Method for detection of pyrethroid resistance in crustaceans and oligonucleotide sequences useful in

专利摘要:
The present invention relates to methods for detecting pyrethroid resistance in crustaceans, such as copepods, including copepods belonging to the family Caligidae, and oligonucleotide sequences comprising small nucleotide polymorphism (SNPs) associated therewith.
公开号:DK201600137A1
申请号:DK201600137
申请日:2016-03-03
公开日:2016-04-04
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:

Method for detection of pyrethroid resistance in crustaceans and oligonucleotide sequences useful in detection of pyrethroid resistance.
Field of the invention
The present invention relates to a method for detection of single polymorphisms associated with pyrethroid resistant Lepeophteirus salmonis, and oligonucleotide sequences and kits useful in the method of the present invention. The present invention furthermore provides kits and reagents useful for the detection of pyrethroid resistance associated SNPs.
Background of the invention
Infestations of the parasitic copepod Lepeophtheirus salmonis, 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 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.
Sea lice (Lepeophtheirus salmonis and Caligus spp.) are the major pathogens affecting global salmon farming industry and have a significant impact in many areas. The annual loss has recently been estimated to €300 million (Costello M. J. (2009). The global economic cost of sea lice to the salmonid farming industry. Journal of Fish Diseases. 32. 115-118) and the aquaculture industry relays heavily on a few chemotherapeutants for lice control. Emerging resistance development to these drugs increase the necessity to develop new treatment methods (biological, prophylactic and drugs) and tools to avoid increased loss due to sea lice and to ensure a sustainable salmon farming industry in the future. Control measures have relied upon a limited number of chemotherapeutants since the 1970s. Parasite resistance and reduced efficacy have now been reported for the majority of these compounds (Sevatdal S., Copley L., Wallace C., Jackson D., Horsberg T.E. (2005). Monitoring of the sensitivity of sea lice (Lepeophtheirus salmonis) to pyrethroids in Norway,
Ireland and Scotland using bioassays and probit modeling). Aquaculture 244. 19-27). In many regions, the recent development of resistance to emamectin benzoate has resulted in heavy mortalities, significant economic impact as well as increased prevalence of other infectious diseases (Bravo S., Erranz F., Lagos C. (2009) A comparison of sea lice,
Caligus rogercresseyi, fecundity in four areas in southern Chile. Journal of Fish Diseases. 32. 107-113).
Although various chemotherapeutants have been in use in the aquaculture industry for more than 30 years, it is only during the last 15 years that such use has been part of some kind of integrated pest management system. Management practices include coordinated salmon production within a defined area, use of single year class of fish, limited production period, fallowing, coordinated restocking, use of wrasse, synchronized treatments during the winter and targeting female lice to reduce the impact of settlement during the spring (Pike A., Wadsworth S. L., (2000), Sea Lice: A review. Advances in Parasitology. Academic Press. 44. 232-337).
Further controls are required to progress towards a true integrated pest management (IPM) system common to other forms of food production. One key to succeeding with a IPM is to develop tools for the management of resistance to the medicines in use (Brook K. (2009). Considerations in developing an integrated pest management program for control of sea lice on farmed salmon in Pacific Canada. Journal of Fish Diseases. 32. 59-73). So far, 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).
Previously, non-mendelian inheritance of resistance by reciprocal crosses between resistant and spider mite susceptible of being resistant towards a hydrazine carbazate derivate have been linked to mutations in mitochondrial DNA of the spider mite (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). This represents the only other known example of mutations in mitochondrial DNA linked to resistance towards an insecticide apart from the study presented here in sea lice. The spider mite however showed resistance towards a hydrazine carbazate derivate, and not towards pyrethroids as shown for in sea lice in the present application. Spidermite and sea lice are distantly related. Up to date, no SNPs consequently associated with chemotherapeutant resistance in sea lice have been reported, and particularly, no SNPs in the mitochondrial DNA has been linked to pyrethroid resistance in any species. However, in many 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 (Zlotkin E, (1999), “The insect voltage-gated sodium channel as target of insecticides”, Annu Rev Entomol. 44:429-55).
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 COl, A6, Cytb and 16sRNA of Lepeophtheirus salmonis 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 Lepeoptheirus salmonis (PRLS) is crucial. Detection of PRLS prior to treatment, and the use of such analyses after treatment to evaluate treatment efficacy constitutes an important determinant for the integrated pest management (1PM) in the aquaculture industry.
Summary of invention
The present invention is based on the surprising finding that resistance towards chemotherapeutics commonly used to combat sea lice infestation is linked to mutations in the mitochondrion genome of the sea lice Lepeophtheirus salmonis. 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 Cyt 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 crustaceans, comprising the steps of detecting single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial genome of the crustaceans to be analyzed. 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, in particular copepods belonging to the family Caligidae, in particular species selected from the group consisting of Lepeophteirus salmonis, Caligus elongatus, and Caligus rogercresseyi.
According to one embodiment, the present invention provides a method for detection of pyrethroid resistance in sea lice, comprising the steps of detecting single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial genome 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 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 one embodiment, the pyrethroid resistance linked SNPs mentioned above is detected in Lepeophteirus salmonis.
According to yet another aspect, a method is provided, comprising the steps of: a) collecting sea lice from infested fish or water samples; b) isolating mitochondrial genomic material from any life stage of collected sea lice; 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 nucleotide C, G, T, G, and T (U), or a complementary oligonucleotide thereof, is present in position 8605, 9035, 13957, 14017, and 14065, 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 crustaceans, such as copepods, in particular copepods belonging to the family Caligidae, in particular species selected from the group consisting of Lepeophteirus salmonis, Caligus elongatus, and Caligus rogercresseyi, , 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 of the organism.
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. 4 and 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 a oligonucleotide sequence being homologous to a fragment of an isolated oligonucleotide sequence according to the present invention, 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. 4, andC13957T, A14017G, and C14065T of SEQ ID 5, 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, and T (U), 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. 4and SEQ ID No. 5, and wherein said sequence comprises at least one of the nucleotide C, G, C, A, and T (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: LsHybridG with LsGulen as its maternal strain, and LsHybridV with LTVikna 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 LsGulen 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 (http://www.ncbi.nim.mh.gov/nuccore/NC_0072 j 5,1 ), 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 crustaceans, including copepods, in particular Lepeophtheirus salmonis, and novel SNPs, based on the surprising findings that mutations linked with resistance against pyrethroid in Lepeophtheirus salmonis were 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 in crustaceans, in particular copepods, in particular copepods belonging to the family Caligidae. In particular, it is to be understood that the present method and the present oligonucleotides may be used to determine pyrethroid resistance in copepods affecting farmed fish, such as fish belonging to the family Salmonidae. 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 crustaceans population being susceptible of developing resistance, i.e. taking into account the composition of haplotypes that are determined within a crustaceans population (see 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) 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 5 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 sea lice according to the present invention, the skilled person will acknowledge that various methods commonly used in order to detect polymorphisms within a population of sea lice may be used. Both methods based on genome sequencing, hybridization and enzyme based methods are applicable for determining whether a sea louse 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 (http://shop.perkmeImer.eom/CQnteni/Manuals/MAN AcycloPmneSNPKiuxir). 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.
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:/71ycofs01 lyOommg.edu/-~gcat·-seek/protocols/DNeasv Blood & Tissue Handbook.pdf)
Still another method applicable for detecting SNP is High Resolution Melting Analysis (HRM) enabling rapid detection of SNPs and determination of genetic variation within a population. The first step of a HRM protocol consist often of amplification of the region of interest, using standard nucleotide sequence amplification techniques well known to the skilled person, and wherein the amplification is performed in the presence of a specialized double-stranded DNA binding dye being highly fluorescent when bound to dsDNA and poorly fluorescent in unbound state. This difference provides for the monitoring of the DNA amplification. After amplification, the target is gradually denatured by increasing the temperature in small increments, resulting in a characteristic melting profile. As the amplified DNA is denatured gradually, dye is released, thus resulting in a drop in fluorescence.
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 U S A. 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, flueythrinate, 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 LsGulen) was collected in June 2006 from newly slaughtered rainbow trout from Gulen in western Norway. The pyrethroid resistant strain (hereon also referred to as LsVikna) was collected in February 2009 from a salmon farm in Vikna, North Trondelag, 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 LsGulen as sensitive control.
Prior to the initiation of the present study, /„vGulcn and /„vVikna 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 (LsHybridV), and 4-5 females from the sensitive strain and 4-5 males from the resistant strain (LsHybridG). 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 LTVikna F5 106
LsHybridG P 96
CvHybridV_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). EC50 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 0 17 0 0 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 0 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 0 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 0 12 0 0 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 0 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 0 14 0 0 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 0 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 0 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 0 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 0 24 0 0 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 0 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 0 0.1 17 0 0 0.3 14 14 50 0.5 1 6 85 1 1 10 90 3 O 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 0 0.1 9 00 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 0 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 0 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 (O.lppb), 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 CO/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. 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.chem.qmul.ac.uk/iubmb/misc/naseq.htmlL
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.
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 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.
Table 7. SNP-analyses correlate well with known resistance status. SNP-analyses using SNP14065 correlates with known resistance status in sea lice (L. 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 (L. 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.
SEQUENCE LI STI NG
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<212> DNA
<213> Lepeopht hei r us sal rroni s <400> 1 agccccct at ttaggtgcag 11 aaat aaat at at 11 at 11 aaat t aact a t gt acct at a 60 tgagtcgccc tagaatcaat aaagt t aagg caatgggagc at agt 11 aat tatagtgttg 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 getegea aagttgaatt aggggtaata gggagtatac ggettaacta tagtgtctag 300 ggtaaatcag tattagaage taegtctccc aaagetataa gttctggaca actagettct 360 acccgaagaa ggt at aaagt caagt t cct t t aat aaagaa tattatgaaa at aat acaag 420 11 cgaaagga t agaggact a agt aagat 11 aggeat 11 at aaget t aget aat accaggt 480 ttaggttcta tttaataaaa gatttggaaa gtcaaggagg tgattactta tagagtaagg 540 ttatcttctc cttcaaagta actttttccc tagggagtag ccaggtcage ctttcaggct 600 tataaaaaga ttagaataag cagggaaaat ctttgagtta aatttaaggt ttagaactaa 660 agaggactat tatttttttt tttttttttt tgageatgtc atgaaaagtc taggagagag 720 get gaegage aat ct at cct tctgagcagt 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 et 11 aaat et 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 gegaagea act gaaaat a at 111 at at t aaat agt at c 1140 ccgagaatta aggcaatega gattggaagt aggaaaagga ggaataaatt gagtaaagtt 1200 at agt agaga gaatctcggc t at aagat 11 at agt ggggg gccct ccagc at t gagt aag 1260 caagct aaga at cagat 111 t act aaegt a gget gggt ag t aat t at gcc et 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 et ag aagt t eet gt ggaagagaga 1440 gagaaaactg tageagaage tatagacatg tgacctaegg aggagtaagc aat aataacc 1500 11 cat at eta et t gagt t ae gcaaagcat t at agccaagg et 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 eetgccagga ttatagaccc cataagaggg 1680 gcctetaegt 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 eet at t aaag t at at at aat t at t at 111 g 11 agcaaaaa 11 egt t et gg et 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 get etaacct 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 ttaattcata at aagt t aat 111 gagaaga ggt ct agggg ct agggt get 2340 aattggactt acaagaagca gattttttat tttttggagg gctgtcgaac t aaat act at 2400 eet 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 aaagttagga gt gt 11 eet t tccatttatg 2580 aagt ct agat 11 ageagt aa ggagaagat g ggattgctgg ct gt at 11 aa tgagtttaca 2640 aaaagt eet 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 tagactacct geageaagta 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 ct 11 act caa tacaatcatt ct gt t gagat cgggggt gag agtcacttga agacatcaca 3720 gat t agt aat aggt aaacat caaaat gcat 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 gccaccat 11 tgggtttgaa gegget gegt ggtattgaca ctttgtagat gttgtttgac 4020 t at 1111 at t 111 gggggtt 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 II 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 t caat eet aa t geaggt cat egaaagat at tttetttaea 111 ct t eet a gt ct ct 11 gg 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 ggggt111aa tatteet11c åget111agg agt11aaagg aat eetcaat act agt attg 4800 agagcaaaac taagaatttc aaatttggaa ctagaagtca gagggttggg tgaettttae 4860 t ct at at eet 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 egatttatga ct ct egt t ag gt t gt 11 at t 4980 III agaat gc t agt gt t aat 11 aeagaggg gatgtggtac aat t aat t ct agggt gggat 5040 ggettaggtc 11aggtctta 111act agtt tgttact at a at aataggaa 11cttctaat 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 t ct gagat t aggt t gagt egaaet agcc 111 aggeat t t aat 111 cca t get 1 11 1 1 1 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 tgtgtaggtt cagtaatgta tagaattegg atgt11ctaa tagttaat aa agtgggaagt 5880 gtctcaaggc ttttaaattg aggagggtct tttaaatttt accagttaag tataagetta 5940 11 at act t ct 111 ccat t at aggaggt aga 1111 ggagaa act tet gagt t act t ccggg 6000 agagt 111 at at gt ct 111 g agaagt aaaa at agt gat t a 11 aggt t aat cacat geagg 6060 gggttget ag gagettatct taggaataca ggggtaaaaa aeaggt1111 gagtaaacta 6120 gggtacctaa gagaeataag aatccggctc cccaacatcc tcagaaaatc cttgataagc 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 cct 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 agcttttage taataaaaag ggaattgeaa actccctatt cctcacaggg ttågettgtt 6600 t aggt aat 11 at at aaaat g cccaat t gt g gagt gggt aa ageat et t gc et t aaacat a 6660 tgcctacttg at etcagt11 agtttccaag at getteet c cccagtaat a gaagagt11a 6720 1111 at t cca tgatgtgatt at agt 11 gt t t aggcct t at et 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 agct at accct et t 6900 t at et 11 at t at at at at ae gatgaaataa caaattttga 111 gggggt a 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 ettacttgaa agagatgtgg 7080 et 11 aact 11 acct at t aaa agaat get gc agt t gat agt gt et t caaag gat gt at t ae 7140 at gettggge ggtccctage 11aggggtta aaatagaege tgtgccagga egaattaat a 7200 ccct at caat at at agat 11 eggeegggat t gt at 111 gg gcaat gt t ca 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 et act agaac 7440 gaaaaat t at t ggt t act et caagcacgt a aaggacccaa t aaggt gt et 11111 aggga 7500 ttcttcagcc 111 et 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 et c ccgt agt ggg t et aagggt g geaet act aa 7620 t et gaaat gt et at ccct t a gagat aaggt 11 ggat et t g aagaat 11 et tgacttggac 7680 t gt t ageggg gat aagagt a agagt 11 acc eet t at 11 et t agagggt ga aggt et aat t 7740 gt gt gt at t c acatattgga ggagt gegag gagt t get ca agt aat et cc t aegaagt ag 7800 11111act at eet agt at11 tet11aat 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 aggtetageg gagaccaacc gaactccctt tgat11et et gagggggaaa 7980 gagagttagt gtccggattc aatacagagt atgggggcag ggggtttget ttaattttta 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 gaagge 8160 gggt t act t a ccct eggeae eggt at gået gat t aat aag at t at gt t ga aaat et et 1 1 8220 t accaagaat t et act eggg ggagggggaa t aat 111 aat aat aat aaga at 11 aat t gt 8280 caact at aaa aaagt gat t a t gat et t gea accat aaaga tattgggact et at acct ae 8340 t aagt ggat t 11 gat et gga 11 agt gggt t t åget at aag agt cat cat c egt et t gage 8400 t gt et cagcc gggageat 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 et 11 11 cat agt aa t acct gt at t aat t gggggt 111 ggaaat t 8520 gat t agt t cc 111 aat act g ggggcacct g acat åget 11 cccccgct t a aacaat at aa 8580 gat t t t ggt t 111 aat accc tctttgagtt t at t get t at aagggeat t a gt agaaagt g 8640 gt geaggaae t gggt gaaca gt at accccc ccct gt et t c gggagt 1111 cact ccgggg 8700 ettcagtaga ttttgegatt ttttccctcc acttågetgg agtgtettet ttattagggg 8760 et gt aaact t t at t agaact at t acaaat t t aeggt get t gggact 111 a gt ggggcaaa 8820 t gccaat at t cccct gat cc gttttaatca et get gt get 111 act et t g t et 11 accag 8880 t gt t ageggg agcaattact at act t et t a ctgatcgaaa 111 aaat aca act 111111 g 8940 accctagagg tggaggggac cccattttat accagcattt attttgattt ttcgggcacc 9000 et gaagt 11 a t at t et aat t cttccagggt 11 ggat t aat et et cagat t at t acccaag 9060 aaact t gt aa agat gagget 111 ggt t ege 11 ggt at aat 11 at gcaat a gt agcaat t g 9120 gagt at t ggg at 11 at t gt t t gaget cat c acat gt 11 ae agt t gggt t a gået eagaea 9180 etegagegta 1111aeggea 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 tetgtttett tttaeagtgg gaggattaac aggtattgta ttågetaact 9360 et gcct t gga t at gået 11 g catgacactt act at gt agt t gcccact 11 cact at gt te 9420 11agaatagg agcagttttt gccctaat ag cgggattcac tcactgatac ccccttttga 9480 caggt11aag aatgaactta act ataagga aagcgcagtt tatcctaat a 111attggtg 9540 t aaat 11 aac gt 11111 cct 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 et t acct aa 9660 et 11 at 111 c t gt ggt agt a tttgtaggga t aat tt gaga gt et at agt t aggt t aegae 9720 cagt at t age ggt at t t gat aggggaagat ccat egagt t caaacat aeg t gt cct 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 111cct11cg tact aataag taagtaaaag ccctgagat a 9900 gaaaccaat c t gået t aegt egat et t aac t caaat cat g t aagt aegaa aagt egaaea 9960 gacttaaaat aggacct t et geageaet at at t act t aat t caacat ega ggt cacgaat 10020 at et111aaa taagagetet aaaagtatta egegetgtta tccctaaggt acctatgtgt 10080 agcaaaat aa ttggttcaga at aact aagg 111 et 11 at t at at t aagt g ggctatccca 10140 gccaaacatg gtaaaggtet tagggtetta tegt ettegg agagtat at c agegt11gta 10200 et 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 gege 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 caagt t at aa 11 ct cagaat t aact accca aat aat aaga 10500 gaaget 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 cgt gt t catccttgtc 11 at aget t a gt 111 ct ace 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 gttcgcataa ct at cct at c 10860 aagatggccc ctat acttcg ggcaccctat at111aaagg gggtggccca tgtctagcac 10920 11 aaagct ag tgcactttct ct gccact 11 aaaaat aat t t at t aaat aa aagat t aagg 10980 at aat ct acg at aagct t ct cccat agaga agt aaagcat aaagcagat t aagt t act t a 11040 11 gagaat aa t agcct at at aaaat t ct aa 111 acat gca at gaaat t gg act cgcagt a 11100 t at ggat 11 a ct aaat aaaa aaagaagt ag agat t t t t aa gttctgaata agaaagt gcc 11160 agcat t cgcg 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 gget 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 t gaat at 111 agt aat t agt t caaat t gt c cgtcactctc 11 aggagat a 11580 agt egt agea 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 gget 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 agcac at aggat 111 gagt eet aag gagt t aagaa at t at ct aaa 12240 at aat t at 11 gggct aaagc at ct agt at t t acaaaat ac t cat 111 agt t aaact acca 12300 aat aaagt t a gcct aat aag t aaaaaaagt ggagttggat agttcagtag act 11 gaacg 12360 gattgaagag at aagggcag caagccct at acat get t ca cagact at aa ct gt cacaaa 12420 t aaaaat et g atagttgagt aggt t at act gaagaccgaa aaaaaaat gc aaat aggaat 12480 tatggtaget gtaagagett ctaataaaag tagcctgat a agcaagtgee ct11attaaa 12540 taaaaatcac ctagaggtga ataaaggagg gagagetaac aagtataaac tgetaaggtg 12600 aggtaaaaaa aaaatgagta caagagttgt taaaatagtt ctcatagagt ataaaaacaa 12660 t aget gt t ag et t aaaagat aat ct aat aa agat gt t aaa 11111 act 11 aattatggaa 12720 ggt cct ct 11 t aat cccct a at t at 11 aag gtettgeatt agaegt 11 ca ct gt t aat ga 12780 aaagaagagt 11 ct ct aaat attcagttag t at aagaat t acact 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 eg agetcctatc aattaagtag 111aataaaa 12960 accttaaatt gtaaatttaa aattagattt tctcttaatt aaagccttag ggagtactaa 13020 t gaat t ct 11 at 111 ccaga tttgatgeta aeggat t gac aaact gat ac 11 acct t ct t 13080 tagcaagagg 111attact a tttccaaggt attgattage gggtaataca 11aggetcca 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 tat gaga 11 at 1111 aa gggt t ct aag aatgaatttt ct aggget 1 1 13260 11 ccgt acat 1111 act agg agt aggcaca tctgtgtgag at t aggt at a gggct gcct t 13320 tat gattagg cagtcaaata gtttgttgag tatacaggag agagcaaagg 111gcccacc 13380 tagttccaga gggggctcct agaggtttaa ttcctctttt ggttttaatt gaaagaatta 13440 gaaggt t aat ccgacct ct a accct aaggg 11 egget t at aget 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 agaattetat 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 aegt 111 t at at t at t a aget 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 ggt 111 aacc agcaaaat aa t ct aat t at t 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 at agt11ccc tctacaggtt 11getccaat ccaggttaaa 13920 agaaaaaaaa tcccgataaa agctcaaaac ataageattt tegaagggtt aaatgtagea 13980 tgtaeggegg ctct111at c ccccagcaaa aatggaaaaa aaaacaaaat tcttaeagae 14040 aaagetaaag ccactacccc ccccaactta tttgggatag accgcaaaat ageataagca 14100 aacaaaaaat accattcagg ttgaatgtga geaggagtta ctaaaggatt agctggaata 14160 aagtttteag ggtctget aa taatcaggga gagtaagaaa ctaaagetcc catgagacaa 14220 aaaataagac cgaagccaaa ggegtct11a aaggtat agt aagggtcaaa aggaatetta 14280 t aat agt 11 c ct ggaacccc caat ggat 11 gaggaccct g 11 gaat gaag aaaaat t gt a 14340 tgaget at aa ctccaactgt ggacaaaaga ggaatgaggt aatgaagagc aaagaatege 14400 cttaaagtag ctttgtccac cctaaacccc ccccaaagtc aaattacaat ttegtcccca 14460 at accaggga t gacagaaaa t agat 11 gt a at aaccgt ag ct cct cagaa agatatetgg 14520 ccccagggga geaeatagcc caaaaaagct ctggetatga 11aaaattaa aat egt agag 14580 cctacaactc aaaeaggagt aagettat ae gaagaatagt aaaggccccg acccgtatga 14640 at gt at aaac acat aaaaaa t aacct cacg gt gt t get at gaat agct cg aat t cct cac 14700 ccat aat t aa cat cccgcat gat at ggt ct aeggat aaaa aeget gt aga aat ct ct ct t 14760 gaataatgea tagccaaaaa aattccggtc at aatctggg taataagaca tagcccaagt 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 ct t gt aaget acaacat act act t aat agt 15000 at 11 at t aaa ct 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 ggaggaggct tctgtagt aa ccgcggagtt tggt11aat g 15180 agaact ct ct t aaagct aac cgaaact agg tt gatet tea aaat at 1111 taagattggt 15240 II aaget at g cacaagt t gc t aat at 11 at t aaaagat 11 aaat aagt ag aaagt t taga 15300 III aat act a agaagaat t a ct agagaat a aagccagacc aaat at 11 at 11 aaagat 1 1 15360 agat aagt ag ct aegget ag gt ggt gggga caaggt geat aatcaataca agt gagt t gg 15420 gggggggggg tatggggggg gatta 15445 <210> 2 <21 1> 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 cat gcct t ca 11 at aat ct t tt teat agt a at acct gt at t aat t ggggg gt 11 ggaaat 120 t gat tagt te ct 11 aat act gggggcacct gacat åget t t ccct eget t aaacaat at a 180 agattttggt 1111 aat acc ct ct 11 gagt 11 at t ggt t a t aagggeat t agt agaaagt 240 ggtgeaggaa ctgggtgaac agt atacccc cccctgtctt egggagt111 tcactccggg 300 getteagtag at 111 gegat 11111 ccct c cact t åget g gagt gt ct 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 t cccct gat c egt 111 aat c act get gt gc 1111 act ct t gt ct 11 acca 480 gt gt t agegg gagcaat t ae t at act t ct t act gat egaa at 11 aaat ae aact 111111 540 gaccct agag gt ggagggga t cccat 111 a t accagcat t tattttgatt 111 egggeae 600 eet gaagt 11 at at t ct aat tcttccaggg 111 ggat t aa teteteagat t at t acccaa 660 gaaact t gt a aagat gagge 1111 ggt t cg ct 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 aagattattt 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 ettagaatag gageagtttt tcgacctagg ettagg 1056 <210> 3 <21 1> 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 et t caaact a at aaaagt 11 åget cacaat ccgt et gagt aaggt aagga 120 cagccctact etgaagtata agaeagtaaa aacttggccc aggaaaatat agtttccctc 180 t aeaggt 111 get ccaat cc aggt t aaaag aaaaaaaat c ccgat aaaag et caaaacat 240 aageattttc gaggggttaa atgtageagg taeggeggtt ettttatccc ccagcaaaaa 300 tggaaaaaaa aacaaaattc ttacagacaa ågetaaagee actacccccc ccaacttatt 360 t gggat agac cgcaaaat ag cat aagcaaa caaaaaat ae cat t caggt t gaatgtgagc 420 aggagttact aaaggattag etggaataaa gttttcaggg tet get aat a atcagggaga 480 gtaagaaact aaagctccca tgagacaaaa aataagaeeg aagccaaagg egt et11aaa 540 ggt at agt aa gggt caaaag gaat et 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 agct 11 gt ccaccc t aaacccccc 720 ccaaagt caa at t acaat 11 cgtccccaat accagggat g acagaaaat a gat 11 gt aat 780 aaccgt agct 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 ct aagt ggat tttgatctgg at t agt gggt 11 agct at aa gagt cat cat ccgt ett gag 60 ctgtctcagc egggageat a 111 aggagat t cccaggt 11 at aat gt t at t gt aact get 120 cat gcct t ca 11 at aat ct t tt teat agt a at acct gt at t aat t ggggg gt 11 ggaaat 180 t gat tagt te ct 11 aat act gggggcacct gacat agct t t cccccgct t aaacaat at a 240 agattttggt 1111 aat acc ct ct t egagt 11 at t act t a t aagggeat t agt agaaagt 300 ggtgeaggaa ctgggtgaac agt at atccc cccctgtctt egggagt111 tcactccggg 360 getteagtag at 111 gegat 11111 ccct c cact t agct g gagt gt ct 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 ct t gt ct 11 acca 540 gt gt t agegg gagcaat t ae t at act t ct 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 eet gaagt 11 at at t ct aat tcttccaggg 111 gggt t aa teteteagat 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 ggagt at t gg ggt 11 at t gt 11 gaget cat cacatgttta cagt t gggt t agact eagae 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 11 ct gt 11 ct ttttacagtg ggaggat t aa caggt at t gt 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 caaaat aat c t aat t at 11 a t gt 11 gt gt a at gcgggcag 11 at t acgcc t gt caaat ga 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 aacaaaat t c 11 acagacaa åget aaagee act acccccc ct aact t at t t gggat agac 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 ccaact gt gg acaaaagagg aat gaggt aa 660 t gaagagcaa agaat cgcct t aaagt agct 11 gt ccaccc t aaaaccccc t caaagt caa 720 at t acaat 11 cgt ccccaat accagggat g acagaaaat a gatttgtaat aaccgt agct 780 cct cagaaag at at ct ggcc ccaggggagc acat agccca aaaaagct ct ggctatgatt 840 aaaat t aaaa t cgt agagcc t acaact caa acaggagt aa gcttatacga agaat agt aa 900 aggccccgac ccgtatgaat gt at aaacac at aaaaaat a acct cgcggt gt t get at ga 960 atagctegaa 11cctcaccc 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
<21 1 > 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 rmni 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 (25)
[1] 1. An in vitro method for detection of pyrethroid resistance in one or more crustaceans, comprising the steps of a. analyzing the mitochondrial genomic material of the crustacean, b. detecting one or more single nucleotide polymorphisms (SNP) in the mitochondrial DNA of the crustacean, c. determining that one or more of the detected SNP's is associated with pyrethroid resistance of the crustacean.
[2] 2. A method according to claim 1, wherein the crustaceans is one or more copepods.
[3] 3. A method according to claim 2, wherein the copepod belongs to the family Caligidae.
[4] 4. A method according to claim 4, wherein the copepod is selected from the group consisting of Lepeophteirus salmonis, Caligus elongatus, and Caligus rogercresseyi.
[5] 5. A method according to claim 3, comprising the steps of detecting single nucleotide polymorphism (SNP) associated with pyrethroid resistance in the mitochondrial genome 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 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.
[6] 6. A method according to any of the claims 1-4, wherein the crustacean is a sea lice, and further comprising the steps of: a) collecting sea lice from infested fish or water samples; b) isolating mitochondrial genomic material from any life stage of collected sea lice 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 nucleotide C, G, T, G and T (U), or a complementary oligonucleotide thereof, is present in position 8605, 9035, 13957, 14017, and 14065, respectively.
[7] 7. A method according to claim 6, wherein step c) is performed using a primer selected from the group consisting of SEQ ID No. 6-15.
[8] 8. A method according to claim 6, wherein step c) is performed using at least one probe selected from the group consisting of SEQ ID No. 16-20.
[9] 9. A method according to claim 6, wherein step c) comprises nucleic acid amplification.
[10] 10. A method according to claim 9, wherein the nucleic acid amplification is performed using polymerase chain reaction.
[11] 11. A method according to any of claims 6-10, wherein 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.
[12] 12. A method according to claim 11, wherein said detection reagent is an oligonucleotide probe.
[13] 13. A method according to claim 6, wherein step c) is performed , wherein step c) is performed using SNP specific probe hybridization, SNP specific primer extension, SNP specific amplification, sequencing, 5’ nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis.
[14] 14. A method according to any of the above claims, wherein the pyrethroid is selected from the group consisting of deltamethrin, and cis-cypermethrin.
[15] 15. An isolated oligonucleotide sequence comprising small nucleotide polymorphism (SNP) associated with pyrethroid resistance in crustaceans, wherein said isolated oligonucleotide sequence comprises nucleotides that distinguishes crustaceans which are resistant to pyrethroids from non-resistance to pyrethroids, and wherein the SNP are present in the mitochondrial DNA of the crustaceans.
[16] 16. An isolated oligonucleotide sequence according to claim 15, wherein the crustacean is a copepod.
[17] 17. An isolated oligonucleotide sequence according to claim 16, wherein the copepod belongs to the family Caligidae.
[18] 18. An isolated oligonucleotide sequence according to claim 16, wherein the copepod is selected from the group consisting of Lepeophteirus salmonis, Caligus elongatus, and Caligus rogercresseyi.
[19] 19. An isolated oligonucleotide sequence according to any of the claim 15-18, comprising 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. 4 and 5 or a fragment thereof, and complementary sequences of SEQ ID No. 4and 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.
[20] 20. A oligonucleotide probe or oligonucleotide primer comprising a oligonucleotide sequence being homologous to a fragment of an isolated oligonucleotide sequence according to claim 19, 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. 4, and C13957T, A14017G, and C14065T of SEQ ID5, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.
[21] 21. A probe or primer according to claim 20 comprising at least 8 contiguous nucleotides.
[22] 22. A probe according to claim 20, 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.
[23] 23. A oligonucleotide primer, 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.
[24] 24. A kit for detection of pyrethroid resistance in crustaceans, such as copepods, e.g. in sea lice (Lepeoptheirus salmonis), which comprises at least one oligonucleotide sequence or probe or primer according to any of the claims 15-23.
[25] 25. The use of an isolated oligonucleotide sequence comprising at least 8 contiguous nucleotides of the sequence selected from the group consisting of SEQ ID No. 3 or SEQ ID No. 4, and wherein said sequence comprises at least one of the nucleotide C, G, T, G and T (U), or a complementary oligonucleotide thereof, is present in position 8605, 9035, 13957, 14017, and 14065, respectively, and wherein the numbering of said positions is in accordance with the sequence depicted in SEQ ID No. 1.

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CL2016000299A1|2016-11-04|

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EP3030674B1|2021-04-14|

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WO2015018861A1|2015-02-12|

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|

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AU6928700A|1999-08-25|2001-03-19|Clarity Biosciences, Inc.|Identifying organisms by detecting intronic nucleic acid or encoded proteins|

---

US8178503B2|2006-03-03|2012-05-15|International Business Machines Corporation|Ribonucleic acid interference molecules and binding sites derived by analyzing intergenic and intronic regions of genomes|

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US7901882B2|2006-03-31|2011-03-08|Affymetrix, Inc.|Analysis of methylation using nucleic acid arrays|WO2021136724A1|2020-01-02|2021-07-08|Patogen As|Method for detecting hydrogen peroxide resistance in crustaceans|

法律状态:
2019-05-22| PME| Patent granted|Effective date: 20190522 |

优先权:

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申请号 | 申请日 | 专利标题

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NO20131075|2013-08-06|

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NO20131075|2013-08-06|

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EP2014066895|2014-08-06|

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PCT/EP2014/066895|WO2015018861A1|2013-08-06|2014-08-06|Method for detection of pyrethroid resistance in crustaceans and oligonucleotide sequences useful in detection of pyrethroid resistance.|

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