GB2388544A - Use of semiochemicals to control sea-lice - Google Patents

Abstract

A method for controlling sealice comprises using semiochemicals. The method may be applied to fish farms, in particular salmon farming, and preferably involves the use of attractants to attract, aggregate or arrest sea-lice. The attractants may be in the form of controlled release formulations and comprise isophorone or 6-methyl-5-heten-2-one and may be potentiated by a synergist such as 1-octen-3-ol. The sea-lice may be lured onto a surface treated with a killing or sterilising agent; or they may be lured into traps for monitoring. The attractants may further be used in combination with masking, repellent or deterrent chemicals that stimulate the lice to move away from there host, towards the lure, in a push-pull strategy. Preferred repellents include 2-aminoacetophenone (2-acetoaniline) and 4-methylquinazoline.

Description

GB 2388544 A continuation (72) Inventor(s): Anne Jennifer Mordue John

Anthony Pickett M ichael Alexander Birkett (74) Agent and/or Address for Service: Agrisense BCS Ltd Treforest Ind Estate, PONTYPRIDD, Mid-Glam, CF37 SSU, United Kingdom

SEMIOCHEMICALS FOR SEA LICE MONITORING AND CONTROL

This present invention is concerned with the monitoring and control of sealice of the order Copepoda. specifically relating to the family Caligidae, through the use of signal or 'message 5 bearing' substances (semiochemicals).

Sea lice, Lepeophtheirus salmonis (Copepoda: Caligidae), are ectoparasites on wild and farmed salmonids causing significant losses to the industry in terms of treatment costs and economic damage to the fish. Salmon farmers are largely dependent on using veterinary I O medicines to control this pest but the industry is continually threatened by the development of resistance by the sealice to the treatments. Many of these treatments also have adverse environmental effects that are becoming increasingly important as the salmon farming industry grows.

15 The lifecycle of Lepeoptheirus salmonis comprises of 10 developmental stages, that is, two planktonic nauplius, one free-swimming copepodid, four attached chalimus, two free swimming pre-adult and one adult stage. The free-swimming copepodid is the infective stage of the lifecycle involved in the energetically demanding process of host location and attachment'. In contrast to the nauplius stages, the copepodid is an active swimmer. However 20 with limited food reserves remaining in the yolk sac, it is criticalthat the copepodid locates and attaches to a suitable host in order to initiate the on-host phase'. No further development in the lifecycle of L. salmonis will take place until the copepodid has settled and attached to a host and therefore, it is the role of the copepodid to identify and attach to a suitable host fish in order to complete the lifecycle.

L. salmonis is considered to be circumpolar in distribution in the Northern Hemispheres and causes losses to salmon farmers throughout this region. Considering the wide geographical distribution of the parasite very little information is available in the public domain concerning sea louse behaviour, host location and physiology. Observations in the literature relate 5 principally to behavioural responses of sealice juvenile stages where it has been established that salinity gradients and light are important physical cues. These may aggregate copepodids in steep salinity gradients especially at estuaries or in the upper layers of the water column (positive phototaxis) where hosts may be prevalent2 3 4 5 6 7. Conflicting evidence has shown that copepodids may (cited experiments by Cabral and Fraile8) or may not6 show directed host 10 location behaviour to chemical stimuli9. However, in decapod crustacea chemical cues are extremely common in mediating sexual and reproductive behaviours and sex pheromones are known to be associated with the urine of unmated femalestO 't. Further, the structure and morphology of decapod olfactory sensillae are well known 12, 13.

15 Studies on the role of chemoreception in copepods both free living and parasitic are increasing. Recent studies on the mating biology of a variety of free-living Copepoda show the importance of chemosensory ability in both mate findings t5 i6 7 '8 and prey/food location'920 of copepods. Since it has been found that Copepoda actively select their prey, more studies have focused on the role of semiochemicals in prey location2t 22 23 24 25. There is 20 very little research on host and food location amongst parasitic copepods. However, evidence is mounting for chemotaxis in mate location of free living Copepoda2627 28 29 30 where it has been shown that males indulge in mate guarding behaviour of virgin females.

Some of the structures of the antenna of the copepodid and the adult male of L salmonis and ?5 Caligus elongatus have been described3'32 29. Two olfactory chemoreceptors (aesthetascs)

have been identified on the 1st antennae of L salmonis and C. elongatus3' 3229. Many other chemo- and mechano-receptors have also been found on L. salmonis adult males29. Adult male sea lice have been shown in behavioural studies to display specific chemotactic responses to semiochemicals (behaviour modifying chemicals) originating from salmons 33 34.

5 Both adults and pre-adult stages of L. saimonis are extremely mobile and move actively both on the local fish micro-environment and between fishy 29 30. Adult male lice are particularly mobile in relation to both colonisation and mating behaviours and reassert with ease on different fish. Males transfer between hosts in sea cages which may be important for dispersal to new hosts, and for mate finding.

10 ' The chemical sense of arthropods is very well developed. Semiochemicals (behaviour modifying chemicals) are extremely important modulators of crustacean behaviour. Although other sensory systems e.g. mechanoreception and vision are used in host location, chemoreception plays a vital role in perceiving signals from the environmental 39. Input from 15 environmental cues, which relate to the chemical ecology of the animal in its environment, form an indispensable part of behavioural processes such as mate and host location. Due to the involvement of pheromones, host location cues and other semiochemicals in processes central to survival, have the potential of being manipulated for use in pest control situations and provide a means of using natural compounds in an environmentally friendly and 20 sustainable manner to reduce pest populations.

It would seem likely that host location mechanisms in L. salmonis involve species specific odours (kairomones) from the salmonid host that would elicit behavioural responses by the sea louse. This invention comes from a study focussed on such host location cues used by 95 free-swimming copepodids and adult male L. salmonis.

J ( Semiochemical cues have now been discovered which can be used to monitor and control sealice populations. According to the invention, it has been demonstrated that host odours, in the form of Atlantic salmon conditioned water (SCW) and SCW extracts, stimulate the 5 copepodid stage of L. salmonis, the preadult and adult male sea lice and cause upstream rheotactic responses which direct the sea louse stages towards the source of the odour.

According to the invention, it has been demonstrated that non-host odours, in the form of turbot conditioned water (TCW) and TCW extracts, do not bring about an attraction response.

10 This may indicate that although fish odours may activate sea lice, it is only the salmonid kairomones that switch on orientated movement. Similarly, Halibut condition water (HCW) has also been shown to mask the attraction of copepodids to their host derived attractants.

According to the invention, there is provided the use of two compounds, 2 15 arninoacetophenone and 4 methylquinazoline, which mask the response of free-swimming copepodids to Salmon Conditioned Water.

It has been demonstrated that mate odours, in the form of pre-adult 11 female and male sealice conditioned water, and extracts, stimulate adult sea lice and cause upstream rheotactic 20 responses which direct the sea louse towards the source of the odour The invention has discovered that the semiochemicals responsible for host and mate-locating behaviour comprise small neutral organic molecules, a situation similar to that of semiochemicals used by terrestrial arthropods.

According to the invention, there is provided the use of a compound, 3,5, 5-trimethyl-2 ( cyclohex-2-enone (isophorone) as a fish-derived attractant for adult sealice: 5 The.discovery is surprising in that although isophorone is a known compound used in inks and dyestuffs, it has also been identified in an edible plant, namely as a constituent of cranberry aroma and was present at 0.2% 44 According to the invention, there is provided the use of a compound, 6 methyl-5-hepten-2-

10 one, as a salmonid specific attractant chemical for free-swimming copepodids.

The discovery of the invention means that the amount of control agents, which are often toxic to wauTn-blooded animals, required for the treatment of Atlantic salmon can be restricted considerably. This novel procedure is also advantageous from the ecological point of view, as 15 it is also applicable to those concepts that are based on integrated pest management (IPM) schemes in use for protection of terrestrial agricultural and horticultural systems.

The invention will now be further described by way of example only with reference to the following examples and drawings which are included for the purposes of illustration only and 20 are not to be construed as being limiting on the present invention. Reference is also made to a number of figures in which:

(a Example 1: Semiochemical isolation example Atlantic salmon (Salmo solar L.), 12-24 months old were maintained in artificial seawater at 5 12 C, 32% salinity and 16L:8D illumination. Fish conditioned water was obtained by placing the fish into a circulating flume (20 x 25 x 420 cm) filled with approximately 1001 artificial seawater circulated at a rate of 30 cm sect 34. Standardisation of fish odour in the water was achieved by using the water at a concentration of 8-lOg live fish 1-24h i. Conditioned water was either used immediately or frozen for later use.

A portable closed solid phase extraction (SPE) system was used for extracting polar, neutral and lipophilic molecules from Atlantic salmon conditioned water (1.21 1). The solid phase extraction cartridges were conditioned prior to extraction using HPLC grade methanol (2 ml), followed by displacement by distilled water (2 ml). Following completion of extraction, 15 interference analyses remaining on the cartridges were removed using distilled water (2ml).

The cartridges were then extracted with distilled ethanol (2 ml). The SPE extract of salmon conditioned water (SCW) was separated into 2 fractions, "volatile" and "non-volatile", by distillation under vacuum (0.04 torr) for 24h at 25 C as described previously. The vacuum distillate comprised components with a molecular weight range comparable to those utilised 20 as volatile semiochemicals by terrestrial organisms (volatile fraction) , whilst the residue arising from the vacuum distillation comprised components with insufficient or no volatility (non-volatile fraction). The volatile fraction was diluted with distilled water (50 ml), and extracted with distilled diethyl ether (3 x 10 ml). The organic layers were combined, dried (anhydrous magnesium sulphate), filtered and evaporated under a gentle stream of nitrogen to 25 1001, and stored in tightly capped microvials at-20 C.

The vacuum distillation volatiles were separated on a Hewlett-Packard 5890A gas chromatograph equipped with a cold on-column injector, a flame ionization detector (FID) and a 50m x 0.32mm i.d. HP-1 bonded phase fused silica capillary column. The oven temperature was maintained at 40 C for 2 minutes and then programmed at 10 C mind to 5 250 C. The carrier gas was hydrogen (5psi).

Coupled GC-MS was provided by a capillary GC column (SOm x 0.32mm i.d. HPI) fitted with an on-column injector directly coupled to a mass spectrometer. Ionisation was by electron impact at 70eV, 250 C. The oven temperature was maintained at 30 C for 5 minutes 10 and then programmed at 5 mini to 250CC. Tentative identifications by GC-MS36 were confirmed by peak enhancement with authentic sarnples3' obtained from commercial sources.

Chemicals were diluted in distilled hexane prior to peak enhancement studies. Table 1 shows a list of either the compounds or their mass spectral characteristics.

15 Example 2: Semiochemical isolation example Atlantic salmon (Salmo solar L.) and turbot (Scophthalmus maximus Rafnesque) 12-24 months old were maintained in artificial seawater at 12 C, 32% salinity and 16L:8D illumination. Fish conditioned water was obtained by placing the fish into a circulating flume 20 (20 x 25 x 420 cm) filled with approximately 100 1 artificial seawater circulated at a rate of 30 cm sec' 34. Standardisation of fish odour in the water was achieved by using the water at a concentration of 8-lOg live fish 1-24h I. Conditioned water was either used immediately or frozen for later use.

95 A portable closed solid phase extraction (SPE) system was used for extracting polar, neutral and lipophilic molecules from different preparations of Atlantic salmon (5.0 1) and turbot

conditioned water (1.73 1). Artificial seawater was used as control (5.0 1). The cartridges ( were conditioned prior to extraction using HPLC grade methanol (2 ml), followed by displacement by distilled water (2 ml). Following completion of extraction, interference analyses remaining on the cartridges were removed using distilled water (2ml). The cartridges 5 were then extracted with distilled ethanol (2 ml) and either used for chemical analysis or the eluates made up to the original volume for bioassays by adding artificial seawater. The extracted conditioned water was retained for bioassays. The SPE extracts of salmon conditioned water (SCW) were separated into 2 fractions, "volatile" and "non-volatile", by distillation under vacuum (0.04torr) for 24h at 25 C as described previously. The vacuum 10 distillate/comprised components with a molecular weight range comparable to those utilised as volatile semiochemicals by terrestrial organisms (volatile fraction), whilst the residue arising from the vacuum distillation comprised components with insufficient or no volatility (non-volatile fraction). For bioassays, the fractions were re-dissolved in ethanol and made up to the original volume by adding artificial seawater. For chemical analysis, the volatile 15 fraction was diluted with distilled water (50 ml), and extracted with distilled diethyl ether (3 x 10 ml). The organic layers were combined, dried (anhydrous magnesium sulphate), filtered and evaporated under a gentle stream of nitrogen to lOOhul, and stored in tightly capped microvials at -20 C.

20 The vacuum distillation volatiles were separated on a Hew!ett-Packard 5890A gas chromatograph equipped with a cold on-column injector, a flame ionization detector (FII)) and a 50m x 0.32mm i.d. HP-I bonded phase fused silica capillary column. The oven temperature was maintained at 40 C for 2 minutes and then programmed at 10 C mini' to 250 C. The carrier gas was hydrogen (Spsi).

Coupled GC-MS of the vacuum distillate was provided by a capillary GC column (SOm x 0.32rnrn i.d. HP-1) fitted with an on-colurun injector directly coupled to a mass spectrometer.

Ionisation was by electron impact at 70eV, 250 C. The oven temperature was maintained at 30 C for 5 minutes and then programmed at 5 min-' to 250 C. Tentative identifications by S GC-MS36 were confimned by peak enhancement with authentic sarnples37 obtained from commercial sources. Chemicals were diluted in distilled hexane prior to peals enhancement studies. Table 2 shows a list of either the compounds or their mass spectral characteristics.

Example 3: Behavioural bioassay example Y-tube bioassays allowed the sea lice to exhibit a preference for different stimuli over seawater control and were also modified to monitor activation responses. The Y-tubes were made of glass with a 16 mm diameter bore. She arms were 14cm in length and the main leg was 15.5 cm long (Fig. 1). Water flowed through into each arm from a reservoir at 30 ml 15 mind. Inlets and outlets of the Y- tube comprised of glass pipettes fixed into plastic tops. The tops fitted over the openings on the arm and leg to make water-tight seals. There was a clear demarcation of the flow down each arm and a mixing of the water from each arm in the main leg of the apparatus was demonstrated using food dyes. In double control runs, artificial seawater was introduced into both arms of the Y-tube. When a stimulus was tested, the 20 solution was made up and introduced into one arm whilst seawater alone was introduced to the other, prior to introduction of the sea louse. When salmon conditioned water extract Divas

tested as the stimulus, extracted seawater was used as control. The arm containing the stimulus source was changed over regularly, with washing in-between, to ensure that there was no positional bias. A single male sea louse was introduced into the base of the main leg US of the Y-tube and allowed 10 minutes to respond, typically by moving up the main leg and into one of the arms. Sea louse activity was defined by the degree of movement within the Y q

tube. Behaviour was divided into two categories, low (Fig. la) and high (Fig lb-c) activity.

Frequencies of intermediate categories between Fig la and lb were low but were defined as less (low) or more (high) than twice the length of the leg of the Y-tube. Movement into either arm, regardless of odour, was included in the high activity level (Fig. 1 c). Using the Y-tube 5 behavioural arena both activation responses and directional responses could be measured in adult male sea lice. For taxis, the number of individuals choosing odour to control arm within the I O min of the test were compared with those with seawater control in both anus.

Identified semiochemicals phenol and isophorone, along with the known salmon flesh 10 component l-octen-3-ol40442, were tested in behaviourar bioassays. Solutions in ethanol (O.lmg Elm) were prepared and loll 1-7 seawater was used in the experiments. When all three were bioassayed, phenol and ethanol (the SPE extract solvent) elicited neither activation nor taxis responses. However, both the isophorone and the l-octen-3-ol caused significant activation responses in the lice and also elicited electrophysiological responses from the 15 antennae. Neither of these compounds, at the concentration tested, caused positive taxis in the Ytube behavioural bioassays. Conversely, isophorone did cause significant attraction in a flow through tank arena. These behavioural bioassays have revealed for the first time that L. salmonis show positive upstream rheotaxis as part of their host location behaviour.

20 Almost two thirds of the sea lice under control conditions showed low activity when clean artificial seawater was flowing in both arms of the Ytube. In the presence of salmon conditioned water (SCW) a significant (p<0.001) increase in activity was seen (Table 3).

Very similar results were seen for SCW extract tested against depleted seawater (i.e. extracted SCW), where the animals showed a significant increase in activity (p<0.001). The biological 25 activity of SCW was retained for at least 16 weeks after entrainment if kept at -20 C (Table

3). When the vacuum distillate of the SCW extract i.e. the volatile and non-volatile fractions, were compared to the controls a significant increase in activity was found with the volatile fraction only (p<0.05) (Table 3), thus confirming that host finding semiochemicals for sea lice comprise small, volatile organic molecules similar to that used by arthropods in terrestrial 5 systems. Turbot conditioned water extract (TCW extract) did not cause an activation in the Y-tube arena (Table 3).

The directional response (taxis) of adult male lice was also significantly altered in the presence of salmon odours (Fig. 2). Results for SCW and TCW from Devine et al. (2000) I O were included for comparative purposes. In seawater controls the animals showed no particular preference for either arm' whereas SCW and SCW extract significantly increased the number of lice moving to the odour source compared with the control arm (p<0.001 and <0.05 respectively). The low molecular weight fraction of the SCW extract elicited significant positive rheotaxis response from the adult male sea lice (p<0.05). On the other 15 hand the non-volatile fraction did not attract the animals directionally which is supported also by no activation response to that stimulus. Both fractions were tested at half-strength (Fig. 2).

TCW and TCW extract did not cause any significant attraction of the lice (Fig. 2). x2 analysis was not performed on the results for TCW extract and SCW non-volatile fraction as n numbers were low due to very low activation to the stimuli.

20. - Activation responses of adult male sea lice were significantly increased by isophorone and 1 octen-3-ol (11 of 0.1mg mli in I I seawater) , whereas phenol had no significant effect at the same concentration (Table 4). Ethanol at 0.4%, the concentration used in extract presentation, caused no behavioural response (Table 4). No directional responses were seen for any of the 2S chemicals tested, isophorone, phenol, 1-octen-3-ol at 0.1ppb and ethanol at 0.4%.

Example 4. CoEgepodid directional responses. activation and arrestment example The copepodid of L. salmonis in addition to preadult and adult stages has also been shown to 5 respond significantly by directional movements, towards the source of potential host odours presented in the form of Salmon Conditioned Water (SCW) and SCW volatile fraction (Fig. 3). Further, significant directional responses to specific chemicals identified from the volatile fraction of SCW, namely, isophorone (l Ill of 0. lmg ml in 1 L seawater) and 6 methyl-5 hepten-2-one (11 of 0. Img ml in I L seawater) have also been demonstrated (Fig. 3). It is I 10 noted that 6 methyl-5-hepten-2-one is in fact specific to salmonid host odour. Further, the fact that 6 methyl-5-hepten-2-one was highly attractive to copepodids demonstrates that it could be used as one of several attractants for the copepodid stage of L. salmonis.

Copepodids were observed to increase activity significantly in the presence of fish derived 1: stimuli (Table 8). Salmon and seatrout conditioned water induced widespread changes in behaviour and movement of copepodids, resulting in copepodids increasing the duration spent swimming, swimming speed and distance travelled. Further, the number of turns increased, while meandering was decreased. Seatrout conditioned water (STOW) produced the most prominent responses of all the fish odours tested. However more specifically, isophorone 20 (identified from SCW) also produced increased activation of a similar magnitude to that observed for STCW. It is suggested that the pattern of movement produced represents a modified search pattern in response to host odour which enables the copepodid to increase the search area.

Although Cod and Turbot conditioned water also induced changes in copepodid behaviour and movement these were limited relative to host derived odours. There were no changes in the amount of swimming, velocity of swimming and active turns that as a result produced a different search patterr to that described for host odour cues.

1 -octen-3-ol ( l Ill of 0. lmg mli in 1 L seawater) caused significant activation of adult male sealice and caused patterns of electrical activity in antenna! recordings. However, in experiments with copepodids 1-octen-3-ol caused a significant decrease in swimming bout 10 frequency (Table 8) while other parameters remained the same as SCW, thus suggesting an arrestant type of response (Table 8).

Example 5: Behavou_al bioassay example The tank bioassays demonstrate the preference of sea lice to a single chemical, in a slow release formulation, and their settlement on the slow release vial in a large flow through arena. The arena was a 28cm x 40cm x I Scm deep container with inflow at six points at the front and an outflow at the back to ensure an even flow through the container. Water flowed 20 continuously from a reservoir at 100-150 ml mind. There was a clear equal flow through the system demonstrated using food dyes. The base of the container was white in order to reflect light. The slow release polyethylene vials were wrapped in muslin along with weights to keep the lures at the bottom of the container. Fibre optic light was positioned under and in front of each vial. Individual vials were loaded with Img of isophorone, identified from the salmon 25 extracts. Control vials were prepared with solvent only (hexane). which was allowed to evaporate to dryness before use. In all runs two vials were positioned at the front of the

container, lOcm away from the inlet with gem between the vials. The position of the vial ( containing the stimulus source was exchanged between experiments, to ensure that there was no positional bias. The animals were released downstream by the outflow of the container, 20 adult males in each experiment. The number of lice that settled on the muslin covering the 5 vials were counted at 30 minute intervals and removed.

The responses of adult male sea lice to the lures containing isophorone at l OOOg per lure was significantly different from controls (p<O.OI) and adult male sea lice were found to prefer to settle on the lure with the isophorone stimulus rather than the control (Fig. 4).

10 Example Semiochemical isolation from sealice ate developmental stages of sealice (a. salmons), adult males (AM), adult females (AF) or preadult II virgin females (PF) were collected from salmon farms on the West Coast of Scotland. The lice were kept in fresh seawater from the collection site, on ice, during 15 transport to the University of Aberdeen aquarium. The sealice were maintained separately as males and females in the absence of hosts in 25 I tanks at Aberdeen University at 12 C in artificial aerated seawater of 32%0 salinity. Lice were used from 12h-7 days after collection Water conditioned from preadult II virgin females (PF), adult mated females (AF) and adult 20 males (AM) was subjected to solid phase extraction (SPE). Six individuals of a specific developmental stage and sex were placed Into chambers at the top of both arms of a Y- tube.

Seawater, from a reservoir, flowed through the chambers at 30ml mini'. The seawater from the chambers was collected, in a glass beaker, from the outlet of the Y-tube. This entrained water was fed directly into the SPE set-up. Seawater running over empty chambers divided by 25 mesh was used as a control. Extracts, reconstituted for behavioural bioassays were made up to the original concentration with artificial seawater.

A portable closed solid phase extraction (SPE) system was used for extracting potential semiochemicals from preadult II virgin female conditioned water (5.17 L) for chemical analysis. Artificial seawater (5. 0 L) was used as control. The SPE columns (International 5 Sorbent Technology (IST) Limited, Hengoed, Glamorgan, UK) consisted of 6 ml glass cartridges containing layered phases (C2 (SOOmg) over ISOLUTE ENV+ (200mg) ). The extractions were performed using a VacMaster-10 SPE manifold (IST). The SPE cartridges were conditioned prior to extraction with 2ml HPLC grade methanol, followed by displacement distilled water (2ml). Following completion of extraction, interference analyses 10 remaining on the filters were removed using distilled water (2ml). The contents of the cartridges were then extracted with distilled ethanol (2ml). SPE extracts were separated into 2 fractions by distillation under vacuum (0.04torr) for 24h at 25 C as described previously (Pickett & Griffiths 1980). The SPE extracts of salmon conditioned water (SCW) were separated into 2 fractions, "volatile" and "non-volatile", by distillation under vacuum 15 (0.04torr) for 24h at 25 C as described previously. The vacuum distillate comprised components with a molecular weight range comparable to those utilised as volatile semiochemicals by terrestrial organisms (volatile fraction), whilst the residue arising from the vacuum distillation comprised components with insufficient or no volatility (non- volatile fraction). For bioassays, the fractions were re-dissolved in ethanol and made up to the 20 original extraction volume by adding artificial seawater.

For chemical analysis, the volatile fraction was diluted with distilled water (50 ml), and extracted with distilled diethyl ether (3 x 10 ml). The organic layers were combined, dried (anhydrous magnesium sulphate), filtered and evaporated under a gentle stream of nitrogen to 2: 1001, and stored in tightly capped microvials at -20CC. The vacuum distillation volatiles were separated on a Hewlett-Packard

5890A gas chromatograph equipped with a cold on-column injector, a flame ionization detector (FTD) and a 50m x 0.32mm i.d. HP-I bonded phase fused silica capillary column. The oven 5 temperature was maintained at 40 C for 2 minutes and then programmed at 10 C min to 250 C. The carrier gas was hydrogen (Spsi).

Coupled GC-MS of the vacuum distillate was provided by a capillary GC column (SOm x 0.32mm i.d. HP-I) fitted with an on-column injector directly coupled to a mass spectrometer.

10 lonisation was by electron impact at 70eV, 250 C. The oven temperature was maintained at 30 C for 5 minutes and then programmed at 5 min' to 250 C. Tentative identifications by GC-MS36 were confirmed by peak enhancement with authentic sarnples37 obtained from commercial sources. Chemicals were diluted in distilled hexane prior to peak enhancement studies. Tables 5 and 6 show a list of either the compounds or their mass spectral 1 5 characteristics.

Example 7. Behavioural bioassay example (sealice-sealice) JO A Y-tube arena was situated on top of a light box and a camera mounted above the arena and linked to a VCR traced the behaviour of the lice in the arena. The movements of the lice were recorded on videotape. Y-tube bioassays allowed the sea lice to exhibit a preference for different stimuli compared with seawater controls and also allowed 25 measurement of their activation responses to different stimuli (Ingvarsdottir et al. 2002a).

The glass Y-tubes had a 16mm diameter bore. The arms were 14 cm and the main leg 15.5

em long (Fig. 1). Water flowed through into each arm from a reservoir at 30 ml min i. Inlets and outlets of the Y-tube comprised of glass pipettes fixed into plastic tops. The tops fitted over the openings on the arms and leg to make watertight seals. There was a clear demarcation of the flow down each arm and a mixing of the water from each arm in the main 5 leg of the apparatus was demonstrated using dyes. In double control runs, artificial seawater was introduced into both arms of the Y-tube. The Ytube experiments were set-up in two different ways. Either, six individuals of a specific sex and developmental stage (PF, AF or AM) were placed in a chamber in one arm of the Y-tube and an empty chamber placed in the other arm, with seawater flowing through the chambers in both arms, or a solution was made I O up and introduced into one arm whilst seawater alone was introduced to the other arm. The arm containing the stimulus source was changed over regularly, with washing in-between, to ensure that there was no positional bias. The chamber restraining the animals to one arm of the Y-tube was made up of 2 parts of I cm Perspex tube inserted into the top of the arm with a 200m nylon mesh dividing the first chamber from the arm and another 200m mesh between 15 the tubes to keep the animals within the chamber and insuring water flow over the animals.

Single sea lice were introduced into the base of the main leg of the Ytube and allowed 10 min to respond. Using the Y-tube assay both overall activity and directional responses were measured in adult male sea lice and preadult 1I virgin females. Sea louse activation was defined by the degree of movement within the Y-tube. Behaviour was divided into two 20 categories, low (Fig. I a) and high (Fig I b-c) activity43. Frequencies of intermediate categories between Fig. Ia and lb were low but were defined as less (low) or more (high) than twice the length of the leg of the Y-tube. Movement into either arm, regardless of stimulus, was included in the high activity level (Fig. I c). For the overall activity levels, the number of individuals in low and high activity categories in the presence of different stimuli were 2: compared to those observed with seawater alone. Directional responses (taxis) were

measured by the movement of the louse up the main leg and into one of the arms. For the directional response, the number of individuals choosing stimulus arm to control arm within the 10 minute test period were compared with those with seawater control in both arms.

Statistical differences were assessed by x2 analysis.

The activation responses of adult male sea lice and pre-adult II virgin females (PF) in the Y tube bioassay to stimuli derived from the same or different sex and developmental stages are shown in Table 7. Sixty percent of the adult male lice and pre-adult II virgin females under control conditions showed low activity when clean artificial seawater was presented in both 10 arms of the Y-tube. In the presence of preadult II virgin female conditioned water (PF) a highly significant (P<0.001) increase in adult male activity was observed. Similar results were obtained for PF extract (PcO.05) (Table 7.). When the volatile and nonvolatile fractions of the PE extract, prepared by vacuum distillation, were tested, a significant increase in activity was found with the volatile fraction only (P<0.05) (Table 7.).

The adult males showed increased activity in response to both adult mated females (AF) (P<0.05) and the combined stimulus of AF and PF (P<0.001) presented in separate arms of the Y tube bioassay. Further, adult males were found to demonstrate a significant activation response in the presence of other males (P<0.05). However, there was no increase in activity, 90 in comparison to seawater controls, for PF to adult males (Table 7.) In seawater controls the animals showed no particular preference for either arm (Fig.5 and 7).

Attraction of adult male lice to preadult II virgin females (PF) was clearly demonstrated in the Y-tube experiments (Fig. 5). The PF, PF extract and the PF volatiles (low molecular weight 25 fraction of the PF extract) significantly increased the number of adult male lice moving to the

stimuli as compared to the control arm (Pc0.001, 0.05 and <0.05 respectively) (Fig. 5). No attraction of males was observed to the PF nonvolatile fraction (Fig. 5). Because very few males responded directionally to the higher molecular weight fraction of the PF extract it was not possible to perform %2 analysis on the results.

The adult males were activated by both the adult mated females and other males but did not show significant directional responses to either of these (Table 7, Fig. 6). In a competitive assay in which males were presented with PF or adult females in separate arms of the Y tube, there was a significant response to PF (P<0.05) (Figure 6).

Although the PF did not show an overall significant increase in activity in response to adult males (Table 7.), there was a significant attraction to the males with all 11 of the PF that showed "high activity" levels choosing the treatment arm (P<O.O 1) (Fig. 6).

15 Example 8: Trappma Sealice Laboratory trials of potential sealice traps loaded with either isophorone or pre-adult II virgin females, demonstrated that adult sealice, both males and females were significantly attracted to those traps containing the isophorone and males were significantly attracted pre-adult virgin females, compared to control traps (Fig. 7). Interestingly, the presence of pre-adult II 20 virgin females attracted significantly more sealice than isophorone only, although this may have been due to the fact that males only were used in this experiment.

Example 9: Host masking agents Generally, semiochemicals which cause a masking effect either alone or in combination can 25 best be demonstrated by masking normal attractiveness of attractants e.g. aphid work45 46.

t(1

However, the compounds described here in relation to the copepodid study are completely novel for this activity.

Experiments with the copepodid stage indicate that the addition of nonhost odours in the 5 presence of a host odour may mask the normal attractiveness of the host odours. It has been demonstrated that, with the addition of non-host odours in the form of 2 arninoacetophenone and 4 rnethylquinazoline to SCW, copepodids were activated, but significant directional responses to the host odour were not observed (Fig. 8; Table 9).

Claims (1)

1. / CLAIMS

   1. method for monitoring and controlling malice through the use ot'semiochemicals 2. A method involving an attractant preparation consisting ot' chemicals that attract.

   aggregate or arrest sea lice. The attractant preparation may consist ot' one or more chemicals isolated. characterized and identified l'rom salmon and other salnonil conditioned water, 3. method involving, an attraetant preparation consisting of chemicals that attract.

   aggregate or arrest sea lice. The attraetant preparation may consist of one or more 1() chemicals isolated. eharacterised and identified from male or tamale scalice conditioned water.: 4. method as described in Claim 2 that consists of the chemicals isophorone or 6-methyl-

   5-hepten-2-one either by themselves or h1 combination or in eombblation with other chemicals isolated' charactcrised and identitieLI from salmon or salmonicl conditioned 1 5 water.

   5. A method as described in Claim 4 where the chemicals are used with other chemicals or synergists that potentiate attraction. aggregation or arrestment ot sea lice.

   6. A synergistic chemical as described hi ('[aim 5. wherein the synergist consists of'1-octen 2() 3-1

   7. A method involving the use ot' the chemical attractants described in ('[aims 1 to 6 wherein the attraetants are supplied in a controlled release formulation or device.

   X. A method involving, the use of the chemicals described in Claims I to 6 in traps for monitoring and controlling sea lice populations.

   at,

   9. A method involving the chemicals in Claims I to 6 to lure. aggregate and / or arrest the sea lice onto a surface treated with a killing or sterilising apcnt.

   10. A method involving the chemicals in ('[aims 1 to 6 to lure the sea lice away from their salmon hosts in combination with other masking. repellent or deterrent chemicals that also stimulate the lice to move away from their host hi a push-pull strategy for controlling their populations. 1 1. A method as described in ('[aim 10 where the masking,. repellent or deterrent chemical is 2-aminoacetophenone or methylquinazoline.

   12. A method involving the chemicals in Claims I to 6 where the attractants are used to 1() confuse the lice and prevent them Prom finding each other for mating or their host salmonids for f'eedhg.

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   I X Kelly l.S, Snell 'I W and l.onsdale l).h ('hemical communication during mating ol' the harpacticoid /igriopus jap'nicu.s. Phil. 7un.Y. R. 'c. I.onclB353:737-744 (1998).

   ( 19 Koehl MAR and Strickler JR. (:opepod feeding currents: Food capture at low Reynolds number. Limnal Occuno,r.26:1062-1073(1981) 0() Poulet Did and Ouellet (J. Tile role of amino acids in the chcmosensory swarming, and feeding of marine copepods. J. plankton Re.s. 4:341-361 (1982).

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   22 Van Alstyne Kl,, Effects of phytopiankton taste and smell on t'eeding behaviour of' the copepod (.'entropuge.shumulu.s. Mu'-. Lcol Prog pier. 34:187-190 (1986).

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   25 Kirb,e 'I' and 'I'hygescn IJE-I. Florid motion and solute distribution around sinking: aggregates. 11. Implications for remote detection by colonizing z.ooplankters. Aver ] 5 ECVI. Prog her. 211: I 5-25 (20()1).

   26 Anstensrud Ma Effects of mating on female hehaviour and asymmetric growth in the two parasitic copepods LernaerJeera hrunchilix (1... 1767) (Penncilidac) and Lepeophtheiru.s pecoruli.s (Mullen 1776) (Calig,idac). (.rll.staccunu 59 245-05X (1990).

   0() 27 Anstensrud M. Mate guarding, and mate choice in two co,nepods. Lernaeocer hranchiu/is (1.) (Pcnnellidae) and l.epeo/'htheirus pecorali.s (Muller)(Calig,idae).

   parasitic on floundcr..J. ('rue. Bio/. 12:31-40 (1992).

   28 Ritchie (,, Mordue (Luntz) AJ. Pike AW and Rae GH. Observations on mating and ultrastructure of the reproductive system of l.epeo/'htheirus salmonis Kroycr 2 (C'opepoda:('aliidae). J L'Yp.ll'' /]i'l Eco/ 201 2X$298 (199fi) my))

   99 Hull MQ, The role of scmiochemicals in the behaviour and biology of l. epecJphtheir'..s sulmonis (Kroyer 1837): Potential for control? Ph[) thesis, University ol' Aberdcen.

   Aberdeen, UK (1997) () Hull MQ. Mordue (Luntz) AJ. Pike AW and Rae (J. Patterns ot'pair f'ormation and 5 mating in an ectoparasitic caligid copepod l.ecrhtheiru.s.su/moni.s (Kroyer 1837); implications for its sensory and mating biok,gy Phil. Trun.s. R..S(JC. L(Jnn B 353 753-764 ( 1 998).

   21 Cresty KA, Boxshall GA and NaL;asawa K. Antennulary sensors of' the infective copepodid larva of' the salmon louse, Lepeophtheiru.s.s'lmoni.s (C'opepoda:Caligidac), I O in Pathogens oJ Wil1 and Furmed l i.sh.,Secr Lice ed by Boxshall GA and. Defaye D. Chichcstcr I.llis Horwood. pp 83-9X ( 1993) : I.averack M.; and Hull MQ. Sensorv innervation ol the antennu1e of'the preadult male ( uli,u.s elongutu.s, in Patho,ú,ren.s of Wiicl und armect l ish..;e' Lice ed by Boxshall (,A and Defaye D, Chichester: F. llis Horwood, pp 1 14-122 ( 1993),,, 15 33 Kabata /, ('opepoda (('rustacca) parasitic on l'ishes: I'rohlems and perspectives. Ai' /'ru.s if ol. 1 9: -63 ( 198 1) 34 Devine GJ, lngvarsdottir A, Mordue W. Pike AW. Pickett J. Ducc I and Mordre (I,untz) AJ, Salmon lice, Lepeophtheiru.s sulmonis exhibit specif'ic chemotactic,, rcsponscs to semiochemicals originating, t'rom the salmonid. A/m.sulur..J ('hem 90 Ecol 26 1 833-1847 (2000) 35 Ritchie G' The host transf'er ahility of Leleoph/heiru.Y. sulmoni.s (Copepoda Caligidae) f'rom f'armed Atlantic salmon..561mo s'l'r 1.1 Fi.sh /)i.s. 20:153-157 (1997)

   ( 36 NIST Standard Referencc Oata Hase (Version 3.0.]). (Offce of the Standard Rel'erence Data Base National lnstilte of Stan.lards and 'I'echnology. Gaitersturg,.

   Maryland, USA) (1990) 37 Pickett.IA. CRC-MS in insect pheromone identif'ication: three extreme case histories. in 5 ('h''tJmlU/)hV (J,79 Isolti/i'}n (Ji /n.se' Jr1es LInfl hL'r'mfnL, AR anl Wilson IO. Plenum Press, Ncw York. pp 2()t:) 3()9 ( 1990).

   38 Llot'l'son R. The ultrastructure ot' a chemoreceptor organ in thc head of copepod crustaceans. 4cc' ZOr')/. S(ockh 52:299-315 ( 1971).

   39 Gill (:W and Poulet SA. tJtilization 't'a computcri>.ed microimpedance system t'or 10 study activity of' copepod appendacs. J L'xp../r. BiJl EcJl. 101:193-198 (I')X6) 40 Josephson DB Lindsay RC' and Stuiber DA. The influence of malurity on the volatile aroma compounds from fresh lhacific and Cireat-L.alves Salmon..I Foo1 S'cience f;6:1576-1579 (1991).

   41 Zhang (111, 11irano T 'Su>.uki '1'. 'hirai 'I' and Morishita A. Studies c>n the odour ot',,, 15 fishes. 3. Volatile compounds and their generation in smelt. Nippon.S'ui.sun (iclkkaishi (13ull..Jupan \oc..Sci. Fish.) 58:77,-779 ( 1992). ', 42 Refs,aard ITHI:. Haahr AM and Jensen B. Isolation and quantification ot'volatiles in fish by dynamic headspace sampling, and mass spectromety..J. A,ric. FO'J ('hem. '.,,.' 47:11141 118.

   2() 43 Invarsdottir A'. Birkett MA Duce 1., Cienna R Mordue W.^ Pickett JA. Wadhams 1..1.. and Mordue (Lunt,.) A..l.. 'Semiochemical strateg,ies t'or sea lice control: host location cues. Pest Mcm..'ici. (2002) h Press) 44. Anjou K & Sydow E. 'I'he aroma ot' cranberries: II Vaccinium macrocarpon. Actu ('hem.Sunc! 21 (8) 2()76-2082 ( 1967) X 1

   ( 45. Pettcrsson, J., Pickctt. J.A., Pyc. B.J Quiroz, A., mart. L. F ' Wadhams L l. and Woodcock, C.M. Winter host component reduces colonizatio hy bird cherry oat aphid. Rhopalosiphum padi (L.)(I lomoptera. Aphidae). and other aphids in cereal f'iclds..lournal of ( hemical l colvg. 20, 05(5-2574 (1994 46. Pickett,,l,A Wadhams, L.J. and Woodcock. C'.M. Nonllost interactions in insect chemical ecoloL;y. In: Proceedings of the 1 ' International (.,nferencc on Insects: C'hemical. Physiological and EnvironTnental aspects' Se,Dtember 26-29. 1994, Ladek Zdroj, Poland, ppl26-133. Eds D.Konopinska, (i.(oldsworthy. R.J. Nachman,.T.

   ] () Nawrot, l. Orchard' G. Rosinski and W. Sobotka. ([Jniversity of Wroclaw) (1995) . ... ..

   Table 1. Representation of thc substances isolated, characterised and identifiied from salmon conditioned water in example 1 Scan l;o: Compound 5 5()2 Phenol 591 Isophorone 758 Compound with m/- t'ragments 57. 13Xs 1'33 208 768 Propionic acid' ?-methyl-. 2.2-dimethyl-1-(2-hydroxy-1methyl ethyl)propyl ester It) 779 Propionic acid. 2-methyl-. À-hydroxv-.4. 4-trimethylpesltylester 902 Diethyl phthalatc 917 1'ropanoic acid 2methyl-' 1 -(1 I -dimethylethyl)-2-methyl- 1.3 propanediyl ester 936 'I'ri-n-butyl phosphate 1: 1()52 Diisobutyl phthalate 1()')8 Dibutyl phthalate 1173 C'ompound with m/ fragments 41. 43, 55. 69 83 97. 111. 137 1213 ('ompound with m/ bagments 41. 43. 55, 69. 1 ()'). 137 13()7 ComF, ound witl1 m/ t'rag,ments 41. 43. 57 59, 69. X3 ')7 111. 125 2() 1322 C'ompound with m - Irag,ments 15(). 165, 253.,15. 393 4()X 133X I lexanedioic acid. his (2-ethylhexyl) ester 1551 L)iisooctyl phthalate WT

   Table 2. Representation of the substances isolated' characterised and identified from salmon conditioned water in example 2.

   Sican no: Compound 5 368 Compound with m./ fra,lilents 75, 77. 105 388 Toluenc 404 Compound with n/ fraunelits 41. 43, 55, 70. 84 433 11exanal 464 Contpound with m/ t'ragmcnts 29, 43, 57, 74 1 () 478 C'ompound with m/7 t'ra,,mcats 43, 45, 57. 73, 1 () I 493 Compound with m/- fragmelits 43, 58, 59, 83. 101 512 1-lexamethyl cyclotrisiloxane 555 1,3-Dimethyl benzene 611 2-Butoxyethanol 15 633 Compound with m/ fiagnelits 77. 93. 1 U5 121. 13> 662 Compound with m/ t'raments 43. SS. 71. I I S 1,1 670 13enzaldehyde 724 6-Methyl-S-ilepten-2-one 724 1,henol 2() 733 Hexanoic acid 749 Octanal 771 Compound with m/ f'ragments 63, 79. 91, 1()7. 119 1,5, 149 774 C'ompound with m/ I'ragnents 41. 43, 55..';9, fi3. 71, 99. 111. 126 779 2-Propylpelitcniol 05 7X5 Benzyl alcohol 8()7 1, imonene 824 Acetophetone 850 3,4-Methyl phenol 875 2-Nonanone. 30 891 Nonanal 903 Isophorone 99S 2-E thylDcxanoic acid 933 2- F2thy I pheno l 943 Camphol 5 968.,,4-l-lillyl phenol,,, 1023 Decanal 1027 Phenoxyethanol 1032 Benzothiazole 1048 3-Acetyl-2,5- dimethylthiophene 40 1071 [)imethoxymethylphenylsilane 1121 1 -Adamantol 1128 Bomyl acetate 1131 N.N-Dibutyltorinatnide 1147 4-Ethylacetophenone 45 1153 Compound with m/ t'ragments 127, 253 369 1183 Dihydro-S-propyl- 2(3H)-f'uranone 1189 Compound with m/- fragments 57. 83, 95, 109, 124, 138. 152, 165. 180, 193, 208 1197 Cotnpound with m/ fra,ments 43. 57, 87 1206 Propionic acid, 2-methyl-. 2,2-dimetilyl-1-(2-hydroxy-l-methyl ethyl) propyl ester 5() 1 ? f 1 Compoulid with m/- tra;,ments 122, 150. 165 1226 I'ropionic acid. 2-methyl-.3-hydroxy-2.4.4-tritnethylpentylester 1259 compoutid with m/f'ragments 59. 111, 127 1292 Coinpound with m/: fragments 41. 59, 68. 97 1304 Geranyl acetone 55 1332 Butylated hydroxytoluene 1338 L'otilpound with m/- fra:,lttents 41, 55, 69, 83, 97, 139 I 365 'I ribut! I-n-phosphate :,'\

   Table 2 (continued) 1431 Diethyl pthalate 1412 Benzophenone 1477 Compound with imi: traments43, 57. 71. 85. 1 À5 1491 Compound with m - tragmentS 41, 57 99. 125 155 211 1446 Compound witn m: fragments 4 1, 57, 59. 69, 83. ti7 155') Compound with m,: fragments 107, 135 165. 183. 19X 1565 Compound withm/:fragments73. 1()7. 121. 135- 149. 191 1() 1571 Compound withm':frarments57. 107 121. 149. 191.

   157( Compound uittim:tragullents 107. 121. 135. 149. 191 1584 Compoundwithm iraments 107. 121. 135. 177 1 592 Compound with m.: I ragments 1 ()7. 1 2 I. I 25. 1 49. 1 77 1599 Compound with m/: iragmcnts 77. 107 135. 17() 15 1608 Compound withm/:tragntents 1()7, 121, 135. 149, 177 1623 Compound with mi- tragments 158, 195, 253, 315, 331, 346 1662 Compound with m,': fragments 158, 195 253, 315. 331, 346 1687 Diisobutyl phthalate 1723 Cotnpound with mi: fraunncuts 191. 220. 275. 3()3 90 1764 Dibutyl phthalate l t)t)t) C ompound with n,: tragmenis 1 5(). 1 95. 253, 3 1 5. 393, 4()8 20X2 C ompounti with,K- ftarmctits 253. 315. 393 9365 Diisooctyl phtnalate . . 3t

   ( Table 3: Activation response of adult male sea lice to different fish derived stimuli in a Y-tube arena. *p<0.05; ***p<O.OOI (72 analysis).

   I ? _

   Stimulus Low activity High activity p n ( Do) (%) Control 60.2 39.X - 83 Salmon conditioned water (SCW) 19.() 81.0 *** 63 SC W extract vs. depleted seawater 8.3 91.7 *** 36 SC'W Bronco lor 16 weeks 30.6 6t). 4 *** 49 SCW volatile traction 27.3 72.7 * 11 SC'W non-volatile fraction 60.9 39.1 n.s. 23 Turbot conditioned water ( I ('W) extract 63. 3 36.7 n.s. 3() Delmed as category a, Fig. 1; 2 Defined as categories b-c. Fig. 1.

   3::

   Table 4: Activation response of adult male sca lice to different chemical stimuli in a Y-

   tu hc arena. * p<O.05; * * * p<-().0() 1 ( X analysis) Stimulus Low activity High activity n ( /u) (uX,) C ontrol 6().9 39.8 - X3 Isophorone 3().t) 6t3. 1 *** 55 Phenol 57.1 4.9 n.s. 2X I-octen-3-ol 35. 7 64.3 * 28 Ethanol* 4h.9 53 n.s. 52 ('hemicals were presented at O. l ppb ( 11 (ot O. l mg stimulus in l ml ethanol) in I litre seawater). iDetined as category a ig. 1; I)elined as categories in-c. I:ig. 1. * Ethanol-0.4%.

   ,

   Table 5. Representation of the substances isolated, characteriscd and identifited from female pre-adult 11 conditioned water in example 5.

   Scan no Compound 7 i 13en,.aldehyde 75 iilcilol 749 ()ctanal 7Xh 13enzyl alcohol 797 2-Lthylhexatiol 824 Acetophenone 0 842 Compound with m,': framents 83. X4 846 4-Methylbenzaldehyde X.>O 3- and 4-Methylphenol X:9 Dimethyl benzl alcotiol 871 Methyl benzoate I S X7G compouticl with m, ira.lnents 43, 71. 99. 1 i4 X90 Nonanal 900 13enzaldehyde dimethyl acetal 931 2-Ethylphenoi 943 Camphor 9() 96X 3- and 4-Ethylphenol 983 4Methylacetcphenolle 986 1.1.4-Trialeth) I benzvl alcohol 995 1-(2ilotixyethoxy)ethan,l I O().C; '-lccanoile 05 1008 1,1 4-Trimethyl-3cyclohevene-l-methalic> 1011 2-lsopropylphcnol 1021 Decanal I ()., 2 i3enzothiazole 1044 3- and 4-lsopropvlpilenol ' () 1068 2-Dimethylpropvlhenzelle,''' I I O(J 2-l-Butylphenol 1104 4-l:. thylacetophenone 1117 Compound with m- firagmelits 41, 75, 109. 157 1129 N.N-L)ibutylforamide 3 S 1143 lJndccanal 1155 1 (H)-lsobenzotiranonc 1164 Isobutyl benzoate 1186 Compound with m/ t'ra,melits 57, 83, 95. 109, 138. 152, 166, 193, 208 1204 Propionic acid, 2-melilyl-. 2,2-dimethyl-1(2-hydroxy-1-methyl ethyl)propyl ester 4() 122fi 1'ropionic acicl. 2methyl-. 3-hydtox;-2,4,4-trdnethylpetitylcster 1268 Compound with m/: fragmcilts 43. 57. 85. 1()9. 123. 1'il 12X3 Dimethyl phthalcite 1290 Compound with m/: fragments 59. 97. I X5 1,00 (icranylacetone 45 1 30i) C'ompound with m,: framents 1 1 3, 1 4 1, 1 63 1363 Tributylpilosphate 1405 Compound with m/: fiagmcnts 57. 72, 114, 128, 153, 188, 217 1413 Compound with m/: tragmcuts 57, 77, 105, 123. 149. 196 1428 C'ompoutid with m/2- tragmelits 65, 105. 149, 177 50 1458 Propanoic acid. 2-methyl-, I-(l,l-dUllethylethy1)-2-methyl-1.3- plopallediyl ester 1464 1-lexadecene 1474 He\adecane 1489 TribL'tylphosphate 1494 Conipoulid with n',: ti-agmcuts 5'J, 8.. 111, 213 5 1568 Compound with ''', Iragments 43. 57, 71, 85, 121. 149, 197. 212, 284 162() Compound with mi: fiagmerits 158, 195. 253, 315. 331, 346 1625 Compoulid witl1 m': fiagments 71. 85. 147. 207. 221. 281. 29h --4

   Table 5 (continued) 1649 Octadecene 16S6 Octadecane 5 1657 Compound with m,'_ firmaments 253, 331 1667 Isopropyl myristate 1684 Isobutyl phthalate 1720 Compound with m/- fi-agmcnts ') I. 137. 18 I. 1')7. 940, 303 1761 L) ibutyl phthalate 1 () I 819 Eicosene 1830 Eicosane 1995 Compound with m': fragments 150. 195. 25,. 315 393. 408 2079 Compound with '''a: fragments 15() 195. 253. 315. 3')3. 4()8 2163 ( ompound with m/: fiagnients 129 147. 253. 31. 3')3 I 9360 Diisooctyl pthalatc . l in.)

   Table 6. Representation of the substances isolated, characteriseu and identified from mated female sealice conditioned water in example S. Scan no Compound 08 Hexamethylcyclotrisiloxane 664 Benzaldehyde _: Phenol 744 Octanal 778X l Benzyl alcohol I U 7t5 Octamethylcyclotetrasiloxane 2Ethylhexanol 819 /\cetophenone 8847 Compound with m/ flagmetits 83. 84 4Methylbenzaltlehyde I 867 3- and 4-Methylphenoi 876 Methyl benzoatc 887 Hexamethylcyclotrisilo.xane Nonanal 9" 2-Ethylhexanoic acid 7- Ethvlphenol 20 969 3- and 4-Ethylpl1enol 4-Methylacetopherlonc I ooi 1. 1. 4-Trdttethyl-3-cyclohexene- I -methanol 1019 7-ls:,propylphenol L)ecana I 9.> 1078 Benzothiazole 1042 3- and 4-lsopropylphetiol f 045 3-Acetyl-2,5dintethyithiophene lo796 Dimethoxymethylphenylsilatle 3() 098 Compound with n1/: fragments 69, 83. 98. 1,9, 167 04 4-:thylacetophenone 116 1 Adamatttol 1_7 N.N-Dibutviformarilide :5 7S Compound with n7;'- fra,ments 177..53. 769 1'()4 Compound with mi: fra,mcuts 43. 81. 109. 150 Proptomc acd, -m thy 7 -dimethyi- I -(7 h f27826 D'nPet hnl ahh, 9-methy lydroxy 4 4 tijeth IY I nilCthy] ethyl)propyl ester 1301 (icratlylacetotic 308 Compound withm:tragment.s 113, 141. 163 1,78 Compourld uitil m:: frarmLults 164. 2.53. 397, 34, 1364 Compoutid with m.: t'ra=,nnents 57. 165. 18(). 9().>, 336 1469 I rbutylpho;sphate 4.) 1471 Hexadecane 1649 Octadecene 1689 Isobutyl phthalate 1817 I:;icosene 1994 Compound with '7.': fragmettts 19:. 253' 315. 39,. 408 9360 Cotlpottnd witft m/: fraunLnts 150, 19.. 953, 3 I 5. 393. 408 Dusooctyl pthalate 3:

   Table 7. Activation response of sea lice to mating stimuli in a Y-tube arena *pcO.OS; ***r)c0.001 (x2 analysts).

   Test Odour Lo activity' lligh activity: n p Animal.Source Male 5'eututer control 60.9 39.X 83 lla/e l'reuclull 11 'irgin 1emule 95.5 74.5 47 0.0() (/'L') 1

   Mule PF extruct 39.4 6().6 33 <0.05 Male PF volutile.s 09.4 70.6 17 <0.05 Mule PF non-voluli/e.s 65.() 35.0 90 n.s.

   llule.ll'l muielfemale.s (AF) 4().0 60.0 45 'O.()S 'le Pt un1 AF (in sepurute 13.3 86.7 3() c().OO urm*) I Malc Males 41.7 SX. 3 48 <O.OS I'i ticahater control 63.3 41.7 3h 131: Males 5g.3 41.7 36 n.s.

   Oefined as L:ategory a Fig. 1. Delned as categories in-c. Fig]., :'1

   owl W l G _ a,!:' o 1 1 _o 1' its J _ o 0 | = C | E| - j mu I D of u - E o O El n _ _ c:. g E it_ D U O I 1

   Bayou=/ It's on s | --- - -

   ,;> _.WO_O O _ _:. hi V,C,oO/i 0:=; X X X X _ __ X Tut, I (d _._ _1 i _ __ D _ _ E _ -. 5

   ( Table 9. Activity of copepodids in Y tube in response to chemicals identified from non host spec ies.

   - I est_doll Lw \t ty _%)_ Hi4Activty; (% Control ((I) 24 76 63 anliloacetophe'ole it) X1 4X 4 methyiquin.lzolilic _ 1 1 _ X() _ 66 ..:1

   l Figure Legends Figure I ( a-c) Diagrammatic representation of Y-tube arena and louse activity 5 patterns for activation and taxis behaviour of adult male sea lice. Activation was recorded as (a) low activity with distance moved less than twice the Icngth of' the base of' the Y-tube and (b-c) high activity categories, with distance moved more than twice the length of the base of the Y-tube or choice of arm made, c shows an actual recording of louse movement using F,thovision34 in response to SCW. Taxis was recorded as the number of lice entering the 1 () odour arm vs. the number of lice entering the control arm.

   Figure 2 of 8 Taxis response ol'adult male sea lice to fish derived stimuli in a Y-tube arena. Seawater control n = 78. S;C'W (salmon conditioned water) n= IOS. 8C'W extract n-45. frozen.SC'W 15 n=38. 1'CW (turbot conditioned waler) n=25' 'I'CW extract n=11 SCW volatile fraction n=16 and SCW non-volatile fraction n=]O. The SCW tractions were diluted by half. * p<O.05; *** p<O.O()1 (X analysis). x2 analysis was not performed on the results for 'l'CW extract and SCW non volatile fraction as numbers were low due to very low activation to the stimul i (Table I).

   2() Figure 3 of 8 I'axis response of copepodids to chemicals identified prom salmon conditioned water.

   Percentage choosing seawater control (n--86) ethanol control (n=6()), salmon conditioned water (n=67), salmon conditioned water-volatile fraction (n=36)' isophorone (n=70) and 6 25 methyl-S-hepten-2-one(n=59). *P<O.()S; **13<().01: ***P<0.0001.

   ARC

   ( Figure 4 of 8 Responses of adult male sea lice to single chemical slow release vials loaded with isophorone in hexane in one vial and control with hexane only. Double control. hexane only in both vials n-ti4. isophoronc 100() n-37. * p<-().()1; (X2 analysis), Figure:S of 8 I'axis response of adult male (AM) sea lice to preadult 11 virgin female (PF) derived stimuli in a Y-tube arena. Seawater control n=93. Pl: (preadult 11 virgin 1'cmale conditioned water n=6X, PF extract n=18. 1'1: volatile fraction (<5()() Dalton) n=14. 1'1' non-volatile fraction (>Sob 1() L) alton) n=4. (* p<().O5; *** p<O.OOI (X analysis)). X analysis was not performed on the results for PEP non volatile fraction as n numbers were knew due to very low activation to the stimulus ('I'able I).

   Figure 6 of X. I'axis response of (i) adult male sea lice (AM) to adult mated females (AL), (ii) At' in one arm 15 and PF in the other, (iii) AM to AM, (iv) PEP to AM. Seawater control (n=93 AM. n=l 1 PF), Al: conditioned water n=19' Al: and let conditioned water n-2(). AM conditioned water for males n=20. AM conditioned water for Pl: n 11. *P<() .()S; **P<0.01 (A analysis). I, Figure 7 of 8 (:apture efficiency of the preliminary sea lice trap design. Pcrcentage capture by control (no 2() odour). isophorone. preadult 11 virgin females, isophorone -1ip,ht and light. **P().()1.

   Figure 8 of 8 Taxis response of copcpodids to potential repellents in vertical Y tube. PercLmtare choosing salmon conditioned water control (no) . aminuacetophenonc (n--48) and 4 5 methylquinazoline (n=66). All US.

   (