WO2002083717A1 - Induction of spawning in crustacea - Google Patents

Abstract

An isolated peptide comprising the following amino acid sequence: GLTDGTCRGR MGNREIYKKV DRVCEDCANI FRLPGLEGLC RDRCFYNEWF LLCLKAANRE DEIENFRVWI SILNA an active fragment thereof, or a peptide with substantial sequence identity which serves to inhibit spawning in penaeid prawns.

Description

INDUCTION OF SPAWNING IN CRUSTACEA TECHNICAL FIELD

The present invention is concerned with the induction of spawning in Crustacea, in particular, in those which require eyestal ablation. The present invention has particular application for farmed penaeid prawns, of which by far the dominant species is P. monodon, although P. vannamei, P. stylirostr±s, P. chinensis and P. japonicus are also farmed in significant numbers .

BACKGROUND ART

Worldwide, growth in the penaeid farming industry has been hampered due to adverse environmental impacts, disease and shortages of broodstock. Many of these problems could be overcome by development of fully closed farming systems. Use of a fully recirculating water system mitigates disease problems resulting from intake of contaminated water, and likewise substantially reduces any effluent impacts. These systems are best suited to use with domesticated stock to avoid introduction of wild broodstock of unknown health status and other pathogenic vector organisms.

The penaeid species that have been successfully domesticated on a commercial scale are generally those which mature readily in captivity, often in growout ponds, including P. vannamei, P. styl±rostris and P. japonicus (Howell, 1999; CENIACUA, 1999; Goyard et al., 1999; Preston et al., 1999). Closing the life cycle of P. vaxmamei has facilitated the development of fully closed, biosecure prawn farming systems for this species in

Central and South America (Mclntosh, 2000) .

By contrast P. monodon has proven difficult to domesticate. The animals are harvested from grow-out ponds at 5-6 months as sub-adults and generally do not mature sexually until at least 12 months of age. Keeping large numbers of broodstock in good health for 12 months or more is a significant challenge for P. monodon domestication. Thus, on a commercial scale almost all farmed P. monodon continue to be produced from wild broodstock, with probably less than 1% from captive broodstock. Other key factors which impinge on successful domestication of P. monodon are the difficulty in achieving reliable spawning of female broodstock, the large variability in spawning performance of captive- reared broodstock, the quality of the spermatophores and the quality of resultant larvae. According to industry operators in Australia, the biggest single barrier at present is obtaining spawning from sufficient numbers of captive-reared broodstock. It is economically questionable holding animals in ponds for a full year or more until they mature if only a small proportion spawn.

Spontaneous spawning of intact broodstock is rare. Preferentially wild broodstock are captured with nearly completely developed ovaries (ie stage III- V - ovarian maturation is rated on a scale of I-IV, with I being immature and IV being just pre-spawning) and these may spawn on the first, second or third nights after arrival in the hatchery. If they do not spawn during this time, artificial stimulation of spawning is required. In addition, artificial induction of spawning may be required so that animals spawn at the correct time to synchronise larval production with demand from farms.

At present, the only practical means to stimulate spawning is by the removal of one eyestalk (unilateral eyestalk ablation) , following which the undeveloped ovary matures over 3-7 days leading to ovulation and spawning.

Sperm that have been stored in a specialised pouch known as the thylecum are released at the same time and fertilisation occurs in the water column. With wild broodstock, typically 60-80% of animals spawn following eyestalk ablation. With domesticated broodstock, by contrast, the proportion of animals that spawn can be only 20% (Menasveta et al., 1994) or even lower. Although eyestalk ablation may be a reasonably efficient means to induce spawning in wild broodstock, it also results in a number of other physiological effects, due to the physical stress and the removal of other key hormones, which impact on the longevity and quality of the broodstock. It is standard practise in P. monodon hatcheries to retain only the first and second batches of eggs post-ablation, and to reject subsequent spawnings due to the decline in broodstock condition and egg numbers and quality. Clearly there is need for an improved method of inducing spawning in both wild and domesticated P. monodon and other farmed penaeid prawns, and in Crustacea generally.

The physiological basis for the effect of eyestalk ablation is removal of the source of a hormone that inhibits ovarian development. The eyestalk contains an organ known as the X-organ-sinus gland (XO-SG) complex. The X-organ comprises cell bodies where hormones such as the Crustacean hyperglycemic hormone (CHH) family of hormones are produced and the sinus gland functions as a storage organ from where the hormones are released into the haemolymph (Fingerman, 1992) . These hormones, collectively known as sinus gland peptides, are postulated to include a hormone variously referred to as gonad inhibiting hormone (GIH) , vitellogenesis inhibiting hormone (VIH) or reproductive inhibiting hormone (RIH), but the latter term is used hereinafter. Other hormones in this family are crustacean hyperglycaemic hormone (CHH) and moult inhibiting hormone (MIH) .

Despite the strong sequence homology, the CHH family hormones do fall into two structural classes (Ohira et al., 1997; Lacombe et al., 1999). Type I precursors consist of a signal sequence and CHH precursor related peptide (CPRP) , both of which are sequentially cleaved to release the mature hormone which is 72-73 amino acids long and is amidated at the C-terminus. Type II precursors also have a signal sequence but lack a CPRP. The mature hormones are generally slightly longer (75-78 residues) and are not amidated at the C-terminus (Figure 1) .

To date, all hormones deemed to be CHHs fall into the Type I class, while hormones specified as MIH, VIH/GIH and MOIH form the known Type II hormones. MOIH stands for Mandibular Organ Inhibiting Hormone, referring to the observation that these hormones, which also originate from the sinus gland, inhibit methyl farnesoate (MF) release from the mandibular organ (Wainwright et al., 1996). MIH and MOIH hormones have also been found with a Type I structure e.g. the spider crab MOIH (Liu and Laufer, 1996). However, none of these hormones has been demonstrated to be the putative reproductive inhibiting hormone (RIH) . SUMMARY OF THE INVENTION

The present inventors have been able to identify which of the sinus gland peptides is the putative RIH in P. monodon and to characterise this neuropeptide, and so have been able to develop techniques to induce spawning more effectively than through eyestalk ablation and, moreover, to do so in a manner which is reversible. According to one aspect of the present invention there is provided an isolated peptide comprising the following amino acid sequence:

GLTDGTCRGR MGNREIYKKV DRVCEDCANI FRLPGLEGLC RDRCFYNEWF LLCLKAANRE DEIENFRVWI SILNA (SEQ ID NO: 1) an active fragment thereof, or a peptide with substantial sequence identity which serves to inhibit spawning in penaeid prawns.

The amino acid sequence recited above is the sequence of the putative reproductive inhibiting hormone (RIH) from P. monodon, hereinafter referred to as PmSGPVI or simply as SGPVI, after signal sequence cleavage. The complete peptide has the following sequence:

MHRLALRTWL AIMIVLFATS LSSIASAGLT DGTCRGRMGN REIYKKVDRV

CEDCANIFRL PGLEGLCRDR CFYNEWFLLC LKAANREDEI ENFRVWISILNA (SEQ ID NO: 2), and forms a further aspect of the present invention, along with active fragments thereof and peptides with substantial sequence identity which serve to inhibit spawning in penaeid prawns .

The present invention also envisages peptides containing either of the above sequences which are not active themselves but which may be processed in vivo or in vitro to an active peptide. In particular, to achieve good protein expression and a ready means for purifying the protein, it is advantageous to express the mature SGPVI as a fusion protein. Any suitable protein known for this purpose may be used, but advantageously the protein is maltose binding protein (MBP) , glutathione-S- transferase (GST) , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) . The MBP stabilised protein is particularly advantageous as it allows ready purification of the fusion protein on an amylose column, and endoproteinases are available to cleave the fusion protein from the MBP carrier, to enable release of the mature neuropeptide (Riggs, 1994) .

It will also be appreciated that peptides with a statistically significant alignment of similar regions to the peptide described above are envisaged. Typically, these have a Smallest Sum Probability P(N) of less than l.Oe^"14, and typically between 5.2e^"21 and 9.9e^~47. Such peptides may be expressed as fusion proteins, as described above . The peptides may be produced by direct peptide synthesis, for example, using solid phase techniques, or through the use of recombinant DNA technology.

According to a further aspect of the present invention there is provided an isolated nucleic acid molecule encoding a peptide as described above.

The isolated nucleic acid may be a cDNA having the following sequence, which includes both 5' and 3' untranslated regions:

1 GCCGTCCCAG GATACACTTC CATTTCAGAC ACCTTGATTT TACCTCGTGT

51 CTGCGTGTAT TAACCATGCA CCGTCTAGCA CTTAGGACAT GGTTGGCGAT 101 AATGATTGTA CTATTTGCGA CAAGCCTCTC CAGCATCGCT TCGGCCGGTC

151 TCACAGACGG CACCTGTAGA GGCAGAATGG GTAATCGTGA GATCTACAAG 201 AAAGTTGATC GTGTTTGTGA AGATTGCGCT AATATCTTCC GATTGCCAGG

251 ATTGGAAGGT CTGTGTAGAG ACCGGTGCTT CTACAATGAA TGGTTTCTGC

301 TTTGTCTGAA GGCTGCCAAC AGGGAGGACG AGATCGAAAA TTTCAGAGTG

351 TGGATAAGTA TTTTGAACGC CTAAAATTGA GGGTGAGCCC AGGACCCAAT

401 CCCTTCCTCT CACTTGGCTT CACTCACATG GACCAACATT CACAGCACTC 451 GGCGACCAAG GATAATTATC ATTACAATTC TTAACCGTGT TTATTTTGGT

501 TTTCTGTTAA GTTAATGGTA CACTCCAATG ACATTCCAGT CTGAGCCTAC

551 AAGGTCTGGT TGTCATGCAG AGTATGTATA GGAAAGATTA CATGTGAAAT

601 ATTGTGTGAT CTGCAGTCTA TCACGACCTA GATCCCTGTT TATTTTGCAG

651 TTCATATATT GCACTGATCT GAATTTGGAA AATAAACAAG CTATATCTCT 701 CATGTATTTT AATCTACGGA TATATAGTAC GAGCATTATT GTTCATGTGA

751 TTTGACTACT TCATGTTAGG ATGAACATAA ATAAACATAG CTGAAATACT

801 AAAAAAAAAA AAAAAAAA (SEQ ID NO: 3)

or fragments or homologues thereof, which encode a peptide active in inhibiting spawning in penaeid prawns.

In particular, the cDNA sequence that encodes the complete peptide is envisaged:

ATGCA CCGTCTAGCA CTTAGGACAT GGTTGGCGAT

AATGATTGTA CTATTTGCGA CAAGCCTCTC CAGCATCGCT TCGGCCGGTC TCACAGACGG CACCTGTAGA GGCAGAATGG GTAATCGTGA GATCTACAAG AAAGTTGATC GTGTTTGTGA AGATTGCGCT AATATCTTCC GATTGCCAGG ATTGGAAGGT CTGTGTAGAG ACCGGTGCTT CTACAATGAA TGGTTTCTGC TTTGTCTGAA GGCTGCCAAC AGGGAGGACG AGATCGAAAA TTTCAGAGTG TGGATAAGTA TTTTGAACGC C (SEQ ID NO: 4)

Also envisaged is the cDNA sequence encoding the mature peptide, which has the following sequence: GGTC TCACAGACGG CACCTGTAGA GGCAGAATGG GTAATCGTGA GATCTACAAG

AAAGTTGATC GTGTTTGTGA AGATTGCGCT AATATCTTCC GATTGCCAGG ATTGGAAGGT CTGTGTAGAG ACCGGTGCTT CTACAATGAA TGGTTTCTGC TTTGTCTGAA GGCTGCCAAC AGGGAGGACG AGATCGAAAA TTTCAGAGTG TGGATAAGTA TTTTGAACGC C (SEQ ID NO: 5)

Also envisaged are cDNAs with at least a 70- degree of homology, preferably 80% and more preferably 90% degree of homology with any one of the sequences described above as determined by the BLASTN algorithm using default parameters . The nucleotide sequence of the present invention can be engineered using methods accepted in the art. In particular, they may be cloned into expression vectors and cell systems that contain the necessary elements for transcriptional and translational control of the inserted coding sequence. These elements may include regulatory sequences, promoters, 5 and 3' untranslated regions and specific initiation signals such as an ATG initiation codon and a Shine-Dalgarno consensus sequence.

In order to stably express a peptide or protein as described above, host cells may be transfected with an expression vector comprising a DNA molecule according to the invention. A variety of expression vector/host systems may be utilised, as would be well understood by the person skilled in the art. The expression vectors may be directed by an appropriate signal sequence to direct secretion to the exterior of the cell, whereby a protein which is folded appropriately and in which disulfide bonds have formed is expressed into the culture medium. However, the signal sequence may be omitted in order to ensure sufficient yield. It is preferred in the present invention to use a thioredoxin reductase negative strain of E. coli as the expression host, which permits formation of disulfide bonds in the cytoplasm. In the present invention much higher yields have been achieved through using this technique, including yields of up to 27 mg of fusion protein per litre of culture, which is approaching the maximum possible yield.

The nucleic acid molecules of the present invention may be produced by direct chemical synthesis, using methods known per se to the person skilled in the art, or derived from a natural source using techniques known per se. According to a further aspect of the present invention there is provided an antibody to any one of the peptides or proteins described above, or to an antigenic fragment thereof. In particular, this aspect of the invention is directed to antibodies to a fusion protein described above, particularly a MBP-fusion protein, and to antibodies to fragments of the mature peptides, especially those having the amino acid sequence AANREDEIEN (SEQ ID NO: 6) and DRVCEDAANIFRLPGLEGLCRDR (SEQ ID NO: 7).

Although not wishing to be bound by theory, it is believed that the peptide referred to herein as SGPVI is the reproductive inhibiting hormone. However, all CHH family hormones are highly homologous to each other and increasing evidence suggests that these neuropeptides are multifunctional. For example, a single hormone in the spider crab Libinia emarginata can regulate both synthesis of methyl farnesoate (MF) , which is thought to be a positive effector of gonadal development, and glucose metabolism (Liu and Laufer, 1996) . Purified hormones with demonstrated CHH activity also inhibited protein synthesis in ovarian fragments in P. japonicus (Khayat et al., 1998) . Thus it is also possible that peptides that appear by sequence homology to be Type I crustacean hyperglycaemic hormones from P. monodon exhibit some RIH activity.

To this end, cDNAs encoding five such peptides, termed PmSGPI-V, were isolated (Davey et al. 2000 and Example 1) . Antibodies generated to the peptides SGPI-V may also serve to remove RIH activity and therefore antibodies to these peptides and fragments thereof, specifically an SGPIII-tail fragment having the amino acid sequence ADLHEEYQAH (SEQ ID NO: 8) and an SGPV loop peptide having the amino acid sequence SRLCDDAYNVFREPNVATECRSN (SEQ ID NO: 9) are envisaged.

Therefore, in a further aspect of the invention there is provided an antibody which binds a crustacean hyperglycaemic hormone and thereby serves to induce spawning in Crustacea, in particular, an antibody to an immunogenic region or fragment of one of SGPI-V.

These antibodies may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric and single chain antibodies. For the production of antibodies it is preferred to use the fusion protein described above. For polyclonal antibody production the fusion protein is injected into an appropriate host such as a sheep, rabbit, rat, goat or mouse, typically in admixture with an adjuvant such as Freund's adjuvant. Other suitable adjuvants include mineral gels such as aluminum hydroxide, muramyl dipeptide, trihalose dicormyn mycolate, surface active substances such as monophosphoryl lipid and plant extracts such as Con A.

Monoclonal antibodies may be prepared using techniques known per se in the art for the production of antibody molecules by continuous cell lines in culture, in particular by hybridoma cell lines. Preferably a fusion protein as described above, particularly a maltose-binding fusion protein, is administered to a suitable host such as a mouse and these cells used for hybridoma fusion.

Alternatively, synthetic peptides corresponding to immunogenic regions of the peptides may be synthesised by chemical methods and fused to carrier protein such as BSA or KLH for immunisation. Advantageously, these peptides are selected from the group consisting of:

Antibody fragments may also be generated by methods known per se in the art. For example, F(ab')₂ fragments may be produced by pepsin digestion of an antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')₂ fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

According to a further aspect of the present invention there is provided an agent for inducing spawning in Crustacea through reducing or eliminating activity of RIH in these animals. Typically, the agent reduces or eliminates the activity of RIH through binding the hormone and may, for example, be an antibody to the hormone. Alternatively, the agent may be a chemical compound which binds the hormone or which serves to downregulate the gene encoding RIH. The peptides of the invention are useful in the screening of candidate agents in a variety of techniques such as high throughput screening for compounds having binding affinity thereto.

Accordingly, in a further aspect of the invention there is provided a method of screening for a candidate agent for inducing spawning in Crustacea, comprising the steps of:

(1) providing a candidate agent and the reproductive inhibiting hormone (RIH) ;

(2) determining the nature and extent of interaction; and

(3) selecting agents which reduce or eliminate RIH activity.

According to a further aspect of the present invention there is provided a method for inducing spawning in Crustacea, comprising the step of reducing or eliminating the activity of RIH in these animals. Advantageously, the technique involves administration of an agent as described above, particularly an antibody, more particularly a monoclonal antibody, to the animal. The agent may be administered to the animal in any suitable way. In the case of penaeid prawns, the prawns may be immunised by injection or a slow release depot of mammalian IgG implanted in the prawn to ensure continual infusion of anti-RIH IgG into the haemolymph.

Alternatively, the agent may be interfering RNA, or RNAi. In this technique double-stranded RNA (dsRNA) with substantial identity to a portion of the sequence of the RIH gene is injected into the animal and effectively switches off production of the RIH protein. The double- stranded RNA employed in the present invention advantageously has a sequence corresponding to the cDNA sequence given above for SGPVI, or a portion thereof. It will be appreciated that RNAi is thought to have a catalytic mechanism and therefore very small quantities of RNA can inactivate expression. Moreover, it is speculated that dsRNA unwinds slightly, allowing the antisense strand to base pair with a short region of the target endogenous message and marking it for destruction. Accordingly, dsRNA molecules containing only shorter regions of homology with the cDNA described above may be useful in the method of the invention.

While not wishing to be bound by theory, it is believed that the peptide SGPVI described herein is the reproductive inhibiting hormone. Nevertheless, RNAi with substantial identity to a portion of the sequence of a cDNA encoding one of the peptides SGPI-V may be useful in the present invention either through containing a short region of homology with the cDNA of SGPVI or, although this is not thought to be the case, because the putative RIH is actually one of these peptides. Accordingly, in a further aspect of the invention there is provided an RNAi which is reactive with the mRNA encoding the putative reproductive inhibiting hormone in such a manner as to degrade said mRNA and thereby induce spawning in Crustacea.

Many hormones are peptides, and are generally produced by the glands of the endocrine system such as the pituitary, thyroid, adrenal glands and the gonads. However, not all hormones are produced by endocrine glands. For example, the mucus membranes of the small intestines secrete peptide hormones in order to stimulate secretion of digestive juices from the pancreas. Other hormones are produced in the placenta to regulate aspects of foetal development. Peptide hormones are typically expressed as ^λXprepro" hormones with subsequent cleavage into the biologically active mature form of the hormone. Hormones significantly affect the activity of many cells and tissues in the body through regulation of growth and metabolism, and the sex hormones regulate the development of sexual organs, sexual behaviour, reproduction and pregnancy. In invertebrates, in addition to regulation of general physiological processes, hormones also regulate metamorphosis and the moulting process. In those that reproduce through spawning, such as prawns and other Crustacea, hormonal activity has a controlling influence.

RNAi techniques have apparently not been used in therapy in animals. In particular, there has been no previous demonstration of the downregulation of a peptide hormone with RNAi . The present inventors have now demonstrated downregulation of the reproductive inhibiting hormone (RIH) in prawns, but it will be appreciated that, having shown this effect, the application of RNAi to the downregulation of other peptide hormones is demonstrated irrespective of the species of animal or the nature of the hormone system.

According to a further aspect of the present invention there is provided the use of RNAi in downregulating peptide hormones in an animal.

According to a still further aspect of the present invention there is provided a method of downregulating a peptide hormone in an animal, comprising introducing to the animal a double-stranded RNA substantially identical in sequence in one strand and complementary in the other to a region of the mRNA encoding the peptide hormone.

Throughout this specification and the claims, the words "comprise", "comprises" and "comprising" are used in a non-exclusive sense, except where the context requires otherwise. It is to be clearly understood that any reference herein to a prior art publication does not constitute an admission that the document forms part of the common general knowledge in the art in Australia or in any other country.

MODES FOR PERFORMING THE INVENTION

Preferred embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which: FIG. 1 shows a generic structure for the

CHH/MIH/RIH-like hormones;

FIG. 2 shows two-dimensional PAGE and Edman sequencing of P. monodon sinus gland peptides, a: A silver-stained 2D-PAGE gel of extract from 10 adult P. monodon sinus glands. Spots marked 1-6 indicate proteins that were subjected to Edman protein sequencing, b. Amino acid sequence obtained by Edman degradation for spots 1-6 compared with P. Japonicus CHH (Pej-sgp-III) ;

FIG. 3 shows preprohormone structure of the P. monodon Type I sinus gland peptides. A: Multiple sequence alignment of the deduced P. monodon precursors (Pm-sgp-I, -II, -III, -IV, and -V) with the following CHH family Type I precursors was performed using the PileUp program (Genetics Computer Group, Madison, Wis., 1994): Carcinus maenas (Cam-CHH) , ffomarus americaπus (Hoa-CHH-A and -B_, Libinia emarginata (Lie-MOIH) , Metapenaeus ensis (Mee- CHH), Macrobrachium lanchesteri (Mal-CHH) , Orconectes limosus (Orl-CHH-A and -A*), Penaeus japonicus (Pej-sgp-I, -III, -V, and -VII), and Schistocerca gregaria (Scg- TP) . Within the preprohormone the putative CHH-like hormone is separated from the CPRP by a dibasic cleavage site (outlined box) . The glycine residue for carboxy-terminal amidation is italicized. The translation termination codon is indicated by as asterisk (*) . The gray shaded boxes indicate conserved amino acids. The black shaded boxes indicate the amino acids conserved with Penaeid species;

FIG. 4 shows antisera used at different dilutions, as indicated above the lanes, to probe a western blot of MBP-PmSGPVI fusion protein digested with an endoproteinase to separate MBP from the PmSGPVI neuropeptide. Secondary detection was with an anti-sheep- IgG conjugated to horseradish peroxidase (HRP) using a chemiluminescent substrate for HRP. A strong positive reaction to both MBP and PmSGPVI is evident in both sheep, particularly sheep 2. In addition, a number of other proteins from the E. coli cultures are recognised by the polyclonal sera; and

Figure 5 shows a partial (+ 7 charge state) broadband Electrospray ionisation Fourier transform mass spectrometry (ESI-FTMS) spectrum of the crude extract of a single sinus gland showing the suite of proteins present including the five putative CHH-like hormones (Pm-SGPI, -II, -III, -IV, and V) in the range 8-9 kDa. Inset: Expansion of the Pm-sgp-IV cluster showing the most abundant peak (*), and the barely detectable monoisotopic peak (arrow) .

Example 1

Identification of SGPI-V Animals

Adult wild-caught P. monodon were obtained from North Queensland coastal waters and held in indoor tanks at the Australian Institute of Marine Science. Sea water was sterilised by ozone and UV treatment, aerated with compressed air and maintained as a flow-through sea water system with water exchange every 2 days. Water temperature and salinity were maintained between 28-31°C and 3.3-3.5 %, respectively. Prawns were fed at a rate of 4 % of average body mass per day on a diet of commercial pellets (Chareon Pokphand Industries, Bangkok) . Eyestalks were removed from live prawns by eyestalk ablation using a hot iron to seal the wound.

Two Dimensional Polyacrylamide Gel Electrophoresis (20- PAGE) and Edman Sequencing

Sinus glands were dissected from freshly ablated eyestalks of adult P. monodon under sterile salt water, snap frozen in liquid nitrogen and stored at -80°C until use. Glands were homogenised in 300 μl of 10 % acetic acid in a glass microhomogeniser, the extract was centrifuged at 15,000 g for 10 min at 4°C to remove cell debris and the supernatant collected. For 2D-PAGE, 10 sinus gland equivalents were dried down in a Savant DNA Speed Vac then resuspended in 100 μl of sample buffer (7.4 M urea, 29.2 mM Tris HC1, 8.35 mg/ml DTT and 2.5 % Triton X-100) . First dimension isoelectric focusing was performed with a Multiphor II (Pharmacia) electrophoresis unit on the anodic side of a re-hydrated 3-10 pH immoboline dry strip in accordance with manufacturer's instructions. The running conditions were 300 V for 3 h, 1700 V for 3 h and 3000 V for a further 48 h at 15°C. Second dimension electrophoresis was performed with a Protean II (Bio-Rad) gel rig using a 16 cm discontinuous Tricine-SDS-PAGE with 1.5 mm spacers (Schagger and Von Jagow, 1987) with a 4 % T, 3 % C stacking gel and a 16.5 % T, 3 % C separating gel. The running conditions were 25 mA/gel at 500 V limit for 1 h followed by 40 mA/gel at 1000 V limit for 4 h. After electrophoresis gels were either silver stained according to standard procedures (Heukeshoven and Dernick, 1986) or proteins were electroblotted to PVDF membrane in 10 mM CAPS, 10 % methanol, pH 11.0 at 400 mA for 90 min. The membrane was then stained with 1 % amido black in 10 % acetic acid (Sanchez et al., 1997) and the size of the eyestalk proteins was estimated by comparison of migration distance to molecular weight standards (Novex) . Proteins in the 6-10 kDa range were sequenced by Edman degradation as described (Cordwell et al., 1995; Grant et al., 1997).

Isolation of mRNA from eyestalk tissue and synthesis of cDNA

Total RNA was extracted from 4 eyestalks (1.5 g) , one each from four broodstock females, and from 10 pleopods (3.4 g) five each from two tank-reared males, using the "one-step" total RNA preparation method (Chomczynski and Sacchi, 1987) . Polyadenylated RNA was isolated from total RNA using oligo(dτ) -cellulose spin columns (Pharmacia mRNA purification kit) . Isolated mRNA was then used for double stranded cDNA synthesis using a ZAP Express™ cDNA synthesis kit (Stratagene) .

RT-PCR of CHH-like gene fragments

Two rounds of anchored polymerase chain reaction

(PCR) were carried out on first strand eyestalk and pleopod cDNA. The first round used an oligonucleotide dT primer and a degenerate primer, Pm-CHH3 (5'

ACCCYTCSTGCACSGGCGTCTTCGA 3- - SEQ ID NO: 10) with 100 ng of P. monodon eyestalk cDNA as template. An aliquot (1 μl) of the material amplified in this reaction was then subjected to further amplification using the same 3 ' oligonucleotide dT primer, and a second 5' degenerate primer, Pm-CHH2 (5' TGYTWCAACGTRTTYMGSGARCCCAA 3' - SEQ ID NO: 11). PCR reactions contained 20 mM Tris HC1, pH 8.4, 50 mM KC1, 1.5 mM MgCl₂, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 100 p ol of each primer, Taq DNA polymerase (5 U) (Promega) and deionised water in a final volume of 100 μl. PCR reactions were performed in a thermocycler (Corbett Research, PC-960G) under the following conditions; 94°C for 1 min, 42°C for 1 min, 72°C for 1 min 30 sees, for 5 cycles, then 94°C for 30 sec, 42°C for 30 sec, 72°C for 1 min 30 sees, for 30 cycles. Amplicons were subcloned into either pBSIISK÷ (Stratagene) or pGEM-T (Promega) for sequence analysis. Double stranded plasmid DNA was extracted using a QIAprep Spin Miniprep kit (Qiagen) and templates were sequenced using vector primers by di-deoxy chain termination cycle sequencing using an ABI Big Dye terminator sequencing kit (Applied Biosystems) and analysed on an Applied Biosystems model 377 DNA sequencer. Sequence analysis was performed using the GCG software package (Version 8.01) (Genetics Computer Group, 1994) . Sequencing of 36 independent clones identified three distinct CHH-like precursor gene fragments.

Construction and screening of an eyestalk cDNA library

P. monodon eyestalk cDNA was unidirectionally cloned into the Zap Express™ vector according to the instructions of the manufacturer (Stratagene). The resulting library contained 5 x 10⁵ independent phage and was amplified to 10⁸ p.f.u per ml. Hybridisation probes were generated from 25 ng of each of the three distinct CHH-like precursor gene fragments by random primed synthesis using a NEBlot labelling kit (New England Biolabs) and [ -P33]dCTP (NEN-DuPont) . Probes were heat denatured for 10 min at 95°C immediately prior to use. For isolation of the CHH-like precursor genes, approximately 100,000 clones of the library were screened with the labelled DNA fragments according to standard procedures (Sambrook et al., 1989). Filters were prehybridised for 2 hr at 60°C in hybridising solution (5 X Denhardts, 5 X SSC (1 x SSC: 150 mM NaCl, 15 mM trisodium citrate), 0.1 % w/v SDS, 1 mM EDTA, 200 μg/ml denatured salmon sperm DNA) then all three radiolabelled probes were added and hybridisation was performed for 16 hr at 60°C. Following hybridisation, the filters were washed once at room temperature and twice at 60°C, each for 30 min in 2 x SSC, 0.1 % w/v SDS. Hybridising clones were plaque purified and in vivo excision of the pBK-CMV phagemid vector from the Zap Express™ vector was performed in accordance with manufacturer's instructions (Stratagene). Single colonies were selected, re-screened and then used to inoculate 3 ml overnight liquid cultures for plasmid preparation and DNA sequencing as described in the previous section.

ESI-FTMS analysis of single sinus gland extracts.

Individual sinus glands were dissected from eyestalks in sterile sea water immediately after ablation and placed in a glass microhomogeniser on ice containing lOOμl of 1:1 (v/v) methanol:water plus 3 % acetic acid. The sinus glands were homogenised in this solution for 1-5 min, followed by sonication for 5 min. The homogenate was then centrifuged at 15,000 g for 10 min at 4²C to remove cell debris and the supernatant collected. The crude extract was dialysed against 1:1 (v/v) methanol:water plus 3% acetic acid to remove salts and other low molecular weight contaminants, and then examined without further purification. For ESI-FTMS analysis the dialysed extract was placed in a 250μl syringe and the contents were continuously infused into the external electrospray ionisation (ESI) source (Analytica of Bradford) of a Bruker BioApex 47e FTMS (Bruker Daltonics) via a syringe pump operating at a flow rate of 1 μl/min. Spectra were acquired over a mass to charge ratio (m/z) range of 600 to 4000, and to improve the signal to noise ratio 128 spectra were averaged prior to Fourier transformation. Resolution of 20 to 30 k FWHM (full width half maximum) at m/z 1000 was routinely achieved. All masses were measured with external calibration using 10^"5 M bovine ubiquitin (Mr = 8559.6163 Da) in the same solvent. For comparison between samples, data were collected with all instrument parameters held constant between samples.

Peptide isolation by 2D-PAGE and Edman protein sequencing

2D-PAGE analysis enabled the isolation of peptides for sequence analysis from a total of only 10 sinus glands. Six major proteins in the 6-10 kDa range were identified (Figure 2) which were likely members of the CHH family (Keller, 1992) . These were sequenced directly by Edman degradation and partial amino acid sequence was obtained for spots 1, 2 and 3. The three sequences obtained had high similarity to each other, differing only in the number of amino acids residues sequenced, and showed strong sequence homology to P. japonicus CHH peptides (Figure 2) . Sequence could not be obtained from spots 4, 5, and 6.

Isolation and characterisation of P. monodon CHH-like precursor genes

The amino acid sequence obtained for spot 3 from 2D-PAGE enabled the design of a degenerate oligonucleotide primer, Pm-CHHl, by reverse translation. A second downstream primer, Pm-CHH2, was designed by comparison of cDNA sequences of other CHH genes in GenBank. The two rounds of anchored PCR using oligonucleotide PCR primers, Pm-CHHl and Pm-CHH2 in combination with the oligonucleotide dT primer, generated a PCR amplicon of approximately 650 bp from the P. monodon eyestalk cDNA and no product from pleopod cDNA. Sequence analysis of 36 independent clones of this amplicon identified three distinct amplicons, which showed substantial sequence homology to a P. japonicus CHH (Pej-sgp-III) precursor gene (Ohira et al., 1997a). These fragments were radiolabelled and used for screening the P. monodon eyestalk cDNA library.

Approximately 100,000 reco binant bacteriophage clones were screened and 38 showed positive hybridisation of varying intensity. Thirty of these were successfully purified and characterised and sequence analysis showed that 20 of these clones encoded CHH-like precursor genes. From these, five distinct precursor genes were identified which showed strong sequence similarity with known CHH preprohormone genes. These were designated Pm-sgp I-V and the* GenBank accession numbers are AF104386-AF104390 (see Table 1) .

The length of the cDNA clones encoding Pm-sgp I, II, III, IV and V precursors is 813, 583, 566, 655 and 647 bp excluding the poly A tail with an open reading frame (ORF) in each of 360, 354, 306, 360 and 366 bp respectively. These cDNAs also include 5' and 3' flanking untranslated regions and the 3 ' region of each contains a polyadenylation signal which consists of a GT rich region followed by either AATAAA or AATGAA 12 to 14 nucleotides upstream from the poly(A) tail. As multiple clones with the same sequence were obtained from the library, we presume that these are all full-length cDNA's.

The putative P. monodon pre rohormones, have the distinct organization of Type I CHH precursors. They consist of a signal sequence, CHH precursor related peptide (CPRP) and mature hormone (see Figure 1) . The N- terminal region of each of the putative preprohormones is a hydrophobic domain that represents a probable signal peptide (Von Heijne, 1986) . The most likely site of cleavage of the signal peptide is shown in Figure 3. This is followed by a putative CHH precursor-related peptide (CPRP) which is separated from the mature CHH-like peptide by a dibasic cleavage site, KR (grey shaded box) . The three C-terminal amino acids encoded by the cDNA are VGK. This triplet occurs in almost all other Type I peptides for which the cDNA sequence is known (Figure 3), but the mature peptides terminate in an amidated valine. Thus it is likely that the lysine, which is a basic amino acid, provides an endoproteolytic cleavage site which would expose the glycine residue (italicised in Figure 3) which is required for carboxyamidation of the preceding valine. If these predictions regarding post-translational processing are correct the resultant mature P. monodon sinus gland peptides would be 72 amino acids in length, have six conserved cysteine residues at positions 7, 23, 26, 39, 43 and 52 and would be C-terminally amidated.

The high resolution capability of the ESI-FTMS enabled the neuropeptide complement of individual sinus glands to be resolved in crude extracts. The observed monoisotopic Mr's were compared with those predicted form Pm-SGP I-V from the cDNA sequences. The different masses that could be predicted from the cDNA sequence, depending on the extent of post-translational processing, are given in Table 2. Assuming that each of the mature peptides was subject to cleavage of the signal peptide and CPRP, C- terminal amidation, and contained three disulfide bonds, each of these predicated Mr's were found to coincide with one of the observed monoisotopic Mr's obtained from the ESI-FTMS spectrum (see Table 2 and Figure 5) . Thus, the results confirmed the gene sequence and post-translational modifications. In addition they demonstrated the presence of all five peptides within a single sinus gland, indicating that the different cDNA clones do not simply represent polymorphisms between individuals.

Example 2

Identification of SGPVI The work described in Example 1 did not yield any

Type II hormone (s). We therefore adopted a different approach, namely to design primers against conserved regions on known Type II hormones. The primer sequences used were as follows:

GIH-1F: 5' GGYGTNATGK NYRAYCGKKA C (SEQ ID NO: 12) dTXhol: 5' GAGAGAGAGAGAGAGAGAGAACTAGT CTCGAG(T)ι₈3' (SEQ ID NO: 13)

GIH-2F: 5' CGNGTGTGYR ANGAYTGYNH YAAC 3' (SEQ ID

NO: 14)

GIH-3R: 5'TGTNRWARCA NYNNYTYYTG CA 3' (SEQ ID NO: 15)

The primer designated GIH-1F spans a region where there is an extra amino acid in the Type II hormones compared to the Type I hormones, and hence an extra three bases. Thus this primer was able to distinguish Type II hormone genes from Type I.

The initial clones isolated were termed AIMS- P.jnoz.80-83. They were isolated as PCR fragments using two rounds of PCR on the eyestalk first strand cDNA described in Example 1. The first round used GIH-lF plus dTXhol and the second round GIH-2F and GIH-3R. The reaction product was then cloned directly into PGEM-T Easy using the manufacturer's instructions (Promega).

The inserts of the four clones were as follows (mpl26, 127,128,131 = AIMS-P.mon80, 81, 82 and 83 respectively) . mpl27 CCGGGTGTGT AAGGATTGCG NCAACATCTT CCGACTTCCA GGCTTGGACG (SEQ ID NO: 16) mpl28 TCGGGTGTGT GAGGATCGCG CCAACATCTN CCGACTTCCA GGCTTGGACG

(SEQ ID NO: 17) mpl26 .CGGGNGTGT GAGGATTGCG CTNACATCTT CCGACTTCCA GGCTTGGACG

(SEQ ID NO: 18) mpl31 TCGGGTGTGT GAGGACTGGG .CAACATCTT CCGACTTCCA GGCTTGGGCG

(SEQ ID NO: 19)

mpl27 GCATGTGCAG GAGCCGCTGC TACCACA (SEQ ID NO: 20) mpl28 GCNTGTGCAN GAACCGCTGC TACCACT (SEQ ID NO: 21) mpl26 GCATGTGCAG GAACCGCTGT TTTCACAA (SEQ ID NO: 22) mpl31 GCATGTGCAG GAGCCGCTGC TTCCACAA (SEQ ID NO: 23)

These aligned P. monodon sequences were then used to design another 60 bp oligonucleotide:

PCRPMVIHl: GGGTGTGTGA GGATTGCGSC AACATCTTCC GACTTCCAGG CTTGGACGGC ATGTGCAGGA (SEQ ID NO: 24)

This was used as a probe against the eyestalk cDNA library described in Example 1. Three full-length cDNA clones were identified, AIMS-P. mon 86,87,88. These all had similar insert sizes, of about 820 bp. The vector is pBK-CMV which is created by phage excision from the lambda vector λ-ZAPII. The full insert of AIMS-P. mon86 was determined.

Examination of FTMS profiles of sinus glands revealed that the protein, SGPVI, could be identified. However, the concentrations of this peptide appeared consistently to be one to two orders of magnitude lower than those of the CHH-like molecules identified in Example 1. SGPVI was found to have the sequence laid out above. AIMS-P. on 86 was found to contain a total insert of 800 bp excluding the poly A Tail. This contains a coding region of 306 bp consisting of 81 bp encoding a putative signal sequence and 225 bp encoding the mature peptide. The coding sequence for the mature peptide (ie with signal sequence cleaved off) was then subcloned into pGEM-T Easy to create AIMS-P.mon95. The PCR primers used were: pmVIHfl: TTATCCCCCG GGCTCACAGA CGGCACCTGT (SEQ ID NO: 25) pmVIHrl: TTGCACCCAA GCTTTTAGGC GTTCAAAATA CTTATCC (SEQ ID

NO: 26)

This placed SmaX and Jfindlll cloning sites immediately at either end of the sequence plus additional restriction sites present in the vector polylinker. The sequence was verified to ensure there were no PCR-induced artifacts. The first codon of the mature peptide has been altered from GGT to GGG to enable the Smal site to be placed appropriately. Both codons encode Glycine so the amino acid sequence is unaltered.

This plasmid was then used to provide material for subcloning into protein expression vectors. It was digested with Smal and EcόRX (this site is derived from the adjoining polylinker sequences) , the approximately 225 bp fragment isolated and subcloned into SnaBl, jEcoRI digested expression vector pMALc2g (New England Biolabs) to create plasmid AIMS-P.monl04, a maltose binding protein (MBP) -SGPVI fusion in which the mature SGPVI peptide can be released from the MBP protein by cleavage with Genenase I (see NEB catalog or web site for details) . This was transformed into E. coli strain AD494 (DE3) for protein expression in a manner known per se to the person skilled in the art. This E. coli host is thioredoxin negative, allowing synthesis of proteins with disulfide bond formation in the cytoplasm.

In order to express the SGPVI the following steps were taken: (1) Inoculated 13L culture with ~14ml o/n culture, grew until OD₅₉₅ ~0.5

(2) Induced expression with 0.3mM IPTG for 3hrs

(3) Harvested cells by pelleting at ~4000xg 20min 4°C

(4) Cell pellets were frozen overnight, then thawed on ice and lysed by resuspending in amylose column buffer

[20mM Tris pH7.4, 200mM NaCl, ImM EDTA, ImM Na-azide] + 2mg/ml lysozyme + O.lmg/ml PMSF, incubated on ice 30- 60min, then snap frozen in liquid-nitrogen and thawed to lyse cells (5) Cell debris and insoluble material were then pelleted out by centrifugation at 9000xg 30min at 4°C; and (6) Supernatant was decanted and used for subsequent purification over amylose affinity resin (NEB) .

Amylose Chromatography was then performed essentially as follows as described in NEB pMA, Manual Cat #800, Version 4.0: (1) Crude extract was diluted 1 in 5 with amylose column buffer to ~ 2.5mg/ml total protein;

(2) Resin was washed three times prior to use with amylose column buffer; (3) The washed resin was added to the diluted crude extract and incubated overnight at 4°C with gentle stirring;

(4) The resin was pelleted by gentle centrifugation (~2500xg, 5min, 4°C) and resuspended, then poured into a glass column 2.5cm x 10cm (Pharmacia);

(5) The column was washed with at least 5 volumes of column buffer using a peristaltic pump set to ~ lml/min. (run overnight); and

(6) The MBP-fusion protein was eluted using amylose elution buffer [amylose column buffer + lOmM maltose], also at ~lml/min.

Eluted fractions were analysed by SDS-PAGE analysis.

Example 3

Production of Polyclonal Antisera

For initial passive immunisation trials the MBP- SGPVI fusion protein produced in Example 2 was used to produce polyclonal antisera. Conjugation of small peptides (<15 kDa, such as SGPVI (8.8 kDa) ) to larger carrier molecules often facilitates development of an appropriate immune response in the host organism and so, in this case, MBP is acting as a carrier protein. The polyclonal antisera were produced in sheep, resulting in very large volumes of antisera being available. The sheep serum was found to contain antibodies that specifically recognized SGPVI after the immunization schedule was complete, indicating that the antibody production was successful (Figure 4) . In order to produce the antisera two sheep were injected once with 100μg fusion protein MBP-PmSGPVI in Complete Freund's Adjuvant followed by two boosts of lOOμg each in Incomplete Freund's adjuvant. Sheep serum was screened for production of polyclonal antibodies (pAB) to SGPVI by Western Blot against digested MBP-PmSGPVI (Genenase-I digested Fraction-4) after being concentrated in a speedvac (1ml - ~0.2ml). Results indicated antibodies were a present that recognised SGPVI, as seen in Fig. 4.

The pAB serum reacted with many of the E. coli proteins from a crude cell lysate used to screen for expressed fusion protein. In order to try and remove many of the anti-JS. coli antibodies from the sheep serum a cyanogen bromide activated sepharose column was prepared to which E. coli & MBP proteins were bound. Prior to removal of α-JE. coli+MBP antibodies, Sheep IgG was isolated from the serum using Pierce "ImmunoPure (G) IgG Purification Kit" and desalted. The CnBr- E. coli-MBP resin was incubated with the purified IgG samples overnight before being poured into a BioRad Econo-Column. The flow- through from the column was collected in ~lml fractions and assayed for protein by OD₂6o_/28o» Fractions containing

>0.3mg/ml IgG were pooled and aliquotted as "Pre-adsorbed" IgG stocks and stored at -20°C.

Example 4 Production of Monoclonal Antibodies to SGPVI

Four mice were immunised with intact fusion protein produced in Example 2 (50μg Fusion protein in monophosphorphoryl lipid A/ trehalose dicornynomycolate followed by three further boosts with lOOμg Fusion protein in Incomplete Freund's Adjuvant) . These mice were bled after their third boost, and the serum screened by Western Blot analysis against Genenase-I digested fusion protein. This revealed a positive antibody response indicating that the mice would be suitable to use for hybridoma production.

A screening antigen was prepared using Glutathione-S-Transferase (GST) as the fusion partner. The mouse serum was screened against the new antigen (by Western Blot) and found to recognise the GST-SGPVI protein while not recognising the GST alone. As such the mice were re-boosted and used for fusions. The hybridomas prepared from the MBP-SGPVI immunised mice were screened against both MBP-SGPVI, GST- SGPVI and MBP alone to identify clones that recognised the SGPVI but not the MBP protein. Four clones, 1E9, 13A1, 10F6 and 1D1, were found to specifically recognise the SGPVI and were subsequently cloned by serial dilution to produce single-cell clones. Each of the single-cell clones from the four parent lines were then re-screened against a variety of antigens (MBP-SGPVI, MBP, KLH- SGPVITpeptide, KLH) . Only one of the four parent lines, 13A1, resulted in single cell clones that recognised MBP- SGPVI but not MBP.

The 13A1 single cell clones strongly recognised the MBP-SGPVI fusion protein while not recognising the MBP alone. These hybridomas were also screened against the conjugated synthetic peptide KLH-SGPVI-tail and KLH but failed to recognise these antigens. Since this conjugated peptide only represents a small portion of the SGPVI hormone, these results suggest that the 13A1 monoclonal antibodies do not recognise the tail section of the SGPVI hormone, but some other region of the protein.

Of the other four lines, the 1E9 clones also recognise MBP-SGPVI but not MBP, although these results were not reproducible, with all single cell clones appearing negative to both MBP-SGPVI and MBP when re- tested. They were however also screened against the KLH-

SGPVI-tail peptide and KLH. All three 1E9 single cell clones screened recognised the KLH-SGPVI-tail peptide, however, they each also tested positive to the KLH conjugating protein (although at a slightly weaker level than to the KLH-SGPVI-tail antigen) suggesting that they may possibly be recognising some epitope on the KLH rather than the SGPVI-tail peptide. The 10F6 single cell clones gave variable weak responses when screened against the MBP-SGPVI and MBP antigens - appearing weakly positive to both antigens on initial screening but negative to both on re-screening. When screened against the KLH-SGPVI-tail peptide and the KLH alone, they appeared positive and negative respectively, indicating that they may recognise an epitope of the SGPVI that is not easily accessible on the MBP-SGPVI fusion protein, although this seems unusual as it was the MBP-SGPVI antigen that the monoclonal was raised against. Despite this unusual results, it may prove to be an interesting antibody if it recognises a particular epitope on the synthetic peptide that is not recognised on the native protein. The last of the four parent lines that were single cell cloned is the 1D1 line. Each of the single cell clones from this line resulted in hybridomas that recognised both the MBP-SGPVI and MBP, indicating that these hybridomas actually recognise the MBP rather than the SGPVI. However, these clones also weakly recognised the KLH-SGPVI-tail peptide but not the KLH alone, suggesting that there is some specificity for the SGPVI peptide. These are unusual results, it may be possible that there is an epitope on MBP that is slightly similar to the SGPVI-tail peptide that enables a weak-binding of the MBP-monocloanl to the KLH-SGPVI-tail peptide.

Table-1: Results of Hybridoma Screening by ELISA

Single cell clones from 13A1 that recognised MBP- SGPVI but not MBP are the optimal choice for use in ascites production. These clones are ready to be cultured and used for monoclonal production from both ascites and culture supernatant .

Example 5

Monoclonal Antibody Production Against Synthetic Peptides

As an additional strategy synthetic peptides to immunogenic regions of the sinus gland neuropeptides were produced for immunisation of mice for mAB production.

Peptide sequences were selected based on two main factors:

Hydrophilic Nature: The hydrophilic regions of proteins are usually located on the external face of the protein while the hydrophobic moieties are usually folded to the inside. Therefore, the antigenic regions of the native protein are most likely to be hydrophilic regions of the neuropeptides.

2. Antigenicity: Some regions of proteins are more likely to elicit an antibody response than others, these are referred to as antigenic regions. Programs are available to analyse particular amino acid sequences to predict those areas that are likely to be the most antigenic. These programs were used to analyse each of the CHH- family of neuropeptides.

Ideally the synthesised peptides would be both hydrophilic and antigenic. T.Shih et al 1998 Zoological Science 15:389-397 tried to raise polyclonal antibodies to the tail section of two CHH-like peptides (Pej-SGP-III & IV) . They were able to raise antibodies to the carrier protein (BSA) after three injections (6 wks) , however, it took six months of fortnightly injections (of 200μg) to get a suitable antibody response to the peptides (the titre increased very slowly, hence the long period of boosting) .

When the tail section of these peptides was analysed, it was found that they are actually hydrophilic and not very antigenic. Therefore, to try and avoid this problem of poor antigenic response from the immunised animals, it was decided to select an area other than the tail end of the peptides against which to raise our monoclonals .

Four different peptides were synthesised each conjugated to both BSA and KLH, for immunisations (BSA- peptide) and screening by ELISA (KLH-peptide) .

Twenty to thirty milligrams of each peptide was synthesised to 95% purity. Peptides 3&4 both contain two cysteine residues

(C), which are known to form a disulphide linkage in the native protein (Cys residues 2 and 4) : these residues were therefore joined to form a disulfide bond prior to conjugation to BSA and KLH. The third internal cysteine residue was changed to an alanine (highlighted A) to prevent undesired disulfide bond formation.

Conjugation to BSA and KLH for Peptides 1 & 2 was performed through the addition a carboxy-terminal cysteine residue. This was a straightforward procedure as there were no internal cysteine residues in these peptides. Peptides 3 & 4 however had to be conjugated through the addition of a carboxy-terminal lysine residue due to the internal cysteine residues used to form di- sulfidebonds . Peptide 4 proved to be a problem using this conjugation method due to the internal lysine residue. To overcome this, the internal lysine residue was converted to asparagine (highlighted residue N) which is the residue occurring at this position in SGPI. Production of Monoclonal Antibodies to Peptide-1 (SGPVI- tail)

Three mice were immunised with Peptide conjugated to BSA (300μg Peptide-BSA in muramyl dipeptide followed by two further boosts with lOOμg and then 50μg in lμg Gerbu MM adjuvant . Two of the four mice were then given two further boost using 50μg, then 25μg of the conjugated peptide without any adjuvant) . These mice were bled after their third boost by ELISA against their respective Peptides conjugated to KLH. This revealed a positive but weak antibody response in all mice; therefore the mice received a fourth boost . These mice were once again screened by ELISA using KLH-conjugated Peptide.

None of the mice immunised with Peptide 1 produced a sufficiently high titre for hybridoma production. These mice were discarded and monoclonal production from this peptide was no longer pursued.

Production of Monoclonal Antibodies to Peptide 2 (SGPIII- Tail)

The immunisation regime for Peptide 2 was identical to that performed for Peptide 1. After the fourth boost, these mice were once again screened by ELISA using KLH-conjugated Peptide. Two of the mice screened had suitable antibody titres; the one with the highest titre was subsequently used for hybridoma production monoclonal.

The hybridoma clones from this peptide were screened by ELISA and five positive hybridomas were selected for single cell cloning, resulting in five single cell clones specific for Peptide 2: 1B4 1D9, 10C3 1G7, 13B9 1H8, 1G10 1D9, 8F9 1G2. These hybridomas are now ready for monoclonal production from ascites and cell culture . Production of Monoclonal Antibodies to Peptide-3 (SGPVI- Loop)

Three mice were immunised with Peptide 3 conjugated to BSA (175μg Peptide-BSA in lμg Gerbu MM adjuvant followed a subsequent boost with conjugated peptide alone ie no adjuvant. Further boosts were performed until test bleeds showed suitable antibody titres. The initial hybridoma production from the first mouse was unsuccessful. The final mouse is currently being used for hybridoma production.

Example 6

Induction of Spawning: Trials of passive immunisation and interference RNA

Materials and Methods

Animals

The animals used for this trial were caught from the wild off the Queensland coast and purchased from a commercial fisherman between October 11-30 2000. Males and females were mixed in tanks and mating was allowed to occur naturally. Animals were held through one moult cycle and treatments were generally administered within 1-

3 days after the second moult.

Treatments

The treatments administered are summarised in

Table 3. The antibody treatments were administered by tail muscle injection. The dsRNA treatments were administered by injection into the base of both eyestalks.

Passive immunization

The antibodies used were polyclonal anti-SGPVI antibodies that had been purified initially for IgG molecules and had subsequently been immunoabsorbed against

E. coli proteins as described in example 3. As a control, purified IgG from pre-immune sheep which had also been immunoabsorbed against E. coli proteins was used. The test and control antibodies were stored in 500 μg aliquots in PBS at -20°C.

dsRNA

Plasmids AIMS-P.mon90 and AIMS-P.mon86, the full length cDNA clones respectively of PmSGPII and PmSGPVI cloned in pBK-CMV, were used as templates for RNA synthesis. 50μg of plasmid was linearised either with XbaX or with BamHI to allow transcription of the sense strand or the antisense strand from the T7 and T3 promoters respectively. RNA transcripts were synthesised using the Promega RiboMAX Large Scale RNA Production kit (#P1290, P1300) according to manufacturer's instructions. To prepare dsRNA, the complementary single stranded RNA molecules for each SGP were annealed based on the annealing protocol outlined by Misquitta & Patterson, 1999. Since concentrations of ssRNA from T7 and T3 reactions were similar for each peptide, equal volumes of ssRNA were mixed together in Orosoph.Ha Injection Buffer

{Misquitta & Paterson 1999 5 /id}, heated to 85°C and cooled slowly to 25°C over lhr in a PCR thermocycler. Samples were checked for successful annealing by agarose gel analysis of T7-ssRNA, T3-ssRNA and dsRNA and concentrations of dsRNA were estimated based on quantities of each ssRNA product used for the annealing reaction.

Results:

Application of treatments

An initial difficulty was estimating the dosage of the various treatments. This was constrained in part by the total availability of the biochemicals being used, and also by the possible modes of delivery.

Antibody treatment : There are not many measurements available of the concentrations of CHH family neuropeptides in haemolymph to provide a basis for estimating the quantity to target. However, CHH levels measured in the crab Carcinus maenas varied from 10-250 fmol / ml, with a transient peak at ecdysis of 1500-2000 fmol / ml (Chung, Dircksen, et al. 1999 338 /id) . Assuming a Mr of approximately 8000 Da, this translates to a concentration range of 80 pg / ml to 2 ng per ml, with occasional peaks to 16 ng / ml haemolymph.

Based on these data, we assumed an expected average concentration of SGPVI in haemolymph of the order of 1 ng / ml. The Mr of an IgG molecule (~150,000) is about 20 x that of SGPVI and so an equivalent molar concentration of IgG would be 20 ng / ml. Assuming a prawn is about 100 g, this corresponds to up to 100 ng of RIH circulating in the prawn, which therefore requires 2000 ng or 2 μg IgG to neutralise. The polyclonal antibody contains a mixture of antibodies and hence an estimate had to be made of the proportion of the polyclonal IgG which is actually targeted against SGPVI. Data from Western blot analysis suggest that the enrichment factor for the IgG preabsorbed against E. coli antigens is about 10. A reasonably conservative estimate of the proportion of anti-SGPVI in the preabsorbed antiserum is between 1-10%, requiring doses of 20-200 μg preabsorbed IgG per prawn. To err on the conservative side, a dose rate of 500 μg was selected for injection into the prawns.

dsRNA

Data provided by Tuschl et al. (Tuschl, Zamore, et al. 1999 15 /id) indicate that amounts of approximately 0.2 fmole of dsRNA have been required to cause RNAi in

Drosophila embryos. As the volume of a Drosophila embryo is estimated to be 7.3 nl, this translates to a concentration of approximately 25 nM dsRNA. Assuming that a prawn is lOOg ie approximately 100 ml, this corresponds to 2.5 nmoles of dsRNA being required for the entire prawn or approximately 1 mg of dsRNA of an 850 bp RNA molecule. This amount was somewhat impractical due to the expense of synthesising such large amounts of RNA, and hence the approach of injecting directly into the eyestalk, and hence aiming for a high local concentration of dsRNA was adopted. The treatment applied was injection of 40 μg dsRNA per eyestalk.

The dsRNA treatment was split into two different treatments. In the first, dsRNA specifically against SGPVI was used. In the second, a mixture of dsRNA directed against SGPII and SGPVI was used. The rationale for the latter was that the identity of the RIH is uncertain. There are data from other systems indicating that CHH-like molecules can also have effects on ovarian protein synthesis (Khayat, Yang, et al. 1998 346 /id) . Hence, as it was relatively straightforward to synthesis dsRNA corresponding to one of the five CHH-like sinus gland peptides from P. monodon, it was decided to include a treatment that comprised a mixture of SGPVI and CHH dsRNAs .

Trial results

The results are summarised in Table 4. For the polyclonal antibody treatment, two out of six treated prawns spawned, but one out of the five animals treated with the pre-immune serum also spawned, and two developed to a gonadal index greater than 2, the same number as among the antibody treated animals.

For the RNAi treatment more animals spawned from the two treatments (two out of eight of each) than from the buffer control (one out of eight) . Of greater significance is the number of animals that reached a GI>3, six and five for the SGPVI and SGPVI + SGPII dsRNA treatments respectively, compared to only two for the buffer control.

Discussion: A factor which may have affected the efficiency of the dsRNA treatments is the mode of delivery, namely injection into the eyestalk. It proved very difficult to achieve precise injection, due to the small size of the target tissue, and also the fluid pressure within the eyestalk which caused back-flushing in some cases. In addition, there is the danger of inadvertently effectively causing eyestalk ablation by placing the needle into the X-organ or sinus gland, and hence destroying it. Future work with dsRNA will have to also focus on the delivery mechanism, and it may be necessary to examine injection into the heart as well. Other delivery mechanisms that might be tested include oral ingestion or soaking the animals in a solution containing dsRNA, although the large amounts of dsRNA potentially required for RNAi in a large adult animal such as a prawn may prohibit these approaches. If dsRNA does prove effective in prawns, the required dosage will also have to be carefully evaluated.

The fact that both dsRNA treatments showed an effect indicates that silencing of the SGPVI gene is sufficient to induce ovarian development. Although the full cDNA sequence was used as a template for dsRNA synthesis, the sequence shows no homology at the nucleotide level to that of the other five sinus gland peptide cDNAs that have been characterised, and hence its effect is likely to be highly specific (although this remains to be tested by directly examining effects on gene expression) . The second treatment, with a mixture of the two dsRNAs, may have been effective either due to the sole effect of SGPVI gene silencing, or due to a combined effect of silencing both genes.

The initial results with the polyclonal antiseru are not as promising. However, there are a number of possible reasons for this. One reason is simply that the specific anti-SGPVl antibodies may be too dilute in the polyclonal antiserum, even following immunoabsorption to remove a proportion of non-specific antibodies that recognize E. coli antigens. This factor may be overcome by the use of monoclonal antibodies which are currently under development and should show a high specificity.

Secondly, the persistence of antibodies in prawn haemolymph is known to be a problem due to the fact that prawns have an open circulatory system. In this case the polyclonal antiserum was delivered by injection into tail muscle, and hence the proportion that reached the eyestalk in the haemolymph may have been very small. This may be overcome by the use of slow release mechanisms for antibody delivery.

Table 1

Structure of cDNAs encoding Type I CHH-like hormones from P. monodon

All measurements are in nucleotide lengths . UTR = untranslated region. CPRP = CHH Precursor Related Peptide. The total length of the cDNA is from the presumed transcription initiation to the final residue prior to the poly-A tail. The length given for the coding region for the mature peptide includes the C-terminal two amino acids (GK in all cases) which are cleaved off during the amidation rection and the termination codon. The 3' UTRs of PmSGPII and PmSGPIII show substantial homology, but there is otherwise little obvious homology between the 5' and 3' UTR's of the respective cDNAs.

Table 2

Table 3

No Treatment Type Material Volume Quantity

1 Ablated Control

2 Non-ablated Control

3 SGPVI RNAi TreatdsRNA 20 ul 40 ug per ment per eyestalk eyestalk

4 SGPII and SGPVI TreatdsRNA 20 ul 40 ug per

RNAi ment per eyestalk eyestalk

5 RNAi annealing Control Buffer 20 ul buffer per eyestalk

6 Antibody TreatAnti- 555 ul 500 ug injected ment SGPVI IgG, preabsor bed

7 Pre-immune Control Preimmun 625 ul 500 ug antibody e IgG, injected preabsor bed

NO ANIMALS ARE ABLATED EXCEPT TREATMENT 1

Table 4

Treatment No No spawned %spawned No GI > % GI >

3 3

None 5 0 0 2 40

Ablated 13 11 85 12 92

RNAi SGP VI 8 2 25 6 75

RNAi SGPII + VI 8 2 25 5 62

RNA buffer 8 1 12. 5 2 25

Antibody against 6 2 33 2 33

SGPVI120

Sheep serum 5 1 20 2 40

GI = gonadal index on a scale of 1-4. > 3 means on the verge of spawning .

REFERENCES

The contents of the following documents are incorporated herein by reference:

CENIACUA (1999). Colombia's closed life cycle program for penaeid shrimp: genetic selection and improvement. Global Aquaculture Advocate 2, 71.

Chomczynski, P., and Sacchi, N. (1987). Single step method of RNA isolation by guanidinium thiocynate-phenol- chloroform extraction. Anal Biochem 162:156-159.

Chung, J.S., Dircksen,H., and Webster, S.G. (1999) A remarkable, precisely timed release of hyperglycaemic hormone from endocrine cells in the gut is associated with ecdysis in the crab Carcinus maenas. Proceedings of the National Academy of Sciences USA 96, 13103-13107.

Cordwell, S.J., Wilkins, M.R., Cerpa-Poljak, A., Duncan, M., Williams, K.L., and Humphrey-Smith, I. (1995). Cross-species identification of proteins separated by two- dimensional gel electrophoresis using matrix-assisted laser desorption time of flight mass spectrometry and amino acid composition. Electrophoresis 16:438-443.

Davey,M.L., Hall,M.H., Willis, R.H., Oliver,R.W.A. , Thurn,M.J., and Wilson, K.J. (2000). Five crustacean hyperglycemic family hormones of Penaeus monodom complementary DNA sequence and identification in single sinus glands by electrospray ionization-Fourier transform mass spectrometry. Marine Biotechnology 2, 80-91.

Fingerman,M. (1992) . Glands and secretion. In: Microscopic Anatomy of Invertebrates, F.W.Harrison and A.G.Humes, Eds. New York: John Wiley and Sons In, pp. 345- 394. Genetics Computer Group. (1994) . Program manual for the GCG package, Version 8.01, September 1994, Madison, WI.

Grant, G.A., Crankshaw, M.W. and Gorka, J. (1997) Edman sequencing as a tool for characterization of synthetic peptides. .Meth Enzymol 289:395-419.

Goyard,E., Patrois,J., Peignon, J. -M. , Vanaa,V., Dufour,R., and Bedier,E. (1999). IFREMER's Shrimp Genetics Program. Global Aquaculture Advocate 2, 26-28.

Heukeshoven, J., and Dernick, R. (1986). In:

Elektrophorese Forum 86. (Radla B, J., Ed.) pp. 22-27, Technische Universitat, Munchen.

Howell,C. (1999). Ten years of breeding at Aquamarina de la Costa, Venezuela. Global Aquaculture Advocate 2, 57.

Keller, R. (1992) . Crustacean neuropeptides: structures, functions and comparative aspects. Experientia 48:439-449.

Khayat,M., Yang,W., Aida,K., Nagasawa,H., Tietz,A., Funkenstein, B . , and Lubzens,Ξ. (1998). Hyperglycaemic hormones inhibit protein and mRNA synthesis in in vitro- incubated ovarian fragments of the marine shrimp Penaeus semisulcatus. General and Comparative Endocrinology 110, 307-318.

Lacombe, C. , Greve,P., and Martin, G. (1999). Overview on the sub-grouping of the crustacean hyperglycemic hormone family. Neuropeptides 33, 71-80.

Liu,L. and Laufer,H. (1996) . Isolation and characterization of sinus gland neuropeptides with both mandibular organ inhibiting and hyperglycemic effects from the spider crab Libinia emarginata. Archives of Insect

Biochemistry and Physiology 32, 375-385. McIntosh,R.P. (2000). Changing paradigms in shrimp farming: III. Pond design and operation considerations. Global Aquaculture Advocate 3, 42-45.

Menasveta, P . , Sangpradub, S . , and Piyatirativorakul, S . (1994) . Effects of broodstock size and source on ovarian maturation and spawning of Peπaeus monodon Fabricius from the Gulf of Thailand. Journal of the World Aquaculture Society 25, 141-149.

Misquitta, L. and Paterson,B.M. (1999) . Targeted disruption of gene function in Drosophila by RNA interference (RNA-i) : A role for nautilus in embryonic somatic muscle formation. Proceedings of the National Academy of Sciences USA 96, 1451-1456.

Ohira,T., Watanabe,T., and Nagasaw,H. (1997). Cloning and sequence analysis of a cDNA encoding a crustacean hyperglycemic hormone from the Kuruma prawn Penaeus japonicus. Molecular Marine Biology and Biotechnology 6, 59-63.

Preston,N.P. , Brennan,D.C. , and Crocos,P.J. (1999). Comparative costs of postlarval production from wild or domesticated Penaeus japonicus broodstock. Aquaculture Research 30, 191-197.

Riggs,P. (1994). Expression and purification of maltose-binding protein fusions. In: Current protocols in molecular biology, F.M.Ausubel, R.Brent, R.E.Kingston,

D.D.Moore, J.G.Seidman, J.A. Smith, and K.Struhl, Eds. New York: John Wiley & Sons, pp. 16.6.1-16.6.14.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Sanchez, J-C, Wirth, P., Jaccoud, S., Appel, R.D., Sarto, C, Wilkins, M.R., and Hochstrasser, D.F. (1997). Simultaneous analysis of cyclin and oncogene expression using multiple monoclonal antibody immunoblots. Electrophoresis 18:638-641.

Schagger, H., and Von Jagow, G. (1987). Tricine- sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368-374.

Tuschl, T., Zamore,P.D., Lehmann,R., Bartel,D.P., and Sharp, .A. (1999). Targeted mRNA degradation by double- stranded RNA in vitro. Genes Development 13, 3191-3197.

Von Heijne, G. (1986) . A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 14:4683- 4691.

Wainwright,G. , Webster, S.G. , Wilkinson,M.C. , Chung, J.S., and Rees,H.H. (1996). Structure and significance of mandibular organ-inhibiting hormone in the crab, Cancer pagurus. Involvement in multihormonal regulation of growth and reproduction. Journal of Biological Chemistry 271, 12749-12754.

Claims

1. An isolated peptide comprising the following amino acid sequence:

GLTDGTCRGR MGNREIYKKV DRVCEDCANI FRLPGLEGLC RDRCFYNEWF LLCLKAANRE DEIENFRVWI SILNA an active fragment thereof, or a peptide with substantial sequence identity which serves to inhibit spawning in penaeid prawns .

2. An isolated peptide as claimed in claim 1 further comprising the signal sequence:

MHRLALRTWL AIMIVLFATS LSSIASA.

3. An isolated peptide as claimed in claim 1 expressed as a fusion protein.

4. An isolated peptide as claimed in claim 3 wherein the fusion protein is selected from the group consisting of fusions with maltose binding protein (MBP) , glutathione-S-transferase (GST) , bovine serum albumin (BSA) of keyhole limpet hemocyanin (KLH) .

5. An isolated peptide as claimed in any one of claim 1 to 4 wherein, in considering the identity regions of alignment to the peptide, there is a Smallest Sum Probability P(N) of less than l.Oe^"14.

6. An isolated peptide as claimed in claim 5 wherein the Smallest Sum Probability P(N) is between

5.2e^~21 and 9.9e^~47.

7. An isolated nucleic acid molecule encoding a peptide as defined in any one of claims 1 to 6.

8. An isolated nucleic acid, comprising the nucleotide sequence set forth in SEQ ID NO: 3, or fragments or homologues thereof which encode a peptide active in inhibiting spawning in penaeid prawns.

9. An isolated nucleic acid as claimed in claim 8 selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 4 and the nucleotide sequence set forth in SEQ ID NO: 5.

10. An isolated nucleic acid as claimed in claim 8 with at least a 70% degree of homology as determined by the BLASTN algorithm using default parameters.

11. An isolated nucleic acid as claimed in claim 8 with at least a 80% degree of homology as determined by the BLASTN algorithm using default parameters.

12. An isolated nucleic acid as claimed in claim 8 with at least a 90% degree of homology as determined by the BLASTN algorithm using default parameters.

13. An expression vector comprising a nucleic acid molecule as defined in any one of claims 7 to 12.

14. A host cell comprising an expression vector as claimed in claim 13.

15. An antibody which selectively binds a reproductive inhibiting hormone and thereby serves to induce spawning in Crustacea.

16. An antibody as claimed in claim 15, being an antibody to a peptide as defined in any one of claims 1 to 6, or to an antigenic fragment thereof.

17. An antibody as claimed in claim 16 wherein the antigenic fragment has the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7.

18. An antibody as claimed in either of claim 15 or claim 16 which is an antibody to a fusion protein, in particular, to a fusion protein with maltose binding protein (MBP) , glutathione-S-transferase (GST) , bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) .

19. An antibody as claimed in claim 15 which is an antibody to an immunogenic region of the peptides SGPI-V, or immunogenic fragments thereof.

20. An antibody as claimed in claim 19 wherein the antibody is an antibody to an SGPIII-tail fragment having the amino acid sequence set forth in SEQ ID NO: 8 or an SGPV loop peptide having the amino acid sequence set forth in SEQ ID NO: 9.

21. An antibody as claimed in any one of claims 15 to 20 selected from the group consisting of polyclonal, monoclonal, chimeric, single chain antibodies and antibody fragments.

22. An agent for inducing spawning in Crustacea through reducing or eliminating activity of reproductive inhibiting hormone in these animals.

23. An agent as claimed in claim 22 comprising a antibody as defined in any one of claims 15 to 21.

24. An agent as claimed in claim 23 wherein the agent comprises double stranded RNA (dsRNA) with substantial sequence identity to a portion of the sequence of the mRNA of the reproductive inhibiting hormone.

25. An agent as claimed in claim 24 wherein the dsRNA has substantial sequence identity to a portion of the mRNA encoded by DNA having the nucleotide sequence set forth in SEQ ID NO: 3.

26. A method of screening for a candidate agent for inducing spawning in Crustacea, comprising the steps of: (1) providing a candidate agent and the reproductive inhibiting hormone (RIH) ;

(2) determining the nature and extent of interaction; and

(3) selecting agents which reduce or eliminate RIH activity.

27. An interfering RNA molecule (RNAi) which is reactive with the mRNA encoding the putative reproductive inhibiting hormone (RIH) in such a manner as to degrade said mRNA and thereby induce spawning in Crustacea.

28. A method for inducing spawning in Crustacea, comprising the step of reducing or eliminating the activity of reproductive inhibiting hormone (RIH) in these animals .

29. A method as claimed in claim 28 wherein RIH activity is reduced or eliminated through treatment with an antibody as defined in any one of claims 15 to 21.

30. A method as claimed in claim 28 wherein RIH activity is reduced or eliminated through treatment with RNAi as defined in claim 27.

31. The use of an RNAi in downregulating peptide hormones in an animal.

32. A method of downregulating a peptide hormone in an animal, comprising introducing to the animal a dsRNA substantially identical in sequence in one strand and complementary in the other to a region of the mRNA encoding the peptide hormone.

33. An isolated peptide with the amino acid sequence set forth in the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

34. An isolated nucleic acid with the nucleotide sequence set forth in the group consisting of SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5.