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Field trials to evaluate the efficacy of emamectin benzoate in the control of sea lice, Lepeophtheirus salmonis (Krøyer) and Caligus elongatus Nordmann, infestations in Atlantic salmon Salmo salar L.
Aquaculture 186 Ž2000. 205–219 www.elsevier.nlrlocateraqua-online
Field trials to evaluate the efficacy of emamectin benzoate in the control of sea lice, Lepeophtheirus salmonis žKrøyer/ and Caligus elongatus Nordmann, infestations in Atlantic salmon Salmo salar L. J. Stone a,) , I.H. Sutherland b, C. Sommerville a , R.H. Richards a , K.J. Varma c a
Institute of Aquaculture, UniÕersity of Stirling, Stirling, Scotland, UK b 31r10 Hermitage DriÕe, Edinburgh, Scotland, UK c Schering-Plough Animal Health, Union, NJ, USA Accepted 6 December 1999
Abstract Three field trials were conducted to evaluate the efficacy of emamectin benzoate as a treatment for sea lice, Lepeophtheirus salmonis ŽKrøyer. and Caligus elongatus ŽNordmann., infestations on Atlantic salmon Salmo salar ŽL... Trials were carried out at sea temperatures of 13.0–15.58C and 7.2–8.58C. Salmon naturally infested with sea lice, with mean weights of 438, 513 and 2662 g, respectively, were held in experimental pens on commercial sites. At day y1 or y2, 20 or 30 fish were sampled from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in-feed at a dose of 50 mg kgy1 biomass dayy1 for 7 consecutive days. Sea lice were counted again on days 7, 14 and 21, and comparisons made with untreated control fish. Treatment with emamectin benzoate was effective against chalimus and motile stages of sea lice. In all three trials, treated groups were surrounded by pens of heavily infested fish and L. salmonis numbers increased over time on control fish by 87–284%, whereas over the same period, L. salmonis were reduced on treated fish by 68–98%. In the low temperature trial, reductions were slower but numbers were still 90% lower than on control fish at day 21. At the end of the
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Corresponding author. Marine Environmental Research Laboratory, University of Stirling, Machrihanish, Argyll PA28 6PW, Scotland, UK. 0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 7 4 - 9
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third trial, both control pens were treated with hydrogen peroxide owing to heavy lice burdens. However, L. salmonis numbers rapidly increased again and at day 55, fish treated only with emamectin benzoate still had 80% fewer lice than control fish. In the two summer trials, large numbers of C. elongatus were rapidly reduced by treatment with 82–84% efficacy by day 21. Despite the potential for continuous re-infestation, oral treatment with emamectin benzoate presented an effective means of controlling all parasitic stages of L. salmonis and C. elongatus on farmed salmon, and in one trial, numbers remained lower on treated fish for at least 55 days. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sea lice; Lepeophtheirus salmonis; Caligus elongatus; Salmo salar; Emamectin benzoate
1. Introduction The success of Atlantic salmon, Salmo salar ŽL.., farming operations may be seriously affected by parasitic copepods, commonly known as sea lice, Lepeophtheirus salmonis ŽKrøyer. and Caligus elongatus ŽNordmann.. Control of these parasites is largely dependent on the use of immersion bath treatments. These are carried out using hydrogen peroxide ŽSalartect w , Brenntag, Paramove w , Solvay-Interox. or the synthetic pyrethoids, cypermethrin ŽExcis w , Vericore. and deltamethrin ŽAlphamax w , Alpharma.. The organophosphates, dichlorvos ŽAquagard w , Novartis. and azamethiphos ŽSalmosan w , Novartis. are also used, depending on the regulations of individual countries ŽRoth et al., 1993.. However, immersion treatments are impractical for use on exposed sites and during adverse weather conditions, and the procedures involved are very stressful to fish. With the exception of cypermethrin ŽJakobsen and Holm, 1990., bath treatments are only effective against pre-adult and adult stages of sea lice, allowing chalimus stages to survive and continue the cycle of infestation. Hydrogen peroxide may cause damage to the gills and its use is restricted in summer owing to its toxicity at higher water temperatures ŽThomassen, 1993; Bruno and Raynard, 1994.. Resistance to the organophosphate, dichlorvos has been identified in some populations of sea lice ŽJones et al., 1992.. As a result of these limitations, treatments are being developed that can be administered in feed. Two insect growth regulators, diflubenzuron ŽLepsidon w , Ewos. and teflubenzuron ŽCalicide w , Nutreco. ŽErdal, 1997. are available in Norway as in-feed treatments. Their mode of action is the inhibition of chitin synthesis ŽHorst and Walker, 1996. and activity is therefore restricted to the molting stages of sea lice. Ivermectin has also been used as an in-feed treatment ŽJohnson and Margolis, 1993; Smith et al., 1993; Palmer et al., 1997. but can only be prescribed in the UK under the cascade procedure where authorised products fail to provide effective control ŽAnonymous, 1998.. The efficacy of emamectin benzoate Ž4Y-deoxy-4Y epimethylaminoavermectin B1 . as an in-feed treatment for Atlantic salmon was initially demonstrated in a series of replicated tank trials employing induced infestations of L. salmonis ŽStone et al., 1999.. A dose of 50 mg kgy1 dayy1 for 7 consecutive days was found to be effective against both adult and immature stages of L. salmonis. The sea trials reported here evaluated the efficacy of emamectin benzoate under field conditions, where fish may be continuously exposed to re-infestation. Two trials carried
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out during the summer allowed evaluation of both L. salmonis and C. elongatus, the species commonly infesting farmed fish in the UK. These trials examined treatment of post-smolts in their first year at sea. The third trial evaluated efficacy in April when sea temperatures were lower and allowed assessment of larger fish in their second year at sea.
2. Materials and methods 2.1. Fish and holding conditions Three trials were carried out on commercial salmon farms on the northwest coast of Scotland. The trials were conducted on pontoons of pens stocked with Atlantic salmon, S. salar. Fish were selected from the main ongrowing pens at each site and acclimatised to small net pens of 12, 27 or 102 m3 as described in Experimental design. Fish were naturally infested with L. salmonis and C. elongatus and were exposed potentially to re-infestation by larval sea lice throughout the duration of the trial. In each trial, lice numbers were similar on fish in the surrounding commercial pens to those of the control fish, prior to the start of treatment. All control and treated fish sampled for sea lice evaluation were weighed at each time point Ž N s 20 or 30 fish.. 2.2. Medicated feed The basal ration used was Trouw UK ŽWincham, Cheshire, UK. 4.0 or 8.5 mm salmon feed pellets. Emamectin benzoate Ž4Y-deoxy-4Y epimethylaminoavermectin B 1 . was dissolved in propylene glycol and mixed with fish oil prior to coating the feed pellets. Control feeds were prepared with propylene glycol and fish oil only. Treatment was administered at a nominal dose of 50 mg kgy1 fish biomass per day. All fish were given an initial unmedicated feed at a rate of 0.1–0.25% biomass at the start of the day and medicated or control feeds were given at a rate of 0.4% or 0.5% biomass, 3 to 4 h later. Medicated feed was administered for a period of 7 consecutive days Ždays 0–6.. Feeding rates and methods were identical for all control and treated groups in each trial. 2.3. EÕaluation of sea lice At day y1 or y2, 20 or 30 fish were randomly selected from each pen to determine pre-treatment numbers of sea lice. Each fish was examined using a hand lens or low power microscope and all attached lice removed and fixed in 5% formalin. Lice were identified as L. salmonis or C. elongatus and the number of chalimus I, II, III, IV, pre-adult I, pre-adult II and adult stages recorded. Pre-adult and adult lice were further identified as males or females. Evaluation of sea lice numbers was carried out again at 7, 14 and 21 days from the start of treatment. In the third trial, additional counts of sea lice on five fish per pen in control pen II and treated pen I were conducted by site staff on days 27, 35, 42, 48 and 55. For these counts, chalimus were counted only up to a
208
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maximum of 100 per fish. The occurrence of sea lice damage was determined on sampled fish by visual assessment of typical epidermal lesions. 2.4. Experimental design The three trials were conducted blind with the identity of each treatment and control ration unknown to personnel responsible for feed administration and collection of data. Treatments were randomly allocated to each trial pen. 2.4.1. First trial Two pens, one control and one treated, were used, each holding 180 fish at the start of the trial. Each pen had a volume of 12 m3 and they were located on a pontoon midway between 28 commercial pens, each measuring approximately 1150 m3. All pens were stocked with fish of the same year class in February at a density of 6.0–6.6 kg my3 and the trial was started in August. The sea temperature over the trial period was 13.0–15.58C and salinity 35 ppt. None of the fish on the site had been treated previously for sea lice. On day 8, fish in the surrounding commercial pens were treated for sea lice with dichlorvos ŽAquagard w , Novartis.. The trial pens were surrounded by a fully enclosed tarpaulin for the duration of this treatment, and for 30 min post-treatment, to protect fish from exposure to the treatment compound, which was rapidly flushed from the site by high current speeds Žaverage 8–10 cm sy1 at 2 m depth.. Pre-treatment fish weights were 268 to 625 g Žmean 438 " 72 g S.D... The sample size for parasite enumeration was 30 fish per pen. 2.4.2. Second trial There were two control and two treated pens with 149 fish per pen at the start of the trial. Each pen had a volume of 27 m3 Žstocking density 2.8 kg m3 . and was located on the same pontoon described above with each commercial pen stocked at 6.7 kg m3. The pens were stocked in February and the trial started in September. The sea temperature over the trial period was 13.8–14.28C and salinity 33–35 ppt. Trial fish had been treated previously for sea lice with the organophosphate, dichlorvos ŽAquagard w , Novartis. 27 and 47 days before the start of the trial. Pre-treatment fish weights were 251 to 878 g Žmean 513 " 136 g S.D... The sample size for parasite enumeration was 20 fish per pen. 2.4.3. Third trial The trial consisted of two control and two treated pens with 360 fish per pen at a density of 9.4 kg m3. This trial used larger pens each with a volume of 102 m3 , located on a pontoon of 32 pens of similar size and stocking density. All pens were stocked with fish of the same year class in April of the previous year and fish were 1026–4464 g Žmean 2662 " 597 g S.D.. in weight. There were also three large commercial rearing units located within 1 km of this site. The trial started in April when the sea temperature was 7.2–8.58C and salinity 31–33 ppt. The fish had been treated for sea lice with dichlorvos ŽAquagard w . in the previous summer and with hydrogen peroxide 20 days before the start of the trial. The entire site, including the two control pens, but not the pens treated with emamectin benzoate, was subjected to a bath treatment with hydrogen
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peroxide on day 22 after the end of the trial Žday 21.. The sample size for parasite enumeration was 20 fish per pen. 2.5. Data handling Parasite counts were summarised as chalimus Žcopepodites, chalimus stages I, II, III and IV., motile lice Žpre-adult and adult stages. and total lice Žchalimus and motile stages combined. and the arithmetic means calculated. Parasite count data were subjected to F-tests for homogeneity of variances and a correlation test to examine the normality of distribution. Lice numbers were analysed using the non-parametric STP test ŽSokhal and Rohlf, 1981.. In the two replicated trials, the data were analysed separately for each replicate pen. The percentage reduction in mean total sea lice, over the trial period was calculated as follows:
ž
%Reductions 100 y 100 =
Mean of control or treated replicates at day y1 Mean of control or treated replicates at day 21
/
.
The efficacy or percentage reduction in mean total sea lice, relative to the control groups, was calculated as follows:
ž
%Efficacys 100 y 100 =
Mean of treated replicates Mean of the control replicates
/
.
Owing to the high standard deviation of fish weights and the small sample size, statistical analysis of fish weights was not appropriate in these trials. Specific growth rates ŽSGR. were calculated as follows: SGR s
log e Wt y log e W0 t
where Wt is the mean weight at day 21 and W0 is the mean weight at day y1 or y2.
3. Results 3.1. Feed consumption and fish weights In each trial, good feed consumption was observed in all treated and control pens during and after the medication period. In the first trial, the increase in mean fish weight over the trial period was 5.2 g ŽSGR 0.01%. in the control group and 91.0 g ŽSGR 0.8%. in the treated group. In the second trial, mean weights increased by 3.8 and 35.5 g ŽSGR 0.03% and 0.3%. in the control groups I and II, while treated pens I and II increased by 92.7 and 133.4 g ŽSGR 0.7% and 1.0%.. In the third trial, owing to high fish weights and hence a high standard deviation, the sample size used was too small to provide
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210
conclusive results on fish weights. No adverse effects or fish mortality were attributed to treatment with emamectin benzoate in any of the trials. 3.2. First trial (13.0–15.58C) 3.2.1. First trial: efficacy against L. salmonis The efficacy and mean numbers of L. salmonis per fish are shown in Table 1. Prior to the start of treatment, mean numbers of L. salmonis were similar in the two trial pens. L. salmonis numbers increased in the control group by 52% over the trial period. In the treated group, there was no corresponding increase and, from day y2 to 21, numbers decreased by 86%. At day 21, treated fish had fewer motile and chalimus stages than control fish. At the start of the trial, none of the fish examined was free of L. salmonis. At day 21, the number of L. salmonis on control fish ranged from 4 to 33, while the range on treated fish was 0–11 and 43% of treated fish sampled had no chalimus or motile stages present. 3.2.2. First trial: efficacy against C. elongatus The results for C. elongatus are shown in Table 2. Prior to the start of treatment, the mean numbers of C. elongatus were similar in the two trial pens. From day y2 to 21, the mean number of C. elongatus declined on control fish by only 36%. Over the same period, numbers fell on treated fish by 98%. At the start of the trial, none of the fish sampled was free of C. elongatus. At day 21, 10% of treated fish had no motile C. elongatus and 67% had no chalimus present whereas none of the control fish was free of chalimus or motile stages. Numbers of motile C. elongatus fell between days 7 and 14,
Table 1 First trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 30 fish per pen. Sea temperatures 13.0–15.58C Time
Nominal dose Žmg kgy1 .
Mean total lice Ž"S.D..
Mean chalimus Ž"S.D..
Mean motiles Ž"S.D..
Percent efficacy Žtotal lice.
0
9.1Ž"4.9.
4.4 Ž"3.2.
4.7 Ž"3.2.
50
10.0 Ž"5.3.
5.2 Ž"4.8.
4.8 Ž"2.6.
Day 7
0 50
17.0 Ž"6.0. 9.4 Ž"5.9.
5.8 Ž"2.7. 4.7 Ž"3.4.
11.3 Ž"5.0. 4.8 Ž"3.1.
45
Day 14
0 50
12.8 Ž"6.3. 2.5 Ž"2.3.
1.1 Ž"1.1. 0.5 Ž"0.9.
11.8 Ž"6.0. 1.9 Ž"2.1.
80
Day 21
0 50
13.8 Ž"8.3. 1.4 a Ž"2.3.
1.2 Ž"1.3. 0.2 Ž"0.5.
12.6 Ž"7.9. 1.2 Ž"2.0.
91
Day y2 Žpre-treatment.
a
Includes one fish with 11 lice.
–
J. Stone et al.r Aquaculture 186 (2000) 205–219
211
Table 2 First trial: Efficacy of emamectin benzoate against natural infestations of C. elongatus on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 30 fish per pen. Sea temperatures 13.0–15.58C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. Žtotal lice.
Day y2 Žpre-treatment.
0 50
41.9 Ž"8.3. 41.3 Ž"12.0.
26.2 Ž"8.8. 20.7 Ž"5.9.
15.6 Ž"5.5. 20.6 Ž"8.8.
–
Day 7
0 50
39.3 Ž"10.6. 16.6 Ž"6.1.
21.3 Ž"6.5. 5.8 Ž"2.8.
17.9 Ž"6.1. 10.9 Ž"4.5.
58
Day 14
0 50
24.5 Ž"9.3. 2.6 Ž"3.2.
12.3 Ž"7.5. 1.0 Ž"2.3.
12.2 Ž"5.1. 1.5 Ž"1.3.
89
Day 21
0 50
26.8 Ž"13.4. 4.3 Ž"5.2.
14.9 Ž"9.4. 0.5 Ž"0.9.
11.9 Ž"6.4. 3.8 Ž"4.8.
84
following Aquagard w treatment of the commercial pens but numbers of chalimus had fallen between days 0 and 7, thus a decline in motile numbers was expected. At day 14, 23% of fish in the control group had lesions caused by L. salmonis and C. elongatus while none of the treated fish examined were affected by sea lice damage.
Table 3 Second trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 20 fish per pen. Sea temperatures 13.8–14.28C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y2 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
18.7 Ž"8.1. 20.4 Ž"8.5. 16.2 Ž"6.4. 26.9 Ž"9.4.
3.6 Ž"3.3. 6.5 Ž"4.1. 2.9 Ž"2.6. 8.3 Ž"4.2.
15.1 Ž"6.7. 13.9 Ž"5.7. 13.3 Ž"5.3. 18.6 Ž"8.5.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
30.3 Ž"7.7. 38.3 Ž"14.6. 10.3 Ž"5.4. 14.9 Ž"5.9.
11.5 Ž"5.8. 19.6 Ž"10.3. 2.4 Ž"1.8. 7.5 Ž"4.4.
18.8 Ž"4.8. 18.9 Ž"7.1. 7.9 Ž"4.5. 7.5 Ž"3.3.
63.3
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
31.7 Ž"8.2. 65.9 Ž"34.0. 2.3 Ž"1.7. 4.0 Ž"4.6.
15.6 Ž"5.2. 41.2 Ž"23.1. 0.8 Ž"1.0. 3.3 Ž"3.8.
16.2 Ž"5.2. 24.8 Ž12.6. 1.5 Ž"1.5. 0.8 Ž"1.1.
93.4
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
58.5 Ž"18.9. 92.1 Ž"39.7. 0.3 Ž"0.7. 0.8 Ž"1.2.
26.7 Ž"10.8. 41.5 Ž"26.7. 0 0.6 Ž"0.9.
31.8 Ž"10.7. 99.3 31.8 Ž"20.8. 0.3 Ž"0.7. 0.2 Ž"0.4.
212
J. Stone et al.r Aquaculture 186 (2000) 205–219
3.3. Second trial (13.8–14.28C) 3.3.1. Second trial: efficacy against L. salmonis The efficacy and mean numbers of L. salmonis per fish are shown in Table 3. Prior to the start of treatment, one pen intended for treatment had significantly Ž P - 0.05 and P - 0.01. more L. salmonis than one control pen and the other treated pen. Over the trial period, L. salmonis numbers increased by 284% in the two control groups. In the two treated groups there was no corresponding increase and, from days y2 to 21, numbers declined by 97% on treated fish. At days 7, 14 and 21 both treated pens had significantly Ž P - 0.01. fewer L. salmonis than fish in the two control pens. Although control group I had significantly Ž P - 0.05. fewer lice than control group II at day 21, both groups still had large numbers of lice present and there were no significant Ž P ) 0.05. differences between the two treated replicates at days 14 and 21. At day 21, individual control fish had 26–190 L. salmonis present but the highest number of L. salmonis found on any treated fish was 5 and 67.5% of treated fish had no L. salmonis present. At day 14, the two treated pens had means of only 0.6 and 2.6 copepodites per fish and there was less than 0.8 chalimus present. In contrast, all control fish had large numbers of copepodites and chalimus stages I, II, III and IV present. At day 21, the two control groups had high numbers of chalimus present but there were no surviving
Table 4 Second trial: Efficacy of emamectin benzoate against natural infestations of C. elongatus on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 20 fish per pen. Sea temperatures 13.8–14.28C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y2 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
25.3 Ž"10.4. 29.2 Ž"7.1. 26.3 Ž"10.4. 39.1 Ž"7.6.
13.2 Ž"6.1. 15.8 Ž"6.3. 13.6 Ž"5.6. 22.5 Ž"7.7.
12.2 Ž"6.6. 13.4 Ž"4.4. 12.7 Ž"5.9. 16.6 Ž"5.5.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
20.3 Ž"8.4. 20.4 Ž"7.6. 8.2 Ž"8.2. 14.0 Ž"7.1.
6.5 Ž"3.4. 9.2 Ž"5.0. 3.2 Ž"2.3. 6.3 Ž"3.3.
13.8 Ž"7.3. 11.4 Ž"4.6. 5.1 Ž"2.7. 7.7 Ž"4.7.
45.6
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
11.0 Ž"3.8. 16.9 Ž"10.9. 1.9 Ž"2.1. 3.7 Ž"2.9.
4.8 Ž"2.8. 9.9 Ž"7.9. 0.6 Ž"0.8. 3.3 Ž"2.4.
6.2 Ž"2.8. 7.0 Ž"3.8. 1.3 Ž"1.6. 0.4 Ž"0.8.
79.9
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
10.5 Ž"4.2. 12.6 Ž"4.4. 1.3 Ž"1.2. 2.9 Ž"2.3.
4.6 Ž"2.3. 6.2 Ž"3.2. 0.6 Ž"0.6. 2.5 Ž"2.2.
5.9 Ž"2.9. 6.4 Ž"3.1. 0.7 Ž"0.8. 0.5 Ž"0.8.
81.9
J. Stone et al.r Aquaculture 186 (2000) 205–219
213
chalimus on fish in one treated pen and means of only 0.5 copepodites and 0.1 chalimus IV in the other treated pen. 3.3.2. Second trial: efficacy against C. elongatus At the start of the trial, there was a high mean number of C. elongatus in each pen ŽTable 4.. Treated pen II had significantly Ž P - 0.01. more C. elongatus than the other three pens prior to the start of treatment. Over the trial period, the mean number of C. elongatus declined by 57% on control fish and by 94% on fish in the two treated groups. Numbers in both treated pens were significantly Ž P - 0.01. lower than on control fish at days 14 and 21. At day 21, 84% and 100% of the immature stages remaining on treated fish were copepodites, whereas in the control groups, only 37% and 34% of the immature stages were copepodites. Prevalence of C. elongatus declined from 100% to 35% of treated fish with motile stages and 67.5% with chalimus, whereas none of the control fish sampled were free of chalimus or motile stages. At day y2, the majority of fish sampled in each trial pen had skin lesions caused by sea lice. At day 21, the prevalence of lice damage on treated fish had fallen to only 12.5%, while 77.5% of control fish were still affected.
Table 5 Third trial: Efficacy of emamectin benzoate against natural infestations of sea lice, L. salmonis, on Atlantic salmon, S. salar. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. The mean number of sea lice was determined on days y2, 7, 14 and 21. Sample size N s 20 fish per pen. Sea temperatures 7.2–8.58C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y1 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
24.6 Ž"16.3. 25.7 Ž"13.4. 17.9 Ž"7.4. 24.9 Ž"9.5.
11.4 Ž"11.1. 11.5 Ž"6.9. 9.7 Ž"5.1. 7.7 Ž"4.7.
13.3 Ž"7.1. 14.1 Ž"8.2. 8.2 Ž"3.8. 17.3 Ž"6.8.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
23.3 Ž"14.7. 27.8 Ž"11.1. 13.5 Ž"5.9. 24.7 Ž"12.1.
10.5 Ž"9.4. 10.3 Ž"6.5. 7.1 Ž"4.3. 7.1 Ž"5.4.
12.8 Ž"7.0. 17.6 Ž"7.5. 6.4 Ž"2.8. 17.6 Ž"8.9.
25.4
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
39.5 Ž"21.6. 38.1 Ž"14.3. 6.7 Ž"3.0. 13.3 Ž"5.1.
17.3 Ž"11.1. 18.0 Ž"9.9. 4.6 Ž"2.1. 5.7 Ž"3.9.
22.2 Ž"12.3. 74.2 20.1 Ž"8.1. 2.2 Ž"1.5. 7.6 Ž"3.7.
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
60.9 Ž"26.1. 75.1 Ž"35.7. 5.6 Ž"4.9. 8.3 Ž"3.8.
35.6 Ž"19.2. 43.6 Ž"23.9. 4.3 Ž"3.4. 3.0 Ž"1.8.
25.4 Ž"9.2. 89.7 31.5 Ž"16.2. 1.3 Ž"2.0. 5.3 Ž"3.2.
214
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3.4. Third trial (7.2–8.58C) 3.4.1. Third trial: efficacy against L. salmonis Table 5 shows the efficacy and mean numbers of L. salmonis for the third trial. Prior to the start of treatment there were no significant Ž P ) 0.05. differences in lice numbers between pens.
Fig. 1. Ža and b. Third trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days. Sample size N s 20 fish per pen. Between days 27 and 55, after the trial period, only five fish in control pen II and treated pen I were evaluated. Chalimus were counted only up to a maximum of 100 per fish. Sea temperatures 7.2–8.58C from days y1 to 20 and 8.8–10.98C from days 22 to 55.
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Over the trial period, L. salmonis numbers increased by 170% in the two control groups. There was no corresponding increase on treated fish and, from days y2 to 21, lice numbers decreased by 68%. At days 14 and 21, both treated groups had significantly Ž P - 0.01. fewer L. salmonis than the two control groups. At day 21, the two treated groups still had a higher mean number of lice compared to the results in the two summer trials, although the mean numbers of lice on control fish were also much higher. The number of L. salmonis on any individual control fish was 28–154 while the range on treated fish was only 0–19. At day 21, 20% of fish in the control groups had characteristic lesions caused by sea lice while only 2.5% of treated fish were affected. At the end of the trial Žday 22., all the pens on the site, with the exception of the two pens treated with emamectin benzoate, were subjected to a hydrogen peroxide bath treatment for sea lice. The two pens treated with emamectin benzoate were not treated with hydrogen peroxide because of low lice numbers and improved appearance of the fish. Following treatment with hydrogen peroxide there was a decline in the mean number of motile lice on control fish at day 27 ŽFig. 1a., but chalimus were not reduced and increased to more than 100 per fish between days 35 and 55 ŽFig. 1b.. As a result, at day 55, mean motile lice numbers reached high levels again and a second hydrogen peroxide treatment had to be carried out 5 weeks after the previous treatment. After day 27, lice numbers on fish treated only with emamectin benzoate increased relatively slowly compared to the control group, and at day 55, treated fish still had 78% fewer chalimus and 86% fewer motile lice than control fish. Between days 22 and 55, fish in the surrounding commercial pens also had much higher numbers of lice than fish in the pens treated with emamectin benzoate. Prior to treatment, sea lice damage was recorded on only 0–5% of fish in each pen. At day 21, 20% of control fish had sea lice damage compared to only 2.5% on fish in the two treated groups. 3.4.2. Third trial: efficacy against C. elongatus There were few C. elongatus present during this trial. Mean numbers at the start of the trial were only 0.1–1.0 per fish and at day 21, the means were 0 and 0.8 per fish in the treated groups and 0 and 1.4 per fish in the control groups. 4. Discussion Despite the variability in fish weights at the start of the trial and hence, feed consumption, good efficacy was achieved and no adverse reactions or fish mortalities were attributed to treatment with emamectin benzoate. Treated groups had higher SGR than the control groups, but it is uncertain whether treatment resulted in significant benefits to fish growth as larger samples sizes would be required to detect statistically significant differences. Fish infested with L. salmonis have shown a decreased feed consumption correlated with the appearance of head lesions, although growth rates were unaffected ŽDawson et al., 1997.. No significant differences in growth were found by Grimnes and Jakobsen Ž1996. between fish infested with L. salmonis and un-infested fish, although heavily infested moribund fish had a lower condition factor. Observations from tank ŽStone et al., 1999. and field trials suggest there was an improved feed response in lice-infested fish following treatment with emamectin. Further, improve-
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ments in growth may be a long-term benefit of effective in-feed medication, as bath treatments require 3–4 days starvation and cause increased stress. Oral treatment of Atlantic salmon with emamectin benzoate showed good efficacy against both motile and chalimus stages of L. salmonis and C. elongatus in all three trials, despite the potential for continuous recruitment from adjacent commercial pens. L. salmonis numbers increased over time on control fish by 87–284%, whereas over the same period, numbers were reduced on treated fish by 68–98%. The first sea trial was conducted as a pilot study and replicate pens were not evaluated as they were in the second and third trials, however, useful data were obtained, particularly on C. elongatus, and for this reason, the results are included here. At the start of the second trial, one treated pen had significantly more L. salmonis and C. elongatus than the other pens. Despite this, fish in this pen still had significantly fewer lice than fish in the two control pens at days 14 and 21. Following treatment with emamectin benzoate, 55–80% of fish had no chalimus or motile L. salmonis present. While efficacy against chalimus stages is beneficial in preventing development to the motile stages ŽStone et al., 1999., a rapid reduction in motile lice numbers is also important, as they are more damaging to host fish. Motile lice numbers were reduced by 21–59%, as early as day 7, on treated fish. Oral treatment with ivermectin at a dose of 0.05 mg kgy1 every third day for three or six doses resulted in good efficacy against both chalimus and adult stages of L. salmonis ŽJohnson and Margolis, 1993.. However, ivermectin appears to have a narrow safety margin ŽJohnson et al., 1993; Palmer et al., 1997. while no adverse effects have been reported with emamectin benzoate at up to 3.5 times the therapeutic dose and no mortalities occurred at 7 times the therapeutic dose ŽRoy et al., in press.. Most of the treated fish, sampled at day 21, had fewer L. salmonis than any control fish. This suggests all fish received an efficacious dose, despite variability in fish weights and feeding competition. Most of the L. salmonis present on treated fish at day 21 were either copepodites or small numbers of mature adults. Copepodites represented recent settlement of larval stages and did not appear to develop further. The transfer of motile stages of L. salmonis between salmon has been recorded in tank and field trials ŽBruno and Stone, 1990; Ritchie, 1997; Treasurer and Grant, 1997. and it is possible that some adult lice on treated fish had transferred from untreated fish on the site. Treatment at low temperatures resulted in slower reductions in L. salmonis numbers and a small number of lice persisted on treated fish at day 21. However, treatment was still effective and treated groups had significantly fewer lice compared to control fish. Further sampling showed that lice numbers remained much lower on fish treated with emamectin benzoate for at least 55 days from the start of treatment. Although there was a gradual increase in chalimus numbers on treated fish, there was little increase in motile lice, suggesting that when chalimus settled on treated fish, their development to mature motile stages was impaired. This confirmed observations in tank trials where attached chalimus failed to develop on fish were treated with emamectin benzoate ŽStone et al., 1999.. The settlement of small numbers of copepodites on treated fish, without a corresponding increase in chalimus, also suggests newly recruited lice fail to develop further. Johnson and Margolis Ž1993. also reported slower development of lice on fish treated orally with ivermectin.
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C. elongatus numbers are present in greater numbers when sea temperatures are high ŽHogans and Trudeau, 1989; Tully, 1989.. In the two summer trials, mean numbers of C. elongatus were high initially and declined on control fish over time as sea temperatures fell. In the third trial, carried out in April, there were few C. elongatus present and these probably represented over-wintering populations. Treatment with emamectin benzoate was also effective against C. elongatus and numbers declined more rapidly on treated fish than on control fish. C. elongatus is not host-specific and may be derived from wild fish ŽWootten et al., 1982.. Motile stages of C. elongatus may transfer between individual salmon and, from infested saithe, Pollachius Õirens, to cohabiting salmon ŽBruno and Stone, 1990.. Transfer from wild and farmed fish in the area may account for the persistence of low numbers of motile C. elongatus on fish treated with emamectin. Treated fish also had low numbers of C. elongatus copepodites, although in contrast to the control fish, there were few chalimus present. Again, this suggests treatment prevented further development of C. elongatus copepodites. Bath immersion treatment of the commercial pens with Aquagard w , in the first trial, did not reduce numbers of motile L. salmonis on the control fish, confirming that the tarpaulins protected the trial fish. Hydrogen peroxide is not effective in removing chalimus stages of L. salmonis ŽMcAndrew et al., 1998. and this was evident from lice numbers following treatment of control fish in the third trial. As a result, although motile lice were reduced on control fish following peroxide treatment at day 22, their numbers rapidly increased again, necessitating another peroxide treatment only 33 days later. Motile lice exposed to hydrogen peroxide may recover ŽTreasurer and Grant, 1997; McAndrew et al., 1998. and could potentially re-infest fish and this may also have contributed to the rapid recovery of motile lice numbers on control fish. In each trial, the prevalence of lice damage increased on control fish over time, while the number of lesions on treated fish was reduced and, overall, lice damage was 15–75% lower on treated fish. In all three trials, treated pens were surrounded by large commercial stocks of fish heavily infested with sea lice, creating a large potential source of copepodite stages. Despite this, very good efficacy was achieved against both L. salmonis and C. elongatus and development of newly recruited larval lice was prevented. When entire sites are treated, the potential for re-infestation will be reduced and the recovery of lice numbers may take even longer, allowing the frequency of treatments to be reduced. In-feed treatments will also permit simultaneous medication of all pens on a site and all sites in a single area, thus reducing any cross-infestation that may occur during the time necessary to apply bath treatments to all pens. 5. Conclusion Oral treatment with emamectin benzoate resulted in reductions of both species of lice commonly infesting farmed salmon and, in one study, lice numbers remained low until the final observation at day 55. Treatment was also effective at low temperatures and in this study, emamectin benzoate was more effective than hydrogen peroxide in reducing lice numbers and in preventing the development of newly recruited lice.
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Acknowledgements The authors wish to acknowledge the help of Ronnie Soutar and the staff at the Norsk Hydro farm at Fishnish, Isle of Mull, and Andrew Grant and the staff at the Marine Harvest McConnell field trial unit at Loch Duich. We would also especially like to thank Cam Matthews, Obdulio Andrade-Salas and Dr. James Bron for their support and technical assistance. This work was funded by Schering-Plough Animal Health, the Scottish Salmon Growers Association, and the Crown Estates Commissioners.
References Anonymous, 1998. Amelia No. 8. Veterinary Medicines Directorate, Woodham Lane, Newhaw, Addlestone, Surrey KT15 3NB. Bruno, D.W., Raynard, R.S., 1994. Studies on the use of hydrogen peroxide as a method for the control of sea lice on Atlantic salmon. Aquacult. Int. 2, 10–18. Bruno, D.W., Stone, J., 1990. The role of saithe, Pollachius Õirens, as a host for the sea lice, Lepeophtheirus salmonis and Caligus elongatus. Aquaculture 89, 201–207. Dawson, L., Pike, A.W., Houlihan, D., McVicar, A.H., 1997. Appetite suppression in Atlantic salmon Salmo salar ŽL.. infected with sea lice Lepeophtheirus salmonis Kroyer. In: Houlihan, D., Kiessling, A., Boujard, T. ŽEds.., Voluntary Food Intake in Fish. COST 827 Workshop, Aberdeen ŽUK. 3–6 Apr. 1997. Erdal, J.I., 1997. New drug treatment hits sea lice when they are most vulnerable. Fish Farming Int. 24 Ž2., 9. Grimnes, A., Jakobsen, P.J., 1996. The physiological effects of salmon lice infection on post-smolt Atlantic salmon. J. Fish Biol. Ž48., 1179–1194. Hogans, W.E., Trudeau, D.J., 1989. Preliminary studies on the biology of sea lice, Caligus elongatus, Caligus curtus and Lepeophtheirus salmonis parasitic on cage-cultured salmonids in the lower Bay of Fundy. Can. Tech. Rep. Fish. Aquat. Sci. 1715, 14. Horst, M.N., Walker, A.N., 1996. Biochemical effects of diflubenzuron on chitin synthesis in the post-molt blue crab Ž Callinectes sapidus .. J. Crustacean Biol. 15, 401–408. Jakobsen, P.J., Holm, J.C., 1990. Promising test with new compound against salmon lice. Norsk Fiskeoppdrett., 16–18, January. Johnson, S.C., Kent, M.L., Whittaker, D., Margolis, L., 1993. Toxicity and pathological effects of orally administered ivermectin in Atlantic, chinook and coho salmon and steelhead trout. Dis. Aquat. Org. 17, 107–112. Johnson, S.C., Margolis, L., 1993. Efficacy of ivermectin for control of the salmon louse, Lepeophtheirus salmonis, on Atlantic salmon. Dis. Aquat. Org. 17, 101–105. Jones, M.W., Sommerville, C.S., Wootten, R., 1992. Reduced sensitivity of the salmon louse, Lepeophtheirus salmonis, to the organophosphate dichlorvos. J. Fish Dis. 15, 197–202. McAndrew, K.J., Sommerville, C., Wootten, R., Bron, J.E., 1998. The effects of hydrogen peroxide on different life-cycle stages of the salmon louse, Lepeophtheirus salmonis. J. Fish Dis. 21, 221–228. Palmer, R., Coyne, R., Davey, S., Smith, P., 1997. Case notes on adverse reactions associated with ivermectin therapy of Atlantic salmon. Bull. Eur. Assoc. Fish Pathol. 17 Ž2., 62–67. Ritchie, G., 1997. The host transfer ability of Lepeophtheirus salmonis ŽCopepoda: Caligidae. from farmed Atlantic salmon, Salmo salar L. J. Fish Dis. 20, 153–157. Roth, M., Richards, R., Sommerville, C., 1993. Current practices in the chemotherapeutic control of sea lice infestations: a review. J. Fish Dis. 16, 1–26. Roy, W.R., Rodger, H., Sutherland, I.H. Tolerance of Atlantic salmon Ž Salmo salar . and rainbow trout Ž Oncorhynchus mykiss . to emamectin benzoate, a new orally administered treatment for sea lice. Aquaculture, in press. Smith, P.R., Moloney, M., McElligott, A., Clarke, S., O’Brien, F., 1993. The efficiency of oral ivermectin in
J. Stone et al.r Aquaculture 186 (2000) 205–219
219
the control of sea lice infestations of farmed Atlantic salmon. In: Boxsall, G., DeFaye, D. ŽEds.., Pathogens of Wild and Farmed Fish: Sea Lice. Ellis Horwood, Chichester, pp. 296–307. Sokhal, R., Rohlf, F.J., 1981. In: Biometry. 2nd edn. Freeman, New York, pp. 437–440. Stone, J., Sutherland, I.H., Sommerville, C.S., Richards, R., Varma, K.J., 1999. The efficacy of emamectin as an oral treatment of sea lice, Lepeophtheirus salmonis, infestations in Atlantic salmon, Salmo salar ŽL... J. Fish Dis. 22, 1–10. Thomassen, J.M., 1993. Hydrogen peroxide as a delousing agent for Atlantic salmon. In: Boxsall, G., DeFaye, D. ŽEds.., Pathogens of Wild and Farmed Fish: Sea Lice. Ellis Horwood, Chichester, pp. 290–295. Treasurer, J.W., Grant, A., 1997. The efficacy of hydrogen peroxide for the treatment of farmed Atlantic salmon Salmo salar L. infested with sea lice ŽCopepoda: Caligidae.. Aquaculture 148, 265–275. Tully, O., 1989. The succession of generations and growth of the caligid copepods Caligus elongatus and Lepophtheirus salmonis parasitising farmed Atlantic salmon smolts Salmo salar ŽL... J. Mar. Biol. Assoc. U. K. 69, 279–287. Wootten, R., Smith, J.W., Needham, E.A., 1982. Aspects of the biology of the parasitic copepods L. salmonis and C. elongatus on farmed Atlantic salmonids and their treatment. Proc. R. Soc. Edinburgh, Sect. B: Biol. Sci. 81, 185–197.
Field trials to evaluate the efficacy of emamectin benzoate in the control of sea lice, Lepeophtheirus salmonis žKrøyer/ and Caligus elongatus Nordmann, infestations in Atlantic salmon Salmo salar L. J. Stone a,) , I.H. Sutherland b, C. Sommerville a , R.H. Richards a , K.J. Varma c a
Institute of Aquaculture, UniÕersity of Stirling, Stirling, Scotland, UK b 31r10 Hermitage DriÕe, Edinburgh, Scotland, UK c Schering-Plough Animal Health, Union, NJ, USA Accepted 6 December 1999
Abstract Three field trials were conducted to evaluate the efficacy of emamectin benzoate as a treatment for sea lice, Lepeophtheirus salmonis ŽKrøyer. and Caligus elongatus ŽNordmann., infestations on Atlantic salmon Salmo salar ŽL... Trials were carried out at sea temperatures of 13.0–15.58C and 7.2–8.58C. Salmon naturally infested with sea lice, with mean weights of 438, 513 and 2662 g, respectively, were held in experimental pens on commercial sites. At day y1 or y2, 20 or 30 fish were sampled from each pen to determine pre-treatment numbers of lice. Emamectin benzoate was administered in-feed at a dose of 50 mg kgy1 biomass dayy1 for 7 consecutive days. Sea lice were counted again on days 7, 14 and 21, and comparisons made with untreated control fish. Treatment with emamectin benzoate was effective against chalimus and motile stages of sea lice. In all three trials, treated groups were surrounded by pens of heavily infested fish and L. salmonis numbers increased over time on control fish by 87–284%, whereas over the same period, L. salmonis were reduced on treated fish by 68–98%. In the low temperature trial, reductions were slower but numbers were still 90% lower than on control fish at day 21. At the end of the
)
Corresponding author. Marine Environmental Research Laboratory, University of Stirling, Machrihanish, Argyll PA28 6PW, Scotland, UK. 0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 3 7 4 - 9
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third trial, both control pens were treated with hydrogen peroxide owing to heavy lice burdens. However, L. salmonis numbers rapidly increased again and at day 55, fish treated only with emamectin benzoate still had 80% fewer lice than control fish. In the two summer trials, large numbers of C. elongatus were rapidly reduced by treatment with 82–84% efficacy by day 21. Despite the potential for continuous re-infestation, oral treatment with emamectin benzoate presented an effective means of controlling all parasitic stages of L. salmonis and C. elongatus on farmed salmon, and in one trial, numbers remained lower on treated fish for at least 55 days. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Sea lice; Lepeophtheirus salmonis; Caligus elongatus; Salmo salar; Emamectin benzoate
1. Introduction The success of Atlantic salmon, Salmo salar ŽL.., farming operations may be seriously affected by parasitic copepods, commonly known as sea lice, Lepeophtheirus salmonis ŽKrøyer. and Caligus elongatus ŽNordmann.. Control of these parasites is largely dependent on the use of immersion bath treatments. These are carried out using hydrogen peroxide ŽSalartect w , Brenntag, Paramove w , Solvay-Interox. or the synthetic pyrethoids, cypermethrin ŽExcis w , Vericore. and deltamethrin ŽAlphamax w , Alpharma.. The organophosphates, dichlorvos ŽAquagard w , Novartis. and azamethiphos ŽSalmosan w , Novartis. are also used, depending on the regulations of individual countries ŽRoth et al., 1993.. However, immersion treatments are impractical for use on exposed sites and during adverse weather conditions, and the procedures involved are very stressful to fish. With the exception of cypermethrin ŽJakobsen and Holm, 1990., bath treatments are only effective against pre-adult and adult stages of sea lice, allowing chalimus stages to survive and continue the cycle of infestation. Hydrogen peroxide may cause damage to the gills and its use is restricted in summer owing to its toxicity at higher water temperatures ŽThomassen, 1993; Bruno and Raynard, 1994.. Resistance to the organophosphate, dichlorvos has been identified in some populations of sea lice ŽJones et al., 1992.. As a result of these limitations, treatments are being developed that can be administered in feed. Two insect growth regulators, diflubenzuron ŽLepsidon w , Ewos. and teflubenzuron ŽCalicide w , Nutreco. ŽErdal, 1997. are available in Norway as in-feed treatments. Their mode of action is the inhibition of chitin synthesis ŽHorst and Walker, 1996. and activity is therefore restricted to the molting stages of sea lice. Ivermectin has also been used as an in-feed treatment ŽJohnson and Margolis, 1993; Smith et al., 1993; Palmer et al., 1997. but can only be prescribed in the UK under the cascade procedure where authorised products fail to provide effective control ŽAnonymous, 1998.. The efficacy of emamectin benzoate Ž4Y-deoxy-4Y epimethylaminoavermectin B1 . as an in-feed treatment for Atlantic salmon was initially demonstrated in a series of replicated tank trials employing induced infestations of L. salmonis ŽStone et al., 1999.. A dose of 50 mg kgy1 dayy1 for 7 consecutive days was found to be effective against both adult and immature stages of L. salmonis. The sea trials reported here evaluated the efficacy of emamectin benzoate under field conditions, where fish may be continuously exposed to re-infestation. Two trials carried
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out during the summer allowed evaluation of both L. salmonis and C. elongatus, the species commonly infesting farmed fish in the UK. These trials examined treatment of post-smolts in their first year at sea. The third trial evaluated efficacy in April when sea temperatures were lower and allowed assessment of larger fish in their second year at sea.
2. Materials and methods 2.1. Fish and holding conditions Three trials were carried out on commercial salmon farms on the northwest coast of Scotland. The trials were conducted on pontoons of pens stocked with Atlantic salmon, S. salar. Fish were selected from the main ongrowing pens at each site and acclimatised to small net pens of 12, 27 or 102 m3 as described in Experimental design. Fish were naturally infested with L. salmonis and C. elongatus and were exposed potentially to re-infestation by larval sea lice throughout the duration of the trial. In each trial, lice numbers were similar on fish in the surrounding commercial pens to those of the control fish, prior to the start of treatment. All control and treated fish sampled for sea lice evaluation were weighed at each time point Ž N s 20 or 30 fish.. 2.2. Medicated feed The basal ration used was Trouw UK ŽWincham, Cheshire, UK. 4.0 or 8.5 mm salmon feed pellets. Emamectin benzoate Ž4Y-deoxy-4Y epimethylaminoavermectin B 1 . was dissolved in propylene glycol and mixed with fish oil prior to coating the feed pellets. Control feeds were prepared with propylene glycol and fish oil only. Treatment was administered at a nominal dose of 50 mg kgy1 fish biomass per day. All fish were given an initial unmedicated feed at a rate of 0.1–0.25% biomass at the start of the day and medicated or control feeds were given at a rate of 0.4% or 0.5% biomass, 3 to 4 h later. Medicated feed was administered for a period of 7 consecutive days Ždays 0–6.. Feeding rates and methods were identical for all control and treated groups in each trial. 2.3. EÕaluation of sea lice At day y1 or y2, 20 or 30 fish were randomly selected from each pen to determine pre-treatment numbers of sea lice. Each fish was examined using a hand lens or low power microscope and all attached lice removed and fixed in 5% formalin. Lice were identified as L. salmonis or C. elongatus and the number of chalimus I, II, III, IV, pre-adult I, pre-adult II and adult stages recorded. Pre-adult and adult lice were further identified as males or females. Evaluation of sea lice numbers was carried out again at 7, 14 and 21 days from the start of treatment. In the third trial, additional counts of sea lice on five fish per pen in control pen II and treated pen I were conducted by site staff on days 27, 35, 42, 48 and 55. For these counts, chalimus were counted only up to a
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maximum of 100 per fish. The occurrence of sea lice damage was determined on sampled fish by visual assessment of typical epidermal lesions. 2.4. Experimental design The three trials were conducted blind with the identity of each treatment and control ration unknown to personnel responsible for feed administration and collection of data. Treatments were randomly allocated to each trial pen. 2.4.1. First trial Two pens, one control and one treated, were used, each holding 180 fish at the start of the trial. Each pen had a volume of 12 m3 and they were located on a pontoon midway between 28 commercial pens, each measuring approximately 1150 m3. All pens were stocked with fish of the same year class in February at a density of 6.0–6.6 kg my3 and the trial was started in August. The sea temperature over the trial period was 13.0–15.58C and salinity 35 ppt. None of the fish on the site had been treated previously for sea lice. On day 8, fish in the surrounding commercial pens were treated for sea lice with dichlorvos ŽAquagard w , Novartis.. The trial pens were surrounded by a fully enclosed tarpaulin for the duration of this treatment, and for 30 min post-treatment, to protect fish from exposure to the treatment compound, which was rapidly flushed from the site by high current speeds Žaverage 8–10 cm sy1 at 2 m depth.. Pre-treatment fish weights were 268 to 625 g Žmean 438 " 72 g S.D... The sample size for parasite enumeration was 30 fish per pen. 2.4.2. Second trial There were two control and two treated pens with 149 fish per pen at the start of the trial. Each pen had a volume of 27 m3 Žstocking density 2.8 kg m3 . and was located on the same pontoon described above with each commercial pen stocked at 6.7 kg m3. The pens were stocked in February and the trial started in September. The sea temperature over the trial period was 13.8–14.28C and salinity 33–35 ppt. Trial fish had been treated previously for sea lice with the organophosphate, dichlorvos ŽAquagard w , Novartis. 27 and 47 days before the start of the trial. Pre-treatment fish weights were 251 to 878 g Žmean 513 " 136 g S.D... The sample size for parasite enumeration was 20 fish per pen. 2.4.3. Third trial The trial consisted of two control and two treated pens with 360 fish per pen at a density of 9.4 kg m3. This trial used larger pens each with a volume of 102 m3 , located on a pontoon of 32 pens of similar size and stocking density. All pens were stocked with fish of the same year class in April of the previous year and fish were 1026–4464 g Žmean 2662 " 597 g S.D.. in weight. There were also three large commercial rearing units located within 1 km of this site. The trial started in April when the sea temperature was 7.2–8.58C and salinity 31–33 ppt. The fish had been treated for sea lice with dichlorvos ŽAquagard w . in the previous summer and with hydrogen peroxide 20 days before the start of the trial. The entire site, including the two control pens, but not the pens treated with emamectin benzoate, was subjected to a bath treatment with hydrogen
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peroxide on day 22 after the end of the trial Žday 21.. The sample size for parasite enumeration was 20 fish per pen. 2.5. Data handling Parasite counts were summarised as chalimus Žcopepodites, chalimus stages I, II, III and IV., motile lice Žpre-adult and adult stages. and total lice Žchalimus and motile stages combined. and the arithmetic means calculated. Parasite count data were subjected to F-tests for homogeneity of variances and a correlation test to examine the normality of distribution. Lice numbers were analysed using the non-parametric STP test ŽSokhal and Rohlf, 1981.. In the two replicated trials, the data were analysed separately for each replicate pen. The percentage reduction in mean total sea lice, over the trial period was calculated as follows:
ž
%Reductions 100 y 100 =
Mean of control or treated replicates at day y1 Mean of control or treated replicates at day 21
/
.
The efficacy or percentage reduction in mean total sea lice, relative to the control groups, was calculated as follows:
ž
%Efficacys 100 y 100 =
Mean of treated replicates Mean of the control replicates
/
.
Owing to the high standard deviation of fish weights and the small sample size, statistical analysis of fish weights was not appropriate in these trials. Specific growth rates ŽSGR. were calculated as follows: SGR s
log e Wt y log e W0 t
where Wt is the mean weight at day 21 and W0 is the mean weight at day y1 or y2.
3. Results 3.1. Feed consumption and fish weights In each trial, good feed consumption was observed in all treated and control pens during and after the medication period. In the first trial, the increase in mean fish weight over the trial period was 5.2 g ŽSGR 0.01%. in the control group and 91.0 g ŽSGR 0.8%. in the treated group. In the second trial, mean weights increased by 3.8 and 35.5 g ŽSGR 0.03% and 0.3%. in the control groups I and II, while treated pens I and II increased by 92.7 and 133.4 g ŽSGR 0.7% and 1.0%.. In the third trial, owing to high fish weights and hence a high standard deviation, the sample size used was too small to provide
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conclusive results on fish weights. No adverse effects or fish mortality were attributed to treatment with emamectin benzoate in any of the trials. 3.2. First trial (13.0–15.58C) 3.2.1. First trial: efficacy against L. salmonis The efficacy and mean numbers of L. salmonis per fish are shown in Table 1. Prior to the start of treatment, mean numbers of L. salmonis were similar in the two trial pens. L. salmonis numbers increased in the control group by 52% over the trial period. In the treated group, there was no corresponding increase and, from day y2 to 21, numbers decreased by 86%. At day 21, treated fish had fewer motile and chalimus stages than control fish. At the start of the trial, none of the fish examined was free of L. salmonis. At day 21, the number of L. salmonis on control fish ranged from 4 to 33, while the range on treated fish was 0–11 and 43% of treated fish sampled had no chalimus or motile stages present. 3.2.2. First trial: efficacy against C. elongatus The results for C. elongatus are shown in Table 2. Prior to the start of treatment, the mean numbers of C. elongatus were similar in the two trial pens. From day y2 to 21, the mean number of C. elongatus declined on control fish by only 36%. Over the same period, numbers fell on treated fish by 98%. At the start of the trial, none of the fish sampled was free of C. elongatus. At day 21, 10% of treated fish had no motile C. elongatus and 67% had no chalimus present whereas none of the control fish was free of chalimus or motile stages. Numbers of motile C. elongatus fell between days 7 and 14,
Table 1 First trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 30 fish per pen. Sea temperatures 13.0–15.58C Time
Nominal dose Žmg kgy1 .
Mean total lice Ž"S.D..
Mean chalimus Ž"S.D..
Mean motiles Ž"S.D..
Percent efficacy Žtotal lice.
0
9.1Ž"4.9.
4.4 Ž"3.2.
4.7 Ž"3.2.
50
10.0 Ž"5.3.
5.2 Ž"4.8.
4.8 Ž"2.6.
Day 7
0 50
17.0 Ž"6.0. 9.4 Ž"5.9.
5.8 Ž"2.7. 4.7 Ž"3.4.
11.3 Ž"5.0. 4.8 Ž"3.1.
45
Day 14
0 50
12.8 Ž"6.3. 2.5 Ž"2.3.
1.1 Ž"1.1. 0.5 Ž"0.9.
11.8 Ž"6.0. 1.9 Ž"2.1.
80
Day 21
0 50
13.8 Ž"8.3. 1.4 a Ž"2.3.
1.2 Ž"1.3. 0.2 Ž"0.5.
12.6 Ž"7.9. 1.2 Ž"2.0.
91
Day y2 Žpre-treatment.
a
Includes one fish with 11 lice.
–
J. Stone et al.r Aquaculture 186 (2000) 205–219
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Table 2 First trial: Efficacy of emamectin benzoate against natural infestations of C. elongatus on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 30 fish per pen. Sea temperatures 13.0–15.58C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. Žtotal lice.
Day y2 Žpre-treatment.
0 50
41.9 Ž"8.3. 41.3 Ž"12.0.
26.2 Ž"8.8. 20.7 Ž"5.9.
15.6 Ž"5.5. 20.6 Ž"8.8.
–
Day 7
0 50
39.3 Ž"10.6. 16.6 Ž"6.1.
21.3 Ž"6.5. 5.8 Ž"2.8.
17.9 Ž"6.1. 10.9 Ž"4.5.
58
Day 14
0 50
24.5 Ž"9.3. 2.6 Ž"3.2.
12.3 Ž"7.5. 1.0 Ž"2.3.
12.2 Ž"5.1. 1.5 Ž"1.3.
89
Day 21
0 50
26.8 Ž"13.4. 4.3 Ž"5.2.
14.9 Ž"9.4. 0.5 Ž"0.9.
11.9 Ž"6.4. 3.8 Ž"4.8.
84
following Aquagard w treatment of the commercial pens but numbers of chalimus had fallen between days 0 and 7, thus a decline in motile numbers was expected. At day 14, 23% of fish in the control group had lesions caused by L. salmonis and C. elongatus while none of the treated fish examined were affected by sea lice damage.
Table 3 Second trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 20 fish per pen. Sea temperatures 13.8–14.28C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y2 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
18.7 Ž"8.1. 20.4 Ž"8.5. 16.2 Ž"6.4. 26.9 Ž"9.4.
3.6 Ž"3.3. 6.5 Ž"4.1. 2.9 Ž"2.6. 8.3 Ž"4.2.
15.1 Ž"6.7. 13.9 Ž"5.7. 13.3 Ž"5.3. 18.6 Ž"8.5.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
30.3 Ž"7.7. 38.3 Ž"14.6. 10.3 Ž"5.4. 14.9 Ž"5.9.
11.5 Ž"5.8. 19.6 Ž"10.3. 2.4 Ž"1.8. 7.5 Ž"4.4.
18.8 Ž"4.8. 18.9 Ž"7.1. 7.9 Ž"4.5. 7.5 Ž"3.3.
63.3
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
31.7 Ž"8.2. 65.9 Ž"34.0. 2.3 Ž"1.7. 4.0 Ž"4.6.
15.6 Ž"5.2. 41.2 Ž"23.1. 0.8 Ž"1.0. 3.3 Ž"3.8.
16.2 Ž"5.2. 24.8 Ž12.6. 1.5 Ž"1.5. 0.8 Ž"1.1.
93.4
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
58.5 Ž"18.9. 92.1 Ž"39.7. 0.3 Ž"0.7. 0.8 Ž"1.2.
26.7 Ž"10.8. 41.5 Ž"26.7. 0 0.6 Ž"0.9.
31.8 Ž"10.7. 99.3 31.8 Ž"20.8. 0.3 Ž"0.7. 0.2 Ž"0.4.
212
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3.3. Second trial (13.8–14.28C) 3.3.1. Second trial: efficacy against L. salmonis The efficacy and mean numbers of L. salmonis per fish are shown in Table 3. Prior to the start of treatment, one pen intended for treatment had significantly Ž P - 0.05 and P - 0.01. more L. salmonis than one control pen and the other treated pen. Over the trial period, L. salmonis numbers increased by 284% in the two control groups. In the two treated groups there was no corresponding increase and, from days y2 to 21, numbers declined by 97% on treated fish. At days 7, 14 and 21 both treated pens had significantly Ž P - 0.01. fewer L. salmonis than fish in the two control pens. Although control group I had significantly Ž P - 0.05. fewer lice than control group II at day 21, both groups still had large numbers of lice present and there were no significant Ž P ) 0.05. differences between the two treated replicates at days 14 and 21. At day 21, individual control fish had 26–190 L. salmonis present but the highest number of L. salmonis found on any treated fish was 5 and 67.5% of treated fish had no L. salmonis present. At day 14, the two treated pens had means of only 0.6 and 2.6 copepodites per fish and there was less than 0.8 chalimus present. In contrast, all control fish had large numbers of copepodites and chalimus stages I, II, III and IV present. At day 21, the two control groups had high numbers of chalimus present but there were no surviving
Table 4 Second trial: Efficacy of emamectin benzoate against natural infestations of C. elongatus on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. Sample size N s 20 fish per pen. Sea temperatures 13.8–14.28C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y2 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
25.3 Ž"10.4. 29.2 Ž"7.1. 26.3 Ž"10.4. 39.1 Ž"7.6.
13.2 Ž"6.1. 15.8 Ž"6.3. 13.6 Ž"5.6. 22.5 Ž"7.7.
12.2 Ž"6.6. 13.4 Ž"4.4. 12.7 Ž"5.9. 16.6 Ž"5.5.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
20.3 Ž"8.4. 20.4 Ž"7.6. 8.2 Ž"8.2. 14.0 Ž"7.1.
6.5 Ž"3.4. 9.2 Ž"5.0. 3.2 Ž"2.3. 6.3 Ž"3.3.
13.8 Ž"7.3. 11.4 Ž"4.6. 5.1 Ž"2.7. 7.7 Ž"4.7.
45.6
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
11.0 Ž"3.8. 16.9 Ž"10.9. 1.9 Ž"2.1. 3.7 Ž"2.9.
4.8 Ž"2.8. 9.9 Ž"7.9. 0.6 Ž"0.8. 3.3 Ž"2.4.
6.2 Ž"2.8. 7.0 Ž"3.8. 1.3 Ž"1.6. 0.4 Ž"0.8.
79.9
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
10.5 Ž"4.2. 12.6 Ž"4.4. 1.3 Ž"1.2. 2.9 Ž"2.3.
4.6 Ž"2.3. 6.2 Ž"3.2. 0.6 Ž"0.6. 2.5 Ž"2.2.
5.9 Ž"2.9. 6.4 Ž"3.1. 0.7 Ž"0.8. 0.5 Ž"0.8.
81.9
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213
chalimus on fish in one treated pen and means of only 0.5 copepodites and 0.1 chalimus IV in the other treated pen. 3.3.2. Second trial: efficacy against C. elongatus At the start of the trial, there was a high mean number of C. elongatus in each pen ŽTable 4.. Treated pen II had significantly Ž P - 0.01. more C. elongatus than the other three pens prior to the start of treatment. Over the trial period, the mean number of C. elongatus declined by 57% on control fish and by 94% on fish in the two treated groups. Numbers in both treated pens were significantly Ž P - 0.01. lower than on control fish at days 14 and 21. At day 21, 84% and 100% of the immature stages remaining on treated fish were copepodites, whereas in the control groups, only 37% and 34% of the immature stages were copepodites. Prevalence of C. elongatus declined from 100% to 35% of treated fish with motile stages and 67.5% with chalimus, whereas none of the control fish sampled were free of chalimus or motile stages. At day y2, the majority of fish sampled in each trial pen had skin lesions caused by sea lice. At day 21, the prevalence of lice damage on treated fish had fallen to only 12.5%, while 77.5% of control fish were still affected.
Table 5 Third trial: Efficacy of emamectin benzoate against natural infestations of sea lice, L. salmonis, on Atlantic salmon, S. salar. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days Ždays 0–6.. The mean number of sea lice was determined on days y2, 7, 14 and 21. Sample size N s 20 fish per pen. Sea temperatures 7.2–8.58C Time
Nominal dose Mean total lice Mean chalimus Mean motiles Percent efficacy Žmg kgy1 . Ž"S.D.. Ž"S.D.. Ž"S.D.. relative to controls Žreplicate. Žtotal lice.
Day y1 Žpre-treatment. 0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
24.6 Ž"16.3. 25.7 Ž"13.4. 17.9 Ž"7.4. 24.9 Ž"9.5.
11.4 Ž"11.1. 11.5 Ž"6.9. 9.7 Ž"5.1. 7.7 Ž"4.7.
13.3 Ž"7.1. 14.1 Ž"8.2. 8.2 Ž"3.8. 17.3 Ž"6.8.
–
Day 7
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
23.3 Ž"14.7. 27.8 Ž"11.1. 13.5 Ž"5.9. 24.7 Ž"12.1.
10.5 Ž"9.4. 10.3 Ž"6.5. 7.1 Ž"4.3. 7.1 Ž"5.4.
12.8 Ž"7.0. 17.6 Ž"7.5. 6.4 Ž"2.8. 17.6 Ž"8.9.
25.4
Day 14
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
39.5 Ž"21.6. 38.1 Ž"14.3. 6.7 Ž"3.0. 13.3 Ž"5.1.
17.3 Ž"11.1. 18.0 Ž"9.9. 4.6 Ž"2.1. 5.7 Ž"3.9.
22.2 Ž"12.3. 74.2 20.1 Ž"8.1. 2.2 Ž"1.5. 7.6 Ž"3.7.
Day 21
0 ŽI. 0 ŽII. 50 ŽI. 50 ŽII.
60.9 Ž"26.1. 75.1 Ž"35.7. 5.6 Ž"4.9. 8.3 Ž"3.8.
35.6 Ž"19.2. 43.6 Ž"23.9. 4.3 Ž"3.4. 3.0 Ž"1.8.
25.4 Ž"9.2. 89.7 31.5 Ž"16.2. 1.3 Ž"2.0. 5.3 Ž"3.2.
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3.4. Third trial (7.2–8.58C) 3.4.1. Third trial: efficacy against L. salmonis Table 5 shows the efficacy and mean numbers of L. salmonis for the third trial. Prior to the start of treatment there were no significant Ž P ) 0.05. differences in lice numbers between pens.
Fig. 1. Ža and b. Third trial: Efficacy of emamectin benzoate against natural infestations of L. salmonis on Atlantic salmon. Fish received medicated feed at the rate of 0.5% biomass per day for 7 consecutive days. Sample size N s 20 fish per pen. Between days 27 and 55, after the trial period, only five fish in control pen II and treated pen I were evaluated. Chalimus were counted only up to a maximum of 100 per fish. Sea temperatures 7.2–8.58C from days y1 to 20 and 8.8–10.98C from days 22 to 55.
J. Stone et al.r Aquaculture 186 (2000) 205–219
215
Over the trial period, L. salmonis numbers increased by 170% in the two control groups. There was no corresponding increase on treated fish and, from days y2 to 21, lice numbers decreased by 68%. At days 14 and 21, both treated groups had significantly Ž P - 0.01. fewer L. salmonis than the two control groups. At day 21, the two treated groups still had a higher mean number of lice compared to the results in the two summer trials, although the mean numbers of lice on control fish were also much higher. The number of L. salmonis on any individual control fish was 28–154 while the range on treated fish was only 0–19. At day 21, 20% of fish in the control groups had characteristic lesions caused by sea lice while only 2.5% of treated fish were affected. At the end of the trial Žday 22., all the pens on the site, with the exception of the two pens treated with emamectin benzoate, were subjected to a hydrogen peroxide bath treatment for sea lice. The two pens treated with emamectin benzoate were not treated with hydrogen peroxide because of low lice numbers and improved appearance of the fish. Following treatment with hydrogen peroxide there was a decline in the mean number of motile lice on control fish at day 27 ŽFig. 1a., but chalimus were not reduced and increased to more than 100 per fish between days 35 and 55 ŽFig. 1b.. As a result, at day 55, mean motile lice numbers reached high levels again and a second hydrogen peroxide treatment had to be carried out 5 weeks after the previous treatment. After day 27, lice numbers on fish treated only with emamectin benzoate increased relatively slowly compared to the control group, and at day 55, treated fish still had 78% fewer chalimus and 86% fewer motile lice than control fish. Between days 22 and 55, fish in the surrounding commercial pens also had much higher numbers of lice than fish in the pens treated with emamectin benzoate. Prior to treatment, sea lice damage was recorded on only 0–5% of fish in each pen. At day 21, 20% of control fish had sea lice damage compared to only 2.5% on fish in the two treated groups. 3.4.2. Third trial: efficacy against C. elongatus There were few C. elongatus present during this trial. Mean numbers at the start of the trial were only 0.1–1.0 per fish and at day 21, the means were 0 and 0.8 per fish in the treated groups and 0 and 1.4 per fish in the control groups. 4. Discussion Despite the variability in fish weights at the start of the trial and hence, feed consumption, good efficacy was achieved and no adverse reactions or fish mortalities were attributed to treatment with emamectin benzoate. Treated groups had higher SGR than the control groups, but it is uncertain whether treatment resulted in significant benefits to fish growth as larger samples sizes would be required to detect statistically significant differences. Fish infested with L. salmonis have shown a decreased feed consumption correlated with the appearance of head lesions, although growth rates were unaffected ŽDawson et al., 1997.. No significant differences in growth were found by Grimnes and Jakobsen Ž1996. between fish infested with L. salmonis and un-infested fish, although heavily infested moribund fish had a lower condition factor. Observations from tank ŽStone et al., 1999. and field trials suggest there was an improved feed response in lice-infested fish following treatment with emamectin. Further, improve-
216
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ments in growth may be a long-term benefit of effective in-feed medication, as bath treatments require 3–4 days starvation and cause increased stress. Oral treatment of Atlantic salmon with emamectin benzoate showed good efficacy against both motile and chalimus stages of L. salmonis and C. elongatus in all three trials, despite the potential for continuous recruitment from adjacent commercial pens. L. salmonis numbers increased over time on control fish by 87–284%, whereas over the same period, numbers were reduced on treated fish by 68–98%. The first sea trial was conducted as a pilot study and replicate pens were not evaluated as they were in the second and third trials, however, useful data were obtained, particularly on C. elongatus, and for this reason, the results are included here. At the start of the second trial, one treated pen had significantly more L. salmonis and C. elongatus than the other pens. Despite this, fish in this pen still had significantly fewer lice than fish in the two control pens at days 14 and 21. Following treatment with emamectin benzoate, 55–80% of fish had no chalimus or motile L. salmonis present. While efficacy against chalimus stages is beneficial in preventing development to the motile stages ŽStone et al., 1999., a rapid reduction in motile lice numbers is also important, as they are more damaging to host fish. Motile lice numbers were reduced by 21–59%, as early as day 7, on treated fish. Oral treatment with ivermectin at a dose of 0.05 mg kgy1 every third day for three or six doses resulted in good efficacy against both chalimus and adult stages of L. salmonis ŽJohnson and Margolis, 1993.. However, ivermectin appears to have a narrow safety margin ŽJohnson et al., 1993; Palmer et al., 1997. while no adverse effects have been reported with emamectin benzoate at up to 3.5 times the therapeutic dose and no mortalities occurred at 7 times the therapeutic dose ŽRoy et al., in press.. Most of the treated fish, sampled at day 21, had fewer L. salmonis than any control fish. This suggests all fish received an efficacious dose, despite variability in fish weights and feeding competition. Most of the L. salmonis present on treated fish at day 21 were either copepodites or small numbers of mature adults. Copepodites represented recent settlement of larval stages and did not appear to develop further. The transfer of motile stages of L. salmonis between salmon has been recorded in tank and field trials ŽBruno and Stone, 1990; Ritchie, 1997; Treasurer and Grant, 1997. and it is possible that some adult lice on treated fish had transferred from untreated fish on the site. Treatment at low temperatures resulted in slower reductions in L. salmonis numbers and a small number of lice persisted on treated fish at day 21. However, treatment was still effective and treated groups had significantly fewer lice compared to control fish. Further sampling showed that lice numbers remained much lower on fish treated with emamectin benzoate for at least 55 days from the start of treatment. Although there was a gradual increase in chalimus numbers on treated fish, there was little increase in motile lice, suggesting that when chalimus settled on treated fish, their development to mature motile stages was impaired. This confirmed observations in tank trials where attached chalimus failed to develop on fish were treated with emamectin benzoate ŽStone et al., 1999.. The settlement of small numbers of copepodites on treated fish, without a corresponding increase in chalimus, also suggests newly recruited lice fail to develop further. Johnson and Margolis Ž1993. also reported slower development of lice on fish treated orally with ivermectin.
J. Stone et al.r Aquaculture 186 (2000) 205–219
217
C. elongatus numbers are present in greater numbers when sea temperatures are high ŽHogans and Trudeau, 1989; Tully, 1989.. In the two summer trials, mean numbers of C. elongatus were high initially and declined on control fish over time as sea temperatures fell. In the third trial, carried out in April, there were few C. elongatus present and these probably represented over-wintering populations. Treatment with emamectin benzoate was also effective against C. elongatus and numbers declined more rapidly on treated fish than on control fish. C. elongatus is not host-specific and may be derived from wild fish ŽWootten et al., 1982.. Motile stages of C. elongatus may transfer between individual salmon and, from infested saithe, Pollachius Õirens, to cohabiting salmon ŽBruno and Stone, 1990.. Transfer from wild and farmed fish in the area may account for the persistence of low numbers of motile C. elongatus on fish treated with emamectin. Treated fish also had low numbers of C. elongatus copepodites, although in contrast to the control fish, there were few chalimus present. Again, this suggests treatment prevented further development of C. elongatus copepodites. Bath immersion treatment of the commercial pens with Aquagard w , in the first trial, did not reduce numbers of motile L. salmonis on the control fish, confirming that the tarpaulins protected the trial fish. Hydrogen peroxide is not effective in removing chalimus stages of L. salmonis ŽMcAndrew et al., 1998. and this was evident from lice numbers following treatment of control fish in the third trial. As a result, although motile lice were reduced on control fish following peroxide treatment at day 22, their numbers rapidly increased again, necessitating another peroxide treatment only 33 days later. Motile lice exposed to hydrogen peroxide may recover ŽTreasurer and Grant, 1997; McAndrew et al., 1998. and could potentially re-infest fish and this may also have contributed to the rapid recovery of motile lice numbers on control fish. In each trial, the prevalence of lice damage increased on control fish over time, while the number of lesions on treated fish was reduced and, overall, lice damage was 15–75% lower on treated fish. In all three trials, treated pens were surrounded by large commercial stocks of fish heavily infested with sea lice, creating a large potential source of copepodite stages. Despite this, very good efficacy was achieved against both L. salmonis and C. elongatus and development of newly recruited larval lice was prevented. When entire sites are treated, the potential for re-infestation will be reduced and the recovery of lice numbers may take even longer, allowing the frequency of treatments to be reduced. In-feed treatments will also permit simultaneous medication of all pens on a site and all sites in a single area, thus reducing any cross-infestation that may occur during the time necessary to apply bath treatments to all pens. 5. Conclusion Oral treatment with emamectin benzoate resulted in reductions of both species of lice commonly infesting farmed salmon and, in one study, lice numbers remained low until the final observation at day 55. Treatment was also effective at low temperatures and in this study, emamectin benzoate was more effective than hydrogen peroxide in reducing lice numbers and in preventing the development of newly recruited lice.
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Acknowledgements The authors wish to acknowledge the help of Ronnie Soutar and the staff at the Norsk Hydro farm at Fishnish, Isle of Mull, and Andrew Grant and the staff at the Marine Harvest McConnell field trial unit at Loch Duich. We would also especially like to thank Cam Matthews, Obdulio Andrade-Salas and Dr. James Bron for their support and technical assistance. This work was funded by Schering-Plough Animal Health, the Scottish Salmon Growers Association, and the Crown Estates Commissioners.
References Anonymous, 1998. Amelia No. 8. Veterinary Medicines Directorate, Woodham Lane, Newhaw, Addlestone, Surrey KT15 3NB. Bruno, D.W., Raynard, R.S., 1994. Studies on the use of hydrogen peroxide as a method for the control of sea lice on Atlantic salmon. Aquacult. Int. 2, 10–18. Bruno, D.W., Stone, J., 1990. The role of saithe, Pollachius Õirens, as a host for the sea lice, Lepeophtheirus salmonis and Caligus elongatus. Aquaculture 89, 201–207. Dawson, L., Pike, A.W., Houlihan, D., McVicar, A.H., 1997. Appetite suppression in Atlantic salmon Salmo salar ŽL.. infected with sea lice Lepeophtheirus salmonis Kroyer. In: Houlihan, D., Kiessling, A., Boujard, T. ŽEds.., Voluntary Food Intake in Fish. COST 827 Workshop, Aberdeen ŽUK. 3–6 Apr. 1997. Erdal, J.I., 1997. New drug treatment hits sea lice when they are most vulnerable. Fish Farming Int. 24 Ž2., 9. Grimnes, A., Jakobsen, P.J., 1996. The physiological effects of salmon lice infection on post-smolt Atlantic salmon. J. Fish Biol. Ž48., 1179–1194. Hogans, W.E., Trudeau, D.J., 1989. Preliminary studies on the biology of sea lice, Caligus elongatus, Caligus curtus and Lepeophtheirus salmonis parasitic on cage-cultured salmonids in the lower Bay of Fundy. Can. Tech. Rep. Fish. Aquat. Sci. 1715, 14. Horst, M.N., Walker, A.N., 1996. Biochemical effects of diflubenzuron on chitin synthesis in the post-molt blue crab Ž Callinectes sapidus .. J. Crustacean Biol. 15, 401–408. Jakobsen, P.J., Holm, J.C., 1990. Promising test with new compound against salmon lice. Norsk Fiskeoppdrett., 16–18, January. Johnson, S.C., Kent, M.L., Whittaker, D., Margolis, L., 1993. Toxicity and pathological effects of orally administered ivermectin in Atlantic, chinook and coho salmon and steelhead trout. Dis. Aquat. Org. 17, 107–112. Johnson, S.C., Margolis, L., 1993. Efficacy of ivermectin for control of the salmon louse, Lepeophtheirus salmonis, on Atlantic salmon. Dis. Aquat. Org. 17, 101–105. Jones, M.W., Sommerville, C.S., Wootten, R., 1992. Reduced sensitivity of the salmon louse, Lepeophtheirus salmonis, to the organophosphate dichlorvos. J. Fish Dis. 15, 197–202. McAndrew, K.J., Sommerville, C., Wootten, R., Bron, J.E., 1998. The effects of hydrogen peroxide on different life-cycle stages of the salmon louse, Lepeophtheirus salmonis. J. Fish Dis. 21, 221–228. Palmer, R., Coyne, R., Davey, S., Smith, P., 1997. Case notes on adverse reactions associated with ivermectin therapy of Atlantic salmon. Bull. Eur. Assoc. Fish Pathol. 17 Ž2., 62–67. Ritchie, G., 1997. The host transfer ability of Lepeophtheirus salmonis ŽCopepoda: Caligidae. from farmed Atlantic salmon, Salmo salar L. J. Fish Dis. 20, 153–157. Roth, M., Richards, R., Sommerville, C., 1993. Current practices in the chemotherapeutic control of sea lice infestations: a review. J. Fish Dis. 16, 1–26. Roy, W.R., Rodger, H., Sutherland, I.H. Tolerance of Atlantic salmon Ž Salmo salar . and rainbow trout Ž Oncorhynchus mykiss . to emamectin benzoate, a new orally administered treatment for sea lice. Aquaculture, in press. Smith, P.R., Moloney, M., McElligott, A., Clarke, S., O’Brien, F., 1993. The efficiency of oral ivermectin in
J. Stone et al.r Aquaculture 186 (2000) 205–219
219
the control of sea lice infestations of farmed Atlantic salmon. In: Boxsall, G., DeFaye, D. ŽEds.., Pathogens of Wild and Farmed Fish: Sea Lice. Ellis Horwood, Chichester, pp. 296–307. Sokhal, R., Rohlf, F.J., 1981. In: Biometry. 2nd edn. Freeman, New York, pp. 437–440. Stone, J., Sutherland, I.H., Sommerville, C.S., Richards, R., Varma, K.J., 1999. The efficacy of emamectin as an oral treatment of sea lice, Lepeophtheirus salmonis, infestations in Atlantic salmon, Salmo salar ŽL... J. Fish Dis. 22, 1–10. Thomassen, J.M., 1993. Hydrogen peroxide as a delousing agent for Atlantic salmon. In: Boxsall, G., DeFaye, D. ŽEds.., Pathogens of Wild and Farmed Fish: Sea Lice. Ellis Horwood, Chichester, pp. 290–295. Treasurer, J.W., Grant, A., 1997. The efficacy of hydrogen peroxide for the treatment of farmed Atlantic salmon Salmo salar L. infested with sea lice ŽCopepoda: Caligidae.. Aquaculture 148, 265–275. Tully, O., 1989. The succession of generations and growth of the caligid copepods Caligus elongatus and Lepophtheirus salmonis parasitising farmed Atlantic salmon smolts Salmo salar ŽL... J. Mar. Biol. Assoc. U. K. 69, 279–287. Wootten, R., Smith, J.W., Needham, E.A., 1982. Aspects of the biology of the parasitic copepods L. salmonis and C. elongatus on farmed Atlantic salmonids and their treatment. Proc. R. Soc. Edinburgh, Sect. B: Biol. Sci. 81, 185–197.