataaa gttttcaggg tetgetaata ateagggaga 480 gtaagaaact aaagctccca tgagacaaaa aataagaeeg aagccaaagg egtctttaaa 540 ggt at agt aa gggt caaaag gaat ct t at a at agt 11 eet ggaaccccca atggatttga 600 ggaccct gt t gaat gaagaa aaat t gt at g åget at aact ccaact gt gg acaaaagagg 660 aat gaggt aa t gaagagcaa agaat cgcct t aaagt åget 11 gt ccaccc t aaacccccc 720 ccaaagtcaa attacaattt cgtccccaat accagggatg acagaaaat a gatttgtaat 780 aaccgt åget ccccagaaag at at ct ggcc ccaggggagc acat agccca aaaaagct ct 840 ggct at gat t aaaat t aaaa t cgt agagcc t acaact caa acaggagt aa get t at aega 900 agaat agt aa aggccccgac ccgt at gaat gt at aaacac at aaaaaat a acct egeggt 960 gt t get at ga atagetegaa 11 eet caccc at aat a 996 <210> 4 <211> 1115
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 4 et aagt ggat tttgatctgg at t agt gggt 11 åget at aa gagt cat cat ccgt et t gag 60 et gt et cage egggageat a 111 aggagat t cccaggt 11 at aat gt t at t gt aact get 120 cat gcct t ca 11 at aat et t tt teat agt a at acct gt at t aat t ggggg gt 11 ggaaat 180 t gat tagt te et 11 aat act gggggcacct gaeatagett t cccccgct t aaacaat at a 240 agattttggt 1111 aat acc et et t egagt 11 at t act t a t aagggeat t agt agaaagt 300 ggtgeaggaa ctgggtgaac agt at atccc cccctgtett egggagt111 tcactccggg 360 getteagtag at 111 gegat 11111 ccct c cact t åget g gagt gt et t c 111 at t aggg 420 get gt aaact 11 at t agaac tattacaaat 11 aeggt get t gggact 111 agt ggggcaa 480 at gccaat at t cccct gat c egt 111 aat t act get gt gc 1111 act et t gt et 11 acca 540 gt gt t agegg gagcaat t ae t at act t et t act gat egaa at 11 aaat ae aact 111111 600 gaccct agag gt ggggggga t eet at 111 a t accagcat t tattttgatt 111 egggeae 660 cct gaagt 11 at at t ct aat tcttccaggg 111 gggt t aa tctctcagat t at t acccaa 720 gaaact t gt a aagat gaggc 1111 ggt t cg ct t ggt at aa 111 at gcaat agt agcaat t 780 ggagtattgg ggt 11 at t gt tt gaget cat cacatgttta cagttgggtt agactcagac 840 actegagegt at111aegge agccactata gtaattget a tccctacagg gattaaagta 900 111 agt t gat t agggact 11 at at ggaagt aagat t at t t at accccct c aat 11 at t gg 960 gt ggt agggt ttctgtttct ttttacagtg ggaggattaa caggtattgt at t åget aac 1020 t ct gcct t gg at at gået 11 geat gaeaet t act aegt ag 11 gcccact t t cact at gt t 1080 cttagaatag gggeagtttt tgccctaata gcggg 1115 <210> 5 <21 1> 1255
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 5 caaaataatc t aat t at 11 a tgtttgtgta atgcgggcag 11 at t acgcc tgtcaaatga 60 cttcaaacta ataaaagttt ågetcacaat ccgtctgagt aaggtaagga cagccctact 120 ctgaagtata agaeagtaaa aacttggccc aggaaaatat agtttccctc taeaggtttt 180 getccaatcc aggttaaaag aaaaaaaatc ccgataaaag ctcaaaacat aagtattttc 240 gaggggt t aa at gt ageagg t aeggeggt t ct 111 at ccc ccagcaaaaa t gggaaaaaa 300 aacaaaattc ttacagacaa åget aaagee act acccccc ct aact t at t tgggatagac 360 cgcaaaat ag cat aagcaaa caaaaaat ae cat t caggt t gaat gt gage aggagttact 420 aaaggattag ctggaataaa gttttcaggg tctget aat a atcagggaga gtaagaaact 480 aaagetccca tgagacaaaa aataagaeeg aagccaaagg egtct11aaa ggtat agt aa 540 gggtcaaaag gaatcttata at agt 11 eet ggaaccccca at ggat 11 ga ggaccct gt t 600 gaatgaagaa aaat t gt at g åget at aact ccaactgtgg acaaaagagg aatgaggtaa 660 t gaagagcaa agaat cgcct t aaagt åget 11 gt ccaccc t aaaaccccc t caaagt caa 720 at t acaat 11 egt ccccaat accagggat g acagaaaat a gatttgtaat aaccgt åget 780 eet cagaaag at at ct ggcc ccaggggagc acat agccca aaaaagct ct ggetatgatt 840 aaaat t aaaa t egt agagcc t acaact caa aeaggagt aa gettataega agaat agt aa 900 aggccccgac ccgtatgaat gt at aaacac at aaaaaat a acct egeggt gt t get at ga 960 at ågetegaa 11eetcaccc at aattaaca tcccgcatga tatggtctae ggataaaaac 1020 get gt agaaa 111 ct ct t ga at aat geat a gccaaaaaaa 11 ccggt cat aat 11 gggt a 1080 ataagacata gcccaagtaa agacccaaag ttccaccccg acgaaatatt aactggggtg 1140 ggaagt gt t a gaagaggat t at agat ag aa agagttttat aagaat t cat aaat ggaget 1200 ttctggcaga aaaagt gegt t aat 111 aga aat t aagt at aagagat t ct t gt aa 1255
<210> 6 <211> 18 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 6 11 aaacaat a taagattt 18 <210> 7 <211> 24
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 7 t agaaagt gg t gcaggaact gggt 24
<210> 8 <211> 26 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 8 gggcaccct g aagt 11 at at t ctaat 26 <210> 9
<211> 26 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 9 aaccaaaagc ct cat ct 11 a caagt t 26
<210> 10 <21 1 > 21 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 10 aggttaaaga aaaaaaatcc c 21
<210> 11 <211 > 21 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 11 taaatgtage agtaeggegg t 21 <210> 12 <211> 19
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 12 geaggtaegg eggttettt 19 <210> 13
<211> 28 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 13 get 11 åget t t gt et gt aag aat 111 gt 28 <210> 14 <211> 27
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 14 ttcttacaga caaagctaaa gccacta 27 <210> 15 <211> 25
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 15 agt aact cct get cacat t c aacct 25
<210> 16 <211> 16 <212> DNA
<213> Lepeopht hei r us sal ironi s <400> 16 cct ct t ygag 111 at t 16 <210> 17 <211> 17
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 17 11 ccagggt t t ggr 11 a 17 <210> 18 <211> 19
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 18 caaaacat aa gycattttc 19 <210> 19 <211> 15
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 19 cagcaaaaat ggraa 15 <210> 20 <211> 14
<212> DNA
<213> Lepeopht hei r us sal ironi s <400> 20 ccccccyaac 11 at 14

权利要求:
Claims (23)
[1] 1. An in vitro method for detection of pyrethroid resistance in an arthropod pest, comprising the steps of determining that the pest is a member of an arthropod species that exhibits inheritance of pyrethroid resistance from female members of the species, identifying a polymorphic site corresponding to a single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial DNA of the species, and detecting the nucleotide at the polymorphic site in the organism to be analyzed.
[2] 2. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is present in a coding region of a gene encoding cytochrome B.
[3] 3. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is present in a coding region of a gene encoding the cytochrome oxidase subunit I (COI).
[4] 4. The method according to claim 1, wherein the single nucleotide polymorphism (SNP) is present in a regulatory non-coding part of the mitochondrial DNA.
[5] 5. The method according to one of the preceding claims, wherein the pest is a crustacean.
[6] 6. The method according to one of the preceding claims, wherein the pest is a copepod.
[7] 7. The method according to one of the preceding claims, wherein the pest is a member of a species having ZZ-ZO type sex determination.
[8] 8. The method according to one of the preceding claims, wherein the pest is a Siponostomatoida.
[9] 9. The method according to one of the preceding claims, wherein the pest is a sea lice.
[10] 10. The method according to one of the preceding claims, wherein the pest is L. salmonis, Caligus elongatus or Caligus rogercresseyi.
[11] 11. The method according to one of the preceding claims, further comprising a. collecting a sample of the organism from its natural environment; b. isolating mitochondrial genomic material from the collected organism, c. identifying the nucleotide at the polymorphic site of the isolated genomic material, d. comparing the nucleotide polymorphic site of the isolated mitochondrial genomic material with a standard mitochondrial nucleic acid sequence from a member of the species known to be susceptible to pyrethroids, wherein the collected organism is determined to be resistant to pyrethroids if the nucleotide at the polymorphic site differs from the nucleotide at the polymorphic site of the standard.
[12] 12. The method according to claim 11, wherein step c) comprises nucleic acid amplification carried out by polymerase chain reaction.
[13] 13. The method according to claim 11, wherein step c) comprises contacting mitochondrial DNA sequence of the collected organism with a detection reagent, and determining which nucleotide is present at the polymorphic site.
[14] 14. A method according to claim 11 wherein step c) is performed using SNP specific probe hybridization, SNP specific primer extension, SPN specific amplification, sequencing, 5’ nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, or denaturing gradient gel electrophoresis.
[15] 15. A method for identifying a single nucleotide polymorphism indicative of pyrethroid resistance in an arthropod pest, comprising the steps of: i. determining whether the species to which the organism belongs exhibits incidents of pyrethroid resistance, ii. determining whether said pyrethroid resistance is inherited within the species from the female members to their progeny, iii. in the event said pyrethroid resistance is inherited within the species from the female members to their progeny, then isolating mitochondrial DNA from a member of the species known to be resistant to pyrethroids and comparing said isolated DNA with the mitochondrial DNA of members of the species known to be susceptible to pyrethroids, iv. identifying one or more single nucleotide polymorphisms in the mitochondrial DNA of the resistant member of the species, v. performing a control to determine whether said single nucleotide polymorphisms are indicative of pyrethroid resistance.
[16] 16. The method according to claim 15, wherein the single nucleotide polymorphism (SNP) is present in a coding region of a gene encoding cytochrome B.
[17] 17. The method according to claim 15, wherein the single nucleotide polymorphism (SNP) is present in a coding region of a gene encoding the cytochrome oxidase subunit I (COI).
[18] 18. The method according to claim 15, wherein the single nucleotide polymorphism (SNP) is present in a regulatory non-coding part of the mitochondrial DNA.
[19] 19. The method according to one of claims 15-18, wherein the pest is a crustacean.
[20] 20. The method according to one of claims 15-19, wherein the pest is a copepod.
[21] 21. The method according to one of claims 15-20, wherein the pest is a member of a species having ZZ-ZO type sex determination.
[22] 22. The method according to one of claims 15-21, wherein the pest is a sea lice.
[23] 23. The method according to one of claims 15-22, wherein the pest is L. salmonis, Caligus elongatus or Caligus rogercresseyi.

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同族专利:

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公开号 | 公开日

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WO2015018863A1|2015-02-12|

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EP3030673B8|2017-11-22|

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CA2920752C|2021-04-06|

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CA2920752A1|2015-02-12|

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DK179538B1|2019-02-08|

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EP3030673B1|2017-10-04|

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NO3030673T3|2018-03-03|

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EP3030673A1|2016-06-15|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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US6027876A|1992-12-30|2000-02-22|Kreitman; Martin|Method for monitoring pesticide resistance|WO2021136724A1|2020-01-02|2021-07-08|Patogen As|Method for detecting hydrogen peroxide resistance in crustaceans|

法律状态:
2019-02-08| PME| Patent granted|Effective date: 20190208 |

优先权:

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申请号 | 申请日 | 专利标题

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NO20131073|2013-08-06|

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NO20131073|2013-08-06|

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EP2014066898|2014-08-06|

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PCT/EP2014/066898|WO2015018863A1|2013-08-06|2014-08-06|Screening and identification of resistant markers in crustacean|

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