julie urban, nysm transmission electron micrograph of a...

Endosymbionts of the Auchenorrhyncha

Transmission electron micrograph of a-symbiont in fat bodies of Pyrops candelaria (Wang & Cheung, 1997)

a: alpha symbiont

n: nucleus

m: mitochondria

v: vacuoles

scale = 2 µm

sg: storage granules

Julie Urban, NYSM

o: osmiophilic materials


bm: bacteroid membrane

hm: host cell membrane

Endosymbionts of the AuchenorrhynchaJulie Urban, NYSM

First investigated by Müller (1962) and Buchner (1965) using light microscopy

“Hunger for symbionts” (Buchner, 1965) in Auchenorrhyncha

Cicadomorpha(188 species examined)

Fulgoroidea(217 species examined)

No symbiont 0

Monosymbiont 15

Disymbiont 119

Trisymbiont 74

Tetrasymbiont 8

Pentasymbiont 1

Hexasymbiont 0

10

7

105

50

9

5

2


Coleorrhynchaa

Cercopoidea Cicadoideaa + ßc

a + ßc + Sta + St

a + W → a + H

a + W+ St

Jassoideaa + t

a + t

a + t

a + ta + t

a + t

a + t

Cicadomorpha

H a + H

a + H

H

a + Ha + H

a + H

a + t + a + t +

a + t + B1 + B2

a +

aJassidae

CicadellidaeDeltocephalidae

Membracidae

Ledridae

Ulopidae

a + x Fulgorina

Fulgoroideaa + x

Tettigometrinaa + x + s

a + x

a + x+ x + f +

f + HDelphacidae

a + x a + x+

x + f x B

x + f + f + H

HCixiidae

a + x a + x a + x a + x+

x + f

f + H

f + H+Fulgoridae

a + x+ x + f

f + HJassidae

a + x+

x + f

f + HFlatidae

Fulgoromorpha

(from Buchner, 1965)

(Müller, 1962)

Fulgoridae +

Derbidae + + +

Issidae + + + +

Flatidae +

Ricaniidae +

Tettigometridae +

Delphacidae + + + +

Meenoplidae + +

Nogodinidae +

Eurybrachidae +

Tropiduchidae + + +

Lophopidae +

Dictyopharidae + + +

Acanaloniidae +

Achilidae + +

Cixiidae + +

a + x a + x + others x + f x + f + others f + H

Endosymbionts of Fulgoroidea

older younger

Endosymbionts of Fulgoroidea(Buchner, 1965)

a-symbiont

x-symbiont

organs with companion symbionts

Endosymbionts of Fulgoroidea(Buchner, 1965)

Used PCR assay (bacterial 16S rDNA) to detect endosymbiont within Auchenorrhyncha -- Sulcia muelleri

Endosymbionts of Auchenorrhyncha(Moran et al., 2005)

Membracoidea

Fulgoroidea

Cicadoidea

Cercopoidea

Used PCR assay (bacterial 16S rDNA) to detect endosymbiont within Auchenorrhyncha -- Sulcia muelleri

Endosymbionts of Auchenorrhyncha(Moran et al., 2005)

(from Wu et al., 2006)

(Fig

ure

take

n fr

om M

oran

, 200

7)

Endosymbionts of Auchenorrhyncha(Takiya et al., 2006)

Takiya et al., 2006 n

Endosymbionts of Auchenorrhyncha(Takiya et al., 2006)

(Fig

ure

take

n fr

om M

oran

, 200

7)

Endosymbiont genomes extremely reduced EXCEPTgenes related to host insect’s metabolism are retained:

aphids -- phloem diet

tsetse flies -- blood diet

sharpshooters -- xylem diet

Buchnera -- genes for essential amino acid synthesis

Wigglesworthia -- genes for B vitamin cofactors

Sulcia -- genes for essential amino acid synthesisBaumannia -- genes for vitamins, cofactors absent in xylem

Insect Endosymbiont Genome

Sequencing of Endosymbiont Genomes

Research on Endosymbionts of AuchenorrhynchaEndosymbionts → biology of insect host

-- nutrient provisioning -- host plant shifts

-- other roles, especially in phloem feeders?

Endosymbionts as reproductive manipulators-- Wolbachia, Cardinium

genome sequencing of bacterial endosymbionts of two sharpshooter species and one spittlebug species (Moran & Wing, in progress)

Endosymbionts associated with plant pathogens-- Basses richess (beets) caused by BLO and is vectored by Pentastiridius sp.

-- Endosymbiont origin of BLO? (Sémétey et al., 2007)

Methods

1. Extraction of endosymbiont DNA

3. Figuring out what you’ve got

4. Increase data set -- iterative method

(Urban, 2008; Urban & Cryan, in progress)

2. PCR amplification

Choice of markers

Choice of primers

BLASTing and analyzing sequences

(Photo by R. Rakitov and D. Takiya; taken from Moran, 2007)

Endosymbiont DNA ExtractionCould identify, remove, and extract from bacteriomes

(Photo from Moran, 2006)

Endosymbiont DNA ExtractionCould identify, remove, and extract from bacteriomes

or not knick cuticle of abdomen, lyse in buffer overnight using DNEasy Kit“Poke - n - Soak” method © (pat. pend.)

a-symbiont x-symbiont companion symbionts

PCR AmplificationChoice of markers

16S rDNA -- standard marker for characterizing bacteria

-- 27,654 bacterial 16S rDNA sequences on GenBank (as of 10/27/08)

-- universal and relatively conserved

-- universal primers available

Sulcia: 10CFB_FF (forward) + 1515R

S688DIASP + A1271DIASP also work(Diaspidid prim. symbiont, Gruwell et al., 2007)

PCR AmplificationChoice of markers

protein coding genes -- used for well characterized bacteria

-- wsp, ftsZ in Wolbachia

[Note: yeast-like endosymbionts -- 18S rDNA]

23S rDNA -- less conserved than 16S-- not as well characterized

Figuring out what you’ve gotBLAST search

MP Phylogeny of Sulcia

Strict consensus of > 45,000 ept

Aphrophora quadrinotataClastoptera arizonanaScolops sulcipesScolops sp. Dictyophara sp.1Dictyophara sp.2Rhaphiophora sp.

RentinusdilatatusDesudaba danae

Neolieftinckana fuscata

Saiva nodataPyrops intricataPenthicodes sp.2

Druentia siccaAnecphora torrida

Kalidasa lanataundet. Fulgoridae

Xosophara sp.

Poblicia fuliginosaPoblicia fuliginosaLystra lanataAlphina glaucaAlphina glauca (2)Scaralis sp.1

Odontoptera carrenoiOdontoptera toulgoetiDiareusa annularisAphrodisias cacicaDiareusa imitatrixCathedra serrata

Enchophora sanguineaFulgora laternaria

Phrictus tripartitusPhrictusquinquepartitusPhrictus regalis

Enchophora rosaceaEnchophora stillifera

Enhydria tessellata

Cyrpoptus suavisAmerzanna peruanaAmycle vernalisFlatolystra sp.1

Echetra modesta

Zanna sp.

Weyrauchia fulmineaEpisciusguianensisEpisciusboliviana

Aracynthus sanguineusNeocynthus sp.Stalubra brunnea

-- analysis based on 46 fulgorids, 4 dictyopharids

-- ~ 1400 bp 16S

Dictyophara sp.1Dictyophara sp.2

Scolops sulcipesRhaphiophora sp.

Rhynchomitra microrhinaHyalodictyon sp.

Lappida sp.

Diareusa imitatrixDiareusa annularis

Copidocephala guttataCopidocephala viridiguttata

Aphrodisias cacicaSinuala tuberculata

Echetra modestaWeyrauchia fulminea

Episcius bolivianaEpiscius guianensis

Echetra nebulosa

Neocynthus crassusNeocynthus sp.

Aracynthus sanguineus

Pterodictya reticularisPhenax variegata

Cyrpoptus suavisAmycle vernalis

Amerzanna peruana

Calyptoproctus sp.Paralystra florenciana

Flatolystra distinctaFlatolystra verrucosa

Dilobura conspurcata

Poblicia fuliginosaLystra lanata

Learcha sponsa

Matacosa miscellaScaralis semilimpida

Alphina glaucaScaralis sp.2Scaralis sp.1Scaralis sp.3

Fulgora castresiiFulgora laternaria

Fulgora cearensisCathedra serrata

Phrictus tripartitusPhrictus quinquepartitus

Phrictus regalis

Enchophora sanguinea var.1Enchophora sanguinea var.2

Enchophora nigromaculata


Artacie dufouri

Stalubra brunneaEnhydria tessellata

Odontoptera carrenoiOdontoptera toulgoeti

Rentinus dilatatusEurinopsyche obscurata

Desudaba danaeKalidasa lanata

Druentia siccaundet. Fulgoridae

Birdantis bloteiXosophara sp.

Penthicodes pulchellaPenthicodes atomaria

Penthicodes farinosaAnecphora torrida

Pyrops sp.Pyrops intricata

Saiva nodataNeolieftinckana fuscata

Zanna sp.1Zanna sp.2

Zanna sp.3Tropidocephala sp.

Oecleus sp.Bothriocera eborea

MP

Topology of F

ulgoridae

NW


RentinusdilatatusDesudaba danae





Xosophara sp.






Enhydria tessellata


Echetra modesta

Zanna sp.



MP

Top

olog

y of

Sul

cia

mue

lleri


Rentinusdilatatu sDesudaba danae





Xosophara sp.






Enhydria tessellata


Echetra modesta

Zanna sp.



NW

Increasing Sulcia data set

Increasing Sulcia data set

tRNA

16S 23S

Other Endosymbionts in FulgoridaeUniversal bacterial 16S primers:

10F, 27F, 84F, 35R,1370R, 1495R, 1507R

Alphaproteobacteria: Wolbachia, Rickettsia

Betaproteobacteria

Gammaproteobacteria: highest BLAST similarities toBaumannia (sharpshooter), Secondary endosymbiont of Pseudococcidae (bugs within bugs), & others

Amplified bacteria in multiple lineages:

? weevil

? aphid

? aphid

? sharpshooter (Baumannia)? Paracoccus

? Melanococcus

? Zanna sp.2? Alphina glauca? Weyrauchia fulminea? Amycle vernalis? Undetermined Fulgoridae

? Enchophora stillifera? Birdantis blotei

? Saiva nodata? Penthicodes pulchella? Episcius guianensis

? Zanna sp.1? Xosophara sp.? Neolieftinckana fuscata? Druentia sicca? Echetra nebulosa

? Lystra lanata? Copidocephala viridiguttata

? Cathedra serrata? Phrictus tripartitus? Phrictus regalis? Diareusa imitatrix? Diareusa annularis? Cyrpoptus suavis? Paralystra florenciana

? Scaralis sp.3? Aracynthus sanguineus

? Echetra modesta

? Enhydria tessellata

? Scaralis sp.2? Flatolystra distincta

outgroups

Old World

New World

? -proteobacteria (8 ept)~ 1500 bp 16S

vertical transmission?

x symbiont???

Fulgoridae +

Derbidae + + +

Issidae + + + +

Flatidae +

Ricaniidae +

Tettigometridae +

Delphacidae + + + +

Meenoplidae + +

Nogodinidae +

Eurybrachidae +

Tropiduchidae + + +

Lophopidae +

Dictyopharidae + + +

Acanaloniidae +

Achilidae + +

Cixiidae + +

a + x a + x + others x + f x + f + others f + H

What is this 2nd endosymbiont?

Increasing ? -proteobacteria data setUse alignment to design new primers for 2nd endosymbiont

Map facultative endosymbionts -- test for correlation with other aspects of host insect biology (e.g., waxes, pigmentation,host plant associations)

Increasing endosymbiont data setsUse planthopper-specific primers -- amplify remaining Fulgoridae and Dictyopharidae

-- Sulcia

-- ? -proteobacteria

Test congruence of host and endosymbiont phylogenies

Auchenorrhyncha: “..a fairyland of symbioses” (Buchner, 1965)

Cicadomorpha(188 species examined)

Fulgoroidea(217 species examined)

No symbiont 0

Monosymbiont 15

Disymbiont 119

Trisymbiont 74

Tetrasymbiont 8

Pentasymbiont 1

Hexasymbiont 0

10

7

105

50

9

5

2

Surveyed only ~10% of Auchenorrhynchan species

Acknowledgements

Methodologies based on protocols of Normark lab,UMass Amherst (shared by Matt Gruwell & Ben Normark)



a: alpha symbiont

n: nucleus

m: mitochondria

v: vacuoles

scale = 2 µm

sg: storage granules

Julie Urban, NYSM

Extracting and Amplifying Bacterial Endosymbiont DNA in Planthoppers

Urban, J.M. 2008. A Phylogenetic Investigation of the Planthopper Superfamily Fulgoroidea (Insecta: Hemiptera) with Emphasis on the Family Fulgoridae. Unpublished dissertation, University at Albany.

DNA Extraction

Endosymbiont DNA was extracted from abdominal tissue using Qiagen DNEasy

Kits (Qiagen, Inc., Valencia, CA, USA). Small incisions were made with a scalpel in the

cuticle of the abdomen, and the entire insect was incubated in lysis buffer overnight. For

insects that were too large to fit into a 1.5ml eppendorf tube, the whole abdomen (or part

of the abdomen for very large specimens) was removed and then incubated for extraction.

PCR Amplification

Polymerase chain reaction (PCR) was conducted in 25 µl reactions using Qiagen

DNA polymerase (Qiagen, Inc.) or Amplitaq polymerase. The PCR cycling program was

1 min at 94ºC, 30-35 cycles of 1 min at 51-54 ºC and 1 min. at 72ºC, ending with

incubation at 72ºC for 7 min. Primers that have successfully amplified Sulcia 16S in

Fulgoridae and Dictyopharidae are 10CFB_FF, 1515R, S688DIASP, and A1271DIASP

(primer sequences provided in attached table). Primers that have successfully amplified

other bacterial lineages in Fulgoridae are 10F, 27F, 84F, 35R, 1370R, 1495R, and 1507R

(Urban & Cryan, in progress). All sequences were compared to available sequences on

GenBank using BLAST searches.

References for Primer Sequences Gruwell, M.E., Morse, G.E., and Normark, B.B. 2007. Phylogenetic congruence of

armored scale insects (Hemiptera: Diaspididae) and their primary endosymbionts from the phylum Bacteroidetes. Molecular Phylogenetics and Evolution 44, 267-280.

Moran, N.A., Dale, C., Dunbar, H., Smith, W.A., and Ochman, H. 2003. Intracellular

symbionts of sharpshooters (Insecta: Hemiptera: Cicadellinae) form a distinct clade with a small genome. Environmental Microbiology 5(2), 116-126.

Moran, N.A., Trat, P., and Gerardo, N.M. 2005. Symbiosis and insect diversification: an

ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Applied and Environmental Microbiology 71(12), 8802-8810.

Takiya, D.M., Tran, P.L., Dietrich, C.H., and Moran, N.A. 2006. Co-cladogenesis

spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts. Molecular Ecology 15, 4175-4191.

Zchori-Fein, E., and Perlman, S.J. 2004. Distribution of the bacterial symbiont Cardinium

in arthropods. Molecular Ecology 13, 2009-2016.

Primer Region Orientation Sequence (5' -- 3') Description Reference10CFB_FF 16S forward AGA GTT TGA TCA TGG CTC AGG ATG for Sulcia Moran et al. 200510FF 16S forward AGT TTG ATC ATG GCT CAG GAT AA for Sulcia Takiya et al. 20061515R 16S reverse GTA CGG CTA CCT TGT TAC GAC TTA G for Sulcia Moran et al. 2005S688DIASP 16S forward GGA ATG TAT GGT GTA GCG GTG AAA TGC Diaspidid Bacteroidetes Prim. Gruwell et al. 2007A1271DIASP 16S reverse CAT TGT AGC ACG TGT GTA GCC CAA G Diaspidid Bacteroidetes Prim. Gruwell et al. 2007Ch-F 16S forward TAC TGT AAG AAT AAG CAC CGG C for Cardinium Zchori-Finn & Perlman, 2004Ch-R 16S reverse GTG GAT CAC TTA ACG CTT TCG for Cardinium Zchori-Finn & Perlman, 2004

Primer Region Orientation Sequence (5' -- 3') Description Reference10F 16S forward AGT TTG ATC ATG GCT CAG ATT G universal eubacterial Moran et al. 2003 (Baumannia)27F 16S forward GAG AGT TTG ATC CTG GCT CAG universal eubacterial M.Gruwell (pers. comm.)84F 16S forward CGG CGS AYG GGT GCG TAA C universal eubacterial Moran et al. 2003 (Baumannia)35R 16S reverse CCT TCA TCG CCT CTG ACT GC universal eubacterial Takiya et al. 20061370R 16S reverse CGT ATT CAC CGG ATC ATG GC universal eubacterial Moran et al. 2003 (Baumannia)1495R 16S reverse CTA CGG CTA CCT TGT TAC GA universal eubacterial M.Gruwell (pers. comm.)1507R 16S reverse TAC CTT GTT ACG ACT TCA CCC CAG universal eubacterial Moran et al. 2003 (Baumannia)

Primer Region Orientation Sequence (5' -- 3') Description ReferenceWsp-F wsp forward TGG TCC AAT AAG TGA TGA AGA AAC TAG CTA Wolbachia universal Takiya et al. 2006Wsp-R wsp reverse AAA AAT TAA ACG CTA CTC CAG CTT CTG CAC Wolbachia universal Takiya et al. 2006FtsZF1 ftsZ forward GTT GTC GCA AAT ACC GAT GC Wolbachia universal Takiya et al. 2006FtsZR1 ftsZ reverse CTT AAG TAA GCT GGT ATA TC Wolbachia universal Takiya et al. 2006

Bacteroidetes

General Bacteria

Wolbachia

Note: Many of these primers were based on a list originally assembled by Matt Gruwell for endosymbionts of Diaspididae

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