# Patent

## Title
Growing marine fish in fresh water

## Abstract

The invention relates to methods, compositions and kits for raising marine fish in freshwater. The methods involve adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to the freshwater in an amount sufficient to increase expression and/or sensitivity of at least one PVCR; and adding feed for fish consumption to the freshwater, wherein the feed comprises an amount of NaCl sufficient to contribute to a significant increased level of the PVCR modulator in serum of the marine fish.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 11/057,660, filed Feb. 14, 2005, now issued U.S. Pat. No. 7,182,041, which is a continuation of U.S. application Ser. No. 10/411,076, filed Apr. 9, 2003, now issued U.S. Pat. No. 6,854,422, which is a continuation of International Application No. PCT/US01/31625, which designated the United States and was filed Oct. 11, 2001, published in English, which is a continuation-in-part of U.S. application Ser. No. 09/687,373, filed Oct. 12, 2000, now issued U.S. Pat. No. 6,463,882. The entire teachings of the above applications are incorporated herein by reference.


BACKGROUND OF THE INVENTION
Growing marine fish has been generally limited to coastal regions or seawater tanks. However, many freshwater aquifers exist, for example, in the Midwest as potential environments for the raising of marine fish. Until now, attempts to grow marine fish in freshwater have been unsuccessful.
Growing marine fish in freshwater would provide an opportunity for non-coastal areas to raise marine fish. The ability to grow marine fish in freshwater can provide fresh fish and economic growth to these areas.
Hence, a need exists to determine whether it is possible to adapt a marine fish to freshwater, and if so, understand the biological mechanisms that allow a marine fish to do so. In particular, a need exists to grow marine fish in freshwater.
SUMMARY OF THE INVENTION
The present invention relates to methods of growing marine fish in freshwater by increasing or maintaining expression of a receptor, referred to as the Polyvalent Cation Sensing Receptor (PVCR). The expression and/or sensitivity of the PVCR is modulated or maintained by subjecting the marine fish to at least one modulator of the PVCR. The marine fish are subjected to the modulator when it is added to the freshwater environment, and optionally, to the feed.
In one embodiment, the present invention is directed toward a method of growing marine fish in freshwater comprising adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to freshwater in an amount sufficient to modulate or maintain expression and/or sensitivity of at least one PVCR in one or more tissues; transferring the marine fish to the freshwater and adding feed for fish consumption to the modified freshwater, wherein the feed contains an amount of NaCl sufficient to contribute to modulate or maintain levels of said PVCR modulator in serum of the marine fish. PVCR modulators useful in the present invention include a divalent cation, a trivalent cation, an aminoglycoside, an organic polycation, an amino acid, a Type I Calcimimetic, a Type II Calcimimetic,. 1,25 dihydroxyvitamin D, a cytokine, and macrophage chemotatic peptide-1. The feed suitable in the methods of the present invention contains at least about 1% NaCl by weight and can optionally include a PVCR modulator.
The present invention also encompasses a method of transferring marine fish to freshwater comprising adding at least one Polyvalent Cation Sensing Receptor (PVCR) modulator to the freshwater in an amount sufficient to modulate or maintain expression and/or sensitivity of at least one PVCR in one or more tissues, transferring the marine fish to the freshwater, adding feed for fish consumption to the modified freshwater, wherein the feed contains at least about 1% NaCl by weight. The PVCR modulator can be a PVCR agonist, a divalent cation, a trivalent cation, an aminoglycoside, an organic polycation or an amino acid.
In another embodiment, the present invention is directed toward a method of growing marine fish in freshwater comprising determining the level of at least one PVCR modulator in freshwater, adding said PVCR modulator to the freshwater in an amount sufficient to modulate or maintain expression and/or sensitivity of at least one PVCR in one or more tissues, transferring the marine fish to the freshwater and adding feed for fish consumption to the modified freshwater, wherein the feed contains an amount of NaCl sufficient to modulate or maintain levels of said PVCR modulator in serum of the marine fish (calcium and magnesium). PVCR modulator that can be assessed. The PVCR modulator is added to freshwater such that the freshwater has between about 0.3 mM and about 10.0 mM calcium and between about 0.5 mM and about 10.0 mM magnesium prior to transferring marine fish.
The present invention is also directed to a method of growing marine fish in freshwater having between about 0.3 mM and about 10.0 mM of calcium and between about 0.5 mM and 10.0 mM of magnesium. The method comprises adding feed to the freshwater wherein the feed contains an amount of NaCl sufficient to modulate or maintain levels of said PVCR modulator in serum of the marine fish, wherein modulated or maintained expression of at least one PVCR is modulated or maintained in one or more tissues.
In another embodiment, the present invention is directed toward a method of transferring marine fish to freshwater comprising transferring the marine fish to freshwater having magnesium and calcium in the freshwater in amounts sufficient to modulate or maintain the expression and/or sensitivity of at least one PVCR in one or more tissues and adding feed to the freshwater, wherein the feed contains at least about 1% NaCl by weight.
The present invention is also directed to a method of growing flounder in freshwater comprising transferring flounder to freshwater having at least one PVCR modulator in an amount sufficient to increase or maintain expression and/or sensitivity of at least one PVCR in one or more tissue and adding feed for fish consumption to the freshwater, wherein the feed contains an amount of NaCl sufficient to contribute to a significant increased level of said PVCR modulator in serum of the flounder. The pH of the freshwater should be greater than 7.0.
In another embodiment, the present invention is directed toward an aquatic mixture for providing an environment to transfer marine fish to freshwater, comprising at least one PVCR modulator. An aquatic mixture is a medium suitable for transfer of marine fish to freshwater during aquaculture.
The present invention is also directed to a kit for growing marine fish in freshwater comprising an aquatic mixture for providing an environment to grow the marine fish, wherein the aquatic mixture comprises at least one PVCR modulator; and an aquatic food composition containing a concentration of NaCl between about 10,000 mg/kg and about 100,000 mg/kg.
Surprisingly, it has been discovered that modulated or maintained expression and/or altering the sensitivity of the PVCR allows these marine fish to live and thrive in freshwater. Until the discovery of the present invention, the aquaculture industry was unable to transfer the marine fish to freshwater without subjecting the fish to stress, death and/or disease. Unlike this practice, carrying out the steps of the invention modulates or maintains the expression and/or alters the sensitivity of the PVCR and allows for transfer of the marine fish to freshwater with minimal or no stress, death and/or disease, and unexpectedly, the fish grow. In fact, marine fish that grow in freshwater have a higher fat content, and a milder, less “fishy” taste.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B are diagrams illustrating the partial nucleotide (SEQ ID NO: 1) and amino acid (SEQ ID NO:2) sequences of the PVCR of Cod.
FIGS. 2A and 2B are diagrams illustrating the partial nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences of the PVCR of Haddock.
FIGS. 3A and 3B are diagrams illustrating the partial nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences of the PVCR of Hake.
FIGS. 4A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:7) and amino acid (SEQ ID NO:8) sequences of the PVCR of Halibut.
FIGS. 5A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:9) and amino acid (SEQ ID NO: 10) sequences of the PVCR of Mackerel.
FIGS. 6A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:11) and amino acid (SEQ ID NO:12) sequences of the PVCR of Pollack.
FIGS. 7A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:13) and amino acid (SEQ ID NO:14) sequences of the PVCR of Sea Bass.
FIGS. 8A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:15) and amino acid (SEQ ID NO:16) sequences of the PVCR of Swordfish.
FIGS. 9A-B are diagrams illustrating the partial nucleotide (SEQ ID NO:17) and amino acid (SEQ ID NO:18) sequences of the PVCR of Tuna.
FIGS. 10A-C are diagrams illustrating the partial nucleotide (SEQ ID NO:19) and amino acid (SEQ ID NO:20) sequences of the PVCR of Winter Flounder.
FIG. 11 is a diagram illustrating the partial nucleotide (SEQ ID NO:21) and amino acid (SEQ ID NO:22) sequences of PVCR of Summer Flounder.
FIGS. 12A-D are diagrams illustrating the alignment of the nucleic acids sequences for Cod (SEQ ID NO:1), Haddock (SEQ ID NO:3), Hake (SEQ ID NO:5), Halibut (SEQ ID NO:7), Mackerel (SEQ ID NO:9), Pollock (SEQ ID NO:11), Sea Bass (SEQ ID NO:13), Swordfish (SEQ ID NO:15), Tuna (SEQ ID NO:17), Winter Flounder (SEQ ID NO:19).
FIGS. 13A-C are diagrams illustrating the alignment of the amino acids sequences for Cod (SEQ ID NO:2), Haddock (SEQ ID NO:4), Hake (SEQ ID NO:6), Halibut (SEQ ID NO:8), Mackerel (SEQ ID NO:10), Pollock (SEQ ID NO:12), Sea Bass (SEQ ID NO:14), Swordfish (SEQ ID NO:16), Tuna (SEQ ID NO:18), Winter Flounder (SEQ ID NO:20).
FIGS. 14A-B are diagrams illustrating the nucleic acid sequence of SKCaR (SEQ ID NO:23).
FIG. 15 is a graphical representation illustrating the growth of summer flounder in freshwater that underwent APS Process I and grown in freshwater for a total of 51 days. Samples of body characteristics of flounders were obtained at (1) prior to placement in freshwater; (2) 20 days after placement in freshwater; (3) 30 days after placement in freshwater; and (4) 51 days after placement in freshwater. APS Process I is defined in Example 2.
FIG. 16 is a graphical representation illustrating the growth of summer flounder in seawater for a total of 51 days. Samples of body characteristics of flounders were obtained at (1) prior to placement in seawater; (2) 20 days after placement in seawater; (3) 30 days after placement in seawater; and (4) 51 days after placement in seawater.



DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods for growing or raising marine fish in freshwater. The methods involve modulating or maintaining expression and/or altering the sensitivity of a Polyvalent Cation Sensing Receptor (PVCR) (e.g., at least one PVCR). The invention relates to modulating or maintaining expression of the PVCR that affects the fish's ability to adapt to freshwater.
In particular, the methods of the present invention include adding at least one PVCR modulator to the freshwater, and adding a specially made or modified feed to the freshwater for consumption by the fish. The feed contains a sufficient amount of sodium chloride (NaCl) (e.g., between about 1% and about 10% by weight, or about 10,000 mg/kg to about 100,000 mg/kg) to modulate or maintain levels of the PVCR modulator in the serum. This amount of NaCl in the feed causes or induces the marine fish to drink more freshwater. Since the freshwater contains a PVCR modulator and the fish ingest increased amounts of it, the serum level of the PVCR modulator significantly increases in the fish, and causes increased or maintained PVCR expression and/or altered PVCR sensitivity. A “significant” increase is used herein to refer to a measurable rise in the level or quantity of PVCR or RVCR modulator as compared to a control or reference. Methods of measuring or detecting a significant increase in PVCR or PVCR modulator are disclosed herein and known to one skilled in the art.
The methods of the present invention pertain to adapting marine fish to freshwater. Marine fish are fish that live, at least for most of their adult lives, in seawater. Marine fish include, for example, Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder, and Summer Flounder. The marine fish are adapted to freshwater having a PVCR modulator.
The term “marine fish” is understood by one of skill in the art. The term, “freshwater,” means water that comes from, for example, a stream, river, ponds, public water supply, or from other non-marine sources having, for example, the following ionic composition: less than about 2 mM of magnesium, calcium and NaCl. The phrases “modified freshwater,” “freshwater as modified by the addition of a PVCR” and “PVCR modulator environment” refer to freshwater to which at least one PVCR modulator has been added, as described herein.
The PVCR modulator is added to the freshwater in sufficient amounts to modulate or maintain expression or alter the sensitivity of at least one PVCR. A PVCR has been isolated from various tissue of several types of marine fish using molecular biological techniques. For example, nucleic acid was isolated from tissue samples from various species of marine fish including Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder and Summer Flounder. The nucleic acid was amplified using Polymerase Chain Reaction (PCR) methodology. The amplified DNA was purified, subcloned into vectors, and their sequences were determined, as described in Example 4.
The PVCR, which is located in various tissues (e.g., gill, skin, intestine, kidney, urinary bladder, brain or muscle) of the marine fish, senses alterations in PVCR modulators including various ions (e.g., divalent cations), for example, in the surrounding water, in their serum or in the luminal contents of tubules inside the body, such as kidney, urinary bladder, or intestine. The ability to sense PVCR modulators results in a modulation or a maintenance in the expression of PVCR, thereby allowing the fish to better adapt to freshwater. Modulated or maintained expression of the PVCR can occur, for example, in one or more tissues. As used herein, the “sensitivity” of the PVCR refers to alteration of PVCR expression in response to a change in the concentration of PVCR modulators. PVCR expression can be assessed by measuring or detecting PVCR polypeptide or nucleic acid molecules in a sample by standard methods.
A “PVCR modulator” is defined herein to mean a compound which modulates expression of the PVCR, or modulates the sensitivity or responsiveness of the PVCR, or maintains an already increased PVCR expression level in one or more tissues. Such compounds include, but are not limited to, PVCR agonists (e.g., inorganic polycations, organic polycations and amino acids), Type II calcimimetics, and compounds that indirectly alter PVCR expression (e.g., 1,25 dihydroxyvitamin D in concentrations of about 3,000-10,000 International Units/kg feed), cytokines such as Interleukin Beta, and Macrophage Chemotatic Peptide-1 (MCP-1)). Examples of Type II calcimimetics, which modulate expression and/or sensitivity of the PVCR, are, for example, NPS-R-467 and NPS-R-568 from NPS Pharmaceutical Inc., (Salt Lake, Utah, Pat. Nos. 5,962,314; 5,763,569; 5,858,684; 5,981,599; 6,001,884) which can be administered in concentrations of between about 0.1 μM and about 100 μM feed or water. See Nemeth, E. F. et al., PNAS 95: 4040-4045 (1998).
Examples of inorganic polycations are divalent cations including calcium at a concentration between about 0.3 and about 10.0 mM and magnesium at a concentration between about 0.5 and about 10.0 mM; and trivalent cations including, but not limited to, gadolinium (Gd3+) at a concentration between about 1 and about 500 μM.
Organic polycations include, but are not limited to, aminoglycosides such as neomycin or gentamicin in concentrations of between about 1 and about 8 gm/kg feed as well as organic polycations including polyamines (e.g., polyarginine, polylysine, polyhistidine, polyornithine, spermine, cadaverine, putricine, copolymers of poly arginine/histidine, poly lysine/arginine in concentrations of between about 10 μM and 10 mM feed). See Brown, E. M. et al., Endocrinology 128: 3047-3054 (1991); Quinn, S. J. et al., Am. J. Physiol. 273: C1315-1323 (1997).
Additionally, PVCR agonists include amino acids such as L-Tryptophan, L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine, L-Histidine, L-Leucine, L-Isoleucine, and L-Cystine at concentrations of between about 1 and about 10 gm/kg feed. See Conigrave, A. D., et al., PNAS 97: 4814-4819 (2000). The molar concentrations refer to free or ionized concentrations of the PVCR modulator in the freshwater, and do not include amounts of bound PVCR modulator (e.g., PVCR modulator bound to negatively charged particles including glass, proteins, or plastic surfaces). Any combination of these modulators can be added to the water or to the feed (in addition to the NaCl, as described herein), so long as the combination modulates or maintains expression and/or sensitivity of at least one PVCR.
The PVCR modulator can be administered to the fish in a number of ways. The invention encompasses administration of the PVCR in any way that is sufficient to modulate or maintain the expression and/or alter the sensitivity of the PVCR. In one embodiment, the PVCR modulator is simply added to the freshwater, as described herein. PVCR modulators that are added to the water increase or maintain or decrease expression and/or alter the sensitivity of the PVCR on the skin and gills of the fish, and can be ingested by the fish, in particular, when fish are fed feed having between about 1% and about 10% NaCl (e.g., in concentrations between about 1 and about 10 gm/100 gm feed). In addition to adding NaCl to the feed, the PVCR modulator can also be added to the feed. Amounts and types of PVCR modulators added to the feed are also described herein. Other embodiments include subjecting the fish to the PVCR modulator by “dipping” the fish in the modulator, e.g., organic polycations. The organic polycations can be formulated in such a way as to allow the polycations to adhere to the skin and gills of the fish, in sufficient amounts to increase or maintain expression of the PVCR.
The invention also embodies assessing the amounts of existing PVCR modulator in the freshwater environment and in the serum of fish. PVCR modulators are assessed or measured using methods known in the art. After assessment, the PVCR modulator is added to the water to bring the concentration up to an amount sufficient to modulate or maintain expression and/or sensitivity of at least one PVCR, or sufficient to bring the concentrations of the PVCR modulator within the stated ranges. For example, an aquifer assessed at having only 0.2 mM of calcium needs additional calcium to bring the concentration up to between about 0.3 mM and between about 10.0 mM.
In a preferred embodiment, the present invention is practiced by adding a combination of two PVCR agonists to the freshwater. In particular, calcium and magnesium are added to the freshwater to bring the concentrations of each to between about 0.3 mM and about 10.0 mM of calcium, and between about 0.5 mM and about 10.0 mM of magnesium. In addition to adding calcium and magnesium to the water, these ranges of ion concentrations can be achieved by providing a brackish water (e.g., diluted seawater) environment for the fish.
Calcium and magnesium can come from a variety of sources, that when added to the water, the calcium and/or magnesium levels modulate or maintain expression of the PVCR, and/or are within the stated ranges. Sources of calcium and magnesium can be a mixture of a variety of compounds, or each can come from a substantially uniform or pure compound. Sources of calcium include, for example, Ca(CO3)2, CaCl2, and CaSO4 and sources of magnesium include, for example, MgCl2, MgSO4, MgBr2, and MgCO3.
In one embodiment, the invention includes intermittent (e.g., interrupted) as well as continuous (e.g., non-interrupted) exposure to freshwater having at least one PVCR modulator, while on the NaCl diet. Intermittent exposure to the PVCR can occur so long as the PVCR expression and/or altered sensitivity remains modulated or maintained. Continuous maintenance in or exposure to freshwater having at least one PVCR modulator is shown in Example 2.
The marine fish are transferred from seawater. The term, “seawater,” means water that comes from the sea, or water which has been formulated to simulate the chemical and mineral composition of water from the sea. The major elemental composition of the prepared seawater preferably falls substantially within the range of the major elemental composition of the natural seawater (e.g, having the following ionic composition: greater than 30 mM of magnesium, greater than about 6 mM of calcium, and greater than about 300 mM NaCl). Methods of preparing artificial seawater are known in the art and are described in, for instance, U.S. Pat. No. 5,351,651.
In one embodiment, the marine fish are treated by the methods of the present invention by subjecting the fish to a gradual or step-wise decrease in salinity for a period of time prior to transfer to freshwater, while being fed a NaCl diet. Salinity refers to the ionic concentrations (e.g., calcium, magnesium and sodium) in water. The fish are maintained in a decreasing salinity environment for a sufficient period of time to modulate or maintain expression and/or sensitivity of at least one PVCR. Factors that can influence the length of time to maintain the fish in a decreased salinity prior to transfer to freshwater include, but are not limited to, size of the fish, level of PVCR expression or sensitivity, if any, prior to addition of the PVCR modulator to the freshwater, the fish's ability to excrete the PVCR modulator and ions, and the fish's surface to volume ratio. Therefore, the length of time the fish is maintained can range between about 5 days and about 60 days, and preferably, between about 10 days and about 25 days.
The ionic concentrations of seawater are decreased by between about 10% and about 90%, and preferably, between about 25% and about 50%. Combinations of decreasing salinity and various lengths of exposure to the salinity are encompassed by the invention. For example, as described in Example 2, fish were adapted to 50% seawater (50% salinity of seawater) for 10 days, and then adapted to 25% seawater (25% salinity of seawater) for 15 days, prior to transfer to freshwater. “Adapted” as used herein, refers to a successful transition to the altered aquatic environment. After maintenance in water having decreasing salinity, as compared to seawater, the marine fish are then placed into freshwater having a PVCR modulator, as described herein. The fish can remain and grow in freshwater, modified by the addition of PVCR modulators, indefinitely, so long as there is modulated or maintained expression and/or sensitivity of the PVCR (e.g., maintained in modified freshwater and fed an NaCl diet).
The invention further includes adding feed to the freshwater. The frequency and amount of feed that fish are fed, are taught in the art. Generally, the fish are fed 1-3 times a day, totaling about 0.25-0.5% body weight/day. The feed has enough NaCl to contribute to a modulated or maintained level of the PVCR modulator in the serum of the marine fish. Specifically, the presence of sufficient amounts of NaCl in the feed causes the marine fish to drink more water from the surrounding environment. Although NaCl decreases PVCR sensitivity, the ingestion of freshwater having at least one PVCR modulator causes an overall rise in the serum level of PVCR modulator. The increase in serum levels of PVCR modulator results in a modulation in expression of PVCR.
In another embodiment, the present invention is directed toward an aquatic mixture for providing an environment to transfer marine fish to freshwater, comprising at least one PVCR modulator. An “aquatic mixture” is defined herein to mean a composition that provides a suitable environment for the successful transfer of marine fish to freshwater by the methods of the present invention. The aquatic mixture can be premixed for immediate use in the methods of the present invention. Alternatively, the aquatic mixture can require reconstitution with water. The aquatic mixture when reconstituted yields a solution comprising about 0.3-10 mM Ca2+ and about 0.5-10 mM Mg2+. The aquatic mixture can optionally include an amino acid in an amount between about 1 gm/kg and about 10 gm/kg.
The present invention also relates to an aquatic food composition. An “aquatic food composition” refers to fish feed, as described herein. An aquatic food composition or feed suitable for use in the present invention contains between about 1%-10% of NaCl by weight, or between about 10,000 mg NaCl/kg of feed and about 100,000 mg NaCl/kg of feed (e.g., 12,000 mg/kg). The feed is referred to herein as a “NaCl diet.” The NaCl can be combined with other sodium salts to confer the desired effect of modulating or maintaining PVCR expression, altering PVCR sensitivity and/or inducing the fish to drink more. Hence, as used herein, the term NaCl, includes a substantially pure compound, mixtures of NaCl with other sources of sodium, or other sources of sodium. The feed can further include a PVCR modulator, and in particular a PVCR agonist such as an amino acid. In one embodiment, the feed has between about 1% and about 10% NaCl by weight and an amino acid such as tryptophan in an amount between about 1 and about 10 gm/kg. This embodiment is referred to herein as “APS Process II,” which is further defined in Example 2.
The feed can be made in a number of ways, so long as the proper concentration of NaCl is present. The feed can be made, for example, by reformulating the feed, or by allowing the feed to absorb a solution having the NaCl and optionally, adding a PVCR modulator. Additionally, a top dressing can be added for palatability. Example 3 describes in detail one way to make the feed. Alternate methods of preparing fish feed are know to those of skill in the relevant art.
Another embodiment of the present invention includes feeding marine fish feed having between 1% and 10% NaCl by weight when the fish are maintained in a freshwater environment having between about 0.3 and about 10.0 mM of calcium, and between about 0.5 mM and about 10.0 mM of magnesium. When this embodiment of the present invention is carried out, the levels of calcium, magnesium and/or sodium in the serum of the marine fish is increased, as compared to PVCR expression and/or sensitivity seen in freshwater fish.
In another embodiment, the fish, while in water having decrease salinity, as compared to seawater, or while in the freshwater having the PVCR modulator, are also exposed to a photoperiod. A photoperiod refers to exposing the fish to light (e.g., sunlight, incandescent light or fluorescent light). Preferably, the photoperiod is substantially continuous, or occurs long enough to increase growth. The photoperiod can occur for at least about 12 hours within a 24 hour interval, or for longer periods such as about 14, 16, 18, 20, 22 or preferably, about 24 hours.
The methods of the present invention modulate or maintain the expression and/or sensitivity of the PVCR in marine fish which results in reduced osmotic stress and in reduced mortality. Marine fish cultured in freshwater by methods of the present invention consume feed and exhibit growth. In contrast, marine fish that are not cultured in freshwater by methods of the present invention experience osmotic stress, reduced or no food consumption, and eventually death. The osmotic stress results from differences in the osmotic pressure between the surrounding environment and body compartments of the fish. This disturbs the homeostatic equilibrium of the fish and results in decreased growth, reproductive failure and reduced resistance to disease. The fish that have undergone the steps of the present invention do not experience a significant amount of osmotic stress. As a result, the fish are able to grow. Surprisingly, as described and exemplified herein, marine fish adapted by the present invention grow almost as well as marine fish maintained in seawater (e.g., 53% increased growth in fish subjected to the present invention for 37 days, as compared to 60% increased growth of fish maintained in seawater for 37 days). Additionally, marine fish cultured in freshwater by methods of the present invention exhibit a survival rate that is significantly greater than the rate for marine fish that are transferred directly to freshwater and not subjected to the steps of the present invention (e.g., between about 60% and about 100%). See FIGS. 15 and 16.
The methods of the present invention also decrease the incidence of disease among the marine fish transferred to freshwater. Because the fish treated with the methods of the present invention experience less stress upon transfer to freshwater, their immune functions are stronger, and they are less susceptible to parasitic, viral, bacterial and fungal diseases. Thus, marine fish cultured by methods of the present invention are healthier.
Methods Assessment of the PVCR
The present invention includes methods of detecting the level of the PVCR to determine whether fish are ready for transfer from seawater to freshwater. Methods that measure PVCR levels include several suitable assays. Suitable assays encompass immunological methods, such as FACS analysis, radioimmunoassay, flow cytometry, enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays. Any method known now or developed later can be used for measuring PVCR expression.
Antibodies reactive with the PVCR or portions thereof can be used. In a preferred embodiment, the antibodies specifically bind with the PVCR or a portion thereof. The antibodies can be polyclonal or monoclonal, and the term antibody is intended to encompass polyclonal and monoclonal antibodies, and functional fragments thereof. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
In several of the preferred embodiments, immunological techniques detect PVCR levels by means of an anti-PVCR antibody (i.e., one or more antibodies). The term “anti-PVCR” antibody includes monoclonal and/or polyclonal antibodies, and mixtures thereof.
Anti-PVCR antibodies can be raised against appropriate immunogens, such as isolated and/or recombinant PVCR or portion thereof (including synthetic molecules, such as synthetic peptides). In one embodiment, antibodies are raised against an isolated and/or recombinant PVCR or portion thereof (e.g., a peptide) or against a host cell which expresses recombinant PVC R. In addition, cells expressing recombinant PVCR, such as transfected cells, can be used as immunogens or in a screen for antibody which binds receptor.
Any suitable technique can prepare the immunizing antigen and produce polyclonal or monoclonal antibodies. The art contains a variety of these methods (see e.g., Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature, 266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)). Generally, fusing antibody producing cells with a suitable immortal or myeloma cell line, such as SP2/0, can produce a hybridoma. For example, animals immunized with the antigen of interest provide the antibody producing cell, preferably cells from the spleen or lymph nodes. Selective culture conditions isolate antibody producing hybridoma cells while limiting dilution techniques produce them. Researchers can use suitable assays such as ELISA to select antibody producing cells with the desired specificity.
Other suitable methods can produce or isolate antibodies of the requisite specificity. Examples of other methods include selecting recombinant antibody from a library or relying upon immunization of animals such as mice.
According to the method, an assay can determine the level of PVCR in a biological sample. In determining the amounts of PVCR, an assay includes combining the sample to be tested with an antibody having specificity for the PVCR, under conditions suitable for formation of a complex between antibody and the PVCR, and detecting or measuring (directly or indirectly) the formation of a complex. The sample can be obtained directly or indirectly, and can be prepared by a method suitable for the particular sample and assay format selected.
In particular, tissue samples, e.g., gill tissue samples, can be taken from fish after they are anaesthetized with MS-222. The tissue samples are fixed by immersion in 2% paraformaldehyde in appropriate Ringers solution corresponding to the osmolality of the fish, washed in Ringers, then frozen in an embedding compound, e.g., O.C.T.TM (Miles, Inc., Elkahart, Ind., USA) using methylbutane cooled with liquid nitrogen. After cutting 8-10μ tissue sections with a cryostat, individual sections are subjected to various staining protocols. For example, sections are: 1) blocked with goat serum or serum obtained from the same species of fish, 2) incubated with rabbit anti-CaR or anti-PVCR antiserum, and 3) washed and incubated with peroxidase-conjugated affinity-purified goat antirabbit antiserum. The locations of the bound peroxidase-conjugated goat antirabbit antiserum are then visualized by development of a rose-colored aminoethylcarbazole reaction product. Individual sections are mounted, viewed and photographed by standard light microscopy techniques. One anti-CaR antiserum used to detect fish PVCR protein is raised in rabbits using a 23-mer peptide corresponding to amino acids numbers 214-236 localized in the extracellular domain of the RaKCaR protein (Riccardi et al., P.N.A.S. 92:131-135 (1995); accession number NP 058692). The sequence of the 23-mer peptide is: ADDDYGRPGIEKFREEAEERDIC (SEQ ID NO:24) A small peptide with the sequence DDYGRPGIEKFREEAEERDICI (SEQ ID NO:25) or ARSRNSADGRSGDDLPC (SEQ ID NO:26) can also be used to make antisera containing antibodies to PVCRs. Such antibodies can be monoclonal, polyclonal or chimeric.
Suitable labels can be detected directly, such as radioactive, fluorescent or chemiluminescent labels. They can also be indirectly detected using labels such as enzyme labels and other antigenic or specific binding partners like biotin. Examples of such labels include fluorescent labels such as fluorescein, rhodamine, chemiluminescent labels such as luciferase, radioisotope labels such as 32P, 125I, 131I, enzyme labels such as horseradish peroxidase, and alkaline phosphatase, β-galactosidase, biotin, avidin, spin labels and the like. The detection of antibodies in a complex can also be done immunologically with a second antibody which is then detected (e.g., by means of a label). Conventional methods or other suitable methods can directly or indirectly label an antibody.
In performing the method, the levels of the PVCR are distinct from the control. Varied levels or the presence of PVCR expression, as compared to a control, indicate that the fish or the population of fish from which a statistically significant amount of fish were tested, are ready for transfer to freshwater. A control refers to a level of PVCR, if any, from a fish that is not subjected to the steps of the present invention, e.g., not subjected to freshwater having a PVCR modulator and/or not fed a NaCl diet.
The PVCRs can also be assayed by Northern blot analysis of mRNA from tissue samples. Northern blot analysis from various shark tissues has revealed that the highest degree of PVCRs expression is in gill tissue, followed by the kidney and the rectal gland. There appear to be at least three distinct mRNA species of about 7 kb, 4.2 kb and 2.6 kb. For example, the PVCRs can also be assayed by hybridization, e.g., by hybridizing one of the PVCR sequences provided herein (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or 23), its complement or an oligonucleotide derived from one of the sequences, to a mRNA purified from tissue sample from a fish. Such a hybridization sequence can have a detectable label, e.g., radioactive, fluorescent, etc., attached, to allow the detection of hybridization product. Methods for hybridization are well known, and such methods are provided in U.S. Pat. No. 5,837,490, by Jacobs et al., the entire teachings of which are herein incorporated by reference in their entirety. The design of the oligonucleotide probe should preferably follow these parameters: (a) it should be designed to an area of the sequence which has the fewest ambiguous bases (“N's”), if any, and (b) it should be designed to have a Tm of approx. 80° C. (assuming 2° C. for each A or T and 4 degrees for each G or C).
Stringency conditions for hybridization refers to conditions of temperature and buffer composition which permit hybridization of a first nucleic acid sequence to a second nucleic acid sequence, wherein the conditions determine the degree of identity between those sequences which hybridize to each other. Therefore, “high stringency conditions” are those conditions wherein only nucleic acid sequences which are very similar to each other will hybridize. The sequences can be less similar to each other if they hybridize under moderate stringency conditions. Still less similarity is needed for two sequences to hybridize under low stringency conditions. By varying the hybridization conditions from a stringency level at which no hybridization occurs, to a level at which hybridization is first observed, conditions can be determined at which a given sequence will hybridize to those sequences that are most similar to it. The precise conditions determining the stringency of a particular hybridization include not only the ionic strength, temperature, and the concentration of destabilizing agents such as formamide, but also on factors such as the length of the nucleic acid sequences, their base composition, the percent of mismatched base pairs between the two sequences, and the frequency of occurrence of subsets of the sequences (e.g., small stretches of repeats) within other non-identical sequences. Washing is the step in which conditions are set so as to determine a minimum level of similarity between the sequences hybridizing with each other. Generally, from the lowest temperature at which only homologous hybridization occurs, a 1% mismatch between two sequences results in a 1° C. decrease in the melting temperature (Tm) for any chosen SSC concentration. Generally, a doubling of the concentration of SSC results in an increase in the Tm of about 17° C. Using these guidelines, the washing temperature can be determined empirically, depending on the level of mismatch sought. Hybridization and wash conditions are explained in Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds., John Wiley & Sons, Inc., 1995, with supplemental updates) on pages 2.10.1 to 2.10.16, and 6.3.1 to 6.3.6.
High stringency conditions can employ hybridization at either (1) 1×SSC (10×SSC=3 M NaCl, 0.3 M Na3-citrate·2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (2) 1×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen (fraction V), 1 mM Na2·EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4.7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42° C., with high stringency washes of either (1) 0.3-0.1×SSC, 0.1% SDS at 65° C., or (2) 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS at 65° C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log10M)+0.41(% G+C)−0.61(% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na+), and “L” is the length of the hybrid in base pairs.
Moderate stringency conditions can employ hybridization at either (1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate·2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (2) 4×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (3) 1% bovine serum albumen (fraction V), 1 mM Na2 EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4.7H2O, 4 ml 85% H3PO4 per liter 0.1-2 mg/ml denatured calf thymus DNA at 65° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 42° C., (5) 5×SSC, 5× Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 65° C., or (6) 5×SSC, 5× Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 42° C., with moderate stringency washes of 1×SSC, 0.1% SDS at 65° C. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log10M)+0.41(% G+C)−0.61(% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na+), and “L” is the length of the hybrid in base pairs.
Low stringency conditions can employ hybridization at either (1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate·2H2O (88 g/liter), pH to 7.0 with 1 M HCl), 1% SDS (sodium dodecyl sulfate), 0.1-2 mg/ml denatured calf thymus DNA at 50° C., (2) 6×SSC, 50% formamide, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 40° C., (3) 1% bovine serum albumen (fraction V), 1 mM Na2·EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO4.7H2O, 4 ml 85% H3PO4 per liter), 7% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 50° C., (4) 50% formamide, 5×SSC, 0.02 M Tris-HCl (pH 7.6), 1× Denhardt's solution (100×=10 g Ficoll 400, 10 g polyvinylpyrrolidone, 10 g bovine serum albumin (fraction V), water to 500 ml), 10% dextran sulfate, 1% SDS, 0.1-2 mg/ml denatured calf thymus DNA at 40° C., (5) 5×SSC, 5×Denhardt's solution, 1% SDS, 100 μg/ml denatured calf thymus DNA at 50° C., or (6) 5×SSC, 5× Denhardt's solution, 50% formamide, 1% SDS, 100 μg/ml denatured calf thymus DNA at 40° C., with low stringency washes of either 2×SSC, 0.1% SDS at 50° C., or (2) 0.5% bovine serum albumin (fraction V), 1 mM Na2EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. The above conditions are intended to be used for DNA-DNA hybrids of 50 base pairs or longer. Where the hybrid is believed to be less than 18 base pairs in length, the hybridization and wash temperatures should be 5-10° C. below that of the calculated Tm of the hybrid, where Tm in ° C.=(2× the number of A and T bases)+(4× the number of G and C bases). For hybrids believed to be about 18 to about 49 base pairs in length, the Tm in ° C.=(81.5° C.+16.6(log10M)+0.41(% G+C)−0.61(% formamide)−500/L), where “M” is the molarity of monovalent cations (e.g., Na+), and “L” is the length of the hybrid in base pairs.
Hence, the present invention includes kits for the detection of the PVCR or the quantification of the PVCR having either antibodies specific for the PVCR protein or a portion thereof, or a nucleic acid sequence that can hybridize to the nucleic acid of the PVCR.
Alterations in the expression or sensitivity of PVCRs could also be accomplished by introduction of a suitable transgene. Suitable transgenes would include either the PVCR gene itself or modifier genes that would directly or indirectly influence PVCR gene expression. Methods for successful introduction, selection and expression of the transgene in fish oocytes, embryos and adults are described in Chen, TT et al., Transgenic Fish, Trends in Biotechnology 8:209-215 (1990).
The present invention is further and more specifically illustrated by the following Examples, which are not intended to be limiting in any way.
EXEMPLIFICATION
Example 1 
Polyvalent Cation-Sensing Receptors (PVCRs) Serve as Salinity Sensors in Fish 
Polyvalent cation-sensing receptors (PVCRs) serve as salinity sensors in fish. These receptors are localized to the apical membranes of various cells within the fish's body (e.g., in the gills, intestine, kidney) that are known to be responsible for osmoregulation. A full-length cation receptor (CaR) from the dogfish shark has been expressed in human HEK cells. This receptor was shown to respond to alterations in ionic compositions of NaCl, Ca2+ and Mg2+ in extracellular fluid bathing the HEK cells. The ionic concentrations responded to encompassed the range which includes the transition from freshwater to seawater. Expression of PVCR mRNA is also modulated in fish after their transfer from freshwater to seawater, and is modulated by PVCR agonists.
Using nucleic acid amplification with degenerate primers, partial genomic clones of PVCRs have also been isolated from other fish species, including Cod (FIGS. 1A-B), Haddock (FIGS. 2A-B), Hake (FIGS. 3A-B), Halibut (FIGS. 4A-B), Mackerel (FIGS. 5A-B), Pollock (FIGS. 6A-B), Sea Bass (FIGS. 7A-B), Swordfish (FIGS. 8A-B), Tuna (FIGS. 9A-B), Winter Flounder (FIGS. 10A-10C) and Summer Flounder (FIG. 11). The degenerate oligonucleotide primers used for isolating these clones, except for Winter Flounder, were 5′-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3′ (SEQ ID NO:27) and 5′-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3′ (SEQ ID NO:28), where K is T or G, Y is C or T, and R is A or G. The degenerate oligos were generated by standard methodologies (Preston, G. M., 1993, “Polymerase chain reaction with degenerate oligonucleotide primers to clone gene family members,” in: Methods in Mol. Biol., vol. 58, ed. A. Harwood, Humana Press, pp. 303-312). Nucleic acids from these species were amplified, purified by agarose gel electrophoresis, ligated into an appropriate plasmid vector (Novagen's pT7 Blue or Promega's pGEM-T) and transformed into an appropriate bacterial host strain (Novagens' Nova Blue Competent Cells or Promega's JM 109 competent cells). The plasmids and inserts were purified from the host cells, and sequenced. FIGS. 13A-C shows the deduced amino acid sequences and alignment for the PVCRs from Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna and Winter Flounder.
A winter flounder lambda ZAP cDNA library was manufactured using standard commercially available reagents with cDNA synthesized from poly A+ RNA isolated from winter flounder urinary bladder tissue as described and published in Siner et al. Am. J. Physiol. 270:C372-C381, 1996. The winter flounder urinary bladder cDNA library was plated and resulting phage plaques screened using a 32P-labeled shark kidney calcium receptor cDNA probe under intermediate stringency conditions (0.5×SSC, 0.1% SDS, 50° C). Individual positive plaques were identified by autoradiography, isolated and rescued using phagemid infections to transfer cDNA to KS Bluescript vector. The nucleotide (nt) sequence, FIG. 10A, (SEQ ID NO:19) of the winter flounder PVCR clone was obtained using commercially available automated sequencing service that performs nucleotide sequencing using the dideoxy chain termination technique. The deduced amino acid sequence (SEQ ID NO:20) is shown in FIGS. 10B and 10C. The winter flounder PVCR nucleotide sequence was compared to others aquatic PVCR using commercially available nucleotide and protein database services including GENBANK and SWISS PIR.
Example 2 
Growth of Marine Fish in Freshwater Using the Methods of the Present Invention
Methods:
The following examples refer to APS Process I and APS Process II throughout. APS stands for “AquaBio Products Sciences®, L.L.C.” APS Process I is also referred to herein as “SUPERSMOLT ® I Process” or “Process I.” An “APS Process I” fish or smolt refers to a fish or smolt that has undergone the steps of APS Process I. An APS Process I smolt is also referred to as a “SUPERSMOLT® I” or a “Process I ” smolt. Likewise, APS Process II is also referred to herein as “SUPERSMOLT® II Process” or “Process II.” An “APS Process II” fish or smolt refers to a fish or smolt that has undergone the steps of APS Process II. An APS Process II smolt is also referred to as a “SUPERSMOLT® II” or a “Process II ” smolt.
APS Process I: Marine fish are exposed to or maintained in freshwater containing 0.3-10.0 mM calcium and 0.5-10.0 mM magnesium ions. This water is prepared by addition of calcium carbonate and/or calcium chloride and magnesium chloride to the freshwater. Fish are fed with feed pellets containing 1-7% (weight/weight) NaCl. See Example 3 for further details regarding the feed. Fish are exposed to or maintained in this regimen of water mixture and feed for a total of 30-45 days, using standard hatchery care techniques. Water temperatures vary between 10-16° C. Fish are exposed to a constant photoperiod for the duration of APS Process I. A fluorescent light is used for the photoperiod.
APS Process II: Marine fish are exposed to or maintained in freshwater containing 0.3-10.0 mM calcium and 0.5-10.0 mM magnesium ions. This water is prepared by addition of calcium carbonate and/or calcium chloride and magnesium chloride to the freshwater. Fish are fed with feed pellets containing 1-7% (weight/weight) NaCl and either 2 gm or 4 gm of L-Tryptophan per kg of feed. See Example 3 for further details regarding the feed. Fish are exposed to or maintained in this regimen of water mixture and feed for a total of 30-45 days using standard hatchery care techniques. Water temperatures vary between 10-16° C. Fish are exposed to a constant photoperiod for the duration of APS Process II. A fluorescent light is used for the photoperiod.
Summer Flounders of various weights that were all derived from a single homogenous stock of farm raised animals (Great Bay AquaFarms Portsmouth, N.H.) were transported and placed in artificial seawater (Crystal Sea) within the APS laboratory. These were divided into two groups (n=13) and one maintain in seawater (Seawater Control) for a total of 81 days and fed a standard flounder diet (Corey Feeds, New Brunswick, Canada). The other (Freshwater) was adapted to APS Process I conditions over 30 days consisting of 5 mM Ca2+, 8mM Mg2+ concentrations in the water and a 1.2% NaCl supplemented diet of 70% standard flounder feed (Corey Feeds, New Brunswick, Canada) and 30% ground squid. These flounder were then maintained in APS Process I conditions for a total of 51 days and their growth compared to that exhibited by matched paired summer flounder maintain in seawater.
Flounders were adapted to the APS Process I by the following 30 day schedule:


    1. Maintenance in seawater for 5 days.
    2. Reduce water salinity to 50% seawater for 10 days.
    3. Reduce water salinity to 25% seawater for 15 days.
    4. Place fish in APS Process I water (5 mM Ca2+, 8mM Mg2+ concentrations in the freshwater, pH 7.6-8.0)


Fish were individually tagged using colored elastomer tags their change in weight was determined at specific time points during the 51 day experimental interval.
The feed conversion ratio or FCR is obtained by dividing the body weight gained by a group of fish into the amount of food fed to the group of fish. The more efficient the conversion of food into body weight growth by fish, the smaller the FCR (small amount of food/large weight gain of fish). A very small FCR number (less than 1) encompasses a highly efficient conversion of food into body weight growth which is the goal of aquaculture. By contrast, a large FCR means an inefficient conversion of food into body weight growth and is generally undesirable. A large or poor FCR is undesirable due to the cost of feed and the necessity to use more feed to grow fish to a given weight. The FCR values for fish subjected to the methods of the present invention are generally smaller and more desirable, in some instances (e.g., when fish were fed dry feed), than most industry published values because the present invention eliminates the presence of osmotically damaged fish that tend to increase the overall FCR since they eat food but do not grow. The methods of the present invention, result in a lower FCR, allowing optimal feeding and growth of most fish. The FCR of fish subjected to the present invention is sufficient to maintain growth and feeding of the majority of fish, or preferably increase the growth and feed consumption of the majority of fish. When fish are subjected to the methods of the present invention, they exhibit ranges of FCRs, for example, would include values between about 0.7 and about 7.0. In particular, food consumption or food intake is improved because it is believed that the fish “smell” or “sense” the food with the PVCR in cells of the olfactory lamellae or olfactory bulb.
The specific growth rate (SGR) of the fish was determined by dividing the weight of the fish at the end of the given time point by the starting weight of the fish.
All calculations to obtain feed conversion ratio (FCR) or specific growth rate (SGR) and growth factor (GF3) were performed using standard accepted formulae (Willoughby, S. Manual of Salmonid Farming Blackwell Scientific, Oxford UK 1999).
Results and Discussion:
A marine fish, Summer Flounder, can be adapted and grown under APS Process I conditions for a prolonged interval (51 days) with growth rates similar to that exhibited by matched control Summer Flounder in seawater.
Tables I and II display data obtained from identical groups of summer flounder maintained under either seawater (seawater control) or APS Process I freshwater conditions. Water quality and temperatures (16.3° C. vs 17.9° C. average) were comparable. Flounders were successfully adapted to APS Process I conditions without significant mortalities and their overall appearance did not differ significantly from those matched controls that were maintained in seawater.







TABLE I







Growth of Summer Flounder in Freshwater.









APS Freshwater













Weight
Weight
Weight
Weight
Total Weight


Flounder #
Start
20 days
37 days
51 days
Gained
















116
1
161
145
144
140
−21


118
2
87
94


123
3
60


142
4
94
104
115
112
18


146
5
73
73


221
6
118
135
145
156
38


223
7
105


225
8
96
112
124
133
37


226
9
156
183
203
221
65


227
10
162
176
172
180
18


233
11
205
207
220
244
39


234
12
221
224


235
13
150
161
164
174
24












Average
129.8462
146.7273
160.875
170
27.2


S. Dev.
50.15
48.34065
36.65452
44.75648


p test

0.017186
0.013243
0.0085


Amount

342
315
291
948


Fed (gm)


water ° C.
17.9
19.4
16.4
17.9


(Average)



FCR
3.96



SGR
0.53%
bw/day





Feed conversion ratio (FCR); specific growth rate (SGR)













TABLE II







Growth of Summer Flounder in Seawater.











APS Seawater
Total days 51
Total













Weight
Weight
Weight
Weight
Weight


Fish #
Start
20 days
37 days
51 days
Gained















117
114






118
147
146
168
168
21


120
60
70
91
94
34


122
90
115
142
153
63


126
128
142
174
196
68


127
67
76
93
105
38


130
95
90
93
86
−9


131
92
87
101
104
12


132
93
101
121
127
34


134
174
191
235
236
62


139
116


140
121
138
170
175
54


145
79
87
100
135
56


Average
105.8462
113
135.2727
143.5455
39.36364


S. Dev.
31.997
37.26392
46.82327
47.05181


T test

0.308845
0.041076
0.014804


Amount Fed

301
324
254
879


(gm)


FCR
1.99


SGR
0.6%



bw/day





body weight = bw






Overall mortalities of fish during the 51 day test interval was lower in seawater ( 2/13 or 15.4%) as compared to flounders maintained under APS Process I conditions ( 5/13 or 38.5%). The average weight gained by all flounders maintain under APS Process I conditions (27.2 gm) was less as compared to overall weight gain of the seawater control group (38.4 gm). Significant weight gains were observed in both groups after intervals of 20 days for APS Process I fish and 37 days for flounder maintained in seawater. Thus, the average specific growth rates (SGR) amongst the surviving flounders in APS Process 1 (0.53% body weight per day) were comparable to those maintained in seawater (0.6% body weight per day).
In contrast, 100% of marine fish (Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder and Summer Flounder) die within 72 hours of freshwater transfer.
Comparison of the food conversion ratio (FCR) between flounders maintained in APS Process I vs seawater shows that flounders maintained under APS Process I conditions displayed a significantly greater FCR (3.96), as compared to their matched seawater controls (1.99).
FIGS. 1A-B show the individual weight gain performances of tagged flounders maintained under APS Process I or seawater conditions. It is notable that there are wide variations in individual growth rates such that some flounders (e.g. #9 and #11) exhibited steady and significant growth under APS Process I conditions while others showed poor weight gains (e.g. #10) or even lost weight (e.g. #1). Similar performance characteristics were observed for flounder in seawater although the variation in individual performances were less pronounced as compared to flounders maintained in APS Process I.
Taken together, these data demonstrate that summer flounder can be successfully maintained under freshwater conditions using APS Process I for a prolonged interval (51 days) of time. Under normal conditions, summer flounder growth and survival are normally restricted to approximately 25% seawater whereupon the flounders die if the salinity is further reduced. These data form the basis of culture of summer flounder in freshwater environments distant from the marine environment itself where prices for flounder fillets would more than offset the poorer performance (increased mortalities and poorer FCR and weight gains) as compared to seawater controls.
Transferring marine fish to freshwater using APS Process II is expected to provide even better growth rates, than seen with APS Process I. Salmon and Trout that underwent APS Process II exhibited significant increases in growth rates, as illustrated in related applications, patent application Ser. Nos. 09/687,372; 09/687,476; 09/687,477, all entitled, “Methods for Raising Pre-Adult Anadromous Fish,” and patent application Ser. No. 09/687,373, entitled “Growing Marine Fish in Fresh Water”, all filed on Oct. 12, 2000.
Example 3 
The Feed
Two general methods were used to prepare feed for consumption by fish as part of APS Process I and II. These two processes involve either reformulation of feed or addition of a concentration solution for absorption by the feed followed by a top dressing for palatability. This disclosure describes the methodology to prepare feed using each of these 2 methods.
Methods:
Feed Manufacture for Salmon Experiments
To reformulate feed, the ingredients are as follows: Base Diet was made using the following ingredients and procedure: 30% Squid (liquefied in blender), 70% Corey Aquafeeds flounder diet (powderized in blender). Ingredients were blended into a semi moist “dough” ball. Other ingredients including NaCl or PVCR active compounds were blended into the base diet by weight according to experimental parameters.
Moore Clark standard freshwater salmonid diet (sizes 1.2, 1.5, 2.0, 2.5, and 3.5 mm) can also be used. A top dressing was applied to the pellets such that top dressing is composed of 4% of the weight of the Base Diet. Top dressing is composed of 50% krill hydrolysate (Specialty Marine Products Ltd.) and 50% Menhaden fish oil. The top dressing is added for palatability and sealing of added ingredients Other ingredients can include NaCl, MgCl2, CaCl2 or L-Tryptophan that are added by weight to the base diet by weight, as described herein.
Preparation of Feed Containing 7% (Weight/Weight) NaCl:
For the APS Process I: Solid NaCl or NaCl apportioned at a ratio of 7% of the weight of the Moore Clark standard freshwater salmonid diet weight was added to a volume of tap water approximately 3-4 times the weight of NaCl. The mixture was heated to 60-70° C. with mixing via use of a magnetic stirring bar to dissolve salt. The NaCl solution was then poured into a hand held sprayer and applied to the Moore Clark standard freshwater salmonid diet that is tumbling inside of a 1.5 cubic meter motorized cement mixer. After absorption of the NaCl rich solution, the wetted Moore Clark standard freshwater salmonid diet is spread out thinly on window screening and placed in an enclosed rack system equipped with a fan and 1500 watt heater to expedite drying process. After drying for approximately 6 hr, the dried NaCl-rich pellets are returned to the cement mixer and a top dressing is applied. The feed is stored at room temperature until use.
Preparation of Feed Containing 7% (weight/weight) NaCl+PVCR Agonist (Tryptophan) For the APS Process II: Solid sodium chloride or NaCl apportioned at a ratio of 7% of the weight of the Moore Clark standard freshwater salmonid diet weight was added to a volume of tap water approximately 3-4 times the weight of NaCl. The mixture was heated to 60-70° C. with mixing via use of a magnetic stirring bar to dissolve salt. USP Grade L-Tryptophan was added to the water at either 2 grams or 4 grams for every kg of Moore Clark standard freshwater salmonid diet depending on formulation need. Dilute hydrochloric acid was added to the water with mixing until the tryptophan was dissolved and the pH of solution was approximately 4.0. The NaCl+tryptophan solution was then poured into a hand held sprayer and was then applied to the Moore Clark standard freshwater salmonid diet tumbling inside a cement mixer. After absorption of the NaCl+tryptophan solution, the wetted Moore Clark standard freshwater salmonid diet is then spread out thinly on window screening and placed in an enclosed rack system equipped with a fan and 1500-watt heater to expedite drying process. After drying for approximately 6 hr, the dried NaCl/tryptophan-rich pellets are then returned to the cement mixer and a top dressing is applied. The feed is stored at room temperature until use.
Example 4 
DNA and Putative Protein Sequences from Partial Genomic Clones of Polyvalent Cation Receptor Protein Amplified by PCR from the DNA of Several Species of Marine Fish 
These data provide the partial PVCR genomic sequences isolated in 13 species of marine fish. Each of these nucleotide sequences is unique and thus could be used as a unique probe to isolate the full-length cDNA from each species. Moreover, these nucleotide sequences could form the basis for a specific assay kit(s) for detection of PVCR expression in various tissues of these fish. For example, the kit could optionally include a labeled hybridization probe suitable for in situ hybridization.
The PVCR has been isolated in several species including Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder and Summer Flounder. Sequences of mammalian CaRs together with the nucleotide sequence of SKCaR (FIGS. 14A and 14B) were used to design degenerate oligonucleotide primers to highly conserved regions in the extracellular and transmembrane domains of polyvalent cation receptor proteins using standard methodologies (See GM Preston, “Polymerase chain reaction with degenerate oligonucleotide primers to clone gene family members,” Methods in Mol. Biol. Vol. 58, Edited by A. Harwood, Humana Press, pages 303-312, 1993). Using these primers, cDNA or genomic DNA from various fish species representing important commercial products are amplified using standard PCR methodology. Amplified bands are then purified by agarose gel electrophoresis and ligated into appropriate plasmid vector that is transformed into a bacterial strain. After growth in liquid media, vectors and inserts are purified using standard techniques, analyzed by restriction enzyme analysis and sequenced where appropriate. Using this methodology, nucleotide sequences were amplified.
To generate this sequence data, DNA was isolated from tissue samples of each of the species indicated using standard published techniques. DNA was then amplified using polymerase chain reaction (PCR) methodology including 2 degenerate PCR primers (DSK-F3 (5′-TGT CKT GGA CGG AGC CCT TYG GRA TCG C-3′; SEQ ID NO:29) and DSK-R4; (5′-GGC KGG RAT GAA RGA KAT CCA RAC RAT GAA G-3′ SEQ ID NO:30). Amplified DNAs were then purified by agarose gel electrophoresis, subcloned into plasmid vectors, amplified, purified and sequenced using standard methods.
FIGS. 12A-C show an aligned genomic DNA sequences of 593 nucleotides for 12 marine fish species, each of which codes for an identical region of the PVCR protein. Note that each nucleotide sequence derived from each specific species is unique. However, alterations in the DNA sequences of these genes often occur at common specific nucleotides within each sequence of 593 nucleotides.
FIGS. 13A-C show aligned corresponding predicted protein sequences derived from genomic nucleotide sequences displayed in FIGS. 12A-D. Note that few alterations in the amino acid sequence of this portion of the PVCR occur as a consequence of alterations in the nucleotide sequence as shown in FIGS. 12A-D. All of these changes (e.g., Ala to Val; Arg to Lys; and Cys to Tyr) are known as “conservative” substitutions of amino acids in that they preserve some combination of the relative size, charge and hydrophobicity of the peptide sequence.
All cited references, patents, and patent applications are incorporated herein by reference in their entirety. Also, companion patent application Ser. No. 10/268,051, entitled “Methods for Growing and Imprinting Fish Using Odorant,” filed Oct. 11, 2001; patent application Ser. No. 09/975,553, entitled “Methods for Raising Pre-adult Anadromous Fish,” filed Oct. 11, 2001; International Application No. PCT/US01/31704, entitled “Polyvalent Cation-sensing Receptor Proteins in Aquatic Species,” filed Oct. 11, 2001. Additionally, patent application Ser. No. 09/687,477, entitled “Methods for Raising Marine Fish,” filed on Oct. 12, 2000; patent application Ser. No. 09/687,476, entitled “Methods for Raising Marine Fish,” filed on Oct. 12, 2000; patent application Ser. No. 09/687,373, entitled “Methods for Raising Marine Fish,” filed on Oct. 12, 2000; Provisional Patent Application No. 60/240,392, entitled “Polyvalent Cation Sensing Receptor Proteins in Aquatic Species,” filed on Oct. 12, 2000; Provisional Patent Application No. 60/240,003, entitled “Polyvalent Cation Sensing Receptor Proteins in Aquatic Species,” filed on Oct. 12, 2000, are all hereby incorporated by reference in their entirety. Additionally, application Ser. No. 09/162,021, filed on Sep. 28, 1998, International PCT application No. PCT/US97/05031, filed on Mar. 27, 1997, and application Ser. No. 08/622,738 filed Mar. 27, 1996, all entitled, “Polycation Sensing Receptor in Aquatic Species and Methods of Use Thereof” are all hereby incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes can be made therein without departing from the scope of the invention encompassed by the appended claims.

## Claims

1. A method of growing marine fish in freshwater, comprising:
a) transferring the marine fish to freshwater having at least one Polyvalent Cation Sensing Receptor (PVCR) modulator in an amount sufficient to modulate or maintain expression and/or sensitivity of at least one PVCR in one or more tissues of said marine fish; and
b) adding feed for fish consumption to the freshwater, wherein the feed contains an amount of an agent sufficient to contribute to a significant increased level of said PVCR modulator in serum of the marine fish upon consumption of the feed by said marine fish.

2. The method of claim 1, wherein the agent is NaCl.

3. The method of claim 2, wherein the feed contains about 7% NaCl by weight.

4. The method of claim 3, wherein the feed additionally contains about 0.2% L-Tryptophan by weight.

5. The method of claim 3, wherein the feed additionally contains about 0.4% L-Tryptophan by weight.

6. The method of claim 2, wherein the feed additionally contains L-Tryptophan.

7. The method of claim 2, wherein the feed contains at least about 1% NaCl by weight.

8. The method of claim 1, wherein the feed additionally contains at least one PVCR modulator.

9. The method of claim 1, wherein the at least one PVCR modulator is selected from the group consisting of a divalent cation, a trivalent cation, an aminoglycoside, an organic polycation, an amino acid, a Type I Calcimimetic, a Type II Calcimimetic, 1,25 dihydroxyvitamin D, a cytokine, and macrophage chemotatic peptide-1.

10. The method of claim 1, wherein the at least one PVCR modulator is a PVCR agonist.

11. The method of claim 10, wherein the PVCR agonist is selected from the group consisting of a divalent cation, a trivalent cation, an aminoglycoside, an organic polycation and an amino acid.

12. The method of claim 1, wherein the marine fish is selected from the group consisting of Cod, Haddock, Hake, Halibut, Mackerel, Pollock, Sea Bass, Swordfish, Tuna, Winter Flounder, and Summer Flounder.

13. A method of growing flounder in freshwater, comprising:
a) transferring the flounder to freshwater having at least one PVCR modulator in an amount sufficient to increase expression and/or sensitivity of at least one PVCR in one or more tissues, wherein the freshwater has a pH that is greater than 7.0; and
b) adding feed for fish consumption to the freshwater, wherein the feed contains an amount of NaCl sufficient to contribute to a significant increased level of said PVCR modulator in serum of the flounder.

14. The method of claim 13, wherein the at least one PVCR modulator is selected from the group consisting of a divalent cation, a trivalent cation, an aminoglycoside, a organic polycation, an amino acid, a Type I Calcimimetic, a Type II Calcimimetic, 1,25 dihydroxyvitamin D, a cytokine, and macrophage chemotatic peptide-1.

15. The method of claim 14, wherein the feed contains at least about 1% NaCl by weight.