Discover the Microbes Within

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Discover the Microbes Within! Teacher Enhancement
Workshop on Symbiosis The Marine Biology
Laboratory March 2006
Funded by NASA Astrobiology, NSF FIBR
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Instructors

• Michele Bahr - MBL

• Jack Werren - U Rochester

• Bob Minckley - U Rochester

• Seth Bordenstein - MBL

• George Wolfe Loudon County

• Michael Clark - U Rochester

• Jennifer Wernegreen - MBL

• Laura Baldo UC Riverside

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Why Are WeHere?
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Integrated Set of Lab Exercises

• Field Biology - Insect Collecting

• Biodiversity Identifying Insects

• Mol Biology DNA Extraction

• Mol Biology PCR

• Mol Biology Gel Electrophoresis

• Bioinformatics Sequence analysis

• Mol Evolution Sequence Phylogeny

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Features of These Labs

• Original Research in Lab Exercises

• Students can make new discoveries

• Integrates across Science
• Biodiversity -gt Molecular -gt Evolutionh

• Modular Structure of the Labs

• Teaches Emerging Fields
• Bioinformatics, Mol Pylogeny

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Endosymbiosis

• Contributes to biodiversity
• Can provide novel biochemical capabilities to
  host
• Often allows hosts to occupy habitats that are
  otherwise inaccessible
• Illustrates the coevolution and interdepence of
  organisms

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Inherited Symbionts
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Asymmetric (Uniparental) Inheritance
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Buchnera
Aphid eggs (Uroleucon ambrosiae)
A. Mira
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Co-Divergence of Buchnera and Aphid Hosts
Courtesy of N. Moran
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Must heritable symbionts always be beneficial?
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Reproductive Parasites
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Male-Killing Microsporidea in Culex salinarius
Culex salinarius infected with Amblyospora
salinaria
Amblyospora opacita
Courtesy of J. Becnel
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Wolbachia
Encarsia species
Cardinium hertergii
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Wolbachia Master Manipulators
Courtesy of Bandi Sacchi
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Wolbachia-Rickettsia Ancient Intracellular
Lineage
400MYA
100MYA
T. deion symbiont
C. pipiens symbiont
insect wolbachiae
N. giraulti symbiont
D. melanogaster symbiont

N. vitripennis symbiont
B. malawi symbiont

nematode wolbachiae
D. immitis symbiont
Cowdria ruminantium
Anaplasma marginale
Ehrlichia equi
Ehrlichia platys
Rickettsia rickettsii
Rickettsia prowazekii
Rickettsia typhi
Bartonella vinsonii
Agrobacterium tumefaciens
Rhizobium leguminosarum
Escherichia coli
0.05
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The Wolbachia Pandemic
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The Wolbachia Pandemic
Insects (20-75 !)
Filarial nematodes
Crustaceans
(family Onchocercidae)
Chelicerates
2-6 million insect species are infected with
Wolbachia!! (Werren et al 1995).
Arthropods
Nematodes
(parasites)
(mutualists)
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GEOGRAPHIC PATTERNS
Werren Windsor 2002
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Phylogeny of Wolbachia (ftsZ)
6 SUPERGROUPS (A-F)
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Termites may have their own Wolbachia
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Reproductive Parasites of arthropods
Parthenogenesis Induction
Feminization
Trichogramma sp.
Male-Killing
Armadillidium vulgare
enhancing infected females
Drosophila sp.
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Asymmetric (Uniparental) Inheritance
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Wolbachia Affect Filarial Nematode Development
Embryogenesis
Moulting
Casiraghi et al. 2002
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Antibiotic Curing Suppresses Ovarian Development
in Asobara tabida

Dedeine et al 2002
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Ovaries of Sxlf4/Sxlf4 mutant females
Wolbachia Infected
Uninfected
FERTILE
STERILE
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Cytoplasmic Incompatibility Wolbachia (Sperm
Egg Incompatibility)
Modification
Rescue
m
r
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Paternal chromosomes are improperly condensed and
ultimately lost as a result of CI
Reed and Werren 1995, Tram Sullivan
2001 Picture courtesy of Uyen Tram
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Spread of CI Wolbachia in D. simulans
Spread of Wolbachia in Drosophila simulans in
California (Turelli Hoffmann 1991).
A Infektion mit Wolbachia A
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Bidirectional CI between different Wolbachia
Ok
ma
ra
X
mb
ra
X
ma
rb
Ok
mb
rb
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How Important Are Wolbachia?
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Wolbachia From Genomes to Communities Back
(NSF FIBR 5 Yrs, 7 Institutions)
Genomics Function Ecology Evolution
Tettelin, Stouthamer, Sullivan, DeSalle, Windsor,
Wernegren
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Wolbachia Fibr Clan Spring 1995
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Wolbachia has a fluid genome
Intragenic Intergenic Recombination
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UPGMA Trees
Orf0646
DLNgiralti162'
DLAalbopictus D10'
DLSinvictaA'
DLD ananassea geno
DLDsimulanswRi'
DLNasonia2 1'
DLDinnubilaA'
DLwMel genome'
DLDsimulanswAu'
DLDmelanogasteryw1
DLDrecensA'
A
DLEkuehniellaA'
DLDneotestaceaA'
DLDoreintaceaA'
DLMuniraptorA'
DLNvitripennisA'
DLDsimulanswHa'
DLIsnyderii'
DLGfirmusB'
B
DLAvulgareB C5'
DLEformosaB'
DLTdeionB'
DLTkaykaiB'
DLTtaiwanemma G6'
DLEkuehniellaB'
DLCaltemansB'
DLAsociuswCon'
DLTurticae'
DLOscapulalis D2'
DLNvitripennisB'
DLAencedon607'
DLAencedon625'
DLDsimulnanswMa2'
DLDsimulanswMa1'
DLDsimulanswNo'
DLAsociuswSoc'
DLTconfusumB G3'
DLTconfusumB G4'
DLCulex genome'
DLCpipiensB D3'
DLCquinqueB A1'
0.01
Macro and micro taxonomy are locus dependent
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Concatenated Tree based on MLST gene set
MrBayes, 1000 generations, 2079 bp
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How Do Wolbachia Get Around?
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World-Wide Wolbachia Sampling Plan
AMNH, STRI, ITS
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Wolbachia Diversity Continental Drift
AMNH, STRI, ITS
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Ecuador
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Ghana
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Ants are rich in Wolbachia Why and what do they
do?
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Can Wolbachia Accelerate Host Evolution
Speciation?
1. Enhancing Reproductive Isolation
2. Accelerating Host Gene Evolution
3. Wolbachia Gene Transfers to Host Genome
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Wolbachia Gene Transfers to Hosts

• Callosobruchus chinensis (Kondo et al 2002)
• Anopheles gambiae (Korochkina et al 2005)
• Drosophila spp.
• Nasonia vitripennis

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Wolbachia Cause Reproductive Incompatibility
Between Nasonia Species
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Nasonia and Wolbachia
N. giraulti wAg wBg
N. longicornis wAl wBl1 wBl2
N. vitripennis wAv wBv
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Distribution of Nasonia in North America
?
?
?
?
?
?
?
N. vitripennis
N. giraulti
N. longicornis
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Protocalliphora larvae
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Protocalliphora sialia Wolbachia
wA1 Wolbachia wA2
Wolbachia wA1 and wA2 Wolbachia
wB Wolbachia Not infected
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Chelymoprha alternans
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Distribution of Single Double Infections in C.
alternans
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Using Wolbachia to Teach Biology in an Integrated
Way
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Interactions with the Host Genome
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Nasonia and Wolbachia
N. giraulti wAg wBg
N. longicornis wAl wBl1 wBl2
N. vitripennis wAv wBv
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Haplo-diploid Sex Determination
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N. giraulti CI embryos exhibit chromosome
segregation defects and arrest early in
development
Nuclear cycle 1
3
4
2
9
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Nasonia
Yeast With Wings
N. giraulti
N. longicornis
Haploid Genetics in a Complex Eukaryote
N. vitripennis
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Wolbachia Reinforcement
Telschow et al. 2005
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Host Preference
NG NL are specialists on Protocalliphora larvae
NV is a generalist
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Questions

• How do W move between hosts?
• What genetic changes occur when they move into a
• new host?
• How do W induce CI, PI, SK and Fem?
• How often are W genes transferred to host genomes
  
• what happens to them when they are?
• How do hosts respond evolutionarily to W
  infections?
• Do W accelerate host evolution and speciation?

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Other Examples
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Removal of Wolbachia Reduces Sexual Isolation
Among Experimental Drosophila Populations
Koukou et al 2006
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Wolbachia Cause Reproductive Incompatibility
Between Nasonia Species
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Interspecific incompatibility
Infected
Cured
Relative No. females
Cross (male x female)
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Evolution of Wolbachia in Nasonia
Ag
Ag,Bg N. giraulti
Bgl
Al,Bl1,Bl2 N. longicornis
Al
Av,Bv N. vitripennis
Bv
Av
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Younger Older Nasonia Species
0.2 Mya
N. giraulti
0.8 Mya
younger
N. longicornis
older
N. vitripennis
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Glossina morsitans
Neighbor joining tree of wsp Wolbachia
Glossina brevipalpis
Ephestia cautella
Nasonia giraulti
Ephesita kuhniella
Trichogramma kaykai
Leptopilina heterotoma
Nasonia longicornis
Amitus fuscipennis
Dros simulans coff
Dros melanogaster
Culex pipiens
Drosophila auraria
Muscidfuraz uniraptor
Nasonia vitripennis
Pachycrepoideus dubius
Glossinsa austeni
Protocalliphora
Phelbot papatasi
Ephestia cautella
Leptopilina Australis
Nasonia vitripennis
Telenomus nawai
Teleogryllus taiwanemma
Protocalliphora
Nasonia longicornis
Nasonia giraulti
Trichogramma sibericum
Tribolium confusum
Aedes Albopictus
Wcep
Aedes albopictus
Culex pipiens pallens
Culex pipiens molestus
Drosophila mauritiana
Armadillidium vulgare
Armadillidium vulgare
Oniscus asellus
Porcellio scaber
Neochrysocharis formosa
0.01 changes
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Questions
1. How do W move through global arthropod
communities?

• Are W more related across taxa within regions or
  within
• taxa across regions?

3. Have some W spread globally recently?
4. Do ecologically associated hosts tend to
share related W ?
5. How has continental drift affected the
patterns of W diversity?
7. Where did A and B W originate?
6. What genetic changes occur when W change
hosts or phenotypes?
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Wolbachia MultiLocus Strain Typing
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MLST Gene Set
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Haplotype Dendogram
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Don Windsor
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