Bacterial Endosymbionts from the Genus C

Prévia do material em texto

695 
Bacterial Endosymbionts from the Genus Camponotus 
(Hymenoptera: Formicidae) 
by 
John J. Peloquin1, Stephen G. Miller3 , Stephen A. Klotz2 , Richard 
Stouthammer1 , Lloyd R. Davis, Jr.3 , &John H. Klotz1 
ABSTRACT 
Gram-negative prokaiyotic endosymbionts from follicle cells of eight 
species of North American Camponotus were isolated and their 16S 
rRNA-encoding DNA was amplified, cloned and sequenced. A BLASTn 
similarity search showed that the endosymbiont of Camponotus 
jloridanus is most similar to Candidatus camponotii. Other than this ant 
endosymbiont, the most similar sequences were from various entero-
bacteria commonly found in the gut of insects and other animals. The 
most parsimonious phylogenetic tree generated by PAUP analysis on 
manually aligned sequences supports a monophyletic relationship of 
the ant endosymbionts when the l 6S RNA sequences of two other 
insect-associated enterobacteria (Yersinia pestis, and Buchnera 
aphidicola) are included. The most parsimonious tree does not support, 
however, a monophyletic grouping of endosymbionts of the North 
American species when Old World isolates of Camponotus-associated 
eubacterial sequences are added to the analysis. These results suggest 
that the association and divergence of the endosymbiont with their ant 
host is ancient. 
INTRODUCTION 
The endosymbiotic bacteria of Formicidae were among the first 
discovered in insects (Blochmann 1884, 1886), but their widespread 
distribution in other insects and biological significance has only 
recently become appreciated (Baumann 1998; Heddi et al., 1993; 
Nardon 1999). In Camponotus, prokaiyotic endosymbionts are located 
in the midgut epithelium and follicle cells of the ovarioles within 
specialized mycetocytes. They are gram-negative, nonmotile, variable 
in shape, and lie freely in the cytoplasm without a surrounding 
membrane (Buchner 1965; Dasch et al. 1984). The bacteria are 
transmitted transovarily from the symbiont-rich follicle cells of a queen 
1Department of Entomology, University of California, Riverside , Riverside, CA 92521 
2Department of Medicine, Section of Infectious Diseases, PO Box 245039, Tucson, AZ, 
85724-5039 
3USDA-ARS-CMAVE, Gainesville, FL 32608 
696 Sociobiology Vol. 38, No. 38, 2001 
into her developing oocytes, and from there enter her offspring's follicle 
cells and midgut epithelium during embryogenesis (Buchner 1965; 
Steinhaus 1946). The bacteria's role in the ant is unknown but 
suspected to be nutritional, possibly supplying essential amino acids to 
their host (Dasch et al. 1984). In the past, classification of bacteria has 
been based on morphological and biochemical characteristics. With the 
advent of molecular techniques, microbial systematics has been revo-
lutionized (Woese 1987). We describe electron microscopy of the bacte-
rial endosymbionts and isolation of their DNA from the ovarioles of eight 
species of North American carpenter ants. We then amplified, cloned, 
and sequenced portions of this l 6S rRNA-encoding DNA and used it to 
construct phylogenetic trees 
MATERIALS AND METHODS 
Ants 
Newly mated queens of eight species of Camponotuswere collected in 
the field and maintained in the laboratory on a 10°/o sucrose water and 
cricket diet. Queens of C. castaneus, C. jloridanus, C. impressus, C. 
snellingi, C. socius, and C. tortuganus, were collected in Alachua Co., 
Florida and C. pennsylvanicus and C. noveboracensis in Maine .. 
Ant Tissue extraction 
Prior to dissection, live queens were surface-sterilized by immersion 
for 3-5 minutes in 5.25°/o sodium hypochlorite. Using aseptic technique, 
the ants were transferred with forceps to 10°/o sodium thiosulfate for 3-
5 minutes, then to lOOo/o ethanol for one minute, and finally to sterile 
distilled water where they were rinsed 3 times. Ants were dissected 
under aseptic conditions in sterile Grace's medium (Gibco-BRL). 
I 
PCR, cloning, and DNA sequence analysis 
Excised ovarian tissue was placed in 200µ1 of20mM Tris HCl, 1 OmM 
MgCl (pH= 6.8), then homogenized with a tissue grinder, and sonicated 
for 10 s. DNA was isolated from the homogenate as follows: a) to disrupt 
gram positive bacteria, two units ofmutanolysin (2 µl of 1 U/µl stock) 
were added to the material, mixed, and then incubated at 37°C for 60 
m; b) homogenates were centrifuged 30 sec at 1200g, and the 
supernatant removed; c) 700µ1 of 30mM Tris HCl with 25 mg/ml 
lysozyme was added to the pellet, mixed, and incubated at 37°C for 30 
m; d) 20 µl of 20°/o SDS ( 1I10th volume) was added to the sample and 
mixed, followed by20µ1 of0.2M EDTA (l/lOth volume); f) the sample 
was extracted with buffer-saturated, neutralized phenol. and then with 
24: 1 chloroform/isoamyl alcohol; g) the upper phase was transferred 
Peloquin, J.J. et al.: Endosymbionts of Camponotus 697 
to a clean tube and a final extraction was performed with water-
saturated ether; h) the upper organic phase was removed, then 1/10 
volume of 3. 0 M sodium acetate was added, along with 2 volumes 100% 
ETOH, and the sample was mixed and stored at -20° C. 
DNA encoding eubacterial 16S rRNA was amplified by PCR. Reac-
tions were performed in 500 mL tubes using the 'universal' eubacterial 
16S rRNA PCR primers rl8F (5'-catggctcagattgaacgctggcg-3'), and 
rl494R (5'-cccctacggttaccttgttacgac-3')(Britschgi and Fallon, 1994; 
Cary et al., 1993; Chen et al., 1994; Goldenberger and Altwegg, 1995; 
Lee etal., 1993; Marzach etal., 1998; McCabe et al., 1999; Smith etal., 
1996; Woo et al., 1999). The remainder of the reaction was in 71.5ml 
water, 10 ml lOx PCR buffer (500 mM KCL, lOOmMTris-HCl pH 9.0@ 
25, l.0°/oTritonOX-100, 15mMMgC12), 16mldNTPs(l.25mMofeach 
dNTP stock), 0.5 ml of each primer (200 mM stock), 0.5 ml 5U /ml Taq 
polymerase (Promega, Madison WI), and 1 ml of template DNA. Each 
reaction was overlain with 100 ml mineral oil (Sigma, St. Louis MO) for 
1 mat 94, 1 mat 50, 2 mat 72, repeated for 30 cycles, and finished with 
an extension of 8 m at 72. 
PCR-amplified DNA fragments were separated on 1 o/o TAE agarose 
gels containing ethidium bromide (Sambrook et al., 1989). A 1000-bp 
DNA ladder (Gibco-BRL catalog number 15615-016) was also run as a 
size standard to estimate DNA length by comparison of electrophoretic 
mobility of PCR-amplified fragments. 
PCR products were extracted from the gels, purified, and ligated into 
the pCR-II (Invitrogen, Carlsbad CA) cloning vector. 'One Shot' (lnvitrogen, 
Carlsbad CA) competent cells were transformed with an aliquot of the 
ligation reaction. Transf ormants were analyzed as mini preps (Sambrook 
et al., 1989). Plasmid clones containing appropriately sized inserts were 
chosen for sequence analysis (performed by University of Florida DNA 
Sequencing Core Facility). DNA sequences were acquired from the 
forward and reverse universal priming sites in pCR-II using forward and 
reverse Ml3-universal primers supplied by the Florida sequencing 
facility. Sequences internal to the EcorRv site of pCR-II were obtained 
using primers designed to anneal to conserved eubacterial sequences 
(ie., 773F, 5'-gcgtggggagcaaacagg-3' and 771R, 5'-atcctgtttgctccccacg-
3'). Data obtained from the Core Facility DNA sequencing were encoded 
and sequence comparisons within this data set. and between archived 
data from other insect-associated bacteria (Yersinia pestis, Buchnera 
aphidicola) were performed with applications in the Wisconsin GCG 
package. The various rRNA encoding DNA sequences were aligned then 
and phylogenetic analysis of the aligned DNA sequence data from the 
ant endosymbionts and other bacteria was performed using Phyloge-
698 Sociobiology Vol. 38, No. 38, 2001 
netic Analysis Using Parsimony (PAUP) ver. 4. 08 for power PC (Swofford, 
2001). Because our sequences for the endosymbionts from C. castaneus 
and C. noveboracensiswerelimited and outside the region of the other 
symbiont sequences, we excluded them from PAUP analysis. Sequence 
information from C. castaneus and C. noveboracensis was analyzed by 
a BLASTN similarity search using a server at the National Center for 
Biotechnology Information(http://www. ncbL nlm. nih.gov I BLAST I) to 
find the Genbank-submitted sequences that most closely resembled 
our submitted sequences. 
Electron Microscopy 
Crushed ovaries were cultured in aerobic and anaerobic Bactec 
bottles (Towson, MD), thioglycolate broth, and upon chocolate and 
blood agar at 28°C. Specimens for transmission electron microscopy 
were fixed in glutaraldehyde/paraformaldehyde, rinsed in phosphate 
buffer, dehydrated through gradient alcohol/acetonitrile and embed-
ded in Epon. Thick sections were stained with toluidine blue and thin 
sections (700 angstroms) were stained with lead citrate/uranyl acetate 
, 
and examined with a Joel 1200 Transmission Scanning Electron 
Microscope (Joel Corp., Boston, MA). 
RESULTS 
Electron microscopy 
Fig. 1 shows the intimate intracellular relationship of these bacteria 
with the ovarian tissue of the insect. Gram stain of crushed ovaries 
demonstrated long, filamentous gram-negative bacilli. Electron mi-
croscopy revealed numerous bacilli without cell walls consistent with 
gram-negative bacteria in the follicles surrounded by ground sub-
stance. 
Bacteria 
The endosymbionts of Camponotus are gram-negative pleomorphic 
bacilli. Their appearance in TEM suggests that they lack a cell wall. We 
were unable to culture isolated bacteria under either aerobic or 
anaerobic conditions on any of blood, chocolate, or gram-negative agar, 
Bactec aerobic and anaerobic media, or in thioglycolate broth. 
Phylogenetic analysis of endosymbiont sequences: 
Fig. 2 shows a BLASTn (Altschul et al., 1997) computer sequence 
search comparing the entire data set we had for l 6S rRNA sequence 
from C. jloridanus. The highest scoring sequences were those of 
Camponotus-derived symbiotic bacteria (genbank accession number 
X9255 l). The next most similar sequence in the database was obtained 
Peloquin, J.J. et al.: Endosymbionts of Camponotus 699 
Fig.1. Top: Lightmicroscopeviewofovocyte. 
Black arrow shows follicle epithelium and 
white arrow, bacteria in the ovocyte. Middle: 
Transmission electron photomicrograph of 
the bacteria in an ovocyte. Bottom: Higher 
power view of the bacteria. Black bar in the 
middle and lower panels equals 1µ. 
from Rahnella aquatalis (genbank 
accession number X79937). Almost 
the same high similarity scores were 
seen with Enterobacterial sequences 
isolated from the genera Erwinia, 
Serratia and Proteus. Sequences 
from rRNA of Yersinia pestis, 
Buchnera aphidicola, and our 
Camponotus symbionts were used 
as starting data in a PAUP (Swofford, 
2001) analysis. The most parsimo-
nious trees constructed by PAUP 
after bootstrap analysis (500 sepa-
rate samplings) grouped all of the 
endosymbionts of Camponotus 
within a single clade (Fig. 3), and the 
100 most parsimonious trees also 
grouped them into a monophyletic 
taxon. When l 6S rRNA sequences 
from Old World endosymbionts of 
Camponotus ( C. balzanl C. silivicola, 
C. nawai, C. vitiosus, C. japonicus, 
C. vagus, C. kiusiuensis, C. 
quadrinotatus, and C. rufipes) were 
compared with the bacterial se-
quences from our North American 
species, the most parsimonious trees 
did not show the latter as a mono-
phyletic group. The endosymbionts 
of all Camponotus, however, were 
placed in a monophyletic group with 
reference to Buchnera aphidicola, 
and Yersinia pestis (Fig. 3). 
BLAST analysis of sequences of 
the 16srRNAfrom C. castaneusand 
C. noveboracensis returned se-
quences derived from other 
Camponotus symbionts and 
Enterobacteriacea. The 10 se-
quences in the database most simi-
lar to C. castaneus and C. 
noveboracensis are listed in Table 1. 
700 Sociobiology Vol. 38, No. 3B, 2001 
Fig. 2 starts here. Caption is at the end of the figure. 
BLASTN 2.0.11 [Jan-20-2000] 
Reference: 
Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, 
Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), 
"Gapped BLAST and PSI-BLAST: a new generation of protein database s.earch 
programs", Nucleic Acids Res. 25:3389-3402. 
Query= 
(1098 letters) 
Database: Non-redundant GenBank+EMBL+DDBJ+PDB sequences 
525,629 sequences; 1,520,271,812 total letters 
If you have any problems or questions with the results of this search 
please refer to the BLAST FAQs 
Distribution of 471 Blast Hits on the Query Sequence 
Score E 
Sequences producing significant alignments: 
emblX92551ICCL16SR C.camponotii 16S ribosomal RNA (strain I... 
emblX92550/CCH16SR C.camponotii 16S ribosomal RNA (strain h .. . 
emblX92552ICCR16SR C.camponotii 16S ribosomal RNA (strain r .. . 
emblX79937IRA287R R.aquatilis (2-87) 16S-rDNA 
embjAJ223407jPCA223407 Pectobacterium carotovorum subsp. od .. . 
emblAJ223408IPCA223408 Pectobacterium carotovorum subsp. wa .. . 
gblAF015259IAF015259 Edwardsiella tarda 16S ribosomal RNA g .. . 
embjAJ233411.1IECA233411 Erwinia carotovora 16S rRNA gene( .. . 
emblZ96089IECZ96089 Erwinia carotovora LMG 2404-T 16S ribos .. . 
gblU80199IECU80199 Erwinia carotovora subsp. wasabiae 16S r .. . 
gbjU80197IECU80197 Erwinia carotovora subsp. carotovora 16S .. . 
emb1X79940IRA388R R.aquatilis (3-88) 16S-rDNA 
embjAJ233433.1ISPL233433 Serratia plymuthica 16S rRNA gene .. . 
emblAJ233430.1 ISGR233430 Serratia grimesii 16S rRNA gene (s .. . 
gblU88435IRSU88435 Rahnella sp. 'CDC 21234' 16S ribosomal R .. . 
emblX07652/PVRN16S Proteus vulgaris 16S rRNA gene 
emb1AJ233425.1 IPVU233425 Proteus vulgaris 16S rRNA gene (st... 
emblAJ233435.1 ISPR233435 Serratia proteamaculans 16S rRNA g .. . 
emb/AJ233434.1 ISPR233434 Serratia proteamaculans 16S rRNA g .. . 
dbj!AB004762IAB004762 Unidentified bacteria gene for 16S ri ... 
gblAF130967 .1IAF130967 Pantoea oleae strain A66 16S ribosom .. . 
gblAF008582.1 IAF008582 Proteus mirabilis 16S ribosomal RNA .. . 
dbjlAB009954IAB009954 Unclassified gamma proteobacteria gen .. . 
gblU88434IRSU88434 Rahnella sp. 'CDC 1-576' 16S ribosomal R .. . 
gblM59149/ERWRR16SA Erwinia carotovora 16S ribosomal RNA. 
emblAJ233426.1IRAQ233426 Rahnella aquatica 16S rRNA gene (s .. . 
emblZ96090IECZ96090 Erwinia carotovora LMG 2386 16S ribosom .. . 
emblZ96091jECZ96091 Erwinia carotovora LMG 2466 16S ribosom .. . 
emblAJ233428.1 ISF1233428 Serratia ficaria 16S rRNA gene (st... 
embjAJ233424.1IPF0233424 Pragia fontium 16S rRNA gene (stra .. . 
gbjU80198jECU80198 Erwinia carotovora subsp. betavasculorum .. . 
(bits) 
1138 
1122 
987 
938 
936 
930 
930 
922 
918 
918 
918 
918 
914 
906 
898 
898 
890 
882 
882 
878 
874 
872 
858 
858 
846 
690 
688 
680 
678 
678 
676 
Value 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
Peloquin, J.J. et a/.: Endosymbionts of Camponotus 
gbjU90757jRAU90757 Rahnella aquatilis 16S ribosomal RNA gen... 674 
embjX79939jRAATCR R.aquatilis (ATCC33989) 16S-rDNA 672 
embjX79938jRA339R R.aquatilis (339) 16S-rDNA 672 
embjX79936jRA334R R.aquatilis (334) 16S-rDNA 672 
embjAJ233427 .1 jSEN233427 Serratia entomophila 16S rRNA gene... 670 
gbjAF130914.1jAF130914 Pantoea endophytica strain A42 16S r... 666 
gbjAF130937.1jAF130937 Enterobacter agglomerans strain A69 ... 664 
gbjAF130929.1jAFl30929 Enterobacter agglomerans strain A59 ... 664 
gbjAF130924.1jAF130924 Enterobacter agglomerans strain A54 ... 664 
gbjAF130912.1jAF130912 Enterobacter agglomerans strain A40 ... 664 
gbjAF130898.1jAFl30898 Enterobacter agglomerans strain A 19 ... 664 
gbjAF130884.1jAFl30884 Enterobacter agglomerans strain A 123...664 
embjAJ233422.1 \0PR233422 Obesumbacterium proteus 16S rRNA g... 664 
emb\Z96096IEPZ96096 Erwinia paradisiaca LMG 2542 16S riboso... 664 
gbjAF008581.1 jAF008581 Providencia stuartii 16S ribosomal R... 662 
gbjU78184\KOU78184 Klebsiella oxytoca 16S ribosomal RNA gen... 662 
dbjjAB021400.1 jAB021400 Pseudomonas flectens DNA for 16S rR... 662 
embjZ96081jPAZ96081 Pantoea ananas LMG 2665 16S ribosomal RNA 662 
gbjAF130925.1jAFl30925 Enterobacter agglomerans strain A55 ... 660 
gbjAF130923.1jAFl30923 Enterobacter agglomerans strain A53 ... 660 
gbjU78183jKOU78183 Klebsiella oxytoca 16S ribosomal RNA gen... 660 
gbjAF130952.1jAFl30952 Enterobacter agglomerans strain A92 ... 658 
gbjAF130949.1jAF130949 Enterobacter agglomerans strain A87 ... 658 
gbjAF130948.1jAF130948 Enterobacter agglomerans strain A84 ... 658 
gbjAF130946.1jAFl30946 Enterobacter agglomerans strain A81 ... 658 
gbjAF130945.1jAFl30945 Enterobacter agglomerans strain A80 ... 658 
gbjAF130940.1jAF130940 Enterobacter agglomerans strain A72 ... 658 
gbjAF130938.1jAFl30938 Enterobacter agglomerans strain A70 ... 658 
gbjAF130934.1jAF130934 Enterobacter agglomerans strain A65 ... 658 
gb\AF130928.1\AF130928 Enterobacter agglomerans strain A58 ... 658 
gbjAF130918.1jAF130918 Pantoea agglomerans strain new*47con... 658 
gbjAF130916.1jAFl30916 Pantoea agglomerans strain new• 45con... 658 
gbjAF130887 .1jAF130887 Enterobacter agglomerans strain A20 ... 658 
gb\AF025365jAF025365 Citrobacter freundii 16S ribosomal RNA... 658 
gbjU80209jEUU80209 Erwinia uredovora 16S ribosomal RNA gene... 658 
embjAJ233407.1jBA0233407 Budvicia aquatica 16S rRNA gene (s... 656 
gbjAF130958.1jAF130958 Pantoea endophytica strain A6 16S ri... 654 
gbjAF130944.1 jAF130944 Enterobacter agglomerans strain A79 ... 654 
gbjAF130943.1jAF130943 Enterobacter agglomerans strain A77 ... 654 
gbjAF130892.1jAF130892 Pantoea endophytica strain A13 16S r... 654 
gbjAF130961.1jAFl30961 Enterobacter agglomerans strain A9 1... 652 
gbjAF130911.1jAFl30911 Enterobacter agglomerans strain A38 ... 652 
gbjAF130951.1 jAF130951 Enterobacter agglomerans strain A91 ... 650 
gbjAF130935.1jAFl30935 Enterobacter agglomerans strain A67 ... 650 
gbjAF130933.1jAF130933 Enterobacter agglomerans strain A63 ... 650 
embjAJ011333jYSP011333 Yersinia sp. 16S rRNA gene, isolate... 650 
gbjAF130930.1jAFl30930 Enterobacter agglomerans strain A60 ... 646 
gbjAF130907.1IAF130907 Enterobacter agglomerans strain A29 ... 646 
gb\AF130941.1IAF130941 Enterobacter agglomerans strain A73 ... 644 
gbjAF130931.1 jAF130931 Enterobacter agglomerans strain A61 ... 644 
gbjAF130922.1jAF130922 Enterobacteragglomerans strain A51 ... 644 
gbjAF130905.1jAF130905 Enterobacter agglomerans strain A27 ... 644 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
0.0 
701 
702 Sociobiology Vol. 38, No. 38, 2001 
dbilD78010ID78010 Xenorhabdus poinarii DNA for 16S Ribosoma ... 
gblAF130963.1IAF130963 Pantoea toletana strain A64 16S ribo .. . 
emblAJ233429.11SF0233429 Serratia fonticola 16S rRNA gene( .. . 
gblAF130936.1IAF130936 Enterobacter agglomerans strain A68 .. . 
emblAJ009930.1jEPAJ9930 Erwinia pyrifoliae 16S rRNA, tRNA-G .. . 
embjAJ010485.1jEAM010485 Erwinia amylovora 16S rRNA gene, t... 
embjX83265IEA16SRR E.amylovora 16S rRNA gene 
gbjAF130966.1IAF130966 Pantoea toletana strain A78 16S ribo .. . 
gbjAF130899.1IAF130899 Enterobacter agglomerans strain A21 .. . 
dbjiAB004745jAB004745 Serratia ficaria gene for 16S ribosom ... 
dbjjD78007jD78007 Xenorhabdus bovienii DNA for 16S Ribosoma ... 
emblZ49828IYE320692 Y.enterocolitica gene for 16S ribosomal... 
gblAF130968.1IAF130968 Pantoea toletana strain A75 16S ribo ... 
dbjlD78008ID78008 Xenorhabdus japonicus DNA for 16S Ribosom .. . 
dbjiD78009ID78009 Xenorhabdus nematophilus DNA for 16S Ribo .. . 
gbjM59155jHAFRR16SA Hafnia alvei 16S ribosomal RNA. 
gblAF130942.1IAF130942 Enterobacter agglomerans strain A74 ... 
642 0.0 
638 0.0 
638 0.0 
636 e-180 
632 e-179 
632 e-179 
632 e-179 
630 e-178 
628 e-178 
624 e-176 
622 e-176 
622 e-176 
618 e-175 
617 e-174 
609 e-172 
609 e-172 
589 e-166 
Fig. 2. BlastN results of search using C. floridanus endosymbiont 16S rRNA sequences showing 
sequence similarities to Gram negative Enterobacteria. 
Electron Microscopy 
Bacteria 
DISCUSSION 
The observed lack of cell walls in the endosymbionts is unlike gram-
negative bacteria, but similar to mycoplasma and the wall-less L forms 
of various bacteria. The fact that we were unable to culture them 
suggests that these bacteria require a specific environment to grow 
perhaps uniquely provided by the follicle's intracellular environment, 
as would be expected from an endosymbiont that has co-evolved with 
its host. Buchnera and Wolbachiaendosymbionts show simil~r require-
ments for their growth (Heddi etal., 1999; Johanowicz and Hoy, 1998). 
Phylogenetic analysis of ovarian endosymbionts: 
The BLASTn search in Fig. 2 displaying sequences in decreasing 
similarity strongly suggests that the closest phylogenetic relative of the 
C. florid.anus endosymbiont is Candidatus camponotii (Schroder et al., 
1996), a previously identified bacterial endosymbiont of Camponotus. 
Listed in decreasing order of similarity with these endosymbiont-
derived sequences are ones obtained from various enterobacteria. 
Interestingly, the endosymbionts of Camponotus sp., though morpho-
logically dissimilar to enterobacteria in lacking cell walls, appear by l 6S 
RNA sequences to be most closely related to the Enterobacteriaceae 
commonly found in the gut of insects and other animals. Corroborative 
evidence for this statement can be found in Table 1. As expected, 
sequences of C. castaneus and C. noveboracensis were more similar to 
Peloquin, J.J. et al.: Endosymbionts of Camponotus 703 
Yersinia 
Buchnera 
balzani 
silvicola 
tortuganus * 
snellingi * 
herculeanus * 
vagus 
japonicus 
pennsylvanicus * 
floridanus * 
socius * 
kiusiuensis 
quadrinotatus 
nawa1 
vitiosus 
rufipes 
impressus * 
Fig. 3. Most parsimonious phylogenetic tree constructed by PAUP (default settings) showing the 
polyphyletic relationship of North American Camponotus endosyrnbionts (with *) when compared 
with Old World symbiont sequences. 
Table 1. BLASTn search results, 10 closest sequences in Genbank: 
Camponotus castaneus 
giJ8250193JembJAJ245598.llEOF245598 C. pennsylvanicus symbiont 16S rRNA 
gil121281SlembJX92551.ljCCL16SR C.camponotii 16S rRNA lignaperda str. 
giJ825019llembJAJ245595.llEOF245595 Endosymbiont 16S RRNA C. socius 
gil1212814JembJX92550.1JCCH16SR C.camponotii 16S rRNA heculeanus strain 
giJ7708202JembiAJ25071S.liCHE250715 C. herculeanus symbiont16S rRNA 
giJ8250192lembJAJ245597.llEOF245597 C. rufipes symbiont 16S rRNA 
gil436814iembJX75274.liYPD16SRN Yersinia pestis (D-28) 16S rRNA 
giJ48619JembiX67464.1JYP16SRRN Y.pestis 16S rRNA gene 
giJ1212816JembJX92552.1JCCR16SR C.camponotii 16S rRNA rufipes strain 
giJ8250189JembJAJ245593.1JEOF245593 Endosymbiont C. sericeiventris 
Camponotus noveboracensis 
giJ7708202JembJAJ250715.llCHE250715 Camponotus herculeanus 16S rRNA 
giJ1212814lemblX92550.llCCH16SR C.camponotii 16S rRNA herculeanus str. 
giJ121281SlemblX92551.1JCCL16SR C.camponotii 16S rRNA ligniperda strain 
giJ8439279JembJAJ245596.llEOF245596 Endosymbiont of Camponotus balzani 
gil8250193JembJAJ245598.1JEOF245598 Endosymbiont of C. pennsylvanicus 
giJ8250190!embJAJ245594.llEOF245594 C. castaneus endosymbiont 16S rRNA 
giJ2570284Jgb!US0207.1JERU80207 Erwinia rubrifaciens 16S rRNA 
gil4582065jembjAJ23341E3.llERU233418 Erwinia rubrifaciens 16S rRNAgil2584756JembJZ96098.1JERZ96098 Erwinia rubrifaciens LMG 2709 16S rRNA 
giJ4754850Jgb!AF130918.lJAF130918 Pantoea agglomerans 16S rRNA 
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Peloquin, J.J. et al.: Endosymbionts of Camponotus 705 
other Camponotus symbionts, than to Yersinia pestis, Erwinia 
rubrifaciens, and Panteoa agglomerans. There is mounting evidence 
that many of the Enterobacteriaceae have properties expected of 
bacteria having a mutualistic relationship with their insect host, 
excepting nitrogen metabolism (Epsky etal., 1997; Epsky et al., 1998; 
Hendrichs et al., 1993; Lauzon et al., 1998; MacCollom et al., 1994; 
Peloquin et al., 2000; Prokopy et al., 1993). 
The most parsimonious phylogenetic tree (Fig. 3) generated by the 
PAUP program for the endosymbionts supported a monophyletic rela-
tionship when the l 6S RNA sequences of other insect-associated 
enterobacteria (Yersinia pestis, Buchnera aphidicola) were included 
(Fig. 3). When additional old world isolates of Camponotus-associated 
eubacterial sequences were examined, using Yersinia, andBuchneraas 
outgroups. a monophyletic grouping of the North American ant endo-
symbionts could not be supported. 
These results suggest that the phylogenetic divergence of the endo-
symbionts and association with their ant hosts is ancient. Additional 
sequence information from the ants may provide more details on these 
relationships (Boursaux-Eude and Gross 2000; Sauer et al. 2000). 
Evidence indicates that competition exists between symbiotic prokruy-
otes within the host organism (Charles et al., 1997; Grenier et al., 1994; 
Heddi etal. 1998; Heddi etal. 1999; Nardon et al. 1998). Progenitors of 
extant prokaryotes may have produced antibiotics or other substances 
that displaced or manipulated the original endosymbionts (Fredenhagen 
et al. 1986; Fredenhagen et al. 1987; Jigami et al. 1986; Nardon et al. 
1992). Alternatively, exposure to some environmental factor may have 
cleared the way for inoculation by other bacteria, which then evolved an 
intimate association with their ant hosts. 
Multiple independent acquisition of endosymbionts would be more 
strongly supported if similar results were obtained from PAUP with 
additional data obtained from other ant endosymbionts. At least one 
other study supports a paraphyletic hypothesis of the origins of ant 
symbiotic bacteria (Sameshima et al. 1999). The association between 
endosymbiont and host maybe dynamic and variable, with competition 
occurring amongst them possibly mediated by antibiotic substances 
(Fredenhagen et al. 1986; Fredenhagen et al. 1987). This constant flux 
of endosymbionts may give new capabilities to the host/ endosymbiont 
association, depending on the properties of the new bacteria. The 
endosymbionts may influence the biology of the association consider-
ably more than is accepted in the traditional concept applied to 
metazoans in which the nuclear genome solely determines the biology 
of the host (Heddi et al., 1999). 
706 Sociobiology Vol. 38, No. 38, 2001 
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