Note: Descriptions are shown in the official language in which they were submitted.
CA 02685795 2009-11-12
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.
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CA 02685795 2009-11-12
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
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CA 02685795 2009-11-12
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.
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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).
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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
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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
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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
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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
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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
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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
20000T
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.
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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:
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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
0 0 0 0 0 0 0 0
Praceii5n 2 g' 0 0 1 0 0 0 0 0 1 fraction 3 0
0 0 0 0 0 0 0 0
Fiaccidn3 0 0 0 0 0 0 0 0 0 FrOCC1On 4 0
0 0 0 0 0 0 0 0
Fracci6a4 - 0 0 0 0 1 0 0 0 1 FracclOn 5 0
1 1 0 0 0 1 0 3
FraccIN 5 0 0 0 0 0 1 0 0 1 Fraccien 6 0
1 0 0 1 1 1 1 5
FraccitIn fi 0 0 0 0 0 0 0 0 0 Fraction\ 7
0 1 1 1 1 0 0 1 5
Fraddart7. 0 1 0 1 0 0 1 1 4 Fraccian 8 0
0 1 1 1 1 1 1 6
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
0 0 0 U 0 U 0 0 0
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
1 1 0 0 0 0 3
kFrahlon1i 0 1 1 1 1 0 0 0 4 Fr8Ccitin 13 0
1 1 1 1 1 1 0 6
FrileelOn 1j 0 0 0 0 1 1 1 0 3 FrdoCiOn 14
0 0 0 0 0 0 0 0 0
Fracclikilf 0 1 1 0 1 1 1 0 5 "Fraccieri
15 0 0 0 1 1 1 1 1 5
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
0 0 0 0 0 0 0 0 0
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
1 0 0 0 1 0 0 2
FracciOn 3 0 0 0 1 0 0 0 U 1 FraccIt413
0 0 0 1 0 0 0 0 1
Fraccijn4 0 0 0 1 0 0 0 0 1 Fracclin4
0 0 1 1 1 0 1 1 5
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
0 0 0 0 0 0 1 0 1
Fraccin 8 0 1 1 1 1 1 1 1 7 Fraccidn 8
0 0 1 0 0 0 0 0 1
O
i
Fraction 9 0 1 1 1 1 0 1 0 5 FracClor9
0 0 0 0 1 1 0 1 3
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
1 1 7
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
15 0 0 0 0 0 0 0 0 0
Frattitin 15 0 0 0 0 0 0 0 0 0 Redden 0
1 0 0 0 1 0 0 2
Fraccierilk 0 0 1 1 0 0 0 0 2 FractIOn
17 0 0 0 0 0 0 1 0 - 1 -
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
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CA 02 685 7 95 2009- 11- 12
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
0 0 0 0 0 0 0 0
iraccion 2 o _ o 0 o 0 1 , 0 1 2 Ram& 2 0 0
0 0 0 0 0 0 0
Fraccion 3 0 0 0 0 0 1 1 1 3 Framitin 3 0
0 0 0 0 1 0 1 2
Fraction 4 0 1 0 1 0 0 0 0 2 Fraction 4 0
0 0 0 0 0 0 0 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
1 1 1 0 1 1 1 6
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
1 1 0 1 1 1 1 6
iraccion 9 0 0 0 0 0 1 1 1 3 Fratcion 9 0
0 0 0 0 0 1 0 1
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
0 0 1 0 0 0 0 0 1
Fracclon 12 0 1 1 1 0 0 0 0 3 Fraccion 12
0 1 0 1 1 1 1 1 6
FracclOn 13 0 0 0 0 0 o 0 0 0 Fracclan 13
0 1 0 0 0 0 0 0 1
FracciOn 14 0 0 0 0 0 0 1 0 1 Fraction 14
0 0 0 0 0 0 1 1 2
Fraccion 15 0 1 , 0 0 0 0 0 0 1 Framion 15
0 1 0 1 1 1 0 0 4
Fraccion 16 0 0 0 0 0 0 0 0 o Fraccion 16
0 0 0 0 0 0 0 0 0
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
0 0 0 0 0 0 0 0 0
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
0 1 0 0 0 1 0 2
FracciOn 2 _ 0 0 0 0 0 0 0 0 0 Fraccion 2
0 0 0 0 0 1 0 0 1
Fraccien 3 0 0 0 1 1 1 0 0 3 FracclOn 3
0 1 0 1 1 0 0 0 3
Fraccion 4 0 0 0 0 0 0 1 1 2 Fraccion 4
0 0 0 0 0 0 0 1 1
-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
0 1 0 0 0 0 1 1 3
Fraction 7 0 1 1 0 0 1 0 0 3 Fraccien 7
0 1 1 1 1 1 1 6
Fracciin a 0 0 0 1 1 1 1 0 4 FracciOn 8
0 0 0 0 0 0 1 0 1
-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
0 0 0 0 0 0 1 1 2
Fraccian 12 , 0 1 1 0 1 1 1 1 6 Fracclon
12 0 0 1 1 1 1 1 1 6
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
0 0 0 0 0 0 0 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
0 0 0 0 0 0 0 0 0
FracciOn 17 0 0 0 0 0 0 0 0 0 Fraccion 17
0 0 0 0 0 0 0 0 0
FracciOn 18 0 0 0 0 0 0 0 0 0 Fraccion 10
0 0 0 0 0 0 0 0 0
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
0 0 0 0 0 0 0 0 0
Fraccion 2 0 0 0 0 0 0 0 0 0 Fraccion
2 0 0 0 0 0 0 0 0 0
Ramie') 3 0 0 0 0 0 0 0 0 0 Fraccion 3
0 0 0 0 0 0 0 0 0
Fracclon 4 0 1 0 0 0 0 0 0 1 Fraccifrn
4 0 0 0 0 0 0 0 0 0
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? ,
0 0 0 0 0 1 0 1 2
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
0 1 1 1 0 0 0 0 3
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
0 1 0 1 1 0 1 1 5
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
0 0 0 0 0 0 0 0 0
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
18 0 0 0 0 0 0 0 0 0
13
CA 02 6857 95 2 00 9- 11- 12
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
0 0 0 0 0 1 0 0 1
FractiOtt 0 0 0 1 0 1 1 1 4 FiaCtiott 2 0
0 0 0 0 0 0 0 0
Fractldi 0 0 0 0 0 0 0 0 0 Ftii6Mitiii 3
0 0 0 0 0 0 0 0 0
FtadViin 4 0 0 0 0 00 0 0 0 Fraction 4 0
1 0 0 0 0 0 0 1
FirtiWon 5 0 0 0 0 0 0 0 1 1 Fraction 5._
0 0 1 0 0 0 0 0 1
FrieciOn 6 0 0 0 1 1 1 1 1 5 fr#cclon 6_ 0
0 1 0 0 0 0 0 1
Fraction 7 0 1 1 .õ 0 0 0 0 0 2 NOM-7 0 0
0 0 0 1 1 0 2
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
0 0 0 0 0 1 1 2
Fra:tion 10 0 0 1 1 1 1 0 0 4 fraccian 10
0 0 0 1 0 0 1 1 3
Fetitain 11 0 0 0 0 0 0 1 1 2 Fraction 11
0 0 0 0 1 0 0 0 1
Fratt464'4 0 1 1 0 1 1 1 1 6 Fraction 12
0 1 0 0 0 1 1 1 4
F1111;0614 0 1 1 1 1 1 1 1 7 FractiOn 13
0 0 1 1 1 1 1 1 6
Frifetikin 14 0 0 0 0 0 0 1 , 0 1
tractiOti 14 0 1 0 1 1 1
0 1 5
rrattion 16 0 0 0 0 0 0 0 0 0 FratclOn 15
0 0 0 1 0 0 0 0 1
Friction 16 0 0 0 0 0 0 0 0 0 "irat116 0
0 0 0 1 0 0 0 1
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
0 0 0 0 0 0 0 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
0 0 0 0 0 0 0 0
FractiOn 2 0 0 0 0 1 0 0 0 1 Fintittik2
0 0 0 1 1 0 0 0 2
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
1 1 1 1 0 0 0 4
RaCt;16115 0 1 0 0 0 0 0 0 1 Fraction -5-
0 0 0 0 0 0 1 1 2
ffiettart 6 0 0 0 0 0 1 0 0 1 Friction 6
0 0 1 0 0 1 1 0 3
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-'
0 0 0 0 0 1 0 0 1
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-
0 0 0 0 0 0 0 0 0
Fradd6i11180 0 U 0 0 0 0 0 0 Fraction-18 0 0 0 0 0 0 0 0 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 0 Fraction 1-
0 1 1 0 1 0 0 0 3
FractiOn 2 0 0 1 0 1 0 1 0 3 rFratclpin 2 0
0 0 0 0 0 1 1 2
Fraction 3 0 1 0 0 1 0 0 0 2 FiMitilldn 3 ,
0 1 1 0 0 0 0 0 2
FrattiOn 4 0 0 0 0 0 0 0 0 0 fraailiSn 4 0
0 0 1 1 1 0 0 3
Fraction 5 0 0 0 0 0 0 . 0 0 0 _ FrattiOn 5
0 0 0 1 1 0 0 0 2
Frattian-6 0 1 0 0 0 0 1 0 2 Fraction 6 0
1 1 0 0 0 1 1 4
Fractibn 7 0 0 1 1 1 1 0 1 5 Fraction 7 0
0 1 1 1 0 0 1 4
EttictIOn 8 0 0 1 1 1 1 0 1 5 Frity6n 8
0 1 0 0 0 1 1 0 3
Fratchin 9 0 1 0 0 0 0 1 1 3 Featition 9 0
0 0 0 0 0 0 0 0
Fraction 10 0 0 0 1 1 1 1 0 4 7_ Fraction
10 0 1 1 1 1 1 1 1 7
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
0 0 1 1 0 1 1 0 4
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
0 1 0 0 1 1 1 0 4
FrattiOn 15 0 0 0 0 0 0 0 0 0 FractiOn ti
0 1 1 1 0 1 0 0 4
0 0 0 0 0 0 0 0 0
FrzultiOn16, 0 0 1 1 1 0 0 0 3 Fraction 16
fr,accion 17 - 0 0 0 0 0
1 0 1
FrottiOn17 0 0 0 0 0 0 0 0 0
FraCtIon le 0 0 0 0 0 0 0 0 0 Frattihn 18
0 0 0 0 0 0 0 0 0
14
CA 02 6857 95 2009-11-12
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
0 0 1 1 0 0 1 3
Fraccien 4.. 0 0 1 1 1 0 0 0 3 Fracclon 4
0 0 0 1 0 0 0 0 1
MOW(' 5 0 0 0 0 0 0 0 0 0 Fracclon ..
0 0 0 0 0 1 0 0 1
Frn 5- 0 0 0 0 0 0 0 0 0 fratclOn - 0
1 0 0 0 0 0 0 1
FracciOn 7 = 0 1 0 1 1 0 1 1 5 -Fracciiiti1
0 0 1 0 1 1 0 0 3
Fraccioeti 0 0 1 1 - 1 1 1 1 6 ham& 8 0
1 0 0 0 0 1 1 3
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
11 0 1 0 0 0 1 0 0 2
Fraedlin 12 0 0 1 1 1 1 0 0 4 F_rafcien
12 0 0 0 1 1 0 1 0 3
Freed& 13 0 1 0 0 1 0 1 1 4 Fred& 13 0
0 1 0 1 1 1 1 5
Fraccien 14 0 0 0 0 0 1 0 0 1 Ramon 44 0
1 1 1 1 1 0 0 5
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
19 0 1 0 1 0 0 0 1 3
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
CA 02685795 2009-11-12
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
16
CA 02685795 2009-11-12
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.
17
