Patent Application
20170035034
The present invention relates to methods for moderating crustacean androgenic gland peptide for producing a monosex culture.
Most cultured crustacean species exhibit sexually dimorphic growth patterns, that is, one sex grows larger than the other sex. For instance, in two most economically important penaeid shrimps, Litopenaeus vannamei and Penaeus monodon, females grow larger than males. On the contrary, several cultured species such as the Australian red-claw crayfish Cherax quadricarinatus and giant freshwater prawn Macrobrachium rosenbergii, males grow faster and reach heavier weights than females [1]. The culture of monosex populations (either all-male or all-female) is a preferred approach for gaining higher yields, with the ecological benefit of reducing the risk of invasion by the cultured species [2].
It is also suggested that sexual manipulations may also render sustainable solutions to the environmental problems caused by the presence of invasive crustacean species with detrimental impacts ranging from aggressive competition with native species for food and shelter, to affecting aquaculture facilities and harvests and causing structural damage to river banks [2].
Sexual differentiation in crustaceans is regulated specifically by a nonsteroidogenic male-specific androgenic gland (AG) which can induce a sexual shift through steroid administration [3]. A lot of studies have proved that AG implantation could masculinize females and its removal could feminize males in amphipod, isopod and decapod species [2]. AG was detected in all studied malacostracan crustacean species, its key role in male make it a viable target for sexual manipulation-based biotechnologies [2]. Attempt was made to obtain a monosex culture by microsurgical removal of the AG from juvenile males [2, 5]. However, this biotechnology is hampered by the low success rate of the microsurgery and by lengthy and labor-intensive progeny testing [2, 5].
Recently, insulin-like androgenic gland peptides (IAG) were identified in many crustacean species. The first decapod insulin-like AG-specific peptide encoding cDNA (IAG) was discovered in 2007 and IAGs of nine additional decapod species have been cloned [2, 4]. It was reported that IAG silencing by RNA interference (RNAi) in such decapod species was useful in obtaining a full and functional sex reversal, leading to the production of monosex populations that are the progeny of sexually reversed males (‘neo-females’) [2]. ‘Neo-females’ are genotypic males having female sexual characteristics. IAG silencing allows one to obtain monosex populations without any genetic modification. The intervention is temporal and not transmissible to next generations [2].
Sex reversal can be obtained by either knocking down the IAG gene in males to obtain neo-females, or administering IAG gene to females to obtain neo-males. Four biotechnological routes to achieve monosex populations have been suggested [2]. In species where the males are homogametic (i.e., bear two homologous sex chromosomes, ZZ), knocking down the IAG in males to induce neo-females will enable the production of 100% male populations [6, 7]. IAG knockdown or administration can also be done in heterogametic moiety. However, knocking down the IAG gene in XY males would result in YY males which are not 100% viable. On the contrary, a 100% female population can be achieved by inserting IAG gene to the XY females, which is a desirable approach for crabs [8, 2].
The enhanced yield achievable through monosex population culture has been shown in case studies in a crab species [8], in a crayfish species [9, 10] and in a prawn species [11]. Therefore, attaining monosex population culture of crustacean by moderating IAG gene would be very useful and valuable with respect to both the industry and the environment.
In case where obtaining a full male population is desirable, one may employ RNAi knockdown of IAG gene to produce the ‘neo-females’ which are then crossed with normal homogametic male to produce a 100% male (ZZ) population. However, the procedure of RNAi knockdown is highly complicated.
A common approach for RNAi knockdown is to inject dsRNA for IAG gene into the shrimp and differentiate the male and female shrimp afterwards (as dsRNA may not guarantee a 100% knockdown of the gene, some males remain both genotypically and phenotypically male). However, it is observed that RNAi knockdown is not always effective in inducing sex reversal, as observed in some studies the low sex reversal rate or even absence of sex reversal. Furthermore, even if IAG gene has been successfully knockdown, it may not be easy to differentiate their sex due to their small size. Furthermore, this approach usually involves crosses of two generations to obtain 100% monosex population and only a few numbers of “neo-females” can be obtained for the second crossing with normal male.
Another approach to create a monosex culture is to convert normal female to neomale using recombinant or purified IAG protein. The obtained neomale is then crossed with normal female to give a 100% female population. However, as in the RNAi method, sex reversal rate is not always high enough to produce sufficient amount of neomale for subsequent crossing.
The present invention provides an alternative approach for obtaining a 100% monosex population. The methods described herein improved the current methods by injecting the dsRNA for IAG gene or recombinant IAG protein at an appropriate molt stage of the crustacean at which the crustacean is more responsive to the treatment. Furthermore, the methods described herein only require one single cross of the animal and thereby reduce the trouble of differentiating the sex of the animal after treatment of the dsRNA for IAG.
The present invention provides a nucleic acid molecule and method for moderating crustacean androgenic gland peptide expression for producing a monosex culture.
In one embodiment, the present invention provides a nucleic acid molecule which is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of insulin-like androgenic gland peptides (IAG) of crustaceans. In another embodiment, the dsRNA described herein is a dsRNA corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of shrimp or lobster.
In one embodiment, the present invention provides a method of manipulating sex, reversing sex or changing sex ratio in crustaceans by moderating the expression of androgenic gland peptide in the crustaceans. In one embodiment, the expression of androgenic gland peptide is moderated by injecting a dsRNA for the gene of IAG peptide into the crustaceans, thereby induces silencing of the IAG gene in the crustaceans and alters the sexual characteristics of the crustaceans.
In one embodiment, the present invention provides a method of producing a monosex culture of female crustacean comprising the step of injecting a dsRNA corresponding to the DNA sequence of the IAG peptide into a male crustacean. In one embodiment, the dsRNA to be injected is a dsRNA corresponding to the DNA sequence of the IAG peptide of a shrimp or lobster, thereby producing a monosex culture of female shrimp or lobster.
In one embodiment, the present invention provides a method of producing a monosex culture of female crustacean comprising the step of injecting a dsRNA for the gene of IAG into a female crustacean that had acquired a spermatophore (a sperm sac) after mating with a male.
In one embodiment, the present invention provides a method of manipulating sex, reversing sex or changing sex ratio in crustaceans by introducing a recombinant IAG peptide into the crustaceans, thereby alters the sexual characteristics of the crustaceans. In one embodiment, the crustacean is a shrimp or lobster.
In another embodiment, the present invention provides a method of obtaining a monosex culture of female crustacean by introducing a recombinant IAG peptide into normal females and crossing the resulted offspring with normal females, thereby produces a monosex culture of female crustacean.
Crustaceans such as shrimps and lobsters have two sex chromosomes W and Z. Female gamete bears either W or Z chromosome while the male gamete contains only the Z chromosome. Therefore, female gamete determines the sex of an individual, i.e. an individual having a genotype of WW or ZW would be a female while a genotype of ZZ would give a male.
The present invention provides a nucleic acid molecule and method for manipulating sex, reversing sex or changing sex ratio in crustaceans by moderating the expression of crustacean androgenic gland peptide in crustaceans. In another embodiment, the present invention provides a nucleic acid molecule and method for producing a monosex culture of crustacean.
In one embodiment, the nucleic acid molecule of the present invention is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of crustacean.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of shrimp.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of Penaeus monodon of SEQ ID NO:1.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of Litopenaeus vannamei of SEQ ID NO:2.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of Fenneropenaeus chinensis of SEQ ID NO:3.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of lobster.
In one embodiment, the nucleic acid molecule is a double-stranded RNA (dsRNA) corresponding to the DNA sequence of the insulin-like androgenic gland peptides (IAG) of other crustacean species.
The sequences of dsRNA for IAG gene of Penaeus monodon, Litopenaeus vannamei and Fenneropenaeus chinensis are:
Penaeus monodon
Litopenaeus vannamei
Fenneropenaeus chinensis
In one embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of female shrimp.
In another embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of female Penaeus monodon.
In one embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of female Litopenaeus vannamei.
In one embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of female Fenneropenaeus chinensis.
In one embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of female lobster.
In one embodiment, the crustacean androgenic gland peptide is moderated for producing a monosex culture of other female crustacean species.
In one embodiment, the present invention provides a method of producing a monosex culture of female crustacean comprising the step of injecting a dsRNA for the gene of JAG peptide into a male crustacean. In one embodiment, the dsRNA to be injected is a dsRNA corresponding to the DNA sequence of the gene of IAG of male shrimp or lobster, thereby producing a monosex culture of female shrimp or lobster.
As discussed above, current methods which use dsRNA for knocking down IAG gene are ineffective because the dsRNA is not injected at the time IAG gene is expressed. It is noted that expression of IAG is specific to certain molt stages. Injection of dsRNA at an inappropriate molt stage may induce no sex reversal or result in delay responses. The present method selects an appropriate molt stage for dsRNA injection such that crustacean would be more responsive to the dsRNA, and thereby enhances the efficiency in moderation of IAG and sex reversal.
In one embodiment, the dsRNA for IAG is introduced into the shrimps once the shrimps have molted. In another embodiment, the dsRNA for IAG is introduced into the shrimps for many molt cycles that result in gradual change of sex.
In one embodiment, the time to inject the dsRNA is when the shrimp or lobster is young or sexually undifferentiated.
In one embodiment, dsRNA is injected into male shrimp when the shrimp is about 2.5-3.0 cm long.
In one embodiment, the shrimp is injected with the dsRNA for once. In another embodiment, after the first injection, the shrimp is injected with the dsRNA for a second time when it molts again.
In one embodiment, the present invention provides a method of producing a monosex culture of female crustacean comprising the step of injecting a dsRNA for the gene of IAG into a female crustacean that had acquired a spermatophore (a sperm sac) after mating with a male.
In one embodiment, the female is artificially inseminated with a spermatophore (a sperm sac). The dsRNA for IAG gene can be introduced into the female together with the sperm sac and induces silencing of the IAG gene in the female. When the female spawns, the eggs (which contain either a Z or W chromosome) will be fertilized by the sperm (with Z chromosome). The eggs with W chromosome that are fertilized by the Z sperm will become a normal female (ZW). The eggs with Z chromosome that are fertilized by the Z sperm will turn into a normal male (ZZ). However, since the gene of IAG would be knocked down by the dsRNA, these normal male will turn into a “neo-female”. Accordingly, a 100% female population can be obtained from one single cross of the animal.
In one embodiment, the method of producing a monosex culture comprises the step of artificially inseminating female shrimps or lobsters with a spermatophore (a sperm sac). The sperm sac can be introduced into the shrimps (e.g. P. monodon and F. chinensis) or lobsters when the females have molted.
In another embodiment, the sperm sac can be introduced into the shrimps (e.g. L. vannamei) when the females is about to spawn.
In one embodiment, the dsRNA for IAG gene can be introduced together with the sperm sac into the sperm receptacle by prior mixing the dsRNA solution with the sperm sac ejected from the male. The sperm sac, together with the dsRNA, will remain in the female for silencing the IAG gene. In one embodiment, the female is injected with the sperm sac and dsRNA 2 days before she spawns her egg.
In one embodiment, the female injected with the sperm sac and dsRNA for IAG gene releases the sperm when she ovulates or spawns her egg, leading to the fertilization of the eggs. In one embodiment, the eggs with W chromosome are fertilized by the Z sperm and become a normal female (ZW). In another embodiment, the eggs with Z chromosome are fertilized by the Z sperm and turn into a “neofemale” (ZZ) under the silencing effect of dsRNA for IAG.
In one embodiment, dsRNA for IAG is injected into female that have mated with the male naturally.
In another embodiment, dsRNA for IAG is injected into the thylecum of the female shrimp that have been mated.
In one embodiment, the method of producing a monosex culture of female shrimp or lobster comprises the following steps:
In one embodiment, the sex of the shrimps or lobsters is differentiated using a sex marker after being injected with the dsRNA for IAG.
In one embodiment, the sex of the shrimps is differentiated using a sex marker AFLP after being injected with the dsRNA for IAG.
In one embodiment, the present invention provides a method of obtaining a monosex culture of female crustacean by introducing a recombinant or purified IAG peptide into a normal female and crossing the resulted offspring with a normal male, thereby produces a monosex culture of female crustacean.
In one embodiment, the recombinant or purified IAG peptide from shrimp or lobster is introduced into the female shrimp or lobster.
In one embodiment, the recombinant IAG peptide described herein is expressed in E. coli. In another embodiment, the recombinant IAG peptide described herein is expressed using other appropriate expression system.
In one embodiment, the IAG peptide is the IAG of Penaeus monodon encoded by the DNA sequence of SEQ ID NO:1. In another embodiment, the IAG peptide comprises the amino acid sequence of insulin-like androgenic gland peptide (IAG) of Penaeus monodon of SEQ ID NO:4.
In one embodiment, the IAG peptide is the IAG of Litopenaeus vannamei encoded by the DNA sequence of SEQ ID NO:2. In another embodiment, the recombinant IAG peptide comprises the amino acid sequence of insulin-like androgenic gland peptide (IAG) of Litopenaeus vannamei of SEQ ID NO:5.
In one embodiment, the IAG peptide is the IAG of Fenneropenaeus chinensis encoded by the DNA sequence of SEQ ID NO:3. In another embodiment, the recombinant IAG peptide comprises the amino acid sequence of insulin-like androgenic gland peptide (IAG) of Fenneropenaeus chinensis of SEQ ID NO:6.
In one embodiment, the recombinant IAG peptide comprises the amino acid sequence of insulin-like androgenic gland peptide (IAG) of Macrobrachium rosenbergii (Fresh water shrimp) of SEQ ID NO:7.
In one embodiment, the recombinant IAG peptide comprises the amino acid sequence of insulin-like androgenic gland peptide (IAG) of Jasus edwardsii (Spiny lobster) of SEQ ID NO:8.
Below are the amino acid sequences of IAG gene that may be used in the present invention:
Penaeus monodon (Tiger shrimp, Genbank #GU208677)
Litopenaeus vannamei
Fenneropenaeus chinensis
Macrobrachium rosenbergii (Fresh water shrimp)
Jasus edwardsii (Spiny lobster, Genbank #AIM55892)
In one embodiment, the method of obtaining a monosex culture of female shrimp or lobster comprising the following steps:
In contrast to current methods, the present method introduces IAG protein into the shrimps at the intermolt stage. It is observed that a higher population of shrimps was responsive to IAG protein treatment at intermolt stage and underwent sex reversal in comparison with treatments performed in other stages.
In one embodiment, the IAG peptide is introduced into the shrimps or lobsters for many molt cycles that result in gradual change of sex.
The present invention provides a method of producing a monosex culture of crustacean, comprising the steps of
In one embodiment, the crustacean is a shrimp or lobster.
In one embodiment, the crustacean is Penaeus monodon, Litopenaeus vannamei or Fenneropenaeus chinensis.
In one embodiment, the double-stranded RNA (dsRNA) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-3.
In one embodiment, the crustacean in the monosex culture has homozygous sex chromosomes or heterozygous sex chromosomes.
The present invention provides a method of producing a monosex culture of crustacean, comprising the steps of
In one embodiment, the crustacean is a shrimp or lobster.
In one embodiment, the crustacean is Penaeus monodon, Litopenaeus vannamei Fenneropenaeus chinensis, Macrobrachium rosenbergii or Jasus edwardsii.
In one embodiment, the crustacean insulin-like androgenic gland (IAG) peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-3.
In one embodiment, the crustacean insulin-like androgenic gland (IAG) peptide is an isolated or recombinant protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:4-8.
In one embodiment, the female crustacean obtained from step b) in above method is introduced with the crustacean insulin-like androgenic gland (IAG) peptide for a second time when the female crustacean reaches the intermolt stage of the next molting cycle.
In one embodiment, the crustacean insulin-like androgenic gland (IAG) peptide is introduced in combination with other chemicals, peptides, proteins, or reagents.
The present invention provides a method of producing a monosex culture of crustacean, comprising the step of injecting a double-stranded RNA (dsRNA) comprising the nucleotide sequence of the crustacean insulin-like androgenic gland (IAG) peptide into a male crustacean, thereby obtaining a culture of crustacean having the phenotype of a female crustacean.
In one embodiment, the crustacean is a shrimp or lobster.
In one embodiment, the crustacean is Penaeus monodon, Litopenaeus vannamei or Fenneropenaeus chinensis.
In one embodiment, the double-stranded RNA (dsRNA) comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs:1-3.
This invention will be better understood by reference to the examples which follow. However, one skilled in the art will readily appreciate that the examples provided are merely for illustrative purposes and are not meant to limit the scope of the invention which is defined by the claims following thereafter.
Throughout this application, it is to be noted that the transitional term “comprising”, which is synonymous with “including”, “containing” or “characterized by”, is inclusive or open-ended, and does not exclude additional, un-recited elements or method steps.
To produce an all female shrimp culture, a simple approach is to inject dsRNA for IAG gene into a male shrimp. In one embodiment, the best time to inject the dsRNA is when they are young, or sexually undifferentiated. For example, for P. monodon (largest female sometime reach 23 cm or 180 grams), one should inject them when they are about 2.5-3.0 cm long. Inject all the small shrimp and later determine if all of the shrimp are female. A deviation of the 1:1 ratio.
In another embodiment, juvenile shrimp of 2.5-3.0 cm is selected for sex identification. Twenty male shrimps is selected and injected with dsRNA for IAG (e.g. 1 μg/g of body weight). The control male shrimps are injected with PBS only. The two groups are cultured separately and the same injection procedure is repeated when they molt again. Most of the shrimps injected with the dsRNA should lose the secondary sexual characteristics (e.g. the appendix masculine of the swimming leg will disappear or reduce in size).
Sex markers can be used to differentiate the male from the female for younger shrimp. In one embodiment, the sex marker AFLP described in Staelens, 2008 can be used for Penaues monodon [12]. This method is useful for screening out the male for dsRNA injection when the animal is small.
Shrimp can be artificially inseminated with a spermatophore (a sperm sac). The sperm sac is collected from the male shrimp. dsRNA for the IAG gene is mixed with the collected sperm sac. When the female molts and the exoskeleton is soft, the sperm sac with dsRNA is inserted into the female thylecum (sperm receptacle), preferably 1-2 days after she molted. The sperm sac, together with the dsRNA, will remain in the female body and silence the IAG gene. Ovary will continue to develop for the next 5-6 days. The female is injected with dsRNA for the IAG gene (e.g. 10 ug/g of body weight) 1 to 2 days before she spawns.
When the female spawns, the eggs (which contain either a Z or W chromosome) will be fertilized by the sperm (with Z chromosome). The eggs with W chromosome that are fertilized by the Z sperm will become a normal female (ZW). The eggs with Z chromosome that are fertilized by the Z sperm will turn into a normal male (ZZ). However, since the gene of IAG would be knocked down by the dsRNA, these normal male will turn into a “neofemale”.
dsRNA for IAG can also be injected into female shrimp that have mated with the male naturally. In another embodiment, dsRNA for IAG can be injected into the thylecum of the female that have been mated which requires lower dose of dsRNA.
As illustrated in
Firstly, female crustacean such as shrimps (ZW) are selected during early stage of sexual development at intermolt stage. In one embodiment, shrimps at the early stage of 2-3 cm in total length are selected for injection. In another embodiment, shrimps at the early stage of 3-4 cm in total length are selected for injection.
The selected shrimps are then injected with a recombinant protein for IAG (insulin-like androgenic gland hormone). When the females reach the intermolt stage of the next molting cycle, the females are injected with a recombinant protein for IAG again. In one embodiment, the concentration of recombinant IAG protein used for injection is slightly higher than the physiological concentration of the IAG protein in the shrimps. In another embodiment, the concentration of recombinant IAG protein used for injection is at the range of 5-20 μg/g of body weight. Injection at an early stage would enhance the efficiency of sex reversal with a lower dose of recombinant IAG protein.
The treated females are then cultured until they have completely converted into a functional males (“neomales”) (ZW). This can be observed by the development of a male sex organ such as pestama with the spermatophore.
The “neomales” obtained is then mated with a normal female (ZW) to produce the F1 generation having a genotype of ZZ, ZW, ZW or WW. Accordingly, one-third of the F1 generation will be male (ZZ) and the remaining F1 generation will be female (ZW or WW).
The female progeny having the WW phenotype is desirable and selected using appropriate DNA sex marker. The obtained female progeny (WW) is then mated with a normal male (ZZ), and finally produce all-female progeny having a genotype of WZ.
Sex markers can be used to assess the progress of sex reversal in the treated organisms, or to differentiate male and female organisms. In one embodiment, the sex marker AFLP described in Staelens, 2008 can be used for Penaues monodon [12]. In another embodiment, the sex marker described in Jiang, 2013 can be used for Macrobrachium rosenbergii [13].
This application claims priority of U.S. Ser. No. 61/887,317, filed Oct. 4, 2014. The content of the preceding application is hereby incorporated in its entirety by reference into this application.
| Number | Date | Country | |
|---|---|---|---|
| 61887317 | Oct 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14506690 | Oct 2014 | US |
| Child | 15236356 | US |
