Unique features of immunity and reproduction in seahorses.

a A species tree showing the evolution of specialized male pregnancy and asplenia traits in the seahorse. During pregnancy, embryos implanted in the “pseudoplacenta” of male seahorses are recognized by the paternal immune system. b Four seahorse species, from the major lineages of seahorses were included in the comparative genomic analyses. Newly sequenced species are indicated in red. Ma, million years ago. c The top 30 KEGG pathways of contracted gene families in seahorses. Categories involved in immunity are colored in brown. The figures were created with BioRender.com. The used map was downloaded from a free world map website (https://www.freeworldmaps.net/outline/maps.html). Source data are provided as a Source Data file.

Seahorse-specific mutation of the tlx1 gene.

a Schematic diagram of the involved genes during spleen growth and morphogenesis (modified from previous studies¹⁸). Red color Tlx1, the sequence-specific variation in seahorses; RA, retinoic acid. b Gene structure (upper panel) and multiple amino acid alignments (lower panel) of Tlx1. Seahorse harbors a missense mutation of A (Alanine, Ala) to T (Threonine, Thr) in the homeodomain. c Extended Data examination of the identified tlx1 mutation in 18 seahorse species. All detected seahorses exhibit T (ACT) instead of A (GCT or GCC or GCG), unlike other vertebrates. The figure was created with BioRender.com.

Missense mutation of tlx1 controls asplenia phenotype in zebrafish.

a Genome editing of three zebrafish lines (tlx1^▲, tlx1^A208T and tlx1^A207T) using CRISPR/Cas9 technology. Procedures are detailed in Supplementary Fig. 15. ^▲ represents full knockout of the gene, while ^A208T and ^A207T indicate the specific point mutations at sites 622 and 619 of zebrafish tlx1, respectively. Blue, tlx1^▲; Red, tlx1^A208T; Yellow, tlx1^A207T. The tlx1 nucleotide sequence of genomic DNA of three zebrafish lines was validated by Sanger sequencing. The figure was created with BioRender.com. b, ctlx1^▲ and tlx1^A208T zebrafish exhibited asplenia while tlx1^A207T zebrafish had an intact spleen; the asplenia ratio of individuals was calculated and listed (n = 11~23). The splenic phenotype of other 74 individuals were listed in Supplementary Figs. 16–18. WT wild type. d Differentially expressed gene (DEG) number of tlx1^▲ and tlx1^A208T compared to that of wild-type individuals via comparisons of the brain-, kidney-, intestine-, and liver-transcriptomes. e Volcano map of the shared and group-specific DEGs in the brain of tlx1^▲ and tlx1^A208T compared to that in the wild-type group. Blue, red, and gray represent significantly changed DEGs in tlx1^▲, tlx1^A208T, and both groups, respectively. f GO enrichment of the DEGs between tlx1^▲ and tlx1^A208T illustrate the different in gene expression patterns associated with brain development and function. The enrichment was conducted using the GOseq R package, and corrected P < 0.05 indicated significant enrichment. The size of the circle represents the number of genes in each category. Source data are provided as a Source Data file.

Immunogenomic basis of asplenia and male pregnancy in seahorses.

a Copy numbers of key genes involved in the development and function of DC, MHC, T/B lymphocytes, and complement components 3–4 in seahorses and other vertebrates. DC dendritic cell, MHC major histocompatibility complex. *, variation of ITAM (immunoreceptor tyrosine-based activation motif) region; †, based on partial sequence (low-coverage sequencing and genome assemblies), but featuring the variation of ITAM; #, Total number of MHC II. The brood pouch types are shown on the right. Hippocampus and Syngnathus species exhibit the closed brood pouches. b Schematic map illustrating the hypothesized molecular trade-offs in the male pregnancy of seahorses. Asplenia caused by the tlx1 mutation could decrease the CD5⁺ B cell populations. In addition to the corresponding reduced diversity in antibodies, the classical complement pathway triggered by antibody-antigen-interactions might also be weakened, affecting the downstream events usually triggering allograft rejection. Loss of C4 is expected to be associated with decreased abundance of Treg cells, which orchestrate self-immune tolerance. The shaded icon and dotted line indicate gene loss in seahorse genomes. Treg cell, regulatory T cell. The figures were created with BioRender.com.