CA2685795C

CA2685795C - Attractant bait for the control of parasites in cultured fish, a method of preparation and use thereof

Info

Publication number: CA2685795C
Application number: CA2685795A
Authority: CA
Inventors: Sergio Contreras Lynch
Original assignee: INSTITUTO DE FOMENTO PESQUERO; VETERQUIMICA Ltda Soc; Universidad de los Lagos
Current assignee: INSTITUTO DE FOMENTO PESQUERO; VETERQUIMICA Ltda Soc; Universidad de los Lagos
Priority date: 2009-01-26
Filing date: 2009-11-12
Publication date: 2021-03-23
Anticipated expiration: 2029-11-12
Also published as: GB2467199B; GB2467199A; CA2685795A1; NO339385B1; GB0918958D0; NO20093450L; CL2009000160A1

Abstract

The present invention is related to the control of ectoparasites belonging to the genus Caligus in cultured fish. More specifically, the invention is directed to the isolation of attractant fractions from fish mucus for the control of parasites in aquaculture industry fish. The present invention is further directed to the manufacture of an attractant bait based on the mucus and/or attractant fractions isolated from fish mucus.

Description

ATTRACTANT BAIT FOR THE CONTROL OF PARASITES IN CULTURED FISH, A
METHOD OF PREPARATION AND USE THEREOF
The present invention is related to the control of ectoparasites belonging to the genus Caligus in cultured fish. More specifically, the invention is directed to the isolation of attractant fractions from fish mucus for the control of parasites in aquaculture industry fish. The present invention is further directed to the manufacture of an attractant bait based on the mucus and/or attractant fractions isolated from fish mucus.
BACKGROUND OF THE INVENTION
Fish ectoparasitosis produced by the genus Caligus represent a yet unsolved problem for the salmon culture industry in a national and international scale.
Currently, the national industry can only use a very small selection of antiparasitic products, all of them chemotherapeutic products, represented by emamectin benzoate and other pyretroid or dichlobenzuron products for ectoparasites, all authorized for use in caligidosis treatment. Due to commercial and sanitary restrictions generated by the growing concern about consumer's food safety, the commercialization of emamectin sulfate and chemicals in general will be strongly reduced in the short term, leaving the salmonid producing industry in a very vulnerable position.
The search for alternative solutions is absolutely necessary and urgent for the national aquaculture industry, because yearly losses. around US$350.000 are caused by infestation by parasites of the genus Caligus.
One alternative to chemical products is biological control, which in general terms is defined as an activity that manages a series of natural enemies, also referred as to predators, with the objective to reduce or even completely fight against parasites that affect a given culture. Biological control is an effective risk-free alternative compared to the numerous and growing issues derived from the use of biocide chemicals. The examples of the use of biological controls are plentiful in agriculture, e.g. the control of yellow spiders with Phytoseiulus persimilis and white flies with Encarsia formosa and Verticiffium lecanii.

The use of sexual pheromones has been also applied as a form of biological plague management. A portion of living beings communicate with each other by means of sounds, but the majority communicates by means of odorants, releasing substances called pheromones; blind obedience of an organism to pheromones opens a vast range of possibilities to manage its behavior at will.

Currently, there are two modalities for using sexual pheromones that have been synthesized and commercialized. First, they can be used as attractant agents for traps and baits. The second usage form is "male confusion" produced by flooding or saturating large areas with sexual pheromones.
In other types of biological control, the behavior is determined by the presence or occurrence of stimuli that are predominantly chemical in nature, though physical and mechanical stimuli can also be present. Hence, a chemical substance present in a plant can make the insect to be attracted to the plant itself; in this case, the substance is an attractant. In other cases, the effect can be the opposite; hence, the substance is a repellent.
Some substances stimulate feeding, while some others inhibit feed intake. Part of this behavior is due to stimuli that are produced as communication mechanisms between individuals of the same species. Sent and received messages can induce sexual attraction, alarm, aggregation, orientation and others. The applications of ethological control include the use of attractants in traps and baits, repellents, feeding deterrents and diverse substances with similar effects. As an example, colorful sticky traps attract insects. The selection of specific colors depends on the specific wavelength related to insect vision.
In the case of Caligus parasitosis, studies have been carried out using a biological control methodological approach with Udonella sp., a platyhelminth that is frequently found on ovigerous Caligus rogercresseyi females and was thought to have some type of effect on fecundity and survival of planktonic stages of the parasite. However, as a result of this research, Udonella sp. was found to be an epibiont with a biological relation more neutral than adverse over Caligus.
The behavior of the free life stages of Caligus in southern Chilean cultures have been little investigated, but it is known that small copepods have a

2 positive phototactic orientation and parasites have in general positive tactisms to several organic substances contained in fish mucus.
The use of biological baits has been suggested to control monogenea, trematode, insect and rodent plagues. The principle used is fundamentally based in exploiting the behavior of parasites when confronted to chemicals contained in fish mucus that can stimulate certain parasite receptors, which induce them to adhere to the host's skin (Buchmann et at, 1998; Buchmann et at, 1999).
Therefore, the attractant substance contained in the host skin mucus attracts the parasite and compels it to approach the skin and parasite the host.
Consequently, getting technological solutions directed to new products against fish ectoparasites from natural products based on the chemoattractant properties thereof is a pending challenge. The development of this type of product is proposed in the present patent application to tackle the growing necessity and demand for natural products in worldwide aquaculture production.
BRIEF DESCRIPTION OF THE INVENTION
The present invention is directed to the control of fish ectoparasites, particularly in the salmonid culture industry, in which one of the high-impact productivity-decreasing ectoparasites belongs to the genus Caligus, and to obtaining attractant fractions for parasite control in aquaculture industry fish. More specifically, the present invention is directed to the isolation of attractant fractions for the control of parasites in aquaculture industry fish. The present invention is also directed to the manufacture and use of an attractant bait based on fish mucus or attractant fractions of fish mucus, wherein said use of an attractant bait is directed to the control of parasites in the aquaculture industry.
In the present invention, fractions are isolated from the mucus of susceptible salmonids that are able to attract infesting stages of parasites from the genus Caligus.

3 DESCRIPTION OF THE DRAWINGS
Figure 1 shows a frequency distribution plot of the parasite attraction toward different mucus fractions from rainbow trout, shown as the attraction percentage of each particular fraction (number of parasites attracted by the fraction /
total number of parasites).
Figure 2 shows a frequency distribution plot of the parasite attraction toward different mucus fractions from Atlantic salmon, shown as the attraction percentage of each particular fraction (number of parasites attracted by the fraction /
total number of parasites).
Figure 3 shows a frequency distribution plot of the parasite attraction toward different mucus fractions from Pacific salmon, shown as the attraction percentage of each particular fraction (number of parasites attracted by the fraction /
total number of parasites).
Figure 4 shows a plot of the average small C. rogercresseyi copepods attached to each fish at the end of an experiment in the presence (Tank 7) or absence (Tank 1) of attractant baits manufactured according to one embodiment of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to obtaining mucus extracts or total mucus from different fish species that show attractant properties for the control of fish ectoparasites in the aquaculture industry and particularly in the salmonid culture industry.
In the present invention, fractions are isolated from salmonid mucus that are able to attract infesting stages of parasites from the genus Caligus.
To understand the meaning of "parasites infesting stages", we will refer to one of the most common salmonid parasites, the salmon louse.
Caligus rogercresseyi, ordinarily known as salmon louse, is characterized by having a direct life cycle including:
1) Free larvae life stages (nauplius I, nauplius II and small copepods).

4 2) Parasitary larvae stages attached to a salmon, referred to as chalimus 1 to IV.
3) Adult motile parasite stages able to move on the host surface.
Harmful stages for salmonids are chalimus (2) and adult parasites (3), which stress fish by feeding from their blood and skin.
Nevertheless, it has been established that small copepod stages (1) of the genus Caligus have a positive chemotactism toward specific molecular compounds contained in salmonid mucus, which allows them to find a host, settle on the host and begin their transformation into a chalimus (2).
In the present invention, fractions are isolated from the mucus of susceptible salmonids that are able to attract these infesting stages of parasites from the genus Caligus, i.e. small copepods.
In this sense, the present invention is able to shorten the life cycle of parasites, interfering in the stage when small copepods attach to the skin of salmonids and transform into the non-motile larvae known as chalimus.
With this strategy, chalimus larvae are prevented to reach the adult stage and reproduce. This can be translated in ostensible decreases of parasite charges, which allow controlling this parasitism.
In this way, the present invention provides an effective product with no negative effects to the environment and human health, contrarily to the chemical alternatives in use nowadays.
In the present invention, fractions are isolated from salmonid mucus that are able to attract infesting stages of parasites from the genus Caligus.
Although a specific parasite has been described herein, someone skilled in the art will readily observe that the process of the invention can be applied using mucus from different fish species to obtain products with equivalent properties for the control of water parasites that are specific to the fish from which the mucus is extracted.
In one implementation of the invention, the mucus is obtained directly from different fish species, such as Patagonian blennie or Antarctic rock cod (Eleginops maclovinus), Atlantic salmon (Selma safer), Pacific salmon or coho or silver salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss) and king salmon or Chinook salmon (Oncorhynchus tshawytscha) and the like, for the control of parasites.
In another implementation of the invention, a process is described to obtain attractant fractions isolated from mucus from the abovementioned fish species. The fractions obtained in this way are able to shorten the life cycle of parasites, particularly parasites from the genus Caligus, interfering in the stage when small copepods attach to the skin of salmonids and transform into the chalimus.
In another implementation, the mucus obtained from the abovementioned fish species or the attractant fractions obtained from the mucus are deposited on a substrate or matrix. In this way, infesting parasite stages are prevented to develop on fish but they develop on the substrate or matrix onto which mucus or attractant fractions isolated from mucus were deposited, in order to control parasites.
In another implementation of the invention, a substrate or matrix is described onto which mucus obtained from the abovementioned fish species or attractant fractions obtained from said mucus are deposited. Said substrate or matrix is useful to inhibit the attachment of parasites to fish in the aquaculture industry. In particular, said substrate or matrix prevents the attachment of parasites from the genus Caligus in the infesting stages thereof.
Another implementation of the invention uses said mucus, said attractant fractions and/or substrates or matrixes containing them for the control of ectoparasites, particularly from the genus Caligus in aquaculture.
The present invention describes the manner by which mucus is obtained, fractions with high attractant activity are selected and active components present in these fractions are characterized. Furthermore, the incorporation of mucus or attractant fractions into a substrate or matrix suitable for use as an attractant bait for fish ectoparasites in the aquaculture industry, in particular parasites from the genus Caligus, is described.
The mucus is obtained from any aquaculture species that can be affected by parasite infestation, such as Patagonian blennie (Eleginops maclovinus), Atlantic salmon (SaImo salar), Pacific salmon or coho or silver salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss) and king salmon or Chinook salmon (Oncorhynchus tshawytscha). In particular, the parasite belongs to the genus Caligus. Preferably, the mucus is obtained from cultured salmonid species selected from trout and salmon, such as e.g. Atlantic salmon (Salmo salar), Pacific salmon or coho or silver salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss) and king salmon or Chinook salmon (Oncorhynchus tshawytscha).
The mucus can be obtained from alive or dead fish, at any developmental stage. Preferably, fish from primary harvest plants, sacrifice centers or process centers are used.
In a preferred implementation, the mucus is obtained from sacrificed fish that are treated with distilled water and dried, after which mucus is extracted by scraping the fish skin, avoiding damaging or tearing the skin. Mucus is collected at cold temperatures, preferably less than 6 C, and more preferably between 1-4 C.
This is done in this way in order to avoid degradation of the attractant molecules present in the mucus, which are labile are higher temperatures.
If attractant fractions should be obtained, the mucus is placed in centrifuge tubes. The tubes are centrifuged between 800 and 2,000 g, preferably around 1,500 g, for around 13 to 18 minutes, preferably for 15 to 16 minutes, and more preferably for 15 minutes, at a temperature ranging from 3 to 5 C, more preferably at 4 C. The supernatant is collected and stored in other tubes to be subsequently frozen. This crude extract will be the starting material for the separation of attractant fractions and compounds from salmonid mucus.
For the separation and extraction of attractant compounds from salmonid mucus, the fraction obtained before is centrifuged in a sucrose gradient.
Using sucrose gradient centrifugation, 18 mucus fractions were obtained, which can be observed in Figures 1 to 3.
Sucrose gradients are formed at concentrations ranging from 10 to 80%
sucrose, preferably using at least a gradient having 4 different concentrations, more preferably 6 different concentrations, and most preferably 8 different concentrations.
If a gradient with 8 different concentrations is used, these concentrations differ in 10% sucrose concentration from one to the next, and a given volume of each concentration is placed in a centrifuge tube starting from the most concentrated solution at the bottom of the tube to the most diluted concentration at the top layer.
Said volumes can be equal or different from each other, however equal volumes of each concentration are preferred to form the sucrose gradient. In the ultracentrifugation process, no more than 60% of the total capacity of the tubes should be used. Preferably, 50-ml ultracentrifuge tubes are used wherein 20 to 30 ml sucrose gradients are formed, preferably around 25 ml, and if an 8 concentrations gradient is used, 3 ml of each concentration are used. A sample of the mucus extract is thawed and a volume ranging from 0.5 to 3.0 ml, preferably from 1 to 2 ml, more preferably around 1 ml, is placed at the top of each tube where the sucrose gradient has been formed.
In one implementation of the invention, more than one ultracentrifugation cycle is used, preferably more than 2 cycles and more preferably 3 or more cycles are used, wherein each cycle has the abovementioned characteristics. Preferably, a first centrifugation cycle is carried out at 15,000 rpm at a temperature controlled at 4 C for 60 minutes. Subsequently, the same centrifuge tube is centrifuged at 15,000 rpm at 4 C for 90 minutes and finally a third cycle is carried out at 15,000 rpm at 4 C for 90 minutes.
Once the ultracentrifugation in a sucrose gradient has been performed, the separated fractions are carefully extracted at the border of the tube, preferably between 1 to 3 ml of each fraction, and more preferably around 1.5 ml. These fractions are placed in individual tubes and kept refrigerated between 1 and 4 C. For each identified fraction, protein concentration was measured using the Lowry method.
Subsequently, each of these fractions can be integrated into a semi-solid gel solution, such as agar, gelatin, carrageenan, acrylamide, polyethylenglycol, chitosan and the like prepared in an appropriate buffer with a pH between 6 and 8, such as TRIS, Tricine, HEPES, TES, MOPS or PIPES, among others, or distilled water, with a concentration from 0.5 to 10% of gelifying agent.
To evaluate the attractant ability of each fraction obtained from the sucrose gradient over infesting stage individuals from the genus Caligus, gel-integrated fractions were used. In these assays, gel concentrations ranging from 0.1 to 10%, preferably from 1 to 5%, more preferably from 1 to 2% and most preferably from 1.2 to 1.7% were used. These mixtures were carried out in the presence of a buffer with a pH between 6 and 8, preferably around 7, or simply distilled water.
Buffers that can be used include TRIS, Tricine, HEPES, TES, MOPS or PIPES.
In vitro assays demonstrate that in fractions 5 to 15 (p < 0.25) the parasite behavior was surprisingly favorable, showing a high preference toward said fractions, which indicates a high attraction of the parasite toward the components of said fractions. In particular, it is possible to appreciate that the parasites, in this case small Caligus copepods, approach plates that have a higher attractant quality and do not die during the assay time, which is carried out for more than 30 minutes, preferably for 60 or more minutes.
Another implementation of this invention is an attractant bait manufactured using fish mucus and/or attractant fractions obtained from fish mucus, a gel and a suitable substrate or matrix.
The fish mucus can be obtained from Patagonian blennie (Eleginops maclovinus), Atlantic salmon (Salmo salar), Pacific salmon or coho or silver salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss) and king salmon or Chinook salmon (Oncorhynchus tshawytscha) and the like. Attractant fractions are obtained as described above.
The gel can be selected from agar, gelatin, carrageenan, polyacrylamide or any other suitable gelifying agent that could solidify at a temperature lower than 40'C to prevent the degradations of the active components that are present in the mucus.
The substrate or matrix is a suitable structure able to support the gel mixed with the mucus and/or attractant fractions obtained from the mucus. Said substrate or matrix could have a sponge-like structure and be manufactured from synthetic or natural materials.
For manufacturing the attractant bait, a gel solution is prepared in distilled water or a suitable buffer such as TRIS, Tricine, HEPES, TES, MOPS
or PIPES and the like, at a concentration ranging from 0.5% to 10% by weight, bringing it to a temperature lower than 40 C. At said temperature, the mucus and/or attractant fraction thereof is added in a proportion ranging from 100 pl to 10 ml of mucus or attractant fraction obtained from the mucus for each liter of gel solution.
The mixture is stirred to homogeneity. When still in liquid form, the liquid is added to the suitable substrate or matrix (sponge-like structure) within a mould. Depending on the gel, the temperature is decreased until solidification of the mixture inside the substrate or matrix.
APPLICATION EXAMPLES
EXAMPLE 1: Isolation of mucus from sacrificed fish Sacrificed fish, washed with distilled water and dried with paper, from the species rainbow trout, Atlantic salmon and Pacific salmon were used, and their skins were scraped with a spatula to extract around 9 ml of mucus per processed fish, which was conserved at 4 C.
EXAMPLE 2: Isolation of attractant fractions from mucus The mucus previously obtained as in Example 1 was centrifuged at 1,500 rpm for 18 minutes at 4 C to isolate a mucus extract. The supernatant was removed and frozen. The mucus samples from each species were processed separately during all the previous and successive steps.
An aliquot of the supernatant (1.5 ml) was loaded into ultra-centrifuge tubes wherein a sucrose gradient was preformed with 8 different sucrose concentrations ranging from 10 to 80% of sucrose. The samples were centrifuged following a 3-cycle process. The ultracentrifugation process of the samples placed in the abovementioned tubes was carried out in a refrigerated Kibota model KR

ultracentrifuge, using a first cycle at 15,000 rpm at 4 C for 60 minutes. A
second cycle was carried out at 15,000 rpm at 4 C for 90 minutes, and a third and last cycle was performed at 15,000 rpm at 4 C for 90 minutes.
Each fraction was individually removed and placed in individual containers. An aliquot of said fractions was assayed to evaluate their attractant capacity over species of the genus Caligus.

EXAMPLE 3: Assessment of the effectiveness of the mucus and/or attractant fractions over Caligus parasites 1 ml of each fraction obtained in Example 2 was mixed with 1.5% agar in Iris buffer at pH 7 and was placed in Petri dishes, which were evaluated according to the attraction they exerted on parasites from the genus Caligus.
The series of plates was transported to evaluate the performance of the different fractions separated from the mucus of salmonid fish.
This was carried out for each mucus type (trout, Pacific salmon, Atlantic salmon) and repeated 6 times.
Furthermore, performance assays were carried out to control the technique, i.e. the inclusion of a placebo fractionation (distilled water) instead of mucus and incorporating it in plates.
Separated mucus fractions in Petri dishes were placed in darkened fish tanks containing sterile seawater, together with positive (agar medium with whole mucus) and negative (agar medium without mucus) controls.
50 small live Caligus copepods were placed in each fish tank, taking said moment as the beginning of the experiment.
To measure the attraction level of the different mucus fractions (determining the presence of small copepods on each plate), sample plates were observed under a magnifying glass at different times: 0, 2, 4, 6, 8, 10, 30 and 60 minutes, and then 24 hours after the beginning of the experiment. This last control time allowed observing the change in the physiological development of the parasite (shedding).
The study contemplated the observation of at least 400 plates with different mucus fractions for each studied hydrobiological species, including positive, negative and technical controls. In this way, a statistical evidence can be obtained.
The results of observations made 24 hours after the beginning of the experiments evidenced that fractions obtained from Atlantic salmon develop morphological and physiological changes in parasites.
The results obtained from the performance assays were statistically evaluated in the following manner:

Frequency distribution: a frequency distribution of the occurrences was constructed with the results obtained.
Chi-squared for ratios: significant differences were detected in the attraction level between the different separated mucus fractions.
Tukey test for ratios: it was specifically determined which fraction was significantly different, according to its attraction level.
For all the mucus fractions obtained, observation times are not significantly different between them, i.e. the attraction of the parasite to the fractions is not dependent on the time during which the attractant is present (see Tables 1, 2 and 3 wherein attraction toward the bait is observed (1) or not (0) with the indicated mucus fraction along time).
Table 1. Performance assays of the different mucus fractions from rainbow trout.
Minutos Minutos 0 2 4 6 8 10 30 60 Total 0 2 4 6 0 10 30 60 Total Fraccion 1 0 0 0 0 0 U_ 0 0 0 Fraccit501_ 0 1 0 0 0 0 0 1 2 Fraccidn 2 0 Praceii5n 2 g' 0 0 1 0 0 0 0 0 1 fraction 3 0 Fiaccidn3 0 0 0 0 0 0 0 0 0 FrOCC1On 4 0 Fracci6a4 - 0 0 0 0 1 0 0 0 1 FracclOn 5 0 FraccIN 5 0 0 0 0 0 1 0 0 1 Fraccien 6 0 FraccitIn fi 0 0 0 0 0 0 0 0 0 Fraction\ 7 Fraddart7. 0 1 0 1 0 0 1 1 4 Fraccian 8 0 FratClon 8' 0 0 0 0 - 1 0 0 0 1 Friia1On 9 0 1 0 0 1 1 1 1 , 5 Fracd6ñ,9 0 1 1 1 1 1 1 1 7 Fraccion 10 Fraccian10, 0 0 0 0 0 1 0 0 1 Fraccinin 11 , 0 0 0 0 0 0 0 0 0 FrIcantl 0 0 0 0 0 0 , 0 1 1 Fraccion 12 0 1 kFrahlon1i 0 1 1 1 1 0 0 0 4 Fr8Ccitin 13 0 FrileelOn 1j 0 0 0 0 1 1 1 0 3 FrdoCiOn 14 Fracclikilf 0 1 1 0 1 1 1 0 5 "Fraccieri Fracciarvi,5 0 0 0 1 0 0 0 0 1 Fracci11 0 0 0 0 0 0 0 0 0 TFrattion,16 0 0 0 0 0 0 0 1 1 Fraccidn17 0 0 0 0 0 0 0 0 0 fraritiOn17 0 0 1 0 1 0 1 1 4 PracclOn 18 FracclOrr18 0 0 0 0 0 0 0 0 0 Mlnutos Mlnutos 0 2 4 6 8 10 30 60 Total 0 2 4 6 8 10 38 60 Total FracciOn 1 0 0 0 0 0 0 0 0 0 FracclOn 1 0 0 0 _ 0 0 0 0 0 0 Ham& 2 0 0 0 0 0 0 0 0 0 'FICtfOfl2 0 FracciOn 3 0 0 0 1 0 0 0 U 1 FraccIt413 Fraccijn4 0 0 0 1 0 0 0 0 1 Fracclin4 Fracei5n5 0 0 0 0 0 0 0 0 0 FracciOnl 0 0 1 1 _ 1 0 0 1 4 -Fraccion 6 0 1 0 1 1 1 1 1 6 Fr#1:16116 0 0 0 0 1 1 , 1 1 4 Fracan 7 0 1 0 1 1 1 0 1 5 FraCC16a7 Fraccin 8 0 1 1 1 1 1 1 1 7 Fraccidn 8 O
i Fraction 9 0 1 1 1 1 0 1 0 5 FracClor9 accion 10 0 1 0 1 0 1 1 0 4 Frac:6164110 0 0 0 0 0 0 0 0 0 FrucciOn IT 0 1 1 0 0 0 0 0 2 Fracchinsli 0 0 0 0 0 0 0 1 1 Fractri 12 0 1 1 1 1 1 Fracc16$112 0 1 1 1 1 1 0 0 5 Fraccatt-13 0 0 0 0 0 0 0 0 0 Fraccion13 0 0 0 0 1 1 1 0 3 fracciOn 14, 0 1 0 0 1 0 0 0 2 FraCcrOn 14 0 1 1 0 0 1 1 1 5 Fraction Frattitin 15 0 0 0 0 0 0 0 0 0 Redden 0 Fraccierilk 0 0 1 1 0 0 0 0 2 FractIOn FracciOn 17 0 0 0 0 0 U 0 U 0 FracciOn18 0 0 0 0 0 0 0 0 0-Fraccion180 0 0 0 0 0 0 U 0 Minutos Minutos 0 2 4 6 , 8 10 30 60 Total 0 2 4 6 8 10 30 60 Total Fraction 1 0 0 0 0 0 0 0 0 o FracclOn 1 0 iraccion 2 o _ o 0 o 0 1 , 0 1 2 Ram& 2 0 0 Fraccion 3 0 0 0 0 0 1 1 1 3 Framitin 3 0 Fraction 4 0 1 0 1 0 0 0 0 2 Fraction 4 0 Fraccion 5 0 1 0 1 0 0 0 0 2 Fraccicin 5 0 0 0 , 1 1 0 0 0 2 Raman 6 0 0 0 0 1 1 1 1 4 FracciOn 6 0 Fraccik 7 0 0 1 0 1 0 , 0 0 2 Framidn 7 0 0 1 0 1 _ 1 0 1 4 Fraccitin 8 0 0 1 1 1 0 0 1 4 iraccidn 0 0 iraccion 9 0 0 0 0 0 1 1 1 3 Fratcion 9 0 Fraccion 10 0 1 0 1 1 1 1 0 5 Fraccion 10 o 0 0 1 1 0 0 1 3 Fraccitin 11 0 0 1 1 0 0 1 1 4 Fraccion 11 Fracclon 12 0 1 1 1 0 0 0 0 3 Fraccion 12 FracclOn 13 0 0 0 0 0 o 0 0 0 Fracclan 13 FracciOn 14 0 0 0 0 0 0 1 0 1 Fraction 14 Fraccion 15 0 1 , 0 0 0 0 0 0 1 Framion 15 Fraccion 16 0 0 0 0 0 0 0 0 o Fraccion 16 Fracckin 17 o 1 0 0 0 o 0 0 1 Fraccion 17 0 1 0 0 0 0 0 0 , 1 Fraccien 18 0 0 0 0 0 0 0 0 0 Fraction 18 Table 2. Performance assays of the different mucus fractions from Atlantic salmon.
Minutos Minutos 0 2 4 6 8 10 30 60 Total 0 2 4 6 8 10 30 60 Total FracciOn 1 _ 0 0 1 1 0 0 , 0 0 2 Fraccion 1 0 FracciOn 2 _ 0 0 0 0 0 0 0 0 0 Fraccion 2 Fraccien 3 0 0 0 1 1 1 0 0 3 FracclOn 3 Fraccion 4 0 0 0 0 0 0 1 1 2 Fraccion 4 -Fraccifin 5 0 0 0 0 0 0 0 0 0 Fraction 5 0 _ 0 1 0 , 1 1 0 0 3 Fraction 6 0 1 1 1 0 0 0 1 4 fracciOn 6 Fraction 7 0 1 1 0 0 1 0 0 3 Fraccien 7 Fracciin a 0 0 0 1 1 1 1 0 4 FracciOn 8 -Frau& 9 0 0 0 0 1 0 1 0 2 Fraccien 9 0 , 0 0 0 0 1 0 0 1 Fraccian 10- 0 1 0 0 0 0 0 , 0 1 Fraccion 10 0 0 0 0 0 0 0 0 0 Fraccion 11 0 0 0 1 0 0 0 1 2 FracclOn 11 Fraccian 12 , 0 1 1 0 1 1 1 1 6 Fracclon FracciOn 13 0 0 1 0 1 0 1 1 4 FracciOn 13, 0 1 0 1 1 1 1 1 6 FracciOn 14 0 0 0 0 0 0 0 0 0 From& 14 0 Fraction 15 , 0 0 , 0 0 0 0 0 0 0 FracciOn 15 0 0 1 0 0 0 0 0 1 Fiaccion 16 0 0 0 0 0 0 0 _ 0 0 FracclOn 16 FracciOn 17 0 0 0 0 0 0 0 0 0 Fraccion 17 FracciOn 18 0 0 0 0 0 0 0 0 0 Fraccion 10 Minutos Minutos 0 2 4 6 8 10 30 60 Total 0 2 4 6 8 10 30 60 Total FracciOn1 0 0 0 0 0 0 0 0 0 Fraccion 1 Fraccion 2 0 0 0 0 0 0 0 0 0 Fraccion Ramie') 3 0 0 0 0 0 0 0 0 0 Fraccion 3 Fracclon 4 0 1 0 0 0 0 0 0 1 Fraccifrn Fraccion 5 0 0 0 0 0 0 0 0 0 Fraction

5 0 0 0 0 0 0 0 0 0 Fraccicin 6 0 0 1 0 1 0 0 0 , 2 Fraction 6 0 0 1 , 1 1 0 1 1 5 Fume& 7 0 1 0 0 1 1 1 1 5 Fraction? , Fraction 8 0 0 1 1 1 1 1 1 6 FracciOn 8 0 1 1 0 1 _ 1 1 1 6 Fraction 9 0 0 0 1 0 1 1 1 4 Fraction 9 0 1 1 0 1 1 1 _ 0 5 Fraccion 10 0 1 1 1 0 0 1 0 4 FraCcion 10 BOCCI& 11 0 0 0 0 0 0 0 0 0 Fraccitin 11 0 0 , 0 0 0 0 0 0 0 FracciOn 12 0 0 1 1 1 1 1 0 5 FracciOn 12 0 0 , 1 0 1 1 1 1 5 Fraccion 13 0 0 0 0 0 1 0 1 2 Fraccion 13 FracciOn 14 0 1 0 1 1 0 , 1 1 5 Fraction 14 0 0 0 1 0 0 0 0 1 Fraccion 150 0 0 0 0 0 0 0 0 Fraccion 15 FracciOn 16 0 0 0 0 0 0 0 0 0 Fraccian 16 0 , 0 0 0 0 1 0 0 1 Fraccian 17 0 1 1 0 0 0 0 0 2 Fraccion 17 0 0 0 0 0 0 , 0 0 0 Fraction 18 0 0 0 0 0 0 0 0 0 'Fraccion Minutos Minutos 0 2 4 6 8 10 30 60 Total 0 2 4

6 8 10 30 60 , Total FriCtitin 1 0 1 0 1 1 0 0 o 3 Fracciati 1 FractiOtt 0 0 0 1 0 1 1 1 4 FiaCtiott 2 0 Fractldi 0 0 0 0 0 0 0 0 0 Ftii6Mitiii 3 FtadViin 4 0 0 0 0 00 0 0 0 Fraction 4 0 FirtiWon 5 0 0 0 0 0 0 0 1 1 Fraction 5._ FrieciOn 6 0 0 0 1 1 1 1 1 5 fr#cclon 6_ 0 Fraction 7 0 1 1 .õ 0 0 0 0 0 2 NOM-7 0 0 froetton-8 0 0 0 1 1 0 0 0 2 featitMei 0 ü 0 0 0 0 1 0 1 Fta&ton 9 0 0 0 0 0 0 0 0 0 raccjói9 0 Fra:tion 10 0 0 1 1 1 1 0 0 4 fraccian 10 Fetitain 11 0 0 0 0 0 0 1 1 2 Fraction 11 Fratt464'4 0 1 1 0 1 1 1 1 6 Fraction 12 F1111;0614 0 1 1 1 1 1 1 1 7 FractiOn 13 Frifetikin 14 0 0 0 0 0 0 1 , 0 1 tractiOti 14 0 1 0 1 1 1 rrattion 16 0 0 0 0 0 0 0 0 0 FratclOn 15 Friction 16 0 0 0 0 0 0 0 0 0 "irat116 0 Fraction-17 0 1 1 1 1 1 1 1 7 jj7 0 0 1 0 0 0 0 1 2 Fraccianl II 0 0 0 0 0 0 0 0 0 frattiOn18 0 Table 3. Performance assays of the different mucus fractions from Pacific salmon.
Minutos Minutos 0 2 4 6 8 10 30 60 Total 0 2 4 6 8 10 30 60 Total Futtelon 1 - 0 0 1 1 0 0 1 0 3 'Friation 1 0 FractiOn 2 0 0 0 0 1 0 0 0 1 Fintittik2 RW0%3 0 0 0 1 1 0 0 0 2 FratclOn=3- 0 0 0 0 0 1 1 0 2 Fraction 4 0 0 1 1 1 1 1 0 5 FractiOr4 0 RaCt;16115 0 1 0 0 0 0 0 0 1 Fraction -5-ffiettart 6 0 0 0 0 0 1 0 0 1 Friction 6 Fiata.16n 7 0 0 1 0 0 1 1 0 3 Fr/it'd-on 7 - 0 1 0 1 1 0 0 1 4 FtatiEttio, 8 0 1 1 0 1 1 1 1 6 Fraction_8. 0 0 0 0 0 0 0 0 0 FraOt16ff9 0 0 0 0 1 0 1 0 2 Fractitin-C 0 0 1 0 1 0 1 1 4 Fracci6n'10 0 0 0 0 0 0 0 0 0 Frac,t14014 0 1 0 0 0 0 0 1 2 F1'OctIon'll 0 0 1 0 0 0 0 0 1 tiaaionsii1 0 0 0 0 0 1 0 0 1 Fi4act1tin 12 0 0 1 0 0 0 0 0 1 Frittion 12. 0 1 1 1 1 0 1 1 6 Fratchki13 0 1 1 1 1 1 1 1 7 Fractiort 13.. 0 0 1 1 0 0 1 1 4 Fr#16414 0 0 0 1 1 1 1 1 5 Fractir501: 0 1 0 0 0 0 0 0 1 Fiptttoti 15 0 1 0 0 1 0 0 0 2 rfottion 15-' Ffittion 16 0 0 0 0 0 0 0 0 0 Ftµ
attity1.16 0 0 0 0 0 0 0 0 0 Traction 17 0 0 0 0 0 0 0 0 0 rttidlottli-Fradd6i11180 0 U 0 0 0 0 0 0 Fraction-18 0 0 0 0 0 0 0 0 0 Minutos Minutos 60 Total.. 0 2 4 6 8 10 30 60 Total Fraction 1 0 0 0 0 0 0 0 0 0 Fraction 1-FractiOn 2 0 0 1 0 1 0 1 0 3 rFratclpin 2 0 Fraction 3 0 1 0 0 1 0 0 0 2 FiMitilldn 3 , FrattiOn 4 0 0 0 0 0 0 0 0 0 fraailiSn 4 0 Fraction 5 0 0 0 0 0 0 . 0 0 0 _ FrattiOn 5 Frattian-6 0 1 0 0 0 0 1 0 2 Fraction 6 0 Fractibn 7 0 0 1 1 1 1 0 1 5 Fraction 7 0 EttictIOn 8 0 0 1 1 1 1 0 1 5 Frity6n 8 Fratchin 9 0 1 0 0 0 0 1 1 3 Featition 9 0 Fraction 10 0 0 0 1 1 1 1 0 4 7_ Fraction FrattiOn'll 0 0 0 0 0 0 0 1 1 Fratcion it 0 1 1 1 1 0 0 0 4 lia&Atin 12 0 0 1 1 1 0 0 0 3 Fracciap 12 Friction 13 0 1 0 0 0 1 1 1 4 Fidóti3 cloom000 FraceMn 14 0 1 1 1 1 1 1 1 7 Ftatil6t414 FrattiOn 15 0 0 0 0 0 0 0 0 0 FractiOn ti FrzultiOn16, 0 0 1 1 1 0 0 0 3 Fraction 16 fr,accion 17 - 0 0 0 0 0 FrottiOn17 0 0 0 0 0 0 0 0 0 FraCtIon le 0 0 0 0 0 0 0 0 0 Frattihn 18 Minutes Minutes 0 2 4 6 8 10 30 60 Total 0 2 4 6 8 10 30 60 Total Fracci; 0 1 0 0 0 0 0 0 1 Fracciônl; 0 1 1 1 1 1 o 0 5 00 0 0 0 0 G 00 F = ;=;; I 0 0 _ 0 0 0 0 0 0 0 PricciOn.3 = 0 0 0 1 1 1 0 0 3 iiaccion 3 0 Fraccien 4.. 0 0 1 1 1 0 0 0 3 Fracclon 4 MOW(' 5 0 0 0 0 0 0 0 0 0 Fracclon ..

Frn 5- 0 0 0 0 0 0 0 0 0 fratclOn - 0 FracciOn 7 = 0 1 0 1 1 0 1 1 5 -Fracciiiti1 Fraccioeti 0 0 1 1 - 1 1 1 1 6 ham& 8 0 Frikeion 9 0 1 1 0 1 1 1 1 6 Fir-amide 9õ,,, 0 0 1 0 0 0 1 1 3 FracciOn 10 0 0 0 0 0 0 1 1 2 Fraccion 10 0 1 1 1 1 1 1 0 6 FracciOn 11 0 0 1 _ 1 0 0 0 0 2 Fraccion Fraedlin 12 0 0 1 1 1 1 0 0 4 F_rafcien Freed& 13 0 1 0 0 1 0 1 1 4 Fred& 13 0 Fraccien 14 0 0 0 0 0 1 0 0 1 Ramon 44 0 FeacciOn 15 0 0 0 0 0 0 0 0 0 Fracci6n 15 0 o 0 o 0 o 0 0 Firi16E110 16 0 1 0 0 0 1 0 0 2 FrMiciati Fraccion 17 0 0 0 0 0 0 0 0 0 =Fraccleni47- 0 0 0 0 0 0 0 0 0 Fraccien 18 0 0 0 0 0 0 0 0 o 'Fraccitilfis U o o 000 0 o o On the other hand, differences are observed between different mucus fractions.
As evidenced in Figures 1 to 3, in this assay the behavior of the parasites when confronted to different mucus fractions from rainbow trout shows a definite attraction in certain fractions, particularly fractions 6, 8 and 12 (p < 0.025). A
different situation occurs with mucus from Atlantic and Pacific salmon, since the attractant mucus components are contained in fractions 12 and 13 (p <0.001), and 7 and 13 (p < 0.001), respectively.
EXAMPLE 4: Manufacture of an attractant bait for the control of Caligus parasites in cultured fish To manufacture the attractant bait, mucus obtained indistinctly and directly from fish according to Example 1 and/or attractant fractions obtained as in Example 2 were used.
Agar was dissolved in distilled water at a concentration of 1.5% by weight at a temperature of 90 C. The solution was stirred at room temperature until its temperature reached 40 C. In this moment, an amount of 150 pl of mucus or attractant fractions per liter of liquid agar solution was added. Once an homogeneous liquid was obtained, 1.8 I were deposited inside a round-shaped mould with an approximate diameter of 32 cm that contained a white polypropylene sponge with an approximate diameter of 32 cm and an approximate thickness from 1.5 to 2 cm.
Special care was put to verify that the sponge was completely soaked in the liquid solution. The bait prepared in this way was left to cool to room temperature until the agar solidified. The final product thus obtained is the attractant bait of the present invention.
EXAMPLE 5: Use of an attractant bait for the control of Caligus rogercresseyi parasites in tanks with cultured fish In this study, 1-m3 tanks were used with 30 fish (Atlantic salmon) each according to the following specifications:
- Negative Control: Fish without C. rogercresseyi and without including baits, but under the same experimental management conditions.
- Positive Control: Fish challenged with C. rogercresseyi without including baits.
- Group 1: Fish challenged with C. rogercresseyi including 4 baits.
Individual fish had an initial live weight of 160 g +1- 15 g and were healthy and free from concomitant diseases. The fishes used in this experiment were from a sanitarily certified commercial stock. Before the assays, fish were acclimated into the tank conditions for at least 7 days.
The day when the experiment began, 4 baits from Example 4 were placed in the test tank at least one hour before inoculating small C.
rogercresseyi copepods. 1,500 +1- 200 small copepods were used in the positive control tank and in the Group 1 tank wherein 4 attractant baits according to Example 4 have been placed.
The study of the effectiveness of the baits was performed continually for

7 days in darkened closed tanks with constant oxygenation. In order to avoid dragging out the small copepods, special care was taken regarding water flows in the tanks.
At the end of said period, all animals were sacrificed to count the parasites present on each animal and on each bait. Figure 4 shows the small copepod average attached to each animal in Tank 1 (fish with C. rogercresseyi without attracting baits) compared to Tank 7 (fish with C. rogercresseyi with the attracting baits according to Example 4). According to a Levene test for variance homogeneity and a one-way ANOVA (analysis of variance), significant differences were detected with a confidence level p < 0.05 between both tanks.
The above experiment demonstrates the efficacy of using attractant baits for controlling parasites, in particular C. rogercresseyi, on cultured fish, particularly salmonids.

Claims

1. An attractant bait for ectoparasites of the genus Caligus in a cultured fish, wherein said attractant bait comprises:
.cndot. an attractant comprising at least one of a fish mucus or an attractant fraction obtained from fish mucus, wherein the fish mucus is obtained from a fish species selected from the group consisting of, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch) and rainbow trout (Oncorhynchus mykiss);
.cndot. a gel selected from the group consisting of agar, gelatin, carrageenan, acrylamide, polyethylenglycol and chitosan; and .cndot. a supporting matrix; and wherein said attractant fraction is obtained by a) centrifuging at 800 to 2 000 g the fish mucus for 13 to 18 minutes at a temperature ranging from 3 to 5°C;
b) placing the supernatant of step a) into a sucrose gradient that has 4 to 8 different concentrations with a correlative difference of 10% between each of them, where the lowest concentration of sucrose is 10%;
c) centrifuging the loaded sucrose gradient between 1 and 3 times at a speed ranging from 12 000 to 20 000 rpm for 60 to 120 minutes at a temperature ranging from 3 to 5°C; and d) isolating the different attractant fractions; and wherein .cndot. the attractant bait for the rainbow trout comprises at least one of attractant fraction 6, 8 or 12;
.cndot. the attractant bait for the Atlantic salmon comprises at least one of attractant fraction 12 or 13; and .cndot. the attractant bait for the coho salmon comprises at least one of attractant fraction 7 or 13.

2. The attractant bait according to claim 1, wherein the gel is agar.

3. The attractant bait according to claim 1, wherein the matrix is a sponge-like structure comprising synthetic or natural materials.

4. The attractant bait according to claim 3, wherein the matrix is made from polypropylene.

5. A method to manufacture the bait defined in any one of claims 1 to 4, wherein said method comprises the steps of:
a) isolating mucus from fish and keeping the mucus at a temperature ranging from 1 to 6°C;
b) centrifuging at 800 to 2 000 g the fish mucus of step a) for 13 to 18 minutes at a temperature ranging from 3 to 5°C;
c) placing the supernatant of step b) into a sucrose gradient that has 4 to 8 different concentrations with a correlative difference of 10% between each of them;
d) centrifuging the loaded sucrose gradient between 1 and 3 times at a speed ranging from 12 000 to 20 000 rpm for 60 to 120 minutes at a temperature ranging from 3 to 5°C; and e) isolating the different attractant fractions.
f) preparing a liquid gel selected from the group consisting of agar, gelatin, carrageenan, acrylamide, polyethylenglycol and chitosan;
g) incorporating the attractant fraction from step e) into the gel of step f);
h) incorporating the gel with the attractant fraction from step g) into a suitable matrix; and i) solidifying the gel of step h) wherein .cndot. the attractant bait for the rainbow trout comprises at least one of attractant fraction 6, 8 or 12;
.cndot. the attractant bait for the Atlantic salmon comprises at least one of attractant fraction 12 or 13; and .cndot. the attractant bait for the coho salmon comprises at least one of attractant fraction 7 or 13.

6. Use of the attractant bait defined in any one of claims 1 to 4 or obtained by the method defined in claim 5 for attracting ectoparasite larvae by placing the bait in the sea in fish culture zones.

7. The use according to claim 6, wherein the ectoparasites are Caligus rogercresseyi.