Bacteriophages (phages) are ubiquitous elements in nature, but their ecology and role in animals remains little understood. Sponges represent the oldest known extant animal-microbe symbiosis and are associated with dense and diverse microbial consortia. Here we investigate the tripartite interaction between phages, bacterial symbionts, and the sponge host. We combined imaging and bioinformatics to tackle important questions on who the phage hosts are and what the replication mode and spatial distribution within the animal is. This approach led to the discovery of distinct phage-microbe infection networks in sponge versus seawater microbiomes. A new correlative in situ imaging approach (‘PhageFISH-CLEM‘) localised phages within bacterial symbiont cells, but also within phagocytotically active sponge cells. We postulate that the phagocytosis of free virions by sponge cells modulates phage-bacteria ratios and ultimately controls infection dynamics. Prediction of phage replication strategies indicated a distinct pattern, where lysogeny dominates the sponge microbiome, likely fostered by sponge host-mediated virion clearance, while lysis dominates in seawater. Collectively, this work provides new insights into phage ecology within sponges, highlighting the importance of tripartite animal-phage-bacterium interplay in holobiont functioning. We anticipate that our imaging approach will be instrumental to further understanding of viral distribution and cellular association in animal hosts.

噬菌体（噬菌体）是自然界中普遍存在的元素，但它们的生态学和在动物中的作用仍然知之甚少。海绵代表了现存最古老的动物-微生物共生关系，并与密集且多样化的微生物群落相关。在这里，我们研究噬菌体、细菌共生体和海绵宿主之间的三方相互作用。我们结合成像和生物信息学来解决噬菌体宿主是谁以及动物体内的复制模式和空间分布是什么等重要问题。这种方法导致在海绵和海水微生物组中发现了不同的噬菌体微生物感染网络。一种新的相关原位成像方法（“PhageFISH-CLEM”）将噬菌体定位在细菌共生细胞内，而且定位在吞噬活性海绵细胞内。我们假设海绵细胞对游离病毒粒子的吞噬作用调节噬菌体-细菌比率并最终控制感染动态。噬菌体复制策略的预测表明了一种独特的模式，其中溶原性在海绵微生物组中占主导地位，可能是由海绵宿主介导的病毒粒子清除促进的，而裂解在海水中占主导地位。总的来说，这项工作为海绵内的噬菌体生态学提供了新的见解，强调了动物-噬菌体-细菌三者相互作用在全生物功能中的重要性。我们预计我们的成像方法将有助于进一步了解动物宿主中的病毒分布和细胞关联。