Feed composition for preventing infection or infestation of a parasite in fish

FIELD OF THE INVENTION

The present invention relates to a fish feed and to a composition as given in the preamble of the claims 1 and 17. The invention also relates to a use of a composition and methods of preventing an infection and/or infestation of a parasite in fish.

BACKGROUND TO THE INVENTION

Sea lice (Caligus rogercresseyi) infestation is for the time being one of the most important health challenges for the salmon production industry. This external parasite impairs production efficiency, causes stress and damages to the fish and is as a secondary infection a possible vector for other pathogens like the Infectious Salmon Anaemia virus (ISAv) and the Piscirickettsia salmonis. In Chile, like in many other countries producing Salmonids, infestation rates have increased significantly mainly affecting species like the Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynchus mykiss) and Coho salmon (Oncorhynchus kisutch). In Norway, sea lice infestations are considered as one of the most serious health problems in aquaculture of Salmon. This higher infestation rate is probably a consequence of a combination of factors including the development of extensive resistances of the sea lice to most of the applied chemical antiparasite agents used in treatment today.

Different plant extracts, terrestrial and marine plants, have been assessed for antibacterial, antifungical, antiviral and antioxidant activity for humans and animals. Certain polyunsaturated aldehydes, produced by terrestrial plant or diatoms have recently been described to have inhibiting or toxic effects in some animal species.

It is an important issue to find new compounds and ways to treat and prevent ectoparasites infestations in fish, especially of sea lice. Antiparasitic agents used for sea lice treatment or prophylaxis must be effective, but should at the same time not be toxic or harmful to the fish, other organisms or the environment in general.

Based on the problem described above, the objective of the present invention is thus to provide new effective agents and methods for inhibiting the infestation of sea lice and other copepod parasites in fish, which are not harmful for the fish or to the environment.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to a fish feed comprising conventional feed ingredients such as protein, lipid, carbohydrates, vitamins and minerals, wherein said fee comprises an alkadienal, optionally substituted in one or several positions with fluoride, chloride, hydroxyl or C1-C4 alkyl. Preferable, said alkadienal is not substituted.

Preferable, an alkadiene entity of said alkadienal contains 6 to 16 carbons, more preferable 7-12 carbons.

Preferable, said alkadienal is a decadienal.

Preferable, said alkadienal is 2-4-decadienal. Preferable, said alkadienal is trans-trans-2-4-decadienal. Preferable, said alkadienal is extracted from peanut oil, potato juice, diatoms, algae or marine fungus.

Preferable, the concentration of said alkadienal is 0.0009 wt% or more of the feed wet weight. Preferable, said fish feed is for a species selected from the family of Salmonidae.

Preferable, the Salmonidae is selected from Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), and Artie charr (Salvelinus alpinus).

Preferable, the feed is for a fish in fish farming. Preferably, the fish feed is for use in the inhibition of parasitic infections and/or infestation to a fish fed said fish feed, and/or for the treatment and/or prophylaxis of infection of said fish.

Preferably, the fish feed for use in the treatment and/or prophylaxis of infection and/or infestation of an ectoparasite. Preferably, the fish feed is for use in the treatment and/or prophylaxis of infection and/or infestation of sea lice.

Preferably, the fish feed is for use in the treatment and/or prophylaxis of infection and/or infestation of sea lice in salmonids.

Preferable, the sea lice are a Copepod species belonging to the family of Caligidae, preferably selected from Caligus rogercresseyi, Caligus elongatus, and

Lepeophtheirus salmonis.

A second aspect of the present invention relates to a composition comprising an alkadienal, optionally substituted optionally substituted in one or several positions with fluoride, chloride, hydroxyl or C1-C4 alkyl, for the treatment and/or prophylaxis of a parasite infection and/or infestation in a fish.

Preferable, in said composition is injected intraperitoneally to said fish.

Preferably, said composition is administered to said fish in an amount corresponding to 2.37 mg to 60 mg of said alkadienal per day, more preferable in an amount corresponding to 2.37 mg to 28 mg per day, and more preferably in a concentration of less than 28 mg per day.

Preferably, said composition is administered to said fish orally.

Preferably, said amount of alkadienal given orally to said fish is 0.013 mg to 0.026 mg per day, more preferable in an amount of 0.013 mg to 0.020 mg per day, and more preferably in an amount of 0.020 mg or less per day.

Preferable, said alkadienal is not substituted.

Preferable, an alkadiene entity of said alkadienal contains 6 to 16 carbons, more preferable 7-12 carbons. Preferable, said alkadienal is a decadienal.

Preferable, said alkadienal is 2-4-decadienal.

Preferable, said alkadienal is trans-trans-2-4-decadienal.

Preferable, said alkadienal is extracted from peanut oil, potato juice, diatoms, algae or marine fungus. Preferable, said fish feed is for a species selected from the family of Salmonidae. Preferable, the Salmonidae is selected from Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynchus mykiss), Coho salmon (Oncorhynchus kisutch), and Artie charr (Salvelinus alpinus).

Preferable, said parasite is an ectoparasite. Preferable, said ectoparasite is sea lice.

Preferable, said sea lice are a Copepod species belonging to the family of Caligidae, preferably selected from Caligus rogercresseyi, Caligus elongatus, and

Lepeophtheirus salmonis.

A third aspect of the present invention relates to a use of a composition according to one of the claims 17-32 for the manufacturing of a pharmaceutical composition for the treatment and/or prophylaxis of parasite infection and/or infestation.

Preferable, said parasite is sea lice, and wherein the composition is used for the treatment and/or prophylaxis of a fish, such as a Salmonidae is selected from

Atlantic salmon (Salmo salar), Rainbow trout (Onchorhynchus mykiss). A fourth aspect of the present invention relates to a method for preventing an infection and/or infestation with a parasite in fish characterized in that the fish is administered a composition according to any of the claims 17-32, or fed a feed according to any of the claims 1-16.

Preferable, the amount of ingested trans-trans-2-4-decadienal is at least 0.052 mg/fish, preferably from 0.052 mg to 0.311 mg/fish.

Preferably, the fish are fed said feed for at least 4 days, preferably for a period between 4 and 12 days.

A fifth aspect of the present invention relates to a method for inhibition of the frontal filament eversion in sea lice copepodids and/or for reducing the maturation, the reproductive ability of sea lice and/or the occurrence of sea lice larvae, and/or for reducing the number of sea lice in nauplius II developmental stage, wherein the fish is administered a composition according to any of the claims 17-32, or fed a feed according to any of the claims 1 -16. Preferable, the amount of ingested trans-trans-2-4-decadienal is at least 0.052 mg/fish, preferably from 0.052 mg to 0.311 mg/fish.

Preferable, the fish are fed said feed for at least 4 days, preferably for a period between 4 and 12 days, and more preferable for at least 12 days.

A representative compound is CAS No. [25152-84-5] with the formula:

Surprisingly it was possible by the present invention to provide a new agent useful in the treatment and prophylaxis of ectoparasite infestations, especially caused by sea lice in Salmonids.

While a direct treatment of fish in a water bath comprising said alkadienal

compounds, specifically the trans-trans-2-4-decadienal did not or only to a low degree affects the infestation with sea lice, it was surprisingly possible to affect the sea lice infestation and quantitative amount indirectly by feeding the fish a diet comprising said active alkadienal compound.

From a practical point of view it is an advantage that the fish does not have to be isolated or removed from the cultivation unit such as a sea cage in order to be treated, since the fish can simplify be fed the diet comprising the active compound. When e.g. using a water bath for external treatment, fish have often to be kept or transferred to an isolated/enclosed unit. This affords more handling and is thus more laborious and is furthermore stressful for the fish. In the present invention, it is thus possible to treat the fish in the cultivation units without isolating the fish. By the present invention it was thus possible to identify a new agent suitable for prophylaxis and treatment of ectoparasite infections in fish, which can be

administered directly endogenous to the fish such as by injecting the fish or by oral administration.

It will be appreciated that features of the invention described in the foregoing can be combined in any combination without departing from the scope of the invention. DESCRIPTION OF THE INVENTION

Embodiments of the invention will now be described, by the way of examples with reference to the following diagrams, wherein Figure 1 shows the trans-trans-2-4-decadienal persistency in sea water during a period of 15 days for three temperatures (10°C, 12°C and 14°C).

Figure 2 shows the Specific Feeding Rate (SFR %) in S. salar obtained daily for each treatment with trans-trans-2-4-decadienal and for the control group.

Figure 3 shows estimates of parameters describing the effects of the trans-trans- 2-4-decadienal dose in feed fed to Salmon and of the exposure time on various sea lice (C. rogercresseyi) stages (Figures 3 A-F). "DxT" denotes the mean estimate, thick lines 50% confidence intervals and thin lines 95% confidence intervals.

Figure 4 shows the frontal filament inhibition of copepodids of C. rogercresseyi. The copepodids were collected from sea lice females of the control feed treatment or from the treatment with feed comprising the higher dose of trans-trans-2-4- decadienal (0.0018 wt % of the feed wet weight). Lower cases indicate statistical significant differences. EXPERIMENTAL SECTION

Example 1

Assessment of trans-trans-2-4-decadienal persistency in sea water

The persistency of trans-trans-2-4-decadienal in sea water was tested for three different temperatures (10°C, 12°C and 14°C). 0.5 g/l of trans-trans-2-4-decadienal was diluted in sea water and incubated in triplicate for each temperature treatment in 250 ml bottles with aeration using an incubator for a period of 15 days. Samples (6 ml/ sample/bottle) were taken at the start of the test and after 1 , 3, 6, 10 and 15 days.

Each of the samples was processed as follows: Solid extraction:

Solid extraction was used to isolate the trans-trans-2-4-decadienal compound diluted in water in accordance with the method described in Pino-Marambio et al. (2007). trans-trans-2-4-decadienal was extracted in an EFS (Biotage, Uppsala - Sweden) column with a glass cartridge of 6 ml (layer C2 (500 mg) over ENV+ (200 mg)), using one cartridge per each replica.

Cartridges were prepared previously with 2 ml of methanol HPLC grade and the methanol was removed with water HPLC grade (2 ml). After the extraction of trans- trans-2-4-decadienal, impurities were removed with more water HPLC grade (2 ml). The chemical compounds absorbed in both phases, C2 and ENV+, were eluted with 2 ml of ethanol HPLC grade.

Distillation with vacuum:

Volatile and non-volatile fractions were obtained through a vacuum distillation. Distillation was performed during 24 hours with a pressure of 0.04 torr and ambient temperature (average 20°C). Liquid to liquid extraction:

Volatile (organic) fraction of the distilled was obtained though a liquid to liquid extraction, using diethyl ether (20ml) and distilled ether (3 x 20 ml). Organic phases were mixed and dried with CaSO . The extract obtained was concentrated until 100 μΙ and stored in vials (-18°C) until further analysis. Trans-trans-2-4-decadienal was determined using GC-MS analysis (Pino-Marambio et a/., 2007).

Results:

After 10 days of sea water exposure of the trans-trans-2-4-decadienal solutions, it was observed that in all the temperatures tested the trans-trans-2-4-decadienal disappeared completely in the solutions (Figure 1 ). In the case of the trans-trans-2-4- decadienal solution incubated at 10°C, a higher persistency from the third day until the sixth day was observed, compared with the trans-trans-2-4-decadienal solutions incubated at 12°C or at 14°C (Figure 1 ).

Example 2

Assessment of trans-trans-2-4-decadienal in feed for Salmon against sea lice infestation C. roaercressevi and reproduction.

The effect of trans-trans-2-4-decadienal on the infestation of Salmon S. salar by C. rogercresseyi and on the reproduction of C. rogercresseyi was tested.

Solutions of trans-trans-2-4-decadienal (SIGMA Aldrich (85% of purity)) were prepared with methanol and then added to coated feed for Salmon. The feed composition is given in table 1. The experimental set up of the feeding trial is shown in Table 2. Table 1 : Feed composition (%) used in example 2

Test

Ingredient Control diet diet 1 Test diet 2

Crude Protein (%) 46.9 46.9 46.9

Lipid (%) 22.6 22.6 22.6

Ash (%) 8.3 8.3 8.3

Nitrogen Free Extracts (%) 16.3 16.3 16.3

Phosphorus (%) 1.31 1.31 1.31

Trans-trans-2-4-decadienal

added in coater (%) 0 0.00090 0.00180

Table 2. Experimental set up and dosage regimen used in the testing of trans-trans-

2-4-decadienal in feed for Salmon.

Treatment trans-trans-2-4- Time (days)

decadienal dose (wt %

in feed)

1 (Control) 0 4

2 0.0018 4

3 (Control) 0 12

4 0.0018 12

5 0.0009 8

Three hundred Atlantic salmons (Salmo salar), with an average weight of 150 g, were infested with copepodids of C. rogercresseyi (80 sea lice/fish) in a 1.5 m ³ tank with open sea water flow system and an average temperature of about 13°C.

When 80% of sea lice reached the adult stages, 250 fish were selected for the experimental period, which were allocated in 25 tanks of 350 I (10 fish/tank), in an open sea water flow system with an average temperature of 13°C.

Atlantic salmon were fed for a period of 12 days with an average SFR (Specific feeding rate) of 0.9%. The different test diets containing trans-trans-2-4-decadienal in the concentrations as shown in Table 1 and 2. For each treatment there were 5 replicate tanks. To avoid loss of sea lice larvae during the trial, a mesh (80 pm) was added in the outlet of each tank. The meshes were cleaned daily, collected sea lice larvae were fixed with formaldehyde (5%) and counted subsequently. Larvae in the water were determined at the last day each of the treatments (day 4, 8 and 12, respectively). Additionally, the different life stages of sea lice attached to the Atlantic salmons (25 fish/tank) were counted, in accordance with the days detailed in Table 2 for each treatment. The variables analyzed were the following:

a) Number of mature sea lice females at days 4, 8 and 12.

b) Number of females with eggs strings of sea lice at day 4, 8 and 12.

c) Number of total embryos (eggs) per each string of sea lice at day 4, 8 and 12. d) Number of total larvae of sea lice at day 4, 8 and 12.

e) Number of non-viable larvae of sea lice at day 4, 8 and 12.

f) Number of nauplius II of sea lice at day 4, 8 and 12.

To evaluate the larval viability of sea lice, it was used an in vitro frontal filament inhibition method for copepodids infestation as described in detail below. Ten females with egg strings of sea lice were collected from each tank and then incubated (at 14°C) for 2 days in filtered (1 μητι) sea water. Copepodids were collected and induced for infestation in Petri dishes with agar and salmon mucus.

Frontal filament inhibition method for assessment of copepodids infestation (in vitro): Mucus was obtained directly from Salmo salar. Fish were anaesthetized with AQUI-S (BAYER, Germany) in a bath for about 5 minutes. Mucus was collected by scraping the body of the fish, thereby avoiding any contamination. The collected mucus was frozen in liquid nitrogen and stored at -80°C until further use.

Mucus samples were defrosted at room temperature and filtrated at 100 μηι for the retention of the scales and rest of tissues of fish, if necessary. 1 I of agar comprising mucus as attractant was prepared by dissolving 30 g agar in 1

1 distilled water with heat and constant stirring. 100 ml of the defrosted Salmo salar mucus was added to the obtained solution at less than 35°C followed by mixing. About 60 ml of the obtained mixture were added into Petri dishes (10 cm diameter and 2 cm of depth), and kept until a gel was obtained.

2 Petri dishes with the agar mixed with Salmo salar mucus (9: 1 ) were placed on the bottom of 2 I plastic containers which were filled with 2 I of filtered (0.1 pm) and UV- sterilized sea water and aerated constantly. The tests were carried out using 3 replicates for each concentration, each replicate being placed in one 2 I plastic container. A minimum of 50 collected copepodids from each treatment were deposited per container and left with aeration for 48 h. After 48 h, all copepodids of each container and of the Petri dish were extracted. Sea lice were transferred in Petri dishes containing a mixture of alcohol, distilled water and glycerol (45%, 45% and 10%, respectively) for fixation and dried in an oven at a constant temperature of 35°C for 4 hours. Each of the fixed samples was examined individually under a microscope (12 subsamples per treatment). The presence of the frontal filament in each copepodid fixed on a microscope slide was counted by studying the level of development of the frontal filament.

Results:

The water temperature in the trial varied between 12°C to 13°C and the oxygen saturation varied from 80% to 110%, with an average of 92%.

The Specific Feeding Rate (SFR %) obtained for each treatment was similar in the trial period (Figure 2). The highest average SFR (1.3 %) was recorded at the seventh day for all treatments, while at the first day the lowest SFR (0.57%) was recorded for doses 0.0009% of trans-trans-2-4-decadienal in feed and 0.0018% of trans-trans-2- 4-decadienal in feed, compared to the control (SFR 0.7%). The amount of trans- trans-2-4-decadienal consumed per fish is shown in Table 3. Table 3: Average specific growth rate (SFR), average fish weight, time (days), feed consumed (g), concentration of trans-trans-2-4-decadienal (%) and amount of trans- trans-2-4-decadienal consumed per fish.

trans-trans-2-4- trans-trans-2-4-

Fish Total feed decadienal decadienal

SFR weigth Time consumed in feed (%) consumed/fish

(g) (days) (g) (mg) (mg)

0.90% 160 12 17.28 0.0009 0.0018 0.15552 0.31104

0.90% 160 4 5.76 0.0009 0.0018 0.05184 0.10368

The deleterious effects of trans-trans-2-4-decadienal in the females, embryos and larval stages of Caligus rogercresseyi are shown in Figure 3 A-F. In general, all data analyzed were over dispersed.

Total eggs

There was not found a relevant interaction effect between the trans-trans-2-4- decadienal dose and the exposure time (=number of days when the fish were fed the different diets) but there were observed clear effects of both the trans-trans-2-4- decadienal dose and the exposure time (Figure 3A). For higher trans-trans-2-4- decadienal doses in feed, it was obtained lower quantities of egg per female. While for longer treatments (exposure time), it was obtained higher quantity of eggs, which is related with the time needed for incubation in the egg strings of females.

Gravid females

There were no clear effects of either the trans-trans-2-4-decadienal dose or the exposure time on the gravid females (Figure 3B). There is an indication that the count of gravid females decreased with increasing exposure time. This is explainable by the fact that with increasing time more eggs become mature and larvae leave the egg strings, thereby reducing the number of gravid females. Mature females

The trans-trans-2-4-decadienal dose and exposure time showed clear negative effects on the number of mature females but there seemed to be no interaction between these parameters (Figure 3C). This indicates that the effects are additive. Both, an increasing dose and an increasing exposure time decreased the counts of mature females, increasing the exposure time being about twice as effective. Based on the model (Montgomery, 1997; Myers & Montgomery, 1995), increasing the dose from zero to 0.0018% reduced the number of mature females from about 99 females to 85 females (15% drop). Similarly, increasing the exposure time from 4 to 12 days with dose 0.0018% reduced the number from about 98 females to 73 females (25% drop). Total number of larvae

The trans-trans-2-4-decadienal dose did not show any effect on the total larvae count, while the exposure time affected the total larvae count (Figure 3D). The latter is explainable by the constant release of larvae from the egg strings and their accumulation in the different larvae life stages (nauplius I, nauplius II and copepodids).

Non-viable larvae

A similar effect as for the total larvae count was also observed for the number of non-viable larvae. The trans-trans-2-4-decadienal dose did not show an effect on the number of non-viable larvae, whereas the exposure time clearly increased the number of non-viable larvae (Figure 3E), which is explained in the same way as for the total number of larvae, being an accumulation of non-viable larvae with time.

Nauplius II

The interpretation of the effects on the Nauplius II stage was similar to the one for the total number of larvae and of non-viable larvae. There was no effect of the trans- trans-2-4-decadienal dose and exposure time, due to an accumulation over the time of larvae in the Nauplius II stage (Figure 3F). In this case there was a slight indication of a negative interaction between the dose and exposure time which may indicate that increasing exposure time with trans-trans-2-4-decadienal had an effect on the Nauplius II stage. Frontal filament inhibition (response)

A comparison between the Control group (not exposed to trans-trans-2-4- decadienal) and the group exposed to the higher dose of 0.0018% trans-trans-2-4- decadienal was carried out, using the inhibition of the frontal filament method in copepodids of C. rogercresseyi. A significant difference was observed between the control response and dose response of trans-trans-2-4-decadienal (0.0018%). Thus, there is a highly inhibition of frontal filament (58%) in copepodids which are exposed to trans-trans-2-4-decadienal (Figure 4). Example 3

Assessment of deleterious effects, lethal or sub lethal, of trans-trans-2-4-decadienal injected in Atlantic salmon and its tissues

A LD50 (Lethal dose, 50 %) trial was performed to evaluate the lethal dose of trans- trans-2-4-decadienal for Atlantic salmon (Salmo salar). 138 fish were pit-tagged and allocated in six tanks (23 fish/tank; biomass per tank: 15 kg/m ³ ), each one with a total capacity of 350 I, with an average weight of 75 g/fish. After 7 days, fish were injected once intraperitoneally with 0.1 ml of the following concentrations of trans- trans-2-4-decadienal in a salt serum: 0.57 mg trans-trans-2-4-decadienal /l/day, 1 .13 mg trans-trans-2-4-decadienal/l/day, 5.65 mg trans-trans-2-4-decadienal/l/day, 28.24 mg trans-trans-2-4-decadienal/l/day or 56.47 mg trans-trans-2-4-decadienal/l/day. Non-injected fish were used as a control. Environmental variables were recorded daily, such as sea water temperature (°C) and oxygen saturation in sea water (%). Fish were not fed during the exposure trial (7 days).

Tissue samples of brain, intestine, skin, liver and muscle from salmon were taken per each dose injected 7 days after the first injection. Triplicate samples per each tissue were taken per each dose, pooled and then split in subsamples for histological analysis and for GC-MS analysis of trans-trans-2-4-decadienal. GC-MS analysis:

For sample preparation 0.1 g of fish tissue (liver, muscle, brain, kidney or intestine) was homogenized and placed in a Falcon tube (NALGENE, Centrifuge Tube Oak Ridge™ Sealing Cap P 10 ml). 1 ml of NaCI (1 mg/ml) and 1 ml of hexane (HPLC grade) was added to the sample and stirred for homogenization. Subsequently, samples were centrifuged at 10.000 rpm for 20 min and placed in a freezer (-18 °C) for 1 hour. The supernatant was withdrawn and filtered (DURAPORE membrane GV (PVDF), Ο.22 μιτι, 13 mm diameter, Hydrophilic, 100/CX). The filtered solution was concentrated to 100 μΙ under a Nitrogen stream. 1 μΙ of this solution was injected for GC-MS analysis using a calibrated gas chromatograph (Focus GC) coupled to a mass spectrometer (Thermo) and equipped with a BP-1 capillary column (30 m, 0.22 mm, 0.25 μιη; SGE, Australia). The oven temperature was kept at 40°C for 2 min and then programmed with a gradient at 10°C min ^"1 to 250°C. The carrier gas was helium. The injector temperature and transfer line to the mass detector was kept at 250°C. Electron impact ionization was employed with energies of 70 eV at 250°C.

Results:

During the period of the trial (7 days) no mortality was recorded in Atlantic salmon (S. salar), which were injected with the different trans-trans-2-4-decadienal doses or in the control. Therefore, an LD50 assessment was not relevant for this trial.

Regarding the environmental conditions of the trial, temperature of sea water was in average 12°C with variability less than 1 °C, while oxygen saturation varied from 90% to 100%.

No important changes in the cell structure of tissues sampled were observed (Table 4 and 5) between fish treated with the trans-trans-2-4-decadienal doses and the control. Damages type 2, were observed with more frequency in the liver and in the intestine. No damages were observed in the skin and muscle.

A higher damage (type 3) was observed in the liver, for the trans-trans-2-4- decadienal dose of 28.24 mg/l (Table 4 and 5), with a necrotic area only in one sample. A higher frequency of tissue damages was observed in the liver and intestine, which were principally type 2 (Table 4). Only one case of damage level 1 , was observed in the brain being a mild meningitis (Table 4 and 5). Concentrations of trans-trans-2-4-decadienal detected in each analyzed tissue are shown in Table 6. Trans-trans-2-4-decadienal was found in all the tissues analyzed (liver, brain, intestine, skin and muscle). Higher concentrations were recorded for the intestine tissues. No concentrations of trans-trans-2-4-decadienal were detected in the control tissues.

Table 4. Observed levels of damages in tissues of salmons injected with different doses of trans-trans-2-4-decadienal.

Level of Details Tissue

damage Brain Liver Intestine

0 No effect — — —

1 Low damage in Mild Cells with

tissue without Meningitis eosinophilic

deleterious effects cytoplasm

for the organ. and eccentric

nuclei

2 Damage in tissue Hepatitis Inflammatory with some reaction in deleterious effects the cells of in functionality of periphery. the organ. But Giant cells organs are in the with process of vacuoles regeneration. empty.

3 Damage in tissue Necrotic area

with deleterious

effects in

functionality of the

organ.

4 Morphological

severe damage of

tissue. Endanger

life.

Table 5. Frequency of damage level in tissues analyzed of S. salar, injected with different doses of trans-trans-2-4-decadienal.

Damage (according to Table 4)

Tissue Dose (mg/l) 0 1 2 3 4

Liver 0.00 - 33% - - -

0.57 - - - - -

1.13 - - 33% - -

5.65 - - - - -

28.24 - - 33% 33% -

56.47 - - 67% - -

Brain 0.00 - 33% - - -

0.57 - - - - -

1.13 - - - - -

5.65 - - - - -

28.24 - - - - -

56.47 - - - - -

Intestine 0.00 - - 33% - -

0.57 - - - - -

1.13 - - 100% - -

5.65 - - 100% - -

28.24 - - 67% - -

56.47 - - 100% - -

Skin 0.00 - - - - -

0.57 - - - - -

1.13 - - - - -

5.65 - - - - -

28.24 - - - - -

56.47 - - - - -

Muscle 0.00 - - - - -

0.57 - - - - -

1.13 - - - - -

5.65 - - - - -

28.24 - - - - -

56.47 - - - - - Table 6. Concentration of trans-trans-2-4H_lecadienal detected in each tissue sample of salmon.

Discussion and conclusions:

Regarding the different times of persistency of trans-trans-2-4-decadienal, dissolved in sea water, at 10°C compared to 12°C and 14°C, trans-trans-2-4-decadienal seems persists for a longer time at a lower water temperature. The trans-trans-2-4- decadienal dissolved in sea water persists for a maximum of 10 days in temperatures between 10°C and 14°C. The results of the assessment test for the present invention show that the unsaturated aldehyde, i.e. trans-trans-2-4- decadienal is not a persistent compound in sea water. This is considered as a great advantage and precondition for the suitability of this agent when used in the treatment of fish in an aquatic environment, especially when used in open systems.

A direct administration of trans-trans-2-4-decadienal to the sea water gave little or no effect on the sea lice infestation in Atlantic salmon (results not shown). Surprisingly, it was found in the present invention that it is possible to negatively affect the sea lice population, when trans-trans-2-4-decadienal was added to the feed and fed to the fish infested by said sea lice.

This is an advantage from a practical point of view since the fish does not have to be isolated or removed from the cultivation unit such as a sea cage in order to be treated, since the fish can simplify be fed the diet comprising the active compound. A water baths treatment affords more handling and is thus more laborious and is furthermore stressful for the fish. In the present invention, it is thus possible to treat the fish in the cultivation units without isolating the fish.

In the LD50 assay, no mortality was observed in the salmons injected with trans- trans-2-4-decadienal. There were observed some mild damages in the liver and intestinal tract after treatment with increasing dosages of the unsaturated aldehydes. The damages are considered being reversible.

Regarding the results of the efficiency trial, a lower number of mature females and eggs of Caligus rogercresseyi were obtained when the salmon were fed higher doses of trans-trans-2-4-decadienal in the salmon feed. At the same time, with an increased time of exposure to trans-trans-2-4-decadienal in the feed, a reduced number of gravid and mature females were obtained. A reduction of 25% in the number of mature females of C. rogercresseyi was obtained. It was also demonstrated that trans-trans-2-4-decadienal has a detrimental effect in the gonadical maturity of females of C. rogercresseyi, when Atlantic salmon (S. salar) is treated with unsaturated aldehyde added in the feed. This indicates that trans-trans- 2-4-decadienal effectively reduces the maturation and reproductive ability of sea lice. It was demonstrated that trans-trans-2-4-decadienal also has a detrimental effect in the availability of eggs in the eggstrings of C. rogercresseyi, when Atlantic salmon (S. salar) is treated with unsaturated aldehyde added in the feed. The demonstrated effects on the different sea lice stages will lead to a quantitative reduction of availability of sea lice larvae in the sea cages used in fish farming and, through this, to a reduction of probability of infestation of copepodids to the fish.

The shown inhibitory effect in the frontal filament eversion (mophogenesis) in copepodids obtained from females of fish treated with the 0.0018% dose in feed indicate that trans-trans-2-4-decadienal negatively affects the viability of the copepodids of C. rogercresseyi during the process of infestation. Trans-trans-2-4- decadienal according to the present invention is thus considered being a bioactive compound which is effective in the prevention of the infestation and attachment of the ectoparasite to the skin of fish.

Injected doses of trans-trans-2-4-decadienal, 0.1 mg/l, 0.5 mg/l and 1.5 mg/l in Atlantic salmon (Salmo salar) did not produce mortality. Therefore, the unsaturated aldehyde can be considered as being safe when used in salmon feed.

The intestine was the organ of Atlantic salmon (Salmo salar) that accumulated more trans-trans-2-4-decadienal and a higher frequency of mild damages was present in this organ.

It will be appreciated that the features of the invention described in the foregoing can be modified without departing from the scope of the invention. References:

Montgomery, D. 1997. Design and Analysis of Experiments. John Wiley & Sons, New York, 684 pp.

Myers, R. & D. Montgomery. 1995. Response Surface Methodology. John Wiley & Sons, New York, 700 pp.

Pino-Marambio, J.E., Mordue (Luntz), A.J., Birkett, M.A., Carvajal, J., Asencio, G., Mellado, A., Quiroz, A.E., (2007). Behavioural Studies of Host, Non-Host and Mate Location by the Sea Louse, Caligus rogercresseyi. Boxshall & Bravo, 2000 (Copepoda: Caligidae). Aquae 271 , 70-76.