Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains

Parasitology International 58 (2009) 451–460

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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t

Establishment and characterisation of salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) laboratory strains Lars A. Hamre a,⁎, Kevin A. Glover a, Frank Nilsen a,b a b

Institute of Marine Research, P.O. Box 1870 Nordnes, N-5817 Norway University of Bergen, Department of Biology, Box 7800 N-5020 Bergen, Norway

a r t i c l e

i n f o

Article history: Received 12 June 2009 Received in revised form 24 August 2009 Accepted 25 August 2009 Available online 2 September 2009 Keywords: Sea lice Genetic Inbreeding Microsatellite Mutant Incubator

a b s t r a c t The salmon louse (Lepeophtheirus salmonis (Krøyer 1837)) is an ectoparasitic copepod which represents a major pathogen of wild and farmed salmonid fishes in the marine environment. In order to facilitate research on this ecologically and economically important parasite, a hatchery and culturing system permitting the closure of the life-cycle of L. salmonis in the laboratory was developed. Here, the hatchery system, breeding practices, and selected louse strains that have been maintained in culture in the period 2002–2009 are presented. The hatchery and culture protocol gave rise to predictable hatching of larvae and infections of host fish, permitting the cultivation of specific strains of L. salmonis for 22 generations. Both in- and out-bred lice and mutant colour strains have been established, and some of these strains were characterised by microsatellite DNA markers confirming their pedigree. No evidence of inbreeding depression, fitness or morphological changes was observed in any of the strains cultured. It is suggested that the culturing system, and the strains produced represent a significant resource for future research on this parasite. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Sea lice from the genera Caligus and Lepeophtheirus are important parasites of wild and farmed fish in the marine environment [1]. Throughout the North Atlantic, Lepeophtheirus salmonis (Krøyer 1837) is the most common species observed on salmonid fishes [1,2], and is responsible for significant economic losses in the Atlantic salmon (Salmo salar L.) and rainbow trout (Oncorhynchus mykiss) farming industries [3]. Adult female and male L. salmonis are approximately 10 and 5 mm long respectively, and live on salmonid hosts, feeding on blood, skin and mucus. The life-cycle is characterised by 10 stages, each separated by a moult [4]. This involves 2 planktonic non-feeding nauplii stages, an infective copepodid stage, 4 chalimus stages where the parasite is attached to the host by a frontal filament, 2 motile preadult stages and the final motile adult stage [4]. Eggs are fertilised as they are extruded, and development from fertilisation to adult is 40 to 52 days at 10 °C for male and female lice respectively [4]. Most pair formation occurs in the form of pre-copular guarding when adult males emerge synchronised with pre-adult II females [5]. Female lice are fertilised by 1 male shortly after moulting to the adult stage, but polyandry is common among older females [6]. The number of eggs per pair of egg

⁎ Corresponding author. Tel.: +47 55236995; fax: +47 555238531. E-mail address: [email protected] (L.A. Hamre). 1383-5769/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2009.08.009

strings varies from 107 to 1220 (see Costello [7]). Adult females have been reported to survive under laboratory conditions for up to 191 days at 7.2 °C and produce 11 pairs of egg strings in this period [8]. Mustafa et al. [9] reported females that lived for up to 210 days and produced 10 egg string pairs/female lice. The commercial production of Atlantic salmon and rainbow trout in marine net-pens has expanded radically since the industry was initiated in the late 1960s. This increase in the number and availability of hosts has lead to an increase in the numbers of lice, and the infection pressure experienced by wild and farmed fish [10]. A range of delousing techniques are available to treat farmed fish, however, chemotherapeutants represent the primary method of control. The number of chemotherapeutants available for delousing salmonids reared in net-pens is limited, and resistance to some of these agents has been documented [11–20]. This situation highlights the importance of developing new approaches to control this parasite. At present there are no reports of L. salmonis having been propagated for more than 1 or 2 generations in the laboratory. Furthermore, the majority of published studies on L. salmonis have been conducted with experimental material originating from salmonids collected in farms, slaughterhouses or the wild (e.g. [21–23]). This entails working with an unpredictable supply of lice of unknown genetic background and variable quality. Consequently, a L. salmonis incubator and culturing system was established at the Institute of Marine Research in Bergen, Norway (IMR) starting in 2002, with the specific aim of producing a stable supply of lice with known genetic background for ongoing research. Here, we present the culturing system and L. salmonis strains

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that have been maintained for up to 22 generations in the laboratory. The application of L. salmonis laboratory strains is discussed. 2. Materials and methods 2.1. Overall cultivation design The aim of establishing the L. salmonis laboratory strains was to create a reliable supply of lice for a wide range of scientific applications in the period 2002–ongoing. The breeding strategy chosen for maintenance of each louse strain represented a combination of scientific requirements in addition to practical constraints. Nevertheless, the general culturing protocol involved collecting gravid L. salmonis from Atlantic salmon or rainbow trout at a local slaughterhouse, incubating and hatching eggs, then using the surviving copepodids to infect a group of naive Atlantic salmon maintained in single or replicated tanks. After having successfully infected the salmon, lice were permitted to develop through all attached and motile stages [4,24], and produce egg strings. Egg strings were harvested, incubated and hatched to establish the next generation. The parental lice founding a new strain is termed generation zero (F0), their offspring generation 1 (F1) and so on. This also applies when the new strain is a branch of an existing strain.

2.2. Incubators, host fish and rearing tanks To provide a predictable supply of copepodids, two types of egg incubators were developed: a small incubator suitable for single pairs of egg-strings (Fig. 1A, B) and a large incubator for hatching up to c 50 pairs of egg-strings (Fig. 1C, D). All incubators were fed with particle filtered salt-water water pumped from a depth of 120 m with a yeararound salinity of 34.5 ppt. and temperature 9.5 ± 1 °C. Incubators were designed as flow-through with a respective total volume and water exchange of 76 ml and 30 ml/min for the small incubators, and 2000 ml and 270 ml/min for the large incubators (exact technical specifications for incubator design available upon request). Farmed Atlantic salmon in the size range c 250–1500 g were used for propagation and maintenance of the L. salmonis strains. For all strains in all generations, Atlantic salmon that had not been in contact with L. salmonis were used. Lice and their host fish were stocked in four different types of tanks depending upon the number of lice and fish cultured. 1500 l circular tanks (grey) with conical bottom, square 500 l tanks (grey, 1 × 1 × 0.5 m) with flat bottom, 200 l oval tanks (green) with window and conical bottom and 160 l oval tanks (green) with window and flat bottom were utilised. Water flow was in general 1 l/min per kg fish, but not below c 7 l/min to maintain sufficient circular current in the tanks. All tanks have a centred outlet and were

Fig. 1. Incubators. A) Overview of the small incubator system. The small incubators are placed in racks and the racks are placed in a level-tank (LT) to ensure correct water level in the incubators. The level-tank outlet (LO, see (C)) provide correct water level. Water is supplied from the header tank (H) placed above the racks. B) Details of header tank. C, D) Large incubator. Consist of two buckets; the bottom of upper bucket is removed, and the two buckets are joined, separated by 180 μm plankton mesh. Water enters in the bottom of the lower bucket, and is drained through a plankton mesh (180 μm) covered funnel (FU) in the upper bucket. A level tube is connected to the funnel and attached to the bucket at the desired water level (LTU).

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covered with either transparent lids or mesh. Some of the strains were reared in parallel tanks in some generations. Fish were hand fed to excess using a commercial diet and maintained according to Norwegian animal welfare regulations. 2.3. Specific cultivation details In order to propagate a new generation for a lice strain, egg-strings were collected from host fish either by picking egg-bearing females directly from fish in a tank or from anaesthetized individuals. Direct collection was attempted when small groups of fish were reared in 160 and 200 l tanks. By lowering water level (2–3 times fish height) it was often possible to collect the required number of female lice, remove egg strings and re-introduce the lice without the need for sedation. In larger tanks, or when direct collection was otherwise not possible, egg-bearing females were collected from fish anaesthetized by a mixture of 60 mg/l benzocaine and 5 mg/l methomidate. For any given batch, egg-strings were incubated until the majority of hatched individuals had reached the copepodid stage, which was c 14 days. Copepodid density in the incubator was estimated by plunging a pipette (both ends removed) into a well-stirred beaker of copepodids, closing the upper opening with the thumb and transferring the contents to a measuring cylinder until >200 copepodids were collected. Volume was recorded and the copepodids were strained and flushed (using 70% alcohol with 9.2 g/l of salt, added to prevent copepodid distortion) to a zooplankton counting chamber and counted. Copepodids that appeared non-viable were not counted. Observations from preliminary infections with cultured lice indicated that c 1/3 of the copepodids added to a tank were found on the fish when sampled at the preadult or adult stages. Consequently, the number of copepodids required for infection of the host fish was calculated by the following equation: copinfection = fish× licepreadult/adult × 3. Fish were infected by stopping water flow to the tank and lowering water level to c 2–5 times the height of the fish. Water was aerated using a fine bubble stone connected to an air pump. A quantified and adjusted number of copepodids were distributed evenly in the tank and left for 1 h before water flow was re-instated. The fish were inspected regularly during this procedure. If inactive, the fish were either startled by waving a hand over the tank and/or the water supply was started for some seconds to refresh and stir up the water a few times during infection. Tanks were covered with transparent lids and the lights were on in the laboratory during infection. Post infection, both fish status and lice load were inspected visually on a daily basis. When lice reached the preadult stages, individual fish with heavy infections or lesions were anaesthetized and the number of lice reduced. For most of the louse strains cultured, eggs for propagation of the next generation were collected from a cohort of lice with a minimum age of approximately 70–90 days post infection (DPI) (Table 1). If a strain of lice was present in only one tank, generation x was kept alive, either on fish or in an incubator (adult lice or eggs/copepodids), until generation x + 1 reached the preadult stage (c 20 days). This was done in case of incubator failure or fish disease prior to the emergence of preadult lice. Fish were normally kept at ambient temperature (9.5 ± 1 °C), 12/12 h day/night. 2.4. Evaluation of incubator performance and infection success Incubator performance was evaluated by estimating the number of copepodids produced per egg string (cop/string). This was conducted for egg strings incubated for strain propagation. In addition, performance of the small incubators was also studied by estimating the number of copepodids produced pr incubated egg (cop/egg) in a specific experiment to test these incubators. In this specific incubator test, eggs from 76 lice were incubated in individual small incubators at 10 °C. Egg string length, egg diameter and height, and time to hatching were recorded (inspected at 24 h intervals). Each incubator was

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Table 1 Breeding history of 5 L. salmonis laboratory strains showing the number of egg string pairs or egg strings* that contributed to generation Fx and the age of the females from which the eggs were collected (in brackets) given as days post infection at c 10 °C. LsGulen

LsOslofjord

Ls1a

Ls1rr

Ls1SS

27.06.06 outbred no wild 150* >25 (72) 73 (56) 12 (118) 20 (70) 60 (64) 180 (119) No data No data 30 (55) No data No data No data (present)

06.11.06 outbred no wild 9* 3 (70) 2 + 2* (74) 3 (117) 3 (92) 3 (75) End

27.12.02 inbred no LsSolund F2 1 (70) 3 (95) 4 (73) >50* (89) 5 (92) 4 (85) 1 (126) 1 (169) 7 (155) 2 (75) 1 (86) 1 (73) 1 (94)

15.04.03 inbred red Ls1a F1 1 (95) 9 (74) 24* (194) 6 (244) 7 (123) 25 (187) 25 (68) 3–4 (114) 4 (70) 2 (70) 2 (61) 2–3 (74) 3 (149)

15.04.03 inbred black Ls1a F1 1 (95) 6 (74) 3 (452) 5 (136) 22 (162) 4 (70) 1 (86) end

F13 F14 F15 F16 F17 F18

2 (57**) 3 (46**) 4 (76) 3 (116) 3 (92) 3 (61)

3 (76) 3 (90) 8 (91) 4 (167) 4 (181) 18 (120) (present)

F19 F20 F21 F22

3 (77) 4 (227) 3 (210) 18 (100) (present)

Established Strain type Selection F0 origin F0 F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12

** = elevated temperature, c 15 °C. Note that the table in general does not reflect the number of egg bearing female lice available for strain propagation.

sampled 9 days post hatching. Maturation of egg strings at start of incubation is given as days post fertilisation (DPF), which is estimated according to: DPF = 10-(days to hatching). This was based on a total egg development time of 9 days (10 °C) [4,25] and the fact that time to hatching was recorded at 24 hour intervals. The difference in hatching success between newly fertilised and mature egg strings were estimated from the regression line DPF vs. development success. Details on egg string measurements are given below. The combined infection success of copepodids and lice survival on the host was evaluated by reviewing available infection data from various experiments conducted using these facilities and tanks for strain maintenance where the numbers of lice were not manipulated between infection and sampling. This value represents the percentage of copepodids added at infection that attach and survive on their hosts until a specified age. The data used here come from tanks that vary with respect to lice strain, cop/fish given at infection, fish origin, fish density, tank type and water flow. In general there were more fish in tanks with small fish (c 100–150 g at infection, n = 12–45, 200 and 500 l tanks) than in tanks with large fish (c >250 g at infection, n = 3–39, 160–1500 l tanks). For all incubations conducted in the period 2002+ the temperature in the whole water column of the small incubators was 10.0 ± 0.5 °C; however, a temperature gradient established from top to bottom in the large incubators. At air temperature 19 °C the surface temperature was 13 °C while the inlet water (i.e. bottom of incubator) was 10 °C. Incubator temperature was not measured on a regular basis. All incubator temperatures given were measured in the bottom of the incubators. 2.5. Description of L. salmonis laboratory strains A total of 16 L. salmonis laboratory strains were cultured at IMR in the period 2002–2009, and 8 of these strains exist at present. The

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breeding history details for some selected strains are given in Table 1. With the exception of the LsGulen strain, which was established from L. salmonis collected on rainbow trout, and LsOslofjord established from wild sea trout (Salmo trutta L.) caught in the Oslofjord, all strains reared at IMR were established from L. salmonis collected from farmed Atlantic salmon sampled at slaughterhouses on the west coast of Norway. LsSolund was established 15 July 2002 and represents a discontinued out-bred strain that was cultured for 17 generations. The F0 generation was collected from Atlantic salmon originating from a farm in Solund, Sogn og Fjordane county. A single female from this strain gave rise to the three inbred strains described below. Ls1a represents an inbred strain established 27 December 2002 by selecting the egg strings from one normal looking mother (LsSolund) at 70 DPI. To compensate for potential inbreeding depression, heavy infections were given during the first 5–6 generations (75–100 copepodids/fish added to the tank at infection). Subsequent generations included giving infections of c 45–60 copepodids/fish. The Ls1rr strain was established 15 April 2003 and is a red colour mutant originating from a single Ls1a F1 female louse which produced c 50/50 red and black copepodids. All copepodids from one pair of egg strings were used for infection. At c 300 degree days (8.5 °C) preadult 2 red females and red adult males were sorted out and pooled on fish in a separate tank, likewise with black (i.e. normal) preadult 2 females and adult black males. At 71 DPI eggs from 9 red and 15 black females were incubated in separate incubators. All the red females produced only red offspring, but among the black females, some produced both red and black copepodids and some produced only black. Incubators were sorted according to content, and 2 tanks of fish were infected with copepodids from incubators containing red and black copepodids respectively. The black copepodids gave rise to the Ls1SS strain. LsOslofjord represents a partially outbred strain established 6 April 2006, originating from 9 female lice collected from wild sea trout captured by angling in the inner part of Oslofjord, Norway. LsGulen represents an outbred strain established 27 June 2006, F0 collected from rainbow trout from Gulen, Sogn og Fjordane. LsBindal F1 was established 22 January 2008 to perform a bioassay due to suspicion of emamectin resistance and was later discontinued. 2.6. Morphometric and fitness analysis of strains In order to evaluate whether differences in morphology and fitness develop over time between inbred and outbred strains, lice representing Ls1a F22, Ls1rr F17, LsSolund F5 and LsGulen F7 were selected for analysis. Morphometric measurements of lice and eggs were carried out using the ImageJ image analysis software (http://rsb.info. nih,gov/ij). Images of whole lice with egg strings were obtained using a Canon EOS 30D camera and a 60 mm macro lens (lice placed on wet white absorbent paper, light from below). Close-up images of egg strings were taken using the stereomicroscope at 25× magnification (to measure egg size). Genital segment size varied with maturation and size of the developing oocytes, which correlated with development of the fertilised external eggs (Hamre, unpublished). Repeated measurements of fertilised egg strings developing in the incubators indicated that egg volume increased c. 30% from 1–2 DPF to 8–9 DPF (Hamre, unpublished). Since the mean degree of egg string maturation varied significantly between samples in general, only lice with pigmented embryos (7, 8 or 9 DPF) were included in analysis. Egg strings that had started hatching were excluded. All statistical calculations on morphometric and infection data were carried out using Statistica 8.0 (www.statsoft.com). The relationship between maturation of eggs (DPF) and development success was evaluated by means of Pearson's product moment correlation. Linear regression was used to calculate percent difference in development success of newly fertilised and ready to hatch egg strings.

Morphometric and fitness measurements were compared between strains by one way ANOVA and Tukey honest significance test for unequal N. Binominal confidence intervals was calculated according to Zar [26]. In all tests, a probability level <0.05 was considered significant.

2.7. Genetic characterisation of the lice strains Lice representing LsGulen, LsOslofjorden, LsBindal, Ls1a, Ls1rr and a wild reference sample (from Kyrkseterøra, Trondheim, Norway) were genotyped with 4 microsatellite loci: LsaSTA1, LsaSTA2, LsaSTA5, LSNUIG14 [27]. In addition, available intermediate generation samples were analysed for 3 of the inbred strains (1 discontinued). DNA was extracted from cephalothorax of adult female lice/whole individual adult male lice using a Qiagen DNeasy 96 tissue kit. Male lice gave DNA concentrations 40–100 ng/μl whilst females gave DNA concentrations of 200–400 ng/μl. DNA extractions were diluted 1:10 prior to PCR. PCR amplification was performed as a multiplex in 96 well plates with a total reaction volume of 20 μl. Each reaction consisted of 2 μl DNA, 2.5 mM MgCl, 0.3 mM dNTPs and 0.75 U Go Taq polymerase and 1× reaction buffer. In all cases, forward primers were labelled with ABI fluorescent dyes for fragment detection. Concentrations for each of reverse and forward primer were as follows: LsaSTA1 0.1 μM (6-FAM), LsaSTA2 0.025 μM (NED), LsaSTA5 0.1 μM (PET), LSNUIG14 0.5 μM (6-FAM). Amplification was performed in an Eppendorf Master Cycler using the following programme: 95 °C for 1 min, then 30 cycles of the following: 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s. The programme was finished with a 2 min extension stage at 72 °C, and hold at 4 °C. DNA fragments were separated on an ABI 3730 sequencing machine and sized according to the Applied Biosystem GeneScan™-500LIZ™ size standard. Alleles were scored using automatic binning implemented in the Genmapper software (V4.0) with manual checking before data was exported to a Microsoft Office Excel spread-sheet. Genepop V3.3 [28] was used to estimate Hardy–Weinberg's equilibrium and pair-wise FST values for the wild reference sample and the 2 outbred strains. FSTAT V2.9.3.2 [29] was used to compute allele frequencies for all loci in all samples. Geneclass V1.0.02 [30] was used to perform self-assignment tests. Self-assignment tests were conducted using the leave one out procedure and the Rannala and Mountain [31] method of computation. TFPGA V1.3 [32] was used to calculate observed heterozygosity for all samples across all loci.

3. Results 3.1. Incubator performance Ciliates and other small invertebrates were not observed on eggs in either of the incubator types. Copepodids were not evenly distributed in the incubators and density was highest in the upper layers. Fourteen days post start of incubation, a respective mean of 114 and 153 copepodids/egg string were produced from eggs of wild and laboratory origin in the large incubators (Table 2). The small incubators yielded a mean of 162 copepodids/egg string (lice of various laboratory origin) when incubating one pair of egg strings per incubator in 14 days. In the specific study of small incubator performance, mean development success was 0.73 cop/egg, or 218 cop/egg string (mean string length 17.7 mm). There was a significant correlation between DPF and development success (Pearson's product–moment correlation, p < 0.001, r = 0.44), and newly fertilised eggs had 28% lower development success than mature eggs (Fig. 2). However, there was no significant increase in development success among eggs put in the incubator between 4 and 9 DPF. Pigmentation of the embryo became visible between days 6 and 7 post fertilisation at 10 °C.

L.A. Hamre et al. / Parasitology International 58 (2009) 451–460

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Table 2 Development success measured as copepodids produced per egg string in large and small continuous flow incubators at c 10 °C and measured c 14 days post start of incubation. Incubator type

Lice origin

N incubators

Egg strings/inc

Cop/string (SD)

Large Large Small

Laboratory Wild Laboratory

32 5 361

54 46 2

153 (45) 114 (70) 162 (41)

Estimates are summarized from egg strings incubated for both specific experiments and strain propagation purposes. Egg strings/inc = mean number of egg strings incubated per incubator. Cop/string = mean number of copepodids produced per incubated egg string.

3.2. Infection and survival success of lice In the period 2002–2009, more than 200 separate tanks were infected with L. salmonis copepodids. All were conducted according to the procedures described herein. In this period, infection failure did not occur demonstrating the reliability of the incubation and infectious system. Specific studies investigating the parameters affecting infection and survival success were performed, however, we report our general experiences based on daily observations of lice infection levels along with reviewing various experimental data and data obtained from tanks for strain maintenance on the combined infection and survival success. A highly variable loss of lice was observed between tanks at the preadult and adult stages. In tanks with many small fish (c 120–150 g at infection) the loss of female lice in particular could at times be extremely variable between tanks that were similar with respect to copepodids/fish, fish size and number, tank type and flow. Unfortunately, the full scale of the observed variability is not reflected in the available infection data (Fig. 3). Consequently, a few (4–5) large host fishes (c >250 g at infection) stocked in 160 and 200 l tanks became the preferred paradigm of fish/ tank type for maintaining louse strains. Under such conditions, cohorts of adult female lice often remained on the fish for up to several hundred days in more than sufficient numbers for strain propagation. The highest age of L. salmonis recorded in the laboratory was 15.5 months (3 females). These lice (3 females) carried eggs that produced infective copepodids (see Ls1SS F3 in Table 1). It should be noted that Table 1 in general does not reflect the number of egg bearing females available for strain propagation. The combined infection and survival success measured at 60–70 DPI was typically 30–50% on large fish stocked at low density (c >250 g at infection, n = 3–39, 160 to 1500 l tanks, 20–90 copepodids/fish) and

Fig. 2. Development success of eggs (copepodids/incubated egg) vs. maturation degree measured as days post fertilisation (DPF). The regression DPF vs. development success is significant, p < 0.001, r2 = 0.19, y = 54.87 + 2.93X.

Fig. 3. Combined infection and survival success (CISS) of salmon lice, measured as the percentage of copepodids added at infection that attach and survive on their hosts until a specified age (tanks with small fish = circles, tanks with large fish = triangles).

10–20% in tanks with many small fish (100–150 g at infection, n = 12– 45, 200 and 500 l tanks, 30–60 copepodids/fish). In addition, there were fewer female lice than male lice in tanks with many small fish than in tanks with few large fish (Fig. 4) at 60–70 DPI. Automatic re-infection did not occur in the indoor tanks due to a rapid water exchange flushing the nauplii out, and with some exceptions all cohorts of lice produced were distinct.

3.3. Ls1rr—the red colour mutant strain The Ls1rr F1 generation consisted of approximately 50/50 red and black individuals/copepodids. The adult red females (n = 9) produced only red offspring. Among the black females (n = 15), eight produced both red and black copepodids and seven produced only black. In the red/black group 38% of the copepodids were red (95% binominal ci.: 28–49%). The red phenotype is prominent in embryos, nauplii and copepodids (Fig. 5), at the preadult stages the red phenotype can still be separated from normal phenotypes visually, however older adult red lice can be difficult to separate from normal lice.

Fig. 4. Sex ratio of L. salmonis (% females) vs. days post infection (DPI) in tanks with small (circles) and large (triangles) host fish.

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L.A. Hamre et al. / Parasitology International 58 (2009) 451–460 Table 4 Body size of male lice from the outbred L. salmonis strain LsGulen and the inbred strains Ls1rr and Ls1a. Strain

N

Ct (mm)

CtA (mm2)

Gs (mm)

GsA (mm2)

LsGulen F7 Ls1rr F17 Ls1a F22

23 25 8

3.28 (0.08) 3.04*1 (0.10) 3.12*1 (0.08)

8.97*2 (0.36) 7.54*2 (0.29) 8.12*2 (0.35)

1.37 (0.10) 1.38 (0.09) 1.35 (0.10)

1.29 (0.06) 1.16*1 (0.08) 1.23 (0.06)

The strains were sampled at 80, 87 and 91 DPI, and cultured at 7.7, 9.3 and 9.1 °C respectively. Ct = cephalothorax length, CtA = cephalothorax area, Gs = genital segment length, GsA = genital segment area. Measurements that are significantly different from those of LsGulen are marked *1 and measurements significantly different to both the other groups are marked *2.

Fig. 5. Mature egg string from a Ls1rr F1 female containing normal and red pigmented embryos along with normal (left) and red pigmented (right) copepodids. Scale bar = 0.5 mm.

3.4. Morphology and fitness parameters Female lice from the LsSolund and Ls1a strains displayed significantly longer cephalothorax than females from Ls1rr (p < 0.002 and p < 0.03 respectively) and LsGulen F7 strains (p < 0.0002 and p < 0.0007 respectively) (Table 3). In addition, Ls1rr females displayed a significantly shorter genital segment than LsSolund (p < 0.002) and LsGulen (p < 0.008) females, and both inbred strains had a significantly shorter abdomen than the outbred strains (Ls1a vs. LsSolund and LsGulen: p < 0.02 and p < 0.005 respectively; Ls1rr vs. LsSolund and LsGulen: p < 0.02 and p < 0.05 respectively) (Table 3). Egg string length and egg volume did not differ between strains (Table 3). Egg development success was similar between strains; however, Ls1rr and Ls1a displayed a slightly lower egg development success compared to the outbred strains. Ls1rr males were significantly smaller than both LsGulen and Ls1a males (Table 4). No differences in louse survival or occurrence of anomalies among the present generations of the strains were observed. 3.5. Genetic characterisation of strains In total, 268 lice were genotyped (Table 5). LsaSTA5 displayed the largest amount of genetic variation as determined by number of

alleles observed in the complete data set, followed by LsaSTA1, LsaSTA2 and NUIG14. Only one significant deviation from the Hardy– Weinberg's equilibrium was observed, and this was for LsaSTA5 in LsBindal (p < 0.001). The wild reference L. salmonis strain displayed a total of 47 alleles which included all observed alleles at LsaSTA2 and NUIG14. The 2 outbred louse strains (LsGulen and LsBindal) displayed the next most genetic variation of the samples. The most recent samples collected from the inbred L. salmonis strains in culture displayed very little genetic variation, however, only Ls1a was monomorphic for all loci. LsOslofjord and the inbred strain Ls1rr displayed a total of 8 and 6 alleles respectively for the 4 loci pooled, and both were monomorphic for 2 loci each. Intermediate samples for Ls1a, Ls1rr and the discontinued Ls1SS inbred line all illustrated the loss of genetic variation during the process of inbreeding. Fig. 6 illustrates the genetic variation observed at the 4 loci for 5 L. salmonis strains and the wild reference strain. For the 2 inbred lines, with the exception of alleles at relatively low frequency, distribution of allelic variation was similar for LsaSTA2 and NUIG14. For LsaSTA1 and LsaSTA2, however, the LsGulen outbred strain displayed a more distinct pattern of allele frequencies compared to the wild reference strain and LsBindal. Allelic variation among the 2 presently cultured inbred strains (Ls1a and Ls1rr) and the LsOslofjord was very distinct (Fig. 6). For the 2 inbred strains originating from the same female, both were fixed for the same allele at LsaSTA2, and shared a common allele for LsaSTA1. For LsaSTA5 and NUIG14 however, Ls1a and Ls1rr were distinct. For the LsOslofjord, which is based on a limited number of parents (Table 1), both LsaSTA2 and LsaSTA5 were diagnostic between this strain and the other 2 inbred strains, although common alleles were observed with Ls1rr for LsaSTA1 and NUIG14. Pair-wise FST values were calculated among the wild reference lice sample and the 2 outbred louse strains. Kyrkseterøra lice was genetically more similar to LsBindal (FST = −0.005) than to LsGulen (FST = 0.021), whereas the largest pair-wise difference was observed between LsGulen and LsBindal (FST = 0.035). Self-assignment tests were performed among 5 of the L. salmonis strains presently in culture in order to examine the possibilities for identification of the strains in a mixed culture situation (Table 6). Overall, correct self-assignment was estimated at 98%. Not surprisingly, all individuals originating from inbred strains were correctly identified to their respective strains, and miss-assignment was only observed between the 2 outbred lines.

Table 3 Body size and fitness parameters for adult egg bearing females from the two outbred L. salmonis strains LsSolund and LsGulen and the inbred strains Ls1rr and Ls1a cultured at 9.3, 9.0, 7.7 and 9.1 °C respectively and measured at, 70, 80, 91 and 87 DPI. Strain

N

Ct (mm)

Gs (mm)

Ab (mm)

EggVol (mm3)

ESL (mm)

C/ES

LsSolund F5 LsGulen F7 Ls1rr F17 Ls1a F22

13 11 16 20

4.64 (0.13) 4.34*1 (0.13) 4.43*1 (0.12) 4.57 (0.13)

3.39*2 3.36*2 3.08 3.19

3.06*3 3.05*3 2.78 2.70

0.0143 (0.00081) 0.0155 (0.00055) 0.0147 (0.00147) Not available

17.1 17.4 18.0 17.3

166 167 145 149

(0.25) (0.25) (0.14) (0.14)

(0.19) (0.32) (0.19) (0.21)

(3.8) (2.0) (4.0) (2.1)

Ct = length of cephalothorax, Gs = length of genital segment Ab = length of abdomen, EggVol = egg volume. ESL = egg string length and C/ES = estimated average number of copepodids produced per egg string. Standard deviations are provided in brackets. Measurements that are significantly different from those of LsSolund and Ls1a are marked *1, measurements significantly different from Ls1rr are marked *2 and measurements significantly different from Ls1a and Ls1rr are marked *3.

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Table 5 Genetic variation represented by number of alleles of past, present and intermittent laboratory strains of L. salmonis reared at IMR at 4 microsatellite loci. Strain

Kyrkseterøra

LsGulen

LsBindal

LsOslofjord

Ls1a

Ls1a

Ls1rr

Ls1rr

Ls1rr

Ls1SS

Ls1SS

All

Generation N observations Strain definition

N/A 47 W

6 46 OLS

1 30 OLS

4 30 OLS

21 29 ILS

3 10 ILS

15 26 ILS

6 20 ILS

2 10 ILS

2 5 ILS

5 15 ILS

N/A 268 N/A

Genetic data LsaSTA1 LsaSTA2 LsaSTA5 NUIG14 All loci

13 (0.85) 9 (0.62) 20 (0.94) 5 (0.69) 47 (0.77)

9 (0.86) 3 (0.41) 11 (0.82) 4 (0.67) 27 (0.69)

9 (0.83) 3 (0.47) 17 (0.91) 4 (0.69) 33 (0.73)

4 1 1 2 8

1 (0) 1 (0) 1 (0) 1 (0) 4 (0)

3 (0.57) 2 (0.32) 3 (0.45) 2 (0.18) 10 (0.38)

2 (0.50) 1 (0) 2 (0.48) 1 (0) 6 (0.25)

2 (0.44) 2 (0.15) 2 (0.44) 2 (0.14) 8 (0.30)

3 (0.61) 2 (0.50) 2 (0.50) 2 (0.26) 9 (0.46)

3 (0.62) 2 (0.48) 3 (0.62) 2 (0.32) 10 (0.51)

2 (0.36) 2 (0.42) 3 (0.65) 2 (0.49) 9 (0.48)

15 (0.82) 9 (0.53) 21 (0.89) 5 (0.64) 50 (0.72)

(0.74) (0) (0) (0.49) (0.31)

Observed heterozygosity is presented in brackets. W = wild reference strain (not cultured), OLS = outbred laboratory strain, ILS = inbred laboratory strain.

A pedigree diagram representing Ls1a, Ls1rr and Ls1SS is presented (Fig. 7). Among the founder mother and her descendants, 7 different alleles were observed for LsaSTA5 demonstrating that the founder mother had mated with a minimum of 3 males. No evidence for multiple paternity was found for daughter 2, and new alleles did not appear past the second generation in any of these strains demonstrating that mixing of strains did not occur. 4. Discussion 4.1. Evaluation of the culturing system The incubators presented here represent an advance in rearing technology for L. salmonis, and sea lice in general. These incubators have permitted, for the first time, the laboratory culture of genetically characterised colour mutant, in- and out-bred strains of L. salmonis for as much as 22 generations. To our knowledge, no other studies documenting the laboratory culture of L. salmonis, or any other sea lice species, beyond 1–2 generations exist. A range of incubator systems have been described for fish louse eggs, including stagnant systems [4,21,25,33–37], systems with plankton mesh immersed in flowing water [4,22,38–40] and flow through systems [41,42]. Prior to development of the incubation system presented here, both stagnant and floating systems were used for incubation of L. salmonis eggs in the wet laboratory facilities at IMR. These were similar to bottles described by Heuch [35] with daily water

exchange kept at 10 °C, or floating systems similar to Johnson [4]. Using these systems we experienced a high variability in both development success and quality of copepodids. The incubators presented here provide a more predictable hatching and infection success than other methods tested, demonstrating that these incubators provide a suitable environment for the egg strings to develop. In an early study of the life-cycle of L. salmonis collected in the Pacific, Johnson and Albright reported development success (cop/egg) to be on average 0.35 and 0.28 in stagnant and floating systems respectively [4]. This is much lower than the success reported here (0.73). In many studies, only mature egg strings have been used for production of copepodids [23,36,38,39,43]. Hatching success of immature unpigmented egg strings was generally lower than that of mature dark egg strings, as has been reported for C. elongatus [33], and L. salmonis here. These results indicate that egg development in incubators is sub-optimal compared to remaining attached to the mother on a fish, thus the observed difference in development success between newly fertilised eggs and mature egg strings is probably a good indicator of incubator performance. However, it is important to note that it is physically more difficult to remove newly fertilised egg strings as opposed to mature egg strings from lice. Consequently, it is possible that the greater hatching success for mature egg strings, as opposed to newly fertilised egg strings, may be in part a consequence of this differential handling. The combined infection and survival success measured at 60– 70 DPI was lower in tanks with many small fish than in tanks with few and larger fish. It is our impression that this was predominantly caused

Fig. 6. Allele frequencies for a wild reference population of L. salmonis and 5 laboratory strains at 4 microsatellite loci (LsaSTA1, LsaSTA2, LsaSTA5 and NUIG14) X-axes: W = Wild reference (Kyrkseterøra), Bi = LsBindal (outbred), Gu = LsGulen (outbred), Os = LsOslofjord (partially inbred), 1rr = Ls1rr (inbred) and 1a = Ls1a (inbred). Y-axes: numbering of alleles for each locus. Bubble size indicates allele frequency.

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Table 6 Results of self assignment among 5 laboratory strains of L. salmonis using data from 4 microsatellite loci. Strain

LsGulen

LsBindal

LsGulen LsBindal Ls1a LsOslofjord Ls1rr

45 3

1 27

Ls1a

LsOslofjord

Ls1rr

% correct

26

98 90 100 100 100

30 29

Data represent absolute number of individuals assigned to the different strains using Bayesian assignment and leave one out sub-option in Geneclass V1.0.02. Total correct self-assignment = 98%.

by a higher loss of preadult/adult lice from tanks with small fish. In addition, lice loss from tanks containing small fish was highly variable and loss of female lice was often disproportionately large. Consequently, the amount of egg bearing females available for strain propagation at c 70 DPI from tanks with small fish was unreliable. High death rate and high between tank variations in loss rate of lice from small fish have been reported by other authors [23,44]. A high loss rate of lice from small fish (120–150 g) may partly be explained by density dependent constraints/intraspecific competition since louse intensity per unit area does not tend to increase with size [1,45,46]. Consequently, louse strains were predominantly maintained on small groups of large fish (c >250 g at infection), which permitted harvesting of eggs from female lice as old as 452 DPI (Table 1). These lice were significantly older than those reported by Heuch (191 days) [8] and Mustafa (210 days) [9] and represents the oldest L. salmonis documented. 4.2. Characterisation of strains Genotyping results confirmed the breeding regimes imposed on the L. salmonis strains analysed within. For example, all inbred strains

displayed little or no genetic variation as revealed by number of alleles and heterozygosity, whereas the outbred strains displayed greater variation. Whilst 2 of the outbred strains (LsBindal and LsGulen) displayed the second and third most genetic variation among all samples genotyped, they nevertheless displayed less allelic variation than the wild reference sample from Kyrkseterøra. It is suggested that this is caused by founder effects and genetic drift, both due to a limited number of individuals contributing to each generation. Only 1 of the inbred strains, Ls1a, was monomorphic for all loci screened. However, inbreeding is optimally achieved though full sibling crossing and it takes approximately 20 generations of breeding to establish a fully inbred strain [47]. In the present study, the general inbreeding regime involved using, with some exceptions, 3–4 sibling females contributing to each generation (Table 1). In addition to this, the founder mother of Ls1a proved to be mated by at least 3 males, demonstrated by the fact that altogether 7 alleles were found on the LsaSTA5 locus among her 4 daughters and their children/grandchildren (Fig. 7). Todd et al. demonstrated that it was common for female lice to be polyandrous (dual paternity), but assumed that effective blockage of the copulatory ducts by the first male almost certainly assures single paternity of the first few pairs of egg strings [6]. The founder mother for the Ls1a strain was most likely carrying her second pair of egg strings considering the egg strings were large (data not included) and her age being c. 700 day degrees, thus implying that females do become multiply mated at an early stage. This means that not only does the Ls1a strain consist of a series of half-sibling crossings, the number of parents contributing to each generation was somewhere between at least 2 and 4 times the number of females contributing with eggs. This amounts to a system with a slow rate of inbreeding, and consequently, although Ls1a is monomorphic for the 4 markers analysed here, it is likely that this strain is not fully inbred. Body size varied significantly between the strains. However, observed differences were small and due to the fact that measurements

Fig. 7. Pedigree re-construction for 2 inbred lice strains currently in culture at IMR and 1 discontinued strain. Genetic data (allele fragment size) for mother and daughter 2 represent genotypes (loci separated by blank space), whilst genetic data for all other groups represent alleles present in sample (1 locus per line). For all samples, loci are ordered LsaSTA1, LsaSTA2, LsaSTA5,and NUIG14.

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were not obtained in a controlled experiment, and that a high degree of phenotypic plasticity is displayed by L. salmonis [48,49], it is not possible to identify whether the small differences observed were the result of genetic or environmental factors. Although both inbred strains displayed lower genetic variability compared to the outbred strains, as measured by number of alleles and heterozygosity at 4 microsatellite markers, this was not reflected in an overall lower variability among the morphometric measurements (Tables 3 and 4). The fitness parameters, including their variability, were also similar among the strains (Table 3), except that both inbred strains produced slightly less copepodids/egg string than the outbred strains.

deleterious alleles are effectively purged from the system [63]. If this is the case it may be challenging to find, maintain and study mutations that to any degree compromise lice survival on the host. In summary, the incubator and rearing protocol described within has permitted, for the first time, the establishment and continued culture of defined L. salmonis laboratory strains of different phenotypic and genetic characteristics. Both the facility design, and resultant strains represent a significant resource for the continued development of research into this parasite, both at a basic biological level and in order to develop new measures to control this parasite in the future.

4.3. Application of L. salmonis laboratory strains in research

Acknowledgements

The incubator system and the outbred strains maintained here have been used in a number of studies on L. salmonis, including studies of gene function [50–55], susceptibility [46], and effectiveness of parasiticides [56]. Prior to establishing these laboratory strains, experiments could only be initiated once sufficient lice had been collected at slaughterhouses, and, that enough copepodids were hatched. The established laboratory strains permit a continuous supply of eggs and lice at almost any stage of development and of known genetic background, which in turn allow for more advanced experimental applications. In addition, the demonstrated ability to identify the strains with genetic markers alone or by phenotype (Ls1rr) permits common garden experiments to be conducted. This opens a new niche of experimental possibilities in studies of e.g. host–parasite interactions and/or evolutionary processes. Model species have been of crucial importance in biological research and several species are widely used, ranging from yeasts [57] and nematodes, e.g., Caenorhabditis elegans [58], to Drosophila melanogaster [59], zebrafish (Danio rerio) [60] and mice [61]. Model organisms are often selected for their suitability to address both specific and general biological questions. An important characteristic is that they are relatively easy to maintain, and display a short life cycle enabling rapid experimental manipulation and breeding. This permits the development of strains displaying specific phenotypic and or genetic characteristics (e.g. mutated, inbred or transgenic animals). However, well established model organisms may be unsuitable for addressing systems only present in specialized species. Whereas many bacteria and virus can be grown in artificial media or in cell cultures, most parasite species can only live or develop on or in a particular host species. This makes many parasites particularly difficult to study when access to live animals or particular strains is necessary. Although L. salmonis is a parasite, it fulfils the requirements for a model organism with some limitations. Short generation time enables quick response to breeding, and as demonstrated here, it is possible to establish and maintain trait specific strains of lice in the laboratory over multiple generations and years. The system has allowed us to establish, in addition to the strains presented, both drug resistant strains and sensitive reference strains to study the nature of resistance mechanisms through experimental breeding (Espedal et al. in prep.). L. salmonis is relatively easy to handle, it is flat, and it is sparsely pigmented. This permits the inspection of internal organs of live animals by stereo microscopy. It is available for experimental manipulations, such as RNA interference [62] to study gene function and may serve as a copepod and caligid genomic model. In light of the experiential possibilities available and the fact that the egg production is readily available to inspection and measurements, it is also suggested that L. salmonis has the potential to serve as a copepod-ectoparasite model to study aspects of evolutionary ecology. However, compared to well known model organisms like zebrafish and Drosophila, where high numbers of strains are maintained in small units, facility requirements (tanks and fish) limit the feasibility to maintain high numbers of L. salmonis strains in culture. Furthermore, high natural mortality from the host and few indications of inbreeding depression may suggest that

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