EP4117427A1 - Seawater readiness markers in salmonid fish - Google Patents
Seawater readiness markers in salmonid fishInfo
- Publication number
- EP4117427A1 EP4117427A1 EP21767991.9A EP21767991A EP4117427A1 EP 4117427 A1 EP4117427 A1 EP 4117427A1 EP 21767991 A EP21767991 A EP 21767991A EP 4117427 A1 EP4117427 A1 EP 4117427A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- fish
- genes
- group
- expression
- salmonid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Definitions
- the present invention relates to a method for determining seawater (SW) readiness of salmonid fish.
- the present invention relates to genetic markers useful in determining whether juvenile fish which have been raised from hatching in freshwater (parr) has transformed into a form that will perform well when transferred to SW.
- Wild salmonid fish start life in fresh water streams and rivers, and after having developed through the stages of small fry to the juvenile parr, the fish develops further by undergoing structural and functional transformations from parr to smolt prior to migration from freshwater to seawater.
- the transformation process is called smoltification and consists of a number of complex developmental changes in the biochemistry, physiology, morphology and behavior of the juvenile salmon, critical amongst which is the acquisition of the ability to efficiently maintain water and ionic balance upon entering the sea (cf. Hoar WS (1988), The Physiology of Smolting Salmonids, Fish Physiology, 11, Ed. Hoar and Randal, Cambridge: Academic Press, pp. 275-343, and McCormick et al. (2013), J Exp Biol, 216(7), pp 1142-1151).
- seawater is defined as water with an increased salinity content, achieved naturally e.g. when fish is transferred to seawater enclosures, or by addition of salts or seawater to freshwater.
- RAS Recirculating Aquaculture Systems
- smoltification can be artificially achieved e.g. by exposing juvenile salmon exceeding a minimum size threshold to short photoperiod (SP) for several weeks mimicking the number of hours daylight in the wintertime and thereafter exposing the fish for continuous light (LL).
- SP short photoperiod
- LL continuous light
- the duration of exposure to SP should be at least six weeks long for LL to induce smoltification (cf. Duncan NJ and Bromage N. (1998), Aquaculture, 68(l-4):369-86).
- the underlying causes of this photoperiodic history-dependence remain unknown and untangling the role of SP exposure in smolt development is of considerable interest, since growth rates and hence aquaculture production are slowed during periods of SP exposure.
- One way of reducing the loss is to ensure that fish that are transferred from freshwater tanks to SW are sufficiently matured, more optimized smolts suited for life in SW.
- the present inventors have solved this problem by providing methods for determining seawater readiness identifying genetic markers found to be linked to the photoperiod-dependent developmental transition and performance in SW.
- the provided method can be used to clarify if salmonid fish have matured sufficiently to perform well following transfer to SW.
- the genetic markers used in the method of the present application also have in common that they are all shown to be downregulated in one of the seven major cell types found in gill in salmonid fish, i.e. non-differentiated progenitor cells (NDC). It is noticed that previous suggested markers for smoltification are all markers found in chloride cells of the gills, which is contrary to the markers used according to the present invention.
- NDC non-differentiated progenitor cells
- a method for determining SW readiness of one or more salmonid fish comprising determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, wherein the tested fish is considered saltwater ready when at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is downregulated.
- the in vitro method of the present invention also provides for improving SW performance of salmonid fish after transfer to SW.
- an in vitro method comprising the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression level of one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 in the biological sample of a).
- an in vitro method wherein expression level of at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is determined by measuring the abundance of a RNA transcript of at least one of the said genes.
- an in vitro method comprising the steps of a) determining the expression levels of one or more genes in a biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493; b) normalising said determined expression level against an expression level of a salmonid housekeeping gene.
- the genetic markers used according to the present invention do not show enriched expression in chloride cells relative to pavement cells, red blood cells, vascular cells, pillar cells, mitochondrion rich cells (MRCs, also known as chloride cells), T cells, endothelial cells, goblet cells, neuroendothelial cells, myeloid cells, lymphatic endothelial cells, dendritic cells, accessory cells, fibrocytes, and vascular cells present in salmonid gill tissue.
- MRCs also known as chloride cells
- the expression level of at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is normalised against gene encoding elongation factor 1A.
- the expression level may be determined in a biological sample from one or more salmonid fish, wherein the biological sample is a sample of integumental tissue.
- an in vitro method comprising the steps of: a) providing a sample of integumental tissue from one or more juvenile salmonid fish of a group of fish to be smoltified prior to exposing said fish for smoltification conditions; b) analysing the sample of step a) to provide a base line expression level by determining the expression level of one or more genes identified by its NCBI Gene ID number selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493; c) exposing the remaining group of salmonid fish for smoltification conditions; d) providing a sample of integumental tissue from one or more salmonid fish of the group of fish of step c); e) determining the expression level in the sample of step d) of one or more of the same genes analysed in in step b); f
- the salmonid fish to be tested in the method according to the present invention is selected from a fish to be tested belonging to the genera Salmo, Oncorhynchus, or Salvelinus.
- the salmonid fish to be tested is selected from the group consisting of Atlantic salmon, Coho salmon, Chinook salmon and Sockeye salmon.
- the fish to be testes is Atlantic salmon.
- the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue, skin tissue or fin tissue.
- the expression level is determined in a sample of gill tissue.
- the present invention relates to the use of one or more genes identified by its NCBI Gene ID number and selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, for determining SW readiness of salmonid fish.
- the present invention furthermore relates to the use of one or more of the genes selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, wherein said one or more genes used as markers for saltwater readiness do not show enriched expression in chloride cells relative to pavement cells, red blood cells, vascular cells, pillar cells, mitochondrion rich cells (MRCs), T cells, endothelial cells, goblet cells, neuroendothelial cells, myeloid cells, lymphatic endothelial cells, dendritic cells, accessory cells, fibrocytes, and vascular cells present in salmonid gill tissue.
- MRCs mitochondrion rich cells
- the present invention also provides for the use of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 to determine SW readiness of salmonid fish and to improve SW performance of salmonid fish after transfer to SW.
- kits for use in a method to the present invention, wherein the kit comprises reagents for the measuring of protein expression levels and/or mRNA expression levels of at least one, two, three, four, five, six, or seven genes from the set of genes selected from the group comprising LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493.
- SP refers to short photoperiod regimen applied in the experiment disclosed herein.
- WSP refers to weeks short photoperiod, i.e. 2WSP, 4WSP and 8WSP refers to fish being kept at a short photoperiod regimen for 2, 4 and 8 weeks respectively.
- LL refers to a continuous light regimen applied in the experiment disclosed herein.
- SP-LL refers to a regimen comprising a short photoperiod followed by a period of continuous light applied in the experiment disclosed herein.
- SW refers to fresh water.
- SW refers to seawater defined as water with an increased salinity content, achieved naturally by transfer to seawater enclosures, or by addition of salts to freshwaters.
- saltwater and “seawater” are to be understood to be synonyms in this respect.
- SW is to be understood to refer to “saltwater” and “seawater”.
- Figure 1 shows experimental design to screen for photoperiod-dependent developmental markers, i.e. to identify genes whose expression in the gill was increased according to a photoperiod-dependent developmental transition.
- Figure 3 shows expression ratios for photoperiod-dependent developmental markers. Data are means with 95% confidence intervals shown as error bars for the log(base 10) of the ratio between the indicated numerator (2) genes (LOC106584756, LOC106605916, LOC106562680, or LOC106562681) and the indicated set of denominator (1) genes (LOC106567921, LOC106565346,
- Figure 4 shows expression ratios for photoperiod-dependent developmental markers as for Fig 3 but (2) genes are LOC106591222, LOC106602268, LOC106561379, or LOC106562250.
- Figure 5 shows expression ratios for photoperiod-dependent developmental markers as for Fig 3 but (2) genes are LOC106592537, LOC106608496, LOC106608493 or LOC106609366.
- Figure 6 shows expression ratios for combinations of LL-induced and LL- suppressed NKA alphal subunit genes.
- Data are means with 95% confidence intervals shown as error bars for the log(base 10) of the ratio between the indicated numerator (b) genes (LOC106596208, LOC106602157, or LOC106610479) and the indicated set of denominator (a) genes.
- Black bars show log ratios for indicated numerator / denominator pairings in fish transferred to LL after 2 weeks of short photoperiod, which subsequently performed poorly in seawater.
- Grey bars show log ratios for indicated numerator / denominator combinations in fish transferred to LL after 8 weeks of short photoperiod, which subsequently performed well in seawater.
- Figure 7 includes violin plots showing the expression level of gene markers used in the present method by the population of cells in each cluster at each sample point.
- the cluster key numbers refers to the following cell types: pavement (0), red blood cells (1), vascular cells 1 (2), pillar cells (3), MRCs (FW) (4), T cells (5), endothelial cells 1 (6), goblet cells (7), non-differentiated cells (NDC) (8), vascular cells 2 (9), vascular cells 3 (10), neuroendothelial cells (11), myeloid cells (12), lymphatic endothelial cells (13), MRCs (SW) (14), endothelial cells 2 (15), dendritic cells (16), accessory cells (17), fibrocytes (18), and vascular cells 4 (19).
- the genes shown in the violin plots a) - g) are downregulated in saltwater ready salmonid fish.
- the CIGEN identifier number, Protein ID, NCBI Gene ID Number and gene name is given above each violin plot.
- Violin plot a) - g) refers to the genes having the NCBI Gene ID No.
- Figure 8 Expression of NKA alpha lb subunit (NKAalb(ii)) and FKBP5 RNA in gill filament biopsy samples from Experiment 2. Expression levels (Y axis) are results from qPCR normalised against EFla (housekeeping gene expression). For each smoltification protocol (Photoperiod, LL+salt) expression levels in individual fish are shown, expressed relative to the mean level of expression at Tl.
- Figure 9 Expression of one downregulated (zymogen granule protein 16) and one upregulated (S100A) NDC marker in gill filament biopsy samples from Experiment 2.
- Expression levels (Y axis) are results from qPCR normalised against EFla (housekeeping gene expression). For each smoltification protocol (Photoperiod, LL+salt) expression levels in individual fish are shown, expressed relative to the mean level of expression at TL
- FIG 10. Specific growth rate (SGR) over a 9-week period of growth in SW in fish raised under the two smoltification protocols in Experiment 2 (i.e. from timepoint T3 to timepoint T4). Each point represents growth in one individual fish.
- Figure 11 Schematic illustration of the experimental design of experiment 2. Tl : 25.04.2019, T2: 06.06.2019, T3: 06.06.2019, T4: 25.09.2019.
- Smoltification is a physiological, morphological and behavioral transformation of salmonid fish such as Atlantic salmon ( Salmo salar) juveniles, from a FW parr to a SW smolt.
- This parr-smolt transformation encompasses several changes adaptive for life in SW, of which the development of gill ion secreting capacity and SW osmotic tolerance is the most studied.
- parr are gated into the smoltification pathway when their body weight/energy status exceeds a certain threshold in the autumn. Thereafter they remain in a “resting” state during the dark winter until smoltification is triggered by increasing day length (photoperiod) in the spring.
- smoltification is artificially initiated by light manipulation to stimulate and synchronize smoltification.
- fish are reared in constant light (referred to herein as LL) until smoltification procedures are initiated, that is by exposing the juvenile fish to a daily light - dark cycle with light exposure for 12 hours or less per day to mimic natural conditions where salmonid fish smoltify after a period of exposure to winter photoperiods.
- LL constant light
- SW readiness in research and commercial fish farming is by assessing the gill Na + K + -ATPase (NKA) activity prior to SW transfer and/or changes in plasma chloride (Cl) levels after a short term (24 hr) SW challenge.
- NKA gill Na + K + -ATPase
- Cl plasma chloride
- SW ready smolt would have increased NKA activity and therefore greater ability to regulate chloride levels upon SW transfer.
- determination of the expression level of two subunits of the NKA protein complex have been used to determining SW readiness (McCormick et al. (2009), J. Exp. Biol., 212, pp 3994-4001).
- genes with significantly higher expression in 8WSP fish compared to 2WSP fish were identified.
- the expression level identified for the genes of group (1) and (2) was furthermore ranked based on expression in the two groups.
- Genes from group (1) having the lowest ratio for 8WSP-LL:2WSP- LL expression were considered strong candidates for markers negatively correlated with SW readiness.
- genes from group (2) having the highest value for 8WSP-LL:2WSP-LL expression were considered strong candidates for markers positively correlated with SW readiness.
- NDC non-differentiated progenitor cells
- nucleotide sequence of the identified genetic markers of the present invention are publicly available and identified herein by reference to Gene ID Number of the GenBank of the National Center for Biotechnology Information (NCBI) (https://www.ncbi. nim.nih .gov/gene).
- the sequence of the identified genetic markers are also available in the integrated database of Salmonid Genomic Resources, Salmobase 2.0 (https vVsalmobase.org/) and identified by its CIGENE identifier number (Centre of Integrative Genetics, University of Life Science, As, Norway).
- Table 1 List of photoperiodic-history dependent smolt genetic markers of the present invention, being down-regulated in NDC cells with induction of SW- readiness.
- the genes are identified by CIGENE identifier number, as well as Protein ID number and NCBI Gene ID Number given in GenBank of the National Center for Biotechnology Information (NCBI)
- a method according to the present invention for determining saltwater readiness of one or more salmonid fish, involving analysing the expression level of genes selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
- LOC106608496 and LOC106608493.
- a fish in which the expression of a gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
- LOCI 06608496, and LOCI 06608493 is downregulated is determined as ready to be transferred to saltwater.
- the expression level of the above marker genes is measured using multiplex qPCR, such as RT qPCR, or RNAseq.
- determination of saltwater readiness may be performed by analysing the expression level of at least one of the above genes.
- expression level of at least one of the LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is determined.
- the expression level of at least two of the genes selecting from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
- LOCI 06608496, and LOCI 06608493 are determined, such as at least three of the mentioned group of genes, such as at least four of the mentioned group of genes, such as at least five of the mentioned group of genes, such as at least six of the mentioned group of genes, such as all of the genes of the mentioned group.
- SW ready “sea water ready” or “saltwater readiness” as used herein is to be understood to mean that a salmonid fish is ready for transfer to a container or sea-cage in which the water salinity is in the range typically used by the aquaculture industry for optimizing growth of salmon following the juvenile freshwater phase (15 - 40 ppt). It is furthermore to be understood that a fish being “saltwater ready” has a higher probability of surviving transfer to saltwater, such as SW. Also, it is to be understood that a fish being SW ready has a higher probability of growing faster and be less susceptible to infectious diseases upon transfer to SW. It is furthermore to be understood that fish that are “saltwater ready” has a good SW tolerance, and that the fish has developed a hypo-osmoregulatory capacity.
- the expression “upregulated” should be understood to mean that the expression of the gene in question is increased in SW ready salmonid fish compared with the expression of said level of the gene in question in juvenile salmonid fish.
- the level of expression is increased in a SW ready salmonid fish compared with a juvenile salmonid fish (parr), e.g. when comparing the level of expression of fish prior to and after being exposed to a smoltification regimen commonly used in the salmon aquaculture industry.
- the expression “downregulated” should be understood to mean the expression of the gene in question is decreased in SW ready salmonid fish compared with the expression of said level of the gene in question in juvenile salmonid fish.
- the level of expression is decreased in a SW ready salmonid fish compared with a juvenile salmonid fish (parr), e.g. when comparing the level of expression of fish prior to and after being exposed to smoltification regimen commonly used in the salmon aquaculture industry.
- smoltification conditions or “smoltification regimen” as used herein is to be understood to mean any method or procedure useful for initiating transition of salmonid fish from the juvenile stage (parr) to a SW ready fish (‘smolt’ stage).
- juvenile salmonid fish is as mentioned above exposed to photoperiods with reduced hourly day light followed by a period of constant daylight.
- the skilled person will acknowledge that exposing the salmonid fish to photomanipulation in aquaculture industry provides for mimicking the light changes that wild salmon is exposed to in nature, often also referred to as winter signal.
- the markers identified herein is the result of thorough analysing of the changes of expressions of numerous test fish exposed to a specific photoperiod and which is also shown to perform well after transfer to seawater.
- the genetic markers disclosed herein are identified in fish exposed to 8 weeks of short photoperiod (8WSP), followed by 8 weeks of continuous light (LL) compared with fish exposed to 2 weeks of short photoperiod followed (2WSP) by 8 weeks of continuous light (LL). Furthermore, the genetic markers disclosed in the present invention correlated with fish that performs well in SW, which thus renders the markers reliable as makers for saltwater readiness. Although the markers are identified in fish exposed to the above regimen (cf. also experiment l),also fish that are exposed to a different smoltification regimen or smoltification conditions can be tested for saltwater readiness using the genetic markers identified herein. Reference is made to experiment 2, in which examples of the novel markers are shown to be responsive both to photoperiod- and salt-diet based smoltification regimens.
- Various methods can be used to determine whether a specific gene is upregulated or downregulated in salmonid fish. Measuring the expression level of the above genes (table 1) may for example be performed by well-known mRNA quantification methods or by protein quantification methods. For example, the expression level can be quantified by measuring mRNA or protein levels directly or indirectly from cell lysates made from the biological sample provided from the fish to be tested.
- the level of expression of a gene can be determined measuring non-coding RNA, i.e. miRNA, tRNA, rRNA, snoRNA, or piRNA).
- Suitable mRNA quantification methods include, without limitation to hybridization-based assays, such as microarray analysis, or methods based on real time quantitative reverse transcription PCR (real time qRT PCR).
- Other methods that can be used to determine expression level of one or more genes includes, but are not limited to methods based on hybridisation of a targeted DNA or RNA (nucleic acid based) probe to RNA transcripts of the above genes (e.g. in situ hybridisation, northern blotting, RNase protection assay, reverse transcription-PCR approaches, microarrays, solution hybridisation technologies, e.g. the multiplexed assay for gene expression analysis, nCounter® provided by NonoString Technologies (h ttp s : //www .
- the MinlON technology provided by Oxford Nanopore Technologies (https://nanoporet.ech.com/products/minion)).
- Various methods for measuring expression of marker genes are provided by Fluidigm®
- oligonucleotide sequence should be capable of hybridize to a nucleic acid sequence with a complementary sequence, such as e.g. genomic material, extracted from biological sample provided according to the present method.
- a complementary sequence such as e.g. genomic material
- the genomic material may be e.g. RNA or DNA, e.g. extraction of RNA from samples of gill fragments as described in example 1 below.
- a “probe” or “primer” useful in determining the expression level of the SW readiness biomarkers according to the present method is generally one that contains at least 8 nucleotides and which is capable of hybridizing a nucleic acid with a complementary sequence, and is separated from most other nucleic acids present in the natural source of the nucleic acid, and is thus substantially free of other cellular material.
- an oligonucleotide probe can be a fragment of DNA or RNA of variable length used in a method according to the present invention in order to hybridize to the target sequence, e.g. single-stranded DNA or RNA.
- the oligonucleotide probe may furthermore be labeled with a molecular marker to easily visualize that hybridization have been achieved.
- Molecular markers commonly known to the skilled person may be used, e.g. a radiolabel, and more preferably, a luminescent molecule or a fluorescent molecule enabling the visualisation of the binding of the probe(s) to a target sequence.
- An oligonucleotide probe can hybridize to another nucleic acid molecule, such as the single strand of DNA or RNA originating from a biological sample from one or more salmonid fish to be analysed, under appropriate conditions of temperature and solution ionic strength, cf. e.g. Sambrook et ah, 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.
- An oligonucleotide probe typically comprises a nucleotide sequence which under stringent conditions hybridize to at least 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule, e.g. single-stranded DNA or RNA isolated from a biological sample to be analysed.
- oligonucleotide primers may be used in methods according to the present invention for determination of the expression level of a SW readiness biomarker gene disclosed herein, i.e. belonging to the group (1) genes and group (2) genes listed above.
- An oligonucleotide primer typically comprises a nucleotide sequence at least 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50 (or any other number in- between) or more consecutive nucleotides.
- the 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).
- oligonucleotide primers can be labeled with a molecular marker to enable visualization of the results obtained. Various molecular markers or labels are available.
- An oligonucleotide primer typically comprises the appropriate number of nucleotides allowing that said primer align with the target sequence to be analyzed.
- Probes and primers useful in determining expression level of the SW readiness biomarkers disclosed herein can be constructed based the common general knowledge of the skilled person and based on the known sequence of said biomarker genes, cf. reference to Gene ID number thereof in table 1.
- expression level of a gene may be determined at the protein level, e.g. by suitable protein quantification methods known in the prior art, including immune assays such as ELISA, Western blot etc. utilizing antibodies that bind to proteins encoded by the genes of interest and as listed in table 1 above. Antibodies that recognize and bind to proteins encoded by the genes listed in table 1 can be manufactured using methods well known to the skilled person. Also, several applicable immune assay methods are available, for example immune assay can be developed for determining expression levels of the genes of interest using ELISA development kits, such as the kit available from MABTECH (https:// www. inabtech.com/knowledge-cemer/product-guide/elisa-ldts/elisa- deveiopment-kits), LSBio
- the expression level of one or more of the genetic markers of the present invention can be determined in one or more fish prior to and after exposing a group of fish to a smoltification regimen, wherein the findings that at least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
- LOCI 06608496, and LOCI 06608493 is downregulated in the fish after being exposed to a smoltification regimen determines that the fish is ready for being transferred to saltwater.
- the expression level of the at least one gene is determined in a selection of a group of juvenile salmonid fish to be exposed to smoltification regimen in order to provide a baseline level of expression of the at least one of the above listed genes. After exposing the salmonid fish to smoltification regimen, the expression level of the same genes is analysed in a biological sample of subsample of the group of fish. The group of fish is furthermore considered SW ready in the case that at least one of the tested genes of the above genes is downregulated.
- the term baseline expression level according to this embodiment is thus to be understood to be the expression level of the above genes in juvenile salmonid fish (parr) prior to being exposed to a smoltification regimen.
- Another method that may be used to determine whether a salmonid fish is ready for transfer to seawater is to provide normalised expression level data with respect to one or more reference or ‘housekeeping’ genes that are known not to change when a juvenile fish (parr) develops to smolt.
- housekeeping genes encoding a protein that are continuously expressed to carry out vital cellular functions irrespective of the stage of the salmonid fish can be used as a control to normalise the measured expression level.
- the skilled person therefore acknowledges that the expression of a housekeeping gene to be used according to the present invention is be independent of the physiological processes involved in smoltification.
- the expression level of the housekeeping genes is thus the same or similar among the fish tested and irrespective of whether the fish is a juvenile parr or a smolt.
- the normalisation should ensure accurate comparison of expression of a marker gene of interest between different test samples.
- housekeeping genes include but are not limited to those described in Olsvik et al, 2005, BMC Mol. Biol., “Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic Salmon”, vol 6, pp 21 (doi 10.1186/1471.2199-6-21).
- An applicable housekeeping gene to be used in determining the expression level of the gene markers disclosed in the present application is e.g. the Arctic salmonid gene encoding elongation factor 1A, such as e.g. EF I AA and EF I AB.
- LOCI 06608496, and LOCI 06608493 and b) one or more housekeeping genes in a sample from the fish to be analysed is measured, and wherein the measurement of the amount of RNA transcribed from b) are used to normalise the expression the genes of a).
- the housekeeping gene refers to a gene whose expression is used to calibrate or normalise the measured expression of the gene of interest (e.g. marker genes disclosed herein).
- the housekeeping gene or “normalising gene” refers to a gene whose expression is used to calibrate or normalise the measured expression of the gene of interest (e.g. marker genes disclosed herein).
- One or more housekeeping genes can be used.
- a combined normalising gene set can be provided.
- the overall expression of the one or more housekeeping genes can be represented by a “normalised value” that are determined by well-known methods, and which can form the basis for determining whether the expression level of one or more of the marker genes disclosed herein is considered downregulated (or upregulated) in a sample of a fish that are subject to method of the present invention. That is, if the normalisation show that one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is downregulated, the group of fish from which one or more test fish is taken is considered to be ready for transfer to saltwater.
- a group of salmonid fish is considered ready for transfer to seawater if 5-25 of test fish selected from said group of salmonid fish show downregulated expression of one or more of the genes selected from the group LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493.
- the expression level of the genetic markers identified herein is determined in a biological sample taken from a selection of fish analysed for SW readiness.
- the biological sample is according to one embodiment of the invention gill tissue, in particular primary a sample originating from gill filaments.
- the gills have a pivotal role in the energy demanding regulation of water and ionic fluxes and undergo extensive differentiation during the smoltification process in order to enable the migration to a salty environment. A number of physiological changes occur in this process, such as inter alia a shift from an ion-absorbing freshwater type to an ion- secreting salt water type of the gill complement of mitochondria rich cells (Evans et al. (2005), Physiol.
- the size and number of gill samples taken per fish to be tested may vary. The skilled person will acknowledge that the size of the sample should be adapted to the amount of storage solution used.
- the size of the gill sample is about 2- 15 mm 3 , such as about 5-10 mm 3 , such as about 2-5 mm 3 , such as about 2-3 mm 3 .
- Gill samples is furthermore commonly taken from the second gill arch and stored in a suitable preservative solution, such as RNAlater, at refrigerator temperature (e.g. about 4 °C). If the gill samples are not to be analysed within a few days, the sample may be stored in a freezer. Alternatively, gill samples may be rapidly frozen, for example but not limited to, placing on dry ice, on a cooled metal block, or immersion in liquid nitrogen.
- smoltification characteristics can be determined at the same time, such as parr marks, silver coloring, and/or form of the fin edges. Also, the weight and length of the fish is commonly measured when taking samples for determination of smolt status. Also, injuries, or signs of illness of the fish from which the samples are taken should be registered.
- the determination may involve testing of from 5-25 fish of the population, such as from 5-20 fish, or from 5-10 fish.
- the skilled person will acknowledge that the number of fish needed may depend upon e.g. the size of the population, the point within the smolt window at which the sampling of test fish are taken, cf. that the smolt window may vary from location to location, the strain of salmonid fish and the genotype thereof.
- the number of fish to be tested may also depend upon the process and conditions used to induce smoltification type of smoltification regimen used.
- gill samples are used for determining level of expression of genetic markers disclosed herein, also other tissues can be used in the method according to the present invention.
- the experiments have been performed using Atlantic salmon ( Salmo salar), it is to be understood that the genetic markers may as well be used to determine SW readiness in other salmonid fish, such as members of the genera Salmo (e.g. Salmo trutta ), Oncorhynchus (e.g. Oncorhynchus mykiss, i.e. rainbow trout), Salvelinus (e.g. Salvelinus salvelinus, i.e. arctic charr).
- Salmo e.g. Salmo trutta
- Oncorhynchus e.g. Oncorhynchus mykiss, i.e. rainbow trout
- Salvelinus e.g. Salvelinus salvelinus, i.e. arctic charr
- kits comprising means for carrying out the method of the invention.
- the kit according to the invention typically comprises reagents for measuring protein expression levels and/or mRNA expression level of at least one, two, three, four, five, six, or seven genes from the set of genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
- LOCI 06608496 and LOCI 06608493.
- Reagents that can be comprised in a kit according to the present invention is e.g. primers or probes suitable for measuring expression levels of the mentioned genetic markers.
- means for analysing expression level using PCR methods can be included in said kit, such as enzymes, buffers, dNTPs, etc.
- Atlantic salmon ( Salmo salar , Linnaeus, 1758, of the AquaGen commercial stain, Trondheim, Norway) were used for both experiments, and were raised from hatching in FW, on continuous light (LL, > 200 lux at water surface) at 4 - 10°C. Fish were fed continuously with pelleted salmon feed (Skretting, Stavanger, Norway).
- the juvenile salmon were distributed among eight 300 L circular tanks with FW at 7 °C and LL and left to acclimate for one week.
- the total number of fish in each tank ranged from 150 to 200, depending on the number of fish to be sampled during the experiment in each tank and the need to avoid density-dependent social stress effects.
- RNAlater® Sigma-Aldrich, St. Louis, Missouri, USA
- RNA samples were frozen at -80 °C until further processing.
- Sequencing libraries were prepared using the TruSeq Stranded mRNA HS kit (Illumina, San Diego, California). Library mean length was determined by a 2100 Bioanalyzer using the DNA 1000 Kit (Agilent Technologies, Santa Clara,
- Cutadapt (28) was used to remove sequencing adapters, trim low quality bases, and remove short sequencing reads using the parameters -q 20 -O 8 -minimum-length 40 (version 1.8.1). Quality control of the reads were performed with FastQC software (29). Mapping of reads to reference genome was done using STAR software (ver. 2.4.2a) (30). HTSEQ-count software (version 0.6. lpl) (31) was used to generate read count for annotated genes.
- the inventors used empirical Bayes quasi-likelihood F-tests to contrast gene expression in 2WSP and 8WSP fish which had been returned to LL for 4 weeks.
- the output was filtered using a false discovery rate (FDR) ⁇ 0.01 and a log2-fold change >
- FDR false discovery rate
- Table 3 Primer sequences for target genes.
- RNA samples from were ethanol-precipitated and DNAse-treated according to the manufacturer’s protocol (TURBO DNA-free Kit, Thermo Fisher).
- cDNA was constructed using the High-Capacity RNA-to-cDNA kit (Thermo Fisher, Waltham, Massachusetts, USA), following the recommended protocol.
- Primers (table 3) were designed to target all splice variants of the target genes, while not picking up ohnologue and paralogue duplicates of the targeted genes.
- Primer3 (34, 35) and ApE software (v2.0.51) were used for designing primers, and primers were checked against both the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) database using BLAST (36) and the SalmoBase database (37) for non-target hits.
- Primer specificity was confirmed by melt-curve analysis, and amplicon size verified by agarose gel electrophoresis.
- a subset of samples was pooled and diluted and analyzed by qPCR. Amplification efficiencies fell between 90% and
- Real-time quantitative PCR analysis was performed using a BioRad CFX Connect Real-Time instrument (Hercules, California, USA), and SYBR Green detection. Reactions were carried out on 96-well plates, with 20 ng RNA cDNA equivalent, 250 nM forward and reverse primer, and lx Sso Advanced Universal SYBR Green Supermix (BioRad, Hercules, California, USA), in a total volume of 20 pL. After initial heating (95°C, 30 sec.), amplification was carried out under the following conditions: 95 °C for 10 sec., and primer-specific annealing temperature for 1 min. over 40 cycles. A melting curve analysis was completed at the end of each run (0.5 °C intervals at 3 sec., from 65 °C to 95 °C).
- Condition factor was calculated as where W is wet body mass (g), and L is fork length (cm).
- SGR Specific growth rate
- Wt and WT are mass (g) at the beginning and end of the period of extended SW exposure, respectively.
- FCR feed conversion ratio
- gill NKA activity is considered a good indicator of osmoregulatory capacity and smolt status we also examined how this trait was influenced by photoperiodic history figure 6). In contrast to plasma osmolality and chloride levels, gill NKA activity did not change significantly under chronic exposure to SP (SPC group). However, the development of NKA activity following return of fish to LL was highly dependent on photoperiodic history (p ⁇ 0.001, for time x photoperiod regime, by two-way ANOVA, supplemental material S3).
- genes whose expression following exposure to LL for 4 weeks was significantly different depending on duration of prior exposure to SP i.e. genes (1) with significantly higher expression in 8WSP fish compared to 2WSP fish and (2) genes with significantly higher expression in 2WSP fish than in 8WSP fish. Significance was defined as a false
- Genes in both group (1) and (2) categories above were ranked based on the ratio between expression in the two groups. Genes from the (1) group with the lowest ratio for 8WSP-LL:2WSP-LL expression were considered strong candidates for markers negatively correlated with smolt readiness. Genes from the (2) group with
- Figures 3 to 5 show relative expression of all combinations of upregulated (2) genes expressed as a ration for down regulated (1) genes. Across this dataset as a whole it can be seen that the difference in this expression ratio between 8WSP fish which grew well in sea water and 2WSP fish which did not grow in SW is typically more than 10-fold. This indicates that these ratios are sensitive indicators for the transition to a SW ready state. Reference is also made to table 2. A calculated ratio of expression level of the relevant pair of genes in a tested salmonid fish representing a group of fish that have been exposed to a smoltification regimen is found to be ready for transfer to SW if the calculated ratio is below the thresholds indicated in Table 2.
- Figure 6 shows the corresponding expression ratios for combinations of up- regulated and down-regulated NKA-subunit genes. Consistent with the NKAalb profiling in Figure 2, this shows that differences in this expression ratio are much lower, providing a much less sensitive predictor of subsequent performance.
- the 20 cell types are shown on the x axis of the violin plots, the y axis reports a symmetrical histogram for the expression of a given gene within its cell population (scaled and loglp normalised, i.e. natural log of l+[expression level], where the 1+ is used in order not to get a negative value for an expression that is between 1 and 0).
- the breadth of each violin plot reports the number of cells that express the chosen gene at a particular expression level - the wider the plot, the higher the proportion of cells that express the chosen gene at that level.
- the identified marker genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, and as used according to the present invention have in common that all are shown to be downregulated in NDC cells after being exposed to smoltification regimen described above.
- the experiment was carried out at the Aquaculture Research Station in Tromso (ARST), Norway.
- the fish were Atlantic salmon of the AquaGen strain QTL- innOva SHIELD (AquaGen, Trondheim, Norway), obtained as fertilized eggs and hatched in February 2018 at ARST. After hatching, fish were reared at 4 °C, in continuous darkness. From start-feeding (22.05.2018) the fry was kept at 10-12 °C until August, then, temperature was decreased gradually to 4 °C until start of the experiment. From start-feeding to the start of experiment fish were kept under continuous light. Fish were fed continuously and in excess with commercial extruded salmon feed using automatic feeders (Skretting, Stavanger, Norway).
- NKA alpha 1 subunit (NKAlb(ii)) expression have been used for determination of smoltification in the prior art.
- prolyl isomerase 5 (FKBP5) have been suggested as marker for smoltification in Pacific salmonids (Houde et ah, supra).
- FKBP5 prolyl isomerase 5
- expression level of NKAlb(ii), FKBP5, LOC106605916 zymogen granule membrane protein 16 like protein and LOC106570104 protein SlOO-Al-like were determined in fish exposed to the experimental design of experiment 2. The expression level was measured by qPCR and normalised against EFla (housekeeping gene).
- NKAlb(ii) and FKBP5 is not as reliable as smoltification markers compared with the markers disclosed herein.
- the expression level of NKAalb at time point T3 are not significantly higher than in unsmoltified fish at Tl.
- the widespread in FKBP5 expression levels across fish at all sampling points under both smoltification protocols shows that FKBP5 is not applicable as a reliable smoltification marker.
- figure 9 shows a marked decline in zymogen granule protein levels from Tl to T3 and marked increase in SI 00 levels over the same period, contrasting with the patterns shown in Figure 8.
- the present inventors have identified a number of marker genes that may be used to determine whether salmonid fish is ready for transfer to saltwater.
- the marker genes are shown in the table below. Table 5 List of photoperiodic-hi story dependent smolt genetic markers. Group 2are down-regulated with induction of SW-readiness. Group 1 are up-regulated with induction of SW-readiness.
- the genes are identified by CIGENE identifier number, as well as Protein ID number and NCBI Gene ID Number given in GenBank of the National Center for Biotechnology Information (NCBI)
- an in vitro method for determining saltwater readiness of one or more salmonid fish, comprising a) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985, wherein the tested fish is considered SW ready when
- At least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 is downregulated; and/or (ii) at least one gene selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 is upregulated.
- the disclosed in vitro method comprises the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 in the biological sample of a).
- LOC106606117, LOC106570104, and LOC106589985 is determined by measuring the abundance of a RNA transcript of at least one of the said genes.
- said at least one gene can be measured using qPCR or high throughput RNA sequencing technologies.
- an in vitro method comprising the steps of a) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921 , LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985; b) normalising said determined expression level against an expression level of a salmonid housekeeping gene.
- a method is disclosed herein, wherein the housekeeping gene is a gene encoding elongation factor 1A.
- a method is disclosed herein, wherein the expression level is determined in a biological sample from one or more salmonid fish, and wherein the biological sample is a sample of integumental tissue.
- a method comprising the steps of: a) providing a sample of integumental tissue from one or more juvenile salmonid fish of a group of fish to be smoltified prior to exposing said fish for smoltification conditions; b) analysing the sample of step a) to provide a base line expression level by determining the expression level of one or more genes identified by its NCBI Gene ID number selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985; c) exposing the remaining
- At least one gene selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 is upregulated compared with the base line level determined in step b).
- a method is disclosed herein, wherein the expression level of at least one gene belonging to the group (i) and at least one gene belonging to group (ii) is determined.
- saltwater readiness is determined by analysing the expression level of at least one of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 (also referred to as group (2) herein) and at least one of the genes selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 (also referred to as group (1) herein).
- the expression level of e.g. at least two genes of group (1), such as at least three, or at least four and so forth; and at least two genes of group (2), such as at least three, or at least four, and so forth may be analysed in order to determine saltwater readiness.
- any number and combination of the genes of group (1) and the genes group (2) can be used according to additional aspect disclosed herein.
- a method wherein determination of SW readiness is performed by analysing the within fish expression ratio of a pair of genes comprising at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 and at least one gene selected from the group consisting of LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985.
- a method is disclosed herein, wherein the method comprises the steps of: a) providing a sample of integumental tissue from one or more salmonid fish to be tested; b) analyzing the sample from step a) by measuring the expression level of the following group of genes identified by its NCBI Gene ID number:
- a method is disclosed herein, wherein the fish to be tested belong to the genera Salmo, Oncorhynchus, or Salvelinus.
- a method is disclosed herein, wherein the fish to be tested is selected from the group consisting of Atlantic salmon, Coho salmon, Chinook salmon and Sockeye salmon.
- the fish to be tested is Atlantic salmon.
- the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue, skin tissue or fin tissue.
- a method is disclosed herein, wherein the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue.
- the present application also disclose the use of one or more genes identified by its NCBI Gene ID number and selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928,
- LOC106606117, LOC106570104, and LOC106589985 for determining sea water readiness of salmonide fish.
- the use of at least two genes are disclosed, wherein at least a first gene identified by its NCBI Gene ID number is selected from the groups consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366; and wherein at least a second gene identified by its NCBI Gene ID number is selected from the group consisting of LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985.
- kits for use in the method of the additional aspect comprises reagents for the measuring of protein expression levels and/or mRNA expression levels of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen genes from the set of genes selected from the group comprising LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366,
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Abstract
The present invention relates to a method for determining salt water readiness of salmonid fish, such as e.g. Atlantic salmon. In particular, the present invention relates to genetic markers useful in determining whether juvenile fish (parr) has transformed into a migratory form (smolt) that are ready for transfer to salt water.
Description
Title: Seawater readiness markers in salmonid fish
Field of invention
The present invention relates to a method for determining seawater (SW) readiness of salmonid fish. In particular, the present invention relates to genetic markers useful in determining whether juvenile fish which have been raised from hatching in freshwater (parr) has transformed into a form that will perform well when transferred to SW.
Background of invention
Wild salmonid fish start life in fresh water streams and rivers, and after having developed through the stages of small fry to the juvenile parr, the fish develops further by undergoing structural and functional transformations from parr to smolt prior to migration from freshwater to seawater. The transformation process is called smoltification and consists of a number of complex developmental changes in the biochemistry, physiology, morphology and behavior of the juvenile salmon, critical amongst which is the acquisition of the ability to efficiently maintain water and ionic balance upon entering the sea (cf. Hoar WS (1988), The Physiology of Smolting Salmonids, Fish Physiology, 11, Ed. Hoar and Randal, Cambridge: Academic Press, pp. 275-343, and McCormick et al. (2013), J Exp Biol, 216(7), pp 1142-1151).
In natural settings, smoltification is stimulated by declining day length (photoperiod) in the autumn followed by increasing photoperiod in spring, causing a cascade of physiological responses mediated by changes in circulating endocrine signals (cf. Hoar WS (1988), supra , Stefansson et al. (2008), Smoltification. In:
Fish Larval Physiology. Ed. Finn RN, Kapoor BG, New Dehli: Science Publishers, Inc. Enfield (NH) & IBH Publishing Co. Pvt. Ltd., McCormick SD, et al. (2007) Aquaculture. 273(2):337-44, and McCormick SD (2001), Am Zool. 2001;41(4):781- 94).
The aquaculture industry depends on this photoperiod-dependent developmental transition for the production of seawater tolerant juvenile salmon to be transferred from fresh water to seawater in which rapid growth can take place. It is noticed that when referring to seawater in this respect, seawater is defined as water with an increased salinity content, achieved naturally e.g. when fish is transferred to seawater enclosures, or by addition of salts or seawater to freshwater. Today, further farming of smoltified fish is performed both in seawater cages and also in land-based Recirculating Aquaculture Systems (RAS) facilities.
The above mentioned so-called smoltification can be artificially achieved e.g. by exposing juvenile salmon exceeding a minimum size threshold to short photoperiod (SP) for several weeks mimicking the number of hours daylight in the wintertime and thereafter exposing the fish for continuous light (LL). Based on observations of
SW performance, it has been shown that the duration of exposure to SP should be at least six weeks long for LL to induce smoltification (cf. Duncan NJ and Bromage N. (1998), Aquaculture, 68(l-4):369-86). The underlying causes of this photoperiodic history-dependence remain unknown and untangling the role of SP exposure in smolt development is of considerable interest, since growth rates and hence aquaculture production are slowed during periods of SP exposure.
In Norway, fish farmers experience about 12-14% loss through mortality after SW transfer (Hjeltnes et al. (2019), Fiskehelserapporten 2018 [Fish Health Report 2018], Veterinary Institute, University of Oslo). Also, surviving fish may suffer from welfare issues and grow poorly. In particular, early maturation of Atlantic salmon can result in decreased growth, inefficient feed conversion, reduced product quality, and also increased susceptibility to pathogens.
One way of reducing the loss is to ensure that fish that are transferred from freshwater tanks to SW are sufficiently matured, more optimized smolts suited for life in SW.
In a report by Aime Lee S. Houde et al. (2019) in Conservation Physiology, 7, pp 1- 21, a subset of candidate genes is suggested to be potentially useful in staging smoltification and classify juveniles as pre-smolt, smolt or de-smolt. The experiments reported by Houde et al. did not directly explore the expression of these markers in response to photoperiod treatments or assess their expression in relation to long term SW performance of fish in order to provide biomarkers that may be useful to determine the SW readiness of salmonid fish.
In a report by Seear et al. (2010), in Marine Biotechnology, 12, pp. 126-140, a list of genes that are twofold upregulated in gill, kidney and brain tissue and twofold downregulated in gill and kidney tissue, respectively, of smolts are listed. The analysed samples were taken from 15 parr sampled in February, and 15 fish where sampled when considered ready for seawater transfer in late April. As with the experiment reported in Houde et al., no data showing the survival of fish after transfer to seawater is shown. Thus, the prior art does not disclose gene markers that are shown to be upregulated or down regulated in fish that are shown to perform well after transfer to seawater.
There is therefore still a need for a method for determining whether a salmonid fish has undergone the smoltification process and become sufficiently ready for transfer to SW in order to improve survival rate, ensure optimal growth, and improve fish health after transferring smolts to SW in the aquaculture industry.
Summary of invention
To be able to optimize smolt production and identify genetic factors that contribute to variation in smolt development, there is a need for robust and precise tools useful
in determining when smolts are SW ready. The present inventors have solved this problem by providing methods for determining seawater readiness identifying genetic markers found to be linked to the photoperiod-dependent developmental transition and performance in SW. The provided method can be used to clarify if salmonid fish have matured sufficiently to perform well following transfer to SW.
The successful identification of genetic markers useful in identifying salmonid fish that are ready to be transferred to seawater is a result of the controlled trial set up comparing fish being exposed to various photoperiodic regimen as well, and importantly, combined with the prolonged seawater test of the trial set up (cf. Fig.
1). The specific trial set up have revealed a set of new gene markers, which have in common that they are considered more reliable compared with genetic markers suggested in the prior art.
The genes identified as markers by the present inventors are thus unified by the specific experimental design used to discover them. Duncan and Bromage (1998), (1998), Aquaculture, 68(l-4):369-86 specifically explored the importance of the duration of exposure to short photoperiods, which they refer to as the ‘different periods of constant short days’, for subsequent SW performance following return to LL. Although the importance of short photoperiods was examined already in 1998, no one has explored different photoperiod regimen, including short periods, in order to identify reliable smoltification markers. No previous studies have used this knowledge to identify predictive markers for smoltification. That is, no previous smolt markers have been identified based on the criterion that their expression during smolt development depends on the duration of prior exposure to short / winter photoperiods (defined as less than 12-h light / 24-h). Thus, while a number of markers are reportedly associated with smolt performance, none of these were identified based on a photoperiodic history-dependent response to LL. In addition, as mentioned above, the prior art markers are neither linked with performance after transfer to seawater. Therefore, although one may trace some difference in expression of certain genes at the beginning of and at the end of a winter signal period, it is acknowledge that it cannot be established that the fish is sufficiently adapted to life in the sea in the absence of a correlation with the fish's survival and growth after transfer to saltwater. Due to the correlation with the fish’s survival and performance in SW, the present method furthermore provides a tool for ensuring improved performance of smolt in SW compared with the prior art markers and methods.
In addition, the genetic markers used in the method of the present application also have in common that they are all shown to be downregulated in one of the seven major cell types found in gill in salmonid fish, i.e. non-differentiated progenitor cells (NDC). It is noticed that previous suggested markers for smoltification are all
markers found in chloride cells of the gills, which is contrary to the markers used according to the present invention.
According to a first aspect, a method is provided for determining SW readiness of one or more salmonid fish, comprising determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, wherein the tested fish is considered saltwater ready when at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is downregulated.
According to the first aspect, the in vitro method of the present invention also provides for improving SW performance of salmonid fish after transfer to SW.
According to one embodiment of the first aspect, an in vitro method is provided, comprising the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression level of one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 in the biological sample of a).
According to another embodiment of the first aspect, an in vitro method is provided, wherein expression level of at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is determined by measuring the abundance of a RNA transcript of at least one of the said genes.
According to another embodiment of the first aspect of the invention, an in vitro method is provided comprising the steps of a) determining the expression levels of one or more genes in a biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493; b) normalising said determined expression level against an expression level of a salmonid housekeeping gene.
According to one embodiment of the first aspect, the genetic markers used according to the present invention do not show enriched expression in chloride cells relative to pavement cells, red blood cells, vascular cells, pillar cells, mitochondrion
rich cells (MRCs, also known as chloride cells), T cells, endothelial cells, goblet cells, neuroendothelial cells, myeloid cells, lymphatic endothelial cells, dendritic cells, accessory cells, fibrocytes, and vascular cells present in salmonid gill tissue.
According to another embodiment of the first aspect, the expression level of at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is normalised against gene encoding elongation factor 1A.
According to another embodiment of the first aspect, the expression level may be determined in a biological sample from one or more salmonid fish, wherein the biological sample is a sample of integumental tissue.
According to another embodiment of the first aspect, an in vitro method is furthermore provided comprising the steps of: a) providing a sample of integumental tissue from one or more juvenile salmonid fish of a group of fish to be smoltified prior to exposing said fish for smoltification conditions; b) analysing the sample of step a) to provide a base line expression level by determining the expression level of one or more genes identified by its NCBI Gene ID number selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493; c) exposing the remaining group of salmonid fish for smoltification conditions; d) providing a sample of integumental tissue from one or more salmonid fish of the group of fish of step c); e) determining the expression level in the sample of step d) of one or more of the same genes analysed in in step b); f) comparing the expression level of the genes analysed in step b) and step e), wherein the tested fish is considered SW ready when at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, is downregulated compared with the base line level determined in step b.
According to the first aspect, the salmonid fish to be tested in the method according to the present invention is selected from a fish to be tested belonging to the genera Salmo, Oncorhynchus, or Salvelinus.
According to yet a further embodiment of the first aspect, the salmonid fish to be tested is selected from the group consisting of Atlantic salmon, Coho salmon, Chinook salmon and Sockeye salmon. According to one embodiment, the fish to be testes is Atlantic salmon.
According to the first aspect, the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue, skin tissue or fin tissue. In a preferred embodiment, the expression level is determined in a sample of gill tissue.
According to a second aspect, the present invention relates to the use of one or more genes identified by its NCBI Gene ID number and selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, for determining SW readiness of salmonid fish.
According to the second aspect, the present invention furthermore relates to the use of one or more of the genes selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, wherein said one or more genes used as markers for saltwater readiness do not show enriched expression in chloride cells relative to pavement cells, red blood cells, vascular cells, pillar cells, mitochondrion rich cells (MRCs), T cells, endothelial cells, goblet cells, neuroendothelial cells, myeloid cells, lymphatic endothelial cells, dendritic cells, accessory cells, fibrocytes, and vascular cells present in salmonid gill tissue.
The present invention also provides for the use of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 to determine SW readiness of salmonid fish and to improve SW performance of salmonid fish after transfer to SW.
According to a third aspect, a kit is provided for use in a method to the present invention, wherein the kit comprises reagents for the measuring of protein expression levels and/or mRNA expression levels of at least one, two, three, four, five, six, or seven genes from the set of genes selected from the group comprising LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493.
Abbreviations
SP refers to short photoperiod regimen applied in the experiment disclosed herein.
WSP refers to weeks short photoperiod, i.e. 2WSP, 4WSP and 8WSP refers to fish being kept at a short photoperiod regimen for 2, 4 and 8 weeks respectively.
LL refers to a continuous light regimen applied in the experiment disclosed herein.
SP-LL refers to a regimen comprising a short photoperiod followed by a period of continuous light applied in the experiment disclosed herein.
FW refers to fresh water.
SW refers to seawater defined as water with an increased salinity content, achieved naturally by transfer to seawater enclosures, or by addition of salts to freshwaters. Thus, when referring to fish being ready for transfer to saltwater, it is to be understood that the fish is ready to be transferred to seawater. Thus, the term “saltwater” and “seawater” are to be understood to be synonyms in this respect. The abbreviation “SW” is to be understood to refer to “saltwater” and “seawater”.
List of Figures
Figure 1 shows experimental design to screen for photoperiod-dependent developmental markers, i.e. to identify genes whose expression in the gill was increased according to a photoperiod-dependent developmental transition.
Figure 2. Upper panel: Plasma osmolality after 24-h SW) challenge tests at the indicated sampling points. Data are mean ± SEM of n= 9-10 fish per sample point. **, significantly higher values than at day 1 and at four and eight weeks after return to LL, p<0.01. Lower panel: Sodium potassium ATPase alphalb subunit expression in fish exposed to SP for different durations prior to return to LL Data are normalised mRNA abundance, mean ± SEM of n= 6 fish per sampling point. *, ** significantly higher expression than LL and SP values at the corresponding time point, p<0.05, 0.01, respectively. Where error bars do not appear, errors lie within the symbol. The dashed line represents the SP control group.
Figure 3 shows expression ratios for photoperiod-dependent developmental markers. Data are means with 95% confidence intervals shown as error bars for the log(base 10) of the ratio between the indicated numerator (2) genes (LOC106584756, LOC106605916, LOC106562680, or LOC106562681) and the indicated set of denominator (1) genes (LOC106567921, LOC106565346,
LOC106569928, LOC106606117, LOC106570104, LOC106589985). Black bars show log ratios for indicated numerator / denominator pairings in fish transferred to LL after 2 weeks of short photoperiod, which subsequently performed poorly in seawater. Grey bars show log ratios for indicated numerator / denominator combinations in fish transferred to LL after 8 weeks of short photoperiod, which subsequently performed well in seawater.
Figure 4 shows expression ratios for photoperiod-dependent developmental markers as for Fig 3 but (2) genes are LOC106591222, LOC106602268, LOC106561379, or LOC106562250. Figure 5 shows expression ratios for photoperiod-dependent developmental markers as for Fig 3 but (2) genes are LOC106592537, LOC106608496, LOC106608493 or LOC106609366.
Figure 6 shows expression ratios for combinations of LL-induced and LL- suppressed NKA alphal subunit genes. Data are means with 95% confidence
intervals shown as error bars for the log(base 10) of the ratio between the indicated numerator (b) genes (LOC106596208, LOC106602157, or LOC106610479) and the indicated set of denominator (a) genes. Black bars show log ratios for indicated numerator / denominator pairings in fish transferred to LL after 2 weeks of short photoperiod, which subsequently performed poorly in seawater. Grey bars show log ratios for indicated numerator / denominator combinations in fish transferred to LL after 8 weeks of short photoperiod, which subsequently performed well in seawater.
Figure 7 includes violin plots showing the expression level of gene markers used in the present method by the population of cells in each cluster at each sample point. The cluster key numbers (in brackets) refers to the following cell types: pavement (0), red blood cells (1), vascular cells 1 (2), pillar cells (3), MRCs (FW) (4), T cells (5), endothelial cells 1 (6), goblet cells (7), non-differentiated cells (NDC) (8), vascular cells 2 (9), vascular cells 3 (10), neuroendothelial cells (11), myeloid cells (12), lymphatic endothelial cells (13), MRCs (SW) (14), endothelial cells 2 (15), dendritic cells (16), accessory cells (17), fibrocytes (18), and vascular cells 4 (19). The genes shown in the violin plots a) - g) are downregulated in saltwater ready salmonid fish.
The CIGEN identifier number, Protein ID, NCBI Gene ID Number and gene name is given above each violin plot.
Violin plot a) - g) refers to the genes having the NCBI Gene ID No.
LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, respectively.
Figure 8. Expression of NKA alpha lb subunit (NKAalb(ii)) and FKBP5 RNA in gill filament biopsy samples from Experiment 2. Expression levels (Y axis) are results from qPCR normalised against EFla (housekeeping gene expression). For each smoltification protocol (Photoperiod, LL+salt) expression levels in individual fish are shown, expressed relative to the mean level of expression at Tl.
Figure 9. Expression of one downregulated (zymogen granule protein 16) and one upregulated (S100A) NDC marker in gill filament biopsy samples from Experiment 2. Expression levels (Y axis) are results from qPCR normalised against EFla (housekeeping gene expression). For each smoltification protocol (Photoperiod, LL+salt) expression levels in individual fish are shown, expressed relative to the mean level of expression at TL
Figure 10. Specific growth rate (SGR) over a 9-week period of growth in SW in fish raised under the two smoltification protocols in Experiment 2 (i.e. from timepoint T3 to timepoint T4). Each point represents growth in one individual fish.
Figure 11 Schematic illustration of the experimental design of experiment 2. Tl : 25.04.2019, T2: 06.06.2019, T3: 06.06.2019, T4: 25.09.2019.
Detailed description of the invention
Smoltification is a physiological, morphological and behavioral transformation of salmonid fish such as Atlantic salmon ( Salmo salar) juveniles, from a FW parr to a SW smolt. This parr-smolt transformation encompasses several changes adaptive for life in SW, of which the development of gill ion secreting capacity and SW osmotic tolerance is the most studied. Under natural conditions, parr are gated into the smoltification pathway when their body weight/energy status exceeds a certain threshold in the autumn. Thereafter they remain in a “resting” state during the dark winter until smoltification is triggered by increasing day length (photoperiod) in the spring. As mentioned above, in fish farming, smoltification is artificially initiated by light manipulation to stimulate and synchronize smoltification. For example, fish are reared in constant light (referred to herein as LL) until smoltification procedures are initiated, that is by exposing the juvenile fish to a daily light - dark cycle with light exposure for 12 hours or less per day to mimic natural conditions where salmonid fish smoltify after a period of exposure to winter photoperiods.
Today, the prevailing method for determining SW readiness in research and commercial fish farming is by assessing the gill Na+K+-ATPase (NKA) activity prior to SW transfer and/or changes in plasma chloride (Cl) levels after a short term (24 hr) SW challenge. These markers have been developed based on the idea that SW ready smolt would have increased NKA activity and therefore greater ability to regulate chloride levels upon SW transfer (McCormick et al. (2013), supra). Furthermore, determination of the expression level of two subunits of the NKA protein complex (the alpha la and alpha lb subunits) have been used to determining SW readiness (McCormick et al. (2009), J. Exp. Biol., 212, pp 3994-4001).
Although these markers clearly distinguish parr from larger fish that have the capacity to smoltify, the present inventors have found that these common smolt markers do not distinguish between different smolt development states and surprisingly are poor predictors of long term SW performance, cf. figure 6.
In order to identify genetic makers based on the photoperiod-dependent developmental transition, useful in determining whether salmonid fish are ready to be transferred to saltwater, following an acclimatization period, salmonid fish were exposed to short photoperiods (SP) (i.e. 8-h light / day, LD8:16) for different lengths of time followed by a period of continuous light (LL) (cf. Figure 1 and Example 1 below). Samples of gills where taken for analysis prior to the short photoperiod and after the LL period. Thereafter, the fish were transferred to SW. As control, age-matched fish only exposed to SP conditions were used. Long term performance was also measured by monitoring feed consumption, fork length and body mass over time after transferring a number of the test fish to SW.
At the end of the experiment, fish were killed, and gill samples were collected for gene expression analysis. The inventors identified genes whose expression following exposure to LL for 4 weeks was significantly different depending on duration of prior exposure to SP, i.e. (1) genes with significantly higher expression in 8WSP fish compared to 2WSP fish, and (2) genes with significantly higher expression in 2WSP fish compared with 8WSP fish. The expression level identified for the genes of group (1) and (2) was furthermore ranked based on expression in the two groups. Genes from group (1) having the lowest ratio for 8WSP-LL:2WSP- LL expression were considered strong candidates for markers negatively correlated with SW readiness. Opposite, genes from group (2) having the highest value for 8WSP-LL:2WSP-LL expression were considered strong candidates for markers positively correlated with SW readiness.
The experiments and analysis disclosed in the experimental part of the present specification thus revealed a set of genes that are upregulated and a set of genes that are downregulated, respectively, in fish performing well after being exposed to SW (table 1 below) and are identified as useful gene markers in determination of whether a salmonid fish is ready to be transferred to SW.
In addition, the inventors have found that 7 of the downregulated genes have in common that they are all expressed in non-differentiated progenitor cells (NDC) as shown in the violin plots of figure 7, and further that they are downregulated in smoltified fish that performs well after transfer to seawater.
The nucleotide sequence of the identified genetic markers of the present invention are publicly available and identified herein by reference to Gene ID Number of the GenBank of the National Center for Biotechnology Information (NCBI) (https://www.ncbi. nim.nih .gov/gene). The sequence of the identified genetic markers are also available in the integrated database of Salmonid Genomic Resources, Salmobase 2.0 (https vVsalmobase.org/) and identified by its CIGENE identifier number (Centre of Integrative Genetics, University of Life Science, As, Norway).
Table 1. List of photoperiodic-history dependent smolt genetic markers of the present invention, being down-regulated in NDC cells with induction of SW- readiness. The genes are identified by CIGENE identifier number, as well as Protein ID number and NCBI Gene ID Number given in GenBank of the National Center for Biotechnology Information (NCBI)
In particular, a method according to the present invention is provided for determining saltwater readiness of one or more salmonid fish, involving analysing the expression level of genes selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOC106608496, and LOC106608493. According to this aspect, a fish in which the expression of a gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493 is downregulated is determined as ready to be transferred to saltwater.
According to one aspect the expression level of the above marker genes is measured using multiplex qPCR, such as RT qPCR, or RNAseq.
According to the present method, determination of saltwater readiness may be performed by analysing the expression level of at least one of the above genes. Thus, according to one embodiment, expression level of at least one of the LOC106605916, LOC106584756, LOC106562680, LOC106562681,
LOC106562250, LOC106608496, and LOC106608493 is determined. According to another embodiment of the first aspect of the invention, the expression level of at least two of the genes selecting from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493 are determined, such as at least three of the mentioned group of genes, such as at least four of the mentioned group of genes, such as at least five of the mentioned group of genes, such as at least six of the mentioned group of genes, such as all of the genes of the mentioned group.
The expressions ‘SW ready, “sea water ready” or “saltwater readiness” as used herein is to be understood to mean that a salmonid fish is ready for transfer to a container or sea-cage in which the water salinity is in the range typically used by the aquaculture industry for optimizing growth of salmon following the juvenile freshwater phase (15 - 40 ppt). It is furthermore to be understood that a fish being “saltwater ready” has a higher probability of surviving transfer to saltwater, such as SW. Also, it is to be understood that a fish being SW ready has a higher probability of growing faster and be less susceptible to infectious diseases upon transfer to SW. It is furthermore to be understood that fish that are “saltwater ready” has a good SW tolerance, and that the fish has developed a hypo-osmoregulatory capacity.
As used herein, the expression “upregulated” should be understood to mean that the expression of the gene in question is increased in SW ready salmonid fish compared with the expression of said level of the gene in question in juvenile salmonid fish. For example, the level of expression is increased in a SW ready salmonid fish compared with a juvenile salmonid fish (parr), e.g. when comparing the level of expression of fish prior to and after being exposed to a smoltification regimen commonly used in the salmon aquaculture industry.
As used herein, the expression “downregulated” should be understood to mean the expression of the gene in question is decreased in SW ready salmonid fish compared with the expression of said level of the gene in question in juvenile salmonid fish. For example, the level of expression is decreased in a SW ready salmonid fish compared with a juvenile salmonid fish (parr), e.g. when comparing the level of expression of fish prior to and after being exposed to smoltification regimen commonly used in the salmon aquaculture industry.
The expression “smoltification conditions” or “smoltification regimen” as used herein is to be understood to mean any method or procedure useful for initiating transition of salmonid fish from the juvenile stage (parr) to a SW ready fish (‘smolt’ stage). Commonly, juvenile salmonid fish is as mentioned above exposed to photoperiods with reduced hourly day light followed by a period of constant daylight. The skilled person will acknowledge that exposing the salmonid fish to photomanipulation in aquaculture industry provides for mimicking the light changes that wild salmon is exposed to in nature, often also referred to as winter signal. The
markers identified herein is the result of thorough analysing of the changes of expressions of numerous test fish exposed to a specific photoperiod and which is also shown to perform well after transfer to seawater.
The genetic markers disclosed herein are identified in fish exposed to 8 weeks of short photoperiod (8WSP), followed by 8 weeks of continuous light (LL) compared with fish exposed to 2 weeks of short photoperiod followed (2WSP) by 8 weeks of continuous light (LL). Furthermore, the genetic markers disclosed in the present invention correlated with fish that performs well in SW, which thus renders the markers reliable as makers for saltwater readiness. Although the markers are identified in fish exposed to the above regimen (cf. also experiment l),also fish that are exposed to a different smoltification regimen or smoltification conditions can be tested for saltwater readiness using the genetic markers identified herein. Reference is made to experiment 2, in which examples of the novel markers are shown to be responsive both to photoperiod- and salt-diet based smoltification regimens.
Various methods can be used to determine whether a specific gene is upregulated or downregulated in salmonid fish. Measuring the expression level of the above genes (table 1) may for example be performed by well-known mRNA quantification methods or by protein quantification methods. For example, the expression level can be quantified by measuring mRNA or protein levels directly or indirectly from cell lysates made from the biological sample provided from the fish to be tested.
The level of expression of a gene can be determined measuring non-coding RNA, i.e. miRNA, tRNA, rRNA, snoRNA, or piRNA).
Examples of suitable mRNA quantification methods include, without limitation to hybridization-based assays, such as microarray analysis, or methods based on real time quantitative reverse transcription PCR (real time qRT PCR). Other methods that can be used to determine expression level of one or more genes includes, but are not limited to methods based on hybridisation of a targeted DNA or RNA (nucleic acid based) probe to RNA transcripts of the above genes (e.g. in situ hybridisation, northern blotting, RNase protection assay, reverse transcription-PCR approaches, microarrays, solution hybridisation technologies, e.g. the multiplexed assay for gene expression analysis, nCounter® provided by NonoString Technologies (h ttp s : //www . nano st ri n g . c cm /) . Also approaches based on nucleotide sequencing following by mapping of reads and counting transcripts may be used (e.g. RNAseq technology by Illumina® (https:/7¾ni¾a.illumina.com/techniqnes/sequeiiein¾/ma sequencing html 9langsel=/no/L PACBIO® ( http s : //www .pacb.com /) . The MinlON technology provided by Oxford Nanopore Technologies (https://nanoporet.ech.com/products/minion)). Various methods for measuring expression of marker genes are provided by Fluidigm®
(h ttp s : /'/'www . f 1 i d i gm . c om/) . The skilled person will acknowledge that any known
method for measuring expression level of a marker gene may be used to determine the expression level of the marker genes disclosed herein, and thus to clarify whether the fish being tested is ready for transfer to seawater.
If expression level is to be measured using a method involving oligonucleotide sequences, such as probes or primers, the skilled person will acknowledge that the oligonucleotide sequence should be capable of hybridize to a nucleic acid sequence with a complementary sequence, such as e.g. genomic material, extracted from biological sample provided according to the present method. The skilled person will understand that the genomic material may be e.g. RNA or DNA, e.g. extraction of RNA from samples of gill fragments as described in example 1 below.
A “probe” or “primer” useful in determining the expression level of the SW readiness biomarkers according to the present method is generally one that contains at least 8 nucleotides and which is capable of hybridizing a nucleic acid with a complementary sequence, and is separated from most other nucleic acids present in the natural source of the nucleic acid, and is thus substantially free of other cellular material.
The skilled person will acknowledge that an oligonucleotide probe can be a fragment of DNA or RNA of variable length used in a method according to the present invention in order to hybridize to the target sequence, e.g. single-stranded DNA or RNA. The oligonucleotide probe may furthermore be labeled with a molecular marker to easily visualize that hybridization have been achieved. Molecular markers commonly known to the skilled person may be used, e.g. a radiolabel, and more preferably, a luminescent molecule or a fluorescent molecule enabling the visualisation of the binding of the probe(s) to a target sequence.
An oligonucleotide probe can hybridize to another nucleic acid molecule, such as the single strand of DNA or RNA originating from a biological sample from one or more salmonid fish to be analysed, under appropriate conditions of temperature and solution ionic strength, cf. e.g. Sambrook et ah, 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. An oligonucleotide probe typically comprises a nucleotide sequence which under stringent conditions hybridize to at least 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50 (or any other number in-between) or more consecutive nucleotides in a target nucleic acid molecule, e.g. single-stranded DNA or RNA isolated from a biological sample to be analysed.
Also, oligonucleotide primers may be used in methods according to the present invention for determination of the expression level of a SW readiness biomarker gene disclosed herein, i.e. belonging to the group (1) genes and group (2) genes listed above. An oligonucleotide primer typically comprises a nucleotide sequence at least 8, 10, 12, 14, 16, 18, 20, 22, 25, 30, 40, 50 (or any other number in- between) or more consecutive nucleotides. 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). Also, oligonucleotide primers can be labeled with a molecular marker to enable visualization of the results obtained. Various molecular markers or labels are available. An oligonucleotide primer typically comprises the appropriate number of nucleotides allowing that said primer align with the target sequence to be analyzed.
Probes and primers useful in determining expression level of the SW readiness biomarkers disclosed herein can be constructed based the common general knowledge of the skilled person and based on the known sequence of said biomarker genes, cf. reference to Gene ID number thereof in table 1.
Alternatively, expression level of a gene may be determined at the protein level, e.g. by suitable protein quantification methods known in the prior art, including immune assays such as ELISA, Western blot etc. utilizing antibodies that bind to proteins encoded by the genes of interest and as listed in table 1 above. Antibodies that recognize and bind to proteins encoded by the genes listed in table 1 can be manufactured using methods well known to the skilled person. Also, several applicable immune assay methods are available, for example immune assay can be developed for determining expression levels of the genes of interest using ELISA development kits, such as the kit available from MABTECH (https:// www. inabtech.com/knowledge-cemer/product-guide/elisa-ldts/elisa- deveiopment-kits), LSBio
(http s : /'/'www.l sbi o. com/products/ei i sakits/ dev el opm entki t) .
The skilled person will furthermore acknowledge that the findings that the genes identified as saltwater readiness markers by the present inventions can be utilized in various methods for determining whether salmonid fish is ready for transfer to SW.
According to one embodiment, the expression level of one or more of the genetic markers of the present invention can be determined in one or more fish prior to and after exposing a group of fish to a smoltification regimen, wherein the findings that at least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493, is downregulated in the fish after being exposed to a smoltification regimen determines that the fish is ready for being transferred to saltwater.
According to one embodiment, the expression level of the at least one gene is determined in a selection of a group of juvenile salmonid fish to be exposed to smoltification regimen in order to provide a baseline level of expression of the at least one of the above listed genes. After exposing the salmonid fish to smoltification regimen, the expression level of the same genes is analysed in a biological sample of subsample of the group of fish. The group of fish is furthermore considered SW ready in the case that at least one of the tested genes of the above genes is downregulated. The term baseline expression level according to this embodiment is thus to be understood to be the expression level of the above genes in juvenile salmonid fish (parr) prior to being exposed to a smoltification regimen.
Another method that may be used to determine whether a salmonid fish is ready for transfer to seawater, is to provide normalised expression level data with respect to one or more reference or ‘housekeeping’ genes that are known not to change when a juvenile fish (parr) develops to smolt. The skilled person will acknowledge that housekeeping genes encoding a protein that are continuously expressed to carry out vital cellular functions irrespective of the stage of the salmonid fish can be used as a control to normalise the measured expression level. The skilled person therefore acknowledges that the expression of a housekeeping gene to be used according to the present invention is be independent of the physiological processes involved in smoltification. The expression level of the housekeeping genes is thus the same or similar among the fish tested and irrespective of whether the fish is a juvenile parr or a smolt. The normalisation should ensure accurate comparison of expression of a marker gene of interest between different test samples.
Examples of such so-called housekeeping genes include but are not limited to those described in Olsvik et al, 2005, BMC Mol. Biol., “Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic Salmon”, vol 6, pp 21 (doi 10.1186/1471.2199-6-21). An applicable housekeeping gene to be used in determining the expression level of the gene markers disclosed in the present application is e.g. the Arctic salmonid gene encoding elongation factor 1A, such as e.g. EF I AA and EF I AB.
According to one aspect of the present method, the amount of RNA transcribed from a) one or more genes selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493 and b) one or more housekeeping genes in a sample from the fish to be analysed is measured, and wherein the measurement of the amount of RNA transcribed from b) are used to normalise the expression the genes of a).
The skilled person will acknowledge that when referring to housekeeping genes that may be used to normalise the expression of the marker genes according to the
present invention, the housekeeping gene, or “normalising gene” refers to a gene whose expression is used to calibrate or normalise the measured expression of the gene of interest (e.g. marker genes disclosed herein). One or more housekeeping genes can be used. When using more than one housekeeping gene, a combined normalising gene set can be provided.
The overall expression of the one or more housekeeping genes can be represented by a “normalised value” that are determined by well-known methods, and which can form the basis for determining whether the expression level of one or more of the marker genes disclosed herein is considered downregulated (or upregulated) in a sample of a fish that are subject to method of the present invention. That is, if the normalisation show that one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is downregulated, the group of fish from which one or more test fish is taken is considered to be ready for transfer to saltwater. According to one aspect of the invention, a group of salmonid fish is considered ready for transfer to seawater if 5-25 of test fish selected from said group of salmonid fish show downregulated expression of one or more of the genes selected from the group LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493.
The expression level of the genetic markers identified herein is determined in a biological sample taken from a selection of fish analysed for SW readiness. The biological sample is according to one embodiment of the invention gill tissue, in particular primary a sample originating from gill filaments. The gills have a pivotal role in the energy demanding regulation of water and ionic fluxes and undergo extensive differentiation during the smoltification process in order to enable the migration to a salty environment. A number of physiological changes occur in this process, such as inter alia a shift from an ion-absorbing freshwater type to an ion- secreting salt water type of the gill complement of mitochondria rich cells (Evans et al. (2005), Physiol. Rev., 85(1):97-177, Pelis et ah, (2012), Am. J. Physiol. Regul. Integr. Comp. Physiol., 280(6), pp. R1844-R52). Also, the distribution of mitochondrial rich cells shifts from the lamellae to the gill filament itself (Hiroi and McCormick (2012), Respir. Physiol. Neurobiok, 184(3), pp. 257-68). The changes in the gills when salmonid fish develops from juvenile fish (parr) to a SW ready smolt make the gill a preferred sample for gene expression level analysis. However, other integumental tissues (including but not limited to skin or fin tissue) that are exposed to different salt concentrations when the fish enters sea water can also be used in the present methods.
The size and number of gill samples taken per fish to be tested may vary. The skilled person will acknowledge that the size of the sample should be adapted to the amount of storage solution used. For example, the size of the gill sample is about 2-
15 mm3, such as about 5-10 mm3, such as about 2-5 mm3, such as about 2-3 mm3. Gill samples is furthermore commonly taken from the second gill arch and stored in a suitable preservative solution, such as RNAlater, at refrigerator temperature (e.g. about 4 °C). If the gill samples are not to be analysed within a few days, the sample may be stored in a freezer. Alternatively, gill samples may be rapidly frozen, for example but not limited to, placing on dry ice, on a cooled metal block, or immersion in liquid nitrogen.
When taking gill samples for determination of smoltification status, other smoltification characteristics can be determined at the same time, such as parr marks, silver coloring, and/or form of the fin edges. Also, the weight and length of the fish is commonly measured when taking samples for determination of smolt status. Also, injuries, or signs of illness of the fish from which the samples are taken should be registered.
When determining smoltification status of a population of Salmonid fish that have been exposed to a smoltification regimen, such as photomanipulation regimen commonly used with the smolt breeders, the determination may involve testing of from 5-25 fish of the population, such as from 5-20 fish, or from 5-10 fish. The skilled person will acknowledge that the number of fish needed may depend upon e.g. the size of the population, the point within the smolt window at which the sampling of test fish are taken, cf. that the smolt window may vary from location to location, the strain of salmonid fish and the genotype thereof. The number of fish to be tested may also depend upon the process and conditions used to induce smoltification type of smoltification regimen used.
Although gill samples are used for determining level of expression of genetic markers disclosed herein, also other tissues can be used in the method according to the present invention.
Furthermore, although the experiments have been performed using Atlantic salmon ( Salmo salar), it is to be understood that the genetic markers may as well be used to determine SW readiness in other salmonid fish, such as members of the genera Salmo (e.g. Salmo trutta ), Oncorhynchus (e.g. Oncorhynchus mykiss, i.e. rainbow trout), Salvelinus (e.g. Salvelinus salvelinus, i.e. arctic charr).
According to one aspect of the present invention, a kit is provided comprising means for carrying out the method of the invention. The kit according to the invention typically comprises reagents for measuring protein expression levels and/or mRNA expression level of at least one, two, three, four, five, six, or seven genes from the set of genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493. Reagents that can be comprised in a kit according to the present invention is e.g. primers or probes suitable for measuring expression levels of the mentioned genetic markers. Also, means for analysing
expression level using PCR methods can be included in said kit, such as enzymes, buffers, dNTPs, etc.
Examples Example 1
Materials and Methods Fish
Atlantic salmon ( Salmo salar , Linnaeus, 1758, of the AquaGen commercial stain, Trondheim, Norway) were used for both experiments, and were raised from hatching in FW, on continuous light (LL, > 200 lux at water surface) at 4 - 10°C. Fish were fed continuously with pelleted salmon feed (Skretting, Stavanger, Norway).
Experimental set-up
The experimental design is summarized in Figure 1. All experimental groups were fed pellet salmon feed continuously and in excess with automatic feeders for eight hours a day, corresponding to the light phase under SP.
1400 fish at approximately 11 months old (beginning on 05.01.2017) were included in the test, weighing an average of 40.3 g (s.d. ± 9.7 g, n=10). The juvenile salmon were distributed among eight 300 L circular tanks with FW at 7 °C and LL and left to acclimate for one week. The total number of fish in each tank ranged from 150 to 200, depending on the number of fish to be sampled during the experiment in each tank and the need to avoid density-dependent social stress effects.
After an initial sampling at the last day of acclimation under continuous light (Day 1), fish in all tanks were transferred to SP. One group of fish remained on SP for 16 weeks (SPC group), while the three other groups were kept on SP for two, four or eight weeks (2WSP, 4WSP and 8WSP groups, respectively; collectively termed the SP-LL groups). LL exposure then continued for a further 8 weeks. All treatments were run in duplicate tanks. After the initial sampling, all SP-LL groups were sampled on the last day of SP, and at four- and eight-weeks post-SP. For the SP-LL groups the two post-SP sampling points corresponded to 196 and 392 degree-days (°d) after re-entering LL. At each of these sampling points, samples were also collected from the SPC group.
24-hours salt-water challenge (SWC)
24-h prior to each FW sampling point, randomly selected fish (n=6 for exp.l, and n=10 for exp.2) were transferred to 100 L tanks supplied with full strength SW
(7°C, 34%o salinity) for 24 hours. No feed was given during this 24-h period. There were no mortalities during the SWCs. After 24 h, the fish were netted out and lethally anesthetized (10 L water container, SW, Benzocaine, 150 ppm), followed by blood sampling, decapitation and tissue dissection as described below.
Blood sampling and tissue dissection
Following lethal anesthesia (in 10 L water container, FW or SW as appropriate, Benzocaine, 150 ppm), body masses (± 0.5 g) and fork lengths (± 0.1 cm) were recorded (For FW, n=6 for exp.l and n=10 for exp.2). Blood was collected from the caudal vein into 2 ml Lithium-heparinized vacutainers (BD vacutainers®, Puls Norge, Moss, Norway), and placed on ice until it was centrifuged (6000 x g ) for 10 min. The plasma fraction was collected and stored at -20°C for later analysis of osmolality and chloride concentration. Fish were then decapitated and dissected. After decapitation, the operculum on the right side of the head (caudal view) was removed and primary gill filaments were collected and placed in RNAlater® (Sigma-Aldrich, St. Louis, Missouri, USA) for transcript and qPCR analyses. Samples were stored at 4 °C for 24 h, and then kept frozen at -80°C until further processing.
Prolonged SW exposure following smolt induction
Following maintenance on LL for eight weeks, 30 randomly selected fish from each of the SP-LL groups (fifteen from each duplicate tank) were netted out and anaesthetized (Benzocaine, 60 ppm), and fork length and body mass was measured. After recovery fish were placed in 300 L, circular tanks supplied with full strength SW (34 %o) at 7°C and continuous light. Fish were fed pelleted salmon feed continuously and in excess by automatic feeders. The amount of feed eaten was monitored daily by collection of feed remnants from the tank outlet sieve. After 15 days in SW, fork length and body mass were again recorded as above, before returning the fish to the SW tanks for a further 15 days. On day 30 of SW exposure, all fish were anaesthetized with benzocaine (150 ppm, Sigma-Aldrich, St. Louis, Missouri, USA), after which fork length and body mass were recorded, and the fish decapitated. No fish died during the prolonged SW exposure. Fish from the SPC group were not subjected to extended SW exposure for animal welfare reasons associated with the anticipated lack of SW tolerance.
Analyses
Plasma osmolality and chloride levels
Thawed plasma samples were analysed using a Fiske One-Ten Osmometer (Fiske Associates, Massachusetts, USA, ± 4 mOsm kg-1) and a Chloride Analyzer from CIBA Corning Diagnostics (Essex, England, ± 2.2 mmol L-l).
RNA extraction
Gill tissue was disrupted using TissueLyser II (QIAGEN, Hilden, Germany), and total RNA was extracted using the RNeasy Plus Universal Mini Kit (QIAGEN, Hilden, Germany). RNA concentrations were measured using a NanoDrop ND2000c
spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA samples were frozen at -80 °C until further processing.
Transcriptome sequencing
Sequencing libraries were prepared using the TruSeq Stranded mRNA HS kit (Illumina, San Diego, California). Library mean length was determined by a 2100 Bioanalyzer using the DNA 1000 Kit (Agilent Technologies, Santa Clara,
California, USA) and library concentration was determined with the Qbit BR Kit (Thermo Scientific, Waltham, Massachusetts, USA). Each sample was barcoded using Illumina unique indexes. Single-end lOObp sequencing of sample libraries was carried out on an Illumina HiSeq 2500 at the Norwegian Sequencing Center (University of Oslo, Oslo, Norway).
Cutadapt (28) was used to remove sequencing adapters, trim low quality bases, and remove short sequencing reads using the parameters -q 20 -O 8 -minimum-length 40 (version 1.8.1). Quality control of the reads were performed with FastQC software (29). Mapping of reads to reference genome was done using STAR software (ver. 2.4.2a) (30). HTSEQ-count software (version 0.6. lpl) (31) was used to generate read count for annotated genes.
Transcriptome analysis
Analysis of differential gene expression was performed with package edgeR (ver. 3.14.0) using R (ver. 3.4.2) and RStudio (ver. 1.0.153). Raw counts were filtered, setting an expression level threshold of a minimum of one count per million reads (cpm) in five or more libraries. The counts were scaled by applying trimmed means of M-values (TMM) scaling. The data was fitted with a quasi -likelihood negative binomial generalized log-linear model.
The inventors used empirical Bayes quasi-likelihood F-tests to contrast gene expression in 2WSP and 8WSP fish which had been returned to LL for 4 weeks.
The output was filtered using a false discovery rate (FDR) < 0.01 and a log2-fold change > |1|. This generated a list of genes whose expression following exposure to LL for 4 weeks was significantly different depending on duration of prior exposure to SP (i.e genes (1) with higher expression in 8WSP fish compared to 2WSP fish and (2) genes with higher expression in 2WSP fish than in 8WSP fish).
Table 3 Primer sequences for target genes.
Real-time quantitative PCR
RNA samples from were ethanol-precipitated and DNAse-treated according to the manufacturer’s protocol (TURBO DNA-free Kit, Thermo Fisher). cDNA was constructed using the High-Capacity RNA-to-cDNA kit (Thermo Fisher, Waltham, Massachusetts, USA), following the recommended protocol.
Primers (table 3) were designed to target all splice variants of the target genes, while not picking up ohnologue and paralogue duplicates of the targeted genes. Primer3 (34, 35) and ApE software (v2.0.51) were used for designing primers, and primers were checked against both the National Center for Biotechnology Information (NCBI, Bethesda, Maryland, USA) database using BLAST (36) and the SalmoBase database (37) for non-target hits. Primer specificity was confirmed by melt-curve analysis, and amplicon size verified by agarose gel electrophoresis. In order to establish primer amplification efficiencies a subset of samples was pooled and diluted and analyzed by qPCR. Amplification efficiencies fell between 90% and
110%.
Real-time quantitative PCR analysis was performed using a BioRad CFX Connect Real-Time instrument (Hercules, California, USA), and SYBR Green detection. Reactions were carried out on 96-well plates, with 20 ng RNA cDNA equivalent, 250 nM forward and reverse primer, and lx Sso Advanced Universal SYBR Green Supermix (BioRad, Hercules, California, USA), in a total volume of 20 pL. After initial heating (95°C, 30 sec.), amplification was carried out under the following conditions: 95 °C for 10 sec., and primer-specific annealing temperature for 1 min. over 40 cycles. A melting curve analysis was completed at the end of each run (0.5 °C intervals at 3 sec., from 65 °C to 95 °C).
Data analysis and statistics Condition factor (CF) was calculated as
where W is wet body mass (g), and L is fork length (cm).
Specific growth rate (SGR) was calculated as
where Wt and WT are mass (g) at the beginning and end of the period of extended SW exposure, respectively. Similarly, the feed conversion ratio (FCR) over the same period was calculated by dividing the total amount of ingested food per tank (g, dry weight) by the increase in total biomass for each tank.
The Ct values of target genes were normalised against EF1A (38, 39) using the AACt method described by Livak and Schmittgen (2001) in Methods, 25(4), pp 402- 408.
GraphPad Prism (ver. 7.03) was used for statistical computation of one- and two- way ANOVAs for physiological measurements and relative mRNA content for both exp.l and exp.2. Summary statistics are given as mean ± standard deviation (S.E.M.).
Effects of photoperiod regime (treatment) and time (i.e. time passed after returning to continuous light for SP-LL groups) were assessed by two-way ANOVA, and Tukey’s test for post hoc pairwise comparisons. To avoid pseudo-replication of data the initial sampling point (day 1), which is common for all groups, was excluded from the ANOVA analysis. Data from this sampling point is provided in figures for reference, and a one-way ANOVA was performed to test for any significant differences between the initial sampling point and all other samplings, applying Dunnetf s test for multiple comparisons. The statistical significance threshold was set to p<0.05.
Results
To characterize the requirement for exposure to SP for induction of expression of the cluster 3 genes, juvenile salmon were exposed to two, four or eight weeks of SP, before being returned to LL (2WSP, 4WSP, 8WSP, respectively), and their short - and long-term SW-tolerance and gene expression were assessed.
Hypo-osmoregulatory capacity
The ability to hypo-osmoregulate during a 24-h SW challenge was not dependent upon duration of prior exposure to SP (Figure 2) The fish were able to hypo- osmoregulate efficiently on day 1 of the experiment, and this ability was lost within two weeks of transfer to SP, as shown by the increased levels of plasma osmolytes compared to day 1 fish. The dynamics of re-establishment of hypo-osmoregulatory capacity following return to LL did not differ between the SP-LL groups, which developed smolt-like hypo-osmoregulatory capacity within four weeks of re-
entering LL. The SPC group spontaneously regained its ability to osmoregulate towards day 86 after having spent more than 12 weeks under SP. Though slightly higher levels were measured on day 113, plasma osmolality levels of the SPC group were not statistically different from those measured on day 1, nor at the endpoints of the SP-LL groups. Chloride plasma levels followed a similar pattern as described for plasma osmolytes.
NKA-activity
Because gill NKA activity is considered a good indicator of osmoregulatory capacity and smolt status we also examined how this trait was influenced by photoperiodic history figure 6). In contrast to plasma osmolality and chloride levels, gill NKA activity did not change significantly under chronic exposure to SP (SPC group). However, the development of NKA activity following return of fish to LL was highly dependent on photoperiodic history (p<0.001, for time x photoperiod regime, by two-way ANOVA, supplemental material S3). In fish exposed to SP for two weeks no significant rise in gill NKA activity was seen during the subsequent eight weeks of LL exposure, while in the 8WSP group NKA activity rose approximately five-fold over eight weeks of LL exposure (p<0.001, two-way ANOVA). In 4WSP fish, an intermediate response was observed, with NKA activity rising some two-fold over the post-SP phase. Gill NKA activity does not appear do predict performance in 24-h SW challenges.
Growth performance during extended exposure to SW
In order to assess long-term SW performance of fish from each of the SP-LL photoperiod regimes, fish from each of the SP-LL groups were transferred to SW tanks for 28 days. Initial weights at the point of transfer to SW did not differ significantly between the groups (table 2), and there were no differences in CF. Contrastingly, subsequent SW growth performance was highly dependent on duration of prior exposure to short photoperiod (p=0.003 for time x photoperiod regime interaction, two-way ANOVA). During the four weeks in SW, fish transferred from the 2WSP group showed no significant increase in body mass, while over the same period body mass increased in the 4WSP and 8WSP groups by 19.6% and 27.3%, respectively. Moreover, fish in the 8WSP group grew significantly more, and at a higher SGR (table 2) than fish from the 4WSP group (P<0.01 for final weight comparison). Total dry weight feed intake was 860 g, 944 g, and 1277 g in the 2WSP, 4WSP, and 8WSP treatment groups, respectively, leading to feed conversion ratios (FCR) of 8.27, 2.31 and 2.11 (table 4).
Table 4 Information on age and weight of fish during the prolonged SW stay. Significance as determined by two-way ANOVA. SGR -Specific growth rate, FCR - Feed conversion ratio
5 In addition to differences in food intake, and weight gain, moderate changes in CF were observed. Upon transfer to SW the fish in the 8WSP group had a significantly lower CF than those in the 2WSP group (p<0.05), while an intermediate value was seen in the 4WSP fish. Only the fish in the 2WSP group showed a significant decrease in CF over the four weeks in SW (p<0.001).
10 RNA profiling of gill tissue
Using qPCR, with normalisation to EFla, the induction of NKA alb expression by re-entering LL was not dependent on photoperiodic history, with all three SP-LL groups showing elevated (and equal) mRNA levels, compared with pre-SP levels, after four weeks of LL exposure (p>0.001, one-way ANOVA) (Figure 2).
15 To identify novel candidate markers, we looked for genes whose expression following exposure to LL for 4 weeks was significantly different depending on duration of prior exposure to SP (i.e. genes (1) with significantly higher expression in 8WSP fish compared to 2WSP fish and (2) genes with significantly higher expression in 2WSP fish than in 8WSP fish). Significance was defined as a false
20 discovery rate (FDR) or < 0.01 (1%) using the EdgeR algorithm.
Genes in both group (1) and (2) categories above were ranked based on the ratio between expression in the two groups. Genes from the (1) group with the lowest ratio for 8WSP-LL:2WSP-LL expression were considered strong candidates for markers negatively correlated with smolt readiness. Genes from the (2) group with
25 the highest value for the 8WSP-LL:2WSP-LL expression ratio were considered strong candidates for markers positively correlated with smolt readiness. The genes in the (1) and (2) categories are listed in Table 1
To further filter these lists we looked at normalised counts data for gene expression in individual fish in the 8WSP and 2WSP groups. For each gene under each
30 condition we calculated Minimum, Maximum, mean and 95% confidence interval (95CI) for the mean (based on a normalised t distribution). Genes for which no overlap in expression was observed across all individual samples in the two groups were retained for further analysis. This produced lists of 12 group (1) genes
showing reduced responsiveness in the 8WSP group and 6 group (2) genes showing increased responsiveness in the 8WSP group, i.e. this procedure for defining novel markers selected 18 genes from a genome of more than 46,000 genes (Lien et al,2016, Nature, vol. 533, doi: 10.1038/naturel7164).
Figures 3 to 5 show relative expression of all combinations of upregulated (2) genes expressed as a ration for down regulated (1) genes. Across this dataset as a whole it can be seen that the difference in this expression ratio between 8WSP fish which grew well in sea water and 2WSP fish which did not grow in SW is typically more than 10-fold. This indicates that these ratios are sensitive indicators for the transition to a SW ready state. Reference is also made to table 2. A calculated ratio of expression level of the relevant pair of genes in a tested salmonid fish representing a group of fish that have been exposed to a smoltification regimen is found to be ready for transfer to SW if the calculated ratio is below the thresholds indicated in Table 2.
Figure 6 shows the corresponding expression ratios for combinations of up- regulated and down-regulated NKA-subunit genes. Consistent with the NKAalb profiling in Figure 2, this shows that differences in this expression ratio are much lower, providing a much less sensitive predictor of subsequent performance.
Cell cluster specific expression of saltwater readiness-associated genes
By comparing the cell fingerprints of samples taken from fish of the group exposed to 8WSP in the above experimental design. The fingerprints were sorted into 20 groups, each group representing a different gill cell type. Violin plots where furthermore used to show where a chosen individual gene is expressed within this complex dataset, i.e. whether the genes of interest are expressed in all cell types or selectively enriched in just one or two or more cell types. The obtained violin plots in figure 7 shows expression level of the marker genes of interest in samples taken at different time points during the smoltification regimens described above, i.e. at T1 (before start of WSP period), T2 (end of WSP period), T3 (end of second LL period) and T4 (end at SW period).
The 20 cell types are shown on the x axis of the violin plots, the y axis reports a symmetrical histogram for the expression of a given gene within its cell population (scaled and loglp normalised, i.e. natural log of l+[expression level], where the 1+ is used in order not to get a negative value for an expression that is between 1 and 0). In other words, the breadth of each violin plot reports the number of cells that express the chosen gene at a particular expression level - the wider the plot, the higher the proportion of cells that express the chosen gene at that level.
As can be seen from the violin plots presented in figure 7, the identified marker genes selected from the group consisting of LOC106605916, LOC106584756,
LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, and as used according to the present invention have in common that all are shown to be downregulated in NDC cells after being exposed to smoltification regimen described above.
Example 2
Material and methods Experimental fish
The experiment was carried out at the Aquaculture Research Station in Tromso (ARST), Norway. The fish were Atlantic salmon of the AquaGen strain QTL- innOva SHIELD (AquaGen, Trondheim, Norway), obtained as fertilized eggs and hatched in February 2018 at ARST. After hatching, fish were reared at 4 °C, in continuous darkness. From start-feeding (22.05.2018) the fry was kept at 10-12 °C until August, then, temperature was decreased gradually to 4 °C until start of the experiment. From start-feeding to the start of experiment fish were kept under continuous light. Fish were fed continuously and in excess with commercial extruded salmon feed using automatic feeders (Skretting, Stavanger, Norway).
Experimental design
On Monday, 25.3.2019, at total of 1080 salmon parr were distributed amongst six 300 L tanks (180 fish per tank). Of these, 15 fish per tank were anesthetised in 50 ppm benzocaine and individually marked with Floy tags (Floy Tag & Mfg. Inc., Seattle, US; www.floytag.com). During the first week of a four-week acclimatisation period, temperature was increased by 1 °C per day to a temperature of 10 °. The experimental design is illustrated in Fig. 11. On 23.04.2019, three of the six tanks (SP group) were subjected to a short photoperiod with 9:15 LD (SP) whilst the other three tanks were kept at 24:0 LD (LL). Lights were switched on at 10 am, and lights went off at 7 pm with 15 minutes dimming period. Fish were fed ad libitum (control feed) between 10 am and 6:30 pm. On 06.06., photoperiod treatment was changed to 24 h light and dietary treatment was started for the LL group (LL + dietary treatment). On 22.07., 85 fish, including all tagged fish, were transferred to SW. Fish were acclimated for 2 days to SW temperatures of 8 °C. During the FW phase, all individually tagged fish from the two treatments were measured (fork length) and weighed three times: (25.04.2019, after 6 weeks on SP/LL control 06.06.2019, and 6 weeks after the return to LL 18.07.2019). Eventually, body mass and fork length were measured after 9 weeks in seawater, on 25.09.2019. The specific growth rate (SGR) over the 9-week period of growth after transfer to SW for the fish the above described smoltification regimen is shown in figure 10, each point representing the growth of one individual fish.
On each of these time points all individually tagged fish were anesthetised in 50 ppm benzocaine, weighed, and fork length measured and transferred back to their original tank. In addition to body mass and fork length measurements, a gill biopsy consisting of a few filaments from the first gill arch on the left side was sampled from five individuals (same individuals at all time points) per tank and stored in RNAlater for later gene expression analysis.
Determination of expression level
NKA alpha 1 subunit (NKAlb(ii)) expression have been used for determination of smoltification in the prior art. Also, prolyl isomerase 5 (FKBP5) have been suggested as marker for smoltification in Pacific salmonids (Houde et ah, supra). In order to compare the prior art markers with markers revealed in experiment 1, expression level of NKAlb(ii), FKBP5, LOC106605916 zymogen granule membrane protein 16 like protein and LOC106570104 protein SlOO-Al-like were determined in fish exposed to the experimental design of experiment 2. The expression level was measured by qPCR and normalised against EFla (housekeeping gene).
The results show that NKAlb(ii) and FKBP5 is not as reliable as smoltification markers compared with the markers disclosed herein. As seen in figure 8, the expression level of NKAalb at time point T3 (smoltified fish) are not significantly higher than in unsmoltified fish at Tl. Furthermore, the widespread in FKBP5 expression levels across fish at all sampling points under both smoltification protocols shows that FKBP5 is not applicable as a reliable smoltification marker.
With the genetic markers identified in the present application, the results show a clear difference in expression at Tl and T2, compared to T3, cf. figure 9. In particular, figure 9 shows a marked decline in zymogen granule protein levels from Tl to T3 and marked increase in SI 00 levels over the same period, contrasting with the patterns shown in Figure 8.
Additional aspects disclosed in the present application:
The present inventors have identified a number of marker genes that may be used to determine whether salmonid fish is ready for transfer to saltwater. The marker genes are shown in the table below. Table 5 List of photoperiodic-hi story dependent smolt genetic markers. Group 2are down-regulated with induction of SW-readiness. Group 1 are up-regulated with induction of SW-readiness. The genes are identified by CIGENE identifier number, as well as Protein ID number and NCBI Gene ID Number given in GenBank of the National Center for Biotechnology Information (NCBI)
According to an additional aspect disclosed herein, an in vitro method is provided herein for determining saltwater readiness of one or more salmonid fish, comprising a) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985, wherein the tested fish is considered SW ready when
(i) at least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 is downregulated; and/or
(ii) at least one gene selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 is upregulated.
According to one embodiment of the above additional aspect, the disclosed in vitro method comprises the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 in the biological sample of a).
According to the addition aspect, the expression level of at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921 , LOC106565346, LOC106569928,
LOC106606117, LOC106570104, and LOC106589985 is determined by measuring the abundance of a RNA transcript of at least one of the said genes.
For example, said at least one gene can be measured using qPCR or high throughput RNA sequencing technologies.
According to an embodiment of this additional aspect, an in vitro method is disclosed herein, comprising the steps of a) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921 , LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985; b) normalising said determined expression level against an expression level of a salmonid housekeeping gene.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the housekeeping gene is a gene encoding elongation factor 1A.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the expression level is determined in a biological sample from one or more salmonid fish, and wherein the biological sample is a sample of integumental tissue.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the method comprises the steps of: a) providing a sample of integumental tissue from one or more juvenile salmonid fish of a group of fish to be smoltified prior to exposing said fish for smoltification conditions; b) analysing the sample of step a) to provide a base line expression level by determining the expression level of one or more genes identified by its NCBI Gene ID number selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366, LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985; c) exposing the remaining group of salmonid fish for smoltification conditions; d) providing a sample of integumental tissue from one or more salmonid fish of the group of fish of step c); e) determining the expression level in the sample of step d) of one or more of the same genes analysed in in step b); f) comparing the expression level of the genes analysed in step b) and step e), wherein the tested fish is considered ready for salty water when (i) at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 is downregulated compared with the base line level determined in step b); and/or
(ii) at least one gene selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 is upregulated compared with the base line level determined in step b).
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the expression level of at least one gene belonging to the group (i) and at least one gene belonging to group (ii) is determined.
Also, a method according to this additional aspect is disclosed, wherein saltwater readiness is determined by analysing the expression level of at least one of the genes selected from the group consisting of LOC106605916, LOC106584756,
LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 (also referred to as group (2) herein) and at least one of the genes selected from the group consisting of LOCI 06567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985 (also referred to as group (1) herein). It is thus to be understood that according to this additional aspect, the expression level of e.g. at least two genes of group (1), such as at least three, or at least four and so forth; and at least two genes of group (2), such as at least three, or at least four, and so forth may be analysed in order to determine saltwater readiness. Thus, any number and combination of the genes of group (1) and the genes group (2) can be used according to additional aspect disclosed herein.
According to another embodiment of the additional aspect, a method is disclosed, wherein determination of SW readiness is performed by analysing the within fish expression ratio of a pair of genes comprising at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366 and at least one gene selected from the group consisting of LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985.
Analysis of the data presented herein have enabled the calculation of a maximum threshold value that can be used to determine whether a fish is ready to be transferred to salt water or not. These calculation provides basis for an embodiment according to the additional aspect, and the disclosure of a method where expression levels of at least one of the upregulated genes identified herein as a marker for SW readiness and the expression level of at least one of the down regulated genes identified as a marker for SW readiness are measured in a biological sample of a fish to be tested, said at least one upregulated gene and at least one downregulated gene makes up a pair of genes, and wherein the expression ratio of the at least one pair provides a maximum threshold value. According to this additional aspect, a fish is considered SW ready if the calculated expression ratio of the at least one pair of genes is below the maximum threshold value shown in the table below.
The basis of these ratio thresholds is established from measurements of expression in SW-ready fish compared to incompletely smoltified fish, where maximum thresholds are the upper 95% confidence limits for the expression ratio in SW-ready fish. As shown in Figures 3, 4and 5, the maximum limits for these expression ratios are generally at least 10-fold below the lower limits for the same ration in incompletely smoltified fish.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the method comprises the steps of: a) providing a sample of integumental tissue from one or more salmonid fish to be tested; b) analyzing the sample from step a) by measuring the expression level of the following group of genes identified by its NCBI Gene ID number:
(1) at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366; and
(2) at least one gene selected from the group consisting of LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985; c) calculating the ratio of expression of one or more pairs of genes, wherein a pair comprises a first gene selected from group (1) and a second gene selected from group (2); and wherein a salmonid fish is determined to be ready for salty water when the relative expression of a pair of genes are below the following threshold values:
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the fish to be tested belong to the genera Salmo, Oncorhynchus, or Salvelinus.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the fish to be tested is selected from the group consisting of Atlantic salmon, Coho salmon, Chinook salmon and Sockeye salmon. According to one embodiment of the additional aspect, the fish to be tested is Atlantic salmon.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue, skin tissue or fin tissue.
According to yet another embodiment of the above additional aspect, a method is disclosed herein, wherein the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue. According to a second additional aspect, the present application also disclose the use of one or more genes identified by its NCBI Gene ID number and selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493,
LOC106609366, LOC106567921, LOC106565346, LOC106569928,
LOC106606117, LOC106570104, and LOC106589985 for determining sea water readiness of salmonide fish.
According to the second additional aspect disclosed herein, the use of at least two genes are disclosed, wherein at least a first gene identified by its NCBI Gene ID number is selected from the groups consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, and LOC106609366; and wherein at least a second gene identified by its NCBI Gene ID number is selected from the group consisting of LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985.
According to a third additional aspect, a kit for use in the method of the additional aspect is disclosed herein, wherein the kit comprises reagents for the measuring of protein expression levels and/or mRNA expression levels of at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen genes from the set of genes selected from the group comprising LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106591222, LOC106602268, LOC106561379, LOC106562250, LOC106592537, LOC106608496, LOC106608493, LOC106609366,
LOC106567921, LOC106565346, LOC106569928, LOC106606117, LOC106570104, and LOC106589985.
SEQUENCE LISTING
<110> Universitetet i Troms0 - Norges Arktiske Universitet <120> Method for determining saltwater readiness in salmonid fish <130> P27051EP00 <160> 4
<170> Patentln version 3.5
<210> 1 <211> 20 <212> DNA <213> Atlantic salmon <400> 1 aggctgctga gatgggtaag 20
<210> 2 <211> 20 <212> DNA <213> Atlantic salmon <400> 2 agcaacgata agcacagcac 20
<210> 3 <211> 20 <212> DNA <213> Atlantic salmon <400> 3 gggtgtgggc atcatttctg 20
<210> 4 <211> 20 <212> DNA <213> Atlantic salmon <400> 4 catccaactg ttcggctgac 20
Claims
1. An in vitro method for determining saltwater readiness of one or more salmonid fish, comprising the step of determining the expression levels of one or more genes in a biological sample, wherein the genes are selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493, wherein the tested fish is considered saltwater (SW) ready when at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOCI 06608493 is downregulated.
2. In vitro method according to claim 1, comprising the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression level of one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 in the biological sample of a).
3. In vitro method according to any of the above claims, wherein the expression level of at least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250,
LOCI 06608496, and LOCI 06608493 is determined by measuring the abundance of an RNA transcript of at least one of the said genes.
4. In vitro method according to any of the above claims, wherein the expression of at least one gene selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is measured using qRT-PCR, or high throughput RNA sequencing technologies.
5. In vitro method according to any of the above claims, comprising the steps of a) determining the expression levels of one or more genes in said biological sample, wherein the genes are selected from the group consisting of LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493;
b) normalising said determined expression level against an expression level of a salmonid housekeeping gene; wherein the tested fish is considered saltwater ready when one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOCI 06608493 is found to be downregulated based on the normalising of expression level of said salmonid housekeeping gene.
6. In invitro method according to any one of the above claims, comprising the steps of a) obtaining a biological sample of a salmonid fish exposed to smoltification conditions; b) determining the expression level of one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 in the biological sample of a); c) determining the expression of one or more housekeeping genes in the biological sample of a); d) normalising the expression of one or more genes one or more of the genes of step e) using the expression of the one or more housekeeping genes of c); wherein the fish from which the biological sample is obtained is considered saltwater ready when one or more of the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 is found to be downregulated based on the normalising of expression level of said salmonid housekeeping gene.
7. In vitro method according to any of the claims 5 and 6, wherein the housekeeping gene is a gene encoding elongation factor 1A.
8. In vitro method according to any of the above claims, wherein the expression level is determined in a biological sample from one or more salmonid fish, and wherein the biological sample is a sample of integumental tissue.
9. In vitro method according to any of the above claims, wherein the method comprises the steps of:
a) providing a sample of integumental tissue from one or more juvenile salmonid fish of a group of fish to be smoltified prior to exposing said fish for smoltification conditions; b) analysing the sample of step a) to provide a base line expression level by determining the expression level of one or more genes identified by its NCBI Gene ID number selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493; c) exposing the remaining group of salmonid fish for smoltification conditions; d) providing a sample of integumental tissue from one or more salmonid fish of the group of fish of step c); e) determining the expression level in the sample of step d) of one or more of the same genes analysed in in step b); f) comparing the expression level of the genes analysed in step b) and step e), wherein the tested fish is considered ready for salty water when at least one gene selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and
LOCI 06608493 is downregulated compared with the base line level determined in step b).
10. In vitro method according to any of the above claims, wherein the fish to be tested belong to the genera Salmo, Oncorhynchus, or Salvelinus.
11. In vitro method according to claim 10, wherein the fish to be tested is selected from the group consisting of Atlantic salmon, Coho salmon, Chinook salmon and Sockeye salmon, preferably Atlantic salmon.
12. In vitro method according to any of the above claims, wherein the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue, skin tissue or fin tissue.
13. In vitro method according to claim 12, wherein the sample from one or more salmonid fish to be tested is a sample of integumental tissue selected from the group consisting of gill tissue.
14. Use of one or more genes identified by its NCBI Gene ID number and selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 as marker for determining sea water readiness of salmonid fish.
15. Use according to claim 14 wherein the genes selected from the group consisting of LOC106605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, and LOC106608493 used as markers for saltwater readiness do not show enriched expression in mitochondrion rich cells (MRCs, also known as chloride cells) relative to pavement cells, red blood cells, vascular cells, pillar cells,, T cells, endothelial cells, goblet cells, neuroendothelial cells, myeloid cells, lymphatic endothelial cells, dendritic cells, accessory cells, fibrocytes, and vascular cells present in salmonid gill tissue.
16. Kit for use in a method according to any of the claims 1 - 13, wherein the kit comprises reagents for the measuring of protein expression levels and/or mRNA expression levels of at least one, two, three, four, five, six, seven, eight, or nine genes from the set of genes selected from the group comprising LOCI 06605916, LOC106584756, LOC106562680, LOC106562681, LOC106562250, LOC106608496, LOC106608493.
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| EP20162777 | 2020-03-12 | ||
| PCT/NO2021/050064 WO2021182973A1 (en) | 2020-03-12 | 2021-03-12 | Seawater readiness markers in salmonid fish |
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| CA (1) | CA3171141A1 (en) |
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| WO2021182973A1 (en) | 2021-09-16 |
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