Signatures and likely sources of the male pregnancy microbiome in wild bay pipefish (Syngnathus leptorhynchus).

backgroundUnderstanding the origin and structure of microbiomes and their associations with ecologically significant host traits is essential for understanding the evolution of host-microbe interactions. These interactions support a wide range of physiological processes important for development, survival, and reproduction. Syngnathid fishes (seahorses, pipefish, and seadragons) represent a compelling system for investigating host-microbiome interactions due to their unique evolution of male pregnancy. Males harbor a fitness-critical brood pouch that provides embryos with protection, osmoregulation, and nutrient exchange through a placenta-like structure, all while requiring the male to modulate his immune system to accommodate developing offspring. These features create a tightly regulated internal environment where microbial interactions could be especially influential to supporting a successful pregnancy. While we have some understanding of the physiological and genetic factors underlying brood pouch development and maintenance, the role of the microbiome and host-microbe interactions in male pregnancy has remained underexplored across the broader diversity of Syngnathidae species and geographic regions. To investigate this relationship further, and for the first time sampling microbiota from a wild syngnathid population, we characterized microbiomes of the bay pipefish (Syngnathus leptorhynchus) using high-throughput 16S rRNA gene sequencing. We quantified microbial community diversity and composition across the brood pouch, embryos, ovaries, gills, intestines, and outer skin body tissues, focusing on sex-specific (brood pouch, embryo, ovary) and sex-shared (gill, intestine, skin) tissues, and variation in pregnancy stage (non-pregnant, early, mid, and late pregnancy).

resultsWe found that the male brood pouch microbiome was distinct from all other body sites (ovaries, embryos, gills, intestines, and outer skin) in community composition, and in that it exhibited the highest richness and phylogenetic diversity of microbes of any site on average, possibly supporting a specialized environment for embryonic development. Moreover, we found that microbial diversity was lower in non-pregnant brood pouches compared to each pregnancy stage (non-pregnant, early, mid, and late pregnancy) but found no significant differences among the pregnancy stages. Female ovaries had the lowest microbial richness and phylogenetic diversity compared to nonpregnant brood pouches, pregnant brood pouches, and embryos. Source tracking analysis using fast expectation-maximization for microbial source tracking (FEAST) indicated that the male outer skin serves as a significant microbial source for both the pregnant brood pouch and developing embryos, establishing a strong paternal influence on the offsprings’ microbial communities. Overall, we identified Proteobacteria, Bacteroidota, Cyanobacteria, Planctomycetota, and Actinobacteriota as the dominant phyla spanning all surveyed bay pipefish tissue sites, consistent with previous teleost fish microbiome studies. Analysis of core microbiome and indicator species further revealed that sequences classified as Methylotenera_A_557637 (two species), GCA-2862085 sp., Yoonia_491068 sp., Pla163 sp007750655, and Roseibacillus_B sp. show relatively high abundance and specificity with respect to the male brood pouch, suggesting that these taxa may have functional connections to the biology of male pregnancy.

conclusionsThese findings reveal insights into the microbial ecology of a unique reproductive system in its natural environment, highlighting the paternal microbiome’s potential functional role in shaping the developing offspring. Our results also indicate a likely influence of both environmental and host-specific factors in shaping the bay pipefish microbiome, but there is need for future research on the functional implications of these microbial communities, especially in the brood pouch during pregnancy, and with respect to offspring viability and fitness.