The genes were divided into functional groups based on the available gene ontology data obtained from the Ensembl genome browser versions 91 and 95, The Zebrafish Information Network (ZFIN) and literature (pie chart on the left). Immune response genes were further divided into subcategories (pie chart on the right). The numbers in the sectors indicate the number of genes in each functional group.

Normalized read counts in wild type zebrafish larvae injected with KCl (unchallenged AB) or ~500 cfu of S. pneumoniae (challenged AB). The genes which had normalized read counts of ≥20 after infection and were induced by ≥3-fold are shown. Each dot represents the normalized read count of a sample of 10 larvae. The differential expressions did not reach statistical significance.

A) Survival of three mutant zebrafish lines, mutant94, mutant450, and mutant14, is compromised in pneumococcal infection compared to wild type (AB) fish and other mutant lines (mutant445 and mutant12). In the experiment, mutant and AB embryos were infected with 35–440 cfu of S. pneumoniae at 2 dpf. The graphs represent the results from three (mutant94, mutant450, mutant14, AB) or one survival experiment (mutant445 and mutant12) (n = 24–48 in each). B) Bacterial burden in mutant94 larvae is elevated after pneumococcal challenge compared to AB and other hypersusceptible mutant lines. Embryos were infected with 505 cfu of S. pneumoniae at 2 dpf and bacterial counts were determined at 18 hpi. The circles represent bacterial counts in a single larva from one experiment (n = 8). Median bacterial count is depicted with a line. The statistical comparison of difference was calculated with log-rank (Mantel-Cox) test in A, and with Kruskal-Wallis test with Dunn’s multiple comparisons test in B. cfu = colony forming units.

The figure shows normalized read counts for crp2-1, il1b, il6, and tnfa in wild type larvae injected with KCl (unchallenged AB), wild type larvae infected with ~500 cfu of S. pneumoniae (challenged AB), and mutant94 larvae infected with ~500 cfu of S. pneumoniae (challenged mutant94) at 18 hpi. Each dot represents the normalized read count in a sample of 10 larvae. The differential expressions did not reach statistical significance.

A) A schematic presentation of the zebrafish crp2-1 (crp2, ENSDARG00000056498) gene structure and the gRNA target site for CRISPR-Cas9 mutagenesis. The target site sequence with depicted gRNA target sequence and protospacer adjacent motif (PAM) site were verified with Sanger sequencing. B) Successful mutagenesis in the embryos injected with target specific gRNA and Cas9 protein was verified with heteroduplex mobility assay. In the heteroduplex mobility assay, multiple bands on polyacrylamide gel after heteroduplex formation indicate the presence of insertion/deletion mutations at the target site. C) Schematic representation of the structure of wild type (WT) Crp2-1 protein and the structure of Crp2^tpu6 mutant protein after introduction of +23 nucleotide insertion and premature stop codon by CRIPSR-Cas9 mutagenesis. D) Homozygous crp2^tpu6/tpu6 larvae have normal morphology under standard laboratory conditions. The panel shows an overview and a representative image of a single larvae of 2 dpf wild type (WT (crp2^tpu6)) and homozygous (crp2^tpu6/tpu6) larvae of F3 generation. Images were taken with Lumar V.12 fluorescence stereomicroscope with an exposure time of 100 ms. E) crp2-1 expression is induced in WT (crp2^tpu6) larvae at 24 hpi. F) crp3 expression is induced in homozygous crp2^tpu6/tpu6 larvae at 24 hpi. In E and F, the expression relative to eef1a1l1 expression was measured with the 2^-ΔCt method from the pools of five WT or mutant larvae infected with 241 cfu of S. pneumoniae. The line depicts the median expression level, and the statistical analyses were conducted with Kruskal-Wallis test with Dunn’s multiple comparisons test. Comparisons were only done to 0 h time point of the same gene.

The figure shows the survival of wild type (WT (crp2^tpu6)), heterozygous (crp2^tpu6/+) and homozygous (crp2^tpu6/tpu6) larvae after pneumococcal infection. In the experiment, 1,646 cfu of S. pneumoniae was injected into the bloodstream of 2 dpf larvae and the survival was monitored until 5 dpf. Data were collected from a single experiment with the group sizes (n) indicated in the figure. The statistical comparison of difference was calculated with log-rank (Mantel-Cox) test.

A) Percentage of surviving and normally developing uninjected zebrafish larvae, larvae injected with 4 ng of RC morpholino, and larvae injected with different doses (1 ng, 2 ng or 4 ng) of crp3 SB morpholino. In the experiment, RC or crp3 SB morpholino was microinjected into the yolk sac of AB zebrafish embryos at the 1–4 cell stage and their survival and development was followed for 5 days. The percentage represents the live (black) and morphologically normal (gray) larvae in each group (n = 31–50) at 5 dpi. B) crp3 transcript levels in RC morpholino and crp3 SB morpholino injected AB zebrafish larvae at 0–5 dpi. In the experiment, 4 ng of RC or crp3 SB morpholino was microinjected into the yolk sac of AB zebrafish embryos at 1–4 cell stage and pools of 3–5 larvae (n = 7–8) were collected at specific time points. crp3 expression levels were measured with qPCR from technical duplicates and normalized with eef1a1l1 expression. A dot/circle represents the relative gene expression in a single pool of embryos/larvae, which was calculated using the 2^-ΔCt method, and the line depicts the median expression level. C) The survival of RC or crp3 SB morpholino injected homozygous (crp2^tpu6/tpu6) larvae after pneumococcal infection. In the experiment, 4 ng of RC or crp3 SB morpholino was microinjected into the yolk sac of crp2^tpu6/tpu6 embryos of F3 generation at 1–4 cell stage, and at 2 dpi the larvae were infected with 296 cfu of S. pneumoniae. The survival of infected larvae was monitored until 5 dpf. Data were collected from a single experiment, with the group sizes (n) indicated in the figure. The statistical comparisons of difference were calculated with Kruskal-Wallis test with Dunn’s multiple comparison in B, and with log-rank (Mantel-Cox) test in C. dpi = days post injection.