Abstract:
The present invention relates to a novel crustacean androgenic gland hormone. More specifically, the invention relates to an androgenic gland hormone derived from  Penaeus monodon . The invention relates further to the use of the hormone to influence the sex ratio in prawn and shrimp cultures, and to set up monosex cultures. It is also related to the use of the hormone and its gene or fragments thereof in sex determination.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This is a national phase entry under 35 U.S.C. §371 of International Patent Application PCT/EP2009/052587, filed Mar. 5, 2009, published in English as International Patent Publication WO 2009/109615 A1 on Sep. 11, 2009, which claims the benefit under Article 8 of the Patent Cooperation Treaty to U.S. Provisional Patent Application Ser. No. 61/033,830, filed Mar. 5, 2008. 
     
    
     TECHNICAL FIELD 
       [0002]    The present invention relates to a novel crustacean androgenic gland hormone. More specifically, the invention relates to an androgenic gland hormone derived from  Penaeus monodon . The invention relates further to the use of the hormone to influence the sex ratio in prawn and shrimp cultures, and to set up monosex cultures. It also relates to the use of the hormone and its gene or fragments thereof in sex determination. 
       BACKGROUND 
       [0003]    Shrimp and prawn cultivation and trade is a very important activity all over the world. The main species under cultivation are  Penaeus monodon  (Giant tiger prawn, Jumbo tiger prawn, Jumbo tiger shrimp, Black tiger prawn, Blue tiger prawn, Grass shrimp . . . ), mainly cultivated in Asia, with an aquaculture production of about 600,000 tons in 2003; and  Penaeus vannamei  (Whiteleg shrimp, white shrimp), mainly cultivated in the Americas and in China and Thailand, with an aquaculture production that is comparable to  P. monodon . For those species, aquaculture is far more important than capture. 
         [0004]    Increasing demands for aquaculture production mean increasing pressure for the development of more efficient production systems. 
         [0005]    As most  Penaeus  sp. are sexually dimorphic (Hansford and Hewitt, 1994), a lot of effort has been paid in setting up monosex cultures. In that respect, several groups have tried to develop reliable sex markers (Khamnamtong et al., 2006). Alternatively, monosex cultures could be obtained by in vitro secretion of androgenic sex hormone from the androgenic gland, as disclosed in U.S. Pat. No. 6,740,794. Although this may be an interesting approach, it is hampered by the fact that the structure of the androgenic gland hormone, or of the gene encoding, is not known. Recently, Manor et al. (2007) described an insulin-like gene that is exclusively expressed in the androgenic gland of the male Australian Crayfish  Cherax quadricarinatus . This may be an interesting candidate gene, possibly coding for the androgenic gland hormone, but its effect has not yet been demonstrated. Moreover, no homologues in prawns or shrimps are known today. Searching for homologous sequences is hampered by the fact that only a limited number of prawn and shrimp sequences are available in the databases, and by the fact that the interspecies homology is probably low, if any. 
         [0006]    Surprisingly, in an AFLP-based transcriptomic profiling experiment in the black tiger shrimp ( Penaeus monodon ), we identified two androgenic gland-specific DNA fragments homologous to isopod “androgenic gland hormone” and crayfish “insulin-like androgenic gland” factor. By further cDNA sequencing and RACE experiments, we identified four mRNAs, termed PmAGH1, PmAGH2, PmAGH3 and PmAGH4, most probably originating from a single gene by alternative splicing. Even more surprisingly, treatment of genetically female juvenile shrimp using recombinant Pm_AGH1, Pm_AGH2, PmAGH3 and/or PmAGH4, had masculinizing effects. Genetically male juvenile shrimp were feminized by dsRNA-mediated knock-down of Pm_AGH1, Pm_AGH2, PmAGH3 and/or PmAGH4. 
       DISCLOSURE 
       [0007]    A first aspect of the invention is an isolated protein selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 and SEQ ID NO:12, or a mutant or variant thereof. Preferably, the protein is isolated from a Penaeus species, more preferably it is isolated from  Penaeus monodon . Mutants and variants as described herein are mutants and variants with at least 50% identity, as measured in a BLASTp (Altschul et al., 1997). Preferably, the mutants and variants are biologically active, as measured by their hormone effect (masculinization; or feminization when acting as a dominant negative compound). Even more preferably, the mutants and variants are retaining the masculinization capacity of the isolated proteins with SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO:12. 
         [0008]    Another aspect of the invention is a nucleic acid fragment, encoding a protein according to the invention. “Nucleic acid fragment,” as used herein, can be any nucleic acid including, but not limited to, messenger RNA, single-stranded DNA, double-stranded DNA, including genomic DNA, possible with its intron-exon organization. In its simplest form, the nucleic acid fragment is limited to the coding sequence, but it may comprise other sequences such as, but not limited to, 5′ and 3′ untranslated sequences, regulatory sequences such as promoter and terminator sequences, and introns. Preferably, the nucleic acid fragment comprises SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8 or SEQ ID NO:11 (coding sequences). 
         [0009]    Still another aspect of the invention is a nucleic acid fragment, comprising SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7 or SEQ ID NO:10 (mRNA) or SEQ ID NO:13 (gDNA), or its complement, or a functional fragment thereof. “A functional fragment” as used herein is, as a non-limiting example, a fragment that can be used as primer for detection, or a fragment that can be used as antisense RNA or as RNAi for down-regulating the expression. 
         [0010]    Another aspect of the invention is the use of a protein according to the invention to modify the sex ratio in prawns or shrimps. Preferably, the prawns or shrimps belong to the family of Penaeidae, even more preferably, the prawn or shrimp is a Penaeus species, most preferably, the prawn or shrimp is  Penaeus monodon.    
         [0011]    Still another aspect of the invention is the use of a nucleic acid fragment according to the invention to modify the sex ratio in prawns or shrimps. Preferably, the prawns or shrimps belong to the family of Penaeidae, even more preferably, the prawn or shrimp is a Penaeus species, most preferably, the prawn or shrimp is  Penaeus monodon.    
         [0012]    Yet another aspect of the invention is the use of a nucleic acid sequence according to the invention to determine sex in prawns or shrimps. Preferably, the prawns or shrimps belong to the family of Penaeidae, even more preferably, the prawn or shrimp is a Penaeus species, most preferably, the prawn or shrimp is  Penaeus monodon . Indeed, on the basis of the sequence presented, it is obvious for the person skilled in the art to develop primers that can be used to measure gene expression of the androgenic gland-specific gene. 
         [0013]    Yet another aspect of the invention is a method of setting up a monosex culture in prawns or shrimps, comprising the use of a protein and/or a nucleic acid according to the invention. Preferably, the prawns or shrimps belong to the family of Penaeidae, even more preferably, the prawn or shrimp is a  Penaeus  species, most preferably, the prawn or shrimp is  Penaeus monodon . Indeed, by injecting the protein into young post-larval animals, females can be converted into males. Alternatively, expression and/or injection of anti-sense RNA, dsRNA and/or RNAi will repress the expression of the androgenic gland-specific protein, and feminizes the animals. As an alternative, antibodies, preferably single chain antibodies or camelid antibodies or antibody fragments such as nanobodies against the proteins of the invention, can be used to feminize the prawns or shrimps. Using the purified protein, or fragments thereof, the generation of antibodies is a process known to the person skilled in the art. 
         [0014]    Another aspect of the invention is a method of setting up a monosex culture in prawns or shrimps, comprising sex determination using a nucleic acid according to the invention. Preferably, the prawns or shrimps belong to the family of Penaeidae, even more preferably, the prawn or shrimp is a  Penaeus  species, most preferably, the prawn or shrimp is  Penaeus monodon . Alternatively, a protein according to the invention could be used in sex determination too, especially in combination with an antibody against the protein according to the invention, as described above. Such antibody can be used, as a non-limiting example, in an ELISA assay to test the concentration of androgenic gland-specific protein present in the animal. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWING 
         [0015]      FIG. 1 : Schematic overview of the genomic DNA encoding the androgenic gland hormone, and the messenger RNAs derived from it by alternative splicing. 
       
    
    
     DETAILED DESCRIPTION 
     Examples 
     Materials and Methods to the Examples 
     Sampling 
       [0016]    Tissue was dissected at the base of the fifth walking leg, where the androgenic gland is supposed to be situated, from mature males. Control tissue was dissected from the identical location in females. Muscle samples were taken from the tail of adult males and females. All samples were immediately snap frozen in liquid nitrogen and stored at −70° C. until RNA preparation. 
         [0000]    cDNA-AFLP 
         [0017]    Snap frozen androgenic gland samples (n=4), control surrounding tissue samples (n=2) and muscle samples (n=6) were pulverized using mortar and pestle under liquid nitrogen. Total RNA was isolated using the TRIzol reagent (Invitrogen). RNA quality was assessed spectrophotometrically (Nanodrop) and by gel electrophoresis. High quality RNA (2.5 μg) was converted to double-stranded cDNA using a biotinylated oligo-dT primer. After purification of the ds cDNA (Qiaquick PCR purification kit, Qiagen), the quality and yield were analyzed by gel electrophoresis. 
         [0018]    AFLP templates were prepared by restriction digest of the ds cDNA using BstYI. The 3′ end fragments were isolated using strepavidin-coated magnetic Dynabeads (Dynal) and further digested using MseI. Restriction site-specific adapters were ligated to the ends of the fragments. 
         [0019]    The adapter-ligation mixture was diluted two-fold to a total volume of 100 μl. Amplification using a BstYI-specific primer with either a T (BstYI-T+0) or C (BstYI-C+0) at its 3′ end in combination with an MseI-specific primer divided the set of fragments in two. Specific amplification of 5 μl of the 600-fold diluted pre-amplification mixtures was performed using the  33 P labelled BstYI-C/T primer with one additional selective nucleotide at its 3′ end in combination with the MseI primer with two selective nucleotides. In this setting, 128 primer combinations were needed to screen the full set of transcript fragments. 
         [0020]    After amplification, the samples were separated on a 5% polyacrylamide sequencing gel. A  33 P labelled 10 bp sizing standard was included. After running and vacuum-drying on whatmann paper, the gel was scanned in a phosphor-imager. 
         [0000]    Sequence Analysis of cDNA-AFLP Fragments. 
         [0021]    When cDNA-AFLP fragments were to be cut from the gel for sequence analysis, the gel was exposed on a Biomax MR autoradiogram (Kodak). Gels were overlaid with the developed film and the relevant fragments were cut from the gel. DNA was dissolved in TE buffer (10 mM Tris, 0.1 mM EDTA) and reamplified using the selective BstYI and MseI primers. Amplification was checked by gel electrophoresis and fragments were directly sequenced using either the selective BstYI or MseI primer. 
       Homology Search Using Obtained Sequences. 
       [0022]    The non-redundant protein sequence database and an in-house compiled DNA sequence database containing all available DNA sequences from invertebrates were searched for sequences homologous to the sequence of the cDNA-AFLP fragment. An in-house-developed algorithm first searched the protein database for sequences homologous to the translated (in all six possible reading frames) query sequence (BLASTX search). When no homology was found, the invertebrate DNA database was searched for homologous DNA sequences (BLASTN search). When homology was identified to a DNA sequence, a BLASTX search was performed with this hit. When no homology was found in the invertebrate DNA database, a TBLASTX search was performed, in which homology was searched in the translated DNA database using the translated query sequence. 
       RACE Analysis of Putative Androgenic Gland Hormone Genes. 
       [0023]    Rapid Amplification of cDNA Ends (RACE) analysis was performed using the BD SMART RACE cDNA amplification kit (BD Biosciences). High-quality total RNA (1 μg) from androgenic gland tissue was converted to 5′ RACE-ready cDNA and 3′ RACE-ready RACE cDNA according to the manufacturer&#39;s instructions. 5′ and nested 5′ RACE were performed using AGHRACE_Rev1 (5′-TATGACAGAAACCGCCAGGGGAGAC-3′ (SEQ ID NO:14)) and AGHRACE_Rev2 (5′-AGGTACCGGGTCCTCGCAATACTCC-3′ (SEQ ID NO:15)) primers, respectively. 3′ and nested 3′ RACE were performed using AGHRACE_Fw1 (5′-CTGGCGGTTTCTGTCATATGCGATG-3′ (SEQ ID NO:16)) and AGHRACEFw2 (5′-GTCTCCCCTGGCGGTTTCTGTCATA-3′ (SEQ ID NO:17)) primers, respectively. Only one distinct fragment was amplified in both the 5′ and nested 5′ RACE while two distinct fragments were amplified in both the 3′ and 3′ nested RACE. Fragments were isolated from the gel and cloned using the TOPO TA cloning kit for sequencing (Invitrogen). Cloned fragments were sequenced using standard T7 and SP6 primers. Sequences were manually curated and assembled using the software packages phredPhrap and consed (Ewing et al., 1998; Ewing and Green, 1998; Gordon et al., 1998). 
         [0024]    Based on the consensus cDNA sequence obtained in the RACE experiments, primers were designed for amplification of the full-length cDNA. Forward primers AGHRT — 3 (5′-ACACAGGACAGGGCAAGTTC-3′ (SEQ ID NO:18)) and AGHRT — 1 (5′-TCCCTCCACAAAAACCACTC-3′ (SEQ ID NO:19)) were used in combination with reverse primers AGHRT — 12 (5′-TCTGGGGCCTATTAATCGAA-3′ (SEQ ID NO:20)) and AGHRT 16  (5′-ACGTTCGGAAAATCGAAAGAT-3′ (SEQ ID NO:21)). The sizes of the PCR fragments were according to what was expected from the RACE experiments and sequencing of the fragments confirmed the sequences obtained previously by RACE. 
       Genomic Sequencing of Putative Androgenic Gland Hormone Genes. 
       [0025]    Using primers AGHFULL2_FW (5′-GTGCTGCCACACACAGGAC_-3′ (SEQ ID NO:22)) and AGHPC3_FW (5′-GCACGCCCTCGTCAAG-3′ (SEQ ID NO:23)) in combination with primer AGHFULL2_REV (5′-TCAAGAATTACTCTGGGGCCTA-3′ (SEQ ID NO:24)) and primer AGH_indel1 (5′-CCAGTACCGCCTGCATCC-3′ (SEQ ID NO:25)) in combination with primer AGH_indel2 (5′-GGCTCACCAGGTGGTCTCT-3′ (SEQ ID NO:26)), we PCR amplified overlapping fragments of the AGH gene on genomic DNA. Additional sequence information was obtained using the Genome Walker Universal Kit (Clontech). Genomic DNA was isolated from several female shrimp and pooled. DNA libraries were prepared according to the manufacturer&#39;s instructions and primary 5′ and 3′ amplification reactions were performed using primers AGHRACE_REV1 and AGHRACE_FW1 (see above), respectively. Nested amplification reactions were performed using primers AGHRACE_REV2 and AGHRACE_FW2 (see above), respectively. A partial genomic sequence, including the complete 3′ UTR region of the mRNA sequences up until the polyA-tail, was assembled using the software packages phredPhrap and consed. 
       Cloning and Recombinant Protein Expression Putative Androgenic Gland Hormone Genes. 
       [0026]    The full-length coding sequence was expressed in a yeast host (Pichia pastoris), to ensure correct folding and glycosylation of the recombinant protein. 
         [0000]    dsRNA Production. 
         [0027]    Using PCR primers AGH_CDS_T7_Fw (5′-TAATACGACTCACTATAGGGATGCCCACTCAGCTGCTT3′ (SEQ ID NO:27)) and AGH_CDS T7_Rev (5′-TAATACGACTCACTATAGGGCTAAGGTACCGGGTCCTCG-3′ (SEQ ID NO:28)), we generated a PCR fragment incorporating a T7 promotor sequence at both. This fragment was used as a T7 RNA polymerase template for dsRNA production using the Megascript RNAi kit (Ambion). 
         [0000]    Sex Reversal Experiments in Juvenile  P. monodon  Shrimp by Injection of Recombinant AGH. 
         [0028]    Groups of young post-larval animals (PL30-PL60) are injected with 120 ng of the recombinant AGH every third day. Group 1 receives injections covering the period of PL30 to PL60. Group 2 receives injections covering the period of PL30 to PL45. Finally, group 3 receives injections covering the period of PL45 to PL60. At PL150, phenotypic sex is assessed by visual inspection and histology while genotypic sex is determined at the DNA level using a PCR-based sex marker as disclosed in PCT/EP2007/054041. 
         [0000]    Sex Reversal Experiments in Juvenile  P. monodon  Shrimp by Injection of AGH-Specific dsRNA. 
         [0029]    Groups of young post-larval animals (PL30-PL60) are injected with 0.5 μg of AGH dsRNA every third day. Group 1 receives injections covering the period of PL30 to PL60. Group 2 receives injections covering the period of PL30 to PL45. Finally, group 3 receives injections covering the period of PL45 to PL60. At PL150, phenotypic sex is assessed by visual inspection and histology while genotypic sex is determined at the DNA level using a PCR-based sex marker as disclosed in PCT/EP2007/054041. 
       Example 1 
     Identification of  Penaeus monodon  Androgenic Gland Hormones 
       [0030]    Transcript profiling of all possible cDNA-AFLP primer combinations (128 PC) was performed on androgenic gland tissue samples (male; n=4), surrounding tissue control samples (female; n=2) and female and male muscle samples (n=3 each). By visual inspection of the cDNA-AFLP gels, we selected 355 androgenic gland specific fragments. We obtained sequence information for 254 (71.6%) of these fragments. Homology searches indicated that at least 47 (18.5%) of these were identical (BLASTX alignment showing at least 75% identities) to bacterial sequences ( Propionibacterium acnes ). This is most likely due to a bacterial infection of the androgenic glands in the sampled animals. 
         [0031]    Two cDNA-AFLP fragments were found to be homologous to “insulin-like androgenic gland factor” from the red-claw crayfish ( Cherax quadricarinatus ): BC4M21M263.5 and BC4M34M404.4. 
         [0032]    RACE analysis, followed by full-length cDNA amplification and sequencing resulted in the identification of four cDNA sequences PmAGH1 (SEQ ID NO:1), PmAGH2 (SEQ ID NO:4), PmAGH3 (SEQ ID NO:7) and PmAGH4 (SEQ ID NO:10). Genomic sequencing of a part of the gene (SEQ ID NO:13) indicated that all four of these cDNAs originate by alternative splicing from a single gene. 
         [0033]    Fragment BC4M21M263.5 corresponds to a 234 bp BstYI-MseI restriction fragment originating from PmAGH1 and PmAGH3, while fragment BC4M34M404.4 corresponds to a 372 bp BstYI-MseI restriction fragment originating from PmAGH2 and PmAGH4. 
         [0034]    Homology search with the full cDNA sequences revealed no significant homology on the nucleotide level. On the protein level, homology was again found with the crayfish “insulin-like androgenic gland factor” (32% identity) and to a lesser extent with the androgenic gland hormones from the isopods  Armadillidium vulgare, Porcelio scaber  and  Porcelio dilatatus  (between 29% and 26% identity). 
       Example 2 
     Sex Reversal in Juvenile  P. monodon  Using Recombinant Pm_AGH1, Pm_AGH2, Pm_AGH3 and/or Pm_AGH4 
       [0035]    Administration of recombinant PenMon_AGH1 and PenMon_AGH2 to juvenile  P. monodon  is able to masculinize genetic females. 
       Example 3 
     Sex Reversal in Juvenile  P. monodon  Using DsRNA-Mediated Knock-Down of the Androgenic Gland Hormone Gene 
       [0036]    Administration of dsRNA targeting PenMon_AGH1/2 is able to knock down gene expression of both variants and feminized genetically male juvenile  P. monodon.    
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