# Patent

## Title
Methods of raising crustaceans in low salinity water

## Abstract

The present invention relates to methods of raising crustaceans, comprising maintaining crustaceans in low salinity water having a Calcium-sensing Receptor (CaSR) modulator(s), and adding feed that contains an agent that contributes to modulation of a crustacean CaSR to the water.

## Background
RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 12/612,518, filed Nov. 4, 2009 now U.S. Pat. No. 7,909,001, which is a continuation of International Application No. PCT/US2008/005675, which designated the United States and was filed on May 2, 2008, published in English, which claims the benefit of U.S. Provisional Application No. 60/927,559, filed on May 4, 2007 and U.S. Provisional Application No. 61/123,666, filed on Apr. 9, 2008. The entire teachings of the above applications are incorporated herein by reference.


BACKGROUND OF THE INVENTION
The production of farmed shrimp and prawns for human consumption, also known as shrimp aquaculture, is a major global food-producing industry that produces greater than 1.5 million tonnes of farmed shrimp on an annual basis. However, current methods of raising shrimp have become unreliable and unstable in many instances, primarily due to disease outbreaks and difficulties meeting various production parameters on a consistent basis.
In the United States and other countries, traditional shrimp farming methods are typically performed at facilities in coastal or brackish waters. However, the high cost of coastal land, the lack of availability of brackish waters, as well as environmental and land stewardship regulations that are not consistent with traditional shrimp farming methods present significant challenges to the continued practice of these traditional methods.
Thus, there is a need for new methods of raising shrimp and other crustaceans (e.g., lobsters, crabs, crayfish).
SUMMARY OF THE INVENTION
The present invention is based on Applicants' discovery of new methods of raising crustaceans under low salinity conditions.
The present invention encompasses, in one embodiment, a method of raising crustaceans in low salinity water, comprising maintaining crustaceans in low salinity water having one or more Calcium-sensing Receptor (CaSR) modulator(s) that are present in an amount sufficient to modulate at least one CaSR in the crustaceans. The method further includes adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent that is present in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the crustaceans, after the crustaceans have consumed the feed. In one embodiment, the agent in the feed is present in an amount sufficient to contribute to an increased level of one or more CaSR modulator(s) in a tissue of the crustaceans. In another embodiment, the feed contains a CaSR modulator. In a particular embodiment, the crustaceans are shrimp.
The invention also provides, in another embodiment, a method of raising crustaceans in low salinity water, comprising adding one or more Calcium-sensing Receptor (CaSR) modulator(s) to low salinity water in an amount sufficient to modulate at least one CaSR in the crustaceans and transferring the crustaceans to the low salinity water containing the one or more CaSR modulator(s). The method also includes the step of adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the crustaceans, after consumption of the feed by the crustaceans.
In yet another embodiment, the invention relates to a method of raising crustaceans, comprising adding one or more Calcium-sensing Receptor (CaSR) modulator(s) to low salinity water in which crustaceans are maintained, wherein the one or more CaSR modulator(s) are added in an amount sufficient to modulate at least one CaSR in tissue of the crustaceans. In addition, the method includes the step of adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the crustaceans, after consumption of the feed by the crustaceans.
In an additional embodiment, the invention relates to a method of raising crustaceans, comprising providing crustaceans that are maintained in low salinity water with a feed that contains one or more Calcium-sensing Receptor (CaSR) modulator(s) in an amount sufficient to modulate at least one CaSR in tissue of the crustaceans, and an agent in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the crustaceans, after consumption of the feed by the crustaceans.
In a further embodiment, the invention provides a method of raising shrimp in low salinity water, comprising maintaining shrimp in low salinity water having at least two Calcium-sensing Receptor (CaSR) modulators, each present CaSR modulator present in an amount sufficient to modulate at least one CaSR in the shrimp. Feed that contains an agent in an amount sufficient to contribute to modulation of at least one CaSR in a tissue of the shrimp is added to the low salinity water containing the CaSR modulators. In a particular embodiment, the CaSR modulators include Ca2+ and Mg2+. In another embodiment, the feed contains at least one CaSR modulator (e.g., tryptophan).
The present invention provides methods of raising crustaceans in low salinity waters, such as the ground waters under a significant portion of the United States or in tanks supported by recirculation aquaculture systems (RAS) equipment, thereby facilitating the development of inland crustacean aquaculture. The methods of the invention also yield crustaceans having highly desirable characteristics, such as improved growth, meat quality, reduced disease, increased survivorship, and/or timing of molting, relative to prior methods of raising crustaceans.



BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 are autoradiographs of filters that were probed with 32P-labeled SalmoKCaR II DNA under reduced stringency hybridization conditions, showing the detection of bacteriophage plaques containing the LobCaR gene. The heavy black circles present on duplicate filters after primary and secondary screening (see panels labeled “Lob 1 Primary” and “Lob 1 Secondary,” respectively) show specific hybridization of the 32P-labeled SalmoKCaR II DNA probe to a single positive plaque from among hundreds of other plaques present on the filter. Final selection of the single pure plaque results in 32P-labeling of all the plaques present on the filter after tertiary screening (see panel labeled “Lob 1 Tertiary”).
FIG. 2 is a schematic diagram depicting the identification and subcloning of 2 restriction endonuclease fragments of 4 kb and 5 kb that contain the LobCaR sequence. Using standard subcloning and DNA sequencing methods, the 4 kb and 5 kb pieces of recombinant DNA were excised from the phage vector and ligated into plasmids for full length DNA sequencing analysis.
FIG. 3 is a schematic diagram of the LobCaR gene showing the locations of 6 exons (labeled), 7 introns, as well as 7 transmembrane (TM) domains.
FIG. 4A is an autoradiograph of a filter containing a lawn of λ bacteriophage plaques containing recombinant DNA from shark kidney CaSR(SKCaR), which was hybridized with a 32P-labeled SKCaR DNA probe and then washed 2×30 min with 0.1×SSC, 0.1% SDS at 65° C.
FIG. 4B is an autoradiograph of a filter containing a lawn of λ bacteriophage plaques containing recombinant lobster genomic CaSR (LobCaR) DNA, which was hybridized with a 32P-labeled SKCaR DNA probe and then washed 2×30 min with 0.5×SSC, 0.1% SDS at 65° C.
FIG. 4C is an autoradiograph of a filter containing a lawn of λ bacteriophage plaques containing recombinant human parathyroid CaSR (HuPCaR) DNA, which was hybridized with a 32P-labeled SKCaR DNA probe and then washed 2×30 min with 0.1×SSC, 0.1% SDS at 65° C.
FIG. 5A is a micrograph of a histology section of intestine from the spiny lobster (Panulirus interruptus), which was probed with rabbit pre-immune serum.
FIG. 5B is a micrograph of a histology section of intestine from the spiny lobster (Panulirus interruptus), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cell types.
FIG. 5C is a micrograph of a histology section of antennal gland from the spiny lobster (Panulirus interruptus), which was probed with rabbit pre-immune serum.
FIG. 5D is a micrograph of a histology section of antennal gland from the spiny lobster (Panulirus interruptus), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cell types.
FIG. 5E is a micrograph of a histology section of testis from the spiny lobster (Panulirus interruptus), which was probed with rabbit pre-immune serum.
FIG. 5F is a micrograph of a histology section of testis from the spiny lobster (Panulirus interruptus), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrows designate representative immunostaining in specific cells.
FIG. 6A is a micrograph of a histology section of intestine from the American lobster (Homarus americanus), which was probed with pre-immune serum.
FIG. 6B is a micrograph of a histology section of intestine from the American lobster (Homarus americanus), which was probed with SDD antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cell types.
FIG. 6C is a micrograph of a histology section of antennal gland from the American lobster (Homarus americanus), which was probed with pre-immune serum.
FIG. 6D is a micrograph of a histology section of antennal gland from the American lobster (Homarus americanus), which was probed with SDD antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cell types.
FIG. 6E is a micrograph of a histology section of testis from the American lobster (Homarus americanus), which was probed with pre-immune serum.
FIG. 6F is a micrograph of a histology section of testis from the American lobster (Homarus americanus), which was probed with SDD antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cells.
FIG. 6G is a micrograph of a histology section of gill tissue from the American lobster (Homarus americanus), which was probed with pre-immune serum.
FIG. 6H is a micrograph of a histology section of gill tissue from the American lobster (Homarus americanus), which was probed with SDD antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrow designates representative immunostaining in specific cells.
FIG. 7A is a micrograph of a histology section from the northern acorn barnacle (Semibalanus balanoides), which was probed with pre-immune serum.
FIG. 7B is a micrograph of a coincident histology section (relative to FIG. 7A) from the northern acorn barnacle (Semibalanus balanoides), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrows designate immunostaining in cirri (thoracic feeding appendages).
FIG. 7C is a micrograph of a histology section from the northern acorn barnacle (Semibalanus balanoides), which was probed with pre-immune serum.
FIG. 7D is a micrograph of a histology section (relative to FIG. 7C) from the northern acorn barnacle (Semibalanus balanoides), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray hue. The arrows designate immunostaining in epithelial cells lining the GI tract. The arrows designate immunostaining in gut tissue.
FIG. 7E is a micrograph of a histology section from the northern acorn barnacle (Semibalanus balanoides), which was probed with pre-immune serum.
FIG. 7F is a micrograph of a histology section (relative to FIG. 7E) from the northern acorn barnacle (Semibalanus balanoides), which was probed with LOB-1 antiserum. Localization of immunoreactive LobCaR protein is detected as a dark gray horseradish peroxidase reaction product. The arrows designate immunostaining in epithelial cells lining the GI tract. The arrows designate immunostaining in gut tissue.
FIG. 8A is a western blot of specific white shrimp (Litopenaeus vannamei) tissues probed with (immune) or without (preimmune) SDD antibody. The arrow shows the positive staining for SDD in the Atlantic salmon kidney positive control (lane 1), as well as in shrimp anterior (lane 2) and posterior gill (lane 3) that is the same as that shown in FIG. 8B.
FIG. 8B is a western blot of specific white shrimp (Litopenaeus vannamei) tissues probed with (immune) or without (preimmune) LobCar antibody. The arrow shows the positive staining for SDD in the Atlantic salmon kidney positive control (lane 1), as well as in shrimp anterior (lane 2) and posterior gill (lane 3) that is the same as that shown in FIG. 8A.
FIG. 9 is an immunoblot depicting at least 3 major bands in both barnacle (Semibalanus balanoides) (Lanes 1, 3, 5 and 7) and star lobster (Panulirus guttatus) (Lanes 2, 4, 6, and 8) fractionated protein extracts that were recognized by anti-SDD LobCaR antiserum (IMMUNE; Lanes 1, 2, 5 and 6), but not by preimmune antiserum (PreImmune; Lanes 3, 4, 7 and 8). A reduction in the intensity of high molecular weight bands (denoted by brackets on Lanes 1 and 2) was observed in lanes fractionated under reducing conditions compared to nonreducing conditions.
FIG. 10 is an autoradiogram of a genomic Southern hybridization blot showing CaSR gene structure in crustaceans, mollusks and teleost fish. Purified genomic DNA prepared from crustacean (lobster), mollusc (mussel) and teleost fish, (i.e., goldfish (Fish #1) and guppy (Fish #2)) was digested with either ECO R1 (lanes marked “1”), BAM H1 (lanes marked “2”) or a combination of ECO R1+BAM H1 (lanes marked “3”), subjected to agarose electrophoresis fractionation, transferred to a membrane and hybridized under high stringency conditions using a full length 32P-labeled LobCaR DNA as a probe. The leftward pointing arrows indicate specific DNA fragments from the crustacean, mollusc and teleost fish genomes that hybridize to the LobCaR DNA probe.
FIG. 11 is an autoradiogram of a genomic Southern hybridization blot showing the presence and structure of CaSR gene homologs in amphioxus (Amph), horseshoe crab (Limulus polyphemus) (H. Crab), sea urchin (Urchin), tunicate and two species of shrimp (Glass shrimp (Palaemonetes spp.) and Sand shrimp (Crangon septemspinosa)). Purified genomic DNA prepared from each of these species was digested with either ECO R1 (lanes marked “1”) or a combination of ECO R1+BAM H1 (lanes marked “2”), subjected to agarose electrophoresis fractionation, transferred to a membrane and hybridized under reduced stringency conditions using a full-length 32P-labeled LobCaR DNA probe. Specific DNA fragments that hybridize to the LobCaR DNA probe are indicated by leftward pointing arrows.
FIGS. 12A and 12B are an alignment of amino acid sequences of candidate CaSR proteins obtained from publicly available databases with the amino acid sequence of SalmoKCaR-2 (Sal 2), used as a probe to isolate LobCaR, and the amino acid sequence of LobCaR (Lobster) (SEQ ID NO:3). Amino acid sequences included in the figure are from Anemone (Nematostella vectensis), Tunicate (Ciona intestinalis), Urchin (Stongylocentrotus purpuratus) and Nematode (Caenorhabditis elegans). Identical amino acid residues at the same position are shown in black and differing amino acids at the same position are shown in light gray. Highly conserved cysteine residues (C) that may be involved in appropriate folding of the expressed protein are shown in medium gray.
FIG. 13A is a micrograph of a histology section from the posterior gill of the shrimp Litopenaeus vannamei, which was probed with Lob-1 antiserum (immune) or in an adjacent section that was not stained with Lob-1 (preimmune). A medium gray in cells located at the presumptive osmoregulatory tissues at the base of gill filaments represents a deposition product that indicates specific staining (see arrows).
FIG. 13B is a micrograph of a higher magnification histology section from the posterior gill of the shrimp Litopenaeus vannamei, which was probed with Lob-1 antiboty (LobCaR) or stained for Na/K atpase in an adjacent section. A darker gray (see arrows) indicates positive staining showing colocalization of LobCaR and Na/Katpase at the base of gill filaments in tissues that are putatively involved in ionoregulation, not respiration (see arrows).
FIG. 13C is a micrograph of a histology section from the eyestalk of the shrimp Litopenaeus vannamei, which was probed with Lob-1 antiserum. Specific staining is present in the integumentary epithelium, presumptive endocrine tissue and presumptive neural sheath tissue (see arrows).
FIG. 13D is a micrograph of a histology section from the intestine of the shrimp Litopenaeus vannamei, which was probed with Lob-1 antiserum. Specific staining is detected in the intestinal epithelial cells and in the lining of gastrointestinal tract (see arrows). The dark staining within the lumen is non-specific staining of fecal material.
FIG. 14 is a graph depicting the relationship of shrimp postlarval survivorship to inland well water ionic composition. Percent survivorship is shown for shrimp raised in well waters with differing sodium/calcium ratios. The encircled data point indicates 95% survivorship in artificial seawater at 4 ppt, which had a [log(Na/Ca)] ratio of 1.49 in this particular study (values taken from tables in Saoud, I, D. Davis, and D. Rouse, 2003. Suitability studies of inland well waters for Litopenaeus vannamei culture. Aquaculture 217:373-383).
FIG. 15A is a graph depicting the effect of adding CaSR modulators to the water and feed (SS & diet group) on molting of the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (control 1, control 2).
FIG. 15B is a graph depicting the effect of adding CaSR modulators to the water and feed (SS & diet group) on survival of the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (control 1, control 2).
FIG. 15C is a graph depicting the effect of adding CaSR modulators to the water and feed (ions & diet group) on growth of the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (control 1, control 2).
FIG. 16A is a graph depicting the effect of adding CaSR modulators to the water and feed (ions & diet group) on levels of Ca2+ and K2+ in the hemolymph of the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (C1, C2).
FIG. 16B is a graph depicting the effect of adding CaSR modulators to the water and feed (ions & diet group) on hemolymph osmolarity in the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (C1, C2).
FIG. 16C is a graph depicting the effect of adding CaSR modulators to the water and feed (ions & diet group) on Na/K atpase activity in the shrimp Litopenaeus vannamei. No CaSR modulators were added to the water or feed in either control group (C1, C2).
FIG. 17 is a graph depicting the concentrations of various amino acids in tail muscle of shrimp raised for greater than 2 weeks under each of 3 experimental condition standardized to the glycine concentration in each sample (n=3 per treatment mean±SEM): 1) low salinity water (4 ppt), 2) normal seawater (32 ppt), and 3) low salinity water (4 ppt) supplemented with CaSR modulators, as well as feed containing the CaSR modulator tryptophan (SS). These data show that the invention has specific effects on amino acids that may be important to osmoregulation or taste, specifically the amino acids outlined (His, Phe, Leu) show that the method leads to amino acid concentrations that are more like shrimp adapted to seawater as compared to those adapted to standard low salinity water.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
As used herein “standard seawater” or “normal seawater” refers to water having a total salt concentration of about 30 to about 34 parts per thousand (ppt). The concentrations of major ionic constituents in standard seawater are typically about 450 mM Na+, about 10 mM Ca2+, about 50 mM Mg2+ and about 10 mM K+ at a pH of about 7.9 to about 8.3.
As used herein, the term “low salinity water” refers to water having a total salt concentration that is significantly less than that of standard seawater, but no less than 0.5 ppt. In a preferred embodiment, “low salinity water” refers to water having a total salt concentration that is about 0.5 ppt to about 12 ppt, and more preferably, about 0.5 ppt to about 5 ppt.
As used herein, “low salinity water with altered ionic conditions” refers to low salinity water, wherein the ratios of the major cations (e.g., Na+, Ca2+, Mg2+, K+, Sr2+) in the water differ significantly from those in standard seawater. In a particular embodiment, “low salinity water with altered ionic conditions” refers to low salinity water, having Na/Ca and/or Mg/Ca ratios that are significantly lower than those found in standard seawater, which typically has a Na/Ca ratio of about 45 and a Mg/Ca ratio of about 5, on a molar/molar basis.
The term “calcium-sensing receptor” or “CaSR” refers to any multimodal G protein coupled receptor (GPCR) that senses extracellular levels of calcium ions. CaSRs are also known in the art as “polyvalent cation-sensing receptors,” “polyvalent cation receptors” and “PVCRs,” and these terms are used interchangeably herein.
“CaSR modulator” refers to any agent that binds to and modulates the expression, sensitivity, activity, signalling and/or physiological function of a CaSR protein in one or more tissues of a crustacean. A “CaSR modulator” can be, for example, a CaSR agonist or a CaSR antagonist. The term “CaSR modulator” encompasses primary receptor ligands as well as allosteric modulators of a CaSR protein and includes compounds belonging to the following functional classes: calcimimetics (e.g., a Type-I calcimimetic, a Type-II calcimimetic) and calcilytics. Preferred calcimimetic and calcilytic CaSR modulators include, for example, polyvalent cations (e.g., divalent cations, trivalent cations, organic polycations), L-amino acids (e.g., L-aromatic amino acids, L-kynurenines), peptides, phenylalkylamines, polyaromatic hydrocarbons, substituted piperidines and substituted pyrrolidines.
As used herein, the “sensitivity” of the CaSR refers to alteration of CaSR expression in response to a change in the concentration of CaSR modulators. CaSR expression can be assessed by measuring or detecting CaSR polypeptide or nucleic acid molecules in a sample by standard methods.
A “significant” increase is used herein to refer to a measurable rise, particularly a significant rise, in the level or quantity of a CaSR modulator(s) in one or more tissues of the crustaceans, as compared to a control or reference.
As used herein, “effective amount” refers to an amount of a CaSR modulator that significantly alters (e.g., increases, decreases) the behavior or physiology of a crustacean.
“Crustacean” refers to any arthropod animal belonging to the subphylum Crustacea. 
The term “peptide” refers to a naturally-occurring or synthetic compound that includes from about 2 to about 100 amino acid residues that are joined together by covalent bonds (e.g., peptide bonds, non-peptide bonds). Such peptides are typically less than about 100 amino acid residues in length and are preferably about 2 to about 10 amino acid residues in length. Peptides can be linear or cyclic and can include unmodified and/or modified amino acid residues. In a preferred embodiment, the peptide comprises amino acids that are joined by peptide bonds. The term “peptide” also encompasses peptidomimetics.
As used herein, the term “polypeptide” refers to a polymer of amino acids of any length and encompasses proteins, peptides, and oligopeptides.
As used herein, the terms “chimeric polypeptide” or “fusion polypeptide” refer to polypeptides derived from the fusion of a first amino acid sequence with a second amino acid sequence, wherein the first and second amino acid sequences are not present in a single naturally-occurring polypeptide chain.
As used herein, “functional equivalent” refers to a variant polypeptide that possesses the biological activity of a native polypeptide and/or a functional domain thereof.
As used herein, the term “antibody” refers to a polypeptide having affinity for a target, antigen, or epitope, and includes both naturally-occurring and engineered antibodies. The term “antibody” encompasses polyclonal, monoclonal, human, chimeric, humanized, primatized, veneered, and single chain antibodies, as well as fragments of antibodies (e.g., Fv, Fc, Fd, Fab, Fab′, F(ab′), scFv, scFab, dAb). (See e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).
As defined herein, the term “antigen binding fragment” refers to a portion of an antibody that contains one or more CDRs and has affinity for an antigenic determinant by itself. Non-limiting examples include Fab fragments, F(ab)′2 fragments, heavy-light chain dimers, and single chain structures, such as a complete light chain or a complete heavy chain.
As used herein, “specifically binds” refers to a probe (e.g., an antibody, an aptamer) that binds to a target protein (e.g., a CaSR, a LobCaR polypeptide) with an affinity (e.g., a binding affinity) that is at least about 5 fold, preferably at least about 10 fold, greater than the affinity with which the probe binds a non-target protein.
“Target protein” refers to a protein to be detected (e.g., using a probe comprising a detectable label).
“Detectable label” as used herein refers to any moiety that is capable of being specifically detected, either directly or indirectly, and therefore, can be used to distinguish a molecule that comprises the detectable label from a molecule that does not comprise the detectable label.
As used herein, the term “nucleic acid” refers to polynucleotides, including naturally occurring polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The term should also be understood to include analogs of either RNA or DNA that are made from nucleotide analogs (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA)). The nucleic acids described herein can be single-stranded (sense, antisense) or double-stranded polynucleotides and can include unconventional modifications to any portion of the molecule, including, for example, the sugar phosphate backbone or the base portion of one or more nucleotides. Furthermore, the nucleic acids of the present invention may be naturally-occurring, recombinant or synthetic.
As used herein, an “isolated nucleic acid” refers to a nucleic acid that is separated from nucleotide sequences that normally flank the nucleic acid as it occurs in nature (e.g., in an organism's genome). The term also applies to nucleic acids that are substantially purified from other components that naturally accompany the nucleic acid (e.g., RNA, DNA, protein) in a cell. Such isolated nucleic acid molecules include, for example, a recombinant DNA molecule that has been incorporated into a vector or plasmid, or into the genome of a host prokaryote or eukaryote, or which exists as a separate molecule (e.g., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) that is independent of other sequences. The term also includes a recombinant nucleic acid that is part of a hybrid gene encoding additional polypeptide sequence. In vivo and in vitro RNA transcripts of the present invention are also encompassed by “isolated” nucleic acid molecules.
A “substantially purified nucleic acid” is one that is separated from at least about 75%, 80%, 90%, or 95% of the components which naturally accompany the nucleic acid in its naturally occurring state, as assessed on a per-weight basis or a per-mole basis.
As used herein, the term “percent (%) identical” refers to sequence identity between two nucleotide sequences or between two amino acid sequences. Identity can be determined by comparing a position in each of two or more sequences, which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same nucleotide base or amino acid, the molecules are considered to be identical at that position. When the equivalent site is occupied by the same or a similar amino acid residue (e.g., similar in steric or electronic nature), then the molecules are referred to as homologous or similar at that position. Expression as a percentage of homology, similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences (i.e., % identity=# of identical positions/total # of positions×100). Various alignment algorithms or programs are known to those of skill in the art and may be used to determine percent identity. A non-limiting example of such an algorithm is described in Karlin, et al. Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated into the BLASTN and BLASTX programs (version 2.2) as described in Schaffer, et al. Nucleic Acids Res., 29:2994-3005 (2001). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information) can be used. In one embodiment, the searchable database is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62; and Gap Costs have an Existence of 11 and an Extension of 1.
The phrase “specifically hybridizes” refers to the specific association of two complementary nucleotide sequences (e.g., DNA/DNA, RNA/RNA, DNA/RNA, DNA/PNA) in a duplex under stringent conditions. The association of two nucleic acid molecules in a duplex occurs as a result of hydrogen bonding between complementary base pairs.
As used herein, the term “isolated polypeptide” refers to a polypeptide that is purified from, or otherwise substantially free of, other proteins and cellular material normally associated with the native protein in a cell or cell lysate. The term “isolated polypeptide” encompasses individual preparations of a protein comprising, in one embodiment, less than 20% (by dry weight) contaminating protein, and, in another embodiment, comprises less than 5% contaminating protein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of biology or chemistry (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry). Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods.
The present invention is based, in part, on the identification of CaSR proteins in crustaceans. As described herein, a gene encoding a CaSR, referred to herein as LobCaR, has been isolated and cloned from tissue of the spiny lobster Panulirus interruptus using molecular biological techniques, as described in Example 1. Using nucleic acid probes based on the LobCaR sequence and antibodies that recognize a peptide in the LobCaR protein, CaSR proteins were detected in various tissues of different species of lobster (Example 3), shrimp (Examples 5 and 7) and other crustaceans (Example 4). Furthermore, the addition of CaSR modulators to low salinity water and shrimp feed increased the incidence of molting, growth, and survivorship (Example 9), and influenced the hemolymph composition and amino acid composition in the tail muscle of shrimp raised in the low salinity water (Example 10).
Methods of Raising Crustaceans
The present invention relates to methods of raising crustaceans in low salinity water. The methods involve modulating the expression, sensitivity, activity, signalling and/or physiological function of a Calcium-sensing Receptor (CaSR) (e.g., at least one CaSR) in one or more tissues of the crustaceans. The invention relates to modulating the CaSR(s) that affects the ability of crustaceans to adapt to, grow in and/or survive in low salinity water, including low salinity water with altered ionic conditions.
In particular, the methods of the present invention include maintaining crustaceans in low salinity water having one or more Calcium-sensing Receptor (CaSR) modulator(s) that are present in an amount sufficient to modulate at least one CaSR in the crustaceans, and adding a specially made or modified feed to the low salinity water for consumption by the crustaceans. The CaSR modulator(s) can be present in the water, the feed, or both. In one embodiment, the method involves adding one or more CaSR modulator(s) to low salinity water that already contains crustaceans. In another embodiment, the crustaceans are transferred to low salinity water to which the CaSR modulator(s) have already been added. In yet another embodiment, the method involves adding a feed that contains one or more CaSR modulator(s) to low salinity water in which crustaceans are maintained. When CaSR modulators are added to both the low salinity water and the feed, the CaSR modulator in the feed can be the same as, or different from, the CaSR modulator in the water. When the CaSR modulator in the water and feed is the same, the water or feed may contain an additional CaSR modulator(s) that is not present in the other.
The feed contains a sufficient amount of an agent that contributes to modulation of at least one CaSR in a tissue of the crustaceans after the crustaceans have consumed the feed. In certain embodiments, the agent in the feed can enhance the ability of a CaSR modulator(s) to modulate the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR in one or more tissues of the crustaceans. Preferably, the CaSR modulator is increased in a tissue of the crustacean that contains a CaSR, such as, for example, hemolymph, a gastrointestinal tissue, a hepatopancreatic tissue, an endocrine tissue, a neural or neuromuscular tissue, a reproductive tissue, an osmoregulatory tissue, and an integumentary tissue. Methods of measuring or detecting a significant increase in a CaSR modulator are disclosed herein and/or known to one skilled in the art. In a particular embodiment, the agent in the feed that contributes to modulation of the at least one CaSR in a tissue of the crustaceans is 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) or another sodium salt.
The methods of the present invention pertain to raising crustaceans in low salinity water. Particularly suitable crustaceans for the methods of the invention are motile crustaceans, such as, for example, shrimp, prawns, spiny lobsters, clawed lobsters, crayfish and crabs. In a preferred embodiment, the crustaceans are shrimp. Exemplary species of shrimp include, but are not limited to, Litopenaeus vannemei, Penaeus monodon, P. stylirostris, P. chinensis, P. japonicus, P. indicus, P. merguiensis and Machrobrachium rosenbergii. In another preferred embodiment, the crustaceans are lobsters (e.g., spiny lobsters, clawed lobsters). Suitable species of lobster include, species of lobster belonging to one of the following genera: Homarus, Nephrops, Jasus, Panulirus, and Palinurus, among others.
The crustaceans are maintained in low salinity water containing CaSR modulators in sufficient amounts to modulate the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR in one or more tissues of the crustaceans. The CaSR, which is located in various tissues (e.g., gill, intestine, antennal gland, testis, lymphoid organ, eyestalk) of crustaceans, senses alterations in levels of CaSR modulators, including various polyvalent ions (e.g., divalent cations), for example, in the surrounding water, in hemolymph or in the luminal contents of tubules or hemal spaces inside the body, such as antennal gland, hepatopancreas, or intestine. The ability to sense CaSR modulators results in a modulation of the CaSR, thereby allowing the crustaceans to better grow in low salinity water. Modulation of the CaSR can occur, for example, in one or more tissues (e.g., ionoregulatory tissues, osmoregulatory tissues).
The modulation of CaSRs by CaSR modulators allows for, or assists in, one or more of the following functions in crustaceans: sensing or adapting to at least one CaSR modulator in tissues (e.g., hemolymph) or in the surrounding environment; altering the behavioral response to sensory stimuli, especially olfaction and gustation; altering osmoregulation or divalent cation homeostasis; altering one or more endocrine pathways; and altering chemosensory signal concentration or composition. As used herein, the “sensitivity” of the CaSR refers to either an alteration of CaSR expression in response to a change in the concentration of CaSR modulators or an alteration in the ability of the CaSR to respond to various ligands that stimulate its action to interact in specific ways with other cellular signal transduction pathways. CaSR expression can be assessed by measuring or detecting CaSR polypeptide or nucleic acid molecules in a sample by standard methods. Suitable assays and techniques for assessing the expression sensitivity, activity, signaling and/or physiological function of a CaSR are known in the art, and include those described herein.
CaSR modulators include both CaSR agonists (e.g., calcimimetics) that increase the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR, and CaSR antagonists (e.g., calcilytics) that decrease the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR. Calcimimetic CaSR modulators include, for example, Type 1 calcimimetics and Type II calcimimetics (e.g., NPS-R-467 and NPS-R-568 from NPS Pharmaceutical Inc., (Salt Lake, Utah, U.S. 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)).
CaSR modulators encompass primary receptor ligands for a CaSR, as well as allosteric modulators of a CaSR (e.g., aromatic amino acids, tryptophan derivatives, peptides). CaSR modulators can be naturally occurring (e.g., isolated from a natural source), synthetic (e.g., produced by standard chemical synthesis techniques), or recombinant (e.g., produced by bio fermentation).
Suitable CaSR modulators for use in the methods of the invention include, but are not limited to, polyvalent cations (e.g., inorganic polycations, organic polycations) and amino acids. Examples of inorganic polycations are divalent cations including calcium (e.g., at a concentration between about 0.3 and about 12.0 mM) and magnesium (e.g., at a concentration between about 0.5 and about 30.0 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 (e.g., 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 (e.g., 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). In a particular embodiment, the organic polycation is hexacycline.
Additionally, CaSR modulators include amino acids, such as L-amino acids. The L-amino acids can be unmodified or modified (e.g., halogenated). Examples of suitable L-amino acids are L-Tryptophan, L-Tyrosine, L-Phenylalanine, L-Alanine, L-Serine, L-Arginine, L-Histidine, L-Leucine, L-Isoleucine, and L-Cystine (e.g., at concentrations of between about 1 and about 10 gm/kg feed). See Conigrave, A. D., et al., PNAS 97: 4814-4819 (2000). In a particular embodiment, the L-amino acid is an aromatic amino acid. In a preferred embodiment, the L-amino acid is L-tryptophan. CaSR modulators further include tryptophan-pathway metabolites and tryptophan derivatives, such as, for example, kynurenine, 3-OH kynurenine, xanthurenic acid, quinolic acid and kynurenic acid.
In addition, suitable CaSR modulators for use in the present invention include peptides. Such peptides are typically less than about 100 amino acid residues in length, and are preferably about 2 to about 10 amino acid residues in length. The peptide can comprise any suitable L- and/or D-amino acid, for example, common α-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g., β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine, statine), and unusual amino acids (e.g., citrulline, homocitruline, homoserine, norleucine, norvaline, ornithine, kynurenine). The amino, carboxyl and/or other functional groups on a peptide can be free (e.g., unmodified) or protected with a suitable protecting group. Suitable protecting groups for amino and carboxyl groups, and methods for adding or removing protecting groups are known in the art and are disclosed in, for example, Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, 1991. The functional groups of a peptide can also be derivatized (e.g., alkylated) using art-known methods.
The peptide can comprise one or more modifications (e.g., amino acid linkers, acylation, acetylation, amidation, methylation, halogenation, terminal modifiers (e.g., cyclizing modifications)), if desired. The peptide can also contain chemical modifications (e.g., N-methyl-α-amino group substitution). In addition, the peptide can be an analog of a known and/or naturally-occurring peptide, for example, a peptide analog having conservative amino acid residue substitution(s). These modifications can improve various properties of the peptide (e.g., solubility, binding), including its ability to modulate a CaSR in a crustacean.
Peptide CaSR modulators can be linear, branched or cyclic, e.g., a peptide having a heteroatom ring structure that includes several amide bonds. In a particular embodiment, the peptide is a cyclic peptide. Such peptides can be produced by one of skill in the art using standard techniques. For example, a peptide can be derived or removed from a native protein by enzymatic or chemical cleavage, or can be synthesized by suitable methods, for example, solid phase peptide synthesis (e.g., Merrifield-type synthesis) (see, e.g., Bodanszky et al. “Peptide Synthesis,” John Wiley & Sons, Second Edition, 1976). Peptides can also be produced, for example, using recombinant DNA methodologies or other suitable methods (see, e.g., Sambrook J. and Russell D. W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).
Peptides can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using methods of combinatorial chemistry, and can be screened using any suitable method to determine if the library comprises peptides with a desired biological activity. Such peptides can then be isolated using suitable methods.
Peptide CaSR modulators can also be peptidomimetic compounds. For example, polysaccharides can be prepared that have the same functional groups as peptides. Peptidomimetics can be designed, for example, by establishing the three dimensional structure of a peptide agent in the environment in which it is bound or will bind to a target molecule. The peptidomimetic comprises at least two components, the binding moiety or moieties and the backbone or supporting structure.
The binding moieties are the chemical atoms or groups that will react or form a complex (e.g., through hydrophobic or ionic interactions) with a target molecule, for example, a crustacean CaSR. For example, the binding moieties in a peptidomimetic can be the same as those in a peptide or protein antagonist. The binding moieties can be an atom or chemical group that reacts with the receptor in the same or similar manner as the binding moiety in the peptide antagonist. For example, computational chemistry can be used to design peptide mimetics of CaSR binding site, for instance, a ligand binding site. Examples of binding moieties suitable for use in designing a peptidomimetic for a basic amino acid in a peptide include nitrogen-containing groups, such as amines, quarternary ammonia moieties, guanidines and amides or phosphoniums. Examples of binding moieties suitable for use in designing a peptidomimetic for an acidic amino acid include, for example, carboxyl, lower alkyl carboxylic acid ester, sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acid or ester thereof.
The supporting structure is the chemical entity that, when bound to the binding moiety or moieties, provides the three dimensional configuration of the peptidomimetic. The supporting structure can be organic or inorganic. Examples of organic supporting structures include polysaccharides, polymers or oligomers of organic synthetic polymers (such as, polyvinyl alcohol or polylactide). It is preferred that the supporting structure possesses substantially the same size and dimensions as the peptide backbone or supporting structure. This can be determined by calculating or measuring the size of the atoms and bonds of the peptide and peptidomimetic. In one embodiment, the nitrogen of the peptide bond can be substituted with oxygen or sulfur, for example, forming a polyester backbone. In another embodiment, the carbonyl can be substituted with a sulfonyl group or sulfinyl group, thereby forming a polyamide (e.g., a polysulfonamide). Reverse amides of the peptide can be made (e.g., substituting one or more-CONH-groups for a-NHCO-group). In yet another embodiment, the peptide backbone can be substituted with a polysilane backbone.
These compounds can be manufactured by known methods. For example, a polyester peptidomimetic can be prepared by substituting a hydroxyl group for the corresponding α-amino group on amino acids, thereby preparing a hydroxyacid and sequentially esterifying the hydroxyacids, optionally blocking the basic and acidic side chains to minimize side reactions. Determining an appropriate chemical synthesis route can generally be readily identified upon determining the chemical structure.
Peptidomimetics can be synthesized and assembled into libraries comprising a few to many discrete molecular species. Such libraries can be prepared using well-known methods of combinatorial chemistry, and can be screened to determine if the library comprises one or more peptidomimetics which have the desired activity. Such peptidomimetic antagonists can then be isolated by suitable methods.
In addition, CaSR modulators include phenylalkylamines. Methods of synthesizing, isolating and/or preparing phenylalkylamines are known in the art. Suitable phenylalkylamines for use in the methods of the invention include, but are not limited to, MC 0100 and MC 0106 compounds described herein (See, Examples 14 and 15 and FIGS. 29A and B).
CaSR modulators can also be substituted piperidines and substituted pyrrolidines. methods of synthesizing, isolating and/or preparing substituted piperidines and substituted pyrrolidines are known in the art. Suitable substituted piperidines and substituted pyrrolidines for use in the methods of the invention include, but are not limited to, substituted piperidines and substituted pyrrolidines described in U.S. Pat. Nos. 7,265,145 and 7,307,171.
CaSR modulators also include compounds that indirectly alter CaSR expression (e.g., 1.25 dihydroxyvitamin D (e.g., in concentrations of about 3,000-10,000 International Units/kg feed), cytokines such as Interleukin Beta, and Macrophage Chemotactic Peptide-1 (MCP-1)).
The molar concentrations of CaSR modulators referred to herein are free or ionized concentrations of the CaSR modulator in the low salinity water, and do not include amounts of bound CaSR modulator (e.g., CaSR 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 agent (e.g., NaCl), as described herein), so long as the combination modulates at least one CaSR.
The CaSR modulator can be administered to the crustaceans in a number of ways. The invention encompasses administration of the CaSR modulator in any way that is sufficient to modulate the CaSR. In one embodiment, the CaSR modulator is simply added to the low salinity water, as described herein. CaSR modulators that are added to the water can, for example, modulate the CaSR on the integument and gills of the crustaceans, and/or can be ingested by the crustaceans. Alternatively or in addition, the CaSR modulator can be added to the feed. Amounts and types of CaSR modulators added to the feed can be any CaSR modulator described herein. In a particular embodiment, tryptophan is added to the feed. Other embodiments include subjecting the crustaceans to the CaSR modulator by “dipping” the crustaceans in the modulator, e.g., organic polycations. The CaSR modulators can be formulated in such a way as to allow the polycations to adhere to the integument and gills of the crustaceans, in sufficient amounts to increase or maintain expression of the CaSR. Alternatively, a particular organism that is a standard food for a crustacean may be enriched as to its content of CaSR modulator(s) and then fed to the crustacean whereupon such CaSR modulators contained in the prey or standard food are released and available to bind to CaSR proteins present in the gastrointestinal tract of larval, juvenile or adult crustaceans.
The invention also embodies assessing the amount of an existing, or background, level of a CaSR modulator in the low salinity water environment and/or the tissues of crustaceans before a CaSR modulator is added to the water and/or feed. CaSR modulators are assessed or measured using methods known in the art. After the existing level of a CaSR modulator in the water is assessed, the CaSR modulator is added to the water to increase or decrease the concentration to an amount sufficient to modulate at least one CaSR, or sufficient to bring the concentrations of the CaSR modulator within the stated ranges.
In a preferred embodiment, the present invention is practiced by adding a combination of two or more CaSR modulators to the low salinity water. In a particular embodiment, calcium and magnesium are added to the low salinity water (e.g., 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 30.0 mM of magnesium).
Calcium and magnesium can come from a variety of sources that are known to those of skill in the art. For example, sources of calcium and magnesium can be mixtures of a variety of compounds that contain calcium and magnesium, or can be substantially uniform or pure compound. Sources of calcium include, for example, Ca(CO3)2, CaCl2, and CaSO4, while 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 of crustaceans to low salinity water having at least one CaSR modulator. Intermittent exposure to the CaSR can occur provided that the CaSR remains modulated.
The invention further includes adding feed to the low salinity water. The feed contains a sufficient amount of an agent that contributes to modulation of at least one CaSR in a tissue of the crustaceans after the crustaceans have consumed the feed. In certain embodiments, the agent in the feed enhances the ability of a CaSR modulator(s) to modulate the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR in one or more tissues of the crustaceans. For example, the agent in the feed can increase the level of one or more CaSR modulators in a tissue of the crustaceans after the crustaceans have consumed the feed (e.g., via gill uptake) and/or directly modulate the expression, sensitivity, activity, signalling and/or physiological function of at least one CaSR in one or more tissues of the crustaceans after the crustaceans have consumed the feed. Thus, the agent in the feed can be, in some embodiments, a CaSR modulator. In a particular embodiment, the agent in the feed that contributes to modulation of the at least one CaSR in a tissue of the crustaceans is 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) or another sodium salt or source of sodium. Other suitable agents include, but are not limited to Ca2+, K+, Mg++, Sr2+, and tryptophan.
The frequency and amount of feed that crustaceans are fed, are taught in the art. Generally, the crustaceans are fed at least about 1-10 times a day, depending on life history stage, size and species of crustacean. The feed has enough CaSR modulator to contribute to a modulation of a CaSR in a tissue (e.g., hemolymph) of the crustaceans. Without wishing to be bound by any one theory, it is believed that, in certain embodiments, the presence of sufficient amounts of NaCl in the feed causes the crustaceans to take up more water from the surrounding environment. The ingestion or gill uptake of water having one or more CaSR modulators causes an overall rise in the level of CaSR modulator in tissues (e.g., hemolymph) of the crustaceans. The increase in levels of CaSR modulator in the crustaceans' tissues results in modulation of CaSRs in those tissues.
The present invention also relates to a feed for crustaceans. A suitable feed for use in the present invention contains between about 1%-10% NaCl by weight, or between about 10,000 mg NaCl/kg of feed and about 100,000 mg NaCl/kg of feed (e.g., about 12,000 mg NaCl/kg). The NaCl can be combined with other sodium salts to confer the desired effect of modulating a CaSR in the crustaceans. Hence, as used herein, the term NaCl, includes a substantially pure compound, and mixtures of NaCl with other sources of sodium. The feed can further include a CaSR modulator as described herein. In a particular embodiment, the feed includes an amino acid (e.g., tryptophan). In one embodiment, the feed has between about 1% and about 10% NaCl by weight and an amino acid in an amount between about 1 and about 10 gm/kg.
The feed can be made in a number of ways, provided that the proper concentration of the agent that contributes to modulation of the at least one CaSR in a tissue of the crustaceans (e.g., 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 CaSR modulator. Additionally, a top dressing can be added for palatability. By contrast, specific live feeds or other organisms used as feeds can be enriched for their content of CaSR modulators that, in turn, may be ingested by a crustacean. Subjecting live feed organisms to either specific environmental conditions or diets containing CaSR modulators that can be accumulated in such organisms also provides a means to provide a crustacean with dietary CaSR modulators. Alternate methods of preparing fish feed are known to those of skill in the relevant art.
Another embodiment of the present invention includes feeding crustaceans feed having between about 1% and about 10% NaCl by weight when the crustaceans are maintained in a low salinity environment.
According to the invention, the crustaceans are raised in low salinity water. Salinity refers to the sum of ionic concentrations (e.g., calcium, magnesium and sodium, etc.) in water. In a particular embodiment, the low salinity water has a total salt concentration in the range of about 0.5 ppt to about 12 ppt. For example, the total salt concentration of the low salinity water can be about 0.5, 1, 2, 3, 4, 5, 10 or 12 ppt. In a preferred embodiment, the low salinity water has a total salt concentration in the range of about 0.5 ppt to about 5 ppt. Low salinity water used in the methods of the invention can have altered ionic conditions, wherein the ratios of the major cations (e.g., Na+, Ca2+, Mg2+, K+, Sr2+) in the low salinity water differ significantly from those in standard seawater. For example, the ratio of Na/Ca and/or Mg/Ca can be significantly lower (e.g., due to increased calcium concentration) than those found in standard seawater (i.e., a Na/Ca ratio of about 45, a Mg/Ca ratio of about 5). Methods of assessing the concentrations and ratios of ions in water of varying salinities are known in the art.
The crustaceans can be raised under standard rearing conditions that are known in the art. Such conditions include recirculating, partial flow through, and flow-through rearing conditions (e.g., hatchery, pond, raceway), among others. Other conditions include extensive, intensive, and semi-intensive culture conditions. In a particular embodiment, the crustaceans are raised under recirculating conditions.
The methods described herein can be practiced on crustaceans at various stages of commercial aquaculture production. The different stages of commercial aquaculture production are know to those of skill in the art and include, for example, broodstock phase, reproductive phase, spawning phase, hatchery phase, settlement phase, larval phase, postlarval phase, juvenile growout phase, adult growout phase, harvest phase, and finishing phase.
The methods of the present invention modulate CaSRs in crustaceans raised in low salinity water, thereby resulting in improved growth and survivorship of the crustaceans, as well as other benefits. For example, the methods of the invention can also result in one or more of the following benefits for crustaceans raised in low salinity water: improved nutrient retention, improved food conversion, reduced food waste, decreased molt associated mortality (e.g. molt timing or frequency), increased size uniformity, increased disease resistance and/or immune function, improved quality or quantity of reproductive output (e.g., of broodstock shrimp), an increase in site specific settlement behavior of larvae, improved flesh or shell characteristics (e.g., smell, taste, texture), reduced competitive behavior, a decrease in stress-induced impairments, improved health benefits to humans consuming the crustacean, and improved shell and by-product characteristics to facilitate post-processing of wastes, among others.
LobCaR Nucleic Acid Molecules, Vectors and Host Cells
The present invention also provides isolated nucleic acid molecules comprising the full length genomic sequence of a CaSR gene (SEQ ID NO: 1) of the spiny lobster (Panulirus interruptus), referred to herein as the LobCaR gene, as well as isolated nucleic acids containing the putative protein coding sequence of the LobCaR gene (SEQ ID NO:2). The invention also relates to isolated nucleic acid molecules that encode a LobCaR polypeptide having the amino acid sequence of SEQ ID NO:3. Furthermore, nucleic acids that encode LobCaR homologs, variants, derivatives and fragments are also encompassed by the invention.
One of skill in the art will recognize that, due to degeneracy of the genetic code, a number of possible nucleotide sequences, in addition to those depicted by SEQ ID NO:1 and SEQ ID NO:2, will encode the same LobCaR polypeptide. Accordingly, The isolated LobCaR nucleic acid molecules of the present invention include nucleic acids with nucleotide sequences that are homologous to the LobCaR gene sequences described herein (e.g., SEQ ID NO:1, SEQ ID NO:2). As used herein, the term “homologous” or “analogous” when referring to a LobCaR nucleic acid sequence means that the nucleic acid sequence has sufficient identity to a nucleic acid sequence encoding the LobCaR protein of SEQ ID NO:3, such that the poylpeptide encoded by that sequence possesses one or more biological activities of the native LobCaR polypeptide. For example, a homologous nucleic acid molecule can be produced with “silent” changes or mutations that do not affect the function or biological activity of the encoded LobCaR polypeptide. Examples of such “silent” changes or mutations include additions, deletions or substitutions. Also encompassed by the present invention are nucleic acid sequences that encode LobCaR polypeptides that exhibit greater, or lesser, biological activity than the LobCaR protein of SEQ ID NO:3.
In certain embodiments, the present invention is directed to nucleic acid molecules that are at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2, or to nucleotide sequences complementary to SEQ ID NO: 1 or SEQ ID NO:2. In a particular embodiment, the isolated nucleic acid molecules are at least 80% identical to SEQ ID NO: 1, the complement of SEQ ID NO:1, SEQ ID NO:2, or the complement of SEQ ID NO:2.
Also encompassed by the present invention are nucleic acid molecules (e.g., DNA, RNA) that have nucleotide sequences that are substantially complementary to the DNA sequences encoding LobCaR.
Stringency conditions for hybridization refer to conditions, such as temperature and buffer composition, which determine the degree of identity that a first nucleic acid sequence and a second nucleic acid sequence must share in order for those sequences to hybridize to each other. Therefore, high stringency conditions are those conditions under which only very similar nucleic acid sequences (e.g., nucleic acid sequences that share greater than or equal to 90% identity) will hybridize to each other. 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 under which a given sequence will hybridize to other similar sequences. 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 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. Suitable 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 include, for example, (1) 1×SSC (10×SSC=3 M NaCl, 0.3 M Na3-citrate2H2O (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 albumin (fraction V), 1 mM Na2EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO47H2O, 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 (100x=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.
Moderate stringency conditions can employ hybridization at either (1) 4×SSC, (10×SSC=3 M NaCl, 0.3 M Na3-citrate2H2O (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 albumin (fraction V), 1 mM Na2.EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO47H2O, 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 (100x=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(log10 M)+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-citrate2H2O (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 albumin (fraction V), 1 mM Na2EDTA, 0.5 M NaHPO4 (pH 7.2) (1 M NaHPO4=134 g Na2HPO47H2O, 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 (100x=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 typically 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(log10 M)+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.
In certain embodiments, the present invention relates to nucleic acid molecules that hybridize with full-length and/or partial LobCaR nucleic acid sequences under conditions of high stringency. In one embodiment, the invention provides nucleic acid molecules that hybridize under conditions of high stringency to the full-length genomic LobCaR gene sequence (i.e., SEQ ID NO: 1 or the complement thereof) and nucleic acid molecules that hybridize to the coding nucleotide sequence for the LobCaR protein (i.e., SEQ ID NO:2 or the complement thereof). Examples of suitable conditions of high stringency are known to those of skill in the art and include, for example, those described herein.
In some embodiments, nucleic acid molecules that hybridize to LobCaR nucleic acid sequences (e.g., SEQ ID NO:1, SEQ ID NO:2) are capable of hybridizing to at least about 200 continuous nucleotides of the LobCaR sequence or more (e.g., 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 continuous nucleotides) under high stringency conditions.
The LobCaR nucleic acids of the present invention can be used for one or more of the following purposes without limitation: (1) to produce recombinant CASR proteins (e.g., LobCaR polypeptides), which can be assessed, for example, for structure determination and/or activity, and can also be used to obtain antibodies that bind to the CASR protein; (2) to serve as a basis for comparison with the nucleotide sequences of other CASRs in order to determine one or more of the following: conserved sequences, unique nucleotide sequences for normal and altered receptors, and suitable target sites for antisense nucleic acids, ribozymes, and/or PCR amplification primers; (3) as hybridization probes to detect presence and/or level of a LobCaR nucleic acid in a sample; (4) as PCR primers to amplify particular nucleic acid sequences (e.g., sequences to be used as hybridization probes); and (5) to identify and isolate additional CASR homologs in other species (e.g., other invertebrate species).
Thus, in certain embodiments, a LobCaR nucleic acid, or a fragment thereof, is used as a probe to detect (e.g., via hybridization) the expression level of a LobCaR nucleic acid in a sample (e.g., a biological sample). Methods for identifying a nucleic acid by hybridization are routine in the art (see Current Protocols In Molecular Biology, Ausubel, F. M. et al., Eds., John Wiley & Sons: New York, N.Y., (1997). For example, using a LobCaR detection probe, LobCaR nucleic acids can be detected by Northern blot analysis of mRNA isolated from a biological sample using standard protocols that are known to those of skill in the art. In a particular embodiment, the LobCaR probe is detectably labeled. For example, the nucleic acid probe can be labeled with, e.g., a radionuclide such as 3H, 32P, 33P, 14C, or 35S, a heavy metal, or a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody), a fluorescent molecule, a chemiluminescent molecule, an enzyme or the like.
In other embodiments, LobCaR nucleic acids and fragments thereof are used to identify and isolate CASR homologs in other invertebrate organisms (e.g., crustaceans). For example, a DNA library (e.g., cDNA library, genomic DNA library) from an invertebrate organism can be screened using a labeled LobCaR nucleic acid sequence to identify CASR genes, for example, as described in e.g., Ausubel, et al., Eds., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1997). The sequences of LobCaR homologs can then be compared to determine differences and similarities between them.
Accordingly, the invention provides isolated fragments (e.g., nucleic acid probes) of the LobCaR nucleic acid molecules described herein. Such fragments can be at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% the length of a LobCaR nucleic acid sequence (e.g., SEQ ID NO:1, SEQ ID NO:2) and are useful as primers and as probes for assays and techniques described herein. In particular embodiments, LobCaR fragments include at least about 50 continuous nucleotides of the LobCaR sequence or more (e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, or 4000 continuous nucleotides).
In one embodiment, LobCaR probes (e.g., oligonucleotide probes) are designed to have a Tm of approximately 80° C. (assuming 2° C. for each A or T and 4 degrees for each G or C).
The present invention further encompasses using primers based on the LobCaR nucleic acid sequences described herein to detect LobCaR nucleic acid molecules by Polymerase Chain Reaction (PCR) methods that are well known in the art. Methods and protocols for performing PCR are described generally in the following: PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al., Nucleic Acids Res., 19:4967 (1991); Eckert et al., PCR Methods and Applications, 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987, & Supp. 49, 2000; and U.S. Pat. No. 4,683,202).
In one embodiment, the level of a LobCaR nucleic acid (e.g., mRNA) in a sample (e.g., tissue sample, cell sample) is determined by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), a technique that is well known in the art. Briefly, RNA is extracted from the tissue of interest, poly A+ RNA is isolated from the extract by standard methods and the isolated poly A+ RNA reverse transcribed. Subsequently, a PCR amplification reaction is performed with LobCaR-specific primers using the products of the reverse transcription reaction and the presence of the predicted LobCaR product is determined, for example, by detecting the product of the RT-PCR reaction (e.g., via agarose gel electrophoresis). The RT-PCR product can be a nucleic acid molecule having part or all of a LobCaR nucleotide sequence. The RT-PCR product can optionally be detectably labeled (e.g., radioactively labeled, fluorescently labeled) and the presence or amount of LobCaR product can be determined using an appropriate detection means (e.g., autoradiography). Alternative methods of labeling and quantifying the RT-PCR product are well known to one of skill in the art (see Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience 1987, & Supp. 49, 2000.
The present invention also includes kits for detecting and/or quantifying a LobCaR nucleic acid molecule comprising nucleotide probes (e.g., PCR primers) that can hybridize to the LobCaR nucleic acid under conditions of appropriate stringency.
In addition to RT-PCR, Northern blotting and other RNA blotting hybridization techniques, the levels of LobCaR nucleic acids can be detected by in situ hybridization. This technique requires fewer cells than Northern blotting, and involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled nucleic acid probes. The practice of the in situ hybridization technique is described in more detail in U.S. Pat. No. 5,427,916, the entire disclosure of which is incorporated herein by reference. Suitable probes for in situ hybridization of a LobCaR nucleic acid molecule can be produced, as described above, from the nucleic acid sequences of SEQ ID NO:1 or SEQ ID NO:2.
The invention also provides vectors (e.g., phages, viral vectors, non-viral vectors) and plasmids containing one or more of the LobCaR nucleic acid sequences described herein (e.g., SEQ ID NO:1, SEQ ID NO:2). Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or are readily prepared by a skilled artisan. Exemplary vectors are described, for example, in Ausubel, F. M., et al., Current Protocols in Molecular Biology, (Current Protocol, 1994) and Sambrook et al., “Molecular Cloning: A Laboratory Manual,” 2nd ED. (1989). Vectors and plasmids containing the LobCaR gene or gene fragments can be used, for example, to generate hybridization probes for detecting and measuring the level of LobCaR gene expression in tissues and/or for identifying LobCaR homologs in other invertebrates; (2) to express the LobCaR mRNA or protein in vitro or in vivo; and (3) to generate transgenic non-human animals or recombinant host cells.
A LobCaR nucleic acid sequence can be inserted into a suitable vector using standard molecular cloning techniques and readily available starting materials known to those of skill in the art. In a particular embodiment, the vector is an expression vector, which contains genetic elements (e.g., regulatory sequences) that are necessary to direct expression of the LobCaR sequence when introduced into an appropriate host cell. Expression vectors generally comprise a promoter that is operably-linked with a portion of the isolated nucleic acid that encodes or specifies a gene product. Numerous expression vectors are known in the art and are commercially available including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In particular, expression systems containing the requisite control sequences, such as promoters and polyadenylation signals, and preferably enhancers, are readily available and known in the art for a variety of different hosts (see e.g., Sambrook, et al., Molecular Cloning a Laboratory Manual, Second Ed. Cold Spring Harbor Press (1989)).
A vector or plasmid containing a LobCaR nucleotide sequence may be provided to a cell so as to stimulate production of the active LobCaR polypeptide in vivo. For this purpose, various techniques and reagents have been developed. For example, a number of viral vectors have been developed that allow for transfection and, in some cases, integration of the virus into the host cell. See, for example, Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science 243, 375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Non-viral delivery methods such as administration of the DNA via complexes with liposomes may also be used.
In one embodiment, the present invention encompasses host cells transformed with the plasmids or vectors described herein. LobCaR nucleic acid molecules can be inserted into a construct which can, optionally, replicate and/or integrate into a recombinant host cell, by known methods. The host cell can be a eukaryotic cell or prokaryotic cell and includes, for example, yeast (such as Pichia pastorius or Saccharomyces cerevisiae), bacteria (such as E. coli or Bacillus subtilis), insect cells (such as baculovirus-infected SF9 cells) or mammalian cells (somatic or embryonic cells, Human Embryonic Kidney (HEK) cells, Chinese hamster ovary cells, HeLa cells, human 293 cells and monkey COS-7 cells).
A nucleic acid molecule can be incorporated or inserted into the host cell by known methods. Examples of suitable methods of transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. “Transformation” or “transfection” as used herein refers to the acquisition of new or altered genetic features by incorporation of additional nucleic acids, e.g., DNA. “Expression” of the genetic information of a host cell is a term of art which refers to the directed transcription of DNA to generate RNA which is translated into a polypeptide. Methods for preparing such recombinant host cells and incorporating nucleic acids are described in more detail in Sambrook et al., “Molecular Cloning: A Laboratory Manual,” Second Edition (1989) and Ausubel, et al. “Current Protocols in Molecular Biology,” (1992), for example.
In one embodiment of the invention, the nucleic acid constructs are delivered to cells by transfection, i.e., by delivery of a “naked” nucleic acid or a nucleic acid that is complexed with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system of this invention is a lipid-complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA constructs may first be optimized for expression (e.g., inclusion of an intron in the 5′ untranslated region and elimination of unnecessary sequences (Felgner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be accomplished using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat. Genet. 5:135-142, 1993 and U.S. Pat. No. 5,679,647 by Carson et al.
In certain embodiments of the invention, a LobCaR nucleic acid is delivered to a cell (e.g. a host cell) as a viral vector, such as a recombinant retrovirus (e.g., lentivirus), adenovirus, adeno-associated virus (AAV), herpes simplex derived vector, hybrid adeno-associated/herpes simplex viral vector, influenza viral vector, especially those based on the influenza A virus, and alphavirus, for example the Sinbis and semliki forest viruses). The LobCaR nucleic acid can also be provided to a host cell as recombinant bacterial or eukaryotic plasmid, among others.
The host cell is then maintained under suitable conditions that allow for expression and recovery of the expressed LobCaR proteins. Generally, the cells are maintained in a suitable buffer and/or growth medium or nutrient source for growth of the cells and expression of the gene product(s). The growth media are not critical to the invention, are generally known in the art and include sources of carbon, nitrogen and sulfur. Examples include Luria broth, Superbroth, Dulbecco's Modified Eagles Media (DMEM), RPMI-1640, M199 and Grace's insect media. The growth media can contain a buffer, the selection of which is not critical to the invention. The pH of the buffered Media can be selected and is generally one tolerated by or optimal for growth for the host cell.
The host cell is maintained under a suitable temperature and atmosphere. Alternatively, the host cell is aerobic and the host cell is maintained under atmospheric conditions or other suitable conditions for growth. The temperature should also be selected so that the host cell tolerates the process and can be for example, between about 13 and 40° C.
LobCaR Polypeptides
The present invention also encompasses isolated LobCaR polypeptides encoded by the LobCaR nucleic acid molecules described herein. Such polypeptides include, for example, an isolated LobCaR polypeptide comprising the amino acid sequence of SEQ ID NO:3. In particular embodiments, a LobCaR polypeptide of the invention is encoded by a LobCaR nucleic acid molecule having the sequence of SEQ ID NO:1 or 2.
An isolated LobCaR polypeptide can be prepared by a variety of techniques that are known to those of skill in the art. For example, the LobCaR polypeptide can be expressed in an in vitro expression mixture using a polynucleotide that encodes a LobCaR protein (e.g., SEQ ID NO:1, SEQ ID NO:2), or it may be expressed in a human or non-human cell that contains an expression vector comprising a LobCaR-encoding nucleic acid molecule, and subsequently purified therefrom. Alternatively, a LobCaR polypeptide can be purified using, for example, standard chromatographic techniques from a naturally occurring source of LobCaR protein (e.g. a tissue or cell sample from an invertebrate organism).
Other forms of the LobCaR polypeptides encompassed by the present invention include those that are “functionally equivalent” to native LobCaR protein of SEQ ID NO: 3.
One of skill in the art will recognize that certain changes in the amino acid sequence of a protein/polypeptide can occur without disrupting one or more functions of that protein/polypeptide. For instance, the skilled artisan would reasonably expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e., conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. (see, for example, Biochemistry, 2nd ed., Ed. by L. Stryer, W.H. Freeman and Co., 1981).
Whether a change in the amino acid sequence of a polypeptide results in a functional homolog can be readily determined by assessing the ability of the variant polypeptide to produce a response in a cell that is identical or similar to a response produced by the wild-type protein. For instance, variant forms of LobCaR polypeptides can be assessed, e.g., for one or more functions performed by native LobCaR polypeptides. Accordingly, the term “LobCaR polypeptide” or “LobCaR protein” also encompasses conservative and other variants (e.g., allelic variants, species variants) of the LobCaR polypeptide depicted by SEQ ID NO:3. In general, these variant polypeptides are also referred to as “analogs”.
Accordingly, in some embodiments, the present invention relates to isolated LobCaR polypeptides having at least about 70% (e.g., 75%, 80%, 85%, 90% or 95%) identity to SEQ ID NO:3. In a particular embodiment, the invention relates to isolated LobCaR polypeptides having at least about 90% identity to SEQ ID NO:3.
The skilled artisan also will recognize that the activity of a protein/polypeptide may reside in one or more functional domains within the protein/polypeptide and, in certain cases, these functional domains will retain their activity outside the context of the full-length form of the protein. Therefore, the term “LobCaR polypeptide” also encompasses derivatives and biologically-active fragments of the LobCaR polypeptide having the sequence shown as SEQ ID NO:3.
Biologically-active LobCaR protein derivatives and fragments may be derived from a full-length LobCaR polypeptide (e.g., following enzymatic digestion, for example, by a protease). Isolated peptidyl portions of LobCaR polypeptides also can be obtained by screening recombinant polypeptides that are produced from nucleic acid fragments that encode such polypeptides. In addition, LobCaR polypeptide fragments can be synthesized chemically using techniques known in the art, such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
LobCaR polypeptides may further comprise post-translational modifications or non-amino acid elements, such as hydrophobic modifications (e.g. polyethylene glycols or lipids), poly- or mono-saccharide modifications, phosphates, and acetylations, among others.
In addition, mimetics (e.g., peptide or non-peptide agents), which are able to mimic one or more activities of a LobCaR polypeptide are also encompassed by the invention. For example, amino acid residues of a LobCaR polypeptide may be mapped, using standard techniques (e.g., scanning mutagenesis), to determine which ones affect one or more activities of a LobCaR protein. Using this information, peptidomimetic compounds can be generated which mimic those residues involved in the activities and/or interactions of interest. Non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine (e.g., see Huffman et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings (Garvey et al., in Peptides: Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson et al., (1986) J. Med. Chem. 29:295; and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill., 1985), b-turn dipeptide cores (Nagai et al., (1985) Tetrahedron Lett 26:647; and Sato et al., (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols (Gordon et al., (1985) Biochem Biophys Res Commun 126:419; and Dann et al., (1986) Biochem Biophys Res Commun 134:71).
LobCaR polypeptides can also be fusion or chimeric polypeptides. Fusion proteins can be manufactured according to known methods of recombinant DNA technology. For example, fusion polypeptides can be expressed from a nucleic acid molecule comprising sequences which code for all or a portion (e.g., a biologically active portion) of the LobCaR polypeptide and its fusion partner (e.g., all or a portion of an immunoglobulin molecule). For example, fusion polypeptides can be produced by the insertion of nucleic acid sequences encoding the fusion partners into a suitable expression vector, phage vector, or other commercially available vector. The resulting construct can be introduced into a suitable host cell for expression. Upon expression, the fusion proteins can be isolated or purified from a cell by means of an affinity matrix or other technique known to those of skill in the art. These cells, or the fusion polypeptides produced from them, can be utilized in a variety of screening assays.
In certain embodiments, the LobCaR polypeptides and/or LobCaR fragments of the invention are reactive with, and bind to, any one of the LobCaR antibodies described herein. In a particular embodiment, a LobCaR polypeptide can be bound by an antibody that binds to an epitope contained in the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
Antibodies that Bind LobCaR Polypeptides
The present invention also encompasses antibodies (e.g., polyclonal antibodies, monoclonal antibodies) that are reactive with, and bind to, any of the LobCaR polypeptides described herein. In a particular embodiment, the antibodies specifically bind LobCaR polypeptides or fragments thereof.
Methods for making and selecting antibodies are well known to those of skill in the art, as evidenced by standard reference works such as: Harlow, et al., Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory N.Y. (1988); Klein, Immunology: The Science of Self-Nonself Discrimination (1982); Kennett, et al., Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses (1980); and Campbell, “Monoclonal Antibody Technology” in laboratory techniques in biochemistry and molecular biology.
Polyclonal antibodies are derived from the sera of animals (e.g., rabbits, mice, guinea pigs) that have been immunized with antigen. Methods for producing such polyclonal antibodies are well known in the art and may involve either injecting the full-length LobCaR protein itself or a portion thereof, or, alternatively, injecting short peptides made to correspond to different regions of the LobCaR polypeptide into an appropriate animal.
Monoclonal antibodies can be prepared using hybridoma technology (Kohler, et al., Nature 256:495 (1975); Hammerling, et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y. pp. 563-681 (1981)). In general, this technology involves, for example, immunizing an animal, usually a mouse, with either intact LobCaR protein or a fragment thereof. The splenocytes of the immunized animals are extracted and fused with suitable myeloma cells (e.g., SP2/O cells) to produce a hybridoma. After fusion, the resulting hybridoma cells are selectively maintained in culture medium (e.g., HAT medium) and then cloned by limiting dilution (Wands, et al., Gastroenterology 80:225-232 (1981)). The cells obtained through such selection are then tested to identify clones which secrete antibodies capable of binding to LobCaR with the desired specificity using a suitable assay (e.g., ELISA).
Anti-LobCaR antibodies can be raised against appropriate immunogens, such as, for example, isolated and/or recombinant LobCaR polypeptides, analogs or fragments (e.g., synthetic peptides), as described herein. Such antibodies can also be raised against a host cell that expresses recombinant LobCaR protein. In addition, cells expressing recombinant LobCaR protein, such as transfected cells, can be used as immunogens or to screen for antibodies that bind the LobCaR polypeptide. In one embodiment, anti-LobCaR antibodies are raised against a peptide sequence from a LobCaR protein (e.g., SEQ ID NO:3). Suitable peptides immunogens include peptides having the amino acid sequence of SEQ ID NO:4 or SEQ ID NO:6.
In certain embodiments, the antibodies of the invention are detectably labeled. 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, magnetic beads and the like. The detection of antibodies in a complex can also be done immunologically with a second antibody which can then be detected (e.g., by means of a label). Conventional methods or other suitable methods can directly or indirectly label an antibody. Labeled primary and secondary antibodies can be obtained commercially or prepared using methods know to one of skill in the art (see Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.).
The invention also encompasses kits that comprise an antibody that binds to a LobCaR polypeptide as described herein for use in, for example, detecting the presence of a LobCaR polypeptide in a sample.
The antibodies of the present invention may be used in methods for determining the level of a LobCaR polypeptide in a sample (e.g., a biological sample from an invertebrate organism, such as, a tissue sample, a cell sample, a blood sample). For example, the LobCaR antibodies described herein may be used to detect the presence of LobCaR proteins and homologs in a sample using standard immunological methods that are known to those of skill in the art. Such methods include Western blot analysis, FACS analysis, radioimmunoassays, flow cytometry, immunochemistry (e.g., immunocytochemistry, immunohistochemistry), enzyme-linked immunosorbent assays (ELISA) and chemiluminescence assays, among others. Radioimmunoassays, or immunometric assays, are also known as “two-site” or “sandwich” assays (see Chard, “An Introduction to Radioimmune Assay and Related Techniques,” in: Laboratory Techniques in Biochemistry and Molecular Biology, North Holland Publishing Co., N.Y. (1978)). In a typical immunometric assay, a quantity of unlabeled antibody is bound to a solid support that is insoluble in the fluid being tested, e.g., blood, lymph, cellular extracts, etc. After the initial binding of antigen to immobilized antibody occurs, a quantity of detectably labeled secondary antibody is added to permit detection and/or quantitation of bound antigen (see, e.g., Radioimmune Assay Method, Kirkham, et al., ed. pp. 199-206, E&S Livingstone, Edinburgh (1970)). Many variations of these types of assays are known in the art and may be employed for the detection of the LobCaR polypeptides.
Antibodies to LobCaR polypeptides may also be used in the purification of either the intact receptor or fragments of the receptor (see generally Dean, et al., Affinity Chromatography, A Practical Approach, IRL Press (1986)). Typically, antibody is immobilized on a chromatographic matrix such as Sepharose 4B. The matrix is then packed into a column and the preparation containing LobCaR polypeptide is passed through the column under conditions that promote binding, e.g., under conditions of low salt. The column is then washed, and bound LobCaR polypeptide is eluted using a buffer that promotes dissociation from antibody, e.g., buffer having an altered pH or salt concentration. The eluted LobCaR polypeptide may be transferred into a buffer of choice, e.g., by dialysis, and either stored or used directly.
Screening Methods for LobCaR Modulators
The present invention also includes methods for identifying and assaying compounds that modulate the expression level and/or activity of an invertebrate CaSR nucleic acid molecule or protein (e.g., a LobCaR nucleic acid molecule or polypeptide). In determining whether compounds are LobCaR modulators, one can measure changes in the expression level of one or more of the LobCaR genes or proteins described herein, or changes that occur in one or more intracellular signal transduction pathways (e.g., a pathway involved in the sensing and/or processing of stimuli) involving the LobCaR polypeptide.
In a particular embodiment, a compound that modulates the expression level or activity of an invertebrate CaSR protein is identified by contacting a cell that expresses a LobCaR polypeptide (e.g., a polypeptide comprising the amino acid sequence of SEQ ID NO: 3) with a test compound and subsequently detecting the expression level or activity of said polypeptide in the host cell, wherein an alteration in the expression level or activity of the nucleic acid or polypeptide relative to a suitable control (e.g., a cell that expresses a LobCaR nucleic acid molecule and/or polypeptide that has not been contacted with the test compound). An alteration (e.g., a decrease, an increase) in the expression level or activity of the LobCaR nucleic acid or polypeptide in the presence of the test compound, relative to the expression level of the same molecule in the control, indicates that the test compound modulates the expression of an invertebrate CASR protein.
Suitable test compounds for the screening methods of the invention include, but are not limited to, compounds belonging to any of the classes of CaSR modulators described herein. Such test compounds may be naturally occurring, or may be produced using synthetic methods known in the art.
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
Identification and Characterization of the LobCaR Gene
A reduced stringency DNA-DNA hybridization approach was used to identify and isolate the LobCaR gene. A 32P-labeled probe containing the cDNA sequence encoding the complete amino acid sequence of an Atlantic salmon CaSR, SalmoKCaR II, which had been previously isolated and described in U.S. Pat. Nos. 6,979,558, 6,951,739, 6,979,559, and 7,101,988, was utilized to probe a commercially-available lobster genomic library (Stratagene FIX II Lobster Genomic Library catalog #946630). A total of 1 million plaques on filters were screened and washed under reduced stringency hybridization conditions that included hybridization and washes using 2×SSC, 0.1% SDS at 53′C. A total of 2 positive plaques were identified (FIG. 1) that appeared to be identical upon purification and mapping by restriction enzyme analysis.
The positive plaque containing the LobCaR gene was then characterized using standard subcloning and DNA sequencing methods. FIG. 2 shows a diagram of a DNA restriction endonuclease map of the DNA insert of the λ bacteriophage that contains the LobCaR gene sequence. The combination of restriction enzyme analyses and DNA-DNA hybridization analyses of the resulting fragments, together with DNA sequencing, demonstrated that the LobCaR sequence was contained within two restriction endonuclease fragments of 4 kb and 5 kb, respectively.
DNA sequence analysis of the entire 4 kb DNA fragment and a portion of the 5 kb DNA fragment revealed that the majority of the LobCaR gene was contained within the 4 kb DNA fragment. The remaining portions of the gene, which code for portions of the extracellular domain of LobCaR, are contained in the 5 kb DNA fragment. The complete sequence of the LobCaR gene (SEQ ID NO:1) is composed of a total of 6 exons that are interspersed between a total of 7 introns (FIG. 3).
To determine whether the LobCaR DNA sequence (SEQ ID NO:1) hybridizes to other known CaSR DNA sequences under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.), a hybridization experiment was performed. Individual filters were lifted from respective agar plates containing a pure lawn of bacteriophage λ plaques that possessed either the LobCaR DNA sequence (FIG. 4B) or the sequence for the shark kidney CaSR (FIGS. 4A and 4C, respectively) (see U.S. Pat. No. 6,337,391, International Patent Application No. PCT 0934407, German Patent No. 69733763.4-08, and Australian Patent No. 755847). Filters 1 and 2 were initially hybridized to a 32P-labeled SKCaR probe under very low stringency conditions (2×SSC, 0.1% SDS at 53° C.), while filter 3 was initially hybridized to 32P-labeled human parathyroid CaSR (HuPCaR) (see U.S. Pat. Nos. 6,688,938; 5,763,569; 5,858,684; and 5,962,314; Australia Patent No. 711247; Canadian Patent No. 2,115,828; Europe Patent Nos. 0 637237; 1,296,142; and 1,366,764). The filters were then washed under the stringency conditions described below followed by autoradiographic exposure to detect the presence of hybridized 32P-labeled SKCaR or HuPCaR to filter DNA.
As expected, the washing of filter #1 containing SKCaR λ bacteriophage plaques under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.) did not result in the removal of 32P-labeled SKCaR probe. This is indicated by the presence of 32P-labeled plaques after exposure to x-ray film for 3 hr of exposure (FIG. 4A). By contrast, significant DNA-DNA hybridization remains only after washing the filter with the LobCaR DNA sequence under reduced stringency (0.5×SSC, 0.1% SDS at 65° C.) (FIG. 4B). This DNA-DNA hybridization is only detectable after an 8 hr autoradiographic exposure that is greater than 2.5 times longer than the exposures performed on the filters shown in Panels 1 and 3. Washing of the filter containing the LobCaR sequence under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.) removes all 32P-labeled SKCaR DNA and no plaques are detected (data not shown). Finally, the washing of a filter with SKCaR in λ bacteriophages under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.) removes all 32P-labeled HuPCaR DNA and no plaques are detected (FIG. 4C).
Thus, the LobCaR DNA sequence (SEQ ID NO:1) does not hybridize to either (SalmoKCaR 2) or elasmobranch (SKCaR) CaSR DNA sequences under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.), suggesting that LobCaR DNA sequence is sufficiently different from both finfish (SalmoKCaR 2) and elasmobranch (SKCaR) CaSR DNA sequences.
Example 2
Comparison of the Amino Acid Sequence of the Putative LobCaR to Known CaSRs from Other Species
A comparison of the percent identity (exact match) between aligned amino acid sequences of the putative LobCaR and other known CaSRs was performed (Table 1). The putative amino acid sequence of LobCaR (SEQ ID NO:3) is 64.3% identical to human parathyroid calcium receptor (HuPCaR) and 65.7% identical to the shark kidney CaSR(SKCaR). By contrast, LobCaR is approximately 83.5% identical to SalmoKCaR-2.







TABLE 1







Comparison of putative LobCaR amino acid sequence


aligned to other CaSRs.


PerCent Amino Acid Identities


for Various Calcium Sensing Receptors or Polyvalent


Cation Sensing Receptors As Compared to the Lobster


Polyvalent Cation Sensing Receptor (LobCaR)


Consisting of 944 Amino Acids









AA Identical to



Lobster PVCR



(LobCaR)










Name of CaSR or PVCR
# of Amino Acids
Number of AA
%













Human Calcium Sensing
1078
693 of 1078
64.3


Receptor (HuPCaR)


Shark Kidney Polyvalent
1027
675 of 1027
65.7


Cation Sensing Receptor


(SKCaR)


Atlantic Salmon Kidney
941
788 of 944*
83.5


Polyvalent Cation Sensing


Receptor (SalmoKCaR-2)





*% identity calculation for SalmoKCaR-2 vs. LobCaR is performed using LobCaR as denominator since it is 3 amino acids longer than SKCaR-2.






These data are consistent with the findings that the LobCaR DNA sequence only hybridizes to SalmoKCaR-2 or SKCaR under reduced stringency DNA-DNA hybridization conditions (FIGS. 1 and 4). These data also indicate that the LobCaR gene, and CaSR genes in crustaceans in general, are sufficiently divergent in structure from all other known CaSRs that have been previously described, thereby suggesting that crustacean CaSRs have different functions than CaSRs in other organisms. This conclusion is supported by the fact that functionally-equivalent beta actin proteins from six highly divergent organisms have greater than 95% amino acid identity and, for most organisms, greater than 98% identity (Table 2). In contrast to actin that is incorporated into actin filaments possessing a highly conserved structure, CaSRs are expressed in multiple cell types where the structural and functional characteristics between such cells is very different and these cells are exposed to differing extracellular environments.







TABLE 2







Comparison of human beta actin amino acid sequence


aligned to other beta actin proteins from various species


obtained using publicly available DNA and protein databases.


PerCent Amino Acid Identities for Various Beta Actin Proteins


As Compared to the Beta Actin Protein From Humans Consisting


of 375 Amino Acids









AA Identical to



Human Beta Actin










Name of Beta Actin
# of Amino Acids
Number of AA
%













Xenopus laevis (African
375
374 of 375
99.73


clawed frog)


Triakis scyllium (banded
375
374 of 375
99.73


hound shark)


Ornithodoros moubata (soft
376
371 of 376
98.7


tick insect)


Salmo salar (Atlantic salmon)
375
370 of 375
98.7


Callinectes sapidus
376
369 of 376
98.1


(Blue Crab)


Strongylocentrotus purpuratus
376
362 of 376
96.3


(Sea Urchin)









Example 3
Expression of the LobCaR Gene in Lobsters is Tissue-Specific
To determine whether the amino acid sequence of SEQ ID NO:3 is the LobCaR protein, and to demonstrate that the LobCaR gene is expressed in various tissues of lobsters, we utilized two separate polyclonal antibodies that were created for both immunocytochemistry analyses and immunoblotting experiments. One antibody, called LOB-1, was raised against a 19 amino acid peptide (SEQ ID NO:4) of LobCaR. The other antibody, called SDD, was raised previously against a 16 amino acid peptide (SEQ ID NO:6) from salmon CaSR. To produce the polyclonal antibodies, the individual peptides were synthesized, conjugated to an antigenic protein (keyhole limpet hemocyanin) and injected intradermally into rabbits. Rabbit polyclonal antisera were produced using standard methods and techniques that are generally known to those of skill in the art. For the SDD antibody, the peptide sequence used to create the antibody differs from the corresponding LobCaR amino acid sequence (SEQ ID NO:5) by a single amino acid, (N vs. K) (Table 3). Thus, the SDD antiserum is reactive to both the LobCaR protein and its corresponding CaSR homolog in Atlantic salmon.







TABLE 3





Amino acid sequences of two peptides used for


the creation of rabbit polyclonal antisera


capable of recognizing LobCaR proteins in


lobsters and other invertebrate organisms.

















Lobster
LOB-1:
SVNGKKGAKLSIDKTKIL




(SEQ ID NO: 4)


 


Lobster

SDDEYGRPGIEKFEKENEM




(in LobCaR CaSR)




(SEQ ID NO: 5)


 


Salmon
SDD:
SDDEYGRPGIEKFEKEKEM




(in SalmoKCaR-2 CaSR)




(SEQ ID NO: 6)









Immunolocalization studies using the LOB-1 and SDD antibodies were performed on various tissues from different species of lobster. Immunoreactive LobCaR protein was detected in cells present within the intestine, antennal gland and testis of spiny lobsters (Panulirus interruptus) using the LOB-1 antiserum (FIGS. 5B, D, F). Exposure of identical histology sections from these same sample tissues to rabbit preimmune control antiserum, which was collected prior to immunization of the rabbits with the LOB-1 peptide, displays minimal or no reactivity (FIGS. 5A, C, E).
A similar study was performed using tissues from another lobster species, the American lobster (Homarus americanus). SDD antiserum (FIGS. 6B, D, F, H), but not its pre-immune counterpart (FIGS. 6A, C, E, G), localized LobCaR protein to specific cells in lobster intestine (FIG. 6B), antennal gland (FIG. 6D), testis (FIG. 6F) and gill (FIG. 6H). The pattern of antibody localization of LobCaR immunoreactive protein is similar in common tissues tested from both lobster species, including intestine, antennal gland and testis. These data show that LobCaR protein is selectively expressed in lobster cells.
Example 4
Antibodies that Recognize LobCaR can Detect the Localization of CaSR Proteins in Other Crustaceans and Invertebrates
LOB-1 and SDD antibodies were utilized as probes to determine the presence and location of LobCaR homologs in other crustaceans and/or invertebrates, we performed immunolocalization studies using the LOB-1 and SDD antibodies described in Example 3 in various life stages of another crustacean, the northern acorn barnacle (Semibalanus balanoides) (FIGS. 7A-F). Immunostaining was detected in selected cells of the barnacle that included the cirri (thoracic feeding appendages) (FIG. 7B) and epithelial cells lining the GI tract (FIG. 7D).
To demonstrate that antibody reagents produced from peptides containing amino acid sequences present in LobCaR are useful as probes to further characterize the structure, function and roles of CaSRs in invertebrates, immunoblotting analyses were performed after sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) fractionation of proteins from barnacles (Semibalanus balanoides) and star lobsters (Panulirus guttatus) under non-reducing (no addition of sulfhydryl reducing agents) or reducing (addition of sulfhydryl reducing agents) conditions (FIG. 9). Exposure of membranes containing fractionated proteins to immune anti-SDD LobCaR antiserum (IMMUNE), but not preimmune (PreImmune) antiserum, resulted in at least three major bands in lanes containing barnacle and lobster extracts (Lanes 1, 2 and 5, 6). The intensity of the larger molecular weight bands (denoted by the brackets) was diminished in lanes fractionated under reducing conditions as compared to nonreducing conditions. The apparent reduction in the molecular weight of these immunoreactive bands is similar to that observed in various other CaSR proteins in both terrestrial (Ward, D. et al. J. Biol. Chem. 1997) as well as aquatic organisms.
Example 5
Identification of CaSR Genes in Various Invertebrate Organisms Using Complete or Partial LobCaR Gene Sequences as Probes
The complete and partial DNA sequences of the LobCaR gene (e.g., SEQ ID NOS: 1 and 2) can be utilized as probes to determine both the presence and structure of CaSR genes in invertebrates using standard genomic DNA-DNA hybridization techniques. Because the DNA sequence of the LobCaR gene is more homologous to other invertebrate and teleost fish CaSR genes than CaSR genes form other organisms (see Table 1), DNA-DNA hybridization techniques using the LobCaR gene DNA can be used to identify the presence and structure of CaSR genes in various invertebrates under both high and reduced stringency conditions, due to lack of significant cross-hybridization with CaSR genes from other organisms (see FIGS. 4A-C).
DNA-DNA hybridization analyses were performed to assess the utility of the LobCaR DNA in identifying and assessing the structure of CaSR genes in various invertebrates. Genomic DNA from crustacean (lobster), mollusc (mussel) or teleost fish (goldfish and guppy) was subjected to restriction endonuclease digestion with either ECO R1, BAM H1 or a combination of ECO R1+BAM H1, and fractionated using agarose gel electrophoresis. The resulting DNA contents of the agarose gel was transferred to a membrane and probed with LobCaR DNA (SEQ ID NO:1) under high stringency conditions (0.1×SSC, 0.1% SDS at 65° C.). FIG. 10 shows the resulting autoradiogram where labeled bands represent specific DNA-DNA hybridization between 32P-labeled LobCaR DNA and DNA from invertebrate or teleost fish genomes coding for CaSR homologs in these organisms.
A similar DNA-DNA hybridization analysis was performed under reduced stringency conditions using 32P-labeled LobCaR DNA as a probe and genomic DNA from cephalochordate (amphioxus), an ancient invertebrate (horseshoe crab, Limulus polyphemus), sea urchin, tunicate and two species of decapod shrimp (FIG. 11), revealing the existence of CaSR homologs of the LobCaR gene in these species.
These data indicate that complete and partial DNA sequences of the LobCaR gene (e.g., SEQ ID NOS: 1 and 2) can be utilized as probes to determine the presence and structure of putative CaSR genes in invertebrates using standard genomic DNA-DNA hybridization techniques. Information gained from these studies can be used not only to select what species and class of invertebrate organism to conduct scientific or commercial development studies on, but also to guide the direction of future functional assessment using knowledge of both the size and presence of specific fragments of CaSR gene homologs present in invertebrate species.
Example 6
Identification of Putative CaSR Genes and Protein Sequences in Publicly-Available Databases Using LobCaR Nucleotide and Amino Acid Sequences
Interactive websites that allow for computer searches of databases containing genomic sequences have been developed for multiple organisms, including the cnidarian starlet sea anemone (Nematostella vectensis), the tunicate sea squirt (Ciona intestinalis), the echinoderm purple sea urchin (Strongylocentrotus purpuratus), and nematode round worm (Caenorhabditis elegans). However, the utility of searches in these databases are limited by the lack of appropriate sequence probes to search them. Therefore, the nucleic acid and amino acid sequences of LobCaR (e.g., SEQ ID NOS:1, 2 and 3) can be used to search these databases and others to identify candidate CaSR genes and proteins that can be studied further.
Accordingly, the complete LobCaR amino acid sequence (SEQ ID NO:3) or portions thereof were used to identify candidate CaSR proteins in publicly available databases. FIG. 12 shows an alignment of SEQ ID NO:3 with amino acid sequences of candidate CaSR proteins, which were not identified as calcium sensing receptor proteins (CaSRs) in their respective databases. Rather, these sequences were labeled as either “weakly similar to” (Ciona intestinalis) or “predicted peptide similar to CaSR” (sea urchin), or mislabeled as a “ANF receptor” (Nematostella vectensis) or “metabotropic glutamate receptor-7” (C. elegans) in their respective databases. However, comparison of SEQ ID NO:3 from lobster with these candidate sequences from various databases shows that these amino acid sequences are actually CaSR homologs.
These claims can be further substantiated using the nucleic acid sequences of LobCaR (e.g., SEQ ID NOS:1 and 2) to perform DNA-DNA hybridization studies as described in Examples 1 and 4, or by performing immunolocalization studies on sections or immunoblot analyses on SDS-PAGE fractionated protein mixtures as described in Example 3. Taken together, these data indicate that SEQ ID NOS:1, 2 and 3 can be used, either individually or in various combinations, to identify and characterize candidate CaSR proteins in various species of widely divergent invertebrate organisms.
Example 7
Proteins Reactive with LobCaR Antiserum are Expressed in Nutrient Transporting and Osmoregulatory Tissues of the Shrimp Litopenaeus vannamei 
To determine whether putative CaSR proteins in shrimp localize to nutrient transporting and osmoregulatory tissues, sections of intestine and posterior gill from the shrimp Litopenaeus vannamei were stained with LobCaR antiserum. Specific staining was observed in cells lining the lumen of the intestine (FIG. 13D) and at the base of secondary filaments in tissues of the gill that are putatively involved in ionoregulation rather than respiration (FIGS. 13A & B). Similar results were obtained when using the SDD antiserum. Staining was also present in eyestalk tissues as well as integumentary epithelium (FIG. 13C). Putative CaSR proteins in shrimp gill osmoregulatory tissues also colocalize Na/K atpase, an enzyme that is crucial to osmoregulation in this species.
Example 8
Relationship of Shrimp Postlarval Survivorship to Inland Well Water Ionic Composition
The relative 48 hr survivorship of shrimp postlarvae (PL20) in southern U.S. well waters with various ionic compositions was tested. Percent (%) survivorship varied from 0-100%, depending on the ionic composition of the water (FIG. 14). All well waters with Na+/Ca2+ ratios that were less than those found in artificial seawater (at or about 45), which are generally indicative of higher Ca2+ concentrations than in artificial seawater, showed good survivorship, whereas well waters with higher Na+/Ca2+ ratios than artificial seawater showed uniformly poor survivorship (values taken from tables in Saoud, I, D. Davis, and D. Rouse, 2003). Suitability studies of inland well waters for Litopenaeus vannamei culture. Aquaculture 217:373-383). Thus, the ratio of Na+ to Ca2+, both CaSR modulatory compounds, appears to have a significant influence on survivorship of shrimp.
Example 9
Addition of CaSR Modulators to Water and Diet Induces Multiple Changes in Shrimp Physiology
To test the effect of CaSR modulators on shrimp physiology, three groups of Litopenaeus vannemei shrimp were tested for 4 weeks. Two control groups (C1 and C2) were held under 4 ppt recirculating water conditions, whereas an experimental group (SS & diet) was held under identical conditions, except that CaSR modulators were added to the water (3 mM CaCl2, 1 mM MgCl2) and the feed (5% NaCl, 0.4% Tryptophan). The experimental group showed an elevated incidence of molting (FIG. 15A), a reduction of mortality (FIG. 15B) and increased growth (FIG. 15C) relative to both control groups. The experimental group also showed elevated levels of certain ions in the hemolymph as compared to controls (FIG. 16A), as well as increased hemolymph osmolarity (FIG. 16B). Gill Na/K atpase activity was also increased in the experimental group (FIG. 16C).
Example 10
The Addition of CaSR Modulators to Low Salinity Water and Feed Alters the Concentrations of Various Amino Acids in Shrimp
Shrimp cultured in low salinity water often exhibit undesirable differences in texture and taste relative to shrimp raised in sea water. These differences are influenced in part by amino acid concentrations in the muscle. To assess the effect of CaSR modulators on the amino acid composition of the shrimp tail muscle, shrimp were raised for more than 2 weeks under 3 experimental conditions: 1) low salinity (4 ppt seawater), 2) normal seawater (32 ppt) and 3) low salinity water (4 ppt) supplemented with calcium and magnesium, as well as feed supplemented with tryptophan. Differences in amino acid concentrations in tail muscle were observed for each treatment group (FIG. 17), indicating that the presence of CaSR modulators in the water and feed alter the amino acid of the shrimp tail muscle. Notably, the addition of CaSR modulators to the water and feed produced shrimp tail muscles with concentrations of at least some amino acids that are closer to those found in shrimp raised in normal sea water.
The relevant teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. Also, the teachings of companion International Patent Application No.: Ser. No. 12/612,549, entitled “Methods of Inhibiting Biofouling by Aquatic Biofouling Organisms,” filed concurrently on May 2, 2008, are hereby incorporated by reference in their entirety.
While this invention has been particularly shown and described with references to example and preferred embodiments thereof, it will be understood by those skilled in the art that various changes may be made therein without departing from the scope of the invention encompassed by the appended claims and equivalents.

## Claims

1. A method of raising crustaceans in low salinity water, comprising:
a) maintaining crustaceans in low salinity water having one or more Calcium-sensing Receptor (CaSR) modulator(s) in an amount sufficient to modulate at least one CaSR in the crustaceans; and
b) adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent in an amount sufficient to contribute to an increased level of the one or more CaSR modulator(s) in a tissue of the crustaceans, after consumption of the feed by the crustaceans.

2. The method of claim 1, wherein the crustaceans are shrimp.

3. The method of claim 2, wherein the shrimp are selected from the group consisting of Litopenaeus vannemei, Penaeus monodon, P. stylirostris, P. chinensis, P. japonicus, P. indicus, P. merguiensis and Machrobrachium rosenbergii.

4. The method of claim 1, wherein the crustaceans are spiny lobsters or clawed lobsters.

5. The method of claim 4, wherein the spiny lobsters or clawed lobsters belong to a genus selected from the group consisting of Homarus, Nephrops species, Jasus, Panulirus, and Palinurus.

6. The method of claim 1, wherein the low salinity water has a salinity of about 0.5 ppt to about 5 ppt.

7. A method of raising crustaceans in low salinity water, comprising:
a) adding one or more Calcium-sensing Receptor (CaSR) modulator(s) to low salinity water in an amount sufficient to modulate at least one CaSR in the crustaceans;
b) transferring the crustaceans to the low salinity water containing the one or more CaSR modulator(s); and
c) adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent in an amount sufficient to contribute to an increased level of the one or more CaSR modulator(s) in a tissue of the crustaceans, after consumption of the feed by the crustaceans.

8. The method of claim 7, wherein the agent in the feed is NaCl.

9. The method of claim 8, wherein the feed contains at least about 1% NaCl by weight.

10. The method of claim 7, wherein the feed contains one or more CaSR modulator(s).

11. The method of claim 7, wherein the crustaceans are shrimp, spiny lobsters or clawed lobsters.

12. The method of claim 7, wherein the crustaceans are raised under recirculating conditions.

13. A method of raising crustaceans, comprising:
a) adding one or more Calcium-sensing Receptor (CaSR) modulator(s) to low salinity water in which crustaceans are maintained, wherein the one or more CaSR modulator(s) are added in an amount sufficient to modulate at least one CaSR in tissue of the crustaceans; and
b) adding feed for crustacean consumption to the low salinity water containing the one or more CaSR modulator(s), wherein the feed contains an agent in an amount sufficient to contribute to an increased level of the one or more CaSR modulator(s) in a tissue of the crustaceans, after consumption of the feed by the crustaceans.

14. The method of claim 13, wherein the agent in the feed is NaCl.

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

16. The method of claim 13, wherein the feed contains one or more CaSR modulator(s).

17. The method of claim 13, wherein the crustaceans are shrimp, spiny lobsters or clawed lobsters.

18. The method of claim 13, wherein the crustaceans are raised under recirculating conditions.

19. A method of raising crustaceans, comprising providing crustaceans that are maintained in low salinity water with a feed that contains:
a) one or more Calcium-sensing Receptor (CaSR) modulator(s) in an amount sufficient to modulate at least one CaSR in tissue of the crustaceans, and
b) an agent in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the crustaceans, after consumption of the feed by the crustaceans.

20. The method of claim 19, wherein the agent in the feed is present in an amount sufficient to contribute to an increased level of the one or more CaSR modulator(s) in a tissue of the crustaceans.

21. The method of claim 19, wherein the agent in the feed is NaCl.

22. The method of claim 21, wherein the feed contains at least about 1% NaCl by weight.

23. The method of claim 19, wherein the feed contains one or more CaSR modulator(s).

24. The method of claim 19, wherein the crustaceans are shrimp, spiny lobsters or clawed lobsters.

25. The method of claim 19, wherein the crustaceans are raised under recirculating conditions.

26. The method of claim 19, wherein the CaSR modulators in the feed are provided via a live feed organism selected from the group consisting of artemia, polychaetes, and other crustaceans.

27. A method of raising shrimp in low salinity water, comprising:
a) maintaining shrimp in low salinity water having at least two Calcium-sensing Receptor (CaSR) modulators, each present CaSR modulator present in an amount sufficient to modulate at least one CaSR in the shrimp; and
b) adding feed for shrimp consumption to the low salinity water containing the at least two CaSR modulators, wherein the feed contains an agent in an amount sufficient to contribute to modulation of the at least one CaSR in a tissue of the shrimp, after consumption of the feed by the shrimp.

28. The method of claim 27, wherein the at least two CaSR modulators are Ca2+ and Mg2+.

29. The method of claim 28, wherein the Ca2+ is present in the water at a concentration of about 0.3 mM to about 12 mM and the Mg2+ is present in the water at a concentration of about 0.5 mM to about 30 mM.

30. The method of claim 27, wherein the feed contains at least one additional CaSR modulator.

31. The method of claim 30, wherein the at least one additional CaSR modulator is tryptophan.