The rearing experiments described here were conducted at the Institute of Marine Research’s experimental facility in Bergen, Norway. This laboratory has a quarantine facility with permit issued by the Norwegian Food Safety Authority (NFSA) to conduct experiments on marine pathogens and includes chlorination of all waste-water to ensure that no pathogens are released to the natural environment. Lice were cultured using well-established rearing protocols for L. salmonis[30 (link)]. During the experiments Atlantic salmon were kept in running seawater and fed commercial fish feed ad libitum. This experiment was conducted in accordance with Norwegian legislation for use of animals in research, and was approved by the Norwegian Animal Research Authority (research permit nr. 2009/186329).
Lice were cultured by infecting Atlantic salmon (Salmo salar) with copepodids from Atlantic and Pacific strains of L. salmonis. The Atlantic strain (LsAtl) was established by mixing approximately equal numbers of copepodids from the previously described LsGulen and LsOslofjord strains [30 (link)]. The Pacific strain (LsPac) was established from copepodids derived from adult female L. salmonis collected at a commercial salmon farm located close to the town of Campbell River (British Columbia, Canada). The Pacific lice were transported to Norway in thermal flasks containing full strength salinity seawater. A permit to import these lice was obtained from the NFSA (Additional file1 ). Unfertilized L. salmonis from the LsPac F1 and the LsAtl F2 generations were sorted according to sex at the pre-adult II stage and used immediately (LsAtl) or kept separate (LsPac) on previously uninfected fish until used in the crossing experiment.
To obtain hybrid strains we crossed LsPac females with LsAtl males and LsAtl females and LsPac males using a similar tank rearing system to that described by Hamre and Nilsen [31 (link)]. Briefly, each cross consisted of two (in one instance one) females and one male which were placed on a single uninfected Atlantic salmon that was isolated in its own tank. This allowed definite control of the parent material and offspring generation. As the unfertilized females were in different stages of development (pre-adult II and adults), the time required to obtain fertilized egg strings from the two hybrid strains were dissimilar. After 15 (LsPac females and LsAtl males) and 35 (LsAtl females and LsPac males) days, females bearing egg strings, and the males used to fertilize them in the single fish tanks, were harvested. These sampled adults were stored in individual tubes containing 100% ethanol, and were indexed so that both parents and egg strings could be subsequently matched. The egg strings from the sampled females were incubated in separate incubators after removal of approximately 3-5mm of both egg strings which was stored in 100% ethanol. The parents and the 3-5mm section of egg strings were genotyped (described below) to validate the crosses by parentage assignment before the resulting copepodids were used to infect previously uninfected Atlantic salmon to produce the LsAtlPac (copepodids from LsAtl females and LsPac males) and LsPacAtl (copepodids from LsPac females and LsAtl males) F1 hybrid strains. The F1 hybrid strains were reared separately in replicate fish tanks (Figure1 ).
The F1 generation hybrids were allowed to develop, fertilize and reproduce naturally on Atlantic salmon in their respective tanks. Approximately 3 months after infection with F1 hybrid copepodids, eggstrings from F1 hybrid females that had been fertilized by F1 hybrid males were harvested and incubated in separate containers for LsAtlPac and LsPacAtl. Copepodids arising from these egg strings were thereafter used to infect groups of previously uninfected Atlantic salmon in order to establish the LsAtlPac F2 and LsPacAtl F2 hybrid generation in two replicate tanks for each strain. The resulting F2 hybrid generations were allowed to develop until the pre-adult stage to allow sex determination before the experiments were terminated. Numbers of adults contributing to each generation were recorded. In addition, the numbers of copepodids that were used to propagate the F2 generation was estimated by counting an aliquot, and the numbers of pre-adults harvested from the F2 generation upon termination of the experiment, were determined by counting and sex determining all the lice present.
Lice were cultured by infecting Atlantic salmon (Salmo salar) with copepodids from Atlantic and Pacific strains of L. salmonis. The Atlantic strain (LsAtl) was established by mixing approximately equal numbers of copepodids from the previously described LsGulen and LsOslofjord strains [30 (link)]. The Pacific strain (LsPac) was established from copepodids derived from adult female L. salmonis collected at a commercial salmon farm located close to the town of Campbell River (British Columbia, Canada). The Pacific lice were transported to Norway in thermal flasks containing full strength salinity seawater. A permit to import these lice was obtained from the NFSA (Additional file
To obtain hybrid strains we crossed LsPac females with LsAtl males and LsAtl females and LsPac males using a similar tank rearing system to that described by Hamre and Nilsen [31 (link)]. Briefly, each cross consisted of two (in one instance one) females and one male which were placed on a single uninfected Atlantic salmon that was isolated in its own tank. This allowed definite control of the parent material and offspring generation. As the unfertilized females were in different stages of development (pre-adult II and adults), the time required to obtain fertilized egg strings from the two hybrid strains were dissimilar. After 15 (LsPac females and LsAtl males) and 35 (LsAtl females and LsPac males) days, females bearing egg strings, and the males used to fertilize them in the single fish tanks, were harvested. These sampled adults were stored in individual tubes containing 100% ethanol, and were indexed so that both parents and egg strings could be subsequently matched. The egg strings from the sampled females were incubated in separate incubators after removal of approximately 3-5mm of both egg strings which was stored in 100% ethanol. The parents and the 3-5mm section of egg strings were genotyped (described below) to validate the crosses by parentage assignment before the resulting copepodids were used to infect previously uninfected Atlantic salmon to produce the LsAtlPac (copepodids from LsAtl females and LsPac males) and LsPacAtl (copepodids from LsPac females and LsAtl males) F1 hybrid strains. The F1 hybrid strains were reared separately in replicate fish tanks (Figure
The F1 generation hybrids were allowed to develop, fertilize and reproduce naturally on Atlantic salmon in their respective tanks. Approximately 3 months after infection with F1 hybrid copepodids, eggstrings from F1 hybrid females that had been fertilized by F1 hybrid males were harvested and incubated in separate containers for LsAtlPac and LsPacAtl. Copepodids arising from these egg strings were thereafter used to infect groups of previously uninfected Atlantic salmon in order to establish the LsAtlPac F2 and LsPacAtl F2 hybrid generation in two replicate tanks for each strain. The resulting F2 hybrid generations were allowed to develop until the pre-adult stage to allow sex determination before the experiments were terminated. Numbers of adults contributing to each generation were recorded. In addition, the numbers of copepodids that were used to propagate the F2 generation was estimated by counting an aliquot, and the numbers of pre-adults harvested from the F2 generation upon termination of the experiment, were determined by counting and sex determining all the lice present.
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