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

Abstract

Symbiotic associations are dynamic systems influenced by both intrinsic and extrinsic factors. Here we describe for the first time the developmental and seasonal changes of the funicular bodies in the bryozoan Dendrobeania fruticosa, which are unique temporary organs of cheilostome bryozoans containing prokaryotic symbionts. Histological and ultrastructural studies showed that these organs undergo strong seasonal modification in the White Sea during the ice-free period. Initially (in June) they play a trophic function and support the development of a large population of bacteria. From June to September, both funicular bodies and bacteria show signs of degradation accompanied by development of presumed virus-like particles (VLPs); these self-organize to hollow spheres inside bacteria and are also detected outside of them. Although the destruction of bacteria coincides with the development of VLPs and spheres, the general picture differs considerably from the known instances of bacteriophagy in bryozoans. We broadly discuss potential routes of bacterial infection in Bryozoa and question the hypothesis of vertical transfer, which, although widely accepted in the literature, is contradicted by molecular, morphological and ecological evidence.

Figure 1

General view of a colony branch of Dendrobeania fruticosa with orange oocytes visible inside autozooids and embryos in ovicells (A) (collected on 23 August 2021), and colony fragment (B) (developing polypide buds inside young zooids on the tip of the branch shown by arrows, some ovicells marked by asterisks) (collected on 23 June 2021) (stereomicroscope).

Figure 2

Schemes of fragments of colonies collected in June (B), June–August (C) and September (A), and showing position of autozooids containing FBs at different stages of development and degradation. In (A) and (C) distal tips of the branches are not growing. Distal tip of the fragment in (B) shows zooidal buds, indicating a growing branch. (D) Vertical columns show schematic depictions of longitudinal sections of autozooids (left) and partial FBs (right) at consecutive stages of development (1–8) and degradation (corresponding to Table 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. BB brown body, FB funicular body, P polypide, PB polypide bud.

Figure 3

General view of funicular bodies inside the zooidal cavity in Dendrobeania fruticosa (A–D, collected on 23 June 2018, E, collected on 31 September 2019) (longitudinal stained sections, light microscopy). (A) Mature non-modified FB (arrow) situated between the basal zooidal wall and the degenerating polypide; part of the funicular cord is sandwiched between the FB and the polypide. (B) Zooid showing two cross-sectioned parts of one lobed FB below growing polypide bud, and part of the second FB to the right (all FBs shown by arrows); a large funicular cord runs above both FBs. (C) Mature non-modified FB situated on basal wall of zooid and connected to thin processes of funicular cells. (D) Enlarged view of two parts of one FB shown in (B) (in C and D FBs show the initial stage of degradation, visible as large ‘vacuoles’ potentially reflecting either cell degradation or a fixation artifact). (E) Collapsing FT. bw basal wall, dp degenerating polypide, fc funicular cord, fm frontal membrane, fp forming polypide bud, gy gymnocyst, ts tentacle sheath, zc zooidal cavity.

Figure 4

Early funicular body of Dendrobeania fruticosa (collected on 23 June 2018) (TEM). (A) Whole view of early-stage funicular body. (B) Partial view of cavity and wall of early-stage FB showing external and inner cell layers (arrowheads: multivesicular bodies; arrow: adherens junction with Z-curve between cells). (C) Bacterial symbionts and microvilli formed by cells of inner layer. Inset: enlarged area showing bacterial pili (arrow). ec cells of external layer, fc cells of funicular cords, ic cells of inner layer, n nucleus, rer rough endoplasmic reticulum, zc zooidal cavity.

Figure 5

Scheme of a mature non-modified funicular body of Dendrobeania fruticosa (corresponding to those collected on 23 June 2018) showing two-layered wall structure, thick funicular cord (to the left), and thin processes of funicular cells adjacent to the external cell layer, and bacteria inside the FB internal cavity. Inset: enlarged area showing cells of both external and inner layers and bacteria with cytoplasmic processes in between (not shown in the larger scheme). ec cells of external layer, fc cells of funicular cord, ic cells of inner layer, zc zooidal cavity.

Figure 6

Ultrastructure of mature non-modified funicular body of Dendrobeania fruticosa (collected on 23 June 2018, also shown in Fig. 3C) (TEM). (A) Part of FB with two-layered wall and adjacent funicular cells; non-modified bacteria fill the internal space. (B,C) Area of internal cavity with bacterial symbionts and microvilli formed by cells of inner layer. (D,E) Details of ultrastructure of cells of inner layer; extensive RER, Golgi apparatuses and large multivesicular bodies in various stages of development are visible (inset and E: presumed exocytosis shown with arrowheads) (arrow: adherens junction with Z-curve between cells of inner layer in B and D). aG Golgi apparatus, bw basal wall, ec cells of external layer, ep epithelium of body wall, fc cells of funicular cords, ic cells of inner layer, m mitochondrion, mv multivesicular body, n nucleus, rer rough endoplasmic reticulum, zc zooidal cavity.

Figure 7

Ultrastructure of funicular bodies of Dendrobeania fruticosa (collected on 23 June 2018) showing the initial stage of degradation (TEM). (A) Part of FB densely filled with modified bacteria; large expanded parts of RER shown by arrows (also visible in C and E) (inset: peripheral area of FB wall showing cells of both external and inner layers and forming ‘interlayer’ space (asterisks) containing electron-dense bodies; scale bar 500 nm). (B,C) Bacteria and cells of inner layer with presumed phagosomes (arrowheads) containing bacteria-like content. (D,E) Presumed phagosomes (shown by arrowhead in E) with bacteria-like content shown at higher magnification. (F) Peripheral area of FB wall showing cells of external and inner layers with an interlayer space (ILS) (asterisks) between them containing electron-dense bodies and ‘fibrils’ sometimes in groups (also visible in inset). aG Golgi apparatus, ec cells of external layer, fc funicular cell, ic cells of inner layer, rer rough endoplasmic reticulum, zc zooidal cavity.

Figure 8

Degradation stages of the funicular bodies and their bacterial symbionts in Dendrobeania fruticosa (TEM). (A) Middle-advanced stage showing remaining bacteria inside the cavity of FB near its inner cells; peripheral part of FB with two cell layers and ILS (asterisk here and elsewhere) inbetween are visible in the right upper corner (19 August 2020). (B) Area of FB at the late-advanced stage showing cavity devoid of bacteria and its wall consisting of inner and external cell layers with greatly expanded ILS; inner layer consists of collapsing cells with cytoplasm of various electron density, some still having long and branching cytoplasmic processes; ‘double’ membrane (arrow)—part of collapsing cell, is visible between two cells (inset: enlarged fragment of ILS with tightly packed ‘globules’; scale bar 500 nm) (31 September 2019). (C) Area of FB at the late-advanced stage of FB degradation showing remaining bacteria collapsing inside FB cavity (31 September 2019). ec cells of external layer, fc funicular cell, ic cells of inner layer, zc zooidal cavity.

Figure 9

Stages of formation of putative virus-like particles in funicular bodies at late-advanced stages of degradation (collected on 23 August 2021 and 31 September 2019) (TEM). (A–C) VLP and ‘self-constructed’ spherical complexes inside bacteria (23 August 2021); (D) VLP in symbiont-containing cavity near bacteria and in ILS between two FB cell layers (asterisk) (inset: probable perforation of the inner cell layer acting as a passage for VLPs transfer to ILS shown by arrowheads, scale bar 200 nm) (23 August 2021); (E,F) Self-assembling/constructing of spherical complexes (arrows) inside ILS (31 September 2019). (G) ‘Globules’ (arrowheads) and ‘fibrils’ inside ILS (to the right), and filaments in FB inner cavity (to the left) divided by inner layer cell; (H) ‘globules’ (arrowheads) and ‘fibrils’ inside ILS under higher magnification. ec cells of external layer, ic cells of inner layer, zc zooidal cavity.

Figure 10

Schemes of funicular bodies of Dendrobeania fruticosa at various stages of degradation (from left to right: early-, middle- and late-advanced), showing changes in their structure accompanied by bacterial degradation and putative VLP appearance (magnified sector in B) and self-construction of spherical complexes inside bacteria (magnified sector in C) and inside ILS. Note continuous ‘double’ membrane between internal cavity of funicular body and intercellular space, remaining after collapse of the inner cell layer. ec cells of external layer, fc funicular cell, ic cells of inner layer, zc zooidal cavity.