Fig. 1

Experimental design. The grandparental generation (F0) was vaccinated using a combination of heat-killed immunological novel Vibrio spp. and Tenacibaculum maritimum (F0-bacteria), or were left naïve (F0-N) as control. Immune-challenged mature pipefish were used in following mating design: 1. Control: [♀F0-naïve x ♂F0-naïve]; 2. Paternal: [♀F0-naïve x ♂F0-bacteria]; 3. Maternal: [♀F0-bacteria x ♂F0-naïve] and 4. Biparental: [♀F0-bacteria x ♂F0-bacteria] and kept according to their mating pairs (families) in separate 36 × 80 L semi-flow through aquaria (16 family replicates per parental bacteria treatment and eight per control group; 56 families). F1-individuals were crossed within former parental treatment groups but left immunologically naïve (out of each of the four grandparental treatment groups five families were chosen to do F1-crosses resulting in 20 F1-families). In spring 2014, F2-juveniles were exposed one-week post birth to the same heat-killed Vibrio (F2-V+) and Tenacibaculum (F2-T+) bacteria used for the F0-generation or left naïve (F2-N) (per F1-crossing four families produced F2-offspring resulting in 16 F1-families). Out of each family 12 individuals were chosen for the direct immune challenge. Per F2-offspring treatment (F2-V+, F2-T+, F2-N) four individual replicates were used; resulting in a total of 192 samples