Real-time quantitative PCR analysis of rainbow trout immune related gene expressed during exposure to Diplostomum pseudospathaceum

Marker-assisted selective breeding of fish with higher levels of resistance towards specific pathogens has shown successful, but. However, the impact of host genotype on multiple pathogen infections are is still poorly investigated. This study examined the resistance in rainbow trout (Oncorhynchus mykis) towards infection with the eye fluke Diplostomum pseudospathaceum. We used genetically selected rainbow trout, carrying SNPs associated with resistance towards the parasitic ciliate Ichthyophthirius multifiliis, and exposed the fish to eye fluke cercariae. We showed that fish partly resistant to I. multifiliis were more susceptible to eye fluke invasion. Expression The expression of immune relevant genes (encoding innate and adaptive factors) was also affected as these genotypes responded less strongly to a secondary fluke infection. The complexity of genome architecture in disease resistance towards multiple pathogens is discussed. A total of 200 rainbow trout (body weight 14.3-17.7 g, body length 10.2-11.5 cm) were used for the study. Two groups of rainbow trout with high (QTL fish) and low (non-QTL fish) frequency of SNPs associated with I. multifiliis resistance, were hatched from eyed eggs at the disease free recirculated Bornholm Salmon Hatchery, Nexø, Denmark and subsequently reared to the fingerling stage. For this purpose, the first group (QTL-fish) was produced by using sperm from three male genotyped parents carrying SNPs AX-89947214 (Omy17) and AX-89960822 (Omy16), and the other group (non-QTL fish) was produced by using sperm from three other male parents negative for these SNPs. In both cases, sperm was used to fertilize a common pool of eggs stripped from a total of 30 outbred females. Processes of hatching and subsequent rearing of fry to the fingerling stage did not differ between groups of QTL fish and non-QTL fish. From each group (QTL and non-QTL fish) we randomly gathered 100 rainbow trout and transported them (3 h duration) from the hatchery to the infection facility at the University of Copenhagen. Fish were then accommodated and acclimatized 14 d in identical aerated glass tanks with internal biofilters (25 fish per 60 L water, total tank volume 80 L), which were placed in a temperature temperature-controlled room (water temperature constant at 12°C, pH 7.6). We used 30% water change per day to maintain ammonia levels below 0.25. Fish were fed by pelleted feed (1% of fish biomass per day). All fish were genotyped with respect to the two relevant QTLs, one on chomosome 16 and one on chromosome 17. Fish being double heterozugous were excluded from qPCR analysis. Exposure of fish to cercariae was conducted in duplicate for each group (QTL fish and non-QTL fish). Group 1. A total of 100 fish (50 QTL and 50 non-QTL fish) was exposed to 240 cercariae/fish during a 48 h period starting from day 0. Group 2. Fish (10 QTL and 10 non-QTL) were kept un-infected until exposure on day 10, at which time the previously exposed fish (Group 1) also were exposed (re-exposure, see below). This group was considered as a control for second exposure in Group 1. Group 3. A total of 80 fish (40 QTL-fish and 40 non-QTL fish) were kept as uninfected time-point control (not exposed) throughout the experiment. Re-exposure (lasting 72 h) to cercariae (480 cercariae/fish) was initiated on day 10 on Group 1 and Group 2. Samples comprised 2x5 fish per group per time-point, comprising eyes for enumeration of metacercariae, gill and spleen for gene expression and fins for genotyping. Time-points were 5h post exposure (5hpe), 4, 7, 20 days post exposure (dpe) (Group 1 and 3) and 20 dpe for Group 2. An additional sampling was done for group 1 at 27 dpe (for enumeration of metacercaria only). Thus, these samples were not subjected to Real-time quantitative PCR analysis.