Transcript The Gut Microbiota of Helix aspersa

The Gut Microbiota of Helix aspersa
Smith, P. N., Baltzley, M. J., Boomer, S. M.
Western Oregon University, Monmouth, OR
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Buttiauxella Significance
The Effects of Penicillin on the Gut Microbiota of H. aspersa
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Day 1
Pseudomonas sp.
Day 2
Day 7
Day 21
Penicillin
Buttiauxella sp.
MacConkey Plates
Day 1
Day 2
Day 7
Day 21
Untreated
Kluyvera sp./Hafnia
Stenotrophomonas
FISH Microscopy
In 1996, Müller et al. isolated six new species of Buttiauxella from the
intestines of terrestrial gastropods, leading his team to propose that the
intestines of snails and slugs represented, “an ecological niche and
source” for Buttiauxella.
Our work supports and expands these findings:
• Buttiauxella represented 33% of the Helix gut microbiota as shown
using metagenomic analysis (vs. 0.005% on the food source).
• 21% of MacConkey-retrieved colonies were Buttiauxella, further
supported by the following analysis:
‒ Phenotypic tests: Lactose (+), Glucose (+), Oxidase (-), Citrate (+)
‒ FISH microscopy: hybridization of Gammaproteobacteria probe
‒ 16S rRNA sequence analysis (1000 bp) of representative colonies*
*We have isolated 2 Buttiauxella cultures one in
99% similar to B. izardii (shown below) and the
other is 99% similar to B. brennerae.
Nutrient Plates
FISH Microscopy
Day 1
The Gut Microbiota of Helix aspersa. The gut microbiota plays an
important role in the host organism’s well-being, contributing to the
host’s immunity and metabolism. An individual’s total gut microbiota is
dynamic, fluctuating in response to changes in diet and environmental
stressors; however, a host often has a subset of gut microorganisms,
known as the core gut microbiome, which is consistent among
individuals in a population. To study the gut microbiota, we are using the
common garden snail, Helix aspersa, as our model organism. In the past,
culture-based studies have been used to identify bacteria from the gut of
Helix aspersa raised on processed food sources, which can alter the gut
microbiota. Little has been done using metagenomics to determine the
natural gut microbiota or identify a core microbiome. We analyzed 16S
bacterial diversity in the feces of wild-caught snails using highthroughput Illumina sequencing of the V1 and V2 variable regions. Our
results show a gut microbiota dominated by Gammaproteobacteria,
particularly members of genus Buttiauxella. This finding was notable in
that several early culture-based studies identified this genus as snail
specific. More recent DNA-based work has identified this genus at low
levels in soil and water environments. Given these data, we assessed the
microbial community present on the snail food source, confirming low
levels of Buttiauxella and high levels of Pseudomonas. This finding
suggests that Helix selectively uptake microbes from their food-source
and/or avoid potentially harmful Pseudomonas. We are using FISH
microscopy and plating techniques to study changes in the gut
microbiota of individual snails in response to changes in their feeding
regimen, and exposure to antibiotics or bacterial stressors.
Microbiota Dynamics Study
Percent of colonies on Mac Plates
The Core Gut Microbiome of Helix aspersa
Abstract
Gammaproteobact
eria
Day 2
Conclusions
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Lettuce
Helix aspersa feeding on lettuce. Image from: http://www.snailworld.com/garden-snail/. Lettuce: A metagenomic analysis of the
bacteria present on lettuce food source showed 68% Pseudomonas
and 0.005% Buttiauxella
Class
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Gammaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Alphaproteobacteria
Betaproteobacteria
Betaproteobacteria
Actinobacteria
Bacteroidetes
Unclassified
OTU
Buttiauxella noackiae
Buttiauxella sp.
Enterobacter sp
Enterobacter asburiae
Erwinia billingiae
Erwinia rhapontici
Pantoea sp.
Pectobacterium wasabiae
Serratia sp.
Kluyvera intermedia
Hafnia sp.
Tatumella terrea
Klebsiella sp.
Rahnella sp.
Rahnella aquatilis
Pseudomonas sp
Rhizobium sp
Sinorhizobium sp
Sphingomonas sp
Variovorax sp
Duganella zoogloeoides
Microbacterium sp
Chryseobacterium sp
Cyanobacterium sp
A metagenomic assessment of the bacterial population on the organic
lettuce food source found it was dominated by Gammaproteobacteria (93%).
The most prevalent genus was Pseudomonas (86%), with Buttiauxella
(0.005%).
Day 7
Buttiauxella
sp.
Snail 2 Snail 3 Snail 7 Snail 8
57.47
58.25
9.56
0.56
1.82
1.63
0.34
0.02
2.11
3.07
1.64
1.03
0.55
2.11
0.03
0.23
8.59
2.10
9.65
0.70
0.14
18.96
1.14
0.17
0.12
4.54
0.24
1.75
0.21
0.12
0.01
0.00
0.02
0.05
0.00
0.00
0.12
0.23
0.24
0.06
0.49
0.05
0.19
0.00
0.03
0.05
0.00
0.16
0.03
0.01
0.17
0.72
1.50
3.07
8.78
0.64
0.02
0.01
0.54
0.61
0.26
0.48
17.96
49.27
0.05
0.07
1.10
2.29
0.15
0.27
0.01
0.00
0.03
0.17
0.99
3.03
0.06
0.02
0.64
0.51
0.12
0.10
4.95
5.55
0.17
0.09
0.03
0.12
0.13
0.10
0.06
0.09
22.73
0.01
22.01
2.72
• Confirming these data, the Gammaproteobacteria probe hybridized with
61% of cells that were stained using DAPI.
• Our findings confirm some previous studies (Watkins and Simkiss 1990,
culture-based/Helix) but not others (Van Horn et al., 2011, 16S/Planorbid;
Cardoso et al., 2012, 16S/Achatina).
Our microbiota dynamics study suggested that penicillin treatment selected
for a homogenous population of Pseudomonas using MacConkey media.
Within a week, the original population (including Buttiauxella) has returned
and remained stable for the three weeks of the experiment.
• These data were consistently observed in the 6 snails treated with
penicillin, but not in the untreated snails.
• Pseudomonas is linked with drug resistance and pathogenicity in snails.
Drug resistance was shown in snails by Watkins and Simkiss, 1990.
• Future work includes metagenomics analysis and assessment using FISH
probes specific to Pseudomonas, and Buttiauxella.
Day 21
C
This work represents the first metagenomic assessment of the gut
microbiome of H. aspersa. Our assessment of four snails shows a gut
microbiome dominated by Gammaproteobacteria (79%). Of these, the most
prevalent were Buttiauxella (34%) and Pseudomonas (23%).
Gammaproteobact
Kluyvera
intermedia
Six H. aspersa were fed 2 g of lettuce soaked in 10 ml of 20 mg/ml penicillin-G in
water for the first two days and then 2 g of lettuce soaked in 10 ml of water for the
remaining 19 days. Two untreated H. aspersa were fed 2 g of lettuce soaked in 10 ml
of DI water every day for 21 days. Day 1 samples were taken before the penicillin
treatment was started. Day 2 samples were taken 24 hours after starting the
penicillin treatment. Day 7 samples were taken 4 days after ending the penicillin
treatment. Day 21 samples were taken 18 days after the penicillin treatment had
ended. Using a stepwise dilution, 10 ng of feces were plated on MacConkey agar,
which is selective for Gram (-) Gammaproteobacteria. Colonies were counted and
identified phenotypically, and using 16S sequencing. 100 µg of feces was analyzed
using FISH microscopy. The samples were labeled with a Gammaproteobacteria
probe (green) and counterstained with the DNA stain DAPI (blue). Scale bars are 10
microns, and all images were captured with a Zeiss LSM-710 Confocal Microscope.
Taken together, these data suggest that the gut microbiota of H. aspersa is
not reflective of the bacteria they ingest, but is selectively maintained.
• Cardoso et al., 2012 speculated that the land snail Achatina selected its
microbiota to survive and adapt to different habitats.
Citations
Cardoso, Alexander M., et al. 2012. "Gut bacterial communities in the giant land
snail Achatina fulica and their modification by sugarcane-based diet." PloS one
7.3: e33440.
Van Horn, D. J., et al. 2011. "Complex intestinal bacterial communities in three
species of planorbid snails." Journal of molluscan studies: eyr038.
Watkins, B., and K. Simkiss. 1990. "Interactions between soil bacteria and the
molluscan alimentary tract." Journal of molluscan studies 56.2: 267-274.
Special Thanks to
Drs. Boomer and Baltzley. The Walker Award for providing
funding for this research and travel to Louisiana. The Charlotte
Mangum Student Support Program. Dr. Coddington and
Willamette University for providing access to their confocal
microscope. Western Oregon University for providing
equipment, lab space, and resources. Dr. Stephen Taylor for
assisting with poster plotting.