SPECIFICATION

FIELD OF THE INVENTION

The claimed invention relates to the aquaculture field. It describes the composition, formulation and preparation of an antiparasitic agent of biological origin, for the treatment of an infestation caused by parasites (sea lice) of the Caligidae family in fish.

I. AIM OF THE INVENTION

The claimed invention relates to the preparation of an aquaculture antiparasitic product based on the use of Bacillus thuringiensis, particularly Bacillus thuringiensis spores and/or recombinant proteins, for the treatment of the infestation caused by parasites of the Caligidae family in aquaculture. It also refers to the preparation of a composition suitable for use in the treatment of the infestation caused by parasites of the Caligidae family in fish. In another embodiment, the claimed invention refers to the preparation of a formulation based on Bacillus thuringiensis spores and/or recombinant proteins, for the treatment of the infestation caused by parasites of the Caligidae family in fish.

Moreover, the field of the invention includes a method of treatment against the infestation caused by Caligus, by administrating Bacillus thuringiensis spores and/or recombinant proteins to fish, which is possible to be administered through diverse routes, such as through feed, by injection or immersion bath. The invention includes the development of a product of biological origin to be applied in fish, for controlling parasites of the Caligidae family, based on Bacillus thuringiensis spores combined with Cry recombinant proteins.

Introduction 1. Sea Lice

Copepods are the most common parasites in wild fish and farm fish. The term "sea lice" is used to define the species of copepods belonging to the Caligidae family, which affect salmon farming worldwide, generating a parasitism called caligidosis (Johnson et ah, 2004).

The species mainly affecting salmonid farming centers belong to the genera Caligus rogercresseyi and Lepeophtheirus salmonis, in the Southern and Norther Hemisphere, respectively (Boxshall and Bravo 2000, Burka et al., 2012). However, there are other copepod families relevant as parasites in aquaculture, belonging to the families Ergasilidae (15%) Lernaepodidae (8%), and Lernanothropidae (5%).

Caligidosis, which is a disease caused by the copepods Caligus rogercresseyi and Lepeophtheirus salmonis, is a persistent problem in the aquaculture industry, having no concrete and effective solution to date. Currently, this disease affects more than 90% of the salmon industry, especially in Chile and Norway. As reported in year 2017 by the Institute Tecnologico del Salmon (Intesal), annual losses due to caligidosis in Chile are estimated in more than 300 million dollars. A similar situation was recently reported by the Sea Lice Research Center (SLRC), indicating that by 2019 there were losses of about 300 million euros in Norway. The main problems caused by this disease relate to delayed growth of the parasitized fish, increase in stress, and the susceptibility of fish to be attacked by other opportunistic pathogens (Johnson et ah, 2004).

The life cycle of L. salmonis consists of 5 phases. The first phase has two planktonic stages, called nauplii I and II. Thereafter, the second phase corresponds to the copepodite stage, which is the infesting phase of the parasite where a host fish must be found. Once attached to the host, the following stages are developed: chalimus (I and II, fixed stages), preadult (I and II, mobile stages) and mature adults (Costello, 2006).

The life cycle of C. rogercresseyi is similar to the aforementioned life cycle of L. salmonis, and it is characterized by having eight development stages: 3 planktonic stages (free life stages), and 5 parasitic stages (stages attached to the host) (Gonzalez et ah, 2003). The first three planktonic stages are named nauplius I, nauplius II, and copepodite. The copepodite is the infesting stage that must attach to the fish to keep developing to become adult. For attachment purposes, the parasite develops a frontal filament which allows it to puncture and feed from the mucus and the skin of the fish. Thus, four immobile parasitic stages attached to the fish are generated, called chalimus I, II, II and IV, which are finally differentiated in adult females and males.

It has been reported that the attachment of these parasites to fish causes wounds, stress and immunosuppression. Therefore, fish become susceptible to acquire diseases due to secondary infections of opportunistic pathogens, causing diseases in fish and leading to their death.

2. Chemical control of Caligus

From the past decade, the control of C. rogercresseyi and L. salmonis is made by using antiparasitics based on chemical compounds. For example, antiparasitics administered orally, such as emamectin and ivermectin benzoate, the mechanism of action thereof consists in activating the glutamate-gated chloride channels in nerve cells and muscle cells, hyperpolarizing cells, causing death of the parasite due to flaccid paralysis. Currently, the use of ivermectin is forbidden and several derivatives thereof were removed from the market because they had negative effects on the marine environment. The oral antiparasitic products are currently administered during the first third of the productive cycle of salmonids, wherein the control for the rest of the fattening period is mainly supported by the use of drugs administered by immersion.

Among the compounds that can be applied by immersion, there are pyrethroid-based drugs, such as deltamethrin and cypermethrin, which act by activating the voltage-gated sodium channels, increasing permeability of nerve cells to sodium, hyperpolarizing the cell and causing paralysis (Sordehmd, 2012).

Immersion or bath treatments with hydrogen peroxide (H2O2) are also practiced, which causes mechanical paralysis due to the formation of bubbles in the hemolymph and tissues of the parasite. Furthermore, it induces peroxidation of lipids in the cell membrane, inactivating enzymes and inhibiting DNA replication. H2O2 is only used for controlling preadult and adult stages of the sea lice, since it is not effective during the larval stages (Chavez-Mardones el al., 2015).

The chemical compound most used nowadays (years 2016 to 2019) is the organophosphate medicament azamethiphos, which penetrates into the cuticle of insects and acts by inhibiting the acetylcholinesterase activity, leading to the death of the insect.

However, it has been reported that sea lice build resistance toward pharmacological chemical products after repeated applications, becoming tolerant to treatments. For example, the application of emamectin was authorized for the first time in Chile in year 2000, wherein said treatment allowed a reduction of more than 90% in the sea lice population. Nevertheless, in year 2005 a loss of sensitivity in C. rogercresseyi to this parasite was first observed, despite the administration of the recommended dose, and to this day said lice is resistant to the drug, managing to reproduce at high rates in salmons (Bravo S. et ah, 2013). Recently, the first cases of appearance of resistance to azamethiphos have been also described, both in Chile and in Norway.

The problem of the appearance of resistance to sea lice towards chemical products after a certain time, together with the need of reducing the environmental impact, and the costs of culturing salmon, make it very necessary to include alternative therapeutic tools to the ones currently registered and in use, so as to allow rotation of active ingredients, and advance in the research and development of non-chemical (mechanical, biological and genetic) alternatives allowing supporting the control of this disease in an integrated manner. Therefore, the claimed invention discloses the development of a treatment of biological origin, innocuous to the environment, wherein its mechanism of action depends on several active compounds, without generating long term resistance, unlike the chemical treatments currently used.

3. Treatment of biological origin of Caligus

It is known that the treatment of particular pests or parasites with compounds of biological origin has many advantages over the treatment with compounds of chemical origin. Some of the most important advantages are that a biological compound is biodegradable, having thus a low environmental impact and no lack period. Moreover, it generates low resistance, since it is composed of many active compounds and it is species -specific, preventing damage to other species in the environment. An example of sea lice control of biological origin in salmons was described in the Northern Hemisphere, wherein in order to control Lepeophtheirus salmonis, the use of fish of the Labridae family was proposed, which feed on ectoparasites (Groner et ah, 2013). However, this type of fish cannot be used in salmonid cultures in Chile, as they inhabit temperate regions.

In Chile, researches have been made to assess the potential of rock cod ( Eleginops maclovinus ) in the control of caligidosis in salmons, wherein the obtained results have shown 48.8% efficacy relative to the control and a preference of rock cod to only eat C. rogercresseyi in adult stage. Furthermore, the use of other fish for controlling these parasites implies a disadvantage to the industry, since this increases the density of cultured fish, and thus their stress due to overcrowding.

4. Bacillus thuringiensis for control of Caligus

In the farming industry, Bacillus thuringiensis (Bt) is one of the most used microorganisms in control of biological origin. Mainly, this bacterium has been used for controlling insect populations affecting agricultural crops and for controlling insects transmitting diseases, such as dengue and malaria (Bravo, A. et ah, 2013).

B. thuringiensis is an anaerobic, facultative and chemoorganotrophic Gram-positive bacterium, which forms spores with entomopathogenic properties. Bt has been used as bio -controller, because during the sporulation stage it produces an inclusion of parasporal crystal proteins with insecticidal activity. There are hundreds of Bt subspecies producing, mainly during sporulation, one or more parasporal inclusions, each composed of one or several related insecticidal proteins thousands of gene sequences have been identified, corresponding to Cry proteins in different strains of B. thuringiensis, being classified in terms of the identity of their primary sequence into 74 groups of Cry proteins. Among these, there are delta-endotoxins, which have a varied genetic identity: crylA(a), crylA(b), crylA(c), crylB, crylC, crylD, cry2A, cry2B, cry3A, cry4A, cry4B, cry4C, cry4D, CytA. Besides delta-endotoxins, Bt also produces other proteins, such as a-endotoxins, b- endotoxins, hemolysins, phospholipases, and chitinases (Soberon el al, 2010; Schiinemann el al, 2014).

The parasporal crystals of B. thuringiensis can be composed of more than five different proteins, depending on the strain. In the literature, it has been described that these proteins can have activity against different groups of insects belonging to the orders lepidoptera, diptera, coleoptera, hymenoptera, hemiptera, isoptera, orthoptera, siphonoptera, thysanoptera, and activity has even been seen against some nematodes and gastropods. The main factor for the activity of these proteins as endotoxins is the characteristic basic pH of the intestine of these insects, which allows the crystals to solubilize. On the other hand, proteases specific to the intestine of each insect make cuts on the different Cry proteins, allowing the formation of the activated toxin, capable of binding to receptors located on the apical membrane of the intestinal epithelium. All these factors define the range of species that can affect a Cry protein and are related to the specificity of action of each toxin. The parasporal protein of B. thuringiensis, when ingested by the insect, is activated and interacts with the midgut epithelium of the larvae, causing a disorganization of membrane permeability, which causes the death of the insect (Soberon et al, 2010; Schiinemann el al, 2014). Bt spore suspensions or inclusions have been commercially used for several decades in agriculture and it comprises spraying spores at frequent intervals in order to maintain an effective level of the biopesticide protein. Most of the commercially available formulations are based on mixtures of parasporal crystals belonging to B. thuringiensis subsp. kurstaki (Btk) being effective against different pest species, and it is generally used against young lepidopteran larvae. There are Bt formulations with different strains of significant commercial interest, such as: HD-1, SA-11, SA- 12, PB 54, ABTS-351 and EG2348 (Riui etal, 2015).

Currently, there are commercial formulations of B. thuringiensis spores authorized by the Agricultural and Livestock Service (SAG) of the Chilean Ministry of Agriculture, which are used for the biological control of insects in agriculture. These products are Dipel® WG, Javelin® WG and Betk-03®.

The claimed invention demonstrates that a formulation or composition of spores and Cry proteins belonging to B. thuringiensis, each separately or in combination is capable of controlling the Caligus infestation in aquaculture. Hence, the invention relates to the use of B. thuringiensis spores and proteins for preparing an antiparasitic composition or formulation for controlling Caligus. In addition to this field, the invention encompasses a method for controlling Caligus in aquaculture, which comprises administering to fish spores or products thereof belonging to B. thuringiensis for the control of the Caligus infestation.

II. BACKGROUND OF THE INVENTION

PRIOR ART

In the prior art there are alternative proposals for methods of treating Caligus infestations, in addition to the treatments authorized and used to date. For example, document WO02054873 A2 refers to the possibility of using microorganisms in the control of Caligus in aquaculture, through the application of a microbiological control agent, other than a fungus, in or proximal to the site of such infestations. Within this proposed solution, it is pointed out that the control of the Caligus infestation can be given by bacteria; however, there is no indication that it could be with B. thuringiensis and the examples are restricted to viruses and protozoa. Furthermore, it has been described that sucking insect pests in animals can be controlled with Bt toxin or spores. For example, document AU588849 B2 describes a method for controlling lice using a composition derived from Bacillus. The lice treated are from sheep, cattle or other animals, and the examples verify the action of sporulated Bacillus in sheep lice. Along the same line, document W02006096905 proposes the use of a bacterium of the genus Bacillus or a combination of cells and cellular components in the manufacture of a medicine for the treatment and prevention of sucking lice infestations in a patient, said medicine being administered to the hair or plumage of a mammal or bird. Preferably, lice are of the order Phthiraptera and of the family Pediculidae, and Bacillus is of the thuringiensis species. Also disclosed is a composition for killing human lice and/or their eggs, which comprises a bacterium of the genus Bacillus or a combination of cells and a carrier for topical application. This document proposes the treatment of human, animal or bird lice with B. thuringiensis and not the treatment in an aquatic environment of lice of the Caligidae family that infect fish. It should be noted that the examples in W02006096905 have no experimental evidence to support the method claimed in the present invention. Also, dated earlier, US5273746 discloses a method of controlling chewing lice of the order Phthiraptera , wherein said method comprises administering an effective amount of a B. thuringiensis toxin to a host harboring lice, or directly onto said lice. In particular, the strain is selected from the group consisting of B. thuringiensis PS192N1, PS36A (NRRL B-18929), PS71M3, PS81F, PS92J, PS86A1, PS204G6, PS81I, PS81GG, PS201T6, PS84C3, PS211B2, PS91C2 (NRRL B-18931), PS40D1, and PS192M4. This document describes that the strains of B. thuringiensis are safe for use in urban areas, and can be used in aquatic environments without damaging other species; however, it proposes the treatment of lice of the order Phthiraptera , instead of the lice present in an aquatic environment of the order Siphonostomatoida, to which the Caligidae family belongs, which infect fish. Unlike the preceding document, US5273746 provides experimental evidence demonstrating the insecticidal capacity of a preparation with B. thuringiensis for sheep lice.

In agriculture, for the use of Bt to be approved, it is required that it is not toxic to marine organisms that are in contact with wastewater from agricultural products. In the study by Yan-Liang Li el al. (2013), the toxicity of the Cry 1 Ac protein for two aquatic invertebrates Chironomus dilutus and Hyalella azteca is assessed. It is concluded that C. dilutus is more sensitive than H. azteca, at concentrations of environmental relevance, and that the risk is limited for aquatic organisms, because the environmentally relevant concentrations were much lower than the LC50 dose.

No literature has been found that uses formulations with Bt bacteria or parts thereof for the control of aquatic pests that infect fish.

Moreover, the behavior of Bt crystals on pests of aquatic animals has neither been reported. The fact that Bt is effective in lice of terrestrial animals does not allow concluding in an evident manner that it can be effective in lice of marine animals. Furthermore, considering that the name "lice" groups a series of sucking organisms of different orders, and that the different Bt toxins have an important degree of toxicity specific for different organisms of the order dipteran or lepidopteran.

Further documents related to the prior art of the claimed invention are the following:

Document CN104397034 A refers to an eco-friendly pesticide, the composition of which contains veratridine, matrine, sodium laurate, cheletryne, surfactant, ethanol and Bacillus thuringensis (Bt), with a broad insecticidal spectrum for application in aquaculture. The document describes that 3- 4 parts of Bt diluted 20-30 times are used in the insecticide. In this case, the composition contains a mixture that is different from the one proposed in the claimed invention, indicating that its use is insecticidal and not to combat sea lice.

Document WO2012149549 A2 discloses microbiocidal compositions based on Bacillus strains that are administered by feeding with the capacity to control bacterial infections in aquaculture (farmed fish or crustaceans). Although this document describes in its specification that the strain may be Bt, its use is to control bacterial infections in aquaculture and not to combat sea lice. Nor does it indicate the use of a composition such as the one disclosed in the present application.

Document JP2005154405 A discloses the use of Bacillus thuringiensis to control fungi in farmed fish or crustaceans, especially Fusarium solani. As described in the document, the aim is to control fungi and not sea lice in salmonids. Nor does it indicate the use of a composition such as that described in the present application.

III. SUMMARY OF THE INVENTION

The claimed invention aims to solve the problem of controlling parasites of the Caligidae family, which affects salmon farming worldwide, providing an environmentally friendly solution, based on the treatment of biological origin, to the absence of effective treatments in the control of this parasitic infestation. Currently, neurotoxic chemical compounds are used, such as emamectin benzoate, pyrethroids and organophosphate compounds (azamethyphos), which damage the environment. Moreover, sea lice ( Caligus ) build resistance to these products after a certain time of application. The claimed invention proposes using a formulation based on Bacillus thuringiensis spores in combination with recombinant Cry proteins (recombinant endotoxins), which does not damage the environment. In addition, in agriculture it has been indicated that biocontrol products based on this bacterium do not produce long-term resistance because their action is through several components, not only an active compound. The product developed in the claimed invention is applied to seawater for the treatment of salmons by immersion baths or by injection or orally.

An advantage of the claimed invention is that the Bt-based product developed is active against larval (copepodites) and adult Caligus, while the current chemical treatments available in the industry only cause mortality of adult Caligus. This occurs because said treatments usually affect the nervous system of the parasites, which is not developed in larvae. With the antiparasitic composition developed, based on a combination of B. thuringiensis spores and recombinant Cry proteins of the same bacterium, 100% mortality of adult and copepodite Caligus.

IV. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Graph showing the survival of copepodites against different doses of Bt spores. It exhibits the survival curves of copepodites treated with different concentrations of spores from the strains ABTS-351, SA-11, and a mixture of Chilean native strains (N1-N2-N3). The resulting mortality was plotted after 240 min of treatment.

Figure 2: Graph showing the survival of adult Caligus rogercresseyi treated with spores of Bacillus thuringiensis subsp. Kurstaki strains ABTS-351 and ATCC-33679. It exhibits the survival percentage of adult Caligus treated with 2xl0 ⁹ CFU/mL spores of Bt.

Figure 3: Graph showing the survival of adult C. rogercresseyi treated with Bt ABTS-351 spores produced in bioreactor. It exhibits the survival percentage of adult parasites treated with spores at a dose of 2xl0 ⁸ CFU/mL in in vitro assays. Figure 4: Graph showing the survival of copepodites treated with Bt ABTS-351 spores produced in bioreactor. The graph exhibits the survival percentage of larvae treated with Bt spores at a dose of 2.8x10 ^s CFU/mL in in vitro assays. Figure 5: SDS-polyacrylamide gel electrophoresis 10% of the moieties of protein CrylAb expressed in E. coli BL21 (DE3) and collected by Ni-NTA agarose affinity chromatography. It shows the molecular size marker AccuRuler RGB Plus (Maestrogen), ft corresponds to flowthrough (unretained flow in the column), and L is the resin wash control. Moieties eluted with 50 mM of imidazole (1-1 to 1-3) and 300 mM of imidazole (2-1 to 2-3) are observed.

Figure 6: SDS-polyacrylamide gel electrophoresis 10% of the moieties of protein CrylAb, produced in H. polymorpha and collected by Ni-NTA agarose affinity chromatography. M represents the molecular size marker AccuRuler RGB Plus (Maestrogen), ft corresponds to flowthrough (unretained flow in the column), and L is the resin wash control. Moieties eluted with 50 mM of imidazole (1-1 to 1-3) and 300 mM of imidazole (2-1 to 2-3) are observed.

Figure 7: Survival curve of C. rogercresseyi larvae treated with proteins Cryl, Cry2 and Cry3. The survival curve is observed when copepodites are treated with 300 pg/mL of each Cry protein. U/T corresponds to the untreated control, B/C corresponds to the buffer control, while proteins CrylAb, Cry2 and Cry3 are shown in the figure with their respective names.

Figure 8. C. rogercresseyi larvae treated with Cry proteins. The images were taken with an optical microscope with a lOx objective. (A) shows an untreated larva, which has only had contact with seawater. (B) shows a larva treated with the CrylAb protein (300 pg/mL), and (C) shows a larva treated with a Cryl/Cry2 combination (300 pg/mL) The arrows represent the presence of bubble- shaped structures.

Figure 9. Survival curve of C. rogercresseyi adults treated with proteins Cryl, Cry2 and Cry3. The survival curve with a dose of 300 pg/mL of proteins Cryl, 2 and 3 is observed. U/T corresponds to the untreated control, B/C corresponds to the buffer control, while proteins Cry lAb, Cry2 and Cry 3 are shown with their respective names.

Figure 10. Survival curve of C. rogercresseyi adults treated with proteins Cryl and Cry4. The survival curve with a dose of 300 pg/mL of each Cry protein. U/T corresponds to the untreated control, and the Cry proteins are indicated with their respective names.

Figure 11. C. rogercresseyi female adults treated with 300 pg/mL of protein Cryl. (A) shows an untreated ovigerous female observed with the stereoscopic lens, wherein the white square corresponds to the intestinal-anal area. (B) corresponds to a lOx zoom with an optical microscope of the intestinal-anal area of an ovigerous female. In (C) ovigerous sacs of a female treated with the CrylAb protein 300 pg/mL are observed with a xlO zoom,

Figure 12. Comparison of the in vitro antiparasitic activity of recombinant Cryl proteins. (A) shows the survival curve of C. rogercresseyi adults treated with 150 pg/mL of CrylAb proteins expressed in E. coli BL21, as compared to CrylAb expressed in H. polymorpha. (B) shows the survival curve of C. rogercresseyi adults treated with 150 pg/mL of proteins CrylAb and CrylAc both produced in E. coli BL21. Figure 13. Survival curve of C. rogercresseyi larvae treated with a combination of proteins

Cry 1, 2 and 3. The survival curves of larvae treated with combinations of Cryl/Cry2 and Cryl/Cry3 is shown, at a dose of 300 pg/mL of each protein, for 360 minutes. U/T corresponds to the untreated control.

Figure 14. Survival curve of C. rogercresseyi adults treated with a combination of proteins Cryl and Cry4. The survival curve of adult Caligus treated with a Cryl/Cry4 combination at a dose of 150 pg/mL of each protein for 360 minutes is shown. U/T corresponds to the untreated control, and the Cry proteins are indicated with their respective names.

Figure 15. Survival curve of Caligus rogercresseyi adults, treated with B. thuringiensis spores, supplemented with the CrylAb protein produced in E. coli. The survival percentage of adult parasites treated with lxlO ⁸ CFU/mL of spores obtained in reactor, combined with recombinant CrylAb in different amounts is shown.

Figure 16. Survival curve of Caligus rogercresseyi larvae treated with B. thuringiensis spores combined with the CrylAb protein produced in E. coli. The survival percentage of copepodites treated with lxlO ⁸ CFU/mL of spores obtained in bioreactor in different amounts of recombinant CrylAb protein is shown.

Figure 17. Survival curve of C. rogercresseyi adults treated with B. thuringensis spores combined with the CrylAb protein produced in H. polymorpha. The survival curves of Caligus adults treated with lxlO ⁸ CFU/mL of Bt spores obtained in bioreactor supplemented with the CrylAb protein expressed and purified from H. polymorpha is shown. Figure 18. Comparison of the effect of Bt spores on Caligus adults in in vivo and in vitro assays. (A) shows the attachment percentage of Caligus adults per fish after an immersion treatment of 60 min with Bt ABTS-351 spores {in vivo). U/T corresponds to the control of fish that received immersion treatment without the antiparasitic. 100% corresponds to the parasitic load of fish before the immersion treatment. (B) shows the survival percentage of Caligus adults treated with spores at a dose of lxlO ⁸ CFU/mL in vitro. The untreated control corresponds to Caligus kept in Petri dishes with seawater.

Figure 19. Comparison of the effect of the combination of Bacillus thuringiensis spores and a protein extract containing CrylAb, on the survival of Caligus in in vivo and in vitro assays.

(A) shows the survival percentage of attached Caligus per fish, after the treatment of parasitized fish with Bt spores at a dose of lxlO ⁹ CFU/mL combined with CrylAb produced in H. polymorpha, without purification (420 pg/mL protein extract) (in vivo). 100% corresponds to the parasitic mean load of fish before the immersion treatment and the untreated control corresponds to fish subjected to a bath without antiparasitic compounds. (B) shows the survival percentage of Caligus adults treated with Bt spores supplemented with CrylAb without purification, in vitro. The untreated control corresponds to Caligus kept in Petri dishes with seawater.

Figure 20. Effect of CrylAb injection on caligus survival in in vivo assays. It shows the percentage of caligus attached to the fish after injection treatment with CrylAb purified from E. coli. The untreated control corresponds to fish injected with physiological serum. 100% corresponds to the number of caligus attached to the fish before the injection (counting prior to the treatment for each condition). Figure 21. Gradual evolution of the effect of Bt spores and CrylAb protein on adult L. salmonis, in vitro. (A) Control group. It shows no effect on activity for control sea lice (without treatment). (B) Bt Spores. Development of inactivity for adult after continuous exposure to Bt Spore (10 ⁹ CFU/ml) over a period of 360 minutes after initial exposure. First sign of reduced activity is seen 180 minutes post exposure (reduced movement); no change/development over the course of the experiment. (C) Bt Spores + CrylAb. Development of inactivity for adult lice after continuous exposure to Bt Spores (10 ⁹ CFU/ml) + CrylAb (150 pg/mL) over a period of 360 minutes after initial exposure. First sign of reduced activity is seen 120 minutes post exposure, reaching complete inactivity by 180 minutes (3 hours). Score: 0 - no effect; 1 - reduced movement; 2 - no movement, but grip of forceps; 3 - no movement and no grip of forceps.

Figure 22. Gradual evolution of the effect of Bt spores and CrylAb protein on L. salmonis copepodites, in vitro. It shows the gradual evolution of mortality or effect on L. salmonis untreated control group (A) and a group treated with Bt spores and CrylAb at 150 pg/ml (B) indicated according to the score: 0, 1, 2 and 3, wherein 0 - no effect; 1 - reduced movement; 2 - reduced movement and copepodites at the center of the wells; 3 - no movement.

V. DETAILED DESCRIPTION OF THE INVENTION

The claimed invention relates to the development of an aquaculture antiparasitic composition or formulation based on a combination of Bacillus thuringiensis spores and recombinant Cry proteins of the same bacterium, as well as Bacillus thuringiensis spores and recombinant Cry proteins on their own, for the control of sea lice of the Caligidae family, through the treatment of infested fish by immersion baths, by injection or orally. The infested fish may belong to the families Salmonidae, Eleginopsidae, Atherinopsidae, Paralichthyidae, or Cichlidae. In particular, the Salmonidae family fish may be of the genus Salmo or Oncorhynchus . More particularly, said fish may be of the species Salmo salar and/or Oncorhynchus mykiss and/or Oncorhynchus kisutch, commonly called Atlantic salmon, rainbow trout, or coho salmon, respectively.

They may also belong to the Eleginops maclovinus species, commonly called rock cod, to the Basilichthys australis species, commonly called silverside, to the Paralichthys adspersus species, commonly called dover sole, or to the Oreochromis aureus species, commonly called tilapia.

The Caligus (or caligus) parasite mentioned in the claimed invention belongs to the Caligidae family and it may belong to the genera Caligus and Lepeophtheirus, particularly it may belong to the Caligus rogercresseyi, Lepeophtheirus salmonis species, among other species.

The composition or formulation according to the claimed invention has been developed to be acceptable for veterinary use.

In the claimed invention the culture and sporulation conditions of Bt were characterized and standardized. Then, the antiparasitic activity of Bt on Caligus was assessed. The obtained results showed that Bt spores have antiparasitic activity against Caligus adults and larvae, in in vitro assays, producing in both cases 100% mortality in 180 minutes of treatment, while by 60 minutes of treatment a 50% mortality is reached for adult caligus, and more than 60% mortality for larvae. Moreover, it was demonstrated that the combination of spores and Cry proteins produces more than 70% mortality of adult and larval caligus in 30 minutes of treatment, and 100% mortality is reached in 90 minutes. In vivo assays demonstrated that the combination of spores and CrylAb reduces the parasitic load of the caligus infested fish, observing a similar effect to the one obtained in in vitro assays.

Optimization of Bt culture conditions The spores in the claimed invention come from the bacterium B. thuringiensis subspecies kurstaki strains ABTS-351 and ATCC-33679. The culture medium used contains Peptone lOgr/Lt, NaCl 5gr/Lt, Glucose 34gr/Lt, yeast extract 20gr/Lt, MnCh 20mg/L, ZnCh 40mg/L. It was seeded with 20% pre-inoculum and incubated for 8 days at 28°C. Obtaining Cry proteins

In another embodiment of the claimed invention, the antiparasitic activity of some Cry proteins in Caligus was assessed. To such purpose, encoding gene sequences were selected from the following Cry proteins based on the information available in the literature and in the NCBI (National Center for Biotechnology Information) Pubmed site available in the Internet (ncbi.nlm.hih.gov/pubmed): CrylAb (GenBank: AY319967.1), CrylAc (GenBank: AD064600.1), Cry2 (GenBank: AM490199.1), Cry3 (Lambert y cols., 1992), and Cry4 (GenBank: ACR43758.2). Next, the encoding genes of Cryl (CrylAb, CrylAc), Cry2 (Cry2Ad), Cry3, and Cry4 were cloned in different expression systems. A modality of the expression system for these proteins can be prokaryotic, such as Escherichia coli or Bacillus subtilis other modality of the expression system can be eukaryotic, such as Hansenula polymorpha or Trichoderma asperellium. Subsequently, the recombinant proteins were purified and their caligus killing activity was assessed.

The encoding DNA sequences used in the claimed invention, as well as the recombinant protein sequences obtained as described are specified in Table I:

Table I: Identification of encoding DNA sequences and recombinant Cry proteins used in the claimed invention. Once the Cry proteins were purified, their antiparasitic effect was determined. The results showed that the CrylAb protein at a dose of 300 pg/mL causes about 30% mortality of copepodites and adults after 60 minutes of treatment, reaching 100% mortality after 120 minutes of in vitro treatment. Proteins Cry2 and Cry4, at a dose of 300 pg/mL, are also active against Caligus, but with greater action times. 100% mortality of adult caligus is reached in 240 minutes of treatment with Cry2, and 90% mortality of adult caligus treated with Cry4 up to 360 minutes. On the other hand, Cry3 is inactive at a dose of 300 pg/mL, since the parasites treated with this protein remain viable, like the untreated control, suggesting thus that the action of Cryl, Cry2, and Cry4 against Caligus is specific. In an additional embodiment of the claimed invention it was determined if there is a synergistic effect between these proteins potentiating the antiparasitic activity. To this end, treatment assays were performed for C. rogercresseyi larvae with combinations of Cry proteins at a ratio of 1:1. The combinations used were Cryl/Cry3 and Cryl/Cry2, at a dose of 300 pg/mL each and Cryl/Cry4 at a dose of 150 mg/mL each. The assays showed that the Cryl/Cry2 combination is the most effective, since with Cryl/Cry2 at a dose of 300 pg/mL each, a larval mortality is reached near to the sum of the effects of both proteins separately. On the other hand, using a combination of 150 pg/mL of Cryl/Cry4 proteins allows reaching a slight increase in mortality of adult caligus over the mortality reached by each of the proteins separately. Furthermore, the Cryl/Cry3 combination does not evidence an increase in the effect observed for each of the proteins on their own.

Combined effect of Bt spores with Cry proteins

In another embodiment of the claimed invention, a combination of spores with recombinant Cry protein was tested. Larvae and adults of Caligus were treated in vitro with a composition or formulation of Bt spores at a concentration of lxlO ⁸ CFU/mL together with a CrylAb protein at different concentrations (25, 50, 100 and 150 pg/mL, respectively). Both for larvae and for adults, with the combination of spores and CrylA protein 100% mortality is reached in a lower time that with spores or Cry proteins on their own. The combination of spores and CrylAb protein at a dose of 150 pg/mL causes more than 70% mortality of adult caligus in 30 minutes of treatment, and 100% mortality is reached by 90 minutes. In larvae it is observed that the combination of spores with CrylAb at a concentration of 150 pg/mL have a better effect, reaching about 80% mortality in 30 minutes and 100% mortality in 90 minutes. Both for larvae and for adults of C. rogercresseyi, the combination of spores at a concentration of lxlO ⁸ CFU/mL with increasing concentrations of CrylAb, from 25 pg/mL to 150 pg/mL cause an incremental increase in mortality of C. rogercresseyi. In adults, a synergy is clearly observed in the antiparasitic effect of the composition consisting of spores and CrylAb from the concentration of CrylAb of 50 pg/mL, while in larvae the synergy is observed with the combination of spores and CrylAb at a concentration of 150 pg/mL. In all cases it can be appreciated that the synergistic effect of the combination as compared to each separate component causes a greater mortality of C. rogercresseyi, either larvae or adults. The synergistic effect was confirmed by the method described by Colby (1967), wherein it was observed that the expected survival percentage for the combination of spores and Cry protein duplicates the one obtained experimentally; therefore, a synergistic effect against adult Caligus was evidenced.

Another embodiment of the claimed invention corresponds to assays with a combination of Cry protein and different doses of spores, which showed that using a dose of lxl0 ⁶ CFU/mL to lxlO ⁹ CFU/mL combined with 150 ug/mL of CrylAb results in 75% to 100% mortality of caligus in 300 minutes of in vitro treatment.

Immersion baths in salmons: In vivo assays

In order to assess the effectiveness of the formulation of spores on their own and combined with the Cry protein, on parasites attached to the fish, in vivo assays were performed, wherein it was demonstrated that there is a similar effect to the one observed in in vitro treatments.

The first test consisted in the treatment by immersion with Bt spores. To this end, fish from the Salmo salar species infested with Caligus rogercresseyi were used, which were treated for 60 minutes with B. thuringiensis spores of the strain ABTS-351 obtained in bioreactor, reaching 27% mortality, similar to the percentage achieved with the same dose of these spores in in vitro assays.

Thereafter, an assay was performed, which consisted in the treatment by immersion of S. salar fish parasitized with a high load of C. rogercresseyi , to assess the antiparasitic effect of Bt spores of the strain ABTS-351 obtained in bioreactor, supplemented with a protein extract containing CrylAb. For that purpose, the fish were treated for 60 minutes with the composition of spores combined with the unpurified CrylAb protein, reaching 38% mortality. This result is also similar to the one observed in vitro.

Embodiments of the invention

In one embodiment, the invention relates to an aquaculture parasiticidal composition or formulation, comprising at least one of: a) spores of Bacillus thuringiensis subsp. Kurstaki strain ABTS-351, and/or ATCC-33679; and/or b) one or more recombinant proteins selected from CrylAb, Cry 1 Ac, Cry 2 and Cry 4.

The claimed invention, in an additional aspect, refers to an aquaculture parasiticidal composition or formulation as described above, comprising one or more recombinant proteins selected from CrylAb, Cry 1 Ac, Cry 2 and Cry 4.

In a preferred embodiment, the aquaculture parasiticidal composition or formulation comprises spores of a Bt strain var. Kurstaki ABTS-351 and/or ATCC-33679; and one or more recombinant proteins selected from CrylAb, Cry 1 Ac, Cry2 and Cry4.

Moreover, the aquaculture parasiticidal composition or formulation as described above comprises the recombinant protein CrylAb (SEQ ID NO: 2).

In addition, the aquaculture parasiticidal composition or formulation as described above comprises the recombinant protein CrylAc (SEQ ID NO: 4).

Furthermore, the aquaculture parasiticidal composition or formulation as described above comprises the recombinant protein Cry2 (SEQ ID NO: 6). In a further aspect, the aquaculture parasiticidal composition or formulation as described above comprises the recombinant protein Cry4 (SEQ ID NO: 8). In a preferred embodiment, the aquaculture parasiticidal composition or formulation as described above comprises spores of a Bt strain var. Kurstaki ABTS-351 and/or ATCC-33679.

In an additional aspect, the claimed invention refers to an aquaculture parasiticidal composition or formulation as described above, wherein the spores are present in a range between lxlO ⁶ and lxlO ¹⁰ CFU/mL.

In another preferred embodiment, the aquaculture parasiticidal composition or formulation as described above comprises the recombinant protein in a range between 25 and 300 pg/mL. Another aspect of the invention relates to a veterinary aquaculture parasiticidal composition or formulation, which comprises the aquaculture parasiticidal composition or formulation as described above, and a veterinarily acceptable carrier, selected from maltodextrin, sucrose, sorbitol and/or gelatinized corn starch. A preferred embodiment of the claimed invention relates to a veterinary aquaculture parasiticidal composition or formulation as described in the preceding paragraph, wherein the composition is present in a form selected from the group consisting of immersion bath, injectable formulation, or feed formula. The claimed invention also refers to the use of a veterinary aquaculture parasiticidal composition or formulation as described above, for preparing a veterinary medicament useful for treating fish infested with the sea lice parasite, wherein the sea lice belongs to the genera Caligus and Lepeophtheirus.

In a preferred embodiment, the claimed invention refers to the use of a veterinary aquaculture parasiticidal composition or formulation for preparing a veterinary medicament useful for treating fish infested with the sea lice parasite, wherein the sea lice belongs to the genera Caligus and Lepeophtheirus.

Another aspect of the claimed invention relates to an aquaculture parasiticidal kit comprising one or more containers containing spores of Bacillus thuringiensis subsp. Kurstaki strain ABTS-351 and/or ATCC-33679, and/or one or more recombinant proteins selected from CrylAb, Cry 1 Ac, Cry2 and Cry4, preferably with a veterinarily acceptable carrier, and an instruction for use insert.

It should be noted that the results of the claimed invention are different from the knowledge previously existing in the prior art in relation to the required dose, formulation thereof, the medium where it is used (seawater), the directions for use, and the organism to be controlled. Hence, the formulation developed in the claimed invention contains Bt spores or recombinant Cry proteins or a combination of both. It is soluble (or homogenizable) in seawater. It is administered through different routes, including immersion baths. It is applied to control parasites (both copepodites and adults) of Caligus in fish, all of which is not derived in an obvious manner from the prior art.

VI. EXAMPLES In the examples described next for the claimed invention, wherein Bt spores alone or together with recombinant Cry proteins are used, they are based on the use of a composition when they are applied combined in the different assays for controlling caligus, or when applied as a formulation wherein the Bt spores and the recombinant Cry proteins can be used separately and/or combined, as required, for controlling caligus.

Example 1: In vitro antiparasitic activity of Bacillus thuringiensis spores against Caligus rogercresseyi. 1.1. Effect of Bt spores on the survival of copepodites

In order to assess the antiparasitic efficacy of Bacillus thuringiensis (Bt) spores against caligus in larval stages (copepodites), the effect of spores from different strains of the bacterium were compared to different doses. The strains used correspond to Bacillus thuringiensis subsp. Kurstaki strain ABTS-351, SA-11, strain ATCC-33679, and to a mixture of three Chilean native strains of Bacillus thuringiensis (N1-N2-N3) (Figures 1 and 2).

In order to carry out the bioassays different amounts of spores were used, which were added to Petri dishes containing about 25 copepodites in 1.5 mL of seawater. The toxicity of the spores was evaluated during the treatment time (0, 30, 60, 90, 120, 180, 240, 300 and 360 min), verifying the mortality of the larvae by means of a stereoscopic lens. The experiment was performed using the following spore doses: lxlO ² , lxlO ⁴ , lxlO ⁶ , lxlO ⁸ , lxlO ¹⁰ CFU/mL. The spores were resuspended in seawater for each of the aforementioned doses. Each dose and condition were assayed in duplicate, and an untreated control was incorporated to confirm the viability of larvae. The resulting mortality was plotted after 240 minutes of treatment (Figure 1). It is observed that the spores from all Bt strains assayed, at doses greater than lxlO ⁹ CFU/mL, affect the survival of copepodites. Doses over lxlO ⁹ CFU/mL cause 100% mortality in 240 minutes of treatment.

Based on the survival graph relative to different spore doses (CFU/mL), the doses causing 50% mortality of copepodites (LD50) was calculated. The spores from the strain ABTS-351 are the ones showing a lower lethal dose and, therefore, they are more active against caligus. The results are summarized in Table 1.

TABLE 1: LD50 against Caligus Rogercresseyi larvae

^~ B. thuringiensis ABTS-351 SA-11 N1-N2-N3 spores

1.2. Effect of Bt spores on the survival of Caligus rogercresseyi.

B. thuringiensis strain ATCC-33679 was used to assess the effect thereof on adult caligus as compared to spores from the strains ABTS-351. The antiparasitic effect of the spores of Bt strain ATCC-33679 on adult caligus was analyzed. For this purpose, 10 motile adult parasites were placed in Petri dishes, in a final volume of 1.5 mL of seawater. The spores of Bt strain ATCC- 33679 were used at a dose of 2xl0 ⁹ CFU/mL, as compared to the same dose of spores of strain ABTS-351 obtained under the same conditions. Each assay was carried out in duplicate. Figure 2 shows the caligus survival results, wherein it is observed that spores of strain ATCC-33679 and the spores of strain ABTS-351 reach between 30% and 40% mortality of adult caligus in 300 minutes. Therefore, it is suggested that the spores of strain ATCC-33679 have an antiparasitic activity similar to that of the spores of strain ABTS-351, when comparing the spores produced under the same laboratory conditions. For both strains, the bacteria were grown in LB liquid medium and then their sporulation was induced in T3 medium (Peptone lOgr/Lt, NaCl 5gr/Lt, Glucose 34gr/Lt, yeast extract 20gr/Lt, MnCF 20mg/Lt) for 5 days at 28°C under stirring (200 rpm). The cultures were centrifuged at 5000 rpm for 30 minutes at 4°C, and the supernatant was removed, the spores were resuspended in 15 mL of sterile water, frozen at -80°C and then were subjected to lyophilization for 24 hours. A yield of 1.85xl0 ⁹ CFU/g of spores was obtained.

1.3. Culture of Bt strain ABTS-351: Production of spores under controlled conditions in bioreactor and control of C. rogercresseyi

An optimization of growth, sporulation and Cry production was performed by controlling different variables, such as pH, p02 and temperature, concentration of salts, culture time, etc. Eighteen different variables were assessed, and the experimental conditions in bioreactor were selected with better sporulation yield. The culture medium used contains Peptone lOgr/Lt, NaCl 5gr/Lt, Glucose 34gr/Lt, yeast extract 20gr/Lt, nCF 20mg/L, and ZnCF 40mg/L. It was seeded with 20% pre inoculum and incubated for 8 days at 28°C.

The resulting spores of Bt strain ABTS-351 were freeze-dried and used in in vitro bioassays at a dose of 2xl0 ⁸ CFU/mL against adult caligus and against larval stages of the parasite.

Figure 3 shows the survival of adult caligus, treated with Bt spores produced under the optimized experimental conditions. It was observed that these spores cause 100% mortality of adult caligus at a dose of 2xl0 ⁸ CFU/mL. Therefore, when the sporulation yield and production of Cry crystals in the spores was optimized, a better caligus killing activity in adults was obtained, which allowed reducing between 10 and 100 times the active dose. Similar results were obtained by treating caligus larvae (copepodites) with the spores obtained in bioreactor. Figure 4 shows the survival of copepodites treated with Bt spores produced under optimized fermentation conditions. It was observed that with a dose of 2.8x10 ^s CFU/mL, spores cause 100% mortality of larvae in 180 minutes, while in 60% of treatment more than 60% mortality is achieved.

Example 2: In vitro antiparasitic activity of recombinant Cry proteins against C. rogercresseyi

Some Cry proteins present in B. thuringiensis subs. Kurstaki were selected to obtain them in recombinant systems that overexpress proteins.

2.1. Expression of Cry proteins in E. coli BL21 (DE3)

To this end, pET21b expression vectors were synthesized with the gene sequence of different Cry proteins. The gene sequences encoding for proteins Cryl (CrylAb and Cry 1 Ac), Cry2 (Cry2Ad), Cry 3, and Cry4 were selected, which were inserted into the multiclonation site between the Ndel and Xhol restriction sites. With these vectors, E. coli BL21 (DE3) cells were transformed, wherein protein expression was induced with IPTG, and were purified by affinity chromatography of the histidine tag present in the recombinant peptide, using a Ni-NTA agarose column. The proteins were solubilized in an alkaline buffer (0.05M NaiCC , 0.005M b-mercaptoethanol, pH 10).

The Cry (CrylAb, CrylAc, Cry2Ad, Cry3, and Cry4) proteins were obtained from inclusion bodies or insoluble moiety of E. coli, after homogenizing and breaking open the cells by sonication, they were centrifuged and the pellet was treated with a basic solution at a pH of 10 (50 mM Na ₂ C0 ₃ , 5 mM b-mercaptoethanol) and then resuspended under constant stirring at 37°C for 2 hours. Once resuspended, the pellet was centrifuged in a Sorvall centrifuge at 6000 rpm for 1 hour using a SS-34 rotor (4300 G) to obtain the proteins from the supernatant. The proteins were purified by affinity chromatography, using a Ni-NTA agarose resin and imidazole 50 mM and 300 mM to elute the protein moieties. The moieties containing the protein of interest were quantified according to the Bradford method and analyzed by 10% SDS-PAGE.

Figure 5 shows the elution profile of the protein CrylAb (130 kDa). The protein elutes with 50 mM imidazole, as observed in moieties 1-1 and 1-2, reaching a purity between 82.9% and 85.1%. The experimental yield for CrylAb was 0.6 mg of protein/gram of bacterial pellet.

2.2. Expression of CrylAb in Hansenula polymorpha

The B. thuringiensis CrylAb protein was expressed in Hansenula polymorpha yeast using the pFPMT-M-CRYlA-6his plasmid to transform the strain RB11. These transformed yeasts showed an intracellular accumulation of CrylA, reaching comparatively high levels of up to ~ 30% of the total cell protein.

Thereafter, the yeasts were subjected to fermentation in order to produce biomass of the strain Hansenula polymorpha RBll/pFPMT-M-CrylA-6his, which produces intracellular Bacillus thuringiensis CrylAb. At the end of the fermentation, an optical density at 600 nm of about 330 OD was reached, corresponding to a biomass yield of 94 g dcw/F (dew: dry celular weight). The yeast was grown in YP/YNB culture broth with glycerol (20 g/1 yeast extract, 40 g/1 soybean peptone, 3.4 g/1 YNB without ammonium sulfate, 10 g/1 (NH ₄ ) ₂ S0 ₄ , 30 g/1 glycerol, pH 6), and a cell paste (dew) was collected by decantation. With the cell paste a cell disruption mechanical method was standardized to break open the yeast cells and obtain an extract of soluble proteins, which contained about 25% of recombinant CrylAb, relative to the total protein content of the extract. In the solubilization of the protein extract, an alkaline buffer (50 mM sodium carbonate pH 10, 5 mM b-mercaptoethanol, 10% glycerol, and protease inhibitors). The total protein yield for the protein extracts was 50.53 mg of total protein/g of cell paste.

In order to determine the antiparasitic activity of CrylAb produced by the strain Hansenula polymorpha RBll/pFPMT-M-CrylA-6his, the protein was purified, which contained a histidine tag (about 5 mg total protein/mL), from a 10 mL aliquot of protein extract supernatant, by means of an affinity chromatography using a Ni-NTA agarose resin in alkaline buffer. The protein moieties were collected by imidazole washings (50-300 mM). Figure 6 shows the purification results of CrylAb from Hansenula polymorpha. The protein elutes with 50 mM imidazole, as observed for the moieties 1-2 and 1-3, and 300 mM imidazole in the moieties 2-1 and 2-2.

2.3. Antiparasitic activity of recombinant Cry proteins with C. rogercresseyi larvae

In order to verify if the Cry proteins expressed in E. coll BL21 (DE3) and purified have any effect on C. rogercresseyi , in vitro treatments were performed using caligus larvae.

The treatment of larvae was carried out using 300 pg/mL of CrylAb, Cry2 and Cry3 proteins. With CrylAb protein in 300 minutes of treatment 100% larval mortality was obtained (Figure 7). When the larvae were treated with Cry2 protein, about 90% mortality was obtained in 300 minutes, while the larvae treated with Cry 3 protein showed no mortality.

In order to determine larval mortality, the larvae were observed in a stereoscopic lens (Lieder) with a 4x zoom, which allow distinguishing an apparent lack of motility from the death of the copepodite. Moreover, after these larvae were treated, they were observed in an optical microscope (Motic AE2000) with a lOx zoom, which allow noticing more detailed structures both internally and externally, due the translucid nature of these organisms. Therefore, it was possible to observe some "bubble-like" structures within those copepodites treated with the CrylAb protein or with a combination of Cryl/Cry2, which are not observed in untreated larvae, as shown in Figure 8. Hence, these results demonstrate that Cryl and Cry2 proteins have an antiparasitic effect against C. rogercresseyi larvae, and that they can cause 100% mortality during this stage of development. It was also evidenced that the Cry3 protein has no antiparasitic activity against the C. rogercresseyi sea lice.

2.4. Antiparasitic activity of Cry proteins against Caligus rogercresseyi adults In order to demonstrate if the Cry proteins expressed in E. coli BL21 (DE3) and purified by affinity chromatography have antiparasitic activity on adult caligus, in vitro assays were carried out using 300 pg/mL of CrylAb, Cry2, Cry3 and Cry4 proteins, wherein each protein was assessed separately (Figures 9 and 10). As shown in Figure 9, 100% mortality of adult caligus treated with CrylAb is obtained in 2 hours of exposure. The treatment with the Cry2 protein causes 100% mortality in 4 hours of exposure. The treatment with the Cry3 protein does not cause mortality in adult caligus, as observed for larvae treated with this protein. Figure 10 shows that adult caligus treated with the Cry4 protein achieved 90% mortality in 360 minutes with a dose of 300 pg/mL, while in the same in vitro assay, the CrylAb protein causes 100% mortality in 240 minutes.

Upon watching the adult caligus through the optical microscope (Motic AE2000) with a xlO zoom, the presence of small bubbles in the ovigerous sacs of females treated with the CrylAb protein was observed, as well as an apparent loss in the structure of the ovigerous sacs (Figure 11).

Furthermore, the antiparasitic activity of Cry A proteins (CrylAb and Cryl Ac produced in E. coli BL21 and CrylAb produced in H. polymorpha ) was compared. To this end, 10 adult caligus were placed in Petri dishes with seawater and treated with 150 pg/mL of each CrylA protein. Figure 12A shows that both recombinant CrylAb proteins have control over Caligus, regardless of their origin (prokaryotes or eukaryotes), with a range between 60-80% mortality of the parasite. On the other hand, Figure 12B shows that the CrylAb protein is more active against the parasites than Cry 1 Ac.

With the results obtained from bioassays with adult caligus, it was demonstrated that the recombinant proteins CrylAb, Cry 1 Ac, Cry2 and Cry4 have an antiparasitic effect against this parasite species and cause 100% mortality on adult caligus and also on the copepodite stage (larvae), wherein the parasiticidal effect of the CrylAb protein is relevant, expressed either in E. coli or in H. polymorpha.

Example 3. In vitro antiparasitic effect of combinations of the CrylAb protein with other recombinant Cry proteins and with B. thuringiensis spores, against C. rogercresseyi.

3.1. Effect of the combination of Cry proteins against C. rogercresseyi

In order to determine if a combination of Cry proteins may favor the antiparasitic effect against C. rogercresseyi larvae, copepodites were treated with combinations of Cry proteins at a ratio of 1:1, using a dose of 300 pg/mL of each protein. As shown in Figure 13, the Cryl/Cry3 combination causes about 20% mortality in 60 minutes, while the Cry 1/Cry 2 combination causes 50% mortality in the same lapse of time, which is an effect near the sum of both proteins.

The combination of Cryl and Cry4 at a dose of 150 pg/mL in a ratio of 1:1 caused 100% mortality in 360 minutes, and from 180 minutes it shows a slight increase in mortality as compared to the effect of each of the proteins separately. 3.2. Bt spores formulated with the CrylAb protein

3.2.1. Bt spores formulated with the CrylAb protein produced in E. coli BL21.

In order to reduce the action times of the Bacillus thuringiensis spores and to obtain a better antiparasitic effect, a formulation or composition of spores of Bt strain ABTS-351 with the recombinant CrylAb protein was assayed.

Figure 15 shows the effect on adult caligus of the Bt strain ABTS-351 spore formulation (dose lxlO ⁸ CFU/mL) combined with the CrylAb protein produced in E. coli in different doses. It is observed that with the formulation of spores and CrylAb protein 100% mortality is caused in a lower time than with spores or Cry protein on their own. The formulation of spores lxlO ⁸ CFU/mL) and CrylAb (150 pg/mL) causes more than 70% mortality of adult caligus in 30 minutes of treatment, and 100% mortality in 90 minutes.

Figure 16 shows the effect on copepodites of supplementation of Bt strain ABTS-351 spores with the CrylAb protein, produced in E. coli. In larvae it is observed that the spore formulation (dose of lxlO ⁸ CFU/mL) combined with 150 pg/mL of CrylAb cause about 80% mortality in 60 minutes of treatment, while spores on their own cause less than 30% mortality in the same time of exposure. In 90 minutes, the formulation of spores and CrylAb protein causes 100% mortality of caligus.

3.2.2. Bt spores formulated with CrylAb produced in H. polymorpha

The antiparasitic activity of supplementation of Bt strain ABTS-351 spores (at a dose of lxlO ⁸ CFU/mL) with the purified CrylAb protein purified from Hansenula polymorpha, at a dose of 150 pg/mL, was assessed in vitro by comparing it to the effect observed with the CrylAb protein and spores on their own. In these assays, 10 caligus specimens were used per Petri dish, in duplicate, using a final volume of 1.5 mL of seawater. The obtained results are shown in Figure 17. It is observed that the supplementation of spores with CrylAb purified from H. polymorpha causes 100% mortality of parasites in 90 minutes of treatment, and near 90% mortality in 60 minutes of treatment.

It can be appreciated that both for larvae and for adults of C. rogercresseyi the spore formulation at a concentration of lxlO ⁸ CFU/mL combined with increasing concentrations of CrylAb, from 25 pg/mL to 150 pg/mL causes an incremental increase in C. rogercresseyi mortality. In adults it is clearly observed a synergy in the antiparasitic effect of the formulation consisting of spores and CrylAb from a concentration of 50 pg/mL, while in larvae the synergy is observed with the combination consisting of spores and CrylAb at a concentration of 150 pg/mL. In both cases, it can be appreciated that the combined effect of the formulation over each component separately causes a greater C. rogercresseyi mortality, in either larvae or adults.

The synergistic effect proven in the claimed invention is a surprising technical effect of the combination, which cannot be attributed or derived from the separate effects of the spores or the CrylAb protein.

3.2.3. Bt spores at different concentrations combined with the recombinant CrylAb protein

The antiparasitic activity of the supplementation of Bt strain ABTS-351 spores at different concentrations (lxlO ⁶ to lxlO ⁹ CFU/mL), with the CrylAb protein at the concentration of 150 pg/mL was assessed in vitro. Table 2 shows the results of the treatments, which evidence that using a dose of lxlO ⁶ CFU/mL to lxlO ⁹ CFU/mL combined with CrylAb (150 pg/mL) allowed achieving 75% and 100% mortality of caligus. Table 2: Mortality of adult caligus in 300 minutes of in vitro treatment when using a combination of Bt spores at different concentrations with CrylAb at the concentration of 150 pg/mL.

Example 4. Assessment of synergy between the parasiticidal effect of spores and CrylAb protein: Colby's method

Due to the significant increase in antiparasitic activity obtained when the parasites are treated using a formulation of spores combined with the CrylAb protein, the existence of a synergistic effect of the formulation was evaluated, using Colby's method (Colby, 1967).

Colby's method is based on a mathematical formula that allow predicting the expected response from a combination of herbicides. This formula is as follows:

If X = percentage of growth inhibition produced by herbicide A, at an R concentration and Y = percentage of inhibition of herbicide B, at an S concentration and E = percentage of inhibition of herbicides A and B, at an R+S concentration

Then E = X + ^Y(1

1 ⁰ 0 ⁰ 0 ^~x)

E = X +Y - — 100

If the growth percentages are considered according to the variables:

Xi = growth percentage relative to the control with herbicide A, at an R concentration Yi = growth percentage relative to the control with herbicide B, at an S concentration Ei = growth percentage relative to the control with herbicides A and B, at an R+S concentration Then, the equation is simplified as follows:

Ei = 100 - E

Xi = 100 - X Yi = 100 - Y

YY

Ei = 100 - (X + Y - — _l otr )

Ei = 100

Ei =^ 100

Using this latter equation, growth can be considered as percentage of parasite survival. Therefore, this equation allows predicting the expected survival value for the combination of two components of a formulation. If the expected survival value is greater than the observed experimental value, this means that there is synergy between the components; on the contrary, when the expected survival value is lower than the one observed, then antagonism is observed. In the claimed invention, the expected survival value of caligus treated with a combination of spores at a dose of lxlO ⁸ CFU/mL and CrylAb protein at a concentration of 150 pg/mL was considered. The survival percentages of adult caligus were assessed in 60 and 90 minutes of treatment, using an average of 5 replicates of in vitro treatments. For example, when the equation is replaced with the survival values in 90 minutes for CrylAb and spores, the following is obtained: i 100 100 33.03 % exp ^r ected survival

The expected survival percentage is 33.03%, this value is higher (almost double) than the one observed experimentally (16%); therefore, it is demonstrated that the formulation of spores with CrylAb has a synergistic effect against adult caligus. A similar result of synergy can be observed in 60 minutes of treatment. The results obtained in 60 and 90 minutes are shown in Table 3. Table 3. Percentages of adult caligus survival in 60 and 90 minutes of treatment and determination of the synergistic response

Time CrylAb Spores Spores+CrylAb Synergistic

(min) (150 (lxlO ⁸ Observed Expected effect* pg/mL) CFU/mL) effect effect

^~ 90 45% 73.4% 16% 33.03% Yes

* If the expected effect is greater than the observed effect, according to the method described by Colby, 1967, there is synergy.

Example 5. In vivo antiparasitic effect of Bt spores and formulation of spores with CrylAb protein in salmons

In order to carry out a treatment of parasitized salmons with Bt spores and combination of spores with CrylAb protein, greater amounts of spores and recombinant protein are needed than the amounts used in in vitro bioassays. Hence, firstly a pilot scale of the fermentation process was made using a 300 L bioreactor, to obtain spores of Bacillus thuringiensis subsp. kurstaki ABTS- 351. The biomass resulting from the pilot-scale fermentation was retrieved by microfiltration and dried by lyophilization. The final product achieved spore levels of 7xl0 ¹⁰ CFU/g and a total of 2.5 Kg of dry product was obtained.

For the larger-scale production of CrylAb, the protein overexpression system in Hansenula polymorpha was used. The yeasts were fermented, obtaining a protein extract containing a total of 55.56 g of protein. The resulting protein extract contains 30% of the recombinant CrylAb protein according to the estimations based on the polyacrylamide gel analysis using the Gel Pro Analyzer program, version 3.1.

After the larger-scale production of spores and proteins, the in vivo assay was performed to determine the antiparasitic activity of spores, and spores combined with protein extract containing CrylAb on adult caligus attached to the fish.

5.1. In vivo antiparasitic effect of Bt spores

The fish ( S . salar, about 300 g) were subjected to an immersion treatment with Bt strain ABTS- 351 spores at a dose of lxlO ⁸ CFU/mL, in 500 L tanks, and they were compared to control fish that received an immersion treatment only with seawater.

Before starting the assay, a counting of caligus per fish was made, to which end the fish were anesthetized before the counting, and then recovered in seawater with saturated oxygen. The parasitized fish were homogeneously distributed in the tanks, for each tank to have a mean of 6.7 caligus/fish before the bath (about 67 total caligus per tank). Once the fish were recovered, the seawater volume was reduced in the tanks to 200 L, and then they were subjected to the bath for 60 minutes with Bt spores. The available oxygen was measured throughout the assay, which was kept in about 8 ppm. Finally, the fish were removed from the tanks and the caligus specimens per fish were counted after the treatment. No mortality or side effects was observed in the treated fish. Table 4 summarizes the results obtained.

Table 4: Results of the antiparasitic bath of fish with Bt strain ABTS-351 spores, under controlled conditions.

Figure 18 shows the survival percentages of caligus attached to the fish, after the immersion treatment with spores. A 27% decrease in the parasitic load of fish treated with Bt spores was obtained after 60 minutes of treatment, a similar result to the one observed in vitro with spores produced under the same conditions.

5.2. In vivo antiparasitic effect of Bt spores combined with the CrylAb protein

For this in vivo antiparasitic efficacy assay, fish of the Salmo salar species were used, weighting about 350 g, maintained in 500 L tanks (30 fish per tank), infested with a high load of Caligus rogercresseyi (47.4 caligus/fish). The fish were subjected to an immersion (bath) treatment for 60 minutes, reducing the volume of the tanks to 120 L, with the formulation of Bt strain ABTS-351 spores at a dose of lxlO ⁹ CFU/mL combined with CrylAb produced in Hansenula polymorpha, without purification (protein extract at a concentration of 420 pg/mL). The antiparasitic effect of the formulation was compared to a control tank, wherein the tank volume was reduced to 100 L, and then seawater was gradually added for about 5 minutes, to complete 120 L, and the fish were maintained there for 60 minutes. Once the immersion treatments were finished, about 60 minutes after, the total tank volume (500 L) was completed and after 2 hours of recovery, 15 fish were sampled from each tank to count the parasites attached.

Table 5 shows a summary of the results obtained after the treatment of infested fish with a high parasitic load of Caligus rogercresseyi , with the formulation of Bt spores supplemented with the protein extract (420 pg/mL), containing 30% of CrylAb, equivalent to about 126 pg/mL of CrylAb protein.

Table 5: Results of the antiparasitic bath in parasitized S. solar fish with Bt spores combined with CrylAb proteins (420 pg/mL), under controlled conditions.

* Pre-treatment = 711 mean parasites counting 15 fish (average N°caligus/fish = 47.4)

Figure 19 shows the survival percentage of caligus attached per fish after treatment with the formulation of Bt strain ABTS-351 spores supplemented with protein extracts containing CrylAb without purification, compared to the survival percentage of caligus from the control tank. It was observed that the control has a caligus loss due to manipulation of about 10%, while the treatment with the formulation of spores and protein extract causes almost 38% mortality of caligus. This result is similar to the one observed in vitro with spores and proteins produced under the same conditions (see Figure 19B).

Example 6. Innocuity of Bacillus thuringiensis in fish

6.1. Effect of Bt spores on salmonids

In order to rule out that Bt is toxic to fish, an assay was carried out to assess the effect of the spores on salmonids. To this end, different amounts of Bt strain ABTS-351 were administered, both in the feed and in a 90-minute immersion treatment (bathed with Bt) of S. salar fish of about 40 g, kept in 207 L tanks with fresh water. The administration of spores in the feed was repeated daily, for 11 consecutive days, while the immersion treatment was conducted on 6 non-consecutive days. Table 6 shows a summary of the tested doses and mortality caused by the treatments. Table 6: Effect of Bt on Salmo salar

None of the treatments caused mortality on fish after 11 days of record. Those fish treated with Bt spores showed neither side effects, nor physiological differences relative to the untreated control fish.

6.2. Effect of CrylA on salmonids

On the other hand, assays were performed to verify the innocuity of the CrylA (CrylAb and Cry 1 Ac) protein, produced in E. coli, and also of the CrylAb protein produced in H. polymorpha. Proteins CryAb and CrylAc were administered by injection, and the CrylAb protein was also administered by immersion and in the feed of fish, kept in 209 L tanks with fresh water. Under none of the conditions assayed there was an appearance of side effects observed at the doses tested. 6.2.1. Injection of recombinant CrylAb and CrylAc

The Cry protein toxicity assessment assay was carried out by injecting 300 pg of CrylAb (Test 1) and 300 pg of Cry Ac (Test 2) (contained in a 100 pL injection volume), which corresponds to a dose of about 150 pg/mL in the serum of 30 g fish ( S . salar). As control, 10 fish were injected with 100 pL alkaline buffer (buffer where CryAb is resuspended) (Cl) and 10 fish were injected with 100 pL Tris buffer (buffer where CrylAc is resuspended) (C2). The injected fish were kept under observation for 15 days. Over the course of the assay, the behavior and appetite of fish were daily evaluated, and 3 fish were subjected to necropsy at the beginning and end of the assay in order to assess the possible damage and toxicity of the administrated protein. After 15 days of observation, there was no fish mortality or appearance of side effects, indicating that proteins CrylAb and CrylAc are innocuous to fish at the administered doses (Table 7).

Table 7. Observation of Salmo salar treated with CrylAb and CrylAc by injection.

6.2.2. Immersion bath with H. polymorpha extract containing CrylAb An immersion bath was prepared for a total of 10 fish weighing about 100 g each, which were treated for 60 minutes with protein extract of H. polymorpha yeast containing CrylAb at a concentration of 420 mg/L in a final volume of 20 L, and they were compared to a control of untreated fish and fish bathed with (5%) alkaline buffer. The fish were kept under observation in recirculation tanks for 10 days post treatment, wherein they showed normal appetite and behavior (Table 8).

Table 8. Observation of S. salar behavior for 10 days after CrylAb bath.

6.2.3. Oral administration of yeast expression CrylAb

In order to determine if the recombinant CrylAb protein produced in H. polymorpha is safe when mixed with fish feed, 300 pg CrylAb were daily administered per fish (corresponding to 67 mg of H. polymorpha yeast/fish/day) and a total of 20 S. salar fingerlings weighting 30-40 g were daily fed for 15 days (Table 9). As control, 20 fingerlings were fed with normal feed for the same period. After 15 days of oral treatment, no fish mortality and no side effects are observed, suggesting the innocuity of H. polymorpha expressing CrylAb. Table 9. Observation of S. salar fish behavior fed for 15 days with H. polymorpha expressing CrylAb.

Example 7: In vivo antiparasitic effect of CrylAb, administered by injection to salmons

In order to demonstrate that the antiparasitic formulation, in particular the CrylAb protein, can be administered by different routes to salmons, this test assessed the efficacy against C. rogercresseyi, of the CrylAb protein produced in E. coli, in an injectable formulation.

The CrylAb protein was administered by injection to 4 Atlantic salmon post-smolts weighting about 120 g, infested with caligus. Each fish received a 130 pL injection of a 9.3 mg/mL solution of CrylAb purified from E. coli (1.2 mg CrylAb were injected per fish) and its effect was compared to 4 control fish, which were injected with the same volume of physiological serum (0.9% NaCl). The fish were kept at 12°C ± 1°C, with 70% oxygen saturation. Prior to the injection, the fish were infested with an average of about 35-45 larvae (copepodites) per fish. The injection was applied when most of the lice were in the developmental Chalimus stage, and twelve (12) days after the injection, the antiparasitic efficacy of injectable CrylAb was assessed, by counting the sea lice attached to the fish, in Chalimus and mobile adult stages. To perform the counting of caligus/fish, the fish were anesthetized with benzocaine and the number of caligus attached to the fish was registered, as well as those arranged in the sampling container. The measurement of the antiparasitic effect of Cry 1 Ab in an injectable formulation was determined in terms of the counting of mobile lice (adult) and chalimus attached to each fish post treatment.

It was demonstrated that, after 12 days post injection, the fish treated with CrylAb in an injectable formulation show a decrease in the number of lice attached, relative to the control untreated fish kept only in sea water (see Figure 20 and Table 10). While in the untreated control caligus keep reproducing, reaching 128% relative to the initial number of parasites attached, in the treated fish an 86% reduction of parasites attached is observed relative to the initial number.

Table 10. Count of total caligus (Chalimus and adult) attached to the fish under each condition.

Example 8. In vitro antiparasitic effect of combinations of CrylAb protein and B. thuringiensis spores against Lepeophtheirus salmonis (L. salmonis).

In order to demonstrate that the antiparasitic formulation or composition has an effect on other pathogenic species, such as Lepeophtheirus salmoni , the effect of the formulation or composition of spores of Bt strain ABTS-351 with recombinant CrylAb protein was tested in in vitro assays.

8.1. Assessment of the antiparasitic effect against adult L. salmonis

The in vitro antiparasitic activity of the complementation of spores of Bt strain ABTS-351 was determined in different doses: lxlO ⁷ , lxlO ⁸ , lxlO ⁹ and lxlO ¹⁰ CFU/mL, with the CrylAb protein purified from E. coli, in the following doses: 25 pg/mL, 75 pg/mL and 150 pg/mL, as compared to the effect observed with CrylAb and spores alone. In this assay, 10 specimens of adult L. salmonis were used per plate, in a final volume of 2 mL of sea water. Once the treatment is applied, the plates are observed, verifying the motility of parasites each 30 minutes, over 6 hours. The obtained results are shown in Figure 21. Bt spores alone cause a slight effect on sea lice, which reduce their motility (score 1) after 120 minutes of treatment (Figure 21B). However, when sea lice are treated with Bt spores at a dose of lxlO ⁹ CFU/mL in combination with the CrylAb protein at a dose of 150 pg/mL, the adult L. salmonis sea lice specimens are completely inactive (score 3), and their mortality is verified in 180 minutes of treatment (Figure 21C). On the other hand, the control group specimens showed no effects on their activity (Figure 21A). 8.2. Assessment of the antiparasitic effect against L. salmonis copepodites

The effect of spores of Bt strain ABTS-351 and the CrylAb protein on L. salmonis copepodites was assessed in vitro. In this assay, 10 L. salmonis copepodites were used per plate, in a final volume of 2 mL of sea water. Spores were used at a dose of lxlO ⁸ , lxlO ⁹ and lxlO ¹⁰ CFU/mL alone and combined with CrylAb purified from E. coli, at the following doses: 25 pg/mL, 75 pg/mL and 150 pg/mL. The behavior and motility of larvae was registered each 30 minutes, over 6 hours of treatment. The obtained results are shown in Figure 22, which exhibits that Bt spores at a concentration of lxlO ⁹ CFU/mL in combination with the CrylAb protein (150 pg/mL) cause mortality of L. salmonis larvae. From 60 minutes of treatment, copepodites sunk to the bottom of the wells, showing a reduced motility, wherein the only movement observed was similar to a mild shiver (short distance movement). This happened almost simultaneously in all copepodites treated with Bt spores at the highest dose and with the combination of Bt spores and CrylAb protein. The same effect was not observed with the CrylAb protein alone.

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