This invention relates to methods for the control of sea lice and to apparatus for use in such methods. In particular, the invention relates to methods which may be used to control sea lice infestations in the farming of salmon and other fish.

In fish farming, large numbers of fish are held in a confined area such as in tanks or net cages until they are ready for slaughter. Under such conditions, diseases and parasite infections can lead to substantial losses.

Sea lice are a group of marine ectoparasites which infest both wild and farmed fish. Especially amongst farmed fish, sea lice numbers are high and this can lead to severe damage of the fish stock. Sea lice are crustaceans of the family Caligidae. Those which are primarily responsible for infestations in marine fish are of the genera Lepeophtheirus and Caligus, especially Lepeophtheirus salmonis and Caligus elongates.

In Europe, it is the salmon louse {Lepeophtheirus salmonis) which is the most common and which poses one the biggest challenges to commercial salmon farming. This sea louse attaches to the fish and causes damage by feeding on the mucous, epidermal tissue and blood of the host. In some cases, damage may also be caused to the underlying tissues. Excessive ectoparasitic infection can lead to death of the fish. Even at low levels of infection, the fish lose weight and take much longer to reach the necessary size for slaughter. Infected fish also have an unpleasant appearance which greatly reduces their market value.

In Norway alone, it is estimated that it will cost the industry up to one billion

Norwegian Kroner to control sea lice infestations. This loss arises from the reduced growth of the fish caused by reduced health and conventional sea lice treatments; reduced market value of the fish; the cost of conventional chemical treatments; increased workload for the fish farmers; and increased fish mortality.

Chemicals traditionally used to control sea lice include insecticides. Whilst these can be effective, there are major disadvantages which limit their use. These chemicals are harmful to many marine organisms and are undesirable in the environment. Development of resistance to such agents following long term use represents an additional problem which necessitates the introduction of new chemicals. This negative cycle has to be broken for the fish farming industry to be perceived as sustainable.

The present invention relates to the use of semiochemicals (behaviour modifying chemicals) to control sea lice infestation. More specifically, the invention is aimed at trapping the sea lice larvae using these chemicals as an attractant before they are able to reach the fish and cause any damage.

The lifecycle of sea lice has several moulting and life stages. Sea lice have three free living stages (nauplii larvae) where they mainly drift with the sea currents before they attach to the fish. The third life stage is the copepodite stage. This is the infective stage where the copepodite can swim freely and is involved in the energetically demanding process of host location and attachment. Until such time as the larvae attach to the fish (i.e. at about 0.7 mm in size and below), they live off nutrition from the egg. This gives the lice a time window in which to find a host dependent on the energy reservoir from the egg and their level of activity. The distance which they are able to swim at this stage is limited by their size. No further development in the lifecycle of the sea lice will take place until the copepodite has settled and attached to a suitable host fish.

The present invention is based on the recognition that sea lice larvae can effectively be trapped before they attach to the fish. This moves the fight against sea lice out of the remit of the fish farm which can focus on fish production. Other positive effects on fish farming include avoiding the need to use harmful chemicals to treat fish once they become infested. Methods which involve the use of a sea lice trap are also much less costly than those which are presently used, particularly those which require the use of expensive chemical agents.

What the inventor has recognised is that if one can get the sea lice to use their energy to swim towards and attach to a different substrate (i.e. other than the fish), they will consume their energy and lose the ability to find and attach to the fish species which is to be protected. In this way, the numbers of sea lice reaching populations of fish in, for example, fish farms can be greatly reduced. This reduces the damage to the fish from sea lice infestations.

Specifically, the inventor has found that substances obtained either directly or indirectly from a host species can be used as a semiochemical to lure or attract sea lice thereby reducing their ability to infest fish.

The inventor has found that sea lice can effectively be trapped by loading these substances onto a substrate, in particular a substrate which mimics the mucosal skin surface of the fish. The substances (semiochemicals) which are used provide a means of controlling the sea lice which does not use harmful chemicals that affect the environment and which contributes to improved fish health, well-being, growth and market quality.

Viewed from one aspect the invention thus provides the use as an attractant for sea lice of a substance which is obtained from a host fish species. More specifically, there is provided the use of such a substance as an attractant for free-swimming copepodites.

Methods of controlling sea lice (e.g. preventing sea lice infestations in a host species) which involve the use of a substance obtained from a host species form a further aspect of the invention.

The methods herein described may be used for the control of all types of sea lice in fish. The term "sea lice" is generally used for species of copepods within the order Siphonostomatoida, family Caligidae. There are 36 genera within this family which include approximately 42 Lepeophtheirus and 300 Caligus species. The methods of the invention are particularly suitable for use in controlling those sea lice which are of the genera Lepeophtheirus (e.g. Lepeophtheirus salmonis) and Caligus (e.g. Caligus curtus, Caligus elongatus).

Host species, and thus the species which can be protected by employing the methods herein described, include both wild and farmed fish. Those belonging to the Salmonidae family are preferred as host species and include Salmo salar, Salmo trutta (brown or sea trout), Salmo clarkii, Oncorhynchus gorbuscha, Oncorhynchus keta, Oncorhynchus nekra, Oncorhynchus kisutch, Oncorhynchus tshawytscha, Oncorhynchus mason, Oncorhynchus mossambicus, Oncorhynchus mykiss (rainbow trout) and Salvelinus species. Other examples of suitable host species include carp, whitefish, sea bass, sea bream, grey mullet, gilthead sea bream, sole, plaice, cod and halibut.

Preferred host species include Atlantic salmon, Pacific salmon and seawater trout, particularly preferably Atlantic salmon.

Substances which may be used as an attractant in the methods of the invention may be obtained from any chosen host species, including any of those species described above. Since many sea louse species are specific to the host genera, the host species from which the attractant is obtained should be chosen

accordingly. Substances which may be used at the attractant include any product or extract derivable from the host fish.

Typically, the attractant will comprise waste material (i.e. by-products) from a fish slaughterhouse (e.g. from salmon slaughterers) or any component which may be derived from such waste. These materials are considered to provide a natural smell at the correct concentration which stimulates positive taxis to sea lice. Waste material produced following slaughter of fish generally includes skin mucous, skin, blood, muscle, bones, etc. Any waste product which contains smell active fractions may be used. However, preferred materials for use in the invention include skin mucous and skin from filleting.

The waste materials may be used directly, i.e. without further processing. However, these will generally be subjected to further processing using methods and materials which are known in the art. For example, the entire waste or any component of the waste material may be homogenised using conventional homogenisation techniques. The resulting homogenates may, if desired, be separated by methods such as centrifugation. Other processing techniques include digestion using enzymes. For example, trypsinated material (a peptide-containing product) may be used. Alternatively, attractant materials may be purified or extracted from samples of waste fish material using suitable methods known in the art. It is envisaged that a number of different fractions or extracts may be used. Both organic and water- soluble extracts may be used, although those which are soluble in water are generally preferred (since the attractants will be capable of leaching into water in use). Suitable fractions may be obtained using known techniques such as liquid chromatography (LC) and may be used alone or as combinations (i.e. mixtures) of different fractions.

The attractant should be used in an amount which is effective to control sea lice infestation. The amount may be determined taking into account factors such as the nature of the attractant used, the species and distribution of the sea lice, etc. It is the concentration of the attractant in the surrounding water that matters and this will be dependent on other factors such as the nature of any gel or other vehicle in which this may be incorporated (see below). The attractants will generally be active when present at the nanomol to picomol (10 ^"9 to 10 ^"12 ) level of concentration. When provided in a release vehicle (e.g. a gel), the attractant will therefore typically be present at a concentration in the μητιοΙ to mmol range.

The natural attractant materials may be used alone in the methods herein described or in combination with other chemicals known to attract sea lice. Examples of such chemicals include host and non-host kairomones such as 6-methyl-5-hepten-2-one, 2-aminoacetophenone, isophorone and 4-methylquinazoline. Appropriate amounts of these chemicals may be determined by those skilled in the art whereby to obtain the desired response from the sea lice. It is envisaged that these will generally be present at a concentration in the μΐηοΙ to mmol range when provided in a suitable release vehicle (e.g. a gel).

Preferably, the attractant herein described will be provided in the form of a controlled release formulation. Such formulations may be obtained by incorporation of the attractant into a suitable gel or other substance (release vehicle) capable of providing a delayed release of the chemical when submerged in sea water or fresh water. Examples of suitable gels are those which are further described herein. T/GB2011/050862

6

In a preferred embodiment of the invention the attractant is provided in or on a suitable substrate which can be submerged in the water and which serves to function as a trap for the sea lice. Such structures serve not only as a support for the attractant, but also as a resting place for the larvae.

Viewed from a further aspect the invention thus provides an apparatus for use in controlling sea lice which comprises a substrate having bound thereto an attractant as herein described.

Suitable substrates which either incorporate the attractant or otherwise act as a support for the attractant may be of any shape or size to suit the particular application and may be made of any material which is capable of being submerged in water (especially seawater) for extended periods of time. The substrate may be rigid or flexible depending on the area in which this is to be located, but preferably this will be flexible so that this does not provide an obstruction in the water. The substrate may comprise a material having an open structure (thereby allowing the free flow of water) or may be solid in structure. Preferred substrates are those having a large surface area and which therefore maximise the exposure of the sea lice to the attractant. A large surface area may be provided by a material having an open, porous structure, e.g. a woven material or netting. Other types of structure which may be used include those which comprise lengths of intertwined or spun threads or fibres, e.g. ropes, strings, etc.

Both natural and synthetic materials may be used to form the substrate. Preferred materials which may be used include textile materials (e.g. linen, canvas), netting, mesh and semi-permeable membranes. Semi-permeable membranes having a range of different pore sizes may be used provided that the pores are such that the active components (attractants) are able to migrate out of the structure and into the surrounding water.

In a preferred embodiment, the substrate may be provided in the form of a sheet or strips of material (e.g. a linen sheet). The sheet may be any size and shape, but typically this may be provided in the form of strips which are each about 0.1 to 2 metres in width and about 10 metres long. The attractant may be fixed to the surface of the substrate or otherwise

incorporated into its structure in such a way that this will leach out into the surrounding water and attract the sea lice. In one example, the attractant may be adhered to the surface of the substrate. One particular method of achieving this involves the use of a gel in which the attractant material is incorporated. Gels have a number of beneficial properties in that these serve act not only to hold the attractant in place on the surface of the substrate, but also mimic the mucosal skin surface of the host species to which the lice can attach and become immobilised.

Suitable gels may be selected on the basis of their degradation (and thus release) characteristics. For example, these may provide immediate or prolonged release of the attractant. Preferably, these will provide slow release of the attractant into the surrounding water in order to maintain effective levels of the agent in the local environment.

Suitable gels should be non-toxic and may readily be determined by those skilled in the art. These include agars, alginates, pectin, chitosans, gelatins, fish skin gelatins, or any combination thereof. These gelling agents may, if required, be cross-linked using conventional cross-linking agents (e.g. a divalent cation such as Ca ²⁺ ) to achieve the desired structure capable of persistence in sea water.

Typically, the attractant material will be mixed into the gel which is then applied (i.e. coated) in the form of a layer over the surface of the substrate. Alternatively, the substrate material may be dipped into the pre-mixed gel. This technique may be used where the substrate is provided in the form of a mesh or netting in which the individual strands of the material need to be coated.

In other embodiments of the invention it is envisaged that a gel may be used to provide a trap having surface characteristics which mimic the mucosal surface of the fish, but in which the gel need not necessarily contain the attractant. For example, the attractant may be provided within a substrate structure which is then coated with a suitable gel. One example of this embodiment of the invention is a mesh bag in which the attractant is disposed and which is provided with an outer layer of gel. The depth of the gel coating should be chosen such that this readily permits the smell of the attractant to permeate into the surrounding water. Suitable coating thicknesses may range from 1 to 100 mm, preferably from 2 to 5 mm.

Other substances which may be provided in the gel include stabilisers, binders, dispersants, anti-bacterial agents, etc. Where present, any such substances should not interfere with the positive taxis effect of the attractant.

Once attached to the substrate, the sea lice larvae become immobilised; they have used their energy in swimming towards the trap and are therefore unable to move on and infest any fish. The trap may periodically be removed from the water and replaced with a fresh trap.

The substrate may additionally contain a toxin or anti-parasitically effective compound which is capable of further immobilising the sea lice. The choice of toxin may be dependent on the different type or species of sea lice, but will typically be one which is effective against Lepeophtheirus salmonis or Caligus elongatus.

Suitable toxins include any of the chemical agents known in the art for use in the control of sea lice, such as organophosphate and pyrethroid compounds, avermectins or chitin synthesis inhibitors. Examples of organophosphate compounds which may be used include trichlorfon (dimethyl-2,2,2-trichloro-1- hydroxyethyl-phosphonate), dichlorvos (2,2-dichloroethenyl-dimethylphosphate) and azamethiphos. Pyrethroid compounds which may be used include pyrethrum, cypermethrin , cis-cypermethrin and deltamethrin. Amongst the avermectins which may be employed are ivermectin and emamectin benzoate. Examples of suitable chitin synthesis inhibitors are diflubenzuron and teflubexuron. These toxins may be mixed into the gel, optionally in combination with the attractant material, and will generally be used at concentrations which are effective in killing the sea lice. The concentration will depend on the nature of the chemical used, but in general this will be in the range from 0.1 to 100 parts per billion.

Other agents which may be provided on the substrate include anti-fouling agents such as copper.

The surface of the substrate should be sufficiently large so that it will be easy for the copepodites to find. In most cases, it is envisaged that several substrates will be used at strategic points around the fish population which is to be protected. For example, these may be spread across a large area. The particular site and range of the sites will depend on the particular location of the infestation or potential infestation and will take into account factors such as the size of the fish population which is to be protected, e.g. the area of any fish farm. Other factors which have an effect on sea lice numbers should also be taken into account, such as the time of year, etc. Where several substrates are used, these may be placed at a suitable distance from one another so that the copepodite can reach one of the substrates by its own propagation.

When used in the control of sea lice, the substrate materials herein described may be suspended in the water at a chosen point or points proximal to a site of potential infestation, such as a fish farm. Generally, these will be positioned upstream or up current from the fish which are to be protected. For example, these may be placed in a pre-determined pattern at critical points upstream (or up current) from a fish farm or surrounding the fish farm if tidal waves bring water back and forth. When used in controlling sea lice infestations in wild fish, these may be used upstream in a river where wild salmon and sea trout may get infected. Alternatively, the substrate materials may be deployed at critical points identified for lice larvae distribution, for example in a fjord system. In this way, the general stock of sea lice larvae in the area can be reduced. Suitable places for the sea lice traps can be determined based on hydrographic knowledge of the area of deployment.

Since it may not be possible to compete with the fish smell close to a fish farm or in other locations where fish are present in high numbers, it is envisaged that the substrates will be placed at a suitable distance from the fish population. A suitable distance may be a site within 1000 metres, preferably within 500 metres, more preferably within 250 metres from a site of infestation or potential infestation.

In use, the substrate materials (traps) will be suspended in the water at a suitable depth. The sea lice larvae are strongly phototactic and so during the day will generally be present in the surface water, i.e. at a depth of less than 15 metres. Positioning the substrate materials at a depth of 1 to 10 metres will generally be sufficient to attract the larvae. Where more than one trap is used, these will be arranged in a pattern and at a separation which ensures that the sea lice can swim to them and attach. Several different methods may be used to suspend the traps at the chosen points in the water. For example, these may be attached to a buoy or other flotation means and suitably weighted such that these remain in position. Alternatively, these can be suspended from the bottom of the sea or river bed by attaching a weight to the lower surface of the trap and, if necessary, a flotation device to its upper surface. Fixing of the trap to the seabed is particularly suitable in areas with boat traffic. Although the trap will generally be positioned vertically, it can be positioned horizontally by fixing it to the seabed. This is especially the case in shallow areas.

The substrate materials (traps) herein described will have a finite lifetime which will be dependent on the nature of the attractant material used and the method for its deployment, in particular the nature of any gel which is used to attach this to the substrate structure. It is therefore envisaged that the structures will be replaced or replenished with attractant periodically in order to maintain the desired level of control of the sea lice. Typically, it is anticipated that these may be replaced or replenished with fresh attractant every week to every second month, for example every 1 to 4 weeks in the summer and every 1 to 3 months in winter. Once replenished, these may be reinstalled at the treatment site.

The gel material containing the attractant herein described is novel and forms a further aspect of the invention. Viewed from a further aspect the invention thus provides a gel containing an attractant material obtained from a host species. Any combination of gel and attractant material as hereinbefore described may be used. Preferred products are those which are provided as controlled release formulations.

In a further embodiment of the invention one or more light sources may be positioned either on the substrate or in close proximity in order to promote taxis of the sea lice towards the trap. The number and positioning of the sources of light may be determined according to factors such as the intensity of the light source and the turbidity of the water which surrounds the target. Where a single light source is used, this may be positioned at or just below the surface of the water above the target structure in order that this illuminates the structure from above. Alternatively, a string of lights may be positioned on or around the structure. Where the structure is a length of textile material, these may run along the entire length of the structure. It is envisaged that the lights would generally only be switched on at night or in low light conditions. Light sources which may be used include diodes which may be powered either by a battery or by solar power. Where solar power is used to generate power, it is envisaged that one or more solar cells may be provided at the surface of the water, for example, on a buoy or other floatation device which is used to position the substrate. Electricity from land may alternatively be used for power.

Embodiments of the invention will now be described with reference to the accompanying figures in which:

Fig. 1 illustrates the positioning of substrates in a tidal water in one particular embodiment of the invention;

Fig. 2 illustrates methods of positioning substrate materials in certain embodiments of the invention;

Fig. 3 illustrates methods for illuminating a substrate material in certain embodiments of the invention; and

Fig. 4 shows the movement of salmon lice larvae into the branches of a Y-tube in the experiment detailed in Example 1 : A denotes experiments in which the water to the lower Y branch passed skin tissue (average of 5 replicates); B denotes experiments in which the water to the lower Y branch passed skin mucus (average of 4 replicates); and C denotes experiments in which clean water passed through both Y branches (average of 3 replicates). In each set of results, the left hand bar denotes the lice movement "In"; that on the right denotes the lice movement "Out".

Fig. 1 shows a plurality of substrate materials (traps) 1 which are provided in the form of lengths of textile material. These act as supports for a gel carrying a sea lice attractant as herein described. The substrate materials 1 are disposed in a tidal water, for example up current from a fish farm. Each substrate material 1 is attached to a buoy 2 which floats on the surface of the water and to an anchor 3 which serves to fix the substrate material in place. The substrate materials are separated by a distance (x) which ensures that the sea lice larvae can swim to at least one of these and become attached. Fig. 2 shows two methods which may be used to locate the substrate materials (traps). In Fig. 2(a) the substrate material 1 is hung from the surface of the water by means of attachment to a buoy 2 and anchor 3. In Fig. 2(b) the substrate material is suspended from the bottom of the sea or river bed by attachment of a floater 4 and weight 5. The trap will normally be positioned vertically as shown in the figure, however, the same kind of attachment can be used for a horizontal positioning, e.g. in shallow water.

Fig. 3 shows embodiments of the invention in which the substrate materials (traps) are illuminated in order to assist in the taxis movement of the sea lice larvae. In Fig. 3(a) a light source 6 is placed at the surface of the water above the substrate material 1. In Fig. 3(b) a string of lights 7 is disposed along the length of the substrate material 1.

The present invention is further illustrated by way of the following non-limiting Example:

Example 1

Materials:

The test chemical/tissue was put in net bags made out of plankton mesh with a mesh size of 100 μιη. The bag was placed in a 250 ml flask with seawater.

Another flask contained clean seawater. A peristaltic pump was used to pump the water to the test systems. All experiments were carried out at 0°C in a climate- controlled room.

Mature females with egg strings were collect from salmon. The egg strings were removed from the females and inoculated in seawater with air bubbling at 10°C in a climate-controlled room. When hatched larvae had developed to the copepodite (free-swimming) stage they were picked out for use in the following experiments.

A salmon was killed by a blow to the head and hung on a support with the head up while the skin mucus was carefully scraped off with a spatula. The fish was then gutted and filleted. Both the skin mucus and fish skin were stored in a fridge until use. The test was performed on the same day. In all experiments the behaviour of the copepodites were filmed on video, which afterwards was analysed.

Methods:

Activation experiments were carried out in two small plastic trays with water flow through. One tray contained clean water (control); the other contained water that had been exposed to the test chemical/tissue (exposed). The behaviour of the copepodites was then compared between control and 'exposed'.

Tests for positive chemotactic response were performed in a Y-tube. The flow pattern of water in the tube was first established by testing with coloured water. Once a satisfactory flow pattern had been established, 'exposed' and clean water (control) was pumped into each of the Y ends. All the ends of the Y-tube were covered by mesh so that the copepodites could not escape. In this system both activity and degree of chemotactic behaviour of the copepodites could be studied.

10-12 lice larvae were added to the bottom of the Y-tube. The number of larvae that entered each branch of the Y-tube was recorded, as well as the numbers leaving the tube. This gave a clear picture of preferred tube by the larvae. The results are shown in Fig. 4.

Results:

Water which had been exposed to both the skin mucus and pieces of fish skin were found to activate the larval behaviour. The copepodites showed positive chemotaxis towards both materials.

In the Y-tube experiments, three levels of lice larvae activity were seen:

1. Low activity level

- when only clean water was passed into both branches of the Y-tube.

2. Slightly increased activity level - in the control branch of the Y-tube (due to an induced general activity from the exposure in the mixing zone). Highly increased activity level

- in the 'exposed' branch of the Y-tube. ificant difference was observed in the results from skin mucus and fish skin.