First evidence of virus-like particles in the bacterial symbionts of Bryozoa

Abstract

23Bacteriophage communities associated with humans and vertebrate animals have been 24 extensively studied, but the data on phages living in invertebrates remain scarce. In 25 fact, they have never been reported for most animal phyla. Our ultrastructural study 26 2 showed for the first time a variety of virus-like particles (VLPs) and supposed virus-27 related structures inside symbiotic bacteria in two marine species from the phylum 28 Bryozoa, the cheilostomes Bugula neritina and Paralicornia sinuosa. We also 29 documented the effect of VLPs on bacterial hosts: we explain different bacterial 30 'ultrastructural types' detected in bryozoan tissues as stages in the gradual destruction 31 of prokaryotic cells caused by viral multiplication during the lytic cycle. We speculate 32 that viruses destroying bacteria regulate symbiont numbers in the bryozoan hosts, a 33 phenomenon known in some insects. We develop two hypotheses explaining exo-and 34 endogenous circulation of the viruses during the life-cycle of B. neritina. Finally, we 35 compare unusual 'sea-urchin'-like structures found in the collapsed bacteria in P. 36 sinuosa with so-called metamorphosis associated complexes (MACs) known to trigger 37 larval metamorphosis in a polychaete worm. 38 39 Importance 40 Complex symbiotic systems, including metazoan hosts, their bacterial symbionts and 41 bacteriophages are widely studied using vertebrate models whereas much less is 42 known about invertebrates. Our ultrastructural research revealed replication of the 43 viruses and/or activation of virus related elements in the bacterial symbionts inhabiting 44 tissues of the marine colonial invertebrates (phylum Bryozoa). The virus activity in the 45 bacterial cells that are believed to be transmitted exclusively vertically is of a special 46 importance. In addition, in the bacterial symbionts of one of the bryozoan hosts we 47 observed the massive replication of the structures seemingly related to the 48 Metamorphosis associated complexes (MAC). To our knowledge, MACs were never 49 reported in the animal prokaryotic symbionts. Our findings indicate that Bryozoa may be 50 new suitable model to study the role of bacteriophages and phage-related structures in 51 the complex symbiotic systems hosted by marine invertebrates.52 3In the marine realm, the large variety of viruses are found across diverse taxa, 78 including protists and various invertebrates such as sponges, cnidarians, flatworms, 79 polychaetes, mollusks, crustaceans and echinoderms (reviewed in Johnson

Seasonal dynamics of a complex cheilostome bryozoan symbiosis – vertical transfer challenged

“…Their development following the total disappearance of bacteria in FBs indicates their bacteriophage nature. If so, our observations support the idea that viruses control the number of symbionts in their bryozoan host 20 , as has been reported in some insects 85,95 . 1): 1-distal zooidal bud and young autozooid with a polypide bud inside (no FBs); 2 -young autozooid with functional polypide and early developing FB; 3 -young autozooid with functional polypide and mature FB; 4 -autozooid with brown body, polypide bud and mature FB at initial stage of degradation; 5 -autozooid with brown body, functional polypide and FB at early-advanced stage of degradation; 6 -autozooid with brown body and FB at mid-advanced stage of degradation; 7 -autozooid with brown bodies and FB at late-advanced stage of degradation; 8 -autozooid with brown bodies and two FBs at terminal stage of degradation.…”

“…Our TEM study of FBs in D. fruticosa showed that they are not swollen parts of the funicular cords, as was stated by Vishnyakov with co-authors for B. neritina 20 . It is more likely that the funicular cords and processes of their cells contact the external cell layer of FBs.…”

“…Combined, all these data imply that bacteria move from FBs to the ooecial vesicle, accumulate there, and then somehow enter the brood cavity, which contains a larva, either through or in-between the epithelial cells and the cuticle of the ooecial vesicle. Findings of bacteria inside larvae and adult colonies of two species from the non-related genus Watersipora 62,68 further strengthened the hypothesis of vertical transfer, which has subsequently been widely accepted by many authors 20,30,47,78 . Despite extensive TEM studies, no bacteria have been found inside the funicular cords in B. neritina (Vishnyakov & Ostrovsky, unpublished data), which contradicts the data of Sharp and coauthors 40 obtained by fluorescent microscopy Accordingly, it was suggested that coelomocytes carry symbionts to the ooecial vesicle instead 20 .…”

“…TEM data showed no communication between the FB cavity and lacunae of the funicular cords in the studied species, in particular in B. neritina (Vishnyakov & Ostrovsky, unpublished data), Aquiloniella scabra 25 and D. fruticosa (this study). Coelomocytes (and presumed peritoneal cells) with bacteria embedded in their cytoplasm were indeed recorded inside the ooecial vesicle in B. neritina 20 . Nevertheless, extensive TEM studies of B. neritina ovicells at various stages of placental development (Vishnyakov & Ostrovsky, unpublished data) have not revealed bacteria between placental cells adjoining a developing embryo.…”

“…It was suggested that these structures serve as sites for incubation and multiplication of bacteria 25 , which can later move to the funicular cords and travel inside them towards incubation chambers containing a larva, thereby corroborating the idea of their vertical transfer 47 . In addition, collapsing bacteria and a variety of virus-like particles were recently detected in the funicular bodies of two bryozoan species 20 .…”