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
Transmission electron micrograph of a-symbiont in fat bodies of Pyrops candelaria (Wang & Cheung, 1997)
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
Endosymbionts of the Auchenorrhyncha
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
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
Enchophora rosaceaEnchophora stillifera
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
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
MP
Top
olog
y of
Sul
cia
mue
lleri
Aphrophora quadrinotataClastoptera arizonanaScolops sulcipesScolops sp. Dictyophara sp.1Dictyophara sp.2Rhaphiophora sp.
Rentinusdilatatu sDesudaba 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
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?
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)
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
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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