Interacting effects of sea louse (Lepeophtheirus salmonis) infection and formalin-killed Aeromonas salmonicida in Atlantic salmon skin transcriptome

Lepeophtheirus salmonis (sea lice) and bacterial co-infection threatens wild and farmed Atlantic salmon performance and welfare. The present microarray-based study examined the dorsal skin transcriptome response to formalin-killed Aeromonas salmonicida bacterin (ASAL) in pre-adult sea lice-infected and non-infected Atlantic salmon to fill the existing knowledge gap and aid in developing anti-co-infection strategies. To this aim, sea lice-infected and non-infected salmon were intraperitoneally injected with either phosphate-buffered saline (PBS) or ASAL (i.e., 4 injection/infection groups: PBS/no lice, PBS/lice, ASAL/no lice, and ASAL/lice). The analysis of the dorsal skin transcriptome data [Significance Analysis of Microarrays (5% FDR)] identified 345 up-regulated and 2,189 down-regulated DEPs in the comparison PBS/lice vs. PBS/no lice, and 82 up-regulated and 3 down-regulated DEPs in the comparison ASAL/lice vs. ASAL/no lice. The comparison ASAL/lice vs. PBS/lice identified 272 up-regulated and 11 down-regulated DEPs, whereas ASAL/no lice vs. PBS/no lice revealed 27 up-regulated DEPs. The skin transcriptome differences between the co-stimulated salmon (i.e., ASAL/lice) and PBS/no lice salmon accounted for 1,878 up-regulated and 3,120 down-regulated DEPs. Groups of 35 salmon smolts [238.9 ± 45.2 g; mean weight ± standard deviation (SD)] were allocated in four 620-L tanks and exposed to Lepeophtheirus salmonis (sea lice) copepodids (at a ratio of 50 lice/fish). In parallel, groups of 15 salmon from the same population were distributed into two 620-L tanks to serve as no-lice infection controls. Four weeks after sea lice exposure, when lice were at the pre-adult stage, lice-infected salmon and non-infected salmon were subjected to an intraperitoneal injection of either phosphate-buffered saline (PBS), a solution of polyriboinosinic polyribocytidylic acid (pIC) or a PBS suspension of formalin-killed Aeromonas salmonicida (ASAL). For each tank holding infected salmon, 6 fish were injected with PBS, 6 with pIC, and 6 with ASAL. For each tank with non-infected salmon, 4 fish were injected with PBS, 4-5 fish with pIC, and 4-5 fish with ASAL. Samples of dorsal skin around louse attachment sites and adjacent intact skin were collected at 24 h post-injection. The microarray experiment analyzed the general skin transcriptome response to sea lice and ASAL co-stimulation, so it only used dorsal skin samples adjacent to louse attachment sites. The analysis employed a common reference microarray experiment design. The test RNA samples included in the experiment came from 6 PBS/no lice, 6 PBS/lice, 6 ASAL/no lice, and 6 ASAL/lice (24 samples in total). The common reference was prepared using pools of equal quantities of RNA from the 24 selected salmon for the present study, plus 6 pIC/no lice and 6 pIC/lice salmon. This design will allow future analyses of the shared molecular responses of Atlantic salmon dorsal skin to ASAL and pIC IP-injections, alone or in combination with lice infection. variable - other: PBS/no lice variable - other: ASAL/no lice variable - other: PBS/lice variable - other: ASAL/lice biological replicate: PBS_NoLice_T30F1, PBS_NoLice_T30F3, PBS_NoLice_T30F4, PBS_NoLice_T33F2, PBS_NoLice_T33F3, PBS_NoLice_T33F4 biological replicate: ASAL_NoLice_T30F2, ASAL_NoLice_T30F3, ASAL_NoLice_T30F4, ASAL_NoLice_T33F2, ASAL_NoLice_T33F3, ASAL_NoLice_T33F5 biological replicate: PBS_Lice_T12F6, PBS_Lice_T2F2, PBS_Lice_T2F5, PBS_Lice_T5F3, PBS_Lice_T5F4, PBS_Lice_T8F4 biological replicate: ASAL_Lice_T12F1, ASAL_Lice_T12F4, ASAL_Lice_T2F2, ASAL_Lice_T5F4, ASAL_Lice_T8F5, ASAL_Lice_T8F7