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Title: Forever competent: Deep-sea bivalves are colonized by their chemosynthetic symbionts throughout their lifetime
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Wentrup et al.
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Table S1: Summary of observations made in this study of symbiont abundance and distribution in gill tissues of juvenile and adult
Bathymodiolus azoricus and B. puteoserpentis using FISH and TEM, and our interpretations of these observations. Because both the sulfur- and
methane-oxidizing symbionts were involved in the initial colonization of gill cells, we do not distinguish between these symbionts in this table.
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6a
Observations
Budding zone and the youngest filaments lacked
symbionts.
(FISH and TEM analyses; Figure 1c and 2a)
Symbionts were first observed in the 7th – 9th filament
(counting from the budding zone). Filaments that were
ontogenetically older than the 7th to 9th filament
contained progressively more and more symbionts.
(FISH and TEM analyses; Figure 1c and 2a)
Within a filament the two putative filament growth
regions (ventral and dorsal ends of the lamellae) lacked
symbionts.
(FISH analyses; Figure 1c, e and S1a, b, c)
The hemolymph lacunae lacked symbionts.
(FISH analyses; Figure 1h)
In newly colonized bacteriocytes the first symbionts
occurred in the apical region close to the externally
facing host cell membrane.
(FISH and TEM analyses; Figure 1h and 2d)
In the posterior growth zone, descending and ascending
lamellae of a given filament are formed at the same
time.
(FISH analyses; Figure 1c)
The first symbionts occurred in the descending
lamellae, while the corresponding ascending lamella of
a given filament lacked symbionts.
(FISH analyses; Figure 1c)
Interpretation
Newly formed gill cells in the posterior growth zone do not
contain symbionts. Symbiont colonization proceeded from
ontogenetically older to ontogenetically younger filaments.
These observations indicate that symbiont colonization can
only occur after host cells have reached a certain
differentiation stage.
Newly formed gill cells in the ventral and dorsal growth
regions also do not contain symbionts. This indicates, as
observations 1 and 2) that symbiont colonization is influenced
by developmental factors.
This indicates that the symbionts do not colonize newly
formed gill cells internally via the hemolymph system.
This suggests that symbiont cells are acquired from the
exterior.
Descending and ascending lamellae of a given filament are
formed at the same time in Bathymodiolus, as known from
other bivalves (Neumann and Kappes, 2003). The gradients in
colonization patterns described in 6a) - 6e) occurred in
filaments of the same ontogenetic age. This suggests that
symbiont colonization is not solely determined by
developmental factors. We hypothesize that self-infection best
explains the observed gradients because the first colonized gill
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Remarks
We were not able to resolve how
symbiont numbers increased after
initial colonization of newly formed
filaments, e.g. through continuous
uptake of symbionts, intracellular
symbiont proliferation, and/or
bacteriocyte proliferation (see
Discussion).
This has been proposed in earlier
studies based on morphological and
molecular observations (see
Discussion).
We have no explanation for why the
descending lamella is colonized before
the ascending lamella of a filament.
6b
6c
6d
6e
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In newly formed descending lamellae, symbionts
occurred on the anterior and not the posterior side of a
given filament.
(TEM and FISH analyses; Figure 1c and 2a)
In older descending lamellae, symbiont abundance
increased progressively dorsoventrally and laterally.
(TEM and FISH analyses; Figure 1c and 2a)
In newly formed ascending lamellae, symbionts first
occurred in gill cells at the abfrontal end.
(FISH analyses; Figure 1c, g)
In older ascending lamellae, symbiont abundance
increased progressively from the abfrontal to the frontal
end on both the anterior and posterior sides of a given
filament.
(FISH analyses; Figure 1c, g)
The ciliated frontal ends of descending and ascending
lamellae lacked symbionts.
(FISH analyses; Figure 1c, e, g)
Gill cells of young uncolonized filaments were
columnar and their apical ends were densely covered by
microvilli, while bacteriocytes at an early colonization
stage with a few symbionts had almost no microvilli.
Ontogenetically older, fully-developed bacteriocytes
with numerous symbionts lacked microvilli completely.
(TEM; Figure 2)
cells were always those closest to already colonized gill
tissues. If environmental infection was the main colonization
mode we would expect a more random colonization pattern
without a gradient (Figure 5).
This is most likely due to the specific function of ciliated cells
and mucus cells at the frontal edges of lamellae.
We hypothesize that the invasion of the symbionts into host
cells induces morphological changes in the host cells.
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This is known for all symbiotic
bivalves.
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Table S2: Summary of observations made in this study of symbiont abundance and distribution in gill tissues of adult “C”. ponderosa clams
using FISH, and our interpretations of these observations. As all other investigated vesicoymid clams, “C”. ponderosa only harbors sulfuroxidizing symbionts.
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Observations
Budding zone lacked symbionts.
(Figure 3b)
Ventral growth zone of filaments lacked symbionts.
(Figure 3a, b)
Symbionts were abundant in ontogenetically older gill
filaments.
(Figure 3a)
The gill’s hemolymph lacunae did not contain
symbionts.
(not shown)
Symbionts were observed in the interfilamental
junctions.
(Figure 3a)
Symbionts occurred at the dorsal ends of gill filaments.
(Figure 3b)
Interpretation
Newly formed gill cells in the posterior growth zone do not
contain symbionts. This indicates that symbiont colonization
can only occur after host cells have reached a certain
differentiation stage and is influenced by developmental
factors.
Remarks
This indicates that the symbionts do not colonize newly
formed gill cells internally via the hemolymph system.
We hypothesize that the symbionts use these gill tissue
bridges to spread from ontogenetically older, colonized
bacteriocytes to newly formed, symbiont-free gill cells.
We do not know if the gills of
vesicomyid clams lack growth zones at
their dorsal ends.
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References
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Neumann, D., and Kappes, H. (2003) On the growth of bivalve gills initiated from a
lobule-producing budding zone. Biol Bull 205: 73-82.
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