Teleost adaptive immune systems have evolved with more flexibility than previously assumed. A particularly enigmatic system to address immune system modifications in the evolutionary past is represented by the Syngnathids, the family of pipefishes, seahorses and seadragons. These small fishes with their unique male pregnancy have lost the spleen as an important immune organ as well as a functional major histocompatibility class II (MHC II) pathway. How these evolutionary changes have impacted immune cell population dynamics have up to this point remained unexplored. Here, we present the first immune cell repertoire characterization of a syngnathid fish (Syngnathus typhle) using single-cell transcriptomics. Gene expression profiles of individual cells extracted from blood and head-kidney clustered in twelve putative cell populations with eight belonging to those with immune function. Upregulated cell marker genes identified in humans and teleosts were used to define cell clusters. While the suggested loss of CD4+ T-cells accompanied the loss of the MHC II pathway was supported, the upregulation of specific subtype markers within the T-cell cluster indicates subpopulations of regulatory T-cells (il2rb) and cytotoxic T-cells (gzma). Utilizing single-cell RNA sequencing this report is the first to characterize immune cell populations in syngnathids and provides a valuable foundation for future cellular classification and experimental work within the lineage.

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Characterization of Pipefish Immune Cell Populations Through Single-Cell Transcriptomics

Abstract The repeated evolution of the same traits in distantly related groups (convergent evolution) raises a key question in evolutionary biology: do the same genes underpin convergent phenotypes? Here, we explore one such trait, viviparity (live birth), which, qualitative studies suggest, may indeed have evolved via genetic convergence. There are >150 independent origins of live birth in vertebrates, providing a uniquely powerful system to test the mechanisms underpinning convergence in morphology, physiology, and/or gene recruitment during pregnancy. We compared transcriptomic data from eight vertebrates (lizards, mammals, sharks) that gestate embryos within the uterus. Since many previous studies detected qualitative similarities in gene use during independent origins of pregnancy, we expected to find significant overlap in gene use in viviparous taxa. However, we found no more overlap in uterine gene expression associated with viviparity than we would expect by chance alone. Each viviparous lineage exhibits the same core set of uterine physiological functions. Yet, contrary to prevailing assumptions about this trait, we find that none of the same genes are differentially expressed in all viviparous lineages, or even in all viviparous amniote lineages. Therefore, across distantly related vertebrates, different genes have been recruited to support the morphological and physiological changes required for successful pregnancy. We conclude that redundancies in gene function have enabled the repeated evolution of viviparity through recruitment of different genes from genomic “toolboxes”, which are uniquely constrained by the ancestries of each lineage.

/pdf/different-genes-are-recruited-during-convergent-evolution-of-2ln5fj8v.pdf

Different Genes are Recruited During Convergent Evolution of Pregnancy and the Placenta

Viviparity has originated independently more than 150 times in vertebrates, while the male pregnancy only emerged in Syngnathidae fishes, such as seahorses. The typical male pregnancy seahorses have closed sophisticated brood pouch that act as both uterus and placenta, representing an excellent model system for studying the evolutionary process of paternal immune protection. Phylogenetic analysis indicated that the hampII gene family has multiple tandem duplicated genes and shows independent lineage-specific expansion in seahorses, and they had the highest ratio of nonsynonymous substitutions to synonymous substitutions (dN/dS) in the seahorse phylogenetic branch. The expression levels of hampIIs in the brood pouch placenta were significantly higher during pregnancy than non-pregnancy. Both LPS stimulation test in vivo and cytotoxicity test in vitro proved the immunological protection function of hampIIs against pathogen infection in seahorse. Besides, seahorse hampII peptides exhibit weaker antibacterial function, but stronger agglutination and free endotoxin inhibition. We assumed that the modified immunological function seemed to be a trade-off between the resistance to microbial attack and offspring protection. In brief, this study suggests that the rapid co-option of hampIIs contributes to the evolutionary adaption to paternal immune care during male pregnancy.

/pdf/hepcidin-gene-co-option-balancing-paternal-immune-protection-2dslk95z.pdf

Hepcidin Gene Co-Option Balancing Paternal Immune Protection and Male Pregnancy

Pregnancy, the post‐fertilization period when embryos are incubated within the body, is a dynamic multistage process that has convergently evolved in many vertebrates. To increase independence from environmental fluctuations and protect offspring from predation, challenges had to be initially overcome. The most obvious, when considering such an intimate relationship between the parent and its semi‐allogenic offspring, was the pressing need to dodge immunity‐associated embryo rejection. In mammals, immunological tolerance was found to be dependent on the active modulation of the immune system. Even though supporting much of the current knowledge on vertebrate pregnancy, mammals lack extant transitional stages that could help reconstruct the evolutionary pathway of this fascinatingly complex reproduction mode. In this issue of Molecular Ecology, Parker et al. selected an untraditional model—the seahorse and pipefish family, whose species evolved male pregnancy across an almost continuous gradient of complexity, from external oviparity to internal gestation. By contrasting gene expression profiles of syngnathids with distinct brooding architectures, this study allowed for the observation of subtle evolutionary adaptations, while confirming the existence of remarkable similarities to “female” pregnancy (e.g., the evolution of male pregnancy in pouched species occurred alongside immune downregulation, and inflammation seems vital during early pregnancy stages). In a world where the debate on sex‐roles takes centre stage, Parker et al.'s appeasing results hint at the fact that the strongly convergent evolution of vertebrate pregnancy was seemingly unaffected by which sex carries the burden of gestation.

Mom and dad are not that different after all: Immune modulation as a prerequisite for the evolution of pregnancy

Microbial profiles and immune responses in seahorse gut and brood pouch under chronic exposure to environmental antibiotics.

The unique male pregnancy in pipefishes and seahorses ranges from basic attachment (pouch-less species: Nerophinae) of maternal eggs to specialized internal gestation in pouched species (e.g. Syngnathus and Hippocampus) with many transitions in between. Due to this diversity, male pregnancy offers a unique platform for assessing physiological and molecular adaptations in pregnancy evolution. These insights will contribute to answering long-standing questions of why and how pregnancy evolved convergently in so many vertebrate systems. To understand the molecular congruencies and disparities in male pregnancy evolution, we compared transcriptome-wide differentially expressed genes in four syngnathid species, at four pregnancy stages (nonpregnant, early, late and parturition). Across all species and pregnancy forms, metabolic processes and immune dynamics defined pregnancy stages, especially pouched species shared expression features akin to female pregnancy. The observed downregulation of adaptive immune genes in early-stage pregnancy and its reversed upregulation during late/parturition in pouched species, most notably in Hippocampus, combined with directionless expression in the pouch-less species, suggests immune modulation to be restricted to pouched species that evolved placenta-like systems. We propose that increased foeto-paternal intimacy in pouched syngnathids commands immune suppression processes in early gestation, and that the elevated immune response during parturition coincides with pouch opening and reduced progeny reliance. Immune response regulation in pouched species supports the recently described functional MHC II pathway loss as critical in male pregnancy evolution. The independent co-option of similar genes and pathways both in male and female pregnancy highlights immune modulation as crucial for the evolutionary establishment of pregnancy. 

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/mec.16333

Haishan Yin

Papers

Immunological tolerance in the evolution of male pregnancy