Bacterial Symbionts of the Earthworm, Eisenia foetida

1
Symbiotic bacterial colonization of the
earthworm, Eisenia foetida
Seana K. Davidson D.A. Stahl laboratory Dept.
Civil and Environmental Engineering
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Members of the Crassiclitellata (Earthworms)
Sam James
12-16 families 8000 species
3
Knop reported bacteria in the nephridia of
earhworms ( 1926)
nephridia
sv
t
sr
after Emile Deyrolle
Nephridia are excretory organs
Are bacterial symbionts involved in the excretion
process?
4
Bacteria detected in the nephridia of earhworms
by FISH (fluorescent in situ hybridization)
intake
pore
Bacteria(red)
Bacteria
ampulla
LSCM images of 16S rRNA FISH
100 mm
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Phylogeny of earthworm nephridial bacteria
16S rRNA gene
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Co-Speciation of Host and Symbiont
mitCOI phylogeny of earthworm hosts
16S rRNA phylogeny of Acidovorax-like symbionts
Allolobophora thaleri
Allolobophora jessyensis
Allolobophora georgii G1
Lumbricus terrestris 4.7-1 (US)
Lumbricus terrestris (Germany)
Lumbricus castaneus - LC
Lumbricus festivus (Germany)
Lumbricus rubellus DQ092902
Eisenia foetida (Germany)
Eisenia foetida (USA)
Octolasion lacteum DQ092910
3
3
Courtesy of A. Schramm
Solid lines symbiont and host sequences from the
same worm individuum Dashed lines host sequences
from databases or different individuals
7
Phylogenies suggest a co-evolving
association Questions How are the bacteria
transmitted? When during development does the
host acquire the symbiont? How is specificity
determined during colonization?
8
The entire life cycle of Eisenia foetida is
maintained in the lab
N. Pinel
9
Embryo development
0-3 days
8-10 days
20 days
mid development (segments appear)
Emerge fully colonized
_at_ 21-22C
colonized juvenile
ampulla
pore
100 µm
10
Bacteria are transferred during egg capsule
formation
Symbiont cells dominate the capsule bacterial
population
5 ?m
other bacteria are also present
11
When and How does colonization occur during
embryogenesis?
500 mm
12
FISH of whole embyros at
10 day
12 -13 day
14-15 day
1 mm
500 mm
(Acidovorax, Eubacteria, Actinobacteria probes)
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15 d
10 d
14
Symbiont migration into nephridia
Eub Probe
Sym Probe
15
dorsal
ventral
16
(No Transcript)
17
Other bacteria are abundant in the egg capsules
5 ?m
FISH Acidovorax (yellow) and Eubacteria (red)
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There are bacteria in the lumen of the embryos
12 day embryo
200 mm
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As embryos develop, bacteria are retained in
portion that becomes midgut
15 day
What is their fate?
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What is the source and identity of bacteria
incorporated into the capsules? Which of these
are persistent or transient?
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Colonization of the earthworm

• is initiated during early development
• coincides with differentiation of segments
• involves a specialized structure that recruits
  bacteria
• Migration into the nephridia likely involves
  signals from the host--chemotaxis?.

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Thanks
Collaborators Andy Schramm University of Aarhus,
Denmark Sam James University of Kansas George
Brown EMBRAPA Brazil
University of Washington David Stahl Nic Pinel

This work was funded by NSF IOB 0345049 NSF
DEB 0516520 NIH NRSA to SKD.
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Putative bacterial symbionts are members of the
Microbacteriaceae within Actinobacteria
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LSCM FISH with Actinobacteria probe and TEM of
nephridia
probe HGC 69a