Measuring the immune system of the three‐spined stickleback – investigating natural variation by quantifying immune expression in the laboratory and the wild

Abstract Current understanding of the immune system comes primarily from laboratory‐based studies. There has been substantial interest in examining how it functions in the wild, but studies have been limited by a lack of appropriate assays and study species. The three‐spined stickleback (Gasterosteus aculeatus L.) provides an ideal system in which to advance the study of wild immunology, but requires the development of suitable immune assays. We demonstrate that meaningful variation in the immune response of stickleback can be measured using real‐time PCR to quantify the expression of eight genes, representing the innate response and Th1‐, Th2‐ and Treg‐type adaptive responses. Assays are validated by comparing the immune expression profiles of wild and laboratory‐raised stickleback, and by examining variation across populations on North Uist, Scotland. We also compare the immune response potential of laboratory‐raised individuals from two Icelandic populations by stimulating cells in culture. Immune profiles of wild fish differed from laboratory‐raised fish from the same parental population, with immune expression patterns in the wild converging relative to those in the laboratory. Innate measures differed between wild populations, whilst the adaptive response was associated with variation in age, relative size of fish, reproductive status and S. solidus infection levels. Laboratory‐raised individuals from different populations showed markedly different innate immune response potential. The ability to combine studies in the laboratory and in the wild underlines the potential of this toolkit to advance our understanding of the ecological and evolutionary relevance of immune system variation in a natural setting.

The procedure to produce to create primary cell culture is as follows: -Dissagregate cells by passing whole spleen or head kidneys through a 40μM cell strainer and suspend in 5ml of RPMI --Centrifuge cell suspension for 2 minutes at 400rfc to pellet cells -Pour off excess RPMIand re-suspend cells. Top up with 4ml RPMI --Repeat centrifugation and re-suspension step twice, using RMPIfor the first and RPMI + for the second.
-After final centrifugation, remove excess liquid with a pipette, and re-suspend cells in 400μl of RPMI + From the 400μl cell suspension, count the number of cells present in 20μl by staining with Trypan Blue. Adjust the concentration of cells in each sample to give 4 x 10 6 cells/ml. Cells should be cultured in a 100μl final volume, giving 40,000 cells/well. Culture cells in 96-well flat bottomed cell culture plates. Add 75μl of cell suspension to 4 wells for each sample (2 control wells, 2 treatment wells). To the control wells, add 25μl additional RPMI + . To the treatment wells, add 25μl of 50μg/ml Zymosan (Sigma-Aldrich Z4250).
Plates should be placed into an incubator at 20 o C, with 5% CO 2 and saturated humidity.
For Zymosan stimulated cells, culture time should be approximately 24 hours, to allow an immune response to develop. Zymosan is a general antagonist of the immune system, here being used to promote an innate immune response. The use of other antagonists is possible, and combined with for longer culture times, allows for the activation of other parts of the immune system. Cells have been successfully cultured for up to 92 hours.
When removed from incubator, cells can be stored at -80 o C until ready to extract RNA.

Effect of Sampling Order on Immune Gene Expression
To ensure that the time which fish were held before processing and the order in which they were processed did not affect the expression of immune system genes, the correlation between sampling order and each of the immune groupings was calculated. Values were calculated using the 'corr.test' function of the 'psych' package in R v.3.1.2, with the Holm correction applied for multiple comparisons.
No significant correlations were found between sampling order and innate expression (r 2 =-0.28, p=0.09), Th1-type expression (r 2 =0.11, p=1), Th2-type expression (r 2 =0.12, p=1), FoxP3a expression (r 2 =0.07, p=1), and TGFβ expression (r 2 =-0.11, p=1), as shown in figure S1. 1 'Short Name' and 'Full Name' refer to names used for genes in the ENSEMBL genome database. 'Response' indicated the arm of the immune response for which each gene is a marker. 'Sequence' denotes whether the gene was found in the stickleback genome, with 'Full' genes being named in the genome, and 'Partial' being found using homology to the gene sequence from another fish species, with corresponding ENSEMBL Gene ID numbers. 'Amplification' showed whether any primers amplified for a given gene, whilst 'Assay' indicated whether a fully working assay could be produced. '**' denotes a gene where 2 copies where present in the genome, and '***' a gene where three copies were present.