Promotion of Synchronous Sexual Maturation and Spawning in Aquatic Animals

[0001] REFERENCE TO PRIOR APPLICATIONS

[0002] This application claims priority of U.S. Provisional Patent Application No. 63/366,841, filed 22 June 2022, the entire contents of which are incorporated herein by reference.

[0003] TECHNICAL FIELD

[0004] This invention broadly relates to recombinant algae that express a gonad stimulating hormone, for promoting sexual maturity and/or spawning in aquatic animals such as fish.

[0005] BACKGROUND

[0006] Aquaculture is a worldwide, multi-billion dollar industry. Managed fisheries and other aquaculture provide food, nutrition and employment, particularly in parts of the world that often experience food insecurity. The Food and Agriculture Organization of the United Nations estimates that global seafood production has reached about 179 million metric tons in 2018, with a value estimated at 250 billion USD. Of the total production, approximately 156 million metric tons were used for human consumption, while the remaining 22 million metric tons were utilized in non-food uses, mainly fishmeal and fish oil. The World Bank’s Agriculture and Environmental Services Division predicts that by 2030, more than 62% of the seafood consumed on the market will be farmed seafood.

[0007] Commercially successful aquaculture requires reliable spawning and fry production. Many fish and other commercially valuable marine animals do not mature sexually under typical aquaculture conditions and environmental controls are expensive, time consuming and often unreliable. Even when animals retain their natural reproductive capacities, it is preferable that farmed aquatic animals mature sexually, then spawn, within predictable timelines. For example, tight timing of sexual maturation and spawning enables fish farmers to know when the eggs are coming and they can plan the larval rearing runs and growout, and tight timing also allows for efficient management of the different nutrients needed at various times during the animals’ reproductive life cycle.

[0008] Sexual maturation and spawning in marine animals are naturally controlled by hormones, in particular, gonadotropin releasing hormone (GnRH), a ~10-amino acid residue neuropeptide in fish. Oral administration of exogenous peptides to any animal in a manner that preserves activity is difficult. The anatomy and physiology of the intestinal tract of marine animals is significantly different from higher vertebrates, requiring specialized dosage forms. The easiest route, oral administration, is not practical because overly large doses are required to compensate for degradation of the peptide in the acidic environment of the stomach and further degradation by stomach and intestinal peptidases. In research studies, GnRH has been administered by rectal and more commonly by intraperitoneal and muscular injections (including, slow-release implants), for example, to carp and sturgeon and activity was maintained. However, these dosing regimens are labor intensive and stressful to the fish, and for very large species not practical. Furthermore, recombinant GnRH is typically administered in a non-native form. Thus, there is a need for an efficient method of gonad stimulating hormone (eg. GnRH) administration to promote sexual maturation and induce spawning of economically important marine animals.

[0009] DESCRIPTION OF THE INVENTION

[0010] Provided herein are methods and compositions for the promotion of sexual maturation and/or spawning in aquatic animals. An exemplary protocol is shown in Figure 1. The methods involve oral delivery to the aquatic animals of algae that recombinantly express a gonad stimulating hormone, such as gonadotropin-releasing hormone (GnRH). In some embodiments, an orally administered gonad stimulating hormone, such as a polypeptide comprising, essentially consisting of, or consisting of GnRH, is absorbed in the intestinal tract and reaches the bloodstream for subsequent delivery to the pituitary to stimulate the release of gonadotropins to regulate reproductive cycles.

[0011] Provided herein is a method of inducing / promoting sexual maturity in a collection of aquatic animals comprising feeding the animals recombinant algae that express a gonad stimulating hormone that promotes sexual maturity. Further disclosed is a method of spawning induction in a collection of aquatic animals comprising feeding the animals an effective amount of recombinant algae that express a gonad stimulating hormone that promotes oocyte maturation, ovulation and spawning.

[0012] Recombinant algae engineered to express a gonad stimulating hormone, such as a polypeptide comprising, consisting essentially of, or consisting of GnRH (ie. the decapeptide or functionally similar neuropeptide), are also provided, as is recombinant algae biomass formulated as aquatic animal feed. It is thought that recombinant algae of the present invention provide a protective vehicle for expressed gonad stimulating hormones to pass through the stomach, so by the time the gonad stimulating hormones reach the intestines, they can be absorbed and reach their receptors as active hormones.

[0013] According to a first aspect of the present invention, there is provided a method of inducing sexual maturity in one or more sexually immature aquatic animals, comprising feeding the one or more animals an effective amount of algae that express a gonad stimulating hormone that promotes sexual maturity.

[0014] According to a second aspect of the present invention, there is provided a method of collective spawning induction in a collection of sexually mature aquatic animals, comprising feeding the animals an effective amount of algae that express a gonad stimulating hormone that promotes spawning.

[0015] According to a third aspect of the present invention, there is provided a method of collectively inducing sexual maturity and spawning in a collection of aquatic animals, comprising feeding the animals algae that express a gonad stimulating hormone that promotes sexual maturation in an amount effective to promote sexual maturation, and then in an amount effective to promote spawning.

[0016] According to a fourth aspect of the present invention, there is provided recombinant algae comprising an expression construct comprising a nucleotide sequence encoding a gonad stimulating hormone for an aquatic animal, operatively linked to an expression control sequence.

[0017] According to a fifth aspect of the present invention, there is provided an alga-produced recombinant, isolated or purified gonad stimulating hormone.

[0018] According to a sixth aspect of the present invention, there is provided a recombinant, isolated or purified nucleic acid encoding the gonad stimulating hormone according to the fifth aspect of the present invention.

[0019] According to a seventh aspect of the present invention, there is provided a recombinant expression cassette comprising at least one expression control sequence operatively linked to at least one nucleotide sequence encoding the gonad stimulating hormone according to the fifth aspect, or to the nucleic acid according to the sixth aspect of the present invention.

[0020] According to an eighth aspect of the present invention, there is provided an expression vector comprising the gonad stimulating hormone according to the fifth aspect, the nucleic acid according to the sixth aspect, or the expression cassette according to the seventh aspect of the present invention.

[0021] According to a ninth aspect of the present invention, there is provided a recombinant alga:

[0022] (1) that comprises the gonad stimulating hormone according to the fifth aspect;

[0023] (2) that expresses the gonad stimulating hormone according to the fifth aspect;

[0024] (3) that comprises the nucleic acid according to the sixth aspect;

[0025] (4) that comprises the expression cassette according to the seventh aspect; or

[0026] (5) that comprises the expression vector according to the eighth aspect of the present invention.

[0027] According to a tenth aspect of the present invention, there is provided a composition comprising the recombinant algae according to the fourth aspect, the gonad stimulating hormone according to the fifth aspect, the nucleic acid according to the sixth aspect, the expression cassette according to the seventh aspect, the expression vector according to the eighth aspect, or the recombinant alga according to the ninth aspect of the present invention. [0028] According to an eleventh aspect of the present invention, there is provided aquatic animal feed comprising the recombinant algae according to the fourth aspect, the recombinant alga according to the ninth aspect, or the composition according to the tenth aspect of the present invention.

[0029] According to a twelfth aspect of the present invention, there is provided:

[0030] a method of promoting sexual maturity in one or more sexually immature aquatic animals, comprising feeding the one or more animals an effective amount of recombinant algae that express a gonad stimulating hormone that promotes sexual maturity; or

[0031] recombinant algae that express a gonad stimulating hormone for use in promoting sexual maturity in one or more sexually immature aquatic animals, wherein said use comprises the step of feeding the one or more animals an effective amount of the recombinant algae to promote sexual maturity; or

[0032] use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for promoting sexual maturity in one or more sexually immature aquatic animals, wherein said aquatic animal feed is formulated for feeding an effective amount of the recombinant algae to the one or more animals to promote sexual maturity.

[0033] According to a thirteenth aspect of the present invention, there is provided:

[0034] a method of collective spawning induction in a collection of sexually mature aquatic animals, comprising feeding the animals an effective amount of recombinant algae that express a gonad stimulating hormone that promotes spawning; or

[0035] recombinant algae that express a gonad stimulating hormone for use in collective spawning induction in a collection of sexually mature aquatic animals, wherein said use comprises the step of feeding the one or more animals an effective amount of the recombinant algae to promote spawning; or

[0036] use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for promoting collective spawning in a collection of sexually mature aquatic animals, wherein said aquatic animal feed is formulated for feeding an effective amount of the recombinant algae to the one or more animals to promote spawning.

[0037] According to a fourteenth aspect of the present invention, there is provided:

[0038] a method of collectively promoting sexual maturity and spawning in a collection of aquatic animals, comprising feeding the animals recombinant algae that express a gonad stimulating hormone that promotes sexual maturation in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning; or [0039] recombinant algae that express a gonad stimulating hormone for use in collectively promoting sexual maturity and spawning in a collection of aquatic animals, wherein said use comprises the step of feeding the animals the recombinant algae in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning; or

[0040] use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for collectively promoting sexual maturity and spawning in a collection of aquatic animals, wherein said aquatic animal feed is formulated for feeding the animals the recombinant algae in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning.

[0041] According to a fifteenth aspect of the present invention, there is provided a recombinant gonad stimulating hormone (polypeptide) or a nucleic acid encoding the recombinant gonad stimulating hormone (polypeptide), having an amino acid or nucleotide sequence as shown or substantially as shown in the accompanying Figures or Table of Sequence Listings.

[0042] Context permitting, the first to fifteenth aspects of the invention can have any one or more of the features described hereafter. It is to be appreciated that, context permitting, a feature described for a product (eg. alga, algae, recombinant gonad stimulating hormone, polypeptide, nucleic acid, composition, expression cassette, expression vector, or animal feed) can be a feature of a method, process or use, and vice-versa.

[0043] I. Aquatic Animals

[0044] Aquatic animals used in the methods of this invention include any animal whose sexual maturation, ovulation or spawning can be modulated using a gonad stimulating hormone and, in particular, GnRH. Such animals include, without limitation, vertebrates, echinoderms, crustaceans and mollusks. Aquatic animals used in this method typically are fish consumed by people as food. However, aquatic animals for ornamental and conservation purposes are also contemplated. In some embodiments, the animals are marine animals.

[0045] Exemplary vertebrates include, without limitation, cartilaginous (Chondrichthyes) and bony (Osteichthyes) fish (e.g., Teleostei), amphibians, reptiles and mammals. Teleosts used in the methods of this disclosure include, without limitation, carp, tilapia, salmon, bream, catfish, trout, snake head, salmon, tuna, cod, halibut, groupers, mullet, trevally, and sturgeon.

[0046] Exemplary crustaceans include, without limitation, lobsters, crabs, shrimp, and prawns. [0047] Exemplary mollusks include, without limitation, octopus, squid, snails, scallops, edible and pearl oysters and clams.

[0048] Most marine fish species of commercial importance are iteroparous, that is, they spawn more than once during their lives. Furthermore, they are gonochoristic, that is, their sexes are separate, they possess no sexual dimorphism, and exhibit external fertilization without parental care. Exceptions include, for example, Pacific salmonids, capelin and eels, which are semelparous; that is, they spawn once in their lives and die. Some fish are viviparous, that is, their embryos develop inside the ovary, with internal fertilization of eggs.

[0049] Some fish are synchronous responders, that is, all the oocytes develop and ovulate at the same time. Other fish are group-synchronous, that is, populations of oocytes develop at different times. Other fish are asynchronous spawners, that is, oocytes of all stages of development are present without dominant populations.

[0050] II. Gonad Stimulating Hormones

[0051] Methods of promoting sexual maturity and/or spawning of aquatic animals involve administering to the animals an effective amount of one or more gonad stimulating hormones, produced recombinantly in algae. If more than one is administered, then these can be different from each other. That is, they need not be sequence identical. More than one type of gonad stimulating hormone or subunits thereof or receptor-active sequences thereof can be expressed. [0052] Any gonad stimulating hormone(s) that can promote sexual maturity or spawning in the particular animal in which it is used can be used in the methods of this disclosure. For clarity, a ‘gonad stimulating hormone’ may act on the animal’s gonads directly or indirectly, eg. via the pituitary.

[0053] Throughout this specification, the terms ‘peptide’, ‘polypeptide’ and ‘protein’ are used interchangeably to refer to a polymer of amino acid residues. However, an oligopeptide typically consists of between about 2 to 20 amino acids, a peptide typically consists of between about 2 and 50 amino acids, and a polypeptide or protein typically has about 50 or more amino acids. A protein can be formed from one or more peptides and/or polypeptides joined together. For simplicity, the use of the term ‘polypeptide’ in this disclosure encompasses oligopeptides, peptides, polypeptides and proteins, unless the context of use requires a different interpretation of that term.

[0054] The gonad stimulating hormone can be a peptide, polypeptide or protein, for example (hereafter a ‘gonad stimulating hormone polypeptide’, for ease of reference).

[0055] The gonad stimulating hormone / gonad stimulating hormone polypeptide can be post- translationally modified or not. Potential post-translational modifications include proteolytic cleavage, a C-terminal amide and/or an N-terminal pyro group.

[0056] Throughout this disclosure, the term ‘nucleic acid’ means a 'polynucleotide', 'oligonucleotide', or 'nucleic acid molecule', and generally means a polymer of DNA or RNA, which can be single stranded or double-stranded, synthesized or obtained (e.g., isolated and/or purified) from natural sources. Nucleic acids include nucleotide / polynucleotide sequences that comprise the bases A, G, C, T or U. [0057] Gonad stimulating hormones, such as gonadotropin releasing hormones, may include a terminal amide group. However, recombinantly produced gonadotropin releasing hormones may not include this terminal group, yet still be functional. Similarly, such hormones may also include an amino pyro group, which may not be present in the recombinant hormone. Accordingly, amino acid sequences described herein for gonad stimulating hormones, particularly gonadotropin releasing hormones, optionally include such modifications, unless explicitly excluded.

[0058] In some embodiments, the gonad stimulating hormone has a native amino acid sequence; that is, an amino acid sequence found in nature. In some embodiments, the gonad stimulating hormone has a non-native amino acid sequence; that is, an artificial, synthetic, nonnatural or mutated amino acid sequence not found in nature, a product of genetic engineering. [0059] In some embodiments, the gonad stimulating hormone is encoded by a native nucleotide sequence; that is, a nucleotide sequence found in nature. In some embodiments, the gonad stimulating hormone is encoded by a non-native nucleotide sequence; that is, a mutated or codon-optimised sequence not found in nature, a product of genetic engineering. Preferably, the nucleotide sequence or nucleic acid is codon optimised for expression in the algal host. Codon optimisation for the host alga may lead to a higher level of expression of the encoded gonad stimulating hormone.

[0060] Some gonad stimulating hormones are normally expressed as pre-proteins, proproteins, or pre-pro-proteins, and so may require processing to function as hormones. For example, GnRH may be expressed as a pre-pro-protein and may be processed to produce a hormone-active (decapeptide or similar) form. The gonad stimulating hormone can be in the form of a pre-protein or pre-pro-protein.

[0061] In some embodiments, the recombinant algae can express the gonad stimulating hormone in a hormone-active form, whereby it is capable of exerting its gonad stimulating effect from the time that it is expressed by the algae. In some embodiments, the recombinant algae can express the gonad stimulating hormone in a hormone-inactive form, whereby the inactive form is required to be processed in order to be able to exert its gonad stimulating effect. That is, the gonad stimulating hormone can be expressed by algae in a hormone-precursor form, whereby it lacks hormonal activity until further processed.

[0062] For example, the inactive hormone-precursor form can be converted to its active form: (i) when formulated as aquatic animal feed; (2) when fed to the aquatic animal; (3) within the gut or intestine of the aquatic animal; (4) within the bloodstream of the aquatic animal; (5) within the pituitary gland of the aquatic animal; (6) within the gonads of the aquatic animal, or, any one or more of the aforementioned (context permitting). Typically, this could involve one or more post-translation modifications such as proteolytic cleavage, addition of a C- terminal amide, and/or addition of an N-terminal pyro group.

[0063] In some embodiments, the gonad stimulating hormone / polypeptide comprises one or more proteolytic cleavage sequences, sites, motifs or domains. For example, a Gly-Lys-Arg (GKR) cleavage site may be present. This cleavage site may also enable amidation of a cleaved gonad stimulating hormone.

[0064] In some embodiments, the gonad stimulating hormone, either in the hormone-active form or in the hormone-precursor inactive form, can comprise, consist of, or essentially consist of a sequence, motif or domain that is capable of binding to or otherwise activating a hormone receptor (ie. a ‘hormone receptor-activating sequence’ or ‘hormone receptor-activating sequence polypeptide’). In some embodiments, the gonad stimulating hormone, either in the hormone-active form or in the hormone-precursor inactive form, can comprise the hormone receptor-activating sequence as well as at least one further peptide, polypeptide or protein sequence covalently bound to the N-terminus and/or C-terminus of the hormone receptoractivating sequence (ie. producing a ‘fusion protein’). In some embodiments, the gonad stimulating hormone can be a fusion protein. In some embodiments, the at least one further peptide, polypeptide or protein sequence can be a sequence, motif or domain that is normally found in the pre-protein, pro-protein or pre-pro-protein form of the gonad stimulating hormone. For example, in some embodiments, the sequence, motif or domain can be a signal peptide of the gonad stimulating hormone, or part or truncation thereof. For example, in some embodiments, the sequence, motif or domain can be a domain of the gonad stimulating hormone, or part or truncation thereof. For example, in some embodiments, the sequence, motif or domain can be the C-terminus of the gonad stimulating hormone, or part or truncation thereof.

[0065] In some embodiments, the at least one further peptide, polypeptide or protein sequence may not be a sequence, motif or domain that is normally found in the gonad stimulating hormone. That is, it may be exogenous. For example, in some embodiments, the sequence, motif or domain can be an exogenous signal peptide or part thereof, or an exogenous translation initiation sequence containing an initiation codon (Met). For example, in some embodiments, the sequence, motif or domain can be of exogenous origin and provide a particular function or functions, such as: greater polypeptide stability; greater expression; protection for reaching its hormone receptor; greater binding affinity to its hormone receptor; or, an ability to cross from the gut or intestine into the bloodstream, or from the bloodstream into the pituitary gland or gonads.

[0066] In some embodiments, the gonad stimulating hormone / polypeptide comprises one or more protein tags, which are peptide sequences added for various purposes. One or more protein tags can be added to the C-terminus (a ‘C-terminal tag’), N-terminus (an ‘N-terminal tag’), and/or internally (an ‘internal tag’). The tag can be, for example, one or more of: an affinity tag for purification using an affinity technique (eg. a strep-tag, or poly(His) tag); a solubilization tag to assist in the proper folding in proteins and keep them from aggregating in inclusion bodies (eg. thioredoxin); a chromatography tag for altering chromatographic properties of the protein (eg. FLAG-tag or polyglutamate tag); an epitope tag for high-affinity antibodies (eg. Myc-tag, HA-tag); a tag enabling specific enzymatic modification, chemical modification or coupling to another peptide, polypeptide or protein; or, a tag that enables removal or cleavage by specific proteolysis.

[0067] In some embodiments, the gonad stimulating hormone comprises one or more flexible linker sequences, motifs or domains. An example of a linker is a glycine repeat, such as G-G- G-G (SEQ ID NO: 1).

[0068] The gonad stimulating hormone, the gonad stimulating hormone polypeptide, the hormone receptor-activating sequence thereof, or the hormone receptor-activating sequence polypeptide thereof, can have any suitable amino acid sequence. It will typically depend on the type of aquatic animal to which the gonad stimulating hormone is fed. In the case of fish, preferably the gonad stimulating hormone has an amino acid sequence derived from a vertebrate and, more particularly, a fish or a bird. In certain embodiments, the gonad stimulating hormone can be derived from an invertebrate.

[0069] In some embodiments, the gonad stimulating hormone / the gonad stimulating hormone polypeptide comprises, consists essentially of, or consists of a gonad stimulating hormone / gonad stimulating hormone polypeptide sequence as shown or substantially as shown in any one of Figures 3, 5, 6, 9, 12, 17, 19 and 21, or elsewhere in this specification, such as in the Table of Sequence Listings, particularly any one of SEQ ID NOs: 2-85, 99, 101, 104, 105, 107, 109, and 113 to 118 (or hormone receptor-activating sequence thereof). In some embodiments, the gonad stimulating hormone / the gonad stimulating hormone polypeptide has characteristics as depicted in any one of Figures 4, 7, 8, 15, 16, 18 and 20.

[0070] Any suitable nucleic acid or nucleotide sequence can encode the gonad stimulating hormone. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the gonad stimulating hormone is expressed. In some embodiments, a nucleic acid or nucleotide sequence encodes a gonad stimulating hormone / gonad stimulating hormone polypeptide (or hormone receptor-activating sequence thereof) as listed in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-85, 99, 101, 104, 105, 107, 109 and 113 to 118. In some embodiments, the gonad stimulating hormone / gonad stimulating hormone polypeptide has an encoding nucleotide sequence as shown or substantially as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOs: 100, 103, 106, 108, 110, 111 and 112. In some embodiments, a nucleic acid or nucleotide sequence encodes a gonad stimulating hormone / gonad stimulating polypeptide as shown in the Figures, particularly any one of Figures 3, 4, 5, 6, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20 and 21. In some embodiments, a nucleic acid or nucleotide sequence encodes the gonad stimulating polypeptide and flanking expression control sequence(s) as shown or substantially as shown or depicted in the Figures, particularly any one of Figures 4, 6, 7, 8, 9 and 15. In some embodiments, a nucleic acid or nucleotide sequence encodes the gonad stimulating polypeptide and flanking expression control sequence(s) as shown or substantially as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 100, 103 and 112. In some embodiments, the expression cassette has a nucleotide sequence as shown or substantially as shown in Figure 6 or 9, or SEQ ID NO: 100 or 103. In some embodiments, the expression cassette is as shown or substantially as shown in any one of Figures 4, 6, 7, 8, 9 and 15.

[0071] By “substantially as shown”, with respect to a nucleotide or amino acid sequence, it is meant that the sequence can differ from that shown yet still perform the required function. The difference can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% or more of the bases/residues shown.

[0072] A. Gonadotropin Releasing Hormones

[0073] In some embodiments, the gonad stimulating hormone can comprise, consist essentially of, or consist of a gonadotrophin releasing hormone (GnRH) in either a hormone-active form or in a hormone precursor-inactive form. (Hereafter, a ‘GnRH receptor-activating hormone’ or ‘GnRH-based hormone’.) GnRH is a hormone that acts on the pituitary gland, and is responsible for the release of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In some embodiments, the GnRH-based hormone / the GnRH-based hormone polypeptide / the GnRH receptor-activating hormone / the GnRH receptor-activating hormone polypeptide comprises, consists essentially of, or consists of a GnRH peptide / GnRH receptoractivating sequence / GnRH receptor-activating sequence peptide having a sequence as shown in any one of Figures 3, 5, 6, 9, 12, 17, 19 and 21, or elsewhere in this specification, such as in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-81, 99, 101, 104, 105, 107, 109 and 113 to 115, or any other GnRH receptor-activating sequence that is capable of activating the GnRH receptor. In some embodiments, the GnRH receptor-activating hormone / the GnRH receptor-activating hormone polypeptide is in a hormone-active form comprising, consisting essentially of, or consisting of a sequence as shown in Figure 3 or elsewhere in this specification, such as in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-81, 99, 101, 104, 105, 107, 109 and 113-115, or any other GnRH receptor-activating sequence that is capable of activating the GnRH receptor. In some embodiments, the GnRH receptor-activating hormone / the GnRH receptor-activating hormone polypeptide is in a horm one-precursor inactive form comprising a sequence as shown in Figure 3 or elsewhere in this specification, such as in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-81, 99, 101, 104, 105, 107, 109 and 113 to 115, or any other GnRH receptor-activating sequence that is capable of activating the GnRH receptor.

[0074] For the sake of convenience, the GnRH peptides shown in Figure 3 and elsewhere herein may each be referred to as ‘GnRH’, ‘GnRH peptide’, ‘GnRH receptor-activating sequence’ or ‘GnRH receptor-activating sequence polypeptide’, context permitting.

[0075] The GnRH peptide can have any suitable amino acid sequence, whether naturally occurring (native), genetically altered, or post-translationally modified or not. The GnRH peptide can be GnRHl (GnRHI), GnRH2 (GnRHII), GnRH3 (GnRHIII), or a non-natural GnRH peptide, as shown in Figure 3 and elsewhere in this specification, such as in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-81, 99, 101, 104, 105, 107, 109 and 113 to 115, for example. Typically, the GnRH peptide is in the form of a functionally active decapeptide, although this need not be the case.

[0076] Certain forms of the GnRH peptide have the amino acid sequence of Formula (I):

[0077] Q - H - W - S - X ¹ - X ² - X ³ - X ⁴ - P - G, wherein:

[0078] X ¹ is a naturally occurring amino acid;

[0079] X ² is a naturally occurring amino acid;

[0080] X ³ is a naturally occurring amino acid; and

[0081] X ⁴ is a naturally occurring amino acid. (SEQ ID NO:2)

[0082] Note that the first amino acid Q (glutamine) can be deamidated via phosphate activated glutaminase to E (glutamic acid). Hence, either amino acid can be included as the first amino acid.

[0083] In another embodiment, the GnRH peptide has the amino acid sequence of Formula (I), wherein:

[0086] X ³ is L, N, M or W; and

[0087] X ⁴ is S, R, N, Y or L. (Formula (I)A) (SEQ ID NO:3)

[0088] In a further embodiment, the GnRH peptide has the amino acid sequence of Formula (I), wherein:

[0089] X ¹ is H, F, I or Y; [0090] X ² is G or D;

[0091] X ³ is L, N or M; and

[0092] X ⁴ is S, R or N. (Formula (I)B) (SEQ ID NO:4)

[0093] In one embodiment, the GnRH peptide has the amino acid sequence of Formula (I), wherein:

[0094] X ¹ is H or Y;

[0095] X ² is G;

[0096] X ³ is W; and

[0097] X ⁴ is Y or L. (Formula (I)C) (SEQ ID NO:5)

[0098] Certain forms of the GnRH peptide have the amino acid sequence of Formula (A):

[0099] Q - H - X ¹ - S - X ² - X ³ - X ⁴ - X ⁵ - P - G, wherein:

[0100] X ¹ is a naturally occurring amino acid;

[0101] X ² is a naturally occurring amino acid;

[0102] X ³ is a naturally occurring amino acid;

[0103] X ⁴ is a naturally occurring amino acid; and

[0104] X ⁵ is a naturally occurring amino acid. (SEQ ID NO:6)

[0105] Note that the first amino acid Q (glutamine) can be deamidated via phosphate activated glutaminase to E (glutamic acid). Hence, either amino acid can be included as the first amino acid.

[0106] In some embodiments, the GnRH peptide has the amino acid sequence of Formula (A), wherein:

[0107] X ¹ is W or Y;

[0108] X ² is H, F, L, or Y;

[0109] X ³ is G, or Q;

[0110] X ⁴ is L, M or W; and

[0111] X ⁵ is S, R, N, K or L. (Consensus Formula (A)l) (SEQ ID NO:7)

[0112] In a further embodiment, the GnRH peptide has the amino acid sequence of Formula (A), wherein:

[0113] X ¹ is W;

[0114] X ² is H;

[0115] X ³ is G;

[0116] X ⁴ is W; and

[0117] X ⁵ is Y. (Consensus Formula (A)2) (SEQ ID NO:8)

[0118] In another embodiment, the GnRH peptide has the amino acid sequence of Formula (A), wherein: [0119] X ¹ is W;

[0120] X ² is Y or H;

[0121] X ³ is G or D;

[0122] X ⁴ is W; and

[0123] X ⁵ is L or K. (Consensus Formula (A)3) (SEQ ID NO:9)

[0124] In one embodiment, the GnRH peptide has the amino acid sequence of Formula (A), wherein:

[0125] X ¹ is W;

[0126] X ² is Y;

[0127] X ³ is G, H or K;

[0128] X ⁴ is L; and

[0129] X ⁵ is R. (Consensus Formula (A)5) (SEQ ID NO: 10)

[0130] Certain forms of the GnRH peptide have the amino acid sequence of Formula (B):

[0131] Q - N - X ¹ - X ² - X ³ - S - X ⁴ - X ⁵ - X ⁶ - X ⁷ - P - G, wherein:

[0132] X ¹ is a naturally occurring amino acid;

[0133] X ² is a naturally occurring amino acid;

[0134] X ³ is a naturally occurring amino acid;

[0135] X ⁴ is a naturally occurring amino acid;

[0136] X ⁵ is a naturally occurring amino acid;

[0137] X ⁶ is a naturally occurring amino acid;

[0138] X ⁷ is a naturally occurring amino acid; and

[0139] the Q - N preceding X ¹ is optional. (SEQ ID NO: 11)

[0140] Note that the first amino acid Q (glutamine) can be deamidated via phosphate activated glutaminase to E (glutamic acid). Hence, either can be included as the first amino acid.

[0141] In one embodiment, the GnRH peptide has the amino acid sequence of Formula (B), wherein:

[0142] X ¹ is Q or Y;

[0143] X ² is Y or H;

[0144] X ³ is F or W;

[0145] X ⁴ is Y, D, L, N or K;

[0146] X ⁵ is G, K, A, E, Q, C or Y;

[0147] X ⁶ is V, L, F, H, Y or W; and

[0148] X ⁷ is H, A, S, M, T, K, W, Q or R. (Consensus Formula (B)4) (SEQ ID NO: 12)

[0149] In some embodiments, the GnRH peptide is derived from a marine animal, in particular, from a teleost fish such as salmon. [0150] Exemplary amino acid sequences for the GnRH peptide include those shown below.

[0151] Salmon: QHWSYGWLPG (SEQ ID NO: 113), optionally with an N-terminal pyro group.

[0152] Goldfish: QHWSHGWLPG (SEQ ID NO:72), optionally with a C-terminal amide. The first Q is interchangeable with E.

[0153] Whitefish: EHWSYGMNPG (SEQ ID NO:73), optionally with a C-terminal amide.

[0154] Catfish: EHWSHGLNPG (SEQ ID NO:74), optionally with a C-terminal amide.

[0155] Urochordata: HWSKGYSPG (SEQ ID NO:75), optionally with a C-terminal amide.

[0156] Octopus: NYHFSNGWHPG (SEQ ID NO:76), optionally with a C-terminal amide.

[0157] Tunicate: Q/EHWSDYFKPG (SEQ ID NO: 58; SED ID NO:77), optionally with an N- terminal pyro group, optionally with a C-terminal amide, or optionally with both an N-terminal pyro group and a C-terminal amide. The first E and Q are interchangeable.

[0158] Sea pineapple H. roretzr. QHWSYGFSPG (SEQ ID NO:78).

[0159] Sea pineapple H. roretzr. QHWSYGFLPG (SEQ ID NO: 79).

[0160] Note, as used herein, particularly in the Table of Sequence Listings “pyro” is an N- terminal pyro group”; and, “a” is a C-terminal amide.

[0161] In some embodiments, the GnRHII/GnRH2 peptide is derived from a chicken GnRH, having the (chicken-II) sequence: E/QHWSHGWYPG (SEQ ID: 114; SEQ ID: 115), optionally with an N-terminal pyro group, optionally with a C-terminal amide, or optionally with both an N-terminal pyro group and a C-terminal amide. As depicted, the first E can be a Q.

[0162] In some embodiments, the GnRH-based hormone / the GnRH receptor-activating hormone / polypeptide comprises the GnRH peptide and one or more of the following peptides, polypeptides or proteins (context permitting):

[0163] 1. at least one GnRH-associated peptide (‘G AP’ );

[0164] 2. a truncated GAP, preferably about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the residues of the at least one GAP;

[0165] 3 a cleavage site adjacent the -terminus of the GnRH peptide;

[0166] 4 a cleavage site adjacent the C-terminus of the GnRH peptide;

[0167] 5 a respective cleavage site adjacent the N-terminus and C-terminus of the GnRH peptide;

[0168] 6 at least one protein tag;

[0169] 7 a translation initiation sequence encoding Met;

[0170] 8 a translation termination sequence having a stop codon;

[0171] 9. at least one cleavage site for a protease;

[0172] 10. at least one flexible linker; [0173] 1 1 a signal peptide; and

[0174] 12 a truncated signal peptide, preferably about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the residues of the signal peptide.

[0175] Any suitable nucleic acid or nucleotide sequence can encode the GnRH-based hormone / GnRH receptor-activating hormone / GnRH-based hormone. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the GnRH receptor-activating hormone is expressed. In some embodiments, a nucleic acid or nucleotide sequence encodes the GnRH receptor-activating hormone / GnRH receptor-activating hormone polypeptide of any one of Figures 3, 5, 6, 9, 12, 17, 19 and 21. In some embodiments, a nucleic acid or nucleotide sequence encodes the GnRH receptor-activating hormone / GnRH receptoractivating hormone polypeptide as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 2-81, 99, 101, 104, 105, 107, 109 and 113 to 115.

[0176] In some embodiments, the GnRH receptor-activating hormone / GnRH receptoractivating hormone polypeptide has a nucleotide sequence as shown or substantially as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOs: 100, 103, 106, 108, 110, 111 and 112. In some embodiments, a nucleic acid or nucleotide sequence encodes the GnRH receptor-activating hormone / GnRH receptor-activating hormone polypeptide shown in the Figures, particularly any one of Figures 3, 4, 5, 6, 7, 8, 9, 12, 15, 16, 17, 18, 19, 20 and 21.

[0177] In some embodiments, a nucleic acid or nucleotide sequence encodes the GnRH receptor-activating hormone polypeptide and flanking expression control sequence or expression control sequences as shown or substantially as shown or depicted in the Figures, particularly any one of Figures 4, 6, 7, 8, 9 and 15. In some embodiments, a nucleic acid or nucleotide sequence encodes the GnRH receptor-activating hormone polypeptide and flanking expression control sequence or expression control sequences, as shown or substantially as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOS: 100, 103 and 112. In some embodiments, the expression cassette comprises a GnRH receptor-activating hormone and has a nucleotide sequence as shown or substantially as shown in Figure 6 or 9, or SEQ ID NO: 100 or 103. In some embodiments, the expression cassette comprising a GnRH receptor-activating hormone is as shown or substantially as shown in any one of Figures 4, 6, 7, 8, 9 and 15.

[0178] B. Gonadotropins

[0179] In some embodiments, the gonad stimulating hormone / polypeptide can be or can have the function of a gonadotrophin, such as a follicle stimulating hormone (FSH), a luteinizing hormone (LH), a relaxin-like gonad-stimulating substance, or a gonad-stimulating substance. In some embodiments, the gonad stimulating hormone /polypeptide can comprise, consist essentially of, or consist of a gonadotrophin, such as a follicle stimulating hormone (FSH), a luteinizing hormone (LH), a relaxin-like gonad-stimulating substance, a gonad-stimulating substance, or one or more subunits or polypeptide chains thereof, or a hormone receptoractivating sequence or hormone receptor-activating sequence polypeptide thereof.

[0180] 1. Gonad-Stimulating Substance

[0181] In certain embodiments, the gonad stimulating hormone has a sequence derived from an invertebrate, for example, a relaxin-like gonad-stimulating substance / polypeptide (RGP) from echinoderms (Mita M. Relaxin-like gonad-stimulating substance in an echinoderm, the starfish: a novel relaxin system in reproduction of invertebrates. Gen Comp Endocrinol. 2013 Jan 15; 181 :241-5. doi: 10.1016/j.ygcen.2012.07.015) (SEQ ID NO:81), or a gonad-stimulating substance / polypeptide from oysters. (See, e.g., Vu Van In, Nikoleta Ntalamagka, Wayne O’Connor, Tianfang Wang, Daniel Powell, Scott F. Cummins, Abigail Elizur, Reproductive neuropeptides that stimulate spawning in the Sydney Rock Oyster (Saccostrea glomerata), Peptides, Volume 82, 2016, Pages 109-119, ISSN 0196-9781, https://doi.Org/10.1016/j.peptides.2016.06.007) (see oyster SEQ ID NOs: 116, 117 and 118). The starfish (Patina pectinifera) relaxin-like gonad-stimulating substance / polypeptide consists of identical A and B chains/subunits (see SEQ ID NO: 81).

[0182] In some embodiments, the gonad stimulating hormone comprises, consists essentially of, or consists of a relaxin-like gonad-stimulating peptide (RGP), or a hormone receptoractivating sequence or hormone receptor-activating sequence polypeptide thereof. (Hereafter, a ‘RGP -based hormone’.)

[0183] The RGP -based hormone / RGP -based hormone polypeptide can have any suitable amino acid sequence. It will typically depend on the type of aquatic animal to which the RGP- based hormone is fed. The RGP -based hormone / RGP -based hormone polypeptide can have an amino acid sequence as shown in the Table of Sequence Listings, particularly SEQ ID NO:81, or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof.

[0184] Any suitable nucleic acid or nucleotide sequence can encode the RGP -based hormone / RGP -based hormone polypeptide. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the RGP -based hormone is expressed. In some embodiments, a nucleic acid or nucleotide sequence encodes the RGP -based hormone / RGP -based hormone polypeptide as shown in the Table of Sequence Listings, particularly SEQ ID NO:81, or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof. [0185] In some embodiments, the gonad stimulating hormone comprises, consists essentially of, or consists of a gonad stimulating substance (GSS), or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof. (Hereafter, a ‘GSS- based hormone’.)

[0186] The GSS-based hormone / GSS-based hormone polypeptide can have any suitable amino acid sequence. It will typically depend on the type of aquatic animal to which the GSS- based hormone is fed. The GSS-based hormone / GSS-based hormone polypeptide can have an amino acid sequence as shown in the Table of Sequence Listings, particularly any one of SEQ ID NOs: 116, 117 and 118, or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof.

[0187] Any suitable nucleic acid or nucleotide sequence can encode the GSS-based hormone / GSS-based hormone polypeptide. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the GSS-based hormone is expressed. In some embodiments, a nucleic acid or nucleotide sequence encodes the GSS-based hormone / GSS-based hormone polypeptide as shown in the Table of Sequence Listings, particularly SEQ ID NOs: 116, 117 and 118, or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof.

[0188] 2. Follicle Stimulating Hormone

[0189] In some embodiments, the gonad stimulating hormone comprises, consists essentially of, or consists of a follicle stimulating hormone (FSH), or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof. (Hereafter, a ‘FSH- based hormone’.)

[0190] FSH in most vertebrates is a 35.5 kDa glycoprotein heterodimer, comprising two polypeptide units, alpha and beta. The alpha subunit has 96 amino acids in most vertebrates. Beta subunits vary in size and can be about 111 amino acids. The follicle-stimulating hormone alpha subunit of Danio rerio has the identifier NCBI Gene ID: 402987 (SEQ ID NO:82). The follicle-stimulating hormone beta subunit of Danio rerio has the identifier NCBI Gene ID: 402919 (SEQ ID NO:83).

[0191] The FSH-based hormone / FSH-based hormone polypeptide can have any suitable amino acid sequence. It will typically depend on the type of aquatic animal to which the FSH- based hormone is fed. The FSH-based hormone / FSH-based hormone polypeptide, can have the amino acid sequences as shown in the Table of Sequence Listings, particularly SEQ ID NOs:82 and 83, or a respective hormone receptor-activating sequence or hormone receptoractivating sequence polypeptide thereof. Both alpha and beta subunits of FSH, or respective hormone receptor-activating sequence thereof, can be expressed by the algae in the same expression construct or in separate expression constructs.

[0192] Any suitable nucleic acid(s) or nucleotide sequence(s) can encode the subunits of the FSH-based hormone / FSH-based hormone polypeptide, or respective hormone receptoractivating sequence thereof. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the FSH-based hormone is expressed. In some embodiments, one or more nucleic acids or nucleotide sequences encode the FSH-based hormone subunits / FSH-based hormone polypeptide subunits as shown in the Table of Sequence Listings, particularly SEQ ID NOs:82 and 83, or a respective hormone receptoractivating sequence or hormone receptor-activating sequence polypeptide thereof.

[0193] 3. Luteinizing Hormone

[0194] In some embodiments, the gonad stimulating hormone comprises, consists essentially of, or consists of a luteinizing hormone (LH), or a hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof. (Hereafter, an ‘LH-based hormone’.)

[0195] LH is a heterodimeric glycoprotein comprising an alpha subunit and a beta subunit. The alpha subunit has 96 amino acids in most vertebrates and is the same subunit that makes FSH. The beta subunit varies in size based on species and is about 120 amino acids. The LH alpha subunit of Danio rerio has the identifier NCBI Gene ID: 402987 (SEQ ID NO: 84). The LH beta subunit of Danio rerio has the identifier NCBI Gene ID: 402917 (SEQ ID NO:85). As mentioned, the alpha subunits of LH and FSH are identical.

[0196] The LH-based hormone / LH-based hormone polypeptide can have any suitable amino acid sequence. It will typically depend on the type of aquatic animal to which the LH-based hormone is fed. The LH-based hormone / LH-based hormone polypeptide, can have the amino acid sequences as shown in the Table of Sequence Listings, particularly SEQ ID NOs:84 and 85, or a respective hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof. Both alpha and beta subunits of LH, or respective hormone receptor-activating sequence thereof, can be expressed by the algae in the same expression construct or in separate expression constructs.

[0197] Any suitable nucleic acid(s) or nucleotide sequence(s) can encode the subunits of the LH-based hormone / LH-based hormone polypeptide, or respective hormone receptoractivating sequence thereof. In some embodiments, the nucleic acid or nucleotide sequence is codon optimised for the algal host in which the LH-based hormone is expressed. In some embodiments, one or more nucleic acids or nucleotide sequences encode the LH-based hormone subunits / LH-based hormone polypeptide subunits as shown in the Table of Sequence Listings, particularly SEQ ID NOs: 84 and 85, or respective hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof.

[0198] III. Vectors and Expression Systems

[0199] Recombinant gonad stimulating hormones / polypeptides are expressed through expression constructs comprising an expression control sequence(s) operatively linked with the nucleotide / polynucleotide sequence(s) encoding the particular gonad stimulating hormone(s). Such expression constructs can be incorporated into algae through the use of an appropriate vector(s).

[0200] A. Expression Constructs /Expression Cassettes

[0201] An expression construct /expression cassette includes a polynucleotide / nucleotide comprising an expression control sequence operatively linked with a heterologous nucleotide sequence (i.e., a sequence to which the expression control sequence is not normally connected to in nature) that is to be the subject of expression. An expression vector includes a polynucleotide / nucleotide comprising an expression construct/cassette and sequences sufficient for replication in a host cell or insertion into a host chromosome or genome. However, the expression vector can replicate without insertion into a chromosome of the host cell. That is, it can remain as an extra-chromosomal element. Plasmids and viruses are examples of expression vectors. The term “expression control sequence” generally refers to a nucleotide sequence that regulates transcription and/or translation of a nucleotide sequence operatively linked thereto. Exemplary expression control sequences include promoters, enhancers, repressors (transcription regulatory sequences) and ribosome binding sites (translation regulatory sequences).

[0202] In another embodiment the expression control sequence is inducible, for example, photo inducible.

[0203] A nucleotide sequence is “operatively linked” to an expression control sequence when the expression control sequence functions in a cell to regulate transcription of the nucleotide sequence. This includes promoting transcription of the nucleotide sequence through an interaction between a polymerase and a promoter.

[0204] The nucleic acid encoding a gonad stimulating hormone disclosed herein includes any nucleotide sequence encoding the prescribed amino acid sequence. This includes any plurality of nucleotide sequences that encode the same amino acid sequence due to the degeneracy of the genetic code. Preferably, the nucleotide sequence encoding the gonad stimulating hormone(s) is codon optimised for the algal host.

[0205] In nature, gonadotropin releasing hormones are often produced as part of a larger polypeptide which is cleaved to produce the operational peptide / hormone receptor-activating sequence. However, in some embodiments, expression constructs / cassettes of the present disclosure do not require extra amino acid sequences; for example, a gonadotropin-releasing hormone can be expressed as a decapeptide or a 11- or 12- amino acid residue peptide, for example.

[0206] B. Vectors

[0207] A vector / expression vector typically comprises an expression construct / expression cassette. Vectors / expression vectors comprise any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and/or eukaryotic cells and/or integrated into a genome or chromosome. They include, without limitation, plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, lentiviral vectors, Adeno Associated viral vectors and the like. The term "plasmid" as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA. In certain embodiments the plasmid may contain sequences for integration into a host chromosome or genome, such as a chloroplast or nuclear genome.

[0208] “ Transfection” refers to the introduction of new genetic material into a cell. It includes transformation (the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane), transduction (the introduction of foreign DNA by a bacteriophage virus into a host cell) and conjugation.

[0209] In some embodiments, the expression vector comprises a polynucleotide operably linked to one or more control elements, such as a promoter and/or a transcription terminator. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operatively linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, operably linked sequences are contiguous and, in the case of a secretory leader, contiguous and in reading phase. Linking is often achieved by ligation at restriction enzyme sites. If suitable restriction sites are not available, then synthetic oligonucleotide adapters or linkers can be used as is known to those skilled in the art. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, (1989) and Ausubel et al., Short Protocols in Molecular Biology, 2nd Ed., John Wiley & Sons (1992).

[0210] The expression construct / expression cassette is contained in a vector suitable for transformation of algal (host) cells, e.g., microalgae. Appropriate vectors include, without limitation, plasmids and viral vectors. The vectors can target the nucleus or the chloroplast of the cell.

[0211] Plasmids are useful for targeting the chloroplast of algal cells. One such system is described by Wang et al. (Wang K, Cui Y, Wang Y, Gao Z, Liu T, Meng C, Qin S. Chloroplast Genetic Engineering of a Unicellular Green Alga Haematococcus pluvialis with Expression of an Antimicrobial Peptide. Mar Biotechnol (NY). 2020 Aug;22(4):572-580. doi: 10.1007/sl0126-020-09978-z. Epub 2020 Jun 13. PMID: 32535692). A target gene to be expressed can be operatively linked to an rbcL promoter. The plasmid can include sequences that target chloroplast or nuclear DNA sequences for incorporation into the host chromosome. For example, Wang et al. describe use of 16S-trnI, psbA promoter, bar, rcbL terminator, rcbL promoter, anti, psbA terminator, and trnA-23S sequences to target the plasmid for incorporation into the chloroplast genome of H. pluvialis. In other embodiments, the target gene can be placed under transcriptional control of a ribosomal RNA promoter such as the 35S promoter.

[0212] In another embodiment the geminiviral vector pBYR2e can drive the expression of transgenes under the control of the 35S promoter along with the suppressor P19 RNA silencing gene, allowing for higher protein expression. Recombinant proteins expressed with this system in the above algae species have been reported to produce proteins ranging from 0.43 to 3.6% of the total soluble protein (TSP) within the algae. (See, e.g., Malla A, Rosales-Mendoza S, Phoolcharoen W ., Vimolmangkang S. Efficient Transient Expression of Recombinant Proteins Using DNA Viral Vectors in Freshwater Microalgal Species. Front Plant Sci. 2021 Apr 7;12:650820. doi: 10.3389/fpls.2021.650820. PMID: 33897742; PMCID: PMC8058379).

[0213] In some embodiments, the expression elements can be selected from one or more of: an atpA promoter, an atpA 5’ untranslated region, a 16S promoter, an rbcL 5’ untranslated region, and, an rbcL 3’ untranslated region.

[0214] In some embodiments, the expression construct includes homologous recombination sequences which integrate the expression cassette in a site-specific manner between the psbA and rrn5 intergenic region of an algae chloroplast. (See Wipawee Dejtisakdi, Stephen M. Miller, Overexpression of Calvin cycle enzyme fructose 1,6-bisphosphatase in Chlamydomonas reinhardtii has a detrimental effect on growth, Algal Research, Volume 14, 2016, Pages 116-126, ISSN 2211-9264, https://doi.Org/10.1016/j.algal.2016.0L003).

[0215] Currently, three main methods are used for microalgae chloroplast transformation as follows: biolistic method, glass bead method, and electroporation. Wang et al. describes use of the biolistic method.

[0216] IV. Recombinant Algae Producing Gonad Stimulating Hormones

[0217] Provided herein are recombinant algae that express gonad stimulating hormones / polypeptides. Algal cells comprise at least one expression construct / expression cassette comprising at least one expression control sequence operably linked with at least one nucleotide sequence encoding at least one gonad stimulating hormone (or subunit thereof). Such expression constructs can be integrated into a chromosome of an algal cell, including a chloroplast chromosome or a nuclear chromosome.

[0218] Preferably, the (expression) vector targets the algal chloroplast genome, such that the expression construct / cassette is integrated into the chloroplast genome.

[0219] In the case of gonadotrophins such as follicle stimulating hormone (FSH) and luteinizing hormone (LH), each of these hormones comprises alpha and beta subunits. Both subunits of FSH (or respective hormone receptor-activating sequence thereof) can be expressed in the algal cells by way of the same expression construct / cassette, or separate expression constructs / cassettes. Both subunits of LH (or respective hormone receptor-activating sequence thereof) can be expressed in the algal cells by way of the same expression construct / cassette, or separate expression constructs / cassettes. If expressed together, they may be separated using a linker and/or cleavage sequence, as discussed elsewhere in this disclosure.

[0220] These subunits, once expressed, can combine to form the relevant hormone - FSH or LH, or hormone receptor-activating sequence. This also applies to relaxin-like gonadstimulating substances and other gonad-stimulating substances comprising different subunits.

[0221] Gonad stimulating hormones, including GnRH and gonadotrophins expressed in recombinant algae and orally delivered to aquatic animals using the methods described here, can cross into the bloodstream and exert hormonal effects in the target tissue(s) / organs of aquatic animals.

[0222] In certain embodiments, the alga (singular) or algae (plural) comprise microalgae. Microalgae are distinguished from macroalgae, such as seaweed or kelp (brown algae).

[0223] A. Exemplary Microalgae

[0224] Non-limiting examples of genera of microalgae that may be used to produce gonad stimulating hormones include, without limitation, Cyanophyta, Prochlorophyta, Rhodophyta, Chlorophyta, Heterokontophyta, Tribophyta, Glaucophyta, Chlorarachniophytes, Euglenophyta, Euglenoids, Haptophyta, Chrysophyta, Cryptophyta, Cryptomonads, Dinophyta, Dinoflagellata, Pyrmnesiophyta, Bacillariophyta, Xanthophyta, Eustigmatophyta, Raphidophyta, Phaeophyta, and Phytoplankton. A microalga may also be a microalgae species including, but not limited to, Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis salina, Nannochloropsis occulata, Scenedesmis dimorphus, Scenedesmus obliquus, Dunaliella tertiolecta, or Haematococcus pluvialis.

[0225] Additional non-limiting examples of microalgae species that can be used with the methods of the present disclosure include: Achnanthes orientalis, Agmenellum spp., Amphiprora hyaline, Amphora coffeiformis, Amphora coffeiformis var. linea, Amphora coffeiformis var. punctata, Amphora coffeiformis var. taylori, Amphora coffeiformis var. tennis, Amphora delicatissima, Amphora delicatissima var. capitata, Amphora sp., Anabaena, Ankistrodesmus, Ankistrodesmus falcatus, Boekelovia hooglandii, Borodinella sp., Botryococcus braunii, Botryococcus sudeticus, Bracteococcus minor, Bracteococcus medionucleatus, Carteria, Chaetoceros gracilis, Chaetoceros muelleri, Chaetoceros muelleri var. subsalsum, Chaetoceros sp., Chlamydomas perigranulata, Chlor ella anitrata, Chlor ella antarctica, Chlorella aureoviridis, Chlorella Candida, Chlorella capsulate, Chlorella desiccate, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolate, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. actophila, Chlorella infusionum var. auxenophila, Chlorella kessleri, Chlorella lobophora, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella ovalis, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella protothecoides var. acidicola, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris fo. tertia, Chlorella vulgaris var. autotrophica, Chlorella vulgaris var. viridis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris fo. tertia, Chlorella vulgaris var. vulgaris fo. viridis, Chlorella xanthella, Chlorella zofmgiensis, Chlorella trebouxioides, Chlorella vulgaris, Chlorococcum infusionum, Chlorococcum sp., Chlorogonium, Chroomonas s^.,Chrysosphaera sp., Cricosphaera sp., Crypthecodinium cohnii, Cryptomonas sp., Cyclotella cryptica, Cyclotella meneghiniana, Cyclotella sp., Dunaliella sp., Dunaliella bardaw il, Dunaliella bioculata, Dunaliella granulate, Dunaliella maritime, Dunaliella minuta, Dunaliella parva, Dunaliella peircei, Dunaliella primolecta, Dunaliella salina, Dunaliella terricola, Dunaliella tertiolecta, Dunaliella viridis, Dunaliella tertiolecta, Eremosphaera viridis, Eremosphaera sp., Ellipsoidon sp., Euglena spp., Franceia sp., Fragilaria crotonensis, Fragilaria sp., Gleocapsa sp., Gloeothamnion sp., Haematococcus pluvialis, Hymenomonas sp., Isochrysis aff galbana, Isochrysis galbana, Lepocinclis, Micractinium, Micractinium, Monoraphidium minutum, Monoraphidium sp., Nannochloris sp., Nannochloropsis salina, Nannochloropsis sp., Navicula acceptata, Navicula biskanterae, Navicula pseudotenelloides, Navicula pelliculosa, Navicula sapr ophila, Navicula sp., Nephrochloris sp., Nephroselmis sp., Nitschia communis, Nitzschia alexandrine, Nitzschia closterium, Nitzschia communis, Nitzschia dissipata, Nitzschia frustulum, Nitzschia hantzschiana, Nitzschia inconspicua, Nitzschia intermedia, Nitzschia microcephala, Nitzschia pusilia, Nitzschia pusilia elliptica, Nitzschia pusilia monoensis, Nitzschia quadrangular, Nitzschia sp., Ochromonas sp., Oocystis parva, Oocystis pusilia, Oocystis sp., Oscillatoria limnetica, Oscillatoria sp., Oscillatoria subbrevis, Parachlor ella kessleri, Pascheria acidophila, Pavlova sp., Phaeodactylum tricomutum, Phagus, Phormidium, Platymonas sp., Pleurochrysis camerae, Pleurochrysis dentate, Pleurochrysis sp., Prototheca wickerhamii, Prototheca stagnora, Prototheca portoricensis, Prototheca moriformis, Prototheca zopfii, Pseudochlorella aquatica, Pyramimonas sp., Pyrobotrys, Rhodococcus opacus, Sarcinoid chrysophyte, Scenedesmus armatus, Schizochytrium, Spirogyra, Spirulina platensis, Stichococcus sp., Synechococcus sp., Synechocystisf, Tagetes erecta, Tagetes patula, Tetraedron, Tetraselmis sp., Tetraselmis suecica, Thalassiosira weissflogii, and Viridiella fridericiana.

[0226] In one embodiment, the preferred species of algae is Haematococcus, Chlorella, Chlamydomonas, Nannochloropsis, Porphyridium, o Phaeodactylum.

[0227] Even more preferably, the microalgal species is Haematococcus pluvailis, Chlamydomonas reinhardtii, Chlorella vulgari, Porphyridium purpureum, or Phaeodactylum tricornutum, most preferably Chlamydomonas reinhardtii.

[0228] B. Production of Gonad Stimulating Hormones

[0229] The algae that may be used to produce gonad stimulating hormones can be grown on land, for example, in ponds, aqueducts, or in closed or partially closed bioreactor systems. The algae can also be grown directly in water, for example, in an ocean, sea, lake, river, reservoir, and the like. The algae may be grown in culture systems of different volumes. In one embodiment, the algae can be grown, for example, in small scale laboratory systems. Small scale laboratory systems refer to cultures in volumes of less than about 6 liters. In an embodiment, the small-scale laboratory culture may be 1 liter, 2 liters, 3 liters, 4 liters, or 5 liters. In another embodiment, the small-scale laboratory culture may be less than one liter. In yet another embodiment, the small-scale laboratory culture may be 100 milliliters or less. In one embodiment the culture may be 10 milliliter or less. In another embodiment the culture may be 5 milliliters or less.

[0230] Alternatively, the culture systems may be large scale cultures, where large scale cultures refer to growth of cultures in volumes of greater than about 6 liters, or greater than about 10 liters, or greater than about 20 liters. Large scale growth can also be growth of cultures in volumes of 50 liters or more, 100 liters or more, or 200 liters or more. Large-scale growth can be growth of cultures in, for example, ponds, containers, vessels, or other areas, where the pond, container, vessel, or area that contains the culture is for example, at least 5 square meters, at least 10 square meters, at least 200 square meters, at least 500 square meters, at least 1,500 square meters, at least 2,500 square meters, in area, or greater.

[0231] The present disclosure further provides for production of gonad stimulating hormones in very large-scale culture systems. A very large-scale liquid culture system may be from 10,000 to 20,000 liters. In an embodiment, the very large-scale culture system may be from 10,000 to 40,000 liters or from 10,000 to 80,000 liters. In another embodiment, the very large- scale culture system may be from 10,000 to 100,000 liters or from 10,000 to 150,000 liters. In yet another embodiment, the culture system may be from 10,000 to 200,000 liters or from 10,000 to 250,000 liters. The present disclosure also includes culture systems from 10,000 to 500,000 liters or from 10,000 to 600,000 liters. The present disclosure further provides for culture systems from 10,000 to 1,000,000 liters.

[0232] In embodiments of the present disclosure, the culture system may be a pond, either natural or artificial. In one embodiment, the artificial pond may be a raceway pond. In a raceway pond, the algae, water, and nutrients circulate around a “racetrack.” Means of motivation, such as paddlewheels, provide constant motion to the liquid in the racetrack, allowing for the organism to be circulated back to the surface of the liquid at a chosen frequency. Paddlewheels also provide a source of agitation and oxygenate the system. CO2 may be added to a culture system as a feedstock for photosynthesis through a CO2 injection system. These raceway ponds can be enclosed, for example, in a building or a greenhouse, or can be located outdoors. In an embodiment, an outdoor raceway culture system may be enclosed with a cover, or exposed.

[0233] Alternatively, microalgae producing gonad stimulating hormones can be grown in closed structures such as bioreactors, where the environment is under stricter control than in open systems or semi-closed systems. A photobioreactor is a bioreactor which incorporates some type of light source to provide photonic energy input into the reactor. The term bioreactor can refer to a system closed to the environment and having no direct exchange of gases and contaminants with the environment. A bioreactor can be described as an enclosed, and in the case of a photobioreactor illuminated, culture vessel designed for controlled biomass production of liquid cell suspension cultures. Examples of bioreactors include, for example, glass containers, stainless steel containers, plastic tubes, tanks, plastic sleeves, and bags. In the case of photobioreactors, examples of light sources that can be used include, for example, fluorescent bulbs, LEDs, and natural sunlight. Because these systems are closed everything that the organism needs to grow (for example, carbon dioxide, nutrients, water, and light) must be introduced into the bioreactor. [0234] In a preferred embodiment, the bioreactor utilizes the control system described in WO 2020/082119 (Spanton), the entire contents of which are incorporated herein. Briefly, the control system provides a controller for controlling a plurality of LED lights, wherein the controller is adapted to control the spectrum and intensity of the LED lights. The controller is adapted to control the flickering frequency of the LED lights.

[0235] The algae used in the methods of the present disclosure are genetically modified to produce recombinant gonad stimulating hormone. Because algae do not naturally produce gonad stimulating hormone (such as GnRH-based hormones), for the purposes of this disclosure, all of the gonad stimulating hormone produced by algae in the present disclosure are considered to be recombinant.

[0236] V. Aquaculture

[0237] Animals whose sexual maturation or spawning is to be regulated by the methods described herein typically will be raised in aquaculture conditions. For example, animals can be raised in tanks, ponds or in enclosed areas in large bodies of water such as oceans or lakes. The collection of animals to be treated can be a plurality of animals including at least 10, at least 100 at least 1000 or at least 10,000. Animals for whom sexual maturation is sought typically are sexually immature. Broodstock fish are those that are designated for seed production. The mature broodstock will be treated with gonad stimulating hormone (such as GnRH-based hormones) either as an injectable or via oral delivery. Broodstock, unlike production fish, are maintained at lower density (of about 4 kg/cubic meter) and fed high quality food.

[0238] VI. Induction / Promotion of Sexual Maturation

[0239] Provided herein are methods of inducing / promoting sexual maturation in an aquatic or marine animal. In particular, provided herein are methods of collectively inducing / promoting sexual maturation in a plurality of aquatic or marine animals such that sexual maturation time of the animals is synchronized. Sexual maturation is considered synchronized if a majority of the animals reach sexual maturity within a span of about one month. Such methods comprise feeding the animals recombinant algae producing one or more gonad stimulating hormones as disclosed herein for period of time sufficient for the animals to achieve sexual maturity. Because the animals eat the algae, the gonad stimulating hormones are administered through an oral route. Sexual maturation includes undergoing pubertal development, gonad maturation, ovulation, and spermiation.

[0240] Animals are fed a diet comprising recombinant algae expressing gonad stimulating hormones.

[0241] Feeding can involve depositing recombinant algae into the water where the animals are being kept. In some embodiments, the recombinant algae are in the form of dewatered or dried biomass. In some embodiments, the recombinant algae are frozen or freeze-dried. In some embodiments, the recombinant algae are dewatered or dried and then formulated into aquatic animal feed, such as fish pellets or wafers. This may involve combining dewatered or dried recombinant algae biomass with one or more ingredients used in making aquatic animal feed. In some embodiments, the recombinant algae are frozen or freeze-dried and then formulated into aquatic animal feed, such as fish pellets or wafers. This may involve combining frozen or freeze-dried recombinant algae biomass with one or more ingredients used in making aquatic animal feed.

[0242] Because the gonad stimulating hormones are thought protectable by the recombinant algal cells / biomass, they can reach the intestine of fish or other animals and be absorbed into the bloodstream. The amount of recombinant algae provided to the animals depends on the particular animal and can be determined by the practitioner based, at least in part, on the amount of gonad stimulating hormone expressed by the recombinant algae. In the case of bony fish, the amount of hormone to be ingested can be about 50 pg to about 100 pg hormone (e.g., GnRH-based hormones) per kg of the aquatic animal, including all numerical values between 50 to 100 and sub-ranges therein. In the case of sexual maturation, the recombinant algae will be administered, typically, over the course of weeks or months. For example, the recombinant algae can be administered for between one week and four months, e.g., between one week and four weeks, between one month and four months (including all sub-ranges therein) or throughout the life of the animals.

[0243] VII. Induction / Promotion of Spawning

[0244] Provided herein also are methods of inducing / promoting spawning in a marine or aquatic animal, that is, the release of eggs or sperm from the animal. In particular, provided herein are methods of collectively inducing / promoting spawning in a plurality of marine or aquatic animals such that spawning time of the animals is synchronized. Such methods comprise feeding the animals recombinant algae producing one or more gonad stimulating hormones as disclosed herein for a period of time sufficient for the animals to achieve final oocyte maturation, ovulation and spawning.

[0245] Animals typically will be raised in aquaculture and in numbers as described above. Animals for whom spawning is sought typically are sexually mature. However, in some methods the process may combine induction / promotion of sexual maturation followed by induction / promotion of spawning.

[0246] Animals are fed a diet comprising recombinant algae expressing gonad stimulating hormones. The amount of recombinant algae provided to the animals depends on the particular animal and can be determined by the practitioner based, at least in part, on the amount of gonad stimulating hormone(s) expressed by the recombinant algae. In the case of bony fish, for oral delivery, the amount of gonad stimulating hormone to induce / promote spawning can be about 500 pg to about 1 mg or to about 6 mg gonad stimulating hormone (e.g., GnRH-based hormone) per kg fish, including all numerical values between 500 pg to 6 mg and sub-ranges. In the case of spawning, the recombinant algae will be administered, typically, over the course of days or weeks (including all sub-ranges therein). For example, the recombinant algae can be administered for between one day and four weeks, e.g., between one day and seven days, or between one week and four weeks (including all sub-ranges therein), depending on the biology of the fish, for example, whether the fish is a synchronous or asynchronous spawner.

[0247] VIII. Recombinant Gonad Stimulating Hormone

[0248] Some embodiments provide for an alga- or algae-derived recombinant, isolated or purified gonad stimulating hormone. The term “isolated” requires that the gonad stimulating hormone be removed from its original environment, in this case being the algal biomass or most of the algal biomass. The term “purified” does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. The term “purified” is further used herein to describe the gonad stimulating hormone which has been separated from other cellular components of algae including, but not limited to, polypeptides or polynucleotides, carbohydrates, lipids, etc. [0249] The term “recombinant” refers to (i) molecules that are constructed outside living cells by joining natural or synthetic nucleic acid segments to nucleic acid molecules that can replicate in a living cell, or (ii) molecules that result from the replication of those described in (i) above. For purposes herein, the replication can be in vitro replication or in vivo replication.

[0250] IX. Aquatic Animal Feed and Compositions

[0251] Some embodiments provide for aquatic animal feed or compositions comprising recombinant gonad stimulating hormone or recombinant algae biomass comprising expressed gonad stimulating hormone. Any suitable form of animal feed or composition can be used.

[0252] In some embodiments, the feed or composition comprises dewatered or dried recombinant algae biomass. In some embodiments, the feed or composition comprises frozen or freeze-dried recombinant algae biomass. In some embodiments, the feed or composition comprises dewatered or dried recombinant algae biomass formulated into animal feed, such as pellets or wafers. In some embodiments, the feed or composition comprises dewatered or dried recombinant algae biomass combined with one or more ingredients typically used in making aquatic animal feed. In some embodiments, the feed or composition comprises frozen or freeze- dried recombinant algae biomass formulated into animal feed, such as pellets or wafers. In some embodiments, the feed or composition comprises frozen or freeze-dried recombinant algae biomass combined with one or more ingredients typically used in making aquatic animal feed or other compositions. Typical ingredients include one or more of various meal (eg. fish meal, blood meal, canola meal, soybean meal), fats and oils, cereals, carbohydrates, proteins, amino acids, antioxidants, salt, vitamins, minerals, binders / binding agents, flavours, humectants, preservatives, colours, food acids, stabilisers, emulsifiers, gelling agents, and thickeners.

[0253] In some embodiments, the feed or composition comprises algae-derived recombinant, isolated or purified gonad stimulating hormone combined with one or more ingredients typically used in making aquatic animal feed, such as pellets or wafers, or other compositions (eg. as explained in the preceding paragraphs).

[0254] EXEMPLARY EMBODIMENTS

[0255] Exemplary embodiments are described the numbered paragraphs below.

[0256] 1. A method of inducing sexual maturity in one or more sexually immature aquatic animals, comprising feeding the one or more animals algae that express a hormone that promotes sexual maturity.

[0257] 2. A method of collective spawning induction in a collection of sexually mature aquatic animals, comprising feeding the animals an effective amount of algae that express a hormone that promotes spawning.

[0258] 3. A method of collectively inducing sexual maturity and spawning in a collection of aquatic animals, comprising feeding the animals algae that express a hormone that promotes sexual maturation in an amount effective to promote sexual maturation, and then in an amount effective to promote spawning.

[0259] 4. The method of any of paragraphs 1, 2, or 3, comprising collectively inducing sexual maturity or spawning in a plurality of aquatic animals.

[0260] 5. The method of paragraph 4, wherein the plurality of animals comprises at least any of 10, 25, 50, 100, 200, 500, 1000 or 10,000 animals.

[0261] 6. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the aquatic animals are bony or cartilaginous fish.

[0262] 7. The method of paragraph 6, wherein the fish are selected from carp, tilapia, salmon, bream, catfish, trout, snake head, salmon, tuna, cod, halibut, and sturgeon.

[0263] 8. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the aquatic animals are selected from mollusks and crustaceans.

[0264] 9. The method of paragraph 8, wherein the mollusks and crustaceans are selected from lobster, shrimp, prawns, octopus, squid, snails, scallops, and clams. [0265] 10. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the animals are raised in aquaculture.

[0266] 11. The method of paragraph 10, wherein aquaculture comprises raising fish in a cage, a tank, a pond or a pen.

[0267] 12. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the hormone is a gonadotropin releasing hormone (GnRH).

[0268] 13. The method of paragraph 12, wherein the GnRH has the following Formula (I):

[0269] Q - H - W - S - X ¹ - X ² - X ³ - X ⁴ - P - G, (I)

[0270] wherein:

[0271] X ¹ is a naturally occurring amino acid;

[0272] X ² is a naturally occurring amino acid;

[0273] X ³ is a naturally occurring amino acid; and

[0274] X ⁴ is a naturally occurring amino acid. (SEQ ID NO:2)

[0275] 14. The method of paragraph 13, wherein:

[0280] 15. The method of paragraph 13, wherein:

[0285] 16. The method of paragraph 13, wherein:

[0286] X ¹ is H or Y;

[0287] X ² is G;

[0288] X ³ is W; and

[0289] X ⁴ is Y or L (“GnRH2”). (SEQ ID NO:5)

[0290] 17. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the hormone is a relaxinlike gonad-stimulating peptide (RGP), a gonad stimulating substance (GSS), a follicle stimulating hormone (FSH) or a luteinizing hormone (LH).

[0291] 18. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the algae are selected from Haematococcus pluvailis, Chlamydomonas reinhardtii and Chlor ella vulgar i.

[0292] 19. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the algae comprise a recombinant expression construct comprising an expression control sequence operatively linked to a nucleotide sequence encoding the hormone. [0293] 20. The method of paragraph 19, wherein the expression construct is integrated into a nuclear or chloroplast chromosome of the algae.

[0294] 21. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the effective amount is effective to release follicle stimulating hormone (FSH) in the one or more animals.

[0295] 22. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the effective amount is effective to release luteinizing hormone (LH) in the one or more animals.

[0296] 23. The method of any of paragraphs 1, 2, 3, 4, or 5, further comprising feeding the animals algae in a dose effective for the induction of spawning in a controlled and synchronized spawning event.

[0297] 24. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein the animals exhibit asynchronous gonad maturation, and the effective amount is effective to release luteinizing hormone (LH) in the animals, e.g., over a prolonged period of time.

[0298] 25. The method of any of paragraphs 1, 2, 3, 4, or 5, comprising administering about 50 to about 100 pg GnRH per kg fish.

[0299] 26. The method of any of paragraphs 1, 2, 3, 4, or 5, comprising administering about 500 pg to about 1 mg or about 6 mg GnRH per kg fish.

[0300] 27. The method of any of paragraphs 1, 2, 3, 4, or 5, comprising administering about 50 to about 100 pg GnRH per kg fish to induce sexual maturation, and about 500 pg about 1 mg or about 6 mg GnRH per kg fish to induce spawning.

[0301] 28. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein feeding comprises a plurality of feeds over the course of days or weeks until the animals achieve sexual maturity.

[0302] 29. The method of paragraph 28, wherein the plurality of feeds is over the course of at least one week or at least one month.

[0303] 30. The method of paragraph 28, wherein the plurality of feeds is over the course of at least one week or at least one month.

[0304] 31. The method of any of paragraphs 1, 2, 3, 4, or 5, wherein feeding comprises a plurality of feeds over the course of days or weeks until sexual maturation and/or spawning is/are induced.

[0305] 32. A recombinant algae comprising an expression construct comprising a nucleotide sequence encoding a gonad stimulating polypeptide for an aquatic animal, operatively linked to an expression control sequence.

[0306] 33. The recombinant algae of paragraph 32, wherein the algae are selected from Haematococcus pluvailis, Chlamydomonas reinhardtii and Chlor ella vulgar i.

[0307] 34. The recombinant algae of paragraph 32, wherein the expression construct is comprised in a plasmid comprising sequences for incorporation into a chloroplast chromosome. [0308] 35. The recombinant algae of paragraph 32, wherein the expression control sequence is inducible. [0309] 36. The recombinant algae of paragraph 32, wherein the expression control sequence is photo-inducible.

[0310] 37. The recombinant algae of paragraph 32, wherein the gonadotropin stimulating polypeptide is an GnRH having the following Formula (I):

[0311] Q - H - W - S - X ¹ - X ² - X ³ - X ⁴ - P - G, (I)

[0312] wherein:

[0313] X ¹ is a naturally occurring amino acid;

[0314] X ² is a naturally occurring amino acid;

[0315] X ³ is a naturally occurring amino acid; and

[0316] X ⁴ is a naturally occurring amino acid. (SEQ ID NO:2)

[0317] 38. The method of paragraph 37, wherein:

[0322] 39. The method of paragraph 37, wherein:

[0327] 40. The method of paragraph 37, wherein:

[0328] X ¹ is H or Y;

[0329] X ² is G;

[0330] X ³ is W; and

[0331] X ⁴ is Y or L. (SEQ ID NO:5)

[0332] 41. The recombinant algae of paragraph 32, wherein the expression comprises a 35S promoter of algal rRNA.

[0333] 42. The recombinant algae of paragraph 37, wherein the GnRH has an amino acid sequence of a native GnRH.

[0334] 43. The recombinant algae of paragraph 37, wherein the GnRH is not post translationally modified.

[0335] Further exemplary embodiments are described the numbered paragraphs below.

[0336] 1. A method of inducing sexual maturity in one or more sexually immature aquatic animals, comprising feeding the one or more animals an effective amount of algae that express a gonad stimulating hormone that promotes sexual maturity.

[0337] 2. A method of collective spawning induction in a collection of sexually mature aquatic animals, comprising feeding the animals an effective amount of algae that express a gonad stimulating hormone that promotes spawning.

[0338] 3. A method of collectively inducing sexual maturity and spawning in a collection of aquatic animals, comprising feeding the animals algae that express a gonad stimulating hormone that promotes sexual maturation in an amount effective to promote sexual maturation, and then in an amount effective to promote spawning.

[0339] 4. The method of paragraph 1 or 3, comprising collectively inducing sexual maturity in a plurality of aquatic animals, or the method of paragraph 2 or 3, comprising collectively promoting spawning in a plurality of aquatic animals,

[0340] 5. The method of paragraph 4, wherein the plurality of animals comprises at least any one of 10, 25, 50, 100, 200, 500, 1000 or 10,000 animals.

[0341] 6. The method of any one of paragraphs 1, 2, 3, 4 and 5, wherein the aquatic animals are:

[0342] - bony or cartilaginous fish, preferably selected from carp, tilapia, salmon, bream, catfish, trout, snake head, salmon, tuna, cod, halibut, and sturgeon; or

[0343] - mollusks or crustaceans, preferably selected from lobster, shrimp, prawns, octopus, squid, snails, scallops, and clams.

[0344] 7 The method of any one of the preceding paragraphs, wherein the animals are raised in aquaculture, preferably comprising raising fish in a cage, a tank, a pond or a pen.

[0345] 8. The method of any one of paragraphs 1 to 7, wherein the algae are selected from Haemalococciis. Chlorella, Chlamydomonas, Nannochlor opsis, Porphyridium, and Phaeodactylum.

[0346] 9. The method of any one of paragraphs 1 to 8, wherein the algae comprise a recombinant expression construct comprising an expression control sequence operatively linked to a nucleotide sequence encoding the gonad stimulating hormone.

[0347] 10. The method of paragraph 9, wherein the expression construct is integrated into a nuclear or chloroplast chromosome of the algae.

[0348] 11. The method of any one of the preceding paragraphs, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a gonadotropin releasing hormone (GnRH) or a GnRH-based hormone which may or may not be post-translationally modified.

[0349] 12. The method of paragraph 11, wherein the GnRH or GnRH-based hormone has the following Formula (I):

[0350] Q - H - W - S - X ¹ - X ² - X ³ - X ⁴ - P - G, (I)

[0351] wherein:

[0352] X ¹ is a naturally occurring amino acid; [0353] X ² is a naturally occurring amino acid;

[0354] X ³ is a naturally occurring amino acid;

[0355] X ⁴ is a naturally occurring amino acid; and

[0356] the Q is optionally E. (SEQ ID NO:2)

[0357] 13. The method of paragraph 12, wherein:

[0362] 14. The method of paragraph 12, wherein:

[0366] X ⁴ is S, R or N (“GnRHl”). (SEQ ID NO:4)

[0367] 15. The method of paragraph 12, wherein:

[0368] X ¹ is H or Y;

[0369] X ² is G;

[0370] X ³ is W; and

[0371] X ⁴ is Y or L (“GnRH2”). (SEQ ID NO:5)

[0372] 16. The method of any one of paragraphs 11 to 15, wherein the GnRH or GnRH-based hormone comprises, consists essentially of, or consists of a sequence as shown in Figure 3 or a sequence as represented by any one of SEQ ID NOs: 13 to 80 and 113 to 115.

[0373] 17. The method of any one of paragraphs 1 to 10, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a gonadotrophin, a relaxin-like gonad-stimulating peptide (RGP), a gonad stimulating substance (GSS), a follicle stimulating hormone (FSH), or a luteinizing hormone (LH), or at least one subunit thereof, or at least one hormone receptor-activating sequence thereof, or an RGP -based hormone as defined herein, a GSS-based hormone as defined herein, a FSH-based hormone as defined herein, or an LH- based hormone as defined herein.

[0374] 18. The method of any one of paragraphs 1 to 16, wherein:

[0375] - the effective amount is effective to release follicle stimulating hormone (FSH) in the one or more animals; or

[0376] - the effective amount is effective to release luteinizing hormone (LH) in the one or more animals.

[0377] 19. The method of any one of paragraphs 1 to 18 when dependent on paragraph 2 or paragraph 3, wherein the dose effective amount induces spawning in a controlled and synchronized spawning event.

[0378] 20. The method of any one of paragraphs 1 to 18, wherein the animals exhibit asynchronous gonad maturation, and the effective amount is effective to release luteinizing hormone (LH) in the animals, optionally over a prolonged period of time.

[0379] 21. The method of any one of paragraphs 1 to 16 and 18 to 20 wherein the animals are fish and the gonad stimulating hormone comprises, consists essentially of, or consists of GnRH or a GnRH-based hormone, said method comprising:

[0380] - administering about 50 pg to about 100 pg GnRH or GnRH-based hormone per kg fish; or

[0381] - administering about 500 pg to about 1 mg or about 6 mg GnRH or GnRH-based hormone per kg fish.

[0382] 22. The method of any one of paragraphs 1 to 16 and 18 to 21 when dependent on paragraph 1 or paragraph 3, wherein the animals are fish and the gonad stimulating hormone comprises, consists essentially of, or consists of GnRH or GnRH-based hormone, said method comprising administering about 50 pg to about 100 pg GnRH or GnRH-based hormone per kg fish to induce sexual maturation.

[0383] 23. The method of any one of paragraphs 1 to 16 and 18 to 21 when dependent on paragraph 2 or paragraph 3, wherein the animals are fish and the gonad stimulating hormone comprises, consists essentially of, or consists of GnRH or GnRH-based hormone, said method comprising administering about 500 pg to about 1 mg or about 6 mg GnRH or GnRH-based hormone per kg fish to induce spawning.

[0384] 24. The method of any one of the preceding paragraphs when dependent on paragraph

1 or paragraph 3, wherein feeding comprises a plurality of feeds over the course of days or weeks until the animals achieve sexual maturity.

[0385] 25. The method of any one of the preceding paragraphs when dependent on paragraph

2 or paragraph 3, wherein feeding comprises a plurality of feeds over the course of days or weeks until the animals achieve spawning.

[0386] 26. The method of paragraph 24 or paragraph 25, wherein the plurality of feeds is over the course of at least one week or at least one month.

[0387] 27. Recombinant algae comprising an expression construct comprising a nucleotide sequence encoding a gonad stimulating hormone for an aquatic animal, operatively linked to an expression control sequence.

[0388] 28. The recombinant algae of paragraph 27, wherein the algae are selected from Haemalococciis. Chlorella, Chlamydomonas, Nannochlor opsis, Porphyridium, and Phaeodactylum .

[0389] 29. The recombinant algae of paragraph 27 or paragraph 28, wherein the expression construct is comprised in a plasmid comprising sequences for incorporation into a chloroplast chromosome.

[0390] 30. The recombinant algae of any one of paragraphs 27 to 29, wherein the expression control sequence is inducible.

[0391] 31. The recombinant algae of paragraph 30, wherein the expression control sequence is photo-inducible.

[0392] 32. The recombinant algae of any one of paragraphs 27 to 31, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of GnRH or a GnRH-based hormone having the following Formula (I):

[0393] Q - H - W - S - X ¹ - X ² - X ³ - X ⁴ - P - G, (I)

[0394] wherein:

[0395] X ¹ is a naturally occurring amino acid;

[0396] X ² is a naturally occurring amino acid;

[0397] X ³ is a naturally occurring amino acid;

[0398] X ⁴ is a naturally occurring amino acid; and

[0399] the Q is optionally E. (SEQ ID NO:2)

[0400] 33. The method of paragraph 32, wherein:

[0408] X ³ is L, N, or M; and

[0409] X ⁴ is S, R or N. (SEQ ID NO:4)

[0410] 35. The method of paragraph 32, wherein:

[0411] X ¹ is H or Y;

[0412] X ² is G;

[0413] X ³ is W; and

[0414] X ⁴ is Y or L. (SEQ ID NO:5)

[0415] 36. The recombinant algae of any one of paragraphs 32 to 35, wherein the GnRH or GnRH-based hormone comprises an amino acid sequence of a native GnRH. [0416] 37. The recombinant algae of any one of paragraphs 32 to 36, wherein the GnRH or GnRH-based hormone is not post-translationally modified.

[0417] Further exemplary embodiments are described the numbered paragraphs below.

[0418] 1. An alga-produced recombinant, isolated or purified gonad stimulating hormone.

[0419] 2 A recombinant, isolated or purified nucleic acid encoding the gonad stimulating hormone of paragraph 1.

[0420] 3. A recombinant expression cassette comprising at least one expression control sequence operatively linked to at least one nucleotide sequence encoding the gonad stimulating hormone of paragraph 1, or to the nucleic acid of paragraph 2.

[0421] 4. An expression vector comprising the gonad stimulating hormone of paragraph 1, the nucleic acid of paragraph 2, or the expression cassette of paragraph 3.

[0422] 5. The expression vector of paragraph 4, comprising insertion sequences for incorporation of the expression cassette into a chloroplast or nuclear chromosome of the alga.

[0423] 6. The expression cassette of paragraph 3, wherein the at least one expression control sequence is inducible, preferably photo-inducible; and/or the expression cassette is codon optimised for expression in the alga.

[0424] 7 A recombinant alga:

[0425] (1) that comprises the gonad stimulating hormone of paragraph 1;

[0426] (2) that expresses the gonad stimulating hormone of paragraph 1;

[0427] (3) that comprises the nucleic acid of paragraph 2;

[0428] (4) that comprises the expression cassette of paragraph 3 or 6; or

[0429] (5) that comprises the expression vector of paragraph 4 or 5.

[0430] 8. The recombinant alga of paragraph 7, wherein the recombinant alga is selected from Haemalococciis. Chlorella, Chlamydomonas, Nannochlor opsis, Porphyridium, and Phaeodactylum.

[0431] 9. A composition comprising the gonad stimulating hormone of paragraph 1, the nucleic acid of paragraph 2, the expression cassette of paragraph 3 or 6, the expression vector of paragraph 4 or 5, or the recombinant alga of paragraph 7 or 8.

[0432] 10. Aquatic animal feed comprising the gonad stimulating hormone of paragraph 1, the recombinant alga of paragraph 7 or paragraph 8, or the composition of paragraph 9.

[0433] 11. The gonad stimulating hormone of paragraph 1, the nucleic acid of paragraph 2, the expression cassette of paragraph 3 or 6, the expression vector of paragraph 4 or 5, the recombinant alga of paragraph 7 or paragraph 8, the composition of paragraph 9, or the aquatic animal feed of paragraph 10, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a hormone receptor-activating sequence that is capable of activating a hormone receptor.

[0434] 12. The gonad stimulating hormone of paragraph 11, the nucleic acid of paragraph 11, the expression cassette of paragraph 11, the expression vector of paragraph 11, the recombinant alga of paragraph 11, the composition of paragraph 11, or the aquatic animal feed of paragraph 11, wherein the hormone receptor-activating sequence further comprises a peptide, polypeptide or protein sequence covalently bound to the N-terminus and/or C-terminus of the hormone receptor-activating sequence.

[0435] 13. The gonad stimulating hormone of paragraph 11 or 12, the nucleic acid of paragraph 11 or paragraph 12, the expression cassette of paragraph 11 or paragraph 12, the expression vector of paragraph 11 or paragraph 12, the recombinant alga of paragraph 11 or paragraph 12, the composition of paragraph 11 or paragraph 12, or the aquatic animal feed of paragraph 11 or paragraph 12, wherein the gonad stimulating hormone comprises one or more of the following features:

[0436] (a) the gonad stimulating hormone comprises a terminal amide group;

[0437] (b) the gonad stimulating hormone lacks a terminal amide group;

[0438] (c) the gonad stimulating hormone comprises an amino pyro group;

[0439] (d) the gonad stimulating hormone lacks an amino pyro group;

[0440] (e) the gonad stimulating hormone comprises both a terminal amide group and an amino pyro group;

[0441] (f) the gonad stimulating hormone lacks both a terminal amide group and an amino pyro group;

[0442] (g) the gonad stimulating hormone comprises a native amino acid sequence;

[0443] (h) the gonad stimulating hormone comprises an artificial amino acid sequence;

[0444] (i) the gonad stimulating hormone is expressed in a hormone-active form by the alga, whereby the hormone-active form is capable of exerting a gonad stimulating effect from the time of expression by the alga;

[0445] (j) the gonad stimulating hormone is expressed in a hormone-inactive form by the alga, whereby the hormone-inactive form is required to be processed in order to be able to exert a gonad stimulating effect in the animal;

[0446] (k) the gonad stimulating hormone comprises one or more proteolytic cleavage sequences, sites, motifs or domains;

[0447] (1) the gonad stimulating hormone comprises one or more flexible linker sequences, motifs or domains;

[0448] (m) the gonad stimulating hormone comprises one or more protein tags;

[0449] (n) the gonad stimulating hormone comprises an initiation codon; and [0450] (o) the gonad stimulating hormone comprises a termination codon.

[0451] 14. The gonad stimulating hormone of paragraph 11, 12 or 13, the nucleic acid of paragraph 11, 12 or 13, the expression cassette of paragraph 11, 12 or 13, the expression vector of paragraph 11, 12 or 13, the recombinant alga of paragraph 11, 12 or 13, the composition of paragraph 11, 12 or 13, or the aquatic animal feed of paragraph 11, 12 or 13, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a gonadotropin releasing hormone (GnRH), a GnRH-based hormone as defined herein, a gonadotrophin, a relaxin-like gonad-stimulating peptide (RGP), a gonad stimulating substance (GSS), a follicle stimulating hormone (FSH), or a luteinizing hormone (LH), or at least one subunit thereof, or at least one hormone receptor-activating sequence thereof, or an RGP -based hormone as defined herein, a GSS-based hormone as defined herein, a FSH-based hormone as defined herein, or an LH- based hormone as defined herein.

[0452] 15. The gonad stimulating hormone of paragraph 14, the nucleic acid of paragraph 14, the expression cassette of paragraph 14, the expression vector of paragraph 14, the recombinant alga of paragraph 14, the composition of paragraph 14, or the aquatic animal feed of paragraph

14, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a gonadotropin releasing hormone (GnRH) or a GnRH-based hormone as defined herein.

[0453] 16. The gonad stimulating hormone of paragraph 15, the nucleic acid of paragraph 15, the expression cassette of paragraph 15, the expression vector of paragraph 15, the recombinant alga of paragraph 15, the composition of paragraph 15, or the aquatic animal feed of paragraph

15, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a GnRH peptide having the amino acid sequence of Formula (A):

[0454] Q - H - X ¹ - S - X ² - X ³ - X ⁴ - X ⁵ - P - G, wherein:

[0455] X ¹ is a naturally occurring amino acid;

[0456] X ² is a naturally occurring amino acid;

[0457] X ³ is a naturally occurring amino acid;

[0458] X ⁴ is a naturally occurring amino acid;

[0459] X ⁵ is a naturally occurring amino acid; and

[0460] the Q is optionally E. (SEQ ID NO:6)

[0461] 17. The gonad stimulating hormone of paragraph 16, the nucleic acid of paragraph 16, the expression cassette of paragraph 16, the expression vector of paragraph 16, the recombinant alga of paragraph 16, the composition of paragraph 16, or the aquatic animal feed of paragraph

16, wherein:

[0462] X ¹ is W or Y;

[0463] X ² is H, F, L or Y; [0464] X ³ is G, or Q;

[0465] X ⁴ is L, M or W; and

[0466] X ⁵ is S, R, N, K or L. (Formula (A)l) (SEQ ID NO:7)

[0467] 18. The gonad stimulating hormone of paragraph 16, the nucleic acid of paragraph 16, the expression cassette of paragraph 16, the expression vector of paragraph 16, the recombinant alga of paragraph 16, the composition of paragraph 16, or the aquatic animal feed of paragraph 16, wherein:

[0468] X ¹ is W;

[0469] X ² is H;

[0470] X ³ is G;

[0471] X ⁴ is W; and

[0472] X ⁵ is Y. (Formula (A)2) (SEQ ID NO:8)

[0473] 19. The gonad stimulating hormone of paragraph 16, the nucleic acid of paragraph 16, the expression cassette of paragraph 16, the expression vector of paragraph 16, the recombinant alga of paragraph 16, the composition of paragraph 16, or the aquatic animal feed of paragraph 16, wherein:

[0474] X ¹ is W;

[0475] X ² is Y or H;

[0476] X ³ is G or D;

[0477] X ⁴ is W; and

[0478] X ⁵ is L or K. (Formula (A)3) (SEQ ID NO:9)

[0479] 20. The gonad stimulating hormone of paragraph 16, the nucleic acid of paragraph 16, the expression cassette of paragraph 16, the expression vector of paragraph 16, the recombinant alga of paragraph 16, the composition of paragraph 16, or the aquatic animal feed of paragraph 16, wherein:

[0480] X ¹ is W;

[0481] X ² is Y;

[0482] X ³ is G, H or K;

[0483] X ⁴ is L; and

[0484] X ⁵ is R. (Formula (A)5) (SEQ ID NO: 10)

[0485] 21. The gonad stimulating hormone of paragraph 15, the nucleic acid of paragraph 15, the expression cassette of paragraph 15, the expression vector of paragraph 15, the recombinant alga of paragraph 15, the composition of paragraph 15, or the aquatic animal feed of paragraph 15, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a GnRH peptide having the amino acid sequence of Formula (B): [0486] Q - N - X ¹ - X ² - X ³ - S - X ⁴ - X ⁵ - X ⁶ - X ⁷ - P - G, wherein:

[0487] X ¹ is a naturally occurring amino acid;

[0488] X ² is a naturally occurring amino acid;

[0489] X ³ is a naturally occurring amino acid;

[0490] X ⁴ is a naturally occurring amino acid;

[0491] X ⁵ is a naturally occurring amino acid;

[0492] X ⁶ is a naturally occurring amino acid;

[0493] X ⁷ is a naturally occurring amino acid;

[0494] the Q - N preceding X ¹ is optional; and

[0495] the Q is optionally E. (SEQ ID NO: 11)

[0496] 22. The gonad stimulating hormone of paragraph 21, the nucleic acid of paragraph 21, the expression cassette of paragraph 21, the expression vector of paragraph 21, the recombinant alga of paragraph 21, the composition of paragraph 21, or the aquatic animal feed of paragraph 21, wherein:

[0497] X ¹ is Q or Y;

[0498] X ² is Y or H;

[0499] X ³ is F or W;

[0500] X ⁴ is Y, D, L, N or K;

[0501] X ⁵ is G, K, A, E, Q, C or Y;

[0502] X ⁶ is V, L, F, H, Y or W; and

[0503] X ⁷ is H, A, S, M, T, K, W, Q or R. (Formula (B)4) (SEQ ID NO: 12)

[0504] 23. The gonad stimulating hormone of paragraph 15, the nucleic acid of paragraph 15, the expression cassette of paragraph 15, the expression vector of paragraph 15, the recombinant alga of paragraph 15, the composition of paragraph 15, or the aquatic animal feed of paragraph 15, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a GnRH peptide comprising an amino acid sequence as shown in Figure 3 or any one of SEQ ID NOs: 13 to 80 and 113 to 115.

[0505] 24. The gonad stimulating hormone of paragraph 23, the nucleic acid of paragraph 23, the expression cassette of paragraph 23, the expression vector of paragraph 23, the recombinant alga of paragraph 23, the composition of paragraph 23, or the aquatic animal feed of paragraph 23, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a GnRH peptide having the sequence E/QHWSDYFKPG (SEQ ID NO: 77; SEQ ID NO: 58), optionally with an N-terminal pyro group, optionally with a C-terminal amide, or optionally with both an N-terminal pyro group and a C-terminal amide.

[0506] 25. The gonad stimulating hormone of paragraph 23, the nucleic acid of paragraph 23, the expression cassette of paragraph 23, the expression vector of paragraph 23, the recombinant alga of paragraph 23, the composition of paragraph 23, or the aquatic animal feed of paragraph 23, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of a GnRHII peptide having the sequence E/QHWSHGWYPG (SEQ ID NO: 114; SEQ ID NO: 115), optionally with an N-terminal pyro group, optionally with a C-terminal amide, or optionally with both an N-terminal pyro group and a C-terminal amide.

[0507] 26. The gonad stimulating hormone of any one of paragraphs 15 to 25, the nucleic acid of any one of paragraphs 15 to 25, the expression cassette of any one of paragraphs 15 to 25, the expression vector of any one of paragraphs 15 to 25, the recombinant alga of any one of paragraphs 15 to 25, the composition of any one of paragraphs 15 to 25, or the aquatic animal feed of any one of paragraphs 15 to 25, wherein the gonad stimulating hormone comprises a GnRH peptide and one or more of the following peptides, polypeptides or proteins:

[0508] 1. at least one GnRH-associated peptide (‘GAP’);

[0509] 2. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the residues of the at least one GAP;

[0510] 3 a cleavage site adjacent the N-terminus of the GnRH peptide;

[0511] 4 a cleavage site adjacent the C -terminus of the GnRH peptide;

[0512] 5 a respective cleavage site adjacent the N -terminus and C-terminus of the GnRH pepti e;

[0513] 6 at least one protein tag;

[0514] 7 a translation initiation sequence encoding Met,

[0515] 8 a translation termination sequence having a stop codon;

[0516] 9 at least one cleavage site for a protease;

[0517] 10. at least one flexible linker;

[0518] 11 a signal peptide; and

[0519] 12. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99% of the residues of the signal peptide.

[0520] 27. The gonad stimulating hormone of paragraph 26, the nucleic acid of paragraph 26, the expression cassette of paragraph 26, the expression vector of paragraph 26, the recombinant alga of paragraph 26, the composition of paragraph 26, or the aquatic animal feed of paragraph 26, wherein the gonad stimulating hormone comprises, consists essentially of, or consists of the amino acid sequence of Figure 5, Figure 6, Figure 9, Figure 12, Figure 17, Figure 19 or Figure 21, or any one of SEQ ID NOs: 99, 101, 104, 105, 107 or 109.

[0521] 28. The gonad stimulating hormone of paragraph 14, the nucleic acid of paragraph 14, the expression cassette of paragraph 14, the expression vector of paragraph 14, the recombinant alga of paragraph 14, the composition of paragraph 14, or the aquatic animal feed of paragraph 14, wherein:

[0522] - the gonad stimulating hormone comprises, consists essentially of, or consists of the RGP -based hormone as defined herein, or the relaxin-like gonad-stimulating peptide (RGP), or at least one hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof;

[0523] - the gonad stimulating hormone comprises, consists essentially of, or consists of the GSS-based hormone as defined herein, or the gonad stimulating substance (GSS), or at least one hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof;

[0524] - the gonad stimulating hormone comprises, consists essentially of, or consists of the FSH-based hormone as defined herein, or the follicle stimulating hormone (FSH), or at least one hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof; or

[0525] - the gonad stimulating hormone comprises, consists essentially of, or consists of the LH-based hormone as defined herein, or the luteinizing hormone (LH), or at least one hormone receptor-activating sequence or hormone receptor-activating sequence polypeptide thereof.

[0526] 29. The aquatic animal feed of any one of paragraphs 10 to 28, comprising recombinant algae biomass.

[0527] 30. The aquatic animal feed of paragraph 29, wherein the algae biomass is dewatered, dried, frozen, freeze dried and/or formulated as animal feed, such as pellets or wafers, preferably for fish.

[0528] 31. A method of promoting sexual maturity in one or more sexually immature aquatic animals, comprising feeding the one or more animals an effective amount of recombinant algae that express a gonad stimulating hormone that promotes sexual maturity.

[0529] 32. Recombinant algae that express a gonad stimulating hormone for use in promoting sexual maturity in one or more sexually immature aquatic animals, wherein said use comprises the step of feeding the one or more animals an effective amount of the recombinant algae to promote sexual maturity.

[0530] 33. Use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for promoting sexual maturity in one or more sexually immature aquatic animals, wherein said aquatic animal feed is formulated for feeding an effective amount of the recombinant algae to the one or more animals to promote sexual maturity.

[0531] 34. A method of collective spawning induction in a collection of sexually mature aquatic animals, comprising feeding the animals an effective amount of recombinant algae that express a gonad stimulating hormone that promotes spawning.

[0532] 35. Recombinant algae that express a gonad stimulating hormone for use in collective spawning induction in a collection of sexually mature aquatic animals, wherein said use comprises the step of feeding the one or more animals an effective amount of the recombinant algae to promote spawning.

[0533] 36. Use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for promoting collective spawning in a collection of sexually mature aquatic animals, wherein said aquatic animal feed is formulated for feeding an effective amount of the recombinant algae to the one or more animals to promote spawning.

[0534] 37. A method of collectively promoting sexual maturity and spawning in a collection of aquatic animals, comprising feeding the animals recombinant algae that express a gonad stimulating hormone that promotes sexual maturation in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning.

[0535] 38. Recombinant algae that express a gonad stimulating hormone for use in collectively promoting sexual maturity and spawning in a collection of aquatic animals, wherein said use comprises the step of feeding the animals the recombinant algae in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning.

[0536] 39. Use of recombinant algae that express a gonad stimulating hormone in the preparation of aquatic animal feed for collectively promoting sexual maturity and spawning in a collection of aquatic animals, wherein said aquatic animal feed is formulated for feeding the animals the recombinant algae in an amount effective to promote sexual maturity, and then in an amount effective to promote spawning.

[0537] 40. The method of paragraph 31 or 37, the recombinant algae of paragraph 34 or 38, or the use of paragraph 33 or 39, wherein the recombinant algae comprise a plurality of the recombinant alga as defined in any one of paragraphs 7, 8, and 11 to 28, or a plurality of the recombinant alga formulated as the composition as defined in any one of paragraphs 9 and 11 to 28, or a plurality of the recombinant alga formulated as the aquatic animal feed as defined in any one of paragraphs 10 to 28.

[0538] 41. The method of paragraph 31, 37 or 40, the recombinant algae of paragraph 32, 38 or 40, or the use of paragraph 33, 39 or 40, comprising collectively promoting sexual maturity in a plurality of aquatic animals.

[0539] 42. The method of paragraph 34, 37 or 40, the recombinant algae of paragraph 35, 38 or 40, or the use of paragraph 36, 39 or 40, comprising collectively inducing spawning in a plurality of aquatic animals. [0540] 43. The method of paragraph 31, 34, 37, 40, 41 or 42, the recombinant algae of paragraph 32, 35, 38, 40, 41 or 42, or the use of paragraph 33, 36, 39, 40, 41 or 42, wherein the aquatic animals are bony fish, cartilaginous fish, mollusks or crustaceans.

[0541] BRIEF DESCRIPTION OF THE DRAWINGS

[0542] The accompanying drawings, which are incorporated herein and form a part of the specification / disclosure, illustrate exemplary embodiments and, together with the written description, further serve to enable a person skilled in the pertinent art to make and use these embodiments and others that will be apparent to those skilled in the art. The invention will be more particularly described in conjunction with the following drawings wherein:

[0543] Figure 1 shows an exemplary protocol for producing recombinant algae for oral feeding to aquatic animals.

[0544] Figure 2 shows an agarose gel electrophoresis demonstrating successful amplification of DNA containing the GnRH cassette (1220 bp). The wildtype ‘wt’ strain (negative control) shows no DNA amplification as expected, a plasmid (positive control) shows successful amplification, while a set of modified strains show either successful or unsuccessful transformation (e.g., G110 vs. G3070 respectively).

[0545] Figure 3 shows exemplary amino acid sequences of native GnRH produced by paralogous GnRH genes 1 (I), 2 (II) and 3 (III), as well as synthetic versions, and consensus sequences. Note that the first Q of each peptide can be replaced with E. SEQ ID NOs are indicated.

[0546] Figure 4. Schematic representation of expression Cassette 1. The atpA expression elements drive the expression of a polynucleotide sequence encoding an expressible polypeptide comprising a GnRH decapeptide and a GnRH-associated peptide (‘GAP’), and the spectinomycin resistance gene aadA. The atpA promoter is shown in yellow, the atpA 5’ untranslated region is shown in pink, the expressible polypeptide encoding ‘GnRH’ is shown in orange, the aadA coding region is shown in dotted orange, and the atpA 3’ untranslated region is shown in violet.

[0547] Figure 5. Amino acid sequence of the expressible polypeptide of Clone 1/Cassette 1 and Clone 2/Cassette 2, with important regions and features indicated. SEQ ID NO:99.

[0548] Figure 6. Nucleotide (SEQ ID NO: 100) and amino acid sequences of the codon optimised expression cassette of Clone 1/Cassette 1, with important regions and features indicated. Expressible polypeptide of Clones 1 and 2 - SEQ ID NO: 101. aadA protein sequence - SEQ ID NO: 102. Nucleotide sequence of the expressible polypeptide of Clone 1 and 2 - SEQ ID NO: 111. Nucleotide sequence of 5' UTR, expressible polypeptide of Clone 1 and 2, and 3' UTR - SEQ ID NO: 112. atpA promoter and 5’ UTR: 1-596 of SEQ ID NO: 100; GnRH+GAP - expressible polypeptide ORF: 597-821 of SEQ ID NO: 100; atpA 3’ UTR: 822-1189 of SEQ ID NO: 100; atpA promoter and 5’ UTR: 1190-1784 of SEQ ID NO: 100; aadA gene ORF: 1785-2573 of SEQ ID NO: 100; atpA 3’ UTR:2574- 2940 of SEQ ID NO: 100.

[0549] Figure 7. Whole plasmid map comprising expression Cassette 1 ligated into a p322- BSKSII backbone.

[0550] Figure 8. Schematic representation of expression Cassette 2. The atpA promoter and 5’ untranslated region (shown in yellow), and 3’ untranslated region (shown in orange) drive the expression of a polynucleotide sequence encoding an expressible polypeptide comprising a GnRH decapeptide and a GnRH-associated peptide (‘GAP’) (‘GnREF, shown in green). The expression elements adjacent the aadA coding region (shown in blue) include a 16S promoter (shown in pink), an rbcL 5’ untranslated region (shown in aqua), and an rbcL 3’ untranslated region (shown in darker aqua).

[0551] Figure 9. Nucleotide sequence (SEQ ID NO: 103) and amino acid sequence of the codon optimised expression cassette of Clone 2/Cassette 2, with important regions and features indicated. Expressible polypeptide of Clone 2 - SEQ ID NO: 101. aadA protein sequence - SEQ ID NO: 102. atpA promoter and 5’ UTR: 1-596 of SEQ ID NO: 103; GnRH+GAP - expressible polypeptide: 597-821 of SEQ ID NO: 103; atpA 3’ UTR: 822-1188 of SEQ ID NO: 103; 16S promoter: 1189-1407 of SEQ ID NO: 103; rbcL 5’ UTR: 1408-1595 of SEQ ID NO: 103; aadA gene: 1596-2384 of SEQ ID NO:103; rbcL 3’ UTR: 2385-2822 of SEQ ID NO: 103.

[0552] Figure 10. Analysis of expression Cassette 1 integration into the Ct chloroplast using PCR. (A) shows a GnRH specific amplicon band of 126 bp (‘Clone 1’), similar to the plasmid positive control (‘plasmid 1’). The wild-type (’WT’) and water control (‘NC’) were negative for GnRH. (B) shows the internal psbD control, indicating the presence of chloroplast DNA. [0553] Figure 11. Analysis of expression Cassette 2 integration in Ct chloroplast using duplex PCR. Clone 2 was GnRH positive while the WT (wild-type control) and NC (water control) were negative for GnRH. The psbD internal control confirms the presence of chloroplast DNA in the samples.

[0554] Figure 12. LC-MS/MS detection of recombinant GnRH in Clones 1 and 2 (SEQ ID NO: 104). The region detected by MS is shown in grey, and blue lines represent the individual tryptic peptides detected.

[0555] Figure 13. GnRH receptor activation assay. No peptide control - treated without added GnRH. Positive control GnRH-analogue GnRH (LHRHa, sourced from Syndel, USA), amidated GnRH (GenicBio Limited, China) and non-amidated GnRH (GenicBio Limited, China) were assayed at different concentrations (113 pg, 11.3 pg, 1.1 pg and 113 ng). White bars show receptor activation by Clones 1 and 2 (10 pL total protein lysate of each). Data below the dotted line represent background activity and data above the dotted line represent receptor activation.

[0556] Figure 14. Mullet pituitary assay - (luteinizing hormone) LH ELISA. Pituitary gland cells negative control and background signal-cells from mullet, LH from inside cells, cells without GnRH added and WT 137c. Positive control -analogue GnRH (sourced from Syndel, USA) 130, 13 and 1.3 ng. White bars show the LH level of cells treated with Clone 1 (10 pl total protein lysate) and Clone 2 (10 pl total protein lysate). Data below the dotted line represent the background LH level and data above the dotted line represent active LH secretion.

[0557] Figure 15. Expression cassettes VI, V2, V3 and V4 for expressing recombinant GnRH. [0558] Figure 16. The expressible polypeptide of Cassettes VI and V4, comprising tuna decapeptide GnRH, GAP, FLAG, cleavage site and linker.

[0559] Figure 17. Nucleotide (SEQ ID NO: 106) and amino acid (SEQ ID NO: 105) sequences of the expressible polypeptides of Cassettes VI and V4.

[0560] Figure 18. The expressible polypeptide of Cassette V2 comprising tuna decapeptide GnRH, GAP, FLAG, cleavage site and linker.

[0561] Figure 19. Nucleotide (SEQ ID NO: 108) and amino acid (SEQ ID NO: 107) sequences of the expressible polypeptide of Cassette V2.

[0562] Figure 20. The expressible polypeptide of Cassette V3, comprising chickenll decapeptide GnRH, GAP, FLAG, cleavage site and linker.

[0563] Figure 21. Nucleotide (SEQ ID NO: 110) and amino acid (SEQ ID NO: 109) sequences of the expressible polypeptide of Cassette V3.

[0564] SEQUENCE LISTING

[0565] Table of Sequence Listings

[0566] EXAMPLES

[0567] The following Examples describe the preparation and recombinant expression of GnRH in microalgae, and the use of that recombinant microalgae for promotion of sexual maturation or spawning in aquatic animals such as fish. Exemplary GnRH peptide sequences are shown in Figure 3 and include the GnRH decapeptide produced by paralogous GnRH genes 1, 2 and 3. In some instances, expressible polypeptides (fusion proteins) comprising the GnRH (deca)peptide together with other covalently bonded sequences are referred to simply as ‘GnRH’.

[0568] An exemplary protocol is shown in Figure 1 and includes the steps of growing microalgae, transformation of that microalgae with an expression construct encoding a GnRH- containing polypeptide, selection of recombinant microalgae, sequencing of selected recombinant microalgae, improving growth of that recombinant microalgae using an optimised lighting regime, recombinant peptide quantification using an ELISA assay or qPCR, scale-up growth of the recombinant microalgae, dewatering or freeze-drying of recombinant microalgae biomass, and formulation of pellets/fish feed containing recombinant microalgae biomass for feeding to fish. Orally administered GnRH-based hormone is absorbed in the intestinal tract of the fish and reaches the bloodstream for subsequent delivery to the pituitary to stimulate the release of gonadotropins to regulate reproductive cycles.

[0569] Example I - Preparation of Recombinant Algae

[0570] Chlamydomonas reinhardtii (Cr) 137c was grown in 1 L TAP (Tris Acetate Phosphate) liquid media for 5 days at room temperature shaking at 100 rpm under flashing LED lights. Cells were counted visually with a light microscope, then centrifuged, resuspended and added to TAP media plates with a concentration of roughly 100,000,000 cells / plate.

[0571] Lyophilized GnRH-containing plasmid DNA was resuspended in filter sterilized distilled water to 1 pg / pL. 50 pL of 60 mg / mL gold nanoparticles were mixed with 10 pL plasmid DNA, 50 pL of 2.5 M CaCh, 20 pL of 0.1 M spermidine and incubated at room temperature for 10 minutes. Nanoparticles were centrifuged for 1 min at 4,000 g, the supernatant removed and nanoparticles washed with 150 pL 70% ethanol, followed by a second wash with 150 pL of 100% ethanol. Nanoparticles were resuspended in 60 pL of 100% ethanol then loaded onto flyer discs and placed in desiccating chambers to dry. [0572] Once Cr TAP plates and plasmid DNA in flyer discs had dried, algae cultures were bombarded with nanoparticles using a GJ- 1000 High Pressure Gas Gene Gun (Nanbai) with helium gas. Bombarded Cr cultures on TAP media plates were incubated overnight at room temperature, shaking at 100 rpm under flashing LED lights to recover, before transferring to selection plates containing 10 pg / mL spectinomycin.

[0573] After 3 weeks of growth, single colonies were isolated and sub-cultured to establish transgenic clones carrying the transgene. 5-10 clones of each plasmid were isolated, and successful transformation verified by conventional PCR before testing recombinant algae activity. The following primers were used for PCR amplification of the target GnRH cassette.

[0574] Table 1 - Primer sequences

[0575] Algae DNA was extracted using the Chelex DNA extraction method, as follows: 1. 1 mL of algae culture was transferred into a 1.5 mL microfuge tube and centrifuged at 10,000 rpm for 1 minute. All of the supernatant/media was removed using a pipette. 2. The algae pellet was resuspended with 50 pL of 6% Chelex solution and vortexed well to mix. 3. The suspended pellet was incubated in a heat block at 100°C for 30 minutes, before being cooled in the fridge for 20 minutes. 4. The suspended pellet was vortexed well to mix, then centrifuged at 10,000 rpm for 1 minute. 5. The supernatant was transferred to a fresh tube (~40 pL), without extracting the Chelex beads. 6. DNA purity was confirmed and the concentration was determined by absorbance at 260 nm. 7. PCR was performed using NEB Hotstart 2x Master mix (M0496L), in 50 pL reaction volumes and 2 pL of extracted plasmid DNA. 8. The following PCR program was used:

[0576] 95°C - 30 sec

[0577] 95°C - 30 sec - 1

[0578] 50°C - 30 sec x 5 cycles

[0579] 68°C - 1 :30 min — | [0580] 95°C - 30 sec - 1

[0581] 68°C - 1 :30 min — | x 30 cycles

[0582] 68°C - 5 min

[0583] 10°C - hold

[0584] 9. Agarose gels were run to confirm the length of the amplified PCR product (1220 bp), before sending samples for DNA sequencing and initiating tests of recombinant algae activity. (See Figure 2.)

[0585] Example II - Testing Recombinant Algae Activity

[0586] Algae productivity was 0.25-3 mg (GnRH)/g (algae) dry weight.

[0587] Example III - Method for Inducing Sexual Maturity

[0588] Once recombinant cultures are scaled up, they are dewatered with centrifugal methods or freeze-dried and mixed into fish feed at various rates to form the dosage pellet (see Example V). These pellets are administered according to species and dose rate to promote gametogenesis, sexual maturity.

[0589] Example IV - Method for Spawning Induction

[0590] Once recombinant cultures are scaled up, they are dewatered with centrifugal methods or freeze-dried and mixed into fish feed at various rates to form the dosage pellet (see Example V). These pellets are administered according to species and dose rate to promote synchronous spawning events.

[0591] Example V - Dosage Pellets

[0592] The dosage pellets can be of any suitable formulation. Examples of suitable formulations are shown below:

[0593] Formulation 1. 30 % dewatered recombinant algae biomass + 60% fish meal + 5% fish oil + 5% binder.

[0594] Feed 10 gram of Formulation 1 per kg fish/day. This comprises about 25% of the daily feed intake required per 1 kg fish/day, required for gametogenesis.

[0595] Formulation 2. 60 % dewatered recombinant algae biomass + 30% fish meal + 5% fish oil + 5% binder.

[0596] Feed 10 gram Formulation 2 per kg fish/day. This comprises about 25% of the daily feed intake required per 1 kg fish/day, required for spawning.

[0597] Formulation 3. 90% dewatered recombinant algae biomass+ 5% fish oil + 5% binder. [0598] A suitable dosage regime for initiating gametogenesis, sexual maturity, is as follows: [0599] Feed 10g of Formulation 1 per kg fish/day for 1-4 weeks or 4-12 weeks depending on the species. This formulation comprises approximately 25% of the daily intake of broodstock. [0600] A suitable dosage regime for instigating a synchronous spawning event is as follows: [0601] Feed 10g of Formulation 2 per kg fish/day for 2-7 days or 8-14 days depending on the species. This formulation comprises of approximately 25% of the daily intake of broodstock.

[0602] Notes:

[0603] Gametogenesis - 3 mg/kg/day = 3 g dewatered algae (based on 1 mg/g).

[0604] Spawning - 6 mg/kg/day - 6 g dewatered algae (based on 1 mg/g).

[0605] Feed rate for broodstock - 4% of body mass, 40 g per day of feed or more per 1 kg fish.

[0606] Example VI - Preparation of Recombinant Algae Expressing GnRH

[0607] Expression vector constructs

[0608] Further expression vectors were constructed using standard cloning techniques and gene synthesis (by Genscript Biotech Corporation or Twist Bioscience).

[0609] Two different expression vector constructs (DNA plasmids) were prepared for expressing GnRH. Figure 4 shows a first expression cassette (‘Cassette 1’) which includes a nucleotide sequence encoding an expressible polypeptide comprising a GnRH decapeptide and a GnRH-associated peptide (‘GAP’). Hereafter, the GnRH decapeptide and GAP will be referred to as the ‘expressible polypeptide’. The amino acid sequence of the expressible polypeptide is shown in Figure 5, with important regions and features indicated. The nucleotide and amino acid sequences of the expressible polypeptide are shown in Figure 6, with important features indicated.

[0610] For expression, the construct has regulatory elements, including an atpA promoter / 5’ UTR upstream of the expressible polypeptide and an atpA 3’ UTR downstream of the expressible polypeptide.

[0611] As seen in Figure 7, the construct further comprises a selection marker, including a polynucleotide sequence encoding aadA for spectinomycin resistance. An atpA promoter / 5’ UTR regulatory element is located upstream of aadA and an atpA 3’ UTR is located downstream of aadA.

[0612] A backbone of the expression construct comprises p322-BSKSII. The expression cassette is inserted at a BamHI restriction site of p322-BSKSII. The expression construct also includes homologous recombination sequences which integrate the cassette in a site-specific manner between the psbA and rrn5 intergenic region of the Cr chloroplast.

[0613] Figure 8 shows a second expression cassette (‘Cassette 2’) which includes a polynucleotide sequence encoding an expressible polypeptide comprising a GnRH decapeptide and a GnRH-associated peptide (‘GAP’). Hereafter, the GnRH decapeptide and GAP will be referred to as the ‘expressible polypeptide’.

[0614] The amino acid and nucleotide sequences of the expressible polypeptide of Cassette 2 are shown in Figure 9, with important regions and features indicated. For expression, the construct has regulatory elements, including an atpA promoter / 5’ UTR upstream of the expressible polypeptide and an atpA 3’ UTR downstream of the expressible polypeptide.

[0615] The construct further comprises a selection marker, including a polynucleotide sequence encoding aadA for spectinomycin resistance. 16S promoter and rbcL 5’ UTR regulatory elements are located upstream of aadA, and an rbcL 3 ’UTR is located downstream of aadA.

[0616] A backbone of the expression construct comprises p322-BSKSII. The expression cassette is inserted at a BamHI restriction site of p322-BSKSII. The expression construct also includes homologous recombination sequences which integrate the cassette in a site-specific manner between the psbA and rrn5 intergenic region of the Chlamydomonas chloroplast.

[0617] Example VII - Algae Transformation-Particle Bombardment

[0618] Cr strain 137c was grown in 1 L TAP liquid media to an early log phase of l-2xl0 ⁶ cells/mL prior to bombardment. A gene gun (GJ-1000) was used to incorporate each expression vector construct/plasmid DNA into the microalgae. Small gold particles coated with the plasmid DNA were bombarded at high velocity at a plate of Cr culture. This method forced the DNA into the microalgae cells and into the chloroplasts, where chloroplast DNA recombination takes place. The bombarded cells were then recovered and plated on TAP media containing the antibiotic spectinomycin, thereby selecting for transgenic colonies.

[0619] The particle bombardment procedure was optimised for Cr strain 137c. A stock solution of gold particles (0.6 pm) was prepared at a concentration of 60 mg/mL and particle/DNA binding was performed as described previously (Sanford, Smith and Russell (1995). Optimizing the Biolistic Process for Different Biological Applications. Recombinant DNA Methodology II. R. Wu. Boston, Academic Press: 485-511. DOI: 10.1016/0076- 6879(93)17086-k; Ismagul, Yang, Maltseva, Iskakova, Mazonka, Skiba, Bi, Eliby, Jatayev, Shavrukov, Borisjuk and Langridge (2018). "A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions." BMC Plant Biology 18(1): 135. DOI: 10.1186/sl2870-018-1326-1).

[0620] Single transgenic colonies were isolated and grown to establish cultures carrying potentially successful transformants.

[0621] Validation of the integration of expression cassette using PCR

[0622] Successful transformation was verified by conventional colony PCR using GnRH primers and psbD chloroplast internal control primers as shown in Table 2.

[0623] Table 2 - Primers used for gene integration validation in transgenic clones

[0624] Integration of GnRH expression Cassettes 1 and 2 was confirmed in clones showing the expected amplicon size of 126 bp. For expression Cassette 1, see Figure 10. The Figure shows a GnRH specific amplicon band of 126 bp (‘Clone 1’), similar to the plasmid positive control (‘plasmid 1’). The negative control (‘NC’) and wildtype (‘WT 137c’) were each GnRH negative.

[0625] Example VIII - qPCR Quantification of mRNA Expression

[0626] qPCR for determining mRNA expression of Clone 1 and Clone 2 was carried out using conventional methods. RNA was extracted using the TRIzol/chloroform method, as described in Poong et al., 2017 (Poong, S.-W., Lim, P.-E., Lai, J.W.-.-S. and Phang, S.-M. (2017), Optimization of high quality total RNA isolation from the microalga, Chlorella sp. (Trebouxiophyceae, Chlorophyta) for next-generation sequencing. Phycological Res., 65: 146-150. https://doi.org/10. l l l l/pre.12165)). Cell lysis was carried out using a tissueLyser (Qiagen) for 2 min at 30 Hz. The extracted RNA was subjected to treatment with TURBO DNase™ (TURBO DNA _: //vc™ Kit; Invitrogen), as recommended by the manufacturer. Next, cDNA was synthesised using the iScript cDNA synthesis kit (Bio-Rad), as recommended by the manufacture. Finally, qPCR was carried out using iTaq Universal SYBR Green Supermix (Bio-Rad), as recommended by the manufacturer.

[0627] The primers used for qPCR are shown in Table 3.

[0628] Table 3. Primers used for qPCR of Clones 1 and 2

* Housekeeping gene from Sanchez-Tarre and Kiparissides (Sanchez-Tarre, V., & Kiparissides, A. (2021). The effects of illumination and trophic strategy on gene expression in Chlamydomonas reinhardtii. Algal Research),' acxl (acetyl-CoA carboxylase subunit a); annealing temp = 60°C.

[0630] qPCR amplification conditions:

[0631] GnRH

[0632] 95°C - 1 min

[0633] 95°C - 10 sec — - — - --4

[0634] 60°C - 40 sec (camera) -I x40

[0635] Melt curve (65°C to 95°C in 0.5°C increments)

[0636] End

[0637] Acx 1

[0638] 95°C - 1 min

[0639] 95°C - 10 sec - i

[0640] 60°C - 40 sec i x40

[0641] 86°C - 10 sec (camera) —

[0642] Melt curve (65°C to 95°C in 0.5°C increments)

[0643] End

[0644] The qPCR results demonstrated that Clone 1 and Clone 2 had significant GnRH expression compared to the wildtype controls which had undetectable GnRH expression. The results are shown in Table 4. The data show GnRH mRNA copies per pL and normalised GnRH to the Acxl housekeeping gene.

[0645] Table 4. Quantification of GnRH mRNA expression using qPCR on Clone 1 and Clone 2. Acxl was used as a housekeeping gene

[0646] Example IX - GnRH polypeptide detection using LC-MS/MS

[0647] Total protein was extracted from Clones 1 and 2 using sonication at 30% amplitude for 5 minutes of 20 seconds ON/20 seconds OFF cycles on ice. Total protein was trypsinized and analysed by LC-MS/MS (QTOF X500R), briefly as addressed below.

[0648] Clones were cultured in 10 mL TAP media and incubated under LED lights with shaking (120 RPM) for 5 days. The cells were pelleted and resuspended in 1 mL RIPA buffer (Invitrogen) containing a proteinase inhibitor cocktail (Sigma). The cells were lysed using sonication to break open the cells, to release the proteins. The cell lysis solution was centrifuged at 10,000x g and supernatant containing the total soluble proteins (TSP) was collected.

[0649] The proteins were concentrated using acetone precipitation. Four volumes of ice cold 80% acetone were added to the TSP supernatant and incubated at -80°C to facilitate protein precipitation. Following centrifugation at 10,000 x g, the protein pellet was washed 3 times with cold 80% acetone.

[0650] The dried TSP pellet was resuspended in urea buffer and trypsin digestion was carried out overnight. Following digestion, the tryptic peptides were concentrated using Sep-Pak c-18 column (Waters). The tryptic peptide solutions were passed through the Sep-Pak cl 8 column and washed. Then peptides were eluted and the solution was dried using a Savant SC250EXP vacuum centrifuge (Thermo Fisher).

[0651] Cleaned-up protein pellet samples were resuspended in 0.1% formic acid and LC- MS/MS sequenced.

[0652] Based on the results, Clones 1 and 2 were found to contain the expressible polypeptide encoding the GnRH decapeptide. It could not be determined if the expressible polypeptide had been cleaved to release the decapeptide. However, the expressible polypeptide was tested for successful receptor binding and expression levels in later Examples.

[0653] Sequenced peptides of the expressible polypeptide regions detected by MS are shown in Figure 12. The sequenced peptides correspond to the GnRH decapeptide and GAP.

[0654] For expression of Cassette 2, see Figure 11. The Figure shows a GnRH specific amplicon band of 126 bp (‘Clone 2’). As expected, the positive control (‘WT 137c’) amplified the correct size for psbD but was GnRH negative, and the negative control (‘NC’) was also GnRH negative.

[0655] Example X - GnRH Receptor Activation Assay [0656] Protein lysates of Clones 1 and 2 were prepared by sonication in PBS or TBS containing a proteinase inhibitor cocktail (Sigma). GnRH receptor activation assays were carried out as described by Nocillado et al., 2022 (Josephine Nocillado, Peter Palma, Tianfang Wang, Evelyn Grace de Jesus-Ayson, Berta Levavi-Sivan, Abigail Elizur, Intracellular production of recombinant GnRHl in yeast, Pichia pasloris. and its potential as oral treatment to advance gonadal development in juvenile orange-spotted grouper, Epinephelus coioides, Aquaculture, Volume 554, 2022, 738115, ISSN 0044-8486, http s : //doi . or g/ 10 , 1016/i , aquaculture.2022 ,738115.

[0657] (https://www.sciencedirect.eom/science/article/pii/S00448486 22002319)) .

[0658] A luciferase reporter assay was performed to determine the bioactivity of algae recombinant GnRH in vitro. The recombinant tilapia GnRH type 3 receptor in pEGFP-Nl expression vector (tGnRHR3) (Avitan A, Zelinger E, Levavi-Sivan B. Homologous desensitization and visualization of the tilapia GnRH type 3 receptor. Gen Comp Endocrinol. 2007 Aug-Sep;153 (1-3): 182-8. doi: 10.1016/j.ygcen.2007.04.003. Epub 2007 Apr 11. PMID: 17507014) was utilised as heterologous receptor. The cAMP-driven CRE-Luc reporter (PGL4.29; Promega) was used as cAMP is involved in GnRH receptor signal transduction (Levavi-Sivan B, Yaron Z. Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia. Mol Cell Endocrinol. 1992 Jun; 85(3): 175-82. doi: 10.1016/0303-7207(92)90256-6. PMID: 1378800). The transient transfection protocol was as described in Avitan et al. (2007) and Palma et al. 2019 (Palma P, Nocillado J, Superio J, Ayson EGJ, Ayson F, Bar I, Elizur A. Gonadal response of juvenile protogynous grouper (Epinephelus fuscoguttatus) to long-term recombinant follicle- stimulating hormone administration. Biol Reprod. 2019 Mar l;100(3):798-809. doi: 10.1093/biolre/ioy228. PMID: 30371741) with modification according to Lipofectamine LTX & Plus reagent (Invitrogen) as the transfection reagent. Briefly, COS-7 cells (40-50% confluence in 8.5 cm petri dish) were transfected with 3 pg each of tGnRHR3 and CRE-Luc reporter plasmids. After incubation for 24 h at 37°C, cells were collected and seeded into 12- well plate containing 1 mL media per well. After another incubation for 24 h at 37°C, the cells were serum-starved for 2 h at 37°C and then ligands were added. The increasing doses of rGnRHl tested were serially diluted from 0 to 2 ng/mL. Three different forms of GnRH standards were used: a synthetic GnRH analogue (Pyr-His-Trp-Ser-Tyr-DAla-Leu-Arg-Pro- NHEt) or LHRHa decapeptide (Syndel, USA), which is amidated at the carboxy terminal; a carboxyl terminal amidated GnRH decapeptide termed GnRH-amide (GenicBio Limited, China); and, a non- amidated GnRH having a free -OH in the carboxyl terminal (GenicBio Limited, China). See Table 5. Synthetic GnRH were used as positive controls at concentrations 0-113 pg/mL. The amino acid sequence of algae recombinant GnRH and the synthetic GnRH standard were the same. The treatments were in duplicate wells. Luciferase activity was determined using Promega's kit as described in Palma et al. (2019).

[0659] Table 5. Synthetic GnRH peptide sequences

[0660] Testing of protein lysates from Clones 1 and 2 in a receptor activation assay showed that both Clones 1 and 2 induced the GnRH receptor above background levels. This can be seen in Figure 13. The receptor activation assay showed that recombinant GnRH expressed by transgenic Chlamydomonas was bioactive and could bind and trigger the GnRH receptor in vitro.

[0661] Example XI - LH Induction Assay- Mullet Pituitary Cells

[0662] Protein lysates of Clones 1 and 2 were prepared largely as described in Example X. An LH-induction assay of mullet pituitary cells was carried out largely as described in Dennis et al., 2020 (Peter Dennis L, Nocillado J, Palma P, Amagai T, Soyano K, Elizur A. Development of a giant grouper Luteinizing Hormone (LH) Enzyme-Linked Immunosorbent Assay (ELISA) and its use towards understanding sexual development in grouper. Gen Comp Endocrinol. 2020 Sep 15;296: 113542. doi: 10.1016/j.ygcen.2020.113542. Epub 2020 Jul 13. PMID: 32585213; https://www.sciencedirect.eom/science/article/pii/S001664802 0302951#b0295) .

[0663] Pituitary glands cells primary culture was prepared from mature mullet. Freshly harvested pituitary glands were placed on ice then washed several times with an antibiotic cocktail in PBS. The pituitary tissue was then dissociated to acquire single cells by chopping tissue to 1 mm pieces and treating with trypsin/EDTA/glucose solution. The cell suspension was diluted to 100,000 cells/mL in L-15 +10% FBS media and 1 mL was loaded into each well in 48 well plates. After cells had recovered and grown for 3-4 days, the media was changed to L-15 (500 pl) and treatments were added in triplicates. A synthetic GnRH analogue (Pyr-His-Trp-Ser-Tyr-DAla-Leu-Arg-Pro-NHEt) or LHRHa decapeptide (Syndel, USA) was used at 113, 11.3, 1.1 pg and 113 ng per mL. Wild type Cr 137c lysate (10 pL) was a negative control. 10 pl of each clone lysate was used. ‘Cells from mullet’ contained media only and sample ‘no GnRH added’ was treated with buffer. All cells were treated for 6 hours then the supernatant was aspirated into clean 1.5 mL tubes and snap frozen before freeze-drying. Group ‘LH from inside cells’ were cells lysed by mechanical means to release internal LH.

[0664] All samples were analysed for LH by competitive ELISA for giant grouper LH as previously described in Dennis et al., 2020. Recombinant GnRH of Clones 1 and 2, expressed in Chlamydomonas, led to the secretion of LH significantly higher than the negative controls (lysed pituitary cells, pituitary cells without GnRH, and wild type Chlamydomonas') . This is shown in Figure 14.

[0665] In vertebrates, endogenous GnRH release causes the downstream secretion of LH via the pituitary gland. The results of the pituitary assay confirm that the recombinant GnRH expressed in microalgae is able to trigger the GnRH receptor and the receptor activation has biological activity, triggering the cascade leading to LH secretion.

[0666] Conclusion

[0667] In Examples VI to XI, expression constructs /vectors were designed to express GnRH- based hormones/peptides in Chlamydomonas chloroplast, resulting in Clones 1 and 2. We showed that an expressible polypeptide comprising a GnRH decapeptide and GAP could be integrated into the chloroplast genome, and that the cassette could express the GnRH decapeptide-GAP mRNA, as evidenced by qPCR. The presence of GnRH decapeptide-GAP polypeptide was confirmed by LC-MS/MS analysis. The GnRH receptor activation assay showed that Clone 1 and Clone 2 were each able to activate the GnRH receptor significantly higher than the control. The pituitary assay confirmed the receptor assay, demonstrating that the GnRH decapeptide deriving from Clone 1 and Clone 2 was able to trigger the release of LH. This reinforces the notion that the receptor activation caused by recombinant GnRH also triggers the downstream pathways which lead to the release of LH.

[0668] Different expression constructs/cassettes for recombinant GnRH can be used to trigger the release of LH. Different expressible polypeptide sequences can be used. These may or may not include GAP. These can comprise a variety of GnRH decapeptide sequences, both naturally occurring and genetically modified/mutated. The expression constructs/cassettes can be expressed in microalgae other than Chlamydomonas. Codon optimised sequences and endogenous or synthetic expression/regulatory elements for the particular microalgal host in use can be used.

[0669] Example XII - Further Expression Vector Constructs Having GnRH

[0670] This Example describes the preparation of various expression vector constructs, each of which includes a nucleotide sequence encoding an expressible polypeptide comprising a GnRH decapeptide, a dibasic cleavage site, a GnRH-associated peptide (‘GAP’), a FLAG-tag, and a linker. Hereafter, the GnRH decapeptide, dibasic cleavage site, and GAP will be referred to as ‘pre-cleaved GnRH’. The FLAG-tag is connected to either the GnRH decapeptide or GAP by way of a linker. Hereafter, the pre-cleaved GnRH together with the linker and FLAG- tag will be referred to as the ‘expressible polypeptide’.

[0671] For expression, each construct has regulatory elements, including an atpA promoter / 5’ UTR upstream of the expressible polypeptide and an atpA 3’ UTR downstream of the expressible polypeptide.

[0672] Each construct further comprises selection markers, including polynucleotide sequences encoding aadA for spectinomycin resistance, and a DCMU chloroplast resistance mutation. An atpA promoter / 5’ UTR regulatory element is located upstream of aadA and an rbcL 3’ UTR is located downstream of aadA.

[0673] A backbone of each construct comprises P322 cleaved at the Btrl restriction site.

[0674] Various expression cassettes are shown in Figure 15.

[0675] The expressible polypeptide of Cassettes VI and V4 comprising tuna decapeptide GnRH and GAP, is shown in Figure 16.

[0676] The nucleotide and amino acid sequences of Cassettes VI and V4 are shown in Figure 17.

[0677] The expressible polypeptides of Cassette V2, comprising tuna decapeptide GnRH and GAP, is shown in Figure 18.

[0678] The nucleotide and amino acid sequences of Cassette V2 are shown in Figure 19.

[0679] The expressible polypeptide of Cassette V3, comprising chickenll decapeptide GnRH and GAP, is shown in Figure 20.

[0680] The nucleotide and amino acid sequences of Cassette V3 are shown in Figure 21.

[0681] Advantages of the present invention, particularly as exemplified, include the following: [0682] - biologically active quantities of gonad stimulating hormones can be produced by recombinant algae, for convenient feeding/oral administration to aquatic animals;

[0683] - the gonad stimulating hormones need not be isolated from the recombinant algae biomass, so there can be minimum processing of the biomass;

[0684] - the recombinant algae biomass may serve as a protective vehicle to deliver the recombinant hormones to the intestine of the animal, for absorption into the bloodstream of that animal;

[0685] - the recombinant algae biomass can be readily formulated into feed pellets or wafers, especially for fish; and

[0686] - in addition to providing gonad stimulating hormones, the recombinant algae biomass is a good source of nutrition.

[0687] It should be understood that the description and the drawings are not intended to limit the invention to the particular form disclosed, but to the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. Further modifications and alternative embodiments of various aspects of the invention will be apparent to those skilled in the art in view of this description. Accordingly, this description and the drawings are to be construed as illustrative only and are for the purpose of teaching those skilled in the art the general manner of carrying out the invention. It is to be understood that the forms of the invention shown and described herein are to be taken as examples of embodiments. Elements and materials may be substituted for those illustrated and described herein, parts and processes may be reversed or omitted, and certain features of the invention may be utilized independently, all as would be apparent to one skilled in the art after having the benefit of this description of the invention. Changes may be made in the elements described herein without departing from the spirit and scope of the invention as described in the following claims. Headings used herein are for organizational purposes only and are not meant to be used to limit the scope of the description. [0688] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0689] As used herein, the following meanings apply unless otherwise specified. The words “can” and “may” are used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). The words “include”, “including”, “includes”, “have” and “having” and the like mean including, but not limited to. Unless the context requires otherwise, the terms “comprise”, “comprises” and “comprising”, or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated. The term "consisting essentially of' refers to the inclusion of recited elements and other elements that do not materially affect the basic and novel characteristics of a claimed combination. By “consisting essentially of’ in the context of an amino acid sequence is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- or C-terminus. The singular forms “a,” “an,” and “the” include plural referents. Thus, for example, reference to “an element” includes a combination of two or more elements, notwithstanding use of other terms and phrases for one or more elements, such as “one or more.” The phrase “at least one” includes “one”, “one or more”, “one or a plurality”, and, therefore, contemplates the use of the term “a plurality”. The term “or” is, unless indicated otherwise, non-exclusive, i.e., encompassing both “and” and “or.” The term “any of’ between a modifier and a sequence means that the modifier modifies each member of the sequence. So, for example, the phrase “at least any of 1, 2 or 3” means “at least 1, at least 2 or at least 3”. The term “about” refers to a range that is 10% but more preferably 5% plus or minus from a stated numerical value within the context of the particular usage.

[0690] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [0691] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with other pieces of prior art by a skilled person in the art.