Deltamethrin resistance in the sea louse Caligus rogercresseyi (Boxhall and Bravo) in Chile: bioassay results and usage data for antiparasitic agents with references to Norwegian conditions



The sea louse Caligus rogercresseyi is a major threat to Chilean salmonid farming. Pyrethroids have been used for anticaligus treatments since 2007, but have shown reduced effect, most likely due to resistance development. Pyrethroid resistance is also a known problem in Lepeophtheirus salmonis in the Northern Hemisphere. This study describes the development of deltamethrin resistance in C. rogercresseyi based on bioassays and usage data for pyrethroids in Chilean aquaculture. These results were compared to bioassays from L. salmonis from Norway and to Norwegian usage data. Available deltamethrin bioassay results from 2007 and 2008, as well as bioassays from Norway, were collected and remodelled. Bioassays were performed on field-collected sea lice in region X in Chile in 2012 and 2013. Bioassays from 2007 were performed prior to the introduction of pyrethroids to the Chilean market. Both the results from 2008 and 2012 showed an increased resistance. Increased pyrethroid resistance was also indicated by the increased use of pyrethroids in Chilean aquaculture compared with the production of salmonids. A similar trend was seen in the Norwegian usage data. The bioassay results from Chile from 2012 and 2013 also indicated a difference in the susceptibility to deltamethrin between male and female caligus.


Sea lice (Copepoda, Caligidae) are a major concern for salmonid aquaculture worldwide. In Chile, Caligus rogercresseyi (Boxhall and Bravo) is the parasite which is causing the greatest concern, while the salmon louse, Lepeophtheirus salmonis (Krøyer), is the most important sea louse of farmed salmonids in the Northern Hemisphere (Johnson et al. 2004). Sea lice are both a health and welfare problem for farmed fish. In the Northern Hemisphere, sea lice may affect wild salmonids if effective control measures are not implemented in the fish farms (Costello 2009a; Jackson et al. 2013). Sea lice cause skin lesions that can lead to secondary infections, as well as acting as vectors for other infections. In addition, sea lice can cause both stress and discomfort for the fish (Mackinnon 1993; Nolan, Reilly & Bonga 1999; Nolan et al. 2000; Johnson et al. 2004; Costello 2006). For the fish farmer, sea lice are a major economic burden. In addition to the substantial cost of sea lice treatments, skin lesions and reduced weight gain may reduce the slaughter value of the fish. (Carvajal, Gonzalez & George-Nascimento 1998; Bravo 2003; Costello 2009b). Environmental costs are also an issue due to the fact that most sea lice treatments include the release of treatment agents into the surrounding area. These agents are administered because they are toxic to the parasites, thus possibly placing non-target organisms also at risk (Clark et al. 1989; Ernst et al. 2001; Burridge et al. 2010).

In Chilean salmonid farming, parasitic copepods have been an issue for more than 30 years. The first identified sea louse was Caligus teres (Wilson) in 1983 (Reyes & Bravo 1983) followed by reports of Caligus flexispina (Lewis) from farmed salmonids in 1994 (González & Carvajal 1994). Finally C. rogercresseyi was described in 2000 (Boxshall & Bravo 2000). Today, C. rogercresseyi is the sea louse that represents the most prominent problem. C. rogercresseyi is not host specific and has hosts among a range of wild fish (e.g. Eleginops maclovinus, Odonthestes regia, Paralichthys microps) as well as farmed salmonid fish (Carvajal et al. 1998; Bravo et al. 2006; Gonzalez et al. 2012). The parasite has also been found in farmed tilapia (Oreochromis mossambicus) (Bravo, Boxshall & Conroy 2011).

Close location of fish farms can result in the spread of sea lice copepodits causing infection in other farms (Molinet et al. 2011; Bravo, Nuñez & Silva 2013). If treatments are not synchronized, post-treatment reinfection may quickly occur. The temperature of the sea water in Southern Chile facilitates a rapid life cycle for the parasite. This combined with the parasites reproducing capacity increases the challenges presented by the parasites (Bravo, Erranz & Lagos 2009a; Bravo 2010b). C. rogercresseyi is also suggested to have played a part in one of the other major salmon infections of recent times, infectious salmon anaemia (ISA) (Valdes-Donoso et al. 2013). C. rogercresseyi may possibly have been both a vector for the pathogen as well as compromising the immune system of the fish making them more susceptible to other infections (Braden et al. 2012).

Control of sea lice in Chile is mainly dependent on chemical treatments and has through the years been dependent on one treatment agent at a time. From the end of the 1990s throughout 2007, emamectin benzoate (EMB) was the leading agent used in sea lice treatments (Bravo, Silva & Monti 2012), while the pyrethroids deltamethrin and cypermethrin have dominated from 2008 till 2012. Both reliance on chemical treatments alone and the use of a sole treatment agent are important factors in the development of sea lice resistance towards chemotherapeutants (Denholm et al. 2002). In L. salmonis, resistance to EMB, pyrethroids, organophosphates and hydrogen peroxide has been reported (Roth et al. 1996; Treasurer, Wadsworth & Grant 2000; Sevatdal & Horsberg 2003; Jones et al. 2012). In C. rogercresseyi, resistance to EMB has been reported since 2008 (Bravo, Sevatdal & Horsberg 2008; Bravo et al. 2013). Signs of resistance to pyrethroids have been seen since 2008, and one report exists on low treatment efficacy on a farm in region XI (Bravo et al. 2013). In 2013, the government permitted the use of the organophosphate azamethiphos as an anticaligus treatment due to the reduced effect of other anti–sea lice agents.

Reduced treatment efficacy may arise as a result of suboptimal treatments or due to parasite resistance towards the agent. Resistance monitoring using biological, biochemical and molecular methods is employed to distinguish between the two causes. If the genetic cause of resistance is unknown, as is the case for pyrethroid-resistant C. rogercresseyi, toxicological tests called bioassays may be used to monitor resistance (Denholm et al. 2002).

The aim of this study was to retrospectively examine the development of deltamethrin resistance in the sea lice C. rogercresseyi and to look for potential gender differences in deltamethrin susceptibility, in region X (41°46′S72°56′W; 43°07′S73°38′W) in Chile, from the time the chemical was introduced until 2013. Available bioassay results and usage data for antiparasitic treatment agents were used to achieve this aim, and these results were compared with bioassay results from known sensitive and resistant strains of L. salmonis from Norway and with Norwegian usage data.

Materials and methods

Sea lice

The sea lice for the Chilean bioassays from 2007, 2008, 2012 and 2013 came from six different salmonid farms in region X in Chile. The bioassays from 2007 were performed prior to the introduction of pyrethroids in Chile, while all the other bioassays were performed in farms with reduced effect of pyrethroid treatments. In 2012 and 2013, six of the eight 24-h bioassays were performed using sea lice from the same farm. During this period, three deltamethrin treatments were applied at the farm: one between the first and the second assay and two between the second and the third bioassay. The two 30-min bioassays from 2012 were performed on sea lice from two of the same farms as the 24-h bioassays. The parasites were collected from sedated Atlantic salmon, Salmo salar (Linnaeus), at the sea farms, transported in cooled sea water to the laboratory and exposed to deltamethrin in bioassays within 5 h. For two of the bioassays in 2008, gravid females were collected at the sea farms and brought to the Aquaculture Institute of the Universidad Austral de Chile in Puerto Montt. The egg strings were then hatched, and sea lice were cultivated on rainbow trout, Oncorhynchus mykiss (Walbaum). Adult sea lice were used in all the bioassays.

30-min bioassays

The three 30-min bioassays from 2007 and the three from 2008 were performed according to Sevatdal & Horsberg (2003). The concentrations of deltamethrin used were between 0 and 3 μg L−1. The concentrations were achieved by preparing a work solution with the formulation AMX (deltamethrin 10 mg mL−1, Pharmaq AS) in sea water and subsequently diluting this in fresh sea water to achieve the correct concentrations. The 30-min bioassays in 2012 were also performed according to Sevatdal & Horsberg (2003), with the exception that the concentration range was between 0 and 14 μg L−1. All bioassays were performed in polystyrene boxes with approximately 10 sea lice in each box. Two boxes of parasites were exposed to each concentration. Upon completion of the 30-min exposure to deltamethrin, rinsing and subsequent holding at 12 degrees Celsius for 24-h in fresh sea water, the parasites were observed and characterized according to gender (in 2008 and 2012) and as inactivated (not able to suck to the surface of the box or swim in a straight line) or alive. One of the results from 2007 and two of the results from 2008 have previously been presented in a dissertation thesis (Bravo 2010a) as well as on a poster (Bravo & Sevatdal 2009b). These data were remodelled for the current study.

24-h bioassays

The 24-h bioassays in 2012 and 2013 were performed according to Helgesen and Horsberg (2013) with slight modifications. Approximately 1 L of filtered (100-μm filter) sea water was filled into six one-litre glass bottles (item number 215-1786, VWR). Sea lice were picked from the fish and distributed randomly to the glass bottles until all bottles held approximately 30 parasites. Gender of the parasites was not taken into account when distributing the parasites to the bottles. The sea lice were kept in a cooler until the test was initiated. The time from when the first sea louse was picked until the test started was between 2.5 and 5 h. The test started with adjusting the water level in each bottle to exactly 1 L. Water was subsequently taken out from the bottles and replaced with the same volume of working solution. The working solution was prepared in a polystyrene bottle by adding 20 μL AMX to 1 L filtered sea water. The bottle containing the working solution was vigorously stirred, and the appropriate amounts were immediately added to the respective glass flasks. Concentrations used in the bioassays varied between 0 and 4 μg L−1. Following the addition of deltamethrin to the various glass bottles, the water was stirred and aerated with an aquarium pump through an air hose and a long cannula through the lid of the bottles. The bottles were kept for 24 h in a cooling cabinet at 12 °C.

The results of the bioassays were read by turning the bottles upside down three times and then moving them in a circle with a diameter of 20 cm ten times, before all the water was poured out into a plastic beaker with a square base. This procedure was repeated once. The beaker was tilted slightly and the parasites were gently pushed to the bottom. The lice that were still attached to the walls of the glass bottles or the beaker and those that swam in a straight line were considered alive. The rest of the lice were considered inactivated or dead. The gender of the sea lice was also recorded.

Bioassays from Norway

The Chilean bioassay results were compared to bioassays of L. salmonis from Norway. To represent the different degrees of sensitivity to pyrethroids, the Norwegian assays were selected from a larger pool of data, based on similarities in methodology. Thus, they are not fully representative for the country as a whole. The 2000 results were presented in the study by Sevatdal et al. (2005a), and the results from LS A, LS B and LS R came from the study by Helgesen & Horsberg (2013). All bioassays were performed on pre-adult L. salmonis. The results from LS A, LS B and LS R from 2009 to 2012 were from sea lice from three different farms, all strains tested both in a 30-min bioassay and in a 24-h bioassay. The bioassays from 2000 were performed on sea lice picked directly from the fish farm, while the other bioassays were from sea lice strains held in a continuous culture in the laboratory. All data were remodelled for the current study.

Usage of pyrethroids for sea lice treatments

The data for usage of antiparasitic agents between 2007 and 2012 in Chilean aquaculture were kindly provided by the National Service for Fisheries and Aquaculture, Chile (Sernapesca). The production of Atlantic salmon and rainbow trout in tons round weight between 2007 and 2012 was also provided by Sernapesca (2013).

The data for usage of antiparasitic agents between 1998 and 2012 in Norwegian aquaculture were provided by the Norwegian Institute of Public Health (Norwegian Institute of public Health 2013). The production data for salmonids in Norwegian aquaculture were provided by Statistics Norway (2013).

The treatment doses for bath treatments were calculated based on the assumption that during treatment, there were 50 kg fish m−3 water and using the following dosage: cypermethrin 0.015 g m−3 water, deltamethrin 0.003 g m−3 water, azamethiphos 0.1 g m−3 water and hydrogen peroxide 1500 g m−3 water. The density of the fish during bath treatments was provided by fish health personnel based on field experience. The treatment doses for the oral treatments were calculated using the recommended dosage for one complete treatment: diflubenzuron 42 mg kg−1, teflubenzuron 70 mg kg−1 and EMB 0.35 mg kg−1.


All bioassay data were modelled using probit modelling in JMP (SAS Institute Inc.), and EC50 values (the concentration immobilizing 50% of the parasites) were calculated. Ninety-five per cent confidential intervals were subsequently calculated. Results from male and female sea lice were calculated collectively apart from the 24-h bioassays from Chile, where the results were calculated for each gender separately. All bioassays had a control group mortality of <20%, except for one bioassay from Chile in 2007 and one 24-h bioassay from Chile in 2012.

The Cochran–Mantel–Haenszel chi-square test (CMH test) with continuity correction was used to compare the mortality of male and female sea lice in each of the bioassays where data on gender was available.


The 30-min bioassays from 2007 showed EC50 values of 0.14, 0.17 and 0.24 μg L−1, while the ones from 2008 showed EC50 values of 0.70, 1.8 and 1.9 μg L−1. The two bioassay results from 2012 showed EC50 values of 0.98 and 1.5 μg L−1. The 30-min bioassays from Norway had EC50 values between 0.06 and 2.8 μg L−1. LS A is from a remote farm without any pyrethroid treatment history. Therefore, it cannot be seen as representative for the whole country. The results are presented in Fig. 1.

Figure 1.

Results from bioassays with 30-min exposure to deltamethrin performed at two sites in region X in Chile in March 2007, two sites in the same region in October and November 2008 and two sites in December 2012. These bioassays were performed on adult Caligus rogercresseyi picked directly from the sites, apart from the two last results from 2008 which were performed on cultivated sea lice. The Norwegian bioassays were performed on pre-adult sea lice taken from different sites. These results are from Lepeophtheirus salmonis picked directly from site in 2000 and cultivated in the laboratory for the other results. LS R and LS B were laboratory strains with known reduced sensitivity to deltamethrin, while LS A was a laboratory strain fully sensitive to deltamethrin. The dots indicate the EC50 level, and the lines on both sites indicate the 95% confidential interval.

The six EC50 values from the 24-h bioassays from 2012 and 2013 from the same farm varied between 0.46 and 1.5 μg L−1 for the male sea lice and 1.0 and 2.1 μg L−1 for the female sea lice. The results from the other two farms were EC50 values of 0.19 and 0.5 μg L−1 for the male sea lice and 0.61 and 0.58 μg L−1 for the female sea lice. The results from Norway on pre-adult L. salmonis showed EC50 values of 0.26 and 0.46 μg L−1 from LS B and LS R and 0.01 μg L−1 from LS A. LS A is from a remote farm without any history of treatment with pyrethroids. Therefore, it cannot be considered as representative for the whole country. The results are presented in Fig. 2.

Figure 2.

Results from bioassays with 24-h exposure to deltamethrin performed at three sites in region X in Chile between December 2012 and February 2013. The bioassays from Norway are performed on sea lice from three different sites. These results are from laboratory cultivated pre-adult Lepeophtheirus salmonis, while the bioassays from Chile were performed on adult male Caligus rogercresseyi picked directly at the sites. LS B and LS R are strains with known reduced sensitivity to deltamethrin, while LS A was a fully sensitive strain. The dots indicate the EC50-level, and the lines on both sites indicate the 95% CI. An * indicates that the results are from the same farm. This farm was treated with pyrethroids three times in this period; once between the first and the second bioassay and two times between the second and third bioassay. The results are presented chronologically in the order the bioassays were performed.

The sensitivity data for male and female C. rogercresseyi indicated a difference in sensitivity between the sexes. Using the Cochran–Mantel–Haenszel test with continuity correction for all bioassays where parasite gender was recorded, gender had a significant (P < 0.05) effect on the combined results for both the 30-min and 24-h bioassays from Chile performed in 2012 and 2013. For the individual bioassays, the gender effect was significant (P < 0.05) for 5 of 8 of the 24-h bioassays. Females were more resistant than males. No gender effect was demonstrated in the 2008 bioassays. Gender was not recorded in 2007. Based on the data from Norway where gender was recorded, no significant effect was demonstrated. The results from Chile are presented in Fig. 3 and Table 1.

Table 1. The results shown are P-values from Cochran–Mantel–Haenszel (CMH) tests performed to assess gender differences in susceptibility to deltamethrin in bioassays with Caligus rogercresseyi
Cochran–Mantel–Haenszel test with continuity correction
BioassaysGender recordedP-value
  1. In all bioassays with significant difference (P < 0.05), female sea lice were more resistant than males.

  2. The tests were performed with continuity correction on all the bioassays where parasite gender was recorded.

  3. CMH tests were also performed on all bioassay results classified according to time period and type of bioassay.

  4. The sites are from region X and the dates of the tests are given.

  5. Statistical significant differences are marked with an asterisk.

30-min 2007
Site A, 08.03.2007No 
Site A, 08.03.2007No 
Site B, 12.03.2007No 
30-min 2008
Site C, 27.10.2008Yes0.16
Site C, 03.11.2008Yes0.51
Site D, 05.11.2008Yes0.93
2008 combined 0.11
30-min 2012
Site A, 05.12.2012Yes0.07
Site E, 13.12.2012Yes0.14
2012 combined 0.01*
24-h 2012/2013
Site A, 04.12.2012Yes<0.01*
Site E, 06.12.2012Yes<0.01*
Site A, 06.12.2012Yes<0.01*
Site F, 20.12.2012Yes<0.01*
Site A, 22.01.2013Yes0.12
Site A, 29.01.2013Yes0.01*
Site A, 02.02.2013Yes0.11
Site A, 03.02.2013Yes0.55
24-h 2012/213 combined <0.01*
Figure 3.

The figure shows EC50 values from 24-h bioassays performed on male and female Caligus rogercresseyi from three different sites in region X in Chile in December 2012 to February 2013. Male and female sea lice were exposed to deltamethrin in the same bottles, and the gender was determined following the exposure period.

In Chile, the pyrethroid deltamethrin was introduced in 2007, while the introduction of cypermethrin occurred in 2010. Combined use of the pyrethroids was 105 kg in 2008; this declined in 2009 and 2010, but increased substantially in 2011 and 2012 to 382 and 874 kg, respectively. The use of EMB peaked in 2007, but declined thereafter. The chitin synthesis inhibitor diflubenzuron was to a certain extent used between 2008 and 2012. The organophosphate azamethiphos was introduced in Chile in 2013. Hydrogen peroxide was not used in the period 2007–2012. The production of salmon and rainbow trout in Chile declined between 2008 and 2010 due to outbreaks of infectious salmon anaemia, but production levels increased again in 2011 and 2012.

In Norway, the pyrethroid cypermethrin was introduced in 1996, while the introduction of deltamethrin took place in 1998. Combined usage of the pyrethroids peaked in 2012 with 353 kg. Usage of EMB reached its peak in 2011 and declined substantially in 2012. Chitin synthesis inhibitors were most frequently used in 2009, 2010 and 2012. The organophosphate azamethiphos was reintroduced in Norway in 2008 and usage peaked in 2012. Hydrogen peroxide was reintroduced in 2009 and its use peaked in 2011. The production of salmonids in Norway has steadily increased over the years. The usage and production data are presented in Table 2.

Table 2. The table shows the use of antiparasitic agents in Chilean and Norwegian aquaculture in kilograms active substance between the years 2007–2012 (Chile, data provided by Sernapesca) and 1998–2012 (Norway, data provided by the Norwegian Institute of Public Health)
CHILE (kg)         200720082009201020112012
Azamethiphos         000000
Hydrogen peroxide         000000
Diflubenzuron         01623878363928152167
Teflubenzuron         000000
Cypermethrin         00029.7341.6677
Deltamethrin         5.2105.231.734.339.9197
Emamectin benzoate         906285654749219
Salmon and trout production (tonnes)         493 448538 258353 570343 477488 813660 990
NORWAY (kg)199819992000200120022003200420052006200720082009201020112012
  1. It also shows the production of Atlantic salmon and rainbow trout in Chile (data from Sernapesca) and the production of Atlantic salmon, rainbow trout and arctic char in Norway (data from Statistics Norway) in the same time period.

  2. The 2012 production data are preliminary for both countries.

Hydrogen peroxide00000000000308 0003 071 0003 144 0002 538 000
Emamectin benzoate03.5301220233239607381412210536
Salmon, trout and char production (tonnes)409 238473 848488 840506 882546 373578 747627 639650 667693 488822 080823 428937 401994 6051 124 6151 312 274

In Chile, the total biomass of Atlantic salmon and rainbow trout treated for C. rogercresseyi has been higher than the slaughter volume for each of the given years. In other words, the fish have been treated several times each year. Since 2008, the majority of fish biomass has been treated with pyrethroids and the relative treatment intensity has increased. This is evident in Fig. 4. In 2012, the biomass of salmonid fish treated with pyrethroids was more than nine times the slaughter volume of these species.

Figure 4.

The graphs show the combined production of Atlantic salmon and rainbow trout in Chile (solid line), the biomass of these species treated against sea lice with all available treatments (stippled line) and with pyrethroids only (dotted line) per year between 2007 and 2012. See text for assumptions made in the calculations.

In Norway, the total biomass of Atlantic salmon, rainbow trout and arctic char treated for salmon lice (L. salmonis) has been approximately the same level as the slaughter volume up until 2007. From 2008, the treated biomass has been higher than the slaughter volume, indicating that the fish have been treated several times each year. In 2012, salmonid fish biomass treated against salmon lice was close to four times the slaughter volume of these species. Approximately 50% of the treated biomass was treated with pyrethroids, as shown in Fig. 5.

Figure 5.

The graph shows the combined production of Atlantic salmon, rainbow trout and arctic char in Norway (solid line), the biomass of these species treated against sea lice with all available treatments (stippled line) and with pyrethroids only (dotted line) per year in the time range 1998 and 2012. See text for assumptions made with respect to calculations.


Due to the fact that the data available from 2007 and 2008 originated from region X in Chile, this region was chosen as the main area for this study. Furthermore, this region is one of the two largest salmonid-producing regions in Chile (Sernapesca 2013). With respect to Norway, only selected bioassay results were included for comparison with the results from Chile. In this article, bioassay results are compared between sea lice collected from sea farms and laboratory reared sea lice, between results obtained at different years and between different countries and different species of sea lice. This was considered valid because the same type of equipment, the same chemical treatment agent and the same two protocols for bioassays were used. In the field, parasites are found on salmonid species and exposed to the same treatment agent in the same concentrations in both countries. Threshold values for sea lice resistance will therefore be expected to be similar in the two countries. The sea lice for the bioassays came from sites with differences in temperature and salinity of the sea water (data not recorded). All bioassays were, however, performed at 12 °C. As all bioassays except two showed a control group mortality of <20%, this procedure was considered acceptable.

With regard to C. rogercresseyi, native sensitivity was determined based on samples which were collected prior to the introduction of pyrethroids in Chile. Native sensitivity level in L. salmonis was determined based on samples which were collected shortly after the introduction of pyrethroids in Norway. The EC50 values for both of these species were very similar, ranging from 0.14 to 0.24 μg L−1 (C. rogercresseyi) and 0.06 to 0.22 μg L−1 (L. salmonis), even though the results were obtained from two different copepods and from two different life stages (pre-adult L. salmonis and adult C. rogercresseyi). Resistance levels given as EC50 values may not be compared accurately between the two countries, but as the parasites experience the same challenge regarding concentration of deltamethrin in a regular field sea lice treatment, EC50 values necessary for resistance is believed to be in the same range.

In Chile, the results for pyrethroids showed a 3–13 times decrease in sensitivity to deltamethrin from 2007 to 2008 when compared to the EC50 values from the bioassays. The 2008 bioassays were performed in farms reporting treatment failures approximately 1.5 years after the first bioassays (Bravo 2010a). These bioassays are, however, not an exact measurement of resistance development in region X due to the fact that each assay was performed on a limited number of parasites from one farm. The limitation in the number of parasites included in each bioassay is caused by practical issues concerning bioassay equipment and handling procedures.

The Chilean bioassay results from 2008 were from two different farms, with one result from sea lice used in a bioassay directly from the farm and one from first generation of cultivated sea lice from the same farm. The last result was from cultivated sea lice from another farm. The EC50 result from the cultivated sea lice was 2.5 times higher than from sea lice collected directly at the site. There was no control group mortality in either of the bioassays to explain the difference. The difference might therefore be explained by different sensitivity in the two sea lice samples taken for the bioassays (Robertson et al. 1995). Adult sea lice from sea farms were most likely of variable age, while adults from cultivated sea lice were of the same age. There is no knowledge on differences in susceptibility to deltamethrin between adult C. rogercresseyi of different ages, although such a difference is unlikely. All the results from 2008 show however a decreased sensitivity level towards deltamethrin compared with the values from 2007.

In Norway, decreased sensitivity towards pyrethroids was first observed in L. salmonis in 1998 (Sevatdal & Horsberg 2000), 4 years after pyrethrin and 2 years after pyrethroids (cypermethrin) were introduced to the market (Grave et al. 2004). Resistance to EMB was discovered in Chile in 2006, more than 6 years after the substance was introduced to the market (Bravo et al. 2008).

The results from 30-min bioassays conducted in 2012 showed no increase in resistance compared with the results from 2008, but between 4 to 10 times as resistant sea lice as in 2007. Unfortunately, no bioassay results from 2009, 2010 or 2011 were available. However, pyrethroid resistance might possibly have developed in both strength and extent between 2008 and 2012, without these limited bioassay results illustrating this development. Due to the limited number of assays, variation in sensitivity could not be determined. Despite this, the increase in use of pyrethroids from 2008 till 2012, compared with the more limited increase in the production of Atlantic salmon and rainbow trout in the same period, points in the direction of a more widespread resistance.

There are similarities between the results from the 30-min bioassays from Chile in 2007, the results from Norway in 2000 and the result from LS A in 2012. The Norwegian strains from 2000 were collected from farms reporting no treatment failures after pyrethroid treatments. The result from LS A was from a sea lice strain where a deltamethrin laboratory treatment demonstrated an almost complete efficacy (Helgesen & Horsberg 2013). However, as this strain was collected from a remote area with no previous history of pyrethroid treatments in the region, it cannot be considered representative of the overall pyrethroid sensitivity status in Norway. The 30-min bioassay results from Chile in 2008 and 2012 were in the same range as the Norwegian results from LS B and LS R from 2010 and 2011, which probably more adequately reflects the current overall pyrethroid sensitivity status in Norwegian salmon lice. Sensitivity was reduced 10–21 times compared with the 2000 results. These Norwegian results were from two strains where the treatment effect of deltamethrin in the laboratory was approximately 70% (Helgesen & Horsberg 2013). These results were from a Norwegian laboratory treatment with a different species and a different life stage of sea lice than in Chilean field conditions. The comparison between interspecies bioassay results was still performed because the results from sensitive sea lice were similar and because the salmonid species treated for sea lice are the same in both countries. This means that the same maximum concentrations of deltamethrin are valid to avoid toxicity among fish, and sea lice in both counties must survive this dose to be considered resistant.

The 24-h bioassays from Chile from three different farms all showed EC50 values for the male sea lice in the same range or above the values for the Norwegian resistant strains (approximately 70% treatment efficacy) and 19 to 150 times as high as the Norwegian sensitive strain. The EC50 values for the female C. rogercresseyi were even higher. The data from the 24-h bioassays indicated that resistance at a farm increased with each new treatment, but the data also showed varying EC50 values within a farm without any selection pressure from treatments. A bioassay is performed on a selection of parasites, and one result can therefore not be interpreted as the exact level of sea lice resistance on the farm at that given time point. The connection between bioassay results and treatment efficacy requires further study.

In 2012 and 2013, two types of bioassays were performed because the 24-h bioassays were also used in a different study. There was very little difference in the EC50 values for the female sea lice with respect to 30-min exposure and 24-h exposure to deltamethrin: 1.8 and 1.3 μg L−1 for the 30 min exposure and 1.7 and 0.6 μg L−1 for the 24 h exposure. These results may suggest a persistent mechanism for resistance.

The gender difference in susceptibility to deltamethrin in C. rogercresseyi was surprising. This was seen both in the 24-h bioassay and in the 30-min bioassay from 2012 (all data were combined) with lower sensitivity in females than in males. Similar effects have been indicated from parasite counts after EMB treatments in Chile (Bravo et al. 2009a). However, the latter results are from sea lice counts at fish farms and could also be the result of a longer life span in females than in males (Bravo 2010b). Female L. salmonis were found to be less sensitive to hydrogen peroxide than other mobile stages (males and pre-adults) by Treasurer et al. (Treasurer et al. 2000). Roth et al. (1996) found indications of female L. salmonis being more azamethiphos resistant than the other mobile stages. L. salmonis show the same trend regarding pyrethroid sensitivity, with adult females being more resistant than males (S. Sevatdal, unpublished data). However, this was not the case for pre-adult parasites. Westcott et al. (2008) found the opposite tendency on pre-adult L. salmonis and susceptibility to EMB, with pre-adult females being more susceptible than pre-adult males (Westcott et al. 2008). Results from field-collected adult sea lice might be partly influenced by age, as the age of field-collected adult sea lice is unknown. Future pyrethroid bioassays on C. rogercresseyi should though include only one gender, equal amounts of both genders or EC50 values should be calculated individually for each gender. The major issue of gender differences in pyrethroid susceptibility in C. rogercresseyi is, however, found in the field, where females are biologically the most important gender.

The development of reduced sensitivity towards a therapeutic agent in arthropod pests is closely linked to the amount used and the application frequency (Denholm & Devine 2013). Table 2 shows the usage of antiparasitic agents in Chilean aquaculture from 2007 to 2012 and in Norwegian aquaculture from 1998 to 2012. The Chilean data clearly show how pyrethroids replaced EMB as the leading treatment agent during 2007 to 2012. The relative use of pyrethroids was 68% in 2008 and 89% in 2012 (data not shown). This change most likely occurred due to the development of EMB resistance in C. rogercresseyi, as shown by Bravo et al. (2008, 2012, 2013). EMB treatment of sensitive parasites is easier to perform and provides long-lasting protection (Stone et al. 1999, 2000); thus, this would most likely have been the preferred treatment if the efficacy was sufficient.

The Norwegian data show a more diverse use of treatments during the years 1998 to 2012. However, pyrethroids have also been used extensively in Norway. From 1998 to 2007, more than 70% of salmonid fish biomass, treated against salmon lice, was treated with pyrethroids. In 2008, the figure was 74%, but dropped to 55% in 2012 (data not shown). The main reason for the decrease was most likely widespread pyrethroid and EMB resistance in addition to the reintroduction of azamethiphos treatment. Initially this treatment had a very good efficacy. The more intense treatment regime in Chile compared with Norway and the pyretroids' larger share of the overall treatments might have contributed to the more rapid development of a severe pyrethroid resistance in Chile.

Figure 4 clearly demonstrates an increase in the relative use of pyrethroids as the total amount of salmonids treated with pyrethroids has increased more than the annual production of salmonids. The decrease in the absolute use of pyrethroids seen in 2009 and 2010 compared with the two previous years might be explained by the overall decrease in the production of Atlantic salmon and rainbow trout during these years (Sernapesca 2013). These species are most susceptible to C. rogercresseyi infection (Gonzalez, Carvajal & George-Nascimento 2000). In 2009 and 2010, production of Atlantic salmon and rainbow trout was reduced more in region X than in region XI, and farms in region X were found to have a higher infestation of sea lice in 2007. This information was provided by a surveillance programme (Yatabe et al. 2011). An increased use of diflubenzuron in 2009 and 2010 might also have contributed to this development. The increase in the amount of salmonids treated with pyrethroids from 2010 to 2011 and more intensively from 2011 to 2012 may partly be explained by the increase in the production of Atlantic salmon and rainbow trout, but some of the increase is most likely the result of pyrethroid resistance. A similar increase in usage was seen in the use of EMB from 2006 to 2007 with this increase also possibly linked to resistance development (Bravo et al. 2012). The assumptions made to create Figs 4 and 5 regarding crowding of the fish during treatment were most likely incorrect for several of the treatments performed, but as the same assumptions were made for each treatment and there are no reason to expect that this has changed during the years, the comparison between the years still was considered valid. If the crowding of the fish was strongly overestimated, a lower proportion of the fish was treated each year than stated in the figures. This, however, would not change the inter-relationship between the proportions of fish treated from year to year.

Figure 5 shows the corresponding graphs for Norway during the years 1998 to 2012. In Norway, the absolute use of pyrethroids increased from 2008 to 2010, dropped in 2011, but increased substantially in 2012, despite the fact that the relative use of pyrethroids compared with other products decreased. Several explanations for the increased use of anti–sea lice treatments can be proposed. The first is the change in regulation where a lower number of adult female parasites are permitted per fish. This number is currently 0.5 adult females per fish. If this number is exceeded, it is required that a treatment normally has to be conducted. Another explanation is the coordinated spring actions against sea lice where practically all farms must perform synchronized treatments (Norwegian Food Safety Authority 2012). But the major cause is most likely a widespread reduced sensitivity and hence reduced treatment efficacy for several of the available treatments (pyrethroids, EMB, azamethiphos). This results in more frequent treatments and a higher usage of the antiparasitic agents.

Despite the fact that the frequency of sea lice pyrethroid resistance seems to have increased, based on the usage data for the chemicals, the EC50 values in the bioassays presented have not increased dramatically for C. rogercresseyi from 2008 to 2013. The reason for this might be that resistance comes at a cost as has been reported for the Australian sheep blowfly [Lucilia cuprina (Wiedemann)] (Mckenzie, Whitten & Adena 1982), and it is therefore contra-indicated for the parasite to become more resistant than needed to survive the treatments. Pyrethroid treatments cannot be performed with an elevated dose because the products are also toxic to fish (Haya 1989; Richterova & Svobodova 2012). Another reason why pyrethroid resistance did not lead to a more increased use of pyrethroids before 2011 and 2012 might be that sensitive parasites have contributed to the re-infection of the fish post-treatment. Re-infection might have arisen from already present free swimming copepodits, from parasites on fish that were not treated because skirt treatments are not 100% effective (Nilsen et al. 2010) or from parasites on nearby farms that were not treated simultaneously. Thus, it can be speculated if more efficient bath treatments with enclosed tarpaulins instead of skirts and synchronized treatments of neighbouring farms with the same agent may actually accelerate resistance development. This, however, remains to be clarified.

The deltamethrin resistance shown in this article, both from the bioassay results and by the development in pyrethroid treatments, is caused by evolutionary factors where the most resistant parasites survive each treatment. This development is increased by frequent treatments with substances acting on the same mechanism in the parasite, as they are likely to select for the same resistance mechanism (Denholm et al. 2002). This is the case for the two pyrethroids: deltamethrin and cypermethrin. Two possible underlying causes of resistance could be either a target site mutation in the voltage-gated sodium channel and/or increased detoxifying mechanisms (Denholm & Devine 2013). In L. salmonis, enhanced metabolism has been demonstrated to be the most probable mechanism (Sevatdal et al. 2005b), although target site mutations in the voltage-gated sodium channels have also been proposed (Fallang et al. 2005). Studies to investigate the underlying mechanism in C. rogercresseyi should be initiated as this knowledge is useful for the development of methods for resistance testing and for the development of new anticaligus treatments.

Given that both pyrethroids and EMB have become relatively ineffective in Chilean aquaculture, new treatment agents are imminently required. This has been recognized by Sernapesca which has permitted the use of the organophosphate azamethiphos since 2013. Azamethiphos has, however, no effect on the chalimus stages of the parasite, and as a result, more frequent treatment with azamethiphos, compared with pyrethroids, is required (Grant 2002). To avoid a rapid development of azamethiphos resistance, there is an urgent need for additional new treatment agents, better integrated parasite management as well as ways of reducing the sea lice burden without the use of chemicals. Examples of possible chemical-free anti–sea lice agents include vaccines (Carpio et al. 2011) as well as cleaner fish (Treasurer 2002). However, while cleaner fish are currently used to combat sea lice in the Northern Hemisphere, no suitable cleaner fish have yet been discovered in Chile.

The bioassay results presented in this article reveal evidence of deltamethrin resistance in region X in Chile since 2008. The sensitivity level of the parasites in this region is approximately equal to that of deltamethrin-resistant L. salmonis in Norway. Furthermore, the data also indicate a difference between genders in C. rogercresseyi. This is in respect to their susceptibility to deltamethrin, with female sea lice being more resistant than males. Based on available data regarding the usage of antiparasitic agents in Norwegian and Chilean aquaculture, there is a strong indication of a development of pyrethroid resistance in Chile, in particular in the period from 2011 to 2012. The available data are, however, not extensive enough to document the entire development of deltamethrin resistance in C. rogercresseyi in region X. Therefore, neither the development in the strength of deltamethrin resistance nor the extent to which it has developed can be substantiated. A larger study on resistance based on bioassay results should therefore be initiated to determine the level of deltamethrin resistance in the whole salmonid-producing area of Chile. A retrospective survey on treatment results should also be performed to procure a clearer picture of the resistance development.


We would like to thank The Research Council of Norway and The Norwegian Seafood Research Fund for funding the project PrevenT (NFR 199778/S40). We would like to thank Pharmaq for giving us access to the bioassay results from 2007, Mainstream Chile for facilitating the work in 2012/2013 and Sernapesca for providing usage and production data. We would like to thank Felipe Campos, Rodrigo Pérez and David Garrido for participating in the data collection in 2012 and 2013 and Josephine Prendergast for proofreading the manuscript.

Conflict of interests

One of the co-authors, Julio Mendoza, is working as a fish health manager for an aquaculture company in Chile. He or his company have, however, not put any restrictions on which data to publish. None of the other authors have any potential conflict of interest.